Growth of Candida utilis on ethanol and isopropanol

Candida utilis grew on ehtanol and an ethanol-isopropanol-water (22:2:1 vols) mixture but not on isopropanol alone. Acetone accumulated in all cultures containing isopropranol but its presence in the alcohol mixture did not lower growth rate or yield significantly when compared with growth experiments on ethanol alone. Growth rate and yield declined at ethanol concentrations greater than 1% (v/v) and 0.3% (v/v) respectively. In a 0.3% (v/v) alcohol mixture, acetate was found only during the exponential growth phase. In a 3% (v/v) mixture, acetate and ethyl acetate accumulated during growth whereas acetaldehyde was present only during the exponential growth phase.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Analysis of Candida utilis genomic DNA,homologous to cDNA of chicken A1 (1) collagen gene

Genomic fragments, homologous to chicken A1(1) collagen cDNA encoding triple-helical domain, were revealed by Southern analysis in various fungi. Such a genomic fragment from Candida utilis was cloned and sequenced. Analysis of the obtained DNA sequence revealed the 119 bp segment, which has possibly originated from the 54bp module common for the fibrillar collagen genes of higher eukaryotes.     

Continuous cultivation of the yeast Candida utilis at different dilution rates on pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h^–1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, g_x/g_s) and (2.96, g_x/l/h) at a dilution rate of 0.425 and 0.475 h^–1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h^–1. The maintenance coefficient attained a value of (0.093, g_s/g_x/h). An increase in dilution rate produced a higher protein content of the biomass.     

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y_glucose and Y_O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

The kinetics and regulation of M-xylose transport in Candida utilis

Low-affinity (K _m=67.6±3.2 mM) and high-affinity (K _m=1.9±1.2 mM) D-xylose transport occur in Candida utilis grown, respectively, on D-glucose or D-xylose. Starvation of glucose-grown cells decreases the K _m value (10.5±2.6 mm). The high-affinity system appearing during starvation required protein synthesis and it was inactivated when cells were exposed to glucose, by a process independent of protein synthesis. High-affinity transport was accompanied by transient alkalinization of yeast suspensions, indicating that it is a proton symport, whereas low-affinity transport was not. Both systems, however, were inhibited by metabolic inhibitors and by replacing H₂O in the transport assay with D₂O, indicating that both may be proton symports. Glucose and acetic acid also inhibited both high-and low-affinity xylose transport.S.G. Kilian, B.A. Prior and J.C. du Preez are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, Republic of South Africa     

Application of a two-stage temperature control strategy for enhanced glutathione production in the batch fermentation by Candida utilis

In batch culture for glutathione production with Candida utilis, a higher temperature (30 °C) was required to hasten cell growth while a lower temperature (26 °C) was needed to increase the production of glutathione. A two-stage temperature control strategy was used to enhance both the yield and the productivity of glutathione. As a result, glutathione production was increased by 5% and 23% of that at 26 °C and 30 °C, respectively, and the intracellular glutathione content reached 2.5% (w/w).     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

Molecular cloning and characterization of the alcohol dehydrogenase ADH1 gene of Candida utilis ATCC 9950

The alcohol dehydrogenase gene (ADH1) of Candida utilis ATCC9950 was cloned and expressed in recombinant Escherichia coli. C. utilis ADH1 was obtained by PCR amplification of C. utilis genomic DNA using two degenerate primers. Amino acid sequence analysis of C. utilis ADH1 indicated that it contained a zinc-binding consensus region and a NAD(P)⁺-binding site, and lacked a mitochondrial targeting peptide. It has a 98 and 73% identity with ADH1s of C. albicans and Saccharomyces cerevisiae, respectively. Amino acid sequence analysis and enzyme characterization with various aliphatic and branched alcohols suggested that C. utilis ADH1 might be a primary alcohol dehydrogenase existing in the cytoplasm and requiring zinc ion and NAD(P)⁺ for reaction.     

