WO2010042478A2

WO2010042478A2 - Modular experimental platform for microorganism physiology and scale-up studies

Info

Publication number: WO2010042478A2
Application number: PCT/US2009/059643
Authority: WO
Inventors: Mark Holl; Jeff Houkal; Rhett Martineau; Greg Bessette; Raveender Vannela; Chao Zhou; Hyun Woo Kim; Jie SHENG; Sindhuja Sadayandi; Daniel Bank; Juan Vela; Bruce Rittmann; Paul Westerhoff; Deirdre Meldrum
Original assignee: Arizona Board Of Regents For And On Behalf Of Arizona State University
Priority date: 2008-10-07
Filing date: 2009-10-06
Publication date: 2010-04-15
Also published as: WO2010042478A3

Abstract

A highly-configurable, modular system for scale-up studies of photosynthetic microorganisms to produce products such as biofuels includes various components that can be altered or configured to change parameters within the system.

Description

DESCRIPTION

MODULAR EXPERIMENTAL PLATFORM FOR MICROORGANISM PHYSIOLOGY AND SCALE-UP STUDIES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Patent Application Serial No. 61/103,489, filed October 7, 2008, entitled "Modular Experimental Platform for Microorganism Physiology and Scale-Up Studies", the entire disclosure of which is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

Embodiments of the present invention relate generally to a system and method for growing microorganisms under controlled conditions. In particular, embodiments of the present invention concern the use of a highly-configurable, modular platform for scale-up studies of photosynthetic microorganisms to produce products such as biofuels, high- value chemical compounds, vitamins, and nutraceuticals.

II. Description of Related Art

Existing benchtop reactors are in general not designed for such a degree of user configurability nor for the express purpose of studying culture methods. In existing systems, topics such as reactor sterilization (or sanitization), temperature regulation, gas delivery, mixing, nutrient delivery, and others can not be readily investigated on a scale with greater relevance to larger cell culture systems. For these larger volumes, common methods of sterilization (or sanitization) and reactor operation cannot be used and instead techniques and methods need to be developed.

SUMMARY

Embodiments of this disclosure comprise an apparatus for the cultivation of cells, e.g., photosynthetic marine microorganisms, and can be used potentially for the cultivation of any type of cells in suspension. The apparatus is capable of delivering the required conditions for optimal experimental growth, including light regime, nutrient and/or mixing gases, temperature, liquid or solid nutrients, experimental compounds via liquid or gas streams, and mechanical mixing. The apparatus supports chemical and other sensing in a highly configurable manner, including the measurement of parameters regarding the growth and/or progress of the cell culture in terms of culture pH, turbidity, dissolved gases, headspace gases, temperature, liquid and gas flow rates, mixing intensity, and others. Sampling and actuator access to the culture vessel is made through multiple modular plugs which are easily placed and removed, which can contain fittings to the requisite instruments and are easily modified for this purpose. This modularity enables a great degree of flexibility and configurational control by the user. The culture vessel is constructed with plate geometry, enabling controlled light delivery and measurement. Access ports are located on the top and front/rear faces of the reactor. The reactor reservoir nominally contains a liquid volume of approximately 17.5 L, but the reactor has been designed to enable easy replacement of the reservoir ends to increase the depth of the reactor and thus its volume. Increasing the depth of the reactor can also increase the number of available access ports. In principle the design elements of the invention may be adapted to reactors of arbitrary experimental benchtop size.

In certain embodiments, the reservoir plates are constructed preferably of glass or polycarbonate but can be made from any transparent material with suitable biocompatibility and resistance to sterilization solutions including bleach, sodium hydroxide, ethanol, and others. The reservoir top/bottom and front/rear walls are constructed of any machinable material with suitable biocompatibility, material toughness, and resistance to sterilization agents, and may consist of polycarbonate, acetal, ABS, Teflon, and others. Instrument plugs are comprised of DeMn or any machinable material with suitable biocompatibility, material toughness, and resistance to sterilization solutions. In specific embodiments, the system comprises numerous subsystems including gas delivery, temperature control, mechanical mixing, light delivery, and others. Temperature control can be accomplished using a water jacket, ensuring light access to the reactor and maximizing the heat exchange surface area. Maximizing the heat exchange surface area minimizes trauma to the microorganisms, and mechanical mixing can be accomplished using magnetically driven shafts with impellers. A magnetic drive reduces potential failures due to leakage and promotes a biocompatible materials interface. Gas delivery is accomplished via mass flow controllers, and light is delivered via dimmable fluorescent bulbs. Spectral signature of said fluorescent bulbs can be selected for maximum efficacy in relation to the organism and study design. In certain embodiments, the system is suited to cultivate cyanobacteria, microalgae, or algae, but can be used also to cultivate any cell type that can grow in suspension, photosynthetic or otherwise. In non-photosynthetic applications the transparent walls provide a window into the process that may be used to assess other investigative criterion of interest - including the assessment of bioflims, for example. Cells can be grown to produce biomass; perform biochemical reactions including bioremediations, chemical transformations, and production of gases; respond to environmental stimuli in a sensing capacity.

