The University of Manchester
Control of Algae in Fuel Storage Ponds
A thesis submitted to The University of Manchester for the degree of
Doctor of Engineering
in the Faculty of Science and Engineering
2019
Julija Konovalovaite
School of Earth and Environmental Sciences
1
Table of Contents
Table of Contents ...... 2
List of figures ...... 7
List of tables ...... 12
List of images ...... 13
Abbreviations ...... 15
Abstract ...... 17
Declaration ...... 19
Copyright statement ...... 20
Acknowledgements ...... 21
1 Introduction ...... 23
1.1 Project context and relevance to the industry ...... 23
1.2 Magnox reactor fuel cycle ...... 25
1.2.1 Spent Magnox fuel storage ...... 25
1.3 The First Generation Magnox Fuel Storage pond ...... 26
1.3.1 History of the pond ...... 26
1.3.2 Structure and processes ...... 27
1.3.3 Conditions in the pond ...... 28
1.3.4 Pond simulant medium ...... 31
1.4 Studies of microbial diversity in First Generation Magnox Storage Pond . 33
1.5 Cyanobacterium Pseudanabaena catenata ...... 34
1.5.1 Cyanobacteria ...... 34
1.5.2 Pseudanabaena catenata ...... 37
1.6 Unwanted algal growth treatment methods ...... 39
1.6.1 Nutrient removal ...... 39
1.6.2 Sonication ...... 40
2 1.6.3 Photocatalysts ...... 42
1.6.4 Oxidation ...... 42
1.6.5 Halogenation ...... 44
1.6.6 Coagulation ...... 45
1.6.7 Filtration ...... 46
1.6.8 Electrochemical methods ...... 47
1.6.9 Barley Straw ...... 48
1.6.10 Biofilm removal ...... 49
1.6.11 Flushing ...... 50
1.6.12 Biocides ...... 50
1.6.13 Selection of appropriate methods for bloom control...... 52
1.7 Rig development ...... 55
1.8 Research objectives...... 57
2 Algal growth in batch cultures ...... 58
2.1 Abstract ...... 58
2.2 Literature review ...... 58
2.2.1 Cyanobacterial adaptations to micro and macro nutrient availability .. 58
2.2.2 Adaptations to highly alkaline conditions ...... 62
2.2.3 Pseudanabaena catenata ...... 63
2.3 Aims of batch culture experiments ...... 67
2.4 Materials and methods ...... 68
2.4.1 Algal culture ...... 68
2.4.2 Effect of reduced inorganic nutrient concentrations: initial experiments 69
2.4.3 Reduced inorganic nutrients- variation in sodium acetate supplements 69
3 2.4.4 Buffered pH experiments ...... 70
2.4.5 Progressive reduction in inorganic nutrient concentrations ...... 71
2.5 Results and discussion...... 73
2.5.1 Impact of nutrient availability ...... 73
2.5.2 Pond water simulation experiment: addition of a carbon source ...... 77
2.5.3 Impact of organic carbon availability at reduced inorganic nutrient concentrations ...... 83
2.5.4 Buffered pH experiment ...... 86
2.5.5 Growth of P. catenata at progressively reduced inorganic nutrient concentrations ...... 88
2.6 Conclusions ...... 101
3 Continuous culture flushing experiments ...... 102
3.1 Abstract ...... 102
3.2 Literature review ...... 102
3.3 Aims of continuous culture experiments ...... 105
3.4 Materials and methods ...... 106
3.4.1 Continuous culture experiments ...... 106
3.4.2 Cell counting ...... 107
3.4.3 Continuous culture: reduced inorganic nutrients ...... 108
3.4.4 Total inorganic carbon / Total organic carbon ...... 108
3.5 Results and discussion...... 110
3.5.1 Continuous culture experiment flushed with BG11 medium ...... 110
3.5.2 Repeated continuous culture experiment ...... 116
3.5.3 Low nutrient flushing experiment ...... 120
3.5.4 Repeated low nutrient experiment ...... 127
3.5.5 Growth rate analysis in continuous cultures ...... 130
4 3.6 Conclusions ...... 133
4 Biocide addition experiments ...... 134
4.1 Abstract ...... 134
4.2 Literature review ...... 134
4.3 Aims ...... 136
4.4 Materials and methods ...... 137
4.4.1 Mexel® 432 experiment ...... 137
4.4.2 Spectrus ® NX1422 biocide trial ...... 140
4.4.3 Repeated experiment ...... 143
4.5 Conclusions ...... 150
5 Prokaryote species succession...... 151
5.1 Abstract ...... 151
5.2 Literature review ...... 151
5.3 Aims ...... 153
5.4 Materials and methods ...... 155
5.4.1 DNA sequencing ...... 155
5.4.1.1 DNA extraction ...... 155
5.4.2 PCR amplification ...... 156
5.4.3 Gel electrophoresis ...... 157
5.4.4 DNA sequencing ...... 158
5.5 Results and discussion...... 160
5.5.1 Repeated continuous culture ...... 160
5.5.2 Low nutrient continuous culture ...... 166
5.5.3 Biocide experiments ...... 170
5.6 Conclusions ...... 177
6 Discussion and Summary ...... 178
5 6.1 Discussion ...... 178
6.2 Summary ...... 180
6.3 Applicability of results to site operations ...... 181
6.4 Conclusions ...... 183
6.5 Scope for future work ...... 184
6.5.1 Effects of changes in growth conditions ...... 185
6.5.2 Experiments with pond culture samples ...... 185
6.5.3 Further biocide trials ...... 185
6.5.4 Continuous culture and scaling experiments ...... 186
6.5.5 Effects of proposed treatment methods on the pond inventory and SIXEP 186
7 References ...... 187
8 Appendix ...... 224
8.1 Appendix 1 ...... 224
8.2 Appendix 2 ...... 227
8.3 Appendix 3 ...... 232
8.4 Appendix 4 ...... 237
6 List of figures Figure 1.1 Nitrate and phosphate concentrations, and volume of water used to flush the pond during the months of May-October in 2014 ...... 29 Figure 1.2. Nitrate, phosphate, and blue green algae concentrations during May- October months in 2014 ...... 30 Figure 2.1. Diagram of ion chromatography process using suppressor and conductometric detector ...... 72 Figure 2.2. Change in turbidity (expressed as absorption) during the first pond water simulant experiment. The error bars show the standard error of the mean value, n=3...... 74 Figure 2.3. Change in chlorophyll a concentrations over the length of the pond water simulant experiment. The error bars show the standard error of the mean value, n=3...... 75 Figure 2.4. Changes in turbidity (absorption) during repeated low nutrient experiment growing P. catenata in BG11 as control, and comparison with Anabaena. The error bars show the standard error of the mean value, n=3...... 78 Figure 2.5. Changes in chlorophyll a concentration during repeated low nutrient experiment growing P. catenata in BG11 as control, and comparison with Anabaena. The error bars show the standard error of the mean value, n=3...... 80 Figure 2.6. Changes in turbidity during repeated low nutrient availability experiment with organic carbon dosing. The error bars show the standard error of the mean value, n=3...... 84 Figure 2.7. Changes in chlorophyll a concentration during repeated low nutrient availability experiment with organic carbon dosing. The error bars show the standard error of the mean value, n=3...... 85 Figure 2.8. Changes in turbidity and control culture pH during the buffered BG11 medium experiment. The error bars show the standard error of the mean value, n=3...... 86 Figure 2.9. Change in chlorophyll a concentrations during buffered BG11 medium experiment. The error bars show the standard error of the mean value, n=3...... 87 Figure 2.10. Growth cycles of all the cultures with progressively lower nutrient concentrations. The error bars show the standard error of the mean value, n=3.The
7 experiments were not continued once two consecutive samples showed reduction in culture concentration...... 91 Figure 2.11. Change in pH during growth cycles of nutrient reduction experiment. The error bars show the standard error of the mean value, n=3. The experiments were not continued once two consecutive samples showed reduction in culture concentration...... 92 Figure 2.12. Changes in external phosphate concentrations. The error bars show the standard error of the mean value, n=3...... 93 Figure 2.13. Changes in external nitrate concentrations. The error bars show the standard error of the mean value, n=3...... 93 Figure 2.14. Culture growth cycles of repeated experiment growing P. catenata in BG11 medium with reduced nitrate and phosphate concentrations. The error bars show the standard error of the mean value, n=3...... 95 Figure 2.15. Change in chlorophyll a concentration during growth cycles of P. catenata grown in progressively lower nutrient concentrations. The error bars show the standard error of the mean value, n=3...... 100 Figure 3.1. Changes in turbidity and pH values during high nutrient continuous culture experiment in flushed and taken directly from the flask samples. The error bars show the instrument error, n=1. The red lines mark changes in culture conditions...... 111 Figure 3.2. Change in chlorophyll a and carotenoid concentrations of flushed and sampled continuous culture over the length of the experiment. The error bars show 5% error, n=1...... 112 Figure 3.3. Changes in turbidity and pH values during repeated high nutrient continuous culture experiment. The error bars show the instrument error, n=1. The red lines mark changes in culture growth conditions ...... 116 Figure 3.4. Change in chlorophyll a and carotenoid concentrations in repeated continuous culture in flasks A and B. The error bars show the 5% error, n=1...... 117 Figure 3.5. Total and inorganic carbon concentrations in duplicate flasks A and B during repeated high nutrient continuous culture experiment. The error bars show the variance coefficient, n=1...... 119
8 Figure 3.6. Changes in turbidity and pH values during the first reduced nutrient continuous culture. The red lines mark the start of flushing and increases to medium pH. The error bars show the instrument error, n=1...... 121 Figure 3.7. Change in chlorophyll a and carotenoid concentrations over the length of the first low nutrient experiment. Error bars show the 5% error, n=1...... 123 Figure 3.8. Change in phosphate and nitrate concentrations during the low nutrient continuous culture experiment. The error bars mark standard mean error, n=1. .. 124 Figure 3.9. Changes in total and inorganic carbon concentrations during low nutrient continuous culture experiment. The error bars show the standard error of the mean, n=1...... 125 Figure 3.10. Change in turbidity and pH values over the length of the repeated low nutrient experiment, the flushing with pH 11medium started on day 8. The error bars show the instrument error, n=1...... 128 Figure 3.11. Variations in chlorophyll a and carotenoid concentrations in repeated low nutrient continuous culture experiment. The error bars show 5% error, n=1. 129 Figure 4.1. Changes in turbidity during the Mexel biocide dosing experiment. The dosing of biocide was done on day 9. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed...... 138 Figure 4.2. Changes in chlorophyll a concentrations during the Mexel® biocide dosing experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed...... 139 Figure 4.3. Change in turbidity during the Spectrus® biocide trial with dosing on day 9. The error bars represent the standard error of the mean value, n=3. The red dot marks the day biocide was dosed...... 141 Figure 4.4. Changes in chlorophyll a concentrations during Spectrus® biocide dosing experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed...... 142 Figure 4.5. Changes in culture turbidity during repeated biocide trial with dosing on day 13. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed...... 145
9 Figure 4.6. Changes in chlorophyll a concentrations during repeated biocide experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed...... 146 Figure 5.1. Changes in culture composition during the continuous culture experiment in duplicate A ...... 165 Figure 5.2. Changes in culture composition during the continuous culture experiment in duplicate B ...... 165 Figure 5.3. Change in culture composition during the low nutrient continuous culture experiment, day 0- at the start of the experiment, 7- before flushing started, day 26- before medium pH increase to 8.5, day 35 -before medium pH increase to 10, day 45- before medium pH increase to 11, day 54- before medium pH increase to 11.5, day 66- before medium pH increase to 12, day 84- the last day of the experiment...... 169 Figure 5.4. Changes in control culture composition during the experiment ...... 173 Figure 5.6. Changes in culture composition before and 2 days after biocide dosing ...... 174 Figure 5.7. Changes in culture composition before and 2 days after biocide dosing ...... 174 Figure 5.5. Changes in culture composition before, 2 days after biocide dosing, and at the end of the experiment...... 174 Figure 5.8. Changes in culture composition before, 2 days after biocide dosing, and at the end of the experiment ...... 175 Figure 5.9. Changes in culture composition before and 2 days after biocide dosing ...... 175 Figure 5.10. Changes in culture composition before and 2 days after biocide dosing ...... 175 Figure 8.1. Changes in pH values during pond water simulant experiment. The error bars show the standard error of the mean, n=3...... 227 Figure 8.2. Changes in pH values during pond water simulation experiment with carbon source addition. The error bars show the standard error of the mean, n=3...... 227
10 Figure 8.3. Changes in average pH values during the organic carbon availability experiment. The error bars show the standard error of the mean, n=3...... 228 Figure 8.4. Changes in average pH values during buffered pH experiment. The error bars show the standard error of the mean, n=3...... 228 Figure 8.5. Change in carotenoid concentration during repeated Mexel® 432 biocide trial with biocide dosing on day 15. The error bars show the standard error of the mean, n=3...... 243 Figure 8.6. Change in carotenoid concentration during Spectrus® NX 1422® biocide experiment with dosing on day 9. The error bars show the standard error of the mean, n=3...... 244 Figure 8.7. Change in pH during the first Spectrus® biocide dosing experiment. The error bars show the standard error of the mean, n=3...... 244 Figure 8.8. Change in carotenoid concentrations during the repeated Mexel® 432 and Spectrus® NX® 1422 experiment with biocides dosed on day 13. The error bars show the standard error of the mean, n=3...... 245 Figure 8.9. Change in pH during the repeated biocide experiment with both the Mexel® and Spectrus® biocides dosed on day 13. The error bars show the standard error of the mean, n=3...... 245
11 List of tables
Table 1.1 Organic carbon concentrations in 2009-2012 ...... 29 Table 1.2. Pond water simulant medium recipe at highest nutrient modification ... 32 Table 1.3 Analyte concentrations in the pond over year 2014 ...... 32 Table 1.4. Pond water simulant medium nutrient concentrations (highest concentration modification) ...... 32 Table 2.1. Nitrate and phosphate concentrations in reduced nutrient BG11 medium ...... 70 Table 2.2. Volumes of buffer used to adjust medium pH ...... 71 Table 2.3.Nitrate and phosphate concentrations in modified BG11 media ...... 71 Table 2.4. The three pond water simulant modifications with the same nitrate and phosphate availability as measured in pond...... 73 Table 2.5. List of all modifications, nitrate, phosphate concentration, nitrogen and phosphorus ratio, and nitrate and phosphate ratio ...... 99 Table 3.1. Comparison of P. catenata doubling times in different continuous cultures in BG11 and reduced nutrient media. CCBG11- First continuous culture grown in BG11 medium, CCA- duplicate A of repeated high nutrient continuous culture, CCB- duplicate B of repeated high nutrient continuous culture, CCRNA- first reduced nutrient continuous culture, CCRNB- repeated reduced nutrient continuous culture...... 132
12 List of images Image 1.1 The experimental set up showing batch experiments on the left and continuous culture (high nutrient continuous culture at the time) on the right...... 56 Image 2.1. From left to right: P. catenata in pond simulant medium, P. catenata in RNBG11 medium, Anabaena, and control P. catenata in BG11 medium on day 4. .. 83 Image 4.1. Cultures 2 days after dosing, from left to right: Spectrus® NX 1422 with 100, 50, 20 ppm, Mexel® 432 100, 50, 20 ppm and control ...... 147 Image 8.1. P. catenata grown in pond simulant medium in lowest nutrient concentrations on day 0 (top) and day 13 (bottom), before the addition of BG11 medium ...... 229 Image 8.2. P. catenata in pond water simulant medium with the highest nutrient concentrations on day 0 (left) and day 13 (right), before the addition of BG11 medium ...... 230 Image 8.3. Cultures grown in reduced nutrient BG11 medium (top) and pond water simulant medium dosed with acetate (bottom) on day 18 ...... 231 Image 8.4.Reduced nutrient continuous culture on day 0 (top), day 7 before culture flushing (bottom) ...... 232 Image 8.5. Reduced nutrient continuous culture on day 26 before pH increase to 8.5 in medium (top), and on day 54 (left) before culture was started to be flushed at pH 11.5 ...... 233 Image 8.6.Reduced nutrient continuous culture on day 66 (top) before flushing with pH 12 medium, and on the last day (84) of the experiment (bottom) ...... 234 Image 8.7.Repeated low nutrient continuous culture on day 0 (top), day 8 (bottom), before flushing with pH 11 medium...... 235 Image 8.8. Repeated low nutrient continuous culture on day 17 at the end of the experiment ...... 236 Image 8.9. Control culture of P. catenata on day 13 ...... 237 Image 8.10. Culture to be dosed with 20 ppm (top), and 50 ppm (bottom) of Mexel® 432 biocide before dosing on day 13 ...... 238 Image 8.11. Culture to be dosed with 20 ppm (top) and 50 ppm (bottom) of Spectrus® NX 1422 biocide before dosing on day 13 ...... 239
13 Image 8.12. Control (top), culture dosed with 20 ppm Mexel® 432 (bottom) biocide on day 15, 2 days after dosing with biocide ...... 240 Image 8.13. Culture dosed with 20 ppm Spectrus® NX 1422 biocide on day 15, 2 days after dosing with biocide ...... 241 Image 8.14. Control (top), and P. catenata dosed with 50 ppm of Mexel® 432 (bottom) on day 20, last day of the experiment ...... 242 Image 8.15. Cultures dosed with 50 ppm Spectrus NX® 1422 biocide on day 20 (bottom), last day of the experiment ...... 243
14 Abbreviations FGMSP - First Generation Magnox Pond
LWR - Light water reactor
BWR - Boiling water reactor
AGR - Advanced gas reactor
PUREX - Plutonium uranium redox extraction
DNA - Deoxyribonucleic acid
NTU - Nephelometric Turbidity Unit
MPFC - Magnetic polyferric chloride
PFC - Polyferric chloride
CTAB - N-cetyl-N-N-N-trimethyl ammonium bromide
RN BG11 - Reduced nutrient Blue Green medium
SDS - Sodium dodecyl sulfate
PCR - Polymerase chain reaction
OTU - Operational taxonomic unit
NDIR - Non-dispersive infrared
SIXEP - Sellafield ion exchange effluent plant
FHP - Fuel handling plant
IPP - Independent pond purge
ATP- Adenosine triphosphate
UV-B - Ultraviolet radiation (280-315 nm)
UV-C - Ultraviolet radiation (100-280 nm)
PAC - Polyaluminium chloride
TOC - Total organic carbon 15
TIC - Total inorganic carbon
TC - Total carbon rRNA - Ribosomal ribonucleic acid
16 Abstract
The First Generation Magnox Storage pond is one of the most hazardous and high risk facilities on the Sellafield site, due to the inventory of Magnox fuel, sludge and other wastes, and the age and condition of the pond structure. Retrieval of the waste and its transfer to safer modern storage is one of the highest priority programmes on the site. This already complicated task can be further hindered by the appearance of microbial blooms during the warmer months of the year. The blooms can cause significant loss of visibility, severely hampering the majority of waste retrieval operations such as those implemented through the operation of remotely operated vehicles. The resultant delays to waste retrieval risks extending the FGMSP programme for several years. One of the primary colonisers of the pond has been identified as a cyanobacterium Pseudanabaena catenata. The aim of this research project was to better understand growth of P. catenata in laboratory cultures that mimic pond conditions, to determine if changes can be made to the pond environment that could reduce the impact of the bloom events or prevent blooms from occurring, and to investigate whether biocides could be used for microbial growth control. Pseudanabaena catenata was grown in batch cultures in pond simulant medium to determine how cultures behave in low nutrient and highly alkaline environments. Further experiments included those using cultures grown in batch conditions in progressively lower nutrient concentrations, to discover the optimum nutrient ratio for growth and to better understand the adaptation processes supporting growth in the pond environment. Batch experiments were concluded with Mexel® 432 and Spectrus® NX 1422 biocide trials for bloom removal. Continuous culture experiments were used to assess the ability of P. catenata to adapt to flushing, nutrient renewal in high and low nutrient concentrations, and increases in pH due to sodium hydroxide dosing. Turbidity, cell and pigment concentrations, and culture pH were used to evaluate culture growth and activity. Information about nutrient requirements was obtained using ion chromatography. The culture used for these experiments was not axenic, and therefore changes in microbial community composition caused by biocide dosing, flushing, or sodium hydroxide dosing were determined by 16S rRNA gene
17 sequencing. The results presented provide insight into how biomass concentrations may be controlled by reduced nutrient levels, and the optimum nitrogen and phosphorus ratios supporting growth. The biocide dosing experiments helped to conclude that the lowest effective dose for bloom reduction was the same for both biocides trialled at 50 ppm. The continuous culture experiments helped to identify the highest doubling rates for the cultures grown in low and high nutrient concentrations, and effects of flushing, and the highest pH (11.5) that cultures can adapt to without reductions in cell concentrations. Results from the continuous culture experiments also suggest that flushing combined with increasing the pH values of the ponds could be used for bloom control. However, this may be only effective when the change is sudden, making it crucial to time this control strategy to the start of the bloom.
45,582 words
18 Declaration
No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.
19
Copyright statement
i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance Presentation of Theses Policy with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses
20 Acknowledgements
To my supervisors Prof Jonathan R Lloyd, Dr Genevieve Boshoff, Dr David Sigee, Dr Jon Pittman for all the help and support, reading my thesis on trains and planes, understanding that I was a clueless engineer at the start of the EngD and helping me to take the baby steps into the world of microbiology, and leading me to become still, but less, clueless engineer, and answering all my random questions of the day.
To Dr Adam Qaisar for proofreading, dog sitting, and all the distractions I may not have necessarily needed, which included thrashing a Porsche at Silverstone on the day of hand in.
To Dr Jay Dunsford and Dr Jonathan Dodds for proof reading and dealing with slightly hyper-active me when I just bombarded them with questions and text to read.
Simon Kellet for understanding when nothing was happening and all the slow progress and providing information and guidance about the industry.
FGMSP team- for information and help provided, answering emails, and that very useful site visit.
Christopher Boothman, Alastair Bewsher, and Dr Alex Baidak- for doing or helping me with analytical techniques that as far as I was concerned were “magic”, even though I still think that DNA sequencing is.
Lynn Foster- for help with cultures, sharing important papers, and not minding me poking my nose in her project just because I found it so interesting.
Norma Watson- for listening, supporting me through most of the EngD, helping with paperwork and not retiring until I was nearly done with my lab work. And once she did, still checking up on me.
Effluents Team at NNL Workington from taking over duties of smiling and nodding politely while I moaned about my EngD and offloaded all the information they really “needed” to know once Norma left.
21 Dr Gareth Shepphard and Dr Dominic Rhodes- for being my “personal libraries” about historical information and inviting a non-drinker to countless whisky nights.
Geomicrobiology group- for help and support, and welcoming me as if I was always based in their offices even though I would show up once a blue moon
To staff at NNL- for treating me like one of their own, listening to me complaining, losing all hope when things weren’t going right, being happy for me when they did, and totally “not” making fun of my shed, which is awesome, by the way.
DCF residents- for trying to make me a bit more social and included in Manchester university community in Cumbria and help during the last months or so before hand in deadline answering all the practical about it.
And finally, Mom and sister, and the extended family and friends- for being OK with even less communication than normal, not complaining about me visiting them once a year (at least it was for Christmas), and two last weeks before handing in of radio silence.
22 1 Introduction
1.1 Project context and relevance to the industry The UK has many nuclear sites that need to be decommissioned, most of them being non-operational nuclear power plants. However, one of the familiar and most complicated is the Sellafield site. Over its lifetime, the site has moved from an ordinance factory, to plutonium production facility, later to a commercial nuclear power plant site, and finally, a reprocessing facility. In the present day, it is a site used for storing and preparing the majority of the UK’s high level and spent fuel waste for disposal, while also undergoing decommissioning of legacy facilities. In 2016/17, out of £3.243bn of funding, the National Decommissioning Authority (NDA) allocated £2.002bn to Sellafield Ltd, and another £2bn for the 2017/2018 financial year, highlighting the complexity and cost of work being undertaken (Annon, 2017a).
The First Generation Magnox Storage Pond (FGMSP) is one of the most heavily contaminated locations on site and considered one of the highest hazard and risk facilities in the United Kingdom. It is one of the main decommissioning priorities due to the radioactivity of the pond and its inventory. It is also located in one of the most congested areas of Sellafield, complicating retrieval operations and limiting changes that could be done to this historic structure. For FGMSP to be fully decommissioned, all the inventory in the pond, including sludge, must be removed and treated accordingly, with the objective of making the waste safer for storage on site before eventual disposal. Sludge retrievals are planned to run between 2015 and 2027, and will cost an estimated £340 million alone (Annon, 2017b). Any delays to the sludge or inventory retrievals could result in delays to the decommissioning programme and increased costs of the project. The FGMSP can experience blooms during the warmer months of the year causing loss of visibility and affecting remotely operated vehicle operations. One of the microorganisms identified as a primary coloniser of the pond is Pseudanabaena catenata, found in samples taken during bloom and non-bloom periods (Evans, 2014; Foster, 2014).
23 The aim of the project is to better understand the growth of P. catenata under various nutrient conditions, explore the behaviour of this organism in continuous culture, and identify possible methods of bloom prevention and control, including the use of biocides. The research outcomes could serve as a basis for better understanding microbial growth in the FGMSP and identify areas of pond management that could be improved. It could also result in bloom prevention, or minimised disruption to pond operations, leading to reduced operational downtime and limiting financial implications. The research could also lead to further understanding of the microorganism and its behaviour in low nutrient conditions, highly alkaline environments, and the ability to adapt to sudden changes in the environment.
24 1.2 Magnox reactor fuel cycle Magnox reactors use natural uranium metal fuel (Knott, 2014; Wiesenack, 2012). It is manufactured by mixing uranium(IV) fluoride (UF4) with magnesium, followed by heating to 600°C. The molten metal formed at the bottom of the furnace is allowed to cool down and is re-melted to cast it into fuel rods,that are machined to the required shape and inserted into magnesium alloy tubes (Wiesenack, 2012). Fuel rod designs differ from plant to plant, but the key features are similar. The fuel rods have “fins” with grooves covering the rod for better heat dissipation. Up to thirteen fuel elements are used in a fuel channel, eight being the most common number (Knott, 2014). The fuel cladding in the UK is a mixture of magnesium and small amounts of aluminium or zirconium (Dawson and Phillips, 2012). Like the fuel rods, the reactor designs are different at each site. The first reactors had external heat exchangers at external buildings with exposed ductwork, and steel was used for the pressure vessels. Later designs moved to enclosed pre-stressed concrete pressure vessels and were further improved by moving heat exchangers inside the reactor (Dawson and Phillips, 2012). Magnox reactors used carbon dioxide gas to provide cooling and graphite as a moderator. The carbon dioxide gas is used to extract heat produced in the core and is pumped through to a heat exchanger. The gas is then used to generate steam which is sent to a steam turbine to generate electricity (Knott, 2014). Early Magnox reactor designs were designed for fuel to be inserted horizontally. Later reactors were designed with vertical channels, and are only able to be refuelled after reactor shut down. As with refuelling, with time reactor design changed for it to be re-fuelled on-load. The current strategy of Magnox reactor decommissioning is to remove everything surrounding the bioshield and empty the vaults after removal of fuel, leave the main reactor building into “period of care and maintenance” for activity levels to drop, and once that is achieved, to demolish all the structures. (Dawson and Phillips, 2012)
1.2.1 Spent Magnox fuel storage The chosen storage method for spent Magnox fuel is to store it in water filled fuel ponds. Open top mild steel skips were used, except for Hunterston A where aluminium skips were used to store the fuel. Due to the fact that fuel elements are
25 clad in magnesium, the ponds must be kept at alkaline conditions: >pH11 to avoid combustion of the cladding and cooled to a temperature around 13°C. If stored for a long-time, fuel can corrode, especially if conditions are deviating significantly from the optimum. The danger of fuel corrosion is that fission products/fissile material can contaminate cooling pond water. The first cooling ponds were open to air resulting in ingress of dust and debris, and difficulties in controlling conditions in the pond. At certain locations, foaming due to high winds became an issue, with added problems of airborne activity spreading to the surrounding area; later built ponds were roofed. Ponds required clean up plants to be built. The plants were designed with sand bed filters to remove particulate matter in the form of magnesium and aluminium hydroxide particulates, and strontium. The effluents are then treated in ion exchange beds to remove soluble caesium and strontium isotopes. Some ponds were fitted with ion resin exchange cartridges with pre- and post-filters as means of controlling activity levels. Pond water treatment creates several wastes; resins from ion exchangers, sludges mostly containing magnesium and aluminium hydroxides. (Dawson and Phillips, 2012)
1.3 The First Generation Magnox Fuel Storage pond
1.3.1 History of the pond The pond was designed to be used as a cooling and de-canning facility for Magnox fuel before reprocessing. The first part of the First Generation Magnox Fuel Storage Pond was constructed during the second half of the 1950’s, with commissioning being completed in 1959. It consisted of an inlet facility and wet bay for fuel de- cladding. There were two extensions added to the pond, one in 1964, and another in 1977 due to the expansion of the Magnox programme. The extensions added dry de-cladding facilities known as cells. In 1973-1974 fuel was stored for longer periods than it was intended in the design of the pond due to the outage of fuel processing. The increased storage time resulted in corrosion of the Magnox fuel cladding forming sludges. The plant ceased operations in 1986 after the newly constructed Fuel Handling Plant was commissioned. The pond and the associated facilities were then left in a care and maintenance state and now is being prepared for decommissioning. At present, the pond contains Magnox fuel, sludge, and other
26 intermediate level wastes. The first fuel and sludge retrievals started in 2015. The pond is located in a congested area on the Sellafield site, making design changes and decommissioning of the plant more complicated. Even though the pond is highly radioactive and is kept at alkaline pH it can experience blooms. The blooms caused by photosynthetic microorganisms can appear in summer months reducing visibility and causing delays in planned in-pond operations. Some of the most heavily impacted operations are those using Remotely Operated Vehicles and Skip Handler Machine due to the reduced visibility. Current decommissioning efforts are focused on removal of fuel and sludge from the pond. The sludge is sent to a Sludge Packaging Plant, where it is safely stored in buffer storage vessels. Fuel with related wastes is moved to the Fuel Handling Plant or to Self Shielded Boxes. Once the pond has been emptied of wastes, water will be removed, treated and remaining structures demolished (Jowsey, 2017).
1.3.2 Structure and processes The pond itself is divided into sub-ponds and holds total 14000 m3 water, is 5 m deep. It is purged to prevent radioactive build-up and for pond chemistry management with reused pond water from the Fuel Handling Plant (FHP) and with Independent Pond Purge (IPP). The flow rate of only the IPP can be regulated, purge from the FHP can only be on or off. The residence time in the pond is determined by the total volume received in 24 hours. With 24 hours of flushing with both purge streams operating the shortest residence time is ~10 days. To prevent corrosion of the Magnox fuel cladding, pH is kept at 11-11.6. The independent pond purge provides caustic dosing to keep pond at the required pH, and to cancel out carbonation. The Water Treatment Plant provides both, demineralised water and caustic feed separately, to the IPP, where they are mixed to reach values of 11.5 for purge water feed. After passing through the pond, water is sent to sandbed filters to remove particulate matter as part of Sellafield Ion eXchange Effluent Plant (SIXEP) process where Cs-137, and Sr-90 are removed from the effluent using ion exchangers. The highly alkaline pH also helps to reduce the solubility of Magnox fuel (Bradford et al., 1976). Magnesium is mostly found as brucite (Mg(OH)2), magnesium cladding corrosion product. Magnesium is a competing ion in the ion
27 exchanger and can lead to early breakthrough of Sr-90 and Cs-137. Concentrations under 1 mg/l are enough to disturb the operations and highest target concentration in the pond is 0.2 mg/l (Kellet, 2017). The processed liquor can be disposed into the sea, provided it meets Environment Agency approved discharge permit (Annon, 2010a). The pond has several probes strategically placed for best coverage that measure pH, temperature, chlorophyll a concentrations, blue green algae concentrations, fluorescent dissolved organic matter, conductivity, and turbidity, and are located around 1 m below the water surface. In addition, pond liquor samples are taken for additional analysis and some of the analytes monitored can be found in Table 1.3. The radioactivity of pond water samples limits the number of samples that can be collected. The least amount of data is available for nitrate and phosphate concentrations, with significant gaps in the dataset as it is not considered necessary for pond radioactivity management. This makes understanding changes in conditions in the pond more difficult and harder to estimate nutrient availability for the microorganisms. The year 2014 was picked as a data set to be used as basis for experiment design, as in a three-year period, nitrate and phosphate concentrations were highest in 2014, and a bloom occurred. In addition, culture composition analysis was also done in 2014.
1.3.3 Conditions in the pond From Table 1.3 it can be seen that average and median values of the micro and macro nutrients are of similar concentrations, except for nitrate and phosphate. By comparing the highest and lowest values over the year of all the monitored ions, concentrations vary significantly over the year, but most of the data shows concentrations close to average and median values. Compared to the three media (recipes can be found in the Appendix 1) the micro and macro nutrient concentrations are significantly lower in the pond. The availability of organic carbon is also quite low in the pond, as seen from . There is a significant variation between the highest and lowest values over the three-year period (2009-2012). Concentrations were towards the lower range in the more recent years. In addition, values below 7 ppm are indicated to be below method detection limit. The temperature data is significantly different between sample temperature and probe
28 data. Sample temperature seems to be closer to ~20°C, whereas probe data would be closer to 16°C. This may be due to the different locations of sampling, indicating lower temperatures deeper in the pond.
Daily flushing volume vs nitrate and phosphate concentrations 9 1600
8 1400 7
1200 3 6 1000 5 800 4 600
3 Volume received Volume received m
Nitrate/phosphatemg/l 400 2
1 200
0 0 11-04 01-05 21-05 10-06 30-06 20-07 09-08 29-08 18-09 08-10 28-10 Date Nitrate Phosphate Volume received
Figure 1.1 Nitrate and phosphate concentrations, and volume of water used to flush the pond during the months of May-October in 2014
Table 1.1 Organic carbon concentrations in 2009-2012
TOC (ppm) DOC (ppm) Highest 56 38 Average 6.581152 5.125654 Median 4 3 Lowest 0 0
From the available data it can be observed, that the most significant increases in nitrate and phosphate concentrations seem to coincide with decreasing total purge volume received (see Figure 1.1). It should be noted, that the significantly higher values could be due to scaling of the results, as samples are diluted for analysis. However, the samples would indicate available, and not total phosphate and nitrate concentrations, therefore, the results have been used as a worst-case scenario. The
29 Figure 1.1 also indicates the gaps in data making it difficult to determine the nutrient availability in the pond. However, during a period of low purge volumes received at the end of July-start of August, no increase in nutrient concentrations was measured, however, one of the reasons could be the start of the bloom event, shown in Figure 1.2.
Nitrate, phosphate vs blue greens
9 0.8
8 0.7
7 0.6 6 0.5 5 0.4 4 0.3 3
2 0.2 Blue algae green µg/l Nitrate/phosphatemg/l 1 0.1 0 0 11-04 01-05 21-05 10-06 30-06 20-07 09-08 29-08 18-09 08-10 28-10 Date Nitrate Phosphate Blue greens
Figure 1.2. Nitrate, phosphate, and blue green algae concentrations during May-October months in 2014
It is not known if microorganisms, especially ones capable of photosynthesis are evenly dispersed in the pond or tend to be closer to the surface and the ambient light. The bloom event (e.g. increase in microorganism concentration), also followed a period of low flushing, as assumed from the data. It can also be seen, that there was a significant delay between increase in blue green concentration using a probe, and the highest nitrate and phosphate concentrations measured earlier in the year (Figure 1.2). However, there are clear gaps in the data, making it impossible to say whether there were no further increases in nutrient concentrations. The bloom appeared in the pond quite quickly, judging from the data available, when concentrations increased from ~0.21 to ~0.67 µg/l in 12 days. Using these values, the doubling time estimate would be ~10.36 days, assuming no purging, due to the low volume of water received during this period. It is important
30 to stress, that due to the limited data and variation in measured concentration of blue green algae with time, this is a rough estimate that could only be used to compare experimental data with conditions in the pond for general representation of applicability. Once the bloom started to develop, the volume of purge water was increased resulting in a decrease in blue green algae concentration. It took over 20 days of purging at high volume to return to blue green algae concentration to levels as observed in the early summer.
1.3.4 Pond simulant medium A pond simulant medium was designed from the available data. Year 2014 was used as a basis to determine the lowest, average, and highest concentrations of nutrients as found in the pond with average values used for the medium. Three media recipes were used to determine the most common micro and macro nutrients required: Z8, Jaworski’s, and BG11 medium, as they were most reported in literature for growing P. catenata. Table 1.3 shows pond water chemistry, and Jaworski’s, BG11, and Z8 (see Appendix 1) media chemistry in unmodified forms. In addition to the monitored micronutrients, iron, copper, cobalt, manganese, and molybdenum were added to the medium recipe to prevent nutrient limitation. Iron, copper, and cobalt concentrations were chosen to be the lower concentration from the media recipes of the three media. Molybdenum loading was chosen from Z8 medium. To enable adjustment of the pH sodium carbonate was used adjust to sodium concentration to required values, but sodium hydroxide could be used instead if high pH of the medium is required. The final recipe and nutrient concentrations can be seen in Table 1.2 and Table 1.4.
