Bioreactor Culture Research Articles

In vitro adventitious roots of hemp in bioreactor culture to efficiently produce non-cannabinoids and cannabidiol phytochemicals

In vitro adventitious roots of hemp in bioreactor culture to efficiently produce non-cannabinoids and cannabidiol phytochemicals

Contact-Free Online Monitoring of Bioreactor Cell Cultures with Magnetic Resonance Relaxometry.

Frequent, low-latency measurements of bioreactor culture growth are critical for achieving maximum culture efficiency and productivity. Typical cell density and viability measurements are made by manually removing a sample from the culture, but this approach is both slow and unsuitable for small culture volumes, which cannot support frequent destructive sampling. In this work, automated magnetic resonance relaxometry measurements of a sealed bioreactor system are used to estimate the cell density and provide qualitative information about the culture in near real-time. The system detects variations in cell density in minutes, enabling rapid intervention that would be impossible with the once-daily measurements taken by a traditional sampling-based culture analysis system.

Optimization and scaling up of extracellular polysaccharide production by submerged culture of Ganoderma lucidum on starch-containing medium using response surface methodology and laboratory bioreactors of various designs.

Basidiomycetes, known for their production of bioactive compounds, traditionally use simple sugars for fermentation. However, their ability to degrade complex plant polysaccharides through enzyme secretion presents potential for the use of renewable raw materials. This study focused on the optimization of exopolysaccharide (EPS) production and efficient substrate consumption by Ganoderma lucidum using response surface methodology (RSM). Using an optimized medium containing 15 g⋅L-1 wheat starch, 0.375 g⋅L-1 NH4Cl, and 0.75 g⋅L-1 CaCl2 (C/N ratio of 40), a significant increase in EPS concentration from 121.1±10.2 mg⋅L-1 to 229.0±20.3 mg⋅L-1 and starch degradation degree (SDD) from 9.1% to 57.6% was achieved after 9 d of submerged cultivation. Scale-up experiments were conducted in both column and stirred tank bioreactors, employing submerged and immobilized cultivation modes. Submerged cultivation in the column bioreactor yielded the highest process desirability of 0.56, achieving EPS concentration of 192.5±5.4 mg⋅L-1 and 60.2% starch degradation degree within 7 d. These results highlight the potential of the used column bioreactor for efficient and rapid EPS production. Notably, bioreactor experiments revealed local maxima in EPS content at specific time points, suggesting that cell wall degradation, potentially induced by shear stress, may contribute to the release of polysaccharides into the culture broth.

Enhancing monoclonal antibody production efficiency using CHO-MK cells and specific media in a conventional fed-batch culture.

Chinese hamster ovary (CHO) cell lines, derived as subclones from the original CHO cell line, are widely used hosts for current biopharmaceutical productions. Recently, a highly proliferative host cell line, CHO-MK, was established from the Chinese hamster ovary tissue. In this study, we assessed the fundamental culture characteristics and capabilities of CHO-MK cells for monoclonal antibody (mAb) production using specified chemically defined media. To achieve this, we established fed-batch cultures of model CHO-MK cells in shake flasks and ambr15 and 2 L bioreactors under various conditions. The mAb-producing CHO-MK cell line A produced 12.6g/L of antibody within 7days in the fed-batch culture using a 2 L bioreactor, with a seeding density of 1 × 106 cells/mL. This performance corresponded to a space-time yield of 1.80g/L/day, representing a productivity level that could be challengingly attained in fed-batch cultures using conventional CHO cells. In addition, when we subjected six different mAb-producing CHO-MK cell lines to fed-batch culture in the ambr15 bioreactor for 7days, the antibody production ranged between 5.1 and 10.8g/L, confirming that combining CHO-MK cells and specified media leads to enhanced versatility. These discoveries underscore that CHO-MK cells combined with specified media might represent a next-generation production platform, which could potentially respond to an increasing demand for antibody drugs, reducing production costs, and shortening antibody drug development times. This study is expected to serve as a benchmark for future production process development using CHO-MK cells.

Quantitative Analysis of Lactobionic Acid in Bioreactor Cultures and Selected Biological Activities.

