Bioreactor Culture Research Articles

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.

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.

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.

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 6h 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 cultures leading to the production of 78.7 gPHB/L, 56.9% (w/w) PHB content and 2.3g/(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.

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.

Development of novel osteochondral scaffolds and related in vitro environment with the aid of chemical engineering principles

In tissue engineering, collaboration among experts from different fields is needed to design appropriate cell scaffolds and the required three-dimensional environment. Osteochondral tissue engineering is particularly challenging due to the need to provide scaffolds that imitate structural and compositional differences between two neighboring tissues, articular cartilage and bone, and the required complex biophysical environments for cultivating such scaffolds. This work focuses on two key objectives: first, to develop bilayered osteochondral scaffolds based on gellan gum and bioactive glass and, second, to create a biomimetic environment for scaffold characterization by designing and utilizing novel dual-medium cultivation bioreactor chambers. Basic chemical engineering principles were utilized to help achieve both aims. First, a simple heat transport model based on one-dimensional conduction was applied as a guideline for bilayer scaffold preparation, leading to the formation of a gelatinous upper part and a macroporous lower part with a thin, well-integrated interfacial zone. Second, a novel cultivation chamber was developed to be used in a dynamic compression bioreactor to provide possibilities for flow of two different media, such as chondrogenic and osteogenic. These chambers were utilized for characterization of the novel scaffolds with regard to bioactivity and stability under dynamic compression and fluid perfusion over 14 d, while flow distribution under different conditions was analyzed by a tracer method and residence time distribution analysis.

Application of genetic code expansion to regulate the synthesis of poly(lactate-co-3-hydroxybutyrate) in Escherichia coli

Genetic codon expansion has the potential to introduce a variety of unnatural amino acids to specific sites within target proteins. In this study, genetic codon expansion was employed to regulate the enzyme expression in metabolic pathways. Firstly, a purple protein from Actinia tenebrosa was selected as the candidate to be engineered. Bringing in UAG stop codon caused premature termination of translation, while expressing orthogonal aminoacyl-tRNA synthetase and tRNA from Methanococcus jannaschii restored translation at UAG site. However, leakage expression was observed without addition of unnatural amino acids, still it can be decreased by increasing numbers of UAG mutations. Subsequently, poly(lactate-co-3-hydroxyburyrate) [P(LA-3HB)] biosynthesis pathway was constructed in Escherichia coli, and propionyl-CoA transferase was mutated to harboring one or two more stop codons. With genetic codon expansion tools, the function of propionyl-CoA transferase was restored, promoting cells to synthesize P(LA-3HB) copolymer. Moreover, the lactate monomer content was regulated ranging from 0 to 33.42 mol% by altering the addition time of inducers. Finally, the strain accumulated 27.09 g/L P(25.1 mol% LA-3HB) in 5-L bioreactor cultivation. This is the first report on metabolic engineering of polyhydroxyalkanoate biosynthesis through genetic codon expansion and would provide helpful strategies to achieve dynamic regulation of multiple metabolic pathways.

Agitated and temporary immersion bioreactor cultures of Reynoutria japonica Houtt. as a rich source of phenolic compounds

Reynoutria japonica Houtt. (Japanese knotweed) is an invasive plant belonging to the Polygonaceae family. However, being native to East Asia, it has been used in natural medicine for ages because of its broad range of biological activity. Although R. japonica is known as a rich source of phenolic compounds, plant biomass collected from the field may be contaminated with toxic elements like heavy metals, and the level of metabolite accumulation depends on environmental conditions. Therefore, the aim of this study was to derive Japanese knotweed tissue cultures and investigate biomass production and phenolic compound synthesis in in vitro conditions. Plants were cultivated in a traditional agar-solidified medium, in a liquid medium with rotary shaking (agitated culture), and in a temporary immersion bioreactors Plantform™, as well as in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted to determine the sum of phenolic compounds, as well as the quantity of selected phenolic acids, catechins, and other flavonoids. Results have shown that agitated cultures and temporary immersion bioreactors increased biomass accumulation compared to solid medium cultures. Tissue cultures of R. japonica had increased synthesis of phenolic compounds compared to plants from ex vitro conditions. Shoots and roots from agitated cultures were 2.8- and 3.3-fold richer in catechins, respectively, compared to plants cultivated in soil. Based on the obtained results it can be concluded that agitated and bioreactor cultures are the best source of Japanese knotweed biomass rich in valuable secondary metabolites.