31 Table 1.2. Pond water simulant medium recipe at highest nutrient modification
Reagent CaCl2.2H H3BO3 K2HPO4 Na2HPO4. NaNO3 Na2CO3 (CH3CO2)2 MgSO4. EDTAFe CuSO4.5 Co(NO3) MnCl2.4 NaMoO4 Al(OH)3 2O 12H2O Zn.2H2O 7H2O Na H2O 2.6H2O H2O .2H2O mg/l 0.724 0.039 0.916 27.907 3.535 179.955 3.116 0.618 2.529 0.079 0.049 0.18 0.031 0.333
Table 1.3 Analyte concentrations in the pond over year 2014
Al Ca Cl (mg/l) K (mg/l) Mg Na NO3 pH PO4 (mg/l) SO4 Temperature Zn (mg/l) B (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Min 0.052 0.07 0.1 0 0 72.7 0 10.9 0 0 18 0.1 0 Max 0.54 0.37 1.7 0.26 1.12 88.2 2.6 11.6 7.9 0.6 25.3 1.5 0.037 Avg 0.115 0.197 0.383 0.103 0.061 79.108 0.088 11.3 0.212 0.239 21.229 0.928 0.007 Med 0.11 0.19 0.3 0.1 0.04 78.7 0.03 11.3 0 0.2 21.1 0.985 0.007
Table 1.4. Pond water simulant medium nutrient concentrations (highest concentration modification)
Al Ca Cl K Mg Na NO3 PO4 SO4 Zn B Fe Cu Co Mn Mo Concentrations used (mg/l) 0.115 0.197 0.383 0.103 0.061 79.108 2.6 7.9 0.239 0.928 0.007 0.342 0.020 0.010 0.050 0.012
32 1.4 Studies of microbial diversity in First Generation Magnox Storage Pond Photosynthetic microorganisms residing in ponds with seemingly unfavourable conditions are a long-known issue. There have been reports of algae adapting to acidic and radioactive conditions in uranium mines in less than 60 years (García- Balboa et al., 2013), with algae found in ponds with pH up to 10 and high radioactivity (Emery and Mcshane, 1978).
The FGMSP was the subject of two PhD projects characterising the microbial diversity in the pond. The indigenous microbial community was dominated by Proteobacteria, the most abundant genus being Pseudomonas. There were also representatives of Plantomycetes, Nitrosprae, Gemmatimonadetes, Firmicutes, Elustimicrobia, Cyanobacteria, Chloroflexi, Bacteroidetes, Armatimonadetes, Actinobacteria, Acidobactera and other less abundant phyla in samples taken in 2012 when the pond was not in bloom (Evans, 2014). The most dominant cyanobacterium identified was most closely related to Pseudanabaena catenata (Evans, 2014). In samples taken during 2014 bloom the cultures were still dominated by Proteobacteria; Bacteroidetes, Firmicutes, Actinobacteria, Cyanobacteria, were also present (Foster, 2014). The cyanobacterium was identified as Pseudanabaena catenata (Foster, 2014). Due to the repeated presence, it was decided that Pseudanabaena catenata is one of the primary photosynthetic colonisers in the pond and would be used as the main organism of interest for this project. Later characterisation showed dominance of Proteobacteria, and presence of Bacteroidetes, Planctomycetes, Cyanobacteria, Actinobacteria in samples collected during a 2016 bloom. Again the most dominant cyanobacterium was classified as Pseudanabaena catenata confirming it as an important microorganism in the ponds ecosystem (Foster, 2017).
33 1.5 Cyanobacterium Pseudanabaena catenata
1.5.1 Cyanobacteria Cyanobacteria are oxygenic photosynthetic bacteria that lack a nucleus and internal membrane systems (Sigee, 2005). The main components of the cyanobacterial cell are thylakoids, deoxyribonucleic acid (DNA), 70S ribosomes, gas vacuoles, polyglucan granules, cyanophycin granules, lipid droplets, and polyhedral bodies (Bold, 1985). They can be found in unicellular, colonial or filamentous forms with the size varying from 0.5 to 40 μm in diameter (Bellinger and Sigee, 2011; Darley, 1982; Madigan et al., 2010). Some filamentous cyanobacteria can branch; true branching is continuous from the same filament and false branching is caused by a break in the filament (Drouet, 1968). The cell morphology varies greatly with environmental conditions (Darley, 1982). Cyanobacterial cells produce mucilaginous sheaths to bind the cells together as colonies or filaments (Fritsch, 1945; Madigan et al., 2010). The contents of the cell are enclosed within a plasma membrane (Fogg, 1973). Below the outer membrane there is a periplasmic space separated by the peptidoglycan layer, housing transport proteins (Gantt, 1994; Nikaido and Saier, 1992). A peptidoglycan layer serves a protective function as well as controlling the shape of the cell by its rigidity (Gantt, 1994).
The intracytoplasmic membranes house the photosynthetic apparatus of cyanobacteria, similar to chloroplast membranes found in algae and plants (Carr and Whitton, 1982). Cyanobacteria have thylakoids, flattened membranous sacks, but unlike in algae they are not enclosed forming organelles (Lang and Rae, 1967). In most cases, the photosynthetic apparatus is located in thylakoids and only in rare cases the plasma membrane can act as a photosynthetic membrane (Gantt, 1994). The thylakoid membrane is covered in phycobilisomes, light harvesting antennae (Vasil, 2012). Polypeptides making the phycobilisomes are phycobiliproteins, linker polypeptides, and phycobilisome associated proteins (Sidler, 1994). The exact protein composition varies depending on species of cyanobacteria and environmental conditions: nutrient availability, lighting, temperature (Rai, 2001; Sidler, 1994). The size of light-harvesting system is light quality dependent to protect the photosynthetic apparatus, it gets smaller with more intense light (Rai,
34 2001). Ratio between chlorophyll a/phycobiliproteins are used to assess the system size (Rai, 2001).
During photosynthesis, red and blue visible light is absorbed by the chlorophylls, while green, yellow and orange light is absorbed using phycobilins (Sidler, 1994). They can extend the absorbed light range for chlorophyll a light at 430-665 nm (Sidler, 1994). The maximum absorption of phycoerythrin is at 565 nm, and the biliprotein light absorption range is between 500 and 650 nm (Carr and Whitton, 1982). In species with chromatic adaptation, green light stimulates production of phycoerythrin, and in red light, phycocyanin (Carr and Whitton, 1982). Phycobiliprotein would be synthesised during dark periods if cyanobacteria had been illuminated with coloured light prior; red and green colour light cause phycoerythrin and phycocyanin respectively (Fujita and Hattori, 1960a; Fujita and Hattori, 1960b; Fujita and Hattori, 1962). Carotenoids, other pigments found in cyanobacteria have two main functions: to protect the cell from photo oxidative damage in the photosynthetic apparatus and for light harvesting (Fogg, 1973; Hirschberg and Chamovitz, 1994). Carotenoids are either carotenes- hydrocarbons or xanthophylls- oxygen containing derivatives (Fogg, 1973).
Maximum photosynthetic rates rise with temperature (Carr and Whitton, 1982). Under very high light levels the rate of photosynthesis can be reduced due to photorespiration and photo oxidative damage (Carr and Whitton, 1982). The damage increases with the length of high light intensity conditions due to increase in concentrations of oxidants (Carr and Whitton, 1982). It has been suggested that cyanobacterial cell growth does not differ significantly if the culture is grown under continuous or intermittent illumination (Fogg, 1973).
Cyanobacteria can contain gas vacuoles. They are cylindrical in shape, but variable in length depending on species and are made from hollow membranes, stacked closely together (Bowen and Jensen, 1965). The gas vacuole size in a culture is related to the volume of gas required for the cells to buoyant (Fogg, 1941). It is thought that gas vacuoles form from small gas vesicles, then grow lengthwise and fill with gas (Waaland and Branton, 1969; Walsby, 1969). This has been seen in both, new forming vacuoles and vacuoles after collapsing (Bowen and Jensen;
35 Lehmann and Jost, 1971; Walsby, 1969). The membrane of the vacuole is permeable to gases (Walsby, 1969) especially oxygen, argon, nitrogen, carbon dioxide and monoxide, hydrogen, and methane (Walsby, 1971).
Cyanobacteria prefer to accumulate nutrients before starting a growth phase (Jia et al., 2013). It is reported, that the reaction of cyanobacteria to nutrient limitations depends on species, time of day and season, and growth medium (Domingues et al., 2011; Fujita, 1985; Gao et al., 2013; Martínez et al., 2012; Qi et al., 2013; Stehfest et al., 2005; Xu et al., 2013). Cyanobacterial cells often experience bleaching when pigments degrade or after division with stopped pigment synthesis due to nutrient deficient conditions (Grossman et al., 1994).
Growth of cyanobacteria outside optimal temperature conditions will result in lower yields, but only extreme temperatures at the low or high ends of the spectrum can stop cell growth (Weiss et al., 1985). Lipid production and saturation, and reaction rates are most affected by changes in temperature and response will depend on the species (Cade-Menun and Paytan, 2010). Low temperatures could cause physical damage in cyanobacteria by ice formation and also slow biochemical reactions. (Fogg, 1973). The reaction to changes in temperature and lighting conditions is species specific, with pronounced variations in the severity of the reaction (Cade-Menun and Paytan, 2010; Jia et al., 2013). It has been suggested that cyanobacteria can synthesise stress protein after being exposed to elevated temperatures (Borbely and Suranyi, 1988; Borbely et al., 1985).
Cyanobacteria are reported to be very susceptible to UV-B (320-290 nm) light, which can affect growth, pigments, photosynthesis, enzymes required for nitrogen uptake, and CO2 uptake (Sinha and Hader, 1998; Rajeshwar P. Sinha et al., 1995; R. P. Sinha et al., 1995; Sinha et al., 1996). Phycobiliproteins can be bleached as they are very sensitive to UV light (Aráoz and Häder, 1997; Rajeshwar P. Sinha et al., 1995; Sinha et al., 1996). Under weak UV-B stress, increased production of phycobiliproteins has been reported; it was suggested that this may be a stress response and a protective measure (Aráoz and Häder, 1997) as phycobiliproteins can prevent 99% of UV-B radiation from reaching DNA (Rai, 2001). Some cultures experience ammonium uptake reduction of up to 10% under UV-B light (Rai, 2001).
36 It also has been reported that nitrogenase activity was weakened after a few minutes and completely stopped within 35-55minutes of UV-B light in some cyanobacterial species (Kumar et al., 1996). It has also been shown that cyanobacterial cells are more UV resistant when visible light is also present compared to grown in dark conditions (Fogg, 1973).
To survive in highly radioactive environments cyanobacteria have adapted to use Mn2+ to form peroxide rather than superoxide, which is used to prevent and minimise damage to cellular proteins (Seckbach et al., 2013). However, it has been reported, that alkaline environments may have reduced levels of bioavailable Mn2+, therefore, algal defence against radiation can be less effective (Seckbach et al., 2013).
1.5.2 Pseudanabaena catenata Pseudanabaena catenata is a filamentous non heterocystous cyanobacterium (Loza et al., 2014). It is a highly adaptable species that was found in various climatic conditions: from tropical (Dos Santos and Calijuri, 1998) to tundra (Richter et al., 2015), and in various environments: lakes in Brazil (Dos Santos and Calijuri, 1998), French alps (Cellamare et al., 2010) and High Atlas mountains (Oudra et al., 2009), river Nile (Mohamed et al., 2006), attached to travertine (Pentecost et al., 1997), or in peat and calcareous sediment in a tarn in Yorkshire (Round, 1953). The culture abundance varied from dominant (Dos Santos and Calijuri, 1998; Hurtado et al., 2008) in high nutrient conditions, to background cultures in low nutrient availability (Cellamare et al., 2010; Kalff and Watson, 1986; Padisák et al., 2003; Pentecost et al., 1997; Richter et al., 2015; Sompong et al., 2005). It has mainly been reported in brackish and freshwater environments (Acinas et al., 2008); oligotrophic (Padisák et al., 1998), mesotrophic (Cellamare et al., 2010), and eutrophic (Patterson and Wilson, 1995; Richter et al., 2015) water bodies. They can be found in benthic (Padisák et al., 2003), periphytic (Padisák et al., 2003), hypolimnion (Gervais et al., 2003) zones, in one case it has been observed to be attached to Microcystis aeruginosa colonies for buoyancy (Patterson and Wilson, 1995). The pH of water bodies was mainly neutral or slightly alkaline (Hurtado et al., 2008; Mercado, 2003; Oudra et al., 2009; Padisák et al., 1998; Pentecost et al., 1997). However,
37 Pseudanabaena sp. was also found in alkaline lake with pH values increasing up to 10.77 without any alkaline pH input and mainly due to microbial photosynthetic activity (López-Archilla et al., 2004). In addition, some gliding motility has been observed as well (Acinas et al., 2008).
Pseudanabaena catenata when grown in liquid medium was suspended in the culture, but a “biofilm” layer could be seen on the flask walls and base of older cultures (Khan et al., 2017). When grown on agar, no colonial gliding or motility was observed (Khan et al., 2017). When grown under varying daylight and temperature regimes, it reached fastest growth rate at 15°C, and 12:12 dark-light regime (Khan et al., 2017). The slowest growth was during 24-hour illumination, and when grown at 25°C. Under 24-hour illumination P. catenata growth was least affected by temperature (4°C, 15°C, 25°C), and most by illumination (Khan et al., 2017). Pseudanabaena catenata varies in morphology depending on temperature and length of illuminating period, when the largest dimensions in cell length and width were observed under 12:12 h light-dark illumination. However, longer illumination periods and increased temperatures can cell dimensions. (Khan et al., 2017)
Species of the Pseudanabaena genus can adapt chromatically, adjusting the pigments produced for the most efficient light absorption (Acinas et al., 2008; Grossman et al., 1993; Kehoe and Gutu, 2006). However, not all the strains were capable of it as some isolates had phycoerythrin and phycocyanin, some only had phycocyanin (Acinas et al., 2008). Under continuous green or red light illumination Pseudanabaena reached steady state in pigment concentration in 10 days for both wavelengths (Stomp et al., 2008). However, compared to non-flexible cultures adapted to only red or green light, P. catenata had lower critical light intensity (Stomp et al., 2008). In addition, for P. catenata to change the pigment production when light was changed from green light to red, it took about 7 days , and 8 to 9 days from red to green (Stomp et al., 2008). In experiments with mixed cultures, Pseudanabaena catenata coexisted with cultures adapted to a narrower wavelength when light was continuous. Under fluctuating light conditions Pseudanabaena catenata outcompeted other cultures (Stomp et al., 2008). Under fast light fluctuation, Pseudanabaena produced a mixture of pigments to cope with
38 changing in available light, with light changing twice a day (Stomp et al., 2008). Under slow fluctuations, with light changing every 4 days, pigment composition varied with changes in light, but would not shift to one pigment completely (Stomp et al., 2008). Under light fluctuations every 28 days, Pseudanabaena changed pigment composition completely, resulting in fluctuation in population density as well (Stomp et al., 2008). It was also noted, that chromatic adaptation for this species is only beneficial if there is sufficient time for the pigment composition adjustment (Stomp et al., 2008).
1.6 Unwanted algal growth treatment methods There are numerous methods available for algal and cyanobacterial growth prevention and removal. Unfortunately, due to the specific pond conditions and limitations in applicability most of them would not be effective. The following section will discuss a selection of control strategies, and the reasoning why certain ones have been chosen to be used in the experimental programme.
1.6.1 Nutrient removal Nutrient limitation should be considered as a first method of algal bloom control as it is a preventative method (Sigee, 2005). It could be achieved by limiting loading or removing the available nutrients. There are several chemical compounds that could be used for nutrient removal. Copper(II) sulfate (CuSO4) can reduce nitrogen uptake in low cell densities in concentrations as low as 0.005 mg/l (Elder and Horne, 1978;
Peterson et al., 1995). It was also observed that addition on CuSO4 increases cell lysis in cyanobacteria which increases with concentration, and damage to cells increases with longer exposure, however, the effects last only for up to 3 days (Fan et al., 2013b; Fitzgerald, 1964). Coagulation can also be one of the means to reduce nutrients in water. Ferric sulfate, aluminium sulfate and polyaluminium chloride
(PAC) have all been shown to reduce ortho-phosphate (PO4) and phosphorous significantly with and without aid of anionic polyacrylamide (Aguilar et al., 2005).
- However, PO4 binding is weaker in alkaline conditions compared to more acidic conditions, thus limiting the application in the pond (Boisvert et al., 1997). Granular activated carbon showed best results in adsorption of organic nitrogen and
39 phosphorous, whereas calcium carbonate based reagent performed better in removing inorganic nitrogen (Wendling et al., 2013).
Not only chemical, but also biological methods are used to reduce nutrient availability. Lake bacteria have been shown to compete with algae and can reduce nutrient concentration and reduce algal growth due to the lowered nutrient availability, however, aeration reduces the effectiveness of the removal efficiency
(Schmack et al., 2012). Bacteria also improve NOx and NH4 removal with no significant difference if aeration is used, but algal population shortly recovers and continuous dosing may be required due to nutrient recycling in the system (Schmack et al., 2012). It was also noted, that high pH and oversaturation with dissolved oxygen may be more favourable to algae and limit the effectiveness of bacterial treatment (Schmack et al., 2012).
1.6.2 Sonication Sonication has been used as a control method for the prevention of algal and cyanobacterial growth (Mason and Phillip, 2002; Rajasekhar et al., 2012b; Wu et al., 2012), however, sonication has been shown to have a maximal effect on cyanobacterial cells with gas vacuoles (Tang et al., 2004). Sonication disrupts growth by affecting the cell membrane, photosynthesis, cell multiplication, metabolic activity as well as collapsing gas vacuoles in cyanobacterial cells (Ahn et al., 2003; Hao et al., 2004a; Hao et al., 2004b; Jong Lee et al., 2000; Lee et al., 2001; Rajasekhar et al., 2012a; Rajasekhar et al., 2012b; Tang et al., 2004; Wu et al., 2012; Xu et al., 2006; Zhang et al., 2006a; Zhang et al., 2006b; Zhang et al., 2006c). The principle of sonication is based on ultrasonic radiation with frequency higher than 20 kHz (Annon) generating cavitation bubbles (Rajasekhar et al., 2012b). During collapse of the bubbles, the temperature can reach up to 5000°C and pressure can increase up to 100 MPa (Suslick, 1990). In addition, sonication is also capable of generating free radicals that have damaging effects on the algal cell wall and cause intracellular matter leakage resulting in cell death (Tang et al., 2004). However, the generation of free radicals is limited to the period of sonication as they are short lived, highly reactive species (Ahn et al., 2003; Tang et al., 2004). Numerous studies have concluded that the efficiency of treatment by sonication depends on several
40 factors including: frequency (kHz), power supplied per volume of growth medium and the exposure time (Hao et al., 2004a; Hao et al., 2004b; Joyce et al., 2010; Lee et al., 2001; Rajasekhar et al., 2012a; Rajasekhar et al., 2012b; Suslick, 1990; Zhang et al., 2006a; Zhang et al., 2006c).
Even though sonication can remove a significant proportion of cyanobacteria from suspension, the effects are short lived and cell growth can recover afterwards (Ahn et al., 2003; Hao et al., 2004b; Jong Lee et al., 2000; Rajasekhar et al., 2012a; Tang et al., 2004; Zhang et al., 2006c). The culture growth stage also has an impact on the treatment efficiency with the best results obtained during exponential or stable stages of algal growth [193, 196]. The time of day the treatment is applied can significantly affect its effectiveness as cells are most susceptible after cellular division (Ahn et al., 2003) and illumination was shown to be necessary for cyanobacteria to regenerate gas vacuoles after sonication (Jong Lee et al., 2000). The effects of sonication can be observed for prolonged periods of time as sedimentation can continue for more than a week after a treatment (Rodriguez- Molares et al., 2014). Power was deemed to be the controlling factor for photosynthesis disruption, with sensitivity being species specific (Joyce et al., 2010; Lee et al., 2001; Rajasekhar et al., 2012b). In addition, in some species sonication can cause a minor decrease in the chlorophyll a content of a cell (Ahn et al., 2003; Hao et al., 2004b; Xu et al., 2006; Zhang et al., 2006c). Although reactions to sonication of filamentous algae are similar it also depends on species and morphology with cell lysis being observed at both high and low frequency ranges (Purcell et al., 2013a).
Sonication was most effective when combined with other types of treatments (Ahn et al., 2003; Gavand et al., 2007; Heng et al., 2009; Nakano et al., 2001; Tabrizi and Mehrvar, 2004; Waite et al., 2003; Zhang et al., 2006a). Sonication coupled with PAC treatment increased efficiency with increasing coagulant dose under optimal conditions of 8-9 pH and coagulant dose of 3 mg/l, and 1 minute sonication time (Zhang et al., 2006a; Zhang et al., 2009). Water samples from natural environments has increased dose requirements of PAC compared to synthetic water used in laboratory studies, due to the natural water chemistry (Zhang et al., 2009).
41 However, coagulation enhanced by sonication would pose a problem of increased waste for disposal.
1.6.3 Photocatalysts As algae and cyanobacteria require light for growth, photocatalysts could be used for growth control (Obregón Alfaro et al., 2010; Peller et al., 2007). Using non-toxic and otherwise stable compounds could provide a long-term solution, as catalyst losses in the process are minimal. There have been several compounds tested for algal bloom control, including TiO2, WO3, Bi2MoO6, Bi2WO6, Bi2W2O9. The catalysts can be used in various forms- they can be immobilised on a non-reactive surface or used in a powdered form (Peller et al., 2007). It has been observed that the oxidation takes place at the surface of the catalyst; therefore, the most appropriate form of treatment requires maximised available surface area of the catalyst for maximum exposure (Hong and Otaki, 2006; Obregón Alfaro et al., 2010; Peller et al.,
2007). The TiO2 used as a photocatalyst mode of action in algal removal is oxidation, with a capability to damage the cell and cause intracellular compounds, including DNA, to leak out of the cell, as well as photosynthetic activity and lowering chlorophyll content, and breaking up colonies (Fahl et al., 1995; Hong et al., 2005; Kim and Lee, 2005; Metzler et al., 2011; Peller et al., 2007). The suppression of algal cell growth is dose, length of treatment, oxygen availability, and species dependant (Hong and Otaki, 2006; Jae-Jin et al., 2008; Kim and Lee, 2005; Obregón Alfaro et al., 2010; Tao et al., 2010). Timing of the treatment in the growth cycle can affect results further, with younger and older cells being more susceptible to photocatalytic treatment (Metzler et al., 2011). Efficiency of treatment with TiO2 and WO3 also increases with the use of Pt and Ir as treatment aids (Linkous et al., 2000).
1.6.4 Oxidation Use of oxidants was also proposed for microbial growth removal. Ozone, potassium permanganate, as well as hydrogen peroxide were used in numerous studies to assess suitability for treatment. Ozone is favoured due to the generation of radicals at >pH 8 that are more reactive compared to the parent compounds (Cloete et al., 1992; Cloete et al., 1998; Fan et al., 2013b; Li et al., 2011). At higher pH, the primary
42 initiator of ozone decomposition is the hydroxide (OH-) anion, increasing in more alkaline conditions (Li et al., 2011; Widrig et al., 1996). Even at low concentrations ozone can affect vital parts of the cell: prevent biofilm formation and remove bio fouling, damage cell wall, induce cell lysis, and release of intracellular matter, full effects seen more than 24 hours later (Fan et al., 2013b; Fan et al., 2014; Huang et al., 2006; Li et al., 2011; Miao and Tao, 2009; Videla, 2002). Solubility of ozone decreases with increasing temperature (Roth and Sullivan, 1981), however, the removal rate of algae increases (Farooq et al., 1977). This could pose a problem for algal control using ozone during warmer months, as both effects must be assed to understand the requirements for sufficient removal. Ozone has several drawbacks: itit is unstable in water and treatment efficiency is affected by water chemistry, it can lead to corrosion and reactions with organic compounds (Cloete et al., 1998; Huang et al., 2006; Videla, 2002). It was noted, that oxidation should be timed at the start of the bloom when cell density is low (Fan et al., 2013b). Ozonation can cause algae to form clusters in some species as a defence response and protection mechanism to minimise the damage (Li et al., 2011).
Hydrogen peroxide is capable of damaging the cell membrane and inducing cell lysis (Fan et al., 2014). It could be used in systems with metals present, but concentration should be kept relatively low, 50-100 ppm reported to be substantial to reduce the microorganism concentration (Fan et al., 2013b; Videla, 2002). The effects of hydrogen peroxide increase with increasing concentration and treatment results can be seen after 2 days, although, after day 3, the number of intact cells starts to recover (Fan et al., 2013b). Hydrogen peroxide also leaves a high residual concentration after treatment (Fan et al., 2013b).
Potassium permanganate was shown to be effective in algal growth treatment, in some cases resulting in cell lysis, the effects of peroxidation being concentration dependent (Chen and Yeh, 2005; Fan et al., 2013a; Fan et al., 2013b; Fan et al., 2014; Wang Female et al., 2013). The effectiveness of potassium permanganate increases with increasing dose (Chen and Yeh, 2005; Fitzgerald, 1964), although the concentration of potassium permanganate above 3 mg/l can have damaging effects on the cyanobacterial cells (Chen and Yeh, 2005; Fan et al., 2013a; Fan et al., 2013b;
43 Fan et al., 2014; Wang Female et al., 2013). Reduction in cell density was observed only with 10 mg/l concentration (Fan et al., 2013a; Fitzgerald, 1964). In addition, potassium permanganate has been shown to be more efficient in algal removal compared to hydrogen permanganate alone (Chen and Yeh, 2005).
1.6.5 Halogenation Chlorine as an algicide can be used in gaseous form, with the mode of action for chlorine compounds being formation of oxidizing agents (Maranda et al., 2013; Ou et al., 2011). It was proposed, that the mechanisms of chlorination appear in the following order: penetration, internal degradation and release of intracellular matter (Ou et al., 2011). Chlorine can damage the cell membrane leading to cell lysis and release of intracellular compounds, cause changes to cell size and shape (Daly et al., 2007; Fan et al., 2014; Ma et al., 2012a; Ou et al., 2011). Effects of chlorine are also species dependent, and efficiency is affected by contact time and water chemistry (Daly et al., 2007; Junli et al., 1997; Ou et al., 2011). The initial chlorine concentration drops significantly in few minutes after the introduction with the largest drop during the first minutes followed by a slower chlorine release (Daly et al., 2007; Fan et al., 2013b; Ma et al., 2012a). It is then quickly dispersed and residual chlorine is present an hour after if initial concentration is higher than 4 mg/l, depending on the culture concentration (Fan et al., 2013b). In addition, higher chlorine doses lead to increased consumption (Ma et al., 2012a). Hypochlorous acid is more reactive compared to hypochlorite due to the formation of free chlorine (Ou et al., 2011). In alkaline pH it forms hypochlorous ions that have very limited biocidal properties (Cloete et al., 1998; Videla, 2002). In addition, use of chlorine compounds can also affects the water pH (Maranda et al., 2013; Ou et al., 2011).
Bromine is less sensitive to changes in pH and is less volatile compared to chlorine, but only 50% of hypobromous acid is present at pH higher than 8.7 (Videla, 2002). Bromine compounds are less affected by ammonia compared to chlorine (Videla, 2002). As with oxygen compounds, chlorine and bromine compounds can lead to corrosion as well as interfere with other compounds in water bodies (Videla, 2002).
44 1.6.6 Coagulation Coagulants work by changing surface charge and bonding particles or organisms into flocs to force precipitation (Shen et al., 2011). Their effectiveness can be affected by pH, as well as temperature (Aguilar et al., 2005; Divakaran and Sivasankara Pillai, 2002; Fast et al., 2014; Jiang et al., 2010; Smith and Davis, 2012; Widrig et al., 1996; Wu et al., 2011). Furthermore, excessive doses can results in decreased efficiency (Beach et al., 2012; Fast et al., 2014; Gerde et al., 2014; Jiang et al., 2010). In low pH coagulants surface is negatively charged and at high pH the surface is positively charged, although the charge is coagulant specific (Aguilar et al., 2005). It was also noted, that the required coagulant dose is more surface charge rather than surface area dependent (Henderson et al., 2008). Furthermore, coagulant dose increase is required for higher cell concentrations (Fast et al., 2014; Gerde et al., 2014; Wyatt et al., 2012; Zheng et al., 2012). In addition, ionic strength and salinity can also affect the coagulant effectiveness (Chen et al., 1998; Gerde et al., 2014; Pan et al., 2011; Zheng et al., 2012). No damage to the cells or no lysis was observed after most coagulants were used (Chow et al., 1998; Divakaran and Sivasankara Pillai, 2002; Pan et al., 2011; Pierce et al., 2004; F. Sun et al., 2013; Sun et al., 2012; Zheng et al., 2012). For best results sufficient mixing is required to evenly disperse coagulants (F. Sun et al., 2013; Sun et al., 2012).
Polyaluminium chloride (PAC), aluminium sulfate are more effective in more acidic conditions (Aguilar et al., 2005; Wu et al., 2011), chitosan, potassium ferrate, cationic starch DS05 in conditions close to neutral (Aguilar et al., 2005; Divakaran and Sivasankara Pillai, 2002; Fast et al., 2014; Gerde et al., 2014), and magnesium salts in alkaline conditions (Smith and Davis, 2012). Performance of the coagulants can be enhanced by use pre-treatment methods, but pre-treatment periods can result in cell lysis, release of extracellular material and reduced efficiency (Chen and Yeh, 2005; Chen et al., 2009; Ma and Liu, 2002; Ma et al., 2012b; Ma et al., 2012c; Paralkar and Edzwald, 1996; Wang Female et al., 2013; Zhao and Zhang, 2011).
The mode of action of aluminium sulfate (Al2(SO4)3) is via formation of aquametal complexes that lead to mono and polynuclear species that adsorb to negative colloid surfaces (Fast et al., 2014). As with most coagulants, the efficiency increased
45 with treatment time (Fast et al., 2014; Sridhar et al., 1988). Another coagulant chitosan works by binding to algal cells, bridging and adsorbing to other particles to form flocs (Beach et al., 2012; Fast et al., 2014). Chitosan efficiency was reported to be species dependant (Pan et al., 2011). Clays can also be used as coagulants, but it would require doses of up to 200 mg/l which would pose a waste disposal problem (Jiang and Kim, 2008). PAC and chitosan aids with sand work by attracting cells, bridging smaller flocs together and acting as ballast to improve settling (Pan et al., 2011). N-Cetyl-N-N-N-trimethylammonium bromide (CTAB) is a cationic catcher used in algae flotation (Chen et al., 1998). It is may be used in alkaline conditions as pH increase to 9 resulted in the efficiency of CTAB to drop only 5% in otherwise identical conditions (Chen et al., 1998).
Polyferric chloride (PFC) mode of action is charge neutralisation (Jiang et al., 2010). Magnetic polyferric chloride (MPFC) combines effects of magnetic nanoparticles and PFC through adsorption by formation of Fe3O4-cell complex (Jiang et al., 2010). Polyferric chloride used in algae flocculation speciates according to pH with cationic forming below 8 and anionic above it (Wyatt et al., 2012). It was also shown, that in alkaline conditions MPFC has better settling rate compared to non-magnetic coagulant (Jiang et al., 2010). In addition, ferric chloride reduces water pH due to formation of ferric hydroxide (Wyatt et al., 2012).
1.6.7 Filtration The efficiency and performance of filtration membranes are dependent on the running set up: membrane material, pore size; algal cell concentrations, and algal cell characteristics (Rossignol et al., 1999). Microfiltration and ultrafiltration membrane efficiency is also temperature dependant (X. Sun et al., 2013). Increasing pressure can also have a positive effect on the efficiency of filtration, but the performance will start to decrease again once the optimum pressure has been surpassed, due to damage to the membranes, depending on membrane in use (Babel and Takizawa, 2010; X. Sun et al., 2013). Membrane pore size may not be the most important parameter as algal cells can form a cake on the membrane; acting as cell size determining filter (Castaing et al., 2010; Rickman et al., 2012; X. Sun et al., 2013). In addition, attention should be paid to irreversible fouling that could be
46 caused by internal deposition (Castaing et al., 2010; Castaing et al., 2011; Rickman et al., 2012). The irreversible part of fouling in organic filtration membranes does not increase after numerous treatments and provided stable filtration of algae (De Gouvion Saint Cyr et al., 2014). Fouling is also dependant on the algae filtered as organic polymers and excreted organic matter may be one of the main causes of fouling (Babel and Takizawa, 2010; Castaing et al., 2011; Liang et al., 2008). In mixed culture media, bacteria and debris are the main causes of membrane fouling (Castaing et al., 2011; De Gouvion Saint Cyr et al., 2014).
The membrane material also plays a key role as hydrophilic membranes may perform better than hydrophobic membranes due to possible excessive fouling (Rossignol et al., 1999; X. Sun et al., 2013). Hydrophobic properties of membranes are also dependant on the angle a membrane is used at according to the water flow (Nurra et al., 2014). As zeta potential (ζ ) changes with pH range for membranes, it is important to choose one that will be least prone to fouling in the algal growth medium according to pH (Nurra et al., 2014). Some ultrafiltration membranes are also pH sensitive with best performance at pH close to neutral and decreasing efficiency and increasing fouling in alkaline and acidic conditions (Dong et al., 2006; Gao et al., 2012). Due to membrane material response to pH, choice of filtration membrane should be made according to material properties with less importance to the cut off size (Nurra et al., 2014). Vibrating membrane modules have been shown to increase filtration membrane efficiency in a cross-flow set up (Nurra et al., 2014).
1.6.8 Electrochemical methods Electric fields generated using aluminium electrodes can also be used to remove cyanobacteria and algae (Alfafara et al., 2002; Sridhar et al., 1988). It has been shown that an applied electric field can damage algae cells without clear damage on membrane, therefore, reducing cell lysis (Gao et al., 2010a). Electrolysis may also destroy or change electron transfer between phycocyanins and chlorophyll a (Xu et al., 2007). Due to the electrical field created by the electrodes and aluminium in the water algae form flocs and rise to the surface forming a mat after the treatment (Sridhar et al., 1988). In highly alkaline environments the main
47 mechanism of algae removal is thought to be enmeshment and sweeping flocculation (Gao et al., 2010b). With increasing current density or power treatment time drops significantly and chlorophyll a removal increases using aluminium electrodes (Gao et al., 2010a; Gao et al., 2010b; Xu et al., 2007). In addition, increase in temperature aides, but increase in pH and culture density decreases effectiveness of aluminium electrodes (Alfafara et al., 2002; Gao et al., 2010b).Aluminium electrodes seem to be more effective in cyanobacterial removal compared to iron electrodes (Gao et al., 2010b). Electro flotation compared to flocculation- flotation was reported to have half the removal efficiency (Alfafara et al., 2002; Gao et al., 2010a). For flocculation, the electrode placement is preferred to be below the algae to optimise contact between the algae and the hydrogen bubbles generated for flotation (Alfafara et al., 2002). It was also noted that a slight temperature increase may happen around the electrodes as well as a significant increase in pH when the system is not continuous (Alfafara et al., 2002; Gao et al., 2010b). Total organic matter could also decrease slightly due to flocculation as well (Alfafara et al., 2002). Removal efficiency can be improved by mixing the liquid at low current densities (Alfafara et al., 2002).
Ti-RuO2 anodes and graphite anodes were also shown to inhibit algal growth (Xu et al., 2007). In addition, no significant difference was observed between the graphite and steel cathodes used with Ti-RuO2 (Xu et al., 2007). It was also noted that the effect of this electrochemical treatment was based on cell oxidation (Xu et al., 2007). It was suggested, that an anode has more of an effect on the cell inhibition than a cathode (Xu et al., 2007).
1.6.9 Barley Straw Barley straw has been shown to be an effective method for removal of cyanobacteria, and more so compared to algae (Ferrier et al., 2005; Purcell et al., 2013b). Barley straw has a specific pattern of inhibition: the lag phase where there is no effect on the algae growth inhibition and algal culture restoration (Murray et al., 2010). It was reported, that the method of algal control using barley straw was a suppression of new cell growth (Murray et al., 2010). The effects of barley straw are species specific, and may result in shift in culture composition, rather than
48 complete removal (Ferrier et al., 2005; Iredale et al., 2012; Purcell et al., 2013b). On the other hand, barley straw can have promotional effects on some species of algae (Ferrier et al., 2005). In addition, sterile, filtered, or unfiltered medium may have an effect on the barley straw treatment (Ferrier et al., 2005). It was concluded that the dosage and preparation of barley straw also affects the inhibition of algae and cyanobacteria (Ball et al., 2001; Ferrier et al., 2005; Iredale et al., 2012; Murray et al., 2010). Barley straw showed the best results when used after 8 weeks of rotting and 4 weeks after dosing of pre-rotten barley (Iredale et al., 2012; Murray et al., 2010). The straw size also has influence on inhibition of cyanobacteria as smaller pieces have a reduced lag phase (Iredale et al., 2012). The increase in treatment temperature had a positive effect on algal inhibition as well (Iredale et al., 2012). It was also noted, that UV light can act as an aid in cyanobacterial growth inhibition and the straw would become more effective earlier compared to the samples treated without UV light, speculated to be due to hydrogen peroxide production (Iredale et al., 2012). Timing of the treatment should be planned for straw inhibition to be effective during the exponential algal growth phase if prediction of bloom event is possible (Murray et al., 2010).
