Ecological impact evaluation by constructing in situ microcosm with porous ceramic arrowhead.

In recent years, bioremediation has been used as an effective technique for the cleaning of polluted sites. However, bioremediation treatment efficacy varies considerably; thus, characterization of indigenous pollutant-degrading soil microorganisms and assessment of the changes in microbial composition by pollutants are essential for designing efficient bioremediation methods. In this study, an ecological impact evaluation method that is cost-efficient and has low contamination risk was developed to assess the indigenous microbial composition. An "in situ microcosm" was constructed using a porous ceramic arrowhead. Phenol, a common environmental pollutant, was used to assess the evaluation efficacy of this method. Our data showed that phenol gradually percolated into the soil adjacent to the arrowhead and stimulated unique indigenous microorganisms (Bacillus sp., Streptomyces sp., and Cupriavidus sp.). Furthermore, the arrowhead approach enabled efficient evaluation of the ecological impact of phenol on soil microorganisms. Thus, the arrowhead method will contribute to the development of bioremediation methods.

Potential use of methane fermentation digested slurry as a low-cost, environmentally-friendly nutrient for bioethanol production from crude glycerol by Klebsiella variicola TB-83D.

A methane fermentation digested slurry (MFDS) was evaluated as a substitute for the commercial nutrient, yeast extract (YE), in ethanol production from glycerol by Klebsiella variicola strain TB-83D. In pH-controlled fed-batch cultures, partial replacement of YE by MFDS did not reduce ethanol productivity significantly. However, non-sterilized MFDS had negative effects on glycerol fermentation by this strain. Although ethanol production decreased when YE was completely replaced by sterilized MFDS, the use of crude glycerol and sterilized MFDS achieved a yield of 14.6 g/L ethanol. This is the first study to report the use of MFDS as the sole nutrient for ethanol production from glycerol, which contributes to the development of a low-cost glycerol biorefinery derived from the biodiesel fuel industry.

Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering.

To improve the ethanol tolerance of the Klebsiella variicola strain TB-83, we obtained the streptomycin-resistant, ethanol-tolerant mutant strain TB-83D by a ribosome engineering approach. Strain TB-83D was able to grow in the presence of 7% (v/v) ethanol and it showed higher ethanol production than strain TB-83. Examination of various culture conditions revealed that yeast extract was essential for ethanol production and bacterial growth. In addition, ethanol production was elevated to 32g/L by the addition of yeast extract; however, ethanol production was inhibited by formate accumulation. With regard to cost reduction, the use of corn steep liquor (CSL) markedly decreased the formate concentration, and 34g/L ethanol was produced by combining yeast extract with CSL. Our study is the first to improve ethanol tolerance and productivity by a ribosome engineering approach, and we found that strain TB-83D is effective for ethanol production from glycerol.

Ethanol production from glycerol-containing biodiesel waste by Klebsiella variicola shows maximum productivity under alkaline conditions.

Biodiesel fuel (BDF) waste contains large amounts of crude glycerol as a by-product, and has a high alkaline pH. With regard to microbial conversion of ethanol from BDF-derived glycerol, bacteria that can produce ethanol at alkaline pH have not been reported to date. Isolation of bacteria that shows maximum productivity under alkaline conditions is essential to effective production of ethanol from BDF-derived glycerol. In this study, we isolated the Klebsiella variicola TB-83 strain, which demonstrated maximum ethanol productivity at alkaline pH. Strain TB-83 showed effective usage of crude glycerol with maximum ethanol production at pH 8.0-9.0, and the culture pH was finally neutralized by formate, a by-product. In addition, the ethanol productivity of strain TB-83 under various culture conditions was investigated. Ethanol production was more efficient with the addition of yeast extract. Strain TB-83 produced 9.8 g/L ethanol (0.86 mol/mol glycerol) from cooking oil-derived BDF waste. Ethanol production from cooking oil-derived BDF waste was higher than that of new frying oil-derived BDF and pure-glycerol. This is the first report to demonstrate that the K. variicola strain TB-83 has the ability to produce ethanol from glycerol at alkaline pH.

Cultivation characteristics of denitrification by thermophilic Geobacillus sp. strain TDN01.

Nine thermophilic denitrification bacteria were isolated from field soil, mud, and spa samples. The alignment of 16S rDNA showed that all were identical to the genus Geobacillus. Two of the bacteria produced N2O and N2 gas and the other seven strains produced N2 gas from nitrate. We examined the growth substrates for Geobacillus TDN01 and determined that sodium succinate, pyruvate, formate, acetate, glycerol, glucose, sucrose, and cellobiose well supported growth of the isolate. Growth occurred under the following concentration of NO3- and phosphate: 10-60 mmol/L, and 0.1-50 mmol/L, respectively. Thermophilic TDN01 grown on sodium succinate accumulated nitrite. A time course of denitrification by Geobacillus TDN01 in a jar fermentor revealed that maintaining a pH of around 7 is important for denitrification without accumulating NO2. The NO3- and NO2- consumption ratios of Geobacillus were 44-75 and 9-41 times higher, respectively, than those of Pseudomonas stutzeri JCM 5965T.

Functional analysis of the thermophilic denitrifying bacterium Geobacillus sp. strain TDN01 in continuous culture.

The thermophilic denitrifying bacterium Geobacillus sp. strain TDN01 was examined to determine the effects of nitrogen and carbon sources and nitrate and nitrite concentrations on denitrification in a batch culture. The specific nitrate removal rate was 12 times higher with ammonia than without ammonia. The consumption rates of nitrate and succinate were proportional. Furthermore, the growth rates with 120 and 150 mM nitrate were only slightly lower than those with 60 mM and did not cause notable growth inhibition. Denitrification ability in continuous culture was analyzed based on the data for batch culture. The maximum specific growth rate micromax and substrate saturation constant KS in the Monod equation were determined by gradually changing the dilution rate. The maximum denitrification rate was six times higher than that of mesophilic bacteria.

Novel plasmid transformation method mediated by chrysotile, sliding friction, and elastic body exposure.

Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 10(7) per microg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.