Differential effects of hydrogen peroxide and ascorbic acid on the aerobic thermosensitivity of yeast cells grown under aerobic and anoxic conditions

We have previously demonstrated that in aerobically‐grown cells of the yeast Saccharomyces cerevisiae, hydrogen peroxide (H₂O₂) increases and ascorbic acid decreases cellular thermosensitivity, as determined by the inducibility of a heat shock (HS)‐reporter gene. In this work, we reveal that the aerobic thermosensitivity of anaerobically‐grown yeast cells also increases in the presence of H₂O₂, albeit differentially between cells with two different lipid profiles. In comparison to aerobically‐grown fermenting cells treated with the same H₂O₂ concentration, both these types of anaerobically‐grown cells were found to be considerably less sensitive to aerobic heat shock and considerably more thermotolerant. Paradoxically, and in contrast to ascorbate‐pretreated aerobically‐grown yeast cells, when anaerobically‐grown cells were heat‐shocked aerobically in the presence of the same ascorbic acid concentration, they exhibited increased thermosensitivity and decreased intrinsic thermotolerance with respect to their untreated counterparts. These findings are discussed with respect to what is currently known about the redox and physiological status of yeast cells grown aerobically and cells reoxygenated following anoxic growth. Copyright © 2009 John Wiley & Sons, Ltd.     

Media for the aerobic resuscitation of Campylobacter jejuni

The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (lamda) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The h of cells grown aerobically in NB2 containing both FBP and blood was similar to that obtained in the same medium incubated in a microaerobic environment (P > 0.05). However, the X was longer during aerobic growth when low numbers of cells (approximately 1 log CFU/ml) in late exponential phase were used as the initial inoculum. The best recovery of stressed C. jejuni was observed in NB2 supplemented with FBP and blood and incubated aerobically. Enrichment in media incorporating FBP and 5% laked horse blood is a simple, convenient, and time-saving method to replace microaerophilic incubation methods for the resuscitation of C. jejuni.     

FORMATION OF N-PROPYL ALCOHOL BY CELL-FREE EXTRACTS OF SACCHAROMYCES CEREVISIAE*

n-Propyl alcohol formation from α-ketobutyric acid was obtained in cell-free extracts of Saccharomyces cerevisiae var. ellipsoideus and the optimal conditions for the formation were determined. In the presence of NAD? the concentration of n-propyl alcohol formed was the same with extracts of yeast grown either aerobically or anaerobically, but in the presence of NADP more n-propyl alcohol was formed with extracts of aerobic yeast. There was no evidence for an increase in an NADP-specific alcohol dehydrogenase, and the increased formation of n-propyl alcohol by extracts of aerobic yeast with added NADP was attributed to a greater reductive capacity of the extracts of aerobic cells in the presence of NADP. No system of enzymes for the formation of n-propyl alcohol was found other than those involved in ethyl alcohol formation. However, alcohol dehydrogenase from aerobic cells had a greater specificity for n-propyl alcohol, compared with ethyl alcohol, than the enzyme from anaerobically grown cells. This difference may be part of the explanation for greater n-propyl alcohol formation in aerobic cultures.     

Growth of Escherichia coli and Salmonella typhimurium on Beef Tissue at 25°C

At 25°C, growth of Escherichia coli and Salmonella typhimurium on beef was influenced by type of tissue, pH, gaseous atmosphere, and physiological state of the cells used to inoculate the tissue. These organisms grew after only a short lag period, both aerobically and anaerobically, on beef fatty-tissue, and on high pH muscle (pH ≥ 6). The lag period was considerably extended on low pH muscle (pH ≤ 5.7) incubated aerobically. On low pH lean tissue stored anaerobically at 25°C for 24 hr, cells from aerobically grown broth cultures did not grow whereas cells from anaerobically grown cultures grew after an extended lag. These results suggest that during the cooling of hot-boned meat growth of E. coli and salmonellae is more likely on fatty tissue or muscle of high pH than on lean tissue of low pH.     

