Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability

The biotech industry is, nowadays, facing unparalleled challenges due to the enhanced demand for biotechnology-based human therapeutic products, such as monoclonal antibodies (mAbs). This has led companies to improve substantially their upstream processes, with the yield of monoclonals increasing to titers never seen before. The downstream processes have, however, been overlooked, leading to a production bottleneck. Although chromatography remains the workhorse of most purification processes, several limitations, such as low capacity, scale-related packing problems, low chemical and proteolytic stability and resins' high cost, have arisen. Aqueous two-phase extraction (ATPE) has been successfully revisited as a valuable alternative for the capture of antibodies. One of the important remaining questions for this technology to be adopted by the biotech industries is, now, how it compares to the currently established platforms in terms of costs and environmental impact. In this report, the economical and environmental sustainability of the aqueous two-phase extraction process is evaluated and compared to the currently established protein A affinity chromatography. Accordingly, the ATPE process was shown to be considerably advantageous in terms of process economics, especially when processing high titer cell culture supernatants. This alternative process is able to purify continuously the same amount of mAbs reducing the annual operating costs from 14.4 to 8.5 million (US$/kg) when cell culture supernatants with mAb titers higher than 2.5 g/L are processed.     

转基因植物表达重组蛋白的研究进展

植物表达系统的一些潜在优点 ,如重组蛋白的高积累水平 ,糖基化 ,细胞内的定位和自然储藏的稳定性是目前植物生产重组蛋白系统研究成为热点的主要原因 .在研究和选择转基因植物表达系统的过程中 ,转化 ,转化后 ,翻译 ,翻译后等环节都会影响到最终产物的数量和质量 ,因此应该了解基因表达的规律 ,以制定植物生产重组蛋白合适的策略 ,重组蛋白积累水平是关键 ,但其它因素如植物的选择 ,转基因植物的处理 ,下游加工等同样重要 .某些情形下 ,仅下游加工的成本一项就影响到特定植物表达系统的实际应用价值 .     

Current status of technical protein refolding

The expression of heterologous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. To fully exploit the production capacity of the cells, efficient strategies for further processing have to be developed. While in lab scale matrix assisted refolding techniques, especially of histidine-tagged proteins have become very popular, in production scale refolding by dilution is still predominant due to its simplicity. However scaling up dilution processes leads to large volumes and low protein concentration. This is a heavy burden both for liquid handling and for subsequent downstream processing steps. Process development aims to operate at uniform, reproducible conditions, to reduce costs to a minimum and to guarantee the required quality of the product. The general refolding kinetics, exploration of appropriate refolding conditions are reviewed. The major refolding operations such as dilution, matrix assisted refolding, pressure driven refolding or continuous refolding applications are discussed in view of industrial applicability.     

The bioseparation needs for tomorrow

Will we replace oil with wheat or corn as a feedstock for producing natural plastic? The success of biotechnology for bulk product manufacturing will heavily depend on engineering solutions in the downstream processes in which separation and purification have a crucial role with respect to commercial development. Development of efficient bioseparation methods is important for a broad range of business areas including pharmaceuticals, nutrition and health products, bio-based materials and crop protection chemicals. Depending on the value of the end product and the scale of production, the processing required varies significantly. Key factors that have an impact on the choice of separation strategy include process throughput, particle size of the product and impurities and the desired end-product concentration. The development of efficient, economical and selective separation methods will be required for successful commercialization of bioprocesses. Despite this well-recognized need, there are relatively few available methods for commercial implementations. Development of novel mechanical systems for selective separation of solid and liquid mixtures must become a top priority for current research investment to reduce the reliance on expensive chromatographic and thermal separation methods.     

Biofilms and their impact on the food industry

Biofilm could be defined as a complex communities of microorganisms seen affixed to surfaces, they form clusters without sticking to any surface and buried firmly in an extracellular matrix (ECM). This matrix is formed by microorganisms in the formation of either extracellular polymeric substances (EPSS) or extracellular polymer. Many reviews have addressed the negative consequences of biofilm production in the food industry, among which we talk about biofilms being responsible for spoilage microorganisms and foodborne pathogens such as Listeria monocytogenes, Bacillus cereus etc. These contamination could be linked to biofilms presence in the processing plant. Although researches have tried conferring solutions to these challenges in the food industry, however, in this review we have tried to focus on the positive impact of biofilms formed in the food industry. It is critically expedient while trying to find the solution to the challenges of biofilm in the food industry to develop and give a major focus on the advantages and positive impact biofilm has in the food industry, which has been greatly neglected. Hence in this article, we have highlighted some positive impacts of biofilms formed in the food industry, like enhancing plant health and productivity of food products, as an agent of water and wastewater treatment in the food industry, as a tool in reducing the amount of excess sludge in the wastewater treatment plant. The development of edible biofilms, fermented food products and the production of biodegradable food packaging are also part of biofilms beneficial roles in the food industries.     

