John M. Woodley

Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme can improve its stability and enable its reuse multiple times, resulting in better performance and commercial viability. Consequently, biocatalysis is being widely applied in the production of pharmaceuticals and some commodity chemicals. Moreover, its broader application will be further stimulated in the future by the emerging biobased economy.

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.

Biocatalysis is an emerging area of technology, and to date few reports have documented the economics of such processes. As it is a relatively new technology, many processes do not immediately fulfill the economic requirements for commercial operation. Hence, early-stage economic assessment could be a powerful tool to guide research and development activities in order to achieve commercial potential. This study discusses the cost contribution of the biocatalyst in processes that use isolated enzymes, immobilized enzymes, or whole cells to catalyze reactions leading to the production of chemicals. A methodology for rapidly estimating the production cost of the biocatalyst is presented, and examples of how the cost of the biocatalyst is affected by different parameters are given. In particular, it is seen that the fermentation yield in terms of final achievable cell concentration and expression level as well as the production scale are crucial for decreasing the total cost contribution of the biocatalyst. Moreover, it is clear that, based on initial process performance, the potential to reduce production costs by several orders of magnitude is possible. Guideline minimum productivities for a feasible process are suggested for different types of processes and products, based on typical values of biocatalyst and product costs. Such guidelines are dependent on the format of the biocatalyst (whole-cell, soluble enzyme, immobilized enzyme), as well as product market size and value. For example commodity chemicals require productivities in the range 2000−10000 kg product/kg immobilized enzyme, while pharmaceutical products only require productivities around 50−100 kg product/kg immobilized enzyme.

The pharmaceutical industry requires synthetic routes to be environmentally compatible as well as to fulfill the demands of process economics and product specification and to continually reduce development times. Biocatalysis has the potential to deliver ‘greener’ chemical syntheses, and in this review some of these opportunities are outlined and outstanding challenges presented. Future development will require research targeted towards increased commercial availability of key enzymes, as well as the improvement of enzyme stability and substrate repertoire, to fully realize the potential of biocatalysis for making pharmaceutical processes greener. The pharmaceutical industry requires synthetic routes to be environmentally compatible as well as to fulfill the demands of process economics and product specification and to continually reduce development times. Biocatalysis has the potential to deliver ‘greener’ chemical syntheses, and in this review some of these opportunities are outlined and outstanding challenges presented. Future development will require research targeted towards increased commercial availability of key enzymes, as well as the improvement of enzyme stability and substrate repertoire, to fully realize the potential of biocatalysis for making pharmaceutical processes greener.

Biocatalytic processes continue to find increasing application in industry. Therefore enzyme immobilization has also become of increasing importance as a means of allowing enzyme containment within reactors operating in continuous mode or else separation of enzyme after use in (fed-)batch reactors, as well as potential recycle. Whilst much has been reported in the scientific literature about enzyme immobilization methods, in many cases the protocol leads to losses in enzyme activity. In this review we outline the reasons for loss of activity during immobilization and highlight suitable diagnostic tests to elucidate the precise cause and thereby methods to restore activity. The need for standardized reporting of immobilization methods is also emphasized as a means of benchmarking alternative approaches.

The aerobic oxidation of 5-hydroxymethylfurfural, a versatile biomass-derived chemical, is examined in water with a titania-supported gold-nanoparticle catalyst at ambient temperature (30 °C). The selectivity of the reaction towards 2,5-furandicarboxylic acid and the intermediate oxidation product 5-hydroxymethyl-2-furancarboxylic acid is found to depend on the amount of added base and the oxygen pressure, suggesting that the reaction proceeds via initial oxidation of the aldehyde moiety followed by oxidation of the hydroxymethyl group of 5-hydroxymethylfurfural. Under optimized reaction conditions, a 71% yield of 2,5-furandicarboxylic acid is obtained at full 5-hydroxymethylfurfural conversion in the presence of excess base.

Abstract The synthesis of 5‐(hydroxymethyl)furfural (HMF) in ionic liquids is a field that has grown rapidly in recent years. Unique dissolving properties for crude biomass in combination with a high selectivity for HMF formation from hexose sugars make ionic liquids attractive reaction media for the production of chemicals from renewable resources. A wide range of new catalytic systems that are unique for the transformation of glucose and fructose to HMF in ionic liquids has been found. However, literature examples of scale‐up and process development are still scarce, and future research needs to complement the new chemistry with studies on larger scales in order to find economically and environmentally feasible processes for HMF production in ionic liquids. This Minireview surveys important progress made in catalyst development for the synthesis of HMF in ionic liquids, and proposes future research directions in process technology.

Abstract Biocatalytic transamination is being established as key tool for the production of chiral amine pharmaceuticals and precursors due to its excellent enantioselectivity as well as green credentials. Recent examples demonstrate the potential for developing economically competitive processes using a combination of modern biotechnological tools for improving the biocatalyst alongside using process engineering and integrated separation techniques for improving productivities. However, many challenges remain in order for the technology to be more widely applicable, such as technologies for obtaining high yields and productivities when the equilibrium of the desired reaction is unfavorable. This review summarizes both the process challenges and the strategies used to overcome them, and endeavors to describe these and explain their applicability based on physiochemical principles. This article also points to the interaction between the solutions and the need for a process development strategy based on fundamental principles. Biotechnol. Bioeng. 2011; 108:1479–1493. © 2011 Wiley Periodicals, Inc.

Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field.

Despite the expanding presence of microscale technology in chemical synthesis and energy production as well as in biomedical devices and analytical and diagnostic tools, its potential in biocatalytic processes for pharmaceutical and fine chemicals, as well as related industries, has not yet been fully exploited. The aim of this review is to shed light on the strategic advantages of this promising technology for the development and realization of biocatalytic processes and subsequent product recovery steps, demonstrated with examples from the literature. Constraints, opportunities, and the future outlook for the implementation of these key green engineering methods and the role of supporting tools such as mathematical models to establish sustainable production processes are discussed.

Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed.

With the current pressing need to rise to the ambition of net zero targets to mitigate carbon emissions and climate change impacts, sustainable processing has never been more critical. Bioprocessing has all the desirable attributes to respond to the sustainable processing challenge: use of cheap, renewable resources, nature-inspired, highly selective biocatalysts operating optimally under mild conditions and reduced energy consumption/carbon footprint. With bioprocessing productivity being far from ideal to meet the large-scale need for food, drugs, biofuels and bio-based chemicals, there has been tremendous interest of late in developing intensified bioprocesses, with significant advancement achieved in tailoring and utilising the technologies in the toolbox traditionally applied in chemical process intensification. This review highlights the wide range of activities currently on-going in bioprocess intensification, focusing on upstream, bioreactor/fermentation and downstream separation steps. Great strides have been made in biocatalyst engineering and high density cell immobilisation for significant productivity enhancement, which, in conjunction with elegant process innovations such as novel bioreactor technologies and in-situ product separations, are enabling bioprocesses to become more competitive than ever before. The future prospects of bioprocess intensification are promising but there are still challenges that need to be overcome to fully exploit this technology.

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.

Biocatalytic processes for the manufacture of small, highly functionalized molecules frequently have limited productivity. A common reason for this is the presence of the reaction products that can cause inhibitory or toxic effects (making poor use of the enzyme) or promote unfavourable equilibria (giving low conversions). In each case, the product needs to be removed as soon as it is formed in order to overcome these constraints and hence increase the productivity of the biocatalytic process. Here, we review the need for in situ product removal and the process research required for its implementation.

Biocatalysis is coming of age, with an increasing number of reactions being scaled-up and developed. The diversity of reactions is also increasing and oxidation reactions have recently been considered for scale-up to commercial processes. One important chemical conversion, which is difficult to achieve enantio- or enantiotopo- selectively, is the Baeyer-Villiger (BV) oxidation of ketones. Using cyclohexanone monooxygenase to catalyse the reaction produces optically pure esters and lactones with exquisite enantiomeric excess values. Recently, these enzymes and their many applications in synthetic chemistry have been explored. The scale-up of these conversions has been examined with the idea of implementing the first commercial Baeyer-Villiger monooxygenase-based process. Here, we review the state-of-the-art situation for the scale-up and exploitation of these enzymes.

In recent years, process intensification (PI) has attracted considerable academic interest as a potential means for process improvement, to meet the increasing demands for sustainable production. A variety of intensified operations developed in academia and industry creates a large number of options to potentially improve the process but to identify the set of feasible solutions for PI in which the optimal can be found takes considerable resources. Hence, a process synthesis tool to achieve PI would potentially assist in the generation and evaluation of PI options. Currently, several process design tools with a clear focus on specific PI tasks exist. Therefore, in this paper, the concept of a general systematic framework for synthesis and design of PI options in hierarchical steps through analyzing an existing process, generating PI options in a superstructure and evaluating intensified process options is presented. For each step, different tools and methods will be needed. In this paper, a knowledge base tool storing and retrieving necessary information/data about intensified processes/equipments has been highlighted including metrics for performance evaluation. The application of the main concepts is illustrated through an example involving the operation of a membrane reactor.

