الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Alginate producing bacteria, such as species of Azotobacter are used for alginate production by fermentation. Alginates are used widely as thickeners, stabilizers, gelling agents, and emulsifiers in the textile, paper making and pharmaceutical industries as well as in food industries. The optimal conditions for cell growth and alginate biosynthesis are known to be quite different. In Egypt, studies concerning alginate are limited and it is not produced on large scale. In view of these facts, the present work was achieved to study alginate production by alginate producing bacteria. The study included isolation and selection of most efficient alginate producing bacteria from different soil samples, nutritional and environmental factors supporting their efficiencies to produce alginate. A part of the study concentrated on the use of some dairy industrial wastes such as (whey) as a substrate for alginate production on large scale.
Results could be summarized as follows: -
1. Five isolates of genus Azotobacter out of twenty-eight isolate gave the highest alginate production in Clementi et al (1995) medium (based medium). These isolates were isolated from different soil samples.
2. Cultivation media were compared as alginate productive cultivation media by selected Azotobacter isolates. Clementi et al (1995) medium was the preferable medium for the propagation and alginate production by tested Azotobacter isolates.
3. Azotobacter isolates was completely identified as Azotobacter vinelandii and Azotobacter chrococcum according to Bergye’s Manual of Systematic Bacteriology using their morphological and physiological characteristics.
The most efficient alginate producing bacteria were completely identified by 16S rRNA gene sequences of two Azotobacter isolates were amplified and subjected to 16S rRNA gene sequencing and the results showed that bacterial isolates were mainly the members of two Azotobacter species, namely, Azotobacter vinelandii MH249041 (B10) and Azotobacter chroococcum MH249629 (M3).
4- Alginate production in based medium:
• Sucrose at 5.0 % concentration was the most favorable carbon source for alginate production by Azotobacter vinelandii which increased alginate concentration about 1.6 fold as compared with glucose in the same medium whereas the most favorable carbon source for alginate production by Azotobacter chrococcum was Sucrose 4.5% concentration.
• The mixture of 6.0 & 8.0 gL-1 yeast extract and 0.8 gL-1 of ammonium sulphate was the best nitrogen source for alginate production were being 7.15 & 6.23 gL-1 on productive medium by Azotobacter vinelandii and Azotobacter chrococcum, respectively after 5 days at 30 °C using shake flasks as a batch culture.
• Five concentrations of Na2HPO4 ranged from 1.0 to 3.0 gL-1 were added to productive medium for alginate fermentation. Maximum alginate yield and productivity were attained at 2.5 gL-1 Na2HPO4 being 15.7% and 0.065 gL-1h-1, respectively by Azotobacter vinelandii. Maximum alginate yield and productivity were attained respectively at 2.5 gL-1concentrations of Na2HPO4 being 14.53 % and 0.055 by gL-1h-1 by Azotobacter chrococcum.
• Five level of initial pH were used to study the alginate production by Azotobacter vinelandii and Azotobacter chrococcum. The highest growth and alginate parameters were observed at initial pH value of 7.0. Whereas increasing the initial pH to 8.0 led to decrease the alginate concentration.
• Inoculum sizes of Azotobacter vinelandii and Azotobacter chrococcum ranged from 1 - 10 % (O.D. 1.3- 0.9), respectively were tested. The inoculation with 7 % standard inoculum gave the highest alginate concentration being 8.69 gL-1, 7.37 gL-1, respectively.
• The agitation of 170 rpm was the optimum for highest alginate yield and productivity being (0.072 gL-1h-1 & 17.38%) produced by Azotobacter vinelandii and (0.061 gL-1h-1 & 16.38%) produced by Azotobacter chrococcum, respectively.
• Maximum alginate production 9.38, 8.23gL-1, (CDW) 9.54, 8.73 gL -1 and yield 18.76, 18.29 % were attained by Azotobacter vinelandii and Azotobacter chrococcum respectively, at 32ºC. These production parameters were higher than that achieved by the same strains at fermentation temperatures of 28, 30, 35, 37 ºC.
• The fermentation period of 72 hrs was the optimum time for highest alginate concentration and productivity (10.57 gL-1 & 0.147 gL-1h-1) produced by Azotobacter vinelandii and (9.68 gL-1 & 0.134 gL-1h-1) by Azotobacter chrococcum on based medium using shake flasks as a batch culture.
5- Alginate production in whey production medium: -
• Different lactose concentrations were investigated for alginate production by both Azotobacter vinelandii and Azotobacter chrococcum after 5 days at 30 °C using shake flasks as a batch culture. The highest alginate production (gL-1) was obtained by Azotobacter vinelandii at 5.0 % concentration of lactose and Azotobacter chrococcum at 4.5 % concentration lactose being 1.2 gL-1 & 0.976 gL-1, respectively.
• All tested organic nitrogen sources (three sources) gave higher alginate production than those obtained in the case of inorganic nitrogen sources (ammonium sulphate, ammonium nitrate). The yeast extract at 2 gL-1was the best nitrogen source, which gave the highest values of alginate concentration, yield and productivity by Azotobacter vinelandii were being 1.09 gL-1and 2.18% & 0.009 gL-1h-1, respectively & by Azotobacter chrococcum were being 1.299 gL-1 & 2.9 % and 0.011 gL-1h-1, respectively during the experiments. Ammonium nitrate was the best inorganic nitrogen source at 1.5 gL-1 for both strains which gave the heighst alginate concentration and yield were being (1,34 gL-1 & 2.68%), respectively by Azotobacter vinelandii and (1.326 gL-1 & 2.95%), respectively by Azotobacter chrococcum.
