CN114807243A - Method for producing yeast cell by ultrahigh-concentration continuous ethanol fermentation - Google Patents
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Abstract
The invention discloses a method for producing yeast cells by ultrahigh-concentration continuous ethanol fermentation, which comprises the following steps: culturing ethanol producing bacteria; preparing a saccharification liquid of wheat and corn; performing ultrahigh-concentration continuous ethanol fermentation by using ethanol producing bacteria; and weakening the fermentation parameter oscillation of the ethanol producing bacteria in the continuous ethanol fermentation process by using product in-situ separation (gas stripping method or membrane separation method) to perform the step of stable and continuous production of the ethanol with ultrahigh concentration. The method effectively realizes the ultrahigh-concentration continuous ethanol fermentation of the single-stage tank on the premise of not increasing the equipment investment, controls the residual sugar concentration to be lower than 1g/L, meets the industrial production requirements, and provides a new technical support for the industrial production of the ultrahigh-concentration continuous ethanol fermentation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for producing yeast cell ultrahigh-concentration continuous ethanol by fermentation, in particular to a method for stably producing yeast cell ultrahigh-concentration continuous ethanol by fermentation coupling product in-situ separation (gas stripping or membrane separation).
Background
Fuel ethanol is one of the most important bio-energy products and is currently produced primarily from saccharine, starchy, and lignocellulosic feedstocks. After the starchy raw materials are used for producing fuel ethanol, the raw material cost still accounts for about 60 percent of the total production cost and is in the top position after the byproduct reduction is deducted, and the energy consumption cost accounts for about 30 percent, and is the second time. The development of cheap raw material resources, particularly lignocellulose biomass represented by various crop straws, is a fundamental way for solving the problem of high cost of raw materials for producing fuel ethanol, but because the biomass has strong resistance to cellulose hydrolysis, the cost for obtaining sugar from the biomass is far higher than that of starch raw materials, and the hydrolysis sugar contains 20-30% of pentose and is difficult to be effectively utilized by strains with excellent ethanol fermentation performance, so that the production cost of the fuel ethanol is far higher than that of the glucide and starch raw materials, and the industrial large-scale production cannot be realized. Therefore, the development of an energy-saving technology for producing fuel ethanol and the reduction of energy consumption in the production process have very important significance.
The high-concentration fermentation aiming at improving the ethanol concentration at the fermentation end point can not only reduce the energy consumption of rectification operation of fermented mash, but also reduce the total amount of waste grain liquid in fuel ethanol production and save the energy consumption for treating the waste grain liquid. At present, in the fermentation of starchy raw materials ethanol at home and abroad, the volume ratio concentration of continuously fermented ethanol is generally 11-12%, and the intermittent fermentation is slightly higher and can reach 13-15%. High-concentration fermentation with ethanol concentration of more than 15% at the fermentation end point, even ultra-high-concentration fermentation with ethanol concentration of 20% at the fermentation end point, is always a research direction of common attention in academia and industry, and people try to improve the ethanol concentration at the fermentation end point continuously from the aspects of improvement of fermentation process, addition of nutrient components of culture medium, optimization of fermentation devices and the like. However, achieving such a high ethanol concentration leads to an increase in the residual sugar concentration at the end of fermentation, affects the sugar alcohol yield (the sugar alcohol yield is calculated from the total sugar entering the fermentation system in industrial production, and the increase in the residual sugar concentration at the end of fermentation leads to a significant decrease in the sugar alcohol yield), does not significantly prolong the fermentation time, increases the chance of contamination by infectious microbes, causes loss of sugar and ethanol produced by fermentation, is the biggest challenge in the development of basic research and application technologies, and is also the root cause of the failure of VHG technology to be practically applied in industrial production so far.
Therefore, the development of ultra-high concentration continuous ethanol fermentation is an important direction for the industrial development of fuel ethanol. DPBayrock, canada, utilized a multi-stage series reactor for short-time ultra-high concentration continuous ethanol fermentation with good results, as detailed in: bayrock DP, Ingleew WM.2001.application of multistage continuous transfer for production of fuel alcohol by high-throughput transfer technology, journal of Industrial Microbiology and Biotechnology 27, 87-93.
However, researches find that the saccharomyces cerevisiae shows long-period and large-amplitude oscillation behavior in the process of carrying out long-time ultrahigh-concentration continuous ethanol fermentation, and find that the saccharomyces cerevisiae has obvious influence on the ethanol fermentation performance of the saccharomyces cerevisiae, and the details are as follows: bai FW, Chen LJ, Anderson WA, Moo-Young M.2004.parameter environments in very high quality media conversion and the air authentication on multi-stage packed column biorator system, Biotechnology and Bioengineering 88: 558. fig..