The system can provide a platform which is fully flexible to allow extensive user configurability in terms of sensor and actuator capabilities for extensive physiological characterization of cultivated organisms. As well, complex physical phenomenon can be investigated owing to the ease with which complex instrumentation can be integrated in a custom modular manner. As an example, biofilm formation may be investigated on different materials and materials with treatments by using the modular caps as insertion points for test coupons. Finally, the system can also facilitate the study of large scale cultivation methods themselves.

In specific embodiments, the system provides: 1) full modularity of actuators and sensors enables flexible configuration for more complete experimental control, 2) ability to test materials interactions with cultures, 3) an expandable culture vessel/reservoir; and 3) a reactor volume that is suitable for investigating problems and challenges associated with large-scale cultivation of photosynthetic organisms.

Specific embodiments of the system may be used for the cultivation of algae, microalgae, and cyanobacteria for biotechnological applications (by-product harvesting, including food supplements, biofuel feedstocks, fertilizers, aquaculture feedstocks, etc.) and sensor applications (on-line, continuous biosensors). Specific embodiments may comprise a modular cell cultivation system including: a reservoir comprising an inner volume; a light source configured to emit light and illuminate the inner volume of the reservoir; a mixer extending into the inner volume of the reservoir; a gas delivery system; a temperature control system; a sensor system configured to sense one or more parameters in the inner volume of the reservoir; a harvesting extraction module; and a sample extraction module. In certain embodiments, the reservoir can be configured so that a user can increase or decrease the inner volume. The reservoir may comprise a pair of side plates and a pair of replaceable end plates. In specific embodiments, the temperature control system may comprise a water jacket proximal to the inner volume of the reservoir and an external thermal control apparatus. In certain embodiments, the reservoir may comprise panels that allow the inner volume to be visible from outside of the reservoir. The light source may be configured to increase or decrease the intensity of the emitted light, and in specific embodiments, the light source may comprise a dimmable fluorescent bulb and/or a light emitting diode (LED). In specific embodiments, the light source is configured to effect spectral wavelength control. In certain embodiments, light source is configured to increase or decrease the intensity of the emitted light.

In specific embodiments, the light source may comprise an array of light emitting diodes (LEDs) configured to emit one or more wavelengths of light. The mixer may comprise a magnetically driven shaft and an impeller in certain embodiments. The sample extraction module may comprise a septum in particular embodiments. Particular embodiments may also comprise multiple controllable light sources.

In certain embodiments, the harvesting extraction module may comprise a valve configured to seal off the inner volume of the reservoir from the outside environment when the valve is in the closed position. The valve may be configured to allow a fluid contained within the inner volume to be removed from the inner volume when the valve is in the open position. In certain embodiments, the sensor system can be configured to measure a parameter selected from the group consisting of: pH, turbidity, dissolved chemical species, dissolved gases, dissolved chemical species, headspace gases, temperature, liquid flow rate, gas flow rate, and mixing intensity.

In specific embodiments, the sensor system may comprise a plurality of sensors and actuators configured to work collectively to control daily cycles of temperature, illumination, and nutrient delivery, and to track the simultaneous effect of these controlled process trajectories. The gas delivery system may comprise one or more mass flow controllers in particular embodiments.

