聚乙烯醇降解细菌筛选及其降解特性

doi:10.13560/j.cnki.biotech.bull.1985.2018-1045

摘要/Abstract

摘要： 从聚乙烯醇泡棉的堆肥降解过程中筛选出可以有效降解聚乙烯醇的菌株,并研究其对聚乙烯醇的降解效果和降解特性。将聚乙烯醇泡棉经过3年的堆肥降解,利用傅里叶转换红外光谱（Fourier transform infrared spectroscopy,FTIR）检测泡棉的分子结构变化;采用Finley法从降解后的聚乙烯醇泡棉中筛选降解效果较好的菌株,通过菌落形态和细胞染色以及16S rRNA基因序列比对进行鉴定;通过紫外分光光度计检测菌株在降解聚乙烯醇材料过程中的菌体浓度变化和聚乙烯醇降解程度,并将筛选菌株对两种醇解度的聚乙烯醇降解效果进行对比。结果表明,经过3年的堆肥降解,聚乙烯醇泡棉分子结构中出现了新的羧基和羟基;筛选得到了四株菌,即Bacillus sp.DG01、Bacillus sp.DG02、Paenibacillus sp.DG03、Paenibacillus sp.DG04。这4株筛选菌株对3.0 g/L PVA1788的降解率分别为67.27%、74.99%、56.74%和59.70%,对3.0 g/L PVA1799的降解率分别为58.27%、54.47%、43.32%和46.59%。PVA泡棉在堆肥降解过程中发生了明显的基团变化,4株菌的生长与聚乙烯醇的降解过程呈耦联关系,且4株菌株整体上对较低醇解度的PVA降解效果更好。

关键词: 聚乙烯醇, 筛选, 生物降解, 红外光谱, 醇解度

Abstract: The aim of this work is to isolate and identify the strains which can effectively degrade polyvinyl alcohol（PVA）from the composting degradation process of PVA foams and further to study the degradation effects and characteristics of these strains on PVA. First,the PVA foam was degraded after 3 years composting,and we used Fourier Transform Infrared Spectroscopy（FTIR）to detect the molecular structure change of the foam. Then we used Finley method to screen the strains with better degradability from the degraded foams. Further we combined morphological characteristics,gram staining experiment and 16s rRNA gene sequence alignment to identify the strains. Finally we employed UV ultraviolet spectrophotometer to measure the degradation rates of PVA by various strains and the OD₆₀₀ of cells in shake flask,and we also compared the degradability of PVA by the screened strains at 2 alcoholysis degrees. The results showed new carboxyl groups and hydroxyl groups appeared in the molecular structure of the PVA foam after 3 years composting degradation;4 strains were screened and identified,including Bacillus sp. DG01,Bacillus sp. DG02,Paenibacillus sp. DG03 and Paenibacillus sp. DG04. The degradation rates by these 4 isolated strains at 3.0 g/L PVA1788 were 67.27%,74.99%,56.74% and 59.70%,respectively,and the degradation rates at 3.0 g/L PVA1799 were 58.27%,54.47%,43.32%,and 46.59%,respectively. The PVA foams presented obvious group changes after the composting;the growth of 4 strains was coupled with the degradation of PVA,and the low alcoholysis degree of PVA was more beneficial to the biodegradation.

Key words: polyvinyl alcohol, screen, biodegradation, FTIR, alcoholysis degree

刘亚兰, 段梦洁, 林晓珊, 张毅. 聚乙烯醇降解细菌筛选及其降解特性[J]. 生物技术通报, 2019, 35(6): 91-98.

LIU Ya-lan, DUAN Meng-jie, LIN Xiao-shan, ZHANG Yi. Identification and Characterization of Bacteria Degrading Polyvinyl Alcohol[J]. Biotechnology Bulletin, 2019, 35(6): 91-98.

