Screening Of Bacteria With High-temperature Resistant And PHAs-producing And The Synthesis Of PHAs Used Alcohol Fermentation Liquid

As a new kind of polymer materials that would replace traditional petrochemical synthetic plastics, polyhydroxyalkanoates(PHAs) is drawing more and more attention from the researchers. With its renewability, biodegradability and biocompatibility, PHAs plays a promising role in the pharmaceutical, fine chemical industry and so on. The accumulation of PHAs occurs in microorganisms when they are living in the circumstance of unbalanced nutrients, such as poor nitrogen, rich carbon, hypoxia form. Sugar and tapioca starch are the pillar industries in Guangxi, however, the wastewater during the producing cause huge pressure in both pollution treatment and environment protection. The wastewater is quite good culture medium for microbes, which is full of carbon and can be synthesized to PHAs cheaply. In this study, we applyied the wastewater of sugar production as carbon source to produce of PHAs after screening and enriching the PHAs-producing bacteria. Our results provide necessary data and foundation for the industrial production of PHAs and wastewater treatment of both sugar and starch production.In this study, we used Nile blue rapid staining and gradient warming to obtained a PHAs-producing bacteria strain named Guangxinu-5 that can resist up to 60℃. This strain was identified as Gram-positive according to its morphological characteristics and Gram staining; and was further identified as a strain of Bacillus cereus according to 16S rDNA sequencing and comparison. PHAs-producing gene was identified by cloning and sequencing of the phaC gene (PHAs synthetase gene); PHAs extracted from the bacteria was analyzed by FT-IR/NMR and was identified as polyhydroxybutyrate (PHB).The opimized conditions for Guangxinu-5 are as followed:Optimum carbon source is sodium acetate, nitrogen source is ammonium chloride, carbon nitrogen ratio is 30, pH value is 7, culture temperature is 50℃, stirring speed when cultured is 1000 r/min. When culturing after optimization, PHAs production rate of Guangxinu-5 reached 2.675 g/L, which was 2.04 times before optimization.When alcoholic fermentation liquid as the carbon source to culture Guangxinu-5 with alcohol fermentation liquid 50 times (cultured after 72 h, NH4CL=0.687g/L、55℃), the highest synthesis rate of PHAs is 213.72 mg/L.The total nitrogen, phosphorus, reducing sugars, CODcr and turbidity of sucrose fermentation broth decreased by 39.66%、75%、86.1%、20.38% and 40.27%,respectively。When cultured in amplified (small fermentation tank 10 liters) continuous circulating(8 times)with alcohol fermentation liquid as the carbon source(no additional carbon source), the first training compared with the eighth training, PHAs yield increased from 0.229g/L to 1.818g/L; however, CODcr values decreased from 2932 mg/L to 1935.2 mg/L with biomass and PHAs content increasing.Our research shows that high-temperature tolerated PHAs bacterial can produce PHAs using wastewater of sugar production as a cheap carbon source in a closed apparatus with no use to lowering the density of nitrogen or lifting oxygen. It greatly released the pressure in both pollution treatment and environment protection in a view of industry, as this method utilized the alcohol fermentation liquid while deducing the content of the organic material.