Shake Flask System Research Articles

A parametric optimization for leveraging the potential of ammonia modified magnetic pine cone hydrochar for Cr (VI) contaminated wastewater treatment

The present study evaluated the biosorption potential of magnetic pine cone hydrochar (MPHC) for chromium removal from wastewater. Initially, three different methodologies were followed for the synthesizing magnetic hydrochar and were tested for chromium removal and with a maximum removal of 91.3%, ammonia-modified MPHC showed its tremendous potential for Cr (VI) removal. Further, the hydrochar was characterized physically, chemically, magnetic, and morphological and a Box-Behnken experimental design was used for examining the effect of all the five parameters for Cr (VI) removal from the simulated wastewater system. Ammonia-modified MPHC showed a removal efficiency of 98.46% under the optimized batch shake flask system with the initial chromium concentration (55 mg/L), MPHC dose (550 mg/L), temperature (40 °C) and reaction time (120 min) and iron-MPHC ratio (30%). Here, the maximum Cr (VI) adsorption capability (qe) was found to be 154 mg/g. Further, the effect of different bed height, inlet pollutant flow rate, and inlet pollutant concentration during the continuous packed bed biosorption study showed a significant removal. The breakthrough curves for Cr (VI) removal obtained via the continuous flow-through column study confirmed that MPHC in the fixed-bed column suits Cr (VI) removal.

Agro-Industrial Wastes as Potential Substrates for Rhamnolipid Production by Pseudomonas aeruginosa USM-AR2.

Rhamnolipid has gained much attention in various fields owing to its distinctive functional properties compared to conventional chemical surfactants, which are mostly derived from petroleum feedstock. Production cost is one of the main challenges in rhamnolipid production, particularly when using refined substrates. One possible solution is to use agro-industrial wastes as substrates for rhamnolipid production. This is a promising strategy due to their abundance and commercially low value, while simultaneously alleviating an agro-industrial waste management problem in the environment. This study aims to evaluate agro-industrial wastes from local crops as possible low-cost alternative substrates for rhamnolipid production by a local isolate, Pseudomonas aeruginosa USM-AR2. Various liquid wastes, namely sugarcane molasses, rice washing water, overly mature coconut (OMC) water, empty fruit bunch (EFB) steam effluent, palm sludge oil (PSO) and palm oil mill effluent (POME) were screened as the main carbon source supplementing mineral salt medium (MSM) in the fermentation of P. aeruginosa USM-AR2. Batch fermentation was carried out in a shake flask system, agitated at 200 rpm and incubated at room temperature, 27 ± 2°C for 120 h. Among the substrates tested, PSO exhibited the highest biomass at 20.78 g/L and rhamnolipid production at 1.07 g/L. This study has shown the potential of agro-industrial wastes in Malaysia as an alternative resource for rhamnolipid production, transforming them into value added products, while reducing the amount of wastes discharged into the environment.

Assessment of cultivation parameters influencing pectinase production by Aspergillus niger LFP-1 in submerged fermentation

BackgroundPectinase is helpful in food and beverage industries, particularly in the preparation of fruit juice, the extraction of vegetable oil, and the fermentation of coffee. The current work aimed to screen Aspergillus niger LFP-1, a recently identified fungal strain, for its ability to produce pectinase and to ascertain the contribution of various physicochemical factors to pectinase production. ResultsThe primary and secondary pectinase activity screenings by Aspergillus niger LFP-1 were performed using pectin screening agar and shake flask system, respectively. The finding revealed that the locally isolated strain is able to secrete favourable pectinase production. Before improvement, the pectinase production was 0.88 ± 0.09 U/mL. However, the improved conditions such as 6 days of the cultivation period, agitation speed of 150 rpm, inoculum size of 1 × 106 spores/mL, 2.5% (w/v) citrus pectin, and 0.4% (w/v) ammonium nitrate could significantly increase pectinase production up to 7.41 ± 0.24 U/mL, representing an 88% increase. In this study, supplementing 2.5% (w/v) citrus pectin to the culture medium as a carbon source increased enzyme production by up to 3.07 ± 0.17 U/mL. Meanwhile, 0.4% (w/v) ammonium nitrate was used as a nitrogen source yielding the highest enzyme activity with a value of 6.86 ± 0.07 U/mL. ConclusionThus, the locally isolated fungal strain, A. niger LFP-1 has outstanding pectinase-producing capability and can be utilized for the commercial production of pectinase. The improved cultural conditions significantly increase pectinase production and shorten the incubation period from 8 days (before improvement) to 6 days (after improvement).

