Abstract:
A process for producing, recovering and purifying lactate from a fermentation broth so that it can be efficiently and economically converted to lactic acid is disclosed. The process comprises growing a high lactate producing, microorganism on an inexpensive substrate until a whole broth containing about 75 to about 90 grams of lactate salt is obtained. The whole broth including the viable cells is then electrodialyzed in a unit having anionic-permeable and cationic-permeable membranes to cause the lactate salt to concentrate in an aqueous liquid which can be treated to convert the lactate salt to lactic acid.

Description:
FIELD OF THE INVENTION 
     The present invention relates to the recovery and purification of organic acid salts from fermentation broths More particularly, it relates to the recovery and purification of lactate salts from fermentation broths by electrodialysis. 
     BACKGROUND OF THE INVENTION 
     Lactic acid is a specialty chemical used in the food and chemical industries. It is a potential commodity chemical because it can be used to make polymers or hydrogenated to make propylene glycol and other three carbon chemical intermediates. Currently, lactic acid is expensive to make by fermentation because the fermentation produces a crude lactate salt from which the cells and protein have to be removed. To be an economical source of lactic acid, the fermentation has to employ a microorganism which produces high concentrations of lactate from inexpensive substrates. In addition, the recovery, separation and recycle of viable cells and nutrients to the fermentor is required because high cell density and fermentation productivity and low nutrient consumption are important. 
     Electrodialysis (ED) is a well known separation process where ionized compounds are separated from non-ionized or weakly ionized compounds in aqueous solutions based on transport through ion exchange membranes in an electric field. The process has been used in a commercial scale in the chlor-alkali, desalination, metal-processing, waste-water treatment, pharmaceutical and food processing industries. Since in a fermentation broth the lactate salt is ionized, whereas the carbohydrates and proteins and amino acids are either non-ionized or weakly ionized, recovery and purification of lactate salts from a fermentation broth by electrodialysis is feasible 
     Recently several papers have been published on the recovery and the purification of lactate from fermentation broths. In the published processes, the broth was either thoroughly cleaned to remove cells and proteins prior to electrodialysis or when the whole broth, containing cells, was used the membrane fouled and led to a loss of efficiency. 
     Prigent (1) disclosed a method for production of lactate from fermenting whey where the broth was filtered by ultrafiltration to remove the cells and then electrodialyzed. Hongo, Nomura and Iwahara (2) used a whole broth of Lactobacillus delbrueckii IFO 3534 in their electrodialysis apparatus, but discovered that the efficiency was poor due to membrane fouling. These workers (3) then devised a complicated fermentation process where the cells were immobilized in alginate beads and the cell-free broth was fed to the electrodialyzer. The described processes are not practical for commercial use because bacterial cells are very small and removing the cells by high speed centrifugation and/or ultrafiltration is expensive. Cell immobilization in alginate beads also is very expensive and not readily adaptable to commercial use. 
     For a fermentation process to be economically attractive to produce lactic acid as a commodity chemical, it must be simple and inexpensive. Ideally, the microorganism should grow on low cost substrates and nutrients; the rate of fermentation should be high (high productivity); the lactate concentration in the fermentation broth should be high; and, an inexpensive recovery and purification process should be available. 
     SUMMARY OF THE INVENTION 
     It is a general object of the present invention to disclose a new and improved process for the production of organic acid salts and their recovery and purification from whole fermentation broths. 
     It is a further object to disclose an inexpensive fermentation process for producing lactate salts on a low cost medium using a novel strain of Lactobacillus acidophilus. 
     It is a further object to disclose an electrodialysis process for the recovery and purification of lactate salts from whole fermentation broth without having to first remove the microbial cells. 
     It is another object to disclose an improved process for producing lactate salts by fermentation which includes recycling the microbial cells back to the fermentor where they can produce additional lactate. 
     The manner in which these and still other objects are accomplished will become apparent to those skilled in the art from the following description and accompanying drawings. 
