CN103911401A - Method for reinforcing biological carbon sequestration - Google Patents
Method for reinforcing biological carbon sequestration Download PDFInfo
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- CN103911401A CN103911401A CN201310751205.9A CN201310751205A CN103911401A CN 103911401 A CN103911401 A CN 103911401A CN 201310751205 A CN201310751205 A CN 201310751205A CN 103911401 A CN103911401 A CN 103911401A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
At least one product selected from acid and alcohol is generated by a microbial fermentation method, including providing a gas zymolyte and a fluid medium and making the microbial generate at least one product selected from acid and alcohol, wherein the fluid medium includes lactic acid and/or salt thereof, and the gas zymolyte includes at least one carbon source selected from Co and Co2.
Description
The application requires to enjoy the U.S. Provisional Application No.61/749 submitting on January 4th, 2013,010 right of priority.
Technical field
The present invention relates to a kind of method of strengthening biological solid carbon.
Background technology
Nearest climatic anomaly, comprise that hurricane has been that global temperatures rising is relevant with Global warming with arid.A major cause of Global warming is the increase due to atmospheric carbon dioxide levels.In fact, it is reported in the past in 200 years CO in atmosphere
2concentration rises to 368ppm from about 280ppm.Therefore, exploitation can effectively reduce CO
2discharge or catch CO from atmosphere
2method be that people are always interested.
Utilize organism by CO
2the method that is converted into organic compound is commonly called carbonic acid gas biological fixation.The most outstanding example of carbonic acid gas biological fixation comprises use photosynthetic microorganism, and as micro-algae and blue-green algae, it uses Driven by Solar Energy carbon fixation approach.
Except photosynthetic microorganism, chemoautotrophy microorganism (chemoautotrophic microorganisms) (class is oxidized by mineral compound instead of photosynthesis obtains the microorganism of its nutrition) has represented the potential surrogate of one for carbon fixation method.
The main non-photosynthetic carbon dioxide fixation approach that chemoautotrophy microorganism is used is Wood-Ljungdahl(WL) path (Fig. 1).It is used that WL path is generally anaerobion, comprises clostridium aceticum (Clostridium aceticum), from producing and ethanol clostridium (Clostridium autoethanogenum), food carbonoxide clostridium (Clostridium carboxidivorans), clostridium difficile (Clostridium difficile), Young clostridium (Clostridium ljungdahlii), hot acetic acid Moore Salmonella (former Clostridium thermoaceticum, Moorella thermoacetica, Clostridium thermoaceticum), have a liking for hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), autotrophy desulfurization bacterium (Desulfobacterium autotrophicum), the blue De Shi clostridium of Wamsteeker (Clostridium sticklandii), Clostridium thermoautotrophicum (Clostridium thermoautotrophicum), formic acid clostridium aceticum (Clostridium formicoaceticum), Clostridium magnum (Clostridium magnum), methyl alcohol bacillus aceticus (Acetobacterium carbinolicum), triumphant 5 bacillus aceticuss (Acetobacterium kivui) and Wu Shi bacillus aceticus (Acetobacterium woodii).
Although some produce acetic acid clostridiums by through engineering approaches for example, for example, in order to production bulk chemical (butyrates, acetate, acetoin and acetone) and biofuel (butanols and ethanol), but still need to develop and can improve anaerobic organisms by CO
2metabolism is the method for useful chemical substance and/or biofuel ability.
Summary of the invention
Therefore, the invention provides for microorganism fermentation and comprise CO
2the method of gaseous substrate, described method comprises:
Provide a gaseous substrate and a liquid nutrient medium to microorganism, wherein said liquid nutrient medium comprises lactic acid and/or its salt, and wherein said gaseous substrate comprises CO
2, and
Make microorganisms be selected from least one product of acid and alcohol.
The present invention also provides a kind of method that improves carbon capture rate in microorganism fermentation, comprising:
For microorganism provides a gaseous substrate and a liquid nutrient medium, wherein said liquid nutrient medium comprises lactic acid and/or their salt, and wherein said gaseous substrate comprises and is selected from CO and CO
2at least one carbon source, and
Make microorganisms be selected from least one product of acid and alcohol.
The present invention other feature and advantage will this specification sheets below part set forth, and some part is apparent in specification sheets, or can know by practice of the present invention.Can recognize or obtain described feature and advantage by the key element and the combination that particularly point out in the appended claims.
Should be understood that, above general remark and following detailed description are only all exemplary and explanat, are not the restrictions to the present invention for required protection.
