Microbial Community Analysis of Mesophilic

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Microbial Community Analysis of Mesophilic Anaerobic Protein Degradation Process Using Bovine Serum Albumin (BSA)-Fed Continuous Cultivation YUEQIN TANG,1 TORU SHIGEMATSU,1* SHIGERU MORIMURA,1 AND KENJI KIDA1,2

Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto City, Kumamoto 860-8555, Japan1 and Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto City, Kumamoto 860-8555, Japan2

Received 15 September 2004/Accepted 16 November 2004

Two mesophilic anaerobic chemostats, one without added Ni2+ and Co2+ (chemostat 1) and the other with added Ni2+ and Co2+ (chemostat 2), were supplied with synthetic wastewater containing bovine serum albumin (BSA) as the sole carbon and energy source in order to study the capacity of protein degradation, microbial community structure and the effects of the addition of trace metals. Volatile fatty acids and ammonia were the main products of chemostat 1, while methane, –1 CO2 and ammonia were the main products of chemostat 2, and critical dilution rates of 0.15 d and 0.08 d–1 were obtained, respectively. Fluorescence in situ hybridization (FISH) with archaeal and bacterial domain-specific probes showed that archaeal cells were very limited in chemostat 1 while large populations of several types of archaeal cells were present in chemostat 2. Phylo- genetic analyses based on 16S rRNA gene clonal sequences, DGGE, and quantitative real-time polymerase chain reaction (PCR) showed that, within the domain Archaea, methanogens affiliated with the genera Methanosaeta and Methanoculleus were predominant in chemostat 2. Within the domain Bacteria, rRNA genes obtained from chemostat 1 were affiliated with the three phyla; Firmicutes (43%), Bacteroidetes (50%) and Proteobacteria (7%). A total of 56% of rRNA genes obtained from chemostat 2 was affiliated with the three phyla, Firmicutes (32%), Bacteroidetes (11%) and Proteobacteria (13%) while 44% of rRNA genes remained unclassified. Phylogeneti- cally distinct clones were obtained in these two chemostats, suggesting that different protein degradation pathways were dominant in the two chemostats: coupled degradation of amino acids via the Stickland reaction in chemostat 1 and uncoupled degradation of amino acids via syntrophic association of amino acid degraders and hydrogenotrophic methanogens in chemostat 2.

[Key words: bovine serum albumin (BSA), anaerobic degradation, methane fermentation, phylogenetic analysis]

In addition to carbohydrates and lipids, proteins are re- mented to volatile fatty acids and finally converted to meth- garded as the main organic substrates in anaerobic digestion ane and CO2 by an association of microorganisms. Species of sludge and wastewaters. The protein composition of sus- from genera such as Clostridium (5–8), Peptostreptococcus pended solid (SS) of domestic sewage is reportedly more (9–11), Acidaminobacter (12), Aminomonas (13), Therma- than 40% (1). Industries that process food such as whey, naerovibrio (14), Sporanaerobacter (15), Sedimentibacter cheese, casein and fish also typically produce wastewater (16) and Aminobacterium (17, 18) have been shown to containing significant amounts of protein. However, proteins degrade amino acids anaerobically. Most anaerobic amino have been demonstrated to degrade more slowly than carbo- acid degrading bacteria are affiliated with the phylum Firmi- hydrates under anaerobic conditions (2). It has also been cutes (the low G+C gram-positive bacteria). In anoxic envi- documented that the anaerobic degradation of protein-rich ronments, the oxidation of certain amino acids is endergonic wastes is often incomplete (3–5). These findings imply the under standard conditions and only proceeds if the reduc- importance of protein degradation processes in anaerobic ing equivalents produced are removed by a biological or a ecosystems. chemical electron scavenger. This is possible by interspe- Under methanogenic conditions, proteins are hydrolyzed cies hydrogen transfer in the presence of hydrogen utilizing to peptides and amino acids, which are subsequently fer- microorganisms, and when chemical compounds, such as amino acids (Stickland reaction) (5), act as the final electron * Corresponding author. e-mail: [email protected] acceptor. It has been considered that amino acids are prefer- phone: +81-(0)96-342-3668 fax: +81-(0)96-342-3679 entially fermented via the Stickland reaction during anaero-

