Yoshiko Okamura

Evaluation of marine sediments as microbial sources for methane production from brown algae under high salinity

Various marine sediments were evaluated as promising microbial sources for methane fermentation of Saccharina japonica, a brown alga, at seawater salinity. All marine sediments tested produced mainly acetate among volatile fatty acids. One marine sediment completely converted the produced volatile fatty acids to methane in a short period. Archaeal community analysis revealed that acetoclastic methanogens belonging to the Methanosarcina genus dominated after cultivation. Measurement of the specific conversion rate at each step of methane production under saline conditions demonstrated that the marine sediments had higher conversion rates of butyrate and acetate than mesophilic methanogenic granules. These results clearly show that marine sediments can be used as microbial sources for methane production from algae under high-salt conditions without dilution.

Improved methane production from brown algae under high salinity by fed-batch acclimation

Here, a methanogenic microbial community was developed from marine sediments to have improved methane productivity from brown algae under high salinity. Fed-batch cultivation was conducted by adding dry seaweed at 1wt% total solid (TS) based on the liquid weight of the NaCl-containing sediment per round of cultivation. The methane production rate and level of salinity increased 8-fold and 1.6-fold, respectively, at the 10th round of cultivation. Moreover, the rate of methane production remained high, even at the 10th round of cultivation, with accumulation of salts derived from 10wt% TS of seaweed. The salinity of the 10th-round culture was equivalent to 5% NaCl. The improved methane production was attributed to enhanced acetoclastic methanogenesis because acetate became rapidly converted to methane during cultivation. The family Fusobacteriaceae and the genus Methanosaeta, the acetoclastic methanogen, predominated in bacteria and archaea, respectively, after the cultivation.

Bacterial community structure and predicted alginate metabolic pathway in an alginate-degrading bacterial consortium

Methane fermentation is one of the effective approaches for utilization of brown algae; however, this process is limited by the microbial capability to degrade alginate, a main polysaccharide found in these algae. Despite its potential, little is known about anaerobic microbial degradation of alginate. Here we constructed a bacterial consortium able to anaerobically degrade alginate. Taxonomic classification of 16S rRNA gene, based on high-throughput sequencing data, revealed that this consortium included two dominant strains, designated HUA-1 and HUA-2; these strains were related to Clostridiaceae bacterium SK082 (99%) and Dysgonomonas capnocytophagoides (95%), respectively. Alginate lyase activity and metagenomic analyses, based on high-throughput sequencing data, revealed that this bacterial consortium possessed putative genes related to a predicted alginate metabolic pathway. However, HUA-1 and 2 did not grow on agar medium with alginate by using roll-tube method, suggesting the existence of bacterial interactions like symbiosis for anaerobic alginate degradation.

Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid

Thraustochytrid production of polyunsaturated fatty acids and xanthophylls have been generally sourced from crop-derived substrates, making the exploration of alternative feedstocks attractive since they promise increased sustainability and lower production costs. In this study, a distinct two-stage fermentation system was conceptualized for the first time, using the brown seaweed sugar mannitol as substrate for the intermediary biocatalyst Gluconobacter oxydans, an acetic acid bacterium, along with the marine thraustochytrid Aurantiochytrium sp. to produce the value-added lipids and xanthophylls. Jar fermenter culture resulted in seaweed mannitol conversion to fructose with an efficiency of 83xa0% by G. oxydans and, after bacteriostasis with sea salts, production of astaxanthin and docosahexaenoic acid by Aurantiochytrium sp. KH105. Astaxanthin productivity was high at 3.60xa0mg/L/day. This new system, therefore, widens possibilities of obtaining more varieties of industrially valuable products including foods, cosmetics, pharmaceuticals, and biofuel precursor lipids from seaweed fermentation upon the use of suitable thraustochytrid strains.

Semi-continuous methane production from undiluted brown algae using a halophilic marine microbial community

Acclimated marine sediment-derived culture was used for semi-continuous methane production from materials equivalent to raw brown algae, without dilution of salinity and without nutrient supply, under 3 consecutive conditions of varying organic loading rates (OLRs) and hydraulic retention time (HRT). Methane production was stable at 2.0gVS/kg/day (39-day HRT); however, it became unstable at 2.9gVS/kg/day (28-day HRT) due to acetate and propionate accumulation. OLR subsequently decreased to 1.7gVS/kg/day (46-day HRT), stabilizing methane production beyond steady state. Methane yield was above 300mL/g VS at all OLRs. These results indicated that the acclimated marine sediment culture was able to produce methane semi-continuously from raw brown algae without dilution and nutrient supply under steady state. Microbial community analysis suggested that hydrogenotrophic methanogens predominated among archaea during unstable methane production, implying a partial shift of the methanogenic pathway from acetoclastic methanogenesis to acetate oxidation.

Dysgonomonas alginatilytica sp. nov., an alginate-degrading bacterium isolated from a microbial consortium.