The development of biorefineries means that a key feedstock for many new processes will be sugars in various forms, such as glucose or fructose. From these feedstocks a range of chemicals can be synthesized using heterogeneous catalysis, immobilized enzymes, homogeneous catalysts, soluble enzymes, fermentations or combinations thereof. This presents a particularly interesting process integration challenge since the optimal conditions for each conversion step will be considerably different from each other. Furthermore, compared to oil-based refineries the feedstock represents a relatively high proportion of the final product value and therefore yield and selectivity in these steps are of crucial importance. In this paper using the conversion of glucose to 2,5-furandicarboxylic acid and associated products as an example, alternative routes will be compared with respect to achievable selectivity, and achievable yield.

In this paper we propose a biorefinery optimization model that can be used to find the optimal processing route for the production of ethanol, butanol, succinic acid and blends of these chemicals with fossil fuel based gasoline. The approach unites transshipment models with a superstructure, resulting in a Mixed Integer Non-Linear Program (MINLP). We consider a specific problem based on a network of 72 processing steps (including different pretreatment steps, hydrolysis, fermentation, different separations and fuel blending steps) that can be used to process two different types of feedstock. Numerical results are presented for four different optimization objectives (maximize yield, minimize costs, minimize waste and minimum fixed cost), while evaluating different cases (single product and multi-product).

Biocatalysis has been attracting increasing interest in recent years. Nevertheless, most studies concerning biocatalysis have been carried out using single enzymes (soluble or immobilized). Currently, multiple enzyme mixtures are attractive for the production of many compounds at an industrial level. In this review, a classification of multienzyme-catalyzed processes is proposed. Special emphasis is placed on the description of multienzyme ex-vivo systems where several reactions are carried out by a combination of enzymes acting outside the cell. Furthermore, reaction and process considerations for mathematical modeling are discussed for the specific case where the synthetic reactions are carried out in a single reactor, the so-called multienzyme 'in-pot' process. In addition, options for multienzyme 'in-pot' process improvements via process engineering and enzyme immobilization technology are described. Finally, enzyme modification via protein engineering is also discussed, such that a better compatibility of the enzymes in the reactor is achieved as a means of assisting the implementation of multienzyme 'in-pot' processes.

Studies on the HCl-catalysed microwave-assisted dehydration of highly concentrated aqueous fructose (27 wt%) to 5-hydroxymethylfurfural (HMF) revealed a significant increase in the fructose conversion rate over the conventional heated systems. Water, being the most benign solvent and therefore ideal for green and sustainable chemistry, normally is a poor solvent for the dehydration process resulting in low HMF selectivities and yields. However, reaction at 200 degrees C with microwave irradiation with a short reaction time of only 1s resulted in good HMF selectivity of 63% and fructose conversion of 52%, while prolonged irradiation for 60s (or more) resulted in nearly full fructose conversion (95%) but lower HMF yield (53%). Decreasing the fructose concentration significantly improved the HMF selectivity, but possibly made the production route less attractive from an industrial point of view due to the resultant low throughput.

Scientific progress in the field of enzyme modification today enables the opportunity to tune a given biocatalyst for a specific industrial application. Much work has been focused on extending the substrate repertoire and altering selectivity. Nevertheless, it is clear that many new forthcoming opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required concentrations, and stability. Finally, future research directions are discussed, including the integration of biocatalysis with neighboring chemical steps.

Abstract This paper summarizes the main findings of a round‐table discussion held to examine the key bottlenecks in the further application and industrial implementation of in‐situ product removal (ISPR) techniques.** It is well established that ISPR can yield great benefits for processes limited by inhibitory or toxic products, as well as unstable products or reactions that are thermodynamically unfavorable. However, several issues for industrial implementation were revealed in the discussion. Most notably implementation will be dependent on (1) research into the appropriate process structure, (2) methods to achieve process robustness, (3) systematic selection methods for separation operations and (4) the nature of the product market. Here, these four issues will be discussed as a basis for future work in this area. Copyright © 2007 Society of Chemical Industry

With increasing emphasis on renewable feed-stocks and green chemistry, biocatalytic processes will have an important role in the next generation of industrial processes for chemical production. However, in comparison with conventional industrial chemistry, the use of bioprocesses in general and biocatalysis in particular is a rather young technology. Although significant progress has been made in the implementation of new processes (especially in the pharmaceutical industry) no fixed methods for process design have been established to date. In this paper we present some of the considerations required to scale-up a biocatalytic process and some of the recently developed engineering tools available to assist in this procedure. The tools will have a decisive role in helping to identify bottlenecks in the biocatalytic development process and to justify where to put effort and resources.

Process intensification (PI) has the potential to improve existing as well as conceptual processes, in order to achieve a more sustainable production. PI can be achieved at different levels, that is the unit operations, functional, and/or phenomena level. The highest impact is expected by looking at processes at the lowest level of aggregation which is the phenomena level. In this paper, a phenomena based synthesis/design methodology incorporating process intensification is presented. Using this methodology, a systematic identification of necessary and desirable (integrated) phenomena as well as generation and screening of phenomena based flowsheet options are presented using a decomposition based solution approach. The developed methodology as well as necessary tools and supporting methods are highlighted through a case study involving the production of isopropyl acetate.

In recent years, biocatalysis has started to provide an important green tool in synthetic organic chemistry. Currently, the idea of using multi-enzymatic systems for industrial production of chemical compounds becomes increasingly attractive. Recent examples demonstrate the potential of enzymatic synthesis and fermentation as an alternative to chemical-catalysis for the production of pharmaceuticals and fine chemicals. In particular, the use of multiple enzymes is of special interest. However, many challenges remain in the scale-up of a multi-enzymatic system. This review summarizes and discusses the technology options and strategies that are available for the development of multi-enzymatic processes. Some engineering tools, including kinetic models and operating windows, for developing and evaluating such processes are also introduced.

The pharmaceutical industry is under growing pressure to increase efficiency, both in production and in process development. This paper discusses the central role of Process Systems Engineering (PSE) methods and tools in pharmaceutical process development and innovation, and searches for answers to questions such as: Which PSE methods can be applied readily? Where is more method development needed? The paper covers key subjects for development of economically and environmentally sustainable pharmaceutical processes, including Process Analytical Technology in its broadest sense, continuous pharmaceutical manufacturing and green processes, and is illustrated with a series of short examples taken from the literature and ongoing research projects.

Mechanistic models are based on deterministic principles, and recently, interest in them has grown substantially. Herein we present an overview of mechanistic models and their applications in biotechnology, including future perspectives. Model utility is highlighted with respect to selection of variables required for measurement, control and process design. In the near future, mechanistic models with a higher degree of detail will play key roles in the development of efficient next-generation fermentation and biocatalytic processes. Moreover, mechanistic models will be used increasingly in the frame of multi-objective decision-making under uncertainty and to promote increased selectivity of products. Mechanistic models are based on deterministic principles, and recently, interest in them has grown substantially. Herein we present an overview of mechanistic models and their applications in biotechnology, including future perspectives. Model utility is highlighted with respect to selection of variables required for measurement, control and process design. In the near future, mechanistic models with a higher degree of detail will play key roles in the development of efficient next-generation fermentation and biocatalytic processes. Moreover, mechanistic models will be used increasingly in the frame of multi-objective decision-making under uncertainty and to promote increased selectivity of products.

Synthesis of 5-hydroxymethylfurfural (HMF) from hexoses has been studied extensively in the scientific literature. However, a process has yet to be implemented at industrial scale. In this paper the simultaneous dehydration of glucose and fructose was investigated, in order to develop a process allowing the use of the cheapest available source of fructose: high fructose corn syrup. The dehydration was catalyzed by hydrochloric acid and conducted in acetone–water mixtures, which ensured good selectivity towards HMF and eliminated precipitation of polymer by-products (insoluble humins). Through a detailed experimental investigation a reaction network was proposed, and subsequently the corresponding kinetic model was fitted to experimental data in order to obtain estimates of the reaction kinetic parameters. The kinetic model is capable of predicting the formation of HMF along with the important by-products: soluble humins, glucose dimers, anhydroglucose, and formic acid. The reaction conditions in four different reactor configurations were optimized and compared using the kinetic model. It was found that a recirculating reactor setup is preferable, where the equilibrium controlled by-products (anhydroglucose and glucose dimers) are recirculated to the dehydration reactor. The model predicts an HMF selectivity of close to 70% in a recirculating reactor at conditions where HMF degradation is avoided.

Chemical industry is facing global challenges such as the need to find sustainable production processes. Process intensification as part of process synthesis has the potential to find truly innovative and more sustainable solutions. In this paper, a computer-aided, multi-level, multi-scale framework for synthesis, design and intensification of processes, for identifying more sustainable alternatives is presented. Within the framework, a three stage work-flow has been implemented where, in the first “synthesis” stage an optimal processing route is synthesized through a network superstructure optimization approach and related synthesis tools. In the second, “design” stage, the processing route from the first stage is further developed and a base case design is established and analyzed. In the third, “innovation” stage, more sustainable innovative solutions are determined. The application of the framework is illustrated through a case study related to the production of di-methyl carbonate, which is an important bulk chemical due to its multiplicity of uses.

