Downstream Processing Techniques Research Articles

A critical review on scale-up strategies of biosurfactant production and its applications

Abstract Eco-friendly, biodegradable, microbially produced surfactants, known as biosurfactants, have been intensively studied for their ability as potential substitutes for their synthetic counterparts. Currently, the commercialization of biosurfactants compared to synthetic (chemical) surfactants remains an economic challenge due to high production costs and lower yields. This review details the challenges and better strategies for industrial scale-up of biosurfactant production and downstream processing techniques. An analysis of the use of low cost, renewable substrates, parameter optimization, strain improvement, bioreactor engineering is discussed. The role of various engineering factors that help in scale-up of biosurfactant production such as drag coefficient, Reynolds number, Enrichment ratio, Eӧtvӧs number have also been considered in this review article. The review article demonstrates the potential activity of integrated separation processes and applications of biosurfactants, bringing biosurfactants into mainstream commercialization.

Selective enzymes separation from the fermentation broth of Yarrowia lipolytica using aqueous two-phase system based on quaternary ammonium compounds

Aqueous two-phase systems (ATPS) are considered efficient and sustainable downstream processing techniques in extracting and separating enzymes. In this work, we evaluated the use of ATPS based on quaternary ammonium (tetrabutylammonium bromide ([N4444]Br), tetrabutylammonium chloride ([N4444]Cl), choline chloride (ChCl), and betaine) + potassium phosphate buffer (pH 7) to separate selective lipase and protease from the fermentation broth with Y. lipolytica. Cultivation in YPD medium supplemented with 200 mM potassium phosphate buffer (pH 7) in the preparation of the culture medium positively influenced the production of lipase and protease, whereby led to lesser pH variations and better values of biomass production (15.29 g p.s. L-1), lipolytic (455.96 U L-1), and proteolytic activity (23.70 U L-1). The presence of the culture medium enhanced the two-phase region, and the phase separation followed the order of ammonium compounds hydrophobicity: [N4444]Br > [N4444]Cl > betaine > ChCl. Lipase was partitioned mainly to the ammonium-rich phase and protease migrates preferentially to the salt-rich phase in all systems studied. Remarkable extraction efficiencies of 100% for lipase and 96.87% for protease were achieved in a single step for [N4444]Cl-based ATPS. Furthermore, a high level of purification was achieved with values of 10.55 and 1.69 for lipase and protease, respectively. According to the remarkable results, quaternary ammonium-based ATPS can be considered an alternative and efficient platform to separate lipase from protease, obtaining two high-value-added compounds selectively.

Astaxanthin as a King of Ketocarotenoids: Structure, Synthesis, Accumulation, Bioavailability and Antioxidant Properties.

Astaxanthin (3,3-dihydroxy-β, β-carotene-4,4-dione) is a ketocarotenoid synthesized by Haematococcus pluvialis/lacustris, Chromochloris zofingiensis, Chlorococcum, Bracteacoccus aggregatus, Coelastrella rubescence, Phaffia rhodozyma, some bacteria (Paracoccus carotinifaciens), yeasts, and lobsters, among others However, it is majorly synthesized by Haematococcus lacustris alone (about 4%). The richness of natural astaxanthin over synthetic astaxanthin has drawn the attention of industrialists to cultivate and extract it via two stage cultivation process. However, the cultivation in photobioreactors is expensive, and converting it in soluble form so that it can be easily assimilated by our digestive system requires downstream processing techniques which are not cost-effective. This has made the cost of astaxanthin expensive, prompting pharmaceutical and nutraceutical companies to switch over to synthetic astaxanthin. This review discusses the chemical character of astaxanthin, more inexpensive cultivating techniques, and its bioavailability. Additionally, the antioxidant character of this microalgal product against many diseases is discussed, which can make this natural compound an excellent drug to minimize inflammation and its consequences.

Marine yeast (Candida sake) cultured on herring brine side streams is a promising feed ingredient and omega-3 source for rainbow trout (Oncorhynchus mykiss)

