| Plastics are widely used in daily production activities due to their superior mechanical properties,such as polyethylene,polypropylene.However,plastics can stabily exist in the environment for a long time without being degraded by microorganisms,which brings serious "white pollution" and secondary environmental pollution.Therefore,biodegradable materials have attracted widespread attention from researchers.Polylactic acid/polybutylene adipate-co-terephthalate(PLA/PBAT)is a popular biodegradable material around the world.It is prepared by blending or copolymerization polylactic acid(PLA)with excellent biodegradability and polybutylene adipate-co-terephthalate(PBAT)with good mechanical properties.However,previous studies on the biodegradation of PLA/PBAT films mainly focused on the degradation properties of materials,but studies on development of PLA/PBAT-degrading microorganism and enhancement and optimization of biodegradability of PLA/PBAT were limited.In this paper,we used farmland soils from in Shandong and Xinjiang covered with PLA/PBAT mulch films for many years,and isolated,screened and identified a variety of PLA/PBAT-degrading bacteria and molds.The protease and lipase activities were initially studied by protease experiment and lipid hydrolysis experiment to determine the best protease and lipase-producing microorganisms as the next co-cultivation research object.Furthermore,using protease activity and lipase activity as response values,the co-culture medium and co-culture conditions of the above-mentioned PLA/PBAT-degrading microorganism were studied by a single factor method,and m was optimized by response surface methodology.In addition,we studied the degradation effect of PLA/PBAT films by the co-culture system.The morphological characterization and structural test of PLA/PBAT films before and after degradation and the analysis of the composition of the degradation solution were used to study the degradation mechanism of the co-culture system.The main research contents and results of this study were as follows:(1)Gradient dilution method was used to isolate,screen,and identify(morphological observation and 16S rRNA and ITS sequence determination)PLA/PBAT-degrading bacteria and from farmland soils in Shandong and Xinjiang.Among them,there were 5 strains of degrading bacteria and 2 strains of degrading molds in Shandong soil,which were significantly more than 2 strains of degradation bacteria and 1 strain of degrading molds in Xinjiang soil.Based on the hydrolytic circle method,it was preliminarily judged that the protease-producing activity of P.mendocina was the highest,and the lipase-producing activity of A.elegans was the highest.Morphological observation,physiological and biochemical experiments,and phylogenetic tree construction were used to further identify P.mendocina and A.elegans.(2)The effects of exogenous nutrition sources on the enzyme-producing activities of P.mendocina and A.elegans were studied by single factor experiments combined with the folin-ciocalteus method and p-nitrophenol method.The results showed that both microorganisms could directly take terephthalic acid,succinic acid,and lactic acid as nutrition sources,and participate in the metabolism of microorganisms.The co-cultivation medium was obtained as follows:100 mL of screening medium was added with 2wt%glucose and 1wt%tryptone.Besides,the single-factor method was used to optimize the culture conditions.The results showed that the optimal conditions were:the initial pH was 7.1,and the inoculation amount was 1mL,and the culture temperature was 30℃.Moreover,the effects of inoculation ratio,inoculation time and inoculation method on the enzyme-producing activity in the co-culture system were investigated by response surface methodology.The results showed that the best protease and lipase activities of the co-culture system were respectively 40.46 U/mL and 11.50 U/mL under the condition which were the inoculation ratio of P.mendocina to A.elegans is 1:1,and the inoculation time was 5.2 days,and the inoculation method was simultaneous inoculation.(3)Study on the degradation performance of PLA/PBAT films by the co-culture system of P.mendocina and A.elegans,The results indicated that P.mendocina and A.elegans could respectively secrete proteases and lipases to catalyze and hydrolyze the ester bonds in PLA and PBAT of PLA/PBAT films,and produce various degradation products such as oligomers and monomers.These degradation products were used as the growth substrate of microorganisms to synergistically degrade PLA/PBAT.And the degradation rate of the co-culture system was 18.95 wt%in 5 days,which was significantly higher than the degradation effect of PLA/PBAT by a single microorganism. |