Accompanying drawing has shown illustrative embodiments disclosed by the invention, is included and form in the part of this specification sheets.
Brief description of the drawings
Fig. 1 has shown Wood-Ljungdahl(WL) path.
Fig. 2 has shown and has been exposed to CO
2and H
2mixed gas in the growth curve of Young clostridium (Clostridium ljungdahlii) BCRC17797, (a) experimental group (containing the Sodium.alpha.-hydroxypropionate of 2.5g/L in substratum), (b) sodium ion control group (containing the NaCl of 1.52g/L in substratum), (c) additive-free control group, as described in experimental example part.
Fig. 3 has shown and in substratum, exists 2.5g/L Sodium.alpha.-hydroxypropionate can induce some gene relating in lactic acid permease and Wood-Ljungdahl path.As described in embodiment part, it is expressed increase at double compared with additive-free control group.
Fig. 4 has shown and in substratum, exists 2.5g/L Sodium.alpha.-hydroxypropionate can induce some gene relating in lactic acid permease and Wood-Ljungdahl path.As described in embodiment part, it is expressed increase at double compared with sodium ion control group.
Fig. 5 shown with respect to additive-free control group, and the expression of some gene in two experimental group (Sodium.alpha.-hydroxypropionate of 1g/L and 2.5g/L), as described in embodiment part.
Fig. 6 has shown that cultivation is after 96 hours, and the output of acetic acid in experimental group, additive-free control group (only having synthetic gas) and sodium ion control group, as described in embodiment part.
Fig. 7 has shown that cultivation is after 96 hours, and the output of ethanol in experimental group, additive-free control group (only having synthetic gas) and sodium ion control group, as described in embodiment part.
Fig. 8 has shown that cultivation is after 90 hours, and with respect to additive-free control group, the expression of some gene in the experimental group that comprises 1g/L Sodium.alpha.-hydroxypropionate, as described in embodiment part.
Embodiment
Detailed description exemplary embodiment below with reference to the accompanying drawings, to those having ordinary skill in the art will appreciate that.Theory of the present invention can embody with various forms, and is not limited to illustrative embodiments set forth in the present invention.
The invention provides for microorganism fermentation and comprise CO
2the method of gaseous substrate, described method comprises:
Provide a gaseous substrate and a liquid nutrient medium to microorganism, wherein said liquid nutrient medium comprises lactic acid and/or their salt, and wherein said gaseous substrate comprises CO
2, and
Make microorganisms be selected from least one product of acid and alcohol.
The present invention also provides a kind of method that improves carbon capture rate in microorganism fermentation, comprising:
Provide a gaseous substrate and a liquid nutrient medium to microorganism, wherein said liquid nutrient medium comprises lactic acid and/or their salt, and wherein said gaseous substrate comprises and is selected from CO and CO
2at least one carbon source, and
Make microorganisms be selected from least one product of acid and alcohol.
In some embodiments, described microorganism is selected from and can passes through Wood-Ljungdahl(WL) path is CO
2and/or CO is converted into any anaerobic organisms of at least one product that is selected from acid and alcohol.
In some embodiments, described microorganism is selected from chemoautotrophy microorganism.
In some embodiments, described microorganism is selected from photosynthetic microorganism, its through genetic engineering modified to can pass through Wood-Ljungdahl(WL) path is CO
2and/or CO is converted at least one product that is selected from acid and alcohol.
In some embodiments, described microorganism is selected from clostridium aceticum (Clostridium aceticum), from producing and ethanol clostridium (Clostridium autoethanogenum), food carbonoxide clostridium (Clostridium carboxidivorans), clostridium difficile (Clostridium difficile), Young clostridium (Clostridium ljungdahlii), hot acetic acid Moore Salmonella (former Clostridium thermoaceticum, Moorella thermoacetica, Clostridium thermoaceticum), have a liking for hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), autotrophy desulfurization bacterium (Desulfobacterium autotrophicum), the blue De Shi clostridium of Wamsteeker (Clostridium sticklandii), Clostridium thermoautotrophicum (Clostridium thermoautotrophicum), formic acid clostridium aceticum (Clostridium formicoaceticum), Clostridium magnum (Clostridium magnum), methyl alcohol bacillus aceticus (Acetobacterium carbinolicum), triumphant 5 bacillus aceticuss (Acetobacterium kivui) and Wu Shi bacillus aceticus (Acetobacterium woodii).