     The present invention basically comprises growing a microorganism capable of producing at a cell concentration of 2.5×10 10  to 3.0×10 10  cells/ml least about 2 grams of lactate per liter of broth per hour on an inexpensive substrate at a pH of about 5.0 to about 5.7 and a temperature of about 35° to about 45° C. until the broth contains about 75 to about 90 grams of lactate per liter of broth. The whole broth is then strained to remove larger then cell particles and electrodialyzed in a unit containing anionic permeable membranes and cationic permeable membranes under conditions which cause the lactate ions to leave the broth and to concentrate in an aqueous liquid from which the lactate can be removed and converted to lactic acid using known techniques and equipment. The viable microbial cells from the whole broth are recycled to the fermentor where they economically produce additional lactate. 
     The preferred microorganism is a strain of Lactobacillus acidophilus IAM 3532; it produces high yields of lactate at cell concentrations of about 2.5×10 10  to about 3.0×10 10  cells per ml. The preferred membranes are the anionic exchange and cationic exchange membranes available from Asahi Glass Co. under the names SELEMION AMV and CMR, respectively. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The drawing is a schematic diagram of an electrodialysis apparatus in which the preferred embodiment of the method of the present invention may be carried out. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The drawing schematically illustrates the electrodialysis apparatus 10 which includes a cell stack 11, a fermentor 12, a screen 13, a feed solution injection, recirculation and removal system 14, a concentrate recirculation and removal system 15, an anolyte rinse system 16 and a catholyte rinse system 17. 
     It will be appreciated the apparatus illustrated in the drawing is for purposes of illustration and modifications thereof can be made by those skilled in the art without departing from the scope of the present invention. 
     Cell stack 11 may be any known type of membrane assembly such as a plate and frame assembly wherein a plurality of suitably perforated flow distribution gaskets 18 support and seal the peripheries of a plurality of cation-permeable membranes 19 and anion-permeable membrane 20 in parallel spaced relation to form a series of parallel cells 21 and end cells 22 and 23. Each cell 21 is defined by a pair of membranes 19 and 20 and a gasket 18. The end cells 22 and 23 are respectively defined by a membrane 19 and a membrane 20 and end caps 24. Disposed within end cell 22 is a suitable anode 25 and a cathode 26 is disposed in the opposite end cell 23. Anode 25 and cathode 26 are connected respectively to the positive and negative terminals of a suitable power source (not shown) through leads 27 and 28. Cell stack 11 also includes suitable couplings (not shown) for admitting and removing liquids from each of cells 21. The components of the cell stack 11 can be held in an abutting relation by suitable clamps or tie rods (not shown). 
     The membranes 19 are anion-permeable and cation-impermeable and the membranes 20 are cation-permeable and anion-impermeable membranes. Suitable materials for use as membranes 19 and 20 include anion and cation exchange resins having active ion capture sites, respectively. The preferred membranes 19 are fabric reinforced microheterogeneous interpolymer membranes, such as the anion exchange membranes manufactured by Asahi Glass Co. SELEMION AMV membranes, while the preferred membranes 20 are the SELEMION type CMR cation exchange membranes available from the same source. The membranes are described in U.S. Pat. No. 4,678,553 and others. Properties for these two membranes are set forth in Table I. 
     In the preferred practice of the invention, the whole broth is pumped from the fermentor 12 through the screen 13 which removes larger than cell size particles. The whole broth, minus those particles, is transported via system 14 to the cells 21 where it is subjected to an electrical current which causes the lactate ions to migrate through the anionic-permeable membrane 19 into an 1.0M ammonium lactate solution which is circulating in the system 15 on the other side of the membrane 19. The lactate enriched ammonium lactate solution, also called the concentrate, is transmitted to a second water splitting electrodialysis apparatus (not shown) to convert the lactate to lactic acid. The feed solution, minus the lactate, but including the viable cells is recirculated via the system 14 to the fermentor. While the electrodialysis is going on the electrodes are rinsed with a suitable electrolyte solution such as 0.1 to 0.4M Na 2  SO 4  in water circulated by the rinse systems 16 and 17. The process can be operated on either a batch or continuous basis. 
