CN104031861A - Method for preparing resting cells in process of preparing ethanol by utilizing anaerobic fermentation of synthesis gas - Google Patents

Abstract

The invention belongs to the technical field of preparing ethanol by anaerobically fermenting synthesis gas by virtue of microbes and particularly relates to a method for preparing resting cells of microbes in the process of preparing ethanol by anaerobically fermenting synthesis gas by virtue of microbes. The method comprises the following steps: activating microbial strains; enriching and growing under anaerobic condition; centrifuging; collecting cell precipitates; performing resuspension culturing and the like. According to the preparation method, a culture medium can overcome interference caused by cell growth in the cell liquid culturing process by specially optimizing, the growing phases of anaerobic microbial resting cells are consistent, and the cells can better perform energy metabolism, so that the efficiency of conversion from converting synthesis gas into ethanol can be improved, and the industrialization of convertion from synthesis gas to ethanol can be promoted.

Description

Utilize the preparation method of resting cell in synthetic gas anaerobically fermenting ethanol production process

Technical field

The invention belongs to and utilize microorganism by synthetic gas anaerobically fermenting ethanol production technical field, be specifically related to the preparation method that a kind of microorganism utilizes microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process.

Background technology

The energy is the important substance basis of human survival and development, is also the focus of current International Politics, economy, military affairs, diplomacy concern.Energy consumption becomes increasingly conspicuous on the impact of ecotope, resolves energy problem, not only will focus on balance between supply and demand, also will pay close attention to the ecological environment problem bringing thus.

Ethanol is a kind of liquid fuel of high-quality, can be used alone, and also can join in gasoline and use as a kind of clean energy.In prior art, utilizing grain raw material to adopt fermentation process is the common method that obtains ethanol, but because grain raw material cost is higher, thereby ethanol is used the restriction that is always limited to ethanol production as fuel.Utilizing synthetic gas anaerobism ethanol production is a new study hotspot in recent years, is also the method for thinking that at present feasibility is higher.

Synthetic gas refers to that a kind of main component is CO, CO ₂, H ₂and N ₂deng the gas mixture of gas, by coal, oil, Sweet natural gas, coke-oven gas, refinery gas, mud and biomass etc., transform and obtain.In physical environment, some microorganisms can utilize synthetic gas to grow as sole carbon source and the energy and metabolism produces ethanol, and this provides theoretical basis for the recovery energy of synthetic gas resource and the exploitation of bio-ethanol.

Existing research thinks, what can utilize that synthetic gas produces ethanol is the special anaerobion of a class.This quasi-microorganism cell can be both in the continuous build phase of biomass while utilizing synthetic gas to produce ethanol, can be also in the stable resting cell stage of biomass.

Resting cell refers to does not have exogenous nutrition provisioning, and the energy metabolism of maintaining thalline by consuming Inner nutrition material, does not carry out growth and breeding, the cell in starvation, dormant state.Various endonuclease capables in resting cell play a role and ceaselessly carry out energy metabolism, and can recover under optimum conditions growth again.

Resting cell fermentation method is to using the whole cell of microorganism as catalysts, the advantage that many normal cell fermentation methods such as have reaction conditions gentleness, selectivity is strong, pollution-free, cost is low, by product is few, the microorganism doubling time is short, biomass accumulation is fast, and enzyme amount that the unit time produces is many, transformation efficiency is high, condition is easily controlled, separation and purification is easier to do not possess.Thereby in synthetic gas anaerobically fermenting ethanol production process, the reasonable application of resting cell phase is the important channel of improving alcohol yied.And in prior art, there is not yet the comparatively suitable preparation method who utilizes the microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process for microorganism.

Summary of the invention

The object of the invention is to provide a kind of microorganism to utilize the preparation method of microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, thereby the whole microorganism cells in quiescent stage of a large amount of preparations, the microorganism cells in this stage only carries out energy metabolism, is beneficial to synthetic gas is converted into ethanol.

