Novel aerobic anoxygenic phototrophic bacterium Jannaschia pagri sp. nov., isolated from seawater around a fish farm.

The genus Jannaschia is one of the representatives of aerobic anoxygenic phototrophic (AAP) bacteria, which is a strictly aerobic bacterium, producing a photosynthetic pigment bacteriochlorophyll (BChl) a. However, a part of the genus Jannaschia members have not been confirmed the photosynthetic ability. The partly presence of the ability in the genus Jannaschia could suggest the complexity of evolutionary history for anoxygenic photosynthesis in the genus, which is expected as gene loss and/or horizontal gene transfer. Here a novel AAP bacterium designated as strain AI_62T (= DSM 115720 T = NBRC 115938 T), was isolated from coastal seawater around a fish farm in the Uwa Sea, Japan. Its closest relatives were identified as Jannaschia seohaensis SMK-146 T (95.6% identity) and J. formosa 12N15T (94.6% identity), which have been reported to produce BChl a. The genomic characteristic of strain AI_62T clearly showed the possession of the anoxygenic photosynthesis related gene sets. This could be a useful model organism to approach the evolutionary mystery of anoxygenic photosynthesis in the genus Jannaschia. Based on a comprehensive consideration of both phylogenetic and phenotypic characteristics, we propose the classification of a novel species within the genus Jannaschia, designated as Jannaschia pagri sp. nov. The type strain for this newly proposed species is AI_62T (= DSM 115720 T = NBRC 115938 T).

Flavobacterium okayamense sp. nov. isolated from surface seawater.

Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA-DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T).

A newly developed solution for the preservation of short-chain fatty acids, bile acids, and microbiota in fecal specimens.

Recent studies have revealed that the gut microbiome affects various health conditions via its metabolites, including short-chain fatty acids (SCFAs) and bile acids (BAs). In the analysis of these, appropriate collection, handling, and storage of fecal specimens are required, and convenient specimen handling processes will facilitate their investigation. Here, we developed a novel preservation solution, "Metabolokeeper®", to stabilize fecal microbiota, organic acids including SCFAs, and BAs at room temperature. In the present study, we collected fecal samples from 20 healthy adult volunteers and stored them at room temperature with Metabolokeeper® and at -80°C without preservatives for up to four weeks to evaluate the usefulness of the novel preservative solution. We found that microbiome profiles and short chain fatty acid contents were stably maintained at room temperature with Metabolokeeper® for 28 days, while the bile acids were stably maintained for 7 days under the same conditions. We conclude that this convenient procedure to obtain a fecal sample for collecting the gut microbiome and gut metabolites can contribute to a better understanding of the health effects of fecal metabolites produced by the gut microbiome.

Roseomonas fluvialis sp. nov., an aerobic bacteriochlorophyll a-containing freshwater bacterium isolated from river epilithic biofilm.

A strictly aerobic bacteriochlorophyll a-containing alphaproteobacterium, designated strain S08T, was isolated from a biofilm sampled at Tama River in Japan. The non-motile and rod-shaped cells formed pink-beige pigmented colonies on agar plates containing organic compounds and showed in vivo absorption maxima at 798 and 866 nm in the near-infrared region, typical for the presence of bacteriochlorophyll a. The new bacterial isolate is Gram-negative, oxidase-negative and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S08T was closely related to species in the genus Roseomonas. The closest phylogenetic relative of strain S08T was Roseomonas lacus TH-G33T (98.2 % sequence similarity). The major cellular fatty acids were C16 : 0, C18 : 1 2-OH and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The predominant respiratory quinone was ubiquinone-9. The major polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an aminolipid. The G+C content of the genomic DNA was 70.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain S08T and the related Roseomonas type strains were all far lower than the cut-off value for the delineation of species. The results of polyphasic comparisons showed that strain S08T was clearly distinguishable from other members of the genus Roseomonas. Therefore, we propose a new species in the genus Roseomonas, namely, Roseomonas fluvialis sp. nov. The type strain is S08T (=DSM 111902T=NBRC 112025T).

Long-term D-Allose Administration Favorably Alters the Intestinal Environment in Aged Male Mice.

