Design,Construction And Application Of A Microbe-enzyme Combined System For The Degradation Of Polyethylene Terephthalate Plastics

Polyethylene terephthalate（PET）is one of the most widely used plastics,and its waste causes serious harms to the ecological environment.Compared with traditional incineration and landfill,biodegradation of PET has attracted much attention because of its green and environmental protection characteristics.The rate of enzymatic degradation is fast,but the degradation is incomplete,and the accumulation of degradation products will increase the environmental pressure.Conversely,microorganisms have a low degradation rate but are able to completely convert PET into environmentally sound substances.The combined degradation system of microorganisms and enzymes can not only realize the complementary advantages of bacteria and enzymes but also completely degrade PET,which provides a new perspective for the development of effective PET degradation methods.At present,most PET-degrading enzymes are high-temperature enzymes,and the optimum catalytic temperature is close to the degradation temperature of PET（60°C-70°C）.However,the optimum growth temperature of PET-degrading bacteria（35°C-40°C）is very different from that of PET-degrading temperature.Therefore,the acquisition of thermophilic PET-degrading bacteria is the key to the construction of a microbe-enzyme combined system to efficiently degrade PET.This topic had obtained thermophilic PET-degrading bacteria through separation and screening,and combined them with PET-degrading enzyme ICCG obtained from a metagenomic library of branch and leaf compost to select the thermophilic degrading bacterium with synergistic effect.On this basis,the topic constructed the microbe-enzyme combined PET-degrading system and explored its degradation characteristics and the synergistic mechanism of the bacterium and enzyme.The main research contents are as follows:（1）Screening and identification of thermophilic PET-degrading bacteria.PET waste collected from hot springs in Tengchong,Yunnan Province and microbial community composition was obtained by total genome extraction and sequencing.The result showed that the microbial community diversity was low and the dominant bacteria were Acinetobacter,Bacillus,Dietzia and Psychrobacter.A total of 5 thermophilic strains were obtained that could use PET as a nutritional source through separation and screening.After identification,the strain JQ1 and JQ4 was Paenibacillus thermoaerophilus;the strain JQ3 was Bacillus thermoamylovorans;the strain JQ2 and JQ5 was Anoxybacillus rupiensis.Among them,B.thermoamylovorans JQ3 combined with ICCG degraded 65.1%of the PET film,while the degradation rates of other strains combined with ICCG were all below 5%.B.thermoamylovorans JQ3 had a synergistic degradation effect with ICCG on PET.Therefore,B.thermoamylovorans JQ3 was selected as the optimized degrading bacterium for further study.（2）The evaluation of the degradation characteristics of B.thermoamylovorans JQ3.B.thermoamylovorans JQ3 was a gram-positive bacterium,rod-shaped,with flagella.Its optimal growth p H,temperature,and Na Cl concentration was 7.0,50℃,and 0.5%,respectively.B.thermoamylovorans JQ3 could degrade PET at a rate of 0.043 mg?d^-1,as well as mono（2-hydroxyethyl）terephthalate（MHET）at 81.8%of 84 h and bis（hydroxyethyl）terephthalate（BHET）at 98%of 12 h.During the degradation of PET,the highest viable count of B.thermoamylovorans JQ3 was 4.5×10⁷ CFU?m L^-1 at 3 d,and then showed a small range of fluctuation state.The peak of esterase activity was observed at 2 d,about 365 U?L^-1,then decreased to a trough(272 U?L^-1)at 5 d,and then stabilized.Moreover,B.thermoamylovorans JQ3 could erode the surface of PET through colonization and biofilm formation behavior.（3）Construction,optimization and application of the microbe-enzyme degradation system.Two types of PET degradation system were established by combination of B.thermoamylovorans JQ3 and ICCG（the tandem system:PET films were treated sequentially by bacteria and enzymes;the parallel system:PET films were treated by a combination of bacteria and enzymes）.Among them,the degradation rate of PET in the tandem system was only 13.5%,which was caused by the degrading enzyme ICCG,rather than the synergistic result of B.thermoamylovorans JQ3 and ICCG.The parallel system showed significant synergistic degradation effect of PET,about 100%of degradation rate,which was 8 times of the sum of the bacterium and the enzyme treatment alone.In the parallel system,the surface of PET was continuously eroded to form holes until it was completely degraded.The further study indicated that the parallel system could break chain of PET by attacking the carbonyl group and alkoxy group of PET to degrade PET.Meanwhile,the parallel system could also degrade MHET and BHET without synergistic effect.The parallel system had the best degradation effect on PET and could completely degrade PET in 10 d under 100.0μg?m L^-1of ICCG,2.5×10⁸ CFU?m L^-1 of B.thermoamylovorans JQ3,50 mg?m L^-1 of PET,and inorganic salt medium of buffer system.The scale-up treatment of PET was investigated in a 3 L reactor,results showed that the degradation rate reached 100%at 4 d and time was greatly shortened.（4）Whole genome and transcriptional level analysis of B.thermoamylovorans JQ3.Genome of B.thermoamylovorans JQ3 was 3800242 bp with 37.44%of（G+C）s%,and127494 bp of N50 length.Based on gene function annotation and evolutionary tree analysis,six potential PET-degrading enzymes（Lip4＿120,Est8＿89,Est11＿32,Est18＿23,Ces19＿14and Ces39＿5）were selected from the genome.Through homologous modeling and molecular docking,their catalytic centers were easy to bind MHET and BHET,and the long chain 2-HE（MHET）₅.These enzymes were heterologous expressed in Escherichia coli BL21（DE3）for functional verification.Results indicated six enzymes showed degradation effects on PET,MHET and BHET.Among them,Ces39＿5 and ICCG exhibited a synergistic degradation effect,and the accumulation of the ultimate product TPA was the highest,about 25μmol?L^-1.During the degradation of PET by B.thermoamylovorans JQ3 and ICCG,the transcriptional level of multiple potential degrading enzymes showed an up-regulated trend by reverse transcription-polymerase chain reaction（RT-PCR）analysis.Of these,the transcriptional level of Lip4＿120 and Ces39＿5 was increased by 18%and 30%respectively,compared with that of cultivation alone.