A preparation method who utilizes microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, comprises the following steps:

(1) by microbial strains activation, enrichment and growth under anaerobic condition;

Described microbial strains is: clostridium carboxidivoransp7, clostridium strainp11, clostridium. Ljungdahliior clostridium autoethanogenumdSM10061;

Described actication of culture adopts activation medium, and activation medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NH ₄cl 0.7 ~ 1.2g, NaCl 0.7 ~ 1.2g, MgCl ₂0.2 ~ 0.5g, KH ₂pO ₄0.5 ~ 0.9g, K ₂hPO ₄1.0 ~ 2.0g, Tryptones 1 ~ 3g, yeast extract paste 0.5 ~ 1.5g, FeCl ₃2.0 ~ 3.0g, cellobiose 0.5 ~ 1.5g, L-cys 0.5 ~ 0.9g, wood sugar 2 ~ 6g, mineral element solution 0.5 ~ 1.5mL, surplus is water; During preparation substratum, cellobiose filters and adds after medium sterilization;

Described mineral element solution, take 1L capacity as example, fills a prescription as follows: ZnCl ₂0.05 ~ 0.1g, MnCl ₂0.05 ~ 0.15g, H ₃bO ₃0.004 ~ 0.008g, CoCl ₂0.1 ~ 0.3g, CuCl ₂0.001 ~ 0.003g, NiCl ₂0.015 ~ 0.03g, Na ₂moO ₄0.02 ~ 0.04g, is dissolved in the FeCl of 10mL HCl ₂1.0 ~ 2.0g, surplus is water; Described water is preferably deionized water; During preparation, first FeCl ₂be dissolved in HCl, then dilute with water, then add other salt, final volume constant volume;

Described enrichment and growth adopts growth medium, and growth medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NaCl 1.0 ~ 1.5g, NH ₄cl 1.0 ~ 2.0g, KCl 0.10 ~ 0.30g, MgSO ₄0.2 ~ 0.5g, CaCl ₂0.04 ~ 0.08g, KH ₂pO ₄0.20 ~ 0.50g, yeast extract paste 0.2 ~ 0.6g, L-cys 0.1 ~ 0.3g, MES 4 ~ 8g, trace element solution 10mL, vitamin solution 10mL, surplus is water, pH4.5 ~ 6.5; During preparation, after adding MES, being adjusted in of pH carry out immediately;

Described trace element solution, take 1L capacity as example, fills a prescription as follows: N (CH ₂cOOH) ₃1.00 ~ 3.00g, MgSO ₄0.50 ~ 1.50g, (NH ₄) ₂sO ₄feSO ₄6H ₂o 0.5 ~ 1.0g, CoCl ₂6H ₂o 0.10 ~ 0.30g, ZnSO ₄7H ₂o 0.10 ~ 0.30g, CuCl ₂2H ₂o 0.01 ~ 0.03g, NiCl ₂6H ₂o 0.01 ~ 0.03g, Na ₂moO ₄2H ₂o 0.01 ~ 0.03g, Na ₂o ₄se (sodium selenate) 0.01 ~ 0.03g, Na ₂wO ₄2H ₂o 0.01 ~ 0.03g, surplus is water; During preparation, N (CH ₂cOOH) ₃finally add, after adding, the NaOH solution with 10.0mol/L regulates pH to 5.0 ~ 7.0 immediately;

Described vitamin solution, take 1L capacity as example, fills a prescription as follows: VB ₆8.00 ~ 12.00g, VITMAIN B1 4.00 ~ 6.00g, VB ₂3.00 ~ 7.00g, calcium pantothenate 3.00 ~ 7.00g, Thioctic Acid 4.00 ~ 6.00g, C ₉h ₁₁o ₂n (phenylalanine) 4.00 ~ 6.00g, VB ₃4.00-6.00g, VB ₁₂3.00 ~ 7.00g, vitamin H 1.00 ~ 3.00g, folic acid 1.00 ~ 3.00g, surplus is water; During preparation, use 0.22 μ m strainer filtration sterilization after solution preparation completes;

