Fed-batch Mode Research Articles

Green utilization of sorghum straw by coupled hydrothermal pretreatment and cellulase hydrolysis for bacillomycin D production under fed-batch mode

The present work explored the coupled hydrothermal pretreatment and cellulase hydrolysis (HPCH) for production of bacillomycin D from sorghum straws by Bacillus subtilis NS-174. The results indicated that HPCH treated sorghum straws had weaker absorption, heavier mass loss and higher crystallinity, and displayed loose and destructive fiber structures. Degradation of hemicellulose and lignin contributhed to yielding glucose and 68.94g/L of glucose was obtained in HPCH treated hydrolysate. High level of glucose in pretreated sorghum straw hydrolysate was helpful for improving bacillomycin D production. When performed by 3rd round (feeding at 144h) fed-batch fermentation, bacillomycin D production and production rate were obtained to be 2351.07mg/L and 113.36mg/L.h in B. subtilis NS-174, respectively.

Highly-efficient co-production of microbial lipid and magnesium ammonium phosphate from N-acetyl-D-glucosamine

The valorization of chitin-rich wastes into chemicals and biofuels holds immense economic and environmental benefits. Here, N-acetyl-D-glucosamine (GlcNAc), the basic structural unit of chitin, was firstly described for co-producing microbial lipid and magnesium ammonium phosphate (MAP). Due to the strong substrate inhibition of GlcNAc, a fed-batch culture mode was successfully adopted to achieve high cell density by Cutaneotrichosporon oleaginosum. When a phosphate limitation strategy was applied, cell mass, lipid titer, content, yield, and productivity were 102.7 g/L, 74.2 g/L, 72.2 %, 21.4 g/100 g, and 0.69 g/L/h, respectively. The ammonium ion was efficiently precipitated by forming MAP with a removal rate around 95.4 %. The lipid samples showed high similarity to vegetable oil, which emerged as high-quality precursor for biodiesel production. This study offers a promising strategy for full conversion of GlcNAc into lipid and slow-release fertilizer, which provides an attractive technical route for turning the chitin-rich materials into valuable products.

De novo biosynthesis and nicotinamide biotransformation of nicotinamide mononucleotide by engineered yeast cells.

β-Nicotinamide mononucleotide (NMN) is a precursor of NAD+ in mammals. Research on NAD+ has demonstrated its crucial role against aging and disease. Here two technical paths were established for the efficient synthesis of NMN in the yeast Pichia pastoris, enabling the production of NMN from the low-cost nicotinamide (NAM) or basic carbon sources. The yeast host was systematically modified to adapt to the biosynthesis and accumulation of NMN. To improve the semi-biosynthesis of NMN from NAM, nicotinamide phosphoribosyltransferases were expressed intracellular to evaluate their catalytic activities. The accumulation of extracellular NMN was further increased by the co-expression of an NMN transporter. Fine-tuning of gene expression level produced 72.1 mg/L NMN from NAM in flasks. To achieve de novo biosynthesis NMN, a heterologous biosynthetic pathway was reassembled in yeast cells. Fine-tuning of pathway nodes by the modification of gene expression level and enhancement of precursor generation allowed efficient NMN synthesis from glucose (36.9 mg/L) or ethanol (57.8 mg/L) in flask. Lastly, cultivations in a bioreactor in fed-batch mode achieved an NMN titre of 1004.6 mg/L at 165 h from 2 g NAM and 868 g glucose and 980.4 mg/L at 91 h from 160 g glucose and 557 g ethanol respectively. This study provides a foundation for future optimization of NMN biosynthesis by engineered yeast cell factories.