1.6.10 Biofilm removal The algaecide Diuron® can be effective for the algal biofilm removal. Diuron® affected photosynthesis of the biofilm from the first day and throughout the laboratory studies, and caused a shift in culture composition (Ricart et al., 2009). It was observed, that chlorophyll a concentration increased with increasing Diuron® concentration to adjust for decreased efficiency of chloroplasts (Ricart et al., 2009). No increase in tolerance to the algaecide could be seen over the course of the study (Ricart et al., 2009).
Copper can also be used to reduce algal biofilm population, but it was observed, that algae present in biofilm is less sensitive compared to planktonic algae by up to 100 times (Barranguet et al., 2000). The higher resistance has been attributed to a dense colony surrounded by mucus as well as biofilm metal binding sites on the cell surface and the matrix (Barranguet et al., 2000). Thickness of the biofilm also has an effect on the resistance to copper as in thicker biofilms copper penetration is
49 slower and surface area compared to volume is lower compared to thinner biofilms (Barranguet et al., 2000).
1.6.11 Flushing Flushing can be used to reduce the concentration of limiting nutrients as well as to reduce the algal concentration due to increased water exchange (Cooke et al., 2005; Pfafflin and Ziegler, 2006). With significantly increased water volumes, dilution can be achieved which can have a similar effect without the increase in water movement (Cooke et al., 2005). Dilution works best in waters with low nutrient concentration and the water used for flushing should have lower nutrient concentrations than the water body with algal cultures (Cooke et al., 2005). In high nutrient concentration waters flushing is effective if the cell growth rate is lower than the cell loss rate due to flushing (Cooke et al., 2005). Vertical mixing can occur during treatment which would lead to re-suspension of particles from the bottom of the water body (Cooke et al., 2005). The rate of water exchange has to increase significantly compared to initial flushing rate to have a sizeable effect, 10-15% has been reported to be a sufficient change (Welch and Patmont, 1980). Flushing should also be used continuously as water quality can deteriorate once the flushing is stopped (Cooke et al., 2005). Furthermore, flushing seems to be more effective on cyanobacteria compared to algae (Hudnell et al., 2010).
1.6.12 Biocides Biocides could be one of the most applicable approaches for algal and cyanobacterial bloom control. This type of treatment also benefits from vast underpinning research and the wide range of commercially available biocides. However, a huge choice of biocides also has several major drawbacks- biocide will have a different mode of action, and varying species removal depending on dosing regimens (Marvá et al., 2010) and effectiveness, according to water chemistry and pH (Videla, 2002). Algicides can be categorised according to their mode of action on algae, for example: action on reproduction, inhibition of photosynthesis, cell division and cell growth (Neuwoehner et al., 2008).
Some of the algicides found naturally include quaternary ammonium salts. Their mode of action on algae is via damage to the cell membrane (Cloete et al., 1992).
50 The inhibition of cell growth increases with increasing concentration of ammonium salts and longer periods of treatment (Kong et al., 2013). Streptomyces sp. KY-34 fermentation liquid with an unknown active compound was also shown to have the ability to inhibit cyanobacteria growth and even to reduce chlorophyll a and photosynthetic pigments with concentration of 3% and higher (Kong et al., 2013). Some damage to the cell membrane was observed with it being detached after prolonged periods of treatment (Kong et al., 2013). It is also thought that higher concentrations of the algaecide could suppress protein production in algal cells (Kong et al., 2013).
Sulfonylurea herbicides are more effective for cyanobacterial control compared to algae with effective concentrations as low as 1 nM (Nyström et al., 1999). Sensitivity of algae to these herbicides are not only concentration, but also species dependant and dosage can vary up to three orders of magnitude (Nyström et al., 1999). The mode of action for these compounds is based on inhibition of production of branch-chained amino acids (Nyström et al., 1999), although, sulfonylurea herbicide effectiveness can be reduced by high concentrations of amino acids and water pH (Nyström et al., 1999). Copper(II) sulfate has also been reported to reduce algal growth. Copper(II) sulfate will only become effective when concentrations of available copper ion (Cu2+) rises to toxic levels (Elder and Horne, 1978; Masuda and Boyd, 1993). The effects of copper biocides are affected by water chemistry in the water body and the availability of copper to cultures before the treatment (De Oliveira-Filho et al., 2004; Elder and Horne, 1978). For example, the speciation of copper in water is very sensitive to changes in the pH (Elder and Horne, 1978; Masuda and Boyd, 1993). At high pH, copper will form copper(II) carbonate (CuCO3) therefore, reducing algicidal properties (Masuda and Boyd, 1993).Treatments with copper are more effective at low pH as Cu2+ solubility decreases with increasing pH as well (Masuda and Boyd, 1993; United States. Public Health Service. Division of Water Pollution et al., 1959).The co-application of ethylenediaminetetraacetic acid (EDTA) as an aid to copper(II) sulfate was less efficient compared to copper(II) sulfate alone (Elder and Horne, 1978). It was also observed that in nutrient rich conditions, the effects of copper(II) sulfate with or
51 without the addition of EDTA were very limited (Elder and Horne, 1978). High organic carbon concentrations can also reduce the effectiveness of the treatment further due to chelation (Elder and Horne, 1978). Water bodies with increased nitrogen concentrations also have reduced effectiveness of treatment with copper(II) sulfate (Elder and Horne, 1978).
1.6.13 Selection of appropriate methods for bloom control Although numerous treatment methods are available, most of them are unsuitable, or have limited applicability to site operations. Even though nutrient removal would be the preferred option, the pond is open to air and is in a flow through system arrangement, therefore limiting the nutrient ingress control options. Sonication could be an attractive option, as it does not involve any chemical means. The use of sonication could have no or very limited effects on the pond inventory and water chemistry. However, if use of sonication would lead to cell lysis it would result in release of nutrients back into the pond to be recycled within the microbial community. Furthermore, if only the gas vesicles are damaged the cells could settle before they are flushed out, the microorganisms could regrow vacuoles and resurface, limiting the treatment efficiency. Photocatalysts would not be a viable option in powdered form as it would be flushed out or may act as a catalysts to unwanted chemical reactions in the pond. In addition, the effects on the sandbed filters and ion exchanger are also unclear. Immobilised photocatalysts would require a large contact area, therefore would have to be suspended in the water, possibly reducing available light and visibility for the remote vehicles operating in the pond. In addition, to be effective in the pond large amounts of the catalyst would be required resulting in a significant cost. Oxidation and halogenations would not be viable options due to the pH of the pond and possible chemical reactions with the pond inventory.
Even though coagulation can be used either to settle or aid cyanobacterial flocculation and floating, coagulants are pH sensitive. However, there are coagulants that perform better in alkaline environments than others. The major disadvantage of coagulants is the large doses of coagulants required, especially if they are physical, rather than chemical. Addition of physical coagulant would
52 increase sludge loading in the pond, addition of chemical coagulants could lead to unwanted chemical reactions. Furthermore, not only microorganisms, but also suspended solids could floc and increase loading on the treatment plants downstream. Most of the coagulants do not damage the cyanobacterial cells either, thus the growth of the unwanted organisms could recover. Finally, the coagulated and settled or floating flocs would have to be collected and separated, introducing additional operations into the pond management.
Due to the size of the pond filtration would have to be applied in the outlet of the plant and water recycled back to the pond to decrease unwanted microorganism concentration. In addition, either the filter area or flow rate through the filter would have to be significant to have any effect on the pond, resulting in increase in running costs. During the bloom events, filtration units may clog or would need to be backwashed at an increased rate, increasing waste to be disposed of. Furthermore, depending on the membrane cut off size additional suspended particles could also be removed, resulting in increased activity loading in the filtration system.
Electrochemical methods would not be applicable to site operations either, due to the size of the pond, long treatment times, large number of probes required for effective treatment, and current increasing running costs. Another possible treatment option- barley straw have some major drawbacks to pond application as well: large quantity of straw required due to the size of the pond, unpredictable window of effectiveness, and due to the presence of bacterial species, it could lead to increase in nutrient availability into the pond. Biofilm removal would only be applicable to the walls of the pond where some biofilm may be present, but the chemicals used may not be effective on microorganisms in suspension.
Flushing could be one of the microorganism concentration reductions methods, that could be applied on site, but it also has some limitations. To be effective, the flow rate has to be significant, therefore, it would increase loading on the plant downstream. Furthermore, the volume of water required for flushing, may not be available due to existing plant limitations. In addition, flushing would have to be applied right at the start of the bloom to minimise microbial growth. On the other
53 hand, flushing could also reduce the nutrient loading in the pond, thus reducing not only concentration of microorganisms, but also nutrient availability, provided water used for flushing has lower nutrient concentration.
Biocides, one of the suggested options, also have several drawbacks: their effectiveness is species specific, they may not be compatible with abatement process, could only cause a shift in culture composition, rather than reduction in total microorganism abundancy. Furthermore, if used often, the species could develop resistance to the biocide or increase minimum required concentration. However, they can be effective on a variety of species at the same time, and could have long lasting effects, long enough for the flow through system to be cleared.
After evaluation of the numerous available options, it was decided, that changes to pond conditions, specifically increase in pH could be one of the options explored to reduce cyanobacterial growth. Although the species is adapted to alkaline conditions, it is possible, that a slight increase in pH would be an effective means of microorganism control. Increase in pH would also have very limited or no impact on the pond, it’s inventory, or the downstream plant. Another option that could be used is increased flushing rate, which could be achieved by simply increasing the volume received into the pond during warmer months of the year. Due to the possible nutrient and microorganism concentration reduction as a result of increased flushing rate, it could be used as a preventative measure as well. And finally, biocides, were also chosen and one of the options, due to wide availability, numerous modes of action, and possibility of using them not only as a preventative, but as bloom removal option. Furthermore, the three chosen strategies can be employed not only on their own, but also in combination increasing probability of effectiveness.
54 1.7 Rig development At the start of the project it was thought that the experiments proposed could be done in large raceways, but as more research was done about the organisms found in the pond, it was decided there was limited information available about the culture behaviour and experiments should be done on a smaller scale. The research was based at the National Nuclear Laboratory Workington Laboratory. As the facilities available were within rig hall, rather than a microbiology laboratory, all the required equipment had to be purchased and the right growth conditions ensured for the experimental programme. At the start of the experimental programme cultures were grown without using aseptic techniques, as the pond is open to air, it was thought it would mimic the growth conditions better. However, this led to repeated contamination issues about two weeks after inoculation resulting in unusable data. Once aseptic techniques were used (autoclaving medium and equipment and use of a Bunsen burner for sterile inoculation and sampling), contamination issues were reduced significantly. In addition, several different flask shapes were used: from round flat-bottomed boiling, to conical, to Fernbach at various stages of the project. The temperature measurement moved from measuring at 4 different points directly in 4 flasks used in the experiments to a separate flask due to very low difference between the earlier results, but this led to reduced contamination as well. At the start of the project cultures were not mixed either, which resulted in culture sinking to the bottom of the flask. Orbital shakers were employed for smaller volume, and magnetic stirrers for larger flasks to keep the culture in suspension. Due to the limited temperature regulation possibilities in the growth room and overheating during warmer weather, a combination of heater and fan was used to limit the temperature fluctuations and ensure similar temperature range between the experiments. The growth conditions added some limitation in comparing results of different experiments, however, due to the temperature fluctuations they are better mimicking conditions in open air.
55
Image 1.1 The experimental set up showing batch experiments on the left and continuous culture (high nutrient continuous culture at the time) on the right.
56 1.8 Research objectives The primary aim of this research was to better understand the behaviour of P. catenata in conditions simulating those found in the FGMSP. Further aims were to identify changes in the pond conditions that could prevent bloom formation or possible methods of bloom control. The main objectives of the project were:
1. To understand the culture behaviour and growth in pond simulant medium, or if it was not possible, in a medium representing the nitrate and phosphate concentrations found in the pond. In addition, to determine how high pH affects culture growth, simulating possible pond conditions. The results would serve as a basis for further experiments.
2. To determine the optimum nitrogen and phosphorus ratios and concentrations required by P. catenata for maximum growth.
3. To assess the effectiveness of two biocides that have the potential to be used as a method of bloom prevention and control at reducing the growth of P. catenata.
4. To assess the impact of batch and continuous culture on the growth of P. catenata, and any issues that could arise due to scaling-up. To determine how the photosynthetic culture reacts to changes in medium pH, and if increases in culture pH coupled with flushing could be used to control growth.
5. To determine if culture composition changes due to biocide dosing or change in continuous culture pH values.
57 2 Algal growth in batch cultures
2.1 Abstract The aim of this set of experiments was to determine the optimum nitrogen and phosphorus ratio for P. catenata growth and to determine if nutrient concentrations and media with pH comparable to pond would support culture growth in batch conditions. The experimental results showed, that cultures grown in medium simulating nutrient concentrations in the FGMSP are only able to support culture growth for less than 5 days, however, reduction in inoculating concentration can prolong the growth time to 7 days. It was also shown, that the micro nutrient concentration or additional organic carbon sources had no effect on culture growth compared to control. The pH of inoculation medium was also shown to be important, as buffered growth medium of pH 10, 11, and 12 prevented culture growth in BG11 medium. Anabaena sp. has been tested as a substitute to Pseudanabaena catenata but was deemed not suitable due to different growth pattern and pigment composition. The optimum nutrient concentration experiments revealed that under these experimental conditions the highest culture concentration was achieved at 547.14 mg/l of nitrate and 21.81 mg/l phosphate concentrations, and 17.38 N:P ratio.
2.2 Literature review
2.2.1 Cyanobacterial adaptations to micro and macro nutrient availability Phosphorus and nitrogen loading are controlling factors in bloom formation, however, a short term increase in concentration or seasonal loading may have a larger impact compared to annual loading (Paerl, 2008). Cells growing in nutrient- limiting conditions can have reduced cell sizes, reduction in photosynthetic activity as well as increased production of lipids, although, this will depend on the species present and available nutrients (Dean et al., 2010; Gao et al., 2013; Pancha et al., 2014; Vanucci et al., 2012). Growth rate is also dependant on nitrogen and phosphorus storage, as well as on light availability (Cade-Menun and Paytan, 2010; Pedersen and Borum, 1996; Qi et al., 2013). Some species show increased sensitivity to nitrogen limitation compared to phosphorous or iron, providing
58 indication of the growth limiting nutrient (Gao et al., 2013; Vanucci et al., 2012). The nutrient availability can influence culture composition, due to different reactions to nutrient shortage or surplus and changes in growth medium chemistry (Fujita, 1985; Schneider et al., 2013).
Cyanobacteria primarily use ammonium and nitrate for nitrogen, but urea and amino acids can be used as alternative sources (Rai, 2001). However, the absence of external ammonia can lead to leakage due to gradient formation (Rai, 2001). It has been suggested that cyanobacteria, like other prokaryotes, have an ability for cyclic
+ retention of NH3/NH4 (Kleiner, 1985). Both, nitrate and nitrite, are reduced to ammonia inside the cell, and genes for nitrate/nitrite uptake are repressed by the presence of ammonia (Flores and Herrero, 2004). Cyanobacteria can also take up urea by energy dependent transport systems and degrade it to NH3 and CO2 by urease (Antia et al., 1991; Flores and Herrero, 2004). Some of the amino acids (arginine, glutamine, asparagine and proline) can also be used as a source of nitrogen in some of the cyanobacteria as well (Flores and Herrero, 2004; Singh et al., 1991).
Some filamentous cyanobacteria have specialised structures, heterocysts, to uptake
N2 (Rai, 2001). They appear under highly nitrogen limited conditions and change to produce nitrogenase and protect it from oxygen (Adams and Carr, 1989; Buikema and Haselkorn, 1993). Cyanobacteria that do not form heterocysts may be able to fix nitrogen (N2) by the use of nitrogenase, that could be irreversibly inactivated by oxygen, but it can be overcome by fixing nitrogen under anaerobic conditions (Rai, 2001). Cyanobacteria use this method for obtaining nitrogen only in extreme conditions as it is very energy intensive (Rai, 2001). Other species of cyanobacteria that do not fix N2 can degrade their phycobiliproteins and chlorophyll a, although after nitrogen is available they can be regenerated (Rai, 2001). Several unicellular organisms separate N2 uptake from photosynthesis, specifically at night (Fay, 1992).
Nitrogen limitation can cause significant decreases in chlorophyll a content in a cell (Fujita, 1985; Martínez et al., 2012). In addition, the content of total carotenoids can decrease in some species in nitrogen-limiting conditions and can be observed due to the changes in culture colour from green to pale yellow (Pancha et al., 2014).
59 Nitrogen depletion can also cause increased storage of carbohydrates or lipids to reroute photosynthetic products into low nitrogen content compounds until conditions are favourable for growth, while protein production and content in the cell decrease (Dean et al., 2010; Pancha et al., 2014; Stehfest et al., 2005). Furthermore, some organisms may consume carbohydrates synthesised in nitrogen-limiting conditions in the stationary phase of growth (Dean et al., 2010). Increased accumulation of phosphorous has also been observed as one of the reactions to nitrogen limitations (Dean et al., 2010). It has also been suggested, that cyanobacteria can grow and uptake nitrate even after there is no available phosphorus, as long as its content in the algal cell has sufficient reserves (Droop, 1983).
Two different mechanisms of algal phosphorus uptake are thought to be used: surface adsorption and internal uptake (Yao et al., 2011). It was observed in some phosphorus starved species that extraction of phosphate from the growth medium by cells can double compared to non-starved cells (Azad and Borchardt, 1970). In addition, due to a prolonged starvation period and lower phosphorous availability once the P concentration has increased, the growth lag phase can last proportionally longer than in non-limited media, until cells return to normal growth (Azad and Borchardt, 1970).
The effects of phosphorus limitation can be delayed by several days in species with a high phosphorus storage capacity, by using it to sustain cell growth (Vanucci et al., 2012). Polyphosphate granules are used by cyanobacteria for phosphorus storage and can be found in exponentially growing cells, although could be seen in some nitrogen or sulphur deficient cells as well (Grossman et al., 1994). It was also reported that algal species can store excess phosphorus after the extracellular concentration reaches a species specific concentration in nutrient rich conditions, instead of it being used for growth (Azad and Borchardt, 1970). Furthermore, once phosphorous concentrations reach a minimum luxury concentration, algae growth has no correlation with phosphorus uptake (Azad and Borchardt, 1970). The luxury uptake of phosphorus as well as the uptake for cell growth are light dependent (Azad and Borchardt, 1970). It was also found, that cells convert inorganic
60 phosphorus to organic forms to prevent the loss of luxury uptake (Azad and Borchardt, 1970). Algae have also adapted to prevent the loss of stored phosphorous during prolonged periods of reduced light or dark conditions (Azad and Borchardt, 1970).
Shortage of phosphate has a limiting effect on cell quantity, but not on the cell growth rate (Azad and Borchardt, 1970; Weiss et al., 1985). Phosphorus limitation effects are also confined to disturbance of photosynthetic electron transfer rather than chlorophyll a content (Stehfest et al., 2005). Observations during laboratory experiments showed that algae at low cell densities even with available phosphorus would leave some as residual phosphorus (Azad and Borchardt, 1970). In addition, in higher temperatures than the species specific optimum, residual phosphorus is still present with high phosphorus concentrations irrespective of algae growth rates (Azad and Borchardt, 1970). Phosphate can also precipitate with increased concentration of Ca2+ and Mg2+ ions and become one of the major limiting factors for algal growth (Rai, 2001).
The ability of algae to uptake and assimilate inorganic carbon has been shown to be species specific (Huertas et al., 2000). In addition, cyanobacteria may stop the take up of nitrate due to reduced availability of CO2 (Turpin, 1991). Production of pigments in algae is also dependent on carbon concentrations (Huertas et al.,
2000). At pH 7 to 9 algae and cyanobacteria rely on carbonic anhydrase for CO2
- conversion from HCO3 (Rai, 2001). As iron containing nitrogenase is used for nitrogen reduction, it has been suggested that low iron availability is linked to nitrogen limitation (Hardie et al., 1983a; Rai, 2001; Sherman and Sherman, 1983).
Iron is also important for the synthesis of phycobilisome complexes and phycobiliproteins (Straus, 1994). Some cyanobacteria under iron deplete conditions increased polyglucoside granule production, increased frequency in heterocysts and membrane damage (Douglas et al., 1986). Other studies showed that although growth of the culture continued at the same rate as in non-iron deficient condition, cells were 50 to 30% shorter and increased glycogen storage was reported (Sherman and Sherman, 1983). Another study showed damage to membranes and ribosomes, accumulation of polysaccharide granules under iron limited conditions
61 (Hardie et al., 1983b). Iron deficiency can also have an impact on the cell health, in low iron conditions volume of the cells was 13.4% lower compared to higher iron conditions, in addition, reduction in culture growth rate was also observed (Wang et al., 2015). Under low iron conditions, cyanobacteria can change a metaloprotein containing iron into one that does not with limited effect on photosynthetic efficiency (Bryant, 1986; Fillat et al., 1991; Laudenbach and Straus, 1988; Sandmann et al., 1990). Furthermore, under iron limiting conditions cyanobacterial capacity for iron uptake may increase (Grossman et al., 1994) as well as new protein concentrations(Scanlan et al., 1989). The importance of iron for cell growth is more pronounced in cyanobacteria compared to algae (Morton and Lee, 1974).
2.2.2 Adaptations to highly alkaline conditions Organisms for which optimum growth is observed in the range of pH 10-11 while growth is inhibited at near neutral pH are called alkaliphiles (Rai, 2001). The most extreme case was reported to be the cyanobacterium Plectonema nostocorum which was found in waters at pH 13 (Seckbach, 2007). Alkali-tolerant organisms can grow at pH 9 and above, but best growth is expected at near neutral (Rai, 2001). The carbon supply in alkaline waters is almost unlimited due to the inorganic species present, and high pH waters can be very productive environments for algae and cyanobacteria (Rai, 2001). Dissolved carbon is present only in carbonate or bicarbonate forms in alkaline environment, and cells are required to adapt to be able to use it (Huertas et al., 2000; Rai, 2001). Cyanobacteria must regulate the uptake of inorganic carbon and ions to maintain an optimal physiological balance to grow (Summerfield and Sherman, 2008). Moving of CO2 is mostly present in pH range between 5 and 8, bicarbonate range is pH 7-11 as a carbon source (Shiraiwa et al., 1993). It has been shown that cyanobacteria have a mechanism to concentrate carbon that could be found in the plasma membrane (Badger and Price, 1992; Price and Badger, 1989; Thielmann et al., 1990). This mechanism is
+ supressed at high CO2 concentrations (Rai, 2001). Due to high, mostly Na concentrations in alkaline waters a bicarbonate pump is the preferred option for obtaining carbon (Rai, 2001).
62 Algal survival in high pH environments depends on an ability to regulate intracellular pH, which is maintained by regulating the pH gradient across the plasmalemma (Summerfield and Sherman, 2008). The need for Na+ ions also increases with increasing pH over 9, due to the need to move H+ into the cells and monovalent ions out of the cell (Seckbach, 2000; Seckbach et al., 2013). It has been suggested that a hydrogen gradient is generated along the plasma membrane by a hydrogen pump in high sodium concentrations (Rai, 2001). Alkaliphilic cyanobacteria have also adapted to high sodium concentrations and use it for gradients across plasma membrane for ion transport and homeostasis (Rai, 2001). Alkaliphilic and alkali-tolerant cyanobacteria despite needing sodium for growth also exclude it by keeping the sodium concentration inside the cell lower than outside (Rai, 2001). It has also been suggested that cyanobacteria are better adjusted for surviving in alkaline conditions, therefore outcompeting algae (Caraco and Miller, 1998; Richmond et al., 1982; Vonshak et al., 1983).
2.2.3 Pseudanabaena catenata Pseudanabaena species require high N/P ratios for optimum growth, and reach highest biomass levels at N/P ratios range of 1:5 and 1:10 ,and fastest growth rates at ratios of 1:10 and 1:12 (Liu and Vyverman, 2015b). The same research showed that Pseudanabaena spp. had the highest daily removal rate of nitrogen compared to Cladophora sp. and Klebsormidium sp. (Liu and Vyverman, 2015b). During the experiment P. catenata removed nitrogen at a rate of 10.6 mg/l d-1 and contained the most nitrogen and phosphorous compared to Cladophora sp. and Klebsormidium sp. (Liu and Vyverman, 2015b). Phosphate removal was the most effective at N:P ratios of 1:7 and above for P. catenata (Liu and Vyverman, 2015a). Pseudanabaena catenata is capable of hydrolysing phosphate from organic phosphorus compounds, and is mostly activated by phosphorus deficiency (Carr and Whitton, 1982). In phosphorus deficient conditions some of the often observed changes in cell composition; falls in protein content, and increases in carbohydrate levels are less or not noticeable at all in P. catenata: (Carr and Whitton, 1982). No phosphatase activity was detected in P. catenata in phosphorus deficient conditions (Healey and Hendzel, 1979).
63 Similar results were obtained in other studies where P. catenata was the dominant species in a lake with high nitrogen loading and showed high affinity towards ammonia and nitrate, the most common nitrogen sources (Xavier et al., 2007). Compared to a lake nearby with different culture composition and nutrient availability, but similar location, nitrogen removal was higher, thought to be mainly due to P. catenata dominance in the mixed culture (Xavier et al., 2007). In addition, other experiment showed P. catenata outcompeting Cryptomonas sp., Flagelates, Novicula sp., Oscilatoria sp., Dinobryon sp., Achnanthes sp. when additional nitrate and phosphate were added into a natural lake water in laboratory conditions (Moss, 1973). However, dominance was reduced and beforementioned species took over when trace elements and vitamins were added, suggesting it was a limiting factor to other species in the mixed culture (Moss, 1973). Furthermore, it illustrates that P. catenata requires lower trace element concentrations or is more resilient to the shortage of micro nutrients (Moss, 1973). However, compared to another lake with similar climatic conditions with slightly higher pH, P. catenata was not dominant against Notzschia palea, Anacystis sp., Rhodomonas sp., Achnanthe sp., Cyrptomonas sp., Flagelates in any of the experiments, and required nitrate, phosphorus, and trace elements to form a significant part of the culture composition, suggesting increased micro nutrient requirement in more alkaline conditions (Moss, 1973).
Another experiment observing the abundance of P. catenata in a natural environment provided some mixed and contradictory results to previously discussed studies. In a lake with high N:P ratio P. catenata was one of the dominant species against Gleocapsa sp., Microcystis sp., Raphidiopsis sp., Oscilatoria sp., Dactylopsis sp., and others, widely spread throughout the lake, with concentration increasing in spring-autumn, and decreasing in winter (Tian et al., 2012). Mean N:P ratio for the whole year was reported to be 34.43, higher than in the previous studies discussed (Tian et al., 2012). However, in this experiment the P. catenata concentration correlated with TN, TP, N:P ratio negatively, but positively to temperature, BOD and COD (Tian et al., 2012). It could be that when nutrients were not the main limiting factor, temperature and BOD, COD had more influence on
64 species growth. Pseudanabaena catenata has also been shown to be closely affected by pH and alkalinity (Rauch et al., 2006)
Other Pseudanabaena sp was one of the dominant species during laboratory experiments undertaken between 14 and 19 °C and N:P ratios between 16 and 80 was one of the dominant species in mixture with Nitzschia microcephala, Berkeleya rutilans, Melirosa nummuliodes, Novicula cincta, Berkeleyarutilans, but Nitzschia capitelata had even higher proportion of biovolume (Hillebrand, 2011). In addition to the same trend, the species dominance increased slowly towards the second half of the 50 day experiment (Hillebrand, 2011). Growth of another Pseudanabaena species was shown to be positively correlated to phosphorus and ammonia (Xiao et al., 2011). When grown in mixed culture, Pseudanabaena galeata became one of the dominant species after nitrogen enrichment in nearly 20 days after inoculation (Ferragut and De Campos Bicudo, 2010). In mixed culture grown in lake water with added nutrients, two Pseudanabaena sp were the most abundant in high light and phosphorus enriched or nitrogen and phosphorus enriched cultures (Aguilera et al., 2017). In low light N+P enriched cultures, Pseudanabaena sp. abundance was lower compared to only P enriched cultures (Aguilera et al., 2017). In unmodified lake water there was no significant difference in low or high light and culture concentration (Aguilera et al., 2017). The species concentration was the highest between days 5 and 8 during the 11 day experiment. Interestingly, abundance of Pseudanabaena sp. decreased, except for P enriched cultures in higher illumination.
It seems that Pseudanabaena species, although found in environments from low to high nutrient availability, tend to be the dominant cultures in high nutrient environments. Furthermore, several studies identified strong affinity towards ammonia and nitrate, suggesting high nitrate concentrations required for optimum growth. Availability of phosphorus and light intensity also have been shown to have an impact on culture growth, with higher concentration or abundance in more intense light when culture was phosphorus enriched. It may be that more intense illumination results in higher phosphorus removal rate for internal consumption.
From the available literature it is clear that the key to prevention of microorganism growth is nutrient limitation. Although P. catenata has higher affinity towards
65 nitrogen compounds, phosphorus limitation would also result in growth prevention. Deviation from the optimum N:P ratio results in reduction of culture growth, hence the experiments were designed to determine the optimum nutrient ratio for P. catenata growth. The results of the experiments will also help to define medium mimicking conditions in the pond to be used later in experimental systems aimed at microorganism growth control strategies for FGMSP.
66 2.3 Aims of batch culture experiments • The aim of the first experiment was to determine if a pond water simulant could support culture growth, and to determine the maximum biomass cultures would achieve. • The aim of the second experiment was to determine if organic carbon availability or starting inoculum concentration may have an impact on culture growth. In addition, Anabaena culture was assessed if it could be used as a substitute to P. catenata in further experiments. • The aim of the repeated organic carbon addition experiment was to determine if addition of organic carbon would improve the culture growth in medium with low nutrient availability. • The aim of the buffered pH experiment was to determine the effect of alkaline pH on P. catenata culture growth and the highest culture concentration that would be achieved. • The progressive nutrient reduction experiment was used to determine the optimum nitrate and phosphate ratio for P. catenata growth.
67 2.4 Materials and methods
2.4.1 Algal culture Pseudanabaena catenata (NIVA-CYA 152) culture was obtained from NIVA Culture Collection of Algae (Norway) in Z8 medium. The culture was grown at the University of Manchester in BG11 medium. Anabaena sp. 1403/13A was obtained from Culture Collection of Algae and Protozoa (UK) and maintained in BG11 medium.
For triplicate cultures, all measurements errors were calculated as standard error:
푠 푆퐸푥̅ = √푛
Equation 2.1 For determination of standard error of the mean
Where SEx is standard error of the mean, s- standard deviation, and n is the number of samples.
Triplicate non-axenic cultures of Pseudanabaena catenata were grown aerobically in 50ml plastic vented culture bottles (Fisher, UK) containing 30 ml of growth media. Cultures grown in BG11 medium (See Appendix 1) were used as control. Pond water simulant media (see Table 1.2) pH was adjusted to 7 by adding hydrochloric acid. All the stock cultures were washed before inoculating by centrifuging using Boeco (Germany) centrifuge at 2000 rpm for 30 minutes, removing supernatant, adding the equivalent of growth medium, and shaking to resuspend the cultures. Cultures were kept in a growth cabinet1 with fluorescent lights under continuous 200 PAR illumination at 24°C, measured with a light meter2, and on an orbital shaker3 moving at 250 rpm. Algal growth was determined as increase in chlorophyll a concentration, using the method of Wintermans (Wintermans and De Mots, 1965). Chlorophyll was extracted by centrifuging samples at 14000 rpm using Sigma (Germany) micro centrifuge for 10 minutes, and removing the supernatant afterwards. Chlorophyll was extracted from the leftover pellet by adding 70% ethanol mixture with water and leaving the mixture to stand for at least 4 hours. Samples were centrifuged again, and supernatant removed to
1 Panasonic MLR 352-PE , Panasonic Biomedical BV 2 Light Scout, Spectrum Technologies, USA 3 Stuart, UK 68 be analysed. Absorption measurements4 were taken at 665 nm and 750 nm wavelength and chlorophyll concentrations were calculated using formula (Jespersen and Christoffersen, 1987):
Ve - total solvent volume (ml), A - absorbance at 665 nm-absorbance at 750 nm, Vs - total volume of sample (litres), L- cell path length (cm), f- (1/specific extraction
V f A Chl − a (g l −1 ) = e Vs l
Equation 2.2. Equation used to determine chlorophyll a concentration coefficient) *1000. The specific extraction coefficient for chlorophyll a is 83.41 g/cm (Wintermans and De Mots, 1965).
2.4.2 Effect of reduced inorganic nutrient concentrations: initial experiments A non-axenic P. catenata culture was grown in triplicate in BG11 medium as control, modified BG11 to highest pond water nitrate and phosphate concentrations (Table 2.1 ), and modified pond water simulant spiked with 3 mM of sodium acetate. Anabaena culture was grown in BG11 medium. Culturing and analytical techniques employed were as mentioned above in section 2.4.1.
2.4.3 Reduced inorganic nutrients- variation in sodium acetate supplements Pseudanabaena catenata was inoculated into 4 different BG11 medium modifications, each in triplicate. Reduced nutrient BG11 medium (having the same concentration of nitrate and phosphate as highest concentration measured in the pond) was used as control, and reduced nutrient BG11 with 3, 10, and 20 mM of added sodium acetate were used for comparison. As in previous experiments, cultures were washed before the experiment and the same analytical procedures were followed (Section 2.4.1).
4 Spectrophotometer, Jenway, UK
69 Table 2.1. Nitrate and phosphate concentrations in reduced nutrient BG11 medium
Concentrations (mg/l) Nitrate 2.6 Phosphate 7.9 2.4.4 Buffered pH experiments Pseudanabaena catenata were grown in 500 ml conical flasks containing 200 ml medium volume in triplicate. BG11 medium acted as a control, and at 3 different pH values of 10, 11, and 12. The BG11 media were buffered using sodium carbonate- bicarbonate buffers (See Table 2.2) and adjusted to required pH using sodium hydroxide. Cultures were grown under 16:8 light-dark 250 PAR fluorescent illumination provided by Growth Technology T5HO lights (UK) as measured with a Light Scout light meter by Spectrum Technologies (USA) on an orbital shaker Stuart (UK) at 100 rpm. Temperature varied in range of: (17-26°C) and was data logged using Squirrel (Grant instruments, UK) data logger every 30 minutes. Absorption was measured at 600, 680, and 750 nm wavelengths using HACH DR 3900 (Germany) spectrophotometer using 1 cm path length quartz cuvettes, pH values were measured using a Hanna HI8424 pH meter (Romania). Chlorophyll was extracted by centrifuging samples at 5000 rpm for 20 minutes using a Thermo Scientific Heraeus (Germany) centrifuge. Supernatant was removed, and 100% acetone was added to the pellet and shaken to mix the pellet with acetone and left to stand for at least 4 hours. The sample was then centrifuged again and the supernatant removed for analysis. The concentration of chlorophyll was calculated using the formula:
퐶푎 = 11.75퐴662 − 2.35퐴645
퐶푏 = 18.61퐴645 − 3.96퐴662
1000퐴470 − 2.27퐶푎 − 81.4퐶푏 퐶 = 푥+푐 227
Equation 2.3. Equation used to determine chlorophyll a and total carotenoid concentrations using 100% acetone as a solvent. (Lichtenthaler and Wellburn, 1983)
Ca-chlorophyll a concentration
70 Cb- chlorophyll b concentration
Cx+c- total carotenoids
A- absorption at a specified wavelength
Chlorophyll b values were used to determine total carotenoids.
Table 2.2. Volumes of buffer used to adjust medium pH
pH NaHCO3 0.1M ml Na2CO3x10H2O 0.1M ml 10.5 20 80 10.8 10 90 2.4.5 Progressive reduction in inorganic nutrient concentrations Pseudanabaena catenata cultures were inoculated in modified BG11 media (See Table 2.3 for all the modifications of nitrate and phosphate used) in sequentially lower nutrient concentrations. Triplicate cultures were grown in 500 ml conical flasks containing 200 ml medium volume. Absorption, pH, and chlorophyll analysis was completed as in buffered pH experiment (Section 2.4.4). Supernatant was used for ion chromatography. Supernatant was centrifuged at 10000 rpm using Sigma (Germany) centrifuge for 15 minutes, to separate left over cell matter and supernatant removed again for ion chromatography.
