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Microbial Community Analysis of Mesophilic 中国科技论文在线 http://www.paper.edu.cn JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2005, The Society for Biotechnology, Japan Vol. 99, No. 2, 150–164. 2005 DOI: 10.1263/jbb.99.150 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 affili- ated 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 deg- radation 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- 150 转载 中国科技论文在线 http://www.paper.edu.cn VOL. 99, 2005 MICROBIAL COMMUNITIES OF BSA-FED CHEMOSTAT CULTIVATIONS 151 bic treatment of protein-rich wastewater or waste because mixed amino acids are readily supplied by protein hydroly- sis. Despite this knowledge of protein and amino acid degra- dation 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 bio- logical techniques have successfully been applied to ana- lyze microbial communities in several ecosystems, includ- ing anaerobic digesters. Although there are several reports about the anaerobic treatment of protein-rich wastewaters concerning the optimization of treatment processes or mor- phological 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 treat- ment 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
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