Isolation And Fermentation Optimization Of PHA Producing Strain In Industrial Wastewater Activated Sludge And Bioinformatics Analysis Of PHA Synthase

Polyhydroxyalkanoates(PHAs)are a kind of intracellular polyester synthesized by bacteria,and is widely distributed in natural bacteria.Nowadays,white pollution and the shortage of oil resources caused by the traditional plastic industry are becoming increasingly serious.Thus,as an environment-friendly plastic,more and more attention has been focus on PHA,but high production cost limits its widely used.It has been reported that the activated sludge contains a large amount of PHA-producing bacteria,which provides a prerequisite for reducing the production cost of PHA and the resource utilization of surplus sludge.In this paper,a PHA producing strain UJN1 was screened from the propylene oxide saponification wastewater activated sludge.UJN1 was then identified by Biolog microbiological identification and 16 S rDNA sequencing.It was identified as Brevundimonas vesicularis.Afterwards,the PHA synthase gene(phaC)was amplified by PCR,and the bioinformatics analysis of this gene was carried out to study the basic physicochemical properties and catalysis mechanism of PHA synthetase.Finally,the PHA fermentation of Brevundimonas vesicularis UJN1 was optimized for medium and cultivation conditions to increase PHA titer and reduce production costs.The main contents and results of this paper are as follows:1.Screening of PHA-producing strains.The fresh sludge obtained from the industrial wastewater treatment plant was firstly domesticated,and then Nile Blue staining was explored to obtain 19 PHA producing strains.Through batch fermentation,the dry cell weight was measured and the PHA yield was determined by gas chromatography.The strain with the highest growth rate and PHA accumulation was selected and named UJN1.2.Identification of UJN1.The strain UJN1 was subjected to physiological and biochemical experiments.The results showed that it was a gram-negative non-intestinal bacterium.Then,the GN-2 microbial identification plate was selected for Biolog microbiological identification of this strain.The genomic DNA of UJN1 was also extracted for 16 S r DNA sequencing.The results of BLAST sequence and Biolog showed that UJN1 had 99% identity with Brevundimonas vesicularis.3.Bioinformatics analysis of phaC.The phaC gene of Brevundimonas vesicularis UJN1 was obtained by PCR and sequenced successfully.The bioinformatics analysis was performed on the open reading frame of phaC,and the physical and chemical properties of hydrophobic,transmembrane region,signal peptide,domain analysis and protein structure of PhaC was also explored.The results show that the isoelectric point of PhaC is 5.40 and the length of ORF is 1731 bp,which contains 576 amino acids.Among the 20 kinds of amino acids that make up the protein,alanine(Ala)accounted for the highest proportion of 14.2%;the instability index(II)was 36.13,indicated that it was a stable protein and had good hydrophilicity.The mechanism of PHA synthesis of the protein was revealed from the molecular level,which provided theoretical basis for further functional identification,expression regulation and transgene research of phaC.4.Optimization of the batch fermentation of Brevundimonas vesicularis UJN1.The medium compositions(carbon source,nitrogen source,C/N)and fermentation conditions(culture time,initial pH,fermentation temperature,shaker speed,inoculation amount,liquid volume,etc)were optimized for UJN1.A multi-factor interaction experiment was carried out to obtain the optimum fermentation conditions after single factor influence experiment.The optimum ratio of sucrose to nitrogen source ammonium chloride was 100 / 1.04,the initial pH was 6.7 and the optimum culture temperature was 33.4 ?.After 48 h of cultivation,the PHA could be accounted for 34.1% of the dry weight.These results showed an increased synthesis efficiency of PHA,which layed a theoretical foundation for large-scale synthesis of PHA using Brevundimonas vesicularis UJN1.