الفهرس 
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NaCl toleranceOnly 14 pages are availabe for public view
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Abstract
Cheese whey is a by-product of dairy industries, produced in large amounts and has a high polluting load, therefore representing a significant environmental problem all over the world. The lactose in cheese whey can be used as a substrate for bio-ethanol production by microbial fermentation, and its anaerobic digestion offers an excellent approach in terms of both energy conservation and pollution control, in addition to that this is one of the least expensive carbon sources for ethanol production.
The present study aimed to detect potent wild yeast strains to ferment the lactose in cheese whey, and their molecular identification. Two steps needed to be carried out to realize this target.
The first step:
1. Isolation and selection of lactose fermenting yeasts according to acid formation using litmus milk approach.
2. Evaluation of selected isolates for three economically important characters:
a. Lactose tolerance.
b. NaCl tolerance.
c. Thermotolerance.
3- Performance of selected isolates for lactose fermentability and bio-ethanol production.
The Second Step:
1- Molecular Genetic Identification of selected isolates by using sequence analysis of the D1/D2 domain of the 26S rRNA gene.
2- Molecular Genetic Differentiation between the selected isolates by using RAPD.
The results showed that:
The first step: Isolation, Selection and Evaluation of yeast isolates:
1- Four isolates (Z-2, Z-5, Z-13 and Z-39) were selected from thirty nine isolated yeast strains based on their lactose fermentability.
2- Evaluation of selected isolates for three economically important characters:
a. Lactose tolerance:
Results of lactose tolerance indicated that the growth of the selected strains Z-2, Z-5 and Z-13 showed normal growth at concentrations of 2%, while the growth was moderate at concentration of 10% and 20%, then the growth was faint at concentrations of 40% and 60%. On the other hand strain Z-39 exhibited moderate growth only at 2% while it couldn’t be occurred at the higher concentrations.
b- NaCl tolerance:
Results of NaCl tolerance indicated that the growth of the three selected strains Z-2, Z-5 and Z-13 showed Moderate growth at concentrations of 4% and 6%, and then exhibited by faint growth at concentration 8% and no growth at 10%. On the other hand, strain Z-39 exhibited faint growth only at 4% while it couldn’t be occurred at the higher concentrations.
C. Thermotolerance:
The results showed that there was a significant variation between these isolates in terms of their ability to tolerate thermal exposure.
Z-39 isolate showed the highest survival percentage (95.00% ± 0.5) at 45°C, while the Z-13 showed the lowest survival percentage (33.50% ± 0.5) at the same temperature.
3- lactose fermentation and bio-ethanol Production:
Isolate Z-5 was the highest ethanol producer by 54.2 ml/L, while, Z-13 produced 50.8 ml/L and Z-2 produced less amount of ethanol by 40 ml/L. On the other hand isolate Z-39 was unable to produce ethanol at all.
The second step:
1- Sequencing of 26S rRNA gene and phylogenetic analysis:
BLAST analysis of the 26S rRNA gene sequence of the isolate, Z-2, was revealed it to be a perfect match with that of Pichia ohmeri type strain with one base substitution (99% similarity).
While the isolate Z-5, was revealed it to be a perfect match with that of Kluyveromyces lactis type strain with no base substitutions (100% similarity), and Z-13 showed a 100% 26S ribosomal DNA sequence similarity to Kluyveromyces marxianus.
Phylogenetic trees were constructed; As a result, the two strains Z-5 and Z-13 were clustered with other Kluyveromyces species, indicating a phylogenetic position within the genus Kluyveromyces. Therefore, the two strains were designated as K. lactis the coded strain Z-5 and K. marxianus strain with code no. Z-13 respectively, whereas strain Z-2 was designated as Pichia ohmeri.
2- RAPD analysis:
RAPD-PCR for the four strains were performed by using five different primers, the results of DNA fingerprints gave distinct band profiles which indicated a clear differentiation of the four considered strains. The four selected strains were regrouped in two clusters; one of them included Z-5 and Z-13, and the second cluster contained Z-2 and Z-39.
The importance of the present study can be concluded according to the following aspects:
1- It introduces a solution for the environmental pollution resulted from the whey, and its utilization to obtain source of renewable clean energy.
2- Results indicated that the nature represents a worthy source for the yeasts that play an important role in biotechnology, since some of yeasts are used in bio-ethanol production from whey lactose.
3- There is a significant relation between the three important economic characters, namely lactose tolerance, NaCl tolerance and thermotolerance, that affect on strain efficiency of fermentation.
4- Study of molecular genetic analysis using 26S rRNA, and RAPD/PCR, could lead to accurate identification for the yeast isolates in comparison to traditional identification tests.
5- Results showed that genus Kluyveromyces exists allover milk products and appear to be highly efficient lactose fermentor and bio-ethanol producer.