Practical deployment of automation to expedite aqueous two-phase extraction.

The feasibility of bioprocess development relies heavily on the successful application of primary recovery and purification techniques. Aqueous two-phase extraction (ATPE) disrupts the definition of "unit operation" by serving as an integrative and intensive technique that combines different objectives such as the removal of biomass and integrated recovery and purification of the product of interest. The relative simplicity of processing large samples renders this technique an attractive alternative for industrial bioprocessing applications. However, process development is hindered by the lack of easily predictable partition behaviours, the elucidation of which necessitates a large number of experiments to be conducted. Liquid handling devices can assist to address this problem; however, they are configured to operate using low viscosity fluids such as water and water-based solutions as opposed to highly viscous polymeric solutions, which are typically required in ATPE. In this work, an automated high throughput ATPE process development framework is presented by constructing phase diagrams and identifying the binodal curves for PEG6000, PEG3000, and PEG2000. Models were built to determine viscosity- and volume-independent transfer parameters. The framework provided an appropriate strategy to develop a very precise and accurate operation by exploiting the relationship between different liquid transfer parameters and process error. Process accuracy, measured by mean absolute error, and device precision, evaluated by the coefficient of variation, were both shown to be affected by the mechanical properties, particularly viscosity, of the fluids employed. For PEG6000, the mean absolute error improved by six-fold (from 4.82% to 0.75%) and the coefficient of variation improved by three-fold (from 0.027 to 0.008) upon optimisation of the liquid transfer parameters accounting for the viscosity effect on the PEG-salt buffer utilising ATPE operations. As demonstrated here, automated liquid handling devices can serve to streamline process development for APTE enabling wide adoption of this technique in large scale bioprocess applications.

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation.

BACKGROUND: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. RESULTS: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. CONCLUSIONS: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial 'toolkits' to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry.

Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid.

Large amounts of pectin-rich biomass are generated worldwide yearly, which can be hydrolysed by pectinases to obtain bio-based chemical building blocks such as D-galacturonic acid (GalA). The aim of this work was to investigate thermophilic pectinases and explore their synergistic application in the bioconversion of pectic substrates into GalA. Two exo-polygalacturonases (exo-PGs) from Thermotoga maritima (TMA01) and Bacillus licheniformis (BLI04) and two pectin methylesterases (PMEs) from Bacillus licheniformis (BLI09) and Streptomyces ambofaciens (SAM10) were cloned and expressed in Escherichia coli BL21 (DE3), purified and fully characterised. These pectinases exhibited optimum activity at temperatures above 50 °C and good stability at high temperature (40-90 °C) for up to 24 h. Exo-PGs preferred non-methylated substrates, suggesting that previous pectin demethylation by PMEs was necessary to achieve an efficient pectin monomerisation into GalA. Synergistic activity between PMEs and exo-PGs was tested using pectin from apple, citrus and sugar beet. GalA was obtained from apple and citrus pectin in a concentration of up to 2.5 mM after 4 h reaction at 50 °C, through the combined action of BLI09 PME with either TMA01 or BLI04 exo-PGs. Overall, this work contributes to expand the knowledge of pectinases from thermophiles and provides further insights into their application in the initial valorisation of sustainable pectin-rich biomass feedstocks.

The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. RESULTS: Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. CONCLUSIONS: This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Development of a miniature bioreactor model to study the impact of pH and DOT fluctuations on CHO cell culture performance as a tool to understanding heterogeneity effects at large-scale.

Understanding the impact of spatial heterogeneities that are known to occur in large-scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0-7.5 and DOT 10%-30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale-up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large-scale bioreactors.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.

The roles of diol dehydratase from pdu operon on glycerol catabolism in Klebsiella pneumoniae.

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350 rpm found to be optimal. In the fed-batch fermentation, 9.3 g/L of 1,2-propanediol was produced after 144 h of cultivation, and the substrate conversion ratio was 0.2 g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3 g/L of 1,2-propanediol was produced in fed-batch fermentation.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production.

BACKGROUND: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. RESULTS: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. CONCLUSIONS: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae.

