Bacterial leaching of zinc from flotation tailings of the Zawar Mines Plant, Udaipur, India was investigated in the laboratory using Thiobacillusferrooxidans in batch experiments. With pH maintained in the range of 3 to 4, zinc extraction was 70-78% in 24-28 days at 5% pulp density. Zinc extraction due to bacterial leaching was about 60% as much as with combined chemical and bacterial leaching. Settled raw sewage appeared to be a good source of nutrients (nitrogen and phosphorous) for bacterial leaching and a 2:1 sewage-tap water mixture at pH 4 produced 65% zinc extraction in 28 days.
Aluminum-tolerant (adapted) strains Alr-1, Alr-2, Alr-3 and Alr-4 of Azospirillum brasilense RAU 3051 (from an acid soil) were isolated through step-wise transfer to higher levels of aluminum. One of the strains (Alr-3) showed more growth and greater nitrogenase activity at low pH and in 100μg AlCl3ml-1 than parental and other Al-tolerant strains. These tolerant strains also showed cross-resistant to neomycin but not to streptomycin except Alr-1. Aluminum-adapted strains also tolerated elevated levels of manganese (75-130μg MnCl2ml-1). It has been demonstrated that the response of inoculation with Al-adapted strains was variable with different genotypes of finger millet in an acid soil. An initial low level of nitrogen was essential for maximum associative N2 fixation and grain and straw yield. Also it appears that acid-adapted strains may be most suitable for nitrogen economy in acid soils having various pH and associated factors of acidity.
A mixture of 11 kinds of thermophilic bacilli was cultured on a medium containing municipal sewage sludge and filter paper. Two types of thermophilic cellulases appeared in the culture liquid. Both had an optimum temperature of 60°C. One is a neutral cellulase with an optimum pH of 7.5 and the other is an alkali cellulase with an optimum pH at 10.0. After exposure to pH 12 at 4°C for 24h, the alkali cellulase still retained 90% of its activity. The alkali cellulase activity was stimulated by 1mM Zn2+, but was considerably inhibited by 1mM Cu2+ or 1mM Hg2+. Thus, the alkali cellulase was specified as a new type of thermophilic cellulase having strong alkali tolerance.
Two strains, Fe1 and Fe2, of iron-oxidizing bacteria from acid drainage water at the Matsuo mine showed dissimilar characteristics of red-brown colonies on FeSO4-9K silica gel plates. The Fe1 colonies were large and irregular. The Fe2 colonies were minute and circular. These bacteria, identified as Thiobacillus ferrooxidans on the basis of their physiological and chemotaxonomical properties (fatty acid composition, ubiquinone type, and DNA base composition), were different strains. Fe2 differed from Fe1 in its extremely low oxidizing activity of sulfur and thiosulfate. Two strains, S2 and S3, of sulfur-oxidizing bacteria were separately isolated on Na2S2O3-9K silica gel plates and agar plates, respectively. However, S2 and S3 formed pale brown and pale yellow colonies on the silica gel and agar plates, respectively. Since these two strains were identical in their properties, they were judged to be quite similar or the same strain. From their morphological, physiological, and chemotaxonomical properties, they were identified as Thiobacillus thiooxidans. The oxidation characteristics of both sulfur and thiosulfate by the strains of iron- and sulfur-oxidizing bacteria were also investigated.
Carbohydrae complex antigen, which was common among the nonproteolytic Clostridium botulinium, was purified from the hot-formamide extract of the cell wall by means of a sequence of ionexchange and gel filtration chromatography. The antigen consisted of glucose, glucosamine, galactosamine, D-alanine, and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. Neither ribitiol nor glycerol appeared. In quantitative precipitin inhibition tests, α-methyl glucoside and β-methyl glucoside gave 85% and 70% inhibition respectively at an equivalence point of the antigen-antibody reaction. D-Alanine or glucosamine did not inhibit the reaction. Partial acid hydrolysis of the antigen, by which about a half of the glucose moiety was eliminated, resulted in a complete loss of the antigenic activity.
The quinone composition of 10 sludge samples consisting of plant-derived activated sludges, laboratory activated sludges, and photosynthetic sludges was analyzed by high-performance liquid chromatography, thin-layer chromatography, and mass spectrometry. The quinone profiles of these sludges showed taxonomic divergence among the sludge bacterial communities. The information could be enhanced by numerical analysis of the profiles using the ‘overlap’ coefficient and mean linkage algorithm. This analytical system provided quantitative information about the population shift in the indigenous bacterial flora of sludge as affected by physico-chemical or biological stress. The quinone profile method with numerical analysis is quite useful for investigating the bacterial community dynamics over time and space, without the necessity of culturing.
