Evaluation of acidogenesis products’ effect on biogas production performed with metagenomics and isotopic approaches

Abstract Background During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system. Results Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities. Conclusions In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock.

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Structure and function of the bacterial and fungal gut flora of Neotropical butterflies

10.1101/128884 ◽

2017 ◽

Cited By ~ 3

Author(s):

Alison Ravenscraft ◽

Michelle Berry ◽

Tobin Hammer ◽

Kabir Peay ◽

Carol Boggs

Keyword(s):

Amino Acids ◽

Microbial Communities ◽

Bacterial Species ◽

Microbial Community Composition ◽

Structure And Function ◽

Feeding Guilds ◽

Gut Flora ◽

Microbial Composition ◽

Host Diet ◽

And Function

AbstractThe relationship between animals and their gut flora is simultaneously one of the most common and most complex symbioses on Earth. Despite its ubiquity, our understanding of this invisible but often critical relationship is still in its infancy. We employed adult Neotropical butterflies as a study system to ask three questions: First, how does gut microbial community composition vary across host individuals, species and dietary guilds? Second, how do gut flora compare to food microbial communities? Finally, are gut flora functionally adapted to the chemical makeup of host foods? To answer these questions we captured nearly 300 Costa Rican butterflies representing over 50 species, six families and two feeding guilds: frugivores and nectivores. We characterized the bacteria and fungi in guts, wild fruits and wild nectars via amplicon sequencing and assessed the catabolic abilities of the gut flora via culture-based assays.Gut communities were distinct from food communities, suggesting that the gut environment acts as a strong filter on potential colonists. Nevertheless, gut flora varied widely among individuals and species. On average, a pair of butterflies shared 21% of their bacterial species and 6% of their fungi. Host species explained 25-30% of total variation in microbial communities while host diet explained 4%. However, diet was still relevant at the individual microbe level—half of the most abundant microbial species differed in abundance between frugivores and nectivores. Diet was also related to the functional profile of gut flora: compared to frugivores, nectivores’ gut flora exhibited increased catabolism of sugars and sugar alcohols and decreased catabolism of amino acids, carboxylic acids and dicarboxylic acids. Since fermented juice contains more amino acids and less sugar than nectar, it appears that host diet filters the gut flora by favoring microbes that digest compounds abundant in foods.By quantifying the degree to which gut communities vary among host individuals, species and dietary guilds and evaluating how gut microbial composition and catabolic potential are related to host diet, this study deepens our understanding of the structure and function of one of the most complex and ubiquitous symbioses in the animal kingdom.

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Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel

BioMed Research International ◽

10.1155/2015/932934 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 18

Author(s):

C. Varrone ◽

T. M. B. Heggeset ◽

S. B. Le ◽

T. Haugen ◽

S. Markussen ◽

...

Keyword(s):

Crude Glycerol ◽

Degradation Efficiency ◽

Anaerobic Sludge ◽

Growth Media ◽

Fed Batch ◽

Microbial Composition ◽

Fermentation Products ◽

Substrate Degradation ◽

Microbial Cultures ◽

Mixed Microbial Cultures

Objective of this study was the selection and adaptation of mixed microbial cultures (MMCs), able to ferment crude glycerol generated from animal fat-based biodiesel and produce building-blocks and green chemicals. Various adaptation strategies have been investigated for the enrichment of suitable and stable MMC, trying to overcome inhibition problems and enhance substrate degradation efficiency, as well as generation of soluble fermentation products. Repeated transfers in small batches and fed-batch conditions have been applied, comparing the use of different inoculum, growth media, and Kinetic Control. The adaptation of activated sludge inoculum was performed successfully and continued unhindered for several months. The best results showed a substrate degradation efficiency of almost 100% (about 10 g/L glycerol in 21 h) and different dominant metabolic products were obtained, depending on the selection strategy (mainly 1,3-propanediol, ethanol, or butyrate). On the other hand, anaerobic sludge exhibited inactivation after a few transfers. To circumvent this problem, fed-batch mode was used as an alternative adaptation strategy, which led to effective substrate degradation and high 1,3-propanediol and butyrate production. Changes in microbial composition were monitored by means of Next Generation Sequencing, revealing a dominance of glycerol consuming species, such asClostridium,Klebsiella, andEscherichia.

