MXPA99011552A - Culturing apparatus and cultivating method - Google Patents

Abstract

Se expone un aparato de cultivo (100) que incluye un lecho de cultivo (110) en fase sólida y permeable al gas, colocado dentro de un espacio interior de un recipiente (108). El lecho de cultivo comprende un sustrato sólido que incluye un agente biológico colocado sobre el mismo. Una entrada de gas (115, 116) permite la introducción selectiva de gas hacia el recipiente (108), bajo presión suficiente para provocar que el gas fluya a través de por lo menos una porción del lecho (110). También se expone un método para cultivar un agente biológico. El método comprende proporcionar un lecho de cultivo permeable al gas, hacer fluir un gas a través del lecho y recuperar por lo menos una porción del agente biológico. El gas de preferencia es un gas que contiene oxígeno, mediante lo cual el flujo de gas a través del lecho proporciona transferencia de oxígeno por transferencia másica convectiva hacia el agente biológico. El aparato y el método de la invención permiten un"pulsado de presión"del gas a través del lecho.

Description

CULTURE DEVICE AND GROWING METHOD RELATED APPLICATION This application claims the priority of the Provisional Patent Application of the States United Serial No. 60 / 082,332, filed on April 20, 1998, the contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION The invention relates in general to the field of cultivation of biological agents. More specifically, the invention is directed to a method for culturing a biological agent and a useful culture apparatus in relation to the cultivation of a biological agent.

BACKGROUND OF THE INVENTION It is well known that certain biological products (such as enzymes, organic acids, vitamins, extracellular proteins, amino acids, antibiotics and the like) can be produced by biological agents, in particular by bacteria and other prokaryotic organisms such as yeasts, molds and fungi, as well as eukaryotic species such as plant or animal cells. Many efforts have been made to increase the amount of desirable biological products that are produced by these P1745 / 99MX biological agents, by making it easier to grow them. As an example, during the Second World War, experts in the art sought to accelerate the production of penicillin in view of the high demand for the treatment of battle wounds. These efforts gave rise to the then new technology of submerged fermentation. This process involves immersing the biological agents (ie fungal mycelia in the production of penicillin) in a tank containing a liquid medium that functions as a nutrient solution. The biological agents excrete or produce the desired biological product in the liquid culture medium. After a period of cultivation (usually lasting several days), the liquid is filtered and the biological product is extracted from it. Other culture methods submerged in liquid medium are known in the art. The techniques for cultivating biological agents in submerged liquid medium, for example submerged fermentation, have not been totally satisfactory. For example, a very important problem, especially with aerobic processes, is that the liquid in which the biological agent is immersed prevents the transfer of oxygen to the biological agents that are to be cultivated. In addition, the use of liquid media causes environmental concern due to the problems P1745 / 99MX associated with the disposal thereof. More recently, solid phase processes have been employed for the cultivation of biological agents. For example, the formation of compost is a technique that involves the aerobic bacterial decomposition of solid organic waste. In general, the growth of a biological agent, for example mycelium, on a solid substrate occurs more easily than when the biological agent is immersed in a liquid. As a result, biological agents grow more rapidly when exposed to ambient air compared to when agents are immersed in the liquid. It is considered that biological agents that are grown on solid substrate surfaces absorb oxygen directly from the ambient atmosphere. In processes that use a liquid medium to cultivate the biological agents, the transfer of oxygen to the cells is therefore relatively impeded. Consequently, the use of solid phase processes reinforces growth and other metabolic activities compared to liquid phase processes. Despite the advantages associated with solid-phase processes for the cultivation of biological agents, these processes have not fully met with success. For example, solid substrates in a solid phase process are usually packaged to P1745 / 99MX form a bed that acts as a good thermal insulator. In many solid phase processes, for example in the degradation of grass clippings and leaves by the formation of compost, the heat is generated by the metabolic activity of the biological agents that are inside the solid phase. The heat, not being able to dissipate quickly, causes that the temperature increases inside the pile of compost. This increase in temperature helps exterminate microorganisms and insects, which is desired in the formation of garden waste compost. However, if the solid phase process is intended to be used at high levels of solid waste digestion and / or for the manufacture of biological products, the increase in temperature may undesirably tend to prematurely terminate the biological process. Another problem that occurs in the solid phase processes is related to the supply of oxygen to the porous beds, the supply is abnormally necessary for the growth of biological agents. In general, at least a portion of the biological agent in a bed will be placed inside the bed, where it is not exposed to ambient oxygen. In this way, the oxygen can only be supplied to the agent by means of a small molecular diffusion through the bed, the diffusion will be presented in a slower way than P1745 / 99 X normally desired and this rate can limit the growth of the biological agent. In addition, solid substrates and biological agents normally form large aggregates that further impede the flow of oxygen to the solid phase, further reducing the supply of oxygen to the biological agent. A general object of the invention is to provide a culture method and apparatus that allow the cultivation of biological agents, so that the desired biological products can be produced more easily than with conventional techniques. Another general objective of the invention is to provide a method and apparatus for cultivation, where oxygen is supplied to a biological agent within a culture bed.

THE INVENTION It has now been found that biological agents can be grown in a culture apparatus that includes a gas permeable bed, placed inside a vessel and that provides a convective flow of gas through the bed. The convective flow of gas through the bed provides a relatively improved heat transfer and / or mass to and from the bed, compared to conventional growing processes. This thermal transfer and / or improved mass has been P1745 / 99MX found reinforces the growth of biological agents within the bed. According to one embodiment of the invention, the container is provided with a gas inlet that allows the introduction of gas into the container. The gas permeable bed contains a biological agent and a suitable substrate to grow the biological agent. The gas is introduced through the gas inlet into the vessel under sufficient pressure to cause gas to flow through at least a portion of the bed. Desirably, the bed includes a gas outlet and both the gas inlet and the gas outlet are hydraulically coupled with valves operating between a closed state and at least one open state. An operator can open and close the valves to thereby modify the flow of gas through the container and thereby moderate the temperature of the bed within a desired range of temperatures. More preferably, the valves operate in order to operate the vessel in a "pulse pressure" mode wherein the gas flow through the apparatus varies cyclically. In accordance with another aspect of the present invention, a method for culturing a biological agent is provided. The method includes the step of providing a gas permeable culture bed that includes a substrate that is biologically conducive.

