Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation
  • C12M25/18—Fixed or packed bed
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/14—Pressurized fluid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure

Definitions

• the present invention relates generally to the field of culturing biological agents. More specifically, the invention is directed towards a method of cultivating a biological agent and towards a culturing apparatus useful in connection with the cultivation of a biological agent.
• the liquid is filtered off, and the biological product is extracted therefrom.
• Other submerged liquid media cultivating methods are known in the art . Techniques for growing biological agents in submerged liquid media, such as submerged fermentation, have not been fully satisfactory. For example, a significant problem, especially with aerobic processes, is that the liquid in which the biological agent is immersed hinders the transfer of oxygen to the biological agents to be cultivated. In addition, use of the liquid media raises environmental concerns because of problems associated with the disposal thereof.
• solid phase processes for cultivating biological agents have been employed.
• composting is a technique that involves aerobic bacterial decomposition of solid organic waste.
• growth of a biological agent, such as mycelia, on a solid substrate occurs more readily than when the biological agent is submerged in liquid.
• biological agents grow more quickly when exposed to ambient air, as compared to when such agents are submerged in liquid.
• biological agents that are cultivated on solid substrate surfaces absorb oxygen directly from the ambient atmosphere. In processes that utilize a liquid medium for culturing the biological agents, the transfer of oxygen to the cells is thus relatively hindered. Accordingly, the use of solid phase processes enhances
• the solid substrates in a solid phase process is usually packed to form a bed which acts as a good heat insulator.
• heat is generated by the metabolic activities of the biological agents disposed within the solid phase.
• the heat not being able to dissipate quickly, causes the temperature to increase inside the composting pile. This temperature increase helps to kill microorganisms and insects, which is desired in the composting of yard waste.
• the temperature increase undesirably may tend to terminate the biological process prematurely.
• Another problem suffered by solid phase processes relates to the delivery of oxygen into the porous beds, which delivery is often necessary for the growth of biological agents.
• oxygen can only be supplied to the agent by slow molecular diffusion through the bed, which diffusion will occur more slowly than is often desired and which may be rate limiting in the growth of the biological agent.
• biological agents can be cultivated in a culturing apparatus which includes a gas- permeable bed disposed within a vessel and which provides for convective flow of gas through the bed.
• the convective flow of gas through the bed provides for relatively enhanced heat and/or mass transfer to and from the bed as compared with conventional culturing processes. This enhanced heat and/or mass transfer has been found to enhance the growth of biological agents within the bed.
• the vessel is provided with a gas inlet that permits the introduction of gas into the vessel.
• the gas-permeable bed contains a biological agent and a substrate suitable for growing the biological agent .
• the gas is introduced through the gas inlet into the vessel under pressure sufficient to cause gas to flow through at least a portion of the bed.
• the bed includes a gas outlet, and both the gas inlet and gas outlet are fluidically coupled to valves operable between a closed
• valves 4 state and at least one open state .
• An operator may open and close the valves to thereby modify the flow of gas through the vessel and to thereby moderate the temperature of the bed within a desired temperature range.
• the valves are operated so as to operate the vessel in a "pressure pulsing" mode, wherein gas flow through the apparatus is cyclically varied.
• a method of cultivating a biological agent includes the step of providing a gas-permeable culturing bed that includes a substrate that is biologically conducive for culturing of a biological agent, flowing a gas through the bed so as to cause the gas to come into convective thermal contact with at least a portion of the biological agent and/or to cause convective mass transfer with the biological agent, and recovering at least a portion of the biological agent .
• the biological agent preferably has an aerobic activity, and the gas preferably is an oxygen-containing gas.
