Bench-top Bioreactor Research Articles

Biogas production and electricity generation from a quail manure wastewater treatment system per water depth

Coturniculture, as an activity which demands low investment and quick return, is shown to be a possibility for the rural family producer. Concomitant to this, we highlight the fact that the use of liquid quail farming waste, aimed at generating energy by anaerobic digestion, can mean a viable and promising technology for obtaining biogas from confined animal production systems. The growing demand for energy establishes that new energy sources are better used, and a great opportunity for their growth may be the use of biomass in anaerobic digestion systems, in which the organic substrate is degraded and transformed into energy and biofertilizer. The method applied was Biochemical Potential of Methane, through benchtop bioreactors with a volume of 250 mL, and in mesophilic conditions. Waste was used as inoculum from the manure tank of the quail egg production farm. Thesubstrates used to compose the treatments were liquid quail farming waste from the water depth treatment system, with 15, 30 and 45 days of deposition. In the results obtained, it was verified that the best mono digestion used was inoculum+substrate of 30days of deposition, with water retention time of 45 days, showing a higher production accumulated in biogas (0.00078476 Nm3) and CH4 (0.000575 Nm3) as well as the highest biogas potential of 0.0043 Nm3 (kg substrate)-1. When converted into electrical energy, by means of a motor generator, using as fuel the biogas produced by the liquid quail farming waste, the value of 104.64 kwh (45 days)-1 was obtained.

High concentration of spores and colony forming units of the biocontrol agent Beauveria bassiana via optimization of submerged cultivation

Liquid cultivation for the production of mycoinsecticides has received recent attention by the industry, due to the many advantages over the solid-state cultivation process. However, important variables must be considered to enable the production of the fungus propagules in the liquid medium. In this study, the optimization of the culture medium and the influence of pH and dissolved oxygen (DO) parameters on the production of spores of Beauveria bassiana in liquid process was investigated. The strain of B. bassiana IBCB 868 was growth in shake flasks followed by experimental design to find the optimal medium condition for spores production. Optimization of the culture medium was performed using a Plackett-Burman design followed by a central composite rotational design (CCRD). The optimized conditions resulted in high concentrations of submerged spores (2.21 × 109 SS mL−1) and colony forming units (1.52 × 109 CFU mL−1) on the third day of cultivation. There was an increase of 165.05% and 210.16% in SS and CFU production, respectively, in the optimized condition when compared to the most favorable culture medium initially tested. Benchtop bioreactor assays with the optimized medium were performed and the influence of pH and DO was investigated. It was observed that the pH fixed at 4.5 and 10% DO in 24 h, followed by 50% DO until 48 h and 30% DO until the end of the cultivation provided the highest concentrations of SS (1.98 × 109 SS mL−1) and CFU (1.9 × 109 CFU mL−1) after four days of growth.

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms.

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM's multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Post-Consumer Poly(ethylene terephthalate) (PET) Depolymerization by Yarrowia lipolytica: A Comparison between Hydrolysis Using Cell-Free Enzymatic Extracts and Microbial Submerged Cultivation.

Several microorganisms have been reported as capable of acting on poly(ethylene terephthalate) (PET) to some extent, such as Yarrowia lipolytica, which is a yeast known to produce various hydrolases of industrial interest. The present work aims to evaluate PET depolymerization by Y. lipolytica using two different strategies. In the first one, biocatalysts were produced during solid-state fermentation (SSF-YL), extracted and subsequently used for the hydrolysis of PET and bis(2-hydroxyethyl terephthalate) (BHET), a key intermediate in PET hydrolysis. Biocatalysts were able to act on BHET, yielding terephthalic acid (TPA) (131.31 µmol L-1), and on PET, leading to a TPA concentration of 42.80 µmol L-1 after 168 h. In the second strategy, PET depolymerization was evaluated during submerged cultivations of Y. lipolytica using four different culture media, and the use of YT medium ((w/v) yeast extract 1%, tryptone 2%) yielded the highest TPA concentration after 96 h (65.40 µmol L-1). A final TPA concentration of 94.3 µmol L-1 was obtained on a scale-up in benchtop bioreactors using YT medium. The conversion obtained in bioreactors was 121% higher than in systems with SSF-YL. The results of the present work suggest a relevant role of Y. lipolytica cells in the depolymerization process.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.

