DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Predation increases multiple components of microbial diversity in activated sludge communities.

Protozoan predators form an essential component of activated sludge communities that is tightly linked to wastewater treatment efficiency. Nonetheless, very little is known how protozoan predation is channelled via bacterial communities to affect ecosystem functioning. Therefore, we experimentally manipulated protozoan predation pressure in activated-sludge communities to determine its impacts on microbial diversity, composition and putative functionality. Different components of bacterial diversity such as taxa richness, evenness, genetic diversity and beta diversity all responded strongly and positively to high protozoan predation pressure. These responses were non-linear and levelled off at higher levels of predation pressure, supporting predictions of hump-shaped relationships between predation pressure and prey diversity. In contrast to predation intensity, the impact of predator diversity had both positive (taxa richness) and negative (evenness and phylogenetic distinctiveness) effects on bacterial diversity. Furthermore, predation shaped the structure of bacterial communities. Reduction in top-down control negatively affected the majority of taxa that are generally associated with increased treatment efficiency, compromising particularly the potential for nitrogen removal. Consequently, our findings highlight responses of bacterial diversity and community composition as two distinct mechanisms linking protozoan predation with ecosystem functioning in activated sludge communities.

Do microplastics mediate the effects of chemicals on aquatic organisms?

Microplastics are ubiquitous in both marine and freshwater ecosystems, where they can act as a physical contaminant, as well as interact with chemicals present in the environment. It has been suggested that chemical contaminants can sorb to microplastics, such that microplastics act as a vector for chemicals into aquatic biota and enhance their negative effects. It has been repeatedly suggested that the main factors underpinning the binding of chemicals to microplastics are hydrophobic partitioning and the size of microplastic particles. Therefore, we used the hydrophobicity of chemicals, as log Kow, as well as the size of microplastic particles to conduct a quantitative analysis of published results to evaluate the influence of microplastics on chemical toxicity. We collated data from 39 laboratory studies that assessed the effects of microplastics, chemicals and their combination on several ecotoxicological responses of freshwater and marine organisms. Each chemical was assigned the relevant octanol / water partition coefficient (log KOW) as a measure of its hydrophobicity, and the mean size of microplastics particles used in each study was recorded. We found no effect of log KOW or the size of microplastic particles on the interaction between microplastics and chemicals with regards to any of the relevant ecotoxicological responses (behaviour, growth, survival and cellular) considered in this study. These findings are significant in showing that the effect of microplastics on the toxicity of chemicals is more complex than just considering hydrophobicity of chemicals and size of microplastics. We call for more mechanistic experiments to motivate a robust risk assessment and mitigation of microplastic toxicity in the environment.

Prebiotic Galactooligosaccharide Supplementation in Adults with Ulcerative Colitis: Exploring the Impact on Peripheral Blood Gene Expression, Gut Microbiota, and Clinical Symptoms.

Prebiotics may promote immune homeostasis and reduce sub-clinical inflammation in humans. This study investigated the effect of prebiotic galactooligosaccharide (GOS) supplementation in colonic inflammation. Seventeen patients with active ulcerative colitis (UC) consumed 2.8 g/d GOS for 6 weeks. At baseline and 6 weeks, gene expression (microarray), fecal calprotectin (ELISA), microbiota (16S rRNA), short-chain fatty acids (SCFAs; gas-liquid chromatography), and clinical outcomes (simple clinical colitis activity index (SCCAI), gastrointestinal symptom rating scale (GSRS), and Bristol stool form scale (BSFS)) were measured. Following prebiotics, clinical scores (SCCAI), fecal calprotectin, SCFAs, and pH were unchanged. Five genes were upregulated and two downregulated. Normal stool proportion (BSFS) increased (49% vs. 70%, p = 0.024), and the incidence (46% vs. 23%, p = 0.016) and severity (0.7 vs. 0.5, p = 0.048) of loose stool (GSRS), along with urgency (SCCAI) scores (1.0 vs. 0.5, p = 0.011), were reduced. In patients with a baseline SCCAI ≤2, prebiotics increased the relative abundance of Bifidobacterium from 1.65% (1.97) to 3.99% (5.37) (p = 0.046) and Christensenellaceae from 0.13% (0.33) to 0.31% (0.76) (p = 0.043). Prebiotics did not lower clinical scores or inflammation but normalized stools. Bifidobacterium and Christensenellaceae proportions only increased in patients with less active diseases, indicating that the prebiotic effect may depend on disease activity. A controlled study is required to validate these observations.

Diversity of dimethylsulfide-degrading methanogens and sulfate-reducing bacteria in anoxic sediments along the Medway Estuary, UK.

