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WO2019003240A1 - Nouveau bioréacteur pour la production en masse de champignons mycorhiziens arbusculaires - Google Patents

Nouveau bioréacteur pour la production en masse de champignons mycorhiziens arbusculaires Download PDF

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Publication number
WO2019003240A1
WO2019003240A1 PCT/IN2018/050266 IN2018050266W WO2019003240A1 WO 2019003240 A1 WO2019003240 A1 WO 2019003240A1 IN 2018050266 W IN2018050266 W IN 2018050266W WO 2019003240 A1 WO2019003240 A1 WO 2019003240A1
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Prior art keywords
bioreactor
vessel
mist
culture
mister
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Alok ADHOLEYA
Gunjan Mukherjee
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ENERGY AND RESOURCES INSTITUTE (TERI)
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ENERGY AND RESOURCES INSTITUTE (TERI)
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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F9/00—Fertilisers from household or town refuse
    • C05F9/04—Biological compost
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • the present invention relates to a novel gas-phase (mist) bioreactor for the in vitro production of arbuscular mycorrhizal fungi (AMF's), especially cultivating the fungal spores and roots using transformed plant roots in a limited aseptic space and the bioprocess using the said developed bioreactor where liquid nutrient medium steadily dispersed in a form of nutrient mist and drains out after condensation and precipitation and recycles at a regular intervals.
  • AMF's arbuscular mycorrhizal fungi
  • Arbuscular mycorrhizal fungi are beneficial symbiotic fungi in that they colonize the cells of roots of plants and stimulate absorption of essential plant nutrients.
  • Mycorrhizal fungi is proved to be a kind of microbial fertilizers due to their stimulating actions on plant growth which is confirmed by many research reports.
  • the actions include: (1) promoting resistance of infected plants to pathogens; (2) enhancing capability of drought resistance; (3) suppressing absorption of nocuous elements; and (4) connecting with other plants via hyphae.
  • Mycorrhiza can be applicable to a wide range of plants from floricultural, horticultural, silvicultural, to agricultural species including legumes, tree and plant species, except a few like mustard (Brassica sp.) and Sugar beet (Beta vulgaris). Finished product has easy storage at ambient temperature and prolonged shelf life at room temperature.
  • Various methods to cultivate AMF includes (1) Potted cultivating method (2) Hydroponic cultivating method (3) Aeroponic cultivating method and (4) Transformed root organ cultivating method (Adholeya et al. 2005, Patent 857/DEL/1999, Patent 2765/DEL/2009).
  • Hydroponic cultivating method is to cultivate symbiotic host plants inoculated with AMF in a specialized hydroponic device with nutrient liquid in which the host plants and AMF inoculums therein are submerged to enhance the growth of mycorrhizae and sporulation of AMF.
  • efficiency of AMF production for this method is better than the method used for potted plants, frequently refreshing the nutrient liquid is needed to prevent from serious problem of adulterated microbial contaminants.
  • the soaked state of the host plants and AMF therein caused by aquatic environment in this method is not the normally and naturally growth condition for AMF which results in the quantity of AMF sporulation failing to increase.
  • Aeroponic cultivating method is to cultivating symbiotic host plants inoculated with AMF in a specialized aeroponic container where vaporized nutrient liquid is provided to the plants and AMF therein. Cultivating large quantities of AMF propagules becomes possible even in the soilless situation. But the deficiencies of this method comprise the need of building a specific aeroponic container, the requirement of huge amount of vaporized nutrient liquid with necessary work to watch and refresh them, and failure to avoid the problem of adulterated microbial and pathogen contaminants.
  • Transformed root organ cultivating method uses isolated plant roots genetically transformed by the Ri plasmid of Agrobacterium rhizogenes (Tepfer, 1984, Cell, Vol. 37, 959-967) to be able to grow rapidly and independently and be inoculated with pure AMF to become a symbiotic root system (Mugnier and Mosse, 1987, Phytopathology, Vol. 77, 1045-1050). It is an advanced cultivating method to acquire AMF propagules without adulterated microbial contaminants in specialized cultivating circumstances after pure AMF are inoculated into the transformed root organ.
