Naoshi Fujimoto

Environmental conditions associating microcystins production to Microcystis aeruginosa in a reservoir of Thailand.

ABSTRACT Three heptapeptide toxins, microcystin-RR, microcystin-RY and microcystin-LR, which can cause health problems in animals and humans were monitored in Bang Phra Reservoir, Thailand using reversed-phase high performance liquid chromatography. The concentrations of the three toxins in the reservoir varied greatly depending on location and time water samples were collected. Water quality parameters such as light intensity, temperature, pH, dissolved oxygen, suspended solid, chemical oxygen demand, dissolved organic carbon, total nitrogen, total phosphorus, ammonia, nitrate, phosphate, total dissolved nitrogen, total dissolved phosphorus and chlorophyll-a were also measured in parallel with microcystin determinations. Relationships among water quality parameters, toxins and chlorophyll-a were established. Toxin concentration increased in proportion to increases in total phosphorus, fraction of dissolved phosphorus, but was inversely correlated with water pH and total suspended solids. The other measured parameters in the study showed no correlations to toxin level in reservoir water. Significant correlations between chlorophyll-a and suspended solids, phosphate, nitrate and ammonia were observed suggesting that nitrogen and phosphorus are the two major nutrients governing growth of algae in the reservoir. This relationship suggests that algal production as well as toxin concentration are dependant on nutrient levels in the water body, since both measured light intensity and temperature level was favorable for algal growth. A small algal bloom observed in the rainy season of each year (lasting for only a couple of months) paralleled measured increases in toxin concentration, chlorophyll-a, TP and TN in the water column. Toxin level in the water column remain detectable for 3--4 months period following the initiation of algal bloom. Results indicate that major blooms are likely to occur following the raining season which usually occurs near the end of October when runoff would increase nutrient level entering the reservoir. This study also demonstrated that an ongoing field-monitoring program is needed in these lakes and reservoirs for predicting toxic level of microcystin production for use in risk assessment and for alerting the public to potential health hazards. Concentration of toxin in the reservoir can perhaps be controlled by reducing non point source nutrient input within the watershed.

Development of a 16S rRNA Gene Primer and PCR-Restriction Fragment Length Polymorphism Method for Rapid Detection of Members of the Genus Megasphaera and Species-Level Identification†

ABSTRACT The genus Megasphaera is relevant to the environment, human health and food, and renewable energy for the future. In this study, a primer set was designed for PCR-restriction fragment length polymorphism (RFLP) analyses to detect and identify the members of Megasphaera. Direct detection and identification were achieved for environmental samples and isolates.

Effects of temperature change on algal growth

Abstract The acclimation to temperature of two algae, Microcystis viridis and Selenastrum capricornutum, was examined in batch culture experiments. Both algae acclimated promptly to an increase in temperature, but S. capricornutum acclimated slowly to a decrease in temperature relative to M. viridis.

Hydrogen fermentation using lactate as the sole carbon source: Solution for ‘blind spots’ in biofuel production

Lactate is not believed to be a suitable substrate for hydrogen fermentation, although glucose and starch are. To eliminate this ‘blind spot’ in hydrogen fermentation, we showed that it is feasible to produce hydrogen with lactate as the sole carbon source by using mixed microflora as well as a single culture. Microflora were isolated from a methane fermentation system, and their hydrogen productivity, fermentation properties, and diversity were analyzed. Since previous studies used inoculum pretreatment to improve efficiency, we used both pretreated and non-pretreated samples. The hydrogen yield of the non-pretreated microflora sample was 0.43 mol/mol lactate, which is approximately 49 times the values reported previously. Megasphaera elsdenii was found to be the main lactate utilizing-hydrogen producing bacteria (LU-HPB) in this sample, but it is not heat-shock tolerant. Thus, heat-shock pretreatment reduces microbial diversity, including lactic acid bacteria (LAB) and LU-HPB, in the inoculum. This is another ‘blind spot’ of hydrogen fermentation from lactate. The hydrogen yield of M. elsdenii was approximately 0.4 mol/mol lactate. Considering the balance of metabolites in the reaction, even if the substrate was completely consumed for lactic acid fermentation, 0.8 mol hydrogen per mol of glucose may be recovered via homo-type lactic acid fermentation. Thus, LU-HPB do not require inoculum and/or substrate pretreatment and may improve the utilization of recalcitrant substrates such as xylose by cooperating with LAB. To our knowledge, this is the first study to identify and characterise a microbial hydrogen fermenter that can use lactate as the sole carbon source.

Examination and Comparison of Microbial Diversity in Field-Scale Sewage Sludge Composters

