Immobilization of microbial cells in a mixed matrix of silicone polymer and calcium alginate gel: epoxidation of 1-octene by Nocardia corallina B-276 in organic media.

Immobilization of Nocardia corallina B-276 was examined for production of 1,2-epoxyoctane from 1-octene in the presence of n-hexadecane as the organic solvent. Hydrophobic silicone polymer was the most suitable material for entrapment of the cells. Coentrapment of aqueous reaction medium into the silicone polymer matrix improved the epoxide productivity. It was also effective to immobilize the cells in a mixed matrix composed of silicone polymer and calcium alginate gel involving the reaction medium. In the organic monophase, the amount of epoxide accumulated with the cells immobilized in an almost equivolumetric composite of both materials was 2 and 7 times the amounts in the silicone and alginate single matrices, respectively, and it became larger than with the free cells in the aqueous-organic two-liquid phase after a longer period of batch operation. The use of such an optimized composite matrix enabled us to perform a relatively simple operation of the continuous three-phase bioreactor.     

固定化小球藻产氧及光合速率的研究

通过对固定化小球藻产氧及光合速率的研究,进一步解决水产养殖水体溶解氧不足的问题,为生物增氧技术投入使用奠定基础.固定化具有较高的生物量及生物活性,在研究中被广泛使用.运用固定化技术对固定化小球藻产氧及光合速率进行了研究,结果表明,固定化小球藻具有较好的生物量及生物活性,海藻酸钠+羧甲基纤维素钠+硅藻土作为固定化基质有利...     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Silicone-immobilized biocatalysts effective for bioconversions in nonaqueous media.

A hydrophobic silicone polymer could be effectively applied to immobilization of two kinds of biocatalysts operating in organic media. Horse liver alcohol dehydrogenase, which was solubilized in a small amount of water, or deposited on water-filled hydrophilic particles, was immobilized in this material. This configuration of the preparation involving finely dispersed aqueous phase permitted a simple packed-bed operation for the enzymatic oxidation of alcohol and reduction of aldehyde with a coupled-substrate NAD(H) recycling in n-hexane. Another example was the immobilization of Nocardia corallina which catalysed epoxidation of liquid alkenes such as 1-tetradecene, 1-octene, and styrene in the presence of n-hexadecane. In order to adjust the hydrophobicity-hydrophilicity balance of the support, it was effective to immobilize the cells in a mixed matrix composed of silicone polymer and Ca-alginate gel. The optimum composition of the mixed matrix, which yielded the highest productivity of epoxide, was 80-90% silicone + 20-10% alginate for the production of 1,2-epoxytetradecane, 40-50% silicone + 60-50% alginate for 1,2-epoxyoctane, and almost 0% silicone + 100% alginate for styrene oxide. This significant change of the optimum composition was primarily associated with the degree of substrate inhibition.     

Mathematical modeling of the integrated process of mercury bioremediation in the industrial bioreactor

The mathematical model of the integrated process of mercury contaminated wastewater bioremediation in a fixed-bed industrial bioreactor is presented. An activated carbon packing in the bioreactor plays the role of an adsorbent for ionic mercury and at the same time of a carrier material for immobilization of mercury-reducing bacteria. The model includes three basic stages of the bioremediation process: mass transfer in the liquid phase, adsorption of mercury onto activated carbon and ionic mercury bioreduction to Hg(0) by immobilized microorganisms. Model calculations were verified using experimental data obtained during the process of industrial wastewater bioremediation in the bioreactor of 1 m³ volume. It was found that the presented model reflects the properties of the real system quite well. Numerical simulation of the bioremediation process confirmed the experimentally observed positive effect of the integration of ionic mercury adsorption and bioreduction in one apparatus.     

Immobilization of mycobacterial cells onto silicone--assessing the feasibility of the immobilized biocatalyst in the production of androstenedione from sitosterol

Silicone rubbers are hydrophobic, a feature that may prove advantageous if this material is to be used as immobilization matrix in bioconversion systems where hydrophobic species are present, such as sterols and mycobacterial cells. Mycobacterium sp. cells with sitosterol side chain cleavage activity were accordingly effectively adsorbed onto silicone and the potential application of the concept was assessed by matching the behavior of the resulting immobilized biocatalyst with free cells and Celite immobilized cells. Mass transfer, kinetics, thermal and storage stability characterization of a biotransformation system based in the use of the silicone immobilized biocatalyst was performed. The feasibility of biocatalyst reutilization was tentatively explored.     