(2) microbial inoculum of growing after stablizing in step (1) is centrifugal, collecting cell precipitation;

It is stable that described growth stabilizes to biomass; Be that biometric measurement regularly adopts ultraviolet spectrophotometer at OD ₆₀₀while measuring under wavelength, biomass is in stablizing invariant state;

Described centrifugal be the centrifugal 10 ~ 30min in rpm6000 ~ 10000; When centrifugal, preferably adopt NG3 substratum to wash, centrifugal collecting precipitation again after washing, this process repeats 1 ~ 3 time, guarantees that growth medium does not impact follow-up cultivation, and guarantees that the microorganism cells of collecting is more consistent vegetative period; Described NG3 substratum is compared with growth medium, lacks CaCl ₂with yeast extract paste composition;

(3) step (2) collection is obtained to cell precipitation and be suspended in NG3 substratum, during suspension, in the cell precipitation of centrifugal acquisition in equal volume growth medium, be suspended in the NG3 substratum of equal volume;

Described NG3 substratum is compared with growth medium, lacks CaCl ₂with yeast extract paste composition;

Anaerobism is cultivated 1 ~ 2d, to microorganism cells biomass in NG3 substratum be in growth medium during 1.0 ~ 1.5 times of microorganism cells biomasss, finish to cultivate, cultivate and finish the microorganism cells that rear gained bacterium liquid culture is quiescent stage.

In the described preparation method who utilizes microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, the preparation of NG3 substratum comprises the following steps:

(1) by NG3 formula, prepare culture medium solution;

Described culture medium solution adopts anaerobism bottle to hold;

(2) the NG3 culture medium solution of being prepared by step (1) is placed in anaerobic culture box, adds deoxidation indicator, deoxidation 20 ~ 36h;

Described deoxidation indicator is that massfraction is 0.1% resazurin solution, and addition is 500uL/L;

(3) substratum after step (2) deoxidation is filled with to the emptying oxygen of High Purity Nitrogen, is then placed in Autoclave, in pot subject to sterilization, after water boiling, at anaerobism bottleneck, plug aseptic syringe needle, sealing Autoclave, under 115 ~ 125 ℃ of conditions, sterilizing 15 ~ 30min.

The preparation method of cell quiescent stage anaerobion provided by the present invention, substratum is through special optimization, overcome the interference that in bacterium liquid culturing process, thalli growth itself causes, the anaerobion resting cell of preparation is more consistent vegetative period, can make cell carry out better energy metabolism, thereby improve the transformation efficiency that synthetic gas is converted to ethanol, promote to utilize the carrying out of synthetic gas ethanol conversion industrialization.

Embodiment

Below in conjunction with embodiment the present invention will be further explained explanation.

Embodiment

With preparation clostridium autoethanogenumthe quiescent stage cell of DSM10061 is example, and concrete preparation method comprises the following steps:

(1) experimental strain clostridium autoethanogenumdSM10061 buys from German DSMZ, by the bacterial strain activation of buying, enrichment and growth under anaerobic condition in anaerobic box; In enrichment and growth process, every 12h sampling, sample uses ultraviolet spectrophotometer at OD ₆₀₀under wavelength, measure, after growth is stable, carry out again next step operation; After the about 96h of enrichment culture, microorganism cells enters the stationary phase of growing, and biomass is stable no longer to be increased;

Described actication of culture adopts activation medium, and activation medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NH ₄cl 0.7 ~ 1.2g, NaCl 0.7 ~ 1.2g, MgCl ₂0.2 ~ 0.5g, KH ₂pO ₄0.5 ~ 0.9g, K ₂hPO ₄1.0 ~ 2.0g, Tryptones 1 ~ 3g, yeast extract paste 0.5 ~ 1.5g, FeCl ₃2.0 ~ 3.0g, cellobiose 0.5 ~ 1.5g, L-cys 0.5 ~ 0.9g, wood sugar 2 ~ 6g, mineral element solution 0.5 ~ 1.5mL, surplus is water; During preparation substratum, cellobiose filters and adds after medium sterilization;

