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

Morphological and biochemical changes in Phaeodactylum tricornutum triggered by culture media: Implications for industrial exploitation

Phaeodactylum tricornutum is a polymorphic marine diatom, displaying three main morphotypes: fusiform, triradiate and oval. It is of great interest for industrial biotechnology as a natural rich source of valuable eicosapentaenoic acid (EPA) and fucoxanthin. Changing culture conditions such as temperature and salinity has been shown to elicit morphological changes in P. tricornutum. However, limited information is available about the conditions that can be used for controlling cell morphology and growth of a particular cell morphotype with high biomass productivity. While the phenomenon of pleiomorphy is intrinsically interesting, there has not been a systematic study linking this behavior to the ability of P. tricornutum to perform as a platform for industrial biotechnology. In this study, the effects of culture medium and culture age on morphological and biochemical changes in P. tricornutum were investigated. Mann and Myers' medium was identified as eliciting significant morphotype conversion from fusiform to oval in P. tricornutum. Liquid cultures containing >90% oval cells were obtained and well-maintained in this medium under constant shaking condition, allowing high dry biomass concentration (0.73 g L−1) to be achieved. Biochemical composition analyses revealed that higher protein (% dry weight) was obtained from oval cell cultures compared to fusiform cell cultures maintained in f/2 medium over 21 days cultivation. Meanwhile, pigment was markedly accumulated in oval cell cultures whereas lipid and carbohydrate were highly accumulated in fusiform cell cultures. This work offered a novel way to regulate cell morphology of P. tricornutum and provided significant implications for upstream cultivation strategies to optimise manufacture of different classes of product in P. tricornutum

Ultra scale-down approaches to enhance the creation of bioprocesses at scale: impacts of process shear stress and early recovery stages

The sensitivity of biological materials to shear stress conditions encountered during large-scale bioprocessing makes successful scale up from the bench challenging. Ultra scale-down technologies seek to use just millilitre quantities to enhance our understanding of the impact of the process environment as a basis for process optimisation. They can help speed translation of new biological discoveries to market and reduce risks encountered in scale up. They are important both as process discovery tools and as preparative tools to yield material for study of subsequent stages. In this review the focus is on the early recovery stages post fermentation or cell culture and in particular the use of continuous-flow and dead-end centrifugation integrated with preparative stages (e.g. flocculation) and subsequent depth filtration. Examples range from therapeutic antibodies, to rationally engineered (synthetic biology) host strains, to stem cells for therapy

Design and parallelisation of a miniature photobioreactor platform for microalgal culture evaluation and optimisation

Miniature photobioreactors (mPBr) represent a potential platform technology for the high-throughput, phototrophic cultivation of microalgae. This work describes the development and characterisation of a novel orbitally shaken twin-well mPBr, and its scale-out to a 24-well microplate format, suitable for optimisation of microalgae culture conditions. Fluid hydrodynamics, oxygen mass transfer coefficient (kLa) and light intensity distribution in the mPBr were first investigated as a function of orbital shaking frequency. High speed video analysis of the shaken wells indicated rapid fluid flow and good mixing while measured kLa values varied between 20 and 80 h−1. Light intensity variation across the scaled-out platform was in the range ±20 μmol m−2 s−1. The use of the mPBr platform was demonstrated for optimisation of conditions for the batch cultivation of Chlorella sorokiniana. Using a modified tris-base phosphate (TBP) medium, the highest biomass concentration and productivity achieved were 9.2 g L−1 and 2.5 ± 0.2 g L−1 d−1 respectively at 5% CO2 with a light intensity of 380 μmol m−2 s−1. In general, cell growth rate and yield increased with increasing shaking frequency (up to 300 rpm) while culture conditions had limited impact on pigment production. Overall, these results demonstrate the application of the mPBr for rapid optimisation of phototrophic culture conditions and establishment of high cell density cultures

Macroalgal biorefinery concepts for the circular bioeconomy: A review on biotechnological developments and future perspectives

