Fed-batch Approach Research Articles

Maximizing the mRNA productivity for in vitro transcription by optimization of fed-batch strategy

In vitro transcription (IVT) is the main manufacturing method to produce mRNA vaccines. In this study, a fed-batch strategy was systematically optimized for IVT process with dual goals of achieving a high reaction rate and maximizing the final mRNA yield simultaneously. Initially various experimental conditions were investigated including Mg2+, nucleotide triphosphates (NTP), dithiothreitol (DTT), spermidine, as well as the temperature and ionic strength. It was found that the concentrations of Mg2+ and NTP had a significant impact on IVT process. Subsequently, under the optimized conditions, dividing the IVT reaction into three distinct phases was proposed to enable more efficient transcription. By optimizing the concentrations of Mg2+ and NTP in two replenishment processes, our fed-batch strategy resulted in the production of 367.8 μg of mRNA with reduced dsRNA byproducts within 180 min. This was achieved under conditions of a final volume of 30 μL, 250 U T7 RNA polymerase (RNAP), and 2 μg DNA template. In conclusion, the IVT process using a fed-batch approach, rather than an excessive one-time NTP input, is an efficient method to improve productivity given a fixed amount of T7 RNAP and DNA template.

Sustainable production of yeast oil from rice straw hydrolysate by Pseudozyma parantarctica through fed-batch cultivation for bio-polyurethane foam formation

Rice straw serves as an alternative feedstock for yeast oil (YO) production and bio-polyurethane (BPU) foam formation. This study optimized yeast growth and YO production by Pseudozyma parantarctica, utilizing rice straw hydrolysate (RSH) as the primary carbon source. Fed-batch strategies were explored to enhance YO production efficiency and its conversion into BPU foam. The optimized conditions proved highly effective in yielding biomass and YO from RSH-OPM. The fed-batch approach significantly improved biomass, YO production, and oil content to 7.35 ± 0.18 g/L, 3.77 ± 0.09 g/L, and 51.21 ± 0.15 % (w/w), respectively. The main fatty acid (FA) compositions of YO consisted of C:16 to C:18 FAs for over 92 %. RSH-based YO was successfully transformed into both rigid and semi-rigid BPU foams. This research demonstrated the feasibility and potential of using RSH as a low-cost and renewable carbon source for YO production and BPU foam formation.

A strategic media engineering and fed-batch approach enhances violacein production by Chromobacterium violaceum MTCC2656

A strategic media engineering and fed-batch approach enhances violacein production by Chromobacterium violaceum MTCC2656

Engineering cannabinoid production in Saccharomyces cerevisiae.

Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomycessp. strainCL190. The production was significantly increased by introducing a fusion protein consisting of ERG20WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56mgL-1 . The implementation of the cannabinoid synthase genes enabled the production of Δ9 -tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed-batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2mgL-1 and 117mgL-1 , respectively.

Intensification and techno-economic analysis of a biocatalytic process for coniferol production from ferulic acid: An innovative strategy for lignin valorization

Coniferol, as a natural monolignol, acts as a chemical building block for various high-value compounds. However, the current petrochemical synthesis of coniferol and its limited scalability raise environmental and cost concerns. A biocatalytic method for coniferol production from its natural precursor, ferulic acid, was previously developed using an engineered cell factory that co-expresses carboxylic acid reductase and aldo-keto reductase. The present study focused on investigating the limiting factors of coniferol production and optimizing the process using the design of experiments and fed-batch approaches. By introducing novel parameters, including an inoculum of 0.25 g/L cells and a starting concentration of 5.0 mM ferulic acid, followed by two fed-batch additions of 2.0 mM ferulic acid and 3.7 g/L glycerol, coniferol production was enhanced by a factor of 2 (reaching 1.6 g/L). The scale-up process in a 1 L bioreactor and its intensified technical and economic analysis demonstrated its cost-effectiveness. Considering two different purchase prices for the ferulic acid of $5.0 and $650.0 per kg, the payback time was estimated at 3.3 and 3.2 years, respectively, and the minimal selling price of coniferol to obtain a IRR of 30% was calculated as $4.2 and $5.0 per gram, which is significantly lower than the current market price. In conclusion, the findings illustrate a biocatalytic approach for producing valuable compounds with potential applications in various industries, including cosmetics and pharmaceuticals. Furthermore, this approach contributes to developing sustainable and profitable biorefinery processes, paving the way for future initiatives focused on biocatalytic lignin valorization.