Establishment of Gypsophila paniculata root culture for biomass, saponin, and flavonoid production

Baby’s breath (Gypsophila paniculata L.) roots are valuable as pharmaceuticals due to the content of triterpenoid saponins and other bioactive phytochemicals. However, the long root harvest period and fluctuation of these constituent’s content are among the constraints to traditional agricultural production. The present study shows an efficient and reliable liquid root culture of G. paniculata cv. ‘Perfecta’ established in a shake flask system using different auxins, media, and sugars. Hairy root (HR) induction in G. paniculata through Rhizobium rhizogenes-mediated transformation was also investigated. α-Naphthaleneacetic acid (NAA) was optimized for adventitious roots (AR) biomass, saponin, and flavonoid production at 1 or 2 mg/L compared to IBA. Full strength Gamborg’s medium (B5) recorded higher saponin content; however, the highest yield of total saponin and total flavonoids was achieved by full strength Murashige and Skoog's (MS) medium. Sucrose was more essential for root growth and accumulated total saponins and flavonoids rather than fructose and glucose. Polymerase chain reaction (PCR) analysis showed that G. paniculata HR carried rolC gene of R. rhizogenes A4 strain in its genome but not virD2 gene. Compared to non-transformed root, saponin content of leaf and stem-derived HR was 2.7 and 2.3-fold, while total flavonoid content was 2.1 and 2.0-fold, respectively. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was also higher in HR extracts than in non-transgenic roots. This study established an efficient protocol for G. paniculata root cultures for sustainable production of important natural saponins and flavonoids.

Sunlight–Triggered Synergetic Facilitation of Hematite and Lactobacillus: A Vital Enhanced Carbon Conversion Pathway in Nature

Sunlight–triggered mineral–microbial interaction is extensively involved in the material circulation and energy flow process in the global system, accompanied by element cycle including carbon, nitrogen, and sulfur. Here, the phenomenon of enhanced carbon conversion efficiency between Lactococcus lactis (L. lactis) and natural hematite was demonstrated for the first time. In dual–chamber system, the average speed of glucose consumption would promote above 109% and the electrons transfer efficiency was promoted 17.9%, which compared with ‘L. lactis (Dark).’ It indicated a burgeoning electron transfer pathway between the L. lactis and hematite under light illumination. To simulate a blend system in nature, there was also almost 2 times promotion under the light condition in shake–flask system. Furthermore, for further close to natural environments about sunlight triggered facilitation, the systems of the hybrid colony (Lactobacillus)–hematite and hybrid colony–red soil were structured. Notably, glucose consumption in light–hematite condition (0.38 ± 0.085 g/L) was 2.23 times higher than ‘L. lactis (Dark)’ condition, which verified that the facilitated carbon source consumption process of Lactobacillus occurring in nature was prevalent widely. Nevertheless, it was an underappreciated carbon conversion pathway in nature, our research may have great significance for a more comprehensive understanding of the carbon cycle in nature.

Bioprocess development as a sustainable platform for eco-friendly alkaline phosphatase production: an approach towards crab shells waste management

