Physiological and DNA characterization of Candida maltosa, a hydrocarbon-utilizing yeast.

Selected yeast classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake, Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

Mechanism of uptake of liquid hydrocarbons by microorganisms

Growth rates of Candida tropicalis were studied in two different fermentors. One was the ordinary shaker flask containing both the aqueous culture medium and liquid hydrocarbons. The other was a specially designed rotating disk-type fermentor containing only the aqueous culture medium, into which vapors of n-paraffins from C₆ to C₁₈ were supplied continuously without forming the liquid hydrocarbon phase. The specific growth rates of Candida tropicalis in the rotating disk fermentor, under such conditions that supply of hydrocarbon vapor was sufficient, showed good agreement with those in the shaker flask. This seems to indicate that hydrocarbon uptake by Candida tropicals by direct contact with liquid hydrocarbon is negligible.     

Characteristics of hydrocarbon uptake in cultures with two liquid phases

In hydrocarbon fermentation, the efficiency of hydrocarbon uptake by cells ins one of the keys to the economical production of single-cell protein. This work is concerned with characterization of cultures with two liquid phases for understanding the hydrocarbon uptake process by cells. Batch cultivation of Candida lipolytica was carried out in shaking flasks and in a tower fermentor with motionless mixers. Micorscopic observation and cell and hydrocarbon concentration distribution in batch cultivation showed that some cells are attached to the large oil drops ad others are free from them. Interfacial tension between oil and water and Sauter mean drop size decreased as cultivation proceeded. On the basis of the experimental results, the process of hydrocarbon uptake by cells is discussed.     

Cultivation of the yeastCandida lipolytica on hydrocarbon

The ability of the yeastCandida lipolytica 4-1 to oxidize and utilize various pure aliphatic hydrocarbons occurring in gas oil was studied. It was found that the given strain ofCandida lipolytica oxidized n-alkanes without adaptation, starting with heptane, and utilized them for growth, starting with nonane. Isoalkanes with a single methyl group in the side chain were also oxidized and utilized for growth, but less than the corresponding n-alkanes. The site of the methyl group in the isoalkane chain influences its conversion to biomass. Branched chains at both ends of the isoalkane molecule prevent its utilization for growth ofCandida lipolytica. 1-olefines are also oxidized and utilized for growth, though less than the corresponding n-paraffins. Alkylaromatic hydrocarbons are oxidized from amylbenzene up to decylbenzene, which is utilized only slightly for growth of the yeast.     

侵袭性热带念珠菌感染流行病学及药物敏感性

全球范围内,随着抗肿瘤药物、免疫抑制剂和广谱抗菌药物的使用,真菌感染的发病率显著提高,其中念珠菌感染占到绝大多数。目前热带念珠菌已经成为非白念珠菌中最常见的病原菌。我国热带念珠菌的临床分离率及对氟康唑及伏立康唑的耐药率都明显高于世界平均水平。但是,相比于白念珠菌,关于热带念珠菌的研究及相关临床信息相对较少。该文就侵袭性热带念珠菌感染危险因素、流行病学以及药物敏感性进行全面的综述。     

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in Candida tropicalis cell suspensions.

The metabolism of glucose and xylose was studied as a function of oxygenation in suspensions of Candida tropicalis by 31P and 13C nuclear magnetic resonance spectroscopy. Both the rate of carbohydrate metabolism and the cytoplasmic pH were independent of the rate of oxygenation in cells metabolizing glucose. However, these two parameters were markedly dependent on the rate of oxygenation in C. tropicalis cells metabolizing xylose. For example, the cytoplasmic pH in fully oxygenated xylose-metabolizing cells was 7.8 but decreased to 6.3 in anoxic cells. In general, suspensions of cells consuming xylose had a lower rate of sugar uptake, a more acidic cytoplasmic pH, lower levels of sugarphosphomonoesters (SP) and ATP, higher levels of intracellular Pi, a more alkaline vacuolar pH, and a lower rate of extracellular Pi assimilation and polyphosphate synthesis than cells consuming glucose. These observations indicate that C. tropicalis metabolizing xylose is less energized than glucose-metabolizing cells. On both carbon sources, however, an inverse correlation between intracellular levels of SP and Pi was observed. Also, uptake of extracellular Pi correlated with the synthesis of polyphosphates within the cells. During anoxia, Pi was not taken up, and polyphosphates were hydrolyzed instead to fulfill the cells' requirements for phosphate.     