Table 2.3.Nitrate and phosphate concentrations in modified BG11 media
BG11 modification Nitrate concentration (mg/l) Phosphate concentration (mg/l) Unmodified 1094.27 21.81
547NO3 547.14 21.81
273NO3 273.57 21.81
109NO3 109.43 21.81
21NO3 21.89 21.81
10NO3 10.94 21.81
5NO3 5.47 21.81
2.6NO3 2.6 21.81 RNBG11 2.6 7.9
2.6PO4 2.6 2.6
71 The Dionex ICS5000 (Thermo Fisher Scientific, USA) ion exchange chromatography system was used for the sample analysis. Two stationary phase columns were : Dionex AG18 Guard Column, and DionexAS18 Analytical Column. The packing used in the columns is isethylvinylbenzene/ divinylbenzenein with 7.5 µm particle size. The set up used for ion exchange chromatography was ion suppression with conductivity detector.
Eluent Eluent pump Injection valve Precolumn
Analytical column Data recording Conductometric Suppressor detector
Figure 2.1. Diagram of ion chromatography process using suppressor and conductometric detector
The eluent used was 36 mM KOH produced by injection of concentrated KOH intro high purity water. Analyte ions together with eluent passed through 250 mm analytical column at 0.25 ml/min, where the ion exchange process takes place. The analyte ions were then transported into electronic suppressor at 23 mA. The signal recorded is related to conductivity of the analyte ions.
72 2.5 Results and discussion
2.5.1 Impact of nutrient availability The pond water simulant was created to imitate conditions found in the pond, resulting in high sodium concentrations and highly alkaline medium, and the nitrate and phosphate concentrations used can be found in Table 2.4. The pH of the pond water simulant medium was adjusted with hydrochloric acid to pH 7, to prevent cumulative stress effects of increased pH and low nutrient concentrations. The positive control cultures in BG11 medium grew to turbidity values comparable to other experiments (see results in the following sections).
Cultures grown in the pond simulant medium showed mixed results. Those inoculated into the pond water simulant with the average (0.1 mg/l nitrate, and 0.2 mg/l phosphate) and the lowest (0.03 mg/l nitrate and 0.01 mg/l phosphate) nutrient concentrations grew quickly from starting average turbidity values of 0.136 and 0.14 to 0.243 and 0.226 respectively, but for a brief period (day 0 to 2). However, the culture grown in the highest (2.6 mg/l nitrate and 7.9 mg/l phosphate) nutrient concentration pond water simulant showed no growth and decreased in concentration during the same period of time from 0.133 to 0.026 as measured as turbidity (see Figure 2.2). After a rapid increase in turbidity values, cultures grown in the pond water simulant decreased in turbidity values quickly down to 0.036 (measured at 600 nm) for the average nutrient and 0.054 for the lowest nutrient concentrations from day 2 to day 6 and did not recover afterwards.
Table 2.4. The three pond water simulant modifications with the same nitrate and phosphate availability as measured in pond.
Pond Simulant Medium (describing nutrient Nitrate (mg/l) Phosphate (mg/l) availability) Highest 2.6 7.9 Average 0.1 0.2 Lowest 0.03 0.01
73 Changes in turbidity and pH during the pond water simulant experiment 3
2.5
2
1.5
1 Absorptionat 600nm 0.5
0 0 5 10 15 20 25 Days Pond medium highest concentration (2.6 mg/l nitrate, 7.9 mg/l phosphate) Pond medium average concentration (0.1 mg/l nitrate, 0.2 mg/l phosphate) Pond medium lowest concentration (0.03 mg/l nitrate, 0.01 mg/l phosphate) BG11 medium as control
Figure 2.2. Change in turbidity (expressed as absorption) during the first pond water simulant experiment. The error bars show the standard error of the mean value, n=3.
The pH values (see Appendix 2 Figure 8.1) of the control increased from inoculation to day 6 from 7.89 to 9.36, followed by a slight decrease to 8.9 and increased again to 9.74. The highest pH values were observed on day 16, the same day as the highest turbidity. The measured pH values of all the pond water simulant cultures also showed an initial increase, rising from day 0 to day 2 for all the pond simulant media values from 7.44 to 7.83 for the highest, from 7.52 to 8.14 for average, and from 7.4 to 8.03 for the lowest nutrient availability (see Table 2.4 for nutrient concentrations). The values decreased by day 6 to 7.17, 7.11, and 7.09 for highest, average, and lowest nutrient availability respectively, and suddenly increasing on day 16 to 8.55, 8.44, and 8.54 respectively, and decreasing again to values ~8 for all the cultures grown in all three pond simulant modifications. The increase in pH values by day two is most likely due to the growth of the cultures in the average and the lowest nutrient availability pond simulant medium, however, pH increased even in cultures with the highest nutrient availability pond simulant medium where
74 no growth was observed (see Figure 2.2). In addition, the increase from day 13 to 16 does not coincide with an increase in turbidity, however, it could be due to addition of BG11 medium (see below). Although the pH values of cultures grown in the pond water simulant media increased, the values reached were lower compared to control at 8.78 on day 2, while the cultures grown in the pond simulant medium increased in turbidity.
Changes in chlorophyll a concentration during pond water simulant experiment 8
7
6
µg/ml 5
4
3
Concentration 2
1
0 0 5 10 15 20 25 Days Pond medium highest concentration (2.6 mg/l nitrate, 7.9 mg/l phosphate) Pond medium average concentration (0.1 mg/l nitrate, 0.2 mg/l phosphate) Pond medium lowest concentration (0.03 mg/l nitrate, 0.01 mg/l phosphate) BG11 medium as control
Figure 2.3. Change in chlorophyll a concentrations over the length of the pond water simulant experiment. The error bars show the standard error of the mean value, n=3.
The change in chlorophyll a concentrations (Figure 2.3) followed similar trend to the turbidity (absorption) values. However, the decrease in chlorophyll a concentrations was sharper compared to the turbidity values for control cultures. It can be seen in Figure 2.2, the growth of control cultures towards the end of the experiment was not uniform between the triplicate cultures (size of the error bars), some continuing to grow, some decreasing in biomass concentration. As with turbidity, chlorophyll a concentrations in the cultures grown in pond water simulant increased and decreased sharply. However, chlorophyll a concentrations at the highest turbidity point for the cultures grown in all three pond simulant medium
75 modifications were lower than on day 0. In addition, chlorophyll a values of the culture grown in the pond simulant with the highest nutrient availability followed the same trend as other pond simulant cultures, but at lower values. Chlorophyll a concentrations in the pond water simulant cultures dropped below detection limit from day 13 and were not measured any further.
To determine if any viable cells were still present in the cultures, some BG11 medium was added into the pond simulant medium cultures on day 16. No increase was observed in turbidity values. Some residual turbidity was measured throughout the experiment, but as it can be seen from microscopy images of the cultures (See Appendix 2), no long filaments (characteristic of P. catenata when in growth phase) were observed.
Despite the cultures being inoculated in the highest nutrient availability pond simulant medium there was no observable growth, although two other cultures grown in lower nutrient concentrations did increase in turbidity values. It could be due to increase in culture pH or sodium concentration. However, the cultures are found in slightly alkaline environments (pH above 8) (López-Archilla et al., 2004; Mercado, 2003; Padisák et al., 1998) and a the highest pH value reached in any of the three pond water simulant modifications was 8.5, lower than the highest control culture pH value of 9.74. Thus, a potential reason could be culture sensitivity to high pH when grown in low nutrient availability. Furthermore, all the cultures were inoculated from the same starting culture, therefore, there should not be a significant difference as was seen between the cultures grown in the 3 pond water simulant modifications. The experiment provided even more interesting results when chlorophyll a and carotenoid concentrations did not increase in any of the cultures on day 2, following increase in turbidity, as seen in the control. Contrary to increase in turbidity they decreased, the increase was observed on day 6, after decrease in turbidity values. In addition, the chlorophyll a concentrations were lower than at the day of inoculation, despite the increase in turbidity values. These results would suggest, that either cultures used the nutrients for cell growth, and not pigment production as suggested by (Grossman et al., 1994). It may also be due to bleaching observed in cultures grown in low
76 nutrient availability (Grossman et al., 1994) due to reduced photosynthetic activity and loss of chlorophyll a and carotenoids (Fujita, 1985; Pancha et al., 2014), or more specifically low nitrogen availability (Martínez et al., 2012). Natural variations during sampling at such low concentrations may also be the reason for such low values. However, the increase in chlorophyll a concentrations after decrease in the turbidity values could be explained by photosynthetic pigment regeneration, as suggested by Rai et al. (Rai, 2001). The results of P. catenata growth in pond simulant medium may also suggest, that the strain used in this experimental work is not adapted to growing at low nutrient availability, as it was obtained from a culture collection. Thus when grown in nutrient concentrations equivalent to what has been found in FGMSP, it may be more sensitive to nutrient limitation, resulting in low concentration and limited photosynthetic pigment production .
2.5.2 Pond water simulation experiment: addition of a carbon source To determine if organic carbon availability may have an impact on culture growth, pond water simulant with the highest nutrient availability was spiked with sodium acetate. In addition, it was decided to lower the inoculum concentration due to the short growth period in the previous experiment. This would help identify if starting culture inoculation was too high to be supported by the low available nutrient concentrations compared to unmodified BG11 medium. Reduced nutrient BG11 medium, containing the same concentration of nitrate and phosphate as highest concentrations found in the pond, was used to determine if low micro nutrient availability was influencing culture growth in the pond simulant media. A culture of Anabaena sp. was also grown to determine differences in growth speed and pigment composition between P. catenata and Anabaena. In addition, the experiment was used to investigate if Anabaena could be used as a substitute for P. catenata as preliminary experiments with P. catenata proved challenging, with poor growth in several early experiments (data not shown). As previously, P. catenata
77 grown in BG11 medium was used as control. The pond water simulant medium spiked with acetate pH was not adjusted.
Changes in turbidity during repeated low nutrient experiment 1.8
1.6
1.4
1.2
1
0.8
0.6
Absorptionat 600nm 0.4
0.2
0 0 2 4 6 8 10 12 14 16 18 Days P. catenata in BG11 as control
Anabaena sp. in BG11
P.catenata in reduced nutrient BG11 (2.6 mg/l nitrate, 7.9 mg/l phosphate)
P. catenata in pond water simulant with highest nutrient conentration spiked with acetate (2.6 mg/l nitrate, 7.9 mg/l phosphate)
Figure 2.4. Changes in turbidity (absorption) during repeated low nutrient experiment growing P. catenata in BG11 as control, and comparison with Anabaena. The error bars show the standard error of the mean value, n=3.
As in the previous experiment, P. catenata grown in pond water simulant, but with added acetate did not grow at the start of the experiment. However, there was a significant increase in turbidity after day 7 from 0.027 to 0.248 on day 17. Pseudanabaena catenata grown in reduced nutrient BG11 medium (RN BG11) reached higher turbidity values than P. catenata grown with acetate in pond simulant medium at the start of the experiment, with highest turbidity value of 0.134 on day 7 compared to 0.035. Furthermore, the cultures grown in RN BG11 medium followed the same growth pattern as cultures grown in pond simulant media in the previous experiment, with measurable increase in turbidity at the start of the experiment, followed by a decrease until the end of the experiment. The lower innoculum concentration allowed some results to be observed in RN BG11
78 cultures: the growth cycle was longer as it increased from 2 days to reach the highest value to 7 days, and slower compared to the previous experiment, resulting in easier to observe growth. The control cultures grew in a comparable maner to other experiments, although a decrease in culture turbidity started earlier than previously, day 11 rather than 16, and the highest turbidity value was lower at 1.29 compared to the previous experiment highest value of 2.24. The changes were attributed to slightly unpredictable culture growth manner and natural variations.
It was decided not to use Anabaena, a robust and well-studied model organism, as a model system since the growth dynamics shown by changes in turbidity in
Figure 2.4 and chlorophyll a concentration in Figure 2.5 differed markedly from P. catenata. With experience, growth of P. catenata became more reproducible. In addition, the behaviour of the Anabaena in medium was different compared to P. catenata; Anabena tended to clump when shaken and woud not separate very easily when left to stand.
The pH values of the control cultures fluctuated, but were lower than in the previous expriment (see Figure 8.2). The pH values of the Anabaena cultures despite decreasing on day 2 from 8.77 to 7.89 increased slightly on day 4 to 9. 40 and remained stable throughout the experiment at ~9.5, with a slight decrease on the last day of sampling to 9.21, as seen in Figure 8.2. Cultures grown in reduced nutrient BG11 medium behaved differently to cultures grown in high nutrient media, the pH decreased from inoculation value of 8.23 towards pH value of 7. There was a slight increase in pH on day 7 to 7.47, the same day when highest turbidity was measured for the cultures grown in RN BG11 medium. Cultures grown in pond water simulant spiked with acetate started at much higher pH values at 10.4, that decreased during the experiment, as for cultures grown in reduced nutrient BG11. After a sharp fall from inoculation to day 2, pH values fluctuated between pH 8 and 8.5.
79 Changes in chlorophyll a concentration during repeated reduced nutrient experiment 6
4 µg/ml
2 Concentration
0 0 2 4 6 8 10 12 14 16 18 P. catenata in BG11 as control Days
Anabaena sp. in BG11
P.catenata RN BG11 (2.6 mg/l nitrate, 7.9 mg/l phosphate)
P. catenata in pond water simulant medium with highest nutrient concentration dosed with acetate (2.6 mg/l nitrate, 7.9 mg/l phosphate)
Figure 2.5. Changes in chlorophyll a concentration during repeated low nutrient experiment growing P. catenata in BG11 as control, and comparison with Anabaena. The error bars show the standard error of the mean value, n=3.
Despite an outlying data point on day 9, most cultures followed the same patterns in chlorophyll a concentartion as in turbidity values. As can be seen from Figure 2.5, Anabaena compared to P. catenata contains significantly more chlorophyll a, the highest concentration of chlorophyll measured for P. catenata was 1.09 µg/l . compared to 5.05 µg/ml for Anabaena. This is more pronounced on day 11, when turbidity values of P. catenata were higher at 1.29 compared to 1.01 for Anabaena sp., however, concentrations of chlorophyll a in the Anabaena cutures at 4.26 µg/ml were nearly 4 times higher that of P. catenata at 1.09 µg/ml. The difference in chlorophyll a concentration may be due to the pigments produced by each of the microorganisms, as P. catenata can produce a range of pigments depending on the available light wavelength (Acinas et al., 2008; Grossman et al., 1993; Kehoe and Gutu, 2006).
Although P. catenata cultures grown in pond simualnt medium spiked with acetate did not increase in turbidity values on day 4, chlorophyll a concentrations did from
80 below the limit of detection to 0.215 µg/ml. An increase in turbidity values towards the end of the experiment was observed in the cultures grwon in the pond simulant medium from 0.027 on day 7 to 0.248 on day 17, as well as chlorophyll a concentrations from below detection limit to 0.211 µg/ml. The culture grown in reduced nutrient BG11 medium followed the trend of the culture spiked with acetate. Chlorophyll a peaked at day 4 and 17 at 0.179 and 0.239 µg/ml concentration, although turbidity values on the day 17 were decreasing. It is not clear if the fluctuations of chlorophyll a concentrations noted were due to the cell growth or natural variation in the samples. The P. catenata cultures grown in pond water simulant medium looked pale white and turbid at the end of the experiment, despite the increase in pigment concentration. It may be due to other species present in the culture, or nutrients being used for pigment production, but not neceserrily chlorophyll a.
As can be seen in Image 8.2 compared to Image 8.3, at the end of the experiment, cultures grown in pond water simulant with acetate seemed to have more cells per filament compared to the previous experiment cultures grown in pond water simulant with varying nutrient avilability. The cultures for both, the reduced nutrient BG11 medium and the pond water spiked with acetate, looked similar, suggetsing that organic carbon may have had an impact on culture growth in pond simulant medium compared to the previous experiment.
As reduction in starting concentration to 0.05 turbidity yielded easier to observe results and longer growth cycles compared to the previous experiment, it was decided to lower starting inoculation of the P. catenata when grown in low nutrient media in later experiments. The cultures did grow in reduced nutrient BG11 medium, as did cultures grown in the average and lowest nutrient availability pond simulant media in the previous experiment, suggesting that micro nutrients did not influence the culture growth. From the observations in study done by (Moss, 1973) it is implied that P. catenata may require lower micro nutrient concentrations or is more resistant to lack of micro nutrients supporting the results of this experiment. It took 7 days for cultures grown in RN BG11 medium to reach the highest turbidity value, suggesting there are enough nutrients for culture growth. However, the
81 highest turbidity value reached by cultures grown in RN BG11 medium was lower at 0.134 compared to 0.224 reached by cultures grown in pond simulant medium in the previous experiment. The addition of sodium acetate to the pond water medium resulted in some growth of the cultures. Although, turbidity values did not start to increase until day 9, and they did not decrease as suddenly as in the previous experiment. Furthermore, the turbidity of cultures grown in pond simulant medium spiked with acetate reached higher values compared to the cultures grown in reduced nutrient (RN) BG11 medium. However, how the culture grown in RN BG11 medium reached similar chlorophyll a concentrations with very low turbidity values is not clear. Pseudanabaena limnetica and planctonica has been reported to uptake dissolved organic carbon in a natural lake with low phosphate concentrations (Znachor and Nedoma, 2009). Thus, suggestion that organic carbon was used to supplement photosynthesis may be applicable to explain the positive impact of sodium acetate addition on the P. catenata growth in pond simulant medium with lower organic carbon availability than in reduced nutrient BG11 medium.
There was a significant difference between behaviour of control and Anabaena cultures grown for comparison with P. catenata. Although Anabaena was inoculated at lower turbidity values, the culture grew faster, compared to the control. This is best seen from day 11 onwards, when turbidity of control cultures started to decrease, and Anabaena cultures continued to grow, as observed by increase in turbidity. Due to the behaviour of the Anabaena culture when stirred, it was decided that organisms are sufficiently different and could not be used as a substitute. Compared to the previous experiment P. catenata grown as a control started to decrease in turbidity values and pigment concentrations earlier and at lower maximum values, providing an indication of how poorly reproducible the culture behaviour can be under identical conditions. It must be noted, that the stock culture may have been different, but it should not cause such a significant difference in the culture growth.
82
Image 2.1. From left to right: P. catenata in pond simulant medium, P. catenata in RNBG11 medium, Anabaena, and control P. catenata in BG11 medium on day 4.
2.5.3 Impact of organic carbon availability at reduced inorganic nutrient concentrations Due to the increase in turbidity in pond simulant medium and reduced nutrient BG11 medium in previous experiment it was decided to determine if an increase in cell growth observed was due to the organic carbon availability. To evaluate the effects of organic carbon availability reduced nutrient BG11 medium and lower inoculation concentrations were used.
The organic carbon was provided in a form of sodium acetate with concentrations of 3, 10, and 20 mM. Reduced nutrient BG11 was used as a control. All cultures grew to comparable concentrations to the previous experiment cultures grown in reduced nutrient BG11 medium. However, the cultures grown with higher acetate concentrations, 10 and 20 mM, reached lower turbidity values compared to control as seen in Figure 2.6. The highest average turbidity reached by control, and growth media with added 3, 10, and 20 mM of sodium acetate were 0.138, 0.138, 0.113, and 0.122 respectively. Although, the length of the growth cycle was slightly shorter, 13 days rather than 16 as seen in the earlier experiment. At the end of the experiment the final average concentrations of cultures grown with and without acetate were similar at 0.029, 0.023, 0.026, 0.035 for control, 3, 10, and 20 mM acetate dosed cultures respectively. The pH values were somewhat different at the start of the experiment, but that may be due to the acetate concentration as seen in Figure 8.3. All pH values followed the same pattern, they decreased after
83 inoculation by day 5, increased afterwards on day 8, and decreased by the end of the experiment to values below 7.
Changes in turbidity over the lenght of the organic carbon addition experiment
0.14
0.12
0.1
0.08
0.06
0.04 Absorptionat 600nm 0.02
0 0 2 4 6 8 10 12 14 Days Reduced nutrient BG11 as control Reduced nutrient BG11 3 mM sodium acetate
Reduced nutrient BG11 10 mM sodium acetate Reduced nutrient BG11 20 mM sodium acetate
Figure 2.6. Changes in turbidity during repeated low nutrient availability experiment with organic carbon dosing. The error bars show the standard error of the mean value, n=3.
The chlorophyll a concentrations mostly followed the same trends as turbidity, reaching the highest average concentrations on day 5, and decreasing in chlorophyll concentration until day 13, the end of the experiment. Although cultures dosed with 3 mM of acetate had similar turbidity as control, chlorophyll a concentration reached lower concentration on day 5 of 0.095 µg/ml compared to 0.111 µg/ml for control. In addition, culture dosed with 10 mM of acetate had a sudden drop in chlorophyll concentration on day 8 from 0.111 to 0.047 µg/ml, when it is not seen in the turbidity values. The cultures spiked with 10 mM of acetate also reached similar chlorophyll values to ones of control, even though turbidity values were the lowest during the experiment.
84 Chlorophyll a concentration during the organic carbon addition experiment 0.14 0.12
0.1 µg/ml µg/ml 0.08 0.06 0.04
Concentration 0.02 0 0 2 4 6 8 10 12 14 Days Reduced nutrient BG11 as control Reduced nutrient BG11 3 mM acetate Reduced nutrient BG11 10 mM acetate Reduced nutrient BG11 20 mM acetate
Figure 2.7. Changes in chlorophyll a concentration during repeated low nutrient availability experiment with organic carbon dosing. The error bars show the standard error of the mean value, n=3.
Although sodium acetate had a positive impact on cultures grown in pond simulant medium, no significant effect was observed on cultures grown with additional organic carbon in RN BG11 medium. The results would suggest that the additional organic carbon has no effect on the cultures grown in RN BG11 medium and is only beneficial to the cultures grown in pond water simulant medium. Alternatively, lower inoculation concentrations and other changes to the cultures grown in the pond simulant had a more of an effect on the culture growth. The results are interesting, as it was reported that glucose and dichloroacetate had a positive effect on Pseudanabaena sp. grown in BG11 medium, although with higher nitrate and phosphate concentrations (Kirkwood et al., 2003). However, other carbon sources were removed from the medium in the study, therefore it may be that due to organic carbon already present in BG11 medium in this experiment, additional sources did not contribute to significant difference in availability. For more repeatable results, it was decided to use the modified RN BG11 medium in further experiments. The changes in chlorophyll a concentrations also provided interesting results. Although chlorophyll concentrations were very similar at the peak turbidity value, the concentration in control sample at the end of the experiment was markedly different. It could be due to an error in sampling, or cultures grown in
85 media with additional organic carbon as culture growth decreases, loose photosynthetic pigments quicker. This assumption would also mean, that cultures grown in media with additional organic carbon, decompose quicker than culture grown in RN BG11 medium.
2.5.4 Buffered pH experiment All the cultures grown in buffered medium showed no growth and turbidity was fluctuating within the starting inoculum turbidity and below detection limit range (Figure 2.8). Visually cultures looked pale and clumped together at the bottom of the flask. As it can be seen from the later experimental data, cultures during growth stage adjust the medium pH in the range of 10-11, therefore, no observable growth is surprising to see at medium buffered at pH 10. As it can be seen the pH of the medium slowly decreased with time Figure 8.4, higher pH decreasing faster. The control culture did not grow to concentrations comparable to other experiments; turbidity value towards the end of the experiment started to decrease at turbidity value 0.609 compared to the highest absorpion values from previous experiments at 1.29 and 2.24. However, due to the length of the experiment it may be that it is an insignificant drop due to natural variation. The experiment was finished by day 18 due to no observable growth in cultures grown in buffered BG11 medium.
Changes in turbidity during the buffered pH experiment 0.7 10.5 0.6 10 9.5 0.5 9 0.4 8.5 0.3 8 7.5 0.2 pHof control
7 Absorptionat 600nm 0.1 6.5 0 6 0 5 10 15 20 Days BG11 (control) BG11 pH10 BG11 pH11 BG11 pH12 BG11 (control) pH
Figure 2.8. Changes in turbidity and control culture pH during the buffered BG11 medium experiment. The error bars show the standard error of the mean value, n=3.
86 As with turbidity values, pigment concentration did not increase towards the end of the experiment, see Figure 2.9. The values of all pigments fluctuated throughout the experiment, reaching values above the day 0 values, but as with turbidity, no significant increase was observed. The chlorophyll a concentration of control cultures increased and decreased following turbidity values, although, slower and in increments compared to other experiments.
Chlorophyll a concentrations during the buffered pH experiment 1.2
1
0.8
0.6
0.4
Concentrationµg/ml 0.2
0 0 5 10 15 20 Days BG11 (control) BG11 pH10 BG11 pH11 BG11 pH12
Figure 2.9. Change in chlorophyll a concentrations during buffered BG11 medium experiment. The error bars show the standard error of the mean value, n=3.
Despite the control cultures (P. catenata in BG11 medium) growing, none of the buffered cultures increased in turbidity. This was not expected, as the cultures were not nutrient limited, and the only changes were the addition of sodium carbonate- bicarbonate buffers to increase the medium pH. These results could be due to the increase in culture pH or increased concentration in sodium. However, the addition of buffer only added up to 50 mg/l of sodium, to already existing 414 mg/l in unmodified BG11 medium. In addition, sodium can be used for ion transport and homeostasis in alkaliphilic cyanobacteria (Rai, 2001). Pseudanabaena catenata may not be alkaliphilic species, but it may use sodium for the same purpose and should not be sensitive to relatively low increase in sodium concentration. As Pseudanabaena sp were found in a lakes with pH up to 10.77 (Ballot et al., 2005; López-Archilla et al., 2004) it is unlikely that pH 10 or 11 itself would be the cause of
87 lack of growth. The most likely reason seems to be the sudden change in the medium pH. Although it can be seen from other experiments, when growing cultures self-adjust medium pH above values of 10, the sudden change in pH may result in decrease in turbidity values. In addition, cultures at the start of the experiment tend to increase their pH within few days, therefore, it is unclear, why it is preventing the culture growth, especially at pH 10. The results of this experiment also contradict observations of a study growing Pseudanabaena sp. isolated from a lake in pH adjusted media (using HCl or NaOH) (Gao et al., 2018). The Pseudanabaena sp. growth was not observed only if pH medium pH was constantly adjusted to be below 5, and the optimum pH was reported to be between 7 and 9, and most importantly, culture growth was still observed at pH 11. The results would suggest, that the culture is more sensitive to buffered medium compared to dosing to adjust the medium pH. Although, the results of the study by (Gao et al., 2018) were obtained using a different strain of Pseudanabaena, under different growth conditions, the inoculum concentration or growth stage of the culture during the experiment are not described. Thus, the results are not directly comparable, but can provide some indication of Pseudanabaena adaptations to alkaline pH. The results of this experiment would suggest, that the culture may be more sensitive to alkaline pH, thus differing from P. catenata found in the FGMSP. However, this may be applicable only to freshly inoculated cultures, due to sudden change in medium pH.
2.5.5 Growth of P. catenata at progressively reduced inorganic nutrient concentrations To determine the optimum nitrate and phosphate ratio for P. catenata an experiment was set up with decreasing nutrient availability. The highest turbidity values reached were used to determine the BG11 medium modification that is closest to the optimum nutrient ratio at these physical growth conditions (For full table of modifications see Table 2.3). To prevent cumulative effects nitrate and phosphate concentrations were lowered separately.
The culture that achieved the highest turbidity values of 2.33 was one grown with half the nitrate (547 mg/l) compared to BG11 medium (see Figure 2.10). Cultures
88 grown in BG11 reached second highest turbidity values of 1.74, however, the growth cycle length was longer at 62 days compared to 59 days for culture grown in 547 mg/l nitrate concentration. The growth cycle of cultures grown in BG11 medium was 60 days to highest turbidity values compared to 55 days for modification with half the nitrate (547 mg/l). All the other cultures reached lower turbidity values than cultures grown in BG11 medium and had shorter growth cycles with reduction of nitrate. From the Figure 2.10, it can also be seen that reduction of phosphate did not have any impact on turbidity values or growth cycle length.
From the ion chromatography results of nutrient concentrations in external medium (Figure 2.12 and Figure 2.13) it can be seen, that all cultures grown with more than 109 mg/l nitrate used all the available phosphate. Once cultures were inoculated in medium with concentrations below 109 mg/l nitrate, phosphate was thought to be in surplus due to detectable concentrations found in the growth media. The media of cultures grown with nitrate concentrations at 5.47 mg/l of nitrate or less at the end of the experiment still contained most of the phosphate compared to concentration at the start of the experiment.
Data also suggest, that on day 0 in most of the cultures phosphate concentrations were lower than compared to concentrations that should be in BG11 medium (21.81 mg/l), which was tested as control. It may be due to measurement error and changes following autoclaving, or due to adjustment in dilution factor. Increase in phosphate concentrations can also be observed in cultures grown in 109.5, 54.7, 5.4, and 2.6 mg/l nitrate containing media towards the end of the experiment as well. Unlike phosphate, only cultures grown in BG11 medium had surplus nitrate in the medium at the end of the experiment. All the other cultures had no detectable nitrate by the time the highest turbidity values were reached. However, even though turbidity values started to decrease in culture grown in BG11, nitrate concentrations continued to decrease.
The pH of cultures with starting nitrate concentration of 54.7 mg/l and above reached pH values above 10, and fluctuated at low amplitude, however, they decreased before reaching the highest turbidity values. Cultures grown in media
89 with nitrate concentartions below 54.7 mg/l reached progressively lower pH values with each lower nitrate concentration. A sharp increase in pH can still be observed, but due to shorter growth cycle the highest pH values were reached earleir (see Figure 2.11) and would decrease shortly afterwards, without the fluctuation phase. It seems that the cultures increase the medium pH up to a certain value, and adjusts it while in the growth phase.
90 Changes in turbidity during nutrient reduction experiment BG11 3 547 mg/l NO3
2.5 274.5 mg/l NO3
109.4mg/l NO3 2 54.7 mg/l NO3
21.98 mg/l NO3 1.5
10.9 mg/l NO3
Absorption at 600nmat Absorption 1 5.47mg/l NO3
2.6 mg/l NO3 0.5
2.6mg/l NO3 7.9 mg/l PO4
0 2.6mg/lNO3 7.9mg/l PO4 from 0 10 20 30 Days 40 50 60 BG11 stock Figure 2.10. Growth cycles of all the cultures with progressively lower nutrient concentrations. The error bars show the standard error of the mean value, n=3.The experiments were not continued once two consecutive samples showed reduction in culture concentration.
91 Change in pH during nutrient reduction experiments BG11 11 547 mg/l NO3
10.5 274.5 mg/l NO3
10 109.4mg/l NO3
9.5 54.7 mg/l NO3
9 21.98 mg/l NO3 pH 8.5 10 mg/l NO3
8 5.47mg/l NO3
7.5 2.6 mg/l NO3
2.6mg/l NO3 7.9 7 mg/l PO4
2.6mg/lNO3 6.5 7.9mg/l PO4 from BG11 stock 0 10 20 30 Days 40 50 60
Figure 2.11. Change in pH during growth cycles of nutrient reduction experiment. The error bars show the standard error of the mean value, n=3. The experiments were not continued once two consecutive samples showed reduction in culture concentration. 92 Change in phosphate concentration during nutrient reduction experiment 20.00 BG11
18.00 547 mg/l NO3
16.00 274 mg/l NO3
14.00 109 mg/l NO3
12.00 54.7 mg/l NO3
10.00 21.98 mg/l NO3
8.00 10 mg/l NO3 Phosphatemg/l 6.00 5.47 mg/l NO3
4.00 2.6 mg/l NO3
2.00 7.9 mg/l NO3
0.00 2.6 mg/l NO3 0 10 20 30 40 50 60 7.9mg/l PO4 2.6 mg/l PO4 Days
Figure 2.12. Changes in external phosphate concentrations. The error bars show the standard error of the mean value, n=3.
Change in nitrate concentrationduring nutrient reduction experiment BG11 1000.00 547 mg/l NO3
900.00 274 mg/l NO3
800.00 109 mg/l NO3
700.00 54.7 mg/l NO3
600.00 21.98 mg/l NO3
500.00 10 mg/l NO3
Nitratemg/l 400.00 5.47 mg/l NO3
300.00 2.6 mg/l NO3
200.00 7.9 mg/l NO3
100.00 2.6 mg/l NO3 7.9mg/l PO4 0.00 2.6 mg/l PO4 0 10 20 30 40 50 60 Days
Figure 2.13. Changes in external nitrate concentrations. The error bars show the standard error of the mean value, n=3.
93 Chlorophyll a concentrations in Figure 2.15 show some similarities with turbidity data, but also some significant differences. The difference between culture grown in BG11 and ones grown in half the nitrate concentration was even more pronounced in reference to chlorophyll a concentrations. Unlike the turbidity values, the difference between chlorophyll a concentrations is less pronounced between cultures grown in nitrate concentrations of 109 and 54.7 mg/l. Once starting nitrate concentrations were lower than 54.7 mg/l, chlorophyll a concentrations were significantly lower and proportional to turbidity values. In addition, for cultures grown in unmodified BG11 chlorophyll a concentrations did not seem to increase in the same pattern as turbidity values and remained somewhat stagnant between days 25 and 62. Furthermore, chlorophyll a concentration in cultures grown at 547 mg/l NO3 dropped sharply on day 52, even considering the error range. Both, the BG11 and 547 mg/l nitrate cultures decreased in chlorophyll a concentration on day 57 in their growth cycle. In addition, cultures grown in 274 mg/l NO3 reached the highest concentration of chlorophyll a not at the same day as the highest turbidity values were measured.
The stock culture used for culture inoculation seemed to have some impact on the turbidity values. The culture inoculated into RN BG11 medium from culture grown in unmodified BG11 medium reached higher concentration measured in turbidity compared to culture inoculated into the same medium, but from low nutrient stock. It is unclear though, if the cyanobacteria stored nutrients, or the low volume of high nutrient concentration medium used for inoculation was the contributing factor.
To determine if phosphate concentration reduction in low nutrient medium resulted in increase in turbidity values an additional experiment was conducted. Three BG11 medium modifications were chosen: with 2.6 mg/l nitrate and 21.98, 7.9, and 2.6 mg/l of phosphate with all cultures inoculated from the same stock.
As in previously reported results, cultures grown in the medium with the lowest phosphate concentration reached higher turbidity values (0.070) compared to the other experiments run at the same time (0.047 and 0.049), however, they were not higher than experiments run previously (Figure 2.14). In addition, reduction in
94 phosphate did not seem to have any impact on the culture growth. Cultures grown in RN BG11 modification with the highest turbidity values of 0.049 did not reach higher turbidity values or as high as in the previous experiment of 0.068. However, the shape of the growth curve illustrates few different growth patterns observed in low nutrient cultures. Some cultures have a slower growth phase at the start of the experiment, and later move towards exponential growth as seen in culture grown in RN BG11, or cultures start to grow immediately and have shorted growth cycles.
Changes in turbidity during repeated low nutrient availability experiment 0.08
0.07
0.06
0.05
0.04
0.03
Absorptionat 600nm 0.02
0.01
0 0 5 10 15 20 Days
2.6 mg/l NO3, 21.98 mg/l PO4 2.6 mg/l NO3, 7.9 mg/l PO4 2.6 mg/l NO3, 2.6 mg/l PO4
Figure 2.14. Culture growth cycles of repeated experiment growing P. catenata in BG11 medium with reduced nitrate and phosphate concentrations. The error bars show the standard error of the mean value, n=3.
The reported optimum N:P ratio in literature for Pseudanabaena catenata was 9 (Healey and Hendzel, 1979), which meant that below that ratio, cultures would become limited by one or the other nutrient. The results somewhat confirm this, as cultures grown at 8.69 and lower ratios were achieving lower turbidity values. However, the highest culture turbidity values were achieved by cultures growing at a N:P ratio of 17.38, and not at a higher ratio either. This result is closer to a study reporting P. catenata becoming one of the dominant species when N:P ratio was 16, with dominance diminishing when N:P ratio was increased to 80 (Xiao et al., 2011). Another study, evaluating nutrient availability on the growth of
95 Pseudanabaena sp. reported that the fastest culture growth rate was achieved at 230.4 mg/l total nitrogen and 7.12 mg/l total phosphorus, equal to unmodified BG11 medium nutrient concentrations (Gao et al., 2018). The results somewhat support the findings of this experiment, although the results are not directly comparable, as growth rate, rather than the highest concentration achieved by the cultures are being compared, in addition to difference in the growth conditions. However, the same study reported that total phosphorus concentration above 5.12 mg/l inhibited Pseudanabaena sp. growth. The latter results contradict findings of this experiment, as no reduction in growth rate was observed due to increase in phosphorus concentration, at least in growth media with low nutrient availability. The data also suggests that total nitrogen availability, rather than ratio is more important, since cultures grown at the lowest nitrate concentrations, even with reduction in phosphate did not reach significantly higher turbidity values. The findings are somewhat supported by (Gao et al., 2018; Xavier et al., 2007) as reportedly P. catenata has high affinity towards ammonia and nitrate and it was dominant species in a lake with average N/P ratio of 34.43 (Tian et al., 2012).
