ÐAn anaerobic microbial consortium developed in our laboratory showed dechlorination of spiked po... more ÐAn anaerobic microbial consortium developed in our laboratory showed dechlorination of spiked polychlorinated biphenyls (PCBs), such as Aroclor 1254 and a de®ned congener 2,3,4,5,6-penta-chlorobiphenyl (CB) in the presence and the absence of lake sediment. Glucose and methanol were used as carbon and energy sources. Highly chlorinated congeners (containing >5 chlorines) such as penta-, hexa-and heptachlorobiphenyls were preferentially dechlorinated both in the presence and the absence of sediment with a simultaneous change in the distribution of relative amounts of tri-and tetra-chlorobiphenyl congeners. The dechlorination pattern and rates observed in both sediment and sediment free conditions were similar. In 24 weeks, almost 70% of the PCBs were dechlorinated without accumulation of any speci®c PCB congeners. Monochlorobiphenyls were dechlorinated at a higher rate than di-and tetrachlorobiphenyls. #
A polychlorinated biphenyl (PCB)-dechlorinat-pond sediments has been studied extensively and re-i... more A polychlorinated biphenyl (PCB)-dechlorinat-pond sediments has been studied extensively and re-ing anaerobic microbial consortium, developed in a gran-cently reviewed (Abramowicz, 1990; Bedard, 1990; ular form, demonstrated extensive dechlorination of Bedard and Quensen, 1995). Attempts to isolate stable PCBs present in Raisin River sediments at room (20 to PCB-dechlorinating anaerobic microorganisms for bio-22C) and at a relatively low (12C) temperature. Highly augmentation or further microbiological studies were chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog unsuccessful. In some instances, enrichments of either comparison showed that tri-, tetra-, penta-, hexa-, and pure or mixed cultures were found to lose their activity heptachlorobiphenyl compounds decreased signifi-when transferred to another matrix (Champine et al., cantly, and mono-and dichlorobiphenyl compounds in-1995; May et al., 1992). creased. After 32 weeks of incubation at 12C, the predom-Both higher and lower chlorinated PCBs can be effec-inant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24-and tively dechlorinated and degraded by anaerobic and 24-2-CB did not accumulate at room temperature, sug-aerobic sequential processes, respectively (Abramo-gesting a further dechlorination of these congeners. Pre-wicz, 1990; Alder et al., 1993; Bedard and Quensen, dominantly meta dechlorination (i.e., pattern M) was cata-1995; Ye et al., 1992). Therefore, in a two-step process, lyzed by the microbial consortium in the granules. highly chlorinated PCBs were first dechlorinated to less Dechlorination in the control studies without granules was not extensive. This study is the first demonstration chlorinated compounds under anaerobic conditions. In of enhanced reductive dechlorination of sediment PCBs addition, aerobic microorganisms oxidatively dechlori-by an exogenous anaerobic microbial consortium.
Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an ... more Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaer-obic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H 2-CO 2), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobac-terium formicicum. The performance of an upflow anaerobic sludge blanket reactor depends on the formation of granules of high settle-ability (13). The physicochemical factors that influence granu-lation have been studied extensively (12). Methanogens were suggested to be key species in the formation of the granules on volatile fatty acids (VFAs) as major substrates: (i) Methano-saeta (Methanothrix) species play an essential role in granula-tion (7, 12); (ii) Methanobrevibacter arboriphilicus produces extracellular polypeptides to induce granulation (18); and (iii) Methanosarcina species produce initial aggregates as nuclei to form granules (4a). We recently confirmed the role of meth-anogens in formation of granules by using defined microbial cultures consisting of Methanosaeta sp., Methanobacterium for-micicum, Methanosarcina mazeii, and two syntrophic fatty acid degraders (27). However, the role of individual microbial species in the development of polymeric structure in the granules is not well understood. Bacterial extracellular polymer (ECP) contributed to the adhesion between different species of methanogens and syn-trophic acetogenic bacteria present in the granules, improving their long-term stability (2, 8, 17). The ECP has been observed in different types of granules by scanning electron microscopy and transmission electron microscopy (TEM) (5, 9, 10, 17). The ECP has been shown to be composed of extracellular polysaccharides (EPS) (5, 7) and polypeptides (18). In this study, we focused on the composition of ECP from anaerobic methanogenic granules and the EPS from two prevalent meth-anogens isolated from the granules (25). We show that the ECP of Methanobacterium formicicum and Methanosarcina mazeii is similar in composition to the ECP from the granules, thus probably playing an important role in the development of granules. MATERIALS AND METHODS Methanogenic granules and methanogen cultures. The methanogenic granules used in this study were obtained from a laboratory-scale upflow reactor fed with a fatty acid mixture consisting of acetate, propionate, and butyrate. These granules consisted mainly of methanogenic and syntrophic acetogenic bacteria (22, 25). Methanobacterium formicicum T1N (DSM 6298) and Methanosarcina mazeii T18 (DSM 6300) were isolated from these granules (25). Media and growth conditions. Methanobacterium formicicum T1N and Meth-anosarcina mazeii T18 were grown in a basal medium (15) buffered with potassium phosphate (20 mM) plus 0.01% (vol/vol) vitamin solution (23) under anaerobic conditions at 37C unless otherwise stated. For the growth of Meth-anobacterium formicicum, the medium was supplemented with 1 mM sodium sulfide and 4 mM sodium acetate. Either formate (40 mM) or a gas mixture of H 2 plus CO 2 (80:20, 1.5 atm in the headspace) was used as carbon and energy source. Methanosarcina mazeii was grown in the basal medium supplemented with 30 mM sodium acetate and 12 mM sodium bicarbonate. The vials (excluding those using H 2-CO 2 as substrate) were pressurized to 0.3 atm with a mixture of N 2 and CO 2 (95:5). The pH of the medium was 7.0. TEM. The cells collected on a Millipore filter were placed in a fixative consisting of 3% glutaraldehyde plus 0.5% Alcian blue in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room temperature. The fixed samples were rinsed six times for 1 h in 0.2 M cacodylate buffer (pH 7.2). The rinsed samples were postfixed with 1% OsO 4 plus 1% LaNO 3 in 0.2 M s-collidine (pH 7.2) at room temperature and dehydrated through a graded series of ethanol solutions followed by propylene oxide. The samples were embedded in Poly/Bed 812 (Polysciences, Inc., War-rington, Pa.). Thin sections were cut with a diamond knife mounted in an LKB ultratome and then stained with uranyl acetate and lead citrate as described by Shea (19). TEM examination was performed on a Philips CM-10 electron microscope at an acceleration voltage of 80 kV. Extraction of extracellular polymers. The cells were harvested from stationary phase cultures by centrifugation (10,000 g at 4C) for 20 min. The polymer was not removed by shaking the cells in phosphate saline solution (11). Therefore , phenol (20% vol/vol) was used to extract the ECP from the cells, while leaving the cells intact. The cells were extracted with 150 ml of 20% (vol/vol) phenol in double-distilled water at 50C with shaking and intermittent sonication for 45 min and then centrifuged at 4C (12,000 g for 20 min). The aqueous layer was removed and saved, the same volume of water was added to the phenol layer, and the sample was again extracted at 50C for 45 min. The aqueous layer was removed and combined with that from the first extraction. The supernatant was placed in dialysis bags (molecular weight cutoff, 12,000 to 14,000) and was extensively dialyzed against water for 2 days. The supernatant was lyophilized to dryness and designated crude ECP. Polysaccharide isolation and purification. The polymer was purified by gel filtration on a Bio-Gel P100 column (Bio-Rad Laboratories, Richmond, Calif.) and eluted with 0.1% formic acid at room temperature. Carbohydrates in the * Corresponding author. Mailing address: MBI International,
The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and is... more The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain TIN, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobu-tyrate degradation by the culture. The reversible isomer-ization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isornerization was coupled with syntrophic bu-tyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure b2000 Pa) or a measurable level of formate (10 pM or higher). Significant formate (more than 1 rnM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. 0 1996 John Wiley & Sons, Inc.
