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Activated zeolite—suitable carriers for microorganisms in anaerobic digestion processes?

Abstract

Plant cell wall structures represent a barrier in the biodegradation process to produce biogas for combustion and energy production. Consequently, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required. Here, we show that natural activated zeolites (i.e. trace metal activated zeolites) represent eminently suitable mineral microhabitats and potential carriers for immobilisation of microorganisms responsible for anaerobic hydrolysis of biopolymers stabilising related bacterial and methanogenic communities. A strategy for comprehensive analysis of immobilised anaerobic populations was developed that includes the visualisation of biofilm formation via scanning electron microscopy and confocal laser scanning microscopy, community and fingerprint analysis as well as enzyme activity and identification analyses. Using SDS polyacrylamide gel electrophoresis, hydrolytical active protein bands were traced by congo red staining. Liquid chromatography/mass spectroscopy revealed cellulolytical endo- and exoglucanase (exocellobiohydrolase) as well as hemicellulolytical xylanase/mannase after proteolytic digestion. Relations to hydrolytic/fermentative zeolite colonisers were obtained by using single-strand conformation polymorphism analysis (SSCP) based on amplification of bacterial and archaeal 16S rRNA fragments. Thereby, dominant colonisers were affiliated to the genera Clostridium, Pseudomonas and Methanoculleus. The specific immobilisation on natural zeolites with functional microbes already colonising naturally during the fermentation offers a strategy to systematically supply the biogas formation process responsive to population dynamics and process requirements.

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References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402

    Article  CAS  Google Scholar 

  2. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56(6):1919–1925

    CAS  Google Scholar 

  3. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotech 23:257–270

    Article  CAS  Google Scholar 

  4. Banks CJ, Zhang Y, Jiang Y, Heaven S (2012) Trace element requirements for stable food waste digestion at elevated ammonia concentrations. Biores Technol 104:127–135

    Article  CAS  Google Scholar 

  5. Bayer EA, Shimon LJW, Shoham Y, Lamed R (1998) Cellulosomes—structure and ultrastructure. J Struct Biol 124:221–234

    Article  CAS  Google Scholar 

  6. Bischofsberger W, Böhnke B, Seyfried CF (2004) Mikrobiologische Grundlagen des anaeroben Abbaus – Überblick und Organismen. In: Dauber S (ed) Anaerobtechnik. Springer, Berlin, Germany, pp 23–49

    Google Scholar 

  7. Bolstad AI, Jensen HB, Bakken V (1996) Taxonomy, biology and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev 9:55–71

    CAS  Google Scholar 

  8. Bougrier C, Dognin D, Laroche C, Rivero JC (2011) Effects of additives solutions of trace elements on anaerobic digestion of maize silage. 1st International Conference on Biogas Microbiology. Leipzig, Germany, September 14–16, Proceedings P-10, p 75

  9. Bruni E, Jensen AP, Angelidaki I (2010) Comparative study of mechanical, hydrothermal, chemical and enzymatic treatments of digested biofibers to improve biogas production. Biores Technol 101(22):8713–8717

    Article  CAS  Google Scholar 

  10. Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M (1999) The domainspecific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. System Appl Microbiol 22:434–444

    Article  CAS  Google Scholar 

  11. Dees C, Ringelberg D, Scott TC, Phelps TJ (1995) Characterisation of the cellulose degrading bacterium NCIMB 10462. Appl Biochem Biotechnol 51:263–274

    Article  Google Scholar 

  12. Fernández N, Montalvo S, Fernández-Polanco F, Guerrero L, Cortés I, Borja R, Sánchez E, Travieso L (2007) Real evidence about zeolite as microorganisms immobilizer in anaerobic fluidized bed reactors. Process Biochem 42(4):721–728

    Article  Google Scholar 

  13. Goldstein JI, Newbury DE, Echlin P, Joy DC, Lyman CE, Lifshin E, Sawyer L, Michael JR (2003) Scanning electron microscopy and X-ray microanalysis. Kluwer Academic/Plenum Publishers, New York

    Book  Google Scholar 

  14. Goodwin JAS, Wase DAJ, Forster CF (1990) Effects of nutrient limitation on the anaerobic up flow sludge blanket reactor. Enzyme Microbial Technol 12:877–884

    Article  CAS  Google Scholar 

  15. Holper J, Lesjak M, Heinzel U, Boos B (2005) Zeolith in der Biogasgewinnung. Sonn. 05450052.5 [EP 1 577 269 A1]. European Patent, Styria, Austria

    Google Scholar 

  16. Jarrell KF, Stark M, Nair DB, Chong JPJ (2011) Flagella and pili are both necessary for efficient attachment of Methanococcus maripaludis to surfaces. FEMS Microbiol Lett 319(1):44–50

    Article  CAS  Google Scholar 

  17. Karita S, Sakka K, Ohmiya K (1997) Cellulosomes, cellulase complexes, of anaerobic microbes: their structure models and functions. In: Onodera R, Itabashi H, Ushida K, Yano H, Sasaki Y (eds) Rumen microbes and digestive physiology in ruminants. Japan Science Society Tokyo/Karger, Basel, pp 47–57

    Google Scholar 

  18. Kang D, Gho YS, Suh M, Kang C (2002) Highly sensitive and fast protein detection with Coomassie Brilliant Blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bull Korean Chem Soc 23(11):1511–1512

