High Methanogenic Activity Research Articles

Microbial community signature of high-solid content methanogenic ecosystems

In this study, changes in bacterial and archaeal communities involved in anaerobic digestion processes operated with high solid contents were investigated. Batch tests were performed within a range of total solids (TS) of 10–35%. Between 10% and 25% TS, high methanogenic activity was observed and no overall specific structure of active bacterial communities was found. At 30% and 35%, methanogenesis was inhibited as a consequence of volatile fatty acids accumulation. Here, a specific bacterial signature was observed with three main dominant bacteria related to Clostridium sp., known for their ability to grow at low pH. Additionally, archaeal community was gradually impacted by TS content. Three archaeal community structures were observed with a gradual shift from Methanobacterium sp. to Methanosarcina sp., according to the TS content. Overall, several species were identified as biomarkers of methanogenesis inhibition, since bacterial and archaeal communities were highly specific at high TS contents.

Evaluation of Process Performance and Sludge Properties of an up-flow staged Sludge Blanket (USSB) reactor for Treatment of Molasses Wastewater

A combination of an acidification reactor and an up-flow staged sludge bed (USSB) reactor wasapplied to treatment of molasses wastewater. The USSB, equipped with three gas solid separators, wasselected because of superior organic removal by means of good retention of granular sludge. The combinedsystem was continuously operated at mesophilic (35°C) conditions for 600 days. The USSB achieved a highorganic removal rate of 37 kgCOD/m3 day (organic loading rate of 43 kgCOD/m3 day with 86.2% CODremoval). The USSB retained high sludge concentration of approximately 58 gVSS/L based on the reactorvolume. The retained granular sludge had good settle-ability with a sludge volume index (SVI) of 4 to 20 ml/gSS. The retained sludge possessed a sufficient level of methanogenic activities for acetate (1.2 gCOD/gVSS day) and for hydrogen (1.7 gCOD/gVSS day). Analysis of microbial community revealed that genus Methanobacterium as hydrogen utilizing methanogen and order Methanosarcinales as acetate utilizingmethanogen were detected in the retained sludge of the USSB reactor. The superior performance of the USSB was attributed to good retention of a large amount of granular sludge with high methanogenic activity.

Enhancement of starting up anaerobic digestion of lignocellulosic substrate: fique’s bagasse as an example

In Colombia there are 20,000ha of fique fields (Furcraea sp., family Agavaceae), that produce around 93,400tons of fique’s bagasse per year. These residuals are disposed into rivers and soil causing pollution. According to physicochemical characteristics, the lignocellulosic residues from fique crops (fique’s bagasse) are appropriate carbon source to biogas production. Anaerobic digestion from fique’s Bagasse (FB) requires a specialized microbial consortium capable of degrading its high lignocellulosic concentration. In this study, the capacities of seven microbial consortia for biomethane potential (BMP) from FB were evaluated. Inoculum of ruminal liquid achieved high hydrolytic activity (0.068gCOD/gVSSday), whereas pig waste sludge inoculum showed high methanogenic activity (0.146gCOD/gVSSday). Mixtures of these two inoculums (RL+PWS) showed the best yields for biomethane potential (0.3m3CH4/KgVSad).

High-rate treatment of molasses wastewater by combination of an acidification reactor and a USSB reactor

