Performance of tapered column packed-bed bioreactor for ethanol production

A tapered column type of bioreactor system packed with immobilized Saccharomyces cerevisiae was used to study the bioreactor performance as a function of design and operating variables. The performance of tapered column bioreactor system was found to be better than that of the conventional cylindrical column reactor system for the ethanol fermentation. The new bioreactor design alleviated problems associated with carbon dioxide evolution and provided a significantly better flow pattern for both liquid and gas phases in the bioreactor without local channelling. A mathematical simulation model, which takes into account of the axial convection and dispersion, interphase mass transfer, and apparent kinetic design parameters, was developed. The effect of radial concentration gradients on the bioreactor performance was found to be insignificant. For the reactor system studied, the maximum ethanol productivity obtained was 60 g ethanol/L gel/h, and the maximum glucose assimilation rate was 140 g glucose/L gel/h. One of the most important findings from this study was that the apparent kinetic parameters change at the glucose concentration of 2 g/L This change was found to be due to the changes in yeast physiology and metabolism. The values of V(m) (') and V(m) (') decreased from 0.8 to 0.39 g ethanol/g cell/h and from 97mM to 11mM, respectively. The substrate inhibition constant was estimated as 0.76M and the product inhibition constant was determined as 113 g ethanol/L The degree of product inhibition showed practically a linear relationship with an increasing ethanol concentration. Based on the hydro-dynamic analysis of the bioreactor system, it was found that the Peclet number, N(Pe) was not a strong function of the flow velocity at low flow rates through the bioreactor system, but its value decreased somewhat at an interstitial velocity greater than 0.03 cm/s. The tapered column bioreactor system showed a much better flow pattern of gas and liquid phases within the reactor, thereby providing a more homogeneous distribution of gas-liquid-solid phases in the reactor without any phase separation.     

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h⁻¹ dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.     

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.     

Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.     

Mycophenolic acid production in solid-state fermentation using a packed-bed bioreactor

Mycophenolic acid (MPA) was produced from Penicillium brevicompactum by solid-state fermentation (SSF) using pearl barley, and submerged fermentation (SmF) using mannitol. It was found that SSF was superior to SmF in terms of MPA concentration (1219 mg/L vs. 60 mg/L after 144 h fermentation), and the product yields were 6.1 mg/g pearl barley for SSF and 1.2 mg/g mannitol for SmF. The volumetric productivities were 8.5 and 0.42 mg/L h for SSF and SmF, respectively.The optimum solid substrate of SSF for MPA production was pearl barley, producing 5470 mg/kg compared with wheat bran (1601 mg/kg), oat (3717 mg/kg) and rice (2597 mg/kg). The optimum moisture content, incubation time and inoculum concentrations were 70%, 144 h and 6%, respectively. Neither the addition of mannitol or (NH4)₂HPO₄ nor adjustment of media pH within the range of 3–7 significantly enhanced MPA production.MPA production by SSF using a packed-bed bioreactor was performed and an increased maximum production of MPA 6.9 mg/g was achieved at 168 h incubation time. The higher volumetric productivity and concentrations makes SSF an attractive alternative to SmF for MPA production.     

Denitrification performance and microbial diversity in a packed-bed bioreactor using PCL as carbon source and biofilm carrier

Polycaprolactone (PCL) was used as both carbon source and biofilm support for denitrifying bacteria in a packed-bed bioreactor. The denitrification performance and microbial diversity were investigated. The microbial community of biofilm developed on the surface of PCL in the reactor was analyzed by pyrosequencing method. The experimental results showed the average nitrate removal efficiency reached 93 % at stable operation. ESEM observation and FTIR analysis were conducted to characterize the PCL structure before and after microbial utilization. For the microbial community, Betaproteobacteria predominated, and most of the PCL-degrading denitrifying bacteria assigned to the family of Comamonadacea. Denitrifying bacteria accounted for more than 20 % in the total population, indicating that PCL is a good carrier and carbon source for biological denitrification.     

Solid substrate fermentation for cellulase production using palm kernel cake as a renewable lignocellulosic source in packed-bed bioreactor