An anaerobic methanogenic microbial consortium , developed in a granular form, exhibited extensiv... more An anaerobic methanogenic microbial consortium , developed in a granular form, exhibited extensive dechlorination of defined polychlorinated bi-phenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphe-nyl was dechlorinated to biphenyl via 2,3,4,6-tetra-chlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dech-lorination products 2,4,6-CB, 2,4-CB, 2-CB and biphe-nyl were 8.1, 41.2, 3.0 and 47.8 M respectively, from an initial 105 M 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the de-chlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium.
Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized... more Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized C-labeled PCP to CH and CO. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of CH and CO from [C]PCP. 2,4,6-trichloro-phenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chloro-phenol or 2-chlorophenol to phenol at pH 7.0-7.2.
An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic... more An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic coculture with a methanogen. The culture formed dense aggregates when Methanobacterium formicicum was the methanogenic partner, but the culture was turbid when Methanospirillum hungatei was the partner. In contrast, a propionate-degrading, lemon-shaped bacterium (strain PT) did not form aggregates with Methanobacterium formicicum unless an acetate-degrading Methanosaeta sp. was also included in the culture. Fatty acid-degrading methanogenic granules were formed in a laboratory-scale upflow reactor at 35C fed with a medium containing a mixture of acetate, propionate, and butyrate by using defined cultures of Methanobacterium formicicum T1N, Methanosaeta sp. strain M7, Methanosarcina mazei T18, propionate-degrading strain PT, and butyrate-degrading strain BH. The maximum substrate conversion rates of these granules for acetate, propionate, and butyrate were 43, 9, and 17 mmol/g (dry weight)/day, respectively. The average size of the granules was about 1 mm. Electron microscopic observation of the granules revealed that the cells of Methanobacterium formicicum, Methanosaeta sp., butyrate-degrading, and propionate-degrading bacteria were dispersed in the granules. Methanosarcina mazei existed inside the granules as aggregates of its own cells, which were associated with the bulk of the granules. The interaction of different species in aggregate formation and granule formation is discussed in relation to polymer formation of the cell surface. The formation of anaerobic granules in upflow anaerobic sludge blanket reactors is important for the reactor to operate at a high chemical oxygen demand (COD) removal rate, and the granule formation phenomenon is believed to be substantially based on microbial self-immobilization (8, 9, 16, 17). Bacterial species that play an essential role in cell-cell aggre-gation are the key to understanding the phenomenon of granule formation. The key species may be defined as ones which (i) form dense aggregates by themselves in an anaerobic reactor and/or (ii) provide a binding surface for other bacteria which cannot form aggregates and granules by themselves. Therefore, the species having the ability to aggregate are likely to be the prevalent microorganisms in the granules. Methanogens have already been hypothesized to be key species in granule formation. Several proposals about the microbial mechanisms of granule formation with volatile fatty acids (VFAs) as major substrates can be summarized as follows. (i) The Methanothrix species plays an essential role in granulation (3, 5, 13). (ii) The hydrogen-utilizing methanogen Methanobre-vibacter arboriphilicus AZ produces extracellular polypeptides to induce granule formation under high-H 2 partial pressure conditions (10). (iii) Methanosarcina cells produce initial aggregates as nuclei to form granules (2). These proposals and mechanisms, however, have not been validated experimentally. Microcolonies of syntrophic acetogens in granular structures have been observed within anaerobic granules (3). Syntrophic microcolonies consisting of acetogens and methanogens were major structural components of granules developed on brewery wastewater (15). The role of syntrophic acetogens in granule formation has not been clearly elucidated. In this study, the aggregate-forming behaviors of various defined methanogenic and syntrophic fatty acid-degrading cultures were analyzed for potential granule-forming syntrophic associations. We report here the formation of anaerobic granules with defined cultures and the stability and VFA-degrading performance of the granules. MATERIALS AND METHODS Bacterial strains. All of the methanogens and syntrophic fatty acid degraders utilized in this study were isolated from methanogenic granules developed on a fatty acid mixture containing acetate, propionate, and butyrate (18). The following methanogens were used in this study: Methanobacterium formicicum T1N (DSM 6298) and Methanospirillum hungatei BD, with either formate or H 2-CO 2 as the substrate; Methanosaeta sp. strain M7, a mesophilic acetate-utilizing meth-anogen; and Methanosarcina mazei T18 (DSM 6300), with H 2-CO 2 , acetate, and methanol as substrates. The following syntrophic acetogenic cultures were used: an obligate anaerobic, spore-forming, rod-shaped, butyrate-degrading strain, BH, and a lemon-shaped, spore-forming, propionate-degrading strain, PT. Strain BH is morphologically similar to Syntrophospora bryantii strains. Both of these syntrophic acetogens were isolated with Methanobacterium formicicum T1N as a partner for syntrophic fatty acid metabolism. Medium. The phosphate-buffered basal medium (6) was used for the growth of cells in anaerobic 158-ml serum vials, which were sealed with butyl rubber stoppers. For the growth of syntrophic fatty acid-degrading cultures, each vial contained 50 ml of phosphate-buffered basal medium supplemented with 15 mM phosphate, 40 mM sodium bicarbonate, 1% vitamin solution (vol/vol) (14), and 1 mM sodium sulfide as a reductant, except for the culture containing Methano-saeta sp. strain M7, which contained 0.3 mM sodium sulfide. The substrate concentration was 15 mM sodium butyrate or 15 mM propionate. Before inoculation , the vials were pressurized with 1 atm (101.29 kPa) of N 2-CO 2 (95:5) mixture gas, and the pH in each vial was adjusted to 7.0 to 7.1 with a 2.5 M KH 2 PO 4 or K 2 HPO 4 solution when necessary. For reactor experiments, liquid medium (16 liters) was prepared anaerobically in a 20-liter carboy. The medium composition was (per liter of distilled water): MgCl 2 6H 2 O, 0.32 g; CaCl 2 2H 2 O, 0.32 g; NaCl, 0.8 g; NH 4 Cl, 0.8 g; KH 2 PO 4 , 0.32 g; Na 2 S 9H 2 O, 0.08 g; resazurin, 0.002 g; and trace element solution, 5 ml (6). The concentrations of acetate, propionate, and butyrate were adjusted by addition of the mixtures of the sodium salt and acid forms of the volatile acids. The medium pH was 4.7. Aggregate formation by defined cultures. A methanogenic culture (Meth-anobacterium formicicum T1N), two syntrophic butyrate-degrading cocultures (butyrate-degrading strain BH plus Methanobacterium formicicum T1N and * Corresponding author.