Certain embodiments may also comprise a method of using any of the previously- described systems. In specific embodiments, the method may include: culturing photosynthetic cells in the inner volume of the reservoir; utilizing the gas delivery system to introduce a gas into inner volume of the reservoir; utilizing the mixer to mix the photosynthetic cells and the gas; utilizing the light source to illuminate a portion of the inner volume of the reservoir; utilizing a sensor in the sensor system to measure the value of a parameter in the inner volume of the reservoir; and removing a portion of the photosynthetic cells from the reservoir. In certain embodiments, an automated system for nutrient delivery and makeup water introduction is attached to the reactor for automated operations. In specific embodiments, an automated system for biomass harvest is attached to the reactor for automated operations.

Certain embodiments may also comprise adjusting the gas delivery system and altering the value of the parameter measured by the sensor. Particular embodiments may also comprise adjusting the mixer and altering the value of the parameter measured by the sensor. Particular embodiments may also comprise adjusting the light source and altering the value of the parameter measured by the sensor. In certain embodiments, removing a portion of the photosynthetic cells from the reservoir comprises manipulating the harvest extraction module to allow the photosynthetic cells to be removed from the inner volume. Certain embodiments may encompass one or more previously defined embodiments for the purpose of simulating daily (24 hour) processing cycles. Certain embodiments may enable non-naturally occurring conditions for the purpose of optimized growth of genetically modified organisms in conditions optimally suited to their synthetically designed physiologies.

In particular embodiments, removing a portion of the photosynthetic cells from the reservoir comprises penetrating a septum of the sample extraction system and withdrawing a portion of the photosynthetic cells. Particular embodiments may comprise replacing a component of the reservoir and increasing or decreasing the inner volume of the reservoir.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or system of the invention, and vice versa. Furthermore, systems of the invention can be used to achieve methods of the invention.

The term "conduit" or any variation thereof, when used in the claims and/or specification, includes any structure through which a fluid may be conveyed. Non-limiting examples of conduit include pipes, tubing, channels, or other enclosed structures.

The term "reservoir" or any variation thereof, when used in the claims and/or specification, includes any body structure capable of retaining fluid. Non-limiting examples of reservoirs include ponds, tanks, lakes, tubs, or other similar structures.

The term "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

The terms "inhibiting" or "reducing" or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as

"have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an assembled view of an exemplary embodiment according to this disclosure.

FIG. 2 is an exploded view of the embodiment of FIG. 1. FIG. 3 is an exploded view of a component of the embodiment of FIG. 1.

FIG. 4 is an exploded view of a component of the embodiment of FIG. 1.

FIG. 5 is an exploded view of a component of the embodiment of FIG. 1.

FIG. 6 is an exploded view of a component of the embodiment of FIG. 1.

FIG. 7 is an exploded view of a component of the embodiment of FIG. 1. DESCRIPTION OF EXEMPLARY EMBODIMENTS

Referring initially to FIGS. 1-7, a cell cultivation system (CCS) 100 comprises a reservoir 110, an electrical control system 115, a light system 120, a plurality of mixers 130, a gas delivery system 140, and a temperature control system 150. CCS 100 further comprises a harvesting extraction module 170 and a sample extraction module 180, as well as a sensor system 160 with a plurality of sensors 161-165.

As shown in the exploded view of FIG. 3, sensors 161-165 extend into the inner volume of reservoir 110 and can therefore monitor conditions and parameters during a cell cultivation experiment. Sensors 161-165 can be configured to measure a variety of different parameters, including, for example, pH, turbidity, dissolved gases, headspace gases, temperature, liquid and gas flow rates, mixing intensity. Sensors 161-165 can also be configured so that a sensor may be replaced without removing the contents of reservoir 110. For example, sensors 161-165 may extend into ports that automatically seal when a sensor is removed from the port. Sensor system 160 allows a user of CCS 100 to monitor a variety of conditions during the cultivation of cells within reservoir 110. In certain embodiments, the operation of CCS 100 can be adjusted or manipulated and the effect on a specific parameter can be monitored. As discussed below, certain variables may be altered while cell cultivation is ongoing within reservoir 110, e.g., pH, temperature, mixing intensity, gas flow rates, etc. Other variables (e.g., reservoir 110 volume) may require the cultivation process to be stopped and re-started in order for the variable to be changed. In certain embodiments, sensors 161- 165 can be configured to work collectively to track daily cycles including, e.g., determining the effects of temperature on both the sensor responses and the actual chemistry of the reservoir.

