WO1985001064A1

WO1985001064A1 - Continuous fermentation process

Info

Publication number: WO1985001064A1
Application number: PCT/US1984/001345
Authority: WO
Inventors: Munir Cheryan; Mohamed Abd El-Fattach Mehaia
Original assignee: Munir Cheryan; Mehaia Mohamed Abd El Fattach
Priority date: 1983-08-25
Filing date: 1984-08-17
Publication date: 1985-03-14
Also published as: EP0154647A4; EP0154647A1

Abstract

A process for the continuous fermentation of milk or whey products and by-products from which a substantial portion of the high molecular weight components has been removed comprising the steps of: (a) continuously introducing one or more milk or whey products or by-products from which a substantial portion of the high molecular weight components has been removed to an agitated fermentation vessel including one or more microorganisms capable of metabolizing lactose, glucose or galactose to produce one or more metabolic products of low molecular weight and carbon dioxide; (b) continuously passing a portion of the contents of the fermentation vessel through a filtration module to produce a permeate fraction and a retentate fraction, the filtration module including a filtration membrane having a mean pore size sufficiently small to prevent passage of substantial amounts of carbon dioxide through the membrane and having a molecular weight cutoff above the molecular weight of all or some of the one or more metabolic products of low molecular weight; (c) recovering from the permeate fraction all or some of the one or more metabolic products of low molecular weight; and (d) recycling the retentate fraction to the fermentation vessel.

Description

CONTINUOUS FERMENTATION PROCESS

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a continuous fermenta- tion process for converting milk or whey products and by¬ products to useful end products, such as, alcohols and other organic fuels, specialty chemicals, and pharma¬ ceuticals. In particular, the invention relates to the continuous fermentation of milk or whey products and by- products from which a substantial portion of the high molecular weight, components has been removed through the use of synthetic membranes having specific physical and chemical characteristics.

2. Description of the Prior Art Disposal of cheese whey is a major problem in the

United States and around the world. About 42 billion pounds (20 billion kg) are produced in the U.S. alone annually, half of which is discarded. See Baker, R.W.,

11th Annual Meeting of the Whey Products Institute, Chi- cago, IL., April 21-23, 1982. This is not only a serious environmental problem, but it is also an unnecessary waste of excellent quality protein and a potential energy source. A major hindrance to utilization of whey is its high lactose content (compared to its protein). Ultra- filtration is now almost a standard unit operation in many

OMPI progressive dairy plants throughout the world; it permits the separation of the high-quality protein and the pro¬ duction of whey protein concentrates with varying amounts of lactose and protein. However, ultrafiltration of whey, or milk for that matter, generates vast quantities of "permeate", which is the fraction that permeates the membrane. It typically contains about 5-6.5% total sol¬ ids, 90% of which is lactose and the. rest nonprotein nitrogenous material, mineral salts and other small co - ponents that can pass through the pores of the ultra- filtration membrane. In many cases, the amount of per¬ meate generated can equal the amount of whey itself; in fact, it is usually more since diafiϊtration is usually necessary to increase the protein content of the re- tentate. Thus, dairy plants utilizing ultrafiltration', whether for treating milk or whey, are still left with a disposal problem.

The production of alcohol and other materials from milk or whey permeate is an attractive alternative. See Delaney, R.A.M., 1981, Recent developments in the uti¬ lization of whey. Cult. Dairy Prod. J., 16(2):ll-22; Friend, B.A. and Shahani, K.M. , 1979, Whey fermentation, N.Z.J. Dairy Sσi. Technol., 14:143-152; and Sandbach, D.M.L., 1981, Production of potable grade alcohol from whey, Cult. Dairy -Prod. J., 16(4):17-22. Raw material costs usually make up the major portion of the final alcohol cost, and further, it is crucial to obtain a high

_ OMPI _ sAf IPO Λ conversion of raw material (feedstock) to fermentable sugars. See Maiorella, B., Wilke, C.R. and Blanch, H.W. , 1981, Alcohol production and recovery. Adv. Biochem. Engr., 20:43-92. Whey or milk permeate thus has a key advantage over other feed stocks such as cellulosic ma¬ terials in that the carbohydrate is already in a readily fermentable form and, considering the otherwise high cost of disposal, its contribution to the rawmaterial cost is negligible and may even be negative. No further pretreat- ment is necessary, not even sterilization since permeate from an ultrafiltration plant is, at least theoretically, sterile.

