Fed-batch System Research Articles

CFD-guided scaling of Pseudomonas putida fermentation

This study evaluated the scale-up of Pseudomonas putida fed-batch fermentation from a 2 L benchtop-scale stirred bioreactor to a 200 L pilot-scale tank by using a validated computational fluid dynamic (CFD) model. One of the major concerns in this fermentation process is the potential reduction in mixing quality with increasing scale, leading to lower yield or product quality. For a low-risk scale-up, a multiphase Euler-Euler CFD model was developed that simulated the hydrodynamics of the fed-batch system at various filling volumes, representing different stages of the fermentation process. The model was validated using experimental data of mixing time and mass transfer coefficient. The hydrodynamic model was then coupled with a Monod kinetic model of P. putida ‘s fermentation. Response surface methodology was used to generate a performance map of the pilot bioreactor at various aeration, agitation, and bioreactor filling volumes. The study considered different established scale-up approaches, such as constant tip speed and aeration rate across scales, constant kLa, as well as constant power to unit of liquid volume (P/V). The performance of the bioreactor was assessed, and the optimum operating ranges of the input parameters were obtained at different stages of the fermentation. Using performance map the possibility of substrate and oxygen gradient formation, and the gradient severity inside the pilot bioreactor at different working volumes were evaluated.

Automated yeast cultivation control using a biosensor and flow cytometry.

Effective microbial bioprocessing relies on maintaining ideal cultivation conditions, highlighting the necessity for tools that monitor and regulate cellular performance and robustness. This study evaluates a fed-batch cultivation control system based on at-line flow cytometry monitoring of intact yeast cells having a fluorescent transcription factor-based redox biosensor. Specifically, the biosensor assesses the response of an industrial xylose-fermenting Saccharomyces cerevisiae strain carrying the TRX2p-yEGFP biosensor for NADPH/NADP+ ratio imbalance when exposed to furfural. The developed control system successfully detected biosensor output and automatically adjusted furfural feed rate, ensuring physiological fitness at high furfural levels. Moreover, the single-cell measurements enabled the monitoring of subpopulation dynamics, enhancing control precision over traditional methods. The presented automated control system highlights the potential of combining biosensors and flow cytometry for robust microbial cultivations by leveraging intracellular properties as control inputs.

Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.

Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440.

p-Coumaric acid (p-CA) is a valuable compound with applications in food additives, cosmetics, and pharmaceuticals. However, traditional production methods are often inefficient and unsustainable. This study focuses on enhancing p-CA production efficiency through the heterologous expression of tyrosine ammonia-lyase (TAL) from Rhodobacter sphaeroides in Pseudomonas putida KT2440. TAL catalyzes the conversion of L-tyrosine into p-CA and ammonia. We engineered P. putida KT2440 to express TAL in a fed-batch fermentation system. Our results demonstrate the following: (i) successful integration of the TAL gene into P. putida KT2440 and (ii) efficient bioconversion of L-tyrosine into p-CA (1381 mg/L) by implementing a pH shift from 7.0 to 8.5 during fed-batch fermentation. This approach highlights the viability of P. putida KT2440 as a host for TAL expression and the successful coupling of fermentation with the pH-shift-mediated bioconversion of L-tyrosine. Our findings underscore the potential of genetically modified P. putida for sustainable p-CA production and encourage further research to optimize bioconversion steps and fermentation conditions.

Photosynthetic distribution inside a cylindrical photobioreactor for Botryococcus braunii: A fed-batch culture application

In this study, the physiological responses and kinetic parameters of Botryococcus braunii were analyzed during a highly irradiated fed-batch culture in a cylindrical photobioreactor. This work includes a model of photon distribution throughout the bioreactor, coupled with an analysis of photosynthetic performance to simulate the level of photosaturation, photolimitation, or photoinhibition during cell growth culture. Under this simulation, the kinetic and physiological behavior was evaluated during four cycles of fed-batch culture. Physiological responses showed cellular photoacclimation to high irradiance conditions, increasing the Electron Transport Rate (ETR) values 2.5 times from cycle 1 to cycle 4, while photoprotection mechanisms such as quantum yield YII and non-photochemical quenching NPQ remained efficient. The total pigment content was low compared to other studies, and a gradual decrease in Chl a, Chl b, and carotenoids was evident between each fed-batch cycle (a 25 % reduction from cycle 1 to cycle 4). Kinetics showed a gradual increase in productivity, growth rates, and nutrient consumption between cycles. The maximum biomass concentration as dry weight (4.86 ± 0.26gL−1) was obtained in cycle 4, which also showed the highest values of productivity (0.45gL−1d−1), specific growth rate (0.134 d−1) and consumption rate (NO3, PO4). The results correlate the engineering approach to the physiological characteristics of the species, indicating that it is possible to establish an efficient fed-batch system for microalgae production.

