不同预处理条件对海水养殖废弃物发酵产氢的影响

【目的】利用海水养殖场有机废弃物厌氧发酵产氢,可在减少有机污染物的同时获取氢气。【方法】以海水养殖场有机废弃物为底物,比较嗜热酶(S-TE)、酸、碱、灭菌、微波不同预处理方法对厌氧发酵产氢效果的影响,并对发酵过程中底物性质变化[SCOD、可溶性蛋白质、可溶性糖、pH、VFAs(挥发性脂肪酸)和乙醇]进行探讨。【结果】灭菌预处理产氢效果最好,产氢率为22.0 mL/g VSS,酸处理的效果最差,产氢率为7.6 mL/g VSS。可溶性糖大量消耗之后,氢气不再产生。接种S-TE预处理污泥的底物能更多地释放营养物质,并在整个发酵过程中保持较为稳定的pH值。发酵过程中产生的VFAs主要成分是乙酸,在发酵后期出现乙醇。【结论】灭菌预处理是海水养殖场有机废弃物厌氧发酵产氢的最佳预处理方法,可溶性糖为这一过程主要的营养来源。     

Feasibility of hydrogen production in thermophilic mixed fermentation by natural anaerobes

The biological sludge from an animal wastewater treatment plant was treated to enrich hydrogen-producing mixed bacteria, and effects on hydrogen yield were investigated during anaerobic fermentation at 55 degrees C. Enrichment of hydrogen-producing bacteria was conducted at pH adjustment of inocula to 3 and 5 with and without additional heat treatment (NHT and HT). The enriched mixed bacteria were cultivated at initial pHs of 5, 6, and 7 with synthetic organic wastewater containing different levels of nitrogen (2.0 and 0.8 g/l as total nitrogen) under static batch conditions. The main effects of heat treatment and enrichment pH were significant on hydrogen production. There was no significant effect of different nitrogen concentrations on hydrogen production. The methane-free biogas contained hydrogen levels of up to 64% for a fermentative condition that showed maximum hydrogen evolution (at culture pH 5 after enrichment at pH 5 with HT). The dominating intermediate metabolites were acetate, n-butyrate, and ethanol. Yields of produced hydrogen were significantly dependent upon levels of n-butyrate.     

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.     

Effects of pH and hydraulic retention time on hydrogen production versus methanogenesis during anaerobic fermentation of organic household solid waste under extreme-thermophilic temperature (70 degrees C)

Two continuously stirred tank reactors were operated with household solid waste at 70 degrees C, for hydrogen and methane production. The individual effect of hydraulic retention time (HRT as 1, 2, 3, 4, and 6 days) at pH 7 or pH (5, 5.5, 6, 6.5, 7) at 3-day HRT was investigated on the hydrogen production versus methanogenesis. It was found that at pH 7, the maximum hydrogen yield was 107 mL-H(2)/g VS(added) (volatile solid added) but no stable hydrogen production was obtained as after some time methanogenesis was initiated at all tested HRTs. This demonstrated that sludge retention time alone was not enough for washing out the methanogens at pH 7 under extreme-thermophilic conditions. Oppositely, we showed that keeping the pH level at 5.5 was enough to inhibit methane and produce hydrogen stably at 3-day HRT. However, the maximum stable hydrogen yield was low at 21 mL-H(2)/g VS(added).     

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Hydrogen and methane production from desugared molasses by a two‐stage thermophilic anaerobic process was investigated in a series of two up‐flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen‐producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H₂/day/L, corresponding to a yield of 132 mL H₂/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH₄/day/L, corresponding to a yield of 239 mL CH₄/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H_2, 38.7% CO₂, and 44.8% CH₄, containing approximately 15% energy by hydrogen is viable to be bio‐hythane.     

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.     

