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The effects of different mole fractions of hydrogen and carbon dioxide on the combustion characteristics of a premixed methane–air mixture are experimentally and numerically investigated. The laminar burning velocity of hydrogen-methane-carbon dioxide-air mixture was measured using the spherically expanding flame method at the initial temperature and pressure of 283 K and 0.1 MPa, respectively. Additionally, numerical analysis is conducted under steady 1D laminar flow conditions to investigate the adiabatic flame temperature, dominant elementary reactions, and NO formation. The measured velocities correspond with those estimated numerically. The results show that increasing the carbon dioxide mole fraction decreases the laminar burning velocity, attributed to the carbon dioxide dilution, which decreases the thermal diffusivity and flame temperature. Conversely, the velocity increases with the thermal diffusivity as the hydrogen mole fraction increases. Moreover, the hydrogen addition leads to chain-branching reactions that produce active H, O, and OH radicals via the oxidation of hydrocarbons, which is the rate-determining reaction. Furthermore, an increase in the mole fractions of hydrogen and carbon dioxide decreases the NO production amount.

Natural gas with H2 is widely used for lean-burn combustion, which leads to NOx emission as the main problem for it. For decreasing NOx emission and increasing thermal efficiency, the investigation on seeking the influence of H2 fractions on the mixture of CH4 and CO2 was conducted. Firstly, the ignition timing was decided through thermal efficiency and brake mean effective pressure (BMEP) for CH4 only. Then, combustion characteristics of CH4, CH4+CO2 and CH4+CO2+H2 were compared with volume percentage of H2 changing from 5% to 30%. Finally, the H2 injection strategy was checked between closed and open valve injections. Among these discussions, thermal efficiency, power output, BMEP and fuel consumption were evaluated. Results show that CO2 addition decreases power output and BMEP, leading to much more fuel consumption and lower thermal efficiency. When H2 is added, at the rich mixture conditions (λ＜1.0), power output and thermal efficiency decrease sharply as the mixture is enriched. However, at the lean-burn conditions (λ＞1.0), the decrease in flow rate of lower heating value (LHV) and increase in power output finally result in the higher efficiency with H2 addition. Moreover, when λ＞1.0, both low fuel consumption and high efficiency can be obtained with H2 addition to achieve the high BMEP. Furthermore, the open valve injection could obtain higher thermal efficiency, power output and BMEP with lower fuel consumption, suggesting that the H2 injection strategy should be well controlled with the ignition timing.

Gas fermentation is one of the promising bioprocesses to convert CO2 or syngas to important chemicals. Thermophilic gas fermentation of volatile chemicals has the potential for the development of consolidated bioprocesses that can simultaneously separate products during fermentation. This study reports the production of acetone from CO2 and H2, CO, or syngas by introducing the acetone production pathway using acetyl–coenzyme A (Ac-CoA) and acetate produced via the Wood–Ljungdahl pathway in Moorella thermoacetica. Reducing the carbon flux from Ac-CoA to acetate through genetic engineering successfully enhanced acetone productivity, which varied on the basis of the gas composition. The highest acetone productivity was obtained with CO–H2, while autotrophic growth collapsed with CO2–H2. By adding H2 to CO, the acetone productivity from the same amount of carbon source increased compared to CO gas only, and the maximum specific acetone production rate also increased from 0.04 to 0.09 g-acetone/g-dry cell/h. Our development of the engineered thermophilic acetogen M. thermoacetica, which grows at a temperature higher than the boiling point of acetone (58°C), would pave the way for developing a consolidated process with simplified and cost-effective recovery via condensation following gas fermentation.

In this study, marine sediment (MS) was successfully used as a source of methanogenic bacteria for the anaerobic digestion (AD) of chicken manure (CM). Using MS showed high production in liquid and semi-solid conditions. Even in solid conditions, 169.3 mL/g volatile solids of chicken manure (VS-CM) was produced, despite the accumulation of ammonia (4.2 g NH3-N/kg CM). To the best of our knowledge, this is the highest methane production from CM alone, without pretreatment, in solid conditions (20%). Comparing MS to Ozouh sludge (excess activated sewage sludge) (OS), using OS under semi-solid conditions resulted in higher methane production, while using MS resulted in more ammonia tolerance (301 mL/gVS-CM at 8.58 g NH3-N/kg). Production optimization was carried out via a response surface methodology (RDM) model involving four independent variables (inoculum ratio, total solid content, NaCl concentration, and incubation time). Optimized methane production (324.36 mL/gVS-CM) was at a CM:MS ratio of 1:2.5 with no NaCl supplementation, 10% total solid content, and an incubation time of 45 days.