Improvement of Process Stability of Microbiological Quinoline Degradation in a Three‐Phase Fluidized Bed Reactor

The biological degradation of quinoline by suspended and immobilized Comamonas acidovorans was studied under continuous and discontinuous operating conditions in a three‐phase fluidized bed reactor. C. acidovorans degrades quinoline into biomass and carbon dioxide. Quinoline and the intermediates of its metabolic pathway are found only by quinoline shockloads. The continuous degradation of quinoline by suspended biomass was only possible, if the dilution rate was less than the growth rate (μ_max =0.42 h^–1) and the concentration of a shockload was less than 1 kg/m³. A concentration greater than 1 kg/m³ led to an irreversible damage of the cells. Hence, two different carrier materials were used for immobilization by attachment, to increase the stability of the process. Using immobilization of biomass on carriers decouples the hydrodynamic retention time and the growth rate of the microorganisms. A comparison of the carrier material showed no differences with respect of activity and stability of the biofilm. The process stability of a three‐phase fluidized bed reactor was increased by immobilized biomass. The degradation of toxic shockloads was only possible with immobilized biomass. A dynamic model has been developed to describe the concentration profile of quinoline, 2‐hydroxyquinoline as metabolite and the suspended biomass. A comparison of the measured and calculated values showed good agreement.     

Immobilized biocatalytic process development and potential application in membrane separation: a review

Biocatalytic membrane reactors have been widely used in different industries including food, fine chemicals, biological, biomedical, pharmaceuticals, environmental treatment and so on. This article gives an overview of the different immobilized enzymatic processes and their advantages over the conventional chemical catalysts. The application of a membrane bioreactor (MBR) reduces the energy consumption, and system size, in line with process intensification. The performances of MBR are considerably influenced by substrate concentration, immobilized matrix material, types of immobilization and the type of reactor. Advantages of a membrane associated bioreactor over a free-enzyme biochemical reaction, and a packed bed reactor are, large surface area of immobilization matrix, reuse of enzymes, better product recovery along with heterogeneous reactions, and continuous operation of the reactor. The present research work highlights immobilization techniques, reactor setup, enzyme stability under immobilized conditions, the hydrodynamics of MBR, and its application, particularly, in the field of sugar, starch, drinks, milk, pharmaceutical industries and energy generation.     

固定化米根霉发酵制L-乳酸

采用海藻酸钙包埋法固定化米根霉（Ｒｈｉｚｏｐｕｓｏｒｙｚａｅ）,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单、动力消耗低、反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。利用它进行重复使用固定化米根霉的间歇发酵或连续发酵制备Ｌ 乳酸,整个过程一般可持续两周以上。固定化米根霉的产酸速率达１６～１８ｇ／Ｌ ｂｅａｄ．ｈｒ,得率为７０～８０％,反应器生产能力约为传统搅拌罐的３倍。采用海藻酸钙包埋法固定化米根霉在三相流化床生物反应器中进行发酵可以有效地提高Ｌ 乳酸的生产效率,具有良好的工业应用前景。     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

High density cultivation of Dictyostelium discoideum in a rotating polyurethane foam-bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In order to achieve high cell density cultivation, polyurethane foam (PUF) with high porosity was introduced as new matrix for the immobilization of D. discoideum. The results showed that about 88–93% cells of D. discoideum were adsorbed onto the PUF particles after 100 min equilibrium between adsorbed and free cells, and the highest immobilization rate was achieved by adding the same quantity of PUF matrix with the thin cylinder style. Furthermore, polyurethane foam was used as the immobilization matrix in a rotating PUF-bed bioreactor system. With batch cultures in the rotating bed bioreactor, the concentration of immobilized cells in the PUF carrier increased to 4.2 × 10⁷ cells ml⁻¹ after 167 h cultivation, which was about fourfold higher than the maximal cell density in the conventional free-cell culture. Further studies showed that the cells of D. discoideum were not just simply adsorbed on the surfaces, but actively attached to the surfaces through their network of pseudopodia or filopodia. The present work is very promising to improve the productivity of recombinant proteins in D. discoideum with high cell density in this novel rotating bed bioreactor.     

Enhancement of biogenic sulfide production in a packed-bed bioreactor via critical inoculum design and carrier material selection

Sulfate reducing bacteria (SRB) are commonly used in environmental bioprocesses for the treatment of acid mine drainage and sulfate wastewaters. Biogenic H(2)S is also a potential source of H(2) fuel with the recent development of H(2)S splitting technologies. In this study, a sulfate reducing packed bed bioreactor (PBR) capable of rapidly achieving high volumetric productivities was developed using a novel method of rational inoculum design and the selection of improved biomass carrier materials. An inoculum with initial composition of approximately 95% Desulfovibrio desulfuricans (ATCC 7757) and 5% SRB consortium was designed based on the pure strain's superior immobilization potential and the SRB consortium's superior kinetics. Diatomaceous earth (DE) pellets, porous glass beads, polyurethane foam and bone char were evaluated as potential biomass carrier materials. The DE pellets immobilized the most biomass and were employed in two packed bed bioreactor fermentations. Using the designed inoculum and DE pellets, a packed bed bioreactor achieved a volumetric productivity of 493 mol H(2)S m(-3) day(-1) (based on a 308 mL working volume) with a dissolved sulfide concentration of 9.9 mM. This occurred after 8.3 days of operation and represents a tenfold reduction in the start-up period compared to other sulfate reducing PBRs described in the literature.     

