High Cell Density Cultivation Research Articles

Economic optimization of expression of soluble human epidermal growth factor in Escherichia coli.

Human epidermal growth factor (hEGF) has multiple biological functions, such as promoting cell proliferation, differentiation, and migration. In addition, it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-extracellular engineered strain of expressed hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli. Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10mg/L of soluble hEGF, a final cell density of 57.4 ± 2g/L DCW (OD600 141.1 ± 4.9), and hEGF productivity of 14.3mg/L/h were obtained using a bioreactor at 32°C for 20h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.

High-level recombinant protein production with Corynebacterium glutamicum using acetate as carbon source.

In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate-utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3-fold higher fluorescence level for acetate-based eYFP production with T7 expression strain MB001(DE3) pMKEx2-eyfp compared to MB001 pEKEx2-eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass-specific production for T7 RNAP-dependent eYFP production using acetate as carbon source. Furthermore, high-level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH-coupled online feeding control, resulting in a final protein titer of 2.7g/L and product yield of 4g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low-cost acetate into high-value bioproducts, such as recombinant proteins.

Selumetinib - a potential small molecule inhibitor for osteoarthritis treatment.

Objectives: Osteoarthritis (OA) is a common disease that mainly manifests as inflammation and destruction of cartilage and subchondral bone. Recently, necroptosis has been reported to play an important role in the development of OA. Selumetinib displays a contrasting expression pattern to necroptosis-related proteins. The present study aimed to investigate the potential therapeutic effects of selumetinib in OA process. Methods: In vitro experiments, interleukin-1β (IL-1β) was used to induce necroptosis of chondrocytes. We used high-density cell culture, Western Blot and PT-PCR to observe the effect of different concentrations of selumetinib on the extracellular matrix of cartilage. Afterwards, we visualized the effect of selumetinib on osteoclast formation by TRAP staining and F-actin rings. In vivo experiment, we induced experimental osteoarthritis in mice by surgically destabilizing the medial meniscus (DMM) while administering different concentrations of selumetinib intraperitoneally. Results: Selumetinib promoted cartilage matrix synthesis and inhibited matrix decomposition. We found that selumetinib exerted a protective function by inhibiting the activation of RIP1/RIP3/MLKL signaling pathways in chondrocytes. Selumetinib also inhibited the activation of RANKL-induced NF-κB and MAPK signaling pathways in BMMs, thereby interfering with the expression of osteoclast marker genes. In the DMM-induced OA model, a postsurgical injection of selumetinib inhibited cartilage destruction and lessened the formation of TRAP-positive osteoclasts in subchondral bone. Conclusion: Selumetinib can protect chondrocytes by regulating necroptosis to prevent the progression of OA and reduce osteoclast formation. In summary, our findings suggest that selumetinib has potential as a therapeutic agent for OA.

Mineral medium design based on macro and trace element requirements for high cell density cultures of Priestia megaterium DSM 509

To assess the suitability of a mineral medium selected from the literature for high cell density cultivations of Priestia megaterium DSM 509 (ultimately for producing PHA), fermentations were planned and tested in a (2 L) bioreactor. After few hours of a batch culture, growth was limited despite an efficient oxygenation and the relative abundance of macronutrients, suggesting trace elements (TE) deficiencies. They had to be increased to achieve higher growth rates and cell densities. Growth was improved by adding up to 10 times more TE as maximum growth rate increased from 0.20 to 2.36 g L−1 h−1, but eventually growth limitations occurred. Major and trace minerals were therefore determined by ICP-OES. These analyses showed that the selected medium was deficient mainly in Co, Mn and Zn. Requirements for 11 minerals could then be assessed and used to formulate a new mineral medium. Tested in a fed-batch culture assigned to a specific growth rate of 0.2 h−1, it allowed P. megaterium to grow in 8 h during the batch phase without being limited until the exhaustion of glucose. In the fed-batch phase, high cell densities of 53.6 g L−1 were reached after 10 h with a space-time yield of 2.96 g L−1 h−1.