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Effects of phosphorus removal chemicals upon methane production during anaerobic sludge digestion

Canadian Journal of Civil Engineering ◽

10.1139/l86-005 ◽

1986 ◽

Vol 13 (1) ◽

pp. 33-38 ◽

Cited By ~ 6

Author(s):

Warren B. Kindzierski ◽

Steve E. Hrudey

Keyword(s):

Anaerobic Digestion ◽

Ferric Chloride ◽

Methane Production ◽

Phosphorus Removal ◽

Anaerobic Sludge ◽

Methane Formation ◽

Chemical Toxicity ◽

Methane Generation ◽

Sludge Digestion ◽

Volatile Solids

Aluminum sulphate (alum) and ferric chloride are commonly employed to aid phosphorus removal in wastewater treatment. Previous studies have indicated that these chemical coagulants produce sludges that adversely affect anaerobic digestion. The objective of this study was to assess the magnitude of the effects chemical coagulants have upon methane generation in anaerobic digestion. Methane production was monitored and concentrations of aluminum or iron present during batch digestion of chemically precipitated sludge were measured.Both alum and ferric chloride addition to activated sludge produced a sludge that demonstrated reduced methane production in batch anaerobic digestion. Neither metal inhibited methanogenesis of an acetate supplement, suggesting that chemical toxicity was not a likely explanation for overall reductions in methane formation. Considering the experimental results and the findings of others, reduced methane generation is most likely caused by physical isolation of degradable substrate by the coagulant floc, which causes an overall reduction in conversion of sludge volatile solids to methane.

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Shifts in microbiota species and fermentation products in a dietary model enriched in fat and sucrose

Beneficial Microbes ◽

10.3920/bm2013.0097 ◽

2015 ◽

Vol 6 (1) ◽

pp. 97-111 ◽

Cited By ~ 23

Author(s):

U. Etxeberria ◽

N. Arias ◽

N. Boqué ◽

M.T. Macarulla ◽

M.P. Portillo ◽

...

Keyword(s):

Gut Microbiota ◽

Bacterial Species ◽

Intestinal Barrier ◽

Short Chain Fatty Acids ◽

Gas Chromatography Mass Spectrometry ◽

Intestinal Barrier Function ◽

Microbial Composition ◽

Fermentation Products ◽

Inflammatory Status ◽

Male Wistar Rats

The gastrointestinal tract harbours a ‘superorganism’ called the gut microbiota, which is known to play a crucial role in the onset and development of diverse diseases. This internal ecosystem, far from being a static environment, can be manipulated by diet and dietary components. Feeding animals with high-fat sucrose (HFS) diets entails diet-induced obesity, a model which is usually used in research to mimic the obese phenotype of Western societies. The aim of the present study was to identify gut microbiota dysbiosis and associated metabolic changes produced in male Wistar rats fed a HFS diet for 6 weeks and compare it with the basal microbial composition. For this purpose, DNA extracted from faeces at baseline and after treatment was analysed by amplification of the V4-V6 region of the 16S ribosomal DNA (rDNA) gene using 454 pyrosequencing. Short-chain fatty acids, i.e. acetate, propionate and butyrate, were also evaluated by gas chromatography-mass spectrometry. At the end of the treatment, gut microbiota composition significantly differed at phylum level (Firmicutes, Bacteroidetes and Proteobacteria) and class level (Erisypelotrichi, Deltaproteobacteria, Bacteroidia and Bacilli). Interestingly, the class Clostridia showed a significant decrease after HFS diet treatment, which correlated with visceral adipose tissue, and is likely mediated by dietary carbohydrates. Of particular interest, Clostridium cluster XIVa species were significantly reduced and changes were identified in the relative abundance of other specific bacterial species (Mitsuokella jalaludinii, Eubacterium ventriosum, Clostridium sp. FCB90-3, Prevotella nanceiensis, Clostridium fusiformis, Clostridium sp. BNL1100 and Eubacterium cylindroides) that, in some cases, showed opposite trends to their relative families. These results highlight the relevance of characterising gut microbial population differences at species level and contribute to understand the plausible link between diet and specific gut bacterial species that are able to influence the inflammatory status, intestinal barrier function and obesity development.

