Patent Application
20140193871
Recent climate anomalies, including hurricanes and droughts, have been associated with global warming, i.e., an increase in global temperature. A major cause of the global warming has been attributed to the increase in atmospheric CO2 concentration. Indeed, it has been reported that the atmospheric CO2 concentration increased from around 280 ppm to 368 ppm over the past 200 years. Thus, there is an ongoing interest in developing processes that could efficiently reduce CO2 emission or capture CO2 from the atmosphere.
Processes using living organisms to convert CO2 into organic compounds are generally referred to as carbon dioxide biofixation. The most prominent example of carbon dioxide biofixation involves the use of photosynthetic microorganisms, such as microalgae and cyanobacteria, which use energy from sunlight to drive a carbon fixation pathway.
Besides photosynthetic microorganisms, chemoautotrophic microorganisms (ones that obtain their nourishment through the oxidation of inorganic chemical compounds as opposed to photosynthesis) represent a possible alternative for use in carbon biofixation processes.
A major non-photosynthetic carbon dioxide fixation pathway used by chemoautotrophic microorganisms is the Wood-Ljungdahl (WL) pathway (
Although some acetogenic Clostridia have been engineered to produce commodity chemicals (e.g., butyrate, acetate, acetoin, and acetone) and biofuels (e.g., butanol and ethanol), there remains a need to develop methods that may improve anaerobic organisms' ability to metabolize CO2 into useful chemicals and/or biofuels.
Accordingly, provided herein are processes for microbial fermentation of a gaseous substrate comprising CO2, the processes comprising:
providing a gaseous substrate and a liquid medium to a microorganism, wherein the liquid medium comprises lactic acid and/or salts thereof, and wherein the gaseous substrate comprises CO2, and
allowing the microorganism to produce at least one product chosen from acids and alcohols.
Also provided herein are processes of improving efficiency of carbon capture in microbial fermentation, comprising:
providing a gaseous substrate and a liquid medium to a microorganism, wherein the liquid medium comprises lactic acid and/or salts thereof, and wherein the gaseous substrate comprises at least one carbon source chosen from CO and CO2, and allowing the microorganism to produce at least one product chosen from acids and alcohols.
Additional features and advantages of the present disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed invention.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the present disclosure.
Below, exemplary embodiments will be described in detail with reference to accompanying drawings so as to be understood by a person having ordinary skill n the art. The inventive concept may be embodied in various forms without being limited to the exemplary embodiments set forth herein.
Provided herein are processes for microbial fermentation of a gaseous substrate comprising CO2, the processes comprising:
providing a gaseous substrate and a liquid medium to a microorganism, wherein the liquid medium comprises lactic acid and/or salts thereof, and wherein the gaseous substrate comprises CO2, and
allowing the microorganism to produce at least one product chosen from acids and alcohols.
Also provided herein are processes of improving efficiency of carbon capture in microbial fermentation, comprising:
providing a gaseous substrate and a liquid medium to a microorganism, wherein the liquid medium comprises lactic acid and/or salts thereof, and wherein the gaseous substrate comprises at least one carbon source chosen from CO and CO2, and allowing the microorganism to produce at least one product chosen from acids and alcohols.
In some embodiments, the microorganism is chosen from any anaerobic organisms capable of converting CO2 and/or CO into at least one product chosen from acids and alcohols via Wood-Ljungdahl (WL) pathway.
In some embodiments, the microorganism is chosen from chemoautotrophic microorganisms.
In some embodiments, the microorganism is chosen from photosynthetic microorganisms genetically engineered to be capable of converting CO2 and/or CO into at least one product chosen from acids and alcohols via Wood-Ljungdahl (WL) pathway.
In some embodiments, the microorganism is chosen from Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium difficile, Clostridium ljungdahli, Moorella thermoacetica (formerly Clostridium thermoaceticum), Methanobacterium thermoautotrophicum, Defulfobacterium autotrophicum, Clostridium sticklandii, Clostridium thermoautotrophicum, Clostridium formicoaceticum, Clostridium magnum, Acetobacterium carbinolicum, Acetobacterium kivui and Acetobacterium woodii.