A major challenge for the aquaculture industry is the supply of sustainable feeds. A promising model to achieve this is to utilize circular flows where feed ingredients, such as single cell protein, are cultivated using side streams of the food industry. The aim of this study was to evaluate the marine yeast Candida sake, produced on herring brine side streams, as a source of protein and immune stimulant in feed for salmonid fish. The dry C. sake product contained 54% protein (3.3% lysine and 0.8% methionine) and 13% lipids (1.1% eicosapentaenoic, EPA, and 1% docosahexaenoic acid, DHA). Four experimental diets were designed and tested in a 9-week feeding trial using juvenile rainbow trout (Oncorhynchus mykiss). A control diet containing both fish and plant-based ingredients constituted the base feed to which 20% (to evaluate effects on digestibility, growth and intestinal physiology), 20% heat-treated (to evaluate effects of downstream processing) and 3% (to evaluate immune stimulatory properties, replacing 3% soy protein concentrate) C. sake was added. The apparent digestibility coefficient of C. sake for protein, fat and gross energy was above 80%, and for amino acids above 90% regardless of treatment, suggesting a high bioavailability of C. sake. All three yeast containing diets performed equally to the control regarding specific growth rate, feed conversion ratio and functional intestinal health. These results suggest that C. sake is a promising alternative protein source for circular feeds in the salmonid industry. The presence of EPA and DHA represents an added value. The heat treatment increased the apparent digestibility coefficient of dry matter by 8% but decreased amino acid digestibility by on average 3%, indicating that heat treatment may not be the optimal downstream processing technique. Furthermore, the inclusion of 3% C. sake increased the intestinal lamina propria width and TGF-β transcription, indicating an immune stimulating effect. Future research is needed to understand these immune modulatory effects of C. sake supplementation.

Tandem enrichment of serum exosomes and exosomal RNA with titanium dioxide

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.

Near-Infrared Spectroscopy can Predict Anatomical Abundance in Corn Stover

Feedstock heterogeneity is a key challenge impacting the deconstruction and conversion of herbaceous lignocellulosic biomass to biobased fuels, chemicals, and materials. Upstream processing to homogenize biomass feedstock streams into their anatomical components via air classification allows for a more tailored approach to subsequent mechanical and chemical processing. Here, we show that differing corn stover anatomical tissues respond differently to pretreatment and enzymatic hydrolysis and therefore, a one-size-fits-all approach to chemical processing biomass is inappropriate. To inform on-line downstream processing, a robust and high-throughput analytical technique is needed to quantitatively characterize the separated biomass. Predictive correlation of near-infrared spectra to biomass chemical composition is such a technique. Here, we demonstrate the capability of models developed using an “off-the-shelf,” industrially relevant spectrometer with limited spectral range to make strong predictions of both cell wall chemical composition and the relative abundance of anatomical components of the corn stover, the latter for the first time ever. Gaussian process regression (GPR) yields stronger correlations (average R2v = 88% for chemical composition and 95% for anatomical relative abundance) than the more commonly used partial least squares (PLS) regression (average R2v = 84% for chemical composition and 92% for anatomical relative abundance). In nearly all cases, both GPR and PLS outperform models generated using neural networks. These results highlight the potential for coupling NIRS with predictive models based on GPR due to the potential to yield more robust correlations.

Improvement of Production of L-Methioninase from Pseudomonas stutzeri by Partitioning Study using Polymeric Phase System

Pseudomonas stutzeri MTCC 101 was explored as new bacterial strain for secreting anticancer L-methioninase. The partitioning study of this enzyme was further performed using Aqueous two-phase system (ATPS) and Triple phase system. Polyethylene glycol (PEG 4000) and magnesium sulfate (MgSO4) was selected as suitable ATPS system for its partitioning with higher partition coefficient (1.92) and 80% yield. Triple phase partition system (TPP) using n-butanol: ammonium sulphate showed its lower degree of yield (51.6%). ATPS phase components of PEG 4000 and MgSO4 was supplemented with its production media for highly recovery of L-methioninase. The culture condition (pH, temperature, inoculum volume, incubation period and agitation speed) during extractive fermentative production of L-methioninase was optimized using Response surface methodology (RSM). This model showed good adequacy with high value of coefficient of determination (R2) as 99.62%. The experiment was validated under statistically optimized culture condition with higher value of yield as 94.909%. This improved rate of yield of L-methioninase during extractive fermentative condition at optimized cultute conditions were significant finding for this research work. This extractive fermentative approach could be used as low cost-effective downstream processing techniques for its large-scale production in industry.

Aqueous polymeric phase system as low-cost downstream processing technique for extraction of D-glucuronic acid

The large-scale availability of glucuronic acid for industrial sectors is limited due to involving costly conventional purification strategies. Here, aqueous two-phase system (ATPS) was used as potent economical platform for selective recovery of D-glucuronic acid. Paenibacillus apiarus MTCC 2192 produced extracellular D-glucuronic acid with maximum yield (0.657 mg/ml) during late log phase. D-glucuronic acid was extracted after treating cell free extract with ATPS system consisting 10% PEG and 15% Potassium hydrogen phosphate (PHP) salt. It showed maximum recovery (82.97%) with lower concentration (5%) of high molecular weight of PEG 6000. The increased concentration of PHP-salt from 10 to 25% (w/w) and variation of pH (4–9) favours high recovery of D-glucuronic acid in top phase due to removal of majority of inhibitory protein component. ATPS with 5% PEG 6000 and 25% PHP-salt showed higher recovery of D-glucuronic acid (84.20%) at pH-4 and could be used as low-cost bio-separation tool to fulfil its great demand.

