Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Preparation of Komagataeibacter xylinus Inoculum for Bacterial Cellulose Biosynthesis Using Magnetically Assisted External-Loop Airlift Bioreactor

The aim of this study was to demonstrate the applicability of a novel magnetically assisted external-loop airlift bioreactor (EL-ALB), equipped with rotating magnetic field (RMF) generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200 × higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible water-related properties, mechanical strength, nano-fibrillar structure and total crystallinity index. The lack of any negative impact of inoculum preparation method using RMF-assisted EL-ALB on BC properties is of paramount value for its future applications, including use as a biomaterial in tissue engineering, wound healing, and drug delivery, where especially BC liquid capacity, nanostructure, crystallinity, and mechanical properties play essential roles.

Effect of Gas Holdup on CO2 Biofixation by Spirulina sp. in the 20-liter Airlift Bioreactor

The rise of CO2 concentration in the Earth is a major environmental problem, which cause global warming. To solve this issue, several methods have been applied, but among these solutions using microalgae is an eco-friendly and cost-effective way of reducing carbon dioxide, as they can efficiently sequestrate CO2 and produce biomass as valuable products. In this study, hydrodynamic parameters, bubble sizes and carbon dioxide uptake were investigated in an airlift bioreactor. Experiments were studied at two different superficial gas velocities (0.185 and 0.524 cm/s) for Spirulina sp. microalgae into a 20-liter airlift bioreactor to find out the amount of carbon dioxide sequestration and cyanobacterial biomass. The highest efficiency of carbon dioxide removal and maximum dry weight of Spirulina sp. were achieved 55.48% and 0.86 g/L respectively at 5% CO2 (v/v) and superficial velocity of 0.185 cm/s. This experiment was conducted in 7 days, light intensity (2600 lux/m2), temperature (30\(\pm\)2 °C) and a light-dark cycle (12–12), which all were constant. The hydrodynamic parameters studied by Spirulina sp. demonstrated a capability of CO2 sequestration in this airlift photobioreactor.

Pilot-Scale Airlift Bioreactor with Function-Enhanced Microbes for the Reduction of Refinery Excess Sludge

A pilot-scale airlift bioreactor (ALBR) system was built and operated continuously for refinery excess sludge (RES) reduction. Combined ALBR and function-enhanced microbes (composed of photosynthetic bacteria and yeast) were integrated into the system. The pilot-scale ALBR was operated for 62 days, and the start-up time was 7 d. Continuous operation showed that the sludge reduction efficiency was more than 56.22%, and the water quality of the effluent was satisfactory. This study focused on investigating the effects of hydraulic retention time (HRT) on the stability of the system and the effect of sludge reduction. Under different HRT conditions of 40, 26.7, 20, and 16 h, the sludge reduction rates reached 56.22%, 73.24%, 74.09%, and 69.64%, respectively. The removal rates of chemical oxygen demand (COD) and total nitrogen (TN) decreased with decreasing HRT, whereas the removal rate of NH4+-N increased. The removal rate of total phosphorus (TP) was approximately 30%. Results indicate that the ALBR and function-enhanced microbe system can reduce sludge and treat sewage simultaneously, and the effluent is up to the national emission standard. Addition of function-enhanced microbes can promote the degradation of petroleum hydrocarbon substances in the sludge, especially alkanes with low carbon numbers. This study suggests that the optimal HRT for the system is 16 h. The total operation cost of the ALBR combined with the function-enhanced microbe system can be reduced by 50% compared with the cost of direct treatment of the RES system.

