الفهرس 
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Abstract
Inorganic phosphate (P) and potassium (K) are major essential nutrient for plant growth and development, however that most soils have low availability. To overcome this problem it is necessary to apply high amounts of P and K fertilizers, however this is inefficient and costly. Insoluble rocks (phosphate or potassium) are a natural, cheap and clean compound but unfortunately it is a poor P or K fertilizer which may be converted to soluble by the action of some microorganisms.
Therefore, the present work has been focused to study the production of fermented solutions by most rocks solubilizing microbial strains in order to use in some agricultural applications. Some microorganisms are capable of accelerate the dissolution of RP in their fermented solutions produced and namely P- solubilizing microorganisms (PSM). In order to improve the biotechnological application of PSM in fertilizer industry to either produce more soluble P fertilizer or enhance the effectiveness of the direct use of RP as P fertilizer, it is necessary to know the impact of some critical factors.
The study included isolation, selection and identification of the most efficient phosphate and potassium solubilizing microorganisms, detection of some nutritional and environmental factors supporting their efficiencies to solubilize phosphate. A part of the study was carried out to use some available agro-industrial residues as cheap substrates for phosphate solubilization in order to production of fermented solution from RP using submerged culture. The fermented solution produced was applied as a mineral source for microbial growth and foliar spray fertilizer for agricultural bean.
Results could be summarized in the following points:
1- One hundred and eight phosphate solubilizing isolates were isolated from different sources with the percentage of 36.15%, 33.33%, 18.51%, and 12% from different rhizosphere plants, rock phosphate soil, soil and rock phosphate powder mine, respectively. Bacterial and fungal isolates were presented with the percentage of 88.9% and 11.1% from the total isolates, respectively. All fungal isolates were more efficient for phosphate solubilization (clear zone around the microbial growth) than bacterial isolates and recorded the highest degree of solubilization. No significant differences between K solubilization activities of all bacterial isolates (20 isolates) in solid culture which recorded the same value of clear zone diameter (6mm) and Khandeparkar’s ratio D/d (1.2). Whereas significant differences between fungal isolates resulting clear zone diameter ranged from 6 to 8 mm and Khandeparkar’s ratio ranged from 1.2 to 1.6.
2- Four out of 108 isolates were selected as the most efficient Ps isolates which gave the highest values of phosphate solubilization index (PSI) and phosphate solubilization efficient (PSE%) in Pikovskaye’s agar medium containing tri-calcium phosphate (TCP) as the source of insoluble phosphate (qualitative estimation). These isolates were Rs22, Rs7, RPf10 & Bf6 which recorded PSE of 320%, 300%, 126% and 125%, respectively. At quantitative estimation, there isolates recorded the maximum rock phosphate solubilization efficiency (RPSE%) being 4.53%, 4.45%, 1.07% and 0.83% by Bf6, RPf10, Rs22 and Rs7, respectively in Pikovskaye’s medium supplemented with 5% RP after 10 day of 30°C using shake flasks as a batch culture.
3- The two bacterial isolates were phenotypic identified as Serratia sp. Rs22 and Rs7 according to Brenner et al (2005). The fungal isolates were phenotypic identified as Aspergillus sp. Bf6 and RPf10 according to Barnetl and Hunter (1988).
4- Factors affecting rock phosphate (RP) solubilization
The original carbon sources of the basal medium (Pikovskaye’s medium) was replaced by different carbon sources (8 sources) to study their effect on RP solubilization. Glucose proved to be the best carbon sources for each of Aspergillus sp. Bf6, Aspergillus sp. RPf10, Serratia sp. Rs7 and Serratia spRs.22 isolates. The suitable concentration of the best carbon source (glucose) for the highest solubilization of RP by tested bacterial isolates was 10g glucose L-1 and 12.5g & 15g glucose L-1 for fungal isolates Bf6 & RPf10, respectively.
Yeast extract was the most favorable nitrogen source for RP solubilization by tested fungal isolates with 1.5gL-1 and 2gL-1 concentrations for Aspergillus sp. Bf6, Aspergillus sp. RPf10, respectively. Tryptone was the best nitrogen source for Serratia sp. Rs7 and Serratia sp. Rs22, at concentration of 1.25gL-1.
Some agro-industrial by-products and wastes such as bagasse, corn cobs, black sugar cane molasses, olive cake wastes, rice straw, sugar beet waste and whey were used as a carbon source for RP solubilization by the most efficient P solubilization isolates Aspergillus sp. Bf6, Aspergillus sp. RPf10, Serratia sp. Rs7 and Serratia sp. Rs22 using shake flasks as a batch culture. These wastes were used in there treatments, as a whole medium (T1), as a carbon source in modified Pikovskaye’s medium (T2) and their acid hydrolysis as a carbone source in the same medium (T3). The results revealed that the maximum RP solubilization activity was obtained by Aspergillus sp. Bf6 and Aspergillus sp. RPf10 on sugar beet waste as whole medium supplement with 5% RP which increased the fermentation period from 10 to 12 days and the RPs content 1.04 and 1.01 fold, respectively, comparing to control treatment (modified Pikovskaye’s medium No.1 & 2). Regarding Serratia sp. Rs22 and Serratia sp. Rs7, the maximum RP solubilization activity was attained in whey as whole medium supplement with 5% RP which resulted to loss 19.8% and 14.9% of their RP solubilization content, respectively and increased the fermentation period from 8 to 10 days compering to control (modified Pikovskaye’s medium No3).