Novel bio-catalytic degradation of endocrine disrupting compounds in wastewater.

Against the backdrop of towering ecological health implications of estrogen pollution and the inefficacies associated with cost-intensive treatment techniques, this study recorded the earliest attempt of developing an inexpensive bacterial laccase-based biocatalysts for biodegradation of EDCs (Endocrine disrupting compounds), particularly estrogens. First, a central composite design was used to investigate the interactive effects of pH (6.0-8.0), inoculum size (100-500 U/mL), and copper (Cu) (25-75mg/L) on laccase activity and estrogen degradation respectively. Thereafter, biocatalysts was synthesized comprising laccase and glass beads or silver impregnated clay granules (SICG), which was further used to treat estrogen infused aquatic matrices under different reaction conditions. Maximum laccase activities and estrogen removal for the two tested laccases were 620 U/mL (85.8-92.9%) and 689.8U/mL (86.8-94.6%) for Lysinibacillus sp. BP1 and Lysinibacillus sp. BP2, respectively, within 72h, under conditions of optimal inoculum size and/or Cu concentration. Apart from a higher estrogen removal rate compared to free laccased, the biocatalysts were more resistant to temperature, pH and other environmental perturbations, and had enhanced storage ability and reusability. In comparison to clay, beads had a higher potential for recyclability and were more stable under certain experimental factors such as pH, reuse, and temperature, as well as storage conditions. Immobilized enzymes were able to remove 100% of E2, as well as over 90% of E1 and EE2, in 24h, indicating that they could be scaled up to benchtop bioreactor levels.

Optimization of an adenovirus-vectored zoster vaccine production process with chemically defined medium and a perfusion system.

ObjectivesCells grown in chemically defined medium are sensitive to shear force, potentially resulting in decreased cell growth. We optimized the perfusion process for HEK293 cell-based recombinant adenovirus-vectored zoster vaccine (Ad-HER) production with chemically defined medium.MethodsWe first studied the pseudo-continuous strategies in shake flasks as a mimic of the bioreactor equipped with perfusion systems. Using design of experiment (DoE) in shake flasks, we obtained the regression models between Ad-HER titer/virus input–output ratio and three production process parameters: time of infection (TOI), multiplicity of infection (MOI), and virus production pH (pH). We then confirmed the effect of Pluronic F68 (PF-68) at 3.0 g/L on HEK293 cell growth and Ad-HER production in shake flasks and a 2 L benchtop bioreactor.ResultsThe optimized process was scale-up to a 2 L benchtop bioreactor with the PATFP perfusion system, which yielded cell density of 7.4 × 106 cells/mL and Ad-HER titer of 9.8 × 109 IFU/mL at 2 dpi, comparable to the bioreactor with a ATF2 system.ConclusionThis optimization strategy could be used to develop a robust process with stable cell culture performance and adenovirus titer. Increasing PF-68 concentration in chemically defined medium could protect cells from shear stress generated by perfusion system.

Optimization of the fermentation parameters to maximize the production of cellulases and xylanases using DDGS as the main feedstock in stirred tank bioreactors

Lignocellulolytic enzymes such as cellulase and xylanases are needed on the industrial scales for low-cost production of biofuels and the other value-added products from lignocellulosic biomass such as distillers' dried grains with solubles (DDGS). Optimization of fermentation variables such as agitation, aeration, and inoculum size for fungal enzyme production by submerged fermentation can enhance the enzyme production levels. Therefore, this research focused on the statistical optimization of fungal inoculum size (1–10%), aeration (0.5-2vvm) and agitation rates (100–500rpm) through response surface methodology (RSM) to obtain highest amounts of cellulases and xylanases. Aspergillus niger (NRRL 330) was grown in dilute acid treated DDGS supplemented with peptone, yeast extract, and ammonium sulfate in benchtop bioreactors to produce these enzymes. The optimal fermentation results were determined to be 6.5% inoculum size, 310 rpm agitation rate and 1.4 vvm aeration, which revealed increases of cellulase activity from 0.6 to 0.82 IU/ml and xylanase activity from 3.99 to 52.76 IU/ml after optimization. The results prove the need for optimization of culture conditions such as aeration, agitation and inoculum size for higher production of hydrolytic enzymes by A. niger using DDGS as the main feedstock.

Membrane Chromatography-Based Downstream Processing for Cell-Culture Produced Influenza Vaccines.

