Bioreactors, systems comprising the same and methods of use thereof

The method of any preceding claim, wherein the internal cavity of the bioreactor vessel is a sterile environment.

The method of any preceding claim, wherein, during the culturing, spraying, and/or removing steps, the bioreactor vessel is present in a closed system.

The method of any preceding claim, wherein culturing the plant explant comprising the bacteria in the bioreactor vessel comprises providing temperature and/or light conditions sufficient to maintain and/or grow the plant explant comprising the bacteria.

The method of any preceding claim, wherein the dispensing inlet comprises a spray nozzle (e.g., an atomizing nozzle) and spraying the media onto the plant explant comprising the bacteria comprises spraying the media through the spray nozzle and onto the plant explant comprising the bacteria. 9 The method of claim 8, wherein the spray nozzle is configured to produce a mist, atomized spray, and/or aerosol.

The method of any preceding claim, wherein spraying the media onto the plant explant comprising the bacteria comprises washing contaminants and/or metabolites (e.g., bacteria and/or phenolics) off the plant explant comprising the bacteria.

The method of any preceding claim, wherein spraying the media onto the plant explant comprising the bacteria comprises spraying the media in an amount sufficient to maintain the plant explant comprising the bacteria in a damp state.

The method of any preceding claim, further comprising repeating the spraying step one or more times and repeating the removing step one or more times, optionally wherein removing the waste media via the drainage outlet occurs prior to a subsequent spraying step.

The method of any preceding claim, wherein spraying the media onto the plant explant comprising the bacteria comprises spraying the media onto the plant explant comprising the bacteria for about 1, 5, or 10 seconds to about 15, 20, or 30 seconds every hour.

The method of any preceding claim, wherein spraying the media onto the plant explant comprising the bacteria comprises spraying about 10, 15, or 15 mL to about 20, 25, 30, 40, or 50 mL of media onto the plant explant comprising the bacteria

The method of any preceding claim, wherein the method is carried out for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, optionally wherein the method is carried out for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days in a closed system (e.g., without exposing the plant explant comprising the bacteria to the external environment).

The method of any preceding claim, further comprising providing a bioreactor assembly that comprises the bioreactor vessel, a medium tank fluidly connected to the dispensing inlet, and a positive pressure source operatively associated with the medium tank and the dispensing inlet, the positive pressure source configured to force the media from the medium tank through the dispensing inlet and into the upper compartment.

The method of any preceding claim, wherein the permeable member comprises a membrane or a screen.

The method of any preceding claim, wherein the bioreactor vessel further comprises a sensor positioned to sense an operational parameter of the bioreactor vessel, and the method further comprises sensing the operation parameter of the bioreactor vessel.

The method of any preceding claim, wherein the bioreactor vessel is a first bioreactor vessel and the plant explant comprising the bacteria is a first plant explant comprising first bacteria, and the method further comprises culturing a second plant explant comprising a second bacteria in a second bioreactor vessel, optionally wherein the second bioreactor vessel is fluidly connected to the medium tank and/or to the drainage receptacle.

The method of any preceding claim, further comprising supplying carbon dioxide into the internal cavity.

The method of any preceding claim, wherein the bioreactor vessel further comprises a vent (e.g., a one-way vent), optionally wherein the vent comprises a filter.

The method of any preceding claim, wherein the bioreactor vessel, medium tank, and/or the drainage receptacle are autoclavable and/or configured to be sterilized, optionally wherein the bioreactor assembly is autoclavable and/or configured to be sterilized.

The method of any preceding claim, wherein culturing the plant explant comprising the bacteria is devoid of culturing the plant explant comprising the bacteria on a solid media (e.g., solid growth media), optionally wherein the bioreactor vessel is devoid of a solid media (e.g., solid growth media).

The method of any preceding claim, further comprising recycling the waste media to provide recycled media, optionally wherein recycling the waste media comprises filtering the waste media to provide the recycled media.

The method of any preceding claim, wherein, following culturing for a period of time (e.g., 7 days) the method increases the survival rate of the plant explants comprising the bacteria compared to a control method (e.g., a method of culturing plant explants comprising bacteria on a solid media) comprising culturing for the same period of time.

The method of any preceding claim, wherein the method increases the processing rate (i.e., amount of plant material processed (e.g., cultured) over a period of time) of plant explants comprising bacteria compared to the processing rate of plant explants comprising bacteria for a control method (e.g., a method of culturing plant explants comprising bacteria on a solid media).

The bioreactor assembly of any one of claims 37-46, wherein the bioreactor vessel is a first bioreactor vessel, and bioreactor assembly further comprises a second bioreactor vessel that is fluidly connected to the medium tank and/or to the drainage receptacle.

• The bioreactor assembly of any one of claims 37-47, wherein the internal cavity has a volume of about 500, 600, 700, 800, or 900 mL to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 L.

The bioreactor assembly of any one of claims 37-48, further comprising a CO2 source fluidly connected with the internal cavity.

The bioreactor assembly of any one of claims 37-49, further comprising a timer operatively connected with the negative pressure source and/or the positive pressure source, the timer configured to induce the negative and/or positive pressure sources to operate intermittently.

The bioreactor assembly of any one of claims 37-50, wherein the drain is positioned below the lower compartment, such that fluid from the lower compartment may drain gravimetrically from the lower compartment to the drain.

The bioreactor assembly of any one of claims 37-51, further comprising a filter in fluid communication with the medium tank and dispensing inlet and positioned on the fluid path between the medium tank and dispensing inlet.

The bioreactor assembly of any one of claims 37-52, further comprising a vent (e.g., a one-way vent) in the internal cavity, optionally wherein the vent comprises a filter.

The bioreactor assembly of any one of claims 37-53, wherein the bioreactor vessel, medium tank, and/or the drainage receptacle are autoclavable and/or configured to be sterilized, optionally wherein the bioreactor assembly is autoclavable and/or configured to be sterilized.