While the earth’s biodiversity has been studied in detail, to date, microbes have been completely omitted from meta-analytical studies of biodiversity data sets (Balmford & Bond, 2005; Diaz et al., 2006). In fact, biodiversity data sets are far from being considered a comprehensive global resource (Collen et al., 2008). Since the origin of prokaryotes on Earth over 3.5 billion years ago, the extent of evolutionary diversification within this group has been truly immense (DeLong & Pace, 2001; Payne et al., 2009). Microbial communities play important biological roles, such as the global cyclical change of materials in various environments (Diaz et al., 2006). As a result, existing biodiversity includes a vast and largely undiscovered diversity of microbes, which are probably very important for the sustainability of ecosystems (Swift et al., 2004). Hence, detailed investigation to characterize the global biodiversity of microbes is a very important task. Microbes have always formed a major component of global biodiversity, either as producers (e.g., phototrophic blue-green algae) or decomposers (e.g., heterotrophic bacteria) (Naeem et al., 2000). Furthermore, in the future, they may serve as producers of useful alternative energy sources (Ohnishi et al., 2010). For example, phototrophic microorganisms use the energy from light for the production of biomass, which is an energy source stored in all living organisms. In fact, microbial decomposers are used in industry to convert microbial biomass and organic waste materials, such as domestic garbage, into biofuels, such as methane, ethanol, and hydrogen (Swift et al., 2004; Kayhanian et al., 2007). Microbes are also used for bioremediation, which is the cleanup of pollution caused by human activities (Jorgensen et al., 2000). In this process, various microbes have been isolated from nature, which are capable of degrading spilled oil, solvents, and other environmentally toxic pollutants. Furthermore, the breadth of microbial diversity on Earth provides genetic resources that offer solutions for environmental and energy issues, and research in this area is currently expanding. Considering the serious environmental and energy issues that humans face today, a better understanding of the ecophysiology of environmental microbes is warranted to address problems such as resource depletion and environmental pollution.

Development of a Solid-State Fermentation System for Producing Bioethanol from Food Waste

従来よりバイオエタノールの製造には液体発酵法が広く採用されている．本法は，エタノールを蒸留した後に発生する蒸留廃液の処理が大きな問題となっている．この蒸留廃液の排出を極力抑制することを可能にする新規の発酵法としてエタノール固体発酵システムを考案した．乾燥生ごみにおいて良好に生育し，かつ糖化酵素生産に適した麹菌として麦味噌用の Aspergillus oryzae KBN650を選出した．本麹菌株の乾燥生ごみにおける糖化力の発現は，培養温度 30℃，初発水分含量 50％が最適であった．また，乾燥生ごみのエタノール固体発酵に適した酵母として焼酎酵母 Saccharomyces cerevisiae A30を選出した．本菌株は乾燥生ごみと生ごみ麹の混合割合が等量ずつで初発水分含量が60％において高いエタノール生成能を示した． 以上の知見に基づき，試料調製，生ごみ麹製造，糖化発酵，蒸留の４つの工程から構成されるエタノール固体発酵装置を製作した．この装置では，乾燥生ごみ 9.6kgから 99％エタノール換算で0.6kgのエタノールが生産され，全糖消費量に対するエタノール収率は73.6％であった．この時，従来型の液体エタノール発酵法の蒸留時に排出される蒸留廃液の排出はなく，固形残渣として排出された．

Bio-hythane production from cassava residue by two-stage fermentative process with recirculation

The two-stage hythane fermentation of cassava residue low in protein, rich in iron, and deficient in nickel and cobalt, resulted in failure after long-term operation, showing a radical decrease in methane production along with an increase in volatile fatty acids (VFAs) accumulation in the second stage. Based on the gap between theoretical demand and existing content of nutrients, the effect of their additions on hythane fermentation was validated in the repeated batch experiment and continuous experiment. The proliferation of hydrolysis bacteria, acidogens, and hydrogen producing bacteria and methanogens was guaranteed by sufficient N (0.7g/L), S (30mg/L), Ni (1.0mg/L), and Co (1.0mg/L), and the metabolism of a sustainable hythane fermentation was recovered. In this optimal nutrient combination of above trace elements, the highest hythane yield (426m3 hythane with 27.7% of hydrogen from 1ton of cassava residue) was obtained.

Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing

In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

Enrichment Culture of Hydrogen Fermentation Microorganisms and Analysis of Microbial Communities

本研究では, 生ごみを発酵基質として安定的に水素を生産する微生物群の探索を試みた. 下水汚泥コンポスト中の微生物群を用いることで, 安定した生ごみの水素発酵が可能であることが確認された. 集積培養過程の微生物相をFISH法とPCR-DGGE法により解析した結果, Clostridium 属の菌種が水素発酵の主役を担っているものと考えられた. また, この他にBacillus 属グループの菌種の存在が確認された. Bacillus 属グループの菌種は生ごみの水素発酵過程においてClostridium 属の水素発酵能の安定化に寄与するものと考えられた. また, 集積した微生物群を用い, pH6.0に制御して生ごみの水素発酵を行った場合の水素収率は2.03mol-H2/mol-hexoseであった. 下水汚泥コンポスト中の複合微生物群は生ごみを基質とした水素発酵システムの植種微生物源として有効であるものと考えられた.

Isolation of Bacteria Lysing Musty Odor Producing Alga Phormidium tenue and Its Lytic Characteristics

An alga-lytic bacterium, Pseudomonas KA6, was isolated from Lake Kasumigaura by a double-layer method. the filtrate of Pseudomonas KA6 culture lysed Phormidium tenue promptly. It was found that the growth of P. tenue is controlled by the addition of the filtrate 5 % and more. The lysis of P. tenue occurred efficiently at 25-35°C and pH 7-9. the cell of P. tenue in the stationary and endogenous phases was lysed promptly compared with that of P. tenue in the logarithmic phase. The filtrate lysed Oscillatoria sp. and Anabaena planktonica immediately as well as P. tenue; however, the filtrate lysed Microcystis aeruginosa slowly, and did not lyse green algae and diatoms. It was suggested that lytic compounds in the filtrate of KA6 culture affect only cyanobacteria in a water environment.