WO2010032260A1

WO2010032260A1 - Stacked basket bioreactor for solid state fermentation

Info

Publication number: WO2010032260A1
Application number: PCT/IN2009/000441
Authority: WO
Inventors: Sameer Sudhir Kulkarni; Sudhir Narayan Kulkarni; Eruch Pesi Gorwalla
Original assignee: Sameer Sudhir Kulkarni
Priority date: 2008-08-05
Filing date: 2009-08-05
Publication date: 2010-03-25
Also published as: WO2010032260A4

Abstract

The invention concerns a stacked basket bioreactor for solid-state fermentation comprising two or more all over perforated baskets which are stacked one above the other and which comprise a circular base plate (1 ) with concentric inner and outer wall (2), pellets of solid substrate (5) packed inside each basket, a jacket (10), a lid (14) with a plurality of ports (17, 18, 19), and a central supply pipe (11 ).

Description

STACKED BASKET BIOREACTOR FOR SOUP STATE FERMENTATION

DESCRIPTION OF INVENTION

AIMS OF SOLID STATE FERMENTATION

Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence of free water. The absence of free water makes the system quite different from submerged liquid fermentations and makes SSF superior for the production of some products.

The commercially successful large scale SSF processes are for the production of soy sauce koji and other traditional fermented foods, citric and gluconic acids, and fungal enzymes such as cellulases, amylases, Upases, and pectinases (1,2). In addition, fungal spores have been produced by SSF for use in steroid transformations and as inocula for production of blue-vein cheeses.

MICROBIOLOGICAL ASPECTS

Bacteria, yeasts and fungi can grow on solid substrates, and find application in SSF processes. Filamentous fungi are the best adapted for SSF and dominate in research works owing to their physiological, enzymological and biochemical properties. The hyphal mode of fungal growth and their good tolerance to low water activity (a_w) and high osmotic pressure conditions make fungi efficient and competitive in natural microflora for bioconversion of solid substrates.

ADVANTAGES OF SSF OVER SUBMERGED FERMENTATION (SmF)

1. Unlike SSF, SmF require stringent Aseptic conditions. This is due to the fact that other competing Microorganisms have a fairly high chance of growth in Liquid Medium and so risk of contamination is higher in SmF.

2. SSF has been shown to produce a more stable product.

3. Low yield in SmF as compared to SSF process thus requiring large volumes of Equipments.

4. In SmF High volumes of water is consumed resulting in high downstream processing costs. However in SSF there is limited consumption of water consumption due to low Water Activity 5. In SmF there are limitations to the solubility of Oxygen in the Liquid medium thus requiring high volumes of Air leading to higher compression costs. The growth mediums being solid exclude the problem of oxygen solubility.

6. High power consumption as most SmF' s are continuously Agitated. Ideally the SSF operation is preferred to be static.

7. Extremely high volumes of polluting Effluents in SmF as compared to SSF.

STATE OF THE ART

U. S Patent Documents United States Patent 6197573 United States Patent 6620614

SSF bioreactors may be of static (fixed-bed) or agitated (stirred) design. Tray and packed bioreactors are most commonly used among the former, whereas rotating drums, periodically agitated tanks, and rocking and fluidized-bed bioreactors are examples of the latter. Most SSF bioreactors operate in batch form, although water and nutrients are added periodically in many systems.

a. Tray Bioreactor - Trays, one of the simplest types of SSF bioreactor, have been employed in the Far East for many years. The main characteristics of this type of bioreactor are as follows:

1. A set of trays are covered with a thin layer of solid substrate.

2. Trays are kept inside a chamber, normally under controlled environment.

3. Forced air is not commonplace, although trays can be perforated allowing air to circulate smoothly through their bases in order to regulate the temperature and provide oxygen.