Biological conversion of lignin-derived ferulic acid from wheat bran into vanillin

Lignin is a promising feedstock for producing vanillin, one of the most extensively used flavor enhancers. However, the biotransformation performance of lignin derivatives into vanillin is still unsatisfactory. In this study, an efficient conversion strategy of lignin into vanillin was established by employing engineered Saccharomyces cerevisiae as a whole-cell biocatalyst. Optimization of cell culture media and whole-cell bioconversion improved the production efficiency of vanillin. The vanillin titer reached 15.3 mM with a molar yield of 71 % in fed-batch fermentation mode, while incorporating in-situ product separation, demonstrated a remarkable 2.6-fold increase. The whole-cell bioconversion, coupled with in-situ separation, successfully converted real lignin hydrolysate into a record vanillin titer of 21.1 mM, equivalent to 1.8 mg of vanillin per gram of wheat bran biomass. The whole-cell bioconversion process integrated in-situ product separation, represents a sustainable approach for vanillin production and offers a promising pathway for lignin valorization.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Adaptation of a microbial consortium to pelagic Sargassum modifies its taxonomic and functional profile that improves biomethane potential.

In recent years, pelagic Sargassum has invaded the Caribbean coasts, and anaerobic digestion has been proposed as a sustainable management option. However, the complex composition of these macroalgae acts as a barrier to microbial degradation, thereby limiting methane production. Microbial adaptation is a promising strategy to improve substrate utilization and stress tolerance. This study aimed to investigate the adaptation of a microbial consortium to enhance methane production from the pelagic Sargassum. Microbial adaptation was performed in a fed-batch mode for 100days by progressive feeding of Sargassum. The evolution of the microbial community was analyzed by high-throughput sequencing of 16S rRNA amplicons. Additionally, 16S rRNA data were used to predict functional profiles using the iVikodak platform. The results showed that, after adaptation, the consortium was dominated by the bacterial phyla Bacteroidota, Firmicutes, and Atribacterota, as well as methanogens of the families Methanotrichaceae and Methanoregulaceae. The abundance of predicted genes related to different metabolic functions was affected during the adaptation stage when Sargassum concentration was increased. At the end of the adaptation stage, the abundance of the predicted genes increased again. The adapted microbial consortium demonstrated a 60% increase in both biomethane potential and biodegradability index. This work offers valuable insights into the development of treatment technologies and the effective management of pelagic Sargassum in coastal regions, emphasizing the importance of microbial adaptation in this context.

Impact of lowering nitrogen content in pig manure through low crude protein diets on anaerobic digestion process stability, biogas yields, and digestate composition

Lowering crude protein in pig diets can reduce nitrogen (N) excretion and alter manure characteristics. Anaerobic digestion (AD) offers potential for converting pig manure into biogas and bio-based fertilizers (i.e., digestate). However, limited research exists on the effects of N content in pig manure on AD when pigs are fed diets with varying crude protein levels. This study investigated how lowering N content in pig manure through low crude protein diets may affect AD process stability, biogas generation, and digestate properties. Manures from different dietary treatments, named as control (CON), low N (LN), and very low N (VLN), with Total Kjeldahl Nitrogen concentrations of 5.87, 5.42, and 5.15 g/L, respectively, were investigated. Daily biogas production, composition (CH4, CO2, and H2S), and digestate properties were monitored over 13 fed-batch cycles (25 ± 4 days per cycle). The experiment was conducted at 20 ± 1 °C, a condition suited for milder climate regions, using six single-stage digesters operated in sequencing fed-batch mode. Data were analyzed by ANOVA using PROC MIXED with repeated measures. Results showed that the differences in N content in pig manure due to the three dietary treatments had a limited impact on biogas generation, with specific methane yields remaining similar over time. CH4 concentrations remained stable between 60 and 65 %, ensuring high-quality biogas despite dietary variations. Differences between treatments became more pronounced with increased organic loading rates (OLRs) due to variations in the amount of volatile solids fed. AD also remained stable (Total Volatile Fatty Acids/Total Alkalinity <0.25) even at an OLR of 2.15 g of chemical oxygen demand L−1 day−1, highlighting AD's robustness at lower temperatures. Digestate samples contained essential minerals beneficial for plant growth. More research is needed to explore varied manure compositions and feeding strategies to better understand the interactions of animal nutrition with AD.

