Growth of yeast Candida utilis in crotonaldehyde containing media

Summary The inhibitory influence of the higher concentration of 20butenal, crotonaldehyde was followed during the batch and long-term continuous fermentation of Candida utilis growing on synthetic ethanol. Most crotonaldehyde is removed from the medium by biotransformation. Crotonaldehyde inhibits the growth, lengthens the lag phase and decreases the biomass yield and the content of crude proteins in the biomass. The yeast C. utilis is capable of growing on media containing very high concentrations of inhibitor in the in-flow during continuous cultivation. Uncharacteristic transport oscillations of the content of crotonaldehyde were observed for which acidic groups on the cell membrane are probably responsible. A sensitive method which is suitable for measuring very low concentration of crotonaldehyde in aqueous solutions is described. Crotonaldehyde acts as an uncompetitive inhibitor with slight mixed type of inhibition. An equation describing the kinetics of inhibition was derived.     

Growth and physiology of a yeast cultivated in batch and continuous culture systems

Inoculum size has been found to affect significantly the maximum attainable specific growth rate during batch cultivation ofCandida utilis. Lower inoculum size resulted in an increased growth rate and relatively longer lag. The culture is found to be most active in the beginning of the exponential phase as regards its RNA synthesis rate. Batch data were used for predicting the conditions of the yeast population in single-stage continuous culture system. Predicted and the experimental values showed a reasonable agreement. In single-stage chemostat the physiology of the yeast was studied on the basis RNA, DNA and protein synthesis rates at various growth rates. The results indicate that the productivity of cells and the rate of synthesis of macromolecules is highest at the dilution rate values of 0.33 to 0.35 hr⁻¹. In order to attain so-called unrestricted conditions of growth a pluristage pluristream continuous system was employed. It is assumed that under such conditions the specific growth rate and the synthetic activity of yeasts may reach its maximum on a given medium. The results presented do not show such conditions of growth under the experimental conditions employed (D ₁=0.35 hr⁻¹ andD ₂=0.2 to 1.7 hr⁻¹) withCandida utilis cultivated on beet molasses medium. Second stage of a two-stage two-stream continuous system is constantly fed with the cells from the foregoing stage; this category of cells on entering the new conditions of the second stage is expected to show some adaptation period. Experiments are reported to this effect.     

Russian Article pp. 255–266

The growth of the yeast Candida utilis VKM-Y-2332 was investigated during the cultivation on a mineral medium with addition of ethanol in the regime of the chemostat and the pH-auxostat. The composition of the macroelements of the biomass produced by Candida utilis VKM-Y-2332 was studied. Stoichiometric equations of the growth process of the examined yeast strain on ethanol were presented. The growth of Candida utilis on ethanol and glucose was also characterized by the way of comparision.     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

Evaluation of the SCP from Opuntia juice

Summary In deseeded opuntia juice medium batch and continuous cultivation ofCandida utilis produced 12 and 19.9gl^–1 yield of the SCP respectively. Its biological value(72), digestibility coefficient(70) and protein score(58) indicated it to be a good protein.     

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l^− 1 in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional–integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.     

Effect of aeration and mixing in various types of cultivation devices on the cell cycle of budding yeasts

When investigating the effect of aeration capacityK _L a of a cultivation device on the cell cycle of daughter cells ofCandida utilis it was found that the length of a phase (S + G₂) of the cell cycle is influenced by the rate of oxygen transfer. An increase ofK _L a, of a cultivation device achieved by increasing the specific output of mechanical energy for air dispersion and mixing may lead to cell damage and to changes in the cell cycle. The effect of high intensity of aeration and mixing is thus invalidated.     

Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells

Recent progress in enzymatic (R)-phenylacetylcarbinol (PAC) production has established the need for low cost and efficient biocatalyst preparation. Pyruvate decarboxylase (PDC) added in the form of Candida utilis cells showed higher stability towards benzaldehyde and temperature in comparison with partially purified preparations. In the presence of 50 mM benzaldehyde and at 4°C, a half-life of 228 h was estimated for PDC added as C. utilis cells, in comparison with 24 h for the partially purified preparation. Increasing the temperature from 4 to 21°C for PAC production with C. utilis cells resulted in similar final PAC levels of 39 and 43 g l⁻¹ (258 and 289 mM), respectively, from initial 300 mM benzaldehyde and 364 mM pyruvate. The overall volumetric productivity was enhanced 2.8-fold, which reflected the 60% shorter reaction time at the higher temperature. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4 and 21°C, respectively, and benzyl alcohol (a potential by-product from benzaldehyde) was not formed.     

