Microbial Reclamation of Alkaline Sodic Soils
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IN
Microbial Reclamation of Alkaline Sodic Soils
Thesis submitted for the degree of I)octor of PhilosoPhY at the Universrty of Adelaide bY
Simon Paul Odelt
Deparbnent of Soil and Water Waite Agricultural Institute Glen Osmond, South Australia
2000
1l TABLE OF CONTENTS
Page
ABSTRACT x
STATEMENT xiv
ACKNOWLEDGEMENTS xv
LIST OF'FIGT]RES xvi
LIST OF TABLES xxi
ABBREVIATIONS xxiv
CHAPTER 1. Introduction I
CHAPTER 2. Literature Review
2.1 Classification and formation of sodic soils 4
2.1.1 Classification of sodic soils 4
2.1.2 Natural formation of sodic soils 5
2.1.3 AnthroPogenic sodic soils 5
2.1.3.I Indiscriminate clearing and over-grazrng of native vegetation 5
2.I.3.2 Inigation of crops with poor quahty water 6
2.2 Chemical reclamation of alkaline sodic soils 6
2.2.1 Sodic soil and gYPs 6
2.2.2 Inigation 8
2.2.3 Iron PYrite I
10 2.2.4 Water soluble PolYmers
2.2.5 Heating 11
2.3 Biological reclamation of alkaline sodic soils l1
2.3.1 Organic matter and the biological reclamation of sodic soil 1l 2.3.2 Limitations of organic matter treatment t4
2.3.3 Types of organic matter t4
2.3.4 Farmyard manure mixtures t4
2.3.5 Acid cheese whey l6
2.3.6 Sewage sludge t7
2.3.7 Straw t9
2.3.8 Crops and other plants 20
2.3.9 Bagasse 22
2.3.10 Molasses 23
2.3.11 Green waste 24
2.4 Microbiology of sodic soils 24
2.4.1 Effect of alkaline conditions and NaCl on the microflora
of sodic soils 24
2.4.2 Effect of soil moisture content on the microflora of sodic soils 25
2.4.3 Effect of gypsum on the microflora of sodic soils 26
2.4.4 Effect of green manure on the microflora of sodic soils 26
2.4.5 Effect of glucose on the microflora of sodic soils 27
2.5 Methods for studying the microbial populations within soils 27
2.5.t Molecular techniques 28
2.5.2 BIOLOG 29
2.5.3 Immunological methods 29
2.5.4 Fatty Acid Methyl Ester (FAME) analysis 30
2.6 Conclusion 31
ll CHApTER 3. Diversity, Alkalitolerance, Halotolerance, and Acid-Producing
Capabilities of Microorganisms Cultured from an Australian Alkaline Sodic Soil 3.1 Introduction 32
3.2 Materials and Methods 33
3.2.1 Soil ProPerties 35 3.2.2 Microbiological culture media and solutions 35
3.2.3 Enumeration of non-filamentous bacteria,
filamentous bacteria (actinomycetes) and frrngi 35
3.2.4 selection of non-filamentous bacteria to be used in alkali
and NaCl tolerance assays 37
3.2.5 selection of filamentous bacteria (actinomycetes) to be
used in preliminary alkati and Nacl tolerance assays 38
3.2.6 Selection of fungi for morphological characterisation 38
3.2.7 Alkali tolerance of filamentous and non-filamentous
Watchem soil bacteria 38
3.2.8 NaCl tolerance of of filamentous and non-filamentous
Watchem soil bacteria 39
3.2.9 combined alkali and Nacl tolerance of filamentous
and non-filamentous \ÙVatchem soil bacteria 39
3.2.10 Assay for acid production by non-filamentous
bacterial isolates 39
3.2.11 Fatry Acid Metþl Ester (FAME) identification of
bacterial isolates 40
3.2.12 Identification of filamentous bacterial isolates 4l 3.2.13 Ftngal identification 4l
lll 3.3 Results 42
3.3.1 Enumeration of fi lamentous bacteria (actinomycetes),
non-filamentous bacteria, and fungi 42
3.3.2 Alkalitolerance and halotolerance of individual
non-fi lamentous bacteria 43
3.3.2.1 Identifrcation of highly halotolerant non-filamentous
bacteria by FAME analYsis 45
3.3.2.2 Identification of moderately haloalkalitolerant
non-filamentous bacteria by FAME analysis 46
3.3.2.3 Identifi cation of highly haloalkalitolerant non-filamentous
bacteria by FAME analYsis 47
3.3.3 Classification non-filamentous bacteria with a
FAME profile similaritY of <30io 48
3.3.4 Acid production by non-filamentous bacteria:
FAME identified isolates 5l
3.3.5 Acid production by non-filamentous bacteria :
Unidentified isolates 52
3.3.6 Alkali and NaCl tolerance of filamentous Watchem soil
bacteria (actinomycetes): Preliminary studies 54
3.3.7 Identification of filamentous bacteria (actinomycetes) 55
3.3.8 Fungal identification 57
3.4 Discussion 60
3.4.1 Alkalitolerance of the non-filamentous watchem soil bacteria 60
3.4.2 Halotolerance of the non-filamentous Watchem soil bacteria 65
lV 3.4.3 Acid production by the non-filamentous Watchem soil
bacterial isolates 68
3.4.4 Alkalitolerance of filamentous bacteria (actinomycetes)
isolated from the V/atchem soil: Preliminary studies 69
3.4.5 Halotolerance of filamentous bacteria (actinomycetes)
isolated from the Watchem soil: Preliminary studies 70
3.4.6 Fungal identification 7T
3.5 Conclusion 73
CHAPTER 4. volatile Fatty Acid Analysis in Alkaline sodic Soils: Method
Development
4.r Introduction 75
4.2 Method development 76
4.2.1 Instrumentation 76
4.2.2 Determination of chromatography conditions 77
4.2.3 Selection of the MSD analysis mode 78
4.2.4 Selection of ions to monitor 79
4.2.5 Construction of calibration curves 79
4.2.6 Selection of an internal standard 80
4.2.7 Solvent formulation 83
4.2.8 Optimisation of the extraction method 85
4.2.9 Optimum GC-MSD configuration and extraction method 87
v CHAPTER 5. Microbial Reclamation of an Älkaline Sodic Soil Using the Model
Organic Substrate Glucose 5.1 Introduction 88
5.2 Materials and methods 88
5.2.1 Soil properties 89
5.2.2 Preparation of soil samples 89
5.2.3 Physical and chemical analyses 90
5.2.3.1 Soil pH and EC 90
5.2.3.2 Soluble cation analysis 90
5.2.3.3 Determination of total carbonate 9l
5.2.3.4 Determination of water soluble carbonate and bicarbonate 9l
5.2.3.5 Measurement of microbial glucose utilisation 9T s.2.3.6 Volatile fatty acid analysis of soil samples 93
5.2.4 Microbiological analyses 93
5.2.4.1 FAME analysis of soil samples 93
5.2.4.2 Methane analysis of the soil headspace 94
5.2.5 Statistical analyses 94
5.3 Results and Discussion 95
5.3.1 Microbial utilisation of glucose 95
5.3.2 Interpretation of whole soil FAME profiles 96
5.3.3 Volatile fatty acid (VFA) and FAME analysis
of soil samples: W4 treatment 102
5.3.4 Volatile fatty acid (VFA) and FAME analysis
of soil samples: 2W4 treatment 104
vl 5.3.s Volatile fatty acid (VFA) and FAME analysis
of soil samples: 2W2treatment 108
5.3.6 Volatile fatty acid (VFA) and FAME analysis
of soil samples: W2 treatment 110 s.3.7 Volatile fatty acid (VFA) and FAME analysis
of soil samples:Aerobic Control t12 s.3.8 Volatile fatfy acid (VFA) and FAME analysis
of soil samples:Anaerobic Control 113
5.3.9 Methane analysis of the soil headspace 115
5.3.10 Soil pH 115
5.3.11 Total carbonate/ Soluble Carbonate and Bicarbonate 118 s.3.12 Soluble Calcium 12l
5.3.13 Soluble Sodium r23
5.3.14 Soil EC 124
5.4 Conclusion t25
CHAPTER 6. Microbial Reclamation of an Alkaline Sodic Soil Using "Complex"
Organic Substrates 6.1 Introduction 127
6.2 Materials and Methods 130
6.2.1 Soil properties 130
6.2.2 Ameliorant evaluation experiment 131
6.2.3 Amelioration of the Watchem soil using
complex organic ameliorants 132
6.2.4 Total Carbon analYsis I32
v11 6.2.5 Interpretation of whole soil FAME profiles t34
6.3 Results and Discussion 135
6.3.1 Ameliorant evaluation experiment 135
6.3.1.1 Ameliorant evaluation experiment: Control 135
6.3.1.2 Ameliorant evaluation experiment: Wheat straw 135
6.3.1.3 Ameliorant evaluation experiment: Sheep manure r37
6.3.1.4 Ameliorant evaluation experiment: Molasses 138
6.3.1.5 Ameliorant evaluation experiment:
Wheat straw and Sheep manure t39
6.3.1.6 Ameliorant screening experiment:
Wheat straw and Molasses t4t
6.3.r.7 Ameliorant screening exPeriment:
Super phosphate, Ammonium nitrate, Molasses
and Wheat straw r42
6.3.1.8 Ameliorant screening exPeriment:
Selection of ameliorants for further study r43
6.3.2 Small-scale amelioration experiment r45
6.3.2.1 Total Carbon analysis t45
6.3.2.2 Volatile Fatty Acid production and
Soil FAME analysis: Control 146
6.3.2.3 Volatile Fatty Acid production and
Soil FAME analysis: Molasses treatment t47
6.3.2.4 Volatile Fatty Acid production and
Soil FAME analysis: Super treatment 150
v111 6.3.2.5 Soil pIV Total Carbonatel
Soluble Carbonate and Bicarbonate t54
6.3.2.6 Soluble Calcium t56
6.3.2.7 Soluble Sodium 158
6.3.2.8 Soil EC 160
6.4 Conclusion 161
CHAPTER 7. General I)iscussion
7.1 Microbial populations within alkaline sodic soils 163
7.2 Microbial reclamation of alkaline sodic soils r66
REFERENCES 173
APPEI\IDIX T. Microbiological growth media and solutions 208
APPEI\DIX 2. FAME protocol for individual bacterial isolates 210
lx Abstract
Alkaline sodic soils cover approximately 60% of Australia's cropping regions. These soils constitute a problem because their high pH and Na* levels cause clay swelling and dispersion, leading to a reduction in crop yields. Losses due to sodic soils are estimated at
$1.4 billion per year, and remediation of these soils to increase yields would be extremely beneficial to local economies. One potential remediation method involves the stimulation of microbial populations in sodic soils to produce acid, thereby decreasing the soil pH and releasing calcium, which helps to prevent soil dispersion. However, little is known about the microbial populations/diversity in sodic soils, md the reactions that occur during biological based remediation. Therefore, the aims of this study were twofold.
The first objective was to determine the alkalitolerance, halotolerance and acid-producing capabilities of microbial populations cultured from alkaline sodic soils. The second objective was to investigate the microbial reactions involved during the amelioration of alkaline sodic soils with glucose, and soils ameliorated with complex organic substrates such as molasses and wheat straw.
The experiments conducted in this thesis focussed upon a typical alkaline sodic subsoil
yo collected from Watchem, Victoria, Australia (pH 9.6,8C I dS m'r , 6.5 CaCO3)' Low numbers of microorganisms were isolated from this soil, with an average of 7 x 104, 5.56 x
lga, and 1.2 xl03 colony forming units of non-filamentous bacteria, filamentous bacteria
(actinomycetes), and fungi being isolated per gram of air-dried Watchem soil, respectively. Fatty acid methyl ester (FAME) analysis of 216 of the non-filamentous bacteria isolated
ûom the Watchem soil revealed that the majorþ belonged to the genus Bacillus (80%).
Streptomyc¿s spp. (51%), and Penicillium spp. (66%), were the most common genera of actinomycetes and fi.¡rgi isolated from the soil, respectively. Alcaligenes rylosorydar4.s \¡ras the only Gram-negative bacterium identifred in this study.
Alkalitolerance screening of the 216 non-filamentous bacteria and 49 actinomycetes isolated from the Watchem soil demonstrated that this subset of microorganisms was highly alkalitolerant, with 96% of the non-filamentous bacteria and 70% of the actinomycetes studied being capable of growth on nutrient agar between pH 9 and 12.
However, whilst all of the non-filamentous bacterial isolates studied were alkalitolerant, their halotolerance levels were found to be dependent upon the concentration of NaCl used in the isolation medium. Tolerance to high concentrations of NaCl at a high pH was also dependent upon the concentration of NaCl used in the isolation medium. These differences in halotolerance can be attributed to different bacterial species being selected on each isolation medium.
preliminary testing of the halotolerance levels of the Watchem soil actinomycete population found that lTYo of the 49 isolates studied were able to grow on agar containing
1.5M NaCl. None of the actinomycetes tested could grow on agar containing lM NaCl,
adjustedtopH 11.
xt V/ith the exception of Micrococcus spp., all of the non-filamentous bacterial genera identified in the Watchem soil via FAME analysis were found to be capable of producing acid from glucose in vitro.In contrast, only l2Yo of the tmidentified isolates were found to be capable of producing acid from glucose in vitro.
Glucose treatment of the Watchem soil was shown to lead to the production of acetic acid and butyric acid, possibly by stimulating the activity of Clostridium spp. Addition of the t), ,,complex" organic substrates molasses (50 t ha and molasses (25 t ha-l) in conjunction t), with wheat straw (10 t ha super phosphate (100 kg hal; and ammonium nitrate (80 kg
r), ha to the Watchem soil initially favoured the growth of fungi. It was not until the fungi became nutrient limited that Clostridium spp. \¡/ere detected in the soil. The aerobic culturable microbial community of the Watchem soil was therefore, found to be unimportant for acid production in the Watchem soil environment. As a consequence of more carbon being metabolised aerobically by fungi than anaerobically by Clostridium
spp., the rate of VFA production in soils amended with "complex" substrates was much
lower than that measured in the glucose-amended soil.
'When watered to a 60Yo moisture content (waterlogged conditions) and treated with
glucose at a rate of either 2Yo (wlw), or 4%o (w/w), sufficient acid was produced in the
Watchem soil, via microbial reactions, to decrease the soil pH. This led to the release of
Ca2* equivalent to between 2 and 2.5 t h{r of glpsum, eflectively ameliorating the
V/atchem soil. No significant release of Ca2* was observed in the Watchem soil treated
with 2Yo (w/Ð or 4Yo (dw) glucose, watered to a 30o/o moisture content (water holding
capacity), suggesting that anaerobic conditions, which facilitate acid production, were not
achieved
x1l Whilst acetic and butyric acid were produced in the Watchem soil treated with "complex" organic substrates, the effects of these acids are thought to have been masked by the high levels of Ca2* introduced with the molasses component of the "complex" organic amendments. In both the soil treated with glucose and the soil treated with "complex" organic substrates, large quantities of substrate were required to obtain a small ameliorative effect on the Watchem soil, limiting the economic feasibilþ of microbial reclamation in dry-land farming systems.
xiii Declaration
of any other Degree This thesis contains no material which has been accepted for the award
and belief, this thesis or Diploma in any University. To the best of the author's knowledge
person, except where due contains no material previously published or written by another reference is made in the text of the thesis'
the Universþ Library' I give my consent to this copy of my thesis, when deposited in being available for photocopying and loan'
xlv Acknowledgements
your excellent Thanks to Dr. P. Rengasamy, Dr. Chris Franco, and Dr. Ian Singleton for promise won't supervision, guidance, ild friendship throughout this project. Chris, I I
make you read my thesis cover-to-cover again!
Thank you also to the Grains Research and Development Corporation for firnding the
research presented in this thesis, as well as providing my scholarship'
their Special thanks to Alla Baklan, Bruce Hawke, Debbie Miller, and Colin Rivers for
excellent technical assistance, advice, and muscles!
Special thanks also to Mum and the girls, Ian and Margaret for your support and
encouragement.
think Finally I would like to thank my wife Belinda, and my new baby boy Lachie. Just
Bel, since I started this almost 5 years ago' we have gone from being boyfriend and your love' girlfriend, to being engaged, then married, and no\¡¡ we're parents! Thanþou for
support, and most of all, your infinite patience'
And Lachie, your birth has been the best incentive of all to get this thesis finished. I can in now look forward to spending time with you and your mum, rather than being stuck
front of the computer!!!
xv LIST OF F'IGURES
Figure 1.1 Distribution of sodic soils in Australia 1
Figure 2.1 Amelioration of sodic soils via the addition of gypsum 6
Figwe2.2a Proposed mechanism for the microbial reclamation
of alkaline sodic soils 13
Figtre2.2b Degradation pathways of complex organic materials t3
Figtre 3.1 Structure of the microbial population survey 34
Figure 3.2 Effect of isolation media pH on numbers of
non-filamentous bacteria isolated from the Watchem soil 42
Figure 3.3 Effect of isolation media NaCl concentration
on numbers of non-filamentous bacteria isolated
from the Watchem soil 43
Figure 3.4 Photograph showing 6 non-filamentous bacterial isolates
from the Watchemsoil growing on nutrient agar
ranging in pH from7-12 44
Figure 4.1 Chromatogram showing well separated VFA standards 78
Figure 4.2 Fragmentation pattem of Octanoic acid 79
Figure 4.3 Calibration curve for Pentanoic acid 80
Figure 4.4 Chromatogram showing position of the internal standard
dodecanal in relation to VFA Peaks 81
Figure 4.5 Fragmentation pattem of Dodecanal 82
90 Figure 5.1 Schematic of the pot used for the glucose experiment
Figure 5.2 The concentration of reducing sugars (glucose) in the
Watchem soil throughout the 60-day incubation period 96
xvl Figure 5.3 vFA production in the watchem soil watered to wHC and treated
103 with 4%o glucose (wÐ over the 60-day incubation period
Figure 5.4 Relative Yo change in the concentration of FAMEs
representing Gram-positive bacteria (Gr)'
Clostridum spp. (C), and frrrgi (F), in the W4 treatment,
over the 60-day incubation period 103
Figure 5.5 Volatile fatty acid production in Watchem soil watered
to 2WHC and treated wtlh4% glucose (w/w)
over the 60-day incubation period 104
106 Figure 5.6 Metabolic pathways for organic acid-production from pynrvate
Figure 5.7 Relative Yo change in the concentration of FAMEs
representing Gram-positive bacteria (Gf)'
Clostridum spp.(C), and tungi (F)' inthe 2W4
treatment, over the 60-day incubation period 107
Figure 5.8 Volatile fatty acid production in Watchem soil
watered to 2WHC and treated with2% glucose (øÐ
over the 60-day incubation period 109
Figure 5.9 Relative %o change in the concentration of FAMEs
representing Gram-positive bacteria (Gf)'
Clostridum spp.(C), and frrngi (F), in the2W2 treatment,
over the 60-day incubation period I l0
Figure 5.10 Volatile fatty acid production in Watchem soil watered
to WHC and treated with2Yo glucose (w/Ð
over the 60-day incubation period 111
xvll Figure 5.11 Relative %o chatge in the concentration of FAMEs
representing Gram-positive bacteria (Gr)'
Clostridum spp. (C), and fungi (F), in the W2
treatment, over the 60-day incubation period tt2
Figure 5.12 Relative %o change in the concentration of FAMEs
representing Gram-positive bacteria (Gr),
Clostridum spp. (C), and fungi (F), in the
aerobic control, over the 60-day incubation period 113
Figure 5.13 Relative %o chatge in the concentration of FAMEs
representing Gram-positive bacteria (Gl)'
Clostridum spp. (C), and fungi (F), in the anaerobic
control, over the 60-day incubation period t14
Figure 5.14 The effect of glucose-treatment on the pH of the
'Watchem soil over the 60-day incubation period 116
Figure 5.15 The relationship between total soil VFA concentration and
the pH of the V/atchem soil following glucose-treatment 116
Figure 5.16 The effect of glucose-treatment on the percentage of
soluble carbonate present in the Watchem soil
over the 60-day incubation Period 118
Figure 5.17 The relationship between the total soil VFA concentration
and the percentage of soluble carbonate present in
the Watchem soil, following glucose treatment 119
Figure 5.18 The relationship between soil pH and the percentage
of soluble carbonates present in the Watchem soil,
following glucose-treatment 119
xvlll Figure 5.19 The effect of glucose-treatment on the percentage
of soluble calcium present in the Watchem soil
over the 60-day incubation Period t22
Figure 5.20 The relationship between the total soil VFA concentration
and the level of soluble calcium present in the
Watchem soil, following glucose-treatment 122
Figure 5.21 The relationship between the percentage of soluble carbonate
and the level of soluble calcium in the Watchem soil,
following gluco se-treatment t23
Figwe 5.22 The effect of glucose-treatment on the percentage of
soluble sodium present in the Watchem soil
over the 60-day incubation Period 124
Figure 5.23 The effect of glucose-treatment on the EC of the
Watchem soil over the 60-day incubation period t25
Figure 6.1 Stages in the selection of complex organic ameliorants 130
Figure 6.2 The effect of Molasses-treatment, Super-treatment, and
distilled water (control) on the concentration of total carbon
in the Watchem soil, over the 90-day incubation period 145
Figure 6.3 Relative o/o change in the concentration of FAMEs
Representing Gram-positive bacteria (Gr)'
Clostridum spp. (C), and frrngi (F), in the Control,
over the 90-day incubation Period t47
Figure 6.4 Identity and concentrations of VFAs produced in the
Watchem soil following Molasses treatment,
over the 90-day incubation Period 148
xix Figure 6.5 Relative %o chmge in the concentration of FAMEs
representing Gram-positive bacteria (Gl-),
Clostridum spp. (C), and frurgi (F), in the Molasses-treatment,
over the 90-day incubation Period 148
Figure 6.6 Identity and concentrations of VFAs produced
in the Watchem soil following Super-treatment,
over the 90-day incubation Period 151
Figure 6.7 Relative %o chatge in the concentration of FAMEs
representing Gram-positive bacteria (Gr),
Clostridum spp. (C), and fungi (F), in the Super-treatment,
over the 90-day incubation Period t52
Figure 6.8 The effect of Molasses-treatment, Super-treatment, and
distilled water (Control) on the pH of the Watchem soil,
over the 90-day incubation Period 155
Figure 6.9 The effect of Molasses-treatment, Super-treatment, and
distilled water (Control) on the concentration of soluble C**
in the Watchem soil, over the 90-day incubation period t57
Figure 6.10 The effect of Molasses-treatment, Super-treatment, and
distilled water (Control) on the concentration of soluble Na*
in the Watchem soil, over the 90-day incubation period 159
Figure 6.11 The effect of Molasses-treatment, Super-treatment, and
distilled water (Control) on the EC of the Watchem soil,
over the 90-daY incubation Period 160
XX LIST OF TABLES
Table2.l Maximum acceptable limits for heavy metals in sewage t9
destined for land application in NSIW, Australia
Table 3.1 Bacterial tolerance of 216 non-filamentous Watchem soil
bacterial isolates to alkali and NaCl 44
Table 3.2 Identification of non-filamentous bacteria able to gro\¡/ on
up to 2M NaCl G'}j'7 .4), but not on lM NaCl at pH 11,
by FAME analysis 45
Table 3.3 Identification of non-filamentous bacteria unable to
grow on 2M NaCl (pH 7.4),but able to grow on
lMNaCl atpH 11, byFAME analYsis 46
Table 3.4 Identification of non-filamentous bacteria able to grow
on both 2M NaCl (pH 7.4) and lM NaCl media at pH I 1,
by FAME analysis 47
Table 3.5 Classification of non-filamentous bacteria with a FAME
profile similarity of <30Yo able to grow on up to
2M NaCl þH 7.4), but not on lM NaCl at pH 11 49
Table 3.6 Classification of non-filamentous bacteria with a FAME
profile similarity of <30o/o unable to grow on 2MNaCl
49 OH 7.4), but able togrow on lMNaCl at pH 11
Table 3.7 Classification of non-filamentous bacteria with FAME
profile similarþ of < 30Yo able to grow on both 2M NaCl 5l G)H1 .4), and on lM NaCl at PH 11
xxr Table 3.8 Acid producing strains of non-filamentous bacteria
identified in the Watchem soil 52
Table 3.9 Acid production by non-filamentous bacteria with a FAME
profile similarity of <30%o able to grow on up to
53 2M NaCl G'H7 .4), and on lM NaCl at pH 11
Table 3.10 Acid production by non-filamentous bacteria with a FAME
profile similarity of <30orlo ableto gto\¡/onupto 2MNaCl
53 G,H7.4),but, not on lM NaCl at PH l1
Table 3.11 Acid production by non-filamentous bacteria with a FAME
profile similarity of <30%o unable to grow on up to
2M NaCl (f)H1 .4), but, able to gtow on lM NaCl at pH 11 54
Table 3.12 Alkalitolerance and halotolerance of 49 actinomycete isolates
from the'Watchem soil 54
Table 3.13 Classification of actinomycetes based upon spore morphologies 56
Table 3.14 Morpholo gical classification of actinomycete s unable
to be identifred using the slide cultr¡¡e technique 57
Table 3.I5a Hyphal and spore morphology of non-Penicillium fungal isolates 58
Table 3.15b Hlphal and spore morphology of firngal isolates
identified as Penicillium species 59
Table 4.1 Effect of the organic solvent composition on
% VFA recovery from an aqueous phase 84
Table 4.2 Effect of the extraction method on VFA recovery 86
Table 4.3 VoYaiation between replicates for each extraction method 86
89 Table 5.1 Treatments used in the glucose experiment
xxll Table 5.2a FAME profiles of Bacillus sPP.
isolated from the Watchem soil 99
Table 5.2b FAME profiles of non-Bacillus bacterial spp.
isolated from the Watchem soil 99
100 Table 5.2c FAME profiles of Clostridium species
100 Table 5.2d FAME profiles of fungi isolated from the Watchem soil
Table 6.1 Physical and chemical properties of the molasses
used in this studY r28
Table 6.2 The effect of distilled water on the Watchem soil pH
over the 90-day incubation Period 135
Table 6.3 The effect of wheat straw treatment on the Watchem soil pH
136 over the 90-day incubation Period
Table 6.4 The effect of sheep-manure treatment on the Watchem soil pH r37 over the 90-day incubation Period
Table 6.5 The effect of molasses treatment on the Watchem soil pH
over the 90-day incubation Period 138
Table 6.6 The effect of wheat-straw and sheep-manure treatment
on the Watchem soil pH over the 90-day incubation period t40
Table 6;7 The effect of wheat-straw and molasses treatment on the
Watchem soil pH over the 90-day incubation period t4l
Table 6.8 The effect of super phosphate, ammonium nitrate'
molasses, and wheat-straw treatment on the watchem soil pH
over the 90-day incubation Period 142
xxlll ABBREVIÄTIONS
EC: Electrical conductivity (dS nt:t)
ESP Exchangeable sodium percentage
FAME: Fatty acid methyl ester
GC-MSD: Gas chromatograph with a mass selective detector
GC: Gas chromatograph
NA: Nutrient agar
PDA: Potato dextrose agar
SAR: Sodium adsorption ratio
TSBA: Tryptic soya broth agar
W Ultra violet
VFA: Volatile fatty acid
YME: Yeast, malt extract medium
xxlv !.rii
CHAPTER 1
Introduction
being Sodic soils cover an estimated33 7o of Australia's surface, the worst affected areas
sparse grazing lands of the intensely farmed parts of southern and eastern Australia, and the
(Figure (Northcote and Skene, t972)' Queensland and'Western Australia 1.1)
l- l Sodic Soils
Figure 1.1. Distribution of sodic soils in Australia
to clay swelling The high concentration of exchangeable sodium found in sodic soils leads
structure' As soil pore and eventually dispersion when the soil is wet, destroying soil pore
drainage, easily turn structure is destroyed, these soils exhibit poor water infiltration and
anaerobic, and when dry, become hard-setting' and olsson, All of this leads to a reduction in crop yields (Lax et al., 1994; Rengasamy
soils with high pH (pH > 1993;Chorom et aL,lgg4). The above effects are enhanced in negative charge 8.4). Firstly, clay particles in alkaline conditions exhibit a higher net (sAR), thus disperse compared to neutral soils with the same Sodium Adsorption Ratio
crop nutrient deficiency more readily (chorom et aI., lgg4). Secondly, a high pH leads to toxicity problems eg', through the precipitation of metal ions, eg., Ct* , as well as nutrient in alkaline sodic the increased availability of boron (Gratram et al, 1992)' For example' of South Eastern soils, such as those found in the'Wimmera and Southern Mallee regions to Australia (which comprise some I million hectares of land), wheat yields in relation
above average rainfall were reported to be less than 50 Vo of their potential in average and
(Carter et al', 1992)' It is seasons (around 2 tonnes hal and 4 tonnes hal, respectively)
to around $ 550 estimated that the income in this region would increase from $ 400 million
1994)' million per annum if the average rate increased to 3 tonnes hal (Rengasamy,
gypsum (CaSO+'2HzO)' At present, treatment of sodic soils is generally by the addition of clay particles' When applied to sodic soil, gypsum causes the exchange of Na* for Ca2* on
particles no longer swell or As Ca2* ions are less readily hydrated than Na* ions, the clay
however, is expensive disperse upon wetting, improving the drainage of the soil. Gypsum alternative sodic soil when applied to a large area, and not a finite resource, therefore,
reclamation strategies should be investigated'
proton injection, which forms one such alternative remediation strategy could be biological to sodic soil and the basis of this thesis. In this approach, organic wastes are added acids (VFAs) by soil conditions altered to promote the production of volatile fatty
microorgantsms.
2 The acid produced by these microorganisms may then releas e C** bound in insoluble
CaCO:, which can then exchange with Na* at clay sutfaces. The aims of this thesis therefore, are:
l) To study the microbial populations present in alkaline sodic soils, with particular reference to their tolerance to alkaline conditions and NaCl, and acid-producing capabilities.
sodic soil 2) To compare the microbial processes leading to acid production in alkaline
manure, molasses, treated with glucose, and more complex organic materials such as sheep and wheat straw.
3) To quantify the effects of microbially-synthesised acids on the physical and chemical properties of alkaline sodic soils, and,
4) To identify environmental conditions that optimise microbial acid production in the
alkaline sodic soil environment.
3 CHAPTER 2
Literature Review
2.L Classification and formation of sodic soils
2.1.1 Classification of sodic soils
In Australia, soils are considered sodic if Na* ions constitute 6 or more percent of total
(ESP) > Sodic cations bound to clay particles, ie., the exchangeable sodium percentage is 6'
(pH 8) soils may be classified as being acidic (pH < 6), neutral (pH 6 to 8), or alkaline >
et based on the pH values of 1:5 soil:water extracts (Rengasamy and Olsson,l99l;Ford aL,1993). The pH of sodic soils is controlled by NazCO:-HzO-COz and CaCO¡-HzO-COz systems operating within the soil. Variations in the Ca2* ion activity, or the ion activity ratio (ca2*)t''l (H*),will therefore, alter the soil pH (Ponnamperuma, 1972).
Alkaline sodic soils, which comprise 73 Vo, 63 7o, and 40 Vo of the agricultural lands in
rWales Victoria, South Australia, and New South respectively (Ford et al., 1993; Naidu and
prevalent type of sodic soil' Rengasamy , 1993; McKenzie et al., lgg3), are the most
followed by neutral and acidic sodic soils. Alkaline sodic soils (pH > 8), which include
1972), calcareous earths, red duplex soils, and cracking grey clays (Northcote and Skene,
areas with are typically high in bicarbonate ions (HCO3-). Neutral sodic soils are found in
while acidic an annual rainfall of up to 550 mm, and are generally high in cl-, and so¿2- ,
leachable Ca2* sodic soils are found in areas of high rainfall (> 550 mm), and are low in
and Mg2* (Rengasamy and Olsson, 1991). 2.1.2 Naturalforrnation of sodic soils
main sources: Sodium found in naturally formed sodic soils is thought to be derived from 3 precipitation of l) the atmosphere, where deposition of sodium is known to occur via the sodium salt- water vapour containing sodium salts (Isbell, 1983), and aeolian transport of
Feldspar, rich dust (McTainsh et a1.,1990), 2) the weathering of sodium-rich rocks such as
sodium from and 3) by the movement of ground water (Chartres, 1993). Accumulation of
any of these sources leads to the formation of sodic soils.
2.1.3 Anthropogenic sodic soils
Human conversion of highly-productive agricultural soil to almost infertile, sodium-rich
soil has been calculated to be occurring at the rate of 10 million hectares per annum
worldwide (Szabolcs, 1989). In Australia alone, water-logging and salinity were estimated
basin by to have reduced productivity on dryland and irrigated farms in the Murray-Darling
is thought to 65 million dollars in 1988 (Meyer, 1995). The loss of quality agricultural land
clearing have occurred by two main mechanisms, the first of these being the indiscriminate
of crops and over-g nzingof native vegetation, and the second of these being the irrigation
with poor quality water. These are discussed in more detail below.
2.1.3.1 Indíscriminate clearing and. over-grazing of nøtive vegetation
water table to The indiscriminate clearing and over-grazing of native vegetation causes the
leach to rise, a consequence of which is that sodium-containing salts, in particular Nacl, soil is often the soil surface, and due to water evaporation, accumulate. This sodium-rich
via sheet and unable to support the growth of plants, making it prone to further degradation
gully erosion (Dregne, 1986).
5 2.1.3.2 lrrigatíon of crops with poor quality water
The inigation of farmland with poor quality, slightly-saline water leads to the formation of sodic soils. This is because Cl- ions present in the water are readily leached, sodium ions become bound to the cation exchange sites present on the surface of clay particles, ie., watering a soil with saline water causes an increase in the exchangeable sodium percentage
(ESp) of the soil (Mantell et al., 1985). This problem is likely to increase if more farmers tap into saline-sodic water supplies (Rengasamy and Olsson, 1993).
2.2 Chemical reclamation of alkaline sodic soils
Traditionally, sodic soils have been reclaimed through chemical means. Described below are several of these methods.
2.2.1 Soilic soil ønd gYpsum
Gypsum (CaSO¿.2HzO) has been shown to be beneficial to sodic soils, as when it is
combined with soil, Ca2* ions are introduced, which can then exchange with Na* ions
adsorbed to the surface of clay particles (Figure 2.1).
Na+ Na+ Sodic Na+ Clay Gypsum Na+ Particle t (free Ca2*) Na* Na+
Ca2* Ca2*
Ca2* Clay free Na+, which is Particle leached Ca2* Ca2*
Figure 2.1. Amelioration of sodic soils via the addition of gYPsum.
