Discussion
The world’s population is expected to reach more than 9 billion people by 2050 (29); consequently, there will be concerns about the nutritional adequacy, especially for animal-derived protein, of such a population (30). These concerns force mankind to attempt finding alternative protein sources to replace or supplement plant proteins (29). Since, plant-based proteins require the use of arable land (31) and fish meals are based on catch of wild fish stocks (29), other sources of protein with high nutritional value need to be identified and developed.
Single cell proteins (SCP), produced by bacteria, algae or fungi, are one of microbial proteins which have been considered by researchers as suitable sources of nutritional protein (29); due to highly nutritious, cheap, and rapidly synthesized properties (32). Among SCP producing microorganisms, yeasts are preferred candidates because of their proper characteristics; the yeasts have rapid growth and high protein content as well as low risk of contamination. Also their cell size and flocculation abilities makes them easy to harvest (29). Yeasts are capable of providing vitamins and a well-balanced source of amino acids (33), whereas, contain lower amounts of nucleic acids in comparison with bacteria; that is an advanced property for human food and animal feed ingredients (29, 32). Yeasts are able to convert low-cost and readily available industrial organic by-products to high quality protein and lipids, efficient for animal feed as well as for human consumption. Furthermore, because of yeast’s ability to bind metal ions from the culture medium, they may be applied as a source of protein production and mineral preparations that can be easily utilized by humans (29).
In order to produce yeast biomass as a source of SCP, identification of yeast strains with optimal properties is of tremendous value. The Saccharomyces cerevisiae is a promising biosorbent yeast which is considered as a model system to accumulate metals in fairly high concentrations; and due to this ability, it is widely employed in many branches of industry (34, 35). The uptake and accumulation of zinc, as a required element for catalytic, regulatory and structural role in many proteins (36), by S. cerevisiae has been proven (18). Considering this, our present study focused on S. cerevisiae as well-studied yeast, obtained from alcohol factory’s effluent (figure 1), to produce zinc-enriched SCP under optimal conditions.
Numerous parameters could affect the absorption of zinc by yeast cells including: cell physiology, cell surface properties as well as chemistry of the metal ions and physicochemical impacts of the environment (18). In current study, we used different concentrations of zinc metal in SDB culture medium to find optimum zinc concentration to obtain a high yield of yeast biomass and zinc biosorption; simultaneously, the effect of incubation time on yeast growth was also investigated. Using the AAS method, the maximum uptake of zinc by yeast cells was observed at 24 h after inoculation; suggesting that, the increment of incubation time more than 24 h, did not positively affect biosorption (table 1). The maximum dry matter content as well as highest growth rate of yeast biomass were observed in 25 µg/ml of zinc in SDB medium and after 24 h incubation (figure 2 and table1); suggesting 25 µg/ml as optimal concentration of zinc for S. cerevisiae growth.
The strong changes in expression level of Zrt1 and Fet4, as zinc transporters, in response to zinc concentrations and 24 h incubation ofS. cerevisiae in SDB medium, versus control medium (SDB without the addition of zinc) was observed, indicating the validity of obtained results. The Zrt1 expression reached a maximum level of 25 µg/ml of zinc concentration; also increment of Fet4 expression in present of 25 µg/ml of zinc was considerable. While the maximum level of Fet4 expression was observed in the presence of 50 µg/ml of zinc (figure 3). Hence, our observation was in accordance with previous study that mentioned uptake of extracellular zinc in S. cerevisiae , in severe zinc limitation, using the high-affinity zinc transporter, Zrt1; whereas, this yeast can partially cope with low zinc conditions by Fet4 as a low-affinity transporter protein for zinc, iron, and copper (19). Therefore, relative quantification of Zrt1 and Fet4 expression in our study, verified at 24 h incubation with 25 µg/ml of zinc concentration, as optimum time and zinc concentration for S. cerevisiae yeast, respectively (figure 3).
Due to the pH-dependency of zinc uptake, optimum pH for zinc biosorption by yeast cell is very important (37); in a study conducted by Chen and Wang in 2007, pH 5.8 was identified as optimum pH for zinc biosorption by S. cerevisiae (35). On the basis of this report we used the same pH for yeast cultivation. After identifying the optimal conditions (25 µg/ml of zinc and 24 h incubation) for S. cerevisiae , an optimum pH of the medium as a critical parameter for zinc uptake by yeast cell was evaluated. By screening of growth rate, zinc uptake and expression level of Zrt1 and Fet4 in S. cerevisiae in different pH values (pH 3-6) under optimal conditions (25 µg/ml of zinc and 24 h incubation) an optimum pH was obtained. By increasing the pH of the medium from 3 to 6, cellular zinc occurrence and yeast growth rate significantly increased and the highest increment were obtained in pH 6, almost similar to results in pH 5.8 (table 2). The maximum Fet4 transcript level was observed in pH 4, while the equal increment of Zrt1 and Fet4 expression was observed in pH 6 (figure 4). As a result, our study approved the previous report of direct dependence of biosorption efficiency to pH of medium (38-40).
The crude protein content of the yeast cells could be affected by the strain, growth culture medium and growth conditions. So, the total protein content of yeast cells, under optimal conditions of 25 µg/ml of zinc at pH 6 after 24 h incubation was evaluated. The results of this estimation showed that protein content of S. cerevisiae biomass was above 50% (w/w).The value obtained in our study was within protein levels, considered reasonable in the context of SCP production. Our results also augment other studies about the protein contents in yeasts which normally vary between 45 and 55% (29-31, 41).