The functional role of lipids in hydrocarbon assimilation

The yeast Candida tropicalis utilizes both glucose and hydrocarbons as sole carbon sources. When grown on hydrocarbons, the cells contain twice as much lipid as when grown on glucose. In transient continuous culture experiments, following a substrate change from glucose to hexadecane, an adaption phase occurred. During this phase the lipid concentration per cell increased greatly. It is proposed that a high cellular lipid concentration is necessary for hydrocarbon assimilation, and this is not just a reflection of the lipophilic nature of the substrate.     

Factors influencing hydrocarbon degradation in three freshwater lakes

The mixed microbial flora of 3 lakes in Ohio with differing histories of hydrocarbon pollution was examined in relation to the ability to use hydrocarbons. Weathered kerosene was spiked with naphthalene, pristane, 1,13-tetradecadiene, andn-hexadecane and added to water-sediment mixtures from the 3 lakes, and utilization of the 4 marker hydrocarbons was measured. Each of the marker hydrocarbons was metabolized; naphthalene was the most readily used and pristane was the most resistant. Values for dissolved oxygen suggest that oxygen did not limit hydrocarbon degradation in the water column at any site examined. Nutrient addition studies indicated that nitrogen and phosphorus limited hydrocarbon degradation at all sites examined. Maximum numbers of heterotrophic bacteria were detected when the water temperature was 10°C or higher. The data indicate that temperature limits hydrocarbon degradation in the winter, except at a site which had been impacted by an oil spill and which received chronic inputs of hydrocarbons and nutrients. In samples from that site, all 4 marker hydrocarbons were degraded at 0°C. Results of temperature and nutrient-addition experiments suggest that different seasonal populations of hydrocarbon users are selected at that site, but not at other lake sites.     

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.     

Fatty acid uptake in Candida tropicalis: induction of a saturable process.

The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and Vmax values of 56 microM and 15 nmol/(min.mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate.     

Assimilation of liquid hydrocarbon by microorganisms. II. Growth kinetics

The growth kinetics of a microorganism with high affinity for liquid hydrocarbon which has a low solubility in water was investigated for Candida intermedia IFO 0761 in our previous work.⁶ The microorganism contained a hydrocarbon pool in and/or on the cell. The transfer of water-soluble substrates to the cell was not the rate-limiting step in the growth of C. intermedia accompanied by clump formation with liquid hydrocarbon. The operating conditions necessary for the oxygen supply for the growth were adequate for the growth of C. intermedia on n-tetradecane. The saturation kinetics was valid for the specific growth rate of C. intermedia and specific concentration of hydrocarbon per unit cell mass; the specific growth rate was expressed by the following equation:      

Diploid formation of Candida tropicalis via protoplast fusion.

Haploid auxotrophic mutants were produced from Candida tropicalis, and protoplast usion was induced by polyethylene glycol. The resulting nutritional complementation was due to heterokaryon formation and, at a much lower frequenty, to spontaneous diploidization. During cultivation, heterokaryotic clones regularly gave rise to heterozygous diploids from which, in turn, haploids could be isolated. The technique of protoplast fusion gives an opportunity for genetic analysis of this and similarly asexual fungal species.     

