Study of a Cell-Recycle Membrane Fermentor for the Production of

Research Institute on Membranes and Modeling of Chemical Reactors c/o ... appraised in terms of bacterial growth, lactic acid concentration, productiv...
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Ind. Eng. Chem. Res. 2002, 41, 433-440

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Study of a Cell-Recycle Membrane Fermentor for the Production of Lactic Acid by Lactobacillus bulgaricus Lidietta Giorno,* Katarzyna Chojnacka,† Laura Donato, and Enrico Drioli Research Institute on Membranes and Modeling of Chemical Reactors c/o University of Calabria, via P. Bucci 17/C, 87030 Rende (CS), Italy

The aim of this experimental work was to study a cell-recycle membrane fermentor for the production of lactic acid. Initially, the fermentation and the ultrafiltration processes were studied separately; afterward, they were combined to obtain a hybrid system. Batch fermentation lasted for about 25 h, giving a yield of about 62% and a productivity of about 0.43 g/(L h). The fermented broth coming from these experiments was used to characterize the polyamide (PA) and polysulfone (PS) membranes with different nominal molecular-weight cutoffs used for the filtration step. The behavior of the permeate flux as a function of transmembrane pressure was investigated. The hydraulic resistance of membrane and fouling components was evaluated. The highest contribution to the reduction of flux was due to reversible fouling. This is because the experiments were carried out at a low axial velocity (in the range between 0.35 and 0.650 m/s) so that the cells would not be damaged and aeration would not occur (as this is an anaerobic fermentation). Although the PA membranes were fouled by adsorption, the relative flux reduction was lower for this type of membrane (0.90) than for PS 100 kDa (0.93) and PS 0.1 µm (0.97). Furthermore, it was found that the cells in the exponential growth phase had higher fouling effects compared to those in the dead phase. The efficiency of the hybrid system as a whole was appraised in terms of bacterial growth, lactic acid concentration, productivity, production yield, and flux decline with time. Introduction Carboxylic acids are an important group of additives having extensive uses in the food industry. They function as antioxidants and stabilize the pH, thus preserving the organolectic properties of foods.1 In particular, lactic acid is widely applied in foods,2 chemical stocks, medicines, and environmentally friendly packaging materials.3 Lactic acid is produced mainly by fermentation.4 Traditional batch fermentation has been studied and developed on an industrial scale for many years; nevertheless, some problems still must be solved for better performance to be obtained. For example, issues of inhibition by final products, high labor costs because of start-up and shut-down procedures, low productivity, low cell density, etc., need to be overcome.5 The use of membrane processes, and particularly membrane reactors, can contribute to improve the process efficiency.6-8 In the production of lactic acid by anaerobic fermentation, strains of so-called homolactic organisms such as Lactobacillus delbru¨ ckii or L. bulgaricus are usually used. These organisms form only one isomer of lactic acid from the carbohydrate supplied. Yields of lactic acid in the range of 80-90% of the amount of carbohydrate supplied have been reported.9,10 Many studies have been carried out using this microorganism. Voelskow and Sukatsch11 described a fed * Corresponding author: Lidietta Giorno, Research Institute on Membranes and Modeling of Chemical Reactors c/o University of Calabria, via P. Bucci 17/C, 87030 Rende (CS), Italy; Tel. +39 0984 492040; Fax +39 0984 402103; E-mail: [email protected]. † On leave from Institute of Chemical Engineering, Wroclaw University of Technology, ul. Norwida 4/6, 50-373 Wroclaw, Poland.

batch process for D-lactic acid production from whey by L. bulgaricus. Whey medium, containing 60 g/L, was used with further additions of whey (fed batch fermentation). The final concentration of D-lactic acid was 115 g/L. Hartmeier et al.12 used immobilized and extractive systems for lactic acid production. Cells of L. bulgaricus and L. delbru¨ ckii were used in a column reactor filled with porous carrier material. Microorganisms were used in simple column reactors filled with raschig rings of sintered glass. Lactose was used as the substrate. In continuous operation, a volumetric productivity of 8.7 g/(L h) was achieved at a dilution rate of 3.5 h-1. Timmer et al.10 studied the economic feasibility of the process of lactic acid production, which is primarily limited by production capacity and product concentration and, to a lesser extent, by productivity, mainly by organic acid inhibition resulting in the energy uncoupling of anabolism and catabolism. Because of this inhibition, the maximum lactic acid concentration that the authors obtained was 50 g/L. The lactic acid concentration in the membrane cell-recycle reactor ranged from 17 to 84 g/L at dilution rates of 0.25-0.75 h-1. The authors also found that lactic acid production during the stage of constant biomass only occurs by maintenance metabolism. The viability of the cells decreases rapidly after the amount of biomass becomes constant. However, lactic acid production by maintenance processes is constant, although the amount of viable cells decreases. Nonviable cells seemed to contribute to a large extent to lactic acid production in membrane cell-recycle reactors. These observations permitted the authors to conclude that four categories of cells exist in the broth: viable and respiring cells, nonviable and respiring cells, dead cells, and lysed cells.

