Yuichi Ishii

Effective isolation of bacterioplankton genus Polynucleobacter from freshwater environments grown on photochemically degraded dissolved organic matter

Effective isolation of freshwater bacterioplankton belonging to genus Polynucleobacter from a shallow eutrophic lake and its tributary was achieved by size-selective filtration with a 0.7-mum pore filter and cultivation on R2A agar medium. Partial 16S rRNA gene analysis showed that over 80% of all the strains were highly similar to the Polynucleobacter cluster. Essential medium components for effective cultivation are pyruvate, yeast extract and peptone, whereas soluble starch and glucose are not necessary. Isolate KF001 (affiliated with Polynucleobacter subcluster D) has a strict requirement for organic acids as carbon sources, and we hypothesize that the Polynucleobacter cluster of bacteria could utilize compounds formed via photochemically dissolved organic matter (DOM) degradation for growth. Because organic acids form from solar radiation of DOM in aquatic environments, carbon sources that are typical products of DOM photochemical degradation were added to the medium. These compounds were readily utilized by KF001 in this study. Finally, we observed the stimulation of strain KF001 activity by photochemical degradation of natural lake water. Our findings suggest a carbon flow of DOM photoproducts to Polynucleobacter in the freshwater microbial loop.

Spatial variation of phosphorus fractions in bottom sediments and the potential contributions to eutrophication in shallow lakes

Spatial variation of phosphorus fractions in bottom sediment, pore water and overlying water in three shallow eutrophic lakes, Nishiura, Kitaura and Sotonasakaura, Japan, and the contributions of the fractional P to mobilization of phosphorus from sediment were examined in this study. The vertical distributions of dissolved inorganic phosphorus (DIP) concentrations in overlying and pore water differed with lake and sampling site. In particular, DIP was high in pore water in the surface layer of the sediment for the middle to downlake areas of Lake Kitaura. DIP release flux calculated from a gradient of the concentrations at the sediment–water interface was high compared with other sites. The distribution of fractional P content in sediments was highly variable. The citrate–dithionite–bicarbonate–non-reactive phosphorus (CDB–NRP) fraction, in particular, differed greatly among the three lakes. According to correlation in the ratios between CDB–NRP and loss on ignition, sediments of these lakes were classified in three clusters. The CDB–NRP fraction was suggested to play a role in DIP release from sediment. The possibility of nitrate concentration playing a role in the control of DIP release was considered.

Ecological niche separation in the Polynucleobacter subclusters linked to quality of dissolved organic matter: a demonstration using a high sensitivity cultivation-based approach

The free-living, cosmopolitan, freshwater betaproteobacterial bacterioplankton genus Polynucleobacter was detected in different years in 11 lakes of varying types and a river using the size-exclusion assay method (SEAM). Of the 350 strains isolated, 228 (65.1%) were affiliated with the Polynucleobacter subclusters PnecC (30.0%) and PnecD (35.1%). Significant positive correlations between fluorescence in situ hybridization and SEAM data were observed in the relative abundance of PnecC and PnecD bacteria to Polynucleobacter communities (PnecC + PnecD). Isolates were mainly PnecC bacteria in the samples with a high specific UV absorbance at 254 nm (SUVA(254) ), and a low total hydrolysable neutral carbohydrate and amino acid (THneutralCH + THAA) content of the dissolved organic matter (DOM) fraction, which is known to be correlated with a high humic content. In contrast, the PnecD bacteria were abundant in samples with high chlorophyll a and/or THneutralCH + THAA concentrations, indicative of primary productivity. With few exceptions, differences in the relative abundance of PnecC and PnecD in each sample, determined using a high-sensitivity cultivation-based approach, were due to DOM quality. These results suggest that the major DOM component in the field, which is allochthonously or autochthonously derived, is a key factor for ecological niche separation between PnecC and PnecD subclusters.

Growth rates and tolerance to low water temperatures of freshwater bacterioplankton strains: ecological insights from shallow hypereutrophic lakes in Japan

To investigate differences in culturable freshwater bacterioplankton between summer and winter, we examined specific bacterioplankton in the low temperature surface water of two shallow, hypereutrophic, temperate lakes in Japan over two winters, and compared our results with the previously published data. We used the size-exclusion assay method (SEAM), a cultivation-based approach that simply and effectively isolates typical freshwater bacterioplankton. The specific clusters detected in the winter samples were the Rhodoferax sp. BAL47 cluster (LimA and LimC), a predominant and ubiquitous freshwater lineage, and the LiUU-5-340 cluster. To confirm tolerance to low water temperature of winter-specific groups, we also compared growth rates at 5°C among several pure strains of typical freshwater bacterioplankton clusters belonging to the Rhodoferax sp. BAL47, Polynucleobacter necessarius, GKS98, LiUU-5-340, and IRD18C08 of the class Betaproteobacteria, and the Luna-1 and Luna-2 of the class Actinobacteria. Specific detectability of freshwater bacterioplankton clusters/subclusters by SEAM in the winter sample substantially correlated with the low temperature-specific growth characteristics of each isolate. Response to water temperature is a key control factor in freshwater bacterioplankton assemblage composition. These results provide important insights into the specific response to water temperature of several ubiquitous culturable freshwater bacterioplankton clusters inhabiting a temperate climate zone.

BIOLOGOCAL INTERACTION EXERTED ON MATERIAL CYCLE IN TIDAL FLAT

Material cycle in tidal flat was investigated from the viewpoint of biological interaction with experimental microcosm system. The experiment, in this paper, was executed using macrophytes and macrobenthos living in tidal flat, which is located in Tokyo Inner Bay.As the result, it was clarified that the living things with different life style exerted the influence on the concentration of dissolved nutrient salts in water under the individual biological treatment process in each kinds.Moreover, for the reason of coexistence due to living things with different life style, the dissolved nutrient salts in water layer was suppressed into 1/4 times as much as the concentration produced by a single kind. It was considered, in conclusion that the utilization of biological interaction depending on various living things is important for purification of water quality in tidal flat.