6 Release of calcium leads to a decrease in 1) the percentage of exchangeable Nan ions presenr in the soil (ESP), 2) soil pH (through the precipitation of HCO-¡ and CO32- complexes) (Gupta and Abrol, 1990), and 3) soil slaking (C** ions are not as readily hydrated as Na* ions) (Lehrschet aL, 1993). Whilstbeneficial to most soils, gypsum has several drawbacks, including cost when applied at high rates, suitability for use in certain soil types (eg., acidic soils), and long-term availability.
with l) Cost-associated problems: As the gypsum requirement for sodic soils increases increasing soil pH (Gupta and Abrol, 1990), the amount required for total reclamation of alkaline sodic soils is too expensive for most farmers. Many farmers, therefore, only purchase enough gypsum to temporarily change the soil Na*/Ca2* balance, so repeated application is necessary. Sometimes, they apply less expensive, low-grade gypsum containing high concentrations of NaCl, leading to soil salinity in the long-term.
2) Soil suitability: Although calcareous sodic soils can be reclaimed with gypsum, alkaline sodic soils generally contain sufficient calcium to facilitate desodification' However, the calcium is in the form of CaCO¡ which is insoluble above pH 8.5 (Rengasamy, personal communication).
These soils may be suitable for microbial reclamation, as calcium will become available for
8.5. exchange with sodium on the surface of clay particles when the soil pH falls below
Addition of gypsum to acidic soils however, is not recommended, as gypsum, being acidic'
will further acidify the soil (Carter 1986).
3) Long-term availability: Gypsum is used for a wide variety of purposes, including the
a finite manufacture of plaster board and plaster of Paris. As it must be mined, gypsum is
7 to resource, therefore, it is important to find alternate methods for introducing free calcium soils.
2.2.2 Inigation
Inigation of sodic soils with saline water can increase crop quality and decrease soil pH and ESp (Dubey and Mondal, 1993). This is because Na* in water increases the electrolyte concentration in soil, and thus, increases water permeability (Cass and Summer, 1974;
Mohite and Shingte, 1981). For complete desodification, however, leaching is essential. If proper leaching does not occur there is the danger of forming saline-sodic soil. Due to the naturally low water infiltration and drainage rates of sodic soil, leaching can only be effectively achieved via frequent inigation of the soils with small amounts of water, in combination with drainage systems (Dahiya and Anlauf, 1990).
Although viable for small areas of land, it is unlikely that inigation will ever become a viable method for the desodification of large inland areas currently used for dryland farming due to the high costs involved, and, more importantly, the availability of suitable quality inigation water.
2.2.3 lron pyrite
Low-grade iron pyrite (FeSz, 15 - 30 7o sulphur) is an inexpensive by-product of coal and
iron mining activities. Whilst the spontaneous oxidation of pyrite is extremely slow under
aerobic conditions, pyrite can be rapidly oxidised by the sulphur-oxidising bacterium
reactions: Thiobacillus ferrooxidans into sulphuric acid, as is shown in the chemical
FeSz + 3 ll2}2+ HzO -> Fe2* + 2SO+2- + 2H+, then
FeSz + 14Fe3* + 8HzO + 15Fe2+ + 2SO¿2- + 16H+
8 3 and 5 These reactions occur best under moist conditions, at an optimum pH of between
the (Sharma and Varma, 1991). Above this pH, oxidation can be enhanced by inoculating pyrite with a pure culture of Thiobacillus ferroxidans'
pyrite oxidation Several researchers have attempted to hamess the protons generated during
pH and to dissolve carbonates present in alkaline sodic soils, thus decreasing the soil releasing calcium ions into the soil solution, ie.,
CaCOs + 2HzSO¿ -> C** + SO¿2- + HzO + COz
The calcium ions released into the soil solution from this reaction may then displace sodium from the soil and improve the soil structure, as was shown Figure 2'l'
Most experiments to date have found that when applied on an equal sulphur basis, the
Sharma and amending abilities of gypsum and pyrite are similar. For example, in work by
7.2) at Gupra (1986), pyrite applied to the surface of an alkaline sodic soil (pH = 8'4, EC =
between 92 between 0 and 75 Vo of the gypsum requirement of the soil, was found to be
the soil with divalent and 9g.g Vo as effective as equivalent levels of gypsum at enriching
(1984), and cations. Similar results have been reported by Singh et aI. (1978), Tiwari et al.
Alam et aL (1986), and more recently, Sharma and Swarup (1997).
gypsum and pyrite Verma and Abrol (1980), who conducted comparative studies between
Sharma and concluded that pyrite was only one-quarter as effective as gypsum. Given that
sodic soil it Swarup (lggi) found that for pyrite to function effectively as a ameliorant in
Verma and must contain between 6 - 8 Vo soluble sulphur, it is possible that the pyrite
9 acid- Abrol used was too low in soluble sulphur to allow the establishment of sulphuric producing Thiobacillur spp. prior to adding it to the soil'
Thiobacillur spp. have an optimum pH of 3 - 5 (Sharma and Varma and, 1991)' and are
soils. The only way only likely to be able to grow within acidic microsites in alkaline sodic
grow on and oxidise such microsites can be formed is for large numbers of Thiobacillu.s to
This can be some of the pyrite into sulphuric acid prior to being applied to the soil.
and incubating it achieved by watering the pyrite to increase its soluble sulphur content
pyrite as a reclaimant of before use (Sharma and Swarup, 1997). The prospect of using iron water is alkaline sodic soils in dryland farming areas is therefore, highly limited since
often in low supPlY.
2.2.4 Water-soluble PolYmers soluble polymer In experiments by Zahow and Amrhein (1992), the addition of the water to polyacrylamide (anionic and cationic forms), and a cationic guar (seaweed)-derivative
significantly sodic soils ranging in ESP from 8 to 35 at atate of 50 mg kg-l, were found to
spontaneous increase the hydraulic conductivity of soils with an ESP < 15 by reducing
when the polymers clay dispersion. For soils with ESP > 15, this effect was observed only
rates of were added to the soil along with gypsum. Improvement in the water infiltration
reported (Levy sodic soils resulting from anionic polyacrylamide application has also been
et aI. ,1995)
conductivity of Although water soluble polymers are useful for increasing the hydraulic
due to their high cost' sodic soils, they are unlikely to ever be applied at a field scale level
10 2.2.5 Heatìng chorom (1996), in his phD thesis on the effects of pH and particle charge on sodic soils,
a showed heating alkaline sodic soils to > 200oC leads to fixation of Na*, and therefore, reduction in soil dispersibility. \Mhilst the heating of sodic soils is beneficial for improving soil structure, the costs in terms of labour, and the energy required to heat whole fields, limits the commercial viability of this method. Furthermore, this method is likely to cause severe damage to soil microflora through direct heating, as well as the destruction of organic nutrients.
2.3 Biological reclamation of sodic soils
2.3.1 Organic matter and biological reclannation of sodít soil
Considerable research, conducted primarily in India, has demonstrated the beneficial effect of adding organic matter to sodic soils, in particular to alkaline and highly sodic soils
(Swarup, 1986; Tomar et aI., 1987; VarmaandMathur, 1990; Gillet a1.,7991; Swarup,
I99I: Swarup, 1992; Dubey and Mondal,1993; More, 1994)'
in Organic materials have the advantage over inorganic materials for remediation of soils,
cheese whey' that, 1) they are readily available, usually as by-products of industry, eg', acid
2) there are many forms available, from garden mulch to sewage sludge, and 3) the
materials used for organic amendments are cheaper, and not as dependent on rain for
arid to incorporation into the soil as fertiliser, and are thus, particularly suited for use in
semi-arid lands, such as those found in Australia (Dennis and Fresquez,1989).
waste Furthermore, farms utilising organic material decrease the output of nutrient-rich
into aquatic environments.
11 Organic amendments are known to improve the structure of sodic soils, as well as increase the amounts of Fe, Mn, and Zn available to crops (Puttaswamygowda et aI., 1973; Swarup,
1985a, lgg2). The ameliorative effect of organic matter can be attributed to 1) the biotransformation of organic matter into organic acids and COz, and 2) an increase in microbial biomass and soil enzyme activities (Schnurer and Rosswall, 1982; Dick et al.,
1988). More details of these mechanisms are given below:
l) The biotransformation of organic matter into organic acids and COz by microorganisms in alkaline sodic soil containing CaCO¡ causes a reduction in soil pH, and eventually a reduction in the ESp. Organic acids, and aqueous, dissolved COz (carbonic acid) react with
CaCO¡ within the soil, freeing Ca2* ions. These Ca2* ions may then exchange with Na* ions adsorbed to clay particles, allowing Na* ions to be leached down further into the soil profile, thus decreasing the ESP (læhrsch et aI., 1993).
To increase production of organic acids, in particular VFAs, and to maintain acid levels
necessary for successful soil amelioration, regular addition of fresh, readily degradable
organic matter is necessary because organic acids will be mineralised by soil
microorganisms (Tsutsuki and Ponnamperuma, 1937). Anaerobic conditions will further
promote VFA production (Ponnamperuma, 1972). The mechanisms involved in microbial
reclamation of sodic soils are better explained in Figures 2.2a. and2'2b'
12 Aeroblc conditio¡u
CO¡+H¡O, then COz+HrO ê H*+ HCO¡', then 2H'+nativeCaCO¡ + Ca2'+CO2+E2O
+ C¡Hr¿Oc
Microorganlsnu Anaerobic conditioru
Organic acids, then
Organic acids + native C¡CO¡ Ð Cat++ COr + E¡O
Ca2* relessed in the sbove reactions is then able to exchange Na+ from the surface of clay particles. Ca2t prcvents soll disperslon, and, therefore, lnproves soll aggregate ståbllity.
ßigrure2.2a hoposed mechanism for the microbial rcclamation of alkaliñê sodic soils.
Complex polymers
Cellulolylic and other hydrolytic Eydrolysls bac¡ería Monomers
Sugars, Amino acids
Fctmenlative bocleria Ferme¡tation
H2 + CO2 Acetâte Propionate, Butyrate
Ace¡ogctLs Acetogenesis H t -pmducing, lotty acid oxidising bactcrío (synrrcphs) Fermentetlon
Acetâte Methanogens
H2 + CO2 Acetate
Methanogens
Mcthanogens
Methanogenesis
CIù
Figure 2.2b. Degradation pathways of complex organic materials (from Brock and Madigan' 1991).
13 quantities 2) Anincrease in microbial biomass and soil enzyme activities leads to increased
of fungal hyphae and bacterial polysaccharides, which can bind micro- and macro-
Tisdall and aggregates, preventing slaking and dispersion of soil (Harris et al', 1964:
Olsson l99l)' Oades, 1982;Lynch and Elliot, 1983; Rengasamy and '
2.3.2 Límitations of organíc matter treøtment
weak, Organic matter in alkaline sodic soils is easily degraded, highly soluble, and forms
can increase soil readily dispersible bonds with Na+, and thus, when added to a sodic soil,
dispersibility (Gupta et aI., 1984; Oades, L984;Rengasamy and Olsson, l99l)'
horizons within the To decrease soil dispersibility and to prevent the formation of organic
with more soil, it is suggested that organic matter should be added to soil in conjunction
strongly bonding cations, eg. ca2* and Mg2* (Rengasamy and olsson, 1991)'
2.3.3 Types of organic matter
therefore, it The choice of organic matter available for sodic soil reclamation is very broad,
is important to select materials that are readily available - preferably on-site to reduce in moisture transport costs, inexpensive, and if to be applied to dry-farmed areas, high
suitable, or potentially and"/or moisture retaining. Described below are examples of wastes
suitable, for reclamation of sodic soils.
2.3.4 Farmyard. manure mixtures
sources of organic Farmyard manure, like crop waste, is one of the most readily obtainable
each cow produces matter on farms. Each pig produces up to 3.2 kg of waste per day, and
between 24.5 and 46.6kgof waste per day (Haug, 1993)'
t4 With such an output it would be advantageous to utilise this resource for the reclamation of sodic soils, especially considering manures contain both readily available nutrients, which encourage rapid microbial growth (hence acid, COz, hyphae, and polysaccharide production), and more stable, bulking organic matter (which can reduce effects of mechanical disturbance and rainfall).
In experiments by Gaffar et at. (1992), farmyard manure, in particular, chicken manure, was found to decrease the sodium adsorption ratio (SAR) in a sodic soil, to a depth of 60 cm. The decrease in SAR thus, improved the growth of sorghum planted in this soil. Gaffar and co-workers went further to show that soil treated with farmyard and chicken manure experienced a reduction in PH.
In separate experiments by Mbagwu (lgg2), poultry manure' on this occasion combined with rice shavings, was found to decrease bulk density and increase macroporosity, volumetric water retention, available water capacity, cumulative infiltration and time to reach infiltration capacity in a degraded sodic ultisol (soil pH unknown)'
The most effective organic amendments in a calcareous soil are those that produce the
highest amount of COz (Robbins, 1936). It follows then, that pig manure, which when
decomposing releases 69 CO2 kg soil-I, is a more valuable sodic soil reclaimant than
4.5g chicken manure, cow manure, and horse manure, which only release 59 CO2 kg soil-l,
co2 kg soil-rand 2.5gCOzkg soil-r' respectively (Ajwa and Tabatabai,1994)'
For example, Pagliai and Sequi (1982), found pig slurry to increase soil porosity
substantially more than that of the other manures tested'
15 soil In another study, by Bernal et aL (1992), the application of pig slurry on a calcareous of low clay content was found to increase the cation exchange capacity (CEC) of the soil
possibly and decrease the soil pH. These effects were enhanced by high slurry application, due to the liberation of exchangeable ammonium, and its subsequent nitrification - an acidogenic reaction. One disadvantage of this method however, is that high application of
contaminate slurry leads to increased leaching of No¡- through the soil, which can then groundwater (KandeleÍ et aI., 1994).
The reclaiming effects of manure on alkaline sodic soils can be further increased by pre-
that incubating manure before application to the soil. For example, Swarup (1986), showed
rice farmyard manure submerged for 30 days and then mixed with a sodic soil improved yield. In a separate experiment by Tomar et at. (1987), the application of farmyard manure
15 weeks with rock to a wheat crop sown in a sodic soil, this time incubated aerobically for phosphate and pyrites, led to an improvement in yield'
to be Whilst a large portion of the yield improvements reported in these studies are likely
VFAs and due to a nutrient response, some of the improvements may also be attributable to
in the COz, formed during the anaerobic incubation of the manure, reacting with carbonates
soil and decreasing the soil pH and ESP (Swarup, 1986; Tomar et aI', 1987)'
2,3.5 Acid cheese whey
Utah State Considerable research has been conducted by Jones, Hansen, and Lehrsch of
University, and Robbins of the USDA Agricultural Research Service, U.S.A., into the
et al', effect of acidic cottage cheese whey on sodic soil (Robbins and Lehrsch,1992; Jones
1993a& b). Acid cheese whey is made during the production of soft cheeses.
T6 This inexpensive by-product is low in pH, and high in soluble calcium. Furthermore, lactose and proteins within the whey are readily available for attack by microorganisms for
conversion to COz and organic acids (Summers and Okos, 1982). Cottage cheese whey
has been shown to reclaim sodic soils by lowering soil pH, ESP, and SAR, and by
(Robbins improving aggregate stability and infiltration, leading to increased crop yields
and læhrsch, 1992; Jones et aL, 1993a & b). Although shown to be extremely useful for
ameliorating sodic soils, the high cost associated with transporting acid cheese whey is
likely to restrict its use to dairy farming areas.
2.3.6 Sewage sludge
In 1989, polprasert estimated that the quantity of sewage produced in some European and
uS cities to range between l.l and 1.5 kg per person per day, and in developing countries
population to range between 1.1 and 1.8 kg per person per day. In Australia (approximate
lg million), this adds up to between 20 - 27 million kg of sewage per day. Polprasert
(19g9), went further to predict that the global production of biosolids by humans would
double by the year 2000 due to population growth, stricter wastewater treatment
requirements, and improvements in treatment works operation, making Sewage disposal a
major problem.
Although at present it is legal to discharge untreated sewage into the ocean in Australia,
this practice is beginning to impact upon the marine environment. One alternative disposal
by method may be to use sewage as an organic fertiliser, as was demonstrated in the study
Dennis and Fresquez (1989).
soil, In this work, sewage sludge was found to act as a low-grade slow-release fertiliser for
releasing N and P even after 2 years.
17 This sewage sludge was also found to decrease soil pH, the extent to which this occurred being proportional to sewage application (Dennis and Fresquez,1989).
In a study by Metzger et al. (1987), sewage sludge added at 5 Vo to soil was found to rapidly increase the numbers of water stable aggregates, caused by a surge in microbial growth, in particular fungi. Similarly, in experiments conducted by Lax et aI. (1994), the addition of municipal solid waste was found to improve the water holding capacity and infiltration of a saline soil, thus improving yields of tomatoes planted within this soil.
The capacity of sludge to increase the structural stability of a given soil is dependent on the properties of the sludge, eg., the C:N ratio, and stability, as well as the properties of the
soil, eg., texture, CaCO¡, and organic mattercontent (Pagliai et aL, 1981). V/hilst sewage provides organic material, it may also contain high levels of heavy metals (Mclaren and
Smith, 1996).
For example, Tiller et al., (lgg7) predicted that sewage sludge applied to Australian
agricultural soils would have a potential sludge-cadmium loading of between 2 and 4
in tonnes per annum. This is of particular concern, as many of our agricultural soils are low
organic matter and have a low CEC and pH. Under these circumstances the sorption of
levels, heavy metals is inhibited, hence these metals can accumulate in plant tissues at toxic
making crops unsaleable (Ross et al., 1991). Shown in Table 2'l ate the maximum
NSW, acceptable limits for heavy metals in sewage destined for land application in
Australia (Adapted from Ross et aI., 1991).
Furthermore, sewage may contain high levels of pathogenic organisms, eg', Ascaris
(Straub ¿f Iumbricolordes (human tapeworm) and microorganisms, eg., Salmonella typhi
18 aI., lgg3). With careful selection of the sewage source, and by pre-treating the sewage in a digester that raises the temperature above 50oC (the lethal temperature for most pathogenic microorganisms), sewage may be used with relative safety, potentially as a sodic soil reclaimant.
Table 2.1 Maximum acceptable limits for heavy metals in sewage destined for land application in NSW, Australia. (Adapted from Ross ¿f al.,l99l).
Maximum Maximum Maximum Maximum Element sludge conc. annual loading cumulative conc. in topsoil (mg kg't) (kg ha'l)" loading (kg ha'l)" (mg kg't)o
Arsenic 15 4 20 20 Cadmium 8 o.25 2.5 2.5 Chromium 500 20 100 100 Copper 1200 20 100 50 Iæad 300 5 50 100 Mercury 7.5 o.2 I 0.5 Nickel r00 l0 50 50 5 Selenium 25 1 5 Zinc 1800 50 250 200 for soil of 1.33 g " Values quoted as kg ha-' are calculated for the toP 75mm of soil using a bulk density b topsoil cm'3. Pollutant loadings quoted in kg ha'l are numerically equivalent to concentrations in expressed as mg kg'l when using the above assumptions.
2.3.7 Straw
Crop residues constitute the most readily available source of organic matter for cultivated
soils - the most abundant of these is straw (Dev and Bhardwaj, 1991). Straw is a plant-
(25 - derived material made of lignocellulose, a composite of lignin (10 - 30 7o), cellulose
45 Vo),and hemicellulose (24 - 50 7o) (Betts et aI.,1992). Due to the recalcitrance of lignin,
straw is difficult to degrade, and can therefore, persist in soil for long periods of time.
Straw degradation at field temperatures is thought to be chiefly due to fungal activity,
though actinomycetes such as Amycolata, Micromonospora, Nocardia, Rhodococcus' of Streptomyces, and the thermophilic Thermomonospora - all shown to be capable
t9 (Trojanowski et al', degrading the lignin component of straw, may also play active roles
and Ball, 1977; Haidler et al., 1978; McCarthy and Broda , 1984; Ball et aL, 1989; Trigo
t994).
shown that Previous studies by Barzegar et at. (1997), and Nelson and Oades (1997) have
the addition of straw residues to sodic soils can increase microbial polysaccharide levels
and hence, improve soil aggregate stability.
of Whilst useful for improving soil carbon levels and aggregate stability, the application
straw to soils under wet conditions however, can retard plant establishment and decrease (particularly crop yields (Cochran et al., lgii). This is due to the production of acetic acid mM under anaerobic conditions), which is toxic to plants at concentrations of around 5
(Lynch, 1977 , 1981b).
prior to To resolve this problem, the soil should be allowed to "rest" for a couple of months
(Cochran et al', sowing, to give the acetic acid time to volatilise or be metabolised to COz
1977 Harper and LYnch, 1981).
2,3.8 Crops and other Plants in plants Depending on the nitrogen source added, eg., NtIa* or NO¡-, nitrogen metabolism
can lead to the production of tf ions. ie., NI{4* + NH3 + H*
freeing plants producing large quantities of H* ions may therefore, be able to facilitate the
soils. Several of calcium ions from minerals such as calcium carbonate in alkaline sodic reclaimants' studies have demonstrated plants as being potentially valuable sodic soil
20 Detailed below are examples of plants used both successfully, and non-successfully for reclamation of sodic soils
l) Rice: Rice is noted for its tolerance to sodium, high pH, and waterlogging, and thus is an ideal crop for alkaline sodic soils. As rice is grown under waterlogged conditions (which restricts its use to irrigated farms), degradation of organic materials occurs under anaerobic conditions, the by-products from which are VFAs and COz. VFAs and COz are able to solubilise native CaCO3, which leads to displacement of Na+ from clay particles and a reduction in soil pH, and hence, an improvement in soil properties (Abrol and Bhumbla,
1979; Swarup, 1985b).
2) Other grasses: In experiments by Qadir et al., (1996), the cultivation of the alkali and salt-tolerant grass Leptochloafusca (commonly known as Kallar grass) on an alkaline sodic
greater soil (pH= 9.1, EC = 7 dS m-t; wu. shown to cause a decrease in pH and EC slightly than that observed for soil treated with 507o of its gypsum requirement. These effects were most pronounced during rapid plant growth, suggesting soil amelioration was due to the solubilisation of native CaCO¡, HzCO¡, COz, and exudates produced by active roots'
Furthermore, these reclaiming effects can be increased under field conditions by excessive watering, which encourages leaching of Na*'
3) Algae: In an experiment by Rao and Burns (1991), blue-green algae, which are known to
tolerate excess sodium, and to grow extensively on the soil surface in wet seasons, were
trialed as a way to mobilise Ca2* in alkaline sodic soils. The blue-green algae however,
were found to produce insufficient organic acids to decrease the soil pH and solubilise
of alkaline native CaCO¡ , even after 17 weeks, leading to the conclusion that reclamation
2l sodic soils with blue-green algae is untenable and not comparable with effective chemical amendments such as gypsum.
4) I-egumes and Oil seeds: Studies by several researchers have shown that legumes such as
Subterranean clover (Triþtium subterraneum) and Serradella (Ornithopus sativus), or oil seed crops such as Canola can directly acidify soils by releasing protons from the roots when an excess of cation over anion uptake occurs (Williams, 1980; Haynes, 1983; Jarvis and Robson, 1983 a,b; Liu et a1.,1989; McKenzie et a1.,1995: Mc Lay et aI., 1997;Tang et aI., 1998).
This acidifying ability, which correlates with the ash alkalinity of the plant material, is greatest in legumes that obtain most of their nitrogen from nitrogen fixation (Robson,
1983). The planting of legumes, or oil seed crops such as Canola, may then prove to be a useful method for decreasing soil pH and solubilising the CaCO¡ deposits present in many alkaline sodic soils.
2.3.9 Bagasse
Sugarcane trash, or bagasse, is the fibrous material left after sugar cane processing
(polprasert, 1989). When introduced to soil, bagasse should improve soil bulk density, and
(and as it contains readily utilisable carbon (sucrose), it should encourage microbial growth
in anaerobic conditions increase VFA production). Sugarcane bagasse incorporated with
soil at 5 t.h1l was found valuable at increasing nitrogen fertiliser utilisation and nitrogen
incorporation into crops, leading to increased yields of wheat (Dev and Bhardwaj, 1991).
22 2.3.10 Molasses
moisture, Molasses is a liquid waste produced during the manufacture of sugar. High in and sucrose, as well as calcium, molasses has been shown to be a highly effective in reclaimant of sodic soils. For example, Perez Escolar (1966) found complete success
reclaiming an alkaline saline-sodic Fe clay (pH 8.2, EC" 66.7 Mmhos) from southwestern
puerto Rico with 2SVo (vlv) molasses. Perez Escolar (1966) proposed that the reclaiming
effect of the molasses was due to VFAs being produced during the microbial
decomposition of the molasses in the soil solubilising native soil carbonates, and releasing
Ca2* into the soil solution.
In another study, Weber and Van Rooyen (lg7l), when investigating the reclaiming ability t) "Little of molasses meal (44. t.ha on alkaline sandy clay loam soils in the semi-arid
Karoo" region of South Africa over a 5-month period, found molasses meal treatment to
a 5.6Vo lead to a >l0OTo increase in water infiltration , a 33Vo increase aggregate stability,
These decrease in relative bulk density, and a 54Vo decrease in the modulus of rupture'
ameliorants ameliorative effects were far superior to those of the well-known sodic soil r), t), (46t.ha gypsum (20 t.ha potassium sulphate (21.5 t.har), sulphur (4 t.ha and manure
t) (1966) no applied to soils in the same area. Unlike the study of Perez Escolar however,
significant change was recorded in the soil pH or SAR after molasses meal treatment,
was due to therefore,'Weber and Van Rooyen (1971) concluded that the reclaiming effect
greater than the aggregating powers of polysaccharides present in the molasses meal being
being the dispersing forces of Na* ions adsorbed to the soil particles, rather than VFAs
native produced during the microbial decomposition of the molasses in the soil solubilising
soil carbonates
23 2,3.11 Green waste h 1986, the US EPA reported that the US discards 23.8 million tons of green waste per annum - up to 7O Vo of this being grass clippings. As grass is easily compacted and moist, decomposition is usually rapid and readily tums anaerobic, releasing VFAs, and NH3
(Haug, lgg3). The introduction of green wastes to alkaline sodic soils, then, might be useful for decreasing the soil pH, as well as increasing soil nutrient levels.
2.4Microbiology of Sodic Soils
So far, the literature review has focussed upon the effects of chemical and organic ameliorants on the physical and chemical properties of sodic soils. The last section of this literature review will discuss the impacts of chemical and organic amendments on the microbiological properties of sodic soils, as well as methods for the study of microbial populations within soils.
2.aJ Effect of atkaline condítions and NaCl on the microflora of sodíc soils
'When investigating the influence of the isolation medium composition on the total bacterial count obtained from three increasingly alkaline and saline sodic soils, Bhardwaj
(1974) found that using media with a high concentration of salts and a high pH led to significantly higher bacterial counts compared to those obtained on neutral, low-salt media.
Giambiagi and Lodeiro (1989) later demonstrated that the number of halophilic bacteria isolated from sodic soils could be increased by using an isolation medium containing both high levels of NaCl and nitrogen.
Gupta and Bajpai (1,974), observed that maximum microbial counts of soils with a
saturation extract pH ranging from 6 - 9.9, and an EC ranging from 1 - 84 occurred in the
neutral, non saline soils, with soil pH influencing the total count more than the EC. 24 Furthermore, Gupta and Bajpai (1974), observed a reduction in bacteria, fungi and
Azotobacter, but an increase in numbers of actinomycetes and bacterial spores as the soil
EC increased.
2.a.2 Effect of soil moísture content on the mìcroflorø of sodíc soíls
In a study comparing the effects of soil moisture content on the microbial biomass, respiration rate, and total and spore-forming bacterial counts of a gypsum treated and a non-treated alkaline sodic soil (Yrigoyen and Giambiagi,1994), it was observed that:
1) The biomass of both the control and treated soils increased with increasing humidity,
2) The respiration levels of both the control and treated soils increased with increasing humidity, however at the lowest soil moisture level (hygroscopic limit), the control soil possessed a significantly higher respiration rate than the treated soil, and,
3) Higher numbers of bacteria were isolated from the treated soil than the control soil'
Furthermore, numbers of microorganisms were observed to increase in proportion to the humidity of the treated soil, but plateau between the permanent wilting point and saturation levels in the control soil.
yrigoyen and Giambiagi (1994) explained the differences between the two soils by
suggesting that the microflora in the non-treated soil were tolerant to NaCl and low levels
of moisture, whereas, the microflora of treated soil had evolved towards more moisture and
salt-sensitive organisms.
25 2.4.3 Effect of gypsum on the miuoflora of sodic soils microflora of Gypsum has been demonstrated as having a mostly deleterious effect on the
gypsum to sodic soils. For example Tyurin et aL (1960), observed that the application of
as microbial sodic soils can cause a temporary decrease in N mineralisation rate, as well
(11.2 t'ha-t activity. Similarly, Carter (1986) found that the long-term gypsum treatment ) of of four acid sodic soils (ranging in pH from 4.5 to 6.5) reduced the overall level
microbial N by 10 to 43Vo, increased the c:N ratio of the biomass, and reduced
mineralisable N by lO to 547o.
(pH 8.4) with 4 Gasoni et aI. (1986), demonstrated that treatment of an alkaline sodic soil
bacteria and t.ha-l gypsum, followed by sulphuric acid treatment, decreased the number of
number of fungi isolated from the soil by 72 Vo and 4O Vo, respectively. However, as the
soil by microorganisms in the soil could be increased to approximately that of the untreated that this adding ground vicia villos¿ and Medicago polymorpha leaves, it is thought sulphuric acid, decline in microbial numbers was more due to the destructive effects of
(1976), found that than from the gypsum treatment. Converse to these studies, Bajpai et aI'
and numbers gypsum application increased the total microbial biomass, microbial activity
of bacteria within sodic soils.
2.a.a Effect of green rnanure on the mícroflorø of sodic soíls microflora of alkaline Green manuring has been shown to have a beneficial effect on the
of the green manure sodic soils. For instance, in work by Rao and Pathak (1996), addition pH from 8.4 to 10, and in Sesbania cannabina to several alkaline sodic soils ranging in
well as decrease soil pH ESP from 3 to 73, was found to increase soil C and N levels, as
and ESP levels, stimulating the biological activity of the soils'
26 2.a.5 Effect of glucose on the microflora of sodic soils
Recently, in experiments by Chorom and Rengasamy (1997), the addition of glucose to an alkaline sodic soil (57o caco¡, pH 9.4) incubated under waterlogged conditions was
soil demonstrated to lead to an increase in microbial activity, followed by a reduction in pH. Although not quantified, the authors attributed this pH decrease to the soil microbial population metabolising the glucose into VFAs, which then solubilised native soil carbonates.
2.5 Methods for studying the microbial populations within soils
Traditionally, the study of soil microbial populations has focussed upon the phenotypic
that characterisation of isolated strains (Torsvik et a1.,1990). Recent evidence suggests culturable soil microorganisms may represent a tiny, possibly ecologically unimportant in portion of the overall diversity present in most soils (Cavigelli et a1.,1995)' For example,
4000 a DNA reassociation kinetics study, Torsvik et al., (1990), detected approximately different bacterial species in a 30 gram sample of forest soil taken from Southern Norway.
Of these, less than l%o wete culturable.
The consequence of using isolate-based methods, therefore, is that functionally
a particular unimportant soil bacteria may easily be misjudged as playing important roles in
plate soil environment because they were the dominant species on a bacterial isolation
(Ammann et al.,1996;Garland and Mills, 1991). Furthermore, studies of this type provide
give no little information on interactions between populations in the environment, and
interest (Holben and sense of identity or diversity of populations performing functions of
Harris, 1995).
27 To better understand the role and significance of microorganisms in ecosystem functioning, it is suggested that a multifaceted approach should be used such as combining a taxonomic study with a study on nutrient cycling, ie., examine the functional diversity of the soil microbial population (Zak et aI., 1994). Described below are some methods that may be of use in conjunction with microbial isolation for studying soil microbial populations.
2.5.1 Molecular techniques
Advances in molecular biology, such as restriction fragment length polymorphism (RFLP) analysis, and the polymerase chain reaction (PCR) amplification and sequencing of 165 rRNA genes, have enabled the detection and classification of never before described microorganisms in natural environments (Pace et aI., 1986; Fuhrman et aI., 1992; Bull ¿r al., 1992; Stackebrandt et a1.,1993; Amann et al,l996; Borneman et al., 1996).
Molecular techniques have several advantages over traditional isolation-based techniques, because: l) there is no need for culturing microorganisms, therefore studies can be performed in situ, and, non-culturable microorganisms are not excluded (Pace et aI., 1986;
Torsvik et al., 1990; Stackebrandt et ø1.,1993; Amann et a1.,1996; Thomas, 1996)'
2) oligonucleotide probes can be synthesised to bind to targets with a wide range of
specificities, from domain, to strain, therefore, these probes may be used to study either
single populations, or, multiple populations, simultaneously (Pace et aI., 1986; Holben and
Tiedje, 1988; Hugenholtz andPace, 1996), 3) relationships can be distinguished between
organisms from a pool of DNA in an ecosystem (Bull et aI., 1992; Holben et al., 1988)'
and 4) it is possible to detect genetic rearangements or gene transfers (Holben and Tiedje,
1988).
28 Molecular techniques, such as the sequencing of 165 rRNA genes, however, are limited by the fact that only approximately 6000 prokaryote 165 rRNA sequences are stored on
soil cannot databases. As a consequence, a large percentage of sequences retrieved from the be matched to known microrganisms (Pace et aL, 1986; Amann et a1.,I996). Furthermore,
the extraction and purification of rRNA and DNA from soils can be difficult due to
interactions with the soil matrix (Felske et aI., 1996; Gray and Herwig' 1996)'
2.5.2 BIOLOG
BIOLOG is a bacterial identification system which classifies bacteria by their ability to
degrade 95 different carbon sources in a multi-well plate. This system may also be used to
determine the functional diversity of whole soil microbial populations by inoculating the
BIOLOG plates with a diluted soil suspension and observing the levels of activity on
certain substrates (Garland and Mills, l99l;Zak et aI., 1994; Bossio and Scow, 1995).
Although BIOLOG can be used to profile the functional diversity of whole soil microbial
communities, it does not provide the identifies of the soil microorganisms. Furthennore,
this method may exclude important populations of microorganisms which are not capable
of growth on BIOLOG medium (Garland and Mills, l99l; Zak et aI., 1994; Bossio and
Scow, 1995).
2.5.3 Immunological tnethods
The staining of microorganisms using Monoclonal antibodies tagged with fluorescent dyes,
method or reporter genes such as lux and green fluorescent protein (GFP), is another useful
for studying microorganisms in situ (Thomas, 1996)'
29 IVhile this technique enables the number and location of specific groups of microorganisms to be determined within the soil matrix, it is first necessary to raise antibodies against the microorganisms of interest. This is a particularly difficult task when non-culturable microorganisms are the object of study. Furthermore, problems may be experienced with the tagging system. For example, lux is dependent on the energy state of cell and may not
(Thomas, 1996). be detectable in resting cells, and GFP does not function in some bacteria
2.5.4 Fatty Acid Methyl Ester (FAME) analysís
Fatty acid methyl ester (FAME) analysis is a relatively recent method for the identification of culturable medical and environmental microorganisms (Federle, 1986; Vestal and
White, 1989; Baath et a1.,1992; Tunlid and White, 1992: Frostegard et a1.,1993; Cavigelli et aI., 1995;Wander et a1.,1995).
