الفهرس 
Microorganisms producing biosurfactantsOnly 14 pages are availabe for public view
 |  | from | 152 |  |  |
| | | from | 152 | | |
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.