Mechanosensitive channels play an important role in the adaptation of cells to hypo-osmotic shock. Among members of this channel family in Escherichia coli, the exact function and physiological role of the mechanosensitive channel homolog YbdG remain unclear. Characterization of YbdG's physiological role has been hampered by its lack of measurable transport activity. Using a nitrosoguanidine mutagenesis-aided screen in combination with next-generation sequencing, here we isolated a mutant with a point mutation in ybdG. This mutation (resulting in a I167T change) conferred sensitivity to high osmotic stress, and the mutant cells differed from WT cells in morphology during hyperosmotic stress at alkaline pH. Interestingly, unlike the cells containing the I167T variant, a null-ybdG mutant did not exhibit this sensitivity and phenotype. Although I167T was located near the putative ion-conducting pore in a transmembrane region of YbdG, no change in ion channel activities of YbdG-I167T was detected. Of note, introduction of the WT C-terminal cytosolic region of YbdG into the I167T variant complemented the osmo-sensitive phenotype. Co-precipitation of proteins interacting with the C-terminal YbdG region led to the isolation of HldD and FbaA, whose overexpression in cells containing the YbdG-I167T variant partially rescued the osmo-sensitive phenotype. This study indicates that YbdG functions as a component of a mechanosensing system that transmits signals triggered by external osmotic changes to intracellular factors. The cellular role of YbdG uncovered here goes beyond its predicted function as an ion or solute transport protein.

Ammonia is an important fertilizer feedstock and an expected next-generation hydrogen carrier. Thus, it is necessary to ensure effective production of ammonia from the waste biomass. In this regard, chicken manure was treated in an autoclave under hydrothermal reaction conditions, and the ammonia release rate was determined in the temperature range of 250-400 °C for holding times ranging from 2 to 120 min. A reaction network for ammonia production was proposed, and the reaction rate constants were determined. A nitrogen yield as high as 0.8 was obtained, corresponding to a hydrogen potential of 88.1 billion m3/year from chicken manure. Consequently, chicken manure was identified as a potentially favorable feedstock for ammonium production.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton–bacteria interactions.

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.

Anaerobic digestion has many excellent functions; it only benefits from energy recovery, but also reduces environmental impact by resource recycling and can be a component in preventing global warming and encouraging carbon recycling. Therefore, a two-phase anaerobic digestion method that divides anaerobic digestion into volatile fatty acids production and gas generation reaction was developed. In this system, pig manure is co-digested with rice straw in a dry anaerobic digestion system. Fluidized bed technology was originally developed for aerobic biochemical oxygen demand removal, nitrification and denitrification at the beginning of the 1970s, and was later introduced to anaerobic treatment processes as anaerobic fluidized bed. The up-flow anaerobic sludge blanket is a high efficiency anaerobic wastewater treatment process developed in the Netherlands in the 1970s. Anaerobic digestion is classified as either wet or dry anaerobic digestion by TS content in the digester.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell’s function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

The activation of microbes, which are needed to initiate continuous methane production, can be accomplished by fed-batch methanization. In the present study, marine sediment inoculum was activated by batch mode methanization with repetition of substrate addition using defined organic matter from sugar, protein, or fat at seawater salinity to investigate the potential for application of the activation method to various types of saline waste and microbial community compositions. All substrates had methane potentials close to the theoretical value except for bovine serum albumin (BSA) whose methane potential was lower, but the maximum methane potential reached the value during repeated methanization. Beta diversity analysis revealed that substrate (especially BSA)-fed and non-fed cultures had distinct microbial community compositions. Bacterial members depended on substrate. Thus, marine sediment inocula activated via the methanization method can be used to effectively treat various types of saline waste.

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

In situ mass spectrometry analysis was conducted for the hydrothermal decomposition of mannitol at different temperatures and residence times in a batch reactor. The temperature was varied in the range of 140–220 °C, and the residence time was varied between 5 and 20 min. The products of mannitol decomposition included furfural, 5-hydroxymethylfurfural, phenol, formic acid, and levulinic acid. At high temperatures, the mass spectrum showed peaks at m/z 46 and 116 that corresponded to formic acid and levulinic acid, respectively. Conventional high-performance liquid chromatography could not detect these acids. Quantification of these products and the rate constants for the mannitol decomposition network were also obtained. The reaction rate coefficients were determined by fitting the experimental results.

This is the first work that describes the application of mass spectrometry to the real-time observation of liquid products of hydrothermal reactions in a continuous reactor. A real-time mass spectrometric analysis (real-time MS analysis) system was developed and designed to elucidate products of glucose decomposition during the hydrothermal treatment process. This was achieved by connecting a continuous-flow reactor with a quadrupole mass analyzer using custom-built connection fittings. The effects of residence time and temperature of hydrothermal treatment on glucose decomposition products were investigated. The temperatures were between 140 and 220 °C, and reaction times were between 5 and 20 min. The real-time MS method investigated the products of glucose decomposition, and cold samples were characterized using high-performance liquid chromatography (HPLC). At 140 °C, glucose decomposition is not complete, which was confirmed by a mass spectrum of a glucose standard. At a temperature of 180 °C or abov...

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.

Simultaneous enzymatic saccharification and comminution (SESC) was used for large-scale anaerobic digestion of wood lignocellulose to generate methane and unmodified lignin. During SESC, 10% aqueous mixture of powdered debarked wood from various species was subjected to bead milling with hydrolytic enzymes to generate particles below 1 μm. This slurry was directly used as a cosubstrate for anaerobic digestion in a 500 L stirred-tank reactor. Temperature and hydraulic retention time (HRT) were maintained at 50 °C and 30 days, respectively. At stable operation periods, an average yield of 224 L of methane per kg of cedar was attained. Comparable yields were achieved with red pine, elm, oak, and cedar bark. High-throughput microbial analysis established the presence of a relevant community to support the elevated level of methane production. The stability of the unmodified lignin in anaerobic digestion was also confirmed, allowing for its recovery as an important by-product.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h

Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 ± 0.06 and 0.40 ± 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars.

Mechanosensitive channels play an important role in the adaptation of cells to hypo-osmotic shock. Among members of this channel family in Escherichia coli, the exact function and physiological role of the mechanosensitive channel homolog YbdG remain unclear. Characterization of YbdG's physiological role has been hampered by its lack of measurable transport activity. Using a nitrosoguanidine mutagenesis-aided screen in combination with next-generation sequencing, here we isolated a mutant with a point mutation in ybdG. This mutation (resulting in a I167T change) conferred sensitivity to high osmotic stress, and the mutant cells differed from WT cells in morphology during hyperosmotic stress at alkaline pH. Interestingly, unlike the cells containing the I167T variant, a null-ybdG mutant did not exhibit this sensitivity and phenotype. Although I167T was located near the putative ion-conducting pore in a transmembrane region of YbdG, no change in ion channel activities of YbdG-I167T was detected. Of note, introduction of the WT C-terminal cytosolic region of YbdG into the I167T variant complemented the osmo-sensitive phenotype. Co-precipitation of proteins interacting with the C-terminal YbdG region led to the isolation of HldD and FbaA, whose overexpression in cells containing the YbdG-I167T variant partially rescued the osmo-sensitive phenotype. This study indicates that YbdG functions as a component of a mechanosensing system that transmits signals triggered by external osmotic changes to intracellular factors. The cellular role of YbdG uncovered here goes beyond its predicted function as an ion or solute transport protein.

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3′-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe

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.

Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein ( mcp ) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l -malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA . While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l -malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l -malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

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 95.1 %, 94.1 % and 92.1 % 16S rRNA gene sequence similarity, respectively. The optimal growth temperature and pH for strain HUA-2T were 35 °C and pH 8.0, 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 ( = DSM 100214T = HUT 8134T).