Term " comprises CO
2gaseous substrate " and similar terms be understood to include wherein carbonic acid gas (CO
2) can be used for any gaseous substrate of microorganism growth and/or fermentation.Similarly, term " comprises and is selected from CO and CO
2the gaseous substrate of at least one carbon source " be understood to include wherein CO and/or CO
2can be used for any gaseous substrate of microorganism growth and/or fermentation.
In some embodiments, gaseous substrate can be the CO that comprises obtaining as the by product of commercial run or from other sources such as such as waste gas sent off from automobiles
2and/or the waste gas of CO.Exemplary commercial run includes but not limited to: petroleum refining process, coal gasification, electrical production, production of carbon black, ammonia production, methanol production and coke production.According to comprising CO
2and/or the gaseous substrate composition of CO, before being introduced into fermentation, may from gaseous substrate, remove unwanted impurity, as dust granules.For example, can use known method to filter or purge gas substrate.
In some embodiments, described gaseous substrate can further comprise and is selected from hydrogen (H
2) and nitrogen (N
2) at least one gas.
In some embodiments, described gaseous substrate is to comprise CO
2, CO and H
2synthesis gas (" synthetic gas ").Synthetic gas can produce by the steam reformation of Sweet natural gas or by the gasification of various organic materials biological example matter, organic waste, coal, oil, plastics or other carbonaceous material.
In some embodiments, described gaseous substrate can comprise the CO of 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% volume
2.In some embodiments, described gaseous substrate can comprise 20% to 80% CO
2.As nonrestrictive example, described gaseous substrate can comprise 20% CO
2with 80% H
2.
Conventionally CO,
2and/or CO will offer microorganism with gaseous form.But, in some embodiments, can provide the CO of liquid form
2and/or CO.For example, by a liquid with comprising CO
2gaseous substrate carry out saturatedly, then this liquid is added in bio-reactor.
In some embodiments, can provide reductibility chemical substance and/or electrode as for CO to microorganism
2fixing electron source.
In some embodiments, can provide at least one electron source for CO to microorganism
2fixing, wherein said at least one electron source can be selected from hydrogen (H
2), reductibility carbon compound (non-limitative example: CO, formic acid and methyl alcohol), sulphur compound (non-limitative example: H
2s and S) and nitrogen compound (non-limitative example: NH
3).
In some embodiments, in order to improve, carbon is caught and/or CO
2fixing efficiency, provides the liquid nutrient medium that comprises lactic acid and/or its salt to microorganism.
In some embodiments, the liquid nutrient medium providing to microorganism can comprise 0.5g/L to 3g/L lactic acid and/or its salt.As nonrestrictive example, the liquid nutrient medium providing to microorganism can comprise, for example 0.5g/L, 1g/L, 1.25g/L, 1.5g/L, 1.75g/L, 2g/L, 2.25g/L, 2.5g/L, 2.75g/L or 3g/L lactic acid and/or its salt.
In some embodiments, the liquid nutrient medium providing to microorganism can comprise that the lactic acid of q.s and/or its salt are with induction and Wood-Ljungdahl(WL) expression of the gene that relates in path.
For example, in some embodiments, the amount that lactic acid and/or its salt exist can be enough to induce the expression of at least one gene, and described gene is selected from
et al., Clostridium ljungdahlii represents a microbial production platform based on syngas(2010) described in Wood-Ljungdahl path in CLJU_c37670, the 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 the CLJU_c09110 that relate to.
In some embodiments, with respect at the same terms but for the microorganism of cultivating there is not lactic acid and/or its salt in the situation that, the amount that lactic acid and/or its salt exist can be enough to induce at least one gene relating in Wood-Ljungdahl path to increase to express approximately 1.5 times or more than.
In some embodiments, with respect at the same terms but for the microorganism of cultivating there is not lactic acid and/or its salt in the situation that, the amount that lactic acid and/or its salt exist can be enough to induce at least one gene relating in Wood-Ljungdahl path to increase to express approximately 1.5 times or more than.As nonrestrictive example, with respect at the same terms but for the microorganism of cultivating there is not lactic acid and/or its salt in the situation that, the amount that lactic acid and/or its salt exist can be enough to induce at least one gene relating in Wood-Ljungdahl path to increase to express approximately 1.5 times to approximately 10 times.
In some embodiments, with respect at the same terms but for the microorganism of cultivating there is not lactic acid and/or its salt in the situation that, the lactic acid existing in liquid nutrient medium and/or the amount of its salt can be enough at least one gene that induction is selected from codH, codH β, codH δ, codH γ, coos1 and metF to be increased and expresses approximately 1.5 times to approximately 10 times.As nonrestrictive example, with respect at the same terms but for the microorganism of cultivating there is not lactic acid and/or its salt in the situation that, the lactic acid existing in liquid nutrient medium and/or the amount of its salt can be enough at least one gene that induction is selected from codH, codH β, codH δ, codH γ, coos1 and metF to be increased and expresses approximately 1.5 times to approximately 8 times.