Conventional fermentation technology, however, is highly inefficient, owing to the high volume requirements for^' conventional batch fermentors which result in high financing and depreciation costs. In addition, the con¬ tinual start up/shut down nature of the batch process makes it difficult to automate and results in high labor costs and inconsistency of products. Improving pro- ductivity of fermentors requires the development of "high-rate" processes, which essentially means (a) main¬ taining a very high microbial cell concentration in the fermentor at all times, (b) maximizing the dilution rate, or minimizing the residence time, in the fermentor, and (c) rapid and continuous removal of inhibitory end-prod¬ ucts. _t

Continuous processes are inherently more efficient but the classic "continuous culture" technique is more suited to production of microorganisms rather than con¬ tinuous production of the products of metabolism. It also suffers from the disadvantage of "washout" which limits the dilution (i.e., production) rate. Immobilization of cells on solid matrices eliminates theproblem of washout, but there is the added step of immobilization. In addi¬ tion, high pressure drops in packed bed reactors and gas hold-up have been reported to be potential problems with immobilized cell systems.

OMPI SUMMARY OF THE INVENTION In view of the present state of the art regarding the utilization of milk or whey products and by-products, it is an object of the present invention to provide a process for converting these materials into various use¬ ful end products, such as, alcohols and other organic fuels, specialty chemicals and pharmaceuticals.

More particularly, it is an object of this inven¬ tion to produce such end products by continuously fer- menting milk or whey products and by-products.

It is an additional object of the invention to provide a process for the continuous fermentation of milk or whey products and by-products, which process does not suffer from the various disadvantages, described above, of continuous culture and immobilization processes.

It is a further object of the invention to provide a fermentation process wherein the fermentation micro¬ organisms are recycled and the products removed in such a way that the process can use milk or whey products and by- products as source materials and still operate for long periods of time at high dilution rates and productivities. To achieve these and other objects, the invention provides a process for the continuous fermentation of milk or whey products and by-products from which a substantial portion of the high molecular weight components has been removed comprising the steps of: (a) continuously intro¬ ducing one or more milk or whey products or by-products

OMPI WIPO ^ :;. from which a substantial portion of the high molecular weight components has been removed to ah agitated fer¬ mentation vessel including one or more microorganisms capable of metabolizing lactose, glucose or galactose to produce one or more metabolic products of low molecular weight and carbon dioxide; (b) continuously passing a portion of the contents of the fermentation vessel through a filtration module to produce a permeate fraction and a retentate fraction, the filtration module including a filtration membrane having a mean pore size sufficiently small to prevent passage of substantial amounts of carbon dioxide through the membrane and having a molecular weight cutoff above the molecular weight of all or some of the one or more metabolic products of low molecular weight; (c) recovering from the permeate fraction all or some of the one or more metabolic products of low molecular weight;- and (d) recycling the retentate fraction to the fermenta¬ tion vessel.

In certain preferred embodiments of the invention, the feed stock for the process is milk or whey permeate obtained from the ultrafiltration ,of milk or whey, the fermentation microorganism is Kluyveromyces fragilis and the end product is ethanol.

As used herein the expression "milk or whey prod- ucts or by-products" is intended to encompass all types of products derivable from milk or whey, including, but not limited to, whole milk and whole whey, fractions of whole milk and whole whey, however obtained, and whole milk and whole whey or fractions of whole milk and whole whey which have been subjected to various processing steps now known subsequently developed in the dairy industry.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the overall configuration of the process of the present invention.

Figures 2 and 3 show the kinetics of fermentation of whey permeate by Kluyveromyces^' fragilis using the process of the present invention for cell concentrations (x) of 3.8 and 90 gm dry wt/liter.

