A comparison of the patterns of extracellular proteins produced by the high alpha-toxin-secreting organism Staphylococcus aureus (Wood 46) during aerobic and anaerobic growth

Staphylococcus aureus (Wood 46) was grown aerobically and anaerobically in supplemented 3% (w/v) Tryptone Soya Broth medium for 24 h at 37 degrees C. Although the bacterial density achieved was 9 times higher in the aerobic culture, the exoprotein produced per unit of bacterial dry weight was only 1.4 times higher than in the anaerobic culture. However, the SDS-PAGE patterns of extracellular proteins were quite different: the aerobic products occurred almost exclusively in the mol. wt range 15-30000 compared with 30-60000 for those produced anaerobically. The only major component common to both preparations was alpha-toxin which accounted for 2.4 times more of the total exoprotein under aerobic than under anaerobic conditions.     

The effect of glucose on the expression of extracellular protein genes by Staphylococcus aureus strain V8

The bacteria from overnight cultures (20 h) of S. aureus V8 and exp negative mutant K6812-1, grown, aerobically, in 3% (w/v) Tryptone Soya Broth, at 37 degrees C, were resuspended in fresh medium, in the case of the parent strain +/- 1% (w/v) glucose, without change in bacterial density. During a 6 h incubation period there was an approximate doubling of bacterial density, to the same level, in each case. However, exoprotein production by the mutant was only 20% that of the parent whilst the addition of glucose to the V8 strain resulted in a tenfold reduction in the exoprotein formed. SDS-polyacrylamide gel electrophoresis showed that the exoprotein patterns of both organisms after 6 h incubation were the same as those observed in the overnight cultures whilst the presence of 1% (w/v) extra glucose changed the pattern produced by the parent to one similar to that of the mutant. The results showed that conditions which lead to the rapid formation of glucose catabolites produced an effect consistent with the inhibition of the activity of the exp gene product.     

Determination of the efficiency of oxidative phosphorylation in continuous cultures of Aerobacter aerogenes.

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for Ymax/ATP and a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures. It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes. For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at mu values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with mu became higher and lower respectively above the mu value of 0.53. Using the YATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and alpha-ketoglutarate were produced. With the YATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose.
Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Prevalence of oxygen-intolerant microorganisms in primary bile acid 7α-dehydroxylating mouse intestinal microflora

The in vitro 7α-dehydroxylation of cholic and chenodeoxycholic acids by mixed cultures of mouse cecal microorganisms was studied. Conventional anaerobic techniques and rigorous oxygen-free anaerobic experimental conditions were compared. It was found that the total number of anaerobic oxygen-intolerant microorganisms was about 10 times higher than that of anaerobic microorganisms that tolerate oxygen. Among the anaerobic 7α-dehydroxylating microorganisms, the oxygen-intolerant ones are about 1,000 to 10,000 times more numerous than the oxygen-tolerant ones. It can be concluded that the 7α-dehydroxylating activity is more common among oxygenintolerant than oxygen-tolerant anaerobic microorganisms.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose. Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

The isolation and characterization of peroxisomes (microbodies) from baker's yeast,Saccharomyces cerevisiae

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for Y _ATP ^max and a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures. It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes. For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at μ values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with μ became higher and lower respectively above the μ value of 0.53. Using the Y _ATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and α-ketoglutarate were produced. With the Y _ATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.     

A comparative study of the formation of extracellular proteins by Aeromonas salmonicida at two different temperatures

Aeromonas salmonicida was grown in a supplemented 3% (w/v) tryptone soya broth medium at 10 degrees C, a temperature at the lower end of the range over which furunculosis has been observed to occur in the field, and 25 degrees C, the optimum temperature for growth. Similar bacterial densities in the range 2.35 +/- 0.05 mg dry wt/ml were achieved in the two cultures at the beginning of the stationary phase of the growth cycle, after 125 h at 10 degrees C and 18 h at 25 degrees C. At this point, at the higher temperature 1.5 times more exoprotein was formed, 80 +/- 2.8 micrograms/ml compared with 54 +/- 1.7 micrograms/ml. Exoprotein contained the same proportion of haemolysin at both temperatures and twice as much protease at the higher temperature. The most marked difference was in an unidentified 100 kD protein which was formed in a 10-fold greater amount at 10 degrees C.     

