Frank Baganz

Miniature bioreactors: current practices and future opportunities

This review focuses on the emerging field of miniature bioreactors (MBRs), and examines the way in which they are used to speed up many areas of bioprocessing. MBRs aim to achieve this acceleration as a result of their inherent high-throughput capability, which results from their ability to perform many cell cultivations in parallel. There are several applications for MBRs, ranging from media development and strain improvement to process optimisation. The potential of MBRs for use in these applications will be explained in detail in this review. MBRs are currently based on several existing bioreactor platforms such as shaken devices, stirred-tank reactors and bubble columns. This review will present the advantages and disadvantages of each design together with an appraisal of prototype and commercialised devices developed for parallel operation. Finally we will discuss how MBRs can be used in conjunction with automated robotic systems and other miniature process units to deliver a fully-integrated, high-throughput (HT) solution for cell cultivation process development.

Accelerated design of bioconversion processes using automated microscale processing techniques

Microscale processing techniques are rapidly emerging as a means to increase the speed of bioprocess design and reduce material requirements. Automation of these techniques can reduce labour intensity and enable a wider range of process variables to be examined. This article examines recent research on various individual microscale unit operations including microbial fermentation, bioconversion and product recovery techniques. It also explores the potential of automated whole process sequences operated in microwell formats. The power of the whole process approach is illustrated by reference to a particular bioconversion, namely the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one for the production of optically pure lactones.

Chitosan flocculation to aid the harvesting of the microalga Chlorella sorokiniana

Microalgae are an attractive feedstock for biofuel production, however the harvesting of algal biomass from very large volumes of culture broth represents a major technological and economic challenge. One low cost strategy for addressing this challenge involves the use of flocculation as an initial dewatering step. In this study, flocculation of the green microalga Chlorella sorokiniana was explored in detail using the natural compound, chitosan as flocculant. Results show that clarification efficiency of the process can reach above 99% below pH 7. The optimal chitosan dosage (approximately 10 mg per gram algal dry weight) is determined primarily by cell concentration rather than cell age, lipid content or composition of the medium. Furthermore, the impact of flocculation on the subsequent dewatering process was determined and it was shown to reduce the volume to be processed by 20-50 folds, and significantly reduce energy input and material costs of centrifugation or filtration operations.

Characterization and Application of a Miniature 10 mL Stirred-Tank Bioreactor, Showing Scale-Down Equivalence with a Conventional 7 L Reactor

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred‐tank bioreactor and evaluate its potential as a scale‐down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5α producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen‐limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.

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 W m−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.

Combined remediation and lipid production using Chlorella sorokiniana grown on wastewater and exhaust gases

Substitution of conventional feedstock with waste based alternatives is one route towards both remediation and reducing costs associated with production of algal biomass. This work explores whether exhaust gases and wastewater can replace conventional feedstock in the production of biomass from Chlorella sorokiniana. Exhaust gases were used to augment production in final effluent, anaerobic digester centrate or in standard medium. Cultures were grown in 1L bottles under illumination of 80 μmol m(-2) s(-1). The results showed an average μmax ranging between 0.04 and 0.07 h(-1), whilst the final biomass yield in different media ranged between 220 and 330 mg L(-1). Lipid yield was increased over time to 31 mg L(-1). CO2 addition resulted in complete nitrogen removal between 48 and 96 h in both final effluent and centrate. The results also indicated that levels of carbon monoxide, carbon dioxide and nitrogen oxides in the exhaust gases can be reduced by between 20% and 95%.

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.

Microfluidic approaches for systems and synthetic biology

Microfluidic systems miniaturise biological experimentation leading to reduced sample volume, analysis time and cost. Recent innovations have allowed the application of -omics approaches on the microfluidic scale. It is now possible to perform 1.5 million PCR reactions simultaneously, obtain transcriptomic data from as little as 150 cells (as few as 2 transcripts per gene of interest) and perform mass-spectrometric analyses online. For synthetic biology, unit operations have been developed that allow de novo construction of synthetic systems from oligonucleotide synthesis through to high-throughput, high efficiency electroporation of single cells or encapsulation into abiotic chassis enabling the processing of thousands of synthetic organisms per hour. Future directions include a push towards integrating more processes into a single device and replacing off-chip analyses where possible.

Characterization and multi-step transketolase-ω-transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube

The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral molecules. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase (TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to L-erythrulose (ERY), and the second unit of the IEMR with immobilized TAm converted ERY into ABT using (S)-α-methylbenzylamine (MBA) as amine donor. With initial 60mM (HPA and GA each) and 6mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 min at the lowest flow rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for determination of enzyme kinetics.

Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli

The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 μmol/min/g(dcw)) than on n-octane (21 μmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.