Thermophilic Anaerobic Sludge Research Articles

Inhibitory Effects of Ammonia on Archaeal 16S rRNA Transcripts in Thermophilic Anaerobic Digester Sludge

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH4+-N/L), but did not change much at lower ammonia concentrations (3 g NH4+-N/L) compared with the 0 g NH4+-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.

Moorella caeni sp. nov., isolated from thermophilic anaerobic sludge from a methanol-fed reactor.

Strain AMPT has been previously suggested as a strain of the species Moorella thermoacetica Jiang et al. 2009 (based on the high 16S rRNA gene identity, 98.3 %). However, genome-based phylogenetic analysis of strain AMPT reveals that this bacterium is in fact a novel species of the genus Moorella. Genome relatedness indices between strain AMPT and Moorella thermoacetica DSM 521T were below the minimum threshold values required to consider them members of the same species (digital DNA-DNA hybridization, 52.2 % (<70%); average nucleotide identity, 93.2 % (<95%)). Based on phylogenetic and phenotypic results we recommend that strain AMPT (DSM 21394T=JCM 35360T) should be classified as representing new species, for which we propose the name Moorella caeni sp. nov.

The Start-Up of Continuous Biohydrogen Production from Cheese Whey: Comparison of Inoculum Pretreatment Methods and Reactors with Moving and Fixed Polyurethane Carriers

This article presents the results of the start-up of continuous production of biohydrogen from cheese whey (CW) in an anaerobic filter (AF) and anaerobic fluidized bed (AFB) with a polyurethane carrier. Heat and acid pretreatments were used for the inactivation of hydrogen-scavengers in the inoculum (mesophilic and thermophilic anaerobic sludge). Acid pretreatment was effective for thermophilic anaerobic sludge to suppress methanogenic activity, and heat treatment was effective for mesophilic anaerobic sludge. Maximum specific yields of hydrogen, namely 178 mL/g chemical oxygen demand (COD) and 149 mL/g COD for AFB and AF, respectively, were obtained at the hydraulic retention time (HRT) of 4.5 days and organic load rate (OLR) of 6.61 kg COD/(m3 day). At the same time, the maximum hydrogen production rates of 1.28 and 1.9 NL/(L day) for AF and AFB, respectively, were obtained at the HRT of 2.02 days and OLR of 14.88 kg COD/(m3 day). At the phylum level, the dominant taxa were Firmicutes (65% in AF and 60% in AFB), and at the genus level, Lactobacillus (40% in AF and 43% in AFB) and Bifidobacterium (24% in AF and 30% in AFB).

A novel thermophilic anaerobic granular sludge membrane distillation bioreactor for wastewater reclamation.

Membrane distillation (MD) has a high heat requirement. Integrating MD with thermophilic bioreactors could remedy this problem. A laboratory-scale thermophilic anaerobic granular sludge membrane distillation bioreactor (ThAGS-MDBR) was used to treat wastewater with a high organic loading rate (OLR). Waste heat from ThAGS was used directly for the MD process to reduce energy consumption. The result demonstrated that the ThAGS-MDBR system achieved a high-efficiency removal of chemical oxygen demand (more 99.5%) and NH4+-N (96.4%). Furthermore, the highest methane production from the proposed system was 332mL/g CODremoved at OLR of 16kg COD/m3/day. Specifically, an aggregate of densely packed diverse microbial communities in anaerobic granular sludge was the main mechanism for the enhancement of bioreactor tolerance with environmental changes. High-quality distillate water from ThAGS-MDBR was reclaimed in one step with total organic carbon less than 1.7mg/L and electrical conductivity less than 120μS/cm. Furthermore, the result of the DNA extraction kit recorded that Methanosaeta thermophila was a critical archaea for high COD removal and bioreactor stability.