For accelerate growth of microorganism and/or CO
2and/or CO fixes, except described lactic acid and/or its salt, can also be suitable for to liquid nutrient medium supplement extra nutrition or the composition of bacterial growth.For example, also can comprise and be enough to make the VITAMIN of microorganism growth, salt, extract and/or mineral substance for the substratum of culturing micro-organisms.
In some embodiments, for CO
2and/or the fixing means of CO is desired fermentation to occur (as from CO
2to acid) condition under carry out.Admissible reaction conditions comprises redox potential, stir speed (S.S.) (if using continuous stirred tank reactor (CSTR)), inoculation level, the maximum gas concentration of substrate of pressure, temperature, gas flow rate, flow rate of liquid, medium pH, substratum, to guarantee the CO in liquid phase
2or other component is unrestricted, and make production concentration maximize to avoid product to suppress.
In some embodiments, described method can be carried out in any applicable bio-reactor, substrate can contact with one or more microorganisms therein, for example, for example hollow-fiber membrane bioreactor of continuous stirred tank reactor (CSTR), immobilized cell reactor, airlift reactor, bubbling column reactor, membrane reactor or trickle bed reactor, the biological reactor of monoblock type or loop reactor.
In some embodiments, the producible product of zymotechnique of the present invention includes but not limited to alcohol and/or acid.Exemplary acid includes but not limited to formic acid, acetic acid, propionic acid, butyric acid, vinylformic acid, lipid acid; And exemplary alcohol includes but not limited to methyl alcohol, ethanol, acetone, propyl alcohol, butanols and 2,3-butanediol.
In tunning, the term " acid " that the present invention uses comprises carboxylic acid and relevant carboxylate anion.For example, term " acetic acid " comprises the mixture of independent free acetic acid, acetate and free acetic acid and acetate.
Term " fatty acid " " comprise the carboxylic acid with long aliphatic chain.In some embodiments, aliphatic chain can be linear or branch, and saturated with undersaturated.In some embodiments, aliphatic chain can comprise for example 4 to 12 carbon atoms, 5 to 11 carbon atoms, or 8 to 10 carbon atoms.
At least one product that is selected from acid and alcohol in fermentation process can reclaim with any currently known methods.
As nonrestrictive example, can be by reclaiming ethanol as the method for fractionation or evaporation and extraction fermentation from fermented liquid.
As nonrestrictive example, can use the adsorption system that comprises activated charcoal filter recovery of acetic acid salt from fermented liquid.In this case, can first use suitable separating unit from fermented liquid, to remove microorganism.Then by acellular, pillar containing the substratum of acetate by containing activated carbon with absorption acetate.Acetate with the form of sour form (acetic acid) instead of salt (acetate) more easily by charcoal absorption.Therefore, before active carbon column, can be by being reduced to and being less than approximately 3 containing the pH value of acetate and acellular substratum, so that most acetate is converted into the form of acetic acid.
Further, as nonrestrictive example, the butyric acid existing in fermented liquid can be by extracting to reclaim and purifying with fatty amine, as pungent in Alamine 336(tri-/decyl amine; Alamine336
cognis, Cincinnati, OH, USA), or other water-insoluble solvent.
Consider from specification sheets and the disclosed practice of the present invention, other embodiments of the present invention are apparent to those skilled in the art.Specification sheets and embodiment are only as exemplary example, and the real scope and spirit of the present invention are limited by following claim book.
Embodiment
[embodiment 1]
Microorganism
Clostridium ljungdahlii BCRC 17797(DSM No.13528; BCRC No.17797) preserve and research centre (Taiwan) purchased from Taiwan Biological resources.Known this bacterial strain be utilize in atmosphere as CO; CO
2and H
2gas mixture; Or synthesis gas (CO and H
2) etc. carbon source by Wood-Ljungdahl path (Fig. 1) growth with produce metabolite.See
et al, Clostridium ljungdahlii represents a microbial production platform based on syngas, Proc Natl Acad Sci USA, 107 (29), pp.13087-13092 (2010).
Material
The PETC basic medium of improvement, comprises (every liter): protease protein peptone (10g), yeast extract (3g) and resazurin stoste (0.5mL, 0.1%, w/v).See Cottera JL 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).
