Cross-Reference to Related Applications

[0001] This application is a non-provisional of US provisional patent application 62/633,937, filed on 22 February 2018, to which it makes a priority claim.

Technical Field

[0002] The disclosed embodiments of the present invention relate to a reactor system, especially one that is adapted for conducting fermentation reactions, especially aerobic fermentations that would be expected to result in generation of foam, which is collected.

Background

[0003] In many chemical reactions, especially biological reactions, and more particularly in aerobic fermentation reactions, a persistent foam is generated, at least during a portion of the reaction time. This may occur for a variety of reasons, some of which are not fully understood. In some of these reactions, oxygen has to be continuously sparged into the bulk fermentation liquor, either as air or as concentrated oxygen, to satisfy the demand of certain types of aerobic bacteria. In fact, the aeration gas volume introduced to meet the oxygen demand of some of these aerobic bacteria is often sufficiently high that the associated foam causes metabolic shifts and release of products for which the bacteria are not highly suited.

[0004] For terminology clarity, the foaming phenomenon dealt with here typically occurs in a batch-type reaction system. The reactor vessel is typically a closed vessel with at least a majority of the volume occupied by the bulk liquor, where an active agent, particularly a specially-selected bacterial strain, reacts with at least one reactant in the bulk liquor. The reaction products are released into the bulk liquor. In some cases, as in the well-known fermentation of glucose to ethanol by yeast, the produced carbon dioxide forms bubbles that rise to the upper surface of the bulk liquor, where the bubbles will break, releasing the carbon dioxide into a headspace of the reactor. However, in some instances, the interaction of buoyancy and surface tension forces in the bubble at the interface of the bulk liquor and the headspace cause the bubble to not burst, establishing a persistent foam. This persistent foam has a gas volume to liquid volume ratio that is much higher than that found in the bulk liquor. Because of this high ratio of gas to liquid, the liquid phase in the persistent foam is essentially web-like. Left unchecked, this persistent foam can expand to substantially fill the headspace. Bubbles of air (or oxygen) introduced into the bulk liquor can also establish a persistent foam in the same manner as the bubbles of carbon dioxide can in an anaerobic fermentation.

[0005] The resolution of persistent foam by introducing surface active agents at the top of the bulk liquor is well-known, but it is extremely undesirable in bioreactions and fine chemical reactions. The agents can be deleterious to the product and will need to be removed during isolating the products of the reaction.

[0006] In bioreactors of this type, it is also known that the reaction often proceeds from a starting material through a metabolic pathway in which one or more metabolites are produced before the final product, which in many cases is the desired product. However, in certain circumstances, there may be an interest in isolating one or more of these intermediate metabolites, as well as other products that may be released by the active microorganism. It is desirable to facilitate rapid, and at least semiautomatic, monitoring and measuring of these metabolites or other products. In some cases, the desired metabolite may be present during a short stage of the growth cycle of the producing microorganism. This short-lived event may be missed when the analyst uses standard bioreactors or fermenters. The short life of the metabolite produced at a stage of microbial growth may be destroyed by degradative enzymes released at a later stage of the growth of the same microorganism. Therefore, relying on conventional flask or bioreactor fermentation will not allow isolating the short-lived intermediate metabolite. An invention to remove and separate these metabolites not only provides the means to mass-produce novel bioreaction products, it also minimizes end-product feed-back inhibition, thus speeding the metabolic processes leading to these products.

[0007] It is thus an object of the present invention to provide a bioreactor system that will physically deal with persistent foams, determine the presence of short-lived metabolites, and facilitate the isolation and purification of these metabolites. These objectives are not presently met by the known prior art bioreactors. Summary

[0008] This and other unmet advantages are provided by the apparatus and method described and shown in more detail below.

[0009] In one embodiment, the apparatus for producing a target metabolite in a bioreaction, comprises a reactor vessel, a water bath, a foam collector and a reaction control console. The reactor vessel is sized to contain a bulk liquor in which the bioreaction is conducted with a headspace maintained above the bulk liquor. The water bath is arranged for controlling temperature in the reactor vessel, under control of the reaction control console, which is in communication with at least the reactor vessel and the water bath to monitor and control the bioreaction. The foam collector is arranged to receive a foam formed at an interface of the bulk liquor and the headspace and to break the received foam into a condensed foam liquid.

[0010] In some embodiments, the apparatus further comprises a fraction collector unit, arranged for withdrawing and analyzing samples from the reactor vessel; as well as a cooling system, arranged for controlling temperature in the fraction collector.

[0011] In many embodiments, the apparatus further comprises one or more analytical probes, arranged in the reactor vessel, means for agitating the bulk liquor and means for injecting one or more gases into the bulk liquor.

[0012] In at least some embodiments, the method for producing and concentrating a target metabolite from a bioreaction has the steps of: conducting the bioreaction with a selected microorganism in a bulk liquor of a reactor system having a headspace above the bulk liquor, under conditions predetermined to induce a persistent foam at an interface of the bulk liquor and the headspace; determining the presence of the target metabolite in the persistent foam; producing a condensed foam liquid by removing the persistent foam from the bioreactor and breaking the foam; and isolating the target metabolite from the condensed foam liquid.

[0013] In these embodiments, the target metabolite is preferentially partitioned into the persistent foam.

[0014] In one specific example, the target metabolite is paenibacillin or

amyloliquecidin GF610, both are newly-discovered antimicrobial peptides. [0015] The step of producing the condensed foam liquid is preferably achieved in a foam collector.

[0016] The step of determining the presence of the target metabolite is achieved by at least one of: elapsed time of the bioreaction; measurement of at least one operating parameter of the bulk liquor selected from the group consisting of: dissolved oxygen, pH, temperature and turbidity; and a positive reaction to a predetermined bioindicator.

[0017] In many embodiments, the step of producing the condensed foam liquid comprises the sub-step of filtering the condensed foam liquid to remove the

microorganisms.

[0018] In some embodiments, a foam collection device of the type described herein may be efficaciously retrofitted to prior art bioreactors to isolate and concentrate a target moiety generated in a bioreaction.

[0019] In achieving these advantages, the device and method intentionally induces or enhances foaming. By doing this, it has been seen that metabolites, such as, for exemplary reasons only, paenibacillin can be partitioned into the foam and separated therefrom. The separation has heretofore been difficult, due to the scarcity or physico chemical properties of the metabolites. Paenibacillin is a potent antimicrobial peptide which is produced in micromolar quantities that can only be separated and purified from a medium through complex chromatographic techniques. Another example of metabolites that are readily partitioned in the foam is amyloliquecidin GF610, a newly- discovered two-component antimicrobial lantibiotic.

Brief Description of the Drawings

[0020] A better understanding of the disclosed embodiments will be obtained from a reading of the detailed description and the accompanying drawings wherein identical reference numerals refer to identical parts and in which:

FIGURE 1 is a schematic depiction of an embodiment of the bioreactor system incorporating the inventive concept;

FIGURE 2 is a schematic depiction of the control instrumentation of the bioreactor system; FIGURE 3 is a detailed side view of a fraction collector portion of the bioreactor system;

FIGURE 4 is a side view of a bioindicator tube;

FIGURE 5 is a sectional perspective view of a foam collector vessel;

FIGURE 6 is a side elevation view of an analytical probe; and

FIGURE 7 is a graph in which light absorbance at 880 nm is plotted against time for an experiment demonstrating use of the bioreactor system for producing paenibacillin.

Detailed Description of a Preferred Embodiment

[0021] The present invention is premised upon the discovery that, at least in certain selected circumstances, the metabolite products that are desired to be isolated can be partitioned in the liquid portion of a persistent foam. Thus, a bioreactor system that can determine the presence of the desired metabolite and can isolate a persistent foam from the bulk liquor provides capabilities not previously available in the prior art. The determination of the presence of the metabolite is based upon one or more of several grounds, including, but not limited to, rapid bioassay and/or predetermined biokinetics.

[0022] Some of the desired products are beneficial antimicrobial agents which may be produced by a microbial strain during a short stage within the growth cycle of the producing microorganism. The modifications provided by the embodiments of the present invention allow samples to be collected at precise intervals during the

bioreaction cycle (e.g., growth of a microorganism). These samples are then processed in real time, and the presence of any active ingredient is determined by combining the sample with an appropriate bio-indicator. For example, the presence of a new

antimicrobial agent synthesized at, for example, the 8 ^th hour of a 24-hour fermentation is facilitated by collecting a sample around that time, micro-filtering the sample, and mixing the filtered sample with a bacterium that is sensitive to the antimicrobial agent, that is, a bio-indicator. In the preferred embodiments, all of these steps are automated and under control of a processor unit.

[0023] The partition of certain bioactive metabolites in the persistent foam may occur by a preferential production in the foam, by a physical process that favors the presence of the metabolite at the interface between the bulk liquor and the headspace, or by another mechanism that is not fully understood. In any case, the metabolites formed during aerated fermentations (by highly aerobic bacteria) tend to be amphiphilic agents (e.g., antimicrobial peptides). While the prior art, both in theory and practice, encourages steps to avoid foam formation during all types of fermentations, the embodiments taught herein collect the foam and return it safely and aseptically to the fermenting bulk liquor. Furthermore, the embodiments can collect the foam separately and allow the agents present in the foam to be harvested from this more concentrated source. The embodiments of the system disclosed here allow the detection and collection of rare active metabolites that have affinity for gas-aqueous interfaces in fermentations.

[0024] The embodiments of the bioreactor system are preferably provided with an optical probe comprising a precisely-spaced light emitter and detector. While the optical probes of this type are known, they are used in the embodiments of the present invention in a novel manner. The probe is immersed into the bioreactor to sense, in real time, the progression of the biological activity. The probe then sends a signal to a monitor or computer for viewing, storage or processing. The probe signal can be converted into a useful parameter such as optical density. When the bioreactor is used to grow a microorganism, the probe readings correspond to medium turbidity which is a measure of cell density. Other useful probes in this aspect are probes that measure changes in redox potential during bioreaction.

[0025] Fermenters and other bioreaction equipment are often heavily laden with features, many of which are rarely used. Flowever, these systems lack the ability to monitor important secondary metabolites, such as antimicrobial agents, which are often synthesized at certain stages of microbial growth cycle and released from the producer cell in minute amounts. Fermentation processes are often so complex that intricate tasks, as those just described, are difficult to monitor or control. It is also often difficult to get the microorganism to respond in the same way with each fermentation because so many factors affect production of secondary metabolites. Obvious fermentation parameters such as air flow, temperature, and substrate feed are relatively easy to control, but so many other parameters are yet to be discovered and tracked. [0026] As shown in the schematic depictions in FIGURES 1 and 2, a preferred bioreactor system 10 of the inventive concept integrates a number of components into a tower enclosure 20. Physical components in the tower 20 include a heated water bath 22, a refrigerated compartment to contain a fraction collector 23, an air pump 24, a peristaltic pump 25, and a console 30, which is depicted in Fig. 2. The console 30 preferably comprises a programmable logic controller (PLC) 32 with a touch-screen for instruction entry. Apart from the tower enclosure 20 is a reactor assembly 40 with a reactor jar 42, equipped with stirring means, preferably a magnetic drive agitator 44.

The reactor assembly 40 is preferably attached to the tower enclosure 20 through quick-connect fittings.

[0027] The console 30 is the main user interface, and it provides an interface to control bioreaction temperature, air flow, stirring speed and fraction collection. In one embodiment, the control unit 32 uses a Horner XL6 programmable logic controller, commercially available from Horner APG Canada, of Calgary, Alberta. Inputs/outputs on the PLC include two Pulse Width Modulation (PWM) outputs to control the fraction collector motors. Also included are analog inputs for temperature sensors, an RS-232 communication port to communicate with the water bath controller, and digital outputs for miscellaneous controls such as air pump and stirring motor. PLC 32 is connected to various probes, such as thermocouple 100a, pH probe 100b and light absorbance measuring probe 100c, as well as to air pump 24 and magnetic stirrer 44, to monitor and manage the bioreaction.

[0028] Beyond the control functions provided by the console 30, the tower enclosure 20 provides air and heat to the reactor assembly 40. Specifically, the air pump 24 has an air inlet 50, which preferably filters the incoming air, a flow control valve 52 and a flow meter 54. Output air passes via conduit 56 through a top of the reactor jar 42 and is injected below a surface of the bulk liquor 60 in the reactor jar.

[0029] In a similar manner, the water bath 22 is a part of a recirculating water system that supplies heated water to a reactor jacket 46, with the system also containing a water pump 48. Conduits 62 and 64 provide water flow to and from the reactor jacket 46. In many aspects of the invention, the water bath 22 is housed in the tower enclosure. The water bath 22 should include a thermostat, which is provided with a control interface to the PLC 32. In preferred embodiments, only the ports to fill the bath with water and to connect it to the bioreactor for fluid circulation are visible on the tower 20. By providing a sealed system, the water bath 22 allows the unit to be moved without the water splashing out and ruining any electronics.

[0030] The fraction collector 23 is housed in the tower enclosure 20, preferably in a refrigerated compartment thereof. The fraction collector 23 is schematically depicted in Fig. 1 by a test tube 70, the peristaltic pump 25, and the conduit 66 which draws samples from the bulk liquor 60 through the peristaltic pump to the test tube. More detail of an exemplary embodiment of the fraction collector 23 is shown in FIGURE 3. This embodiment accommodates a rack 72 for accommodating a plurality of test tubes 70, preferably up to seventy-two such test tubes of a typical size of 13-mm. In the depicted embodiment, a filling arm 74 is precisely positioned above a test tube by stepper motors under the control of the PWM outputs of the PLC 32. The fraction collector 23 is embedded into an insulated enclosure equipped with a controllable cooling means, preferably a Peltier device 75, to maintain temperatures of around 4°C. The Peltier device is electrically powered and generates a hot and a cold side, each of which is fixed to a heat exchanger and two DC brushless fans used to move the heated or cooled air. As the first fan removes the hot air to the exterior of the tower enclosure, the second fan circulates air in the fraction collector past the cooling plate of the Peltier device.

[0031] In a preferred version of the fraction collector 23, a speedy bioassay is achieved by collecting the fractions directly into a bioindicator tube 70a that is partially filled with a suitable bioindicator. Such a bioindicator tube 70a is depicted in FIGURE 4. A good example of a bioindicator 76 for the bioindicator tube 70a is a cell suspension of a bacterium that is sensitive to the antimicrobial produced during the fermentation. Examples of bacterial bioindicators include Escherichia coli K12 and Listeria innocua.

A top portion of the bioindicator tube 70a is fitted with a microfilter cartridge 78 to separate microbial cells from the collected fraction of the fermentation liquor that potentially contains the desired secondary metabolite. This design gives quick indication of when the antimicrobial compound is produced in the microbial growth cycle. In at least some of the embodiments, LUER-LOK-type fittings are used to connect each bioindicator tube with a dispensing tube of the fraction collector 23. A vertical axis on the fraction collector allows for this quick coupling. A rack cover may be used to hold all the tubes and filters, so that the fraction rack can easily detach after filling is complete.

[0032] A preferred manner of removing a desired fraction volume from the bioreactor is a peristaltic pump 25, which may be located exterior to the tower. The peristaltic pump 25 is controlled by the PLC 32. Tubing will be connected directly from the bioreactor vessel 42 to the fraction collector 23.

[0033] Referring back to Figs. 1 and 2, details of the bioreactor assembly are shown. The bioreactor vessel or jar 42 is, in preferred embodiments, primarily made out of glass, with sufficient volume to contain about 6 liters of the bulk liquor 60. A lid 80 of the bioreactor would preferably be stainless steel, or another material for the ability to be sterilized or autoclaved. The lid 80 may be clamped onto a flange at the top of the bioreactor vessel, such as by using an O-ring gasket to seal the facing surfaces. The lid 80 is provided with a plurality of openings for microbial inoculation, sample collection, probe and thermocouple insertion, foam collector interface, etc. The bioreactor jar 42 will be easily disconnected from the tower 20 for autoclaving. A reactor jacket 46 may be provided for selectively heating or cooling the bioreactor vessel 42. Input and output ports of the water jacket may be coupled directly to a compression fitting for connecting the water jacket to the reservoir in the bioreactor tower.

[0034] The preferred manner of agitating the bulk liquor 60 in a sealed bioreactor vessel 42 may be a stirring propeller in the bioreactor, coupled magnetically to a stirring motor 44 external to the bioreactor vessel. Such a system eliminates a rotational seal that would tend to leak over time. The magnetic stirring motor 44 allows for a stationary seal on the lid and will have minimal leaking after repeated autoclaving. The motor 44 will be easily detached so the unit with the impeller in place can easily be autoclaved.

[0035] A known problem in conducting aerobic fermentation is the need to provide air, with or without enhanced oxygen content, into the fermenting medium. As described above, the tower enclosure 20 has an air pump 24 for providing this air to the reactor jar 42. Excess air also needs to be vented out to avoid pressurization of the bioreactor vessel. Adding air while stirring the bulk liquor of a bioreactor can lead to the problematic or desirable foaming described above.

[0036] The preferred embodiments of the present invention are provided with a foam collector 90, located on or near the lid of the bioreactor vessel 42, as seen in more detail in FIGURE 5. It is desirable to provide a jacket 92 to the foam collector 90 for temperature control. Heating may be used to maintain condensed foam in the collector 90 at the fermentation temperature, and cooling may be used to assist in breaking a persistent foam into a condensed form. As seen in Fig. 1 , a conduit 94 allows foam from the reactor vessel 42 to enter the top 96 of the foam collector, where an opening 97 is used to vent pressure that may build up during this process. A float valve 98, located inside the collector 90, can be selectively opened or closed as a selected amount of condensed foam accumulates in the collector. In this manner, condensed foam may be either returned to the bulk liquor 60 through conduit 99 or drawn off for analysis or separation of desired product. This foam collector can also be manually plugged to allow foam collection by tightening float adjustment screw 101 , pushing the float to the closed position. Collected foam 103 can then be retrieved by syringe through the septum 102 on the foam collector lid.

