Abstract:
A process is described for the creation of an inoculum for an organism capable of photosynthesis of a target compound within a closed bioreactor, involving steps of transfer of a sterile growth medium under pressure without exposure to atmosphere. The process is useful for the creation of inocula for the growth of cyanobacteria to produce target chemical products, such as ethanol, in closed photobioreactors.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/852,169, filed Mar. 15, 2013, the disclosure of which is incorporated herein by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
       [0002]    This invention was made in part with United States government support under the Department of Energy grant number DE-EE0002867. The government has certain rights in this invention. 
     
    
     REFERENCE TO SEQUENCE LISTING 
       [0003]    Not Applicable. 
       TECHNICAL FIELD 
       [0004]    This invention relates to the staged creation of inocula of bacterial species suitable for scaling to commercial production closed bioreactors. The process described herein is preferably directed to the creation of inocula for the use of cyanobacteria to produce target chemical products in photobioreactors. 
       BACKGROUND OF INVENTION 
       [0005]    Cyanobacteria form a phylogenetically coherent group of gram-negative prokaryotes that are capable of oxygenic photosynthesis, wherein their photosystems PSI and PSII extract and transfer electrons from water molecules to electron acceptors and generate oxygen as a co-product. They are capable of fixing carbon from CO 2  under aerobic conditions. 
         [0006]    As photoautotrophic organisms, the rates of photosynthesis and growth of cyanobacteria are directly affected by the physical parameters of the environment. 
         [0007]    In the wild, the competitive success of cyanobacteria depends on a continual fine-tuning of growth rate in order to exploit the changing nutritional environment. To cope with depleted nutrients and exploit those that are plentiful, the cyanobacteria undergo transitions from exponential to arithmetic (linear) growth into non-growth (stationary) physiological states. 
         [0008]    The duration of the exponential and linear growth phase in culture depends upon the size of the inoculum, starting density of the inoculum, growth rate, environmental conditions, and capacity of the medium to support microbial growth. Cyanobacterial growth does depend on light intensity. The dependence on external light intensity is impacted by culture density. 
         [0009]    It has been reported by Foster that wild cyanobacteria grow optimally in the range of 15-75 μE m −2  s −1  and batch cultures progress from a lag phase into an exponential growth phase. This is typically followed by a period of linear growth that continues until the culture reaches the non-growing stationary phase. Linear growth in bacteria occurs when there are perturbations in the environment such that a critical nutrient is regulated arithmetically. In cyanobacteria, linear growth is most often associated with light limitation caused by self-shading of cells as cultures reach a certain cell density J. S. Foster, et al., Arch. Microbiol, (2007) 187:265-279. The optimal light range may be broader than indicated by Foster, such as 15-300 μE m −2  s −1 . 
         [0010]    In 1999, Deng and Coleman disclosed the introduction of new genes into the cyanobacterium  Synechococcus  PCC 7942 to create a novel pathway for fixed carbon utilization which created the target chemical product ethanol. M.-D. Deng and J. R. Coleman, Appl. Envir. Microbiology (1999) 65: 523-528. Related patents are R. P. Woods, et al. U.S. Pat. No. 6,306,639 and U.S. Pat. No. 6,699,696. Other target chemical products have been identified; see for example, U.S. Pat. No. 7,794,969 and U.S. Pat. No. 8,183,027. 
         [0011]    In the production of target chemical products, such as ethanol, from microorganisms, such as cyanobacteria, an inoculum of the microorganism is needed so as to provide a population of such microorganism, suitable for scaling up to levels amenable to commercial scale production. In the case of specialty chemicals, produced in low amounts, this inoculum might be cultured in a vessel so that the cell density increases to a cell density suitable for reaching a production level that meets overall productivity metrics. [See for example PCT/US2011/022790, MICROORGANISM PRODUCTION OF HIGH-VALUE CHEMICAL PRODUCTS, AND RELATED COMPOSITIONS, METHODS AND SYSTEMS; see separately Example 1 of PCT/GB2012/050194] In the case of commodity chemicals, such as biofuels, inoculum scale-up might proceed in several stages. 