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bic treatment of protein-rich wastewater or waste because mixed amino acids are readily supplied by protein hydrolysis. Despite this knowledge of protein and amino acid degradation that was primarily obtained by investigating pure culture assays, the contribution of these microorganisms to protein degradation in anaerobic ecosystems remains un- known. Because only a fraction of the total microorganism population in an ecosystem can be cultured (19), the actual diversity of protein and/or amino acid degraders remains unclear. Culture-independent methods using molecular biological techniques have successfully been applied to ana- lyze microbial communities in several ecosystems, including anaerobic digesters. Although there are several reports about the anaerobic treatment of protein-rich wastewaters concerning the optimization of treatment processes or morphological observation (3, 4, 20–23), there have not been any reports concerning the analysis of microbial community structure among anaerobic protein digesters to date. In this study, using anaerobic digested sludge as the source FIG. 1. Operation schedules for chemostats 1 (a) and 2 (b). Ar- of microorganisms, two chemostats supplied with synthetic rows refer the times FISH, and DNA extraction for 16S rRNA gene wastewater containing bovine serum albumin (BSA) as the analyses were conducted. sole carbon and energy source were constructed as treatment models for protein-rich wastewater. The capacity for Fluorescence in situ hybridization (FISH) A 0.5-ml por- P L protein degradation, microbial community structure and the tion of culture broth was centrifuged at 16,000 g for 5 min at 4 C effects of the addition of trace metals were then investi- and the resulting pellet was used as the sample for FISH. FISH was carried out according to the method of Amann (24). EUB338 gated. (S-D-Bact-0338-a A-18) (25), which was 5>-end labeled with Cy5 (Amersham Bioscience, Piscataway, NJ, USA), and ARC915 (S-D-Arch-0915-a-A-20) (26), which was 5 >-end labeled with Cy3 MATERIALS AND METHODS (Amersham Bioscience), were used for the detection of bacterial and archaeal cell, respectively. A confocal laser scanning micro- Synthetic wastewater Synthetic wastewater with BSA as the scope FV300 (Olympus, Tokyo) was used for microscopic obser- sole carbon and energy source was prepared without Ni2+ and Co2+ vation. as follows (g/l): BSA (albumin, bovine, general grade; Nacalai DNA extraction, PCR amplification and cloning DNA Tesque, Kyoto), 17; KH2PO4, 0.3; KHCO3, 4.0; NH4Cl, 1.0; NaCl, from the microbial community was extracted as previously re- 0.6; MgCl2 6H2O, 0.82; CaCl2 2H2O, 0.08; cystein–HCl H2O, ported (27). Amplification of 16S rRNA gene from the extracted 0.1; 10 ml of the trace element solution of DSMZ medium 318 community DNA was performed by PCR using AmpliTaq Gold [Deutsche Sammlung von Mikroorganismen und Zellkulturen (Applied Biosystems, Foster City, CA, USA) according to the manu- GmbH: Catalogue of Strains 2001. http://www.dsmz.de/dsmzhome. facturer’s instructions (100 ng template DNA, 1PPCR buffer, 2.5 htm] without NiCl26H2O and CoCl26H2O; and 10 ml of the vita- units AmpliTaq Gold polymerase, 250 M dNTPs and 40 pmol of min solution of DSMZ medium 318 without B12. For the synthetic each primer in a 100- l reaction volume). The primer sets Ar28F 2+ 2+ wastewater with Ni and Co , NiCl26H2O and CoCl26H2O were (5>-TGGTTGATCCTGCCAGAGG-3>) and 1490R (5>-GGTTACC added to give Ni2+ and Co2+ concentrations of 0.10 and 0.12 mg/l, TTGTTACGACTT-3>), and Eu27F (5>-AGAGTTTGATCCTGGC respectively. The pH of the synthetic wastewater was adjusted to TCAG-3>) and 1490R, were used to amplify 16S rRNA gene from 7.0 with HCl. The total organic carbon (TOC) concentration of the the total-community DNA and targeted Archaea and Bacteria, re- synthetic wastewater was 8000 mg/l. spectively. The reactions were performed using a Gene Amp PCR Operation of BSA-fed chemostats Two anaerobic chemo- System 2400 (Applied Biosystems) with the following cycle con- stats were operated using two completely stirred tank reactors ditions: preincubation at 95LC for 9 min; and 30 cycles of 95LC (CSTRs), each with a working volume of 1.7 l and were mixed for 1 min, 50LC for 1 min and 72LC for 2 min. The amplified 16S thoroughly using a magnetic stirrer. The temperature during the rRNA gene fragments were cloned into a pT7Blue T-vector plas- continuous cultivation was maintained at 37LC by immersing the mid (Novagen, Madison, WI, USA) using the DNA Ligation Kit reactors in a thermostated water-bath, and pH was maintained ver. 2 (Takara, Kyoto). Libraries of clones were constructed using automatically at 7.0 by feeding 1 N HCl solution through a pump Eschericha coli DH5-competent cells (Takara) according to the which was controlled by a pH controller. A 1.2-l portion of meso- manufacturer’s instructions. philic digested sludge from the Kumamoto-Hokubu sewage treat- Sequencing and phylogenetic analysis Cloned 16S rRNA ment plant (Kumamoto City) was washed twice with a synthetic genes were prepared from randomly selected recombinants and wastewater without BSA, Ni2+ and Co2+ under anaerobic condi- were used as templates for sequencing with a CEQ8000 genetic tions, was diluted to 1.7 l, and was then placed in each CSTR. Syn- analysis system (Beckman Coulter, Fullerton, CA, USA) and a thetic wastewaters containing BSA as the sole carbon source, with CEQ DTCS-Quick Start Kit (Beckman Coulter) in accordance or without Ni2+ and Co2+ for chemostat 2 and chemostat 1, respec- with the manufacturer’s instructions. About 1400 bases were se- tively, were fed into the CSTR. Biogas was collected in a gas- quenced for all clones selected. All sequences were checked manu- holder. Figure 1 summarizes the time schedule for dilution rate ally for chimeric artifacts using the CHIMERA_CHECK program changes of chemostat 1 and chemostat 2. ver. 2.7 of the Ribosomal Database Project II (RDP-II) (28). 中国科技论文在线 http://www.paper.edu.cn 152 TANG ET AL. J. BIOSCI. BIOENG.,