Simultaneous nitrification and denitrification using an autotrophic membrane-immobilized biofilm reactor

AIMS: To develop a laboratory-scale autotrophic membrane-immobilized biofilm reactor to remove nitrogen from drinking water. METHODS AND RESULTS: A polyvinyl alcohol (PVA) immobilized biofilm, attached to the surface of a silicone tube, was used as the basis of a bioreactor for simultaneous nitrification and denitrification of water. The bioreactor was aerated with air to supply oxygen for nitrification. Pure hydrogen was supplied to the silicone tube and diffused through the membrane wall to feed the biofilm for autotrophic denitrification. The bioreactor was effective for the simultaneous nitrification and denitrification of water after a short period of acclimation, while the biofilm exhibited good resistance to the inhibition of denitrification by dissolved oxygen; the denitrification rate decreased by only 8% as the dissolved oxygen increased from 2 mg l(-1) to saturation. CONCLUSIONS: By using PVA crosslinked with sodium nitrate to entrap nitrifying and denitrifying sludge on the surface of a silicone tube, a novel bioreactor for simultaneous nitrification and denitrification was developed. In addition to performing as an immobilizing agent to strengthen the biofilm, PVA protected the denitrifying microorganisms to reduce the inhibition by dissolved oxygen under aerobic condition. Therefore, nitrification and denitrification occurred simultaneously within the biofilm. Furthermore, the immobilization technique shortened the acclimation period of the bioreactor. SIGNIFICANCE AND IMPACT OF THE STUDY: The described space saving and simple to operate bioreactor for nitrogen removal performed autotrophic denitrification to solve the problem of residual carbon in heterotrophic denitrification, and thus is suitable for removing nitrogen from drinking water.     

Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads

Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC₅₀ for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD⁺ in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.     

Development of a single-pass ceramic matrix bioreactor for large-scale mammalian cell culture

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via sllicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up. (c) 1992 John Wiley & Sons, Inc.     

Development of bioreactors for the culture of surface immobilized plant cells

The scaleup of the technique of plant cell surface immobilization was performed successfully in specifically designed laboratory size bioreactors. The immobilizing matrix was formed into a vertically wound spiral providing for a high immobilizing area-to-volume ratio (0.8-1.2 cm(-1)). A modified airlift and a mechanically stirred vessel delivered a best bioreactor performance characterized by low biomass frothing and highly efficient plant cell attachment and retention (>/=96%). The growth of Catharanthus roseus cells investigated in these bioreactors was found not to be mass transfer limited. It required mild mixing and aeration levels (k(L)a approximately 10-15 h(-1)). The biomass formation pattern of surface immobilized plant cells generally exhibited a linear growth phase followed by a stationary phase characterized by the presence of residual carbohydrates in the medium, contrary to suspension cultures. This behavior was found to depend on the plant cell type and/or line cultured, as well as on the inoculum age. The space restriction and unidirectional growth of the SIPC biofilm combined with the limited availability of essential intracellular nutrients rapidly accumulated from the medium by the stationary phase inoculated plant cells all likely contributed to the culture behavior.     

Use of surface-immobilized Trichoderma in batch and fed-batch fermentations

Needle-punch polyester was shown to be an effective support material for the immobilization of Trichoderma reesei Rut C30. When used as a resident inoculum for a batch process, the immobilized Trichoderma was very stable and resulted in a reduced rate of biomass generation in the bulk liquid phase as compared to cultures inoculated with free mycelium. Fed-batch fermentations with the immobilized Trichoderma produced ca. 80% of the activity of those using free cells; however, the activity was more stable and the crude enzyme broth produced had a greatly reduced biomass concentration.     

Biotechnological production of xylitol in a three-phase fluidized bed bioreactor with immobilized yeast cells in Ca-alginate beads

Cells of Candida guilliermondii entrapped in Ca-alginate beads were used for xylitol production, from concentrated hemicellulose hydrolyzate of sugarcane bagasse, in a fluidized bed bioreactor (FBR). The maximum xylitol concentration 28.9 g xylitol/L was obtained at a high aeration rate of 600 mL/min after 70 h of fermentation, indicating that the use of high aeration rate in this system is favored for better oxygen transfer into the immobilized cells. The specific xylitol productivity and the xylitol yield were of 0.4 g xylitol/L.h and 0.58 g xylitol/g xylose respectively. The immobilization efficiency at the end of the fermentation was of 65 %. After 90 h of fermentation xylitol productivity and yield decreased to 0.25 g xylitol/L.h and 0.47 g xylitol/g xylose respectively, indicating the beginning of xylitol consumption by the yeast. The use of FBR system with immobilized cells presented high xylitol yield and productivity.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Vegetable sponge as a matrix to immobilize micro-organisms: a trial study for hyphal fungi, yeast and bacteria

The vegetable sponge of Luffa cylindrica was studied as a matrix for the immobilization of hyphal fungi, yeast and bacteria. All were observed to be entrapped within the sponge. When the various immobilized systems were subcultured in their respective fresh nutrient media, the hyphal fungi showed an increase in biomass with no cellular release and secondary colony formation. The immobilized yeast and bacteria released cells into the medium. Advantages of the reticulated biostructure as an immobilization matrix are discussed.