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Abstract Productions of microbial nanoparticles and biosurfactant have received great attention because of their broad applications in the fields of medicine, agriculture, biotechnology and waste management. Having a great potential in this applied field, the genus Streptomyces was employed for production of nanoparticles and biosurfactant, to be applied in foliar fertilization and bioremediation of petrol, respectively, which may reduce the need for chemical fertilizers and surfactant which have deleterious effect on the environment. This study aimed to maximized nanoparticles synthesis and biosurfactant production by the selected local isolates of Streptomyces and using these substances as fertilizers and in bioremediation of petrol. To achieve this goal, the following steps were performed: 1. Isolation of different local actinomycetes from various arid soils. 2. Purification and maintenance of the isolated actinomycetes. 3. Studying of some morphological and biochemical properties of the purified actinomycetes. 4. Screening of the purified streptomycete isolates according to their biological activities (nanoparticles and biosurfactant production). 5. Identification of the most active actinomycete isolate(s). 6. Studying the environmental and nutritional conditions affecting nanoparticles and biosurfactant production by the most active strains. 7. Applications of the produced nanoparticles and biosurfactant in fertilization and degradation of waste in soil.Results can be summarized as follows: Fifty actinomycete isolates were obtained from arid soils at 19 different locations of Egyptian desert. All isolates were identified according to their culture, morphological and physiological characteristics. They were found to belong to genus Streptomyces. Part I: Nanoparticles synthesis by Streptomyces isolates 1- For nanoparticles synthesis, based on optical density of the MnSO4 solution, 18 isolates, out of the 50 Streptomyces isolates, showed relatively high optical densities of 0.4 or more at 350nm, and, thereby, were selected for measurement of particle size using Zeta-seizer potential. Results showed a great variation in particle size between isolates ranging between 110-1375 nm 2- Streptomyces isolate 9g3 synthesized the smallest manganese particle size of 110nm, and thus was selected for further studies. 3- Streptomyces isolate 9g3 was completely identified based on its cultural, morphological, physiological, biochemical and molecular characteristics, to be a strain of Streptomyces ramulosus. 4- The characteristics of manganese nanoparticles (MnNPs), synthesized by Streptomyces ramulosus 9g3, were investigated by different analytical techniques, such as UV- visible spectrophotometer, TEM, EDX, XRD and FTIR. 5- Absorption of aqueous MnSO4, treated by Streptomyces ramulosus 9g3 for4 days, was measured at wavelengths from 200 to 900 nm, using UV- visible spectrophotometer and the maximum absorption was obtained at 350 nm, which corresponds to MnSO4. 6- TEM images showed different sizes of MnNPs which arose from the biodegradation of MnSO4 by Streptomyces ramulosus 9g3. The diameter of these nanoparticles ranged from 60-110 nm. 7- EDX spectrum showed the presence of strong signal from manganese atoms. Moreover, the presence of optical absorption peak in the range of 6 to 8 KeV is typical for the absorption of metallic manganese nanocrystallites 8- X-ray diffraction (XRD) further confirmed the generation of manganese. Inspection of the XRD patterns of vacuum dried manganese nanoparticles revealed the existence of sharp diffraction lines at low angles (2° to 99°). The manganese nanoparticles exhibited peaks at 2θ=38°, 44°, 64° and 73° that can be indexed to the (101), (220), (222) and (311) facets of manganese, respectively. These results confirming the results of TEM examination where the same figure was recorded. 9- FTIR showed the absorption in the region of 1000 to 1200 cm-1 confirming the presence of C-O or O-H and the absorption in the region of 2800cm-1 to 3200cm-1 confirming the presence of O-H and CHO functional groups. The absorption in the region of 1200 to 1500cm-1 corresponds to C=O. The absorptions in the region 2300 – 2900 cm-1, confirmed the presence of carbonyl (-C=O) groups. The absorption in the region 1600 to1900 cm-1 confirmed the presence of N-H. The presence of these chemical groups, i.e. C-O, O-H, CHO, C=O, - C=O and N-H indicate amide linkage of proteins of biological origin. 10- Many factors affecting manganese nanoparticle size by Streptomyces ramulosus9g3 such as: manganese sulfate concentration, reaction time, mycelium age, initial pH, agitation rate, reaction temperature and inoculum size. Manganese nanoparticles size became smaller as more control factors were applied. The size was 110 nm when only MnSO4 concentration was studied, while was reduced to 38 nm when all factors used were applied. Therefore, smallest Mn particle size could be obtained when reaction conditions were MnSO4 concentrations 0.0001g/l, reaction time 4 days, age of mycelium 5 days, initial pH 7, agitation rate 200 rpm, reaction temperature 30±2°C, mycelium inoculum size 6 g/l .Part II: Biosurfactant production by Streptomyces isolates. 