Figure 4 shows the long-term stability of the process of the present-.invention for the conversion of whey permeate to ethanol by Kluyveromyces fragilis. The dilution rate employed was 2.4 hr and the initial cell concentration was 40 gm dry wt/liter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As known in the art, typical commercial fermenta¬ tion processes generally include five major steps:

• (1) collection and hauling of raw materials to the fermentation site;

(2) pretreatment of the raw materials;

(3) hydrolysis of the pretreated raw materials to readily fermentable sugars;

(4) conversion of the sugars to fuels, chemicals or other products by fermentation; and

(5) separation of the products from the fermentor broth, generally be distillation.

The above general scheme applies to any fermenta¬ tion process. The relative importance or need of. the individual steps will differ depending on the rawmaterial and final product. This invention relates specifically to improving the productivity of fermentation step (4).

The overall configuration of the process of the present invention is shown in Figure 1. Feed or substrate is pumped continuously by pump 2 into a suitable reaction vessel 4 that acts as the fermentor, to which has been previously added an inoculum of actively growing culture. The fermentor is connected via a suitable pump 6 to an appropriate membrane module 8 in a semi-closed loop con- figuration. -In operation, the contents of the fermentor are pumped continuously through the membrane module and

recycled back to the fermentor. The products of fer¬ mentation are continuously withdrawn as permeate or ul- t afiltrate. The rate of feed flow into the fermentor is matched exactly to the permeate outflow rate (flux) to ^' keep the total reaction or fermentation volume constant. The parameters affecting the productivity and long-term operation of the process of the present inven¬ tion are as follows: Feed or Substrate As described above, the feed or substrate for the process of the present invention is milk or whey products and by-products from which a substantial portion of the

. high molecular weight components has been removed. The high molecular weight components of milk and whey products and by-products, if notpreviously removed, can be removed by a variety of method^'s, known in the art, including ultrafiltration, salt precipitation, heat denaturation, and the like. The high moiecular weight components must be removed from the feed stock since they will not pass through the membranes of membrane module 8 and, in gen¬ eral, are not metabolized by the fermentation culture. Accordingly, as more and more feed stock is added to fermentor 4, the high molecular weight components will accumulate in the fermentor and eventually bring the fermentation process to a halt. Particularly preferred substrates for the process of the present invention are milk and whey permeates obtained from the ultrafiltration of milk or whey. These substrates already have the high molecular weight com- ponents removed and thus, in many cases, can be used directly in the fermentation process without pretreat- ment. Also, as discussed above, these materials are basically waste products of the dairy industry and thus are available in large quantities at essentially no, or at most a nominal, cost.

Some microorganisms, e.g., Kluyveromyces fragi¬ lis, are capable of transforming lactose directly into useful end products, e.g., ethanol. For these organisms,, pre-hydrolysis of the lactose to glucose and galactose is not required and the whey or milk product or by-product can be used directly as the fermentation substrate. For other microorganisms, such as Saccharomyces cerevisiae, how¬ ever, it will be necessary to hydrolyze the lactose to glucose and galactose. The hydrolysis can be performed by various techniques known in the art, such as, by chemical or enzymatic hydrolysis.

In some cases, it may be necessary to adjust the concentration of the whey or milk products and by-products to the optimum for fermentation by a particular micro- organism. For example, the substrate can be diluted if necessary by adding water, or concentrated if necessary by evaporation or ultrafiltration or reverse osmosis or any such suitable method. In general, the feed is preferably sterilized prior to pumping into the fermentor. This can be done either by conventional heat sterilization or by the rela¬ tively newer technique of membrane filtration known as cold sterilization using microfilters with a pore size of 0.45 for yeasts and 0.22 for bacteria. In many cases, the milk or whey product or by-product used as the feed source for the fermentation will already be sterile when supplied, and thus, additional sterilization prior to fermentation will not be required. Microorganisms and Products

The choice of microorganism for use in the fer¬ mentation process depends upon the product to.be produced, the viability of the organismunder-the process conditions and the ability of the organism to metabolize the sugar component (lactose or glucose and galactose) of the milk or whey product or by-product. Various microbes can be used. For example, for the production of ethanol from unmodified cheese or milk whey, the preferred microbe is Kluyveromyces fragilis. If the lactose in cheese whey is hydrolyzed to its constituent monosaccharides, as for example, by a suitable enzyme or by acid, microbes such as Saccharomyces cerevisiae or Zymomonas mobilis can be used. Other products that can be produced with this invention include fuels and organic solvents such as acetone, butanol, 2, 3-butylene glycol, lactic acid and other fermentation products, in particular those products that are produced by heterofermentative microbes, i.e., those products where gas is bi-product of the reaction. Such microbes include those of the genus Clospridium Bacillus, Lactobacillus, Aerobactor and the like.

