Screening of the Xanthan and Guar Gum Degrading Microbes
|Keywords||Xanthan Guar Gum Polysaccharide Degradation Oligosaccharide Enzyme Breaker Screening|
Xanthan and guar gum are important working-fluid thickeners used in oil-exploiting process and that they are effectively and safely depolymerized, which is called break, is on of key working procedures in exploiting. For high-temperature oil field, chemical breaker is usually used, but it does not work well for the oil field of low tempetature for its inefficient breaking-ability and malignant environmental effects. Enzymatic break provides a safe and effective method for low-temperature oil field. Screening of microbes able to depolymerize xanthan and guar gum and obtaining enzyme with effective and safe breaking ability are preconditions of developing enzyme breaker. On other hand, oligosaccharide shows much of physiological activities such as Antimicrobial, antitumor, Gastrointestinal -Microecology- improving and so on. The enzymes able to hydrolyze xanthan and guar gum is key of reseaching and developing xantho-oligosaccharide and guar-oligosaccharide.In order to develop enzyme breakers to get through difficulties in break process of low-temperature oil field’s exploiting and obtain enzymes able to hydrolyze xanthan and guar gum for oligosaccharide-producing , mass screening work was done in the thesis. 4 strains of xanthan degrading microbes X1, X2, X4, X11 and 2 strains of guar gum degrading microbes G3 and G5 are separated from soil, of which X1,X2,X4,X11 and G5 were mold and G3 was Gram-negative bacteria. Four xanthan degrading microbes X1, X2, X4 and X11 did not show effective viscosity-ruducing ability during of fermentation in xanthan medium. X4, which performed better and could endure high temperature of 45℃, was chosen for further research. Through preliminary optimization of the culture conditions, the medium using organic nitrogen sources and the initial pH value at 6.0 , cultivation temperature at 30℃, the viscosity of the medium reduced by 80% at 60h, the viscosity-reducing ability was much improved. Through breeding of X4 by UV mutation, the selected positive mutant strains X4.14 and X4.15 showed better viscosity-reducing ability than X4. The viscosity of medium reduced to 13.4% and 13.1% at 60h’s fermentation.Degradation mechanism of X4 was preliminary studied. The fermentation broth of X4 could change the viscosity and the transparence of the xanthan substrate. The polysaccharide in transparent broth produced during fermentation was separated and analyzed by MS and paper layer chromatography, and the result indicated that polysaccharide might produced by eliminating the mannose residue at the branch end of xanthan. During the fermentation stage,the viscosity of the fermentation broth was gradually decreased and none of reductive sugar was detected, which indicated that enzyme responsible for degrading xanthan might be cellulase with low activity. The finally presumed degradation mechanmism was that, the mannose residue at the branch end of xanthan was cut down by certain enzyme firstly, which made the stable double-helix conformation of xanthan destroyed, then, the backbone was gradually hydrolyzed by cellulase.G3 and G5 exhibited excellent guar gum-degrading ability. The relative viscosity of the guar gum medium was reduced completely from 6.5 almost to 1 by G3 and G5 at 36h and 24h of cultivation time, respectively. They may both produce guar gum-depolymerizing endoase, which can hydrolyze the guar gum rapidly.Peptone was the best for G3 in the tested nitrogen sources. By using peptone the bacteria grow quickly, both the the time of reductive sugar produced in fermentation broth and the quantities of reductive sugar were larger than it using others. For G5, cultured in medium using NH4Cl as nitrogen source, the pH values of broth dropped to 3.0 at which G5 could endure and had enzymatic activity but reductive sugar in broth was almost none, which mean the reductive sugar produced by hydrolyzing guar gum was consumed rapidly by the greatly increasement of mycelia. Hereby, NH4Cl was the best for G5 in the tested nitrogen sources.How the initial pH values of medium affected the the microorganisms was studied. The suitable pH value of mediun for G3 was alkalescent and neutral and the best initial pH value of medium was 8.0 for liquid fermentation. Meanwhile, The suitable pH value of mediun for G3 was acidic and neutral and the best initial pH value of medium was 6.0 for liquid fermentation.The G3 and G5 both could endure high temperature of 45℃and endure basic and acidic environments respectively, which show their potential for enzyme breaker used in oil-field with different environments.