    Article  CAS  Google Scholar 

  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  20. Lebuhn M, Liu F, Heuwinkel H, Gronauer A (2008) Biogas production from mono-digestion of maize silage-long-term process stability and requirements. Wat Sci Tech 58(8):1645–1651

    Article  CAS  Google Scholar 

  21. Lieber A, Kiesel B, Babel W (2002) Microbial diversity analysis of soil by SSCP fingerprinting technique using TGGE Maxi System. In: Merbach W, Hütsch BW, Augustin J (eds) Teubner. Ökophysiologie des Wurzelraumes, Stuttgart, pp 61–65

    Google Scholar 

  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin-Phenol reagents. J Biol Chem 193:265–275

    CAS  Google Scholar 

  23. Maestrojuán GM, Boone DR, Xun L, Mah RA, Zhang L (1990) Transfer of Methanogenium bourgense, Methanogenium marisnigri, Methanogenium olentangyi, and Methanogenium thermophilicum to the genus Methanoculleus gen. nov., Emendation of Methanoculleus marisnigri and Methanogenium, and description of new strains of Methanoculleus bourgense and Methanoculleus marisnigri. Int J Syst Bacteriol 40(2):117–122

    Article  Google Scholar 

  24. Onyenwoke RU, Vadim VK, Anatolly ML, Wiegel J (2007) Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park. Int J Syst Evol Microbiol 57:2191–2193

    Article  CAS  Google Scholar 

  25. Pobeheim H, Munk B, Johansson J, Guebitz GM (2010a) Influence of trace elements on methane formation from a synthetic model substrate for maize silage. Biores Technol 101(2):836–839

    Article  CAS  Google Scholar 

  26. Pobeheim H, Munk B, Mueller H, Berg G, Guebitz GM (2010b) Characterization of an anaerobic population digesting a model substrate for maize in the presence of trace metals. Chemosphere 80(8):829–836

    Article  CAS  Google Scholar 

  27. Potivichayanon S, Sungmon T, Chaikongmao W, Kamvanin S (2011) Enhancement of biogas production from bakery waste by Pseudomonas aeruginosa. World Acad Sci Eng Technol 56:529–532

    Google Scholar 

  28. Schwarz WH (2001) The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56(5–6):634–649

    Article  CAS  Google Scholar 

  29. Schwieger F, Tebbe C (1998) A new approach to utilize PCR-SSCP for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64(12):4870–4876

    CAS  Google Scholar 

  30. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68(5):850–858

    Article  CAS  Google Scholar 

  31. Stahl DA, Amann R (1996) Development and application of nucleic acid probes. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 205–248

    Google Scholar 

  32. Takashima M, Speece RE (1989) Mineral nutrient requirements for high-rate methane fermentation of acetate at low SRT. J Wat Poll Cont Fed 61:1645–1650

    CAS  Google Scholar 

  33. Van Gylswyk NO, van der Toorn JJTK (1986) Enumeration of Bacteroides succinogenes in the rumen of sheep fed maize-straw diets. FEBS Lett 38(4):205–209

    Google Scholar 

  34. Weiß S, Tauber M, Somitsch W, Meincke R, Müller H, Berg G, Guebitz GM (2010) Enhancement of biogas production by addition of hemicellulolytic bacteria immobilised on activated zeolite. Wat Res 44(6):1970–1980

    Article  Google Scholar 

  35. Weiß S, Zankel A, Lebuhn M, Petrak S, Somitsch W, Guebitz GM (2011) Investigation of mircroorganisms colonising activated zeolites during anaerobic biogas production from grass silage. Biores Technol 102(6):4353–4359

    Article  Google Scholar 

  36. Wichern M, Gehring T, Fischer K, Andrade D, Luebken M, Koch K, Gronauer A, Horn H (2009) Monofermentation of grass silage under mesophilic conditions: measurements and mathematical modeling with ADM 1. Biores Technol 100(4):1675–1681

    Article  CAS  Google Scholar 

  37. Yu Y, Lee C, Kim J, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89(6):670–679

    Article  CAS  Google Scholar 

  38. Zheng D, Alm EW, Stahl DA, Raskin L (1996) Characterization of universal small-unit rRNA hybridization probes for quantitative molecular ecology studies. Appl Environ Microbiol 62:4504–4513

    Google Scholar 

  39. Zverlov VV, Hiegl W, Köck DE, Kellermann J, Köllmeier T, Schwarz WH (2010) Hydrolytic bacteria in mesophilic and thermophilic degradation of plant biomass. Eng Life Sci 10(6):528–536

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was performed within the Austrian Centre of Industrial Biotechnology ACIB and has been kindly supported by IPUS GmbH (Austria), the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT–Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Many thanks go to contributors of the collaborating organisations and institutes (FELMI-ZFE, ZMF and LfL).

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Correspondence to G. M. Guebitz.

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Weiß, S., Lebuhn, M., Andrade, D. et al. Activated zeolite—suitable carriers for microorganisms in anaerobic digestion processes?. Appl Microbiol Biotechnol 97, 3225–3238 (2013). https://doi.org/10.1007/s00253-013-4691-6

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Keywords

  • Biogas
  • Zeolites
  • Hemicellulases
  • Cellulases
  • Microbial community
  • Grass silage