Enhanced plasmid stability and production of hEGF by immobilized recombinant E. coli JM101

Recombinant Escherichia coli JM101 was immobilized with porous polyurethane foam (PUF) particle as supporter matrix for human epidermal growth factor (hEGF) production. Flask culture showed that cell immobilization in PUF can improve cell growth and hEGF expression. A bubble column and a three-phase fluidized bed bioreactor by self-design was further applied to produce hEGF, respectively. The results demonstrated that PUF is a feasible immobilized supporter material with good biocompatibility. Immobilization could also decrease the probability for segregational plasmid loss and overgrowth of plasmid-free cells. Cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. hEGF productivity was enhanced from 8.73 mg/l h of free-culture to 11.4 mg/l h of immobilized cultivation.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous 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 this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment

Summary Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 × 10⁸ cells/ cm³ PUF and 3.5 × 10⁷ cells/ cm³ PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 × 10⁷ cells/cm³ culture vessel volume was obtained in a packed-bed culture of Vero cells. Offprint requests to: K. Funatsu     

High density culture of anchorage-dependent animal cells by polyurethane foam packed-bed culture systems

Summary Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of polyurethane foam (PUF) and grew to a high cell density (1.1 × 10⁸ cells/cm³ PUF and 4.2 × 10⁷ cells/cm³ PUF, respectively) in a PUF-plates packed-bed culture system. This density of Vero cells was twice that obtained previously with a PUF-particles packed-bed culture system. A maximum cell density of 6.7 × 10⁷ cells/cm³ culture vessel volume was obtained in a PUF-disc packed-bed culture of Vero cells. From the cell density of CHO-K1, growing in a monolayer on the surface of PUF and a petri dish, per bulk volume of PUF, we estimated that a surface area to volume ratio of PUF plates effective for cell growth was about 109 cm²/cm³.Offprint requests to: K. Funatsu     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

Ferrous iron oxidation by foam immobilized Acidithiobacillus ferrooxidans: Experiments and modeling

Ferrous iron bio‐oxidation by Acidithiobacillus ferrooxidans immobilized on polyurethane foam was investigated. Cells were immobilized on foams by placing them in a growth environment and fully bacterially activated polyurethane foams (BAPUFs) were prepared by serial subculturing in batches with partially bacterially activated foam (pBAPUFs). The dependence of foam density on cell immobilization process, the effect of pH and BAPUF loading on ferrous oxidation were studied to choose operating parameters for continuous operations. With an objective to have high cell densities both in foam and the liquid phase, pretreated foams of density 50 kg/m³ as cell support and ferrous oxidation at pH 1.5 to moderate the ferric precipitation were preferred. A novel basket‐type bioreactor for continuous ferrous iron oxidation, which features a multiple effect of stirred tank in combination with recirculation, was designed and operated. The results were compared with that of a free cell and a sheet‐type foam immobilized reactors. A fivefold increase in ferric iron productivity at 33.02 g/h/L of free volume in foam was achieved using basket‐type bioreactor when compared to a free cell continuous system. A mathematical model for ferrous iron oxidation by Acidithiobacillus ferrooxidans cells immobilized on polyurethane foam was developed with cell growth in foam accounted by an effectiveness factor. The basic parameters of simulation were estimated using the experimental data on free cell growth as well as from cell attachment to foam under nongrowing conditions. The model predicted the phase of both oxidation of ferrous in shake flasks by pBAPUFs as well as by fully activated BAPUFs for different cell loadings in foam. Model for stirred tank basket bioreactor predicted within 5% both transient and steady state of the experiments closely for the simulated dilution rates. Bio‐oxidation at high Fe²⁺ concentrations were simulated with experiments when substrate and product inhibition coefficients were factored into cell growth kinetics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

NaCS‐PDMDAAC immobilized cultivation of recombinant Dictyostelium discoideum for soluble human Fas ligand production

Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post‐translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly‐dimethyl‐diallyl‐ammonium chloride (NaCS‐PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi‐continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled‐up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS‐PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10⁷ cells mL^?1 (equivalent to a maximum cell density of 2.22 × 10⁸ cells mL^?1 in capsules) and a hFasL concentration of 130.40 μg L^?1 (equivalent to a hFasL concentration of 1434.40 μg L^?1 in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10⁷cells mL^?1 (equivalent to a maximum cell density of 1.89 × 10⁸ cells mL^?1 in capsules) and a hFasL concentration of 106.10 μg L^?1 (equivalent to a hFasL concentration of 1167.10 μg L^?1 in capsules) were obtained after ～170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS‐PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large‐scale applications. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:424–430, 2015     

