Towards synthetic microbial consortia for bioprocessing

The use of microbial consortia for bioprocessing has been limited by our ability to reliably control community composition and function simultaneously. Recent advances in synthetic biology have enabled population-level coordination and control of ecosystem stability and dynamics. Further, new experimental and computational tools for screening and predicting community behavior have also been developed. The integration of synthetic biology with metabolic engineering at the community level is vital to our ability to apply system-level approaches to building and optimizing synthetic consortia for bioprocessing applications. This review details new methods, tools and opportunities that together have the potential to enable a new paradigm of bioprocessing using synthetic microbial consortia.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Designing a blueprint for next-generation stem cell bioprocessing development

The creation of a blueprint for stem cell bioprocess development that it is easily readable and shareable among those involved in the construction of the bioprocess is a necessary step toward full-fledged bioprocess integration. The blueprint provides the culturing tools and methodologies, designed to highlight knowledge gaps within biological sciences and bioengineering. This review highlights a blueprint for stem cell bioprocessing development using a landscape architecture approach that can aid the development of culture technologies and tools that satisfy the demands for stem cell-derived products for use in clinical and industrial applications. This work is intended to provide insights to cell biologists, geneticists, bioengineers, and clinicians seeking knowledge outside of their field of expertise and fosters a leap from a reductionist approach to one, that is, globally integrated in stem cell bioprocessing.     

Bio-production of lactobionic acid: Current status,applications and future prospects

Lactobionic acid has appeared on the commercial scene as a versatile polyhydroxy acid with numerous promising applications in the food, medicine, pharmaceutical, cosmetics and chemical industries. This high value-added bio-product has recently received growing attention as a bioactive compound, providing an excellent chemical platform for the synthesis of novel potentially biocompatible and biodegradable drug delivery vehicles. Recent advances in tissue engineering and nanomedicine have also underlined the increased importance of this organic acid as a key biofunctionalization agent. The growing commercial relevance of lactobionic acid has therefore prompted the development of novel systems for its biotechnological production that are both sustainable and efficient. The present review explores recent advances and studies related to lactobionic acid bio-production, whether through microbial or enzymatic approaches, highlighting the key bioprocessing conditions for enhanced bio-production. Detailed overviews of the current microbial cell factories as well as downstream processing methodologies for lactobionic acid production are also presented. Furthermore, the potential prospects and current applications of this polyhydroxy acid are also discussed, with an emphasis on the role of lactobionic acid as a key platform in the development of novel drugs, biomaterials, nanoparticles and biopolymer systems.     

Use of immobilised biocatalysts in the processing of cheese whey

Food processing industry operations need to comply with increasingly more stringent environmental regulations related to the disposal or utilisation of by-products and wastes. These include growing restrictions on land spraying with agro-industrial wastes, and on disposal within landfill operations, and the requirements to produce end products that are stabilised and hygienic. Much of the material generated as wastes by the dairy processing industries contains components that could be utilised as substrates and nutrients in a variety of microbial/enzymatic processes, to give rise to added-value products. A good example of a waste that has received considerable attention as a source of added-value products is cheese whey. The carbohydrate reservoir of lactose (4–5%) in whey and the presence of other essential nutrients make it a good natural medium for the growth of microorganisms and a potential substrate for bioprocessing through microbial fermentation. Immobilised cell and enzyme technology has also been applied to whey bioconversion processes to improve the economics of such processes. This review focuses upon the elaboration of a range of immobilisation techniques that have been applied to produce valuable whey-based products. A comprehensive literature survey is also provided to illustrate numerous immobilisation procedures with particular emphasis upon lactose hydrolysis, and ethanol and lactic acid production using immobilised biocatalysts.     

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.     

常压室温等离子体诱变育种与微生物液滴培养筛选技术应用进展

安全和高效的微生物突变及高通量筛选技术是微生物功能发掘、功能创制和生物产业技术创新的重要方向及重要支撑.有效的生物育种技术及高通量筛选技术成为该领域研究人员的关注点.其中,常压室温等离子体(atmospheric and room temperature plasma,ARTP)因具有活性粒子种类多、操作可控性强、基因...     