D-Allose, a C3 epimer of D-glucose, has potential to improve human health as a functional food. However, its effect on the intestinal environment remains unknown. Aged humans progressively express changes in the gut, some of which deleteriously affect gastrointestinal health. In this study, we profiled the intestinal microbiome in aged mice and analyzed organic acids produced by bacteria in cecum contents after long-term ingestion of D-allose. D-Allose did not significantly change organic acid concentration. However, long-term ingestion did significantly increase the relative abundance of Actinobacteria and reduce the relative abundance of Proteobacteria. These results suggest that oral D-allose improves the proportion of favorable intestinal flora in aged mice. D-Allose significantly decreased the relative abundance of Lachnospiraceae bacteria, but increased the relative abundance of Bacteroides acidifaciens and Akkermansia muciniphila. Thus, D-allose might serve as a nutraceutical capable of improving the balance of gut microbiome during aging.

Fecal short-chain fatty acids and obesity in a community-based Japanese population: The DOSANCO Health Study.

In Western populations, fecal concentrations of short-chain fatty acids (SCFAs) are positively correlated with the prevalence of obesity. However, gut microbiota involved in the production of SCFA varies between races. Our purpose was to investigate the associations between fecal SCFAs and the prevalence of obesity in a community-based Japanese population. We classified a total of 568 participants aged ≥18 into four quartiles of fecal concentrations of SCFA subtypes (acetate, butyrate, and propionate) and total SCFAs to compare the prevalence of obesity, defined as a body mass index ≥ 25.0 kg/m2. Using the first quartile SCFA group as a reference, the prevalence ratios of obesity were calculated for each SCFA group through a log-binomial regression model adjusted for major potentially confounding factors including age, sex, exercise habits, total energy intake, and total dietary fiber intake. In the study population, the prevalence of obesity was 35.8%. The prevalence ratios (95% confidence intervals) of obesity in the second, third, and fourth quartile groups of fecal total SCFAs were 1.30 (0.89-1.89), 1.74 (1.23-2.47) and 1.70 (1.19-2.41), respectively, after adjusting for the confounders. Similar positive associations were observed for every subtype. The prevalence ratios (95% confidence intervals) in the fourth quartile groups of fecal acetate, butyrate, and propionate were 1.41 (1.02-1.97), 2.16 (1.49-3.14), and 1.97 (1.35-2.89), respectively, after adjusting for the confounders. In conclusion, our results demonstrated that fecal SCFA concentrations of every subtype were positively associated with the prevalence of obesity in a community-based Japanese population.

Effect of Daikenchuto On Spontaneous Intestinal Tumors in ApcMin/+ Mice.

Daikenchuto (TU-100) is herbal medicine which predominantly contains ginger, Japanese pepper, and ginseng. We investigated whether TU-100 can affect the composition of gut flora and intestinal tumor development using ApcMin/+ mice, a murine model of intestinal tumor. Bacterial 16S rRNA sequencing and short-chain fatty acid analysis were performed on faecal samples. Tumor number and size were analysed. Any change in gene expression of the tumor tissues was assessed by real-time PCR. Principal coordinate analysis (PCoA) showed that the faecal microbiota cluster of TU-100-fed mice was different from the microbiota of control mice. However, no significant difference was observed in the concentration of short-chain fatty acids, tumor number, and gene expression levels between the two groups. Our data showed that TU-100 can affect the intestinal environment; however, it does not contribute in tumor progression or inhibition in our setting.

Coprobacter secundus subsp. similis subsp. nov. and Solibaculum mannosilyticum gen. nov., sp. nov., isolated from human feces.