  • the most advanced method is Transformed root organ cultivation. This method uses isolated plant roots genetically transformed by the Ri plasmid of Agrobacterium rhizogenes (Tepfer, 1984, Cell, Vol. 37, 959-967) to be able to grow rapidly and independently and be inoculated with pure AMF to become a symbiotic root system (Mugnier and Mosse, 1987, Phytopathology, Vol. 77, 1045-1050). It is an advanced cultivating method to acquire AMF propagules without adulterated microbial contaminants in specialized cultivating circumstances after pure AMF are inoculated into the transformed root organ. [0012] Mugnier et al. in U.S. Pat. No.
  • the transformed root organ cultivating method can use liquid (solution) medium to cultivate the root system too.
  • liquid (solution) medium to cultivate the root system too.
  • reactor/growth chambers Three types of reactor/growth chambers have been described including the submerged style, rotating drum, and the airlift style as follows:
  • the submerged style indicates cultivating transformed root organs in shallow liquid medium by still placed cultivation (Nuutila et al, 1995, Plant Cell Rep., Vol. 14, 505-509).
  • the depth of the liquid nutrient medium cannot be large in order to prevent the root organs from the asphyxiant counteraction that is not good for growth. Therefore the submerged style is unable to contribute to the incremental reproduction of AMF propagules on a mass-produced scale.
  • Rotating drum also called the vibrating style, indicates cultivating the root organs in a predetermined quantity of the liquid medium and adding the amount of dissolved oxygen in the solution by spinning stir or vibration. The stress caused by spinning stir and vibration will inhibit the growth of root organs and decrease the incremental reproduction rate of AMF at the same time.
  • the airlift style indicates cultivating root organs in a container full of liquid nutrient medium and releasing bubbles continuously from the bottom of the container to facilitate breathing of root organs (Jolicoeur et al, 1999, Biotechnology and Bioengineering, Vol. 63, No. 2, 224-232).
  • the method which obviously overcomes the asphyxiant problem met in the submerged style and is free from the excess mechanical stress arising in the rotating drum style is a more feasible way in all methods adopting liquid medium. But sterile air should be injected persistently into the container, and the injection process usually costs high and allows for adulterated microbial contamination to occur.
  • both of the root organs and AMF therein are submerged continuously in the liquid medium during the process, and the liquid-full container is not a normal growth circumstance to the root organ and AMF though a large quantity of dissolved oxygen is obtainable in the container by injected air. Therefore the unit efficiency is lower than the submerged style.
  • Declerck et al in Patent application No. PCT/EP2009/050434 dated 15 Jan 2009 describes about a semi-hydroponics bioreactor to grow pre-mycorrhizal plant.
  • the whole pre-mycorrhizal plant needs to transfer to plastic tubular hydroponics bioreactor.
  • Aseptic transferred of whole plant into such reactor is a question and maintaining sterility for longer duration in such system is a problem.
  • the main objective of the invention is to provide a novel bioreactor to maximize aseptic mass production and up scaling of AM fungi by cultivating the fungal spores and propagules in a transformed hairy root of plant.
  • the bioreactor includes a sterilizable vessel made up of stainless steel or glass, misting forming device and mist controlling system, with an option of a special type of inoculation assembly, basket to grow culture and culture holding mesh.
  • Another objective of the present invention is to provide novel and improved systems/apparatus for the growth and cultivation of AM Fungi affording efficient oxygen and nutrient distribution capacity throughout the reactor vessel, superior to conventional fermentors or bioreactors.
  • Another objective of the present invention is to provide improved oxygen and nutrient supply at low shear to prevent damage of shear-sensitive tissues of transformed hairy root to produce AM fungal propagul.es which would be contamination free, simple, inexpensive and effective using the novel pilot scale bioreactor.
  • the present invention relates to a novel gas-phase (mist) bioreactor for in vitro aseptic mass production and up scaling of arbuscular mycorrhizal (AM) fungi by cultivating the fungal spores in a transformed hairy root of plant in a limited aseptic space and the bioprocess using the bioreactor comprising of: sterilizable vessel made of stainless steel or glass; mist forming device; mist controlling system, and with an option of a special type of inoculation assembly, basket to grow culture and culture holding mesh,
  • liquid nutrient medium steadily floods in a form of mist inside the vessel and then drains out and recycles at a regular intervals.