The aim of this study was to quantitatively analyse lactobionic acid obtained from bioreactor cultures using whey as a liquid medium with bacteria of the Pseudomonas taetrolens species. The most important culture parameters affecting the production of the acid are indicated and evaluated. The highest lactobionic acid yield was 37.42 g/L, selecting the appropriate strain (Pseudomonas taetrolens 4') and environmental conditions (2% lactose concentration in the bioreactor). The amount of lactose and lactobionic acid was determined by high-performance liquid chromatography. Microorganism analysis was also carried out using a flow cytometer with imaging to study the metabolic activity of microorganisms during lactobionic acid production. In addition, during the study, Bifidobacteria were microencapsulated with lactobionic acid and their survival was evaluated in an in vitro model of the gastrointestinal tract, checking the prebiotic properties of the acid. The highest number of viable cells in the microcapsules after digestion was obtained using the Bifidobacterium bifidum strain DSM 20082. The antagonistic activity of lactobionic acid was also analysed.

Cell Adhesion and Local Cytokine Control on Protein-Functionalized PNIPAM-co-AAc Hydrogel Microcarriers.

Achieving adequate cell densities remains a major challenge in establishing economic biotechnological and biomedical processes. A possible remedy is microcarrier-based cultivation in stirred-tank bioreactors (STBR), which offers a high surface-to-volume ratio, appropriate process control, and scalability. However, despite their potential, commercial microcarriers are currently limited to material systems featuring unnatural mechanical properties and low adaptability. Because matrix stiffness and ligand presentation impact phenotypical attributes, differentiation potential, and genetic stability, biotechnological processes can significantly benefit from microcarrier systems tailorable toward cell-type specific requirements. This study introduces hydrogel particles co-polymerized from poly(N-isopropylacrylamide) (PNIPAM) and acrylic acid (AAc) as a platform technology for cell expansion. The resulting microcarriers exhibit an adjustable extracellular matrix-like softness, an adaptable gel charge, and functional carboxyl groups, allowing electrostatic and covalent coupling of cell adhesive and cell fate-modulating proteins. These features enable the attachment and growth of L929 mouse fibroblast cells in static microtiter plates and dynamic STBR cultivations while also providing vital growth factors, such as interleukin-3, to myeloblast-like 32D cells over 20 days of cultivation. The study explores the effects of different educt compositions on cell-particle interactions and reveals that PNIPAM-co-AAc microcarriers can provide both covalently coupled and diffusively released cytokine to adjacentcells.

From coffee waste to nutritional gold: bioreactor cultivation of single‐cell protein from Candida sorboxylosa

AbstractBACKGROUNDIndustrial effluents are continuously discharged into the environment. These wastewaters contain valuable compounds that can be reused for biotechnological applications. Coffee wastewater (CWW) is a powerful effluent that can be used for single‐cell protein (SCP) production reaching important content (up to 80%). Several yeast species can be used for SCP production, but Candida species are commonly applied for this purpose (17 species reported including the novel C. sorboxylosa). In addition, SCP can be produced in bioreactors under controlled conditions under three operation modes. Thus, batch mode is frequently used but continuous mode presents interesting advantages in economic terms, although it has been poorly applied in SCP production.RESULTSThe initial evaluation under batch operation mode showed that volumetric oxygen transfer coefficient (kLa of 101 h−1) improved biomass production (1.39 g L−1) and SCP yield (59.9%) in C. sorboxylosa. Thus, continuous mode was established at selected kLa and feeding with optimized medium composed of 87.5% (v/v) CWW, 1.38 g L−1 yeast extract, and 7.24 g L−1 (NH4)2SO4, in order to provided necessary nutrients. In this sense, the process presented higher values in dry cell weight and SCP productivity (0.57 and 0.29 g L−1·h, respectively), achieving a 3.35‐ and 2.90‐fold increase in biomass and protein productivity, respectively, compared to batch mode. The SCP from C. sorboxylosa exhibited an interesting essential amino acid profile under continuous mode (33.704%).CONCLUSIONThe bioprocess highlights several advantages during bioreactor cultivation, including: (i) reduced energy consumption for temperature control; (ii) successful establishment of an initial continuous operation mode with promising performance; and (iii) SCP from C. sorboxylosa exhibited a notable composition of essential amino acids, which could be beneficial for potential use in animal feed. © 2024 Society of Chemical Industry (SCI).

Comparison Between Simple Batch and Fed-Batch Bioreactor Cultivation of Recombinant BCG.

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process.

