Multiple Applications of Enzymes Induced by Algal Biomasses from a New Bacillus Isolate to Saccharify Algae and Degrade Chemical Dyes

  • Yanwen Wu
  • Haipeng Guo
  • Jinchi Zhang
  • Xuantong Chen
  • Mingjiang Wu
  • Wensheng Qin
Original Paper


To find a multifunctional lignocellulolytic enzyme-producing strain, ten bacterial isolates from paper mill wastewater were tested for their carboxymethyl cellulose (CMC) hydrolytic ability. Bacillus sp. TPF-1, which exhibits the highest hydrolytic ability, was selected to produce lignocellulolytic enzymes using various biomass types as carbon sources. The highest CMCase (9.12 U/mL) and xylanase (102.55 U/mL) activities were obtained by green algae, and the maximum laccase activity (7037.28 U/L) was induced by Sargassum fusiforme. CMCase and xylanase showed the highest activities at 55 and 50 °C, respectively, with the same optimum pH of 5.4. The laccase exhibited optimum temperature of 40 °C and retained 60% more activity at 80 °C in extreme acid conditions (pH 2.2). To explore the multiple applications of these enzymes, crude enzymes induced by green algae were used to saccharify untreated algae. The reducing sugar produced by crude enzymes and commercial cellulase was 23 and 14% higher than that of the control, respectively, and it was 48% higher using crude enzymes with commercial cellulase (72 h). Additionally, the laccase induced by S. fusiforme was tested to decolorize two chemical dyes under an acidic condition (pH 2.2). The highest decolorization rates were 56.13 and 62.14% for Coomassie brilliant blue R-250 and Congo Red, respectively, in the presence of hydroxybenzotriazole monohydrate.


Bacillus CMCase Algae Laccase Xylanase 



We acknowledge the financial support from BioFuelNet Canada, Lakehead University, National Special Fund for Forestry Scientific Research in the Public Interest of China (Grant No. 201504406), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 15KJA220004), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Compliance with Ethical Standards

Conflict of interest

No conflict of interest exits in the submission of this manuscript. We declare that we do not have any commercial or associative interest that represents a conflict of interest in the work submitted.

Supplementary material

12649_2018_277_MOESM1_ESM.docx (104 kb)
Supplementary material 1 (DOCX 103 KB)


