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

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    Activated zeolitesuitable carriers for microorganismsin anaerobic digestion processes?

    S. Wei & M. Lebuhn & D. Andrade & A. Zankel &M. Cardinale & R. Birner-Gruenberger & W. Somitsch &B. J. Ueberbacher & G. M. Guebitz

    Received: 12 July 2012 /Revised: 2 January 2013 /Accepted: 3 January 2013 /Published online: 23 February 2013# Springer-Verlag Berlin Heidelberg 2013

    Abstract Plant cell wall structures represent a barrier in thebiodegradation process to produce biogas for combustionand energy production. Consequently, approachesconcerning a more efficient de-polymerisation of celluloseand hemicellulose to monomeric sugars are required. Here,we show that natural activated zeolites (i.e. trace metalactivated zeolites) represent eminently suitable mineralmicrohabitats and potential carriers for immobilisation ofmicroorganisms responsible for anaerobic hydrolysis of bio-polymers stabilising related bacterial and methanogeniccommunities. A strategy for comprehensive analysis ofimmobilised anaerobic populations was developed thatincludes the visualisation of biofilm formation via scan-ning electron microscopy and confocal laser scanning

    microscopy, community and fingerprint analysis as wellas enzyme activity and identification analyses. UsingSDS polyacrylamide gel electrophoresis, hydrolytical ac-tive protein bands were traced by congo red staining.Liquid chromatography/mass spectroscopy revealed cellulo-lytical endo- and exoglucanase (exocellobiohydrolase) as wellas hemicellulolytical xylanase/mannase after proteolytic di-gestion. Relations to hydrolytic/fermentative zeolite colonis-ers were obtained by using single-strand conformationpolymorphism analysis (SSCP) based on amplification ofbacterial and archaeal 16S rRNA fragments. Thereby, domi-nant colonisers were affiliated to the genera Clostridium,Pseudomonas and Methanoculleus. The specific immobilisa-tion on natural zeolites with functional microbes already

    Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-4691-6) contains supplementary material,which is available to authorized users.

    S. Wei :M. Cardinale :G. M. GuebitzInstitute of Environmental Biotechnology,Graz University of Technology, Petersgasse 12,8010 Graz, Austria

    M. Lebuhn :D. AndradeBavarian State Research Centre for Agriculture,Vttinger Strae 38,85354 Freising, Germany

    A. ZankelInstitute for Electron Microscopy, Graz University of Technology,Steyrergasse 17,8010 Graz, Austria

    R. Birner-GruenbergerProteomics Core Facility, Center for Medical Researchand Institute of Pathology, Medical University of Graz,Stiftingtalstrasse 24,8010 Graz, Austria

    W. SomitschEngineering Consultant, Wiedner Hauptstrasse 90/2/19,1050 Vienna, Austria

    B. J. UeberbacherIPUS Mineral- & Umwelttechnologie GmbH, Werksgasse 281,8786 Rottenmann, Austria

    S. Wei :G. M. Guebitz (*)ACIB Austrian Centre of Industrial Biotechnology, Petersgasse 14,8010 Graz, Austriae-mail:

    Present Address:G. M. GuebitzDepartment of Agrobiotechnology Tulln, Institute ofEnvironmental Biotechnology, University of Natural Resourcesand Life Science, Konrad Lorenz Str. 20,3430 Tulln, Austria

    Appl Microbiol Biotechnol (2013) 97:32253238DOI 10.1007/s00253-013-4691-6

  • colonising naturally during the fermentation offers a strategyto systematically supply the biogas formation process respon-sive to population dynamics and process requirements.

    Keywords Biogas . Zeolites . Hemicellulases . Cellulases .

    Microbial community . Grass silage


    In order to stabilise and optimise biogas formation fromenergy plants several approaches were investigated to avoidunfavourable acidification, ammonia accumulation etc. de-creasing the production of methane. Adequate availability ofessential trace elements for the bacterial community wasidentified to be essential (Takashima and Speece 1989;Goodwin et al. 1990). Pobeheim et al. (2010a) showed thatthe addition of mixed trace elements increased methaneyields by up to 30 % in batch digestion of maize silage.Furthermore, effective removal of volatile solids (around73 %) has been reported for maize silage operated complete-ly stirred tank reactors (CSTR), when higher concentratedsolutions of trace metals were added (Bougrier et al. 2011).Enhancement of methane production (up to 20 %), was alsoobserved for Co and Ni addition which was corroborated byrisen cofactor F420 activities and dominance of hydrogeno-trophic Methanoculleus sp. (Pobeheim et al. 2010b). Theimportance of Co, Se and other trace metals was also ob-served for stable food waste digestion. Here, the accumula-tion of volatile fatty acids (VFAs) can only be avoided,when digesters are supplemented with a certain concentra-tion of Se, Mo, Co and W (Banks et al. 2012).