Pilot Scale Production of Single Cell Oil by Apiotrichum brassicae and Pichia kudriavzevii from Acetic Acid and Propionic Acid

Volatile fatty acids can be used as a cheap carbon source for biotechnological lipid production with oleaginous yeasts, but one factor limiting their large-scale use is their inherent cytotoxicity. Developing a suitable cultivation strategy can help mitigate the adverse effect volatile fatty acids have, since these effects are strongly dependent on concentration and pH. This work shows that, by employing a pH-stat fed-batch approach for the cultivation of Apiotrichum brassicae V134 and Pichia kudriavzevii V194, lipid contents above 56 g/100 g dry cell weight and dry cell weight concentrations above 30 g/L can be reached. Furthermore, volumetric lipid productivities up to 0.29 g/Lh could be achieved using acetic and propionic acid as a sole carbon source. It was also demonstrated that the developed process is robust and scalable. Scale-up to the 500 L scale resulted in a similar lipid yield, dry cell weight (31–37 g/L), and single cell oil content (56 g/100 g dry cell weight–58 g/100 g dry cell weight). The main fatty acid present in the produced lipids was oleic acid (36–43%), but also odd-numbered fatty acids, especially heptadecanoic acid (7–15%), were present. Additionally, different methods for the pretreatment of biomass prior to lipid extraction were assessed, and the iodine value (48), peroxide value (7.3), and acid value (4.3) of the extracted single cell oil were determined.

A novel bacteriocin against multiple foodborne pathogens from Lacticaseibacillus rhamnosus isolated from juice ferments: ATF perfusion-based preparation of viable cells, characterization, antibacterial and antibiofilm activity

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I–K–K–V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25–121 °C and pH 2–10. It significantly reduced cell viability of bacterial pathogens (p < 0.001), and strongly inhibited their biofilm formation. Scanning electron microscopy demonstrated deformation of pathogenic cells caused by CLK_01, leading to cytoplasmic content leakage and bacterial death. Summarily, we employed ATF perfusion to obtain viable L. rhamnosus, and presented that bacteriocin CLK_01 could serve as a promising biopreservative for controlling foodborne pathogenic bacteria and their biofilms.

In-silico analysis of a halophilic bacterial isolate-Bacillus pseudomycoides SAS-B1 and its polyhydroxybutyrate production through fed-batch approach under differential salt conditions

Polyhydroxybutyrate (PHB) is a natural biopolymer and a viable substitute for petroleum-derived polymers that possess immense potential for diverse applications. In the present study, PHB was produced by a halophilic bacteria identified as Bacillus pseudomycoides SAS-B1 by 16S rRNA gene sequencing. The bacterial genome was evaluated through complete genome sequencing, which elucidated a 5,338,308 bp genome with 34.88 % of G + C content and 5660 genes. Other genome attributes were analyzed such as functional profiling, gene ontology, and metabolic pathways. Genes involved in PHB biochemical pathway were identified such as phaA, phaB, and phaC. Furthermore, sodium-dependent transporters and other ATP-binding genes were identified in the genome that may be involved in sodium uptake during saline conditions. The PHB production by B. pseudomycoides SAS-B1 was examined under differential salt conditions. The PHB yield was increased from 3.14 ± 0.02 g/L to 6.12 ± 0.04 g/L when salinity was increased upto 20 g/L with intermittent feeding of glucose and corn steep liquor. FTIR, NMR, and GC–MS studies elucidated the presence of desired functional groups, molecular structure, and monomeric compositions of PHB respectively. Further, TGA revealed the thermal stability of the recovered PHB upto (220−230) °C and has a crystallinity index of upto 33 ± 0.5 % as confirmed by XRD analysis.