4. In some cases the solid bed is agitated gently, either manually or automatically, using simple mechanical devices.

The main drawback of this type of bioreactor is that it is highly labor intensive, so that automation of large-scale processes is very difficult. Also, because the solid layer in the tray must be thin and the trays cannot be too close to one another, very large chambers are required to achieve industrial levels of production. Industrial applications of this type of reactor include production of mushrooms (Agaricus bisporus) and tempeh as well as aseptic production of soy sauce koji mold spore inoculum.

b. Rotating Drums. Features of rotating drums used as bioreactors include the following:

1. A partially inclined cylinder is filled with substrate up to a predefined level.

2. Smooth cylinder rotation mixes the solid bed; this process is usually enhanced with baffles.

3. Aeration is occasionally provided by means of tubes placed in the free space of the cylinder or in its shaft.

Rotation is controlled to avoid damaging the microorganisms or causing substrate agglomeration or particle breakup. The control of temperature in this type of bioreactor is very difficult and becomes more difficult when increasing the scale of the reactor.

c. Fluidized-Bed Bioreactors. This bioreactor is a vertical cylinder inside which the solid substrate rests on a perforated plate. Air forced through the plate at high flow rates suspends the solid in the air, causing the solid-air mixture to behave like a fluid. The necessary air for keeping the fluidized bed up is conducted in a circulation. The air must be kept with an exactly calculated moisture content. This procedure requires a lot of energy for keeping the fluidized bed up.

d. Packed-Bed Bioreactors. Packed-bed bioreactors are normally cylindrical tanks, filled with a solid substrate suspended on a perforated surface through which air is forced axially through the bed. The inlet air conditions (temperature, humidity, and flow rate) are generally regulated. In larger systems, cooling fluid is forced through double walls, coils, or plates (Zymotis Bioreactor) immersed in the solid bed to overcome the difficulty of removing metabolic heat. The main advantage of this construction is large quantities of substrate can be processed without significant investment in capital cost.

The main disadvantage of this type of bioreactors is its propensity for heterogeneous growth that hinders scaling up to industrial size.

e. Stirred Bioreactors. Stirred bioreactors are probably the type of SSF bioreactor most widely used at the industrial scale. Here the solid bed is agitated by screws, palettes, or other mechanical devices. Agitation is intermittent and is varied during the process. These bioreactors may be either vertical or horizontal. Problems for the use of large amounts of substrate are inevitable for this type of reactor, as these amounts can no longer be moved evenly without causing destructions in the structure of the substrates.

f. Modular Fermenters of Biocon (United States Patent 6197573) & Prophyta Biologischer Pflanzenschutz (United States Patent 6620614): These two bioreactors use a number of thin beds coupled with cooling plates oriented normal to the air flow. The difference between the two bioreactors is that in the Prophyta design the same air passes through each successive bed while in the Biocon' s Plafractor design the air is introduced separately into each bed. Also the Plfractor incorporates mixing arms for each tray or module to mix the contents during fermentation. Even though these designs employ cooling plates for the fermentation temperature control & modular construction, the major drawback of such designs is the complicated assembly leading towards increased Bioreactor downtimes. Also in the Plafractor design the mixing arms included in each module & dedicated valves for each set of modules further complicates the construction & increases the overall cost of this particular Bioreactor. SUMARY OF THE PRESENT INVENTION

The primary task of the present invention was to develop a SSF Bioreactor because the Packed Bed Bioreactor is most suitable for a large number of Industrial applications. The SSF Bioreactor abolishes the major disadvantages of the Packed Bed Bioreactor, which are highlighted as below.

1. Due to the natural granular form of the solid substrate, the dense packing of the bed and due the subsequent formation of the Aerial hyphae during the cultivation period, the hyphae that grow into the inter-particle spaces are not disrupted or squashed onto the particle surface, and therefore these hyphae represent an extra impediment to air flow, increasing the pressure drop. This leads to higher compression costs.

2. Channeling is a potential problem in packed beds. Channeling is problematic because air will flow preferentially through the cracks, such that in the regions of the bed where the particles are bound together, there will be no bulk flow, such that Oxygen transfer will be limited to diffusion and heat transfer will be limited to conduction.

