Release of protein from Bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill

Release of protein from a suspension of bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill has been studied. Protein release on disruption in the mill is a first-order rate process. The rate constant is dependent on at least six parameters. Increased disruption efficiency was obtained at higher agitator speeds, greater loading of bead attritive elements and lower rates of upward recycle of yeast suspension through the mill. An increase in temperature from 5 to 42°C was accompanied by a reduction in disruption efficiency of approximately 20%. With optimal values of the parameters examined the throughput of the mill is 5.32 kg/hr of soluble protein for 90% disruption.     

Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO₂ and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.     

Experiences with a 20 litre industrial bead mill for the disruption of microorganisms

Suspensions of several yeast strains and bacterial species were disrupted in a continuously operating industrial agitator mill of 22.7 litre internal working volume. The influence of agitator speed, flow rate, concentration of microorganisms in the slurry, packing density of glass beads and bead diameter on the disruption process was studied using baker's yeast (Saccharomyces cerevisiae). Cell disintegration was followed by assaying the appearance of protein and the activities of d-glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate:NADP⁺ oxidoreductase, EC 1.1.1.49] and α-d-glucosidase [α-d-glucoside glucohydrolase, EC 3.2.1.20] in the soluble fraction. The best operating conditions for the disintegration of baker's yeast with respect to activity yield appeared to be at a rotational speed of 1100 rev/min, a flow rate of 100 litre h^?1 and a cell concentration of 40% (w/v). The location of the desired enzyme in the cell is of importance for the choice of bead diameter and packing density of the glass beads. Temperature increase and power consumption during disintegration are also strongly influenced by the bead loading in the mill. With optimized parameters, 200 kg baker's yeast can be processed per hour with a degree of disintegration >85%. The disruption process in the mill was found to be very effective for several yeast species tested, e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Candida boidinii. The usefulness of the Netzsch LME 20-mill for the disruption of bacteria species was demonstrated with Escherichia coli, Brevibacterium ammoniagenes, Bacillus sphaericus and Lactobacillus confusus. As expected, the mill capacity for bacterial disruption was significantly smaller than for the yeast. Between 10 and 20 kg per h bacteria may be processed, depending on the organism.     

Mechanical disintegration of microorganisms in an industrial homogenizer

Disintegration of microorganisms in a continuously working industrial homogenizer has been studied. The homogenizer consists of rotating discs in a cylinder filled with glass beads. Different parameters for disintegration of baker's yeast were investigated. The disintegration process is a first-order reaction and it is influenced by the flow rate of the suspension and by the agitator speed. At a flow rate of 200 liters/hr about 85% of the yeast cells can be disrupted in a single pass through the disintegrator. This type of disintegrator can be used for disruption of cells in order to produce single-cell protein, active enzymes and other valuable cell components.     

Investigation of some methods for increasing the digestibility in vitro of microalgae

In order to increase the availability of the cell bound protein in Scenedesmus algae, mechanical, enzymatic, and chemical methods of degrading the cell wall structure were investigated. Mechanical treatment involved the use of a ball-mill. The algae suspension together with glass beads was milled in a water-cooled chamber equipped with rotating disks. The enzyme tested was a cellulolytic enzyme (Meicelase) and the chemical employed was hydrogen peroxide. In the ball-mill experiments a complete disintegration was achieved ina disintegrator, working with batches. Trails wwere also performed with a continuous disintegrator and the depedence of disintegration on bead size and flow rate was studied. The disintegration determined by microscropic cell count was compared to the increase of the pepsin digestibility. The meicelase treatment caused a slight increase of the pepsin digestibility, as measured after 3 hr pepsin incubation. No increase of the pepsin disgestibility could be detected with hydrogen peroxide treatment. After the ball-mill disintegration 95% of contaminating bacteria were killed and yields of extractable proteins were higher. The capacity of availble continuous ball-mills is such that they could be used on a pilot-plant scale and the energy cost of disintegration would be of the same magnitude as that of separation.     

Disintegration of yeast Saccharomyces cerevisiae in the vertical perl mill with a bell-shaped impeller

Investigation of disintegration of yeast Saccharomyces cerevisiae in the laboratory batch perl mill with a bell-shaped impeller was carried out. The number of non-damaged cells, changing in time was determined using hemocytometer (Thom's chamber).To describe kinetics of the disintegration process the differential equation was applied: where N _p the number of non-damaged cells in the sample, [number of cells/ml] t time, [s] m,k constants.The effect of three operating parameters: rotation frequency of the impeller shaft n, filling of the mill with disintegrating elements (ballotini) S _k and the initial concentration of yeast cells in the suspension C ₀ on the process of disintegration was analyzed.For S _k =0.5, m=1 and dependence of constant k on the rotation frequency of the impeller and suspension concentration were obtained. For S _k =0.6 and 0.7 the values of m were higher than 1. The effect of rotation frequency of the impeller and filling of the mill, with ballotini on constant k and exponent m was determined.List of Symbols a, b constants - a ₁, b ₁, c ₁, d ₁ constants - C ₀ initial concentration of suspension g/ml - C concentration of cell suspension g/ml - k constant disintegration rate 1/s; N ₀ ^1-m /s - m variable in the equation - N ₀ initial number of cells no. of cells/ml - N _p number of non-damaged cells no. of cells/ml - r process rate g/ml·s - X(t) disintegration degree % - , variables in the equation - z variable in the equation - S _k degree of filling the mill with disintegrating elements     

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.     