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3–62.5% viral recovery was achieved, with the removal of 86.3–96.5% host cell DNA and 95.5–99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

Bioprocess development as a sustainable platform for eco-friendly alkaline phosphatase production: an approach towards crab shells waste management

BackgroundThere are substantial environmental and health risks associated with the seafood industry's waste of crab shells. In light of these facts, shellfish waste management is critical for environmental protection against hazardous waste produced from the processing industries. Undoubtedly, improved green production strategies, which are based on the notion of "Green Chemistry," are receiving a lot of attention. Therefore, this investigation shed light on green remediation of the potential hazardous crab shell waste for eco-friendly production of bacterial alkaline phosphatase (ALP) through bioprocessing development strategies.ResultsIt was discovered that by utilizing sequential statistical experimental designs, commencing with Plackett–Burman design and ending with spherical central composite design, and then followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor, an innovative medium formulation could be developed that boosted ALP production from Bacillus licheniformis strain ALP3 to 212 U L−1. The highest yield of ALP was obtained after 22 h of incubation time with yield coefficient Yp/s of 795 U g−1, which was 4.35-fold higher than those obtained in the shake-flask system. ALP activity has a substantial impact on the volatilization of crab shell particles, as shown by the results of several analytical techniques such as atomic absorption spectrometry, TGA, DSC, EDS, FTIR, and XRD.ConclusionsWe highlighted in the current study that the biovalorization of crab shell waste and the production of cost-effective ALP were being combined and that this was accomplished via the use of a new and innovative medium formulation design for seafood waste management as well as scaling up production of ALP on the bench-top scale.

A sustainable and effective bioprocessing approach for improvement of acid phosphatase production and rock phosphate solubilization by Bacillus haynesii strain ACP1

There is indeed a tremendous increase in biotechnological production on a global scale, more and more innovative bioprocesses, therefore, require to perform ideally not only in a small lab- but also on large production scales. Efficient microbial process optimization is a significant challenge when accomplishing a variety of sustainable development and bioengineering application objectives. In Egypt's mines, several distinct types of rock phosphate (RP) are utilized as a source of phosphate fertilizers in agriculture. It is more ecologically beneficial to utilize RP bio-solubilization than acidulation. Therefore, this work aimed to strategically scale up the acid phosphatase (ACP) production and RP bio-solubilization by the newly-discovered Bacillus haynesii. The use of consecutive statistical experimental approaches of Plackett–Burman Design (PBD), and Rotatable Central Composite Design (RCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor revealed an innovative medium formulation. These approaches substantially improved ACP production, reaching 207.6 U L−1 with an ACP yield coefficient Yp/x of 25.2 and a specific growth rate (µ) of 0.07 h−1. The metals Na, Li, and Mn were the most efficiently released from RP during the solubilization process by B. haynesii. The uncontrolled pH culture condition is the most suitable setting for simultaneously improving the ACP and organic acids production. The most abundant organic acid produced through the cultivation process was lactic acid, followed by glutamic acid and hydroxybenzoic acid isomer. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of RP particles.

Scalable expansion of iPSC and their derivatives across multiple lineages

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for “ready-to-use” cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci.

Among the prospective biocontrol agents, the saprophytic filamentous fungus Clonostachys rosea is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for C. rosea. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of C. rosea by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (Bemisia tabaci biotype B) and Sclerotinia sclerotiorum (causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 109 conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 109 submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 104/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 109 conidia/ml and 4.35 × 108 colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of C. rosea biomass was estimated at 3.4 × 1010 conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of C. rosea inhibited 100% germination of S. sclerotiorum sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC50 values of 3.2 × 104 CFU/g soil and 1.5 × 107 CFU/ml, respectively. Therefore, the submerged liquid culture of C. rosea may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of B. tabaci and S. sclerotiorum.

