Title of Invention

"AN ACOUSTIC MIST BIOREACTOR"

Abstract

An acoustic mist bioreactor which comprising transducer (A) as mount on which the reactor vessel (B) characterized in that the said transducer being placed at a distance of 1-4 cm from the bottom and the said bioreactor (B), the said bioreactor(B) having a glass support (C) with a two tier autoclavable nylon mesh arrangement such as herein described, for placing the hairy root cultures (D), the said bioreactor(B) being connected to a medium reservoir (E) with a silicone tube through a peristaltic pump (F) , the bioreactor (B) is also being connected with a silicone tube to carry the filtered air (PTFE filter 0.22µm) (G) form the compressor (H) to the bottom of the said bioreactor (B) through air sparger (I), the said bioreactor (B) also being provided with an out let for the air (J) which is connected with a silicone tube where the other end of this silicon tube is opened inside a flask (K) containing a moisture absorbent.

Full Text

The present invention relates to a device for an acoustic mist bioreactor. In particular this bioreactor uses an acoustic field to generate the nutrient medium mist for enhanced mass transfer in hairy root cultures. The major utility of this invented acoustic mist bioreactor is in plant biotechnology for example: a) For the biomass production of cultured plant tissues eg. hairy root cultures for obtaining phytochemicals, which can be used in food, chemical, and pharmaceutical industries. b) For the biomass production of plant tissue and cell cultures for obtaining phytochemicals, which can be used in food, chemical, and pharmaceutical industries. c) For mass propagation of plants. Acoustic mist bioreactor offers following advantages: a) Efficient mist generation, which in turn results in efficient mass transfer. b) Enables to run the bioreactor at higher biomass densities, which results in higher productivities. c) High-energy efficiency, since acoustic transducers are high voltage, low energy drawing devices. d) Simplicity in operation and ease of scale-up. Reference may be made to Mc Kelvey et a/., 1993 where they have used a trickle reactor in which the hairy roots of Hyoscymus muticus were placed on a chromatography paper supported by stainless steel mesh and the medium was sprayed on to the hairy roots. They observed reduced growth rate as it reduces contact of roots with the gas phase limiting the oxygen mass transfer. Williams and Doran (2000) used a liquid dispersed bioreactor for the scale up of Atropa belladona hairy roots, where the liquid medium is sprayed on the growing hairy roots on the vertical stainless steel support. They found that even though gas-liquid oxygen transfer is rapid, significant oxygen limitations were observed. They also observed that the roots growing near the bottom of the vessel trapped and which led to the poor contact with gas phase indicating poor liquid mixing within the root bed. Mist distribution and guidance mechanism is lacking in the bioreactor used. Droplet size is big resulting in higher thickness of the film around the root. They have observed that lack of solid-liquid mass transfer to be the main draw back. Reference may be also made to Buer et a/., 1996 and Chatterjee et a/., 1997 wherein they reported the similar principle of using an acoustic field for the mist production for the cultivation of hairy root cultures. These reported designs employed acoustic window for the generation of the mist, resulting in the mist bioreactor, which is a single unit. This design comprised of an acoustic window (polyurethane material) attached to the bioreactor base. The drawback of this design was impeded transduction on repeated autoclaving. They also reported a lot of frictional losses and non-uniform distribution of mist in their design of the bioreactor. The main object of the present invention is to provide a device for acoustic mist bioreactor, which obviates the drawbacks as detailed above. Another object of the present invention is to de link the mist generation system from the bioreactor vessel so that the bioreactor vessel can be taken out and autoclaved separately which prevents contamination. Another object of the present invention is to give a mist distribution by properly designing the mist guidance in the bioreactor. In the drawing accompanying this specification Figure 1 represents a design for acoustic mist bioreactor. Accordingly the present invention provides an acoustic mist bioreactor which comprising transducer (A) as mount on which the reactor vessel (B) characterized in that the said transducer being placed at a distance of 1-4 cm from the bottom and the said bioreactor (B), the said bioreactor(B) having a glass support (C) with a two tier autoclavable nylon mesh arrangement such as herein described, for placing the hairy root cultures (D), the said bioreactor(B) being connected to a medium reservoir (E) with a silicone tube through a peristaltic pump (F) , the bioreactor (B) is also being connected with a silicone tube to carry