This simple, rapid, inexpensive process works on the principle that most genera and some species of microorganisms contain a unique combination of long-chained fatty acids in their cell wall (Cavi gelli et aI., 1995).Individual isolates may therefore, be identified by extracting and methylating the fatty acid component of their cell wall, separating the
individual FAMEs on a gas chromatograph, and comparing their profile to profiles of
known type-strains of microorganisms previously entered on a database. Matches of greater
than3OTo are considered an acceptable limit for the positive identification of environmental
microorganisms (Haldeman et al., 1993).
Below this level, microorganisms can be said to be similar to their closest match, but are
not that species. One limitation of FAME therefore, is that it may not be possible to
identify some isolates as they are not present on the MIDI database.
30 The composition of whole soil microbial populations may also be studied using FAME
analysis, by monitoring the concentration of certain marker fatty acids. For example, the
concentration of the FAMEs iso 15:0 and anteiso 15:0 can be used to monitor the
populations of Gram-positive bacteria (O'leary and Wilkinson, 1988), l8:2 ol6c and 18:3
coóc can be used as markers for fungi (Federle, 1986; Vestal and White, 1989; Frostegard ¿/
aI., ¡993;Bardgett et al., 1996;Zogget aI., 1997), and cyclo 17:0 and cyclo 19:0 can be
used as markers for Gram-negative bacteria (Frostegard et aI.,1993;Zogg et aL.,1997).
Whilst whole soil FAME analysis eliminates the need to isolate microorganisms and
provides a snapshot of the microbial diversity present at the time of sampling, the fact
many fatty acids are common to different microorganisms, and that there are hundreds of
different fatty acids in environmental samples, especially in agricultural soils, often make it
difficult to interpret results (7*,lles et ø1., 1992; Cavigelli et al., 1995; Wander et a1.,1995).
Despite the limitations of FAME, its simplicity, speed, and low cost made it ideal for use in
this thesis.
2.6 Conclusion
The literature on sodic soil reclamation studies has focussed primarily on the effects of
ameliorants on the physical and chemical properties of sodic soils. However, there is a
dearth of research on changes to the microbiological properties of sodic soils, especially
those brought on by organic amendments, or the microbial reactions taking place during
reclamation. Furthermore, the literature review revealed that little work has been performed
on investigating the microbial diversity of sodic soils. This aims of this thesis are
therefore, to address these issues.
3T CHAPTER 3
Diversity, Alkalitolerance' Halotolerance, and Acid-
Producing Capabilities of Microorganisms Cultured
from an Australian Alkaline Sodic Soil
3.1 Introduction
The literature review revealed a number of "gaps" in our knowledge of microorganisms inhabiting sodic soils. Firstly, it is still unclear as to what extent the microbial flora of sodic
1974; soils can tolerate alkaline and saline conditions (Bhardwaj, 1974; Gupta and Bajpai,
Giambiagi and Lodeiro, 1989). Secondly, the groups of microorganisms involved in the
detailed degradation of organic ameliorants have not been characterised. ThirdlY, no
organic analyses of the microbial reactions which take place during the degradation of matter have been conducted. Lastly, whilst the effects of microbial reactions on the physical and chemical properties of sodic soils have been investigated (Swarup, 1985a;
Robbins, 1986; Swarup, 1986; Tomar et a1.,1987; Gaffar, 1992; Mbagwu, 1992; Chorom
the and Rengasamy, lggT), it is not known how these reactions effect the structure of microbial communities present within sodic soils.
of Study of the characterisation of microbial populations within alkaline sodic soils is
ions, alkaline interest, because with their high pH (> s.5) and high concentration of sodium
sodic soils are predicted to select a range microorganisms with unusual metabolic
the capabilities and growth characteristics. More importantly though, identification of groups of microorganisms within alkaline sodic soils might provide a clue as to which microorganisms are responsible for acid production following organic matter treatment'
With this information, it may be possible to adapt the remediation strategy to suit the
or growth of these acid-producing microorganisms, eg., provide specific types of nutrients, apply organic ameliorants to fields during seasons where soil moisture and temperature levels are favourable.
The study of the microbial reactions which take place in alkaline sodic soils during their
to reclamation with organic materials is also important because soil microbes are thought play key roles in the cycling of nutrients, decomposition of wastes and residues' and In detoxification of environmental pollutants (Bossio and Scow, 1995; Zak et aL, 1996)'
addition, soil microbes are involved in changes to the soil structure, including the
formation of soil aggregates (Lynch, 1981; Tisdall and Oades,1982; Lynch and Elliot,
l9g3). Changes to soil microbial communities resulting from agricultural practices, soil ecosystem management, and global change may then affect the quality of the
environment (Bossio and Scow, 1995).
Therefore, the objectives of the study presented in this chapter were to identify the
potential culturable microbial population present within an alkaline sodic soil, including
whether the acid-producing (carbonate solubilising) microorganisms, and, to determine
high pH and Na* levels in the soil had led to the selection of microorganisms with in Figure alkalitolerant and halotolerant properties. This study was carried out as shown
3.1
33 Alkaline sodic soil 1pH, l Na*, CaCo¡ present
I
I I
Microbial Isolation Using different selective agars to encourage diversity, some simulating soil properties (high pH, NaCl)
Obtain Pure cultures 49 filamentous bacteri a,216 non-filamentous bacteria, 40 fungi
Identification of cultures Biochemical screening FAME analysis of selected bacteria Alkali tolerance (PH 7 -12) Morphological characterisation NaCl tolerance (0.5-2.0M NaCl) of filamentous bacteria and fungi Acid production by non-filamentous bacteria
Figure 3.1. Structure of the microbial population survey presented in this ChaPter.
34 3.2 Materials and Methods
3.2.1 Soil propertíes
The soil used in this study was a grey alkaline sodic clay (45Vo clay,9.5Vo silt,45.5Eo sand, pH 9.60, E.C. I dS m-r (:10mM NaCl L-r ),5.6Vo CaCO¡, 4.2 mgtotal C. g-r air-dried soil,
463¡tgtotal N g-r air-dried soil) collected from Watchem, Victoria, Australia at a depth of
l0-25cm and sieved to <2mm. This soil was selected for use in this study due to its high pH and sodicity.
3.2.2 Microbíologícal culture medía and solutíons
The composition of the microbiological culture media and reagents used in this study are
presented in Appendix l.
3.2,3 Enumeratíon of fiIamentous bacteria (actìnomycetes), non-filntnentous bacteria
andfungí
Viable counts of non-filamentous bacteria, actinomycetes and fungi were assessed in the
Watchem soil using microbial plate counts. An isolate-based approach, though limited in
its ability to examine more than l7o of the total soil microbial community (Cavigelli et aI.,
1995) was used in this study, as it enabled the simple, inexpensive, and rapid assessment of
the alkalitolerant, halotolerant and acid producing properties of the microorganisms
inhabiting the Watchem soil.
To do this, I g of air-dried soil was added to 9 ml of sterile saline (pH7.4), and sonicated
for 30 seconds to resuspend aggregated or attached microorganisms. One hundred
microlitre volumes of appropriately-diluted sonicated soil suspension were then spread
onto 10 replicate agar plates of each of the following selective media:
35 a) For isolation of non-filamentous alkaliphilic and alkalitolerant bacteria, half-strength nutrient agar (NA) adjusted to pH 7, 8, g, 10, 11, and 12 was used. The pH of each agar was adjusted with 5M NaOH solution prior to autoclaving, then checked after autoclaving using pH indicator striPs.
b) For isolation of non-filamentous halophilic and halotolerant bacteria, half-strength NA, pH7.4,was used. NaCl was added to this agar, to give final NaCl concentrations of 0.5,
1.0, 1.5, and 2.0 M.
c) For isolation of anaerobic bacteria, modified brain heart infusion (BHD agar was used. jar Following inoculation, the agar plates were incubated in a BBL GasPak anaerobic
(Becton-Dickinson, Maryland, USA), into which was placed a COz[Hz gas-generating envelope (Becton-Dickinson, Maryland, USA).
(5 d) For isolation of actinomycetes, starch salts agar (ISP-4) supplemented with rifampicin
pg d-l) was used.
(2O e) Initially, for isolation of fungi, Czapekdox agar supplemented with rifampicin at ¡tg
mfl) was used. This medium however, led to the isolation of low numbers of
morphologically different fungi. As it was thought interesting to investigate as wide a
diversity of culturable microorganisms as possible, it was decided that additional selective
media would be used to obtain fungi. Therefore, 100 pl volumes of sonicated Watchem
soil suspension were plated onto 3 replicate plates of each of the following isolation media:
1) half-srrength potato dextrose agar (PDA) + rifampicin (20 pg mfr ), pH 10, 2) half-
+ strength PDA + rifampicin (20 ttg ml-r ) + lM NaCl , pH 10, 3) half strength PDA
36 rifampicin (2O tte-t-t ) + I M NaCl, 4) half-strength PDA + naladixic acid (20 pg ml-r ),
5) Czapek dox agar + rifampicin (20 pg d-t ), pH 10, 6) Czapek dox agar + rifampicin
(ZO tted-r ) + 1 M NaCl, pH 10, 7) Czapekdox agar + rifampicin (20 pg d-l ) + 1 M
NaCl, and 8) Czapekdox agar + naladixic acid (20 pg d-t ).
When necessary, the media were adjusted to pH 10 with 5 M NaOH solution prior to autoclaving, then checked after autoclaving using pH indicator strips.
Cycloheximide was added at 100 pg ml-r to all media used to isolate bacteria and filamentous bacteria, to deter fungal growth. Due to the use of a relatively rich media base for the isolation of non-filamentous bacteria (half-strength nutrient agar) from the
'Watchem soil, numbers of bacteria growing on each plate were counted after only 3-5 days incubation at 25oC, before crowding could occur. It is therefore, possible that this approach may have led to the exclusion of slower growing microorganisms from the study. Numbers of actimomycetes and fungi were counted after 5 days incubation at25oC.
s.2.4 Selection of non-filamentous bacteria to be used in alkali and NaCl tolerance screens
80 non-filamentous bacteria isolated on each of a) half-strength nutrient agar, pH 7, b) half-
strength nutrient agar, pH I 1, and c) half-strength nutrient agar pH 7 .4 + 1.5 M NaCl were
selected for further study as follows: 4 agar plates of each of the described media were
selected at random from the 10 replicates. These plates were divided so that each section
contained 20 non-filamentous bacteria. All non-filamentous bacteria from a particular
section were then streaked onto nutrient agar slants (pH 7.4), and incubated at 25oC until
confluent growth was observed. The 216 surviving isolates were then stored at 4oC until
required. 37 3.2.5 Selectíon of fitamentous bacterín(actínomycetes) to be used in preliminary alkali and NaCl tolerance screens
Due to budgetary and time constraints, it was decided that comprehensive screening of the
alkalitolerant and halotolerant properties of the W'atchem soil microbial community would
only be conducted on the non-filamentous bacterial isolates. Therefore, only 49
actinomycetes displaying distinctly different morphological characteristics from each other,
isolated from a limited range of selective media were selected for further study. These
cultures were streaked onto YME agar slants (pH 7.3) and stored at4"C until required.
3.2.6 Selection of fungi for morphological characterisation
After incubating the isolation plates at 25oC for 3 days, 40 fungi displaying different
morphological characteristics (approximately 5 from each medium described in section
3.2.3 e) were streaked onto PDA slants (pH 5.6) and stored at 4oC until required.
3.2.7 Alkali tolerance of filamentous and non-filnmentous Watchetn soíl bacteria
The alkali tolerance of the 216 non-filamentous bacteria and 49 filamentous bacteria
(actinomycetes) isolated from the Watchem soil was determined by streaking the isolates
onto plates containing half-strength nutrient agar, and YME agar, respectively, adjusted
prior to autoclaving to pH 7,8,9,10, 11, and 12 with 5 M NaOH. The pH of each agar was
then checked after atitoclaving using pH indicator strips. Bacterial growth was scored out
of 3 (3 for vigorous growth, 0 for no growth) after 3 days incubation at 25oC fot the non-
filamentous bacteria, and 5-10 days incubation at 25oC for the actinomycetes.
38 3.2.8 NaCl tolerance of thefitamentous and non-filamentous Watchem soil bacteria
The NaCl tolerance of the 216 non-filamentous bacteria and 49 filamentous bacteria
(actinomycetes) isolated from the Watchem soil was determined by streaking the isolates onto plates containing half-strength nutrient agar, and YME agar, respectively, with NaCl concentrations of 0.5 M, 1.0 M, 1.5 M, and 2 M (all pH 7 .4). Bacterial growth was scored out of 3 (3 for vigorous growth, 0 for no growth) after 3 days incubation (non-filamentous bacteria only) at 25oC on half-strength nutrient agar containing 0.5 M NaCl, and after 5-10 days incubation at 25oC for the actinomycetes and non-filamentous bacteria streaked across media containing higher NaCl levels.
3.2.9 Combined. alkatí and NaCl tolerance filamentous and non-fiIamentous Watchem soil bacteria
The ability of the 216 non-filamentous bacteria and 49 filamentous bacteria
(actinomycetes) isolated from the Watchem soil to survive high NaCl concentrations at an alkaline pH was tested by streaking the isolates onto half-strength NA + 1 M NaCl, pH
1lx, and YME + I M NaCl, pH 11, respectively. Bacterial growth was scored out of 3 (3 for vigorous growth, 0 for no growth) after 5-10 days incubation at 25oC.
*All media were adjusted to pH 11 with 5 M NaOH prior to autoclaving, and the pH of the
medium was re-checked after autoclaving using pH indicator strips.
3.2.10 Assay for acid production by non'fiInmentous bacteríal isolates
This assay was conducted to determine which of the non-filamentous Watchem soil
bacterial isolates subjected to FAME analysis (see section 3.2.71) had acid-producing
capabilities when supplied with glucose. The Watchem soil bacteria were inoculated into 2
replicate test tubes containing 7 ml of sterile half-strength nutrient broth (Oxoid)'
39 supplemented with 2Vo (wlv) glucose and the pH indicator bromocresol purple (20pg ml-t
), adjusted to a final PH of 7.2
Following inoculation, the tubes were incubated at 25oC with minimal disturbance. Acid production, signified by the bromocresol purple indicator turning from purple to yellow
(pH = 5), was scored in each tube after 24hrs, then again after 48 hrs.
3.2.11 Fatty Acíd Methyl Ester (FAME) identification of non'fi'lamentous bacterial
isolates
kr this study, characterisation by FAME analysis was performed on 3 groups of non-
filamentous sodic soil bacteria, namely, 1) those capable of growth on media containing up
to 2MNaCl at pH/.4, but not on media containing I M NaCl at pH Il,2) those capable
of growth on media containing 1 M NaCl at pH l l, but not on media containing 2 M NaCl
at pH 7.4, and 3) those capable of growth on media containing I M NaCl at pH 11 and up
to ZMNaCI at pH7.4. These populations were selected because they were expected to be
diverse due to their different growth capabilities.
FAME analysis was performed according to the protocols provided by MIDtrM (Microbial
ID, Inc., Newark, Delaware, USA) as shown in Appendix 2. FAME profiles were matched
to those of known organisms in the MIS-TSBA aerobe database v 3.9 (Microbial ID, Inc.,
Newark, Delaware, USA). A match of greater than30Vo was considered an acceptable limit
for positive bacterial identification. Such a level was deemed appropriate for the positive
identification of environmental microorganisms by Haldeman et aI' (1993)'
40 3.2.12 ldentificøtíon of filamentous bacterial isolates
The morphological characteristics of the 49 filamentous bacteria (actinomycetes) isolated
from the Watchem soil were studied in order to identify them to the genus level, using the
slide culture technique (Waksman, 1950; Wellington and Toth, 1994). To accomplish this,
cultures were streaked onto YME agar plates, and sterile glass coverslips were pushed into
the centre of the streaks. Plates were incubated at 25oC until the actinomycetes were
observed to sporulate (approximately 14 days). Coverslips were then removed and
observed under 40x magnification.
3.2.1 3 Fangal identificatìon
pure fungal cultures were inoculated onto the centre of agar plates containing PDA and
incubated for 7 days at 25oC. Samples of hyphae, plus sporulating structures, were then
taken of each isolate and teased apart on a clean microscope slide. The fungi were then
identified to genus level based upon their hyphal and spore morphology, observed under a
microscope at 40x magnification (Larone, 1987). Fungal spore colours and pigment colours
were also recorded.
4t 3.3 Results j.J.I Enumeration of fiIamentoas bacteria (actinomycetes), non-fi.Iamentous bacterìa, andfungi
An averag e of 7 x 10a cfu of non-filamentous bacteria was isolated g-r of air-dried
Watchem (Figure 3.2). Highest numbers were isolated at pH 9 (9.6 x 104 cfu of non- filamentous bacteria g-r air-dried soil), with a small decrease occurring at pH 12
(3.86 x 10a cfu of non-filamentous bacteria g-r air-dried soil)'
1 .50 x 10s
=o o tto
Y 00 x o5 aú o Ð o CL (lt o .J 5.00 x 1oa (ú ¡¡ o
(.,
0 7 I 910 11 12 pH of the growth medium
Figure 3.2. Effect of isolation media pH on numbers of non'filamentous bacteria isolated from the Watchem soil.
A total reduction in the number of non-filamentous bacteria however, was observed
to occur between 0.5 M and 2.0 M Nacl (all media pH7.g (Figure 3.3). Plate counts
to determine the numbers of (facultative) anaerobic bacteria, actinomycetes, and
fungi in the Watchem soil yielded 2.03 x 104, 5.56 x 10a, and 1.2x103 cfu g-r air-
dried soil, respectivelY.
42 1.25x 105
=o o 1.00x 105 tto Y (! 7.50 x 104 o g! o Ê G 5.00x lOa Lo IJ cl ,t¡ o 2.50x 104 (t=
2 0 0.5 1 1.5 NaCl conc. of growth medium (M) Figure 3.3. Effect of isolation media NaCl concentration on numbers of non' filamentous bacteria isolated from the \{atchem soil'
3.3.2. Alkalítolerance and halotolerance of indiví.d.uøl non-filamentous bacteria
growth All isolates tested (n - 216) were found to be highly alkalitolerant, capable of on half-strength nutrient agar ranging in pH from 7 to 12 (Table 3.1, Figure 3.4).
Halotolerance levels, however, were found to be dependent on the NaCl concentration of the medium they were isolated on. For example, of the non-
NaCl filamentous bacteria isolated on half-strength nutrient agar containing 1.5 M
(pH 7.4), 627o tolerated 2 M NaCl (pH 7.4). In contrast, only 22Vo and 397o of bacteria isolated on media containing 0.02 M NaCl, at pH 7 and 11, respectively' were tolerantto2 M NaCl (pH 7.a) (Table 3.1)'
Nacl Tolerance to NaCl at a high pH was also found to be influenced by the
isolated on concentration of the initial isolation medium (Table 3.1). Of the bacteria
1.5M NaCl agar (pH 7.4), 72Vo toletated the agar at pH 11 containing lM NaCl,
43 whereas only 27Vo and 427o of bacteria originating from pH 11 and pH 7 nutrient agars (both containing 0.02M NaCl), grew on this medium, respectively.
pH 10 pH l1 pH 12
Figure 3.4. Photograph showing 6 non-filamentous bacterial isolates from the \ilatchem soil growing on nutrient agar ranging in PH from 7'12.
Isolation pH 7 pH 10 PIJL2 1.0M 1.5M 2.0M 1.0M medium NaCl NaCl NaCl NaCl, pH 11 pH7 100 100 99 96 45 22 42 (0.02M NaCl)
27 pH 11 100 100 100 100 60 39 (0.02M NaCl)
62 12 1.5M 100 100 9'.1 100 94 NaCl (pH 7.2)
Table 3.1. Bacterial tolerance of 2L6 non-filamentous Watchem soil bacterial isolates to alkali and NaCl.
44 3.3.2.1 Identificatûon of highty halotolerant non-fi,lamentous bacterin by FAME analysís
36 non-filamentous bacteria were found capable of growing on media containing up
to 2M NaCl (pH 7.4),btttnot on media containing lM NaCl at pH 11 (Table 3'2)' Of
these highly halotolerant bacteria,2l were isolated at pH 1l'
FAME analysis of the highly halotolerant bacteria indicated 10 different bacterial
species, 6 of which belonged to the genus Bacillus (Table 3.2). Alcaligenes
study) (1 isolate), ryIoxydans (the only Gram-negative isolate identified in this
Micrococcus kristina¿ (1 isolate), and Bacillus sphaericus (2 isolates) were unique to
this group. A high percentage of bacteria isolated at pH I I could not be identified
against the MIS database (38Vo) (Table 3.2).
Bacterial species # Isolates # Isolates # Isolates from 1.5M from pH 7 from pII 11 NaCl nutrient agar nutrient agar nutrient agar (pH 7.4)
0 AIc ali g ene s xylo s ory dans 0 I Arthrobacter orydans 0 2 0 0 Arthrobacter Pascens 0 2 Bacillus atrophaeus 0 1 4 Bacillus brevis 0 0 J 0 B acillus chitinosporous 0 1 Bacillus megaterium 0 1 1 Bacillus sphaericus 0 2 0 0 B ac illus thurin giensis 1 I Micrococcus kristinae 0 1 0 0 Unidentified 1 8 Not tested I 2 4 Table 3.2. Identification of non-filamentous bacterial isolates able to grow on up to2MNaCl (p[7.4),but not on lM NaCl at pH 11, by FAME analysis.
45 3.3.2.2 Identificatíon of moderately haloalkalitolerant non'filamentous bacteria by FAME analysís
FAME analysis of the non-filamentous bacteria capable of growth on I M NaCl agar at pH 11, but not on media containing 2 M NaCl (pH 1 .Ð identified 15 species. Of these, Arthrobacter globiformis (l isolate), Aureobacterium esteromaticum (1 isolate), Micrococcus roseus (1 isolate), and Micrococcus varians (1 isolate) were unique to this group (Table 3.3). BaciUus atrophaeus was the most commonly isolated bacterium (7 isolates), the majority of which were obtained on nutrient agar containing 1.5 M NaCl (PH 7.4).
Bacterial species # Isolates # Isolates # Isolates from from pII T from pH 11 1.5M NaCl nutrient agar nutrient agar nutrient agar /J'It7.4\ 0 Arthr ob ac t e r g I o b iþ rmi s 0 I Arthrobacter oxydans 0 2 0 Arthrobacter pascens 0 2 0 0 A ur e ob act e r ium e s t e r omati c um I 0 I B ac ill us amy IoIi quefac iens 0 0 6 Bacillus atrophaeus 1 0 Bacillus brevis I I 3 Bacillus cereus 2 0 0 Bacillus chitinosporus 0 I 0 Bacillus megaterium I 0 0 Bacillus subtilis I I 2 0 B acillus thuring iensis 3 0 Micrococcus roseus 1 0 0 Micrococcus varians 0 1 0 0 P aenibacillus gordonae I 0 Unidentified 5 3 3 Not tested 0 2 4 grow Table 3.3. Identification of non'filamentous bacterial isolates unable to on 2M NaCl (pH7. ),but able to grow on 1M NaCl at pH 11, by FAME analysis.
46 3.3.2.9 ld¿ntûfication of híghty hatoatkalitolerant non-filamentous bactería by
FAME analysis
FAME analysis of the non-filamentous bacteria capable of growth on both the nutrient agars containing2 M NaCl (pH 7.a) and on the agar containing I M NaCl at pH ll led to the identification of 13 different bacterial species (Table 3.4). Bacillus circulans (l isolate), Bacillus filocolonicns (l isolate), Bacillus licheniþrmis (l isolate), Brevibacterium iodinum (! isolate), and Micrococcus luteus (2 isolates) were found to be unique to this group. Bacillus subtilis and Bacillus atrophae¿¿.t were the most commonly isolated bacteria (10 and 9 isolates respectively), all of which were isolated from nutrient agar containing 1.5M NaCl (pH 7'4)'
Bacterial species # Isolates # Isolates # Isolates from from pH 7 from pH 11 1.5M NaCl nutrient agar nutrient agar nutrient agar (pH 7.4)
3 B a c ill u s amy I o I i quefac i en s 0 1 Bacillus atrophaeus 0 0 9 Bacillus brevis I 0 4 Bacillus cereus 1 0 0 Bacillus circulans I 0 0 0 B ac illus filo c olonicus I 0 Bacillus lichendormis 0 0 1 Bacillus megaterium 2 0 I Bacillus subtilis 0 0 10 Bacillus thuringiensis I 0 0 1 B rev ib ac t e rium io dinum 0 0 0 Micrococcus luteus 1 1 P aenib ac illus g o rdonae 0 0 1 Unidentified 3 5 J Not tested 0 3 1 Table 3.4. Identiflrcation of non-fÏlamentous bacterial isolates able to grow on both 2M NaCl (pH 7.a) and lM NaCl media at pH 11, by FAME analysis.
47 A high proportion (7l.4Vo) of bacteria isolated at pH 11 were unidentifiable using the current MIS database, as compared to 27.2Vo isolated at pH 7 and9.l%o on 1.5 M
NaCl (pH 7.a). A similar result was shown earlier in this Chapter (Table 3.2) for the highly halotolerant bacteria.
3.3.3 Ctassification of non-fiInmentous bacteria wíth a FAME profile sirnílarity of <307o
As shown in Tables 3.2,3.3, and3.4, several of the non-filamentous bacteria isolated from the'Watchem soil could not be identified using FAME analysis because they displayed a level of similarity of less than 30Vo* to any of the bacteria in the current
MIDI database. * This value is considered to be the minimum cut-off for the positive identification of environmental microorganisms (Haldeman et a|1993).
Nevertheless, further analysis of their FAME profiles revealed that these "unknown" bacterial isolates could be categorised in terms of their closest matches on the MIDI database, as well as by their morphological characteristics. By using this method of classification, the highly halotolerant cluster of bacteria (Table 3.5), were found to
display similarity to 8 different bacterial species, with Bacillus coagulans,
Arthrobacter pasCens, Micrococcus roseus, Micrococcus lylae, Arthrobacter
orydans, and Rathayibacter rathayi-hke bacteria being unique to this group. One
isolate (no. 103), with pale orange/pink coloured, round, shiny colonies was found to
have no similarity to any bacteria in the current MIS database.
48 7o Isolate Source Colony Characteristics Nearest FAME ID Similarity
0 103 pH ll Pale orangeþink, round, shinY Unidentified 104 pH 1l Milky pink, round, shinY Bacillus coagulans 13.3 106 pH ll Pale orange, round, shinY B acillus p sychrophilus 4.8 l4l pH l1 Milky white, round, shinY Arthrobacter pascens 16.7 145 pH 1l Yellow, round, shinY C e llulomonas flav i g ena 19.7 151 pH l1 Milky white, round, shinY Micrococcus roseus 5.7 7.8 r52 pH 11 Milky white, round, shinY Micrococcus roseus 162 pH 11 Milky white, round, shinY Micrococcus lylae 14.3 164 pH l1 Milky white, round, shinY Arthrobacter orydans 20.7 286 pH7 Pink cream, brown Pigment, Rathayibacter rathayi 2.7 irresular outside edge Table 3.5. Classification of non'filamentous bacteria with a FAME ProfÏle similarity of < 30Vo able to groril on up to 2M NaCl (pH 7.4), but not on lM NaCl at pH 11.
Bacteria clustering into the moderately haloalkalitolerant group (Table 3.6) also
demonstrated similarities to 8 known bacterial species, with Bacillus epiphytus,
and Bacillus filocolonicus, Bacillus megaterium, Curtobacterium fløccltmfaciens
Tatlockia micdadei- like bacteria being exclusive members'
To Isolate source colony characteristics Nearest FAME ID Similarity
94 pH l1 Pale orange, round, shinY Cellulomonas flavigena 21.9 18.3 101 pH ll Milky pink, round, shinY Tatlockia micdadei t73 pH 1l Cream/white, round, mucoid Bacillus megaterium 1.8 241 1.5M Gold/orange, irregular, shinY B acillus p sychroPhilus I 1.1 25.9 247 1.5M Cream, irregular, shinY B ac illus filo c o I onicu s 260 l.5M Milky yellow, round, shinY Curtobacterium 10.9 flaccumfaciens 28.8 274 pH7 Cream, round, very shinY Bacillus firmus 281 p}l7 Cream, round, very shinY Bacillus firmus 28.2 282 pH7 Cream, round, very shinY Bacillus firmus 26.7 9.4 343 p}l7 Cream, irregular, shinY B acillus filocolonicus 350 p}l7 Transoarent, cream, shinv Bacillus ep 17.3 Table 3.6. Classification of non'filamentous bacteria with a FAME Profile simitarity of < 307o unable to grow on 2M NaCl (pH 7'4)' but able to grow on lM NaCl at pH 11.
49 Ten bacterial isolates were found to cluster into the highly haloalkalitolerant group
(Table 3.7). In this cluster, similarities were detected with 7 known bacterial species including Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus firmus, Bacillus psychrophilus, Bacillus sphaericus, and Paenibacillus pabuli. paenibacillus pabuli, Bacillus circulans, Bacillus amyloliquefaciens, Bacillus brevis,
and Bacillus sphaericuslike bacteria were found to be unique to this cluster of isolates.
Three bacteria, isolated at pH 11, displayed a common morphology type - burnt
orange in colour, round, and shiny (Table 3.7). Isotates 97 and 109 were determined
to be simil ar to Bacillus psychrophilus, and isolate 108 was determined to be similar
to Bacillus circulans. Given the low percentage-similarity of these bacteria to the
known bacterial species (all < l87o), and the fact all were found to have similar
FAME profiles (See Table 5.2ainChapter 5), there is a high likelihood that these are
the same bacterial species. Similarly, isolates 274, 281 and 282 (Table 3.6) are
on the thought to be the same bacterium, because: a) all three bacteria were isolated
pH 7 medium, b) they all share the same morphological characteristics, c) they all
all share the same growth characteristics on high pH and salty media, and lastly, d)
three bacteria share similar FAME profiles (Table 5.2ain Chapter 5)'
50 Vo Isolate Source Colony Characteristics Nearest FAME ID Similarity
r.2 95 pH 11 Pale milky pink, round, Paenibacillus Pabuli shiny 8.1 97 pH l1 Burnt orange, round, shinY B acillus p sychroPhilus 11.5 108 pH 11 Burnt orange, round, shinY Bacillus circulans 4.3 109 pH ll Burnt orange, round, shinY Bacillus psychroPhilus iens 28.3 183 1.5M Cream, round, mucoid B ac illus amyloliquefac 27.6 188 1.5M Cream, round, mucoid Bacillus brevis 2.r 267 1.5M Milky yellow, round, shinY Bacillus sphaericus 293 1.5M Cream, very shiny, round Bacillus firmus 23.3 294 1.5M Cream, very shiny, round Bacillus firmus 8 340 1.5M Cream. very shinv, round Bacillus firmus 18.7 Table 3.7. Classification of non'filamentous bacteria with FAME ProfÏle similarity or < 30vo able to gro\ü on both 2M NaCl (pH 7.4)' and on lM NaCl at pH 11.
3.3.4 Acìd productìon by non-filamentous bacteria: FAME identifieil isolates
Of the 93 non-filamentous bacterial isolates identified via FAME analysis, 83 were found capable of producing acid in half strength nutrient broth (Oxoid) supplemented with 2Vo (w/v) glucose (Table 3.8). The isolates identified as Bacillus thuringiensis
the indicator and, Bacillus cereus were found to be rapid acid producers, turning bright yellow within 24 hours. None of the 4 Micrococcas species isolated from the
Watchem soil produced any detectable acid from glucose, even after 48 hours of incubation
5l Isolate Bacterial species Acid producer +/-
142 Alc ali g ene s ryIo s orydans + + 155 Ar thr ob ac t e r g I o b iþ rmi s 136,147,163 Arthrobacter orydans 144,152 Arthrobacter Pascens + 339 Aur e ob a c t e r ium e s t e rar omati c um 2ll Bacillus licheniþrmis + 134, 179,216,232,238 B ac illus amyloliquefaciens + 105, 181,192, 197,200, Bacillus atroPhaeus + 201,22r,223,227,229, 234,235,236,242,243, 246,249,251,285 159, 177, 182, 191,204, Bacillus brevis + 205,208,228,231,239, 268,280,287 306,317,331 Bacillus cereu's* + 17I Bacillus chitinosporus + 308 Bacillus circulans + 289 Bacillus fiIocolonicus 116,257,258,284,290 Bacillus megaterium + 123,128 Bacillus sphaericus + 125,176,178, 185, 190, Bacillus subtilis + 217,222,224,226,245, 248,262,265,337 * + 137 ,310,312,323,325 B acillus thuringiensis 240 B r ev ib act e rium i o dinum* + 175 Micrococcus kristinae 9r,344 Micrococcus luteus 345 Micrococcus roseus 110 Micrococcus varians + 253,332 P aenib ac illu s R o rdonae Table 3.8. Acid producing strains of non-filamentous bacteria identiflred in the Watchem soil. t = strong acid producer after ?Ahrs'
3.3.5 Acid production by non-fitamentous bactería: Unidentifi'ed isolates
V/here the majority of identified non-filamentous bacterial isolates were found to be
capable of acid production in the glucose-enriched broth, only ll'SVo of the
unidentified isolates tested were found to produce a positive result (Tables 3'9' 3.10
and 3.11).
52 Interestingly, all of the unidentified acid-producing non-filamentous bacteria were found isolated on half-strength nutrient agar containing l.5M NaCl (pH 7'4), and
11 (Table 3'9)' capable of growth on both 2 M NaCl (pH 7.4), and lM NaCl at pH to be a particularly One Bacillus firmus-like bacterium (isolate 340), was found yellow strong acid producer, changing the nutrient broth from deep purple to bright
in colour within the first 24 hours of incubation (Table 3.9).
Acid Isolate source colony characteristics Nearest FAME ID producer +l-
95 pH l1 Pale milky pink, round, shinY Paenibacillus Pabuli 97 pH ll Burnt orange, round, shinY Bacillus p sy chroPhilus r08 pH 1l Burnt orange, round, shinY Bacillus circulans p us 109 pH 11 Burnt orange, round, shinY B ac illus sy chr oPhil i en s + 183 l.5M Cream, round, mucoid B ac illus amyl oliquefac + 188 1.5M Cream, round, mucoid Bacillus brevis 267 1.5M Milky yellow, round, shinY Bacillus sphaericus 293 1.5M Cream, very shinY, round Bacillus firmus 294 1.5M Cream, very shinY, round Bacillus firmus + 340 1.5M Cream, shiny, round Bacillus firmus* a FAME profile Table 3.9. Acid Production bY non'filamentous bacteria with similarity of <30Vo able to grow on both 2M NaCl (pH7.4), and lM NaCl at PH 11. * = high acid producer at 24 hrs.
