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Applied Microbiology and Biotechnology

, Volume 103, Issue 21–22, pp 9169–9180 | Cite as

Role of extracellular polymeric substances in biofilm formation by Pseudomonas stutzeri strain XL-2

  • Xue Song Ding
  • Bin ZhaoEmail author
  • Qiang An
  • Meng Tian
  • Jin Song Guo
Environmental biotechnology

Abstract

Pseudomonas stutzeri strain XL-2 exhibited significant performance on biofilm formation. Extracellular polymeric substances (EPS) secreted by strain XL-2 were characterized by colorimetry and Fourier transform infrared (FT-IR) spectroscopy. The biofilm growth showed a strong positive correlation (rP=0.96, P<0.01) to extracellular protein content, but no correlation to exopolysaccharide content. Hydrolyzing the biofilm with proteinase K caused a significant decrease in biofilm growth (t=3.7, P<0.05), whereas the changes in biofilm growth were not significant when the biofilm was hydrolyzed by α-amylase and β-amylase, implying that proteins rather than polysaccharides played the dominant role in biofilm formation. More specifically, confocal laser scanning microscopy (CLSM) revealed that the extracellular proteins were tightly bound to the cells, resulting in the cells with EPS presenting more biofilm promotion protein secondary structures, such as three-turn helices, β-sheet, and α-helices, than cells without EPS. Both bio-assays and quantitative analysis demonstrated that strain XL-2 produced signal molecules of N-acylhomoserine lactones (AHLs) during biofilm formation process. The concentrations of C6-HLS and C6-oxo-HLS were both significantly positively correlated with protein contents (P<0.05). Dosing exogenous C6-HLS and C6-oxo-HLS also resulted in the increase in protein content. Therefore, it was speculated that C6-HLS and C6-oxo-HLS released by strain XL-2 could up-regulate the secretion of proteins in EPS, and thus promote the formation of biofilm.

Keywords

Biofilm formation Extracellular polymeric substances (EPS) Extracellular proteins N-Acylhomoserine lactones (AHLs) Pseudomonas stutzeri 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 51208534) and Technical Innovation and Application Demonstration Project of CQ CSTC (grant no. cstc2018jscx-msybX0308).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animal performed by any of the authors.