In this paper, a systematic framework for novel and sustainable synthesis-design of processing routes is presented along with the associated computer-aided methods and tools. In Stage 1, superstructure optimization is used to determine the optimal processing route(s). In Stage 2, the design issues are resolved and targets for improvement are identified through the use of integrated tools. In Stage 3, new alternatives are generated using the selected route and the previously identified targets. In addition to the various computer-aided tools, two special tools are presented: (1) a database employing a specially developed knowledge representation system, and (2) Super-O, a software interface that guides users through the formulation and solution of synthesis problems. Super-O transfers data between the different tools, including a library of generic models, representing a wide range of processing options. Application of the synthesis and design stages is highlighted through two case studies (biorefinery and carbon capture-utilization).

The combination of two cofactor self-sufficient biocatalytic cascade modules allowed the successful transformation of cyclohexanol into the nylon-6 monomer 6-aminohexanoic acid at the expense of only oxygen and ammonia. A hitherto unprecedented carboxylic acid capping strategy was introduced to minimize the formation of the dead-end intermediate 6-hydroxyhexanoic acid. For this purpose, the precursor ε-caprolactone was converted in aqueous medium in the presence of methanol into the corresponding methyl ester instead of the acid. Hence, it was shown for the first time that esterases--specifically horse liver esterase--can perform the selective ring-opening of ε-caprolactone with a clear preference for methanol over water as the nucleophile.

Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in particular, certain insect AFPs show very high antifreeze activity. Recent studies have shown AFPs to be promising candidates as green and environmentally benign inhibitors for gas hydrate formation. Here we show that an insect antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), the most potent protein yet found for freezing inhibition, can inhibit methane hydrates as effectively as the synthetic polymeric inhibitor polyvinylpyrrolidone (PVP). In high pressure rocking cell experiments, onset hydrate nucleation temperatures and growth profiles showed repeatable results. RmAFP1 clearly showed inhibition of hydrates compared to amino acids (l-valine and l-threonine) and the protein bovine serum albumin (BSA). This indicates that proteins or amino acids do not generally inhibit hydrate formation. The promising performance of RmAFP1 as a new green kinetic hydrate inhibitor could further the development and increased production of green hydrate inhibitors.

Today enzymatic catalysis is widely used in the pharmaceutical industry and is expanding fast into the fine and specialty chemicals sector, driven by the need for evermore sustainable chemistry. This review highlights the increasing emphasis now placed on achieving better process performance metrics to reflect the demand for cost-effective and scalable processes, ready for direct implementation into industry. The review also highlights other developments at the frontiers of this field including flow chemistry and multi-step enzymatic reactions, which benefit sustainability.

Biocatalytic oxidation reactions have the potential to substitute many chemically catalyzed oxidations in the pharmaceutical and fine chemical industry due to their superior regio- and stereoselectivity and low environmental impact. Galactose oxidase (GOase) has been shown to be a promising biocatalyst for the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones, respectively. However, GOase requires a number of additives to sustain its catalytic function, such as the enzyme catalase for degradation of the byproduct hydrogen peroxide as well as single-electron oxidants to reactivate the enzyme upon loss of the amino acid radical in its active site. In this work, the addition of catalase, single-electron oxidants, and copper ions was investigated systematically in order to find the minimum concentrations required to obtain a fully active GOase. Furthermore, it was found that the concentration and type of buffer is essential for the activity of GOase, which was significantly more active in sodium phosphate buffer than in other buffers investigated. Enzyme stability and oxygen requirements are of crucial importance for the implementation of oxidase based processes. GOase was shown to be completely stable for 120 h in buffer with stirring at 25 °C, and the activity even increased 30% if the enzyme solution was also aerated in a similar experiment. The high Km for oxygen of GOase (>5 mM) relative to the solubility of oxygen in water reveals a trade-off between supplying oxygen at a sufficiently high rate and ensuring a high degree of enzyme utilization (i.e., ensuring the highest possible specific rate of reaction). Nevertheless, the good stability and high activity of GOase bode well for its future application as an industrial biocatalyst.

Micropollutants present in water have many detrimental effects on the ecosystem. Membrane technology plays an important role in the removal of micropollutants, but there remain significant challenges such as concentration polarization, membrane fouling, and variable permeate quality. The work reported here uses a multifunctional membrane with rejection, adsorption, and catalysis functions to solve these problems. On the basis of mussel-inspired chemistry and biological membrane properties, a multifunctional membrane was prepared by applying “reverse filtration” of a laccase solution and subsequent “dopamine coating” on a nanofiltration (NF) membrane support, which was tested on bisphenol A (BPA) removal. Three NF membranes were chosen for the preparation of the multifunctional membranes on the basis of the membrane properties and enzyme immobilization efficiency. Compared with the pristine membrane, the multifunctional membrane exhibited significant improvement of BPA removal (78.21 ± 1.95%, 84.27 ± 7.30%, and 97.04 ± 0.33% for NT103, NF270, and NF90, respectively), all of which are clearly superior to the conventional Fenton treatment (55.0%) under similar conditions and comparable to soluble laccase coupled with NF270 membrane filtration (89.0%). The improvement would appear to be due to a combination of separation (reducing the enzymatic burden), adsorption (enriching the substrate concentration as well as prolonging the residence time), and lastly, catalysis (oxidizing the pollutants and breaking the “adsorption saturation limits”). Furthermore, the synergistic effect of the polydopamine (PDA) layer on the enzymatic oxidation of BPA was confirmed, which was due to its enhanced adsorption and electron transfer performance. The multifunctional membrane could be reused for at least seven cycles with an acceptable activity loss, demonstrating good potential for removal of micropollutants.

Enzyme catalyzed reactions are rapidly becoming an invaluable tool for the synthesis of many active pharmaceutical ingredients. These reactions are commonly performed in batch, but continuous biocatalysis is gaining interest in industry because it would allow seamless integration of chemical and enzymatic reaction steps. However, because this is an emerging field, little attention has been paid towards the suitability of different reactor types for continuous biocatalytic reactions. Two types of continuous flow reactor are possible: continuous stirred tank and continuous plug-flow. These reactor types differ in a number of ways, but in this contribution, we focus on residence time distribution and how enzyme kinetics are affected by the unique mass balance of each reactor. For the first time, we present a tool to facilitate reactor selection for continuous biocatalytic production of pharmaceuticals. From this analysis, it was found that plug-flow reactors should generally be the system of choice. However, there are particular cases where they may need to be coupled with a continuous stirred tank reactor or replaced entirely by a series of continuous stirred tank reactors, which can approximate plug-flow behavior. This systematic approach should accelerate the implementation of biocatalysis for continuous pharmaceutical production.

A multi-criteria strategy to identify sustainable bio-succinic acid production processes on a commercial scale.

The application of conventional organic solvents has been essential in several steps of bioprocesses in order to achieve sufficient economic efficiency. The use of organic solvents is frequently used either to partly or fully replace water in the reaction medium or as a process aid for downstream separation. Nowadays, manufacturers are increasingly requested to avoid and substitute solvents with hazardous potential. Therefore, the solvent selection must account for potential environmental hazards, health and safety problems, in addition to fulfilling the ideal characteristics for application in a process. For the first time, criteria including Environment, Health and Safety (EHS), as well as the technical requirements for reaction and separation have been reviewed, collected and integrated in a single organic solvent screening strategy to be used as a guideline for narrowing down the list of solvents to test experimentally. Additionally, we have also included a solvent selection guide based on the methodology developed in the Innovative Medicines Initiative CHEM21 (IMI CHEM21) project and applied specifically to water-immiscible solvents commonly used in bioprocesses.

Properties of ionic liquids (ILs) are required for the design of products and processes involving ILs.Although innumerable ILs may be generated through the combination of a variety of cations, anions and substituents, only a small part of them have been reported to exist (have been synthesized).The available experimental data are generally limited and sometimes even contradictory.A detailed knowledge about the properties of ILs is critically important, especially for ILs not yet available.Based on collected experimental data from numerous literature sources, a series of group contribution models have been developed for estimating various properties (density, heat capacity, viscosity, surface tension, melting point) of ILs.To evaluate the predictive capability of the proposed or employed group contribution models, nearly 70% of the datasets (i.e.training sets) are used for correlation, and then the remaining datasets (i.e.test sets) not included in the training sets are used for prediction.The calculation results show that the proposed group contribution models can predict the properties of studied ILs with sufficient accuracy.These property estimation models can both be used easily, and also provide estimation of important properties for previously unstudied ILs, some of which may be considered as potential solvents in many industry applications.

