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.     

The application of cell recycle to continuous fermentative lactic acid production

Summary A continuous stirred tank reactor (CSTR) with cell recycle was used to produce lactic acid from glucose usingLactobacillus delbreuckii NRRLB445. A volumetric productivity of 76 gm/1-h was obtained with an effluent concentration of 35 gm/1 lactic acid and a residual glucose concentration of less than 0.02 gm/l.     

Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.     

Efficiency of lactic acid production by Lactobacillus helveticus in a membrane cell recycle reactor

Abstract: An economic evaluation is presented of lactic acid production in a membrane cell recycle reactor. From this evaluation it is concluded that the economic feasibility of the process is primarily limited by production capacity and product concentration and to a lesser extent by productivity. In membrane cell recycle reactor experiments and batch cultivation experiments with Lactobacillus helreticus , it is shown that the economic feasibility of the process using this organism is limited by organic acid inhibition resulting in energy uncoupling of anabolism and catabolism. Due to this inhibition, the maximum lactic acid concentration that can be obtained in the membrane reactor process is 50 g I^1—. Furthermore it is shown that not only the fermentative conversion of lactose into lactic acid but also the hydrolysis of lactose into glucose and galactose is an important process. The β-galaetosidase activity needed for the hydrolysis is generated during the exponential growth phase of Lb. helveticus     

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.     

Unstructured models for growth and lactic acid production during two-stage continuous cultures of Lactobacillus helveticus

During lactic acid fermentation, seed culture is usually carried out without pH control, while culture is carried out at pH controlled at the optimal value to overcome inhibitory effects. The Luedeking–Piret expression was therefore previously modified by introducing additional terms involving the undissociated form of the lactic acid, the main inhibitory species, in case of batch cultures without pH control or involving the residual lactose concentration to account for the carbon substrate limitation, responsible for cessation of production during batch cultures of Lactobacillus helveticus at controlled pH. Both expressions were also merged to deduce a generalized model. Both models, as well as the Luedeking–Piret model, were developed to describe continuous two-stage culture of L. helveticus. By considering the parameter values obtained from the fitting of batch culture data, both modified Luedeking–Piret models showed interesting predictive potential. Indeed, some rather reliable predictive calculated values were recorded in both stages; the residual standard deviations were 0.5 and less than 8.8 for the biomass and the product concentrations at steady-state in the culture stage (second stage). The optimization of the parameters for growth- and non-growth-associated parameters improved the fitting in the culture stage, leading to residual standard deviations below 2.6 for lactic acid concentrations at steady-state.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Unsteady-state operation of continuous fermentor for enhancement of cell mass production

The transient behavior of continuous fermentation is studied concentrating on the time scale intrinsic to the system. The time scale is the time required for the fermentorto reach a stable steady state after the disturbance of cell mass is introduced. When the cell concentration is disturbed from the steady-state value, in particular, at the dilution rate near washout, the transient period becomes extended significantly, and the steady state is resumed sluggishly. This sluggish transient behavior could be turned to an advantage for enhancing the cell mass output rate. The proposed transient operation is a continuous fermentation whereby a positive disturbance in the cell mass is introduced, so that the cell concentration is higher than the steady-state value for an extended transient period. It is shown that a significantly higher cell mass production than that from the steady-state continuous fermentation can be achieved. Simple experiments were performed to demonstrate the improvement of cell (Candida utilis) productivity.     

Process engineering for a membrane recycle fermentor

Membrane recycle fermentors are used successfully on laboratory scale to increase the efficiency of fermentation processes. The design of a process on larger scale however is obstructed by the lack of relevant data in literature. Compared to a stand-alone fermentor a membrane recycle fermentor presents new features which must be considered in the design. These features include the use of high density cultures, the additional volume in the membrane section and the circulation of the broth. In this theoretical study these aspects are analyzed with the characteristic time concept, in case of an ethanol fermentation integrated with microfiltration. The analysis shows that depending on the reactor configuration used concentration gradients can be expected. These gradients may decrease the efficiency of the fermentation, or can be advantageous, for example by letting the substrate conversion approach completion in the membrane section.     

Effective lactic acid production by two-stage extractive fermentation

For effective microbial lactic acid production using Lactobacillus delbrueckii, two-stage extractive fermentation was examined. Extractants were screened from the viewpoints of a high distribution coefficient for lactic acid and less toxicity toward the microorganism. Even if the extractant showed some toxicity toward the microorganism, it was found that a reduction of toxicity was possible by back-extraction using oleyl alcohol. As a result, 40% Alamine 336 diluted with oleyl alcohol, and oleyl alcohol, were selected as the extractant and the back-extractant, respectively. After two-stage extraction by these extractants, the growth rate was improved by the removal of lactic acid. This method was then applied to continuous extractive fermentation using a jar-fermentor. During 4-h extraction, lactic acid accumulation in the broth was significantly suppressed, while the cell growth and glucose consumption rates were also found not to be reduced. After 24 h, the cell concentration attained an OD₆₆₀ of 14, which corresponded to a level 1.25 times higher than that of the control culture without extraction. Total lactic acid productivity was 1.4 times level compared with the control culture.     

