Applied Microbiology and Biotechnology

, Volume 103, Issue 3, pp 1455–1464 | Cite as

Heavy water-labeled Raman spectroscopy reveals carboxymethylcellulose-degrading bacteria and degradation activity at the single-cell level

  • Oladipo Oladiti Olaniyi
  • Kai Yang
  • Yong-Guan Zhu
  • Li CuiEmail author
Methods and protocols


Biodegradation of cellulose-rich substrates is an indispensable process for soil carbon replenishment in various ecological niches. Biodegradation of cellulose has been studied extensively via an enzyme assay to quantify the amount of cellulase with a view to identify effective cellulose degraders. However, a bulk enzyme assay undermines the role of physiological heterogeneity between cells; it is therefore imperative to opt out for a more effective method such as single-cell Raman spectroscopy combined with heavy water (D2O) to reveal active cellulose degraders. Cellular incorporation of D2O-derived D produces a new C-D Raman band which can act as a quantitative indicator of microbial activity. In this study, metabolic responses of seven cellulose-degrading bacteria to carboxymethylcellulose (CMC) and glucose were evaluated via the C-D Raman band. On the basis of % C-D, CMC was demonstrated to be most efficiently metabolized by Bacillus velezensis 2a-9 and Providencia vermicola 5a-9(b). Metabolic activity between individual cells of B. velezensis and P. vermicola towards CMC ranged from approximately 8 to 27% and 6 to 16%, respectively, clearly indicating heterogeneous degradation activities among isogenic populations. Linear correlation between % C-D and specific endoglucanase activity validated Raman results on CMC-degrading activity. Also, % C-D obtained from bacteria cultivated with only glucose was around 60% higher than that obtained from CMC, indicating the preference of bacteria for simple sugar glucose than CMC. In conclusion, Raman spectroscopy combined with heavy water is a sensitive analytical technique to reveal cellulose degraders and their degrading activities.


Single-cell microbiology Raman spectroscopy Heavy water isotope labeling Cellulose biodegradation Heterogeneous degradation activity 



This study was funded by the National Key Research and Development Program of China (2017YFD0200201, 2017YFE0107300), Natural Science Foundation of China (21777154), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB15020302, XDB15020402), CAS President’s International Fellowship Initiative (PIFI) for 2017, and K.C.Wong Education Foundation.

Compliance with ethical standards

This research does not involve the use of human or animals.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

253_2018_9459_MOESM1_ESM.pdf (684 kb)
ESM 1 (PDF 683 kb)


