Selection and Study of Potent Lactose-Fermenting Yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen strains from milk products, showing maximum potency, fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11–12, and 10–12%, respectively (4°C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30°C for 2–3 days, the ethanol concentration was 4–5%.     

Isolation and characteristics of the lactose-fermenting yeasts Candida kefyr

Screening of lactose-fermenting yeast strains has been conducted among 162 strains isolated from various plants and 28 strains isolated from cheese. Four yeast strains fermented lactose and were identified as Candida kefyr. The specific β-galactoside activity of the studied strains was 1501–2113 IU/g dry biomass. The ability of strains C. kefyr C24 and C30 to produce ethanol from lactose was significantly inhibited by the increase in substrate concentration (100 g/L).     

Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions (approximately 60 degrees C) was studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of approximately 0.93 g g(-1). Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO2 and H2. The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO2 and H2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose was converted to ethanol by C. thermolacticum.     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Protein enrichment of potato processing waste through yeast fermentation

Potato starch obtained from waste waters of chips manufacturing was used as a fermentation substrate for yeast protein enrichment. Among 18 yeast strains, 6 strains were screened according to their biomass yield and protein content after fermentation for 16 h at 30 degrees C in an aerated glucose-based liquid media (4.5 Ls). Using concentrated media (25% solids) made from potato starch pre-hydrolyzed with malt flour and batch-fermented for 20 h at 26 degrees C under aerobic conditions, Candida utilis ATCC 9256 was the most efficient protein-forming strain. Scaled-up at the 100 Ls level, the aerobic batch process was improved under fed-batch conditions with molasses supplementation. After drying, fermented starch contained 11-12% protein, including 7-8% yeast protein.     

Isolation of thermotolerant ethanologenic yeasts and use of selected strains in industrial scale fermentation in an Egyptian distillery

An enrichment and isolation program for new ethanol-producing thermotolerant yeasts as well as a screening program of some known thermotolerant strains resulted in the selection of several strains capable of growth at 40-43 degrees C. Among these strains four grew and fermented sugar cane molasses at 43 degrees C under batch conditions with sugar-conversion efficiencies >94% and ethanol concentrations 6.8-8.0% (w/v). The two best-performing strains, a Saccharomyces cerevisiae F111 and a Kluyveromyces marxianus WR12 were used in eight 87.5 m(3) fermentation runs (four using each strain) for industrial ethanol production in an Egyptian distillery using sugar cane molasses. Mean ethanol production was 7.7% and 7.4% (w/v), respectively, with an added advantage of cooling elimination during fermentation and higher ethanol yields compared to the distillery's S. cerevisiae SIIC (ATCC 24855) strain in use. The isolate S. cerevisiae F111 was subsequently adopted by the distillery for regular production with significant economical gains and water conservation.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Ethanol fermentation in a continuous tower fermentor

A mutant of Saccharomyces cerevisiae, which forms large, multicellular flocs in liquid culture, rapidly fermented media containing high concentrations of glucose (100-180 g/L) in a continuous nonaerated tower fermentor at 30 degrees C. The fermentor operated continuously for seven months. Batch and tower fermentor data were fitted to a kinetic model incorporating linear ethanol inhibition and Monod dependence on glucose. Conversion, ethanol yield, and ethanol productivity were related to the apparent fermentation time for initial glucose concentrations of 130 and 180 g/L. Productivities of 8-12 g ethanol/L h were achieved through the yeast bed giving conversions exceeding 90% of the theoretical yield.     

Continuous production of ammonium lactate by Streptococccus cremoris in a three-stage reactor

Batch and continuous fermentation studies were performed to optimize the production of ammonium lactate from whey to optimize the production of ammonium lactate from whey permeate. The product known as fermented ammoniated condensed whey permeate (FACWP) is a very promising animal feed. After an initial screening of four strains which produce predominantly L(+)- lactic acid, the desired isomer [D(-)-lactic acid is toxic], Streptococcus cremoris 2487 was chosen for further study. In batch mode, pH between 6.0 and 6.5 and 35 degrees C provided optimum incubation conditions. To stimulate a plug flow reactor, three CSTRs (continuous stirred tank reactors) were connected in tandem. For a 7.5-h retention time, 1.6-fold and 1.3-fold higher productivities were obtained for three-stage than for the single- and two-stage reactors, respectively. Various retentions times were examined (5, 7.5, and 10 h; 5g/L yeast extract). Although maximum lactate productivity occurred at a 5-h residence time (5.38 g/L H. 75% lactose utilization), lactose utilization was more complete at 7.5 h (4.38 g/L h productivity, 91% lactose utilization and a productivity, 91% lactose utilization). Retention time was increased to 15 h to obtain 95.9% lactose utilization and a productivity of 2.42g/L h for 2g/L yeast extract. Based on this lower yeast extract concentration, it was determined that ammonium lactate production and subsequent concentration by 11-fold would yield a product (FACWP) 17% more than soybean meal (crude protein contents are equivalent, 44%) at current market prices.     

Cellulolytic cocci isolated from the cecum of guinea pigs (Cavia porcellus).

