Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester

Abstract

Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)–fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5±1.4% of total 4′, 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8±0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0±1.2%) and Chloroflexi (3.7±0.8%). The probe MX825-hybridized Methanosaeta (7.6±0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8±0.6%) and Methanobacteriaceae (2.7±0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR–FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6±4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [¹⁴C]glucose-, [¹⁴C]propionate-, [¹⁴C]butyrate- and [¹⁴C]acetate-utilizing microorganism community, and accounted for 27.7±4.3%, 29.6±7.0%, 34.5±7.6% and 18.2±9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively.

Introduction

Anaerobic digestion is the biological decomposition process regulated by mutual metabolic interactions among at least three functional groups of microorganisms in the absence of oxygen. The first community of microorganisms hydrolyzes complex polymeric substances mainly lipids, cellulose and protein to fundamental structural building blocks such as glucose and amino acids. The second microbial community subsequently ferments these products (e.g. glucose and amino acids) to fatty acids, acetate and hydrogen. This acidogenesis process was reported to be most important and a critical step among decomposition processes in anaerobic digesters (Mawson et al., 1991). Finally, the third community converts acetate and hydrogen to methane and carbon dioxide (methanogenesis). Stable operation of anaerobic digester requires that these functional microbial communities to be in dynamic equilibrium.

The most susceptible members in anaerobic sludge digester are known to be the propionate-, butyrate- or acetate-utilizing microorganisms. Accumulation of these organic acids will cause pH depletion, which has a drastically adverse impact upon the entire microbial consortia and methane production. Moreover, the diversities and distributions of these functional groups and their contributions to the overall anaerobic digesting processes are still poorly understood. A better understanding of diversities and population sizes of those functional microbial communities is required to enhance the performance and stability of anaerobic sludge digester.

Some studies have depicted the microbial community structures of anaerobic digester sludge (Godon et al., 1997; Chouari et al., 2005) and UASB granules (Sekiguchi et al., 1998) using a 16S rRNA approach. Due to the limitation of 16S rRNA gene-cloning analysis in quantifying the system (Head et al., 1998), fluorescent in situ hybridization (FISH) (Harmsen et al., 1996; Sekiguchi et al., 1999) and dot blot hybridization (Chouari et al., 2005) techniques have been used to quantify those community structures. However, no study has comprehensively described functional community structures of the major trophic groups in a full-scale anaerobic sludge digester yet. To analyze the functional microbial community structure, appropriate analytical techniques that can distinguish the in situ metabolic function without isolation and enrichment must be sought and applied. The microautoradiography combined with FISH technique (MAR–FISH) is currently the most widely applied tool for directly linking phylogenetic identification with in situ metabolic activity of microorganisms within a complex microbial community at a single-cell resolution (Lee et al., 1999; Okabe et al., 2004). MAR–FISH is, however, generally limited by the lack of taxonomic resolution of FISH probes and availability of appropriate FISH probes. Therefore, additional technique such as micromanipulation followed by 16S rRNA sequencing analysis is also of necessity to identify intriguing cells that cannot be identified by MAR–FISH (Thomsen et al., 2004).

The objective of this study was, therefore, to characterize and quantify functional community structures of major trophic groups: glucose-, propionate-, butyrate- and acetate-utilizing microbial communities in a full-scale anaerobic sludge digester. First, phylogenetic differentiation (identification) of bacterial and archaeal communities in anaerobic digester sludge was performed by 16S rRNA gene-cloning analysis and FISH with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes. Second, the community structures (diversity and relative abundance) of these major trophic groups were quantitatively analyzed by MAR–FISH.