It is to be understood that the foregoing list of products and microorganisms is not to be considered re¬ strictive of the present-invention. Rather, the invention encompasses the use of any microorganism which can metab¬ olize lactose, glucose or galactose to produce useful low molecular weight products and carbon dioxide. Fermentor

The fermentor is basically a suitable vessel op- erated, most preferably, as a "continuous stirred tank reactor" (CSTR) . Constructions for such vessels are well known in the art. In general, the vessel should have the following characteristics and controls, all of which are well known and generally available in the art: (a) an internal volume large enough to accom¬ modate the required initial charge of feed and inoculum of microorganisms, plus all the controls and accessories as described here¬ inafter; (b) means for agitation, such as a stirrer, de¬ signed to efficiently and thoroughly mix the ■ fermentor contents;

OMPI

^?^ o§ (c) means for temperature measurement and con¬ trol;

(d) means for pH measurement and control;

(e) means for introducing sterile air or nitrogen into the vessel and its contents, and means for measuring volumetric flow of said gases;

(f) means for removal of air, nitrogen and gas¬ eous products of fermentation;

(g) entry ports" for feed substrate and recycled fermentor contents from the membrane module, and an exit port to the membrane module and pump. Membrane Module and Pump

The parameters of the membrane module, in par- ticular, its mean pore size, are critical to the success of the present invention. Also, the rate at which the contents of the fermentor are pumped through the membrane module play an important role in the. long-term stability of the process. The membrane module should meet the following criteria:

(1) It must be of the cross-flow or tangential- flow type, with one entry for the feed and two outlets (one for the retentate and one for the permeate) . This is as opposed to the "dead¬ end" membrane module construction with only one inlet (for the feed) and one exit (for the permeate) which affords almost no polari¬ zation control and consequently will rapidly plug up. (2) The membrane module should be constructed of materials that can withstand the chemicals in the system, such as ethanol or butanol or other products, depending on the fermenta- tion. Membranes made of polysulfone, poly- tetrafluoroethylene and the like are suit¬ able. (3) The pore size of the membrane should be such that it will permit the passage of the prod¬ ucts of fermentation, but not the microorgan- isms, and, most importantly, not the gas produced during the fermentations. A by¬ product of almost all fermentations is the production of gas such as carbon dioxide. Indeed, in a typical sugar to ethanol fer- mentation, one mole of sugar is converted to

2 moles of ethanol and 2 moles of carbon dioxide. This carbon dioxide gas will escape from the system either through a gas exit port in the fermentor, or through the mem- brane into the permeate outlet, if the pore

_. OMPI ^ ^~ WIPO^~ A>j size of the membrane is large enough and the surface tension of the recycled retentate low enough. If gas does escape through the membrane, it is at the expense of product. Indeed, if the pore size is large enough essentially all that will be found in the permeate is gas.

For example, a cartridge manufactured by Gelman Sciences, Ann Arbor, MI and sold under the trademark "Acroflux" has been tested in the process of Figure 1. This cartridge has a 0.2 mean pore size and a rated bubble point pressure of 30-35 psi with water. When used in the process of the present invention, even pressures as low as 4-6 psi, depending on cell and ethanol concentration in the system, were found to result in substantial gas escape through the membrane, at the expense of liquid product. For comparison, hollow fiber membranes marketed by Amicon Corporation and Ro icon Corporation, both of Mas¬ sachusetts, USA, were also used in the present process. These fibers have much smaller pore sizes, on the order of 10 to 100 angstroms, with correspondingly higher bubble point pressures. Using these membranes, gas escape through the membrane module was much less, negligible in most cases, and gas escape from the system occured through the fermentor exit ports. In general, it has. been found preferable to use membranes having mean pore sizes below approximately 100 angstroms and most preferably below about 50 angstroms to avoid the problems of gas escape through the membrane module.