3. During Aeration i.e. the axial or vertical flow of Air through the Packed Bed, Oxygen gets consumed as the air progresses upwards. Therefore the top region of the Bed shall have air consisting of lower amounts of Oxygen concentration, thus limiting the vertical height of the Bed.

4. By release of metabolic heat, there are temperature gradients, both axial as well as radial, inside the bed, which can be evacuated through an increased air-circulation (evaporation coldness) in such reactors. However the temperature of Air, as it progresses unidirectionally upwards, continues to rise. This leads to lower temperature gradients in the top regions of the Bed due to which the Heat transfer in these regions is not appreciable, again limiting the vertical height of the bed.

5. Another way of relinquishing the heat is by providing cooling fluid jacket on the side walls of the bioreactor. However for solid particles which have low thermal conductivity and low heat capacity the provision of this arrangement shall be quite ineffective. Also by incorporating such cooling surfaces, the cost of the Packed Bed Bioreactor increases significantly.

6. For SSF operation, where the spent substrate has to be discarded after the completion of the Fermentation process and the Bioreactor has to be filled with fresh load of substrate for fermentation, the downtimes during which the Bioreactor remain idle in-between these steps is comparatively high for operations at large scale.

Therefore, it is an objective of this invention to provide an improved packed bed bioreactor which is easily adaptable for reactions involving solid substrates for solid state fermentation. It should include the following features.

1 : Avoiding foreign contamination inside the Bioreactor by maintaining strict Aseptic conditions during the Fermentation process.

2. Provide High surface area for proliferation of microorganisms.

3. Evacuate the Heat without drying out the substrate

4. Provide free flow of air through the packed Bed without much restriction during the fermentation process in order to minimize the compression costs

5. Provide good aerodynamics for the uniform passage of Air during the fermentation process to avoid channeling of Air and to maintain uniform air flow.

6. Guarantee uniform Oxygen supply to all the growing microorganisms.

7. Avoid intermixing of substrate particles during the fermentation process

8. Provide a design which is viable for scale up.

9. Reduce the Bioreactor downtimes and reduce the labor for loading and unloading of Bioreactor.

10. To design a cost efficient Bioreactor, without compensating any of the above mentioned points. INVENTIVE CHARACTER OF PRESENT INVENTION

The inventiveness of this present Invention lies in

1. Forming Pallets from aggregates Solid substrate. The palletized media is large enough so that the forced air can pass freely in between them and therefore increase Convection Heat transfer within the bed and prevent channeling.

2. By using palletized media channeling is prevented, thus lower rate of air circulation are required & there is no likelihood of the bed drying out.

3. High Bioreactor average volumetric productivities by increasing the ratio of the surface area of solid substrate available for the propagation of microorganisms to the total volume of the Bioreactor.

4. Utilizing Radial flow of Air and other Nutrients during the cultivation process in order to decrease the distance of flow through the Bed as compared to the column of same dimensions utilizing axial flow of Air or other Nutrients.

5. Ease in scale-up of SSF because the performance of a Fermenter using the palletized media will be the same whether a small or a large Fermenter is used since the air spaces between the pellets will remain the same.

6. Formation of fungal mats or caking of the bed is eliminated, because in palletized media the inter-pellet distance between two pellets is greater than the inter-particle distance of granules in a granular substrate medium.

7. Ease in loading & unloading of substrate particles by using Baskets, packed with the palletized media, stacked on above the other.

8. Simplicity in construction & operation.

DESCRIPTION OF DRAWINGS

Fig. 1 - schematic drawing of the Baskets.

Fig. 2 - section of the assembled Bioreactor laden with the Baskets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to the Drawings, it is seen in the embodiment of a Bioreactor illustrated in Fig. 2 the overall construction of the Equipment.

As shown in Sheet No.l, the Bioreactor consists of plurality of Baskets as shown in Fig. 1. Each Basket consists of a Circular Base 1, cylindrical outer pipe or wall 2 and a cylindrical Inner Pipe or Wall 3. As per the properties of the fluids and the Process requirements the Inner wall, the outer wall and the base are perforated with plurality of holes or perforations 4. In other preferred embodiment the cylindrical walls could be constructed from Wire screen, Knitted wire mesh, Filter screen or porous filter material. Yet in other preferred embodiment the Baskets may be rectangular, oval or prismatic in shape.