P1745 / 99MX to cultivate a biological agent, to flow a gas through the bed in order to cause the gas to be placed in thermal convective contact with at least a portion of the biological agent and / or to cause convective mass transfer with the biological agent and recover at least a portion of the biological agent. The biological agent preferably has an aerobic activity and the gas is preferably an oxygen-containing gas.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a culture apparatus according to the invention; Figure 2 is a graphical representation of the variance of the pressure within the container of the apparatus shown in Figure 1 with respect to time, when operated in a pressure pulsation mode according to a preferred embodiment of this invention; Figure 3 is a representative illustration of the flow of gas through the culture bed; Figure 4 is a flow diagram illustrating the possible control logic for the operation of a gas outlet valve in the culture apparatus shown in Figure 1; Figure 5 is a flow diagram that P1745 / 99MX illustrates the possible control logic for the operation of a gas inlet valve in the culture apparatus shown in Figure 1; Figure 6 is a flowchart illustrating another possible control logic for operating a gas outlet valve in the culture apparatus shown in Figure 1; Figure 7 is a flow chart illustrating another possible control logic for the operation of a gas inlet valve in the culture apparatus shown in Figure 1; Figure 8 is a flowchart illustrating another possible control logic for the operation of a gas outlet valve in the culture apparatus shown in Figure 1; Figure 9 is a flow diagram illustrating another possible control logic for the operation of a gas inlet valve in the culture apparatus shown in Figure 1; Figure 10 is a flow diagram illustrating another possible control logic for the operation of a gas outlet valve in the culture apparatus shown in Figure 1; Figure 11 is a flow chart illustrating another possible control logic for the operation of a gas inlet valve in the culture apparatus shown in Figure 1; Figure 12 is a flowchart that P1745 / 99MX illustrates another possible control logic for the operation of a gas outlet valve in the culture apparatus shown in Figure 1; Figure 13 is a flow chart illustrating another possible control logic for the operation of a gas inlet valve in the culture apparatus shown in Figure 1.

DESCRIPTION OF THE PREFERRED MODALITIES The invention in general is directed towards the cultivation (ie cultivation) of any biological agent that can be grown on a substrate and, therefore, the invention is contemplated with utility in relation to both aerobic and anaerobic culture. of biological agents, for example by fermentation. By "cultivation" and "cultivation" is considered an increase in the amount of biological agent as a result of the growth of the biological agent on the substrate or together with the substrate. It is contemplated that a biological agent can be cultivated in a desirable manner because of its ability to produce a biological product (for example the penicillin product is obtained from mycelial cells). "Cultivation" (and "cultivation") of the biological agent therefore aims to encompass obtaining a desired biological product directly, as well as obtaining the biological agent itself. Examples of biological agents that can P1745 / 99MX cultured and recovered according to the invention include bacteria, yeasts, spores, fungi, molds, plant and animal cells and in general any biological agent that is known or will be discovered in the future. The agent of preference is an aerobic agent, but it can also be an agent having an anaerobic activity. The biological products that can be recovered include enzymes, amino acids, vitamins, organic acids, extracellular proteins, antibodies and, in general, any biologically produced material. For example, the invention can be used in relation to the cultivation of cellulase-producing biological agents, this culture being preferably achieved on a cellulosic substrate. The cellulose cellulose hydrolyzates include three enzymes, endoglucanase (EC 3.2.1.74, commonly known as Cx), exoglucanase (EC 3.2.1.91, also known as C ^ and cellobiase (EC 3.2.1.21) .These enzymes work together to convert The activities of these enzymes are summarized below: Endoglucanase: Gn Gn- + Gm and Gm Gm-p + Exoglucanase: Gp? Gp-2 Celobiase: G, • - »2GX where G represents a unit of anhydroglucose and can vary from approximately 2,000 to 10,000 P1745 / 99MX in cellulose (Gn therefore represents cellulose); where n, m and p are integers in n > m > p. In this series of reactions, the endoglucanase breaks the cellulose molecule into two anhydroglucose chains; exoglusanase breaks down cellobiose (G2, composed of two anhydroglucose units) from a linear anhydroglucose chain; and cellobiase converts cellobiose into glucose. Among many microbes that produce cellulase, those that belong to Tri choderma are known for their high productivity of mixtures of the three components of the cellulase. Aspergillus niger is also a producer of the cellulase enzyme, although some strains of A. niger is known to produce cellobiase only. The invention is not limited to the culture of the above biological agents, but is contemplated as generally applicable to the cultivation of any suitable biological agent. The invention generally contemplates both a culture apparatus and a method for growing a biological agent. The apparatus is generally shown in Figure 1. In relation to it, the apparatus 100 includes a gas source 101, and this source can be, for example, an air compressor 102, an oxygen tank 103 or any other Adequate source of gas. When the biological agent has an aerobic activity, the gas of preference is a gas containing oxygen, for example oxygen Purified P1745 / 99MX or air, but it is further contemplated that a different gas may be used in connection with the invention in some embodiments. For example, it may be desirable to periodically flood the container with an inert gas, for example nitrogen gas. In this case, the source 101 may include a compressed nitrogen tank (not shown). The gas optionally, but preferably, is filtered through a filter 105 and humidified in the humidifier 106, any of which may, if desired, be provided with a heating and / or cooling mechanism, for example a coil jacket which contains heating and / or cooling coils (not shown). After leaving the humidifier 106, air passes into a culture vessel 108 which includes a gas permeable culture bed 110 positioned within an interior space defined by a wall 112 thereof. The culture bed 110 comprises a substrate that includes a biological agent placed thereon, the substrate is biologically favorable for the growth of the biological agent. Preferably, the substrate is selected to be compatible with the biological agent to be cultivated and, therefore, for example when cellulase enzymes are to be grown, the substrate may comprise recycled paper fibers, wood chips or other sources that They contain cellulose. Other substrates Suitable P1745 / 99MX which are used in conjunction with the invention include grains, for example wheat bran, comminuted corn, grains of brown rice and other organic materials such as soluble proteins. More generally, any substrate that provides physical and nutritional support for the biological agent and that is adequately porous to allow the passage of gas can be used. By "gas permeability" is meant that the bed allows gas to flow therethrough in a convective thermal communication with at least a portion of the biological agent in the bed and / or in a convective mass transfer communication with at least a portion of the biological agent. The substrate may comprise an inorganic material (for example a diatomaceous earth) in admixture with a nutritive substrate such as for example urea, grains or other suitable nutrients. The selection of a particular substrate for a specific biological agent to be cultivated is contemplated within the skill level of the connoisseurs of this field. The substrate is provided in the form of a solid phase substrate, by which a porous or gas permeable substrate is contemplated which is preferably wet (ie, has sufficient moisture to promote growth of the biological agent) but which is not submerged in a bathroom P1745 / 99MX liquid. Preferably, the substrate is initially provided in the form of several discrete and wet packages, the substrate packages are packed into the bed with sufficient void volume to allow the bed to be permeable to at least the compressed gas. It is contemplated that the substrate packages will be formed of a cohesive and friable mass after the biological agent has been allowed to grow for a sufficient period of time, and therefore can no longer be identifiable as discrete packages. The container 108 shown in Figure 1 is equipped with two gas inlets 115, 116, each of which has a gas inlet valve 115A, 116A, controlled by a respective valve actuator 115B, 116B. The container is also equipped with a gas outlet 117 and a gas outlet valve 117A which is operated by a gas outlet valve actuator 117B. The valve actuators can furthermore be controlled by a controller 120, as discussed in more detail below. The controller, valves and accompanying valve actuators are considered optional in relation to the invention and, therefore, the container may be equipped with no valves, one, two or all three valves illustrated at 115A, 116A and 117A, or It can be equipped with more valves and valve actuators, if desired.