Described mineral element solution, take 1L capacity as example, fills a prescription as follows: ZnCl ₂0.05 ~ 0.1g, MnCl ₂0.05 ~ 0.15g, H ₃bO ₃0.004 ~ 0.008g, CoCl ₂0.1 ~ 0.3g, CuCl ₂0.001 ~ 0.003g, NiCl ₂0.015 ~ 0.03g, Na ₂moO ₄0.02 ~ 0.04g, is dissolved in the FeCl of 10mL HCl ₂1.0 ~ 2.0g, surplus is water; Described water is preferably deionized water; During preparation, first FeCl ₂be dissolved in HCl, then dilute with water, then add other salt, final volume constant volume;

Described enrichment and growth adopts growth medium, and growth medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NaCl 1.0 ~ 1.5g, NH ₄cl 1.0 ~ 2.0g, KCl 0.10 ~ 0.30g, MgSO ₄0.2 ~ 0.5g, CaCl ₂0.04 ~ 0.08g, KH ₂pO ₄0.20 ~ 0.50g, yeast extract paste 0.2 ~ 0.6g, L-cys 0.1 ~ 0.3g, MES 4 ~ 8g, trace element solution 10mL, vitamin solution 10mL, surplus is water, pH4.5 ~ 6.5; During preparation, after adding MES, being adjusted in of pH carry out immediately;

Described trace element solution, take 1L capacity as example, fills a prescription as follows: N (CH ₂cOOH) ₃1.00 ~ 3.00g, MgSO ₄0.50 ~ 1.50g, (NH ₄) ₂sO ₄feSO ₄6H ₂o 0.5 ~ 1.0g, CoCl ₂6H ₂o 0.10 ~ 0.30g, ZnSO ₄7H ₂o 0.10 ~ 0.30g, CuCl ₂2H ₂o 0.01 ~ 0.03g, NiCl ₂6H ₂o 0.01 ~ 0.03g, Na ₂moO ₄2H ₂o 0.01 ~ 0.03g, Na ₂o ₄se 0.01 ~ 0.03g, Na ₂wO ₄2H ₂o 0.01 ~ 0.03g, surplus is water; During preparation, N (CH ₂cOOH) ₃finally add, after adding, the NaOH solution with 10.0mol/L regulates pH to 5.0 ~ 7.0 immediately;

Described vitamin solution, take 1L capacity as example, fills a prescription as follows: VB ₆8.00 ~ 12.00g, VITMAIN B1 4.00 ~ 6.00g, VB ₂3.00 ~ 7.00g, calcium pantothenate 3.00 ~ 7.00g, Thioctic Acid 4.00 ~ 6.00g, C ₉h ₁₁o ₂n 4.00 ~ 6.00g, VB ₃4.00-6.00g, VB ₁₂3.00 ~ 7.00g, vitamin H 1.00 ~ 3.00g, folic acid 1.00 ~ 3.00g, surplus is water; During preparation, use 0.22 μ m strainer filtration sterilization after solution preparation completes;

(2) microbial inoculum of growing after stablizing in step (1) is centrifugal, collecting cell precipitation;

It is stable that described growth stabilizes to biomass; Biometric measurement regularly adopts ultraviolet spectrophotometer at OD ₆₀₀under wavelength, measure;

Described centrifugal be the centrifugal 10 ~ 30min in rpm6000 ~ 10000; When centrifugal, preferably adopt NG3 substratum to wash, centrifugal collecting precipitation again after washing, this process repeats 1 ~ 3 time, guarantees that growth medium does not impact follow-up cultivation, and guarantees that the microorganism cells of collecting is more consistent vegetative period; Described NG3 substratum is compared with growth medium, lacks CaCl ₂with yeast extract paste composition;

(3) step (2) is collected and obtains cell precipitation and be suspended in NG3 substratum, during suspension, by the cell suspension of one bottle of (80mL/ bottle) growth medium centrifugation acquisition in one bottle of (80mL/ bottle) NG3 substratum,

Described NG3 substratum is compared with growth medium, lacks CaCl ₂with yeast extract paste composition;

Anaerobism is cultivated 1 ~ 2d, to microorganism cells biomass in NG3 substratum be in growth medium during 1.0 ~ 1.5 times of microorganism cells biomasss, finish to cultivate, cultivate and finish the microorganism cells that rear gained bacterium liquid culture is quiescent stage.

