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

Yoshida

et al. 2017

Appl Environ Microbiol

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For the efficient production of target metabolites from carbohydrates, syngas, or H 2 -CO 2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.KEYWORDS thermophilic, acetogen, Moorella, transformation, biomass, sugar, syngas, fermentation T he use of various forms of renewable energy and industrial bulk chemicals is increasingly desired because of concerns about the depletion of fossil resources and the existence of global warming/climate change. Solar, wind, waterpower, and biomass energies are abundant forms of renewable energy that are available all over the world, and they are promising as sources that could meet the global demand for energy as electricity and heat. However, the number and amount of renewable resources for producing bulk substrates are very limited. Presently, bulk chemicals used to produce, for example, plastics and paints, are produced from naphtha.

“…Ultrapure water containing 0.1% (vol/vol) phosphoric acid was used as the mobile phase at a flow rate of 0.7 ml/min. Crotonate was used as an internal standard (33).…”

Section: Methodsmentioning

confidence: 99%

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073

Iwasaki

Yoshida

et al. 2017

Appl Environ Microbiol

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“…It was also demonstrated that marine sediments collected from various tidal flats in Japan exhibited high methanogenic activity from brown algae under condition of 0.51 M NaCl while mesophilic granules from upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater lost the methanogenic activity under the saline condition (20). This observation suggested that marine sediments in tidal flats were promising sources of methanogenic microbial consortia that could be applied for the anaerobic treatment of organic waste with high salinity.…”

mentioning

confidence: 96%

“…However, in the study, all tested marine sediments showed activity in acetoclastic methanogenesis lower than that in hydrolytic/acetogenesis, acidogenesis from butyrate and hydrogenotrophic methanogenesis. Furthermore, to determine whether the methane production from the brown alga by marine sediments was sustainable, when the culture was diluted to 10% (v/v) with fresh medium containing the alga and salt to conduct subculture, acetoclastic methanogenesis was significantly weakened, suggesting low microorganism growth rates compared to the other steps (20). To overcome this constraint and practically use the marine microbial community, the microbial physiology of a variety of marine methanogenic microbes needs to be elucidated to optimize them further, and the appropriate bioreactor configuration for high-throughput treatment needs to be determined.…”

mentioning

confidence: 99%

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

Journal of Bioscience and Bioengineering

Suehira

Miura

et al. 2016

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A marine sediment collected from Hiroshima Bay was cultured in artificial seawater, containing 0.51 M NaCl and 60 mM acetate and was found to exhibit active methane production at 37°C. Following four successive serial dilutions of cultures in medium containing 0.51 M NaCl, 60 mM acetate, and antibiotics, the well-acclimated methanogen was found to exhibit growth over a range of NaCl concentration (between 0 M and 2.06 M). The specific growth rates of the highly enriched methanogen, termed strain HA, in the absence of NaCl and in the presence of 1.54 M NaCl were estimated to be 0.037 h(-1) and 0.027 h(-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 0.51 M 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 70 mM/d under condition of 0.51 M NaCl and successful methane production even under 1.54 M NaCl.

“…The concentration of methane was analyzed by gas chromatography (GC-8A; Shimadzu Corp., Kyoto, Japan) equipped with a thermal conductivity detector and a stainless steel column packed with activated carbon, at 70 . Argon was used as the carrier gas 4) . The concentration of acetate was quantified by high-performance liquid chromatography (LC-2000 Plus HPLC; JASCO Corp., Tokyo, Japan) equipped with a refractive index detector (RI-2031 Plus; JASCO Corp.), Shodex RSpak KC-811 column (Showa Denko K.K., Kanagawa, Japan), and a guard column (Shodex RSpak KC-G; Showa Denko K.K.)…”

Section: Chemical Analysismentioning

confidence: 99%

“…at 60 . Ultrapure water containing 0.1 % (v/v) phosphoric acid was used as the mobile phase at a flow rate of 0.7 mL/min 4) .…”

Section: Chemical Analysismentioning

confidence: 99%

High-rate Fermentation of Acetate to Methane under Saline Condition by Aceticlastic Methanogens Immobilized in Marine Sediment

Kobayashi

Miura

et al. 2016

J. Jpn. Petrol. Inst.

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, which is impossible without immobilization of the cells in the sediment. Using scanning electron microscopy, we observed Methanosaeta-like filamentous microorganisms and Methanosarcina-like coccoid microorganisms in granule-like structures. The results demonstrated that marine sediment is not only a promising microbial resource of halophilic aceticlastic methanogens, but also a supporting material to fix aceticlastic methanogens in a fixed-bed reactor.