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).