The term “gaseous substrate comprises CO2” and like terms should be understood to include any gaseous substrate in which carbon dioxide (CO2) is available to the microorganism for growth and/or fermentation. Similarly, the term “the gaseous substrate comprises at least one carbon source chosen from CO and CO2” should be understood to include any gaseous substrate in which CO2 and/or CO is available to the microorganism for growth and/or fermentation.
In some embodiments, the gaseous substrate may be a CO2 and/or CO-containing waste gas obtained as a by-product of an industrial process, or from another source such as from automobile exhaust fumes. Exemplary industrial processes include but not limited to: petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing. Depending on the composition of the CO2 and/or CO-containing gaseous substrate, it may be desirable, prior to introducing it to the fermentation, to remove undesired impurities, such as dust particles from the gaseous substrate. For example, the gaseous substrate may be filtered or scrubbed using known methods.
In some embodiments, the gaseous substrate may further comprise at least one gas chosen from hydrogen (H2) and nitrogen (N2).
In some embodiments, the gaseous substrate is a synthesis gas (“syngas”) comprising CO2, CO, and H2. Syngas can be produced by steam reforming of natural gas or gasification of various organic materials such as biomass, organic waste, coal, petroleum, plastics, or other carbon containing materials.
In some embodiments, the gaseous substrate may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by volume of CO2. In some embodiments, the gaseous substrate may comprise from 20% to 80% of CO2. As a non-limiting example, the gaseous substrate may comprise 20% of CO2 and 80% of H2.
In general, CO2 and/or CO will be provided to the microorganism in a gaseous state. However, in some embodiments, CO2 and/or CO can be provided in a liquid. For example, a liquid may be saturated with the gaseous substrate comprising CO2 and that liquid is then added to a bioreactor.
In some embodiments, reduced chemical species and/or electrode(s) may be provided to the microorganism as an electron source for CO2 fixation.
In some embodiments, at least one electron source may be provided to the microorganism for CO2 fixation, wherein the at least one electron source may be chosen from hydrogen (H2), reduced carbon compounds (as non-limiting examples: CO, formate, and methanol), sulfur compounds (as non-limiting examples: H2S and S), and nitrogen compounds (as a anon-limiting example, NH3).
In some embodiments, to improve the efficiency of carbon capture and/or CO2 fixation, a liquid medium comprising lactic add and/or salts thereof is provided to the microorganism.
In some embodiments, the liquid medium provided to the microorganism may comprise 0.5 g/L to 3 g/L lactic acid and/or salts thereof. As non-limiting examples, the liquid medium provided to the microorganism may comprise, for instance, 0.5 g/L, 1 g/L, 1.25 g/L, 1.5 g/L, 1.75 g/L, 2 g/L, 2.25 g/L, 2,5 g/L, 2.75 g/L, or 3 g/L lactic acid and/or salts thereof.
In some embodiments, the liquid medium provided to the microorganism may comprise lactic acid and/or salts thereof in an amount sufficient to induce the expression of gene(s) involved in the Wood-Ljungdahl (WL) pathway.
For example, in certain embodiments, the lactic acid and/or salts thereof may be present in an amount sufficient to induce the expression of at least one gene chosen from CLJU_c37670, CLJU_c37570, CLJU_c37550, CLJU_c37580, CLJU_c06990, CLJU_c20040, CLJU_c08930, CLJU_c07020, CLJU_c37650, CLJU_c37640, CLJU_c37630, CLJU_c37610, CLJU_c37560, and CLJU_c09110, involved in the Wood-Ljungdahl pathway as described in Köpke M, at al., Clostridium ljungdahlii represents a microbial production platform based on syngas. (2010).
In certain embodiments, the lactic acid and/or salts thereof may be present in an amount sufficient to induce about a 1.5 fold or more increase, relative to the microorganism cultured under the same condition but without the presence of lactic acid/or salts thereof, of at least one gene involved in the Wood-Ljungdahl pathway.