It should be noted that, during preparation NG3 substratum, preparation according to the following steps:

(1) by NG3 formula, prepare culture medium solution;

Described culture medium solution adopts anaerobism bottle to hold;

(2) the NG3 culture medium solution of being prepared by step (1) is placed in anaerobic culture box, adds deoxidation indicator, deoxidation 20 ~ 36h;

Described deoxidation indicator is that massfraction is 0.1% resazurin solution, and addition is 500uL/L;

(3) substratum after step (2) deoxidation is filled with to the emptying oxygen of High Purity Nitrogen, is then placed in Autoclave, in pot subject to sterilization, after water boiling, at anaerobism bottleneck, plug aseptic syringe needle, sealing Autoclave, under 115 ~ 125 ℃ of conditions, sterilizing 15 ~ 30min.

By above-mentioned prepared clostridium autoethanogenum(DSM10061) resting cell and conventional utilization clostridium autoethanogenum(DSM10061) carry out synthetic gas anaerobically fermenting ethanol production and contrast, fermentation results is as following table.

By above table, can find out, no matter be normal fermentation, or supplement synthetic gas fermentation, no matter liquid amount is how many, utilize resting cell compared to normal fermentation method, ethanol production all has comparatively significantly raising, thereby for utilizing anaerobion that synthetic gas fermentative production of ethanol has been established to good experiment basis.

In general, the present invention is directed to part anaerobion and utilize the microorganism culturing link in synthetic gas ethanol conversion process to do special optimization design, more for current research clostridium carboxidivoransp7, clostridium strainp11, clostridium. Ljungdahlii, clostridium autoethanogenumthe substratum of DSM10061 bacterial classification is optimized design, by optimizing corresponding culture medium prescription, make to prepare a large amount of, the anaerobion resting cell of uniformity comparatively, by these resting cells are inoculated, anaerobically fermenting, through check, resting cell utilizes synthetic gas ethanol conversion output higher than conventional cell 20%, thereby for rational exploitation and utilization anaerobion, synthetic gas fermentative production of ethanol is laid a good foundation.

Claims (3)

1. a preparation method who utilizes microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, is characterized in that, the method comprises the following steps:

(1) by microbial strains activation, enrichment and growth under anaerobic condition;

Described enrichment and growth adopts growth medium, and growth medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NaCl 1.0 ~ 1.5g, NH ₄cl 1.0 ~ 2.0g, KCl 0.10 ~ 0.30g, MgSO ₄0.2 ~ 0.5g, CaCl ₂0.04 ~ 0.08g, KH ₂pO ₄0.20 ~ 0.50g, yeast extract paste 0.2 ~ 0.6g, L-cys 0.1 ~ 0.3g, MES 4 ~ 8g, trace element solution 10mL, vitamin solution 10mL, surplus is water, pH4.5 ~ 6.5;

Described trace element solution, take 1L capacity as example, fills a prescription as follows: N (CH ₂cOOH) ₃1.00 ~ 3.00g, MgSO ₄0.50 ~ 1.50g, (NH ₄) ₂sO ₄feSO ₄6H ₂o 0.5 ~ 1.0g, CoCl ₂6H ₂o 0.10 ~ 0.30g, ZnSO ₄7H ₂o 0.10 ~ 0.30g, CuCl ₂2H ₂o 0.01 ~ 0.03g, NiCl ₂6H ₂o 0.01 ~ 0.03g, Na ₂moO ₄2H ₂o 0.01 ~ 0.03g, Na ₂o ₄se 0.01 ~ 0.03g, Na ₂wO ₄2H ₂o 0.01 ~ 0.03g, surplus is water;

