Free University of Brussels

FACULTEIT WETENSCHAPPEN

VAKGROEP TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN DIENST MICROBIËLE INTERACTIES

SCK•CEN DEPARTEMENT RADIOPROTECTIE LABORATORIUM VOOR MICROBIOLOGIE EN RADIOBIOLOGIE

Academiejaar: 2004-2005

Microbial characterization of the nitrifying packed-bed pilot reactor in the Micro-Ecological Life Support System Alternative (MELiSSA)

Promotor: Prof. Dr. P. Cornelis Scriptie voorgedragen tot het behalen van de graad Licentiaat in de Biologie Co-promotor: Dr. Ir. L. Hendrickx Benny PYCKE

FACULTEIT WETENSCHAPPEN

VAKGROEP TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN DIENST MICROBIËLE INTERACTIES

SCK•CEN DEPARTEMENT RADIOPROTECTIE LABORATORIUM VOOR MICROBIOLOGIE EN RADIOBIOLOGIE

Academiejaar: 2004-2005

Microbial characterization of the nitrifying packed-bed pilot reactor in the Micro-Ecological Life Support System Alternative (MELiSSA)

Promotor: Prof. Dr. P. Cornelis Scriptie voorgedragen tot het behalen van de graad Licentiaat in de Biologie Co-promotor: Dr. Ir. L. Hendrickx Benny PYCKE

Acknowledgements

First, I would like to thank my promoter, Prof. Dr. Pierre Cornelis, and Prof. Dr. Max Mergeay for allowing me to work at SCK•CEN, and for the advice that they have given me over the year. All my appreciation goes out to my mentor, Dr. Ir. Larissa Hendrickx, whom I would like to thank for all the assistance and advice during my stay at SCK•CEN, and for helping me master what there was to learn in a microbiological laboratory.

I also owe many thanks to the researchers at the Free University of Barcelona for lending their support to this research.

I would also like to thank everyone in the SCK•CEN Radiobiology and Microbiology laboratory (Annik, Fabienne, Felice, Greet, Hanane, Ilse, Iris, Jasmine, Joris, Joyce, Louis, Marcella, Mieke, Monica, Natalie, Nicolas, Patrick, Paul & Paul, Rafi, Sarah, Sébastien & Sébastien, and Werner) and the VITO Biology laboratory (An, Annemie, Leni, Karolien, and Winnie) for the pleasant work atmosphere and providing assistance when I was in need, and especially Arlette for all the help with and advice about several molecular techniques.

In addition, I would like to thank everybody who lives at the dormitory and made my stay a pleasant one, Aurélie, James, Ruth, Sandor, etc.

Finally, I owe my thanks to my parents and girlfriend for supporting me during this whole period.

Benny.

Table of contents

LIST OF FIGURES ...... 7

LIST OF TABLES...... 9

ABSTRACT ...... 10

SAMENVATTING ...... 11

ABBREVIATIONS...... 12

A. INTRODUCTION...... 14

B. STATE OF KNOWLEDGE...... 16

PART I: LIFE SUPPORT ...... 16

1. LIFE SUPPORT SYSTEMS ...... 16 1.1. General introduction...... 16 1.2. Micro-Ecological Life-Support System Alternative (MELiSSA) ...... 16 PART II: NITROGEN RECYCLING IN NATURAL AND ENGINEERED SYSTEMS...... 20

1. NITROGEN AND MICROORGANISMS...... 20 1.1. Introduction ...... 20 1.2. Nitrogen cycle...... 20 1.3. Aerobic nitrification...... 24 1.3.1. Autotrophic nitrification...... 24 1.3.2. Classification of autotrophic nitrifying bacteria...... 24 1.3.3. The sensitiveness of the AMO enzyme...... 26 1.3.4. Heterotrophic nitrification...... 26 2. NITROGEN RECYCLING IN BIOREACTOR CIII ...... 28 2.1. Nitrosomonas europaea ATCC 19718 ...... 28 2.2. Nitrobacter winogradskyi Agilis ATCC 25391 ...... 30 PART III: THE BACTERIAL COMMUNITY OF NITRIFYING REACTORS ...... 33

1. BACTERIAL COMMUNITIES IN ENGINEERED ENVIRONMENTS...... 33 1.1. General introduction...... 33 1.2. Bacterial communities in nitrifying reactors and wastewater treatment plants...... 34 1.2.1. Community composition in nitrifying reactors and wastewater treatment plants ...... 34 1.2.2. Spatial patterns in nitryifying reactors ...... 36 2. ANALYSIS OF BACTERIAL COMMUNITIES ...... 37 2.1. Approaches to bacterial community analysis...... 37 2.2. Analysis by means of DGGE...... 38 PART IV: HORIZONTAL GENE TRANSFER ...... 40

1. BACTERIAL CONJUGATION...... 40 1.1. Mechanisms of Horizontal Gene Transfer (HGT)...... 40 1.2. Bacterial conjugation...... 41 1.2.1. Properties and implications ...... 41 1.2.2. Requirements for bacterial conjugation...... 42 1.2.3. Plasmids ...... 44 2. EXOGENOUS PLASMID ISOLATION ...... 46 C. OBJECTIVES...... 48

D. MATERIAL AND METHODS ...... 49

1. BACTERIAL STRAINS, CULTURE CONDITIONS, BACTERIAL MEDIA, AND ANTIBIOTICS ...... 49 1.1. Bacterial strains...... 49 1.2. Plasmids...... 49

1.3. Growth conditions...... 50 1.4. Bacterial media...... 50 1.4.1. 1-2-3 medium...... 50 1.4.2. 284-medium ...... 51 1.4.3. 869-medium ...... 51 1.4.4. Co-culture medium (COC)...... 51 1.4.5. KM1 ...... 51 - 1.4.6. Nitrite (NO2 ) medium...... 52 1.4.7. Nitrosomonas minimal medium (NMM) ...... 52 1.4.8. Purity Check Medium (PCM) ...... 52 1.4.9. Skinner & Walker (SW) inorganic medium...... 52 1.4.10. SOB medium...... 53 1.4.11. SOC medium...... 53 1.4.11. Antibiotics...... 53 2. AMMONIA ANALYSIS ...... 53 2.1. Determination of ammonia content in media...... 54 3. NUCLEIC ACID PREPARATION AND EXPERIMENTS ...... 55 3.1. Preparation of genomic DNA from Bacteria ...... 55 3.1.1. Miniprep of N. europaea pure culture Genomic DNA ...... 55 3.1.2. Fast-prep DNA extraction ...... 55 3.1.3. Determination of DNA purity and DNA concentration ...... 56 3.2. DNA-based molecular analyses...... 56 3.2.1. Polymerase Chain Reaction (PCR) ...... 56 3.2.3. Denaturing Gradient Gel Electrophoresis (DGGE)...... 58 3.2.4. Gels, buffers and visualisation ...... 59 3.2.5. PCR Cloning Transformation...... 60 3.2.6. DNA sequencing ...... 61 3.3. Preparation of total RNA from Bacteria...... 61 3.3.1. Total RNA isolation ...... 61 3.3.2. Determination of RNA purity and RNA concentration...... 62 3.4. RNA-based molecular analyses ...... 62 3.4.1. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) ...... 62 4. DETECTION OF BACTERIAL CONJUGATION ...... 62 4.1. Triparental exogenous plasmid isolation...... 62 E. RESULTS...... 64

1. ORIGIN OF REACTOR SAMPLES AND SAMPLE PREPARATION ...... 64 2. AMMONIA ANALYSES ...... 66 2.1. Reactivation of lyophilized samples...... 66 2.2. Detection of ammonia consumption of reactivated lyophilized samples...... 67 2.2. Detection of ammonia consumption of reactivated frozen samples ...... 69 3. COMMUNITY ANALYSIS ...... 70 3.1. DGGE-based total community analysis...... 70 3.1.1. DGGE-based total-community analysis...... 70 3.1.2. DGGE-based in silico clustering analysis ...... 71 3.1.3. Ammonia-oxidizing community analysis...... 74 3.1.4. Culture-based whole-community analysis ...... 76 3.2. DGGE-based active community analysis...... 80 3.3 Identification of community members ...... 81 3.3.1. Identification of isolates...... 81 3.3.2. Identification through clone screening...... 84 4. DETECTION OF PLASMIDS ...... 85 4.1. PCR-based plasmid detection ...... 85 4.2. Detection of mobilizing plasmids...... 87 4.2.2. Initial screening of the bioreactor samples...... 87 4.2.3. Exogenous isolation of mobilizing plasmids...... 88 F. DISCUSSION ...... 90

G. PERSPECTIVES...... 99

H. REFERENCES ...... 101

APPENDIX I: LIST COMPRISING THE USED SUBSTANCES AND SCALES, AND THEIR ORIGIN.. 117

6 List of figures Figure 1. General concept of the MELiSSA loop and the interactions between the different compartments (Pérez et al., 2004)...... 17

Figure 2. Schematic view (a) and picture (b) of the nitrifying packed-bed pilot plant at UAB. (1) packed-bed section with immobilized culture, (2) bottom section for aeration, liquid distribution and instrumentation, (3) top section for gas disengagement, (4) gas sparger, (5) gas exit condenser, (6) gas closed loop, connected to controlled oxygen/nitrogen supply to control oxygen level, (7) liquid feed, (8) liquid recirculation, (9) liquid outlet, (10) acid addition, (11) base addition, (12) T° probes, (13) oxygen probes, (14) pH probes, (15) cold finger, (16) hot finger (Gòdia et al., 2004)...... 19

Figure 3. The biogeochemical nitrogen cycle and its comprising transformations (Pidwirny, 2004) ...... 21

Figure 4. The microbial nitrogen cycle (Ye & Thomas, 2001). The anammox reaction is not depicted on this figure. Immobilization is the incorporation of ammonia into biomass...... 21

Figure 5. The phylogenetic relationship between autotrophic among AOB and NOB (both indicated in bold) (Teske et al., 1994). AOB are found in both β-Proteobacteria and γ-Proteobacteria, where NOB are found in α- Proteobacteria, γ-Proteobacteria and δ-Proteobacteria...... 25

Figure 6. View of the Nitrosomonas europaea inner-membrane, where ammonia oxidation occurs through action of the membrane-bound AMO and periplasmic HAO (Poughon et al., 2001). The top section is the Nitromonas europaea periplasm, where HAO is located. The cytoplasm is the section depicted below the (inner-)membrane; within this membrane, AMO is embedded...... 28

Figure 7. Theoretical model for the energy metabolism of Nitrobacter sp. from oxidation of nitrite. X is unknown (Poughon et al., 2001). The motive enzyme, nitrite oxidase, is located in the inner-membrane...... 30

Figure 8. Response of N. agilis and N. winogradskyi to mixed culture conditions determined by studying their respective growth in co-culture conditions (Fliermans et al., 1974)...... 32

Figure 9. Schematic drawing of the community structure of the biofilms in the Kölliken RBC and of the main nitrogen conversion reactions (Egli et al., 2003). Aerobic AOB and NOB appeared to grow on the outer shell of the biofilm. Further inwards, where the anoxic zone is located, the anammox bacteria were observed...... 37

Figure 10. The exogenous plasmid isolation principle by triparental mating. (a) The transfer of the Mob+ Tra+ plasmid from the unidentified plasmid-harbouring helper cell that is present in the microbial community to the donor cell, (b) the donor cell transfers its plasmids to the plasmid-free recipient, the indigenous 'donor plasmid' (Mob+ Tra-) is transferred through action of the 'mobilizing helper-plasmid' (Mob+ Tra+) to the chosen plasmid- free recipient, (c) after selection, only the recipients carrying a selectable marker gene, that harbour the 'donor plasmid' carrying the particular R-gene (i.e. the gene encoding a chosen selectable function); can subsequently be isolated...... 47

Figure 11. Scheme of sections (N0-N7) in which the CIII bioreactor was divided after dismantling...... 65

Figure 12. Flasks containing lyophilized samples (N0-N7) incubated for 7 days in SW medium. (a) First inoculation, (b) second inoculation...... 67

Figure 13. Ammonia content of SW-medium supernatant after 7 days of growth of the lyophilized samples + harvested from the CIII pilot reactor. C : sterile medium, N0-N7: CIII lyophilized sample. Error bars indicate the standard deviation...... 68

Figure 14. Ammonia content of COC-medium supernatant after 7 days of growth of the lyophilized samples + harvested from the CIII pilot reactor. C : sterile medium, N0-N7: CIII lyophilized sample. Error bars indicate the standard deviation...... 69

Figure 15. Ammonia content of SW medium supernatant after 2.5 days of growth of the frozen samples + harvested from the CIII pilot reactor. C : sterile medium, N0-N7: CIII lyophilized sample. Error bars indicate the standard deviation...... 70

Figure 16. DGGE profile of uncultured lyophilized and uncultured frozen samples. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene...... 71

Figure 17. Similarity analysis performed on the uncultured frozen samples and the N. europaea DGGE profiles using the BioNumerics™ program, yielding: UPGMA dendrogram from DGGE analysis, DGGE gel, and percentages similarity between the samples based on the 16S rRNA gene in uncultured frozen samples...... 73

Figure 18. DGGE profiles of samples grown 284 Gluc medium, COC medium, and SW medium targeting the amoA functional gene (using amoA-1F and amoA-2R primers). L = VITO ladder, N = N. europaea (Neu) amoA genes...... 74

Figure 19. DGGE amoA-profile of the lyophilized uncultured samples, lyophilized samples cultured in 284 Gluc, COC medium, and SW medium using the optimized amoA-1F* primer. L = VITO ladder, N = N. europaea (Neu) amoA genes...... 75

Figure 20. DGGE profiles for the lyophilized samples grown in 869 medium and PCM, targeting the 16S rRNA gene. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene...... 77

Figure 21. DGGE profiles of the lyophilized samples grown in 284 Gluc-, COC-, SW-medium, targeting the 16S rRNA gene. Bands #16N4 and #16N5 are located in the red square, band #1607 in the yellow square, #1618 in the green square, and #1619 in the blue square. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene...... 78

Figure 22. DGGE profiles of the lyophilized samples grown in 1-2-3 medium, targeting the 16S rRNA gene. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene...... 79

- Figure 23. DGGE profile of frozen samples grown in SW medium and NO2 medium and amplified with 16S rRNA-targeting primers. The red square indicates the presence of Neu-associated bands in samples N0 and N1. 80

Figure 24. DGGE migration profile of the frozen uncultured samples run together with pure cultures isolated from sample N4 grown on NMM solid medium. L = VITO ladder, N = N. europaea (Neu) 16S rRNA gene. .... 82

Figure 25. DGGE profile of five CIII isolates targeting the amoA-gene using the optimized amoA-1F* primer. L = VITO ladder, N = N. europaea (Neu) amoA genes...... 82

Figure 26. Agarose gel electrophoresis of the PCR-based replicon-specific plasmid detection using the IncP (a) oriT- and trfA2-targeting and (b) traG-targeting primers on lyophilized samples grown in 284 Gluc medium, 869 + - medium, and PCM. N0-N7: frozen samples, C : positive control, C : negative control, L: 100 bp ladder, L: 1kb ladder...... 86

List of tables Table 1. A list of completely sequenced microbial genomes with nitrogen cycle pathwaysa ...... 23

Table 2. Cellular functions possibly encoded by plasmids; adapted (Frost, 2000)...... 42

Table 3. Used strains ...... 49

Table 4. Plasmids used as positive control for the Inc-group PCR (Couturier et al., 1988) and for the exogenous plasmid isolation (Bossus, 2005)...... 50

Table 5. Overview of all used antibiotics and their properties (L. Janssen, pers. comm.)...... 53

Table 6. Primers used for the amplification of broad host range IncQ, IncP, IncN en IncQ plasmids...... 57

Table 7. Primers used for the DGGE analysis...... 58

Table 8. Primers used for the DNA sequencing ...... 61

Table 9. Media used for culturing specific subfraction of bacteria residing in the nitrifying reactor (CIII) of MELiSSA...... 66

Table 10. Features of the isolates that were obtained through plating...... 83

Table 11. Predicted importance of organisms in the CIII pilot plant of MELiSSA regarding ammonia oxidation 84

Table 12. Replicon-specific primers and optimisations...... 85

Table 13. Screening of bioreactor samples for growth on a range of solid media and for presence of antimicrobial resistances...... 87

Abstract

After a test trial of four years at the Free University of Barcelona (Spain), the MELiSSA CIII packed-bed pilot plant proved to be a functionally stable nitrifying bioreactor by efficiently converting high loads of ammonia to nitrate. The packed-bed bioreactor was dismantled and biomass stored as lyophilized and frozen samples originating from eight positions along the vertical feed-axis of the bioreactor. The nitrifying activity of the reactivated samples was tested using a colorimetric indophenol ammonia analysis; the bacterial community was analyzed using a PCR-based Denaturing Gradient Gel Electrophoresis (DGGE) approach, targeting both 16S rRNA- and ammonia monooxygenase (amoA)-genes in lyophilized and frozen samples, reactivated lyophilized samples, and on cDNA originating from reactivated frozen samples. The presence of mobilizing plasmids was screened using a PCR-based IncP- specific approach and a qualitative exogenous mobilizing plasmid isolation approach, based on replica mating. The indophenol ammonia analysis demonstrated the absence of nitrifying activity in seven of the eight inoculated lyophilized samples, demonstrating a sensitivity of ammonia-oxidizing bacteria due to the lyophilization process. The frozen samples showed normal nitrifying activity. DGGE analysis on lyophilized samples demonstrated the CIII bioreactor community to be enriched in diversity, and yielded complex community profiles. Eight isolates were obtained from the nitrifying lyophilized sample and six were identified. Both gram-positive as gram-negatives were detected in the bioreactor, among which some might have a stabilizing effect on the functionally stable reactor. Approximately six organisms were observed to be active in ammonia-rich and nitrite-rich media. Surprisingly, no N. europaea was detected on cDNA of ammonia-enriched medium cultures, despite the observed nitrifying activity in the ammonia analysis. The PCR-based screening for IncP- plasmids demonstrated that nearly no IncP-plasmids were present, and no traG-harbouring plasmids were observed. No transconjugants could be detected with the qualitative triparental exogenous plasmid isolation, and as of yet no mobilizing plasmids could be detected in the bioreactor. The results indicate that the CIII nitrifying packed-bed pilot plant was a functionally stable ammonia-converting reactor, with high bacterial diversity and nearly devoid of mobilizing-plasmid-harbouring organisms.

Samenvatting Na een proefperiode van vier jaar aan de Vrije Universiteit van Barcelona (Spanje), bleek de nitrificerende MELiSSA CIII biofilm reactor functioneel stabiel te zijn, door gedurende de gehele proefperiode grote hoeveelheden ammoniak om te zetten tot nitraat. De bioreactor werd ontmanteld, de biomassa werd in acht delen verdeeld over de verticale as van de reactor, gelyofiliseerd en ingevroren. De nitrificerende activiteit van de gereactiveerde stalen werd getest gebruikmakend van een colorimetrische indofenol ammoniak analyse; de bacteriële gemeenschap werd geanalyseerd met Denaturing Gradient Gel Electrophoresis (DGGE) door specifieke amplificatie van het 16S rRNA- en het functionele ammoniak monooxygenase (amoA)-gen in gelyofiliseerde en ingevroren stalen, gereactiveerde gelyofiliseerde stalen, en op cDNA afkomstig van gereactiveerde ingevroren stalen. De aanwezigheid van mobiliserende plasmiden werd onderzocht door middel van IncP-plasmide specifieke PCR en een kwalitatieve exogene mobiliserende plasmide isolatie methode, gebaseerd op replica mating. De indofenol ammoniak analyse toonde een afwezigheid aan van nitrificatie in zeven van de acht geinoculeerde gelyofiliseerde stalen, wat wees op een gevoeligheid van ammoniakoxiderende bacteriën voor het vriesdroog proces. De ingevroren stalen vertoonden allemaal een normale nitrificerende activiteit. De DGGE analyse van gelyofiliseerde stalen wees op een aangerijkte diversiteit in de CIII bioreactor na de proefperiode van vier jaar, en toonde een complexe bacteriële gemeenschap. Acht pure culturen werden geïsoleerd uit het gereactiveerde nitrificerende staal en zes daarvan konden worden geïdentificeerd. Zowel gram-positieve als gram-negatieve bacteriën werden gedetecteerd in de bioreactor, waarvan sommige een mogelijke stabiliserende werking konden gehad hebben op de functionaliteit van de reactor. Zes organismen bleken actief te zijn in ammoniakrijk en nitrietrijk medium, maar N. europaea werd niet onder de actieve bacteriën geobserveerd. De PCR-screening naar IncP- plasmiden toonde aan dat er vrijwel geen IncP-, en geen traG-dragende plasmiden aanwezig zijn in de gereactiveerde gelyofiliseerde bioreactor stalen. Met de kwalitatieve triparentale exogene plasmide isolatiemethode werden geen transconjuganten gedetecteerd in de stalen afkomstig uit de bioreactor. De resultaten tonen aan dat de nitrificerende CIII biofilm reactor een functioneel stabiele reactor was, met een grote microbiële diversiteit die vrijwel geen mobiliserende plasmide-dragende organismen bevatte.

Abbreviations ADRA Amplified ribosomal DNA Restriction Analysis ALS Advanced Life Support System AMO Ammonia MonoOxygenase amoA Functional ammonia monooxygenase gene AOB Ammonia-Oxidizing Bacteria ATCC American Type Culture Collection BAS Biofilm Airlift Suspension BLSS Biological Life Support System bp basepair

CX Compartment X of the MELiSSA loop Cd Cadmium cDNA complementary DNA CEEF Closed Ecology Experiment Facility CELSS Closed Ecological Life Support System CES Closed Ecological System Co Cobalt COC Co-Culture DGGE Denaturing Gradient Gel Electrophoresis DNA DeoxyriboNucleic Acid ESA European Space Agency FISH Fluorescent in situ Hybridization Gluc Gluconate HAO HydroylAmine Oxidoreductase HGT Horizontal Gene Transfer Inc Incompatibility Km Kanamycin MELiSSA Micro-Ecological Life Support System Alternative MMO Methane MonoOxygenase MPN Most Probable Number

NX Sample originating from position X in the CIII bioreactor Nal Naladixic acid napA nitrate reductase gene NASA National Aeronautics and Space Administration

Neu Nitrosomonas europaea Ni Nickel n.k. not known NMM Nitrosomonas Minimal Medium NOB Nitrite-Oxidizing Bacteria PCM Purity Check Medium PCR Polymerase Chain Reaction PP Parallel Plater Rcf Relative centrifugal force Rif Rifampicin RISA rRNA Intergenic Spacer Analysis Rpm Rounds per minute RNA RiboNucleic Acid rRNA ribosomal RNA Sm Streptomycin SSCP Single Strand Conformation Polymorphism SW Skinner & Walker Te Tetracycline TGGE Thermal Gradient Gel Electrophoresis T-RFLP Terminal-Restriction Fragment Length Polymorphism UAB Universitat Autonoma de Barcelona UCBR Ultra-Compact Biofilm Reactor UPGMA Unweighted Pair Group Method with Arithmetic Mean VITO Vlaamse Instelling voor Technologisch Onderzoek WHO World Health Organisation

13 Introduction A. Introduction The 'Micro-Ecological Life Support System Alternative' (MELiSSA) is a project conceived for long-term missions providing a bioregenerative self-sustainable living environment for the crew, irrespective of the destination or location, i.e. Earth or beyond. This bioregenerative life-support system comprises several functions, ranging from food delivery and waste recycling, to air revitalisation; and is composed of five interlinked compartments, of which four are microbial bioreactors. Each reactor performs specific bioconversions (hydrolization, mineralization, nitrification, oxygen production) and with all reactors interlinked, the MELiSSA will enable the support of the crew for a period of several years.

When a regenerative Biological Life Support System (BLSS), such as the MELiSSA, is used, it will occupy an important position during the mission. A range of problems comes to mind that will have to be studied thoroughly before such a system can be dependable. Several of these complications are innate to microbial organisms and since they cannot be circumvented, countermeasures should be developed to avoid risks at all costs. These complications are e.g. horizontal gene transfer of virulence genes to non-pathogenic strains, the stable (involuntary) integration of a pathogenic or a process-inhibiting organism in one of the bioreactors, and mutations in the MELiSSA strains due to cosmic radiation or simply by long time culturing.

This thesis reports on the molecular analysis and characterization of the nitrifying consortium of the MELiSSA loop bioreactor after a run of four years at the Free University of Barcelona (UAB) under supervision of the team of Dr. F. Gòdia. The nitrifying reactor was inoculated with a Nitrosomonas europaea ATCC 19718 and Nitrobacter winogradskyi ATCC 25391-co- culture and was fed with sterilized ammonium-containing synthetic medium, optimally composed to allow growth of the N. europaea-N. winogradskyi-co-culture. Microscopic investigation after three years revealed the presence of other organisms; the two originally inoculated bacteria (N. europaea and N. winogradskyi), however, were still present in large numbers. With the addition of other microbial species, the complexity of the system is increased. As a result, the monitoring of genetic stability has been complicated. Because of the bioreactor's stability during four years and because all previous attempts to successfully cultivate N. europaea and N. winogradskyi axenically together failed; the pilot reactor at UAB has presented itself as an important object to investigate the composition of the bacterial community of a stable nitrifying reactor which will be fed with a high ammonia load. The

14 Introduction reactor at UAB could be a model reactor for the improvement of the concept of Compartment III in the MELiSSA, which will result in a optimally performing nitrifying reactor.

The goal of this thesis was to analyse the nitrifying performance, characterize the microbial population and assess the genetic safety (by possible dissemination of plasmids carrying undesired genes) of a stable nitrifying biofilm; by respectively investigating (i) the nitrification rate using colorimetric ammonia and nitrite analyses, (ii) the microbial community using PCR-based detection coupled with denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA- and the functional ammonia monooxygenase (amoA)-gene in whole-community, community isolates and cultured fractions, and (iii) the ability to perform horizontal gene transfer in controlled conditions as a community using an exogenous plasmid isolation procedure, and in parallel a PCR-based plasmid detection. The DGGE analysis, was followed by clone screening and sequencing (targeting the 16S rRNA-gene) to identify various strains and determine their possible role in the reactor (necessary, detrimental, opportunistic, or supportive). The present study, therefore, gives a first glimpse on the stability and usefulness of this community and its constituents within the MELiSSA system.

The section on the 'State of Knowledge' is divided into four parts. It will offer the reader a synthesis of the MELiSSA project (PART I, chapter 1), provide the necessary background information on the role of nitrification in the nitrogen cycle (PART II, chapter 1), on nitrifying organisms in the CIII bioreactor (PART II, chapter 2). Subsequently, it will address previous microbiological studies performed on nitrifying reactors (PART III, chapter 1) and how such studies were performed (PART III, chapter 2). Furthermore, some aspects concerning horizontal gene transfer and its detection through exogenous plasmid isolation are reviewed in a final chapter (PART III, chapter 1 and 2, respectively).

'Material and methods' comprises the experimental and technical aspects of this work. The 'Results' are described in the following section where after the main observations are reviewed and linked with the literature in the section, 'Discussion'.

The final section of this thesis gives a summary of future work on this subject.

15 State of knowledge B. State of knowledge PART I: LIFE SUPPORT 1. Life support systems 1.1. General introduction In 1985, the European Space Agency (ESA) started developing life support technology, driven by the desire to play an important role in the up-coming space station era. ESA decided to participate in interplanetary missions, involving long distances, where delivery of water, food and oxygen from Earth is no longer feasible, neither technically or economically. Therefore, life support systems must as much as possible be regenerative (Gòdia et al., 2002), making the crew largely independent of Earth. These life-support activities consist mainly of air revitalization, air quality control and monitoring, water management, waste management and food production (Tamponnet & Savage, 1994).

Since 1961 a range of bioregenerative testbeds have been investigated. The simplest testbeds consisted of two compartments with gas exchange between CO2 and O2 producing organisms (BIOS 1-2). More advanced testbeds additionally guaranteed biological water purification and food production in BIOS 3 and in the US Closed Ecological Life Support System (CELSS). In addition, the complete Earth-based ecological system for water recovery, plant growth and domestic animal cultivation comprised the Biospheres and the Japanese Closed Ecology Experiment Facility (CEEF)(Eckhart, 1996). Similar to NASA's Advanced Life Support (ALS), MELiSSA is a regenerative system, containing biological elements that satisfy the need for air revitalisation, water regeneration, and solid waste reclamation, and closes the food cycle (Sakano et al., 2002; Pérez et al., 2004). To date, several studies are performed to guarantee MELiSSA's performance, to determine its safety and reliability, and furthermore, improve the recycling yield. In addition, specific spatial requirements (that determine MELiSSA's weight and size) must be met, while retaining the appropriate energy in- and output needed to sustain a crew of five (Fulget et al., 1999).

1.2. Micro-Ecological Life-Support System Alternative (MELiSSA) The MELiSSA is a self-regenerative life-support system for manned space missions based on an ecosystem containing microorganisms, higher plants and a human crew. In addition, the MELiSSA can help to gain understanding of the behaviour of closed artificial ecosystems and can help drive the development of the technology needed for construction of a BLSS for long- term manned space missions, extraterrestrial settlements or even Antarctic bases (Mergeay et

16 State of knowledge al., 1988). This life-support system will be active for nearly the whole duration of the mission (i.e. several years for a mission to Mars), during which it will provide a food source for the crew, degrade biological waste, and revitalize the atmosphere. MELiSSA will thus provide several essential aspects needed to sustain life. Its proper functioning must never become compromised.

Figure 1. General concept of the MELiSSA loop and the interactions between the different compartments (Pérez et al., 2004).

The objective of the MELiSSA is the production of edible biomass in the form of algae and higher plants. These photosynthetic organisms grow on the effluent flowing from the previous bioreactors, and are thus cultured until ready for consumption. In addition, both plants and cyanobacteria are able to revitalize the atmosphere by consuming the excess carbon dioxide and by producing oxygen during photosynthesis.

MELiSSA is a closed loop of five interconnected compartments (Figure 1) and is driven by human waste (faeces, urine, carbon dioxide and several minerals). Each compartment represents an important step in the bioconversion of wastes into reusable material and every compartment can be considered an independent entity performing specific biotransformations.