Searches for similar sequences were performed using the BLASTN used as standards for the quantification of the genera Methano- program (29). Multiple alignments were generated with the Clustal saeta, Methanosarcina, Methanoculleus and Methanospirillum, re- X program ver. 1.8 (30) and phylogenetic trees were constructed spectively. using the MEGA program ver. 2.1 (31) based on evolutionary dis- Other analysis methods All of the following parameters of tances that were calculated using the Neighbor-Joining method the culture broth in the reactor, except for total solids (TS), total (32) and Kimura’s two-parameter model (33). Bootstrap resampl- volatile solids (TVS), suspended solids (SS) and volatile sus- ing analysis (34) for 500 replicates was performed in order to esti- pended solids (VSS), were analyzed in supernatants obtained after mate degrees of confidence in tree topologies. Identical sequences centrifugation at 10,000 rpm for 10 min. TS, TVS, SS and VSS were recognized by analysis of phylogenetic trees and manual were analyzed in accordance with standard methods (36). Soluble comparisons, in which sequences that were more than 99.8% simi- total organic carbon (TOC) was analyzed using a TOC auto ana- lar were defined as identical, and such sequences were used for lyzer (TOC-V; Shimadzu, Kyoto) according to the testing methods further phylogenetic analysis as an operational taxonomic unit for industrial wastewater, JISK0102-1986 (36). Volatile fatty acids + (OTU). Four libraries of clones were constructed with total-com- (VFAs) were analyzed as described previously (37). NH4 and – munity DNA that had been extracted from culture broth from the NO3 were measured using the HACH methods (HACH, Loveland, chemostats. An archaeal 16S rRNA gene library (BSA1A library) CO, USA) (38). Methane content of the biogas was measured us- and a bacterial 16S rRNA gene library (BSA1B library) were pre- ing a gas chromatograph equipped with a thermal conductivity de- pared from the community DNA from chemostat 1 at the dilution tector (TCD) (GC323; GL Sciences, Tokyo) and a packed column rate of 0.15 d–1. Twenty-three clones from BSA1A library and 44 (Porapack N; GL Sciences). The protein concentration of culture clones from BSA1B library were sequenced. And, an archaeral broth was measured using the Lowry–Folin method (39). 16S rRNA gene library (BSA2A library) and a bacterial 16S rRNA Nucleotide sequence accession numbers Archaeal and bac- gene library (BSA2B library) were prepared from chemostat 2 at terial 16S rRNA gene sequences obtained in this study were depos- the dilution rate of 0.08 d–1. Thirty-nine clones from BSA2A li- ited with the DDBJ and are available under the accession nos. brary and 54 clones from BSA2B library were sequenced. AB175336 to AB175393. Denaturing gradient gel electrophoresis (DGGE) The V3 regions of 16S rRNA genes from the community DNA extracted from the chemostats were amplified by PCR and the amplified RESULTS products were used for DGGE analysis. PCR was performed using Continuous anaerobic degradation of BSA using AmpliTaq Gold (Applied Biosystems) in a total volume of 100 l in 0.2-ml reaction tubes, and the GeneAmp PCR system 2400 anaerobic chemostats Continuous anaerobic degrada- (Applied Biosystems). The primer sets and GC clamp used were tion of BSA was carried out for about one year using syn- the same as those described by Øvreås et al. (35). For amplifica- thetic wastewater containing BSA as the sole carbon and 2+ tion of bacterial rRNA gene, PRBA338F and PRUN518R were energy source at 37LC. Synthetic wastewater without Ni used as primers. The thermal profile was as follows: incubation at and Co2+ was fed into chemostat 1, while that with Ni2+ and 95LC for 9 min; and then 30 cycles of incubation at 95LC for 1 min, Co2+ was fed into chemostat 2. The dilution rate (D) of 55LC for 1 min and 72LC for 2 min. Archaeal rRNA gene was ob- chemostat 1 was varied in steps from 0.01 to 0.2, as shown tained by nested PCR with a thermal profile similar to that used for on the schedule in Fig. 1. The concentrations of TOC, VFA, bacterial rRNA gene, with the following exceptions: there were 35 ammonia and VSS were stable at a dilution rate between cycles of amplification and the annealing temperature was set at 0.05 and 0.15 d–1, indicating that chemostat 1 was at steady- 53.5LC. For nested PCR, PRA46F and PREA1100R primers were used to produce fragments of rRNA gene, and these fragments state at the dilution rates (Fig. 2a). The critical dilution rate (Dc), where the system was able to maintain stable condi- were then used as a template for the second PCR with primers –1 PARCH340F and PARCH519R. DGGE was performed with a tions, was 0.15 d . The VSS concentration at a dilution rate –1 DCode Universal Mutation Detection System (Bio-Rad Laborato- of 0.15 d was maintained at about 1.6 g/l for about 270 d. ries, Hercules, CA, USA). The amplified fragments of rRNA gene The measured outputs of carbon and nitrogen accounted for were loaded onto a 10% (w/v) polyacrylamide gel in 0.5PTAE about 100% of the inputs (Table 1 and 2), and the system (pH 8.0). Gel prepared with a denaturing gradient ranging from was thought to be in the stable condition. High concentra- 30% to 60% was used for analysis of archaeal rRNA gene while tions of VFAs (8000–12,000 mg/l) and ammonia (2600–3000 gel prepared with a denaturing gradient ranging from 20% to 60% mg/l) were detected at this dilution rate (Tables 1 and 2). was used for analysis of bacterial rRNA gene. Electrophoresis was Under steady-state conditions at a dilution rate of 0.15 d–1, performed at 60LC, first for 20 min at 25 V and then at 130 V for 8 h and 12 h for analysis of archaeal and bacterial rRNA gene, re- the protein degradation ratio was approximately 75% and spectively. Representative clones from 16S rRNA gene libraries the main products of degradation were VFAs and ammonia were analyzed simultaneously to determine the correlation be- (Fig. 2a). Negligible amounts of gas were produced. Acetic tween the bands detected in DGGE and the clones obtained in the acid, propionic acid, butyric acid and iso-valeric acid ac- rRNA gene libraries. counted for most of the VFA produced (Table 1). The result Real-time quantitative PCR For detection and quantifica- suggested that acetogenesis and methanogenesis were com- tion of methanogens by real-time quantitative PCR, the TaqMan pletely inhibited by the lack of Ni2+ and Co2+. The dilution fluorogenic PCR system (Applied Biosystems) was used and the rate of chemostat 2 (with Ni2+ and Co2+) was varied in steps method was the same as reported previously (27). The primer/probe from 0.01 to 0.2, as shown on the schedule in Fig. 1. The sets for the genera Methanosaeta, Methanosarcina, Methano- concentrations of TOC, VFA, ammonia and VSS were sta- culleus and Methanospirillum were MS1b/SAE835R/SAE761TAQ, –1 MB1b/SAR835R/SAR761TAQ, AR934F/MG1200b/MCU1023TAQ ble at a dilution rate between 0.05 and 0.08 d , indicating (27) and AR934F/MG1200b/MSP1025TAQ (5 >-GAATGATAGTC that chemostat 2 was at steady-state at the dilution rates GGGATGAAGACTCTA-3>, 1000–1025, E. coli position), respec- (Fig. 2b). Based on the fermentation results, the Dc at which –1 tively. The cloned 16S rRNA gene fragments, EA02 (AB092914), BSA could be converted to methane was only 0.08 d (Fig. DA16 (AB092915), AA01 (AB092916), BA03 (AB092917), were 2B). The VSS concentration at a dilution rate of 0.08 d –1 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 153

+ FIG. 2. Effect of dilution rate on TOC, VFA, NH4 , and VSS concentrations and gas production rate in chemostat 1 (a) and chemostat 2 (b).