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process. Key Points • A biological route of 2,3-dihydroxyisovalerate production was setup. • Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae. • Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell. • 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation.

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles.

Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine-metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn2+ (6.9-fold), EII was stimulated by Mn2+ (3.7-fold), while EIII was slightly stimulated by Zn2+ , Ca2+ , and Mg2+ . DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2-fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019.

An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes.

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm2 of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.

Continuous enzymatic hydrolysis of sugar beet pectin and l-arabinose recovery within an integrated biorefinery.

Sugar beet pulp (SBP) fractionated by steam explosion, released sugar beet pectin (SB-pectin) which was selectively hydrolysed using a novel α-l-arabinofuranosidase (AF), yielding monomeric l-arabinose (Ara) and a galacturonic acid rich backbone (GABB). AF was immobilised on an epoxy-functionalised resin with 70% overall immobilisation yield. Pretreatment of SB-pectin, to remove coloured compounds, improved the stability of the immobilised AF, allowing its reutilisation for up to 10 reaction cycles in a stirred tank reactor. Continuous hydrolysis of SB-pectin was subsequently performed using a packed bed reactor (PBR) with immobilised AF. Reactor performance was evaluated using a Design of Experiment approach. Pretreated SB-pectin hydrolysis was run for 7 consecutive days maintaining 73% of PBR performance. Continuous separation of Ara from GABB was achieved by tangential flow ultrafiltration with 92% Ara recovery. These results demonstrate the feasibility of establishing a continuous bioprocess to obtain Ara from the inexpensive SBP biomass.

Data on a thermostable enzymatic one-pot reaction for the production of a high-value compound from l-arabinose.

The dataset presented in this article is related to the research article entitled "One-pot, two-step transaminase and transketolase synthesis of l-gluco-heptulose from l-arabinose" (Bawn et al., 2018 in press) [1]. This article presents data on initial experiments that were carried out to investigate new thermostable transketolase (TK) activities with l-arabinose. Transaminase (TAm) sequences from an in-house library of thermophilic strains were analyzed to compare homologies to characterized TAms with desired activity. DNA and amino acid sequences are presented for all the enzymes investigated. Calibration curves for products of the TK and TAm reactions are also presented along with chromatographic analysis of the various one-pot reactions.

One-pot, two-step transaminase and transketolase synthesis of l-gluco-heptulose from l-arabinose.

The use of biocatalysis for the synthesis of high value added chemical building blocks derived from biomass is becoming an increasingly important application for future sustainable technologies. The synthesis of a higher value chemical from l-arabinose, the predominant monosaccharide obtained from sugar beet pulp, is demonstrated here via a transketolase and transaminase coupled reaction. Thermostable transketolases derived from Deinococcus geothermalis and Deinococcus radiodurans catalysed the synthesis of l-gluco-heptulose from l-arabinose and ß-hydroxypyruvate at elevated temperatures with high conversions. ß-Hydroxypyruvate, a commercially expensive compound used in the transketolase reaction, was generated in situ from l-serine and α-ketoglutaric acid via a thermostable transaminase, also from Deinococcus geothermalis. The two steps were investigated and implemented in a one-pot system for the sustainable and efficient production of l-gluco-heptulose.

An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

Centrifugal partition chromatography in a biorefinery context: Optimisation and scale-up of monosaccharide fractionation from hydrolysed sugar beet pulp.

The isolation of component sugars from biomass represents an important step in the bioprocessing of sustainable feedstocks such as sugar beet pulp. Centrifugal partition chromatography (CPC) is used here, as an alternative to multiple resin chromatography steps, to fractionate component monosaccharides from crude hydrolysed sugar beet pulp pectin. CPC separation of samples, prepared in the stationary phase, was carried out using an ethanol: ammonium sulphate (300gL-1) phase system (0.8:1.8v:v) in ascending mode. This enabled removal of crude feedstream impurities and separation of monosaccharides into three fractions (l-rhamnose, l-arabinose and d-galactose, and d-galacturonic acid) in a single step. Throughput was improved three-fold by increasing sample injection volume, from 4 to 16% of column volume, with similar separation performance maintained in all cases. Extrusion of the final galacturonic acid fraction increased the eluted solute concentration, reduced the total separation time by 24% and removed the need for further column regeneration. Reproducibility of the separation after extrusion was validated by using multiple stacked injections. Scale-up was performed linearly from a semi-preparative 250mL column to a preparative 950mL column with a scale-up ratio of 3.8 applied to mobile phase flow rate and sample injection volume. Throughputs of 9.4gL-1h-1 of total dissolved solids were achieved at the preparative scale with a throughput of 1.9gL-1h-1 of component monosaccharides. These results demonstrate the potential of CPC for both impurity removal and target fractionation within biorefinery separations.