As it was expected, the turbidity values and chlorophyll a concentration decreased with reduction of nutrients, except for cultures grown in 547 mg/l nitrate. The cultures grown in medium with half the nitrate found in unmodified BG11 medium (547 mg/l) reached the highest turbidity values, and chlorophyll a concentrations. This is surprising, as there was no difference between BG11 and this 547 mg/l BG11 medium phosphate concentration. However, even though cultures were growing at the same time for some period of time, they were not inoculated on the same day. Although, this should have only a small impact on the difference between the cultures. In addition, the maximum growth yield of the culture grown in 547 mg/l nitrate was reached more quickly compared to the cultures grown in BG11 medium. This could mean that although the turbidity values of cultures grown in BG11 were lower, the growth cycle and stationary growth phase could have been longer. In addition, as seen from previous experiments, P. catenata is not the most reproducible culture and there can be significant variations between the cultures
96 grown in the same conditions even in triplicate. Therefore, it should not be surprising, that cultures did not grow to the same concentrations.
It is worth also mentioning, that subsequent reductions in nitrate concentration by 50% did not directly translate to reduction in turbidity values. The cultures turbidity values decreased, but not by 50% compared to the higher nitrate concentrations suggesting a not directly linear relationship. In addition, reduction in phosphate in growth media with low nitrate concentrations did not show significant difference in the highest culture concentrations measured by turbidity. Also although with reduction of phosphate culture turbidity values increased, during the repeated experiment they were similar to turbidity values of cultures grown in higher phosphate concentrations. This could mean, that there is some variation in the highest turbidity values that can be achieved as with cultures grown in high nutrient conditions.
However, the stock culture may have some impact on the culture concentration. This difference can be seen from the data of culture inoculated with stock grown in BG11 medium into RN BG11 medium compared to cultures inoculated into the same medium from stock grown in low nutrient medium. The pH values of the cultures also provide additional insight into effects of nutrient availability: it seems that cultures grown in lower nutrient medium below 54.7 mg/l nitrate were not able to adjust the medium pH as much as other cultures, even though they were increasing in turbidity values at a similar rate. It could be the growth rate of cultures, or the required minimum concentration of cells to be able to adjust the medium pH. One of the reasons for this could be that the results of carbon dioxide being consumed for growth, at higher cell concentration, more carbon dioxide is required, thus increasing the culture pH more. The lower concentration cultures did increase the pH of the medium, but not to such high values. This information could be used in non-buffered near neutral pH environments, where increase in the pH values could predict increase in the P. catenata concentration before it is visible. It is concentration dependant, therefore background concentrations in large lakes may not be significant, but at higher concentrations cultures can have a greater impact. Furthermore, from observations during my experimental work, a decrease
97 in pH values below ~9.5 in high concentration cultures could signal either an upcoming decrease in culture concentration or a slower growth phase. The decrease in pH in most of the cultures can be seen before cultures reached the highest turbidity values.
The data obtained from ion chromatography analyses shows that cultures grown in BG11 medium are phosphate limited, and there was still nitrate left at the end of the experiment. On the other hand, once the starting nitrate concentration was lowered to 109 mg/l nitrate and below, the cultures becomes nitrate limited, and some phosphate was still present in the medium at the end of the experiment. Cultures grown with 547 and 247 mg/l of nitrate could not be clearly distinguished in terms of which nutrients were limiting as in both cultures nitrate and phosphate levels were not detected anymore at the same time in their growth cycles. From the literature, the often reported N:P ratio by weight is 9 as Healey and Hendzel (Healey and Hendzel, 1979), and similar results by Liu and Vyverman (Liu and Vyverman, 2015b), and both medium modifications fall around the reported optimum ratio, see Table 2.5. However, these cultures were grown under different light and temperature conditions which could impact on the optimum ratio, although, it does confirm affinity of P. catenata to high nitrate: phosphate ratio. The low nutrient experiments also showed, that although the N:P ratio increased, there was no significant difference between the cultures, suggesting, there are minimum nutrient concentrations required for significant culture growth.
98 Table 2.5. List of all modifications, nitrate, phosphate concentration, nitrogen and phosphorus ratio, and nitrate and phosphate ratio
Modification NO3 mg/l PO4 mg/l N:P NO3:PO4
BG11 1094.29 21.81 34.75 50.17
547.14 NO3 547.14 21.81 17.38 25.09
273.57 NO3 273.57 21.81 8.69 12.54
109.43 NO3 109.43 21.81 3.48 5.02
54.71 NO3 54.71 21.81 1.74 2.51
27.35 NO3 27.36 21.81 0.87 1.25
10.94 NO3 10.94 21.81 0.35 0.50
5.471 NO3 5.47 21.81 0.17 0.25
2.6 NO3 2.60 21.81 0.06 0.12 RN 2.60 7.90 0.30 0.33
2.6 PO4 2.60 2.60 0.93 1
99 Changes in chlorophyll a concentrations during nutrient reduction experiments BG11 6 547 mg/l NO3
274.5 mg/l NO3 5
109.4mg/l NO3
4 54.7 mg/l NO3
21.98 mg/l NO3 3 10.9 mg/l NO3
Concentrationµg/ml 2 5.47mg/l NO3
2.6 mg/l NO3
1 2.6mg/l NO3 7.9 mg/l PO4
2.6mg/lNO3 0 7.9mg/l PO4 0 10 20 30 40 50 60 from BG11 stock Days
Figure 2.15. Change in chlorophyll a concentration during growth cycles of P. catenata grown in progressively lower nutrient concentrations. The error bars show the standard error of the mean value, n=3.
100 2.6 Conclusions The experiments described here provided additional insight and supported studies by other authors: P. catenata reaches the highest turbidity and chlorophyll a concentrations in high nutrient and high N:P ratio media. Although the exact ratio varies with each experiment, and most likely physical growth conditions, as shown when compared to ratios reported in the literature, they are somewhat comparable. In addition, cultures can adjust the pH of the medium to alkaline values above 10, most likely due to carbon dioxide consumption, although this is dependent on culture concentration and growth stage dependent. However, cultures grown in buffered alkaline media did not exhibit any growth suggesting sensitivity to sudden changes in pH or sodium concentration. In addition, organic carbon availability experiments did not provide consistent results and seem to have a limited effect on the cultures growing in low nutrient availability medium, contrary to what some reported studies would suggest. The results also showed that reduced nutrient BG11 medium modification can be used to mimic nutrient availability in the FGMSP and would be suitable for further continuous culture experiments. In addition, unmodified BG11 medium could be used to evaluate culture growth in continuous culture with high nutrient availability, and nutrient renewal.
101 3 Continuous culture flushing experiments
3.1 Abstract The experiments were set up to examine changes in culture growth with nutrient renewal and compare to batch culture growth results. The experiment results could be used to determine if flushing or pH adjustment are viable methods of bloom control with unlimited nutrient supply. The experiment results showed that continuous culture grown in BG11 medium has slower culture growth compared to batch cultures. And contrary to batch culture experiments, cultures continued to grow in medium of pH 11.5. However, purging with pH 12 or 12.5 medium was able to reduce culture concentration. Cultures grown in low nutrient (RN BG11) medium grew faster compared to batch cultures. However, as with cultures grown in unmodified BG11, purging with pH 12 medium resulted in reduction in culture concentration. It was also shown, that sudden increase in pH coupled with purging was more effective compared to slow increase in pH.
3.2 Literature review There have been several authors suggesting bloom management by increasing mixing, disturbance, and flushing as means of preventing or controlling microorganism blooms in natural environments (Mitrovic Simon et al., 2005; Paerl, 2008; Verspagen Jolanda et al., 2006; Webster Ian et al., 2000). If species are reported to be sensitive to flushing rates, increased flow rate may be used as a means of control, as in the case of Anabaena circinalis. A discharge of > 300 ml/ day helped to prevent development of a bloom in Lower Darling River weir pool (Mitrovic et al., 2011). However, if a bloom has formed overnight, mixing may not be sufficient to reduce it, in addition, it was suggested that lower flow or discharge may not prevent bloom formation, but move it downstream (Mitrovic et al., 2011). Natural seasonal variations in rainfall may also have an impact on microbial communities due to reduced retention time, preventing bloom formation of species sensitive to flushing (Khuantrairong and Traichaiyaporn, 2008). Similar observations were made during lake Albufera studies during 1980-88, when longer periods of reduced flushing resulted in highest concentrations of microbial growth (Romo and
102 Miracle, 1994). In addition, contrary results of concentration decrease was observed in winter and autumn months possibly due to reduced illumination and increased rainfall, and increase in water level (Romo and Miracle, 1994).
Another study modelling flushing in lake coves to reduce the blooms of Prymnesium parvum reported that flushing may be a viable option for bloom control. As long as the inflow from the main lake is lower than the flushing rate used it could reduce the bloom up to 65-80% (Lundgren et al., 2013). In addition, intermittent flushing was shown to have limited applicability, as microorganism would recover during downtime (Lundgren et al., 2013). The study also suggested flushing using deepwater, thus reducing the need for external water source (Lundgren et al., 2013). Another study showed even more links of cyanobacteria, specifically Microcystis aeruginosa, and water column stagnation and water residence time (Romo et al., 2012). Less mixing along the water column, more stagnant layers, and increase in residence time resulted in larger colonies (Romo et al., 2012). In lake Albufera flow rates of 10 m3/s or higher were able to reduce concentration of M. aeruginosa (Romo et al., 2012).
An experiment done on small pilot ponds showed how nutrient loading can affect the microbial growth combined with increase in water retention time. Mixed culture grown in pond with C:N:P ratio of 104:10:1 increased in concentration less compared to cultures grown in pond with C:N:P ratio of 9:7:1 when residence time decreased from 7 to 4 days (Cromar and Fallowfield, 1997). The experiment suggested that cultures in grown in high nutrient environment will be less sensitive to dilution and flushing, as they are not nutrient limited. However, cultures grown in low nutrient environment will be more sensitive to flushing due to nutrient limitation and lower culture concentration. For cultures growing in low nutrient environments flushing with water of similar or higher nutrient concentration may increase nutrient availability. A study of 40 lakes in Brazil linked lack of precipitation, increased temperature, and water column stability to higher cyanobacterial biovolume, but lower biovolume of diatoms (Brasil et al., 2016). Another model and data comparison study showed Aphanizomenon and Anabaena chlorophyll levels increased due to decrease in flow and increase in temperature
103 (Elliott, 2010). In addition, the PROTECH model used showed how warm, stable water column, and flushed at low rate water body would favour cyanobacterial growth (Elliott, 2010). However, a diatom species Asterionella was affected by temperature and flushing less, and more by light limitation due to the season and deep mixing (Elliott, 2010). Although, due to low nutrient inflow in low flow conditions internal phosphorus supply in the lake and ability to fix nitrogen made Aphanizomenon and Anabaena dominant species during warmer months (Elliott, 2010).
The flow in hydroelectric reservoirs was shown not only affect the growth of microorganisms and nutrient availability directly by nutrient renewal, but also by creation of gradients in larger slower flushing reservoirs (Rangel et al., 2012). The reduction in nutrient availability may have arisen due to sedimentation and prevention of nutrient release (Rangel et al., 2012).
Pseudanabaena catenata is reported to be quite adaptable to light deficient conditions, but seems to be sensitive to flushing, as other species in S1 functional group according to (Reynolds et al., 2002). During study of La Maggiore made in 1996 Pseudanabaena sp. increased in abundance during late summer- early autumn months when highest temperatures, most stratification, and most stable water column were observed (Morabito et al., 2003). During a yearlong study of a lake in Australia, abundance of P. catenata decreased with lake remediation measures resulting in decrease in nitrogen and phosphorus availability, but it was though that P. catenata was so abundant due to low water residence time (~14 days) as well (Soares et al., 2011).
During flushing experiments, Pseudanabaena catenata became the main culture when cultures were non continuously flushed once a day, and growth medium contained silica (Gaedeke and Sommer, 1986). During experiments with non- continuous flushing every two days, P. catenata was the main species in the mixed culture for both: media with and without silica (Gaedeke and Sommer, 1986). The biomass concentration increased with each dilution, and towards the end of the period between flushing (Gaedeke and Sommer, 1986). At longer dilution intervals, P. catenata was one of the main species at disturbances every seven, and fourteen
104 days (Gaedeke and Sommer, 1986). Surprisingly it also seems that P. catenata thrives in conditions with higher flow rate, possibly due to renewal of nutrients (Xavier et al., 2007).
Under continuous mixed culture with Pseudanabaena sp. phosphorus removal was most efficient during daylight hours and removal rate decreased during the dark hours (Sukačová et al., 2015). During 24 hour illumination phosphorus removal rate was the highest at the start of the experiment (Sukačová et al., 2015). In addition, during high rate phosphorus removal media pH increased to values above 10, but decreased markedly during dark hours (Sukačová et al., 2015).
3.3 Aims of continuous culture experiments • Continuous culture experiments with unmodified BG11 medium were set up to determine if P. catenata cultures would reach higher turbidity values compared to batch cultures due to nutrient renewal. • Continuous culture experiments with unmodified BG11 were used to assess the impact of purging (dilution rate) and increase in pH as methods of bloom control. • Continuous culture experiments with reduced nutrient BG11 medium were set up to determine if nutrient renewal would have a positive effect on culture growth compared to batch experiments. • Continuous culture with reduced nutrient BG11 medium experiment was used to evaluate culture response to flushing and increase in pH when grown in lower nutrient availability. • The results of the continuous culture experiments would also provide information about the cultures maximum growth rate in low and high nutrient availability. • To gain information about effective dilution rates that could be used to adjust the purging regimes in the FGMSP. • The experimental setup would also offer steady state reproducible systems that could be used as a platform to manipulate medium compositions and culture pH.
105
3.4 Materials and methods
3.4.1 Continuous culture experiments The experiment was set up to evaluate culture growth in almost unlimited nutrient availability due to high nutrient concentration and nutrient renewal. Pseudanabena catenata was grown in a 1800 ml Fernbach flask containing 700 ml BG11 medium. It was grown at the same 16:8 light-dark regime as in batch cultures under fluorescent lights on a magnetic stirrer (Frisher Scientific Isotemp, China) operating at 50 rpm. Once the culture reached a 0.778 turbidity value, the culture was flushed with BG11 medium using Cole Parmer Materflex (USA) peristaltic pump. The flow rate was set at 80% doubling rate of the last sampling point. Once the culture reached a steady state, pH of the medium was adjusted by sodium hydroxide dosing, adjusting it to pH 11, 11.5, and 12 every 14-21 days to increase the pH of the growth culture. The medium pH was measured before changing the supply and by weekly monitoring. Absorption, pH, and pigment concentrations were measured as stated in previous experiments (Section 2.4.4 ). A sample retrieved from the flask and flushed out samples were used for analysis and comparison. Cell counts were done using Sedgewick-Rafter counting chamber (Pyser-SGI, UK), counting 10 squares in a grid pattern. During repeated experiments flushing was started at 0.700 and 0.673 turbidity values. The pH of the medium was adjusted every 22 days, representing doubling rate of the repeated cultures.
Chlorophyll a and pigments were extracted by filtering 1.5 ml of sample through Whatman GF/ A or C (China) filters using a 100 ml Buchner flask and filter funnel. The filter was removed, placed into a glass bottle, acetone was added and packed using a glass rod. The samples were covered with parafilm and bottle lid, wrapped in aluminium foil, and left in a fridge for at least 4 hours. The acetone was then transferred into a centrifuge tube, spun at 5000 rpm using Thermo Scientific Heraeus (Germany) for 20 minutes, and procedure followed as described in section (2.4.4).
106 The chemostat flushing rate was calculated by using doubling rate of the culture:
푙표푔푥푡 − 푙표푔푥0 푙푛푥푡 − 푙푛푥0 휇 = = 푙표푔푒(푡 − 푡0) (푡 − 푡0)
Equation 3.1. Equation used to calculate culture growth rate
Where µ - culture exponential growth rate, xt - culture concentration at a time t, x0 - culture concentration at t=0, t is time. Loge = 0.43429 (Schlegel and Zaborosch, 1993).
푙푛2 푡 = 푑 µ
Equation 3.2. Equation used to calculate culture doubling time
Where td is doubling time. At steady state µ=D, where D is the dilution rate.
푓 퐷 = 푉
Equation 3.3. Equation to calculate dilution rate of the vessel
Where f - flow rate, and V - total volume. From these equations, we can find that
푙푛2 푙푛2 푓 푙푛2×푉 휇 = , therefore, = and flow rate can be calculated: 푓 = = 휇 × 푉 푡푑 푡푑 푉 푡푑
Equation 3.4. Equation to calculate flushing flow rate
3.4.2 Cell counting Cells were counted using Sedgewick-Rafter counting chamber (Pyser-SGI Ltd., UK). It holds 1 ml volume in a shallow central chamber with a 1000 square grid, and has to be covered with a thick slide. The samples were mixed before pipetting into a chamber with slide covering diagonally. The cover slip was rotated with care to remove all air bubbles. Cells were allowed to settle and counted manually with a tally counter. Ten squares were counted in total in a pre-determined grid pattern. The cells touching left and bottom sides of the square were not counted to avoid overcounting. The cell concentration was determined by using equation:
1000 × 퐶 × 퐷 푇 = 푁
Equation 3.5. Equation used to calculate culture concentration.
107 Where T is total number of organisms per millilitre, N is squares counted, C is total number of organisms counted, and D is dilution factor (Bellinger and Sigee, 2015)
3.4.3 Continuous culture: reduced inorganic nutrients Reduced nutrient BG11 medium was inoculated with P. catenata stock culture grown in RN BG11 medium. The culture was grown in 1800 ml Fernbach culture flasks in 700 ml medium. The culture was stirred using a magnetic stirrer, at the same conditions as high nutrient continuous culture. Once the turbidity value had reached 0.050, close to highest concentrations measured in batch cultures grown in the same medium, the culture was flushed using Cole Parmer Materflex (USA) pump. Once steady state conditions were reached, the pH of the RN BG11 medium was adjusted to 8.5, 10, 11, 11.5, and 12 using sodium hydroxide every 9-10 days (approximately three complete volume changes). Absorption, pH, chlorophyll, and cell counts were measured as stated in previous experiment (Sections 2.4.4 and 3.4.1- cell counting.). A repeated reduced nutrient culture followed the same methodology as above, but the culture was flushed with RN BG11 medium with pH adjusted to 11.
3.4.4 Total inorganic carbon / Total organic carbon The Total Organic Carbon (TOC) was calculated by subtracting Total Inorganic Carbon (TIC) concentration from Total Carbon (TC) concentration.
TOC= TC-TIC
An analytic Jena Multi N/C 2100S (Germany) total carbon analyser was used to determine total and total organic carbon concentrations. Defrosted samples were first syringe filtered through 0.2 µm Millex PTFE filters (Japan or Ireland) into a clean centrifuge tube to remove any intact cell matter. The filtered samples were diluted by a factor of 4 or 20, for high nutrient cultures, to 4 ml of total sample volume. BG11 and RN BG11 media were also analysed to determine the baseline total and inorganic carbon concentrations. Two separate injections were used for total and inorganic carbon and were repeated between two and 4 times. A 0.25 ml sample volume was used per measurement. The furnace was set to 800°C, and 160 ml/min oxygen flow rate. The TIC concentration was determined by acidifying the
108 sample with 10% phosphoric acid. The number of injections was determined by coefficient of variance value, which was set to be below 5%. The coefficient of variance can be calculated using formula:
휎 푐 = × 100% 푣 휇
Equation 3.6. Equation to calculate variance
Where cv is the coefficient of variance in percentage, σ is the standard deviation, and µ is the mean.
109 3.5 Results and discussion
3.5.1 Continuous culture experiment flushed with BG11 medium A culture of P. catenata was inoculated and grown under batch conditions at the start of the experiment. Flushing started on day 57 at 0.845 ml/h until day 95, at 50% culture growth rate (Figure 3.1). The fluctuations in turbidity and pH values during the same period were mostly due to failed magnetic stirrer. In addition, the sharp fall in turbidity values between days 75 and 95 from 0.848 to 0.652 was due to clogged tubing when no access to the culture was possible. The flushing was stopped, excess media volume was removed, and culture was allowed to recover. Flushing of the culture was restarted at the same rate on day 97. The flow rate was increased on day 101 to 3.44 ml/h. This resulted in a sharp drop in turbidity and cell concentration from 0.575 on day 101 to 0.308 on day 117. From day 117 flow rate was reduced to 2.49 ml/h to prevent culture from being completely flushed out. The lower flow rate resulted in culture reaching steady growth. From day 103 samples were also taken directly from the flask for comparison with the flushed out samples.
No significant differences in turbidity or cell concentration was observed between flushed and pipetted samples, except when the outlet tube clogged up later in the experiment. However, the pH values of samples obtained from outlet tube were lower compared to samples taken directly from the flask. It may be due to the tubing, or available CO2. The spikes in flushed sample turbidity values on days 136, 143, 157 were due to clogged up tubing during sampling. From day 140, pH of the medium used for culture flushing was increased to 11 by addition of sodium hydroxide, but there was no observable effect on the culture. The medium pH was increased to 11.5 on day 154 resulting in a slow and gentle decline after flushing at higher pH has started. Absorption values of 0.252 for samples and 0.287 for flushed samples decreased to 0.223 and 0.215, respectively, over 12-day period. On day 166 medium pH was increased to 12, the turbidity decreased sharply after increase in medium pH from 0.223 and 0.215 for samples and flushed samples to 134 and 0.138 respectively during the 7-day period. On day 173 pH of the medium used to flush the culture was increased to 12.5, turbidity continued to decrease at the same
110 rate as after previous increase in pH from 0.134 to 0.138 to 0.067 and 0.062 respectively. From day 178 onwards turbidity values remained somewhat stable, fluctuating at lower amplitude below starting inoculation values. The experiment ended on day 192. Change in absorption and pH during continuous culture experiment
Start of Flushing Flushing restared pH 11 pH 12 0.9 flushing stopped 13 Flowrate pH 11.5 pH 12.5 reduced 0.8
12 0.7
0.6 11
0.5 pH 0.4 10
0.3 Absorption at 600 nm 600 at Absorption 0.2 9
0.1
0.0 8 0 20 40 60 80 100 120 140 160 180 200 Days Sampled Flushed Flushed pH Sampled pH Figure 3.1. Changes in turbidity and pH values during high nutrient continuous culture experiment in flushed and taken directly from the flask samples. The error bars show the instrument error, n=1. The red lines mark changes in culture conditions.
The pH values surged in the first few days after inoculation to 10.09 on day 13 and fluctuated slightly until stirrer failures resulted in higher amplitude fluctuations. While turbidity values dropped between day 75 and 95, the pH readings were mostly unchanged, ranging from 10.32 to 10.23. Once the culture flushing resumed on day 97 pH readings stabilised and fluctuations remained in the same range (9.7 to 10.47) until day 173. After culture turbidity value decreased below 0.114 coinciding with medium pH increase to 12.5 the pH readings increased from 10.45 to 11.88 in ~10 days, the final pH value being 12.09. The culture pH values were not affected by flushing medium pH values of 11 and 11.5. However, once culture was flushed with medium of pH 12, pH increased slightly, and 12.5 resulted in a sharp
111 increase in culture pH and continued to slowly increase right to the end of the experiment.
Change in chlorphyll a and carotenoid concentrations during continuous culture experiment 1.6
1.4
1.2
1.0
0.8
0.6
Concentration (µg/ml) Concentration 0.4
0.2
0.0 0 20 40 60 80 100 120 140 160 180 200 Days Cl a flushed Cl a sampled Carotenoid flushed Carotenoid sampled
Figure 3.2. Change in chlorophyll a and carotenoid concentrations of flushed and sampled continuous culture over the length of the experiment. The error bars show 5% error, n=1.
The chlorophyll a and carotenoid concentrations fluctuated more, compared to the turbidity values, especially at the start of the experiment, until flushing started (Figure 3.2). In addition, the increase in pigment concentrations was not as fast and fluctuations were more pronounced compared to turbidity values. The highest concentrations of chlorophyll a and carotenoids were reached on day 61 and 59 of 1.54 and 1.088 µg/ml respectively. Although turbidity values later fluctuated at a similar range, chlorophyll a and carotenoid concentrations measured later were all lower. The decrease in turbidity between days 75 and 95, resulted in increase in chlorophyll a values from 0.987 to 1.128 µg/ml, but decrease in carotenoid concentration from 0.649 to 0.504 µg/ml. The reduction in chlorophyll a concentrations was slower compared to the decrease in turbidity during the same
112 period. In addition, the highest turbidity value measured on day 75, was not replicated in chlorophyll a and carotenoid concentrations. The outliers on days 117, 147, and 157 could be attributed to sampling error as no similar concentrations were measured later in the experiment.
There is a sharp decrease in chlorophyll a and carotenoid values from day 108 (0.592 to 0.296 µg/ml for flushed and 0.733 to 0.296 µg/ml for sampled chlorophyll a) for two days, coinciding with the decrease in turbidity values after the medium flow rate increased. Compared to the turbidity values, the drop is sharper and chlorophyll a values were below starting concentrations, unlike with turbidity values. The same could be said about carotenoid concentrations as they followed turbidity, and chlorophyll a values, just at lower concentrations throughout the experiment. However, from day 124, the difference between chlorophyll a and carotenoid concentrations was a lot less significant. The spikes in turbidity of flushed samples on days 134, 139, 143, and 157 are not seen in the either chlorophyll a or carotenoid concentrations. As with turbidity, chlorophyll a and carotenoid concentration continuously decreased from day 164, with a slight increase on day 185. The pigment concentrations did not recover and continued to drop until the end of the experiment even further below the starting concentrations of 0.587 µg/ml.
Although there were numerous disturbances during the first continuous culture experiment, the data obtained provided useful information. There was a stationary growth phase at the start of the experiment, suggesting cultures needed to adapt to the new environment, followed by quite rapid growth (turbidity increased from 0.129 to 0.302 over 7 days). However, it took nearly 40 days for the culture to reach turbidity values close to 0.8, suggesting that scaling of the culture is not straightforward in high nutrient conditions. Another observation about the culture growth is the behaviour when it is not mixed- stirrer failures resulted in decrease in turbidity and cell concentration. There may be several reasons for the outcome: self-shading, or poorer nutrient circulation, availability of CO2 at the bottom of the flask. Either way, the results would suggest that P. catenata culture grown in these conditions requires well mixed environment for culture to grow. Which is in
113 contradiction to studies reporting P. catenata thriving in more stagnant and stratified waters, as well as low light availability (Morabito et al., 2003; Reynolds et al., 2002). In addition, Pseudanabena catenata was observed growing suspended in liquid media by (Khan et al., 2017). In addition, (Gao et al., 2018) reported that mixing rate did not have any significant effect on the growth rate of Pseudanabaena sp. was grown in batch conditions, including non-mixed cultures. However, the difference in the response could be due to batch/ continuous culture conditions, and the culture volume, as 300 ml of medium were used in the study (Gao et al., 2018) versus 700 ml in this experiment.
However, the light provided during the experiment is not as bright when compared to bright summer sunshine above 1000 PAR (measured outside the facility with the same light meter). Therefore, shading and access to light may not have such an impact during the summer months in an uncovered pond, especially when such a large surface area is available.
The overflow events due to clogging of the outlet tube also highlighted that culture reaction will depend on the flow rate and the length of time the culture was overflowing. As can be seen between days 75 and 95, culture was overflowing, but at a slow rate for a long period of time. This resulted in decrease in the turbidity values and cell concentration. However, due to clogged tubing events later in the experiment, when cultures were flushed at higher rate and overfilled for a shorter period, there was less of an effect on the culture, as can be seen from samples obtained straight from the flask. These results would suggest, that these events did not dilute the culture sufficiently in the time flask was filling up, or culture was growing fast enough to offset the increase in the volume. However, with increase in volume in the flask, residence time in the flask should be increasing, thus possibly promoting the culture growth. The positive effect of increase in residence time on cyanobcaterial growth was reported in natural environments (Elliott, 2010; Romo and Miracle, 1994), thus supporting the results of this experiment. Although, the culture was not monitored during that time and the concentration measured after two weeks could have been at the end of a growth cycle.
114 From the experimental data it can also be seen that a flow rate of 3.44 ml/h was sufficient to reduce culture concentration, hence the flushing rate had to be reduced for cultures to reach stationary growth. The results would indicate that the culture growth rate was slower than the flushing rate, thus the flushing rate may be sufficient to prevent culture growth even though the nutrient concentration in the medium is the same or higher compared to nutrient concentration in the flask as suggested by (Cooke et al., 2005) and observed in large lakes. Once flushing was combined with increase in pH up to 11, no noticeable effect has been observed. Similar observation was made during a study of Pseudanabaena sp. growth at pH varying from 3 to 11 and maintained using HCl or NaOH dosing in batch conditions (Gao et al., 2018). Pseudanabaena sp. cultures increased in concentration at pH 11, in medium dosed with NaOH to maintain the pH of 11 and cultures with medium adjusted to pH 11 at the start of the experiment, but not maintained. In addition, as in batch part of this experiment, non-dosed cultures naturally increased the pH between 10 and 11 as the culture concentration increased (Gao et al., 2018). Once the medium pH increased to 11.5 and above the increase in pH caused a decrease in culture concentration. The results would suggest, that the culture is pH sensitive, but above certain values.
The results are also in contradiction to the results obtained from the buffered medium experiment, where cultures were sensitive even to medium pH of 10. However, the conditions were changed slowly in the continuous culture experiment and P. catenata could adapt to slowly increasing pH. Furthermore, the concentration of the culture when pH increased was higher in continuous culture compared to the buffered pH experiment, and cultures were in a different growth stage. It would not be surprising, if freshly inoculated cultures are more sensitive to changes in pH due to a sudden change in conditions, compared to well established cultures. The results following flushing with pH 12.5 medium also add some weight to suggestion, that P. catenata is capable of regulating medium pH. There may be several reasons for limited effect of increase in flushing medium pH: the medium pH has to be above a certain value to have an effect on the culture, culture below a
115 certain concentration cannot adjust the medium pH as efficiently, or due to the cell death, culture pH was not adjusted by metabolism of the organisms anymore.
3.5.2 Repeated continuous culture experiment The repeated experiment was set up to determine the repeatability and reliability of the previously completed continuous culture experiment. As in the first experiment, cultures were inoculated and grown in batch conditions until turbidity values reached ~0.6-0.7. At the start of the experiment, the cultures grew well, no disturbances were observed as seen in Figure 3.3. There was a sharp increase in turbidity between inoculation and day 10 from 0.101 and 0.108 to 0.359 and 0.345 for flasks A and B. The culture growth slowed down slightly afterwards, but still continued at a steady rate. As in previous experiments, exponential growth was not observed, and cultures were growing closer to a more linear fashion.
Change in turbidity and pH during repeated continuous culture experiment Start of flushing pH 11 pH 11.5 pH 12 0.8 11.0
0.7 10.5
0.6 10.0
0.5 9.5
0.4 9.0 pH
0.3 8.5
Absorption at 600 nm 600 at Absorption 0.2 8.0
0.1 7.5
0.0 7.0 0 20 40 60 80 100 120 140 160 180 200 Days Culture A pH A Culture B pH B
Figure 3.3. Changes in turbidity and pH values during repeated high nutrient continuous culture experiment. The error bars show the instrument error, n=1. The red lines mark changes in culture growth conditions
116 Flushing with unmodified BG11 medium started on day 38 at 1.325 ml/h flow rate and ~22 days residence time. The turbidity values decreased over the first week from 0.700 and 0.673 to 0.565 and 0.589 for flasks A and B respectively, and then slowly stabilised. Flushing at increased medium pH to 11 started on day 94 resulting in a slow drop in turbidity between days 94 (0.385 and 0.445) and 113 (0.339 and 0.383). However, both cultures recovered by day 120, when medium used for flushing pH was increased to 11.5. The turbidity values fluctuated and decreased slightly over the next 27 days. After the increase in medium pH to 12 on day 147 the turbidity values decreased sharply from of 0.324 and 0.398 to 0.206 and 0.183 on day 170. The cultures somewhat recovered over next 7 days to 0.279 and 0.251 turbidity. However, the culture concentration fell again, and it was decided to end the experiment due to time pressure and comparable results to the previous experiment.
Changes in chlorophyll a and carotenoid concentraitons during repeated continuous culture experiment 1.4
1.2
1.0
0.8
0.6
Concentration µg/ml Concentration 0.4
0.2
0.0 0 20 40 60 80 100 120 140 160 180 200 Days Cl a A Cl a B Carotenoids A Carotenoids B Figure 3.4. Change in chlorophyll a and carotenoid concentrations in repeated continuous culture in flasks A and B. The error bars show the 5% error, n=1.
117 Although turbidity and cell concentration increased continuously between day 0 and 38, there was a decrease in chlorophyll a and carotenoid concentration between days 13 and 20. The chlorophyll a concentration decreased from 0.380 and 0.470 µg/ml to 0.314 0.352 µg/ml. In contrast to turbidity values and cell concentration, another decrease in chlorophyll a and carotenoid concentration can be seen on day 42, when values decreased by 0.333 µg/ml, although, only for Flask A. It may be attributed to a sampling error as concentration recovered the next sampling day. A dip in carotenoid concentration can also be seen on day 50 in Flask A, but not for chlorophyll a. A slight decrease in cell concentration in Flask A was recorded on that day. As in the previous experiment, after flushing started the difference between chlorophyll a and carotenoid concentrations decreased. In addition, as observed earlier, there were some fluctuations in chlorophyll a concentrations. From the Figure 3.4 it can be seen, that carotenoid concentrations seem to be more stable compared to chlorophyll a. The fluctuations between days 66 and 91 coincided with fluctuations in cell concentrations, however, chlorophyll a concentrations fluctuated at a higher amplitude compared to carotenoid concentrations. The increase in pigment concentrations between days 111 and 128 is due to increase and cell concentration, as well as on days 140, and 162. Contrary to the previous experimental results, the decrease in pigment concentrations was not as sharp, turbidity values stayed above starting concentration. However, as previously noted, when flushed with medium pH adjusted to 12 pigment values dropped close to ones at the start of the experiment. The slight increase in turbidity values at the end of the experiment was repeated in pigment concentrations as well.
118 Total and inorganic carbon concentrations during repeated continuous culture experiment 160
140
120
100
80
60
Concentrationmg/l 40
20
0 90 100 110 120 130 140 150 160 170 180 190 Days Total carbon flask A Total carbon flask B Total inorganic carbon A Total inorganic carbon B
Figure 3.5. Total and inorganic carbon concentrations in duplicate flasks A and B during repeated high nutrient continuous culture experiment. The error bars show the variance coefficient, n=1.
There is a clear trend in increase in the total carbon and total inorganic carbon concentrations throughout the experiment (Figure 3.5). The increase in total inorganic carbon followed the increase in medium pH. The difference between the total and inorganic carbon stayed between 26 and 37 mg/l throughout the experiment, higher concentrations measured in duplicate B samples. The higher microorganism concentration may be the reason for slightly higher total and inorganic carbon concentrations compared to ones of flask A. In addition, the lowest total organic carbon concentration was measured on day 147 and recovered on day 185 for both flasks. It should also be mentioned, that BG11 medium total carbon concentration was measured to be 9.86 mg/l ±226.8 µg/ml, and total inorganic carbon 5.4 mg/l ±211.1 µg/ml.
The repeated continuous cultures also grew slower compared to the batch culture growth in the same medium and at a similar rate compared to the first continuous culture. As reported in earlier experimental results, pH rose just after inoculation to values fluctuating ~ 10.5 from 7.98 and 8.2 in 6 days and decreased slightly after flushing to ~pH 10. Medium pH did not have a significant effect on the culture pH,
119 as it can be seen from Figure 3.3. The culture pH did slowly creep up as during the experiment, but did not reach the same pH values measured in cultures grown in batch conditions, suggesting P. catenata growth and slow adjustment to the new conditions.
Contrary to the results of the previous continuous culture experiment, the culture pH values during the repeated continuous culture experiment did not reach the pH values above 11.5. It could be due to the medium pH used, as the highest pH value the flushing medium was adjusted to in the second experiment was 12, rather than 12.5. Or it could be due to the culture turbidity towards the end of the experiment being higher compared to the first continuous culture experiment. However, as in the earlier experiment, flushing combined with sodium hydroxide dosing was able to reduce culture growth in high nutrient availability. Continuous flushing compared to flushing with interruptions seemed to aid reduction in turbidity at lower pH, as reduction in turbidity was observed from flushing at pH 11.5.
3.5.3 Low nutrient flushing experiment The reduced nutrient continuous culture experiment was set up to determine if culture grown at low nutrient concentrations, but with nutrient renewal would reach higher concentrations compared to cultures grown in batch conditions. In addition, as in previous experiment, sodium hydroxide dosing was used to evaluate cultures adaptation to elevated pH and sodium concentration.
As in previous experiments, culture was inoculated, although with much lower inoculum concentration, and grown in batch conditions. The turbidity values increased and went from 0.004 to 0.051 in 7 days (see Figure 3.6), as in batch culture experiments. As the highest concentrations achieved in batch conditions in earlier experiments were close to those measured by day 7, it was decided to start flushing at 10 ml/h flow rate (80% of growth rate maximum) to prevent cell death. As in continuous culture experiments, turbidity values decreased over the 12 following days to 0.23, but did recover somewhat by day 26 to 0.26 when culture flushing at increased medium pH started.