The contents of this article are based on the results of discussions the corresponding author has had since 2015 with the co-authors, who are members of academia and industry in Europe, on the scope and significance of chemical and biochemical engineering as a discipline. The result is a multi-layered view of chemical and biochemical engineering where the inner-layer deals with the fundamental principles and their application; the middle-layer deals with consolidation and expansion of the principles through a combination of science and engineering, leading to the development of sustainable technologies; and the outer-layer deals with integration of knowledge and collaboration with other disciplines to achieve a more sustainable society. Through this multi-layered view several important issues with respect to education, research and practice are highlighted together with current and future challenges and opportunities.

Abstract 5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O 2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

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.

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process (1000μl) for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant kLa values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells (800μl) to conical flasks (100 ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures.

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.

Using biocatalysis for some chemical synthesis steps has unique advantages such as achieving higher product selectivity under ambient process conditions. However, a common limitation with such systems is the inhibition or toxicity posed by the starting substrate as well as limited aqueous solubility in many cases. In this review, we discuss the supply of substrate to bioconversions. The delivery of substrate via an auxiliary, which may be water-miscible, or a second phase such as a water-immiscible organic solvent, adsorbing resin, or a gas, is examined through recent examples in the field. Finally, guidelines for experimental planning and process considerations are suggested to facilitate the choice of substrate delivery method and accelerate process development.

All refineries are characterized by mixed feedstocks and a range of products, but biorefineries in particular can involve a very diverse range of ‘bio’ related material (of variable quality) as a feedstock. The subsequent processing steps may involve heterogeneous catalysis, immobilized enzymes, homogeneous catalysts, soluble enzymes, fermentations or combinations thereof, alongside purification steps. This presents a particularly interesting process integration challenge since the optimal conditions for each process step will be considerably different to each other, there are large temperature gradients through the process and a significant amount of water is used. The basic question to address is thus which product to produce and which sequence of unit operations to apply to obtain the highest profit for the biorefinery. Use of a systematic methodology to analyze and improve processing routes for a specific biorefinery product is therefore a useful first step in the evaluation of the biorefinery product tree. In this paper, we will illustrate the use of a systematic methodology for design and analysis using bioethanol production as an example. More specifically, we will consider a well-known bioethanol production route, analyze it with respect to cost, operation and sustainability, and based on these, generate new alternatives with respect to waste reduction (water) and efficient downstream separation.

The next generation of process engineers will face a new set of challenges, with the need to devise new bioprocesses, with high selectivity for pharmaceutical manufacture, and for lower value chemicals manufacture based on renewable feedstocks. In this paper the current and predicted future roles of process system engineering and life cycle inventory and assessment in the design, development and improvement of sustainable bioprocesses are explored. The existing process systems engineering software tools will prove essential to assist this work. However, the existing tools will also require further development such that they can also be used to evaluate processes against sustainability metrics, as well as economics as an integral part of assessments. Finally, property models will also be required based on compounds not currently present in existing databases. It is clear that many new opportunities for process systems engineering will be forthcoming in the area of integrated bioprocesses.

Abstract Nowadays, production of bulk and commodity chemicals from renewable feedstocks is widely debated and investigated as an alternative to the fossil platform. The conversion of biomass necessitates the development of a new generation of catalysts that enable new kinds of reactions from a different chemical platform under different conditions than those conventionally employed. Indeed, new process and catalyst concepts need to be established. Both enzymatic catalysis (biocatalysis) and heterogeneous inorganic catalysis are likely to play a major role and, potentially, be combined. One type of combination involves one‐pot cascade catalysis with active sites from bio‐ and inorganic catalysts. In this article the emphasis is placed specifically on oxidase systems involving the coproduction of hydrogen peroxide, which can be used to create new in situ collaborative oxidation reactions for bulk chemical production.

Biocatalysis is already established in chemical synthesis on an industrial scale, in particular in the pharmaceutical sector. However, the wider implementation of biocatalysis is currently hindered by the extensive effort required to develop a competitive process. In order that resources spent on development are used in the most efficient manner for these challenging systems, a holistic view on process development and a more in-depth understanding of the underlying constraints (process related as well as biocatalyst related) are required. In this concept article a systematic approach to solve this problem is proposed, involving the use of process tools and methods to assist in development.

Abstract In recent years, much interest has been shown in the use of multi‐enzyme cascades as a tool in organic synthesis. Such enzymatic cascades can provide added value to a synthetic scheme by starting from cheaper raw materials or making more valuable products. Additionally, they can be used to help shift the equilibrium of otherwise thermodynamically unfavourable reactions to give a higher conversion of the target product. By coupling an energetically unfavourable reaction with a more favourable one, the multi‐enzyme cascade mimics the approach taken in nature in metabolic pathways. Nevertheless, it can be challenging to combine several engineered enzymes in vitro for the conversion of non‐natural substrates. In this mini‐review we focus on enzyme coupling reactions as a tool to alleviate thermodynamic constraints in synthetically useful biocatalytic reactions. The implications of thermodynamic parameters such as the equilibrium constant on the multi‐enzyme cascades and the conventional methods of equilibrium shifting are also discussed in addition to methods used to estimate such values.

Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to “test” for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.

A systematic methodology for design of tailor-made blended products has been developed. In tailor-made blended products, one identifies the product needs and matches them by blending different chemicals. The systematic methodology has four main tasks. First, the design problem is defined: the product needs are identified, translated into target properties and the bounds for each target property are defined. Secondly, target property models are retrieved from a property model library. Thirdly, a mixture/blend design algorithm is applied to obtain the mixtures/blends that match the design targets. The result is a set of blends that match the constraints, the composition of the chemicals present in the blend, and the values of the target properties. Finally, the mixture target property values are verified by means of rigorous models for the properties and the mixtures. In this paper, the methodology is highlighted through two case studies involving gasoline blends and lubricant base oils.

The further implementation of new bioprocesses, using biocatalysts in various formats, for the synthesis of chemicals is highly dependent upon effective process intensification. The need for process intensification reflects the fact that the conditions under which a biocatalyst carries out a reaction in nature are far from those which are optimal for industrial processes. In this paper the rationale for intensification will be discussed, as well as the four complementary approaches used today to achieve bioprocess intensification. Two of these four approaches are based on alteration of the biocatalyst (either by protein engineering or metabolic engineering), resulting in an extra degree of freedom in the process design. To date, biocatalyst engineering has been developed independently from the conventional process engineering methodology to intensification. Although the integration of these two methodologies has now started, in the future synergistic integration should enable many new opportunities for bioprocesses for the production of chemicals.

In this study, the biocatalytic membranes were prepared by ‘reverse filtration’ of laccase and subsequently various mussel-inspired coating strategies: single dopamine (DA) deposition, DA/polyethyleneimine (PEI) co-deposition, and DA/Cu2+ co-deposition, where nanofiltration (NF) membranes were used as the matrix to further exploit the potential of the biocatalytic membranes. Such prepared biocatalytic membranes were enzymatically active on both sides, making it possible to construct a bifacial enzymatic membrane reactor (EMR) for highly efficient micro-pollutants removal (taking bisphenol A (BPA) as an example). Compared with the single polydopamine (PDA) coated membrane, the biocatalytic membranes prepared by DA/PEI and DA/Cu2+ co-depositions exhibited much better performances in terms of enzyme loading, activity and permeability as well as the stability of immobilized enzyme. The BPA removal efficiency was highest for the EMR with the PDA/Cu2+ coated membrane attributed to copper-enhanced electron transfer, while it was lowest for the EMR with the PDA/PEI coated membrane due to the high diffusional resistance of the dense PDA/PEI layer. Meanwhile, the mechanism for performance deterioration of biocatalytic membrane during BPA treatment was revealed, and it was found that the trade-off between BPA removal efficiency and long-term stability could be broken by applying the bifacial EMR with PDA/Cu2+ coated membrane in flow-through mode, since the pressure-induced convective mass transfer improved the substrate accessibility to enzyme together with products removal.

Abstract Energy storage will be essential for balancing the renewable energy systems of tomorrow, especially if excess electricity from wind and solar power requires immediate utilization. The use of biogas as a carbon source can generate carbon dioxide‐neutral carbon‐based energy carriers, such as methane or methanol. The utilization of biogas today is limited to the generation of heat/power or biomethane (first‐generation upgrading); both processes disregard the potential of the coproduced carbon dioxide during the fermentation process. By using renewable energy, biogas upgrading systems can convert carbon dioxide into hydrocarbon‐based high‐energy‐density fuels, which can replace fossil‐based fuels for applications in which they are hard to decarbonize. The possibilities for the future utilization of biogas are discussed, and the terminology for “second‐generation upgrading” is introduced to help research and development within this field. It is believed that second‐generation upgrading of biogas will have a huge potential for dynamic energy storage.

As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional measurement techniques focus on a single performance metric (such as the total turnover number), here, it is argued that three metrics (achievable product concentration, productivity, and enzyme stability) are required for an accurate assessment of scalability.

