Cost-effective production of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> using simple netting bag solid bioreactor

A novel simple solid state fermentation method, netting bag bioreactor (Φ 120 × 800 mm), was developed and used to cultivate Bacillus licheniformis as probiotics. High spore yield (1.2 × 10¹¹ CFU/g dry substrate) has been obtained by using this method. Comparing to the tray bioreactor and the packed bed bioreactor for Bacillus fermentation, the netting bag method was more cost-effective, time- and space-saving and the material cost is also as low as ca. US $293 per 1,000 kg spores. Thus, netting bag SSF can be widely applied to produce probiotic bacteria in developing areas.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

A solid-state bioreactor coupled with forced aeration and pressure oscillation

A novel design of a solid-state bioreactor, operated with periodic pressure oscillation coupled with forced aeration through the medium, gave efficient control of temperature. The evaluation of the bioreactor assembly with respect to temperature and cellulase production by Penicillium decumbens JUA10 showed that, at 4 atm and the bed depth of 6 cm, the maximal temperature variation in the reactor was +1.5 °C at a set value of 30 °C compared with +6.8 °C in a static tray system. The highest cellulase and -glucosidase activities were 15 IU g^–1 and 51 IU g^–1 substrate dry matter at 96 h, respectively, while only 10 IU g^–1 and 24 IU g^–1 were obtained in the static tray culture system.     

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor

Aims: To improve the α‐amylase production in solid‐state fermentation (SSF) condition utilizing a new bioreactor (NB) system. Methods and Results: In NB system, 20 g of wheat bran moistened with liquid medium in 1 : 1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α‐amylase activity of 22 317 Ugds^?1 was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2‐cm bed height with air flow rate of 0·1 l min^?1 g^?1 wheat bran at 32°C and initial medium pH of 6. Conclusions: Within a short incubation period, significantly high α‐amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain. Significance and Impact of the Study: The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α‐amylase production.     

Gas phase H<Subscript>2</Subscript>S product recovery in a packed bed bioreactor with immobilized sulfate-reducing bacteria

An N₂ strip gas was used in a packed bed sulfate-reducing bioreactor to recover the dissolved sulfide product and improve sulfate conversion. The highest volumetric productivity obtained was 261 mol H₂S m⁻³ d⁻¹. Lowering the initial pH of the medium from 7 to 6 increased the H₂S content of the strip gas from 3.6 to 5.8 mol%. The ratio of strip gas to liquid flow rates (G/L) was found be to a suitable basis for scaling the process. Calculations indicated that modest G/L values (<10²) were required to recover the residual dissolved sulfide in a downstream stripping column.     

Conidia production by <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor

Conidia of Beauveria bassiana CS-1, which have the potential for the control of the diamondback moth (Plutella xylostella), were produced by solid-state fermentation (SSF) using a packed-bed bioreactor with rice straw and wheat bran. As the packing density and the bed height were increased, the production of conidia decreased. In a packed-bed bioreactor under no aeration and no addition of polypropylene (PP) foam (control), the total average of conidia was 4.9 × 10⁸ g^-1. The production of conidia was affected more by the addition of PP foam as an inert support than forced aeration and was approx. 23 times higher than that of the control. The total average of conidia produced by B. bassiana was 1.1–1.2 × 10¹⁰ g^-1 . Revisions requested 6 September 2004/2 November 2004; Revisions received 1 November 2004/8 December 2004     

Supermacroporous polymer‐based cryogel bioreactor for monoclonal antibody production in continuous culture using hybridoma cells

