Patent Grant
9200297
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 23, 2013, is named 06720.0370_SL.txt and is 58,791 bytes in size.
Synthesis gas (“syngas”) is a fuel gas mixture comprising primarily carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (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.
Catalytic processes have been developed to convert syngas into a variety of fuels and chemicals such as methane, methanol, formaldehyde, acetic acid and ethanol. Microorganisms have also been used to convert syngas into fuels and chemicals. For instance, syngas has been converted into liquid fuel and chemicals by acetogenic Clostridia microorganisms such as Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium carboxidivorans, and Clostridium ragsdalei.
Anaerobic bacteria, such as those from the genus Clostridium, produce ethanol from CO, CO2, and H2 via the acetyl-CoA biochemical pathway (also known as the Wood-Ljungdahl pathway) as shown in
Microbial processes used for converting syngas are generally referred to as syngas fermentation. Compared to chemical catalytic processes, syngas fermentation processes may be conducted at lower temperature and pressure, have higher reaction specificity, and do not require a specific ratio of CO to H2.
There remains, however, an ongoing need to discover and develop additional microorganisms that are capable of producing chemicals or biofuels by syngas fermentation. In particular, it would be advantageous to use bacterial species that may grow on different types of substrates and also provide good yields of products of interest.
One embodiment of the present disclosure is to provide an isolated biologically pure culture of Clostridium tyrobutyricum ITRI04001 suitable for converting, for example, syngas or a gas mixture comprising primarily CO and/or CO2 to fuels such as butanol and/or chemicals such as volatile fatty acids (e.g., butyric acid). For instance, in some embodiments, the bacteria disclosed herein may convert CO and/or CO2 to volatile fatty acids such as formic acid, acetic acid, lactic acid, propanoic acid, butyric acid, and mixtures thereof.
Another embodiment of the present disclosure is to provide a process for producing volatile fatty acids such as formic acid, acetic acid, lactic acid, propanoic acid, butyric acid, and mixtures thereof, comprising culturing a microorganism having the genotypic characteristics of ITRI04001 in a medium, providing at least one substrate comprising at least one carbon source chosen from CO and CO2 to the microorganism, and recovering volatile free acid from the culture.
Additional objects 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 present disclosure. The objects and advantages of the present disclosure 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 invention as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain embodiments of the invention.
Below, exemplary embodiments will be described in detail with reference to accompanying drawings so as to be easily understood by a person having ordinary skill in the art. The inventive concept may be embodied in various forms without being limited to the exemplary embodiments.
Reference will now be made in detail to exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings.
The term “acid” as used here includes both carboxylic acids and the associated carboxylate anion. The ratio of molecular acid to carboxylate in the fermentation broth may dependent upon the pH of the system.
The term “volatile free acids” refers to fatty acids with a carbon chain of six carbons or fewer. For instance, volatile fatty acids include formic acid (formate), acetic acid (acetate), lactic acid (lactate), propanoic acid (propionate), and butyric acid (butyrate).
Anaerobic Bacterial Species
The present disclosure provides an isolated anaerobic bacterial species capable of producing volatile free acids from relatively common substrates. In some embodiments, the disclosed bacteria can produce volatile fatty acids such as formic acid, acetic acid, lactic acid, propanoic acid, butyric acid, and mixtures thereof. In some embodiments, the bacteria disclosed herein may produce salts chosen from formate salts, acetate salts, lactate salts, propionate salts, butyrate salts, and mixtures thereof.
In some embodiments, the disclosed isolated anaerobic bacterial species relate to an acidogenic Clostridium, C. tyrobutyricum comprising 16S rRNA sequences as set forth in SEQ ID NO:1, or any nucleotide sequence with at least 80%, such as 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:1.
In some embodiments, the disclosed isolated anaerobic bacterial species is an isolated biologically pure culture of Clostridium tyrobutyricum ITRI04001, deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH with accession number DSM 27751.
In some embodiments, the isolated anaerobic bacteria species is an isolated biologically pure culture of Clostridium tyrobutyricum having the genotypic characteristics of ITRI04001.