Enhancing Protein Content in Wild-Type Saccharomyces cerevisiae via Random Mutagenesis and Optimized Fermentation Conditions.

Single-cell protein (SCP) derived from microorganisms is widely recognized as a viable alternative protein source for the future. Nevertheless, the commercialization of yeast-based SCP is hampered by its relatively low protein content. Therefore, this study aimed to enhance the protein content of Saccharomyces cerevisiae via random mutagenesis. To achieve this, S. cerevisiae KCCM 51811, which exhibited the highest protein concentration among 20 edible S. cerevisiae strains, was selected as a chassis strain. Subsequently, a KCCM 51811 mutant library was constructed (through UV irradiation) and screened to isolate mutants exhibiting high protein content and/or concentration. Among the 174 mutant strains studied, the #126 mutant exhibited a remarkable 43% and 36% higher protein content and concentration, respectively, compared to the parental strain. Finally, the #126 mutant was cultured in a fed-batch system using molasses and corn-steep liquor, resulting in a protein concentration of 21.6 g/l in 100 h, which was 18% higher than that produced by the parental strain. These findings underscore the potential of our approach for the cost-effective production of food-grade SCP.

Hypoxia-activated ADCC-enhanced humanized anti-CD147 antibody for liver cancer imaging and targeted therapy with improved selectivity.

Therapeutic antibodies (Abs) improve the clinical outcome of cancer patients. However, on-target off-tumor toxicity limits Ab-based therapeutics. Cluster of differentiation 147 (CD147) is a tumor-associated membrane antigen overexpressed in cancer cells. Ab-based drugs targeting CD147 have achieved inadequate clinical benefits for liver cancer due to side effects. Here, by using glycoengineering and hypoxia-activation strategies, we developed a conditional Ab-dependent cellular cytotoxicity (ADCC)-enhanced humanized anti-CD147 Ab, HcHAb18-azo-PEG5000 (HAP18). Afucosylated ADCC-enhanced HcHAb18 Ab was produced by a fed-batch cell culture system. Azobenzene (Azo)-linked PEG5000 conjugation endowed HAP18 Ab with features of hypoxia-responsive delivery and selective targeting. HAP18 Ab potently inhibits the migration, invasion, and matrix metalloproteinase secretion, triggers the cytotoxicity and apoptosis of cancer cells, and induces ADCC, complement-dependent cytotoxicity, and Ab-dependent cellular phagocytosis under hypoxia. In xenograft mouse models, HAP18 Ab selectively targets hypoxic liver cancer tissues but not normal organs or tissues, and has potent tumor-inhibiting effects. HAP18 Ab caused negligible side effects and exhibited superior pharmacokinetics compared to those of parent HcHAb18 Ab. The hypoxia-activated ADCC-enhanced humanized HAP18 Ab safely confers therapeutic efficacy against liver cancer with improved selectivity. This study highlights that hypoxia activation is a promising strategy for improving the tumor targeting potential of anti-CD147 Ab drugs.

Coupling of CO2 Electrolysis with Parallel and Semi-Automated Biopolymer Synthesis - Ex-Cell and without Downstream Processing.

Important improvements have been achieved in developing the coupling of electrochemical CO2 reduction to formate with its subsequent microbial conversion to polyhydroxybutyrate (PHB) by Cupriavidus necator. The CO2 based formate electrosynthesis was optimised by electrolysis parameter adjustment and application of Sn based gas diffusion electrodes reaching almost 80 % Faradaic efficiency at 150 mA cm-2. Thereby, catholyte with the high formate concentration of 441±9 mmol L-1 was generated as feedstock without intermediate downstream processing for semi-automated formate feeding into a fed-batch reactor system. Moreover, microbial formate conversion to PHB was studied further, optimised, and successfully scaled from shake flasks to semi-automated bioreactors. Therein, a PHB per formate ratio of 16.5±4.0 mg g-1 and a PHB synthesis rate of 8.4±2.1 mg L-1 OD-1 h-1 were achieved. By this process combination, an almost doubled overall process yield of 22.3±5.5 % was achieved compared to previous reports. The findings allow a detailed evaluation of the overall CO2 to PHB conversion, providing the basis for potential technical exploitation.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16

BackgroundExisting plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2.ResultsIn this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml.ConclusionThe new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Succinic acid production from softwood with genome-edited Corynebacterium glutamicum using the CRISPR-Cpf1 system.

Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock-out mutant of the lactate dehydrogenase 1 gene (ΔldhA-6) and co-expressed the succinic acid transporter (Psod:SucE- ΔldhA) using the CRISPR-Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid. Upon evaluating a 1%-5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82g L-1 succinic acid production over 6h. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:SucE-ΔldhA] produced 17.70g L-1 succinic acid in 6h. In the fed-batch system, 39.67g L-1 succinic acid was produced over 48h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.

Osmotic Stress Alleviation in Saccharomyces cerevisiae for High Ethanol Fermentations with Different Wort Substrates

High-gravity fermentation, used for ethanol production from sugarcane, corn, and mixed substrates, offers several benefits. Yeast, a rapidly multiplying unicellular microorganism, can be adapted for high sugar and ethanol tolerance on a lab scale. However, different substrates can enhance fermentation efficiency. Our study consisted of two experiments. In the first, we compared simple batch feeding with a fed-batch system for yeast selection in high-gravity fermentation. We ran eight cycles with increasing initial sugar contents (50 to 300 g L−1). No significant differences were observed in the first seven cycles, but in the eighth, the fed-batch system showed lower glycerol and fructose contents and higher cell viability than the simple batch system. In the second experiment, we used the fed-batch system with 300 g L−1 from sugarcane, corn, and mixed wort. The results showed that mixed wort produced higher ethanol contents and greater fermentation efficiency compared to corn and sugarcane as substrates. In conclusion, our findings indicate that the fed-batch system is more suitable for high-gravity fermentation on a lab scale, and the combination of sugarcane juice and corn can enhance fermentation efficiency, paving the way for integrating these substrates in industrial ethanol production.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.Graphical

Identification of optimal flow rate for culture media, cell density, and oxygen toward maximization of virus production in a fed-batch baculovirus-insect cell system.

In recent times, it has been realized that novel vaccines are required to combat emerging disease outbreaks, and faster optimization is required to respond to global vaccine demands. Although, fed-batch operations offer better productivity, experiment-based optimization of a new fed-batch process remains expensive and time-consuming. In this context, we propose a novel computational framework that can be used for process optimization and control of a fed-batch baculovirus-insect cell system. Since the baculovirus expression vector system (BEVS) is known to be widely used platforms for recombinant protein/vaccine production, we chose this system to demonstrate the identification of optimal profile. Toward this, first, we constructed a mathematical model that captures the time course of cell and virus growth in a baculovirus-insect cell system. Second, the proposed model was used for numerical analysis to determine the optimal operating profiles of control variables such as culture media, cell density, and oxygen based on a multiobjective optimal control formulation. Third, a detailed comparison between batch and fed-batch culture was perfromed along with a comparison between various alternatives of fed-batch operation. Finally, we demonstrate that a model-based quantification of controlled feed addition in fed-batch culture is capable of providing better productivity as compared to a batch culture. The proposed framework can be utilized for the estimation of optimal operating regions of different control variables to achieve maximum infected cell density and virus yield while minimizing the substrate/media, uninfected cell, and oxygen consumption.

Sugar Alcohol Sweetener Production by Yarrowia lipolytica Grown in Media Containing Glycerol.

Most of the world's annual production of mannitol is by chemical means, but, due to increasing demand for natural sweeteners, alternative production methods are being sought. The aim of the study was to screen Yarrowia lipolytica yeast strains and select culture conditions for the efficient and selective biosynthesis of mannitol from glycerol. From 21 strains examined in the shake-flask culture for mannitol biosynthesis from glycerol (100 g/L), three strains were selected-S2, S3, and S4-and further evaluated in batch bioreactor cultures with technical and raw glycerol (150 g/L). The best production parameters were observed for strain S3, which additionally was found to be the most resistant to NaCl concentration. Next, strain S3 was examined in batch culture with regard to the initial glycerol concentration (from 50 to 250 g/L). It was found that the substrate concentrations of 50 and 75 g/L resulted in the highest mannitol selectivity, about 70%. The fed-batch culture system proposed in this paper (performed in two variants in which glycerol was dosed in four portions of about 50 or 75 g/L) resulted in increased mannitol production, up to 78.5 g/L.

