CN109988757B - Method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast - Google Patents

Abstract

The invention discloses a method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast, which comprises the steps of fixing a fiber material in a spherical grid as an immobilized medium, simultaneously filling the immobilized medium in a fermentation tank, fixing the yeast on a fiber carrier through the adsorption effect, carrying out fermentation, discharging to another fermentation tank after 2-4 batches of fermentation are finished and the alcohol content of each batch of fermentation reaches 8-10% (v/v), supplementing the material to the full tank, and continuing discharging the material to another empty tank after the alcohol content reaches 8-10% (v/v). And by analogy, performing semi-continuous fermentation. The immobilization method has the advantages of low production cost, convenient operation, stable process, high fermentation strength and high product yield, and can ensure that the immobilized yeast has lasting and higher activity.

Description

Method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast

Technical Field

The invention belongs to the technical field of industrial biology, and particularly relates to a surface immobilized yeast semi-continuous fermentation technology.

Background

Due to the over-exploitation of fossil energy and other non-renewable energy sources, the energy crisis has become the first problem to be solved in the development of the world today. The search for new alternative energy sources has become the key point of energy strategy in these years. Ethanol is not only a renewable fuel, but also can be mixed with gasoline in any proportion to relieve the pressure of the demand for petroleum, and is a good energy substitute. At present, fuel ethanol production enterprises generally adopt a large inoculation amount (20-35%) mode to carry out free fermentation, the concentration of bacteria is high (>2.5 hundred million/ml), the consumed sugar is high, and waste is caused to a certain extent; in addition, the mechanical stirring and product inhibition effect in the later fermentation period lead to higher yeast death rate, insufficient fermentation aftereffect, long fermentation period and higher residual total sugar level, thereby leading to lower starch utilization rate. Meanwhile, during the yeast expansion and fermentation process, the bacterial infection is easy to occur, thereby further reducing the utilization rate of starch.

The immobilized yeast has a long research history and has obvious advantages, for example, the immobilized yeast adsorbed by a carrier has obvious advantages of flora, so that the immobilized yeast is not easy to be infected by bacteria in the fermentation process, the fermentation speed is high, and the total sugar consumption is more thorough. In addition, the immobilized yeast can be repeatedly utilized, so that the manpower, material resources and financial resources in the yeast expansion and accompany process are saved, and the continuous fermentation is facilitated. However, the number of cases put into production is very small, and the main problems are: high cost of the immobilized medium, low mechanical strength, poor adsorption effect and mass transfer effect, easy degradation of the activity of the continuous fermentation yeast, easy contamination of the bacteria and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a surface immobilized yeast semi-continuous fermentation technology to solve the problems of poor ethanol production effect of immobilized yeast fermentation and the like in the prior art.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a surface immobilized yeast semi-continuous fermentation technology comprises the following steps:

(1) filling the spherical latticed carrier filled with the adsorption material into a layered fermentation tank with a circulating pipeline;

(2) pumping yeast wine into the fermentation tank in the step (1), circularly culturing and adsorbing and fixing the saccharomyces cerevisiae on the adsorption material of the spherical latticed carrier;

(3) discharging the yeast mash after the adsorption and fixation of the saccharomyces cerevisiae, pumping the yeast mash into a fermentation culture medium for immobilized saccharomyces cerevisiae fermentation, and replacing the fermentation culture medium with a new fermentation culture medium after the first 2-4 batches of fermentation are finished;

(4) after 2-3 batches of fermentation are stable, discharging 50-100% of the fermentation liquor when the alcohol content of each batch of fermentation reaches 8-10% (v/v), continuing the after-fermentation in another empty fermentation tank, and supplementing a new fermentation medium to the immobilized fermentation tank until the tank is full. When the alcohol content reaches 8-10% (v/v), discharging the materials, continuing to perform after-fermentation in another empty fermentation tank, and feeding the materials to a full tank. Performing semi-continuous fermentation by the same method, and replacing one bacteriostatic agent every 2-3 batches;

in the step (1), the adsorption medium is any one or a combination of several of plant fiber, animal fiber and chemical synthetic fiber.

In the step (1), the spherical meshes are made of polypropylene or polyethylene.

In the step (1), the adsorption medium is in a square or round sheet shape, and the ratio of the side length to the diameter of the spherical grid is 0.6-0.8: 1; wherein, 2-5 pieces of adsorption media are arranged in one spherical grid.

In the step (1), the height-diameter ratio of the fermentation tank is 1.5-2.5: 1; in each fermentation tank, the absolute dry amount of the adsorption medium is 2-8 g/L.