10.1021/ie010201r CCC: $22.00 © 2002 American Chemical Society Published on Web 10/10/2001

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The decrease in viability and the constant biomass and lactic acid productivity clearly indicate the existence of viable and nonviable cells, both of which are metabolically active. The same phenomenon was observed during the experiments performed in the present work. Moueddeb et al.13 developed a membrane bioreactor in which microorganisms were confined and fixed in the pores of two organic membranes. The reactor was tubular and contained two coaxial porous alumina tubes. The bacteria were fixed into the macroporous support. This membrane reactor could be operated for 90 h, resulting in total substrate conversion at high contact time. However, a continuous decrease of the permeate flux was observed as a result of membrane plugging by bacterial population growth. Mehaia et al.14 immobilized L. bulgaricus in the shell side of an industrial hollow-fiber UF module. Acid whey permeate containing 46 g/L of lactose supplemented with 10 g/L of yeast extract was fed through the tube side at dilution rates of 0.2-2.5 h-1. At a cell concentration of 100 g/L, the productivity was 1.5-5 g/(L h). They also produced lactic acid from whey permeate, which was obtained by ultrafiltration of cottage cheese whey and supplemented with yeast extract, by L. bulgaricus in a high-performance membrane bioreactor configured in the cell-recycle mode.15 At a cell concentration of 10 g/L, the productivity of lactic acid was 35 g/(L h). Increasing the cell concentration to 30 g/L enabled the use of a dilution rate of 1 h-1 with complete substrate utilization. All of these case studies show the possibility of improving lactic acid bacteria fermentation by separating inhibitory metabolites, prolonging the exponential growth phase, and achieving high biomass productivity. Continuous cross-flow filtration allows for the removal of lactate and the complete recycling of bacterial cells to the fermentor. However, some problems are associated with this method, for example, the gradual decrease of the permeate flux with cell growth.16 Membrane techniques are effective for considering athermal separation, causing no damage to the cells. However, the performance of membrane operations is diminished by concentration polarization and fouling phenomena. Fouling is a consequence of the (ir)reversible deposition of retained particles, colloids, suspensions (i.e., bacterial cells) and macromolecules (i.e., proteins). Although transmembrane pressure (TMP) is the driving force for the permeation, the flux increases with pressure only up to a limiting value of TMP, above which no further improvements of flux are obtained. This TMP value depends on the chemicophysical properties of the suspension and membrane and on the crossflow velocity. High axial velocity (e.g., Reynolds’ number) is recommended for controlling concentration polarization and improving the permeate flux. Nevertheless, high shear rate can damage cells and reduce the biocatalyst efficiency. In the present work, a study on a cell-recycle membrane fermentor working at low axial velocity (laminar flow regime) has been carried out. The choice was dictated by the necessity of not affecting the cell integrity, to study the performance of continuous fermentation under conditions that make it comparable to the batch process. Using broth containing about 18 g/L of glucose, batch fermentation was carried out to investigate the performance of cells in the traditional system. The same type of broth and the same cellular

strain were then used to investigate the continuous fermentation in a cell-recycle membrane fermentor. The performance of ultrafiltration and microfiltration membranes in terms of permeate flux as a function of TMP and the contribution of hydraulic resistance of reversible and irreversible fouling were investigated. Ultrafiltration membranes made of polysulfone (100 kDa cutoff) showed higher steady-state permeate fluxes than ultrafiltration membranes made of polyamide (50 kDa cutoff) and microfiltration membranes made of polysulfone (0.1 µm pore size). On the other hand, PA membranes showed the lowest relative flux reduction. Materials and Methods Microorganism and Medium. In all experiments, a strain of Lactobacillus bulgaricus from DSM was used. This microorganism produces D-lactic acid as a growthassociated chemical from glucose.9 The medium for the cultivation contained 18 ((1) g/L of glucose (MRS medium from Difco, Detroit, MI). The pH of the medium during fermentation was kept constant at 6.0 with 1 M Na2CO3. Analytical Methods. The amount of cells was determined from optical density measurement at 660 nm using a spectrophotometer (UV-160A, UV-visible, Shimadzu). The concentration of lactic acid was determined using a D-7000 HPLC system from Hitachi with an UV detector (λ ) 254 nm). The analytical conditions were as follows: Sumichiral OA 5000 column, 5 µm, 150 × 4.6 mm, from Chrompack; column temperature 25-28 °C, pressure 85-90 bar; mobile phase water/2-propanol 95/5 v/v with 2 mM CuSO4; flow rate 1 mL/min. A glucose (GO) Assay Kit from Sigma was used to evaluate the glucose concentration. Protein content was measured with a BCA protein assay reagent test from Pierce. Viscosity was measured with a ball viscometer from Haake, Paramus, NJ. The reported values are the results of an average of six measurements for each sample. Membranes Used. Polyamide membranes with nominal molecular-weight cutoff (NMWCO) of 50 kDa (PA 50 kDa), having inner and outer diameters of 1.10 and 2.00 mm, respectively, were kindly provided by Berghof, Eningen/Reutlingen, Germany. Polysulfone membranes with a cutoff of 100 kDa (PS 100 kDa), having inner and outer diameters of 0.90 and 1.75 mm, respectively, and polysulfone membranes with a pore size about 0.1 µm (PS 0.1 µm), having inner and outer diameters of 1.00 and 1.77 mm, respectively, were both obtained from Romicon. The membranes were characterized by measuring the pure water permeability (permeate flux vs TMP) and were then washed with cleaning solvents (according to the indicated procedure reported below) and characterized again. This allowed to establish whether the cleaning procedure caused any change to the membrane performance. Pure water means bidistilled water filtered with 0.2 µm membrane. Washing Procedure. The following procedure was used for back-flushing cleaning: (1) Rinse with NaOH (0.5 N) at 40 °C for 20 min, (2) rinse with pure water four times for 10 min each, (3) rinse with sodium hypochlorite (NaClO) solution 1% (v/v) for 10 min, and (4) rinse with pure water four times for 10 min each. Equipment, Operation Mode, and Calculations Batch Fermentor. The chemostat culture was performed