Bioelectrochemical vs hydrogenophilic approach for CO2 reduction into methane and acetate

Methane and acetate production through CO2 reduction has been tested by using chemoautotrophic microorganisms under bioelectrochemical and hydrogenophilic conditions. For the methanogenic tests, a thermophilic anaerobic sludge has been used as inoculum while the thermal treatment of the anaerobic sludge allowed the acetogenic inoculum selection. The optimal pH has been selected through methane and acetate hydrogenophilic tests at three different pH values (5.5, 6.5, 7.5) while three different cathodic potentials (-0.7, −0.9, −1.1 V vs SHE) have been tested in bioelectrochemical experiments by using the optimum pH value. Both methane and acetate production showed higher efficiency by the use of the bioelectrochemical system instead of the corresponding hydrogenophilic tests, i.e. during the −0.9 V vs SHE bioelectrochemical condition, the efficiency for methane and acetate reached the 95 and 88% while in the hydrogenophilic tests the efficiency resulted considerably lower, with average values of 11% and 16% for methane and acetate, respectively. The higher efficiency of the bioelectrochemical system has been also confirmed by the determination of methane and acetate production rate which shown a 45 and 66% increase with respect the hydrogenophilic tests. The higher efficiency of bioelectrochemical tests has been justified by assuming a higher hydrogen mass transfer from the electrode surface to the attached biofilm. Moreover, the bioelectrochemical tests suggested the presence of a direct electron uptake for methane production, while for the acetate production, a hydrogen mediated mechanism appeared necessary. Finally, the microbial community characterization by the DGGE analysis highlighted the effectiveness of the thermal treatment for acetogens selection by methanogens inhibition.

Mesophilic, thermophilic and hyperthermophilic acidogenic fermentation of food waste in batch: Effect of inoculum source

Distinctions in hydrolysis and acidogenesis were examined for a series of anaerobic batch reactors inoculated with three different anaerobic mixed cultures (mesophilic, thermophilic and hyperthermophilic anaerobic sludge) and operated at the temperature of inoculum’s origin and additionally at 70 °C. Hyperthermophilic temperatures led to increased hydrolysis rates during the start-up stage but a rapid drop in pH limited the overall hydrolysis efficiency, indicating the importance of pH control to sustain the high reaction rates at higher temperatures. No significant difference (P > 0.05) was observed among hydrolysis efficiencies obtained for different reactors which ranged between 27 ± 3% and 40 ± 14%. The highest fermentation yield of 0.44 g COD of fermentation products/g VSS-CODadded was obtained under thermophilic conditions, followed by mesophilic (0.33 g COD ferm. prod./g VSS-CODadded) and hyperthermophilic conditions (0.05–0.08 g COD ferm. prod./g VSS-CODadded). Fermentative performance was better at mesophilic and thermophilic conditions as indicated by improved production of volatile fatty acids (VFA). VFAs accounted for 60–71% of the solubilised matter at thermophilic and mesophilic conditions. Acetic acid formed the primary VFA (70%) at mesophilic temperatures, while butyric acid was the major VFA at thermophilic (60%) conditions. Hyperthermophilic conditions led to increased production of lactic acid, which comprised up to 32% of the solubilised matter. Overall, the results indicate that different operating temperatures may not significantly affect the substrate degradation efficiency but clearly influence the biotransformation pathways.

Improvement of methanogenic activity of anaerobic digestion using poly(l-lactic acid) with enhanced chemical hydrolyzability based on physicochemical parameters

Because packing bags and disposable items of poly (l-lactic acid) (PLLA) waste are discharged together with other organic waste including garbage, anaerobic co-digestion of PLLA and other organic waste is required. However, because of low hydrolyzability of PLLA products, the chemical hydrolyzability must be improved for PLLA treatment during anaerobic digestion. This study aimed to assess weight-average molecular weight (Mw) and crystallinity (Xc), to determine the chemical hydrolyzability of PLLA, for PLLA treatment during anaerobic digestion. Moreover, the possibility of anaerobic co-digestion of the PLLA after improvement of chemical hydrolyzability and other organic waste was also discussed. Detectable methanogenic activity of the mesophilic and thermophilic anaerobic sludges of PLLA occurred in the Mw range of 6,800 to 16,500, and 6,800 and 38,000, respectively. The methanogenic activity of mesophilic and thermophilic anaerobic sludge was higher with PLLA with a high crystallinity (Xc = 39.9–46.1%) than with nearly amorphous PLLA (Xc = 0.3–3.5%). The maximum methanogenic activity of anaerobic sludge using PLLA with an Xc of approximately 40–45% and with a Mw of 10,300 and 16,500 for mesophilic and thermophilic anaerobic sludge were 0.013 gCOD·gVS−1·d−1 and 0.13 gCOD·gVS−1·d−1, respectively. A survey on the possibility of anaerobic co-digestion of PLLA after improvement in chemical hydrolyzability based on Mw and Xc and organic wastes revealed that thermophilic conditions at 55 °C are more advantageous than mesophilic conditions at 37 °C.