• Fig. 1 is a schematic representation of a culturing apparatus in accordance with the invention
• Fig. 2 is a graphical representation of the variance of the pressure within the vessel of the apparatus shown in Fig. 1 with time, when operated in a pressure pulsing mode in accordance with a preferred mode of the invention;
• Fig. 3 is a representational illustration of gas flow through the culturing bed
• FIG. 4 is a flow diagram illustrating a possible control logic for operation of a gas outlet valve in the culturing apparatus shown in Fig. 1 ;
• Fig. 5 is a flow diagram illustrating a possible control logic for operation of a gas inlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 6 is a flow diagram illustrating another possible control logic for operation of a gas outlet valve in the culturing apparatus shown in Fig . 1 ;
• Fig. 7 is a flow diagram illustrating another possible control logic for operation of a gas inlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 8 is a flow diagram illustrating another possible control logic for operation of a gas outlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 9 is a flow diagram illustrating another possible control logic operation of a gas inlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 10 is a flow diagram illustrating another possible control logic for operation of a gas outlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 11 is a flow diagram illustrating another possible control logic for operation of a gas inlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 12 is a flow diagram illustrating another possible control logic for operation of a gas outlet valve in the culturing apparatus shown in Fig. 1;
• Fig. 13 is a flow diagram illustrating another possible control logic for operation of a gas inlet valve in the culturing apparatus shown in Fig. 1.
• the invention generally is directed towards the culturing (i.e., cultivating) of any biological agent that can be grown on a substrate, and thus, for example, the invention is contemplated to find utility in connection with both aerobic and anaerobic culturing of biological agents, such as by fermentation.
• culturing and “cultivating” are contemplated an increase in the amount of the biological agent as a result of growth of the biological agent on or in conjunction with the substrate.
• a biological agent may be desirably cultivated for its ability to produce a biological product (e.g., the product penicillin is obtained from mycelial cells) .
• “Culturing” (and “cultivating”) of the biological agent thus are also intended to encompass obtaining a desired biological product directly, as well as obtaining the biological agent itself.
• biological agents that may be cultured and recovered in accordance with the invention include bacteria, yeasts, spores, fungi, molds, plant and animal cells, and generally any biological agent as may be known or as hereinafter may be discovered.
• the agent preferably is an aerobic agent, but also may be an agent that has an anaerobic activity.
• the biological products that may be recovered include enzymes, amino acids, vitamins, organic acids, extracellular proteins, antibodies, and, in general, any biologically produced material.
• the invention may be employed in connection with the cultivation of cellulase-producing biological agents, which cultivation preferably is accomplished on a cellulosic substrate.
• hydrolyzing cellulases include three enzymes, endoglucanase (EC 3.2.1.74, commonly known as C x ) , exoglucanase (EC 3.2.1.91, also known as C x ) , and cellobiase (EC 3.2.1.21). These enzymes work together to convert cellulose into glucose. The activities of these enzymes are summarized below:
• G 2 - 2G ⁇ wherein G represents an anhydroglucose unit, and can range from about 2,000 to 10,000 in cellulose (G n thus representing cellulose) ; and wherein n, m, and p are integers wherein n > m > p.
• endoglucanase cleaves the cellulose molecule into two anhydroglucose chains; exoglucanase cleaves cellobiose
• G 2 composed of two anhydroglucose units
• cellobiase converts the cellobiose to glucose.
• Aspergilous niger also is a producer of cellulase enzymes, although some strains of A . niger are known to be producers of cellobiase only.
• the invention is not limited to cultivation of the foregoing biological agents, but instead is contemplated to be generally applicable to the cultivation of any suitable biological agent.
• the invention generally contemplates both a culturing apparatus and a method for cultivating a biological agent.
• the apparatus is generally shown in
• the apparatus 100 includes a source 101 of gas, which source may be, for example, an air compressor 102, an oxygen tank 103, or any other suitable gas source.
• the gas preferably is an oxygen containing gas, such as purified oxygen or air, but it is further contemplated that a different gas may be used in connection with the invention in some embodiments.
• the source 101 may include a tank of compressed nitrogen (not shown) .
• the gas optionally but preferably is filtered through a filter 105 and is humidified at humidifier 106, either of which may, if desired, be provided with a heating and/or cooling mechanism, such as a coiled jacket containing heating and/or cooling coils (not shown) .
• humidifier 106 either of which may, if desired, be provided with a heating and/or cooling mechanism, such as a coiled jacket containing heating and/or cooling coils (not shown) .
• the air passes into a culturing vessel 108 that includes a gas- permeable culturing bed 110 disposed within an interior space defined by a wall 112 thereof.
• the culturing bed 110 comprises a substrate that includes a biological agent disposed thereon, the substrate being biologically conducive for the growth of the biological agent.
• the substrate preferably is selected for compatibility with the biological agent to be cultured, and thus, for example, when cellulase enzymes are being cultured, the substrate may comprise recycled paper fibers, wood chips, or other cellulose- containing source .
