G.J. Lye

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale‐up

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale‐up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164–176]. In order to enable the predictive scale‐up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale‐up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, Pg/Pug, was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631–2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215–224]. A correlation relating the measured oxygen mass transfer coefficient, kLa, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale‐up studies at matched kLa (0.06–0.11 s−1) and Pg/V (657–2,960 Wu2009m−3) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant kLa was shown to be the most reliable basis for predictive scale‐up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of kLa values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development. Biotechnol. Bioeng. 2008;100: 1144–1155.

pH control in microwell fermentations of S. erythraea CA340: influence on biomass growth kinetics and erythromycin biosynthesis

Abstract The creation of microscale fermentation procedures could have significant benefits at all stages of fermentation process development from discovery through to process optimisation. For both microbial and mammalian fermentations, pH is a vital process parameter as it has a marked affect on cell growth rate, viability and product synthesis. In this work, we describe the influence of various pH control strategies on growth and erythromycin synthesis by Saccharopolyspora erythraea CA340 at the 7xa0l scale and show that the effects can be reproduced in pH-controlled microscale fermentations (thousandfold scale translation). At the 7xa0l scale the implementation of base only or full pH control (NaOH and H3PO4 additions) significantly increased both the maximum growth rate and biomass concentrations attained compared to fermentations without pH control. There was over a twofold increase in erythromycin biosynthesis and the ratio of erythromycin A (EA) to erythromycin C (EC) increased from 2:1 to 6:1 (base only pH control) to 11:1 (full pH control). In order to measure pH during microscale fermentations, a specially designed microtitre plate was built that allowed the insertion of a micro-pH probe into each well (total well volume 7xa0ml). This, allowed manual base only pH control to be implemented in microwell fermentations which enhanced both the maximum specific growth rate and the maximum biomass concentration. Total erythromycin synthesis and the ratio of EA:EC were also significantly enhanced. This work has demonstrated the benefits of implementing pH control in microscale fermentations and now allows the specification of an automated pH control system.

Immobilised enzyme microreactor for screening of multi-step bioconversions: Characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.

“α,α’-Dihydroxy Ketones and 2-Amino-1,3-diols: Synthetic and Process Strategies using Biocatalysts

There is increasing interest in the use of biocatalysts in synthetic applications due to their ability to achieve highly stereoselective and atom efficient conversions. Recent developments in molecular biology techniques and synthetic biology have also enhanced potential applications using non-natural substrates. Here we review strategies to alpha,alpha-dihydroxy ketones and 2-amino-1,3-diols via the use of transketolase, a carbon-carbon bond forming biocatalyst, and the enzyme transaminase which converts aldehydes and ketones to amines. Using an integrated strategy we have investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with miniaturization studies and modelling to achieve rapid and predictive process scale-up.

Design and Characterization of a Prototype Enzyme Microreactor: Quantification of Immobilized Transketolase Kinetics

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200‐μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His6‐tagged enzymes via Ni‐NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop‐flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK‐catalysed synthesis of L‐erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis–Menten model. Results show that the TK kinetic parameters in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol min–1, Km(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the kcat for the microreactor of 4.1 × 105 s−1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His6‐immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell‐based systems for TK bioprocess characterization.

Extraction of erythromycin-A using colloidal liquid aphrons: Part II. Mass transfer kinetics

An emerging technique for the recovery of microbial secondary metabolites, such as antibiotics, is the use of colloidal liquid aphrons (CLAs) in pre-dispersed solvent extraction (PDSE) processes. Knowledge of the extraction kinetics and the limiting mass transfer resistances will be vital for efficient process design and operation. In this work, the rates of erythromycin extraction using CLAs and conventional aqueous-organic, two-phase systems have been investigated. The CLAs used were formulated from 1% w/v Softanol 120 in decanol and 0.5% w/v SDS in water. The rate of erythromycin extraction with CLAs dispersed in well-mixed systems was found to be extremely rapid with equilibrium being achieved within 15 s or less. Overall erythromycin mass transfer coefficients, K-o, were typically 6.3 x 10(-6) m s(-1) for extraction experiments, and 1.0 x 10(-6) m s(-1) for stripping experiments. The rapid rates of erythromycin transfer were attributed to the small size of the dispersed CLAs, typically 5 mum diameter, and hence the large interfacial area available for mass transfer of around 15 x 10(3) m(2) m(-3). Experiments over a range of Reynolds numbers indicated that both the extraction and stripping processes were probably under mixed control of an interfacial resistance and boundary-layer diffusion. To investigate the influence of the surfactants used for aphron formulation on erythromycin extraction, further experiments were performed using aqueous-organic, two-phase systems in a non-dispersive stirred cell. The measured K-o values were of the same order of magnitude as in the case of experiments with dispersed CLAs. When present individually, or together with SDS, the non-ionic Softanol surfactant was seen to retard erythromycin extraction rates in all cases. In contrast, the presence of SDS was found to enhance K-o values by a factor of 2-4 compared to surfactant-free systems. This may be attributed to a specific interaction between individual SDS acid erythromycin molecules and/or the generation of interfacial turbulence at a microscopic level. Further investigations are underway to elucidate the mechanism responsible. The concentration of either surfactant was also found to significantly affect the measured K-o values. The prediction of K-o values using existing correlations, and the implications of the experimental results on contactor design and operation are also discussed