Protoplast fusion between a petite strain ofCandida utilis andSaccharomyces cerevisiae respiratory-competent cells

Prototrophic RD mutant cells ofCandida utilis NRRL-Y-1084 and auxotrophic mutant respiratory-competent cells ofSaccharomyces cerevisiae 4003-5Ba his ₄ leu ₂ can ^S meth ₂ trp ₅ ade ₁ ura ₃ gal were turned into protoplasts to be further fused with the aid of polyethylene glycol (PEG) and Ca²⁺ ions. Minimal medium containing glycerol as the carbon source was employed for fusion product selection. The respiratory-competent fusion products, mainly oval cells, resembledCandida utilis and had the fermentative abilities of this strain (dextrose, sucrose, raffinose). Five fusion products were analyzed as to their ability to metabolize dextrose, xylose, cellobiose, trehalose, glycerol, succinic acid, citric acid, salycin, and maltose. Fusion products partially restored the respiratory-competentCandida utilis capacity to grow by use of these carbon compounds, and none of theSaccharomyces cerevisiae fermenting abilities were found. Our results would suggest either a partial recombination between parental mitochondria or some occurring phenomenon affecting the cell, membrane function after somatic fusion without concomitant nuclear fusion.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

The effective expression of xylanase gene in <Emphasis Type="Italic">Candida utilis</Emphasis> by 18S rDNA targeted homologous recombination in pGLR9K

In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml⁻¹ in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis.     

Influence of the initial glycerol concentration on the specific growth rate of Candida utilis cultivated on a synthetic medium containing glycerol as the main carbon source

Summary The following equation correlates the specific growth rate () and the initial concentration of glycerol (S) in batch cultivation of Candida utilis IZ 1840 when S varies from 15 to 75 g L^-1 µ 0 273 - 0.00193 S     

Influence of glucose concentration on the physiology and lipid composition of some yeasts

The fatty acid contents of the “Crabtree-positive” yeastsSaccharomyces cerevisiae, S. carlsbergensis andS. delbrueckii decreased with increasing concentrations of glucose in the medium. These lower values were due to a lower content of sterol esters and phosphatides inS. cerevisiae, and of sterol esters inS. carlsbergensis. In contrast the fatty acid contents of the Crabtree-negativeS. fragilis, Schwanniomyces occidentalis andCandida utilis increased with increasing concentrations of glucose and inCandida utilis this was due almost entirely to a higher content of triglycerides. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

The study of a strain ofCandida utilis in the course of continuous cultivation on ethanol with special respect to biotin requirement

It was found using a long-term continuous cultivation in a synthetic ethanol medium that a strain ofCandida utilis can permanently form a sufficient amount of growth factors. This fact is confirmed both by high yields of biomass and steady level of biotin in the yeast mass. Composition of proteins is the same as in yeasts grown on other carbon sources.     

Repression of Pyruvate Kinase in Candida utilis and Rat Liver by Sclerin through Energy Charge

Though sclerin (SCL) only slightly inhibited the activity of pyruvate kinase (PK) in crude extract of Candida utilis, markedly repressed the level of that in the growing cells in a glucose medium. The repression of PK was largely recovered by 2,4-dinitrophenol (DNP), and SCL rather raised the level in the cells growing on gluconate.

SCL also slightly inhibited the activity of a partially purified PK from rat liver, and, when orally administered, or incubated with the liver slices, obviously lowered the level of PK in the liver and liver slices. The effect of SCL in the liver slices was reversed by DNP. SCL stimulated the oxidative phosphorylation in mitochondria prepared from the fresh liver, and served to maintain the activity in the liver slices during incubation.

Both activity of PK from Candida utilis and rat liver was remarkably inhibited by adenylate energy charge in vitro.

It is concluded that SCL represses the level of PK in these cells and tissues through a high energy charge by stimulating the oxidative phosphorylation.     

Repeated batch cultivation of the microalga Spirulina platensis

Summary The cultivation of photosynthetic microorganisms such as the microalga Spirulina platensis can provide an alternative source of food. The water in Mangueira Lagoon (Rio Grande do Sul state, southern Brazil) has several required nutrients for the growth of Spirulina and could be added to culture medium to reduce the cost of producing S. platensis. Although little studied, repeated batch cultivation is a very useful technique because it has a better cost–benefit ratio than other cultivation methods. In a series of runs, we studied the influence of cell concentration, renewal rate and strain on the specific growth rate and biomass productivity of S. platensis during repeated batch cultivation, the runs taking place in 2-l Erlenmeyer flasks for 2160 h at 30 °C and a light intensity of 2500 lux under a 12 h photoperiod. The three factors studied had a significant (P < 0.05) effect on the results (specific growth rate and productivity). Using Zarrouk’s medium, the highest specific growth rate (μ_X) was 0.111 day⁻¹ while the biomass productivity (P _X) was 0.0423 g l⁻¹ day⁻¹, while Mangueira Lagoon water supplemented with 10% Zarrouk’s medium gave μ_X = 0.113 day⁻¹ and a productivity P _X = 0.0467 g l⁻¹ day⁻¹. These values were two to three times higher than the results obtained in batch cultivation, indicating that the repeated batch cultivation of S. platensis is attractive and convenient.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.     

NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeasts

Summary The kinetics and enzymology of d-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD⁺-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD⁺/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase.     

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO₃ ⁻ and NH₄ ⁺ variation in the media might partially explain the pH stabilization.