Synthesis of a green polyurethane foam from a biopolyol obtained by enzymatic glycerolysis and its use for immobilization of lipase NS-40116

The use of green sources for materials synthesis has gained popularity in recent years. This work investigated the immobilization of lipase NS-40116 (Thermomyces lanuginosus lipase) in polyurethane foam (PUF) using a biopolyol obtained through the enzymatic glycerolysis between castor oil and glycerol, catalyzed by the commercial lipase Novozym 435 for the PUF formation. The reaction was performed to obtain biopolyol resulting in the conversion of 64% in mono- and diacylglycerol, promoting the efficient use of the reaction product as biopolyol to obtain polyurethane foam. The enzymatic derivative with immobilized lipase NS-40116 presented apparent density of 0.19 ± 0.03 g/cm³ and an immobilization yield was 94 ± 4%. Free and immobilized lipase NS-40116 were characterized in different solvents (methanol, ethanol, and propanol), temperatures (20, 40, 60 and 80 °C), pH (3, 5, 7, 9 and 11) and presence of ions Na⁺, Mg⁺⁺, and Ca⁺⁺. The support provided higher stability to the enzyme, mainly when subjected to acid pH (free lipase lost 80% of relative activity after 360 h of contact, when the enzymatic derivative lost around 22%) and high-temperature free lipase lost 50% of relative activity, while the immobilized remained 95%. The enzymatic derivative was also used for esterification reactions and conversions around 66% in fatty acid methyl esters, using abdominal chicken fat as feedstock, were obtained in the first use, maintaining this high conversion until the fourth reuse, proving that the support obtained using environmentally friendly techniques is applicable.

     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Effects of surface treatment and process parameters on immobilization of recombinant yeast cells by adsorption to fibrous matrices

The effects of surface properties of Saccharomyces cerevisiae strains 468/pGAC9 and 468 on adhesion to polyethyleneimine (PEI) and/glutaraldehyde (GA) pre-treated cotton (CT), polyester (PE), polyester + cotton (PECT), nylon (NL), polyurethane foam (PUF), and cellulose re-enforced polyurethane (CPU) fibers were investigated. Process parameters (circulation velocity, pH, ionic strength, media composition and surfactants) were also examined. 80%, 90%, and 35% of the cells were adsorbed onto unmodified CT, PUF, and PE, respectively. PEI-GA pre-treated CT and alkali treated PE yielded 25% and 60% cell adhesion, respectively. Adsorption rate (K_a) ranged from 0.06 to 0.17 for CT and 0.06-0.16 for PE at varied pH. Adhesion increased by 15% in the presence of ethanol, low pH and ionic strength, and decreased by 23% in the presence of yeast extract and glucose. Shear flow and 1% Triton X-100 detached 62% and 36% nonviable cells from PE and CT, respectively, suggesting that cell immobilization in fibrous-bed bioreactors can be controlled to optimize cell density for long-term stability.     

Immobilization of animal cells in fixed bed bioreactors

Immobilization of animal cells has become a highly popular means of achieving high-density animal cell cultures. The advantages of immobilization are that it stabilizes cells in culture and enables long-term culture periods to be achieved. Immobilization increases cell productivity by increasing the usable substrate surface area for anchorage-dependent cells, or by facilitating perfusion of anchorage-independent cells. A method for production of secreted biological products from anchorage-dependent and independent cells is described. The method is based on immobilization of animal cells within the polymeric matrix of polyurethane foam, packed in a fixed bed bioreactor.     

Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor

-d-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of -d-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.     

