Resultat från BRC:s process- och teknikforskning


  • Scope: Co-digestion of activated sludge and fibre sludge at kraft pulp mills is a promising option to implement biogas production in the pulp and paper industry.
  • Highlights: A lab-scale study run for 800 days shows that fibre sludge is a promising new substrate for biogas production. The use of sludge recirculation decreases the retention time in the digesters, greatly lowering investment costs and allowing for high-rate treatment of the large wastewater flows. The addition of activated sludge stabilizes the production process, and potentially lowers the wastewater treatment costs for the mills.

Ekstrand E.-M., Karlsson M., Truong X.-B., Björn A., Karlsson A., Svensson B. H. and Ejlertsson J. (2016). High-rate anaerobic co-digestion of kraft mill fibre sludge and activated sludge by CSTRs with sludge recirculation. Waste Management, 56, 166-72.


  • Scope: Biochemical methane potential (BMP) tests are commonly used to analyze the biodegradability and methane potential of organic material. In this study, various mixing strategies were applied to evaluate the influences of mixing on BMP tests.
  • Highlights: The effects of mixing strategies are prominent for the most viscous substrate, both the highest methane potential and highest maximal daily specific methane production were obtained at highest mixing intensity. However, the organic removal efficiencies among all test samples were not affected by mixing, which may be an evidence that mixing helps the releasing of gas bubbles trapped in the liquid. In addition, mixing is not necessary or the manually shake once per day is farily enough during the BMP test when the digster content is quite dilute.

Bing, W., Björn, A., Strömberg, S., Achu Nges, I., Nistor, M., Liu, J. (2016). Evaluating the influences of mixing strategies on biochemical methane potential (BMP) test. Journal of Environmental Management (Accepted).


  • Scope: Chemical thermo-mechanical pulp (CTMP) mills holds a large biomethane potential in their wastewater. Their broadened market has involved increased bleaching and utilisation of different raw materials. Therefore, the main aim of this study was to obtain and maintain a stable anaerobic digestion (AD) process, with a high methane yield and total organic carbon (TOC) reduction, when digesting CTMP wastewater, from different production protocols including shifts in raw material and bleaching.
  • The results showed that variations in raw material (aspen, birch and spruce) and consequently in TOC-loading (3.6-6.6 kg TOC m-3 and day-1) was feasible with methane production of 360 to 500 NmL g TOC-1; the highest yield occurred with effluent from the production of birch- followed by aspen- and spruce pulp. Applications will call for measures to optimize the methane production e.g. by tailored micronutrient additions and prolonged campaigns. The higher methane production from HW wastewaters compared to SW, together with rapid worldwide increase in production of bleached HW CTMP compared to bleached SW CTMP, means that there is an increasing incentive globally for biomethane production from CTMP wastewaters.

Madeleine Larsson, Xu-Bin Truong, Annika Björn, Jörgen Ejlertsson, Bo H. Svensson, David Bastviken, Anna Karlsson (2016). Anaerobic digestion of wastewater from the production of bleached chemical thermo-mechanical pulp – Higher methane production for hardwood than softwood. Journal of Chemical Technology & Biotechnology (In Press)


  • Scope: Codigesting fats, oils, and greases with municipal wastewater sludge can greatly improve biomethane recovery at wastewater treatment facilities. This study utilized recently-developed quantitative PCR assays for syntrophic LCFA-degrading bacteria along with 16S amplicon sequencing to relate changes in microbial community structure to LCFA accumulation during transient loading increases to an anaerobic codigester receiving waste restaurant oil and municipal wastewater sludge.
  • Highlights: Enhanced biomethane production from high loadings of fats, oils and greases can be achieved by promoting a higher biomass of slow-growing syntrophic consortia, such as with longer digester solids retention times. This work also demonstrates the potential for controlling the loading rate of fats, oils, and greases based on the analysis of the codigester community structure, such as with quantitative PCR measurements of syntrophic LCFA-degrading bacteria abundance.

