A mesoscopic stochastic model for the specific consumption rate in substrate-limited microbial growth.

The specific consumption rate of substrate, as well as the associated specific growth rate, is an essential parameter in the mathematical description of substrate-limited microbial growth. In this paper we develop a completely new kinetic model of substrate transport, based on recent knowledge on the structural biology of transport proteins, which correctly describes very accurate experimental results at near-zero substrate concentration values found in the literature, where the widespread Michaelis-Menten model fails. Additionally, our model converges asymptotically to Michaelis-Menten predictions as substrate concentration increases. Instead of the single active site enzymatic reaction of Michaelis-Menten type, the proposed model assumes a multi-site kinetics, simplified as an apparent all-or-none mechanism for the transport, which is controlled by means of the local substrate concentration in the close vicinity of the transport protein. Besides, the model also assumes that this local concentration is not equal to the mean substrate concentration experimentally determined in the culture medium. Instead, we propose that it fluctuates with a mostly exponential distribution of Weibull type.

Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal.

Kinetic and Energetic Parameters of Carob Wastes Fermentation by Saccharomyces cerevisiae: Crabtree Effect, Ethanol Toxicity, and Invertase Repression.

Carob waste is a useful raw material for the second-generation ethanol because 50% of its dry weight is sucrose, glucose, and fructose. To optimize the process, we have studied the influence of the initial concentration of sugars on the fermentation performance of Saccharomyces cerevisiae. With initial sugar concentrations (S0) of 20 g/l, the yeasts were derepressed and the ethanol produced during the exponential phase was consumed in a diauxic phase. The rate of ethanol consumption decreased with increasing S0 and disappeared at 250 g/l when the Crabtree effect was complete and almost all the sugar consumed was transformed into ethanol with a yield factor of 0.42 g/g. Sucrose hydrolysis was delayed at high S0 because of glucose repression of invertase synthesis, which was triggered at concentrations above 40 g/l. At S0 higher than 250 g/l, even when glucose had been exhausted, sucrose was hydrolyzed very slowly, probably due to an inhibition at this low water activity. Although with lower metabolic rates and longer times of fermentation, 250 g/l is considered the optimal initial concentration because it avoids the diauxic consumption of ethanol and maintains enough invertase activity to consume all the sucrose, and also avoids the inhibitions due to lower water activities at higher S0.

A simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies.

UNLABELLED: We propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: Mathematical models used to predict microbial growth are based on liquid cultures data. Models describing growth on solid surfaces, highlighting the differences with liquids cultures, are scarce. In this work, we have demonstrated that a single Gompertz equation describes accurately the increase of the yeast colonies, up to the point where they reach their maximum size. The model can be used to quantify the differences in growth kinetics between solid and liquid media. Moreover, as all its parameters have biological meaning, it could be used to build secondary models predicting yeast growth on solid surfaces under several environmental conditions.

Acute and chronic stress increase salivary cortisol: a study in the real-life setting of a national examination undertaken by medical graduates.

Spanish medical graduates who apply for a medical specialty training position (MIR) must take an examination that will shape their future personal and professional lives. Preparation for the test represents an important stressor that persists for several months. The aim of this study was to elucidate the stress pattern of this group and evaluate possible changes in the circadian rhythm of cortisol release in medical graduates preparing for this test. A repeated-measures longitudinal study was performed, measuring the salivary cortisol concentrations in 36 medical graduates (13 males and 23 females; mean age of 24.2 years) on five sampling days. Five cortisol samples were collected from 07:00 to 21:00 h in order to monitor changes in the circadian rhythm. On all sampling days (except on the day of the official examination), anxiety and psychological stress were evaluated with the Spanish versions of the State-Trait Anxiety Inventory (STAI) and the Perceived Stress Scale (PSS). During the study period, participants showed higher levels of anxiety than the Spanish reference population as well as a progressive increase in self-perceived stress. A significant increase in salivary cortisol concentration was observed in both chronic (study and examination preparation) and acute (examinations) situations. Our results suggest that the cortisol awakening response (CAR) may be a good indicator of anticipatory stress but is unaffected by long-term examination preparation. Comparison of results between the official examination day and the mock examination days yielded evidence that learning may modulate the behavior of the hypothalamic-pituitary-adrenal axis.

A model of the specific growth rate inhibition by weak acids in yeasts based on energy requirements.

