Applied Biochemistry and Biotechnology

, Volume 167, Issue 2, pp 270–284 | Cite as

Isolation, Screening, and Optimization of the Fermentation Conditions of Highly Cellulolytic Bacteria from the Hindgut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

  • Ping Sheng
  • Shengwei Huang
  • Qi Wang
  • Ailing Wang
  • Hongyu ZhangEmail author


From the hindgut contents of Holotrichia parallela, 93 cellulolytic bacterial isolates were isolated after enrichment in carboxymethyl cellulose medium. Among these isolates, a novel bacterium, designated HP207, with the highest endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the 16S ribosomal DNA analysis, morphological characteristics, and biochemical properties. The production of the endoglucanase was optimized by varying various physical culture conditions using a submerged fermentation method. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.432 U mL−1 in bacterial cultures was obtained, higher than those of the most widely studied bacteria and fungi, which are the attractive candidates for the commercial producer of cellulase. And the crude endoglucanase enzyme was also highly thermostable; approximately 55 % of the original activity was maintained after pretreatment at 70 °C for 1 h. Thus, from the present study, the bacterium can be added up to the database of cellulolytic bacteria.


Holotrichia parallela Cellulolytic bacteria Pseudomonas sp. HP207 Endoglucanase 



This research was supported by the National Natural Science Foundation of China (no. 30671404), the Special Fund for Agro-scientific Research in the Public Interest (no. 201003025), the earmarked fund for Modern Agro-industry Technology Research System of China (no. CARS-27), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (no. 200805040010). The authors thank Xiaoxue Li and Cong Li for comments on the manuscript.