Acid Isolate source colony characteristics Nearest FAME ID producer +l- N.D r03 pH l1 Pale orangeþink, round, shinY Unidentified to4 pH l1 Milky pink, round, shinY Bacillus coagulans N.D N.D 106 pH 11 Pale orange, round, shinY B ac illu s p sy chr o Philus t4l pH 11 Milky white, round, shinY Arthrobacter Pascens s av i g ena 145 pH 1l Yellow, round, shinY C e llulomona fl roseus 151 pH 11 Milky white, round, shinY Micrococcus roseus r52 pH 11 Milky white, round, shinY Micrococcus lYlae r62 pH 11 Milky white, round, shinY Micrococcus 164 pH l1 Milky white, round, shinY Arthrobacter orydans N.D 286 pH7 Pink cream, brown Pigment, Rathayibacter rathaYi outside edge a FAME Table 3.10. Acid Production by non-filamentous bacteria with Profile similarity of < 30Vo able to grow on up to 2M NaCl (pH 7.4)' but not on lM NaCl at pH 11. N.D = not determined'
53 Acid Isolate Source Colony Characteristics Nearest FAME ID producer +l- 94 pH l1 Pale orange, round, shinY Cellulomonas flavigena 101 pH ll Milky pink, round, shinY Tatlockia micdadei 173 pH 11 Creamy white, round , mucoid Bacillus megaterium 241 l.5M Golden orange, irregular, shinY Bacillus psychroPhilus 247 1.5M Cream, irregular, shinY Bacillus filocolonicus 260 r.5M Milky yellow, round, shinY Curtobacterium flaccumfaciens 274 pH7 Cream, round, very shinY Bacillus firmus 281 pH7 Cream, round, very shinY Bacillus firmus 282 pH7 Cream, round, very shinY Bacillus firmus 343 pH7 Cream, irregular, shinY Bacillus fiIocolonicus 350 oH7 Transoarent, cream. shinv Bacillus a profile Table 3.11. Acid Production by non-fÏlamentous bacteria with FAME similarity of < 30Vo unable to grow on 2M NaCl (pH7.4), but able to grow on 1M NaCl at pH 11.
3.3.6 Atkati and NaCl tolerance of filamentous Wøtchem soíl bøcteria
(actínomycetes) : Prelilninary studies preliininary studies of the alkalitolerance and halotolerance levels of actinomycetes
isolated from the'Watchem soil are shown in Table 3.12 (tolerance levels are shown
for only 46 isolates, as isolates7,12, and 14 did not grow on YME agar)' Seventy
four percent of the isolates were observed to grow at pH 12, and no isolates grew on
YME agar containing either 2M NaCl, or lM NaCl at pH 1l (Table 3.12).
Growth pH 7 pH 10 PHl2 1.0M 1.5M 2.0M 1.0M Medium NaCl NaCl NaCl NaCl, pH 11
0 0 7o Tolerant 100 96 70 47 t7 Isolates
Table 3.12. Alkalitolerance and halotolerance of 49 actinomycetes isolated from the Watchem soil.
54 j. 3.7 I dentific atio n of filam e ntous b acteria (actinorny c etes )
Although 49 actinomycetes were examined in this study, only 30 could be classified
using the slide morphology technique as many isolates were unable to grow on
oatmeal agar. Of the isolates, 17 were classified as streptomycetes (SIVo), 11 were
classified as streptoverticilliates (28.57o), and 2 were classified as Micromonospora
species (67o) (Table 3.13).
While the morphologies of the classified actinomycetes were found to vary very
little, with 5l7o producing white-coloured aerial mycelia, most isolates could readily
be distinguished from one another by the characteristics of their substrate mycelia
(Table 3.13). Pigment production was restricted to 2O7o (6) of the isolates - 3
Streptomyces species and 3 streptoverticilliates (Table 3.13)'
Morphological descriptions of the unclassified actinomycetes are shown in Table
3.14.
55 # Isolation Substrate Aerial Diffusible Genus medium mycelia mycelia pigment colour
I A Cream/grey White Streptomycete 2 A Yellow White Yellow Streptomycete 8 A Cream White Streptomycete 12 B Dark orange Micromonospora lWhite 13 B Cream Streptomycete t4 B Orange Micromonospora 43 A Dark brown Brown Brown Streptomycete M A Purple Grey Streptoverticilliate 48 C Dark purple Grey Streptomycete 57 E Brown Grey Streptoverticilliate 58 E Orange White/yellow Yellow Streptomycete 59 E Cream Cream Streptomycete 61 E White, convex White Streptomycete 63 D Cream, wrinkled V/hite Streptomycete 64 D Cream V/hite Streptomycete 65 B Brown, waxy White Orange Streptoverticilliate 67 F White, wrinkly White Streptomycete 69 B Yellow cream White Streptoverticilliate 70 B Brown, waxy White Orange Streptoverticilliate 72 A Grey cream White Streptomycete 74 A Yellow Yellow Streptoverticilliate 77 A Cream Orange Streptoverticilliate 78 A Orange cream White Streptoverticilliate 80 A Cream Grey/white Streptomycete 88 A Grey/green White Streptomycete 9l A Brown White Streptomycete gro*n B1 A Brown, skin-like Brown Streptoverticilliate I A Orange cream White Streptoverticilliate L A Cream Grey Streptoverticilliate Y A Orange cream V/hite Streptomycete
56 # Isolation Substrate mycelia Aerial Diffusible medium mycelia pigment colour
7 A Grey cream, wrinkly 11 B Apricot l5 B Yellow, skinJike White 49 C Dark brown Grey 50 c Cream Grey 52 B White Grey 54 B Cream Brown 62 E Cream, wrinkly 7l A Purple V/hite Brown 8l A Brown, skinlike Brown 85 A Brown, skin-like Brown t17 A Tan, raised Brown B A Dark grey green, wrinklY Brown H A Yellow, skin-like Yellow K A Creamy white, waxy M A Yellow, skin-like Yellow R A Cream Grey S A Brown, skinJike Brown X A Cream, skin-like Table 3.14. Morphological characterisation of actinomycetes unable to be identifïed using the slide culture technique. Isolation medium A'E are as follows: A= ISP4 + 20 ¡tg ml'r rifampicin + 100 Pg ml'l cycloheximide, B= ll4 Strength PDA + 20 pg ml-r rifampicin, C= ll4 Strength PDA + 20 ttg ml-1 rifampicin, pH 8, ,E= ll2 Strength nutrient agar + 20ltg ml-l rifampicin + 100 pg mlr cycloheximide, PH 8.
3.3.8 Fungal identificatíon
Penicillum was the most common fungal genus found in the watchem soil, making
tp 23 of the 35 identified isolates (65.7Vo). V/ithin this genus, 8 subtypes were
identified (based upon similar morphological characteristics). More specifically these pink were isolates with a) yellow substrate hyphae, b) cream substrate hyphae, c)
green substrate hyphae, d) reddishtinged hyphae, e) white aerial hyphae, Ð flat yellow substrate hyphae, g) blue-green aerial hyphae, and h) white aerial hyphae and
spores (Table 3.15b).
57 Rhizopus was the next most coÍlmon genus (ll. %o), followed by Fusarium (8.6Vo),
Gliomastix and Ulocladium (5.7Vo), and Gliocladium (2-9Vo) (Table 3.15a)' The
remaining 5 fungi used in this study could not be classified to the genus level due to
an inability to maintain them as pure cultures (infected by fungal mites).
Isolate Isolation Colony Morphology Genus medium 10 A V/hite aerial hyphae, black spores Rhizopus l1 B Purple substrate hyphae, white aerial Fusarium hyphae l3 B V/hite aerial hyphae, black spores Rhizopus l5 c Flat, dark green/black substrate hyphae Gliomastix t9 D Dark green spores, white outer edge, flat Unidentified 23 E Peach substrate hyphae, white aerial hyphae Acremonium 29 F Dark green, flat substrate hYPhae Ulocladium old 15 G White aerial hyphae, dark green spores Fusarium old t7 G Ma¡oon substrate and aerial hyphae, green Gliocoladium spores old 19 E Dark green, flat substrate hyPhae Ulocladium otd27 H White aerial hyphae, black spores Rhizopus old 28 I White aerial hyphae Fusarium otd32 E White aerial hwhae, black spores Rhizopus Table 3.15.a. Hyphal and spore morphology of non-P¿nicíllium fungal isolates. Isolation medium A-K are as follows: ll4 Strength PDA + lM NaCl' PH 10' pH 10, D= B= 1il4 Strength PDA + 20 pg-.it"-picin, ml-r Nat¡dixic^= acid, C= Czapek dox, Czapek dox + 20 ttg -t-i E= 1/4 Strength PDA + 20pg ml'l rifampicin , F= ll4 strength PDA + 20 pg ml-r rifampicin, pH 10, G= ISP4 + 20¡rg ml-r rifampicin,H= !14 Strength Nutrientagar,pHll, I= Czapek dox.
58 Isolate Isolation Colony MorphologY Genus medium 2 A Grey substrate hyphae with red edge Penicillium 3 A Yellow substrate hyPhae Penicillium 4 A White aerial hyphae Penicillium 5 A White aerial hyphae, green spores Penicillium 6 A Creaml grey substrate hYPhae Penicillíum 8 A Pink substrate hyphae Penicillium t2 B V/hite aerial hyphae, dark green spores Penicillium t4 B Flat, green substrate hYPhae Penicillium t7 C White aerial hyphae, green sPores Penicillium 22 D Cream substrate hyphae Penicillium 24 E Yellow substrate hyphae, white aerial hyphae Penicillium 25 E Green/yellow substrate hYPhae Penicillium 26 E Peach substrate hyphae, green aerial hyphae Penicillium 27 E White aerial hyphae, dark green spores Penicillium old 2 F \Mhite aerial hyphae, green sPores Penicillium old 7 G White aerial hyphae, green sPores Penicillium old 8 G Yellow substrate hyphae Penicillium old ll G Yellow substrate hyphae Penicillium old 12 G Cream substrate hyphae, white aerial hyphae, Penicillium green spores old 13 G Fluffy, white hyphae, green spores Penicillium old 20 E Dark blue-green, aerial hYPhae Penicillium otd24 G White aerial hyphae, yellow spores Penicillium old 26 G White aerial hyphae Penicillium Table 3.15.b. Hyphal and spore morphology of fungal isolates identified as Penicíllium species. Isolation medium A-K are as follows: ì¡= ll4 Strength PDA + lM NaCl, B= LlA Strength PDA + 20 Pe ml'l Naladixic acid, C= Czapek dox + lM NaCl, pH 10, D= Czapek dox + 20 ¡tgml'l rifampicin, E= 1/4 Strength PDA 20 ml-1 + 20 ¡rg ml'l rifampicin, pII 10, F= Czapek dox, G= ISP4 + lrg rifampicin,K= ll4 Strength Nutrient agar' pII 11.
59 3.4. Discussion
3.4.1 Atkalìtolerance of the non-fil.amentous'watchem soíl bactería
pH 7 and 12 Microbial plate counts using half-strength nutrient agar adjusted to between culturable non- with concentrated NaoH, indicated that a high proportion of the alkalitolerant, with filamentous bacterial community within the'watchem soil was highly
g soil-r at pH 9 (highest bacterial numbers only being reduced from 9.6 x 10a cfu of bacteria at pH 12' numbers were recorded at this pH) to 3.86 x 10a cfu of bacteria g soil-r
this, with 99Eo krdividual testing of 216 non-filamentous bacterial isolates later confirmed
agar with a pH of the isolates tested being found capable of growth on half strength nutrient of 12.
bacteria isolated from Such broad-range alkalitolerance has been reported previously in cultured the alkaline sodic soils by Bhardwaj Q97Q. In his experiments, Bhardwaj (Karnal: pH 10'5' EC microorganisms from three increasingly saline alkaline sodic soils
values from saturation 52.5; Mathura: pH 9.0, EC 72;Indore: pH 10'4, F,C79.8 - all EC the bacterial extract) onto nutrient agars ranging in pH from 7 to 11.5. After counting
similar bacterial colonies emerging after 7 days of incubation at 30o c, Bhardwaj observed However' bacterial counts to those of this study for the three soils between pH 7 and 9'5'
was higher than 9'5, with numbers decreased rapidly when the pH of the isolation medium
no bacteria at all being isolated from the Mathura soil at pH 11.5.
study compared to this The lower rate of bacterial alkalitolerance observed in Bhardwaj's
that were isolated' The study is thought to be due to the different suites of microorganisms
depth of 0-15cm (topsoil)' difference may be due to Bhardwaj using soils sampled from a
(subsoil). Subsoils where in this study the soil used was sampled from a depth of 10-25cm
Bone and Balkwill' 1988) have a lower nutrient status than topsoils (Beloin et aI',1988; 60 the and, in the case of alkaline sodic soils, where water often cannot infiltrate through
probably much upper surface due to clay swelling (Rengasamy and Olsson, 1993), are
diverse array drier. Thus, where the microflora of topsoils are known to consist of a highly of metabolically active Gram-positive and Gram-negative bacteria, as well as autotrophs
as the Watchem soil and fungi (Bone and Balkwill, 1988), the microflora of subsoils, such of microbial used in this study, would be expected to contain a much smaller diversity types (Bone and Balkwill, 1988), and consist of mainly Gram-positive spore-forming
recognised for their bacteria such as Bacillus, and species such as Arthrobacter, which are
ability to survive carbon-limited terrestrial environments (Boylen and Ensign, l97O;
Poindexter, 1981; Kieft et a1.,1997).
Whilst it is unknown whether the microorganisms isolated by Bhardwaj (1974) were individual consistent with those of a "typical" topsoil (Bhardwaj did not characterise
isolated from bacterial isolates), MIDI-FAME analysis of the 216 non-filamentous bacteria
the Watchem soil revealed mainly bacterial species that are associated with the subsoil
environment, ie., Bacillus spp. and Arthrobacter spp' (80Vo and lOTo of MIDI-FAME
identified non-filamentous bacterial isolates, respectively).
in this It is not surprising that Bacillus spp. were the most common bacterial spp' isolated Gibson in study, because since the first recorded observation of alkaliphilic Bacilli by
and the 1934, Bacillus spp. have become recognised as the most regularly encountered
1989; Kroll, best studied genus of alkaliphilic microorganisms (Ikulwich and Guffanti, on the potential 1990; Nielsen et a1.,1995). The majority of these studies have focussed for industrial applications of alkaliphilic and alkalitolerant bacilli, for example searching
(Horikoshi and alkalitolerant cellulases, cr-amylases, lipases and proteases for detergents
Akiba, 1982; Sharp and Munster, 1988; Ito,1997ilto et aI', 1998)'
61 However, some researchers have concentrated on explaining the specific mechanisms by
which bacilli are able to tolerate high pH environments. Some of these tolerance
mechanisms are discussed below:
Mechanism 1 - Adjustment of the external pH: Horikoshi and Akiba (1982) found that their some alkaliphilic bacilli have the ability to adjust the pH of their growth medium to
optimum pH by excreting acids when the external pH reaches their upper pH limit' and
hydrolysing proteins to raise the pH of the growth medium when the pH becomes overly
acidic
As was shown in the results of the acid-production assay (Tables 3.8 - 3.11), the majority
of the bacterial isolates tested (the majority of which are Bacillus spp.) showed acid-
producing capabilities when cultured in l/2-strength nutrient broth supplemented with 27o
(w/v) glucose (pH 7), and thus, have the potential to acidify their surroundings to their
to basify optimum pH. Whether the Watchem soil microbial community also has the ability
acidic media is not known.
that Mechanism 2 - Active transport of Na* and H* ions : one important characteristic
alkaliphilic and alkalitolerant bacteria possess that is not found in neutrophilic bacteria is pH the ability to adjust their cytoplasmic pH to at least two units below the external
(Krulwich and Guffanti, 1983; Krulwich et al., 1997)'
This ability is postulated to be mediated by a secondary electrogenic cation*ÆI+ antiport pumped the situated in the bacterial cell membrane, through which H* ions are into
(Guffanti et 1978, bacterial cell at the expense of monovalent cations, normally Na* al',
Guffanti et al., L979;Guffanti et a|.,1986; Kdwich et al', 1997)' 62 Thus, the majority of alkaliphilic and alkalitolerant bacteria are dependent on Na* for
maintaining their internal pH (Guffanti et aL, 1986; Krulwich et al., 1997). Recently
though, Kitada et al. (1997), isolated an alkalitol erant Bacilløs with a K'/H* antiport. Na+
has also been shown to be necessary for germination and uptake of amino acids in some
alkaliphilic bacilli (Kudo and Horikoshi, 1983; Kitada et al., 1989).
Given that the soil used in this study was slightly saline as well as alkaline, it is possible
that the bacteria in the Watchem soil may have evolved a similar dependence on sodium
ions to maintain their internal pH at near-neutral levels. The existence of such antiports in
the bacteria isolated in this study is therefore, thought worthy of further investigation.
Mechanism 3 - Specialised cell membrane and cell wall composition: Compositional
studies of the membrane lipids from two obligately alkaliphilic and two alkalitolerant
Bacillus species by Clejan et al. (1986) found that where unsaturated fatty acids eg., 16:1
comprised tp to 9O7o of the fatty acids present in the cell wall of the obligately alkaliphilic
isolates, the alkalitolerant isolates contained only between 0 and 2Vo wsaturated fatty
acids, and up to 767o branched chain fatty acids. Furthermore, the average chain lengths of
the fatty acids were shorter in the alkalitolerant bacteria than in the obligate alkaliphiles
(Clejan et al., 1986).
Subsequent research by Dunkley et aL (1991), found that the reason for these differences in
membrane structure could be attributed to the obligate alkaliphiles having high levels of
fatty acid desaturase activity, whereas, the alkalitolerant bacteria had no detectable activity.
63 Alkaliphiles are also known to have a reduced level of basic amino acids eg., lysine,
acid and arginine, and histidine, and higher levels of acidic components eg., teichuronic teichuronopeptides within their cell walls (Krulwich et aI., 1997).
> 80Vo branched Interestingly, all of the bacterial species identified in this study possessed
upon the fatty acids eg,, iso 15:0 and anteiso 15:0, within their cell membranes' based
(See Tables average cell membrane composition of each species as per the MIS database
of Clejan et al. (1986).It 5.2a and 5.2b inChapter 5), therefore, supporting the observation
possess cell walls would be interesting to confirm whether the bacteria in this study also
rich in acidic comPonents.
by Mechanism 4 - High level expression of respiratory chain components: In experiments
oF4 was Guffanti et aI. (19g6), the facultarively alkaliphilic (alkalitolerant) Bacill¿¿s strain
found to incorporate high levels of the respiratory chain components cytochrome type-a
then fell in and type-c in its cell membranes when cultured at pH 10.5. These components
role of the concentration when the bacterium was cultured at pH 7.5. Although the exact
cytochromes in bacterial alkalitolerance is not yet fully understood (Kroll, 1990)'
therefore' one cytochromes are involved in the transfer of electrons within living cells,
and reason for the high level of cytochromes in the cell membranes of alkaliphilic
alkalitolerant bacteria could be to drive Na*/H* antiporters.
coloured Given that bacteria containing high levels of cytochromes are typically highly
were cream or white (Kroll, 1990), and the majority of the bacteria identified in this study
in determining in colour, it is thought that this mechanism is probably of low importance
the alkalitolerance of the Watchem soil bacterial population.
64 3.4.2 Halotolerance of the non-filannentous Watchem soil bacteria
to tolerate a The ability of the non-filamentous \ilatchem soil bacteria isolated in this study
by the wide range of NaCl concentrations (0.02 M - 2 M) was found to be influenced the composition of the medium from which the bacteria were isolated, with 227o of
compared bacteria isolated at pH 7 (O.02 M NaCl) displaying wide-range NaCl tolerance,
and on media containing to 39Vo and 62Vo of the bacteria isolated at pH l l (0.02 M NaCl)
1.5 M NaCl (pH 7.4), respectively. Bacillus atrophaeus and Bacillus subtilis were
particularly halotolerant, with 90Vo and 86Vo of these bacterial isolates being isolated on
(Tables 3.2,3'3, half-strength nutrient agar (pH 7,4) containing 1.5M NaCl, respectively
and 3.4).
bacterial The reason for the different levels of halotolerance appears to be that unique (0'02 M populations were isolated from each medium. For instance, the pH 7 medium (0'02 M NaCl) was favourable for the growth of Bacillus thuringiensis, the pH l1 medium (pH NaCl) was favourable for the growth Arthrobacter spp., and the 1.5M NaCl medium
(Tables and 7.4) was favourable for the growth of B. atrophaeus and B. subtilis 3.2,3'2
isolated 3.4). The ability of different selection media to affect the types of microorganisms
(1982), the addition is well documented. Furthermore, in an experiment by Quesada et aI.
predominantly of 100 g of NaCl per litre of agar was found to lead to the isolation of was shown Gram-positive aerobic rods, and the addition of 200 g of NaCl per litre of agar
to lead to the isolation of Gram-positive cocci'
this study As was indicated earlier in the discussion, a proportion of the bacteria in were not including Arthrobacter spp., Micrococc¡ls spp'' and Bacillus thuringiensis'
1.5 M NaCl, isolated directly from the Watchem on half-strength nutrient agar containing
pure culture (Tables yet, they could tolerate up to 2 M NaCl once they were obtained as a
65 3.2,3.3,and 3.4). This could be because the 2 M NaCl was inhibitive to the germination of bacterial spores, such as are produced by Bacillus spp., and the re-activation of resting
Arthrobacter and Micrococcøs cells directly from the soil, but not to healthy, growing
cells
Halotolerant Bacillus, Arthrobacter and Micrococctls spp. have previously been isolated
al., from saline and hypersaline soils (Quesada et aL, 1982; Zahtan et al., 1992l' Zahran et
1995; Zahran,lggT). For example, Quesada et al. (1982), observed that of the 43 Bacilli,
14 Arthrobacter, and l8 Microcci, they isolated from hypersaline soils ranging in NaCl
concentration from 57o to lO.|Vo (w/w), 30Vo, 43Vo and 44Vo respectively, were capable of
growing in a nutrient broth containing 250 g.l-r NaCl (4.3M)'
Bacteria inhabiting saline environments are understood to have evolved three methods of
haloadaptation, firstly, the accumulation of K*, secondly, the production of compatible
solutes, and thirdly, modifications to the structure of cell envelopes and membranes
(Csonka, 1989; Thiemann and Imhoff, l99l;Zahran, 1997). These are discussed in more
detail below:
K* accumulation: The basis of this mechanism is to accumulate K* within the cytoplasm
until the internal solute concentration reaches that of the surrounding medium, thereby
preventing the efflux of water from the cell (Zahtan,1997)'
(Oren, This mechanism is known to be used only by anaerobic heterotrophic eubacteria
1986; Zahran, lggT), halophilic archaebacteria eg., Haloanaerobium (Oren, 1991)' and
K+ is unlikely sulphate reducing bacteria (Olivier et al., lgg4). Therefore, accumulation of
66 and to be responsible for the halotolerance exhibited by the aerobic eubacteria eg., Bacilli
Arthrobacr¿r isolated in this study.
which Compatible solute production: Compatible solutes are defined as organic osmolytes with are responsible for maintaining the osmotic balance of cells, and are compatible
cellular metabolism (Brown, 1976;Zahtan,lgg7). Examples of compatible solutes include
glycine betaine' sucfose, glutamate, glutamine, y-aminobutyrate, ectoine, trehalose, proline,
protection and choline (Csonka, 1989; Wohlfarth et ø1., 1990). These compounds provide
potential, to bacterial cells against high osmotic pressures by decreasing their internal water
which enables the cells to restore their volume and turgor pressure to pre salt-stressed
levels (Brown and Simpson,1972; Csonka, 1989).
or Compatible solutes can be synthesised following genetic induction (Smith et al., 1994a),
either selectively transported from the external environment (Wohlfarth et aI., 1990). By
that mechanism however, both the type and the concentration of the compatible solute
osmotic accumulates within the bacterial cell is known to be influenced by the level of
and the stress, the temperature, the cellular growth phase, the carbon sources available,
Smith ¿r presence of osmolytes in the surrounding growth medium (Wohlfarth et a1.,1990; of al., I994b; Zahran, lggT). For example, Wohlfarth et al. (1990), found that incubation
halmephilum ín a the bacteri a Deleya halophila, Halomonas elongata, and Flavobacterium of glucose medium led to the accumulation of the amino acid ectoein, whereas incubation of glycine the same bacteria in a medium containing yeast extract led to the accumulation
betaine.
adopted by Given that the compatible solute strategy of osmoregulation is known to be Arthrobacter' and eubacteria (Zharan, lggT),which includes the bacterial genera Bacillus, 6l Micrococcøs, it is likely that this is the major mechanism by which the bacteria isolated in this study were able to cope with high osmotic stress. Characterisation of the osmolytes involved would therefore, be interesting.
Cellular changes: The third mechanism by which halophilic bacteria are understood to
tolerate osmotic stress is to alter the composition of their cell membranes. Gram-positive
bacteria for example, are known to be able to increase the concentration of certain
phospholipid components when exposed to high levels of NaCl (Kates, 1986). This has the
effect of increasing the net negative charge of the cell membrane, which increases its
stability (Thiemann and Imhoff, 1991), and restricts the permeability of the membrane to
ions (Kanemasa et aI., 1972; Ohno et aI., 1976; Ohno et al., 1979). Furthermore, Imhoff
and Thiemann (1991), demonstrated that in high NaCl, the purple, phototrophic bacterium
Ectothiorhodospira changed from having a cell membrane rich in C-18 fatty acids to one
rich in C-16. This was hypothesised to have increased the fluidity of the membrane.
possible Given that 92 of the 93 identified bacteria in this study were Gram-positive, it is
that they have also adapted the ability to alter the composition of their cell membranes in
response to osmotic stress. It would be interesting to confirm this in future studies.
3.4.3 Aciit production by the non-filamentous Watchem soil bacteríal isolates
bacteria Approximat ely 70Vo of the identified, and l2Vo of the unidentified non-filamentous
isolated in this study were found to be capable of producing acid from glucose in vitro
(Tables 3.g - 3.11). These results were expected given thatBacilløs and Arthrobacter,the
known major genera of bacteria identified in this study (Tables 3.2, 3'3, and 3'4), ne both
none of the to be capable of producing acid from glucose (Bergey, 1984). Interestingly, from Micrococcøs isolates identified from the Watchem soil were able to produce acid
68 glucose, even though they are known to be metabolically capable of doing so (Bergey, l9S4). Potentially the media used in the fermentation study lacked nutrients that are essential for Micrococcus to produce acid from glucose. This however, requires further investigation.
3.4.4 Alkalitolerance of Jitamentous bøcteria (actinomycetes) ìsolated from the
Wøtchem soil: Preliminary studies
Preliminary screening of the 46 actinomycetes isolated from the Watchem soil for¡nd 70% were capable of tolerating pH values between 7 and 12. T\e existence of alkaliphilic and alkalitolerant actinomycetes in soils has at least been known since 1947, when Johnstone, investigating the dry, alkaline (pH S.7 to 9.2) calcium carbonate-rich soils of the Bikini atoll, found that the microflora of these soils consisted of up to 95%o actinomycetes.
Since then, Mikami et al. (1982) have isolated alkaliphilíc Streptomyces species (the predominant actinomycete species detected in this study), from soils in Japan, with pH optima ranging between pH 9-9.5, but which could tolerate up to pH 11.5. Also, in studies by Tsujibo et al. (1988 , I990a,1990b), alkalitolerant actinomycetes belonging to the genus
Nocardiopsl,s were isolated from soils in Mino city, Neyagawa city, and Soraku-gun, Japan, with one isolate (Nocardiopsis dassonviltei, OPC-15) showing antibacterial activity against
Proteus miribalis and Bacillus subtilis, one isolate (Nocardiopsis dassonvil/ei, subspecies
alba, OPC-18) producing three types of xylanases under alkaline conditions, and one
isolate (Nocardiopsis dassonvitlei, OPC-210) producing two types of alkaline serine
proteases.
More recently, Groth et at. (1997) described a novel alkaliphilic actinomycete, Bogoriella
caseilytica in an alkaline sodic soil þH 10) taken from near Lake Bogoria in the Kenyan-
69 between pH 7 Tanzanian rift valley. This bacterium was found to be capable of growth '2
species was found and 10.1, with optimal growth occurring at pH 9-10. FurtheÍnore' this
up to 8Vo NaCl to be halotolerant between 0-I8Vo NaCl (0 - 3M NaCl), with concentrations
not affecting growth.
well understood, V/hilst the mechanisms involved in bacterial alkalitolerance are relatively
actinomycetes' there appears to be no literature on the alkalitolerance mechanisms of
potentially, they possess the same basic alkalitolerance mechanisms to non-filamentous
excreting bacteria, ie., the ability to adjust the pH of their external environment by
and the ability to acids/trydrolysing proteins, the active transport of H* ions into the cell,
is yet to be manipulate the structures of their cell walls and cell membranes. This however,
confirmed.
the Watchem 3.4.5 Høtotolerance of filamentous bacteria (actínomycetes) isolateil from
soil: Initíal studies
l77o were capable Halotolerance screening of the actinomycetes in this study revealed that
halotolerance of tolerating between 0.5 and 1.5M NaCl. Like alkalitolerance, broad-range
research by has also been reported previously in actinomycetes isolated from soils. In from Killham and Firestone (1984), for example, two streptomyces species isolated
were found to be alkaline saline-sodic soils in california (s. griseøs and s. califomrcøs) 0'75M NaCl' capable of growth in media containing between 0 - |M NaCl and 0 - the marine respectively. Salt-tolerant actinomycetes have also been isolated from
Jensen et al" l99l; environment (Barcina et aI., 1987; Goodfellow and Haynes, 1984;
1988; Odell, 1994)' Pisano et a1.,1989; Pisano et a1.,1986; Weyland and Helmke,
to The mechanisms involved in the halotolerance of actinomycetes are poorly understood'
One potential mechanism, observed by Killham and Firestone (1984) is the accumulation
of compatible solutes within the cytoplasm. In their research, Killham and Firestone
(19g4), found that when cultured in increasingly saline solutions Streptomyces species
began to accumulate the neutral amino acids proline, glutamine and alanine as a compatible
solute, and, above a threshold of 0.75M NaCl, K* began to accumulate. It may also be
possible that the isolates in this study utilise similar compatible solutes'
3.4.6 Fungal identification
Spore morphology testing of the fungi isolated from the'Watchem soil revealed 7 different
fungal genera - Penicillium, Acremonittm, Fusarium, RhiZopus, Ulocladium, Gliomastix'
Fusarium, and Gliocladium. Similar genera of fungi including Penicillium, Ulocladium,
the Dead and Acremoniumhave also been identified in the saline surface soils from around
being a Sea valley in Israel (Steiman et aI., 1995; Guiratd et aI., 1995), with Penicillium
major component of the areas mycoflora (Steiman et aI., 1995). Furthermore, Acremonium,
the Fusarium, and Rhizopl,rs species have been isolated from alkaline soils taken from
Makkah district in Saudi Arabia (Ramadani and Aggab,1993)'
(1995), Interestingly, none of the fungi isolated by Steiman et al. (1995), or Guiraud et al' fungi demonstrated strict halophilicity, only halotolerance. Additionally, none of the
isolated by Ramadani and Aggab (1993), displayed strict alkaliphilicity, only strictly alkalitolerance. The fungi isolated in this study were also found not to require
was alkaline or saline conditions, but were able to tolerate high pH and high NaCl. This
isolated on demonstrated when the fungi isolated on high pH growth media, and the fungi
growth media containing a high level of NaCl, were able to be maintained on ll2 strength
(Tables 3.14a and potato dextrose agar, which has a pH of 5.6 and contains no NaCl 7l lWatchem 3.14b; Appendix l). The mechanisms by which the soil fungal isolates might have been able to tolerate the alkaline and saline media are discussed below:
to Mechanism I - Adjustment of the external pH: Fungi such as Fusarium spp', are known
(Horikoshi and be able to adjust the pH of their external environment to their own optimum
Akiba, lgg2). As Fusariutn was one of the fungal genera isolated in this study, it may also
high be possible that the fungi in the Watchem soil have adapted this strategy for surviving pH conditions.
Mechanism 2 - Specialised cell membrane and cell wall composition: The majority of fungal cell walls consist of long chains of pl-3 glucan (Hiura and Tanimura, 1991).
Horikoshi and Akiba (1g82),however observed that some Fusarium spp. shorten the chain
is length of p1-3 glucan in response to alkaline conditions. Whilst the significance of this
p1-3 uncertain (Hiura and Tanimura,lggl),it is possible that the shortened glucan chains
in improve the integrity of the cell wall. As Fusarium was one of the fungal genera isolated of this study, this mechanism may also be important in determining the structural integrity fungal cells within the alkaline Watchem soil.
Mechanism 3 - Compatible solutes: Osmophilic yeasts are known to adapt to saline
glycerol conditions via the accumulation of low molecular weight organic solutes such as
that (Brown and Simpson,1972; Andre et a1.,1988; Meikle et al., 1983). It is likely then,
to osmotic the fungi isolated in this study also accumulate compatible solutes in response
study accumulate stress. It would be interesting to determine if the fungi isolated in this
glycerol as well, or whether other osmol¡es are accumulated.
