A cartridge oxygenator for mammalian cell culture in a stirred tank bioreactor

Summary Tubing made of membrane with high oxygen permeability is often used in supplying oxygen to animal cell culture bioreactors. We have fabricated the tubing into a cartridge configuration. Such an arrangement allows damaged tubing to be replaced conveniently and eases the maintenance of such an oxygenator in bioreactors.     

Scale-up of biotransformation process in stirred tank reactor using dual impeller bioreactor

The gas-liquid mass transfer coefficient K(L)a in the fermenter is a strong function of mode of energy dissipation and physico-chemical properties of the liquid media. A combination of disc turbine (DT) and pitched blade turbine down flow (PTD) impellers has been tested in laboratory bioreactor for gas hold-up and gas-liquid mass transfer performance for the growth and biotransformation medium for an yeast isolate VS1 capable of biotransforming benzaldehyde to L-phenyl acetyl carbinol (L-PAC) and compared with those in water.Correlations have been developed for the prediction of the fractional gas hold-up and gas-liquid mass transfer coefficient for the above media. The mass transfer coefficient and respiration rate have been determined in the shake flask for the growth as well as for biotransformation medium. These results, then have been used to optimize the operating parameters (impeller speed and aeration) for growth and biotransformation in a laboratory bioreactor. The comparison of cell mass production and L-PAC production in the bioreactor has been done with that obtained in shake flask studies.     

Determination of the average shear rate in a stirred and aerated tank bioreactor

A method for evaluating the average shear rate () in a stirred and aerated tank bioreactor has been proposed for non-Newtonian fluids. The volumetric oxygen transfer coefficient (k _L a) was chosen as the appropriate characteristic parameter to evaluate the average shear rate (). The correlations for the average shear rate as a function of N and rheological properties of the fluid (K and n) were obtained for two airflow rate conditions (ϕ_air). The shear rate values estimated by the proposed methodology lay within the range of the values calculated by classical correlations. The proposed correlations were utilized to predict the during the Streptomyces clavuligerus cultivations carried out at 0.5 vvm and four different rotational impeller speeds. The results show that the values of the average shear rate () varied from 437 to 2,693 s⁻¹ by increasing with N and flow index (n) and decreasing with the fluid consistency index (K).     

In situ filtration of Anchusa officinalis culture in a cell-retention stirred tank bioreactor

Summary The effect of agitation and aeration on filtration of Anchusa officinalis culture in a stirred tank bioreactor integrated with an internal filter unit was investigated. Increases in suction head of the pump that drove the filtration process were measured at impeller speeds of 100 and 200 rpm. Surprisingly, suction head attained at 200 rpm was about 40% higher than at 100 rpm. Direct observation of the cake deposition process in the reactor using a dilute cell suspension revealed that the filter cake formed at 100 rpm was thicker, but less compact. Aeration at 0.4 vvm was shown to have little effect on the filtration rate, since the bulk fluid flow was dominated by the impeller hydrodynamics. The initial flux can be recovered by filter backwashing with compressed air at a flow rate of 0.6 vvm for a duration of 5 minutes.     

Combined effect of hydrodynamic and interfacial flow parameters on lysozyme deactivation in a stirred tank bioreactor

The dynamic environment within a bioreactor and in the purification equipment is known to affect the activity and yield of enzyme production. The present research focuses on the effect of hydrodynamic flow parameters (average energy dissipation rate, maximum energy dissipation rate, average shear rate, and average normal stress) and the interfacial flow parameters (specific interfacial area and mass transfer coefficient) on the activity of lysozyme. Flow parameters were estimated using CFD simulation based on the k-epsilon approach. Enzyme deactivation was investigated in 0.1, 0.3, 0.57, and 1 m i.d. vessels. Enzyme solution was subjected to hydrodynamic stress using various types of impellers and impeller combinations over a wide range of power consumption (0.03 < P(G)/V < 7, kW/m3). The effects of tank diameter, impeller diameter, blade width, blade angle, and the number of blades on the extent of deactivation were investigated. At equal value of P(G)/V, epsilon(max), and gamma(avg), the extent of deactivation was dramatically different for different impeller types. The extent of deactivation was found to correlate well with the average turbulent normal stress and the mass transfer coefficient.     

Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110

Nitrilases constitute an important class of hydrolases, having numerous industrial applications. The present work aims to address the production of nitrile hydrolyzing enzymes from Pseudomonas putida MTCC 5110 in a 6l bioreactor. Effect of various physico-chemical conditions and process parameters like pH, temperature, aeration and agitation rates and inducer concentration was studied. Further, the enzyme activity was enhanced by adopting the inducer feeding strategy. Various biochemical engineering parameters pertaining to the cultivation of P. putida in different physico-chemical conditions were reported. Finally, segregation of growth phase from the enzyme production phase allowed significant reduction in total fermentation time.     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.     

Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes

The total operating costs of small-scale monoclonal antibody production were calculated for two different upstream options and general downstream procedure based on protein A chromatography. The upstream options were a spin-filter equipped stirred-tank bioreactor (STR) and a hollow fiber bioreactor (HFB). Both the bioreactors were operated in perfusion mode. The total operating costs of the processes were 6,900 €/g for STR option and 6,400 €/g for the HFB option. In the both systems, the costs were dominated by expenses derived from the downstream section (almost 80%) that was almost identical in the both systems. In the upstream section, the investment depreciation was the largest cost item. The lower total costs of the HFB option were a result of lower investment costs and more concentrated product that led into savings also in downstream section. This study brings out the HFB as on viable alternative for stirred-tank bioreactor, especially in small-scale diagnostic monoclonal antibody production.     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds

Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB). A mathematical model describing this system was developed and tested using the experimental results. A model microbial culture consisting of Pseudomonas sp. JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used. The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations. Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored. Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds.     

The response of a bacterial biofilm community in a simulated industrial cooling water system to treatment with an anionic dispersant

The effect of a dispersant on the microbial community in a simulated open recirculating cooling water system was determined by continuous operation of the system over two consecutive periods of 196 and 252 d, respectively. An open recirculating cooling water system feeding a modified Robbin's Device with synthetic cooling water to simulate the environment of an industrial cooling water system was set up. Planktonic and biofilm (mild steel and Nylon(R)) samples were taken weekly in 1997 (196-d period) and fortnightly in 1998 (252-d period). Each biofilm was scraped off and diluted in 10-ml 1 x phosphate-buffered saline (PBS). Serial dilutions were performed and plated onto R2A agar (pH 8.0) to obtain the predominant culturable bacteria. The diversity was determined by allocating groups according to colony morphology, diameter and colour. Diversity was calculated according to the Shannon-Weaver Index. During the first run (1997), dispersant was added on day 57 to a final concentration of 15 mg l-1 for 49 d, stopped for 49 d and dosed at 30 mg l-1 for 41 d. The second run entailed adding dispersant to a final concentration of 30 mg l-1 on day 98 for 70 d, stopping dosing for 56 d and resuming dosing at 30 mg l-1 for another 28 d. The 2-year evaluation period demonstrated that the biofilm-removing action of the dispersant decreased to a point where it was not effective at all. Our results showed that the synthetic dispersant evaluated was only effective initially, but was ineffective in controlling biofouling on Nylon, and to a lesser degree on mild steel at the recommended (15 mg l-1) as well as at double the recommended concentration in the long term. The release of cells from biofilms observed when dispersant dosing was terminated, supports the notion that a community attaching in the presence of the surface active agent was selected for. The decreased efficacy may therefore be due to a selection of strains able to remain attached and/or attach in the presence of the dispersant as demonstrated by shifts in the biofilm communities on both Nylon and mild steel.     

Stability and change in estuarine biofilm bacterial community diversity

Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain Bacteria in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Fla. Community profiling was achieved by using 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the benthos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T-RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in dissolved oxygen.     

An analysis of the bacterial community in a membrane bioreactor fed with photo-Fenton pre-treated toxic water

A photo-Fenton-membrane bioreactor (MBR) coupled system is an innovative tool for the treatment of wastewater containing high quantities of contaminants. In this paper, wastewater with 200 mg l^?1 of dissolved organic carbon (DOC) of a selected mixture of five commercial pesticides: Vydate^®, Metomur^®, Couraze^®, Ditimur-40^®, and Scala^® was treated by combining photo-Fenton and MBR. The effect of photo-treated pollutants on MBR operation was investigated by studying the population changes that occurred with time in the activated sludge of the biological system. Pre-treatment with photo-Fenton was carried out (only up to 34% of mineralization of DOC) and, after MBR treatment, 98% of biodegradation efficiency was obtained. During the biological treatment, little changes in the activated sludge population were detected by DGGE analysis, maintaining acceptable biodegradation efficiency, which points out the robustness of the MBR treatment versus changes in feed composition.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor

Applied Microbiology and Biotechnology - The hydrogen (H2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the...     

