Shake Flask Cultures Research Articles

Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19

BackgroundDeveloping effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines.ResultsThis work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting.ConclusionsOur study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.Graphical

Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymatic activities, and transcriptomics on cellulose. From plate cultures, the Δxyr1 mutant had reduced growth on D-xylose, xylan, and cellulose, and from shake-flask cultures with cellulose, the Δxyr1 mutant had ~90% lower β-glucosidase activity, and no detectable β-xylosidase or cellulase activity. The comparison of the transcriptomes from 18 h shake-flask cultures on D-fructose, without a carbon source, and cellulose, showed major effects of XYR1 deletion whereby the Δxyr1 mutant on cellulose was transcriptionally most similar to the cultures without a carbon source. The cellulose induced 43 plant biomass-degrading CAZymes including xylanases as well as cellulases, and most of these had massively lower expression in the Δxyr1 mutant. The expression of a subset of carbon catabolic enzymes, other transcription factors, and sugar transporters was also lower in the Δxyr1 mutant on cellulose. In summary, T. harzianum XYR1 is the master regulator of cellulases and xylanases, as well as regulating carbon catabolic enzymes.

The Development and Characterisation of a P(3HB-co-4HB)-Bioactive Glass-Graphene Hydrogel as a Potential Formulation for Biomedical and Therapeutical Translation.

The clinical management of wounds is known to be a significant challenge: not only does the dressing need to ensure and provide the appropriate barrier and healing characteristics, but consideration of patient compliance concerning comfort, functionality, and practicality also needs to be included. The poly(3-hydroxybutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) copolymer, isolated from Cupriavidus malaysiensis USM1020 (C. malaysiensis USM1020), was produced in the presence of excess carbon sources (1,4-butanediol and 1,6-hexanediol) using either a shake flask cultivation process or a bioreactor fermentation system. P(3HB-co-4HB) is widely known to be biodegradable and highly biocompatible and contains a tuneable 4HB monomer molar fraction, which is known to affect the final physicochemical properties of the intracellular copolymer. In this paper, we describe not only the fabrication of the polymeric gel but also its optimised profiling using a range of physical and mechanical techniques, i.e., SEM, FTIR, DMA, DSC, and WCA. The further enhancement of the gel through additional functionalisation with sol-gel-derived bioactive glass and liquid-exfoliated graphene was also investigated. The biocompatibility and biological characterisation of the substrates was assessed using murine osteoblasts (MC3T3), human primary dermal fibroblasts (HDFs), human fibroblast (BJ) cells, and standard cell culture assays (i.e., metabolic activity, LDH release, and live/dead staining). In short, P(3HB-co-4HB) was successfully isolated from the bacteria, with the defined physico-chemical profiles dependent on the culture substrate and culturing platform used. The additional enhancement of the copolymer with bioactive glass and/or graphene was also demonstrated by varying the combination loading of the materials, i.e., graphene resulted in an increase in tensile strength (~11 MPa) and the wettability increased following the incorporation of bioactive glass and 0.01 wt% graphene (WCA ~46.3°). No detrimental effects in terms of biocompatibility were noticed during the 7 days of culture in the primary and established cell lines. This study demonstrates the importance of optimising each of the individual components within the biocomposite and their relationship concerning the fine-tuning of the material's properties, thus targeting and impacting the endpoint application.

Fine-Tuning the Expression of the Glycolate Biosynthetic Pathway in Escherichia coli Using Synthetic Promoters

Glycolate plays an important role as a platform chemical in both polymeric material and cosmetic industries. However, the microbial production of glycolate often encounters challenges associated with unbalanced metabolic flux, leading to a notably low titer. Additionally, the use of expensive inducers, such as IPTG, contributes to an increase in the overall production cost. To address these issues, the key enzymes involved in the glycolate biosynthetic route, including citrate synthase (gltA), isocitrate lyase (aceA), isocitrate dehydrogenase kinase/phosphatase (aceK) and glyoxylate reductase (ycdW), were overexpressed in E. coli under the control of inducible promoters with varying strengths in order to determine the optimal combination. Subsequently, the glycolate pathway was further modulated by replacing inducible promoters with various constitutive synthetic promoters. Through this systematic optimization, the best strain, named Mgly4T1562, produced 3.02 g/L glycolate with 97.32% theoretical yield in shake-flask cultivation. The titer further increased to 15.53 g/L in a fed-batch experiment. Notably, this study marks the first successful utilization of synthetic promoters in tuning the glycolate biosynthetic pathway for glycolate biosynthesis. The strategy presented in this research holds significant promise for facilitating the cost-effective and industrially viable production of glycolate without the need for expensive inducers.

A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future.

Budding and explosive membrane vesicle production by hypervesiculating Escherichia coli strain ΔrodZ.