• Other substrate suitable for use in conjunction with the invention includes grains, such as wheat bran, cracked corn, whole grain rice, and other organic materials such as soluble proteins. More generally, any substrate that provides physical and
• the bed allows gas to flow therethrough in convective thermal communication with at least a portion of the biological agent in the bed and/or in convective mass transfer communication with at least a portion of the biological agent.
• the substrate may comprise an inorganic material (such as a diatomaceous earth) in admixture with a nutritive substrate such as urea, grain, or other suitable nutrient. The selection of a particular substrate for a given biological agent to be cultivated is contemplated to be within the level of ordinary skill in the art .
• the substrate is provided in the form of a solid phase substrate, by which is contemplated a porous or gas-permeable substrate that preferably is wet (i.e., has sufficient moisture to promote the growth of the biological agent) , but that is not submerged in a liquid bath.
• the substrate is initially provided in the form of wet discrete plural packings, the substrate packings being packed in the bed with sufficient void volume to allow the bed to be permeable to at least compressed gas. It is contemplated that the substrate packings will form a friable cohesive mass after the biological agent has been allowed to grow for a sufficient length of time, and thus no longer may be identifiable as discrete packings.
• the vessel 108 shown in Fig. 1 is equipped with two gas inlets 115, 116, each having a gas inlet valve 115A, 116A, controlled by a respective valve actuator 115B, 116B.
• the vessel also is equipped with a gas outlet 117 and a gas outlet valve 117A which is operated by a gas
• valve actuator 117B 11 outlet valve actuator 117B.
• the valve actuators further may be controlled by a controller 120, as discussed in more detail hereinbelow.
• the controller, valves and accompanying valve actuators are deemed optional in connection with the invention, and thus the vessel may be equipped with none, one, two or all three of the illustrated valves 115A, 116A, and 117A, or may be equipped with further valves and valve actuators if desired.
• the gas passes into the vessel 108 via one or both of gas inlets 115, 116.
• the gas inlet or inlets preferably are charged with water vapor prior to introducing gas to thereby avoid drying out of the culturing bed.
• gas leaving the humidifier 106 passes along either or both of paths 118, 119 through respectively valves 115A and 116A and gas inlets 115 and 116 (the paths 118, 119 being shown in broken lines as optional alternatives) .
• the vessel 108 preferably includes a head space 121 proximal a boundary 122 of the bed 110 and a bottom space 124 proximal another boundary 126 of the bed 110.
• the bed 110 may rest on a screen 127 within the vessel 108, the screen 127 permitting fluid flow therethrough but not permitting solid contents of the bed 110 to pass into the bottom space 12 .
• Entering gas may pass either into the head space 121 via gas inlet 115, or the bottom space 124 via gas inlet 116 (or via optional path 128 via gas inlet 115, path 128 being shown in broken lines as an optional alternative) .
• gas is introduced to the vessel in a manner such that gas flows through at least a portion of the bed 110. It is contemplated in preferred embodiments of the invention that the gas will flow through the
• the bed 110 comprises discrete plural particles 130 of substrate with a biological agent disposed 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 arrow 136.
• the first and second boundaries 131 and 135 are preferably but not necessarily coextensive with the boundaries 122, 126 between the bed 110 and the head space 121 and bottom space 124 respectively (as shown in Fig. 1) .
• gas may be introduced into the bottom space 124 of the vessel 108 through gas inlet 116 under a pressure greater than ambient pressure. The gas will flow from the bottom space 124 through the bed 110 into the head space 121 and out the gas outlet 117.
• the vessel is pressurized by introducing gas at the head space through gas inlet 115 with the gas outlet valve 117A being closed. Gas will flow into the bed 110, even if the vessel is not equipped with a bottom space. The gas outlet valve 117A then may be opened to allow gas to escape from the vessel 108.
• the biological agent is disposed on the bed, i.e., on a surface of the bed or within the bed.
• the biological agent is homogeneously dispersed throughout the bed. While it is not intended to limit the invention to a particular theory of operation, it is
• the passage of gas through the bed may also affect heat transfer and temperature within the bed.
• increasing the flow of oxygen to the bed may cause the temperature within the bed to increase or decrease. It is believed that the increase in oxygen flow rate will cause the metabolic activity of microorganisms with in the bed to increase (thus tending to increase the temperature within the bed) but also increasing the convection of heat away from the bed (thus tending to decrease the temperature within the bed, so long as the gas is at a temperature lower than that of the bed) .