     
                                           TABLE 1__________________________________________________________________________TYPES AND PROPERTIES OF SELEMION MEMBRANES             CMR   AMV             Strongly                   Strongly             acidic                   basic             cation-                   anion-             permeable                   permeableDESIGNATION       membrane                   membraneType              (Na.sup.+ type)                   (Cl.sup.- type)                              REMARKS__________________________________________________________________________    T Na.sup.+             Over 0.92                   --         Calculated from the                              membrane potentional:    T Cl.sup.-             --    Over 0.94  0.5/1.0 mol/P NaClTransport                          solution at 25° C.number   TCa.sup.++  + Mg.sup.++             Under 0.04*      Measured by electro-                              dialysis of sea                              water at 2A/dm.sup.2Resistance perunit area    X-cm.sup.2             2.0-2.3                   2.0-3.0    Measured by 1000                              Hz-AC 0.5 mol/P                              NaCl at 25° C.Thickness    mm       0.13-0.15                   0.12-0.15Bursting stengthkg/cm.sup.2       3-4   3-5Use      General desalination/                   General desalination/    concentration  concentration    Sea water concentration__________________________________________________________________________ *After treatment 
    
     The practice of the invention is further illustrated by the following example. 
     EXAMPLE 
     MATERIALS AND METHODS 
     Electrodialysis Unit 
     The electrodialysis apparatus consisted of a stack of the type shown in the drawing which comprised an alternating series of anion and cation selective membranes separated by flow distribution gaskets. The membranes were bound on one end by an anolyte compartment and an anode on the other end by a catholyte compartment and cathode. The stack pack for evaluation was provided by HPD Inc. (Naperville, IL) and contained membranes manufactured by Asahi Glass Co. (Japan). The stack pack contained the following: 9 cell pairs 
     anion membrane--AMV 
     cation membrane--CMR 
     effective area--178 cm 2   
     electrolyte--0.1 to 0.4M Na 2  SO 4   
     The unit consisted of three independent flowchannels fed to the electrodialyzer stack pack. The three streams were: 
     (1) the feed stream--whole broth from the fermentor 
     (2) concentrate stream--initially a 1.0M ammonium lactate solution which is further enriched by lactate from the feed product 
     (3) electrolyte--0.1 to 0.4M sodium sulfate 
     From each reservoir, material was pumped through a valve, rotameter, and pressure gauge, to the stack pack, and then back to the reservoir. Another set of five gallon containers was located below each reservoir for removal purposes. The whole unistrut frame was on wheels so it could be easily moved. 
     The electrical current was supplied by a Hewlett Packard (HP) regulated DC power supply model 62688. It was connected to the anode and cathode of the membrane stack which could produce 0-20 amperes and deliver 0-50 volts. Another voltmeter was used to measure the voltage drop across the membranes (excluding electrodes). Two platinum wires were inserted between eight cell pairs of membranes and then connected to the voltmeter. 
     Fermentation Broth 
     The feed material, ammonium lactate fermentation broth of L. acidophilus IAM 3532, was produced using a fermentor in the pilot plant. 
     The anaerobic fermentation was performed at 39° C. in the fermentor with a working volume of 150 L for 69 hours. The media contained approximately 10% dextrose (Cerelose), 2% corn steep liquor, and 0.2% corn oil. A 4% inoculum was used. The pH was maintained between 5.3-5.5 by addition of ammonium hydroxide on a demand basis. Agitation speed was maintained at 100 rpm. 
     For one set of experiments the cells in the fermentation broth were removed by processing the broth through a high speed Westphalia centrifuge. The broth was then filtered with supercel precoat in a vacuum filter. 
     For the second set of experiments freshly prepared whole broth using the same media and similar fermentation conditions (pH=5.3-5.5, temp. 35°-40° C.) was used for the whole broth experiments. The broth was screened through a 200  mesh wire screen to remove larger than cell size particles before electrodialysis. 
     Permissible Current Density (PCD) Determination 
     Before making actual runs, permissible current density (PCD) was determined to obtain a safe range for operating current density. For these runs, only two reservoirs were used. One reservoir supplied broth to both the diluting and concentrating compartments and then was pumped back to the reservoir. The second reservoir was for the electrolyte. 