Biological production and recovery of 2,3-butanediol using arabinose from sugar beet pulp by Enterobacter ludwigii

Sugar beet pulp (SBP) is a major byproduct from the sugar industries and consists of >20% w/w arabinose. The current work evaluated the potential of Enterobacter ludwigii assimilating pure arabinose and arabinose rich hydrolysate from SBP pellets for 2,3-butanediol (BDO) production. The hydrolysate was obtained through dilute acid pretreatment (DAP) with sulphuric acid. The process was optimized for acid and solid loading to obtain a hydrolysate free from furan derivatives. The effect of different levels of substrate (10–60 g/L) using pure arabinose was conducted in shake flask experiments, followed by co-fermentation with small amounts of glucose and SBP hydrolysate. After flask cultivations, BDO fermentations were carried-out in a bench-top bioreactor in batch and fed-batch modes using pure arabinose as well as SBP hydrolysate. The fed-batch culture led to BDO production of 42.9 and 35.5 g/L from pure arabinose and SBP hydrolysate with conversion yields of 0.31 and 0.29 g/g, respectively. Finally, BDO accumulated on pure arabinose and SBP hydrolysate were recovered using an aqueous two-phase extraction system. The recovery yield of BDO accumulated on arabinose and hydrolysate was ∼97%. The work demonstrated the feasibility of using SBP as a suitable feedstock for manufacturing BDO.

The efficient development of a novel recombinant adenovirus zoster vaccine perfusion production process

The adenovirus vector vaccines induce humoral and cellular immune responses and have been used to develop vaccines for effective prevention of life-threating viruses, such as Ebola and Coronaviruses. High demand of vaccines worldwide requires optimization of the production process. Perfusion process increases cell concentration and volumetric productivity, so that it becomes the commonly used strategy in vaccine production In this study, we optimized and developed a perfusion process for the adenovirus-based zoster vaccine production efficiently. We first tested different perfusion strategies in shake flasks, showing semi-continuous strategies for optimal HEK 293 cell growth. We then evaluated three empirical key process parameters (cell concentration at the time of infection (VCC), multiplicity of infection (MOI), virus production pH) by the design of experiment (DoE) method, from which the robust setpoint (VCC 1.04 × 107 cells/mL, MOI 9, and virus production pH 7.17) was confirmed in both shake flask and 2 L benchtop bioreactor. In the bioreactor, we compared the performances of two perfusion systems, the commercially-available XCell ATF® system and a novel peristaltic pump-driven alternating tangential flow perfusion system (PATFP system) that we developed. During cell cultivation stage, both perfusion systems have comparable performances regarding viable cell concentration and cell viability. At 2 dpi, the PATFP system resulted in an adenovirus titer of 2.1 × 1010 IFU/mL and cell-specific virus yield of 2,062 IFU/cell, reaching 75% and 77% of values for XCell ATF® system. This study demonstrates the perfusion process to be superior strategy for adenovirus-based vaccine production compared to the batch-mode strategy (1,467 IFU/cell). Furthermore, our PATFP system shows potential to be comparable to the XCell ATF® system, and it would become an alternative perfusion strategy for the vaccine production.

Bacterial Cellulose Synthesis by Gluconacetobacter xylinus: Enhancement via Fed-batch Fermentation Strategies in Glycerol Media

Bacterial cellulose (BC) is an abundant polysaccharide, which is secreted by several genera of bacteria. It has remarkable characteristics, which include high purity, high tensile strength, high biocompatibility and non-toxic. The main feature that differentiates BC and plant cellulose (PC) is the absence of contaminants such as lignin, hemicellulose and pectin. However, the main drawbacks in producing BC are low yield and expensive carbon source. Due to that, this study was carried out to enhance BC volumetric productivity in fed-batch operation mode using glycerol as a carbon source. BC was produced in fill-and-draw and pulse-feed fed-batch cultures of Gluconacetobacter xylinus DSM 46604 in a 3-L bench-top bioreactor. The fed-batch fermentation trials were conducted in agitated and aerobic conditions at 30 ºC. For fill-and-draw fed-batch culture, a total of 24.2 g/L of BC accumulated in the bioreactor after 9 days, which corresponded to a yield and productivity of 0.2 g/g and 2.69 g/L/day, respectively. Pulse-feed fed-batch fermentation resulted in a yield and volumetric productivity of 0.38 g/g and 2.71 g/L/day, respectively. The pulse-feed fed-batch culture proved to be a better fermentation system for utilizing glycerol, which is a low-cost and abundant carbon source.
 HIGHLIGHTS
 