In the step (1), 2-4 layers are arranged in the fermentation tank, each layer is separated by a grating, the aperture of the grating is 0.4-0.6 times of the diameter of the spherical carrier, and the spherical carrier is arranged on the grating.

In the step (2), the yeast is an artificial semisynthetic culture medium, and any one or a combination of several of corn, cassava or aged grain saccharified liquid and molasses, and the number of yeast is 1.8-3.5 hundred million/ml;

wherein the content of the first and second substances,

the formula of the artificial semisynthetic culture medium is 25-200 g/L of glucose, 2-20 g/L of peptone, 2-10 g/L of yeast extract, 0.2-1 g/L of magnesium sulfate and Ph4.0-4.5;

in the saccharification liquid of corn, cassava or aged grain, the mass ratio of material to water is 2.5-3: 1, 0.1-0.5g/l of urea is taken as an auxiliary material;

the initial total sugar of the molasses is 30-200 g/L, and no auxiliary materials are added;

wherein the circulating culture and fixation means circulating culture at 30-35 ℃, and the circulating amount is 0.5-4 BV.

In the step (3), the completion of the seed fixation means that the residual reducing sugar is reduced to below 2g/l, and the amount of free yeast is reduced by 1.0-2.0 hundred million/ml.

In the step (3), the fermentation liquor is any one or a combination of several of an artificially prepared culture medium, corn, cassava or aged grain saccharified liquid and molasses;

wherein the content of the first and second substances,

the formula of the artificial semisynthetic culture medium is 200-250 g/L of glucose, 3-10 g/L of peptone, 3-10 g/L of yeast extract, 0.1-1 g/L of magnesium sulfate and 4.0-4.5 of pH;

in the saccharification liquid of corn, cassava or aged grain, the mass ratio of material to water is 2.5-3: 1; 0.2-0.3g/l of auxiliary material urea;

the initial total sugar of the molasses is 200-260 g/L;

the fermentation conditions of the immobilized yeast are as follows: the temperature is 32-35 ℃, and the circulation amount is 0.5-2 BV.

In the step (3), after the single batch fermentation is finished, the apparent sugar is reduced to be less than 0.3 DEG Bx, and the reducing sugar is reduced to be less than 2 g/L.

In the step (4), the stable fermentation means that the difference of the fermentation periods of 2-3 continuous batches is not more than 6 hours, and the concentration of free cells is reduced to be less than 0.9 hundred million/ml.

Pumping new fermentation conditions of a fermentation tank in the step (4): the temperature is 32-36 ℃, the circulation amount is 0.5-1BV, and the fermentation end point is the same as that of 2-3 batches of fermentation before the immobilized fermentation.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

1. the immobilization technology adopted by the invention has the advantages of low material cost and no toxicity; the surface area of the adsorption medium is large, and the adsorption effect is good;

2. the invention adopts an immobilization mode with low filling density and large gaps among carriers, and has stronger mass transfer effect compared with other filling type adsorption or crosslinking carriers. The mass transfer effect is better;

3. compared with intermittent fermentation, the semi-continuous fermentation adopted by the invention has higher efficiency, the whole fermentation period is shortened by fast reaction in an immobilized fermentation tank in the early stage, and the starch is converted by pumping into an empty tank for post-fermentation in the later stage.

4. The semi-continuous fermentation can ensure that the immobilized yeast is always in higher activity, simultaneously avoids the phenomenon of easy bacterial contamination in the continuous fermentation process, and is suitable for industrial application;

drawings

Fig. 1 is a schematic structural diagram of a spherical mesh.

FIG. 2 is a schematic flow chart of the present invention;

1 mash tank, 2 immobilization tank, 3 empty fermentation tank, 4 empty fermentation tank connecting pipes, 5 centrifugal pump, 6 cooling water inlet and 7 cooling water outlet.

Detailed Description

Example 1 semi-continuous fermentation of bamboo fiber immobilized yeast to produce ethanol as fuel using synthetic medium as fermentation raw material

Firstly, fixing cotton fibers (the thickness is 0.22cm) cut into 2.5cm side lengths on a central axis (the length of a spherical hole edge is 0.8cm) of a spherical grid made of polypropylene and having the diameter of 4.5cm as shown in figure 1 to prepare an immobilized medium; the immobilization medium was then placed in a 100L reactor with an aspect ratio of 2:1 at a loading of 2 g/L. The cultured strain was then added to the fermentor and was fixed for 42h at a flow rate of 80L/min. Until the adsorption concentration of the thalli is 2.0 hundred million/ml. The waste liquid is then discharged. Then, a fermentation medium (glucose concentration of 220g/L, fermentation broth pH4.2) was fed in at a liquid loading of 75% for cyclic fermentation at a temperature of 34 ℃ and a flow rate of 800L/h. The concentration of free yeast in the solution is reduced to about 0.8 hundred million/ml from three batches of fermentation starting, and the fermentation tends to be stable. When the alcohol content of the fermentation liquor reaches 9% (v/v), the fermentation liquor is discharged to an empty tank for post-fermentation, the average fermentation period is about 12 hours after 10 batches of fermentation, the time is shortened by 4 hours compared with the immobilized batch fermentation, the conversion rate of the sugar alcohol is equivalent, the speed is 12 times faster than that of free single batch fermentation, and the conversion rate of the sugar alcohol is improved by 6 percent.

Example 2 a cassava liquefied mash culture medium is used as a fermentation raw material to produce fuel ethanol by adopting bamboo fiber immobilized yeast semi-continuous fermentation.

Firstly, column fibers (with the thickness of 0.2cm) cut into 18cm diameter are fixed in a spherical grid (the length of a spherical hole edge is 5cm) made of polyethylene and with the diameter of 25cm as shown in the figure, and an immobilized medium is prepared; the immobilization medium was then placed in a 20 ton reactor with an aspect ratio of 2.5:1 at a loading of 2.2 g/L. The cultured strain was then added to the fermentor and fixed for 48h at a flow rate of 20 tons m 3/h. Until the concentration of the free thallus in the solution is reduced by about 1.8.2 hundred million/ml. Then preparing liquefied cassava mash: adding liquefying enzyme (15U/g dry weight of cassava) at a ratio of 1:2.4, liquefying at 85 ℃ for 2 hours, press-filtering, and discharging the immobilized seed waste liquid. Then cassava liquefied mash is added, saccharifying enzyme (180U/g dry weight of cassava) and bacteriostatic agent are added in an amount of 10ppm, the liquid loading amount is 80%, and circulating fermentation is carried out, wherein the temperature is 33 ℃, the pH value of the fermentation liquid is 4.3, and the flow rate is 10m 3/h. And (3) completely discharging the fermentation liquor to an empty tank for post-fermentation when the concentration of free thalli is reduced to 0.9 hundred million/ml and the alcohol content reaches 10% (v/v) from the fourth batch. The average fermentation period of 20 batches of fermentation is 42h, the conversion rate and the yield of the sugar alcohol are respectively 92.6 percent and 2.9g/L/h, the yield is improved by 16 percent compared with the batch fermentation, and the yield is improved by about 40 percent compared with the free fermentation.

Example 3 production of Fuel ethanol by semi-continuous fermentation of immobilized Yeast Using molasses Medium as fermentation raw Material

Firstly, the carrier preparation and immobilization method (total sugar 180g/L molasses is used as seed culture medium) the same as example 2, after the immobilization, the acidified molasses is diluted to sugar concentration 245g/L and pumped into an immobilization fermentation tank for circular fermentation, wherein the temperature is 35 ℃, the fermentation liquid pH is 4.0, and the flow rate is 10m 3/h. And (3) when the fermentation is carried out for the third batch stably, the concentration of free bacteria is 0.85 hundred million/ml, and the alcohol content reaches 10% (v/v), discharging 50% of the materials to an empty tank for post-fermentation, then feeding the materials to the original volume, continuously discharging the materials to the empty tank filled with 40% of fermentation liquor, and pumping the fermentation liquor of the immobilized fermentation tank to a second empty tank after the tank is filled. The average fermentation period of 15 batches of fermentation is 40 hours, which is 10 hours faster than that of immobilized batch fermentation, and the sugar-alcohol conversion rate is improved by 2.2 percent.

Example 4 production of Fuel ethanol by semi-continuous fermentation of immobilized Yeast Using molasses as fermentation raw Material

The carrier preparation and immobilization method (using 200g/L total sugar molasses as seed culture medium) as in example 2, after the immobilization, the acidified molasses was diluted to 230g/L sugar concentration and pumped into the immobilization fermentation tank for circular fermentation, wherein the temperature is 34 ℃, the fermentation broth pH is 4.2, and the circulation amount is 15m 3/h. And when the fermentation batch is stable in the third batch, the concentration of free bacteria is 0.9 hundred million/ml, and the alcohol content reaches 8 percent (v/v), continuously discharging (the alcohol content is maintained at 8 percent (v/v)) to an empty tank for post-fermentation, and continuously feeding. When the liquid in the later fermentation tank reaches 80%, the fermentation liquid in the immobilized fermentation tank is pumped into a second empty tank. The average fermentation period of 25 batches of fermentation is 36 hours, which is 12 hours faster than that of immobilized batch fermentation, and the sugar-alcohol conversion rate is improved by 2.6 percentage points.