P1745 / 99MX The gas passes into the container 108 through one or both of the gas inlets 115, 116. The gas inlet or the gas inlets are preferably charged with steam prior to the introduction of the gas in order to prevent drying of the culture bed. In the illustrated embodiment, the gas exiting the humidifier 106 passes along one or both paths 118, 119, respectively through the valves 115A and 116A and the gas inlets 115 and 116 (the paths 118, 119 are shown in interrupted lines as optional alternatives). The container 108 preferably includes a head space 121 close to a boundary 122 of the bed 110 and a bottom space 124, close to another boundary 126 of the bed 110. For example, the bed 110 may rest on a screen 127 inside the container 108, the screen 127 allows the fluid to flow through but does not allow the solid content of the bed 110 to pass into the bottom space 124. The incoming gas may pass either into the head space 121 through the inlet of gas 115, or to the bottom space 124 by the gas inlet 116 (or by an optional path 128 through the gas inlet 115, path 128 is shown in interrupted lines as an optional alternative). In operation, the gas is introduced to the container in such a way that it flows through at least a portion of the bed 110.

P1745 / 99MX contemplates in the preferred embodiments of the invention that the gas will flow through the entire bed in order to maximize the benefits that can be achieved according to the invention. It is contemplated that the gas will not be able to flow through the entire bed, for example when the bed contains occlusions or when the gas inlet and outlet are positioned to cause the gas flow to be only through a portion of the gas. bed. As shown in Figure 3, the bed 110 comprises discrete plural particles 130 of substrate with a biological agent placed thereon. Gas flows through a first boundary 131 of the bed, as represented by arrow 132, through at least a portion 133 of the bed, as represented by arrow 134, and through a second boundary 135 of the bed , as represented by the arrow 136. The first and second boundary 131 and 135 preferably, but not necessarily, are coextensive with the boundaries 122, 126 between the bed 110 and the head space 121 and the space 124, respectively ( as shown in Figure 1). For example, the gas can be introduced into the bottom space 124 of the container 108 through the gas inlet 116, under a pressure greater than ambient pressure. The gas will flow from the lower space 124 through the bed 110 to the head space 121 and out of the gas outlet P1745 / 99 X 117. In another embodiment, the container is pressurized by introducing gas into the headspace, through a gas inlet 115, where the gas outlet valve 117A is closed. The gas will flow to the bed 110, even if the container is not equipped with a bottom space. The gas outlet valve 117A is then opened to allow gas to escape from the container 108. The biological agent is placed on the bed, ie on a bed surface or inside the bed. Preferably, the biological agent is dispersed homogeneously through the bed. While it is not intended to limit the invention to a particular theory of operation, it is considered that passage of gas through the culture bed will improve mass transfer and / or heat transfer within the bed, as a result of convective transfer. of oxygen through the bed. For example, in the case of the aerobic growth of a biological agent, the biological agent disposed within the interior portions of the bed or growing within them will be able to breathe more easily than if the gas were not allowed to flow through the bed, as result of the convective transfer of oxygen through the bed. It is therefore believed that the volatile metabolites will be allowed to escape from the bed by convective mass transfer.

