Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules

Abstract

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH(4), H(2), pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH(4), H(2), pH, and ORP revealed that acid and H(2) production occurred in the upper part of the granule, below which H(2) consumption and CH(4) production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H(2) was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH(4) in the inner layer. We determined the effective diffusion coefficient for H(2) in the anaerobic granules to be 2.66 x 10(-5) cm(2) s(-1), which was 57% in water.

FIG. 1.

Phylogenetic tree representing affiliation of 16S rRNA clone sequences of Bacteria retrieved from granule samples (OTU numbers). The tree was generated by using nearly full-length 16S rRNA gene sequences and the neighbor-joining method. The scale bar represents 5% estimated divergence. The numbers at the nodes are bootstrap values (1,000 replicates) with more than 50% bootstrap support.

FIG. 2.

Phylogenetic tree representing affiliation of 16S rRNA clone sequences of Archaea retrieved from granule samples (OTU numbers). The tree was generated by using nearly full-length 16S rRNA gene sequences and the neighbor-joining method. The scale bar represents 5% estimated divergence. The numbers at the nodes are bootstrap values (1,000 replicates) with more than 50% bootstrap support.

FIG. 3.

Confocal laser scanning microscope images of thin sections of the anaerobic granules showing the in situ spatial organization of bacteria and archaea. (A) DAPI-stained and differential interference contrast images. (B) FISH with TRITC-labeled probe ARC915 and the FITC-labeled EUB338-mixed probe. (C) FISH with FITC-labeled probe GNSB-941. (D) FISH with TRITC-labeled probe ARC915 and FITC-labeled probe BET42a. (E) FISH with TRITC-labeled probe ARC915 and FITC-labeled probe LGC354. (F) FISH with TRITC-labeled probe ARC915 and FITC-labeled probe ALF968. (G) FISH with TRITC-labeled probe MX825 and FITC-labeled probe HGC69A. Scale bars indicate 200 μm (panels A and B) and 50 μm (panels C through G). For panels B through G, TRITC-labeled probes are red and FITC-labeled probes are green.

FIG. 4.

Concentration profiles of pH, ORP, CH₄, and H₂ in the anaerobic granule. Each profile value is the average of three measurements, and the error bars represent the standard deviations of triplicate measurements. Zero on the vertical axis corresponds to the surface of the granule.

FIG. 5.

Spatial distributions and magnitudes of the net volumetric production rates of CH₄ and H₂. The rates were calculated based on the corresponding concentration profiles shown in Fig. 4. Each profile value is the average of three measurements, and the error bars represent the standard deviations of triplicate measurements. Negative values indicate consumption rates. Zero on the vertical axis corresponds to the surface of the granule.

FIG. 6.

A typical transient H₂ concentration profile measured at the center of the granule inhibited by chloroform. Points (○) indicate H₂ concentrations measured, and the solid line is a theoretical curve.