Now as shown in Sheet No.2, the Pellets of Solid substrate 5 are placed or packed uniformly between the Inner Cylindrical Wall 3 and the Outer Cylindrical Wall 2. The Pellets of Solid substrate 5 for Solid substrate fermentation could be Pellets of cassava, or rice bran or wheat bran etc. The Pallets are essentially spherical in shape. However the Pallets may be formed of any other geometrical shape which provides for higher viable surface area for cultivation of microorganisms. In another preferred embodiment the porosity of the Pallets could also be increased, by lowering the Bulk Density of the pallets, which could provide higher surface area viable for the cultivation of microorganisms.

The flow of Nutrient Medium or Air is from the Inner Pipe 3, passes through holes 4 into the solid bed of pallets 5 and exits from the outer pipe 2 horizontally. In other preferred embodiment, the flow of Nutrient Medium is from the outer Pipe 2, passing through the solid bed of pellets 5 and exit from the inner pipe 3 horizontally. The flow so induced within each basket is Radial flow. The content of the basket, which is located at the top, is tightly covered with lid 6. Additionally the Inner Pipe 3, of the Top Basket, is also sealed with circular plate 7. These arrangements are provided, so that the fluids don't bypass this top bed.

For Bioreactors requiring designs for large production capacities, the plurality of baskets Fig. 1, which are filled with the Pellets of Solid substrate 5, are stacked one above the other as shown in Fig. 2 and the rubber gasket 8 prevents the fluids or the nutrient medium or Air to bypass the bed. The stacking of baskets Fig. 2, filled with solid pallets 5, also prevents the pallets located at the bottom of each basket from getting crushed. This is because the hydrostatic vertical force experienced by the pallets located at the bottom of the basket is limited to the vertical height of each basket rather than the overall vertical height of the stacked assembly. At the same time it is easier to load each individual basket, evenly with the requisite quantity Pallets of Solid substrate 5. Those skilled in the art will realize that due to such tight packing of Pellets of Solid substrate 5, the porosity of the Bed is evenly maintained so that there is no channeling through the Bed.

In other preferred embodiment the number and the diameter of holes 4 in each Basket is varied from the lowermost Basket to the topmost Basket. They increase from the Bottom to the top. Those skilled in the art will realize that for taller Bioreactors there is a decrease in the Pressure of the fluid Medium as it travels upward through the Central supply Pipe due to friction. This results in uneven fluid distribution through each Basket. Those skilled in art will understand that such misdistributions can be prevented by varying the Area required for the Fluid to pass through it that is by varying the Diameter and the Number of Holes 4 in the Inner Pipe of each basket.

The Baskets are stacked one above the other Fig. 2, by inserting these individually inside the Bioreactor assembly Fig. 2. The Bioreactor shell consists of the Outer wall or Shell 10, the top cover Hd 14 and the bottom Base 13. The stacked assembly of baskets rests on the Base 13. These baskets are held firmly by the Bolting Device 9, fixed to the Shell 10, which firmly holds the stacked basket assembly during the process operation. In other preferred embodiment the length of the Bolts, through the bolting device 9 may be varied, in order to accommodate requisite number of baskets which are stacked one above the other. The Bolting device also serves to maintain a positive seal in between the Baskets by tightly compressing the Rubber Gasket 9 in between two succeeding baskets.

The Bioreactor Base 13 is secured to the Shell 10 with Flange 12. This Base consists of a Central Supply Pipe 11. Nutrient Medium, Air or Extraction fluid is fed through ports 20 & 21 which are mounted on this Central Supply pipe as shown in Fig. 2. The Inner pipe 3 of each Basket is concentric to the Outer Cylindrical Wall. The Baskets, which are stacked one above the other, are all concentric to the Central Supply Pipe 11. In nutshell the Central Supply Pipe 11, the Inner pipe 3 and Outer pipe 4 of each individual stacked Basket and the Bioreactor shell 10 are all concentric.