Engineering the cell wall reactive groups of Plant Growth Promoting Rhizobacteria by culture strategy for heavy metal removal

This research delved into the effects of nutrient limitation on the level of sporulation and the cadmium adsorption capacity of the bacterium Bacillus sp. isolated from the rhizosphere of endemic soils in the Region of Valparaiso, Chile. The bacteria were subjected to nitrogen limitation in fed-batch mode and were compared to bacteria grown in batch culture without nutrient limitation. The cultures were carried out in a 3 L bioreactor with an external nitrogen supply of ammonium at a flow of 0.123 L h−1. The specific maximum growth rate was 0.42 h−1 in batch and 0.45 h−1 in the exponential phase of the fed-batch.The analysis of sporulation did not show any significant difference between the biomass coming from the fed-batch and batch cultures.It was found that maximum cadmium adsorption capacity varied with culture strategy. The dry biomass grown without nutrient limitation exhibited a maximum adsorption capacity for cadmium of 65.0 mgCd g−1biomass. Conversely, the limited biomass achieved a lower cadmium adsorption capacity of approximately 36.0 mgCd g−1biomass. FTIR analysis showed that nitrogen limitation induced changes in the composition of the outer cell wall, specifically an increase of deacetlylated polysaccharides, reducing the relative amount of secondary amines and proteins from the peptidoglycan matrix. Amino groups from acetylated polysaccharides and proteins have been associated elsewhere with greater cadmium affinity, which could explain the poor results obtained with the nitrogen-restricted biomass. This study shows that new physiological states displaying different adsorption capabilities were effectively obtained by engineering the cell coverage of the bacteria using varying culture strategies. The fed-batch culture proved to be a valuable tool for studying PGPR strains for biosorption and other applications. Exploring diverse nutrient limitations and other pollutants in this bacterium and other members of the PGPR family offer great opportunities to tailor biosorption strategies based on specific conditions, ultimately contributing to sustainable environmental solutions.

Engineering of Saccharomyces cerevisiae towards synthesis of linalool using linalool synthase from Magnolia champaca

Linalool is one of the commercially important fragrance molecule usually extracted from Lavandula angustifolia (lavender) and Ocimum basilicum (basil) plants. In the present study, efforts were made to produce this molecule in microbial system to meet demand-supply imbalance. Linalool synthase (LIS) gene from Magnolia champaca (Mc) and Coriandrum sativum (Cs) were successfully cloned and expressed in Saccharomyces cerevisiae CEN PK2–1 C. It was observed that expression of full-length LIS (fLIS) resulted in lesser linalool when compared to truncated LIS (tLIS) devoid of plastid signal for both Mc and Cs. In terms of linalool yield, MctLIS resulted in 1.27-fold higher linalool when compared to CstLIS. Later, when two more genes viz., TPI1 and ALD6 which presumably increase sterol pathway flux were overexpressed, actually resulted in lower linalool and increased acetate production. However, multicopy expression of MctLIS and tHMG1 in this strain has reversed the above phenomenon due to presumptive push-pull strategy. Finally, this engineered strain was cultivated in the 2 L bioreactor in fed-batch mode to obtain 10.85 µg/mL of linalool. Docking studies of homology model of MctLIS with geranyl pyrophosophate (GPP) revealed V387, Y361, T434, R427 and R249 as key interactions sites. The study reports the linalool production using LIS gene from Magnolia champaca for the first time and could be a potential chassis for further studies.

Modulating antibody N-glycosylation through feed additives using a multi-tiered approach.

Glycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product.

Using Extracted Sugars from Spoiled Date Fruits as a Sustainable Feedstock for Ethanol Production by New Yeast Isolates.