In some embodiments, the isolated anaerobic bacterial species comprises at least two groups of genetic sequences encoding key enzymes for producing volatile fatty acids from syngas: one coding for acetyl-CoA synthesis from syngas, and another coding for production of volatile fatty acids such as formic acid, acetic acid, lactic acid, propanoic acid, butyric acid, and mixtures thereof. The enzymes of the acetyl-CoA pathway include formate dehydrogenase, formyl-THF synthetase, methenyl-THF cyclohydrogenase, methylene-THF dehydrogenase, methylene-THF reductase, methyltransferase, and CO dehydrogenase/acetyl-CoA synthase.
The genetic sequence encoding for the production of formic acid, acetic acid, lactic acid, propanoic acid, and butyric acid are as follows:
For formic acid or formate synthesis: pfl (SEQ ID NO:11), pflA (SEQ ID NO:12), pflD (SEQ ID NO:13)
For acetic acid or acetate synthesis: pta (SEQ ID NO:14), ack (SEQ ID NO:15), ctf (SEQ ID NO:16)
For lactic acid or lactate synthesis: ldh (SEQ ID NOs 17-19), dldh (SEQ ID NO:20)
For propanoic acid or propionate synthesis: pct (SEQ ID NOs 21, 22)
For butyric acid or butyrate synthesis: thl (SEQ ID NO:23), hbd (SEQ ID NOs 24, 25), crt (SEQ ID NOs 26, 27), bcd (SEQ ID NO:28), ptb (SEQ ID NO:29).
In some embodiments, the isolated biologically pure culture of Clostridium tyrobutyricum comprising sequences as set forth in SEQ ID NOs 1 through 30, or at least 80%, such as 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NOs 1 through 30.
The isolated anaerobic bacterial species disclosed here may be cultivated with any culture media, substrates, conditions, and processes generally known in the art for culturing anaerobic bacteria.
For example, the disclosed isolated anaerobic bacterial species can be cultured under anaerobic conditions. “Anaerobic conditions” means the level of oxygen (O2) is below 0.5 parts per million in the gas phase.
Further as an example, ITRI04001 can be cultivated in Clostridium Growth Medium (CGM) comprising at least one carbon source chosen from glucose, xylose, fructose, lactate, and acetate.
Processes for Producing Volatile Fatty Acids
The isolated anaerobic bacterial species disclosed here, such as ITRI04001, have the ability under anaerobic conditions to produce volatile fatty acids from substrates comprising, for example, CO+H2, or H2+CO2, or CO+H2+CO2. The CO or CO2 may provide the carbon source and the H2 or CO may provide the electron source for the reactions producing volatile fatty acids.
Thus, the present disclosure further provides a process for producing volatile free acid comprising:
culturing a microorganism having the genotypic characteristics of ITRI04001 in a medium;
providing at least one substrate comprising at least one carbon source chosen from CO and CO2 to the microorganism; and
recovering at least one free volatile free acid.
In some embodiments, the medium does not comprise glucose, xylose, fructose, lactate, or acetate.
In some embodiments, the medium comprises at least one carbohydrate chosen from glucose, xylose, fructose, lactate, and acetate.
In some embodiments, the at least one substrate further comprises at least one gas chosen from N2 and H2.
In some embodiments, the at least one substrate comprises CO and H2.
In some embodiments, the at least one substrate comprises CO2 and H2.
In some embodiments, the at least one substrate comprises CO, CO2, and H2.
In some embodiments, the at least one substrate is a “waste” gas such as syngas, oil refinery waste gases, steel manufacturing waste gases, autothermal reforming of natural gas, and coal gasification.
In some embodiments, the at least one substrate is syngas.
Syngas may be provided from any known source. In one embodiment, syngas may be sourced from gasification of carbonaceous materials. Gasification involves partial combustion of biomass in a restricted supply of oxygen, and the resultant gas may comprise mainly CO.
In some embodiments, syngas suitable for used in the processes disclosed here comprises CO, CO2, H2, and N2.
In some embodiments, syngas may comprise CO in an amount ranging from 10% to 100%, relative to the total moles of the syngas. For instance, the syngas may comprise from 20% to 90%, from 30% to 80%, or from 40% to 70%, relative to the total moles of the syngas.
