Weekly Observations of Estuarine Microbial Assemblages during Summer in the Inner Part of Ariake Bay, Japan; Microbial Water-sediment Coupling in Turbid Shallow Waters.

Estuarine microbial assemblages are altered by a number of environmental factors, and knowledge of these changes is essential for understanding the functions of microbes in estuarine ecosystems. The aims of the present study were to examine the relationship between microbial assemblages in the water column and sediment surface, and to identify the environmental factors that influence the short-term dynamics of microbial assemblages in these two zones in summer in the inner part of Ariake Bay. The microbial assemblage of each sample consisted of a mean of 71.1% operational taxonomic units (OTUs), which commonly occurred in the water column and sediment surface, although their relative composition markedly differed between the two zones. In the water column, spatiotemporal changes in microbial assemblages correlated with several environmental factors, such as the nitrogen content in suspended particles, turbidity, and salinity. On the other hand, temporal changes in the sediment's microbial assemblages were governed by a single environmental factor, namely, the oxygen reduction potential. These results suggest that the composition of microbial assemblages in the water column and sediment surface differed even in highly turbid brackish waters with high sediment resuspension, and the environmental factors contributing to the change in the assemblage composition also differed between the water column and sediment.

Isolation of sake yeast strains from Ariake Sea tidal flats and evaluation of their brewing characteristics.

Screening for new sake yeasts can expand the sensory diversity of sake, due to their production of metabolites that characterize sake's aroma and taste. In this study, mud from tidal flats in the Ariake Sea was screened for Saccharomyces cerevisiae strains with ethanol productivity suitable for sake brewing, and the brewing characteristics of isolated strains were evaluated. Five strains (H1-1, H1-2, H1-3, H3-1, and H3-2) classified as S. cerevisiae were isolated. Karyotype analysis by pulsed-field gel electrophoresis showed that five isolated strains were closely related to sake yeast strains (K7, K701, K9, K901, and Y52) instead of laboratory yeast strain. Results of small-scale brewing tests including sake yeast strains K701, K901, and Y52 showed that the five isolated strains have fermentation activity comparable to sake yeast strains. Principal component analysis (PCA) revealed that the five isolated strains produce higher levels of ethyl caproate and lower levels of acidic compounds than sake yeasts. In addition, isolated strains H3-1 and H3-2 produce higher levels of isoamyl acetate and lower levels of acetic acid than other isolated strains. Consequently, five S. cerevisiae strains that have high fermentation activity and differ from common sake yeast strains in terms of brewing characteristics were successfully isolated from the Ariake Sea.

Development of Monascus purpureus monacolin K-hyperproducing mutant strains by synchrotron light irradiation and their comparative genome analysis.

Monascus purpureus have been used for making koji and other fermented foods and supplements. M. purpureus characteristically produces monacolin K (MK), a secondary metabolite that competitively inhibits cholesterol synthesis. Synchrotron light irradiation was applied to induce mutation in the strain KUPM5 to improve the MK-producing ability of M. purpureus strain KUPM5. Screening by a bioassay utilizing sensitivities to yeast Saccharomyces cerevisiae from 936 colonies allows isolating three mutant strains: SC01, SC02, and SC03. These mutant strains and the parental strain KUPM5 were subjected to make koji using rice, and their metabolites were compared. All strains SC01, SC02, and SC03 in koji showed higher production of MK than the strain KUPM5. Particularly, the SC02 strain produced MK threefold higher than KUPM5 and maintained the production capabilities of other metabolites, including red, yellow, and orange pigments, mycelial contents, and α-amylase activity comparable to those of the strain KUPM5. Comparative genome analysis among strain KUPM5 and the mutants revealed that synchrotron light irradiation introduced mutations in approximately 90% of the total genes, including SNV, MNV, and indel mutations. The frequencies of SNV substitution in the whole genome occupied 68.96% of all mutations, of which 92.38% were transversions and 7.62% were transitions. This study, therefore, proved the synchrotron light irradiation was highly efficient for the strain improvement of a filamentous fungus, M. purpureus, and provided insights into the properties of mutation in the fungus by this mutagen.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Genomic diversity of 39 samples of Pyropia species grown in Japan.