Described vitamin solution, take 1L capacity as example, fills a prescription as follows: VB ₆8.00 ~ 12.00g, VITMAIN B1 4.00 ~ 6.00g, VB ₂3.00 ~ 7.00g, calcium pantothenate 3.00 ~ 7.00g, Thioctic Acid 4.00 ~ 6.00g, C ₉h ₁₁o ₂n 4.00 ~ 6.00g, VB ₃4.00-6.00g, VB ₁₂3.00 ~ 7.00g, vitamin H 1.00 ~ 3.00g, folic acid 1.00 ~ 3.00g, surplus is water;

(2) microbial inoculum of growing after stablizing in step (1) is centrifugal, collecting cell precipitation;

It is stable that described growth stabilizes to biomass;

Described centrifugal be rpm 6000 ~ 10000,10 ~ 30min;

(3) step (2) collection is obtained to cell precipitation and be suspended in NG3 substratum, during suspension, in the cell precipitation of centrifugal acquisition in equal volume growth medium, be suspended in the NG3 substratum of equal volume;

Described NG3 substratum is compared with growth medium, lacks CaCl ₂with yeast extract paste composition;

Anaerobism is cultivated 1 ~ 2d, to microorganism cells biomass in NG3 substratum be in growth medium during 1.0 ~ 1.5 times of microorganism cells biomasss, finish to cultivate, cultivate and finish the microorganism cells that rear gained bacterium liquid culture is quiescent stage.

2. the preparation method who utilizes as claimed in claim 1 microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, is characterized in that, described in step (1), microbial strains is: clostridium carboxidivoransp7, clostridium strainp11, clostridium. Ljungdahliior clostridium autoethanogenumdSM10061;

Described actication of culture adopts activation medium, and activation medium is liquid nutrient medium, take 1L capacity as example, fills a prescription as follows: NH ₄cl 0.7 ~ 1.2g, NaCl 0.7 ~ 1.2g, MgCl ₂0.2 ~ 0.5g, KH ₂pO ₄0.5 ~ 0.9g, K ₂hPO ₄1.0 ~ 2.0g, Tryptones 1 ~ 3g, yeast extract paste 0.5 ~ 1.5g, FeCl ₃2.0 ~ 3.0g, cellobiose 0.5 ~ 1.5g, L-cys 0.5 ~ 0.9g, wood sugar 2 ~ 6g, mineral element solution 0.5 ~ 1.5mL, surplus is water;

Described mineral element solution, take 1L capacity as example, fills a prescription as follows: ZnCl ₂0.05 ~ 0.1g, MnCl ₂0.05 ~ 0.15g, H ₃bO ₃0.004 ~ 0.008g, CoCl ₂0.1 ~ 0.3g, CuCl ₂0.001 ~ 0.003g, NiCl ₂0.015 ~ 0.03g, Na ₂moO ₄0.02 ~ 0.04g, is dissolved in the FeCl of 10mL HCl ₂1.0 ~ 2.0g, surplus is water.

3. the preparation method who utilizes as claimed in claim 1 microorganism resting cell in synthetic gas anaerobically fermenting ethanol production process, is characterized in that, in step (3), the preparation of NG3 substratum comprises the following steps:

(1) by NG3 formula, prepare culture medium solution;

Described culture medium solution adopts anaerobism bottle to hold;

(2) the NG3 culture medium solution of preparation in step (1) is placed in to anaerobic culture box, adds deoxidation indicator, deoxidation 20 ~ 36h;

(3) substratum after step (2) deoxidation is filled with to the emptying oxygen of High Purity Nitrogen, is then placed in Autoclave, in pot subject to sterilization, after water boiling, at anaerobism bottleneck, plug aseptic syringe needle, sealing Autoclave, under 115 ~ 125 ℃ of conditions, sterilizing 15 ~ 30min.