In certain embodiments, the lactic acid and/or salts thereof may be present in an amount sufficient to induce about a 1.5 fold or more increase, relative to the microorganism cultured under the same condition but without the presence of lactic acid/or salts thereof, of at least one gene involved in the Wood-Ljungdahl pathway. As a non-limiting example, the lactic acid and/or salts thereof may be present in an amount sufficient to induce about a 1.5 fold to about a 10 fold increase, relative to the microorganism cultured under the same condition but without the presence of lactic acid/or salts thereof, of at least one gene involved in the Wood-Ljungdahl pathway.
In some embodiments, the lactic acid and/or salts thereof may be present in the liquid medium in an amount sufficient to induce about a 1.5 fold to about a 10 fold increase, relative to the microorganism cultured under the same condition but without the presence of lactic acid and/or salts thereof, of at least one gene chosen from codH, codH beta, codH delta, codH gamma, coos1 and metF. As a non-limiting example, the lactic acid and/or salts thereof may be present in the liquid medium in an amount sufficient to induce about a 1.5 fold to about an 8 fold increase, relative to the microorganism cultured under the same condition but without the presence of lactic acid and/or salts thereof, of at least one gene chosen from codH, codH beta, codH delta, codH gamma, coos1 and metF.
To increase the growth of the microorganism and/or CO2 and/or CO fixation, in addition to lactic acid and/or salts thereof, the liquid medium may be supplemented with additional nutrients or ingredients suitable for growing the bacteria. For example, the medium used for culturing the microorganism may also comprise vitamins, salts, extracts, and/or minerals sufficient to permit growth of the microorganism.
In some embodiments, the process for CO2 and/or CO fixation may be carried out under conditions for the desired fermentation to occur (e.g., CO2 to acids), Reaction conditions that can be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO2 or other components in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
In some embodiments, the processes may be carried out in any suitable bioreactor in which the substrate can be contacted with one or more microorganisms, such as a continuous stirred tank reactor, an immobilized cell reactor, a gas-lift reactor, a bubble column reactor, a membrane reactor such as a Hollow Fiber Membrane Bioreactor or a trickle bed reactor, monolith bioreactor, or loop reactors.
In some embodiments, fermentation processes described herein may produce products including but not limited to alcohols and/or acids. Exemplary acids include but are not limited to formic acid, acetic acid, propionic acid, butyric acid, acrylic acid, fatty acids; and exemplary alcohols include but are not limited to methanol, ethanol, acetone, propanol, butanol, and 2,3 butanediol.
In the context of fermentation products, the term “acid” as used herein includes both carboxylic acids and the associated carboxylate anion. For example, the term “acetic acid” includes free acetic acid, acetate salt alone, and a mixture of free acetic acid and acetate salt.
The term “fatty acid” includes any carboxylic acids with a long aliphatic chain, In some embodiments, the aliphatic chain can be linear or branched, and saturated or unsaturated. In some embodiments, the aliphatic chain may contain, for example, from 4 to 12 carbon atoms, 5 to 11 carbon atoms, or 8 to 10 carbon atoms.
At least one product chosen from acids and alcohols of the fermentation processes may be recovered using any known methods.
As a non-limiting example, ethanol may be recovered from a fermentation broth by methods such as fractional distillation or evaporation, and extractive fermentation.
Also as a nonlimiting example, acetate may be recovered from a fermentation broth by using an adsorption system involving an activated charcoal filter. In this case, a suitable separation unit may first be used to remove microorganisms from the fermentation broth. The cell free acetate-containing medium is then passed through a column containing activated charcoal to adsorb the acetate. Acetate in the acid form (acetic acid) rather than the salt (acetate) form is more readily adsorbed by activated charcoal. Thus, the pH of the cell free acetate-containing medium may be reduced to less than about 3 before it is passed through the activated charcoal column, to convert the majority of the acetate to the acetic acid form.