The Bioreactor is tightly closed with Lid 14, thus isolating the contents of the Bioreactor from the Outer environment. This results in Aseptic environment to prevail inside the Bioreactor, during the Cultivation of Microorganisms on the Pellets of Solid substrate 5. The Lid 14 also mounts various ports via 17, 18 & 19 for supply of Nutrient & Inoculum or for recovery of the extraction fluid and Air.

A circular ring 15 with plurality of Spray Nozzles 16, constructed from a hollow Pipe is mounted inside the Lid 14 and placed concentric to the Bioreactor shell 10. Inoculum, which is pumped through port 17, passes through this ring 15 into the annular region between the Bioreactor Shell 10 and the Outer cylindrical pipe 2 of the stacked baskets. This arrangement is so provided to uniformly distribute all the Liquid in this annular region, so that the Liquid penetrates through all the Baskets containing pellets of Solid substrate 5, at an equivalent rate.

Port 20 is used for sending air/steam inside the Bioreactor and port 19 is used for recovering the air after it has traveled through the Bed. In another referred embodiment vacuum may also be applied at port 20. Port 21 is used for pumping the extraction fluid or any other liquid medium inside the Bioreactor. The spent extraction fluid is recovered from port 18. In another preferred embodiment this extraction may be recovered from pluralities of port 22.

BIOREACTOR OPERATION

A. FILLING THE BIOREACTOR WITH A SOLID PARTICLES

The individual empty baskets may be washed and sterilized, in other Equipments like

Washer and Sterilizer (Not Shown), prior to loading them with Pellets of Solid substrate 5.

The baskets are then loaded with the requisite quantity of Pellets of Solid substrate 5.

Then each basket is stacked one above the other inside the Bioreactor by inserting each Basket individually inside the Bioreactor.

In another preferred embodiment, all these baskets could be stacked outside the

Bioreactor and the entire assembly of stacked baskets may be loaded inside the

Bioreactor shell. The Lid 14 is then tightly closed to isolate the contents within the

Bioreactor from the surrounding environment.

B. STERILIZATION OF SOLID PELLETS

For certain operations where it is absolutely necessary to sterilize the solid particles 5 inside the Bioreactor, the Bioreactor if first vacated of the air inside by applying

Vacuum through port 20.

Steam is then charged inside the Bioreactor through port 20 and this is maintained at sterile temperature for a definite period, in order to ensure that the Pellets of Solid substrate 5 are very well sterilized. Once the sterilization cycle is complete, cold sterile air is allowed in through port 20 to cool the substrate to required fermentation temperature.

The Pellets of Solid substrate are now ready for Inoculation. C. SOLID STATE FERMENTATION

Inoculation: Under strict aseptic conditions, Inoculum is pumped inside the Bioreactor through port 17, in order to cultivate the bacteria or the Fungus. The inoculum, which is used for the inoculation of the Fermenter, consists of, but not limited to, a highly concentrated suspension of small germinable units (preferably of spores, conidiospores or bacterial germs) of the microorganisms to be cultivated. The Baskets are completely submerged inside the Liquid Inoculum. The Inoculum is allowed to stay in the Bioreactor for a specific period, so that the solid substrate pellets 5 adsorb the Inoculum.

Cultivation: The residual Inoculum is then drained from the Bioreactor and Sterile Air of requisite humidity and temperature is continuously passed across this bed in order to provide Oxygen to the growing cells.

In other preferred embodiment the Bioreactor could be operated in fed-batch mode i.e intermittently supplying Nutrient Medium to the growing Fungus.

Control of Fermentation Temperature: The process of fermentation releases the Metabolic Heat. The heat so generated must be removed efficiently so as to prevent the increase of temperature of the solid bed. By circulating air at a required temperature through the bed and venting it out through port 19. The released heat is thus extracted efficiently via convection.