References

  1. Adav SS, Lee DJ, Tay JH (2008) Extracellular polymeric substances and structural stability of aerobic granule. Water Res 42:1644–1650.  https://doi.org/10.1016/j.watres.2007.10.013 CrossRefPubMedGoogle Scholar
  2. Aguilera A, Souza-Egipsy V, San Martin-Uriz P, Amils R (2008) Extraction of extracellular polymeric substances from extreme acidic microbial biofilms. Appl Microbiol Biotechnol 78:1079–1088.  https://doi.org/10.1007/s00253-008-1390-9 CrossRefPubMedGoogle Scholar
  3. Ando DJ (2005) Infrared spectroscopy: fundamentals and applications. Wiley, ChichesterGoogle Scholar
  4. Badireddy AR, Korpol BR, Chellam S, Gassman PL, Engelhard MH, Lea S, Rosso KM (2008) Spectroscopic characterization of extracellular polymeric substances from Escherichia coli and Serratia marcescens: suppression using sub-inhibitory concentrations of bismuth thiols. Biomacromolecules 9:3079–3089CrossRefGoogle Scholar
  5. Badireddy AR, Chellam S, Gassman PL, Engelhard MH, Lea AS, Rosso KM (2010) Role of extracellular polymeric substances in bioflocculation of activated sludge microorganisms under glucose-controlled conditions. Water Res 44:4505–4516.  https://doi.org/10.1016/j.watres.2010.06.024 CrossRefPubMedGoogle Scholar
  6. Barth A, Zscherp C (2002) What vibrations tell us about proteins. Q Rev Biophys 35:369–430CrossRefGoogle Scholar
  7. Beech I, Hanjagsit L, Kalaji M, Neal AL, Zinkevich V (1999) Chemical and structural characterization of exopolymers produced by Pseudomonas sp. NCIMB 2021 in continuous culture. Microbiology 145:1491–1497.  https://doi.org/10.1099/13500872-145-6-1491 CrossRefPubMedGoogle Scholar
  8. Blosser RS, Gray KM (2000) Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. J Microbiol Methods 40:47–55CrossRefGoogle Scholar
  9. Cao B, Shi L, Brown RN, Xiong Y, Fredrickson JK, Romine MF, Marshall MJ, Lipton MS, Beyenal H (2011) Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics. Environ Microbiol 13:1018–1031.  https://doi.org/10.1111/j.1462-2920.2010.02407.x CrossRefPubMedGoogle Scholar
  10. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilm: a common cause of persistent infections. Science 284:1318–1322CrossRefGoogle Scholar
  11. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298CrossRefGoogle Scholar
  12. De Kievit TR, Gillis R, Marx S, Brown C, Iglewski BH (2001) Quorum-sensing genes in Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl Environ Microbiol 67:1865–1873CrossRefGoogle Scholar
  13. Du GC, Yu J (2002) Green technology for conversion of food scraps to biodegradable thermoplastic polyhydroxyalkanoates. Environ Sci Technol 36:5511–5516.  https://doi.org/10.1021/es011110o CrossRefPubMedGoogle Scholar
  14. Dube CD, Guiot SR (2019) Characterization of the protein fraction of the extracellular polymeric substances of three anaerobic granular sludges. AMB Express 9:23.  https://doi.org/10.1186/s13568-019-0746-0 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Feng H, Ding Y, Wang M, Zhou G, Zheng X, He H, Zhang X, Shen D, Shentu J (2014) Where are signal molecules likely to be located in anaerobic granular sludge? Water Res 50:1–9.  https://doi.org/10.1016/j.watres.2013.11.021 CrossRefGoogle Scholar
  16. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633.  https://doi.org/10.1038/nrmicro2415 CrossRefPubMedGoogle Scholar
  17. Frølund B, Palmgren R, Keiding K, Nielsen PH (1996) Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res 30:1749–1758.  https://doi.org/10.1016/0043-1354(95)00323-1 CrossRefGoogle Scholar
  18. Gaudy AF (1962) Colorimetric determination of protein and carbohydrate. Ind Water Wastes 7:17–22Google Scholar
  19. Gilbert KB, Kim TH, Gupta R, Greenberg EP, Schuster M (2009) Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR. Mol Microbiol 73:1072–1085CrossRefGoogle Scholar
  20. Huang H, Peng C, Peng P, Lin Y, Zhang X, Ren H (2019) Towards the biofilm characterization and regulation in biological wastewater treatment. Appl Microbiol Biotechnol 103:1115–1129.  https://doi.org/10.1007/s00253-018-9511-6 CrossRefPubMedGoogle Scholar
  21. Inhülsen S, Aguilar C, Schmid N, Suppiger A, Riedel K, Eberl L (2012) Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111. Microbiologyopen 1:225–242CrossRefGoogle Scholar
  22. Jennings LK, Storek KM, Ledvina HE, Coulon C, Marmont LS, Sadovskaya I, Secor PR, Tseng BS, Scian M, Filloux A, Wozniak DJ, Howell PL, Parsek MR (2015) Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix. Proc Natl Acad Sci USA 112:11353–11358.  https://doi.org/10.1073/pnas.1503058112 CrossRefPubMedGoogle Scholar
  23. Karunakaran E, Biggs CA (2011) Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins. Appl Microbiol Biotechnol 89:1161–1175CrossRefGoogle Scholar
  24. Liang Z, Li W, Yang S, Du P (2010) Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge. Chemosphere 81:626–632.  https://doi.org/10.1016/j.chemosphere.2010.03.043 CrossRefPubMedGoogle Scholar