Escherichia coli produces extracellular vesicles called outer membrane vesicles. In this study, we investigated the mechanism underlying the hypervesiculation of deletion mutant ΔrodZ of E. coli. RodZ forms supramolecular complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. Because mreB is an essential gene, an expression-repressed strain (mreB R3) was constructed using CRISPRi, in which the expression of mreB decreased to 20% of that in the wild-type (WT) strain. In shaken-flask culture, the ΔrodZ strain produced >50 times more vesicles than the WT strain. The mreB-repressed strain mreB R3 showed eightfold higher vesicle production than the WT. ΔrodZ and mreB R3 cells were observed using quick-freeze replica electron microscopy. As reported in previous studies, ΔrodZ cells were spherical (WT cells are rod-shaped). Some ΔrodZ cells (around 7% in total) had aberrant surface structures, such as budding vesicles and dented surfaces, or curved patterns on the surface. Holes in the PG layer and an increased cell volume were observed for ΔrodZ and mreB R3 cells compared with the WT. In conditions of osmotic support using sucrose, the OD660 value of the ΔrodZ strain increased significantly, and vesicle production decreased drastically, compared with those in the absence of sucrose. This study first clarified that vesicle production by the E. coli ΔrodZ strain is promoted by surface budding and a burst of cells that became osmotically sensitive because of their incomplete PG structure.

Bacillus thuringiensis VP4: A potential keratinase producer isolated from poultry waste soils of Himachal Pradesh

Keratinase enzymes are protein hydrolyzing enzymes belonging to the serine hydrolase group and act specifically on keratin proteins to degrade them. The current study was conducted to isolate best keratinase producer bacterial strains. Four Bacillus species (B. cereus PP1- OP748781, B. cereus JP1- OP748782, B. cereus TP1- OP748783 and B. thuringiensis VP4- OP420549), were isolated from the poultry farms of Himachal Pradesh. These strains were evaluated for keratinase production by using chicken feathers in shake flask culture. Among four isolates, Bacillus thuringiensis VP4 showed highest enzymatic activity 343 U/ml. The molecular weight of the keratinase was observed 45 KDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimization of enzyme production was done with isolate Bacillus thuringiensis VP4 at various parameters such as temperature, pH and effect of metal ions. The maximum keratinase production was observed at 35°C temperature and pH 10.0. The isolated strain Bacillus thuringiensis VP4 might be used to produce poultry compost through degradation of chicken feathers and poultry waste in short time which may also be helpful for farmers and overcoming environmental pollution.

Development of user-friendly protocols for single-cell arachidonic acid production in developing countries

Arachidonic acid (ARA) is a sorely needed nutrient and an effective and safe schistosomicide and tumoricide. Consumption levels are low in developing countries because of unawareness of its critical impact on health and limited commercial availability. The research work aims to encourage production in developing countries via providing user-friendly definitive protocols for polyunsaturated fatty acids production by the fungus Mortierella alpina and ARA isolation. Fatty acids content was determined by gas chromatography-flame ionization detector. The optimal cultivation conditions included culture medium to flask volume 1:5; shake-flask culture at 25 °C, then at 20 °C, and stationary aging at 10 °C, each over 7 days, while addition of trace minerals and vitamins was dispensable. The ARA yield attained >1.8 g per liter culture. Repeat experiments led to the selection of an optimal lipid extraction method from dried biomass, and efficacious urea complexation-based protocol for obtention of high ARA yield and purity.

Research on the targeted improvement of the yield of a new VB12-producing strain, Ensifer adhaerens S305, based on genomic and transcriptomic analysis

BackgroundVitamin B12 (VB12) has a wide range of applications and high economic value. In this study, a new strain with high VB12 production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB12 production via fermentation in recent years, the directional modification of the S305 strain to obtain a strain suitable for the industrial production of VB12 has great potential and commercial value.Results16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) analysis showed that the high-yielding VB12 strain was a E. adhaerens strain and that its VB12 synthesis pathway genes were highly similar to related genes of strains of this and other species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB12 yields of the S305 strain were more than double those of the Casida A strain under different medium components. Multiple genes with significantly upregulated and downregulated transcription were identified by comparing the transcription intensity of different genes through transcriptome sequencing. KEGG enrichment analysis of the porphyrin metabolism pathway identified 9 significantly upregulated and downregulated differentially expressed genes (DEGs) in the VB12 synthesis pathway, including 7 transcriptionally upregulated genes (cobA, cobT, hemA, cobJ, cobN, cobR, and cobP) that were episomally overexpressed in the Casida A strain. The results showed that the VB12 yield of the overexpressed strain was higher than that of the wild-type strain. Notably, the strains overexpressing the cobA and cobT genes exhibited the most significant increases in VB12 yield, i.e., 31.4% and 24.8%, respectively. The VB12 yield of the S305 strain in shake-flask culture was improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the strain.ConclusionPhylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains were quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB12 synthesis pathway could increase the yield of VB12, further improving the VB12 yield of the E. adhaerens S305 strain.