Microbial production of short and medium chain esters: Enzymes,pathways, and applications

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.     

MINLP models for the synthesis of optimal peptide tags and downstream protein processing

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.     

Recovery of succinic acid from fermentation broth

Succinic acid is of high interest as bio-feedstock for the chemical industry. It is a precursor for a variety of many other chemicals, e.g. 1,4-butandiol, tetrahydrofuran, biodegradable polymers and fumaric acid. Besides optimized production strains and fermentation processes it is indispensable to develop cost-saving and energy-effective downstream processes to compete with the current petrochemical production process. Various methods such as precipitation, sorption and ion exchange, electrodialysis, and liquid–liquid extraction have been investigated for the recovery of succinic acid from fermentation broth and are reviewed critically here.     

Two-phase systems: potential for in situ extraction of microalgal products

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted.Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40°C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60°C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

Microalgae autoflocculation: an alternative to high-energy consuming harvesting methods

With increasing concerns regarding energy and environment, algae biofuel is generating considerable interest around the world. Nevertheless, the harvesting step required before downstream biomass processing is a major bottleneck. Commonly employed methods include addition of chemicals or use of mechanical equipment that increase dramatically the biofuel production cost. This review deals with naturally occurring processes that can help offset those costs by causing microalgae flocculation. Interaction theories are briefly reviewed. In addition, operational parameters such as pH, irradiance, nutrients, dissolved oxygen, and temperature effect on microalgae flocculation are evaluated. Finally, microalgae flocculation is also considered from an ecological point of view by taking advantage of their interaction with other microorganisms.     

Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens

The production of pharmaceutical proteins in plants provides a valuable alternative to other traditional eukaryotic expression systems from economic and safety perspectives. The moss Physcomitrella patens allows the expression and secretion of complex target proteins into a simple aqueous maintenance medium, which facilitates downstream processing by rendering it less complex. To address the question of whether the addition of protein-stabilizing substances enhances the recovery of a target protein secreted into the culture medium, several additives at different concentrations were tested in a small-scale screening system. Although polyvinylpyrrolidone (PVP) and human serum albumin (HSA) showed a significant impact on protein levels, supplementation of the medium with these substances was accompanied by certain limitations in upstream processes, such as foam formation (HSA), and in downstream processes, such as reduced binding efficiency on chromatography columns (PVP), respectively. In order to reap the benefit of the enhancing effect and to avoid the given negative aspects, we developed a new strategy based on the recombinant expression of HSA in plants that are already capable of expressing a target protein. First, we analysed the expression and secretion of recombinant HSA in transiently and stably transformed wild-type (WT) plants. HSA was then co-expressed in Physcomitrella plants transgenic for human vascular endothelial growth factor (VEGF). Even with high expression levels of recombinant human VEGF (rhVEGF), the co-expression of recombinant HSA (rHSA) resulted in 48%-102% higher recovery of the target protein without concomitant negative effects on the upstream process. This strategy enables the enhanced recovery of target protein and does not require the addition of foreign components directly to the culture medium.     

Integrated two‐liquid phase bioconversion and product‐recovery processes for the oxidation of alkanes: Process design and economic evaluation

Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two‐liquid phase bioreactor systems. In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non‐water soluble substrates. The apolar products typically accumulate in the emulsified apolar phase. We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two‐liquid phase media. Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1‐alkanols from n‐alkanes on a 10 kiloton/yr scale, taking the conversion of n‐octane to 1‐octanol as a model system. Here, we describe overall designs of fed‐batch and continuous‐fermentation processes for the oxidation of octane to 1‐octanol by Pseudomonas oleovorans, and we discuss the economics of these processes. In both systems the two‐liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n‐octane is dissolved, while the cells are present in the aqueous phase. In one system, multiple‐batch fermentations are followed by continuous processing of the product from the separated apolar phase. The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product. Fewer fermentors were required and a higher space‐time‐yield was possible for production of 1‐octanol in a continuous process. The overall performance of each of these two systems has been modeled with Aspen software. Investment and operating costs were estimated with input from equipment manufacturers and bulk‐material suppliers. Based on this study, the production cost of 1‐octanol is about 7 US$kg⁻¹ when produced in the fed‐batch process, and 8 US$kg⁻¹ when produced continuously. The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed‐batch process and slightly higher upstream costs for continuous fermentation. The largest cost contribution was due to variable production costs, mainly resulting from media costs. The organisms used in these systems are P. putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further. Hence, such cells need a second carbon source, which in these systems is glucose. Although the continuous process is about 10% more expensive than the fed‐batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed‐batch fermentation by decreasing the dilution rate while maintaining near constant productivity. Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 84: 459–477, 1999.     