         [0012]    In the case of inocula to create cultures for open systems, published US application 20100304456 lays out some guidelines: 
         [0013]    It is preferred that (1) the amount of biomass provided by the Closed Systems to inoculate the Open Systems should be equal to more than 5% of the carrying capacity of the aggregate Open Systems; (2) the growth rate of the species being cultivated is greater than approximately one and a half doublings per day (i.e. cell biomass doubles about every 16 hours); and that (3) no culture be maintained in any Open System for a period of more than 5 days. The combination of these three limitations assures that, under any circumstances, the culture should attain a biomass of the desired microbe that is equal to at least approximately 90% of the carrying capacity in 5 days or less. This is important for several reasons. First, a culture that is inoculated at a relatively high cell concentration (i.e. greater than 5% of carrying capacity) will dominate the medium compared to any unwanted cells that may have inadvertently been introduced. Second, because most species grow at rates substantially less than 1 doubling every 16 hours (1.5 doublings per day), a species that is capable of growing this rapidly will outpace most potential competitors. Third, the combination of the large inoculum (greater than 5% of carrying capacity) and high growth rate (greater than 1 doubling every 16 hours) assures that, within 5 days, the total biomass will be very near carrying capacity. These conditions are important to (1) reducing the risk of contamination, and (2) promoting the production of total biomass or the biosynthesis or production of oil. First, a potential contaminant would have to have a large inoculum and would have to grow more rapidly than the desired species to dominate the culture medium within 5 days. Second, oil production in particular is favored in cultures that are near carrying capacity because resources become limiting to growth once the culture passes 50% of carrying capacity. By limiting resources favorable to growth, one generally stimulates the biosynthesis of oil. 
         [0014]    Paragraph 81 of US 20110217692 shows the risks of contamination. 
         [0015]    Paragraph 82 of US 20110287541 discusses amounts stored for use as inocula. 
         [0016]    Earlier art mentions the preparation of inocula ultimately for use in open, rather than closed, systems. For example, H. W. Blanche, Current Opinion in Biotechnology (2012) 23:390-395; E. Olguin, Biotechnology Advances (2012) 30:1031-1046; J. Quinn, Bioresource Technology (2012) 117: 164-171; I. Christenson, Biotechnology Advances (2011) 29:686-702 [discussing Cellana]. 
         [0017]    The present invention is directed to the creation of inocula suitable for introduction into closed bioreactor systems. The present invention provides a method for rapid scale-up of inoculum by monitoring of optical density, and control thereof. An embodiment of invention permits the method to proceed by minimizing exposure of the inoculum to ambient air. 
       SUMMARY OF INVENTION 
       [0018]    An aspect of the present invention is a method for the staged growth of inoculum wherein the optical density of the culture at 750 nm is maintained between 0.01 and 8.0 by controlled addition of a liquid medium, which can be sterile salt water. 
         [0019]    An aspect of the present invention is a system which allows for the staged growth of inoculum in the absence of exposure to air, thereby minimizing problems with contamination. An embodiment of this system is given in  FIG. 1 . 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0020]      FIG. 1 . An embodiment of the inoculum transfer procedure. 
           [0021]      FIG. 2 . An embodiment of the inoculum transfer procedure. 
           [0022]      FIG. 3 . Schematic layout of inoculum embodiment. 
           [0023]      FIG. 4 . Schematic layout showing scale-up to 4,500 liter bioreactor. 
           [0024]      FIG. 5 . Schematic layout of inoculum embodiment showing surge tank. 
       
    
    
       [0025]    The numbered figure elements for  FIGS. 1 and 2  are:
         100 : Air Pump     101 : Check Valve     102 : Quick Disconnect Coupling     103 : Tubing Cap     104 : Culture/Media filled line     105 : ⅜″ Santoprene Line to 80 L Inoculum bag     106 : Luer Lock Air Pump Connection Fitting     107 : 2 Micron Filter     108 : Ball Valve     109 : Liquid Trap     110 : Humidifier     111 : CO2 Delivery     112 : Air Filled Line     113 : CO2 Delivery Line     114 : 3 way valve     200 : 1 L Vessel     300 : 20 L Nutrient Carboy     400 : Exhaust Trap     501 ;  502 ;  503  etc.: 5 L Vessel       
 
         [0045]    The numbered figures elements for  FIG. 3  are:
         1 : Air inlet to pressurize vessel     2 : 1 L culture with 500-900 ml culture volume     3 : Large scale up vessel 1 (20 L, 80 L, or 500 L vessel)     4 : Large scale up vessel 2 (80 L, 500 L, or inoculated PBR)     5 : Media 1 (typically 20 L carboy with seawater and nutrients) to fill #3     6 : Media 2 (typically 20 L carboy with seawater and nutrients) to fill #4     7 : Overflow waste (if necessary)     8 : Sample port     9 : Peristaltic pump for better control on transfer without adding pressure to system       
 
         [0055]    #3 (paragraph 26) can be operated continuously to maintain an OD of 1.0 or lower. If culture reached full capacity, overflow can be turned over to new vessel (i.e., #4) or can be wasted in dump container (#7). 
