Microbial ecologists have investigated roles of species richness and diversity in a wide variety of ecosystems. Recently, metagenomics have been developed to measure functions in ecosystems, but this approach is cost-intensive. Here we describe a novel method for the rapid and efficient reconstruction of a virtual metagenome in environmental microbial communities without using large-scale genomic sequencing. We demonstrate this approach using 16S rRNA gene sequences obtained from denaturing gradient gel electrophoresis analysis, mapped to fully sequenced genomes, to reconstruct virtual metagenome-like organizations. Furthermore, we validate a virtual metagenome using a published metagenome for cocoa bean fermentation samples, and show that metagenomes reconstructed from biofilm formation samples allow for the study of the gene pool dynamics that are necessary for biofilm growth.

The seasonal change of bacterial community structure in biofilms on the surface of reed (Phragmites australis) was investigated for about three years (from 2005 June to 2008 March) in Lake Biwa by comparing it with that in surrounding lake water. The community structure in biofilms was different from that in the lake water throughout the seasons and years. The community structure in lake water was similar in the same seasons of different years, corresponding to similar environmental factors (i.e., temperature, dissolved oxygen, and light intensity) and nutrient ion concentrations at the same season. However, the community structure in the biofilms was not similar in the same season of different years. This seems to be due to the formation of new biofilms on sprouted reeds in every early summer and the high nutrient concentrations and bacterial density in subsequently formed biofilms. Although the community structure in the biofilms changed along with the seasonal change, the bacteria belonging to Bacillus and Paenibacillus were detected in any season. This study revealed the possibility that the bacterial community structure in the initial stage of the biofilm formation govern the subsequent seasonal change of the community structure in biofilms.Key words: biofilm, bacterial community, seasonal change, Lake BiwaIn natural environments, microorganisms attach to surfaces and form biofilms. Biofilms are ubiquitous in aquatic environments and have been proposed and confirmed to have the ability to trap and retain ions, including nutrient ions (7,10,39).In Lake Biwa, where water temperature, dissolved oxygen (D.O.), and light intensity change seasonally with similar patterns every year, we have studied the biofilms on the surfaces of reeds and stones for about three years (from June 2005 to March 2008), and revealed that the interior of the biofilms is rich in nutrients, hundreds to thousands of times greater in nutrient ion concentrations, compared with the surrounding lake water. Furthermore, we have clarified for the first time that the nutrient ion concentrations in biofilms showed a similar seasonal change every year (high in winter and low in summer) synchronized with the change of lake water (39). We found that the extracellular polymeric substances (EPS) of biofilms have electric charges (17), and in a preliminary experiment, that biofilms accumulated the nutrient ions from surrounding water (unpublished data). The biofilms seem to accumulate the nutrient ions by the ion exchange process as proposed other study (10), and form a nutrient-rich environment showing similar seasonal change to the lake water. The higher nutrient ion concentrations in biofilms and the seasonal change of the concentrations may influence the microbial community structure and activity in the biofilms. To investigate microbial community structures in biofilms by considering the internal environment of biofilms will lead to the understanding of the ecology of natural biofilms.Several studies, although based on a short period (one year at...

In biofilms, the matrix of extracellular polymeric substances (EPSs) retains water in the interstitial region of the EPS. This interstitial water is the ambient environment for microorganisms in the biofilms. The nutrient condition in the interstitial water may affect microbial activity in the biofilms. In the present study, we measured the concentrations of dissolved organic nutrients, i.e., saccharides and proteins, contained in the interstitial water of biofilms formed on the stones. We also analyzed the molecular weight distribution, chemical species, and availability to bacteria of some saccharides in the interstitial water. Colorimetric assays showed that the concentrations of saccharides and proteins in the biofilm interstitial water were significantly higher (ca. 750 times) than those in the surrounding lake waters (p < 0.05). Chromatographic analyses demonstrated that the saccharides in the interstitial waters were mainly of low molecular-weight saccharides such as glucose and maltose, while proteins in the interstitial water were high molecular-weight proteins (over 7000 Da). Bacterial growth and production of EPS occurred simultaneously with the decrease in the low molecular-weight saccharide concentrations when a small portion of biofilm suspension was inoculated to the collected interstitial water, suggesting that the dissolved saccharides in the interstitial water support bacterial growth and formation of biofilms.