A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

Microbial biomass production from enzymatically saccharified organic municipal waste and present microbial inhibitors

AbstractThe study investigated the potential of the organic fraction of municipal solid waste (OFMSW) for microbial biomass production. The compositional analysis of OFMSW showed richness in sugars, proteins, lipids, organic acids, and ethanol, suggesting promising cheap cultivation feedstock if inhibitory compounds are sustainably detoxified. The enzymatic hydrolysis with Cellic® CTec3 and AMG® 300 L BrewQ (Novozymes A/S) demonstrated excellent saccharification of sugar polymer, reaching 92% glucan hydrolysis and 70% xylan hydrolysis. However, higher enzymatic dosages led to a rise in the total organic acids content, potentially causing increased microbial inhibition. Full hydrolysate and hydrolysate after solids removal were cultivated with seven robust microbial strains. Cultivation on hydrolysate with solids showed consumption of sugars and organic acids solely by commercial backer yeast Saccharomyces cerevisiae. Removal of solids from hydrolysate resulted in increased performance of tested strains, showing consumption of measured organic acids and ethanol by S. cerevisiae, Yarrowia lipolytica DSM 8218, and Cutaneotrichosporon oleaginosus ATCC 20509. Remarkably, the investigation of biomass production revealed superior cell mass formation and detoxification by S. cerevisiae, resulting in 18.9 g of biomass/L hydrolysate with 50% of crude protein (w/w) in shake flasks and 13.2 g/L of hydrolase with 46% of crude protein (w/w) in a 5-L bioreactor. Furthermore, bioreactor cultivation confirmed organic acids and ethanol conversion into biomass, highlighting S. cerevisiae’s suitability for utilizing OFMSW for microbial biomass production. These findings contribute to advancements in biowaste-to-fodder conversion, promoting the development of a more sustainable circular economy. Graphical abstract

Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors

BackgroundWhile pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified.MethodsThis study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture.ResultsThe robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology.ConclusionsThis study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.

Overexpression of the transcriptional activators Mxr1 and Mit1 enhances lactic acid production on methanol in Komagataellaphaffii

A bio-based production of chemical building blocks from renewable, sustainable and non-food substrates is one key element to fight climate crisis. Lactic acid, one such chemical building block is currently produced from first generation feedstocks such as glucose and sucrose, both requiring land and water resources. In this study we aimed for lactic acid production from methanol by utilizing Komagataella phaffii as a production platform. Methanol, a single carbon source has potential as a sustainable substrate as technology allows (electro)chemical hydrogenation of CO2 for methanol production. Here we show that expression of the Lactiplantibacillus plantarum derived lactate dehydrogenase leads to L-lactic acid production in Komagataella phaffii, however, production resulted in low titers and cells subsequently consumed lactic acid again. Gene expression analysis of the methanol-utilizing genes AOX1, FDH1 and DAS2 showed that the presence of lactic acid downregulates transcription of the aforementioned genes, thereby repressing the methanol-utilizing pathway. For activation of the methanol-utilizing pathway in the presence of lactic acid, we constructed strains deficient in transcriptional repressors Nrg1, Mig1-1, and Mig1-2 as well as strains with overrepresentation of transcriptional activators Mxr1 and Mit1. While loss of transcriptional repressors had no significant impact on lactic acid production, overexpression of both transcriptional activators, MXR1 and MIT1, increased lactic acid titers from 4 g L−1 to 17 g L−1 in bioreactor cultivations.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

P-380 Improvement of follicle growth and health during dynamic culture of bovine ovarian cortical tissue (BOCT) is associated to a higher remodeling of the extracellular matrix

