title: Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.
abstract: Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Kohrs
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Heyer
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Magnussen
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: benndorf@mpi-magdeburg.mpg.de.
authors.forename: D
authors.initials: D
authors.lastname: Benndorf
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstraße 1, 39106 Magdeburg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Muth
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Behne
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstraße 1, 39106 Magdeburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Rapp
authors.affiliation: Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Technology Assessment and Substance Cycles, Max-Eyth-Allee 100, 14469 Potsdam, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Kausmann
authors.affiliation: Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Technology Assessment and Substance Cycles, Max-Eyth-Allee 100, 14469 Potsdam, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Heiermann
authors.affiliation: Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Bioengineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Klocke
authors.affiliation: Otto von Guericke University, Bioprocess Engineering, Universitätsplatz 2, 39106 Magdeburg, Germany; Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstraße 1, 39106 Magdeburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Reichl
mesh_terms: D019843:Archaeal Proteins / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D056804:Biofuels; D019149:Bioreactors; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification*; D007525:Isoelectric Focusing / Q000379:methods; D054892:Metagenome; D008697:Methane / Q000096:biosynthesis*; D017018:Methanomicrobiales / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019640:Methanosarcinales / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059013:Microbial Consortia / Q000502:physiology; D010944:Plants / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014866:Waste Products
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D056804:Biofuels; D005656:Fungal Proteins; D020543:Proteome; D014866:Waste Products; D008697:Methane
keywords: Anaerobic digestion; Community profiling; Fractionation; GeLC-MS/MS; KEGG pathway analysis; Liquid isoelectric focusing; Metaproteomics