• the rate at which the bed temperature is caused to increase as a result of microorganism activity may be more than, less than, or equal to, the rate at which the temperature is caused to decrease as a result of convection of heat away from the bed.
• the flow rate of the gas, and other factors the temperature within the bed may be caused to increase or to decrease by increasing the oxygen flow rate.
• the effect of the flow rate preferably is empirically determined for a given apparatus and process.
• heat may be removed from the bed by flowing an inert gas (such as nitrogen) through the bed.
• an inert gas such as nitrogen
• non-humidified gas or gas that has a low humidity, may be introduced to thereby cause evaporation of water vapor from the bed and to thereby remove latent heat from the bed.
• the gas inlet may not be charged with water vapor prior to introducing gas into the bed, thus causing evaporative cooling of the bed.
• the gas may be cooled prior to entering the bed, or the vessel may be equipped with cooling coils (not shown) .
• the apparatus is operated in a "pressure pulsing" mode.
• pressure pulsing in one embodiment is contemplated cyclically pressurizing and depressurizing the vessel.
• An example of the pressure profile within the vessel generated in accordance with such a pressure pulsing is shown in Fig. 2 (pressure being given as gauge pressure) .
• Pressure pulsing also encompasses cyclical increasing and subsequently decreasing the cyclical flow rate of gas through the bed.
• cyclical is contemplated repeating the pressurization/depressuriza- tion or increase in flow rate/decrease in flow rate operations at least once, and more preferably, at least five times, after the initial pair of operations is completed.
• the pair of operations may be
• the pressure pulsing may be accomplished using various embodiments of the apparatus shown in Fig. 1, as well as in any other suitable manner.
• the vessel may be equipped with a gas inlet 115 with no gas inlet valve, and a gas outlet with gas outlet valve 117A.
• Pressure pulsing then may be accomplished by closing the gas outlet valve 117A, allowing the pressure within the vessel to build and preferably to hold when the pressure within the vessel reaches that pressure of the gas incoming through the gas inlet 115, subsequently opening the gas outlet valve to thereby allow the pressure within the vessel to decrease to ambient pressure, and repeating this operation.
• gas may enter the vessel through gas inlet 116, which may or may not be equipped with a gas inlet valve, or may come in through gas inlet 115 via optional path 128.
• gas enters the vessel through both gas inlets 115 and 116, at least gas inlet 116 of which is equipped with a gas inlet valve 116A. Pressure pulsation may be accomplished by periodically opening and closing gas inlet valve 116A to allow respectively greater and smaller amounts of gas to enter the vessel 108.
• the gas inlet and outlet valves 115A, 116A and 117A may be manually operated.
• each valve is equipped with a valve actuator 115B, 116B, and 117B respectively, each of which modifies the state of its respective valve (for example, by fully opening or fully closing each valve or by incrementally increasing or decreasing the amount of fluid that may flow through said valve) .
• valve actuators may be integral with the vessel or may be remote from the vessel.
• the valve may be associated with the gas source (such as the valve on a pressure vessel) , or may be associated with the compressor.
• the valve actuator may be, for example, a switch for actuating the compressor (the compressor thus serving as a valve) .
• the valve actuator may be, for example, a solenoid actuator, the gas valve thus comprising a solenoid valve.
• the valve and actuator may be any other suitable devices.
• the valve actuators may be controlled by a controller 120.
• the apparatus is equipped with a temperature sensor 140 measuring the temperature of the bed, and/or one or more pressure sensors 141, 142, measuring a pressure within the vessel 108 (it being contemplated that the pressure reported in the head space by pressure sensor 141 may differ from that reported in the bottom space by pressure sensor 142) .
• the controller 120 communicates with the temperature sensor 140 via line 144, and communicates with the one or more pressure sensors via lines 145, 147.
• the controller further communicates with valve actuators 115B, 116B, 117B via lines 148, 149, and 150 respectively .
• the controller may be any electronically or otherwise operated mechanism.
• the controller may comprise simple control logic circuitry, such as a wired circuit, or may comprise a timer.
• the controller comprises a microprocessor or microcontroller 152 including a timer 153, a data bus 154, and an I/O interface 155 via which the sensors and
• valve actuators communicate with the microprocessor or microcontroller 152.