Five strains of anaerobic, gram-variable cellulolytic cocci, belonging to the genus Ruminococcus, were isolated from the cecum of a guinea pig. They differed from most previously described strains of cellulolytic ruminococci as follows. (i) Lactate was the major fermentation product; lesser amounts of formate and ethanol and a trace of succinate were also produced, along with an uptake of acetate. (ii) No growth occurred at 30 degrees C; however, good growth was observed at 38 and 45 degrees C, (iii) Glucose, cellobiose, cellulose, xylose, arabinose, xylan, sucrose, and lactose were fermented by all strains. Rumen fluid was required for growth in a complete medium containing all nutrients previously found to be required by species in this genus. Limited growth occurred when rumen fluid was replaced by yeast extract, and maximum, but delayed, growth occurred when a water extract of alfalfa was added to the complete medium. No qualitative differences were found in the cell wall amino acids and sugar composition of these strains as compared to Ruminococcus flavefaciens and Ruminococcus albus; however, cell walls of the guinea pig strains appeared to contain a higher proportion of glucose.     

Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha

Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up to 45 degrees C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.     

Optimization of fermentation conditions for ethanol production from whey

Summary Optimal conditions for ethanol production in 7% whey solutions by the yeast Candida pseudotropicalis ATCC 8619 included initial pH of 4.57 and 30°C. Complete fermentation of the available lactose took place without supplementary nutrients; additions of nitrogen or phosphorus salts, yeast extract or corn steep liquor resulted in increased yeast production and lower ethanol yields. A positive correlation was observed between increases in yeast inocula and lactose utilization and ethanol production rates; 8.35 g/l of ethanol was obtained within 22 h by using yeast inoculum of 13.9 g/l. No differences in fermentation rates or ethanol yields were observed when whole or deproteinized whey solutions were used. Concentrated whey permeates, obtained after removal of the valuable proteins from whey, can be effectively fermented for ethanol production.     

Lantana camara for fuel ethanol production using thermotolerant yeast

AIM: Evaluation of Lantana camara's use as feedstock for fuel ethanol production. METHODS AND RESULTS: Lantana camara plant material was hydrolysed with 1% sulfuric acid for 18 h at room temperature, followed by heat treatment of 121 degrees C for 20 min. Hemicellulosic hydrolyzate was separated and used for detoxification by ethyl acetate and overliming. Cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 55 degrees C. Using 15% (dw/v) substrate 73 g l(-1) total reducing sugars were obtained to give 78.7% hydrolysis of carbohydrate content. Acid and enzyme hydrolyzates were mixed equally and used for fermentation with thermotolerant Saccharomyces cerevisiae (VS(3)). Yeast fermented L. camara hydrolyzate well with a fermentation efficiency of 83.7% to give an ethanol yield of 0.431 +/- 0.018 g ethanol pre g sugar and productivity of 0.5 +/- 0.021 g l(-1) h(-1). CONCLUSIONS: Even though inhibitors were present in L. camara hydrolyzate, maximum sugars were utilized by thermotolerant yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of L. camara for fuel ethanol production with improved strains and detoxification can be recommended.     

Comparison of yeast strains for batch ethanol fermentation of cheese-whey powder (CWP) solution

AIMS: To test the suitability of cheese whey powder (CWP) solution for ethanol fermentation and to compare performances of different Kluyveromyces marxianus strains for ethanol fermentation from CWP solution. METHODS AND RESULTS: Batch ethanol fermentation of cheese whey (CW), CWP and lactose solutions with the same initial sugar contents were compared by using two different K. marxianus strains and the CWP solution was found to be the most suitable substrate. CWP solution was fermented to ethanol using three different yeast strains and DSMZ-7239 was found to be the most suitable one yielding the highest rate and extent (3.3%, v/v) of ethanol formation. CONCLUSIONS: CWP solution and K. marxianus strain of DSMZ-7239 were found to be more suitable for ethanol fermentation with the highest ethanol yield when compared with the other substrates and the yeast strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: CWP can be used as a concentrated form of CW for ethanol fermentations with considerable advantages.     

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Selection of yeast strain and fermentation conditions for high-yield ethanol production from lactose and concentrated whey

A total of 65 yeast strains were screened for their ability to grow and ferment lactose in a standard DURHAM tube test at 30 °C. Based on the kinetic parameters for lactose and whey lactose fermentations in shake flask cultures, the strain Candida psedotropicalis 65 was chosen for further studies. Some of the cultural parameters affecting ethanolic fermentations on lactose were standardized. At an initial lactose concentration of 100–120 g/l in the medium containing concentrated whey or lactose, at 40 °C and within 48 h, the selected strain reached an ethanol concentration of 41–59 g/l, an ethanol productivity of 1.3–3.0 g/l/h, a lactose consumption of 99%, an ethanol yield 0.4–0.49 g/g and a biomass yield of 0.027 g/g.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20 degrees C for 24 h and then stored at either 20 degrees C or 5 degrees C for 96 h. Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20 degrees C and 5 degrees C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10(7)-10(9)/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10(3)-10(6) and 10(2)-10(5) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Higher maximum numbers, 10(9)-10(10), of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10(5)-10(8) and 10(4)-10(7) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.