Screening Of Phosphate-accumulating Organisms And Study Of Polyphosphate Promote The PHB Accumulation

Poly-β-hydroxybutyrate(PHB) is a group of polyesters produced and naturally accumulated by a large number of bacteria as intracellular granules in response to unfavorable growth conditions and nutrient imbalance.Poly-β-hydroxybutyrate represent an interesting alternative to synthetic polymers due to many advantages.Not only they are biodegradable and biocompatible,but they can also be produced from renewable resources.This study was conducted by using the activated sludge from sewage treatment plant as a simple,three effective phosphate-accumulating organisms(PAOs) were screened.The identification of these PAOs according to their physiological,biochemical properties and 16SrDNA gene sequence,the result showed that they are Acinetobacter baumannii, Pseudochrobactrum saccharolyticum,Pseudomonas fluorescens,the strains number are YPS-1, YPS-2,YPS-3.YPS-2 was selected to continue the experiments of optimization.Aerobic/anaerobic twostage craft were designed for PHB compound and accumulation.Take cell growth and phosphorus accumulate rate as indicators,the experiment was advanced through the shake flask fermentation,the optimal results were as follows:carbon source is CH₃COONa,nitrogen source is(NH₄)₂SO₄,phosphorus source is KH₂PO₄.Under the aerobic stage,cell growth and phosphorus accumulate/release rate were investigated using the single-factor experiment and the orthogonal design experiment method.The optimal medium concentration was as follows: carbon source is 1%,nitrogen source is 0.1%,phosphorus source is 100 mg/L.The optimal culture conditions were as follows:medium pH value is 7,the temperature is 30℃,for 10%of volume,incubation time is 72h.The result of phosphorus accumulate conditions provides a theoretical basis for research of typical cycle.It is important for phosphate release and PHB in anaerobic culture.The study of cell growth,phosphorus release rate and PHB accumulation under four nutritional conditions(limit N,limit P,limit N and P,complete nutrition),found that the nutritional balance in favor of cell growth,but the condition,lack of nitrogen and phosphorus is conducive to phosphate release quantity and the accumulation of PHB.This shows that,PHB needs to be accumulated in the unbalanced growth conditions.During the aerobic/anaerobic bacterial growth with a typical cycle,YPS-2 grows strongly, while it has almost no growth in anaerobic stage.Phosphorus accumulation and phosphorus release increased with time,but the anaerobic phosphorus release will take longer.There are certain linear relations among phosphorus,PHB and time,they are increased with time,but the slopes of them were not same.Under the aerobic/anaerobic alternating optimum conditions, accumulation of phosphorus more than 70mg/L,phosphorus release more than 45mg/L,the quality of stem cell units in the proportion of PHB more than 55%.It better achieved biological phosphorus removal and accumulation of PHB.In order to improve the production of PHB,make the cells pre-treatment,found that SDS/EDTA mixture more effective than others.Conditions of PHB extraction were investigated using the orthogonal design experiment method.The optimal conditions were as follows:concentration of NaClO is 30%,NaClO/CCl₄(V/V)=3:1,temperature is 40℃,time is 24h.Compared with the non-optimized,the production of PHB increased significantly,it is two times the size of former.Infrared spectrum analysis for Samples and standards show that samples containing PHB,the result can be used to further qualitative analysis and improve the yield of PHB.