This study focuses on investigating sugar recovery from spoiled date fruits (SDF) for sustainable ethanol production using newly isolated yeasts. Upon their isolation from different food products, yeast strains were identified through PCR amplification of the D1/D2 region and subsequent comparison with the GenBank database, confirming isolates KKU30, KKU32, and KKU33 as Saccharomyces cerevisiae; KKU21 as Zygosaccharomyces rouxii; and KKU35m as Meyerozyma guilliermondii. Optimization of sugar extraction from SDF pulp employed response surface methodology (RSM), varying solid loading (20-40%), temperature (20-40 °C), and extraction time (10-30 min). Linear models for sugar concentration (R1) and extraction efficiency (R2) showed relatively high R2 values, indicating a good model fit. Statistical analysis revealed significant effects of temperature and extraction time on extraction efficiency. The results of batch ethanol production from SDF extracts using mono-cultures indicated varying consumption rates of sugars, biomass production, and ethanol yields among strains. Notably, S. cerevisiae strains exhibited rapid sugar consumption and high ethanol productivity, outperforming Z. rouxii and M. guilliermondii, and they were selected for scaling up the process at fed-batch mode in a co-culture. Co-cultivation resulted in complete sugar consumption and higher ethanol yields compared to mono-cultures, whereas the ethanol titer reached 46.8 ± 0.2 g/L.

Non-sterile cultivation of Yarrowia lipolytica in fed-batch mode for the production of lipids and biomass.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry.

From yeast screening for suitability as single cell protein to fed-batch cultures.

Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gxgs-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Increasing urine nitrification performance with sequential membrane aerated biofilm reactors

This study aimed to investigate whether separating organics depletion from nitrification increases the overall performance of urine nitrification. Separate organics depletion was facilitated with membrane aerated biofilm reactors (MABRs). The high pH and ammonia concentration in stored urine inhibited nitrification in the first stage and therewith allowed the separation of organics depletion from nitrification. An organics removal of 70 % was achieved at organic loading rates in the influent of 3.7 gCOD d−1 m−2. Organics depletion in a continuous flow stirred tank reactor (CSTR) for organics depletion led to ammonia stripping through diffused aeration of up to 13 %. Using an MABR, diffusion into the lumen amounted for 4 % ammonia loss only. In the MABR, headspace volume and therefore ammonia loss through the headspace was negligible. By aerating the downstream MABR for nitrification with the off-gas of the MABR for organics depletion, 96 % of the ammonia stripped in the first stage could be recovered in the second stage, so that the overall ammonia loss was negligibly low. Nitrification of the organics-depleted urine was studied in MABRs, CSTRs, and sequencing batch reactors in fed batch mode (FBRs), the latter two operated with suspended biomass. The experiments demonstrated that upstream organics depletion can double the nitrification rate. In a laboratory-scale MABR, nitrification rates were recorded of up to 830 mgNL−1 d−1 (3.1 gN m−2 d−1) with ambient air and over 1500 mgNL−1 d−1 (6.7 gN m−2 d−1) with oxygen-enriched air. Experiments with a laboratory-scale MABR showed that increasing operational parameters such as pH, recirculation flow, scouring frequency, and oxygen content increased the nitrification rate. The nitrification in the MABR was robust even at high pH setpoints of 6.9 and was robust against process failures arising from operational mistakes. The hydraulic retention time (HRT) required for nitrification was only 1 to 2 days. With the preceding organics depletion, the HRT for our system requires 2 to 3 days in total, whereas a combined activated sludge system requires 4 to 8 days. The N2O concentration in the off-gas increases with increasing nitrification rates; however, the N2O emission factor was 2.8 % on average and independent of nitrification rates. These results indicate that the MABR technology has a high potential for efficient and robust production of ammonium nitrate from source-separated urine.

Optimization of biotransformation process for the synthesis of pyrazine-2-carboxylic acid hydrazide using acyltransferase activity of Bacillus smithii IITR6b2 in batch and fed-batch mode

