The bioprocess TEA calculator: An online technoeconomic analysis tool to evaluate the commercial competitiveness of potential bioprocesses

Techno-economic analysis connects R&D, engineering, and business. By linking process parameters to financial metrics, it allows researchers to understand the factors controlling the potential success of their technologies. In particular, metabolic and bioprocess engineering, as disciplines, are aimed at engineering cells to synthesize products with an ultimate goal of commercial deployment. As a result it is critical to be able to understand the potential impact of strain engineering strategies and lab scale results on commercial potential. To date, while numerous techno-economic models have been developed for a wide variety of bioprocesses, they have either required process engineering expertise to adapt and/or use or do not directly connect financial outcomes to potential strain engineering results. Despite the clear value of techno-economic analysis, these challenges have made it inaccessible to many researchers. I have developed this online calculator (https://bioprocesstea.com OR http://bioprocess-tea-calculator.herokuapp.com/) to make the basic capabilities of early-stage techno-economic analysis of bioprocesses readily accessible. The tool, currently focused on aerobic fermentation processes, can be used to understand the impact of fermentation level metrics on the commercial potential of a bioprocess for the production of a wide variety of organic molecules. Using the calculator, I review the commercially relevant targets for an aerobic bioprocess for the production of diethyl malonate.     

Bioprocess engineering: now and beyond 2000

Abstract: Bioprocess engineering may be defined as the translation of life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is a critical link from discovery to commercialization. Current bioprocess engineering is primarily focused on biopharmaceutical products of high dollar value per gram such as erythropoietin or growth hormones. However, other products of current interest include ethanol, amino acids, organic acids, antibiotics, and specialty chemicals. Current challenges for increased use of bioprocesses for producing bulk and semi-bulk chemicals include both technical and infrastructural barriers. Technical barriers are easy to identify and at times can be overcome by engineering improvements or changes brought about radical developments in science (e.g. recombinant DNA). Infrastructural barriers, such as raw-material substitutions or educational limitations are more difficult to define and change. Recently the National Academy of Sciences examined barriers to bioprocess engineering and issued a report entitled: "Putting Biotechnology to Work: Bioprocess Engineering". A key recommendation was the establishment of a coordinated long-range plan of research, development, training and education in bioprocess engineering involving participation by industry, academe and the federal government. The report was the first national analysis devoted entirely to bioprocess engineering and covered new topics such as space bioprocess engineering. Other topics covered by the author include the current state of the US chemical industry and future directions in three promising areas of bioprocess engineering environmental bioprocess engineering, marine bioprocess engineering and microsystem bioprocess engineering.     

Apoptosis: A mammalian cell bioprocessing perspective

Apoptosis is a form of programmed and controlled cell death that accounts for the majority of cellular death in bioprocesses. Cell death affects culture longevity and product quality; it is instigated by several stresses experienced by the cells within a bioreactor. Understanding the factors that cause apoptosis as well as developing strategies that can protect cells is crucial for robust bioprocess development. This review aims to a) address apoptosis from a bioprocess perspective; b) describe the significant apoptotic mechanisms linking them to the most relevant stresses encountered in bioreactors; c) discuss the design of operating conditions in order to avoid cell death; d) focus on industrially relevant cell lines; and e) present anti-apoptosis strategies including cell engineering and model-based optimization of bioprocesses. In addition, the importance of apoptosis in quality-by-design bioprocess development from clone screening to production scale are highlighted.     

Cytochromes P450: exploiting diversity and enabling application as biocatalysts

The remarkable chemical reactivity and substrate range displayed by cytochromes P450 (P450s) renders them attractive as potential catalysts for a host of challenging chemical reactions in industry. The opportunities afforded by these biocatalysts are increased by the availability of greater diversity provided by the genomic resource and the variant libraries of well-known P450s produced by rational and random engineering techniques. The exploitation of this enormous diversity will require novel tools in screening, to identify enzyme reactions of interest, and also in the enabling of these valuable activities through protein engineering and bioprocess optimisation.     

Machine learning in bioprocess development: from promise to practice

Fostered by novel analytical techniques, digitalization, and automation, modern bioprocess development provides large amounts of heterogeneous experimental data, containing valuable process information. In this context, data-driven methods like machine learning (ML) approaches have great potential to rationally explore large design spaces while exploiting experimental facilities most efficiently. Herein we demonstrate how ML methods have been applied so far in bioprocess development, especially in strain engineering and selection, bioprocess optimization, scale-up, monitoring, and control of bioprocesses. For each topic, we will highlight successful application cases, current challenges, and point out domains that can potentially benefit from technology transfer and further progress in the field of ML.     