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in 2.5-L stirred fermentor (Bioindustrie Mantovane BM 3000) using about 2 L of medium. The bioreactor containing MRS broth was sterilized in situ. Sterile 1 M Na2CO3 during chemostat and batch cultivation controlled the pH value at 6 ((0.1). Inoculum consisted of 2.5% (v/v) of a 24-h-old culture of L. bulgaricus. The culture temperature was maintained at 37 °C and agitation with 100-120 rpm. The yield was calculated as

Y ) (glactic acid/gglucose) × 100

(1)

Lactic acid is produced mainly from glucose, but also from other organic compounds present in the broth. Therefore, the yield calculated in this way can be higher than 100%. This method of yield evaluation was used to simplify the procedure, because of problems associated with analysis of total carbon available for the bacteria. Ultrafiltration Unit. An ultrafiltration process was used to clarify the fermentation broth by separating the cells and macromolecules (recycled to the retentate stream) from small molecules (recovered in the permeate). A study of the effect of transmembrane pressure (TMP) was carried out using a cross-flow ultrafiltration system (equipped with capillary membranes) in the recycle mode of operation, where the permeate was recycled back to the retentate stream to maintain the properties of the feed solution constant. The effect of TMP on the permeate flux during ultrafiltration of the fermented broth was studied by carrying out experiments starting from both low and high TMP values. Therefore, in one case, the permeate flux as a function of TMP was measured starting from low TMP values and going to higher values until the flux reached a plateau, and then, the TMP was reduced again. In the other case, the procedure was the opposite, starting from high values of TMP, slowing to lower values, and then increasing it again. For each series of experiments, a new membrane module was used. These procedures had the aim of evaluating the limiting value of TMP above which further increases did not lead to improvements of the permeate flux and determining the effects of TMP on fouling for the different type of membranes. This allowed us to determine whether it is better to start ultrafiltration directly at high values of pressure or to increase it gradually, depending on the fouling mechanism. As reported in the literature, for microfiltration composite membranes (an R-alumina filtering layer on an alumina support with a 0.2-µm mean pore diameter) the behavior of flux versus TMP is very different for increasing TMP than for decreasing TMP.17 For both types of procedures, before the experiments with the fermentation broth were started, the pure water flux as a function of TMP was measured for the membranes both when new and after being conditioned with washing solutions. This characterization gave the initial performance of the membrane. At the end of the experiments, the membranes were rinsed with distilled water, and the pure water permeability was measured again; this allowed us to evaluate the irreversible fouling. Afterward, they were washed using the indicated procedure and characterized again; this gave a measure of the possibility of regenerating the membrane.

Figure 1. Continuous membrane fermentor apparatus with ultrafiltration cell-recycle system.