Use of in situ solid-phase adsorption in microbial natural product fermentation development

It has been half a century since investigators first began experimenting with adding ion exchange resins during the fermentation of microbial natural products. With the development of nonionic polymeric adsorbents in the 1970s, the application of in situ product adsorption in bioprocessing has grown slowly, but steadily. To date, in situ product adsorption strategies have been used in biotransformations, plant cell culture, the production of biofuels, and selected bulk chemicals, such as butanol and lactic acid, as well as in more traditional natural product fermentation within the pharmaceutical industry. Apart from the operational gains in efficiency from the integration of fermentation and primary recovery, the addition of adsorbents during fermentation has repeatedly demonstrated the capacity to significantly increase titers by sequestering the product and preventing or mitigating degradation, feedback inhibition and/or cytotoxic effects. Adoption of in situ product adsorption has been particularly valuable in the early stages of natural product-based drug discovery programs, where quickly and cost-effectively generating multigram quantities of a lead compound can be challenging when using a wild-type strain and fermentation conditions that have not been optimized. While much of the literature involving in situ adsorption describes its application early in the drug development process, this does not imply that the potential for scale-up is limited. To date, commercial-scale processes utilizing in situ product adsorption have reached batch sizes of at least 30,000 l. Here we present examples where in situ product adsorption has been used to improve product titers or alter the ratios among biosynthetically related natural products, examine some of the relevant variables to consider, and discuss the mechanisms by which in situ adsorption may impact the biosynthesis of microbial natural products.     

Current developments in microbial control of insect pests and prospects for the early 21st century

The role of microbial control in crop and forest protection and the abatement of insects of medical and veterinary importance has expanded considerably with the discovery and development of new microbial control agents and genetic improvement in bacterial and viral pathogens, and improvements in formulation, application options and compatibility with other interventions. A synopsis of the literature regarding the current use of bacteria, viruses, fungi, protozoans and nematodes as microbial control agents is presented along with speculation on their potential in the early 21st century. The most widely used of all microbial control agents isBacillus thuringiensis. The isolation within the past two decades of new strains that are larvicidal for certain Diptera and Coleoptera has increased the utility of the bacterium considerably. Further improvements in efficacy and broadening of its host range are in progress with the isolation of strains with new toxins and the manipulation ofB. thuringiensis genes that encode toxin production using both recombinant and nonrecombinant methods. Genetic manipulation of these genes has also enabled their incorporation into crop plants. The development and commercial availability of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae expands the options for the control of insects, especially those with soil inhabiting stages. The results of natural epizootics of fungi and viruses often obviate the requirement for additional interventions. Breakthroughs in understanding the genetics ofBaculovirus and subsequent gene manipulation have increased their virulence and utility. Improved production methods that utilize insect cell culture technology may enable affordable use ofBaculovirus in the not too distant future. Fungi continue to offer the only control options using entomopathogens against plant sucking insects. Although fungi have great potential for development as microbial control agents, only a few have been used on an operational scale. Some factors that might limit the full range of entomopathogen potential, including development of resistance, are discussed. Because of their selectivity and minimal environmental impact, microbial control agents will be ideal components of integrated pest management programs in the early 21st century and beyond. However, if they are used merely as replacements for chemical pesticides, then eventually these agents will face some of the same fate as the chemicals they replace, particularly with respect to resistance.     

The renaissance of continuous culture in the post-genomics age

The development of continuous culture techniques 60 years ago and the subsequent formulation of theory and the diversification of experimental systems revolutionised microbiology and heralded a unique period of innovative research. Then, progressively, molecular biology and thence genomics and related high-information-density omics technologies took centre stage and microbial growth physiology in general faded from educational programmes and research funding priorities alike. However, there has been a gathering appreciation over the past decade that if the claims of systems biology are going to be realised, they will have to be based on rigorously controlled and reproducible microbial and cell growth platforms. This revival of continuous culture will be long lasting because its recognition as the growth system of choice is firmly established. The purpose of this review, therefore, is to remind microbiologists, particularly those new to continuous culture approaches, of the legacy of what I call the first age of continuous culture, and to explore a selection of researches that are using these techniques in this post-genomics age. The review looks at the impact of continuous culture across a comprehensive range of microbiological research and development. The ability to establish (quasi-) steady state conditions is a frequently stated advantage of continuous cultures thereby allowing environmental parameters to be manipulated without causing concomitant changes in the specific growth rate. However, the use of continuous cultures also enables the critical study of specified transition states and chemical, physical or biological perturbations. Such dynamic analyses enhance our understanding of microbial ecology and microbial pathology for example, and offer a wider scope for innovative drug discovery; they also can inform the optimization of batch and fed-batch operations that are characterized by sequential transitions states.     

Insect cell culture for industrial production of recombinant proteins

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.     

