A plate method for screening of bacteria capable of degrading aliphatic nitriles

A novel indicator plate method was developed for screening of aliphatic-nitrile-degrading bacteria. Isolated bacteria were tested for utilization of acetonitrile as sole source of carbon and nitrogen with the release of ammonia. The released ammonia causes increase of the pH of the medium. Phenol red indicator is used for detection of ammonia based on colour change of the indicator dye from red to pink. The liberation of ammonia from aliphatic-nitrile-utilizing bacteria is also studied in plates containing other indicators such as bromothymol blue and phenolphthalein. The usefulness of the indicator plate is demonstrated for bacteria that degrade certain aliphatic nitriles. Bacteria degrading nitriles as a nitrogen source can also be isolated with a medium containing additional carbon source. This plate method would be useful in isolation and screening of bacteria for degradation of aliphatic nitriles and also for production of nitrile-hydrolyzing enzymes.     

Bacterial bioassay of microgram to subnanogram quantities of glucose

A simple and highly sensitive bioassay for the measurement of glucose is described. The method is based on the fact that the yields of cultured bacteria depend on the amount of glucose input, which is a limiting factor in Davis minimal medium. The number of cells cultured under fixed conditions was shown to be dependent on the amount of glucose in the culture medium. Therefore, the concentration of glucose can be measured by counting the cell number after culturing the bacteria. With Davis medium depleted of sodium citrate, we used this method to detect a subnanogram level of glucose, although with the accuracy of order of magnitude estimation. In the microgram per milliliter range of glucose, the cell number can be measured optically, with proliferation being proportional to the input glucose. The high selectivity for glucose of this method is based on the preferential usage of glucose as a carbon and energy source by the bacteria adopted. The feasibility of applying this method to other substances is discussed.     

Bacteria of the genus Acinetobacter. Methods for their isolation and primary identification

A liquid accumulation medium and a solid isolation medium for primary identification of bacteria of the genus Acinetobacter have been proposed. Both media have identical composition and contain ethanol as the only source of carbon and energy and sodium ammonium phosphate as the source of nitrogen. Besides, the modification of Baumann's medium with sodium acetate as the source of carbon and potassium nitrate as the source of nitrogen has been developed. This modified medium has simplified composition and is not inferior to the above-mentioned media in selectivity, though its use gives less satisfactory results. The two proposed media, both liquid and solid, are recommended for wide use.     

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

Purple Sulfur Bacteria Control the Growth of Aerobic Heterotrophic Bacterioplankton in a Meromictic Salt Lake

In meromictic Mahoney Lake, British Columbia, Canada, the heterotrophic bacterial production in the mixolimnion exceeded concomitant primary production by a factor of 7. Bacterial growth rates were correlated neither to primary production nor to the amount of chlorophyll a. Both results indicate an uncoupling of bacteria and phytoplankton. In the chemocline of the lake, an extremely dense population of the purple sulfur bacterium Amoebobacter purpureus is present year round. We investigated whether anoxygenic phototrophs are significant for the growth of aerobic bacterioplankton in the overlaying water. Bacterial growth rates in the mixolimnion were limited by inorganic phosphorus or nitrogen most of the time, and the biomass of heterotrophic bacteria did not increase until, in autumn, 86% of the cells of A. purpureus appeared in the mixolimnion because of their reduced buoyant density. The increase in heterotrophic bacterial biomass, soluble phosphorus concentrations below the detection limit, and an extraordinarily high activity of alkaline phosphatase in the mixolimnion indicate a rapid liberation of organically bound phosphorus from A. purpureus cells accompanied by a simultaneous incorporation into heterotrophic bacterioplankton. High concentrations of allochthonously derived dissolved organic carbon (mean, 60 mg of C(middot)liter(sup-1)) were measured in the lake water. In Mahoney Lake, liberation of phosphorus from upwelling purple sulfur bacteria and degradation of allochthonous dissolved organic carbon as an additional carbon source render heterotrophic bacterial production largely independent of the photosynthesis of phytoplankton. A recycling of inorganic nutrients via phototrophic bacteria also appears to be relevant in other lakes with anoxic bottom waters.     