    Activated zeolites as used in this study combine tracemetal activation on the surface with an high ion-exchangecapacity for supplementation with cationic macro-elementssuch as Na+, K+, Mg2+ or Ca2+ and trace elements as well,that zeolites can be loaded with (Holper et al. 2005).Consequently, these materials potentially provide an excel-lent operational environment for microorganisms participat-ing either in the biomass hydrolysis or methane formationprocess. Recent studies demonstrated the capability of thesezeolites to be colonised by certain microbial populationsduring anaerobic batch-wise operation and laboratory-scaled mono-grass silage or vinasses operated bioreactors(Wei et al. 2011; Fernndez et al. 2007). Furthermore, itwas shown that microorganisms acting on biomass asdegraders, obtained by selective cultivation, can be immo-bilised on zeolite surfaces. These populations can provide aspecific enzymatic activity as addition to the natural consor-tium activities, e.g. hydrolytic activity to increase recalci-trant biomass degradation (plant cell walls) and thus resultin higher methane yields in batch-culture experiments asdemonstrated before (Wei et al. 2010). This effect could

    not be seen with enzymatic treatment alone as reported byBruni et al. (2010) applying commercially available laccasesor mixture of cellulases and hemicellulases to digested bio-fibres. Consequently, there is strong need to identify zeoliteimmobilised organisms and their enzymes in order to exploitthis unique system to improve biogas formation. Here, weshow that a combination of sophisticated microscopic [con-focal laser scanning microscopy (CLSM)-fluorescence insitu hybridization (FISH), scanning electron microscopy(SEM)], genetic [(single-strand conformation polymor-phism analysis (SSCP)] and biochemical [liquid chromatog-raphy/mass spectroscopy (LC-MS) protein identification]methods can lead to a comprehensive mechanistic under-standing of the function of zeolite immobilised anaerobicpopulations.

    Materials and methods

    Semi-continuous fermentation and analogue batch operation

    The activated zeolites used (IPUS GmbH, Rottenmann,Austria) consisted of a natural zeolitic tuff containing >85 %clinoptilolite, which was crushed to a grain size below2.5 mm. The material was loaded with Fe, Ni, Co, Mo,Se, Cu and Zn as trace metal elements to enhancemicrobial activity (Holper et al. 2005). Powderous par-ticles of

  • carried out in mineral salt medium, which had the followingcomposition (in milligramme per litre): MgSO47H2O, 9.4;CaSO42H2O, 4.7; Na2HPO42H2O, 752; KH2PO4, 63.92;NH4CL, 18.8. After mixing, 0.47-ml trace element solutionwas added and pH adjusted to 7.2 by using 1 M HCl. Traceelement solution was composed of (in milligramme per litre):Na2EDTA2H2O, 2,740; ZnSO47H2O, 100; MnCl24H2O,25.6; H3BO3, 300; CoCl26H2O, 200; CuCl22H2O, 10;NiCl22H2O, 20; Na2MO42H2O, 900; Na2SeO35H2O, 30.4;FeSO47H2O, 1,000. In order to induce production of hydro-lytic enzymes, the following model substrate was added fed-batch-wise to the mineral salt medium in a concentration of1%DM (w/v): microcrystalline cellulose (Fluka), 30%; xylanfrom Birchwood (Roth, Karlsruhe, Germany), 50 %; ligninwith low sulfonate content (Sigma-Aldrich, St. Louis, MO,USA), 14.5 %; pectin C (Roth), 0.5 % and starch (Roth), 5 %.The re-activation experiment was carried out over a totalperiod of 55 days at temperatures according to the formerbatch-fermentation experiments, i.e. 35 and 45 C respective-ly. Samples were taken as duplicates at regular intervals.

    PCR-based SSCP and sequencing analysis

    The total bacterial community DNAwas extracted by usinga DNA extraction kit for soil samples (FastDNA Spin Kitfor Soil, MP Biomedicals, Solon, OH, USA) following themanufacturers recommendations. To collect zeolite-immobilised microorganisms, 0.1 g of zeolite was rinsedthree times with 500 l of 1 PBS (pH8.0) before applyingthe DNA extraction procedure. Free microorganisms werecollected from sample supernatants (1.0 ml) representing thetotal sludge inoculation phase. Samples were centrifuged for15 min at 16,750g to collect the cells. Amplification ofbacterial 16S rRNA gene fragments was carried out usingthe bacterial primer pair Com-1 (5-CAG CAG CCG CGGTAA TAC-3) (Schwieger and Tebbe 1998) and Unibac-II-927rP (5-CCC GTC AAT TYM TTT GAG TT-3) for anamplicon size of 412 bp according to Lieber et al. (2002).Amplification of archaeal 16S rRNA gene fragments wasperformed using the primer pair ARC-787f (5-ATTAGATACC CSBGT AGTCC-3) and ARC-1059r (5-GCCATGCACC WCCTC T-3) for an amplicon size of 273 bpfollowing Yu et al. (2005), using a Biometra T personal/gradient system (Biometra, Gttingen, Germany). After pu-rification of PCR-generated DNA products using the GeneClean Turbo Kit (Qbiogene, Heidelberg, Germany) follow-ing the manufacturers recommendations, DNA-content wasdetermined before SSCP analysis. Therefore, 1 l of puri-fied PCR-products were analysed using a micro-volumespectrophoto