A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans.

The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed. This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale. It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps. At the cultivation stage, the specific productivity of the E. coli in units per gram per hour is four times that of the S. lividans while the total biomass yields are of the same order. The initial volume for downstream processing of S. lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E. coli, however, the recoverable yield from the E. coli is a fifth of that obtained from the S. lividans and requires three additional stages prior to chromatography. Even with these stages the final specific activity is 64% of the S. lividans. The results indicate the need to consider the whole process when making such comparisons.     

Impacts of nutritional technology on feeds offered to horses: A review of effects of processing on voluntary intake,digesta characteristics and feed utilisation

Advances in feed processing technology applied to diet systems for ruminant livestock have been used extensively in the equine feed industry. The translation of these technologies is an important area of interest for the feed processing industry servicing the various sectors of the equine industry such as feeds for the racing, meat, milk and urine production, as well as supplements for leisure horse use. However, there are few reviews examining impacts of feed processing technologies on the processes controlling voluntary intake or utilisation of processed feeds by horses. In this paper, some of the specific features of feeds and impacts of feed processing on factors controlling meal pattern, frequency and size, and digestive physiology will be addressed. Three main areas are examined in this review, being impacts of feed processing on processes of “information gathering” (sensory and nutritional knowledge) by the horse, eating behaviour of the horse offered processed feeds (notably issues of preference and control of short-term ingestion rate), and implications of constraints of digestive physiology, process and function on voluntary intake and digestibility of processed feeds. The review highlights areas of future research and development for nutritional technology to increase knowledge of interactions between equine physiology and feed processing to enhance efficiency of capture of nutrients and maintain the welfare of horses managed in the housed environment.     

The negative impact of the environment on methylation/epigenetic marking in gametes and embryos: A plea for action to protect the fertility of future generations

Life expectancy has increased since World War II, and this may be attributed to several aspects of modern lifestyles. However, now we are faced with a downturn, which seems to be the result of environmental issues. This paradigm is paralleled with reduced human fertility, decreased sperm quality, increased premature ovarian failure, and diminished ovarian reserve syndromes. Endocrine disruptor chemicals and other toxic chemicals, herbicides, pesticides, plasticizers, to mention a few, are a rising concern in today's environment. Some of these are commonly used in the domestic setting: cleaning material and cosmetics and they have a known impact on epigenesis and imprinting via perturbation of methylation processes. Pollution from polyaromatic hydrocarbons, particulate matter <10 and <2.5 μm, and ozone released into the air, all affect fertility. Poor food processing management is a source of DNA adduct formation, which impairs the quality of gametes. An important concern is the nanoparticles that are present in food and are thought to induce oxidative stress. Now is the time to take a step backward. Global management of the environment and food production is required urgently to protect the fertility of future generations.     

Technological Change and the Environmental Impacts of Food Production and Consumption: The Case of the UK Yogurt Industry

This article examines the changing food production and consumption system of yogurt in the United Kingdom, focusing on the environmental impact of the yogurt processing stage from a technological perspective. Based on interviews with key industry stakeholders, we describe the evolution of the yogurt industry since the 1950s, characterized by a shift toward industrialized mass production and increased product diversity. We explain how new technologies, sociopolitical trends, and industrial strategies have shaped the evolution of the industry over this period. Moreover, we argue that the environmental impact of the current yogurt production and consumption system has been determined to a large extent by technological change. The article explores the future of the yogurt production and consumption system, considering the environmental implications of increased production of more differentiated yogurt products and how technological change may mitigate some of those impacts. The analysis reveals that to appreciate the environmental impact of the yogurt industry, policy makers should not focus simply on the primary stage of production but on all stages of the yogurt production and consumption system. Minimizing waste and improving resource efficiency at the yogurt processing stage are found to be particularly important. Transformation to an alternative yogurt production and consumption system with substantially lower environmental impacts requires significant commitment to change across the industry and the wider sociotechnical landscape.