72 3.5 Conclusion
the soil Although the use of selectiv e agar media to survey the microbial population of from'Watchem, Victoria, Australia, would have led to the isolation of that further that this population is highly adapted to high pH and Na* concentrations, and investigation of this community using a multi-faceted approach should be conducted' The culturable microbial population of the Watchem soil was found to contain an average of 7 x lga cfu of non-filamentous bacteria g-t of air-dried soil. This population was 93 bacteria dominated by Gram-positive bacteria, with Bacillils spp. comprising 80Vo of the identified in this study, followed by l}Vo Arthrobacter spp., 5Vo Micrococcus spp',ZVo paenibacillas spp., and lfto Brevibacterium and Aureobacterium. Only one Gram-negative investigation bacterium, Alcaligenes ryIosorydan.î, was isolated from the soil. Preliminary that of the actinomycete and fungal communities within the Watchem soil indicated (5lVo of members of the genus Streptomyc¿s were the most common actinomycete isolated isolates), and that 667o ofthe fungal isolates belonged to the genus Penicillium' All of the non-filamentous bacterial isolates tested were found to be highly alkalitolerant culturable between pH 7 and l2.Initial studies investigating the alkalitolerance of the tolerant to the actinomycete population however, found only TOVo of these isolates to be same pH range. was found to NaCl tolerance of the culturable non-filamentous bacterial population studied be influenced by the composition of the medium from which the bacteria were isolated. at pH 7 pH 1 1 , and on media 22Vo, 39To , and 62Vo of the non-filamentous bacteria isolated , between 0'02 M containing 1.5 M NaCl, respectively, displayed wide-range NaCl tolerance 73 particularly halotolerant, and 2 M NaCl. Bacillus atrophaeus and Bacillus subtilis were with 90Vo and 86Vo of these isolates, respectivelY, from half-strength nutrient agat containing l.5M NaCl. Other bacterial genera such as Micrococcøs and Arthrobacter' agar although not isolated directly from the V/atchem soil on half-strength nutrient containing 2M NaCl, were found to tolerate 2M NaCl once subcultured' With the exception of Micrococcas spp., all of the genera of non-filamentous bacteria producing acid identified in the watchem soil via FAME profile analysis were capable of from glucose. In contrast only l27o of the unidentified non-filamentous bacteria isolated from the'Watchem soil were found to be capable of producing acid from glucose' 74 CHAPTER4 volatite Fatfy Acid Analysis in Alkaline sodic soils: Method development 4.1 Introduction Volatile fatty acids (VFAs) such as acetic, propionic and octanoic acid are formed in soils during the microbiological decomposition of organic materials (Ponnamperuma, 1972; Tsutsuki and ponnamperuma, 1937). In alkaline sodic soils it is hypothesised that these microbially-synthesised acids solubilise natural lime deposits, leading to a release of Ca2* which can displace Na* from the surface of clay particles and improve soil water handling characteristics (see Figure 2.2a n Chapter 2). The basis of this research is to generate protons via the microbial production of fatty acids to effect such changes and improve soil structure. Therefore, it is important to quantiff the amount of VFAs produced in alkaline sodic soils following organic matter treatment in order to identiff organic ameliorants and treatment regimes that are most suitable for use at a field-scale level' (C1 VFAs are readily extractable from soil into water as they are polar molecules highly use a gas polar - Cs moderately polar). Water, however, is an inappropriate solvent for in oxidation chromatograph fitted with a mass selective detector (GC-MSD), as it causes of the the detector filament, and the swelling and decomposition of the stationary phase in column. A GC-MSD was selected for use in this study instead of a traditional GC fitted with a flame ionisation detector (FD), due to both its higher sensitivity, and its ability to conclusively identiff individual compounds within a mixture based upon their retention time, and ion fragmentation Pattem' in Furthermore, existing methods, designed for a GC-FID were deemed unsuitable for use and this study because 1) different columns were used (Paul and Beauchamp, 1989; Chang VFAs were Sanders, |993;Baziramakenga et al., 1994; Cummins and Wells, 1997),2) the 1989; analysed whilst they were dissolved in an aqueous phase (Paul and Beauchamp, Bazhamakeîga et al., 1994), and 3) these methods were designed for the analysis of free rWatchem acids, which are expected to occtu in extremely low levels in the highly alkaline soil (Paul and Beauchamp, 1989; Chang and Sanders,L993;Baziramakenga et al., 1994)' ìVith these constraints, it was necessary to develop a new method for the extraction and to analysis of VFAs from the Watchem soil. Described in this chapter are the steps taken develop this method. 4.2 Method DeveloPment 4.2.1 Instrumentøtion volatile fatty acid samples were analysed using a Hewlett Packard model 6890 gas 6890 mass chromatograph (Palo Alto, CA, USA) fitted with a Hewlett Packard model column (IIP- selective detector (MSD) and a spliVsplitless injector. A fused-silica capillary attached l0 m of inert Innowax, 25 m x 0.2 mm I.D., 0.2 ¡rm film thickness) to which was was used fused-silica pre-column (0.2 mm I.D.) (J&W Scientific Inc', Folsom, CA, USA) to separate the fattY acids. 76 Helium, with a punty of 99.999% (BOC gas Australia) was used as a carrier gas at a flow Packard MSD rate of I ml minute-I. The GC and MSD were controlled using the Hewlett Chemstation software package version 4.00'00 1989-1995' 4. 2. 2 D etermin stíon of chr omatogr øphy condítions pl a 40 To determine appropriate chromatography conditions for separating vFAs, 1 of ¡tg 3- mfl VFA standard mixture containing acetic, propionic, 2-methyl propionic, butyric, methyl butyric, pentanoic, hexanoic, heptanoic and octanoic acids (Ultra Scientific, North into Kingstown, RI, USA), made in diethyl ether (BDH, Victoria, Australia), was injected (temperature the GC using a Hewlett-packard 6890 autoinjector. Oven ramping conditions program) and the split ratio (amount "split ofP into waste, compared to the fraction of peaks sample entering the column) were altered over several runs until 8 well separated octanoic acid were detected by the mass selective detector (MSD), in order from acetic to (Figure 3.1). Analyses were performed in scan mode (scan was restricted to compounds with an atomic mass between 50 and 150 units). using a The final chromatography conditions were: lpl of sample was injected into the GC The 10:1 split ratio, ie., 10 parts was split to waste, I part was injected into the column' 200oC at a oven was ramped from an initial temperature of 70oC to a final temperature of temperature rate of 20oC per minute. The injector temperature \¡/as 200oC and the detector 2900c 77 e 75000 d 70000 g €¡5000 toooo f h 5500c, 50000 a 45000 c ¿toooo .ts5000 3000c' b =5000 20000 't sooo 1 0000 sooo o 4 c¡ 7 o7 e Figure 4.1. Chromatogram showing well separated VF'A standards. a = acetic acid, b = p"opanoic acid, c= 2-methyl propanoic acid, d = butanoic acid, e = 3-methyl ¡utanìic acid, f = pentanoic acid, g = hexanoic acid, h: heptanoic acid, i: octanoic acid. 4.2.3 Selectìon of the MSD analysis mode Mass selective detectors are able to detect compounds in 2 different modes, scan mode and selected ion monitoring (Snvr¡ mode. In the first of these, scan mode, the MSD scans for all compounds within a certain range of molecular weights (in the case of the 6890 system, compounds with molecular weights between 1.2 and 700 can be detected). This mode, therefore, is useful for detecting unknown compounds in a mixture, but, as it scans over a wide range of molecular weights, is relatively insensitive' In the second mode, selected ion monitoring (SM) however, the MSD focuses upon detecting specific ion fragments, which are produced following the electron ionisation of the compounds within the detector. By directing the MSD to only "look" for ions unique to mode, compotrnd(s) of interest, the sensitivþ of the instrument is increased markedly. This therefore, is useful for detecting individual compounds within a mixture. Given the improved sensitivity, it was decided that all soil VFA analyses would be performed with the GC-MSD in SIM mode. 78 4.2.4 Selection of ions to monitor 2-methyl To select appropriate ions for the MSD to monitor, ionisation pattems of acetic, propionic, butyric, 3-methyl butyric, pentanoic, hexanoic, heptanoic and octanoic acids were downloaded from the NIST MS Chemstation library (May 1995 edition) software provided with the 6890 GC-MSD system (see Figure 4.2 for example). Subsequent examination of the ionisation patterns determined that all VFAs of interest could be and detected within the soil samples by seeking out ions 4I mlz, 43 mlz, 45 m/2,60 mlz, 74 m/2. AÞun,tEncã #e415: OctãnÞlc Acld 9000 €rooo 7000 7 5000 5000 4000 3000 2000 1 ì 1 000 3 3 1 o m.4F-> Figure 4.2,Fragmentation pattern of Octanoic acid' 4.2.5 Construction of calibratìon cutves were To enable the quantification of VFAs within the soil samples calibration curves (acetic, propionic, 2- constructed for each acid. To do this, lpl of a VFA standard mixture metþl propionic, butanoic, 3-methyl butanoic, pentanoic, hexanoic, 3-metþl pentanoic' the GC at and octanoic acids) dissolved in dietþl ether was injected in triplicate into Chapter 4'2'2' concentrations ranging from 5-80 pg mll and separated as described in 79 All compounds were found to produce a linear relationship, from which, the calibration curves were constructed (see Figure 4.3 for example). He> 7 (t 1 z z ,. .A.Dorrnt coef of Det (r^2) g - 998, Figure 4.3. Calibration curre for Pentanoic 4.2.6 Selection of an ìnternal standard For accurate quantification of VFAs in soil samples it was decided that an intemal standard would be used. An aldehyde compound was considered to be particularly appropriate for use as an intemal standard in this study because: a) Aldehydes are highly volatile therefore, they are suitable for GC analysis (Morrison and Boyd, 1992), b) Aldehydes contain polar carbonyl groups thus, will be miscible with the same organic solvents used for the VFAs, c) The presence of the polar carbonyl group in aldehyde compounds makes them suitable for separation on a HP Innowax column, d) Under the chromatography conditions used in this study, there is unlikely to be any cross reaction with the VFAs 80 made from (Morrison and Boyd, lgg2), and, e) No aldehydes were detected in extracts blank soil samples. (acetic, To select the intemal standard, lpl of a standard mixture containing VFAs propionic, 2-methyl propionic, butyric, 3-methyl butyric, pentanoic, hexanoic, heptanoic and octanoic acids) and aldehydes (formaldehyde, acetaldehyde, propanal, acrolein' butanal, methacrolein, 2-methylpropanal, 2-methylbutanal, pentanal, hexanal' 2- and methylpentanal, heptanal, octanal, nonanal, trndecanal, dodecanal, tridacanal, ether at a tetradecanal) (Ultra Scientific, North Kingstown, RI), dissolved in diethyl performed in concentration of 50 pg ml-r, was injected into the GC-MSD. Analysis was chromatogtam scan mode to enable the detection of all aldehyde compounds. The resulting times (ie' not was visually inspected to identiff aldehyde compounds with unique retention acids. coeluting with any VFAs), with elution times between those of acetic and octanoic use as the Dodecanal, with an elution time of 6.35 minutes (Figure 4.4) was selected for intemal standard. Subsequent examination of the ionisation pattern of dodecanal ions 57 mJz,68 determined that dodecanal could be detected in SIM mode by monitoring mJz and82 mlz (Figure 4.5). 81 ,A.ÞundancÈ 75000 a 70000 rt tr 65000 fit 5(1000 C'o b 55000 T' 50000 o ¿15000 o 40000 35000 30000 =5000 20000 I 5000 I OOOO 5000 o 4 4 7 a o3 -f lmË--' Figure 4.4. Chromatogram showing position of the internal standard dodecanal in reiation to Vf'.A. peaks. a = 3-methyl butanoic acid, b = pentanoic acid' Abundance 57 t2 st 1000 mtZ=> Figure 4.5. tr'ragmentation pattern of Dodecanal at a All future VFA analyses wers performed using dodecanal as the internal standard atarate of 1 pl of concentration of 20 ttgml-l. This was added to the solvent mixture prior to dodecanal standard (2%o dodecanal in chloroform w/v) to I ml of solvent, extracting VFAs from the soil. 82 4.2.7 Solvent formulation VFAs from To identiff a suitable organic solvent or organic solvent mixture for extracting in water were the soil solution, 3 ml volumes of a 40 ppm VFA standard mixtt[e prepared the following extracted for 30 minutes on a rotary shaker set 60 rpm with 2 ml of each of (DClvÐ (Sigma- solvent mixtures: a) 100% Dietþl Ether, b) 100% Dichloromethane Aldrich chemical co., castle Hill, Australia), d) 50:50 DCM:Ether, d) 30:70 DCM:Ether, 35:65 DCM:Ether' e) 70:30 DCM:Ether, f) 40:60 DCM:Ether, g) 60:40 DCM:Ether, and h) 50 600 pL of each extract was then removed to a clean GC vial containing approximately mode on the GC- mg of anhydrous NazSO¿ to remove traces of water, and analysed in SIM MSD as described in Chapter 4.2.6.411samples \¡'/ere analysed in duplicate' ability to Diethyl ether was selected as a solvent due to its high volatilþ, as well as its with solvate highly polar organic molecules eg., acetic acid, whilst remaining immiscible DCM was water (ie.,dietþl ether forms 2 phases with water) (Monison and Boyd, 1992). water, as well selected as a solvent again, due to its high volatility and immiscibility with acid (Morrison and as for its ability to solvate less polar organic molecules eg., octanoic Boyd, lggz).By combining these solvents, it was hypothesised that a solvent ratio could be interest (ie., those formulated that was favourable for the extraction of all of the VFAs of between C2 and C8). phase are shown in The effects of solvent composition on VFA extraction from an aqueous due to its inability to Table 4.1. pure diethyl ether was found to be an unsatisfactory solvent acids. Furthermore, recover the higher molecular weight hexanoic, heptanoic and octanoic was also the volatilþ of the ether made sample handling difficult. Pure dichloromethane lower molecular found to be an tmsatisfactory solvent, as it was unable to recover the 83 weight acetic and propanoic acids, and, being denser than water, dichloromethane settled at the base of the extraction tubes, impeding solvent recovery. After considering both the phase separation characteristics (ie., the miscibility of the solvents with each other, and the abilþ of the solvent mix to separate from the aqueous phase), and the extraction efficiency of several dichloromethane:diethyl ether combinations over the range of VFAs, it was concluded that a mixture of dichloromethane: diethyl ether (35: 65) would be used for extracting VFAs from the Watchem soil solution (Table 4.1). Ratio of: Percentage of each acid recovered DCM Ether Acet. Prop. 2]Ù,d- But. 3M- Pent. Hex. Hept. Oct. Prop. But. 100 0 -2.0 1.6 4.0 3.3 9.7 49.8 74.0 70.5 50.1 103.8 75.5 80 20 5.2 18.1 58.3 45.9 85.7 96.8 rt4.7 60.7 70 30 4.3 t7.8 s6.6 4t.0 76.3 93.2 99.5 89.3 N/A* 50 50 N/A* N/A* N/A* N/A,I. N/A* N/A* N/A* N/A* 114.1 t06.7 7t.7 35 65 13.7 37.2 97.9 71.3 t20.5 t12.8 83.6 30 70 t6.2 44.7 r07.5 83.5 1 27 I t22.3 130.2 115.9 20 80 16.6 43.3 101.1 75.5 1 16.1 111.0 tt2.7 100.6 68.5 t22.2 106.0 73.9 0 100 16.8 47.9 tt5.7 88.8 135.5 tt3.9 Table 4.1. Effect of the organic solvent composition on 7o VFA recovery from an aqueous phase. * N/A: DCM, Ether and aqueous phases all separate, so not analysed on GC-MSD. Recoveries >1007o are due to use of an extraction method with a high variability. 84 4.2.8 Optimisøtion of the extraction method Having identified a solvent mixture for extracting VFAs from an aqueous solution, it was necessary to develop and optimise a method for extracting VFAs from Watchem soil solutions. To do this 2 g of sieved sterile soil (<2mm) was weighed into 18 glass test tubes and spiked with 120 pl of VFA standard mix (500pg mfr acids Cz - Cs in sterile distilled water) + 480 pl sterile distilled water. After incubating the soil ovemight at 25oC to allow equilibration between the acids and capped soil, 5 ml of sterile distilled water was added to each test tube. The tubes were then tightly and mixed ovemight at25oC on a rotary shaker' test To increase the solubility of the acids, 250 pl of methanol was added to half of the tubes prior to shaking. To investigate whether decreasing the solution pH increased VFA recovery, a second set of 18 test tubes was prepared as above, and the pH of each tube adjusted to 2 prior to shaking, using dilute HCl. Following shaking the test tubes were centrifuged at 2000 rpm for 15 minutes to remove the soil particles. Supernatant (3 ml) was then added to a clean glass test tube, (re)adjusted (35:65) to pH 2 with dilute HCI and extracted with 2 ml of Dichloromethane:Diethyl ether removed for either a) 10 min, b) I hf or c)2hrs. 600 pl of each extract (top layer) was then and to a clean GC vial containing 50 mg of anhydrous NazSO¿ to remove traces of water added to the analysed on the GC-MSD as described previously (Section 4.2.2). Dodecanal measured in solvent mixture at20 ¡tgml-l served as an intemal standard. All samples were triplicate. 85 Optimum VFA recovery was recorded in the tubes that were incubated for 2 hrs, to which acetic methanol had been added (Table 3.2). For example, extraction of the highly-polar (2hrs acid was improved from 4 % (lo minutes extraction, no methanol) to 14 % extraction, methanol added). The reproducibility of the extraction method was also maximised by extracting the methanol-treated samples for 2 hrs, with the variation in acid o/o (Table 4.3)' recovery being less than 5 for all acids but acetic and octanoic acid Acidification of the soil solutions to pH 2 p.'jlor to overnight shaking was found to have no beneficial effect over acidiffing the soil solutions immediately before solvent extraction. Y VT'A 10 min. lOmin + I hour I hour * 2 hours 2 hours * MeOII MeOH MeOH Acetic 4.0 12.5 3.3 9.2 6.3 14.0 Propanoic 14.5 30.0 26.7 30.0 26.7 37.0 2-Methyl 36.0 46.5 52.5 48.5 49.2 60.3 Propanoic Butanoic 39.0 49.5 s6.0 51.5 s 1.3 62.5 3-Methyl 52.7 68.0 74.0 70.8 69.2 86.3 Butanoic Pentanoic 64.7 75.0 73.0 74.8 75.0 80.2 42.7 Hexanoic 56.7 61.0 57.3 64.7 6r.2 Heptanoic 53.5 62.2 59.3 59.5 56.2 63.8 Octanoic 37.5 46.0 43.7 40.7 36.8 47.0 Table 4.2.E:flectof the extraction method on VFA recovety. VFA 10 min. lOmin + t hour I hour * 2 hours 2 hours * MeOH MeOH MeOII Acetic 87.5 20.0 t14.7 17.6 t22.8 15.6 Propanoic 91.5 t9.2 4t.9 t6.4 28.2 t.4 7.1 3.7 2-Methyl 21.6 1 5 I t2.5 t9.6 Propanoic Butanoic t9.9 14.0 13.9 20.5 8.4 2.4 2.4 3-Methyl 18.6 1 1.1 I 5 1 20.8 9.6 Butanoic Pentanoic 6.2 4.1 5.0 13.8 11 2 4.2 Hexanoic 4.9 3.4 4.3 13.2 9.7 4.9 Heptanoic 4.1 5.8 6.3 1 4. I I 1.1 3.0 Octanoic 13.5 t2.4 10.4 11.6 0.21 7.7 Table 4.3. o/o Variation between replicates for each extraction method. 86 4.2.9 Optimum GC-MSD conJiguration and utraction method The final GC-MSD configuration and extraction method was as follows: To extract the VFAs, 2 g of soil, 5 ml of distilled water and 250 pl of methanol were added to a 15 ml test tube and shaken end over end ovemight. Following shaking, the test tubes were centrifuged at 2000 rpm for 20 minutes at 4oC. 3 ml of the resulting supernatant was then added to a clean 15 ml glass test tube and acidified to pH 2 using dilute HCI (pH was tested using indicator strips). 2 ml of freshly prepared solvent (dichloromethane:diethyl ether, 35:65, spiked with dodecanal to a final concentration of 20 ttgml-l was then added to each test tube, and after capping tightly, the tubes were shaken end over end for 2 hotus. After the allowing aqueous and solvent phases to separate, 600 pL of the solvent (top) phase, containing the VFAs, was removed from each tube and placed into a clean GC vial containing approximately 50 mg of anhydrous NazSO¿. Following capping, all GC vials were sealed with parafilm and stored at -15oC to prevent sample volatilisation. To analyse the VFAs, lpl of sample was injected into the GC using a 10:1 split ratio. The oven was ramped from an initial temperature of 70oC to a final temperature of 200oC, at a l. rate of 20oC min The injector temperature was 200oC and the detector temperature 280oC. Samples were detected by a Hewlett-Packard MSD 58904 mass spectrometer using an ionisation voltage of 90eV. The GC-MS was connected to a Hewlett-Packard Vectra MS Chemstation computer for data and acquisition and analysis by SIM (ions 41ml2, 43m/2, 45m1z, 57m/2,60m/z,68mlz,74mlz and 82mlz measured). Quantification of VFAs was performed using the internal standard dodecanal (2}pgmfl).VFA concentrations were determined by calculating the ratio between the peak areas of dodecanal and the VFAs. 87 CHAPTER 5 Microbial reclamation of an alkaline sodic soil using the model organic substrate glucose 5.L Introduction Incubation of alkaline sodic soils treated with glucose has been shown to lead to a (SAR), as well reduction in the soil pH, soluble carbonate, and the sodium adsorption ratio Chorom and as an increase in the EC and hydraulic conductivity of the soil (Chorom, 1996; properties are Rengasamy ,lgg7). Although these changes to the soil physical and chemical of thought to be the result of biological reactions, specifically, the microbial metabolism 1997)' glucose into volatile fatty acids (VFAs) (Chorom , 1996; Chorom and Rengasamy, this has not been Proven. The aim of the experiment described in this chapter, therefore, is to confirm if the changes Chorom and in the soil physical and chemical properties observed by Chorom, (1996), and identify the microbial Rengasamy , (lgg7), are indeed microbially based, and, if so, to populations, and reactions responsible for the changes' 5.2 Materials and Methods 5.2.1 Soil properties The properties of the soil used for this experiment are the same as those described previously in ChaPter 3.2.1. 5.2.2 Preparation of Soíl SamPles with distilled 25Og air-dried Watchem soil samples were treated with glucose and mixed the microbial water as shown in Figure 5.1. The major focus of this work was to examine soil reactions which took place in both moist (WHC) and waterlogged (2WHC) Watchem following glucose treatment. For this reason, following mixing, the soil samples were easier to placed into non-leaching pots (Figure 5.1) in which the soil moisture levels were pots maintain, then incubated at Z5oC.It is acknowledged however, that using non-leaching potentially encouraged Na* ions released from soil in the upper portion of the pot to be 7 readsorpted by soil in the lower portion of the pot. All pots were watered to weight every on days using distilled water. Three pots from each treatment were removed for analysis days 0, 3,6, ll, 19,40, and 60' Treatment Final soil glucose level Final soil moisture level (vo)*'^ (Vo wlw) Aerobic Control 0 30 w2 2 30 w4 4 30 Anaerobic Control 0 60 x WHC* 2W2 2 60 2W4 4 60 * on a Table 5.L. Treatments used in the glucose experiment. 7o Glucose was calculated the soil to (w/w) basis. ^ Glucose was pre-dissolved in the distilled water used to moisten 2 x WHC ensufe even mixing. WHC = Water holding capacity. * A watering regime of was used to promote anaerobic conditions, ie. the soil was waterlogged. 89 E 80mm E O (o Lid Sealed Pot Ventilation holes (8mm diameter) Figure 5.1. Schematic of the pot used for the glucose experiment. mixed until Soil samples, taken from the top to the base of each pot using a corer, then homogenous, were then subjected to the following physical, chemical and microbiological analyses: 5.2.3. Physical and Chemical analyses 5.2.3.1 SoiI pH and EC soil pH and electrical conductivity (EC) levels were determined in 1:5 soil:water Conductivity suspensions using an Orion EA 940 pH Meter, and, an Orion model 170 Meter, respectivelY. 5.2.3.2 Soluble Cation AnalYsis analysing 1:5 Concentrations of soluble cations (Ct* and Na* ) were determined by Flame soil:water extracts (shaken end over end overnight), with a Corning Clinical #42 flltet paper Photometer, model 410C. All samples wele filtered through Whatman prior to analysis to remove particulate matter. 90 5.2.3.3 Determinøtíon of total carbonate (sieved < 2mm) was Total carbonate was determined as follows: 59 of air-dried soil (standardised against 1 weighed into a 250 ml bottle. To this was added 100 ml of lM HCI and filtered M anhydrous Naz CO¡). The soil was then shaken end over end overnight transferred to a clean beaker through a Whatman #l filter paper. Ten ml of filtrate was then phenolphthalein indicator along with 25 ml of distilled water. After adding 3 drops of 0.I7o against HCI) (in ethanol), the solution was titrated against 0.5 M NaOH (standardised lM (Rayment and Higginson, 1992)' 5.2.3.4 Determination of water soluble carbonate and bicarbonate dry soil Water soluble carbonate and bicarbonate was determined as follows: Ten grams of end over (sieved < 2mm) and 50 ml distilled water were added to a 100 ml bottle, shaken was then end ovemight and filtered through a Whatman #1 filter paper. Ten ml of filtrate titrared againsr 0.01 M HCI (Standardised against 0.01 M NazCO¡). Phenolphthalein indicator indicator was used for the determination of soluble carbonate, and methyl orange 1992). was used for the determination of soluble bicarbonate (Rayment and Higginson, 5.2.3.5 Measuretnent of microbial glucose utilisation Microbial utilisation of glucose was monitored using the Somogyi-Nelson reducing sugars and Tabatabai (1993) to assay (Nelson, lg44). This assay was shown previously by Deng sugars in soils' Before be highly sensitive and accurate for the determination of reducing the assay the following reagents (A-D) were prepared: tartrate, 20 g NaHCO¡' 2009 Reagent A: 25 g Anhydrous NazCO¡ ,25 gsodium potassium anhydrous NazSO¿ / L distilled water. 9l Reagent B: 30 g CuSOa.5HzO / 200 ml distilled water. To this was added 4 drops of concentrated HzSO¿. Reagent C: 25 g Ammonium molybdate was dissolved in 450 ml distilled water. To this was added 2l ml concentrated HzSO¿. In a separate beaker, 3 g Na2HAsO¿.7HzO was dissolved in 25 ml of distilled water. The arsenate solution was then slowly added to the molybdate solution, with gentle mixing, to make reagent C, and diluted to 500 ml. This was then stored at25oC in the dark to prevent the formation of a precipitate. Reagent D: (Prepared fresh). 25 mlreagent A was combined with 1 ml reagent B Standards: Glucose solution (12.5, 25, 50, 100 and 200 ¡tg ml-l , prepared in distilled water) Procedure: Two grams of soil sample was combined with 5 ml distilled water in a test tube and shaken end over end for t hour. After shaking, the test tubes were centrifuged at 2000 rpm for 20 minutes at 4oC. Three mls of the resulting supernatant was then placed into a test tube containing 0.3 g Dowex HCR-S strongly acidic cation exchange resin (Sigma Chemical Company). After allowing the resin to settle to the base of the test-tube, I ml of appropriately diluted cation exchange resin-treated sample was then combined with I ml reagent D, placed into a boiling water bath for 20 minutes, then cooled in a cold water bath to room temperature. One ml of reagent C was then added to each sample and mixed until effervescence ceased. After standing for 20 minutes (to allow colour development) the samples were dilute d to 25 ml using distilled water and then absorbence levels read on a Shimadzu UV-1601 UV-VIS spectrophotometer at a wavelength of 660 nm' 92 5.2.3.6 Volatile fatty acíd analysis of soil samples Volatile fatty acids were measured in all soil samples as described in Chaptet 3-2.9 5.2.4 Microbiological analyses 5.2.4.1 FAME analysis of soíl samples Fifty grams of soil sample, taken from the top of the pot to the bottom, was removed from each replicate pot, following which, all samples from a particular treatment were combined into one 150 g sample. Six grams of this combined soil sample was weighed into a pyrex tube. To this was added 6 ml of Reagent | (45g NaOH, 150 ml Methanol, 150 ml MilliQ water - Saponification). Following vortexing, the mixture was placed into a water bath at 100oC for 30 minutes, cooled, then centrifuged at 2000 rpm for 3 minutes. Three grams of the resulting supernatant was then removed into a clean test tube, spiked with 75 ¡tl of 2-5 mM C19 standard (nondecanoic acid), combined with 6 ml of Reagent 2 (325 ml 6 N }JCl,275 ml Methanol - Methylation), vortexed, then heated in water bath at 80oC for 10 minutes. After cooling the tubes rapidly on ice, 3 ml of Reagent 3 (2O0 ml HPLC-grade Hexane, 200 ml HPLC-grade Methyl-tertiary Butyl ether - Extraction) was added to each test tube, and the tubes shaken end over end for 10 minutes. After allowing the aqueous and solvent phases to separate, the solvent (top) phase was removed into a clean test tube. To this was added 4 ml of Reagent 4 (10.8 g NaOH, 900 ml MilliQ water -Wash). After shaking end over end for 5 minutes, and allowing the phases to separate, 750 pl of top phase was then removed to a clean GC vial for analYsis. 93 FAME extracts were separated on a Hewlett-Packard 5890 series tr Plus gas chromatograph as described in Chapter 2. Peaks were then examined using principal component analysis software (MlS-Microbial ID,Inc., Newark, Delaware, USA)' Samples with a total peak area of < 50000 units were concentrated under a stream of nitrogen and re-analysed. 5.2.4.2 Methane analysis of the soil heødspace This analysis was performed to detect any loss of VFAs via metabolism by methanogenic bacteria such as Methanobacterium, Methanococctts, and Methanothrix (Brock and Madigan, 1991). These acids can be metabolised as shown: CH3COOH (acetic acid) + CH¿+ COz To analyse for methane production, 10 g of freshly sampled soil was placed into a 20 ml Hungate tube, following which the headspace gases were allowed to equilibrate for I hr at Z1oC. Headspace gas samples were removed from the test tube with a gas-tight syringe and injected into a Perkin Elmer "Autosystems" gas chromatograph equipped with a flame ionisation detecror (Ftr)) and a Porapack Q 80/100 column. Ultra high purity helium was used as a carrier gas at a flow rate of 25 ml min-I. Injector and oven temperatures were both 60oC and the FID temperature was 250oC. 5.2.5 Statistical analYses All data were statistically analysed using the package 'Genstat for Windows, version 5.0'. Differences between day 0 and 60 values for each treatment were tested using a l-way ANOVA without blocking. Differences between treatments on days 0 and 60 were tested using a2-way ANOVA without blocking. 94 5.3 Results and Discussion 5.3.1 Microbial utílisation of glucose For the first 11 days of incubation, glucose utilisation rates were found to be low in all treatments. Following this lag period however, glucose levels were observed to fall rapidly by 72Vo,99Vo, and B3Vo in the treatments W2 (soil watered to a 307o moisture content and treated with2Vo glucose (w/w)),2W2 (soil watered to a60Vo moisture content and treated with27o glucose (w/w)), and2W4 (soil watered to a 607o moisture content and treated with 47o glucose (w/w)), respectively (Figure 5.2)' These results suggest that the microorganisms within the Watchem soil were dormant upon treatment with glucose. This is consistent with the finding in Chapter 3, that the Watchem soil microflora contains largely fungi, actinomycetes, and Bacilli, all of which produce spores. Additionally, the lag period may be the result of the high C:N ratio generated following the addition of glucose to the soil (26:1 in the W2 and 2W2 treatments, 44:1 in the 2W4 treatment), inhibiting the rapid growth of microorganisms (Haug, 1993). Whilst glucose was metabolised in the W2, 2W2, and 2W4 treatments, no significant decrease in glucose concentration was observed in V/4 (soil watered to a 307o moisture content and treated with 4Vo glucose (w/w)). Furthermore, sample variation was high for this treatment (Figure 5.2). For example, on day 6, glucose levels were found to vary between 8364 and 18014 pg glucose g-r air-dried soil' It is therefore, hypothesised that while conditions were favourable for the microbial utilisation of glucose in the W2,2W2, and,2W4 treatments, in the W4 treatment, the high osmotic effect caused by adding 4Vo glucose (w/w) to the soil and watering it to a 3O7o moisture content, was inhibitory. 95 The high variation in glucose concentration between the replicates in the W4 treatment may then be attributable to the presence of highly osmo-alkalitolerant strains of microorganisms in some pots and not others. Such highly osmo-alkalitolerant microorganisms were reported to be present in the'Watchem soil previously in Chaptet 2, agat when 27Vo of bacterial isolates studied were found capable of growing on nutrient containing I M NaCl (= 6Vo NaCl) at pH l1' 30000 rw2c EI2W2G rW4G ø2W4G È 25000 E'ì l- Ê o 20000 (E tr o .J 15000 (,o o o o(, 10000 =Et o U' 5000 0 60 0 3 6 11 19 40 lncubation time (daYs) Figure 5.2. The concentration of reducing sugars (glucose) in the Watchem soil throughout the 60'day incubation period. (Values for the controls were omitted from this figure as no glucose was added to these treatments) day 0' Interestingly, for all treatments, only half of the glucose added was extractable on that For example, adding 4Vo g\tcose to the Watchem soil (w/w), it would be expected - 40000 pg glucose g-1 air-dried soil would be detected in the W4 and 2W4 treatments only 20000 pg glucose g-r air-dried soil was detected' 96 Given that glucose molecules contain several hydroxyl groups, it is possible that a proportion of the glucose was bound to the highly charged clay particles or cations present in the soil, eg., the Ca2* in calcium carbonate; the remaining glucose existing in an equilibrium state between the soil particles and the soil solution. 5.3.2 Interpretation of whole soil FAME profiles Average FAME profiles for each of the 24 species of non-filamentous bacteria identified in the Watchem soil (Chapter 3) were analysed from the MIDI-FAME TSBA database as shown in Tables 5.2a and 5.2b. Similar tables were constructed for fungi of the genera Acremonium, Alternaria, Penicillium, Ulocladium, and Rhizopus which were detected in 'Watchem the soil (Chapter 3) (Table 5.2c), and for bacteria of the genus Clostridium' which were predicted to increase in numbers in the anaerobic treatments 2W2 and 2W4 (Table 5.2d). After analysing each table for FAMEs that were common or unique to each set of microorganisms, it was decided that the following FAMEs would be used for markers when interpreting whole soil extracts: a) iso 15:0: Changes to the concentrations of this FAME was deemed to represent changes to the population of Gram-positive bacteria, including Bacillus, Micrococcus, and Arthrobacter which were shown. to be present in the V/atchem soil in Chapter 3. This FAME has also been used as a marker for Gram-positive bacteria in studies by Federle (1986), O'Leary and Wilkinson (1988), Wander et al. (1995), and Zelles et aI' (1995). to represent b) anteiso l5:0: Changes in the concentration of anteiso l5:0 were also deemed Arthrobacter' changes in Gram-positive bacteria including Bacillus, Micrococcøs, and 97 Additionally, this FAME might detect changes in the population of Clostridram species such as Clostridium biþrmentans subgroup A. This marker FAME was used previously as a marker for Gram-positive bacteria in studies by O'l,eary and Wilkinson (1988), and 7-elles et aL (1995). the c) l6:1 coTc: Monounsaturated fatty acids with the <¡7c unsaturation are synthesised via anaerobic desaturase pathway (Scheuerbrandt and Bloch, 1962). This pathway is found in all strictly anaerobic bacteria, eg., Clostidium, and Gram-negative aerobes such as Pseudomonøs (Borga et al., 1994; Kidd-Haak et aI., 1994; Zogg et aI., 1997)' Changes to the concentration of 16:1 c¡7c in combination with the Gram-positive bacteria markers iso l5:0 and anteiso 15:0 were therefore, interpreted as representing changes to the population of Clostridium spp. in the Watchem soil. Alternatively, changes to this marker in combination with Gram-negative markers such as cyclo 17:0 and cyclo 19:0 (Frostegatd et al., 1993;Zogg et aI., 1997) were interpreted as changes within the population of Gram- negative bacteria. c) 18:3 o6c: This FAME was selected as a marker for the fungal population of the Watchem soil, including Rhizop¡rs spp. and17.753 d) 18:l cogc (or 18:1 rollc): FAMEs 18:l algc and 18:l collc elute at 17'767 minutes respectively. Both have a window of error of 0.008 minutes. It is therefore, possible that 18:l r¡9c can be misnamed 18:1 collc. 