Anaerobic microbial methylation of inorganic tin in estuarine sediment slurries

Estuarine sediment slurries and microorganisms were examined for the ability to methylate inorganic tin. Under controlled redox conditions, tin was methylated only in oxygen-free sediment slurries. Monomethyltin usually comprised greater than 90% of the alkyltin products formed, although dimethyltin was also produced. Autoclaved anoxic sediments did not produce organotins. Several bacterial cultures, most notably sulfate-reducing bacteria isolated from anoxic estuarine sediments, formed monoand dimethyltin from inorganic tin in the absence of sediment. The results suggest that inorganic tin methylation in estuarine environments is an anaerobic process catalyzed primarily by sulfate-reducing microorganisms.     

Use of oxygen-permeable silicone rubber pouches for growing mass cultures of bacteria

Growth of most bacteria often involves the use of expensive incubated shaker systems. In this report, oxygen-permeable silicone rubber pouches, with oxygen permeability over 100 times higher than other polymers, were employed for growing bacterial cultures. With little, if any, agitation oxygen-permeable silicone rubber pouches produced bacterial growth rates equivalent to growth rates obtained in shaker flasks. The silicone rubber pouch described has a glass cuvette integrated into its design that permits readings of bacterial density without opening the pouch. One can sterilize and store powdered bacterial culture medium in silicone rubber pouches ; therefore, bacterial cultures can be initiated by simply adding water and bacteria.     

Occurrence of the thiosulphate sulphurtransferase producers in the population of mesophilic heterotrophic bacteria and microfungi of spruce humus

The activity of thiosulphate sulphurtransferase (rhodanese, EC 2.8.1.1) in randomly isolated bacteria and mieromycetes of the humus horizon in a spruce forest was followed. Bacteria isolated on soil extract agar (70 cultures) did not yield unambiguous results due to poor growth of the cultures. Of 63 bacterial cultures on meat-peptone agar almost 80 % of the isolates produced the enzyme. Sixty-three % of isolates had a concentration higher than 5 nkat/L and 35 % higher than 20 nkat/L. The soil rhodanese activity can be explained by their presence. None of 30 isolates of micromycetes produced thiocyanate.     

A comparative study of the formation of extracellular proteins by Aeromonas salmonicida at two different temperatures

Aeromonas salmonicida was grown in a supplemented 3% (w/v) tryptone soya broth medium at 10°C, a temperature at the lower end of the range over which furunculosis has been observed to occur in the field, and 25°C, the optimum temperature for growth. Similar bacterial densities in the range 2.35 × 0.05 mg dry wt/ml were achieved in the two cultures at the beginning of the stationary phase of the growth cycle, after 125 h at 10°C and 18 h at 25°C. At this point, at the higher temperature 1.5 times more exoprotein was formed, 80 × 2.8 μg/ml compared with 54 × 1.7 μg/ml. Exoprotein contained the same proportion of haemolysin at both temperatures and twice as much protease at the higher temperature. The most marked difference was in an unidentified 100 kD protein which was formed in a 10-fold greater amount at 10°C.     

The potential of biological methane generation from chicken manure

The potential for biological methane generating from the manure of laying hens was investigated in the laboratory. Fresh manure was collected, analyzed, and used to prepare medium for bacterial growth. At 55°C and under anaerobic conditions, methanogenic cultures were initiated by incubating the medium with different inoculations from various natural environments. Since there were no significant differences in gas production among these initiated cultures after 40 days of acclimation, they were mixed to maintain a genetic pool. The mixed culture was then challenged with different retention times (RT) and different volatile solid (VS) concentrations for the selection of optimal conditions and cultures. The conditions were finally selected to be 4-day RT and 6% VS for the maximal rate of gas production. The optimal pH and temperature were determined to be 7.5 and 50°C, respectively. Under such conditions the selected culture produced total gas at a rate 4.5 L/L day and methane (70% of total gas) 3.2 L/L day. The chicken manure therefore was able to support the methane yield at 270 L/kg of VS, a value comparably higher than other kinds of livestock wastes.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures.