The liquefying compartment (i.e. the first compartment, CI) is responsible for the biodegradation of the raw waste material and faeces generated by the crew. The CI- thermophilic anaerobic bacteria generate volatile fatty acids, ammonia and carbon dioxide produced during anaerobic fermentation. The species performing this transformation are a

17 State of knowledge mixed culture of various strains isolated from human intestinal flora. The CI-effluent is fed to Rhodospirillum rubrum, which is the anaerobic photoheterotrophic bacterium of the second compartment (CII). In this bioreactor, the compounds formed in CI are mineralized. The third compartment (CIII) is a nitrifying compartment which is responsable for the nitrogen transformation from ammonia (mainly originating from urea) into nitrate, via nitrite by Nitrosomonas europaea and Nitrobacter winogradskyi. Nitrate is a highly soluble form of nitrogen and is the nitrogen source for the final and fourth compartment, the 'oxygen and food producing' compartment. The fourth compartment comprises both algae (CIva; Spirulina platensis) and higher plants (CIVb; wheat, soybean, potato, lettuce, rice, onion, spinach and tomato as candidate crops) and consumes the carbon dioxide produced in compartments CI,

CII and the CO2 produced by the crew itself. The oxygen produced during the photosynthetic carbon fixation can be used to drive the oxidation of ammonia and deliver the necessary oxygen for the crew (Savage et al., 2001).

Due to the MELiSSA system's complexity, a range of problems comes to mind that might undermine its functioning. These problems originate from four different angles, (i) engineering problems (e.g. technical failures, defect control systems, small reactor volumes) causing a reduced reactor efficiency, (ii) problems innate to the MELiSSA's biological components (e.g. genomic evolution, contamination, horizontal gene transfer), (iii) problems caused by consumer waste (e.g. pathogens, viruses, pharmaceuticals), and (iv) problems caused by external physical parameters (e.g. cosmic radiation, microgravity, vibration, acceleration)(Hendrickx et al., accepted for publication). These problems need to be resolved before the MELiSSA system can become dependable.

The nitrifying reactor was first run at bench-scale (total volume: 0.62 dm³) and later scaled-up to pilot plant scale (total volume: 8.1 dm³). This work presents research performed on samples originating from the nitrifying packed-bed pilot reactor (Figure 2) that has run for four years at the Free University of Barcelona (UAB), Spain (Gòdia et al., 2002; Pérez et al., 2004).

The CIII reactor was inoculated with a co-culture of N. europaea (ATCC 19718) and N. winogradskyi Agilis (ATCC 25391). Both members of the co-culture are very slow growing; therefore, the risk of wash-out is significant when growing these nitrifying bacteria in a reactor. To prevent wash-out, both species were immobilized on Biostyr® (expanded polystyrene) beads with an average diameter of 4.1 mm. Both N. europaea and N. winogradskyi show a natural tendency towards immobilization. Initially the cells attach to the

18 State of knowledge polystyrene surface because the micro-roughness of the beads protects the cells against shear- stress effects produced by liquid and gas flows. Because of the beads and the attached bacteria, the pilot reactor had a solid volume of approximately 3.9 dm³ (48% of total reactor volume) and a specific surface area of 705 m² m-3 relative to the total reactor volume. The increasing biofilm thickness lead to mass transfer limitations of nutrients in and out of the biofilm matrix, and biofilm development was found to be heterogeneous along the vertical axis of the reactor (Pérez et al., 2004).

(a) (b)

Figure 2. Schematic view (a) and picture (b) of the nitrifying packed-bed pilot plant at UAB. (1) packed- bed section with immobilized culture, (2) bottom section for aeration, liquid distribution and instrumentation, (3) top section for gas disengagement, (4) gas sparger, (5) gas exit condenser, (6) gas closed loop, connected to controlled oxygen/nitrogen supply to control oxygen level, (7) liquid feed, (8) liquid recirculation, (9) liquid outlet, (10) acid addition, (11) base addition, (12) T° probes, (13) oxygen probes, (14) pH probes, (15) cold finger, (16) hot finger (Gòdia et al., 2004).

19 State of knowledge PART II: NITROGEN RECYCLING IN NATURAL AND ENGINEERED SYSTEMS 1. Nitrogen and microorganisms 1.1. Introduction Nitrogen can be considered an ambiguous element. On the one hand, nitrogen is an essential element, is present in lots of essential biomolecules, and can determine growth rate; on the other hand, nitrogen can be over-accessible to these organisms in certain environments and subsequently cause serious environmental problems (Taiz & Zeiger, 2002). Nitrogen pollution is one of the causative agents of the eutrophication of water bodies (e.g. lakes). The need for wastewater treatment and bioremediation processes has long been acknowledged to solve the problems of nitrogen pollution by both fertilizer and industry. The high ammonia- and nitrite loads in these lakes can be eliminated by nitrification and subsequent denitrification, causing the nitrogen to be transformed to atmospheric nitrogen, subsequently reducing the nutrient load of the lake (Painter, 1986). These biotransformations are therefore important components of wastewater treatment plants, bioremediation projects and are an integral part of bioreactors (Ye & Thomas, 2001).

1.2. Nitrogen cycle

Nitrogen is an element that is abundant in both the atmosphere (with 78% N2) and ground (soil and aquatic systems). For the most part, the gaseous reservoir of nitrogen is not available to living organisms. Whereas, in terrestrial and aquatic ecosystems nitrogen occurs in a wide range of compounds and is readily available, irrespective of pH (Taiz & Zeiger, 2002). The biogeochemical nitrogen cycle (Figure 3) is a complicated network comprising atmospheric, biological and industrial nitrogen fixation; nitrification (ammonia-oxidation to nitrite and nitrite-oxidation to nitrate), anammox (anaerobic ammonia oxidation), denitrification (bioconversion of soluble nitrogen compounds to gaseous nitrogen compounds), immobilization by bacteria and fungi (microbial absorption and assimilation of ammonia or nitrate); ammonification (mineralization), plant acquisition, volatilization, ammonia fixation, and nitrate leaching. Furthermore, nitrogen is able to ascend the food chain through feeding and predation in the form of biomolecules (Taiz & Zeiger, 2002). Several nitrogen pathways are highly dependant on microbial activities (Hovanec & DeLong, 1996). The corresponding transformations are presented below.

20 State of knowledge

Figure 3. The biogeochemical nitrogen cycle and its comprising transformations (Pidwirny, 2004)

The microbial nitrogen cycle (Figure 4) comprises nitrogen fixation, nitrification, anammox, denitrification, and immobilization; and contains all known biological conversions of nitrogen compounds performed by microorganisms. Nitrogen can be both energy source and nitrogen source for microorganisms by incorporating nitrogen in biomass (Figure 4).

Figure 4. The microbial nitrogen cycle (Ye & Thomas, 2001). The anammox reaction is not depicted on this figure. Immobilization is the incorporation of ammonia into biomass.

21 State of knowledge Nitrogen fixation can occur in association with plants (by Rhizobium sp., Frankia sp., Nostoc sp., Anabaena sp., Acetobacter sp.) and as free-living bacteria (by e.g. Nostoc sp., Anabaena sp., Azospirillum sp., Bacillus sp., Clostridium sp., and Rhodospirillum sp.). Biological nitrogen fixation (through action of nitrogenase) accounts for the most of the overall nitrogen fixation, converting atmospheric nitrogen in ammonia. Biological nitrogen fixation thus is a key process in the biogeochemical nitrogen cycle (Taiz & Zeiger, 2002). N. europaea and other ammonia oxidizing bacteria (AOB) participate together with N. winogradskyi and other nitrite oxidizing bacteria (NOB) in the biogeochemical nitrogen cycle in a process that is termed nitrification (the biological conversion of reduced nitrogen in the + form of ammonia [NH3] of ammonium [NH4 ] to nitrite and nitrate). Nitrifiers (AOB plus NOB) thus significantly increase the nitrogen accessibility for higher plants, and are of enormous interest in the treatment of polluted soils and have a potential use in bioremediation of e.g. hydrocarbon-polluted sites (Deni & Penninckx, 1999). Anammox is a recently identified pathway performed by a lithoautotrophic planctomycete (member of the order Planctomycetales). The recently discovered planctomycete is provisionally classified (based on 16S rRNA-gene sequences) as Candidatus Kuenenia stuttgartiensis, and constitutes the dominant fraction of the biofilm of which it was extracted. This novel bacterium grows extremely slowly, dividing only once every two weeks, and was reported to have a novel hydroxylamine oxidoreductase (Strous et al., 1999). Anammox is an alternative way of oxidizing ammonia to nitrate, in which ammonia and nitrite are anaerobically converted to dinitrogen gas. Although the natural occurrence of anammox bacteria is still relatively obscure, they have been detected at quite different locations (Jetten et al., 1998; Hellinga et al., 1999; Schmid et al., 2000; Devol, 2003; Kuypers et al., 2003). What is more, the anammox reaction might provide an alternative for the nitrogen removal in wastewater treatment systems (Strous et al., 1999; Ye & Thomas, 2001). Denitrification has been observed for a wide range of microorganisms. The presence of nitrate reductase was reported in Pseudomonas fluorescens YT101, Pseudomonas sp. G-179, Pseudomonas isachenkovii, and Rhodobacter sphaeroides f. sp. denitrificans. The dissimilatory reduction of nitrate to gaseous forms of nitrogen is commonly carried out by either a membrane-bound nitrate reductase or a periplasmic nitrate reductase. The role of these two types of enzymes in the denitrification process varies, depending on the organism. In addition to the two types of nitrate reductase, two types of nitrite reductase exist; that catalyze the conversion of nitrite to nitric oxide. One type is the cytochrome cd1 nitrite

22 State of knowledge reductase, and the other type is the copper-containing nitrite reductase, both appear to be present in Methylomonas sp. strain 16a. In addition, several types of nitric oxide reductase exists, which have been found in several Neisseria species, Synechocystis sp. PCC6803, Ralstonia eutrophus, and Methylomonas sp. strain 16a. In the some Fungi, the enzyme nitric oxide reductase is present as well, e.g. Fusarium oxysporum. Denitrification is a process in which nitrate is converted to nitric oxide, nitrous oxide or dinitrogen gas, and allows microbes to gain energy under oxygen-limiting conditions. The gasses formed during denitrification, NO and N2O, are 'green house gasses', while the soluble forms of oxidized nitrogen compounds readily flow to ground- and surface waters (Ye & Thomas, 2001). Furthermore, it is interesting to mention that investigation of the anaerobic metabolism of Bacillus subtilis has shown that the bacterium can perform dissimilatory nitrate reduction to ammonia, via nitrite. B. subtilis thus uses nitrate or nitrite as electron acceptors to support anaerobic growth (Tiedje, 1988). It has been shown that ammonia oxidizers can switch from nitrification to oxygen-limited autotrophic nitrification-denitrification (OLAND) in order to survive and maintain active while residing in oxygen-limited sediments (Kuai & Verstraete, 1998). During OLAND the cells oxidize ammonia to nitrite, which is subsequently used as an e-acceptor to form dinitrogen gas. The production of dinitrogen gas has an additional advantage, since it can be used as a means of transport out of the sediment (up the water column) into an aerobic zone, which is favorable for nitrification (Philips et al., 2002).

Table 1. A list of completely sequenced microbial genomes with nitrogen cycle pathwaysa (Ye & Thomas, 2001) Species Pathway or enzymes Nitrosomonas europaea Ammonia oxidation Dissimilatory nitrite and nitric oxide reductases Methylomonas sp. 16a Ammonia oxidation Dissimilatory nitrite and nitric oxide reductases Neisseria menigitidis Dissimilatory nitrite and nitric oxide reductases Synechocystis sp. PCC6803 Cytochrome b nitric oxide reductase Bacillus subtillis strain 168 Dissimilatory nitrate reduction to ammonia Pseudomonas aeruginosa PAO Denitrification Rhodobacter sphaeroides Denitrification Nitrogen fixation Paracoccus denitrificans ATCC 19367 Denitrification Heterotrophic nitrification P. denitrificans SANVA100 Denitrification Azoarcus tolulyticus Tol-4 Denitrification aSituation in 2001

The existence of dissimilatory nitrite and nitric oxide reductases in nondenitrifying bacteria, as revealed by genome sequences (Table 1), indicates a broader physiological role for these

23 State of knowledge enzymes, which could range from anaerobic metabolism to detoxification (Ye & Thomas, 2001).

1.3. Aerobic nitrification 1.3.1. Autotrophic nitrification Nitrification occurs in a wide range of environments, such as soils (Deni & Penninckx, 1999), ocean water, freshwater lakes (Solorzano, 1969), wastewater (Grunditz & Dalhammar, 2001), and aquaria (Hovanec & DeLong, 1996); and is mainly due to autotrophic bacteria. The MELiSSA-project needs to apply autotrophic nitrification because (i) Rhodospirillum rubrum mineralizes all organic carbon sources and, (ii) by depleting all organic carbon sources specificity and selectivity is improved, lowering contamination risk in CIII by heterotrophic organisms. It is interesting to note that many AOB have either a whole or partial denitrification pathway (Table 1). For example, N. europaea appears to possess the copper-type dissimilatory nitrite and cytochrome bc1 nitric oxide reductase, but not the dissimilatory nitrate and nitrous oxide reductases (Whittaker et al., 2000). These findings might have repercussions for the

MELiSSA CIII bioreactor and its ammonia conversion efficiency.

1.3.2. Classification of autotrophic nitrifying bacteria In freshwater systems, the bacterial genera responsible for the oxidation of ammonia and nitrite are presumed to be predominantly the genera Nitrosomonas and Nitrobacter, resp.; both of which belong to the class Proteobacteria (Wheaton et al., 1994). More recent studies, based on 16S rRNA sequence analysis, show that (i) all known autotrophic AOB of the β- Proteobacteria occurring in terrestrial environments belong to a monophyletic lineage represented by members of only two genera: Nitrosomonas spp. and Nitrosospira spp. (Briones et al., 2002), and (ii) ammonia-oxidizing and nitrite-oxidizing bacteria belong to separate lineages within the Proteobacteria (Figure 5). Autotrophic AOB bacteria form a distinct group within the β-Proteobacteria, and are affiliated with the iron-oxidizing bacterium Gallionella ferruginea and the photosynthetic bacterium Rhodocyclus purpureus (Hovanec & DeLong, 1996); with the exception of the autotrophic AOB bacterium Nitrosococcus oceanus, which is a marine species that belongs to the γ-proteobacterial lineage. Chemolithotrophic NOB appear to be phylogenetically widespread in the class of Proteobacteria, occurring in the α-, δ-, and γ-clades (Nitrobacter, Nitrospina, and Nitrococcus, respectively). Phylogenetic analyses of the α-proteobacterial lineage have shown

24 State of knowledge that Nitrobacter winogradskyi is most closely related to Bradyrhizobium japonicum and Rhodopseudomonas palustris (Figure 5) (Gibson et al., 1980; Orso et al., 1994).

Figure 5. The phylogenetic relationship between autotrophic among AOB and NOB (both indicated in bold) (Teske et al., 1994). AOB are found in both β-Proteobacteria and γ-Proteobacteria, where NOB are found in α-Proteobacteria, γ-Proteobacteria and δ-Proteobacteria.

PCR primers targeting the whole nitrite-oxidizing bacteria (NOB) group are not available at present; but 16S rRNA-gene-targeting primers, targeting specific genera, have been used instead (Cébron et al., 2003). In addition to the 16S rRNA-gene approach, a functional gene- based PCR assay has been developed to assess the diversity of naturally occurring ammonia- oxidizing communities (Rotthauwe et al., 1997). This approach targets the 491-bp sequence of the amoA-gene which encodes the active-site of the ammonia monooxygenase enzyme (encoded by the amoCAB-operon) (McTavish et al., 1993). The functional amoA gene can be a basis of taxonomy alike, yielding similar, although not identical, evolutionary relationships (Rotthauwe et al., 1997; Stephen et al., 1999; Purkhold et al., 2000; Purkhold et al., 2003). In addition, Rotthauwe et al. (1997) were able to obtain 25 different partial amoA gene sequences from various environmental and culture strains, which have resulted in the generation of a database, which Purkhold et al. (2003) subsequently updated with 12 novel betaproteobacterial sequences.

25 State of knowledge 1.3.3. The sensitiveness of the AMO enzyme The ammonia-removing process is particularly susceptible to inhibition by a wide range of (mostly chemical) compounds at low concentrations (in the order of several micromolar). When nitrification is inhibited, generally it is because the motive enzyme, ammonia monooxygenase (AMO) is inhibited. AMO is a membrane-bound protein that mediates the conversion of ammonia to hydroxylamine, which is the first step in the nitrification reaction, and is relatively easy inhibited (Hooper & Terry, 1973). First, because AMO contains copper in its active site; the presence of metal binders or chelating agents will reversibly inhibit its activity, e.g. thiourea and allylthiourea through affinity inhibition (Hooper & Terry, 1973). Both products bind a range metals but have a high affinity for copper (Cu), the co-factor of the AMO enzyme. Another method of AMO-inhibition is competitive inhibition. In view of the fact that AMO is capable of oxidizing a wide range of reduced compounds, such as sulphur, aliphatic, aromatic and halogenated molecules as alternate substrates, AMO is readily inhibited; leaving only a trace activity to oxidize ammonia. These forms of inhibition must thus be easily and sensitively traced (Ilzumi et al., 1998), specifically in wastewater treatment plants. Finally, AMO can be inhibited irreversibly by a wide range of compounds, such as acetylene, that acts as a suicide substrate for many monooxygenases (McTavish et al., 1993).

The ammonia monooxygenase (amoCAB) operon seems to have a high degree of sequence similarity to the methane monooxygenase enzyme (MMO). MMO appears to exist in a cytoplasmic form in addition to a membrane-bound form in contrast to AMO, of which only a membrane-bound form has been observed. Both AMO and MMO have low substrate specificity, they can both degrade many carbohydrates and halogenated carbohydrates next to ammonia and methane. In addition, they both show a similar inhibitor profile (McTavish et al., 1993).

1.3.4. Heterotrophic nitrification Heterotrophic nitrification was first reported in 1894 for a fungus (Stutzer & Hartleb, 1894). Later, it was likewise thought to be restricted to old bacterial cultures and stationary phase cultures and to be only a minor component in the biogeochemical nitrogen cycle (Alexander, 1977). A wide range of organisms is capable of performing heterotrophic nitrification: bacteria (e.g. Alcaligenes faecalis, A. eutrophus strain TUD, Pseudomonas aeruginosa, several Bacillus species and Arthrobacter sp. (Papen et al., 1989; Ye & Thomas, 2001)); plus

26 State of knowledge several fungi (Lang & Jagnow, 1986). In case of heterotrophic nitrification, the process of - NO2 generation can be coupled to NO and N2O production (Papen et al., 1989), but it is never coupled to energy generation. Even though heterotrophic nitrification was thought to be of minor importance, it can occur in situ and even be the dominant nitrifying reaction in certain environments (Killham, 1986), like in acidic soils (Prosser, 1989), heath and conifer forest soils (Van de Dijk & Troelstra, 1980; Schimel et al., 1984).

27 State of knowledge

2. Nitrogen recycling in bioreactor CIII The bacterial genera performing the oxidation of ammonia to nitrate belong to the Nitrosomonadaceae and Bradyrhizobiaceae; Nitrosomonas and Nitrobacter respectively being the best studied organisms performing it (Wood, 1986; Bock et al., 1989; Brock & Madigan, 1991; Abeliovich, 1992). This biological process is carried out in two steps, the first being the oxidation of ammonia to nitrite by N. europaea, the second being the oxidation of nitrite to nitrate by Nitrobacter winogradskyi. Because the substrate consumption rate of Nitrosomonas is lower than of Nitrobacter, nitrification will be rate limited by ammonia oxidation (Mendum et al., 1999). Both are part of the nitrifying consortium (i.e. members of the biofilm) in the MELiSSA loop and are described in detail below.

2.1. Nitrosomonas europaea ATCC 19718 N. europaea is a motile gram-negative bacterium that extracts all its energy from the aerobic oxidation of ammonia to nitrite, which is the first step of the nitrification reaction (Figure 6). Ammonia monooxygenase (AMO) performs the oxidation of ammonia to hydroxylamine, and the periplasmic enzyme hydroxylamine oxidoreductase (HAO) performs the subsequent oxidation of hydroxylamine to nitrite (McTavish et al., 1993).

28 State of knowledge - + NH3 + O2 + 2e + 2H → NH2OH + H2O (AMO) - + - NH2OH + H2O → NO2 + 5H + 4e (HAO)

This bacterium can be found oxidizing ammonia to nitrite in several places such as soil, sewage, freshwater, the walls of buildings and on the surface of monuments especially in polluted areas where air contains high levels of nitrogen compounds.

N. europaea is able to grow in oxic and anoxic conditions with hydrogen and/or organic compounds such as organic acids, sugars and amino acids as electron donors and nitrite as the electron acceptor (Clark & Schmidt, 1966; Clark & Schmidt, 1967; Martiny & Koops, 1982; Bock et al., 1995; Hommes et al., 2003). However, the most important carbon source is carbon dioxide, which is obtained through fixation. Finally, only some mineral salts are needed to complete the metabolic need of this bacterium (Chain et al., 2003).

Grunditz and Dalhammar (2001) developed assays to detect nitrification inhibition and were able to characterise N. europaea with respect to temperature, pH and cell activity. Optimum temperature was 35°C, optimum pH was 8.1 and there seemed to be a significant (linear) relationship between the nitrification rate and the cell concentration in the studied interval.

It has been demonstrated that a solid phase (e.g. soil particles or Biostyr® beads) is not necessary for the growth of nitrifying bacteria in culture solution (Aleem & Alexander, 1958). However, when N. europaea cells are grown with a support to form a biofilm, nitric oxide (NO) appears to be a signal to induce biofilm formation (Schmidt et al., 2004).

Under ammonia-limited conditions, ammonia-assimilating heterotrophic bacteria and/or plant roots repress the ammonia-oxidizing activity of N. europaea (Verhagen & Laanbroek, 1991). In addition, Grunditz and Dalhammar (2001) observed a slight linear decline in cell activity when the harvested cell cultures were kept refrigerated in liquid media at +3°C before use in the bioassays. Whether nitrite has a stimulating or inhibiting effect on ammonia oxidation in N. europaea is still ambiguous (Stein & Arp, 1998; Laanbroek et al., 2002). However, nitrite can be reduced by N. europaea to NO by nitrite reductase (Whittaker et al., 2000), and previously it was demonstrated that NO indirectly stimulates aerobic ammonia oxidation by Nitrosomonas eutropha cells (Zart et al., 2000). N. europaea shows higher temperature

29 State of knowledge sensitivity than N. winogradskyi (Wijffels & Tramper, 1995). Therefore, if the temperature is too low, one can expect ammonia accumulation instead of nitrite accumulation. Cloned luciferase-encoding operons were transferred by conjugation to a natural Nitrosomonas sp. Thereby, conjugation was established as a tool for gene transfer into Nitrosomonas strains (Ludwig et al., 1999). It is interesting to remark that some Nitrosomonas europaea carry plasmids, one of them is the putative ParE plasmid, encoding a stabilization protein (Burall et al., 2004).

2.2. Nitrobacter winogradskyi Agilis ATCC 25391 Members of the genus Nitrobacter are characterised by their chemolithotrophic metabolism. They use the conversion of nitrite to nitrate as their sole source of energy for growth and cell synthesis (Figure 7).

In natural circumstances Nitrobacter sp. is aquatic (in rivers or on sediments), but can also be found terrestrially (e.g. humid agricultural soils). In these agricultural soils, a progressive accumulation of nitrogenous and food-waste solids is observed. These compounds are circulated continuously in a closed, aerated ditch below feeding cattle. Herein, Nitrobacter sp. lives of cattle waste.

Because Nitrobacter can use carbon dioxide as a carbon source they are classed as facultative autotrophic organisms (Nelson, 1931). However, Nitrobacter (α-Proteobacteria) can grow heterotrophically, while the remaining (known) NOB, Nitrospina (δ-Proteobacteria), Nitrococcus (γ-Proteobacteria), and Nitrospira (Nitrospira phylum) are unable to grow

30 State of knowledge heterotrophically, and are subsequently classed as obligate autotrophic bacteria (Ehrich et al., 1995). Nutritional studies have indicated that Nitrobacter requires iron, phosphorous and magnesium (Aleem & Alexander, 1960), calcium (Kingma-Boltjes, 1935) for growth. (Finnstein & Delwiche, 1965) discovered that molybdenum is an essential micronutrient; without it, Nitrobacter grows only with very low yields.

Grunditz and Dalhammar (2001) were able to characterize Nitrobacter winogradskyi with respect to temperature, pH and cell activity. Its optimum temperature was 38°C, optimum pH was 7.9 and there seemed to be a significant linear relationship between the nitrification rate and cell concentration. The cell activity decreased slightly with storage time.

Furthermore, it has been demonstrated that a solid phase in culture solution is not necessary for the growth of nitrifying bacteria and the activity of the nitrite-oxidizing bacteria can be easily followed by observing the disappearance of the substrate, the formation of nitrate or by the consumption of oxygen (Aleem & Alexander, 1958).

Various pesticides can affect the autotrophic nitrite-oxidizing metabolism (Stojanovic & Alexander, 1958). In addition, growth can be significantly inhibited by the ammonium ion in alkaline soils causing nitrite to accumulate (Stojanovic & Alexander, 1958), as well as by the accumulation of nitrate (Gould & Lees, 1960). In culture solutions, Nitrobacter can be inhibited by a range of chemical substances (Lees & Simpson, 1957). What is more, the reaction seems to be inhibited by low concentrations of cyanide and several other inhibitors like p-chloromercuribenzoate, iodoacetic acid and 2,4-dinitrophenol (Aleem & Alexander, 1958).

Nitrobacter was used in the MELiSSA nitrifying reactor because it is an excellent model organism for the ammonia-to-nitrate reaction (Fliermans et al., 1974). Furthermore, the choice of N. winogradskyi above Nitrobacter agilis seems logical because the former quickly outgrows the latter when growing both in a mixed culture (Fliermans et al., 1974) (Figure 8). N. winogradkyi is used as part of biological water detoxifying filters in wastewater treatment plants (Malhautier et al., 1998), in bioremediation projects (Deni & Penninckx, 1999) and in aquaria (Hovanec & DeLong, 1996) (as is N. europaea). Nitrobacter is however, not

31 State of knowledge commonly observed in wastewater treatment plants (Hovanec & DeLong, 1996; Wagner et al., 1996).

Figure 8. Response of N. agilis and N. winogradskyi to mixed culture conditions determined by studying their respective growth in co-culture conditions (Fliermans et al., 1974).

32 State of knowledge PART III: THE BACTERIAL COMMUNITY OF NITRIFYING REACTORS 1. Bacterial communities in engineered environments 1.1. General introduction Species richness (the number of species within a community) and species evenness (the relative sizes of species populations within a community) are two essential parameters for defining total community structure and diversity (Ward et al., 1990). The main quest is unravelling how diversity and dynamics of communities contribute to ecosystem stability. It is clear that there exists a parallel between maintaining process stability in natural and engineered environments (e.g. bioreactors). However, until recently several methodological limitations have hindered the experimental characterization of complex microbial communities in engineered and natural environments (Briones & Raskin, 2003). Long have cultivation-based analyses been the only means of analyzing communities, unravelling only a tiny fraction of the total microbial community. With the introduction of molecular tools, this situation changed completely, providing new insights into microbial ecology (Dabert et al., 2002). However, cultivation-based techniques remain an important aspect of microbial community analyses (Garland et al., 2001).

Engineered environments (such as bioreactors) offer a setting that is generally more manageable, in terms of studying ecological processes, than most natural systems. Studying these engineered systems might provide better ways to describe, predict, develop, and maintain Closed Ecological Systems (CES) stably. In addition, it can teach us how engineered systems can recover from unstable periods. Even though ecosystems are in a state of equilibrium, the individual populations, composing these ecosystems, are generally varying at the level of population size, even under stable-ecosystem conditions (Huisman & Weissing, 2002). However, this nonequilibrium behaviour of population size, in functionally stable conditions, is not always observed (LaPara et al., 2002). When a stable ecosystem comprises metabolically flexible populations, the size of these populations might remain relatively stable while maintaining a stable ecosystem. The functional (or metabolic) flexibility of a population may compensate the need for higher population diversity, and subsequently dampen the need for fluctuations in population size (Briones & Raskin, 2003). Population diversity alone does not drive ecosystem stability; what is more, the requisite is the presence of multiple pathways (functional redundancy) to achieve a metabolic product. Parallel processing of a substrate is associated to more stable ecosystems, in contrast to systems were serial processing of a substrate predominate. Ecosystem stability is thus

33 State of knowledge depending on functional redundancy, which is ensured by the presence of a reservoir of species able to fulfil the same ecological function (Peterson et al., 1998). Even though ecosystem stability is more important than the persistent presence of a species, however population persistence is perceived as the most important property for the former; because the population persistence is closely related to fitness of individuals, and it is the fitness parameters that provide the information needed to manage natural communities (Grimm et al., 1992).

1.2. Bacterial communities in nitrifying reactors and wastewater treatment plants 1.2.1. Community composition in nitrifying reactors and wastewater treatment plants A wide range of organisms performs the oxidation of ammonia in aquatic and terrestrial environments, wastewater treatment plants, and reactors alike (Table 2). Even though the composition of a bacterial community (in e.g. soils, plants, and reactors) is a product of its environment, a natural tendency in bacterial community composition might be observed, e.g. the recurrence of certain bacterial genera for a specific reaction in different environments. The main players expected in these habitats are autotrophic AOB and NOB, anammox bacteria, heterotrophic nitrifiers, and several opportunistic bacteria such as heterotrophic bacteria (Egli et al., 2003) that thrive on organic carbon that originates from extracellular polymers and from dead microbial cells (Burrell et al., 1998).

When designing a system for nitrogen removal several issues will have to be considered. From wastewater treatment systems it was observed that nitrification can be optimized when operating under a low or an organic carbon free environment (Lee et al., 2004). The presence of organic carbon stimulates the growth of heterotrophic organisms, which out-compete autotrophic nitrifiers due to their higher growth rate and biomass yields (Harremoes, 1982). The inhibitory effect of organic matter towards nitrification has been reported in airlift reactors such as the Biofilm Airlift Suspension (BAS) reactor (van Benthum et al., 1998) and Ultra-Compact Biofilm Reactor (UCBR) (Ong et al., 2003). As a result, the competition of heterotrophic organisms leads to a reduction in the specific nitrification activity of the nitrifying biofilm. Therefore, many developed nitrifying bioreactors operate under low organic carbon content or without organic carbon (Egli et al., 2003; Pérez et al., 2004). Using FISH analysis on communities grown on low carbon containing influent, Egli et al. (2003) observed that the main groups of microorganisms, constituting the biofilm, were AOB from the Nitrosomonas europaea/eutropha lineage (20-30%), anaerobic ammonia-oxidizing

34 State of knowledge bacteria of the "Candidatus Kuenenia stuttgartiensis" type (20-30%), filamentous bacteria from the phylum Bacteroidetes (7%), and NOB from the genus Nitrospira (<5%). In addition, through clone screening of the 16S rRNA-gene, Egli et al. (2003) discovered several limited populations of Sphingomonas sp. as well as several uncultured bacteria (Egli et al., 2003).

