Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Identification of some chromosomes of the brewing yeast Saccharomyces uvarum

Abstract Chromosomal DNA molecules of Saccharomyces uvarum and Saccharomyces cerevisiae were separated using Orthogonal Field Alteration Gel Electrophoresis (OFAGE). Hybridization with specific probes of S. cerevisiae chromosomes allowed the identification of seven chromosomes of S. uvarum . The majority of the studied chromosomal DNA molecules show the same OFAGE mobility as the corresponding molecules of S. cerevisiae , with some minor differences.
Hybridizations with two distinct bands of S. uvarum were observed with each URA1 (marker of chromosome XI) and ARG80 (marker of chromosome XIII) probes, demonstrating the presence of at least two copies of these genes in the brewing yeast.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Isolation and characterization of a methyl chloride utilizing,strictly anaerobic bacterium

From sludge obtained from the sewage digester plant in Stuttgart-Möhringen a strictly anaerobic bacterium was enriched and isolated with methyl chloride as the energy source. The isolate, which was tentatively called strain MC, was nonmotile, gram-positive, and occurred as elongated cocci arranged in chains. Cells of strain MC formed about 3 mol of acetate per 4 mol of CH₃Cl consumed, indicating that the organism was a homoacetogenic bacterium fermenting methyl chloride plus CO₂ according to: The organism grew with 2–3% methyl chloride in the gas phase at a doubling time of near 30 h. Dichloromethane was not utilized. The bacterium also grew on carbon monoxide, H₂ plus CO₂, and methoxylated aromatic compounds. Optimal growth with methyl chloride was observed at 25°C and pH 7.3–7.7. The G+C-content of the DNA was 47.5±1.5%. The methyl chloride conversion appeared to be inducible, since H₂ plus CO₂-grown cells lacked this ability. From the morphological and physiological characteristics, the isolate could not be affiliated to a known species.     

我国十省(市)十字花科蔬菜芜菁花叶病毒(TuMV)株系分化研究 Ⅰ.用Green氏方法划分株系

Provvidenti,R.(1980)和Green,S.K.(1985)先后用同一套鉴别寄主谱,研究了美国和我国台湾省的TuMV株系分化情况。本文用Green的方法,对由十省(市)7,982份病样中筛选出的19个TuMV主流分离物,在同一条件下进行了鉴定。结果表明,属于C_1株系的有黑_3分离物;属于C_4株系的有京_2、京_3、冀_2、宁_1(南京)、粤_1和川_1共6个分离物;属于C_5株系的有黑_1、辽_1、京_1、冀_1、沪_1、鲁_1、鲁_2共7个分离物。未检出C_2和C_3株系。黑_2、沪_2、宁_2和秦_1(陕西)共4个性状相近的分离物和冀_3分离物尚不能按Green的标准归类,暂分别定为C_(3-2)和C_6株系。此外,对Green氏方法应用于我国十字花科蔬菜TuMV株系分化研究中存在的问题作了讨论。     

Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains

Gram-positive, non-spore-forming, non-acid-fast, rod-shaped aerobic bacteria with the ability to desulfurize dibenzothiophene (DBT) or dibenzosulfone (DBTO₂) were isolated from soil samples contaminated with fossil fuels. Using a bioavailability method, cells with the desired DbtS⁺ phenotype were enriched. Modified fluorescence and colorimetric assays were used for the initial detection of 2-hydroxybiphenyl (OH-BP) in microtiter plates; subsequently, isolates were grown in wells of microtiter plates and screened for the production of desulfurization product. Fluorescence under UV light and the production of colored product in the phenol assay were used as presumptive indications of production of OH-BP. Confirmation of the presence of OH-BP was achieved with HPLC, UV-absorbance, and mass spectrometry. Nutrient utilization and fatty acid composition (as discerned with Biolog plates and gas chromatography, respectively) were used to identify presumptively the strains as Rhodococcus erythropolis; colony and cell morphology may not be consistent with the identification achieved by nutrient utilization and fatty acid composition. The desulfurization end product, OH-BP, can not be used as carbon source by the tested strain, N1-36.     

Microbes in food processing technology

Abstract: There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-haled methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.     

Methyl chloride metabolism of the strictly anaerobic,methyl chloride-utilizing homoacetogen strain MC

The methyl chloride metabolism of the homoacetogenic, methyl chloride-utilizing strain MC was investigated with cell extracts and cell suspensions of the organism. Cell extracts were found to contain all enzyme activities required for the conversion of methyl chloride or of H₂ plus CO₂ to acetate. They catalyzed the dechlorination of methyl chloride with tetrahydrofolate as the methyl acceptor at a rate of 20 nmol/min × mg of cell protein. Also, the O-demethylation of vanillate with tetrahydrofolate could be measured at a rate of 40 nmol/min × mg. Different enzyme systems appeared to be responsible for the dehalogenation of CH₃Cl and for the O-demethylation of methoxylated aromatic compounds, since cells grown with methoxylated aromatic compounds exhibited a significantly lower activity of CH₃Cl conversion than methyl chloride grown cells and vice versa. In addition, ammonium thiocyanate (5 mM) completely inhibited CH₃Cl dechlorination, whereas the consumption of vanillate was not affected significantly. The data were taken to indicate, that the methyl chloride dehalogenation is catalyzed by a specific, inducible enzyme present in strain MC, and that tetrahydrofolate rather than the corrinoid-protein involved in acetate formation is the primary acceptor of the methyl group in the dechlorination reaction.     

Use of Local Raw Materials for the Production of Bacillus sphaericus Insecticide in Ghana

Locally available raw materials were used as fermentation media for the preparation of an effective bacterial insecticide of Bacillus sphaericus in Ghana. The choice of materials for media production was based on their availability, their cost and how well they supported growth and sporulation of the bacterium. The materials used were anchovy (Engraulis encrasicolus), spent grain from breweries, bambara beans (Vigna subterranea), sprout maize (Zea mays) and B. sphaericus strain IAB 881. The larvicidal activities of each final whole culture of the insecticides prepared were evaluated against third and fourth instar larvae of the mosquito species Culex quinquefasciatus. The levels of larvicidal activity exhibited by preparations using the raw materials were similar to those where the bacterium was cultured in a synthetic medium, with median lethal concentrations ranging from 0.30 10- 5 to 0.68 10-6. Cell counts were in the range 11 108 - 36 108 colony-forming units ml-1, and spore counts were between 29 107 and 61 107 ml-1.     

Ploidy and strain differences in seed germination of Glycine wightii at different pH levels

Summary Seeds from 27 wild strains (18 tetraploids and 9 diploids) of Glycine weightii were germinated at a pH range of 5 to 8. The differences in germination (%) between all the strains were highly significant but between pH levels they were only nearly significant (P=0.067) with no interaction between pH levels and strains. Mean germination (%) for all tetraploids seems to be slightly higher ( 2%) than that for all diploids, especially at pH's 5, 7 and 8 but this may be due to the significantly longer time ( one day) it took tetraploids to complete germination. The apparent inverse relationship between seed weight and germination (%) was not significant.Mean germination time was highly significant for strains, pH's and their interaction. Increasing mean germination (%) resulted in decreasing mean germination time among strains. Large seeds took less time to germinate especially those from some of the tetraploid strains. This indicates that it is possible to produce a variety with high germination (%), fast germination rate and possibly large seeds. If the marked difference in pH tolerance among strains will prove to be mainly hereditary, then it will be also possible to select for either specific pH tolerance or tolerance at a wide range of pH.