Evaluation of different nutrient limitation strategies for the efficient production of poly(hydroxybutyrate‐co‐hydroxyvalerate) from waste frying oil and propionic acid in high cell density fermentations of Cupriavidus necator H16

Because of its application potential and biodegradability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate;PHBV), a member of the polyhydroxyalkanoates (PHA) biopolymer family, is one of the most extensively studied PHA. High PHBV productivity with a significant amount of hydroxyvalerate (HV) content is very appealing for commercial scale production. The goal of this study was to investigate the efficiency of various defined limitation strategies, namely nitrogen, phosphorus, and oxygen-limitation, for high yield PHBV production by Cupriavidus necator H16 with increased HV unit using waste frying vegetable oil (WFO) and propionic acid (PA) in a high cell density culture (5 L bioreactor). With optimized WFO and PA feeding, highest PHBV harvest (121.7 ± 2.59 g/L; HV 13.9 ± 0.44% (w/w)) and volumetric productivity (2.03 ± 0.04 gPHBV/L·h) were obtained in oxygen-limited operation, while highest HV content (19.8 ± 0.28 wt%) and yield coefficient (0.43 ± 0.017 gHV/gPA) were observed during phosphorus-limited cultivation. Although nitrogen limitation is widely applied in the production of PHA, nitrogen-limited cultivation had the lowest cell dry matter, PHBV production, volumetric productivity, oil-to-HB and PA-to-HV yield coefficients for the given conditions. The results of the present study demonstrate the highest PHBV yield together with the highest HV content using WFO as main carbon source and PA as the HV precursor ever reported in the literature.

RSPO3 is a novel contraction-inducible factor identified in an “in vitro exercise model” using primary human myotubes

The physiological significance of skeletal muscle as a secretory organ is now well known but we can only speculate as to the existence of as-yet-unidentified myokines, especially those upregulated in response to muscle contractile activity. We first attempted to establish an “insert-chamber based in vitro exercise model” allowing the miniature but high cell-density culture state enabling highly developed contractile human myotubes to be readily obtained by applying electric pulse stimulation (EPS). By employing this in vitro exercise model, we identified R-spondin 3 (RSPO3) as a novel contraction-inducible myokine produced by cultured human myotubes. Contraction-dependent muscular RSPO3 mRNA upregulation was confirmed in skeletal muscles of mice subjected to sciatic nerve mediated in situ contraction as well as those of mice after 2 h of running. Pharmacological in vitro experiments demonstrated a relatively high concentration of metformin (millimolar range) to suppress the contraction-inducible mRNA upregulation of human myokines including RSPO3, interleukin (IL)-6, IL-8 and CXCL1. Our data also suggest human RSPO3 to be a paracrine factor that may positively participate in the myogenesis processes of myoblasts and satellite cells. Thus, the “insert chamber-based in vitro exercise model” is a potentially valuable research tool for investigating contraction-inducible biological responses of human myotubes usually exhibiting poorer contractility development even in the setting of EPS treatment.

Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization

Acidic and chemical inhibitor stresses undermine efficient lactic acid bioproduction from lignocellulosic feedstock. Requisite coping treatments, such as detoxification and neutralizing agent supplementation, can be eliminated if a strong microbial host is employed in the process. Here, we exploited an originally robust yeast, Saccharomyces cerevisiae BTCC3, as a production platform for lactic acid. This wild-type strain exhibited a rapid cell growth in the presence of various chemical inhibitors compared to laboratory and industrial strains, namely BY4741 and Ethanol-red. Pathway engineering was performed on the strain by introducing an exogenous LDH gene after disrupting the PDC1 and PDC5 genes. Facilitated by this engineered strain, high cell density cultivation could generate lactic acid with productivity at 4.80 and 3.68 g L−1 h−1 under semi-neutralized and non-neutralized conditions, respectively. Those values were relatively higher compared to other studies. Cultivation using real lignocellulosic hydrolysate was conducted to assess the performance of this engineered strain. Non-neutralized fermentation using non-detoxified hydrolysate from sugarcane bagasse as a medium could produce lactic acid at 1.69 g L−1 h−1, which was competitive to the results from other reports that still included detoxification and neutralization steps in their experiments. This strategy could make the overall lactic acid bioproduction process simpler, greener, and more cost-efficient.