A combination of an acidification reactor and an up-flow staged sludge bed (USSB) reactor was applied for treatment of molasses wastewater containing a large amount of organic compounds and sulfate. The USSB reactor had three gas-solid separators (GSS) along the height of the reactor. The combined system was continuously operated at mesophilic temperature over 400 days. In the acidification reactor, acid formation and sulfate reduction were effectively carried out. The sugars contained in the influent wastewater were mostly acidified into acetate, propionate, and n-butyrate. In addition, 10–30% of influent sulfur was removed from the acidification reactor by means of sulfate reduction followed by stripping of hydrogen sulfide. The USSB achieved a high organic loading rate (OLR) of 30 kgCOD m−3 day−1 with 82% COD removal. Vigorous biogas production was observed at a rate of 15 Nm3 biogas m−3 reactor day−1. The produced biogas, including hydrogen sulfide, was removed from the wastewater mostly via the GSS. The GSS provided a moderate superficial biogas flux and low sulfide concentration in the sludge bed, resulting in the prevention of sludge washout and sulfide inhibition of methanogens. By advantages of this feature, the USSB may have been responsible for achieving sufficient retention (approximately 60 gVSS L−1) of the granular sludge with high methanogenic activity (0.88 gCOD gVSS−1 day−1 for acetate and as high as 2.6 gCOD gVSS−1 day−1 for H2/CO2). Analysis of the microbial community revealed that sugar-degrading acid-forming bacteria proliferated in the sludge of the USSB as well as the acidification reactor at high OLR conditions.

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30 degrees C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH(4)/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.

Evaluation of Influent Medium Composition and Concentration in a Batch Reactor for Effective Startup of UASB Reactors

This study demonstrated the use of batch bioreactor experiments as an evaluation tool to determine appropriate influent medium composition and concentration for effective startup of an upflow anaerobic sludge blanket (UASB) reactor. Using seed sludge from a domestic treatment plant, the weight ratio of glucose and volatile fatty acids (VFA) present in the influent synthetic medium was varied and the batch performance was assessed in terms of pH variation during the course of the experiment, chemical oxygen demand (COD) reduction, specific substrate uptake rates, methanogenic activity, and methane yield. The performance was also evaluated by increasing the concentration of influent COD. Medium composition with glucose to VFA weight ratio at or below 2:8 and COD concentration at 11g∕L was determined to be optimum for effective startup of the UASB reactors. Utilization of the optimized influent medium provided a pH variation from 6.5 to 7.8 over the length of the UASB reactor and resulted in granule formation, high methanogenic activity, and methane yield. The evaluation method provided a practical approach to determine the applicability of seed sludge from a particular source and the desired influent characteristics for reduced startup duration in UASB processes.

Changes in process performance and microbial characteristics of retained sludge during low-temperature operation of an EGSB reactor

A lab-scale expanded granular sludge bed (EGSB) reactor was seeded with granular sludge and operated to investigate the influence of temperature decrease on both process performance and the microbial community structure of the granular sludge. Synthetic wastewater containing sucrose and volatile fatty acids was used as feed. The EGSB reactor was brought online at a starting temperature of 15°C and was reduced stepwise to a final temperature of 5°C. The reactor exhibited sufficient COD removal efficiency between 10°C and 15°C. However at 5°C serious deterioration of process performance was observed. The methane-producing activity of the retained sludge increased when it was compared to the activity of the seed sludge (day 0) during 10°C to 15°C operation. When hydrogen fed, sludge showed much higher methanogenic activity as compared with seed sludge activity at test temperatures of 15°C and 20°C on day 196 of reactor operation. At this time, proliferation of the genus Methanospirillum in the retained sludge was observed and a decrease in Methanobacterium species was also measured. Throughout the experiment, the genus Methanosaeta was detected in abundance and the community structure of the Domain Bacteria was stably maintained. The sugar-degrading acid-forming bacteria, Lactococcus and Anaerovibrio were detected in the retained sludge throughout the experiment as well and the propionate-degrading acetogen Syntrophobacter fumaroxidans was also detected, although its population size decreased at 5°C.

Treatment of domestic wastewater by using a microaerobic membrane bioreactor

The performance of an innovative process, a microaerobic membrane bioreactor seeded with anaerobic granular sludge, was investigated for simultaneous removal of organic substances and nitrogen from domestic synthetic wastewaters. Total COD removal efficiency of more than 93% was achieved, unaffected by the changes in influent COD and HRT throughout the entire operation. Nitrogen was removed through the simultaneous nitrification and denitrification process. The N-removal rate of the system was 20–45 mg N L −1 d −1 at a nitrogen loading rate of 26–67 mg N L −1 d −1. Nitrification was the rate-limiting step. The average TN removal efficiency increased from 77% at a COD/TN ratio of 19.5 to 87% at a ratio of 7.7. The batch assay with granules can achieve 92% COD removal and 84% TN removal, whereas the COD and TN removal declined to only 84% and 67%, respectively, after the granules were homogenized. The microaerobic granules also remained at a relatively high methanogenic activity. The test results showed that cake layer resistance was the major resistance to the membrane filter.

