Whey is the aqueous fraction of milk generated as a by-product of cheese manufacturing which is produced in large amounts. The main solute in cheese whey is lactose, present at a concentration of about 3-8% (Speer, 1998). Other components are protein, salts and vitamins that are present in minor amounts. The low concentration of these components makes their recovery uneconomical. Because of it’s high organic content with high BOD, whey dumped directly to the environment is causing serious contamination problems (Ben-Hassan and Ghaly, 1994; Cristiani-Urbina et al. 2000; Roostita and Fleet, 1996). As a solution, bioconversion of whey into SCP or ethanol has been performed in several countries (Mawson, 1994; Gonzales Siso, 1996; Grba et al. 2002). The use of whey for the production of yeast biomass has the advantages that it is a simple treatment process, and the final discharge of the whey is facilitated since the pollutant load is significantly reduced and the whey lactose is converted into yeast biomass. The SCP could be produced from whey using yeasts such as Kluyveromyces, Candida and Trichosporon, as they are naturally able to metabolize lactose (Galvez et al. 1990; Mansour et al. 1993). However, it has been observed that in aerated cultures of Kluyveromyces fragilis, K. lactis a change in cellular metabolism from oxidative to a mixed oxidative-fermentative state can occurred. This change causes the production of by-metabolic products such as alcohol, aldehydes, esters, etc., which reduce the yields of biomass on whey (Beausejour et al. 1981; Inchaurrondo et al. 1994; Cristiani-Urbina et al. 1997). To avoid this undesirable effect, the mixed yeast culture has been used (Carlotti et al. 1991; Cristiani-Urbina et al. 2000).

The Kluyveromyces Species have been most widely studied for SCP production from whey (Mansour et al. 1993; Moresi et al. 1989; Grba et al. 2002). We evaluated the ability of different strains of Kluyveromyces lactis and Kluyveromyces marxianus for consumption of lactose and yeast biomass production. In order to increase the biomass yield and BOD removal of whey, the use of the mixed culture of Kluyveromyces strains and S. cerevisiae is proposed in this research.

In a search for yeast strains capable of lactose fermentation, 30 different yeast strains were isolated by using MEB containing 0.1gl^-1 chloramphenicol and YGCA. Among them, 11 strains were found to ferment lactose (M1-M11). These strains were identified by morphological and physiological properties using the standard taxonomic key outlined by Kurtzman and Fell, 1998. From these, six strains were identified as Kluyveromyces lactis, four strains as Kluyveromyces marxianus and one as Candida versatilis.Comparing to other studies it seems that K. lactis, K. marxianus var. marxianus and its anamorph, C. kefyr, are some of the most predominant and important yeast species in milk (Gadaga et al. 2000). As for the other yeast strains isolated and identified in this study, C. versatilis has been reported in yogurt, cheese and other dairy products (Deak et al. 1996; Jakobsen and Narvhus, 1996; Gadaga et al. 2000). K. marxianus and K. lactis are used in different biotechnology applications (Shah et al. 1993; Caballero et al. 1995; Gonzales Siso, 1996; Klaus, 1996). The thermotolerance of K. marxianus which was also identified among the isolated yeasts in the present study, could be used in very rapid processes of ethanol production, which might compensate for it’s lower tolerance to ethanol compared to S. cerevisiae. Among the yeast strains Kluyveromyces fragilis (K. marxianus) have been the most widely studied for the production of yeast biomass from whey (Beausejour et al. 1981; Moresi et al. 1989; Grba et al. 2002) and large-scale processes for producing Kluyveromyces biomass have been in operation for several years (Litchfield, 1983). K. lactis has been used for it’s industrial potential in the production of b-galactosidase enzyme which could be used to reduce the lactose content of milk (Suarez et al. 1995).

Enzyme activity of b -galactosidase in isolated yeast strains was measured (Table 1). The enzyme lactase, b-galactosidase, hydrolyzes lactose into glucose and galactose which are more soluble, and sweeter than lactose. This enzyme is produced by many microorganisms that use lactose as energy source (In and Jin, 1998; Uhlig, 1998). Comparing to other studies it seems that the enzyme activity from Kluyveromyces species is higher than other species (Cavaille and Combes, 1995; Inchaurrondo et al. 1998; Santos et al. 1998; Rech et al. 1999). Among isolated yeast strains, the M2 strain (K. lactis) was found to have the highest enzyme activity, 8183 unit/ml.