Methane is a powerful greenhouse gas but the microbial diversity mediating methylotrophic methanogenesis is not well-characterized. One overlooked route to methane is via the degradation of dimethylsulfide (DMS), an abundant organosulfur compound in the environment. Methanogens and sulfate-reducing bacteria (SRB) can degrade DMS in anoxic sediments depending on sulfate availability. However, we know little about the underlying microbial community and how sulfate availability affects DMS degradation in anoxic sediments. We studied DMS-dependent methane production along the salinity gradient of the Medway Estuary (UK) and characterized, for the first time, the DMS-degrading methanogens and SRB using cultivation-independent tools. DMS metabolism resulted in high methane yield (39%-42% of the theoretical methane yield) in anoxic sediments regardless of their sulfate content. Methanomethylovorans, Methanolobus and Methanococcoides were dominant methanogens in freshwater, brackish and marine incubations respectively, suggesting niche-partitioning of the methanogens likely driven by DMS amendment and sulfate concentrations. Adding DMS also led to significant changes in SRB composition and abundance in the sediments. Increases in the abundance of Sulfurimonas and SRB suggest cryptic sulfur cycling coupled to DMS degradation. Our study highlights a potentially important pathway to methane production in sediments with contrasting sulfate content and sheds light on the diversity of DMS degraders.

Bioconversion of food waste to volatile fatty acids: Impact of microbial community, pH and retention time.

Bio-based production of materials from waste streams is a pivotal aspect in a circular economy. This study aimed to investigate the influence of inoculum (three different sludge taken from anaerobic digestors), pH (5 & 10) and retention time on production of total volatile fatty acids (VFAs), VFA composition as well as the microbial community during anaerobic digestion of food waste. The highest VFA production was ∼22000 ± 1036 mg COD/L and 12927 ± 1029 mg COD/L on day 15 using the inoculum acclimated to food waste at pH 10 and pH 5, respectively. Acetic acid was the dominant VFA in the batch reactors with initial alkaline conditions, whereas both propionic and acetic acids were the dominant products in the acidic condition. Firmicutes, Chloroflexi and Bacteroidetes had the highest relative abundance in the reactors. VFA generation was positively correlated to the relative abundance of Firmicutes.

A comprehensive study of volatile fatty acids production from batch reactor to anaerobic sequencing batch reactor by using cheese processing wastewater.

Volatile fatty acids (VFAs) has great potential for closed-loop production in dairy industries via resource recovery from waste-streams. In the current study, the transition of VFA production from batch reactor to anaerobic sequencing batch reactor (ASBR) by using cheese industry wastewater under alkali pH was evaluated with respect to seed sludge structure, microbial diversity and reactor type. The transition from the batch reactor to the ASBR demonstrated that the maximum VFA production yield (g COD/g SCOD) was comparable in two reactors (batch: 0.97; ASBR: 0.94), whereas, the dominant acid type was different (batch: 49% lactic acid; ASBR: 80% propionic acid). There was a significant correlation between the productions of butyric acid with Gracilibacteraceae and Desulfovibrionaceae; propionic acid with Desulfovibrionaceae and Synergistaceae; lactic acid with Pseudomonadaceae and Rhodocyclaceae. The high VFA production efficiency can be achieved by long term reactor operation, which enables the shift from industrial waste-streams to biorefineries.

Volatile fatty acid production from semi-synthetic milk processing wastewater under alkali pH: The pearls and pitfalls of microbial culture.

Volatile fatty acids (VFA) are one of the most promising sustainable and environmentally friendly bioproduct owing to their wide usage area and high market demand. For this reason, in this study, the evaluation of VFA production from pure and mixed bacterial cultures was aimed. Three different mixed cultures with C. aceticum, C. butyricum and P. acidipropionici as pure cultures were used for inoculation of milk processing wastewater fermentation under pH 10 for 15 days. The mixed culture fermentation had the highest VFA production efficiency whereas the highest amount of acetic, butyric and propionic acid productions were obtained by C. aceticum, C. butyricum and P. acidipropionici, respectively. Also, the mixed cultures demonstrated faster pH regulation and acclimation than the pure cultures tested. Therefore, development of synthetic cultures may offer a useful approach to produce VFA mixtures with one-dominant acid type and with high production efficiency.

Profiling of Active Microorganisms by Stable Isotope Probing-Metagenomics.