In addition, professor Walter Borzani also found long-period, large-amplitude oscillatory behavior in the research work using saccharomyces cerevisiae continuous ethanol fermentation of molasses (Blackstrap mortases, containing a large amount of ash and other impurities) with a substrate sugar concentration of 20%, as detailed in: borzani W.2001.variation of the ethanol yield reducing catalysis concentration conversion in undistributed hydrolysis of sugar-cane blackstrap molases. world Journal of Microbiology and Biotechnology 17: 253-258; unfortunately, Walter Borzani only reported experimental phenomena, analyzing the effect on ethanol fermentation yield, and did not develop a weakening strategy for this oscillation phenomenon. At present, a corresponding oscillation weakening strategy is developed aiming at oscillation behaviors under the condition of ultrahigh-concentration continuous ethanol fermentation, and the oscillation is weakened better by an immobilization method under the condition of a multistage series reactor, but the multistage tank series operation has the defect of increasing the investment of equipment and the difficulty of operation and maintenance. In addition, no report or patent technology about a strategy capable of stabilizing high-concentration ethanol fermentation is found.
Disclosure of Invention
The invention provides a method for performing stable yeast cell ultrahigh-concentration continuous ethanol fermentation production by a fermentation coupling product in-situ separation (gas stripping or membrane separation) technology.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
a method for producing yeast cells by ultrahigh-concentration continuous ethanol fermentation, namely a method for weakening parameter oscillation of ethanol producing bacteria in the ultrahigh-concentration continuous ethanol fermentation process by coupling gas stripping or a membrane separation device in the continuous ethanol fermentation process, comprises the following steps:
(1) culturing ethanol producing bacteria;
(2) performing ultrahigh-concentration continuous ethanol fermentation by using ethanol producing bacteria; and
(3) and (3) realizing the stable and continuous production of the ultrahigh-concentration ethanol by fermentation in the process (2) by using a gas stripping method or a membrane separation step.
The ethanol producing strain is preferably industrial saccharomyces cerevisiae or gene engineering flocculating yeast BHL 01.
Wherein, the step (2) uses an ultrahigh concentration continuous ethanol fermentation device, the gas stripping method of the step (3) uses a gas stripping device, and the membrane separation method uses a membrane separation device; the gas stripping device or the membrane separation device is coupled with the ultrahigh-concentration continuous ethanol fermentation device, when fermentation parameters in fermentation liquor of the fermentation device have 1-10 complete oscillation periods, the gas stripping device or the membrane separation device is started, gas is introduced into the ethanol fermentation liquor, and the oscillation behavior of ethanol production bacteria in the continuous ethanol fermentation process is weakened by a gas stripping method.
The initial sugar concentration in the continuous fermentation medium used in the ultrahigh-concentration continuous ethanol fermentation in the step (2) is 250g/L-350g/L, and the optimal concentration is 280 g/L; the dilution rate of the continuous fermentation medium is 0.012-0.036h -1 Preferably 0.027h -1 。
The sugar in the culture medium is glucose, or saccharified solution of semen Tritici Aestivi and semen Maydis. In a continuous ultra-high concentration ethanol fermentation process, the fermentation parameters of the yeast cells, including residual sugars (including glucose, xylose, arabinose), ethanol, biomass in the fermentation broth, exhibit a large oscillation in periodicity.
And (3) in the gas stripping process of the step (3), the gas is nitrogen, air or fermentation self-production gas such as carbon dioxide, the raw material liquid for gas stripping is fermentation liquid of ethanol producing bacteria, and the gas forms small bubbles in a fermentation system under the shearing action of a stirring paddle. When the fermentation parameters of the fermentation liquid under the condition of ultrahigh-concentration continuous fermentation present 1-10 complete oscillation cycles, starting the gas stripping device, and introducing gas stripping gas into the ethanol fermentation liquid, wherein the flow rate of the gas introduced into each 1.5 liters (the effective volume of continuous fermentation) of the ethanol fermentation liquid is 300L/h, and the optimal flow rate is 200L/h. After the gas is introduced for about 100 hours, the ethanol concentration in the fermentation liquor can be maintained at about 50g/L, and the residual sugar concentration in the fermentation liquor is lower than 1 g/L. And when the introduction of the gas stripping gas is stopped, the continuous fermentation system recovers to the periodic oscillation behavior before gas stripping, and the oscillation period exceeds 100 h.