     The unit was operated at low current and the voltage was recorded every 30 seconds for a period of 30 minutes to two hours. If the voltage remained constant over time, the procedure was repeated at a higher current. This procedure was continued until a point was reached when the voltage began to increase with time denoting the onset of polarization or fouling. The current density at that point was the PCD for that operating condition (salt concentration, pH, temperature, and linear velocity). 
     Runs were made at 4, 5, 6, 7, 10 and 12.5 amps each for 30 minutes using broth containing 1M ammonium lactate at 50° C. and 5 cm/sec linear velocity. The initial operating current and current density (12 amp, 67 mamp/cm 2 ) was decided upon based on the PCD results. 
     Batch Electrodialysis Description 
     The system was operated in a batch mode. Thus, the lactate broth was continually being demineralized. The electrodialysis process continued until the solution was demineralized to a desired degree. The following is a description of the operating procedure. 
     Charging 
     1. Drain the system (Note: the system should be filled with demineralized water at all times to keep membranes from drying out) and close the valves. 
     2. Fill the diluting reservoir with the solution to be demineralized, 6-14 liters of ammonium lactate broth. 
     3. Fill the concentrating reservoir with one liter of ammonium lactate of a concentration similar to that of the diluting compartment. This ensures good initial conductivity and circulation rate in the concentrating compartment. (Ammonium lactate was prepared from Fisher Lactic Acid and pH adjusted to 5.2 with ammonium hydroxide). 
     4. Fill the electrolyte reservoir with 4-6 liters of 0.1 to 0.4M Na 2  SO 4  or other suitable electrolyte. 
     5. Open valves and turn on pumps. Allow liquid in all three lines to flow at a rate that the floats in the rotameter are barely lifted. 
     6. Open valves allowing pressure in the lines to build up gradually such that the pressure differential between any two lines at no time exceeds 1 psig. 
     7. Continue opening valves until the desired flowrate is reached. 
     8. Turn on steam line to heat the diluting stream to desired temperature. 
     Membrane Equilibration 
     1. Turn on power supply to a reading of 0.2 amperes. 
     2. Continue running the system for 10 minutes. 
     Batch Operation 
     1. Set valves to desired flow rates. 
     2. Set power supply for desired current (the desired current was previously determined based on the permissible current density). 
     3. During the run, data collected included: current, voltage drop across total unit, voltage drop across eight cell pairs, volume levels in the diluting and concentrating compartments, pH of the diluting and electrolyte compartments, conductivity of the diluting compartment, and temperature of the diluting compartment. 
     4. The unit was operated at a constant current until the resistance started to increase thus increasing the voltage drop across the unit. At that time, a maximum voltage was set and the current was allowed to decrease until the end of the run. 
     5. Samples were taken at thirty minute intervals in addition to initial and final samples. 
     Analytical Methods 
     Conductivity was measured using a portable conductivity meter (Cole Parmer model 1484-10). 
     Lactate concentration reported is the anion concentration and was measured after appropriate dilution and acidification by an HPLC method using a 1 ft. long HPX87 H+ column of BioRad (Calif. U.S.A.). 
     Amino Acids--a semiquantitative assay method to measure L-amino acids in presence of ammonium salts was developed based on two coupled enzymatic reactions, L-amino acid oxidase and peroxidase. This method allowed rapid determination of L-amino acids using L-leucine as a standard. Total and free amino acids for important samples were determined by Hazelton Laboratories (Wis. U.S.A.) using standard methods of ion exchange chromatography. 
     Definition of Terms 
     Current Density--current per unit area of available membrane through which current passes. The higher the applied current density, the lower cell area required to achieve a specific degree of ion transfer. 
     Current Efficiency--ratio of the number of equivalents of lactate transferred to the number of Faradays of electricity passed through the membrane. 
     Flux--volume transferred through the membrane in a specific time and available area. 
     Data and calculation details from an electrodialysis run are presented in Tables 2-4. 