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Microbial Communities and Physicochemical Characteristics of Traditional Dajiang and Sufu in North China Revealed by High-Throughput Sequencing of 16S rRNA

Frontiers in Microbiology ◽

10.3389/fmicb.2021.665243 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tingting Liang ◽

Xinqiang Xie ◽

Jun Ma ◽

Lei Wu ◽

Yu Xi ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Microbial Communities ◽

Malic Acid ◽

Bacterial Communities ◽

North China ◽

Bacterial Species ◽

Microbial Community Composition ◽

Microbial Composition ◽

Bacterial Phyla

The process of soybean fermentation has been practiced for more than 3,000 years. Although Dajiang and Sufu are two popular fermented soybean products consumed in North China, limited information is available regarding their microbial composition. Hence, the current study sought to investigate, and compare, the physicochemical indicators and microbial communities of traditional Dajiang and Sufu. Results showed that the titratable acidity (TA), and salinity, as well as the lactic acid, and malic acid contents were significantly higher in Sufu samples compared to Dajiang. Furthermore, Sufu samples contain abundant sucrose and fructose, while the acetic acid content was lower in Sufu compared to Dajiang samples. Moreover, the predominant bacterial phyla in Dajiang and Sufu samples were Firmicutes and Proteobacteria, while the major genera comprise Bacillus, Lactobacillus, Tetragenococcus, and Weissella. Moreover, Dajiang samples also contained abundant Pseudomonas, and Brevundimonas spp., while Halomonas, Staphylococcus, Lysinibacillus, Enterobacter, Streptococcus, Acinetobacter, and Halanaerobium spp. were abundant in Sufu samples. At the species level, Bacillus velezensis, Tetragenococcus halophilus, Lactobacillus rennini, Weissella cibaria, Weissella viridescens, Pseudomonas brenneri, and Lactobacillus acidipiscis represented the major species in Dajiang, while Halomonas sp., Staphylococcus equorum, and Halanaerobium praevalens were the predominant species in Sufu. Acetic acid and sucrose were found to be the primary major physicochemical factor influencing the bacterial communities in Dajiang and Sufu, respectively. Furthermore, Bacillus subtilis is strongly correlated with lactic acid levels, L. acidipiscis is positively correlated with acetic acid levels, while Staphylococcus sciuri and S. equorum are strongly, and positively, correlated with malic acid. Following analysis of carbohydrate and amino acid metabolism in all samples, cysteine and methionine metabolism, as well as fatty acid biosynthesis-related genes are upregulated in Dajiang compared to Sufu samples. However, such as the Staphylococcus, W. viridescens, and P. brenneri, as potentially foodborne pathogens, existed in Dajang and Sufu samples. Cumulatively, these results suggested that Dajiang and Sufu have unique bacterial communities that influence their specific characteristics. Hence, the current study provides insights into the microbial community composition in Dajiang and Sufu samples, which may facilitate the isolation of functional bacterial species suitable for Dajiang and Sufu production, thus improving their production efficiency.

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Higher-Order Interactions Dampen Pairwise Competition in the Zebrafish Gut Microbiome

mBio ◽

10.1128/mbio.01667-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Deepika Sundarraman ◽

Edouard A. Hay ◽

Dylan M. Martins ◽

Drew S. Shields ◽

Noah L. Pettinari ◽

...

Keyword(s):

Microbial Communities ◽

Species Interactions ◽

Bacterial Species ◽

Higher Order ◽

Microbial Composition ◽

Intestinal Microbes ◽

Larval Zebrafish ◽

Microbial Species ◽

Species Abundances ◽

Multiple Species

ABSTRACT The microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health, and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multispecies consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germfree, to allow the introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, interbacterial interactions become weaker, suggesting that higher-order interactions in the vertebrate gut stabilize complex communities. IMPORTANCE Understanding the rules governing the composition of the diverse microbial communities that reside in the vertebrate gut environment will enhance our ability to manipulate such communities for therapeutic ends. Synthetic microbial communities, assembled from specific combinations of microbial species in germfree animals, allow investigation of the fundamental question of whether multispecies community composition can be predicted solely based on the combined effects of interactions between pairs of species. If so, such predictability would enable the construction of communities with desired species from the bottom up. If not, the apparent higher-order interactions imply that emergent community-level characteristics are crucial. Our findings using up to five coexisting native bacterial species in larval zebrafish, a model vertebrate, provide experimental evidence for higher-order interactions and, moreover, show that these interactions promote the coexistence of microbial species in the gut.