Some Pyropia species, such as nori (P. yezoensis), are important marine crops. We conducted a phylogenetic analysis of 39 samples of Pyropia species grown in Japan using organellar genome sequences. A comparison of the chloroplast DNA sequences with those from China showed a clear genetic separation between Japanese and Chinese P. yezoensis. Conversely, comparing the mitochondrial DNA sequences did not separate Japanese and Chinese P. yezoensis. Analysis of organellar genomes showed that the genetic diversity of Japanese P. yezoensis used in this study is lower than that of Chinese wild P. yezoensis. To analyze the genetic relationships between samples of Japanese Pyropia, we used whole-genome resequencing to analyze their nuclear genomes. In the offspring resulting from cross-breeding between P. yezoensis and P. tenera, nearly 90% of the genotypes analyzed by mapping were explained by the presence of different chromosomes originating from two different parental species. Although the genetic diversity of Japanese P. yezoensis is low, analysis of nuclear genomes genetically separated each sample. Samples isolated from the sea were often genetically similar to those being farmed. Study of genetic heterogeneity of samples within a single aquaculture strain of P. yezoensis showed that samples were divided into two groups and the samples with frequent abnormal budding formed a single, genetically similar group. The results of this study will be useful for breeding and the conservation of Pyropia species.

Breeding sake yeast and identification of mutation patterns by synchrotron light irradiation.

Sake yeast is one of the important factors that characterize the aroma and taste of sake. To obtain sake yeast strains with different metabolic capabilities from other strains, breeding of a sake yeast is an effective way. In this study, sake yeast strain Y5201 was mutagenized by synchrotron light irradiation to obtain the mutant strains showing different brewing characteristics from parental strain Y5201, and comparative genome analysis between strain Y5201 and mutant strains was performed to identify mutation points and patterns induced by synchrotron light irradiation. Screening with the drug-resistant and fermentation tests selected the nine mutants (C18, C19, C29, C50, C51, C52, C54, T25, and T49) from the mutagenized Y5201 cells. Principal component analysis results based on the analysis of the small-scale brewing test metabolites showed that the mutant strain C19 was different from other strains, which had higher productivity of ethyl caproate and isoamyl acetate than those of the Y5201. Comparative genome analysis revealed that mutants by synchrotron light irradiation had a higher diversity of single nucleotide substitutions and a higher frequency of Indel (insertion/deletion) in these DNA than ethyl methanesulfonate and UV irradiation. These results suggest that synchrotron light irradiation is an effective and unique mutagen for yeast breeding.

Genetic diversity and population structure of razor clam Sinonovacula constricta in Ariake Bay, Japan, revealed using RAD-Seq SNP markers.

The razor clam Sinonovacula constricta is a commercially important bivalve in Japan. The current distribution of this species in Japan is limited to Ariake Bay, where the fishery stock is declining. It is necessary to understand the genetic population structure of this species in order to restore the fishery stock while preserving the genetic diversity of the clam. Here, we report for the first time the genetic population structure of S. constricta in Ariake Bay, Japan. Paired-end restriction site-associated DNA sequencing (RAD-Seq) analyzed samples of S. constricta collected from seven mudflats located along Ariake Bay. Two different genetic populations exist in Ariake Bay, one inhabiting wild habitats and the other inhabiting the transplanted area of artificial seedlings. Our results suggest that genetic differentiation occurred between these two populations (Fst value = 0.052), and a high level of genetic differentiation is maintained between the two groups. In the future, monitoring the interbreeding status of the two genetically distinct populations and the genetic differentiation within each population is important for conserving the genetic diversity of S. constricta in Japan.

Induction of mutation in Monascus purpureus isolated from Thai fermented food to develop low citrinin-producing strain for application in the red koji industry.

Red koji is produced from cultivating rice with Monascus strains that contain various types of fungal secondary metabolites, such as red pigments and monacolin K. Monascus strain also produces citrinin-a mycotoxin. In this study, Monascus purpureus KUPM5 isolated from the Thai fermented food, sufu, was mutagenized to reduce its citrinin production using UV irradiation, NTG treatment, and a combination of UV and NTG. Screening of the mutants using plate bioassay based on the inhibitory effect against Bacillus subtilis enables the selection of 10 mutants. The mutant strains KS301U and KS302U showed an 80% reduction in citrinin production in red koji compared with the wild type (wt), and maintained the ability to produce red pigments similar to the wild type. Activities of enzymes, α-amylase, protease, and lipase, from red koji extract produced by the mutant strain KS302U, were higher than those of the wt, whereas those of the mutant strain KS301U were similar to those of the wt. Consequently, strains KS301U and KS302U were successfully selected as strains suitable for producing red koji and fermented food.

A novel process for direct production of acetone-butanol-ethanol from native starches using granular starch hydrolyzing enzyme by Clostridium saccharoperbutylacetonicum N1-4.