Oxygen effects on methane production and oxidation in humid tropical forest soils

AbstractWe investigated the effects of oxygen (O2) concentration on methane (CH4) production and oxidation in two humid tropical forests that differ in long‐term, time‐averaged soil O2 concentrations. We identified sources and sinks of CH4 through the analysis of soil gas concentrations, surface emissions, and carbon isotope measurements. Isotope mass balance models were used to calculate the fraction of CH4 oxidized in situ. Complementary laboratory experiments were conducted to determine the effects of O2 concentration on gross and net rates of methanogenesis. Field and laboratory experiments indicated that high levels of CH4 production occurred in soils that contained between 9±1.1% and 19±0.2% O2. For example, we observed CH4 concentrations in excess of 3% in soils with 9±1.1% O2. CH4 emissions from the lower O2 sites were high (22–101 nmol CH4 m−2 s−1), and were equal in magnitude to CH4 emissions from natural wetlands. During peak periods of CH4 efflux, carbon dioxide (CO2) emissions became enriched in 13C because of high methanogenic activity. Gross CH4 production was probably greater than flux measurements indicated, as isotope mass balance calculations suggested that 48–78% of the CH4 produced was oxidized prior to atmospheric egress. O2 availability influenced CH4 oxidation more strongly than methanogenesis. Gross CH4 production was relatively insensitive to O2 concentrations in laboratory experiments. In contrast, methanotrophic bacteria oxidized a greater fraction of total CH4 production with increasing O2 concentration, shifting the δ13C composition of CH4 to values that were more positive. Isotopic measurements suggested that CO2 was an important source of carbon for methanogenesis in humid forests. The δ13C value of methanogenesis was between −84‰ and −98‰, which is well within the range of CH4 produced from CO2 reduction, and considerably more depleted in 13C than CH4 formed from acetate.

Dynamics of methane in mesotrophic Lake Biwa, Japan

AbstractAs a part of a core project of IGBP (International Geosphere‐Biosphere Programme), distribution, production, oxidation and transport processes of methane in bottom sediments and lake water in a mesotrophic lake (Lake Biwa) have been studied with special reference to the spatial heterogeneity of each process. In this study, we attempted to synthesize previously reported results with newly obtained ones to depict the methane dynamics in the entire lake. The pelagic water column exhibited subsurface maxima of dissolved methane during a stratified period. Transect observation at the littoral zone suggested that horizontal transportation may be a reason for the high methane concentration in epilimnion and thermocline at the offshore area. Tributary rivers and littoral sediments were suggested to be the source. Observations also showed that the internal wave caused resuspension of the bottom sediment and release of methane from the sediment into the lake water. The impact of the internal waves was pronounced in the late stage of a stratified period. The littoral sediment showed much higher methanogenic activity than the profundal sediments, and the bottom water of the littoral sediments had little methanotrophic activity. In the profundal sediment, most of the methane that diffused up from the deeper part was oxidized when it passed through the oxic layer. Active methane oxidation was also observed in the hypolimnetic water, while the lake water in the epilimnion and thermocline showed very low methane oxidation, probably due to the inhibitory effect of light. These results mean a longer residence time for methane in the epilimnion than in the hypolimnion. Horizontal inflow of dissolved methane from the river and/or littoral sediment, together with the longer residence time in the surface water, may cause the subsurface maxima, which have also been observed in other lakes and in the ocean.