• the sensors provide signals to the controller to thereby communicate temperature or pressure data to the microprocessor or microcontroller 152, and the microprocessor sends control signals to the valve actuators 115B, 116B, and 117B for modifying the state of the gas inlet and/or outlet valves.
• the microprocessor or microcontroller or the logic circuitry may be programmed via any suitable manner for accomplishing pressure pulsation. For example, if the pressure pulsation is accomplished with a microcontroller or microprocessor, via opening and closing the gas outlet valve, one suitable control program is diagrammatically illustrated in Fig. 4. At step 160, a delay register in the microprocessor or microcontroller is reset with a closed reference time, i.e., the length of time that the gas output valve should remain closed. After the timer has indicated the passage of this amount of time, the microprocessor or microcontroller, at step 161, sends an open valve signal to the gas outlet valve actuator.
• a closed reference time i.e., the length of time that the gas output valve should remain closed.
• the delay register is reset with an open reference time, i.e., a value indicating the amount of time that the gas outlet valve should remain open. Subsequently, after such time has passed, a close valve signal is sent to the gas outlet valve actuator at step 162.
• the open reference time and closed reference times preferably are empirically determined for the given apparatus and method, and may take into account the amount of time required for the valve actuator to accomplish respectively opening and closing of the valve. It is further contemplated that an operator may terminate the control loop at any time desired. If pressure pulsation is to be accomplished via
• Fig. 5 is one appropriate program for the microprocessor or microcontroller, steps 164-167 corresponding substantially to steps 160-163 shown in Fig. 4. It should be understood that the control programs shown in Figs. 4 and 5 and in the subsequent figures, while illustrated as control programs for a microcontroller or microprocessor, may be implemented by logic circuitry or by other suitable control mechanisms.
• the temperature within the bed may increase as the rate of metabolic activity within the bed increases.
• the heat may cause premature termination of the growth of the biological agent, for example, if the temperature reaches an undesirably high level.
• the microprocessor or microcontroller may be programmed in accordance with the control logic shown diagrammatically in Fig. 6 with respect to the control of a gas outlet valve.
• a reference temperature, or desired maximum operating temperature of the vessel is obtained, for example, by receiving user input or a stored memory variable.
• the microprocessor or microcontroller receives a signal from the temperature sensor, and at step 172, this temperature received is evaluated as against the reference temperature. If the received temperature is not yet as great as the reference temperature, after a delay 173 control passes to step 171. If, on the other hand, the temperature within the bed has reached or exceeded the
• a signal is sent to the gas outlet valve actuator at step 174 to thereby cause the gas outlet valve to open and to thereby cause depressurization of the vessels.
• a delay 175 another signal is sent to the gas valve actuator at step 176 to thereby cause the gas valve to close.
• Control passes to step 171 after another delay 177. It is contemplated that the delays at steps 173, 175, and 177 and the reference temperature may be empirically determined for a given apparatus and biological agent.
• the reference temperature preferably defines or is below the maximum temperature within the desired activity range of the biological agent.
• the microprocessor or microcontroller may be used to control a gas inlet valve actuator.
• the program is comparable to that shown in Fig. 6 except that a close valve signal is sent at step 178 and an open valve signal is sent at step 179.
• a further alternative program is shown in Fig. 8.
• a gas outlet valve is caused to open when the temperature within the bed has reached a first reference temperature, and the gas outlet valve is caused to close when the temperature within the bed has fallen to a second reference temperature.
• the first and second reference temperatures are obtained as step 180, such as by receiving input from a user or by retrieving data valves from memory storage.
• Steps 181-185 are comparable to steps 171-175 respectively of the embodiment shown in Fig. 6.
• a signal is again received from the temperature sensor at step 186.
• this temperature is compared to the second reference
• Fig. 9 illustrates a similar embodiment wherein the microprocessor or microcontroller is used to control a gas inlet valve actuator. In this embodiment, a close valve signal is sent at step 191 and an open valve signal is sent at step 192.
• the valve may include one or more pressure sensors 141, 142.
• the vessel includes one pressure sensor, which preferably is located in the head space when the gas inlet is in the bottom space of the vessel and is preferably located in the bottom space of the vessel when the gas inlet is in the head space of the vessel.