Growth strategies of Chlorella vulgaris in seawater for a high production of biomass and lipids suitable for biodiesel

Chlorella vulgaris is a freshwater microalga that synthesises large amounts of saturated lipids, which makes it suitable for production of bioenergy and biofuels. Since its cultivation usually requires freshwater, it competes with agriculture, economic development and ecological conservation for this limited natural resource. This study investigated the possibility of the partial replacement of freshwater by seawater (50 %) in the growth medium for a more sustainable biomass and lipid production. Chlorella vulgaris 211-11b was cultivated as shake-flask cultures in Bold's Basal Medium (BBM) formulated with 50 % freshwater and 50 % seawater under photoautotrophic, mixotrophic and heterotrophic conditions for eight days with glucose as organic carbon source in the latter two cases. The alga's best growth performance and highest lipid contents (49 % DW−1), dominated by palmitioleic and oleic acid, occurred under mixotrophic rather than photoautotrophic and heterotrophic conditions. This study demonstrates a more economic and ecologically sustainable biomass and lipid production of C. vulgaris by saving 50 % freshwater, which is available for other purposes.

Modeling heterogeneity in a cell culture using a coupled population balance-oxidative stress model

Population balance models (PBMs) were formulated to describe the evolution in time of the cell population in terms of growth and oxidative stress. Flow cytometry data was used to gain insight into the distribution of important quantities (e.g., cell size, intracellular concentrations of metabolites) over the entire cell population. A coupled population balance-oxidative stress model was developed to predict distributions in cell size and intracellular glutamate, ROS, NADPH and NADP+ concentrations in shake flask cultures of B. pertussis. The major advantage of using a PBM is that it accounts for the distributions and can predict the heterogeneity of the cell population with respect to experimental conditions that are averaged out in bulk models. When comparing the coupled population balance – oxidative stress model to the bulk oxidative stress model, it is apparent that the PBM provides much better predictions of the intracellular ROS concentration. We hypothesize that due to the nonlinear relations between cell growth and oxidative stress, intracellular and cell surface quantities can be better modeled with population balance models.

Enhanced Biodegradation of Crude Oil in Seawater by Using Microbial Consortia

A biodegradation study was performed to assess the biodegradation of crude oil in seawater by consortium of Candida tropicalis RETL-Cr1, Pseudomonas aeruginosa BAS-Cr1 and Chromobacterium violaceum MAB-Cr1. Two independent experiments using different growth substrates, 5 mM glucose and 5% (v/v) crude oil, were undertaken to compare the microbial growth profile. The study was carried out using shake flask culture at 30°C, agitated 200 rpm. Microbial growth profile was monitored by measuring the optical density (OD600) on hourly and weekly basis. Biodegradation efficiency and rate were quantified by comparing the initial and final crude oil concentration, whereas the degradation of selected aliphatic hydrocarbons was quantified by comparing the initial and final area in chromatogram. The biodegradation ratios were monitored using Gas Chromatography Mass Spectrometry (GC-MS) method. Present finding showed that glucose was completely utilized at T10. However, the consortia can growth in crude oil even after 28 days. Overall biodegradation efficiency is 69.74% while the overall biodegradation rate is 24.85 g/L/d. The consortia could degrade 37% of n-alkane in crude oil after 28 days. Besides, biodegradation ratios shown that biodegradation had took place throughout the degradation period. It can be concluded that the consortia have high potential to degrade crude oil efficiently.

Biodisintegration of human mucin protein by protease produced from Escherichia coli AJ55 isolated from Urinary Tract Infection of Iraqi pa-tients

Proteases have various applications in the food, pharmaceutical, medicine, pathogenicity of some pathogenic bacteria, and detergent sectors as well as meeting the needs of approximately 60% of the global enzyme industry, whereas they catalyze the breakdown of protein molecules into peptides and amino acids. Production and purification of protease enzyme by the isolate Escherichia coli AJ55 was scrutinized in the present study. Cultivation optimum conditions, were various complex medium, carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the total protease production in shake flask culture of E.coli AJ55. The nutrient broth supplemented with 2% glucose and 2% yeast extract, with a pH of 7.0 and incubated at 37 °C for 24 hours, better conditioned for producing the maximum production of protease. Escherichia coli AJ55 proteolytic enzyme was separated and purified using ion-exchange chromatography on a DEAE-Cellulose column and Sephadex G-150 gel after being precipitated with 0-70% saturated ammonium sulfate. Protease that had been partially purified had a yield of 34%, a purification fold of 13.4, an activity of 12.16 U/ml, a protein concentration of 0.005 mg/ml, and a specific activity of 2432 U/mg. By using gel filtration chromatography on a Sephadex G-150 gel, partially purified protease was examined for its ability to cleave the mucin protein. The findings of mucin biodegradation showed that the five fractions of the small peptides were produced after treatment of mucin with partially purified protease.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g−1 h−1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