All treatments of sugar beet wastes, bagasse, rice straw and corncobs failed to support RP solubilization by both isolates of Serratia sp. Rs7 and Rs22. Also corn cobs showed negative effect on RPs actitivity in all treatments for both fungal isolates (Aspergillus sp. Bf6 and RPf10).
Different concentrations of RP ranged from 1 to 12gL-1 were tested for maximum P solubilization activity by all tested isolates. Increasing the RP concentration from 5.0 to 7.0 gL-1 or to 10 gL-1 in whey or sugar beet waste media led to increase RP solubilization activity (RPs content) 1.26 & 1.22 fold by Serratia sp. Rs22 and Serratia sp. Rs7 or 2.14 & 2.04 fold by Aspergillus sp. Bf6 and Aspergillus sp. RPf10, respectively. Therefore, whey medium containing 7gL-1 and sugar beet waste media containing 10gL-1 RP were the preferable medium for RP solubilization by tested bacterial and fungal isolates.
Different levels of pH values were studied. pH 5.5 was found to be the most favorable one for RP solubilization and RPSE% after 12 days of incubation period by fungal isolates Aspergillus sp. Bf6 (182.4 µgP ml-1 & 6.08%) and RPf10 (196 µgP ml-1 & 6.53%), whereas the pH 7.0 was the best value for bacterial isolates Serratia sp. Rs7 (15.0 µgP ml-1 & 0.71%) and Rs22 (16.9 µgP ml-1 & 0.8%).
Different degrees of incubation temperatures ranged from 20 to 35°C were tested. 30°C was the optimum fermentation temperature which recorded the highest values of RP solubilization by Aspergillus sp. Bf6 and Aspergillus sp. RPf10 being 182.2 and 196.04 µgP ml-1 with RPSE 6.07 and 6.53%, respectively. The corresponding figures obtained by Serratia sp. Rs7 and Serratia sp. Rs22 were 15.05 & 17.36 µgP ml-1 for RPs content and 0.72 and 0.83 % for RPSE, respectively.
Inoculum sizes ranging from 1 to 5% were examined. The inoculation with 3% gave the highest RPs content being 15.99, 17.95, 183.4 and 196.17 by Serratia sp. Rs7, Rs22, Aspergillus sp. Bf6 and RPf10, respectively.
from the above mentioned results, it could be concluded that the tested RP solubilizing fungal (Aspegillus sp. Bf6 and Aspergillus sp. RPf10) and bacterial cultures (Serretia sp. Rs7 and Serretia sp. Rs22) recorded the maximum RPs activity in sugar beet waste and whey media supplemented with 10 and 7 gL-1 insoluble RP at pH 5.5 and 7.0 then inoculated with 3% standard inoculum and incubated at 30°C for 12 and 10 days, respectively. Whereas, tested fungal cultures resulted higher RPs activity than bacterial culture. At this treatment of sugar beet waste, Aspergillus sp. RPf10 gave RPs content and RPSE% (196.17 & 6.54) higher than Aspergillus sp. Bf6 (183.40 & 6.11). So, Aspergillus sp. RPf10 was selected as a good P solubilizing organism
5- The most efficient RP solubilization isolate (Aspergillus sp. RPf10) was identified with 18S rDNA gene sequencing. The strain of Aspergillus sp. RPf10 was close to the gene sequencing of Aspergillus tubingensis USMI03 strain.
6- The fermented solution produced by Aspergillus tubingensis RPf10 containing higher amounts of citric acid (543.39 µgP ml-1), growth promoting (IAA 11.96 mgL-1) and micro or macro elements (N,P,K,Mn,Zn & Cu) as well as higher phosphatase activity than that produced by tested isolates.
7- Some application of Aspergillus tubingensis RPf10 fermented solutions.
As a nutrient solution of microbial growth
Using the Aspergillus tubingensis RPf10 fermented solution as a whole medium led to decrease the growth rate of tested bacterial isolates and fungal isolates except Tricoderma sp. which gave the same growth on fermented solution treatments comparing to control. Whereas the tested yeast isolates recorded the same growth rate on fermented solution treatments as whole media and higher growth rate on fermented solution + glucose treatment compering to control. Also, the latter treatment resulting higher growth of some tested bacteria (Bacillus megaterium and Pseudomonas fluoresons and the same growth rate of other bacteria (E.coli). On contrast the growth of Rhizopus sp. was decreased on fermented solution treatments than control. So, it could be suggested the using of Aspergillus tubingensis RPf10 fermented solution as a whole medium or as a mineral source for microbial growth which varied from one microorganism to another.
As nutrient solution for the improvement of bean plant growth.
In a pot experiment, Aspergillus tubingensis RPf10 fermented solution at different P concentrations (from 49.25 to 197.01µgP ml-1) were applied as foliar spray during 45 day of bean plants cultivation at different treatments. The treatment T7 irrigated with Hoagland and foliar spray with non filtered fermented solution (presence of fungus) at 197.01 µgP ml-1 could be more favorable than that irrigated with Hoagland and foliar spray with filtered fermented solution (T6) and than Hoagland solution only (T5) for plant growth, as increased the plant hight, yield & P content about 1.05, 0.99 & 1.04 fold and 2.53, 1.97 & 2.87 fold after 45 days, respectively suggesting its potential use as fertilizer.