       DETAILED DESCRIPTION OF THE INVENTION 
     Distinctions Over Art 
       [0056]    Relative to US published application 20100304456, which teaches an inoculum equal to more than 5% of the carrying capacity of the final system, the present invention teaches as low as 1%. Further, with initial OD 750  of as low as 0.1, the present invention can achieve a doubling time of as low as 9 hours, and on average 12-14 hours. 
       Example 1 
     Sequential Grow Up of Inoculum 
       [0057]    The initial inoculation of the inoculum scale-up system is performed in the sterile laminar flow hood by transferring 100 mL of culture into the 1 L bottle in the scale-up system (using a sterile pipet). This culture is then diluted with sterile MBG11 media until there is approximately 600-900 mL in the bottle. All further transfers are performed according to inoculum scale-up system protocols (i.e. pressurization of culture bottle or media bottle and opening of valves without breaking the sterile envelope). 
       Transfer to 5 L Bottle 
       [0058]    In the 1 L bottle, there will be approximately 900 mL of culture. When this volume reaches an ideal optical density at 750 nm (OD 750 ) of 4.0 (or higher, provided the culture is not in stationary phase), it is to be transferred to the previously connected three 5 L bottles. Each 5 L bottle receives 300 mL of inoculum. The 5 L bottles are then topped with 2 L of MBG11, from the previously attached sterile media carboy. This top-off results in an OD 750  of approximately 0.6. When this 2 L of culture reaches an OD 750  of 2.0, it receives another 2 L of media (resulting in a final volume of 4 L at OD 750  of 1.0). Media may be varied to extend exponential and linera growth phase such that the optical density at 750 nanometers can exceed 4.0. 
       Transfer to 80 L Flat Panel Reactor 
       [0059]    When the culture in a single 5 L bottle reaches an OD 750  of 3.0 (or higher, provided the culture is not in stationary phase), it is transferred into an 80 L reactor by fusing the tubing on the bottles and the reactor with the sterile tube fuser. 20 L of media is then added (also using the sterile tube fuser) for an OD 750  of 0.5. When this volume reaches an OD 750  of 2.0, an additional 20 L of media is added for an OD 750  of 1.1. Once again, when this culture reaches an OD 750  of 2.0, it receives the final 40 L of media (OD 750  of 1.1). 
       Transfer to 500 L Reactors 
       [0060]    When the 80 L reactor reaches an ideal OD 750  of 3.5, it is transferred into a 500 L reactor by means of the sterile tube fuser with 420 L MBG11 media (resulting in an OD 750  of 0.6). 
       Transfer to 4,500 L Reactors 
       [0061]    When a 500 L reactor reaches an OD 750  of 4.5, it is transferred to a 4,500 L commercial photobioreactor with the additional 4000 L of MBG11 media (final OD 750  of 0.5). 
       Example 2 
     Protecting Inoculum from Contamination 
       [0062]    In previous work, the transfer of inoculum into 1 liter and 5 liter containers was done in a fume hood, which required extra time and space. The repeated need to use the fume hood represented a bottleneck in the process of growing up inoculum. Furthermore, more frequent manipulations increased the risk of contamination. One has to do work sterilely, and any time one opens up a flask there is always a risk of contamination. 