Abstract Study question Is improvement of follicle growth and health during dynamic culture in Perifusion Bioreactor (PB) associated to a healthier condition and remodeling of extracellular matrix? Summary answer The dynamic culture in PB allows the extracellular matrix (ECM) network rearrangement improving follicle growth and viability compared to static culture What is known already Over the past two decades, in vitro folliculogenesis has achieved moderate success in large mammals, emerging as a distinctive and reliable method for obtaining secondary follicles. Recently, although dynamic culture in bioreactors has been demonstrated to enhance follicle progression, quality, and viability compared to static culture, less emphasis has been devoted to ECM remodeling. Ovarian tissue ECM plays a crucial role in cell–cell, and cell-ECM interactions involved in follicle activation and growth. The comprehension of ECM function deepens our understanding of follicular development and holds significant promise for future reproductive technologies Study design, size, duration Bovine ovaries (age 8-24 months) were collected at slaughterhouse. BOCT strips (1x1x0.5mm) from the same ovary were cultured in groups of 10 for 14 days in PB (dynamic culture) and conventional dishes (CD, static culture). At the end of culture, collagen thickness and packing density was assessed through PicroSirius red (PSR) staining, follicle stages and quality through histology, and follicle viability through live-dead far-red and propidium iodide labeling under confocal microscopy Participants/materials, setting, methods Neosynthesis and remodeling of collagen fibers was analyzed on PSR-stained samples under polarized light measuring the color of each pixel. A color threshold was set to isolate the four colors seen in PSR-stained samples under polarized light: green, thin fibers (neosynthesized); yellow, mid-sized fibers (low assembly); orange, mid-sized fibers (medium assembly); red, mature thick fibers (high assembly). The color threshold was set as follows: red 2–9, yellow 39-51, orange 10–38, green 52–128 Main results and the role of chance In fresh tissue, analysis of PSR-stained samples showed the following values: Red 1.35; Orange 4.93; Yellow 0.18; Green 0.15. At Day 14, compared to the static culture, culture in PB, exhibits a significantly higher production of green (PB 0.22 vs CD 0.02 P < 0.01) and yellow (PB 0.56 vs CD 0.26 P < 0.05) fibers indicating an improved neosynthesis and remodeling of collagen and ECM health condition. Such higher ECM remodeling after dynamic culture is associated to a significantly higher percentage of secondary follicles (PB 15.4– CD 4.1%, P < 0.01 ), superior follicle quality (grade I/II, PB 70,5 - CD 28.4%, P < 0.01) and viability (PB 70 – CD 41.1%, P < 0.01). PSR analysis suggests that the continuous nutrients and oxygen supply, catabolites removal, and biomechanical stimulation during dynamic PB culture, stimulates the deposition of newly synthesized collagen which in turn, promotes follicle growth Limitations, reasons for caution Although the bovine is a suitable reproductive model for human, such findings should be replicated on human ovarian tissue Wider implications of the findings The aim of ovarian tissue culture is to promote the growth of healthy secondary follicles that can be isolated for further culture to generate MII oocytes. The present findings suggest that adoption of dynamic ovarian tissue culture could enhance the outcome of folliculogenesis in vitro Trial registration number not applicable

A Novel β-Galactosidase from Kluyvera intermedia and its Potential for Hydrolyzing Lactose in Milk

ABSTRACT A new β-galactosidase (EC 3.2.1.23) discovered in Kluyvera intermedia (β-gal-Kli) was recombinantly produced in Escherichia coli BL21 (DE3). The bioreactor cultivation resulted in a maximum β-gal-Kli activity of 124.6 ± 25.5 μkat oNPGal,37 °C/Lculture. The β-gal-Kli was purified 4.5fold to a specific activity of 172.3 μkat oNPGal,37 °C/gprotein. The β-gal-Kli showed maximum activity at pH 6.5 and 50°C and was sufficiently active and stable at an industrially relevant temperature of 8°C (half-life of 63 days). The β-gal-Kli was compared to a commercially available β-galactosidase (opti-lactase LX2) for lactose hydrolysis in milk (45.3 ± 0.9 glactose/L) at 8°C. With the activity of 2.7 nkatlactose/mLmilk, “lactose-free” (lactose <0.1 g/L) was realized by β-gal-Kli after 72 h, while 192 h was needed for opti-lactase LX2. Additionally, the galactooligosaccharide synthesis was observed with both these two enzymes. In conclusion, the β-gal-Kli showed its potential for the production of “lactose-free” dairy products.