The present invention relates generally to methods and devices for the production of bioplastics and resins. More specifically, it relates to the biosynthesis of polyhydroxyalkanoate (PHA) bioplastics and resins using bacteria that can use gaseous alkanes and alkenes for PHA synthesis.
Conventional microbial production of bioplastic polyesters is performed using fermenting liquid-phase bioreactors. The production typically involves cycling through two growth phases. First, pure or mixed cultures of bacteria are grown under balanced growth conditions, i.e., with sufficient carbon feedstock and nutrients for cell division. Next, the biomass is grown under unbalanced conditions, i.e., with sufficient feedstock (typically a sugar) but lacking one or more essential nutrients (e.g., N or P). During this period of unbalanced growth, many bacteria produce polyhydroxyalkanoate (PHA) granules, e.g., polyhydroxybutyrate (PHB) and/or polyhydroxyvalerate (PHV). In conventional techniques for such biosynthesis of PHA, both phases of production involve submerged (liquid-phase) growth, i.e., all of the feedstock is supplied through the aqueous phase. Cells containing PHA granules can be harvested by filtration or centrifugation and lysed. Dissolution of the PHA into a separate liquid phase (organic solvents, supercritical CO2, etc.) may then be used to remove the residual non-PHA cell debris. Alternatively, dissolution of the non-PHA phase (e.g., acid-base treatment) can be used to isolate undissolved PHA. There are, however, limitations associated with method for biosynthesis of PHA. It would be desirable to increase growth rates so that bioplastics can be more rapidly produced, readily switch feedstocks to modify the composition of PHA produced, decrease water requirements for PHA production, and increase the energy efficiency of PHA biosynthesis.
Gaseous carbon substrates offer several advantages for growth of PHA-producing microorganisms, but their low aqueous solubility limits culture density, specific growth rate, and PHA production rate. Maintenance of high substrate levels in the water phase leads to inefficient use of substrates and high demand for energy.
The present inventors have recognized that terrestrial plants solve a similar problem with minimal energy investment by extracting carbon and oxygen from the gas phase. Motivated by this insight, the inventors have developed a similar strategy for bioreactor design to enable an efficient, low-energy means of growing and harvesting bioplastic-rich biomass.
In contrast with prior methods in which the substrates used to produce polyhydroxyalkanoates (PHAs) are delivered through liquid phase fermentations, the present invention provides methods in which gas substrates (methane, propane, butane, etc.) are delivered in gas phase to microbial biofilms in a high solids fermentation. The gas phase delivery of these substrates addresses the mass transfer problem associated with use of poorly soluble gaseous substrates and provides a simple and practical way to produce bioplastics.
This technique significantly increases the rate of mass transfer to cells, enabling more rapid production of the bioplastic, enables delivery of diverse gas phase substrates for co-polymer production, and improves opportunities for PHA extraction. Diffusion of a substrate through the gas phase is 10,000 times faster than diffusion through liquid water, and does not require the high energy inputs necessary for mass transfer through liquid.
According to preferred embodiments of the invention, the unbalanced growth conditions are established in a high solids fermentation, thus avoiding the mass transfer limitations associated with delivery of poorly soluble substrates, such as methane and oxygen into a water phase. In one specific implementation, type II methanotrophs are grown under the appropriate selection conditions (i.e., balanced growth), separated from the liquid phase, then transferred to a chamber. In the chamber, gas phase substrates (methane, propane, butane, oxygen) are delivered under unbalanced conditions, i.e., where nitrogen and other nutrients required for balanced growth are not present, and the gas phase substrates are consumed to produce bioplastic granules inside the cells. The cells containing these granules are then lysed and the bioplastic powder recovered.
Production of PHA is thus a two-step process: (1) balanced growth in which cells accumulate, and (2) unbalanced growth in which cells expand as PHA accumulates within the cells. In a preferred embodiment, submerged growth is carried out for step (1) and solid-phase fermentation for step (2), but both steps can also be carried out in a high solids fermentation
Embodiments of the invention provide methods for producing polyhydroxyalkanoate (PHA). The methods include providing a chamber containing microorganisms in the form of moist biofilms, delivering gas phase carbon substrates to the chamber for production of PHA in a high-solids fermentation, and extracting PHA from the microorganisms. The production of PHA takes place during unbalanced growth conditions in the chamber. In some cases, the method may include delivering nutrients to the chamber for balanced growth of the microorganisms in the chamber. The nutrients may be delivered in liquid phase. The moist biofilms may have various forms such as films covering moist particles in the chamber or films covering moist membrane sheets in the chamber. The nutrients may be delivered to the biofilms through the membrane sheets.
The methods of the present invention provide a simple and economic technique to produce diverse polyhydroxyalkanoate (PHA) bioplastics and resins from low-cost gaseous substrates, such as biogas methane derived from organic wastes. The methods and devices of the present invention provide low-cost production of bioplastics that can replace conventional synthetic plastics and resins derived from petrochemical feedstocks.
Embodiments of the present invention provide for improvements in the production of polyhydroxyalkanoates (PHAs) by communities of microorganisms in bioreactors. During unbalanced growth, a nutrient such as N or P limits growth and biopolymer accumulates inside the cells. In contrast with conventional methods, in embodiments of the present invention the unbalanced growth occurs in a high solids phase in which the carbon feedstock provided as a gas. In the context of the present invention, “high solids” refers to the use of biomass within a chamber that is filled predominately with gas rather than liquid fluids, where the biomass is composed of at least 20% solid phase biomass by volume in an attached or immobilized state within the chamber.