The overall characteristics of flux reduction during ultrafiltration can be described by18

J)

TMP µR

(2)

where J is the permeation flux, L h-1 m-2; TMP is the transmembrane pressure, bar; µ is the viscosity, Pa s; and R is the overall hydraulic resistance, m-1. The hydraulic resistance due to the membrane (Rm) and that due to the polarization and fouling (Rf) give the overall hydraulic resistance (R)

R ) Rm + Rf

(3)

Rf, the polarization and fouling hydraulic resistance, is given by the resistance due to reversible fouling (Rr), which accounts for the reversible fouling and polarization effects, and by the resistance due to irreversible fouling (Ri), which is the fouling not removed after rinsing with water

Rf ) Rr + Ri

(4)

Rm is calculated from the slope of the plot of the pure water flux, Jw0, versus TMP; Ri is calculated from the slope of the plot of the pure water flux, Jw*, versus TMP measured after the experiment and after the membrane has been rinsed with water. The slope of the plot of the broth flux versus TMP in the linearity range determines Rf. The reversible fouling hydraulic resistance (Rr) is calculated as the difference between the overall resistance (Rf) and the resistance due to the irreversible fouling (Ri). For the calculation of the different resistances, the contribution of membrane resistance was, in general, negligible. The relative flux reduction (RFR) was calculated as

RFR ) 1 -

J* Jw0

(5)

where J* is the flux during operation and Jw0 is the pure water flux at the beginning of the experiments. Hybrid System. The system was obtained by combining the batch fermentor to the ultrafiltration unit (Figure 1). The ultrafiltration system was sterilized with 70% ethanol in water and then rinsed with sterile water (bidistilled water, filtered with a 0.2-µm membrane filter and sterilized at 124 °C for 20 min). It was verified that the ethanol did not significantly change the pure water permeate flux.

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Figure 2. Results from batch fermentation experiments. Behavior of growth curve and glucose and lactic acid concentrations.

Figure 3. Decrease of protein concentration during batch fermentation.

A peristaltic pump (Masterflex model 7518-10) was used to add new medium to the fermentor to maintain a constant working volume (2 L). The medium and the tubes connected to the bottle were sterilized in an autoclave at 124 °C for 20 min. Results and Discussion Batch fermentation of glucose with the production of lactic acid by L. bulgaricus and ultrafiltration of the fermented broth were studied separately. This was done to investigate the microorganism growth rate, lactic acid productivity, effect of fermentation broth interactions with the membranes, limiting value of TMP, fouling properties, etc. Afterward, the process was carried out in continuous mode using the hybrid membrane cellrecycle fermentor. Batch Fermentation. Batch fermentation was performed at 37 °C in the 2.5-L fermentor using 1.9-2 L of working volume. The pH was kept constant in the range of 5.9-6.1 with sterile 1 M Na2CO3. The agitation rate was 80-120 rpm. The broth was sterilized inside the fermentor for 20 min at 120 °C. After sterilization and cooling, the broth was inoculated with a 24-h-old culture (2-2.5% v/v) of L. bulgaricus. The fermentation took place for 24-35 h. Each hour, sampling was performed to analyze the cell growth the protein, glucose, and lactic acid concentrations. The growth curve from batch experiments, as well as the behavior of the lactic acid and glucose concentrations, is shown in the Figure 2. The initial glucose concentration was about 17 g/L, and the final concentration of lactic acid was 10.8 g/L, which indicates an average yield of about 62% and an average productivity 0.43 g/(L h). The amount of protein during the fermentation time is reported in Figure 3. Because this is an anaerobic fermentation, high mixing reduces the cell growth. This was confirmed by carrying out batch fermentation using the hybrid system, in other words, by circulating the broth through the ultrafiltration unit and recycling both the retentate and permeate streams back to the fermentor. It was observed that the growth rate was not affected for values of axial flow rate up to 1 m/s. Ultrafiltration in Recycle Mode. After a batch fermentation experiment was finished, the broth was collected and used for ultrafiltration experiments, where the

Figure 4. Summary of the behavior of the steady-state flux as a function of TMP for PA 50 kDa membranes starting from low (increasing and then decreasing) or high (decreasing and then increasing) TMP values.

permeate was recycled to the retentate stream to keep the volume and the broth composition constant. To make the experiments comparable, for each series of experiments, the ∼2 L of fermented broth were split into four samples of equal volume (about 400 mL) and each one was used as the initial feed solution for an UF experiment. For each type of membrane, the experiments were carried out starting from low (or high) TMP value and increasing (or decreasing) it, as described in the Ultrafiltration Unit section. Each series of experiments was carried out with a new module having the same properties (type of membrane, membrane area, and pure water permeability) with the same conditions of broth composition and temperature. The steady-state values of flux from these experiments were then plotted vs TPM. Figures 4-6 show the behavior obtained with membranes PA 50 kDa, PS 100 kDa, and PS 0.1 µm, respectively. The operating conditions for the various systems are reported in Table 1. As can be seen, for the PA 50 kDa membrane, although starting from low TMP values led to a slightly higher permeate flux, the difference is not very significant. This means that adsorption, reversible fouling, and concentration polarization phenomena strongly affected the process from the very beginning. This is mainly because the experiments were carried out at low axial flow rate to avoid shear effects on cells and aeration during continuous ultrafiltration. Therefore, starting from low or high pressure values does not make a difference.

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Figure 5. Behavior of flux as a function of TMP for PS 100 kDa. The results were obtained starting from low values of TMP, increasing it, and then reducing it again.