CCS 100 provides a user with the flexibility to adjust many different parameters and variables that can affect the cultivation of microorganisms within reservoir 110. For example, reservoir 100 comprises a pair of side plates 101 and 102, as well as end plates 103 and 104. In the embodiment shown, end plates 103 and 104 are replaceable and can be interchanged with different end plates (not shown) that increase the volume contained within reservoir 100. By changing the end plates 103, 104 for those with a different configuration, a user can change the volume contained within reservoir 110. In certain embodiments, end plates 103 and 104 are also transparent, so that a user can see any contents contained within the inner volume. Other variables besides the volume of reservoir 110 may also be adjusted in CCS 100. For example, light system 120 comprises a plurality of light sources 121 that can be adjusted to increase or decrease the intensity of light emitted from light sources 121. In certain embodiments, light sources 121 comprise dimmable fluorescent bulbs, while in other embodiments, light sources 121 comprise light emitting diodes (LEDs) or Xenon bulbs. In certain embodiments, light source 121 is configured to emit a single color (e.g., wavelength) of light, while in other embodiments, light source 121 is configured to emit multiple wavelengths of light. By incorporating adjustable light sources, a user can increase or decrease the intensity and/or the wavelength of the light directed towards the inner volume of reservoir 110. In addition, in certain embodiments reservoir 110 comprises spectrally- selective materials that can filter light from light source 121 so that only certain wavelengths reach the inner volume of reservoir 100. Such flexibility can allow a user to easily study the effect of light intensity and wavelengths on the cultivation of photosynthetic cells within reservoir 110. As seen in the exploded view of FIG. 3 and the detailed view of FIG. 4, mixers 130 comprise a shaft 131 with a series of blades or propellers 132 distributed along the shaft. Mixers 130 also comprise a drive motor 133 and a magnetic coupling 134 with an internal magnet 135 and an external magnet 136. Magnetic coupling allows drive motor 133 to be mounted outside of reservoir 110 and eliminates the need for shaft 131 to extend through reservoir 110. This configuration can decrease the likelihood of a leak developing in reservoir 110. As shown in FIG. 3, mixers 130 extend into reservoir 110 so that propellers 132 are distributed throughout the inner volume of reservoir 110. Mixers 130 can be operated to rotate so that propellers 132 mix the contents of reservoir 110.

Referring specifically now to FIG. 5, harvesting extraction module 170 comprises a pair of fluid fittings 171 and 172, a fluid control shaft 173, a handle 174, and a locking mechanism 175. Handle 174 can be manipulated so that fluid control shaft 173 is positioned to allow the contents from reservoir 110 to be withdrawn from one of the fluid fittings 171, 172. In addition, fluid control shaft 173 can be positioned so that cleaning solution can be flushed through fluid fittings 171, 172. Referring now to FIG. 6, sample extraction module 180 comprises a septum 181 disposed between a plug 182 and a cylinder clamp 183. Septum 181 can be constructed of a self-sealing material (e.g., silicone, latex, neoprene, etc.) that can allow a needle (or other sampling device) to penetrate the septum and withdraw material from reservoir 110. In the embodiment shown in FIG. 7, temperature control system 150 comprises a fluid (e.g., water) jacket assembly 151 that allows chilled or warmed fluid to be circulated proximal to the inner volume of reservoir 110. Fluid jacket assembly 151 comprises a series of frames 152-155 that can be assembled to form a fluid jacket that allows a fluid to circulate within the jacket. Fluid jacket assembly also comprises a pair of fluid entrance fittings 156 and exit fittings 157 to allow fluid to enter and exit the jacket. A pump (not shown) can be used to circulate fluid from the jacket to a heating/cooling exchanger or other fluid temperature control device. In other embodiments, temperature control system 150 may comprise a second heating or cooling source (not shown) that can be used to create a thermal gradient in the inner volume of reservoir 110. The effect of such a gradient on the cultivation of photosynthetic cells within reservoir 110 can then be monitored via monitoring system 160.

Claims

1. A modular cell cultivation system comprising: a reservoir comprising an inner volume; a light source configured to emit light and illuminate the inner volume of the reservoir; a mixer extending into the inner volume of the reservoir; a gas delivery system; a temperature control system; a sensor system configured to sense one or more parameters in the inner volume of the reservoir; a harvesting extraction module; and a sample extraction module.

2. The modular cell cultivation system of claim 1 wherein the reservoir is configured so that a user can increase or decrease the inner volume.