ATCC1754 salts solution, comprises (every liter): NH
4cl(20g), KCl(2g), MgSO
47H
2o(4g), NaCl(16g), KH
2pO
4(2g) and CaCl
2(0.4g).
Vitamin solution, comprise (every liter): vitamin H (2mg), folic acid (2mg), pyridoxol (10mg), vitamin (5mg), riboflavin (5mg), nicotinic acid (5mg), calcium D(+) pantothenic acid (5mg), vitamin B12 (5mg), p-aminosallcylic acid (5mg) and Thioctic Acid (thioctic) are (5mg).
Trace element solution comprises (every liter): nitrilotriacetic acid(NTA) (2g), MnCl
24H
2o(1.3g), FeSO
47H
2o(0.4g), CoCl
26H
2o(0.2g), ZnSO
47H
2o(0.2g), CuCl
22H
2o(0.02g), NiCl
26H
2o(0.02g), Na
2moO
42H
2o(0.02g), Na
2seO
3(0.02g) and Na
2wO
42H
2o(0.025g).
The PETC substratum (mPETC) of improvement, pH6.8: mend with 50ml ATCC1754 salts solution in the PETC basic medium of 1 liter of improvement, the vitamin solution of 10ml and the trace element solution of 10ml are with preparation mPETC.Then prepared substratum is divided and installed in serum bottle, then autoclaving 15～20 minutes under 121 DEG C, 15psi.
The air-tight bottle of cultivating for anaerobism: by 200ml jaw serum bottle rubber stopper seal, rubber plug is further in position hermetic closed to guarantee with aluminium jaw.
Method
Clostridium ljungdahlii first with the mPETC that contains 10g/L fructose cultivate based at 37 DEG C in an above-mentioned air-tight bottle anaerobism cultivate.Work as OD
600when value reaches 1 to 1.5 scope, will cultivate based under 3500rpm centrifugal 210 seconds.Gained cell precipitation thing is resuspended in to the mPETC without fructose, and then further at 37 DEG C, anaerobism is cultivated 1 hour to remove the sugared substrate of any remnants.
45ml mPETC substratum is added in above-mentioned jaw serum bottle, then cover with aluminium foil and autoclaving 15 minutes under 121 DEG C, 15psi.After cooling, the described bottle containing substratum transferred in anaerobic room, and is added halfcystine-HCl(50%w/v of 50 μ l to substratum) to remove any dissolved oxygen.
For experimental group, further to adding sodium lactate solution in substratum to obtain the about 0.022M of 2.5g/L() lactic acid concn.
For sodium ion control group, to adding NaCl solution in substratum to obtain the about 0.026M of 1.52g/L() NaCl concentration.This control group is whether the existence that sodium ion in Sodium.alpha.-hydroxypropionate is tested in design plays a role in carbon dioxide fixation.Therefore, in this experimental group, add the sodium ion of same molar.
For additive-free control group, in substratum, add appropriate water.
Then removed the Clostridium ljungdahlii culture of residual sugar to the serum bottle inoculation 5ml of the each 45ml of containing mPETC substratum.Sampling on this aspect is labeled as T=0 sample.Then bottle is hermetic closed to guarantee by rubber stopper seal further aluminium coating matter jaw lid.
First contain 80%N with injection needles handle
2and 20%CO
2gaseous mixture purge the space of air-tight bottle inner top with 10psi pressure.Be inserted into rubber plug with another injection needles discharges the gas in bottle simultaneously.Purge mixed gas after approximately 30 seconds, in bottle, obtain the anaerobic condition of filling carbonic acid gas.
Then, further purge another kind of containing 80%H via injection needles with 20psi pressure to the space of bottle inner top
2and 20%CO
2gaseous mixture to replace previous gaseous mixture.After approximately 30 seconds, remove syringe needle, the pressure of bottle interior accumulates to about 20psi.Then bottle is moved on to the brooder of 37 DEG C and be placed on that hunting speed is provided is on the shaking table of 100rpm.
Every 12 hours, discharge the gas of bottle inner accumulated with syringe needle, and purge and upgrade to the headspace in bottle by above-mentioned same procedure, to supplement gaseous carbon source under stable pressure condition.
Use with the syringe of syringe needle and collect liquid sample.Measure OD
600afterwards, by liquid sample under 12,000rpm centrifugal 10 minutes.The cell precipitation thing obtaining is stored in 700 μ l TRIzol solution and is extracted and gene expression analysis for follow-up RNA.By Agilent 1100 series of high efficiency liquid chromatographs (HPLC) for supernatant liquor and Aminex HPX-87H(300mm x7.8mm) tubing string analyzes.