Anaerobic methanogenic granules were stored under anaerobic condition at 4 ° and 22°C for 1-to 18... more Anaerobic methanogenic granules were stored under anaerobic condition at 4 ° and 22°C for 1-to 18-month period to evaluate the effect of storage on degradation of volatile fatty acids (VFAs), including acetate, propionate and butyrate, and on methane production. The length of storage period affected the activities of different microbial trophic groups. During storage at 22°C, the degradation rates for all the three acids decreased gradually. At low temperature (4°C), reduction in degradation rates of acetate and propionate was relatively slower than that at 22°C. Reduction in butyrate degradation rate was faster (by 45%) during the first month of storage at 4°C, but the rate declined afterwards. Nevertheless, the granules maintained, although at reduced level, their metabolic activities for all three VFAs even after storage for 18 months. Higher decay coefficients were obtained at 22°C than those at 4°C. For a relatively short period (1-5 months), granules can be stored at ambient temperature (approx. 20-22°C) with limited loss in their VFA degradation rates. However, granules can maintain higher levels of VFA degradation rates when they are stored at low (4°C) rather than at ambient temperature. Reactor studies indicated that the granules can completely recover their original VFA degradation rates in three days when stored for 31 d at 22°C. The granules stored at 22°C for 9 months were used successfully as inoculum to start a laboratory-scale reactor. The original VFA degradation rates of the granules were achieved after 15-20 d of reactor operation at 35°C.
Syntrophic degradation of normal-and branched-chain fatty acids with 4 to 9 carbons was investiga... more Syntrophic degradation of normal-and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum TIN, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methyl-valerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded. Branched-chain fatty acids, such as isobutyrate and 2-meth-ylbutyrate, are intermediates of the anaerobic digestion of proteins (9). Both have also been detected during simultaneous degradation of acetate, propionate, and butyrate by methanogenic granules (19, 22). These branched-chain fatty acids can be converted either to acetate or completely to CO2 by some sulfate-reducing bacteria via sulfate reduction (14, 16, 17). 2-Methylbutyrate can be converted to acetate, propionate, and methane by syntrophic butyrate degraders such as Syntro-phospora bryantii (13) and strains NSF-2 and SF-1 (12), together with H2-utilizing methanogens. The syntrophic conversion of isobutyrate in the absence of sulfate has been studied with digested sludges (1, 15, 25). A reversible isomerization between butyrate and isobutyrate indicates that butyrate is an intermediate of anaerobic isobutyrate degradation (15). The appearance of isobutyrate during syntrophic butyrate degradation is due to the isomerization performed by some organisms involved in isobutyrate degradation. The mechanism of formation of 2-methylbutyrate is unknown. To date, only one syntrophic butyrate-degrading culture, the straight rod-shaped Syntrophospora-like strain SF-1, has been reported to use isobutyrate (12). However, the utilization of isobutyrate by this strain was questioned (6, 14). We isolated a defined triculture that would syntrophically degrade butyrate and isobutyrate, perform reciprocal isomerization between butyrate and isobutyrate, and convert acetate and H2 (or formate) to methane (4, 21). Recently, an anaerobic bacterium , strain WoG13, which grows on dicarboxylic acid glu-tarate and which also performs reciprocal isomerization of butyrate and isobutyrate has been isolated (8). Isobutyrate can * Corresponding author. Mailing address: Michigan Biotechnology Institute, 3900 Collins Rd.,. Electronic mail address: [email protected]. be degraded by a triculture consisting of strain WoG13, Syntrophomonas wolfei, and Methanospirillum hungatei. In this triculture, strain WoG13 isomerized isobutyrate to butyrate and S. wolfei together with Methanospirillum hungatei converted butyrate to acetate and methane. Our triculture, consisting of a syntrophic isobutyrate-bu-tyrate degrading rod (strain IB) along with Methanobacterium formicicum TiN and Methanosarcina mazei T18, was isolated from syntrophic methanogenic granules (21). This triculture can also utilize 2-methylbutyrate, valerate, and other branched-and normal-chain fatty acids, with up to nine carbon atoms, as substrates and can synthesize 2-methylbutyrate during syntrophic butyrate degradation in the presence of propi-onate. In the present communication, we describe the isomer-ization and syntrophic conversion of isobutyrate, butyrate, and other normal-and branched-chain fatty acids by this defined triculture. (Preliminary results of this study have been presented at annual meetings of the American Society for Microbiology [4, 20].) MATERIALS AND METHODS Chemicals and gases. All chemicals used were of analytical grade and were obtained from Sigma Chemical Co., St. Louis, Mo., or Mallinckrodt Inc., Paris, Ky. A gas mixture of N2-CO2 (70:30) was obtained from Union Carbide Corp. (Linde Div., Warren, Mich.) and passed over heated (350°C) copper filings to remove traces of 02-Sources of cultures. The syntrophic isobutyrate-butyrate-degrading strain IB was isolated by us earlier (21). It was isolated from syntrophic methanogenic granules developed on a volatile fatty acid mixture consisting of acetate, propionate, and butyrate, along with Methanobacterium formicicum TiN 2220
Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium f... more Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species o n H 2-C O 2. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the a m o u n t H 2 released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoe-thane sulfonate, M. formicicum synthesized formate from H2 plus HCO~ or produced H2 from formate to a steady-state level at which point the Gibbs free energy (AG') available for formate synthesis or H2 production was approximately-2 to-3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H 2 pressure. The relative rates of conversion of formate and H2 were apparently controlled by the AG' available for formate synthesis, hydrogen production, methane production from formate and methane production from H2. Results from 14C-tracer tests indicated that a rapid isotopic exchange between HCOO-and HCO~ occurred during the growth of M.formicicum on H2-CO 2. Data from metabolism of 14C-labelled formate to methane suggested that formate was initially split to H2 and HCO~ and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on Hi-CO z was inhibited although production of methane was not. Formate synthesis from H 2 w a s also inhibited.
Two types of mesophilic methanogenic granules (R-and F-granules) were developed on different synt... more Two types of mesophilic methanogenic granules (R-and F-granules) were developed on different synthetic feeds containing acetate, propionate and bu-tyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H2-CO2 were related to the feed composition used for their development. These granules p e r f o r m e d a reversible reaction between H2 production from formate and formate synthesis from H2 plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and buty-rate during butyrate or isobutyrate degradation. Valer-ate and 2-methylbutyrate w e r e produced and consumed during propionate-butyrate degradation. The respective apparent Km (mM) for various substrates in disrupted R-and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an o p t i m u m temperature range from 40 to 50°C for H2-CO2 and formate utilization and 40 ° C for acetate, propionate and butyrate utilization and a similar o p t i m u m pH.
Two types of methanogenic gianules capable of high chemical oxygen demand removal rates were deve... more Two types of methanogenic gianules capable of high chemical oxygen demand removal rates were developed in laboratory-scale upflow reactors at 35 ° C. One granule type (R-granules) had a rod-type Metha-nothrix-like species as the predominant species whereas the other (F-granules) had a filament-type M. soehn-genii-like acetate-utilizer as the predominant species. These two types of granules were compared in terms of operational performance, physical-chemical characteristics and microbial population. The R-granules had a higher density [65-70 vs 39-43 g suspended solids (SS)/1], specific gravity (1.03 vs 1.01) and specific volu-metric methane production rate (180 vs 120 1 CH4/I granules per day) than the F-granules. Acetate, pro-pionate and butyrate degraders in both types of granules had similar specific growth rates. The most probable number enumeration indicated that both types of granule had the same population levels (cells/g SS) in terms of methanogens (H2-CO2-, formate-and acetate-utilizing), and syntrophie acetogens. Hydrolyticrfer-mentative bacteria were present in greater number in the F-granules than in the R-granules. The R-granules had a higher cell density than the F-granules. The differences in operational performance were due mainly to their different microbial composition, especially the predominant acetate-utilizing methanogens in the granules. The long-filamentous M. soehngenii-like rods in the F-granules appeared to be responsible for their lower density and large-sized granules.
Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.... more Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28°C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99%o was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14CJPCP to 14CH4 and 14Co2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP. Pentachlorophenol (PCP) is one of the biocides that was widely used in the United States, mainly for the preservation of wood and wood products. Along with other chlorophe-nols, PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency (16). Under aerobic conditions , PCP can be degraded by bacteria (2, 8, 29, 34) and fungi (23, 33). Aerobic organisms such as Flavobacterium spp. (8, 34, 37) and Rhodococcus spp. (2, 12, 35) have been successfully used in pilot-scale and field studies for the treatment of PCP-contaminated wastewater and groundwater. PCP can also be completely mineralized to methane and CO2 by anaerobic microorganisms (27). Reductive dechlori-nation of PCP occurs prior to complete mineralization in digested sludges and soils (3, 24, 26-29, 31, 41). Combined systems of an anaerobic fluidized bed plus trickling-filter or aerated lagoons were used to treat chlorophenolic waste from the paper pulp bleaching process (12, 35). Chlorophe-nols were removed from the wastewater by 50 to 60%, and mineralization of added PCP to CO2 was observed by use of radiotracer assay. However, only the overall system removal of chlorophenols was reported, and the role of anaer-obic fluidized bed in dechlorination was not clear. PCP removal or dechlorination was reported in semi-continuous-flow, stirred-sewage sludge digestors (11), in a bioreactor which was partially packed with glass beads to treat a mixture of meta-, ortho-, andpara-chlorophenols (19), in an anaerobic fixed-film reactor (13), and in upflow anaerobic sludge blanket (UASB) reactors with anaerobic granular sludge (14, 28, 42). In some cases, the dechlorination of PCP was not carried out completely, resulting in the appearance of lesser chlorinated phenols (28, 42). The volumetric PCP loading rates of the anaerobic reactor systems mentioned above were ca. 2.2 mg of PCP per liter of reactor volume per day or less. We have developed methanogenic PCP-degrading gran-* Corresponding author. 389 ules on a synthetic wastewater containing PCP, acetate, propionate, butyrate, and methanol in a laboratory-scale UASB reactor at 28°C (5, 6). The maximum PCP removal rate of the granules was as high as 14.6 mg/g of volatile suspended solids (VSS) per day, and a stable volumetric PCP removal rate of 90 mg of PCP per liter per day was achieved. The purpose of this article was to examine the feasibility of the development of methanogenic granules with high dechlorinating activity and to investigate the performance of the granules in treating wastewaters containing high PCP concentrations in a laboratory-scale UASB reactor system. MATERIALS AND METHODS Chemicals and gases. All chemicals (analytical grade) except PCP were obtained from Sigma Chemical Co. (St. Louis, Mo.); PCP was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Nitrogen gas and gas mixtures of N2-CO2 (95:5 and 70:30) were obtained from Linde Division, Union Carbide Corp. (Warren, Mich.), and passed over heated (370°C) copper filings to remove traces of 02. Analytical methods. Methane, methanol, and volatile fatty acids (VFA) were determined by using gas chromatography as described elsewhere (43, 44). PCP in solution was determined with high-performance liquid chromatography (HPLC). Samples (1.0 ml) were mixed with 0.5 ml of acetonitrile on a vortex mixer, centrifuged for 5 min at 5,500 x g with an Eppendorf 5415 centrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.), and filtered through 0.45-,um-pore-size syringe filters (Acrodisc LC13; Gelman Sciences Co., Ann Arbor, Mich.). A Waters HPLC system, consisting of the model 501 pump, the Lambda-Max model 481 UV detector, and the model 740 data module, was used. Samples were injected by using a Rheodyne 7010 injector fitted with a 50-,ul loop. Separation was accomplished with a Waters Radial-Pak C-18 column. The mobile phase consisted of acetonitrile and 5% aqueous acetic acid (8:2, vol/vol). The flow rate of Vol. 59, No. 2
The microbial species composition of metha-nogenic granules developed on an acetate-propionate-bu... more The microbial species composition of metha-nogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (101Z/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (101~/g dry weight), but low numbers of hydrolytic-fermentative bacteria (109/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospiril-lure hungatei strain BD, and a non-filamentous, bamboo shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading spore-forming oval-shaped species, strain PT, and a propion-ate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophi-cally with an H2-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, acetate and butyrate but they were involved in propionate degradation.
Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contai... more Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35°C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-C02. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens. Methanogenic granules are self-immobilized consortia of methanogens, syntrophic acetogens, and hydrolytic-fermen-tative bacteria that convert soluble organic matter to methane and CO2. The published work on the granule formation, microbial composition, substrate conversion potentials, and microbial structures of granules has focused on methanogens and syntrophic acetogens (7-11, 15, 23, 32, 39). This is likely due to the observation that sulfate-reducing bacteria (SRB) are present at much lower levels than syntrophic acetogens in granules treating sugar wastewater (10). Considerable information as to the methanogenic degradation of acetate, formate, and H2-CO2 in granules has been reported (7, 8, 11). Little work, however, has been performed to evaluate the conversion of other common substrates such as ethanol, propionate, and butyrate. SRB are quite diverse in terms of metabolic activities, morphotypes, trophic properties, and substrate affinities. In the presence of sulfate, acetate can be oxidized to CO2 by some pure SRB cultures; propionate, butyrate, and other volatile fatty acids (VFAs) can be oxidized completely to * Corresponding author. CO2 or converted to acetate or acetate plus propionate (in the case of odd long-chain acids with five or more carbon atoms); branched fatty acids such as isobutyrate, isovaler-ate, and 2-methylbutyrate can also be oxidized completely to CO2 or incompletely to acetate (35, 37). Hydrogen and formate can be utilized by many SRB as electron donors for sulfate reduction (35). Acetate and methanol are degraded via sulfate reduction by a coculture consisting of Desulfo-vibrio vulgaris and Methanosarcina barkeri (22). Methanol can be also degraded to CO2 via sulfate reduction by a coculture consisting of Desulfovibrio vulgaris and various homoacetogens (6, 12). In the absence of sulfate, certain SRB such as Desulfovibrio spp. may grow together with H2-utilizing methanogens to convert ethanol or lactate to acetate syntrophically (18, 34). The existence of syntrophic associations between H2-producing SRB and H2-consuming methanogens in lake sediments was suggested (5). No mention of syntrophic catabolism of VFAs by SRB in granular systems has been reported. VFAs such as propionate and butyrate are thought to be converted only by obligate syntrophic acetogens in concert with H2-utilizing methano-gens (3, 19, 20, 28, 35). In this study, methanogenic granules grown on brewery 3438
High performance biomethanation granules with operational specific COD removal rates of 7 kg COD ... more High performance biomethanation granules with operational specific COD removal rates of 7 kg COD removedl kg SS/d were obtained by ecoengineering conventional, granular, UASB digester sludge using a designed protocol of starvation and selection on a defined volatile fatty acid (VFA) based mineral medium. Addition of low (0.15 m M) sulfate levels to this VFA medium increased the maximum shock-load COD removal rate of the ecoengineered biomethanation granules to 9 kg COD/kg SS/d with specific acetate, propionate, and butyrate removal rates of 111, 28, and 64 mol/g SS/d. Addition of moderate (26 m M) calcium levels inhibited growth and altered the structure of granules. The general cellular, growth, stability , and performance features of these ecoengineered granules are described and discussed in relation to their use as improved biomethanation starter cultures.