3. The modular cell cultivation system of any of claims 1-2 wherein the reservoir comprises a pair of side plates and a pair of replaceable end plates.

4. The modular cell cultivation system of any of claims 1-3 wherein the temperature control system comprises a water jacket proximal to the inner volume of the reservoir and an external thermal control apparatus.

5. The modular cell cultivation system of any of claims 1 -4 wherein the reservoir comprises panels that allow the inner volume to be visible from outside of the reservoir.

6. The modular cell cultivation system of any of claims 1-5 wherein the light source is configured to increase or decrease the intensity of the emitted light.

7. The modular cell cultivation system of any of claims 1-6 wherein the light source is configured to effect spectral wavelength control.

8. The modular cell cultivation system of claim 7 wherein the light source is configured to increase or decrease the intensity of the emitted light.

9. The modular cell cultivation system of any of claims 1-8, further comprising multiple controllable light sources.

10. The modular cell cultivation system of any of claims 1 -9 wherein the light source comprises a dimmable fluorescent bulb.

11. The modular cell cultivation system of any of claims 1 -9 wherein the light source comprises a light emitting diode (LED).

12. The modular cell cultivation system of any of claims 1-9 wherein the light source comprises an array of light emitting diodes (LEDs) configured to emit one or more wavelengths of light.

13. The modular cell cultivation system of any of claims 1-12 wherein the light source comprises any combination of one or more dimmable fluorescent bulbs and one or more light emitting diodes.

14. The modular cell cultivation system of any of claims 1-13 wherein the mixer comprises a magnetically driven shaft and an impeller.

15. The modular cell cultivation system of any of claims 1-14 wherein the sample extraction module comprises a septum.

16. The modular cell cultivation system of any of claims 1-15 wherein the harvesting extraction module comprises a valve configured to seal off the inner volume of the reservoir from the outside environment when the valve is in the closed position.

17. The modular cell cultivation system of claim 16 wherein the valve is configured to allow a fluid contained within the inner volume to be removed from the inner volume when the valve is in the open position.

18. The modular cell cultivation system of any of claims 1-17 wherein the sensor system is configured to measure a parameter selected from the group consisting of: pH, turbidity, dissolved chemical species, dissolved gases, headspace gases, temperature, liquid flow rate, gas flow rate, and mixing intensity.

19. The modular cell cultivation system of claim 1-18 wherein the sensor system comprises a plurality of sensors and actuators configured to work collectively to control daily cycles of temperature, illumination, and nutrient delivery, and to track the simultaneous effect of these controlled process trajectories.

20. The modular cell cultivation system of any of claims 1-19 wherein the gas delivery system comprises one or more mass flow controllers.

21. A method of cultivating cells, the method comprising: providing the system of any of claims 1-20; culturing photosynthetic cells in the inner volume of the reservoir; utilizing the gas delivery system to introduce a gas into inner volume of the reservoir; utilizing the mixer to mix the photosynthetic cells and the gas; utilizing the light source to illuminate a portion of the inner volume of the reservoir; utilizing a sensor in the sensor system to measure the value of a parameter in the inner volume of the reservoir; and removing a portion of the photosynthetic cells from the reservoir.

22. The method of claim 21 whereby an automated system for nutrient delivery and makeup water introduction is attached to the reactor for automated operations.

23. The method of any of claims 21 -22 whereby an automated system for biomass harvest is attached to the reactor for automated operations.

24. The method of any of claims 21 -23 further comprising: adjusting the gas delivery system and altering the value of the parameter measured by the sensor.

25. The of any of claims 21 -24 further comprising: adjusting the mixer and altering the value of the parameter measured by the sensor.

26. The method of any of claims 21-25 further comprising: adjusting the light source and altering the value of the parameter measured by the sensor.

27. The method of any of claims 21-26 wherein removing a portion of the photosynthetic cells from the reservoir comprises manipulating the harvest extraction module to allow the photosynthetic cells to be removed from the inner volume.

28. The method of any of claims 21-27 wherein removing a portion of the photosynthetic cells from the reservoir comprises penetrating a septum of the sample extraction system and withdrawing a portion of the photosynthetic cells.

29. The method of any of claims 21-28 further comprising replacing a component of the reservoir and increasing or decreasing the inner volume of the reservoir.