The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge.

The fungicidal effect of low-temperature plasma generated by positive direct current discharge and its influence on the growth dynamics was evaluated on three micromycete species and yeast in water suspensions. The fungicidal effect was lower than analogous bactericidal effect and differs substantially among various fungal species. Together with the cidal effects, the slower growth of exposed fungal spores was observed.

Biodegradation of methyl tert-butyl ether using bacterial strains.

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen. The biochemical oxygen demand of the cultures was measured using OxiTop, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).

A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model--towards an international standard.

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

Sample processing effect on polymerase chain reaction used for identification of Campylobacter jejuni.

Model samples of Campylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.

Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay.

A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples. Pre-enrichment of samples was combined with filtration through a membrane for the removal of food debris. The IMS-PCR assay combines selective extraction of bacteria by specific antibodies with primer specific PCR amplification that enables to detect Salmonella in non-fatty food samples in 24 h. In comparison with conventional cultural methods, the IMS-PCR is a rapid and specific method. Combined with filtration bags, it partially reduces the negative effects of the food matrix and allows the quick detection of Salmonella cells. The shortened protocols for Salmonella spp. detection described here can improve considerably current methodologies.

Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

Analysis of PCB-degrading bacteria: physiological aspects.

Several aerobic co-cultures capable of co-metabolising polychlorinated biphenyls (PCBs) were acquired by cultivation on biphenyls (BP). The source of micro-organisms was PCB-contaminated soil taken from various sites in the Czech Republic. Several bacterial strains (Gram-negative rods) were isolated, and their capacity to degrade Delor 103 (a PCB mixture containing di- to hexachlorobiphenyls) was analysed. This study was focused on co-culture 319 and isolate 2. The growth parameters of both those cultures were studied on BP; for isolate number 2 the specific growth rate mu = 0.122 (h-1) was calculated. The degradation of the individual congeners was estimated and resulted in more than 50% of the degradation of nearly all congeners during a 2-week experiment. Toxicity of Delor 103 on the vitality of the cells was followed by using viable plate count. The viability of the tested strain was preserved in the 100 times higher Delor 103 concentration compared with conditions in degradation experiments.

Screening and Separation of Microorganisms Degrading PCBs.

We performed an assay to assess the polychlorinated biphenyl (PCB) degradative capability and congener specificity of aerobic microorganisms. Microbial strains were isolated and separated from different types of soils in the Czech Republic, and their PCB-degrading abilities were compared. An industrial mixture of PCB congeners ranging from dichloro- to hexachlorobiphenyl and representing various chlorination patterns was used throughout. The PCB degradative ability of microorganisms was determined by gas chromatography after 7 days of incubation. The degree of degradation was found to depend on the number of chlorine substituents.

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles of Saccharomyces cerevisiae.

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures of Saccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0 degrees C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits features envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.

Turnover of canavanine-containing proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3-4 h after a shift to fresh ethanol-based medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.

Regulation of extracellular proteins and alpha-amylase secretion by temperature in Bacillus subtilis.

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Functional half-life of the alpha-amylase mRNA of Bacillus licheniformis.

Some aspects of the regulation of alpha-amylase synthesis in Bacillus licheniformis CCM 2205 were investigated. The effect of actinomycin D and chloramphenicol was studied at the level of RNA transcription and translation. alpha-amylase synthesis in Bacillus licheniformis CCM 2205 was practically not altered during the first 20 min after the addition of actinomycin D, although RNA synthesis was almost completely blocked. In contrast to RNA polymerase inhibitor, chloramphenicol stopped immediately the synthesis of alpha-amylase. By using the least squares method the mean half-life of alpha-amylase mRNA was calculated to range from 7.5 to 8.4 min. the mean half-life of cell protein mRNA was determined to range from 2.6 to 3.8 min. Having in mind the immediate effect of chloramphenicol on the alpha-amylase synthesis, it can be concluded that de novo protein synthesis is required in the case of actinomycin D resistant residual synthesis.

Effect of temperature on alpha-amylase formation and DNA replication in Bacillus subtilis.

The effect of temperature on extracellular alpha-amylase synthesis and chromosomal and plasmid DNA replication in Bacillus subtilis A18 carrying plasmid pMI10 was studied. The specific growth rate mu increased with elevated temperature up to 42.5 degrees C, while the activities of alpha-amylase per population dry mass decreased. No obvious quantitative changes of 14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40 degrees C were found.

Kinetics of alpha-amylase production in a continuous culture of Bacillus licheniformis.

As found during continuous cultivation of Bacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of alpha-amylase production is proportional to growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose.

Kinetics of alpha-amylase production in a batch and a fed-batch culture of Bacillus subtilis.

Analysis of the kinetics of alpha-amylase production in a batch and a fed-batch culture of Bacillus subtilis made it possible to derive a kinetic model of the process describing mutual interactions between growth and production. The specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing sugars in the medium also inhibit growth. The overall production of alpha-amylase is a result of an equilibrium between the rate of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly proportional to the specific growth rate (characterizing the physiological state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium.

Comparison of growth rates of two clones of Bacillus subtilis, producer of alpha-amylase.

In a population of the productive Bacillus subtilis strain the production of alpha-amylase differentiates at the cell level. Individual cells in the population were analyzed and the production of isolates was tested. The mean specific activity of alpha-amylase in the productive group after the isolation was 5.0 U/mg dry substance and in the nonproductive group 1.5 U/mg dry substance. This ratio remained unchanged during long-term observations and after repeated transfers. In selected strains of both groups the specific growth rate was determined in a synthetic medium containing various amounts of casein hydrolyzate. The detected differences in the growth rate between the productive and the nonproductive clones are determined by amino acid concentration.

Production of extracellular enzymes in continuous culture.

The production of bacterial enzymes in batch fermentations is compared with results obtained in continuous culture. When studying the production of alpha-amylase in Bacillus subtilis it was found that instability of the enzyme synthesis was due to nonhomogeneity of the population rather than to "the culture's history" (i.e. succession of several physiological states necessary for the enzyme production). The plasmid contained in the production clone was found to be the factor responsible for the alpha-amylase production. Predominance of the production clone or of the nonproduction one depends on the cultivation conditions used. As compared with batch cultivation the continuous production yields higher enzyme concentrations under optimal conditions and the fermentor productivity may be four to five times higher.

[Experimental verification of the results of the mathematical modeling of the process of formation of alpha-amylase-nonproductive mutants of Bacillus subtilis under continous cultivation]. / Eksperimental'naia proverka rezul'tatov matematicheskogo modelirovaniia protsessa obrazovaniia na produktivnykh po al'fa-amilaze mutantov Bacillus subtilis v usloviiakh nepreryvnogo kul'tivirovaniia.

When Bacillus subtilis produces alpha-amylase in the course of continuous cultivation, it is difficult to maintain the activity at a constant level. This may be due to the formation of nonproductive mutants. Individual cells in the population have been analysed in the course of the continuous process. The composition of the population changes depending on time and the composition of the growth medium. Semisynthetic media cause selection of mutants which synthesize the enzyme at a low rate. In contrast, complex media which are more enriched in the sources of carbon and nitrogen induce accumulation of mutants with a high activity.