Differential location of regulatory and nonregulatory trehalases inCandida utilis cells

The isolation of vacuoles by density gradient centrifugation of protoplast lysates fromCandida utilis cells showed a high specific activity for nonregulatory trehalase in vacuoles whereas the regulatory trehalase activatable by phosphorylation behaves as a cytoplasmic enzyme. The vacuolar trehalase is a glycoprotein that can be precipitated by Con A-Sepharose. Treatment of this enzyme with endo H reduced its reactivity with the lectin without loss of enzyme activity and decreased its apparent molecular weight by gel filtration.Abbreviations cAMP adenosine 3, 5-cyclic monophosphate - Con A Concanavalin A - CP buffer 10mM sodium citrate-phosphate pH 6.8 - endo H endo--N-acetyl glucosaminidase H - PMSF phenyl-methyl-sulphonylfluoride - PNPG p-nitrophenyl--glucoside - PNPP p-nitrophenyl-phosphate     

A note on the kinetics of uptake of d-glucose by the food yeast,Candida utilis

Unlike other yeasts so far investigated, the d-glucose carrier of Candida utilis (strain NCYC 737) appears to change affinity for d-glucose according to its exogenous concentration. When the concentration of d-glucose was <0.4 mM, the apparent K _m 0.2 mM; at >0.4 mM, the K _m 10 mM.     

The influence of different carbon sources and medium osmolarity on the potassium requirements of Candida utilis NCYC 321, growing in continuous culture

In order to study the influence of different carbon sources on the K⁺-requirements of Candida utilis NCYC 321, this yeast was grown at several different dilution rates in potassium-limited continuous cultures with either glucose, glycerol, ethanol, citrate or lactate serving as the carbon and energy source.It was found that the nature of the carbon source profoundly influenced the cellular potassium content, especially at low dilution rates, but that these differences could not be correlated with any differences in relative growth rate (i.e., / _max. And although small amounts of potassium seemingly were needed to serve in osmoregulation and in the cotransport of some acidic carbon sources (lactate and citrate), these requirements were negligible.Independent of carbon source, a strong correlation existed between the intracellular potassium concentration and the yield value on oxygen (Y _O), and between cellular potassium concentration and growth rate. From these two correlations it was concluded that potassium probably was involved mainly in processes associated with ATP synthesis in this yeast.Finally the effect of the addition of NaCl to the medium was tested with glucose-containing cultures that were either carbon- or potassium-limited. Up to a concentration of 20 g/l, NaCl was without influence on Y _O, Y _glucose and q _{O 2}, but effected a slight increase in the cellular potassium content of the potassium-limited cells and a decrease in that of the glucose-limited cells.     

Fermentation ofCandida utilis for uricase production

Summary Conditions for the production of microbial uricase byCandida utilis were studied. For the selected strain, hypoxanthine proved to be the most effective inducer of uricase formation. The highest values of biomass as well as uricase activity in the mechanically agitated fermentor were obtained under the following conditions: 50 h, rotation impeller speed 7 s^–1, air flow rate 1.25×10^–5 m³s^–1, concentration of inducer 0.1%.List of symbols b width of baffle, m - c length of baffle, m - D diameter of cylindrical fermentor, m - d diameter of impeller, m - d ₁ diameter of impeller disc, m - Fr _m impeller Froud number - g gravitional acceleration, ms^–2 - H height of batch surface above bottom, m - H ₂ height of impeller disc above bottom, m - h height of impeller blade, m - Kp _g flow rate number - L length of impeller blade, m - N rotational speed of impeller, s^–1 - Re _m impeller Reynolds number - T time, h - V volume of batch, m³ - V _g air (gas) flow rate, m³s^–1 - x mass fraction of the dry matter of cells - x ₀ initial value of the mass fraction of the dry matter of cells - _r volume fraction of the dry matter of cells - <eta<₁ viscosity of pure liquid, Pa s - viscosity of batch (suspension of microbial suspension), Pa s - density of batch, kg m^–3     

Rubidium as a probe for function and transport of potassium in the yeast Candida utilis NCYC 321, grown in chemostat culture

