Exploring the Effect of Cu2+ on Sludge Hydrolysis and Interaction Mechanism between Cu2+ and Xylanase by Multispectral and Thermodynamic Methods

  • Xiangyuan Zhang
  • Hong Liu
  • Lei Ju
  • Chunguang LiuEmail author


Although hydrolysis of sludge can be improved by enzymes, it not only depends on the nature and concentration of hydrolase but also rests with factors affecting the activity of hydrolase, such as heavy metals. In this research, the impact of Cu2+ on sludge hydrolysis on xylanase is studied with respect to the concentration and components of soluble organic matter in sludge using three-dimensional fluorescence spectra. Results showed that Cu2+ exposure not only inhibited the hydrolysis of sludge due to the denaturation of xylanase but also affected the components of soluble organic matter in sludge. In order to illuminate the interaction mechanism between Cu2+ and xylanase, UV–Vis, steady-state fluorescence, circular dichroism, synchronous fluorescence, light scattering spectra, and isothermal titration calorimetry techniques were applied. The results show that Cu2+ spontaneously interacts with xylanase by a hydrophobic bond, hydrogen bond, or van der Waals force with two binding sites in the activity region of xylanase. This interaction not only causes a looser skeleton structure of xylanase and a more hydrophobic microenvironment of tyrosine and tryptophan residues but also leads to the static quenching of fluorophore due to the formation of complexes (xylanase-Cu2+). This work establishes a new strategy to investigate the interaction between enzymes and heavy metals at a molecular level, which is helpful for clarifying the bioactivities of heavy metals.


Xylanase Sludge hydrolysis Heavy metals Three-dimensional fluorescence spectra Isothermal titration microcalorimetry 



This study has been supported by the National Natural Science Foundation of China (21507071), the Fundamental Research Funds of Shandong University (2014BT013 and 2015JC030), and China Post-doctoral Science Foundation funded project (2014M560555 and 2015T80720).

Supplementary material

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ESM 1 (DOCX 1.71 MB)


