BY-PRODUCTS TECHNOLOGY

Module 3. Processing and utilization of whey

Lesson 26
FERMENTED PRODUCTS FROM WHEY-1

26.1 Introduction

Fermentation for the large-scale utilization of whey was first investigated in 1930s and 1940s. The industrialization of these technologies has been slow. Whey contains lactose as a major component of whey solids in addition to proteins, minerals and water soluble vitamins, which make it excellent growth medium for various microorganisms. Numerous microbial processes have been developed recently to utilize whey for the production of some useful products of industrial importance such as single cell protein, baker’s yeast, ethyl alcohol, methane (biogas), organic acids, vitamins etc. ( and Table 26.1). In this lesson, production of single cell protein, baker’s yeast, enzyme (β-galactosidase), ethyl alcohol and methane is discussed.

Table 26.1 Fermented products from whey

26.2 Food Yeast Production

26.2.1 Single-cell-protein (SCP)

Many scientists believe that single-cell protein production is possible solution to meet out the shortage of protein. Many yeasts such as Candida spp., Saccharomyces fragilis, Torula spp., Rhodotorula spp. use whey, ethyl alcohol, starches, n-paraffins, sulphite waste, etc. as raw material for production of SCP.

Yeast biomass has been produced commercially by aerobic fermentation of lactose from whey or whey permeates by Kluyveromyces fragilis, strains of K. marxianus var. lactis or var. marxianus. This transformation is presented as:

The resulting product, acetaldehyde, participates in the krebs cycle in protein synthesis. For its growth and development, this yeast requires lactose, oxygen, and presence of particular nutritive salts and other component, which are added to the medium.

In some cases, ethanol is also produced as a by-product by varying the aeration rate during the fermentation. The requirements of a suitable culture strains are as follows:

• High specific growth rate and biomass yield to ensure a high productivity.

• The strains must not be affected by whey proteins if they are present.

• The strain must be suited to continuous culture.

• The strains must be acid resistant. To control contamination it is necessary to operate at a low pH or to wash the yeast at frequent intervals with acid to remove contaminants.

• Large cell size and uniform morphology to aid cell separation and concentration.

• Adequate protein content and acceptability in feeding trials.

The ‘Bel’ process , developed in France in 1950s, is a frequently cited example of this fermentation. Here, strains of K. marxianus var. lactic or var. marxianus are those most commonly grown for the production of SCP. Sweet cheese whey is first deproteinated and diluted to a lactose concentration of 20-24 kgm^-3. Whey is limiting in nitrogen sources for yeast growth, so ammonium salts are added to maintain high nitrogen content and growth rate and trace metals (Fe, Cu, Mn, and Zn) may also be added. The continuous fermentation is operated at a dilution rate of 0.33 h^-1 and a temperature of 39°C. There is continuous mixing and aeration of the culture. For a fermenter of 23 m³ capacity, about 1800 m³ air is pumped per hour. The fermentation period is 4 hours and the substrate addition of deproteinated fresh whey constitutes 5,600-6,000 l/h. The residual sugar level is about 1 kgm^-3, the biomass yield is 0.55-0.6 kg yeast (dry basis) per kg lactose metabolized and the biomass productivity is approximately 4.5 kgm^-3 h^-1. The yeast is separated and concentrated in two-stage washing and centrifugation process, plasmolyzed to render the yeast protein more accessible and dried.

The composition and uses of biomass produced from whey are similar to those of other food yeasts. The crude protein content is about 50% on dry basis and the only significant limitation is of the sulphur-containing amino acids. The protein value is similar to casein. “Protibel” the yeast-protein compound obtained by the Bel process is made up of about 50% protein, 30% carbohydrates, 6% lipids and 8% minerals. This can be very well used in foodstuffs.

26.2.2 Baker’s yeast

Baker’s yeast or a yeast autolysate can also be obtained by whey fermentation. The yeast autolysate may be used to replace yeast extract in microbiological media. Two processes have been developed for the production of baker’s yeast to overcome the limitation of S. cerevisiae not being able to utilize lactose. In the first, the lactose is hydrolyzed using β-galactosidase, and the glucose and galactose are consumed simultaneously by the yeast in continuous culture. The second process utilizes a two-stage fermentation system. In the initial stage, lactic acid bacteria convert lactose to lactate which is used in subsequent fermentation by the yeast. Although the baker’s yeast so produced appears comparable in quality to that from the conventional process, only very limited quantities are manufactured by those methods.

26.3 β-Galactosidase

Lactase is an enzyme which catalyses hydrolysis of the galactosidic linkage of lactose. Lactase occurs naturally in some plants and in intestine of various animals. Additionally it is prepared by a wide variety of molds, yeast and bacteria. The production of enzyme β-galactosidase or lactase has been, of late, the subject of considerable interest. Whey seems to provide the best medium for the production of this enzyme. The enzyme β-galactosidase or lactase can be produced from selected strains of Kluyveromyces spp. following growth on diluted whey.

