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2.8.2. Thermal stability investigation of immobilized lipase MG10
To test the irreversible thermal inactivation of both form of lipase, the enzyme solution was incubated at revealed temperature for 3 h. At different time interval, aliquots were picked up and examined for remaining activity.
Storage stabilities of the free and coated-MGO-magnetic CLEAs enzyme were also examined by placing enzyme solutions in phosphate buffer (100 mM, pH 7.5) without substrate at 4 °C. Every 2 days, cMGO-CLEAs lipase was picked up by a magnetic and washed by distilled water. After that, the activity of both forms of enzymes was measured as described formerly. The residual enzyme activities were dignified by calculating the original lipase activity as 100%.

2.8.3. Investigation of Kinetic parameters
Kinetic factors of both free and coated-MGO-magnetic CLEAs lipase were examined using diverse concentrations of substrate in 100 mM phosphate buffer (pH 7.0) at 45 °C. In both forms, 2 mg of lipase was used in assay reaction. The amounts of Vmax, Km factors for free and coated-MGO-magnetic CLEAs lipase were considered from line Waver-Burk plot of the initial reaction rates equivalent to different substrate concentrations.

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2.9. Biodiesel construction
Enzymatic transesterification reactions were carried out by free and cMGO-CLEAs lipase and maintained for 48 h with a stirring speed of 160 rpm. The reaction consists of 0.4 g oil (oil from Ricinus communis), methanol (1:3 molar ratio between R. communis oil and methanol) and 0.2% enzyme (free or correspond lipase on support) (w/w, based on the oil weight, g). At diverse time intervals (6, 12, 24 and 28 h), 100 µl of reaction blend was picked up and diluted with the same volume of n-hexane solvent. Afterward, the sample was gathered and the upper layer (10 µL) was performed to gas chromatography (GC) investigation for biodiesel measurement (Ji et al., 2010; Wang et al., 2017; Malekabadi et al., 2018).
3. Results and discussion
3.1. Screening and identification of bacterial producing the methanol-tolerant lipase

Lipase producing bacteria were screened in enrichment culture medium supplemented with olive oil as a sole source of carbon. Furthermore, methanol (30%, v/v) was also used to acquire the methanol tolerant lipase. The clear area around the colonies on the tributyrin agar plate was evaluated as lipase production. The greatest lipolytic strains were also examined on the olive oil plate complemented with phenol red, as a pH indicator. Results showed this isolate was a strain which displayed the maximum pink area around the colony. The 16S rDNA gene of MG isolate was amplified and sequenced (Genbank Accession No. MF927590.1) and compared by BLAST investigation to other bacteria in the NCBI database. The results proposed a near relationship between MG10 isolate and the other members of the Enterobacter genus with a extreme sequence homology (99%) to Enterobacter cloacae. The phylogenetic tree (Fig. 1) designated that the strain MG10 was associated with Enterobacter species and used for the following study.

3.2. Purification and immobilization of the lipase
Cell free supernatant of MG10 stain was exposed to ammonium sulfate precipitation (85% saturation) and Q-sepharose chromatography. Lipase MG10 was eluted from the Q-Sepharose column with a 19.5-fold purification and a 38.1 % yield, and it displayed a specific activity of 442.6 U/mg. This yield of MG10 lipase was analogous to the lipase of S. maltophilia (33.9%) (Li et al., 2013) and lower than lipase from P. aeruginosa PseA (51.6%) (Gaur et al., 2008), but greater than lipase of B. licheniformis (8.4 %) (Sharma and Kanwar, 2017). SDS–PAGE analysis of the MG10 lipase shown that it has a single band about 33 kDa, which it is dissimilar with the other Enterobacter cloacae.
Results of protein measurement with Bradford technique displayed that protein loading on these coated magnetite nanomaterials was succeeded. Moreover, the results of determination of protein loading on these nanomaterials shown that, immobilization efficiency was achieved about 73%. mGO-CLEAs lipase was spread in phosphate buffer. After a magnet was positioned sidewise, mGO-CLEAs Lipase showed fast response (60 seconds) to the peripheral magnetic field. It incomes that the magnetic CLEAs-Lip particles were shown suitable magnetic concern even though layers of CLEAs-Lipase were covered on their surfaces, wherein it is significant in term of lipase immobilization.

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2. PURPOSE:
The purpose of this report is to investigate the Effectiveness of the transportation in the area and to find out the solutions to solve the complaints received from the residence of Qauia.

3. SCOPE:
Data was collected from:
3a. 10 survey questionnaires – carried out through door to door interview
3b. Social media – the same survey was posted on a forum group set up for this matter whereby the residents of Qauia have been observing the effectiveness of transportation in the area
4. FINDINGS:
This is carried out from 100 residence and it’s a simple yes and no questionnaires survey
According to the survey the transport system in the area is not effective
Sample questionnaires
Yes No Total Remarks
01 Is Shore Bus Transport the only company providing service in Qauia? 100% 100 Shore bus is the only company
02 The bus service runs every day? 90% 10% 100 On some public holidays no
03 The bus service always on time? 65% 35% 100 Not every day, most of the time late
04 Bus conditions 90% 10% 100 Some of their bus needs replacement
05 Any taxi base? 100% 100 1 taxi base
06 Do you think another bus company should provide service in the area? 100% 100 Yes as they are slack for most of the time especially during peak hours
07 Are they well organized and consistent with their pickup routine / timetable? 40% 60% 100 Clash happens most of the time with drivers
08 Is bus he only means of transport in the area? 90% 10% 100 09 Is it very hard to get transport in the area? 80% 20% 100 Not everyone can afford to pay taxi as all rely on bus as it is cheap
10 Is the Shore Bus service in the area very effective? 40% 60% 100 Not very effective as demanded by the residents of Qauia
EMA MANOA-s11006456