Volatile fatty acid formation in an anaerobic hybrid reactor

A laboratory scale model reactor was used to investigate volatile fatty acid (VFA) formation during anaerobic degradation in an anaerobic hybrid reactor. The purpose of the study was to determine the role of upflow anaerobic sludge blanket (UASB) and anaerobic filter (AF) regions of the hybrid reactor on bio-degradation of organic compounds using VFA as an operational parameter. Acetic, propionic, butyric, and valeric acid concentrations of samples taken from the reactor effluent, fixed bed region and the upper part of the sludge bed region were analysed. VFA formation was monitored at two phases. In phase-I, 10 kg COD m −3 per day organic loading rate (OLR) ( C o=10,000 mg COD l −1 and θ H=1 day) and in phase-II 7.6 kg COD m −3 per day OLR ( C o=15,000 mg COD l −1 and θ H=2 days) was applied. The average acetic acid concentrations of the upper end of the reactor and sludge bed region were 150 and 354.9 mg l −1, respectively. These results showed that VFA concentrations increase from the top to the bottom of the reactor, indicating higher methanogenic activity in the upper part and higher acetogenic activity in the bottom of the reactor. pH measurements confirmed this result.

Variations in organic geochemistry and lithology of a carbonate sequence deposited in a backplatform basin (Triassic, Hungary)

The variations in the organic matter quantity and quality were studied with respect to the mineral composition of the carbonate sequences accumulated on a gentle slope (Zl-1 well) and at the toe of the slope (Rzt-1 well) located between a Late Triassic carbonate platform and a backplatform basin. Parallel variations observed in mineral composition and organic geochemical features of the successions appeared to be controlled by the change in climate and by sea-level fluctuations. The repetitive sea-level changes resulted in a variation in the carbonate-rich basin facies and in the mineralogically heterogenous slope and toe-of-slope ones. According to Rock Eval pyrolysis, organic petrography and carbon isotope ratios, the immature organic matter is of predominantly marine origin and composed of mainly liptinites in both of the studied boreholes. The results of the GC and GC/MS analyses of the saturated hydrocarbon fractions of bitumens together with the composition of kerogen pyrolysates reveal a predominant algal input with a minor variable bacterial and subordinate terrestrial contribution for the Rzt-1 borehole. In the Zl-1 borehole a significantly higher proportion of the bacterial biomass contributed to the organic precursors. The δ13C values and the composition of the kerogen pyrolysates together with the results of the maceral analysis and GC data suggest a relatively higher, but moderate, higher plant derived contribution in the slope facies and at the top of the toe-of-slope facies. The elementary composition of kerogens and Rock Eval data display type II-S kerogen in the basin and the slope facies, and type I-II-S one in the toe-of-slope facies. Variations in the hydrogen content of the organic matter mainly reflect variations in the preservation conditions and in primary productivity. The presence of the 2,6,10,15,19-pentamethyleicosane and the extremely low pristane/phytane ratios indicate a relatively high methanogenic bacterial activity and strongly anoxic depositional conditions in the Rzt-1 well, especially in two most organic-rich toe-of-slope facies.

Ammonia inhibition on thermophilic aceticlastic methanogens

The inhibition effects of total ammonia nitrogen (TAN) on aceticlastic methanogenic activity using biomass from thermophilic anaerobic reactors were investigated in this study. Anaerobic Toxicity Assays (ATA) were conducted after the biomass was acclimated to different levels of TAN. The TAN background concentrations in the reactors were 400, 1,200, and 3,050 mg/L. The results from ATA showed: 1) high TAN concentrations could cause inhibition of aceticlastic methanogens; 2) biomass acclimated to higher TAN concentrations could alleviate the inhibition effect due to the increase of TAN concentration; 3) the lethal TAN concentration for methanogens was approximately 10,000 mg/L regardless of the background TAN concentration; 4) ATA results also revealed the role played by pH. At a given TAN concentration, methanogenic activity varied with the pH values. The highest methanogenic activity was always observed at a pH of 7.0-7.5. 5) It was also observed that acclimation could increase the pH tolerance range, which made methanogens less sensitive to pH changes.

Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor.

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH(4)/g of volatile suspended solids [VSS].day or 1.1 g of CH(4) chemical oxygen demand/g of VSS.day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.