the filtered air (PTFE [poly tetra fluro ethylene] filter 0.22µm) (G) form the compressor (H) to the bottom of the said bioreactor (B) through air sparger (I), the said bioreactor (B) also being provided with an out let for the air (J) which is connected with a silicone tube where the other end of this silicon tube is opened inside a flask (K) containing a moisture absorbent. In an embodiment of the present invention the bioreactor vessel comprises of autoclavable vessel made up of polycarbonate. In an another embodiment of the present invention a two-tier membrane anchorage comprising of stainless steel wire mesh, nylon net is used for the root support. In an another embodiment of the present invention an air outlet is used to counter the respiratory build up, and an inlet is used for the media replenishment from the reservoir. In yet another embodiment of the present invention wherein an air sparger is used with a PTFE air filter (0.22 urn). In yet another embodiment of the present invention wherein the mist generation is by applying acoustic field through transparent bioreactor vessel bottom. In yet another embodiment of the present invention the height of 1-4cm is used for placing the bioreactor over transducer for maximum mist generation. In yet another embodiment of the present invention wherein mist guidance configurations used are glass cylinders, inverted cones and draft tubes . The present invention deals with a device for acoustic mist bioreactor which comprises of a polycarbonate jar (300 ml) as a bioreactor vessel, having a two-tier membrane anchorage for the root support, with an air out let to counter the respiratory build up, an air sparger with a PTFE air filter (0.22 urn) and an inlet for the media replenishment from the reservoir. The bioreactor is kept on an acoustic transducer (Holmes Co Ltd.) of high frequency approximately 1.2 MHz creating high-energy ultra sound waves. These waves are propelled through the vessel bottom and then through liquid medium to generate a mist, which can be made to condense onto growing hairy root mass, which has been resting on the anchorage, forming a film which will enhance mass transfer. The mist eventually coalesces into bigger droplets and trickle down through the hairy root mass. The acoustic field intensity was found to vary with the distance from the transducer, hence the height at which the bioreactor vessel is to be placed for maximum mist generation was optimized. This was observed to be 2.5 cm. The rate of mist generation was found to be independent of the volume of the nutrient media. However, keeping in mind the requirement to the growth of hairy roots for 15 days in the present bioreactor vessel of 300 ml capacity, a 100 ml of nutrient medium was employed. The bioreactor to nutrient medium ratio may be varied based on the hairy root productivity in pilot runs. An inoculum size that has been standardized in conventional bioreactors for the faster growth rate of transformed roots is employed in this bioreactor also. The novelty of the device given in the present invention lies in delinking the bioreactor vessel from the mist generation mechanism, which need not be repeatedly autoclaved therefore facilitating the autoclaving of the bioreactor vessel separately by taking it out from the unit. This design will facilitate contamination free run of bioreactor. Another novel aspect of the present invention lies in exploiting the transparent nature of the bioreactor vessel (polycarbonate) material to acoustic energy for the generation of the mist. Another novel aspect of the present invention lies in exploiting the variation of the acoustic field strength with actual distance from the transducer. EXAMPLE 1 100 ml of MS basal liquid media was taken in a 300 ml capacity acoustic mist bioreactor vessel. An inoculum size of 850 mg fresh weight of transformed roots of Beta vulgaris was inoculated in the acoustic mist bioreactor and the total biomass was harvested after a culture period of 15 days. During the whole culture period the media volume in the reactor was maintained to 100 ml by replenishing the media from the media reservoir and an air flow rate of 30 ml min"1 was kept during the whole culture period. Parameters viz., specific growth rate, and total betalaine content were calculated to check the reactor's performance. We have found out that this design works out very well for the growth of transformed root cultures of Beta vulgaris, due to efficient mass transfer which was evident by the high specific growth rate (0.06 day-1) (Table 1) of the cultured roots in acoustic bioreactor which was high as compared to bubble column and pressure mist bioreactors (0.04 and 0.03 day -1) respectively (Table 1). It is encouraging to note that the specific growth rate of acoustic mist bioreactor is even higher than shake flask. It may be noted that normally the design of bioreactor is considered efficient if it can achieve the specific growth rate equal to that of a shake flask. Table 1: Performance of hairy root cultures of Beta