Mixing characteristics of draft tube airlift bioreactor using the electrical resistance tomography

In this work, mixing characteristics in terms of mixing time, hydrodynamics (liquid circulation velocity and gas hold up) and shear rate were performed in the downcomer of a draft tube airlift bioreactor with different geometries (i.e., Ad/Ar between 0.38 – 2.31 and bottom clearances between 0.003-0.00 m). Newtonian (water and 34.5% coalescing sugar solution) and on Newtonian (0.2% and 0.5% xanthan gum solutions) with different viscosities were used as the liquid phase. Compressed air was used as the gas phase which was introduced through cross and circular shaped sparger configurations at superficial velocities Ugr = 0.00165-0.00807 m/s. The combined effects of geometric parameters (Ad/Ar, bottom clearances), sparger configuration, and liquid viscosity on mixing characteristics have been presented. Results showed that the increase in superficial gas velocity (Ugr) corresponds to an increase in energy generated, and thus decreases in mixing time. However, the increase in Ugr corresponds to the increase in liquid circulation velocity, gas holdup and shear rate values. Moreover, bottom clearances and draft tube diameters show effects on flow resistance frictional losses which affect results of mixing parameters investigated. The influence of sparger configurations on mixing time and liquid circulation velocity is significant due to their effect on gas distribution. Mixing time decreased to about 40% in air-water media using the cross shaped sparger. Results obtained with cross shaped sparger showed even and uniform distribution of gas, which provided better mixing as compared to the circular shaped sparger configuration. However, the sparger configuration effect on shear rate is not as significant (about 20% reduction in shear rate values using the cross shaped sparger). The effect of fluid viscosity had a significant influence on both mixing times and circulation velocity, especially in the coalescing media of sugar and xanthan gum solutions. Results from this work will help to develop a clear pattern for operation and mixing that can help improving [sic] several industrial processes, especially the ones related to emerging fields of technology such as the biotechnology industry.

Mixing characteristics of draft tube airlift bioreactor using the electrical resistance tomography

In this work, mixing characteristics in terms of mixing time, hydrodynamics (liquid circulation velocity and gas hold up) and shear rate were performed in the downcomer of a draft tube airlift bioreactor with different geometries (i.e., Ad/Ar between 0.38 – 2.31 and bottom clearances between 0.003-0.00 m). Newtonian (water and 34.5% coalescing sugar solution) and on Newtonian (0.2% and 0.5% xanthan gum solutions) with different viscosities were used as the liquid phase. Compressed air was used as the gas phase which was introduced through cross and circular shaped sparger configurations at superficial velocities Ugr = 0.00165-0.00807 m/s. The combined effects of geometric parameters (Ad/Ar, bottom clearances), sparger configuration, and liquid viscosity on mixing characteristics have been presented. Results showed that the increase in superficial gas velocity (Ugr) corresponds to an increase in energy generated, and thus decreases in mixing time. However, the increase in Ugr corresponds to the increase in liquid circulation velocity, gas holdup and shear rate values. Moreover, bottom clearances and draft tube diameters show effects on flow resistance frictional losses which affect results of mixing parameters investigated. The influence of sparger configurations on mixing time and liquid circulation velocity is significant due to their effect on gas distribution. Mixing time decreased to about 40% in air-water media using the cross shaped sparger. Results obtained with cross shaped sparger showed even and uniform distribution of gas, which provided better mixing as compared to the circular shaped sparger configuration. However, the sparger configuration effect on shear rate is not as significant (about 20% reduction in shear rate values using the cross shaped sparger). The effect of fluid viscosity had a significant influence on both mixing times and circulation velocity, especially in the coalescing media of sugar and xanthan gum solutions. Results from this work will help to develop a clear pattern for operation and mixing that can help improving [sic] several industrial processes, especially the ones related to emerging fields of technology such as the biotechnology industry.

Preparation of Komagataeibacter xylinus inoculum for bacterial cellulose biosynthesis using magnetically assisted external-loop airlift bioreactor

The aim of this study was to demonstrate the applicability of a novel magnetically-assisted external-loop airlift bioreactor (EL-ALB), equipped with RMF generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200x higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible mechanical strength, nanostructure and total crystallinity index. The results obtained in this study may find multiple applications in any biotechnological processes requiring a high-quality bacterial inoculum.