参考文献

[1] Peix A, Ramírez-Bahena MH, Velázquez E.The current status on the taxonomy of Pseudomonas revisited:an update[J]. Infection, Genetics and Evolution, 2018, 57:106-116.
[2] Scales BS, Dickson RP, LiPuma JJ et al. Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[J]. Clinical Microbiology Reviews, 2014, 27(4):927-948.
[3] Goldberg JB, Hancock RE W, Parales RE, et al.Pseudomonas 2007[J]. Journal of Bacteriology, 2008, 190(8):2649-2662.
[4] Haas D, Défago G.Biological control of soil-borne pathogens by fluorescent pseudomonads[J]. Nature Reviews Microbiology, 2005, 3(4):307-319.
[5] Weller DM, Landa BB, Mavrodi OV, et al.Role of 2, 4-diacetylphlo-roglucinol-producing fluorescent pseudomonas spp. in the defense of plant roots[J]. Plant Biology, 2007, 9(1):4-20.
[6] Kittinger C, Lipp M, Baumert R, et al.Antibiotic resistance patterns of Pseudomonas spp. isolated from the River Danube[J]. Frontiers in Microbiology, 2016, 7:586.
[7] Zhou Y, Xu YB, Xu JX, et al.Combined toxic effects of heavy metals and antibiotics on a Pseudomonas fluorescens strain Zy2 isolated from Swine wastewater[J]. International Journal of Molecular Sciences, 2015, 16(2):2839-2850.
[8] Martinez JL, Sánchez MB, Martínez-Solano L, et al.Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems[J]. FEMS Microbiology Reviews, 2009, 33(2):430-449.
[9] Tian T, Wu XG, Duan HM, et al.The resistance-nodulation-division efflux pump EmhABC influences the production of 2, 4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24[J]. Microbiology, 2010, 156(1):39-48.
[10] Wetmore KM, Price MN, Waters RJ, et al.Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons[J]. MBio, 2015, 6(3):e00306-15.
[11] Price MN, Wetmore KM, Waters RJ, et al.Mutant phenotypes for thousands of bacterial genes of unknown function[J]. Nature, 2018, 557(7706):503-509.
[12] Zhao C, Zhao J, Wang W, et al.Expression of MLAA34-HSP70 fusion gene constructed by SOE-PCR[J]. Pakistan Journal of Pharmaceutical Sciences, 2017, 30:1125-1127.
[13] King EO, Ward MK, Raney DE.Two simple media for the demonstration of pyocyanin and fluorescin[J]. The Journal of Laboratory and Clinical Medicine, 1954, 44(2):301-307.
[14] Schwalbe R, Steele-Moore L, Goodwin AC, Antimicrobial susceptibility testing protocols[M]. Boca Raton:CRC Press, 2007.
[15] Yan Q, Wu XG, Wei HL, et al.Differential control of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by sigma factor RpoS and the GacS/GacA two-component regulatory system[J]. Microbiological Research, 2009, 164(1):18-26.
[16] Yan X, Yang R, Zhao RX, et al.Transcriptional regulator PhlH modulates 2, 4-diacetylphloroglucinol biosynthesis in response to the biosynthetic intermediate and end product[J]. Applied and Environmental Microbiology, 2017, 83(21):e01419-17.
[17] Hellman LM, Fried MG.Electrophoretic mobility shift assay(EMSA)for detecting protein-nucleic acid interactions[J]. Nature Protocols, 2007, 2(8):1849-1861.
[18] 魏海雷, 张力群. 荧光假单胞杆菌2p24中生防相关调控基因gacS的克隆和功能分析[J]. 微生物学报, 2005, 45(3):368-272.
[19] Zhang W, Zhao Z, Zhang B, et al.Posttranscriptional regulation of 2, 4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24[J]. Applied and Environ Microbiology, 2014, 80(13):3972-3981.
[20] Wei HL, Zhang LQ.Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J]. Antonie Van Leeuwenhoek, 2006, 89(2):267-280.
[21] Li X, Gu GQ, Chen W, et al.The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in Pseudomonas fluorescens 2P24[J]. Microbiological Research, 2018, 206:159-167.
[22] Liu P, Zhang W, Zhang LQ, et al.Supramolecular structure and functional analysis of the type III secretion system in Pseudomonas fluorescens 2P24[J]. Frontiers in Plant Science, 2016, 6:1190.
[23] Martinez-Hackert E, Stock AM.The DNA-binding domain of OmpR:crystal structures of a winged helix transcription factor[J]. Structure, 1997, 5(1):109-124.
[24] Stock AM, Robinson VL, Goudreau PN.Two-component signal transduction[J]. Annual Review of Biochemistry, 2000, 69(1):183-215.
[25] Groisman EA.Feedback control of two-component regulatory systems[J]. Annual Review of Microbiology, 2016, 70(1):103-124.
[26] Li YC, Chang CK, Chang CF, et al.Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element[J]. Nucleic Acids Research, 2014, 42(13):8777-8788.