Study of the cellulase enzyme system of some native cellulolytic bacteria for degradation of lignocellulosic wastes

Document Type : Research Paper

Authors

1 scientific staff

2 University of TehranDepartment of Soil Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

3 Soil and Water Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Cellulolytic bacteria with cellulase enzyme system play an important role in decomposing lignocellulose wastes and accelerating the composting process. For this purpose, a study was carried out on some native cellulolytic bacteria isolated from different sources in Soil and Water Research Institute. Twenty eight isolates were selected based on morphological characteristics and cellololytic test, in specific medium (B.H.M). Spot cultivation on B.H.M medium and observation of halo diameter as the cellulolytic characteristics of the isolates led to the separation of 12 isolates. The maximum and minimum diameter of a clear zone around the colony of MAL and HSE isolates were 31 and 8 millimeters respectively. Also the maximum and minimum proportion of clear zone diameter to colony were observed in TER and CPL isolates. The activity of endoglucanase, exoglucanase and betaglucosaidase among 12 isolates showed that the least amount of endoglucanase was belong to SAD (0.17 U.ml-1) and the most one was NTL (2.55 U.ml-1) isolates. The maximum activity of betaglucosidase in NTT isolate was 0.31 U.ml-1 and the maximum exoglucanase enzyme activity was belong to HPN isolate (0.35 U.ml-1)and minimum was belong to BSB isolate (0.18 U.ml-1). Analysis of variance showed that the differences between the isolates with the highest and lowest triple cellulase activity was significant (especially endoglucanase (at one percent statistic level but was not significant between some isolates. There was a direct relation between cellulase activity and the enzyme compound. NTT isolate showed the highest activity of beta-glucosidase enzymes and it was the second and the third in terms of endoglucanase and exoglucanase production, respectively. It had the highest activity of three enzymes (3.21 unit) and identified and registered as Bacillus methyltrophicus

Keywords


  1. رجب خانی،ز. زمانی، م. مطلبی، م. عنصری دیزج یکان، ح. 1387. بهینه سازی تولید آنزیم بتا یک و چهار اندوگلوکاناز (سلولاز) قارچ Aspergillus niger(R4)و همسانه سازی ژن.eglB  مجله زیست شناسی ایران،جلد 22 ،شماره 4.
  2. عصاره، ر.ح. شهبانی ظهیری و س.عشقی، 1393، جداسازی و شناسایی باکتری­های بومی تجزیه کننده سلولز از خاک، مجله پژوهش­های سلولی و ملکولی (مجله زیست شناسی ایران)، جلد 27، شماره 1.
  3. Atlas, R.M. 2004. Handbook of Microbiological Media, CRC Press, Boca Raton, Fla, USA.
  4. Bhat, M.K.and Bhat, S. 2000.Cellulose degrading enzymes and their potential industrial applications. Biotechnology Adventure. 15: 583-620.
  5. Bushnell, D.L. and Haas, H.F. 1941. The utilization of certain hydrocarbons by microorganisms, Kansas Agricultural Experiment Station. 199: 653–673.
  6. Colowick, S.P. and Kaplan, N.O. 1955. In: Methods in Enzymology. Academic Press INK London. pp: 149-158.
  7. Enari, T.M. 1983. Microbial cellulases. In: Microbial Enzymes & Biotechnology. Applied science publications, London. pp: 183-223
  8. Gained, S. et al. 2005. Biodegradation study of crop residues as affected by exogenous inorganic nitrogen fungal inoculants. Journal of Basic Microbiology. 4: 301-310.
  9. Hankin, L. Anagnostakis, S.L. 1977. Solid media containing carboxymethylcellulose to detect Cx cellulase activity of microorganisms. Journal of Genetic Microbiology. 98: 109-115.
  10. Han, S.J., Yoo, Y.J., Kangs, H.S. 1995 Characterization of a bifunctional cellulase and its structural gene. The cel gene of Bacillus SP. D04 has exo and endoglucanase activity. Journal of Biological Chemistry. 43: 260-270.
  11. Hemati A. Aliasgharzad, N. Khakvar, R. 2018 In vitro evaluation of lignocellulolytic activity of thermophilic bacteria isolated from different composts and soils of Iran, Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.04.010
  12. Kasana, R., Salvan, R., Dhar, H., Dutt, S., Gulati, A., 2008. A Rapid and Easy Method for the Detection of Microbial Cellulases on agar Plates Using gram’s iodine. Current, Microbiology. 57:503- 507.
  13. Li W, Zhang WW, Yang MM, Chen YL (2008) Cloning of the ThermostableCellulase Gene from Newly Isolated Bacillus subtilis and its expression inEscherichia coli. Molecular. Biotechnology. 40:195–201.
  14. Lynd, L.R., Weimer, P.J., Van, Z.Y., W.H., Pretorius, I.S. 2002 Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews. 66: 506–577.
  15. Miller, G. L. 1959. Use of the dinitrosalicylic acid reagent for the determination of reducing sugars. Analytical Chemistry. 31: 426-428.
  16. Sadhu, S., Maiti, T.K. 2013 Cellulase production by bacteria: A Review. British microbiology research journal. 3:235-258.
  17. Saffari,H., Pourbabaee,A. A., Asgharzadeh, A., Besharati, H. 2016. Isolation and identification of effective cellulolytic bacteria in composting process from different sources Archive of . Agronomy.and  Soil Science. DOI:10.1080/03650340.2016.1198006
  18. Schwarz, W.H.2001. The cellulosome and cellulose degradation by anaerobic bacteria. Applied Microbiology and Biotechnology. 56: 634-649.
  19. Stewart, B. J., Leatherwood, J.M. 1976. Derepressed synthesis of cellulase by Cellulomonas. Journal of Bacteriology. 128: 609-615.
  20. Teather, R. M. and Wood, P. J. 1982. Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rument. Applied Environmental Microbiology. 12: 777-780.
  21. Teeri, T. 1997. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends in Biotechnology. 15: 160-167.
  22. Van, Dyk, J.S., Pletschke, B.I., 2012. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes factors affecting enzymes, conversion and synergy. Biotechnology advances. 30: 1458–1480.
  23. 23-Vargas-Garcia, M.C., et al. 2007. Waste management. 27: 1099-1107.
  24. Vargas-Gracia, M.C., Surez-Estrella, F.F., Lopez, M.J., Moreno, J., 2010. Microbial population dynamics and  enzyme activities in composting processes with different starting materials. Waste Management. 30: 771–778.
  25. Zhang, Y-HP , Hong, J. , Ye, X. 2009. Cellulase assays. Methods of Mollecular Biology. 581: 213-231.