An obligately anaerobic, Gram-stain-negative, rod-shaped bacterium, designated strain 2CBH44T , was isolated from the fecal sample of a healthy Japanese man. This strain was initially assigned as a novel species of the genus Coprobacter based on the 16S rRNA gene sequence similarities compared with other Coprobacter species. The 16S rRNA gene sequence analysis revealed strain 2CBH44T had relatively low 16S rRNA gene sequence similarity (97.5%) to Coprobacter secundus 177T . However, strain 2CBH44T showed 96.9% average nucleotide identity value with C. secundus 177T , indicating that strain 2CBH44T and C. secundus 177T belong to the same species. On the other hand, the digital DNA-DNA hybridization value between strain 2CBH44T and C. secundus 177T was 73.5%, indicating that strain 2CBH44T is a subspecies of C. secundus. Another anaerobic, Gram-stain-variable, rod-shaped bacterium, designated strain 12CBH8T , was also isolated from human feces. Strain 12CBH8T had significantly low 16S rRNA gene sequence similarities (<92.0%) to the validated bacterial species within the family Oscillospiraceae. The percentage of conserved protein values between the genome of strain 12CBH8T and that of the validated related taxa were <50%, suggesting that strain 12CBH8T belongs to a novel genus. On the basis of the collected data, strain 2CBH44T represents a novel subspecies of C. secundus, for which the name Coprobacter secundus subsp. similis subsp. nov. (type strain 2CBH44T = JCM 34079T = DSM 111570T ) is proposed. Strain 12CBH8T represents a novel species of a novel genus, for which the name Solibaculum mannosilyticum gen. nov., sp. nov. (type strain 12CBH8T = JCM 34081T = DSM 111571T ) is proposed.

Veillonella nakazawae sp. nov., an anaerobic Gram-negative coccus isolated from the oral cavity of Japanese children.

Two strains of previously unknown Gram-negative cocci, T1-7T and S6-16, were isolated from the oral cavity of healthy Japanese children. The two strains showed atypical phenotypic characteristics of members of the genus Veillonella, including catalase production. Sequencing of their 16S rRNA genes confirmed that they belong to genus Veillonella. Under anaerobic conditions, the two strains produced acetic acid and propionic acid as metabolic end-products in a trypticase-yeast extract-haemin medium containing 1 % (w/v) glucose, 1 % (w/v) fructose and 1 % (v/v) sodium lactate. Comparative analysis of the 16S rRNA, dnaK, rpoB and gltA gene sequences revealed that the two strains are phylogenetically homogeneous and comprise a distinct, novel lineage within the genus Veillonella. The sequences from the two strains shared the highest similarity, at 99.9, 95.8, 96.9 and 96.7 %, using the partial 16S rRNA, dnaK, rpoB and gltA gene sequences, respectively, with the type strains of the two most closely related species, Veillonella dispar ATCC 17748T and Veillonella infantium JCM 31738T. Furthermore, strain T1-7T shared the highest average nucleotide identity (ANI) value (94.06 %) with type strain of the most closely related species, V. infantium. At the same time, strain T1-7T showed the highest digital DNA-DNA hybridization (dDDH) value (55.5 %) with the type strain of V. infantium. The two strains reported in this study were distinguished from the previously reported species from the genus Veillonella based on catalase production, partial dnaK, rpoB and gltA sequences, average ANI and dDDH values. Based on these observations, the two strains represent a novel species, for which the name Veillonella nakazawae sp. nov. is proposed. The type strain is T1-7T (JCM 33966T=CCUG 74597T).

Fluviibacter phosphoraccumulans gen. nov., sp. nov., a polyphosphate-accumulating bacterium of Fluviibacteraceae fam. nov., isolated from surface river water.

Three aerobic, Gram-stain-negative, non-motile, rod-shaped bacteria, designated as strains SHINM1T, ICHIJ1 and ICHIAU1, were isolated from surface river water (Saitama Prefecture, Japan). Phylogenetic analyses based on 16S rRNA and 40 marker gene sequences revealed that the strains formed a distinct phylogenetic lineage within the order Rhodocyclales. The three strains shared 100 % 16S rRNA gene similarity. Growth occurred at 15-30 °C and pH 6.0-9.5, but not in the presence of ≥1.0 % (w/v) NaCl. The isolates stained positive for intracellular polyphosphate granules. The major cellular fatty acids were C16 : 0, summed feature 2 (C12 : 1 aldehyde and/or iso-C16 : 1 I and/or C14 : 0 3-OH), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine and an unidentified phospholipid. The predominant quinone system of strain SHINM1T was ubiquinone-8 and its DNA G+C content was 56.7 mol%. Genome sequencing of the three isolates revealed a genome size of 2.29-2.43 Mbp and average nucleotide identity by orthology values of ≥98.9 %. Based on the results of phenotypic and phylogenetic analyses, strains SHINM1T, ICHIJ1 and ICHIAU1 represent a novel species of a new genus, for which the name Fluviibacter phosphoraccumulans gen. nov., sp. nov. is proposed, within a new family, Fluviibacteraceae fam. nov. of the order Rhodocyclales. The type strain is SHINM1T (=JCM 32071T=NCIMB 15105T).

