Sadatoshi Meguro

Integrated delignification and simultaneous saccharification and fermentation of hard wood by a white-rot fungus, Phlebia sp. MG-60.

We propose a new process of unified aerobic delignification and anaerobic saccharification and fermentation of wood by a single microorganism, the white-rot fungus Phlebia sp. MG-60. This fungus is able to selectively degrade lignin under aerobic solid state fermentation conditions, and to produce ethanol directly from delignified oak wood under semi-aerobic liquid culture conditions. After 56 d aerobic incubation, 40.7% of initial lignin and negligible glucan were degraded. Then under semi-aerobic conditions without the addition of cellulase, 43.9% of theoretical maximum ethanol was produced after 20 d. Changing from aerobic conditions (biological delignification pretreatment) to semi-aerobic conditions (saccharification and fermentation) enabled the fermentation of wood by solely biological processes. This is the first report of ethanol production from woody biomass using a single microorganism without addition of chemicals or enzymes.

Direct ethanol production from cellulosic materials by the hypersaline-tolerant white-rot fungus Phlebia sp. MG-60.

White-rot fungus Phlebia sp. MG-60 was identified as a good producer of ethanol from several cellulosic materials containing lignin. When this fungus was cultured with 20 g/L unbleached hardwood kraft pulp (UHKP), 8.4 g/L ethanol was produced after 168 h of incubation giving yields of ethanol of 0.42 g/g UHKP, 71.8% of the theoretical maximum. When this fungus was cultured with waste newspaper, 4.2g/L ethanol was produced after 216 h of incubation giving yields of ethanol of 0.20 g/g newspaper, 51.1% of the theoretical maximum. Glucose, mannose, galactose, fructose and xylose were completely assimilated by Phlebia sp. MG-60 with ethanol yields of 0.44, 0.41, 0.40, 0.41 and 0.33 g/g of sugar respectively. These results indicated that Phlebia sp. MG-60 was a good candidate for bioethanol production from cellulosic materials.

Purification and characterization of polyphenol oxidase from Henry chestnuts (Castanea henryi)

Polyphenol oxidase from Castanea henryi nuts was partially purified. Some characteristics of the enzyme were then determined to help devise measures for the prevention of undesirable enzymatic browning during storage and processing of the nuts. Preparation of acetone powder extracts of the nuts, ammonium sulfate precipitation, dialysis, and gel filtration resulted in 37-fold purification of the enzyme with a yield of 13%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the excitation spectrum confirmed the enzyme preparation to be homogeneous. The approximate molecular weight of the enzyme was determined by gel filtration to be 69 kD. The enzyme catalyzed the oxidation of catechol and pyrogallic acid as substrates but did not affect cresol or tyrosine. Using catechol as substrate, p-nitrophenol, thiourea, orcinol, and naphthol showed strong inhibition. The optimal pH and temperature for the enzyme were pH 5.0 and 40°C, respectively. The enzyme proved heat labile. When the enzyme was incubated at 70°C for 30 min, the remaining activity of the enzyme was only 8%. Possible approaches to applying the results of this study to the prevention of enzymatic browning in the production of C. henryi nuts is also discussed.

Five heavy metals in propagules of ten mangrove species of China

Five heavy metals in the propagules of 10 mangrove species in China have been investigated. The results revealed that the levels of five heavy metals in most of the propagules were lower than the background levels of the soil. The levels of copper, manganese, zinc, cadmium, and lead in the propagules varied at about 2.1–7.8 μg/g, 3.9–28.0 μg/g, 5.7–60.0 μg/g, 0.014–0.057 μg/g, and 0.018–0.038 μg/g, respectively. On average, the levels of five heavy metals were in the order Zn > Mn > Cu > Cd > Pb. The biological absorption coefficients were 0.02–1.30 for copper, manganese, zinc, and cadmium and about 0.0007–0.0020 for lead in propagules, showing the following order: Zn > Cd > Cu > Mn > Pb. The above results indicate that the five heavy metals have low accumulation in most of the propagules of 10 mangrove species and are at safe levels for the effective utilization of mangrove resources.

Effect of chemical factors on integrated fungal fermentation of sugarcane bagasse for ethanol production by a white-rot fungus, Phlebia sp. MG-60

Bioethanol production through integrated fungal fermentation (IFF), involving a unified process for biological delignification with consolidated biological processing by the white-rot fungus Phlebia sp. MG-60, was applied to sugarcane bagasse. Initial moisture content of the bagasse was found to affect biological delignification by MG-60, and 75% moisture content was suitable for selective lignin degradation and subsequent ethanol production. Additives, such as basal media, organic compounds, or minerals, also affected biological delignification of bagasse by MG-60. Basal medium addition improved both delignification and ethanol production. Some inorganic chemical factors, such as Fe(2+), Mn(2+), or Cu(2+), reduced bagasse carbohydrate degradation by MG-60 during delignifying incubations and resulted in increased ethanol production. The present results indicated that suitable culture conditions could significantly improve IFF efficiency.

Coexisting Curtobacterium Bacterium Promotes Growth of White-Rot Fungus Stereum sp.

White-rot basidiomycetes are the main decomposers of woody biomass in forest ecosystems. Little is known, however, about the interactions between white-rot fungi and other microorganisms in decayed wood. A wood-rotting fungus, Stereum sp. strain TN4F, was isolated from a fruit body, and its coexisting cultivable bacteria were isolated from its substrate; natural white-rot decayed wood. The effects of bacteria on fungal growth were examined by confrontational assay in vitro. A growth-promoting bacterium for this Stereum strain was identified as Curtobacterium sp. TN4W-19, using 16SrRNA sequencing. A confrontational assay revealed that Curtobacterium sp. TN4W-19 significantly promoted the mycelial growth of Stereum sp. TN4F in the direction of the bacterial colony, without direct contact between the mycelium and bacterial cells. This is the first report of a positive interaction between a white-rot fungus and a coexisting bacterial strain in vitro.

Cultivation of shiitake in sugi wood meal II: effects of seasoning treatment for wood meal on mycelial growth

The effects of seasoning treatment for fresh sugi wood meal on the mycelial growth of shiitake was investi-gated. The mycelial growth of shiitake in sugi wood meal increased to the same level as that in extract-free sugi wood meal on the 12th day and to approximately 70% of that in konara wood meal on the 28th day of the seasoning treatment. A drastic decrease in extracts of fresh sugi wood meal occurred during the early stage of treatment, and the neutral fraction of methanol extracts decreased to less than 2%. The drainage of sugi wood meal media significantly increased during the later stage of treatment. No changes in the chemical components of sugi wood meal were detected during the seasoning treatment. The mycelial growth of shiitake in the fully seasoned sugi wood meal, which was prepared for commercial mushroom cultivation, was at almost the same level as that in the extract-free sugi wood meal. All of the seasoned sugi wood meals contained a neutral fraction of less than 1%. These results suggested that seasoning treatment can promote mycelial growth of shiitake in sugi wood meal by eliminating inhibitors and improving the physical properties of sugi wood.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60

BackgroundThe white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase.FindingsIn the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder.ConclusionsThis first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.