  • gas-phase (mist) bioreactor is in- situ sterilizable stainless bioreactor having the capacity of the reactor as 25 litres and having all parts of the bioreactor made of SS316 which are in contact with the culture or media.
  • Another embodiment of the present invention is that the vessel made of glass is sterilized by using autoclave.
  • One embodiment of the present invention is that the capacity of glass bioreactor is 7 litres.
  • the in- situ sterilizable stainless bioreactor comprises of insulated vessel made of stainless steel SS316 with 240 Grit internal and 180 Grit the external surface finishing and has spiral jacket made of SS304, having the H/D ratio of the vessel 2: 1 and the top plate of the vessel is made of SS316 and is bolted to the shell through Rothman Clamps and bottom dish is welded to the shell.
  • the H/D ratio of the glass vessel is 3: 1.
  • Further embodiment of the present invention is that the working temperature range of the vessel is 110°-140°C and working pressure is 0.5-3.0 bar.
  • the glass bioreactor may be operated in normal atmospheric pressure.
  • top plate of the stainless steel vessel consists of 10 ports.
  • the bottom part of the stainless steel vessel consists of drain valve and harvesting/sampling port and pH, DO, and temperature probes are attached to it. Further, pH probe is connected to pH transmitter and can be set through programmable logic controller (PLC); 25 mm DO probe is connected with DO Transmitter; CO2 and ethylene gas analyser are connected to the exhaust line of the vessel.
  • PLC programmable logic controller
  • 25 mm DO probe is connected with DO Transmitter
  • CO2 and ethylene gas analyser are connected to the exhaust line of the vessel.
  • Another embodiment of the present invention is that in the mist bioreactor system, one peristaltic pump is connected with media line to form mist inside the reactor and other extra pump is used for cleaning of vessel.
  • the other embodiment of the present invention includes that the mycorrhizal fungi may be grown in a basket or may be in a funnel shape single conical structure instrument.
  • Another embodiment of the present invention includes an inoculation assembly made up of stainless steel SS316, the assembly is closed cylindrical in shape with a flip disk is attached inside the assembly and the assembly can be fitted with the Triclover Clamp (TC) end of the top plate of bioreactor and can be sterilized separately after removing from the reactor to transfer culture into pre sterilized assembly.
  • TC Triclover Clamp
  • the embodiment of the present invention includes the mist forming device, which consists of an atomizing spray nozzle or ultrasonic mister.
  • the preferred embodiment of the present invention includes that Atomizing spray nozzle is attached to side wall of the vessel.
  • the nozzle may be attached to top plate of the reactor.
  • the spray nozzle is consists of air and media line to generate mist inside the reactor vessel.
  • Another embodiment of the present invention consists of an Ultrasonic Mister, which may be used instead of atomizing nozzle.
  • the Mister device may be fitted with a special triclover clamp (TC) joint on the top plate of the bioreactor.
  • TC triclover clamp
  • the mister is capable of providing a volumetric throughput of 3-4 L mm " ' and of providing droplets having a diameter of approximately 15-20 ⁇ . This droplet Size may vary between 5 ⁇ and 25 ⁇ .
  • air atomizing Misting device in Glass Mist- Bioreactor is fitted with a special arrangement inside the bottom of the bioreactor as per design described in the detailed description wherein air atomizing Misting system can be capable of providing a volumetric throughput of 0.1-2.0 ml min -1 and of providing droplets having a diameter of between 5 ⁇ and 25 ⁇ .
  • the said mister includes a pulsed output having a duty cycle and the mist is in form of droplets.
  • Another embodiment of the present invention includes that the total bioprocess is automatic operable type with process control by PLC, which has provision to set all the parameters in the desired process limit and also to display and record the data by Supervisory Control and Data Acquisition (SCAD A).
  • SCAD A Supervisory Control and Data Acquisition
  • the said gas includes ambient air, ethylene, carbon dioxide or other gases.