120 The medium pH was adjusted to 8.5, resulting in increase in turbidity to 0.051 on day 28, and a sharp decrease to 0.028 by day 31. The medium pH increased further on day 35 to 10, when turbidity was 0.04, as culture somewhat recovered. The culture turbidity values increased after an increase in medium pH over the following two days to 0.049. On day 42 the turbidity decreased to 0.045, as culture slowly adapted to increase in pH. The medium pH was increased further to 11 on the same day, which resulted in an increase in turbidity to 0.051 on day 45 and then a decrease to 0.036 on day 47. On day 54, culture turbidity recovered to 0.052 and medium pH was adjusted to 11.5. As with previous increase in pH, turbidity values improved to 0.057 on day 56, and then sharply decreased to 0.044 on day 59. By day 66 the culture reached the highest turbidity of 0.062 and medium pH was increased for the final time to 12. The last increase resulted in a very sharp drop in turbidity to 0.036 in 3 days, culture did not start to recover until day 80 to turbidity value 0.039. The experiment ended on day 84 and culture turbidity of 0.044. Changes in absorption and pH during low nutrient availability continuous culture experiment
Start of flushing pH 8.5 pH 10 pH 11 pH 11.5 pH 12 0.07 14
0.06
12 0.05
0.04
10
pH
0.03
0.02
Absorption at 600 nm 600 at Absorption 8
0.01
0.00 6 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Days Absorption pH Figure 3.6. Changes in turbidity and pH values during the first reduced nutrient continuous culture. The red lines mark the start of flushing and increases to medium pH. The error bars show the instrument error, n=1.
121 The pH of the culture grown in medium with low nutrient availability was significantly lower compared to the cultures in high nutrient availability conditions. Therefore the medium pH was adjusted slowly to allow culture in low nutrient availability medium to adapt to the significant changes in pH and prevent culture stress. The culture pH, unlike in previous continuous culture experiments, did not increase significantly while the culture was grown in batch conditions and fluctuated around pH 7. Furthermore, flushing with medium that was not pH adjusted did not seem to have any effect on the culture either, besides one reading on the day 21 of pH 8.24. Once medium pH was adjusted to 8.5, a clear increase in culture pH can be seen with a slight delay of 2-3 days to 8.41 on day 31. Only one increase in medium pH did not result in an increase in culture pH, when flushed with pH 10 medium. Higher medium pH values changed culture pH quicker. Increase in medium pH to 11 resulted culture pH of 9.7, increase to 11.5 increased culture pH to values ~10.5, and medium pH 12 caused culture pH to increase to pH values ~11.8. In addition, compared to continuous culture experiments with high nutrient medium, pH in feed medium had a more significant impact on culture pH and shorter culture adjustment period.
122 Changes in chlorophyll a and carotenoid concentrations during low nutrient availability continuous culture experiment
0.08
0.06
0.04
0.02 Concentration µg/ml Concentration
0.00
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Days Chlorophyll a Carotenoids Figure 3.7. Change in chlorophyll a and carotenoid concentrations over the length of the first low nutrient experiment. Error bars show the 5% error, n=1.
As in previous experiments, carotenoid concentrations were more stable compared to chlorophyll a concentrations. The pigments followed the same trends as turbidity and cell concentration, as seen in Figure 3.7. The chlorophyll a and carotenoid concentrations increased from day 0 to day 7 from 0.0117 and 0.0101 to 0.0446 and 0.0293 µg/ml respectively. Although on day 7, turbidity and cell concentration values were higher compared to day 11, the pigment concentration remained stable. The peak in chlorophyll a concentration (0.0728 µg/ml) on day 47 is on a low point in turbidity, and not a peak in cell concentration either. A peak in chlorophyll on days 56-61 (0.0728-0.0634 µg/ml) also coincided with a decrease in cell concentration and turbidity. From day 66 onwards, as with turbidity and cell concentration, chlorophyll a concentration decreased from 0.0564 to 0.0047µg/ml by day 69, and carotenoid concentration to decreased from 0.0439 µg/ml (the highest concentration in the experiment) to 0.0177 µg/ml, and below detection limit by day 74. The pigment concentrations dropped sharply after flushing medium
123 pH was adjusted to 12, however, pigment concentrations did not recover as seen in turbidity and cell concentrations. The results would suggest either damage to the photosynthetic apparatus, or cells multiplying with reduced or delayed photosynthetic pigment production. In addition, the pigment concentrations did not recover over 13-day period.
Change in nitrate and phosphate concentrations during
7.00 reduced nutrient continuous culture experiment
6.00
5.00
4.00
3.00
2.00 Concentration(mg/l) 1.00
0.00 0 10 20 30 40 50 60 70 80 Days Phosphate Nitrate
Figure 3.8. Change in phosphate and nitrate concentrations during the low nutrient continuous culture experiment. The error bars mark standard mean error, n=1.
During the low nutrient continuous culture experiment nitrate concentration in culture medium decreased from the inoculation (2.12 mg/l) and did not increase until the last day of the experiment (1.73 mg/l) as seen in Figure 3.8. The nitrate was still detectable, indicating presence, but at very low concentrations during most of the experiment. The phosphate concentration slowly decreased from 5.71 mg/l on inoculation day until the day 35 to 5.25 mg/l. The phosphate concentration then plummeted until the end of the experiment to 0.070mg/l. As P. catenata requires relatively high N:P ratio for optimal growth, the results indicate that nearly all the nitrate was used by the culture. However, once the medium pH increased to 10, the culture may have had an increase in required phosphate, resulting in increase in culture turbidity (see Figure 3.6). The increase in nitrate concentration in culture medium on day 84 would indicate a reduced need for nitrate and paired with decrease in chlorophyll a and carotenoid concentrations, stress in culture.
124 Total and inorganic carbon concentrations during reduced nutrient continuous culture experiment 40 35 30 25
20 mg/l 15 10 5 0 0 10 20 30 40 50 60 70 80 Days Total carbon Total inorganic carbon
Figure 3.9. Changes in total and inorganic carbon concentrations during low nutrient continuous culture experiment. The error bars show the standard error of the mean, n=1.
The total carbon concentrations (Figure 3.9) fluctuated during the experiment, from 14.14 mg/l at the start of the experiment, decreased on days 7 and 26 to 6.83 and 5.8 mg/l respectively. The concentration shot up to 21.49 mg/l on day 35, and despite a decrease in culture concentration, increased further to 36.41 mg/l on day 66, and decreased on the last day of the experiment to 29.09 mg/l. The total inorganic carbon concentration fluctuated, but general trend of increase with increase in culture and medium pH can be observed, despite the decrease on day 66. Unlike the previous experiment, the total carbon concentration varied significantly, with lower organic carbon concentrations measured on days 7, 26, 45, and 84, the last day of the experiment.
Due to the lower inoculating concentration, doubling time of the first continuous culture grown in low nutrient medium was just over 1 day. This resulted in a high calculated flow rate and shorter time for adaptations. In addition, the low nutrient continuous culture seemed to be affected by the increase in pH more, as clear stages of increase in pH coinciding with increase in medium pH can be seen in Figure 3.6. These observations support previous suggestions, that cultures with lower concentrations are no able to adjust the medium pH as effectively as cultures with higher concentrations. Furthermore, it seems that there is a certain
125 concentration of P. catenata that is required to be able to adjust the medium pH. Contradicting results of the continuous culture experiments in high nutrient medium turbidity values of the culture increased with time and reached higher values when culture was flushed compared to the batch conditions before flushing. It would suggest that nutrient renewal can increase culture concentrations in continuous cultures with low nutrient availability as observed by (Cromar and Fallowfield, 1997; Xavier et al., 2007). In addition, after the decrease in concentration, culture seemed to be able to recover from flushing and increase in medium pH. It may be due to the lower culture concentration- less cells to recover in number, or due to shorter generation time resulting in adaptation to higher flow rate.
However, the pigment concentration decreased significantly and did not recover until the end of the experiment. It is suggested, that cells were not producing pigments and focusing on multiplying as suggested by (Fujita, 1985; Pancha et al., 2014). Although nitrogen concentrations in the growth medium were very low during most of the experiment, cultures did not show signs of nitrogen defficiency as observed by (De Loura et al., 1987), as concentration of carotenoids in the culture was not significantly higher compared to chrolophyll a. It may be due to the fact that nitrogen, although at low concentration compared to BG11 medium, but was still present and constantly renewed due to flushing. Although, it should be noted that the differences in growth conditions and Pseudanabaena strain should be taken into account when comparing the results of the two studies. It is also worth noting, that during cell counting filaments were shorter than earlier in the experiment, only few cells length, and more transparent that normal. Although, the experimental results show that turbidity values did increase, due to visual observations of cell culture health it was decided to end the experiment with assumption that flow rate and sodium dosing combination was able to prevent healthy culture growth. The change in cell appearance corresponds to observations made during buffered pH batch culture experiment, providing additional evidence to support the suggestion, that cultures at lower concentrations are more sensitive to more alkaline conditions.
126 3.5.4 Repeated low nutrient experiment The reduced nutrient continuous culture experiment was repeated with modifications, to determine, if flushing and sudden increase in pH would have the same impact as slow adaptation to changing conditions.
As in the previous experiment, the culture was inoculated with low concentration stock culture and grown in batch conditions. Unlike in the previous experiment, growth was slower for the first 3 days (0.003 to 0.005 turbidity), but later on culture went into nearly exponential growth phase as seen in Figure 3.10 and Table 3.1. Despite the delay in growth, as previously, it reached turbidity of 0.048 in 8 days and flushing with RNBG11 medium with pH adjusted to 11 started at 17.6 ml/h. or 80% of the growth rate. There was a sharp decrease in turbidity values, over the two following days to 0.015 and they did not recover until the end of the experiment on day 17. As observed previously, the pH values did fluctuate slightly (6.65-7.64), but no significant increase was observed while culture was growing in batch conditions. There pH did increase after flushing started, when pH value jumped from 6.69 to 10.81 in two days. The results would suggest, that flushing combined with sudden and sharp increase in medium pH has a more significant effect compared to flushing and slow increase in medium pH. The decrease in culture concentration may also be due to sudden increase in sodium concentration.
127 Changes in absorption and pH during repeated low nutrient availability continuous culture experiment
Flushing at pH 11 0.06
10
0.04
8
pH
0.02 Absorption at 600 nm 600 at Absorption
6
0.00 0 5 10 15 20 Days Absorption pH Figure 3.10. Change in turbidity and pH values over the length of the repeated low nutrient experiment, the flushing with pH 11medium started on day 8. The error bars show the instrument error, n=1.
During the experiment chlorophyll a and carotenoid concentrations followed changes turbidity. There was a slight decrease in concentrations on day 3 from 0.0117 and 0.005 µg/ml for chlorophyll a and carotenoids respectively, to 0.002 µg/ml for both pigments. The concentrations increased sharply onwards to 0.039 µg/ml chlorophyll a on day 6 and 0.025 µg/ml carotenoids on day 8 (Figure 3.11). The highest concentration of carotenoids was measured on the same as turbidity and cell concentration, chlorophyll a concentration started to decrease by then. After flushing at pH 11 started, chlorophyll a concentration decreased below limit of detection and did not recover until the end of the experiment. Carotenoid concentration did decrease, but slower compared to chlorophyll a concentrations.
128 Changes in chlorophyll a and carotenoid concentrations during repeated low nutrient availability continuous culture experiment
0.04
0.02 Concentration (µg/ml) Concentration
0.00
0 2 4 6 8 10 12 14 16 18 20 Days Chlorphyll a Carotenoids
Figure 3.11. Variations in chlorophyll a and carotenoid concentrations in repeated low nutrient continuous culture experiment. The error bars show 5% error, n=1.
Unlike in the first low nutrient continuous culture experiment, some slower growth was observed at the start, however the culture moved into exponential growth from day 3. It may due to preference of cyanobacteria to store nutrients before growth (Jia et al., 2013), it may be more pronounced in culture grown in low nutrient availability. This resulted in an ever-higher flow rate and shorter residence time compared to earlier high nutrient availability continuous culture experiments. When response of cultures to sodium hydroxide dosing is compared between slow increase in medium pH and a fast one, the sudden change seemed to be more effective at preventing growth. This is illustrated as low and high nutrient cultures continued to grow until medium of pH 12 was introduced, compared to flushing with pH 11 medium without time for adaptation. As with the previous experiment, cultures at lower concentration seem to be more sensitive to sudden changes and increase in pH compared to cultures of higher concentration. The most likely
129 explanation would be the lack of ability to adjust the growth medium pH due to low cell culture cell concentration.
3.5.5 Growth rate analysis in continuous cultures As it can be seen from the Table 3.1, even at the start of the growth cycle, cultures grown in high nutrient concentrations grew slower, compared to cultures grown in low nutrient concentration medium. The fastest growth rates between the cultures grown in high, 3.3 days doubling time, and low nutrient medium, 0.91 days doubling time, also differ significantly, resulting in different flow rates used for culture flushing. However, cultures were inoculated with different starting concentrations between growth media. As seen from the Figure 3.3 repeated high nutrient continuous culture grew faster up to day 8, afterwards, as confirmed by the cell counts and doubling rate, culture growth slowed down. It was observed that self-shading and increase in water level may result in slower growth (Romo and Miracle, 1994), as culture concentration in high nutrient availability cultures increased, available light decreased. It would also support the need for mixing and decrease in concentration when cultures were not mixed due to uneven cell distribution and availability of light. However, the suggestion somewhat contradicts the results of another study, with the reported fastest growth rate of Pseudanabaena sp. at 27 μmol photons m−2s−1 (Gao et al., 2018), much lower than 250 µmol photons m-2 s-1 (PAR) light intensity used during this experiment, thus self-shading is unlikely to be impacting culture growth significantly. However, culture grown in low nutrient medium reached turbidity values higher than cultures grown in batch conditions, suggesting adaptation to higher flow due to nutrient renewal (Cromar and Fallowfield, 1997). However, the results of high nutrient flushing experiments contradict some of the results of (Cromar and Fallowfield, 1997) as cultures were seen to be recovering at flushing medium of pH 12, and adjustment of flushing medium to pH 11 did not result in reduction in culture concentration. The suggested reason behind the culture behaviour is most likely to be the nutrient availability, which is similar or higher in the flushing medium compared to the growth medium, resulting in nutrient renewal. The suggestions of (Cooke et al., 2005) support the experimental results, as medium for flushing
130 should have lower nutrient availability to be effective, or flow rate and resulting cell loss rate should be higher than the cell doubling rate.
131 Table 3.1. Comparison of P. catenata doubling times in different continuous cultures in BG11 and reduced nutrient media. CCBG11- First continuous culture grown in BG11 medium, CCA- duplicate A of repeated high nutrient continuous culture, CCB- duplicate B of repeated high nutrient continuous culture, CCRNA- first reduced nutrient continuous culture, CCRNB- repeated reduced nutrient continuous culture.
Days CCBG11 Days CCA CCB Days CCRNA Days CCRNB 0-4 39.75463 0-2 3.718715 4.026068 0-3 1.382825 0-3 -5.06085 4-7 -62.1957 2-6 2.59544 3.894522 3-5 2.269696 3-6 1.380788 7-11 6.828273 6-8 7.7406 4.326431 5-7 1.601667 6-8 0.918658 11-13 3.31987 8-10 -17.1354 8.258439 13-15 45.76981 10-13 8.883191 -62.9633 15-18 24.82157 13-15 15.67839 5.236184 18-20 -32.1442 15-17 52.94125 24.7196 20-22 4.045634 17-20 30.10622 114.4759 22-25 47.02394 20-22 11.33539 9.693364 25-27 74.37281 22-24 7.417496 42.69382 27-29 7.401719 24-27 -32.7758 90.86794 29-32 26.79738 27-29 7.917325 62.69149 32-34 14.97273 29-31 162.8 5.855449 34-36 10.75649 31-34 147.3448 -29.1519 36-42 267.5143 34-36 6.860163 75.71066 42-46 29.39326 36-38 505.2218 6.718437 46-48 -8.38351 48-50 5.567073 50-53 6.834769 53-55 -5.92393 55-57 10.55135
132 3.6 Conclusions The results of the continuous culture experiments in high nutrient conditions would suggest that flushing may be an option for culture control if the flow rate is high enough to offset the culture growth rate and culture would not have enough time to adjust. Flushing coupled with increase in pH seems to be a more effective option, specifically flushing with medium adjusted to pH 12, as it seemed to be effective during both high nutrient availability experiments. During the repeated high nutrient continuous culture experiment, lower flow rates may have allowed the culture to have more time to adapt to changes in conditions, resulting is some recovery after an increase in medium pH and the slight rebound in turbidity values towards the end of the experiment. The experiments also highlighted either scaling or self-shading issue affecting culture growth in high nutrient availability and culture concentration. The slow doubling rate of continuous cultures resulted in lower flushing rate, thus prolonging time for the culture to adapt to changing conditions. The suggestion is supported by the low nutrient continuous culture experiments, is that a sudden change in flow rate and increase in pH to 11 were more effective that flushing alone or flushing with 11.5 pH medium.
133 4 Biocide addition experiments
4.1 Abstract The objective of these experiments was to determine if two selected biocides- Mexel® 432 and Spectrus® NX 1422 have any impact on the growth of P. catenata. The results obtained have a potential to be applied as a method of algal control in storage ponds during bloom-time. It was not clear if the biocides would be used as a preventative measure or means of removing a bloom, therefore, the experiments were set up to determine if biocides would be able to disrupt growth of microorganisms later in the bloom. For both of the biocides trialled, concentrations of 50 ppm were effective at reducing culture concentration and growth prevention for at least 7 days. There was no significant difference between 50 and 100 ppm doses. Biocide doses of 20 ppm were able to disrupt culture growth, but cultures recovered within 2 days between sampling. Thus, the optimum dose is likely to be between 20 and 50 ppm.
4.2 Literature review There have been numerous studies detailing the use of biocides to prevent movement of invasive species (Jellyman et al., 2011), use in industrial settings to remove fouling (Konstantinou and Albanis, 2004), and prevent microbiological bloom formation (Shao et al., 2013). This literature review is focused on the biocides that have been effective against photosynthetic microorganisms and their possible effects on the microorganism growth.
Some of the commonly used biocides, Diuron® and Irgarol®, have shown photosynthetic damage capabilities (Neuwoehner et al., 2008). Diuron® was also able to affect cell viability of Fahrenholzia pinnata, Cylindrotheca closterium, and Thalassiosira pseudonana in concentrations over 10 µg/ml (Silkina et al., 2012). At concentrations of 5 µg/l Diuron® showed reduction in Tetraselmis suecica cell growth, but 95% of the cells were functioning and no significant mortality was observed (Stachowski-Haberkorn et al., 2013). Diuron® effects were the most uniform on diatoms, with least variation observed between different species (Silkina et al., 2012). In addition, after treatment of algae with 25 µg/l of Diuron®,
134 algae placed in fresh medium did not recover and most diatom cells were non- viable (Silkina et al., 2012). During long-term exposure experiments it was shown that 2 out of 3 cultures can develop a resistance to Diuron® in 25-32 generations (Stachowski-Haberkorn et al., 2013). It was also observed, that during repeated short term experiments after a year, resistance was still present in the developed cultures (Stachowski-Haberkorn et al., 2013). Furthermore, although cells evolved to be resistant to Diuron®, cell size was smaller compared to non-developed cells, and higher chlorophyll concentrations were measured compared to cells that were not exposed to the herbicide (Stachowski-Haberkorn et al., 2013). It is thought that Diuron® may still have an effect on photosynthesis, but not on cell growth (Neuwoehner et al., 2008). The main effects of another biocide, Tributyltin, were shown to be a disturbance of algae cell reproduction and inhibition of cell growth (Neuwoehner et al., 2008).
Simazine® is a herbicide capable of completely stopping cell growth at concentrations of 1.5 µg/l depending on the algal species present- it is most effective on Scenedesmus intermedius out of three species tested (Marvá et al., 2010). Whereas the concentration of Diquat® required to reach the same effect is several times higher at 60 µg/l for the same species (Marvá et al., 2010). Some algal strains can evolve to become resistant to these herbicides, with Scenedesmus intermedius strains even having a reduced growth rate with Simazine® being absent in the growth medium (Marvá et al., 2010). It was also noted that cells in mutated cultures were larger where samples were collected from areas that encounter these herbicides compared to areas where no herbicides are used (Marvá et al., 2010). Glutaraldehyde also showed good results in algae growth inhibition at concentrations over 1 mg/l for Pseudokirchneriella subcapitata (Sano et al., 2005b).The effectiveness of the biocide increased with higher concentrations (Sano et al., 2005b). Glutaraldehyde affects microorganisms by reacting with primary amines and sulfhydryl groups which leads to damage of algal cell wall (Sano et al., 2005b).
The synthetic biocide Mexel® 432/336 showed good results in small scale trials and was effective against a broad spectrum of organisms (La Carbona et al., 2010).
135 PeraClean® synthetic biocide has also been tested, but require much higher concentrations of 30.9 mg/l- compared to 10.1 mg/l or less for Mexel® 432 and 1.6 mg/l for Seacleen® for the same species Tetraselmis suecica and Alexandrium minutum (La Carbona et al., 2010). The effectiveness of removal also depends on the mix of species in the algal culture for Seakleen®, Peraclean®, and Vibrex® biocides (Gregg and Hallegraeff, 2007). Tetraselmis suecica was more resistant to the three biocides compared to Alexandrium catenella, Gymnodinium catenatum, Chatonrlla marina. In addition, Seakleen®’s effectiveness increased with longer treatment times due to longer exposure time, whereas Peraclean® and Vibrex® effectiveness was not exposure time dependant (Gregg and Hallegraeff, 2007). However, these algicides are persistent, and it can take up to 15 weeks to drop to concentrations that do not inhibit the culture growth (Gregg and Hallegraeff, 2007). Degradation of Peraclean® was slowed down in low light and is slower with prolonged dark periods (Gregg and Hallegraeff, 2007). The activity of these biocides on algae was reduced by lower temperatures (6°C compared to 17°C) (Gregg and Hallegraeff, 2007). In addition, Vibrex® may have limited applicability due to the use of hydrochloric acid as an activator by the manufacturer (Gregg and Hallegraeff, 2007).
It is important to determine if the biocide decays and how long it takes for it to reach concentrations that are not toxic (Sano et al., 2005a). In addition, persistence of biocides also has to be taken into account to prevent damage or increased running costs of the equipment (Kim et al., 2006).
4.3 Aims • To investigate if two biocides, Mexel® 432 and Spectrus® NX 1422, would be effective at reducing growth of P. catenata in batch cultures • To determine the optimum dose of the effective biocide(s)
136 4.4 Materials and methods Pseudanabaena catenata cultures were grown in 200 ml BG11 medium, contained in 500 ml conical flasks, in triplicate. Cultures were grown under 16:8 light-dark 250 PAR fluorescent illumination provided by Growth Technology T5HO lights (UK) as measured with a Light Scout light meter by Spectrum Technologies (USA) on an orbital shaker Stuart (UK) at 100 rpm. Temperature varied in range of: (17-26°C) and was data logged using Squirrel (Grant instruments, UK) data logger every 30 minutes. Absorption was measured at 600, 680, and 750 nm wavelengths using HACH DR 3900 (Germany). Photosynthetic pigment analysis was done as in previous experiments, see Section 2.4.4. Cultures not dosed with biocides were used as controls. Once a culture turbidity at 600 nm reached values around 0.600-0.700 they were dosed with either Mexel ® 432 (Mexel Industries SAS, France) or Spectrus® NX 1422 (GE Water, USA) at concentrations of 1, 5, 10, 20, 50, and 100 ppm.Results and discussion
4.4.1 Mexel® 432 experiment It appears that only the highest concentration of biocide was able to slow down culture growth for 3 days (Figure 4.1). Very limited growth was observed between days 9 (dosing) and 12 as turbidity stayed around 0.555-0.567, but cultures slowly recovered afterwards. The response of the culture dosed with 20 ppm was uniform across the triplicate flasks. Cultures dosed with 1 and 5 ppm biocide concentrations increased in turbidity values faster after dosing compared to control and cultures dosed with 10 ppm biocide. Although after day 9 cultures dosed with 1 and 5 ppm biocide grew somewhat faster, at the end of the experiment on day 17 the difference in concentrations between the control and 1, 5, and 10 ppm Mexel® dosed cultures was lower compared to day 12.
137 Culture growth during Mexel 432 dosing experiment 1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3 Absorption at 600 nm 600 at Absorption 0.2
0.1 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 Days BG11 (control) Mexel 20 ppm Mexel 10 ppm Mexel 5 ppm Mexel 1 ppm Figure 4.1. Changes in turbidity during the Mexel biocide dosing experiment. The dosing of biocide was done on day 9. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed.
The chlorophyll a concentrations did not follow the general trend seen in the turbidity values (Figure 4.2). Although culture dosed with 20 ppm of biocide had a stagnant growth period between days 9 and 12, chlorophyll a concentrations increased from 1.185 to 1.560 µg/ml during the same period. However, there was a delayed stagnant period where chlorophyll a concentration went from 1.560 µg/ml on day 12 to 1.570 µg/ml on day 15. Furthermore, cultures dosed with 10 ppm had a significant increase in chlorophyll a concentrations from day 9 (dosing) to day 12 from 1.288 to 2.358 µg/ml. However, the concentrations decreased towards the end of the experiment to 1.415 µg/ml for chlorophyll a on day 17. It is worth noting that chlorophyll a concentration decreased for the cultures at the end of the experiment (day 17) including control - this decrease may be due to an error in sampling or slight change in environmental conditions.
138 Change in chlorophyll a concentration during Mexel® 432 dosing experiment 2.4 2.2 2.0 1.8 1.6
(µg/ml) 1.4 a 1.2 1.0 0.8 Chlorophyll Chlorophyll 0.6 0.4 0.2 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 Days BG11 (control) Mexel 20 ppm Mexel 10 ppm Mexel 5 ppm Mexel 1 ppm
Figure 4.2. Changes in chlorophyll a concentrations during the Mexel® biocide dosing experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed.
Carotenoid concentrations steadily increased in all the cultures until day 9 (see Appendix 4). The trend continued until the end of the experiment on day 17 except for cultures dosed with 10 ppm biocide. The carotenoid concentration increased faster from day 9 to 15 by 0.503 µg/ml, but decreased to 1.220 from 1.394 µg/ml by day 17 in cultures dosed with 10 ppm biocide. The carotenoid concentrations in these cultures seemed to closely follow the variations in chlorophyll a concentrations.
The lower biocide concentrations (1, 5, and 10 ppm) did not have a significant effect on the P. catenata, as indicated by standard error bars, and reached the highest turbidity values of all the cultures in the experiment. It is unlikely, that the biocide had a positive effect on the growth - as these cultures were at higher turbidity values before dosing, therefore, it is most likely that the biocide did not have an effect on the culture growth. The 20 ppm dose of Mexel® 432 biocide had limited effectiveness on culture growth, although it was dosed into a high
139 concentration culture, it only slowed down the growth for a few days, and later cultures can be seen recovering and continuing to grow. From the culture responses to the varying biocide doses none of them was effective to significantly disrupt the culture growth.
4.4.2 Spectrus ® NX1422 biocide trial The same doses were used for Spectrus® biocide trial as with Mexel® for comparable results. During the Spectrus® biocide trial only a 20 ppm dose had an effect on culture growth, as in the previous experiment after dosing on day 9 (Figure 4.3). However, compared to the Mexel® biocide, the culture concentration (measured as absorption) decreased by day 11 from 0.641 to 0.584. As in previous experiments, the cultures recovered and continued to grow from day 11 onwards. However, the response of the culture to the biocide in triplicate was nonuniform, with 2 out of 3 flasks decreasing in turbidity, while one continued to grow. There was only a slightly slower growth phase in one of the flasks. In Mexel® biocide trials cultures recovered quickly, and turbidity values moved towards those of the unaffected cultures, whereas in trial with Spectrus® biocide P. catenata continued to grow parallel to the unaffected cultures. After the decrease in average turbidity values the cultures resumed growth at similar rate as prior to biocide dosing. During the Spectrus® dosing experiment there was no significant difference between control and low biocide doses, indicating that low biocide concentrations are not likely to be effective in reducing culture growth.
140 Culture growth during Spectrus® NX 1422 dosing experiment 1.6
1.4
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Absorption at 600 nm 600 at Absorption 0.4
0.2 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 20 22 Days BG11 (control) Spectrus 20 ppm Spectrus 10 ppm Spectrus 5 ppm Spectrus 1 ppm
Figure 4.3. Change in turbidity during the Spectrus® biocide trial with dosing on day 9. The error bars represent the standard error of the mean value, n=3. The red dot marks the day biocide was dosed.
Chlorophyll a concentrations followed the same trends as turbidity as seen in Figure 4.4. However, chlorophyll a concentrations tended to be more varied and erratic, but general trends in concentration in cultures can be seen. The chlorophyll a concentration decreased from 1.135 on day 9 to 0.918 µg/ml on day 11 for culture dosed with 20 ppm Spectrus ® biocide. In addition, after a jump in chlorophyll a concentration on day 7, there was a period until day 11 when chlorophyll a concentration remained quite stable for control cultures - a large error is due to non-uniform culture response. Although culture dosed with 20 ppm biocide did recover chlorophyll a concentrations from day 11 to 13, the concentration increased only by 0.1786 µg/ml over the next 6 days. Another observed decrease in chlorophyll a was from day 15 to day 17 in cultures dosed with 5 ppm biocide (from 2.13 to 1.71 µg/ml).
Changes in carotenoid concentration resembled the turbidity trends closely as seen in (Appendix 4). The only difference in the response to biocide dosing was the
141 carotenoid concentration - rather than decreasing after dosing on day 9, it increased at a lower rate compared to other cultures (from 0.774 to 0.810 µg/ml) when dosed with 20 ppm biocide. As with turbidity, the increase in concentration continued at a similar pace to other cultures from day 11.
The change in pH followed the same pattern in all the cultures: after inoculation the pH increased sharply until day 5, from ~7.3 to just above 10.5, and fluctuated above 10.5 until day 9 (see Appendix 4). After dosing on day 9, pH in all the cultures decreased on day 11. The most significant pH decrease was observed in the cultures dosed with 20 ppm – to an average of 10, while the pH decreased to values just below 10.5 for all the other cultures. From day 13 pH increased in in all the cultures and fluctuated around ~10.7 until the end of the experiment on day 21.
Change in chlorophyll a concentration during Spectrus® NX 1422 dosing experiment 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4
Chrolophyll a concentration (µg/ml) concentration a Chrolophyll 0.2 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 20 22 Days BG11 (Control) Spectrus 20 ppm Spectrus 10 ppm Spectrus 5 ppm Spectrus 1 ppm
Figure 4.4. Changes in chlorophyll a concentrations during Spectrus® biocide dosing experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed.
Spectrus® biocide was slightly more effective in reducing the culture growth, although as in the previous experiment, only cultures dosed with 20 ppm of the
142 biocide showed any measurable effects. However, compared to the previous experiment the chlorophyll a concentration decreased, and carotenoid stagnated during the same period - decrease in turbidity was observed in cultures dosed with 20 ppm of Spectrus® biocide. In addition, as in the previous experiment, it appears that a 20 ppm dose is not sufficient to disrupt culture growth for longer periods of time and beyond recovery. However, the pH in cultures dosed with 20 ppm biocide also decreased with a decrease in turbidity and pigments. The same trend but at lower amplitude was observed in other cultures as well. It could be due to the biocide dosing as it directly affected the pH of the medium, or it could be due to an indirect effect of decrease in culture concentration. As the control was not dosed with a biocide the latter may be more likely.
4.4.3 Repeated experiment As the highest biocide doses of 20 ppm trialled earlier were not successful in disrupting culture growth for longer periods, and a decrease in concentration was marginal, higher doses of biocide (50 and 100 ppm) were used for further study. As in previous experiments, cultures dosed with 20 ppm of Mexel® and Spectrus® biocides decreased in turbidity values within 2 days of dosing (days 13 and 15) as seen in Figure 4.5. The cultures recovered from day 17 and grew at similar rate until day 20, the last day of the experiment. In addition, the culture response was not uniform, as only one out of three cultures dosed with Spectrus® biocide decreased in turbidity. The cultures dosed with 20 ppm Mexel biocide had rather more uniform response after dosing, from day 13 to day 15, as average turbidity decreased from 0.626 to 0.608, but it was measured in all three flasks.
Cultures dosed with 50 and 100 ppm Mexel® and Spectrus® biocides were affected for longer period. After 50 ppm Mexel® biocide dosing the turbidity from 0.586 to 0.304, and for cultures dosed with 100 ppm decreased from 0.611 to 0.303. The results looked similar in cultures dosed with 50 and 100 ppm Spectrus® biocide as turbidity decreased from 0.667 to 0.297 and from 0.706 to 0.287 respectively. Compared to cultures dosed with 20 ppm of either of the biocides the turbidity values did not recover even a week after dosing. The lowest turbidity values at the end of the experiment were measured in cultures dosed with 50 ppm Spectrus®
143 biocide at average value of 0.213. All the cultures fell into two groups: continuing to grow (control and cultures dosed with 20 ppm of either biocide), and cultures that decreased in turbidity (cultures dosed with 50 and 100 ppm Mexel® and Spectrus ® biocides).
As in previous experiments - the pH at the start of the experiment rose from between pH 8.5 to 9, to ~10.5 for all the cultures on day 3. The pH fluctuated around ~10.6 until dosing on day 13 - the control and culture dosed with 20 ppm Mexel® continued to fluctuate around this value until the end of the experiment. There was a slight decrease in culture pH on day 15 for cultures dosed with 20 ppm Spectrus® biocide - pH decreased from 10.66 to 10.01. However, on day 17 the pH recovered to an average value of 10.66. The decrease in pH was observed at the same time as decrease in turbidity. However, the response was not uniform in the triplicate cultures as only one flask decreased in pH, resulting in a large error. Furthermore, the pH decreased significantly in cultures dosed with 50 and 100 ppm biocide doses. From day 13 to day 15 the pH fell from ~10.6 to 8.37 in cultures dosed with 50 ppm doses and 8.21 and 8.24 in cultures dosed with 100 ppm doses. The pH did increase by day 20, but only up to values around 8.5 for all the cultures dosed with 50 ppm or more.
144 Culture growth during repeated Mexel® 432 and Spectrus® NX 1422 dosing experiment 1.0
0.9
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0.7
0.6
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0.3 Absorption at 600 nm 600 at Absorption 0.2
0.1 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 20 22 Days BG11 (Control) Mexel 20 ppm Mexel 50 ppm Mexel 100 ppm Spectus 20 ppm Spectrus 50 ppm Spectrus 100 ppm
Figure 4.5. Changes in culture turbidity during repeated biocide trial with dosing on day 13. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed.
Chlorophyll a concentrations followed the same pattern as turbidity values (see Figure 4.6), however, as in the previous experiments the results were more erratic. Although the chlorophyll a concentration increased over the duration of the experiment some lower concentrations were measured, even in control cultures, on day 15. A slow growth period can be seen from day 3 to day 9. As with turbidity measurements, the cultures dosed with 20 ppm Spectrus® biocide did not react to the biocide uniformly. Only one of three cultures had higher chlorophyll a concentration. In addition, in one of the cultures chlorophyll a concentration decreased to less than a third of previously measured concentration in prior to dosing (0.714 to 0.213 µg/ml). The decrease in chlorophyll a concentration in cultures dosed with 20 ppm Mexel® biocide was more uniform and average concentration decreased from day 13 to 15 from 0.773 to 0.687 µg/ml. All the
145 cultures dosed with 20 ppm of either biocide and control recovered and chlorophyll a concentration increased towards the end of the experiment. The highest average chlorophyll a concentration was achieved by cultures dosed with 20 ppm Spectrus® biocide at 1.217 µg/ml concentration.
Change in chlorophyll a concentration during repeated Mexel® 432 and Spectrus® NX 1422 dosing experiment 1.4
1.2
1.0
(µg/ml) 0.8 a
0.6
0.4 Chlorophyll Chlorophyll
0.2 Dosing 0.0 0 2 4 6 8 10 12 14 16 18 20 22 Days BG11 (Control) Mexel 20 ppm Mexel 50 ppm Mexel 100 ppm Spectrus 20 ppm Spectrus 50 ppm Spectrus 100 ppm
Figure 4.6. Changes in chlorophyll a concentrations during repeated biocide experiment. The error bars represent the standard error of the mean value, n=3. The red line marks the day biocide was dosed.