Ziels R, Karlsson A, Beck DA, Ejlertsson J, Shakeri Yekta S, Björn A, Stensel HD, Svensson BH (2016) Microbial community adaptation influences long-chain fatty acid conversion during anaerobic codigestion of fats, oils, and grease with municipal sludge. Water Research 103: 372-382.


  • Scope: In anaerobic digestion environments, the fraction of metals available for microbial uptake is controlled by interactive physicochemical and biological processes, which enable the transport and association of metal species to the cell membrane and further internalization of the metals by the cells This study deals with the interrelationship between overall chemical speciation of S, Fe, Co, and Ni in relation to metals bio-uptake processes in a number of laboratory- and full-scale continuous stirred tank biogas reactors.
  • Highlights: Prevailing sulfidic conditions restrict accessibility of metals for microorganisms. However, sulfidic conditions do not essentially prevent metal acquisition by microorganisms. It is demonstrated that relative S to Fe content regulates chemical speciation and bio-uptake of Co and Ni.

Shakeri Yekta S, Skyllberg U, Danielsson Å, Björn A, Svensson BH (2016) Chemical Speciation of Sulfur and Metals in Biogas Reactors – Implications for Cobalt and Nickel Bio-uptake Processes. Journal of Hazardous Material (In press).


  • Scope: There are several ways of optimizing an anaerobic digestion process, of which the administration of trace elements is one of the most promising. This study used semi-continuous laboratory scale biogas reactors to simulate the effects of trace-element addition in different combinations, while degrading the organic fraction of municipal solid waste and slaughterhouse waste.
  • Highlights: Combined addition of Fe, Co and Ni was superior to the addition of only Fe, Fe and Co or Fe and Ni. However, the addition of only Fe resulted in a more stable process than the combined addition of Fe and Co, perhaps indicating a too efficient acidogenesis and/or homoacetogenesis in relation to a Ni-deprived methanogenic population. The higher stability was supported by observations of differences in viscosity, intraday VFA- and biogas kinetics as well as by the 16S rRNA gene and 16S rRNA of the methanogens.

Moestedt J, Nordell E, Shakeri Yekta S, Lundgren J, Martí M, Sundberg C, Ejlertsson J, Svensson BH, Björn A (2016). Effects of trace element addition on process stability during anaerobic co-digestion of OFMSW and slaughterhouse waste. Waste management 47: 11-20.


  • Scope: The ecophysiology of long-chain fatty acid (LCFA)-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated.
  • Highlights: Microbial community dynamics were followed using qPCR and Illumina-based high throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid showed: that increased mineralization rates of LCFA were a result of quantitative shifts within the microbiome towards a higher abundance of LCFA-degrading Syntrophomonas bacteria and methanogenic archaea; the newly developed qPCR assays may thus serve as a novel method to determine the fatty acid β-oxidization potential of AD reactors based on the concentration of syntrophic β-oxidizing bacteria within the community. Such an approach may lead to better anaerobic digester feeding strategies that result in stable elevated methane production from waste fats, oils, and grease.

Ryan M. Ziels, David A. C. Beck, Magalí Martí, Heidi L. Gough, H. David Stensel, Bo H. Svensson (2015). Monitoring the Dynamics of Syntrophic β-Oxidizing Bacteria During Anaerobic Degradation of Oleic Acid by Quantitative PCR. FEMS Microbiology Ecology 91 Issue 4 (2015) ISSN: 0168-6496 Online ISSN: 1574-6941.