18:1 togc is found in Clostridium and fungi (Tables 4.2c and 4.2d), as well as in Gram-positive bacteria such as Corynebacterium (Kidd-Haak, 1994). 98 Changes to the concentration of this marker were therefore, interpreted only after careful examination of soil samples for potential signs of anaerobiosis, ie., if the soil was blackened by sulphides and had the odour of HzS and VFAs. In this case, increases in 18:1 Additionally, 18:2 co6c was used as a marker for fungi (Federle, 1986; Vestal and White, 1989; Frostegard et a1.,1993; Bardgett et al., 1996;Zogg et aI., 1997), and cyclo 17:0 and cyclo 19:0 were used as markers for Gram-negative bacteria (Frostegard et aI-, 1993; Zngg et aI.,1997). 99 o 2 o Ê o ao o o o o o À 6 E o o c a 3 o ì o 6 o s6 o o 6 (t ! I o(ft 6 lr ¡ (t o o (, (, o 9 ! õ I (, C' o o o o o I o o (t o b o ¡\ o 6 o o C Organlsm o o c o o o o É o o 6 o o o ¡\ g c c o 3 (¡ o I N N I 6 I I I z ¡ ì ¡ o G 6 ¡ ¡ Ì ì o Ì c, o I ct o o ì o c, o Ct o o o o o cl C' ct o o o ct ct o ct t o I ct t o e e 6 ¡o @ @ (o @ o @ ro È ?- F È ¡\ t\ È t\ @ € o o ö C' 6¿ N dt ç, (f' a rl !t h o ro o n n + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Bacillus æreus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Bacillus limus + + + + + + + + + + fuc¡ilus licheniíomis + + + + + + + + + + + + + + Becithts meoaterium + + + + + + + Bac¡ltus sohaedcus + + + + + Bacüus sublilis + + + + + + + + + + + + + + Becillus lhurino¡ensis + + + + + + + + Table 4.2a: FAME profiles of Bacillas species isolated from the lVatchem soil. .9 o o õ o p o o o E o o c, p, o an .2 6 (, o lr I - C' o o (, (, o (.t o o o o 6 o o) o¡ o) Organism - - - o o .D o o o gto Ê o o o o Ê lt an c o o o tt, (l. ct N nt 6 6 ì I o N z 3 o Ì ì ì ¡ o Ì o o o o o o o o o o o o o o (t o o c I I (oI (o aO t\ l\ 1\ 1.- o ao I N (\l N ci¡ ci, rf st r,o r¡' ¡¡t r¡' ro to to @ ao t- 0 + + + + + + + + + + + + + + + + + Arthrobacter + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + esteromaticum + + + + + + + + + + + + Brevibacterium iodinum + + + + + + + + + Microcaccus + + + + + + + + + + + + Micrococcus luteus + + + + + + + + + + + + Micræoccus roseus + + + + + + Micrococcus varians + + + + + + + + + + + + + + + + + + + + + + Table 4.2b. FAME proFrles of non'Bacillus bacterial species isolated from the Watchem soil. oO + + + + + + + - + + + + + + + + + + + + Ocsrt*t tm til e/'7Êrta'Ê st.b A + + + + + + + + + + + A 67 iö tm ¿i I enþ nlaß - B + + + + + Sþ + + + + + + + + + + + + + + + + + + + -sLÖ + + + + + + + + + + + + + + + + + + + + + + + + + + AAriúunbutwiatn-s.OB + + + + + + + + + + + + + + + + ClcEifrk nnddvl¡am + + + + + + + + dd¡furt"mølñtpp.tls + + + + + + + + Table 4.2c. FAME profiles of Clostridium species. o co o ll o ol N o o (t F. Organlsm - @ (0 o ì 3 ctt (l' o N o) o o) o ¡ Ì o I o o ôl ql= c, (o (o r(o ¡.- l\ oo o @ o I + + + + + + + + Penicillium - sub A + + + + Penicillium - sub B + + + + + + + + + + Ulocladium + + + + + o Table 4.2d. FAME profiles of fungi isolated from the Watchem soil. 5.3.3 Votatíle fatty acíd (VFA) and FAME analysis of soil samples: W4 treatment Only acetic acid was found in the soil treated with 4Vo glucose and incubated aerobically (W4), throughout the 60 day incubation period' As would be predicted from the low rates of glucose utilisation described previously for this treatment, the average level of acetic acid found in the soil was very low (3 pg acid g of air-dried soil-l). Further, the acetic acid concentration varied greatly between each replicate, as is demonstrated by the large standard deviations shown in Figure 5.3. Despite the low level of glucose utilisation and VFA production in the W4 treatment, FAME analysis of the whole soil revealed that microbial numbers increased throughout the 60 day incubation period. FAME 18:1 otllc? (18:l rogc) increased the most (85Vo), on day 40 (Figure 5.4). Given that the concentration of 16:l ro.lc was also observed to increase on day 40, and that no significant increase occurred in either the concentration of the fungal marker' 18:3 toóc, or the Gram-negative bacteria markers cyclo 17:0 and cyclo 19:0 (Figure 5.4), it is thought that the increase in I 8: 1 c¡l lc ? ( I 8: I rogc) is likely to be due to Clostridium spp., not fungi or Gram-negative bacteria. Furthermore, as the soil in the V/4 treatment was not expected to be totally anaerobic under the watering regime used (the soil was watered to a 307o moisture content), it is thought that the Clostridium spp. may have established themselves in anaerobic microsites throughout the soil. The random nature in which anaerobic microsites would occupy the soil could therefore, account for some of the variation presented observed in the VFA measurements. These findings support the hypothesis earlier in this chapter (see section 5.3.1) that the high osmotic pressure in the W4 treatment t02 was inhibitory to the growth and activity of the majority of microorganisms within the 'Watchem soil, in particular the aerobic population. 't2 EAcetic acid 10 â'õ U' tt I o ItI (ú 6 o ctt E) a 4 o Ê (,o 2 lr 0 0 3 6 11 19 60 -¿ lncubation time (daYs) Figure 5.3. VFA production in the Watchem soil watered to WHC and treated with 47o glucose (Ww) over the 60-day incubation period. 90 115:0 lso 80 E 1õ:0 antelso Ë70 t6:'l w7c o I t) ? ¡¡¡ 60 tr 18:1 w11c = E¡ 18:3 w6c ll 50 'õ .rì 40 tr .E Eo30 s 920 g &. 10 F 0 -10 lncubation time (daYs) Figure 5.4. Relative 7o change in the concentration of FAMEs representing Gram' po-ritio" bacteria (G+), Clostridum spp. (C), and fungi (F), in the W4 treatment' over the 60-day incubation Period. 103 5.3.4 Volatíle fatty aci.d (VFA) and FAME analysis of soil samples: 2W4 treatment In contrast to the W4 treatment, in the soil treated with 47o glucose incubated anaerobically (2W4), seven different VFAs were detected, acetic, propanoic, 2-methyl propanoic, butyric, pentanoic, hexanoic and octanoic acid (Figure 5.5). Butyric acid, which peaked on day 60 at a concentration of 1778 pg acid g of air-dried soil-r, was found to be the dominant type of VFA produced. The detection of high levels of butyric acid in the soil is not unexpected, because although fermentation of glucose to acetate is the most energy efficient way of producing ATp (Thauer et al., 1977 Crabbendam et aI., 1985), butyrate is often preferentially formed by fermentative bacteria such as Clostridium to reduce the total amount of acid and Hz released into their environment, which are both detrimental to their growth (Beauchamp et al., 1989). 2500 B Acetic acid I Propanoic acid â 2000 El 2-Methyl ProPanoic acid 'õ tr Butyric acid at tt I Pentanoic acid o 1 500 E Hexanoic acid It I I Octanoic acid o o Et 1000 ct) = d Ê o 500 C' l! 0 0 3 6 11 19 40 60 -500 lncubation time (daYs) Figure 5.5. Volatile fattY acid production in Watchem soil watered to 2WHC and treated with 47o glucose (w/w) over the 60-day incubation period. 104 VFA production in the 2W4 treatment began only after l1 days incubation, which was also the time (Figure 5.2) when glucose levels began to decrease. VFA production for this treatment peaked on day 60 at 2078pg acid g of air-dried soil-r (Figure 5.5). As was also shown for the W4 treatment, the standard deviations for some of the VFAs in the 2W4 treatment were quite large. This was particularly evident on day 40, where the butyricacidconcentrationineachreplicatevariedbY+54.gvo,and,ismostprobablydueto different bacterial species becoming dominant in each replicate. In stoichiometric terms, under anaerobic conditions, approximately twice as much acid should be detected in the soil as glucose used. This was not the case however, as the 15502 pg g-t soil of glucose utilised produced a cumulative total of only 4431 ¡tg acid g of air- dried soil-r of VFAs (Figures 5.5 and 5.6). Several explanations for this anomaly include: 1) some of the VFAs were volatilised into the atmosphere, 2) some of the VFAs sorbed to soil particles, so were not extractable, 3) conditions were not sufficiently anaerobic to prevent a proportion of the glucose being converted via aerobic respiration to COz and HzO (Brock and Madigan, 1991), 4) the GC- MSD was not sensitive enough to detect all of the VFAs produced, and, 5) some of the VFAs eg., acetate, were metabolised by soil microorganisms via the glyoxylate cycle (Brock and Madigan, 1991), or converted to methane (see section 5.2.4'3)' 105 CH¡CH¡COOH \h ?ropimi< ocid HrlCOr Su< co, CH¡CCX¡COOH CH¡{.€Htll¡ I il o ooH KeY role ol PYruvale ¡n A..lold.htd. Ac.læGGlic o.¡d princrpal formantations (l) Lsctic Ac.loin (Sîreptococcus, Al l*r,,t Loslic xid LactobÐciilug l2l I *nf Hl yo6sls, tew bacterrs) I | +2f Hì coholic lmany I (3) Mixed sc¡d (most Entercbac?eùa- CH¡CHOH I ¡ulyr¡c ocid CH¡CHCHCH¡ caaal. 14) ButEn€diol (Enlercbscted Etho¡ol /l ù¡lono Butyric (Closttidium) (6) Prooionic OH OH {5} a.ttont l+co2 (Propion¡bacteiluml !utonodiol lFpro?oñoll (from Figure 5.6. Metabolic path\ryays for organic acid'production from pyruvate Davis et a1.,1990). that Given the high concentration of butyric acid detected in the soil, it was predicted numbers of FAME analysis of the whole soil would detect a large increase in the increased by Clostridium*. The Clostridiummarkers 16:l ttl,ic and 18:1 c¡9c however, both of anteiso less than 37o, whichcould be attributed to random efÏor, and, the concentration subgroup A (Table l5:0, a marker found in Clostridium such as Clostridium biþrmentans 5.2d),increased by 6.4Vo after 60 days of incubation (Figure 5.7). *Although representatives from other bacteria genera such as Butyrivibrio and pseudobutyrivibrio, both from the rumen (Hespell et aI., 1993; Moore et al., 1994: Forster from the et al.,1996;Van Gylswyk et aL,1996); Porphyromonqs aîd Fusobacterium'both known to produce oral cavity of humans (Moore et aI., 1994); and Tisseriella ate also able to survive in the butyric acid from glucose, it is thought unlikely that these would be spp', watchem soil due to nutritional constraints and the high pH of the soil' clostridium (souza et al"l974:Li et al', however, have been found to grow under high pH conditions 1993). 106 following These results suggest that as nitrogen was probably limited in the Watchem soil glucose was glucose treatment, rather than being incorporated into new biomass, the be the metabolised primarily to butyric acid. Butyric acid has previously been shown to major metabolite produced in nitrogen-limited soil incubated under anaerobic conditions by Swerts et aI. (1996)' incubation FAME analysis detected no Gram-negative bacteria throughout the 60-day period (Figure 5.7), which is consistent with the finding in Chapter 2,that Gram-positive bacteria accounted for 99Vo of the bacteria isolated from the Watchem soil. 7 I15:0 lso 6 tr 15:0 antelso d I16:1 w7c o(, 5 t¡l tr 18:'l wllc ? = L 4 N 18:3 w6c .; E¡) 3 õ o c s 2 o c C'F 'l so É, 0 40 60 0 3 6 11 19 -1 lncubation time (daYs) Figure 5.7. Relative Vo change in the concentration of FAMEs representing Gram' over positive bacteria (G+)' Clostrí'dum spp, (C), and fungi (F), in the 2W4 treatment' the 60-day incubation Period. t07 5.3.5 Volatile fatty acid (VFA) and. FAME analysis of soil samples: 2W2 treatment Overall VFA production for the 2W2 treatment was found to be around half that of the 2W4 treatment. This was expected given that approximately twice as much glucose was utilised in the 2W4 treatment. Butyric acid was the dominant VFA, with the highest levels on day 40 at 851 pg acid g of air-dried soil-r , again indicating that Clostridium spp. might be involved (Figures 5.8 and 5.9). As was previously shown for the 2W4 treatment however, there was no significant increase in the level of the Clostridiummarkers 16:l a7c and l8:l anteiso 15:0 increased by only 5.7Vo after 40 days of incubation (Figure 5'9)' These results also suggest that the Watchem soil was nitrogen limited. Therefore, instead of being converted to new biomass, the carbon added to the soil was metabolised by the existing microbial population into VFAs. One further observation of note for tbe 2W2 treatment was that the variation in VFAs detected increased with increasing incubation time. For example, on day 11, when a significant level of VFA production began, only acetic and butyric acids were detected. However, on day 60, five VFAs - acetic, propionic, butyric, and hexanoic acid were detected (Figure 5.8). This change is thought to be due to a succession in Clostridium spp. Although absolute numbers of microorganisms were found to remain relatively stable throughout the 60 day incubation period (Figure 5.9), competition for available nutrients would still have soil occurred between the different species and strains of microorganisms in the Watchem (Deacon, 1984), some of which produced different VFAs' 108 For example, amongst the genus Clostridium, C. biftrmentans produces formic and acetic acid, C. propionicus produces propionic acid, and C. butyricum produces butyric acid (Brock and Madigan, tggl). This succession in the microbial population is best increased demonstrated by the FAME results between days 40 and 60, when peak 16: I oíc from 0 to 4.5Vo, and anteiso 15:0 decreased from 5.7Vo to 4.9Vo (Figure 5'9)' 1200 e Acetic acid I Propanoic acid â 1000 tr Butyric acid 'õ tr Pentanoic acid at !t I Hexanoic acid .9 800 €I (ú o ct) 600 Et d E 400 o o l! 200 0 60 0 3 6 11 19 40 Incubation time (daYs) and Figure 5.8. Volatile fatty acid Production in Watchem soil watered to 2\ilHC treated with 27o glucose (ilw) over the 60'daY incubation period. the 60-day Again, FAME analysis failed to detect any Gram-negative bacteria throughout Chapter 3, that 99vo incubation period (Figure 5.9), which is consistent with the finding in Gram-positive. of the non-filamentous bacteria isolated from the Watchem soil were 109 6 115:0 lso 5 tr 15:0 antelso d w7c tr I16:1 (,o 4 tr 18:1 wllc ? t¡¡ tr 18:3 w6c = lr 3 'õ E'l tr 2 tG' o s 1 F (, cl 0 õ 3 6 É, 0 1 -2 Incubation time (daYs) Figure 5.9. Relative 7o change in the concentration of FAMEs representing Gram' positive bacteria (G+), Clostridum spp. (C), and fungi (F), in the 2W2 treatment' over the 60-day incubation Period. treatment 5.3.6 Volatile fatty aci^d (vFA) and FAME ønalysis of soil samples: w2 The soil in the W2 treatment was watered to a moisture content of 307o (WHC), therefore, as not all soil pore spaces would be occupied by water, it was hypothesised that the majority of the glucose would be metabolised aerobically into COz and HzO' Results of the VFA analysis of the soil refuted this hypothesis however, as maximum VFA production occurred along with a rise in the relative percentage of the Clostridium marker levels of acetic 1g:1 rollc? (1g:l g air-dried soil-l and and butyric acid were detected on these days (60.83 pg acetic acid of g air-dried 2g.5 pg butyric acid g of air-dried soil-r on day 40,and 86.19pg acetic acid of respectively)' soil-r and 29.6 ¡tgbutyric acid g of air-dried soil-r on day 60, 110 These results suggest that adding 2Vo glucose (w/w) to the Watchem soil and watering it to a 30Vo moisture content created an osmotic pressure that inhibited the growth of aerobic, but not anaerobic microorganisms, most likely Clostridium spp. Such selective inhibition of the aerobic Watchem soil microbial community was observed previously in the soil treated with 4Vo glucose (w/w), watered to a 30Vo moisture level. Again, as the soil in the V/2 treatment was not expected to be totally anaerobic under the watering regime used (the soil was watered to a 30Vo moisture content), it is thought that the osmo-alkalitolerant Clostridium spp. may have established themselves in anaerobic microsites throughout the soil. This could also explain the high variation observed in the concentration of VFAs detected in the soil (as much as 1007o). 100 @ Acetic acid I Propanoic acid tr Butyricacid 80 acid 'õâ o It .g 60 tt¡ (! 340 cttt d (,520 lr 0 0 3 b 11 19 40 -20 lncubation time (daYs) Figure 5.10. Volatile fatty acid production in Watchem soil watered to WHC and treated with}Vo glucose (w/w) over the 60'day incubation period. 111 FAME analysis failed to detect any Gram-negative bacteria (represented by changes in the concentration of cyclo 17:0 and cyclo 19:0) throughout the duration of the experiment (Figure 5.11), which is consistent with the finding in Chapter 3, that 99Vo of the non- filamentous bacteria isolated from the Watchem soil were Gram-positive' 60 I l5:0 lso 50 tr 15:0 ante¡so d tr I'16:l w7c o ,i 40 tr 18:1 wl tc ? = @ l8:3 w6c l¡. .= 30 o cEl E20G o ñ o .Z 10 s c o F F É. F 0 0 3 6 11 19 40 60 -10 lncubation time (daYs) Figure 5.1L. Relative 7o change in the concentration of FAMEs representing Gram- positive bacteria (G+)' Clostridum spp. (C), and fungi (F), in the W2 treatment' over the 60-day incubation Period. Control 5.3.T Volatíle fatty acid (VFA) anit FAME analysis of soil samples:Aerobic No VFAs were detected in the aerobic control, W, throughout the 60 day incubation period. This was expected given that no additional nutrients were input into the system. Other than represent the a 507o increase in 18:1<¡l1c? (18:1ro9c) occurring at day 40, which may establishment of Clostridium spp. in anaerobic microsites throughout the soil, changes to the levels of the other marker FAMEs were all less than ZVo, and therefore, could be attributed to natural variation between the soil samples (Figure 5.I2)' 112 6.00 l'15:0 lso 5.00 tr l5:0 antelso ço o 116:1 w7c IJ 4.00 l¡¡ S l8:1 w1'lc ? = lr 3.00 I l8:3 w6c .E o c gE') 2.OO fil o s 1.00 o F s 0.00 o 0 3 6 11 19 40 60 É, -1.00 -2.00 lncubation time (daYs) Figure 5.12. Relative 7o change in the concentration of FAMEs representing Gram- positive bacteria (G+)' Clostridum spp. (C), and fungi (F), in the aerobic control, over the 60-day incubation period. Control 5.3.g Voløtile fatty acid (VFA) and FAME ønalysis of soit samples:Anaerobic VFA analysis of the anaerobic control, 2W, detected no VFA production throughout the 60 day incubation period. Again, this was expected as no additional nutrients were input into the system. Whilst the general trend of all marker FAMEs was to decrease' all of the 27o, and decreases in the relative percentage change in FAME concentration were below thus, could be the result of natural variation within the soil samples (Figure 5.13). 113 2 I15:0 lso d 1'5 tr l5:0 antelso Ê o 18:1 C' tr w'llc ? t¡¡ 118:3 w6c ¡r=l .= o P 0'5 G o :g G+ ä0 0 11 õaú É -o.s -1 lncubation time (daYs) Figure 5.1,3. Relative Vo change in the concentration of FAMEs representing Gram- positive bacteria (G+)' Clostri'dum spp. (C), and fungi (F), in the anaerobic control, over the 60-day incubation period. tt4 5.3.9 Methane analysis of the soil headspace ( results differ from those throughout the 60 day incubation period, for all treatments. These alkaline sodic soil with of Chorom (1996), who found that the incubation of a submerged production of 135 similar physical and chemical properties to the watchem soil, led to the t soil treated with pg L I methane treated with2fto glucose (w/w), and 55 pg L methane for was employed in both studies' 47o (wtw) glucose. As the same methane analysis method study is that the most probable explanation for methane only being detected in Chorom's anaerobic the submerged conditions used by Chorom (1996) produced sufficiently to a 3O7o or conditions to promote the growth of methanogens, whereas watering the soil 6OVo moisture level did not. 5.3.10 Soil pH w2 The pH of the watchem soil was observed to decrease in the 2w4, 2W2 and were significant at treatments (2.1, 1.7 and 0.35 pH units, respectively. All pH reductions pH of the V/4 treatment the 5Vo level). No significant change however, was observed in the or both controls (W and 2W) (Figure 5.14). with higher Soil pH levels were found to correlate well with the soil VFA concentration, (Figure 5'15)' soil VFA concentrarions leading to larger decreases in soil pH ß2 = 0.9128) 115 IW trw2 IW4 tr2W a2w2 tr2W4 10.00 9.50 9.00 I Él o U' 8.50 8.00 7.50 0 3 6 11 19 40 60 lncubation time (daYs) Figure 5.14. The effect of glucose-treatment on the pH of the Watchem soil over the 60-day incubation Period. 10 R2 = 0.9128 95 I I 'õ U' a I 7.5 7 21 00 0 350 7oo 1050 1400 1750 Total soil VFA conc. (ug/g of air'dried soil ) pH the Figure 5.1.5. The relationship between total soil VFA concentration and the of 'Watchem soil following glucose'treatment. A polynomial function \ryas used to fit this data as it produced the best fit. Based upon the observations of Chorom (1996), and Yan et al' (1996), the longevity of these pH decreases, brought about by the solubilisation of native soil carbonate and bicarbonate, however, are likely to be temporary. 116 (wlw) For example, Chorom (1996), found that the addition of between 2Vo and 6Vo (5l%o clay,25Vo glucose to a red alkaline sodic subsoil from Two Wells, South Australia under anaerobic sand, 24Vo silt, pH g.4, EC 0.35 dS ma , O.45Eo Carbon, 5vo caco3) 40 days, conditions led to the soil pH decreasing to approximately pH 5.5 for the first be inversely following which the pH began to increase. This pH increase was found to smaller proportional to the amount of glucose added, ie., higher glucose levels led to a increase in pH. (w/w) to the similarly, Yan et aI. (1996), found glucose, when added at a rate of 0.57o 2Vo total upper layer of a grassland soil derived from loess (pH 6, l6Vo clay,36Vo WHC, the first 4 carbon) and incubated under aerobic conditions, caused a I unit pH decrease in after days days of incubation, then the soil pH increased to its initial value of pH 6 7 incubation. soil following Such pH increases can be caused by a build up of basic organic anions in the the reaction between VFAs and soil carbonates. Shown below is the acid-carbonate reaction for butyric acid as an example: H*- Butyrate- + CaCO 3 + Ca2* + Butyrate- (basic) + COz r HzO' Following this it is likely that a further increase in soil pH occurs due to the microbial equation below' decarboxylation of these basic organic anions, which, as is shown in the et 1996)' requires protons (Helyar and Porter, 1989; Barekzai and Mengel,1993;Yan aI'' R-CO-COO- + H+ + R-CHO + COz tt7 5.3.11 Total carbonatel soluble carbonate and. Bícørbonate Soluble carbonate concentrations were observed to increase in the anaerobic treatments, l.06%o, respectively on 2W2 and 2V/4 from approximately O.25Vo on day O, to OS4EI and as as for day 60, and remained static at0.26Vo for the aerobic treatments W2 and'W4, well observed in all the controls W and 2V/ (Figure 5.16). The percentage of soluble carbonate concentration treatments was observed to correlate strongly with both the total soil VFA (Figure 5.18). (R, = Ogs3¿)(Figure 5.11),and the soil pH (R2 = 0.9281) 1.25 ¡w trw2 1.00 lw4 tr2W o (ú a2w2 L o El2W4 ¡¡ 0.75 oo 0) ¡¡ f õ 050 U' s o.25 0.00 0 3 619 40 60.00 lncubation time (daYs) Figure 5.16. The effect of glucose' treatment on the percentage of soluble carbonate present in the Watchem soil over the 60'day incubation period. 118 't.2 1 a o R2 = 0.9532 o'B Eo € a! ? 06 ¡¡ a õ 'ns o.4 a a o.2 0 1750 21 00 0 350 7oo 1050 1400 Total soil VFA conc. (ug/g of air'dried soil) Figure 5.17. The relationship between the total soil VFA concentration and the percentage of soluble carbonate present in the Watchem soil, following glucose treatment. (Combined results from W2, W4,2W2 and 2W4 treatments). A straight line function was used to fit this data as it produced the best fit' 1.2 R2 = 0.9281 o o o 0.8 Ê, o a .o oG' g 0.6 ¡t a E o Ø a s 0.4 a o a 0.2 0 9.5 't0 7 7.5 8 8.5 9 SoilpH Figure 5.18. The relationship between soil PH and the percentage of soluble carbonates present in the Watchem soil, following glucose'treatment. (Combined used to results from W2, W4,2W2 and 2W4 treatments). An exponential function was fit this data as it produced the best fit. 119 was Total carbonate levels in all treatments, however, remained unchanged at 5'67o' This the unexpected, given that it is necessary for carbonate to be consumed in order for results reductions in pH observed in the W2, 2W2, and 2W4 treatments to occur. These carbonate in suggest that the sensitivity of the method used to determine the level of total this study was not adequate. the 60 No changes were observed in the concentration of soluble bicarbonate throughout (including day incubation period, as insufficient bicarbonate was present in any soil sample the Controls) to enable detection. 120 5.3.12 Soluble Calcium or either of the No change was observed in the soluble calcium levels of the V/2 treatment, (Figure 5'19)' However, two controls (w and 2w) throughout the 60-day incubation period 483 pg ml-l soluble calcium levels were observed to increase by 387 Frg ml-l and (equivalent to 1.9 t hat and 2.4 t ha-r of applied gypsum (Rengasamy, personal (Figure similar communication)) in the 2W2 and 2'W4 treatments, respectively 5'19)' A (1989) to level of gypsum (2.24 t ha-r¡ was previously shown by Carter and Pearen horizon of a significantly reduce the sodium adsorption ratio (SAR) in the A, B, and C 40 t ha-l of glucose sodic clay for a period of 6 years. Since the equivalent of 20 that and release was applied to the soil in the 2W2 and 2W4 treatments respectively, in order to 'Watchem glucose treatment is approximat ely 2.5 t hal equivalent of gypsum into the soil, of unlikely to be cost-effective as a sole ameliorant for alkaline sodic soils. The addition lower amounts of glucose in conjunction with gypsum under waterlogged conditions further though, might be a viable alternative. It would be interesting to assess this in studies. pg ml-t Soluble calcium levels were observed to decrease by a marginally significant 53 was over the 60-day incubation period (p = 0.33) (Figure 5.19). No corresponding decrease suggests observed in the percentage of soluble carbonate for this treatment though, which that the decrease is due to errors in measurement' soil The concentration of soluble calcium was found to correlate well with both the total of soluble VFA concentration (R2 = 0.9675) (Figure 5.2o), as well as the total percentage carbonate (Rt = 0.9493) (Figure 5'21). t2t 900 E rw ó1 trw2 tr 700 o rw4 a! E2W Ê (,o .AN2 o 500 .J tr2W4 E I(! o o 300 ¡¡ õ U' 100 40 60 0 3 619 lncubation time (daYs) Figure 5.19. The effect of glucose-treatment on the percentage of soluble calcium present in the Watchem soil over the 60'day incubation period. 800 700 600 R2 = 0.9675 =E d) f 500 ç 'õ 400 a Eo o ¡t 300 a õ c, 200 100 0 1750 2100 0 350 700 1 050 1400 Total soil VFA conc. (ug/g of air'dried soil ) Figure 5.20. The relationship between the total soil VFA concentration and the level of soluble calcium Present in the Watchem soil, following glucose'treatment. (Combined results from W2, W4,2W2, and 2W4 treatments). A straight line function was used to fit this data as it produced the best fit 122 1.2 R2 = 0.9493 1 o (! Ê, 0.8 o .E¡ o.E g 0.6 ¡¡ õ U' 0.4 s o.2 0 600 700 100 200 3oo 400 500 Soluble Calcium (pg.ml { ) Figure 5.21. The relationship between the percentage of soluble carbonate and the level of soluble calcium in the Watchem soil, following glucose'treatment. (Combined was used to fit results from W2, W4, 2W2, and 2V/4 treatments). A straight line function this data as it produced the best fit. 5.3.13 Soluble Sodiutn Microbial production of VFAs not only led to an increase in soluble calcium levels in the was ZWZ and 2W4 treatments, it also led to an increase in soluble sodium levels. This pg particularly evident in the 2W2 treatment, where soluble sodium increased from 1200 ml-r to2160 pg ml-I, and in the 2W4 treatment, where soluble sodium increased from 1267 pg ml-t to 2270 pg mfr (Figure 5.22). Both increases were highly significant (p<0.001). V/hilst the majority of the sodium is likely to have come from solubilisation of sodium sodium carbonate present in the soil, a proportion of the sodium may be attributable to sodium exchanging from clay particles. No significant change was observed in the soluble levels of the W2 and W4 treatments, and both controls, which supports the previously presented data for soil carbonate (Figure 5.22)' r23 2800 TW E ó nw2 1 lw4 Ê o E2W 2300 llt a2W2 tr2W4 o cu o (,, 1800 E E o u, g tt 1300 õ tt, 800 0 3 6 19 40 60 lncubation time (daYs) Figure 5.22. Tlnre effect of glucose'treatment on the percentage of soluble sodium present in the Watchem soil over the 60'day incubation period. 5.3.14 Soíl EC (Rowell, As the EC of a soil is determined from its total concentration of soluble salts lggs),it was not surprising to observe that significant increases occurred in the EC levels of the 2w2 and2w4 treatments - 2W2 increasing from an EC of 0.723 ds m-r to 1.515 dS (Figure The m-r, and 2W4 increasing from an EC of 0.671 dS m-l to 1.891 dS m-l 5.23). (1996) increases in EC in this study are appreciably lower than those observed by Chorom for similar glucose application levels, where 2Vo and 47o glucose treatments led to (1996), increases in EC of 4.63 dS m-1, and 5.19 dS m-1, respectively. However, Chorom incubated his soil under strictly anaerobic conditions, and his experiment was conducted more glucose for a period of 320 days, compared to 60 days. Therefore, it is probable that reacted with was metabolised into VFAs (Brock and Madigan, 1991), which would have larger amounts of carbonate, releasing more cations into the soil solution' 124 and No significant changes were observed in the EC levels of the w2 and w4 treatments observed in both controls (w and 2w). This was expected as no significant changes were the levels of soluble Na* and Ca2* of these treatments (Figure 5'23). 2.2 rw 2 trw2 rw4 1.8 s2W N 2W2 .2W4 E 1.6 Øt, o 1.4 u¡ o U' 1.2 1 08 0.6 60 0 3 6 11 19 40 Incubation time (daYs) Figure 5.23. The effect of glucose'treatment on the EC of the Watchem soil over the 60-day incubation period. 5.4 Conclusion With the exception of the 4Vo ghtcose treatment incubated at a 3O7o moisture content, addition of glucose to the Watchem soil was found to lead to the production of VFAs. Little increase was observed in the number of soil microorganisms following glucose treatment of the soils, probably due to nitrogen limitation' Butyric acid was the most abundant VFA detected in the soil, followed by acetic acid. Trace amounts of propionic acid, pentanoic acid, and hexanoic acid were also detected, t25 Controls. As The amount of vFAs produced were in the order 2w4 > 2W2 > W2 > V/4 > butyric acid was the major acid produced, it suggests that clostridium spp' were results the responsible for VFA production in the soil. This is also supported by the of microbial FAME analyses. The genera of bacteria identified as acid-producers in the to population study in Chapter 2, ie., Bacillus and Arthrobacter, therefore, did not appear be important acid producers in the'Watchem soil environment. Such a finding reinforces that culturable soil microorganisms may represent a tiny, possibly ecologically unimportant portion of the overall diversity present in most soils (Cavi gelli et aL,1995)' Acids in the 2WZ and 2W4 treatments were produced at levels sufficient to solubilise -2'5 native soil carbonate, and decrease the pH, leading to a release of Ca2* equivalent to 2 t ha-l of gypsum. The extent of the pH decrease was lower than that reported by Chorom (1996) for submerged soil treated with the same levels of glucose (ZVo and 4Vo w/w), within suggesting that in this study, VFA production occurred within anaerobic microsites the soil. This would also account for the variability in VFA concentrations observed between the replicates. Whilst calcium was introduced into the soil solution following the microbial fermentation 20 t ha-r of the glucose in the 2W2 and2'W4 treatments, this required application levels of and 40 t ha-l of glucose, respectively, therefore, glucose treatment is not cost-effective when used as a sole ameliorant for alkaline sodic soils r26 CHAPTER Ó Microbial reclamation of an alkaline sodic soil using "complex" organic substrates 6.1 Introduction Watchem was In the previous chapter, addition of glucose to the alkaline sodic soil from generation volatile fatty shown to lead to changes in the soil microbial population, the of Furtherrnore, acids, and consequently, to the solubilisation of calcium carbonate in the soil. anaerobic it was established that the generation of VFAs from glucose was enhanced under conditions. (1986), Tomat et al' As was discussed in the literature review, in experiments by Swarup (1987), Gaffar et aI. (1992), and Mbagwu (lgg2), the addition of more complex organic properties of matter, eg., farmyard manure and straw, can also beneficially change the the alkaline sodic soils, including the pH and ESP. Little however, is understood about mechanisms involved behind these changes. Therefore, this chapter describes two experiments. The first of these experiments, an .'complex" combinations of evaluation of organic ameliorants, was conducted to identify sodic soil from organic matter that, when incorporated with, or applied to, the alkaline watchem, victoria, Australia,led to a decrease in the soil pH. b) The ,,complex" organic ameliorants used in this study were a) sugarcane molasses, following wheat straw, and c) sheep manure. These ameliorants were selected for the reasons and soluble a) Sugarcane molasses: Sugarcane molasses has both a high carbohydrate mineral content (Table 6.1), and was identified as having good potential to ameliorate is readily alkaline sodic soils (see the literature review - Chapter 2.4.10). Furthermore, it benefits of available in Australia. Thus, it was decided that the potential ameliorative molasses would be evaluated. PhvsicaUChemical Value pH* 5.26 EC (dS m'l)* 14.6 Moisture content (7o) 22.72 Total Carbon (Vo)s 36 Total Nitrogen (7o) 1 Soluble Calcium (Pg ml't ) 6500 Soluble Sodium (ue ml-r ) 2500 x Table 6.1. Physical and chemical properties of the molasses used in this study. pH and EC were determined from 1:5 raw molasses :water extracts. I Total carbon was determined using the rapid titration method of Walkley and Black (Allison et al-,1965) matter b) Wheat straw: Wheat straw is one of the most readily available sources of organic in dry-land farming regions. Straw contains both readily degradable nutrients, eg., sugars, (Betts et aI', as well as complex nutrients, eg., lignocellulose, cellulose and hemicellulose lgg2), and should sustain microorganisms over the long-term. Furthermore, when incorporated, straw will increase the bulk density of the soil, increase microbial polysaccharide levels and improve soil aggregate stability (Barzegat et a1.,1997l' Nelson and Oades, 1997), as well as help retain moisture' organic c) Sheep manure: Sheep manure is one of the most readily available sources of matter in dry-land farming regions, and contains high concentrations of easily degradable to contain a organic matter (Frostegard et al., l9g7). Furthermore, sheep manure is likely (Frostegard et al., large number of microorganisms that originate from the gut microflora lggT),many of which are capable of producing vFAs (Cooper and cornforth' 1978; Patni 128 may prevail in and Jui, 1985; Kirchmann and Lundwall, 1993). These microorganisms phosphate and favourable conditions in the soil and increase VFA production. Super phosphorus ammonium nitrate were also included in the study as supplementary sources of and nitrogen, resPectivelY. ameliorant One single component and one multi-component treatment identified in the soil were evaluation experiment as having good potential as amendments for alkaline sodic essentially a then selected for further investigation in the second experiment, which was 'Watchem these two small-scale amelioration of the soil. The relationship between experiments is explained more clearly in Figure 6'1' 129 ium nitrate added to Molasses, Wheat s soil pH recorded watchem soil and c' in the soil pH were on days 0, 45 and 90. Two combinations resulting in a large decrease then selected for further studY Watchem bated for 90 daYs at25o C. Soil samPles Days 0, 10, 25, 45, 65, 90. Bioloeical analvsis PhvsicaVChemical analvses FAME extract of soil to detect pH and EC changes in the microbial population Volatile fattY acid Production Soluble carbonate and bicarbonate Total carbonate Soluble cations ttcomplex" Figure 6.1. Stages in the selection of organic ameliorants. 