Allocation of ATP to synthesis of cells and hydrolytic enzymes in cellulolytic fermentative microorganisms: bioenergetics, kinetics, and bioprocessing

Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control.     

Bioprocessing of plant cell cultures for mass production of targeted compounds

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered. This paper is dedicated to Prof. Dr. Mladenka P. Ilieva on the occasion of her 70th birthday.     

New era of optogenetics: from the central to peripheral nervous system

Abstract

Optogenetics has recently gained recognition as a biological technique to control the activity of cells using light stimulation. Many studies have applied optogenetics to cell lines in the central nervous system because it has the potential to elucidate neural circuits, treat neurological diseases and promote nerve regeneration. There have been fewer studies on the application of optogenetics in the peripheral nervous system. This review introduces the basic principles and approaches of optogenetics and summarizes the physiology and mechanism of opsins and how the technology enables bidirectional control of unique cell lines with superior spatial and temporal accuracy. Further, this review explores and discusses the therapeutic potential for the development of optogenetics and its capacity to revolutionize treatment for refractory epilepsy, depression, pain, and other nervous system disorders, with a focus on neural regeneration, especially in the peripheral nervous system. Additionally, this review synthesizes the latest preclinical research on optogenetic stimulation, including studies on non-human primates, summarizes the challenges, and highlights future perspectives. The potential of optogenetic stimulation to optimize therapy for peripheral nerve injuries (PNIs) is also highlighted. Optogenetic technology has already generated exciting, preliminary evidence, supporting its role in applications to several neurological diseases, including PNIs.     

土壤微生物在法庭科学领域中的应用研究进展

土壤物证在法庭科学领域的应用由来已久,主要是基于其外观、颜色、元素组成、矿物学等理化特性及土壤夹杂物等的比对检验。近年来,随着高通量测序技术的发展,法医土壤微生物检验不再完全依赖于传统培养技术,而是直接挖掘分析土壤中的全部微生物DNA信息,并将这些信息应用于法庭科学领域内的样本比对、土壤物证区域环境推断和溯源研究等,从而凸显出土壤微生物物证在案件侦查和法庭诉讼方面的巨大价值和应用潜力。本文通过综述国内外有关法医土壤微生物研究的最新进展,指出了土壤微生物多样性检验在法庭科学领域的应用潜力,分析了法医土壤微生物群落多样性的影响因素,最后探讨了法医土壤微生物研究中存在的问题和未来的发展方向。     

Environmental microbiology through the lens of high-throughput DNA sequencing: Synopsis of current platforms and bioinformatics approaches

The incursion of High-Throughput Sequencing (HTS) in environmental microbiology brings unique opportunities and challenges. HTS now allows a high-resolution exploration of the vast taxonomic and metabolic diversity present in the microbial world, which can provide an exceptional insight on global ecosystem functioning, ecological processes and evolution. This exploration has also economic potential, as we will have access to the evolutionary innovation present in microbial metabolisms, which could be used for biotechnological development. HTS is also challenging the research community, and the current bottleneck is present in the data analysis side. At the moment, researchers are in a sequence data deluge, with sequencing throughput advancing faster than the computer power needed for data analysis. However, new tools and approaches are being developed constantly and the whole process could be depicted as a fast co-evolution between sequencing technology, informatics and microbiologists. In this work, we examine the most popular and recently commercialized HTS platforms as well as bioinformatics methods for data handling and analysis used in microbial metagenomics. This non-exhaustive review is intended to serve as a broad state-of-the-art guide to researchers expanding into this rapidly evolving field.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Continuous integrated manufacturing for biopharmaceuticals: A new paradigm or an empty promise?

Continuous integrated bioprocessing has elicited considerable interest from the biopharma industry for the many purported benefits it promises. Today many major biopharma manufacturers around the world are engaged in the development of continuous process platforms for their products. In spite of great potential, the path toward continuous integrated bioprocessing remains unclear for the biologics industry due to legacy infrastructure, process integration challenges, vague regulatory guidelines, and a diverging focus toward novel therapies. In this article, we present a review and perspective on this topic. We explore the status of the implementation of continuous integrated bioprocessing among biopharmaceutical manufacturers. We also present some of the key hurdles that manufacturers are likely to face during this implementation. Finally, we hypothesize that the real impact of continuous manufacturing is likely to come when the cost of manufacturing is a substantial portion of the cost of product development, such as in the case of biosimilar manufacturing and emerging economies.