The ultrahigh-concentration continuous ethanol fermentation can obviously reduce the energy consumption of fuel ethanol production, but the continuous ethanol fermentation of yeast cells under the VHG condition shows a long-period and large-amplitude oscillation behavior, so that the ethanol fermentation performance is seriously influenced, and the stable operation of a rectifying device is not facilitated, so that the ultrahigh-concentration continuous ethanol fermentation technology cannot be practically applied to industrial production.
The method provided by the invention effectively realizes the ultrahigh-concentration continuous ethanol fermentation of the single-stage tank without increasing equipment investment, controls the residual sugar concentration to be lower than 1g/L, meets the industrial requirements, and provides a new technical support for the industrial production of the ultrahigh-concentration continuous ethanol fermentation.
Drawings
FIG. 1 is a schematic view of a continuous air or nitrogen stripping ethanol fermentation apparatus according to the present invention;
FIG. 2 is a schematic structural view of a continuous self-produced gas-stripping ethanol fermentation apparatus according to the present invention;
FIG. 3 is a data plot of fermentation parameter oscillations during continuous ethanol fermentation of free yeast cells;
FIG. 4 is a data curve of fermentation parameter oscillation during continuous ethanol fermentation of flocculated yeast cells;
FIG. 5 is a data plot of fermentation parameters before and after nitrogen stripping in a free cell continuous ethanol fermentation system;
FIG. 6 is a data plot of air-gas-advanced post fermentation parameters in a free cell continuous ethanol fermentation system;
FIG. 7 is a data curve of fermentation parameters before and after stripping of the self-generated gas in the flocculation cell continuous ethanol fermentation system.
Detailed Description
The embodiments are described in detail below with reference to the accompanying drawings and technical solutions.
Cultivation of ethanol producing bacteria
The ethanol producing strain is industrial saccharomyces cerevisiae (Saccharomyces cerevisiae) or gene engineering flocculating yeast BHL 01. The gene engineering flocculation yeast BHL01 is a constitutively expressed flocculation yeast strain obtained by introducing flocculation genes into industrial saccharomyces cerevisiae.
For the sake of brevity, industrial saccharomyces cerevisiae, referred to herein as free yeast; the gene engineering flocculating yeast BHL01, referred to as flocculating yeast for short.
Inoculating the strain cultured in the culture dish into a growth culture medium in a shake flask, and performing shake culture in a constant temperature shaking table at 25-37 ℃ and 200-300rpm for 15-25h, wherein the strain culture condition is preferably that the strain is cultured for 20h under shaking conditions of the shaking table at 30 ℃ and 300 rpm.
Preparation of saccharified liquid of wheat and corn
The preparation of the saccharified liquid is carried out in a stirring tank, degermed and peeled corn flour or wheat flour and warm water at about 60-65 ℃ are prepared into starch slurry according to the proportion of 1:2.5, alpha-amylase is added according to the proportion of 0.6mL per kilogram of corn flour or wheat flour, the temperature is raised to 85-90 ℃, the temperature is kept for liquefaction for 1-1.5 hours, then the temperature is lowered to 60-65 ℃, saccharifying enzyme is added according to the proportion of 1.2mL per kilogram of corn flour, the temperature is kept for saccharification for 10-15 hours, and then the saccharified liquid is obtained by filtration and used for the whole fermentation system.
Ultra-high concentration continuous ethanol fermentation by ethanol production bacteria
Inoculating the seed culture solution containing ethanol producing bacteria obtained in the above step into a fermentation tank 3 filled with 1.5L of batch fermentation culture medium according to the inoculation amount of 10% by volume, performing batch fermentation culture under the fermentation conditions of 30 ℃, 300rpm and pH4.5, and adjusting the temperature and pH value of the fermentation liquid by using a temperature regulator 7 and a pH regulator 8 arranged on the fermentation tank 3. The pH value of the fermentation liquid during the fermentation is preferably controlled to 4.5, and is adjusted by adding a 2mol/L aqueous solution of sodium hydroxide to the fermentation liquid through a pH adjuster 8. Compressed air is introduced into the fermentation tank 3 in the fermentation process, the flow of the compressed air is controlled through the rotor flow meter 5b, the aeration rate is controlled to be 0.05vvm, and the humidity of the air is regulated through a device 6b filled with water before the air is introduced into the fermentation tank, so that the air introduced into the fermentation tank 3 contains saturated water vapor. The residual sugar and ethanol concentration in the fermentation broth were measured every 12 h.