[0037] It will also be desirable to provide the bioreactor vessel 42 with at least one probe in contact with the bulk liquor 60 for monitoring and analyzing conditions of the bioreactions being conducted. A typical probe 100 of this type is depicted in FIGURE 6. A number of commercially available probes will be useful for this purpose, many of which will be multi-functional. Temperature may be measured by a thermocouple 100a, pH by a pH probe 100b, absorbance by an absorbance meter 100c and these will be tracked with respect to time through connection to PLC 32, as shown in Fig. 2.

Dissolved oxygen is another parameter that can be monitored. The probe 100 is easily disconnected and can be capped and autoclaved. This allows a proper probe (or plurality of probes) 100 to be used, appropriate to the reaction being conducted. The probe 100 also comes with a display meter that will display current reading and allow real time monitoring of the fermentate process.

[0038] A fermentation run was carried out using Paenibacillus polymyxa OSY-DF, a highly aerobic bacterium that produces the antimicrobial peptide, paenibacillin. [0039] In the experiment conducted, the growth of the microorganism was successfully recorded using the bioreactor system described herein. Two liters of reconstituted milk powder (10%) was used as the growth medium for paenibacillin production. Airflow was set at 2.3 liters per minute and the stirring speed was 72 rpm. The temperature was maintained at 30 ^Q C. Data were collected at the rate of one set of readings per minute. A sample of the data, particularly, absorbance at 880 nm, is presented in FIGURE 7, covering a period of approximately 21 hours. These data reveal that the microorganism’s growth occurred in a bi-phasic manner, and the production of paenibacillin occurred mainly at the middle of the second phase. The ability of the bioreactor disclosed herein to monitor the growth and production in a turbid medium, such as the milk, is considered to be unprecedented.

[0040] In conducting a separate experiment, the fermentate and foam were collected separately from the fermenter and the foam collector, respectively. Both the fermentate and the foam (in the form of condensed foam liquid) were centrifuged to remove bacterial cells. The cell-free centrifugation supernatants were mixed with XAD-7HD resin (10%) and absorption of paenibacillin to the resin was allowed during 24 hours of holding. The resin was washed and the paenibacillin was eluded from the resin, using 75% ethanol. Most of the ethanol was evaporated, and residues were resuspended in water resulting in 2x concentrated fermentation extract and foam extract. Each of the two extracts was spotted (10 pi each) onto a lawn of an indicator bacterium ( Listeria innocua ) embedded in a layer of soft agar. Review of the agar after an appropriate incubation period showed that the fermentation extract provided a clear zone of inhibition, but that the zones appearing where the foam extract was spotted to the agar are distinctly much larger and clearer. It is qualitatively estimated that the antimicrobial activity in the foam extract is on the order of at least 20 times greater than that observed in the fermentate extract.

[0041] This result was quantitatively confirmed in another experiment. In that experiment, a fermentation run of approximately 24 hours duration was conducted at 30 ^Q C, in which Tryptic Soy Broth was fermented with Paenibacillus polymyxa OSY-DF. In a first run, in which the foam collector was not used, paenibacillin concentration in the final bulk solution was 32 pg/ml. However, in a second run conducted using the foam collector, the paenibacillin concentration in the bulk solution was only 4 pg/ml, with the concentration in the foam collector was 132 pg/ml, a concentration 33 times greater than the concentration in the bulk solution.

[0042] In an experiment using a Bacillus velezensis GF610 which produces the two- component antimicrobial lantibiotic, amyloliquecidin GF610, the foam contained the antimicrobial agent is a concentrated form whereas the bulk medium contained the agent at much lower concentration.

[0043] While the foregoing has described laboratory scale bioreactor applications of the invention and particularly the collection and condensation of persistent foam to concentrate products found in the liquid phase thereof, the concepts may be adapted to non-biological reactions carried out in an agitated bulk liquor, such as for the production of fine chemicals. In such a case, it may be desirable to scale-up the size of the reactor vessel to accommodate volumes on the order of 600 liters of bulk liquor instead of the 6-liter capacity described here. Other variations and modifications will be known to one of skill in the art and still be within the scope of the claimed invention.