After a test period of 91 days, the NASA Advanced Life Support project's ammonia-oxidizing community within the bioreactor was analyzed, targetting 16S rRNA gene, amoA, and nitrous oxide reductase (nosZ, denitrifying bacteria). The community obtained through T-RFLP analysis consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. In addition, fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified. Temporal shifts in the species composition of total bacteria in ammonia-oxidizing and denitrifying bacteria in the TFB were also detected. Therefore, they suggested that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation (Sakano et al., 2002).

In wastewater treatment systems, the microbial community comprises only a small fraction of autotrophic bacteria; however, these autotrophs are responsible for the bulk of the nitrification (Randall, 1992). What is more, in several studies Nitrobacter could never be detected in wastewater treatment plants, even though it was present in sewage sludge (Wagner et al., 1996). Therefore, it was hypothesized that other bacteria were responsible for nitrite oxidation (Hovanec & DeLong, 1996; Wagner et al., 1996). Therefore, a study was performed on a nitrite-oxidizing bioreactor, where a batch reactor was inoculated with wastewater sludge (Burrell et al., 1998). Burell et al. (1998) observed 16 heterotrophs (6 gram positive and 10 gram negative) in the seed sludge by culture-dependent methods, but no autotrophic microorganisms. 16S rRNA-gene-based clone libraries revealed that the seed sludge comprised a complex microbial community. The microbial seed sludge community was dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G+C, gram-positive bacteria (10%). Four percent of their clones were closely related to the autotrophic NOB Nitrospira moscoviensis. After an aerated run of six months, fed only with mineral salts and nitrite, bacteria closely related to the autotrophic NOB N. moscoviensis (89%) dominated the nitrite-oxidizing bioreactor. Two clone sequences appeared to be closely related to the Nitrobacter-genus. Subsequently, Nitrospira strains are now hypothesized to be the (previously unknown) NOB in wastewater treatment systems, sewage sludge, aquaria, and

35 State of knowledge bioreactors (Burrell et al., 1998; Hovanec et al., 1998; Juretschko et al., 1998; Schramm et al., 1998).

Even though heterotrophic bacteria can inhibit nitrification (in case of low dissolved oxygen and substrate concentration) their presence can be advantageous. Because heterotrophs grow on the outer layer of the biofilm, and subsequently protect the autotrophs by protecting them from detachment (Furumai & Rittmann, 1994). Therefore, Nogueira et al. (2002) developed two nitrifying biofilm reactors, differing in hydraulic retention time. These nitrifying reactors were operated to allow pure nitrification (in an initial phase), and subsequently shifted to combined nitrification and organic carbon removal (of acetate) (Nogueira et al., 2002). No AOB belonging to the Nitrosospira-cluster were detected with 16S rRNA FISH probes, and while most AOB were most likely affiliated with the Nitrosomonas europaea/eutropha group, Nitrosococcus mobilis was absent. Consistent with other studies performed on other nitrifying bioreactors Nogueira et al. (2002) found Nitrospira-like bacteria to be the dominating population of NOB in the bioreactor. Members of the genus Nitrobacter were only detected (in small amounts) after the shift to operation with acetate addition (Nogueira et al., 2002). This led to the hypothesis that Nitrospira thrives at low nitrite concentrations, while Nitrobacter can compete successfully only in environments with relatively high nitrite concentrations (Schramm et al., 1999). Regarding the heterotrophs, Proteobacteria of the alpha-, beta- and gamma-subclasses and bacteria belonging to Cytophaga-Flavobacterium- cluster were detected in both reactors (Nogueira et al., 2002).

1.2.2. Spatial patterns in nitryifying reactors When analyzing a bioreactor (with a unidirectional feed and flow-through), several spatial patterns are to be expected. Egli et al. (2003) observed a vertical decreasing gradient of oxygen, bicarbonate, and ammonia in the biofilm, but also smaller horizontal gradients of nitrite, since the nitrite oxidizers population has to rely on the nitrite produced by the aerobic ammonia oxidizers. In addition to this pattern, the structural composition (Figure 9) of the biofilm correlated very nicely to other related biofilm systems (Helmer-Madhock et al., 2002) (Koch et al., 2000). Structural analysis of the biofilm demonstrated that the aerobic nitrifiers were at the top and the anammox bacteria in the lower layer of the biofilm.

36 State of knowledge

2. Analysis of bacterial communities 2.1. Approaches to bacterial community analysis Bacterial communities can be studied on two levels, organism-based detection (i.e. culture- dependant detection and antibody-based detection) and biomolecular detection (i.e. rRNA probes and PCR-based detection methods, etc.). Within the molecular approaches two different strategies can be used depending on whether or not the sequences of the bacteria are known (Dabert et al., 2002). The molecular probe approach includes Fluorescent in situ Hybridisation (FISH) and dot-blot hybridization, where the knowledge of the target-sequence is prerequisite for probe design. The category of molecular tools not requiring prerequisite knowledge of the microbial community being studied comprises Terminal-Restriction Fragment Length Polymorphism (T-RFLP), Amplified Ribosomal DNA Restriction Analysis (ADRA), Denaturing Gradient Gel Electrophoresis (DGGE), Thermal Gradient Gel Electrophoresis (TGGE), Single Strand Conformation Polymorphism (SSCP), and rRNA Intergenic Spacer Analysis (RISA) . The microbial community is displayed as a specific pattern of peaks (SSCP, T-RFLP), or as a specific pattern of bands (ADRA, T/DGGE, RISA) (Dabert et al., 2002). In addition, to observe the active (live) population of the microbial community, it is possible to target the whole population mRNA; by combining whole RNA extraction, reverse transcriptase PCR, followed by 16S rRNA-gene amplification (or of another arbitrary chosen gene). All previous methods targeted the bacteria on gene-level; however, recently, researchers are experimenting with analyses on a genomic level by means

37 State of knowledge of Amplified Fragment Length Polymorphism (AFLP). This approach yields equivalent results and was already successfully applied (Franklin & Mills, 2003; Franklin et al., 2005).

In natural samples, nitrifiers are commonly analysed using the most-probable-number (MPN) technique (Matulewich et al., 1975). The MPN procedure is used for statistical estimation of the numbers of chemoautotrophic nitrifiers present in the environment. But, the accuracy and sensitivity of the MPN for quantification of nitrifying bacteria is unknown, because no standard exists and this quantification provides no information on the nature of the bacteria, nor on the species diversity (Fliermans et al., 1974). Often, antibodies or rRNA-targeted oligonucleotide probes are used for in situ analyses in order to avoid the limitations of the most-probable-number technique. The problem with antibodies, however, is the limitation in serological diversity of cells living within the same ecosystem. Furthermore, the organisms need to be isolated prior to antibody development (Belser & Schmidt, 1978). This means that uncultivable nitrifiers are undetectable when using antibody-related detection. However, genus-specific monoclonal antibodies, which are used to probe nitrite oxidoreductase, can overcome the problem of serological diversity in the study of nitrite oxidizers (Bartosch et al., 1999).

2.2. Analysis by means of DGGE DGGE combines PCR with rRNA-gene-based phylogeny and has already proven its worth in exploring the microbial environments and identification of uncultured organisms (Muyzer et al., 1993; Muyzer et al., 1996; Araya et al., 2003). It is a culture-independent method of obtaining a genetic fingerprint of the microbial community composition. DGGE allows the separation of DNA molecules (e.g. 16S rRNA-gene amplicons) of the same length but with different base sequences. The DGGE separation is based on the electrophoretic mobility of partially denatured double stranded DNA molecules in a polyacrylamide gel (6 or 8%) which contains an increasing linear gradient of DNA denaturing agents (20%-50% or 35%-65%), like urea or formamide (Muyzer et al., 1996). With DGGE, total community DNA can be used as template for a specific PCR amplification (with the forward primer containing a GC-clamp) of the 16S rRNA-gene fragments with universal (e.g. 16S rRNA-gene targeting primers) or more specific primers (e.g. only targeting the AOB or an individual species). The DGGE analysis of PCR-amplified 16S rRNA-genes was already successfully applied in the study of genetic diversity of different microbial

38 State of knowledge communities in a broad range of environments, including experimental bioreactors (Muyzer et al., 1993). The functional ammonia monooxygenase (amoA), which is an AOB-specific gene, can also be used to analyze bacterial communities mainly comprised of ammonia-oxidizing bacteria. The gene is of specific interest because of the major role it plays in the oxidative conversion of ammonia and a broad range of carbohydrates and halogenated carbohydrates (McTavish et al., 1993).

39 State of knowledge PART IV: HORIZONTAL GENE TRANSFER 1. Bacterial conjugation 1.1. Mechanisms of Horizontal Gene Transfer (HGT) Horizontal gene transfer (HGT) is the acquisition of nucleic acids, independent of vertical gene transfer (from parent to descendant) and is a frequently occurring phenomenon in natural and engineered systems alike. The disseminated nucleic acids can carry genes that confer phenotypic traits to their hosts. In view of the MELiSSA project, HGT (and specifically conjugation) is a very important factor in the spread of virulence and antibiotic resistance genes. Many pathogenetic traits are encoded on mobile genetic elements (Miller & Day, 2004). Via transposition and integration events, the genes carried by nucleic acids (e.g. plasmids) can integrate in to the chromosome of the new host. In addition, it has been found that pathogenicity islands may not be exclusively encoded on the bacterial chromosome. Pathogenicity islands are examples of how gene acquisition by horizontal gene transfer can generate sequence diversity in bacterial pathogens and enable them to evolve new virulence traits (Mahenthiralingam, 2004).

HGT works through diverse systems during which microorganisms can obtain additional nucleic acids. The first mechanism is transformation, which is the uptake of a naked soluble DNA fragment (Griffith, 1928), and is regarded as the simplest form of gene transfer. Some bacterial species prefer the uptake of linear fragments, while others prefer circular DNA. Mostly transformation occurs e.g. after a cell has died and has released its contents (comprising DNA) into the environment; however, some bacterial species, like Bacillus, sporadically engage in rolling-circle replication, and eject these single-stranded fragments. These actively ejected DNA fragments can be taken up by another bacterium and made double-stranded by their new host. It is possible that the non-self DNA becomes stably integrated into the genome, resulting in the acquisition of nucleic acids independent of its ancestors. Subsequently, this new DNA can be passed on to the host's descendents (Milkman, 2004). Secondly, transduction is the transfer of genetic material from a bacterial cell to another by means of a bacteriophage and occurs when a bacterium becomes infected by a bacteriophage. Transduction comes in two forms: generalized and specialized. In generalized transduction, the degraded host DNA (which is degraded by the virus) is mistakenly incorporated in a novel phage head, which in turn can be injected into a new host. In specialized transduction, the host

40 State of knowledge receives some or all of an integrated or episomal phage together with some flanking former- host-chromosomal DNA (Milkman, 2004). Finally, conjugation is a form of sexual reproduction seen in some algae, bacteria, and some ciliate protozoans. Bacterial conjugation is a process of genetic recombination between two organisms where cell-cell contact, via a cytoplasmic bridge between them, is required. In gram-negative bacteria the cytoplasmic bridge is the pilus, which is formed by an outgrowth of one or both individuals. The pilus links the two bacteria and through this tube, the nucleic acids are transferred to the recipient, through action of a type IV secretion system (encoded by the plasmid itself)(Frost, 2000).

1.2. Bacterial conjugation 1.2.1. Properties and implications Bacterial conjugation (BC) is one of the most important means of horizontal gene transfer between microorganisms. Conjugative elements include plasmids, conjugative transposons, which can either exist as a separate genomic entity or be stably incorporated into the host chromosome to allow chromosome mobilization (Frost, 2000). The process is widespread among bacteria and can be observed both within the same genus as beyond (e.g. broad-host- range plasmids). Furthermore, several bacteria, like Agrobacterium tumefaciens, harbour plasmids that have adapted to cross taxonomic boundaries (i.e. nucleic acid transfer from bacteria to yeast or higher plants).

Bacterial conjugation is a common means of escaping selective pressure by providing additional cellular functions to the host; thus, giving a selective advantage, when the host bacterium undergoes such pressure (Bergstromm et al., 2000). Consequently, this process has had a major influence on the evolution of bacterial genomes, and in the diversification and speciation of bacteria (Bergstromm et al., 2000; Ochman et al., 2000; Gogarten et al., 2002). Because of the horizontal exchange of derived features, these cellular functions can quickly become common in the bacterial population (e.g. antibiotic resistances in hospitals). Unlike the acquisition of antibiotic resistance, adoption of a pathogenic lifestyle (as a result of virulence gene acquisition) usually coincides with a fundamental change in the microorganism's ecology (Ochman et al., 2000). Because of lateral gene transfer, closely related bacteria can have very diverse life-styles and properties, whereafter genes acquired by HGT become the species-specific traits (Ochman et al., 2000). Table 2 gives an overview of possibilities for derived cellular functions (Frost, 2000).

41 State of knowledge Even though bacterial conjugation has been studied primarily in liquid media, most naturally occurring populations live in biofilm communities. In view of the MELiSSA CIII packed-bed bioreactor, it is interesting to mention that it has been shown that bacterial conjugation occurs within biofilms (Christensen, 1998), and that some conjugative plasmids contribute directly to the capacity of the bacterial cell to form a biofilm (Ghigo, 2001).

Table 2. Cellular functions possibly encoded by plasmids; adapted (Frost, 2000) A. BY ALL PLASMIDS 1. Self-replication

B. BY SOME PLASMIDS 1. Self-transfer 2. Resistance to antimicrobial agents a. Antibiotic; e.g. bacitracin, aminoglycosides, chloramphenicol, etc. b. Synthetic chemotherapeutic agents c. Heavy metals; e.g. Cd, Hg, Ni 3. Pigment production 4. Toxin production 5. Catabolic functions; e.g. lactose 6. Phage sensitivity and resistance 7. Antibiotic production 8. Bacteriocine production 9. Induction of plant tumor 10. Production of H2S 11. Host-controlled restriction and modification 12. Biofilm induction

Traditionally, bacterial conjugative gene flow was considered to be the mobilization of a plasmid from the original donor to the recipient strain. However, gene flow in two directions was reported with high frequency, in some cases of the same order of magnitude as the frequencies of the traditional gene flow, for broad-host-range IncP1 plasmids (Mergeay et al., 1987) and was also reported for a range of other plasmids, of differing Inc-groups (Heinemann & Ankenbauer, 1993; Ramos-Gonzalez et al., 1994). This process was called retrotransfer or gene capture, and was used to describe the process where the original host of a conjugative plasmid, inherits (captures) genetic traits (either chromosomal markers or plasmids) from the mating partner, free of conjugative plasmids.

1.2.2. Requirements for bacterial conjugation Before conjugation can occur in gram-negative bacteria, a range of requirements has to be met. A plasmid can only perform conjugation if several specific regions are located on the plasmid itself. Conjugative / mobilising plasmids (60-500 kb) contain regions like the oriT, which is called the nic-site, with which several proteins (encoded by the transfer (tra) and mobilizing (mob) sites) interact through DNA-binding motifs. These specific regions make up

42 State of knowledge more than half of the (unmodified / natural) plasmid coding capacity. The relaxosome is a protein-nucleic acid complex and is formed through the binding of single strand binding proteins to the plasmid's nic site. This relaxosome is then mobilized and migrates to the membrane-bound transport machinery, where it binds to each other and forms the transferosome. The mobilizable plasmids (<15kb) contain an oriT-site, but lack tra genes. The oriT can be subject of activation by Tra proteins of other (mobilizing) plasmids that work in trans. The transfer-frequency of these mobilizable plasmids is nearly the same as the transfer- frequency of the mobilizing plasmids. The non-mobilizable plasmids carry neither oriT-site nor tra-genes. Furthermore, plasmid mobilization appears to be Inc-group independant, and mob-dependant. Once the mob-sequence is lost, the possibility of mobilising the plasmid is lost as well (Frost, 2000).

In addition to the need of specific plasmid-borne sequences to perform bacterial conjugation, intimate contact between the two bacteria is needed. Therefore, the pilus, which is present on the surface of cells, is formed in gram-negative bacteria that harbour a conjugative plasmid expressing the tra genes. Many different types of pili have been described in gram-negative bacteria. Long, thin, and flexible pili are ideal for mating in liquids (like F-like plasmids); whereas short, rigid pili are thought to be ideal for mating on surfaces like solid medium or plant roots (e.g. IncP plasmid RP4) (Koraimann, 2004).

Different physiological factors are needed to perform the conjugation with the highest efficiency. Research clearly indicates the existence of a temperature optimum at which one can best perform the conjugation. Other factors like the oxygen level, nutrient availability and growth phase are very important as well. In summary, one can say for aerobic organisms, that one gets the best results for the conjugation when the bacteria are grown with good aeration, in a nutrient-rich environment and in a shallow temperature range (Frost, 2000).

Sometimes, bacterial conjugation can be inhibited or can be reduced by plasmids themselves. Some conjugative elements encode an exclusion system that reduces redundant plasmid transfer between cells harbouring the same or closely related plasmids (Minkley Jr. & Ippen- Ihler, 1977). Such redundant plasmid transfer is thought to be deleterious to the donor cell (e.g. lethal zygosis) (Skurray & Reeves, 1974). In addition, multiple matings with a single recipient cell results in its death because of severe membrane and cell wall damage (Skurray

43 State of knowledge & Reeves, 1974). The influx of large amounts of single-stranded DNA additionally causes SOS response induction (Higashitani et al., 1995).

In F plasmids, the exclusion genes are traT and traS, their respective gene products (TraTF and TraSF) reduce the stabilization of mating pairs and block the DNA import from the potential donor cell, respectively (Minkley Jr. & Ippen-Ihler, 1977; Finlay & Paranchych, 1986). The former reaction is known as surface exclusion, the latter as entry exclusion. Entry exclusion genes have also been reported e.g. for RP4 (trbK) (Haase et al., 1996), the IncN plasmid pKM101 (eex) (Winans & Walker, 1985). In addition, entry exclusion not only reduces redundant transfer but also protects high-density F+ cell populations from potentially lethal matings with other donor cells (Skurray & Reeves, 1974).

1.2.3. Plasmids Mobile genetic elements (MGE) are a range of genetic elements that can be interexchangeable between bacteria (e.g. plasmids) or can change location within the bacterial genome (e.g. transposons, mini-transposons, genomic islands). MGE often endow their hosts with a supplementary response-mechanism, to enable the bacterium to react efficiently to environmental stress (Thomas & Smalla, 2000), and together with rearrangements in the genome can enable the bacterium to adapt and evolve (Bergstromm et al., 2000). Of all MGE, plasmids probably represent the most important reservoir for both gene transfer and capture. Plasmids are small, double-stranded and circular DNA molecules that can carry different kinds of factors. These factors have the ability to provide e.g. an antibiotic resistance to the host (R factors) or can be the causative agent of gene transfer (F factors). The latter were the first to be discovered in plasmids and appeared to spread in the bacterial population (Lederberg, 1952 & Hayes, 1953). Plasmids were most intensively studied in bacteria, and the existence of analogs in Eukarya is known.

Plasmids endow a small metabolic burden to their host, and their presence is based on a trade- off principle. To co-exist stably and to minimize this metabolic load, plasmids control their own regulation, so that the copy number of a given plasmid is relatively fixed under defined cell growth conditions. To maintain this steady state in copy number, plasmids operate a self- encoded negative control system, that is able to 'sense' and adjust the plasmid copy number. Actually, three types of plasmid copy number control systems are recognized, and work by means of (i) directly repeated sequences (iterons) that complex with replication initiator

44 State of knowledge proteins, (ii) antisense RNAs that hybridize to a complementary region of an essential RNA, and are called countertranscribed (ct) RNAs; and (iii) ctRNA combined with proteins.

Identification and classification should be based on genetic characteristics that are universally present and relatively static. When two closely related plasmids are introduced simultaneously into the same cell, one will be eliminated during the bacterial growth phase; this phenomenon is termed incompatibility. Thus, according to the specificity of their replication machinery, conjugative antibiotic resistance plasmids from gram-negative bacteria have been classified into about 20 incompatibility groups. The incompatibility (Inc) is the result of a close relationship in copy-number-control system that regulates the number of a specific plasmid per cell (Datta, 1975). A final point considering the classification of plasmids is to emphasize that actually there is no naming convention with real biological meaning. This is probably due to the fact that plasmids do not appear to have a single phylogenetic history and therefore can not be assigned a classic taxonomy, but they can move independently through the bacterial population without great difficulty, and are relatively plastic, able to lose or gain genes over time (Molbak et al., 2003). Little is known about the majority of the plasmids. The most studied plasmids originate from frequently used strains and strains with clinical importance (Götz et al., 1996). Furthermore, the lack of information is mostly due to the inability to culture most bacteria traditionally. However, these can now be studied since the development of methods for extraction of nucleic acids are independent of cultivation. In addition to these techniques, PCR enables the researchers to analyze total community DNA by the use of MGE specific primers (Götz et al., 1996).

A significant part of the plasmid comprises genes responsible for transfer of the plasmid, although not present in all plasmids; these genes can also interact with other non-self sites, and subsequently mobilize other (mobilizable) plasmids. Conjugative transfer of plasmid DNA is initiated and terminated at a specific site termed the origin of transfer, oriT. In the way oriT encodes transfer of the plasmid, lots of regions have been found encoding gene products utilized for replication, e.g. trfA2, korA, rep, oriV, and repB (Götz et al., 1996). Amplification of parts of the latter genes thus allows a screening for the presence of specific plasmids in different environments (Götz et al., 1996).

45 State of knowledge 2. Exogenous plasmid isolation Plasmids have become very important tools in gene technology and science e.g., as cloning vectors and expression vectors. The use of these vectors is often straightforward because of the specific traits they harbour. In natural samples, however, the plasmids and the respective traits they carry are unknown. The exogenous plasmid isolation is a technique that allows the detection of transfer of plasmids, that potentially play a role in the distribution of traits in a bacterial population, and is applicable to consortia and pure cultures alike. We know that plasmids are present in nearly all bacterial species (Amabile-Cuevas & Chicurel, 1992), but irrespective of the traits plasmids carry, plasmids can be mobilizing (Tra+, Mob+), mobilizable (Tra-, Mob+) or non-mobilizable (Tra-, Mob-).

Exogenous plasmid isolation (Figure 10) is a natural way to detect plasmid translocation to a chosen (plasmid-free) recipient, without the need of cultivating or isolating individual strains from the consortium. Once the triparental mating has taken place, the plasmid-free recipient becomes host to a plasmid with known antimicrobial resistances or metabolic pathways (the 'donor plasmid') and has probably become host to an unknown mobilizing plasmid (the 'helper plasmid'). The plasmid-harbouring recipient can be selected now from the other bacteria (donors, unknown bacteria, and plasmid-free recipients). After selection, the recipient strain is screened for presence of the mobilizing plasmid (originating from the consortium) and the mobilizable plasmid (which carries the selectable markers) (Top et al., 1994).

The triparental exogenous plasmid isolation technique has been used successfully many times before, to isolate plasmids and examine the potential of bacteria from various environments to mobilize a plasmid. These environments are mostly natural environments, like river epilithon (Hill et al., 1992), wheat rhizosphere (Smit et al., 1998; van Elsas et al., 1998), and in polluted soils and sludges (Top et al., 1994).

46 State of knowledge (a)

(b) (c)

Figure 10. The exogenous plasmid isolation principle by triparental mating. (a) The transfer of the Mob+ Tra+ plasmid from the unidentified plasmid-harbouring helper cell that is present in the microbial community to the donor cell, (b) the donor cell transfers its plasmids to the plasmid-free recipient, the indigenous 'donor plasmid' (Mob+ Tra-) is transferred through action of the 'mobilizing helper-plasmid' (Mob+ Tra+) to the chosen plasmid-free recipient, (c) after selection, only the recipients carrying a selectable marker gene, that harbour the 'donor plasmid' carrying the particular R-gene (i.e. the gene encoding a chosen selectable function); can subsequently be isolated.

47 Objectives C. Objectives The work reported herein was destined to determine the diversity of bacteria present in the MELiSSA nitrifying packed-bed pilot reactor once its pilot period had expired, in view of the fact that several additions to the co-culture were expected to have settled indefinitely and without compromising reactor stability or integrity. These microorganisms were expected despite the fact that the reactor has run axenically (Pérez et al., 2004). The microbial community was analysed using PCR-based detection coupled with denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA- and the functional ammonia monooxygenase (amoA)-gene in whole-community, cultured fractions, cDNA of cultured fractions and community isolates. This approach, combined with clone screening and sequencing (targeting the 16S rRNA-gene) to identify various strains and determine their possible role in the reactor (necessary, detrimental, opportunistic, or supportive).

The second part of the thesis comprises the assessment of horizontal gene transfer within the community, by examining the ability of the community to perform bacterial conjugation with non-pathogenic strains in controlled conditions. The detection of plasmids is performed through PCR-amplification of plasmid-specific regions in an initial stage of the research, followed by the detection of mobilizing plasmids present in the consortium (through exogenous plasmid isolation and replica mating). These conjugation experiments are important to determine the stability and integrity of the reactor because some gene products might compromise its proper functioning.

48 Material and methods D. Material and methods 1. Bacterial strains, culture conditions, bacterial media, and antibiotics 1.1. Bacterial strains In addition to the uncharacterised nitrifying pilot plant community and its isolates, several strains were used in this study (Table 3). Some were used as positive controls for the Inc- specific plasmid PCR (CM1282, CM1286, CM1287, CM1288, CM1291, and CM1292), others for the triparental exogenous plasmid isolation (AE815, CM404, CM1120, and CM1962) or for the replica mating (AE2547). All strains possessed a selective marker-gene that allowed them to be cultured axenically (Table 3). The plasmids are listed and described in Table 4.

Table 3. Used strains Strain Species Features and plasmids Source AE815 Cupriavidus metallidurans CH34 Rif+, GFP+, plasmidless (Springael, 1991) + + - + AE2547 Cupriavidus metallidurans CH34 Ap , Tc , Phe , GFP C. Lodewyckx, Belgium CM404 Escherichia coli Km+, Nm+, Tra+; pRK2013 M. Davidson, Athens, USA CM1120 Escherichia coli Sm+, plasmidless Gibco BRL, Belgium CM1282 Escherichia coli Tc+, pULB2431 (Couturier et al., 1988) CM1286 Escherichia coli Ap+, Tc+, pULB2432 (Couturier et al., 1988) CM1287 Escherichia coli Kn+, pULB2420 (Couturier et al., 1988) CM1288 Escherichia coli Ap+, Tc+, pULB2424 (Couturier et al., 1988) CM1291 Escherichia coli Tc+, pULB2426 (Couturier et al., 1988) CM1292 Escherichia coli IncX plasmid (Couturier et al., 1988) CM1962 Escherichia coli Km+, pMOL222 M. Mergeay, Belgium TOP10 Escherichia coli hscR, Z∆M15 Invitrogen, Germany

1.2. Plasmids For the PCR-mediated plasmid detection, several plasmids were used that enabled Inc-group identification, and are described below (Table 4). The pMOL222 plasmid was used for the exogenous plasmid isolation; this plasmid is a pKT240 (IncQ, Tra-) derivative containing the czc and ncc operons, which encode high-level resistances to Ni, Cd, and Co (Dong et al., 1998). pMOL222 is a Tra- plasmid, and the transfer to the indigenous community will be dependent on mobilization by a cointroduced mobilizing plasmid (Smets et al., 2003).

49 Material and methods

Table 4. Plasmids used as positive control for the Inc-group PCR (Couturier et al., 1988) and for the exogenous plasmid isolation (Bossus, 2005) Plasmid Origin Inc group Antibiotic resistance Source located on the vector pMOL222 pKT240 IncQ Mob+, NiR, CdR, CoR Mergeay, M. pRK2013 pRK2013 IncP Tra+, KmR, NmR (Figurski & Helinski, 1979) pULB2439 R387 IncK AmpR (Couturier et al., 1988) pULB2432 R46 IncN AmpR, TcR (Couturier et al., 1988) pULB2420 RK2 IncP KmR (Couturier et al., 1988) pULB2424 R1162 IncQ AmpR, TcR (Couturier et al., 1988) pULB2426 Rsa IncW TcR (Couturier et al., 1988) pULB2405 R6K IncX AmpR (Couturier et al., 1988)

1.3. Growth conditions Cupriavidus sp. were selectively cultured at 30°C in 284 Gluconate in an obscured shaking incubator, whereas the Escherichia coli strains were selectively cultured at 37°C in 869 medium in a shaking incubator. Bioreactor samples were lyophilized or incubated in -80°C in 20% glycerol for storage. For analysis, bioreactor samples were grown in a range of media: 1- 2-3 medium, COC medium, SW medium (AOB-targeting media), 284 Gluconate medium

(soil bacteria-targeting medium), 869 medium, PCM (heterotroph-targeting media), and NO2- medium (NOB-targeting medium; media content is described in 1.4.).

1.4. Bacterial media All media were autoclaved for 15 min at 121°C and for solid media, 1.5% (w/v) Select Agar (Cat. no. 30391.023; Invitrogen™, Paisley, Scotland) was added, unless stated otherwise.

1.4.1. 1-2-3 medium (Hommes, pers. comm.)

'Part 1' is comprised of 900 ml MilliQ water to which, 3.3 g (NH4)2SO4 (=50 mM), 0.41 g

KH2PO4, 0.75 ml of a 1 M MgSO4 stock solution, 0.2 ml of a 1 M CaCl2 stock solution, 0.33 ml of a 30mM FeSO4/50mM EDTA stock solution, and 0.02 ml of a 50mM CuSO4 stock solution was added.

'Part 2' is comprised of 400 ml MilliQ water to which, 40.82 g KH2PO4, and 3.6 g NaH2PO4 was added. When all salts were dissolved, pH was adjusted to 8.1 with a 10 N NaOH solution; subsequently, the volume was adjusted to 500 ml with MilliQ water. The solution 'Part 2' was autoclaved in 100 ml fraction in 250 ml flasks.