② when the concentration of the residual sugar in the fermentation liquor in the fermentation tank 3 is lower than 1g/L, starting to feed from the storage tank1, feeding into fermenter 3 via peristaltic pump (2 in FIG. 1, 2a in FIG. 2) for 0.012-0.036h -1 The continuous fermentation culture medium is fed at the dilution rate, continuous fermentation culture is carried out, and fermentation parameters in the fermentation liquid are measured every 12 hours. The continuous fermentation culture conditions are the same as the fermentation conditions in the first step. The dilution rate of the continuous fermentation medium is preferably 0.027h -1 。
The continuous fermentation system controls the fermentation volume through an overflow port on the fermentation tank 3, and the fermentation liquid flows into the waste liquid tank 4 through the overflow port.
After the ultrahigh-concentration continuous ethanol fermentation is carried out, the detected fermentation parameters fluctuate greatly, and stable continuous oscillation behavior is shown after about 100-130 h.
The carbon source in the continuous fermentation medium is glucose or saccharification liquid of wheat and corn, and the initial sugar concentration is 250-300g/L, preferably 280 g/L.
The fermentation parameters include residual sugar, ethanol and biomass in the fermentation broth.
In the traditional ethanol fermentation process, due to the inhibition effect of high-concentration ethanol on cells in the later stage of fermentation, batch fermentation of high-concentration substrates can be only carried out, continuous high-concentration ethanol fermentation is difficult to realize, and although continuous fermentation is carried out industrially, fed-batch culture media are culture media containing low-concentration sugar. Therefore, the ultra-high concentration continuous ethanol fermentation is an important direction of industrial ethanol fermentation, and is greatly improved compared with the traditional ethanol fermentation process. However, the oscillation phenomenon of ethanol producing bacteria in the continuous ultrahigh-concentration ethanol fermentation process can not stably convert the substrate into ethanol in the fermentation process, and easily causes great substrate loss and energy consumption, so that weakening the oscillation phenomenon is particularly important for producing industrial ethanol by ultrahigh-concentration continuous fermentation. The invention effectively weakens the oscillation phenomenon of the yeast cells by utilizing the method of gas stripping in high-concentration continuous ethanol fermentation.
Weakening fermentation parameter oscillation behavior of ethanol producing bacteria in fermentation process by gas stripping or membrane separation method
After 1-10 complete fermentation parameter oscillation periods, starting a gas stripping device while not changing the continuous ethanol fermentation condition, introducing gas into the fermentation liquid at the speed of 100-300L/h through a rotor flow meter 5a, splitting large bubbles into a plurality of small bubbles under the shearing action of a stirring paddle, and carrying out ethanol gas stripping in a gas phase along with the bubbles flowing into a condensing device to be collected due to the ethanol transfer action between two phases driven by the ethanol concentration gradient of the gas-liquid two phases.
When the gas stripping method is utilized, the introduced gas is air, nitrogen or fermentation self-produced gas, wherein the continuous gas stripping ethanol fermentation device shown in the figure 1 is adopted when the introduced gas is air or nitrogen; when the gas is introduced for fermentation and self-production of gas, the ethanol fermentation device for continuous self-production of gas stripping shown in figure 2 is adopted. When the membrane separation method is used, the fermentation-membrane separation coupling device shown in fig. 3 and the hydrophilic polyvinyl alcohol (PVA) -phosphorylated Chitosan (CS) composite matrix membrane are used to heat and evaporate the bacteria liquid, the mixed steam enters the membrane, and the ethanol molecules with membrane selectivity permeate the membrane and are collected by the condensing device.
In FIG. 1, the flow rate of the gas introduced is controlled by a rotameter 5a and the humidity is adjusted by a device 6a before the gas is introduced into the fermentation tank 3 so that the gas introduced into the fermentation tank 3 contains saturated water vapor. The gas in the fermentation tank 3 carries away part of ethanol and is condensed in the condensation pipe 11 and recovered into the collection bottle 9, because the ethanol in the fermentation liquid is removed, the toxic damage of the ethanol to cells is reduced, the toxic action of the ethanol production bacteria is relieved, the fermentation system tends to be stable after the gas is stripped for a period of time, the ethanol concentration in the fermentation tank 3 is maintained at a certain concentration, the ethanol concentration is maintained at 47.36 +/-8.15% g/L when the ethanol production bacteria are free yeast, the ethanol concentration is maintained at 75.56 +/-4.67% g/L when the ethanol production bacteria are flocculation yeast, and meanwhile, the residual sugar concentration in the fermentation liquid is also maintained at a level lower than 1g/L, and the continuous fermentation state which is similar to a steady state is maintained. The fermentation system does not gradually recover to the previous periodic oscillation behavior until after the gas stripping operation is stopped (see fig. 5, 6, 7). The low-temperature condensate flows into the condensation pipe 11 through the water inlet 12, the condensation pipe 11 is kept at the temperature range of 0-5 ℃, the water outlet 13 is formed, and the collecting bottle 9 is kept at the low temperature state in the condensation jacket 10.
