Development of thin-film photo-bioreactor and its application to outdoor culture of microalgae

Photosynthetic microalgae have received much attention as a microbial source of diverse useful biomaterials through CO₂ fixation and various types of photo-bioreactors have been developed for efficient microalgal cultivation. Herein, we developed a novel thin-film photo-bioreactor, which was made of cast polypropylene film, considering outdoor mass cultivation. To develop optimal design of photo-bioreactor, we tested performance of three shapes of thin-film photo-bioreactors (flat, horizontal and vertical tubular shapes) and various parts in the bioreactor. Collectively, vertical tubular bioreactor with H/D ratio 6:1 and cylindrical stainless steel spargers showed the most outstanding performance. Furthermore, the photo-bioreactor was successfully applied to the cultivation of other microalgae such as Chlamydomonas reinhardtii and Chlorella vulgaris. The scalability of photo-bioreactor was confirmed by gradually increasing culture volume from 4 to 25 L and the biomass productivity of each reactor was quite consistent (0.05–0.07 g/L/day) during the cultivation of H. pluvialis under indoor and outdoor conditions. Especially, we also achieved dry cell weight of 4.64 g/L and astaxanthin yield of 218.16 mg/L through long-term cultivation (100 days) under outdoor condition in 15 L photo-bioreactor using Haematococcus pluvialis, which means that the astaxanthin yield from outdoor cultivation is equal or superior to that obtained from controlled indoor condition. Therefore, these results indicate that we can apply this approach to development of optimal photo-bioreactor for the large-scale culture of microalgae and production of useful biomaterials under outdoor condition.     

Production of extracellular protease from crude substrates with dregs in an external-loop airlift bioreactor with lower ratio of height to diameter

Bacillus subtilis AS1.398 was cultivated in a 11.5-L total volume external-loop airlift bioreactor with a low height-to-diameter ratio of 2.9 and a riser-to-downcomer diameter ratio of 6.6 for the production of protease from crude substrates with dregs. The influence of aeration rate, liquid volume, and sparger hole diameter on protease production was investigated. An average of 8197 u/mL protease activity was obtained after a total fermentation time of 32 h in the external-loop airlift bioreactor with a liquid volume of 8.5 L, air flow rate of 1.5 vvm, and sparger hole diameter of 1.5 mm. The addition of one stainless steel sieve plate in the riser of the airlift bioreactor increased productivity of protease. After 32 h of fermentation, an average of 8718 u/mL protease activity was achieved in the external-loop airlift bioreactor with one sieve plate and an air flow rate of 1.2 vvm, liquid volume of 8.5 L, and gas sparger hole diameter of 1.5 mm. This was 9.0% higher than the typical averages of about 8000 u/mL protease activity in the mechanically stirred tank bioreactors of the enzyme factory using the same microorganism. It is possible to make a scale-up of the external-loop airlift bioreactor and feasible to operate it for production of protease from crude substrate with dregs.     

利用响应面方法优化转小鼠金属硫蛋白-I基因聚球藻7002的培养基成分

为实现转小鼠金属硫蛋白基因-I聚球藻7002的高密度培养, 并将其应用于实际的重金属废水处理过程, 首先需要对培养基的成分进行优化。本文利用响应面这一多因素过程优化的有效工具, 通过全因子实验、最陡爬坡实验和中心组合实验, 对转小鼠金属硫蛋白基因-I聚球藻7002培养基的主要成分以及初始pH进行了优化。优化后的培养基组成为: NaHCO3 1.696 g/L, NaNO3 8.57 g/L, 初始pH为8.57, 其他成分同Medium A。优化条件分别在2 L和20 L气升式光生物反应器中得到了验证, 最大细胞浓度分别达到每升4.16 g干重和每升3.12 g干重, 分别比优化前提高了9倍和7倍, 从而为其产业化应用打下基础。     

生物反应器在满天星快繁中的应用

利用2.5 L柱状气升式生物反应器采用接触法进行了满天星(Gypsophila paniculata)增殖培养。结果表明: 外植体接种密度(每个反应器接种外植体个数)为35比密度为25和45更有利于满天星的增殖生长, 平均每个生物反应器内可获得252株生长健壮的不定苗; 光强为90 mmol.m-2.s-1对满天星增殖培养最有利, 过高的光强对不定芽和株高有抑制作用; 通过小孔隙(15 mm)的多孔喷头注入0.1 vvm空气有利于满天星增殖培养。     

Substrate gradient formation in the large-scale bioreactor lowers cell yield and increases by-product formation

A heterogeneous micro-environment was identified in a 12 m³ bioreactor with a height-to-diameter ratio of 2.5. The reactor was aerated by a ring sparger and stirred by three Rushton turbines. E. coli cells were cultivated in minimal medium to a cell density in the order of 30?g/l. Samples of glucose, the growth limiting component fed to the process, were taken at three levels in the bioreactor (top/middle/bottom). These showed that glucose concentration declined away from the feedpoint. The gradients depended on the mixing characteristics of the feedpoint, and concentrations of up to 400 times the mean value were found when feed was added to a relatively stagnant mixing zone. This resulted in up to 20% lower biomass yield compared to the bench scale. Gradients also affected the by-product formation, resulting in acetate formation in the large-scale bioreactor. IPTG induction of a recombinant protein was shown to influence important cell parameters and considerably increased the yield of carbon dioxide per glucose added, indicating an increased maintenance. The product formation rate was, however, not notably affected by the scale-up.     

NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free cultures

Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day(-1) with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be approximately 30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing.     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

Macromixing hydrodynamic study in draft-tube airlift reactors using electrical resistance tomography

The present study summarizes results of mixing characteristics in a draft tube airlift bioreactor using ERT. This technique offers the possibility for noninvasive and nonintrusive visualization of flow fields in the bioreactor and has rarely been utilized previously to analyze operating parameters and mixing characteristics in this type of bioreactors. Several operating parameters and geometric characteristics were examined. In general, results showed that the increase in superficial gas velocity corresponds to an increase in energy applied and thus, to a decrease in mixing time. This generally corresponded to an increase in liquid circulation velocity and shear rate values. Bottom clearances and draft tube diameters affected flow resistance and frictional losses. The influence of sparger configurations on mixing time and liquid circulation velocity was significant due to their effect on gas distribution. However, the effect of sparger configuration on shear rate was not significant, with 20% reduction in shear rates using the cross-shaped sparger. Fluid viscosity showed a marked influence on both mixing times and circulation velocity especially in the coalescing media of sugar and xanthan gum (XG) solutions. Results from this work will help to develop a clear pattern for operation and mixing that can help to improve several industrial processes, especially the ones related to emerging fields of technology such as the biotechnology industry.     

Enhanced astaxanthin production from microalga,Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation

For efficient astaxanthin production from the culture of green microalga, Haematococcus pluvialis, a two-stage mixotrophic culture system was established with stepwise increased light irradiance. By perfusion process, high density biomass (2.47 g/L) was achieved during the vegetative stage due to no detrimental effect of inhibitory metabolites, which was 3.09 and 1.67 times higher than batch and fed-batch processes, respectively. During the induction stage, biomass and astaxanthin were subsequently produced to the very high level 12.3 g/L and 602 mg/L, under stepwise increased light irradiance (150–450 μE/m²/s), respectively. These results indicate that the combinatorial approach of perfusion culture during the vegetative stage and stepwise light irradiation during the induction stage is a promising strategy for the simultaneous production of high concentration of biomass and astaxanthin in microalgae including H. pluvialis.     

Hydrodynamic performance of a three-phase airlift bioreactor with an enlarged degassing zone

The hydrodynamics of biotechnological processes is complex. So far, few studies were made with bioreactors of the airlift type with an enlarged degassing zone.In this work, the influence of solids loading, solids specific gravity and draught tube dimensions on mixing and circulation times and critical air flow rate for an internal loop airlift bioreactor with an enlarged sedimentation/degassing zone is studied.The results indicate that the critical air flow rate as well as the mixing time increase with an increase in solids loading in the bioreactor. Circulation time presents a maximum for a solids load between 5 and 10% (v/v). It is also shown that small variations in solids specific gravity, for values close to that of the liquid, have a significant influence on the critical air flow rate and on the mixing time.An optimal (minimal) value for the circulation time and for the critical air flow rate was obtained for a riser to down comer diameter ratio of 0.46. The minimum mixing time was obtained for a riser to down comer height ratio of 0.80.This work was supported by J.N.I.C.T. (Junta Nacional de Investigação Cientifica e Tecnológica).     

Effects of Various Light-Emitting Diode (LEd) Wavelengths on the Growth of Scenedesmus Obliquus Fachb-12 and Accumulation of Astaxanthin

Given the central role of light in the algal photosynthesis, respiration, cell division, growth and the accumulation of value products, the effects of light-emitting diodes (LEDs) light wavelengths (blue, white, red and green) were studied in Scenedesmus obliquus. Biomass, residual nutrient amount, soluble protein, astaxanthin and reactive oxygen species, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity were analyzed to determine the effects of different monochromatic light wavelengths via biochemical methods. The results showed that blue light wavelength is the optimal light wavelength for phosphorus removal efficiency and the accumulation of biomass and astaxanthin in S. obliquus. Meanwhile, high reactive oxygen species content under the blue light might induce the accumulation of astaxanthin. The high activity of SOD, CAT and POD might participate in clearing the reactive oxygen species to facilitate the growth of microalgae. Furthermore, we found mixed blue/green lights treatment is the most appropriate mixture for the nitrogen removal. Under the blue light treatment, high light intensity and 18L:6D light cycle is the best condition for biomass and astaxanthin accumulation. Optimal nitrogen/phosphorus removal efficiency was observed under a 24L:0D light cycle. These results might provide a foundational data for the optimizing the productivity of high-value metabolites and treatment of wastewater.     