  • Scope / Highlights: The chapter present state of the art on the role of trace elements in anaerobic digestion in terms of metal speciation with special concern on sulfur, microbial interactions, biotechnology applications and modelling approaches. It further defines the challenges within these subtopics.
  1. Fermoso, E. D. van Hullebusch, G. GuibaudAffiliated withThe Hebrew University of Jerusalem Institute of Life Sciences, The Hebrew U, Institute of Life SciencesFaculté des Sciences et Techniques, University of Limoges Groupement de Recherche, Eau, Sol, Environnement, G. Collins , B. H. Svensson, C. Carliell-Marquet, J. P. M. Vink , G. Esposito (2015). Fate of Trace Metals in Anaerobic Digestion. In: Georg GübitzAlexander BauerGuenther BochmannAndreas GronauerStefan Weiss(eds) Biogas Science and TechnologyAdvances in Biochemical Engineering/Biotechnology Vol 151, pp. 171-195.


  • Scope: Anaerobic digestion of alkaline kraft elemental chlorine-free bleaching wastewater in two mesophilic, lab-scale upflow anaerobic sludge bed reactors.
  • Highlights: Resulted in significantly higher biogas production (250 ± 50 vs. 120 ± 30 NmL g TOC IN −1); reduction of filtered total organic carbon (fTOC) (60 ± 5 vs. 43 ± 6%) for wastewater from processing of hardwood (HW) compared with softwood (SW). A stable anaerobic process was maintained with hydraulic retention times (HRTs) between 7 and 14 h. Lowering the HRT (from 13.5 to 8.5 h) did not significantly affect the process. The results show that this type of wastewater is suitable for a full-scale implementation, but the difference in methane potential between SW and HW needs consideration regarding process dimensioning and biogas yield optimization.

Madeleine Larsson, Xu-Bin Truong, Annika Björn, Jorgen Ejlertsson, David Bastviken, Bo H. Svensson and Anna Karlsson (2014). Anaerobic digestion of alkaline bleaching wastewater from a kraft pulp and paper mill using UASB technique. Environmental Technology 36:1489-1498.


  • Scope: Optimal supply of trace elements is a prerequisite for microbial growth and activity in anaerobic digestion bioprocesses. Trace elements in anaerobic digestion bioreactors undergo complex physicochemical reactions and may be present as free ions, complex bound or as precipitates depending on pH, or on the presence of sulfur compounds or organic macromolecules.
  • Highlights: This review provides an overview of the state of the art in approaches, with respect to sampling and analytical procedures, to determine bioavailable trace elements in anaerobic bioprocesses.

van Hullebusch ED, Guibaud G, Simon S, Lenz M, Shakeri Yekta S, Fermoso FG, Duester L, Roussel J, Guillon E, Skyllberg U, Almeida M, Pechaud Y, Garuti M, Frunzo L, Esposito G, Carliell-Marquet C, Collins G. (2014) Methodological approaches for trace element fractionation and speciation analysis to estimate bioavailability in anaerobic digestion ecosystems: an overview. Critical Reviews in Environmental Science and Technology 46: 1324–1366.


  • Scope: Initiation of the different types of metal uptake by microorganisms depends on thermodynamic and kinetic processes, which relates to the properties of available organic and inorganic aqueous metal complexes (e.g., lability, strength and ionic charge state) in contact with the cell membrane. This study investigated the equilibrium chemistry and chemical speciation of S, Fe and metals (Co, Ni, Cu, Zn, Cd, and Pb) in eight full scale continuous stirred tank biogas reactors operating under under sulfidic and ferruginous conditions.
  • Highlights: Iron in the aqueous phase was dominated by Fe(II)-thiol complexes under sulfidic conditions and by Fe(II)-phosphate complexes under ferruginous conditions. Thiols, representing organic functional groups, and sulfide complexes were the major aqueous species of Co(II), Ni(II), Cd(II) and Pb(II) under sulfidic conditions. Under ferruginous conditions, thiol complexes were still important, but carbonate and phosphate complexes in particular dominated the aqueous phase speciation of Co(II) and Ni(II). The aqueous phase speciation of Zn and Cu was dominated by Zn(II)-sulfide and Cu(I)-polysulfide complexes, respectively. The importance of S:Fe molar ratio as a regulating factor for the chemical speciation of metals in biogas reactors was highlighted.