     RESULTS 
     Electrodialysis of Centrifuged and Filtered Broth 
     Batch electrodialysis of centrifuged and filtered broth which contained no cells was run in the ED stack pack. Table 2 shows the results of two different runs. As can be seen from the data, this particular strain of L. acidophilus produces a high lactate anion concentration (75-85 g/l) in the broth using a simple media consisting of a carbohydrate, corn steep liquor and small quantities of crude corn oil. The overall productivity in the batch fermentation was between 2-2.5 g/l hr. Electrodialysis of this broth performed very efficiently with current efficiencies of 90 (±2)% and at high current densities. High lactate concentration in the concentrate product also was achieved due to good flux and low water transfer rate through the membranes. The concentrate product was also highly purified and &gt;90% of the amino acids was retained in the broth while 92% of the lactate was removed from it. 
     Electrodialysis of Whole Fermentation Broth 
     The next set of experiments was made with whole culture lactate broth without cell removal (containing 2.5-3×10 10  cell/ml). The first was a batch run to determine feasibility which was followed by a second batch run at a slightly lower temperature, 45° C., to maintain viability of the cells. In the second run, the electrodialyzed broth was returned for further fermentation. No irreversible fouling was detected during the course of either run. The results of these runs are shown in Table 3. 
     Comparing the whole broth to filtered broth runs, slightly less lactate was removed but the current efficiency remained high (90-91%). Water transfer rates were similar so there was still a 2.5× concentration of product lactate across the membrane. The slightly higher electricity requirement for the last run is due to the lower operating temperature (45° vs. 50° C.). Temperature is directly related to conductance (inverse of resistance) and energy requirement is directly related to resistance. This would be a small penalty to pay to maintain cell viability. 
     During the whole broth electrodialysis run, samples of the whole broth were assayed for viable cells (by plate count on MRS agar media). The cells remained predominantly viable. 
     Whole Broth Recycle Fermentation 
     The spent broth from the electrodialysis apparatus was further recycled to the fermentor and fermented with additional dextrose, corn oil (0.2%), and different concentrations of corn steep liquor (F2-1.4%, F3-2.4%, F4-0%). The fermentation volume was made up to the original broth volume (0.7 liter to 1 liter). The viable cell count for F2 and F3 at the beginning of recycle fermentation was comparable to the cell count in the electrodialyzer feed broth. The recycle fermentations F2 and F3 proceeded rapidly producing 77 and 85 g/l lactate respectively in 24 hours with productivities of 2.2 and 2.5 g/l-hr, respectively. These are comparable to that obtained in typical batch fermentations, 2-2.5 g/l-hr. Recycle fermentation F4 (0% CSL) was slow and required 100 hours to consume the dextrose and produce 90.4 g/1 lactate. This result shows that viable cells from the electrodialysis of whole broth can be recycled and used to ferment more carbohydrates to lactate without losing productivity and the ability to produce high lactate concentration. 
     Protein and Amino Acid Transport 
     An analytical method was developed using the enzymes L-amino acid oxidase and peroxidase in a semiquantitative method for quick analyses of free L-amino acids. This assay is very useful in determining the gross performance of membranes during an electrodialysis run. The amino acid rejection data in TABLES 2 and 3 are based on this assay and clearly show a very high retention of amino acids in the broth and purification of lactate in the product concentrate. 
     To obtain a more detailed understanding of the protein and amino acid transport, samples from key ED runs were analyzed for complete amino acid analysis. The data is summarized in Table 4. The data shows that the product concentrate contains only a small fraction (10-15%) of the total and free amino acids from the feed broth, which means that 85-90% of the proteins and amino acids are rejected. It also shows that electrodialysis of whole broth performs very well for protein and amino acid rejection and a highly purified concentrate can be obtained by a one step process by electrodialysis of whole broth. 
     It will be apparent to those skilled in the art that the process of the present invention is a low cost fermentation process which can produce lactic acid and its derivatives as specialty and commodity chemicals, using low cost nutrients. The process also produces a high lactate product concentration at a high productivity and an economical purification process. 