BackgroundThere are substantial environmental and health risks associated with the seafood industry's waste of crab shells. In light of these facts, shellfish waste management is critical for environmental protection against hazardous waste produced from the processing industries. Undoubtedly, improved green production strategies, which are based on the notion of "Green Chemistry," are receiving a lot of attention. Therefore, this investigation shed light on green remediation of the potential hazardous crab shell waste for eco-friendly production of bacterial alkaline phosphatase (ALP) through bioprocessing development strategies.ResultsIt was discovered that by utilizing sequential statistical experimental designs, commencing with Plackett–Burman design and ending with spherical central composite design, and then followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor, an innovative medium formulation could be developed that boosted ALP production from Bacillus licheniformis strain ALP3 to 212 U L−1. The highest yield of ALP was obtained after 22 h of incubation time with yield coefficient Yp/s of 795 U g−1, which was 4.35-fold higher than those obtained in the shake-flask system. ALP activity has a substantial impact on the volatilization of crab shell particles, as shown by the results of several analytical techniques such as atomic absorption spectrometry, TGA, DSC, EDS, FTIR, and XRD.ConclusionsWe highlighted in the current study that the biovalorization of crab shell waste and the production of cost-effective ALP were being combined and that this was accomplished via the use of a new and innovative medium formulation design for seafood waste management as well as scaling up production of ALP on the bench-top scale.

Effects of trehalose and ergosterol on pinene stress of Candida glycerinogenes.

Pinene is a commercially important monoterpene that can be prepared using engineered bacterial and yeast species; however, high pinene levels can adversely affect the stability and permeability of microbial membranes leading to significantly reduced growth yields. This study reports that the fluidities and permeabilities of cell membranes of Candida glycerinogenes decrease aspinene levels increase resulting in adverse effects on cell growth. Exposure of cells to pinene results in upregulation of the genes encoding ergosterol and trehalose whose production helps stabilize their cell membranes. Exogenous addition of ergosterol and trehalose to pinene-treated cells also reduces the fluidity and permeability of the cell membrane, whilst also reducing production of intracellular reactive oxygen species. This led to the finding that the biomass of yeast cells cultivated in shake flask systems are improved by exogenous addition of trehalose and ergosterol. Overexpression of genes that encode trehalose and ergosterol produced a recombinant C. glycerinogenes strain that was found to tolerate higher concentrations of pinene.

Culturing and Scaling up Stem Cells of Dental Pulp Origin Using Microcarriers.

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Development of a bellows pumping device for enhancing ventilation to shake-flask systems

Shake-flask culture has been developed as an alternative to microbial surface culture. Shake-flask systems are known to affect microbial activity via composition of the headspace and ventilation ability of the culture plug. Gaseous control methods are limited to improvement of monitoring devices for shake-flask. In this study, a novel device was developed to improve ventilation in shake-flask culture without any complicated operations. This device was designed to induce a reciprocating motion of metal balls that results in the activation of the built-in bellows pump to send the outside air forcibly and aseptically into the flask gas phase. The ventilation capacity of the developed nonelectric bellows pump for shake-flask (NeBP-sf) was evaluated using the half-life of CO2 as an index. The maximum CO2 half-life of the NeBP-sf was found to be less than 10 min, and more than 5 times higher than that of the control (about 50 min). The NeBP-sf improved the ventilation capacity of shaking culture, resulting in enhanced growth of Escherichia coli compared with the control. Overall, these findings show that the developed device could have practical applications in reducing the space requirements and does not require additional electrical power to function.

High efficient assimilation of NO₃⁻-N with coproduction of microalgal proteins by Chlorella pyrenoidosa

The aim of this study was to establish a novel technology using microalgae for NO₃⁻ removal from high concentration wastewater and conversion to algal proteins. The effects of cultivation modes and illumination modes on the biomass yield, NO₃⁻ assimilation rate and algal protein yield were first investigated in shaking flasks for mixotrophic cultivation of Chlorella pyrenoidosa, and subsequently the scale-up verification in 5-L photo fermenter was successfully conducted. Fed-batch cultivation without medium recycling was the best cultivation mode in shaking flask system, in which the highest biomass yield (35.95 g/L), the average NO₃⁻ assimilation rate (2.06 g/(L·d)) and algal protein content (up to 42.44% of dry weight) were achieved. By using a staged increase of light intensity as illumination modes, the specific growth rate of cells could be significantly promoted to the highest (0.65 d⁻¹). After a 128-hour continuous cultivation in a 5-L photo fermenter, the highest biomass yield and the average NO₃⁻ assimilation rate were reached to 66.22 g/L and 4.38 g/(L·d) respectively, with the highest algal protein content at 47.13% of dry weight. Our study could provide a photo fermentation technology of microalgae for highly efficient treatment of waste industrial nitric acid and/or high concentration nitrate wastewater. This microalgae-based bioconversion process could coproduce protein-rich microalgal biomass, which facilitates the resource utilization of these type wastewater by trash-to-treasure conversion.