         [0063]    In the present embodiment, depicted schematically as a system in  FIG. 1 , the one liter bottle and the five liter bottles are autoclaved as an interconnected system. The 20 liter carboy is autoclaved separately, but then connected aseptically to system in the hood or by sterile tube-fused connections outside of the hood. There is a transfer of the initial inoculum, in the hood or by sterile tube-fusing, to the one liter container. All connections are made and removed in the hood, which can be mobile, or by remote sterile tube-fused connections. Ports are sprayed with ethanol and opened under flame from a portable burner. An alternative is to steam the ports. With the use of the sterile tube fuser, no further work is done in the hood after the initial 100 mL culture volume added to the 1 L bottle. 
         [0064]    Pressure from a pump is used to move liquid media. Excess gas volume leaves the system through an outlet port fitted with a 0.2 micron filter, so that no bacteria can enter the system. There is a sterile input port, which allows input of things such as vitamin B-12 and neomycin. There is a sterile sampling port in the 1 liter and 5 liter vessels to remove liquid samples, under pump pressure, so that one may determine optical density of the inoculum culture. As the cell density increases within the 1 liter container, sterile sea water liquid is moved from the carboy to the 1 liter container to top off the liquid level in a way to maintain a desired optical density. As the liquid volume within the 1 liter container approaches 1 liter (generally around 900 mL), the inoculum from the 1 liter container is moved via pump pressure to the five liter container. 
         [0065]    The inoculum is allowed to grow in the 5 liter container, with addition of media via pump pressure done in a way to roughly maintain roughly a desired optical density of culture in the 5 liter container. This optical density can be less than 1.0 for higher growth rates. 
         [0066]    There is a sterile tube fuser which allows transfer of inoculum culture from the 5 liter container to an 80 liter container. The liquid level is topped off to maintain a desired optical density. There is a sterile tube fuser which allows transfer of inoculum culture from the 80 liter container to a 500 liter container. 
         [0067]    In further embodiments, there are a plurality of one liter containers operationally connected to a plurality of 5 liter containers operationally connected to a plurality of 80 liter containers operationally connected to a plurality of 500 liter containers. 
       Example 3 
     Staged Inoculum Up to 4,500 Liter 
       [0068]    Using the procedure outline in Example 2, 900 mL of liquid was transferred sequentially to a 1 liter bottle. The 900 mL comprised 150 mL of culture and 750 mL of media and nutrients. 
         [0069]    After obtaining an OD of 2.0, the 900 mL of culture was transferred via pumping according to Example 2 into two 5 liter bottles with 2 liters of media in each. When the OD within the 5 liter bottles reached 1.5, an another liter of media was added to each bottle. When the OD once again reached 1.5, an additional 2 liters of media was added to each 5 liter bottle, for a total volume of 4 liters in each bottle. 
         [0070]    After obtaining an OD of 3.0, the 8 liters of culture was transferred via pumping according to Example 2 into 20 liters of natural salt water within an 80 liter bioreactor supported on a frame. Each time the OD reached 2.0, the volume was doubled until there were 80 liters in the reactor. 
         [0071]    After obtaining an OD of 2.5 in the volume of 80 liters, the culture was transferred via pumping according to Example 2 into a bioreactor of volume 200 liters, which had sequential media additions until a final volume of 500 L was reached. 
         [0072]    After obtaining an OD of 5.0 in a volume of 500 L, the culture was transferred into a bioreactor of volume 4,500 liters. 
         [0073]    The 1 liter, 5 liter, and 80 liter containers had as a carbon source for growth 1% CO 2 . The larger containers utilized 1.75% CO 2 . The 1 liter, 5 liter, and 80 liter containers were cultivated in a greenhouse. The added liquid comprised of BG-11 (with 3 μM EDTA) and Na 2 CO 3  (20 mg/Liter)+K 2 HPO 4  (40 mg/Liter). 
       Example 4 
       [0074]    Using the staging of inocula in a manual procedure and similar to the outline in Example 1, 600 mL of liquid was transferred to a 1 liter bottle. The 600 mL comprised 100 mL of culture and 500 mL of media and nutrients. 
         [0075]    After obtaining an OD of 2.5 or up to 4.0, the 600 mL of culture was transferred via pumping according to Example 1 into 10 liters of natural salt water within an 80 liter bioreactor supported on a frame. The 10 L culture volume was subsequently topped-off to 80 liters with sterile salt water and nutrients over the course of 5 days, while maintaining the OD at levels between 0.5 to 1.0. 