Shakeri Yekta S, Svensson BH, Björn A, Skyllberg U (2014). Thermodynamic modeling of iron and trace metal solubility and speciation under sulfidic and ferruginous conditions in full scale continuous stirred tank biogas reactors. Applied Geochemistry, 47: 61-73.


  • Scope: Formation of sulfide to a varying but significant extent controlles the chemical speciation of Fe, Co, and Ni during anaerobic digestion of stillage. However, despite its regulatory effect, formation of metal-sulfide precipitates does not necessarily prevent microbial uptake of these essential metals. The objective of this study was to further assess major chemical reactions and chemical forms contributing to solubility and speciation of Fe(II), Co(II), and Ni(II) added to sulfur (S)-rich semi-continuous stirred tank biogas reactors.
  • Highlights: The solubility and speciation of supplemented Fe were controlled by precipitation of FeS(s) and formation of the aqueous complexes of Fe-sulfide and Fe-thiol. The relatively high solubility of Co (∼20% of total Co content) was observed presumably due to the formation of compounds with microbial origin. Nickel had lower solubility than Co and its speciation was regulated by interactions with FeS(s) (e.g. co-precipitation, adsorption, and ion substitution) in addition to precipitation/dissolution of discrete NiS(s) phase and formation of aqueous Ni-sulfide complexes.

Shakeri Yekta S, Lindmark A, Skyllberg U, Danielsson Å, Svensson BH (2014). Importance of reduced sulfur for equilibrium chemistry and kinetics of Fe(II), Ni(II) and Co(II) supplemented to semi-continuous stirred tank biogas reactors fed with stillage. Journal of Hazardous Material, 269: 84-89.


  • Scope: The present report reviews a number of fields that are linked to improvements in the biogas production process as based on specifically increased organic loading rates and/or hydraulic retention time; mixing and rheology; improved monitoring and control; and co-digestion with more high-potential substrates.
  • Highlights: g.: The complexity of the chemical reactions controlling the bioavailability of the trace metals is wide, why optimal addition strategies for trace elements needs to be developed. Substrates as food wastes, sewage sludge, cattle manure, certain energy crops and algae are good bases to obtain processes with good nutrient- and trace element balances. The results implies that trace element levels and availability will directly affect the hydrolysis rates as well as rates and degradation pathways for digestion of amino acids, long chain fatty acids and carbohydrates. However, their effect on hydrolysis seems neglected, why studies are needed to map the metals present in active sites and co-factors of enzymes mediating these primary reactions in AD. Further investigations are then needed to elucidate the importance of the identified metals on the different degradation steps of AD aiming at increased degradation rates of polymeric and complex substrates. Further research is also needed for development and design of proper metal additive solutions for application in full scale biogas plants. Recirculation of reactor material as a way to enrich and maintain an active microbial biomass (and, thus, an increase in the substrate turnover rate) in tank reactors has been tested for digestion of fat within BRCs project DP6. The methane yield increased from 70 to 90% of the theoretical potential at a fat-loading rate of 1.5 g VS/L and day. The same strategy has been successful during digestion of fiber sludge from the pulp and paper industry, i.e. the recirculation has been crucial in establishment of low hydraulic retention times. Also degradation of sewage sludge (SS) would likely be improved by recirculation as the retention time of the solid SS is prolonged in such a system. However, this remains to be tested. The recirculation concept also needs to be evaluated in larger scale reactors to form a base to include extra costs and energy consumption vs. the benefits from increased yields. Plug flow reactors might be another possibility as well as membrane reactors, which physically separates the hydrolyzing and methanogenic phases. See the report for further highlights.