Analysis and study of the bioelectric production potential of actinomycete and microbial isolates in industrial glass factory wastewater using a microbial fuel cell

A microbial fuel cell (MFC), a novel technology, is a biochemical catalyzer system that can convert the chemical energy of materials to bioelectric energy. This system can serve as a unique device for the treatment of wastewater. Based on this knowledge, we decided to study the bioenergy production ability of Actinomycete and microbial isolates in industrial glass factory wastewater as the decomposers of organic materials in this wastewater and the generation of Voltage and current in two batches and fed-batch conditions. At the most favorable condition maximum of 1.08 V (current 3.66 mA and power density 2.88 mW/m2), 81.2% chemical oxygen demand was obtained for a fed-batch system. Also, the outcomes of MFC’s essential parameters, for example, pH and TDS, were studied before and after the performance of MFC. The results showed a significant decrease after the operation of the MFC. To realize which Actinomycete were the most powerful bioelectric microorganism, the growth curve and electricity performance of three kinds of Actinomycete was selected. Results showed that the C2 would be more potent because its Voltage of 0.224 V and current of 1.187 mA possessed by it would result in an excellent power density of 141.42 mW/m2.

Enhanced production of nicotinamide mononucleotide by high cell density culture of engineered Escherichia coli

Nicotinamide mononucleotide (NMN), an intermediate product of NAD+ biosynthesis, is commonly found intracellularly in living species. Recently, NMN has attracted great interest for the treatment of different diseases. Here, we developed a high cell density fed-batch culture system to produce NMN at higher concentrations using an engineered E. coil strain. Fed-batch culture using linear feeding strategy exhibited 9.38 ± 0.8, 14.8 ± 0.56, and 5.04 ± 0.21 g/L NMN concentrations, and 59.5 ± 0.44, 68.8 ± 0.49, and 36.2 ± 0.32 g/L dry cell weights, using different concentrations of nicotinamide (NAM) of 3, 5, and 10 g/L, respectively. A maximum NMN concentration of 17.2 ± 1.3 g/L with a productivity of 0.956 g/L/h was achieved using two intermittent additions of 5 g/L NAM after 6 h and 17 h. NMN obtained from the fermentation broth was purified using size exclusion chromatography, generating a yield of 53.9 % NMN. Liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis was additionally used to validate the purity of NMN after purification. The fed-batch fermentation process applied in this study, using cheaper feed-stocks, demonstrated a remarkable enhancement in the production of therapeutically important NMN, making this a promising strategy for industrial-scale production of NMN.

A novel hybrid system for continuous biodegradation and toxicity removal of low molecular weight phthalates

Dimethyl phthalate (DMP) and diethyl phthalate (DEP) are environmental toxicants that can interfere with the endocrine system and cause adverse health impacts by mimicking the natural hormone activity. The present study focused on the biodegradation of DMP and DEP mixture by Cellulosimicrobium bacteria in a bioreactor under batch, fed-batch, continuous and continuous with biomass recycle modes. Biomass recycling was done followed by microfiltration using an indigenous low-cost tubular ceramic membrane. Under the batch mode of operation, maximum degradation values of 85 % and 58 % were observed at 1000 and 2000 mg/L initial concentrations of DMP and DEP, respectively, in mixture, whereas, complete degradation was achieved in the fed-batch system at the same initial concentrations. In continuous operation mode at 24 h hydraulic retention time, complete degradation was achieved for all inlet DMP and DEP concentrations. The biomass recycle operation mode was found to be the best strategy owing to complete degradation of the phthalates, even at very high inlet concentrations and at a short hydraulic retention time (HRT) of 16 h. A high germination index (GI) value of 91.86 % and 0 % brine shrimp mortality further demonstrated the potential of the biomass recycle system for the treatment of phthalate containing wastewater.

Black glucose-releasing silicon elastomer rings for fed-batch operation allow measurement of the oxygen transfer rate from the top and optical signals from the bottom for each well of a microtiter plate

