Abstract There are very few reports about water purification and its influence on indigenous microorganisms by effective microorganisms (EM) in the fields. This study investigated the effect of EM on water purification and microbial community via in vitro and field experiments. In in vitro water purification experiments, we examined values for chemical oxygen demand (COD), total phosphorus, and total nitrogen (TN). In systems in which the active EM solutions were at a concentration of less than 1:2000 of the total water sample, the values after 28 days were equal to or lower than those of the control. In systems using 1:200 and 1:100 active EM solutions, COD and TN values were significantly lower in artificial wastewater compared to control. Conversely, they were significantly higher than the control in the pond water. When the active EM solutions were added to the pond water, total microbial plate counts were higher than the control after 28 days for all concentrations of the EM active solutions. Excessive use of EM caused deterioration of water quality and increase of microbial counts. The results of field experiments for 4 years suggested that EM treatment did not purify the pond water, establish introduced EM, or affect indigenous microorganisms.

ABSTRACT: Hygiene management of domestic refrigerators is an important aspect of food poisoning prevention. The aim of the present study was to confirm the relationship between microbial contamination and hygiene management by measuring microbial levels and investigating temperature and cleaning frequency and method of domestic refrigerators in Japan. We analyzed three internal sections (the egg compartment, bottom shelf, and vegetable drawer) of 100 domestic refrigerators in Japan. Salmonella, Listeria monocytogenes, and Yersinia enterocolitica were not found in any of the refrigerators, but coliforms and Escherichia coli were detected in more than one household, and Staphylococcus aureus was the most frequently isolated pathogen. The prevalences of these microorganisms had similar tendencies in all three sections sampled and were highest in the vegetable drawer. The temperature distribution in the refrigerators was also investigated, and a temperature >6.1°C (improper temperature) was found in 46.2% of the areas surveyed. Only 17% of the respondents cleaned their refrigerators monthly or more often, and this frequency was lower than that reported in other countries. Fifty percent of the respondents used only water to clean the refrigerator, 10% used only an alcohol or disinfecting wipe, and 8% used only a dry cloth. Although no significant correlations were found between microbial contamination and temperatures in refrigerators, correlations were found between microbial contamination and refrigerator cleaning frequency and/or method. To our knowledge, this is the first detailed survey concerning relationships between microbial contamination and hygiene management in domestic refrigerators in Japan. The data obtained can be used to promote food poisoning management in Japanese households.

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvCTM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD+ ratio. On the other hand, introduction of the exogenous Entner-Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.