  • the other embodiment of the present invention includes a bioprocess for aseptic mass production and up scaling of arbuscular mycorrhizal fungi (AMF) using the gas- phase (mist) bioreactor of the present invention.
  • AMF arbuscular mycorrhizal fungi
  • Figure 1 Drawing of the 25 Litre stainless Steel Nutrient Mist Bioreactor (Outer part).
  • Figure 2 Drawing of the 25 Liter stainless steel Nutrient Mist Bioreactor showing inoculation plates inside vessel.
  • Figure 3 Design of the top plate of 25 Liter stainless steel Nutrient Mist Bioreactor.
  • Figure 4 Design of lamp holder with connection detail attached with Vessel in stainless steel Nutrient Mist Bioreactor.
  • Figure 5 A and B Design of culture growing plate inside stainless steel Nutrient Mist-Bioreactor.
  • Figure 6. Design photograph of culture growing plate inside stainless steel Nutrient Mist-Bioreactor.
  • Figure 8. Inoculation assembly of stainless steel Nutrient Mist-Bioreactor (Internal design).
  • Figure 9. Exploded view of Spray Nozzle system: 1 -Retainer ring, 2- Air cap, 3-Fluid cap, 4-Gasket, 5-Spring, 6-Stainless steel ball, 7-Body, 8-Stainer gasket, 9-Stainer screen, 10-Stainer body.
  • FIG. 11 Bioprocess diagram of the operation of pilot scale stainless steel Nutrient Mist bioreactor for production of AM fungi
  • Figure 13 Glass Nutrient Mist Bioreactor with air atomizing system for Mycorrhizal fungal growth.
  • Figure 14 Air atomizing system for Glass Nutrient Mist Bioreactor (Exploded view).
  • a novel gas-phase (mist) bioreactor that reduces gas exchange limitations, does not oxygen limit the growth of arbuscular mycorrhizal (AM) fungal associated hairy root culture.
  • the bioreactor may be in-situ sterilizable made of stainless steel material or made of glass, which is autoclavable.
  • the bioreactor is capable of cultivating the AM fungal spores by using root organs of symbiotic host plant in a limited aseptic space using bioreactor where liquid nutrient medium steadily floods in a form of mist inside the vessel and then drains out and recycles at regular intervals.
  • the description of the bioreactor is mentioned below capable for production of in vitro aseptic mass production of arbuscular mycorrhizal fungi (AMF).
  • the present invention relates to gas-phase (mist) bioreactor for in vitro aseptic mass production of arbuscular mycorrhizal (AM) fungal spores and roots in a transformed hairy root of plant in a limited aseptic space comprising of- a) a sterizable vessel made of stainless steel or glass;
  • liquid nutrient medium steadily floods in a form of mist inside the vessel and then drains out after condensation and recycles at a regular intervals.
  • the bioreactor vessel is a sterilizable vessel made of stainless steel or glass.
  • the stainless steel vessel is in-situ sterilizable.
  • the capacity of the reactor may be 25 Litre.
  • the vessel is made of stainless steel SS316 with spiral jacket with insulation.
  • the H/D ratio of the vessel is 2: 1.
  • Design of the said mist-bioreactor vessel is shown in Figure 1 & 2.
  • the other form of bioreactor vessel is made of glass.
  • the capacity of the reactor may be 7 Litre with the H/D ratio of the vessel may be is 3 : 1.
  • the vessel made of glass is autoclavable. Design of the said mist-bioreactor vessel is shown in Figure 13.
  • the working temperature of the stainless steel vessel is 110-140 Deg C and working pressure range is 0.5-3.0 bar.
  • the top plate of the stainless steel vessel is made of SS316 and is bolted to the shell through Rothman Clamps.
  • the bottom dish is welded to the shell.
  • the top plate of the Stainless Steel Mist Bioreactor consists of 10 ports as follows: Two (2) numbers of inoculation ports extended inside the vessel with SS pipe upto the inoculation Teflon culture disk (Figure 3, PI, P2), 1 No of exhaust port (Figure 3, P3), 1 No of view glass ( Figure 3, P4), 1 No sterile pressure gauge ( Figure 3, P5), 1 no of safety valve (Figure 3, P6), 1 No for CIP cleaning ( Figure 3, P7), 1 No for steam inlet ( Figure 3, P8), 1 No of central port ( Figure 3, P9) is to attach rotated shaft for culture holder, 1 additional port (Figure 3, P10) for other use.