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2.6 GENOME PLASTICITY AND EVOLUTION OF ESCHERICHIA COLI
Like all life forms, new strains of E. coli evolve through the natural biological processes of mutation, gene duplication, and horizontal gene transfer; in particular, 18% of the genome of the laboratory strain MG1655 was horizontally acquired since the divergence from Salmonella. E. coli K-12 and E. coli B strains are the most frequently used varieties for laboratory purposes. Some strains develop traits that can be harmful to a host animal. These virulent strains typically cause a bout of diarrhea that is often self-limiting in healthy adults but is frequently lethal to children in the developing world. (Futadar et al., 2005). More virulent strains, such as O157:H7, cause serious illness or death in the elderly, the very young, or the immunocompromised.
The genera Escherichia and Salmonella diverged around 102 million years ago (credibility interval: 57–176 mya), which coincides with the divergence of their hosts: the former being found in mammals and the latter in birds and reptiles. (Wang et al., 2009). This was followed by a split of an Escherichia ancestor into five species (E. albertii, E. coli, E. fergusonii, E. hermannii, and E. vulneris). The last E. coli ancestor split between 20 and 30 million years ago.
The long-term evolution experiments using E. coli, begun by Richard Lenski in 1988, have allowed direct observation of genome evolution over more than 65,000 generations in the laboratory. For instance, E. coli typically do not have the ability to grow aerobically with citrate as a carbon source, which is used as a diagnostic criterion with which to differentiate E. coli from other, closely, related bacteria such as Salmonella. In this experiment, one population of E. coli unexpectedly evolved the ability to aerobically metabolize citrate, a major evolutionary shift with some hallmarks of microbial speciation.
2.7 INCUBATION PERIOD
The time between ingesting the STEC bacteria and feeling sick is called the “incubation period”. The incubation period is usually 3–4 days after the exposure, but may be as short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. HUS, if it occurs, develops an average of 7 days after the first symptoms, when the diarrhea is improving.

2.7.1 DISCOVERY OF ANTIBIOTICS
• History of antibiotics – 1
19th century:Louis Pasteur & Robert Koch
• History of antibiotics – 2
Plant extracts
– Quinine (against malaria)
– Ipecacuanha root (emetic, e.g. in dysentery)
Toxic metals
– Mercury (against syphilis)
– Arsenic (Atoxyl, against Trypanosoma)
• Dyes
– Trypan Blue (Ehrlich)
– Prontosil (azo-dye, Domagk, 1936)
• History of antibiotics – 3
Paul Ehrlich
• started science of chemotherapy
• Systematic chemical modifications
(“Magic Bullet”) no. 606 compound = Salvarsan (1910)
• Selective toxicity.
• Developed the Chemotherapeutic Index
• History of antibiotics – 4
Penicillin- the first antibiotic – 1928• Alexander Fleming observed the
killing of staphylococci by a fungus (Penicillium notatum)
• observed by others – never exploited
• Florey & Chain purified it by freeze-drying (1940) – Nobel prize 1945
• First used in a patient: 1942
• World War II: penicillin saved 12-15% of lives
• History of antibiotics – 5
Selman Waksman – Streptomycin (1943), was the first scientist who discovered antibiotic active against all Gram-negatives for examples; Mycobacterium tuberculosis
– Most severe infections were caused by Gram-negatives and Mycobacterium
tuberculosis, extracted from Streptomyces – extracted from Streptomyces
– 20 other antibiotics include. neomycin, actinomycin
2.8 CHARACTERISTICS OF ANTIBIOTICS
According to the Oxford Dictionary, the term Antibiotics encompasses medicines (such as penicillin or its derivatives) that inhibit the growth of or destroys microorganisms. Antibiotics are naturally occurring substances that exhibit inhibitory properties towards microbial growth at high concentrations. (Zaffiri, et al., 2012).
-Antibiotics are selective in their effect on different microorganisms, being specific in their action not only against genera and species but even against strains and individual cells. Some of these agents act mainly on gram-positive bacteria, while others inhibit only gram-negative ones.
-Some antibiotics are produced by some organism, from different strains of penicillin.
-Bacteria are sensitive to the antibiotic which enable them to developed resistance after contact, for several periods.

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2.9 ROLE OF ANTIBIOTICS
Based on the clinical use of antibiotics, it may appear that these compounds play a similar role as microbial weapons in nature, yet this seems unlikely due to the fact that the concentrations used in the clinical setting are significantly higher than that produced in nature (Fajardo et al., 2008). Due to experimental evidence, it makes more sense to see antibiotics as small, secreted molecules involved in cell-to-cell communication within microbial communities.
(Martinez, 2008). Diverse Studies have been conducted in which different antibiotics and antibiotic-like structures were administered to different bacterial species at levels below the compounds minimum inhibitory concentrations (MIC). (Fajardo et al., 2008). that was

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