  25. Lv J, Wang Y, Zhong C, Li Y, Hao W, Zhu J (2014) The effect of quorum sensing and extracellular proteins on the microbial attachment of aerobic granular activated sludge. Bioresour Technol 152:53–58.  https://doi.org/10.1016/j.biortech.2013.10.097 CrossRefPubMedGoogle Scholar
  26. Merroun ML, Selenska-Pobell S (2008) Bacterial interactions with uranium: an environmental perspective. J Contam Hydrol 102:285–295CrossRefGoogle Scholar
  27. Moscoso M, Garcia E, Lopez R (2006) Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol 188:7785–7795.  https://doi.org/10.1128/JB.00673-06 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Omoike A, Chorover J (2004) Spectroscopic study of extracellular polymeric substances from Bacillus subtilis: aqueous chemistry and adsorption effects. Biomacromolecules 5:1219–1230.  https://doi.org/10.1021/bm034461z CrossRefPubMedGoogle Scholar
  29. O'Toole G, Kolter R (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304CrossRefGoogle Scholar
  30. O'Toole G, Kaplan HB, kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79CrossRefGoogle Scholar
  31. Pickering KL, Verbeek CJR, Viljoen C (2012) The effect of aqueous urea on the processing, structure and properties of CGM. J Polym Environ 20:335–343CrossRefGoogle Scholar
  32. Quiroz NGA, Hung CC, Santschi PH (2006) Binding of thorium(IV) to carboxylate, phosphate and sulfate functional groups from marine exopolymeric substances (EPS). Mar Chem 100:337–353CrossRefGoogle Scholar
  33. Rasamiravaka T, Labtani Q, Duez P, El Jaziri M (2015) The formation of biofilms by Pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control mechanisms. Biomed Res Int 2015:759348.  https://doi.org/10.1155/2015/759348 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Saleh TA (2011) The influence of treatment temperature on the acidity of MWCNT oxidized by HNO3 or a mixture of HNO3 /H2SO4. Appl Surf Sci 257:7746–7751CrossRefGoogle Scholar
  35. Saleh TA, Al-Shalalfeh MM, Al-Saadi AA (2016) Graphene dendrimer-stabilized silver nanoparticles for detection of methimazole using surface-enhanced Raman scattering with computational assignment. Sci Rep 6:1–12CrossRefGoogle Scholar
  36. Skariyachan S, Sridhar VS, Packirisamy S, Kumargowda ST, Challapilli SB (2018) Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal. Folia Microbiol 63:413–432.  https://doi.org/10.1007/s12223-018-0585-4 CrossRefGoogle Scholar
  37. Tan CH, Koh KS, Xie C, Tay M, Zhou Y, Williams R, Ng WJ, Rice SA, Kjelleberg S (2014) The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules. Isme J 8:1186–1197CrossRefGoogle Scholar
  38. Tretinnikov ON, Tamada Y (2001) Influence of casting temperature on the near-surface structure and wettability of cast silk fibroin films. Langmuir 17:7406–7413.  https://doi.org/10.1021/la010791y CrossRefGoogle Scholar
  39. Wang J, Ding L, Li K, Huang H, Hu H, Geng J, Xu K, Ren H (2018a) Estimation of spatial distribution of quorum sensing signaling in sequencing batch biofilm reactor (SBBR) biofilms. Sci Total Environ 612:405–414CrossRefGoogle Scholar
  40. Wang X, An Q, Zhao B, Guo JS, Huang YS, Tian M (2018b) Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL. Appl Microbiol Biotechnol 102:2019–2030.  https://doi.org/10.1007/s00253-017-8720-8 CrossRefPubMedGoogle Scholar
  41. Yang L, Han DH, Lee BM, Hur J (2015) Characterizing treated wastewaters of different industries using clustered fluorescence EEM-PARAFAC and FT-IR spectroscopy: implications for downstream impact and source identification. Chemosphere 127:222–228CrossRefGoogle Scholar
  42. Yuan SJ, Sun M, Sheng GP, Li Y, Li WW, Yao RS, Yu HQ (2011) Identification of key constituents and structure of the extracellular polymeric substances excreted by Bacillus megaterium TF10 for their flocculation capacity. Environ Sci Technol 45:1152–1157CrossRefGoogle Scholar
  43. Zhang P, Fang F, Chen YP, Shen Y, Zhang W, Yang JX, Li C, Guo JS, Liu SY, Huang Y, Li S, Gao X, Yan P (2014) Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation. Chemosphere 117:59–65CrossRefGoogle Scholar
  44. Zhang Y, Wang F, Zhu X, Zeng J, Zhao Q, Jiang X (2015) Extracellular polymeric substances govern the development of biofilm and mass transfer of polycyclic aromatic hydrocarbons for improved biodegradation. Bioresour Technol 193:274–280.  https://doi.org/10.1016/j.biortech.2015.06.110 CrossRefPubMedGoogle Scholar
  45. Zhao B, Cheng DY, Tan P, An Q, Guo JS (2018) Characterization of an aerobic denitrifier Pseudomonas stutzeri strain XL-2 to achieve efficient nitrate removal. Bioresour Technol 250:564–573.  https://doi.org/10.1016/j.biortech.2017.11.038 CrossRefPubMedGoogle Scholar
  46. Zhou W, Zhang HO, Ma Y, Zhou J, Zhang Y (2013) Bio-removal of cadmium by growing deep-sea bacterium Pseudoalteromonas sp. SCSE709-6. Extremophiles 17:723–731CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of EducationChongqing UniversityChongqingPeople’s Republic of China

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