'Part 3' is 500 ml of a 5% (w/v) Na2CO3 (anhydrous) solution, which was made and autoclaved.

50 Material and methods To make the '1-2-3 medium', a 100 ml aliquot of 'Part 2' was added to an 900 ml aliquot of 'Part1', to which 8 ml of 'Part 3' was added.

1.4.2. 284-medium (Mergeay et al., 1985) To 985 ml MilliQ water, 6.06 g Tris (Tris-hydroxymethyl-aminomethane hydrochloride)/HCl,

4.68 g NaCl, 1.49 g KCl, 1.07 g NH4Cl, 0.43 g Na2SO4, 0.2 g MgCl2.6H2O, and 0.03 g

CaCl2.2H2O were added. To the resulting solution 4 ml of a 1% Na2HPO4 solution, 10 ml of a

48 mg/100 ml Fe(III)NH4 citrate solution and 1ml of SL7 trace solution was added. This SL7 trace solution contained 1.3 ml of a 25% HCl solution, 0.07 g ZnCl2, 0.1 g MnCl2.H20, 0.062 g H3BO3, 0.19 g CoCl2.6H2O, 0.017 g CuCl2.2H2O, 0.024 g NiCl2.6H2O, and 0.036 g

Na2MoO4.2H20 per litre of MilliQ water (Biebl & Phennig, 1981). Subsequently, the pH was adjusted to 7.8 with HCl or NaOH solution. For solid medium 2% (w/v) agar was added instead of the standard 1.5% (w/v) agar. After the addition of agar, 0.2% (w/v) of carbon source (sodium gluconic acid) was added (i.e. 5 ml of a 40% stock solution per litre medium).

1.4.3. 869-medium (Mergeay et al., 1985) To one litre MilliQ water, 40 g Luria Broth (Invitrogen™, Paisley, Scotland) (or 20 g + Tryptone, 10 g Yeast Extract & 10 g NaCl), 2.0 g Glucose D , 0.69 g CaCl2.2H2O was added. Subsequently, the pH was adjusted to 7.0 with a NaOH solution.

1.4.4. Co-culture medium (COC) (Pérez et al., 2004)

To one litre MilliQ water, 1.32 g (NH4)2SO4, 0.68 g KH2PO4, 0.71 g Na2HPO4, 0.0025 g -6 FeSO4.7H2O, 4 x 10 g CuSO4.5H2O, 0.177 g (NH4)6Mo7O27.4H2O, 0.052 g MgSO4.7H2O,

0.8 g NaHCO3, and 0.74 mg CaCl2.2H2O was added. Subsequently, the pH was adjusted to 8.2 with a NaOH solution.

1.4.5. KM1 (Ludwig et al., 1999) To one litre Nitrosomonas Minimal Medium (NMM), 0.05 g proline, and 0.001 g thiamine was added. When necessary, 0.05% (w/v) glucose was added.

51 Material and methods

- 1.4.6. Nitrite (NO2 ) medium (Schmidt & Belser, 1982) The nitrite medium was prepared by aseptically mixing autoclaved (or filter-sterilized) stock solutions. To 986 ml sterile MilliQ water, 1.0 ml (0.85 g/100 ml) KNO2 solution, 1.0 ml (1.34 g/ 100 ml) CaCl2.2H2O solution, 5.0 ml (4.0 g/ 100 ml) MgSO4.7H2O solution, 4.0 ml (3.48 g/100 ml) K2HPO4 solution (0.2 M), 1.0 ml (2.72 g/100 ml) KH2PO4 solution (0.2 M), 1.0 ml of chelated iron solution (containing 0.246 g/ 100 ml FeSO4.7H2O and 0.331 g/100 ml EDTA disodium), and 1.0 ml trace elements solution (containing 0.01 g/ 100 ml NaMoO4.2H2O,

0.02 g/ 100 ml MnCl2, 0.0002 g/ 100 ml CoCl2.6H2O, 0.01 g/ 100 ml ZnSO4.7H20, and 0.002 g/ 100 ml CuSO4.5H2O per litre MilliQ water) was added. The pH was adjusted to approximately 7.2-7.5.

1.4.7. Nitrosomonas minimal medium (NMM) (Koops et al., 1991)

To 998 ml MilliQ water, 0.535 g NH4Cl, 0.054 g KH2PO4, 0.074 g KCl, 0.049 g

MgSO4.7H2O, 0.0147 g CaCl2.2H2O, 0.584 g NaCl, 1 ml of a 0.05% cresol red solution, 1 ml of trace elements solution was added. The trace elements solution contained, per litre MilliQ water, 0.1M HCl, 44.6 mg MnSO4.2H2O, 49.4 mg H3BO3, 43.1 mg ZnSO4.7H2O, 37.1 mg

(NH4)6Mo7O24.4H2O, 173 mg FeSO4.7H2O, and 25.0 mg CuSO4.5H2. Subsequently, the pH of the medium was adjusted to 7.8 with 0.5g CaCO3 (pH increase) or 11.9 g HEPES (pH drop).

1.4.8. Purity Check Medium (PCM) (Schmidt & Belser, 1982) To 200 ml MilliQ water, 2 g glucose, 1 g yeast extract, and 1 g casein hydrolyzate (= Tryptone or Peptone n° 140) was added, resulting in a 10x stock solution.

1.4.9. Skinner & Walker (SW) inorganic medium (Soriano & Walker, 1968)

To one litre MilliQ water, 0.5 g (NH4)2SO4, 0.2 g KH2PO4, 0.04 g CaCl2.2H2O, 0.04 g

MgSO4.7H2O, 0.5 mg iron (as Fe-citrate or Fe-EDTA), and 0.5 mg phenol red was added. Subsequently, the pH was adjusted to 7.5 - 8.0 with a sterile 5% sodium carbonate solution. The recommended and used pH in this study was 7.5 (Grunditz & Dalhammar, 2001).

52 Material and methods

1.4.10. SOB medium (Sambrook et al., 1989) To 950 ml of MilliQ water, 20 g bacto-tryptone, 5 g bacto-yeast extract, and 0.5 g NaCl was added. After all solutes were dissolved, 10 ml of a 250 mM KCl solution (1.86 g KCl/ 100 ml MilliQ water) was added. Thereafter, pH was adjusted to 7.0 with a 5 N NaOH solution (~0.2 ml). The volume was adjusted to one litre. Just before use, 5 ml of a sterile solution of a 2 M

MgCl2 solution was added.

1.4.11. SOC medium (Sambrook et al., 1989) SOC medium was identical to SOB medium, except that it contained 20 mM glucose. After the SOB medium had been autoclaved, it was allowed to cool down and the 20 ml of a sterile 1 M solution of glucose was added. After the sugar had dissolved, the volume was adjusted to 100 ml with MilliQ water, and was sterilized by filtration.

1.4.11. Antibiotics To increase the selectivity of the medium, filter-sterilized antibiotics were added; the respective properties of the utilized antibiotics are listed below (Table 5).

Table 5. Overview of all used antibiotics and their properties (L. Janssen, pers. comm.) Antibiotic Dissolved in Stock Final concentration in Light Fast solution media (µg/ml, unless sensitive spontaneous (%) stated otherwise) degradation during storage

Ampicillin H2O (+ a drop of NaOH) 1 15, 50, 100 - + Kanamycin H2O 1 15, 50, 100 - - Naladixic acid H2O (+ a drop of NaOH) 1 30 - - Nickel H20 1 1 mM, 2 mM - - Rifampicin 1% NaHCO3 1 100 + + Tetracycline H2O 1 20 + -

2. Ammonia analysis Traditionally, nitrification has been studied by chemically monitoring ammonia or nitrite disappearance, nitrite or nitrate production, or a combination of these methods (Hovanec & DeLong, 1996). Substrate usage can be determined either by using colorimetric methods or by using high-pressure liquid chromatography (Bollman & Laanbroek, 2001; Laanbroek et al., 2002). In this study, the colorimetric method using indophenol was used.

53 Material and methods 2.1. Determination of ammonia content in media An intensely blue compound, indophenol, is formed by the reaction of ammonia, hypochlorite, and phenol catalysed by sodium nitroprusside (Solorzano, 1969). It is important to note that this reaction can be inhibited with magnesium and calcium, complexing Mg2+ and Ca2+ with citrate the interference produced by precipitation of these ions at high pH is eliminated. There is no interference from other trivalent forms of nitrogen. Interfering turbidity was removed by centrifugation or filtration prior to analyses. Detection was performed with a computer-controlled (Ascent Software Version 2.4.2.) Multiscan Ascent multiwell spectrophotometer (ThermoLabsystems) for detection at 620 nm, specifically conceived for analyses of microtiter plates, with the light path being somewhat less than 1 cm. The phenol solution was prepared by mixing 11.1 ml liquefied phenol (> 89%) with 95% (v/v) ethyl alcohol to a final volume of 100 ml. The sodium nitroprusside solution was a 0.5% solution; 0.5 g nitroprusside was dissolved in 100 ml MilliQ water. Alkaline citrate was prepared by dissolving 200 g trisodium citrate and 10 g sodium hydroxide in MilliQ water. Then, the solution was diluted to 1000 ml. Finally, the oxidizing solution was a mix of 100 ml alkaline citrate solution with 25 ml sodium hypochlorite, and had to be prepared fresh daily.

Lyophilized and frozen samples were grown COC medium and SW medium and sample supernatant was harvested and kept at 4°C (one day) until the analysis took place. Firstly, 250 µl of sample was applied to a microtiter plate. To these 250 µl sample was added, with thorough mixing after each addition, 10 µl phenol solution, 10 µl sodium nitroprusside solution, and 25 µl oxidizing solution. The samples were covered with parafilm before incubation of at least one hour, during which the colour developed at room temperature (22° to 27°C) in subdued light; the colour remained stable for 24 hours. The absorbance was measured at 620 nm. The standard was prepared by diluting a 10 mg/ml stock solution; each step was a two-fold dilution, and this eight times. The ammonia in the supernatant of the consortium culture was analysed at the same time as the standard, using a microtiter plate. The positive control was sterile unused medium and sterile MilliQ water was used as blank. The samples were diluted identically to the positive control, i.e. three times a ten-fold dilution. The standard curve was plotted against the (known) ammonia concentrations. Sample ammonia concentration was calculated by comparing sample absorbance with the standard curve. The blank was subtracted before calculating the ammonia concentration in the media.

54 Material and methods 3. Nucleic acid preparation and experiments 3.1. Preparation of genomic DNA from Bacteria 3.1.1. Miniprep of N. europaea pure culture Genomic DNA N. europaea DNA was extracted using a modified miniprep CetylTrimethyl AmmoniumBromide (CTAB) protocol (Saghai-Maroof et al., 1984). The N. europaea cell culture was grown in 1 litre of 1-2-3 medium and harvested at OD600 nm = 0.10. The culture samples were divided in several tubes, and were centrifuged in aliquots of 20 ml during 10 min at 5000 rcf. The supernatant was discarded and the pellet was resuspended in 567 µl Tris- EDTA (TE) buffer by repeated pipeting and transferred to microtubes (Eppendorf). Thirty µl of 10% Sodium Dodecyl Sulfate (SDS) and 3 µl of 20 mg/ml proteinase K was added to give a final concentration of 100 µl/ml proteinase K in 0.5% SDS. This solution was mixed thoroughly and was incubated for one hour at 37°C. Subsequently, 100 µl of 5 M NaCl was added and was mixed, followed by the addition of 80 µl of CTAB/ NaCl solution (i.e. a 10% CTAB solution in 0.7 M NaCl). The mixture was mixed and was incubated for 10 min at 65°C. An equal volume (0.7 to 0.8 ml) of chloroform/isoamyl (24:1) alcohol was added, thoroughly mixed and spun 4 to 5 min in a microcentrifuge. The aqueous, viscous supernatant was transferred to fresh microcentrifuge tubes, leaving the interface behind. An equal volume of phenol/chloroform/isoamyl (25:24:1) was added, mixed well, and spun in a microcentrifuge for 5 min. The supernatant was transferred to clean tubes and the residual phenol was extracted twice more with chloroform followed by a centrifugation of 5 min. The supernatant was transferred to fresh tubes, followed by addition of 0.6 volumes isopropanol to precipitate the nucleic acids. The tubes where slightly shaken until the DNA precipitate became clearly visible. At this point, the precipitate was transferred to fresh tubes, 70% ethanol was added, and the precipitate was pelleted by spinning briefly at room temperature. Subsequently, the DNA was washed with 70% ethanol to remove the residual CTAB, and was spun for 5 min at room temperature to precipitate the DNA. The supernatant was carefully removed and the pellet dried for several hours, later to be dissolved in 100 µl sterile MilliQ water.

3.1.2. Fast-prep DNA extraction The culture (2 ml) was spun at 5000 rcf for 10 min. The supernatant was discarded, and the pellet dissolved in 200 µl TE buffer (10mM). Then, 50 µl lysozyme (5 mg/ml) was added followed by the addition of 30 µl RNase (10 mg/ml). This mix was incubated at 30°C for 30 min. The mix was transferred to fast-prep tubes containing sterile glass beads. To this tube, 30

55 Material and methods µl 10% SDS, 200 µl phenol (pH 4.0) and 200 µl chloroform was added. Then, the tube was shaken for 40 s at speed 6.0 and once again for 40 s, with an interval of 1 min. After this, the tube was cooled on ice and was centrifuged for 5 min at 13000 rcf. The aqueous phase (the top one) was transferred to a fresh tube to which 200 µl phenol (pH 4.0) was added. The tube was mixed thoroughly and was spun for 5 min at 13000 rcf. The aqueous phase was transferred to a fresh tube. To this fresh tube, 200 µl chloroform was added; then thoroughly vortexed and spun for 5 min at 13000 rcf. Again, the aqueous phase was transferred to a fresh tube. Subsequently, one-tenth of the volume of the aqueous phase, 3 M ammonium acetate; and 2.5 volumes, 99.8 % ethanol were added. The tube was placed at –20°C overnight. The day after, the sample was centrifuged for 20 min at 12000 rpm at 4°C. The supernatant was removed carefully, and the pellet was washed with 70% ethanol and centrifuged for 5 min at 14000 rpm. The supernatant was removed again and the pellet was dried at room temperature. When dry, the DNA was dissolved in 100 µl sterile MilliQ water.

3.1.3. Determination of DNA purity and DNA concentration All concentrations were determined by means of the Nanodrop® ND-1000 Spectrophotometer (Nanodrop, Wilmington, USA). This spectrophotometer accurately determines the DNA concentration up to 3700 ng/µl (of double stranded DNA) without dilution. To do this, the instrument automatically detects high concentration and utilizes the 0.2 mm path length to calculate the absorbance. The typical reproducibility, when sample range was between 1.5- 100 ng/µl, has a standard deviation of approximately 1.5 ng/µl and, above 100 ng/µl, ranged within the 2% of the sample concentration. In addition, DNA purity was determined using the ratio of sample absorbance at 260 nm and 280 nm (260/280-ratio). A ratio of ~1.8 was regarded as pure DNA. The ratio of sample absorbance at 230 nm and 260 nm was used as a secondary measure of purity. The 230/260 values between 1.8 and 2.2 were regarded as 'pure'. In all experiments, only pure DNA was used.

3.2. DNA-based molecular analyses 3.2.1. Polymerase Chain Reaction (PCR) PCR is a cyclic process for DNA amplification. Due to an exponential increase of the target DNA sequence, after n cycles of denaturation, primer annealing and chain extension/elongation, the yield is 2n-times the target DNA sequence. The PCR reactions were performed with TaKaRa Ex Taq™ Polymerase (TaKaRa, Madison, USA) and the

56 Material and methods corresponding TaKaRa Ex Taq™ 10x buffer. The PCR mix contained (per reaction) 4 µl of 2.5 mM Mixture (TaKaRa), 5 µl of 10x TaKaRa Ex Taq™ buffer, 0.25 µl of forward primer (100 ng/µl; Invitrogen™, Paisley, Scotland), 0.25 µl of reverse primer (100 ng/µl), and 2 µl of TaKaRa Ex Taq™ Polymerase (1 U/µl). Subsequently, 2.5 µl of bacterial colonies, and 36 µl of High Purity (DNA-free) H2O, or 1 µl of DNA sample, and 37.5 µl of high purity water was added. The mix (containing everything except the sample) was prepared under a Labcare PCR Workstation (VWR International, Leuven, Belgium), to prevent contamination by (airborne) template DNA.

The following programs rapid PCR-based detection of plasmids in total-community genomic DNA samples. The PCR programs was 5 min at 94°C, with a hold at 80°C to add the polymerase (hotstart), thereafter, 35 cycles of 1 min at 94°C, 1 min at the primer specific annealing temperature (Table 6), and 1 min at 72°C, followed by a final amplification at 72°C for 10 min (Götz et al., 1996). The primers were Invitrogen™ Costum Primers and were manufactured by Invitrogen™ Ltd., Paisley, Scotland.

Table 6. Primers used for the amplification of broad host range IncQ, IncP, IncN en IncQ plasmids (Götz et al., 1996) Inc group Region Primer sequence Product size Annealing (5' – 3') (bp) temp (°C) IncN rep 1 AGT TCA CCA CCT ACT CGC TCC G 164 55 rep 2 CAA GTT CTT CTG TTG GGA TTC CG

kikA 1 ACT TAC CTT TAT CAA CAT TCT GGC G 329 55 kikA 2 CGA CTG GTT ACT TCC ACC TTC GC

IncP oriT 1 CAG CCT CGC AGA GCA GGA T 110 57 oriT 2 CAG CCG GGC AGG ATA GGT GAA GT

trfA2 1 CGA AAT TCR TRT GGG AGA AGT A 241 57 trfA2 2 CGY TTG CAA TGC ACC AGG TC

korA 1 ATG AAG AAA CGG CTN ACC GA 294 52 korA 2 TTC CTG TTT YYT CTT GGC GTC

traG1 CTG CGT CAC GAT GAA CAG GCT TAC C 762 63 traG2 ACT TCC AGC GGC GTC TAT GTG G

IncQ repB 1 TCG TGG TCG CGT TCA AGG TAC G 1160 64 repB 2 CTG TAA GTC GAT GAT CTG GGC GTT

oriV 1 CTC CCG TAC TAA CTG TCA CG 436 59 oriV 2 ATC GAC CGA GAC AGG CCC TGC

oriT 1 TTC GCG CTC GTT GTT CTT CGA GC 191 61 oriT 2 GCC GTT AGG CCA GTT TCT CG

57 Material and methods 3.2.3. Denaturing Gradient Gel Electrophoresis (DGGE) All DGGE analyses were performed at VITO (Mol, Belgium) based on the adapted protocol of the Ghent research group (Microbiology laboratory, State University Ghent, Belgium) (El Fantroussi et al., 1999). The PCR-mix contained (per sample) 4 µl dNTP Mixture (2.5 mM)(TaKaRa), 5 µl TaKaRa Ex Taq™ 10x buffer, 0.25 µl forward primer (100µM), 0.25 µl reverse primer (100µM) (Table 7), 0.25 µl TaKaRa Ex Taq™ polymerase (5 U/µl) and 39.25

µl High Purity (DNA-free) H2O. Of this mix (prepared under the PCR Workstation), 49 µl was taken per sample and to it 1 µl template DNA was added. The annealing temperatures were specific per primer pair and are given below (Table 7). When the PCR program was completed, 10 µl of sample was taken to perform a 1% agarose gel electrophoresis, and samples were checked for amplification. When amplification had occurred, 10 µl of sample (for a total of 180-300 ng DNA/lane) was mixed with 10 µl loading dye (0.25 ml 2%

Bromophenol Bue, 0.25 ml 2% Xylene cyanol, 7 ml 100% Glycerol and 2.5 ml H2O), and was loaded on the DGGE gel, alongside 20 µl VITO DGGE reference ladder (6 µl ladder and 10 µl loading dye and 4 µl water, with 4 ladders per gel) (Geets et al., 2005). The DGGE gel was run at 60°C at 200V (constant voltage) for 15 minutes and overnight (16 hours) at 120V in an Ingeny phorU (Goes, The Netherlands) containing 7 litres of 1x Tris-Acetate-EDTA (TAE) buffer (prepared from a Biorad 50x TAE, Catalogue no. 161-0773).

Table 7. Primers used for the DGGE analysis Gene Name Primer sequence Product Annealin size (bp) g temp (°C) amoA amoA-1F a 5' - GGG GTT TCT ACT GGT GGT - 3' 491 55 amoA-2R a 5' - CCC CTC KGS AAA GCC TTC TTC - 3' amoA amoA-1F*b 5' - GGG GHT TYT ACT GGT GGT - 3'

16S rRNA P63F 5' - CAG GCC TAA CAC ATG CAA GTC - 3' 495 55 P518R 5' - ATT ACC GCG GCT GCT GG - 3' GC clamp c: 5' - CCG CCG CGC GGC GGG CGG GGC GGG GGC ACG GGG - 3' covalently bound to the 5'-end of the amoA-1F forward primer or to the 16S rRNA-targeting forward primer, EUB63F a (Rotthauwe et al., 1997) b (Stephen et al., 1999) c (Oved et al., 2001)

An initial amplification was performed, yielding the 491 bp fragment, amplification was checked by loading an aliquot (10 µl) of the PCR product on a 1% agarose gel. Thereafter, another PCR was performed on the former amplicons but this time with primers with an attached GC-clamp (Briones et al., 2002). The same reverse primer was used (amoA-2R), and the degenerated GC-clamp forward primer in a total volume of 50 µl, containing one µl template from the former reaction.

58 Material and methods 3.2.4. Gels, buffers and visualisation To dissolve and store the 'pure' DNA samples, MilliQ water or TE buffer (containing 1.21 g/l (10 mM) TrisBase and 0.37 g/l (1 mM) EDTA) was added.

Samples were loaded on 1% agarose gels, containing 1/10000 volume ethidium bromide. The electrophoreses were run in a Tris-Borate-EDTA (TBE) buffer containing 5.5 g boric acid, 0.744 g EDTA and 10.8 g Tris per litre of MilliQ water and also contained 1/1000 volume ethidium bromide, although the latter was only added at the moment of running the gel. For samples, 10 µl were loaded mixed with 2-3 µl of loading dye (4 g sucrose and 2.5 mg bromophenol blue in 6 ml TE buffer). Five microlitre DNA Molecular Weight Marker XIV (100 bp ladder, 250 µg/ml in TE buffer, Roche, Cat. no. 11 721 933 001) was taken and was mixed with 2 µl of loading dye. Positive and negative controls were handled in the same fashion as samples. All agarose gels were run at a constant voltage of 85 V (with a GibcoBRL PS 304 power supply), for 2 hours. The DGGE gels were prepared under a Labcare Ductless Fume Cupboard (Model 2450) and were prepared by mixing an 24 ml 8% acrylamide/bis (37.5:1) solution containing 20%, 35%, 50%, or 65% urea/formamide with 100 µl of 10% Ammonium Per Sulfate (APS, Biorad, Cat. 161-0700) and 5 µl TEMED (N,N,N',N'-Tetra Methyl Ethyleen Diamine; Biorad, Cat. 161- 0801) per gel. The denaturing gels contained 8% acrylamide with a 35 to 60% urea/formamide gradient for the 16S amplification, and an 8% acrylamide with a 20 to 50% urea gradient for the amoA amplification. The gradients were prepared using a system based on communicating vessels. After the denaturing gel had polymerised, a stacking gel was added on top, prepared by mixing 8% acrylamide, 200 µl 10% APS, and 10 µl TEMED.

To visualize the migrated PCR amplicons, the agarose gel was subjected to UV irradiation. The PCR amplicons were detected with a Machine Universal Hood II (Biorad, Segrate, Italy) harbouring a camera, visualised using the respective software (Biorad Quantity One 4.5.0.) and printed with a Mitsubishi P93E Video Copy Processor (Mitsubishi, Nagaokakyo-City, Kyoto, Japan). The migrated DGGE amplicons were fixed by 200 ml TAE containing 0.5% acetic acid, and were left for 5 minutes. After removing the TAE with acetic acid, 100 µl TAE containing 30 µl SYBR® Gold1 was added and was left for 30 minutes in the dark and at room temperature. After removing the buffer, the gel was subjected to UV irradiation, and

1 SYBR® Gold. Nucleic acid gel stain, 10000x concentrate in DMSO. By Molecular Probes, Leiden, The Netherlands. Lot. No. 18E1-3.

59 Material and methods visualised by a Pharmacia Biotech ImageMaster® VDS, through the SYBR® Gold visualising Kenko (Japan) Y2 SY-48 55 mm lens and the Kenko (Japan) Close-Up No. 2 55 mm lens. Pictures were printed by the FTI-500, Fujifilm Thermal Imaging System, and gels were analyzed with GelCompare Software (Applied Maths, Kortrijk, Belgium). DGGE gels were analyzed with BioNumerics™ (AppliedMaths®, Kortrijk, Belgium) for similarity analyses. Bands were detected by the software or selected by visual screening. Pearson correlation and UPGMA analyses were used for determination of similarity.

3.2.5. PCR Cloning Transformation Transformation of the E. coli One Shot® TOP10 Electrocomp™ Cells (Invitrogen, Mannheim, Germany) was performed using the QIAGEN® PCR Cloningplus Kit (QIAGEN® Benelux, Venlo, The Netherlands). The QIAGEN® Cloning Kit takes advantage of the single A overhang at each end of PCR products generated using Taq. The pDrive Cloning Vector is supplied in a linear form with a U overhang at each end and hybridizes with high specificity to such PCR products. The buffer of the Ligation Master Mix was said to provide optimal conditions. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN® Benelux, Venlo, The Netherlands; for amplicon purification with 95% recovery in QIAquick Spin Columns) prior to ligation. The ligation of the PCR amplicons in the pDrive Cloning Vector was performed following the instructions of the QIAGEN® PCR Cloning Handbook (Ligation Protocol, 04/2001). The ligation mix contained 1 µl pDrive Cloning Vector (50 ng/µl), 4 µl PCR product (65 ng), and 5 µl Ligation Master Mix (2x). During the incubation period of 30 minutes at 4°C, the PCR products were incorporated in the pDrive Vector.

The E. coli One Shot® TOP10 Electrocomp™ Cells (Invitrogen) were grown in SOB until an

OD600 of 0.6 was achieved. The cell cultures were kept on ice (4°C), and subsequently were washed four times with cold (4°C) sterile water until the cells became electrocompetent, due to the osmotic effect. These 50 µl of electrocompetent cells were mixed with 2 µl of the ligation mix in a specially conceived cuvette. The electroporation occurred at 2300V, which was maintained approximately 2.5 seconds. After the electroporation the cells were inoculated in 500 µl pre-warmed (37°C) SOC. After one hour, 100 µl of the transformation mixture was inoculated on solid 869 medium (containing 100 µg/ml Amp, and 50 µM IsoPropyl-Beta-D- ThioGalactopyranoside (IPTG) and 80 µg/ml X-gal for blue/white screening of recombinant colonies.

60 Material and methods

The positive (white) clones were stored at -80°C, after dissolving an early stationary phase culture (grown in 869 Amp50) of identical clone cells in a glycerol solution, containing 25% glycerol, 10 mM Tris (pH 8.0), and 2 mM MgSO4.

3.2.6. DNA sequencing DNA sequences were obtained using the BigDye® Terminator v 1.1 Cycle Sequencing Kit (Applied Biosystems, Lennik, Belgium). To optimize the results, the PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN® Benelux, Venlo, The Netherlands), yielding a minimum of 3-10 ng of 'pure' DNA. The sequencing reactions (Table 8) were performed by Arlette Michaux, following manufacturer's instruction and were run in an ABI Prism® 310 Genetic Analyzer (Applied Biosystems, Lennik, Belgium).

Table 8. Primers used for the DNA sequencing Gene Name Primer sequence Product Annealing size (bp) temp (°C) 16S rRNA P338F 5' - ACT CCT ACG GGA GGC AGC - 3' ~180 55 P518R 5' - ATT ACC GCG GCT GCT GG - 3' ~455 55

3.3. Preparation of total RNA from Bacteria 3.3.1. Total RNA isolation Total RNA was extracted with the Promega SV Total RNA Isolation System Kit. Lysis of the bacterial cells occurred due to addition of lysozyme (3 mg lysozyme/ml in 10 mM Tris/EDTA-buffer). The cellular ribonucleases were inactivated with Guanidine ThioCyanate (GTC) and β-mercaptoethanol. GTC in combination with SDS degraded all nucleoprotein complexes, allowing the release of protein-free RNA in the solution. Dilution of cell extracts in the presence of high GTC concentrations allowed selective precipitation of cellular proteins. Subsequently, the RNA was isolated through selective precipitation by means of ethanol, followed by the adsorption of the RNA on the silica surface of the glass fibre membrane. RNase-free DNaseI was added to the silica membrane to remove the contaminating DNA. Subsequently, the bound RNA was purified from contaminating salts, proteins, and cellular impurities by means of several wash steps. In a final step, the total RNA was eluted from the membrane by adding nuclease-free H2O. The resulting RNA solution was stored at -80°C. The experimental procedure is described in the technical manual of the Promega SV Total RNA Isolation System Kit.

61 Material and methods

3.3.2. Determination of RNA purity and RNA concentration All total RNA concentrations were determined with the RNA 6000 Nano Assay for the Agilent 2100 Bioanalyzer. This is a capillary biochip conceived to analyze RNA purity and determine the RNA concentration of a given sample (one chip can analyze up to 12 samples simultaneously). The RNA 6000 ladder (Ambion Inc., cat. no. 7152) was used as ladder. After the chip had run, the supplied software visualised the migrated RNA yielding a gel or peak profile (electropherogram) that showed the 16S and 23S peaks and the extent of eventual RNA degradation that might have occurred during RNA extraction. In addition, the Agilent software calculated the RNA concentration and purity, based on the comparison with the standard ladder.

3.4. RNA-based molecular analyses 3.4.1. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) Reverse Transcriptase (RT) is both an RNA- and DNA-directed DNA polymerase that is used to synthesize single stranded cDNA from mRNA. In addition to polymerase activity, the RT enzyme possesses RNase H activity that enables it to degrade the RNA from RNA/DNA hybrids. RT-PCR was performed with the TaqMan Reverse Transcription Kit (Roche). The master mix per sample contained 38.5 µl RNA template (1 µg/ 100 µl), 2.5 µl Multiscribe RT-polymerase (a recombinant Moloney Murine Leukemia Virus RT, which possibly has RNase H+ function)(50 U/µl), 5 µl Random Hexa primers (50 µM), 2 µl RNase inhibitor (20 U/µl), 20

µl dNTP Mixture (2.5 mM)(TaKaRa), 10 µl (10x) RT-PCR Buffer, and 22 µl MgCl2 (25 mM). The RT-programme comprised one cycle of 10 min at 25°C (primer binding), 30 min at 48°C (RT enzyme activation), and 5 min at 95°C (RT enzyme inactivation). The resulting cDNA was stored at -80°C.

