Alcohol production by selected yeast strains in lactase-hydrolyzed acid whey

Ethanol production by Kluyveromyces fragilis and Saccharomyces cerevisiae was studied using cottage cheese whey in which 80 to 90% of the lactose present had been prehydrolyzed to glucose and galactose. Complete fermentation of the sugar by K. fragilis required 120 hr at 30°C in lactase-hydrolyzed whey compared to 72 hr in nonhydrolyzed whey. This effect was due to a diauxic fermentation pattern in lactase-hydrolyzed whey with glucose being fermented before galactose. Ethanol yields of about 2% were obtained in both types of whey when K. fragilis was the organism used for fermentation. Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K. fragilis, but galactose was fermented only when S. cerevisiae was pregrown on galactose. Slightly lower alcohol yields were obtained with S. cerevisiae, owing to the presence of some lactose in the whey which was not fermented by this organism. Although prehydrolysis of lactose in whey and whey fractions is advantageous in that microbial species unable to ferment lactose may be utilized, diauxie and galactose utilization problems must be considered.     

Saccharomyces cerevisiae Mutants Resistant to Catabolite Repression: Use in Cheese Whey Hydrolysate Fermentation

Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant.     

Amino acid-dependent transport of sugars by Fusobacterium nucleatum ATCC 10953.

Resting cells of Fusobacterium nucleatum 10953 (grown previously in a medium containing glucose) failed to accumulate glucose under aerobic or anaerobic conditions. However, the addition of glutamic acid, lysine, or histidine to anaerobic suspensions of cells caused the immediate and rapid accumulation of glucose. Except for the amino acid-dependent transport of galactose and fructose (the latter being transported at approximately one-third the rate of glucose), no other sugars tested were accumulated by the resting cells. Amino acid-dependent uptake of sugar(s) by F. nucleatum was abolished by exposure of cells to air, and under aerobic conditions the rates of fermentation of glutamic acid and lysine were less than 15% of the rates determined anaerobically. The energy necessary for active transport of the sugars (acetyl phosphate and ATP) is derived from the anaerobic fermentation of glutamic acid, lysine, or histidine. Competition studies revealed that glucose and galactose were mutual and exclusive inhibitors of transport, and it is suggested that the two sugars (Km = 14 microM) are translocated via a common carrier. The products of amino acid-dependent sugar transport were recovered from resting cells as ethanol-precipitable, high-molecular-weight polymers. Polymer formation by F. nucleatum, during growth in medium containing glucose or galactose, was confirmed by electron microscopy.     

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

Butanol from whey ultrafiltrate: batch experiments with Clostridium beyerinckii LMD 27.6

Summary For batch fermentations by Clostridium beyerinckii LMD 27.7 (formerly known as Clostridium butylicum) whey ultrafiltrate, glucose, lactose, and galactose were used as substrates. The aims of the experiments were to find the conditions for butanol production from whey ultrafiltrate and to compare the results with those of other substrates. The conditions necessary for butanol production were established. The mean solvent productivity found on whey ultrafiltrate fermentation was two to three times lower than that found on glucose; the overall solvent yields were comparable. Butanol production from galactose and mixtures of glucose and galactose was also possible.     

The effects of detergents on anaerobic digestion

Summary The anionic detergent sodium dodecylbenzene sulphonate (SDBS) inhibited mesophilic fermentation in anaerobic digesters. Total gas production and methanogenesis from glucose were reduced to half maximal rates at between 20 and 50 ppm SDBS during the initial phase of digestion, and over 20 days the pH declined from 7.4 to 6.0 in inhibited cultures. As well as accentuating the accumulation of propionic, iso-butyric and iso-valeric acids, a most remarkable effect of this anionic detergent observed only at high concentrations (100 ppm) was to divert the pathway of fermentation with transient accumulation of ethanol. Methanogenesis from cellulose was also inhibited. In thermophilic populations degrading glucose, SDBS was less toxic, and ethanol was not produced. Both the non-ionic detergent Tergitol (nonyl phenyl polyethylene glycol ether) and soap were virtually without effect on the mesophilic anaerobic digestion of glucose.     

Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose

The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.     

Fermentation of lactose by yeast cells secreting recombinant fungal lactase.

Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were not observed. However, a typical biphasic growth was observed on a mixture of glucose and galactose under aerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploid distiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higher ethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeast grown under aerobic conditions. A constant proportion (10%) of the population retained the plasmid throughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobic conditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount of lactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partial galactokinase activity.     