The role of pyruvate decarboxylase in the Kluyver effect in the food yeast,Candida utilis

The glucose-fermenting yeast, Candida utilis cannot use the β-D -glucoside, cellobiose, anaerobically, although it is able to do so aerobically. β-Glucoside transport and hydrolysis and pyruvate decarboxylase activities of this yeast were measured aerobically and anaerobically. β-Glucoside transport was five-fold faster aerobically than anaerobically, but there was no corresponding difference in β-glucosidase activity. Pyruvate decarboxylase activity varied greatly, being synthesized de novo in response to the presence of D -glucose and anaerobic conditions and about 50% deactivated on the removal of D -glucose or the addition of air. Activation and deactivation were rapidly reversible. Failure to utilize cellobiose anaerobically, in particular, and the Kluyver effect, in general, probably depends on much reduced glycolytic flux, associated under anaerobic conditions, with (i) lower transport rate, (ii) low substrate affinity of the relevant glycosidase and (iii) deactivation of pyruvate decarboxylase. So, in addition to the complex effects of oxygen, anaerobiosis and specific sugars on induction, repression and derepression, there are fine controls on pyruvate decarboxylase activity, leading to fast activation or deactivation of the enzyme.     

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.     

Staphylococcus aureus growth and enterotoxin A production in an anaerobic environment.

The effects of strict anaerobic conditions on the growth of Staphylococcus aureus and the production of staphylococcal enterotoxin A (SEA) were studied. The growth of S. aureus, a facultative anaerobic bacterium, is slower anaerobically than aerobically. When grown on brain heart infusion broth at 37 degrees C, the anaerobic generation time at mid-log phase was 80 min, compared with 35 min for the aerobic control. In contrast to previous studies demonstrating that staphylococcal cell density was 9- to 17-fold greater in aerobic than in anaerobic cultures, data for a staphylococcal strain implicated in food poisoning showed that the cell density was only two to three times as great in aerobic cultures. Production of SEA was monitored by Western immunoblotting and shown to be growth dependent. With slower anaerobic growth, relatively less toxin was produced than under aerobic conditions. but in both cases SEA was detected after 120 min of incubation. The combined effects of temperature and aeration on S. aureus were also studied. Growth and toxin production of aerobic and anaerobic cultures at temperatures ranging from 14 to 37 degrees C were analyzed. Growth was still observed at low temperatures in both environments. A linear model for S. aureus aerobic or anaerobic growth as a function of incubation temperature was developed from these studies. The model was tested from 17 to 35.5 degrees C, and the results suggest that the model can accurately predict the S. aureus growth rate in this temperature range. The data suggest that anaerobic conditions are not an effective barrier against S. aureus growth.     

The ecology of bacterial spoilage of fresh meat at chill temperatures

At chill temperatures the spoilage flora of meat is composed of psychrotrophs originating largely from the hides of slaughtered animals. Under humid conditions, aerobic floras are usually dominated by pseudomonads while anaerobic floras are dominated by lactobacilli. In both cases growth occurs on low molecular weight soluble components of meat which are attacked in the order glucose, glucose-6-phosphate (Enterobacteriaceae only) and amino acids. Under aerobic conditions spoilage becomes detectable when the bacteria begin to degrade amino acids which remain abundant at the meat surface when growth ceases, probably because of limited availability of oxygen. Under anaerobic conditions growth ceases because the diffusion of fermentable substrates to the surface is not rapid enough to support further growth. Aerobically, there is no interaction between different bacterial species until the maximum cell density is approached; anaerobically, however, lactobacilli produce an antimicrobial agent which inhibits growth of competing species. The composition of spoilage floras can be affected by changes in water activity and the storage atmosphere.     

Chemical studies with silage microorganisms in artificial media and sterile herbages

The fermentation changes in artificial media and in sterile herbages inoculated with strains of yeast, hetero-and homo-fermentative lactic acid bacteria isolated from silage were studied in two experiments. In the first, using malt extract broth, four treatments were employed: aerobic, high level of glucose; aerobic, low level of glucose; anaerobic, high level of glucose; anaerobic, low level of glucose. The pH decreased in all cultures. There were no appreciable differences in pH among the cultures of the heterofermentative lactic acid bacterium, but in the cultures of the homofermentative organism there was a greater decrease in pH aerobically than anaerobically. The lowest pH values in the yeast cultures, occurred in the high glucose treatments both aerobically and anaerobically. In all the yeast cultures, acetic, propionic, butyric, isobutyric and isovaleric acids were present. The heterofermentative lactic acid bacterium produced only acetic acid, the greatest amount occurring under aerobic conditions. Ethanol was detected in the culture of the heterofermentative lactic acid bacterium but not in that of the homofermentative organism. Relatively large amounts of i-pentanol were found in the aerobic yeast cultures. In the second experiment, inoculated grass was ensiled with similar treatments to those used in the first experiment. Two incubation periods, 3d and 10d were used. The pH values in all treatments were high (> pH 5.0). In aerobic conditions, the lactic acid contents after the 10d incubation were higher than after 3d. The losses of water soluble carbohydrates during fermentation were greater in the aerobic than anaerobic treatments. Acetic acid was present in all cultures but the higher volatile fatty acids (C₃-iC₅) were only detected in appreciable amounts in the aerobically treated herbages after 10d. In addition to ethanol, n-propanol and i-butanol were also present in all the 10d anaerobic treatments. i-Pentanol occurred in both the aerobic and anaerobic treated herbages.     