4. Detection of bacterial conjugation 4.1. Triparental exogenous plasmid isolation Bioreactor samples and the used strains were grown overnight in their respective selective media. For this method E. coli CM1962 was used as the plasmid donor, harbouring the mobilizable pMOL222 plasmid containing a NiR-gene. Overnight-cultures CM1962, and -2 AE815, and cultured reactor samples (N0-N7) were spun down, and redissolved in 1 ml 10 M

MgSO4. From each of the cultures small aliquots (drops) were mixed and spotted on empty

62 Material and methods sterile petridishes in aliquots of 60 µl. Thereafter, aliquots of CM1962, AE815, and culture samples were spotted on the solid conjugation medium (869 medium) using the Parallel-plater (PP) with sterilization (by dipping in 98.8% Ethanol and flaming the PP-needles) between each spotted aliquot. The mixing of the three bacterial cultures allowed the mobilization of pMOL222 into AE815 by the action of a mobilizing plasmid present in the sample. After overnight growth on the mating medium, the mating mix patches scraped of the conjugative -2 medium with a loop, were washed twice with 10 M MgSO4, were dissolved in 60 µl aliquots. Small drops of these aliquots were spotted and inoculated on sterile selective medium (284 Gluconate Rif 100 µg/ml Ni 2mM), and several control media (284 Gluc Rif100 µg/ml, 284 Gluc, 869 Sm20 Km50, and 869 medium) using the PP, with sterilization of the PP between each spotting procedure. The transfer of pMOL222 enabled AE815 to grow on the selective medium. After two days of growth, AE815 was scraped of the selective medium using a loop, -2 was washed twice in 10 M MgSO4, was dissolved in 60 µl aliquots, and was spotted and inoculated on sterile selective plates. Subsequently, the DNA could be extracted from the purified plasmid-harbouring AE815. For every target group (defined by the growth medium), the triparental mating of E. coli CM404, which carries a known mobilizing plasmid, CM1962 and AE815 was used as positive control. The negative control was triparental mating between the non-mobilizing plasmid-carrying E. coli CM1120, E. coli CM1962 (mobilizable plasmid carrier), and C. metallidurans AE815. In addition to this negative control, the biparental mating of CM1962 and AE815 was used as an additional negative control. To ensure a significant screening of the triparental mating2 ability of the fraction of the bacterial community, all matings were performed in triplicate.

2 One mating comprised: (i) AE815 x sample x CM1962, (ii) AE815 x sample, and (iii) sample.

63 Results E. Results 1. Origin of reactor samples and sample preparation An axenic mixed culture of Nitrosomonas europaea and Nitrobacter winogradskyi, immobilized by surface attachment on polystyrene beads, was used for nitrification in a packed-bed reactor at pilot-scale. The total volume of the reactor was 8.1 dm3. Basic measurements were obtained on-line by probes (pH, dissolved oxygen, temperature) located at the top and bottom sections and their values were weighted by the control system. During the entire run fixed operating conditions were: pH 8.1, stirring 400 rpm, and light protection (using tin foil). The optimal temperature proved to be 30°C, coinciding with maximal nitrifying activity. The total gas flow-rate (3 dm3 min-1) and as well as liquid flow-rate (0.0028 dm3 min-1) were kept constant. Dissolved oxygen concentration was controlled by adding pure oxygen or nitrogen in the input gas. Exhaust gas was recirculated and an on-off valve regulated pressure in the loop. Oxygen partial pressure in the culture medium was regulated by using oxygen-enriched air if necessary. Ammonia conversion ranged from 95 to 100% when the oxygen concentration was maintained above 80% saturation. The maximal + -2 -1 surface removal rates were measured as 1.91 gN-NH4 m day . Good stability and reproducibility were observed for four years (Pérez et al., 2004). After four years, the pilot reactor still performed ammonia conversion with high efficiency, with the effluent containing - - + 296 mg N-NO3 /l, 0.073 mg N-NO2 /l, and <0.169 mg N-NH4 /l, thus indicating an ammonia conversion of 99% (Pérez, pers. comm.). Sterile COC medium was prepared and fed to the reactor. Although originally intended, optimal axenic reactor processing conditions could not be guaranteed due to difficult sterile valve handling and the use of large media influent volumes. Therefore, it can safely be assumed that other bacteria have entered the reactor and have settled in the biofilm community. However, because of previous failures to maintain an axenic co-culture of Nitrosomonas europaea and Nitrobacter winogradskyi for periods exceeding several months (D. Demey, pers. comm.), the high nitrifying performance of the pilot reactor offered the opportunity to investigate a functionally stable nitrifying reactor, which had run without decreasing nitrifying efficiency for a period of four years. For the analysis of biofilm structural and microbial analysis (research being performed at UAB) as well as the analysis of nitrifying activity and community structure along the flow- axis of the reactor, the bioreactor had to be dismantled. The reactor was dismantled in seven parts and divided using sterile gloves, dishes and instruments (Figure 11). Positions N0 and N1 originated from an identical section, position N0 was the influent-attached compacted biofilm,

64 Results and position N1 was biofilm foam-attached biofilm. However, the operating room was not decontaminated. Hence, it is possible that airborne microorganisms could have entered the samples (Figure 11). To limit the chance of contamination, the dismantling was done quickly; where after the samples immediately were prepared for storage. Fifty ml of each of the eight fractions were incubated at -80°C in 25% glycerol, whereas the rest of the biomass of each of the eight fractions were lyophilized (Pérez, pers. comm.).

N 5

N 0

N7 N6 N5 N4 N3 N2 N1 N0

Figure 11. Scheme of sections (N0-N7) in which the CIII bioreactor was divided after dismantling.

65 Results

2. Ammonia analyses 2.1. Reactivation of lyophilized samples

In order to activate all possible members of the CIII consortium, the samples were cultured in a range of carefully chosen media to enable growth of different fractions of bacteria; AOB, NOB, heterotrophic organisms (Table 9). It is important to remark that, 1-2-3 is a medium specifically conceived for growing N. europaea in pure culture and COC medium is the original medium used for the pilot run of the reactor.

Table 9. Media used for culturing specific subfraction of bacteria residing in the nitrifying reactor (CIII) of MELiSSA Medium Targeted group of bacteria Days of Theoretical capacity to support Theoretical capacity to support (Medium type) growth growth of N. europaea growth of N. winogradskyi 1-2-3 Nitrosomonas europaea 2 ++ - 284 Gluc Soil bacteria 3 + - 869 (Standard complex medium) 3 - - COC AOB & NOB 7 ++ - NMM AOB 7 ++ - - NO2 NOB 7 - ++ PCM (Standard complex medium) 3 - - SW AOB 7 ++ -

Growth was observed for all lyophilized samples (N0-N7) in all media, except the AOB- targeting media. The problem demonstrated itself when growing the lyophilized samples in SW medium. SW medium is an excellent medium to assess the growth of nitrifying bacteria, since it is pink coloured3 at pH 7.5, and the colour is an indicator of nitrification activity. Pink will turn yellow when nitrification occurs (pH diminishes). Hence, in active samples, a colour change to yellow would be expected, as to confirm growth of the nitrifiers. However, since this colour change was only observed in SW-grown N4 samples, the conclusion can be drawn that N4 is the only actively nitrifying lyophilized sample (Figure 12).

3 The colour is caused by the cresol red (pH 7.2-8.8) in the medium and is commonly used as indicator for nitrification.

66 Results

(a) (b)

Figure 12. Flasks containing lyophilized samples (N0-N7) incubated for 7 days in SW medium. (a) First inoculation, (b) second inoculation.

2.2. Detection of ammonia consumption of reactivated lyophilized samples Nitrifying organisms could have been very sensitive to the lyophilization process. Hence, the observed difference in medium-colour (when growing the lyophilized samples in SW medium, Figure 12) between samples N0-3,5-7 and N4 could be caused by the abundant presence of viable nitrifying bacteria in the lyophilized N4 sample in comparison with samples originating from the other sections in the reactor. Therefore, ammonia analyses using the indophenol method were performed on the supernatant of lyophilized samples (N0-N7) grown in SW medium and COC medium, to test and quantify the samples' ammonia oxidation activity. For the standardization of the ammonia analysis, a dilution row of known ammonia concentrations was plotted against the measured OD. The standard curve indicated a linear relation between ammonia concentration and measured OD value. The standard curve for the lyophilized samples grown in SW medium was defined by the equation: y = 0.1088x + 2 0.0151, with R = 0.999. The ammonia content in the supernatant of samples N0-N3 and N5-N7 were not significantly different in comparison with the positive control (Figure 13). N4, however, was significantly different from the positive control, in both one-tail and two-tail two-sample t-Tests with unequal variances, with P(T≤t) = 0.00087 and P(T≤t) = 0,001752, respectively. These results indicate that when grown in SW medium, N4 is the only lyophilized sample actively consuming ammonia after seven days of culture (Figure 13).

67 Results

2 1,8 l /

g 1,6 m (

t 1,4 n e 1,2 nt o

c 1 m

u 0,8 ni o 0,6 m

m 0,4 A 0,2 0 C+ N0 N1 N2 N3 N4 N5 N6 N7 Sample

The same analysis was repeated for lyophilized samples grown in COC medium. The standard curve for the lyophilized samples grown in COC medium was defined by the equation: y = 0,0948x + 0,0156, with R2 = 0,999. As for the analysis performed on the SW supernatant, the ammonia content in the supernatant of samples N0-N3 and N5-N7 grown in COC medium were not significantly different from the positive control (Figure 14). Again, the ammonia content of the N4-sample was significantly different from the positive-control, in both one-tail and two-tail two-sample t-Tests with unequal variances, with P(T≤t) = 0,005165 and P(T≤t)

=0,010329, respectively. Initially, samples originating from section N1 seemed to oxidize ammonia. However, the difference with the sterile unused medium's ammonia content was not significant.

68 Results

5 4,5 l /

g 4 m (

t 3,5 en t 3 n

co 2,5 m u

i 2 n 1,5 mo m

A 1 0,5 0 C+ N0 N1 N2 N3 N4 N5 N6 N7 Sample

Figure 14. Ammonia content of COC-medium supernatant after 7 days of growth of the lyophilized + samples harvested from the CIII pilot reactor. C : sterile medium, N0-N7: CIII lyophilized sample. Error bars indicate the standard deviation.

These results indicate that when the lyophilized samples are grown in an AOB-targeting medium, N4 is the only lyophilized sample actively consuming ammonia after seven days of culture (Figure 13 and 14). The results therefore support the probability of a sensitiveness of nitrifiers to the lyophilization process.

2.2. Detection of ammonia consumption of reactivated frozen samples To determine whether the frozen samples had conserved their nitrifying ability, the ammonia analysis was performed on frozen samples, reactivated by inoculation in SW medium. These samples were grown for 2.5 days in SW medium, until the colour-transition of red-to-yellow occurred in all samples (N0-N7). This colour change was a first indication of nitrifying activity, and could be expected from the nitrifying performance during reactor processing. Likewise, ammonia concentration was quantified using the indophenol method. The standard curve for frozen samples grown in SW medium was defined by the equation: y = 0,1331x + 0.0177 with R2 = 0.9992.

69 Results

1,8 1,6 ) l /

g 1,4 m

( 1,2 t n e 1 nt o

c 0,8 a ni

o 0,6 m

m 0,4 A 0,2 0 C+ N0 N1 N2 N3 N4 N5 N6 N7 Sample

The results in Figure 15 indicate a significantly quicker ammonia usage (P(T≤t)< 0.004 for one-tail t-Test, and P(T≤t) < 0.008 for two-tail t-Test) for the first four samples (N0-N3) in comparison with the latter four (N4-N7). The ammonia content of N4-N7 was not significantly different from the positive control. Since the results indicate that, the first samples oxidize significantly more ammonia than the last four, during the same incubation period, it is probable that there is a higher abundance of AOB in the first four samples relative to the last four. This higher abundance of AOB closer to the bottom of the reactor can be expected due to the relative position of the sample to the influent.

3. Community analysis 3.1. DGGE-based total community analysis 3.1.1. DGGE-based total-community analysis When examining the DGGE profiles obtained after 16S rRNA-gene PCR amplification, the observed total bacterial community profiles in the uncultured lyophilized samples seemed to give only a very weak signal, due to an inefficient DNA extraction performed directly on the lyophilized samples. However, in the uncultured frozen samples the profiles appeared to be relatively complex (Figure 16). The DGGE reference ladder was assembled and supplied by VITO (Mol, Belgium), and is further referred to as 'VITO ladder' (Figure 16) (Geets et al., 2005).

70 Results Firstly, bands #1614, #1615, #1609, # 1606, and #1607 appeared to be on the same position as the five N. europaea (Neu) bands in samples N0 and N1. However, in the following samples

(N2-N7), #1614, and #1615 disappeared, only #1609, #1606 and #1607 remained present in all samples. Whether #1606 and #1607 (and #16N4 and #16N5) are real Neu-associated bands remains to be determined, because of the relatively weak bands in the pure Neu-lane (#16N4 and #16N5). Bands #1605 and #1612 were clearly present in all samples, although less clear in N4 and N5. Band #1616 was absent in N0-N2, but increased gradually in intensity from sample N3 to N5, to diminish again in intensity in samples N6 and N7. Band #1617 was only present in samples N0 and N1, and its presence seemed to be correlated with the presence of Neu-like bands, #1614 and #1615. However, #1617 does not seem to be a Neu-associated band. _Uncultured lyophilized_ _Uncultured frozen_

L N0 N1 N2 N3 N4 N5 N6 N7 N0N1 N2 N3 N4 N5N6 N7 L N #1614 #16N1

#16L1 #16N2 #1615 #1609 #16N3 #16L2 #1605 #1612 #1616 #16L3 #1617 #1606 #16L4 #16N4 #16N5 #1607 #16L5 #1608 #16L6 #16L7

#16L8

#16L9 #16LX

Figure 16. DGGE profile of uncultured lyophilized and uncultured frozen samples. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene.

3.1.2. DGGE-based in silico clustering analysis

To link the differences in ammonia-oxidizing efficiency observed between samples N0-N4 and

N5-N7 with the community composition, a clustering analysis was performed to yield information on a functional gradient along the feed-axis of the CIII bioreactor. Because of the unidirectional feed in the CIII bioreactor, a decreasing gradient of groups of bands (#1609, #1605, #1612, and #1617) can be observed, with high density close to the feed. In addition, an increasing gradient of other bands (#1608 and #1616) can be observed, with high density of the latter bands close to the exhaust. To test the hypothesis, a bioinformatical analysis was

71 Results performed on DGGE profiles of the uncultured frozen samples with the Applied Maths® program, BioNumerics™ (Sint-Martens-Latem, Belgium)(Figure 17). It was observed that the 4 pure N. europaea DGGE profile was most closely related to the N0 and N1 DGGE profiles

(>70% identical), and least related to the N6 and N7 DGGE patterns (<49% identical). There was always a very high relatedness between a sample and the following one (~99% identical), except from N1 to N2 (only 95% identical), and from N3 to N4 (97% identical).

Therefore, the samples can be subdivided into three distinct groups, N0-N1, N2-N3, and finally

N4-N7. However, the analysis might have been biased due to two bands (#1614, #1615) present in Neu, N0 and N1, and surprisingly absent from the rest of the bioreactor positions. Therefore, it is possible that removal of those bands would result in a single group containing

N0-N4. When assessing the mutual correlations within the three groups, samples N0 and N1 appeared a highly related group (98.8% identical); N2 and N3 were observed to be highly related as well (99.3% identical) and N4, N5, N6 and N7 were mutually even more closely related (with 98.1 to 99.7% identical patterns). These results confirm that the reactor can be subdivided in two parts, each with its respective functional group (similar to the results of observed ammonia consumption). The first part comprises the AOB with N0-N1 (a N. europaea fraction, #1606, #1607, and #1609), and N2-N3 (a highly performing AOB fraction, N. europaea-bands plus #1605 and #1612). Whereas the second part of the reactor comprises the NOB, N4-N7 (the presumed NOB fraction, #1608 and #1616). It is important to note that the absence or presence of these bands yielded the division in three parts, and that the presence of certain bands point to vital organisms (not necessarily ammonia oxidizers), absence in the first four samples can point to AO-inhibiting organisms or organisms vital to nitrite oxidation, e.g. N. winogradskyi.

4 The correlation was determined with Pearson correlation and UPGMA (i.e. a pair-wise clustering technique).

72 Results

100

00 9. 1 9

3 0 100 99. 99.

3 5 1 00 9. 8. 8. 1

9 9 9

4 8 7 7 100 97. 97. 95. 95.

3 9 4 2 2 00 9. 7. 8. 7. 7. 1 9 9 9 9 9

1 2 2 0 7 8 100

95. 96. 91. 92. 88. 89.

9 2 5 4 3 9 0 0 ...... 0

8 4 5 0 1 7 9 1 9 9 9 9 9 8 8

5 6 1 6 8 5 9 7 100 70. 73. 58. 61. 52. 54. 46. 48.

0 1 2 3 4 5 6 7 peae ro u

e . N Sample N Sample N Sample N Sample N Sample N Sample N Sample N Sample N

......

6 1 0%]

00. C3 1 -

0 0 10 . 0 on [ i

lat

e 80 r r o on c s r

a e 60 P C316S

73 Results 3.1.3. Ammonia-oxidizing community analysis When inoculating lyophilized samples in ammonia-rich media, ammonia-oxidation was only observed in N4 for SW medium, and in (N1 and) N4 for COC medium (see Results 2.2.). These results suggested that the nitrifiers (and other bacteria) did not survive the lyophilization process. Therefore, samples originating from all positions of the bioreactor (N0-

N7) were cultured in 284 Gluc medium, COC medium, and SW medium. The inoculum was kept minimal (2-10 mg/15ml of medium); where after 14 ml of medium (while avoiding to take much of the original inoculum) was used to extract genomic DNA of the grown community. Initially, the results for the amplification of the amoA gene (using amoA-1F and amoA-2R) showed five bands when run on the DGGE gel instead of the expected two (N. europaea has undergone a duplication of the AMO gene) (McTavish et al., 1993), this was only observed for some samples. The observed five bands were three bands with low migration distance (#AM01, #AM02, #AM03), and two migrating further (#AM04, #AM05). In the DGGE runs, the VITO ladder could not be used any more due to the low migration distance of the amoA amplicons relative to the ladder (Figure 18). The phenomenon of multiple bands (five instead of the expected two) was discussed by several authors (Briones et al., 2002) and was said to be due to non-specific amplification caused by the less efficient amplification of fragments amplified with GC-clamp attached primers. In some samples no DGGE bands were observed, this was due to a failed amplification during PCR (results not shown).

______284 Gluc______COC______SW______- - N0 N1 N2 N3 N4 N5N6 N7 N0 N1 N2 N3 L N4 N5 N6 N7 C C N0 N1 N2 N3 N4N5 N6 N7 N L #AM01 #AM02 #AM03

#AM04 #AM05 Figure 18. DGGE profiles of samples grown 284 Gluc medium, COC medium, and SW medium targeting the amoA functional gene (using amoA-1F and amoA-2R primers). L = VITO ladder, N = N. europaea (Neu) amoA genes.

The results for the ammonia-oxidizing community could be improved by two optimisations found in the literature (Stephen et al., 1999; Briones et al., 2002). From the initial results, it appeared that Nitrosomonas europaea was the only ammonia-oxidizing bacterium. To improve the detection limit, the forward primer was degenerated (from amoA-1F to amoA-

74 Results 1F*) to increase the primers' 'detection range' and subsequently allow the detection of other (formerly) undetected AOB, without losing specificity (i.e. detecting bacteria other than AOB) (Stephen et al., 1999). Although Stephen et al. (1999) did not detect other bacteria than AOB with this primer pair, it remains possible that part of the MMO-gene can be amplified nonetheless, because of the high sequence similarity this gene has with the AMO gene. The efficiency of amplification could be augmented using a two-step amplification. The first, being an amplification of the amoA fragment from total community genomic DNA with the amoA-1F* and amoA-2R primers. Then, a second round of amplification performed on the PCR products of the first round, this time with the GC-clamp attached to the forward primer. This two-step amplification was performed with the purpose to exclude aspecific amplification by the poor performing GC-amoA-1F primer.

____Uncultured samples______284 Gluc______

N0 N1 N2 N3 N4 N5 N6 N7 N0 N1 N2 N3 N4 N5 N6 N7 N #AM06 #AM07 #AMN1

#AM08 #AMN2

#AM09

#AM10 #AMN3 ______COC______SW______

L N0 N1 N2 N3 N4 N5 N6 N7 L N0 N1 N2 N3 N4 N5 N6 N7 N #AM14

#AM15

#AM06 #AM07 #AMN1

#AM08 #AMN2

#AM09 #AM10 #AMN3 Figure 19. DGGE amoA-profile of the lyophilized uncultured samples, lyophilized samples cultured in 284 Gluc, COC medium, and SW medium using the optimized amoA-1F* primer. L = VITO ladder, N = N. europaea (Neu) amoA genes.

After optimisation (Figure 19), the DGGE amoA-patterns of all samples were different for all media in comparison with the Neu-control. Four more bands (#AM06, #AM08, #AM09, and AM10) appeared in the uncultured samples and samples grown in 284 Gluc, COC medium, and SW medium; that were not present in the Neu-control. Although, bands with very low

75 Results intensity (#AMN2 and #AMN3) could be detected in the Neu-profile, with similar migration distance as #AM09 and #AM10. Bands #AM06, #AM08, #AM09, and #AM10 might indicate additional AOB in the CIII bioreactor. Only after sequencing of the respective bands (#AM06, #AM08, #AM09, and #AM10) it will be possible to unambiguously determine whether these bands represent new AOB in the bioreactor. Note that it is possible that #AM06 only is due to too high quantities of DNA loaded on the gel. In addition, #AM09 was more intense in N4 and

N7 than in the rest of the samples when grown in 284 Gluc. The N3 sample grown in SW medium showed a highly different DGGE pattern in comparison to all other samples or Neu- control (Figure 19), but this was probably due to aspecific amplification during PCR (results not shown).

3.1.4. Culture-based whole-community analysis In all positions of the bioreactor, AOB appeared to be present. Possibly, the absence of ammonia consumption in the lyophilized samples was due to a too small fraction of AOB that could still be detected because of the sensitivity of PCR-based analyses. To screen for the presence of bacteria other than AOB and NOB and to analyze the bioreactor community thoroughly, the lyophilized samples (N0-N7) and the fresh samples (N0-N7) were grown in a range of carefully selected media. These media were 1-2-3 medium, COC medium, SW medium (as AOB-targeting media); 284 Gluconate medium (as soil bacteria-targeting medium); and 869 medium, and PCM (as heterotroph-targeting media). The DGGE was performed on lyophilized samples grown in every medium individually with primers targeting the 16S rRNA-gene. Agarose gel electrophoresis confirmed good amplification with 16S rRNA-targeting primers for all samples grown in all media individually. Samples were subsequently loaded on DGGE, with N. europaea (Neu) DNA as positive control, and sterile MilliQ water as a negative control.

Different patterns were observed along the feed-axis (bottom-to-top, N0-N7), and for every medium individually (except PCM and 869 that yielded highly similar patterns). For the lyophilized samples grown in PCM and 869 medium only one band (#1601, Figure 20) was observed, which migrated further than the control Neu-bands. This band might be the same as #1605 (and #1603) (Figure 16 and 21), but this cannot be acknowledged until the PCM- and 284-Gluc-grown samples are run on the same gel. This band might correspond to a heterotrophic bacterium that successfully maintained itself in the CIII bioreactor by thriving on

76 Results organic debris originating from the autotrophic bacteria. The resemblance of DGGE profiles between PCM and 869 can be explained by the high similarity in medium composition.

______869______PCM______- N0 N1 N2 N3 L N4 N5 N6 N7 N C N0 N1 N2 N3 N4 N5 N6 N7 N L

#1601

Figure 20. DGGE profiles for the lyophilized samples grown in 869 medium and PCM, targeting the 16S rRNA gene. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene.

The DGGE patterns of lyophilized samples grown in 284 Gluconate medium yielded the most complex profile. Although N0 and N1 are nearly identical in position, the yielded DGGE profiles differ. In N0, only one band was observed (#1602), while five where observed for N1.

Positions N2 and N3 yielded highly similar profiles when grown in 284 Gluc, which is consistent with the clustering analysis (see Results 3.1.2.). In addition, the difference in migration patterns between samples N4 and N5, N6 and N7, was equally surprising; with a low diversity to high diversity transition (N4 to N5) and a high diversity to low diversity transition

(N6 to N7). However, the low observed diversity in N4 and N7 might be explained by PCR biases caused by DNA sequences visualised in bands, #1603 and #1604 (Figure 21). Finally, several bands were observed in the 284 Gluc-grown samples that were found in COC- and

SW-grown samples as well. Band #1603 is similar to #1605, band #1607 was observed in N5 and N6, #1608 was observed in samples N1-N7, but not in N0. Band #1606 was not observed in the samples grown in 284 Gluc medium. The reason why 284 Gluc yielded the most complex profile is the wide range of bacterial groups that can be grown with the medium. 284 Gluc is able to support growth of soil bacteria in general, and AOB; due to the presence of ammonium (1.07 g/l). It is interesting to note that #1606 and #1607 reside on identical positions as #16N4 and #16N5 in the pure Neu sample. Note that, in case #1606 and #1607 were spotted; #1609, which visualizes the most intense band associated with the DGGE pattern of Neu was never observed with #1606 and #1607 in a sample lane.

77 Results 284 Gluc______COC______SW______

L N0 N1N2N3 N4N5N6 N7 N N0N1 N2 N3L N4 N5N6 N7N N0 N1N2 N3N4 N5N6 N7 N L

#1604 #1609 #1605 #1603 #1618

#1619 #1606 #1607

#1608 #1602

The DGGE profiles of lyophilized samples grown in COC medium and SW medium yielded similar patterns, with several bands appearing at the same height (#1605, #1606, 1607, and #1608). COC-medium-grown samples, however, yielded a slightly more complex profile than samples grown in SW medium, indicating a broader growth support in COC medium. Bands #16N3, #16N4, and #16N5, which corresponded to Neu sequences, were only found in COC- cultivated samples N1 and N4. This however, is a very interesting result; and might explain the strange ammonia usage results (Figure 14), because only in COC-cultured N1 and N4, growth of N. europaea was observed. Interestingly, N1 and N4 cultivated in COC were the only two of the lyophilized samples that performed ammonia oxidation. Although, N1 did not consume ammonia significantly when grown in COC (Figure 14), it can be said that in these positions

(N1 and N4) N. europaea remained the dominant AOB and no other AOB have replaced N. europaea in the bioreactor. It is possible that bands #1607 (yellow square, Figure 21) and #1619 (blue square, Figure 21) enhance nitrification, because their presence coincides with nitrification activity (Figure 13 and 14), band #1618 (green square, Figure 21) possibly has an inhibiting effect on nitrification because its presence coicides with a lack of nitrification activity (Figure 13 and 14). Again, bands mostly migrated further than Neu-controls, but were well in range of the VITO ladder.

Lyophilized samples grown in 1-2-3 medium yielded the most surprising DGGE pattern, with only three different bands (#1609, #1610, and #1611; Figure 22). One (#1609), migrating similarly to the Neu-band (which is only one of the five bands of the Neu-DGGE profile;

78 Results

Figure 16); #1610 migrating further (not identified); and N4 presented only one band (#1611) that migrated differently from #1609 and #1610. Sample N2 also yielded only one band, however, it migrated similarly to #1610. Although sequencing needs to be performed on the bands, it can be assumed that #1609 is the bioreactor-residing N. europaea. Because 1-2-3 medium is quite selective for N. europaea, it is possible that organism #1610 could either be N. winogradskyi; which could thrive on nitrite produced by N. europaea or another autotrophic AOB which has a similar metabolism as N. europaea.

______1-2-3______

L N0 N1 N2 N3 N4 N5 N6 N7 N

#1609

#1611

#1610 Figure 22. DGGE profiles of the lyophilized samples grown in 1-2-3 medium, targeting the 16S rRNA gene. L = VITO ladder, N = N. europaea (Neu) 16S rRNA-gene.

The presence of (an) additional nitrifier(s) using the amoA-targeting primers could be the same as the organism(s) (#1610 and #1611) detected using the 16S rRNA-gene-targeting primers when samples were grown in 1-2-3 medium. However, the DGGE on 1-2-3-medium- grown samples using amoA-targeting primers failed; and hence, it was impossible to indicate the presence of AOB in the selective medium.

Through the differential culturing approach, it was possible to distinguish the different fractions of the bacterial community. 869-medium- and PCM-grown samples yielded only one band on DGGE, but could not be identified on the uncultured gel due to problems with migration in that gel. In 284-Gluc-grown samples, N. europaea and #1608 appeared to be present in all samples (except N0), #1605 and #1607 were mostly present in the higher part of the bioreactor samples. COC- and SW-medium appeared to be media that are more specific.

In SW-grown N4 we observed the presence of #1605, #1607, and #1608; whereas in SW- grown N5 we observed the presence of #1605 and #1608. Because of the observed activity in

79 Results

N4 with the ammonia analyses, it might be possible that #1607 is an organism that enhances the performance of the nitrifying bacteria.

3.2. DGGE-based active community analysis To analyze the active population in the frozen samples, the thawed samples were inoculated in - SW medium and NO2 medium, targeting active populations. After four days of culturing, the total RNA was extracted from the samples and was converted to cDNA. After 16S rRNA- targeted DGGE analysis (Figure 23), one very intense band (#AC01) was observed for the - SW- and NO2 -medium alike. However, all SW-medium-grown samples indicated the presence of a second, less intense band (#AC03); and band #AC02 in the profiles of samples - grown in SW medium (N2-N7; Figure 23). The samples grown in NO2 -medium also indicated the presence of band #AC04, which is found in samples N0 and N1, and of band #AC05, - which is found from samples N2 to N7 in NO2 medium as well as in SW medium. It is interesting to note that #AC01 lies a little higher in sample N0 and N1, but there is no further evidence to conclude them to be different sequences other than the #AC01-sequence.