TABLE 1. The concentration of BSA and volatile fatty acids in influent and effluent, and the gas production in each chemostat Total Iso- Gas Inorganic Carbon Total C Protein Acetate Propionate Butyrate –1 VFA –1 –1 –1 valerate production carbon in SS per day (g l ) –1 (g l ) (g l ) (g l ) –1 –1 –1 –1 –1 (g l ) (g l ) (ml d ) (g l ) (g-C d ) (g-c d ) Chemostat 1 (0.15 d–1) Influent 17.00 0.00 N.D. N.D. N.D. N.D. – 0.5 0.00 2.435 (2.30 g-C d–1) (0.135 g-C d–1) Effluent 4.25 11.10 4.69 1.99 2.02 2.37 120 1.05 0.21 2.600 (0.57 g-C d–1) (1.48 g-C d–1) (0.06 g-C d–1) (0.28 g-C d–1) Chemostat 2 (0.08 d–1) Influent 17.00 0.00 N.D. N.D. N.D. N.D. – 0.50 0.00 1.292 (1.22 g-C d–1) (0.072 g-C d–1) Effluent 1.70 1.07 0.31 0.76 N.D. N.D. 1780 0.78 0.07 1.320 (0.12 g-C d–1) (0.07 g-C d–1) (0.95 g-C d–1) (0.11 g-C d–1) N.D., Not detected. SS, Suspended solid. The calculated amounts of carbon are shown in parentheses.

TABLE 2. The balance of nitrogen element was about 1.7 g/l. The output and input of carbon and nitro- of the BSA-fed chemostats gen were shown in Tables 1 and 2, and the outputs ac- Solid counted almost 100% of inputs. Under steady-state condi- + – Protein-N NH4 -N NO3 -N compo- Total N tions at this dilution rate, the protein degradation ratio was (g-N d–1) (g-N d–1) (g-N d–1) nent-N (g-N d–1) –1 over 90% (Tables 1 and 2). The main products of the BSA (g-N d ) degradation were biogas (about 1050 ml/ld) and ammonia –1 Chemostat 1 (0.15 d ) (about 3000 mg/l), thus suggesting that acetogenesis and Influent 0.735 0.061 0.007 0.000 0.803 methanogenesis had occurred, and approximately 100% of Effluent 0.183 0.620 0.036 0.045 0.884 the TOC was detected in the effluent and the biogas evolved. Chemostat 2 (0.08 d–1) Influent 0.392 0.033 0.004 0.000 0.429 The methane concentration in the biogas was approximately Effluent 0.039 0.335 0.011 0.023 0.408 50%. Fluorescence in situ hybridization (FISH) The two chemostats constructed here showed different properties as described above. This difference would be caused by the 中国科技论文在线 http://www.paper.edu.cn 154 TANG ET AL. J. BIOSCI. BIOENG.,

FIG. 3. Phase-contrast photomicrographs (a, c) and results of FISH (b, d) for microorganisms among the mesophilic anaerobic BSA digesters. Panels a and b show cells grown in chemostat 1. Panels c and d show cells grown in chemostat 2. The florescent probes used were a combination of ARC915 (Archaea, green) and EUB338 (Bacteria, red). The bar represents 10 m.

difference of microbial communities of both chemostats. Domain Archaea In the BSA1A library, 10 OTUs (23 The microbial community analyses on the chemostats were clones) were obtained. All of them were assigned to the therefore conducted. To determine the relative properties of order Methanomicrobiales in the phylum Euryarchaeota bacterial and archaeal cells, Cy5-labeled EUB338 and Cy3- (Fig. 4). Seven OTUs of 14 clones were closely related to labeled ARC915 were used simultaneously in the culture Methanocelleus bourgensis with 98–99% sequence similari- broth at dilution rates of 0.15 d–1 for chemostat 1 and 0.08 d–1 ties (BSA1A-01, one clone; BSA1A-02, one clone; BSA1A- for chemostat 2. As shown in Fig. 3a and 3b, for chemostat 1, 03, eight clones; BSA1A-04, one clone; BSA1A-05, one the number of cells hybridized with the ARC915 probe was clone; BSA1A-06, one clone; BSA1A-07, one clone). Two small and almost all cells hybridized with the EUB338 OTUs of the eight clones were closely related to Methano- probe, thus indicating that bacterial cells were absolutely dominant. Most of the cells hybridized with the EUB338 TABLE 3. Distribution of 16S rRNA gene clones detected probe were rod shaped. In contrast, as shown in Fig. 3c and in the culture broth of the BSA-fed chemostats 3d, for chemostat 2, a significant number of rod-shaped, Chemostat 1 Chemostat 2 filamentous and coccoid cells were hybridized with the (without Ni 2+ and Co2+)a (with Ni 2+ and Co2+)b Taxon (phylum) ARC915 probe, although the majority of cells hybridized Number of Number of Number of Number of with the EUB338 probe. Filamentous cells were dominant OTUs clones OTUs clones among the cells hybridized with the ARC915 probe. Archaea General phylogenetic analysis All the clones se- Euryarchaeota 10 23 9 39 quenced in two archaeral libraries (BSA1A and BSA2A) were Bacteria affiliated with the phylum Euryarchaeota. Among the clones Firmicutes 11 19 10 17 Bacteroidetes 5223 6 analyzed, 10 different sequences (OTUs) were found in Proteobacteria 23 47 BSA1A library and 9 OTUs in the BSA2A library (Table 3). Unclassified –– 424 Among the clones sequenced in the bacterial libraries, 18 Total (Bacteria)18442154 OTUs were found in BSA1B library and 21 OTUs in the a Dilution rate (d–1) 0.15 BSA2B library, and their classification were shown in Table 3. b Dilution rate (d–1) 0.08 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 155

FIG. 4. Phylogenetic tree showing the genetic relationships among the clones affiliated with the order Methanosarcinales and the order Methanomicrobials. The tree was constructed by the Neighbor-Joining method (34) using partial sequences of 16S rRNA gene. BSA1A and BSA2A refer to clones derived from chemostat 1 and chemostat 2, respectively. Numbers of clones with identical sequences are shown in parentheses. The bar represents two substitutions per 100 nucleotide positions. Bootstrap probabilities (36) are indicated at the branch nodes. The DDBJ/EMBL/GenBank accession numbers for reference strains and clones obtained in this study are shown in parentheses in this and other fig- ures. The tree was rooted using Methanobacterium bryantii as the outgroup.

