Biocatalytic utilization of lignin for increased biogas production in a biorefinery setting

In the project BioLiGas, the main goal was to produce biogas from lignin-rich biomass by means of enzymes and microbial communities. The project was led by NMBU with NIBIO and PFI-RISE as partners, and NREL in USA as international partner. An important part of the project was to develop an analysis platform to characterize lignin. This was established at Ås campus with equipment such as NMR, pyrolysis GC-MS and simultaneous thermal analyzer (STA) connected to FTIR, and is unique in the Norwegian context. In this part of the project, Ida Synnøve Aarum completed her doctoral degree entitled "Structural characterization of lignin and some side-products from steam exploded woody biomass". The main raw materials included in the project were spruce and birch, and to improve enzymatic hydrolysis and biogas production, a number of different pretreatments were used, including steam explosion, low temperature sulphonation and hot water extraction. Observed effects were delignification and modification of lignin structure with low temperature sulfonation, while steam explosion and hot water extraction affected lignin structure without de-lignification. The effect of pretreatment on enzymatic hydrolysis was investigated using a commercial cellulase mixture. Pre-treatment with low temperature sulfonation led to the highest sugar yield, probably due to increased enzymatic access since most of the lignin were removed. The possible role of oxidative enzymes in anaerobic digestion of lignin-rich material was also investigated. Lignin-rich birch fractions (73% lignin) were used to test the possible role of peroxidases and laccases in biogas yield. Lignin substrate and manganese peroxidase were added to batch reactors containing methane-producing bacteria, with simultaneous addition of hydrogen peroxide to activate the enzymes. The peroxidases did not result in any increase in biogas production. However, during the preliminary tests, a catalase-like activity was observed in the microbial culture, which probably used the supplied hydrogen peroxide. A treatment of the lignin-rich birch fractions with laccase prior to anaerobic digestion was also investigated. After incubation in a biogas reactor for 30 days, no increase in biogas production could be seen. The third type of oxidative enzymes we tested were lytic polysaccharide monooxygenases (LPMOs) which are also activated by hydrogen peroxide. Here, LPMO activity was observed in the biogas reactors, and in some cases a faster biogas process. These enzymes have the potential to make biogas processes more efficient, but more research is needed. Biogas experiments were performed both in batch flasks and in continuous reactor systems (CSTR). In general, all of the carbohydrates in lignin-rich substrates were converted to biogas, but some of the lignin was also utilized. Particularly pseudo-lignin, benzyl ether and phenylcoumaran appeared to be available to the bacteria in the biogas reactors. However, large parts of the lignin were not biodegradable in biogas reactors, and especially high temperature pretreatment seems to be disadvantageous since it results in more condensed and inaccessible lignin for enzymes and microbes. The project has been an important first step in establishing research on lignin at Ås Campus. It has also been very important for continuity and reinforcement of the biogas research environment at Ås Campus. It should also be mentioned that NorBioLab infrastructure has been central to the implementation of the project.

Prosjektet har styrket Ås campus som et kompetanseområdet innen biogass i Norge. Det har også etablert ligninforskning som et nytt tema på Ås campus. NMBU har nå utdannet sin første PhD student innen temaet lignin, og nye vitenskapelige artikler er publisert. Dette gjør NMBU og forskningspartnerne mer attraktive for fremtidige prosjekter som involverer foredling av lignin, et tema som blir stadig viktigere innen bioøkonomien. Prosjektet har også etablert samarbeid med et internasjonalt ledende miljø innen ligninforskning i USA. Kompetansen bygd opp i dette prosjektet vil være nyttig for biogassprodusenter i Norge og for en bedrift som Borregaard hvor prosessering av lignin er helt sentralt. Resultatene fra prosjektet bidrar til kunnskapsbasen som kreves for å bygge opp en bioøkonomi i Norge.

Lignocellulosic biomass is the only renewable resource that can substitute fossil resources at a large scale. So far the main focus has been to use the carbohydrate fraction of this biomass for production of fuels, chemicals and feed, while the lignin-part has been considered a waste-product. In this project we aim at using lignin-rich biomass for the production of biogas which can be used as a fossil fuel substitute. Biogas is produced from organic materials by a mixed microbial community during an anaerobic digestion process. Lignin is generally thought to be difficult to degrade to biogas, but up to now, studies on this topic is rare. The main topics of this project will be to: 1) establish an analytical platform for lignin characterization, 2) investigate and optimize different pretreament and fractionation methods to maximize biogas production from lignin, 3) apply enzymes to to saccharify the carbohydrate fraction of lignocellulose to create a sugar stream, while optimizing anaerobic digestability of the remaining lignin-rich residue, and 4) Apply oxidative enzymes to increase digestibility of lignin-rich fractions and increase the biogas yield. Additionally, the project will generate generic knowledge on biorefining and biofuel production, with particular focus on lignin processing and lignin valorization. The project is lead by the Norwegian University of Life Sciences (NMBU) and is conducted in close collaboration with Paper and Fibre Research Institute (PFI), Norwegian Forest and Landscape Institute (SoL) and National Renewable Energy Laboratory (NREL) in USA. The project will be aligned with several ongoing projects.