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     

Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.     

Extracellular polymers in granular sludge from different upflow anaerobic sludge blanket (UASB) reactors

Thermal extraction was used to quantify extracellular polymers (ECP) in granules from anaerobic upflow reactors. The optimal time for extraction was determined as the time needed before the intracellular material gives a significant contribution to the extracted extracellular material due to cell lysis. ECP contents of 41 to 92 mg · g^–1 volatile suspended solids of granules were found depending on the type of granular sludge examined. The content of polysaccharides, protein and lipids in the extracted ECP was quantified. Furthermore, the different methyl esters of the lipids were determined and quantified. Lower amounts of polysaccharides and proteins were found in the extracellular material from granules grown on methanogenic and acetogenic substrates compared to granules grown on more complex substrates. In contrast, the lipid content was lower on complex substrates. Changing the feed of an upflow anaerobic sludge blanket reactor from a sugar-containing waste-water to a synthetic waste-water containing acetate, propionate and butyrate resulted in a decrease in both the protein and polysaccharide content and an increase in the lipid content of the extracellular material. Furthermore, the amount of protein and polysaccharides in the ECP found under mesophilic conditions was significantly higher than under thermophilic conditions, while the lipid content was lower.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Effects of Temperature on Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.     

Development of thermophilic methanogenic sludge in compartmentalized upflow reactors

The characteristics and development of thermophilic anaerobic sludge in upflow staged sludge bed (USSB) reactors were studied. The compartmentalized reactors were inoculated with partially crushed mesophilic granular sludge and then fed with either a mixture of volatile fatty acids (VFA) or a mixture of sucrose and VFA. The staged degradation of the soluble substrate in the various compartments led to a clear segregation of specific types of biomass along the height of the reactor, particularly in reactors fed with the sucrose-VFA mixture. Both the biological as well as the physical properties of the cultivated sludge were affected by the fraction of nonacidified substrate. The sludge in the first compartment of the reactor treating the sucrose-VFA mixture was whitish and fluffy, most likely resulting from the development of acidifying bacteria. Sludge granules which developed in the top part of this reactor possessed the highest acetogenic and methanogenic activity and the highest granule strength as well. The experiments also revealed that the conversion of the sucrose-VFA mixture into methane gradually deteriorated at prolonged operation at high organic loading rates (50 to 100 g COD . L(-1) . day(-1)). Stable long-term performance of a reactor can only be achieved by preserving the sludge segregation along the height of the reactor. In the reactor fed solely with the VFA mixture little formation of granular sludge occurred. In this reactor, large differences in sludge characteristics were also observed along the reactor height. Li(+)-tracer experiments indicated that the hydraulic regime in the USSB reactor is best characterized by a series of at least five completely mixed reactors. The formation of granular sludge was found to influence the liquid flow pattern. (c) 1996 John Wiley & Sons, Inc.     

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.     

Thermophilic anaerobic digestion of high strength wastewaters

Investigations on the thermophilic anaerobic treatment of high-strength wastewaters (14-65 kg COD/m(3)) are presented. Vinasse, the wastewater of alcohol distilleries, was used as an example of such wastewaters. Semicontinuously fed digestion experiments at high retention times revealed that the effluent quality of digestion at 55 degrees C is comparable with that at 30 degrees C at similar loading rates. The amount of methane formed per kilogram of vinasse drops almost linearly with increasing vinasse concentrations. This can be attributed to increasing concentrations of inhibitory compounds, resulting in increasing volatile fatty acid (VFA) concentrations in the effluent. The treatment of vinasse was also investigated using upflow anaerobic sludge blanket (UASB) reactors. Thermophilic granular sludge, cultivated on sucrose, was used as seed material. The sludge required a 4-month adaptation period, during which the size of the sludge granules decreased significantly. However, the settling characteristics remained satisfactory. After adaptation, high loading and methane generation rates could be accommodated at satisfactory treatment efficiencies, namely, 86.4 kg COD/m(3) day and 26 m(3) CH(4)(STP)/m(3) day, respectively. As in the semicontinuously fed digesters, the effluent VFA concentrations were virtually independent of the loading rates applied, indicating that the toxicity of the vinasse is more important than the loading rate in determining the efficiency of the conversion of vinasse to methane.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor at pH 6 on an Acetate-Propionate Mixture

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatment of various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by a natural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge is cultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resembling Methanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stable sludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate by lowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operation of the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specific methanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2, respectively.     

Growth kinetics of thermophilic Methanosarcina spp. isolated from full-scale biogas plants treating animal manures

This study determines the growth kinetics of thermophilic strains of Methanosarcina spp. from full-scale thermophilic biogas plants. The complete set of kinetic parameters, including maximum specific growth rate μ(max), half saturation constant K(S), acetate threshold concentration and cell growth yield Y(X/S), were determined for six Methanosarcina strains newly isolated from full-scale reactors and the type strain Methanosarcina thermophila TM-1(T). The kinetic experiments were performed in media supplemented with acetate and activated carbon at the optimum growth temperatures of the individual strains, 50-55 degrees C. The μ(max) values of the isolates were in the range of 0.044-0.064 h(-1), the K(S) ranged from 6.5 to 24.7 mM acetate and the threshold for acetate utilization from 0.11 to 0.40 mM. The cell growth yields of the strains were between 0.78 and 2.97 g dry weight cells mol(-1) acetate. The six isolates exhibited significantly higher μ(max) and had higher affinity to acetate than the type strain M. thermophila TM-1(T). Generally, the affinities of thermophilic Methanosarcina strains tested in this study cover a similar range to those reported in the literature for mesophilic Methanosarcina spp. with acetate as substrate. The strains isolated from plants treating mixtures of animal manures and industrial organic wastes had higher affinity for acetate and lower thresholds than strains isolated from reactors operating solely on manures.     

