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Increasing lignocellulosic feedstock for advanced biofuels can tackle the decarbonization of the transport sector. Dedicated biomass produced alongside food systems with low indirect land use change (iLUC) impact can broaden the feedstock availability, thus streamlining the supply chains. The objective of this study was the design and evaluation of advanced ethanol value chains for the Emilia-Romagna region based on low iLUC feedstock. Two dedicated lignocellulosic crops (biomass sorghum and sunn hemp) were evaluated in double cropping systems alongside food crop residues (corn stover and wheat straw) as sources to simulate the value chains. A parcel-level regional analysis was carried out, then the LocaGIStics2.0 model was used for the spatial design and review of the biomass delivery chain options regarding cost and greenhouse gas (GHG) emissions of the different feedstock mixes. Literature data on bioethanol production from similar feedstocks were used to estimate yields, process costs, and GHG emissions of a biorefinery process based on these biomasses. Within the chain options, GHG emissions were overly sensitive to cultivation input, mostly N-fertilization. This considered, GHG emissions resulted similar across different feedstock with straw/stover (averaging 13 g CO2eq MJ−1 fuel), sunn hemp (14 g CO2eq MJ−1 fuel), and biomass sorghum (16 g CO2eq MJ−1 fuel). On the other hand, the bioethanol produced from biomass sorghum (608 € Mg−1 of bioethanol) was cheaper compared with straw (632 € Mg−1), sunn hemp (672 € Mg−1), and stover (710 € Mg−1). The bioethanol cost ranged from 0.0017 to 0.020 € MJ−1 fuel depending on the feedstock, with operations and maintenance impacting up to 90% of the final cost. In summary, a single bioethanol plant with an annual capacity of 250,000 Mg of biomass could replace from 5% to 7% of the Emilia-Romagna's ethanol fuel consumption, depending on the applied sourcing scenario.

Advanced biofuels are produced from renewable resources that comply with stringent sustainability criteria, and lower GHG emissions than fossil fuels. In the BECOOL/BioValue EU-Brazil cooperation project, novel value chains for advanced bioethanol are developed. Second generation (2G) bio-ethanol represents the most important advanced biofuel from sugar streams, but its production costs are higher than those of other fuels, and innovations are needed to improve efficiency and economics of the process. In this work, beech wood pellets were treated using the FABIOLATM technology, an acetone-based low-temperature organosolv fractionation process. This fractionation results in three main product streams: lignin, a high-purity cellulosic pulp and hemicellulose sugars in solution. The cellulosic pulp was enzymatically hydrolysed producing a glucose-rich (C6) stream and the hemicellulose sugar solution (C5) is conditioned for further biological conversion. The C6 and C5 streams obtained were used for fermentation by the yeasts Saccharomyces cerevisiae and Spathaspora passalidarum, respectively. While the C6 stream was readily fermentable, the C5 stream was only fermentable after dilution. Therefore the C5 stream was subjected to detoxification by activated carbon. Detoxified C5 streams showed higher fermentability and the production of bio-ethanol reached approximately 0.3 g bio-ethanol/gram xylose consumed. Fermentation experiments at 10-L scale confirmed the results obtained at laboratory scale. Combining FABIOLATM and detoxification resulted in efficient C6 and C5 utilization for bio-ethanol production.

The aim of the project “More Fuels from Organic Residues” (MOTOR) is to produce advanced biofuels for the transport sectors where electrification or gaseous biofuels will not generate the reduction of their carbon footprint in the short term. In MOTOR, wet lignocellulosic waste streams that do not compete with the food/feed market are used as a feedstock for a smart fermentation process. In the present study, the utilization of paper sludge as substrate for fermentation for production of fuel precursors and subsequent catalytic conversion to fuels at laboratory scale is described. The steps of the process were assessed experimentally, and based on the data generated, a conceptual process is being developed to explore the economic and sustainability aspects of the new value chain for biofuel.. Results show that solubilization of sugars from paper sludge using existing enzyme cocktails is very limited and needs improvement. On the other hand, the catalytic production of fuel from the fermentation products shows promising results in terms of type of products.

Clostridium beijerinckii has been an organism of great interest for industrial biotechnology for about a century because it can degrade a wide range of sugars to produce acetone, butanol, ethanol, and isopropanol, chemicals used in consumer goods (paints, cosmetics, food ingredients) and as alternatives to transport fuel. Still, C. beijerinckii has other less studied physiological traits, such as the formation of intracellular compartments, spores and microcompartments. Therefore, we decided to study sporulation and microcompartment formation in C. beijerinckii using functional genomics.Sporulation is a cell differentiation process that occurs in all Firmicutes. It is triggered when the cells are exposed to an unfavorable environmental condition (presence of oxygen, high pH variation), and yields spores, metabolically inactive cells resistant to harsh conditions (UV, chemicals, heat, oxygen). In Chapter 1, the research that has been done in the last 30 years on sporulation in solvent-producing clostridia, including the latest updates on the molecular regulation of sporulation in solventogenic clostridia, is presented. Common triggers and regulation mechanisms of sporulation in several solventogenic species are identified, underlining the differences and similarities between species. Potential links in the regulation of sporulation and solvent production are pinpointed. This chapter highlights the need for more studies on signal transduction pathways, transcriptional and post-translational regulation of sporulation, and solvent production. A better understanding of the connections between both physiological phenomena would provide us with relevant targets for strain engineering of solventogenic clostridia.Metabolic engineering of Clostridium used to be tedious and hampered the studies on strain physiology. An efficient and fast engineering tool was highly desired. Chapter 2 describes the adaptation of a CRISPR-Cas9 method for use in Clostridium beijerinckii. The tool

Seaweed biomasses are highly suited as raw material for production of renewable chemicals and fuels because of their special chemical composition (carbohydrates, proteins and minerals) and the possibility of cultivating them at large scale [1]. In the MACROFUELS project, year-round cultivation strategies for seaweeds are being developed. Laminaria digitata (Fig. 1) is one of the most well-studied brown seaweed species, and it has the potential to be cultivated in the North Sea. Brown seaweeds are rich in glucans and mannitol that can be converted to a biofuel such as butanol by fermentation by solventproducing Clostridia [2]. These organisms ferment mannitol and other sugars including glucose and xylose and produce a mix of acetone / isopropanol, butanol and ethanol (ABE/IBE) as end products.

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Poster met onderzoeksinformatie. In dit onderzoek wordt gezocht naar een techniek om biodiesel te produceren uit goedkope biomassa.

Poster met onderzoeksinformatie over de productie van biobutanol uit tarwestro door middel van het aceton-butanol-ethanol (ABE) fermantatieproces.