Result
Fig. 2 has shown bacterial growth index OD in each group of substratum
600variation, (a) experimental group (comprising 2.5g/L Sodium.alpha.-hydroxypropionate), (b) sodium ion control group (NaCl that comprises 1.52g/L), (c) is additive-free control group.In all groups, all Fast Growths of Clostridium ljungdahlii BCRC 17797 in first 70 hours, then their growth rate slows down.
At t=144 hour, OD
600value is: the experimental group that (a) contains Sodium.alpha.-hydroxypropionate reaches 0.71, and (b) sodium ion control group reaches 0.82, and (c) additive-free control group reaches 0.79.Therefore the rate of increase that this that, observe is three groups there is no significant difference.
Analyze the media samples of t=144 hour with HPLC, as shown in table 1, when CO is provided
2during as carbon source, the Clostridium ljungdahlii BCRC17797 growing in mPETC can produce acetic acid.
As shown in table 1, in t=144 hour, the acetate concentration in experimental group is higher than additive-free control group or sodium ion control group.And additive-free control group is almost identical in the acetate concentration of the acetate concentration of t=144 hour and sodium ion control group.
Table 1
The data of table 1 have further shown that the lactic acid concn of experimental group is from 2.92g/L(t=0) drop to 2.25g/L(t=144).This shows that bacterium has consumed a small amount of lactic acid (being 0.67g/L).The lactic acid of 0.67g/L consuming can provide the carbon atom of about 0.022M, and the 6.39g/L acetic acid that produces of the experimental group carbon atom that comprises about 0.213M.This shows that at least 0.191M(0.213 deducts 0.022M) carbon atom from the CO existing in gaseous mixture
2.
As shown in Table 1, cultivate after 144 hours, additive-free control group produces the acetic acid of about 4.97g/L, and it is containing the carbon atom of the 0.166M that has an appointment, and sodium ion control group produces the acetic acid of about 4.91g/L, and it is containing the carbon atom of the 0.164M that has an appointment.Therefore, transformed by experimental group the CO that (fixing) is acetic acid
2amount (0.191M(is that the carbon atom existing in the acetic acid of experimental group generation deducts the carbon atom amount that may be transformed from lactic acid)) at least exceed approximately 15% than additive-free control group (having 0.166M carbon atom in the acetic acid producing additive-free control group) or sodium ion control group (having 0.164M carbon atom in the acetic acid producing in sodium ion control group).This has shown that the lactic acid existing in experimental group substratum has strengthened the carbon dioxide fixation of Clostridium ljungdahlii BCRC 17797.
Use the expression of the specific gene of listing in real-time quantitative polymerase chain reaction (RT-qPCR) quantization table 2.
Table 2
| Gene | Protein expression | Code |
| Lactic acid permease | Lactic acid permease (lactic acid is transported to the enzyme into cell) | CLJU_c21610 |
| codH | CodH/CO desaturase | CLJU_c37670 |
| codHβ | CO desaturase/acetyl-CoA enzyme beta subunit | CLJU_c37550 |
| codHδ | CO desaturase/acetyl-CoA synthetic enzyme δ subunit | CLJU_c37580 |
| codHγ | CO desaturase/acetyl-CoA synthetic enzyme γ subunit | CLJU_c37570 |
| cooS1 | Anaerobic type carbon monoxide dehydrogenase | CLJU_c09110 |
| metF | Methylene tetrahydrofolate reductase | CLJU_c37610 |
Note: CO desaturase is that bacterium passes through WL path conversion CO
2one of enzyme that gas uses; It is also a kind of enzyme for the synthesis of acetyl-CoA.
As shown in Figure 3, with respect to additive-free control group, the lactic acid induction existing in the substratum of experimental group: (i) lactic acid permease increases approximately 11 times, shows that this bacterium responds to lactic acid; (ii) codH, codH β, codH δ and codH γ increase approximately 4 to 8 times; (iii) cooS1 increases approximately 3 times; (iv) metF increases approximately 4 times.
As shown in Figure 4, with respect to sodium ion control group, the lactic acid induction lactic acid permease, codH, codH β, codH δ, codH γ and the metF that in the substratum of experimental group, exist increase approximately 3 to 6 times.This shows that lactic acid can induce the expression of the gene relating in carbon dioxide fixation really.
Embodiment 2
Experimental program described in repetition experimental example 1, difference is, except having additive-free control group, is provided with two experimental group, and one is the lactic acid that has 1g/L in substratum, and another is the lactic acid that has 2.5g/L in substratum.