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.     

Evaluation of productive biofilms for continuous lactic acid production

In white biotechnology research, the putative superiority of productive biofilms to conventional biotransformation processes based on planktonic cultures has been increasingly discussed in recent years. In the present study, we chose lactic acid production as a model application to evaluate biofilm potential. A pure culture of Lactobacillus bacteria was grown in a tubular biofilm reactor. The biofilm system was cultivated monoseptically in a continuous mode for more than 3 weeks. The higher cell densities that could be obtained in the continuous biofilm system compared with the planktonic culture led to a significantly increased space-time yield. The productivity reached 80% of the maximum value 10 days after start-up and no subsequent decline was observed, confirming the suitability of the system for long-term fermentation. The analysis of biofilm performance revealed that productivity increases with the flow velocity. This is explained by the reduced retention time of the liquid phase in the reactor, and, thus, a minor pH drop caused by the released lactic acid. At low flow velocities, the pH drops to a value where growth and production are significantly inhibited. The biofilm was visualized by magnetic resonance imaging to analyze biofilm thickness. To deepen the understanding of the biofilm system, we used a simple model for cell growth and lactic acid production.     

An experimental study of acid production rate controlled operations of a continuous fermentor

The efficacy of acid production rate (APR) controlled operations of a continuous fermentor supporting the growth of a methylotroph, L3, was experimentally examined. Direct digital control of pH at a constant value allowed for on-line estimation of APR during the fermentation. Two types of APR controlled operations were studied. In the first type of operation, the APR was controlled at a constant value according to a predetermined program by manipulating the feed flow rate to the fermentor. Such an operation effectively stabilized the cell mass productivity of a continuous fermentor subjected to disturbances in the feed nutrient concentration. It resulted in a near complete conversion of methanol to yield a cell mass product with very low amounts of unutilized methanol at both steady state and transient fermentation situations. In the second type of operation, the feed flow rate was manipulated to optimize the steady state value of APR during the fermentation. This method shows promise for on-line steady state optimization of cell mass productivity in a continuous fermentor.     

Continuous production of extracellular protease by Bacillus subtilis in a two-stage fermentor

Growth and protease production of Bacillus subtilis in semisynthetic and synthetic media were studied in batch culture and in a two-stage, laboratory scale, continuous fermentor. The amount, of extracellular protease production was measured under specific growth conditions in both stages of the ferment or. At the dilution rates employed, the cells in the first stage of the ferment or produced negligible quantities of protease, and the culture primarily functioned as a continuous inoculum for the second stage of the fermentor. The culture effluent from the second stage of the fermentor contained extracellular protease, on the average, equal to 60 per cent, of the activity that had been found in the supernatant of a 48-hr batch culture grown in a medium having the same composition as that in the continuous fermentor. Extracellular protease was produced in semisynthetic medium by B. subtilis in the two-stage fermentor for as long as 20 days without culture degeneration. Additional studies indicated that continuous protease production could also be achieved in a synthetic medium. The RNA/ protein ratios of cells grown in semisynthetic medium in batch culture and in each stage of the two-stage fermentor were examined. There was a positive correlation between the amount of protease produced by the cells and their RNA/ protein ratio. Techniques employed for continuous production of protease by B. subtilis and the potential use of the method for investigating the control of secondary metabolite synthesis are discussed.     

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.     

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1–2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min⁻¹.     

Performance of a perforated plate column as a multistage continuous fermentor

Microorganisms were continuously cultivated in multistage column consisting of ten perforated plate sections to which medium and air were supplied concurrently from the bottom. At steady state the cell concentration in the various stages was gradationally differentiated from the bottom to the top in the direction of medium flow. RNA content per unit cell concentration at each sage was determined. The cells in the lower stages were higher in RNA content than those from the upper stages. Wash out was observed to occur in the column at dilution rates which do not result in wash out in a single stage chemostat system. A study of the flow characteristics revealed that the overall performance of the plate column was equivalent to that of a multistage system, when hole diameter and hole area to column cross sectional area ratio were properly selected. This was true even in highly aerated conditions. These results indicated that the perforated plates in the column hindred intermixing through the plates, and that each stage functioned as an independent stirred vessel. Industrial and research application of this type fermentor was discussed.     

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.     

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.     

High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor

A recombinant Escherichia coli HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle fermentor. The strain was very stable throughout the whole experiment. The main inhibitory by-product was acetic acid, and cell growth ceased when its concentration was above 14 g/L Cell density could be increased up to 145 g/L dry weight without experiencing by-product inhibition by regulating glucose concentration in the fermentor and by using total membrane recycle. Acetic acid formation was negligible not only when cells were cultured in medium containing no glucose but also when glucose was limited. Dissolved oxygen control as well as glucose limitation was an indispensable condition for minimizing acetic acid formation when the medium contained glucose. Low concentrations of accumulated acetic acid were reused when glucose was limited. Use of highly concentrated medium reduced the membrane surface area required for cell recycle greatly. The recycle fermentor could be operated in various operational modes including partial bleed and repeated recycle culture to give high productivity. Productivity of a repeated recycle system was over 10 times higher than that of a simple batch system.