Extraction of Products: After the necessary products have been secreted, the Extraction fluid is then passed through the Bioreactor through port 21 in order to pull out the released Products. The spent Extraction fluid is retrieved from port 18. In another preferred embodiment the extraction fluid may be sent intermittently during the process of cultivation to recover the products of fermentation. G. POST CULTIVATION

After all the necessary operations are completed, in certain processes, it is important to kill the cells or microorganisms, so that they don't contaminate the environment or taint the operating personnel, when the top lid is opened. Under such cases a sterilizing medium, which could be either Steam or Sterilizing gas like Ethylene

Oxide or Formaldehyde, is passed inside the Bed to kill the cells.

Other Liquid sterilizing mediums can also be passed through the Bed

H. EMPTYING THE BIOREACTOR

Once all the operations are successfully carried out, the baskets are removed one by one, starting from the topmost by opening the Lid. Once all the baskets are removed outside the Bioreactor, the residual pellets may be disposed and the baskets may be washed and reused for another fermentation operation.

Claims

1. A modular structure of Stacked Basket Bioreactor for Solid State Fermentation for cultivation of microorganism on Pellets of Solid Substrate in a contained environment, which comprises of a. Plurality of baskets, which can be stacked one above the other, to form a stack of Baskets, wherein each basket comprises of i. Circular Base, cylindrical outer pipe or wall and a cylindrical Inner Pipe or

Wall which are concentric to each other, ii. Circular Base, cylindrical outer pipe or wall and a cylindrical Inner Pipe or

Wall having perforations or holes. b. Pallets of Solid substrate are packed inside each individual, vertically stacked Basket. c. Bioreactor Shell d. Lid having plurality of Ports e. Bioreactor base having a Central supply pipe.

2. A bioreactor as claimed in claim 1 having a mechanism, by which the Microorganisms cultivating on the pallets of solid substrate packed inside the bioreactor are totally isolated from the outside environment.

3. A bioreactor as claimed in claim 1, which is made up of any corrosion resistant material like Stainless Steel.

4. A bioreactor as claimed in claim 1, wherein a bolting device is provided for firmly holding the stacked baskets.

5. The length of each bolt in the bolting Device as claimed in claim 4, is altered so as to hold any desired number of baskets stacked inside the bioreactor as claimed in claim 1.

6. A bioreactor as claimed in claim 1, wherein Wire screen, Knitted wire mesh, Filter screen or porous filter material can be used instead perforated cylindrical walls in baskets.

7. A bioreactor as claimed in claim 1, wherein the temperature of the solid bed is controlled by circulation of Air through the Bed via convection.

8. A Central Supply pipe as claimed in claim 1 (e), wherein it is aligned concentrically to the inner walls of the Baskets, having plurality of ports in order to supply nutrients and Air during cultivation, and extraction post cultivation.

9. A Bioreactor as claimed in claim 1 (b), wherein the flow of Air or Nutrient medium is in a radial or horizontal direction through the Baskets filled with pallets of solid substrate.

10. A system of Solid State fermentation system as claimed in claim 1 , which comprises of Pellets of Solid Substrate having multiplicity of geometric shapes and sizes.

11. Pellets of Solid Substrate as claimed in claim 10, wherein the solid state medium is selected from the group consisting of: sawdust, rice hulls, ground corn cobs, corn fiber, nut hulls, soybean hulls, maize bran, wheat, vermiculite, diatomaceous earth, and polyurethane foam.

12. A Lid having plurality of ports as claimed in claim 1, in order to evacuate air or extraction fluid from the Bioreactor.

13. A Bioreactor as claimed in claim 1, which comprises of a circular ring with plurality of Spray Nozzles, constructed from a hollow Pipe is mounted inside the Lid as claimed in claim 12 and placed concentric to the Bioreactor shell.

4. A Bioreactor as claimed in claim 1, wherein the holes on the inner and outer cylindrical walls having increasing size of the diameter & number from bottom to the top stacked Baskets.