Following table 3 has shown cultivated after 96 hours, the amount of the lactic acid consuming in experimental group and additive-free control group and the acetic acid of generation.
Table 3
| In substratum, add lactic acid (g/L) | 0 | 1 | 2.5 |
| Lactic acid consumes (g/L) | 0.00±0.00 | 0.23±0.03 | 0.23±0.01 |
| Acetic acid produces (g/L) | 7.23±1.42 | 8.13±1.59 | 9.15±0.06 |
Fig. 5 has shown with respect to additive-free control group, the expression of some gene in two experimental group.
Embodiment 3
Experimental program described in repetition experimental example 1, difference is, except having additive-free control group, is provided with three experimental group (Sodium.alpha.-hydroxypropionate of 0.5g/L, 1g/L and 3g/L) and a sodium ion control group (containing the NaCl of 1.57g/L).
Fig. 6 and Fig. 7 have shown that cultivation is after 96 hours, the acetic acid producing respectively in experimental group, nothing interpolation control group (only having synthetic gas) and sodium ion control group and the amount of ethanol.
Fig. 8 has shown that cultivation is after 90 hours, with respect to additive-free control group, containing the expression of some gene in the experimental group of 1g/L Sodium.alpha.-hydroxypropionate.Following table 4 shows the implication of the enzyme representative of the each CLJU_c numeral shown in Fig. 8.
Table 4
| CLJU_c37670 | CodH, carbon monoxide dehydrogenase |
| CLJU_c37570 | CodH γ, CO desaturase/Acetyl-CoA synthetase γ subunit |
| CLJU?c37550 | CodH β, CO desaturase/Acetyl-CoA synthetase complex body β subunit |
| CLJU_c37580 | CodH δ, CO desaturase/Acetyl-CoA synthetase δ subunit |
| CLJU_c06990 | Fdh α, hydrogenlyase α subunit |
| CLJU_c20040 | Fdh sub α, hydrogenlyase α subunit |
| CLJU_c08930 | FdhH, hydrogenlyase H |
| CLJU_c07020 | FdhD, hydrogenlyase subunit |
| CLJU_c37650 | Fhs, formate-tetrahydrofolate ligase |
| CLJU_c37640 | FchA, cyclisation formyl |
| CLJU_c37630 | FolD, difunctional methylenetetrahydrofolate dehydrogenase/cyclisation anhydroleucovorin |
| CLJU_c37610 | MetF, Methylene tetrahydrofolate reductase |
| CLJU_c37560 | MeTr, methyl tetrahydrofolate: corrinoid/iron-sulphur protein methyltransgerase |
| CLJU_c09110 | CooS1, anaerobic type carbon monoxide dehydrogenase |
Obviously those skilled in the art can carry out various modifications and variations to embodiment disclosed in this invention.Be noted that specification sheets and embodiment are only exemplary.
Claims (18)
1. one kind comprises CO for microorganism fermentation
2the method of gaseous substrate, described method comprises:
Provide a gaseous substrate and a liquid nutrient medium to microorganism, wherein said liquid nutrient medium comprises lactic acid and/or its salt, and wherein said gaseous substrate comprises CO
2, and
Make described microorganisms be selected from least one product of acid and alcohol.
2. the method for claim 1, wherein said gaseous substrate further comprises and is selected from H
2, N
2at least one gas with CO.
3. the method for claim 1, wherein said gaseous substrate comprises the CO of at least 20% volume
2.
4. the method for claim 1, wherein said lactic acid and/or its salt are to exist with the concentration of 0.5g/L to 3g/L.
5. the method for claim 1, wherein with respect to there not being the microorganism of cultivating under the condition of lactic acid and/or its salt, the amount that described lactic acid and/or its salt exist be enough at least one gene that induction is selected from codH, codH β, codH δ, codH γ, coos1 and metF increase express approximately 1.5 times or more than.
6. the method for claim 1, wherein said at least one product is selected from acetic acid, propionic acid, butyric acid, vinylformic acid and lipid acid.
7. the method for claim 1, wherein said at least one product is selected from ethanol, acetone, propyl alcohol, butanols and 2,3-butanediol.
8. the method for claim 1, wherein said microorganism can be by CO
2and/or CO is converted at least one product that is selected from acid and alcohol.
