Tomonori Sonoki

Chemical and biochemical characterisation of biochar-blended composts prepared from poultry manure

The aim of this study was to assess the effect of a 2% (v/v) addition of biochar on the quality of a composting mixture prepared with poultry manure and different local organic wastes (rice husk and apple pomace). Compost quality was evaluated in terms of typical stabilisation indices, the microbial biomass and selected enzymatic activities related to the C, N and P cycles. The main effects of biochar were a 10% increase in C captured by humic substance extraction and a 30% decrease of water-soluble C, due to an enhanced degradation rate and/or the sorption of these labile compounds into the biochar. The urease, phosphatase and polyphenol oxidase activities of the biochar-blended compost were enhanced by 30-40% despite the lower amount of microbial biomass. Denaturing gradient gel electrophoresis revealed a higher diversity of fungi in biochar-amended compost, suggesting a change in microbial composition compared to the unamended compost.

Biochar influences the microbial community structure during manure composting with agricultural wastes.

The influence of biochar derived from a hardwood tree (Quercus serrate Murray) on the dynamics of the microbial community during the composting of poultry manure (PM) and cow manure (CM) was evaluated by phospholipid fatty acid analysis (PLFAs). Changes in the PLFA patterns were related to key composting properties (C/N ratio, temperature, and bulk density) as the major drivers of the dynamics of the microbial community. At the beginning of the process, the fungal biomass was significantly greater in PM and CM than in the respective co-composted materials with biochar (PMB and CMB); this difference declined gradually during the process. In contrast, the Gram+ to Gram- ratio was increased by the presence of biochar. After 12 weeks of composting, factor analysis based on the relative abundances of single PLFAs revealed changes in the microbial community structure which depended on the original organic wastes (CM vs PM).

A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.

Coexistence of Two Different O Demethylation Systems in Lignin Metabolism by Sphingomonas paucimobilis SYK-6: Cloning and Sequencing of the Lignin Biphenyl-Specific O-Demethylase (LigX) Gene

ABSTRACT Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5,5′-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designatedligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilisSYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.

Influence of biochar addition on methane metabolism during thermophilic phase of composting.

CH4 is known to be generated during the most active phase of composting, even in well‐managed composting piles. In this manuscript, we studied the influence of biochar on the CH4 metabolism during composting of cattle manure and local organic wastes. We evaluated the presence of methanogens and methanotrophs in the composting piles quantified by the level of mcrA encoding methyl coenzyme M reductase alpha subunit and pmoA encoding particulate methane monooxygenase. A decrease of methanogens (mcrA) and an increase of methanotrophs (pmoA) were measured in the composting mixture containing biochar during the most active phase of composting. During the thermophilic phase, the mcrA/pmoA ratios obtained in the composting piles with biochar were twofold lower than in the pile without biochar.

Influence of biochar addition on the humic substances of composting manures

Application of biochar (10% v/v) to a manure composting matrix was investigated to evaluate its effect on the chemical composition of humic substances during the composting process. The characteristics of the humic acid (HA) and fulvic acid (FA) fractions were analyzed in compost mixtures originating from two different manures (poultry manure (PM) and cow manure (CM)). The C contents of HA and FA from the manure compost/biochar blends (PM+B and CM+B) were higher than those from PM and CM, with an enhanced recalcitrant fraction, as determined by thermogravimetric analysis. Spectroscopic analysis showed that enrichment of aromatic-C and carboxylic-C occurred in the FA fractions of PM+B and CM+B to a greater extent than in PM and CM. Biochar addition into the composting mixture improved the final compost quality, especially for the light humified fraction (FA).

Enhanced saccharification of rice straw by overexpression of rice exo-glucanase

BackgroundEfficient production of carbon-neutral biofuels is key to resolving global warming and exhaustion of fossil fuels. Cellulose, which is the most abundant biomass, is physically strong and biochemically stable, and these characteristics lead to difficulty of efficient saccharification of cellulosic compounds for production of fermentable glucose and other sugars.ResultsWe transformed rice with overexpressing constructs of rice genes encoding each of three classes of cellulases. The exo-glucanase overexpressing plants showed various abnormalities in leaf such as division of leaf blade, crack on leaf surface, excess lacunae in midrib structure and necrotic colour change. The overexpressing plants also showed sterility. Noticeably, these plants showed enhanced saccharification of stems after maturation. These results indicate that overexpression of the exo-glucanase gene brought about various developmental defects associated with modification of cell wall and enhanced saccharification in rice. On the other hand, endo-glucanase-overexpressing plants could not be obtained, and overexpression of β-glucosidase brought about no effect on plant growth and development.ConclusionsOur results indicate that genetic engineering of cellulosic biomass plants by overexpressing cellulase genes will be one of the approaches to confer enhanced saccharification ability for efficient production of cellulosic biofuels such as ethanol.

Effective Removal of Bisphenol a from Contaminated Areas by Recombinant Plant Producing Lignin Peroxidase

We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. Lignin peroxidase is a well-known enzyme used for the degradation of some environmental pollutants. In the present study, we introduced an extracellular fungal enzyme, the lignin peroxidase of Trametes versicolor , into tobacco plants. Six transgenic plant, designated FLP-1, 2, 3, 4, 5 and 8, produced lignin peroxidase in a crude extract of the root. The FLP-1, FLP-2 and FLP-8 were able to remove 10μmol of bisphenol A g -1 dry weight from hydroponic culture. The efficiency of this removal was approximately 4-fold greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.

Hybrid aspen with a transgene for fungal manganese peroxidase is a potential contributor to phytoremediation of the environment contaminated with bisphenol A

To assess the possible utility of a fungal gene for manganese-dependent peroxidase (MnP) produced by a transgenic plant in phytoremediation, we transformed hybrid aspen with a chimeric gene for MnP. Our gene construct allowed expression of the gene for MnP in plants and relatively high MnP activity was detected in the hydroponic medium in which roots of plants that expressed the transgene had been cultured. Some of our transgenic plants were able to remove bisphenol A from the medium more efficiently than wild-type plants. Our results demonstrate that, without any modification of the coding sequence, a chimeric gene for fungal MnP can be expressed in a woody plant, with secretion of active MnP from roots into the rhizosphere. Our strategy suggests new options using woody plants for phytoremediation.

Specific degradation of β-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes of Sphingomonas paucimobilis SYK-6 produced in recombinant Escherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such asβ-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol-β-aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleavedβ-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated theβ-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result ofβ-aryl ether cleavage of αO-methylumbelliferyl-β-hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report thatβ-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave theβ-aryl ether linkage in synthetic high-molecular-weight lignin.