Figure 6. Behavior of flux as a function of TMP for PS 0.1 µm kDa. The results were obtained starting from low values of TMP (increasing it, and then reducing it again) and from high values of TMP (reducing it, and then increasing it again). Table 1. Operating Conditions during UF/MF Experiments in Recycle Operation Mode with Broth from Batch Fermentation axial membrane membrane velocity TMP flux 2 module area (m ) (m/s) RFR (bar) [L/(h m2)] PA 50 kDa 2.5 × 10-3

0.413

0.90

PS100 kDa 1.9 × 10-3 PS 0.1 µm 5.2 × 10-3

0.655 0.350

0.93 0.98

1.4 0.4 0.4 0.4

30 20 30 17.5

broth viscosity (Pa s) 1.22 × 10-3 1.20 × 10-3 1.20 × 10-3

For the PS 100 kDa membrane the behavior of flux vs TMP has a different shape when the TMP is increased compared to that shown when the TMP is decreased again (Figure 5). This difference is due to the fouling accumulated on the membrane during the “increase of pressure” step. For the PS 0.1 µm membrane, the starting value of the TMP does not make any substantial difference in the behavior of flux vs TMP (Figure 6). The different behavior of the membranes is probably due to the different mechanism of fouling, which depends on the membrane material, pore size, and fluid dynamic conditions. For example, most of the irreversible fouling of the PA 50 kDa membrane is due to adsorption. This was evaluated by measuring the pure water permeability before and after the membranes were put into contact with the broth without any pressure being applied. On the other hand, the irreversible fouling of the PS 100 kDa was mostly due to cake formation. In fact, whereas adsorption had a negligible effect on the permeate flux, the increase of TMP during

Figure 7. Comparison of pure water flux through PA 50 kDa membrane before experiment, after the membrane is rinsed, after the membrane is washed, and during permeate flux with fermentation broth.

broth filtration reduced the permeate flux considerably. The reduction of flux for the PS 0.1 µm membrane was more drastic and was mostly affected by the reversible fouling. The measurements of the pure water flux for the various membranes at the different stages (before the experiment, during the experiment, after being rinsed after the experiment, and after being washed after the experiment) were carried out to evaluate the effect of fouling during ultrafiltration of the fermented broth and the possibility of regenerating the membrane. An example of the linearity of the data from which the permeability and hydraulic resistance were calculated is reported in Figure 7 for the PA 50 kDa membranes. Similar plots were also constructed for the other membranes. Furthermore, the figure shows that the initial flux could be completely recovered after the membrane was washed with the cleaning procedure. Table 2 summarizes the permeability of the different membranes and the hydraulic resistance of the various components for each type of membrane. As can be seen, for all types of membranes, the major contribution to the overall resistance comes from the reversible fouling resistance, which accounts for reversible fouling and concentration polarization. The PS 100 kDa membrane showed higher permeate flux [about 30 L/(h m2)] at low TMP (about 0.5 bar) than the PA 50 kDa [about 20 L/(h m2)] and PS 0.1 µm [about 17.5 L/(h m2)] membranes. The PA 50 kDa membrane showed a lower relative flux reduction (0.90) than membranes PS 100 kDa (0.93) and PS 0.1 µm (0.97). Ultrafiltration in Concentration Mode. A PS 100 kDa membrane module (area 1.5 × 10-3 m2) was used to carry out experiments in concentration mode, where the permeate was recovered separately (and not recycled to the retentate stream). This experiment had the aim of evaluating the effect of broth concentration on membrane fouling. The experiment was carried out using 750 mL of broth, at an axial flow rate of 5.4 L/h and a temperature of 20 °C. Retentate absorbance was measured, and a linear correlation of optical density (660 nm) vs time was observed as the effect of cell concentration during ultrafiltration. A steady-state flux of about 45 L/(h m2) was obtained (Figure 8), with a volume reduction factor (Vpermeate/Vinitial feed) of about 0.45 and a relative flux reduction of 0.87. The higher flux obtained in this experiment compared to the that described in the previous section is due to the less fouling properties of the broth used. In fact, this broth came from a

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Table 2. Hydraulic Resistance of Membrane and Fouling Components for the Different Membranes Used type of membrane

LpW0 a [L/(h m2 bar)]

LpW* b [L/(h m2 bar)]

Lpbroth c [L/(h m2 bar)]

Rmd (m-1)

Rfe (m-1)

Rif (m-1)

Rrg (m-1)

PA 50 kDa PS 100 kDa PS 0.1 µm

285 712 1332

56.95 327.44 618.33

14.153 21.55 9.13

1.3 × 1012 5.1 × 1011 2.7 × 1011

2.1 × 1013 1.4 × 1013 3.3 × 1013

6.3 × 1012 1.1 × 1012 5.8 × 1011

1.5 × 1013 1.3 × 1013 3.3 × 1013

a L W0 ) permeability with pure water at the beginning. b L W* ) permeability with pure water, at the end of the experiment, after p p rinse with water. c Lpbroth ) permeability with broth during experiment. d Rm ) hydraulic resistance due to the membrane. e Rf ) hydraulic resistance due to the overall fouling. f Ri ) hydraulic resistance due to the irreversible fouling. g Rr ) hydraulic resistance due to the reversible fouling.