The synthesis of pyrazine-2-carboxylic acid hydrazide, a heterocyclic hydrazide compound, with negligible production of the by-product (pyrazine-2-carboxylic acid) in a solvent-free reaction condition was developed using the acyltransferase activity of amidase of Bacillus smithii strain IITR6b2. This study involves a greener approach with no hazardous chemicals and one-step biotransformation of pyrazinamide to its acid hydrazide (through hydrazinolysis). The Central Composite Design of the Response Surface Methodology was used to find the factors affecting the synthesis of the pyrazine-2-carboxylic acid hydrazide and pyrazine-2-carboxylic acid to find its true optimum. The four factors, namely pyrazinamide, hydrazine dihydrochloride, temperature, and cell concentrations, were considered during optimization. The optimized reaction conditions from the Central Composite Design for achieving the 32.26 mM of pyrazine-2-carboxylic acid hydrazide involve 40 mM of pyrazinamide, 1000 mM of hydrazine dihydrochloride with 2.5 mg/mL cell concentration at 20 °C. It resulted in an optimum production of pyrazine-2-carboxylic acid hydrazide utilizing the acyltransferase activity of amidase of Bacillus smithii IITR6b2, and the results obtained through the Design of Experiments were validated. The fed-batch biotransformation was carried out with alginate-entrapped whole-cell enzyme with the following parameters: agitation at 200 rpm, a pyrazinamide concentration of 40 mM, and 1000 mM of hydrazine dihydrochloride. The final pyrazine-2-carboxylic acid hydrazide concentration of 126 mM was obtained, corresponding to a 63% molar conversion.

Influence of process parameters on the environmental impact of 2,3-butanediol production by fermentation

Producing bio-based chemicals such as 2,3-butanediol using biological processes can play an important role in combatting global warming and addressing fossil oil depletion. To identify sustainable processes, this study sought to incorporate environmental considerations right from fermentation optimization at laboratory scale. As the literature review revealed discrepancies in Life Cycle Assessment of fermentation processes, the study began with a comprehensive Life Cycle Inventory for 2,3-butanediol fermentation. Five impact categories showed significant normalized values (in person.year): ionizing radiation human health (3.2.10−2), human toxicity cancer effects (7.10−3), freshwater ecotoxicity (2.6.10−3), ozone depletion (2.3.10−3), and human toxicity non-cancer effects (1.8.10−3). The main contributors to these impact categories (hotspots) were identified as: energy demand, carbon source, potassium hydroxide for pH adjustment, antifoam, and nitrogen source. The energy demand for heating was found to represent up to 45 % of the ionizing radiation human health impact. The study also examined the influence on fermentation performance and on environmental impact of: 1) oxygen supply rate in batch mode, and 2) sugar feeding rate in fed-batch mode. When the fermentation and separation steps were considered together, the fermentation conditions producing the highest 2,3-butanediol concentration (74.7 g.L−1) also resulted in the lowest impacts across all environmental impact categories.

Utilization of tofu wastewater and Nannochloropsis oceanica for eutrophication mitigation and eicosapentaenoic acid valorization: Advancing carbon neutrality and resource recycling

Seeking environmentally-friendly and resource-recycling strategies for tofu wastewater (TFW) treatment could effectively mitigate harsh ecological impacts and resource wastage caused by direct discharge. This study investigated the feasibility on the utilization of Nannochloropsis oceanica (N. oceanica) for TFW eutrophication mitigation and eicosapentaenoic acid valorization. The findings demonstrated that employing N. oceanica resulted in significant removal rates for total phosphorus, total nitrogen, ammonia nitrogen, and chemical oxygen demand by 88.60 %, 97.02 %, 94.22 %, and 98.02 % under fed-batch treatment mode. Such nutrients removal efficiency met discharge permit standards while accomplished a superior biomass at 26.37 g/L and EPA yield at 43.5 mg/g by N. oceanica, representing 2.61- and 2.52-fold more than that of batch treatment mode. Furthermore, TFW feeding enhanced the cytomembrane fluidity of N. oceanica and promoted TFW nutrients uptake into cells consequently boosted the nutrients removal efficiency. Simultaneously, TFW feeding upregulated enzymes such as antioxidases, desaturases and elongases responsible for EPA biosynthesis in N. oceanica to collectively protect EPA from oxidation stress and stimulate proactive accumulation. EPA derived from TFW-fed N. oceanica exhibited promising in vitro antihyperlipidaemia efficacy in constructed hyperlipopyte HepG-2 cells. Further, the molecular docking indicated that the hypolipidemic activity partly contributed to that EPA inhibited PPARα signaling to trigger fatty acids β-oxidation and cholesterol transport suppression. The outcomes present a viable route that utilization of microalgae for TFW eutrophication mitigation and EPA valorization to advance carbon neutrality and resource recycling.

