A metagenomic study of the microbial communities in four parallel biogas reactors

Abstract

Background: Biogas is a renewable energy carrier which is used for heat and power production or, in the form of purified methane, as a vehicle fuel. The formation of methane from organic materials is carried out by a mixed microbial community under anaerobic conditions. However, details about the microbes involved and their function are limited. In this study we compare the metagenomes of four parallel biogas reactors digesting a protein-rich substrate, relate microbiology to biogas performance, and observe differences in these reactors' microbial communities compared to the original inoculum culture.

Results: The biogas process performance during the startup phase of four parallel continuous stirred tank reactors (designated R1, R2, R3, and R4) co-digesting fish waste and cow manure was studied. The microbial composition of the inoculum (day 0) and the four reactors at day 59 was studied and compared using 454 FLX Titanium pyrosequencing. In the inoculum and the reactor samples, the Bacteria Clostridium and Syntrophomonas were highly abundant, and the dominating methanogen was the hydrogenotrophic Methanoculleus. Syntrophic prokaryotes frequently found in biogas reactors with high concentrations of ammonium and volatile fatty acids were detected in all samples. The species Candidatus Cloacimonas acidaminovorans of the candidate phylum Cloacimonetes (WWE1) increased in all reactors and was the dominating bacterium at day 59. In particular, this bacterium showed a very high abundance in R1, which distinguished this reactor significantly from the other reactors in terms of microbial composition. Methane production and the reactor slurry characteristics were monitored in the digestion period. Generally all four reactors operated stably and showed rather similar characteristics. The average methane production in the reactors varied between 0.278 and 0.296 L gVS(-1), with the lowest production in R1.

Conclusions: This study showed that four parallel reactors co-digesting manure and fish waste silage operated stably during a startup phase. Several important Archaea and Bacteria degrading the protein-rich substrate were identified. In particular, microorganisms involved in syntrophic methane production seemed to be important. The detailed characterization of the microbial communities presented in this work may be useful for the operation of biogas plants degrading substrates with high concentrations of proteins.

Keywords: Anaerobic digestion; Biofuel; Biogas; Biorefinery; Metagenomic; Methane; Syntrophic oxidation; Taxonomic structure.

Figure 1

Anaerobic process performance in R1, R2, R3, and R4 during 28 days of continuous operation (day 36 to day 59). A) Methane productions, B) % volatile solid (VS) removal, C) pH values, D) NH₄ ⁺ concentrations, E) acetic acid concentration, and F) propionic acid concentration.

Figure 2

Rarefaction curves of taxonomic richness in the samples at the genus and the fully resolved level in MEGAN.

Figure 3

Taxonomic distribution at the domain level in MEGAN. Reads assigned at the domain level given as percentage of total reads in each metagenome. “No hits” are reads without hits in the BLAST search. “Not assigned” are reads with a hit in BLAST, but with no assignment to a taxon due to the settings in MEGAN. “Environmental samples” are reads with hits in other metagenome sequences with unknown biological classification.

Figure 4

Percentage of reads assigned to prokaryotic phyla with more than 0.1% of total reads assigned.

Figure 5

PCA of phyla with Euclidean distance greater than 0.1 from origo. Reads with no hits in the blast search and reads not assigned by MEGAN are excluded. The metagenomic parameters are represented by red arrows. Labels are shown for parameters with Euclidean distance over 0.1 from origo. All metagenome data were given as percentage of total reads.

Figure 6

Percentage of reads assigned to the 44 genera with more than 0.1% reads assigned. Insert shows full scale of the Y-axes.

Figure 7

Abundance shift compared to inoculum at the genus level. This figure shows the fold change in abundant genera (>0.1% in one or more metagenomes) in the reactor samples compared to the inoculum. Fold change values less than 1 were replaced by the negative of their inverse.

Figure 8

PCA of genera with Euclidean distance from origo greater than 0.1. Reads with no hits in the BLAST search and reads not assigned by MEGAN are excluded. The metagenomic parameters are represented by red arrows. Labels are shown for parameters with Euclidean distance over 0.1 from origo. All metagenome data were given as percentage of total reads.

Figure 9

The methanogenesis pathway. Enzymes are shown in blue boxes. Subunits missing in all our datasets (R1, R2, R3, R4, and IN) after search against the KO database at MG-RASTare underlined. Abbreviations used in the figure are Acetyl-Pi: acetyl phosphate; ack: acetate kinase; acs: acetyl-CoA synthetase; cdh: acetyl-CoA decarbonylase/synthase; CO: carbon monoxide; CoA: coenzyme A; CoB: coenzyme B; CoB-S-S-CoM: coenzyme M 7-mercaptoheptanoylthreonine-phosphate heterodisulfide; F420: coenzyme F420; fmd: formylmethanofuran dehydrogenase; Formyl-H4MPT: 5-formyl-5,6,7,8-tetrahydromethanopterin; Formyl-MF: formylmethanofuran; frh: coenzyme F420 hydrogenase; ftr: formylmethanofuran-tetrahydromethanopterin N-formyltransferase; H4MPT: 5,6,7,8-tetrahydromethanopterin; hdr: heterodisulfide reductase; mch: methenyltetrahydromethanopterin cyclohydrolase; mcr: methyl-coenzyme M reductase; mer: 5,10-methylenetetrahydromethanopterin reductase; Methenyl-H4MPT: 5,10-methenyl-5,6,7,8-tetrahydromethanopterin; Methyl-CoM: methylcoenzyme M; Methylene-H4MPT: 5,10-methylenetetrahydromethanopterin; Methyl-H4MPT: 5-methyl-5,6,7,8-tetrahydromethanopterin; mtd: methylenetetrahydromethanopterin dehydrogenase; mtr: tetrahydromethanopterin S-methyltransferase; ppa: inorganic diphosphatase; pta: phosphate acetyltransferase.

Figure 10

Reads assigned to level 2 metabolism subsystems at MG-RAST (KO database).