Attempts to grow the yeast Candida utilis in continuous culture, using media in which all the potassium had been replaced by other monovalent cations, revealed that neither lithium, sodium, caesium nor ammonium ions could functionally substitute for potassium. However, potassium could be effectively replaced by rubidium which gave (on a molar basis, and under conditions where cation availability limited growth) the same yield of cells as did potassium.Comparison of potassium- and rubidium-limited cultures showed them to be virtually identical in all the measured parameters, with the single exception of the maximum growth rate value which was considerably decreased in the rubidium-containing culture (0.35 h^-1 as compared with 0.55 h^-1).When, with variously-limited chemostat cultures, both potassium and rubidium were supplied in equimolar amounts, these ions were taken up by the cells in a ratio that varied with both the growth rate and the nature of the growth limitation. With glucose-, phosphate- or magnesium-limited cultures, the molar ratio K⁺:Rb⁺ was 1:0.6 (at D=0.1 h^-1) and 1:0.17 (at D=0.5 h^-1). In contrast, ammonia-limited cultures took up increased amounts of rubidium when growing at a low rate such that the ratio was 1:1.2, at D=0.1 h^-1, though still 1:0.17 at the higher growth rate value (D=0.5 h^-1).From a comparison of glucose- and ammonialimited cultures growing first with an equimolar mixture of potassium and rubidium, and then with rubidium alone, it was noted that the yield on oxygen was significantly decreased when potassium was absent.These results are discussed in relation to the transport and possible functions of monovalent cations in micro-organisms. It was concluded that, on the basis of these experiments, some objections could be raised against estimation of potassium transport rates by means of the tracer ⁸⁶Rb.     

Purification and characterization of two isoforms from Candida rugosa lipase B

Two isoforms of Candida rugosalipase B (LB1 and LB2) were purified by anionic exchange chromatography. The lipases had the same N-terminal sequence, carbohydrate content and pH and thermal stability but different pIs and significant differences in their activities against different p-nitrophenol esters and triacylglycerides.     

Solid-substrate fermentation ofKloeckera apiculata andCandida utilis on apple pomace to produce an improved stock-feed

Apple pomace was used in solid-substrate fermentation with the yeastsKloeckera apiculata orCandida utilis Y15. A total crude protein content of 7.5% (w/w) was achieved after 72 h for each yeast. The concentration of essential amino acids in the modified apple pomace was more than twice that in the control, enhancing its nutritive value as a stock-feed supplement. The fermentation of pomace as described can be used to reprocess this waste material into a useful value-added product for the agricultural sector.H. Rahmat was and R.A. Hodge, G.J. Manderson and P.L. Yu are with the Biotechnology Group, Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand; H. Rahmat is now with the Department of Chemical Engineering, Queen's University of Belfast, Belfast BT9 5AG, Northern Ireland, UK.     

Influence of specific growth limitation and dilution rate on the phosphorylation efficiency and cytochrome content of mitochondria of Candida utilis NCYC 321

With Candida utilis cells that had been removed directly from a 61 chemostat culture, in steady state, well-coupled mitochondria generally could be isolated. This required a modified snail-gut enzyme procedure that allowed the total processing time to be decreased to 3 h, or less. Examination of these mitochondria in an oxygraph showed the presence of 3 sites of energy conservation when the cells were grown at various dilution rates between 0.1 and 0.45 h^-1 in environments that were, successively, glucose-, ammonia-, magnesium-, phosphate- and sulphate-limited. Potassium-limited cells also apparently possessed 3 sites of oxidative phosphorylation when growing at dilution rates greater than 0.2 h^-1, but only 2 sites when growing at lower dilution rates. Analysis of cytochrome spectra obtained with these intact mitochondria revealed large quantitative (but not qualitative) differences, depending on the environmental conditions under which the yeast had been cultured. In particular, comparison of the ratio of cytochrome b to cytochrome a showed a pattern of change with dilution rate in mitochondria from potassium-limited cells that was distinctly different from those evident in mitochondria from cells that had been limited in their growth by the availability of other nutrients.     

Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.