  1. Cano, R., Pérez-Elvira, S. I., & Fdz-Polanco, F. (2015). Energy feasibility study of sludge pretreatments: a review. Applied Energy, 149, 176–185.CrossRefGoogle Scholar
  2. Chen, H., Chen, Y., Zheng, X., Li, X., & Luo, J. (2014). How does the entering of copper nanoparticles into biological wastewater treatment system affect sludge treatment for VFA production. Water Research, 63, 125–134.CrossRefGoogle Scholar
  3. Donoso-Bravo, A., & Fdz-Polanco, M. (2013). Anaerobic co-digestion of sewage sludge and grease trap: assessment of enzyme addition. Process Biochemistry, 48, 936–940.CrossRefGoogle Scholar
  4. Gaur, R., Tiwari, S., Rai, P., & Srivastava, V. (2015). Isolation, production, and characterization of thermotolerant xylanase from solvent tolerant Bacillus vallismortis RSPP-15. International Journal of Polymer Science, 14, 655–661.Google Scholar
  5. Gauthier, T. D., Shane, E. C., Guerin, W. F., Seitz, W. R., & Grant, C. L. (1986). Fluorescence quenching method for determining equilibrium constants for polycyclic aromatic hydrocarbons binding to dissolved humic materials. Environmental Science & Technology, 20, 1162–1166.CrossRefGoogle Scholar
  6. Gonzalez-Estrella, J., Puyol, D., Gallagher, S., Sierra-Alvarez, R., & Field, J. A. (2015). Elemental copper nanoparticle toxicity to different trophic groups involved in anaerobic and anoxic wastewater treatment processes. Science of the Total Environment, 512, 308–315.CrossRefGoogle Scholar
  7. Hao, F., Jing, M., Zhao, X., & Liu, R. (2015). Spectroscopy, calorimetry and molecular simulation studies on the interaction of catalase with copper ion. Journal of Photochemistry and Photobiology B: Biology, 143, 100–106.CrossRefGoogle Scholar
  8. Hu, Y., Hao, X., Wang, J., & Cao, Y. (2016). Enhancing anaerobic digestion of lignocellulosic materials in excess sludge by bioaugmentation and pre-treatment. Waste Management, 49, 55–63.CrossRefGoogle Scholar
  9. Jayabharathi, J., Thanikachalam, V., Sathishkumar, R., & Jayamoorthy, K. (2012). Fluorescence investigation of the interaction of 2-(4-fluorophenyl)-1-phenyl-1H-phenanthro [9,10-d] imidazole with bovine serum albumin. Journal of Photochemistry and Photobiology B: Biology, 117, 222–227.CrossRefGoogle Scholar
  10. Knob, A., & Carmona, E. C. (2010). Purification and characterization of two extracellular xylanases from Penicillium sclerotiorum: a novel acidophilic xylanase. Applied Biochemistry and Biotechnology, 162, 429–443.CrossRefGoogle Scholar
  11. Kong, Q., Wang, Z.-B., Shu, L., & Miao, M.-S. (2015). Characterization of the extracellular polymeric substances and microbial community of aerobic granulation sludge exposed to cefalexin. International Biodeterioration & Biodegradation, 102, 375–382.CrossRefGoogle Scholar
  12. Kumar, A., Gupta, R., Shrivastava, B., Khasa, Y. P., & Kuhad, R. C. (2012). Xylanase production from an alkalophilic actinomycete isolate Streptomyces sp. RCK-2010, its characterization and application in saccharification of second generation biomass. Journal of Molecular Catalysis B: Enzymatic, 74, 170–177.CrossRefGoogle Scholar
  13. Li, J., Liu, X., Liu, Y., Ramsay, J., Yao, C., & Dai, R. (2011). The effect of continuous exposure of copper on the properties and extracellular polymeric substances (EPS) of bulking activated sludge. Environmental Science and Pollution Research, 18, 1567–1573.CrossRefGoogle Scholar
  14. Li, W.-T., Chen, S.-Y., Xu, Z.-X., Li, Y., Shuang, C.-D., & Li, A.-M. (2014). Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: a comparative study. Environmental Science & Technology, 48, 2603–2609.CrossRefGoogle Scholar
  15. Li, K., Wei, D., Zhang, G., Shi, L., Wang, Y., Wang, B., Wang, X., Du, B., & Wei, Q. (2015). Toxicity of bisphenol A to aerobic granular sludge in sequencing batch reactors. Journal of Molecular Liquids, 209, 284–288.CrossRefGoogle Scholar
  16. Lu, D., Zhao, X., Zhao, Y., Zhang, B., Zhang, B., Geng, M., & Liu, R. (2011). Binding of Sudan II and Sudan IV to bovine serum albumin: comparison studies. Food and Chemical Toxicology, 49, 3158–3164.CrossRefGoogle Scholar
  17. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  18. Moretti, M. M. D. S., Bocchini-Martins, D. A., Nunes, C. D. C. C., Villena, M. A., Perrone, O. M., Silva, R. D., Boscolo, M., & Gomes, E. (2014). Pretreatment of sugarcane bagasse with microwaves irradiation and its effects on the structure and on enzymatic hydrolysis. Applied Energy, 122, 189–195.CrossRefGoogle Scholar
  19. Ochoa-Herrera, V., León, G., Banihani, Q., Field, J. A. & Sierra-Alvarez, R. (2011). Toxicity of copper(II) ions to microorganisms in biological wastewater treatment systems. Science of the Total Environment, 412–413, 380–385.Google Scholar
