Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

Use of response surface methodology to optimise environmental stress conditions on Penicillium glabrum, a food spoilage mould

Fungi are ubiquitous microorganisms often associated with spoilage and biodeterioration of a large variety of foods and feedstuffs. Their growth may be influenced by temporary changes in intrinsic or environmental factors such as temperature, water activity, pH, preservatives, atmosphere composition, all of which may represent potential sources of stress. Molecular-based analyses of their physiological responses to environmental conditions would help to better manage the risk of alteration and potential toxicity of food products. However, before investigating molecular stress responses, appropriate experimental stress conditions must be precisely defined. Penicillium glabrum is a filamentous fungus widely present in the environment and frequently isolated in the food processing industry as a contaminant of numerous products. Using response surface methodology, the present study evaluated the influence of two environmental factors (temperature and pH) on P. glabrum growth to determine 'optimised' environmental stress conditions. For thermal and pH shocks, a large range of conditions was applied by varying factor intensity and exposure time according to a two-factorial central composite design. Temperature and exposure duration varied from 30 to 50 °C and from 10 min to 230 min, respectively. The effects of interaction between both variables were observed on fungal growth. For pH, the duration of exposure, from 10 to 230 min, had no significant effect on fungal growth. Experiments were thus carried out on a range of pH from 0.15 to 12.50 for a single exposure time of 240 min. Based on fungal growth results, a thermal shock of 120 min at 40 °C or a pH shock of 240 min at 1.50 or 9.00 may therefore be useful to investigate stress responses to non-optimal conditions.     

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2)

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.     

A note on estimation of food spoilage yeasts by measurement of adenosine triphosphate (ATP) after growth at various temperatures

The growth at 4 degrees, 10 degrees and 15 degrees C of six psychrotrophic yeasts isolated from foods was compared using total viable counts and ATP measurement. A linear relationship (r greater than or equal to 0.97) was obtained between log10 (number of viable yeasts) and log10 (ATP content) of cultures grown at these temperatures. This relationship was not temperature-dependent. The results are discussed and their significance for the rapid estimation of yeasts in foods is considered.     

A note on estimation of food spoilage yeasts by measurement of adenosine triphosphate (ATP) after growth at various temperatures

The growth at 4, 10 and 15C of six psychrotrophic yeasts isolated from foods was compared using total viable counts and ATP measurement. A linear relationship ( r > 0.97) was obtained between log₁₀ (number of viable yeasts) and log₁₀ (ATP content) of cultures grown at these temperatures. This relationship was not temperature-dependent. The results are discussed and their significance for the rapid estimation of yeasts in foods is considered.     

Effects of treating seed potatoes with trace elements on sprouting and microbial spoilage during storage under tropical conditions

Attempts were made to extend the storage life of seed potatoes by treating them with trace elements as antimicrobial and antisprouting agents. In tests to measure the growth of Fusarium oxysporum, and Erwinia carotovora pv. carotovora it was found to be decreased more by iron, copper and zinc than by boron, manganese and molybdenum. Dipping seed tubers in aqueous solutions of trace element salts decreased both sprouting and microbial spoilage during storage at ambient conditions. Field testing at the end of storage proved that treating seed potatoes with ferrous sulphate did not decrease the yield of tubers or increase their iron content. It is suggested that trace elements may be used to extend the storage life of seed potato tubers, in tropical conditions.     

Mite growth on fungus under various environmental conditions and its potential application to biofilters

The effects of relative humidity, temperature, pH and vapor-phase toluene concentration on Tyrophagus putrescentiae growth on Cladophialophora sp. were tested in controlled environmental chambers. It was observed that the mites were able to reproduce readily at relative humidities between 90% and 97% as well as on porous perlite support material pre-soaked in nutrient media of pH 2.5, 4 and 7. Also, the presence of toluene at gas-phase concentrations of 500 to 2000 mg m⁻³ was found to be non-toxic to the mites. The mites, however, were unable to maintain a large population when the temperature was maintained at 14 °C, and overpopulation of the living space led to declines in mite population over time. Overall, it was found to be relatively simple to cultivate mites that may be used for fungal biomass control measures in biofilter applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

New modified Square Root and Schoolfield models for predicting bacterial growth rate as a function of temperature

Summary A new modified Square Root model and two new modified Schoolfield models were evaluated for their ability to predict the growth rate ofYersinia enterocolitica as a function of temperature. The new Square Root model fits the data better than both the original Square Root model and the Zwietering Square Root model. Both new Schoolfield models, a six-and a four-parameter equation, fit the data better than the original Schoolfield model. The new four-parameter Schoolfield model was developed by removing the term describing low temperature inactivation from the new six-parameter Schoolfield model. Inclusion of the two extra parameters in the new six-parameter Schoolfield model (F=318) did not significantly improve the fit compared to the new fourparameter Schoolfield model (F=488).     

