Chiocchio, Roberto; (2021) Continuous Sugar Beet Pulp Pretreatment and Bioconversion in a Biorefinery Context. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Valorisation of agricultural wastes, such as Sugar Beet Pulp (SBP), for production of biofuels and value-added chemicals, has garnered increasing interest in recent years. Through physicochemical means, lignocellulosic material can be pretreated to release monosacharides which can then be upgraded by fermentative and biocatalytic routes. Previous UCL-led research has examined many aspects of utilisation of waste streams from sugar refineries. Vinasse, a glycerol-rich waste product of bioethanol production, was used as a nutrient source for enzyme production. Sugars from SBP, such as D-glucose, L-arabinose and D-galacturonic acid and which make up approximately 25% w/w, 21% w/w, and 20% w/w of the total pulp weight, respectively, were solubilised through operations such as steam explosion pretreatment and depolymerisation of the released polysaccharides. These SBP monosaccharides were then employed in bioconversion reactions using thermostable enzymes. This Thesis aims to study SBP as a feedstock for the enzymatic production of value added chemicals. It also aims to translate key reactions in the valorisation process from batch mode into a continuous flow process in a scalable, 100 mL, Agitated Cell Reactor (ACR). Initial Residence Time Distribution characterisation of the ACR showed that it provided excellent plug flow properties, equivalent to 13 stirred reactors in series. The ACR was able to handle SBP slurries over a range of solids loadings (1% w/v – 5% w/v) and residence times (3.8 min – 19.0 min). The SBP suspension was shown to be shear thinning with measured viscosities in the range of 0.0011 Pa.s at 1% w/v and 0.0339 Pa.s at 10% w/v. A set of correlations was developed that enable prediction of the feed viscosity as a function of SBP concentration and shear rate. The SBP particle size distribution ranged from 15.0 μm (D10) to 446 μm (D90) with a median size of 128 μm. Studies on the particle flow through the ACR demonstrated that steady state could be achieved, but that larger particles had longer residence times than smaller particles through the ACR. Dilute acid pretreatment (DAP) of SBP was investigated as an alternative to previous work on steam explosion as it would be more compatible with continuous operation. DAP using sulfuric acid at concentrations up to 75 mM and 80 °C was performed. These conditions showed good release of polymeric L-arabinose, which increased with higher temperatures and acid concentrations (70% w/w at 75 mM and 80 °C). Cellulose, which is more heat- and acid-resistant than SBP pectin, was only slightly hydrolysed into D-glucose, creating the potential for selective sugar fractionation. When compared to steam explosion pretreatment, flow DAP in the ACR obtained similar throughputs (3.5 and 3.1 g(L-arabinose).hr⁻¹, respectively), but productivity (throughput in terms of reactor volume) was an order of magnitude higher (3.5 and 25.6 g(L-arabinose).L⁻¹.hr⁻¹). Monomerisation of the polymeric L-arabinose could be achieved in a continuous flow enzyme-membrane as in previously described work. Finally, valorisation of the L-arabinose monomers by a continuous-flow, two-step enzymatic process in the ACR was demonstrated. L-gluco-heptulose is a rare ketoheptose which has potential cancer and diabetes treatment applications. The one-pot two-step production of L-gluco-heptulose using a thermostable transaminase (TAm) and transketolase (TK) both isolated from Deinococcus geothermalis DSM11300 was also carried out in the ACR. The initial goal was to use immobilized TK and TAm enzymes in order to intensify the bioconversion process. While TK could be successfully immobilized on both Nickel-chelated beads and Epoxymethacrylate resin, the TAm immobilization proved challenging with only low levels of retained activity. Consequently, the flow studies were performed with soluble TK and TAm enzymes. ACR bioconversions compared favourably with well-mixed batch reactions yields using the same reaction time (2 hours). Initial studies demonstrated the conversion of model substrates L-arabinose, L-serine and α-ketoglutaric acid into L-gluco-heptulose. Subsequently it was shown that L-gluco-heptulose could be synthesised equally well using SBP-derived L-arabinose. Concentrations of the intermediate product hydroxypyruvic acid (HPA) and L-gluco-heptulose obtained in continuous mode were 2.62 mM and 0.60 mM, respectively using SBP derived L-arabinose and 1.21 mM and 0.31 mM, respectively, using model solutes. This was equivalent throughputs of 170.5 µM.hr⁻¹ and 39.0 µM.hr⁻¹ for the SBP derived L-arabinose and 81.0 µM.hr⁻¹ and 20.0 µM.hr⁻¹ for the model solutes. Higher final L-gluco-heptulose concentrations could be obtained by increasing starting L-arabinose concentrations. The continuous process demonstrated here has clear potential for use within a SBP biorefinery. Future work needs to focus on alternative methods of TAm immobilization to enable process intensification and scale-up to pilot scale in order to demonstrate robust commercial operation.

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