  20. Papadopoulou, A., Green, R. J., & Frazier, R. A. (2005). Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study. Journal of Agricultural and Food Chemistry, 53, 158–163.CrossRefGoogle Scholar
  21. Ross, P. D., & Subramanian, S. (1981). Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry, 20, 3096–3102.CrossRefGoogle Scholar
  22. Sambusiti, C., Rollini, M., Ficara, E., Musatti, A., Manzoni, M., & Malpei, F. (2014). Enzymatic and metabolic activities of four anaerobic sludges and their impact on methane production from ensiled sorghum forage. Bioresource Technology, 155, 122–128.CrossRefGoogle Scholar
  23. Shahrestani, H., Taheri-Kafrani, A., Soozanipour, A., & Tavakoli, O. (2016). Enzymatic clarification of fruit juices using xylanase immobilized on 1,3,5-triazine-functionalized silica-encapsulated magnetic nanoparticles. Biochemical Engineering Journal, 109, 51–58.CrossRefGoogle Scholar
  24. Sheng, G.-P., Xu, J., Luo, H.-W., Li, W.-W., Li, W.-H., Yu, H.-Q., Xie, Z., Wei, S.-Q., & Hu, F.-C. (2013). Thermodynamic analysis on the binding of heavy metals onto extracellular polymeric substances (EPS) of activated sludge. Water Research, 47, 607–614.CrossRefGoogle Scholar
  25. Song, W., Yu, Z., Hu, X., & Liu, R. (2015). Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 137, 286–293.CrossRefGoogle Scholar
  26. Sun, H., Yang, X., Li, M., Han, S., Liu, Y., Tan, X., Liu, C., & Liu, R. (2015). Insights into the effect of N-acetyl-l-cysteine-capped CdTe quantum dots on the structure and activity of human serum albumin by spectroscopic techniques. Journal of Luminescence, 167, 1–7.CrossRefGoogle Scholar
  27. Tervahauta, T., Rani, S., Hernández Leal, L., Buisman, C. J. N., & Zeeman, G. (2014). Black water sludge reuse in agriculture: are heavy metals a problem? Journal of Hazardous Materials, 274, 229–236.CrossRefGoogle Scholar
  28. Valencia, S., Marín, J. M., Restrepo, G., & Frimmel, F. H. (2014). Evaluation of natural organic matter changes from Lake Hohloh by three-dimensional excitation–emission matrix fluorescence spectroscopy during TiO2/UV process. Water Research, 51, 124–133.CrossRefGoogle Scholar
  29. Wang, Y., Qin, J., Zhou, S., Lin, X., Ye, L., Song, C., & Yan, Y. (2015). Identification of the function of extracellular polymeric substances (EPS) in denitrifying phosphorus removal sludge in the presence of copper ion. Water Research, 73, 252–264.CrossRefGoogle Scholar
  30. Wongwilaiwalin, S., Rattanachomsri, U., Laothanachareon, T., Eurwilaichitr, L., Igarashi, Y., & Champreda, V. (2010). Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system. Enzyme and Microbial Technology, 47, 283–290.CrossRefGoogle Scholar
  31. Xie, C., Lu, R., Huang, Y., Wang, Q., & Xu, X. (2010). Effects of ions and phosphates on alkaline phosphatase activity in aerobic activated sludge system. Bioresource Technology, 101, 3394–3399.CrossRefGoogle Scholar
  32. Yang, G.-F., Ni, W.-M., Wu, K., Wang, H., Yang, B.-E., Jia, X.-Y., & Jin, R.-C. (2013). The effect of Cu(II) stress on the activity, performance and recovery on the anaerobic ammonium-oxidizing (Anammox) process. Chemical Engineering Journal, 226, 39–45.CrossRefGoogle Scholar
  33. Yin, Y., Liu, Y.-J., Meng, S.-J., Kiran, E. U., & Liu, Y. (2016). Enzymatic pretreatment of activated sludge, food waste and their mixture for enhanced bioenergy recovery and waste volume reduction via anaerobic digestion. Applied Energy, 179, 1131–1137.CrossRefGoogle Scholar
  34. Yu, S., Zhang, G., Li, J., Zhao, Z., & Kang, X. (2013). Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge. Bioresource Technology, 146, 758–761.CrossRefGoogle Scholar
  35. Zhang, Y. Z., Chen, X. X., Dai, J., Zhang, X. P., Liu, Y. X., & Liu, Y. (2008). Spectroscopic studies on the interaction of lanthanum(III) 2-oxo-propionic acid salicyloyl hydrazone complex with bovine serum albumin. Luminescence, 23, 150–156.CrossRefGoogle Scholar
  36. Zheng, H., Liu, Y., Liu, X., Han, Y., Wang, J., & Lu, F. (2012). Overexpression of a Paenibacillus campinasensis xylanase in Bacillus megaterium and its applications to biobleaching of cotton stalk pulp and saccharification of recycled paper sludge. Bioresource Technology, 125, 182–187.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Xiangyuan Zhang
    • 1
  • Hong Liu
    • 1
  • Lei Ju
    • 2
  • Chunguang Liu
    • 1
    Email author
  1. 1.Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and EngineeringShandong UniversityJinanChina
  2. 2.School of Computer Science and TechnologyShandong UniversityJinanChina

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