Continuous Sugar Beet Pulp Pretreatment and Bioconversion in a Biorefinery Context

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

Thermomechanical Study of a New Concept of Beryllium Protected High Heat Flux Component

Abstract not availableJRC.(IAM)-Institute For Advanced Material

Analysis of Artemisia annua extracts and related products by high performance liquid chromatography-tandem mass spectrometry coupled to sample treatment miniaturisation

Artemisinin, the main antimalarial compound of Artemisia annua L., is currently attracting increasing interest for its antiproliferative properties, but its content is highly variable, depending on several genetic, environmental and processing conditions. Aim of the present study is to analyse the artemisinin content in different plant extracts, to test their in vitro activity on cell proliferation and then to correlate these data to the active principle concentration. For this purpose, an innovative miniaturised sample pretreatment strategy based on microextraction by packed sorbent (MEPS) was developed and coupled to an original advanced method based on liquid chromatography with diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). The method was fully validated, granting consistent data. Good linearity was found over a suitable concentration range, i.e. 5\u20131000ng/mL. Extraction yields (>85%), precision (RSD < 3.5%) and accuracy (recovery 88\u201393%) were all within acceptable levels of confidence. After validation, the method was successfully applied to the determination of artemisinin in A. annua extracts. Analyte content was widely variable (up to twenty-fold) according to the starting material and the extraction procedure, ranging between 5.9\u3bcg/g and 109\u3bcg/mL. The cytotoxic activity of all analysed extracts was also tested on human leukemic cells by viable cell count and cell cycle analysis. Artemisinin concentrations and biological activity were carefully evaluated and the observed antiproliferative effects varied according to artemisinin content in each extract type. This highlights the need to quantitatively analyse the main active constituent of plant extracts and the obtained data have shown to be promising for the choice of the related herbal product dosage

From STRs to SNPs via ddRAD-seq: Geographic assignment of confiscated tortoises at reduced costs

Assigning individuals to their source populations is crucial for conservation research, especially for endangered species threatened by illegal trade and translocations. Genetic assignment can be achieved with different types of molecular markers, but technical advantages and cost saving are recently promoting the shift from short tandem repeats (STRs) to single nucleotide polymorphisms (SNPs). Here, we designed, developed, and tested a small panel of SNPs for cost-effective geographic assignment of individuals with unknown origin of the endangered Mediterranean tortoise Testudo hermanni. We started by performing a ddRAD-seq experiment on 70 wild individuals of T. hermanni from 38 locations. Results obtained using 3182 SNPs are comparable to those previously obtained using STR markers in terms of genetic structure and power to identify the macro-area of origin. However, our SNPs revealed further insights into the substructure in Western populations, especially in Southern Italy. A small panel of highly informative SNPs was then selected and tested by genotyping 190 individuals using the KASP genotyping chemistry. All the samples from wild populations of known geographic origin were genetically re-assigned with high accuracy to the original population. This reduced SNPs panel represents an efficient molecular tool that enables individuals to be genotyped at low cost (less than euro15 per sample) for geographical assignment and identification of hybrids. This information is crucial for the management in-situ of confiscated animals and their possible re-allocation in the wild. Our methodological pipeline can easily be extended to other species

Additional file 2 of Unveiling a hotspot of genetic diversity in southern Italy for the endangered Hermann’s tortoise Testudo hermanni

Supplementary Material

Additional file 1 of Unveiling a hotspot of genetic diversity in southern Italy for the endangered Hermann’s tortoise Testudo hermanni

Supplementary Material