         [0076]    After obtaining an OD of 1.0 in a volume of 80 liters of culture, the 80 L volume of inocula was transferred via pumping according to Example 1 into a bioreactor of volume capacity of 500 liters at an OD containing 100 liters of sterile seawater and media. The total volume of 180 liters with a final OD ranging between 0.2 to 0.5. After obtaining an OD of 1.0 in the initial volume, the initial volume was subsequently topped-off to 500 liters of sterile salt water and nutrients over the course of 7-10 days, while maintaining the OD at levels between 0.5 and 1.0. 
         [0077]    After obtaining an OD of 2.5 in a volume of 500 liters of culture, 500 L of is transferred into a bioreactor of volume 4,500 liters. 
         [0078]    The 1 liter and 80 liter containers had as a carbon source for growth 1.2% CO 2 . The larger containers utilized 1.75% CO 2 . These carbon amounts are dependent on restriction of diffuser and height of water column. The 1 liter and 80 liter containers were exposed to light from Spectralux T5 fluorescent 54 W (about 100 PAR at initiation 0.1 to 0.25 OD; and 400 PAR at OD 2.5 or greater). The added liquid comprised BG-11 (with 304 EDTA) and Na 2 CO 3  (20 mg/Liter)+K 2 HPO 4  (40 mg/Liter) 15 mg/L NaNO 3 . 
       Example 5 
     Relating to Inoculation of Multiple Bioreactors: Treatment with Sterilizing Gas 
       [0079]    Groups of bioreactors should be designed so that a sterilizing gas (such as ozone) can be supplied to all components of the system including each bioreactor and associated piping. 
         [0080]    The piping systems should be free of dead ends and each system should end in a 0.2 μm filter so that the sterilizing gas (e.g., ozone) can be drawn across the entire internal surface of each system. Points where sterilizing gas will be added to the system should be identified and filters sized appropriately to ensure a consistent flow of 25 LPM with less than a 5 PSI pressure drop at these inlet points. 
         [0081]    As a general matter, contamination can be controlled with the use of ozone. As to liquid water, this can be done by injecting concentrated ozone gas into process seawater. As to bioreactors and process lines in need of control, this can be done by treatment with humid ozone gas. Initially obtained sterility can be maintained by keeping the system isolated by 0.2 μm air filters during liquid transfers. 
         [0082]    As to sterilization of seawater by ozone, the TRO [total residual oxidant] content of seawater will degrade from about 10 to about 0 in about 1 to 4 days, depending upon factors including liquid volume, light intensity, and temperature. The TRO of treated seawater should be sufficiently low so as not to interfere with the inoculation process, and preferably about zero. As a practical matter, ozone treated seawater should be allowed to stand so that TRO can decay for no less than 48 hours before inoculation. 
         [0083]    Further as to the use of ozone, pressure must be maintained between 5 and 30 psi in the ozone generator to maintain its functionality. Flowpaths in the seawater sterilization system must be aligned and checked to ensure that pumps do not work against closed valves. 
       Example 6 
     Relating to Inoculation of Multiple Bioreactors: Use of a Manifold 
       [0084]    A manifold can be used to connect multiple photobioreactors (PBR) in series (i.e., header) for inoculating each PBR from a single source culture and making a single connection to each reactor. 
         [0085]    Separately, a manifold may be used in reverse for connecting multiple source cultures; however, a homogeneous stream is not made and tubing diameters must be considered. 
         [0086]    The term manifold can reference a series of connections for linking more than one vessel to a single line that can be used for splitting a large volume of liquid (or gas) into multiple smaller vessels OR conversely, combining multiple small source volumes into a larger reservoir. By connecting multiple vessels to a single line, there is only one connection made to the PBR at time of inoculation. 
