Isolation and Characterization of a New Clostridium sp. That Performs Effective Cellulosic Waste Digestion in a Thermophilic Methanogenic Bioreactor

Abstract

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55°C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 ‫؍(‬ Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.Waste utilization is a major concern in energy development and environmental improvement. In this regard, various attempts have been made to develop effective microbial digestion systems (6). We study the application of thermophilic methane fermentation in the reduction and utilization of municipal solid wastes. Although the operation of a thermophilic reactor consumes more electrical energy than that by a mesophilic reactor, it enables effective degradation of substrates and production of methane in a short fermentation period (1). We expect that the fermentation will contribute to the rapid and efficient processing of municipal solid wastes in urban districts. In Tokyo, half of the combustible waste consists of paper (22). Recently, utilization of the markedly increasing amount of shredded office paper has become a matter of great concern; it is nonrecyclable because shredded cellulose fiber is not suited for papermaking.Conversion of cellulose to methane is mediated by four microbial populations: cellulolytic microbes, noncellulolytic saccharolytic microbes, syntrophic hydrogen-generating bacteria, and methane-producing members of the domain Archaea (2). Generally, the initial hydrolysis is the rate-limiting step in microbial conversion; the efficiency of cellulose degradation markedly affects the methane formation activity in the methanogenic microbial system (2, 11, 15). Therefore, characterization of the cellulose-digesting microbial population is important for the development of efficient fermentation systems. This paper describes the isolation and characterization of a Clostridium sp. that effectively degrades cellulosic wastes. We operated a methanogenic bioreactor that primarily digested office paper and detected the presence of a group of effective cellulose-digesting ...

Unique diversity content of a thermophilic anaerobic microbial consortium that utilizes propionate in a synthetic medium

“…A methanogenic bioreactor was operated at 55°C by continuously feeding artificial municipal wastes (Shiratori et al, 2006). We occasionally observed the occurrence of fermentation breakdown due to acidification of sludge; in some cases, the methanogenic activity never recovered even after prolonged non-feeding incubation.…”

“…Total DNA extracted from the cellular precipitate of the culture broth was subjected to PCR using primers M85FN [5Ј-C(G/T)GC-TCA(G/T)TAACACGTGG; nt 85-101 by M. thermautotrophicus numbering] and M1350R [5Ј-GGCGGT-GTG(C/T)GCAAGGAG; nt 1352-1332 by M. thermautotrophicus numbering] for Archaea, and B8F (5Ј-AGAGTTTGATCCTGGCTCAG; nt 8-27 by E. coli numbering) and 1492R [5Ј-GG(C/T)TACCTTGTTAC-GACTT; nt 1510-1492 by E. coli numbering] for Bacteria. The conditions for PCR, DNA cloning and phylogenetic analyses were described previously (Shiratori et al, 2006). Table 1 summarizes the phylogenetic affiliation of the archaeal and bacterial 16S rRNA gene sequences obtained in this study.…”

“…Previously, we described development and long-term operation of a thermophilic methanogenic bioreactor that digested artificial municipal solid wastes, which mainly contained shredded office paper and typical Japanese food wastes (Shiratori et al, 2006). Although the bioreactor effectively converted the substrates to methane during high performance periods, it occasionally fell into a breakdown phase due to acidification of sludge.…”

“…Sludge collected from the above bioreactor was added at 30%(v/v) to M medium [a minimal medium described previously (Shiratori et al, 2006)] containing 40 mM calcium propionate as the sole carbon source and 1 mM FeCl 2 (pH 7.0). The addition of FeCl 2 was expected to support the stable growth of methanogenic Archaea (Stams et al, 1992).…”

“…1A). The population was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial DNA fragments of 16S rRNA gene using a D-Code system (Bio-Rad) as described previously (Shiratori et al, 2006). The DNA fragments were amplified by PCR using the following primers: 341F (5Ј-CCTACGGGAG-GCAGCAG; nt 341-357 by Escherichia coli numbering) with a 5Ј-terminal GC clamp (5Ј-CGCCCGCCGC-GCGCGGCCGGGCGGGGCGGGGGCAGCGGGGG) and Uni533R (5Ј-TTACCGCGGCTGCTGGCAC; nt 533-515 by E. coli numbering) for Bacteria, and AP5F (5Ј-GGCAACACGCGGGCTACAATGG; nt 1170-1191 by Methanothermobacter thermautotrophicus numbering) with a 5Ј-terminal GC clamp (5Ј-GCGCGCGCGCGCCC-CGCGCCCGTCCCGCCGGGCCCGCGGC) and AP7R (5Ј-GGCGGTGTGTGCAAGGAGCAG; nt 1352-1335 by M. thermautotrophicus numbering) for Archaea.…”