The production of enzyme using permeate as substrate was suggested by a group of workers. Candida pseudotropicalis was grown at 30°C on 2% whey solution supplemented with 0.5-0.1% yeast extract, 0.1-0.2% ammonium sulphate at a pH of 3.5. The enzyme was extracted with chloroform. β-galactosidase is used to hydrolyse lactose to overcome intolerance, and to generate syrups which are sweeter than lactose and which do not crystallize as readily.

26.4 Ethanol Production

The conversion of lactose in whey to bio-ethanol has long been considered as a possible solution for whey bioremediation. Acid whey from the production of lactic and sulphuric casein is used to ferment the lactose to ethanol. In New Zealand, acid casein whey is processed to produce ethanol, which is used in pharmaceuticals, perfumes and inks as well as beverages. The technology to process deproteinated whey into ethyl alcohol was developed in Europe about 20 years ago and was purchased from Ireland by the Anchor Ethanol Company in the late 1970s. Since that time, two plants have been established, at Tirau (using a continuous fermentation process) and Reporoa (using a batch fermentation process). The combined annual production of these two plants is 11,000,000 litres of ethanol. Two further independent distilleries based on whey have subsequently been built in New Zealand, producing an additional 6,000,000 litres. The production of ethanol from whey involves total lactose fermentation and a reduction of BOD value. Various lactose fermenting yeasts are capable of forming ethanol, and strains from K. marxianus, C. Kefyr and Torula cremoris as well as a mixed culture of K. marxianus and Zymomonas mobilis, are particularly efficient in this respect. High ethanol yields (about 80%) can be reached from adapted Kluyveromyces fragilis strains. Ethanol can then be further used as an energy source or to produce vinegar or acetic acid. Ethyl alcohol is produced by the conversion of lactose present in whey.

The yeast is added to ferment the lactose in two reactions: firstly splitting the lactose into the two sugars of which it is composed and secondly fermenting these sugars to ethanol.

Lactose + H₂O → Galactose + Glucose
Galactose + Glucose → 4 Ethanol + 4CO₂

The yeast is then removed from the liquid and the ethanol separated out by distillation to produce eight different grades of ethanol for a variety of industrial uses.

Cheese whey generally contains carbon and nitrogen substrate required for microorganism growth. Lactose is first hydrolyzed by β-galactosidase and the resulting mixture of glucose and galactose is then used as the carbon source by Saccharomyces cerevisiae. Different processes based on continuous fermentation with cell immobolization or cell recycling have been explored for production of ethanol. A recent alternative is to use recombinant yeast grown directly on cheese whey, allowing high yield of ethanol.

In a typical process of ethanol production, deproteinated cheese or acid casein, whey are used as the substrate, and may be concentrated by reverse osmosis or supplemented by other lactose-rich streams to increase the lactose concentration to a maximum of 10-13%. The fermentation employs strains of the yeast Kluyveromyces marxianus var. marxianus. The process may be operated under aseptic conditions using pasteurized serum. Fermentation temperatures are in the range 24-34°C. Typically the fermenter vessels are of 120-150 m³ total volume and some mixing is required to prevent supersaturation of CO₂ in the vessel. Almost complete utilization of the lactose can be achieved and ethanol yield is in the range of 75-85% of the theoretical value of 0.538 kg ethanol per kg lactose metabolized. A maximum ethanol concentration of about 5.5% is achieved by fermentation of concentrated whey streams.

26.5 Methane (Biogas)

The production of methane or biogas via fermentation of whey is a process composed of three successive steps: lactose (and protein) hydrolysis, fermentation and methanogenesis. This complex process involves several mixed bacterial species. According to Fig.26.3, the methanogenic process converts around 90% of hydrolyzed organic matter to biogas: CH₄ and CO₂. The microbial production of methane from whey offers the major benefits of an efficient waste treatment process coupled with the production of convenient energy source. The methane fermentations, or anaerobic digestion, requires a limited input of energy or nutrients and produces very little sludge for ultimate disposal in comparison with conventional aerobic treatment processes.

Fig. 26.3 Anaerobic production of Biogas

Selected references

Guimaraes, P.M.R. 2010. Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for valorization of cheese whey. Biotechnology advances, 28: 375-384.
Puranik, D.B., Jain, P. and Bandopadhyay, S. 1997. Value added commercial products from whey fermentation. Indian Dairyman, 49 (1): 21-24.
Prasad, V. 2007. Whey processing. In: Dairy India-sixth edition, ed. P.R. Gupta and Sharad Gupta. Dairy India Yearbook, New Delhi: 244.
Gandhi, D.N. 1989. Production of some useful products of industrial importance through mi-crobial fermentation of whey. Indian Dairyman, 39 (4): 182-184.
http://www.iei.liu.se/envtech/forskning/forskningsprojekt/synergies-biofuels/synergies-inventory-research/ethanol-synergies-inventory/inventory-of-ethanol-synergies/1.178581/CheeseWheyforEthanolProduction.pdf
http://nzic.org.nz/ChemProcesses/dairy/3H.pdf