Figure 8. Behavior of flux as a function of time and volume reduction factor through PS 100 kDa membrane during ultrafiltration of broth in concentration operation mode. The broth was collected from a batch fermentation that lasted 32 h and had a protein concentration of about 5.5 g/L.

fermentation that lasted 32 h so that the cells were in the death phase, whereas the previous one came from a fermentation that lasted 25 h so that the cells were in the stationary phase. As will also be discussed in the next sections, the bacteria growth phase seemed to have a strong effect on the fouling properties of the broth. Continuous Fermentation in UF Cell-Recycle Membrane Fermentor. Using the apparatus shown in Figure 1, continuous fermentations were performed at 37 °C using 1.8-2 L of working volume. The pH was kept constant in the range of 5.9-6.1 with sterile 1 M Na2CO3. The agitation rate was 80-100 rpm. The broth was sterilized in situ for 20 min at 120 °C before the fermentation took place. After sterilization and cooling, the culture was inoculated with a 24-36-h-old culture of L. bulgaricus (2-2.5% v/v). The fermentation process was carried out in batch mode for 12-14 h to allow for growth of bacteria and production of lactic acid before ultrafiltration was started. Depending on the UF starting time and dilution rate, the concentration of the substrate can be maintained at the value optimal for the microorganism growth. Experiments were carried out using PA 50 kDa membrane samples having surface areas of 2.5 × 10-3, 7.44 × 10-3, and 12.4 × 10-3 m2 and a PS 100 kDa membrane sample with a surface area of 2.9 × 10-3 m2. The fermentation broth volume was kept constant by matching the feed flow rate with the permeate flow rate. Every 2 h sampling was performed to analyze the cell optical density and the protein, glucose, and lactic acid concentrations. The results of the continuous fermentations at the different dilution rates are reported in Figure 9. The conditions of the experiments are summarized in Table 3. In the first experiment, the ultrafiltration started after 12 h from the onset of the fermentation process and lasted for 49 h with a PA 50 kDa module (2.5 ×

Figure 9. Comparison between the behavior of continuous fermentations at different dilution rates.

10-3 m2). After this time (therefore, after 61 h from the beginning of the fermentation), because of the low permeate flux, the module was replaced with a new PA 50 kDa membrane to keep the dilution rate constant during the continuous process. The permeate flux of this second module was higher than that of the first one (Figure 10). This is probably because the foulant properties of the fermentation broth during the growing phase of the bacteria are different from the properties during the nongrowing phase. The difference could be in the protein concentration, which is high during the lag phase, decreases during the exponential growth and stationary phases, and remains almost constant during the death phase (Figure 11). Furthermore, the higher fouling properties, could be due to the difference in physiological conditions, which has a high influence on the cell shape, water content, cell resistance etc. The continuous fermentation was finished after 83.5 h of operation and had a final lactic acid concentration of 16 g/L. During the last hours of fermentation, the cell concentration did not increase while lactic acid concentration was constant, although dilution rate was also constant. This means that new cells were not produced, but that old cells were still metabolically active. This phenomenon is in agreement with the literature data.10 During the 83.5 h of continuous fermentation, 79 g of glucose was consumed and 74.4 g of lactic acid was produced. The total volume used (feed + broth in the fermentor) was 4.65 L. This gives an average productivity of 0.19 g/(L h) [productivity during the batch fermentation was 0.43 g/(L h)] and a production yield of 94% (average yield during batch fermentation was 62.2%). In the batch fermentation, the maximum optical density (660 nm) value was 2.2 and was reached after 20 h, whereas in the continuous fermentation, the value of 3.0 was reached after 22 h. Therefore, the growing phase was 7 h longer during continuous fermentation, and 36% more cells were kept in the fermentor. The fermentation process was negatively affected by the fact

Ind. Eng. Chem. Res., Vol. 41, No. 3, 2002 439 Table 3. Operating Conditions and Overall Results during Continuous and Batch Fermentations type of expta cont 1 cont 2 cont 3 cont 4 batch 1 batch 2 a

type of membr

membr area (m2)

PA 50 kDab 2.5 × 10-3 PA 50 kDac 2.5 × 10-3 PS 100 kDab 2.9 × 10-3 PS 100 kDac 2.9 × 10-3 PA 50 kDa 7.44 × 10-3 PA 50 kDa 12.4 × 10-3

steady TMP flux (bar) [L/(h m2)] RFR 0.8 0.8 0.5 0.5 0.8 0.8

12 20 16 17 10 10

0.95 0.84 0.95 0.95 0.96 0.96

dil rate (h-1)

ferm time (h)

