Scaling DNA data storage with nanoscale electrode wells.

Synthetic DNA is an attractive medium for long-term data storage because of its density, ease of copying, sustainability, and longevity. Recent advances have focused on the development of new encoding algorithms, automation, preservation, and sequencing technologies. Despite progress in these areas, the most challenging hurdle in deployment of DNA data storage remains the write throughput, which limits data storage capacity. We have developed the first nanoscale DNA storage writer, which we expect to scale DNA write density to 25 × 106 sequences per square centimeter, three orders of magnitude improvement over existing DNA synthesis arrays. We show confinement of DNA synthesis to an area under 1 square micrometer, parallelized over millions of nanoelectrode wells and then successfully write and decode a message in DNA. DNA synthesis on this scale will enable write throughputs to reach megabytes per second and is a key enabler to a practical DNA data storage system.

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.