6.2 Materials and Methods 6.2.1 Soil proPerties as those described The properties of the soil used for this experiment are the same previously in ChaPter 3.2.1 130 6,2.2 Ameliorant evaluatíon expertment 'Watchem 2009 samples of soil were combined with 36 different combinations of sugarcane molassesx, wheat straw, sheep manure, super phosphate*, and ammonium nitrate* (see Tables 6.2 - 6.8 for details of amounts added), wetted to water holding capacity using x distilled water (30vo w/w)¡, then mixed thoroughly. These components were pre- + dissolved in the distilled water used to moisten the soil to ensure even distribution. Although in the glucose experiment (Chapter 5) waterlogged conditions (2WHC) were found to enhance the microbial production of VFAs, a30Vo soil moisture content was used for this experiment, as this moisture level is more likely to be easily achieved under dryland farming conditions. As was stated in the previous chapter, the major focus of this work was to examine the microbial reactions which took place in the Watchem soil following organic amendment. For this reason, following mixing, the soil samples were placed into non-leaching pots (Chapter 5, Figure 5.1) in which the soil moisture levels were easier to maintain, and then incubated at 25oC. Again, it is acknowledged that using non-leaching pots potentially encouraged Na* ions released from soil in the upper portion of the pot to be readsorpted by soil in the lower portion of the pot. Furtheflnore, in order to gauge useful levels of .,complex" organic ameliorants in the Watchem soil, a wide range of application levels were tested. Some of these levels however, may be non-feasible at field-scale. After filling, the 216 pots were afranged in a random order across 9 rows, and incubated at pots from 25oC. All pots were watered to weight every 7 days using distilled water' Two each treatment were removed at random on days 2, 45, and 90, and their pH values (30vo served recorded as described in Chapter 5. Soil incubated with distilled water wlw) as a control. 131 ameliorants 6.2.3 Ametioratíon of the Watchem soil using "complex" organic Molasses* (50 t ha-l) lg x 2509 samples of Watchem soil were combined with either a) @)'orb)Wheatstraw(10tha-l)+Molasses*(25tha-l)+Super (Super treatment), wetted to phosphate* (100 kg hal; + Ammonium nitratex (80 kg ha-r) thoroughly' A WHC using distilled water (3OVo wlw = 75 ml per 2509 of soil), then mixed (30Vo w/w 75 ml per third set of 18 soil samples was combined with distilled water only = water used 2509 of soil) (Çq$rc!). x These components were pre-dissolved in the distilled to moisten the soil to ensure even distribution' loss and not allow The mixtures were then were placed into pots designed to limit moisture order leaching (see Figure 5.1 in Chapter 5). The 54 pots were then arranged in a random water across 3 rows, and incubated at 25oC. Pots were watered to weight using distilled testing on days every 7 days. Three pots from each treatment were removed at random for 0, 10,25,45,67 and90. Soil samples from each pot were analysed for pH, EC, soluble cation, total carbonate, their FAME water-soluble bicarbonate and carbonate levels, as well as for changes in profiles, as described in chapter 4. VFA analysis was conducted on the soil samples as of Walkley detailed in Chapter 4.2.g. Total carbon was measured according to the method and Black (Allison et a\.,1965) (below). 6.2.4 Total Carbon anøIYsis using the In the previous chapter, the microbial utilisation of glucose was monitored carbohydrates Somogyi-Nelson reducing sugars assay (Nelson, 1944)' Reducing sugars are eg', that can reduce Fehling's or Tollen's reagent. This includes all monosaccharides, 1987)' glucose, and most disaccharides except sucrose (Morrison and Boyd, 132 to measure As sucrose is the major source of carbon in molasses, it was inappropriate Therefore, carbohydrate utilisation in this experiment using the Somogyi-Nelson method' reagents are changes to the level of total carbon in the soil were monitored. The following used in this assay: water and diluted a) 1N potassium dichromate: 98.08 gK2Cr2O7 was dissolved in distilled to a final volume of 2L. distilled b) 0.5N Ferrous sulphate: 278 gFeSO¿.7HzO was dissolved in a small volume of diluted with water. To this was added 30 ml of concentrated HzSOa. The solution was then distilled water to a final volume o12L. c) o-phenanthroline indicator: 331 g o-phenanthroline monohydrate and I'74 g FeSO¿.7HzO was dissolved in 250 ml of distilled water' this Procedure: 0.5 - lg of soil sample was weighed into a 250 ml Erlenmeyer flask' To was added 10 ml of 1 N potassium dichromate solution and 20 ml of concentrated HzSO¿. After mixing for 1 minute, the solution was allowed to stand for 30 minutes. Following was added to this, 200 ml of distilled water and 10 ml of concentrated orthophosphoric acid was added to each flask. After cooling for 10 minutes, 0.5 ml of o-phenanthroline indicator solution. each flask, and the solutions were titrated against the 0.5 N ferrous sulphate potassium dichromate to which no soil had been added was used as a blank. Total carbon soil was determined using the relationship: 1 ml of dichromate utilised = 3 mg of total carbon. t33 6.2.5 Interpretation of whole soil FAME profiles FAME profiles of the control, molasses and superìfteated soils were interpreted based upon relative percentage changes to the levels of FAMEs iso 15:0, anteiso 15:0, 16:l or7c, l8:3 5.2.4.2. co6c, 18:l rogc (or 18: I crr1lc), l8:2 cúc, and cyclo l7:0 as described in Chapter 134 5.3. Results and Discussion 6, 3. 1. Ameliorant evaluation exp eriment ameliorant screening Due to the large variety of ameliorants evaluated, the results of the experiment is discussed in sub-sections as shown below: 6. 3. I. I Arn eliorant ev aluation exp eriment : C o ntrol the 90-day No significant change was observed in the pH of the control throughout duration of the experiment (Table 6.2). Tieatnrcnt pHday90 Tftatnrent Iævdt/ha incorpolatcd/ on pHday0 pHday45 soil suface 9.17 (0.01) Control None Norp e.2s (0.04) e.31(0.04) Table 6.2. The effect of distilled water on the Watchem soil PH over the 90'daY incubation period. (Values in parenthesis indicate the average deviation from the mean) 6.3.1.2 Ameliorant evaluatíon experiment: Wheat straw the Incorporation of wheat straw to the Watchem soil was found to immediately decrease soil pH, with the highest incorporation rate (50 t ha-t) leading to the largest pH reduction (1.72 units relative to the control) (Table 6.3). During incubation however, the pH of the ha-l wheat straw-treated soil was observed to increase towards the control, with the 50 t wheat straw treatment finishin gO.24 pH units higher than the control after 90 days. These results indicate that the wheat straw contained water-soluble acidic components. Upon incorporation with the soil, the wheat straw released these acidic components, thus the soil pH decreased. 135 is As the soil pH was observed to increase throughout the 90-day incubation period it being so' a suspected that the acidic components within the straw are VFAs' This proportion of the pH incrqase may be attributable to the soil microbial population reaction between decarboxylating basic organic anions remaining in the soil following the 5.3.9, to VFAs in the wheat-straw and soil carbonates. This reaction was shown in Chapter et 1996)' require protons (Helyar and Porter, 1989; Barekzai and Mengel, 1993; Yan aI" into the Additional VFAs, such as acetic acid and butyric acid, may also have been released soil by Clostridium spp. These bacteria have been shown to be able to utilise carbohydrates (Chapman et al', released during the fungal decomposition of cellulose in wheat straw IggZ).Decarboxylation of these acids would account for the remaining pH increase' Ammonification, although enhanced under alkaline conditions (Brock and Madigan, 1991), is not thought to have played a significant role in increasing the soil pH. This is because around wheat straw contains mainly carbon (typical wheat straw has a C:N ratio of 9l:1) (Hadas et al., 1998), and nitrogen becomes limited at a c:N ratios of greater than 30:1 (Haug, 1993). All available nitrogen therefore, is likely to have been immobilised by the microbial population rather than metabolised into ammonia. Treatment pH day 45 pH day 90 Treatment Level t/ha incorporated./ on pH day 0 soil surface (O.r2) e.22 (0.03) 9.16 (0.00) Wheat straw 10 incorporated 9.O4 (0.46) 8.98 (0.04) 9.16 (0.04) Wheat straw 25 incorporated 8.46 (0.03) 8.78 (0.00) 9.41 (0.43) Wheat straw 50 incorporated 7.s3 (0.00) 9.36 (0.00) 9.31 (0.04) Wheat straw l0 on soil 9.07 (0.04) 9.31 (0.02) 9.32 (0.00) Vy'heat straw 25 on soil 9.21 (0.03) 9.36 (0.04) 9.28 (0.16) Wheat straw 50 on soil 9.23 (0.04) (0.04) 9.17 (0.01) Control None None 9.2s 9.31 over the 90- Table 6.3. The effect of wheat straw treatment on the Watchem soil PH deviation from the day incubation Period. (Values in parenthesis indicate the average mean) 136 have little Application of wheat straw to the surface of the Watchem soil was found to present within effect on the soil pH (Table 6.3), possibly because the low levels of acids carbonates' the straw were unable to penetrate through the soil surface and dissolve soil 6.3.1.3 Ameliorant evaluatíon experíment: Sheep manure Treatment pH day 90 Treatment Level Uha incorporated/ on pH day 0 pH day 45 soil surface (0.04) 9.26 (0.O4) 9.2 (0.06) Sheep manure l0 incorporated 9.r6 (0.02) (0.0r) 9.24 (0.00) Sheep manure 25 incorporated 9.1s 9.ll (0.02) 9,1s (0.08) 9.16 (0.02) Sheep manure 50 incorporated 9.r7 (0.06) 9.2s (O.07) 9.26 (0.02) Sheep manure 10 on soil 9.19 (0.0s) 9.18 (0.00) 9.14 (0.07) Sheep manure 25 on soil 9.02 8.ss (0.05) Sheep manure 50 on soil e.09 (0.03) 9.1(0.01) (0.01) Control None None e.2s (0.04) e.31 (0.04) 9.17 90- Table 6.4. The effect of sheep'manure treatment on the Watchem soil pH over the day incubation period. (Values in parenthesi s indicate the average deviation from the mean). Incorporation of sheep manure into the Watchem soil was not found to have any effect on the soil pH throughour the 90-day incubation period (Table 6.4). Interestingly though, the surface application of 50 t ha-l of sheep manure appeared to decrease the pH of Watchem soil from 9.09 to 8.55 over the 90-day incubation period (Table 6.4). The most probable explanation for this is that the large mass of manure applied to the soil became 1985; anaerobic, promoting the growth of VFA-producing microorganisms (Patni and Jui, the Frostegard et al., 1997; Sorensen, 1993). These VFAs might then have leached from however, is manure into the soil, dissolving some of the soil carbonate. This pH decrease likely to be only short-term due to the accumulation of basic organic anions in the soil following the reaction between the VFAs and soil carbonates, and their subsequent protons (Helyar decarboxylation by the soil microbial population; a reaction which requires and Porter, 1989; Barekzai and Mengel,l993;Yan et al', 1996)' 137 6, 3. L4 Amelíorant evaluøtion exp erhnent : Molas s e s was not The molasses used in this experiment was shown to have a pH of 5.26, therefore, it surprising to find that molasses treatment of the Watchem soil caused an immediate reduction in soil pH, with the highest application rate of 50 t ha-r leading to the greatest (2.26 decrease in pH of 2.38 units (relative to the control pH of 9.25), followed by 25 tha-t t however, units decrease), and l0 t ha (1.37 units decrease) (Table 6.5). This pH reduction was found to be only short-term, with the soil pH for all 3 molasses application levels (Table increasing to an average of 0.09 units below the control (pH 9.17) within 90 days 6.s). These results indicate that the molasses used in this experiment also contained water- soluble acidic components, possibly VFAs. A proportion of the observed pH increase may therefore, be attributed to a build up of basic organic anions in the soil following the reaction of the acids with soil carbonates, and their subsequent decarboxylation by soil microorganisms (Helyar and Porter, 1989; Barekzai and Mengel, 1993; Yan et al., 1996)' Additionally, the organic acids may have been produced via microbial fermentation of the the sucrose within the molasses. Decarboxylation of these acids would also contribute to soil pH increase. Treatment pH day 90 Treatment Level t/ha incorporated/ on pH day 0 pH day 45 soil sur{ace 9.18 (0.04) 9.12 (0.06) Molasses l0 incorporated 7.88 (0.23) (0.18) 8.79 (0.04) 9.03 (0.10) Molasses 25 incorporated 6.99 8.72 (0.02) 9.08 (0.06) Molasses 50 incorporated 6.87 (0.00) (0.04) (0.01) Control None None 9.2s (0.04) 9.31 9.L7 Table 6.5. The effect of molasses treatment on the Watchem soil pH over the 90'daY rncubation period. (Values in parenthesis indicate the average deviation from the mean). 138 tnanure 6.3.1.5 Ameliorant evaluation experiment: Wheat straw and Sheep was found to cause an Introduction of wheat straw and sheep manure to the Watchem soil between the amount of immediate decrease in soil pH. As no conelation was observed is likely that the manure incorporated into the soil and the extent of the pH decrease, it previously in this straw was responsible for the introduction of acid. This was noted Chapter in section 6.3.1.2. 25 t ha-l of After initially decreasing the soil pH, the incorporation of both l0 t har and the soil pH toward the sheep manure with 10 t hal wheat straw was found to increase possibly wheat straw both control value of 9.17 by day 90 (Table 6.6). Sheep manure' and and contain vFAs (Cooper and cornforth, 1978; Patni and Jui, 1985; Kirchmann due to soil Lundwall, 1993). A proportion of the pH increases observed could have been the VFAs in these microorganisms decarboxylating the organic anions which remain after and Mengel, ameliorants react with soil carbonates (Helyar and Porter, 1989; Batekzai 1993;Yan et aI., 1996). t wheat straw was Interestingly, the pH of the soil treated with 50 t hal manure and 10 t ha as sheep found to continue decreasing over time (Table 6.6). This finding is unusual 1 50 ha (Table 6'4), and manure alone had no effect on the soil pH when incorporated at t incorporated at a rate lot ha- wheat straw alone increased the pH of the watchem soil when t is that the sheep manure provided extra lTable 6.3). The most likely explanation for this mixture' nitrogen to bacteria colonising anaerobic microsites within the manure/straw microsites allowing carbon in the wheat stfaw to be metabolised into VFAs' Anaerobic incorporation have previously been reported to occur in large clumps of manure following into soil by Frostegatd et aI. (1997). r39 Treatment pH 45 pH day 90 Treatment Levels Uha incorporated/ on pH day 0 day soil surface Wheat straw + (0.00) (0.02) 10, 10 incornorated 8.83 (0.0s) 9.2 9.13 Wheat straw + (0.32) (0.0s) 8.94 (0.02) Sheep manure 10,25 incorporated 8.45 9.03 Wheat straw + (0.12) (0.03) 8.42 (0.41\ SheeD manure r0, 50 incorporated 9.01 9.09 Wheat straw + (0.05) (0.01) (0.02) manure 10. l0 on soil 9.0s 9.21 9.18 Wheat straw + (0.02) 9.1 (0.13) manure 10,25 on soil 8.9s (0.1s) 9.27 Wheat straw + (0.13) (0.09) 8.92 (0.09) Sheep manure 10,50 on soil 9.34 9.r8 (0.01) Control None None 9.2s (0.04) 9.31 10.04) 9.17 Table 6.6. The effect of wheat'straw and sheep'manure treatment on the Watchem soil pH over the 90-day incubation period. (Values in parenthesis indicate the average deviation from the mean). In contrast to the incorporated sheep manure and wheat straw, incubation of the Watchem soil treated with surface applied sheep manure and wheat straw was found to decrease the soil pH (Table 6.6). Since firstly, wheat straw, when applied to the surface of the Watchem soil alone had no affect on soil pH (Table 6.3), and, secondly, the pH reductions observed on day 90 appeared to be related to the amount of manure applied to the soil, it is thought that the manure was responsible for the pH decrease. As was hypothesised earlier in this chapter, it is possible that the surface-applied manure reduced the pH of the Watchem soil because anaerobic microorganisms became established within the clumps of manure (Frostegard et at., 1997). The VFAs produced by these microorganisms possibly leached into the soil, where they dissolved soil carbonates, thus, basic decreasing the pH. This pH decrease however, is only postulated to be temporary, as organic anions would accumulate in the soil following the reaction between the VFAs and soil carbonates (Helyar and Porter, 1989; Barekzai and Mengel,I993;Yan et al., 1996)' 140 6.3.1,6 Ameliorant screening experiment: Wheat straw a.nd Molasses Treatment pH Treatment Level t/ha incorporated/ ol pH day 0 pH day 45 day 90 soil surface Wheat straw + (0.00) (0.06) Molasses 10. 10 incorporated 8.3 (0.08) 9.04 9.05 Wheat straw + (0.0s) (0.04) Molasses t0,25 incorporated 7.23 Q.O4) 8.9 8.84 Wheat straw + (0.16) (0.06) 8.6 (0.00) Molasses 10,50 incorporated 6.52 8.6 Wheat straw + (0.01) (0.15) Molasses 10, 10 on soil 8.35 (0.14) 9.17 9.09 straw + (0.04) (0.1) 1 on soil 7.58 (0.54) 8.91 9.04 Wheat straw + (0.03) (0.09) Molasses 10.50 on soil 6.89 (0.01) 8.87 8.67 Control None None 9.2s t0.04) 9.31 t0.04) 9-1710.011 Table 6.7. The effect of wheat'straw and molasses treatment on the'Watchem soil PH oyer the 90-day incubation period. (Values in parenthesis indicate the average deviation from the mean). Earlier in this chapter, the molasses and wheat straw used in this experiment were found to contain water-soluble acidic components. As expected, the incorporation of both of these (Table amendments into the Watchem soil caused an immediate decrease in the soil pH 6.7). The pH of the surface-treated soil also decreased immediately following treatment' as the molasses, being liquid, was able to penetrate into the soil (Table 6.7)' Subsequent incubation of the molasses and strarw-treated soils however, led to an increase in pH toward the control value (Table 6.7). Assuming that both the molasses and wheat straw contain organic acids eg., VFAs, some of the pH increase observed in soil treated with these ameliorants may have been due to basic organic anions building up in the soil following the reaction of the acids and soil carbonate, and their subsequent decarboxylation by soil microorganisms (Helyar and Porter, 1989; Barekzai and Mengel, 1993; Yan et al., l9e6). t4r 6.3.1.7 Amelíorant screening experiment: super phosphate, Arnmonium nitrate, Molnsses and Wheat straw Incubation of the watchem soil with super phosphate and ammonium nitrate was observed (0.13 (Table to cause a slight reduction in pH relative to the control after 90 days units) 6.g). This pH decrease is likely to have been caused by the ammonium nitrate and super phosphate relieving nitrogen, and phosphorus shortages in the soil, respectively, allowing microorganisms to mineralise more carbon and produce acid. A similar reduction in soil pH was observed following super phosphate, ammonium nitrate and wheat straw treatment of the Watchem soil (Table 6.8). Again, this pH reduction might be attributable to the ammonium nitrate and super phosphate relieving nitrogen, and phosphorus shortages in the soil, respectively, allowing microorganisms to mineralise more carbon and produce acid. Treatment pH 90 Treatment Level t/ha incorporated/ on pH day 0 pH day 45 day soil surface (0.03) 9.04 (0.02) + 0.1,0.08 incorporated 9.17 rc.02\ 9.16 Super + N[I4NO3 + (0,07) (0.03) (0.03) straw 0.1.0.08,10 incorporated 9.08 9.15 9.03 Super + N}I4NO3 + (0.31) (0.06) 9.01 (0.03) Molasses 0.1,0.08,10 incorporated 8.33 9.1 Super + N}I4NO3 + (0.04) (0.04) 0.1,0.08,25 incorporated 7.2 (O.20) 9.03 9.06 Super + N[I4NO3 + (0.41) (0.02) 8.70 (0.13) Molasses 0.1,0.08,50 incorDorated 7.s 8.8s Super + NFI+NO¡ + Molasses + Wheat (0.02) (0,04) 8.98 (0.16) straw 0.1,0.08,10,10 incorporated 7.97 8.96 Super + N[I4NO3 + Molasses + Wheat (0.04) 8.34 (0.40) straw 0.1.0.08,25,l0 incorporated 7.54 (0.r4) 8.7s Super + NFIqNO¡ + Molasses + Wheat (0.03) 8.35 (0.01) straw 0.1.0.08.s0,10 incorporated 6.41 (0.06) 8.44 (0.04) 9.17 (0.01) Control None None e.2s 9.31 10.04) Table 6.8. The effect of super phosphate' ammonium nitrate, molasses' and wheat- straw treatment on the Watchem soil PH over the 90-day incubation period. (Values in parenthesis indicate the average deviation from the mean) t42 found to lead Super phosphate, ammonium nitrate, and molasses treatment of the soil was (Table 6'8)' to an immediate decrease in soil pH, followed by a steady increase in pH Similar effects on soil pH occurred following super phosphate, ammonium nitrate, 'Watchem and molasses and straw treatment of the soil (Table 6.8). Since both molasses wheat straw were a component of these ameliorant mixtures, and both are suspected to contain VFAs, the increase in pH observed in soil treated with these ameliorant after combinations may partly be due to the decarboxylation of organic anions remaining the reaction between VFAs and soil carbonates (Helyar and Porter, 1989; Barekzai and Mengel, 1993;Yan et aI., 1996)' 6.3.1.8 Ameliorant screening experiment: Selection of ameliorants for further study After analysing the results of the ameliorant screen, two ameliorant combinations: a) r) Wheat straw (10 t ha + Molasses (25 t ha-l) + Super phosphate (100 kg ha-r; + Ammonium nitrate (80 kg ha-l), and b) Molasses, incorporated into the'Watchem soil at a rate of 50 t hal were selected for further study. These were selected for the following reasons: a) whear srraw (10 t ha-r) + Molasses (25 t ha-r¡ + super phosphate (100 kg ha-r¡ + Ammonium nitrate (S0 kg ha-r¡: The soil treated with this combination was found to have (pH the lowest pH of all of the 36 treatments tested after the 90-day incubation period 8.34' compared to control value of 9.16). b) V/heat straw ito t ha-t¡ + Molasses (50 t ha-r) + Super phosphate (100 kg ha-r; + Ammonium nitrate (g0 kg ha-l¡ was found to produce the largest initial reduction in the Watchem soil pH (2.84 units relative to the control). However, this treatment was deemed too similar to treatment a) above to warrant further investigation. t43 Molasses, incorporated at a rate of 50 t hal was therefore, selected for further study as it produced the next largest decrease in the initial pH of the Watchem soil (2.37 units relative to the control). Furthermore, as molasses contains nitrogenous compounds, and, has a high soluble mineral content (see Table 6.1), the use of molasses as a sole ameliorant, eliminates the need for fertiliser use. 144 6.3.2 Small-scale amelioration experiment 6.3.2.1 Total Carbon analYsis the Molasses-treated The concentration of total carbon was found to decline rapidly in both Super- soil (ie., molasses, combined with the Watchem soil at atate of 50 t ha-l ), and the nitrate' combined treated soil (ie., wheat Straw, molasses, Super phosphate, and ammonium with the watchem soil at rates of 10 t hal ,25 t ha-r, 100 kg ha-r , and 80 kg ha-r, 1 1'4 respectively) with total carbon in the Molasses-treated soil decreasing by 39.37o from mg C g-r air-dried soil, to 6.92 mg C g'1 air-dried soil, and total carbon in the Super- soil rreated soil falling by 3g¿o from 6.i2mg C g-1 air-dried soil to 4.16 mg C g-r air-dried within the first l0 days of incubation (Figure 6.2)' 12 Ð Control I Molasses o vt ît ¡ SuPer o ï o 'õ Eì o Eì Ê o 6 oG' G' o a 67 90 0 10 25 45 lncubation time (daYs) Figure 6.2. The effect of Molasses'treatment, Super-treatment, and distilled water (Control) on the concentration of total carbon in the Watchem soil, over the 90-daY incubation period. This finding is in contrast to the observation in Chapter 5, where utilisation of the simple 11 days incubation' carbon source, glucose, did not occur in the Watchem soil until after of The most probable explanation for this is that along with carbon, molasses contains r45 nitrogen (Table 5.1), and thus, when it was incorporated into the Watchem soil, the molasses improved the soil C:N ratio (raw molasses has a C:N ratio 36:1, the C:N ratio of the soil was 9:1). In the glucose-treated soil however, it is thought that as only carbon was introduced, the C:N ratio of the soil was increased to a level that only select microorganisms could become metabolically active. Total carbon levels in the control remained constant at 4.2 mg C g-1 air-dried soil, throughout the 90-day incubation period. 6.3.2.2 Volatile Fatty Acíd productíon and Soíl FAME ønalysis: Control As would be predicted from the results of the total carbon assay where no significant change was observed in the soil carbon level, no VFAs were detected in the Control soil throughout the 90-day incubation period. While fluctuations were observed in the concentrations of iso and anteiso 15:0 (representing Gram-positive bacteria), 17:I a7c (possibly representing Clostridium spp.), and l8:3 6c (representing fungi), these were all by less than 27o, and therefore' are postulated to be due to natural variation within the soil samples (Figure 6'3)' 146 1 115:0 lso tr 15:0 antelso o tr 05 t 16:1 w7c o o tr l8:3 w6c t¡¡ = ll 0 .= 25 45 67 90 o 0 10 cËD EGI o -0 5 s o arl o I É, -1.5 Incubation time (daYs) Figure 6.3. Relative Vo change in the concentration of FAMEs representing Gram- positive bacteria (G+)' Clostridum spp. (C), and fungi (F), in the Control, over the 90' day incubation period. 6.3.2.3 Volatíle Fatty Acid. production ønd Soil FAME analysis: Molasses treatment Volatile fatty acid (VFA) analysis of the Molasses-treated soil detected 6 types of acid propionic' throughout the 90-day incubation period, namely, acetic, propionic, 2-Methyl g buryric acid, pentanoic and hexanoic acid (Figure 6.4)' While 46.6 ¡tg acid of air-dried -l molasses (see soil of the acetic acid detected can be attributed to coming directly from the -r acid day 0 on Figure 6.4), the additional 66.6 ¡tg acid g of air-dried soil of acetic -r 46'6 (maximum acetic acid concentration = 113.2 pg acid g of air-dried soil on day 67 - -r¡, the pg acid g of air-dried soil and all of the 5 other types of VFAs detected throughout the remainder of the experiment are likely to be attributable to microbial metabolism of molasses - a hypothesis supported by the soil FAME results (Figure 6.5). 147 180 IAcetic acid 160 ¡ Propionic acid tr 2-Methyl-ProPionic acid â 140 tr Butyric acid o I Pentanoic acid ao t 120 E Hexanoic acid .9 ttI 100 'õ o 80 E) E) 60 d Ê o 40 C' l! 20 0 0 10 25 45 67 90 -20 lncubation time (daYs) Figure 6.4. Identity and concentrations of VFAs produced in the Watchem soil following Molasses treatment, over the 90-day incubation period. 250 I l5:0 antelso C 16:1 w7c qt 6 2oo 118:1 w9c o tr l8:2 w6c ¡¡¡ =lr F .E 150 o E'I tr G o 100 s o G 6, cu É. c 0 67 90 0 10 25 45 lncubation time (daYs) Figure 6.5. Relative Vo change in the concentration of FAMEs representing Gram- positive bacteria (G+)' Clostridum spp. (C), and fungi (F), in the Molasses-treatment, over the 90-day incubation period. 148 Fungal growth was found to be particularly favoured in the Molasses-treated soil, with the concentrations of l8:1 growth, a and 2O4.63Vo, respectively (Figure 6.5). During this period of rapid microbial surface succession in fungal species was observed. For the first 10 days of incubation, the of the molasses-treated soil was covered by white hyphal growth, following which fungi with yellow, grey, and pink hyphal growth became dominant. The succession in the fungal population is reflected in the FAME results in Figure 6.5, where for the first 10 days the fungal marker 18:1 <¡9c was present at a higher concentration than 18:2 a¡6c, after which 18:2 co6c increased in concentration. After the period of rapid microbial growth, numbers of fungi present in the soil were observed to diminish - with all visible traces of fungal growth disappearing between days 67 and90 (Figure 6.5). The decline in fungal growth in the Watchem soil is probably the ol9c result of a nitrogen limitation (Deacon, 1984). Furthermore, as the fungal markers 18:1 and 18:2 c¡6c were not detected in the soil after 67 days incubation (Figure 6.5), it appears that the fate of the dead fungal cells was to become a nutrient source for other soil microorganisms. 'Watchem Given that an abundance of fungal growth was observed on the surface of the soil, it was hypothesised that a large proportion of the carbon in the molasses were metabolised aerobically by fungi via the tricarboxylic acid cycle, the overall equation for which is: Pyruvate + 4NAD + FAD + 3COz + 4NADH + FADH (Brock and Madigan, 1991). r49 The results of the VFA analysis support this hypothesis, as a cumulative total of only 854 -r pg acid g of air-dried soil of VFAs were detected from the 4.5 g of carbon added to the soil via the molasses over the 90-day duration of the experiment (Figure 6.4), compared to -r produced over 60 days from 1.9 g of carbon in g226 ¡tg acid g of air-dried soil of VFAs the Watchem soil incubated with 4Vo glucose (w/w) at 2WHC, where anaerobic bacteria are thought to have dominated (see Chapter 5.3.4). The remaining carbon in the soil is thought to have been utilised by Clostridium spp. This is because butyric acid was found to increase throughout the 90-day incubation period (Figure 6.4). Ctostridium spp. were also suggested in the previous chapter to play an important role in the metabolism of glucose in the Watchem soil. 6.3.2.4 Volatile Fatty Acid production and Soil FAME analysis: Super treatment Treatment of the Watchem soil with wheat straw (10 t ha-r ), molasses (25 t ha-t ), super phosphate (100 kg ha-r), and ammonium nitrate (S0 kg har ) was also found to introduce acetic upon mixing (Figure 6.6). Given that firstly, the molasses used for both the Molasses-treatment and the Super-treatment came from the same jar, secondly, both treatments were combined with the watchem soil simultaneously, and thirdly, the level of acetic acid detected in the Super-treated soil was approximately half that of the soil treated + with 50 t ha-r Molasses (33 t 8 pg acid g of air-dried soil-r compared to 46'6 3 pg acid g -r) of air-dried soil (Figures 6.6 and 6.4), the majority of the acetic acid detected in the Super-treated soil can be attributed to coming from the molasses. A proportion of the acetic acid detected in the soil is also likely to have come from the wheat straw - possibly a by- product of microbial straw degradation. Acetic acid has previously been shown to be produced during the microbial breakdown of wheat straw under anaerobic conditions by Lynch (1977,1981b). 150 The detection of acetic acid in the Watchem soil on day 0 of the experiment thus, confirms the hypothesis presented earlier in this chapter that both the molasses and wheat straw contained VFAs. 90 6 Acetic acid 80 I Propionic acid tr Propionic acid-2 MethYl â70 tr acid 'õq, tt 60 .9 Ësoi J40o Et El \tU = cd20 o lt:10 0 10 67 90 _10 -20 Incubation time (daYs) Figure 6.6. Identity and concentrations of VFAs produced in the Watchem soil following Super-treatment, over the 90'day incubation period. Not all of the VFAs detected in the Watchem soil throughout the 90-day duration of the experiment however, were introduced via the molasses and straw, because firstly, the concentration of acetic acid almost doubled between days 0 and l0 of the experiment (Figure 6.6), and, secondly, the larger propionic, 2-methyl propionic and butyric acids were in detected (Figure 6.6). These VFAs are thought to be of microbial origin, afact reflected the FAME results (Figure 6.7). 151 140 antelso F I15:0 tr 16:1 w7c . 120 TJ 118:1 wgc (Jo tr cyclo 17:0 t¡¡ 100 ¡t= s80 o C't E60 F o s 940 g o É, 20 c c c 0 90 0 10 25 45 67 lncubation time (daYs) Figure 5.7. Relative Vo change in the concentration of FAMEs representing Gram' positive bacteria (G+)' Clostrí'dum spp. (C), and fungi (F)' in the Super'treatment, over the 90-day incubation period. Super-treatment of the V/atchem soil was found to encourage the rapid growth of fungi containing a high concentration of 18:1r¡9c (Figure 6.7). This result differs from the Molasses-treated soil, where initially, fungi containing a high concentration of 18:1 ro9c were dominant, then species containing a high concentration of 18:2 (Figure 6.5). The difference between these fungal populations may be a function of the different nutrient levels/sources added to the soil in the Super treatment, as well as due to fungi inoculated into the soil via the wheat straw growing at the expense of fungal spores already present in the soil. rapid Similarly, as was found with the Molasses-treated soil, after going through a phase of growth, fungal growth was observed to disappear from the Super-treated soil. Although compared the fungi in the Super-treated soil began to decline only after 25 days incubation, r52 decline is to 45 days incubation in the Molasses-treated soil (Figure 5.5), the reason for the thought to be similar - depletion of nutrients, in particular nitrogen. Two pieces of evidence supporting this hypothesis are, firstly, the total carbon level of the Super-treated soil was stable at 4.16 mg C g air-dried soil-r from day 10 of the experiment (Figure 6.2), and, secondly, more complex VFAs such as butyric acid (which is only thought to be produced by Clostrid.ium spp. in the alkaline Watchem soil) increased in the concentration as time progressed (Figure 6.6). The disappearance of the 18:l ol9c from soil is, therefore, postulated to be due to the decomposition of the fungi' As fungi were found to be dominant in the Super-treated soil for the majority of the experiment, it is again postulated that a large proportion of the carbon introduced into the soil would have been metabolised aerobically by fungi via the tricarboxylic acid cycle. The pg g VFA analysis results support this hypothesis, as a cumulative total of only 238.6 acid -r of air-dried soil of VFAs was detected from approximately 4.75 g of carbon added in the (Figure form of molasses and wheat straw over the 90-day duration of the experiment 6.6). Most of the remaining carbon in the Super-treated soil is thought to have been metabolised by Clostridium spp. into VFAs. This is supported by the fact that the levels of markers anteiso l5:0 and 16:l o¡7c (which are found in Clostridium species such Clostridium biftrmentans subgroup A), and the level of butyric acid were observed to increase in concentration throughout the 90-day incubation period (Figures 6'6 and 6'7)' Additionally, fermentative facultatively anaerobic Gram-negative bacteria, 08', Enterobacter and Enterococcus may have metabolised some of the carbon into VFAs, as these organisms, marked by the FAME cyclo 17:0, were detected in the soil between days 153 l0 and 90 of the experiment (Figure 6.7). As no cyclo 17:0 was detected in the Molasses- the straw. treated soil, these organisms are thought to have been introduced into the soil via Such bacteria are known to be both alkalitolerant and halotolerant (Grant and Tindall, 19g6), and are found in a wide variety of habitats from the skins and intestinal tracts of animals, to soil and water (Brock and Madigan, 1991), therefore, it is not surprising that they were able to multiply in the alkaline, sodic conditions of the Watchem soil. to The random nature in which anaerobic microsites are likely to have been created, leading both groups of microorganisms becoming established throughout the soil, might explain the variation observed in the concentrations of VFAs in the soil (Figure 6.6)' Interestingly, no VFAs were detected in the soil on day 90 of the experiment (Figure 6.6). Whether this is due to the microbial population utilising the acids, or due to a physical reason, such as volatilisation or irreversible binding to clay particles however, is unclear. 