ÐAn anaerobic microbial consortium developed in our laboratory showed dechlorination of spiked po... more ÐAn anaerobic microbial consortium developed in our laboratory showed dechlorination of spiked polychlorinated biphenyls (PCBs), such as Aroclor 1254 and a de®ned congener 2,3,4,5,6-penta-chlorobiphenyl (CB) in the presence and the absence of lake sediment. Glucose and methanol were used as carbon and energy sources. Highly chlorinated congeners (containing >5 chlorines) such as penta-, hexa-and heptachlorobiphenyls were preferentially dechlorinated both in the presence and the absence of sediment with a simultaneous change in the distribution of relative amounts of tri-and tetra-chlorobiphenyl congeners. The dechlorination pattern and rates observed in both sediment and sediment free conditions were similar. In 24 weeks, almost 70% of the PCBs were dechlorinated without accumulation of any speci®c PCB congeners. Monochlorobiphenyls were dechlorinated at a higher rate than di-and tetrachlorobiphenyls. #
A polychlorinated biphenyl (PCB)-dechlorinat-pond sediments has been studied extensively and re-i... more A polychlorinated biphenyl (PCB)-dechlorinat-pond sediments has been studied extensively and re-ing anaerobic microbial consortium, developed in a gran-cently reviewed (Abramowicz, 1990; Bedard, 1990; ular form, demonstrated extensive dechlorination of Bedard and Quensen, 1995). Attempts to isolate stable PCBs present in Raisin River sediments at room (20 to PCB-dechlorinating anaerobic microorganisms for bio-22C) and at a relatively low (12C) temperature. Highly augmentation or further microbiological studies were chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog unsuccessful. In some instances, enrichments of either comparison showed that tri-, tetra-, penta-, hexa-, and pure or mixed cultures were found to lose their activity heptachlorobiphenyl compounds decreased signifi-when transferred to another matrix (Champine et al., cantly, and mono-and dichlorobiphenyl compounds in-1995; May et al., 1992). creased. After 32 weeks of incubation at 12C, the predom-Both higher and lower chlorinated PCBs can be effec-inant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24-and tively dechlorinated and degraded by anaerobic and 24-2-CB did not accumulate at room temperature, sug-aerobic sequential processes, respectively (Abramo-gesting a further dechlorination of these congeners. Pre-wicz, 1990; Alder et al., 1993; Bedard and Quensen, dominantly meta dechlorination (i.e., pattern M) was cata-1995; Ye et al., 1992). Therefore, in a two-step process, lyzed by the microbial consortium in the granules. highly chlorinated PCBs were first dechlorinated to less Dechlorination in the control studies without granules was not extensive. This study is the first demonstration chlorinated compounds under anaerobic conditions. In of enhanced reductive dechlorination of sediment PCBs addition, aerobic microorganisms oxidatively dechlori-by an exogenous anaerobic microbial consortium.
Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an ... more Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaer-obic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H 2-CO 2), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobac-terium formicicum. The performance of an upflow anaerobic sludge blanket reactor depends on the formation of granules of high settle-ability (13). The physicochemical factors that influence granu-lation have been studied extensively (12). Methanogens were suggested to be key species in the formation of the granules on volatile fatty acids (VFAs) as major substrates: (i) Methano-saeta (Methanothrix) species play an essential role in granula-tion (7, 12); (ii) Methanobrevibacter arboriphilicus produces extracellular polypeptides to induce granulation (18); and (iii) Methanosarcina species produce initial aggregates as nuclei to form granules (4a). We recently confirmed the role of meth-anogens in formation of granules by using defined microbial cultures consisting of Methanosaeta sp., Methanobacterium for-micicum, Methanosarcina mazeii, and two syntrophic fatty acid degraders (27). However, the role of individual microbial species in the development of polymeric structure in the granules is not well understood. Bacterial extracellular polymer (ECP) contributed to the adhesion between different species of methanogens and syn-trophic acetogenic bacteria present in the granules, improving their long-term stability (2, 8, 17). The ECP has been observed in different types of granules by scanning electron microscopy and transmission electron microscopy (TEM) (5, 9, 10, 17). The ECP has been shown to be composed of extracellular polysaccharides (EPS) (5, 7) and polypeptides (18). In this study, we focused on the composition of ECP from anaerobic methanogenic granules and the EPS from two prevalent meth-anogens isolated from the granules (25). We show that the ECP of Methanobacterium formicicum and Methanosarcina mazeii is similar in composition to the ECP from the granules, thus probably playing an important role in the development of granules. MATERIALS AND METHODS Methanogenic granules and methanogen cultures. The methanogenic granules used in this study were obtained from a laboratory-scale upflow reactor fed with a fatty acid mixture consisting of acetate, propionate, and butyrate. These granules consisted mainly of methanogenic and syntrophic acetogenic bacteria (22, 25). Methanobacterium formicicum T1N (DSM 6298) and Methanosarcina mazeii T18 (DSM 6300) were isolated from these granules (25). Media and growth conditions. Methanobacterium formicicum T1N and Meth-anosarcina mazeii T18 were grown in a basal medium (15) buffered with potassium phosphate (20 mM) plus 0.01% (vol/vol) vitamin solution (23) under anaerobic conditions at 37C unless otherwise stated. For the growth of Meth-anobacterium formicicum, the medium was supplemented with 1 mM sodium sulfide and 4 mM sodium acetate. Either formate (40 mM) or a gas mixture of H 2 plus CO 2 (80:20, 1.5 atm in the headspace) was used as carbon and energy source. Methanosarcina mazeii was grown in the basal medium supplemented with 30 mM sodium acetate and 12 mM sodium bicarbonate. The vials (excluding those using H 2-CO 2 as substrate) were pressurized to 0.3 atm with a mixture of N 2 and CO 2 (95:5). The pH of the medium was 7.0. TEM. The cells collected on a Millipore filter were placed in a fixative consisting of 3% glutaraldehyde plus 0.5% Alcian blue in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room temperature. The fixed samples were rinsed six times for 1 h in 0.2 M cacodylate buffer (pH 7.2). The rinsed samples were postfixed with 1% OsO 4 plus 1% LaNO 3 in 0.2 M s-collidine (pH 7.2) at room temperature and dehydrated through a graded series of ethanol solutions followed by propylene oxide. The samples were embedded in Poly/Bed 812 (Polysciences, Inc., War-rington, Pa.). Thin sections were cut with a diamond knife mounted in an LKB ultratome and then stained with uranyl acetate and lead citrate as described by Shea (19). TEM examination was performed on a Philips CM-10 electron microscope at an acceleration voltage of 80 kV. Extraction of extracellular polymers. The cells were harvested from stationary phase cultures by centrifugation (10,000 g at 4C) for 20 min. The polymer was not removed by shaking the cells in phosphate saline solution (11). Therefore , phenol (20% vol/vol) was used to extract the ECP from the cells, while leaving the cells intact. The cells were extracted with 150 ml of 20% (vol/vol) phenol in double-distilled water at 50C with shaking and intermittent sonication for 45 min and then centrifuged at 4C (12,000 g for 20 min). The aqueous layer was removed and saved, the same volume of water was added to the phenol layer, and the sample was again extracted at 50C for 45 min. The aqueous layer was removed and combined with that from the first extraction. The supernatant was placed in dialysis bags (molecular weight cutoff, 12,000 to 14,000) and was extensively dialyzed against water for 2 days. The supernatant was lyophilized to dryness and designated crude ECP. Polysaccharide isolation and purification. The polymer was purified by gel filtration on a Bio-Gel P100 column (Bio-Rad Laboratories, Richmond, Calif.) and eluted with 0.1% formic acid at room temperature. Carbohydrates in the * Corresponding author. Mailing address: MBI International,
The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and is... more The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain TIN, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobu-tyrate degradation by the culture. The reversible isomer-ization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isornerization was coupled with syntrophic bu-tyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure b2000 Pa) or a measurable level of formate (10 pM or higher). Significant formate (more than 1 rnM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. 0 1996 John Wiley & Sons, Inc.