Membrane Purification Techniques for Recovery of Succinic Acid Obtained from Fermentation Broth during Bioconversion of Lignocellulosic Biomass: Current Advances and Future Perspectives

Recently, the bioconversion of biomass into biofuels and biocommodities has received significant attention. Although green technologies for biofuel and biocommodity production are advancing, the productivity and yield from these techniques are low. Over the past years, various recovery and purification techniques have been developed and successfully employed to improve these technologies. However, these technologies still require improvement regarding the energy-consumption-related costs, low yield and product purity. In the context of sustainable green production, this review presents a broad review of membrane purification technologies/methods for succinic acid, a biocommodity obtained from lignocellulosic biomass. In addition, a short overview of the global market for sustainable green chemistry and circular economy systems or zero waste approach towards a sustainable waste management is presented. Succinic acid, the available feedstocks for its production and its industrial applications are also highlighted. Downstream separation processes of succinic acid and the current studies on different downstream processing techniques are critically reviewed. Furthermore, critical analysis of membrane-based downstream processes of succinic acid production from fermentation broth is highlighted. A short review of the integrated-membrane-based process is discussed, as well, because integrating “one-pot” lignocellulosic bioconversion to succinic acid with downstream separation processing is considered a critical issue to address. In conclusion, speculations on outlook are suggested.

In situ product recovery as a strategy for bioprocess integration and depletion of inhibitory products

AbstractMicrobial growth inhibition by end‐products is an undesirable behavior that causes low yields and reduces the overall productivity and feasibility of fermentative bioprocesses. Classic downstream processing techniques have been explored widely to attenuate this behavior by integrating them with the upstream operations. These in situ extraction techniques appear as an attractive alternative to increase titers and consequently total recovery and/or purification. This also reduces the number of downstream operations and thus the time and final cost of the bioprocess. In the present work, process integration at the earliest stage of a bioprocess – the bioreactor – is presented as an attractive strategy to facilitate primary recovery and in some cases to overcome end‐product microbial inhibition. A careful revision of selected strategies, from classical to hybrid techniques for in situ product recovery, is presented along with pros and cons, to give the reader a general overview of different experimental strategies and selection criteria that can be generally applied to different bioproducts/bioprocesses. © 2021 Society of Chemical Industry (SCI).

A review on the lactic acid fermentation from low-cost renewable materials: Recent developments and challenges

Abstract Recently, lactic acid production from renewable materials has gained enormous attention due to the several functional properties it offers in different fields. The high yield productivity of lactic acid by utilizing economical and easily available substrates has received immense attraction in the petrochemical industry. Biomass or waste materials from various sources have become a significant concern because they cause severe environmental pollution if disposed of improperly. Hence, the employment of an integrated biorefinery platform for waste materials is an ideal option to produce high-value bio-products while remediating the waste. Optical pure lactic acid production through fermentation has gained interest due to its high potential applications in food, pharmaceutical, textiles, and cosmetic industries as well as highly promising packaging materials. The manufacturing of biodegradable bioplastic from polylactic acid materials is a green alternative to that derived from petrochemicals. However, high manufacturing costs have impeded the widespread application of polylactic acid due to the high cost of lactic acid production. This review paper summarizes the most recent advancements and challenges in pure lactic acid production from various substrates and conventional processes, including pretreatment and enzymes hydrolysis and fermentative technologies. Moreover, a detailed analysis of the techno-economic feasibility, downstream processing, and lactic acid purification techniques are also discussed.

Liquid Biphasic Systems for Oil-Rich Algae Bioproducts Processing

Oleaginous algae are nowadays of significance for industrial biotechnology applications and for the welfare of society. Tremendous efforts have been put into the development of economically feasible and effective downstream processing techniques in algae research. Currently, Liquid Biphasic Systems (LBSs) are receiving much attention from academia and industry for their potential as green and effective downstream processing methods. This article serves to review the applications of LBSs (LBS and Liquid Biphasic Flotation System (LBFS)) in the separation, recovery and purification of algae products, as well as their basic working principles. Moreover, cell disruptive technologies incorporated into LBSs in algae research are reported. This review provides insights into the downstream processing in algae industrial biotechnology which could be beneficial for algae biorefinement.