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool.

At times of pandemics, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the situation demands rapid development and production timelines of safe and effective vaccines for delivering life-saving medications quickly to patients. Typical biologics production relies on using the lengthy and arduous approach of stable single-cell clones. Here, we used an alternative approach, a stable cell pool that takes only weeks to generate compared to a stable single-cell clone that needs several months to complete. We employed the membrane, envelope, and highly immunogenic spike proteins of SARS-CoV-2 to produce virus-like particles (VLPs) using the HEK293-F cell line as a host system with an economical transfection reagent. The cell pool showed the stability of protein expression for more than one month. We demonstrated that the production of SARS-CoV-2 VLPs using this cell pool was scalable up to a stirred-tank 2 L bioreactor in fed-batch mode. The purified VLPs were properly assembled, and their size was consistent with the authentic virus. Our particles were functional as they specifically entered the cell that naturally expresses ACE-2. Notably, this work reports a practical and cost-effective manufacturing platform for scalable SARS-CoV-2 VLPs production and chromatographic purification.

Degradation of potato peels using amylase- and pectinase-producing fungal strain in an electrochemical cell and by-product analysis

ABSTRACT Degradation of the waste using amylase- and pectinase-producing fungal strains in Microbial fuel cells (MFC) can be a sustainable and economic strategy for solid waste management. An isolate of the fungal strain Aspergillus niger, which showed amylase and pectinase activities was implemented for potato peel waste degradation. MFC was constructed and the highest OCV was observed using KMnO4 in catholyte (1586 ± 63 mV/m3) with anode graphite electrode-coated MWCNT. MFC in fed-batch mode was also performed by adding 10% of the sample every 24 h, and an improved result was obtained. The power density observed with 100-ohm and 1000-ohm external resistors was 119 ± 7 W/m3 and 42 ± 9 W/m3, respectively. From MFC operation at an optimised condition removal rate of COD, ammoniacal-nitrogen, reducing sugar and TSS were 37.69%, 67.72%, 72.64%, and 65.95% respectively. Sugar and byproduct analysis in digested product was done by HPLC, various value-added products were generated from waste sample.

Mixotrophic cultivation of microalgae using agro-industrial waste: Tolerance level, scale up, perspectives and future use of biomass

Alternative culture media make large-scale microalgae biomass production more viable and cost-effective. The aim of this work was to study the addition of whey as an alternative source of organic nutrients in fed-batch mode for the cultivation of microalgae (Spirulina platensis, Chlorella homosphaera and Scenedesmus obliquus). The microalgae demonstrated acceptance of whey at low concentrations, causing the increasing in the maximum specific growth rates (μmax) and biomass concentrations ([X]max) compared with the cultivation without whey in standard medium (μmax of 0.168 d−1 versus 0.075 d−1, and [X]max of 2.274 g L−1 versus 0.970 g L−1, respectively, for S. platensis). Therefore, a cost savings in cultivation media of up to 32 %, 57 %, and 73 % were possible in the cultivation of S. obliquus, S. platensis, and C. homosphaera, respectively, considering the production of 1 kg of biomass. The addition of whey presented different effects in the levels of intracellular protein considering the species of microalgae: an increasing was observed in C. homosphaera (24.18 to 43.96 g/100 g of biomass) and a decreasing in S. platensis (73.75 to 44.06 g/100 g of biomass), comparing with the controls. Scale-up with a natural light source can affect microalgal productivity and biomass composition in a medium supplemented with whey, because of the influence of irradiation. Optimizing the large-scale production of microalgae using agro-industrial residues can lead to significant advancements in biomass production at reduced costs. These biomasses can be integrated into bioenergy production, biofertilizer manufacturing, and animal feed production. By utilizing waste as a nutritional source and optimizing cultivation processes and biomass generation, a closed-loop cycle is established, aligning with the principles of circular bioeconomy and enhancing the sustainability of the process.