Synthesis of superoxide dismutase,catalase and other enzymes and oxygen and superoxide toxicity during changes in oxygen concentration in cultures of brewing yeast

The physiological effects on brewing yeast, growing in semi-defined wort medium, of a sudden transition from aerobiosis to anaerobiosis were studied. Two yeast strains were examined, used for ale and lager fermentations respectively. The reverse transition (from anaerobiosis to aerobiosis) was also examined. Transitions were applied by changing the sparging gas during growth or in stationary phase, and the effects on the specific activities of certain key enzymes and on the viability of the cultures were examined. Neither type of transition led to significant changes in growth rate, the rate of ethanol production or the specific activities of alcohol dehydrogenase and pyruvate decarboxylase. The most significant change was in the specific activity of CuZn-superoxide dismutase, which showed a rapid increase in activity on transition from anaerobiosis to aerobiosis, and a decrease in activity on the reverse transition. Catalase activity in the ale yeast generally followed that of CuZn-superoxide dismutase, whereas in the lager yeast it remained unchanged by the transitions. The transition from anaerobiosis to aerobiosis caused increases in citrate synthase and Mn-superoxide dismutase, though only after a significant lag period. Aerobic to anaerobic transitions caused a decrease in Mn-superoxide dismutase activity, while citrate synthase remained unchanged. Anaerobically grown cells showed a rapid loss in viability on exposure to oxygen (5–7% in the first hour), while aerobically grown cells were unaffected. When anaerobically grown cells were exposed to 0·25 mM -potassium superoxide, there was an 8% loss of viability within 10 min, whereas aerobic cells were not affected. It is concluded that the toxic effect of oxygen is due to superoxide (or a species derived from it) and that the CuZn-superoxide dismutase (but not the Mn-isoenzyme) plays a role in protecting the cells. The de novo synthesis of the CuZn-enzyme is not always rapid enough to confer full protection.     

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature.     

Factors Affecting Growth and Toxin Production by Clostridium Botulinum Type E on Irradiated (0.3 Mrad) Chicken Skins

A model system (chicken skins with chicken exudate) was used to determine if Clostridium botulinum type E (Beluga) spores, stressed by low dose irradiation, would develop and produce toxin at abuse temperatures of 10 and 30°C in the absence of characteristic spoilage. Unstressed spores germinated, multiplied, and produced toxin on vacuum-packed chicken skins, stored at either 30 or 10°C. Cell numbers increased faster and toxin was evident sooner at 30°C than at 10°C. At 30°C, growth occurred and toxin was produced more slowly when samples were incubated aerobically than anaerobically. When samples were incubated aerobically at 10°C, no toxin was detected within a test period of 14 days. An irradiation dose of 0.3 Mrad at 5°C reduced a spore population on vacuum-sealed chicken skins by about 90%. The surviving population produced toxin at 30°C under either aerobic or anaerobic conditions, at 10°C no toxin was detected even on skins incubated anaerobically. Under the worst conditions (30°C, vacuum packed) toxin was not detected prior to characteristic spoilage caused by the natural flora surviving 0.3 Mrad.     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Increased D-values of Staphylococcus aureus resulting from anaerobic heating and enumeration of survivors

D-values (decimal reduction times) were determined for Staphylococcus aureus ATCC #13565 and 14458 at 56°C in casein peptone soymeal peptone broth + 0·5% yeast extract (CASO-YE) and skim milk. The heating medium was either pre-reduced and survivors enumerated using a strictly anaerobic method, or it was nonreduced and survivors were enumerated using aerobic, thioglycollate overlay, anaerobic jar or strictly anaerobic methods. The D-values obtained for the two strains in both heating media were significantly higher (P < 0·05) when the medium was pre-reduced and enumeration was done anaerobically. Anaerobic enumeration following heating in non-reduced media also resulted in significantly higher D-values except for S. aureus ATCC #13565 heated in skim milk. The thioglycollate overlay enumeration method did not enhance recovery of heat-stressed cells and, for S. aureus ATCC #13565 in CASO-YE, a significantly lower D-values resulted. The anaerobic jar enumeration method also did not enhance recovery of heat-stressed cells, except in the case of S. aureus ATCC #14458 heated in skim milk. Our results suggest that strictly anaerobic conditions enhance the heat-resistance of S. aureus and/or its recovery from heat-injury.     