Zygosaccharomyces bailii, a spoilage yeast, capable of metabolic activity in food environments with low pH, low a(w) and in the presence of weak acid preservatives was chosen for a study on the effect of benzoic acid on growth parameters. In batch cultures, under controlled pH, this food preservative inhibited growth, decreasing the specific growth rate (mu) and the yield coefficient (Y(S)) on glucose. Data obtained at pH 3.5, 4.0 and 4.5 showed that this inhibition was exclusively promoted by the undissociated form of the acid since the effect was independent of pH when the concentration of the acid was expressed in this form. Moreover, the relationship between the values for mu and Y(S), provided evidence that the specific consumption rate of glucose (q(S)) was not affected by benzoic acid, indicating that the inhibition of growth should be completely explained by a decrease of Y(S). The outcome of parallel experiments performed in continuous culture was that the decrease of Y(S) was due to an increase of the maintenance coefficient (m), defined as the fraction of q(S) diverted from growth to cope with stress, represented in this case by the presence of the preservative. Based on these results a model was built, assuming that m increased hyperbolically with the concentration of benzoic acid, from zero in the absence of the acid up to q(S) when growth was completely inhibited. The concentration of the acid, for which m=q(S)/2, is a constant (K(W)), and represents a measure of the tolerance for a preservative, in this case benzoic acid. The simple equation mu/mu(0)=1+W/K(W) predicts the value of mu for a concentration (W) of the preservative, requiring the knowledge of two parameters: the specific growth rate in the absence of the preservative (mu(0)) and K(W). The equation fitted very well the data of the effect of benzoic acid on the specific growth rate of Z. bailii, having K(W)=0.96 mM benzoic acid. The model was also validated with other spoilage yeasts grown in the presence of benzoic acid in microtiter plates in an automated spectrophotometer. The values obtained for K(W) under these conditions confirm Z. bailii as the most tolerant (K(W)=2.1 mM) followed by Pichia sp. (K(W)=0.78 mM), Saccharomyces cerevisiae (K(W)=0.53 mM) and Debaryomyces hansenii (K(W)=0.11 mM).

ATP requirements for benzoic acid tolerance in Zygosaccharomyces bailii.

AIMS: To calculate the energetic requirements for benzoic acid tolerance in Zygosaccharomyces bailii in chemostat experiments. METHODS AND RESULTS: A 5.6-l stirred-tank chemostat was used. The yield of ATP (Y(ATP)) was calculated under nitrogen atmosphere, assuming equimolar ATP and ethanol production. Under these conditions Y(ATP), equal to 20 g mol(-1) of ATP, was not affected by the acid, whereas the maintenance coefficient (m(ATP)) increased from 1.0 mmol of ATP g(-1) h(-1) in the absence of the acid to 4.8 in the presence of 0.67 mmol l(-1) undissociated benzoic acid. These ATP requirements were similar to those found in Saccharomyces cerevisiae with other weak acids. CONCLUSIONS: No significant differences have been found in the energy expended to cope with the acid between sensitive and tolerant species. Therefore, the main difference between tolerant and sensitive species could rely on cellular features that would not need extra energy in terms of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential mechanisms involved in the tolerance to weak acids in yeasts have been extensively studied but their actual relevance has not been assessed. Our results suggest that future efforts should concentrate on nonexpending energy features as membrane permeability and metabolic tolerance in the cytoplasm.

Pentadiene production from potassium sorbate by osmotolerant yeasts.

Some yeast strains belonging to the species Zygosaccharomyces rouxii and Debaryomyces hansenii are capable of spoiling sorbate containing high-sugar foods by producing pentadiene, a volatile compound reported to have 'petroleum-like' odour. Quantification of the diminution of sorbate and the subsequent increase of pentadiene was performed by growing the yeasts in experimental media containing 600 g/l sucrose and different sorbate concentrations. Final sorbate concentrations were notably lower than their corresponding initial ones, and it was found that the higher the initial concentration of sorbate in the media, the higher the amount of pentadiene produced. In all cases, Z. rouxii was able to produce more pentadiene than D. hansenii when expressing pentadiene concentration as a function of cell biomass. These results suggest that pentadiene is a metabolite of sorbate.

Biology of killer yeasts.

Killer yeasts secrete proteinaceous killer toxins lethal to susceptible yeast strains. These toxins have no activity against microorganisms other than yeasts, and the killer strains are insensitive to their own toxins. Killer toxins differ between species or strains, showing diverse characteristics in terms of structural genes, molecular size, mature structure and immunity. The mechanisms of recognizing and killing sensitive cells differ for each toxin. Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. They are also suitable to study the mechanisms of protein processing and secretion, and toxin interaction with sensitive cells. This review focuses on the biological diversity of the killer toxins described up to now and their potential biotechnological applications.