9. the method for claim 1, described microorganism is selected from clostridium aceticum (Clostridium aceticum), from producing and ethanol clostridium (Clostridium autoethanogenum), food carbonoxide clostridium (Clostridium carboxidivorans), clostridium difficile (Clostridium difficile), Young clostridium (Clostridium ljungdahlii), hot acetic acid Moore Salmonella (former Clostridium thermoaceticum, Moorella thermoacetica, Clostridium thermoaceticum), have a liking for hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), autotrophy desulfurization bacterium (Desulfobacterium autotrophicum), the blue De Shi clostridium of Wamsteeker (Clostridium sticklandii), Clostridium thermoautotrophicum (Clostridium thermoautotrophicum), formic acid clostridium aceticum (Clostridium formicoaceticum), Clostridium magnum (Clostridium magnum), methyl alcohol bacillus aceticus (Acetobacterium carbinolicum), triumphant 5 bacillus aceticuss (Acetobacterium kivui) and Wu Shi bacillus aceticus (Acetobacterium woodii).
10. a method that improves carbon capture rate in microorganism fermentation, comprising:
Provide a gaseous substrate and a liquid nutrient medium to microorganism, wherein said liquid nutrient medium comprises lactic acid and/or its salt, and wherein said gaseous substrate comprises and is selected from CO and CO
2at least one carbon source, and
Make described microorganisms be selected from least one product of acid and alcohol.
11. methods as claimed in claim 10, wherein said gaseous substrate further comprises and is selected from H
2and N
2at least one gas.
12. methods as claimed in claim 10, wherein said gaseous substrate comprises the CO of at least 20% volume
2.
13. methods as claimed in claim 10, wherein said lactic acid and/or its salt are to exist with the concentration of 0.5g/L to 3g/L.
14. methods as claimed in claim 10, wherein with respect to there not being the microorganism of cultivating under the condition of lactic acid and/or its salt, the amount that described lactic acid and/or its salt exist be enough at least one gene that induction is selected from codH, codH β, codH δ, codH γ, coos1 and metF increase express approximately 1.5 times or more than.
15. methods as claimed in claim 10, wherein said at least one product is selected from acetic acid, propionic acid, butyric acid, vinylformic acid and lipid acid.
16. methods as claimed in claim 10, wherein said at least one product is selected from ethanol, acetone, propyl alcohol, butanols and 2,3-butanediol.
17. methods as claimed in claim 10, wherein said microorganism can be by CO
2and/or CO is converted at least one product that is selected from acid and alcohol.
18. methods as claimed in claim 10, described microorganism is selected from clostridium aceticum (Clostridium aceticum), from producing and ethanol clostridium (Clostridium autoethanogenum), food carbonoxide clostridium (Clostridium carboxidivorans), clostridium difficile (Clostridium difficile), Young clostridium (Clostridium ljungdahlii), hot acetic acid Moore Salmonella (former Clostridium thermoaceticum, Moorella thermoacetica, Clostridium thermoaceticum), have a liking for hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), autotrophy desulfurization bacterium (Desulfobacterium autotrophicum), the blue De Shi clostridium of Wamsteeker (Clostridium sticklandii), Clostridium thermoautotrophicum (Clostridium thermoautotrophicum), formic acid clostridium aceticum (Clostridium formicoaceticum), Clostridium magnum (Clostridium magnum), methyl alcohol bacillus aceticus (Acetobacterium carbinolicum), triumphant 5 bacillus aceticuss (Acetobacterium kivui) and Wu Shi bacillus aceticus (Acetobacterium woodii).
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| CN105039423A (en) * | 2015-08-27 | 2015-11-11 | 中国科学院天津工业生物技术研究所 | Method for improving concentration of alcohol substances in fermented product of anaerobic gas-feeding microbes |
| TWI641685B (en) * | 2017-05-23 | 2018-11-21 | 國立中興大學 | Culture condition and culture method for growing recombinant carbon-fixing microorganism under anaerobic environment |
| CN112979356A (en) * | 2021-05-24 | 2021-06-18 | 中国农业科学院农业环境与可持续发展研究所 | Application of microbial gene abundance and extracellular enzyme activity in straw returning and carbon fixation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039423A (en) * | 2015-08-27 | 2015-11-11 | 中国科学院天津工业生物技术研究所 | Method for improving concentration of alcohol substances in fermented product of anaerobic gas-feeding microbes |
| TWI641685B (en) * | 2017-05-23 | 2018-11-21 | 國立中興大學 | Culture condition and culture method for growing recombinant carbon-fixing microorganism under anaerobic environment |
| CN112979356A (en) * | 2021-05-24 | 2021-06-18 | 中国农业科学院农业环境与可持续发展研究所 | Application of microbial gene abundance and extracellular enzyme activity in straw returning and carbon fixation |
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