Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing

The growing demand for consumables and energy, combined with increasing consciousness over environmental issues like global warming, faces us with the challenge to find alternatives for fossil resources. Alternative production methods for energy, like windmills, solar panels and hydroelectricity plants, are far developed and have become economically competitive to fossil resourcebased production processes. However, the production of many (bulk) chemicals and products is still dominated by the petroleum industry. One such chemical is lactic acid, a fermentation product of many bacteria and a compound that is gaining interest as a building block for poly lactic acid (PLA). PLA is a polymer used to produce bioplastics, and thereby provides an alternative to petroleumbased plastic production. As described in Chapter 1, economically feasible production of lactic acid is envisioned through consolidated bioprocessing (CBP). In a CBP process, pretreated lignocellulosic biomass is hydrolyzed to fermentable sugars and those sugars are subsequently fermented to desired product in one reaction vessel. The organism of choice for this hydrolyzation and fermentation is preferentially a thermophile, capable of enzyme production and lactic acid fermentation. Species from the genus Geobacillus have many of the desired characteristics, and in Chapter 2 we have enriched and isolated facultative anaerobic (hemi)cellulolytic Geobacillus strains from compost samples. By selecting for growth on both cellulose and xylan, 94 strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible with a transformation efficiency of 1.7×105 CFU/µg of plasmid DNA. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, we demonstrated that strain T12 is capable of direct conversion of beechwood xylan to mainly lactic acid in minimal media. Chapter 3 describes the genome sequencing and several features of G. thermodenitrificans T12. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 kb and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes is similar to those of the denitrification pathways found in strain NG80-2. The host-defence systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system that could potentially be exploited as a genome editing tool for thermophiles. Furthermore, the hemicellulose utilisation (HUS) locus of strain T12 appeared to have orthologues for all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. The HUS-locus associated gene, GtxynA1, encodes an extracellular endoxylanase of strain T12, and belongs to the family 10 glycoside hydrolases (GH10). In Chapter 4, we describe the cloning, expression and characterization of GtXynA1. The recombinant endoxylanase was purified to homogeneity and showed activity between 40°C and 80°C, with an optimum activity at 60°C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60°C. Highest activity was demonstrated towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 can degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endoxylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to be a potential candidate for CBP but lacks the desired endo and exoglucanases needed for the conversion of cellulose. In Chapter 5 we report the heterologous expression of endoglucanases and exoglucanases by G. thermodenitrificans T12, in an attempt to complement the enzymatic machinery of this strain and its suitability for consolidated bioprocessing. A metagenome screen was performed on the metagenome of 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-βglucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having most activity towards p-nitrophenyl-β-dxylopyranoside. Structure modelling of GE40 revealed a protein architecture similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic analysis against 2-HE-cellulose, CM-cellulose and barley β-glucan. In addition, we successfully expressed the earlier characterized Geobacillus sp. 70PC53 endoglucanase celA and the C. thermocellum exoglucanase celK in strain G. thermodenitrificans T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of Geobacillus thermodenitrificans T12 as a host for consolidated bioprocessing. In Chapter 6, we provided more insight in the genetic variation of the hemicellulolytic utilization cluster of G. thermodenitrificans. This variation is far greater than described before and gives ample opportunities for the further development of Geobacillus spp. for hemicellulose degradation. The production of cellulases in Geobacillus species is demonstrated to be successful, and we have expanded on that knowledge with the expression of both endo and exoglucanases from C. thermocellum. However, in line with previous studies, direct cellulose fermentation by geobacilli is not yet achieved, most likely due to insufficient cellulase production and/or secretion. With a rapidly expanding genetic toolbox for thermophiles, now including a thermostable Cas9, we expect that the successful development of Geobacillus spp. for consolidated bioprocessing is just a matter of time.