Generally, the syngas can be added to the fermentation reaction in a gaseous state. However, the present disclosure is not limited to addition of the substrate in this state. For example, a liquid may be saturated with a syngas and that liquid added to the bioreactor.
In some embodiments, syngas may be supplemented with gas from other sources to alter the concentration of gaseous components (e.g., CO, CO2, and/or H2) provided to the microorganism. For example, syngas may be supplemented with gas with a higher level of CO (e.g., steel mill waste gas is enriched in CO) to enrich the level of CO.
For the growth of the microorganism and CO (or CO2)-to-volatile free acid fermentation to occur, in addition to the syngas, the medium may be supplemented with additional nutrients or ingredients suitable for growing the microorganism. Thus, in addition to the at least one carbon source, 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 producing volatile free acids may be carried out under conditions for the desired fermentation to occur (e.g. CO (or CO2)-to-volatile free acids). Reaction conditions that should 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 CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
The processes disclosed herein 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 (CSTR), an immobilized cell reactor, a gas-lift reactor, a bubble column reactor (BCR), a membrane reactor such as a Hollow Fiber Membrane Bioreactor (HFMBR) or a trickle bed reactor (TBR), monolith bioreactor, or loop reactors. Also, in some embodiments of the invention, the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which fermentation broth from the growth reactor is fed and in which most of the fermentation product (e.g. volatile fatty acids) is produced.
In some embodiments, fermentation will be allowed to proceed until a desired level of volatile fatty acids is produced in the culture media. Alternatively, production may be halted when a certain rate of production is achieved, e.g. when the rate of production of a desired product has declined due to, for example, build-up of bacterial waste products, reduction in substrate availability, feedback inhibition by products, reduction in the number of viable bacteria, or for any reasons known to those of skill in the art. In addition, continuous culture techniques are known which allow the continual replenishment of fresh culture medium with concurrent removal of used medium, including any liquid products.
The fermentation will result in fermentation broth comprising volatile free acids, as well as bacterial cells such as ITRI04001.
Free volatile free acid can be removed from the typically aqueous fermentation broth by any known methods such as precipitation, extraction (e.g., organic solvent liquid-liquid extraction), adsorption, dialysis (e.g., electrodialysis), ion exchange, and pressure-driven membrane separation processes. For example, the methods may the ones described in but not limited to Schügerl K., 2000 Biotechnol Adv. 18:581-599; Yang, S.-T. and Lu, C. (2013) Extraction-Fermentation Hybrid (Extractive Fermentation), in Separation and Purification Technologies in Biorefineries, UK. doi: 10.1002/9781118493441.ch15.
For example, precipitation has been widely used for recovering fumaric and lactic acid, which have a low solubility when present in their calcium salt form. As an example, for recovering lactic acid, a conventional fermentation process produces calcium lactate precipitate, which can then be collected and re-acidified.
As another example, the butyric acid present in the 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 claims.
The composition of the Reinforced Clostridial Medium (RCM) and Clostridium Growth Medium (CGM) used in the following tables.
Clostridium Growth Medium (CGM)
Raw milk samples from dairy cattle were collected in Taiwan and subsequently heated at 70° C. for 10 min. After cooling to room temperature, the samples were mixed with Reinforced Clostridial Medium (RCM) supplemented with 20% sodium lactate, and then incubated at 37° C. under anaerobic conditions.
To isolate Clostridia bacterial species, PCR-based assays specific for 16S rRNA sequence with a pair of primers (forward: GCGGCGTGCYTAAYACATGC (SEQ ID NO: 31), and reverse: GGGTTGCGCTCGTTGCRGGA (SEQ ID NO: 32)) was used to evaluate the samples. When the expected size of PCR products was detected in the samples, the samples were then diluted serially and spread on Clostridium Growth Medium (CGM) agar plates containing 5 g/L glucose and 5 g/L sodium lactate. The resulting plates were incubated at 37° C. anaerobically until colonies appeared. Single colonies were picked up from the CGM agar plates and analyzed by PCR-based assays specific for 16S rRNA sequence with a pair of primers: forward: GCGGCGTGCYTAAYACATGC (SEQ ID NO: 31), and reverse: GGGTTGCGCTCGTTGCRGGA (SEQ ID NO: 32). The PCR products were sequenced and analyzed by Basic Local Alignment Search Tool on NCBI website (NCBI BLASTN, database selected: Nucleotide collection, optimized for megablast).