- ______SW______NO2 ______

L N0 N1 N2 N3 N4 N5 N6 N7N0 N1 N2 N3 N4 N5 N6 N7

#AC01 #AC01

#AC02 #AC04 #AC05

#AC03

+ For the organisms active in NH4 -enriched medium (SW), it is highly probable that the detected #AC01 is the same organism as #1605, #AC02 is the same as #1612, #AC03 is located below #1608, and #AC05 is the same as #1606 or #1617. For the organisms active in - the NO2 -enriched medium, #AC01 probably is the same as organism as #1605, #AC04 remains unknown, and # AC05 is the same as organism as #1606 or #1617. Meaning that

80 Results #1605 is an organism that can maintain itself in the presence of high concentrations of ammonia and nitrite.

It is important to note (with regard to the results for ammonia usage) that positions N0 and N1, - do not contain bands, #AC02 and #AC05 when grown in SW medium; or in NO2 medium. - Surprisingly, N0 and N1 appear to have an active N. europaea when grown in NO2 -enriched medium, this might be an indication of ammonia production (through nitrite or nitrate reduction) in these positions of the bioreactor.

3.3 Identification of community members 3.3.1. Identification of isolates

Supernatant originating from SW-grown N4 was inoculated on a nitrification minimal medium (NMM), to isolate the nitrifiers present in this sample. Eight isolates were obtained through plating of SW N4 culture sample on NMM solid medium and discriminated by visual screening (colour, colony morphology). All isolates were purified by sequential plating (Table 10), and the number of purification steps depended on the isolate. The DGGE migration patterns of five of the purified isolates were compared with the migration patterns of the uncultured frozen community (Figure 24). Isolate GG was not detected in the uncultured frozen sample, which might indicate that GG was only representative of a minor population in the CIII bioreactor. Isolate HKW is possibly the same organism as the one indicated by band #1607, and isolate KW possibly is the same organism as the one indicated by band #1605 or #1612. Both HKR and GW were found on the same position as band #1608, which suggests that they are probably closely related.

81 Results ____Frozen samples______Isolates____

N0 N1 N2 N3 N4 N5 N6 N7 L GG HKW KW HKR GW N #16N1

#16N2

#16N3 #1605 #1605 or #1612?

#1612 #1613

#1607 #1607

#1608 #1608

To determine whether the isolated bacteria were AOB, the isolates were screened on the presence of the functional amoA-gene. However, none of the eight isolates gave a signal on the DGGE, demonstrating them not to harbour the amoA gene (Figure 25).

_____Isolates______

L GG HKW KW HKR GW N

Figure 25. DGGE profile of five CIII isolates targeting the amoA-gene using the optimized amoA-1F* primer. L = VITO ladder, N = N. europaea (Neu) amoA genes.

The isolates were purified and genomic DNA was extracted. After PCR amplification using 16S rRNA-gene-targeting primers P63F and P518R, the amplicons were purfied, and were sequenced and identified (using the online NCBI BLAST5 program)(Table 10), as performed by Dejonghe et al. (2003). The identified sequences were amplified with both P338F and P518R primers prior to sequencing. Only six of the eight isolates could be sequenced successfully. The first isolate (GG, #1613) yielded a 502 bp fragment with the reverse primer,

82 Results and yielded highest similarity with Stenotrophomonas sp. (96% identical bases and 2% gaps). The second isolate (HKW, #1607) was identified using both forward and reverse primers, which yielded 172 bp and 489 bp sequences, respectively. Both sequences were identified to be closely related to Nocardioides sp. with 97% identical bases and 0% gaps for the forward primer and 94% identical bases and 1% gaps for the reverse primer. The third isolate (KW, #1605 or #1612) was identified with both forward and reverse primer, yielding 153 bp and 480 bp sequences, respectively. The third isolate was determined to be closely related to Sphingomonas sp. with 100% identical bases for the forward primer, and 93% identical bases and 3% gaps for the reverse primer. The fourth isolate (HKR, #1608) was also identified using both the forward and reverse primers, the forward primer yielded a 160 bp sequence 100% identical to Gordonia nitida and the reverse primer yielded a 431 bp sequence 97% identical to Gordonia sp. The fifth isolate (GW, #1608) gave a sequence for the forward primer, which yielded a 159 bp sequence 98% identical to Gordonia nitida and equally related to Mycobacterium mucogenicum, both with no gaps. The sixth isolate (Grey) was determined to be most closely related to Pantoea sp., with the forward primer yielding a 151 bp sequence of which (only) 25 bases were 100% identical to Pantoea sp. The reverse primer could have given a clearer view on the identity of isolates five and six, however, there was no clear result during sequencing. 'Red' and 'Sun' could not be identified through sequencing because of a technical malfunction during the sequencing process.

Table 10. Features of the isolates that were obtained through plating Assigned code Colony colour Colony morphology and feature(s) Identified as/ closely related to GG Yellow Large, round, flat, fast growing, sticky, Stenotrophomonas sp. heterotroph (?) HKW White Small, round, flat, slow growing, moist Nocardioides sp. KW White Small, round, flat, slow growing, moist Sphingomonas sp. HKR Pink Small, round, flat, slow growing, moist Gordonia sp. GW Pink Large, round, flat, fast growing, moist Gordonia nitida Grey Greyish pink Small, round, flat, slow growing, moist Pantoea sp. Red Dark red Small, round, flat, slow growing, delayed colour n.k. appearance, moist Sun Yellow Small, milled edges, slow growing, dry n.k. N. europaea None (pink) Nearly invisible with the eye, pink (pellet colour), N. europaea extremely slow growing Not known (n.k.) : sequence remains unknown because of an unsuccessful sequencing (due to technical problems)

Table 11. gives an overview of all discussed bands, the proposed role the organisms with which they correspond to might have, and the isolates they correspond to.

5 www.ncbi.nlm.nih.gov/BLAST, 'nucleotide-nucleotide BLAST, (blastn)'

83 Results

Table 11. Predicted importance of organisms in the CIII pilot plant of MELiSSA regarding ammonia oxidation Uncultured Cultured reactor Active reactor Importanced Reactor isolatese reactor samplesb microorganismsc samplesa - #1601 - - - #1602 - - - - #1605 #1603, #1604 #AC01 Potentially supporting (Sphingomonas sp.) #1606 - (#AC05) - - #1607 - - Potentially supporting Nocardioides sp. + #1608 #1610 (#AC03) Potentially NH4 -associated Gordonia sp. #1609 #16N3 - Important Nitrosomonas europaea - #1611 - - - #1612 - #AC02 Potentially supporting (Sphingomonas sp.) - #1613 - - Stenotrophomonas sp. #1614 #16N1 - Important Nitrosomonas europaea #1615 #16N2 - Important Nitrosomonas europaea #1616 - - - - #1617 #1611 (#AC05) - - - - #AC04 - - - - - Important Nitrobacter winogradskyi - - - - Pantoea sp. - #1618 - Potentially inhibiting - - #1619 - Potentially supporting - a: DGGE band number obtained by 16S rRNA-gene amplification on frozen samples b: DGGE band number obtained by 16S rRNA-gene amplification on cultured (in various media) lyophilized samples c: DGGE band number obtained by 16S rRNA-gene amplification on cDNA of cultured frozen samples d: Assigned role on the basis of presence or absence in the various culture conditions and bioreactor positions e: Assigned relatedness due to similar migration pattern with an isolate that was identified as closely related to. (): ambiguous indications

3.3.2. Identification through clone screening Even though none of the isolates proved to be an AOB or NOB, the identification of the other players in the bioreactor remains. Capture of all 16S rRNA-gene amplified sequences of the bacteria present in the bioreactor can be performed via clone screening. Subsequently, these clones can be identified and, based on their absence or their presence and the information found in the literature, a proposition can be made regarding their role (necessary, detrimental, opportunistic, or supportive) in the nitrifying biofilm.

Eight positions (N0-N7) and culture types (medium) were chosen for clone screening, because of their interesting DGGE migration pattern. These samples were 1-2-3-medium-grown N7,

284-Gluc-medium-grown N2, 284-Gluc-medium-grown N6, and COC-medium-grown N4 that contained bacteria identified by targeting the 16S rRNA gene; 284-Gluc-medium-grown N5, and SW-medium-grown N3 contained bacteria that were identified by targeting the amoA - gene. In addition, SW-medium-grown N1 and NO2 -medium-grown N7 contained bacteria that were identified targeting the 16S rRNA gene of their respective cDNA. Sixty-five clones were obtained: 46 clones for 284-Gluc-medium-grown N2, six clones for 284-Gluc-medium-grown

N6, five clones for COC-medium-grown N4 (amoA), one clones for 284-Gluc-medium-grown

84 Results - N5 (amoA), three clones for SW-medium-grown N1 cDNA, and two clones for NO2 -medium- grown N7. The sequencing and identification of the clones is currently in progress.

4. Detection of plasmids 4.1. PCR-based plasmid detection Götz et al. (1996) designed primer systems for amplification of different replicon-specific DNA regions. These primers were designed based on published sequences for broad-host- range plasmids belonging to the Inc-groups: IncN, IncP, and IncQ. Götz et al. (1996) detected PCR products for all IncQ primers, but not in all cases for IncN, and IncP. In an initial experiment with the replicon-specific primers, following Götz' recommendations (Table 12)(Götz et al., 1996), performed on seven different reference plasmids (Table 4), no bands were detected. Thereafter, the PCR program and/or mixture were optimized by slightly adapting the annealing temperatures, and/or using different MgCl2 concentrations in the PCR mixtures instead of those recommended by Götz et al. (1996) (data not shown). Optimisations were performed for IncP-targeting primers (amplifying oriT-, trfA2-, and korA-genes), IncQ- targeting primers (amplifying repB-, oriV-, and oriT-genes), and IncN-targeting primers (amplifying rep-, and kikA-genes; Table 12).

Table 12. Replicon-specific primers and optimisations Targeted Targeted Götz' Optimized Götz' final Optimized Inc group gene annealing annealing [MgCl2] final [MgCl2] temperature temperature IncN rep 55°C 55°C 3.75 mM 1.5 mM kikA 55°C 55°C 3.75 mM 1.5 mM IncP oriT 57°C 57°C 3.75 mM 1.5 mM trfA2 57°C 57°C 3.75 mM 1.5 mM korA 52°C 52°C 3.75 mM 1.5 mM traG 63°C 63°C 3.75 mM 1.5 mM IncQ repB 62°C 64°C 3.75 mM 1.5 mM oriV 57°C 59°C 3.75 mM 1.5 mM oriT 57°C 61°C 4.75 mM 1.5 mM

PCR-based plasmid detection was performed on lyophilized samples grown in 284 Gluc medium, 869 medium, and PCM with primers targeting the IncP oriT-, trfA2-, korA-, and traG-genes. The traG-gene is an IncP-residing gene and was important (in a first phase) to investigate because of its role in the IncP transfer system. The traG-gene encodes the IncP conjugative transfer system between gram-negative bacteria. Secondly, Götz et al. (1996) observed that IncP- (together with IncQ-) specific sequences were the most prevalent in soil and manure slurry samples. Thirdly, the triparental exogenous plasmid isolation experiments used in this study are based on the detection of transfer mediated through the IncP-type

85 Results transfer system. Therefore, it was decided in a first phase to test for the presence of IncP- residing genes.

Of the three tested media (284 Gluc medium, 869 medium, and PCM), only the 284-Gluc- grown samples N4 and N7 yielded positive results when screening for the IncP-plasmidborne oriT-gene (band #IP01)(Figure 26a). When screening for the presence of the IncP- plasmidborne trfA2-gene (band #IP02) was observed, this band was found in samples N5 and

N7. However, no PCR product was detected when screening for the IncP-plasmidborne korA- or traG-genes. The absence of PCR product in the traG-screening experiment is a first indication of absence of IncP-type-mediated conjugative mechanism in the samples (Figure 26b). In all screenings, many aspecific bands were observed; therefore, nested primers have been developed and optimization in currently in progress (L. Hendrickx, pers. comm.).

___869______PCM___ __284 Gluc__ _Uncultured frozen_

+ - + - (a) 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 N C C L1 (b) N0 N1 N2 N3 N4 N5 N6 N7 C C L L1 oriT traG

#IP01 #IP03

trfA2

#IP02

Figure 26. Agarose gel electrophoresis of the PCR-based replicon-specific plasmid detection using the IncP (a) oriT- and trfA2-targeting and (b) traG-targeting primers on lyophilized samples grown in 284 + - Gluc medium, 869 medium, and PCM. N0-N7: frozen samples, C : positive control, C : negative control, L: 100 bp ladder, L: 1kb ladder.

It is interesting to note the differences in patterns in aspecific bands between N0-N3 and N4-

N7, which were the two groups of samples showing high ammonia oxidation activity (N0-N3), and low ammonia oxidation activity (N4-N7) (Figure 15)(see Results 2.2.). The similarity was also observed in the DGGE community analysis. In all media, and for all primer-pairs, the first four samples were highly similar with simple patterns; the last four were often more complex less similar patterns.

86 Results 4.2. Detection of mobilizing plasmids 4.2.2. Initial screening of the bioreactor samples Prior to the triparental mating, bioreactor samples were checked for growth on a range of solid media. In addition, the samples were screened for antimicrobial resistances that might interfere with the triparental mating; undesired resistances were those against nickel and rifampicin (Table 13). The samples were inoculated at 30°C in the dark for two weeks to allow growth of slow-growing autotrophic bacteria.

Table 13. Screening of bioreactor samples for growth on a range of solid media and for presence of antimicrobial resistances

Medium N0 N1 N2 N3 N4 N5 N6 N7 1-2-3 + + + + + + + + 284 Gluc + + + + + + + + 284 Gluc Rif 100 - - - + - - - + 284 Gluc Rif 100 Ni 1 mM ------284 Gluc Rif 100 Ni 2 mM ------869 ++ ++ ++ ++ ++ ++ ++ ++ COC + + + + + + + + KM1 0.05% Glucose ++ ++ ++ ++ ++ ++ ++ ++ NMM + + + + + + + + NMM Km15 + + + + + + + + NMM Nal30 + + + + + + + + NMM Nal30 Km15 + + + + + + + + PCM ++ ++ ++ ++ ++ ++ ++ ++ SW + + + + + + + + - : no growth observed; + : ranging from one colony to relatively good growth; ++ : excessive growth.

Bioreactor samples grew well on 1-2-3 medium, 284 Gluconate medium, 869 medium, COC medium, KM1 medium, NMM, PCM, and SW medium. The fastest growth was observed on 869, KM1 (enriched with glucose), and PCM; these allow growth of faster growing heterotrophic microorganisms, which explained the excessive growth on these media. In addition, members of the community seemed to posses antimicrobial resistance genes against kanamycin and naladixic acid, N3 and N7 appeared to posses a resistance to rifampicin. This screening allowed the use of the selective growth approach, yielding different fractions to perform triparental mating and provided a small overview of antimicrobial properties present in the biofilm community, but needs to be extended; thus, yielding more information with regard to antimicrobial resistances in the bacterial community. It is clear that mobilizable antimicrobial resistances are undesired in the reactor because these properties can propagate to other bacteria, including virulent bacteria.

Although samples N3 and N7 were able to grow on solid media with rifampicin added, no growth was observed on the media enriched with nickel, this indicated the absence of RifR- genes in combination with NiR-genes.

87 Results

4.2.3. Exogenous isolation of mobilizing plasmids To assess which part of the bacterial community is responsible for HGT, lyophilized and frozen samples originating from these eight different positions were grown in a range of media (1-2-3 medium, 284 Gluc medium, 869 medium, NMM, PCM, SW medium). Based on DGGE profiles, COC-medium- and NMM-grown samples, 869-medium- and PCM-grown samples, and 1-2-3-medium- and SW-grown samples were pooled prior to the triparental mating experiment. Hence, a total number of 192 matings6 were needed to be performed. One mating comprised: (i) triparental matings (AE815 x cultured sample x CM1962), (ii) biparental matings (AE815 x cultured sample), (iii) reactivated samples (cultured sample only), (iv) positive control (AE815 x CM404 x CM1962), and (v) negative controls (AE815 x CM1962, and AE815 x CM1120 x CM1962). All were inoculated on the selective media to discriminate between the presence of mobilizable or conjugative and mobilizing NiR-carrying plasmids. The triparental isolation experiment yields information on both, whereas the biparental mating experiments yield information on the presence of conjugative plasmids carrying NiR-genes. Individually inoculating the cultured sample yields information on the prevalence of Ni-resistance genes, irrespective of the fact that they are chromosomal, or plasmidborne genes. Because of the vast number of matings, the classical exogenous isolation method as described by Top et al. (1994) was adapted using a replica mating technique, which was already succesfully used by Beyens et al. (2005) (see Materials and methods), to perform a qualitative screening of mobilizing plasmids.

Because the exogenous plasmid isolation approach was not a quantitative experiment, no standardization had to be performed. The mating experiments were performed in triplicate for

24 hours; where after the mating patches were washed twice with 0.01M MgSO4 and inoculated on the selective media. The qualitative mating experiments were performed on solid media, with on every plate (internal) positive and negative control matings in triplicate. All triparental mating experiments were negative, yielding no transconjugants. In all experiments, positive controls yielded a thick patch of transconjugants, whereas the negative controls were negative in all cases.

6 One mating comprised: (i) AE815 x sample x CM1962, (ii) AE815 x sample, and (iii) sample, subsequently yielding 576 patches to be analysed. All mating were performed in triplicate.

88 Results All mating experiments between AE815 and the samples were negative, yielding no transconjugants, meaning that no conjugative (Mob+, Tra+) NiR plasmids were present in the community.

When plating the COC/NMM-grown samples on 869 Km50 Sm20, an interesting growth pattern was observed. An increasing gradient in abundance of bacterial growth was observed from N0 to N7, only in the COC/NMM-grown fraction of the community. Therefore, these results might give an indication of an increasing prevalence of Sm- and Km-resistance genes in the ammonia-oxidizing bacterial-community, along the feed-axis of the bioreactor. When inoculated on rifampicin containing medium (with or without Ni), no growth was observed for the COC/NMM-grown fraction; indicating that no RifR- or combined NiR- and RifR-genes were present in this fraction. Growth, but no gradient pattern, was observed for PCM/869-medium-grown samples inoculated on 869 Km50 Sm20. In addition, no growth was observed for these samples grown on 284 Gluc medium containing rifampicin or Rif and Ni. These observations indicate that the 869-medium-grown fraction of the community does not contain RifR- (and NiR-)genes. Growth, but no gradient pattern, was observed for the 284-Gluc-grown samples inoculated on 869 Km50 Sm20. In addition, growth was observed on 284 Gluc Rif100 medium for the reactivated samples; one in three positive patches for N0, N1, N3, and N6, and two in three positive patches for N2, N5, and N7. However, no growth was observed on the 284 Gluc Rif100 Ni 2 mM medium was observed. These results indicate that some of the 284-Gluc- reactivated bacteria in the community possess Km-, Sm-, and/or Rif-resistance genes.

In none of the cultured fraction, plasmids could be exogenously isolated, indicating the absence of plasmids that are able to be transferred through action of the IncP-type conjugative system. It is still possible, that other conjugative mechanisms are active in the community of the bioreactor. However, this was not tested in this study. It is important to remark that the possibility of mobilizing plasmids present in the community remains and must be considered, but they were simply not detected. The qualitative isolation approach was performed with relatively small volumes of bacterial cultures. Therefore, it remains possible that a more sensitive method could result in the isolation of mobilizing plasmids from the reactor community.

89 Discussion

F. Discussion

Stability of the CIII bioreactor. The packed-bed CIII pilot plant proved to be a stable nitrifying reactor after having converted high loads of ammonia for four years at the UAB, Barcelona (Pérez et al., 2004). Because of previous failures to maintain an axenic co-culture of Nitrosomonas europaea and Nitrobacter winogradskyi for periods exceeding several months (D. Demey, pers. comm.), microbial additions to the co-culture might have been the source of the observed functional stability. It has been shown that within a consortium (with its members performing a series of bioconversions) some species interact with their fellow members, without having a clear function in the bioconversion itself (Dejonghe et al., 2003). They are thought to have a supporting role, through e.g. excretion of growth factors. This is consistent with previous research regarding the stability of ecosystems and the need of biodiversity (Briones & Raskin, 2003). In addition, recently performed research, regarding the influence of ecological isolation on the structural and functional stability of complex microbial communities, indicates that 'open' systems are much less prone to detrimental environmental (e.g. temperature fluctuations) and biological stresses (e.g. starvation and invasion by an opportunistic pathogen) (Franklin et al., 2005). Therefore, 'open' systems are regarded as more stable than their 'closed' counterparts. In addition, the results indicate that isolation will be an important factor influencing the activity of microbial communities on board spacecrafts. Franklin et al. (2005) proposed a way of mitigating these effects, by either including communities with high diversity or by periodically re-inoculating the systems using diverse inocula transported from Earth.

Observed nitrifying activity of samples originating from the CIII pilot plant. The ammonia conversion analyses provided additional evidence that the CIII microbial consortium was a functionally active nitrifying community. However, the loss of nitrifying activity in seven of the eight reactor positions in the lyophilized samples was most likely due to a sensitiveness of nitrifiers to the lyophilization process. Based on to the persistent ammonia- oxidizing performance of the community in the N4 reactor position, a functional community was observed in that position when growing the lyophilized samples in COC medium. In

COC-grown position N4 these bacteria were #1609 (N. europaea), #1605 (possibly

Sphingomonas sp.), #1608 (possibly Gordonia sp.), and #1619 (not identified). This made N4 a very usefull sample to investigate the composition of a functionally stable community. In

SW-grown lyophilized N4 only three bands were observed, #1605 (possibly Sphingomonas sp.), #1607 (possibly Nocardioides sp.), and #1608 (possibly Gordonia sp.), with surprisingly,

90 Discussion the absence of the N. europaea band. Therefore, it was impossible to determine unambiguously the cause of the observed activity.

The DGGE analysis demonstrated that the CIII co-culture was enriched with a vast number of bacteria. Subsequent, identification demonstrated them to be both gram-negative and gram- positive bacteria. A clustering analysis was performed that showed that the reactor could be 'divided' into two parts. Strangely enough, this separation did not appear to be fluent, but rather sudden in the middle of the reactor. These findings were also observed in the DGGE analyses, where samples N0-N3 were determined to be quite different from N4-N7. This however, does not mean that N. europaea was not present in the higher parts of the reactor. In the DGGE of the frozen samples N0-N3, bands #1605, #1607, #1608, #1609, and #1617 were detected, indicating their potential importance during the first phase of ammonia conversion.

Analysis of the spatial distribution of the CIII bioreactor. In parallel with the research performed at SCK•CEN, Montras et al. (2005) performed oligonucleotide-based Fluorescent In Situ Hybridization coupled with Confocal Laser Scanning Microscopy observeations to gain insight in the spatial distribution of the nitrifying bacterial population of the CIII pilot plant along the bed's vertical axis. The use of probes specifically conceived to target the original members of the co-culture, gave an indication of their relative abundance. Initial qualitative interpretation indicated heterogeneous biofilm distribution in relative cell density of both species along the vertical feed-axis, with a high abundance of N. europaea closer to the feed, and an increasing abundance of Nitrobacter closer to the exhaust. In the middle of the reactor, both N. europaea as well as N. winogradskyi appeared to present. In addition, probing with a general eubacterial probe indicated the abundant presence of viable and active bacteria along the feed-axis. In addition, other bacterial species were found (investigation currently in progress)(Montras et al., 2005). Hence, it can be demonstrated that N. europaea and N. winogradskyi were still prominently present.

Detection of AOB in the CIII pilot plant along the bed length. The ammonia usage analysis on the frozen samples gave an indication of the relative abundance and community structure along the feed-axis of the reactor. These results were consistent with those obtained through FISH analysis. Indeed, if there is a greater relative abundance of AOB at the bottom of the reactor (close to the feed) then one would expect to observe a relatively faster ammonia oxidation there. Based on the FISH analysis, one would hence expect to see a relatively faster

91 Discussion nitrite oxidation at the top of the reactor. Unfortunately, this was not tested. However, the screening for presence of the amoA-gene did not yield any indication of a greater relative abundance of AOB closer to the feed, which is probably due to the DGGE technique. To quantify the abundance of screened sequences, it is necessary to couple the DGGE analysis with a 16S rRNA- or amoA-gene oligonucleotide probe cell-blot hybridization approach (Hastings et al., 1998). Since these hybridization experiments were not performed, no quantitative information can be yielded regarding the relative abundance of AOB along the vertical feed-axis. Therefore, based on the DGGE results, it can only be said that amoA- harboring bacteria are present along the length of the CIII bioreactor.

Analysis of bacteria active in ammonia- and nitrite-rich media. Band #AC01 (possibly Shingomonas sp.) was present in samples originating from all positions along the feed-axis of the bioreactor. Band #AC05 (possibly Gordonia sp.) was only active in all SW-medium- grown samples. Band #AC05 (unknown bacterium) was active in the samples originating - from the upper parts of the reactor, both in SW-medium- and NO2 -medium-grown samples. - Finally, the N. europaea band was only observed in NO2 -medium grown samples, which was a strange observation since N. europaea is an AOB. However, the DGGE analysis performed to identify the active bacteria probably was subject to a bias. This bias was probably caused by the large difference in growth rates between the autotrophic members of the co-culture and the heterotrophic bacteria present in the reactor.

Characterization of the isolates based on literature. Since biofilms may provide a greater diversity of niches compared with activated sludge (Sakano et al., 2002), it was no surprise to find a range of bacteria, other than N. europaea and N. winogradskyi. Pure cultures were established by single colony isolation onto fresh NMM agar plates. It is important to note that not for all bands, yielded in the DGGE analyses, the respective bacterium has been identified. The six isolates have been identified as closely related to Sphingomonas sp., Nocardioides sp., Gordonia sp., Gordonia nitida, Stenotrophomonas sp., and Pantoea sp. and are probably only a small fraction of all bacteria present in the CIII bioreactor. Based on amoA-targeted DGGE, none of the isolates appeared to be autotrophic AOB; still, all are related to bacteria harbouring nitrogen metabolic mechanisms. The alphaproteobacterial genus Sphingomonas was defined as a group of Gram-negative, rod- shaped, chemoheterotrophic, strictly aerobic bacteria that contain glycosphingolipids (GSLs) instead of lipopolysaccharide in their cell envelopes, and typically produce yellow-pigmented

92 Discussion colonies (Yabuuchi et al., 1990). Glucose is assimilated by most strains (Balkwill et al., 1997) and is frequently used as a growth substrate in both complex and defined media. A wide variety of other sugars including arabinose, fucose, galactose, lactose, mannose, melibiose, sucrose, trehalose, and xylose also are commonly assimilated (Balkwill et al., 1997; Denner et al., 2001; Fujii et al., 2001). Sphingomonads are widely distributed in nature, having been isolated from many different aqueous and terrestrial habitats, as well as activated sludge, clinical specimens (Dejonghe et al., 2000; Layton et al., 2000; Tiirola et al., 2002), and a mercury-reducing biofilm reactor (von Canstein et al., 2002). A bacterium closely related to Sphingomonas sp. has been reported in the Biological Wastewater Treatment Reactors of the ALS bioregenerative life support system (Sakano et al., 2002). This ALS has the same goal as the MELiSSA project. The widespread distribution and numerical abundance of these organisms suggest that they play an important role in the cycling of carbon and other nutrients in marine environments. Sphingomonads are metabolically versatile and, thus, are able to utilize a wide range of naturally occurring organic compounds as well as many types of refractory environmental contaminants (Halden et al., 1999). However, relatively little is known regarding the ecology of this diverse group of microorganisms. Some of the sphingomonads (especially Sphingomonas paucimobilis) also play a role in human disease, primarily by causing a range of mostly nosocomial, non-life-threatening infections that typically are easily treated by antibiotic therapy. Owing to their remarkable biodegradative and biosynthetic capabilities, sphingomonads have been utilized for a wide range of biotechnological applications, from bioremediation of environmental contaminants to production of extracellular polymers and have subsequently been found in biological filters (Sakano & Kerkhof, 1998). Sphingomonas sp. was reported as recipient of bacterial conjugation, recieving a catabolic plasmid (Dejonghe et al., 2000; Tiirola et al., 2002). However, the real ecological role of the Spingomonas sp. in the CIII bioreactor remains unknown.

The genus Nocardioides contains Gram-positive, non-acid-fast, aerobic and mesophilic nocardioform actinomycetes that can form a mycelium that fragments into irregular rod- to coccus-like elements. However, not all Nocardioides spp. are reported to form mycelia (Yoon et al., 1998). The morphological heterogeneity found in the genus Nocardioides makes it necessary to use chemical markers to characterize the genus. There are currently five validly described Nocardioides species, namely N. albus, N. jensenii, N. luteus, N. plantarum and N. simplex (Yoon et al., 1998). Nocardioides sp. have been observed in a range of environments,

93 Discussion mostly soils, like aromatic hydrocarbon enriched soils, where Nocardioides sp. had an undetermined role (Greene et al., 2000), and rice paddy soils, where it was observed to be an abundant organism (Hengstmann et al., 1999).

The genus Gordonia (in the family of Gordoniaceae) belongs phylogenetically to the suborder Corynebacterineae, the mycolic acid group within the order Actinomycetales (Stackebrandt et al., 1997), and its classification has changed drastically in recent years. The genus Gordonia comprises 19 validly published species. Members of this genus differ from the genus Nocardia by their ability to reduce nitrate and the absence of a mycelium (Goodfellow & Alderson, 1977). Members of the genus Gordonia are aerobic, non- sporeforming, gram-positive to gram-variable, slightly acid-fast, nonmotile, catalase-positive, nocardioform actinomycetes, and susceptibility to lysozyme has been demonstrated. The term “nocardioform” is morphologically descriptive and refers to mycelial growth with fragmentation into rod-shaped or coccoid elements (Lechevalier, 1989). The colony morphology of Gordonia species varies from slimy, smooth, and glossy to irregular and rough; it may even differ within one species depending on the medium used for growth (Linos et al., 2002). The colors of the colonies cover a broad range, including white, yellow, tannish, orange, red, and pink (Xue et al., 2003), many Gordonia species also produce colonies with reddish pigmentation, indicating their capacity to synthesize significant amounts of carotenoids. The deviations in the 16S rRNA-gene sequences of different Gordonia species occur mostly in two hypervariable regions: between nucleotide positions 136-229 and 996-

1028. Therefore, the detected Gordonia sp. in the CIII bioreactor might only be a closely related bacterium. All species differ from each other in these regions except G. alkanivorans and G. nitida (the latter was identified in the CIII bioreactor). Several Gordonia species were isolated due to their capabilities to degrade or modify aliphatic and aromatic hydrocarbons, halogenated aromatic compounds, benzothiophene, nitrile, polyisoprene, xylene, rubber materials (Arenskötter et al., 2001). Most Gordonia species have been isolated from environmental sources; however, a few are also sporadically associated with human infections. In almost all cases, patients suffered from immunosuppression after a disease, whereafter infection by Gordonia species occurred only secondarily and was caused by G. bronchialis. Gordonia species may play an important role during wastewater treatment and in biofilters. Since the genus Gordonia is closely related to the genera Nocardia and Mycobacterium, members of which cause tuberculosis or leprosy (Aspinall et al., 1995). A Gordonia-produced compound, gordonan, might support the process of infection or the

94 Discussion fixation of Gordonia spp. to a host, as do biofilms produced by P. aeruginosa. However, whether gordonan also has effects on mammalian cells is still unclear (Kondo et al., 2000) and since some members of the genus Gordonia are opportunistic pathogens one must seriously consider the risks associated with these bacteria.

