Characteristics Of R.ornithinolytica 160-1 Produced Flocculant And Its Fermentation Optimization

An efficient bioflocculant producing strain Raoultella ornithinolytica 160-1 was isolated and identified by By 16S r DNA sequencing,physiological and biochemical tests,and MALDI-TOF MS analysis.The Raoultella ornithinolytica160-1 secreted extracellular polysaccharide polymer（EPS-160）was cultured for 24 hours,and then precipitated and extracted with ethanol at 4℃to obtain a crude polysaccharide.The ions and residual medium components were removed through a dialysis bag for further purification A pure polysaccharide of 2.76 g/L was obtained.The purified lyophilized polysaccharides were tested for flocculation activity and the structure and composition of the polysaccharides were examined by different methods.With the assistance of Al³⁺and Fe³⁺,EPS-160 can flocculate kaolin.With the aid of Al³⁺,the flocculation efficiency of EPS-160 is higher.When the Al³⁺concentration was 0.08 mg/m L and the EPS-160 concentration was 43.2 mg/L,the polysaccharide flocculant obtained the highest flocculation efficiency,and the highest efficiency was 97.94%.EPS-160 can be stored in a p H range of 1 to 13 and a temperature range of 40 to 100℃.And EPS-160guarantee high flocculation efficiency in an environment with a p H of 3～10 and 20～100℃.The experimental chemical analysis results showed that EPS-160 is mainly composed of protein（5.7%）and polysaccharide（80.3%）.The molar ratio of glucose,rhamnose and mannose in the polysaccharide components measured by high performance liquid chromatography was 3.1:1:2.4.After adding the optimal concentration of Al³⁺and flocculant,the kaolin solution significantly flocculated,the potential changed from-29.2 m V to-1.94 m V,and obvious flocculation occurred.Kaolin flocculation quickly formed a flake floc and precipitated to the bottom.The results of infrared spectroscopy indicated that functional groups such as hydroxyl,carboxyl and methoxy groups existed in EPS-160.There are three types of polysaccharides with different molecular weights measured by gel permeation chromatography.The molecular weights are 1.72×10³,1.12×10⁴and1.17×10⁶.Zeta potential and IR results show that charge neutralization and bridging are the main flocculation mechanisms of EPS-160.This study explored the fermentation conditions of R.ornithinolytica 160-1.It was determined that fermentation at 150 rpm and 25℃.for 24h could obtain the highest polysaccharide flocculant with high flocculation activity.The response surface was used to optimize the fermentation production of R.ornithinolytica 160-1.When sucrose,（NH₄）₂SO₄and Mg SO₄.7H₂O were 66.41 g/L,12.84 g/L and 0.75 g/L,the maximum yield of EPS-160 was 10.11 g/L.The results of zeta potential measurements and infrared spectrometer suggested that charge neutralizationand bridging werethe main flocculation mechanism.The high flocculation rate（86.79%）was also achieved with a low-cost medium（the starch wastewater）after56h cultivation,indicating that R.ornithinolytica160-1 can be used to treat crop production wastewater,and produce flocculants with low cost fermentation.Based on a high flocculation efficiency,p H tolerance and thermal stability,EPS-160 exhibits its great potentialin industrial application.