Outlet 108 carries bacteria from vessel 100, through pump 109, and into a high solids fermentation chamber 110. Some bacteria may be recirculated through line 105 and pump 107 back into reactor 100. In the high solids fermentation chamber 110, the microorganisms may take the form of moist biofilms on a support 112 within the chamber 110. Gas phase carbon substrates are then delivered to the chamber, taken up by the moist biofilms, resulting in the production of PHA in a high-solids fermentation. For example, biogas and oxygen in gas phase enter the chamber 110 through inlets 114 and 116, respectively. The lack of nutrients in chamber 110 creates unbalanced growth conditions and results in the accumulation of PHA within the bacteria. Extraction of the PHA from the microorganisms is performed by delivering supercritical carbon dioxide or another suitable solvent to the chamber 110 through inlet 118. A ceramic filter 120 in the chamber 110 catches the PHA granules and allows cellular debris to pass down in an underdrain 122 in the chamber where it is collected and then exits the chamber via outlet 124. The cellular debris biomass flows through valve 125. The biomass exiting the system may be used, for example, in methane fermentation. The PHA granules are removed from the chamber 110 via outlet 126 and used for the subsequent production of bioplastic materials.
Solid-state fermentation within chamber 110 involves the use of a support 112 for the growth of the biofilm as well as means for delivery of substrates to the biofilms through the gas phase. The support 112 for biofilm growth may be made of non-biodegradable materials, such as fabrics, ceramics, plastics, and porous membranes, or biodegradable supports, such as wheat straw or cellulosic materials.
Gas phase substrates may be include a variety of substances, including 1) carbon sources such as biogas, natural gas (CH4), propane, butane, ethane, ethylene, CO, CO2, etc., 2) nitrogen sources such as N2 and NH3, 3) electron acceptors such as O2 and N2O, and 4) electron donors such as H2.
In some embodiments, the process may also involve a phase of growing the biofilms in the chamber using liquid phase delivery of nutrients and trace elements. Such liquid phase delivery can be achieved using a percolate spray, mist, or periodic immersion of biomass in a bath or rotating drum, sprinkled permeate, aerosols, or passage of biofilm biomass on a disk or drum that rotates through a bath. Again, a variety of substrates can be delivered providing considerable flexibility in the incubation conditions. Substrates that may be delivered through the liquid phase may include 1) carbon sources such as volatile fatty acids, sugars and other soluble organics, 2) nitrogen sources such as nitrate and ammonium, 3) electron acceptors such as dissolved oxygen and nitrate, 4) trace nutrients such as phosphorus, sulfur, and essential cations, and 5) electron donors such as formate.
In another embodiment, biofilms are attached to the exterior of gas- and water-permeable membrane curtains 300, as shown in
In preferred embodiments, methane biogas is used as a low-cost carbon source for PHA synthesis. Type II methanotrophs are used to produce polyhydroxybutyrate (PHB), a useful bioplastic, when grown with methane under unbalanced growth conditions. Cells grown by conventional means under balanced conditions are harvested then subjected to unbalanced growth conditions in a solid-state fermentation. As described above in relation to
The configuration shown in
Several variations of the above techniques are possible. In one variation, balanced growth may be performed in chamber 202 in a separate phase from unbalanced growth by introducing liquid with nutrients into the chamber, e.g., through inlet 219. In another variation, cells may be harvested from liquid culture in the seed reactor 200 via centrifugation, passage through a bag filter, membrane separation, or dissolved air flotation. In another variation, the cells can be incubated in the presence of different gas phase substrates to create different useful polyhydroxyalkanoates. More specifically, by changing gas phase substrates (from biogas methane to propane or butane, for example) or by modifying the composition of the percolate/bath water composition to include a soluble carbon source, such as propionate or butyrate, during the high solids fermentations, co-polymers, such as PHBV or different PHA molecules such as polyhydroxyhexamoate (PHHx) are produced with different properties than PHB, extending the range of possible applications for the bioplastics that are produced.
In some variants of the above embodiments, submerged growth of cells during balanced growth can be achieved using dispersed-growth suspensions or attached growth, in which cells grow upon a carrier, such as activated carbon particles. Dispersed cells can be harvested by centrifugation, passage through a filter, membrane separation, or dissolved air flotation then subject to high solids fermentation for PHA accumulation. Attached growth cells can be detached, concentrated then subject to unbalanced growth conditions in a high solids fermentation.
The embodiments of the present invention have several important advantages over conventional submerged-only fermentations. First, embodiments of the invention have lower energy expense for delivery of a wide range of substrates during the PHA accumulation phase. In liquid reactors, delivery of substrates is a major operational expense because energy is required for mixing of the liquid. Gas phase diffusion coefficients are 10,000 times higher than liquid phase diffusion coefficients. This translates into much faster rates of mass transfer and less energy inputs for delivery of substrates to the biomass. Second, embodiments of the present invention require smaller reactor volumes than comparable liquid phase reactors. Less space is required for PHA production, less water is required, and PHA-enriched biomass can potentially be extracted within the same tank, minimizing solids and water handling and capital costs. Third, embodiments of the invention allow easy delivery of mixed substrates. Gases can be easily removed from an incubation chamber, and new gases introduced. This enables modification of the fermentations to enable production of PHA block co-polymers or mixed PHA polymers that have improved properties for specific applications.
This application claims priority from U.S. Provisional Patent Application 61/278,682 filed Oct. 8, 2009, which is incorporated herein by reference.
Number | Date | Country | |
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61278682 | Oct 2009 | US |