Newly discovered Synechococcus sp. PCC 11901 is a robust cyanobacterial strain for high biomass production

Cyanobacteria, which use solar energy to convert carbon dioxide into biomass, are potential solar biorefineries for the sustainable production of chemicals and biofuels. However, yields obtained with current strains are still uncompetitive compared to existing heterotrophic production systems. Here we report the discovery and characterization of a new cyanobacterial strain, Synechococcus sp. PCC 11901, with promising features for green biotechnology. It is naturally transformable, has a short doubling time of ≈2 hours, grows at high light intensities and in a wide range of salinities and accumulates up to ≈33 g dry cell weight per litre when cultured in a shake-flask system using a modified growth medium − 1.7 to 3 times more than other strains tested under similar conditions. As a proof of principle, PCC 11901 engineered to produce free fatty acids yielded over 6 mM (1.5 g L−1), an amount comparable to that achieved by similarly engineered heterotrophic organisms.

Optimization of Medium Composition for Inulin Fructotransferase (IFTase) Production by Nonomuraea sp.

Inulin fructotransferase is an enzyme which converts inulin into difructose anhydride III (DFA III). Inulin fructrotransferase was produced by fermentation in medium containing various concentrations of inulin (10 g/L, 20 g/L) as carbon source, yeast extract (1 g/L, 2g/L) as organic nitrogen source and (NH4)2SO4 (2g/L, 5g/L) as inorganic nitrogen source using Nonomuraea sp. ID06-A0189. The aim of the study was to optimize the medium composition using various concentrations of carbon and nitrogen sources for inulin fructotransferase production by Nonomuraea sp. ID06-A0189, through shake flask system. Fermentation was conducted at 30 °C, pH 7.0 and agitated in 130 rpm for three days. The result showed that comparable with those found in another condition the highest enzyme activity was produced in medium using inulin as carbon source with concentration of 10 g/L, yeast extract as organic nitrogen source with concentration of 5 g/L and ammonium sulfate as inorganic nitrogen source with concentration of 2 g/L. Addition of inulin as a carbon source increased the volume of biomass by about 11.5-24 mL. The enzyme activity in the culture supernatant reached 25.3 U/mL after three days incubation.

Screening of Medium Constituents for the Cultivation of Scenedesmus dimorphus UTEX 1237 using 2k Factorial Design Approach

Freshwater microalga, Scenedesmus dimorphus is a beneficial natural resource for producing biofuel and novel bioproducts. S. dimorphus growth greatly depends on physical culture conditions as well as medium constituents. A study was conducted to select the suitable medium components that could promote high biomass production in S. dimorphus UTEX 1237. Screening of growth medium constituents was performed using the two-level factorial design (2k FD) method. S. dimorphus was cultivated in TAPS aquaculture medium employing two types of carbon source (sodium bicarbonate and acetic acid) and three types of nitrogen sources (ammonium chloride, sodium nitrate, and urea). Consequently, medium components that significantly influenced biomass production comprised of acetic acid, ammonium chloride, and urea. Two weeks of S. dimorphus cultivation in shake flask system consisting of 1.5 g/L of acetic acid, 10 g/L of ammonium chloride and 0.4 g/L of urea was found to affect a specific growth rate of 0.0049 h−1 and biomass productivity of 0.05 g/L/day, thus affecting 66.7% improvement over normal TAP formulation.

