Isolation,Identification And PHA Synthesis Properties Of Bacteria From The Activated Sludge Of Propylene Oxide Saponification Wastewater

Polyhydroxyalkanoate?PHA?is a kind of diverse and biodegradable high molecular polymer,which can be synthesized by a variety of microorganisms,and generally exists as a carbon source and energy storage substance in the cell.In microorganisms,PHA exists in the form of hydrophobic particles.In addition,PHA has excellent characteristics such as bio-renewable,bio-degradable,and good biocompatibility.Therefore the industrialized production of PHA is of great significance to the sustainable development of agriculture,industry,medicine and the plastic industry.Propylene oxide?PO?is one kind of the important propylene derivatives,which is mainly used in the production of polyether polyols,propylene glycol,and various nonionic surfactants.At present,the chlorohydrin method is mainly used to produce PO in China.The PO wastewater has the characteristic of high pH,high calcium chloride content,and high COD,and the wastewater contains almost no nitrogen source substances.This results in this activated sludge has a completely different biological community structure compare to other activated sludge.In our previous studies,we have utilized the microbial consortium in the activated sludge to produce PHA with the hydrolyzed acidification solution produced by sludge anaerobic acidification as the culture medium.Analysis of PO saponification wastewater sludge before and after domestication was performed through Illumina MiSeq high-throughput sequencing technology.At the genus level,the unclassified bacterial genus was dominant before and after domestication.In order to better excavate the unidentified bacteria in the activated sludge,we had assembled a summary microfluidic droplet device that could isolate the single cell of microorganisms with high throughput.Many strains were screened from PO saponification wastewater activated sludge through enrichment culture,traditional plate coating and microfluidic droplet technology,including two new species with the 16S rDNA similarity less than 97%.The potential new species lm1^T was classified and identified based on genetics,phenotypic characteristics and cell structure composition.The strain lm1^T was short-rod-shaped,Gram-stain-negative,non-spore-forming,and non-motile.After 5 days incubation on LB medium at 37?,the colonies were red,the surface was convex,round,and the edges were neat.For lm1^T aerobic growth,the optimal growth conditions are at 40?,1.0-2.0%?w/v?NaCl,pH 8.0.The main components of strain lm1^T fatty acids are C_19?:?09?:?0 cyclo?8c,C_18?:?1?7c,and C_16:0.The polar lipid profile was composed of phosphatidylethanolamine,phosphatidylglycerol,phosphatidylcholine,diphosphatidylglycerol,three unidentified aminolipids,one unidentified glycolipid and five unknown lipids.lm1^T contained Q-10 as the predominant respiratory quinone.The G+C content of lm1^T was 64.4 mol%.Phylogenetic analysis based on the 16S rRNA gene sequence showed that Rhodoligotrophos appendicifer JCM 16873^T?96.7%16S rRNA gene sequence similarity?was the most similar to lm1^T,followed by Rhodoligotrophos jinshengii CCTCC AB2013083^T?96.2%?.The average nucleotide identity value and the digital DNA–DNA hybridization value between strain lm1^T and R.appendicifer JCM 16873^T were 73.4%and 14.3%,respectively.Based on polyphasic taxonomic data,strain lm1^T could be classified as a representative of a novel species of the genus Rhodoligotrophos,for which the name Rhodoligotrophos defluvii sp.nov.is proposed.The potential new species L72T was classified and identified based on genetics,phenotypic characteristics and cell structure composition.The strain L72^T was short rod-shaped,with negative Gram stain,aerobic,no spore formation and no flagella.After 7days incubation on LB medium at 30?,the colonies were white,round,convex,smooth,and the edges were neat.The growth temperature range is 25-40?,it can grow under the conditions of 0-3.0%?w/v?NaCl and pH 6.0-8.0,and the best growth conditions are:30?,1.0-2.0%?w/v?NaCl,pH 7.0.The cells are catalase positive and oxidase positive.The main respiratory quinone is Q-10.The main polar lipids are phosphatidylcholine?PC?,glycolipid?GL?,aminophospholipid?APL?,phosphatidylethanolamine?PE?,phosphatidylserine?PS?,phosphatidyldimethylethanolamine?PME?,An unknown lipid?L?and two unidentified phospholipids?PL?.The main fatty acids?>5%of total fatty acids?are C_19:09:0 cyclo?8c?36.5%?,C_18:1?7c?12.2%?,iso-C_15:0?9.7%?,and anteiso-C_15:0?9.4%?.The GenBank accession number of the 16S rRNA gene sequence of propylenella oxidense L72T is MK124547.The whole genome number of strain L72^T is SPKJ00000000 and is deposited in DDBJ/ENA/GenBank.It was determined by genome sequencing that the DNA G+C content of this type of strain was 69.0 mol%.Phylogenetic analysis based on 16S rRNA gene sequence showed that Oharaeibacter diazotrophicus DSM 102969^T had the highest similarity with L72^T,and the OrthoANI value with strain L72^T was 71.5%.According to multiphase taxonomic data,strain L72T could be identified as Methylocystaceae The new genus and new species in the family is named Propylenella oxidense sp.nov.The whole genome sequencing results of R.defluvii lm1^T were analyzed and found to contain phaC_R,phaA_R,and phaB_R,key genes for PHA synthesis.Through Blast comparison analysis,the protein encoded by phaC_R was predicted to belong to type I PHA synthase and the highest similarity is only 65.98%.Meanwihile,it was found that phaCR,phaAR,and phaBR are not adjacent to each other in the genome different from Cupriavidus necator H16^T,a type strain of type I PHA synthase.In order to verify the function of phaC_R in R.defluvii lm1^T,phaC_R was linked to phaA_C and phaB_C of Cupriavidus necator H16^T into pBluescript II SK?-?,and the recombinant plasmid pL1-SK was constructed and transferred into E.coli DH5?.Under the conditions of 37?and 180 rpm,E.coli DH5?/pL1-SK could synthesize PHB with 2%glucose as the carbon source,its cell dry weight is 1.7 g/L,PHB content was 5.5wt%;When sodium and glucose were used as the carbon source,the dry weight of the cell is1.8 g/L,and the PHB content is 5.2 wt%;when sodium propionate and glucose were used as the carbon source,the dry weight of the cell is 1.3 g/L,and the PHB content was 5.9 wt%;when sodium valerate and glucose were used as the carbon source,the cell dry weight was 1.2g/L,and the PHB content was 9.6 wt%;when sodium caproate and glucose were used as the carbon source,the cell dry weight was 1.2 g/L,and the PHB content was 9.6 wt%.In order to further verify the function of phaA_R and phaB_R in R.defluvii lm1^T,phaA_R and phaBR were combined with phaCR into pBluescript II SK?-?to construct the recombinant plasmid pL2-SK and transformed to E.coli DH5?.Under the conditions of 37?and 180 rpm,E.coli DH5?/pL2-SK could synthesize PHB with 2%glucose as the carbon source.The cell dry weight was 1.7 g/L and the PHB content was 8.7 wt%.When sodium and glucose were used as the carbon source,the cell dry weight was 2.3 g/L,and the PHB content was 4.2 wt%;when sodium propionate and glucose were used as the carbon source,the cell dry weight was2.2 g/L,and the PHB content was 4.8 wt%;when sodium valerate and glucose were used as the carbon source,the cell dry weight was 1.4 g/L,and the PHB content was 7.8 wt%;when sodium caproate and glucose were used as the carbon source,the cell dry weight was 1.4 g/L,and the PHB content is 7.8 wt%.