 Komagataeibacter species, which were formerly known as Acetobacter or Gluconacetobacter is one of the Gram-negative BC producers that secretes a large quantity of BC microfibrils extracellularly
 One of the main challenges in bacterial cellulose (BC) production is low productivity and high processing cost
 As fed-batch fermentation is one of the operation modes in bioprocess that can control the microbial growth rate, this operation mode is conducted to enhance the yield of BC, substrate consumption and also volumetric productivity
 Fill-and-draw and pulse feed fed-batch culture were conducted to enhance yield and volumetric productivity. The pulse-feed fed-batch culture resulted to be a favorable operation mode for utilizing glycerol, which is a low-cost and abundant carbon source
 
 GRAPHICAL ABSTRACT

Evaluation of a microbioreactor system as a screening tool for optimizing lentiviral vector process development in suspension culture

With an increasing number of lentiviral vector (LVV)-based cell and gene therapy candidates reaching clinical trials, scalable suspension cell culture processes using stirred tank reactors (STRs) are needed to meet future demands. However, to cost-effectively scale LVV production in STRs requires process development which can be expensive and time consuming to perform in bench-top bioreactors. To address these issues, a multi-parametric approach for process development using a micro scale bioreactor system (Ambr® 15 cell culture system, Sartorius) was assessed. Since the medium exchange process step cannot be linearly or methodically scaled-down from a bench-scale STR to a microbioreactor due to system differences, this study focused on adjusting to those differences by developing and testing three different medium exchange protocols. The implementation of one approach (Process 2.0) using an automated cell settling medium exchange protocol produced results which closely aligned with an established LVV bench-scale process in transfection efficiency and productivity, as well as lowered variability between vessels in the cell culture workstation. In summary, this study demonstrates the suitability of the Ambr 15® system as a process screening tool which has the potential to reduce costs and timelines of the development of scalable LVV production systems in suspension culture.

Biovalorization of raw agro-industrial waste through a bioprocess development platform for boosting alkaline phosphatase production by Lysinibacillus sp. strain APSO

This study highlighted the exploitation of mathematical models for optimizing the growth conditions that give the highest phosphatase productivity from a newfound Lysinibacillus sp. strain APSO isolated from a slime sample. Mathematical models facilitate data interpretation and provide a strategy to solve fermentation problems. Alkaline phosphatase (ALP) throughput was enhanced by 16.5-fold compared to basal medium based on a sequential optimization strategy that depended on two-level Plackett–Burman design and central composite design. The additional improvement for volumetric productivity and specific production yield was followed in a 7 L bench-top bioreactor to evaluate microbial growth kinetics under controlled and uncontrolled pH conditions. The pH-controlled batch cultivation condition neither supported cell growth nor enhanced ALP productivity. In contrast, the uncontrolled pH batch cultivation condition provided the highest ALP output (7119.4 U L−1) and specific growth rate (µ = 0.188 h−1) at 15 h from incubation time, which was augmented > 20.75-fold compared to the basal medium. To the authors’ knowledge, this study is the second report that deals with how to reduce the production cost of the ALP production process via utilization of agro-industrial waste, such as molasses and food waste (eggshell), as a nutrimental source for the improvement of the newfound Lysinibacillus sp. strain APSO ALP throughput.

Enhanced lipid production by addition of malic acid in fermentation of recombinant Mucor circinelloides Mc-MT-2

In our previous work, we reported a novel approach for increasing lipid production in an oleaginous fungus Mucor circinelloides by overexpression of mitochondrial malate transporter protein. This transporter plays a vital role in fatty acid biosynthesis during malate and citrate transport systems in oleaginous fungi. In this study, the controlling metabolic supplementation strategy was used to improve the lipid production by overexpression of malate transporter protein in M. circinelloides strain coded as Mc-MT-2. The effects of different metabolic intermediates on lipid production in batch fermentation by Mc-MT-2 were investigated. The optimal lipid production was obtained at 0.8% malic acid after 24 h of fermentation. Furthermore, in fed-batch bioreactors containing glucose as a carbon source supplemented with malic acid, the highest cell growth, and lipid production were achieved. The resulting strain showed the fungal dry biomass of 16 g/L, a lipid content of 32%, lipid yield of 5.12 g/L in a controlled bench-top bioreactor, with 1.60-, 1.60- and 2.56-fold improvement, respectively, compared with the batch control without supplementation of malic acid. Our findings revealed that the addition of malic acid during fermentation might play an important role in lipid accumulation in the recombinant M. circinelloides Mc-MT-2. This study provides valuable insights for enhanced microbial lipid production through metabolic supplementation strategy in large scale and industrial applications.

Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors.

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cellsas precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity.