Manabu Kanno

Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance

ABSTRACT Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

Discovery of Glycoside Hydrolase Enzymes in an Avicel-Adapted Forest Soil Fungal Community by a Metatranscriptomic Approach

To discover the structural and functional novel glycoside hydrolase enzymes from soil fungal communities that decompose cellulosic biomass, transcripts of functional genes in a forest soil were analyzed. Pyrosequencing of the Avicel and wheat-amended soil cDNAs produced 56,084 putative protein-coding sequence (CDS) fragments, and the most dominant group of putative CDSs based on the taxonomic analysis was assigned to the domain Eukarya, which accounted for 99% of the total number of the putative CDSs. Of 9,449 eukaryotic CDSs whose functions could be categorized, approximately 40% of the putative CDSs corresponded to metabolism-related genes, including genes involved in carbohydrate, amino acid, and energy metabolism. Among the carbohydrate-metabolism genes, 129 sequences encoded glycoside hydrolase enzymes, with 47 sequences being putative cellulases belonging to 13 GH families. To characterize the function of glycoside hydrolase enzymes, we synthesized the putative CelA gene with codon optimization for heterologous expression in Escherichia coli, which was shown to be similar to the structure of plant expansins, and observed stimulation for cellulase activity on Avicel degradation. This study demonstrated that fungal communities adapt to Avicel and wheat decomposition and that metatranscriptomic sequence data can be reference data for identifying a novel gene.

An Oleaginous Bacterium That Intrinsically Accumulates Long-Chain Free Fatty Acids in its Cytoplasm

ABSTRACT Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

Catenisphaera adipataccumulans gen. nov., sp. nov., a member of the family Erysipelotrichaceae isolated from an anaerobic digester.

An obligately anaerobic bacterium, designated strain GK12(T), was isolated from an anaerobic digester in Fukagawa, Hokkaido Prefecture, Japan. The cells of strain GK12(T) were non-motile, non-spore-forming cocci that commonly occurred in chains. 16S rRNA gene sequence analysis revealed that strain GK12(T) was affiliated with the family Erysipelotrichaceae in the phylum Firmicutes and showed 91.8 % sequence similarity to the most closely related species, Faecalicoccus acidiformans. The strain grew at 30-50 °C (optimally at 40 °C) and at pH 5.5-8.5 (optimally at pH 7.5). The main end product of glucose fermentation was lactate. Yeast extract was required for growth. The strain contained C14 : 0, C14 : 0 1,1-dimethoxyalkane (DMA), C16 : 0 DMA and C18 : 0 DMA as the major cellular fatty acids (>10 % of the total). The polar lipid profile was composed of phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The whole-cell sugars were galactose, rhamnose and ribose. The cell-wall murein contained alanine, glutamic acid, lysine, serine and threonine, but not diaminopimelic acid. The G+C content of the genomic DNA was 47.7 mol%. Based on phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Catenisphaera adipataccumulans gen. nov., sp. nov., is proposed to accommodate strain GK12(T) ( = NBRC 108915(T) = DSM 25799(T)).

Detection and isolation of plant-associated bacteria scavenging atmospheric molecular hydrogen

High-affinity hydrogen (H2 )-oxidizing bacteria possessing group 5 [NiFe]-hydrogenase genes are important contributors to atmospheric H2 uptake in soil environments. Although previous studies reported the occurrence of a significant H2 uptake activity in vegetation, there has been no report on the identification and diversity of the responsible microorganisms. Here, we show the existence of plant-associated bacteria with the ability to consume atmospheric H2 that may be a potential energy source required for their persistence in plants. Detection of the gene hhyL - encoding the large subunit of group 5 [NiFe]-hydrogenase - in plant tissues showed that plant-associated high-affinity H2 -oxidizing bacteria are widely distributed in herbaceous plants. Among a collection of 145 endophytic isolates, seven Streptomyces strains were shown to possess hhyL gene and exhibit high- or intermediate-affinity H2 uptake activity. Inoculation of Arabidopsis thaliana (thale cress) and Oryza sativa (rice) seedlings with selected isolates resulted in an internalization of the bacteria in plant tissues. H2 uptake activity per bacterial cells was comparable between plant and soil, demonstrating that both environments are favourable for the H2 uptake activity of streptomycetes. This study first demonstrated the occurrence of plant-associated high-affinity H2 -oxidizing bacteria and proposed their potential contribution as atmospheric H2 sink.

Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion

Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar‐derived LA solution (40–160 g L−1) was demonstrated. When using reagent LA solution with pH5.0–6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg−1. With cedar‐derived LA solution (pH6.0), good agreement in levulinate recovery (88–99%), and energy consumption (0.18–0.22 kWh kg−1) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar‐derived LA. From 0.5 mol L−1 of the ED‐concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L−1 LA solutions (40–160 g L−1).

Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate.

ABSTRACT Burkholderia stabilis LA20W produces trehalose using levulinic acid (LA) as a substrate. Here, we report the 7.97-Mb draft genome sequence of B. stabilis LA20W, which will be useful in investigations of the enzymes involved in LA metabolism and the mechanism of LA-induced trehalose production.