Rhamnolipid Synthesis The Enzyme Rh1a Rh1b Gene Cloning, Expression, Purification And Expression And Applied Research In The Oil Microorganisms

Biosurfactants are produced by a wide variety of microbes and are amphipathic molecules with a hydrophiiic and hydrophobic group. One of the biosurfactants well studied is rhamnolipid produced by Pseudomonas aeruginosa. As the traits of the biosurfactants, biosurfactant can form micelles, enhance the solubility of poorly soluble compounds in water. Most of biosurfactants are biodegradable and less toxic than the chemically synthesized surfactants, which makes them have great potential application chance in enhancing oil recovery and cleaning oil tanks. With the increasing demand for the crude oil, microbial enhanced oil recovery (MEOR) becomes a very interesting technique. The aim of our project is to construct a strain that can produce high amount of rhamnolipid or other biosurfactants by the gene engineer technique. The constructed bacteria that carrying a foreign gene can be used in MEOR or the other fields, such as removal the pollutant from the environment.We collected the crude oil and soil samples from Daqing Oilfield, from which some strains that can use crude oil as carbon source were screened. Also, by using the other screen technique, we screened some bacteria with the capability to produce biosurfactants. Under laboratory conditions, it was demonstrated that when some bacteria grew using crude oil as carbon source, the viscosity of the crude oil was reduced. Most of the stains couldn't be identified by traditional way or the system of Microstation which belong to Pseudomonas sp. and Bacillus sp.. We characterized some of the screened strain. Different strain has different capacity to use different length of alkane. Most of the stains could grow under the conditions of high NaCl concentration (0-10%) or under 60℃. The Mass spectrum analysis showed that some strains produced some acids when it grew by using crude oil as carbon source. The cultures ofbacteria producing the biosurfactant could reduce the interfacial tension between water and oil. With the help of the biosurfactant produced by bacteria, the liquid culture of the bacteria could not only reduce the viscosity of the crude oil greatly, but also prevent the wax deposition on the well wall, the efficiency could be about more than 30%.According to the different aims, the fermentation conditions of strains Lll and DQ2 were optimized. For the work regarding to Lll, we got more fermentation product—biosurfactant with a low oil-water IFT 5.3xlO"3 mN/m by controlling the pH and adding glucose. And for DQ2, we got more cells of the strain, during this fermentatiom, we founded an easy way on testing its fermentation ending. Also, we studied the storage condition for the fermentation product of DQ2 and Lll. All the work provides a good basis for the industry production.Crude oil is a complicated compound containing a lot of hydrocarbons, such as alkanes. The oil-degrading bacteria have great potential in bioremediation. Some bacteria were able to decrease the chemical oxygen demand (COD) of the waste water from Oilfield significantly. UV spectrum also showed the composition change of the waste water after treatment. We found that strain M12 could continuously treat the waste water with a good COD removal efficiency, so we checked the effect of cell immobilization on the COD removal efficiency. Polyvinyl Alcohol (PVA) was chosen to immobilize the bacterial cells and these immobilized cells could be applied in the waste water treatment for many cycles with high COD removal efficiency. By physical absorption, the COD of the waste water could also be reduced. So we also used cinder which was a common waste in China as matrix to filter the waster water after M12 treatment. A continuous waste water treatment system with immobilized cells and a cinder column was developed and this continuous system had a significant effect on decreasing the COD of the waste water from Oilfield. According the application situation, aprocess was designed for treating the waste water from Oilfield.We analyzed the 16SrRNA of some bacteria that could be used in enhancing oil recovery. The sequence of the 16SrRNA of strain DQ2 was amplified and sequenced by PCR. By the sequencing aliment, it was shown that DQ2 had great homology with Bacillus licheniformis. People have a lot of interest in specific detecting the bacteria that are used in MEOR and bioremediation, but so far, it is said no good way in MEOR to analyze the bacterium used. According to the published way in specific detecting environmental microbiology and based upon the sequencing aliment of DQ2 with other homologous strains, a set of specific primers were designed to identify DQ2 when it was used in MEOR. We found that this set of primers had great sensitivity and specificity. In this experiment the detecting procedure was simplified by using colony PCR, namely DQ2 could be specifically detected without purifying the genomic DNA.The model reservoirs were used to test the effect of the screened on the MEOR. We applied the fermentation product of Lll to stimulate the oil from the model reservoirs instead of water, the results showed that the oil recovery could be enhanced by about 11%. When we injected M3 or DQ2 into the model and after a shut—in period process, the oil recovery could be enhanced by more than 6% followed by water stimulation. It was also demonstrated the Lll fermentation product could also act as a kind of wax inhibitor by inhibiting wax deposition which means it can also enhance the oil recovery by prolonging the period of the well cleaning.It was demonstrated that Pseudomonas aeruginosa could produce a large amount of rhamnolipid. As one of some characteristics of this strain may cause some disease, there is some limitation for its industrial application. In 1994 it was published that in this strain rhlA and rhlB played important roles in the synthesis of rhamnolipid. However, in 2003 it was further demonstrated that RhlA is responsible for the synthesis of 3-(3-hydroxyalkanoyloxy) alkanoic acid (HAA) While RhlB isresponsible for transferring TDP-rhanmose to HAA to produce rhanmolipid containing one rhanmose. The RhlC can produce rhanmolipid containing two rhanmose. We subcloned rhlA and rhlB from Pseudomonas aeruginosa into pET system and overexpressed them in E.coli. The recombinant protein RhlA containing a 6-His-Tag was a protein with a molecular weight about 33 kDa. We purified the fusion protein RhlA by using affinity column and sizing column in FPLC. By western blotting with using anti-6-His-Tag antibody, we confirmed that the purified protein was what we wanted. Also, the Fourier transformed infrared spectroscopy showed that the purified protein was folded correctly. Using the same pET expression system, the RhlB was overexpressed and purified by affinity column and ion exchange chromatography. The enzyme assay showed that RhlB was active. The above purified proteins RhlA and RhlB can be used for further structural or functional analysis.We used pVLT as a vector to express RhlA and RhlB in oil-degrading bacteria P22 and P8. The RT-PCR (reverse transcription polymerase chain reaction) showed the expression of RhlA and RhlB after IPTG induction. After introduction of rhlAB, the rhamnolipid production of P22 and P8 was enhanced greatly, which was more than that of Pseudomonas aeruginosa. It was also showed that culture product of P22 and P8 carrying rhlAB could enhance oil recovery greatly under laboratory conditions. We found they had better effect than that of Lll.It is well known that RhlA is responsible for the production of 3-(3-hydroxyalkanoyloxy) alkanoic acid(HAA) which could be a mixture of compound with different chain length. HAA is also a kind of biosurfactant and plays some important role in the quorum sensing of bacteria, moreover, it can be secreted outside of the cells. HAA can also reduce the surface tension. Most of our stains can use crude oil as carbon source, and there are a lot of alkanes in crude oil which can be the substrate of the HAA synthesis, so the effect of RhlA on HAA synthesis insome oil degrading bacteria was studied. We found that after only rhlA was transformed into P22 or P8, the fermentation product of the strains can also have very good effect on MEOR. So these constructed strains carrying the foreign gene rhlA have great potential in industrial application.