Lipid production by Cryptococcus albidus using biowastes hydrolysed by indigenous microbes

The efficiency of Cryptococcusalbidus was evaluated for its abilities to assimilate onion and apple hydrolysates as a medium for lipid production. Onion waste (OW) and apple waste (AW) were hydrolysed at an organic load of 2% total solids by indigenous microbes under mesophilic conditions. The indigenous microbes effectively hydrolysed both wastes giving the highest reducing sugar content of 4.8 g/L and 10.8 g/L with OW and AW hydrolysates, respectively. The microbiome analysis revealed that most of the indigenous microbes belonged to genus Bacillus and a significant population of α-proteobacteria and γ-proteobacteria were also present. Cell retention culture of C. albidus at a dilution rate of 0.01 h⁻¹ resulted in a total dry cell weight (DCW) of 13.5 g/L with an intracellular lipid content of 20.0% at 168 h, corresponding to an enhancement of 3.48-folds and 2.37-folds in DCW and lipid concentration, respectively, as compared to batch fermentation.

     

Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures ofAspergillus niger

Aspergillus niger has been grown in glucose- and maltose-limited continuous cultures to determine the bioenergetic consequences of the production of the extracellular enzyme glucoamylase. Growth yields (g biomass per mol substrate) were high, indicating that growth was very efficient and protein production for biomass was not exceedingly energy consuming. It has been found that the energy costs for the production of this extracellular enzyme is very high. Depending on the efficiency of energy conservation the glucoamylase protein yield on ATP is between 1.3 and 2.6 g protein per mol ATP, which is equal or less than 10% of the theoretical maximum of 25.5. These high energy costs most probably have to be invested in the process of excretion. A comparison between an industrial over-producing strain and the wild typeAspergillus niger showed that this over-producing strain most probably is a regulatory mutant. Two regions of specific growth rates could be determined (one at specific growth rates lower and one at specific growth rates higher than 0.1 h^-1), which are characterized by differences in mycelium morphology and a significant deviation from linearity in the linear equation for substrate utilization. Analysis of the region of specific growth rates higher than 0.1 h^-1 yielded maintenance requirements of virtual zero. It has been concluded that for a good analysis of the growth behaviour of filamentour fungi the linear equation for substrate utilization is not suitable, since it contains no term for the process of differentiation.     

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Ploidy differences in Cryptococcus albidus

Presumed haploid and diploid cultures ofCryptococcus albidus were analysed for their DNA content per cell. A ratio of approximately 1:2 was obtained by relating the DNA content per cell of the two phases to ploidy. As the diplophase formed neither longitudinally septated cells nor ballistoconidia, the earlier suggestions thatCryptococcus is closely related toTremella seems less likely. On the assumption that the metabasidia ofCryptococcus are gastromycetoid, a closer relationship between this genus and the tulasnelloid fungi appears more probable.Microbiology Research Group, South African Council for Scientific and Industrial Research, Pretoria, South Africa.     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Characterization of laccase activity produced by Cryptococcus albidus

This study deals with the characterization of laccase enzyme activity produced by Cryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a number of chemicals. These chemicals can interfere with the proper functioning of the enzymes used for bioremediation. Thus, it is important to study the effect of such interfering solvents, detergents, metal chelators, and other chemicals on enzyme activity before industrial applications. Laccase showed maximum activity at pH 2.5 and temperature 20-30°C when ABTS was used as a substrate. The enzyme followed Michaelis-Menten kinetics: K(m) was 0.8158 mM and V(max) was 1527.74 U/mg. Laccase showed good thermostability with a half-life of 81 min at 25°C, 77 min at 35°C, 64 min at 45°C, 36 min at 55°C, and 21 min at 65°C. There was no effect of sodium dodceyl sulfate (SDS) (0.1-1.0%) and EDTA (0.1-0.5%) on laccase activity. Sodium azide and 2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lower concentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, the activity was completely inhibited at a concentration of 50% or above of acetone, methanol, 1,4-dioxan, and acetonitrile.     

Uptake and utilization of glutamic acid by Cryptococcus albidus

Cryptococcus albidus utilizes glutamate as a sole carbon source. The kinetics of uptake of this amino acid were studied. l-Glutamic acid was taken up by two saturable systems: a high affinity system with a Michaelis constant (K(m)) of 1.15 x 10(-5) M and a V(max) of 0.049 mumol per mg per h and a low affinity system with a K(m) of 2.5 x 10(-3) M and a V(max) of 3.61 mumol per mg per h. Both systems possessed characteristics of active transport which were dependent on temperature and pH and which required metabolic energy. Uptake was inhibited at 37 C but the temperature-sensitive step was reversible. Chemical fractionation of cells with 5% trichloroacetic acid showed that glutamic acid initially entered a soluble pool which decreased after 1 h as the amino acid was incorporated into the protein and nucleic acid fractions of the yeast. Some of the glutamate was completely oxidized and could be recovered as (14)CO(2). Therefore, the amino acid was also used as an energy source.     

