Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in mono or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.

Patchy stomatal behavior during midday depression of leaf CO2 exchange in tropical trees.

We investigated effects of heterogeneous stomatal behavior on diurnal patterns of leaf gas exchange in 10 tree species. Observations were made in middle and upper canopy layers of potted tropical rainforest trees in a nursery at the Forest Research Institute Malaysia. Measurements were taken from 29 January to 3 February 2010. We measured in situ diurnal changes in net photosynthetic rate and stomatal conductance in three leaves of each species under natural light. In both top-canopy and sub-canopy species, midday depression of net assimilation rate occurred in late morning. Numerical analysis showed that patchy bimodal stomatal behavior occurred only during midday depression, suggesting that the distribution pattern of stomatal apertures (either uniform or non-uniform stomatal behavior) varies flexibly within single days. Direct observation of stomatal aperture using Suzuki's Universal Micro-Printing (SUMP) method demonstrated midday patchy stomatal closure that fits a bimodal pattern in Shorea leprosula Miq., Shorea macrantha Brandis. and Dipterocarpus tempehes V.Sl. Inhibition of net assimilation rate and stomatal conductance appears to be a response to changes in vapor pressure deficit (VPD). Variable stomatal closure with increasing VPD is a mechanism used by a range of species to prevent excess water loss from leaves through evapotranspiration (viz., inhibition of midday leaf gas exchange). Bimodal stomatal closure may occur among adjacent stomata within a single patch, rather than among patches on a single leaf. Our results suggest the occurrence of patches at several scales within single leaves. Further analysis should consider variable spatial scales in heterogeneous stomatal behavior between and within patches and within single leaves.

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

Comparison of radiorespirometric Budemeyer assay with ATP assay and mouse foot pad test in detecting viable Mycobacterium leprae from clinical samples.

PURPOSE: This study compares the results of radiorespirometric Buddemeyer assay with adenosine triphosphate (ATP) assay and mouse foot pad (MFP) test to validate the sensitivity of Buddemeyer assay in detecting viable M. leprae in clinical samples. METHODS: Viability was assessed using all the three methods in 60 skin biopsy specimens, including 20 untreated lepromatous leprosy (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid borderline (BT-BB) and 15 treated BT-BB cases. RESULTS: Of the 20 untreated BL-LL cases tested, positivity indicating the presence of viable M. leprae was detected in 85, 60 and 85% with Buddemeyer, ATP and MFP test, respectively. Among the 13 treated BL-LL cases, scores were 61, 54 and 0%; among the 12 untreated BT-BB cases, the scores were 58, 16 and 16% and among the 15 treated BT-BB cases, the scores were 46, 20, 0%, respectively. CONCLUSION: The detection sensitivity (positive scores) with three tests were closely comparable in the two untreated groups of cases. On the other hand, in the two treated groups, a good proportion of cases scored positive in the in vitro tests but none in the MFP test. Among the two in vitro methods, the Buddemeyer assay emerged as a better test, in terms of sensitivity and specificity.

Process-oriented dose assessment model for 14C due to releases during normal operation of a nuclear power plant.

Swedish nuclear utility companies are required to assess doses due to releases of radionuclides during normal operation. In 2001, calculation methods used earlier were updated due to new authority regulations. The isotope (14)C is of special interest in dose assessments due to the role of carbon in the metabolism of all life forms. Earlier, factors expressing the ratio between concentration of (14)C in air and in various plants were used. In order to extend the possibility to take local conditions into account, a process-oriented assessment model for uptake of carbon and doses from releases of (14)C to air was developed (POM(14)C). The model uses part of DAISY which has been developed to model the turnover of carbon in crops. [Hansen, S., Jensen, H.E., Nielsen, N.E., Svendsen, H., 1993. Description of the Soil Plant System Model DAISY, Basic Principles and Modelling Approach. Simulation Model for Transformation and Transport of Energy and Matter in the Soil Plant Atmosphere System. Jordbruksförlaget, The Royal Veterinary and Agricultural University, Copenhagen, Denmark]. The main objectives were to test model performance of the former method, and to investigate if taking site specific parameters into account to a greater degree would lead to major differences in the results. Several exposure pathways were considered: direct consumption of locally grown cereals, vegetables, and root vegetables, as well as consumption of milk and meat from cows having eaten fodder cereals and green fodder from the area around the nuclear plant. The total dose of the earlier model was compared with that of POM(14)C. The result of the former was shown to be slightly higher than the latter, but POM(14)C confirmed that the earlier results were of a reasonable magnitude. When full account of local conditions was taken, e.g. as regards solar radiation, temperature, and concentration of (14)C in air at various places in the surroundings of each nuclear plant, a difference in dose between sites of approximately one order of magnitude was found.

Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous cultures.

The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1). A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1).

Effect of temperature on oxygen stores during aerobic diving in the freshwater turtle Mauremys caspica leprosa.

Oxygen stores available for aerobic diving were studied in the freshwater turtle (Mauremys caspica leprosa) at three constant body temperatures (15 degrees, 25 degrees, and 35 degrees C) and during the thermal transient (30 degrees-15 degrees C) induced by immersion in cold water. The term "aerobic dive limit" has been defined as the maximal duration of the dive before lactate increases. This increase occurs when a critical PO2 value is reached, and it is well characterized at lung level by a sharp increase in the lung apnoeic respiratory quotient. Kinetic analysis of lung gas composition during forced dives at fixed body temperature shows that critical PO2 values rise with temperature and that the postventilatory PO2 at the beginning of a dive decreases, so that the two temperature-dependent factors lead to a significant decrease with temperature in the lung O2 stores available for aerobic diving. During dives with transient body cooling, a natural condition in M. caspica leprosa, temperature equilibration occurs fast enough to expand aerobic scope by bearing the critical PO2 to the same value obtained at a fixed temperature of 15 degrees C. These dives are characterized by reversed CO2 transport (from lung to tissues) and therefore by negative values of the lung respiratory quotient; a decrease in temperature increases CO2 capacitance of tissues, resulting in a fall in PCO2 at constant CO2 content. Because this does not occur in the gas phase, PCO2 difference can lead to diffusion in the direction opposite from normal. This pattern may favour lung-to-tissue O2 transfer, through the Bohr effect. Therefore, the aerobic dive limit is reduced at high temperature not only through a metabolic rate effect but also through a marked decrease in the available O2 stores; fast body cooling (30 degrees-15 degrees C) associated with immersion in cold water extends the O2 stores available for aerobic diving to a level similar to that of immersions at constant body temperatures that are in equilibrium with water temperature.

Ruminococcus hydrogenotrophicus sp. nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces.

A new H2/CO2-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject. The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli. The isolates grew autotrophically by metabolizing H2/CO2 to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds. The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate. The G+C content of DNA of strain S5a33 was 45.2 mol%. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed. The type strain of R. hydrogenotrophicus is S5a33 (DSM 10507). Furthermore, H2/CO2 acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus).

Titration of numbers of human-derived Mycobacterium leprae required to progressively oxidize 14C-palmitic acid and release 14CO2.

Mycobacterium leprae was isolated from skin-punch biopsies of 2 untreated lepromatous leprosy patients. The bacteria were enumerated, diluted 10-fold and cultured in Middlebrook 7H9 medium supplemented with albumin, dextrose, catalase and 14C-palmitic acid. The cultures were incubated at 33 degrees C in a modified Buddemeyer radiorespiratory detection vessel. Those cultures containing at least 10(7) mycobacteria demonstrated a progressive evolution of 14CO2.

Competency of human-derived Mycobacterium leprae to use palmitic acid in the synthesis of phenolic glycolipid-I and phthiocerol dimycocerosate and to release CO2 in axenic culture.

Insufficient numbers of viable Mycobacterium leprae have hampered metabolic studies using human-derived M. leprae. In this study, sufficient numbers of M. leprae were obtained from an untreated lepromatous patient to titrate the effects of pH on the metabolism of 14C-palmitic acid by M. leprae. Catabolic metabolism (oxidation of 14C-palmitic acid and release of 14CO2) was maximal when M. leprae were incubated at 33 degrees C and suspended in Middlebrook 7H9, ADC supplemented medium that had been buffered to maintain a pH of 4.8. Anabolic metabolism (synthesis of 14C-phenolic glycolipid-I and its precursor, 14C-phthiocerol dimycocerosate) was maximal when the pH was maintained at 6.8.