In addition to having a small pore size, it has been found preferable that the membrane module be of the straight through type, with no spacers or wire mesh or other insertions in the feed channel that could cause a "hang-up" or physical blockage of the feed channel at high cell concentrations. Examples of such straight through type modules are hollow fibers and other unobstructed straight tube modules.

In addition to selecting the proper module, it is important to operate the module to avoid "concentration -polarization", that is, the accumulation of retained solutes or particles on the membrane due to convective transport through the membrane. As a result of this effect, a substantial quantity of microbial cells may be adsorbed or "immobilized" on the membrane or trapped within the membrane module itself. For example, when using the Gelman "Acroflux" membrane cartridge, described above, it was determined that with an.initial cell concen¬ tration of 30 gm/liter, after a few hours of operation about 8 grams (dry weight) of yeast were "immobilized" in the module, which had 1000 sq. cms. of membrane area. To avoid concentration polarization, it has been found preferable to pump the contents of the fermentor through the membrane module at high flowrates. Flow rates

' lREA ζ^ OMPI . , ^ . IPO ^j as -high as 70 liters/minute can be used, if necessary, with currently available membranes to counteract the effects of fouling due to concentration polarization. These high flow rates also remove the products of reaction from the system as quickly as possible and thus minimize product inhibition. In conventional batch processes, product inhibition is a major reason for low productivity. Thus recycle flow rates should be kept as high as possible consistent with operating limits of the module itself. For similar reasons, permeate flux should be kept as high as possible consistent with the microbial growth kinetics and conversion efficiencies.

In addition to the foregoing, it has been found desirable to use as high a surface area to volume ratio for the membrane module as possible, so as to maximize fer¬ mentation in the reaction vessel, and minimize it within the membrane module itself. This approach leads to easier and better optimization of the overall system. Bleed Circuit In some fermentations, the desired products of fermentation are "growth-associated", i.e., the micro¬ organisms produce the product only while growing and reproducing. Since fresh feed substrate is being con¬ tinuously pumped into the system and since for these fermentations growth and multiplication of the cells is necessary for continued operation, the microbial cell concentration continually, increases. In general, high cell concentrations are desirable because they lead to high productivity. However, very high cell concentra¬ tions can result in fairly severe polarization problems, even at high flow rates. Accordingly a balance must be maintained between cell concentration and membrane foul¬ ing problems. For growth-associated fermentations it thus is desirable to incorporate a "bleed circuit" into the recycle loop as shown in Figure 1, to selectively bleed out a small portion of the recycled fermentor contents to keep the cell concentration in the system within manage¬ able limits. The bleed liquid, containing microbial cells and product, can be sent to a second membrane module 10, such as a microfilter operated as a dead-end system, to separate the product from the microbial cells, which can then be dried and sold as animal feed. Although shown as part of the return path from module 8, it is to be understood that the bleed circuit can be directly con¬ nected to fermentor 4. Indeed, for fermentations where dead cells and other debris collect at certain locations in the fermentor, e.g., at the top^*, such locations are preferred for attachment of the bleed circuit.

^ Λ EXAMPLES

Without intending to limit it in any manner, the present invention will be more fully described by the following examples which illustrate the continuous fer- mentation of whey permeate by Kluyveromyces fragilis to produce ethanol.

Materials and Methods

Kluyveromyces fragilis NRRL Y-2415 was obtained from the Northern Regional Research Laboratory, USDA, Peoria, Illinois. The culture was maintained on a lac- tose-agar slant culture containing 50 g lactose, 5 g peptone, 3 g malt extract, 20 g agar and 3 g yeast extract in 1 liter of water. All ingredients were obtained from

Difco Laboratories, Detroit, MI. For the experiments described in this paper, the cells were grown aerobically in MYLP broth in shake culture flasks set in a rotary shaker at 28-30 C. After about 20 h the culture was centrifuged aseptically at 1500 G. The yeast cells were added to the fermentor in a paste form. Whey permeate was obtained by ultrafiltration of cottage cheese whey. Whole whey was obtained from a local cottage cheese plant and pasteurized immediately upon receipt at 63^*C for 30 min. The whey was clarified with a screen filter to remove particles larger than 100

^V microns before being processed through a Romicon hollow fiber ultrafiltration unit equipped with a HF15-43-PM50 module (Romicon, Inc., Woburn, MA). This module had a surface area of 1.39 square meters and a nominal molecular weight cut-off of 50,000. The permeate typically con¬ tained a 5.9% w/v total solids, 0.04% w/v total nitrogen (essentially all of it soluble in 12% TCA indicating it was all nonprotein nitrogen), 4.5% w/v lactose and 0.68% w/v ash. This permeate was used with no further treatment as the feed to the fermentors.

