Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle

The performance of an innovative two-stage continuous bioreactor with cell recycle—potentially capable of giving very high ethanol productivity—was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l⁻¹ and ethanol concentrations of 31 and 65 g l⁻¹ were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l⁻¹ in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l⁻¹ h⁻¹) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.     

Performance comparison of ethanol and butanol production in a continuous and closed-circulating fermentation system with membrane bioreactor

Since both ethanol and butanol fermentations are urgently developed processes with the biofuel-demand increasing, performance comparison of aerobic ethanol fermentation and anerobic butanol fermentation in a continuous and closed-circulating fermentation (CCCF) system was necessary to achieve their fermentation characteristics and further optimize the fermentation process. Fermentation and pervaporation parameters including the average cell concentration, glucose consumption rate, cumulated production concentration, product flux, and separation factor of ethanol fermentation were 11.45?g/L, 3.70?g/L/h, 655.83?g/L, 378.5?g/m²/h, and 4.83, respectively, the corresponding parameters of butanol fermentation were 2.19?g/L, 0.61?g/L/h, 28.03?g/L, 58.56?g/m²/h, and 10.62, respectively. Profiles of fermentation and pervaporation parameters indicated that the intensity and efficiency of ethanol fermentation was higher than butanol fermentation, but the stability of butanol fermentation was superior to ethanol fermentation. Although the two fermentation processes had different features, the performance indicated the application prospect of both ethanol and butanol production by the CCCF system.     

Cybernetic model predictive control of a continuous bioreactor with cell recycle

The control of poly-beta-hydroxybutyrate (PHB) productivity in a continuous bioreactor with cell recycle is studied by simulation. A cybernetic model of PHB synthesis in Alcaligenes eutrophus is developed. Model parameters are identified using experimental data, and simulation results are presented. The model is interfaced to a multirate model predictive control (MPC) algorithm. PHB productivity and concentration are controlled by manipulating dilution rate and recycle ratio. Unmeasured time varying disturbances are imposed to study regulatory control performance, including unreachable setpoints. With proper controller tuning, the nonlinear MPC algorithm can track productivity and concentration setpoints despite a change in the sign of PHB productivity gain with respect to dilution rate. It is shown that the nonlinear MPC algorithm is able to track the maximum achievable productivity for unreachable setpoints under significant process/model mismatch. The impact of model uncertainty upon controller performance is explored. The multirate MPC algorithm is tested using three controllers employing models that vary in complexity of regulation. It is shown that controller performance deteriorates as a function of decreasing biological complexity.     

Performance of a cell recycle bubble column fermentor with a membrane filter module for continuous vinegar production

The steady-state performance of a bubble column combined with a membrane filter module for cell separation and recycle is investigated numerically in the case of vinegar fermentation. The one-dimensional dispersion model for describing the longitudinal mixing of the liquid phase is employed. Kinetic expressions and their parameter values are taken from the available literature. Several characteristics of this fermentor system namely the concentration profiles of cells, substrate and product, the viability of viable cells relative to total cells, the washout condition for cells and the productivity of acetic acid are discussed. The average cell viability in the whole column and the critical dilution rate for washout are presented as equations. Low levels of the axial mixing are found to enhance the vinegar productivity. The optimum dilution rate giving the maximum productivity is determined and both are shown as figures with the Peclet number, the recycle ratio and the bleed ratio as parameters.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

Lactic acid from acid whey permeate in a membrane recycle bioreactor

Whey permeate was obtained by ultrafiltration of cottage cheese whey and supplemented with yeast extract. The lactose in the permeate was converted into lactic acid by Lactobacillus bulgaricus in a high-performance membrane bioreactor configured in the cell recycle mode. At a cell concentration of 10 g l⁻¹, optimum productivity of lactic acid was 35 g l⁻¹ h⁻¹. Increasing the cell concentration to 30 g l⁻¹ enabled the use of a dilution rate of 1 h⁻¹ with complete substrate utilization. At 60 g l⁻¹, productivity was over 80 g l⁻¹ h⁻¹ with complete substrte utilization; this is vastly superior to conventional batch fermentations.     

Continuous ethanol production using cell recycle with a settler

Summary A system for a high-density culture of Zymomonas mobilis was tested using a reactor with cell recycle by means of a simple settler. Growth-limiting conditions were created by raising the temperature and lowering the amount of yeast-extract. In this case the biomass production decreased, while the specific ethanol productivity did not change markedly. Under these conditions we succeeded in creating a system with a productivity of 40.5 g.1^-1.h^-1. The choice for optimal design of the settler and optimal conditions has to be made by optimization taking into account economic and whole process considerations.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Biological hydrogen production using a membrane bioreactor

A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production. The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens. Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions. When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas. When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L. This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose). When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased. However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3%. Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm). Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself. Regular backpulsing was essential for maintaining permeate flux through the membrane. Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp. MDA2507 AF238333 (99%). No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used.     