Dietary influence of kefir on microbial activities in the mouse bowel.

AIMS: In this work the microflora present in kefir, a fermented milk product, was studied together with the effect of kefir administration on different groups of indigenous bacteria of mouse bowel. METHODS AND RESULTS: Kefir microflora was composed of lactic acid bacteria, acetic acid bacteria and yeasts. Yeast population was composed of Saccharomyces cerevisiae, S. unisporus, Candida kefir, Kluyveromyces marxianus and K. lactis. The streptococci levels in kefir treated mice increased by 10-fold and the levels of sulfite-reducing clostridia decreased by 100-fold. The number of lactic acid bacteria increased significantly. CONCLUSIONS: The administration of kefir significantly increased the lactic acid bacteria counts in the mucosa of the bowel. Ingestion of kefir specifically lowered microbial populations of Enterobacteriaceae and clostridia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first long-term study about the effects of the kefir administration on the intestinal microflora of mice.

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin.

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.

Performance of mycological media in enumerating desiccated food spoilage yeasts: an interlaboratory study.

Dichloran 18% glycerol agar (DG18) was originally formulated to enumerate nonfastidious xerophilic moulds in foods containing rapidly growing Eurotium species. Some laboratories are now using DG18 as a general purpose medium for enumerating yeasts and moulds, although its performance in recovering yeasts from dry foods has not been evaluated. An interlaboratory study compared DG18 with dichloran rose bengal chloramphenicol agar (DRBC), plate count agar supplemented with chloramphenicol (PCAC), tryptone glucose yeast extract chloramphenicol agar (TGYC), acidified potato dextrose agar (APDA), and orange serum agar (OSA) for their suitability to enumerate 14 species of lyophilized yeasts. The coefficient of variation for among-laboratories repeatability within yeast was 1.39% and reproducibility of counts among laboratories was 7.1%. The order of performance of media for recovering yeasts was TGYC > PCAC = OSA > APDA > DRBC > DG 18. A second study was done to determine the combined effects of storage time and temperature on viability of yeasts and suitability of media for recovery. Higher viability was retained at -18 degrees C than at 5 degrees C or 25 degrees C for up to 42 weeks, although the difference in mean counts of yeasts stored at -18 degrees C and 25 degrees C was only 0.78 log10 cfu/ml of rehydrated suspension. TGYC was equal to PCAC and superior to the other four media in recovering yeasts stored at -18 degrees C, 5 degrees C, or 25 degrees C for up to 42 weeks. Results from both the interlaboratory study and the storage study support the use of TGYC for enumerating desiccated yeasts. DG18 is not recommended as a general purpose medium for recovering yeasts from a desiccated condition.

Role of intrapartum hypoxia in carnitine nutritional status during the early neonatal period.

We analyze markers of carnitine insufficiency and deficiency, lysine (LYS) and methionine (MET), in 39 neonates with intrapartum hypoxia (selection criteria: umbilical artery pH <7.20, lactate >1.8 mmol/l and PaO2 <25 mm Hg), and in 35 healthy newborn infants (control group) in the early neonatal period (1-7 days of life). Free (FC), total (TC) carnitine and acylcarnitines (AC=short-chain+long-chain acylcarnitines) were measured using a radioisotopic micromethod; LYS and MET were determined by high-pressure liquid chromatography. AC and TC plasma concentrations and AC/FC ratio were higher while FC/TC ratio was lower in the hypoxic neonates than in the control group. Hypoxic newborn infants (59%) presented "carnitine deficiency" (FC/TC <0.7) and 60% of them "carnitine insufficiency" (AC/FC ratio >0.4) vs. 31% and 28%, respectively, for the neonates of the control group (p<0.05). In the healthy neonates group, MET correlated with FC/TC and the AC/FC ratio. FC, TC, AC, AC/FC and umbilical artery pH (pHua) were inversely correlated. FC/TC and MET correlated with pHua. We conclude that: (1) an important percentage of newborn infants with intrapartum hypoxia suffer carnitine deficiency and carnitine insufficiency in the early neonatal period, related to MET plasma levels; (2) the carnitine deficiency or insufficiency in the neonate is determined by the degree of intrapartum acidosis.

Production and characteristics of Debaryomyces hansenii killer toxin.

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 degrees C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin.

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.

A chromogenic medium for the detection of yeasts with beta-galactosidase and beta-glucosidase activities from intermediate moisture foods.

