Fermentation of whey and starch by transformedSaccharomyces cerevisiae cells

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains ofSaccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced.l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspective are discussed.     

Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation

The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular ß-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-to-ethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Fed-batch fermentation to produce oligonucleotides from Kluyveromyces marxianus grown on whey

Oligonucleotides (ON) extracted from yeasts are used as antiviral agents, immunostimulators, and flavour enhancers. Fed-batch fermentation of cheese whey by Kluyveromyces marxianus was carried out to produce high biomass yields to extract ON. K marxianus was grown for 20 h in medium containing 5% (w/v) dehydrated whey, at 30°C (pH 4.5), with agitation (350 rpm), and under aeration (1.0–2.0 vvm). After 20 h, media containing 10–15% (w/v) of dehydrated whey were added at different flow rates (180–230 ml/h). Samples were analyzed at 6–8 h intervals for cell count, lactose consumption, and ethanol production. Maximum production of biomass (28.13 g/l), yield (0.58 g/g), productivity (2.42 g/l per h), and specific growth rate (0.63 1/h) were obtained when medium containing 15% (w/v) of whey was added at 180 ml/h under 2 vvm aeration. Fed-batch fermentation converted 95% of whey lactose into biomass.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Optimizing alcohol production from whey using computer technology

This study was undertaken with the major goal of optimizing the ethanol production from whey using computer technology. To reach this goal, a mathematical model that would describe the fermentation and that could be used for the optimization was developed. Kluyveromyces fragilis was the microorganism used to ferment the lactose in the whey into ethanol. Preliminary studies showed that K. fragilis produced about 90% of the theoretical ethanol yield when grown in whey-complemented media. However, when this yeast is grown in nonsupplemented whey media, it does not produce more than 32% of that yield. Comparative batch fermentations of lactose and whey-complemented media showed that whey possibly contains enhancing components for yeast growth and ethanol production. To obtain the mathematical model, the one-to-one effect of the process variables (lactose and yeast extract concentrations, air flowrate, pH, and dilution rate) on the ethanol production were first investigated. Experiments on the pH effect showed that a decrease in pH from 7 to 4 produced an increase in ethanol concentration from 16.5 to 26.5 g/L (50 g/L initial lactose). The results obtained from modeling of the continuous fermentation using the previously listed variables showed that air flowrate, pH, and dilution rate were the process variables that most influence the production of ethanol.     

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.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Whey disposal by deproteinization and fermentation

The non-pollutant plant support material of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) was used for the entrapment of living yeast cells (Kluyveromyces fragilis) which hydrolyse lactose with the subsequent fermentation of glucose and galactose at high cell densities (up to 7.0 × 10⁸/ml support). The stabile yeast-plant cell immobilizates are able to produce ethanol from lactose-containing media (e.g. whey) by batch fermentation (on a rotary shaker) or continuous fermentation (in a turbulence reactor) for several days (at a pH below 4.2 and a temperature of 30°C). The removal of whey proteins by a preceding heat denaturation of whey, high dilution rates, C_So values of 50 to 60 g lactose per litre whey and the preferential use of the K. fragilis strain DSM 7238 were determined as the prerequisites for an optimum continuous fermentation. Economically interesting productivities (P_max ? 15 g ethanol/1 · h, D = 0.72 h^?1) with an actual lactose turnover of 90% were obtained by using these parameters.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Fermentation of sweet whey by ethanologenic Escherichia coli

Whey, an abundant byproduct of the dairy industry, contains large amounts of protein and lactose which could be used for fuel ethanol production. We have investigated a new organism as a candidate for such fermentations: recombinant Escherichia coli containing the genes encoding the ethanol pathway from Zymomonas mobilis. The highest level of ethanol achieved, 68 g/L, was produced after 108 hours in Luria broth containing 140 g lactose/L. Fermentations of lower lactose concentrations were completed more rapidly with approximately 88% of theoretical yields. Reconstituted sweet whey (60 g lactose/L)was fermented more slowly than lactose in Luria broth requiring 144 hours to produce 26 g ethanol/L. Supplementing sweet whey with a trace metal mix and ammonium sulfate reduced the required fermentation time to 72 hours and increased final ethanol concentration (28 g ethanol/L). By adding proteinases during fermentation, the requirement for ammonia was completely eliminated, and the rate of fermentation further improved (30 g ethanol/L after 48 hours). This latter incresed in rate of ethanol production and ethanol yield are presumed to result from incorporation of amino acids released by hydrolysis of whey proteins. The fermentation of sweet whey by ethanologenic E. coil reduced the nonvolatile residue by approximately 70%. This should reduce biological oxygen demand and reduce the cost of waste treatment. Whey supplemented with trace metals and small amounts of proteinase may represent an economically attractive feedstock for the production of ethanol and other useful chemicals.     

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.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus

Summary Investigations have been carried out on lactic acid production by Lactobacillus helveticus CNRZ 303 in whey ultrafiltrate. Addition of beet molasses was investigated with good results, although yeast extract proved to be more effective. The size of inoculum and the preculture medium also played a significant role in determining the amount of lactic acid produced during the fermentation process. High lactose consumption (94.09%), together with good lactic acid production (26.09 g/l) and yield (0.90%), were obtained in whey ultrafiltrate supplemented with 1% (w/v) beet molasses (WUM), with a 10% (w/v) inoculum and peptonized milk as preculture medium. Although these results were similar to those obtained when yeast extract was used as supplement, the maximum volumetric productivities proved to be quite different, and were definitely higher with yeast extract. Offprint requests to: L. Chiarini     

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.     

Improved ethanol production from whey with Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65–70 g/l lactose) by Saccharomyces cerevisiae CCY 10–13–14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/l × h), compared to the fermentation in which the lactose was directly fermented by K. marxianus.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

Enhancing bioethanol production from delactosed whey permeate by upstream desalination techniques

Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so‐called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: (i) simple dilution; (ii) nanofiltration; and (iii) electrodialysis. Lactose consumption, ethanol production and time‐dependent yields were compared between the three methods. A dilution of DWP to 60% v/v led to fermentation taking less than 80 h and yield above 7% AbV (alcohol by volume). After nanofiltration, 7.5% AbV was produced in about 80 h, and after electrodialysis, 11% AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DWP could be enhanced by prior partial desalination.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.