An anaerobic methanogenic microbial consortium , developed in a granular form, exhibited extensiv... more An anaerobic methanogenic microbial consortium , developed in a granular form, exhibited extensive dechlorination of defined polychlorinated bi-phenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphe-nyl was dechlorinated to biphenyl via 2,3,4,6-tetra-chlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dech-lorination products 2,4,6-CB, 2,4-CB, 2-CB and biphe-nyl were 8.1, 41.2, 3.0 and 47.8 M respectively, from an initial 105 M 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the de-chlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium.
Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized... more Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized C-labeled PCP to CH and CO. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of CH and CO from [C]PCP. 2,4,6-trichloro-phenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chloro-phenol or 2-chlorophenol to phenol at pH 7.0-7.2.
An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic... more An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic coculture with a methanogen. The culture formed dense aggregates when Methanobacterium formicicum was the methanogenic partner, but the culture was turbid when Methanospirillum hungatei was the partner. In contrast, a propionate-degrading, lemon-shaped bacterium (strain PT) did not form aggregates with Methanobacterium formicicum unless an acetate-degrading Methanosaeta sp. was also included in the culture. Fatty acid-degrading methanogenic granules were formed in a laboratory-scale upflow reactor at 35C fed with a medium containing a mixture of acetate, propionate, and butyrate by using defined cultures of Methanobacterium formicicum T1N, Methanosaeta sp. strain M7, Methanosarcina mazei T18, propionate-degrading strain PT, and butyrate-degrading strain BH. The maximum substrate conversion rates of these granules for acetate, propionate, and butyrate were 43, 9, and 17 mmol/g (dry weight)/day, respectively. The average size of the granules was about 1 mm. Electron microscopic observation of the granules revealed that the cells of Methanobacterium formicicum, Methanosaeta sp., butyrate-degrading, and propionate-degrading bacteria were dispersed in the granules. Methanosarcina mazei existed inside the granules as aggregates of its own cells, which were associated with the bulk of the granules. The interaction of different species in aggregate formation and granule formation is discussed in relation to polymer formation of the cell surface. The formation of anaerobic granules in upflow anaerobic sludge blanket reactors is important for the reactor to operate at a high chemical oxygen demand (COD) removal rate, and the granule formation phenomenon is believed to be substantially based on microbial self-immobilization (8, 9, 16, 17). Bacterial species that play an essential role in cell-cell aggre-gation are the key to understanding the phenomenon of granule formation. The key species may be defined as ones which (i) form dense aggregates by themselves in an anaerobic reactor and/or (ii) provide a binding surface for other bacteria which cannot form aggregates and granules by themselves. Therefore, the species having the ability to aggregate are likely to be the prevalent microorganisms in the granules. Methanogens have already been hypothesized to be key species in granule formation. Several proposals about the microbial mechanisms of granule formation with volatile fatty acids (VFAs) as major substrates can be summarized as follows. (i) The Methanothrix species plays an essential role in granulation (3, 5, 13). (ii) The hydrogen-utilizing methanogen Methanobre-vibacter arboriphilicus AZ produces extracellular polypeptides to induce granule formation under high-H 2 partial pressure conditions (10). (iii) Methanosarcina cells produce initial aggregates as nuclei to form granules (2). These proposals and mechanisms, however, have not been validated experimentally. Microcolonies of syntrophic acetogens in granular structures have been observed within anaerobic granules (3). Syntrophic microcolonies consisting of acetogens and methanogens were major structural components of granules developed on brewery wastewater (15). The role of syntrophic acetogens in granule formation has not been clearly elucidated. In this study, the aggregate-forming behaviors of various defined methanogenic and syntrophic fatty acid-degrading cultures were analyzed for potential granule-forming syntrophic associations. We report here the formation of anaerobic granules with defined cultures and the stability and VFA-degrading performance of the granules. MATERIALS AND METHODS Bacterial strains. All of the methanogens and syntrophic fatty acid degraders utilized in this study were isolated from methanogenic granules developed on a fatty acid mixture containing acetate, propionate, and butyrate (18). The following methanogens were used in this study: Methanobacterium formicicum T1N (DSM 6298) and Methanospirillum hungatei BD, with either formate or H 2-CO 2 as the substrate; Methanosaeta sp. strain M7, a mesophilic acetate-utilizing meth-anogen; and Methanosarcina mazei T18 (DSM 6300), with H 2-CO 2 , acetate, and methanol as substrates. The following syntrophic acetogenic cultures were used: an obligate anaerobic, spore-forming, rod-shaped, butyrate-degrading strain, BH, and a lemon-shaped, spore-forming, propionate-degrading strain, PT. Strain BH is morphologically similar to Syntrophospora bryantii strains. Both of these syntrophic acetogens were isolated with Methanobacterium formicicum T1N as a partner for syntrophic fatty acid metabolism. Medium. The phosphate-buffered basal medium (6) was used for the growth of cells in anaerobic 158-ml serum vials, which were sealed with butyl rubber stoppers. For the growth of syntrophic fatty acid-degrading cultures, each vial contained 50 ml of phosphate-buffered basal medium supplemented with 15 mM phosphate, 40 mM sodium bicarbonate, 1% vitamin solution (vol/vol) (14), and 1 mM sodium sulfide as a reductant, except for the culture containing Methano-saeta sp. strain M7, which contained 0.3 mM sodium sulfide. The substrate concentration was 15 mM sodium butyrate or 15 mM propionate. Before inoculation , the vials were pressurized with 1 atm (101.29 kPa) of N 2-CO 2 (95:5) mixture gas, and the pH in each vial was adjusted to 7.0 to 7.1 with a 2.5 M KH 2 PO 4 or K 2 HPO 4 solution when necessary. For reactor experiments, liquid medium (16 liters) was prepared anaerobically in a 20-liter carboy. The medium composition was (per liter of distilled water): MgCl 2 6H 2 O, 0.32 g; CaCl 2 2H 2 O, 0.32 g; NaCl, 0.8 g; NH 4 Cl, 0.8 g; KH 2 PO 4 , 0.32 g; Na 2 S 9H 2 O, 0.08 g; resazurin, 0.002 g; and trace element solution, 5 ml (6). The concentrations of acetate, propionate, and butyrate were adjusted by addition of the mixtures of the sodium salt and acid forms of the volatile acids. The medium pH was 4.7. Aggregate formation by defined cultures. A methanogenic culture (Meth-anobacterium formicicum T1N), two syntrophic butyrate-degrading cocultures (butyrate-degrading strain BH plus Methanobacterium formicicum T1N and * Corresponding author.