  • the top plate design drawing is mentioned in Figure 3.
  • One Lamp Holder is attached with Top Plate ( Figure 4).
  • the top plate of the vessel is also made of stain less steel SS 316.
  • the top plate of the vessel is bolted to the glass vessel with Clamps.
  • Bottom part of stainless steel vessel consists of drain valve (Figure 1, Nil) and harvesting/sampling port ( Figure 1, N12).
  • pH, DO and temperature probes are attached to the bottom part of the vessel.
  • pH probe is connected to pH transmitter (pH range 0-14) and can be set through PLC.
  • 25 mm DO probe is connected with DO Transmitter (Range 0-100%).
  • CO2 and ethylene gas analyser are connected to the exhaust line of the vessel.
  • pH, temperature and DO Probes are shown in the reactor design (Figure 1) as N8, N9, N10 respectively.
  • One peristaltic pump (3-30 rpm) is connected with media line to form mist inside the reactor and other extra pump (30-300 rpm) is used for cleaning of vessel.
  • a two tier rack made of perforated Teflon sheet fitted with SS supporting ring (SS 316) is fixed into the 25 L reactor vessel and can be removed easily for cleaning.
  • the central stem of the rack (basket) is made of SS rod (SS 316) connected from the centre of the top plate using mechanical seal.
  • the rack can be rotated by manually rotating the central rod of the rack from outside of the vessel. Diameter of upper and lower racks is 160 mm and 190 mm respectively. The distance between two racks is 190 mm.
  • the perforated Teflon plate for growth of culture was mentioned in Figure 5A showing culture Plate No. 1 and Figure 5B is Plate No. 2.
  • Mycorrhizal fungi may be grown in a funnel shape single conical structure instrument also.
  • the funnel shape may be prepared using SS mesh (SS 316) to grow AM Fungi.
  • the diameter of the cone may be 160 mm and length 100 mm.
  • Such type of conical shape culture holder can create anchor to the mycorrhizal hairy root with the soft wall mesh. The design photograph of such culture basket is shown in Figure 6.
  • a funnel shape single conical structure mesh is attached to grow mycorrhizal fungi.
  • the funnel shape may be prepared using SS mesh.
  • Such type of conical shape culture holder can create anchor to the mycorrhizal hairy root with the soft wall mesh ( Figure 13).
  • Inoculation assembly is made of stainless steel SS 316. Assembly is closed cylindrical in shape with a flip disk is attached inside the assembly. The assembly can be fitted with the TC end of the top plate of Bioreactor. Assembly can be sterilized separately after removing from the reactor to transfer culture into pre sterilized assembly.
  • the design of the assembly is shown in Figure 7.
  • the internal design of the inoculation assembly is shown in Figure 8.
  • Step-1 is inoculation to assembly
  • step-2 is attachment of assembly to main bioreactor for actual inoculation to vessel.
  • the inoculation process is mentioned below.
  • AM fungal associated hairy root to be aseptically cut into small pieces inside laminar hood. Then culture is aseptically transferred to pre-sterilized inoculation assembly. Airflow is maintained at a range of 8-10 LPM into the vessel at the time of inoculation of culture to sterilized bioreactor. The inoculation assembly is attached aseptically to TC end of the reactor. Finally, the culture to be inoculated to bioreactor by flipping the assembly leaver. Mist forming device
  • atomizing spray nozzle is attached to side wall of the vessel.
  • the nozzle may be attached to top plate of the reactor.
  • the spray nozzle is made of brass and in combination of stainless steel SS 316.
  • the spray nozzle is consists of air and media line to generate mist inside the reactor vessel.
  • the attachment of atomizing spray nozzle is shown in Figure 1, N14, and Figure 9.
  • Ultrasonic Mister may be used instead of atomizing nozzle.
  • the Mister device may be fitted with a special triclover clamp (TC) joint on the top plate of the bioreactor as per design ( Figure 10).