Further as a non-limiting example, butyric acid present in a fermentation broth may be recovered and purified by extraction using an aliphatic amine, such as Alamine 336 (tri-octyl/decyl amine; Alamine336 Cognis, Cincinnati, Ohio, USA), or other water-immiscible solvents.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Clostridium ljungdahlii BCRC 17797 (DSM No. 13528; BCRC No. 17797) was purchased from Bioresource Collection and Research Center, Taiwan. This strain was known to grow and generate metabolites by utilizing the carbon source in the atmosphere, such as CO, gas mixture of CO2 and H2, or synthesis gas (CO and H2), through the Wood-Ljungdahl pathway (
Modified PETC Basal Medium containing (per liter): protease peptone (10 g), yeast extract (3 g), and resazurin stock solution (0.5 mL, 0.1%, w/v). See Cottera J L et al, Influence of process parameters on growth of Clostridium ljungdahlii and Clostridium autoethanogenum on synthesis gas, Enzyme and Microbial Technology 44(5), pp. 281-299 (2008).
ATCC 1754 Salt Solution containing (per liter): NH4Cl (20 g), KCl (2 g), MgSO4.7H2O (4 g), NaCl (16 g), KH2PO4 (2 g), and CaCl2 (0.4 g).
Vitamin Solution containing (per liter): biotin (2 mg), folic acid (2 mg), pyridoxine (10 mg), thiamine HCl (5 mg), riboflavin (5 mg), nicotinic acid (5 mg), calcium D(+) pantothenate (5 mg), vitamin B12 (5 mg), p-aminobenzoic acid (5 mg), and lipoic acid (thioctic) (5 mg).
Trace Element Solution containing (per liter): nitrilotriacetic acid (2 g), MnCl2.4H2O (1.3 g), FeSO4.7H2O (0.4 g), CoCl2.6H2O (0.2 g), ZnSO4.7H2O (0.2 g), CuCl2.2H2O (0.02 g), NiCl2.6H2O (0.02 g), Na2MoO4.2H2O (0.02 g), Na2SeO3 (0.02 g), and Na2WO4.2H2O (0.025 g).
Modified PETC Medium (mPETC), pH 6.8: To prepare mPETC, one liter of Modified PETC Basal Medium was supplemented with 50 ml of ATCC 1754 Salt Solution, 10 ml of Vitamin Solution, and 10 ml of Trace Element Solution. The prepared medium was then dispensed into serum bottles and then autoclaved at 121° C., 15 psi, for 15-20 minutes.
Air-tight bottles used for anaerobic culturing: 200 ml crimp-top serum bottle sealed with a rubber stopper, which was further held in place by an aluminum crimp to ensure an air-tight closure.
Clostridium ljungdahlii was first cultured anaerobically with mPETC medium containing 10 g/L of fructose at 37° C. in one of the air-tight bottles mentioned above. When OD600 reached a value ranging from 1 to 1.5, the culture medium was centrifuged at 3500 rpm for 210 seconds. The resulting cell pellet was resuspended with mPETC without fructose and then further cultured anaerobically at 37° C. for one hour to remove any residual sugar substrates.
45 ml of mPETC culture medium was added to one of the crimp-top serum bottles mentioned above, which was then covered with an aluminum foil and autoclaved for 15 minutes at 121° C., 15 psi. After cooling, the medium-containing bottle was moved into an anaerobic chamber, and 50 μl of cysteine-HCl (50% w/v) was added to the medium to remove any dissolved oxygen.
For an experimental group, sodium lactate solution was further added to the culture medium to obtain a lactate concentration of 2.5 g/L (about 0.022 M).
For a sodium ion control group, NaCl solution was added to the culture medium to obtain a NaCl concentration of 1.52 g/L (about 0.026 M). This control group was designed to test whether the sodium ions present in the sodium lactate play any role in carbon dioxide fixation. Thus, the same molar amount of sodium ion as the experimental group was added.
For a no-additive control group, an appropriate amount of water was added to the culture medium.
Each of the serum bottles containing 45 ml of mPETC culture medium was then inoculated with 5 ml of Clostridium ljungdahlii culture that had residual sugar removed. A sample taken at this point was labeled as t=0 sample. The bottles were then sealed with rubber stoppers and further covered with aluminum crimp caps to ensure an air-tight closure.