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Staphylococcal Enterotoxin B and Nuclease Production Under Controlled Dissolved Oxygen Conditions

Enterotoxin B, nuclease, and total exoprotein production by Staphylococcus aureus strain S-6 was studied in a 0.5-liter fermentor system. While these extracellular products were elaborated over a wide range of aeration rates, maximal production occurred within the very narrow range of 125 to 150 cm(3) of air per min. The levels attained at the optimal aeration rate were not increased by maintaining a constant pH, although yield of enterotoxin:cell mass was highest at a constant pH of 7.0. During the growth cycle of the cultures, when aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen (DO) levels, initially set at 100% of saturation, decreased to 5 to 10% 4 to 5 h after inoculation. The oxygen demand of the culture then maintained this level for an additional 4 to 6 h. This interval of low DO was characterized by maximal growth and exoprotein production. When the DO was controlled at a constant value throughout growth (by increasing or decreasing the airflow rate as appropriate), the culture demonstrated different optima for maximal growth and exoprotein production. A constant DO of 100% stimulated growth to extremely high densities, but the accumulation of toxin and nuclease was not observed. On the other hand, maintaining constant DO levels at 50 or 10% raised exoprotein levels higher than those achieved in a culture grown at the optimal aeration rate. Compared to the optimal aeration rate culture, the 10% DO culture yielded 20% more nuclease, 25% more toxin, and 40 to 50% more total exoprotein. These results indicate that it is the DO and not the aeration rate, per se, that is influential in controlling growth, toxin, nuclease, and total exoprotein production.     

Volatilisation of metals and metalloids: An inherent feature of methanoarchaea?

As shown by recent studies, anaerobic members of Archaea and Bacteria are involved in processes that transform ionic species of metals and metalloids (arsenic, antimony, bismuth, selenium, tellurium and mercury) into volatile and mostly toxic derivatives (mainly methyl derivatives or hydrides). Since the fact that these transformations proceed in both environmental settings and in parts of the human body, we have to consider that these processes also interfere directly with human health. The diversity of the volatile derivatives produced and their emission rates were significantly higher in methanoarchaeal than in bacterial strains, which supports the pivotal role of methanoarchaea in transforming metals and metalloids (metal(loid)s) into their volatile derivatives. Compared with methanoarchaea, 14 anaerobic bacterial strains showed a significantly restricted spectrum of volatilised derivatives and mostly lower production rates of volatile bismuth and selenium derivatives. Since methanoarchaea isolated from the human gut (Methanosphaera stadtmanae, Methanobrevibacter smithii) showed a higher potential for metal(loid) derivatisation compared to bacterial gut isolates, we assume that methanoarchaea in the human gut are mainly responsible for the production of these volatile derivatives. The observation that trimethylbismuth ((CH(3))(3)Bi), the main volatile derivative of bismuth produced in human feces, inhibited growing cultures of Bacteroides thetaiotaomicron, a representative member of the human physiological gut flora, suggests that these volatiles exert their toxic effects on human health not only by direct interaction with host cells but also by disturbing the physiological gut microflora.     

Bacterial stress enrichment enhances anaerobic hydrogen production in cattle manure sludge

Methodology was evaluated to selectively enrich hydrogen-producing species present in biological sludge produced during organic wastewater treatment. The influence of bacterial stress enrichment on anaerobic hydrogen-producing microorganisms was investigated in batch tests using serum bottles. Enrichment conditions investigated included application of acute physical and chemical stresses: wet heat, dry heat and desiccation, use of a methanogen inhibitor, freezing and thawing, and chemical acidification with and without preacidification of the sludge at pH 3. For each enrichment sample, cultivation pH value was set at an initial value of 7. After application of selective enrichment (by bacterial stress), hydrogen production was significantly higher than that of untreated original sludge. Hydrogen production from the inocula with bacterial stress enrichment was 1.9–9.8 times greater when compared with control sludge. Chemical acidification using perchloric acid showed the best hydrogen production potential, irrespective of preacidification. Enhancement is due to the selective capture of hydrogen-producing sporeformers, which induces altered anaerobic fermentative metabolism.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.