Perfusion process for CHO cell producing monoclonal antibody: Comparison of methods to determine optimum cell specific perfusion rate

Over twenty-five years ago, perfusion was applied to mammalian cells to produce therapeutic proteins, mainly sensitive ones. Nowadays, other issues such as process intensification make perfusion attractive again. However, it is a complex process to set up compared to the classical fed-batch approach. In this paper, we compared three culture methods to determine the perfusion process parameters for a CHO cell line producing a monoclonal antibody. The push-to-low (PTL) and perfusion-without-bleeding (PWB) methods were performed in bioreactors equipped with a cell retention device, while the semi-perfusion (SP) method was performed in spin tubes. In our culture conditions, the PTL reference method, performed at steady state, indicated a minimum cell specific perfusion rate (CSPRmin) estimated at 100 pL.cell−1.day−1. The PWB and SP methods led to lower CSPRmin estimations related to lower cell metabolic fluxes. We therefore propose a three-step approach to determine the perfusion parameters: (i) SP screening method to estimate the optimum medium and temperature, then (ii) PWB run to determine both the growth rate and the maximum cell concentration and finally, (iii) the perfusion process can be operated according to the previously determined parameters, with a final adjustment of the CSPRmin.

Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring.

The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

Efficient catalytic conversion of biomass derived cellulose to short chain polyols

In efforts to replace petro-derived polyethylene terephthalate (PET) with a ‘green’ equivalent, technologies are being developed and scaled up for biobased production of the two components of PET viz. monoethylene glycol (MEG) and terephthalic acid. Herein, we report work on single-pot catalytic hydrogenolysis of cellulose isolated from lignocellulosic biomass into polyols with the major fraction being monoethylene glycol. A combination of silica-alumina-supported nickel (NSA) and bulk tungsten-based catalyst (HW) was investigated for selective conversion of cellulose to MEG. While raw rice straw showed negligible conversion, the two-step pretreated rice straw performed similar to pure cellulose. Ethylene glycol in up to 60 % yield was obtained over 0.15%NSA-0.2%HW within minutes at a hydrogen pressure lower than reported thus far for similar catalysts. Product buildup was seen to have no effect on the catalyst activity and a fed-batch approach could generate product stream in high concentrations with MEG as a single major product.

Development of an Escherichia coli whole cell biocatalyst for the production of hyperoside.

To produce high concentrations of hyperoside from quercetin using recombinant Escherichia coli with in situ regeneration of UDP-galactose. Sucrose synthase from Glycine max (GmSUS) was co-expressed with UDP-glucose epimerase from E. coli (GalE) in E. coli for regenerating UDP-galactose from UDP and sucrose. Glycosyltransferase from Petunia hybrida (PhUGT) was introduced to synthesize hyperoside from quercetin through the regeneration system of UDP-galactose. Co-expressing with molecular chaperones GroEL/ES successfully enhanced the catalytic efficiency of the recombinant strain, which assisted the soluble expression of PhUGT. By using a fed-batch approach, the production of hyperoside reached 863.7mg L-1 with a corresponding molar conversion of 93.6% and a specific productivity of 72.5mg L-1h-1. The method described herein for hyperoside production can be widely applied for the synthesis of isorhamnetin-3-O-galactoside, kaempferol-3-O-galactoside and other flavonoids.