culleus chikugoensis with 98% sequence similarities Domain Bacteria (BSA1A-08, one clone; BSA1A-09, seven clones). In addi- Phylum Firmicutes (Low G+C gram-positive bacteria) tion, one clone (BSA1A-10) was closely related to Methano- In the BSA1B library, 11 OTUs (19 clones, 43% of total spirillum hungatei with a 96% sequence similarity. clones in the library) were affiliated with the phylum Firmi- In the BSA2A library, nine OTUs (39 clone) were ob- cutes. The OTUs were divided into five phylogenetic groups. tained. Three OTUs (28 clones) were assigned to the order Two groups, six OTUs (13 clone) and one OTU (one clone), Methanosarcinales and six OTUs (11 clones) were as- were assigned to the Clostridium cluster XII (40) and De- signed to the order Methanomicrobiales in the phylum sulfotomaculum cluster I (41), respectively (Fig. 5). Two Euryarchaeaota (Fig. 4). The most dominant sequence was groups, one OTU (one clone) and two OTUs (two clones), closely related to Methanoseata concilii with a 100% se- were closely related to Clostridium cluster XIII and X (40), quence similarity (BSA2A-08, 25 clones). Five OTUs (10 respectively (Fig. 5). Another group, one OTU (two clones), clones) were closely related to Methanoculleus bourgensis was assigned to a cluster containing clone PeH17 (42). Four with 98–99% sequence similarities. One clone (BSA2A-06) OTUs (BSA1B-08 through 11, 11 clones) in Clostridium was closely related to Methanospirillum hungatei with a cluster XII were closely related to Clostridium sp. strain PO 97% sequence similarity. (the nucleotide sequence accession no. AJ002593), isolated from an anaerobic digester, which uses gelatin as a sole 中国科技论文在线 http://www.paper.edu.cn 156 TANG ET AL. J. BIOSCI. BIOENG.,

FIG. 5. Phylogenetic tree showing the genetic relationships among the clones affiliated with the phylum Firmicutes (low G+C gram-positive bacteria). The tree was constructed by the Neighbor-Joining method (34) using partial sequences of 16S rRNA gene. The bar represents two substitutions per 100 nucleotide positions. Numbers in parentheses and numbers at the branch nodes are the same as those in Fig. 4. The tree was rooted using Arthrobacter globiformis as the outgroup. 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 157

source of carbon and energy (Javis, G.N., personal com- assigned to Clostridium cluster III (40) and a cluster con- munication), with 98–100% sequence similarities. OTUs taining clone ML635J-14 (43), respectively (Fig. 5). All 5 BSA1B-06 and 07 in this cluster were closely related to OTUs (BSA2B-01 through 05, nine clones) in the cluster Sporanaerobacter acetigenes with a 94% sequence similar- related to Clostridium cluster X were closely related to ity. OTU BSA1B-05 (one clone) was closely related to Sedi- Aminobacterium mobile, an asaccharolytic mesophilic syn- mentibacter hydroxybenzoicus with a 96% sequence simi- trophic amino-acid degrading bacterium (18), with approxi- larity. OTU BSA1B-01 (one clone) in the cluster related to mately 95% sequence similarity. Four OTUs (BSA2B-06 to Clostridium cluster X was closely related to Aminobac- 09) were not related to any pure cultured microorganisms, terium colombiense, a mesophilic asaccharolytic syntrophic but were related to the uncultured clone ML635J-14 ob- anaerobe that degrades amino acid (17), with a 98% se- tained from Mono Lake (44). quence similarity. Another OTU named BSA1B-02 in this Phylum Bacteroidetes Five OTUs (22 clones, 50% of cluster was closely related to Aminomonas paucivorans, a total clones in library) of the BSA1B library were affiliated mesophilic asaccharolytic amino-acid-degrading bacterium with the phylum Bacteroidetes. Of these, three OTUs (20 (13), with a 92% sequence similarity. clones) were assigned to the order Bacteroidales (Fig. 6). In the BSA2B library, 10 OTUs (17 clones, 32% of total OTUs BSA1B-12 and 13 were closely related to the uncul- clones in the library) were affiliated with the phylum Firmi- tured clone vadin BC27, obtained from an anaerobic reac- cutes. The phylogenetic positions of the OTUs in the tor treating wine distillation waste (45), with approximately BSA2B library were different from those of OTUs from the 95% sequence similarity. The other two OTUs (BSA1B-15 BSA1B library. The OTUs of the BSA2B library were and 16, two clones) were assigned to the order Sphingobac- divided into three phylogenetic groups. Five OTUs (nine teriales. clones) were closely related to Clostridium cluster X (40). In contrast to the BSA1B library, only three OTUs (six One OTU (one clone) and 4 OTUs (seven clones) were clones, 11% of total clones in library) of the BSA2B library

FIG. 6. Phylogenetic tree showing the genetic relationships among the clones affiliated with the phylum Bacteroidetes. The tree was constructed by the Neighbor-Joining method (34) using partial sequences of 16S rRNA gene. The bar represents five substitutions per 100 nucleotide positions. Numbers in parentheses and numbers at the branch nodes are the same as those in Fig. 4. The tree was rooted using Spirochaeta africana as the outgroup. 中国科技论文在线 http://www.paper.edu.cn 158 TANG ET AL. J. BIOSCI. BIOENG.,

FIG. 7. Phylogenetic tree showing the genetic relationships among the clones affiliated with the phylum Proteobacteria. The tree was constructed by the Neighbor-Joining method (34) using partial sequences of 16S rRNA gene. The bar represents two substitutions per 100 nucleotide positions. Numbers in parentheses and numbers at the branch nodes are the same as those in Fig. 4.