Karlsson, A., Björn, A., Shakeri Yekta, S., Svensson, B.H. (2014). Improvement of the Biogas Production Process: Explorative project (2014). Biogas Research Center (BRC) Report, 2014:2. Linköping, Linköping University Electronic Press, 2014, 87 s. URN: urn:nbn:se:liu:diva-112983.


  • Scope: The Swedish pulp and paper industry holds a large and unused potential for biogas production.
  • Highlights: Specifically several kraft pulping streams gave good methane potentials, such as cooking effluents, condensates and composite pre-sedimentation streams. Generally, all wastewater streams from mechanical pulping showed high methane potentials, as did several of the chemical thermo-mechanical pulping effluents. The fibre fraction of kraft pulping mills holds a large and easily accessible methane potential. Challenges in implementing biogas production on the wastewaters relate to inhibitory bleaching chemicals and high wastewater flows.

Ekstrand E.-M., Larsson M., Truong X.-B., Cardell L., Borgström Y., Björn A., Ejlertsson J., Svensson B. H., Nilsson F. and Karlsson A. (2013). Methane potentials of the Swedish pulp and paper industry – A screening of wastewater effluents. Applied Energy, 112, 507-17


  • Scope: The importance of trace metal availability for microbial activities as a prerequisite to obtain efficient and stable biogas processes is widely reported. Assessment of potential bioavailability of the augmented metals provides information regarding the fraction of the supplemented trace metals being available for anaerobic microorganisms. The objective of this study was to determine the effect of sulfide on chemical speciation and bioavailability of Co and Ni in lab-scale semi-continuous stirred biogas tank reactors treating stillage.
  • Highlights: Production of sulfide during the anaerobic digestion of stillage determined the chemical speciation of Co and Ni. Process data showed that both Co and Ni were available for microbial uptake and the extensive Co- and Ni-sulfide precipitation, due to presence of high sulfur content of the stillage, did not inhibit microbial uptake of Co and Ni in the reactors.

Gustavsson J, Shakeri Yekta S, Karlsson A, Skyllberg U, Svensson BH (2013). Potential bioavailability and chemical forms of Co and Ni in biogas process – an evaluation based on sequential and acid volatile sulfide extractions. Engineering in Life Sciences, 13: 572–579.


  • Scope: Addition of Co and Ni often improves the production of biogas during digestion of organic matter, i.e. increasing CH4-production, process stability and substrate utilization which often opens for higher organic loading rates. This article addresses the role of trace elements during anaerobic digestion of stillage from ethanol production from grains.
  • Highlights: Amendment with Co and Ni was necessary to maintain biogas process stability and resulted in increased CH4-production and substrate utilization efficiency. The acetate-utilizing Methanosarcinales dominated during stable process performance, i.e. when both Co and Ni were supplied, while hydrogenotrophic Methanomicrobiales increased together with VFA concentrations under Co or Ni deficiency. The increase was more pronounced at Co limitation. This study demonstrates that there are good possibilities to improve the performance of biogas processes digesting sulfur-rich substrates by supplementation of Co and Ni.

Gustavsson J, Shakeri Yekta S, Sundberg C, Karlsson A, Ejlertsson J, Skyllberg U, Svensson BH (2013). Bioavailability of cobalt and nickel during anaerobic digestion of sulfur-rich stillage for biogas formation. Applied Energy. 112: 473–477.


  • Scope: A deeper understanding of the microbial community structure and function dynamics during different settings of anaerobic digestion is vital to improve process performance. In this study, the microbial community in 21 full-scale biogas reactors, seven digesting sewage sludge (SS; six mesophilic and one thermophilic) and 14 codigesting (CD) various combinations of wastes (ten mesophilic and four thermophilic), was investigated using 454 pyrosequencing of 16S rRNA gene sequences.
  • Highlights: At the phylum level, the foremost populations in the SS reactors included Actinobacteria, Proteobacteria, Chloroflexi, Spirochetes, and Euryarchaeota, while Firmicutes was the most prevalent in the CD reactors. The main bacterial class in all reactors was Clostridia. Acetoclastic methanogens were detected in the SS, but not in the CD reactors. Their absence suggests that methane formation from acetate takes place mainly via syntrophic acetate oxidation in the CD reactors. The microbial composition was mainly governed by the substrate differences and the process temperature.