  1. 1.
    Guo, H., Wu, Y., Hong, C., Chen, H., Chen, X., Zheng, B., Jiang, D., Qin, W.: Enhancing digestibility of Miscanthus using lignocellulolytic enzyme produced by Bacillus. Biores. Technol. 245, 1008–1015 (2017)CrossRefGoogle Scholar
  2. 2.
    Sangrila, S., Maiti, T.: Cellulase production by bacteria: a review. Br. Microbiol. Res. J. 3(3), 235–258 (2013)CrossRefGoogle Scholar
  3. 3.
    Maki, M., Leung, K.T., Qin, W.: The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int. J. Biol. Sci. 5(5), 500–516 (2009)CrossRefGoogle Scholar
  4. 4.
    Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Biores. Technol. 101(13), 4851–4861 (2010)CrossRefGoogle Scholar
  5. 5.
    Otajevwo, F., Aluyi, H.: Cultural conditions necessary for optimal cellulase yield by cellulolytic bacterial organisms as they relate to residual sugars released in broth medium. Mod. Appl. Sci. 5(3), 141–151 (2011)Google Scholar
  6. 6.
    Daniel, G., Nilsson, T.: Developments in the study of soft rot and bacterial decay. In: Bruce, A., Palfreyman, J.W. (eds.) Forest Products Biotechnology, pp. 37–62. Taylor & Francis, London (1998)Google Scholar
  7. 7.
    Kim, H.-J., Lee, Y.-J., Gao, W., Chung, C.-H., Son, C.-W., Lee, J.-W.: Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using orthogonal array method. Biotechnol. Bioprocess Eng. 16(3), 542–548 (2011)CrossRefGoogle Scholar
  8. 8.
    Pandey, S., Singh, S., Yadav, A.N., Nain, L., Saxena, A.K.: Phylogenetic diversity and characterization of novel and efficient cellulase producing bacterial isolates from various extreme environments. Biosci. Biotechnol. Biochem. 77(7), 1474–1480 (2013)CrossRefGoogle Scholar
  9. 9.
    Hung, K.-S., Liu, S.-M., Tzou, W.-S., Lin, F.-P., Pan, C.-L., Fang, T.-Y., Sun, K.-H., Tang, S.-J.: Characterization of a novel GH10 thermostable, halophilic xylanase from the marine bacterium Thermoanaerobacterium saccharolyticum NTOU1. Process Biochem. 46(6), 1257–1263 (2011)CrossRefGoogle Scholar
  10. 10.
    Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V.S., Yazdani, S.S.: Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect-gut bacterium. Appl. Environ. Microbiol. 77(14), 4859–4866 (2011)CrossRefGoogle Scholar
  11. 11.
    Fenel, F., Leisola, M., Jänis, J., Turunen, O.: A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1, 4-β-xylanase II. J. Biotechnol. 2(108), 137–143 (2004)CrossRefGoogle Scholar
  12. 12.
    Verma, P., Madamwar, D.: Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens. World J. Microbiol. Biotechnol. 19(6), 615–618 (2003)CrossRefGoogle Scholar
  13. 13.
    Givaudan, A., Effosse, A., Faure, D., Potier, P., Bouillant, M.-L., Bally, R.: Polyphenol oxidase in Azospirillum lipoferum isolated from rice rhizosphere: evidence for laccase activity in non-motile strains of Azospirillum lipoferum. FEMS Microbiol. Lett. 108(2), 205–210 (1993)CrossRefGoogle Scholar
  14. 14.
    Ruijssenaars, H., Hartmans, S.: A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity. Appl. Microbiol. Biotechnol. 65(2), 177–182 (2004)CrossRefGoogle Scholar
  15. 15.
    Giardina, P., Faraco, V., Pezzella, C., Piscitelli, A., Vanhulle, S., Sannia, G.: Laccases: a never-ending story. Cell. Mol. Life Sci. 67(3), 369–385 (2010)CrossRefGoogle Scholar
  16. 16.
    Saratale, R.G., Saratale, G.D., Chang, J.-S., Govindwar, S.: Bacterial decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. Eng. 42(1), 138–157 (2011)CrossRefGoogle Scholar
  17. 17.
    Vicente, A.I., Viña-Gonzalez, J., Santos-Moriano, P., Marquez-Alvarez, C., Ballesteros, A.O., Alcalde, M.: Evolved alkaline fungal laccase secreted by Saccharomyces cerevisiae as useful tool for the synthesis of C–N heteropolymeric dye. J. Mol. Catal. B 134, 323–330 (2016)CrossRefGoogle Scholar
  18. 18.
    Claus, H.: Laccases of Botrytis cinerea. In: König, H., Unden, G., Fröhlich, J. (eds.) Biology of Microorganisms on Grapes, in Must and in Wine, pp. 339–356. Springer, New York (2017)CrossRefGoogle Scholar
  19. 19.
    Lu, L., Wang, T.-N., Xu, T.-F., Wang, J.-Y., Wang, C.-L., Zhao, M.: Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization. Biores. Technol. 134, 81–86 (2013)CrossRefGoogle Scholar
  20. 20.