A consolidated bio-processing of ethanol from cassava pulp accompanied by hydrogen production

A biphasic fermentation approach was undertaken for the production of ethanol and hydrogen from cassava pulp. The glucose generated by co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense was 13.65±0.45 g L(-1), which was 1.75 and 1.17-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. The accumulated glucose could be utilised rapidly by subsequently inoculated Saccharomyces cerevisiae. An inoculum ratio of 1:1, a thermophilic fermentation of 84 h, and a pulp concentration of 4% proved optimal for ethanol production, fermentation efficiency, and productivity. With these conditions, the ethanol level reached 8.83±0.31 g L(-1) with a fermentation efficiency of 64.95±2.71%. Hydrogen production of 4.06 mmol by the co-culture system was 1.54 and 2.09-fold greater than that produced by mono-cultures of C. thermocellum and T. aotearoense, respectively. This sequential co-culture approach provided a consolidated bio-processing means to produce ethanol and hydrogen from cassava pulp.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier     

pH值和产甲烷抑制剂对两相耦合系统污泥发酵定向产乙酸的影响

采用产氢产乙酸/同型产乙酸两相耦合工艺对剩余污泥进行了半连续式厌氧发酵,主要研究了pH值和产甲烷抑制剂2-bromoethanesulphonate(BES)对耦合系统定向产乙酸的影响.结果表明:碱性pH(pH=10.0)和添加BES都能促进A相乙酸的积累,提高乙酸的产率,同时碱性pH比添加BES更有利于污泥的水解.当...     

Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15

Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3−) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3− also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3− as a buffering agent and the effect of HCO3−-consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens. Rate differences in TCE dechlorination were observed as a result of added varying HCO3− concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3− consumption. Significantly faster dechlorination rates were noted at all HCO3− concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3− concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3− were provided initially. Our study reveals that HCO3− is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3− and the changes in pH exerted by methanogens and homoacetogens.     

Effects of temperature and initial pH on biohydrogen production from food-processing wastewater using anaerobic mixed cultures

This study attempted to determine the optimal temperature and initial cultivation pH by conducting a series of batch tests in stirred-tank bioreactor using fructose-producing wastewater as an organic substrate. The bioreactor temperature was controlled at 35–55°C with an initial pH of 4–8. Hydrogen production efficiency was assessed using specific hydrogen production potential (SHPP) and the maximum specific hydrogen production rate (SHPR_m). Experimental results indicated that temperature and initial pH markedly affected SHPP and SHPR_m, volatile fatty acids distribution as well as the ratio of butyrate/acetate (BHu/HAc). Two-fold higher SHPP and SHPR_m were obtained at thermophilic condition (55°C) than those at mesophilic condition (35°C). The optimal initial pH was 6 for hydrogen production with peak values of SHPP of 166.8 ml-H₂/g-COD and SHPR_m of 26.7 ml-H₂/g-VSS-h for fructose-processing wastewater. Molasses-processing wastewater had a higher SHPP (187.0 ml-H₂/g-COD) and SHPR_m (42.7 ml-H₂/gVSS-h) than fructose-processing wastewater at pH 6. The DGGE profiles indicated that molasses-processing wastewater is a better substrate than fructose-processing wastewater for growth of hydrogen-producing bacteria due to the high staining intensity of bands.     

The metabolism of hydrogen by extremely thermophilic, sulfur-dependent bacteria

Abstract In just the last few years, a group of bacteria have been discovered that have the remarkable property of growing near and above 100°C. These extremely thermophilic organisms, defined here as having the ability to grow at 90°C with optimum growth at 80°C and above, have been isolated mainly from sulfur-rich, marine geothermal environments, both shallow and deep sea. They comprise over a dozen different genera, and except for one novel eubacterium, all may be classified as archaebacteria. The majority of the extremely thermophilic genera metabolize elemental sulfur (S°) and a survey of the various organisms reveals that most of them also depend upon the oxidation of hydrogen gas (H₂) as an energy source. In addition, two extremely thermophilic genera are known that actively produce H₂ as end-products of novel fermentative metabolisms. The enzyme hydrogenase, which is responsible for catalysing H₂ activation and H₂ production, appears to play several roles in electron and energy transfer during the growth of these organisms. Hydrogenase has so far been purified from only one extremely thermophilic species, from Pyrococcus furiosus ( T _opt = 100°C), and hydrogenase activity has been exmained in cell-free extracts of only a few others. However, a comparison of their properties with those of hydrogenases from mesophilic bacteria suggests that (a) the hydrogenase responsible for catalysing H₂ oxidation in extremely thermophilic organisms may be an extremely thermostable version of the mesophilic enzyme, and (b) a new type of 'evolution' hydrogenase, lacking the Ni-S or Fe-S catalytic sites of the mesophilic enzymes, is required for catalysing H₂ evolution at temperatures near and above 100°C.     