Single cell protein and effect of nitrogen supplementation

The interest in industrial waste as substrate for SCP production has increased recently. For this purpose the SCP production by the isolated yeast strains and cheese whey as a culture media was next studied. After preparation of cheese whey, inoculation of yeast strains and incubation in suitable temperature, the biomass yield of the pour culture of the isolated yeast strains was detected. In whey medium without any supplementation, the M2 (K. lactis), M11 (K. marxianus) and M5 (K. lactis) strains had the most SCP production with biomass of 11.79, 11.54 and 11.09 g/l dry biomass yield respectively. Amount of SCP production can be improved with addition of ammonium sulfate as nitrogen supplementation (Cristiani-Urbina et al. 2000). We found that ammonium sulfate (0.8 gl^-1) had significant effect on biomass yield. The amount of produced biomass of M11 strain increases from 11.54 gl-¹ in whey without supplementation to 15.75 gl^-1, in the present of nitrogen supplementation. In this condition, the M11 (K. marxianus) and M2 (K. lactis) strains were found to have the highest biomass yield, 15.75 and 15.35 gl^-1, respectively.

Improvement of biomass yield and BOD removal using mixed culture

Intermediate compounds such as ethanol, esters etc., formed during aerobic growth of K. lactis and K. marxianus, could reduce the yeast biomass yields (Beausejour et al. 1981; Inchaurrondo et al. 1994; Cristiani-Urbina et al. 1997). These intermediate compounds can be metabolized by some other yeast strains. With this regard to increase the biomass yields, the mixed culture of yeast strains has been studied (Carlotti et al. 1991; Cristiani-Urbina et al. 2000). We used mixed culture of isolated yeast strains (M2, M5, M6 and M11) with Saccharomyces cerevisiae for this purpose. The data showed that the biomass yields of the co-cultures of the selected strains and S. cerevisiae were significantly greater than those of the each strains alone. The biomass yield of S. cerevisiae and the M2 strain (K. lactis) co-culture showed a significant increase from 11.79 and 15.35 g/l (using pure culture in whey without supplementation and with ammonium sulfate supplementation, respectively) to 22.38 g/l. The biomass yield obtained in this condition was greater than those reported for K. marxianus (Bainotti et al. 1987), T. cremoris and K. fragilis (Beausejour et al. 1981; Litchfield, 1983; Mansour et al. 1993), for the mixed cultures of K. fragilis, K. lactis, Torulopsis (Pigache et al. 1992) and C. kefyr and C. valida (Carlotti et al. 1991). Increasing demand for protein sources of high nutrition value has stimulated the application of SCP in animal feed. With regards to these results we consider these yeast strains especially in a co-culture to be viable candidates for production of single cell protein from cheese whey.

We have also, studied the BOD removal using pure cultures of M2 (K. lactis) and M11 (K. marxianus) strains and mixed cultures of these yeast strains with S. cerevisiae. When the whey was inoculated by mixed culture of M2 or M11 strains with S. cerevisiae, the BOD removal was significantly increased. A greater BOD removal efficiency (88.5%) was obtained using mixed culture of M2 strain (K. lactis) and S. cerevisiae, decreasing initial BOD from 30000 to 3450 mgl^-1. The BOD removal achieved for the mixed culture was greater than those reported for K. marxianus (Pigache et al. 1992).

Since the S. cerevisiae can not grow in lactose medium, the results indicate that the S. cerevisiae consuming some of the extra cellular metabolites produced during the growth of Kluyveromyces species.

The results of this study suggest that yeast strains M2 (K. lactis) and M11 (K. marxianus) could be viable candidates for SCP and b-galactosidase production and removal of pollutants, from whey. Also the mixed culture of K. lactis and K. marxianus with S. cerevisiae could be used as an attractive alternative for removal whey BOD and obtaining a valuable biomass yield.

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