Biocatalysis is a growing area of synthetic and process chemistry with the ability to deliver not only improved processes for the synthesis of existing compounds, but also new routes to new compounds.

Biocatalytic oxidations can offer clear advantages compared to chemically catalyzed oxidations in terms of chemo, regio and stereoselectivity as well as a reduced environmental impact. One of the most industrially important reactions is the oxidation of alcohols, which can be carried out using alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables the use of molecular oxygen as a substrate, generating either water or hydrogen peroxide as a by-product. The use of these enzymes is now gaining an increased interest, and several different enzymes of both types have been applied for proof-of-concept. In this review, we give an overview of the state-of-the-art, and discuss several important issues for future implementation in a production process.

Separation of monosaccharides and monovalent salts is important in the biorefinery and food industry. Nanofiltration (NF) technology is promising for this purpose but its poor selectivity still needs to be addressed. In this work, the separation of monosaccharides and monovalent salts by NF is improved by regulating the surface charge and pore size distribution of polyamide NF membranes. The carboxyl groups on the polyamide membrane are selectively activated by N-(3-Dimethylaminopropyl)-N′-ethyl carbodiimide (EDC) and N-hydroxy succinimide (NHS), and subsequently molecules with amino groups are grafted on the membrane to reduce the effective pore size and electronegativity. The conditions and grafted molecules are optimized, and polyethyleneimine (600 Da) is selected as the best for enhancing the separation of glucose/fructose and KCl. Such targeted modification is found to reduce the effective mean pore size while maintaining the porosity of the NF270 membrane, due to pore segmentation. This results in a remarkable improvement in glucose/fructose rejection (from 67.96% to 84.14%) and separation factor (from 2.20 to 6.78), with only a 4.70% permeability loss. The modified membrane also maintains separation performance in crossflow filtration and after alkaline cleaning (pH 12), which outperforms the pristine NF270 and those modified by mussel-inspired coating and simple physical adsorption.

A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.

The cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871 has been cloned into Escherichia coli in an L-arabinose inducible vector. The recombinant E. coli containing the L-arabinose inducible CHMO was grown at 1.5 litres under controlled conditions to determine the parameters for growth and induction. It was found that induction with 0.1% (w/v) L-arabinose at late logarithmic phase of growth and growth for a further 2.5 to 3 h gave the optimal CHMO titre ( approximately 3500 U.l(-1,) 630 U. g dry cell weight(-1)). High dissolved oxygen concentrations were shown to be deleterious to the CHMO titre. This influenced the strategy for growth and induction, and was optimal when the oxygen uptake rate was maximized but the dissolved oxygen concentration was zero. Finally, a 300 litre scale fermentation was carried out giving a total CHMO titre of >8 x 10(5) U.

Abstract The performance, in Baeyer–Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept‐2‐en‐6‐one into two regioisomeric lactones and of methyl phenyl sulphide into the corresponding ( R )‐sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems tested, 2‐propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half‐life from 1 day in diluted buffer to 1 week in 1 M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half‐life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g·L −1 ) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water‐organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept‐2‐en‐6‐one (10 g·L −1 ) both lactone products with high optical purity (enantiomeric excess ≥96%), which was not the case when all of the substrate was added in a single batch. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 489–496, 2002.

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5- and 55-L fed-batch processes to oxidize bicyclo[3.2.0]hept-2-en-6-one to its corresponding regioisomeric lactones, (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. By employing a bicyclo[3.2.0]hept-2-en-6-one feed rate below that of the theoretical volumetric biocatalyst activity (275 micromol x min(-1) x L(-1)), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2-0.4 g x L(-1). In this way approximately 3.5 g x L(-1) of the combined regioisomeric lactones was produced with a yield of product on reactant of 85-90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5-fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.

Chiral amino alcohols represent an important class of value-added biochemicals and pharmaceutical intermediates. Chemical routes to such compounds are generally step intensive, requiring environmentally unfriendly catalysts and solvents. This work describes a multidisciplinary approach to the rapid establishment of biocatalytic routes to chiral aminodiols taking the original synthesis of (2S,3S)-2-aminopentane-1,3-diol as a specific example. An engineered variant of Escherichi coli transketolase (D469T) was used for the initial asymmetric ynthesis of (3S)-1,3-dihydroxypentan-2-one from the achiral substrates propanal and hydroxypyruvate. A bioinformatics led strategy was then used to identify and clone an ω-transaminase from Chromobacterium violaceum (DSM30191) capable of converting the product of the transketolase-catalysed step to the required (2S,3S)-2-aminopentane-1,3-diol using isopropylamine as an inexpensive amine donor. Experiments to characterize, optimize and model the kinetics of each reaction step were performed at the 1 mL scale using previously established automated microwell processing techniques. The microwell results provided excellent predictions of the reaction kinetics when the bioconversions were subsequently scaled up to preparative scales in batch stirred-tank reactors. The microwell methods thus provide process chemists and engineers with a valuable tool for the rapid and early evaluation of potential synthetic strategies. Overall, this work describes a concise and efficient biocatalytic route to chiral amino alcohols and illustrates an integrated multidisciplinary approach to bioconversion process design and scale-up.

Biocatalytic Baeyer−Villiger oxidations using oxygen as an environmentally friendly oxidant in aqueous media have been shown to proceed with excellent stereo- and enantioselectivity for a large number of substrates at laboratory scale. These are good starting boundary conditions for process research and development compared to systems with reactive oxidants and flammable organic solvents. In this paper we discuss some of the considerations required to scale up a whole-cell biocatalytic oxidation from the laboratory to pilot-plant (200 L) scale. Issues for fermentation, bioconversion, and product recovery are discussed, supported by data from pilot-plant and scale-down experimentation. A simple fed-batch approach has been used.

Three types of oxygenase biocatalysts are treated in detail in this review: the non-haem iron alkene mono-oxygenases, the haem and vanadium haloperoxidases, and flavin-based Baeyer–Villiger mono-oxygenases. Other oxygenases are briefly included for comparison. Characteristics of the biocatalysts are presented, and the scope and limitations concerning their applicability for the selective introduction of oxygen are discussed. Key issues include catalytic activity, productivity, cloning and expression, as well as process engineering aspects. Various bottlenecks are identified for the different biocatalysts and measures to increase the number of oxygenase reactions in practical use are discussed.

A Green Technology Comparison framework incorporating a life cycle approach and sustainability metrics has been used to compare the performance, and the environment, health, safety, and life cycle impacts of two synthetic methods used to produce 7-aminocephalosporic acid (7-ACA). The routes under study were a chemical synthetic process and a two-enzyme catalyzed process, both starting from the potassium salt of cephalosporin C. Cradle-to-gate life cycle impact estimations were performed using the Fast Life Cycle Assessment of Synthetic Chemistry (FLASC™) tool and following modular gate-to-gate methodology. The results compare the synthetic efficiency, environment, health, safety, and life cycle metrics for a mature chemical process and a more recent but less developed enzymatic process for making 7-ACA. The chemical process has a higher yield, but a significantly lower reaction mass efficiency and half the mass productivity of the enzymatic process. The chemical process uses more hazardous materials and solvents and requires about 25% more process energy than the enzymatic process. When accounting for the cradle-to-gate environmental life cycle, the chemical process has a larger overall environmental impact, mainly derived from the production of raw materials. In comparison to the enzyme-catalyzed process, the chemical process uses approximately 60% more energy, about 16% more mass (excluding water), has double the greenhouse gas (GHG) impact, and about 30% higher photochemical ozone creation potential (POCP) and acidification impact.

Immobilized lipases can be used in biodiesel production to overcome many disadvantages of the conventional base-catalyzed process. However, the glycerol by-product poses a potential problem for the biocatalytic process as it is known to inhibit immobilized lipases, most likely by clogging of the catalyst particles. In this paper, this negative effect was further investigated and confirmed in ethanolysis of rapeseed oil. A dyeing method was developed for in situ visualization of glycerol in order to study its partitioning and accumulation during the ethanolysis reaction. The method was used to illustrate the interaction of glycerol with immobilized lipases and thus provided an aid for screening supports for lipase immobilization according to their interaction with glycerol. Glycerol was found to have great affinity for silica, less for polystyrene and no affinity for supports made from polymethylmethacrylate and polypropylene. It was also found that the immobilization of enzyme on the support influenced the adsorption of glycerol to the surface of the enzyme carrier.

Abstract Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C 7 -C 16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C 7 -C 16 n-alkanes and in the activation of a whole-cell alkane biosensor by C 10 and C 11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C 12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 μmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C 10 -C 16 alkanes and represents the highest reported gain in product yields resulting from its use.