Evaluation of methods for preparing hydrogen-producing seed inocula under thermophilic condition by process performance and microbial community analysis

Five methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid (BESA), load-shock and heat shock treatments) as well as an untreated anaerobic digested sludge were compared for their hydrogen production performance and responsible microbial community structures under thermophilic condition (60 degrees C). The results showed that the load-shock treatment method was the best for enriching thermophilic hydrogen-producing seeds from mixed anaerobic cultures as it completely repressed methanogenic activity and gave the a maximum hydrogen production yield of 1.96 mol H(2) mol(-1) hexose with an hydrogen production rate of 11.2 mmol H(2) l(-1)h(-1). Load-shock and heat-shock treatments resulted in a dominance of Thermoanaerobacterium thermosaccharolyticum with acetic acid and butyric acid type of fermentation while base- and acid-treated seeds were dominated by Clostridium sp. and BESA-treated seeds were dominated by Bacillus sp. The comparative experimental results from hydrogen production performance and microbial community analysis showed that the load-shock treatment method was better than the other four methods for enriching thermophilic hydrogen-producing seeds from anaerobic digested sludge. Load-shock treated sludge was implemented in palm oil mill effluent (POME) fermentation and was found to give maximum hydrogen production rates of 13.34 mmol H(2) l(-1)h(-1) and resulted in a dominance of Thermoanaerobacterium spp. Load-shock treatment is an easy and practical method for enriching thermophilic hydrogen-producing bacteria from anaerobic digested sludge.     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process

The present study investigated a two-stage anaerobic hydrogen and methane process for increasing bioenergy production from organic wastes. A two-stage process with hydraulic retention time (HRT) 3 d for hydrogen reactor and 12 d for methane reactor, obtained 11% higher energy compared to a single-stage methanogenic process (HRT 15 d) under organic loading rate (OLR) 3 gVS/(L d). The two-stage process was still stable when the OLR was increased to 4.5 gVS/(L d), while the single-stage process failed. The study further revealed that by changing the HRT_hydrogen:HRT_methane ratio of the two-stage process from 3:12 to 1:14, 6.7%, more energy could be obtained. Microbial community analysis indicated that the dominant bacterial species were different in the hydrogen reactors (Thermoanaerobacterium thermosaccharolyticum-like species) and methane reactors (Clostridiumthermocellum-like species). The changes of substrates and HRT did not change the dominant species. The archaeal community structures in methane reactors were similar both in single- and two- stage reactors, with acetoclastic methanogens Methanosarcina acetivorans-like organisms as the dominant species.     

Review of solid state fermentation for lignocellulolytic enzyme production: challenges for environmental applications

Within the context of increasing environmental concern, energy production from lignocellulosic substrates is gaining great interest. Enzymes have proven their efficiency in the degradation of the lignocellulosic complex but their use remains limited in environmental applications such as anaerobic digestion mainly due to their prohibitive cost. Therefore, solid state fermentation (SSF) emerges as an interesting alternative for the in situ production of lignocellulolytic enzymes. Various research efforts on the lab scale optimization of SSF are discussed. They are presented according to the type of inoculum used in the process: bacterial species and fungal species under both mesophilic and thermophilic conditions. In general, parameters that impact the SSF process include: substrate type and particle size, substrate pretreatment, inoculum, nutrient supplementation, moisture content, pH, aeration, temperature and mixing. Using different substrates, authors aim at maximizing enzyme production taking into account one to several of the indicated operational parameters. The reviewed research puts forward the adaptation of the operational parameters, enzyme production cost and loading, enzyme mixture quality and efficiency and finally reactor design as the main challenges for environmental large-scale application.     