  • the assembly was designed with standard joint such a way that it can be fit with any other mist-bioreactor.
  • the mister is capable of providing a volumetric throughput in a. range of 3-4 I, mm "'1 and of providing droplets having a diameter of approximately 15-20 p.rn. This droplet size may vary between 5 ⁇ and 25 ⁇ , When independent air flow is required for growth such mister may be used. In such case mist formation is not dependent on air flow and in which smaller droplet size helps in better gas transfer for growth.
  • the size of the droplets provided by mister should offer good aeration and control of the gas phase composition, reduces shear damage to the root bed and also reduce any chemical gradients within the bioreactor vessel during growth of the root bed on culture basket.
  • the mister is fitted on top of the bioreactor and dimensioned such that the headspace distance between the mister and the root bed must be minimum distance of 15-20 em to ensure uniform medium distribution.
  • the mister made front atomizing nozzle can be sterilized alongwith the bioreactor through in- situ sterilization. But once the ultrasonic mister is attached the steam sterilization is avoided.
  • the mister is cold sterilized as follows. The mister is soaked overnight in 70% ethanol followed by pumping (25 mL rain "1 ) 500 ml, of a solution of 1 0% bleach, then 100 ml. sterile water, 500 ml, of a solution of 70% ethanol, and finally a solution of 100 ml, sterile water.
  • An alternative sterilization method involves the following. Briefly, the mister head is washed with tap water to remove any large debris, dried at 60° C, , wrapped in aluminum foil, and heated again at 60° C.
  • a third sterilization procedure may involve gas-phase sterilization using ethylene oxide.
  • the present invention also explains about a bioreactor alongwith culture mesh holder with mist forming device, which is designed as per mist deposition model in which root beds are treated as if they are fibrous filters.
  • Mist cycle is optimized with duty cycle by regulating misting by switching On/Off in scaled up pilot bioreactor.
  • the duty cycle may be changed in case of use of different mister device.
  • Vdep is therefore a non-1 in ear functio of a (packing fraction of root bed).
  • the medium required to support the growth of the root bed (V req , mL d " ; ) depends upon the amount of biomass present, the growth rate ⁇ "" 1 ), the apparent biomass yield of the growth-limiting nutrient Yx/ S (g DW biomass per g nutrient consumed) and the concentration of the limiting nutrient medium Cs (g L '"1 ).
  • Vdep To maintain a desired growth rate ⁇ , Vdep must be greater than or equal to V eq.
  • Air inlet line is used for air feed into the system by means of spray through the mist generators. Air inlet line is connected with in-situ sterilizable absolute filter element (5" size, 0.2 ⁇ ) with full draining stainless steel housing. Extra two supporting pre-sterilized filters (0.2 ⁇ ) are also attached before in-situ sterilisable filter elements. Air compressor is connected with the airline of the bioreactor.
  • Airline of the bioreactor was constructed by serially connecting two numbers of pre-sterilized air filters (2 Nos, 0.2 micron, 1" and 5", filters) before in-situ sterilizable absolute filter element (5" size, 0.2 ⁇ ) with full draining stainless steel housing leading to vessel.
  • Air exhaust line is also connected with in-situ sterilisable absolute filter element (5" size, 0.2 ⁇ ) with full draining stainless steel housing.
  • C02 and ethylene gas analyser are connected to the exhaust line of the vessel. These analysers are connected with isolation valves to avoid steam entry into the analysers.
  • Cooling water circulation line is connected to the bioreactor to control temperature through vessel jacket.
  • the control panel of the bioreactor consists of controllers, transmitters and other accessories.
  • the total fermentation process is automatic operable type with process control by PLC, which has provision to set all the parameters in the desired process limit and also to display and record the data.
  • SCADA Supervisory Control And Data Acquisition
  • the system is designed to suit intellution based SCADA Software and interface system to control the fermenters as required by user.
  • the software is operated through Windows computer.
  • SCADA records all measured data and records developed Algorithm to feed nutrients as per the batch. Algorithm for Mist control:
  • Algorithm is developed for controlled mist cycle during the batch. Program is also developed to regulate mist flow by controlling air and media into the feed.