Seasonal variations in vertical distribution of methanogenic activity and Fe(II) content and relationship between them in wetland rice field soil

Methanogenic activity and Fe(II) content in soil samples collected at different depths of the plow layer in a wetland rice field were determined over a period of 1 y. The methanogenic activity increased at first in the top layer of soil (0–1 cm in depth) during the continuous flooding period of the field and the activity decreased after the period of midseason drainage. In contrast, in the deeper soil layers, the increase in the activity persisted during the period of intermittent irrigation after midseason drainage and the period of the highest methanogenic activity at each depth in the year varied for about 1 month depending on the depth. The Fe(II) content in soil did not change appreciably during the continuous flooding period regardless of the soil depth. However, after midseason drainage, the content significantly varied with depth. Thus, until the midseason drainage, a sharp increase in the methanogenic activity preceded the increase in the Fe(II) content in the surface (0–1 cm) soil layer, while the opposite was observed in the deeper soil layers. Based on these data, the relationship between the methanogenic activity and Fe(II) content in soil was investigated by calculating the correlation coefficients between them and comparing the slopes and constants of regression lines obtained. It was found that although during the cropping season of rice the methanogenic activity depended mainly on the Fe(II) content in soil as a whole, the relationship between the two parameters was different before and after the period of midseason drainage. The relationship between them before the midseason drainage was distinctly different depending on the depth, while after the midseason drainage the relationship was almost the same irrespective of the depth. During the non-cropping season, no correlation was obtained between the two parameters. Most of the correlation coefficients between the methanogenic activity and the soil temperature were low for all the data sets examined except for the deepest soil layer (13–15 cm) during the cropping season. Correlation coefficients between the methanogenic activity and water content in soil were also low.

Two-particle model of anaerobic solid state fermentation

A structured mathematical model of anaerobic solid state fermentation (ASSF) has been developed. Since a stable ASSF requires addition of significant quantities of methanogenic seed sludge and mass-transfer limitation becomes important, the model postulates the existence of two different types of particles inside the fermenting solid mass--so-called "seed" particles with low biodegradability and high methanogenic activity and so-called "waste" particles with high biodegradability and low methanogenic activity. Any particle is assumed to be a completely mixed reactor and mass transfer of solutes between the particles is brought about by diffusion. The model includes multiple-reaction stoichiometry, microbial growth kinetics, material balances, liquid-gas interactions and liquid phase equilibrium chemistry. The theoretical model agrees on the qualitative level with existing experimental studies of ASSF. Hypothetical computer simulations are presented to illustrate the influence of biodegradability and mass transfer intensity on the stability of ASSF. On this basis, possible measures are proposed to prevent accumulation of volatile fatty acids inside the "seed" particles beyond their assimilative methanogenic capacity.

Effects of organic matter application, temperature, and sunshine duration on seasonal and annual variations of methanogenic activity in wetland rice field soil

Methanogenic activity of the soil fractions obtained after soil collection from the plow layer of a wetland rice field treated with rice straw was measured by incubating the soil slurries in vitro and seasonal and annual variations were monitored during consecutive 5 y. In this rice field, after a sharp increase in activity, the high activity was maintained for 1 or 2 months and then the activity rapidly decreased. Although almost the same pattern of variations was repeated every year, the period of increase and decrease in activity was different depending on the year and the variations did not correspond to the growth of the rice plants or water management of the field. Differences in the time of the first sharp increase in methanogenic activity were more related to the cumulative sunshine duration than to the cumulative air temperature. Fe2+ concentration in the soil treated with rice straw increased after flooding of soil and it decreased to lower levels until the middle of September. The period of high Fe2+ concentration in soil corresponded to the period of high methanogenic activity. Methane concentration in soil gas increased rapidly soon after flooding and reached a level of 35 to 50% until the beginning of June. In rice field soil without application of organic matter, the methanogenic activity never reached such high levels as those in soil treated with rice straw. Methanogenic activity was also detected in soil after harvest of rice as well as in winter, although, with some exceptions, the activity was much lower than that during the cropping season. The vertical profiles of methanogenic activity in the plow layer showed that the activity was distributed rather heterogeneously and the pattern of distribution significantly changed depending on the season.