In FIG. 2, the stripping gas is the fermentation self-produced gas, and the flow rate of the introduced gas is adjusted by an air pump 2 b. A trace amount of air was introduced into the fermentation tank 3 through the gas flow meter 5b to maintain the normal growth of ethanol-producing bacteria, and the aeration rate was controlled at 0.05 vvm. The other devices and functions in fig. 2 are the same as in fig. 1.
In the process of gas stripping weakening, when the introduced gas is nitrogen, the concentration of the collected ethanol is higher than that when the introduced gas is air. This is because when the gas is introduced as air, the oxygen in the air provides sufficient oxygen to the yeast cells under the conditions of ultra-high concentration ethanol fermentation, so that the carbon flux flows to the biomass synthesis pathway in large quantities, which may weaken the influence of ethanol stress during the oscillation of fermentation parameters, while the inert gas nitrogen does not influence the ethanol fermentation, so that the obtained ethanol concentration is higher than that when air is introduced.
In the gas stripping weakening process, when the introduced gas is fermented self-produced gas, the problems that when external source gas (air or nitrogen) is used, the gas flow rate is high, and the ethanol in the gas is not condensed thoroughly, so that part of ethanol is discharged along with tail gas to cause loss can be effectively solved, the gas stripping efficiency is improved, and the external source gas supply is saved.
In FIG. 3, during the membrane separation process, the fermentation broth in the strain fermentor 3 enters the membrane reactor 4 under the action of a peristaltic pump. Because the membrane material is hydrophilic, ethanol molecules preferentially permeate the membrane, and other substances are prevented from flowing back into the strain fermentation tank 3 by the membrane. Liquid nitrogen is continuously supplemented in the condensing device 5, so that the evaporated ethanol is liquefied, and finally, ethanol liquid is collected in the condensing device 6.
The flocculation yeast cell is used as ethanol producing bacteria, and the unique flocculation sedimentation characteristic of the flocculation yeast cell maintains high biomass in a fermentation system, so that biomass loss is prevented, the consumption of substrate synthesized biomass is reduced, the energy consumption of biomass removal in the effluent ethanol separation process is reduced, and the yield of ethanol can be effectively improved.
Examples
The present invention will be described in detail with reference to examples.
Ethanol producing bacteria: industrial Saccharomyces cerevisiae (Saccharomyces cerevisiae) was purchased from ATCC (ATCC number:4126) in the United states.
② the gene engineering flocculating yeast BHL01 (the preservation number of the strain is CGMCC 3408, the preservation unit is China general microbiological culture Collection center).
Culture medium:
firstly, shake flask growth culture medium, wherein each liter of culture medium contains 30g of glucose, 10g of yeast powder and 6g of peptone, and the culture medium is sterilized for 20min at 121 ℃.
② batch fermentation culture medium, each liter of culture medium contains 120g of sugar (glucose or saccharification liquid of wheat and corn), 5g of yeast powder and 3g of peptone, and the sterilization is carried out for 20min at 121 ℃.
③ continuous fermentation culture medium, wherein each liter of culture medium contains 280g of sugar (glucose or saccharification liquid of wheat and corn), 5g of yeast powder and 3g of peptone, and the sterilization is carried out for 15min at 110 ℃.
<Cultivation of ethanol producing bacteria and ultrahigh concentration continuous ethanol fermentation>: inoculating industrial Saccharomyces cerevisiae with an inoculating loop, which is stored on a loop slant (4 deg.C), into a 250mL shaking flask containing 100mL shaking flask growth medium, culturing for 20h on a rotary shaker at 300rpm and 30 deg.C, inoculating into a batch fermentation medium in a fermentation tank 3 in an amount of 10% (volume%) of the medium, culturing at 300rpm, 30 deg.C, pH4.5 and aeration amount of 0.05vvm, and adjusting the humidity of air to saturation by an apparatus 6 b. After 15h of cultivation, the residual sugar concentration in the fermentation broth was determined every 12h, when the residual sugar concentration in the fermentation broth was less than 1g/L, according to 0.027h without changing the fermentation conditions -1 Continuously fermenting at the speed of the fermentation medium, and measuring fermentation parameters in the fermentation liquid, namely the biomass, the concentration of ethanol and the concentration of residual sugar every 12 hours. During the continuous fermentation process, the fermentation parameters fluctuate, and a stable continuous oscillation behavior is presented after a period of time.
And (3) oscillating behavior in the gas stripping weakening fermentation process, namely, continuously fermenting the ethanol with ultrahigh concentration to present periodic oscillating behavior, starting a gas stripping device after 2-3 complete oscillating periods, introducing sterile gas at the speed of 200L/h through a flowmeter 5a, keeping a fermentation system stable after a period of time, keeping the state similar to a steady state unchanged, and gradually recovering the fermentation system to the previous periodic oscillating behavior until the gas stripping operation is stopped.
Analysis of the ethanol and sugar concentration determinations used conventional liquid chromatography. The biomass was determined using a constant temperature dry weight method. Comparative example 1 fermentation parameter oscillation behavior in free cell ultra-high concentration ethanol fermentation System
Performing ultrahigh concentration continuous ethanol fermentation in single-stage stirred fermenter, culturing free yeast cells and performing ethanol fermentation according to the above method for 0.027h -1 The dilution rate of (a) continuous fermentation medium was fed into the fermentation broth, and after a certain period of time, the fermentation parameters of the yeast cells exhibited a long-period large-amplitude oscillatory behavior (fig. 3). As shown in FIG. 3, the residual sugar, ethanol and biomass concentrations in the fermentation broth were oscillated at 179.70-92.61g/L, 71.31-31.4g/L and 7.04-2.18g/L for about 130 h.
Comparative example 2 fermentation parameter oscillation behavior in a flocculated cell ultra-high concentration ethanol fermentation System
Performing ultrahigh concentration continuous ethanol fermentation in single-stage stirred fermenter, culturing flocculated yeast cells and performing continuous ethanol fermentation for 0.027h -1 The dilution rate of (a) continuous fermentation medium was fed into the fermentation broth, and after a certain period of time, the fermentation parameters of the yeast cells exhibited a long-period large-amplitude oscillatory behavior (fig. 4). As shown in FIG. 4, the concentrations of residual sugar, ethanol and biomass in the fermentation broth were oscillated respectively at 212.21-59.73g/L, 88.41-25.37g/L and 12.24-3.17g/L for a period of about 130 h.
Example 1 attenuation of oscillations with air in free cell ultra-high concentration ethanol fermentation systems
The culture of free yeast cells and continuous ethanol fermentation were performed as described above using the continuous gas stripping ethanol fermentation apparatus shown in FIG. 1.
On the basis of carrying out ultrahigh-concentration continuous ethanol fermentation in a single-stage stirring tank, starting an air stripping device after 2-3 complete oscillation periods of fermentation parameters of yeast cells, introducing sterile air into the fermentation tank 3 through a flowmeter 5a, and carrying out ethanol air stripping while carrying out continuous ethanol fermentation. Experiments show that the concentration of ethanol in fermentation liquor is greatly reduced by stripping ethanol, the concentration of ethanol collected in a condensation collection bottle 9 in the figure 1 reaches 166.81g/L, and the yield of ethanol reaches about 25%; greatly weakens the oscillation of fermentation parameters in the continuous ultrahigh concentration fermentation process of the single-stage tank, reduces the residual sugar concentration from 140.07 +/-28.22 percent to below 1g/L, meets the industrial requirement of ethanol fermentation, and increases the biomass from 4.02 +/-58.84 percent to 32.37 +/-4.78 percent as shown in figure 5. The fermentation parameters before air stripping and the specific fermentation parameters after air stripping are shown in the following table:
| parameters of oscillation | Residual sugar g/L | Ethanol g/L | Biomass g/L |
| Qi advancing | 140.07±28.22％ | 57.54±37.78％ | 4.02±58.84％ |
| After air stripping weakening | 0.08±26.54％ | 37.03±9.77％ | 32.37±4.78％ |
Example 2 attenuation of oscillations by Nitrogen gas stripping in free cell ultra-high concentration ethanol fermentation systems
The culture of free yeast cells and continuous ethanol fermentation were performed as described above using the continuous gas stripping ethanol fermentation apparatus shown in FIG. 1.
On the basis of the ultrahigh-concentration continuous ethanol fermentation of the single-stage stirring tank, when a complete oscillation period of fermentation parameters of yeast cells appears, starting the gas stripping device, introducing sterile nitrogen into the fermentation tank 3 through the flowmeter 5a, and carrying out ethanol gas stripping while carrying out the continuous ethanol fermentation. The result shows that the concentration of ethanol in the fermentation liquor is greatly reduced by stripping the ethanol, the concentration of the ethanol collected in the condensation collection bottle 9 in the figure 1 reaches 189.07g/L, and the yield of the ethanol reaches about 25%; greatly weakens the parameter oscillation occurring in the continuous ultrahigh concentration fermentation process of the single-stage tank, the residual sugar concentration is reduced to below 1g/L from 134.53 +/-37.33 percent, the industrial requirement of the ethanol fermentation is met, and the biomass is increased to 24.33 +/-4.17 percent g/L from 3.38 +/-71.01 percent g/L, as shown in figure 6. The fermentation parameters before nitrogen stripping and the specific fermentation parameters after stripping are shown in the following table:
| parameters of oscillation | Residual sugar g/L | g/L of ethanol (fermentation liquor) | Biomass g/L |
| Qi advancing | 134.53±37.33％ | 54.86±39.03％ | 3.38±71.01％ |
| After the nitrogen gas stripping is weakened | 0.08±25.72％ | 47.36±8.15％ | 24.33±4.17％ |
Example 3 stripping weakening oscillation in a flocculation Yeast cell ultra-high concentration ethanol fermentation System Using fermentation biogas
The culture of the flocculated yeast cells and the continuous ethanol fermentation were carried out as described above using the continuous self-produced gas stripping ethanol fermentation apparatus shown in FIG. 2.
On the basis of carrying out ultrahigh-concentration continuous ethanol fermentation in a single-stage stirring tank, starting a gas stripping device after a complete oscillation period of fermentation parameters of yeast cells, introducing fermentation self-produced gas into a fermentation tank 3 through an air pump 2b, and carrying out circulating ethanol gas stripping while carrying out continuous ethanol fermentation. Experiments show that the concentration of ethanol in fermentation liquor is greatly reduced by stripping ethanol, the concentration of the ethanol collected in a condensation collection bottle 9 in the graph 2 reaches 294.36g/L, the total ethanol yield reaches 95.01 +/-5.14% g/L, and the ethanol yield reaches about 34.55% (g ethanol/g sugar); greatly weakens the parameter oscillation occurring in the continuous ultrahigh concentration fermentation process of the single-stage tank, reduces the residual sugar concentration from 121.41 +/-62.79% g/L to below 1g/L, meets the industrial requirement of ethanol fermentation, and increases the biomass from 7.76 +/-59.41% g/L to 49.05 +/-11.29% g/L as shown in figure 7. Combining examples 1, 2 and 3, it is known that in the ultra-high concentration continuous ethanol fermentation system of free cells and flocculated cells, the oscillation behavior of yeast cells can be effectively weakened by the continuous stripping of external source gas or self-generated gas, and the fermentation efficiency and the substrate conversion rate can be improved.
The fermentation parameters before and after stripping of the fermentation self-produced gas are shown in the following table:
| parameters of oscillation | Residual sugar g/L | g/L of ethanol (fermentation liquor) | Biomass g/L |
| Qi advancing | 121.41±62.79％ | 62.54±67.11％ | 7.76±59.41％ |
| After the nitrogen gas stripping is weakened | 0.09±27.02％ | 75.56±4.67％ | 49.05±11.29％ |
Example 4 attenuation of oscillations by Membrane separation in free cell ultra-high concentration ethanol fermentation systems
The culture of free yeast cells and continuous ethanol fermentation were performed as described above using the continuous gas stripping ethanol fermentation apparatus shown in FIG. 3.
On the basis of carrying out the ultrahigh-concentration continuous ethanol fermentation of the single-stage stirring tank, starting the membrane separation device after a complete oscillation period of fermentation parameters of yeast cells, introducing the fermentation liquid separated by the membrane device into the fermentation tank 3 through the peristaltic pump 2b, and carrying out membrane separation while carrying out the continuous ethanol fermentation. As the membrane is hydrophilic, ethanol molecules can permeate through the separation membrane, experiments show that the membrane separation greatly reduces the ethanol concentration in the fermentation liquor, the concentration of the ethanol collected in the condensation collection bottle 6 in the figure 3 reaches 790.15g/L, the ethanol yield reaches about 25 percent, and the parameter oscillation occurring in the continuous ultrahigh concentration fermentation process of the single-stage tank is greatly weakened; the residual sugar concentration is reduced to below 1g/L from 132.23 +/-46.39 percent g/L, the industrial requirement of ethanol fermentation is met, and the biomass is improved to 22.51 +/-4.72 percent g/L from 6.97 +/-52.43 percent g/L. The fermentation parameters before and after membrane separation are shown in the following table:
| parameters of oscillation | Residual sugar g/L | g/L of ethanol (fermentation liquor) | Biomass g/L |
| Qi advancing | 132.23±46.39％ | 56.20±32.16％ | 6.97±52.43％ |
| After membrane separation | 0.08±21.23％ | 56.62±9.63％ | 22.51±4.72％ |
Example 5 attenuation of oscillations by Membrane separation in a flocculated Yeast cell ultra-high concentration ethanol fermentation System
The culture of the flocculated yeast cells and the continuous ethanol fermentation were carried out as described above using the membrane separation-fermentation coupling apparatus shown in FIG. 3.
On the basis of carrying out ultrahigh-concentration continuous ethanol fermentation in a single-stage stirring tank, starting a membrane separation device after a complete oscillation period of fermentation parameters of yeast cells, introducing fermentation liquor separated by a membrane device into the fermentation tank through a peristaltic pump 2b, and carrying out membrane separation while carrying out continuous ethanol fermentation. Experiments show that the membrane separation greatly reduces the ethanol concentration in the fermentation liquor, the total ethanol yield reaches 90.42 +/-6.51 percent g/L, the ethanol concentration reaches about 34.80 percent (g ethanol/g sugar), the residual sugar concentration is reduced to below 1g/L from 125.78 +/-51.01 percent g/L, the industrial requirement of the ethanol fermentation is met, and the biomass is improved to 42.51 +/-9.13 percent g/L from 4.68 +/-62.04 percent g/L. In combination with examples 1-5, it is known that in the ultra-high concentration continuous ethanol fermentation system of free cells and flocculated cells, the oscillation behavior of yeast cells can be effectively weakened and the fermentation efficiency and the substrate conversion rate can be improved by gas-phase membrane separation.
The fermentation parameters before and after membrane separation are shown in the following table:
| parameters of oscillation | Residual sugar g/L | g/L of ethanol (fermentation liquor) | Biomass g/L |
| Qi advancing | 125.78±51.01％ | 65.89±61.05％ | 4.68±62.04％ |
| After membrane separation | 0.09±24.35％ | 70.21±5.36％ | 42.51±9.13％ |
Claims (8)
1.A method for producing ethanol by ultrahigh-concentration continuous fermentation of yeast cells is characterized by comprising the following steps:
(1) culturing ethanol producing bacteria;
(2) carrying out ultrahigh-concentration continuous ethanol fermentation by using ethanol production bacteria by adopting an ultrahigh-concentration continuous ethanol fermentation device;
(3) and (3) realizing the stable and continuous production of the ultrahigh-concentration ethanol by fermentation in the process (2) by using a gas stripping method or a membrane separation step. The air stripping method uses an air stripping device, and the membrane separation method uses a membrane separation device; the gas stripping device or the membrane separation device is coupled with the ultrahigh-concentration continuous ethanol fermentation device, when fermentation parameters in fermentation liquor of the fermentation device have 1-10 complete oscillation periods, the gas stripping device or the membrane separation device is started, gas is introduced into the ethanol fermentation liquor, and the oscillation behavior of ethanol production bacteria in the continuous ethanol fermentation process is weakened by a gas stripping method.
2. The method according to claim 1, wherein in the step (1), the ethanol producing bacteria is preferably industrial saccharomyces cerevisiae or genetically engineered flocculating yeast BHL 01.
3. The method of claim 2, wherein the initial sugar concentration in the continuous fermentation medium used in the ultra-high concentration continuous ethanol fermentation of step (2) is 250g/L to 350 g/L.
4. The method according to claim 3, wherein the initial sugar concentration of the continuous fermentation medium used in the ultra-high concentration continuous ethanol fermentation of step (2) is 280 g/L.
5. The method of any one of claims 1 to 4, wherein the dilution rate of the continuous fermentation medium used in the ultra-high concentration continuous ethanol fermentation of step (2) is 0.012 to 0.036h -1 。
6. The method according to claim 5, wherein the dilution rate of the continuous fermentation medium used in the ultra-high concentration continuous ethanol fermentation of step (2) is 0.027h -1 。
7. The method according to claim 3, 4, 5 or 6, wherein in the step (3), the stripping gas is introduced into the ethanol fermentation broth, and the gas flow rate per 1.5L of ethanol fermentation broth is 300L/h; after gas is introduced for about 100 hours, the ethanol concentration in the fermentation liquor is maintained at 50g/L, and the residual sugar concentration in the fermentation liquor is lower than 1 g/L; and when the introduction of the gas stripping gas is stopped, the continuous fermentation system recovers to the periodic oscillation behavior before gas stripping, and the oscillation period exceeds 100 h.
8. The method according to claim 7, wherein in the step (3), the stripping gas is introduced into the ethanol fermentation broth, and the gas flow rate is 200L/h for every 1.5L of the ethanol fermentation broth. After gas is introduced for about 100 hours, the ethanol concentration in the fermentation liquor is maintained at 50g/L, and the residual sugar concentration in the fermentation liquor is lower than 1 g/L; and when the introduction of the gas stripping gas is stopped, the continuous fermentation system recovers to the periodic oscillation behavior before gas stripping, and the oscillation period exceeds 100 h.
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