Effects of pretreatment in a temporary immersion bioreactor on organogenesis efficacy of <i>Lilium candidum</i> L. bulbscales

Our experiment was conducted in two stages, i.e., pretreatment (first stage) and regeneration (second stage). The first stage was carried out in a liquid Murashige and Skoog basal medium (5 µM BAP and 0.05 µM NAA) in a bioreactor with a RITA temporary immersion system under the light of a fluorescent lamp. Explants (bulbscales) were immersed in the medium once a day for 15 minutes (RITA 1×15) or three times a day for 1 (RITA 3×1), 5 (RITA 3×5), and 15 minutes (RITA 3×15) for one to six weeks. For regeneration, the explants were transferred onto a solid medium of the same composition for another six weeks. The bulbscales not exposed to the liquid medium were used as a control. Biomass weight, biomass growth index, number and percentage of dry matter of bulblets, and the content of soluble sugars in the bulblets and in the liquid medium were examined. The bulblets were formed in all combinations from the third week of the culture, and their number increased in the RITA 3×15 combination for both the first and the second stages of the experiment. After the longest, 6-week pretreatment, more bulblets were obtained than in the control. Their fresh weight after six weeks of regeneration was positively associated with extended pretreatment time. This was in contrast with the dry weight of the bulblets, which decreased in the second stage of the experiment along with the extension of its first stage. Prolonged contact of the explants with the liquid medium during the pretreatment resulted in a higher content of soluble sugars in the bulblets at both stages of the experiment. The content of soluble sugars in the liquid medium decreased over time in all tested combinations. After six weeks of bioreactor culture, the lowest level of soluble sugars was observed in the RITA 3×15 combination.

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi.

Plant biotechnology creates opportunities for the cultivation of plants regardless of their natural habitats, which are often protected or difficult to access. Maintaining suspension cell cultures in bioreactors is an advanced part of biotechnological research that provides possibilities for obtaining plant tissue on a large scale. In this study, the suspension culture cultivation of a Chinese endemic plant, Schisandra henryi, in a stirred tank bioreactor was elaborated for the first time. The phytochemical profile of the tissue extracts was determined with UHPLC-MS/MS for the lignans (fifteen dibenzocyclooctadiene lignans, one aryltetralin lignan, and two neolignans) and UHPLC-DAD-ESI-MS3 for the phenolic compounds (procyanidins and their derivatives and catechin). The maximum total lignan content of 1289 µg/100 g DW was detected for the extracts from suspensions cultured in a bioreactor for over 10 days. For the phenolic compounds, catechin was the dominant compound (390.44 mg/100 g DW). The biological activity of the extracts was tested too. To determine antioxidant potential we used DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Molybdenum reduction, and β-carotene bleaching tests. The inhibition activity of the S. henryi extract on the enzymes responsible for skin aging, hyaluronidase and tyrosinase, was assessed with spectrophotometry. The cytotoxic activity of the extracts was estimated on human ovarian SKOV-3, cervical HeLa, and gastric AGS cancer cells and non-cancer, normal fibroblasts by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results showed the great potential of the obtained cell biomass extracts. The results of the antioxidant tests indicated their strong ability to reduce the level of free radicals, similarly to that of ascorbic acid, as well as the weak capacity to protect lipids from oxidation. Moreover, anticancer potential, particularly on the cervical and gastric cancer cells, was confirmed too.

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

Lycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

Genome reduction improves octanoic acid production in scale down bioreactors.

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

Biofertilizer based on <i>Agrobacterium </i>as a key to food security

Background: The concept of food security is contingent upon the fulfillment of two fundamental requirements: physiological and socioeconomic. The former comprises providing sustenance that is both safeguarded and enhanced in line with people’s nutritional needs and priorities, whereas the latter includes the mechanisms by which these needs are supplied. The objective is to ensure a state of robust and healthy well-being. As a sustainable solution, biofertilizers play a pivotal role in addressing the challenges of food security. Biofertilizers represent a cost-effective and environmentally friendly solution, with the potential to enhance soil productivity through nitrogen fixation. They also enhance crop yield by increasing the concentration of nutrients. Access to functional foods derived from biofertilizer-enhanced yields may contribute to improved dietary diversification and overall well-being. Objective: The objective is to isolate and screen the most active nitrogen-fixing bacterium as a basis to a new biofertilizer. Methods: The isolation of nitrogen-fixing bacteria was conducted using the soil sowing technique. The selection of nitrogen-fixing bacteria was based on their morphophysiological characteristics. Bacterial growth was evaluated by viable cell count. Cultivation of the selected bacterium occurred in both flasks (on a shaker) and a bioreactor to compare growth conditions. The stimulatory effect of the selected bacterium on seed germination was assessed based on the final germination rate. The selected bacterium identified through molecular taxonomy. Results: A total of 100 pure cultures were isolated, from which nitrogen-fixing bacteria were selected. The cultivation conditions for these bacteria were optimized regarding pH, temperature and process duration in a flask on a shaker. Strain NB4 exhibited the most favorable development under optimal settings. Additionally, cultivation parameters for this strain were optimized in a laboratory bioreactor. Notably, a bacterial liquid culture water solution comprising 1.5% strain NB4 facilitated complete germination of wheat (Triticum aestivum L.), seeds and produced high-quality sprouts. The 16S rRNA gene sequence of strain NB4 was submitted to the GenBank database under accession number MT670424.1, identifying it as Agrobacterium Pusense RP 1. Conclusion: This research aimed to develop an effective biofertilizer to tackle the pressing issue of food security. The project’s cornerstone was Agrobacterium pusense RP 1, nitrogen-fixing strain isolated and identified through rigorous research. Keywords: Isolation and identification of nitrogen-fixing bacteria, cultivation in bioreactor, germination of wheat seeds, Agrobacterium pusense.

Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9 % lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5 %) and purity (98.1 %) were obtained with 1,3-dioxolane at 80°C and 6 h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor culture leading to the production of 78.7 gPHB/L, 56.9 % (w/w) PHB content and 2.3 g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.

Design, experimental optimization, and flow analysis of a novel bioreactor for dynamic mammalian cell culture at laboratory scale using Box-Behnken design

Improving the quality of dynamic cell culture in laboratories is an important field in bioengineering. In this study, a novel lab-scale bioreactor using a vibrating agitator and a modified flask has been introduced to create a strong mixing at low shear stress. This bioreactor has been optimized using Box-Behnken design based on three dimensionless important structural factors including disc diameter, vibration amplitude, and the height of the disc placement. Three growth indicators including the specific growth rate, the natural logarithm of the maximum cell density, and productivity have been considered as biological responses. The results show that the disc diameter has the most important role in these indicators. If the disc diameter, vibration amplitude, and the height of disc placement are set to 0.24, 0.02, and 0.4 of the flask diameter, respectively, the values of the specific growth rate, the maximum cell density, and productivity at this optimum settings are 0.033 (h−1), 13.11, and 5133 (cells/(mL.h)), respectively. These values of the indicators are high and indicate the better performance of this bioreactor than other lab-scale bioreactors. In addition, investigating Reynolds number in the fluid flow indicates that in the range of 780 up to 1150, growth indices are high.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Investigation of Acetoin Biosynthesis by Bacillus subtilis ACA-DC 1176 Growing on Crude Glycerol in Flask and Bioreactor Trials

Acetoin biosynthesis by two Bacillus subtilis strains valorising crude glycerol was thoroughly explored within a pre-defined range of culture conditions and systems. B. subtilis ACA-DC 1176 stood out for its higher efficiency in acetoin production, prompting an investigation into the potential for enhanced productivity through the evaluation of diverse culture conditions and media compositions. The primary by-products of the biodiesel and corn industries, namely crude glycerol and corn steep liquor, respectively, were successfully employed as the principal carbon and nitrogen sources of the newly developed low-cost culture medium. Furthermore, the results of the various feeding strategies that were tested indicated that the conversion of 2,3-butanediol (BDO) to acetoin occurred exclusively when the concentration of glycerol was below approximately 5 g/L. This seemed to be necessary for the production of NADH, which is essential for maintaining cellular processes. Following the complete depletion of glycerol, acetic acid increased and became the predominant metabolite, while both acetoin and BDO decreased, presumably resulting in ATP generation. This is likely a mechanism employed by the cell to generate energy in the absence of a carbon source. In the fed-batch bioreactor culture, the kinetics of metabolites differed, as there was no conversion of BDO to acetoin at the final depletion of glycerol. At volumetric mass transfer coefficient (kLa) levels exceeding approximately 70 1/h, the production of acetoin was favoured over that of BDO, with the highest observed acetoin/BDO ratio reaching 4.29 g/g. Conversely, at kLa values below approximately 60 1/h, the titres of acetoin and BDO were found to be nearly equal. The final concentrations of acetoin and BDO reached 36.0 g/L and 25.5 g/L, respectively, resulting in a total yield of both (acetoin + BDO) per glycerol consumption of 0.40 g/g. To the best of our knowledge, this is the first study to focus on acetoin production from crude glycerol fermentative valorisation. The study presents new findings regarding the parameters influencing the level of BDO conversion to acetoin. However, further research is required in order to gain a comprehensive understanding of the underlying phenomena and metabolic pathways involved.