Microbial enzyme production in a membrane bioreactor

Summary Erwinia chrysanthemi cells were used to study the possibility of producing bacterial enzymes in a bioreactor coupled with a membrane filtration unit. Continuous fermentations with total cell recycle failed to give good production of pectate lyase (PL). Enzymatic, mechanical and physico-chemical damages were involved in this phenomenon. With a sequential recycle mode, we obtained productivity of 1.5 units·h^–1·1^–1 with a high PL concentration. Protease accumulation occurred when the bioreactor was coupled to a filtration unit. Moreover we have observed no loss of activity due to high shear stress caused by pumping. Offprint requests to: P. Boyaval     

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-rate continuous production of lactic acid by Lactobacillus rhamnosus in a two-stage membrane cell-recycle bioreactor

It is important to produce L(+)-lactic acid at the lowest cost possible for lactic acid to become a candidate monomer material for promising biodegradable polylactic acid. In an effort to develop a high-rate bioreactor that provides high productivity along with a high concentration of lactic acid, the performance of membrane cell-recycle bioreactor (MCRB) was investigated via experimental studies and simulation optimization. Due to greatly increased cell density, high lactic acid productivity, 21.6 g L(-1) h(-1), was obtained in the reactor. The lactic acid concentration, however, could not be increased higher than 83 g/L. When an additional continuous stirred tank reactor (CSTR) was attached next to the MCRB a higher lactic acid concentration of 87 g/L was produced at significant productivity expense. When the two MCRBs were connected in series, 92 g/L lactic acid could be produced with a productivity of 57 g L(-1) h(-1), the highest productivity among the reports of L(+)-lactic acid that obtained lactic acid concentration higher than 85 g/L using glucose substrate. Additionally, the investigation of lactic acid fermentation kinetics resulted in a successful model that represents the characteristics of lactic acid fermentation by Lactobacillus rhamnosus. The model was found to be applicable to most of the existing data with MCRBs and was in good agreement with Levenspiel's product-inhibition model, and the Luedeking-Piret equation for product-formation kinetics appeared to be effective in representing the fermentation kinetics. There was a distinctive difference in the production potential of cells (cell-density-related parameter in Luedeking-Piret equation) as lactic acid concentration increases over 55 g/L, and this finding led to a more precise estimation of bioreactor performance.     

Gas phase acetaldehyde production in a continuous bioreactor

The gas phase continuous production of acetaldehyde was studied with particular emphasis on the development of biocatalyst (alcohol oxidase on solid phase support materials) for a fixed bed reactor. Based on the experimental results in a batch bioreactor, the biocatalysts were prepared by immobilization of alcohol oxidase on Amberlite IRA-400, packed into a column, and the continuous acetaldehyde production in the gas phase by alcohol oxidase was performed. The effects of the reaction temperature, flow rates of gaseous stream, and ethanol vapor concentration on the performance of the continuous bioreactor were investigated. (c) 1993 John Wiley & Sons, Inc.     

Ethanol from hydrolyzed whey permeate using Saccharomyces cerevisiae in a membrane recycle bioreactor

A diauxic fermentation was observed during batch fermentation of enzyme-hydrolyzed whey permeate to ethanol by Saccharomyces cerevisiae. Glucose was consumed before and much faster than galactose. In the continuous membrane recycle bioreactor (MRB), sugar utilization was a function of dilution rate and concentration of sugars. At a cell concentration of 160 kg/m³, optimum productivity was 31 kg/(m³ · h) at ethanol concentration of 65 kg/m³. Low levels of acetate (0.05–0.1 M) reduced cell growth during continuous fermentation, but also reduced galactose utilization.     

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.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

A horizontal bioreactor for ethanol production by immobilized cells

A mathematical model which describes ethanol formation in a horizontal tank reactor containing Saccharomyces cerevisiae immobilized in small beads of calcium alginate has been developed. The design equations combine flow dynamics of the reactor as well as product formation kinetics. The model was verified for 11 continuous experiments, where dilution rate, feed glucose concentration and bead volume fraction were varied. The model predicts effluent ethanol concentration and CO₂ production rate within the experimental error. A simplification of the model is possible, when the feed glucose concentration does not exceed 150 kg/m³. The simplification results in an analytical solution of the design equation and hence can easily be applied for design purposes as well as for optimization studies.     

A horizontal bioreactor for ethanol production by immobilized cells

Continuous ethanol production in a three stage horizontal tank bioreactor (HTR) by yeast cells entrapped in Ca-alginate was about 30% higher than in a vertical type of bioreactor and reached 31 kg/(m³ · h) at 95% glucose utilization. Maximum ethanol productivity obtained was 41.2 kg/(m³ · h), however, with 38% of the glucose fed to the HTR being wasted. The higher performance of the HTR had been mainly accounted for the reduction of the adverse CO₂ gas phase effect and the more pronounced plug-flow character. Glucose and ethanol profiles along the HTR revealed that 50–80% of the overall fermentation activity was present in the first stage. Within a test period of 23 d the HTR showed an excellent operational stability.Compared to other continuous ethanol production processes using entrapped yeast cells the HTR presented here belongs to the top ones.