6.3.2.5 Soit púl Total Cørbonatel Soluble Carbonate and Bicarbonate Both the Super-treatment and the Molasses-treatment of the Watchem soil were found to was cause an immediate decrease in the soil pH (Figure 6.8). As no soluble carbonate detected in either of these treatments immediately following mixing, it is thought that this reduction in pH may have been the result of the acetic acid present in the molasses and wheat straw reacting with carbonate in the soil. Whilst the soluble carbonate level was observed to decrease following the Molasses- rWatchem treatment and the Super-treatment of the soil, the level of total carbonate detected in both treatments remained static throughout the experiment at 5'6Vo' This was unexpected, because it is highly likely that the VFAs introduced into the soil via the r54 molasses, wheat straw and microbial reactions would also have been able to react with the insoluble carbonate fraction within the Watchem soil. This finding supports the hypothesis in the previous chapter, that the method used to determine total carbonate in this study was low in sensitivity. No comment can be made about the role of soluble bicarbonate in this system because insufficient soluble bicarbonate was present in any sample (including the Control) to enable detection. Although the soil pH initially decreased in the Super-treated and Molasses-treated Watchem soil, the overall effect of both treatments was to increase the pH. For example, the Molasses-treated soil decreased from pH 9.58 to pH 8.37 immediately following treatment, then increased to pH 9.16 after 90 days of incubation (Figure 6.8)' Similarly, for the Super-treated soil, the soil decreased from pH 9.58 to pH 8.78 immediately following treatment, and increased to pH 9.19 (Figure 6.8). It should be noted however, that although the pH of both treatments increased over incubation time, their pH was always below the control (Figure 6.8). 10.00 E Control I Molasses ElSuPer 9.60 9.20 ! CL o U' 8.80 8.40 8.00 0 10 25 45 67 90 Incubation t¡me (days) Figure 6.8. The effect of Molasses-treatment, Super-treatment, and distilled water (Controt) on the pH of the Watchem soil, over the 90'day incubation period. 155 One mechanism that may have lead to an increase in the pH of these soils is the build-up of basic organic anions following the reaction between the VFAs and carbonate within the soil, and their subsequent decarboxylation by soil microorganisms (Helyar and Porter, 1989; Barekzai and Mengel,l993;Yan et aI., 1996). No change was observed in the pH or the percentage of soluble carbonate present in the Control. This was expected as no VFAs were detected in any sample throughout the 90-day incubation period. 6.3.2.6 Soluble Calcium The molasses used throughout this study contained 6500 pg ml-r of soluble calcium' It was therefore, calculated that 1300pg mfl (equivalent to 6.5 t ha-r of gypsum (Rengasamy, personal communication)), and 650 pg ml-r of soluble calcium (equivalent to 3.25 t ha-l of gypsum (Rengasamy, personal communication)) would be introduced to the soil solution of the Molasses-treated and Super-treated soils respectively. The Ca2* introduced into the soil solution via these treatments alone, may therefore, be sufficient to facilitate the complete removal of sodium bound to the surface of clay particles in the V/atchem soil (Carter and Pearen, 1989). With the background (control) concentration of soluble calcium being 140 pg ml-r, initial soluble calcium concentrations were predicted to be 1440 pg ml-l in the Molasses-treated soil, and 790 pg ml-t in the Super-treated soil. Initial soluble calcium concentrations for both treatments, however, were much lower than the expected values, with 617 pg ml-l of soluble calcium being recorded in the Molasses-treated soil, and 308 pg ml-r of soluble calcium being recorded in the Super-treated soil (Figure 6'9)' 156 The most probable cause for this discrepancy is that a proportion of the soluble calcium exchanged with other cationic species bound to the surface of clay particles such as sodium, or with other soil components, eg., organic matter' 700 I Control ffi SuPer 600 El Molasses E 500 ól- ^ E 400 '6 G' o 300 o ã õ 200 u, 't00 0 90 0 10 25 45 67 lncubation time (daYs) Figure 6.9. The effect of Molasses-treatment, Super-treatment, and distilled water (Cãntrol) on the concentration of soluble Ca2* in the Watchem soil, over the 90'daY incubation period. Soluble calcium levels in the Molasses-treated and Super-treated soils were not only observed to decline immediately following mixing, they continued to decline throughout the 90-day incubation period. For instance, in the Molasses-treatment, soluble calcium Super treatment, from 308 pg ml-l decreased from 617 Frg ml-l þ 361pg ml-r, and, in the to 225 pg ml-l (Figure 6.9). This is interesting because VFAs were produced in both the treatments (Figures 6.4 and 6.6), which would have solubilised calcium carbonate in soil, releasing calcium ions into the soil solution. These results suggest that the excess the calcium calcium ions introduced into the soil solution initially via the molasses, and ions introduced into the soil following the reaction between VFAs and calcium carbonate, that the were re-precipitated into calcium carbonate. This is supported by the observation r51 pH 8'5 pH of both the Super-treated soil and the Molasses-treated soil was greater than after 0 and25 days incubation, respectively the 90- Soluble calcium levels in the control remained constant at 140 pg d-r throughout the level of day incubation period. This was expected as no changes were observed in soluble carbonate. 6.3.2.7 Soluble Sodium introduced 500 Molasses, and Super treatment of the Watchem soil was calculated to have (Control) pg ml-l, and 250 pg mfr of soluble sodium respectively. With the background sodium concentration of soluble sodium of 1500 pg ml-r, the total day 0 soluble the total concentration in the Molasses-treated soil was predicted to be 2000 pg ml-l, and, 1750 pg day 0 soluble sodium concentration in the Super-treated soil was predicted to be I I in ml-r. However, soluble sodium levels of 2667 pg mf and 2375 pg mf were recorded after treatment the Molasses-treated soil and Super-treated soil respectively, immediately on day 0 (Figure 6.10). sodium The two most probable sources of the additional sodium in the soil are firstly, from such as exchanging with calcium on the surface of clay particles and other soil components the soil' organic matter, and secondly, from the dissolution of sodium carbonate within Both theories are supported by the observation that on day zeto, the measured soluble onto clay calcium levels were lower than the expected levels, ie., calcium was exchanged particles at the expense of sodium, and no soluble carbonate was detected in either the Molasses-treated soil or the Super-treated soil (Figure 6'9)' 158 No significant changes were observed in the soluble sodium levels of either the Molasses- (Figure treated soil or the Super-treated soil throughout the remainder of the experiment 6.10). The most probable explanation for this, is that the amount of calcium ions introduced into the soil via the molasses was indeed sufficient in both treatments to clay displace all of the sodium bound to the exchange sites present on the surface of particles and organic matter. Furthermore, these results suggests that an equilibrium may have been occurring between the displaced sodium ions, those released following the reaction between VFAs and carbonate in the soil, and the sodium ions being re-precipitated into sodium carbonate as the soil pH increased above pH 8'5' Soluble sodium levels remained constant in the control throughout the 90-day incubation period (Figure 6.10). Again, this was expected as no changes were observed in the level of soluble carbonate throughout the 90-day incubation period' 3500 I Control E Super B Molasses 3000 E ó l_ 2500 E .= !, o tL 2000 o õ ¿ o U' 1500 1000 90 0 10 25 45 67 lncubation time (daYs) Figure 6.10. The effect of Molasses-treatment, Super-treatment, and distilled water (Cãnhol) on the concentration of soluble Na* in the Watchem soil, over the 90-daY incubation period. 159 6.3.2.8 Soil EC values of Since high levels of soluble sodium and calcium present in the molasses, the EC the Molasses-treated soil and the Super-treated soil were observed to increase immediately following treatment from approximately 1 dS m-l (control value) to 1.85 dS m-r and l'42 dS m-r, respectively (Figure 6.11). No significant change was observed in the EC values of the Molasses-treated soil or the Super-treated soil, throughout the remaining 90 days of the experiment. These results confirm the hypothesis that the C*+ introduced into the soil via the molasses was sufficient in both treatments to displace all of the Na+ bound to the surface of clay particles and organic matter. Furthermore, these results suggest that an equilibrium reaction occurred Na* between the Ca2* and Na* introduced into the soil via the molasses, the Ca2* and introduced into the soil following the reaction between VFAs and soil carbonate, and the Ca2* and Na* being re-precipitated into carbonate as the soil pH increased' trControl 2.3 I Molasses ESuper T E I ttØ o l¡¡ 1.3 08 90 0 10 25 45 67 Incubation time (daYs) Figure 6.11. The effect of Molasses' treatment, SuPer'treatment, and distilled water (Control) on the EC of the Watchem soil, over the 90'day incubation period. 160 6.4 Conclusion The pH-reducing abilities of 36 different combinations of the "complex" organic substrates molasses, sheep manure, and wheat straw, in conjunction with super phosphate and ammonium nitrate, were evaluated after either being incorporated, or applied to the surface of the alkaline sodic subsoil from Watchem, Victoria, Australia. Two ameliorant combinations, a) molasses, combined with the Watchem soil at a rate of 50 tha'@,andb)wheatstraw,molasses,superphosphate,andammonium I I nitrate, combined with the watchem soil at rates of 10 t ha ,25 th{r, 100 kg ha , and 80 kg hal, respectively (Super treatment), were selected for further study as they were found to cause significant reductions in the soil pH. While amelioration of the Watchem soil by glucose-treatment was found to have been caused by microbiological reactions, amelioration of the Watchem soil by the "complex " organic treatments, Molasses and Super, was found to have been facilitated by the chemical properties of the ameliorants. The equivalent of 6.5 thal and 3.25 thal of gypsum was introduced into the soil solution via the molasses component of the Molasses and Super treatments, respectively, which was enough to completely displace all sodium ions bound to the surface of clay particles. Calcium ions introduced into the soil following the reaction between VFAs and soil carbonate therefore, did not appear to be important for ameliorating the V/atchem soil. The fate of these calcium ions appears to have been re-precipitation into calcium carbonate as the soil pH increased above pH 8'5' 161 Molasses-treatment and Super-treatment of the Watchem soil led to the stimulation of the soil microbial community, and the production of VFAs. Acetic acid and butyric acid were the most prevalent VFAs detected in the soil, again, suggesting the involvement of Clostridium spp. This is also supported by the results of the FAME analyses' Furthermore, pots, as considerable variation was observed in the VFA concentrations between replicate it is thought that the Clostridium spp. became established within anaerobic microsites. Where addition of glucose to the Watchem soil appeared only to stimulate the activity of Clostridium spp. within anaerobic microsites in the soil, Molasses-treatment and Super- treatment of the Watchem soil was found to favour the growth of fungi. The growth of VFA-producing bacteria (Clostridium spp.) was only observed to increase after the fungi disappeared from the soil. This is thought to be due to the fungal population of the Watchem soil rapidly utilising the nitrogen input into the system via the molasses component of both treatments, until a point that nitrogen became limited. Closffidium spp.' were then able to utilise the fungal cells as a nutrient supply, along with the remaining carbon in the soil, producing VFAs. A consequence of the majority of the carbon input into this system being metabolised aerobically by fungi was that VFA production was much lower in the "complex" organic matter-treated soils, than in the glucose-amended soil. Although the gypsum equivalent of 6.5 t ha-r and 3.25 thar of calcium was introduced into this the Watchem soil via the molasses component of the Molasses and Super-treatments, not required high application rates of 50 t ha-l and 25 f hat, respectively' Such rates are cost-effective, therefore, further research to identify levels of "complex" organic ameliorants that can ameliorate alkaline sodic soils by partly chemical, partly microbiological means is required. 162 CHAPTER 7 General Conclusion 7.1 Microbial populations within alkaline sodic soils The microbial population study presented in Chapter 3 of this thesis focussed upon a typical alkaline sodic subsoil collected from Watchem, Victoria, Australia (pH 9.6, EC I dS --t , 6.5 % CaCOù. Low numbers of microorganisms were isolated from the Watchem soil, with an average of 7 x 104, 5.56 x 10a , and 1.2 x103 colony forming units of non-filamentous bacteria, filamentous bacteria (actinomycetes), and 'Watchem fungi isolated per gam of air-dried soil, respectively. FAME characterisation of 93 non-filamentous bacterial isolates, and morphological characterisation of 49 actinomycetes and 40 ñrngi, found that the dominant species isolated from the Watchem soil were non-filamentous Gram-positive bacteria of the genus Bacillus, actinomycetes of the genus Streptomyces, and fungi of the genus Penicillium. As all of these microorganisms are spore-formers, it suggests that the microbial population inhabiting this soil may exist for long periods in a state of dormancy. This is further supported by the finding that l\Yo of the non-filamentous bacteria isolated from the Watchem soil were Arthrobacter spp., which are also recognised for their abilþ to persist in a dormant state within soils (Boylen and Ensign, 1970; Poindexter, 1981 ; Kieft, 1997). Alkalitolerance testing of 216 non-filamentous bacteria and preliminary alkalitolerance testing of 49 actinomycetes also suggest that the microorganisms inhabiting the Watchem soil may be highly adapted to their high pH environment (pH 9.6), with 96Yo of thenon-filamentous bacteria, and70% of the actinomycetes tested, being able to grow on agar between pH 9 and pH 12. Further screening of more actinomycetes isolated on agar media with high pH and NaCl levels, however, is required before direct comparisons can be made between this population, and the non-filamentous bacterial population of the Watchem soil' Whilst ubiquitously atkalitolerant, halotolerance of the non-filamentous bacterial isolates was found to be dependent upon the concentration of NaCl used in the medium on which they were first isolated. For example, 62%o of the bacteria isolated on half-strength nutrient agar containing 1.5 M NaCl (Í,H 7 .4), tolerated 2 M NaCl (ç,H 7.4), compared to 22Yo, and 39% of the bacteria isolated on media containing 0.02 M NaCl at pH 7 and pH 11, respectively. Tolerance to high concentrations of NaCl at a high pH was also found to be influenced by the NaCl concentration of the initial isolation medium, with72% of the non-filamentous bacteria isolated on l.5M NaCl agar þH 7.4) being able to grow on half-strength nutrient agar containing lM NaCl adjusted to pH 11, and only 27%o md 42% of non-filamentous bacteria originating from pH 11 and pH 7 nutrient agar (both containing 0.02M Nacl) respectively, being able to gro\ / on this medium. These differences in halotolerance were determined to be the result of different species being isolated on each medium. t64 Preliminary halotolerance screening of the 49 actinomycete isolates suggested that this population of the \tr/atchem soil was considerably less halotolerant than the non- filamentous bacterial population, with only l7o/o of the isolates tested being capable of growing on agar containing 1.5M NaCl. No isolates were capable of growth on media containing lM NaCl adjusted to pH 11. Again, further screening of actinomycetes isolated on agar media with high pH and NaCl levels is required before direct comparisons can be made between this population and the non- filamentous bacterial population of the Watchem soil' Although the alkalitolerant and halotolerant properties of the bacteria isolated from the Watchem soil was assessed, no work was conducted on characterising the mechanisms behind these properties. Such research would be useful, because the unique growth characteristics of these microorganisms indicate that they may have evolved unusual metabolic capabilities and therefore, could be a source of novel, industrially important, secondary metabolites and enzymes. For these reasons, it would also be interesting to assess the alkalitolerant and halotolerant properties of individual frrngi isolated from the Watchem soil. The results of the acid-production assay showed thatT}Yo of the identified, and l2Yo 'Watchem of the unidentified non-filamentous bacteria isolated from the soil were able to produce acid from glucose. Interestingly, none of the bacteria identified as MicrococcT^s spp. were found to produce acid from glucose, despite the fact that they are known to possess the metabolic pathways to do so (Bergey, 1984). 165 It would therefore, be worthwhile investigating whether the inability of the Micrococcys spp. isolated from the Watchem soil to produce acid from glucose was the result of essential nutrients not being present in the acid-production mediuln' or alternatively, whether this population has evolved unusual metabolic pathways in the high pH Watchem soil environment. FAME analysis of 162 non-filamentous bacteria isolated from the Watchem soil led to the species-level identification of only 76%o, with database limitations preventing characterisation of the remainng 24%. Since this is almost one quarter of the bacterial isolates, it may be worthwhile in future studies to try identiffing the ,1nknown" bacteria using 165 rRNA profiling, though this technique too, is limited by the ability to match the "unknown" isolates to bacteria previously described on a database (Pace et al., 1986; Amann et a1.,1996). Furthermore, 165 rRNA profiling may be useful in identiffing the filamentous bacterial isolates and fungal isolates which could not be classified based upon their morphological characteristics. 7.9 Microbial reclamation of alkaline sodic soils The second major focus of this thesis was to compare the microbial processes leading to acid production in alkaline sodic soils treated with glucose (Chapter 5), and soils treated with more "complex" organic materials such as sheep manure, molasses, and wheat straw (Chapter 6). Furthermore, the impact of microbially-synthesised acids on the physical and chemical properties of alkaline sodic soils was investigated (Chapters 5 and 6). 166 These experiments were conducted using the typical alkaline sodic subsoil collected o/o from Watchem, Victoria, Australia (pH g.6,EC I dS m-l , 6'5 CaCO3)' Both glucose, and "complex" organic substrate treatment of the Watchem soil ie., t) molasses (50 t ha-l), and molasses (25 t ha-t) * wheat straw (10 t ha * super phosphate (100 kg har¡ + ammonium nitrate (S0 kg hal), were found to lead to the production of VFAs. However, while the glucose was utilised slowly, with no glucose metabolism being detected in the soil until after 11 days of incubation, metabolism of the "complex" ameliorants occurred rapidly, with approximately 40Yo of the carbon substrate being utilised within the fust 10 days of incubation. These different rates of substrate utilisation can be attributed to the glucose and ,,complex,' ameliorants stimulating the gfowth and activþ of dif[erent microbial populations within the Watchem soil. For example, in the soil treated with .,complex" ameliorants, nitrogen was introduced into the soil with the molasses component of the ameliorants (the molasses used in the study contained 1olo nitrogen (dÐ), producing a C:N ratio in the soil that was favotrable for the rapid growth of firngi. VFA-producing bacteria were not detected in the soil r¡ntil aftet 67 days of incubation, when nitrogen was likely to have been limited. However, in the glucose treated soil, only VFA-producing anaerobic bacteria were observed to become active. A further factor affecting glucose metabolism in the Watchem soil, is thought to be osmotic pressure, especially in the soil treated with 4%o glucose (w/w), and watered to a30%o moisture content (WHC). r67 In this treatment, FAME analysis showed an increase in microbial numbers (represented by an increase in the Clostridium marker 18:1al9c), but no significant with decrease was observed in the concentration of glucose. In contrast' soil treated the same level of glucose (4o/o wlw), but watered to a 600lo moisture content, showed an83%o reduction in glucose concentration. Given that more carbon source was metabolised aerobically by fungi in the soil treated with the "complex" organic substrates, than anaerobically by bacteria, lower concentrations of VFAs were detected than in the soil treated with glucose' Whilst the concentration of VFAs differed in the soil treated with glucose and the soil treated with "complex" organic substrates, the major VFAs detected, however' were the same - acetic and butyric acid. The detection of these acids suggests that Clostridium spp. \ilere responsible for VFA production in both systems. This hypothesis is supported by the results of the FAME analyses performed on whole soil samples, where increases were observed in the concentrations of the fatty acids l6:lø'lc, and l8:1co9c. Although l6:1co7c is also fowrd in Gram-negative aerobes such as pseudomonas, and 18:lal9c is also found in Gram-positive bacteria such as Corynebacterium,l6:1co7c and l8:1or9c were deemed to represent Clostridium spp. in the Watchem soil because firstly, acetic and butyric acid were detected in the soil in conjunction with an increase in these fatty acids, and, secondly, after considering was other soil parameters, eg., the pH and the potential oxygen level of the soil, it thought that the soil conditions were unfavourable for the growth of the other microorganisms. 168 profiles These results higlìlight that it is often diffrcult to interpret whole soil FAME due to different microorganisms possessing common fatty acids (Zelles et al., 1992; Cavigelli et al., 1995; Wander et a1.,1995). This study however, demonstrated that such diffrculties in interpretation could be partly ovefcome by considering the results of other analyses eg., vFA analysis, and the physical and chemical properties of the soil. When the V/atchem soil was watered to a 600/o moisture content (waterlogged conditions), glucose treatment was found to produce suffrcient levels of VFAs to decrease the soil pH and solubilise native calcium carbonate. These VFA-carbonate reactions were found to lead to the release of 387 pg mrr c** ¡the soil teated with 2% (wlw) glucose, and 483 pg m[r C** nthe soil teated with 4 % (wlw) glucose, I which is equivalent to 1.9 t ha and 2.4 thdr of applied gypsum, respectively. These results confirm that the reductions in soil pH and soluble carbonate, as well as an and increases in soluble Ca2* and EC observed by Chorom (1996), and Chorom were Rengasamy (lgg7) following the glucose treatment of an alkaline sodic soil, fatty indeed, the result of soil microorganisms metabolising the glucose into volatile observed acids (VFAs), and subsequent acid-carbonate reactions. The pH decreases by in the glucose-amended soil in this study however, are lower than those reported (2Yo and Chorom (1996) for submerged soil treated with the same levels of glucose anaerobic 4Yo wlw). This suggests that vFA production probably occurred within microsites distributed throughout the Watchem soil' r69 This is further supported by the observations that soil VFA concentrations were found to vary widely between replicate pots, and no significant increase in soluble calcium levels occcurred in the glucose-treated soil watered to a 30Yo moisture content, where fewer anaerobic microsites are likely to have formed. Whilst microbial reclamation of alkaline sodic soils using glucose as a substrate was shown to be theoretically possible, at the levels that were effective, it would require I the application of between 20 to 40 t ha glucose. Therefore, the use of glucose as a sole ameliorant for alkaline sodic soils is not economically viable. The application of smaller amounts of glucose in conjunction with gypsum, followed by incubation under waterlogged (totally anaerobic) conditions, however, might be a viable alternative worth assessing in future studies. Under such conditions, VFA-production would be increased significantly, therefore, partial amelioration of the soil would be achieved via the C** introduced with the gypsum, following which, partial amelioration would be achieved via microbial reactions. Further characterisation of the growth requirements of the VFA-productng Clostridium-like bacteria detected in this study may also be of benefit, as it may reveal potential avenues to promote their activity in soil, and thus, improve the efficacy of vFA production. In contrast to the Watchem soil amended with glucose, where improvements to soil physical and chemical properties were found to have been facilitated by microbial reactions, amelioration of the Watchem soil treated with "complex" organic substrates was found to have been facilitated by the Ca2* and acetic acid present in the molasses and wheat straw. 170 -r A total of l300pg ml-r of Ca2* and 46.6 pg of acetic acid g of air-dried soil was introduced in the Watchem soil immediately following treatment with molasses at a -r rate of 50 t ha-r, and 650 Frg ml'l of C** and 33 pg of acetic acid g of air-dried soil was introduced into the Watchem soil immediately following treatment with t) molasses (25 t ha * wheat straw (10 t ha-t) * super phosphate (100 kg har; + ammonium nitrate (80 kg hat). Thus, enough Ca2* and acetic acid was introduced in both treatments to completely displace all sodium ions bound to the surface of clay particles, as well as decrease the soil pH. The VFAs produced in the Watchem soil during the metabolism of the "complex" organic substrates, are therefore, thought to have played no significant part in ameliorating the Watchem soil. Instead, these acids are thought to have become involved in the complex 3 part equilibrium, shown below: Reaction 1) VFAs + CaCO¡ + Organic anions (eg., butyrate from butyric acid) + c** , Reaction 2) The organic anions from the above acid-carbonate reaction are then decarboxylated by soil microorganisms, increasing the soil pH (Helyar and Porter, 1989; Barekzai and Mengel,l993;Yan et al., 1996). Reaction 3) The Ca2* released in Reaction 1) is then re-precipitated into CaCO¡ after the soil pH increases above pH 8.5 r7l Although the equivalent of 6.5 t ha'r arrd 3.25 t ha-r of gypsum was introduced into the Watchem soil via the molasses component of the "complex" organic ameliorants investigated in this study, this required large amounts of substrate*, and is therefore, unlikely to be viable on a field-scale level. * It should be noted that in the inital "complex" amendment screening trial, lower levels of organic ameliorants failed to significantly decrease the soil pH (see Chapter 6). Given the high Ca2* and carbon content of molasses though, it may be worthwhile investigating the use of low levels of molasses, incubated under waterlogged conditions. This method may enable amelioration of alkaline sodic soils to be achieved through a combination of chemical reactions, facilitated by the C*t and acetic acid present in the molasses, and microbial reactions, which would be enhanced under the waterlogged (anaerobic) conditions. In conclusion, the studies presented in this thesis revealed that a significant portion of culturable microorganisms inhabiting alkaline sodic soils exhibit a high degree of adaptation to their environment, ie., they are highly alkalitolerant, and many are halotolerant. Furthermore, it was found that the populations within these soils could be stimulated to produce acid via the addition of both simple and complex carbon sources, leading to the solubilisation of calcium carbonate, and the introduction of calcium ions into the soil solution. 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Determination of phospholipid- and lipopolysaccharide-derived fatty acids as an estimate of microbial biomass and community structures in soils. Biol. Fertil. Soils 19: ll5-123. Zogg, G.P., Zak, D.R., Ringelberg, D.B., MacDonald, N.W., Prgitze¡ K.S., and White, D.C. (1997).Compositional and fi,¡nctional shifts in microbial communities due to soil warming. Soil Sci. Am. J. 6I:475-481. 207 Appendix 1 Microbiological growth media and solutions Lemco L Acid production broth: 2.5g Glucose, 49 Bacteriological peptone, 4E [¿b I distilled water, PH7. glycerophosphate, 0'019 Czapeks Dox agar: 29 Nal.[O¡, 0.5g KCl, 0.5g Magnesium water, pH 6.8. FezSO¿ , 0.359 KzSO¿, 30g sucros e, L2g agat |Ldistilled 0'5g Ilumic acid- vitamin (tIvA) agar: 1g Humic acid (dissolved in l0ml 0.1M NaOþ' solution, l5g agarl NazHPO¿, l.7lg KCl,0.059 MgSO¿,0.019 FeSO¿, 10ml Vitamin 980m1distilled water, PH 7.5. solids, Modified Brain-Heart infusion agar: 12.59 Calf brain solids, 59 Beef infusion Yeast extract, t0g agar tL 10g Proteose peptone, 59 NaCl, 29 Glucose,2.5gNa2POl , 59 distilled water, PH7.4. NaCl, l5g agar lL U2 Strength Nutrient agar: 0.5 g Lab I-emco, 1g Yeast extract, 2'5g distilled water, pH adjusted as appropriate with NaOH' agar /L distilled water, oatmeal agar: 20g Rolled oats, loml Trace element solution, l5g pH7.5, 20g Glucose 15g agar IL distilled 1/2 Strength Potato Dextrose agar: 49 Potato extract, water, pH 5.6. 208 pH7 Saline solution: 99 NaCl /L distilled \ryater' '4' Project Agar 4): 10g starch' 29CaCO3' starch salts agar (fnternational streptomycete 2g(NfI¿)zSO¿,lgKzHPO¿,lgMgSOa'lgNaCl'lmgFeSO¿'lmgMnClz'lmgZnSO¿' 15g agar /L distitled water, PH 7'3' / 100m1 2tmgZnSO¿, 2}mgcoclz' 40mg MnSO¿ Trace element solution: 70mg cuso¿, distilled water (100x Stock)' Trypticsoyabrothagar([SBA):30gTSB@ifco)'l5gagarlLdistilledwater'pH7'3 5mg Riboflavin, 5mg Nicotinic acid' Vitamin solution for r{vA: 5mg Thiamine-HCl, acid' ca-pantothenate' 5mg p-aminobenzoic 5mg Pyridoxine-HCl, 5mg Inositiol, 5mg 2.Smgd-biotin / 100m1 distilled water"- 15g agar Malt extract,49 Glucose' 49 Yeast extract' Yeast malt-extract (YME) agar: 10g /L distilled water, PH7 '3' 209 Appendix2 FAME protocol for individual bacterial isolates The ç¡¡adn¡t S¡:€at pattem is rccommended for culturin{ cells on plates for idcntification by the lfls. This stseatinÍ pattem results in ample material for uralysis while confìrming thc presence of a single c¡lony type or pure culture. Puri Chec (i ndi vi dual col oni es) F'lame Here I select a well-lsohted colony from the primary isohtion platÈ the I St€ritize urd coot your inoculartion loop. The loop can be cooled by plunging it into agar in the phte in an area without ury cell coloniæ' ¡ With the sterile inoculation loop, tnnsfer the colony to the IrfISi phte. Spread the colony over the area of quaaranf i touching the entire ring of the loop to the medh so that the rc$on is heavily inoculated. / the ¡ Inocr¡lat¿ quadrant 2 by rotating the loop 90o urd passing the loop edgøthrough ã-¿¡ of quadrurt I twicq ar ãho*tt in ttr. above drawing. Then stseak the rest of quadrant 2 with panllel lines without reentering quadrant 1. the ¡ Stefiliz¿ urd coolyour inoculation loop. The loop can be cooled by plunEin$ it inÛo agar in the plate in an a¡ea without ury cell colonies. the r With the ste¡ile loop, inocr¡late quadrant 3 by passing one edge of the loop through .oã* of quadnr¡l 2 tuice Then streak. th. roiof quadrant 3 with panllcl lincs without reentering quadrant 2:--- loop ¡ Inoculate quadnnt 4 by roäting th. toop 180' urd passing the other edge of the panllel througþ the comer of quadrant 3 twice. then s¡eak the reJt of guadrurt 4 with lincs without reentering quadrant 3. The most st¡ble faUy a{d compositions üe from cultures in the late loE phasa The ltflS l¡U.-y has been deväloped by sàlecting incubation conditions that are most favor¿ble for a m.joåty of microorg-Lms. The following standard culture conditions have been used for most oi the libr¿ry ãnuies. Exceptions a¡e not¿d in the library listinEs in Section 12. Use a small, high-quality incubator in which gowth conditions car¡ be contsolled. Use of a larger incubator that is shared with other users increases the risk of not maintaining proper as very low levels of these -ñd¡tionr. Do not teave ury disinfecting agents in the incubator chemicals in the atmosphere can ret¡rd the growth of organisms on aEar plates. Aerobe Incr¡batio¡ tt¡e stan¿ar¿ incr¡bation conditions for aerobes (ISBA) ue: t 28 È l"C temperature I 24 t 2 houn time The clinical aerobes (CLnÐ require incubation at 35 t l"C. Slore-Gruwin¡ Oñanisn¡ @le,thefäHyacidcompositionsofpre-logphaseculturesmaynotqm be quurtitatively repioducible. Analyses of 24-hour culturæ of slow-growing isolates t..oìt in low ririt tity index values or misidentification. For accurate speciation of such cultures, extended incubation is necessary to obtain quantitative reproducibility. I To analyze and identify those isolates that in the normal'time do not yield enough growth in quadrant 3 for processing ¡ Harvest and process colonies from quadrurts 2 or 1. Use report as a preliminary identification. r Continue to incubate tþe cr¡lh¡re forur additional period of time until confluent growth is obtained. I Afþr additional incr¡bation, hanrestand process colonies from quadrant 3 to confirm the earlier iden tifi cation. Fastidior¡s Or(urisns N@canbeculturedwiththesh¡da¡dconditions.Fastidiousorganisms may require *ti.tr.¿ media or specific atmoçheric conditions. The approprhte culh¡re conditions for these orgurisms are listed ncxt !o the enEiæ in thp St¡ndafd Ubr¿ry listings. The IrfIS can only identis these orgurisms if you r¡sc the indicated cr¡lture conditions. Closely related groups of fastidious orgurlsms have been incubated under the same culture generation condiúons for generation of the libr¿ry-ø entries. The conditions used for library of fastidious orgurlsms will be similar ttt*. conditions necess¡ry for prima¡y isohtion of the sr¡lture. r.--'- 2\0 Preparlng Extracts - Flve Steps }IART'ESÎING Third Qurdr¡nt 4 n LooP Coat Eottm SAPONIFICAT|ON Add 1.0 ¡l Yortex 100 c -Yortex 100'c Reagcnt 11 5-10 scc 5 EI n 5-10 scc 25 ¡in cool t METIx¡.¡--TION O Add 2.0 ¡l Yortcx 80 i 1'C 10 t I ¡in Rcagent 12 5-10 scc Cool R¡pidly (*; 3' (:r:n *^ /St"..¡ ÐffRAGNON r Add 1.25 rl l0 rin Rclove S¡vc Reagent 13 Eotto¡ Phase Top Phasc -f /¡ *¡^1. TYASH d E Add 3.0 ¡l 5 rin Ræve 2/3 - Trcn¡fer C¡P Rcagent 14 Top Phrse to GC Yi¡ì 2 I