Studies On The Synthesization Of Polyhydroxyalkanoate Utlizing Biodiesel Byproduct By Pseudomonas

In recent years, oil crisis and shortage of energy have become increasingly serious problems. Countries around the world are actively developing other renewable energy alternatives such as solar, wind, water, biomass energy. As a kind of extremely promising renewable energy, biodiesel received widespread attention from Academia and industry community around the world. However, in the production of biodiesel, it will produce a large number of by-products (mainly glycerol). According to the information from the National Biodiesel Association of the United States, it is expected that the production of biodiesel would reach 12 million tons all over the world by 2020. Excessive accumulation of by-products will lead to environmental problems as wastewater, waste stream, and it will also bring enormous impact on traditional glycerol market. It has become the key and security to ensure the sustainable development of biodiesel that how to use new techniques and new technologies for processing the by-products of biodiesel, researching and developing new products with high added value.Polyhydroxyalkanoates (PHAs) comprise a large family of bacterial storage polyesters that are accumulated by many microorganisms as intracellular carbon and energy storage materials under C, N source imbalance conditions, which have good thermal plasticity, biocompatibility, and biodegradability could be wildly used in environment-friendly materials, tissue engineering, drug controlled release carrier and other aspects. It is a hotspot that value-added utilization of PHA from biosynthesis, however the high cost of raw materials has restricted its development. Pseudomonas is a kind of good PHA producing strains which can utilize different carbon sources for PHA synthesis, by-product of biodiesel is very good and cheap raw material for PHA synthesis by Pseudomonas. It has important meanings that utilizing biodiesel by-product as raw materials for PHA synthesis.Pseudomonas oleovorans ZJ03 preserved in our lab previously was used as starting strain for UV random mutagenesis. The high-yield strain was screened by Nile Red dye screening assay and further verified by GC method. Type I and II PHA synthase genes from Pseudomonas were cloned and sequenced, which was then expressed in PHA-negative Ralstonia eutropha PHB-4 to confirm gene function, the results as follows:1)Pseudomonas oleovorans ZJ03 was used as starting strain for UV random mutagenesis. High-yield strain was screened by Nile Red dye screening assay by the principle that fluorescent intensity of dye stained PHA was proportional with the PHA yield. Results were further verified by GC method. Twice mutagenesis had been done in this study. For the first time three mutant strains including a forward mutant A1 and two negative mutants A3 and A8 were obtained. The PHA yields of them were 10.98% and 13.67% respectively, and the yield of A1 was just slightly increased compared with wild type. For the second time, a mutant strain with 44.67% PHA content increased from 24.59% was obtained by direct spreading selective plate and picking the colony with higher fluorescent intensity. But the result had a low repeatability and stability, further work was needed to be done to get a stable mutant strain with high PHA yield by condition optimizing.2) Type I PHA synthase gene was cloned from genomic DNA of P. oleovorans ZJ03 by PCR method with primers designed according to the conserved sequence of the published results by sequence alignment. The gene was subcloned to expression vector pBBR1MCS2 which was then transformed into Escherichia coli S17-1 for plasmid conjugation. Gene function was confirmed in PHA-negative strain R. eutropha PHB-4 after conjugation by PHA accumulation in bacterial cells. PHA contents were determined by GC and results were 73.88±3.80% and 63.09±1.18% utilizing fructose and gluconate as substrate respectively. Gene function was verified by the results above.3) Type II PHA synthase genes were cloned from genomic DNA of P. corrugata YF388 by PCR method with primers designed according to the conserved sequence of the published results by sequence alignment. The genes were subcloned to expression vector pBBR1MCS2. After transformed into Escherichia coli S17-1 the plasmids were conjugated to R. eutropha PHB-4. The expressing vectors were constructed successfully by sequencing and double digestion. In summary, high-yield mutant stain was obtained in this study. PHA synthase genes were cloned successfully which were then subcloned into expressing vector and functional expressed in R. eutropha PHB-4. It provided certain basis for site-directed mutagenesis of PhaC genes and chimeric synthases construction to synthesize new kinds of PHA with different monomer composition.