Change in carotenoid concentrations somewhat mimicked trends in turbidity values (see Appendix 4). Carotenoid concentrations increased steadily from inoculation to dosing (day 13). Carotenoid concentrations continuously increased only in control cultures throughout the experiment. The highest concentration on day 13 was in flasks before being dosed with 100 ppm Spectrus® biocide at 1.069 µg/ml, and the lowest in cultures to be dosed with 50 ppm Mexel® biocide at 0.805 µg/ml. Slight decreases in concentrations were measured in cultures dosed with 20 ppm Mexel® (0.859 to 0.829 µg/ml) and Spectrus® (0.898 to 0.807 µg/ml) biocides on day 15. As with turbidity, in cultures dosed with Mexel® biocide concentration decreased slightly in all three flasks. Whereas in cultures dosed with Spectrus® biocide
146 carotenoid concentration decreased only in one flask - from 0.889 to 0.490 µg/ml, resulting in a more pronounced change and larger error.
The cultures dosed with 50 and 100 ppm biocide doses had the sharpest drop in carotenoid concentrations - the lowest on day 15 was in cultures dosed with 100 ppm Spectrus® biocide (1.069 to 0.038 µg/ml). Although concentration of carotenoids did decrease significantly (0.805 to 0.195 µg/ml) in cultures dosed with 50 ppm Mexel® biocide, they were the highest out of the four cultures which experienced significant decrease. By the end of the experiment (day 20) the carotenoid concentration had decreased further in cultures dosed with higher biocide doses to 0.056 and 0.046 µg/ml in cultures dosed with 50 and 100 ppm Mexel® respectively, and to 0.011 and 0.019 µg/ml in cultures dosed with 50 and 100 ppm Spectrus® respectively.
As can be seen from Image 4.1, as in previous experiments, the colour between control and cultures dosed at 20 ppm is not noticeable. However, cultures dosed with higher biocide concentrations showed notable change in colour - cultures appeared pale, but some turbidity remained until the end of the experiment. There is only a small difference between biocides - cultures dosed with Mexel® had more pigmentation on the first sampling day, especially cultures with 50 ppm dose, compared to Spectrus® dosed cultures. The colour did even out towards the end of the experiment.
Image 4.1. Cultures 2 days after dosing, from left to right: Spectrus® NX 1422 with 100, 50, 20 ppm, Mexel® 432 100, 50, 20 ppm and control
147 The results of the experiment had similar outcomes when compared to culture behaviour when 20 ppm of either biocide dose was applied. As previously observed, dosing with 20 ppm Mexel® biocide resulted in a slower growth for 2-3 days with cultures recovering afterwards. Dosing with Spectrus® biocide resulted in a non- uniform response. However, increase in biocide dose lead to a more significant reduction in culture concentration (measured as absorption), and recovery in culture concentration was not observed within 5 days. As similar results were obtained when cultures were dosed with 50 and 100 ppm doses, out of all the concentrations tested 50 ppm is closer to the optimum dose when applied to cultures of comparable concentration. However, the effective dose is more likely to be between 20 and 50 ppm doses.
When the effects of the Mexel® and Spectrus® biocides at the same dose are compared, Spectrus® seems to disrupt the cell growth marginally better due to lower chlorophyll a and carotenoids concentration, and visual observations results 2 days after dosing. It should also be noted, that the reported effective concentrations are for established cultures and maybe lower if biocides were applied earlier in the growth cycle when microorganism concentrations are lower. One study reported that the optimum algaecide dosing time would be at the start of logarithmic growth phase for Heterocapsa circularisquama and Heterosigma akashiwo (Baek et al., 2014). Whereas for Microcystis aeruginosa optimum dosing time was suggested to be prior to the exponential growth phase (Ni et al., 2018). The results of the two studies do agree on the optimum dosing time being early in the growth cycle, when the microorganism concentration is low. However, the optimum timing is species and biocide dependent (Baek et al., 2014). In addition, compared to the results when cultures were dosed with 20 ppm, 50 ppm and higher doses resulted in the culture growth being disrupted at least for a week, providing longer lasting results as well. Furthermore, the decrease in culture pH is likely to be due to the decrease in culture concentration. This assumption is more likely than the direct effect of biocide on the culture pH as the concentration of biocide was only five times higher and a change in pH value is logarithmic.
148 The overall results are also not quite comparable to ones reported in literature of 10.1 mg/l for Mexel® biocide (La Carbona et al., 2010). The effective dose of 50 ppm for the Mexel® biocide for P. catenata although is at the same order of magnitude, it is still significantly higher. The difference is likely to be due to different response in species and dosing at different stages in the growth cycle, as well as different growth conditions. In addition, the effective dose was significantly higher compared to recommended dose in flow through systems, which was closer to the previously reported dose (Annon, 2010b). However, the effective Spectrus® concentration was comparable to recommendation from the manufacturer (Mcnair, 2017). The biocides in this experiment not only reduced the culture concentration, but also chlorophyll a and carotenoid concentrations, suggesting some photosynthesis disruption or degradation of photosynthetic pigments.
Although the results of the experiment showed that the culture growth was stagnant for a week after dosing with 50 and 100 ppm doses, it is not clear if the cultures would recover over longer period of time. However, biocides can have continuing growth inhibiting effects for weeks (Gregg and Hallegraeff, 2007; Mohr et al., 2008). Although, in the event of a bloom, flushing in the FGMSP is likely to be used reducing biocide concentration and effectiveness over longer periods of time. On the other hand, it may not be possible in other natural or man-made water bodies. The experimental results also suggested that the biocides could be effective at alkaline pH of at least 10.5, as measured in the cultures prior to biocide dosing. Although the pH did decrease after dosing, most likely due to reduction in cell concentration and CO2 consumption as suggested by (Mohr et al., 2008), and as observed in batch experiments once culture concentration decreased.
One important factor to consider when using biocides is the possibility of the microorganism resistance to biocides. (Stachowski-Haberkorn et al., 2013) study found that Tetraselmis suecica developed resistance to diuron biocide within 25 generations with the longest doubling time of 2.6 days. (Marvá et al., 2010) found that green microalgae resistance to biocides arose from spontaneous mutations prior to the use of biocides, resulting in re-emergence of the culture growth when cultures where incubated post culture decline. Furthermore, it was also reported
149 that biocide resistant strains after short experiments were still abundant a year later (Stachowski-Haberkorn et al., 2013), reducing the applicability of the biocide for preventative dosing and long term use. Furthermore, biocide dosing may be selective, preventing the growth of biocide sensitive species, but also causing a shift in culture composition due to presence of resistant species (Mohr et al., 2008), thus, resulting in another bloom as suggested by (Crafton et al., 2018) after large scale in-situ study.
4.5 Conclusions Both of the biocides appear to have similar effectiveness on P. catenata with 50 and 100 ppm doses effective in batch cultures for at least 7 days. The reduction in culture concentration may provide time to flush out the leftover cell matter if used in the FGMSP or other pond with sufficient water exchange rate. However, the biocides should be used sparingly due to possibility of microorganisms developing resistance over time. It would not only prove ineffective and expensive, but may also result in harder to control bloom with resistance to at least some classes of biocides. In addition, the effects of biocides on the downstream effluent processing plants should also be assessed before use due to long residence times.
150 5 Prokaryote species succession
5.1 Abstract The aim of the culture DNA sequencing was to determine how culture composition changes with changes in the growth conditions. The data could help identify parameters that have the most influence in preventing growth of the microorganisms causing the loss of visibility. The outcome of the sequencing could be used to target the microorganisms with the most effective treatment and prevention strategies.
5.2 Literature review The First Generation Magnox Storage Pond has a distinct and complex culture composition, as introduced in the previous chapters. However, most of the species found in the pond were also present in the mixed culture obtained for work in this thesis: Flavobacterium macrobrachii, Porphyrobacter colymbi, Sediminibacterium goheungense, Rhodobacter sp., Lacibacterium aquatile, Aminobacter aminovorans, Hydrogenophaga palleroni (Foster, 2017). Given the range of manipulations made to the mixed culture (cultures grown in low and high nutrient availability medium purged with high pH medium or dosed with biocide) to help inform bloom control strategies, it is important to investigate the impact of the culture treatments on microbial community compositions. DNA-based analyses were selected and the results are presented in this chapter. A high-throughput approach was adopted, using the Illumina MiSeq platform to sequence 16S rRNA gene products amplified from the laboratory mixed culture using broad-specificity PCR primers.
In addition to Pseudanabaena catenata, this sequencing technique detected a range of other bacteria in the mixed culture used in the experiments described in this thesis. These organisms were predominantly heterotrophic bacteria which would not be able to fix CO2, and would therefore require other organic carbon inputs into the pond water environment, or the primary colonisation of photosynthetic organisms such as Pseudanabaena catenata. For example, Flavobacteria are photoheterotrophs, and one such organism detected is Flavobacterium macrobracii is an aerobic rod-shaped bacterium which is non-
151 motile and lacks flagella (Sheu et al., 2011). The colour was described as yellow, and cell shape as round (Sheu et al., 2011). The growth temperature was suggested to between 15 and 30°C and pH 7 to 8 range (Sheu et al., 2011). During the initial tests it was shown to be negative for nitrate reduction and carbohydrates assimilation (Sheu et al., 2011). Porphyrobacter are also heterotrophs, and P. colymbi was detected and is a non-spore forming, rod shaped motile bacteria (Furuhata et al., 2013). Colonies have been described as orange pigmented, with best growth temperature range of 20-40°C, and pH range between 7 and 9 (Furuhata et al., 2013). It was not able to reduce nitrate during the characterisation study (Furuhata et al., 2013). Other heterotrophs detected included Sediminibacterium goheungense an aerobic rod-shaped motile bacterium forming orange colour colonies (Kang et al., 2014). The preferred growth temperature was suggested to be between 20 and 42°C and pH 5 to 7 (Kang et al., 2014). In addition, no nitrate reduction was observed (Kang et al., 2014). Lacibacterium aquatile is a bacterium forming creamy white colonies, that grows best within 10-37°C temperature pH 6-9 range (Sheu et al., 2013a). It was tested negative for nitrate reduction as well (Sheu et al., 2013a).
Aminobacter aminovorans is colourless non-nitrate reducing bacterium (Urakami et al., 1992). Hyrogenophaga palleronii is pale to dark yellow rod-shaped bacterium, pigmented due to carotenoids with turbidity maxima at 405 and/or 425, and 445 µm wavelength (Davis et al., 1970). It is aerobic, and can use ammonium salts or nitrate as nitrogen sources with optimum growth temperature of 30°C (Davis et al., 1970).
In addition to the species above, samples of cultures used in these experiments had other microorganisms present in the samples. Flavobacterium chungnamense is rod shaped non-motile cream yellow bacterium. The species grows is temperatures between 10 and 30°C and pH 6.5-9.5, slightly higher compared to F. macrobacii (Lee et al., 2011). It is also not capable of nitrate reduction (Lee et al., 2011). Phreatobacter oligotrophus is an aerobic rod-shaped motile bacterium (Tóth et al., 2014). The colour of colonies was described as whitish and seems even transparent, and preferred growth conditions between 20 and 45°C and pH range of 5.5 to 9.5
152 (Tóth et al., 2014). Another prominent species Gemmobacter lanyuensis is an anaerobic and chemo-heterotrophic non-motile rod shaped bacterium of cream white colour (Sheu et al., 2013b). The growth was shown to occur between 15 and 40 °C and pH 6-9 (Sheu et al., 2013b). Unlike most species described previously, it is able to reduce nitrate (Sheu et al., 2013b). Blastonomas fulva is another rod shaped non-motile yellow bacterium (Lee et al., 2017). The reported growth temperature is similar to other species found in samples between 8 and 37°C and pH range between 6 and 8 (Lee et al., 2017). However, it was also stated that the culture does not seem to grow at pH 5 or 9 (Lee et al., 2017). The species was also not shown to be nitrate reducing (Lee et al., 2017).
Once the species present in the samples are identified and preferred growth parameters are known, behaviour according to changes in growth conditions may be easier to predict. However, mixing, media flow rate, sodium hydroxide concentration, light availability, biocide used and concentration will impact the culture composition and may cause shifts in dominance over time. For example it was illustrated in an experiment when under mixing conditions caused by strong winds Pseudanabaena was significantly reduced in abundance, compared to Planktothrix species (Mischke, 2003). In addition, P. catenata grown in mixed cultures reached higher concentrations in high nutrient conditions compared to with low nutrient availability (Loza et al., 2014). In high nutrient concentrations the concentration increased from day 10 to 20, but in low nutrient concentrations numbers decreased at the same time, and the same abundance was not reached (Loza et al., 2014).
5.3 Aims • To investigate how flushing and change in pH due to sodium hydroxide dosing would affect the culture composition during continuous culture experiments with unmodified BG11 medium • To compare culture compositions in continuous cultures grown in unmodified and reduced nutrient BG11 medium, and the responses of the microbial community to flushing and increase in medium pH
153 • To determine the effect of Mexel® and Spectrus® biocides on culture composition, and identify if P. catenata is one of the species with sensitivity to the biocides
154 5.4 Materials and methods
5.4.1 DNA sequencing Samples of 1.5 ml in volume were taken from the cultures used in experiments, centrifuged at 5000 rpm for 20 minutes. Supernatant was removed and used for total carbon/ total inorganic carbon analysis. The pellet was retained for DNA sequencing and kept frozen to prevent degradation.
5.4.1.1 DNA extraction DNA extraction was done using Qiagen DNeasy PowerSoil Kit (Qiagen, Germany). The process started with sample homogenisation by transferring 0.25 ml of thawed well-mixed sample into a clean 2 ml Bead Solution tube and gently mixed by inverting it. To induce cell lysis, 0.06 ml of reagent C1 containing sodium dodecyl sulfate (SDS) was added. The anionic detergent C1 is used to break down fatty acids and lipids associated with the cell membrane. The chemical lysis agents effects are further enhanced by mechanical shaking with previously added glass beads [330]. The mixture was gently homogenised, then transferred to MoBio Power Lyzer, and shaken at 2500 times per minute, for 45 seconds causing cells to break open. To separate the glass beads and other solids from the liquid containing DNA, samples were centrifuged for 1 minute at 14000 rpm (Sigma 1-14 Centrifuge, Sigma, Germany). After centrifugation 0.55 ml of supernatant was transferred into a clean 2 ml centrifuge tube. 0.25 ml of reagent C2 was added to the supernatant and incubated at 4°C for 5 minutes. Addition of reagent precipitates organic and inorganic compounds, excluding DNA, like humic acid, cell debris, and proteins to remove contaminants and prevent inhibition of analytical techniques to be used later. After incubation tubes were centrifuged at 14000 rpm for 1 minute and 0.6 ml of supernatant was transferred into a clean 2 ml centrifuge tube. An additional solution C3 (0.2 ml) was added into the removed supernatant to help precipitate any contaminants present. Samples were mixed gently and incubated at 4°C for 5 minutes. After incubation, the tubes were centrifuged at 14000 rpm for 1 minute. 0.750 ml of supernatant was transferred into a clean 2 ml microcentrifuge tube and 1.2 ml of C4 solution was added and mixed. An addition of high salt containing solution into supernatant adjusts solutions salt concentration.
155 Then 0.65 ml of mixture was transferred into a MB spin column and centrifuged at 14000 rpm for 1 minute where under high salt conditions DNA binds to the silica membrane of the filter. Centrifugation helps to filter out the non-binding material and remove left over contaminants not removed in the previous steps. The liquid at the bottom of the column was removed and assembled column centrifuged again. As previously, liquid at the bottom was removed. Once the column was reassembled, 0.5 ml of C5 ethanol based wash solution was added to clean DNA to remove salt, humic acid, and other contaminants. Ethanol does not cause DNA to be washed out of the silica filter, but only removes residual contamination. The spin columns were centrifuged at 14000 rpm for 30 seconds. Liquid at the bottom removed, and this step repeated. The columns were centrifuged again at 14000 rpm for 1 minute, with no addition of solution to remove left over supernatant from the column as it can interfere with PCR and gel electrophoresis. The bottom half of the column was discarded and the filter part transferred into a 2 ml centrifuge tube, followed by 0.1 ml of C6 solution to release DNA from the filter into the buffer. The solution was placed on the centre of the white filter membrane to ensure even spread. The DNA that was selectively bound to the silica under high salt conditions is released into the elution buffer which lacks salt. Samples were left to stand for 1 minute and centrifuged for 1 minute at 14000 rpm. The DNA containing supernatant was transferred into a clean tube and stored for further work, filter discarded.
5.4.2 PCR amplification The polymerase chain reaction (PCR) is used to selectively amplify a region of DNA, in this study for sequencing and phylogenetic analysis. Several components are required for the PCR reaction, Taq polymerase, two oligonucleotide primers, and nucleotides. The most commonly used polymerase is Taq, named after Thermus aquaticus, the organism from which it was first isolated (Eckert and Kunkel). The bacterium is found in hot springs, resulting in Taq being thermostable. Two oligonucleotides (primers) hybridize to the DNA, one to each of the strand of the helix, moving in opposite directions during the PCR cycle (Brown, 2016).
156 Two separate PCR amplifications were done, one to determine if DNA extraction was successful and later used for gel electrophoresis, and later one used for DNA sequencing.
For the first amplification a master mix was made up of; 35.7 ml of deionised water, 5 ml of Ex buffer, 4 ml of dNTP, 1 ml of 16s 8F primer, 1 ml of 16s 1492R primer, and 0.3 ml of Tekara Ex Taq, enough for 40 samples. Then 0.047 ml of master mix was mixed with 0.003 ml of sample and amplified using a Techline TC-4000 thermocycler (UK). The procedure started when mixture was heated to 94°C, where hydrogen bonds holding polynucleotides brake and DNA denatured into two single strands. The temperature was then reduced to 50-60°C to allow primers to attach to the complementary single stands in the required positions. The temperature was then increased to 74°C, for the Taq polymerase to synthesise a complementary DNA strand. The cycle then was then repeated to multiply the available DNA strands (Brown, 2016). The multiplied number of DNA strands were then used for further analysis.
The second PCR amplification was performed using Roche FastStart High Fidelity PCR System (Roche Diagnostics Ltd, Burgess Hill, UK) in 50 μl reactions under the following conditions; initial denaturation at 95°C for 2 min, followed by 36 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final extension step of 5 min at 72°C. The PCR products were purified and normalised to ~20 ng each using the SequalPrep Normalization Kit (Fisher Scientific, Loughborough, UK). The PCR amplicons from all samples were pooled in equimolar ratios. The amplified DNA was used for sequencing.
5.4.3 Gel electrophoresis One of the uses of gel electrophoresis is to determine if DNA extraction and PCR have been successful. Gel electrophoresis is based on electrophoresis - using electric charges to separate molecules. DNA is negatively charged, resulting in movement towards positively charged electrode (anode) (Brown, 2016). The rate of movement depends on shape and charge-mass ratio of the molecule. Due to the similar shapes and sizes of DNA molecules, separation is not possible using just electrophoresis. A suitable gel matrix is used to separate the DNA fragments by size
157 (Brown, 2016). Due to the required movement through the pore network within the gel, shorter chains will travel faster and further within the gel. Gel composition is a key factor for DNA separation, as it will be the determining factor of pore size (Brown, 2016). To create the gel base, 1.6 g of agarose was mixed with 100 ml of TAE buffer, and microwaved for 2 minutes. The mixture was allowed to cool down to a temperature cold enough to handle manually, 0.01 ml of Syber Safe DNA gel stain was added, poured into the mould with gel combs and left to stand for approximately 30 minutes. Once the gel became solid, combs were removed and TAE buffer was poured onto the gel tank (~600 ml). A mixture of 0.002 ml of gel loading buffer was mixed with 0.001 ml of sample after PCR amplification and carefully loaded into the wells in the gel. Once all samples were loaded, negative, and positive controls were loaded as the first and last wells. Then electrodes were connected and using BioRAD PowerPAC 300 (USA) 80 V current was applied for 1.5 hours to separate the DNA fragments. Once the process was complete, extra buffer was poured off, and gel removed from the mould. The gel was then placed into a Bio Rad Gel Doc 2000 (USA) to visualise the bands of DNA under UV light.
5.4.4 DNA sequencing Next generation DNA sequencing has made it possible to sequence a larger number of samples cheaper and quicker, and with better accuracy (Fadrosh et al., 2014). Microbial community analysis using the Illumina technology is based on amplification and sequencing of 16S rRNA (prokaryote) and 18S rRNA (eukaryote) genes (Fadrosh et al., 2014). The 16S rRNA gene, which was targeted in this work, has 9 hypervariable regions surrounded by regions with more conserved sequences (Fadrosh et al., 2014). The more conserved the region is, the higher level of taxonomy it represents (Yang et al., 2016). The V4 region is one of the most suitable sub regions use in sequencing due to phylogenic resolution it could provide (Yang et al., 2016). The gene is amplified with region specific primers with adapter sequences. The reverse primers used also contain a twelve base barcode used to pool numerous sequences into one lane (Caporaso et al., 2012). The barcodes are used to distinguish between the end and the start of the amplified regions of products from the same sample. The primers and adapters are used to isolate the
158 required region and to provide information where the region starts and finishes. When the amplified gene is read, the adapters are used to determine where the region starts and finishes, so no additional information would be read, and the barcode determines where the region surrounded by adapters associated with one sample is. The region is read twice to provide an additional set of data and reduce errors. Due to low genetic diversity of the amplified 16S rRNA genes, additional DNA in a form of control library of PhiX DNA form a phage can be used to artificially increase it (Kozich et al., 2013).
Sequencing of PCR amplicons of 16S rRNA genes was done using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) targeting the V4 hyper variable region (forward primer, 515F, 5′-GTGYCAGCMGCCGCGGTAA-3′; reverse primer, 806R, 5′- GGACTACHVGGGTWTCTAAT-3′) for 2 × 250-bp paired-end sequencing (Illumina) (Caporaso et al., 2012; Caporaso et al., 2011). The run was performed using a 4 pM sample library spiked with 4 pM PhiX to a final concentration of 10% following the method as described by Schloss and Kozich (Kozich et al., 2013). Raw sequences were separated into samples by barcodes (up to one mismatch was allowed) using a sequencing pipeline. Quality control and trimming was performed using Cutadapt (Martin, 2011), FastQC (Bioinformatics), and Sickle (Joshi and Fass, 2011). MiSeq error correction was performed using SPADes (Nurk et al., 2013). Forward and reverse reads were incorporated into full-length sequences with Pandaseq (Masella et al., 2012). Chimeras were eliminated using ChimeraSlayer (Haas et al., 2011), and OTU’s were generated with UPARSE (Edgar, 2013). OTUs were sorted by Usearch (Edgar, 2010) at the 97% similarity level, and singletons were removed. Rarefaction analysis was conducted using the original detected OTUs in Qiime (Caporaso et al., 2010). The taxonomic assignment was performed by the RDP classifier (Wang et al., 2007).
159 5.5 Results and discussion
5.5.1 Repeated continuous culture Besides the species identified in the cultures received from the University of Manchester, other species were present as well. The abundance of other species is most likely due to contamination from air and surfaces, and due to the nature of the environment the experiments were completed. In addition, the cultures were subcultured numerous times and transported significant distances, thus increasing the likelihood of contamination.
Before the sequencing results can be discussed, some limitations of the technique must be pointed out. The technique in qualitative, rather than quantitative: the DNA abundance is relative to the sample, rather than absolute, therefore general trends in microorganism concentrations need to be considered (change in turbidity or cyanobacterial cell counts in this work).
The majority of species identified in the cultures grown during the repeated continuous culture experiment were Alphaproteobacteria, Betaproteobacteria, Flavobacteriia, and Cyanobacteria, with some Cytophagia and Sphingobacteriia. During the experiment, the culture composition changed significantly due to introduction of a flushing regime and change in flushing medium pH (Figure 5.1 and Figure 5.2). Although both flasks had the same species present, the species abundance was different, even though they were inoculated from the same stock culture. The difference in culture composition could have some influence in similar but not identical culture behaviour and response to changes in growth conditions. Unfortunately, samples for sequencing were not taken on the day of inoculation of before and after the start of flushing. It is most likely that the difference in species abundance changed slowly over time before the first sample for the sequencing was saved. The first sample was taken on day 94 to obtain a baseline before flushing with adjusted medium pH was started.
Before flushing with medium adjusted to pH 11, the cultures were dominated by Porphyrobacter colymbi, Gemmobacter lanyuensis, Phreatobacter oligotrophus, Pseudanabaena foetida var. intermedia, Sediminibacterium goheungense, and
160 Flavobacterium chungnamense. Although a culture of Pseudanabaena catenata was bought and identified in the previous sequencing run, cyanobacteria present in the cultures was found to be the most closely related to Pseudanabaena feotida var. intermedia. Sequencing can be prone to errors, results may not be a 100% match, and the use of sequenced data library will affect the results. When the sequencing is reported here, the closest relatives are variable at a species level, but genus level identification is reliable - the species level is discussed to relate the preferred growth conditions of microorganisms to experimental results. Therefore, it is most likely that P. catenata rather than P. feotida is the cyanobacterial species in the samples, but it will be referred as such due to the results produced by sequencing. The results show that although Pseudanabaena species are relatively abundant in the culture, it is not the most abundant species. Porphyrobacter colymbi and Flavobacterium chungnamense DNA was more abundant in the sample (see Figure 5.1 and Figure 5.2).
There were some differences in culture compositions in flasks A and B after flushing with pH 11 medium started in samples taken on day 97. Although abundance of P. colymbi decreased in flask A and increased in flask B, the difference in abundance between the flasks decreased. For both of the flasks the abundance was ~42 and 39% in flasks A and B respectively, compared to the previous samples of 54 and 34%. The abundance P. feotida increased in flask A during the same sampling period from 12. to 17%, and stayed stable in flask B as abundance has not changed. The same trend was observed in changes in F. chungnamense as in flask A abundance increased from 16 to 22%, and decreased slightly in flask B from 25 to 23%. There were only minute changes in abundance of less dominant microorganisms in the samples. The only significant changes occurred in Sediminibacterium goheungense when in flask B the abundance decreased from 9 to 7%. There was little change in turbidity of the culture during this period (see Figure 3.3). It seems that if DNA abundance in the samples changes, n the flask A it was at the expense of P. colymbid in the flask A and F. chungnamense, S. goheungense, and other minor species. It seems that flushing at slightly elevated pH even out culture composition,
161 although it is not clear why the same species would react differently in identical conditions, especially as pH values were nearly identical.
On day 120, just before pH in the medium increased to 11.5, and at the end of flushing with pH 11 medium changes in both of the flasks were similar. Abundance of P. colymbi increase to 50% in flasks A and B. P. feotida also increased in abundance, although not as significantly: to 20% in flasks A and B. There was a slight decrease in abundance was of F. chungnamense. The abundance decreased to 11% for A and B. Gemmobacter lanyuensis also decreased in abundance, but only in flask A from 7% to 5% in sample on day 97 and 120 respectively. The turbidity in flasks A and B was significantly different on day 120, however, the species composition was very similar, the pH values were also very close. It seems that the flushing with more alkaline medium either supported growth of P. colymbi and P. feotida, or species like F. chungnamense and G. lanyuensis were more sensitive to increased sodium hydroxide concentration as pH values were similar to ones on day 97. As turbidity in flask B increased, it may be due to increase in P. colymbi and P. feotida concentrations more, than other species. Thus, the total concentration of minor species may have not changed, but the abundance did due to increased total concentration of the beforementioned species.
At the end of flushing with pH 11.5 medium and before the start of flushing with pH 12 medium the composition of cultures in flasks A and B started to change non- uniformly (day 147). Although, some trends were applicable to both cultures. In flasks A and B abundance of F. chungnamense increased to 18 and 17 % respectively, and S. goheungense decreased to 4 and 2 %. In addition, the abundance of P. feotida decreased in both cultures, although more in flask B, compared to A (10 and 17% respectively). In addition, Blastomonas fulva abundance increased from below 2% to 3.34 in flask A and to 1.71% in flask B. Besides the decrease in P. feotida abundance, the most important change was increase of P. colymbi abundance in flask B to 60%, compared to flask a of 50%. As previously, the turbidity values did fluctuate, but they were comparable to the previous sample. In addition, the pH values were not higher either. The change in
162 culture composition could then be explained by adaptation or resistance to sodium concentrations, or better pH regulation within the cell.
At the end of the experiment and after flushing with medium with pH 12 on day 185 culture composition changed significantly compared to the previous samples. While P. colymbi was the dominant species throughout the sampling period, the abundance decreased to 13 and 21% in flasks A and B respectively. The abundance of F. chungnamense also decreased to below 1% in A and B. In addition, another significant change was increase in abundance of B. fulva to 43 and 30% in A and B. Furthermore, species like Alpha proteobacterium, Phreatobacter oligotrophus, and Nesterenkonia sp. became more abundant (4-8%). And finally, the abundance of P. feotida increased to 25 and 27% in flasks A and B respectively. The abundance at the end of the experiment was the highest compared to all the previous samples. Although the pH of the culture has not changed significantly, the turbidity values were lower than any other day of the experiment, except for days 0 and 2. In addition, the cell concentration was also lowest since day 2 of the experiment.
The results of the culture composition on the last day of the experiment therefore have to be interpreted with care due to the decrease in culture concentration. It seems that the B. fulva either outcompeted P. colymbi, or abundance increased due to decrease of P. colymbi concentration, or both of the changes combined. In addition, the same could be said about S. goheungense and other minor species. The general decrease in P. colymbi abundance may have formed a significant part in decrease in turbidity. Therefore, lower concentrations of more minor species would become relatively more abundant. The same could be applied to P. feotida, as cell concentration of P. feotida decreased, but abundance in the culture slightly increased.
There were clear shifts in culture composition in both the flasks, A and B, with increase in medium pH. The species most negatively affected by increase in medium pH and sodium concentrations was P. colymbi. Whereas B fulva seemed to be better adapted to survive, and maybe even outcompete other cultures in more alkaline conditions. Other species more resistant to increase in medium pH were P. oligotrophus, A. proteobacterium, Nesteronkia sp.. Although P. feotida was affected
163 by the increase in medium pH and sodium concentrations, it seemed to be more resilient or better adapted compared to P. colymbi. Although the flushing and sodium hydroxide dosing seemed to increase the P. feotida abundance, when changes in turbidity, cell and pigment concentrations are taken into account, the combined treatment seems to be effective in reducing Pseudanabaena concentrations.
164 Species distribution in high nutrient continuous Species distribution in high nutrient continuous culture A culture B 100% 100%
80% 80%
60% 60%
40% 40%
Abundance Abundance
20% 20%
0% 0% 94 97 120 147 185 94 97 120 147 185 Days Days Other Hydrogenophaga carboriunda strain YZ2 Porphyrobacter colymbi strain TPW-24 Other Blastomonas fulva strain T5 Porphyrobacter colymbi strain TPW-24 Alpha proteobacterium P-20 Blastomonas fulva strain T5 Gemmobacter lanyuensis strain Orc-4 Alpha proteobacterium P-20 Phreatobacter oligotrophus strain PI_21 Gemmobacter lanyuensis strain Orc-4 Phreatobacter oligotrophus strain PI_21 Pseudanabaena foetida var. intermedia NIES-512 Pseudanabaena foetida var. intermedia NIES-512 Sediminibacterium goheungense strain HME7863 Sediminibacterium goheungense strain HME7863 Flavobacterium chungnamense strain ARSA-103 Flavobacterium chungnamense strain ARSA-103 Nesterenkonia sp. Tibet-IBa2 Nesterenkonia sp. Tibet-IBa2 Rhodococcus cerastii strain C5 Rhodococcus cerastii strain C5 Figure 5.1. Changes in culture composition during the continuous Figure 5.2. Changes in culture composition during the continuous culture culture experiment in duplicate A experiment in duplicate B
165 5.5.2 Low nutrient continuous culture The continuous culture grown in low nutrient medium had a lower turbidity and cell concentration. On the day of inoculation, the most abundant organisms were Hydrogenophaga carboriunda at 26%, Sediminibacterium goheungense at 15 %, Preatobacter oligotrophus at 12%, Flavobacterium chungnamense at 10 %, see Figure 5.3. Pseudanabaena feotida abundance was 5% and Gemmobacter lanyuensis 5%. There were other species present, but abundance was below 5%. The turbidity during inoculation was only 0.04, therefore, it is worth considering, that due to low P. feotida concentration as cell counts that other otherwise minor species were relatively more abundant, compared to cultures with higher culture concentrations. The make up of the culture seems to indicate quite an even spread between various species without an overwhelmingly dominant culture.
On day 7, there were significant shifts in dominant cultures once culture reached turbidity values similar to ones in the batch cultures. Some species decreased in abundance significantly and went from being one of the dominant species, to more background cultures. The sharpest decrease was in abundance of H. carboriunda as abundance decreased to 13%, P. oligotrophus decreased to 5%, S. goheungense to 9%, A. proteobacterium to 2 %. The most significant increase in abundance was of P. feotida to 23 %, the highest abundance of all the species on that sampling day. Other species that increased in abundance also were F. chungnamense to 14%, a slight increase, G. lanyuensis to 15%, P. colymbi to 9% from below 5% abundance the previous day. It seems that the increase in turbidity and cell concentration translated into increase in abundance of P. feotida. However, other microorganisms also increased in abundance, as they outcompeted other species in low nutrient environment.
On day 26, the last day before medium pH was increased to 8.5 culture composition underwent some significant changes. The most stable abundance was of P. feotida, which was at 24%, the highest of all the species. However, other two species had similar abundance: F. chungnamense increased further to 20%, and H. carboriunda recovered to 22%. A. proteobacterium also somewhat increased the abundance to 9%, similar to the start of the experiment. S. goheungense abundance decreased
166 even further to 2%, the same could be said about P. oligotrophus with abundance of 3%. All the other minor species were below 5% abundance. It seems that with flushing and renewal of nutrients H carboriunda increased in abundance, even tough the turbidity values decreased. The same was observed in abundance of F. chungnamense and A. proteobacterium.
At the end of flushing with 8.5 medium pH on day 35 and before flushing with pH 10 medium started culture composition changed again. Although P. feotida with 22%, H. carboriunda with 28%, A. proteobacterium with 10% abundance were still the dominant species, S. goheungense decreased in abundance to 1 %. The decrease in abundance also was observed in F. chungnamense to 4%. However, abundance of Aminobacter sp. increased to 16%, and Alpha proteobacterium increased slightly to 10%. It seems that S. goheungense and F. chungnamense were sensitive either to increased pH or sodium concentrations, as turbidity was slightly higher compared to the previous sample. The increase in abundance of Aminobacter sp. may indicate better tolerance or adaptation to higher pH or sodium concentrations.
On day 45, the last day of flushing with pH 10 some less significant composition changes were observed. H. carboriunda at 17%, P. feotida at 18%, Aminobacter sp. at 12% were still the dominant species. However, the fraction of Animobacter sp. decreased compared to the previous sample, as well as of A. proteobacterium to 6%. F. chungnamense abundance increased to 12%. Variovorax ginsengisoli and Brevundimonas dendrificans abundance was forming a significant part for the first time at 9.44% 7.85% respectively. At the end of flushing with pH 11 on day 54 the culture composition looked rather similar with minor variations as seen in Figure 5.3.
After flushing with pH 11.5 medium culture composition showed some major changes on day 66. Although, P. feotida at 21%, F. chungnamense at 12% were still forming a large part of the culture. P. colymbi abundance increased to 26%, other cultures with significant increase were G. lanyuensis to 10%, S. goheungense to 7%, P. oligotrophus to 9%, and Runella slithyformis to 5%. The abundance of H. carboriunda shrank to 4% and Aminobacter sp. to 1% over the 12-day period. The
167 turbidity was the highest on day 66 throughout the whole experiment, the cell counts were not as high, but still significantly higher than the starting concentration. As culture concentration had not decreased by this point, it seems that the changes in culture composition are most likely to be due to the sodium hydroxide dosing and increased pH to 10.7. Although the pH value can be reached by cultures in growth phase, it is due to the elevated sodium concentrations.
And finally, on the last day of the experiment there were more significant shifts in culture composition. P. colymbi was the most abundant species at 37%, followed by G. lanyuensis at 20%. All other species formed much smaller fractions: P. feotida abundance was 10%, Aminobacter sp. of 9%, and F. chungnamense at 9%. All other species were 5% or lower abundance. The change in species coincided with increase in pH and decrease in cell counts and turbidity values. However, the cell counts and turbidity were still above the starting values. The results would suggest that P. feotida abundance decreased due to decreasing concentration in the culture, whereas other cultures were likely to be either better adapter or outcompeted by the increase in pH or sodium concentrations.
168 Species distribution in low nutrient continuous culture
100%
90%
80%
70%
60%
50%
Abundance 40%
30%
20%
10%
0% 0 7 26 35 45 54 66 84 Days Other Hydrogenophaga carboriunda strain YZ2 Variovorax ginsengisoli strain Gsoil 3165 Porphyrobacter colymbi strain TPW-24 Blastomonas fulva strain T5 Alpha proteobacterium P-20 Gemmobacter lanyuensis strain Orc-4 Rhizobium subbaraonis strain JC85 Aminobacter sp. RE 54 Phreatobacter oligotrophus strain PI_21 Brevundimonas denitrificans strain TAR-002 Pseudanabaena foetida var. intermedia NIES-512 Sediminibacterium goheungense strain HME7863 Flavobacterium chungnamense strain ARSA-103 Runella slithyformis strain DSM 19594 Rhodococcus cerastii strain C5 Figure 5.3. Change in culture composition during the low nutrient continuous culture experiment, day 0- at the start of the experiment, 7- before flushing started, day 26- before medium pH increase to 8.5, day 35 -before medium pH increase to 10, day 45- before medium pH increase to 11, day 54- before medium pH increase to 11.5, day 66- before medium pH increase to 12, day 84- the last day of the experiment.