Estimating environmental impacts of early-stage bioprocesses

The use of bioprocesses in industrial production promises resource- and energy-efficient processes starting from renewable, nonfossil feedstocks. Thus, the environmental benefits must be demonstrated, ideally in the early development phase with standardized methods such as life cycle assessment (LCA). Herein we discuss selected LCA studies of early-stage bioprocesses, highlighting their potential and contribution to estimating environmental impacts and decision support in bioprocess development. However, LCAs are rarely performed among bioprocess engineers due to challenges such as data availability and process uncertainties. To address this issue, recommendations are provided for conducting LCAs of early-stage bioprocesses. Opportunities are identified to facilitate future applicability, for example, by establishing dedicated bioprocess databases that could enable the use of LCAs as standard tools for bioprocess engineers.     

Extracellular vesicles concentration is a promising and important parameter for industrial bioprocess monitoring

Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.     

A new microfluidic concept for parallel operated milliliter-scale stirred tank bioreactors

Parallel miniaturized stirred tank bioreactors are an efficient tool for "high-throughput bioprocess design." As most industrial bioprocesses are pH-controlled and/or are operated in a fed-batch mode, an exact scale-down of these reactions with continuous dosing of fluids into the miniaturized bioreactors is highly desirable. Here, we present the development, characterization, and application of a novel concept for a highly integrated microfluidic device for a bioreaction block with 48 parallel milliliter-scale stirred tank reactors (V = 12 mL). The device consists of an autoclavable fluidic section to dispense up to three liquids individually per reactor. The fluidic section contains 144 membrane pumps, which are magnetically driven by a clamped-on actuator section. The micropumps are designed to dose 1.6 μL per pump lift. Each micropump enables a continuous addition of liquid with a flow rate of up to 3 mL h(-1) . Viscous liquids up to a viscosity of 8.2 mPa s (corresponds to a 60% v/v glycerine solution) can be pumped without changes in the flow rates. Thus, nearly all feeding solutions can be delivered, which are commonly used in bioprocesses. The functionality of the first prototype of this microfluidic device was demonstrated by double-sided pH-controlled cultivations of Saccharomyces cerevisiae based on signals of fluorimetric sensors embedded at the bottom of the bioreactors. Furthermore, fed-batch cultivations with constant and exponential feeding profiles were successfully performed. Thus, the presented novel microfluidic device will be a useful tool for parallel and, thus, efficient optimization of controlled fed-batch bioprocesses in small-scale stirred tank bioreactors. This can help to reduce bioprocess development times drastically.     

Industrial bioprocess control and optimization in the context of systems biotechnology

The developments of the systems biotechnology and its application in the industrial process open up new horizons to industrial biotechnology. The unprecedented understanding of the relationships between cellular behaviors and the surrounding environments during the bioprocess has been achieved. In this paper, we review new advances in the strain improvement, bioprocess control and optimization. The holistic viewpoints and ideas applied in industrial bioprocesses and their future prospects are discussed by illustrating some successful cases.     

Temporally segmented modelling: a route to improved bioprocess monitoring using near infrared spectroscopy?

Near infrared spectroscopy (NIRS) was used to monitor an industrial bioprocess for the production of the antibiotic, tylosin, using a segmented modelling approach. Models were built over the entire time course of the fermentation from 0 to 150 h, and also in two distinct phases or segments of the bioprocess from 50 to 100 h (synthetic phase) and from 100 to 150 h (stationary phase). All models were validated externally and the performance of the full range and segmented models compared. The standard error of prediction (SEP) of the segmented models was less in both 50–100 h and 100–150 h and the correlation highest in the 50–100 h range. This would suggest that data segmentation is potentially a useful method of accommodating the impact of the pronounced matrix changes which occur in some bioprocesses in NIRS models for key analytes. While there are many reports on bioprocess monitoring using NIRS, there have been no previous studies on the use of segmented NIR models within a bioprocess as a means of accommodating matrix change.     

Reflections on biosystems motivating supramolecular engineering

Some goals of bioelectronics—interfacing biology and electronics — are the understanding of supramolecular bioprocesses and the construction of supramolecular devices. The principles for the design and fabrication of machineries with functional components of molecular size are inspired by reflecting on biosystems, and it seems important to consider such principles. We first discuss attempts to construct supramolecular machines, and then we consider the bacterial reaction centre as an example where supramolecular engineering helps to elucidate a bioprocess. We then discuss possible mechanisms leading to the emergence of life-like systems in the light of the basic principles used to design supramolecular devices. Finally, we reflect on prospects in molecular engineering inspired by studying the emergence of life-like systems.     

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d -1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.     

Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

Background  

In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP), which provides high-throughput at minimal expenses in time, money and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high-throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalabilty of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a micro-scale. In order to evaluate the scale-up from a microtiter plate scale (200 μL) to a stirred tank fermenter scale (1.4 L), two standard microbial expression systems, Escherichia coli and Hansenula polymorpha, were fermented in parallel at both scales and compared with regard to the biomass and protein formation.     

