Innovative enzyme technology for sustainable biofuels

The conversion of lignocellulosic (non-edible) biomass to second generation biofuels is an important element of the transition to a more sustainable future. The enzymatic saccharification of cellulose to fermentable sugar is a key step and remains a major process cost, in particular for Nordic woody biomass, since technology development has been focused on other raw materials, such as corn stover. This project has been focused on harnessing the potential of a relatively recently discovered class of enzymes called “lytic polysaccharide monooxygenases” (LPMOs). These enzymes have a high potential in developing better enzymatic processes, but, at the start of this project many aspects of these very powerful novel enzymes remained unclear, also when it comes to their industrial application. In this project, we have been developing enzymatic technologies that allow commercially feasible conversion of Nordic woody biomass, primarily spruce, to sugar and, subsequently, biofuels. We have been working on feedstocks pretreated by various methods, including proprietary methods of two industrial partners, Borregaard and St1 Biofuels. We have tried to unravel how LPMOs function under various process conditions, and have investigated how their efficiency and their interactions with the other involved enzymes can be optimized. This has included attempts to develop new LPMO variants with novel (improved) properties using high-throughput mutant screening. The impact of small fractions of hemicellulose remaining in pretreated material and the possible roles of LPMOs in removing these has received particular attention. To increase industrial relevance, we explored enzymeatic saccharification of cellulose in a larger valorization context, not only looking at the resulting sugar streams, but also at the resulting lignin fraction ("hydrolysis lignin"). We have evaluated lignin quality and options for lignin-based creation of additional value. This has included lab-scale thermochemical conversion of lignin to bio-oils and validation of the potential to upgrade these bio-oils to transportation fuels. Highlights from the second half of the project include: (1) We have discovered LPMOs that are capable of removing "recalcitrant hemicellulose" and we have discovered how these LPMOs work together with other enzymes acting on hemicelluloses. This novel knowledge will be assessed in applied settings and may also yield new options for other cellulose valorization, in particular production of cellulose-based polymeric materials. (2) We have published our work on the development of new enzyme mixtures for biomass processing at higher temperatures; the results show that certain enzyme cocktails with higher thermostability can be industrially competitive for certain substrates, such as sulfate-pulped spruce wood. (3) We have finalized (and published) an integrated process that gives better solubilization of cellulose (-> sugar) and better "residual lignin". Residual lignin is what remains after the polysaccharides, such as cellulose, have been solubilized by degradation with enzymes (cellulases and LPMOs). The residual lignin has been characterized and further refined by SINTEF. Importantly, by using a novel approach, we reached unprecedented efficiency in the enzymatic saccharification of spruce cellulose. (4) We have shown how light can be used to manipulate LPMO activity and, by that the efficicny of cellulose saccharification. This work, published in Nature Communications, is particularly important, because these light effects are mediated by lignin, which is present in the biomass. (5) In the final phase of the project, we have made attempts to engineer the substrate specificity of LPMOs to increase our understanding of this aspect and to potentially create better enzymes. The results of this work, including high-throughput mutant screening will not be know before autumn 2023. (6) Together with the FME Bio4Fuels we have done process modelling for some of the biorefining routes addressed in the project. The PhD student on the project defended her thesis on June 24, 2022.

NMBUs ledende fagmiljø i feltet har behold og videreutviklet sin posisjon. Miljøet er respektert verden over og har blant annet produsert både originalpublikasjoner i toppjournaler og ledende oversiktsartikler, som siteres mye, noe som bidrar til å sette NMBU, og dermed Norge på kartet. NMBU er en ettertraktet samarbeidspartner for internasjonal akademia og industri. Likeså har SINTEFs lignin og prosesskompetanse blitt styrket; på grunn av blant annet dette er SINTEF en ettertraktet partner i f eks EU-prosjekter. Prosjektet har også bidratt til å styrke SINTEFs kompetanse innen «high-throughput screening»; denne kompetansen er svært sentral for SINTEFs rolle i flere najsonale og internasjonale forskningsprosjekter. Det er viktig å påpeke at prosjektet har vært helt avgjørende for den delen av FMEen Bio4Fuels som omhandler biologisk (enzymatisk) prosessering av biomasse. Prosjektet har ført til eller muliggjort flere typer (delvis ad hoc) internasjonalt samarbeid, som bidrar til (1) å få flere, bedre og mer relevante resultater, (2) å styrke forskningspartnernes og Norges renomme i feltet, og (3) å styrke grunnlaget for framtidig samarbeid. Prosjektet har gjort at forskningspartnere har mer kompetanse å tilby industrien. Det har vært konkret samarbeid med Borregaard (No) og St1 (Fi) med mål om å implementere nyervervet kunnskap i industrielle prosesser. Videre har prosjektet vært med på å danne grunnlaget for SFI Industriell Bioteknologi (2020-2028), hvor mange norske industrier er med. Resultater fra prosjektet har gitt grunnlag for utvikling av et helt nytt anvendelsesområde for enzymteknologi, nemlig bruk av enzymer i produksjon av cellulose-baserte avanserte materialer. Dette er et viktig område hvor det forventes mer aktivitet i tiden framover. Alt i alt har dette prosjektet styrket Norges kompetanse og internasjonal anseelse innen prosessering av ikke-spiselig biomasse og styrket norsk kompetanse innen industriell bioteknologi. Denne kompetansen er svært viktig for et samfunn på jakt etter økt bærekraft.

The biochemical conversion of lignocellulosic biomass to biofuels holds considerable promise. Indeed, the first commercial so-called second generation biofuel plants are in operation. The enzymatic saccharification step remains a major process cost, in particular for Nordic woody biomass, since technology development has been focused on other raw materials, such as corn stover. Recent research indicates that considerable improvements in enzyme technology are feasible, by adjusting the enzymes itself and by optimizing process conditions. In particular, much work remains to be done to optimize the impact of lytic polysaccharide monooxygenases (LPMOs), which are newly discovered redox enzymes that are a crucial component of today's commercial cellulase cocktails. In this project, we will develop enabling enzymatic technologies for the conversion of Nordic woody biomass (primarily softwood) to biofuels. We will work on feedstocks pretreated by in-house developed steam explosion methods and on feedstocks delivered by two industrial partners using proprietary technologies. We will study LPMO functionality under various process conditions, and investigate how enzyme efficiency can be optimized. Furthermore, we will study how blends of cellulases and hemicellulases can be tailored for our feedstocks. The impact of "recalcitrant" hemicellulose, i.e. small fractions of hemicellulose remaining in pretreated material, will receive particular attention. Simultaneous optimization of enzyme blends and process conditions should then lead to more efficient saccharification processes and, eventually, cheaper biofuels. To secure industrial relevance, we will evaluate the quality of the products (sugars and "hydrolysis lignin"). In particular, we will assess lignin quality and evaluate options for lignin-based creation of additional value. This will include lab-scale thermochemical conversion to bio-oils and validation of the potential to upgrade these to transport fuels.