Understanding the effects of low enzyme dosage and high solid loading on the enzyme inhibition and strategies to improve hydrolysis yields of pilot scale pretreated rice straw

The commercial viability of 2G bioethanol production is dependent on cost-effective and efficient enzymatic hydrolysis of biomass at low enzyme and high dry solid loadings. In the present study, rice straw was subjected to a dilute acid pretreatment in a continuous pilot plant (250 kg/day).It was observed that, the glucan conversion yields were reduced by 26% at high solid loading (20% w/w) with low enzyme dosage of 3 FPU/g w/w of in-house developed enzyme (MRJ16). Thus, multiple approaches were investigated to enhance the glucan conversion yields, a) fed batch approach (splitting biomass or enzyme dosage during process), b) additives supplementation (surfactants, metal ions, non-enzymatic proteins and activated charcoal) and c) enzyme blending with accessory enzymes. Results showed that, the 3-stage fed-batch approach enhanced the hydrolysis yield by 12% in comparison to 14.3% increase with the selected surfactant (0.5% w/w Ecosurf). The selected metal salt (NiCl2, 1 mM) increased the glucan conversion by 13.2%, while, the non-enzymatic proteins (5% w/w yeast hydrolysate and yeast extract) improved the yields to 14.8% and 14.4%, respectively. The activated charcoal at a concentration of 0.5% w/w enhanced the hydrolysis yields by 14.4%. Further, 15.1% increase in sugar yield was obtained by supplementing the in-house produced enzyme (MRJ16) with xylanase from Thermomyces lanuginosus and BGL from Penicillium janthinellum EMS-UV-8. Finally, a unique optimized strategy was developed on the basis of selected approaches and parameters which demonstrated 24.7% higher saccharification in comparison to control. To the best of our knowledge, this is the only study available in literature where; a wide range of strategies were studies on a single feedstock (i.e. rice straw) pretreated at pilot-scale and determine its effect for improved enzymatic hydrolysis. Furthermore, developed process helps us to save (almost 2X) expensive enzyme to achieve same level of glucan conversion (∼62.0%) to make the overall process cost effective by using cheap and easily available additives. The current study also provided an in-depth understanding of enzyme inhibition at low enzyme dosages and high solid loadings in an industrially relevant scenario.

Optimization and Kinetic Modeling of a Fed-Batch Fermentation for Mannosylerythritol Lipids (MEL) Production With Moesziomyces aphidis

Mannosylerythritol lipids are glycolipid biosurfactants with many interesting properties. Despite the general interest in those molecules and the need for a robust process, studies on their production in bioreactors are still scarce. In the current study, the fermentative production of MEL in a bioreactor with Moesziomyces aphidis was performed using a defined mineral salt medium. Several kinetic process parameters like substrate consumption rates and product formation rates were evaluated and subsequently enhanced by increasing the biomass concentration through an exponential fed-batch strategy. The fed-batch approaches resulted in two to three fold increased dry biomass concentrations of 10.9–15.5 g/L at the end of the growth phase, compared with 4.2 g/L in the batch process. Consequently, MEL formation rates were increased from 0.1 g/Lh up to around 0.4 g/Lh during the MEL production phase. Thus, a maximum concentration of up to 50.5 g/L MEL was obtained when oil was added in excess, but high concentrations of residual fatty acids were also present in the broth. By adjusting the oil feeding to biomass-specific hydrolysis and MEL production rates, a slightly lower MEL concentration of 34.3 g/L was obtained after 170 h, but at the same time a very pure crude lipid extract with more than 90% MEL and a much lower concentration of remaining fatty acids. With rapeseed oil as substrate, the ideal oil-to-biomass ratio for full substrate conversion was found to be around 10 goil/gbiomass. In addition, off-gas analysis and pH trends could be used to assess biomass growth and MEL production. Finally, kinetic models were developed and compared to the experimental data, allowing for a detailed prediction of the process behavior in future experiments.

Process Development for Benzyl Alcohol Production by Whole-Cell Biocatalysis in Stirred and Packed Bed Reactors.

The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. A marine bacterium was isolated and selected for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. Enzymatic production routes are of interest for sustainable processes. To overcome benzaldehyde low water solubility, DMSO was used as a biocompatible cosolvent up to a concentration of 10% (v/v). A two-phase system with n-hexane, n-heptane, or n-hexadecane as organic phase allowed at least a 44% higher relative conversion of benzaldehyde than the aqueous system, and allowed higher initial substrate concentrations. Cell performance decreased with increasing product concentration but immobilization of cells in alginate improved four-fold the robustness of the biocatalyst: free and immobilized cells were inhibited at concentrations of benzyl alcohol of 5 and 20 mM, respectively. Scaling up to a 100 mL stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system.