Effect of limitation of bacterial growth with carbon, sulfur and iron on the synthesis of cytochromes, development of cyanide-resistant respiration and super-synthesis of metabolites

The work is concerned with the effect produced by limiting the growth of various bacteria with carbon, sulfur and iron on cytochrome synthesis, development of cyanide-resistant respiration and oversynthesis of metabolites. The cessation of bacterial growth due to the exhaustion of a carbon source was shown to be accompanied with the development of cyanide-resistant respiration though the oversynthesis of metabolites did not occur. If the growth was limited by a sulfur or iron source, the concentration of cytochromes a, b and c fell down as compared with that when the growth was limited by a carbon source, and metabolites were produced and accumulated in the medium. In that case, the respiration of virtually all the bacteria was inhibited by cyanide to a great extent. As was demonstrated for Pseudomonas aeruginosa, the development of cyanide-resistant respiration was inhibited when metabolites accumulated and then the respiration became completely resistant to cyanide as soon as the oversynthesis ceased. Apparently, whatever limits the bacterial growth, the process of oversynthesis inhibits cyanide-resistant oxidase.     

Characterization of indicator bacteria in municipal raw water, drinking water, and new main water samples

Municipal water samples were analyzed by membrane filter (MF) and presence-absence (P-A) tests for pollution indicator bacteria. In four years, 11 514 bacterial cultures were isolated from either raw water, drinking water, or new main water samples submitted to three environmental laboratories. The bacterial species occurring most often in all types of water samples were Escherichia coli (11.6-39.7%), Enterobacter aerogenes (18.1-26.3%), Aeromonas hydrophila (8.8-17.0%), Klebsiella pneumoniae (7.7-10.3%), and Citrobacter freundii (5.9-22.7%). A lactose - lauryl tryptose - tryptone broth was examined as an alternative medium to modified MacConkey broth in the presumptive portion of the P-A test. The intensity of acid and gas production in presumptive positive P-A bottles was compared with the types and frequencies of indicator bacteria shown by confirmatory tests. The results of detecting indicator bacteria following the analysis of 53 130 samples over a 2-year period were arranged by water source (well, lake, river, mixed) and water type (raw or drinking) to determine the influence of these parameters on the recovery of indicator bacteria. A further subdivision of the sample types into raw surface, raw ground, in-plant, plant discharge, reservoir, and distribution samples demonstrated the effect of water treatment practices.     

Dynamics of production of extracellular polysaccharides by nodule bacteria

The dynamics of the accumulation of the extracellular polysaccharides synthesized by nodule bacteria and the possibility of their assimilation by these bacteria as a source of carbon was studied. When nodule bacteria were cultured for 20 days in a medium containing glucose, an increase in the titer of the bacteria and the accumulation of extracellular polysaccharides was observed in the first three days. After this the titer of the nodule bacteria decreased with a decrease in the glucose in the medium, but the amount of extracellular polysaccharide synthesized did not increase. These data suggest that extracellular polysaccharides are not assimilated by nodule bacteria as a source of carbon and evidently are protective substances for the cells.     

细菌生理特性快速检测试剂盒研制

选取各代谢途径中利用某些特定碳源,如蔗糖、葡萄糖、甘露醇等95种碳源作为测试碳源,分别加入96孔酶标板中.选用指示剂噻唑兰(MTT)来鉴定细菌利用特征碳源情况.同时选取不同种类和不同浓度的抗生素,不同起始pH、不同NaCl浓度加入96孔酶标板中,测定细胞是否生长,可以快速地获得该菌的抗生素耐受性谱.试验还从指示剂浓度、培养基的固化、接菌浓度、观察时间等方面对试剂盒进行优化,得到一种最佳的试剂盒鉴定系统BiobiqA(碳源利用谱)和BiobiqB(生理抗性谱).用模式菌株对试剂盒进行测试和验证,结果表明,试剂盒具有操作简便、结果准确、节省成本、节约时间等优点,可以进行细菌生理特性的快速检测.     