Control experiments were also performed with a synthetic medium consisting of (per liter) : 50-100 g lactose (depending on the concentration of lactose needed), 5 g peptone, 3 g yeast extract and 3 g malt extract. This medium was sterilized using a 0.2- micron membrane microfilter (the "Acroflux" capsule manufactured by Gelman Sciences, Ann Arbor, MI.)

The fermentation was conducted in accordance with the process shown in Figure 1 and described above. Spec- ifically, a conventional batch fermentation vessel, op¬ erated as a "continuous stirred tank reactor", was coupled to a membrane module via a suitable pump in a semi closed- loop configuration. During operation, the entire fer¬ mentor vessel contents were pumped through the membrane module. The permeate from this module contained the

- s E , OMPI products of fermentation (ethanol) as well as other feed components that can permeate the membrane, while the retentate, containing the yeast cells, was recycled back to the fermentation vessel. To maintain a continuous operation, the feed (whey permeate or lactose medium) was continuously pumped into the fermentor vessel at the same rate as the permeate flux, thus keeping the total volume of the system constant.

The membrane module used in the fermentation proc- ess was a "short-short" hollow fiber cartridge containing 0.7 square meters of PM-50 fibers (Romicon, Inc., Woburn, MA) . The fibers in this module are unobstructed straight tubes composed of polysulfone, and have a mean pore size and molecular weight cutoff of approximately 30 angstroms and 50,000 daltons, respectively. In^' order to prevent fouling of the membranes in this filter, the contents of the fermentor- vessel were pumped through the filter at rates higher than approximately 10 liters/minute. Total fermentation volumes were typically 0.5-1.0 L. The pH during fermentation was not controlled and was usually 3.9-4.5. The temperature was maintained at 30^*C. Nitro¬ gen was passed through the fermentation vessel to_.maintain anaerobic conditions. Samples were taken aseptically from the fermentation vessel periodically (to measure cell concentration) and from the permeate (to measure ethanol and lactose concentrations). Cell concentrations are expressed herein as yeast cell dry weight per unit volume (g/L). Cell concen¬ trations were measured optically at a wavelength of 525 nm. Cell dry weights were obtained by drying washed cells at 105^*C. Lactose concentration was determined by the meth¬ ods of Nickerson et al and Summer and Somero. See Nickerson, T.A., Vujicic, I.F. and Lin, A.Y., 1976, Col- orimetric estimation of lactose and its hydrolytic prod¬ ucts, J. Dairy Sci., 59:386-390; and Summer, J.B. and Somero, G.F., 1949, Lab Expts. in Biol. Chem. , Academic Press, New York. Ethanol concentration was measured by the alcohol dehydrogenase method as described in Sigma Technical Bulletin No. 332-UV (Sigma Chemical Company) or by gas chromatography (Hewlett-Packard, model 5710A) using a stainless steel column (6' x 1/8") connected to a glass (6" x ϊ/4") pre-colu n packed with 1-1766, 60/80 Carbopack B/5% Carbowax 20M (Supelco) . The temperature of the injector was 100'C and of the detector 200^*C. Nitro¬ gen, at a flow rate of 20 mL/min, was used as the carrier gas and the column oven was operated isothermally at 85"C. Ethanol was measured from a calibration curve using iso- propanol as an internal standard. Both methods gave essentially the same results. Total solids was measured by a gravimetric procedure and nitrogen was measured using the Kjeldahl method. See Association of Official Ana- lytical Chemists, 1980, Official methods of analysis, 13th edition, Assoc. Off. Anal. Chem. , Washington, DC.