Double-blind evaluation of BACTEC and Buddemeyer-type radiorespirometric assays for in vitro screening of antileprosy agents.

Two radiorespirometric assays, the BACTEC 460 and Buddemeyer-type 14CO2 detection systems, were evaluated in a double-blind manner for their ability to discriminate between authentic antileprosy agents and inactive compounds. Freshly harvested, nude-mouse derived Mycobacterium leprae were incubated in axenic media in the presence of coded test solutions prepared in a remote laboratory. Activity was assessed by comparing the rate of 14CO2 evolution from [1-14C]palmitic acid to controls. Breaking the code revealed that both systems demonstrated a dose response to ethionamide, pefloxacin and rifampicin as well as sensitivity to dapsone. Most of the water, ethanol, sucrose, dabsyl chloride and riboflavin negative-control samples failed to effect a significant reduction in radiorespirometric activity. This study confirms the ability of the radiorespirometric assays to function as a primary drug screening system in leprosy.

Oxidation of palmitic acid by Mycobacterium leprae in an axenic medium.

The ability of Mycobacterium leprae to oxidize palmitic acid during incubation in an axenic medium was studied. By using a Buddemeyer-type detection system, partially purified nude-mouse-derived M. leprae was found to produce 14CO2 from 14C-labeled palmitic acid in a linear fashion for at least 1 week. Procedures known to remove residual host tissue did not diminish the rate of 14CO2 evolution, indicating that bacterial metabolism was being measured. Palmitate oxidation was temperature sensitive, with an apparent optimum of 33 degrees C, but pH insensitive. Bacilli exposed to a variety of antileprosy drugs for 1 or 2 weeks displayed significantly reduced rates of 14CO2 evolution upon subsequent addition of 14C-labeled palmitic acid. This activity could be readily detected with 10(6) bacilli, thus indicating its potential for use in clinical susceptibility testing.

The effects of 2,4-dichlorophenoxyacetic acid on respiration, nitrogen and carbohydrate metabolism of Aspergillus terreus Thom.

The effects of 2,4-D on respiration, carbohydrate and nitrogen metabolism have excited much interest in relation to higher plants (Hansen 1946, Hseuth and Lou 1946, SMITH et al. 1947, and Said and Naguib 1955). But the effects of 2,4-D on dungi have been tackled to a much less extent (Guiscafre-Arrilaga 1948, Bever and Slife 1948, Wei and Ling 1948, and Manil and Strazewska 1950). These investigators studied the effects exerted on fungal growth. Said and Naguib (1962) studied the effect of 2,4-D on the carbohydrate metabolism of Fusarium moniliforme. They showed that both sucrose inversion and absorption were retarded in the presence of 2,4-D. In the present investigation, a trial was carried out in order to elucidate the effect of 2,4-D on growth, nitrogen and carbohydrate metabolism of Aspergillus terreus when grown on two different nitrogen sources, namely sodium nitrate and ammonium phosphate.

Radiometric measurement of differential metabolism of fatty acids by Mycobacterium lepraemurium.

An assay system has been developed based on automated radiometric quantification of 14CO2 produced through oxidation of (1--14C) fatty acids by mycobacteria. With this system, the Hawaiian strain of M. lepraemurium was studied using the K-36 buffer as a suspending solution for the organisms along with 5.0 muCi of one of the following fatty acids: acetate, butyric, hexanoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic, and malonic. The 14CO2 production by this organism was greatest with lauric, decanoic, myristic, octanoic, stearic, oleic, linoleic, linolenic, and malonic. The 14CO2 production by this organism was greatest with lauric, decanoic, myristic, octanoic, and stearic acids, in decreasing order. Assimilation studies and radiochromatograms confirmed that most of the oxidized substrates were converted into by-products with no change in those from which no oxidation was found. These data suggest that the radiometric measurement of differential fatty acid metabolism may provide a basis of radiometric identification of M. lepraemurium and assessment of the growth requirements of this organism.