In this paper a multi-level, multi-scale framework for process synthesis-intensification that aims to make the process more sustainable than a base-case, which may represent a new process or an existing process, is presented. At the first level (operation-scale) a conceptual base case design is synthesized through the sequencing of unit operations and subsequently analyzed for identifying process hot-spots using economic, life cycle and sustainability metrics. These hot-spots are limitations/bottlenecks associated with tasks that may be targeted for overall process improvement. At the second level (task-scale) a task-based synthesis method is applied where one or more tasks representing unit operations are identified and analyzed in terms of means-ends for generating intensified flowsheet alternatives. At the third level (phenomena-scale) a phenomena-based synthesis method is applied, where the involved phenomena in various tasks are identified, manipulated and recombined to generate new and/or existing unit operations configured into flowsheet alternatives that target the tasks associated with hot-spots. Every lower-scale or higher-level, generates more alternatives than their corresponding larger-scale. Those alternatives that are able to address the identified hot-spots therefore give innovative and more sustainable process designs that otherwise could not be found from the larger-scales. In this paper, membrane-based operations identified through this framework are highlighted in terms of extension of the combined intensification-synthesis method and its application to generate membrane-based operations. Also, application of the framework is illustrated through a case study involving the production of methyl acetate where membrane-based intensified operations play a major role in determining more sustainable process design alternatives.

The application of lipase catalysis to the production of biodiesel has received much interest during the past several years. Although most of the previous work has involved the use of immobilized enzyme, more recent work has indicated that liquid formulations of lipase can provide a highly competitive option for the conversion of oils and fats to biodiesel. This study investigates the impact of several process parameters on the production of fatty acid ethyl esters from rapeseed oil in a pure batch process on the liquid lipase formulation Callera™ Trans L. Oil conversion in excess of 98% was achieved by combining a 50% stoichiometric excess of ethanol (1.5 equivalents) with 20% (w/w) water relative to the oil. The rate of reaction was directly proportional to the amount of lipase added in this system (500–2000 LU per gram oil). Addition of glycerol to the initial reaction mixture reduced the initial reaction rate, but also improved the final yield of biodiesel by suppressing hydrolysis.

Today, some established working fluids are being phased out due to new international regulations on the use of environmentally harmful substances. With an ever-increasing cost to resources, industry wants to converge on improved sustainability through resource recovery, and in particular waste heat recovery. In this paper, an organic Rankine cycle process and its pure working fluid are designed simultaneously for waste heat recovery of the exhaust gas from a marine diesel engine. This approach can overcome design issues caused by the high sensitivity between the fluid and cycle design variables and otherwise high resource demands, which through conventional methods cannot be addressed. The global optimal design was a 1.2 MW cycle with 2,2,3,3,4,4,5,5-octafluorohexane as the new fluid. The fluid has no ozone depletion potential and a global warming potential under the regulatory limit. By using the simultaneous design approach the optimum solution was found in 5.04 s, while a decomposed approach found the same solution in 5.77 h. However, the decomposed approach provided insights on the correlation between the fluid and cycle design variables by analyzing all possible solutions. It was shown that the high sensitivity between the fluid and cycle design variables was overcome by using the simultaneous approach. Correlation between net power output and the product of the overall heat transfer coefficient and the heat transfer area could further be addressed by employing a new solution strategy including maximum constraints for this product. The use of such constraints resulted in the design of a new fluid (5-chloro-4,5,5-trifluoro-2,3-dimethylpent-2-ene) with a 1.25 MW net power output. Finally, a comparison with conventional fluids was shown where 2,2,3,3,4,4,5,5-octafluorohexane offered an improvement on net power output and economic and environmental metrics.

ABSTRACT In this work, we demonstrate the scale‐up from an 80 L fed‐batch scale to 40 m 3 along with the design of a 4 m 3 continuous process for enzymatic biodiesel production catalyzed by NS‐40116 (a liquid formulation of a modified Thermomyces lanuginosus lipase). Based on the analysis of actual pilot plant data for the transesterification of used cooking oil and brown grease, we propose a method applying first order integral analysis to fed‐batch data based on either the bound glycerol or free fatty acid content in the oil. This method greatly simplifies the modeling process and gives an indication of the effect of mixing at the various scales (80 L to 40 m 3 ) along with the prediction of the residence time needed to reach a desired conversion in a CSTR. Suitable process metrics reflecting commercial performance such as the reaction time, enzyme efficiency, and reactor productivity were evaluated for both the fed‐batch and CSTR cases. Given similar operating conditions, the CSTR operation on average, has a reaction time which is 1.3 times greater than the fed‐batch operation. We also showed how the process metrics can be used to quickly estimate the selling price of the enzyme. Assuming a biodiesel selling price of 0.6 USD/kg and a one‐time use of the enzyme (0.1% (w/w oil ) enzyme dosage); the enzyme can then be sold for 30 USD/kg which ensures that that the enzyme cost is not more than 5% of the biodiesel revenue. Biotechnol. Bioeng. 2016;113: 1719–1728. © 2016 Wiley Periodicals, Inc.

The use of non-aqueous organic media is becoming increasingly important in many biotechnological applications in order to achieve process intensification. Such media can be used, for example, to directly extract poorly water-soluble toxic products from fermentations. Likewise many biological reactions require the supply of oxygen, most normally from air. However, reliable online measurements of oxygen concentration in organic solvents (and hence oxygen transfer rates from air to the solvent) has to date proven impossible due to limitations in the current analytical methods.For the first time, online oxygen measurements in non-aqueous media using a novel optical sensor are demonstrated. The sensor was used to measure oxygen concentration in various organic solvents including toluene, THF, isooctane, DMF, heptane and hexane (which have all been shown suitable for several biological applications). Subsequently, the oxygen transfer rates from air into these organic solvents were measured.The measurement of oxygen transfer rates from air into organic solvents using the dynamic method was established using the solvent resistant optical sensor. The feasibility of online oxygen measurements in organic solvents has also been demonstrated, paving the way for new opportunities in process control. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

The ability of enzymes to operate in organic solvent is now widely accepted and is the basis for extensive research in enzymology. The challenge is to select the solvent media that allows the modulation of enzyme activity. For a rational selection of a solvent, it is necessary to understand the effect of organic solvent molecules on enzyme structure and the enzymatic reaction on a molecular level. To gain such insight, we combined experimental kinetics studies with full atomic molecular dynamics simulations and found a correlation between the activity of Candida antarctica lipase B (CALB) [for the esterification reaction between butyric acid and ethanol at a fixed water activity] and the binding of the solvent/substrate molecules in the active site region of CALB. We have investigated the influence of four organic solvents—hexane (HEX), methyl tertiary butyl ether (MTBE), acetonitrile (ACN), and tertiary butanol (TBU)—on the catalytic activity of CALB for the esterification reaction. The solvents have been chosen on the basis of different polarity/functional groups. Our study shows that these organic solvents do not alter the overall conformation of CALB; rather, the solvent effects on the performance of the enzyme may be ascribed to binding of solvent molecules to the enzyme active site region and the solvation energy of substrate molecules in the different solvents. Polar solvent molecules interact strongly with CALB and compete with the substrate to bind to the active site region, resulting in an inhibitory effect which is also confirmed by the binding free energies for the solvent and substrate molecules estimated from the simulations. Consequently, the catalytic activity of CALB decreases in polar solvents. This effect is significant, and CALB is over 10 orders of magnitude more active in nonpolar solvents (HEX and MTBE) than in the polar solvents (ACN and TBU). TBU molecules show an exceptional behavior because the solvent molecule forms an extensive hydrogen bond network within the CALB active site region suggesting that solvent molecules rich on hydrogen bond acceptors and donors are poor solvents when used for lipase-catalyzed esterification reactions.

ABSTRACT Callera™ Trans L, a liquid formulation of Thermomyces lanuginosus lipase, has recently shown great promise as a cost‐efficient catalyst for methanolysis of triglyceride substrates, specifically in the BioFAME process. However, identifying the right combination of temperature and concentrations of catalyst, water and methanol to realize the full potential of the reaction system has remained a challenge. This study presents an investigation of the impact of temperature, enzyme and water concentration on the reaction, as well as the effect of methanol feed rate for the conversion of rapeseed oil in a fed‐batch reaction system. It was observed that the reaction can be divided into two distinct parts. The first part of the reaction, during which primarily tri‐ and diglycerides are converted, proceeded at a high rate and thus required a high rate of methanol supply. The second part of the reaction, where the remaining di‐ and monoglycerides are converted, proceeded at a much lower rate. Consequently, it is necessary to reduce the methanol feed rate during the latter part of the reaction to avoid inhibition or even inactivation of the enzyme. Since the second part of the reaction occupied most of the 24‐h reaction time, it was concluded that this is the part of the process where further development efforts should be targeted. This point was demonstrated by partially substituting the catalyst with a lipase with a different specificity, which enhanced the performance during the second phase of the reaction. Biotechnol. Bioeng. 2014;111: 2446–2453. © 2014 Wiley Periodicals, Inc.