Optimization of central carbon metabolism by Warburg effect of human cancer cell improves triterpenes biosynthesis in yeast

Optimizing central carbon metabolism (CCM) represents an attractive and challenging strategy to improve the biosynthesis of valuable chemicals due to the complex regulation of the CCM in yeast. In this study, we triggered the similar Warburg effect of cancer cells in yeast strains by introducing the human hypoxia-inducible factor-1 (HIF-1) complex, which regulated the expression of numerous enzymes involved in CCM and redirected the metabolic flux from glycolysis to tricarboxylic acid cycle. This redirection promoted the production of squalene to a 2.7-fold increase than the control strain BY4741. Furthermore, the HIF-1 complex boosted the production of represented endogenous triterpenoid ergosterol to 1145.95 mg/L, and exogenous triterpenoid lupeol to 236.35 mg/L in shake flask cultivation, 10.5-fold and 9.2-fold increase than engineered strains without HIF-1 complex integration, respectively. This study provides a novel strategy for optimizing CCM by HIF-1 mediated Warburg effect of cancer cells to improve biosynthesis of triterpenoids in yeast.

De novo biosynthesis of anticarcinogenic icariin in engineered yeast

Icariin (ICA) has wide applications in nutraceuticals and medicine with strong anticancer activities. However, the structural complexity and low abundance in plants of ICA lead to the unsustainable and high-cost supply from chemical synthesis and plant extraction. Here, the whole biosynthesis pathway of ICA was elucidated, then was constructed in Saccharomyces cerevisiae, including a 13-step heterologous ICA pathway from eleven kinds of plants as well as deletions or overexpression of ten yeast endogenous genes. Spatial regulation of 8-C-prenyltransferase to mitochondria and three-stage sequential control of 4’-O-methyltransferase, 3-OH rhamnosyltransferase, and 7-OH glycosyltransferase expression successfully achieved the de novo synthesis of ICA with a titer of 130 μg/L under shake-flask culture. The ICA synthesis from glucose represents the longest reconstructed pathway of flavonoid in microbe so far. This study provides a potential choice for the sustainable microbial production of number of complex flavonoids.

Characterization and biodegradation of paracetamol by Bacillus pumilus strain PYP2

As we know the recent pandemic, coronavirus disease (COVID-19) due to SARS CoV-2 virus has led to an increase in the consumption of various drugs as medicines by the patients. Paracetamol (acetaminophen, APAP) act as an emerging contaminant classified among the class pharmaceutical and personal care pollutant (PPCP) and is detected in wastewater and sewage systems. The enrichment culture approach was used for the isolation APAP-degrading bacterium wastewater sample. Microscopic examination, biochemical and 16S rRNA sequence analysis showed that the isolate PYP-2 belongs to the Bacillus pumilus strain. Shake flask and batch culture degradation studies have shown that the strain can degrade APAP. Further, the response surface methodology (RSM) plot was used to know the best physical condition for biodegradation by optimization study. The optimum pH of 5.0, temperature of 30 °C, agitation speed of 146 rpm, and APAP 267 mg/L concentration were reported for PYP-2-based degradation. Bacterial biomass kinetic analysis was performed at the best physical condition, and the results showed that the specific growth rate (µ) was 713 mg/L. Oxalic acid, 2-isopropyl-5-methyl cyclohexanone, and phenothiazine were the intermediates of the APAP degradation pathway detected by the GC-MS chromatogram peaks. Therefore, this research has shown that Bacillus pumilus strain PYP-2 has the metabolic capacity to biodegrade APAP, providing new tools for bioremediation.

A procedure to harmonize the hydrodynamic force during microbial cultivation in shaking flasks.

Shake flask cultivation is a routine technique in microbiology and biotechnology laboratories where cell growth can be affected by the hydrodynamic conditions, which depend on the agitation velocity, shaking diameter, and shake flask size. Liquid agitation is implemented inherently to increase aeration, substrate transfer to the cells, and prevent sedimentation, disregarding the role of hydrodynamics in microbial growth and metabolism. Here, we present a simple approach to help standardize the hydrodynamic forces in orbital shakers to increase the experimental accuracy and reproducibility and give students a better knowledge of the significance of the agitation process in microbial growth.

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

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