  1. 1.
    Willis, J. D., Oppert, C., & Jurat-Fuentes, J. L. (2010). Methods for discovery and characterization of cellulolytic enzymes from insects. Insect Science, 17, 184–198.CrossRefGoogle Scholar
  2. 2.
    Immanuel, G., Dhanusha, R., Prema, P., & Palavesam, A. (2006). Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. International Journal of Environmental Science and Technology, 3, 25–34.Google Scholar
  3. 3.
    Baig, M. M. V., Baig, M. L. B., Baig, M. I. A., & Yasmeen, M. (2004). Saccharification of banana agro-waste by cellulolytic enzymes. African Journal of Biotechnology, 3, 447–450.Google Scholar
  4. 4.
    Clarke, A. J. (1997). Biodegradation of cellulose: Enzymology and biotechnology (pp. 23–68). Lancaster: Technomic Pub. Co.Google Scholar
  5. 5.
    Srinivas, R., & Panda, T. (1998). pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cerevisiae. Journal of Industrial Microbiology and Biotechnology, 21, 178–183.CrossRefGoogle Scholar
  6. 6.
    Saratale, G. D., Chen, S. D., Lo, Y. C., Saratale, R. G., & Chang, J. S. (2008). Outlook of biohydrogen production from lignocellulosic feedstock using dark fermentation—A review. Journal of Scientific and Industrial Research, 67, 962–979.Google Scholar
  7. 7.
    Zhang, Y. H. P., Himmel, M. E., & Mielenz, J. R. (2006). Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 24, 452–481.CrossRefGoogle Scholar
  8. 8.
    Lynd, L. R. (1996). Overview and evaluation of fuel ethanol from cellulosic biomass: Technology, economics, the environment, and policy. Annual Review of Energy and the Environment, 21, 403–465.CrossRefGoogle Scholar
  9. 9.
    Lopes, F. N. (2008). Industrial exploitation of renewable resources: From ethanol production to bioproducts development. Journal of Social Biology, 202, 191–199.CrossRefGoogle Scholar
  10. 10.
    Brune, A. (1998). Termite guts: The world's smallest bioreactors. Trends in Biotechnology, 16, 16–21.CrossRefGoogle Scholar
  11. 11.
    Sun, J. Z., & Scharf, M. E. (2010). Exploring and integrating cellulolytic systems of insects to advance biofuel technology. Insect Science, 17, 163–165.CrossRefGoogle Scholar
  12. 12.
    Huang, S. W., Zhang, H. Y., Marshall, S., & Jackson, T. A. (2010). The scarab gut: A potential bioreactor for bio-fuel production. Insect Science, 17, 175–183.CrossRefGoogle Scholar
  13. 13.
    Zhang, H., & Jackson, T. A. (2008). Autochthonous bacterial flora indicated by PCR-DGGE of 16 S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae). Journal of Applied Microbiology, 105, 1277–1285.CrossRefGoogle Scholar
  14. 14.
    Cazemier, A. E., Verdoes, J. C., Van Ooyen, A. J. J., & Op Den Camp, H. J. M. (1999). Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae. Applied and Environmental Microbiology, 65, 4099–4107.Google Scholar
  15. 15.
    Cazemier, A. E., Verdoes, J. C., Reubsaet, F. A. G., Hackstein, J. H. P., Drift, C. V. D., & Op den Camp, H. J. M. (2003). Promicromonospora pachnodae sp. nov., a member of the (hemi)cellulolytic hindgut flora of larvae of the scarab beetle Pachnoda marginata. A Van Leeuwen, 83, 135–148.CrossRefGoogle Scholar
  16. 16.
    Wenzel, M., Schönig, I., Berchtold, M., Kämpfer, P., & König, H. (2002). Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis. Journal of Applied Microbiology, 92, 32–40.CrossRefGoogle Scholar
  17. 17.
    Miller, G. L. (1959). Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  18. 18.
    Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R., Germon, J. C., Soulas, G., et al. (2001). DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and Environmental Microbiology, 67, 2354–2359.CrossRefGoogle Scholar
  19. 19.
    Vladut-Talor, M., Kauri, T., & Kushner, D. J. (1986). Effects of cellulose on growth, enzyme production, and ultrastructure of a Cellulomonas species. Archives of Microbiology, 144, 191–195.CrossRefGoogle Scholar
  20. 20.
    Abou-Taleb, K. A. A., Mashhoor, W. A., Nasr, S. A., Sharaf, M. S., & Abdel-Azeem, H. H. M. (2009). Nutritional and environmental factors affecting cellulase production by two strains of cellulolytic Bacilli. Australian Journal of Basic and Applied Sciences, 3, 2429–2436.Google Scholar
  21. 21.
    Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596–1599.CrossRefGoogle Scholar
  22. 22.
    Crowson, R. A. (1981). The biology of the Coleoptera (p. 802). London: Academic Press.Google Scholar
  23. 23.
    Crowson, R. A. (1954). The natural classification of the families of Coleoptera. England: Glassey.Google Scholar
  24. 24.
    Koyama, M., Iwata, R., Yamane, A., Katase, T., & Ueda, S. (2003). Nutrient intake in the third instar larvae of Anomala cuprea and Protaetia orientalis submarmorea (Coleoptera: Scarabaeidae) from a mixture of cow dung and wood chips: Results from stable isotope analyses of nitrogen and carbon. Applied Entomology and Zoology, 38, 305–311.CrossRefGoogle Scholar
  25. 25.
    Kotchoni, S. O., & Shonukan, O. O. (2002). Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus. World Journal of Microbiology and Biotechnology, 18, 487–491.CrossRefGoogle Scholar
  26. 26.
    Palleroni, N. J. (2010). The Pseudomonas story. Environmental Microbiology, 12, 1377–1383.CrossRefGoogle Scholar
  27. 27.
    Brodey, C. L., Rainey, P. B., Tester, M., & Johnstone, K. (1991). Bacterial blotch disease of the cultivated mushroom is caused by an ion channel forming lipodepsipeptide toxin. Molecular Plant-Microbe Interactions, 4, 407–411.CrossRefGoogle Scholar
  28. 28.
    Young, J. M. (1970). Drippy gill: A bacterial disease of cultivated mushrooms caused by Pseudomonas agarici n. sp. New Zealand Journal of Agricultural Research, 13, 977–990.CrossRefGoogle Scholar
  29. 29.
    Kodama, K., Kimura, K., & Komagata, K. (1985). Two new species of Pseudomonas: P. oryzihabitans isolated from rice paddy and clinical specimens and P. luteola isolated from clinical specimens. International Journal of Systematic and Evolutionary Microbiology, 35, 467–474.Google Scholar
  30. 30.
    Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I., & Kim, S. W. (2004). Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresource Technology, 91, 153–156.CrossRefGoogle Scholar
  31. 31.
    Kotchoni, S. O., & Shonukan, O. O. (2002). Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus. World Journal of Microbiology and Biotechnology, 18, 487–491.CrossRefGoogle Scholar
  32. 32.
    Ariffin, H., Hassan, M. A., Shah, U. K. M., Abdullah, N., Ghazali, F. M., & Shirai, Y. (2008). Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by Bacillus pumilus EB3. Journal of Bioscience and Bioengineering, 106, 231–236.CrossRefGoogle Scholar
  33. 33.
    Narasimha, G., Sridevi, A., Buddolla, V., Subhosh, C. M., & Rajasekhar, R. B. (2006). Nutrient effects on production of cellulolytic enzymes by Aspergillus niger. African Journal of Biotechnology, 5, 472–476.Google Scholar
  34. 34.
    Acharya, P. B., Acharya, D. K., & Modi, H. A. (2008). Optimization for cellulase production by Aspergillus niger using saw dust as substrate. African Journal of Biotechnology, 7, 4147–4152.Google Scholar
  35. 35.
    Kocher, G. S., Kalra, K. L., & Banta, G. (2008). Optimization of cellulase production by submerged fermentation of rice straw by Trichoderma harzianum Rut-C 8230. The Internet Journal of Microbiology, 5.Google Scholar
  36. 36.
    Gautam, S. P., Bundela, P. S., Pandey, A. K., Khan, J., Awasthi, M. K., & Sarsaiya, S. (2011). Optimization for the production of cellulase enzyme from municipal solid waste residue by two novel cellulolytic fungi. Biotechnology Research International, 2011. doi: 10.4061/2011/810425
  37. 37.
    Shanmughapriya, S., Kiran, G. S., Selvin, J., Thomas, T. A., & Rani, C. (2010). Optimization, purification, and characterization of extracellular mesophilic alkaline cellulase from sponge-associated Marinobacter sp. MSI032. Applied Biochemistry and Biotechnology, 162, 625–640.CrossRefGoogle Scholar
  38. 38.
    Dees, C., Ringleberg, D., Scott, T. C., & Phelps, T. (1995). Characterization of the cellulose-degrading bacterium NCIB 10462. Applied Biochemistry and Biotechnology, 51, 263–274.CrossRefGoogle Scholar
  39. 39.
    Shankar, T., & Isaiarasu, L. (2011). Cellulase production by Bacillus pumilus EWBCM1 under varying cultural conditions. Middle-East Journal of Scientific Research, 8, 40–45.Google Scholar
  40. 40.
    Amritkar, N., Kamat, M., & Lali, A. (2003). Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue–cellulose matrices. Process Biochemistry, 39, 565–570.CrossRefGoogle Scholar
  41. 41.
    Ray, A. K., Bairagi, A., Ghosh, K. S., & Sen, K. (2007). Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from fish gut. Acta Ichthyologica et Piscatoria, 37, 47–53.Google Scholar
  42. 42.
    Guedon, E., Desvaux, M., & Petitdemange, H. (2002). Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering. Applied and Environmental Microbiology, 68, 53–58.CrossRefGoogle Scholar
  43. 43.
    Priest, F. G. (1977). Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews, 41, 711–753.Google Scholar
  44. 44.
    Mosier, N., Wyman, C., Dale, B., Elande, R. R., Lee, Y. Y., Holtzapple, M., et al. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96, 673–686.CrossRefGoogle Scholar
  45. 45.
    Kim, J. Y., Hur, S. H., & Hong, J. H. (2005). Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810. Biotechnology Letters, 27, 313–316.CrossRefGoogle Scholar
  46. 46.
    Quiroz-Castañeda, R. E., Balcázar-López, E., Dantán-González, E., Martinez, A., Folch-Mallol, J., & Anaya, C. M. (2009). Characterization of cellulolytic activities of Bjerkandera adusta and Pycnoporus sanguineus on solid wheat straw medium. Electronic Journal of Biotechnology, 12.Google Scholar
  47. 47.
    Christakopoulos, P., Hatzinikolaou, D. G., Fountoukidis, G., Kekos, D., Claeyssens, M., & Macris, B. J. (1999). Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus. Archives of Biochemistry and Biophysics, 364, 61–66.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ping Sheng
    • 1
  • Shengwei Huang
    • 1
  • Qi Wang
    • 1
  • Ailing Wang
    • 1
  • Hongyu Zhang
    • 1
    Email author
  1. 1.State Key Laboratory of Agricultural Microbiology, Institute of Urban and Horticultural Pests, and Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and TechnologyHuazhong Agricultural UniversityWuhanChina

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