MedGA: A novel evolutionary method for image enhancement in medical imaging systems

Medical imaging systems often require the application of image enhancement techniques to help physicians in anomaly/abnormality detection and diagnosis, as well as to improve the quality of images that undergo automated image processing. In this work we introduce MedGA, a novel image enhancement method based on Genetic Algorithms that is able to improve the appearance and the visual quality of images characterized by a bimodal gray level intensity histogram, by strengthening their two underlying sub-distributions. MedGA can be exploited as a pre-processing step for the enhancement of images with a nearly bimodal histogram distribution, to improve the results achieved by downstream image processing techniques. As a case study, we use MedGA as a clinical expert system for contrast-enhanced Magnetic Resonance image analysis, considering Magnetic Resonance guided Focused Ultrasound Surgery for uterine fibroids. The performances of MedGA are quantitatively evaluated by means of various image enhancement metrics, and compared against the conventional state-of-the-art image enhancement techniques, namely, histogram equalization, bi-histogram equalization, encoding and decoding Gamma transformations, and sigmoid transformations. We show that MedGA considerably outperforms the other approaches in terms of signal and perceived image quality, while preserving the input mean brightness. MedGA may have a significant impact in real healthcare environments, representing an intelligent solution for Clinical Decision Support Systems in radiology practice for image enhancement, to visually assist physicians during their interactive decision-making tasks, as well as for the improvement of downstream automated processing pipelines in clinically useful measurements.

An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Potential of Continuous Manufacturing for Liposomal Drug Products.

Over the last several years, continuous manufacturing of pharmaceuticals has evolved from bulk APIs and solid oral dosages into the more complex realm of biologics. The development of continuous downstream processing techniques has allowed biologics manufacturing to realize the benefits (e.g., improved economics, more consistent quality) that come with continuous processing. If relevant processing techniques and principles are selected, the opportunity arises to develop continuous manufacturing designs for additional pharmaceutical products including liposomal drug formulations. Liposome manufacturing has some inherent aspects that make it favorable for a continuous process. Other aspects such as formulation refinement, materials of construction, and aseptic processing need development, but present an achievable challenge. This paper reviews the current state of continuous manufacturing technology applicable to liposomal drug product manufacturing and an assessment of the challenges and potential of this application.

An Easy and Fast Protocol for Affinity Bead-Based Protein Enrichment and Storage of Proteome Samples.

Analysis of dilute protein samples is a challenging task for scientific and industrial labs all over the world. Although there are different methods available that allow for protein enrichment from various biological sources, all of them have serious limitations apart from their advantages. In order to perform highly reproducible and sensitive protein analysis of lowest concentrated samples, we optimized a method to enrich proteins on affinity beads (StrataClean) recently. This chapter describes the general protocol of this strategy, thereby discussing the power as well as the limits of this technique for qualitative and quantitative proteomic studies. Moreover, additional application and protocol variants will be discussed, expanding the number of compatible up- and downstream processing techniques compared to the originally published method. Hence, we evaluated the reduction of time for sample preparation by use of preprimed affinity beads and shorter incubation durations as well as the influence of high concentration of salts or urea in the sample buffer.

PEG-salt aqueous two-phase systems: an attractive and versatile liquid-liquid extraction technology for the downstream processing of proteins and enzymes.

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid-liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG-salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid-liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.

Natural Origin Lycopene and Its “Green” Downstream Processing

Lycopene is an abundant natural carotenoid pigment with several biological functions (well-known for its antioxidant properties) which is under intensive investigation in recent years. Lycopene chemistry, its natural distribution, bioavailability, biological significance, and toxicological effects are briefly outlined in the first part of this review. The second, major part, deals with various modern downstream processing techniques, which are assessed in order to identify promising approaches for the recovery of lycopene and of similar lipophilic compounds. Natural lycopene is synthesized in plants and by microorganisms, with main representatives of these two categories (for industrial production) tomato and its by-products and the fungus Blakeslea trispora, respectively. Currently, there is a great deal of effort to develop efficient downstream processing for large scale production of natural-origin lycopene, with trends strongly indicating the necessity for “green” and mild extraction conditions. In this review, emphasis is placed on final product safety and ecofriendly processing, which are expected to totally dominate in the field of natural-origin lycopene extraction and purification.

Concentration by ultrafiltration and stabilization of phytase produced by solid-state fermentation

Ultrafiltration is an attractive downstream processing technique for concentrating enzymes and could be considered the primary step of purification. However, the efficiency of this process is often limited by protein fouling and shear-induced enzyme inactivation, which decreases permeate flux and results in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the enzyme activity was 14% during operation, indicating that the shear forces generated in the filter have significant influences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol retained 70% of the initial enzyme activity at 70°C after 1h and had more than 3 and 6 months storage half-life at 29°C and 4°C, respectively.

Design of ionic liquids for lipase purification

Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase ( Candida antarctica lipase B – CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C 8 side alkyl chain, the [N(CN) 2] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C 8mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery.