RAPID PRODUCTION OF BIOLOGICAL PRODUCTS BY FERMENTATION IN A DENSELY-PACKED MICROBIAL MEMBRANE REACTOR

This application is a continuation-in-part of copending ^■ Serial No. 442,036, filed November 16, 1982.

The present invention is directed to a method and means for rapid, efficient, and continuous production of biological products by cultivation of microbial plant or animal cells. In particular, the present invention is directed to a method and means for rapid, efficient and continuous production of lactic acid by fermentation in a denselypacked membrane reactor.

Fermentations employing immobilized viable cells have been described by Abbott, "Immobilized Cells", Annual Reports on Fermentation Processes 1 205223 (1977) ; £, 91128 (1978); and by Messing "Immobilized Microbes",

Annual Reports on Fermentation Processes 4, 190238

(1980) . In a conventional hollow fiber fermentor the cells are gently immobilized around the outside of the fibers in the shell space of the device. The micropo- rous walls of the hollow fiber allow the nutrients and products to diffuse through the walls freely, but prevent penetration by the cells. Experimental work with cells in hollow fiber reactors wherein the orga¬ nisms were in a non-viable, densely-packed cell mass around the fibers have been described by Kan et al., Biotechnol., Bioenq. , 20 217230 (1978), Mattiasson et al., Biotechnology Letters, ' 561566 (1981), Inloes et al._r IEC Winter Symposium, Boulder, Colorado, January 1982.

Applicants have disclosed a batch recycle mode of a hollow fiber fermentor in Biotechnology Letters, 4_, ^*483 (1982) and the use of such reactors for producing ethanol using . cerevisiae ATCC 4126 in Lawrence Berkeley Laboratory report No. LBL12602 entitled "Bioconversion of Cellulose", March 1981. Therefore, it is an object of the present invention to provide novel and improved methods in reactors for growing single microbial, plant or animal cέlls, particularly microor¬ ganisms, such as bacteria and yeast, to provide for rapid economic and efficient production of microbiologi¬ cal products, particularly lactic acid.

The present invention provides a method and apparatus for continuous, cultivation of cells to produce micro¬ biological products employing a reactor comprising at least two microporous-walled hollow fibers. Each of the fibers has an open port at one end and a sealed port at the other end and the microporous wall of each fiber defines a cellfree zone in the interior of the fiber and forms a microporous barrier permeable to cell nutrient- containing and cell product-containing medium but impermeable to the cells located in the shell space of the reactor.

According to the present invention, the reactor may be operated by continuously introducing through the open

OMPI port into the cellfree zone of one of the fibers at a pressure greater than the pressure of the nutrient- containing medium within the shell space of the reactor, a nutrient medium stream, whereby the nutrient medium enters the cell-containing shell space by flowing through the walls of the fiber. In the shell space the cells metabolize the nutrients to produce a prod¬ uct-containing spent-nutrient medium. The mixture of product-containing and nutrient-containing medium is continuously withdrawn through the open port from the cell free zone of the second fiber. According to the present invention the mass transfer of the nutrients from the surface of the first fiber throughout the shell space and the mass transfer of the products from the cells located throughout the shell space into the second fiber is enhanced by convective currents, which result in the cells proliferating in the shell space to a cell density of about 50 gram cells DW/L up to about 600 gram cells DW/L.

The reactor will normally accommodate a plurality of each of the types of fibers, i.e., one for the nutrient stream and one for the product stream, which are mounted in a closed housing provided with means for introduction of the nutrient stream under pressure and means for removal of the fluid within the reactor.

A preferred embodiment of the invention is for the production of lactic acid from the lactic acid bacteria L. delbreuckii (NRRLB445) .

In initiating the method according to the present invention, the cells are inoculated into the space surrounding the hollow fibers, while the nutrient stream is directed through the entry ports of the nutrient

O PI IPO stream-accommodating hollow fibers. The nutrients and substrates pass through the pores of the hollow fiber walls into the surrounding volume in the shell space. The nutrient stream is introduced at a pressure greater than the pressure within the reactor therefore there is an qutward flow of nutrient stream along the length of the fibers. The product-containing stream is withdrawn through one or a plurality of hollow fibers at the exit ports thereof. The withdrawal of fluid from the lumen of the exit (product stream-containing) fibers causes- an inward flow towards the surfaces of the exit fiberss Thus the outward flow from the entrance (nutrient stream-containing) fibers and the inward flow to the exit fibers, and the juxtaposition of the entrance and exit fibers to each other within the cell reactor causes convective currents which allow a flow of nutrients to the cells and flow of product away from the cells such that the entire shell space may be utilized and occupied by cells so that the reactor may be operated in a more efficient manner.

The microporous hollow fibers which are employed accord¬ ing to the present invention may be anisotropic or isotropic hollow fibers having a uniform tight mesh structure throughout the entire membrane. A preferred class of hollow fibers may be constructed of polymeric materials which may be hydrophobic, hydrophilic, posi¬ tively or negatively charged, neutral or combinations thereof. The hollow fibers may also comprise ceramic or metallic materials. Generally the outside diameter of such fibers may be at least 75 microns, preferably about 150 microns, and usually less than 1500 microns. The inside diameter of such fibers is preferably at least 25% of the total diameter, however, the range of this dimension may be as high as approximately 90% of the

OMPI total diameter. The wall thickness of hollow fibers may be greater than 15 microns and less than 500 microns. Examples of such fibers include type X10 Celgard* microporous polypropylene hollow fibers produced by Celanese Company. According to the present invention, one, end of each fiber will be sealed by any convenient means, such as, by a thermosetting glue. In usual applications, the microbiological reactor will contain a plurality of entrance fibers and exit fibers in each fiber module. Usually between about 50 to 600 fibers will be utilized in each module, preferably, half of the fibers used for the nutrient stream and the other half used for the product stream.

A wide variety of cells, such as bacteria, yeast, fungi, and mammalian and plant cells may be utilized in accor¬ dance with the present invention. These cells may be naturally-occurring strains or lines, or strains or lines modified by conjugation or by other known genetic engineering techniques. The specific nutrient media and growth environments used in the reactor will depend upon the particular type of cell being cultured. In this respect, the choice of growth environment may be readily determined by those of ordinary skill in the art of growing a particular cell type or microorganism.

The biological products obtainable from the practice of the present invention include natural products such as excreted or nonexcreted proteins. Examples include enzymes, polypeptides, hormones, lymphokines, antibiot¬ ics, toxins, immunoglobluins, amino acids, organic acids, alcohols, ketones, aldehydes, and the like.

A particularly preferred embodiment of the present invention is directed to the use of the homofermentative

OMPI lactic acid bacteria L. delbreuckii (NRRLB445) to produce lactic acid. According to the present invention, high production rates of lactic acid by this bacterium may be attained. The cell mass density of L. delbreuckii attainable may approach 400 to 600 grams per liter.

A further advantage of the present invention is that a reduction in the yield of cell mass is obtainable which is beneficial to attaining maximum product yield based on substrate consumed. This is desirable since within the confined volume of a microbiological reactor, there may be problems resulting from excessive cell growth, since continued cell division increases the pressure to the point where the fibers may collapse. The reduction in the yield of cell mass may occur naturally due to by-product inhibition or cell lysis, or may be further reduced by selectively depriving the culture of growth related nutrients, or by placing the cells in a stress¬ ful environment, such as high temperature, low pH or utilization of a growth inhibitor.

FIGURE 1 is an illustration of a microbiological reactor useful in accordance with the present invention. The reactor comprises a housing 10 accommodated with a main nutrient inlet port 11 and main product exit port 12. The reactor accommodates a fiber module comprising a plurality of fibers for nutrient stream 13 and fibers for product stream 14 for each of the fibers 13 and 14 there is one open end and one sealed end which may be sealed by sealing means 15, which may be conveniently thermosetting glue. The fibers 13 and 14 are embedded in module ends 16. The module comprising 14, 15 and 16 may be replaced as a unit in the reactor. The module is held in place within the reactor by Oring seals 17 and reactor caps 18. The cells may be introduced into the shell space 19 of the reactor through inoculation ports 20. The cells 21 will proliferate within the shell space of the reactor, occupying essentially all available shell space. In the operation of the microbiological reactor, nutrient stream is introduced through main reactor port 11 under pressure and the nutrient stream enters each of the nutrient fibers 13. The nutrient flows through the fiber walls along the length of fibers 13 into the shell space. The cells 21 proliferate to attain a high cell density within the shell space 19 and products are convectively flowed towards the product fibers 14 and withdrawn through main exit port 12.

Referring to FIGURE 2 there is shown a diagram of an apparatus used for the continuous production of biologi¬ cal products according to the present invention. As shown, the apparatus utilizes a single microbiological reactor 30 similar to that described above in connection with FIGURE 1. The nutrient reserve is maintained within tank 31 which is maintained under a nitrogen bubbler 32 and a vent 33 accommodating a 34. Nutrient flows via lines 35 and 36 through pump 37, prefilter 38 into reactor 30. The product stream which exits biologi¬ cal reactor 30 may be monitored by monitoring means 39 which monitors any convenient parameter utilized for analyzing the quality of the product stream, such a pH. The flow of product stream is also monitored by flow meter 40 as it flows into product collecting tank 41 accommodating vent 42. As shown, the prefilter 38 reactor 30 and analyzing means 39 may be maintained within an incubator, indicated by 43 to maintain the optimum temperature for the growth conditions within the reactor. Typical operation conditions for continuous production of biological products may be performed utilizing the apparatus described above in connection with FIGURES 1 and 2 utilizing a hollow fiber module consisting of Amicon Vitafiber* shell (9.5 cm x 0.65 cm diameter) and Celanese Celgard* microporous polypropylene hollow fibers (type X10, 100 micron id, 150' micron od) . A typical hollow fiber module may consist of 108, 216, 300 or 408 fibers. When utilizing bacteria L. delbreuckii (NRRLB445) the fermentor may be typically maintained at 45°C in a constant temperature incubator. The medium is prefiltered to remove ^'all particles which might obstruct the fibers.

A typical nutrient medium for L. delbreuckii may consist of the following:

TABLE A

glucose 5

Yeast extract (Difco Bacto) 3

MgS0₄ 7H₂0 0.6

MnSO₄ H₂O 0.03

FeS0₄ 7H₂0 0.03

Succinic Acid 11.8

NaOH 7.3

K₂H P0₄ 0.2

KH₂ PO₄ 0.2

In a typical operation the hollow fiber fermentor may be sterilized by ethylene oxide and wetted with a sterile solution of 50% v/v ethanol prior to use. The fermentor may be inoculated by injecting a growing cell suspension into the shell space through one of the inoculation ports. For L. delbreuckii, the feed pH will typically be about 6.0, and the pH of the exit stream continuously monitored. A typical medium flow rate will be in the range from about 10 to 10 to 220 mis. per hour. Glucose content of the streams may be determined enzymatically, for example by glucose oxidaseperoxidase methods using an Instrumentation Laboratories model 919 gluoose analyzer. The L (+) and D . (-) lactic acid may be determined enzymatically according to the procedure described in the Sigma Chemical Company Technical Bulletin (726 UV/826 UV) .

In a typical run of the apparatus described in con¬ nection with FIGURES 1 and 2 above, utilizing fiber modules containing 108, 216, 300, and 408 fibers respec- , tively, cell densities at least as high as 200 gm cell DW/L, as shown below, are expected.

TABLE B

CONTINUOUS CULTURE

NUMBERS OF FIBERS 108 216 300 408

Fiber Length (cm) 5.0±.2 7.1±.3 6.6±. 6 6.6+.:

2 Surf >ace Area (cm ) 28 72 93 127

Shell Side Volume (cm³) 2.9 2.8 2.9 2.5

Max.Lactic Acid Prod. Rate (gm/L-h) based on shell vol. 29 73 77 97 based on reactor vol. (3.6 cm ) 23 57 62 67

Specific Productivity* gm Lactic acid gm-DW-h .14 .13 .24 .22

Final Cell Density based on shell vol. 204 580 325 450 γ_IELDS ?n DW cells .96±. .12 .96i :.34 . 88±.08 gm glucose q DW cells* .03 .08 .03 .09 gm glucose

Flow rate (cm^3/h) 50 120-190 150 220 deviation of run (h) 480 145 340 80

^♦based on final cell mass

OMPI