High-Level Expression of Sucrose Isomerase in Bacillus subtilis Through Expression Element Optimization and Fermentation Optimization.

Sucrose isomerase is an important food enzyme that catalyzes the isomerization of sucrose into isomaltulose, a functional sugar widely used in food industry, while the production level of sucrose isomerase in food safe host strains was much lower than industrial requirement. Bacillus subtilis is an excellent host strain for recombinant protein expression, which owns the characteristics of powerful secretory capability and generally recognized as safe state. In this study, the expression of sucrose isomerase in B. subtilis was improved through expression element optimization and fermentation optimization. Firstly, the extracellular chaperone PrsA was overexpressed to enhance extracellular folding of sucrose isomerase, which improved the recombinant expression level by 80.02%. Then, the protein synthesis level was optimized through promoter screening, improving the recombinant expression level by 60.40%. On the basis of strain modification, the fermentation conditions including nitrogen source, carbon source, metal ion, pH and temperature were optimized successively in shake-flask. Finally, the 3 L bioreactor cultivation condition was optimized and yielding a sucrose isomerase activity of 862.86 U/mL, the highest level among the food safety strains. This study provides an effective strategy to improve the expression level of food enzymes in B. subtilis.

Potato and dairy industry side streams as feedstock for fungal and plant cell cultures

Utilization of industrial side streams as nutrient source for cellular agriculture is a promising option to improve the sustainability of the production processes. The aim of the study was to evaluate usability of two liquid food industry side streams, potato cell fluid and acid whey, as nutrient source for production of food ingredients with plant cell cultures (arctic bramble (Rubus arcticus L.) and tobacco BY-2 (Nicotiana tabacum L.)) and filamentous fungi (Paecilomyces variotii, Rhizopus oligosporus and Trichoderma reesei). The side streams were found to contain various sugars and other potential nutrients suitable for the studied organisms. The side streams were used as a sole nutrient source (fungi) or as a replacement of selected nutrients in the growth media (fungi and plant cells) in flask scale cultivations. Acid whey was successfully used as a growth medium for filamentous fungi, but it inhibited plant cell growth presumably due to high organic acid content (14 g/l). Potato side stream was found suitable as a fungal and plant cell growth media supplement, and it was used together with glucose for filamentous fungi, or as a partial replacer of macronutrients for plant cells, in bioreactor cultivations yielding high fungal (up to 36 g/l dry weight) and good plant cell (9.5 g/l dry weight) biomass production. Analyses of the produced biomasses revealed good nutritional value in terms of amino acid (17–27 % of dry matter) and dietary fiber (23–30 % of dry matter) contents, giving good premises for utilization in human nutrition.

Utilization of formic acid by extremely thermoacidophilic archaea species.

The exploration of novel hosts with the ability to assimilate formic acid, a C1 substrate that can be produced from renewable electrons and CO2, is of great relevance for developing novel and sustainable biomanufacturing platforms. Formatotrophs can use formic acid or formate as a carbon and/or reducing power source. Formatotrophy has typically been studied in neutrophilic microorganisms because formic acid toxicity increases in acidic environments below the pKa of 3.75 (25°C). Because of this toxicity challenge, utilization of formic acid as either a carbon or energy source has been largely unexplored in thermoacidophiles, species that possess the ability to produce a variety of metabolites and enzymes of high biotechnological relevance. Here we investigate the capacity of several thermoacidophilic archaea species from the Sulfolobales order to tolerate and metabolize formic acid. Metallosphaera prunae, Sulfolobus metallicus and Sulfolobus acidocaldarium were found to metabolize and grow with 1-2 mM of formic acid in batch cultivations. Formic acid was co-utilized by this species alongside physiological electron donors, including ferrous iron. To enhance formic acid utilization while maintaining aqueous concentrations below the toxicity threshold, we developed a bioreactor culturing method based on a sequential formic acid feeding strategy. By dosing small amounts of formic acid sequentially and feeding H2 as co-substrate, M. prunae could utilize a total of 16.3 mM of formic acid and grow to higher cell densities than when H2 was supplied as a sole electron donor. These results demonstrate the viability of culturing thermoacidophilic species with formic acid as an auxiliary substrate in bioreactors to obtain higher cell densities than those yielded by conventional autotrophic conditions. Our work underscores the significance of formic acid metabolism in extreme habitats and holds promise for biotechnological applications in the realm of sustainable energy production and environmental remediation.