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

From mesophilic to thermophilic conditions: one-step temperature increase improves the methane production of a granular sludge treating agroindustrial effluents

Objectives

To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor.

Results

One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8–2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2–2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions.

Conclusions

More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

     

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

The microbial communities established in mesophilic and thermophilic expanded granular sludge bed reactors operated with sulfate as the electron acceptor were analyzed using 16S rRNA targeted molecular methods, including denaturing gradient gel electrophoresis, cloning, and phylogenetic analysis. Bacterial and archaeal communities were examined over 450 days of operation treating ethanol (thermophilic reactor) or ethanol and later a simulated semiconductor manufacturing wastewater containing citrate, isopropanol, and polyethylene glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR-amplified 16S rRNA gene fragments using denaturing gradient gel electrophoresis revealed a defined shift in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 30°C to 55°C (thermophilic reactor). The addition of copper(II) to the influent of both reactors did not noticeably affect the composition of the bacterial or archaeal communities, which is in agreement with the very low soluble copper concentrations (3–310 μg l⁻¹) present in the reactor contents as a consequence of extensive precipitation of copper with biogenic sulfides. Furthermore, clone library analysis confirmed the phylogenetic diversity of sulfate-reducing consortia in mesophilic and thermophilic sulfidogenic reactors operated with simple substrates.     

Microbial characteristics of methanogenic sludge consortia developed in thermophilic U ASB reactors

The effect of temperature on granulation and microbial interaction of anaerobic sludges grown in thermophilic upflow anaerobic sludge bed (UASB) reactors was investigated at two different temperatures, 55°C (Run 1) and 65°C (Run 2). Each run consisted of two phases. Phase 1 was conducted by feeding acetate for a period of 200 days. In Phase 2, both reactors were fed a mixture of acetate and sucrose for a further 100 days. During Phase 1, no granulation occurred in the sludge of either run. Microscopic observation revealed that the predominant methanogen was Methanothrix in Run 1, whereas Methanobacterium-like bacteria existed to a significant extent in Run 2. The acetate-utilizing methanogenic activity of both sludges increased with increasing test temperature in the range 55–65°C. Since the acetate-grown sludges exhibited far higher H₂-utilizing methanogenic activity than acetate-utilizing methanogenic activity, it is suggested that a syntrophic association of acetate-oxidizing bacteria with hydrogenotrophic methanogens was responsible for a considerable portion of the overall acetate elimination in thermophilic anaerobic sludge. During Phase 2, granules coated with either filamentous bacteria or cocci-type bacteria (both presumably acid-forming bacteria) were successfully established in Run 1 and Run 2, respectively. Since the acetate-utilizing methanogenic activities of the granular sludges were four to five times higher than those of the acetate-grown sludges (Phase 1), the co-existence of these coating bacteria appeared to contribute to the enclosing of acetate consumers inside granules. Correspondence to: S. Uemura     

Strategies to suppress hydrogen-consuming microorganisms affect macro and micro scale structure and microbiology of granular sludge

Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2-bromoethanesulfonate (BES) and with BES + Chloroform, were applied to suppress hydrogen-consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors-R(Heat), R(BES), and R(BES + Chlo)--were inoculated with the treated sludges and fed with synthetic sugar-based wastewater (5 g(COD) L(-1), HRT 20-12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in R(Heat) was under 300 mL H(2) L(-1) day(-1), with a transient peak of 1,000 mL H(2) L(-1) day(-1) after decreasing HRT. In R(BES + Chlo) hydrogen production rate did not exceed 300 mL H(2) L(-1) day(-1) and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In R(BES), there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 ± 200 mL H(2) L(-1) day(-1). This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H(2)-consuming microorganisms can cause changes in the macro- and microstructure of granular sludge, which can be incompatible with the operation of high-rate reactors.     

Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO(2) to Methanogenesis in Cattle Waste at 40 and 60 degrees C

The quantitative contribution of fatty acids and CO(2) to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 muM/min to a peak (49 muM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 muM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 muM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 muM/min) was similar in both digestors. The proportion of CH(4) produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO(2) reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH(4) produced. Acetate synthesis from CO(2) was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 muM/min) than the mesophilic digestor (0.3 to 0.5 muM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

A new post-treatment system for anaerobic effluents containing a high Ca2+ content

A novel reactor concept has been developed that combines Ca²⁺ removal and BOD conversion under mesophilic (30 °C ) as well as under thermophilic (55 °C ) conditions. Soluble Ca²⁺, present in many industrial wastewaters, precipitates as a result of forced aeration in the aerobic bioreactor. The CaCO₃ precipitates, in turn, act as a carrier material for biofilm growth. This resulted in dense rapidly settling sludge granules covered with a biofilm on the surface. These sludge characteristics facilitate the application of hydraulic retention times of 1 h while maintaining a high biological activity in the reactor.