P1745 / 99MX The passage of gas through the bed can also affect the thermal transfer and the temperature inside the bed. For example, in the case of a gas containing oxygen, the increase in oxygen flow to the bed can cause the temperature inside the bed to increase or decrease. It is considered that the increase in the oxygen flow rate will cause the metabolic activity of the microorganisms that are inside the bed to increase (thus tending to increase the temperature inside the bed) but will also increase the heat convection away from the bed (tending Thus, the temperature inside the bed decreases, while the gas is at a lower temperature than the bed.The rate at which the temperature of the bed is increased as a result of the activity of the microorganisms can be greater, less or equal at the rate at which the temperature is decreased as a result of heat convection away from the bed.Therefore, depending on the fermentation step, gas flow velocity and other factors, the temperature of the bed can be made increase or decrease with increasing oxygen flow rate The effect of the flow rate of preference is determined empirically for an ato and specific process. In the case of anaerobic agents within the bed, it is contemplated that an increase in flow velocity will not cause a P1745 / 99MX increase in metabolic activity within the bed. In this case, the increase in gas flow velocity will be expected to cause the bed temperature to decrease if the gas is at a lower temperature than the bed temperature, and increase if the gas is at a higher temperature. If desired, the heat can be removed from the bed by flowing an inert gas (eg, nitrogen) through it. Alternatively, the non-humidified gas or gas having low humidity may be introduced in order to cause evaporation of the water vapor from the bed and thus remove the latent heat thereof. For example, it is possible that the gas inlet is not loaded with water vapor before introducing the gas into the bed, thus causing an evaporative cooling of the bed. Alternatively, the gas may be cooled before entering the bed or the vessel may be equipped with cooling coils (not shown). In a particularly preferred embodiment, the apparatus operates in a "pulsating pressure" mode. By "pulsating pressure" in one embodiment, cyclical pressurization and depressurization of the container is contemplated. An example of the pressure profile inside the container generated according to a pulsating pressure is shown in Figure 2 (pressure that is provided as meter pressure). "Pulsing pressure" also covers increasing and subsequently P1745 / 99 X Cyclically decrease the cyclical flow velocity of the gas through the bed. In any case, by "cyclical" it is contemplated the repeated pressurization / depressurization or increase / decrease operations in the flow velocity, at least once and, preferably, at least five times, after the pair is contemplated initial of operations. In general, the pair of operations can be repeated as many times as desired for a given application. It will be apparent to those skilled in the art that pressure pulsation can be achieved using various embodiments of the apparatus shown in Figure 1, as well as any other suitable form. For example, the container may be equipped with a gas inlet 115 without a gas inlet valve, and a gas outlet with a gas outlet valve 117A. The pulsating pressure can then be achieved by closing the gas outlet valve 117A, allowing the pressure within the container to accumulate and preferably to be maintained when the pressure within the container reaches the pressure of the gas entering through the gas inlet 115. , subsequently opening the gas outlet valve to allow the pressure inside the vessel to decrease to the ambient pressure and repeat this operation. Alternatively, the gas can enter the vessel through the inlet of P1745 / 99MX gas 116, which may or may not be equipped with a gas inlet valve, or may enter through the gas inlet 115 by an optional route 128. In another embodiment of the invention, the gas enters the container at Through the two gas inlets 115 and 116, at least the gas inlet 116 is equipped with a gas inlet valve 116A. Pulsating pressure can be achieved by periodically opening and closing the gas inlet valve 116A to allow larger and smaller amounts of gas respectively to enter the container 108. The gas inlet and outlet valves 115A, 116A and 117A can be operated manually. In a preferred embodiment of the invention, each valve is equipped with a valve actuator 115B, 116B and 117B, respectively, each of which modifies the status of its respective valve (e.g., by fully opening or closing each valve or increasing or decreasing in increments the amount of fluid that can flow through the valve). Valves and valve actuators may be integral with the container or may be remote from it. For example, the valve may be associated with the gas source (for example the valve on a pressure vessel) or may be associated with the compressor. The valve actuator can, for example, be a switch to drive the compressor (the compressor then serves as P1745 / 99MX valve). Alternatively, the valve actuator may, for example, be a solenoid actuator, the gas valve then comprising a solenoid valve. Alternatively, the valve and the actuator may be any other type of suitable device. The valve actuators can be controlled by a controller 120. In a highly preferred embodiment of the invention, the apparatus is equipped with a temperature sensor 140 which measures the temperature of the bed and / or one or more pressure sensors 141, 142, which they measure a pressure inside the container 108 (it is contemplated that the pressure reported in the head space of the pressure sensor 141 may differ from that reported in the bottom space by the pressure sensor 142). In accordance with that embodiment of the invention, the controller 120 communicates with the temperature sensor 140 via line 144 and communicates with one or more pressure sensors via lines 145, 147. The controller also communicates with the actuators of valve 115B, 116B, 117B by lines 148, 149 and 150, respectively. The controller can be any mechanism operated electronically or otherwise. In some cases, the controller may comprise a simple control logic circuitry, for example a wired circuit or may comprise a timer. In one mode, the controller P1745 / 99MX comprises a microprocessor or microcontroller 152 which includes a timer 153, a data bus 154 and an input / output interface 155 by which the sensors and valve actuators communicate with the microprocessor or microcontroller 152. The sensors provide signals to the controller so as to communicate the temperature or pressure data to the microprocessor or microcontroller 152, and the microprocessor sends control signals to the valve actuators 115B, 116B and 117B to modify the status of the inlet and / or outlet valves of the valve. gas. The microprocessor or microcontroller or the logic circuitry can be programmed by any suitable way to achieve pressure pulsation. For example, if the pressure pulsation is achieved with a microcontroller or microprocessor, by opening and closing the gas outlet valve, a control program is illustrated diagrammatically in Figure 4. In step 160, a delay recorder in the microprocessor or microcontroller it is reset with a closed reference time, that is, the length of time that the gas outlet valve must remain closed. After the timer has indicated the passage of this amount of time, the microprocessor or microcontroller, in step 161, sends an open valve signal to the actuator P1745 / 99MX gas outlet valve. In step 162, the delay register is reset with an open reference time, i.e., a value that indicates the amount of time that the gas outlet valve must remain open. Subsequently, after this time has elapsed, a valve closing signal is sent to the gas outlet valve actuator in step 163 and this cycle is repeated. The open reference time and reference times in the closed state of preference are determined empirically from a specific apparatus and method, and the amount of time required for the valve actuator to achieve the respective opening and closing can be taken into account. from valvule. It is further contemplated that an operator may terminate the control cycle at any desired time. If the pressure pulsation is to be achieved by the cyclic opening and closing of the gas inlet valve, the program is shown diagrammatically in Figure 5 in a program suitable for the microprocessor or microcontroller, steps 164-167 essentially correspond to the steps 160-163 shown in Figure 4. It should be understood that the control programs shown in Figures 4 and 5 and in the subsequent Figures, although illustrated as control programs for a microcontroller or microprocessor, may be implemented by the logic circuitry or by P1745 / 99MX any other suitable control mechanism. During the growth of the biological agent, the temperature inside the bed may increase as the speed of metabolic activity within the bed also increases. The heat may cause premature termination of growth of the biological agent, for example, if the temperature reaches an undesirably high level. On the other hand, if the temperature decreases below the desired range of operation of the enzyme, the metabolic activity within the bed may decrease undesirably. When the bed includes a temperature sensor, the microprocessor or microcontroller can be programmed according to the control logic shown diagrammatically in Figure 6 with respect to the control of a gas outlet valve. In step 170 a desired reference temperature or a maximum desired operating temperature of the container is obtained, for example, by receiving a user input or a stored memory variable. In step 171, the microprocessor or microcontroller receives a signal from the temperature sensor and, in step 172, this received temperature is evaluated against the reference temperature. If the received temperature is still not as large as the reference temperature, after a delay 173 the control goes to step 171. On the other hand, if the temperature inside the P1745 / 99MX bed has reached or exceeded the reference temperature, a signal is sent to the actuator of the gas outlet valve in step 174 in order to cause the gas outlet valve to open and thus cause depressurization of the containers. After a delay 175, another signal is sent to the gas valve actuator in step 176 to thereby cause the gas valve to close. The control proceeds to step 171 after another delay 177. It is contemplated that the delays in steps 173, 175 and 177 and the reference temperature can be determined empirically for a specific biological agent and apparatus. The reference temperature preferably defines the maximum temperature or is below it, within the range of activity desired for the biological agent. In an alternative embodiment, as shown in Figure 7, the microprocessor or microcontroller can be used to control a gas inlet valve actuator. The program is comparable to that shown in Figure 6, except that a valve closing signal is sent to step 178 and a valve opening signal to step 179. A further alternative program is shown in Figure 8. In this embodiment, it causes a gas outlet valve to open when the temperature inside the bed has reached a first P1745 / 99MX reference temperature, and the gas outlet valve is made to close when the temperature inside the bed has dropped to a second reference temperature. The first and second reference temperatures are obtained in step 180, for example by receiving the input from a user or recovering data valves from memory storage. Steps 181-185 are comparable to steps 171-175 respectively of the mode shown in Figure 6. After the valve has been opened and after delay 185, a signal from the temperature sensor is received again in step 186. In step 187, this temperature is compared with the second reference temperature to determine if the temperature inside the bed has fallen to or below the second reference temperature. Otherwise, after a delay of 188 the control goes to step 186. On the other hand, if the temperature has dropped to the second reference temperature or below it, the control goes to step 189, where a signal is sent. valve closing signal to the gas outlet valve actuator. After a delay 190, the control goes to step 180. The first and second reference temperatures preferably define the upper or lower temperatures of the desired activity range for the biological agent or remain within P1745 / 99MX of this interval. The temperature of the bed should remain between approximately 30 ° C and 42 ° C for both Tri choderma and A. niger Figure 9 illustrates a similar embodiment wherein the microprocessor or microcontroller is used to control a gas inlet valve actuator. In this embodiment, a valve closing signal is sent to step 191 and a valve opening signal is sent to step 192. As an alternative for the measurement of the temperature within the bed and the opening or closing of the valves in response thereto, the valve may include one or more pressure sensors 141, 142. More preferably, the container includes a pressure sensor that is preferably located in the headspace, when the gas inlet is in the space of bottom of the container and, preferably, it is located in the bottom space of the container when the gas inlet is in the head space of the container. Figures 10 to 13 are comparable to Figures 6 to 9, respectively, and illustrate diagrammatically the programming of the microcontroller or microprocessor, whereby the gas inlet or outlet valves can be opened or closed in response to pressure changes within the container. Steps 170 '-192' are comparable to steps 170-192 in Figures 6 to 9. Figure 10 illustrates the operation of P1745 / 99MX a gas outlet valve, wherein a valve opening signal (in step 174 ') has been provided when the pressure has reached at least one reference pressure, and subsequently a shut-off signal is provided. valve (in step 177 ') after a delay 176'. Figure 11 is comparable to Figure 10 and shows control of a gas inlet valve, where a valve opening signal is provided in step 178 'and a valve closing signal in step 179'. Figure 12 illustrates the operation of a gas outlet valve where a valve opening signal is provided (in step 184 ') when the pressure has reached at least a first reference pressure and a closing signal of valve (in step 189 ') when the pressure has fallen to the second reference pressure or below. Figure 13 is comparable to Figure 12 in the operation of a gas inlet valve, wherein a valve closing signal is provided in step 191 'and a valve opening signal in the passage 192'. The pressures and delays in the following programs can be determined empirically for a specific biological agent or agent. The above description of the programs that can be used together with the pulsating pressure for P1745 / 99MX, no reason is exhaustive and it should be contemplated that those skilled in the art will be able to find other ways to program a microprocessor or microcontroller or to operate an apparatus according to the present invention. For example, instead of closing or opening a valve entirely, it is contemplated that a suitable program could cause the valve actuator to incrementally open or close a valve, as appropriate. It is further contemplated that other microprocessor or microcontroller programs or other logic circuitry or other control schemes may be developed by experts in this field. After growing the biological agent, at least a portion thereof is recovered from the container. By "recovering" the biological agent is meant any action by which a portion of the biological agent or biological product produced by the biological agent is removed from the container, regardless of whether that agent or biological product was initially present in the container or was he cultivated in it. For example, as shown in Figure 1, the container 108 can be provided with a liquid intake port 107 and a drain 109. To recover the biological agent, the sterilized water can be fed into the container through the port.