Sundberg C, Abu Al-Soud W, Larsson M, Alm E, Shakeri Yekta S, Svensson BH, Sørensen SJ, Karlsson A (2013). 454-pyrosequencing analysis of bacterial and archeal richness in 21 full-scale biogas reactors. FEMS Microbiology Ecology. 85: 612-626.


  • Scope: x Biogas production from maize/sugar beet silage was studied under mesophilic conditions in a continuous stirred tank reactor pilot-scale process. While energy crop mono-digestion is often performed with long hydraulic retention times (HRTs), the present study demonstrated an efficient process operating with a 50-day HRT and a corrected total solids (TScorr) based organic loading rate of 3.4 kg/m3
  • Highlights: High performance in crop mono-anaerobic digestion was achieved in pilot-scale. Methane yields were comparable to lab-scale maximum expected yields. Macro-and micronutrients enabled digestion at short hydraulic retention time. Contrary to previous reports, no viscosity problems were encountered. The effluent compiled with Swedish certification standards for bio-fertilizers.

Achu Nges, I., Björn, A., Björnsson, L. (2012). Stable operation during pilot-scale anaerobic digestion of nutrient-supplemented maize/sugar beet silage. Bioresource Technology, 118: 445-454.


  • Scope: This book chapter briefly introduces the area of rheology and presents important parameters for rheological characterization of biogas reactor fluids. Examples are given from investigations on such parameters for lab-scale reactors digesting different substrates.
  • Highlights: The biogas reactor fluids investigated were behaving viscoplasticly, since they had yield stress and one of them was also thixotropic, due to its partial structure recovering. However, the reactor treating slaughterhouse waste was very close to act as a Newtonian fluid. Also, there was a difference in dynamic- and limit viscosities depending on the substrates used. The results demonstrated that similar TS values did not necessarily correspond to similar flow and viscosity behaviours. To encounter problems related to involvement of new substrates and/or co-digestions in existing facilities, investigations for possible viscosity changes are needed. This is important in order to achieve proper designs in relation to possible variation in substrate mixes in conjunction with new constructions, but also to better control material flows in the existing facilities to avoid disturbances in the reactor performance.

Björn, A., Segura del La Monja, P., Karlsson, A., Ejlertsson, J. & Svensson, B.H. (2012). Rheological characterization. Biogas. Editor Sunil Kumar. InTech publisher, Rijeka, Kroatien. ISBN: 979-953-307-221-9.


  • Scope: Dissolved organic matter (DOM) is a key component in the reaction network of anaerobic digestion. DOM composition comprises the dissolved fraction of influent organic matter, which is a part of available nutrient sources, and the intermediates of microbial processes during degradation of composite organic compounds. In this study, DOM was characterized in eight full scale continuous stirred tank biogas reactors using ultrahigh resolution mass spectrometry.
  • Highlights: The biogas reactors treating sewage sludge had distinctly different DOM characteristics compared to the codigesters treating a combination of organic wastes. Furthermore, the samples from thermophilic and mesophilic codigesters had different DOM composition. The DOM characteristics was apparently governed by the substrate differences and the process temperature. However, despite the differences, the eight biogas reactors shared a large number of compositionally linked DOM compounds suggesting that the biochemical processes common under anaerobic condition might contribute to formation of similar DOM compounds.

Shakeri Yekta S, Gonsior M, Schmitt-Kopplin P, Svensson BH (2012). Characterization of dissolved organic matter in full scale stirred tank biogas reactors using ultrahigh resolution mass spectrometry: a qualitative overview. Environmental Science & Technology. 46: 12711-12719.