Polygalacturonase production by Kluyveromyces marxianus: effect of medium composition

A strain of Kluyveromyces marxianus (CCT 3172), isolated from a cocoa fermentation in Brazil, secreted an endopolygalacturonase (PG) when grown under self-induced anaerobic conditions; neither polymethylesterase nor pectate lyase appeared in culture filtrates. Replacing glucose in the medium with sucrose had no effect on PG secretion or ethanol production. Growth in fructose-containing medium retarded secretion of PG and ethanol, but had no effect on growth. Growth and ethanol production in media containing galactose resembled those in fructose-containing medium, although PG secretion was lowered. Growth and PG secretion were considerably retarded in xylose-containing medium, and were similarly affected in media containing different concentrations of glucose. Varying the concentration of ammonium sulphate in media had no effect on growth or PG secretion.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions

Streptococcus mutans JC2 produced formate, acetate, ethanol, and lactate when suspensions were incubated with an excess of galactose or mannitol under strictly anaerobic conditions. The galactose- or mannitol-grown cell suspensions produced more formate, acetate, and ethanol than the glucose-grown cells even when incubated with glucose. The levels of lactate dehydrogenase and fructose 1,6-bisphosphate were not significantly different in these cells, but the level of pyruvate formate-lyase was higher in the galactose- or mannitol-grown cells, and that of triose phosphate was lower in the galactose-grown cells. This suggests that the regulation of pyruvate formate-lyase may play a major role in the change of the fermentation patterns. The cells of S. mutans grown on glucose produced a significant amount of volatile products even in the presence of excess glucose under strictly anaerobic conditions. However, when the anaerobically grown cells were exposed to air, only lactate was produced from glucose. When cells were anaerobically grown on mannitol and then exposed to air for 2 min, only trace amounts of fermentation products were formed from mannitol under anaerobic conditions. It was found that the pyruvate formate-lyase in the cells was inactivated by exposure of the cells to air.     

Lactate removal by anionic-exchange resin improves nisin production by Lactococcus lactis

When lactate was removed from sucrose fermentation in situ, using the anionic-exchange resin Amberlite IRA-67, by Lactococcus lactis growing in batch culture, nisin production increased by two-fold when compared to the alkali pH-controlled fermentation. In comparison to sucrose, lactate removal increased nisin production 1.5-fold and 0.3-fold when galactose and glucose were used as carbon sources respectively.     

Growth of Neocallimastix sp. Strain R1 on Italian Ryegrass Hay: Removal of Neutral Sugars from Plant Cell Walls

The anaerobic fungus Neocallimastix sp. strain R1 was grown for up to 5 days on a medium containing autoclaved Italian ryegrass hay as the carbon source. Culture supernatants and digested cell walls were harvested at 12-h intervals. Supernatants were analyzed for the fermentation products formate and acetate, and residual cell walls were analyzed for dry-matter and neutral-sugar losses. Fungal growth was accompanied by the digestion of plant cell walls and the accumulation of fermentation products in culture media. Dry-matter losses were accounted for by removal of four major neutral sugars (arabinose, galactose, glucose, and xylose) from the plant cell walls. First-order reaction kinetics could be used to describe the loss of each sugar. All cell wall sugars, including arabinose and galactose, which are not fermented by Neocallimastix sp. strain R1 were removed simultaneously. Although the rates of removal of individual sugars were similar, there were significant differences in their extents of removal: the extent of removal of arabinose exceeded that of the other three sugars, and xylose was the least digestible. This study provides the first account of simultaneous (nonpreferential) removal of neutral sugars from plant cell walls by an anaerobic fungus. Although in vitro techniques were used, these results indicate a potentially significant role for the anaerobic fungi as fiber digesters in the rumen.     

An evaluation of D-glucosamine as a gratuitous catabolite repressor of Saccharomyces carlsbergensis.

Summary Glucose represses mitochondrial biogenesis and the fermentation of maltose, galactose and sucrose in yeast. We have analyzed the effect of D-glucosamine on these function, in order to determine if it can produce a similar repression. It was found that glucosamine represses the respiration rate (QO₂) but more rapidly than glucose and to a final level slightly higher than in glucose-treated cells. Derepression of the respiration rate following either glucose or glucosamine repression was similar. A two hour lag was followed by a linear increase in QO₂ to the derepressed level. Both glucose and glucosamine repressed the level of cytochrome oxidase to the same level. Glucosamine was also found to repress maltose and galactose fermentation but not sucrose fermentation. The derepression of maltase synthesis was inhibited by glucosamine. The constitutive synthesis of maltase was repressed by the addition of glucosamine. Glucosamine was judged to produce a repressed state similar to glucose repression in many respects.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

The response by microorganisms to steady-state growth in controlled concentrations of oxygen and glucose. II. Saccharomyces carlsbergensis

The growth of Saccharomyces carlsbergensis in continuous culture has been studied when dissolved oxygen and glucose concentrations were held constant at a series of steady-state levels. Both oxygen and glucose controlled the degree of aerobic metabolism and of ethanolic fermentation. When the glucose uptake rate was low (between 1.2 and 2.8 mmoles per hour per gram of yeast) the relative distribution of glucose between ethanolic and aerobic fermentation was sensitive to oxygen: when dissolved oxygen was near to saturation, glucose metabolism was 0.98 aerobic; when dissolved oxygen was 0.01 saturated, 0.8 of intake glucose metabolism was by ethanolic fermentation. On the other hand when glucose intake was high (between 7.6 and 18.2 mmoles) metabolism was predominately by ethanolic fermentation even when dissolved oxygen concentration was at saturation. The extent, to which catabolism proceeded by an anaerobic or aerobic pathway, as judged by ethanol production, was controlled more by the uptake of glucose than of oxygen.