A selective and differential solid medium for the specific detection of some common yeasts frequently causing spoilage in intermediate moisture foods is described. The principle of the method is based on the detection of two enzymes, beta-glucosidase and beta-galactosidase, using the chromogenic substrates salmon-Gluc and X-Gal. Over 140 yeasts and bacteria were tested, and Debaryomyces hansenii and Kluyveromyces marxianus strains produced salmon and dark blue colonies, respectively, thus permitting their clear discrimination from other yeasts common in intermediate moisture foods. The medium was very satisfactory when intermediate moisture foods were tested.

A mathematical model for toxin accumulation by killer yeasts based on the yeast population growth.

The accumulation of toxin by killer yeast populations is modelled starting from a mechanistic approach that explains the toxin production in terms of yeast population growth, and takes into account the environmental inactivation of the toxin. A modified Richard's general equation for limited growth is used to define the function that describes the toxin produced in relation to the yeast biomass increase. The relationship between the rates of cell and toxin production is explicitly shown, and the implications of the resulting proportionality factor are discussed. The model parameters have been adjusted and the model has been validated using experimental data of growth and toxin accumulation from cultures of Pichia membranaefaciens in two different media. The differences between both types of cultures are analysed on the basis of parameter estimates and the predicted rate of toxin production per cell. The results support the hypothesis that biomass production and toxin synthesis are controlled in different ways; they also suggest that the composition of the medium could have a distinct effect on toxin synthesis. Model assumptions are discussed in comparison with a previous model for killer-sensitive interaction of Saccharomyces cerevisiae strains.

A differential medium for the isolation of Kluyveromyces marxianus and Kluyveromyces lactis from dairy products.

A selective and differential solid medium, called Kluyveromyces Differential Medium (KDM), is described for the isolation of Kluyveromyces marxianus and K. lactis from dairy products. Its discriminative potential is based on the detection of the enzyme beta-galactosidase, in the absence of lactose. Of the more than 95 strains tested, including yeasts, bacteria, and filamentous fungus, only the strains of K. marxianus and K. lactis produced blue colonies on the medium due to the presence of X-Gal/ IPTG. The bacterial strains were not able to grow in KDM. On this basis, the medium was very satisfactory when testing naturally or experimentally contaminated dairy food products. When quality assessment tests were performed, optimal values of productivity (growth and color) and selectivity were obtained for K. marxianus and K. lactis.

Quantitative and ultrastructural changes in glia and pericytes in the parietal cortex of the aging rat.

The frequency of astrocytes, microglia plus oligodendrocytes, and pericytes displaying nuclei was analyzed and quantified in 160-microm-wide strips of the parietal cortex (Par1 region) from young and aged Wistar rats. The study was performed on two groups of rats aged 3-4 and 32-36 months. Quantifications of the glial cell types and pericytes were made in 1-microm-thick sections stained with toluidine blue. Ultrathin sections were also made to analyze the ultrastructural features of these cells during aging. Astrocytes and pericytes increased in number by about 20% and 22%, respectively, with age. These increases were most significant in layers II-IV and V for both cellular types. Clusters of astrocytes were common in these layers of aging rats. The ultrastructural analysis also indicated changes in all cell types that stored inclusions and vacuoles with age, which were particularly abundant in microglial cells. End-feet astrocytes and pericytes surrounding the vascular wall also contained vacuoles and inclusions, and consequently the vascular wall increased in thickness. In conclusion, the aging process increased astrocyte and pericyte populations, but not microglia plus oligodendrocyte populations, in the rat parietal cortex. Although no significant change in nuclear size could be observed in any cell type, all glial cells as well as pericytes underwent morphological ultrastructural changes. These modifications may result from the need to correct possible homeostatic imbalances during aging.

Alanyl-aminopeptidase activity decrease in cerebrospinal fluid of Alzheimer patients.

We analyzed the amino acid concentrations and aminopeptidase activities in plasma and the cerebrospinal fluid (CSF) of Alzheimer patients. Correlations were calculated between these parameters and the degree of cognitive impairment, duration of the disease and age. In comparison with a control group, no changes were found in the levels of amino acids in CSF or plasma of Alzheimer patients. Alanyl-aminopeptidase activity was significantly lower in the CSF of Alzheimer patients, whereas no differences in CSF were detected as regards the remaining aminopeptidases. The plasma/CSF ratio for aminopeptidase activities was higher in AD patients than in controls, although the difference was only significant for alanyl-aminopeptidase. Amino acid ratios did not show this general tendency. The correlations between plasma aspartate and glutamate concentrations and the stage of the disease, measured with the Mini Mental State Examination, were studied. Changes in aminopeptidase activities and their role in protein dysfunction underlying Alzheimer disease are discussed.