Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

Engineering Yarrowia lipolytica for the utilization of acid whey

Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted β-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology.     

Production of β-carotene from deproteinized waste whey filtrate using Mucor azygosporus MTCC 414 in submerged fermentation

The cheese whey, a by-product of dairy industry proved to be an attractive substrate for production of β-carotene. The β-carotene production from Mucor azygosporus MTCC 414 by using deproteinized waste whey filtrate under submerged fermentation was investigated. Various fermentation variables, such as lactose content in whey, initial pH, production temperature, incubation time, and carbon and nitrogen sources played significant role on β-carotene production. Maximum β-carotene production (385 μg/g dcw) was obtained with the whey (pH 5.5) containing 3.5% (w/v) lactose supplemented with soluble starch at (1.0%, w/v) at 30°C after a 5 days incubation. Moreover, unlike other microorganisms which utilize pre-hydrolyzed lactose, this Mucor azygosporus MTCC 414 was found to be capable of utilizing unhydrolyzed lactose present in the whey.     

Coprecipitation of acid cheese whey and tannery waste and recovery of protein

Combining acid cottage cheese whey and the lime–sulfide effluent from tannery unhairing processes spontaneously coprecipitates the whey proteins with the large peptides and proteins of the tannery waste. The floculation of the denatured protein material also carries down the hide pigments, excess lime, and the casein fines from the whey. The clear supernatant contains lactose, sulfur in various states of oxidation, free amino acids, peptides, and ammonium salts, but no detectable macromolecular proteins. The recovered solid products, which contain more than 20% of the original nitrogen, appear to have a good balance of essential amino acids although actual composition varies with the composition of the raw wastes. Feed supplements may possibly by obtained by this method from two presently wasted industrial effluents.     

Efficient lactic acid production from high salt containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre-treatment by proteolytic microorganisms

Lactic acid bacteria have an inefficient proteolytic system. Therefore, cultivation media which may have high protein content are usually supplemented with yeast extract or protein lysates (peptones). These additives might be conveniently replaced by in situ treatment of the cultivation medium with proteolytic enzymes or proteolytic microbes. Lactobacillus salivarius ssp. salicinius, a lactic acid bacterium species that can grow at high salt concentration, was used to ferment lactic acid in cheese whey (with 3 gl(-1) whey protein content) and lactose mother liquor (90 gl(-1) lactose, 9 gl(-1) proteins, 30 gl(-1) minerals). The contribution of protease enzymes or proteolytic microbes to acid production by lactobacilli was examined. Efficient conversion of lactose to lactic acid was obtained in the presence of additional proteolytic activity. Fastest acid production was obtained with the addition of protease enzymes. However, almost equally efficient acid production was obtained by treating the medium with Bacillus megaterium. The results show that fast and complete conversion of lactose to lactic acid can be obtained in dairy by-products without expensive additives.     

Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24

Methylobacterium sp. ZP24, isolated from a local pond, is able to grow in a medium containing 12 g l⁻¹ lactose as a sole source of carbon, giving 5·25 g l⁻¹ biomass yield and poly-3-hydroxybutyrate (PHB) up to 59% of its dry weight in 40 h. The isolate was also able to utilize cheese whey and produce 1·1 g l⁻¹ PHB. Addition of ammonium sulphate increased the production of PHB from whey 2·5-fold. The potential of Methylobacterium sp. ZP24 in PHB production from cheese whey is discussed.     

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.     

Production of a Thermostable beta-d-Galactosidase by Alternaria alternata Grown in Whey

In the course of exploring new microbial sources of extracellular beta-d-galactosidase (EC. 3.2.1.23), Alternaria alternata was found to excrete elevated quantities of a thermostable form of the enzyme when cultivated in whey growth medium. Optimum cultural conditions for maximum enzyme production were a whey lactose concentration of 6%, supplementation of the medium with 0.050 M (NH(4))(2)SO(4), an inoculum size of 10 conidia per ml, and a cultivation time at 28 to 30 degrees C of 5 days. The fungus utilized whey lactose for the production of the enzyme most efficiently, and the observed maximum yield, 280 nanokatals of hydrolyzed o-nitrophenyl-beta-d-galactopyranoside per g of whey lactose, was comparable to maximum yields reported for certain commercial fungi. The optimum pH and temperature of the enzymatic reaction were 4.5 to 5.5 and 60 to 70 degrees C, respectively, and the enzyme lost half of its activity when heated at 65 degrees C for 84 min. These properties make the enzyme particularly suitable for processing acid and less-acid (pH 5 to 6) dairy products and by-products.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

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.     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

Structures and Properties of Gellan Polymers Produced by Sphingomonas paucimobilis ATCC 31461 from Lactose Compared with Those Produced from Glucose and from Cheese Whey

The dairy industry produces large quantities of whey as a by-product of cheese production and is increasingly looking for new ways to utilize this waste product. Gellan gum is reliably produced by Sphingomonas paucimobilis in growth media containing lactose, a significant component of cheese whey, as a carbon source. We studied and compared polysaccharide biosynthesis by S. paucimobilis ATCC 31461 in media containing glucose, lactose (5 to 30 g/liter), and sweet cheese whey. We found that altering the growth medium can markedly affect the polysaccharide yield, acyl substitution level, polymer rheological properties, and susceptibility to degradation. Depression of gellan production from lactose compared with gellan production from glucose (approximately 30%) did not appear to occur at the level of synthesis of sugar nucleotides, which are the donors of monomers used for biosynthesis of the repetitive tetrasaccharide unit of gellan. The lactose-derived biopolymer had the highest total acyl content; the glucose- and whey-derived gellans had similar total acyl contents but differed markedly in their acetate and glycerate levels. Rheological studies revealed how the functionality of a gellan polysaccharide is affected by changes in the acyl substitution.     