The bacterial numeration and an observation of a new syntrophic association for granular sludge

The bacterial numeration and microbial observation were made on granular sludge from laboratory single and two-phase UASB reactors. It was shown that the fermentative bacteria (group I), H2-producing acetogenic bacteria (group II) and methanogenic bacteria (group III) of granular sludge in single UASB reactor were 9.3 × 108−4.3 × 109, 4.3 × 107−4.3 × 108, 2.0−4.3 × 108, respectively, during the granulation process. The sludge of methanogenic reactor exhibited the similar results. That indicates there is no big difference between suspended and granular sludge, and bacterial population for three groups of anaerobic bacteria are similar. The formation of granular sludge depends mainly on the organization and arrangement of bacteria. An observation of granular sludge using electron microscope revealed that the fermentative bacteria and hydrogenotrophic methanogens existed on outer surface of granules, and aceticlastic methanogens and H2-producing acetogenic bacteria occupied the inner layer. A new syntrophic association between Methanosaeta sp. and Syntrophomonas sp. (even plus Methanobrevibacter sp.) was observed. Because Methanosaeta can effectively convert the acetate (the end product of propionate and butyrate) to methane, such a new syntrophic association is supposed to support the degradation of short fatty acids and high methanogenic activity of granular sludge. Based on structural pattern, a hypothesis on mechanism of granulation called “crystallized nuclei formation” is proposed.

Acidification during anaerobic treatment of brewery wastewater

Several experimental tests were performed to study acidification during the anaerobic treatment of brewery wastewater. Batch test results suggested that a pH of 5-6 and a water temperature of 40°C were optimal conditions for acidification. Continuous-flow test results indicated a tendency for the carbohydrate reduction percentage to become higher with longer retention time in the acidification reactor. A high Specific Methanogenic Activity (SMA) of over 1.4 kg COD kg−1 MLVSS d−1 was observed, which the carbohydrate reduction percentage exceeded 75%. However, there was a tendency for the SMA to become lower due to residues of highly concentrated carbohydrates. The carbohydrate reduction percentage dropped to below 60% in the acidification treated water, when the retention time in the acidification reactor was extremely short. For achieving a carbohydrate reduction percentage of more than 75%, the retention time showed a tendency to become longer with higher organic concentration (e.g. COD 2,500-4,000 mg l−1: 3 h, COD 5,000-8,000 mg l−1: 6 h, and COD 10,000-16,000 mg l−1: 12 h).

The bacterial numeration and an observation of a new syntrophic association for granular sludge

The bacterial numeration and microbial observation were made on granular sludge from laboratory single and two-phase UASB reactors. It was shown that the fermentative bacteria (group I), H2-producing acetogenic bacteria (group II) and methanogenic bacteria (group III) of granular sludge in single UASB reactor were 9.3 × 108−4.3 × 109, 4.3 × 107−4.3 × 108, 2.0−4.3 × 108, respectively, during the granulation process. The sludge of methanogenic reactor exhibited the similar results. That indicates there is no big difference between suspended and granular sludge, and bacterial population for three groups of anaerobic bacteria are similar. The formation of granular sludge depends mainly on the organization and arrangement of bacteria. An observation of granular sludge using electron microscope revealed that the fermentative bacteria and hydrogenotrophic methanogens existed on outer surface of granules, and aceticlastic methanogens and H2-producing acetogenic bacteria occupied the inner layer. A new syntrophic association between Methanosaeta sp. and Syntrophomonas sp. (even plus Methanobrevibacter sp.) was observed. Because Methanosaeta can effectively convert the acetate (the end product of propionate and butyrate) to methane, such a new syntrophic association is supposed to support the degradation of short fatty acids and high methanogenic activity of granular sludge. Based on structural pattern, a hypothesis on mechanism of granulation called “crystallized nuclei formation” is proposed.