Abstract The increasing industrial interest in biocatalytic processes is predominantly driven by the need for selective chemistry, with high reaction yield (Y reaction ) and few side reactions, as well as the need for optically pure chiral molecules (in particularly in the pharmaceutical industry). Interestingly, it is often argued that the mild conditions frequently used in biocatalytic reactions (ambient temperature and pressure, neutral pH and aqueous-based media) automatically lead to environmentally-friendly and cost-effective production processes. However, such a conclusion is not justified without the use of adequate tools to evaluate the performance of a process, in particular during process development. Nevertheless, at the early development stage, evaluation of biocatalytic processes is not a trivial task, not only due to the lack of data, but also because at this stage many of the biocatalytic processes are not yet fully optimized. Hence, in this paper we propose the use of a range of tools which can be used to guide process development, research tasks and support decision-making. Three sets of metrics are identified, each for use at different stages of process development (route selection, early development and late development), each with different objectives.

ABSTRACT Biocatalytic oxidation reactions employing molecular oxygen as the electron acceptor are difficult to conduct in a continuous flow reactor because of the requirement for high oxygen transfer rates. In this paper, the oxidation of glucose to glucono‐1,5‐lactone by glucose oxidase was used as a model reaction to study a novel continuous agitated cell reactor (ACR). The ACR consists of ten cells interconnected by small channels. An agitator is placed in each cell, which mixes the content of the cell when the reactor body is shaken by lateral movement. Based on tracer experiments, a hydrodynamic model for the ACR was developed. The model consisted of ten tanks‐in‐series with back‐mixing occurring within and between each cell. The back‐mixing was a necessary addition to the model in order to explain the observed phenomenon that the ACR behaved as two continuous stirred tank reactors (CSTRs) at low flow rates, while it at high flow rates behaved as the expected ten CSTRs in series. The performance of the ACR was evaluated by comparing the steady state conversion at varying residence times with the conversion observed in a stirred batch reactor of comparable size. It was found that the ACR could more than double the overall reaction rate, which was solely due to an increased oxygen transfer rate in the ACR caused by the intense mixing as a result of the spring agitators. The volumetric oxygen transfer coefficient, k L a , was estimated to be 344 h −1 in the 100 mL ACR, opposed to only 104 h −1 in a batch reactor of comparable working volume. Interestingly, the large deviation from plug flow behavior seen in the tracer experiments was found to have little influence on the conversion in the ACR, since both a plug flow reactor (PFR) model and the backflow cell model described the data sufficiently well. Biotechnol. Bioeng. 2017;114: 1222–1230. © 2017 Wiley Periodicals, Inc.

In this study the effect of carbonic anhydrase addition on the absorption of CO2 was investigated in a wetted wall column apparatus. Four different solvents: the primary amine monoethanolamine (MEA), the sterically hindered primary amine 2-amino-2-methyl-1-propanol (AMP), the tertiary amine N-methyl-diethanolamine (MDEA) and the carbonate salt solution K2CO3 were compared in concentrations from 5 to 50 wt% in a temperature range of 298–328 K with and without enzyme. Necessary mass transfer parameters such as liquid side mass transfer coefficient and solvent and enzyme reaction rates were determined and benchmarked to a 30 wt% MEA solution. The study reveals that the addition of the enzyme carbonic anhydrase (CA) dramatically increases the liquid side mass transfer coefficient for MDEA, and K2CO3; AMP has a moderate increase whereas MEA was unchanged. The results confirm that just bicarbonate forming systems benefit from CA. The influence of temperature on the enzyme kinetics and mass transfer coefficients is different for different solvent types. A temperature increase resulted in lower liquid side mass transfer coefficient for MDEA and K2CO3 but in a higher coefficient for AMP. The overall first order enzyme reaction rate (s−1) was linearly dependent on enzyme concentration for MDEA and K2CO3 at 313 K. Temperature and concentration did increase the enzymatic rate constant slightly in the concentration range of 5–15 wt% K2CO3 and significantly between 15 and 20 wt%. The enzymatic reaction rate constant for MDEA decreased with temperature, the solvent concentration had a negligible on it. The enzymatic reaction rate for AMP rose with temperature and was higher for lower solvent concentration.

Abstract Enzyme‐mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube‐in‐tube reactor, operated in the low‐dispersed flow regime to generate time‐series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

In process industries, separation techniques need to be employed to match product quality and purity specifications. Most vapour-liquid based separation techniques involving the separation of close-boiling or azeotropic as well as gaseous mixtures are energy intensive. Downstream separations from bioreactors are, on the other hand, difficult because of relatively small amounts of products in large amounts of reactants and carriers such as water. With growing energy and environmental challenges, novel, sustainable and innovative separation techniques are receiving increasing attention. Because of their non-volatility and other tuneable properties, ionic liquids (ILs) based separation techniques are promising alternatives. In this work, a systematic method that combines group contribution (GC)-based property prediction and IL-based separation process design is presented. That is, the optimal IL molecular structure and the corresponding optimal flowsheet configuration for a specific IL-based separation process are simultaneously identified. Case studies involving the separation of azeotropic mixtures such as ethanol-water and acetone-methanol are presented to highlight the application of the method for synthesis-design of the IL-based separation technology.

The design and development of new routes for the production of sustainable bulk-chemicals requires focus on feedstock, conversion technology and downstream product recovery. This brief article discusses some of the constraints with using fermentation and suggests the removal of some constraints by using microbial biocatalysis or enzyme biocatalysis, which give a number of benefits in the context of the requirements for bulk-chemical production. Some potential process concepts are described, for products in the suitable low-price range. These examples (biodiesel, furfurals and amines) are used to illustrate the power of biocatalysis. Suggestions for future research efforts beyond molecular biology, involving process-based concepts, are also discussed.

Abstract Biotechnological processes are typically perceived to be greener than chemical processes. A life cycle assessment (LCA) was performed to compare the chemical and biochemical synthesis of lactones obtained by Baeyer–Villiger oxidation. The LCA is prospective (based on experiments at a small scale with primary data) because the process is at an early stage. The results show that the synthesis route has no significant effect on the climate change impact [(1.65±0.59) kg g product −1 vs. (1.64±0.67) kg g product −1 ]. Key process performance metrics affecting the environmental impact were evaluated by performing a sensitivity analysis. Recycling of solvents and enzyme were shown to provide an advantage to the enzymatic synthesis. Additionally, the climate change impact was decreased by 71 % if renewable electricity was used. The study shows that comparative LCAs can be used to usefully support decisions at an early stage of process development.

Prediction of thermodynamic behavior is essential for the early design stage of separation processes including solvent selection, process optimization and its performance evaluation.In order to better utilize ionic liquids (ILs) as solvents in gas separation processes, the UNIFAC-IL model of IL-liquid solute systems is extended to IL-gas systems by using experimental data from published works and pseudo-experimental data specifically generated from a calibrated COSMO-RS model.In this work, we consider in the model development a total number of 100 ILs from 6 cation families (i.e.imidazolium, pyridinium, pyrrolidinium, ammonium, phosphonium, guanidium) and 24 anion families (i.e.bis(trifluoromethanesulfonyl) amide, tetrafluoroborate, hexafluorophosphate, dimethylphosphate, heptafluorobutyrate, trifluoroacetate, trifluoromethanesulfonate, methylsulfonate, methylsulfate, ethylsulfate, nitrate, p-toluenesulfonate, 2-(2-methoxyethoxy)ethylsulfate, chloride, bromide, dicyanamide, tetracyanoborate, tris(pentafluoroethyl)trifluorophosphate, lactate, levulinate, saccharinate, succinamate, tetrafluoroethanesulfonate, bis(2,4,4-trime-thylpentyl)phosphinate), and 13 gases including CO 2 , SO 2 , H 2 S, NH 3 , N 2 O, CO, N 2 , O 2 , H 2 , CH 4 , C 2 H 4 , C 2 H 6 , C 3 H 8 .The extended UNIFAC-IL-Gas model consists of two sub-models, namely the UNIFAC-IL-Gas (Exp.)model and the UNIFAC-IL-Gas (Pseudo-Exp.)model.The training and testing of the UNIFAC-IL-Gas (Exp.)model is based on 100% experimental data, while the training of the UNIFAC-IL-Gas (Pseudo-Exp.)model is based on pseudo-experimental data, but its testing is also based on 100% experimental data.

Conventional oxidation catalysts frequently exhibit limited selectivity, restricting their use in industrial syntheses. In particular, for the continuous synthesis of complex active pharmaceutical ingredients the high selectivity of biocatalytic oxidations is attractive. However, due to their dependence on gaseous molecular oxygen, which is poorly water-soluble, such reactions are frequently limited by low dissolved oxygen concentrations. In order to better understand how oxygen limitation influences the effectiveness with which an enzyme can be used, the continuous oxidation of glucose into gluconic acid, by glucose oxidase, was studied in a continuous stirred tank reactor. Results showed that a 3-fold increase in the oxygen content of the feed gas improved the reaction rate by twice as much as a 10-fold increase in enzyme concentration, confirming that the reaction is most sensitive to dissolved oxygen concentrations. Therefore, the enzyme could, on average, be used four times more effectively at an enzyme concentration of 0.1 g·L–1 than at 1 g·L–1, due to higher dissolved oxygen concentrations at steady state. However, at feed gas compositions greater than 60% oxygen, reaction rates began to drop due to enzyme deactivation. It was also found that the measurement of mass transfer coefficients is significantly affected by media composition, which may lead to inaccurate predictions of reaction rate.