Xylanase production from combined Reutealis trisperma with potato dextrose broth by Tricodherma reesei: the effect of pretreatment

Xylanase is an important enzyme in pulp and paper, food, feed, and textile industries. It is produced by fermentation process with Tricodherma reesei as fungi, which grows on several carbon sources such as cellulose and xylan. Reutealis trisperma (kemiri sunan) have been used for biodiesel production after being mechanically pressed. The solid waste, which is called Reutealis trisperma cake (RTC) contains carbon and nitrogen sources which are valuable to be used for xylanase production by T. reesei. Shake flask systems were used to grow T. reesei in medium consists of RTC and Potato Dextrose Broth (PDB). The systems were maintained at 28°C, 200 rpm at initial pH of 6.0 for 84 hours. Two forms RTC used in this study were natural and alkaline pretreated. The RTC fermentations were performed for different concentration of PDB (15g/L and 20 g/L) and also for different concentration of RTC (13.33 g/L; 16.67 g/L and 20 g/L). Pretreated systems gave better yield of xylanase production compared to natural systems. Higher concentration of PDB for pretreated system was found to yield lower xylanase production. The highest xylanase activity (198.1 U/mL) was obtained in media containing 15 g/L PDB and 20 g/L pretreated RTC.

Effect of stirring speed on the production of phenolic secondary metabolites and growth of Buddleja cordata cells cultured in mechanically agitated bioreactor

Shake-flask in vitro culture of Buddleja cordata cells produces large amounts of biomass and synthetizes verbascoside (VB), linarin and hydroxycinnamic acids, bioactive phenolic secondary metabolites (PSMs). In this work, we determined the effect of stirring speed on the growth of and production of PSMs [total phenolic, phenylethanoid glycoside and flavonoid contents (PeC, PeGC and FC, respectively)] by B. cordata cells cultured in two bioreactors. Two different stirring speeds (120 and 400 rpm) were tested in two stirred-tank bioreactors: a 2 L bioreactor equipped with a ring diffuser (B2RD) and a 3 L bioreactor with a sintered diffuser (B3SD). Growth kinetics of B. cordata cells were measured in the bioreactors and shake-flask systems. The stirring speed and type of bioreactor affected phases, parameters of growth and production of PSMs. The highest production of biomass (13.62 g L−1) and PSMs [PeC of 64.63 mg gallic acid equivalents g−1 (mg GAE g−1); PeGC of 119.24 mg VB equivalents g−1 (mg VBE g−1); and FC of 5.02 mg quercetin equivalents g−1 (mg QE g−1)] occurred in B2RD at 400 rpm. These values were similar to the found in shake-flasks system. This work establishes the basis for bioprocess advances of B. cordata focused on the development of a sustainable strategy for the management of natural resources and as a source of bioactive PSMs on a large scale.

Continuous non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22

In-situ extraction of microalgal oil without cell destruction has been proposed as a cost-effective alternative solution for the production of algal biofuel. This study examines the viability and efficiency of n-dodecane in both column and shake flask systems for the continuous extraction of botryococcene from Botryococcus braunii (BOT-22). Botryococcene from B. braunii was found to be non-destructively extracted using n-dodecane without negatively affecting the growth and photophysiology of the microalgae. Recirculation of the culture (23 times per day) through a column system containing n-dodecane with a daily effective extraction time of 276 s resulted in 21% higher botryococcene extraction (25.1 mg g−1 biomass) when compared to a constantly stirred shake flask consisting of both algae culture and n-dodecane together in a vessel. Botryococcene extraction was highest in the first 24 h of extraction when compared to the rest of the extraction period. These results suggest that recirculation of B. braunii culture through a column system containing n-dodecane is efficient for the non-destructive extraction of botryococcene and less toxic compared to other previously tested solvents.

Bioprocess development of a stable FUT8-/--CHO cell line to produce defucosylated anti-HER2 antibody.

In recent years, an increasing number of defucosylated therapeutic antibodies have been applied in clinical practices due to their better efficacy compared to fucosylated counterparts. The establishment of stable and clonal manufacturing cell lines is the basis of therapeutic antibodies production. Bioprocess development of a new cell line is necessary for its future applications in the biopharmaceutical industry. We engineered a stable cell line expressing defucosylated anti-HER2 antibody based on an established α-1,6-fucosyltransferase (FUT8) gene knockout CHO-S cell line. The optimization of medium and feed was evaluated in a small-scale culture system. Then the optimal medium and feed were scaled up in a bioreactor system. After fed-batch culture over 13days, we evaluated the cell growth, antibody yield, glycan compositions and bioactivities. The production of anti-HER2 antibody from the FUT8 gene knockout CHO-S cells in the bioreactor increased by 37% compared to the shake flask system. The N-glycan profile of the produced antibody was consistent between the bioreactor and shake flask system. The antibody-dependent cellular cytotoxicity activity of the defucosylated antibody increased 14-fold compared to the wild-type antibody, which was the same as our previous results. The results of our bioprocess development demonstrated that the engineered cell line could be developed to a biopharmaceutical industrial cell line.