An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

An immunofluorescence method for detection of Vibrio cholerae serovar 01 in aquatic environmental samples and enrichment broths is described. Antiserum specific for the 01 somatic antigen was produced in rabbits and used in an indirect fluorescent antibody method incorporating fluoresceinisothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine isothiocynate conjugated bovine serum albumin as background stain. Comparisons of the immunofluorescent procedure and conventional culture methods for isolation of V. cholerae 01 showed that detection occurred significantly more frequently with the fluorescent antibody system.     

A unified stoichiometric model for oxidative and oxidoreductive growth of yeasts

Yeasts degrade glucose through different metabolic pathways, where the choice of the pathway is dependent on the nature of the limitation in the various substrates. When oxygen is limiting in addition to glucose, yeasts often grow according to a mixture of oxidative and reductive metabolism. Oxygen may be limiting either by supply or by inherent biological restrictions such as the respiratory bottleneck in Saccharomyces cerevisiae or by both. A unified model incorporating both supply and biological limitations is proposed for the quantitative prediction of growth rates, consumption and production rates, as well as key metabolite concentrations during mixed oxidoreductive metabolism occuring as a result of such oxygen limitations. This simple unstructured model can be applied to different yeast strains while at the same time requiring a minimum number of measured parameters. "Estimators" are utilized in order to predict the presence of supply-side or biological limitations. The values of these estimators also characterize the relative importance of oxidative to total metabolism. Results from the aerobic and oxygen-limited chemostat cultures were used to corroborate the model predictions. During these experiments, the heat released by the yeast cultures was also monitored on-line. The model correctly predicted the overall stoichiometry, steady-state concentrations, and rates including heat dissipation rates measured in the various situations of oxygen limitations. Direct continuous measurements such as heat can be used in conjunction with the unified model for on-line proces control. (c) 1992 John Wiley & Sons, Inc.     

Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection

During wine production, yeast cells are affected by several conditions that are adverse to growth (oxidative, osmotic and ethanol stress among others) and they should detect and respond to these conditions, otherwise alcoholic fermentation can be negatively affected. In this work we have analyzed the fermentative behaviour of 14 commercial and non-commercial strains in several synthetic musts. According to the data obtained these strains have been classified into three groups depending on whether or not vinification was completed (and on the amount of residual sugar remaining in the must if it was not). Moreover, we have determined the resistance of these strains to several stress situations under laboratory growth conditions. We have been able to establish a correlation between the groups based on fermentative behaviour and resistance to several stress conditions (especially oxidative and ethanol stress), by applying discriminant analysis to the data obtained in these experiments. Our results indicate a clear relationship between stress resistance and fermentative behaviour and this opens up the possibility of using this information as a criterion for the future selection of wine yeasts.     

Accumulation of pyrophosphate and other energy-rich phosphorus compounds under various conditions of yeast growth

In the cells of hybrid yeast strain Saccharomyces N.C.Y.C. 644 SU₃ (Karlsberg collection), a large amount of pyrophosphate (30–300 mol/g of dry weight) accumulates whatever the aeration conditions and the content of glucose in the medium. The content of pyrophosphate is 10–1000 times higher than that of ATP. At the early and mid-exponential growth phases two maxima of pyrophosphate accumulation are observable. The periods of maximal pyrophosphate accumulation in yeast coincide with those of the minimal content of polymeric acid-soluble polyphosphates and intense budding. In the light of the data obtained, the question is discussed as to the relationship between the metabolism of pyrophosphates and acid-soluble polyphosphates in yeast.     

水体中氮素对水葫芦生长发育的影响

本文通过对水体中氮素营养条件的研究,分析了水体中不同氮素对水葫芦生长的影响,从而为福建省水葫芦的综合治理提供科学依据。结果表明：在水体氮浓度为25～30mg·L^1的范围内,水葫芦生长情况最佳,高于或低于这个范围,水葫芦的生长发育均受到不同程度的抑制。