The effect of hydrodynamic stress on the growth of Xanthomonas campestris cultures in a stirred and sparged tank bioreactor

The specific growth and the xanthan production rates by the bacterium Xanthomonas campestris under different shear levels in shake flasks and in a stirred and sparged tank bioreactor have been studied. The shake flask has been used as a reference for studying the shear effects. An effectiveness factor expressed by the ratio of the observed growth rate and the growth rate without oxygen limitation or cell damage was calculated in both modes of cultures. It was observed that the effectiveness factor was strongly dependent on the operational conditions. A strong oxygen transfer limitation at low stirring rates, indicated by a 54 % decrease in the effectiveness factor was observed. In contrast, at higher stirrer speed, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in the effectiveness factor values for stirrer speeds higher than 500 rpm. Cell morphological changes were also observed depending on the agitation conditions, differences in morphology being evident at high shear stress.     

Nitrate removal from drinking water in a fluidized-bed biological denitrification bioreactor

In most industrially developed countries an increasing degree of nitrification can be observed in potential water reservoirs. High nitrate content is unacceptable by public health standards. Since contamination seems to be unavoidable, the only realistic solution is purification prior to utilization. One of the potential variations is biological denitrification with a highly intensive facility, a fluidized- or expanded-bed bioreactor. Based on laboratory and pilot plant experiments, a detailed analysis is presented on the problems arising and solutions offered in the construction of a purification system meeting high quality requirements of drinking water purification. The crucial point is selection of the denitrifying microorganisms and organic matter required for denitrification, which simultaneously determines the attachment of bacteria to the support material (autoimmobilization), the intensity of nitrate removal and the character of post-purification.     

Response of performance and bacterial community to oligotrophic stress in biofilm systems for raw water pretreatment

Understanding the dynamics of performance and bacterial community of biofilm under oligotrophic stress is necessary for the process optimization and risk management in biofilm systems for raw water pretreatment. In this study, biofilm obtained from a pilot-scale biofilm reactor was inoculated into a pilot-scale experimental tank for the treatment of oligotrophic raw water. Results showed that the removal of NH₄ ⁺–N was impaired in biofilm systems when influent NH₄ ⁺–N was less than 0.35 mg L^?1 or NH₄ ⁺–N loading rate of less than 7.51 mg L^?1 day^?1. The dominant bacteria detected in biofilm of different carrier were obvious distinct from phylum to genus level under oligotrophic stress. The dominant bacteria in elastic stereo media carrier changed from Proteobacteria (51.1%) to Firmicutes (32.7%), while Proteobacteria was always dominant in suspended ball carrier after long-term operation under oligotrophic conditions. Oligotrophic stress largely decreased the functional bacteria for the removal of nitrogen and organics including many genera in Proteobacteria and Nitrospirae, but increased several genera with spore forming organisms or potential bacterial pathogens in ESM carrier mainly including Bacillus, Mycobacterium, Pseudomonas, etc.     

Predominant bacterial genera in granular activated carbon water treatment systems

Granular activated carbon (GAC) beds may be used for removal of dissolved organic matter during the treatment of drinking water. However, they might also change the microbiological quality of the water entering the distribution system either by changing the predominant bacteria or the bacterial density of the treated water. A 3-year pilot plant study of water treatment using GAC beds was conducted at the Baxter Water Treatment Plant in Philadelphia. During the study, bacteria were isolated from the raw water and from the effluents of the GAC treatment units. At the end of the study, bacteria were also isolated from the GAC units and from sand beds operated in parallel with the GAC units. Bacterial genera in the GAC effluents and in the GAC units themselves were similar to those found in the raw water and in the sand beds. Prechlorination and (or) preozonation of the water before GAC treatment had no noticeable effect on the bacterial genera found as compared with GAC unit having no predisinfection. The bacterial genera found in this study were similar to those found in seven other studies of GAC water treatment that used a variety of treatment schemes and a variety of heterotrophic plate count techniques to evaluate bacterial populations. From these several studies it appears that GAC treatment does not change the nature of the bacterial populations associated with drinking water.