were affiliated with the phylum Bacteroidetes. OTUs BSA2B- Spirochaetes (Fig. 8), but was distinct from both. OTUs 11 (one clone) and 12 (4 clones) were assigned to the order BSA2B-18 (one clone) and BSA2B-19 (one clone) showed Bacteroidales and were closely related to the uncultured a significant relationship with the phylum Bacteroidetes clone WCHB1-29 obtained from a hydrocarbon- and chlori- with a bootstrap value of 100. The other two OTUs, OTU nated-solvent-contaminated aquifer (46) (Fig. 6). Another BSA2B-21 (20 clone) and BSA2B-20 (two clones), were ap- OTU (BSA2B-13, one clone) formed a cluster with species parently distinct from the phylum. These OTUs were closely of the order Sphingobacteriales. related to the uncultured clone R5p16 (nucleotide sequence Phylum Proteobacteria Within the Proteobacteria, two accession no. AF482444) obtained from granular sludge con- OTUs (BSA1B-17 and 18, three clones, 7% of total clones cerning fatty acid oxidization, with a 99% and a 93% se- in BSA1B) in the BSA1B library and 4 OTUs (BSA2B-14 quence similarity, respectively. through 17, seven clones, 13% of total clones in BSA2B) in Analysis of community structure by DGGE To the BSA2B library were detected (Fig. 7). OTU BSA1B-17 evaluate the coverage of the microbial communities in the (one clone) was closely related to a sulfate-reducing bac- chemostats by our 16S rRNA gene library analysis, the com- teria, Desulfovibrio desulfuricans, which belongs to the class munity DNAs from the culture broths of chemostat 1 at a Deltaproteobacteria (47), with a 99% sequence similarity. dilution rate of 0.15 d–1 and chemostat 2 at a dilution rate OTU BSA1B-18 (2 clones) was closely related to Comamo- of 0.08 d–1 were analyzed by DGGE. Fragments of approxi- nas testosteroni, which belongs to the class Betaproteo- mately 200 bp in the V3 region of archaeal and bacterial bacteria (47) with a 97% sequence similarity. Four OTUs 16S rRNA gene were amplified from the community DNAs (BSA2B-14 through 17, seven clones) in the BSA2B-library and clones obtained by the rRNA gene library analysis, sep- were closely related to Arcobacter skirrowi, which belongs arately, and used to DGGE. The position of bands derived to the class Epsilonproteobacteria (47), with approximately from the community DNAs and rRNA gene clones were 98% sequence similarity. compared to consider the correlation. Unclassified clones Four OTUs (24 clones) in the At least five and six bands were detected after DGGE BSA2B library were not affiliated with any phyla deter- using the amplified archaeal rRNA gene derived from the mined to date. The phylogenetic position of these in the do- community DNA of chemostat 1 and chemostat 2, respec- main Bacteria was related with the phyla Bacteroidetes and tively (lanes 1 and 2 in Fig. 9). Among these bands, A2 and 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 159

FIG. 8. Phylogenetic tree showing the genetic relationships among the clones unclassified so far. The tree was constructed by the Neighbor- Joining method (34) using partial sequences of 16S rRNA gene. The bar represents five substitutions per 100 nucleotide positions. Numbers in parentheses and numbers at the branch nodes are the same as those in Fig. 4. The tree was rooted using Methanobacterium bryantii as the outgroup.

A3 were common to both chemostats. Band A2, which was At least 21 and 19 bands were detected after DGGE using the dominant band in the two systems, was correlated with the amplified bacterial rRNA genes derived from culture OTUs BSA1A-01 to 07 and OTUs BSA2A-01 to 05, which broths of chemostat 1 and chemostat 2, respectively (lanes 3 were closely related to Methanoculleus bourgensis. Band and 4 in Fig. 9). The band patterns for the two chemostats A3 was correlated with OTU BSA1A-10 and BSA2A-06, were quite different, indicating different community struc- which were closely related to Methanospirillum hungatei. tures. For chemostat 1, two intense bands, B2 and B6, were Band A1, which was also predominant in chemostat 1, was detected. Band B2 was correlated with the predominant correlated with OTUs BSA1A-08 and 09, which were close- OTUs BSA1B-12 to 14 (20 clones), which were closely re- ly related to Methanoculleus chikugoensis. Band A4, which lated to the clone vadin BC27 (45). Band B6 was correlated was the most predominant band in chemostat 2, was corre- with the predominant OTUs BSA1B-08 to 11 (11 clones), lated with OTUs BSA2A-07 through 09, which were close- which were closely related to Clostridium sp. strain PO. ly related to Methanosaeta concilii. These observations sup- Band B7 was correlated with the OTUs BSA1B-04 (two ported the results of archaeal rRNA gene clone library anal- clones) and 05 (one clone). Bands B1, B3, B4, B5, B8 and ysis, which showed that the genus Methanoculleus was pre- B9 were correlated with OTUs BSA1B-16 (one clone), 18 dominant in chemostat 1 and the genera Methanosaeta and (two clones), 06 (one clone), 07 (one clone), 15 (one clone) Methanoculleus were predominant in chemostat 2. All the and 02 (one clone), respectively. For chemostat 2, four in- OTUs obtained in the analysis of archaeal libraries were tense bands, B10, B11, B12 and B18, were detected. Bands detected as bands in DGGE. Two bands from chemostat 1 B10 and B11 were correlated with OTUs BSA2B-20 (two and three bands from chemostat 2 were not correlated to the clones) and 21 (20 clones), which were closely related to rRNA gene clones, indicating the limited coverage of the the clone R5p16. The two bands were detected from a sin- rRNA gene clone libraries. However, the libraries could gle clonal sequence, due to electrophoresis conditions. Band cover most of the archaeal communities, because all the B12 was correlated with OTU BSA2B-14 to 17 (seven bands with no correlation with the rRNA gene clones were clones), which were closely related to Arcobacter skirrowi, not predominant. and OTU BSA2B-18 (one clone) and 19 (one clone). A sin- 中国科技论文在线 http://www.paper.edu.cn 160 TANG ET AL. J. BIOSCI. BIOENG.,

FIG. 9. Results of denaturing gradient gel electrophoresis (DGGE) for the fragments of 16S rRNA gene from Archaea (lanes 1 and 2) and Bac- teria (lanes 3 and 4) in BSA-fed chemostats. Lanes 1 and 3 show results for chemostat 1. Lanes 2 and 4 show the results for chemostat 2. Bands are numbered as A1 to B19. The fragments of representative clones with approximate migratory positions in the DGGE profile are schematically illus- trated beside the result of DNA samples and the ratio of clone in its library is shown in parenthesis.