  1. Akinyele BJ, Fabunmi AO, Olaniyi OO (2013) Effect of variations in growth parameters on cellulase activity of Trichoderma viride NSPR006 cultured on different wood-dusts. Malaysian J Microbiol 9(3):193–200. Google Scholar
  2. Bala JD (2016) Aerobic treatment and biodegradation of palm oil mill effluent by indigenous microorganisms. Ph.D Dissertation, Universiti Sains Malaysia, Penang, MalaysiaGoogle Scholar
  3. Berry D, Mader E, Lee TK, Woebken D, Wang Y, Zhu D, Palatinszky M, Schintlmeister A, Schmid MC, Hanson BT, Shterzer N, Mizrahi I, Rauch I, Decker T, Bocklitz T, Popp J, Gibson CM, Fowler PW, Huang WE, Wagner M (2015) Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells. Proc Natl Acad Sci U S A 112(2):E194–E203. CrossRefGoogle Scholar
  4. Cui L, Chen P, Chen S, Yuan Z, Yu C, Ren B, Zhang K (2013) In situ study of the antibacterial activity and mechanism of action of silver nanoparticles by surface-enhanced Raman spectroscopy. Anal Chem 85:5436–5443. CrossRefGoogle Scholar
  5. Cui L, Butler HJ, Martin-Hirsch PL, Martin FL (2016a) Aluminium foil as a potential substrate for ATR-FTIR, transflection FTIR or Raman spectrochemical analysis of biological specimens. Anal Methods 8(3):481–487. CrossRefGoogle Scholar
  6. Cui L, Zhang YJ, Huang WE, Zhang BF, Martin FL, Li JY, Zhang KS, Zhu YG (2016b) Surface-enhanced Raman spectroscopy for identification of heavy metal arsenic(V)-mediated enhancing effect on antibiotic resistance. Anal Chem 88(6):3164–3170. CrossRefGoogle Scholar
  7. Cui L, Yang K, Zhou G, Huang WE, Zhu YG (2017) Surface-enhanced Raman spectroscopy combined with stable isotope probing to monitor nitrogen assimilation at both bulk and single-cell level. Anal Chem 89(11):5793–5800. CrossRefGoogle Scholar
  8. Cui L, Yang K, Li HZ, Zhang H, Su JQ, Paraskevaidi M, Martin FL, Ren B, Zhu YG (2018) Functional single-cell approach to probing nitrogen-fixing bacteria in soil communities by resonance Raman spectroscopy with 15N2 labeling. Anal Chem 90(8):5082–5089. CrossRefGoogle Scholar
  9. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12):6640–6645. CrossRefGoogle Scholar
  10. Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, Feng JX (2007) Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 75(2):319–328. CrossRefGoogle Scholar
  11. Gusakov AV, Sinitsyn AP (2012) Cellulases from Penicillium species for producing fuels from biomass. Biofuels 3(4):463–477. CrossRefGoogle Scholar
  12. Hatefi A, Makhdoumi A, Asoodeh A, Mirshamsi O (2017) Characterization of a bi-functional cellulase produced by a gutbacterial resident of Rosaceae branch borer beetle, Osphranteria coerulescens (Coleoptera: Cerambycidae). Int J Biol Macromol 103:158–164. CrossRefGoogle Scholar
  13. Heppenstall LD, Strong RJ, Trevisan J, Martin FL (2013) Incorporation of deuterium oxide in MCF-7 cells to shed further mechanistic insights into benzo[a]pyrene-induced low-dose effects discriminated by ATR-FTIR spectroscopy. Analyst 138(9):2583–2591. CrossRefGoogle Scholar
  14. Holt JG, Kneg NR, Sneath PH, Stanly JJ, Williams ST (1994) Bergey’s manual of determinative bacteriology. Wilkins Publishers, BaltimoreGoogle Scholar
  15. Huang WE, Griffiths RI, Thompson IP, Bailey MJ, Whiteley AS (2004) Raman microscopic analysis of single microbial cells. Anal Chem 76:4452–4458. CrossRefGoogle Scholar
  16. Huang WE, Stoecker K, Griffiths R, Newbold L, Daims H, Whiteley AS, Wagner M (2007) Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ Microbiol 9:1878–1889. CrossRefGoogle Scholar
  17. Kotchoni OS, Shonukan OO, Gachomo WE (2003) Bacillus pumilus BpCRI 6, a promising candidate for cellulase production under conditions of catabolite repression. Afr J Biotechnol 2(6):140–146CrossRefGoogle Scholar
  18. Kubryk P, Kolschbach JS, Marozava S, Lueders T, Meckenstock RU, Niessner R, Ivleva NP (2015) Exploring the potential of stable isotope (resonance) Raman microspectroscopy and surface-enhanced Raman scattering for the analysis of microorganisms at single cell level. Anal Chem 87(13):6622–6630. CrossRefGoogle Scholar
  19. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9(4):299–306. CrossRefGoogle Scholar
  20. Kumar BNV, Guo S, Bocklitz T, Rosch P, Popp J (2016) Demonstration of carbon catabolite repression in naphthalene degrading soil bacteria via Raman spectroscopy based stable isotope probing. Anal Chem 88(15):7574–7582. CrossRefGoogle Scholar
  21. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. John Wiley & Sons, New YorkGoogle Scholar
  22. Lester W, Sun SH, Seber A (1960) Observations on the influence of deuterium on bacterial growth. Ann N Y Acad Sci 84:667–677. CrossRefGoogle Scholar
  23. Liang YL, Zhang Z, Wu M, Wu Y, Feng JX (2014) Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. Biomed Res Int 2014:512497. Google Scholar
  24. Lo YC, Saratale GD, Chen WM, Bai MD, Chang JS (2009) Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production. Enzym Microb Technol 44(6–7):417–425. CrossRefGoogle Scholar
  25. Maki M, Leung KT, Qin W (2009) The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5(5):500–516. CrossRefGoogle Scholar
  26. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. CrossRefGoogle Scholar
  27. Sadhu S, Maiti TK (2013) Cellulase production by bacteria: a review. Br Microbiol Res J 3(3):235–258. CrossRefGoogle Scholar
  28. Sheng P, Huang SW, Wang Q, Wang AL, Zhang HY (2012) Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria from the hindgut of Holotrichia parallela larvae (Coleoptera: Scarabaeidae). Appl Biochem Biotechnol 167(2):270–284. CrossRefGoogle Scholar
  29. Soares FL, Melo IS, Dias AC, Andreote FD (2012) Cellulolytic bacteria from soils in harsh environments. World J Microbiol Biotechnol 28(5):2195–2203. CrossRefGoogle Scholar
  30. Song Y, Cui L, Lopez JAS, Xu J, Zhu YG, Thompson IP, Huang WE (2017) Raman-deuterium isotope probing for in-situ identification of antimicrobial resistant bacteria in Thames river. Sci Report 7(1):16648. CrossRefGoogle Scholar
  31. Stöckel S, Meisel S, Elschner M, Melzer F, Rösch P, Popp J (2015) Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff. Anal Bioanal Chem 407(3):787–794. CrossRefGoogle Scholar
  32. Stursova M, Zifcakova L, Leigh MB, Burgess R, Baldrian P (2012) Cellulose utilization in forest litter and soil: identification of bacterial and fungal decomposers. FEMS Microbiol Ecol 80:735–736. CrossRefGoogle Scholar
  33. Tao YF, Wang Y, Huang S, Zhu PF, Huang WE, Ling JQ, Xu J (2017) Metabolic activity-based assessment of antimicrobial effects by D2O-labeled single-cell Raman microspectroscopy. Anal Chem 89(7):4108–4115. CrossRefGoogle Scholar
  34. Vinuselvi P, Kim MK, Lee SK, Ghim CM (2012) Rewiring carbon catabolite repression for microbial cell factory. Biochem Mol Biol Rep 45(2):59–70. Google Scholar
  35. Wang Y, Huang WE, Cui L, Wagner M (2016) Single cell stable isotope probing in microbiology using Raman microspectroscopy. Curr Opin Biotechnol 41:34–42. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Lab of Urban Environment and Health, Institute of Urban EnvironmentChinese Academy of SciencesXiamenChina
  2. 2.Department of MicrobiologyFederal University of TechnologyAkureNigeria
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.State Key Lab of Urban and Regional Ecology, Research Centre for Eco-Environmental SciencesChinese Academy of SciencesBeijingChina

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