       Example 7 
     Relating to Inoculation of Multiple Bioreactors: Use of a Mixing Chamber 
       [0087]    Inoculation of research and commercial photobioreactors requires a large volume or biomass of potentially axenic cyanobacteria culture. Often to meet the volume or biomass demands, multiple cultures are mixed together into a single chamber or linked together in series. Processing a single chamber to meet the capacity of inoculum for sterility and cleanup is not always practical or feasible for the specific inoculation event Linking cultures in series also is not ideal for creating a homogenous culture, largely due to inconsistencies in mixing ratios between the linked cultures. This embodiment describes a practice for and verification of using a mixing chamber for up to three source cultures to create a homogenous stream for inoculation of replicate photobioreactors. More than three source cultures may be used as long as the combined tubing inner diameters (ID) of the mixing chamber (and source culture harvest line) exceed the mixing chamber outlet tubing ID. 
         [0088]    The mixing chamber is positioned as an intermediate vessel between multiple source cultures that flow into the chamber through three separate inlets and an outlet vessel for depositing the mixed stream via a single outlet siphon. The outlet is connected to a peristaltic pump that draws head through the sealed mixing chamber, which creates a siphon from the source cultures at equal rates. The inlets extend further than the outlet siphon into the mixing chamber to allow for adequate mixing below the outlet siphon level. The multiple source cultures enter the mixing chamber via the inlet lines and become homogenized below the outlet siphon level. Once the chamber volume reaches the outlet siphon, the chamber contents are homogenously mixed and the outlet stream is an average of all source culture cell densities. For example, if three source cultures are being used with the following cell densities measured as OD 750 : 1.0, 1.5, and 1.75, the average cell density of the mixed source cultures should be 1.42 OD 750 . Using a 10 L carboy has yielded sufficient results in mixing three 80 L source cultures at varied cell densities into a single homogenous stream. 
         [0089]    The outlet line can be any diameter and more inlets also can be installed on the mixing chamber; however, the outlet diameter cannot exceed the maximum of a single inlet or the sum of tubing ID&#39;s of all inlets combined. The inlet lines are connected to the lid by barbed fittings forming a union through the lid to extend into the chamber from the underside of the lid. The inlet extension into the chamber must be longer than the outlet line that extends through the chamber. The inlet lines must be the same length, although slight variations (&lt;1 cm) in the lengths have not shown to affect results significantly. The outlet line is connected to the barbed fitting on the lid with the largest diameter, and also to the transfer tubing used in the peristaltic pump. The pump draws from the single outlet line coming out of the mixing chamber. The pump drawing force on the siphon tube with a larger diameter than the combined source culture harvest lines (i.e., inlet tubes) provides enough force to siphon from multiple source cultures. The source culture harvest line and mixing chamber inlet tube IDs should match; however may be different as long as the maximum siphon tubing ID does not exceed the combined inlet tubing ID&#39;s and the chamber inlet IDs are not greater than the source culture harvest line IDs. 
       Example 8 
     Relating to Inoculation of Multiple Bioreactors: Use of a Surge Tank 
       [0090]    In order to connect inoculum and nutrients to multiple bioreactors, one may employ an inoculation/nutrient delivery header along with a surge tank. Such a system can comprise a diaphragm pump and a sequence of valves and piping that can direct liquid flow to and from a surge tank (recirculation) through a variety of paths. This recirculation allows for equal liquid distribution to the reactors. 
         [0091]    In one embodiment, the system consists of a fiberglass deck box and a polyethylene surge tank. The deck box contains the pump, piping and valves that allow for the directional control of liquid flow. The plumbing consists of ½ inch schedule 40 PVC, ½ inch PVC ball valves, ½ inch PVC unions, ½ inch schedule 80 threaded tees, ½ inch PVDF and polyethylene barbed fittings. 
         [0092]    The surge tank sits external to the deck box and acts as the re-circulatory reservoir for liquid additions. All santoprene lines on the surge tank are equipped with 25 mm 0.22 μM PTFE Polyfilters; each has a terminal male luer fitting, a three inch section of ¼inch silicone tube with a slide clamp, and a male luer fitting secured to the filters female luer end. The silicone tubing can be attached to santoprene via a barbed polypropylene reducer. All barbed fittings are cable tied in place. This filter and clamp arrangement allows air to be forced through the lines to clear any residual liquid without compromising the sterility of the system. 
         [0093]    One may transfer sterile seawater to the surge tank using a peristaltic pump. One may use a diaphragm pump to transfer inoculum culture during the inoculum scaleup. On may use a diaphragm pump to transfer organisms into the surge tank.