Stimulation of xylanase synthesis in Cryptococcus albidus by cyclic AMP

Cryptococcus albidus secretes a xylanase when induced by xylan or beta-methylxyloside, a non-metabolizable inducer, and production of the enzyme is repressed by xylose. The effect of exogenous cAMP on xylanase production was tested under different growth conditions. The cAMP elicited a 1.5 to 2 fold increase in xylanase production during the induction by xylan and B-methylxyloside but did not relieve the repression observed during growth on xylose. Cyclic AMP also affected the growth rate of the cells and did not modulate the activity of pure xylanase in vitro. A 15-nucleotide sequence located upstream from the xylanase gene could be part of a cAMP regulatory sequence.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

Induction of xylanase by β-methylxyloside in Cryptococcus albidus

Abstract The compound β-methylxyloside (β-MX) was found to induce the production of extracellular xylanase (EC 3.2.1.8) by the yeast Cryptococcus albidus . The induction of xylanase by β-MX requires de novo protein synthesis and proceeds via de novo accumulation of translatable mRNA coding for xylanase as demonstrated by the addition of cycloheximide. In vitro translation of cellular messenger RNA followed by immunoprecipitation with xylanase-specific antibodies reveals that the stimulation leads to the appearance of xylanase-coding mRNA. In vivo labeling experiments show that the β-MX induces specifically the 48 kDa xylanase, and that the addition of xylose in the culture medium reverses the β-MX action by suppressing completely the production of xylanase by the cells.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48 000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40 000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40 000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44 000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.     

Beta-glucanases in the yeast Cryptococcus albidus var. aerius. Production and separation of beta-glucanases in asynchronous cultures.

beta-Glucanases were detected in cell-free extracts of the yeast Cryptococcus albidus var. aerius when grown on glucose as the sole carbon source. The production of beta-glucanases was followed in log-phase cells and stationary-phase cells; the maximal production of beta-(1 leads to 3) and beta-(1 leads to 6) glucanases takes place respectively in log-phase and stationary-phase cells. The results show that there are marked differences in the elution profiles on Sephadex G-50 of fractions containing beta-glucanase from cells grown for 12, 24, 48, 72, and 96 h. The possibility either of replacement changes in fractions containing beta-glucanase activity or of a different synthesis of each beta-glucanase during the growth of the yeast is discussed. The results suggest that all fractions containing beta-glucanases hydrolyze both beta-(1 leads to 3) and beta-(1 leads to 6) linkages. Evidence in support of the conclusion that a low molecular form of beta-glucanase has a molecular weight of 2100 +/- 100 is also shown.     

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

Metabolic aspects of the growth of a pink-pigmented facultative methylotroph in carbon-limited chemostat culture

Summary The regulation of carbon metabolism in a pink-pigmented facultative methylotroph has been studied. In methanol-limited chemostat culture a pH optimum at 7.0 with a narrow growth rate optimum with respect to growth yield and metabolic uncoupling was revealed. The average growth yield was 14±0.036 g·mol^–1 and the organism displayed a low maintenance energy and high maximum specific growth rate. When the carbon concentration in the feed remained constant and the dilution rate increased a deviation from linearity between substrate consumption and growth rate was found at higher growth rates. The addition of a pulse of methanol to a carbon-limited culture showed that anabolism could be dissociated from catabolism with the resulting accumulation of formaldehyde in concentrations which were not lethal. Offprint requests to: F. M. Girio     

beta-Xylosidases in the yeast Cryptococcus albidus var. aerius.

beta-xylosidase activity has been detected in cell-free extracts and in culture fluids when Cryptococcus albidus var. aerius was grown on glucose as the sole carbon source. The enzyme appears to be constitutive. Mild acid treatment of whole cells suggested that the total activity is located in the periplasmic space and some experiments indicated that it is partially associated with the cell walls. DEAE-Sephadex A50 chromatography has shown that there are two different forms of beta-xylosidase in the cell-free extracts, but only one form is present in the supernatants of culture.     

Energy conservation by pyrroloquinoline quinol-linked xylose oxidation in Pseudomonas putida NCTC 10936 during carbon-limited growth in chemostat culture

Abstract When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents, Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate. No other products were formed from this pentose, nor was it incorporated into biomass. The presence of xylose in these cultures resulted in higher Y_glucose and Y_lactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose. As the Y₀ values for growth on glucose or on lactate alone were equal to the Y₀ values with xylose as co-substrate, it is concluded that for flucose- or lactate-limited growth energy conservation by PQQH₂ oxidation is as efficient as by NADH₂ oxidation.     

Overproduction of nitrogenase by nitrogen-limited cultures of Rhodopseudomonas palustris.

Rhodopseudomonas palustris cells grown on limiting nitrogen produced four- to eightfold higher nitrogenase specific activity relative to cells sparged with N2. The high activity of N-limited cells was the result of overproduction of the nitrogenase proteins. This was shown by four independent techniques: (i) titration of the Mo-Fe protein in cell-free extracts with Fe protein from Azotobacter vinelandii; (ii) direct detection of the subunits of Mo-Fe protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (iii) monitoring of the electron paramagnetic resonance spectrum of Mo-Fe protein in whole cells; and (iv) immunological assay of the Fe protein level with an antiserum against the homologous protein of Rhodospirillum rubrum. The derepressed level of nitrogenase found in N2-grown cells was not due to an increased turnover of nitrogenase. The apparent half-lives of nitrogenase in N2-grown and N-limited cells were 58 and 98 h, respectively, but were too long to account for the difference in enzyme level. Half-lives were determined by measuring nitrogenase after repression of de novo synthesis by ammonia and subsequent release of nitrogenase switch-off by methionine sulfoximine. Observations were extended to R. rubrum, Rhodopseudomonas capsulata, and Rhodomicrobium vannielii and indicated that overproduction of nitrogenase under nitrogen limitation is not an exceptional property of R. palustris, but rather a general property of phototrophic bacteria.