    Singh, G., Kaur, K., Puri, S., Sharma, P.: Critical factors affecting laccase-mediated biobleaching of pulp in paper industry. Appl. Microbiol. Biotechnol. 99(1), 155–164 (2015)CrossRefGoogle Scholar
  21. 21.
    Rodriguez, E., Pickard, M.A., Vazquez-Duhalt, R.: Industrial dye decolorization by laccases from ligninolytic fungi. Curr. Microbiol. 38(1), 27–32 (1999)CrossRefGoogle Scholar
  22. 22.
    Maki, M., Broere, M., Leung, K., Qin, W.: Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers. Int. J. Biochem. Mol. Biol. 2(2), 146–154 (2011)Google Scholar
  23. 23.
    Guo, H., Chen, H., Fan, L., Linklater, A., Zheng, B., Jiang, D., Qin, W.: Enzymes produced by biomass-degrading bacteria can efficiently hydrolyze algal cell walls and facilitate lipid extraction. Renew. Energy 109, 195–201 (2017)CrossRefGoogle Scholar
  24. 24.
    Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31(3), 426–428 (1959)CrossRefGoogle Scholar
  25. 25.
    Laemmli, U.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)CrossRefGoogle Scholar
  26. 26.
    Guo, H., Zheng, B., Jiang, D., Qin, W.: Overexpression of a laccase with dye decolorization activity from Bacillus sp. induced in Escherichia coli. J. Mol. Microbiol. Biotechnol. 27(4), 217–227 (2017)CrossRefGoogle Scholar
  27. 27.
    Schinner, F., Von Mersi, W.: Xylanase-, CM-cellulase-and invertase activity in soil: an improved method. Soil Biol. Biochem. 22(4), 511–515 (1990)CrossRefGoogle Scholar
  28. 28.
    Qinnghe, C., Xiaoyu, Y., Tiangui, N., Cheng, J., Qiugang, M.: The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus. Process Biochem. 39(11), 1561–1566 (2004)CrossRefGoogle Scholar
  29. 29.
    Aa, K., Flengsrud, R., Lindahl, V., Tronsmo, A.: Characterization of production and enzyme properties of an endo-β-1, 4-glucanase from Bacillus subtilis CK-2 isolated from compost soil. Antonie Van Leeuwenhoek 66(4), 319–326 (1994)CrossRefGoogle Scholar
  30. 30.
    Xu, F.: Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J. Biol. Chem. 272(2), 924–928 (1997)CrossRefGoogle Scholar
  31. 31.
    Martins, L.O., Soares, C.M., Pereira, M.M., Teixeira, M., Costa, T., Jones, G.H., Henriques, A.O.: Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J. Biol. Chem. 277(21), 18849–18859 (2002)CrossRefGoogle Scholar
  32. 32.
    Kocabas, A., Kocabas, D.S., Bolukbasi, U.B.: One-Step purification and characterization of a low molecular weight xylanase from Aspergillus terreus NRRL 1960. J. Appl. Biol. Sci. 5(2), 61–65 (2011)Google Scholar
  33. 33.
    Chaurasia, P.K., Yadav, R.S.S., Yadava, S.: Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes. Process Biochem. 49(10), 1647–1655 (2014)CrossRefGoogle Scholar
  34. 34.
    Haltrich, D., Nidetzky, B., Kulbe, K.D., Steiner, W., Župančič, S.: Production of fungal xylanases. Biores. Technol. 58(2), 137–161 (1996)CrossRefGoogle Scholar
  35. 35.
    Panwar, D., Srivastava, P.K., Kapoor, M.: Production, extraction and characterization of alkaline xylanase from Bacillus sp. PKD-9 with potential for poultry feed. Biocatal. Agric. Biotechnol. 3(2), 118–125 (2014)Google Scholar
  36. 36.
    Amore, A., Parameswaran, B., Kumar, R., Birolo, L., Vinciguerra, R., Marcolongo, L., Ionata, E., La Cara, F., Pandey, A., Faraco, V.: Application of a new xylanase activity from Bacillus amyloliquefaciens XR44A in brewer’s spent grain saccharification. J. Chem. Technol. Biotechnol. 90(3), 573–581 (2015)CrossRefGoogle Scholar
  37. 37.
    Han, S.J., Yoo, Y.J., Kang, H.S.: Characterization of a bifunctional cellulase and its structural gene The cel gene of Bacillus sp. D04 has exo-and endoglucanase activity. J. Biol. Chem. 270(43), 26012–26019 (1995)CrossRefGoogle Scholar
  38. 38.
    Goyal, V., Mittal, A., Bhuwal, A.K., Singh, G., Yadav, A., Aggarwal, N.K.: Parametric optimization of cultural conditions for carboxymethyl cellulase production using pretreated rice straw by Bacillus sp. 313SI under stationary and shaking conditions. Biotechnol. Res. Int. 2014(12), 651839 (2014)Google Scholar
  39. 39.
    Ladeira, S.A., Cruz, E., Delatorre, A.B., Barbosa, J.B., Leal Martins, M.L.: Cellulase production by thermophilic Bacillus sp: SMIA-2 and its detergent compatibility. Electron. J. Biotechnol. 18(2), 110–115 (2015)CrossRefGoogle Scholar
  40. 40.