The headspaces of the air-tight bottles were first purged with a gas mixture containing 80% N2 and 20% of CO2 under a pressure of 10 psi. Needles were inserted into the rubber stops to allow the gas to escape from the bottles. After about 30 seconds of exposure to the gas mixture, an anaerobic condition filled with CO2 was obtained within the bottles.
Using needles, the headspaces of the bottles were then further purged with another gas mixture containing 80% H2 and 20% of CO2 under a pressure of 20 psi to replace the previous gas mixture. After about 30 seconds, needles were removed and the pressure within the bottles was built up to about 20 psi. The bottles were then moved to a 37° C. incubator and placed on a shaker providing a shaking speed of 100 rpm.
Every 12 hours, a needle was used to release the gas built up within the bottle, and the headspace of the bottle was purged and refreshed under the same process as described above to replenish gaseous carbon source under a stabilized pressure.
Syringes with needles were used to collect liquid samples. After measuring the OD600, the liquid samples were centrifuged at 12,000 rpm for 10 minutes. The resulting cell pellet was stored in 700 μl TRIzol solution for later RNA extraction and gene expression analysis. The supernatant was analyzed with an Agilent 1100 series high-performance liquid chromatography (HPLC) with Aminex HPX-87 H (300 mm×7.8 mm) column.
At t=144 hours, the OD600 value of: (a) the experimental group with sodium lactate reached 0.71, (b) the sodium ion control group reached 0.82, and (c) the non-additive control group reached 0.79. Accordingly, no significant difference was observed in the growth rates of these three groups.
Medium samples taken at t=144 hours were analyzed by HPLC. As shown in Table 1, when CO2 was supplied as the carbon source, Clostridium ljungdahlii BCRC 17797 grown in mPETC was capable of producing acetic acid.
As shown in Table 1, at t=144 hours, the concentration of acetic acid in the experimental group was higher than that in either the no-additive control group or the sodium ion control group. And the concentration of acetic acid in the no-additive control group at t=144 hours was about the same as that of the sodium-ion control group.
As the data in Table 1 further shows, the concentration of lactic acid in the experimental group decreased from 2.92 g/L (at t=0) to 2.25 g/L (at t=144). This indicates that a small amount of lactic acid (i.e., 0.67 g/L) was consumed by the bacteria 0.67 g/L of lactic acid consumed could provide about 0.022 M of carbon atoms, and 6.39 g/L of acetic acid produced in the experimental group contained about 0.213 M of carbon atoms. This indicates that at least 0.191 M (0.213 M minus 0.022 M) of carbon atoms came from CO2 presented in the gas mixture.
As Table 1 above shows, after 144 hours of culture, the sodium-ion control group produced about 4.97 g/L of acetic acid, which contained about 0.166 M of carbon atoms. Thus, the amount of CO2 converted (fixed) to acetic acid by the experiment group (0.191 M (carbon atoms presented in the acetic acid produced by the experimental group minus the amount of carbon possibly converted from lactic acid) was at least about 15% higher than that of the non-additive control group (0.166 M of carbon atoms presented in the acetic acid produced in sodium-ion control group). This shows that lactic acid present in the culture medium of the experimental group improved the carbon dioxide fixation of Clostridium ljungdahlii BCRC 17797.
Real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify the expression of certain genes listed in Table 2.
As shown in
As shown in
The experimental protocol as described in Example 1 was repeated, except that, besides having a no-additive control group, two experimental groups were prepared, one had 1 g/L of lactate in the culture medium and the other had 2.5 g/L of lactate in the culture medium.
Table 3 below shows, after 96 hours of culture, the amount of lactate consumed and acetate produced for the experimental groups and a no-additive control group.
The experimental protocol as described in Example 1 was repeated, except that, besides having a no-additive control group, three experimental groups (0.5 g/L, 1 g/L, and 3 sodium lactate) and one sodium ion control group (containing 1.57 g/L of NaCl) were prepared.
It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only.
This application claims priority to U.S. Provisional Application No. 61/749,010, filed on Jan. 4, 2013.
| Number | Date | Country | |
|---|---|---|---|
| 61749010 | Jan 2013 | US |