vulgaris in Acoustic mist bioreactor. (Table Removed) EXAMPLE 2 In yet another example, experiment was performed by taking 100 ml of MS basal liquid media in a 300 ml capacity acoustic mist bioreactor vessel. An inoculum size of 850 mg of transformed roots of Tagetes patula was inoculated in the acoustic mist bioreactor and the total biomass was harvested after a culture period of 15 days. During the whole culture period the medium volume in the reactor was maintained to 100 ml by replenishing the medium from the medium reservoir and an air flow rate of 30 ml min"1 was kept during the whole culture period. Parameters viz., specific growth rate, and total thiophene content were calculated to check the reactors performance. We have observed while working with hairy root cultures of Tagetes patula that,the specific growth rate (0.06 day" 1) (Table 2) was high in acoustic mist bioreactor as compared to bubble column bioreactor (0.05 day "1) (Table 2). Table 2: Performance of Tagetes patula hairy root cultures in Acoustic mist bioreactor. (Table Removed) The present device of mist bioreactor is contrary to the conventional wisdom of acoustic wave transmission. Conventional knowledge indicates, that acoustic energy can not be transmitted efficiently through a liquid and a solid material placed in the path of the acoustic wave, since they behave as a reflector of the acoustic energy, Contrary to this, in the present invention a solid material (the base of the bioreactor vessel) is shown to transmit the acoustic energy and not reflecting it. Conventional wisdom indicates that energy increases, as the distance from the source of energy (transducer) decreases. In the present invention, it was observed that the misting intensity increased with the increase in the distance up to a stage from the source (transducer). The misting intensity eventually decreased resulting in maxima at a critical distance from the transducer. The main advantages of the present invention are 1) Efficient mist generation, which in turn results in efficient mass transfer. 2) Enables to run the bioreactor at higher biomass densities. 3) High-energy efficiency, since acoustic transducers are high voltage, low energy drawing devices. 4) Separation of acoustic transducer from culture vessel which eliminates contamination risks. 5) Simplicity in operation and ease of scale-up. The major application of this invented acoustic mist bioreactor is in plant biotechnology for example: 1) For the biomass production of hairy root cultures for obtaining phytochemicals, which can be used in food, chemical, and pharmaceutical industries. 2) For the biomass production of plant tissue and cell cultures for obtaining phytochemicals, which can be used in food, chemical, and pharmaceutical industries. 3) For the production of biomass of cultivated plant cells and tissues for varied applications. We Claim: 1. An acoustic mist bioreactor which comprising transducer (A) as mount on which the reactor vessel (B) characterized in that the said transducer being placed at a distance of 1-4 cm from the bottom and the said bioreactor (B), the said bioreactor(B) having a glass support (C) with a two tier autoclavable nylon mesh arrangement such as herein described, for placing the hairy root cultures (D), the said bioreactor(B) being connected to a medium reservoir (E) with a silicone tube through a peristaltic pump (F) , the bioreactor (B) is also being connected with a silicone tube to carry the filtered air (FTFE [poly tetra fluro ethylene] filter 0.22µm) (G) form the compressor (H) to the bottom of the said bioreactor (B) through air sparger (I), the said bioreactor (B) also being provided with an out let for the air (J) which is connected with a silicone tube where the other end of this silicon tube is opened inside a flask (K) containing a moisture absorbent. 2. An acoustic mist bioreactor as claimed in claim 1 wherein in the bioreactor vessel is made up of polycarbonate. 3. An acoustic mist bioreactor as claimed in claims 1-2 wherein a two-tier membrane anchorage is selected from stainless steel wire mesh and, nylon net. 4. An acoustic mist bioreactor as claimed in claims 1-3 wherein an air outlet is used to counter the respiratory build up, and an inlet is used for the media replenishment from the reservoir. 5. An acoustic mist bioreactor as claimed in claims 1-4 wherein mist guidance configuration used is selected from glass cylinders, inverted cones and draft tubes. 6. An acoustic mist bioreactor as claimed in claims 1-4 wherein the acoustic field is generated through transparent bioreactor vessel bottom. 7. An acoustic mist bioreactor as herein described with reference to the examples accompanying the drawings and specifications.

Documents:

283-del-2001-abstract.pdf

283-del-2001-claims.pdf

283-del-2001-correspondence-others.pdf

283-del-2001-correspondence-po.pdf

283-del-2001-description (complete).pdf

283-del-2001-drawings.pdf

283-del-2001-form-1.pdf

283-del-2001-form-18.pdf

283-del-2001-form-2.pdf

283-del-2001-form-3.pdf