Results Wheypermeate was found to be a suitable medium for fermentation by Kluyveromyces fragilis; no supplementa¬ tion with nutrients was necessary for alcohol production, in agreement with Castillo et al. and Mahmoud and Kosikow- ski. See Castillo, F'.J., Izaguirre, M.E., Michelena, V., and Moreno, B., 1982, Optimization of fermentation con- ditions from ethanol production from whey, Biotechnol. Lett., 4:567-72; and Mahmoud, M.M. and Kosikowski, F.V., 1982, Alcohol and single-cell protein production by Kluy¬ veromyces in concentrated whey permeates with reduced ash, J. Dairy Sci., 65:2082-2087. The theoretical con- version of lactose to ethanol is 0.54 g of ethanol per g of lactose consumed. Batch fermentation studies using whey permeate containing 45 g/L lactose resulted in 0.4- 0.45 g ethanol/g lactose, i.e., about 75-83% of the¬ oretical. Using the synthetic lactose medium described above resulted in slightly higher yields of 85-90%. Pro¬ ductivity, defined as the amount of ethanol per unit volume per unit time, was typically about 1.9 - 5 g ethanol/L/h for the batch system, depending on the initial concentration of yeast cells charged to the fermentor. These figures are in general agreement with published literature on whey and whey permeate fermentations. See Castillo, F.J., Izaguirre, M.E., Michelena, V., and Moreno, B., 1982, Optimization of fermentation conditions from ethanol production from whey, Biotechnol. Lett., 4:567-72; Mahmoud, M.M. and Kosikowski, F.V., 1982, Al¬ cohol and single-cell.protein production by Kluyveromyces in concentrated whey permeates with reduced ash, J. Dairy Sci., 65:2082-2087; Moulin, G., Guillaume, M., and Galzy, P., 1980,Alcohol production -by yeast in whey ultrafil- trate, Biotechnol. Bioeng. , 22:1277-1281; and Rajagoplan, K. .and Kosikowski, F.V., 1982, Alcohol from membrane processed concentrated cheese whey, I&EC Product Research & Dev. , 21:82-87.

In a typical experiment, the fermentation vessel

» was filled with whey permeate to the required volume, the yeast cells added to give the final required concen¬ tration, and the feed pump (maintaining a constant flow rate of whey permeate into the system) and ultrafiltration pump turned on simultaneously. Transmembrane pressure and recycling rate were set depending on the dilution rate needed, but in all cases the flow through the membrane module was kept above about 10 liters/minute to avoid membrane fouling. The system was considered to have reached "steady state" after 5 fermentor volumes had passed through the system, which is adequate considering this system was operated as a "continuous stirred tank reactor". The PM-50 membrane was very effective in retaining all the yeast cells in the system; the permeate from the module was clear and contained ethanol, unhydrolysed lactose and other minor components from the whey that were permeable (not analyzed). Due to the lowpore size, very little gas (carbon -dioxide) generated by the yeast or nitrogen (passed through he system to maintain anaerobic conditions) passed through the membrane into the per¬ meate, but instead -.escaped through vents in the fer- mentation vessel.

Figure 2 shows results obtained with the process using a low initial cell concentration of 3.8 g(dry weight)/L. This concentration is equivalent to about q 2x10 cells/milliliter. . The data for continuous systems - is usually plotted in terms of "Dilution Rate" (D), which is the flow rate (L/h) divided by the fermentation volume

(L). Thus higher dilution rates imply higher production rates or smaller fermentor volumes, both of which are desirable. Productivity (PD) of a continuous system is then simply dilution rate (D) x product concentration in thepermeate outlet (P) . As shown in Figure 2, as dilution rate is increased, the concentration of ethanol de¬ creases. Essentially 100% of the lactose was fermented up to a dilution rate of 1.5/h. Productivity, however, increases from 12 to a maximum of 32 g/L/h at a dilution rate of 2.5/h, but would probably decrease at higher dilution rates. This productivity is 5-10 times higher than the productivities, described above, for batch fer¬ mentations of whey permeate performed under otherwise identical conditions. Figure 3 shows data obtained with the process operated at approximately a twenty-fold higher initial cell concentration (^'90 g/L). Higher dilution rates are possible under these conditions, which resulted in pro¬ ductivities of 54-67 g/L/h at dilution rates of 5-13 h The significance of these figures must be stressed. These figures represent productivities 2500- 3000% higher than comparable batch processes, at resi¬ dence times of the order of 6-30 minutes, instead of the hours or days that it would take for conventional batch operations.