Performance of thermophilic anaerobic sludge digester after alkaline‐assisted thermal disintegration optimization using response surface methodology

AbstractThe Box–Behnken statistical experimental design was used to investigate the effects of operating parameters on thermo‐alkaline disintegration of sludge. The optimum disintegration degree was 77.83% at 90°C, 0.2 mol/L NaOH and 25 min retention time. The coefficient of determination (R2) of 97.68% confirms that the model has a very good fitness with the experimental variables. The effects of a thermo‐alkaline disintegration on the performance of the thermophilic anaerobic digestion of sludge were also investigated. The thermo‐alkaline pretreatment showed an initial solubilization effect and methane production (0.73 (L/Lr.day) increased up to around 54% compared to raw sludge (0.48 L/Lr.day). VS removal in disintegrated digester (65.87%) increased of 13.13% compared to conventional sludge digester (52.74%) at the end of the operation. Similarly, methane contents in the biogas of digester fed with disintegrated sludge (71.14%) increased of 6.44% compared to conventional sludge digester (64.7%).

Bioelectrochemical enhancement of anaerobic digestion: Comparing single- and two-chamber reactor configurations at thermophilic conditions

Bioelectrochemical system (BES) can act as an auxiliary technology for improving organic waste treatment and biogas production in anaerobic digestion (AD). For the first time this study directly compared the performance of a single- and a cation-exchange membrane-equipped two-chamber BES-AD systems at thermophilic conditions. The results indicated that an active glucose-fed thermophilic anaerobic sludge could readily (<3days) increase biogas production in both reactor configurations by inserting a carbon electrode poised at −0.8V (vs. Ag/AgCl). However, after a 3-week operation, the biogas production rates from the single- and two-chamber BES reactor decreased due to volatile fatty acids accumulation. Only the two-chamber configuration could enable methane enrichment (98% CH4v/v) in biogas. Overall, this study suggests that integrating bioelectrodes in-situ could not sustainably improve biogas production in a thermophilic AD reactor, and future studies should be directed towards the use of bioelectrodes for improving biogas quality.

Characterization and Genome Analysis of the First Facultatively Alkaliphilic Thermodesulfovibrio Isolated from the Deep Terrestrial Subsurface.

Members of the genus Thermodesulfovibrio belong to the Nitrospirae phylum and all isolates characterized to date are neutrophiles. They have been isolated from terrestrial hot springs and thermophilic methanogenic anaerobic sludges. Their molecular signatures have, however, also been detected in deep subsurface. The purpose of this study was to characterize and analyze the genome of a newly isolated, facultatively alkaliphilic Thermodesulfovibrio from a 2 km deep aquifer system in Western Siberia, Russia. The new isolate, designated N1, grows optimally at pH 8.5 and at 65°C. It is able to reduce sulfate, thiosulfate or sulfite with a limited range of electron donors, such as formate, pyruvate, and lactate. Analysis of the 1.93 Mb draft genome of strain N1 revealed that it contains a set of genes for dissimilatory sulfate reduction, including sulfate adenyltransferase, adenosine-5′-phosphosulfate reductase AprAB, membrane-bound electron transfer complex QmoABC, dissimilatory sulfite reductase DsrABC, and sulfite reductase-associated electron transfer complex DsrMKJOP. Hydrogen turnover is enabled by soluble cytoplasmic, membrane-linked, and soluble periplasmic hydrogenases. The use of thiosulfate as an electron acceptor is enabled by a membrane-linked molybdopterin oxidoreductase. The N1 requirement for organic carbon sources corresponds to the lack of the autotrophic C1-fixation pathways. Comparative analysis of the genomes of Thermodesulfovibrio (T. yellowstonii, T. islandicus, T. àggregans, T. thiophilus, and strain N1) revealed a low overall genetic diversity and several adaptive traits. Consistent with an alkaliphilic lifestyle, a multisubunit Na+/H+ antiporter of the Mnh family is encoded in the Thermodesulfovibrio strain N1 genome. Nitrogenase genes were found in T. yellowstonii, T. aggregans, and T. islandicus, nitrate reductase in T. islandicus, and cellulose synthetase in T. aggregans and strain N1. Overall, our results provide genomic insights into metabolism of the Thermodesulfovibrio lineage in microbial communities of the deep subsurface biosphere.

Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters.

Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1—a Pseudomonas sp.) and thermophilic (Iso T10—a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457–0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130–0.486, P = 0.075–0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and/or horizontal gene transfer between raw sludge bacteria and the digester microbial community.

Influence of biodegradation in thermophilic anaerobic aqueous conditions on crystallization of poly(butylene succinate)

This work investigates the biodegradation of poly(butylene succinate) (PBS) in an environment of thermophilic (55 °C) and mesophilic (37 °C) anaerobic sludge. The thermophilic anaerobic sludge was prepared in a laboratory from mesophilic sludge sourced from a municipal waste water treatment plant and placed under thermophilic conditions. It was confirmed that PBS failed to decompose under mesophilic anaerobic conditions (2 wt. %), while the degree of biodegradation achieved under thermophilic conditions reached 24.8 wt. %. Abiotic hydrolysis of PBS in an environment of phosphate buffer (pH = 7) at 55 °C reached 17.8%, leading to the conclusion that hydrolytic enzymes present in thermophilic anaerobic sludge are involved in the biodegradation process as well as abiotic hydrolysis. Nonisothermal crystallization kinetics of PBS before and after biodegradation was investigated by differential scanning calorimetry (DSC). After biodegradation, a large shift in melting temperature Tm was observed, while shift of crystallization temperature Tc was even more pronounced. Lower Tm means smaller crystals. Crystallization kinetics was approximately 50% slower, which could be explained by lower nucleation density. Scanning electron microscopy (SEM) revealed a beautiful spherulitic structure after biodegradation at elevated temperature. During biodegradation, microorganisms assimilated mostly in the amorphous part of the polymer, and elevated temperature helped healing of crystal imperfections. By X-ray diffraction (XRD), it was found that biodegradation does not have a large effect on crystal form of PBS.

Biodegradation of waste PET based copolyesters in thermophilic anaerobic sludge

A series of poly(ethylene terephthalate-co-lactate) copolyesters with random microstructure was prepared from PET waste beverage bottles and l-lactic acid. Square specimens, sectioned from melt-pressed film, were incubated in thermophilic sludge (55 °C, alkaline environment) for 394 days. The biodegradability of the samples, determined from biogas yield, reached 34–69 % depending on the starting aromatic to aliphatic units' ratio.Water uptake study in sludge and abiotic buffers at 55 °C showed sharp border around 57% of aromatic units in copolyester composition between polymers susceptible and resistant to both hydrolytic attack by hydrolases and the abiotic hydrolysis. Samples biodegraded in sludge and those abiotically aged in buffers were characterized by 1H NMR, ATR-FTIR, SEC, DSC, and TGA analysis to gain an insight into the chain scission mechanism.Aromatic oligomers as model of copolyester degradation intermediates were prepared and proved to biodegrade in sludge as well.

In situ identification of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process.

Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of thermophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system.

A Thermophilic Gram-Negative Nitrate-Reducing Bacterium, Calditerrivibrio nitroreducens, Exhibiting Electricity Generation Capability

To exploit the potential diversity of thermophilic exoelectrogens, two-chamber microbial fuel cells (MFCs) were inoculated with thermophilic anaerobic digester sludge and operated at 55 °C without supplementing with exogenous redox mediator. The MFC generated a maximum power density of 823 mW m(-2) after 200 h of operation. Molecular phylogenetic analyses suggested that the microbial population on the anode was dominated by a species closely related to a thermophilic nitrate-reducing bacterium Calditerrivibrio nitroreducens, for which a strain (Yu37-1) has been isolated in pure culture. Thus, a pure culture of the C. nitroreducens strain Yu37-1 was inoculated into MFC to examine the electricity generation capability. Without an exogenous mediator, MFCs stably produced electricity with a maximum power density of 272 mW m(-2) for >400 h of operation. The MFC current recovered to the original level within few hours after medium replacement, suggesting that the electricity generation was caused by the anodic microorganisms. Cyclic voltammetry indicated that redox systems (E3 and Ec) with similar potentials (-0.14 and -0.17 V) made the main contributions to the exoelectrogenic activities of the sludge-derived consortium and C. nitroreducens Yu37-1, respectively. This study undertook the bioelectrochemical characterization of C. nitroreducens as the first example of a thermophilic Gram-negative exoelectrogen.