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

Abstract This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.

Quantification of kinetics for enzyme-catalysed reactions: implications for diffusional limitations at the 10 ml scale

The effects of different reaction scales [100xa0μl reactions in 96-standard round well (SRW) plates and 10xa0ml reactions in 24-square well (SW) plates] have been investigated using, as a model, transketolase (TK)-catalysed reaction producing l-erythrulose. Reactions were carried out under non-shaking, shaking and at 10xa0ml scale stirring conditions to assess the effect of diffusional limitations. Statistical analysis confirmed the significance of the observed difference in reaction rates under given conditions. Only when the laboratory scale system (10xa0ml) was well mixed did the reaction rate become comparable to that in the microwells, where there is negligible diffusional limitation. These findings have important implications for the scale-up (or scale-down) of enzyme-catalysed reactions.

Thermal profiling for parallel on-line monitoring of biomass growth in miniature stirred bioreactors

Recently we have described the design and operation of a miniature bioreactor system in which 4–16 fermentations can be performed (Gill etxa0al., Biochem Eng J 39:164–176, 2008). Here we report on the use of thermal profiling techniques for parallel on-line monitoring of cell growth in these bioreactors based on the natural heat generated by microbial culture. Results show that the integrated heat profile during E.xa0coli TOP10 pQR239 fermentations followed the same pattern as off-line optical density (OD) measurements. The maximum specific growth rates calculated from off-line OD and on-line thermal profiling data were in good agreement, at 0.66xa0±xa00.04 and 0.69xa0±xa00.05xa0h−1 respectively. The combination of a parallel miniature bioreactor system with a non-invasive on-line technique for estimation of culture kinetic parameters provides a valuable approach for the rapid optimisation of microbial fermentation processes.

Process Selection and Characterisation for the Biocatalytic Hydration of Poorly Water Soluble Aromatic Dinitriles

The biotransformation of poorly water soluble aromatic dinitriles is of industrial and scientific interest. Although processes do exist for the transformation of water soluble nitriles, such as acrylonitrile, no description of a process suitable for the large scale biotransformation of poorly water soluble nitriles appears in the literature. In this work we illustrate a systematic design procedure for optimising the production of 3-cyanobenzamide from 1,3-dicyanobenzene (1,3-DCB). The regio-selective nitrile hydratase (NHase) of the well characterised Rhodococcus R312 strain was initially selected as catalyst. Isolation of the NHase at process scale however was not feasible due to the rigid cell wall of the bacteria and the poor stability of the isolated enzyme. The whole cell form of the biocatalyst was thus used even though the activity of the associated amidase could overmetabolise the amide product into the corresponding acid. To overcome productivity limitations imposed by the characteristically low aqueous solubility of this class of substrate (∼0.34gl-1 in the case of 1,3-DCB) the use of an aqueous-organic two-phase bioreactor was investigated. After screening a wide range of solvents to act as a substrate reservoir toluene was selected as the organic phase due to the most favourable combination of LogP value (2.9) and 1,3-DCB saturation concentration (∼30gl-1). The effects of phase volume ratio (0.05-0.3), wet weight biomass concentration (1.25 — 200gwwl-1) and substrate concentration in the organic phase (5-25 gl-1) were then combined in a process map to define a suitable operating window where the maximum space-time yield of amide formation could be obtained. Compared to a single-phase transformation, the two-phase process yielded 12 times as much of the amide product of which less than 8% w/w was lost due to overmetabolism.