Advanced pathway engineering for phototrophic putrescine production

SummaryThe polyamine putrescine (1,4‐diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2‐based bio‐production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9‐based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5‐fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co‐production of cadaverine and 4‐aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10‐fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

The Increase in the Drug Resistance of Acute Myeloid Leukemia THP-1 Cells in High-Density Cell Culture Is Associated with Inflammatory-like Activation and Anti-Apoptotic Bcl-2 Proteins.

It is known that cell culture density can modulate the drug resistance of acute myeloid leukemia (AML) cells. In this work, we studied the drug sensitivity of AML cells in high-density cell cultures (cell lines THP-1, HL-60, MV4-11, and U937). It was shown that the AML cells in high-density cell cultures in vitro were significantly more resistant to DNA-damaging drugs and recombinant ligand izTRAIL than those in low-density cell cultures. To elucidate the mechanism of the increased drug resistance of AML cells in high-density cell cultures, we studied the activation of Bcl-2, Hif-1alpha, and NF-kB proteins, as well as cytokine secretion, the inflammatory immunophenotype, and the transcriptome for THP-1 cells in the low-density and high-density cultures. The results indicated that the increase in the drug resistance of proliferating THP-1 cells in high-density cell cultures was associated with the accumulation of inflammatory cytokines in extracellular medium, and the formation of NF-kB-dependent inflammatory-like cell activation with the anti-apoptotic proteins Bcl-2 and Bcl-xl. The increased drug resistance of THP-1 cells in high-density cultures can be reduced by ABT-737, an inhibitor of Bcl-2 family proteins, and by inhibitors of NF-kB. The results suggest a mechanism for increasing the drug resistance of AML cells in the bone marrow and are of interest for developing a strategy to suppress this resistance.

Neratinib exerts dual effects on cartilage degradation and osteoclast production in Osteoarthritis by inhibiting the activation of the MAPK/NF-κB signaling pathways

Osteoarthritis (OA) is a degenerative disease caused by the progressive destruction of cartilage and subchondral bone [1]. Studies have shown that by inhibiting the degradation of cartilage cells and the loss of subchondral bone, OA can be prevented and treated. Neratinib, as a small molecule compound with anti-inflammatory and anti-tumor properties, is a very effective inhibitor of IL-1β-induced chondrocyte inflammation and anabolic metabolism. By investigating the effect of neratinib in ATDC5 chondrocytes, the study finds that neratinib reduces inflammation by inhibiting the MAPK and NF-κB signaling pathways, and at the same time reduces pyrolysis (indicated by the results of reverse transcription quantitative PCR and western blotting). For anabolic metabolism, after high-density cell culture, IL-1β-induced catalytic changes and degradation of the extracellular matrix were evaluated by toluidine blue staining. Since osteoclasts are key participants in the process of subchondral bone remodeling in OA, we also studied the effect of neratinib on the maturation of osteoclasts. The results showed that neratinib also acts as an anti-osteoclast agent in vitro. By inhibiting the NF-κB and MAPK pathways, it reduces the expression of osteoclast-related genes, thereby inhibiting RANKL-induced osteoclastogenesis. The results of in vivo animal experiments supported the conclusions from the experiments in vitro. Neratinib inhibited both the destruction of medial meniscus induced cartilage degradation and osteoclast formation, which proves that neratinib has a dual effect, protecting cartilage and inhibiting osteoclast formation. These results indicate that neratinib can be a brand-new latent strategy for the treatment of OA.

Three-dimensional cell culture approach for in vitro immunization and the production of monoclonal antibodies

The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10–12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.

Intensifying Continuous Production of Gag-HA VLPs at High Cell Density Using Stable Insect Cells Adapted to Low Culture Temperature.

Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus–like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (kD = 0.009 vs. 0.005 h−1, for TFF and ATF, respectively). Reducing CSPR to 0.01–0.02 nL/cell.d using ATF had a negligible impact on specific production rates (rHA = 72–68 titer/109 cell.h and rp24 = 12–11 pg/106 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.

Culture media selection and feeding strategy for high titer production of a lentiviral vector by stable producer clones cultivated at high cell density

The growing interest in the use of lentiviral vectors (LVs) for various applications has created a strong demand for large quantities of vectors. To meet the increased demand, we developed a high cell density culture process for production of LV using stable producer clones generated from HEK293 cells, and improved volumetric LV productivity by up to fivefold, reaching a high titer of 8.2 × 107 TU/mL. However, culture media selection and feeding strategy development were not straightforward. The stable producer clone either did not grow or grow to lower cell density in majority of six commercial HEK293 media selected from four manufacturers, although its parental cell line, HEK293 cell, grows robustly in these media. In addition, the LV productivity was only improved up to 53% by increasing cell density from 1 × 106 and 3.8 × 106 cells/mL at induction in batch cultures using two identified top performance media, even these two media supported the clone growth to 5.7 × 106 and 8.1 × 106 cells/mL, respectively. A combination of media and feed from different companies was required to provide diverse nutrients and generate synergetic effect, which supported the clone growing to a higher cell density of 11 × 106 cells/mL and also increasing LV productivity by up to fivefold. This study illustrates that culture media selection and feeding strategy development for a new clone or cell line can be a complex process, due to variable nutritional requirements of a new clone. A combination of diversified culture media and feed provides a broader nutrients and could be used as one fast approach to dramatically improve process performance.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L−1 with a maximal productivity of 78 mg L−1 day−1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

Strain Construction and Process Development for Efficient Recombinant Production of Mannuronan C-5 Epimerases in Hansenula polymorpha.

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Impact of Elevated Levels of Dissolved CO2 on Performance and Proteome Response of an Industrial 2'-Fucosyllactose Producing Escherichia coli Strain.

Large-scale microbial industrial fermentations have significantly higher absolute pressure and dissolved CO2 concentrations than otherwise comparable laboratory-scale processes. Yet the effect of increased dissolved CO2 (dCO2) levels is rarely addressed in the literature. In the current work, we have investigated the impact of industrial levels of dCO2 (measured as the partial pressure of CO2, pCO2) in an Escherichia coli-based fed-batch process producing the human milk oligosaccharide 2′-fucosyllactose (2′-FL). The study evaluated the effect of high pCO2 levels in both carbon-limited (C-limited) and carbon/nitrogen-limited (C/N-limited) fed-batch processes. High-cell density cultures were sparged with 10%, 15%, 20%, or 30% CO2 in the inlet air to cover and exceed the levels observed in the industrial scale process. While the 10% enrichment was estimated to achieve similar or higher pCO2 levels as the large-scale fermentation it did not impact the performance of the process. The product and biomass yields started being affected above 15% CO2 enrichment, while 30% impaired the cultures completely. Quantitative proteomics analysis of the C-limited process showed that 15% CO2 enrichment affected the culture on the protein level, but to a much smaller degree than expected. A more significant impact was seen in the dual C/N limited process, which likely stemmed from the effect pCO2 had on nitrogen availability. The results demonstrated that microbial cultures can be seriously affected by elevated CO2 levels, albeit at higher levels than expected.

Towards high-level protein, beta-carotene, and lutein production from Chlorella sorokiniana using aminobutyric acid and pseudo seawater