The effect of temperature abuse on Clostridium perfringens in cooked turkey stored under air and vacuum

The potential for growth of Clostridium perfringens in aerobic and anaerobic (vacuum) packaged cooked ground turkey was investigated. Samples of autoclaved ground turkey were inoculated with ∼3·0 log₁₀ cfu g^-1 of C. perfringens strain NCTC 8238 or NCTC 8239, packaged and stored at various temperatures. Vegetative growth and heat-resistant spores were enumerated by plating unheated and heated (75°C for 20 min) samples, respectively, on tryptose-sulfite-cycloserine agar. The type of atmosphere influenced the growth of C. perfringens at 15 and 28°C. Both strains grew to about 7 logs within 9 h anaerobically and by 24 h aerobically at 28°C. While aerobic growth was slow at 15°C, mean log₁₀ cfu g^-1 increased anaerobically by 4-4·5 logs by day 8 for both strains. Spores were not found at 4 and 15°C, but were detected as early as 24 h at 2°C under anaerobic conditions in both strains. C. perfringens population stabilized or slowly decreased at 4°C. Cyclic and static temperature abuse of refrigerated products for 5 h will not permit C. perfringens growth. However, temperature abuse of such products for relatively long periods may lead to high and dangerous numbers of organisms. Reheating such products to an internal temperature of 65°C before consumption would prevent food-poisoning since the vegetative cells were killed.     

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild‐type CBS 232.29 strain under similar morphology‐controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween‐80 and sparged with nitrogen gas was most suitable for yeast‐like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24–0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml_culture. Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth‐associated amylase production by the ATCC 1216b (0.63 U/ml_culture) and wild‐type CBS 232.29 (0.33 U/ml_culture) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml_culture), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain. Copyright © 2010 John Wiley & Sons, Ltd.     

Growth and Enterotoxin A Production by Staphylococcus aureus in Precooked Bacon in the Intermediate Moisture Range

Different water activities were obtained in precooked bacon by varying the frying time. Water activity (a_w) correlated best to the moisture, salt and protein content. When stored aerobically at 37°C, S. aureus A100 was capable of rapid growth in precooked bacon at a a_w of 0.84 or above, whereas at 20°C a a_w of 0.88 or higher was required. Under anaerobic storage at 37°C, growth was observed at a a_w of 0.90, and at 20°C slight growth was noted at a a_w of 0.91. The increase in the minimal a_w required for aerobic growth at the lower temperature was reflected in the differences between the isotherms obtained at 37°C and 20°C. The maximum populations achieved were higher for samples stored aerobically. Enterotoxin A (19–821 ng/g) was found in all aerobically stored samples where growth occurred. Enterotoxin A (38–109 ng/g) was also found in all anaerobically incubated samples where the population of S. aureus increased more than one logarithmic cycle.     

Fertilization stimulates anaerobic fuel degradation of antarctic soils by denitrifying microorganisms

Human activities in the Antarctic have resulted in hydrocarbon contamination of these fragile polar soils. Bioremediation is one of the options for remediation of these sites. However, little is known about anaerobic hydrocarbon degradation in polar soils and the influence of bioremediation practices on these processes. Using a field trial at Old Casey Station, Antarctica, we assessed the influence of fertilization on the anaerobic degradation of a 20-year old fuel spill. Fertilization increased hydrocarbon degradation in both anaerobic and aerobic soils when compared to controls, but was of most benefit for anaerobic soils where evaporation was negligible. This increased biodegradation in the anaerobic soils corresponded with a shift in the denitrifier community composition and an increased abundance of denitrifiers and benzoyl-CoA reductase. A microcosm study using toluene and hexadecane confirmed the degradative capacity within these soils under anaerobic conditions. It was observed that fertilized anaerobic soil degraded more of this hydrocarbon spike when incubated anaerobically than when incubated aerobically. We conclude that denitrifiers are actively involved in hydrocarbon degradation in Antarctic soils and that fertilization is an effective means of stimulating their activity. Further, when communities stimulated to degrade hydrocarbons under anaerobic conditions are exposed to oxygen, hydrocarbon degradation is suppressed. The commonly accepted belief that remediation of polar soils requires aeration needs to be reevaluated in light of this new data.