In this work, a new approach for acetone-butanol-ethanol (ABE) production has been proposed. Direct fermentation of native starches (uncooked process) was investigated by using granular starch hydrolyzing enzyme (GSHE) and Clostridium saccharoperbutylacetonicum N1-4. Even the process was carried out under suboptimal condition for activity of GSHE, the production of ABE was similar with that observed in conventional process or cooked process in terms of final solvent concentration (21.3 ± 0.4 to 22.4 ± 0.4 g/L), butanol concentration (17.5 ± 0.4 to 17.8 ± 0.3 g/L) and butanol yield (0.33 to 0.37 g/g). The production of solvents was significantly dependent on the source of starches. Among investigated starches, corn starch was more susceptible to GSHE while cassava starch was the most resistant to this enzyme. Fermentation using native corn starch resulted in the solvent productivity of 0.47 g/L h, which was about 15 % higher than that achieved in cooked process. On the contrary, uncooked process using cassava and wheat starch resulted in the solvent productivity of 0.30 and 0.37 g/L h, which were respectively about 30 % lower than those obtained in cooked process. No contamination was observed during all trials even fermentation media were prepared without sterilization. During the fermentation using native starches, no formation of foam is observed. This uncooked process does not require cooking starchy material; therefore, the thermal energy consumption for solvent production would remarkably be reduced in comparison with cooked process.

Distribution of virulence markers among Vibrio vulnificus isolates of clinical and environmental origin and regional characteristics in Japan.

BACKGROUND: Vibrio vulnificus is an opportunistic human pathogen that is widely distributed in estuarine environments and is capable of causing necrotizing fasciitis and sepsis. In Japan, based on epidemiological research, the incidences of V. vulnificus were concentrated in Kyusyu, mainly in coastal areas of the Ariake Sea. To examine the virulence potential, various genotyping methods have recently been developed. This study aimed to investigate the distribution of virulence markers among V. vulnificus isolates of clinical and environmental origin in three coastal areas with different infection incidences and to determine whether these isolates have the siderophore encoding gene viuB. METHODOLOGY/PRINCIPAL FINDINGS: We examined the distribution of genotypes of the 16S ribosomal ribonucleic acid (rRNA) gene, vvhA, vcg, and capsular polysaccharide (CPS), and the presence of viuB in 156 isolates collected from patients and environmental samples in Japan. The environmental samples were collected from three coastal areas: the Ariake Sea, Ise & Mikawa Bay, and Karatsu Bay. The results showed disparity in the ratios of genotypes depending on the sample origins. V. vulnificus isolates obtained from patients were classified into the clinical type for all genotypes. In the environmental isolates, the ratios of the clinical type for genotypes of the 16S rRNA gene, vvhA, and vcg were in the order of the Ariake Sea>Ise & Mikawa Bay>Karatsu Bay. Meanwhile, CPS analysis showed no significant difference. Most isolates possessed viuB. CONCLUSIONS: Many V. vulnificus belonging to the clinical type existed in the Ariake Sea. Three coastal areas with different infection incidences showed distinct ratios of genotypes. This may indicate that the distribution of clinical isolates correlates with the incidence of V. vulnificus infection.

Membrane-assisted extractive butanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 with 1-dodecanol as the extractant.

A polytetrafluoroethylene (PTFE) membrane was used in membrane-assisted extractive (MAE) fermentation of acetone-butanol-ethanol (ABE) by Clostridium saccharoperbutylacetonicum N1-4. The growth inhibition effects of 1-dodecanol, which has a high partition coefficient for butanol, can be prevented by employing 1-dodecanol as an extractant when using a PTFE membrane. Compared to conventional fermentation, MAE-ABE fermentation with 1-dodecanol decreased butanol inhibition and increased glucose consumption from 59.4 to 86.0 g/L, and total butanol production increased from 16.0 to 20.1g/L. The maximum butanol production rate increased from 0.817 to 0.979 g/L/h. The butanol productivity per membrane area was remarkably high with this system, i.e., 78.6g/L/h/m(2). Therefore, it is expected that this MAE fermentation system can achieve footprint downsizing.

Breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport.

Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.

Production of acetone-butanol-ethanol (ABE) in direct fermentation of cassava by Clostridium saccharoperbutylacetonicum N1-4.

In this work, acetone-butanol-ethanol (ABE) fermentation characteristics of cassava starch and cassava chips when using Clostridium saccharoperbutylacetonicum N1-4 was presented. The obtained results in batch mode using a 1-L fermenter showed that C. saccharoperbutylacetonicum N1-4 was a hyperamylolytic strain and capable of producing solvents efficiently from cassava starch and cassava chips, which was comparable to when glucose was used. Batch fermentation of cassava starch and cassava chips resulted in 21.0 and 19.4 g/L of total solvent as compared with 24.2 g/L of total solvent when using glucose. Solvent productivity in fermentation of cassava starch was from 42% to 63% higher than that obtained in fermentation using corn and sago starches in the same condition. In fermentation of cassava starch and cassava chips, maximum butanol concentration was 16.9 and 15.5 g/L, respectively. Solvent yield and butanol yield (based on potential glucose) was 0.33 and 0.41, respectively, for fermentation of cassava starch and 0.30 and 0.38, respectively for fermentation using cassava chips.