Effect of sparger design on hydrodynamics of a gas recirculation anaerobic bioreactor

The effects of sparger design and gas flow rate on, gas holdup distribution and liquid (slurry) recirculation velocity have been studied in a surrogate anaerobic bioreactor used for treating bovine waste with a conical bottom mixed by gas recirculation. A single orifice sparger (SOS) and a multi-orifice ring sparger (MORS) with the same orifice open area and gas flow rates (hence the same process power input) are compared in this study. The advanced non-invasive techniques of computer automated tomography (CT) and computer automated radioactive particle tracking (CARPT) were employed to determine gas holdup, liquid recirculation velocity, and the poorly mixed zones. Gas flows (Q(g)) ranging of 0.017 x 10(-3) m(3)/s to 0.083 x 10(-3) m(3)/s were used which correspond to draft tube superficial gas velocities ranging from 1.46 x 10(-2) m/s to 7.35 x 10(-2) m/s (based on draft tube diameter). Air was used for the gas, as the molecular weights of air and biogas (consisting mainly of CH(4) and CO(2)) are in the same range (biogas: 28.32-26.08 kg/kmol and air: 28.58 kg/kmol). When compared to the SOS for a given gas flow rate, the MORS gave better gas holdup distribution in the draft tube, enhanced the liquid (slurry) recirculation, and reduced the fraction of the poorly mixed zones. The improved gas holdup distribution in the draft tube was found to have increased the overall liquid velocity. Hence, for the same process power input the MORS system performed better by enhancing the liquid recirculation and reducing the poorly mixed zones.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m^?2 s^?1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574. © 2012 Wiley Periodicals, Inc.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations.     

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.     

Membrane sparger in bubble column,airlift, and combined membrane–ring sparger bioreactors

The bubble column and the two internal loop airlift reactors (riser/downcomer area ratios of 0.11 and 0.58) characterized in this study were equipped with a rubber membrane sparger, which produced small bubbles, giving high mass transfer coefficients. The low mixing intensity in the bubble column was increased by an order of magnitude in the airlift reactors. We designed a novel aeration and mixing system by adding a ring sparger to the membrane sparger in the bubble column and maintained the advantages of both airlift configuration (good mixing properties) and bubble column configuration (efficient aeration, without any internal constructions). The combined membrane–ring sparger system has unique features with respect to the efficiency of utilization of substrate gasses and energy. Model experiments showed that the small bubbles from the membrane sparger do not coalesce with the large bubbles from the ring sparger. If different gases were added through the two spargers it was possible to transfer a hazardous or expensive gas quantitatively to the liquid through the membrane sparger (dual sparging mode). In the combined membrane–ring sparger system the energy input for mixing and mass transfer is divided. Therefore, the energy consumption can be minimized if the flow distribution of air through the membrane and ring sparger is controlled by the oxygen demand and the inhomogeneity of the culture, respectively (split sparging mode). The dual sparging mode was used for mass production of the alga Rhodomonas sp. as the first step in aquatic food chains. Avoiding mechanical parts removes an important risk of malfunction, and a continuous culture could be maintained for more than 8 months. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 452–458, 1999.     

CFD of mixing of multi‐phase flow in a bioreactor using population balance model

Mixing in bioreactors is known to be crucial for achieving efficient mass and heat transfer, both of which thereby impact not only growth of cells but also product quality. In a typical bioreactor, the rate of transport of oxygen from air is the limiting factor. While higher impeller speeds can enhance mixing, they can also cause severe cell damage. Hence, it is crucial to understand the hydrodynamics in a bioreactor to achieve optimal performance. This article presents a novel approach involving use of computational fluid dynamics (CFD) to model the hydrodynamics of an aerated stirred bioreactor for production of a monoclonal antibody therapeutic via mammalian cell culture. This is achieved by estimating the volume averaged mass transfer coefficient (k_La) under varying conditions of the process parameters. The process parameters that have been examined include the impeller rotational speed and the flow rate of the incoming gas through the sparger inlet. To undermine the two‐phase flow and turbulence, an Eulerian‐Eulerian multiphase model and k‐ε turbulence model have been used, respectively. These have further been coupled with population balance model to incorporate the various interphase interactions that lead to coalescence and breakage of bubbles. We have successfully demonstrated the utility of CFD as a tool to predict size distribution of bubbles as a function of process parameters and an efficient approach for obtaining optimized mixing conditions in the reactor. The proposed approach is significantly time and resource efficient when compared to the hit and trial, all experimental approach that is presently used. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:613–628, 2016