Cryogel matrices composed of different polymeric blends were synthesized, yielding a unique combination of hydrophilicity and hydrophobicity with the presence or absence of charged surface. Four such cryogel matrices composed of polyacrylamide–chitosan (PAAC), poly(N‐isopropylacrylamide)–chitosan, polyacrylonitrile (PAN), and poly(N‐isopropylacrylamide) were tested for growth of different hybridoma cell lines and production of antibody in static culture. All the matrices were capable for the adherence of hybridoma cell lines 6A4D7, B7B10, and H9E10 to the polymeric surfaces as well as for the efficient monoclonal antibody (mAb) production. PAAC proved to be relatively better in terms of both mAb production and cell growth. Further, PAAC cryogel was designed into three different formats, monolith, disks, and beads, and used as packing material for packed‐bed bioreactor. Long‐term cultivation of 6A4D7 cell line on PAAC cryogel scaffold in all the three formats could be successfully done for a period of 6 weeks under static conditions. Continuous packed‐bed bioreactor was setup using 6A4D7 hybridoma cell line in the three reactor formats. The reactors ran continuously for a period of 60 days during which mAb production and metabolism of cells in the bioreactors were monitored periodically. The monolith bioreactor performed most efficiently over a period of 60 days and produced a total of 57.5 mg of antibody in the first 30 days (in 500 mL) with a highest concentration of 115 μg mL^?1, which is fourfold higher than t‐flask culture. The results demonstrate that appropriate chemistry and geometry of the bioreactor matrix for cell growth and immobilization can enhance the reactor productivity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Co-metabolic degradation of trichloroethylene by Pseudomonas putida in a fibrous bed bioreactor

Co-metabolic degradation of trichloroethylene (TCE) by Pseudomonas putida F1 was investigated in a novel bioreactor with a fibrous bed. A pseudo-first-order rate constant for TCE degradation was 1.4 h^–1 for 2.4 to 100 mg TCE l^–1. Competitive inhibition of toluene on TCE removal could be prevented in this bioreactor. 90% TCE was removed over 4 h when 95 mg toluene l^–1 was presented simultaneously.     

Modified CelliGen-packed bed bioreactors for hybridoma cell cultures

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG₁ monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen^™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10⁸ cells per cm³ of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.     

Decolorization of Direct Red 28 by mixed bacterial culture in an up-flow immobilized bioreactor

Aerobic mixed bacterial culture comprised of five isolates (Bacillus vallismortis, B. pumilus, B. cereus, B. subtilis and B. megaterium) identified by 16srDNA analysis was developed from wastewater samples from the aeration tank of an effluent treatment plant of a textile and dyeing industry and evaluated for its ability to decolorize azo dye Direct Red 28 in an up-flow immobilized packed bed bioreactor using marble chips as support matrix. The bioreactor was operated under two parameters: an aeration rate of 0.4 and 0.6 mmol/min at a flow rate of 60, 90 and 120 ml/h, respectively. At a constant aeration rate of 0.4 mmol/min and with flow rates of 60, 90 and 120 ml/h, optimum decolorization of 91, 75 and 72% was observed, while at an aeration rate of 0.6 mmol/min and flow rates of 60, 90 and 120 ml/h, optimum decolorization of 93, 78 and 72% was observed over 10 days. The study concluded that across the two aeration rates and the respective flow rates, the higher aeration rate of 0.6 mmol/min along with a flow rate of 60 ml/h was best suited to decolorize Direct Red 28 in the packed bed bioreactor. Spectral changes of the input and output of the bioreactor by UV–visible spectroscopy indicated decolorization of the dye solution by degradation in addition to the visual observation of the biosorption process.     

Production of Trichoderma micropropagules as a biocontrol agent in static liquid culture conditions by using an integrated bioreactor system

ABSTRACT

In this study, we optimised the conditions for the production of micropropagules of Trichoderma harzianum EGE-K38 in static liquid culture in Modified Czapec Medium (MCM) containing 8?g/L glucose in an integrated tray bioreactor system designed by our research group. Incubation temperature, air flow rate, inoculum spore concentration, inoculation size, medium volume and the use of spores or agar plugs containing mycelia as inoculum were individually studied as one factor at a time. The maximum micropropagule count was 5.2?±?0.2?×?10^9?cfu/mL and dry cell weight was 17?±?2?g/L. For the subsequent drying processes, the maximum drying yield percentage ((viable micropropagule counts after drying/viable cells before drying)*100) after drying of micropropagules was 23.30% (cfu/cfu). Results obtained from our integrated tray bioreactor system showed that static liquid culture fermentation offers potential for industrial scale fungal BCAs production.     