There were clear shifts in species abundance with changes in growth conditions due to flushing and increase in culture pH. There were only two species present during the whole experiment with more that 4% abundance: P. feotida, lowest abundance of 5% on day 0, and F. chungnamense, lowest abundance of 4% on day 35%. The result would suggest, that these species are either more tolerant or better adapted to low nutrient, high pH and high sodium concentrations. G. lanyunensis was also present at the start and the end of the experiment, but abundance decreased in samples on days 45 and 54, suggesting either sensitivity at the pH range, or it was
169 outcompeted for nutrients by other species. However, once the pH and sodium concentration increased and more sensitive species decreased in concentration they were able to increase in abundance gain. The same trend was observed in changes in abundance of R. slithyformis and P. olygotrophus. P. colymbi showed a high adaptability to high pH as abundance increased significantly from day 54, the last day of flushing with 11.5 pH medium. Whereas H. carboriunda, B. dendrificans, and V. ginsengisoli decreased in abundance over the length of the experiment, suggesting the species are sensitive to more alkaline pH and higher sodium concentrations. Neither of species showed sensitivity to flushing, as cultures that decreased in abundance by day 26 increased again later in the experiment. P. feotida also seems to be adapted to slightly alkaline pH. As in the high nutrient continuous culture experiments medium of pH 12 or sodium concentrations required to increase the medium pH resulted in decrease in abundance, suggesting P. feotida growth limiting conditions.
5.5.3 Biocide experiments The species composition changed slightly during the experiment, but the dominant species were the same throughout the experiment. P. colymbi abundance decreased with time from 83% on day 13 to 72% on day 20. Whereas the abundance of F. chungnamense increased from 4% to 15% during the same period. P. feotida abundance varied at low proportion, 3% on day 13, 0.40 on day 15, and 1.27% on day 20. All the other species, besides P. oligotrophus with abundance varying from 5 to 7%, were below 3% abundance. It is rather surprising how abundance of P. feotida was so low in the control samples, especially as absorbance values only increased towards the end of the experiment.
As in control cultures, in cultures dosed with 20 ppm Mexel® biocide culture composition varied just slightly. The abundance of P. colymbi increased from 70 to 74% from day 13 to day 20. F. chungnamense abundance decreased slightly from 14 to 13%, and abundance of P. oligotrophus from 8.82 to 6.98% during the same period. The abundance of P. feotida decreased as well, from 2 to 1% from day 13 to day 20. As in control cultures, the abundance of P. feotida was very low. As biocide
170 dosing had very little effect on the turbidity of the cultures dosed with 20 ppm biocide it is worth assuming that the changes were occurring naturally.
Cultures dosed with 50 ppm Mexel® biocide compared to the control and the cultures dosed with 20 ppm biocide had significant shifts in culture composition. The abundance of P. colymbi decreased from 72 to 43% from day 13 to day15. In addition, the abundance of P. feotida decreased from 7 to 0.44% and P. oligotrophus from 7 to 3% during the same period of time. However, F. chungnamense abundance shot up from 8 to 35%. Other cultures like Aminobacter sp, G. lanyuensis, B. fulva abundance increased from 0.05, 2, and 2% to 4, 4, and 5% respectively. As turbidity values decreased during the same period it s reasonable to assume that the decrease in turbidity was due to the effect of Mexel® biocide on P. colymbi, P. feotida, and P. oligotrophus. As turbidity decreased as well, it may be that the species like P. chungnamense may not have resistance to the biocide, but more of a tolerance.
Comparable changes were also observed in cultures dosed with 100 ppm Mexel® biocide dose. The abundance of P. colymbi decreased from 74 to 52%, P. oligotrophus from 7 to 5%. However, P. feotida abundance increased from 2 to 3%, contrary to the results of dosing with 50 ppm Mexel® biocide. As with lower biocide dose abundance of F. chungnamense increased from 11 to 33%. Although most species abundance varied as in cultures dosed with half the biocide dose, the increase in P. feotida concentration is rather surprising. It may be, that the decrease in turbidity was mostly due to the decrease in P. colymbi abundance. However, it may be that most of the species decreased in concentration, but in varying proportions, thus increasing abundance in lower concentration sample.
The cultures dosed with 20 ppm Spectrus® biocide had the most unique changes in culture composition. The abundance of P. colymbi fluctuated from 78, to 56, and 73 % on days 13, 15, and 20. As in other cultures, it was the most abundant species. The abundance of F. chungnamense also fluctuated from 4, to 23, and 16% on the same sampling days. In addition, the abundance of P. feotida varied from 2, to 13, and 0.16% on days 13, 15, and 20. S. goheungense increased throughout the experiment from 1% to 5% from day 13 to day 20. The decrease in abundance of P.
171 colymbi coincided with decrease in turbidity values and increase in abundance of P. feotida. However, on the last day of the experiment the culture abundance retuned to pre-dosing levels. The results would suggest, that P. colymbi is more sensitive to Spectrus® biocide than P. feotida at such low doses. However, once the culture recovered, the abundance of P. colymbi increased again, suggesting short term effects at 20 ppm doses.
Cultures dosed with 50 and 100 ppm Spectrus® had mostly similar changes to culture composition. P. colymbi decreased in abundance from 75 to 35% in cultures dosed with 50 ppm and from 80 to 33% in cultures dosed with 100 ppm. F chungnamense abundance on the opposite, increased from 10 to 43% in cultures dosed with 50 ppm and from 10 to 40% in cultures dosed with 100 ppm Spectrus® biocide. Aminobacter sp. concentration also shot up after dosing from 0.05 to 12% and from 0.04% to 15% in cultures dosed with 50 and 100 ppm Spectrus® doses. P. feotida abundance behaviour was different between cultures dosed with different concentrations. In cultures dosed with 50 ppm the abundance decreased from 5% to 1%, however in cultures with 100 ppm dose the abundance increased from below 1% to 4%. It is important to note that the turbidity values decreased in cultures treated with both doses, therefore the increase in abundance of less abundant species may be due to decrease of dominant species concentration. However, even in that case, F. chungnamense and Aminobacer sp. were more resistant to the biocide.
From all the results combined it is clear that the culture composition will change as the culture concentration increases and nutrients get slowly depleted. However, biocide dosing does have a significant effect on the dominant species in the cultures. Biocides dosed at 20 ppm had limited effect on P. colymbi, although Spectrus® biocide was slightly more effective. The culture used for sequencing was the culture with the lowest turbidity values after dosing with 20 ppm Spectrus® biocide, so the effects were more pronounced. The higher biocide doses of both the biocides provided similar results: P. colymbi seems to be sensitive to both of the biocides, however, F. chungnamense is a lot more resistant. Interestingly, increase
172 in abundance of Aminobacter sp was more pronounced in cultures dosed with Spectrus® biocide rather than Mexel®, suggesting higher sensitivity.
Control culture composition 100% 90% 80% 70% 60% 50% 40% Abundance 30% 20% 10% 0% 13 15 20 Days
Other Porphyrobacter colymbi strain TPW-24 Blastomonas fulva strain T5 Gemmobacter lanyuensis strain Orc-4 Aminobacter sp. RE 54 Phreatobacter oligotrophus strain PI_21 Pseudanabaena foetida var. intermedia NIES-512 Sediminibacterium goheungense strain HME7863 Flavobacterium chungnamense strain ARSA-103
Figure 5.4. Changes in control culture composition during the experiment
173 Composition of cultures Composition of cultures dosed Composition of cultures dosed dosed with 20 ppm Mexel® with 50 ppm Mexel® with 100 ppm Mexel® 100% 100% 100% 90% 90% 90% 80% 80% 80% 70% 70% 70% 60% 60% 60% 50% 50% 50% 40% 40%
40% Abundance
Abundance Abundance 30% 30% 30% 20% 20% 20% 10% 10% 10% 0% 0% 0% 13 15 20 13 15 13 15 Days Days Days Other Other Other Porphyrobacter colymbi strain TPW-24 Porphyrobacter colymbi strain TPW-24 Porphyrobacter colymbi strain TPW-24 Blastomonas fulva strain T5 Blastomonas fulva strain T5 Blastomonas fulva strain T5 Gemmobacter lanyuensis strain Orc-4 Gemmobacter lanyuensis strain Orc-4 Gemmobacter lanyuensis strain Orc-4 Aminobacter sp. RE 54 Aminobacter sp. RE 54 Aminobacter sp. RE 54 Phreatobacter oligotrophus strain PI_21 Phreatobacter oligotrophus strain PI_21 Phreatobacter oligotrophus strain PI_21 Pseudanabaena foetida var. intermedia NIES-512 Pseudanabaena foetida var. intermedia NIES-512 Pseudanabaena foetida var. intermedia NIES-512 Sediminibacterium goheungense strain HME7863 Sediminibacterium goheungense strain HME7863 Sediminibacterium goheungense strain HME7863 Flavobacterium chungnamense strain ARSA-103 Flavobacterium chungnamense strain ARSA-103 Flavobacterium chungnamense strain ARSA-103 Figure 5.7. Changes in culture composition Figure 5.5. Changes in culture composition Figure 5.6. Changes in culture composition before, 2 days after biocide dosing, and at before and 2 days after biocide dosing before and 2 days after biocide dosing the end of the experiment.
174 Composition of cultures dosed Composition of cultures dosed Composition of cultures dosed with 20 ppm Spectrus® with 50 ppm Spectrus® with 100 ppm Spectrus® 100% 100% 100% 90% 90% 90% 80% 80% 80% 70% 70% 70% 60% 60% 60% 50% 50% 50% 40% 40% 40% 30% 30% 30% 20% 20% 20% 10% 10% 10% 0% 0% 0% 13 15 20 13 15 13 15 Days Days Days Other Other Other Porphyrobacter colymbi strain TPW-24 Porphyrobacter colymbi strain TPW-24 Porphyrobacter colymbi strain TPW-24 Blastomonas fulva strain T5 Blastomonas fulva strain T5 Blastomonas fulva strain T5 Gemmobacter lanyuensis strain Orc-4 Gemmobacter lanyuensis strain Orc-4 Gemmobacter lanyuensis strain Orc-4 Aminobacter sp. RE 54 Aminobacter sp. RE 54 Aminobacter sp. RE 54 Phreatobacter oligotrophus strain PI_21 Phreatobacter oligotrophus strain PI_21 Phreatobacter oligotrophus strain PI_21 Pseudanabaena foetida var. intermedia NIES-512 Pseudanabaena foetida var. intermedia NIES-512 Pseudanabaena foetida var. intermedia NIES-512 Sediminibacterium goheungense strain HME7863 Sediminibacterium goheungense strain HME7863 Sediminibacterium goheungense strain HME7863 Flavobacterium chungnamense strain ARSA-103 Flavobacterium chungnamense strain ARSA-103 Flavobacterium chungnamense strain ARSA-103 Figure 5.8. Changes in culture composition Figure 5.9. Changes in culture composition Figure 5.10. Changes in culture composition before, 2 days after biocide dosing, and at before and 2 days after biocide dosing before and 2 days after biocide dosing the end of the experiment
175 From the sequencing results clear trends can be identified in species response to changes in growth conditions. P. colymbi was relatively well adapted to flushing and increase in pH and sodium concentrations in high nutrient availability, however, once medium pH increased to 12.5 the abundance decreased significantly. The response is somewhat expected due to reported pH growth range (Furuhata et al., 2013). The same trend could be seen in changes in abundance of F. chungnamense and G. lanyunensis. On the other hand, B. fulva abundance increased significantly at the same time, after dosing with 12.5 pH medium started, suggesting better adaptation to more alkaline conditions. This is rather surprising as B. fulva was reported not being able to grow in pH 9 or above (Lee et al., 2017). P. feotida seems to be adapted to growing in a wide range of pH values as it formed a significant part of the culture composition throughout the high nutrient flushing experiment.
The culture composition in low nutrient flushing experiment was more varied, suggesting that low nutrient availability prevented domination of a few species and supported larger species diversity. However, there were significant shift in culture composition due to changes in growth conditions, as in the experiment with high nutrient availability. P. feotida, as in the previous experiment was present throughout the experiment with abundance varying between 5 and 21%. H. carboriunda abundance decreased once flushing with medium pH of 11.5 started, the species went from forming a significant part of the culture to more background level. P. colymbi abundance increased towards the end of the experiment, contrary to the results in the high nutrient availability experiment and suggested pH range the culture grows according to (Furuhata et al., 2013). It may be, as nutrient availability increased towards the end of the experiment, the species was able to increase in concentration and abundance.
The biocide experiments provided some understanding of species sensitivity and resistance to the biocides. Low doses of Spectrus® and Mexel® biocides had only very limited or no effect on P. colymbi, the dominating species in the cultures. However, higher doses of 50 and 100 ppm resulted in decrease in abundance of P. colymbi and increase in F. chungnamense. Aminobacter sp. reaction to Spectrus® biocide was surprising as abundance increase significantly in cultures dosed with 50
176 and 100 ppm doses from background levels. P. feotida response was varied, but doe to low abundance in the samples it is harder to evaluate it.
5.6 Conclusions The first major observation made was that P. catenata was not the dominant species according to DNA signatures in these experiments, however, it may be dominant volumetrically, due to the larger cell volume. Furthermore, the sequencing data provided insight how varied culture composition was depending on nutrient availability, and how quickly it could be changed due to variations in growth conditions. In addition, some species did not respond to changes in pH and sodium concentration in the same way in high and low nutrient conditions. The observation would suggest species adaptation to certain growth conditions and more competition between species in low nutrient availability. Moreover, the effectiveness of biocides was also shown to be species specific, although changes in culture composition were mostly the same when dosed with Mexel® and Spectrus® biocides.
177 6 Discussion and Summary
6.1 Discussion The experiments completed form their own separate and distinctive pieces of work. However, there is also a need to discuss the results in a wider context of the whole research programme. First, the scaling of the cultures is not proportional at high nutrient availabilities, as was observed in batch and continuous culture experiments. Once the P. catenata cultures were inoculated into 700 ml of BG11 medium, growth was significantly slower compared to cultures grown in 200 ml BG11 medium under the same growth conditions. The impact on the doubling time due to an increase in culture volume, subsequently impacted on the flushing rate and residence time of the continuous cultures. However, the same was not observed in the cultures grown in RN BG11 medium, as cultures grew as fast as in batch conditions.
Furthermore, the reaction to nutrient renewal due to flushing was distinctly different in cultures grown under low and high nutrient concentrations. Ion chromatography results suggested phosphorus limitation in cultures grown in BG11 medium in batch cultures, and flushing with the BG11 medium would result in constant renewal of phosphorus, even at a 22-day residence time. However, this did not seem to have any impact on the culture growth rate. On the other hand, nutrient renewal in the cultures grown in low nutrient availability resulted in turbidity values higher than ones in batch cultures from the same starting concentration. In addition, the ion chromatography results also showed an increase in nitrate concentrations towards the end of the experiment, either due to decreased uptake, or due to surplus nitrate concentrations. Furthermore, phosphorus concentrations decreased significantly after day 35, suggesting increased uptake rate.
In addition, the continuous cultures showed improved tolerances to an increase in pH and sodium concentrations, when compared to the batch cultures. In batch cultures the pH would seem to be self-regulated, and did not to increase above pH 11, whereas in continuous culture, the cells were still growing even though they
178 were dosed with pH 11.5 and 12 medium. The difference in response to increased pH values may be due to longer time periods for adaptations. The results of DNA sequencing of these cultures also provided interesting results, as species reported not being able to grow at pH 9 or above were dominant once cultures were flushed with pH 12-12.5 medium.
The quick change in pH due to buffering, or flushing, with pH 11 medium during repeated low nutrient continuous culture experiments showed how important slow changes are to a culture’s ability to grow. An increase to pH 11 was sufficient to reduce culture growth, whereas in slow flushing cultures the pH had to increase to 12 or above to have a similar impact.
The DNA sequencing results provided some insight into why the brown-yellow culture colour was so distinct. As most of the bacterial species present were described as pale or bright yellow-orange in the literature, it may be that the colour of the P. catenata cells were not the sole influence of the whole culture colour. In addition, the abundance of yellow-orange coloured bacteria may be the reason behind the more stable carotenoid concentrations noted compared to chlorophyll a. throughout the experiment. If the pigments in the coloured bacteria have similar turbidity ranges and carotenoids to those ofe P. catenata, the carotenoid concentration results would be of the whole microbial community, rather than P. catenata.
The make-up of the culture itself was rather surprising as the proportion of P. catenata in the microbial mix was lower than expected. The growing conditions and number of times the culture was subcultured should also be taken into account when considering the culture composition. In addition, the abundance is reported as DNA found in the sample, rather than actual abundance as cell concentration. In addition, the relative abundance of the species in the samples appears to be relativelyr stable and did not indicate a significant contamination event during the studies.
The DNA sequencing data also provided very distinct culture compositions under differing growth conditions. Compared to high nutrient availability experiments low
179 nutrient continuous culture had a more diverse species composition, and the proportion of species was more even. However, the make-up of batch cultures before biocide dosing was completely different with P. colymbi being the most dominant organism detected in these experiments while its abundance decreased after the biocide dosing. In addition, there were clear shifts in culture composition at the start of flushing, during increases in medium pH, or biocide dosing. However, due to changes in culture concentration (measured as absorption), some of the species may have increased in abundance due to a decrease in relative concentrations of other species. The sequencing data of the biocide experiment may be the best example of this.
6.2 Summary From the data gathered, some points emerge. First, considering the different behaviour of the cultures in buffered and unbuffered medium, and continuous cultures dosed with high pH medium the following can be noted. Over several experiments, unbuffered cultures self-adjusted the pH of the cultures to above pH 10, and the cultures continued growing, with pH values fluctuating but maintained above pH 10. However, cultures in the buffered medium did not grow in cultures buffered at pH 10, 11, or 12. Furthermore, cultures grown in low and high nutrient continuous cultures and flushed with high pH medium were stable until they were flushed with medium pH adjusted to 12. The results contradict each other, although, it has been shown, that a sudden change in pH does prevent culture growth. Another point to note is that changes in microbial composition of the cultures were observed under varying growth conditions - high nutrient continuous cultures and the batch cultures grown for biocide experiments had distinct compositions, they were all dominated by high proportions of close relatives to P. colymbi, F. chungnamense, P. oligotrophus and P. foetida. Although the proportions changed, the species varied only slightly. However, the low nutrient continuous cultures exhibited a different behaviour; the culture was more diverse, although there were changes in species abundance with culture pH. Furthermore, cultures that were grown in BG11 medium were more affected by increases in pH and biocide concentrations, cultures grown in low nutrient continuous culture seemed
180 less effected, and even survived and adapted in more extreme conditions. As discussed earlier, this could be due to a more diverse “syntrophic” community, supporting each other with nutrient exchange. It could also be due to a shorter doubling time enabling quicker adaptations in the low nutrient culture. In addition, although biomass concentrations in the low nutrient culture were lower, the abundance of P. feotida was higher compared to the batch cultures. In addition, the same was observed in high nutrient continuous cultures. It seems that continuous cultures can support a higher abundance of P. feotida compared to the batch cultures. In addition, although the culture collection identified the dominant cyanobacterium as P. catenata, the sequencing data provided results that it was an organism most closely related to P. feotida. This clearly warrants further investigations, including a detailed phylogenetic analysis of the sequences obtained from these experiments.
6.3 Applicability of results to site operations From the data gathered there are several areas where knowledge could be transferred to pond operations. First, the nutrient concentrations found in the pond, if P. catenata is distributed uniformly, should not be able to support the bloom event. However, due to the lack of data it is impossible to determine what are the actual concentrations of nutrients available in the pond. In addition, due to the size and complexity of the pond system, nutrient availability may not be as limited in all areas as the data provided would suggest. Organisms may be able to scavenge nutrients required for their growth in discrete areas. Another observation is that although cultures grown in high nutrient continuous culture grew slower, compared to smaller volume batch cultures, this was not applicable to the cultures grown in low nutrient medium. This could support the hypothesis, that slower growth is due to shading. However, this could have a significant impact on the pond operations; as the results show, just how quickly cultures in low nutrient medium continuous culture can grow from very low concentrations to full bloom. During the final low nutrient experiment, the doubling time was just under a day, requiring high flow rate conditions to prevent culture growth, which could not be implemented in the pond due to the volume of water required, suspension of
181 sludge, capacity of water treatment equipment and other pond specific issues. However, the experiments with continuous culture also showed, that if a culture is allowed to adapt slowly to the increase in medium pH, only pH 12 values will have a significant effect on the culture. On the other hand, when flushing is coupled with a sudden increase in pH, lower pH values were sufficient to reduce culture growth. However, this was applicable to cultures grown in laboratory conditions; cultures growing in the pond are adapted to living in highly alkaline conditions and will most likely need a significant increase in pH. However, this could be used to control bloom events, as residence times would decrease due to prolonged flushing periods, but coupled with a fast increase in pH, the same effect could be simulated. In addition, it relies on cultures being adapted to growing in lower pH waters. This would mean that the pH could be kept just above 11 most of the year, and only suddenly increased to remove the bloom quicker at times when an increase in turbidity is detected by online monitoring, minimising possible effects on the SIXEP and pond infrastructure. However, a prolonged increase in pH could also be used to as a preventative measure. The pond water pH could be increased above pH 12, to try and prevent the bloom with a hope that organisms will not adapt.
The use of biocides could be another way of removing the algal bloom. However, any additional chemical compounds may have an impact in SIXEP or other processes downstream, therefore, the preferred option would be not to use biocides or to use the lowest concentration possible of biocides that are compatible with downstream processes. Both biocides used in the experiments were effective with the culture pH at 10.5, suggesting positive results at higher pH as well. Although the biomass levels were high in these experiments to simulate a bloom, the required biocide concentrations to be effective were at least 50 ppm. However, there are few limitations of the experiment that could mean lower concentrations could be effective in the pond environment. First, these concentrations were used in batch conditions, therefore, a lower dose may be required when used in conjunction with flushing due to the cumulative dose on the organism. In addition, the dose was used for a full bloom event, if the biocide would be used just as an increase in microorganism concentrations is noticed, lower doses may be sufficient
182 to prevent or stop the bloom before the concentration increases significantly. Both of the biocides used prevented the culture from recovering for at least a week, which could be used in bloom removal. Another way of treating the bloom could be reduction in flow rate followed by a high dose of biocide, and increased flow rate to remove the dead cell matter from the pond once the bloom colour has reduced in intensity as seen in the batch culture experiments.
6.4 Conclusions At the start of the project the main objectives were:
1. To understand the culture behavior and growth in pond simulant medium, or if it was not possible, in medium representing the nitrate and phosphate concentrations found in the pond. In addition, to determine how high pH affects culture growth, simulating possible pond conditions. The results would serve as a basis for further experiments.
2. To identify the optimum nitrogen and phosphorus ratios and preferred nutrient concentrations of P. catenata for maximal growth to inform how growth could be controlled in the FGMSP by nutrient limitations.
3. To identify biocides that have potential to be used as a method of bloom prevention and control. The optimum dosage for full bloom removal and effective period of the biocide treatment could be determined from the experimental results.
4. To identify the differences in culture behaviour in batch and continuous culture, and any issues that could arise due to scaling-up. To determine how the photosynthetic culture reacts to changes in medium pH, and if increases in culture pH coupled with flushing could be used to control growth.
Most of the primary objectives set were achieved. The only area that clearly needs additional work is understanding culture growth in the pond simulant medium, due to very mixed results. However, this research added to the understanding of culture growth under varying nutrient regimes. Although the results did not completely support the earlier findings by Healey and Hendzel (1979), but that could be due to
183 different growth conditions. The experiments also highlighted the variable behaviour of the culture even when grown under optimised conditions in the laboratory. The further experiments using Mexel® and Spectrus® biocides provided a better understanding of the potential effects of the biocides on P. catenata, and the dosing required to clear a bloom. The information obtained also gave a better understanding of effects of the biocides on different species present in the cultures grown in this study, which mimic closely the mixed community in the FGMSP (Foster, 2014). In addition, the research highlighted the variations in the response of a representative culture to different nutrient concentrations, medium flow rate, and growth in alkaline conditions.
Growth of the cyanobacterium Pseudanabaena catenata seems to be unpredictable (in laboratory culture), but very adaptable species. Given time, even cultures grown in high nutrient unadjusted pH medium can adapt to low nutrient availability and highly alkaline growth conditions. However, it does not seem to be able to adapt easily to fast changing environments, which could be used as an advantage for bloom control. Due to these findings, some changes in the pond environment were identified as having potential to control growth of P. catenata, as well as two biocides. Although both the biocides required quite high doses, as mentioned before, this dose was tested against high cell density batch cultures. To conclude, this research project provided more information about culture behaviour in batch and continuous cultures, varying nutrient availability, and reaction to changes in medium pH. Furthermore, one of the most important goals of the projects was achieved; from the experimental results, several strategies to changes in pond conditions were identified for prevention of P. catenata growth and bloom control.
6.5 Scope for future work The results of the research completed not only provided valuable information about the culture, but also opened a lot of areas for further investigation. The research areas could expand not only to understanding the behaviour of microbial communities under different growth conditions, but also to effectiveness of the biocide, and effects of proposed methods of control on the pond inventory, structure, and abatement processes.
184 6.5.1 Effects of changes in growth conditions A lot of work is still required to better understand the effects of temperature, available light, and pH on the culture growth. As some experiments gave varying results it would be worth repeating them. Furthermore, to eliminate the possibility that the results of the buffered pH experiments failed due to sodium concentration, other hydroxides or carbonates could be used. Furthermore, the theory of self- shading could be investigated using vessels of varying surface area. In addition, the effects of available light, or temperature on optimum nitrogen and phosphorus ratio, and cultures growing in reduced nutrient media would provide insight into changes in response and nutrient needs depending on the growth conditions. Furthermore, the changes in culture composition could be investigated alongside changes in environmental conditions, to determine the species culture adaptability.
6.5.2 Experiments with pond culture samples Cultures sampled directly from the pond could be used to repeat the experiments completed and determine the differences in behaviour between lab grown and pond cultures. The relationship between photosynthetic and heterotrophic organisms could also be explored further: is it symbiotic, are there key organisms influencing bloom formation besides cyanobacteria. If the latter is true, could the accessory organisms be targeted to prevent/ control bloom formation. Could conditions in the pond be manipulated to disturb the relationships between species.
6.5.3 Further biocide trials There is a lot of scope left in finding the most suitable biocide. Other available biocides that are effective at alkaline pH could be investigated. In addition, although dosage of biocides to remove the bloom has been investigated, the preventative dose is still not clear. The effectiveness of biocides at medium pH values mimicking those in the pond, or effectiveness in pond simulant medium has not been investigated either. Furthermore, continuous culture experiments could also be used to gather more information about the effectiveness of the biocides. The preventative dose required for continuous cultures could be determined in high and low nutrient media, effectiveness at highly alkaline conditions with constant
185 sodium hydroxide dosing, and the required dose to remove a bloom in a continuous culture could be identified.
6.5.4 Continuous culture and scaling experiments Continuous culture experiments could be used to understand the growth dynamics if several species are responsible for the bloom. Experiments with seeding events at different growth stages, or effectiveness of biocides on mixed cultures would provide invaluable information. Scaling experiments should also be used to determine P. catenata behaviour in larger volumes, especially when there is no external stirring and the culture only relies on mixing due to flushing. It would also help to determine if larger volume would lead to higher culture concentrations as assumed happens in the pond.
6.5.5 Effects of proposed treatment methods on the pond inventory and SIXEP As pond water is treated in the SIXEP ion exchange plant before being discharged, effects of increased pH and use of biocides in SIXEP operation should be investigated. Furthermore, the biocides could have an effect on the waste inventory, therefore, the effects of biocides on sludge and other sensitive materials should be carried out in high pH pond simulant medium, due to the drop seen in pH during biocide trials.
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223 8 Appendix
8.1 Appendix 1 BG11 (Stanier et al., 1971) growth medium recipe
NaNO3 1.5 g
K2HPO4 0.04 g
MgSO4·7H2O 0.075 g
CaCl2·2H2O 0.036 g Citric acid 0.006 g Ferric ammonium citrate 0.006 g EDTA (disodium salt) 0.001 g
Na2CO3 0.02 g Trace metal mix 1.0 ml Agar (if needed) 10.0 g Distilled water 1.0 L
Trace metal mix:
H3BO3 2.86 g
MnCl2·4H2O 1.81 g
ZnSO4·7H2O 0.222 g
NaMoO4·2H2O 0.39 g
CuSO4·5H2O 0.079 g
Co(NO3)2·6H2O 49.4 mg Distilled water 1.0 L
224 Jaworski’s medium (Schlösser, 1984)
Ca(NO3)2·4H2O (4.0 g/200 ml) 1.0 ml
KH2PO4 (2.48 g/200 ml) 1.0 ml
MgSO4·7H2O (10.0 g/200 ml) 1.0 ml
NaHCO3 (3.18 g/200 ml) 1.0 ml EDTAFeNa (0.45 g/200 ml) 1.0 ml
EDTANa2 (0.45 g/200 ml) 1.0 ml
H3BO3 (0.496 g/200 ml) 1.0 ml
MnCl2·4H2O (0.278 g/200 ml) 1.0 ml
(NH4)6Mo7O24·4H2O (0.20 g/200 ml) 1.0 ml Cyanocobalamin (0.008 g/200 ml) 1.0 ml Thiamine HCl (0.008 g/200 ml) 1.0 ml Biotin (0.008 g/200 ml) 1.0 ml
NaNO3 (16.0 g/200 ml) 1.0 ml
Na2HPO4·12H2O (7.2 g/200 ml) 1.0 ml Deionized water to 1.0 L
225 Z8 (Staub, 1961)medium
MgSO4·7H2O 0.25 g
NaNO3 0.467 g
Ca(NO3)2·4H2O 59 mg
NH4Cl 31 mg
Na2CO3 0.02 g FeEDTA solution 10 ml Gaffron micronutrients 1.0 ml Deionized water to 1.0 L
FeEDTA solution: Made in two solutions:
Solution A - 2.8 g FeCl3 in 100 ml 0.1 N HCl
Solution B - 3.9 g EDTANa2 in 100 ml 0.1 N NaOH Add 10 ml solution A and 9.5 ml solution B plus water to 1 L.
Gaffron micronutrients:
H3BO3 3.1 g
MnSO4·4H2O 2.23 g
ZnSO4·7H2O 0.22 g
(NH4)6Mo7O24·4H2O 0.088 g
Co(NO3)2·6H2O 0.146 g
VOSO4·6H2O 0.054 g
Al2(SO4)3K2SO4·2H2O 0.474 g
NiSO4(NH4)2SO4·6H2O 0.198 g
Cd(NO3)2·4H2O 0.154 g
Cr(NO3)3·7H2O 0.037 g
Na2WO4·2H2O 0.033 g KBr 0.119 g KI 0.083 g Deionized water to 1 L
226 8.2 Appendix 2
Changes in pH values 10.5
10
9.5
9
8.5
pH 8
7.5
7
6.5
6 0 5 10 15 20 25 Days
BG11 medium as control Pond medium highest concentration Pond medium average concentration Pond medium lowest concentration
Figure 8.1. Changes in pH values during pond water simulant experiment. The error bars show the standard error of the mean, n=3.
Change in pH values 11 10.5 10 9.5 9
pH 8.5 8 7.5 7 6.5 0 2 4 6 8 10 12 14 16 18 Days P. catenata BG11 Anabaena BG11 P.catenata in reduced nutrient BG11 P. catenata in pond medium highest concentration with acetate
Figure 8.2. Changes in pH values during pond water simulation experiment with carbon source addition. The error bars show the standard error of the mean, n=3.
227 Changes in pH 7.9 7.7 7.5 7.3
pH 7.1 6.9 6.7 6.5 0 2 4 6 8 10 12 14 16 Days Reduced nutrient BG11 Reduced nutrient BG11 3 mM acetate Reduced nutrient BG11 10 mM acetate Reduced nutrient BG11 20 mM acetate
Figure 8.3. Changes in average pH values during the organic carbon availability experiment. The error bars show the standard error of the mean, n=3.
Change in pH
12 11.5 11 10.5 10
pH 9.5 9 8.5 8 7.5 0 5 10 15 20 Days BG11 BG11 pH10 BG11 pH11 BG11 pH12
Figure 8.4. Changes in average pH values during buffered pH experiment. The error bars show the standard error of the mean, n=3.
228 Image 8.1. P. catenata grown in pond simulant medium in lowest nutrient concentrations on day 0 (top) and day 13 (bottom), before the addition of BG11 medium
229 Image 8.2. P. catenata in pond water simulant medium with the highest nutrient concentrations on day 0 (left) and day 13 (right), before the addition of BG11 medium
230 Image 8.3. Cultures grown in reduced nutrient BG11 medium (top) and pond water simulant medium dosed with acetate (bottom) on day 18
231 8.3 Appendix 3
Image 8.4.Reduced nutrient continuous culture on day 0 (top), day 7 before culture flushing (bottom)
232 Image 8.5. Reduced nutrient continuous culture on day 26 before pH increase to 8.5 in medium (top), and on day 54 (left) before culture was started to be flushed at pH 11.5
233 Image 8.6.Reduced nutrient continuous culture on day 66 (top) before flushing with pH 12 medium, and on the last day (84) of the experiment (bottom)
234 Image 8.7.Repeated low nutrient continuous culture on day 0 (top), day 8 (bottom), before flushing with pH 11 medium.
235 Image 8.8. Repeated low nutrient continuous culture on day 17 at the end of the experiment
236 8.4 Appendix 4
Image 8.9. Control culture of P. catenata on day 13
237 Image 8.10. Culture to be dosed with 20 ppm (top), and 50 ppm (bottom) of Mexel® 432 biocide before dosing on day 13
238 Image 8.11. Culture to be dosed with 20 ppm (top) and 50 ppm (bottom) of Spectrus® NX 1422 biocide before dosing on day 13
239 Image 8.12. Control (top), culture dosed with 20 ppm Mexel® 432 (bottom) biocide on day 15, 2 days after dosing with biocide
240 Image 8.13. Culture dosed with 20 ppm Spectrus® NX 1422 biocide on day 15, 2 days after dosing with biocide
241 Image 8.14. Control (top), and P. catenata dosed with 50 ppm of Mexel® 432 (bottom) on day 20, last day of the experiment
242 Image 8.15. Cultures dosed with 50 ppm Spectrus NX® 1422 biocide on day 20 (bottom), last day of the experiment
Carotenoid concentration during Mexel 432® experiment 1.6 1.4 1.2 1
0.8 µg/ml 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 16 18 Days BG11 (control) Mexel 20 ppm Mexel 10 ppm Mexel 5 ppm Mexel 1 ppm
Figure 8.5. Change in carotenoid concentration during repeated Mexel® 432 biocide trial with biocide dosing on day 15. The error bars show the standard error of the mean, n=3.
243 Carotenoid concentrations during Spectrus® experiment 2.5
2
1.5 µg/ml 1
0.5
0 0 2 4 6 8 10 12 14 16 18 20 22 Days
BG11 (control) Spectrus 20 ppm Spectrus 10 ppm Spectrus 5 ppm Spectrus 1 ppm
Figure 8.6. Change in carotenoid concentration during Spectrus® NX 1422® biocide experiment with dosing on day 9. The error bars show the standard error of the mean, n=3.
Change in pH over the lenght of the repeated biocide
11.5 experiment 11 10.5 10 9.5
9 pH 8.5 8 7.5 7 6.5 0 2 4 6 8 10 12 14 16 18 20 22 Days Control Spectrus 20 ppm Spectrus 10 ppm Spectrus 5 ppm Spectrus 1 ppm
Figure 8.7. Change in pH during the first Spectrus® biocide dosing experiment. The error bars show the standard error of the mean, n=3.
244 Carotenoid concentrations during repeated biocide trial 1.6
1.4
1.2
1
0.8 µg/ml 0.6
0.4
0.2
0 -3 2 7 12 17 22 Days
BG11 (control) Mexel 20 ppm Mexel 50 ppm Mexel 100 ppm Spectrus 20 ppm Spectrus 50 ppm Spectrus 100 ppm
Figure 8.8. Change in carotenoid concentrations during the repeated Mexel® 432 and Spectrus® NX® 1422 experiment with biocides dosed on day 13. The error bars show the standard error of the mean, n=3.
Change in pH during repeated biocide experiment 11
10.5
10
9.5 pH
9
8.5
8 0 2 4 6 8 10 12 14 16 18 20 22 Days
BG11 (control) Mexel 20 ppm Mexel 50 ppm Mexel 100 ppm Spectrus 20 ppm Spectrus 50 ppm Sectrus 100 ppm
Figure 8.9. Change in pH during the repeated biocide experiment with both the Mexel® and Spectrus® biocides dosed on day 13. The error bars show the standard error of the mean, n=3.
245