Gram-stain-negative, facultatively anaerobic, non-motile, non-spore-forming, rod-shaped bacterium, designated strain HUA-2T, was isolated from an alginate-degrading microbial consortium. Strain HUA-2T was related to Dysgonomonas capnocytophagoides JCM 16697T, Dysgonomonas macrotermitis JCM 19375T and Dysgonomonas mossii CCUG 43457T with 95.1u2009%, 94.1u2009% and 92.1u2009% 16S rRNA gene sequence similarity, respectively. The optimal growth temperature and pH for strain HUA-2T were 35u2009°C and pHu20098.0, respectively. Enzyme production, major fermentation products from glucose, and the major cellular fatty acids were different from those of D. capnocytophagoides CCUG 17966T or other members of the genus Dysgonomonas. Therefore, strain HUA-2T is proposed to represent a novel species of the genus Dysgonomonas, for which we propose the name Dysgonomonas alginatilytica sp. nov. The type strain is HUA-2T (u2009=u2009DSM 100214Tu2009=u2009HUT 8134T).

In situ mass spectrometry of glucose decomposition under hydrothermal reactions

We designed an in situ mass spectrometry (in situ MS) analysis method and developed to identify the products of glucose decomposition under hydrothermal condition for the first time. The in situ MS analysis was performed by coupling a tubular batch reactor with a quadrupole mass analyzer via custom-built connection fittings. The products of glucose decomposition were investigated by in situ MS, mass spectrometry of cold effluent, and high-performance liquid chromatography (HPLC) analysis of cold effluent and the results were compared. At 140 °C, in situ MS and mass spectrometry of cold effluent showed that the decomposition of glucose does not proceed; this was confirmed by comparison with the mass spectral database for glucose. At 180 °C or higher, a clear base fragmentation peak of 5-hydroxymethylfurfural (5-HMF) at position m/z 97 and that of furfural at m/z 96, formic acid (m/z=46) and levulinic acid (m/z=116) were observed by mass spectrometry. No levulinic acid or furfural was observed through conventional HPLC analysis under any condition; only glucose, formic acid, and 5-HMF could be detected. The effectiveness of in situ MS analysis is clear, compared to mass spectrometry analysis of cold effluent and HPLC analysis.

Characterization of a halotolerant acetoclastic methanogen highly enriched from marine sediment and its application in removal of acetate.

A marine sediment collected from Hiroshima Bay was cultured in artificial seawater, containing 0.51xa0M NaCl and 60xa0mM acetate and was found to exhibit active methane production at 37°C. Following four successive serial dilutions of cultures in medium containing 0.51xa0M NaCl, 60xa0mM acetate, and antibiotics, the well-acclimated methanogen was found to exhibit growth over a range of NaCl concentration (between 0xa0M and 2.06xa0M). The specific growth rates of the highly enriched methanogen, termed strain HA, in the absence of NaCl and in the presence of 1.54xa0M NaCl were estimated to be 0.037xa0h(-1) and 0.027xa0h(-1), respectively. The pH and temperature for optimum growth were determined to be 7.0-8.8 and 37°C, respectively. Although cells that had morphology similar to Methanosaeta sp. became dominant in the culture, methane production was still detected in the medium containing 0.51xa0M NaCl and other substrates such as methanol, formate, and methylamine, indicating contamination with other methanogens. The phylogenetic tree based on 16S rRNA gene sequences revealed that the strain HA was closely related to Methanosaeta harundinacea 6Ac and 8Ac(T), with sequence similarity of 98% and 97%, respectively. The continuous removal of acetate with upflow anaerobic filter reactor for industrial use of strain HA determined a methane production rate of 70xa0mM/d under condition of 0.51xa0M NaCl and successful methane production even under 1.54xa0M NaCl.

Isolation and characterization of bacterium producing lipid from short-chain fatty acids

Anaerobic fermentation generates propionic acid, which inhibits microbial growth and accumulates in wastewater containing increased amounts of organic matter. We therefore isolated a propionic acid-assimilating bacterium that could produce triacylglycerol, for use in wastewater treatment. Nitratireductor sp. strain OM-1 can proliferate in medium containing propionic, acetic, butyric, and valeric acids as well as glycerol, and produces triacylglycerol when both propionic and acetic acids or glycerol are present. In composite model wastewater containing acetic acid, propionic acid and glycerol, this strain shows an even higher conversion rate, suggesting that it is suitable for wastewater treatment. Further, nitrogen depletion in medium containing an acetic-propionic acid mixture resulted in the production of the light oil 2-butenoic acid 1-methylethyl ester, but not triacylglycerol. Collectively, our data indicate that strain OM-1 has the potential to reduce accumulation of activated sludge in wastewater treatment and may contribute to the production of biodiesel.

Efficient conversion of mannitol derived from brown seaweed to fructose for fermentation with a thraustochytrid

Macroalgae are a promising biomass feedstock for energy and valuable chemicals. Mannitol and alginate are the major carbohydrates found in the microalga Laminaria japonica (Konbu). To convert mannitol to fructose for its utilization as a carbon source in mannitol non-assimilating bacteria, a psychrophile-based simple biocatalyst (PSCat) was constructed using a psychrophile as a host by expressing mesophilic enzymes, including mannitol 2-dehydrogenase for mannitol oxidation, and NADH oxidase and alkyl hydroxyperoxide reductase for NAD+ regeneration. PSCat was treated at 40xa0°C to inactivate the psychrophilic enzymes responsible for byproduct formation and to increase the membrane permeability of the substrate. PSCat efficiently converted mannitol to fructose with high conversion yield without additional input of NAD+. Konbu extract containing mannitol was converted to fructose with hydroperoxide scavenging, inhibiting the mannitol dehydrogenase activity. Auranthiochytrium sp. could grow well in the presence of fructose converted by PSCat. Thus, PSCat is a potential carbohydrate converter for mannitol non-assimilating microorganism.