Phenolic compounds profiling in shake flask and bioreactor system cell cultures of Scrophularia striata Boiss

Variation in levels of phenolic acids and flavonoids in Scrophularia striata Boiss. cells cultured in both shake flask and bioreactor in vitro systems, was studied at different growth phases. Four phenolic acids (cinnamic, salicylic, coumaric, and caffeic acid), one stilbenoid (resveratrol), and seven flavonoids (diosmin, rutin, kaempferol, catechin, myricetin, quercetin, and luteolin) were analyzed by high-performance liquid chromatography with photodiode array detection. Production of phenolics in the bioreactor was higher than in shake flasks. Catechin was the most abundant flavonoid in both culture systems, while quercetin, which was detected only in the bioreactor, was the lowest amount represented (32.82 μg g−1 DW). Resveratrol accumulation in bioreactor cultures was 59.84-fold higher than that in shake flasks. Moreover, hierarchical clustering analysis based on Pearson’s correlation coefficient confirmed a positive correlation between the growth phase and some metabolites. The flavonoid accumulation increased with the cells’ physiological age in the bioreactor. Principal component analysis showed that the time course of induction of phenolic acids, flavonoids, and a stilbenoid (resveratrol) was significantly correlated. These findings highlight the capacity of S. striata for large-scale production of desired phenolics using a bioreactor system.

Kinetics and Optimization of Lipophilic Kojic Acid Derivative Synthesis in Polar Aprotic Solvent Using Lipozyme RMIM and Its Rheological Study.

The synthesis of kojic acid derivative (KAD) from kojic and palmitic acid (C16:0) in the presence of immobilized lipase from Rhizomucor miehei (commercially known as Lipozyme RMIM), was studied using a shake flask system. Kojic acid is a polyfunctional heterocycles that acts as a source of nucleophile in this reaction allowing the formation of a lipophilic KAD. In this study, the source of biocatalyst, Lipozyme RMIM, was derived from the lipase of Rhizomucor miehei immobilized on weak anion exchange macro-porous Duolite ES 562 by the adsorption technique. The effects of solvents, enzyme loading, reaction temperature, and substrate molar ratio on the reaction rate were investigated. In one-factor-at-a-time (OFAT) experiments, a high reaction rate (30.6 × 10−3 M·min−1) of KAD synthesis was recorded using acetone, enzyme loading of 1.25% (w/v), reaction time of 12 h, temperature of 50 °C and substrate molar ratio of 5:1. Thereafter, a yield of KAD synthesis was optimized via the response surface methodology (RSM) whereby the optimized molar ratio (fatty acid: kojic acid), enzyme loading, reaction temperature and reaction time were 6.74, 1.97% (w/v), 45.9 °C, and 20 h respectively, giving a high yield of KAD (64.47%). This condition was reevaluated in a 0.5 L stirred tank reactor (STR) where the agitation effects of two impellers; Rushton turbine (RT) and pitch-blade turbine (PBT), were investigated. In the STR, a very high yield of KAD synthesis (84.12%) was achieved using RT at 250 rpm, which was higher than the shake flask, thus indicating better mixing quality in STR. In a rheological study, a pseudoplastic behavior of KAD mixture was proposed for potential application in lotion formulation.

Development of a circulation direct sampling and monitoring system for O2 and CO2 concentrations in the gas\u2013liquid phases of shake-flask systems during microbial cell culture

Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO2 and O2 in the gas–liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of Escherichia coli using this set-up indicated that a high concentration of CO2 accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5–9.0 h). By packing a CO2 absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO2 from the headspace. It was posited that removing CO2 from the headspace would suppress increases in the dissolved CO2 concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5–12.0 h) was extended, the U.O.D.580 and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of E. coli cells without changing the composition of the medium, temperature, and shaking conditions.