  • PLC is connected with laptop installed with SCADA software. All the data of the mist- bioreactor is recorded into the laptop through SCADA.
  • Steam Boiler and air compressor are connected with mist-bioreactor system.
  • Steam boiler may be a capacity of 10-15 KW and can produce steam 20-30 kg/hr with maximum pressure of around 5-10 bar.
  • Oil free air compressor (1 HP) is connected with reactor through Polyurethane (PU) tubing. Controlled air pressure distribute further to instrumentation line and process line.
  • Process line is connected to vessel through filter and spray nozzle whereas instrumentation line is used for controlling pneumatically operated valves controlled through PLC.
  • the Stainless Steel Mist-Bioreactor is connected and run as per self-explanatory drawing of the bioprocess mentioned in Figure 11.
  • the media, form of mist is spread inside the reactor vessel.
  • the deposited media from the reactor vessel is transferred to another reservoir for analysis and further re-circulated to main reservoir tank/bottle to reproduce mist in the mist-bioreactor.
  • FIG. 13 The concept drawing of the Glass Mist-Bioreactor is shown in Figure 13.
  • the bioreactor may be operated in normal atmospheric pressure.
  • Media line can be connected with bioreactor by connecting the bottle attached with pre-sterilized Media filters (0.2 u, 1" x2 Nos) with silicon tubing and needle assembly. The needle is pierced aseptically through septum of the Nozzle port to connect media line with Reactor.
  • Sugar containing modified minimal media can be prepared for AM fungal growth in bioreactor. Substantial growth is observed inside the bioreactor. Freshly growing white colour hairy root is observed. Hairy root tips came out from the mesh in branching pattern ( Figure 12).
  • Gas-phase (mist) bioreactor for in vitro aseptic mass production of arbuscular mycorrhizal (AM) fungal spores and roots in a transformed hairy root of plant in a limited aseptic space is unique for mycorrhizal growth which includes the in-situ sterilizable Stainless steel vessel or autoclavable glass.
  • the advantage of use of media in form of mist droplet consumes less nutrient medium than conventional cultivation methods.
  • contamination of the culture medium with bacteria is minimized by the exposure to medium vapor because they are damaged or even filled during the nebulization.
  • Mycorrhizal fungi can be grown in this mist-bioreactor with limited supply of media in four weeks' time.

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Abstract

La présente invention concerne un nouveau bioréacteur pour maximiser la production en masse aseptique et la mise à l'échelle de champignons MA par culture des spores et des racines fongiques dans un chevelu racinaire transformé de plante. Le bioréacteur comprend un dispositif de formation de brume en acier ou en verre, un type spécial d'ensemble d'inoculation, un tamis de maintien de culture et un système de régulation de brume, un milieu nutritif liquide s'écoulant en continu sous forme d'un brouillard à l'intérieur du récipient, puis s'évacuant et se recyclant à des intervalles réguliers. La présente invention concerne de nouveaux systèmes/appareils améliorés pour la croissance et la culture de champignons MA procurant une capacité efficace de distribution d'oxygène et de nutriments partout dans la cuve de réacteur et conférant une alimentation améliorée en oxygène et en nutriments à faible cisaillement pour empêcher l'endommagement de tissus sensibles au cisaillement de chevelu racinaire transformé pour produire des spores fongiques MA qui sont exemptes de contaminations, simples, peu coûteuses et efficaces à l'aide du nouveau bioréacteur.
PCT/IN2018/050266 2017-06-27 2018-04-30 Nouveau bioréacteur pour la production en masse de champignons mycorhiziens arbusculaires Ceased WO2019003240A1 (fr)

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FR3147813A1 (fr) * 2023-04-17 2024-10-18 Synsym Biosciences Sas Méthode de production de micro-organismes et installation de mise en œuvre

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FR3147813A1 (fr) * 2023-04-17 2024-10-18 Synsym Biosciences Sas Méthode de production de micro-organismes et installation de mise en œuvre
CN117223542A (zh) * 2023-11-15 2023-12-15 山东金必来生物科技有限公司 一种丛枝菌根真菌菌丝收集器以及收集方法
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