11- The 50 isolates previously isolated from arid soils were re-tested for their abilities to produce biosurfactants. 12- Thirteen isolates were first excluded since they showed negative results in all of the four tests. Out of the remainder 37 isolates, only 17 isolates showed positive results in the four tests applied (drop collapsing test, para film M test, zone of hemolysis and lipase hydrolysis zone) and hence they were subjected to E24 test. 13- Emulsification index test revealed a great variations between the 17 isolates tested ranging between 14.7 and 31.7% 14- Streptomyces isolate 5S showed the highest E24 being 31.7% and hence was selected for further work. 15- Streptomyces isolate 5S was identified based on its cultural, morphological, physiological, biochemical and molecular characteristics. The genotypic and phenotypic data clearly confirmed that isolate 5S represents a novel species of the genus Streptomyces, for which the name Streptomyces aegyptius sp nov. was proposed and deposited in the Egyptian Microbial Culture Collection (EMCC) under the code number of 1927. 16- Different factors affecting biosurfactant production by Streptomyces aegyptius sp nov. 5S were studied (7 carbone sources, 6 nitrogen sources, 4 waste oils and 5 surfactants). Substitutions ingredients of starch nitrate basal medium were achieved in order to improve biosurfactant production. The recommended medium for enhancing biosurfactant production by Streptomyces aegyptius sp nov. 5S. should contain treated molasses (34.10%), peptone (35.11%), waste oil (38.85%) and tween 80 (39.1%).17- Optimization of biosurfactant production by Streptomyces aegyptius sp nov. 5S was achieved using Response Surface Methodology (RSM). Results indicated that peptone, treated molasses, tween 80, pH, incubation periods and inoculum size positively and significantly affected biosurfactant production using Plackett-Burman design 18- According to ANOVA analysis for RSM, actual combination for best biosurfactant production between the 86 runs composed of the following components: treated molasses (20g/l), peptone (2g/l), tween80 (2 g/l), initial pH (7), incubation period (5 days) and inoculum size (2%) and the predicted biosurfactant activity was 63.4 % (E24). 19- The optimized medium producing the maximum biosurfactant production (ANOVA analysis) was composed of: treated molasses (21.043g/l), peptone (2.403g/l), tween 80 (3g/l), initial pH 7.2, incubation period (5.69days) and inoculum size (2.856%). Part III: Application experiments Two experiments were carried out to test the efficiency of manganese nanoparticles producing S. ramulosus 9g3 as foliar fertilizer for green bean cultivation and the other aimed to use biosurfactant solution and spore suspension of S. ramulosus 9g3 ( 25x108ml-1) for bioremediation of contaminated soil with octane: a- The first trial was performed in greenhouse to apply MnNPs produced by S. ramulosus 9g3 as a foliar fertilization in green bean cultivation and the results can be summarized as follows: 20- Full dose treatment of MnNPs resulted in significantly higher plant height (27.5 cm), number of leaves (5 leaves), number of pods (4 plant-1) and fresh weigh of pods (22.7 g plant-1).21- Full dose treatment of MnNPs increased photosynthetic efficiency since chloroplly A and B were significantly higher than in the other treatments being 0.738 and 0.948 mg-1 wt. respectively. Moreover, significant increase in Mn, Fe and total nitrogen of leave content was observed in MnNPs treatments being 26.72 mgkg-1, 117.30 mgkg-1 and 1.66% respectively. 22- Specific activity of the enzyme SOD in MnNPs full dose treatments was 46.08 Umg-1 while were 34.04 and 29.69 Umg-1 for MnNPs half dose and MnSO4 full dose treatments, respectively. b- The second trial was conducted to evaluate the capability of biosurfactant solution and spore suspension of Streptomyces aegyptius sp nov. 5S in the bioremediation of octane contaminated soil. 23- In sterilized and non-sterilized soil amended with biosurfactant, degradation of octane was significant, direct and faster during the early stage of the experiment up to 30 days being 197 and 208 gkg-1, respectively 24- Degradation of octane in the control treatment did not exceed 20 and 73 gkg-1 of sterilized and non-sterilized soils, respectively. 25- Degradation of octane was slow up to 30 days in both sterilized and non-sterilized treatments being 50 and 84 gkg-1, respectively 26- The highest octane degradation rate was recorded for non-sterilized treatment, being 297 gkg-1, which was significant higher than the other treatments. 27- Finally, the use of biological method for the bioremediation of contaminated soil could be considered as a pure green chemistry as well as completely toxic-free which might participate in reducing environmental pollution. |