Distribution of bacteria with nitrilotriacetate-degrading potential in an estuarine environment.

Attempts to isolate estaurine bacteria capable of metabolizing nitrilotriacetate (NTA) as a sole carbon source from areas within Escambia Bay, Fla., were unsuccessful; however, bacteria from freshwater streams and from estaurine surface microlayers were easily adapted to degradation of NTA in freshwater medium. A Pseudomonas sp. strain (ATCC 29600), capable of growth on NTA as a sole carbon source, metabolized NTA at a reduced rate in a saline medium (15%), compared with a freshwater medium (0 to 15%). Microorganisms capable of degrading NTA exist in estuarine surface microlayers and in fresh subsurface waters just before entering the estuary; these data indicate an interference with NTA catabolism by some unknown factors of the estuarine environment rather than an absence of potential NTA-degrading bacteria.     

Glucosamine measurement as indirect method for biomass estimation of Cunninghamella elegans grown in solid state cultivation conditions

Glucosamine measurement has been tested as the indirect method to estimate the biomass produced by Cunninghamella elegans during solid state cultivation (SSC). The independence of this cell constituent content from the age and the conditions of the culture have been verified. The influence of the medium composition, in particular the nature of the carbon source on glucosamine amount is presented. Glucosamine can be considered as a well-adapted biomass indicator, with the necessity to establish for each medium tested a prior correlation between biomass and glucosamine amount. This correlation should be defined in submerged conditions before applying the biomass estimating method in SSC.     

Automatic method for evaluating the activity of sourdough strains based on gas pressure measurements

A new method is proposed for the evaluation of the activity of sourdough strains, based on gas pressure measurements in closed air-tight reactors. Gas pressure and pH were monitored on-line during the cultivation of commercial yeasts and heterofermentative lactic acid bacteria on a semi-synthetic medium with glucose as the major carbon source. Relative gas pressure evolution was compared both to glucose consumption and to acidification and growth. It became obvious that gas pressure evolution is related to glucose consumption kinetics. For each strain, a correlation was made between maximum gas pressure variation and amount of glucose consumed. The mass balance of CO2 in both liquid and gas phase demonstrated that around 90% of CO2 was recovered. Concerning biomass production, a linear relationship was found between log colony-forming units/ml and log pressure for both yeasts and bacteria during the exponential phase; and for yeasts, relative gas pressure evolution also followed optical density variation.     

Bacterial Growth as a Practical Indicator of Extensive Biodegradability of Organic Compounds

The proportionality of growth, as indicated by turbidity of cultures of Pseudomonas C12B, to the initial concentration of sodium dodecyl sulfate, dodecanol, or a mixture of C₁₀-C₂₀ secondary alcohol sulfates, each provided as sole carbon source in basal mineral salts medium, was demonstrated. Subsequently, the direct correlation of culture turbidity as a growth indicator and degradation of sodium dodecyl sulfate and the C₁₀-C₂₀ compounds was established. Degradation of these detergents was measured by the rise in surface tension and the decrease in methylene blue values, respectively. Turbidimetry was found to be a poor indicator of degradation of dodecanol in the early hours of culture, however, and did not correlate over a significant range with degradation of substrate. Viable cell counts did parallel dodecanol degradation as measured by gas-liquid chromatography. The use of bacterial growth as a reliable, quantitative, and easily measured parameter indicating biodegradability was suggested for those organic compounds which can be shown to serve as a carbon source for a bacterium.     

In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichoderma species

Culture filtrates of Trichoderma viride and Trichoderma harzianum were inhibitory of Fusarium moniliforme and, to a lesser extent, Aspergillus flavus. The degree of inhibition was, however, dependent on the carbon or nitrogen source incorporated into the medium. Scanning electron microscopy revealed the development of abnormal fruiting structures on exposure to some Trichoderma culture filtrate, while macroscopically, growth restriction and, in the case of A. flavus, altered colony colouration were observed. Based on the results of inverted colony culture, it would appear that some isolates of Trichoderma produce inhibitory volatile compounds. The production of possible antibiotics was also demonstrated. The aggressive behaviour (towards A. flavus and F. moniliforme) demonstrated by Trichoderma spp. may be partly explained by the liberation of extracellular enzymes by these fungi. An isolate of T. viride exhibited amylolytic, pectinolytic, proteolytic and cellulolytic activity. Based on the results of the present investigation, Trichoderma spp. are potential candidates for biocontrol of some mycotoxin-producing fungi, but there exists some doubt as to their osmotolerance within the air-dry seed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.     