gle band of the same position was detected from these Real-time quantitative PCR Real-time quantitative OTUs, although the amplified sequence of OTUs BSA2B- PCR experiments were performed in order to quantify the 14 to 17 was different from BSA2B-18 and 19. Band B18 four methanogens using DNA extracted from the culture was correlated with OTU BSA2B-01 (one clone), 03 to 05 broths of chemostat 1 at a dilution rate of 0.15 d–1 and (seven clones), which were affiliated with Aminobacterium chemostat 2 at a dilution rate of 0.08 d–1. For chemostat 1, mobilis. Band B15 was correlated with OTU BSA2B-10 only the 16S rRNA gene of the genus Methanoculleus was (one clone) and 12 (four clones). Band B19 was correlated detected with a copy number of approximately 1.5 P 106 with OTU BSA2B-06 to 07 (five clones). Bands B13, B14, copies/50 ng DNA (Table 4). For chemostat 2, rRNA genes B16 and B17 were correlated with OTU BSA2B-11 (one of the genera Methanosaeta and Methanoculleus were de- clone), 13 (one clone), 09 (one clone) and 02 (one clone), tected, both at a copy number of approximately 2.5 P 106 respectively. These observations supported the results of copies/50 ng DNA. A relatively small amount of rRNA bacterial rRNA gene clone library analysis and almost all gene of the genus Methanosarcina was also detected in OTUs obtained in the bacterial libraries were detected as chemostat 2, although clones affiliated with Methanosarcina bands in DGGE. The trace level-bands, 12 bands from were not obtained in 16S rRNA gene clone libraries. In ad- chemostat 1 and nine bands from chemostat 2, were not dition, 16S rRNA gene of the genus Methanospirillum were correlated to any clones in the rRNA gene libraries. These not detected in the two chemostats, although clones affili- results indicated that the bacterial rRNA gene clone librar- ated with Methanospirillum were obtained in 16S rRNA ies could cover most of the bacterial communities in the gene clone analysis. These data supported the results of 16S chemostats. rRNA gene library and DGGE analysis, which confirmed

TABLE 4. Quantification of 16S rRNA gene of methanogens in the culture broth of the BSA-fed chemostats Primer/probe set Target organism Chemostat 1 a Chemostat 2b MS1b/SAE835R/SAE761TAQ Methanosaeta spp. N.D. 2.36P106 MB1b/SAR835R/SAR761TAQ Methanosarcina spp. N.D. 1.39P105 AR934F/MG1200b/MCU1023TAQ Methanoculleus spp. 1.51P106 2.77P106 AR934F/MG1200b/MSP1025TAQ Methanospirillum spp. N.D. N.D. N.D., Not detected. The values represent the averages of experiments conducted in duplicates. Unit, 16S rRNA gene copies/50 ng-DNA. a Dilution rate (d–1)0.15 b Dilution rate (d–1)0.08 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 161

that, among the Archaea, the genus Methanoculleus and the found to be dominant. The genus Methanosaeta would play genera Methanosaeta and Methanoculleus were predomi- a role in mineralization of acetate produced by amino-acid nant in chemostat 1 and chemostat 2, respectively. Our re- degradation and subsequent acetogenesis. Hydrogen produced sults of real-time quantitative PCR suggested that a signifi- during amino-acid degradation and acetogenesis would be cant population of the genus Methanoculleus (approxi- converted to methane by the genus Methanoculleus in this mately 29% of total methanogens in chemostat 2) existed in chemostat. chemostat 1, which showed almost no methane production In both chemostats, cells belonging to the phylum Firmi- ability. This discrepancy might be caused by the difference cutes were predominant among the Bacteria. Nineteen (43% of population in the two chemostats. From 30 ml of culture in BSA1B library) and 17 (31% in BSA2B library) rRNA broth of chemostats 1 and 2, 39.6 g and 91.5 g of total gene clonal sequences were obtained from chemostat 1 and DNA were obtained, respectively. This means that micro- chemostat 2, respectively. Because most of the strains re- bial concentration in chemostat 2 was 2–3 times higher than lated to protein and/or amino acid degradation are affiliated that in chemostat 1. Because same amount of the extracted with this phylum, the Firmicutes would play a central role DNA was used for the real-time quantitative PCR experi- in protein and/or amino acid degradation in our chemostats. ment, the estimated value for chemostat 1 would be larger Interestingly, our phylogenetic analysis of the rRNA gene than those for chemostat 2 at a same volume of culture clonal sequences affiliated with the Firmicutes showed that broth. the major phylogenetic positions in the Firmicutes differed between clones from chemostat 1 and chemostat 2. Most of DISCUSSION the clones affiliated with Firmicutes from chemostat 1 (6 OTUs, 13 clones) fell into the Clostridium cluster XII (42). Two mesophilic anaerobic BSA-fed chemostats were con- The genus Sporanaerobacter was found to degrade certain structed: one without the trace metals Ni2+ and Co2+ (chemo- amino acids via the Stickland reaction (15). Strains in the stat 1) and the other with Ni2+ and Co2+ (chemostat 2). For genus Sedimentibacter, which was phylogenetically related chemostat 1, the major products of BSA degradation were to this cluster, also reportedly carry out the Stickland reac- volatile fatty acids and ammonia. Acetogenesis and metha- tion (16). nogenesis did not occur in this chemostat. In constrast, ace- In contrast, no clones from chemostat 2 were closely re- togenesis and methanogenesis after BSA degradation pro- lated to the Clostridium cluster XII. A significant number of ceeded in chemostat 2. The population of cells belonging clones affiliated with Firmicutes from chemostat 2 (five to the domain Archaea was very small in chemostat 1, as OTUs, nine clones) were assigned to the cluster containing demonstrated by our FISH experiments, while a significant the genus Aminobacterium. Aminobacterium mobile and number of archaeal cells were detected in chemostat 2. The Aminobacterium colombiense have been demonstrated to archaeal cells in our chemostats were classified into three degrade certain amino acids in syntrophic association with genera of methanogens, Methanoculleus, Methanospirillum hydrogenotrophic methanogens but not to perform the Stick- and Methanosaeta, by analysis of 16S rRNA gene clonal se- land reaction (17, 18). The other four OTUs (seven clones) quences. Methanogens reportedly play an important role in were found to form a cluster with uncultured clones ML635J-14 acetogenesis from fatty acids by hydrogen removal (48) as and ML635J-38, which were obtained from Mono Lake well as in methanogenesis from H2 and CO2 and/or acetate. (44). The metabolic functions of the bacteria in these four The inhibition of acetogenesis and methanogenesis in chemo- OTUs are not understood, because these OTUs formed a stat 1 could, therefore, account for the small populations of cluster distinct from rRNA gene from any of the cultured methanogens in the chemostat. Methanogens require Ni2+ strains in this phylum. These analyses on microbial commu- and Co2+ for their growth and methanogenic activity (49). nities suggest that the major amino acid degradation path- The lack of Ni2+ and Co2+ in chemostat 1 may have inhibited way in chemostat 1 is different from that in chemostat 2. methanogen growth, thus resulting in small populations. The major pathway in chemostat 1 would be the Stickland The lack of a methanogen population in the chemostat reaction, which would be carried out by an amino acid de- would, therefore, cause the inhibition of methanogens-de- grader belonging to the Clostridium cluster XII, while bac- pendent reactions, such as acetogenesis and methanogene- teria closely related to the genus Aminobacterium, which is sis. associated with hydrogenotrophic methanogens, would play In chemostat 1, two genera of hydrogenotrophic metha- a central role in amino acid degradation in chemostat 2. nogens, Methanoculleus and Methanospirillum, were de- Twenty-two (50% in BSA1B library) and 6 (11% in tected by the analysis of 16S rRNA gene clonal sequences. BSA2B library) clones affiliated with the phylum Bactero- These genera could possibly act as hydrogen scavengers idetes were obtained from chemostat 1 and chemostat 2, re- during amino acid degradation and/or acetogenesis. How- spectively. No obvious phylogenetic differences between ever, their metabolic functions among the total microbial the two chemostats were observed. Because most of these flora of the chemostat could essentially be considered negli- clones formed a cluster distinct from the rRNA gene of cul- gible because of their poor populations. In contrast, large tured strains, their metabolic functions could not be eluci- populations of methanogenic archaea were detected in dated. Some strains in this phylum, including Bacteroides chemostat 2 and were classified into three genera; Methano- fragilis, have been shown to have the ability to degrade pro- culleus, Methanospirillum and Methanosaeta. Among the teins and amino acids (50, 51), although data on amino acid three genera, Methanosaeta, an aceticlastic methanogen, degradation by strains belonging to this phylum are cur- and Methanoculleus, a hydrogenotrophic methanogen, were rently very limited. These clones in our chemostats would 中国科技论文在线 http://www.paper.edu.cn 162 TANG ET AL. J. BIOSCI. BIOENG.,