Centrifugal partition chromatography in a biorefinery context: Separation of monosaccharides from hydrolysed sugar beet pulp.

A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid. Dimethyl sulfoxide (DMSO) was selected as an effective phase system modifier improving monosaccharide separation. The best phase system identified was ethanol:DMSO:aqueous ammonium sulphate (300gL(-1)) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200mL column) in ascending mode (upper phase as mobile phase) with a mobile phase flow rate of 8mLmin(-1). A mixture containing all four monosaccharides (1.08g total sugars) in the proportions found in hydrolysed SBP was separated into three main fractions; a pure l-rhamnose fraction (>90%), a mixed l-arabinose/d-galactose fraction and a pure d-galacturonic acid fraction (>90%). The separation took less than 2h demonstrating that CPC is a promising technique for the separation of these sugars with potential for application within an integrated, whole crop biorefinery.

Creation of an ultra scale-down bioreactor mimic for rapid development of lignocellulosic enzymatic hydrolysis processes.

BACKGROUND: Cellulosic bioethanol processes involve several steps, all of which require experimental optimisation. A significant aid to this research would be a validated ultra scale-down (USD) model that could be used to perform rapid, wide ranging screening and optimisation experiments using limited materials under process relevant conditions. RESULTS: In this work, the use of 30 mL shaken conical tubes as a USD model for an enzymatic hydrolysis process is established. The approach is demonstrated for the hydrolysis of distillers' dried grains with solubles (DDGS). Results from the USD tubes closely mimic those obtained from 4 L stirred tanks, in terms of the rate, composition and concentrations of sugars released, representing an 80-fold scale reduction. The utility of the USD approach is illustrated by investigating factors that may be limiting hydrolysis yields at high solids loadings. Washing the residual solids periodically during hydrolysis allowed 100% of the available sugar to be hydrolysed using commercially available enzymes. CONCLUSION: The results demonstrate that the USD system reported successfully mimics the performance of conventional stirred tanks under industrially relevant conditions. The utility of the system was confirmed through its use to investigate performance limitation using a commercially relevant feedstock. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Microscale methods to rapidly evaluate bioprocess options for increasing bioconversion yields: application to the ω-transaminase synthesis of chiral amines.

This work aims to establish microscale methods to rapidly explore bioprocess options that might be used to enhance bioconversion reaction yields: either by shifting unfavourable reaction equilibria or by overcoming substrate and/or product inhibition. As a typical and industrially relevant example of the problems faced we have examined the asymmetric synthesis of (2S,3R)-2-amino-1,3,4-butanetriol from l-erythrulose using the ω-transaminase from Chromobacterium violaceum DSM30191 (CV2025 ω-TAm) and methylbenzylamine as the amino donor. The first process option involves the use of alternative amino donors. The second couples the CV2025 ω-TAm with alcohol dehydrogenase and glucose dehydrogenase for removal of the acetophenone (AP) by-product by in situ conversion to (R)-1-phenylethanol. The final approaches involve physical in-situ product removal methods. Reduced pressure conditions, attained using a 96-well vacuum manifold were used to selectively increase evaporation of the volatile AP while polymeric resins were also utilised for selective adsorption of AP from the bioconversion medium. For the particular reaction studied here the most promising bioprocess options were use of an alternative amino donor, such as isopropylamine, which enabled a 2.8-fold increase in reaction yield, or use of a second enzyme system which achieved a 3.3-fold increase in yield.