Relationship between dementia and gut microbiome-associated metabolites: a cross-sectional study in Japan.

Dysregulation of the gut microbiome is associated with dementia. However, the relationship between microbiome-associated metabolites and dementia has yet to be identified. Outpatients visiting a memory clinic in Japan enrolled in this cross-sectional study; 107 subjects were eligible for the study, 25 of which had dementia. We collected demographics, activities of daily living, risk factors, cognitive function, and brain imaging data. The gut microbiome was assessed using terminal restriction fragment length polymorphism analysis. Concentrations of faecal metabolite were measured. We used multivariable logistic regression analyses to identify whether metabolites were independently related to dementia. The concentrations of metabolites were significantly different between subjects with and those without dementia. Every 1 standard deviation increment in faecal ammonia concentration was associated with around a 1.6-fold risk for the presence of dementia. A higher faecal lactic acid concentration was related to a lower risk of dementia, by around 60%. A combination of higher faecal ammonia and lactic acid concentrations was indicative of the presence of dementia, and had a similar predictive value as traditional biomarkers of dementia. Thus, faecal ammonia and lactic acid are related to dementia, independently of the other risk factors for dementia and dysregulation of the gut microbiome.

Amedibacterium intestinale gen. nov., sp. nov., isolated from human faeces, and reclassification of Eubacterium dolichum Moore et al. 1976 (Approved Lists 1980) as Amedibacillus dolichus gen. nov., comb. nov.

Four strains (9CBEGH2T, 9BBH35, 6BBH38 and 6EGH11) of Gram-stain-positive, obligately anaerobic, rod-shaped bacteria were isolated from faecal samples from healthy Japanese humans. The results of 16S rRNA gene sequence analysis indicated that the four strains represented members of the family Erysipelotrichaceae and formed a monophyletic cluster with 'Absiella argi' strain N6H1-5 (99.4% sequence similarity) and Eubacterium sp. Marseille-P5640 (99.3 %). Eubacterium dolichum JCM 10413T (94.2 %) and Eubacterium tortuosum ATCC 25548T (93.7 %) were located near this monophyletic cluster. The isolates, 9CBEGH2T, 'A. argi' JCM 30884 and Eubacterium sp. Marseille-P5640 shared 98.7-99.1% average nucleotide identity (ANI) with each other. Moreover, the in silico DNA-DNA hybridization (DDH) values among three strains were 88.4-90.6%, indicating that these strains represent the same species. Strain 9CBEGH2T showed 21.5-24.1 % in silico DDH values with other related taxa. In addition, the ANI values between strain 9CBEGH2T and other related taxa ranged from 71.2 % to 73.5 %, indicating that this strain should be considered as representing a novel species on the basis of whole-genome relatedness. Therefore, we formally propose a novel name for 'A. argi' strains identified because the name 'A. argi' has been effectively, but not validly, published since 2017. On the basis of the collected data, strain 9CBEGH2T represents a novel species of a novel genus, for which the name Amedibacterium intestinale gen. nov., sp. nov. is proposed. The type strain of A. intestinale is 9CBEGH2T (=JCM 33778T=DSM 110575T).

Mesosutterella multiformis gen. nov., sp. nov., a member of the family Sutterellaceae and Sutterella megalosphaeroides sp. nov., isolated from human faeces.