Microalgae produced lipids, pigments, lutein, and valuable chemicals under stressful conditions, making them as attractive feedstock for biorefineries. However, high cell density culture of microalgae in harsh condition using seawater is still a challenge. In this study, we used aminobutyric acid (GABA) from an in vitro biotransformation of monosodium glutamate (MSG) to alleviate salinity stress in Chlorella sorokiniana (Cs). Initially, Chlorophyll a/b content was suppressed under the stress of 20 g/L sodium chloride (NaCl), leading to an extreme decline in protein and pigment accumulation. While the biomass and protein of Cs cultured in 10 g/L NaCl reached 3.44 g/L and 1.14 g/L with 5 mM GABA, with 56.3% and 171% increase compared to control. The optimal conditions were 10 mM GABA and 20 g/L NaCl in modified BG11 medium, with a biomass, protein, lutein and β-carotene content of 4.56 g/L, 2.07 g/L, 24 mg/L and 27.48 mg/L, respectively. Finally, protein from Cs was used as a prebiotic for cultivating Escherichia coli Nissle 1917 (EcN) and Lactobacillus rhamnosus ZY (LGG). We provided an effective and sustainable approach for the simultaneous production of biomass and high-end products by microalgae using GABA and seawater for the first time.

Enhanced co-production of medium-chain-length polyhydroxyalkanoates and phenazines from crude glycerol by high cell density cultivation of Pseudomonas chlororaphis in membrane bioreactor

Enhanced co-production of medium-chain-length polyhydroxyalkanoates (mcl-PHA) and extracellular phenazines was assessed through a high cell density cultivation of Pseudomonas chlororaphis subsp. aurantiaca (DSM 19603) in a membrane bioreactor using crude glycerol as a fermentative substrate. A maximum dry cell weight (DCW) of 59.25 ± 0.31 g/L was achieved at 90 h of cultivation with a maximum mcl-PHA and extracellular phenazines concentrations of respectively 19.05 ± 0.04 g/L (32.16% of DCW) and 79.42 ± 0.35 mg/L. mcl-PHA concentration achieved through cell retention culture was 28.43-folds higher than that obtained by batch culture. Fourier transform infrared spectroscopy, gas chromatography–mass spectrometry, and 1H nuclear magnetic resonance analysis identified the produced PHA as a mcl-PHA copolymer of 3-hydroxyhexanoate (0.68%), 3-hydroxyoctanoate (7.76%), 3-hydroxydecanoate (49.18%), 3-hydroxydodecanoate (4.89%), and 3-hydroxytetradecanoate (37.50%). The mcl-PHA exhibited a highly amorphous structure with low crystallinity index (4.19%) and high thermal stability. This is the first report on the enhanced co-production of mcl-PHA and phenazines in a membrane bioreactor.

Fine-tuned regulation of photosynthetic performance via γ-aminobutyric acid (GABA) supply coupled with high initial cell density culture for economic starch production in microalgae

Microalgal starch is considered as renewable and sustainable feedstock for biofuels and biorefinery. High cell density culture is favourable for photoautotrophic starch production in microalgae in the aspects of productivity and economy, but it often encounters low starch content or extra stress exposure that limits the production. This study aimed to economically enhance photosynthetic starch production from CO2 fixation in a green microalga Tetraselmis subcordiformis by regulating photosynthetic stress status with a signalling molecule γ-aminobutyric acid (GABA) combined with the application of high initial cell density culture. By increasing initial cell density (ICD) from the normal of 1.1 g L−1 (NICD) to as high as 2.8 g L−1 (HICD), the starch content, yield, and theoretical productivity were improved by 7%, 63%, and 42%, respectively. The addition of GABA under HICD resulted in 14%, 19%, and 26% of further enhancement in starch content, yield, and theoretical productivity, respectively. GABA exhibited distinct regulatory mechanisms on photosynthesis and stress status under HICD relative to NICD. GABA augmented excessive light energy absorption and electron transfer through photosystem II that reinforced the photoinhibition under NICD, while alleviated the stress reversely under HICD, both of which facilitated starch production by enabling a suitable stress status while simultaneously maintaining a sufficient photosynthetic activity. The increase of ICD and/or GABA supply particularly boosted amylopectin accumulation, leading to the changes in starch composition and was more favourable for fermentation-based biofuels production. Preliminary techno-economic analysis showed that the highest net extra benefit of 9.64 $ m−3 culture could be obtained under HICD with 2.5 mM GABA supply where high starch content (62%DW) and yield (2.5 g L−1) were achieved. The combined HICD-GABA regulation was a promising strategy for economic starch production from CO2 by microalgae for sustainable biomanufacturing.Graphical