     The preferred strain of Lactobacillus acidophilus IAM 3532 grows well on media containing carbohydrates, corn steep liquor and low concentration (0.2%) of crude corn oil. In addition, it produces high concentrations of lactate (75-90 g/l) with a high productivity 2-2.5 g/l hr. 
     During the electrodialysis of whole broth with viable cells no irreversible membrane fouling was observed. Hence, the factors that determine the economic and process feasibility of electrodialysis--high current efficiency, low energy consumption, high selectivity and purification, high flux, high current density could be achieved using whole broth. Other practitioners (1,2,3) have performed electrodialysis of lactate fermentation broth either after extensive and costly ultrafiltration, microporous filtration or centrifugation to remove and recycle cells; or have found irreversible membrane fouling leading to loss of efficiency; or used expensive and complex cell immobilization methods to retain cells in the fermentor and avoid introduction of cells into the electrodialyzer. 
     The combination of the special strain of Lactobacillus acidophilus IAM 3532 and the use of fabric reinforced, microheterogeneous interpolymer membranes (Asahi Glass AMV and CMR) permits simultaneous electrodialysis and cell recycle from whole broth to be achieved without loss of efficiency either in the fermentor or the electrodialysis separator. The mature cells remain viable and are recycled to the fermentor where fermentation continues with addition of carbohydrates and nutrients, and the purified and concentrated lactate from the electrodialyzer can now be used to feed a water splitting electrodialysis system such as the &#34;AQUATECH&#34; system (4) where the lactate salt is converted to lactic acid and alkali. The alkali obtained can be recycled to the fermentor for neutralization, and purified concentrated lactic acid can be used for further processing to make polymers, specialty chemicals or commodity chemical intermediates. 
     
                       TABLE 2______________________________________Comparison of Electrodialysis Runs               Centrifuged and               Filtered Broth               Run I Run II______________________________________Lactate Removal, %    92      92Broth:Initial Lactate Conc., g/l                 81      75Final Lactate Conc., g/l                 9       9Concentrate:Initial Lactate Conc., g/l                 52      72Final Lactate Conc., g/l                 203     196Length of Run, minutes                 150     140Temperature, ° C. (avg.)                 50      50Current Efficiency, % 92      88Initial Current Density, mamp/cm.sup.2                 67      67Flux, gal/hr-ft.sup.2 0.17    0.18Average Water Transfer, ml/Fa                 300     316L-amino acids retained, %(by enzymatic assay)  97      98Electricity Requirement, kwhr/lb                 0.08    0.08______________________________________ 
    
     
                       TABLE 3______________________________________Comparison of Electrodialysis Runs              Whole Broth              Run III                     Run IV______________________________________Lactate Removal, %   82       75Broth:Initial Lactate Conc., g/l                77       76Final Lactate Conc., g/l                18       30Concentrate:Initial Lactate Conc., g/l                65       49Final Lactate Conc., g/l                175      173Length of Run, minutes                140      140Temperature, ° C.                50       45Current Efficiency, %                90       91Initial Current Density, mamp/cm.sup.2                56       56Flux, gal/hr-ft.sup.2                0.17     0.15Average Water Transfer, ml/Fa                350      315L-amino acids retained, %(by enzymatic assay) 97       97Electricity Requirement, kwhr/lb                0.08     0.11______________________________________ 
    
     
                       TABLE 4______________________________________Protein and Amino Acid Transport     Run II     (Centrifuged and                 Run III     Filtered Broth)                 (Whole Broth)     Feed   Product  Feed     Product     Broth  Concen-  Broth    Concen-     (filtered)            trate    (filtered)                              trate______________________________________Volume (1)  10.6     3.75     10.6   3.25Total Amino AcidsConcentration (g/l)       2.93     1.27     4.58   1.47Weight (g)  31.06    4.76     48.55  4.78Rejection (%)        84.7%           90.2%Free Amino AcidsConcentration (g/l)       1.15     0.44     2.41   0.77Weight (g)  12.2     1.65     25.55  2.5Rejection (%)        86.5%           90.2%______________________________________