Claims (4)

1. A method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast is characterized by comprising the following steps:

(1) filling the spherical latticed carrier filled with the adsorption medium into a layered fermentation tank with a circulating pipeline;

(2) pumping yeast wine into the fermentation tank in the step (1), circularly culturing and adsorbing and fixing the saccharomyces cerevisiae on the adsorption material of the spherical latticed carrier;

(3) discharging yeast mash after adsorption and fixation of the saccharomyces cerevisiae, pumping the yeast mash into a fermentation culture medium for fermentation of immobilized saccharomyces cerevisiae, and replacing the fermentation culture medium with a new fermentation culture medium after the first 2-4 batches of fermentation are finished;

(4) after 2-3 batches of fermentation are stable, discharging 50-100% of the materials when the alcohol content of each batch of fermentation reaches 8-10% in volume fraction, continuing to perform after-fermentation in another fermentation tank, supplementing a new fermentation medium to the immobilized fermentation tank until the fermentation tank is full, repeating the steps when the alcohol content reaches 8-10% in volume fraction, and performing semi-continuous fermentation in the same way;

2-4 layers are arranged in the fermentation tank, each layer is separated by a grid, the aperture of the grid is 0.4-0.6 times of the diameter of the spherical carrier, and the spherical carrier is arranged on the grid;

in the step (3), the fermentation medium is any one or a combination of several of an artificial semisynthetic medium, a corn saccharification liquid, a cassava saccharification liquid, an aged grain saccharification liquid and molasses;

wherein the content of the first and second substances,

the formula of the artificial semisynthetic culture medium is as follows: 200-250 g/L of glucose, 3-10 g/L of peptone, 3-10 g/L of yeast extract, 0.1-1 g/L of magnesium sulfate and pH 4.0-4.5;

in the corn saccharification liquid, the cassava saccharification liquid and the aged grain saccharification liquid, the mass ratio of material to water is (2.5-3): 1; 0.2-0.3g/l of auxiliary material urea;

the initial total sugar of the molasses is 200-260 g/L;

the fermentation conditions of the immobilized yeast are as follows: the temperature is 32-35 ℃, and the circulation amount is 0.5-2 BV;

in the step (3), the adsorption and fixation of the saccharomyces cerevisiae means that the residual reducing sugar is reduced to be below 2g/L, and the content of free saccharomycetes is reduced by 1.0-2.0 hundred million/ml;

in the step (3), after the single batch fermentation is finished, the apparent sugar is reduced to be less than 0.3 DEG Bx, and the reducing sugar is reduced to be less than 2 g/L;

in the step (4), the stable fermentation means that the difference of the fermentation periods of 2-3 continuous batches is not more than 6 hours, and the concentration of free cells is reduced to be below 0.9 hundred million/ml;

pumping new fermentation conditions of a fermentation tank in the step (4): the temperature is 32-36 ℃, the circulation amount is 0.5-1BV, and the fermentation end point is the same as that of 2-3 batches of fermentation before the immobilized fermentation;

in the step (1), the adsorption medium is any one or combination of several of plant fiber, animal fiber and chemical synthetic fiber;

in the step (1), the spherical latticed carrier is made of polypropylene or polyethylene.

2. The method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast according to claim 1, wherein in the step (1), the adsorption medium is in a square or round sheet shape, and the ratio of the side length to the diameter of the spherical grid is 0.6-0.8: 1; wherein, 2-5 pieces of adsorption media are arranged in one spherical grid.

3. The method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast according to claim 1, wherein in the step (1), the height-diameter ratio of the fermentation tank is 1.5-2.5: 1; in each fermentation tank, the addition amount of the adsorption medium is 2-8 g/L.

4. The method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast according to claim 1, wherein in the step (2), the yeast comprises any one or a combination of several of an artificial semi-synthetic culture medium, a saccharification liquid of corn, a saccharification liquid of cassava, a saccharification liquid of aged grain and molasses, and the number of saccharomyces cerevisiae in the yeast is 1.8-3.5 hundred million/ml;

wherein the content of the first and second substances,

the formula of the artificial semisynthetic culture medium is as follows: 25-200 g/L of glucose, 2-20 g/L of peptone, 2-10 g/L of yeast extract, 0.2-1 g/L of magnesium sulfate and pH 4.0-4.5;

in the corn saccharification liquid, the cassava saccharification liquid and the aged grain saccharification liquid, the mass ratio of material to water is (2.5-3): 1, 0.1-0.5g/l of urea is taken as an auxiliary material;

the initial total sugar of the molasses is 30-200 g/L, and no auxiliary materials are added;

wherein the circulating culture is circulating culture at 30-35 ℃, and the circulating amount is 0.5-4 BV.

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