0.017 0-61 0.020 61-83.5 0.024 0-42.5 0.024 42.5-62 0.040 0-25 0.070 0-12.5 0-25 0-25

protein (g/L) 17 4 14 10 20 20 17 17

total lactic acid vol (L) (g/L) (g)

total producglucose tivity yield (g) [g/(L h)] (%)

4.65

16.0

74.4

79.0

0.19

94

3.88

16.0

62.1

65.9

0.25

94

4.33 3.38 1.9 1.9

10.5 10.8 10.5 11.1

45.5 36.5 20 21

73.6 57.5 32.3 32.3

0.42 0.86 0.42 0.44

62 63 62 65

cont ) continuous fermentation in the cell-recycle membrane reactor. b First module. c Second module.

Figure 10. Behavior of flux as a function of time for membranes Pa 50 kDa and PS 100 kDa during continuous fermentation at different dilution rates.

Figure 11. Protein concentration during continuous fermentations at different dilution rates.

that glucose decreased too much, because of the low dilution rate. The behavior of the glucose and lactic acid concentrations is reported in Figure 12. The same type of experiment was carried out with PS 100 kDa (2.9 × 10-3 m2) (experiment 2 in Table 3). The TMP was kept constant at 0.5 bar, and a steadystate permeate flux of about 15 L/(h m2) was reached. The dilution rate was 0.024 h-1. After 28.5 h of ultrafiltration (i.e., after 42.5 from the onset of the fermentation), the membrane module was replaced with a new one with the same characteristics to investigate the influence of bacteria growth phase on membrane fouling. The steady-state flux was more or less the same for these two membranes (Figure 10). In fact, in this experiment, during the ultrafiltration time, the bacteria were still in the growing phase because of the higher dilution rate. Therefore, the bacterial growing phase has a stronger influence on membrane fouling than the

Figure 12. Behavior of glucose and lactic acid concentrations during continuous fermentation at a dilution rate of 0.17-0.20 h-1.

other growth phases. This is further validated by the fact that, when the broth containing bacteria in the death phase was filtered, the resulting permeate flux was much higher [45 L/(h m2)] compared to this one [15 L/(h m2)]. As previously discussed, the protein content during the fermentation process might be the main factor affecting the behavior of membrane fouling. To increase the dilution rate, PA 50 kDa membrane modules consisting of 12 and 20 fibers with effective surface areas of 7.44 × 10-3 and 12.4 × 10-3 m2, respectively, were used (experiments 3 and 4 in Table 3). Dilution rates of 0.04 and 0.07 h-1, respectively, were obtained. As expected, with increasing dilution rate, the cellular growth increases (and lasts for longer time, see Figure 9) as does the amount of lactic acid produced. However, very high dilution rates result in low concentrations of lactic acid in the permeate, which is not advantageous for downstream processing.19 In Table 3, the behavior of the lactic acid concentration as a function of dilution rate is reported. The lower yield at higher dilution rates is due to the fact that the glucose that permeated through the membrane was not recycled back to the fermentor. It is expected that 100% conversion can be obtained by recycling the broth after separation of lactic acid. The selective separation of lactic acid by membrane-based solvent extraction through two hollow fiber membrane contactors is currently under investigation. Conclusions A study on the continuous production of lactic acid by L. bulgaricus in a cell-recycle membrane fermentor has been carried out. To avoid negatively affecting the cellular growth by cell damaging and aeration, the ultrafiltration processes

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were carried out under conditions of laminar flow. Polyamide membranes with a cutoff of 50 kDa and polysulfone membranes with a cutoff of 100 kDa and a pore size of 0.1 µm were used. The hydraulic resistance due to the membrane wall and the reversible and irreversible fouling were evaluated. The major contribution to the flux reduction was given by the reversible fouling. The value of its hydraulic resistance was 1 order of magnitude higher than that due to the irreversible fouling and the membrane itself. The high value for reversible resistance (which also accounts for concentration polarization) is due to the need to work at axial velocities lower than 1 m/s (reported in Table 1 for the different membranes). Provided that the limiting transmembrane pressure value was not overcome, the initial transmembrane pressure value did not affect the behavior of permeate flux. The limiting TMP values for PA 50 kDa, PS 100 kDa, and PS 0.1 µm were 0.8, 0.4, and 0.4 bar, respectively. During filtration of broth, the PA 50 kDa membranes showed the lowest relative flux reduction (0.90), although for these membranes, the irreversible fouling was due to adsorption. Different fouling properties of the broth depending on the growth phase step were observed. In particular, broth containing cells in the death phase had lower fouling effects than that containing cells in the exponential growth phase. A correlation between the protein contents in the different growth phases and the fouling properties of the broth was observed. This represents an important factor to consider in the design of overall production systems. The cell-recycle membrane reactor allowed the cellular growth to be increased, thus leading to a higher production of lactic acid compared to the traditional batch reactor. An increase in bacterial growth as a function of the dilution rate was obtained. At higher dilution rates, the concentration of lactic acid decreases. This is an advantage for the reaction system, because product inhibition is reduced; on the other hand, the low lactic acid concentration in the permeate makes its downstream separation more difficult. A compromise between the two opposite needs should be considered. The final aim of this research is to integrate continuous fermentation with membrane-based solvent extraction to selectively separate the lactic acid and recycle the broth, containing unconverted glucose and other nutrients, back to the fermentor, thus approaching total conversion of the raw materials. The knowledge achieved in this work will allow for the optimization of the various parameters in the overall system. Literature Cited (1) Frederick, J. F. Encyclopedia of Food Science and Technology; John Wiley & Sons: New York, 2000; pp 1-6.