Anaerobic methanogenic granules were stored under anaerobic condition at 4 ° and 22°C for 1-to 18... more Anaerobic methanogenic granules were stored under anaerobic condition at 4 ° and 22°C for 1-to 18-month period to evaluate the effect of storage on degradation of volatile fatty acids (VFAs), including acetate, propionate and butyrate, and on methane production. The length of storage period affected the activities of different microbial trophic groups. During storage at 22°C, the degradation rates for all the three acids decreased gradually. At low temperature (4°C), reduction in degradation rates of acetate and propionate was relatively slower than that at 22°C. Reduction in butyrate degradation rate was faster (by 45%) during the first month of storage at 4°C, but the rate declined afterwards. Nevertheless, the granules maintained, although at reduced level, their metabolic activities for all three VFAs even after storage for 18 months. Higher decay coefficients were obtained at 22°C than those at 4°C. For a relatively short period (1-5 months), granules can be stored at ambient temperature (approx. 20-22°C) with limited loss in their VFA degradation rates. However, granules can maintain higher levels of VFA degradation rates when they are stored at low (4°C) rather than at ambient temperature. Reactor studies indicated that the granules can completely recover their original VFA degradation rates in three days when stored for 31 d at 22°C. The granules stored at 22°C for 9 months were used successfully as inoculum to start a laboratory-scale reactor. The original VFA degradation rates of the granules were achieved after 15-20 d of reactor operation at 35°C.
Syntrophic degradation of normal-and branched-chain fatty acids with 4 to 9 carbons was investiga... more Syntrophic degradation of normal-and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum TIN, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methyl-valerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded. Branched-chain fatty acids, such as isobutyrate and 2-meth-ylbutyrate, are intermediates of the anaerobic digestion of proteins (9). Both have also been detected during simultaneous degradation of acetate, propionate, and butyrate by methanogenic granules (19, 22). These branched-chain fatty acids can be converted either to acetate or completely to CO2 by some sulfate-reducing bacteria via sulfate reduction (14, 16, 17). 2-Methylbutyrate can be converted to acetate, propionate, and methane by syntrophic butyrate degraders such as Syntro-phospora bryantii (13) and strains NSF-2 and SF-1 (12), together with H2-utilizing methanogens. The syntrophic conversion of isobutyrate in the absence of sulfate has been studied with digested sludges (1, 15, 25). A reversible isomerization between butyrate and isobutyrate indicates that butyrate is an intermediate of anaerobic isobutyrate degradation (15). The appearance of isobutyrate during syntrophic butyrate degradation is due to the isomerization performed by some organisms involved in isobutyrate degradation. The mechanism of formation of 2-methylbutyrate is unknown. To date, only one syntrophic butyrate-degrading culture, the straight rod-shaped Syntrophospora-like strain SF-1, has been reported to use isobutyrate (12). However, the utilization of isobutyrate by this strain was questioned (6, 14). We isolated a defined triculture that would syntrophically degrade butyrate and isobutyrate, perform reciprocal isomerization between butyrate and isobutyrate, and convert acetate and H2 (or formate) to methane (4, 21). Recently, an anaerobic bacterium , strain WoG13, which grows on dicarboxylic acid glu-tarate and which also performs reciprocal isomerization of butyrate and isobutyrate has been isolated (8). Isobutyrate can * Corresponding author. Mailing address: Michigan Biotechnology Institute, 3900 Collins Rd.,. Electronic mail address: [email protected]. be degraded by a triculture consisting of strain WoG13, Syntrophomonas wolfei, and Methanospirillum hungatei. In this triculture, strain WoG13 isomerized isobutyrate to butyrate and S. wolfei together with Methanospirillum hungatei converted butyrate to acetate and methane. Our triculture, consisting of a syntrophic isobutyrate-bu-tyrate degrading rod (strain IB) along with Methanobacterium formicicum TiN and Methanosarcina mazei T18, was isolated from syntrophic methanogenic granules (21). This triculture can also utilize 2-methylbutyrate, valerate, and other branched-and normal-chain fatty acids, with up to nine carbon atoms, as substrates and can synthesize 2-methylbutyrate during syntrophic butyrate degradation in the presence of propi-onate. In the present communication, we describe the isomer-ization and syntrophic conversion of isobutyrate, butyrate, and other normal-and branched-chain fatty acids by this defined triculture. (Preliminary results of this study have been presented at annual meetings of the American Society for Microbiology [4, 20].) MATERIALS AND METHODS Chemicals and gases. All chemicals used were of analytical grade and were obtained from Sigma Chemical Co., St. Louis, Mo., or Mallinckrodt Inc., Paris, Ky. A gas mixture of N2-CO2 (70:30) was obtained from Union Carbide Corp. (Linde Div., Warren, Mich.) and passed over heated (350°C) copper filings to remove traces of 02-Sources of cultures. The syntrophic isobutyrate-butyrate-degrading strain IB was isolated by us earlier (21). It was isolated from syntrophic methanogenic granules developed on a volatile fatty acid mixture consisting of acetate, propionate, and butyrate, along with Methanobacterium formicicum TiN 2220
Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium f... more Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species o n H 2-C O 2. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the a m o u n t H 2 released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoe-thane sulfonate, M. formicicum synthesized formate from H2 plus HCO~ or produced H2 from formate to a steady-state level at which point the Gibbs free energy (AG') available for formate synthesis or H2 production was approximately-2 to-3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H 2 pressure. The relative rates of conversion of formate and H2 were apparently controlled by the AG' available for formate synthesis, hydrogen production, methane production from formate and methane production from H2. Results from 14C-tracer tests indicated that a rapid isotopic exchange between HCOO-and HCO~ occurred during the growth of M.formicicum on H2-CO 2. Data from metabolism of 14C-labelled formate to methane suggested that formate was initially split to H2 and HCO~ and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on Hi-CO z was inhibited although production of methane was not. Formate synthesis from H 2 w a s also inhibited.
Two types of mesophilic methanogenic granules (R-and F-granules) were developed on different synt... more Two types of mesophilic methanogenic granules (R-and F-granules) were developed on different synthetic feeds containing acetate, propionate and bu-tyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H2-CO2 were related to the feed composition used for their development. These granules p e r f o r m e d a reversible reaction between H2 production from formate and formate synthesis from H2 plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and buty-rate during butyrate or isobutyrate degradation. Valer-ate and 2-methylbutyrate w e r e produced and consumed during propionate-butyrate degradation. The respective apparent Km (mM) for various substrates in disrupted R-and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an o p t i m u m temperature range from 40 to 50°C for H2-CO2 and formate utilization and 40 ° C for acetate, propionate and butyrate utilization and a similar o p t i m u m pH.
Two types of methanogenic gianules capable of high chemical oxygen demand removal rates were deve... more Two types of methanogenic gianules capable of high chemical oxygen demand removal rates were developed in laboratory-scale upflow reactors at 35 ° C. One granule type (R-granules) had a rod-type Metha-nothrix-like species as the predominant species whereas the other (F-granules) had a filament-type M. soehn-genii-like acetate-utilizer as the predominant species. These two types of granules were compared in terms of operational performance, physical-chemical characteristics and microbial population. The R-granules had a higher density [65-70 vs 39-43 g suspended solids (SS)/1], specific gravity (1.03 vs 1.01) and specific volu-metric methane production rate (180 vs 120 1 CH4/I granules per day) than the F-granules. Acetate, pro-pionate and butyrate degraders in both types of granules had similar specific growth rates. The most probable number enumeration indicated that both types of granule had the same population levels (cells/g SS) in terms of methanogens (H2-CO2-, formate-and acetate-utilizing), and syntrophie acetogens. Hydrolyticrfer-mentative bacteria were present in greater number in the F-granules than in the R-granules. The R-granules had a higher cell density than the F-granules. The differences in operational performance were due mainly to their different microbial composition, especially the predominant acetate-utilizing methanogens in the granules. The long-filamentous M. soehngenii-like rods in the F-granules appeared to be responsible for their lower density and large-sized granules.
Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.... more Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28°C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99%o was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14CJPCP to 14CH4 and 14Co2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP. Pentachlorophenol (PCP) is one of the biocides that was widely used in the United States, mainly for the preservation of wood and wood products. Along with other chlorophe-nols, PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency (16). Under aerobic conditions , PCP can be degraded by bacteria (2, 8, 29, 34) and fungi (23, 33). Aerobic organisms such as Flavobacterium spp. (8, 34, 37) and Rhodococcus spp. (2, 12, 35) have been successfully used in pilot-scale and field studies for the treatment of PCP-contaminated wastewater and groundwater. PCP can also be completely mineralized to methane and CO2 by anaerobic microorganisms (27). Reductive dechlori-nation of PCP occurs prior to complete mineralization in digested sludges and soils (3, 24, 26-29, 31, 41). Combined systems of an anaerobic fluidized bed plus trickling-filter or aerated lagoons were used to treat chlorophenolic waste from the paper pulp bleaching process (12, 35). Chlorophe-nols were removed from the wastewater by 50 to 60%, and mineralization of added PCP to CO2 was observed by use of radiotracer assay. However, only the overall system removal of chlorophenols was reported, and the role of anaer-obic fluidized bed in dechlorination was not clear. PCP removal or dechlorination was reported in semi-continuous-flow, stirred-sewage sludge digestors (11), in a bioreactor which was partially packed with glass beads to treat a mixture of meta-, ortho-, andpara-chlorophenols (19), in an anaerobic fixed-film reactor (13), and in upflow anaerobic sludge blanket (UASB) reactors with anaerobic granular sludge (14, 28, 42). In some cases, the dechlorination of PCP was not carried out completely, resulting in the appearance of lesser chlorinated phenols (28, 42). The volumetric PCP loading rates of the anaerobic reactor systems mentioned above were ca. 2.2 mg of PCP per liter of reactor volume per day or less. We have developed methanogenic PCP-degrading gran-* Corresponding author. 389 ules on a synthetic wastewater containing PCP, acetate, propionate, butyrate, and methanol in a laboratory-scale UASB reactor at 28°C (5, 6). The maximum PCP removal rate of the granules was as high as 14.6 mg/g of volatile suspended solids (VSS) per day, and a stable volumetric PCP removal rate of 90 mg of PCP per liter per day was achieved. The purpose of this article was to examine the feasibility of the development of methanogenic granules with high dechlorinating activity and to investigate the performance of the granules in treating wastewaters containing high PCP concentrations in a laboratory-scale UASB reactor system. MATERIALS AND METHODS Chemicals and gases. All chemicals (analytical grade) except PCP were obtained from Sigma Chemical Co. (St. Louis, Mo.); PCP was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Nitrogen gas and gas mixtures of N2-CO2 (95:5 and 70:30) were obtained from Linde Division, Union Carbide Corp. (Warren, Mich.), and passed over heated (370°C) copper filings to remove traces of 02. Analytical methods. Methane, methanol, and volatile fatty acids (VFA) were determined by using gas chromatography as described elsewhere (43, 44). PCP in solution was determined with high-performance liquid chromatography (HPLC). Samples (1.0 ml) were mixed with 0.5 ml of acetonitrile on a vortex mixer, centrifuged for 5 min at 5,500 x g with an Eppendorf 5415 centrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.), and filtered through 0.45-,um-pore-size syringe filters (Acrodisc LC13; Gelman Sciences Co., Ann Arbor, Mich.). A Waters HPLC system, consisting of the model 501 pump, the Lambda-Max model 481 UV detector, and the model 740 data module, was used. Samples were injected by using a Rheodyne 7010 injector fitted with a 50-,ul loop. Separation was accomplished with a Waters Radial-Pak C-18 column. The mobile phase consisted of acetonitrile and 5% aqueous acetic acid (8:2, vol/vol). The flow rate of Vol. 59, No. 2
The microbial species composition of metha-nogenic granules developed on an acetate-propionate-bu... more The microbial species composition of metha-nogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (101Z/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (101~/g dry weight), but low numbers of hydrolytic-fermentative bacteria (109/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospiril-lure hungatei strain BD, and a non-filamentous, bamboo shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading spore-forming oval-shaped species, strain PT, and a propion-ate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophi-cally with an H2-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, acetate and butyrate but they were involved in propionate degradation.
Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contai... more Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35°C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-C02. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens. Methanogenic granules are self-immobilized consortia of methanogens, syntrophic acetogens, and hydrolytic-fermen-tative bacteria that convert soluble organic matter to methane and CO2. The published work on the granule formation, microbial composition, substrate conversion potentials, and microbial structures of granules has focused on methanogens and syntrophic acetogens (7-11, 15, 23, 32, 39). This is likely due to the observation that sulfate-reducing bacteria (SRB) are present at much lower levels than syntrophic acetogens in granules treating sugar wastewater (10). Considerable information as to the methanogenic degradation of acetate, formate, and H2-CO2 in granules has been reported (7, 8, 11). Little work, however, has been performed to evaluate the conversion of other common substrates such as ethanol, propionate, and butyrate. SRB are quite diverse in terms of metabolic activities, morphotypes, trophic properties, and substrate affinities. In the presence of sulfate, acetate can be oxidized to CO2 by some pure SRB cultures; propionate, butyrate, and other volatile fatty acids (VFAs) can be oxidized completely to * Corresponding author. CO2 or converted to acetate or acetate plus propionate (in the case of odd long-chain acids with five or more carbon atoms); branched fatty acids such as isobutyrate, isovaler-ate, and 2-methylbutyrate can also be oxidized completely to CO2 or incompletely to acetate (35, 37). Hydrogen and formate can be utilized by many SRB as electron donors for sulfate reduction (35). Acetate and methanol are degraded via sulfate reduction by a coculture consisting of Desulfo-vibrio vulgaris and Methanosarcina barkeri (22). Methanol can be also degraded to CO2 via sulfate reduction by a coculture consisting of Desulfovibrio vulgaris and various homoacetogens (6, 12). In the absence of sulfate, certain SRB such as Desulfovibrio spp. may grow together with H2-utilizing methanogens to convert ethanol or lactate to acetate syntrophically (18, 34). The existence of syntrophic associations between H2-producing SRB and H2-consuming methanogens in lake sediments was suggested (5). No mention of syntrophic catabolism of VFAs by SRB in granular systems has been reported. VFAs such as propionate and butyrate are thought to be converted only by obligate syntrophic acetogens in concert with H2-utilizing methano-gens (3, 19, 20, 28, 35). In this study, methanogenic granules grown on brewery 3438
High performance biomethanation granules with operational specific COD removal rates of 7 kg COD ... more High performance biomethanation granules with operational specific COD removal rates of 7 kg COD removedl kg SS/d were obtained by ecoengineering conventional, granular, UASB digester sludge using a designed protocol of starvation and selection on a defined volatile fatty acid (VFA) based mineral medium. Addition of low (0.15 m M) sulfate levels to this VFA medium increased the maximum shock-load COD removal rate of the ecoengineered biomethanation granules to 9 kg COD/kg SS/d with specific acetate, propionate, and butyrate removal rates of 111, 28, and 64 mol/g SS/d. Addition of moderate (26 m M) calcium levels inhibited growth and altered the structure of granules. The general cellular, growth, stability , and performance features of these ecoengineered granules are described and discussed in relation to their use as improved biomethanation starter cultures.
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