Complete Genome Sequences of Ralstonia solanacearum Strains Isolated from Zingiberaceae Plants in Japan.

Here, we report the complete genome sequences of three Ralstonia solanacearum strains isolated from Zingiberaceae plants in Japan. The total genome sizes of these strains ranged from 5.87 to 6.05 Mb. Strains MAFF 211472, MAFF 211479, and MAFF 311693 each carried one chromosome and one megaplasmid. MAFF 311693 contained an additional 71.9-kb plasmid.

On-farm solid state simultaneous saccharification and fermentation of whole crop forage rice in wrapped round bale for ethanol production.

BACKGROUND: In an attempt to reduce environmental loading during ethanol production from cellulosic plant biomass, we have previously proposed an on-site solid state fermentation (SSF) method for producing ethanol from whole crops, which at the same time provides cattle feed without producing wastes. During the ensiling of freshly harvested plant biomass with cellulase and glucoamylase, the added yeast and lactic acid bacteria induced simultaneous saccharification and production of ethanol and lactic acid in hermetically sealed containers on-farm. In a previous study, laboratory-scale SSF (using 250 g of fresh rice crop biomass) yielded 16.9 weight % ethanol in dry matter (DM) after 20 days of incubation. In this study, the fermentation volume was scaled up to a normal-sized round bale and the fermentation process (ethanol concentrations of the products) was monitored. The ethanol produced was recovered and the recovery efficiency was evaluated. RESULTS: SSF tests with forage rice round bales using polyethylene-wrapped whole plant materials (cultivar Leaf Star, average of 125.2 kg dry weight) were monitored in the field without temperature control. They yielded 14.0 weight % ethanol and 2.9 weight % lactic acid in DM after six months of incubation, and the ethanol ratio in the bale remained stable for 14 months after processing. SSF tests with three different rice cultivars were conducted for three years. Ethanol recovery from a fermented whole bale (244 kg fresh matter (FM) containing about 12.4 kg ethanol) by one-step distillation using vacuum distillation equipment yielded 86.3% ethanol collected from distilled solution (107 kg of 10.0 weight % ethanol). In addition, an average of 1.65 kg ethanol in 40.8 kg effluent per bale was recovered. Relative nitrogen content was higher in SSF products than in silage made from the same plant material, indicating that fermentation residue, whose quality is stabilized by the lactic acid produced, can be used as cattle feed. CONCLUSIONS: We have successfully demonstrated an efficient on-site ethanol production system with non-sterilized whole rice crop round bale. However, issues concerning the establishment of the ethanol recovery procedure on-site and evaluation of the fermentation residue as cattle feed have to be addressed.

Silage produces biofuel for local consumption.

BACKGROUND: In the normal process of bioethanol production, biomass is transported to integrated large factories for degradation to sugar, fermentation, and recovery of ethanol by distillation. Biomass nutrient loss occurs during preservation and degradation. Our aim was to develop a decentralized ethanol production system appropriate for farm or co-operative level production that uses a solid-state fermentation method for producing bio-ethanol from whole crops, provides cattle feed, and produces no wastes. The idea is to incorporate traditional silage methods with simultaneous saccharification and fermentation. Harvested, fresh biomass is ensiled with biomass-degrading enzymes and yeast. Multiple parallel reactions for biomass degradation and ethanol and lactic acid production are induced in solid culture in hermetically sealed containers at a ranch. After fermentation, ethanol is collected on site from the vapor from heated fermented products. RESULTS: The parallel reactions of simultaneous saccharification and fermentation were induced efficiently in the model fermentation system. In a laboratory-scale feasibility study of the process, 250 g of freshly harvested forage rice with 62% moisture was treated with 0.86 filter paper units/g dry matter (DM) of cellulase and 0.32 U/g DM of glucoamylase. After 20 days of incubation at 28°C, 6.4 wt.% of ethanol in fresh matter (equivalent to 169 g/kg DM) was produced. When the 46 wt.% moisture was gathered as vapor from the fermented product, 74% of the produced ethanol was collected. Organic cellular contents (such as the amylase and pronase degradable fractions) were decreased by 63% and organic cell wall (fiber) content by 7% compared to silage prepared from the same material. CONCLUSIONS: We confirmed that efficient ethanol production is induced in nonsterilized whole rice plants in a laboratory-scale solid-state fermentation system. For practical use of the method, further study is needed to scale-up the fermentation volume, develop an efficient ethanol recovery method, and evaluate the fermentation residue as an actual cattle feed.

DNA Analysis of Ralstonia solanacearum and Related Bacteria Based on 282-bp PCR-Amplified Fragment.

Strains of Ralstonia solanacearum, Pseudomonas syzygii, and the blood disease bacterium (BDB) from different countries were tested for polymerase chain reaction amplification of the 282-bp fragment using the primer pair 759 and 760. These 282-bp fragments from 49 strains of R. solanacearum, six strains of P. syzygii, and two strains of BDB were sequenced. A phylogenetic tree was generated based on the sequence alignment. The R. solanacearum strains were divided into three groups. Group I was composed of strains belonging to biovars 3, 4, 5, and biovar N2 from Japan. Most of the strains from this group were of Asian origin except for two strains from Australia and Guyana (GMI 1000), the type strain. Group II was composed of strains belonging to biovars 1 and 2 and biovar N2 from Brazil. Group III was composed of strains belonging to biovar N2 from Japan and the Philippines. All strains of P. syzygii and BDB clustered in group III. Based on nucleotide differences of the 282-bp fragments, restriction enzyme NlaIII was capable of differentiating R. solanacearum strains into the three groups. Restriction analysis of 165 R. solanacearum isolates from the Philippines using NlaIII showed that all biovar 3 and 4 (group 1) strains had restriction fragments of 116 and 166 bp, strains belonging to biovars 1 and 2 (group 2) showed no restriction, and one strain belonging to biovar 2 (group 3) showed restriction fragments of 54 and 228 bp in size. Thus, NlaIII could be used for rapid differentiation of R. solanacearum strains. Additionally, other restriction enzymes, such as McrI, BsiEI, and MnlI could be used to differentiate R. solanacearum strains from P. syzygii strains.