Stable isotope probing (SIP) provides researchers a culture-independent method to retrieve nucleic acids from active microbial populations performing a specific metabolic activity in complex ecosystems. In recent years, the use of the SIP method in microbial ecology studies has been accelerated. This is partly due to the advances in sequencing and bioinformatics tools, which enable fast and reliable analysis of DNA and RNA from the SIP experiments. One of these sequencing tools, metagenomics, has contributed significantly to the body of knowledge by providing data not only on taxonomy but also on the key functional genes in specific metabolic pathways and their relative abundances. In this chapter, we provide a general background on the application of the SIP-metagenomics approach in microbial ecology and a workflow for the analysis of metagenomic datasets using the most up-to-date bioinformatics tools.

Volatile fatty acids production via mixed culture fermentation: Revealing the link between pH, inoculum type and bacterial composition.

The aim of the study was to investigate the effects of operational parameters, inoculum type and bacterial community on mixed culture fermentation to produce one dominant acid type in the mixture of volatile fatty acids (VFA). The study was performed using three different inocula (large&small granular and slurry) with glucose under various initial pH. The VFA production efficiency reached to 0,97 (gCOD/gSCOD) by granular sludge. VFA composition was changed by initial pH: in neutral conditions, acetic acid; in acidic conditions, acetic and butyric acids, in alkali conditions butyric acid were dominated, respectively. The VFA production was positively affected by the high relative abundance of Firmicutes. On the contrary, a negative correlation was seen between VFA production and the relative abundance of Chloroflexi. The results revealed the physical sludge structure of inoculum was the key factor for production efficiency, whereas, pH was the most important parameter to affect VFA composition.

The study of structure of anaerobic granules and methane producing pathways of pilot-scale UASB reactors treating municipal wastewater under sub-mesophilic conditions.

This study was carried out to investigate the relationship between the methane producing pathways and the characteristics of anaerobic granules treating municipal wastewater. For this purpose, two pilot scale upflow anaerobic sludge blanket reactors with different granule size distribution (1-2 mm and 3-4 mm) were investigated at operating temperatures of 20 °C and 28 °C for 239 days. There was an increased and stable biogas production when temperature was elevated to 28 °C likely due to reduction in methane solubility. Larger granules had multi-layered internal microstructures with higher acetoclastic methanogenic activities (250-437 mL CH4 g-1 VS d-1) than smaller granules (150-260 mL CH4 g-1 VS d-1). The relative abundance of acetoclastic methanogens of larger granules was higher, confirming acetoclastic methane producing pathway was more prominent. However, there was no significant difference in the performance of the two reactors because they were operating below their capacities in terms of organic loading rate to volatile solids ratio.

Microbial Cycling of Methanethiol.

Methanethiol (MT) is an organic sulfur compound with a strong and disagreeable odour. It has biogeochemical relevance as an important compound in the global sulfur cycle, where it is produced as a reactive intermediate in a number of different pathways for synthesis and degradation of other globally significant sulfur compounds such as dimethylsulfoniopropionate, dimethylsulfide and methionine. With its low odour threshold and unpleasant smell, MT can be a significant cause of malodour originating from animal husbandry, composting, landfill operations, and wastewater treatment and is also associated with faeces, flatus and oral malodour (halitosis). A diverse range of microorganisms drives the production and degradation of MT, including its aerobic and anaerobic metabolism. MT producing and degrading organisms are known to be present in terrestrial, freshwater and marine environments but may also be important in association with plant and animal (including human) hosts. This chapter considers the role of MT as an intermediate of the global sulfur cycle and discusses current knowledge of microbial pathways of MT production and degradation.

COD/sulfate ratio does not affect the methane yield and microbial diversity in anaerobic digesters.

Anaerobic digestion of organic matter is the major route of biomethane production. However, in the presence of sulfate, sulfate-reducing bacteria (SRB) typically outcompete methanogens, which may reduce or even preclude methane production from sulfate-containing wastewaters. Although sulfate-reduction and methanogenesis can occur simultaneously, our limited understanding of the microbiology of anaerobic digesters treating sulfate-containing wastewaters constrains improvements in the production of methane from these systems. This study tested the effects of carbon sources and chemical oxygen demand-to-sulfate ratio (COD/SO42-) on the diversity and interactions of SRB and methanogens in an anaerobic digester treating a high-sulfate waste stream. Overall, the data showed that sulfate removal and methane generation occurred in varying efficiencies and the carbon source had limited effect on the methane yield. Importantly, the results demonstrated that methanogenic and SRB diversities were only affected by the carbon source and not by the COD/SO42- ratio.

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere.

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Culture-dependent and culture-independent methods reveal diverse methylotrophic communities in terrestrial environments.