FIG. 10. Possible scheme of protein (BSA) degradation pathways and microorganisms detected in chemostats 1 and 2 as suggested by the results of this study.

play some role in protein and/or amino acids degradation. acids via the Stickland reaction when cultured under pure Twenty-two rRNA gene clones (41% in BSA2B library) culture conditions. However, a large portion of the reducing found only in chemostat 2 formed a cluster with an uncul- equivalents were channeled to methanognesis in the case of tured clone R5p16 and could not be assigned to any phyla. co-culture with a hydrogenotrophic methanogen, Methano- Because the uncultured clone R5p16 was obtained from thermobacter thermautotrophicus strain Z245 (52). More- methanogenic granular sludge concerning fatty acid degra- over, the proteolitic activity of C. proteoclasticus increased dation, these clones in chemostat 2 would be likely to con- in co-culture with M. thermautotrophicus (53). The syn- tribute to acetogenesis. The possible roles of microorga- trophic amino acid degradation would overcome the Stick- nisms detected in our chemostats in protein degradation are land reaction in protein degradation in ecosystems where a summarized in Fig. 10. significant number of hydrogenotrophic methanogens are We demonstrated by microbial community analyses that present, although information on such comparative ecologi- the major amino acid degraders were different in the two cal aspects is very limited at present. chemostats. Surprisingly, no rRNA gene clones closely re- In spite of the advantages for syntrophic degradation of lated to known amino acid degraders performing the Stick- amino acids, the growth rates of syntrophic amino acid de- land reaction were obtained from chemostat 2, into which graders were significantly lower, with an apparent specific 2+ 2+ –1 the trace metals Ni and Co were added and methane was growth rate of 0.08 d , as calculated from the Dc value of the final product. In contrast, a significant number of clones chemostat 2. In contrast, chemostat 1, in which bacteria related to known bacteria capable of syntrophic amino acid performing the Stickland reaction dominated, exhibited a –1 degradation with hydrogenotrophic methanogens were ob- higher Dc value of 0.15 d . It is believed to be difficult for tained from this chemostat. These findings suggest that syn- amino acid degraders to grow rapidly in syntrophic associ- trophic degradation of amino acids is favorable in chemo- ation with hydrogenotrophic methanogens in a CSTR. In stat 2 when compared with the Stickland reaction. It is con- order to induce a higher rate of treatment for protein-rich sidered that the Stickland reaction would dominate in envi- wastewater and waste, a two-step methane fermentation ronments rich in amino acids but that the reducing equiva- process, including VFA production from proteins mainly by lents might be preferentially channeled to methanogenesis the Stickland reaction as a first step, and acetogenesis and in environments with low supplies of amino acids and high methanogenesis as a second step, might be favorable. This methanogenic activity (52). In our chemostats, addition of is supported by several studies that found a two-stage an- the trace metals Ni2+ and Co2+ enabled methanogens to have aerobic process more efficient for the treatment of protein- higher populations in chemostat 2 when compared with rich wastewater (20, 43). chemostat 1. The higher methanogenic activity might lead In conclusion, we cultured protein degrading anaerobic to the dominance of syntrophic amino acid degraders in microbial communities using BSA-fed chemostat cultivation. chemostat 2, thus resulting in syntrophic degradation of Microbial community analysis suggested that the major amino acids being the major pathway. pathway of amino acid degradation was via the syntrophic Some bacteria that are able to degrade amino acids syn- association of amino acid degraders and hydrogenotrophic trophically, are able to perform the Stickland reaction. One methanogens in the presence of the trace metals Ni2+ and of these bacteria, Caloramator proteoclasticus, was reported Co2+ and methane was the final product, and via the Stick- to use glycine as an electron acceptor and degrade amino land reaction in the absence of these metals and volatile 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 163

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