BackgroundIn industrial microbial biotechnology, fed-batch processes are frequently used to avoid undesirable biological phenomena, such as substrate inhibition or overflow metabolism. For targeted process development, fed-batch options for small scale and high throughput are needed. One commercially available fed-batch fermentation system is the FeedPlate®, a microtiter plate (MTP) with a polymer-based controlled release system. Despite standardisation and easy incorporation into existing MTP handling systems, FeedPlates® cannot be used with online monitoring systems that measure optically through the transparent bottom of the plate. One such system that is broadly used in biotechnological laboratories, is the commercial BioLector. To allow for BioLector measurements, while applying the polymer-based feeding technology, positioning of polymer rings instead of polymer disks at the bottom of the well has been proposed. This strategy has a drawback: measurement requires an adjustment of the software settings of the BioLector device. This adjustment modifies the measuring position relative to the wells, so that the light path is no longer blocked by the polymer ring, but, traverses through the inner hole of the ring. This study aimed at overcoming that obstacle and allowing for measurement of fed-batch cultivations using a commercial BioLector without adjustment of the relative measurement position within each well.ResultsDifferent polymer ring heights, colours and positions in the wells were investigated for their influence on maximum oxygen transfer capacity, mixing time and scattered light measurement. Several configurations of black polymer rings were identified that allow measurement in an unmodified, commercial BioLector, comparable to wells without rings. Fed-batch experiments with black polymer rings with two model organisms, E. coli and H. polymorpha, were conducted. The identified ring configurations allowed for successful cultivations, measuring the oxygen transfer rate and dissolved oxygen tension, pH, scattered light and fluorescence. Using the obtained online data, glucose release rates of 0.36 to 0.44 mg/h could be determined. They are comparable to formerly published data of the polymer matrix.ConclusionThe final ring configurations allow for measurements of microbial fed-batch cultivations using a commercial BioLector without requiring adjustments of the instrumental measurement setup. Different ring configurations achieve similar glucose release rates. Measurements from above and below the plate are possible and comparable to measurements of wells without polymer rings. This technology enables the generation of a comprehensive process understanding and target-oriented process development for industrial fed-batch processes.

Improving fed-batch culture efficiency of Rhodiola sachalinensis cells and optimizing flash extraction process of polysaccharides from the cultured cells by BBD–RSM

Fed-batch culture of plant cells can enhance biomass and bioactive compound accumulation compared with batch culture, in which the initial culture medium is one of the controllable factors in the fed-batch culture system. Rhodiola sachalinensis is a valuable medicinal herb and its cell culture is an alternative approach to obtaining plant material. R. sachalinensis cells has been previously cultured in a fed-batch culture system and the initial culture medium has been found to critically affect production yield. Therefore, this study investigated the effect of nitrogen concentration, ammonia and nitrate ratio (NH4+/NO3-), and phosphorus and calcium concentrations in initial culture medium on cell biomass, as well as salidroside and polysaccharide accumulation to improve the fed-batch culture efficiency. Then, the cultured cells were extracted by flash extraction and the extraction conditions were optimized by Box-Behnken Design Response Surface Method (BBD–RSM). Finally, the antibacterial and antibiofilm effects of the extract were examined to further utilize R. sachalinensis cell cultures. The results showed that the fed-batch culture efficiency was obviously improved when the initial culture medium was modified as 30 mM of nitrogen with 5/25 of NH4+/NO3-, 0.94 mM of phosphorus, and 2.3 mM of calcium, at which 277.3 mg/L of salidroside and 4.5 g/L of polysaccharides were obtained In flash extraction, polysaccharide yield was as the response value to optimize extraction process by controlling extraction time, extraction temperature, and liquid–solid ratio in the BBD–RSM experiments. The optimal flash extraction conditions were 55.7 s of extraction time, 56.5 °C of extraction temperature, and 37.0 mL/g of liquid–solid ratio, which the polysaccharide yield reached 12.76% under the optimized conditions. In antibacterial experiment, the cell extract (RCE) inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Salmonella choleraesuis; the biofilm of B. subtilis and S. choleraesuis was scavenged in the groups of 8 mg/mL and 16 mg/mL RCE. The findings provided a basis for further industrial production of cell cultures and a theoretical reference in using R. sachalinensis cells in the production of antibacterial-related products.

Automatic Fed-Batch Cultivation Enhances Microbial Lipid Production from Volatile Fatty Acids

Organic waste is generated worldwide, and its disposal and recycling are becoming a challenge. Due to its high carbon content, however, it may be converted into valuable products. Carbon neutrality is essential, and unstable international oil prices stress the increasing importance of biofuels significantly. Volatile fatty acids (VFA) derived from organic waste can be converted to microbial lipids by oleaginous yeast using it as a carbon source. When VFA is consumed by oleaginous yeast, the pH of the medium rises; hence, acidic agents have to be added to the medium to maintain the broth’s pH. In this study, we enhanced microbial lipid productivity by automatic fed-batch cultivation using VFA as an acidic agent, and the modified cultivation showed 48.9% and 69.0% higher biomass and lipid productivity than manual multi-fed culture. At a VFA concentration of 5 g/L and pH 7.0, a lipid yield of 0.25 g/g alongside lipid productivity of 0.11 g/L/h was obtained from an automatic fed-batch system. Oleic acid accounted for the largest proportion of microbial lipids, and the fatty acid composition was suitable for biodiesel production.