Two novel, obligately anaerobic, Gram-stain-negative, rod or coccoid-shaped bacteria, designated strains 4NBBH2T and 6FBBBH3T, were isolated from faecal samples of a healthy Japanese woman and man. The 16S rRNA gene sequence analysis showed that these strains represent a distinct lineage within the family Sutterellaceae. Strain 4NBBH2T formed a monophyletic branch between the genera Parasutterella and Sutterella, with sequence similarity to Sutterella stercoricanis CCUG 47620T (92.6 %), followed by Sutterella wadsworthensis WAL 7877 (92.4 %), Sutterella parvirubra YIT 11816T (92.1 %) and Parasutterella secunda YIT 12071T (91.8 %). Strain 6FBBBH3T was affiliated to the genus Sutterella, with highest similarity to S. stercoricanis CCUG 47620T (97.1 %), followed by S. parvirubra YIT 11816T (96.6 %) and S. wadsworthensis WAL 7877 (95.2 %). Strains 4NBBH2T and 6FBBBH3T were asaccharolytic. Analysis of fatty acids revealed that strain 4NBBH2T could be differentiated from Sutterella species (including strain 6FBBBH3T) by the presence of a low concentration of C16 : 1ω7c. The major respiratory quinones of strain 4NBBH2T were menaquinone (MK)-6 and methylmenaquinone (MMK)-6, whereas those of strain 6FBBBH3T were MK-5 and MMK-5. The G+C content of the genomic DNA of strains 4NBBH2T and 6FBBBH3T were 56.9 and 62.8 mol%, respectively. On the basis of the collected data, strain 4NBBH2T represents a novel species in a novel genus of the family Sutterellaceae, for which the name Mesosutterella multiformis gen. nov., sp. nov. is proposed. The type strain is 4NBBH2T (=JCM 32464T=DSM 106860T). We also propose a novel Sutterella species, Sutterellamegalosphaeroides sp. nov., for strain 6FBBBH3T (=JCM 32470T=DSM 106861T).

Sprouting as a gardening strategy to obtain superior supplementary food: evidence from a seed-caching marine worm.

Only a handful of non-human animals are known to grow their own food by cultivating high-yield fungal or algal crops as staple food. Here we report an alternative strategy utilized by an omnivorous marine worm, Hediste diversicolor, to supplement its diet: gardening by sprouting seeds. In addition to having many other known feeding modes, we showed using video recordings and manipulative mesocosm experiments that this species can also behave like gardeners by deliberately burying cordgrass seeds in their burrows, which has been previously shown to reduce the loss of seeds to water. These seeds, however, are protected by the seed husk, and we used feeding experiments to show that they were not edible for H. diversicolor until they had sprouted or the seed husk had been artificially removed. Additionally, sprouts were shown to be highly nutritious, permitting higher growth rates in H. diversicolor than the low-quality basal food, detritus. We propose both a proximate cause (seed husk as a physical barrier) and ultimate cause (nutritional demand) for this peculiar feeding behavior. Our findings suggest that sprouting may be a common strategy used by seed-collecting animals to exploit nutrients from well-protected seeds.

Phospholipid-derived fatty acids and quinones as markers for bacterial biomass and community structure in marine sediments.

Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.

Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture.

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.

Increase in Alphaproteobacteria in association with a polychaete, Capitella sp. I, in the organically enriched sediment.

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.

The succession of microbial community in the organic rich fish-farm sediment during bioremediation by introducing artificially mass-cultured colonies of a small polychaete, Capitella sp. I.

We monitored seasonal changes of the abundance and composition of microorganisms in the fish-farm sediment in Kusuura Bay, Amakusa, Japan, using the quinone profiling technique, during bioremediation by introducing cultured colonies of polychaete, Capitella sp. I. In November 2004, approximately 9.2 million cultured worms were transferred to the fish-farm sediment, which increased rapidly, and reached 458.5 gWW/m(2) (528,000 indiv./m(2)) in March 2005. During this fast-increasing period of Capitella, the microbial quinone content of the surface sediment (0-2 cm) also increased markedly, and reached 237 micromol/m(2) in January 2005, although the water temperature decreased to the lowest levels in the year. Particularly, the mole fraction of ubiquinone-10 in total quinones in the sediment, indicating the presence of alpha subclass of Proteobacteria, increased by 9.3%. These facts suggest that the bacterial growth was enhanced markedly by the biological activities of worms in the sediment, and the bacteria played an important role in the decomposition of the organic matter in the sediment.

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction.

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.