Novel Propagation Strategy of Saccharomyces cerevisiae for Enhanced Xylose Metabolism during Fermentation on Softwood Hydrolysate

An economically viable production of second-generation bioethanol by recombinant xylose-fermenting Saccharomyces cerevisiae requires higher xylose fermentation rates and improved glucose–xylose co-consumption. Moreover, xylose-fermenting S. cerevisiae recognises xylose as a non-fermentable rather than a fermentable carbon source, which might partly explain why xylose is not fermented into ethanol as efficiently as glucose. This study proposes propagating S. cerevisiae on non-fermentable carbon sources to enhance xylose metabolism during fermentation. When compared to yeast grown on sucrose, cells propagated on a mix of ethanol and glycerol in shake flasks showed up to 50% higher xylose utilisation rate (in a defined xylose medium) and a double maximum fermentation rate, together with an improved C5/C6 co-consumption (on an industrial softwood hydrolysate). Based on these results, an automated propagation protocol was developed, using a fed-batch approach and the respiratory quotient to guide the ethanol and glycerol-containing feed. This successfully produced 71.29 ± 0.91 g/L yeast with an average productivity of 1.03 ± 0.05 g/L/h. These empirical findings provide the basis for the design of a simple, yet effective yeast production strategy to be used in the second-generation bioethanol industry for increased fermentation efficiency.

Abstract 10701: Suspension Differentiation of Cardiomyocyte Spheroids Improves Quality and Yields

Introduction: Improvements in stem cell viability, differentiation efficiency, and functional activity opened the door for advanced research applications in cardiac regeneration. However, the use of cell-based cardiac therapies, including implantable patches and cell products as well as in-vitro models for mechanistic studies and drug development requires large numbers of high-quality cardiomyocytes (CMs). Typically, differentiation is conducted in 6-well culture plates via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. This monolayer method requires significant manual labor and cannot be easily scaled. Suspension culture and differentiation of free-floating hiPSC aggregates following similar biochemical cues offers advantages for commercial biomanufacturing purposes and provides a more physiological environment for cell development. Hypothesis: We assessed the hypothesis that when hiPSCs are cultured and differentiated in suspension they will produce CMs with improved yield, purity, and quality. Methods: hiPSCs were expanded as aggregates in culture flask using a fed-batch approach then differentiated over a 12 day period with Wnt activation on day 0 and inhibition on day 3. Optimization studies examined purity on day 9 and complete characterization via morphological assessments and CM-specific genes expression was done on day 12 following metabolic purification. All studies were conducted with a minimum of 4 replicates. Results: Optimal conditions for both purity and yield based on cardiac troponin T staining were achieved with an initial cell density of 1.6x10 6 cells/mL, 6 μM CHIR99021, and 55 rpm shaking. After scaling to 30mL culture volume the purity of hiPSC-CMs differentiated via our novel protocol was 98.2±0.8% with yields of 1.47±0.18 million cells/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures. Conclusions: Suspension differentiation of hiPSCs into CMs in a widely available format results in improved biomanufacturing endpoints. Therefore, this method functions as a groundwork for future bioreactor systems to produce the large number of cells needed for clinical applications.