    Ariffin, H., Abdullah, N., Kalsom, M.U., Shirai, Y., Hassan, M.: Production and characterisation of cellulase by Bacillus pumilus EB3. Int. J. Eng. Technol. 3(1), 47–53 (2006)Google Scholar
  41. 41.
    Breccia, J.D., Siñeriz, F., Baigorí, M.D., Castro, G.R., Hatti-Kaul, R.: Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens. Enzyme Microb. Technol. 22(1), 42–49 (1998)CrossRefGoogle Scholar
  42. 42.
    Lin, C., Shen, Z., Qin, W.: Characterization of xylanase and cellulase produced by a newly isolated Aspergillus fumigatus N2 and its efficient saccharification of barley straw. Appl. Biochem. Biotechnol. 182(2), 559–569 (2017)CrossRefGoogle Scholar
  43. 43.
    Meunier-Goddik, L., Penner, M.H.: Enzyme-catalyzed saccharification of model celluloses in the presence of lignacious residues. J. Agric. Food Chem. 47(1), 346–351 (1999)CrossRefGoogle Scholar
  44. 44.
    Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., Ladisch, M.: Features of promising technologies for pretreatment of lignocellulosic biomass. Biores. Technol. 96(6), 673–686 (2005)CrossRefGoogle Scholar
  45. 45.
    Burke, R., Cairney, J.: Laccases and other polyphenol oxidases in ecto-and ericoid mycorrhizal fungi. Mycorrhiza 12(3), 105–116 (2002)CrossRefGoogle Scholar
  46. 46.
    Wang, W., Lu, J.-B., Wang, C., Wang, C.-S., Zhang, H.-H., Li, C.-Y., Qian, G.-Y.: Effects of Sargassum fusiforme polysaccharides on antioxidant activities and intestinal functions in mice. Int. J. Biol. Macromol. 58, 127–132 (2013)CrossRefGoogle Scholar
  47. 47.
    Thanaraj, S., Seshadri, R.: Influence of polyphenol oxidase activity and polyphenol content of tea shoot on quality of black tea. J. Sci. Food Agric. 51(1), 57–69 (1990)CrossRefGoogle Scholar
  48. 48.
    Sisler, E., Evans, H.: A comparison of chlorogenic acid and catechol as substrates for the polyphenol oxidase from tobacco and mushroom. Plant Physiol. 33(4), 255–257 (1958)CrossRefGoogle Scholar
  49. 49.
    Chen, X., Nie, W., Fan, S., Zhang, J., Wang, Y., Lu, J., Jin, L.: A polysaccharide from Sargassum fusiforme protects against immunosuppression in cyclophosphamide-treated mice. Carbohyd. Polym. 90(2), 1114–1119 (2012)CrossRefGoogle Scholar
  50. 50.
    Huang, X., Zhou, H., Zhang, H.: The effect of Sargassum fusiforme polysaccharide extracts on vibriosis resistance and immune activity of the shrimp, Fenneropenaeus chinensis. Fish Shellfish Immunol. 20(5), 750–757 (2006)CrossRefGoogle Scholar
  51. 51.
    Chen, X., Nie, W., Yu, G., Li, Y., Hu, Y., Lu, J., Jin, L.: Antitumor and immunomodulatory activity of polysaccharides from Sargassum fusiforme. Food Chem. Toxicol. 50(3), 695–700 (2012)CrossRefGoogle Scholar
  52. 52.
    Wu, X., Jiang, W., Lu, J., Yu, Y., Wu, B.: Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry. Food Chem. 145, 976–983 (2014)CrossRefGoogle Scholar
  53. 53.
    Baldrian, P.: Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme. Appl. Microbiol. Biotechnol. 63(5), 560–563 (2004)CrossRefGoogle Scholar
  54. 54.
    Majeau, J.-A., Brar, S.K., Tyagi, R.D.: Laccases for removal of recalcitrant and emerging pollutants. Biores. Technol. 101(7), 2331–2350 (2010)CrossRefGoogle Scholar
  55. 55.
    Hirose, J., Nasu, M., Yokoi, H.: Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores. Biotech. Lett. 25(19), 1609–1612 (2003)CrossRefGoogle Scholar
  56. 56.
    Held, C., Kandelbauer, A., Schroeder, M., Cavaco-Paulo, A., Gübitz, G.M.: Biotransformation of phenolics with laccase containing bacterial spores. Environ. Chem. Lett. 3(2), 74–77 (2005)CrossRefGoogle Scholar
  57. 57.
    Kawai, S., Nakagawa, M., Ohashi, H.: Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole. Enzyme Microb. Technol. 30(4), 482–489 (2002)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Yanwen Wu
    • 1
    • 2
  • Haipeng Guo
    • 2
    • 3
  • Jinchi Zhang
    • 1
  • Xuantong Chen
    • 2
  • Mingjiang Wu
    • 4
  • Wensheng Qin
    • 2
  1. 1.Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu ProvinceNanjing Forestry UniversityNanjingChina
  2. 2.Department of BiologyLakehead UniversityThunder BayCanada
  3. 3.School of Marine SciencesNingbo UniversityNingboChina
  4. 4.College of Life and Environmental ScienceWenzhou UniversityWenzhouChina

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