Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester

Abstract

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.

FIG. 1.

Change in reactor performance of the thermophilic methanogenic process: ⋄, pH; ▴, gas production rate; •, acetate concentration; □, propionate concentration. Arrows indicate the sampling points for SSCP analysis and quantitative PCR. Arrows in boldface indicate the sampling points for FISH.

FIG. 2.

Microbial community structure in the thermophilic methanogenic process as determined using PCR-SSCP analysis. (A) Bacterial fingerprint; (B) archaeal fingerprint. Bands 1, 2, and 3 in panel B were closely related to Methanothermobacter thermautotrophicus (accession number AE000940; 100% sequence similarity), Methanosarcina thermophila (M59140; 98% sequence similarity), and Methanoculleus thermophilicus (AB065297; 100% sequence similarity), respectively.

FIG. 3.

Principal-component analysis of the SSCP data. (A) PCA plot for the bacterial fingerprint; (B) PCA plot for the archaeal fingerprint. The successive time points (days) are connected by arrows and indicated by numbers.

FIG. 4.

Transition of the 16S rRNA gene copy numbers of the methanogens Methanoculleus sp. (○), Methanosarcina sp. (□), and Methanothermobacter sp. (▵). Two trials were conducted to analyze each sample. The variations between the duplications were less than 1% of the mean.

FIG. 5.

Fluorescence in situ hybridization of methanogenic microflora viewed by confocal laser scanning microscopy. (A) Day 48; (B) day 63; (C) day 90. The methanogenic microflora was simultaneously hybridized with Alexa488-labeled archaeal probe ARC915 (green) and rhodamine-labeled bacterial probe EUB338 (red). Representative fields of vision are shown. Bars, 20 μm.