• Different levels of sodium phosphate were used. The concentration of 2 gL-1gave the highest concentration of alginate (3.827 gL-1, 3.496 gL-1) for both Azotobacter vinelandii and Azotobacter chrococcum, respectively.
• Different levels of pH values were examined. pH 7.0 was the most suitable pH for alginate production by Azotobacter vinelandii and Azotobacter chrococcum, respectively.
• The inoculation with 9 % of standard inoculum (O.D. 1.6, 1.2) by Azotobacter vinelandii and Azotobacter chrococcum gave the highest alginate concentration were being (4.39, 4.47 gL-1), respectively.
• Agitation of 200 rpm optimized for a higher concentration of alginate and productivity (5.16 gL-1and 0.043 gL-1h-1) produced by Azotobacter vinelandii and 4.67 gL-1and 0.039 gL-1h-1) produced by Azotobacter chrococcum, respectively than the agitation was 170 rpm (control).
• The optimal fermentation temperature of Azotobacter vinelandii and Azotobacter chrococcum was 30° C (control), which gave the highest concentration of alginate.
• Alginate production increased during the fermentation period to record the highest alginate concentration (8.897 gL-1) at 72 hrs by Azotobacter vinelandii flowed by (8.374 gL-1.) by Azotobacter chrococcum.
• The effect of gamma irradiation on survival and alginate productivity. where the calculated D10-values of Azotobacter vinelandii and Azotobacter chrococcum were found to be 0.29, 0.33 kGy, respectively indicates the high sensitivity of these strains to Gamma radiation, gamma irradiation of Azotobacter vinelandii and Azotobacter chrococcum at the dose of 0.5 KGY, increased the alginate concentration (9.37& 9.46 gL-1), respectively.
6- Some properties of produced alginate: -
• Azotobacter chrococcum alginate containing the higher ratio of standard alginate (83.49 %) than Azotobacter vinelandii alginate (66.3 %) samples.
• Emulsifying capability of alginate produced from Azotobacter vinelandii and Azotobacter chrococcum as well as commercial alginate were examined against olive oil and gave the emulsifying capacity of 28.34, 36.85 and 47.23 %, respectively. Moreover, the highest flocculating activity was observed by standard alginate followed by Azotobacter vinelandii and Azotobacter chrococcum alginate.
• A tough worm like gel or firm gel where formed when 10 % calcium chloride or 0.5 g sodium chloride were added to alginate solution for two strains. The crude of alginate was soluble in water and insoluble in ethanol, acetone or isopropanol.
• HPLC chromatogram of Azotobacter chrococcum alginate had higher alginate standard ratio than Azotobacter vinelandii alginate.
• The FTIR spectra of Azotobacter chrococcum alginate and Azotobacter vinelandii alginate gave the range peaks of absorbance as well as the standard alginate.
• The changes in viscosity of ice cream made with different kinds of alginate (standard alginate, Azotobacter vinelandii alginate and Azotobacter chrococcum) and CMC and relation between shear rate and shear stress indicated that CMC has the heighst level of viscosity followed by standard alginate, Azotobacter vinelandii alginate and Azotobacter chrococcum alginate at the same speed were being (168, 147, 138 & 124 cp), respectively.
• Sensory evaluation results of ice-cream made from milk containing CMC and different kinds of alginate (standard alginate, Azotobacter vinelandii alginate and Azotobacter chrococcum alginate) indicated that the appearance and body & texture were non significantly different from the control (CMC). Generally, the addition of Azotobacter vinelandii alginate and Azotobacter chrococcum alginate enhanced the ice-cream quality.
• Generally, it can be used as gelling or solidification agent in different applications. Thus recommendation will be applied after tested their biosafety.
The study concluded that the optimum conditions for bacterial growth and alginate production were varied from one tested strain to anther. The two strains Azotobacter vinelandii and Azotobacter chrococcum were isolated from soil and selected as high efficient alginate producing bacteria. The highest alginate production in shake flask culture was obtained by the two strains in Clementi medium containing sucrose, yeast extract, ammonium sulphate & sodium phosphate and in whey production medium containing lactose, yeast extract, ammonium nitrate & sodium phosphate at pH 7 after 72 hrs fermentation period at 32 and 30°C, respectively.
The calculated D10-values of Azotobacter vinelandii and Azotobacter chrococcum were found to be 0.29, 0.33 kGy, respectively indicates the high sensitivity of these strains to Gamma radiation. The alginate concentration increased at the dose of 0.5 KGY by Azotobacter vinelandii and Azotobacter chrococcum. from these results, it is concluded that, the use of γ-radiation as a mutagenic agent may be useful for production of alginate from Azotobacter vinelandii and Azotobacter chrococcum capable to producing higher quantities of alginate.
HPLC chromatogram of Azotobacter vinelandii alginate had higher alginate standard ratio than Azotobacter chrococcum alginate.
FT-IR spectra of Azotobacter chrococcum alginate and Azotobacter vinelandii alginate have the same range of peaks as well as standard alginate.
CMC in the production of ice- cream has the heighst level of viscosity followed by standard alginate, Azotobacter vinelandii alginate and Azotobacter chrococcum alginate at the same speed.