A Medium for Detecting Phenol-Degrading Bacteria

S ummary . A rapid test for the identification of phenol-degrading bacteria was devised using a medium containing phenol as the sole source of carbon and energy. Degradation of phenol in such a medium was assessed by growth and change in pH value. The results obtained are in agreement with those obtained by methods requiring chemical analysis of phenol in spent culture fluid.     

Isolation of Hydrocarbon-Oxidizing Psychroactive Bacteria from Oil-Polluted Soils

Microorganisms growing on a mineral medium with crude oil and its light fractions as the only source of carbon and energy have been isolated from samples of oil-polluted soils collected in the Usa district (Komi Republic, Russia). Hydrocarbon-oxidizing psychroactive bacteria of the genus Cytophaga, which are clearly capable of consuming crude oil hydrocarbons, have been identified for the first time. A method for cultivating microorganisms on porous plastic is proposed.     

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.     

Wood-eating catfishes of the genus Panaque: gut microflora and cellulolytic enzyme activities

Fresh gut contents of the wood-eating loricariid Panaque and a generalized loricariid Liposarcus sp. had enzymatic activity directed against both cellulose and hemicellulose. Aerobic cultures made from the guts of Panaque exhibited growth on a minimal salts medium containing only crystalline cellulose as a carbon source as well as on a variety of other substrates containing carbon polymers found in wood. Anaerobic cultures made from Panaque guts only grew with glucose as a carbon source. Cultures of whole gut contents grown on a yeast extract basal salts medium had significant cellulolytic activity. However, no culture of individual microbes had significant cellulolytic activity, suggesting that any cellulose breakdown which occurs in loricariid guts is by a consortium of micro-organisms. A variety of aerobes, microaerophiles and facultative anaerobes were found in the guts of Panaque ; several of these bacteria appear to be new species.     

Factors influencing the formation and stability of D-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens.

D-glucoside 3-dehydrogenase specific activity in Agrobacterium tumefaciens was maximal towards the end of the exponential growth phase of batch cultures; over 90% of the activity disappeared within the next 15 h. Manganese ions, although essential for growth of the organism, strongly repressed D-glucoside 3-dehydrogenase synthesis in sucrose medium but had little effect when the carbon source was methyl alpha-D-glucoside. D-Glucoside 3-dehydrogenase activity increased linearly with increasing specific growth rate in chemostat cultures limited by carbon, nitrogen, phosphate or manganese when methyl alpha-D-glucoside was the carbon source. High enzyme activity was found with sucrose as carbon source only when the growth medium was manganese-limited. D-Glucoside 3-dehydrogenase activity disappeared from A. tumefaciens incubated in carbon- and nitrogen-free medium or in nitrogen-free medium containing succinate, but on continued incubation the activity returned and was then stable. The recovery of activity could be prevented by chloramphenicol or erythromycin. Bacteria containing the recovered dehydrogenase activity could not convert sucrose to 3-ketosucrose when oxygen acted as the terminal electron acceptor, but produced 3-ketosucrose at the normal rate in the presence of ferricyanide. D-Glucoside 3-dehydrogenase activity disappeared irreversibly from bacteria incubated in nitrogen-free medium containing sucrose. Loss of activity followed first order kinetics in bacteria taken from nitrogen-, phosphate- or manganese-limited chemostat steady states; an accelerating rate of decay occurred in cells grown under carbon-limitation. 8-Hydroxyquinoline, chloramphenicol, erythromycin, 2,4-dinitrophenol and manganese ions could reduce the rate of decay.