• Figs. 10-13 are comparable to Figs. 6-9, respectively and diagramatically illustrate the programming of the microprocessor or microcontroller whereby the gas inlet or outlet valve may be opened or closed in response to pressure changes within the vessel .
• Steps 170' -192' are comparable to steps 170-192 in Figs 6-9.
• Fig. 10 illustrates operation of a gas outlet
• FIG. 21 in which an open valve signal is given (at step 174') when the pressure has reached at least a reference pressure, and a close valve signal is subsequently given (at step 177') after a delay 176'.
• Fig. 11 is comparable to Fig. 10 and shows the control of a gas inlet valve, wherein an open valve signal is given at step 178' and a close valve signal at step 179'.
• Fig. 12 illustrates operation of a gas outlet valve wherein an open valve signal is given (at step 184') when the pressure has reached at least a first reference pressure and a close valve signal (at step 189') when the pressure has fallen to or below a second reference pressure.
• Fig. 13 is comparable to Fig. 12 in the operation of a gas inlet valve, wherein a close valve signal is given at step 191' and an open valve signal at step 192' .
• the pressures and delays in the foregoing programs may be empirically determined for a given apparatus or biological agent .
• the vessel 108 may be provided with a liquid intake port 107 and a drain 109.
• the biological agent may be recovered via removal of the liquid through the drain 109.
• the biological agent then may be continue to be cultivated. Water or nutrients may be added as may be appropriate .
• the following non-limiting Examples are provided for illustration of the present invention.
• a two liter New Brunswick glass jar fermentor was equipped with a metal mesh screen to hold a wet porous solid bed having a depth of about 10 centimeters.
• the fermentor was equipped with a gas inlet for introducing air to the bottom space of the fermentor (beneath the screen) , a gas outlet valve leading from the head space (above the bed) , a gas outlet solenoid valve (Omega Technologies Co.) and an electrical timer (ChronTrol) serving as a controller. Air was allowed to flow into the bottom space and then upwards through the porous bed into the head space . Pressure pulsation was created by periodically opening and closing the gas outlet valve.
• the bed included a substrate with an Aspergillus niger culture disposed thereon.
• Example 1 was repeated without pressure pulsation. Only the bottom portion of the porous bed near the air inlet became blackened, and the central portion of the bed became tightly packed, with little mycelial growth and even less spore formation.
• the enzyme productivity was calculated to be 806 FPU/hour-liter for cellulase complex from Trichoderma.
• FPU filter paper unit
• One international "filter paper unit” is defined to be the amount of cellulases that can produce one micro-mole of glucose per minute from cellulose.
• a Trichoderma culture was cultivated on recycled paper fibers in a solid phase fermentation in a 1000 ml fermentor (a laboratory Erlenmeyer flask) to produce high potency cellulases.
• the fermentation was accomplished by placing the flask at room temperature on a laboratory bench top. After the fermentation was completed, the whole wet solids were air dried to become the final enzyme product.
• This product contains 246 FPU/gram, from which the productivity of the fermentor can be calculated to be 234 FPU/hour-liter of fermentor volume.
• the productivity of the solid phase fermentation was found to be higher than those of submerged fermentation of different Trichoderma cultures, reported in the literature and collected in the following Table:
• COMPARATIVE EXAMPLES 2-5 A mixture including 200 g corn fiber (14% moisture content, from A.E. Staley, Decatur, IL) , 60 g ground corn, 30 ml corn steeping liquid (A.E. Staley), 4.0 g (NH 4 ) 2 S0 4 , 2.0 g K4 2 P0 4 , and 600 ml water was prepared (final moisture content was 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 solid A . Niger culture in a septic hood.
• the substrate mixture prepared as discussed above was divided and transferred to four 250 ml Erlenmeyer flasks as follows:
• Comparative Example 2 30 g Comparative Example 3 60 g Comparative Example 4 40 g (with 100 ml water) CCoommppaarraattiivvee EExxaammppllee 55 40 g (with 100 ml 5% glucose solution) .
• the mixture of Comparative Examples 2, 4, and 5 were set in a 30° C shaker at 200 rpm to begin fermentation.
• the mixture of Comparative Example 3 was allowed to ferment at ambient temperature without shaking.
• a method for cultivating a biological agent has been provided, and also an apparatus useful in accomplishing same.

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