Diversity and variability of methanogens during the shift from mesophilic to thermohilic conditions while biogas production

Anaerobic digestion (AD) is the most popular path of organic waste disposal. It is often used in wastewater treatment plants for excessive sludge removal. Methanogenic fermentation had usually been performed under mesophilic conditions, but in the past few years the thermophilic processes have become more popular due to economics and sludge sanitation. Methanogens, the group of microorganisms responsible for methane production, are thought to be sensitive to temperature change and it has already been proven that the communities performing methanogenesis under mesophilic and thermophilic conditions differ. But in most cases the research performed on methanogen diversity and changeability was undertaken in two separate anaerobic chambers for meso- and thermophilic conditions. It is also known that there is a group of microorganisms performing AD which are insensitive to temperature. Also the linkage between digester performance and its microbial content and community changeability is still not fully understood. That is why in this experiment we analyzed the bacterial community performing methanogenesis in a pilot scale anaerobic chamber during the shift from mesophilic to thermophilic conditions to point at the group of temperature tolerant microorganisms and their performance. The research was performed with PCR–DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis). It occurred that the community biodiversity decreased together with a temperature increase. The changes were coherent for both the total bacteria community and methanogens. These bacterial shifts were also convergent with biogas production—it decreased in the beginning of the thermophilic phase with the bacterial biodiversity decrease and increased when the community seemed to be restored. DGGE results suggest that among a wide variety of microorganisms involved in AD there is a GC-rich group relatively insensitive towards temperature change, able to adapt quickly to shifts in temperature and perform AD effectively. The studies of this microbial group could be a step forward in developing more efficient anaerobic digestion technology.     

Effect of inhibition treatment, type of inocula, and incubation temperature on batch H2 production from organic solid waste

Two types of induction treatments (heat-shock pretreatment, HSP, and acetylene, Ac), inocula (meso and thermophilic) and incubation temperatures (37 and 55 degrees C) were tested according to a full factorial design 2(3) with the aim of assessing their effects on cumulative H(2) production (P(H), mmol H(2)/mini-reactor), initial H(2) production rate (R(i,H), micromol H(2)/(g VS(i) x h)), lag time (T(lag), h), and metabolites distribution when fermenting organic solid waste with an undefined anerobic consortia in batch mini-reactors. Type of inocula did not have a significant effect on P(H), T(lag), and R(i,H) except for organic acids production: mini-reactors seeded with thermophilic inocula had the highest organic acid production. Concerning the induction treatment, it was found that on the average Ac only affected in a positive way the P(H) and T(lag). Thus, P(H) in Ac-inhibited units (6.97) was 20% larger than those in HSP-inhibited units (5.77). Also, Ac favored a shorter T(lag) for P(H) in comparison with HSP (180 vs. 366). Additionally, a positive correlation was found between H(2) and organic acid production. In contrast, solvent concentration in heat-shocked mini-reactors were slightly higher than in reactors spiked with Ac. Regarding the incubation temperature, on the average mesophilic temperature affected in a positive and very significant way P(H) (10.07 vs. 2.67) and R(i,H) (2.43 vs. 0.76) with minimum T(lag) (87 vs. 459). The positive correlation between H(2) and organic acids production was found again. Yet, incubation temperature did not seem to affect solvent production. A strong interaction was observed between induction treatment and incubation temperature. Thus, Ac-inhibited units showed higher values of P(H) and R(i,H) than that HSP-inhibited units only under thermophilic incubation. Contrary to this, HSP-inhibited units showed the highest values of P(H) and R(i,H) only under mesophilic conditions. Therefore, the superiority of an induction treatment seems to strongly depend on the incubation temperature.     

Thermophilic bioremediation of whey: effect of physico-chemical parameters on the efficiency of the process

A two-stage process for the bioremediation of blue Stilton whey has been developed. It employs both naturally occurring thermotolerant organisms found in whey (lactic acid bacteria and yeast) and a thermophilic isolate (Bacillus sp.). Thermophilic digestion occurred only at neutral pH. Multiple substrates were consumed simultaneously under mesophilic but not thermophilic conditions.     

Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids

An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.