A fatal case of necrotizing fasciitis due to bacterial translocation of Klebsiella oxytoca.

We report a 73-year-old man with hepatocellular cell carcinoma who had eruptions on and severe pain in the lower leg. Within several hours, the patient's skin lesions had progressed markedly. Magnetic resonance imaging findings were consistent with necrotizing fasciitis. Klebsiella oxytoca was isolated from cultures of biopsy samples taken from the leg. The resulting DNA fingerprint pattern revealed that the enteric bacterium was the same as that obtained from the biopsy samples taken from the leg. Furthermore, a dendrogram showed that genetic proximity between samples was extremely high. These results confirmed that translocation of Klebsiella oxytoca as an enteric pathogen caused the necrotizing fasciitis in this patient.

Novel high-efficient butanol production from butyrate by non-growing Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) with methyl viologen.

Non-growing Clostridium saccharoperbutylacetonicum N1-4 hardly produced butanol from only butyrate. As adding glucose to the medium, butyrate utilization and butanol production were stimulated. Addition of 0.1 mM methyl viologen as electron carrier resulted in the highest yield of butanol of 0.671 mol/mol to butyrate and glucose.

Kinetic modeling and sensitivity analysis of acetone-butanol-ethanol production.

A kinetic simulation model of metabolic pathways that describes the dynamic behaviors of metabolites in acetone-butanol-ethanol (ABE) production by Clostridium saccharoperbutylacetonicum N1-4 was proposed using a novel simulator WinBEST-KIT. This model was validated by comparing with experimental time-course data of metabolites in batch cultures over a wide range of initial glucose concentrations (36.1-295 mM). By introducing substrate inhibition, product inhibition of butanol, activation of butyrate and considering the cessation of metabolic reactions in the case of insufficiency of energy after glucose exhaustion, the revised model showed 0.901 of squared correlation coefficient (r(2)) between experimental time-course of metabolites and calculated ones. Thus, the final revised model is assumed to be one of the best candidates for kinetic simulation describing dynamic behavior of metabolites in ABE production. Sensitivity analysis revealed that 5% increase in reaction of reverse pathway of butyrate production (R(17)) and 5% decrease in reaction of CoA transferase for butyrate (R(15)) highly contribute to high production of butanol. These system analyses should be effective in the elucidation which pathway is metabolic bottleneck for high production of butanol.

Utilization of excess sludge by acetone-butanol-ethanol fermentation employing Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564).

Clostridium saccharoperbutylacetonicum N1-4 could not grow or produce butanol in excess sludge medium. However, adding glucose to the excess sludge medium resulted in a specific growth rate and butanol productivity of 0.29 h(-1) and 0.55 g/l/h, respectively, and the final butanol production reached 9.3 g/l. Since the content of suspended solids in medium reduced to less than 50% of the initial content during acetone-butanol-ethanol (ABE) fermentation, the sludge was quantitatively decreased by this fermentation employing this strain.

High production of acetone-butanol-ethanol with high cell density culture by cell-recycling and bleeding.

A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).

High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-Stat continuous butyric acid and glucose feeding method.

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.

Synchronized fresh cell bioreactor system for continuous L-(+)-lactic acid production using Lactococcus lactis IO-1 in hydrolysed sago starch.

An efficient bioreactor, termed a 'synchronized fresh cell bioreactor', was developed and consisted of a pH-dependent substrate feed system coupled with cross flow filtration and turbidity control. The effect of high dilution rate and high cell density coupled with high cell viability on the production of l-lactic acid in continuous culture by Lactococcus lactis IO-1 in enzyme-hydrolysed sago starch medium was investigated. For all changes in dilution rate, cells responded in a synchronized way to the addition of glucose by increasing the rate of biomass formation. Consequently, a glucose-free feed solution was required to maintain the cell concentration at a particular pre-set value. This set-up facilitated the maintenance of the cells in a permanent log phase. At a cell concentration of 15 gl(-1) and a feed glucose concentration of 53 gl(-1), volumetric LA productivities of 8.2, 19.3 and 33.1 gl(-1)h(-1) were obtained at dilution rates of 0.21, 0.50 and 1.1 h(-1), respectively. The respective residual glucose concentrations in the spent medium were 1.90, 0.24 and 3.80 gl(-1). By increasing the cell density, the volumetric productivity increased proportionally. At high cell density, higher dilution rates resulted in lower lactate concentrations in the culture medium resulting in higher productivity. This reactor facilitated efficient operation with high cell viability by maintaining the cells in continuous growth phase for long-term fermentation. Therefore, the growth rate (mu) was calculated according to the Monod equation. Using this system, high specific productivities can be obtained which guarantees high commercial productivity at economical cost with only a small investment for setting up the sago industry.