(2) Boyaval, P.; Corre, C.; Terre, S. Continuous Lactic Acid Fermentation with Concentrated Product Recovery by Ultrafiltration and Electrodialysis. Biotechnol. Lett. 1987, 9 (3), 207-212. (3) Hitomi, O.; Hiyama, K.; Yoshida, T. Kinetics of Growth and Lactic Acid Production in Continuous and Batch Culture. Appl. Microbiol. Biotechnol. 1992, 37, 544-548. (4) Hitomi, O.; Hiyama, K.; Yoshida, T. Noncompetitive Product Inhibition in Lactic Acid Fermentation from Glucose. Appl. Microbiol. Biotechnol. 1992, 36, 773-776. (5) Drioli, E.; Giorno, L. Biocatalytic Membrane Reactors: Applications in Biotechnology and the Pharmaceutical Industry; Taylor & Francis: London, 1999. (6) Matson, S.; Quinn, J. A. Method and Apparatus for Conducting Catalytic Reactions with Simultaneous Product Separation and Recovery. U.S. Patent 4,786,597, 1987. (7) Lopez, J. L.; Matson, S. L.; Stanley, T. J.; Quinn, J. A. Liquid/Liquid Extractive Membrane Reactors. In Extractive Bioconversions; Bioprocess Technologies Series; Mattiasson, B., Holst, O. Eds.; Marcel Dekker: New York, 1990; pp 27-66. (8) Matson, S. L.; Quinn, J. A. Membrane Reactors. In Membrane Handbook; Ho, W., Sirkar, K., Eds.; Van Nostrand Reinhold: New York, 1992. (9) Aiba, Sh.; Humphrey, A.; Millis, N. Biochemical Engineering; Academic Press: New York, 1973. (10) Timmer, J.; Kromkamp, J. Efficiency of Lactic Acid Production by Lactobacillus helveticus in a Membrane Cell Recycle Reactor. Microbiol. Rev. 1994, 14, 29-38. (11) Voelskow, H.; Sukatsch, D. (Hoechst AG). Eur. Pat. 72010, 1983 (12) Hartmeier, W.; Herrfurth, S.; Hembach, T.; Kempken, R. Immobilized and Extractive Systems to Produce Lactic Acid. In Engineering and Food; Elsevier Applied Science: New York, 1990; Vol. 3, Advanced Processes. (13) Moueddeb, H.; Sanchez, J.; Bardot, C.; Fick, M. Membrane Bioreactor for Lactic Acid Production. J. Membr. Sci. 1996, 114, 59-71. (14) Mehaia, M.; Cheryan, M. Immobilization of L. bulgaricus in a Hollow Fiber Bioreactor for Production of Lactic Acid from Acid Whey Permeate. Appl. Biochem. Biotechnol. 1987, 14, 2127. (15) Mehaia, M.; Cheryan, M. Lactic Acid from Acid Whey Permeate in a Membrane Recycle Bioreactor. Enzyme Microb. Technol. 1986, 8, 289-192. (16) King, R. D.; Cheetham, P. S. Food Biotechnology I; Elsevier Applied Science: New York, 1987. (17) Boyaval, P.; Lavenant, C.; Ge´san, G.; Daufin, G. Transient and Stationary Operating Conditions on Performance of Lactic Acid Bacteria Cross-flow Microfiltration. Biotechnol. Bioeng. 1996, 49, 78-86. (18) Aimar, P.; Howell, J. Effect of Concentration Boundary Layer Development on the Flux Limitations in Ultrafiltration. Chem. Eng. Res. Des. 1989, 67, 225-261. (19) Giorno, L.; Leva, L.; Donato, L.; Drioli, E. Development of an Integrated Membrane Process for the Production and Downstream Processing of Carboxylic Acids. In Biotechnology 2000: The World Congress on Biotechnology, Berlin, Germany, Sept 3-8 2000; DECHEMA: Frankfurt/Main, Germany; Vol. 1, pp 440442.

Received for review March 1, 2001 Revised manuscript received July 16, 2001 Accepted July 17, 2001 IE010201R