Applications of PAT‐Process Analytical Technology in Recombinant Protein Processes with Escherichia coli

Monitoring of bioprocesses and thus observation and identification of such processes is one of the main aims of bioprocess engineering. It is of vital importance in bioprocess development to improve the overall productivity by avoiding unintentional limitations to ensure not only optimal process conditions but also the observation of established production processes. Furthermore, reproducibility needs to be improved and final product quality and quantity be guaranteed. Therefore, an advanced monitoring and control system has been developed, which is based on different in‐line, on‐line and at‐line measurements for substrates and products. Observation of cell viability applying in‐line radio frequency impedance measurement and on‐line determination of intracellular recombinant target protein using the reporter protein T‐Sapphire GFP based on in‐line fluorescence measurement show the ability for the detection of critical process states. In this way, the possibility for the on‐line recognition of optimal harvest times arises and disturbances in the scheduled process route can be perceived.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

Analyzing and understanding the robustness of bioprocesses

The robustness of bioprocesses is becoming increasingly important. The main driving forces of this development are, in particular, increasing demands on product purities as well as economic aspects. In general, bioprocesses exhibit extremely high complexity and variability. Biological systems often have a much higher intrinsic variability compared with chemical processes, which makes the development and characterization of robust processes tedious task. To predict and control robustness, a clear understanding of interactions between input and output variables is necessary. Robust bioprocesses can be realized, for example, by using advanced control strategies for the different unit operations. In this review, we discuss the different biological, technical, and mathematical tools for the analysis and control of bioprocess robustness.     

On a general model structure for macroscopic biological reaction rates

Macroscopic modelling of bioprocesses requires the determination of a biological reaction scheme and a kinetic model. The a priori selection of an appropriate kinetic model structure is usually made difficult by the lack of detailed bioprocess knowledge and the profusion of apparently similar biological kinetic laws. Moreover, parameter identification is made arduous and time-consuming by the strong non-linearities involved in kinetic laws. In most cases, these kinetic structures are non-linearizable and no first parameter estimation can be deduced easily. In order to avoid such identification problems, Bogaerts et al. [Bogaerts, Ph., Castillo, J., Hanus, R., 1999. A general mathematical modelling technique for bioprocesses in engineering applications. Syst. Anal. Model. Simul. 35, 87-113] have developed a general linearizable kinetic structure which allows the representation of activation and/or inhibition effects of each component in the culture. This paper further generalizes this structure in order to improve the way saturation effects are taken into account, and in turn, improve the biological interpretation of the model parameters. The main advantage of the proposed structure lies in an associated systematic estimation procedure. The usefulness of the proposed model is tested with simulated as well as with experimental data.     

Regulatory and metabolic network of rhamnolipid biosynthesis: Traditional and advanced engineering towards biotechnological production

During the last decade, the demand for economical and sustainable bioprocesses replacing petrochemical-derived products has significantly increased. Rhamnolipids are interesting biosurfactants that might possess a broad industrial application range. However, despite of 60 years of research in the area of rhamnolipid production, the economic feasibility of these glycolipids is pending. Although the biosynthesis and regulatory network are in a big part known, the actual incidents on the cellular and process level during bioreactor cultivation are not mastered. Traditional engineering by random and targeted genetic alteration, process design, and recombinant strategies did not succeed by now. For enhanced process development, there is an urgent need of in-depth information about the rhamnolipid production regulation during bioreactor cultivation to design knowledge-based genetic and process engineering strategies. Rhamnolipids are structurally comparable, simple secondary metabolites and thus have the potential to become instrumental in future secondary metabolite engineering by systems biotechnology. This review summarizes current knowledge about the regulatory and metabolic network of rhamnolipid synthesis and discusses traditional and advanced engineering strategies performed for rhamnolipid production improvement focusing on Pseudomonas aeruginosa. Finally, the opportunities of applying the systems biotechnology toolbox on the whole-cell biocatalyst and bioprocess level for further rhamnolipid production optimization are discussed.     

Prospects for an HIV Vaccine: Conventional Approaches and DNA Immunization

Abstract

Microbial transglutaminase is an important enzyme in food processing for improving protein properties by catalyzing the cross-linking of proteins. Recently, this enzyme has been shown to exhibit wider potential application in tissue engineering, textiles and leather processing, site-specific protein conjugation and wheat gluten allergy reduction. The production of microbial transglutaminase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering during the last three decades. More recently, studies on the biological mechanism of transglutaminase synthesis have further contributed towards the understanding of microbial transglutaminase production by Streptomyces. This will further facilitate improving the production of recombinant microbial transglutaminase. In this paper, we will review the progress in bioprocess engineering and genetic engineering in microbial transglutaminase production. We will highlight our understanding of the biological mechanisms of microbial transglutaminase synthesis, including biotechnological approaches used based on these biological mechanisms as a way of improving transglutaminase production.We address in addition the future research needs for microbial transglutaminase production.