Enhanced production of laccase in <Emphasis Type="Italic">Coriolopsis rigida</Emphasis> grown on barley bran in flask or expanded-bed bioreactor

In this work, the effect on laccase activity of adding xylidine, veratryl alcohol and copper sulphate to cultures of Coriolopsis rigida under submerged cultivation conditions have been investigated. The highest activities (approximately 6 × 10⁵ nkat/l) were obtained when the inducers copper sulphate (2 mM) and xylidine (10 mM) were added simultaneously. In addition, operating in the optimal conditions, it was possible to maintain the sustained production of laccase (around 3 × 10⁵ nkat/l) for successive repeated batch cultures in an expanded-bed laboratory scale bioreactor. On the other hand, in vitro phenol degradation by laccase obtained in the bioreactor was studied with/without an effective mediator 1-hydroxybenzotriazol (HBT). The presence of a radical mediator plays an important role inducing the degradation of phenol, and without mediator the polymerization of phenol was detected.     

Monoclonal antibody production: viability improvement of RC1 hybridoma cell in different types of bioreactor

The study was done to improve the viability of the RC1 hybridoma cell in order to produce more amount of monoclonal antibody (mAb). By using the optimized media, the cell had been cultured in two bioreactor systems which were the MiniPerm and Stirred Tank bioreactor (ST bioreactor), and the results were compared to the one obtained by using the T-Flask bioreactor which was used as a standard. The results showed that the ST bioreactor was able to improve the viability of the cell to the value of 91.8% which was a little bit better than the one obtained by the MiniPerm bioreactor (88.6%) and far better than that of achieved by the T-Flask bioreactor (76.4%). This was well correlated with the good growth performance of the cell in the ST bioreactor with the specific growth rate (μ) value of 0.0289 h⁻¹ followed by MiniPerm bioreactor with the value of 0.0243 h⁻¹ and then the T-Flask with the value of 0.0151 h⁻¹. The low value of doubling time (t _d ) obtained in the ST bioreactor (24 h) compared to the one obtained in the MiniPerm (29 h) and T-Flask bioreactor (46 h) had also contributed to the higher value of cell viability. As a result a higher concentration of mAb was able to be produced by the ST bioreactor (0.42 g l⁻¹) compared to that of the MiniPerm (0.37 g l⁻¹) and T-Flask bioreactor (0.23 g l⁻¹).     

Laboratory-scale biofiltration of acrylonitrile by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> DAP 96622 in a trickling bed bioreactor

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 μl/min ACN and 0.8 μl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l^?1 h^?1 ACN and 83.52 mg l^?1 h^?1 TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100–200 mg l^?1 h^?1 and TOL inlet load of ~400 mg l^?1 h^?1, with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was ~90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT^® agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.     

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.     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

Simultaneous production of propionic acid and vitamin B12 from corn stalk hydrolysates by Propionibacterium freudenreichii in an expanded bed adsorption bioreactor

Abstract

Vitamin B12 and propionic acid that were simultaneous produced by Propionibacterium freudenreichii are both favorable chemicals widely used in food preservatives, medicine, and nutrition. While the carbon source and propionic acid accumulation reflected fermentation efficiency. In this study, using corn stalk as a carbon source and fed-batch fermentation process in an expanded bed adsorption bioreactor was studied for efficient and economic biosynthesis of acid vitamin B12 and propionic. With liquid hot water pretreated corn stalk hydrolysates as carbon source, 28.65?mg L^?1 of vitamin B12 and 17.05?g L^?1 of propionic acid were attained at 168?h in batch fermentation. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed corn stalk in expanded bed adsorption bioreactor (EBAB), giving 47.6?mg L^?1 vitamin B12 and 91.4?g L^?1 of propionic acid at 258?h, which correspond to product yields of 0.37?mg g^?1 and 0.75?g g^?1, respectively. The present study provided a promising strategy for economically sustainable production of vitamin B12 and propionic acid by P. freudenreichii fermentation using biomass cornstalk as carbon source and expanded bed adsorption bioreactor.     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)