Lipase production by immobilized Candida rugosa cells

Summary Immobilization of Candida rugosa cells on a solid support for extracellular lipase production has been explored. The use of Ca-alginate beads and of mixed matrix of polyurethane foam/Ca-alginate beads enabled us to operate a batch and a continuous four-phase fluidized bed bioreactor. Cells co-entrapped together with polyurethane into Ca-alginate did not show higher lipase production levels than the cells entrapped in Ca-alginate gels. The addition of gum arabic to the medium greatly enhanced lipase production without affecting the hydrodynamic operating conditions significantly. This fact demonstrates that the reactor system is limited in terms of organic substrate dispersion and direct contact with cells. Correspondence to: C. Solà     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Repeated-batch production of glucoamylase using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> immobilized in a fibrous bed bioreactor

The recombinant Saccharomyces cerevisiae strain C468/pGAC9 has an unstable hybrid plasmid pGAC9, which directs production of glucoamylase. A fibrous cotton material with a good adsorption capability for recombinant S. cerevisiae cells was used as the immobilization matrix in an internal loop airlift-driven fibrous bed bioreactor (ILALFBB) system. With batch cultures in the ILALFBB, the fraction of plasmid-carrying cells was 72% after more than 2 days cultivation, which was two times higher than that in the conventional free-cell culture. Correspondingly, a high activity of glucoamylase (GA; 113 U/l) was achieved with a high productivity of 43 U/l/h. The ILALFBB system also maintained a high fraction of viable plasmid-carrying of 74% for glucoamylase production during repeated-batch cultures, achieving a high glucoamylase activity of 140 U/l with a productivity of 19–130 U/l/h in all 14 batches studied during 19.8 days. The stable and long-term glucoamylase production from the ILALFBB was attributed to the effect of cell immobilization on plasmid stability. Plasmid-carrying cells were preferentially retained in the fibrous matrix because of their ability to adhere to the fiber surface and to form cell aggregates higher than those of plasmid-free cells. The repeated batch using immobilized cell of recombinant S. cerevisiae in the ALALFBB system thus provides a feasible method for stable, long-term and high-level production of glucoamylase.     

Towards the production of fungal biocontrol candidates using inert supports: a case of study of Trichoderma asperellum in a pilot fixed bed fermenter

The production of fungal biocontrol agents by solid-state fermentation (SSF) processes in inert supports demands deeper studies in SSF-modelling and SSF-optimisation to cope with scale-up issues. Here, we report the systematic application of fractional factorial and central composite designs to optimise the conidia productivity and maximum specific growth rate of the biological control candidate Trichoderma asperellum strain Th204 using two inert supports: polyurethane foam (PUF) and rice husk (RH), in a pilot 16 L fixed bed fermenter. By using response surface methodology, 2D contour graphs and Spearman’s correlation coefficients, axial temperature, conidia concentration, bed moisture and pressure drop gradients were modelled. C:N ratio and airflow rate were identified as significant factors. Optimal conditions using PUF a C:N ratio of 18.1 and airflow of 0.8?m³?h^?1 were found, with the highest productivity of 3.09?×?10⁷conidia g^?1 initial dry matter h¹. Polynomial models and response surfaces found in this study are advantageous to design strategies to scale-up the SSF process in fixed bed fermenters for fungal biological control candidates.     

Aerobic biodegradation of nitrobenzene by a defined microbial consortium immobilized in polyurethane foam

An aerobic microbial consortium constructed by the combination of Rhodotorula mucilaginosa Z1, Streptomyces albidoflavus Z2 and Micrococcus luteus Z3 was immobilized in polyurethane foam and its ability to degrade nitrobenzene was investigated. Batch experimental results showed that polyurethane-foam-immobilized cells (PFIC) more efficiently degrade 200–400 mg l⁻¹ nitrobenzene than freely suspended cells (FSC). Kinetics of nitrobenzene degradation by PFIC was well described by the Andrews equation. Compared with FSC, PFIC exhibited better reusability (over 100 times) and tolerated higher shock-loadings of nitrobenzene (1,000 mg l⁻¹). Moreover, In the presence of salinity (≤5% NaCl, w/v), phenol (≤150 mg l⁻¹) and aniline (≤50 mg l⁻¹), respectively, degradation efficiency of nitrobenzene by PFIC reached over 95%. Even in the presence of both 100 mg l⁻¹ phenol and 50 mg l⁻¹ aniline, over 75% nitrobenzene was removed by PFIC in 36 h. Therefore, the immobilization of the defined consortium in polyurethane foam has application potential for removing nitrobenzene in industrial wastewater treatment system.