The genus Stenotrophomonas is phylogenetically placed in the class Proteobacteria, where it forms a deep branch located at the root of the gamma-subclass together with the genus Xylella (Moore et al., 1997). Yellow-pigmented bacterial strains, which show chemotaxonomic characteristics similar to those of the genera Stenotrophomonas, have been frequently isolated from biofilters for waste gas treatment (Lipski & Altendorf, 1997). However, Finkmann and collegues (2000) investigated the physiological properties of Stenotrophomonas strains isolated from experimental biofilters fed with ammonia or ammonia and dimethyl disulfide, and showed that the isolates were able to reduce nitrite to nitrous oxide while nitrate was not reduced. The accumulation of nonvolatile nitrification products and the resulting acidification of biofilters and similar systems (e.g. bioreactors) can be reduced by such mechanisms (Finkman et al., 2000). This indicated the presence of a denitrification pathway, which was unknown for strains of the genus Stenotrophomonas. Therefore, they proposed that this strain was a new species, Stenotrophomonas nitritireducens. In CIII an isolate was identified as closely related to Stenotrophomonas sp.. However, the problematic climatic effect of the greenhouse gas, nitrous oxide, needs to be considered in closed loop systems. The S. nitritireducens strains were resistant to fucidin, tetracycline and novobiocin, but susceptible to streptomycin, nalidixic acid and kanamycin. In addition, the Stenotrophomonas genus appears to have pathogenic bacteria in the taxon, Stenotrophomonas maltophilia is a species known to cause hospital-acquired infections and recently implicated in opportunistic infections in HIV- infected patients (Bernard et al., 1996).

The genus Pantoea forms a monophyletic unit, closely related to Erwinia. Pantoea is a gram- negative, nonencapsulated, nonspore-forming ubiquitous straight rod, which can be isolated from diverse geographical and ecological sources such as plant surfaces, buckwheat seeds, human feces and the environment (Gavini et al., 1989). The often yellow pigmented colonies are facultative anaerobic and oxidase negative. The genus Pantoea is divided into seven different species, namely: Pantoea agglomerans, Pantoea ananas, Pantoea citrea, Pantoea dispersa, Pantoea punctata, Pantoea stewartii and P. terrea. Some of these species have only recently been separated from other genera, e.g. the genus Erwinia (Mergaert et al., 1993). The

95 Discussion most prominent species of are P. agglomerans, formerly named Enterobacter agglomerans. Although Pantoea dispersa was initially described in 1989 as a new species of the genus Pantoea (Gavini et al., 1989), there are no reports in the literature of clinically significant infections involving this gram-negative rod. Endophytic bacteria, like the nitrogen-fixing strain P. agglomerans, are prokaryotic organisms that can reside in the internal portion of the host plant but do not trigger harmful reactions or disease symptoms. Instead they, can promote the growth of many field crops by producing plant growth-promoting substances and fixing nitrogen from the atmosphere (Sturz et al., 2000). Moreover, several strains of endophytic bacteria can ameliorate disease development (Benhamou et al., 1996) and induce both biotic and abiotic stress tolerance (Hallman et al., 1997) of inoculated plants. As the bacteria can proliferate inside the plant tissue, they are likely to interact more closely with the host, face less competition for nutrients, and are more protected from adverse changes in the environment than bacteria in the rhizosphere (Reinhold-Hurek & Hurek, 1998). Pantoea citrea is a spontaneous rifampicin-resistant organism and is the causal agent of pink disease in pineapple. Pujol et al. (1998) characterized a cryptic plasmid of 5229 bp, subsequently designated pUCD5000. Based on nucleotide and amino acid sequence analyses, they observed that pUCD5000 contains both replication and mobilization loci (bom and mobCABD) that are similar to those in plasmids pSW100 and pSW200 in Pantoea stewartii and pEC3 in Erwinia carotovora subsp. carotovora, respectively (Pujol & Kado, 1998).

Bacterial communities in bioreactors and wastewater treatment plants. The organisms found in the CIII bioreactor were in correspondance with the detected organisms in other ammonia-oxidizing or nitrite-oxidizing bioreactors. Other researchers have isolated (or cloned) similar bacteria from their reactors or their wastewater treatment plants. Sakano et al. (2002) performed T-RFLP analyses to analyze the bacterial biofilm community and its spatial distribution in the bioreactors of the bioregenerative life support system ALS (Sakano et al., 2002). Therein, Stenotrophomonas nitritireducens were detected, along with several other Proteobacteria, Cytophagales, Planctomytales, in addition to several Nitrospira and Nitrosomonas species and strains. In a nitrite-oxidizing bioreactor, McDevitt et al. (2000) observed Stenotrophomonas africae along with several pseudomonads, acinotobacters, and unclassified gamma proteobacteria by using a nested-PCR approach targeting the nitrate reductase (napA) gene (McDevitt et al., 2000). In the study of the microbiology of a nitrite- oxidizing bioreactor (Burrell et al., 1998), Burrell and collegues observed a Mycobacterium fallax-related organism (the genus Mycobacterium is a genus closey related to the genus

96 Discussion Gordonia) was found together with another unidentified actinomycete. As in the previous studies, a couple of Stenotrophomonas-related organisms were detected. In addition also rhodococci, staphylococci, Acinobacter-, Paracoccus-, Pseudomonas-, and Bacillus-related organisms, several Nitrospira and Nitrobacter strains were detected in the reactor performing nitrite-oxidation (Burrell et al., 1998). In wastewater treatment plants proteobacteria are abundant in the majority of the studies, and often represent more than 50% of the clones. Betaproteobacteria are the most frequently retrieved members of this division. Apart from Proteobacteria, 16S rDNA isolates affiliated to the Bacteroidetes, the Chloroflexi and the Planktomycetes were retrieved in a significant number of studies (Wagner et al., 2002). In municipal activated sludge, Snaidr et al. (1997) observed a high microdiversity of bacteria belonging to the Betaproteobacteria. Furthermore, 3% of the organisms were observed to be Sphingomonas-related bacteria, and 4% were Arcobacter-related populations (Snaidr et al., 1997). For a summary of 16S rRNA-gene-based diversity surveys of wastewater treatment plants and reactors, one is referred to Table 1 in the publication of Wagner and collegues (Wagner et al., 2002). Many organisms residing in the

CIII reactor of MELiSSA still have to be identified. It remains to be elucidated if these organisms and the species composition of the community are similar to the ones found in other ammonia-oxidizing bioreactors as well as in other reactors processing high ammonia loads. Based on the literature, it is important to note that AOB from the N. europaea/N. eutropha-lineage (including Nitrosococcus mobilis) are currently accepted as the most important for ammonia oxidation (Wagner et al., 2002). Regarding NOB, FISH analyses have proven Nitrosospira to be the dominant NOB, and not Nitrobacter as main player in the nitrite-oxidation (Wagner et al., 1996).

Assembly of the most functionally stable ammonia-oxidizing community. Based on the DGGE profiles and the culture-dependant isolation of functional groups, a (temporary7) 'ideal community' can be proposed. This 'ideal community' should be composed of nitrifiers and nitrification-supporting or ecosystem-stabilizing bacteria. A range of bands, observed during 16S rRNA screening, are candidate of an ideal AOB community: (i) #1609, Nitrosomonas europaea, (ii) #1607, Nocardioides sp., (iii) #1606, presently not identified, (iv) #1605 and/or #1612, where one of the two is closely related to Sphingomonas sp.. Band #1617 remains to be identified and subsequently its role in the community remains to be unravelled. However, because #1617 is only present in N0 and N1 and because N2 and N3 appeared to be more

97 Discussion efficient in ammonia usage, it cannot be said unambiguously that #1617 has an important supporting role.

Assembly of the most functionally stable nitrite-oxidizing community. A similar ideal community can be assembled for the NOB; here, only #1616 and #1608 are candidates, both increase in abundance closer to the exhaust and are therefore believed to be growing due to the presence of nitrite or nitrate at these positions in the bioreactor. In the DGGE of the uncultured frozen samples, band #1608 had a similar migration pattern as two isolates, both were identified as closely related to Gordonia sp.. Although N. winogradskyi was not identified in this work, it has been demostrated that Nitrobacter winogradskyi was still prominently present prior to bioreactor dismantling and that it is an important bacterium in the

CIII bioreactor.

Bacterial conjugation in the CIII pilot plant. PCR-based detection of plasmids, by targeting replicon-specific regions, has shown that only in 284-Gluc-grown samples, (N3-)N4-N5 contain IncP-plasmid-harbouring bacteria. Even though the qualitative triparental exogenous plasmid isolation, based on replica mating, has been used successfully before (Beyens et al., 2005), no transconjugants were observed in this study. The fact that no transconjugants were observed might have two causes: (i) the detection limit of the technique is too low and transconjugants might simply not have been detected, (ii) there might be no mobilizing-IncP- type plasmid-harbouring bacteria present in the bioreactor. However, gene transfer might still occur between bacteria present in the bioreactor, although the transfer mechanism might be another than the IncP-type transfer system tested in this study.

Concluding remarks. The culture conditions might have biased the results obtained in this study. Even though it is impossible to re-enact the original bioreactor conditions fully, it might be a good alternative approach to reactivate and culture the CIII pilot plant samples in biofilm promoting conditions. These biofilm promoting conditions can be achieved by e.g. providing a support for the cells and/or allowing them to form a biofilm on Biostyr® beads and mildly shaking the flasks during the incubation period.

7 Temporary: until the community has been fully characterized and all its constituents have been identified.

98 Perspectives

G. Perspectives 1) Full identification of the community. A clone screening approach was applied on a range of carefully selected samples, and the clone screening is still in progress. Only when all the bacteria of the CIII community have been identified, their function has been fully characterized and carefully composed communities have been tested on functional stability, it will be possible to compose the 'ideal' AOB and NOB community.

2) Mutation due to long-time culturing. Continuous bacterial evolution during long-time culturing cannot be neglected and needs to be studied thoroughly (Arjan et al., 2002; Zinser & Kolter, 2004). It has been demonstrated that bacteria face many different environmental stresses; possibly causing numerous important alterations in their genome (Matic et al., 2004). The mutations can be traced and can be detected with Amplified Fragment Length Polymorphism (AFLP) (Janssen, 1996). Thus, AFLP analyses could be performed on isolated N. europaea and N. winogradskyi strains as well as on the original N. europaea ATCC 19718 and N. winogradksyi Agilis ATCC 25391 strains. Comparing the obtained profiles could provide a view on the extent of the mutations that occurred during these four years inside the reactor.

3) Full analysis of HGT carrying undesired genes in the reactor. The genetic stability and safety of the CIII community must be assessed fully. The approach comprises an extended search for antimicrobial resistances, virulence genes, plasmids, PCR and bioassay detection of genetical determinants indicating the presence of other conjugative mechanisms, and HGT related genes.

4) Characterization and behaviour of the (new) CIII bioreactor community under space related conditions. It will be important to study the effect of various (space-related) phenomena. For instance, relatively little information is available in the literature regarding the effects of microgravity on microorganisms. However, a device (Rotating Wall Vessel, RWV) has been tested at SCK•CEN to assess the usefullness of this machine to simulate microgravity (Crabbé et al., 2005). Because the results indicate the induction of genes in the

RWV that are similar to the ones observed in-space, both Rhodospirillum rubrum (CII) and

Arthrospira platensis (CIVb) are currently being studied in the RWV. To date, nothing is known of the behaviour of nitrifying organisms in space. Therefore, this research is of vital importance in view of the in-space application of the MELiSSA system.

99 Perspectives

6) Coupling CIII to the other compartments. The nitrifying packed-bed bioreactor, CIII, has already been tested coupled to CIV, with success (Gòdia et al., 2002). However, for a fully functional MELiSSA loop, all compartments will have to be coupled and remain functionally stable with high performance for periods exceeding several years. The first tests will start in 2006 at VITO (Mol, Belgium).

100 References

H. References Abeliovich, A. (1992) Transformation of ammonia and the environmental impact of nitrifying bacteria. Biodegradation 3: 255-264.

Aleem, M. I. H. and M. Alexander (1958) Cell-free nitrification by Nitrobacter.

Aleem, M. I. H. and M. Alexander (1960) Nutrient and Physiology of Nitrobacter agilis. Applied Microbiology 8: 80-84.

Alexander, M. (1977) Introduction to Soil Microbiology. New York, John Wiley & Sons, Inc.

Amabile-Cuevas, C. F. and M. E. Chicurel (1992) Bacterial Plasmids and Gene Flux. Cell 70: 189-199.

Araya, R., K. Tani, T. Takagi, N. Yamaguchi and M. Nasu (2003) Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis. FEMS Microbiology Ecology 43: 111- 119.

Arenskötter, M., D. Baumeister, M. Berekaa, G. Pötter, R. M. Kroppenstedt, A. Linos and A. Steinbüchel (2001) Taxonomic Characterization of two Rubber Degrading Bacteria Belonging to the Species Gordonia polyisoprenivorans and analysis of hypervariable regions of 16S rDNA sequences. FEMS Microbiological Letters 205: 277-282.

Arjan, J., G. M. de Visser and R. E. Lenski (2002) Long-Term Experimental Evolution in Escherichia coli. Rejection of Non-Transitive Interactions as Cause of Declining Rate of Adaptation. BMC Evolutionary Biology 2(19).

Aspinall, G. O., D. Chatterjee and P. J. Brennan (1995) The Variable Surface of Mycobacteria: Structures, Synthesis of Epitopes, and Biological Properties. Advances in Carbohydrate Chemistry and Biochemistry 51: 169-242.

Balkwill, D. L., R. H. Reeves, J. K. Frederickson, D. C. White, D. B. Ringelberg, D. P. Chandler, M. F. Romine, D. W. Kennedy and C. M. Spadoni (1997) Taxonomic Study of Aromatic-Degrading Bacteria From Deep-Terrestrial-Subsurface Sediments and Description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov. International Journal of Systematical Bacteriology 47(1): 191- 201.

Bartosch, S., I. Wolgast, E. Spieck and E. Bock (1999) Identification of Nitrite-Oxidizing Bacteria with Monoclonal Antibodies Recognizing the Nitrite Oxidoreductase. Applied and Environmental Microbiology 65(9): 4126-4133.

Belser, L. W. and E. L. Schmidt (1978) Serological Diversity within a Terrestrial Ammonia- Oxidizing Population. Applied and Environmental Microbiology 36: 589-593.

Benhamou, N., J. W. Kloepper, A. Quadt-Hallman and S. Tuzon (1996) Induction of Defense-Related Ultrastructural Modifications in Pea Root Tissues Inoculated with Endophytic Bacteria. Plant Physiology 112: 919-929.

101 References

Bergstromm, C. T., M. Lipsitch and B. R. Levin (2000) Natural Selection, Infectuous Transfer and the Existence Conditions for Bacterial Plasmids. Genetics 155: 1505-1519.

Bernard, K., S. Tessler, C. Munro, M. Oughton and S. Tyler (1996) Characterization of Two Clusters of Previously Undescribed Gram-Negative Bacteria, Possibly Genus Stenotrophomonas species nova, abstr. C407. Abstracts of the 96th General Meeting of the American Society for Microbiology, American Society for Microbiology, Washington, D.C.

Beyens, G., L. Hendrickx and S. Eeckelaerts (2005) Exogene Plasmide Isolatie uit Stalen van de Liquificerende Reactor van de MELiSSA loop. Departement Biotechniek, Provinciale Hogeschool Limburg.

Biebl, H. and N. Phennig (1981) Isolation of the Family Rhodospirillaceaea. The Prokaryotes, a Handbook on Habitats, Isolation, and Identification of bacteria. M. P. Starr, J. Stolp, H. G. Truper, A. Balows and H. G. Schlegel. Berlin, Springer-Verlag. 1.

Bock, E., H. P. Koops and H. Harms (1989) Nitrifying bacteria. Autotrophic Bacteria: 81-96.

Bock, E., I. Schmidt, R. Stüven and D. Zart (1995) Nitrogen Loss Due to Denitrifying Nitrosomonas Cells Using Ammonium or Hydrogen as Electron Donors and Nitrite as Electron Acceptors. Arch. Microbiologie 163: 16-20.

Bollman, A. and H. J. Laanbroek (2001) Continuous Culture Enrichments of Ammonia- Oxidizing Bacteria at Low Ammonium Concentrations. FEMS Microbiological Reviews 37: 211-221.

Bossus, A. (2005) MOLCO Bacterial Strain Database. Mol, VITO.

Briones, A. M., S. Okabe, Y. Umemiya, N.-B. Ramsing, W. Reichardt and H. Okuyama (2002) Influence of Different Cultivars on Populations of Ammonia-Oxidizing Bacteria in the Root Environment of Rice. Applied and Environmental Microbiology 68(6): 3067-3075.

Briones, A. M. and L. Raskin (2003) Diversity and Dynamics of Microbial Communities in Engineered Environments and their Implications for Process Stability. Current Opinion in Microbiology 14: 270-276.

Brock, E. and M. Madigan (1991) Lithotrophs: the nitrifying bacteria. Biology of Microorganims. New York, Prentice Hall International Editions: 721-722.

Burall, L. S., J. M. Harro, X. Li, C. V. Lockatell, S. D. Himpsl, J. R. Hebel, D. E. Johnson and H. L. T. Mobley (2004) Proteus mirabilis Genes That Contribute to Pathogenesis of Urinary Tract Infection: Indentification of 25 Signature-Tagged Mutants Attenuated at Least 100- Fold. Infection and Immunity 72(5): 2922-2938.

Burrell, P. C., J. Keller and L. L. Blackall (1998) Microbiology of a Nitrite-Oxidizing Bioreactor. Applied and Environmental Microbiology 64(5): 1878-1883.

Cébron, A., T. Berthe and J. Garnier (2003) Nitrification and Nitrifying Bacteria in the Lower Seine River and Estuary (France). Applied and Environmental Microbiology 69(12): 7091- 7100.

102 References

Chain, P., J. Lamerdin, F. Larimer, W. Regala, V. Lao, M. Land, L. Hauser, A. Hooper, M. Klotz, J. Norton, L. Sayavedra-Soto, D. Arciero, N. Hommes, M. Whittaker and D. Arp (2003) Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithotroph Nitrosomonas europaea. Journal of Bacteriology 185(9): 2759-2773.

Christensen, B. B. (1998) Establishment of New Genetic Traits in a Microbial Biofilm Community. Applied and Environmental Microbiology 64: 2247-2255.

Clark, C. and E. L. Schmidt (1966) Effect of Mixed Culturre on Nitrosomonas europaea Stimulated by the Uptake and Utilization of Pyruvate. Journal of Bacteriology 91: 367-373.

Clark, C. and E. L. Schmidt (1967) Growth Response of Nitrosomonas europaea to Amino Acids. Journal of Bacteriology 93: 1302-1309.

Couturier, M., F. Bex, P. Bergquist and W. K. Maas (1988) Identification and Classification of Bacterial Plasmids. Microbiological Reviews 52(3): 375-395.

Crabbé, A., P. de Boever, M. Mergeay and F. De Coninck (2005) Effect van Gesimuleerde Micrograviteit op het Gedrag van de Bacterie Ralstonia metallidurans CH34. Departement Geneeskunde en Farmacie. Brussel, Vrije Universiteit Brussel: 64.

Dabert, P., J.-P. Delgenès, R. Moletta and J.-J. Godon (2002) Contribution of Molecular Microbiology to the Study in Water Pollution Removal of Microbial Community Dynamics. Re/Views in Environmental Science & Bio/Technology 1: 39-49.

Dejonghe, W., E. Berteloot, J. Goris, N. Boon, K. Crul, S. Maertens, M. Höfte, P. De Vos, W. Verstraete and E. M. Top (2003) Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain. Applied and Environmental Microbiology 69(3): 1532-1541.

Dejonghe, W., J. Goris, S. El Fantroussi, M. Höfte, P. De Vos, W. Verstraete and E. M. Top (2000) Effect of Dissemination of 2,4-Dichlorophenoloxyacitic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons. Applied and Environmental Microbiology 66(8): 3297-3304.

Deni, J. and M. J. Penninckx (1999) Nitrification and Autotrophic Nitrifying Bacteria in a Hydrogen-Polluted Soil. Applied and Environmental Microbiology 65(9): 4008-4013.

Denner, E. B., S. Paukner, P. Kampfer, E. R. B. Moore, W. R. Abraham, H. J. Busse, G. Wanner and W. Lubitz (2001) Sphingomonas pituitosa sp. nov., an exopolysaccharide- producing bacterium that secretes an unusual type of sphingan. International Journal of Systematical Bacteriology 51(3): 827-841.

Devol, A. H. (2003) Solution to a Marine Mystery. Nature 422: 575-576.

Dong, Q., D. Springael, J. Schoeters, G. Nuyts, M. Mergeay and L. Diels (1998) Horizontal Transfer of Bacterial Heavy Metal Resistance Genes and its Applications in Activated Sludge Systems. Water Science and Technology 37(4-5): 465-468.

103 References

Eckhart, P. (1996) Spaceflight, Life Support and Biospherics, Physico-chemical versus bioregenerative life support. L. Markley, W. L. Larson, R. E. Münch and J. F. Shea. Torrance, CA, Microcosm, Inc.: 125-174.

Egli, K., F. Bosschard, C. Werlen, P. Lais, H. Siegrist, A. J. B. Zehnder and J. R. van der Meer (2003) Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon. Microbial Ecology 45: 419- 432.

Ehrich, S., D. Behrens, E. Lebedeva, W. Ludwig and E. Bock (1995) A New Obligately Chemolithotrophic, Nitrite-Oxidizing Bacterium, Nitrospira moscoviensis sp. nov. and its Phylogenetic Relationship. Arch. Microbiologie 164: 16-23.

El Fantroussi, S., L. Verschuere, W. Verstraete and E. M. Top (1999) Effect of Phenyl Urea Herbicides on Soil Microbial Communities Estimated by 16S rDNA Fingerprints and Communtiy-Level Physiological Profiles. Applied and Environmental Microbiology 65: 982- 988.

Figurski, D. H. and E. R. Helinski (1979) Replication of an Origin-Containing Derative of Plasmid RK2 Dependent on a Plasmid Function Provided in trans. Proceedings of the National Academy of Sciences USA 76: 1648-1652.

Finkman, W., K. Altendorf, E. Stackebrandt and A. Lipski (2000) Characterization of N2O- Producing Xanthomonas-Like Isolates From Biofilters as Stenotrophomons nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov., Pseudoxanthomonas broegbernensis gen. nov., sp. nov. International Journal of Systematical Bacteriology 50(273-282).

Finlay, B. B. and W. Paranchych (1986) Nucleotide Sequence of the Surface Exclusion Genes traS and traT from the IncF0 lac Plasmid pED208. Journal of Bacteriology 166(3): 713-721.

Finnstein, M. S. and C. C. Delwiche (1965) Molybdenum as a Micronutrient for Nitrobacter. Journal of Bacteriology 89(1): 123-128.

Fliermans, C. B., B. B. Bohlool and E. L. Schmidt (1974) Autoecological Study of the Chemolithotroph Nitrobacter by Immunofluorescence. Applied Microbiology 27(1): 124-129.

Franklin, R. B., J. L. Garland and A. L. Mills (2005) The influence of ecological isolation on the Structural and Functional Stability of Complex Microbial Communities. 3rd International Workshop on Space Microbiology, Mol, Belgium.

Franklin, R. B. and A. L. Mills (2003) Multi-Scale Variation in Spatial Heterogeneity for Microbial Community Structure in an Eastern Virginia Agricultural Field. FEMS Microbiology Ecology 44: 335-346.

Frost, L. S. (2000) Bacterial Conjugation. Encyclopedia of Microbiology, Academic Press. Volume 1.

Fujii, K., N. Urano, H. Ushio, M. Satomi and S. Kimura (2001) Sphingomonas cloacae sp. nov., a Nonylphenol-Degrading Bacterium Isolated From Wastewater of a Sewage-Treatment Plant in Tokyo. International Journal of Systematical Bacteriology 51(2): 603-610.

104 References

Fulget, N., L. Poughon, J. Richalet and C. Lasseur (1999) MELiSSA: Global Control Strategy of the Artificial Ecosystems by Using First Principles Models of the Compartments. Advances in Space Research 24(3): 397-405.

Furumai, H. and B. E. Rittmann (1994) Evaluation of Multiple-Species Biofilm an Floc Processes Using a Simplified Aggregate Model. Water Science and Technology 34(9): 439- 446.

Garland, J. L., K. L. Cook, J. L. Adams and L. J. Kerkhof (2001) Culturability as an Indicator of Succession in Microbial Communities. Microbial Ecology 42: 150-158.

Gavini, F., J. Mergaert, A. Beji, C. Mielcarek, D. Izard, K. Kersters and J. De Ley (1989) Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov. International Journal of Systematical Bacteriology 39: 337-345.

Geets, J., K. Vanbroekhoven, B. Borremans, J. Vangronsveld, L. Diels and D. van der Lelie (2005) Column Experiments to Assess the Effects of Electron Donors on the Efficiency of in situ Precipitation of Zn, Cd, Co, and Ni in Contaminated Groundwater Applying the Biological Sulfate Removal Technology. Environmental Science and Pollution Research: 1- 17.

Ghigo, J.-M. (2001) Natural Conjugative Plasmids Induce Bacterial Biofilm Development. Nature 412: 442-445.

Gibson, J. E., E. Stackebrandt, L. B. Zablen, R. Gupta and C. R. Woese (1980) A phylogenetic analysis of the purple photosynthetic bacteria. Current Opinion in Microbiology 3: 59-64.

Gòdia, F., J. Albiol, J. L. Montesinos, J. Pérez, N. Creus, F. Cabello, X. Mengual, A. Montras and C. Lasseur (2002) MELiSSA: a Loop of Interconnected Bioreactors to Develop Life Support in Space. Journal of Biotechnology 99: 319-330.

Gòdia, F., J. Albiol, J. Pérez, N. Creus, F. Cabello, A. Montras, A. Mascot and C. Lasseur (2004) The MELiSSA pilot plant facility as an integration test-bed for advanced life support systems. Advances in Space Research 34: 1483-1493.

Gogarten, J. P., W. F. Doolittle and J. G. Lawrence (2002) Prokaryotic Evolution in Light of Gene Transfer. Molecular Biological Evolution 19: 2226-2238.

Goodfellow, M. and G. Alderson (1977) The actinomycete genus Rhodococcus: a home for the rhodochrous complex. Journal of Genetic Microbiology 100: 99-122.

Götz, A., R. Pukall, E. Tietze, R. Prager, H. Tschäpe, J. D. van Elsas and K. Smalla (1996) Detection and Characterization of Broad-Host-Range Plasmids in Environmental Bacteria by PCR. Applied and Environmental Microbiology(July 1996): 2621-2628.

Gould, G. W. and H. Lees (1960) The Isolation and Culture of the Nitrifying Organisms. Canadian Journal of Microbiology 6: 299-307.

105 References

Greene, E. A., J. G. Kay, K. Jaber, L. G. Stehmeier and G. Voordouw (2000) Composition of Soil Microbial Communities Enriched on a Mixture of Aromatic Hydrocarbons. Applied and Environmental Microbiology 66(12): 5282-5289.

Griffith, F. (1928) Significance of Pneumococcal Types. Journal of Hygiene (Cambridge) 27: 113-159.

Grimm, V., E. Schmidt and C. Wissel (1992) On the Application of Stability Concepts in Ecology. Ecological Modelling 63: 143-161.

Grunditz, C. and G. Dalhammar (2001) Development of nitrification inhibition assays using pure cultures of Nitrosomonas and Nitrobacter. Water Research 35(2): 433-440.

Haase, J., M. Kalkum and E. Lanka (1996) TrbK, a Small Cytoplasmic Membrane Lipoprotein, Functions in Entry Exclusion of the IncPalpha Plasmid RP4. Journal of Bacteriology 178(23): 6720-6729.

Halden, R. U., B. G. Halden and D. F. Dwyer (1999) Removal of Dibenzofuran, Dibenzo-p- Dioxin, and 2-Chlorodibenzo-p-Dioxin From Soils Inoculated With Sphingomonas sp. Strain RW1. Applied and Environmental Microbiology 65(5): 2246-2249.

Hallman, J., A. Quadt-Hallman, W. F. Mahaffee and J. W. Kloepper (1997) Bacterial Endophytes in Agricultural Crops. Canadian Journal of Microbiology 43(895-914).

Harremoes, P. (1982) Criteria for Nitrification in Fixed Film Reactors. Water Science and Technology 14: 167-185.

Hastings, R. C., J. R. Saunders, G. H. Hall, R. W. Pickup and A. J. McCarthy (1998) Application of Molecular Biological Techniques to a Seasonal Study of Ammonia Oxidation in a Eutrophic Freshwater Lake. Applied and Environmental Microbiology 64(10): 3674- 3682.

Heinemann, J. A. and R. G. Ankenbauer (1993) Retrotransfer in Escherichia coli conjugation: Bidirectional Exchange or de novo Mating? Journal of Bacteriology 175: 583-588.

Hellinga, C., M. C. van Loosdrecht and J. J. Heijnen (1999) Model Based Design of a Novel Process for Nitrogen Removal from Wastewater. Mathemathical and Computer Model Dyn. 5: 351-371.

Helmer-Madhock, C., M. Schmid, E. Filipov, T. Gaul, A. Hippen, K. H. Rosenwinkel, C. F. Seyfried, M. Wagner and S. Kunst (2002) Deammonification in Biofilm Systems: Population Structure and Function. Water Science and Technology 46: 223-231.