Cytological mechanisms involved in assimilation of n-alkanes by yeasts]

The paper describes cytological mechanism of adaptation of yeasts to assimilation of aliphatic hydrocarbons added to a growth medium as a sole source of carbon. The process was studied by light optical and electron microscopy, employing fluorescent labelling and electron microscopy contrasting. Two types of yeasts were found, which differed by the response of the cell walls to hydrocarbons: those that formed "channels" and those that did not form them. Cytological response to hydrocarbon assimilation was detected also in the mitochondria and canals of the endoplasmic reticulum. Components of the Golgi apparatus may also participate in this process, in particular, in formation of peroxisomes (microbodies). Close contacts of the yeast cells with the hydrocarbon being assimilated is important; assimilation may start in a close vicinity of the cell walls. The rate of flavin production by Candida tropicalis 303 IBFM increases during growth on solid paraffins, beginning with C20-paraffin.     

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.     

Cadmium biosorption by yeast, Candida tropicalis CBL-1, isolated from industrial wastewater

The present study is aimed at assessing the ability of metal-resistant yeast, Candida tropicalis CBL-1, to uptake metal from liquid medium. The minimum inhibitory concentration of Cd(II) against Candida tropicalis CBL-1 was 2,800 mg/L. The yeast could also tolerate Zn(II) (3,100 mg/L), Hg(II) (2,400 mg/L), Ni(II) (2,200 mg/L), Cr(VI) (2,000 mg/L), Pb(II) (1,100 mg/L), and Cu(II) (2,200 mg/L). The yeast isolate showed typical growth curves but lag and log phases extended in the presence of cadmium. The yeast isolate showed optimum growth at 30oC and pH 7. The metal processing ability of the isolate was determined in a medium containing 100 mg/L of Cd(II). Candida tropicalis CBL-1, could reduce Cd(II) 59%, 64% and 70% from the medium after 48, 96 and 144 h, respectively. C. tropicalis CBL-1 was also able to remove Cd(II) 46% and 60% from the wastewater after 6 and 12 days, respectively. Cd produced an increase in glutathione and non-protein thiols level by 37% (17.50±0.8-24.0±1.2) and 18% (3.30±0.7- 3.90±0.8) at 100 mg/L concentration, respectively. Metal tolerance and accumulation together with changes in the GSH status and non-protein thiols under Cd exposure were studied in C. tropicalis.     

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.     

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.     

Sterol content and polyene antibiotic resistance in isolates of Candida krusei, Candida parakrusei, and Candida tropicalis.

Three isolates, one from each species of Candida krusei, C. parakrusei, and C. tropicalis, obtained from infected patients, were more tolerant of significantly higher concentrations of polyene antibiotics than the corresponding reference wild types. The resistant strains isolated had the same sterols as their wild-type counterparts but in lower concentrations.     

Evidence for surfactant production by the haloarchaeon Haloferax sp. MSNC14 in hydrocarbon-containing media

The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60–70 %) and pristane (by 30–50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance study of glucose and xylose metabolism in agarose-immobilized Candida tropicalis.

Candida tropicalis can ferment both hexose and pentose sugars. Here, we have used 31P and 13C nuclear magnetic resonance spectroscopy to study the capacity of this yeast species to metabolize glucose or xylose when immobilized in small (< 1-mm-diameter) agarose beads. Immobilized C. tropicalis metabolizing glucose showed rapid initial growth within the beads. A corresponding drop in the intracellular pH (from 7.8 to 7.25) and hydrolysis of intracellular polyphosphate stores were observed. Although the initial rate of glucose metabolism with immobilized C. tropicalis was similar to the rate observed previously in cell suspensions, a decrease by a factor of 2.5 occurred over 24 h. In addition to ethanol, a significant amount of glycerol was also produced. When immobilized C. tropicalis consumed xylose, cell growth within the beads was minimal. The intracellular pH dropped rapidly by 1.05 pH units to 6.4. Intracellular ATP levels were lower and intracellular Pi levels were higher than observed with glucose-perfused cells. Consumption of xylose by immobilized C. tropicalis was slower than was previously observed for oxygen-limited cell suspensions, and xylitol was the only fermentation product.