  • Scope: Sequential extraction methods are widely used for characterization of chemical forms or fractions of metals in samples from soil, sediment, and sludge. This study investigates the effect of sequential extraction of trace metals on sulfur (S) speciation in anoxic sludge samples from two lab-scale biogas reactors augmented with Fe.
  • Highlights: Changes in chemical speciation of S and Fe occurred during sequential extraction procedure, which suggested indirect effects on trace metals associated to the FeS fraction. Critical limitations for the application of sequential extraction for trace metal speciation analysis outside the framework for which the methods were developed were highlighted.

Shakeri Yekta S, Gustavsson J, Svensson BH, Skyllberg U (2012). Sulfur K-edge XANES and acid volatile sulfide analyses of changes in chemical speciation of S and Fe during sequential extraction of trace metals in anoxic sludge from biogas reactors. Talanta. 89: 470-477



The main components of biological structures, which are, thus, the components degraded also by anaerobic digestion for biogas production are polysaccharides, proteins and lipids. For lipids it is not the hydrolysis rate but the long chain fatty acid (LCFA) products of hydrolysis that is problematic since these are amphiphilic (surface active) and thus “toxic” to the microorganisms in biogas reactors. Thus, anaerobic digestion of FOG (fat, oil and grease) which release LCFA are often inhibited. Therefore, methods to increase the energy rich FOG fraction focuses more on how to alleviate this inhibition rather than increasing rate of hydrolysis.

  • It was found, in batch experiments, that when fat rich substrates like slaughterhouse waste was digested the process was rapidly inhibited. It was further found that this inhibiting substance was not added with the substrate but was a metabolite produced by the anaerobic digestion. Because of the rapid inhibition it was speculated to be caused by hydrolysis of triglyceride fats and production of LCFA.
  • When zeolites was added with the slaughterhouse waste substrate at the start of experiment the lag phase of biogas production was prevented.
  • By spiking the substrates with LCFA the lag phase was significantly increased, but could be made to return to normal (uninhibited) by the addition of zeolites. It was further found that the effect was due to LCFA bound to zeolites and thereby lowered concentration of free LCFA in solution.

Zeolites relieves inhibitory stress from high concentrations of long chain fatty acids Erik Nordell, Anna B. Hansson and Martin Karlsson. Waste management 2013;33:2659-2663.


Many trials of using enzymes for increasing rate and yield of biogas production from sludge produced at waste water treatment plants has been performed. The results have however been inconclusive, often because of various shortcomings (technical, scientific approach or interpretation of data). In order to understand what is really happening, we analyzed both what happened to the enzymes, and the effect on biogas production.

  • All enzymes rapidly lost their activity, both in excess sludge and in anaerobic digester environment.
  • Inactivation could to a large part be assigned to proteolytic degradation of added enzymes.
  • At low biogas production, the biogas production from the added enzymes themselves was significant (which is most often not accounted for).
  • Only proteases provided a high net increase in biogas production. Although the percent increase was significant, this increase was from a low absolute production. Thus, although a high percent increase, too little was gained to advocate the use of proteases at the concentrations necessary.
  • Better enzymes with longer activity life time are needed so that significantly less enzyme need to be added.

Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion. Anna Odnell, Michael Recktenwald, Katarina Stensén, Bengt-Harald Jonsson and Martin Karlsson. Water Research 2016;103:462-471.


Upgrading of biogas to vehicle gas quality is one of the largest CAPEX and OPEX costs of biogas production plants because they rely on either high pressure or high temperatures. The problem of biogas upgrading is the same as in carbon capture and sequestration (CCS), i.e. to separate carbon dioxide from other flue gases. The idea of using enzymes (carbonic anhydrases) to capture and carbon dioxide in CCS has been around for the last two decades and should be applicable also for biogas upgrading. However, the approaches have been slightly stupid, still relying on amine solvents, re-generation with steam etc and are thus very energy intense. In an alternative approach the process could be designed to use alternative enzymes, solvents and processes that is much more energy efficient.