To develop an enzymatic process for L-phenylalanine production through transaminase reaction, we selected from our bacterial culture collection a Citrobacter freuindii strain as an excellent producer of the enzyme. The cells of this bacterium rapidly and efficiently formed L-phenylalanine from phenylpyruvic acid and an amino donor amino acid. With L-glutamic acid as the amino donor, L-phenylalanine production reached 30mg/ml in 1h and 89mg/ml in 24h incubation. The conversion yield to form L-phenylalanine from phenylpyruvic acid was 50 to 80%. With L-aspartic acid as amino donor, the rate of L-phenylalanine production is lower (i.e. 8mg/ml in 1h and 30mg/ml in 4h) but the conversion yield was much higher (80 to 100%). With L-aspartic acid as the amino donor, oxalacetic acid, a putative reaction product, appeared in small amounts. Instead, a much larger amount of pyruvic acid, a well-known decarboxylation product of oxalacetic acid, appeared. From these facts it was concluded that the reaction, which should be reversible, inclines almost completely toward L-phenylalanine formation when L-aspartic acid is used as the amino donor, because oxalacetic acid (the reaction product and also the substrate for the reverse reaction) is eliminated through decarboxylation to form pyruvic acid and other metabolites. The Citrobacter strain had a high aspartase activity to form L-aspartic acid from fumaric acid and ammonia. Coupling the two activities of the transaminase and the aspartase, an enzymatic process for L-phenylalanine production from phenylpyruvic acid and ammonium fumarate was established.
A sensitive method using selective plating media to detect a genetically engineered microorganism (GEM) released into the environment was developed. In experiments, a strain of Pseudomonas sp. capable of assimilating monofluoroacetate (FA) as a sole carbon source was chosen as the GEM model. Two sorts of Japanese paddy soil were used as a microcosm. In a trial when serial 10-fold soil dilutions were spread on minimal FA agar plates and incubated, native oligotrophic microorganisms (105-106CFU/g of dry weight of soil) grew on the plates. So, it was difficult to pick out a few populations of target bacteria from such a high number of background microbial communities. A further examination showed that those indigenous microorganisms colonized even on plates containing only water and agar. For this reason, we tested silica-gel instead of agar as an agent to solidify the selective media. On minimal FA silica-gel plates, the number of indigenous oligotrophic colonies was greatly decreased. When a definite number of target Pseudomonas cells was mixed with the soil dilutions and cultured on minimal FA silica-gel plates, only the target colonies were appeared. We applied the most-probable-number technique to the plate-counting method to attain as low as 4CFU/g of dry weight of soil as the statistical limit of detecting FA-assimilating bacteria. This selective plating method is suitable for environmental monitoring of GEMs which assimilate FA aerobically, as it is on highly sensitive, specific and feasible.
Proteins and slime substances were extracted from the membrane fraction of the actively growing plasmodia of Physarum polycephalum, and analyzed by SDS-PAGE. A glycoprotein with a molecular weight of 120kD had a very high affinity for concanavalin A. The synthesis of this glycoprotein was greatly inhibited by tunicamycin. When the culture entered the stationary phase, the plasmodia produced large amounts of the slime on their surface. In the shaken culture, most of the slime substances were released from the plasmodial surface together with the 120kD glycoproteins. In this growth phase, some gelatinolytic, glycosidase and hemagglutinating activities were also released into the medium. Some of these had appeared in the membrane fraction of growing plasmodia. The results suggest that these biologically active molecules are associated with the plasmodial membrane together with slime substances.
Minimum water activities (aw) for the growth of 35 yeast strains isolated from high-sugar foods and related materials were determined by incubating in broths containing 1% glucose, 0.5% polypeptone, 0.3% yeast ext., 0.3% malt ext. and aw-controlling solutes at 25°C for up to 120 days. Three kinds of sugars (glucose, fructose, sucrose) and sodium chloride were used as aw-controlling solutes. The minimum aw for growth of the yeasts depended on aw-controlling solutes as well as yeast species and strains. Most species showed the highest minimum aw for growth in NaCl-media and about half of the species showed the lowest minimum aw for growth in sucrose-media. One strain of Zygosaccharomyces rouxii had minimum aw for growth as low as 0.67 in fructose-media. Miso ext. and koji ext., rich in inositol, and casamino acid decreased minimum aw for growth of Z. rouxii in NaCl-media. Pre-incubation in the presence of high concentrations of glucose or fructose also decreased minimum aw for growth of yeasts in glucose-media or fructose-media.
In batch culture, addition of acetate stimulated the initial growth of Megasphaera elsdenii in medium containing glucose and Trypticase. As the initial concentration of acetate was increased, butyrate production increased. When acetate was used, the rate of butyrate production increased. Hydrogen production was decreased by adding acetate. Inhibition of hydrogen production by the hydrogenase inhibitor carbon monoxide inhibited the growth of M. elsdenii, but this inhibition was cancelled by adding acetate. In continuous culture, where the growth rate was kept relatively low by limiting the glucose supply, adding acetate similarly increased butyrate production. These results suggest that acetate serves as an electron sink in M. elsdenii.