Adaptation of Psychrophilic and Psychrotrophic Sulfate-Reducing Bacteria to Permanently Cold Marine Environments

The potential for sulfate reduction at low temperatures was examined in two different cold marine sediments, Mariager Fjord (Denmark), which is permanently cold (3 to 6(deg)C) but surrounded by seasonally warmer environments, and the Weddell Sea (Antarctica), which is permanently below 0(deg)C. The rates of sulfate reduction were measured by the (sup35)SO(inf4)(sup2-) tracer technique at different experimental temperatures in sediment slurries. In sediment slurries from Mariager Fjord, sulfate reduction showed a mesophilic temperature response which was comparable to that of other temperate environments. In sediment slurries from Antarctica, the metabolic activity of psychrotrophic bacteria was observed with a respiration optimum at 18 to 19(deg)C during short-term incubations. However, over a 1-week incubation, the highest respiration rate was observed at 12.5(deg)C. Growth of the bacterial population at the optimal growth temperature could be an explanation for the low temperature optimum of the measured sulfate reduction. The potential for sulfate reduction was highest at temperatures well above the in situ temperature in all experiments. The results from sediment incubations were compared with those obtained from pure cultures of sulfate-reducing bacteria by using the psychrotrophic strain ltk10 and the mesophilic strain ak30. The psychrotrophic strain reduced sulfate optimally at 28(deg)C in short-term incubations, even though it could not grow at temperatures above 24(deg)C. Furthermore, this strain showed its highest growth yield between 0 and 12(deg)C. In contrast, the mesophilic strain ak30 respired and grew optimally and showed its highest growth yield at 30 to 35(deg)C.     

耐冷好氧亚硝酸盐型反硝化细菌的鉴定及脱氮特性研究

旨在对获得的具有耐冷高效去除亚硝酸盐氮和总氮的好氧反硝化细菌进行分类地位及除氮特性研究。结合形态学观察、特异性磷脂脂肪酸检测和16S r RNA基因序列分析,对实验室新分离获得的Y-9菌株进行鉴定,在此基础上,研究了温度、转速、p H、接种量、碳源和亚硝酸盐氮浓度等不同条件对该菌脱氮能力的影响。结果显示,菌株Y-9属于恶臭假单胞菌(Pseudomonas putida),还原亚硝酸盐氮的最适温度为15℃,最佳溶解氧水平对应转速为100 r/min,在该溶解氧水平下15℃、48 h内对亚硝酸盐氮和总氮的去除率高达100%和77.13%,最佳p H为7,100 m L反硝化培养基中最适接种量为1 m L OD600为0.5的菌悬液,碳源为柠檬酸钠,适合治理低浓度的亚硝酸盐氮污染水体,但对高浓度的亚硝酸盐氮具有一定耐受性。Y-9为一株耐冷反硝化细菌,在亚硝酸盐污染的养殖水体,尤其是冷水鱼类养殖水体中具有较大的应用潜力。     

Identification of a Component of Crystalline Egg Albumin Bactericidal for Thermophilic Aerobic Sporeformers

During an investigation of the effect of basic and acidic proteins on the growth of thermophilic aerobic sporeformers, crystalline egg albumin was found to be strongly bactericidal. This finding was uncharacteristic of acidic proteins. The bactericidal fraction was heat sensitive and separated from the non-bactericidal albumin fraction during gel filtration on Sephadex G-75. Cells of Micrococcus lysodeikticus and Bacillus stearothermophilus were lysed rapidly by the bactericidal component, leading to its tentative identification as lysozyme. The bactericidal substance possessed an electrophoretic mobility on polyacrylamide gel containing sodium dodecyl sulfate identical to that of crystalline egg white lysozyme. Users of crystalline egg albumin are cautioned that commerical preparations may be contaminated with lysozyme. Destruction of the thermophilic aerobes by lysozyme should be considered when performing counts on egg products.     

Incidence Study of Spores of Clostridium botulinum in Convenience Foods

The objective of this study was to gather data on the incidence of Clostridium botulinum spores in selected consumer-convenience food products. The incidence of spores of C. botulinum in 100 samples of each of four categories of commercially available convenience foods was determined. These categories included (i) "boil-in-the-bag" foods, (ii) vacuum-packed foods, (iii) pressurized foods, and (iv) dehydrated and freeze-dried foods. Of the 400 samples analyzed, one was found to contain the spores of C. botulinum. This occurred in vacuum-packed frank-furters and was identified as type B.     

Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10⁶ CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N₂ atmosphere when mean cell densities increased to 10⁶ CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10⁶ CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

Thermoradiation Inactivation of Naturally Occurring Bacterial Spores in Soil

Samples of soil collected from the Kennedy Space Center near the spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilzation techniques. The inactivation kinetics of the naturally occurring spores in soil were investigated by using dry heat and ionizing radiation, first separately and then simultaneously. Dry-heat inactivation kinetics of spores was determined at 105 and 125 C; radiation inactivation kinetics was determined for dose rates of 660 and 76 krads/h at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C, with a dose rate of 76 krads/h. Combined treatment was found to be highly synergistic, requiring greatly reduced radiation doses to accomplish sterilization of the population.     

Immunomagnetic-Electrochemiluminescent Detection of Bacillus anthracis Spores in Soil Matrices

Rapid (<=1.5-h) detection of anthrax spores in soil suspensions was accomplished by an immunomagnetic electrochemiluminescence method. Strain-dependent detection limits in the range of 10(sup2) to 10(sup5) spores were achieved in buffer. The rank order of sensitivity for the assay in buffer was Sterne > Ames > Vollum 1B. Detection was up to 3 orders of magnitude less sensitive in soil suspensions, and the rank order of sensitivity was altered.     

Methyl Anthranilate, an Inhibitor for the Germination of Spores of Aerobic Bacilli

Methylanthranilate inhibited the germination of spores of aerobic bacilli without affecting growth and sporulation. The inhibition of germination could not be reversed by removal of methylanthranilate.     

The Practical Significance in the Microbiological Examination of Cold Stored Foods of the Allegedly Low Heat Resistance among Psychrotrophic Organisms

Summary: The significance in food microbiology of the observation of Stapert, Sokolski & Northam (1962), that some bacteria occurring in water have such low heat resistances that they would be affected by the warm agar used for pouring plates, was tested. Forty-two samples of 6 different foods stored for 7 days at 3° to enrich their psychrotrophic microflora, were examined using the same medium, but in thin poured plates and on spread plates, respectively, incubated at 14°. The logarithmic average count of the foods was c. 10⁸/g and included about 60% Gram-negative rods and 30% cocci. In 90% of the samples no differences were observed between the results from the two methods of counting. The slightly higher spread plate counts in the remaining instances could be accounted for by the increased disruption of bacterial agglomerations always observed in this procedure. Hence there is no reason to query the results of poured plate counts in general. Where it is yet probably safer to use spread plates, rigorous asepsis in pouring and storing should be observed to prevent the development of colonies of contaminants too small to be detected when the plates are inoculated, but leading to erroneous counts after incubation.     

The Occurrence of Bacillus cereus in some Dried Foods Including Pulses and Cereals

In a survey of 39 dried food samples which represented 12 different pulses and cereals. 22 (56%) were found to be contaminated with Bacillus cereus. The numbers varied between 1 times 102 and 6 times 10⁴ organisms/g. During normal cooking procedures and on storage at room temperature, the B. cereus resident on red lentils and kidney beans increased to a level at which enterotoxin production could become significant. Some physiological characteristics including deoxyribonuc-lease (DNase) and ribonuclease (RNase) secretion and salicin fermentation did not correlate with the ability or otherwise of a strain to cause food poisoning. Nevertheless, serotype 8 strains were prevalent on these foods and these have been implicated in both the diarrhoeal and vomiting-type food poisoning syndromes.     

Occurrence of Bacteriochlorophyll a in a Strain of an Aerobic Heterotrophic Bacterium

We developed a practical preparation procedure for K-252a by methylating K-252b on an industrial scale. The water-insoluble K-252a, which was present in the cell mass, was converted to the water-soluble K-252b Na salt in an alkaline solution. The obtained K-252b was methylated with dimethylsulfate in the presence of potassium carbonate in dimethylacetamide. We have already used this method to manufacture 90 kg of K-252b from the fermentation broth, and regenerated 65 kg of K-252a from K-252b.     

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5°C but exhibited a 1,000-fold decrease after 1 week of incubation at 27°C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27°C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.     

Microbial Transformation of 2,4,6-Trinitrotoluene in Aerobic Soil Columns

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.