Characteristic of ITRI04001
The ITRI04001 colonies grown on CGM agar plates appeared to have irregular margins, brownish yellow color, and slightly raised centers.
When observed under the microscope, ITRI04001 appeared motile and exhibited endospores morphology.
Phylogenetic analysis of the 16S rRNA gene sequence (SEQ 1) of ITRI04001 indicated that ITRI04001 belongs to Clostridium tyrobutyricum. ITRI04001 and Clostridium tyrobutyricum ATCC® 25755 (“ATCC 25755”) shared about 99% of sequence similarity with regard to their 16S rRNA genes.
ITRI04001 was cultivated in CGM medium containing 5 g/L of glucose, xylose, fructose, lactate, or acetate at 37° C., under anaerobic conditions. Growth was monitored by measuring optional density (OD) at a wavelength of 600 nm OD600). After 72 hours, growth of ITRI04001 was observed in all types of media. Accordingly, ITRI04001 can be cultivated using glucose, xylose, fructose, lactate, or acetate as a carbon source.
ITRI04001 was plated on RCM agar containing various antibiotics with different concentrations and then cultivated for 72 hours at 37° C., under anaerobic conditions. Colonies of ITRI04001 were observed on the surface of the agar plates containing apramycin (25 ug/ml), spectinomycin (250 ug/ml), streptomycin (500 ug/ml), or Kanamycin (25 ug/ml).
ITRI04001 was cultivated in CGM medium containing 5 g/L of glucose, and liquid samples of the culture were taken every 24 hours and were analyzed with HPLC.
Growth Rate
After ITRI04001 and ATCC® 25755 were cultivated in RCM medium until mid-log phase. Samples of the cultures were taken out and seeded into CGM medium containing (1) 5 g/L of glucose or (2) 5 g/L of glucose and 5 g/L of lactate at different dilution ratios. Growth of either ITRI04001 or ATCC® 25755 was monitored by measuring OD at 600 nm.
Butyrate Production
ITRI04001 and ATCC® 25755 were inoculated into CGM medium containing 8 g/L of lactate and 5 g/L of glucose at the same dilution ratio on stationary phase and cultivated at 37° C., under anaerobic conditions. After 24 hours of culture, samples of the culture media were taken for measuring the concentrations of butyrate, glucose, and lactate by HPLC.
Flagellin Gene
Two fragments of ITRI04001's flagellin gene (SEQ ID NO:34 and SEQ ID NO:36) were sequenced and compared to similar sequence segments in ATCC® 25755 (SEQ ID NO:33 and SEQ ID NO:35). ITRI04001 and ATCC® 25755 shared about 92% of sequence similarity with regard to the fragment 251/272 and 91% of sequence similarity with regard to the fragment 148/162. See
Syngas fermentation using ITRI04001 was performed in a 200 mL bottle containing 50 mL CGM medium, pH 6.0, and without any carbohydrate. The sole carbon source provided to the ITRI04001 during the fermentation was from syngas comprising 10% H2, 20% CO, 20% CO2, and 50% N2, which was pressurized to 20 psi gauge in the headspace of the bottle. All experiments were performed on a rotary shaker with 100 r.p.m. in 37° C. Culture medium was sampled at 0, 24, 48 and 62 hours culturing time and analyzed with HPLC (Agilent 1100 series with Aminex HPX-87H (300 mm×7.8 mm) column).
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, with a true scope of the disclosure being indicated by the following claims and their equivalents.
This application claims priority to U.S. Provisional Application No. 61/709,294, filed on Oct. 3, 2012.
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| Number | Date | Country | |
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| 20140120591 A1 | May 2014 | US |
| Number | Date | Country | |
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| 61709294 | Oct 2012 | US |