One-carbon compounds such as methanol, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) are significant intermediates in biogeochemical cycles. They are suggested to affect atmospheric chemistry and global climate. Methylotrophic microorganisms are considered as a significant sink for these compounds; therefore, we analyzed the diversity of terrestrial bacteria that utilize methanol, DMS and DMSO as carbon and energy source using culture-dependent and culture-independent methods. The effect of habitat type on the methylotrophic community structure was also investigated in rhizosphere and bulk soil. While thirteen strains affiliated to the genera Hyphomicrobium, Methylobacterium, Pseudomonas, Hydrogenophaga, Rhodococcus, Flavobacterium and Variovorax were isolated, denaturing gradient gel electrophoresis revealed the dominance of Thiobacillus, Rhodococcus, Flavobacterium and Bacteroidetes species. Furthermore, methylotrophic communities that degrade methanol or DMS are not shaped by terrestrial habitat type. Rhizosphere and soil samples showed dominance of Methylophilus spp. and Methylovorus spp. for methanol enrichments; Cytophaga spp., Pseudomonas tremae and Thiobacillus thioparus for DMS enrichments.

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing.

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [(13)C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment.

Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles in situ. In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). Microbial communities involved in DMS degradation were analysed by denaturing gradient gel electrophoresis, high-throughput sequencing of SIP gradient fractions and metagenomic sequencing of phi29-amplified community DNA. Labelling patterns of time course SIP experiments identified members of the Methylophilaceae family, not previously implicated in DMS degradation, as dominant DMS-degrading populations in soil and lake sediment. Thiobacillus spp. were also detected in (13)C-DNA from SIP incubations. Metagenomic sequencing also suggested involvement of Methylophilaceae in DMS degradation and further indicated shifts in the functional profile of the DMS-assimilating communities in line with methylotrophy and oxidation of inorganic sulphur compounds. Overall, these data suggest that unlike in the marine environment where gammaproteobacterial populations were identified by SIP as DMS degraders, betaproteobacterial Methylophilaceae may have a key role in DMS cycling in terrestrial environments.

Effect of sludge age on the bacterial diversity of bench scale sequencing batch reactors.

Sludge age or mean cell residence time (MCRT) plays a crucial role in design and operation of wastewater treatment plants. The change in performance, for example micropollutant removal, associated with changes in MCRT is often attributed to changes in microbial diversity. We operated four identical laboratory-scale sequencing batch reactors (two test and two control) in parallel for 212 days. Sludge age was decreased gradually (from 10.4to 2.6 days) in experimental reactors whereas it was kept constant (10.4 days) in control reactors. The reactor performance and biomass changed in a manner consistent with our understanding of the effect of sludge age on a reactors performance: the effluent quality and biomass declined with decreasing MCRT. The composition of the bacterial and ammonia-oxidizing bacterial communities in four reactors was analyzed using denaturing gradient gel electrophoresis (DGGE), and similarities in band patterns were measured using the Dice coefficient. The overall similarity between the communities in reactors run at different sludge ages was indistinguishable from the similarity in communities in reactors run at identical sludge ages. This was true for both the general bacterial communities and putative AOB communities. The number of detectable bands in DGGE profiles was also unaffected by sludge age (p approximately 0.5 in both cases). Initially, the detectable diversity of activated sludge communities in all four reactors clustered with time, regardless of their designation or sludge age; however, these clusters were only weakly supported by bootstrap analysis. However, after 135 days, a sludge age specific clustering was observed in the bacterial community but not the putative ammonia-oxidizing bacterial community. The mean self-similarity of each reactor decreased, variance increased, and the number of detectable bands in DGGE profiles decreased over time in all reactors. The changes observed with time are consistent with ecological drift. Sludge age has a subtler and slower effect than we anticipated. However, we postulate that sludge age may be more evident in the taxa occurring below the detection limit of DGGE. New sequencing technology may help us address this hypothesis.

Toluene inhibition on an anaerobic reactor sludge in terms of potential activity and composition of acetoclastic methanogens.

The aim of this study was to determine the effect of toluene on an anaerobic sludge taken from a full-scale upflow anaerobic sludge blanket (UASB) reactor in terms of potential activity and composition of acetoclastic methanogens. Specific methanogenic activity (SMA) test results showed that 5%, 9.5%, 14%, 24%, 29%, 38% and 62% inhibition occurred in the potential methane production (PMP) rate of the sludge at toluene concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM and 1 mM, respectively. Fluorescence in situ hybridization (FISH) results showed that relative abundance of archaeal cells was approx. 19% throughout the SMA tests. The anaerobic sludge was dominated by acetoclastic genus Methanosaeta which were slightly affected by increasing toluene concentrations do not have any effect on relative abundance of Methanosaeta spp., which was between 73% +/- 1.6 and 68% +/- 2.1 of the archaeal population.