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Impacts of Low Temperature and Ensiling Period on the Bacterial Community of Oat Silage by SMRT

Microorganisms ◽

10.3390/microorganisms9020274 ◽

2021 ◽

Vol 9 (2) ◽

pp. 274

Author(s):

Xiaomei Li ◽

Fei Chen ◽

Xuekai Wang ◽

Lin Sun ◽

Linna Guo ◽

...

Keyword(s):

Bacterial Community ◽

Single Molecule ◽

Storage Temperature ◽

Bacterial Species ◽

Microbial Composition ◽

Acetic Acid Production ◽

Fermentation Products ◽

Silage Fermentation ◽

Different Temperatures ◽

Storage Temperatures

The objective of this study was to investigate how storage temperatures influence the bacterial community of oat silage during the ensiling process via PacBio single molecule, real-time sequencing technology (SMRT). Forage oat was ensiled at four different temperatures (5 °C, 10 °C, 15 °C, and 25 °C) and ensiling days (7, 14, 30, and 60 days). With the rise in storage temperature, the lactic acid content showed an increased trend. Acetic acid production was observed highest in silage fermented at 5 °C compared with other treatments, and Enterococcus mundtii was also the dominant bacterial species. Lactiplantibacillus pentosus and Loigolactobacillus rennini were exclusively detected in silages at 10 °C, 15 °C, and 25 °C, and dominated the fermentation after 60 days of ensiling at 10 °C and 25 °C, respectively. In addition, L. pentosus, L. rennini, and E. mundtii may be related to changes in the fermentation products due to the differences in ensiling temperature. In conclusion, results of this study improve our understanding of the complicated microbial composition underlying silage fermentation at low temperatures, which might contribute to target-based regulation methods for enhancing silage quality and developing new inoculants.

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BIOGAS PRODUCTION IN THE CONCENTRATED DISTILLERY WASTEWATER TREATMENT

Alternative Energy and Ecology (ISJAEE) ◽

10.15518/isjaee.2018.25-30.051-059 ◽

2018 ◽

pp. 51-59 ◽

Cited By ~ 1

Author(s):

N. Golub ◽

M. Potapova ◽

M. Shinkarchuk ◽

O. Kozlovets

Keyword(s):

Wastewater Treatment ◽

Dry Matter ◽

Ph Value ◽

Biogas Production ◽

Anaerobic Sludge ◽

Methane Formation ◽

Maximum Output ◽

Processing Technologies ◽

Spent Wash ◽

Distillery Spent Wash

The paper deals with the waste disposal problem of the alcohol industry caused by the widespread use of alcohol as biofuels. In the technology for the production of alcohol from cereal crops, a distillery spent wash (DSW) is formed (per 1 dm3 of alcohol – 10–20 dm3 DSW), which refers to highly concentrated wastewater, the COD value reaches 40 g O2/dm3. Since the existing physical and chemical methods of its processing are not cost-effective, the researchers develop the processing technologies for its utilization, for example, an anaerobic digestion. Apart from the purification of highly concentrated wastewater, the advantage of this method is the production of biogas and highquality fertilizer. The problems of biotechnology for biogas production from the distillery spent wash are its high acidity–pH 3.7–5.0 (the optimum pH value for the methanogenesis process is 6.8–7.4) and low nitrogen content, the lack of which inhibits the development of the association of microorganisms. In order to solve these problems, additional raw materials of various origins (chemical compounds, spent anaerobic sludge, waste from livestock farms, etc.) are used. The purpose of this work is to determine the appropriate ratio of the fermentable mixture components: cosubstrate, distillery spent wash and wastewater of the plant for co-fermentation to produce an energy carrier (biogas) and effective wastewater treatment of the distillery. In order to ensure the optimal pH for methanogenesis, poultry manure has been used as a co-substrate. The co-fermentation process of DSW with manure has been carried out at dry matter ratios of 1:1, 1:3, 1:5, 1:7 respectively. It is found that when the concentration of manure in the mixture is insufficient (DSW/manure – 1:1, 1:3), the pH value decreases during fermentation which negatively affects methane formation; when the concentration of manure in the mixture is increased (DSW/manure – 1:5, 1:7), the process is characterized by a high yield of biogas and methane content. The maximum output of biogas with a methane concentration of 70 ± 2% is observed at the ratio of components on a dry matter “wastewater: DSW: manure” – 0,2:1:7 respectively. The COD reduction reaches a 70% when using co-fermentation with the combination of components “wastewater: DSW: manure” (0,3:1:5) respectively.

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Substrate use prioritization by a coculture of five species of gut bacteria fed mixtures of arabinoxylan, xyloglucan, β-glucan, and pectin

10.26686/wgtn.12585620.v1 ◽

2020 ◽

Author(s):

Y Liu ◽

AL Heath ◽

B Galland ◽

N Rehrer ◽

L Drummond ◽

...

Keyword(s):

Gut Microbiota ◽

Dietary Fiber ◽

Plant Cell Wall ◽

Bacterial Species ◽

Short Chain ◽

Emergent Properties ◽

Human Gut ◽

Fermentation Products ◽

Plant Polysaccharides ◽

Human Gut Microbiota

© 2020 American Society for Microbiology. Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, β-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula. Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology. This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

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The treatment of iron oxalate leach liquors in a UASB with sulfate reduction

Water Science & Technology ◽

10.2166/wst.1997.0614 ◽

1997 ◽

Vol 36 (6-7) ◽

pp. 383-390 ◽

Cited By ~ 1

Author(s):

J. E. Teer ◽

D. J. Leak ◽

A. W. L. Dudeney ◽

A. Narayanan ◽

D. C. Stuckey

Keyword(s):

Bacterial Species ◽

Upflow Anaerobic Sludge Blanket ◽

Anaerobic Sludge ◽

Desulfovibrio Vulgaris ◽

Hydrogenotrophic Methanogenesis ◽

Irreversible Effects ◽

High Concentrations ◽

A Minor ◽

Anaerobic Sludge Blanket ◽

Reducing Bacteria

The presence of small amounts of iron (>0.013% Fe) in sand creates problems in the manufacture of high quality glass. Removal by hot sulphuric acid is possible, but creates environmental problems, and is costly. Hence organic acids such as oxalic have been investigated since they are effective in removing iron, and can be degraded anaerobically. The aim of this work was to identify key intermediates in the anaerobic degradation of oxalate in an upflow anaerobic sludge blanket reactor (UASB) which was removing iron from solution in the sulphide form, and to determine the bacterial species involved. 2-bromoethanesulfonic acid (BES) and molybdenum were selected as suitable inhibitors for methanogenic and sulphate reducing bacteria (SRB) respectively. 40mM molybdenum was used to inhibit the SRB in a reactor with a 12hr HRT. Total SRB inhibition took place in 20 hrs, with a complete breakthrough of influent sulphate. The lack of an immediate oxalate breakthrough confirmed Desulfovibrio vulgaris subspecies oxamicus was not the predominant oxalate utilising species. Nevertheless, high concentrations of molybdenum were found to inhibit oxalate utilising bacteria in granular reactors but not in suspended population reactors; this observation was puzzling, and at present cannot be explained. Based on the intermediates identified, it was postulated that oxalate was degraded to formate by an oxalate utilising bacteria such as Oxalobacter formigenes, and the formate used by the SRBs to reduce sulphate. Acetate, as a minor intermediate, existed primarily as a source of cell carbon for oxalate utilising bacteria. Methanogenic inhibition identified that 62% of the CH4 in the reactor operated at 37°C originated from hydrogenotrophic methanogenesis, whilst this figure was 80% at 20°C. Possible irreversible effects were recorded with hydrogenotrophic methanogens.

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