  • The suggested enzymes had a melting temperature of 78 °C. This would necessitate a process temp. of approx. max. 60 °C. This could be achieved by regenerating at reduced pressure of approx. 500 mbar (instead at normal pressure and 120 °C as for amines) and, thus, provide an alternative process that is very much more energy efficient.
  • In an alternative solute, K2CO3, the melting temperature was, however, increased to 83 °C by which process temp. and pressure could be increased and more easily reached, but still only rely on hot water rather than steam.
  • A suggested process solution was designed and additional novel enzyme variants was designed and produced. Several of these enzymes had beneficial properties such as increased melting temperature (up to 93 °C), better folding properties, and better production properties. Thus, showing that rational design for better enzymes can be achieved.
  • All in all it was shown, in theory, that a low energy upgrading process could be designed around available enzymes. The process was however not tested in practice because the project had to be prematurely closed because of financial reasons.
  • “Funnily”, our research report with all suggested process solutions was released in August 2015, and the largest actor working with the problem for approx. 20 years (CO2 solutions Inc, Canada) released information about their new approach utilizing……K2CO3 and low-pressure regeneration…., on 25th of Nov. 2015!

Enzymatic upgrading of biogas. Martin Karlsson and Patrik Nygren. Energiforskrapport 2015:108, pp 1-48. ISBN 978-91-7673-108-6. URL:


Metaproteomics have been suggested to complement metagenomics in identification of novel enzymes for industrial applications, e.g. biogas production. However, no one have suggested how that should be made possible. Our solution to the problem is use constructed environments to reach a number of objectives (full microbial communities, clean environment, tight control over enzyme expression etc.)

  • A full methanogenic microbial community could be maintained on a chemically defined growth medium.
  • Over time, the microbial community reached a metabolic steady-state with presumably low fluctuation of metabolic proteins and proven suppressed expression of extracellular hydrolytic enzymes.
  • From the enzyme suppressed metabolic steady-state the desired enzyme activity could be induced without cross-talk with other activities. That is, protease activity could be induced without the simultaneous increase in cellulase activity, and vice versa.
  • The above opens up for using a metaproteomics-guided approach for targeted bioprospecting of novel enzymes with sought for activity from microbial communities, e.g. the biogas producing microbial community.

Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state. Jutta Speda, Mikaela A Johansson, Bengt-Harald Jonsson and Martin Karlsson. Applied Microbiology and Biotechnology. 2016; 100: 7989-8002.


Hitherto, ALL sample preparation methods for the “complete” protein complement of microbial communities have been performed of the intracellular protein fraction. However, the vast majority of industrially relevant enzymes (cellulases, proteases, lipases etc.) are extracellular enzymes. Therefore it was proven that by maintaining the microbial community in a constructed environment on chemically defined medium, also the approx. 25 % of extracellular enzymes were made accessible.

  • Most “standard” protein precipitation methods such as TCA precipitation were not useful as they precipitated also contaminants, thus hindering downstream analysis. For best result extraction/precipitation methods performed best. Resolubilization was best performed with thiourea/urea.
  • By best methods no pretreatment of sample (concentration) was necessary.
  • No method could by itself capture the complete extracellular protein complement. At best a single method could only capture approx. 35 % of all proteins available. But by combining two best complementary methods approx. 60 % of all proteins can be captured. Basically, this mean that the most results of metaproteomic studies today is BS with regards to stating that “the complete protein complement” was analyzed. However, with our approach we could show that it is possible to capture and analyze the extracellular enzymes, which is an absolute prerequisite to identify novel hydrolytic enzymes.

Assessment of sample preparation methods for metaproteomics of extracellular proteins Jutta Speda, Mikaela A Johansson, Uno Carlsson and Martin Karlsson. Analytical Biochemistry 2017; 23-36.