Insight into the pilot‐scale fed-batch fermentation for production of Enterococcus faecium CW3801 using molasses-based medium

Enterococcus sp. has been used as starters in food fermentation due to their probiotic and antimicrobial properties in food biopreservation. The antimicrobial properties were mainly contributed by the bacteriocin called enterocin. Hence, the availability of a cost-effective pilot-scale cultivation conditions is a necessity for the production of probiotic bacteria. This study aims to investigate optimization of medium composition using sugarcane molasses as a carbon source using response surface methodology and the potential use of fed-batch cultivation for improvement of the cell viability of Enterococcus faecium CW3801 for the use as a probiotic starter culture. Two feeding strategies (ramp and constant) were applied in fed-batch cultivation for enhancement of the production of E. faecium in a 2-L stirred tank bioreactor using the optimized medium and scaled up to a 15-L bioreactor. Optimized fermentation medium which comprised of 10% (v/v) of molasses and 10 g/L of yeast extract at pH 7 yielded maximum cell viability of 29.4 × 1011 CFU/mL with 3900 AU/mL of bacteriocin-like inhibitory substances (BLIS) activity. In the fed-batch, the cell viability (8.4 × 1013) and dry cell weight (6.34 g/L) reached the highest in optimized medium when the ramp (stepwise) feeding was applied. In scaling up to 15-L bioreactor, the growth of E. faecium was achieved at 2.3 × 1013 CFU/mL with the dry cell weight of 5.28 g/L under the same condition. The BLIS in 15-L bioreactor was 6% higher than the 2-L bioreactor. This study demonstrated that molasses and yeast extract are good feedstock for the growth of E. faecium. The E. faecium, a non-vancomycin resistant enterococcus (VRE) was successfully produced by a fed-batch cultivation approach and scaled up to a 15-L bioreactor using a ramp feeding strategy. Results from this study revealed that the fed-batch cultivation using molasses-based medium has industrial potential for the production of probiotics.

Substrate-Flexible Two-Stage Fed-Batch Cultivations for the Production of the PHA Copolymer P(HB-co-HHx) With Cupriavidus necator Re2058/pCB113.

Recent studies of the impact and dimension of plastic pollution have drawn the attention to finding more sustainable alternatives to fossil-based plastics. Microbially produced polyhydroxyalkanoates (PHAs) biopolymers are strong candidates to replace conventional plastic materials, due to their true biodegradability and versatile properties. However, widespread use of these polymers is still hindered by their high cost of production. In the present study, we target high yields of the PHA copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] using a substrate-flexible two-stage fed-batch approach for the cultivation of the recombinant Cupriavidus necator strain Re2058/pCB113. A more substrate-flexible process allows to cope with constant price fluctuations and discontinuous supply of feedstocks on the market. Utilizing fructose for biomass accumulation and rapeseed oil for polymer production resulted in a final biomass concentration of 124 g L–1 with a polymer content of 86 wt% holding 17 mol% of HHx. Productivities were further optimized by operating the biomass accumulation stage in a “drain and fill” modus where 10% of the culture broth was recycled for semi-continuous biomass accumulation, after transferring 90% to a second bioreactor for PHA production. This strategy succeeded in shortening process times rising productivity yields to ∼1.45 g L–1 h–1.

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

ABSTRACT Most of the current industrial processes for L-leucine production are based on fermentation, usually in fed-batch operation mode. Although the culture technology has advanced in recent decades, the process still has significant drawbacks. To solve these problems, we investigated the effects of chemostat culture conditions on the production of L-leucine by Corynebacterium glutamicum CP. The dilution rate, the nitrogen source, and the carbon–nitrogen ratio of the medium were optimized. With the addition of ammonium acetate to the chemostat medium, the initial C/N ratio was adjusted to 57.6, and the L-leucine titer reached the highest level at the optimal dilution rate of 0.04 h−1. Compared with fed-batch culture, the L-leucine titer was reduced (from 53.0 to 24.8 g L−1), but the yield from glucose was increased by 10.0% (from 0.30 to 0.33 mol mol−1) and productivity was increased by 58.3% (from 1.2 to 1.9 g L−1 h−1). Moreover, the titer of the by-product L-alanine was significantly reduced (from 8.9 to 0.8 g L−1). In addition, gene expression levels and activity of key enzymes in the synthesis of L-leucine and L-alanine were analyzed to explain the difference of production performance between chemostat culture and fed-batch culture. The results indicate that chemostat culture has great potential to increase the industrial production of L-leucine compared to current fed-batch approaches.