Two-stage mesophilic anaerobic–thermophilic digestion for sludge sanitation to obtain advanced treated sludge

In the present study, raw, mesophilic anaerobic, and thermophilic anaerobic sludge were analysed to evaluate whether the pathogen content limits established in the “Proposal for a Directive of the European Parliament and of the Council on spreading of sludge on land’’ were satisfied. Salmonella spp., Escherichia coli, and Clostridium perfringens spores were cultivated and pathogenity genes invA and cpa PCR-amplified. Thermophilic anaerobic digestion produced Class A biosolids by eliminating E. coli and Salmonella spp. but did not accomplished the microbial requirements of the future Directive due to the presence of C. perfringens spores (9.6×104CFUsmL−1). Hence, the final goal of this work was to propose a two-stage process capable of removing the spores of C. perfringens to obtain an advanced treated sludge that could be land-applied with no environmental risks. The first stage of the process suggested in this study involved the mesophilic anaerobic digestion of the sludge while the second stage of operation consisted of an aerobic or anaerobic thermophilic digestion.

Complete Genome Sequence of Clostridium clariflavum DSM 19732

Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 protein-coding and 98 RNA genes, for the type strain DSM 19732.

Rheological Characterization of Mesophilic and Thermophilic Anaerobic Digestion Sludge

Rheological Characterization of Mesophilic and Thermophilic Anaerobic Digestion Sludge

Kinetics of inactivation of indicator pathogens during thermophilic anaerobic digestion

Thermophilic anaerobic sludge digestion is a promising process to divert waste to beneficial use, but an important question is the required temperature and holding time to achieve a given degree of pathogen inactivation. In this study, the kinetics of inactivation of Ascaris suum and vaccine strain poliovirus type 1 (PVS-1), selected as indicators for helminth ova and enteric viruses respectively, were determined during anaerobic digestion at temperatures ranging from 51 to 56 °C. Inactivation of both indicator organisms was fast with greater than two log reductions achieved within 2 h for A. suum and three log reductions for PVS-1, suggesting that the current U.S. regulations are largely conservative. The first-order inactivation rate constants k followed Arrhenius relationship with activation energies of 105 and 39 KJ mol −1 for A. suum and PVS-1, respectively indicating that A. suum was more sensitive to temperature. Although inactivation was fast, the presence of compounds in the sludge that are known to be protective of pathogen inactivation was observed, suggesting that composition-dependent time–temperature relationships are necessary.

Investigation of the diversity of homoacetogenic bacteria in mesophilic and thermophilic anaerobic sludges using the formyltetrahydrofolate synthetase gene

Homoacetogenic bacteria are strict anaerobes capable of autotrophic growth on H(2)/CO(2) or CO, and of heterotrophic growth on a wide range of sugars, alcohols, methoxylated aromatic compounds and one carbon compounds, yielding acetate as their sole metabolic end-product. Batch activity tests on anaerobic granular sludge, using H(2)/CO(2) as a substrate and 2-bromoethanesulfonate (BES) as a specific methanogenic inhibitor revealed that H(2)/CO(2) conversion and concomitant acetate production commenced only after a lag period of 60-100 h. This finding suggests that the homoacetogenic population of digester sludge could be maintained by heterotrophic growth on sugars or other organic compounds, rather than by autotrophic growth on H(2)/CO(2). In the present study, two upflow anaerobic sludge bed (UASB) reactors were operated at 37 degrees C and 55 degrees C for two distinct trial periods, each characterised by the application of influents designed to enrich for homoacetogenic bacteria. Specific primers designed for the amplification of the functional gene encoding formyltetrahydrofolate synthetase (FTHFS), a key enzyme in the acetyl-CoA pathway of acetogenesis, were used as a specific probe for acetogenic bacteria. The diversity of acetogens in the granular sludge cultivated in each reactor was revealed by application of FTHFS targeted PCR. Results show that biomass acetogenic composition was dependent upon the operational temperature of the reactor and the substrate supplied as influent.