The imminent need for transition to a circular bioeconomy, based on the valorisation of renewable biomass feedstocks, will ameliorate global challenges induced by climate change, environmental pollution and population growth. A reduced reliance on depleting fossil fuel resources and ensured production of eco-friendly and cost-effective bioproducts and biofuels, requires the development of sustainable biorefinery processes, with many utilising macroalgae as feedstock, showing promising and viable prospects. Nonetheless, macroalgal biorefinery research is still in its infancy compared to lignocellulosic biorefineries that utilise terrestrial plants. This article presents a review on the latest scientific literature associated with the development and status of macroalgal biorefineries, and how bioproducts generated from these bioprocesses have contributed towards the bioeconomy. The fundamental need to understand how the unique biochemical composition of macroalgae fit within a biorefinery concept are explained, alongside discussion of the novel biotechnologies that have been applied. In order to comprehend the increasing significance of this exciting field, the review will also provide insight, for the first time, on the current global funding and intellectual property landscape related to macroalgae and their implementation across the entire biorefinery concept. Imperative areas for further research and development, to bridge the gap between fundamental bioscience in the laboratory and the successful application of compatible biotechnologies at a commercial scale, to boost the macroalgae industry are also covered

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 Dei nococcus radiodurans catalysed the synthesis of L-gluco-heptulose from L-arabinose and β-hydroxypyruvate at elevated temperatures with high conversions. β-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

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity

High-throughput measurement of protein stability in microtiter plates

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C-1/2 +/- 0.05 M, m(G), and DeltaG(H2)O) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability. (C) 2005 Wiley Periodicals, Inc

Engineering characterisation of a shaken, single-use photobioreactor for early stage microalgae cultivation using Chlorella sorokiniana.

This work describes the characterisation and culture performance of a novel, orbitally shaken, single-use photobioreactor (SUPBr) system for microalgae cultivation. The SUPBr mounted on an orbitally shaken platform was illuminated from below. Investigation of fluid hydrodynamics indicated a range of different flow regimes and the existence of 'in-phase' and 'out-of-phase' conditions. Quantification of the fluid mixing time (tm) indicated a decrease in tm values with increasing shaking frequency up to 90 rpm and then approximately constant tm values in the range 15-40 s. For batch cultivation of Chlorella sorokiniana, the highest biomass concentration achieved was 6.6 g L(-1) at light intensity of 180 μmol m2 s(-1). Doubling the total working volume resulted in 35-40% reduction in biomass yield while shaking frequency had little influence on culture kinetics and fatty methyl esters composition. Overall this work demonstrates the utility of the SUPBr for early stage development of algal cultivation processes

Potential of sugar beet vinasse as a feedstock for biocatalyst production within an integrated biorefinery context

BACKGROUND: This work explores the feasibility of vinasse as an inexpensive feedstock for industrial biocatalyst production within the context of an integrated sugar beet biorefinery. As an exemplar, production of CV2025 ω-Transaminase (ω-TAm) in Escherichia coli BL21 was studied. RESULTS: Characterisation of vinasse showed that it comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 cell growth and CV2025 ω-TAm production were feasible in cultures using 17% to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The d-galactose present in vinasse facilitated auto-induction of the pQR801 plasmid enabling CV2025 ω-TAm expression without addition of expensive Isopropyl-β-d-thiogalactopyranoside (IPTG). Assessment of different vinasse pre-processing options confirmed simple dilution of the vinasse was sufficient to reduce the concentration of polyphenols to below inhibitory levels. Optimisation experiments, carried out using a controlled, 24-well microbioreactor platform, showed supplementation of diluted vinasse medium with 10 g L^{−1} yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, maximum biomass concentration, CV2025 ω-TAm volumetric and specific activity, respectively. Investigation into the metabolic preferences of E. coli BL21 when grown in vinasse showed a preference for D-mannitol utilisation before simultaneous metabolism of glycerol, d-xylitol, d-dulcitol and acetate. Scale-up of optimised conditions for batch CV2025 ω-TAm production to a 7.5 L stirred tank reactor (STR) was demonstrated based on matched volumetric mass transfer coefficient (kLa). The results showed good comparability with respect to cell growth, substrate consumption and CV2025 ω-TAm production representing over a 700-fold volumetric scale translation. Further enhancements in CV2025 ω-TAm production were possible in the STR when operated at higher k_{L}a values. CONCLUSION: This work describes the promising application of vinasse for production of microbial enzymes and insights into carbon source utilisation in complex feedstocks. Exploitation of vinasse as a fermentation feedstock could be further extended to other processes involving different microorganisms and target enzymes