P-L745 / 99MX of intake 107, and the biological agent can be recovered by withdrawing the liquid through drain 109. The biological agent then continues to be cultured. Water or nutrients may be added as the case may be. The following non-limiting Examples are provided to illustrate the invention.

EXAMPLE 1 A New Brunswick two-liter glass bottle fermenter was equipped with a metal mesh to hold the porous and moist solid bed having a depth of approximately 10 centimeters. The fermenter was equipped with a gas inlet to introduce air into the bottom space of the fermenter (below the screen), a gas outlet valve leading to the head space (above the bed), an outlet solenoid valve of gas (Omega Technologies Co.) and an electric synchronizer (ChronTrol) that serves as a controller. Air was allowed to flow into the bottom space and then upwards through the porous bed into the headspace. A pressure pulsation was created by periodically opening and closing the gas outlet valve. The bed included a substrate with a culture of Aspergillus niger disposed thereon. With pulsating pressure, the growth of the bacteria was uniform and thick throughout the bed. He P1745 / 99MX porous bed turned totally black due to the thick formation of black spores of the culture of A. niger The entire bed was "loose" and, in fact, it was difficult to remove it completely from the bottle without dropping portions of it. Example 1 was repeated without pressure pulsation. Only the lower portion of the porous bed near the air inlet was blackened and the central portion of the bed was tightly packed, while the mycelial growth was little and there was even less spore formation.

EXAMPLE 2 In an environment identical to that of EXAMPLE 1, sterile water was pumped into the bottle from the bottom upwards, until the top was removed from the porous bed. After the liquid extract was pumped out, some liquid was still drained slowly from the bed into the bottom space. Once the draining was completed, the air flow was reintroduced to start the bioprocess again. Once good growth was observed, the extraction was repeated. The first extraction was done three days after the inoculation. The second extraction was done 24 hours later and the extraction was repeated daily for five days. All the residual mass was removed from the bottle and extracted vigorously with the addition of detergent.

P1745 / 99MX This method of enzymatic production was done with the culture of A. niger for celobiasa and also with the cultivation of Tri choderma for the whole cellulase complex. The results shown are the following: From these results and the measured quantities of the solid enzymatic product, the productivity of the enzyme was calculated as 806 UPF / hour-liter for the cellulase complex from Trichoderma. In the case of A. niger producer of celobiasa, a productivity of 620 International Units / hour-liter was achieved. An international "filter paper" (UPF) unit is defined as the amount of cellulases that can produce a micro mol of glucose per minute from cellulose.

P1745 / 99MX COMPARATIVE EXAMPLE 1 A Tri choderma culture was grown on recycled paper fibers in a solid phase fermentation in a 1000 ml fermenter (a laboratory Erlen eyer flask) to produce high potency cellulases. The fermentation was achieved by placing the flask at room temperature on a laboratory table. After the fermentation was finished, all of the wet solids were air dried to become the final enzyme product. This product contained 246 UPF / gram, from which the productivity of the fermenter can be calculated as 234 UPF / hour-liter volume of the fermenter. The productivity of the fermentation in solid phase was higher than that of the submerged fermentation of different crops Tri choderma, reported in the literature and collected in the following Table: Cellulase Productivity of the Previous Technique in Liquid Phase Fermentation P1745 / 99 X It is therefore observed that the productivity of the method used in Example 2 for the Tri Choderma culture fermentation is more than 500% better than the productivity of the liquid submerged fermentation reported in the prior art and 344% higher to that of Comparative Example 1.

EXAMPLE 3 X COMPARATIVE EXAMPLES 2 TO 5 A mixture that includes 200 g of corn fiber (content of 14% moisture, from AE Staley, Decatur, IL), 60 g of ground corn, 30 ml of maceration liquid of corn ( AE Staley), 4.0 g of (NH4) 2S04, 2.0 g of K2P04, and 600 ml of water were prepared (final moisture content of about 75%). This mixture was autoclaved for 30 minutes at 121 ° C and allowed to cool to form a substrate. To this substrate was added 20 g of solid culture of A. Niger in a septic hood.

EXAMPLE 3 Most of the mixture (80%) of the substrate / biological agent was transferred to a 2 L fermenter with a packed bed with a height of 15 cm equipped with a gas inlet, a gas outlet and a valve output solenoid P174S / 99MX gas. The fermenter operated with the pulsating pressure profile shown in Figure 2. An air inlet was provided for the bottom space and air was allowed to flow continuously into the bed. The gas outlet led from the headspace in the vessel and the gas outlet valve was controlled by a timer. The fermentation was allowed to proceed for 60 hours, after which the substrate became black. Subsequently, 100 ml of water containing 0.2% (NH4) 2S04 was introduced to remove the enzymes by washing. This wash was repeated once a day for three days. The samples of the extract were analyzed every day to determine enzymatic activity. Before the activity analysis, 10 g of the solid sample was mixed with 10 ml of 0.05N citrate buffer pH 4.5 in a 250 ml flask. The mixture was stirred for three hours in a 25 ° agitator, filtered to remove solids and spores and stored in a refrigerator.

COMPARATIVE EXAMPLES 2 TO 5 The substrate mixture prepared as discussed above was divided and transferred to four 250 ml Erlenmeyer flasks in the following manner: Comparative Example 2 30 g Comparative Example 3 60 g Comparative Example 4 40 g (with 100 ml from P - .745 / 99MX water) Comparative Example 5 40 g (with 100 ml of 5% glucose solution). The mixture of Comparative Examples 2, 4 and 5 was adjusted to 30 ° C on a shaker at 200 rpm to start fermentation. The mixture of Comparative Example 3 was allowed to ferment at room temperature without agitation. The following results were obtained: Physical phenomena P1745 / 99MX It is therefore observed that the enzymatic activity per mg of protein obtained according to Example 3 was higher compared to that obtained according to the Comparative Examples. The following results were obtained in relation to Example 3: Production of Glucoamylase During Solid State Fermentation The dry weight of final solids: 195 grams Final solid culture moisture content: 86.9% P1745 / 99MX Volume: 14885.5 ml Production of Cellobiase During Fermentation in Solid State Dry weight of final solid: 195 grams Moisture content of the final solid culture (10. 1. 3D / 10 * 100% = 86.9% * Volume: (195 / 13.1% / 10) * 100 ml = 14885.5 ml Therefore, it is noted that the previous general objects have been satisfied. A method for cultivating a biological agent and also a useful apparatus for achieving this method has been provided.

P1745 / 99MX

Claims (61)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A cultivating apparatus comprising: a container having a wall defining a interior space; a bed of solid phase culture permeable to gas placed inside the interior space, the bed comprises a substrate that includes a biological agent placed on it, the substrate is biologically suitable for the growth of the biological agent; a gas inlet to allow gas to enter the container; and a gas outlet valve hydraulically coupled to the gas inlet and operating between a closed state and at least one open state, thus allowing the selective introduction of the gas through the gas inlet and into the container under sufficient pressure to cause gas to flow through at least a portion of the bed.
2. 2. The apparatus according to claim 1, the substrate comprises plural and described packages, the substrate packages are packed in the bed with enough voids volume to P174S / 99MX allow gas permeability. The apparatus according to claim 1, wherein the interior space includes a first gap proximal to a first boundary of the bed and a second bore proximal to a second boundary of the bed, the gas inlet communicates with the container in the first bore , thus allowing the imposition of a pressure differential between the first gap and the second gap, whereby the gas flows from the first gap through the first boundary, through an intermediate portion of the bed and through the second boundary toward the second hole. 4. The apparatus according to claim 3, wherein the gas inlet communicates with a source of compressed gas, the container is pressurized selectively by the introduction of compressed gas through the gas inlet. The apparatus according to claim 4, wherein the source of the compressed gas is an air compressor. The apparatus according to claim 4, wherein the source of compressed gas is an oxygen tank. The apparatus according to claim 4, further including a gas outlet communicating hydraulically with the container in one of the first and second recesses, and a gas outlet valve that hydraulically couples with the gas outlet, P1745 / 99MX The gas outlet valve operates between a closed state and at least one open state, thus allowing the selective release of gas from the container, through the gas outlet. The apparatus according to claim 7, further comprising a gas outlet valve controller for generating signals in order to modify the state of the gas outlet valve and a gas outlet valve actuator that responds to the signals and that modifies the state of the gas outlet valve in response to it. 9. The apparatus according to claim 8, the gas outlet valve controller comprises a timer, the timer generates a signal to open the gas outlet valve after a first period of time has elapsed. 10. The apparatus according to claim 9, the timer subsequently generates a signal to close the gas outlet valve after a second period of time has elapsed. The apparatus according to claim 8, further including a temperature sensor that measures a temperature inside the bed, the gas outlet valve controller communicates with the temperature sensor and generates a signal to open the outlet valve of the gas. gas, when the temperature has reached a first reference temperature. 12. The apparatus according to claim 11, P1745 / 99 X wherein the gas outlet valve controller subsequently generates a signal to close the gas outlet valve, when the temperature has reached a second reference temperature. 13. The apparatus according to claim 8, which also includes a pressure sensor that measures a pressure inside the container, the controller of the gas outlet valve communicates with the pressure sensor and generates a signal to open the gas outlet valve when the pressure has reached a first reference pressure. 14. The apparatus according to claim 13, wherein the controller of the. The gas outlet valve subsequently generates a signal to close the gas outlet valve when the gas pressure has reached a second reference temperature. The apparatus according to claim 7, further comprising a first gas inlet valve controller for generating signals to modify the state of the gas inlet valve and a gas inlet valve actuator responsive to the signals and which modifies the state of the gas inlet valve in response to the signals. 16. The apparatus according to claim 15, wherein the gas inlet valve controller comprises a timer, the timer generates a signal to close the gas inlet valve after a first period of time has elapsed. P1745 / 99MX time. The apparatus according to claim 16, wherein the timer subsequently generates a signal to open the gas inlet valve after a second period of time has elapsed. 18. The apparatus according to claim 15, further including a temperature sensor that measures a temperature inside the bed, the gas inlet valve controller communicates with the temperature sensor and generates a signal to close the inlet valve of the gas inlet valve. gas when the temperature has reached a first reference temperature. 19. The apparatus according to claim 18, wherein the gas inlet valve controller subsequently generates a signal to open the gas inlet valve when the temperature has reached a second reference temperature. The apparatus according to claim 15, which further includes a pressure sensor which measures a pressure inside the container, the gas inlet valve controller communicates with the pressure sensor and generates a signal to close the inlet valve of gas when a pressure inside the container has reached a first reference pressure. The apparatus according to claim 20, wherein the gas inlet valve controller subsequently generates a signal to open the P1745 / 99MX gas inlet valve when the pressure has reached a second reference pressure. 22. The apparatus according to claim 3, further including a second gas inlet, the second gas inlet is in hydraulic communication with the container in the second recess. The apparatus according to claim 22, wherein the container further includes a second gas inlet valve and a second gas inlet valve controller for generating signals in order to modify the state of the second gas inlet valve, and a second gas inlet valve actuator that responds to the signals and modifies the state of the second gas inlet valve in response thereto. The apparatus according to claim 1, wherein the interior space includes a head space proximal to a first boundary of the bed, the container includes a gas inlet in hydraulic communication with the container in the headspace. 25. The apparatus according to claim 3, wherein the biological agent is selected from the group consisting of bacteria, yeasts, molds, fungi and combinations thereof. 26. A cultivating apparatus comprising: a container having a wall defining an interior space; a solid phase culture bed, P1745 / 99MX permeable to gas, placed inside the interior space, the bed comprises a substrate that includes a biological agent disposed thereon, the substrate is biologically suitable for the growth of the biological agent; a gas inlet that allows the introduction of gas into the container; a gas outlet; and a gas outlet valve hydraulically coupled to the gas outlet and operating between a closed state and at least one open state, thus allowing selective exhausting of the gas from the container, through the gas outlet. 27. The apparatus according to claim 26, the substrate comprises a plurality of discrete packages, the substrate packages are packed in the bed with a sufficient volume of voids to allow gas permeability. The apparatus according to claim 26, wherein the interior space includes a first hole proximal to a first boundary of the bed and a second bore proximal to a second boundary of the bed, the gas inlet communicates with the container in the first bore , thus allowing the imposition of a pressure differential between the first gap and the second gap, whereby the gas flows from the first gap through the first boundary, through P1745 / 99MX an intermediate portion of the bed and through the second boundary to the second hole. 29. The apparatus according to claim 28, where the gas inlet communicates with a source of compressed gas, the container is selectively pressurized by the introduction of compressed gas through the gas inlet. 30. The apparatus according to claim 29, wherein the source of the compressed gas is an air compressor. 31. The apparatus according to claim 29, wherein the source of compressed gas is an oxygen tank. 32. The apparatus according to claim 29, wherein the gas outlet communicates with the container in one of the first or second recesses. The apparatus according to claim 26, further comprising a gas outlet valve controller for generating signals in order to modify the state of the gas outlet valve and a gas outlet valve actuator that responds to the signals and that modifies the state of the gas outlet valve in response thereto. 