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

Reactor properties of a high-speed bead mill for microbial cell rupture

Laboratory and pilot-plant high-speed bead mills of 0.6 and 5 liter capacity and consisting of four and five impellers in series, respectively, were used to follow the batch and continuous disruption of bakers' yeast (Saccharomyces cerevisiae). The mills are not scaled equivalents. Throughputs ranging from 1 × 10^?6m³/sec to 12 × 10^?6m³/sec for the 0.6 liter mill and from 16 × 10^?6m³/sec to 100 × 10^?6m³/sec for the 5 liter mill were used for continuous disruption studies. Variables studied included the effect of impeller tip speed, temperature, and packed yeast concentration (ranging from 15 to75% by weight packed yeast). Disruption kinetics, as measured by the release of soluble protein, followed a first-order rate equation, the rate constant being a function of impeller tip speed and yeast concentration. For continuous disruption studies the bead mills behaved as a series of continuous stirred-tank reactors, each impeller forming a reactor. In the smaller mill a considerable degree of backflow between the reactors was evident. For certain mixing conditions the maximum amount of releasable protein was dependent on the impeller geometry, construction material, and also the concentration of packed yeast. The relative power efficiencies of the two mills are discussed along with possible criteria for scaling of bead mills.     

Restoration of respiratory competence in the yeastCandida utilis after somatic fusion withSaccharomyces cerevisiae

After somatic fusion between a mitochondrial mutant ofCandida utilis andSaccharomyces cerevisiae respiratory competent strain, the complex III of the respiratory chain seems to be restored. Fusion products, FP, synthesizing normal apocytochromeb were recovered and showed normal-shaped mitochondria along the cytoplasm as in theCandida utilis original, respiratory-competent strain.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Preparation of zymosan from yeast cell walls

Cell walls of the yeastSaccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5m Na₂HPO₄, pH 7.8–8.0, as a 5% or 10% suspension, depending on the mode of heating. The suspension was boiled for 3h, purified by repeated washing with water and ethanol and dried. The yield was approximately 1.8% of the starting amount of pressed commercial baker’s yeast.     

Flavin-linked mitochondrial α-glycerophosphate dehydrogenase of Candida utilis

The 150-fold purification of the l-α-glycerophosphate dehydrogenase of Candida utilis electron-transport particles by very mild procedures is described. The active enzyme contains FAD, iron and copper. The function of the metals, if any, is not clear. Its molecular weight is about 5·10⁵. The subunit composition is complex and remains unresolved because the enzyme is contaminated with protease(s). The activity of this enzyme is very low in Saccharomyces cerevisiae unless the cells are grown in glycerol. The NAD-dependent cytoplasmic α-glycerophosphate dehydrogenase is present in C. utilis but could not be demonstrated in glucose-grown S. cerevisiae.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

A continuous,multistage tower fermentor. I. Design and performance tests

The design of a continuous column fermentor with a multiple staging effect is described. The column is divided into four compartments by horizontal perforated plates and is provided with a central agitator shaft driving an impeller in each compartment. A tube at the center of each plate forms a liquid seal around the shaft and also acts as a “downcomer.” The fermentor is normally operated with counter-current flow of gas and medium. Fresh medium is added to the top stage and product is withdrawn from the bottom. The effect of plate and agitator design on fermentor performance was studied in terms of factor such as oxygen transfer rate, gas holdup, and interstage mixing. By proper choice of the design parameters, the fermentor was made to approximate a perfect four-stage cascade in terms of reactor performance. Preliminary experiments were performed with air-water systems, but a more realistic picture of fermentor performance was obtained in experience involving propagation of Escherichia coli. Data for business and substrate concentrations in each stage confirmed the staging effect of the apparatus. The fermentor operated in a stable manner for periods of more than two weeks.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

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.     

Resistance ofSchizosaccharomyces pombe to mechanical strain caused by mixing in continuous culture

Schizosaccharomyces pombe is less resistant to mechanical strain caused by mixing thanCandida utilis orSaccharomyces cerevisiae. The highest utilizable frequency of stirring in the continuous culture is 11.9 Hz.