Palm kernel cake (PKC), is an agro-industrial residue created in the palm oil industry, and large quantities of PKC are produced in Malaysia. Sustainable development of the palm oil industry in Malaysia demands an economical technology for the environmentally friendly utilization of PKC in industrial utility systems. This research was carried out to evaluate the use of PKC in the production of cellulase by the cultivation of Aspergillus niger FTCC 5003 in a laboratory packed-bed bioreactor for seven days. A central composite design was used to perform eighteen trials of solid substrate fermentation under selected conditions of incubation temperature, initial moisture content of substrate, and airflow rate. Experimental results showed that a cellulase yield of 244.53 U/g of dry PKC was obtained when 100 g of PKC was hydrolyzed at an incubation temperature of 32.5°C, an initial moisture level of 60%, and an aeration rate of 1.5 L/min/g PKC. An empirical second-order polynomial model was adjusted to the experimental data to evaluate the effects of the studied operating variables on cellulase production. The statistical model revealed that the quadratic term for initial moisture content had a significant effect on the production of cellulase (P < 0.01). The regression model also indicated that the quadratic terms for incubation temperature and interaction effects between initial moisture content and aeration rate significantly influenced cellulase production (P < 0.05). The empirical model determined that the optimum conditions for cellulase production were an incubation temperature of 31.0°C, an initial moisture content of 59.0% and an airflow rate of 1.55 L/min/g PKC.     

Estimation of Chinese hamster ovary cell density in packed-bed bioreactor by lactate production rate

A method is described for estimating recombinant Chinese hamster ovary (rCHO) cell density in a packed-bed bioreactor by lactate production rate. The lactate production rate, which depended on both the cell numbers and cell growth rate, was modeled by segregating the cell population into two parts: one growing at a maximum specific growth rate and another non-growing. The individual cell in each part had the same lactate production rate. The established rate equation of lactate production matched the experimental data reasonably well and could be used to estimate the cell growth in the batch culture with microcarriers. Furthermore, in the perfusion culture of rCHO cells in a packed-bed bioreactor, the final cell density, 1.3×10¹⁰ cells l^–1, estimated by lactate production rate, was comparable to the direct sample counting of 1.2×10¹⁰ cells l^–1, showing that lactate production rate method would be useful in tracing the cell growth in packed-bed bioreactors.     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.     

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.     

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

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

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

Continuous acetonitrile degradation in a packed-bed bioreactor

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l^–1 reactor day^–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH₃, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l^–1 reactor day^–1 was achieved at a feeding rate of 1.6 g acetonitrile l^–1 reactor day^–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Optimization of tannase production by Aspergillus niger in solid-state packed-bed bioreactor

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett–Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature (30°C), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.     

Biodegradation of nonylphenol in a continuous packed-bed bioreactor

A packed bed bioreactor, with 170 ml glass bead carriers and 130 ml medium, was tested for the removal of the endocrine disrupter, nonylphenol, with a Sphingomonas sp. The bioreactor was first continuously fed with medium saturated with nonylphenol in an attempt to simulate groundwater pollution. At best, nonylphenol was degraded by 99.5% at a feeding rate of 69 ml h^–1 and a removal rate of 4.3 mg nonylphenol day^–1, resulting in a 7.5-fold decrease in effluent toxicity according to the Microtox. The bioreactor was then fed with soil leachates at 69 ml h^–1 from artificially contaminated soil (1 g nonylphenol kg^–1 soil) and a real contaminated soil (0.19 g nonylphenol kg^–1 soil). Nonylphenol was always completely removed from the leachates of the two soils. It was removed by 99% from the artificial soil but only 62% from real contaminated soil after 18 and 20 d of treatment, respectively, showing limitation due to nonylphenol adsorption.     

Continuous production of pediocin by immobilized Pediococcus acidilactici PO2 in a packed-bed bioreactor

 A continuous bioreactor packed with a fibrous matrix was set up. Cells of Pediococcus acidilactici PO2 were inoculated and MRS broth was fed gradually until cell growth and immobilization were achieved. Kinetics of fermentation and production of bacteriocin were investigated at dilution rates ranging from 0.63 day^-1 to 1.58 day^-1 and at pH values that varied between 4.0 and 5.5. A maximum bacteriocin activity of 6400 AU/ml was detected when the medium was fermented at dilution rates of at least 1.19 day^-1 and the pH controlled at 4.5. The maximum bacteriocin productivity was 1.0×10⁷ AUl^-1 day^-1 at a dilution rate of 1.58 day^-1 and pH 4.5. At this high dilution rate, 1.21 g cells/l medium was produced, 95.9% of the glucose in MRS broth was utilized, and 15.1 g lactic acid/l accumulated in the bioreactor effluent. The bioreactor was operated continuously for 3 months without encountering any clogging, degeneration, or contamination problems, indicating good long-term stability of the bioreactor for bacteriocin production. About 94% of the cells in the bioreactor were immobilized, and the remainder were suspended in the medium. According to scanning electron microscopic observations, cell immobilization in the fibrous matrix was attained by natural attachment to fiber surfaces and entrapment in the void volume within the fibrous matrix. In conclusion, conditions for the optimum continuous production of pediocin were defined; this may facilitate the development of large-scale industrial processes for production of this bacteriocin. Received: 25 September 1995/Received revision: 30 November 1995/Accepted: January 1996     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.