Enzymes in living cells are highly dynamic but at the same time regularly confined for achieving efficient metabolism. Inspired by this phenomenon, we have prepared a novel biocatalytic membrane with high enzyme activity and stability by tuning the confinement strength of the membrane to enzyme, which was achieved via modifying the support layer of a polymeric nanofiltration (NF) membrane and reversely filtrating enzyme. A mussel-inspired coating was used to modify the support interior of the NF membrane to enhance charge and steric effects on enzyme, thus stabilizing enzyme in the membrane with little increment in mass transfer resistance for substrate and products (only 20% permeability loss with a high enzyme loading of 1.34 mg/cm2). A suitable confinement strength of the membrane to enzyme could delay the enzyme leakage and endow enzyme with certain mobility for efficient reaction. Thus, the obtained biocatalytic membrane exhibited a negligible decline in BPA removal efficiency for 7 reuse cycles (<3.5%) or 36 h continuous operation (<1%) in flow through mode, resulting in a long-term stability adequate for micropollutant removal. For the first time, enzyme mobility was defined and calculated to quantify the confinement strength of the membrane, which could be used to optimize the microenvironment for enzyme immobilization and predict the performance of the biocatalytic membrane. This work concluded that rationally regulating the enzyme mobility in the membrane and a periodic back-flushing operation for redistribution of enzymes could achieve a long-term stable removal of micropollutant in water by a biocatalytic membrane.

Metrics are a useful way to assess biocatalyst performance and, when compared to techno-economic targets, can help set goals for further enzyme and bioprocess research and development. Here, we outline some of the remaining challenges to ensure wider acceptance of this approach, both in industry and in academia.

Biocatalysis offers many attractive features for the synthetic chemist. In many cases, the high selectivity and ability to tailor specific enzyme features via protein engineering already make it the catalyst of choice. From the perspective of sustainability, several features such as catalysis under mild conditions and use of a renewable and biodegradable catalyst also look attractive. Nevertheless, to be sustainable at a larger scale it will be essential to develop processes operating at far higher concentrations of product, and which make better use of the enzyme via improved stability. In this Concept, it is argued that a particular emphasis on these specific metrics is of particular importance for the future implementation of biocatalysis in industry, at a level that fulfills its true potential.

The ability to predict the phase equilibria behavior is of crucial relevance in the early design stage of biphasic liquid-liquid systems. Ionic liquid-based aqueous biphasic systems (IL-ABS) have demonstrated superior performance in many applications such as the recovery of bio-products and the recycling of hydrophilic ILs from aqueous solutions. In order to better utilize these novel biphasic liquid-liquid systems, modelling studies on phase equilibria behavior are carried out in this work. First, the IL database developed in our previous work is extended to these unconventional biphasic systems. In total, 17,449 experimental binodal data points covering 171 IL-ABS at different temperatures (278.15 K-343.15 K) are collected. Then, all involved IL-ABS are correlated using a popular three-parameter mathematical description and the optimal parameters of each IL-ABS are obtained. Afterwards, we try to build a linear group contribution (GC) model to predict the phase equilibria behavior of IL-ABS, but it fails due to the high complexity of these biphasic systems. For this reason, we finally turn to applying a well-known machine learning algorithm, i.e., artificial neural network (ANN), to build a nonlinear GC model for such a purpose. This model gives a mean absolute error (MAE) of 0.0175 and squared correlation coefficient (R2) of 0.9316 for the 13,789 training data points, and for the 3,660 test data points they are 0.0177 and 0.9195, respectively. The results indicate that the proposed nonlinear ANN-GC model, to some extent, is capable to predict the phase equilibria behavior of IL-ABS. Besides the efforts of building GC models, we also discuss some main issues that govern the phase equilibria behavior of IL- ABS, which could be a guidance in the design of IL-ABS.

The inorganic carbon source (CO2) availability to the microorganisms has a key role in the succinic acid (SA) fermentation process. This study aims to investigate the behavior of the inorganic carbon source for SA production and underline the importance of CO2 in the fermentation process. The effect of pressure was investigated for CO2 availability and its overall impact on the fermentation process. Results obtained demonstrated a significant effect of pressure on yield, productivity and CO2 fixation rate. Industrial waste, rich in maltose, glucose, and sucrose, was used as feedstock combined with a gas mixture of CH4 and CO2, which simulated biogas composition. A final titer for succinic acid of 25.5 ± 2.4 g/L was detected at a headspace gas pressure of 1.4 atm. The yield raised from 0.48 g/g sugars to 0.64 and 0.65 g/g sugars when 1.4 and 1.6 atm pressure was applied, respectively. Overall, this study represents one of the first attempts to explore CO2 availability during succinic acid production, using biogas as a CO2 source and sugars-rich industrial waste as feedstock.

A practical procedure is reported for the use of the enzyme transketolase, from Escherichia coli, for asymmetric carbon-carbon bond synthesis. The reactions with the biocatalyst are conveniently carried out, on a gram scale, in unbuffered aqueous media by employing a pH autotitrator. An improved large scale synthesis of hydroxypyruvate is also reported.

Transketolase has been obtained in greater quantities from an over-expressed E. coli transformant carrying the transketolase gene. Crude extracts of this organism are suitable for use in small scale biotransformations to provide mmol quantities of product. Initial results indicate that the transketolase from E. coli is relatively non-specific for the aldehyde component of the reaction.

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min−1 g−1 dry weight) than that of sonicated cells (500 μmol min−1 g−1 dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l−1 and at product concentrations above 4.5–5 g l−1 the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.

In this article, a recombinant cyclohexanone monooxygenase (CHMO), overexpressed in Escherichia coli has been used to study the oxidation of bicyclo[3.2.0]hept-2-en-6-one to its two corresponding lactones at very high enantiomeric excess. The reaction is a useful model for the study of biocatalytic oxidations to create optically pure molecules. The major limitations to a highly productive biocatalytic oxidation in this case are oxygen supply, product inhibition, and biocatalyst stability. In this article, we investigate the effects of whole cell biocatalyst concentration on the rate of reaction at a range of scales from shake flasks to 75 L bioreactors. At low cell concentrations (<2 gdcw/L) the maximum specific rate (0.65 g/gdcw·h) is observed. However, at higher cell concentrations (> 2 gdcw/L), the reaction becomes oxygen limited and both the specific rate and absolute rate decrease with further increases in cell concentration. The role of oxygen limitation in reducing the rate of reaction with scale was investigated by increasing the maximum oxygen transfer rate in the reactor at a high cell concentration and observing the increase in product formation rate. We propose a qualitative model demonstrating the relationship between oxygen limitation, biocatalyst concentration, and the rate of reaction. This conceptual model will be a useful guide in the industrial scale-up of whole cell mediated Baeyer–Villiger biocatalysis. © 2006 Wiley Periodicals, Inc.

Abstract The equilibrium constant is a critical parameter for making rational design choices in biocatalytic transamination for the synthesis of chiral amines. However, very few reports are available in the scientific literature determining the equilibrium constant ( K ) for the transamination of ketones. Various methods for determining (or estimating) equilibrium have previously been suggested, both experimental as well as computational (based on group contribution methods). However, none of these were found suitable for determining the equilibrium constant for the transamination of ketones. Therefore, in this communication we suggest a simple experimental methodology which we hope will stimulate more accurate determination of thermodynamic equilibria when reporting the results of transaminase‐catalyzed reactions in order to increase understanding of the relationship between substrate and product molecular structure on reaction thermodynamics. Biotechnol. Bioeng. 2012; 109:2159–2162. © 2012 Wiley Periodicals, Inc.

Abstract Effective estimation of parameters in biocatalytic reaction kinetic expressions are very important when building process models to enable evaluation of process technology options and alternative biocatalysts. The kinetic models used to describe enzyme‐catalyzed reactions generally include several parameters, which are strongly correlated with each other. State‐of‐the‐art methodologies such as nonlinear regression (using progress curves) or graphical analysis (using initial rate data, for example, the Lineweaver‐Burke plot, Hanes plot or Dixon plot) often incorporate errors in the estimates and rarely lead to globally optimized parameter values. In this article, a robust methodology to estimate parameters for biocatalytic reaction kinetic expressions is proposed. The methodology determines the parameters in a systematic manner by exploiting the best features of several of the current approaches. The parameter estimation problem is decomposed into five hierarchical steps, where the solution of each of the steps becomes the input for the subsequent step to achieve the final model with the corresponding regressed parameters. The model is further used for validating its performance and determining the correlation of the parameters. The final model with the fitted parameters is able to describe both initial rate and dynamic experiments. Application of the methodology is illustrated with a case study using the ω‐transaminase catalyzed synthesis of 1‐phenylethylamine from acetophenone and 2‐propylamine. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012