34. The apparatus according to claim 33, the gas outlet valve controller comprises a timer, the timer generates a signal to open the gas outlet valve after a first period of time has elapsed. P1745 / 99MX 35. The apparatus according to claim 34, the timer subsequently generates a signal to close the gas outlet valve after a second period of time has elapsed. 36. The apparatus according to claim 33, further including a temperature sensor that measures a temperature inside the bed, the gas outlet valve controller communicates with the temperature sensor and generates a signal to open the outlet valve of the gas. gas, when the temperature has reached a first reference temperature. 37. The apparatus according to claim 36, wherein the controller of the gas outlet valve subsequently generates a signal to close the gas outlet valve, when the temperature has reached a second reference temperature. 38. The apparatus according to claim 33, further including a pressure sensor that measures a pressure inside the container, the gas outlet valve controller communicates with the pressure sensor and generates a signal to open the outlet valve of gas when the pressure has reached a first reference pressure. 39. The apparatus according to claim 38, wherein the gas outlet valve controller subsequently generates a signal to close the gas outlet valve when the gas pressure has reached a second reference temperature. P1745 / 99MX 40. The apparatus according to claim 26, further comprising a gas inlet valve hydraulically coupled to the gas inlet valve and communicating with the container in one of the first and second recesses, the inlet valve of The gas operates between a closed state and at least one open state and further comprises a gas inlet valve controller for generating signals to modify the state of the gas inlet valve and a gas inlet valve actuator that responds to the signals and modifying the state of the gas inlet valve in response thereto, the apparatus further comprises a gas outlet valve controller for generating signals in order to modify the state of the gas outlet valve, and a gas outlet valve actuator that responds to the signals and modifies the state of the gas outlet valve in response thereto. 41. A method for growing a biological agent comprising the steps of: providing a solid phase and gas permeable bed, wherein the bed comprises a substrate that includes a biological agent disposed thereon, the substrate is biologically conducive for the growth of biological agents; flow a gas through the bed causing the gas to come into contact with P174S / 99MX at least a portion of the biological agent; and subsequently recovering at least a portion of the biological agent. 42. The method according to claim 41, wherein the gas is a gas containing oxygen. 43. The method according to claim 41, wherein the biological agent is selected from the group consisting of bacteria, yeasts, molds, fungi and combinations thereof. 44. The method according to claim 41, wherein the biological agent is selected from the group consisting of biological agents producing cellulase enzyme. 45. The method according to claim 44, wherein the substrate is selected from the group consisting of whole grain rice, wheat bran and comminuted corn. 46. The method according to claim 44, wherein the biological agent is selected from the group consisting of Trichoderma and A. Niger. 47. The method according to claim 41, wherein the bed is placed within an interior space defined by a wall of a container, the container includes a gas inlet and a gas inlet valve hydraulically coupled therewith, the valve The gas inlet operates between a closed state and at least one open state, thus allowing the selective introduction of gas into P1745 / 99MX through the gas inlet and into the container under a pressure sufficient to cause the gas to flow through at least a portion of the bed, the method includes the step of opening the valve gas inlet . 48. The method of claim 47, wherein the interior space includes a first proximal to a first edge of the bed gap and a second gap proximal to a second edge of the bed, the gas inlet communicates with the container in a first recess thus allowing the imposition of a pressure differential between the first recess and the second recess, the method includes the step of introducing the gas into the container through the gas inlet, whereby the gas flows from the first hollow through the first boundary, through an intermediate portion of the bed and through the second boundary toward the second bore. 49. The method according to claim 48, wherein the container further includes a gas outlet and a gas outlet valve fluidly coupled therewith, the method comprising the steps of: determining a reference temperature; measure a reference temperature inside the bed; and open the gas outlet valve if the temperature has reached or exceeded the reference temperature. P1745 / 99 X 50. The method according to claim 49, further comprising the steps of: determining a second reference temperature; after opening the gas outlet valve, measure a reference temperature inside the bed; and close the gas outlet valve if the temperature has dropped to the second reference temperature or below it. 51. The method according to claim 48, wherein the container further includes a gas outlet and a gas outlet valve hydraulically coupled thereto, the method comprising the steps of: determining a reference pressure; measure a reference pressure inside the bed; and open the gas outlet valve if the pressure has reached the reference pressure or has exceeded it. 52. The method according to claim 51, further comprising the steps of: determining a second reference pressure; After opening the gas outlet valves, measure a pressure inside the bed; and close the gas outlet valve if the P1745 / 99MX pressure has dropped to the second reference pressure or below it. 53. The method of claim 48 wherein the container further includes a gas outlet and an outlet valve gas hydraulically coupled therewith, and a valve controller gas outlet to generate signals and modify the state of the gas outlet valve, and a gas outlet valve actuator that responds to the signals and modifies the state of the gas outlet valve in response to them, the method includes the step of generating a signal to open the valve of gas outlet. 54. The method according to claim 53, wherein the gas outlet valve controller comprises a timer, the method includes the steps of: introducing gas through the gas inlet to pressurize the container; after the passage of a first period of time, generate a signal to open the gas outlet valve. 55. The method according to claim 54, further comprising the step of generating a signal for closing the gas outlet valve after the passage of a second period of time. 56. The method according to claim 53, wherein the container includes a pressure sensor that P1745 / 99MX measures a pressure inside it, the pressure sensor communicates with the controller, the method includes the steps of: introducing gas through the gas inlet to pressurize the container; after a pressure inside the vessel has reached a first reference pressure, generate a signal to open the gas outlet valve. 57. The method according to claim 56, further comprising the step of generating a signal to close the gas outlet valve after the pressure has decreased to a second reference pressure. 58. The method according to claim 48, wherein the container further includes a second gas inlet communicating with the vessel in the second recess, the vessel further includes a second gas inlet valve, the method comprising the steps of: introducing gas into the vessel through the gas inlet valve and through the second gas inlet valve; determine a reference temperature; measure the temperature inside the bed; and closing the second gas inlet valve if the temperature has reached or exceeded the reference temperature. P1745 / 99MX 59. The method according to claim 58, further comprising the steps of: determining a second reference temperature; after closing the second gas inlet valve, measure a temperature inside the bed; and operate the gas inlet valve if the temperature has dropped to the second reference temperature or below it. 60. The method according to claim 48, wherein the container further includes a second gas inlet communicating with the container in the second recess, the container further includes a second gas inlet valve and an inlet valve controller. gas for generating signals and modifying the state of the second gas inlet valve, and a second gas inlet valve actuator that responds to the signals and modifies the state of the second gas inlet valve in response thereto, the method includes the steps of: introducing gas into the container through the first gas inlet and the second gas inlet; and subsequently generating a signal to close the second gas inlet valve. 61. The method according to claim 41, in P1745 / 99MX where the step of recovering the biological agent comprises recovering a biological product produced by the biological agent. P1745 / 99MX