Hendrickx, L., H. De Wever, V. Hermans, F. Mastroleo, N. Morin, P. Janssen and M. Mergeay (accepted for publication) Microbial Ecology of the Closed Artificial Ecosystem MELiSSA (Micro-Ecological Life Support System Alternative). Research in Microbiology.

Hengstmann, U., K.-J. Chin, P. H. Janssen and W. Liesack (1999) Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically

106 References

Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil. Applied and Environmental Microbiology 65(11): 5050-5058.

Higashitani, N., A. Higashitani and K. Horiuchi (1995) SOS Induction in Escherichia coli by Single-Stranded DNA of Mutant Filamentous Phage: Monitoring by Cleavage of LexA Repressor. Journal of Bacteriology 177(12): 3610-3612.

Hill, K., A. J. Weightman and J. C. Fry (1992) Isolation and Screening of Plasmids from the Epilithon Which Mobilize Recombinant Plasmid pD10. Applied and Environmental Microbiology 58(4): 1292-1300.

Hommes, N., L. Sayavedra-Soto and D. Arp (2003) Chemolithoorganotrophic growth of Nitrosomonas europaea on Fructose. Journal of Bacteriology 185: 6809-6814.

Hooper, A. and K. R. Terry (1973) Specific Inhibitors of Ammonia Oxidation in Nitrosomonas. Journal of Bacteriology 115: 480-485.

Hovanec, T. A. and E. F. DeLong (1996) Comparative Analysis of Nitrifying Bacteria Associated with Freshwater and Marine Aquaria. Applied and Environmental Microbiology 92(8): 2888-2896.

Hovanec, T. A., L. T. Taylor, A. Blakis and E. F. Delong (1998) Nitrospira-like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria. Applied and Environmental Microbiology 64: 258-264.

Huisman, J. and F. J. Weissing (2002) Oscillations and Chaos Generated by Competition for Interactively Essential Resources. Ecological Research 17: 175-181.

Ilzumi, T., M. Mizumoto and K. Nakamura (1998) A Bioluminescence Assay Using Nitrosomonas europaea for Rapid an Sensitive Detection of Nitrification Inhibitors. Applied and Environmental Microbiology 64(10): 3656-3662.

Janssen, P. (1996) Evaluation of the DNA fingerprint method AFLP. Microbiology 142: 1881-1893.

Jetten, M. S. M., M. Strous, K. T. van de Pas-Schooned, J. Schalk, U. G. van Dongen, A. A. van de Graaf, S. Logemann, G. Muyzer, M. C. van Loosdrecht and J. G. Kuenen (1998) The anaerobic ammonia oxidation. FEMS Microbiological Reviews 22: 421-437.

Juretschko, S., M. Timmermann, K.-H. Schmidt, A. Schleifer, H.-P. Pommerening-Röser, H. P. Koops and M. Wagner (1998) Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrosospira- like bacteria as dominant populations. Applied and Environmental Microbiology 64(3042- 3051).

Killham, K. (1986) Heterotrophic nitrification. Oxford, IRL Press.

Kingma-Boltjes, T. Y. (1935) Undersuchungen uber die nitrifizierenden Bacterien. Arch. Microbiologie 6: 79-138.

107 References

Koch, G., K. Egli, J. R. van der Meer and H. Siegrist (2000) Mathematical Modeling of Autotrophic Denitrification in a Nitrifying Biofilm of a Rotating Biological Contactor. Water Science and Technology 41: 191-198.

Kondo, T., D. Yamamoto, A. Yokota, A. Suzuki, H. Nagasawa and S. Sakuda (2000) Gordonan, an acidic polysaccharide with cell aggregation-inducing activity in insect BM-N4 cells, produced by Gordonia sp. Bioscience, Biotechnology and Biochemistry 64(2388-2394).

Koops, H. P., B. Böttcher, U. C. Möller, A. Pommerening-Röser and G. Stehr (1991) Classification of Eight New Species of Ammonia-Oxidizing Bacteria: Nitrosomonas communis sp. nov., Nitrosomonas ureae sp. nov., Nitrosomonas aestuarii sp.nov., Nitrosomonas marina sp. nov., Nitrosomonas nitrosa sp. nov., Nitrosomonas eutropha sp. nov., Nitrosomonas oligotropha sp. nov. and Nitrosomonas halophila sp. nov. Journal of General Microbiology 137: 1689-1699.

Koraimann, G. (2004) Bacterial Conjugation: Cell-Cell Contact-Dependant Horizontal Gene Spread. Microbial Evolution: Gene Establishment, Survival, and Exchange. R. V. Miller and M. J. Day. Washington, D.C., ASM Press: 111-124.

Kuai, L. and W. Verstraete (1998) Ammonium removal by the oxygen-limited autotrophic nitrification-denitrification system. Applied and Environmental Microbiology 64: 4500-4506.

Kuypers, M. M. M., A. O. Sliekers, G. Lavik, M. Schmid, B. B. Jorgensen, J. G. Kuenen, J. S. Sinninghe Damsté, M. Strous and M. S. M. Jetten (2003) Anaerobic Ammonium Oxidation by Anammox Bacteria in the Black Sea. Nature 422: 608-611.

Laanbroek, H. J., M. J. Bär-Gilissen and H. L. Hoogveld (2002) Nitrite as a Stimulus for Ammonia-Starved Nitrosomonas europaea. Applied and Environmental Microbiology 68(3): 1454-1457.

Lang, E. and G. Jagnow (1986) Fungi of a Forest Soil Nitrifying at low pH Values. FEMS Microbiology and Ecology 38: 257-265.

LaPara, T. M., C. H. Nakatsu, L. M. Pantea and J. E. Alleman (2002) Stability of the Bacterial Communities Supported by a Seven-Stage Biological Process Treating Pharmaceutical Wastewater as Revealed by PCR-DGGE. Water Research 36: 638-646.

Layton, A. C., P. N. Karanth, C. A. Lajoie, A. J. Meyers, I. R. Gregory, R. D. Stapleton, D. E. Taylor and G. S. Sayler (2000) Quantification of Hypomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis. Applied and Environmental Microbiology 66(3): 1167-1174.

Lechevalier, H. A. (1989) Nocardioform actinomycetes. Bergey's Manual of Systematic Bacteriology. S. T. Williams, M. E. Sharpe and J. G. Holt. Baltimore, Md., The Williams & Wilkins Co. vol.4.

Lee, L. Y., S. L. Ong and W. J. Ng (2004) Biofilm morphology and nitrification activities: recovery of nitrifying Biofilm Particles Covered with Heterotrophic Outgrowth. Bioresource Technology 95: 209-214.

108 References

Lees, H. and J. R. Simpson (1957) The biochemistry of the nitrifying organisms. Nitrite oxidation by Nitrobacter. Biochemistry Journal 65: 297-305.

Linos, A., M. Berekaa, A. Steinbüchel, K. K. Kim, C. Spöer and R. M. Kroppenstedt (2002) Gordonia westfalica sp. nov., a Novel Rubber-Degrading Actinomycete. International Journal of Systematical Bacteriology 52(1133-1139).

Lipski, A. and K. Altendorf (1997) Identification of Heterotrophic Bacteria Isolatied From Ammonia-Supplied Experimental Biofilters. Systematical and Applied Microbiology 20: 448- 457.

Ludwig, C., S. Ecker, K. Schwindel, H.-G. Rast, K.-O. Stetter and G. Eberz (1999) Construction of a Highly Bioluminescent Nitrosomonas as a Probe for Nitrification Conditions. Arch. Microbiologie 172: 45-50.

Mahenthiralingam, E. (2004) Gene Associations in Bacterial Pathogenesis: Pathogenicity Islands and Genomic Deletions. Microbial Evolution: Gene Establishment, Survival, and Exchange. R. V. Miller and M. J. Day. Washington, D.C., ASM Press: 249-274.

Malhautier, L., V. Degrange, R. Guay, J.-R. Degorce-Dumas, R. Bardin and P. Le Choirec (1998) Estimating size and diversity of nitrifying communities in deodorizing filters using PCR and immunofluorescence. Journal of Applied Microbiology 85: 255-262.

Martiny, H. and H. P. Koops (1982) Incorporation of Organic Compounds into Cell Protein by Lithotrophic, Ammonia-Oxidizing Bacteria. Antonie van Leeuwenhoek 48: 327-336.

Matic, I., F. Taddei and M. Radman (2004) Survival Versus Maintenance of Genetic Stability: a Conflict of Priorities During Stress. Research in Microbiology 155: 337-341.

Matulewich, V. A., P. F. Strom and M. S. Finnstein (1975) Length of incubation for enumerating nitrifying bacteria present in various environments. Applied Microbiology 29: 265-268.

McDevitt, C., P. C. Burrell, L. L. Blackall and A. G. McEwan (2000) Aerobic Nitrate Respiration in a Nitrite-Oxidizing Bioreactor. FEMS Microbiological Letters 2000(184): 113- 118.

McTavish, H., J. A. Fuchs and A. B. Hooper (1993) Sequence of the Gene Coding for Ammonia Monooxygenase in Nitrosomonas europaea. Journal of Bacteriology 175(8): 2436- 2444.

Mendum, T. A., R. E. Sockett and P. R. Hirsch (1999) Use of Molecular and Isotopic Techniques to Monitor the Response of Autotrophic Ammonia-Oxidizing Populations of the Beta-Subdivision of the Class Proteobacteria in Arable Soils to Nitrogen Fertilizer. Applied and Environmental Microbiology 65: 4155-4162.

Mergaert, J., L. Verdonck and K. Kersters (1993) Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea sterwartii (Smith 1898) comb. nov., respectively, and

109 References description of Pantoea stewardii subsp. indologenes subsp. nov. International Journal of Systematical Bacteriology 43: 162-173.

Mergeay, M., P. Lejeune, A. Sadouk, J. Gerrits and L. Fabry (1987) Shuttle Transfer (or Retrotransfer) of Chromosomal Markers Mediated by Plasmid pULB113. Mol Gen Genet 209(61-70).

Mergeay, M., D. Nies, H. G. Schlegel, J. Gerits, P. Charles and F. Van Gijsegem (1985) Alcaligenes eutrophus CH34 Is a Facultative Chemolithotroph with Plasmid-Bound Resistance to Heavy Metals. Journal of Bacteriology 162(1): 328-334.

Mergeay, M., W. Verstraete, G. Dubertret, M. Lefort-tran, C. Chipaux and R. B. Binot (1988) MELiSSA a microorgansims based model for CELLS development. Proceedings of the 3rd Symposium on Space Thermal Control & Life Support System, Noordwijk, The Netherlands.

Milkman, R. (2004) Horizontal Transfer, Genomic Diversity, and Genomic Defferentiation. Microbial Evolution: Gene Establishment, Survival, and Exchange. R. V. Miller and M. J. Day. Washington, D.C., ASM Press.

Miller, R. V. and M. J. Day (2004) Horizontal Gene Transfer in the Real World. Microbial Evolution: Gene Establisment, Survival, and Exchange. R. V. Miller and M. J. Day. Washington, D.C., ASM Press: 173-177.

Minkley Jr., E. G. and K. Ippen-Ihler (1977) Identification of a Membrane Protein Associated with Expression of the Surface Exclusion Region of the F Transfer Operon. Journal of Bacteriology 129(3): 1613-1622.

Molbak, L., A. Tett, D. W. Ussery, K. Wall, S. Turner, M. Bailey and D. Field (2003) The Plasmid Genome Database. Microbiology 149(11): 3043-3044.

Montras, A., L. Hendrickx and J. Pérez (2005) Nitrifying Biofilms in Biological Life Support Systems for Space Missions. 3rd International Workshop on Space Microbiology, Mol, Belgium.

Moore, E. R. B., A. S. Krüger, L. Hauben, S. E. Seal, R. De Baere, R. de Wachter, K. N. Timmis and J. Swings (1997) 16S rRNA Gene Sequence Analyses and Inter- and Intrageneric Relationships of Xanthomonas species and Stenotrophomonas maltophilia. FEMS Microbiological Letters 151: 145-153.

Muyzer, G., E. C. de Waal and A. G. Uitterlinden (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction- amplified genes codeing for 16S rRNA. Applied and Environmental Microbiology 59: 695- 700.

Muyzer, G., S. Hottenträger, A. Teske and C. Wawer (1996) Denaturing gradient gel electrophorsis of PCR-amplified 16S rDNA - A new molecular approach to analyse the genetic diversity of mixed microbial communities. Molecular Microbial Ecology Manual 3.4.4.: 1-23.

110 References

Nelson, D. H. (1931) Isolation and Characterisation of Nitrosomonas and Nitrobacter. Zentr. Bacteriol. Parasitenk. Abt. II 83: 280-311.

Nogueira, R., L. F. Melo, U. Purkhold, S. Wuertz and M. Wagner (2002) Nitrifying an Heterotrophic Population Dynamics in Biofilm Reactors: Effects of Hydraulic Retention Time and the Presences of Organic Carbon. Water Research 36: 469-481.

Ochman, H., J. G. Lawrence and E. A. Groisman (2000) Lateral Gene Transfer and the Nature of Bacterial Innovation. Nature 405: 299-304.

Ong, S. L., L. Y. Lee, J. Y. Hu and W. J. Ng (2003) Nitrogen Removal Using Combined UCBR-Packed Bed System. Journal of Environmental Engineering 129(1): 43-51.

Orso, S., M. Gouy, E. Navarro and P. Normand (1994) Molecular phylogenetic analysis of Nitrobacter spp. International Journal of Systematical Bacteriology 44: 83-86.

Oved, T., A. Shaviv, T. Goldrath, R. T. Mandelbaum and D. Minz (2001) Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil. Applied and Environmental Microbiology 67(8): 3426-3433.

Painter, H. A. (1986) Nitrification in the Treatment of Sewage and Wastewater. Nitrification. J. I. Prosser. Oxford, United Kingdom, IRL Press: 185-211.

Papen, H., R. von Berg, I. Hinkel, B. Thoene and H. Rennenberg (1989) Heterotrophic - - Nitrification by Alcaligenes faecalis: NO2 , NO3 , N2O, and NO Production in Exponential Growing Cultures. Applied and Environmental Microbiology 55(8): 2068-2072.

Pérez, J., J. L. Montesinos, J. Albiol and F. Gòdia (2004) Nitrification by immobilized cells in a Micro-Ecological Life Support System Using Packed-Bed Bioreactors: an Engineering Study. Journal of Chemical Technology and Biotechnology: DOI: 10.1002/jctb.1050.

Peterson, G., C. R. Allen and C. S. Holling (1998) Ecological Resilience, Biodiversity, and Scale. Ecosystems 1(6-18).

Philips, S., S. Wyffels, R. Sprengers and W. Verstraete (2002) Oxygen-limited autotrophic nitrification/denitrification by ammonia oxidisers enables upward motion towards more favorable conditions. Applied Microbiology and Biotechnology 59: 557-566.

Pidwirny, M. (2004) PhysicalGeography.net, Online Textbook of Fundamentals (of Physical Geography).

Poughon, L., C.-G. Dussap and J.-B. Gros (2001) Energy Model and Metabolic Flux Analysis for Autotrophic Nitrifiers. Biotechnology and Bioengineering 72(4): 416-433.

Prosser, J. I. (1989) Autotrophic Nitrification in Bacteria. Advances in Microbial Physiology 30: 125-181.

Pujol, C. J. and C. I. Kado (1998) Characterization of pUCD5000 Involved in Pink Disease Color Formation by Pantoea citrea. Plasmid 40: 169-173.

111 References

Purkhold, U., A. Pommerening-Röser, S. Juretschko, C. Schmidt, H. P. Koops and M. Wagner (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Applied and Environmental Microbiology 66: 5368-5382.

Purkhold, U., M. Wagner, M. Timmermann, A. Pommerening-Röser and H. P. Koops (2003) 16S rRNA and amoA-based Phylogeny of 12 Novel Betaproteobacterial Ammonia-Oxidizing isolates: Extenstion of the Dataset and Proposal of a New Lineage Within the Nitrosomonads. International Journal of Systematical Bacteriology and Evolutionary Microbiology 53: 1485- 1494.

Ramos-Gonzalez, M. I., M. A. Ramos-Diaz and J. L. Ramos (1994) Chromosomal Gene Capture Mediated by the Pseudomonas putida TOL Catabolic Plasmid. Journal of Bacteriology 176: 4635-4641.

Randall, C. W. (1992) Design and Retrofit of Wastewater Treatment Plants for Biological Nutrient Removal. Introduction and Principles of Biological Nutrient Removal. C. W. Randall. Lancaster, Pa, Technomic Publishing Company Inc.: 2-84.

Reinhold-Hurek, B. and T. Hurek (1998) Interactions of Gramineous Plants with Azoarcus spp. and Other Diazotrophs: Identification, Localization, and Perspectives to Study Their Function. Critical Reviews in Plant Science 17: 29-54.

Rotthauwe, J.-H., K.-P. Witzel and W. Liesack (1997) The Ammonia Monooxygenase Structural Gene amoA as a Functional Marker: Molecular Fine-Scale Analysis of Natural Ammonia-Oxidizing Populations. Applied and Environmental Microbiology 63(12): 4704- 4712.

Saghai-Maroof, M. A., K. M. Soliman, R. A. Jorgensen and R. W. Allard (1984) Ribosomal DNA Spacer Length Polymorphism in Population Dynamics. Proceedings of the National Academy of Sciences USA 81: 8014-8018.

Sakano, Y. and L. J. Kerkhof (1998) Assessment of Changes in Microbial Community Structure during Operation of an Ammonia Biofilter with Molecular Tools. Applied and Environmental Microbiology 64(12): 4877-4882.

Sakano, Y., K. D. Pickering, P. F. Strom and L. J. Kerkhof (2002) Spatial Distribution of Total, Ammonia-Oxidizing, and Denitrifying Bacteria in Biological Wastewater Treatment Reactors for Bioregenerative Life Support. Applied and Environmental Microbiology 68(5): 2285-2293.

Sambrook, J., E. F. Fritsch and T. Maniatis (1989) A2. Bacterial Media, Antibiotics and Bacterial Strains. Molecular Cloning, A Labratory Manuel. C. Noran, Cold Spring Harbour Press.

Savage, C. J., G. B. T. Tan and C. Lasseur (2001) ESA Developments in Life Support Technology: Achievements and Future Priorities. Acta Astronautica 49(3-10): 331-344.

Schimel, J. P., M. K. Firestone and K. S. Killham (1984) Identification of Heterotrophic Nitrification in an Sierran Forest Soil. Applied and Environmental Microbiology 48: 802-806.

112 References

Schmid, M., U. Twachtmann, M. Klein, M. Strous, S. Juretschko, M. S. M. Jetten, J. W. Metzger, A. Schleifer and M. Wagner (2000) Molecular Evidence for Genus Level Diversity of Bacteria Capable of Catalyzing Anaerobic Ammonium Oxidation. Systematical and Applied Microbiology 23: 93-106.

Schmidt, E. L. and L. W. Belser (1982) Nitrifying Bacteria. Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties. Madison, WI, USA, Monograph n°9: 1027-1042.

Schmidt, I., P. J. M. Steenbakkers, H. J. M. op den Camp, K. Schmidt and M. S. M. Jetten (2004) Physiologic an Proteomic Evidence for a Role of Nitric Oxide in Biofilm Formation by Nitrosomonas europaea and Other Ammonia Oxidizers. Journal of Bacteriology 186(9): 2781-2788.

Schramm, A., D. De Beer, J. C. van den Heuvel, S. Ottengraf and R. Amann (1999) Microscale Distribution of Populations and Activities of Nitrosospira and Nitrospira spp. Along a Macroscale Gradient in a Nitrifying Bioireactor: Quantification by in situ Hybridization and the Use of Microcsensors. Applied and Environmental Microbiology 65(8): 3690-3696.

Schramm, A., D. De Beer, M. Wagner and R. Amann (1998) Identification and activities in situ of Nitrosospira spp. as Dominant Populations in a Nitrifying Fluidized Bed Reactor. Applied and Environmental Microbiology 64(3480-3485).

Skurray, R. A. and P. Reeves (1974) F Factor-Mediated Immunity to Lethal Zygosis in Escherichia coli K-12. Journal of Bacteriology 117(1): 100-106.

Smets, B. F., J. B. Morrow and C. A. Pinedo (2003) Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response. Applied and Environmental Microbiology 69(7): 4087-4097.

Smit, E., A. C. Wolters and J. D. van Elsas (1998) Self-Transmissible Mercury Resistance Plasmids with Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence of Wheat Roots and Mercury Addition. Applied and Environmental Microbiology 64(4): 1210-1219.

Snaidr, J., R. Amann, I. Huber, W. Ludwig and K.-H. Schleifer (1997) Phylogenetic Analysis and in situ Identification of Bacteria in Activated Sludge. Applied and Environmental Microbiology 63(2884-2896).

Solorzano, L. (1969) Determination of Ammonia in Natural Waters by the Phenolhypchloride method. Limnology and Oceanography 14: 799.

Soriano, S. and N. Walker (1968) Isolation of Ammonia-Oxidizing Autotrophic Bacteria. Journal of Applied Microbiology 31: 493-497.

Springael, D. (1991) Genetica van PCB-Degraderende Bacteriën: Identificatie van een Nieuw Katabolisch Transposon Tn4371. Faculteit Landbouwwetenschappen. Brussel, Vrije Universiteit Brussel: 352.

113 References

Stackebrandt, E., F. A. Rainey and N. L. Ward-Rainey (1997) Proposal for a New Hierarchic Classification System, Achinobacteria classis. nov. International Journal of Systematical Bacteriology 47: 479-491.

Stein, L. Y. and D. Arp (1998) Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea Upon Exposure to Nitrite. Applied and Environmental Microbiology 64: 4094-4102.

Stephen, J.-R., Y.-J. Chang, S. J. MacNaughton, G. A. Kowalchuk, K. T. Leung, C. A. Flemming and D. C. White (1999) Effect of Toxic Metals on Indigenous Soil ß-Subgroup Proteobacterium Ammonia Oxidizer Community Structure and Protection against Toxicity by Inoculated Metal-Resistant Bacteria. Applied and Environmental Microbiology 65(1): 95-101.

Stojanovic, B. J. and M. Alexander (1958) The effect of inorganic nitrogen on nitrification. Soil Science.

Strous, M., J. A. Fuerst, E. H. Kramer, S. Logemann, G. Muyzer, K. T. van de Pas-Schooned, R. Webb, J. G. Kuenen and M. S. M. Jetten (1999) Missing lithotroph identified as new planctomycete. Nature 400(446-449).

Strous, M., J. G. Kuenen and M. S. M. Jetten (1999) Key physiology of anaerobic ammonia oxidation. Applied and Environmental Microbiology 65: 3248-3250.

Sturz, A. V., B. R. Christie and J. Nowak (2000) Bacterial endophytes: Potential Role in Developing Sustainable Systems of Crop Production. Critical Reviews in Plant Science 19: 1- 30.

Stutzer, H. and R. Hartleb (1894) Über Nitratbildung. Zentralbl. Backteriol. Parasitenkunde Infectionskr. Hyg. Abt. 1 Orig. Reihe A 22: 701.

Taiz and Zeiger (2002) Plant Physiology, Sinauer.

Tamponnet, C. and C. Savage (1994) Closed Ecological Systems. Journal of Biological Education 28: 167-173.

Teske, A., E. Alm, J. M. Regan, S. Toze, B. E. Rittmann and D. A. Stahl (1994) Evolutionary Relationships among Ammonia- and Nitrite-Oxidizing Bacteria. Journal of Bacteriology 176(21): 6623-6630.

Thomas, C. M. and K. Smalla (2000) Trawling the horizontal gene pool. Microbiology Today 27: 24-27.

Tiedje, J. M. (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. Biology of Anaerobic Microorganisms. A. J. B. Zehnder. New York, Wiley: 179- 244.

Tiirola, M. A., H. Wang, L. Paulin and M. S. Kulomaa (2002) Evidence for Natural Horizontal Transfer of the pcpB Gene in the Evolution of Polychlorophenol-Degrading Sphingomonads. Applied and Environmental Microbiology 68(9): 4495-4501.

114 References

Top, E. M., I. De Smet, W. Verstraete, R. Dijkmans and M. Mergeay (1994) Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges. Applied and Environmental Microbiology 60(3): 831-839. van Benthum, W. A. J., B. P. Derissen, M. C. van Loosdrecht and J. J. Heijnen (1998) Nitrogen Removal Using Nitrifying Biofilm Growth and Denitrifying Suspended Growth in a Biofilm Airlift Suspension Reactor Coupled with a Chemostat. Water Research 32(7): 2009- 2018.

Van de Dijk, S. J. and S. R. Troelstra (1980) Heterotrophic Nitrification in a Heath Soil Demonstrated by an in-situ Method. Plant Soil 57(11-21). van Elsas, J. D., B. B. McSpadden Gardener, A. C. Wolters and E. Smit (1998) Isolation, Characterization, and Transfer of Cryptic Gene-Mobilizing Plasmids in the Wheat Rhizosphere. Applied and Environmental Microbiology 64(3): 880-889.

Verhagen, F. J. M. and H. J. Laanbroek (1991) Competition for Ammonium Between Nitrifying and Heterotrophic Bacteria in Energy-Limited Chemostats. Applied and Environmental Microbiology 57: 3255-3263. von Canstein, H., S. Kelly, Y. Li and I. Wagner-Döbler (2002) Species Diversity Improves the Efficiency of Mercury-Reducing Biofilms under Changing Environmental Conditions. Applied and Environmental Microbiology 68(6): 2829-2837.

Wagner, M., A. Loy, R. Nogueira, U. Purkhold, N. Lee and H. Daims (2002) Microbial Community Composition and Function in Wastewater Treatment Plants. Antonie van Leeuwenhoek 81(665-680).

Wagner, M., G. Rath, H. P. Koops, J. Flood and R. Amann (1996) In situ Analysis of Nitrifying Bacteria in Sewage Treatment Plants. Water Science and Technology 34: 237-244.

Ward, D. M., R. Weller and M. M. Bateson (1990) 16S rRNA Sequences Reveal Numerous Uncultured Microorganisms in a Natural Community. Nature 345: 63-65.

Wheaton, F. W., J. N. Hochheimer, G. E. Kaiser, J. Kronos, G. S. Libey and C. C. Easter (1994) Nitrification filter principles. Aquaculture water reuse systems: engineering design and management. M. B. Timmons and T. M. Losordo. Amsterdam, Elsevier: 101-126.

Whittaker, M., D. Bergmann, D. Arciero and A. B. Hooper (2000) Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea. Biochim. Biophys. Acta 1459: 346-355.

Wijffels, J. I. and J. Tramper (1995) Nitrification by Immobilized Cells. Enzyme Microbial Technology 17: 482-492.

Winans, S. C. and G. C. Walker (1985) Entry Exclusion Determinant(s) of IncN Plasmid pKM101. Journal of Bacteriology 161(1): 411-416.

Wood, P. (1986) Nitrification as a bacterial energy source. (Nitrification) Special publication for the Society for General Microbiology 20: 63-78.

115 References

Xue, Y., X. Xuesong, P. Zhou, R. Liu, F. Liang and Y. Ma (2003) Gordonia paraffinivorans sp. nov., a Hydrocarbon-Degrading Actinomycete Isolated From An Oil-Producing Well. International Journal of Systematical Bacteriology 53(1643-1646).

Yabuuchi, E. Y., I. Yano, H. Oyaizu, Y. Hashimoto, T. Ezaki and H. Yamamoto (1990) Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov. Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbial Immunology 34(2): 99-119.

Ye, R. W. and S. M. Thomas (2001) Microbial nitrogen cycles: physiology, genomics and applications. Current Opinion in Microbiology 4: 307-312.

Yoon, J.-H., S. T. Lee and Y.-H. Park (1998) Genetic Analyses of the Genus Nocardioides and Related Taxa Based on 16S-23S rDNA Internally Transcribed Spacer Sequences. International Journal of Systematical Bacteriology 48: 641-650.

Yoon, J.-H., S. T. Lee and Y.-H. Park (1998) Inter- and Intraspecific Phylogenetic Analysis of the Genus Nocardioides and Related Taxa Based on 16S rDNA Sequences. International Journal of Systematical Bacteriology 48(187-194).

Zart, D., I. Schmidt and E. Bock (2000) Significance of Gaseous NO for Ammonia Oxidation by Nitrosomonas eutropha. Antonie van Leeuwenhoek 77: 49-55.

Zinser, E. R. and R. Kolter (2004) Escherichia coli Evolution During Stationary Phase. Research in Microbiology 155: 328-336.

116

APPENDIX I: List comprising the used substances and scales, and their origin

Substances Substance Brand Origin Select Agar Invitrogen CaCl2 J.T. Baker Chemicals Deventer Holland CaCO3.2H2O J.T. Baker Chemicals Deventer Holland CaCO3.2H2O Merck Darmstadt Germany Cresolred UCB Belgium (pH 7.0 - 8.8) Cu(II)SO4 J.T. Baker Chemicals Deventer Holland EDTA Merck Darmstadt Germany Fe(II)SO4 Merck Darmstadt Germany Sodium Gluconate Merck-Schuchardt Hohenbrunn Germany Contains [HClO4] >99% D(+)-Glucose (anhydrous) Merck Darmstadt Germany Ethanol Merck Darmstadt Germany KCl Merck Darmstadt Germany KCO3 (anhydrous) J.T. Baker Chemicals Deventer Holland K2HPO4 Merck Darmstadt Germany KH2PO4 Merck Darmstadt Germany KHSO4 Merck Darmstadt Germany Lennox L Broth Base Invitrogen MgSO4.7H2O Merck Darmstadt Germany MgCl2.6H2O Merck Darmstadt Germany NaCl Merck Darmstadt Germany NaCO3 Merck Darmstadt Germany NaHCO3 Merck Darmstadt Germany Na3(PO4).12H2O J.T. Baker Chemicals Deventer Holland Na2HPO4.2H2O VWR Int. (PROLABO) Leuven Belgium NaSO4 Merck Darmstadt Germany NH4Cl Merck Darmstadt Germany NH4SO4 Merck Darmstadt Germany Phenol red BHD Indicators Poole England (pH 6.8 – 8.4) Propanol Merck Darmstadt Germany Tris Gibco BRL Poisley United Kingdom

Scales Brand Type Range Mettler Toledo PB 1502 0.5 g - 1510 g; d = 0.01 g Mettler Toledo PG 802-5 0 g – 810 g; d = 0.01 g Sartorius Handy H110 < 1 g

117