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.     

High-Level Production of Heterologous Protein by Engineered Yeasts Grown in Cottage Cheese Whey

Cottage cheese whey is a cheese industry by-product still rich in proteins and lactose. Its recycling is seldom cost-effective. In this work we show that the lactose-utilizing yeast Kluyveromyces lactis, engineered for production of recombinant human lysozyme, can be grown in cottage cheese whey, resulting in high-level production of the heterologous protein (125 μg/ml).     

Hyper production of enzyme penicillin amidase by locally isolated thermo-tolerantBacillus sp. MARC-0103 from rice starch in cheese whey

The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.     

Buffalo Cheese Whey Proteins,Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.     

Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora

Whey permeate from dairy industry was hydrolyzed enzymatically to cleave its main carbon source, lactose, to glucose and galactose. The hydrolysis products were chosen as carbon sources for the production of poly-3-hydroxybutyric acid (PHB) by Pseudomonas hydrogenovora. In shaking flask experiments, the utilization of whey permeate as a cheap substrate was compared to the utilization of pure glucose and galactose for bacterial growth under balanced conditions as well as for the production of PHB under nitrogen limitation. After determination of the inhibition constant Ki for sodium valerate on biomass production (Ki=1.84 g/l), the biosynthesis of PHA co-polyesters containing 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) units from hydrolyzed whey permeate and valerate was investigated. The application of hydrolyzed whey permeate turned out to be advantageous compared with the utilization of pure sugars. Therefore, fermentation under controlled conditions in a bioreactor was performed with hydrolyzed whey permeate to obtain detailed kinetic data (maximum specific growth rate, mu max=0.291/h, maximum polymer concentration, 1.27 g/l PHB), values for molecular mass distribution (weight average molecular weight Mw=353.5 kDa, polydispersity index PDI=3.8) and thermo analytical data. The fermentation was repeated with co-feeding of valerate (maximum specific growth rate, mu(max)=0.201/h, maximum polymer concentration, 1.44 g/l poly-(3HB-co-21%-3HV), weight average molecular weight M(w)=299.2 kDa, polydispersity index PDI=4.3).     

Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling

This article deals with the production by fermentation of a mycostatic and aromatic food additive based on propionic acid. Membrane bioreactors have been used from laboratory scale up to pilot and industrial production plants. Due to the high cell densities achieved by the sequential recycling mode of operation, a mixed acids solution was rapidly produced from whey permeate. The sterile fermented broth obtained was subsequently concentrated at different levels by evaporation and spray drying according to the projected use. Concentrated Propionibacterium cells (200 g . L(-1) DW) were obtained from the process by periodic bleeds and could be used to good effect as cheese starters, silage preservatives, or probiotics. Propionic acid concentrations from 30 to 40 g . L(-1) were easily achieved with no residual lactose. The highest volumetric productivity was 1.6 g . L(-1) . h(-1) for total acid and 1.2 g . L(-1) . h(-1) for propionic acid with a specific productivity of 0.035 h(-1). (c) 1993 John Wiley & Sons, Inc.     

Evolutionary adaptation of Kluyveromyces marxianus strain for efficient conversion of whey lactose to bioethanol

The aim of the present work is to develop an osmotolerant yeast strain with high lactose utilization and further use it to ferment lactose rich whey permeate for high ethanol titer and to reduce energy consumption. Ethanol production and growth rate of selected MTCC 1389 strain were quite high in whey containing lactose up to 150 g/L but it remains constant in lactose concentration (200 g/L) as cells encountered osmotic stress. Thus, strain MTCC 1389 was used for an adaptation to lactose concentration 200 g/L for 65 days and used further for fermentation of lactose rich whey. Fermentation with an adapted K. marxianus MTCC 1389 strain in laboratory fermenter resulted in ethanol titer of 79.33 g/L which is nearly 17.5% higher than the parental strain (66.75 g/L). Expression analysis of GPD1, TPS1and TPS2 found upregulated in lactose adapted K. marxianus strain as compared to the parental strain. These results suggest that an adapted K. marxianus strain accumulates glycerol and trehalose in response to lactose stress and improve osmotolerance in K. marxianus cells. Thus, the study illustrates that evolutionary engineering is an efficient strategy to obtain a superior biofuel yeast strain, which efficiently ferments four-fold concentrated cheese whey.