The data shown in Figures 2 and 3 represent a pseudo-steady-state, i.e., after at least 5 fermentor volumes had passed through the system.. It is not a true steady state since the ethanol fermentation process is a "Gaden Type-I" fermentation, i.e., it is growth-asso¬ ciated and thus the cells must be growing and reproducing for ethanol to be produced. Hence cell concentration in the system is continuously increasing, which must be taken into account during long-term operation. Figure 4 shows a continuous, nonstop run with the process operated at a dilution rate of 2.4 h and an initial cell concentration of 40 g/L. It took about 5 hours to reach a steady operation as far as lactose consumption and ethanol output were concerned, but as•can be seen in the figure, the cell concentration continuously kept increasing. The system was therefore bled inter¬ mittently to maintain the cell concentration in the range of 40 g/L. As the cell concentration data in Figure 4 shows, the concentration ranged from 40 - 60 g/L and^' averaged about 50 g/L. Under these conditions, essentially no lactose appeared in the permeate and the ethanol concentration averaged 17.3-19.6 g/L (2.1-2.4% v/v) throughout the run. Also, essentially no gas appeared in the permeate. The productivity was 43-47 g ethanol/L/h and there was little problem in maintaining this level of productivity for more than 10 days.

Figure 4 also shows specific growth rate (μ) calculated from the cell concentration vs time data in the same figure. Specific growth rates for this yeast ranged from 0.06-0.1 h~ throughout the run, which is comparable to the value otained by Rajagopalan and Kosikowski. See Rajagoplan, K. and Kosikowski, F.V., 1982, Alcohol from membrane processed concentrated cheese whey, ISEC Product Research & Dev., 21:82-87. Cell viability, as measured by the methylene blue test, remained greater than 90% throughout the run.

Claims

WHAT IS CLAIMED IS:

1. A process for the continuous fermentation of milk or whey products and by-products from which a sub¬ stantial portion of the high molecular weight components has been removed comprising the steps of: (a) continuously introducing one or more milk or whey products or by-products from which a sub¬ stantial portion of the high molecular weight components has been removed to an agitated fer¬ mentation vessel including one or more microor- ganisms capable of metabolizing lactose, glucose or galactose to produce one or more metabolic products of low molecular weight and carbon di¬ oxide;

(b) continuously passing a portion of the contents of the fermentation vessel through a filtration module to produce a permeate fraction and a retentate fraction, the filtration module including a filtration membrane having a mean pore size sufficiently small to prevent passage of substantial amounts of carbon dioxide through the membrane and having a molecular weight cutoff above the molecular weight of all or some of the one or more metabolic products' of low molecular weight;

T ∑P_O-- -

- (c) recovering from the permeate fraction all or some of the one or more metabolic products of low molecular weight;- and

(d) recycling the retentate fraction to the fermentation vessel.

2. The process of Claim 1 wherein the mean pore size of the filtration membrane is less than approximately 100 angstroms.

3. The process of Claim 2 wherein the mean pore size of the filtration membrane is less than approximately

50 angstroms.

4. The process of Claim 1 wherein the membrane module is composed of unobstructed straight tubes.

5. The process of Claim 1 wherein the contents of the fermentation vessel are passed through the membrane module at a flow rate sufficiently high to prevent sig¬ nificant accumulation of the one or more microorganisms on the membrane surface.

6. The process of Claim 5 wherein the membrane module is composed of unobstructed straight tubes of a diameter of approximately one millimeter and the flow rate is greater than approximately ten liters/minute.

7. The process of Claim 1 wherein the one or more milk or whey products or by-products from which a sub- stanti-al portion of the high molecular weight components has been removed comprises milk or whey permeate obtained from an ultrafiltration process. 31

8. The process of Claim 7 wherein the one or more metabolic products of low molecular weight comprises ethanol.

9. The process of Claim 8 wherein the one or more s microorganisms comprises Kluyveromyces fragilis.

10. The process of Claim 1 including the addi¬ tional step of bleeding off a portion of the contents of the fermentation vessel so as to maintain the concen¬ tration of the one or more microorganisms below a prede- termined level.