CN111363765A - Method for preparing lactic acid by fermentation - Google Patents
Method for preparing lactic acid by fermentation Download PDFInfo
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- CN111363765A CN111363765A CN202010465504.6A CN202010465504A CN111363765A CN 111363765 A CN111363765 A CN 111363765A CN 202010465504 A CN202010465504 A CN 202010465504A CN 111363765 A CN111363765 A CN 111363765A
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Abstract
The invention relates to the field of microbial fermentation, and discloses a method for preparing lactic acid by fermentation, which is characterized by comprising the following steps: lactic acid Pediococcus (A), (B), (C)Pediococcus acidilactici) Inoculating to a fermentation medium, introducing trace amount of oxygen-containing gas into the fermentation system for pre-fermentation until the content of Pediococcus acidilactici in the fermentation system reaches OD6008-13; then stopping introducing oxygen-containing gas, and continuing fermentation until the consumption of pentose in the fermentation system reaches 45-55 wt%; wherein the fermentation medium before inoculation contains 40-100g/L glucose and 15-45g/L pentose, and the aeration quantity of the oxygen-containing gas is 0.005-0.05vvm in terms of oxygen. The method provided by the invention can be used for lactic acid fermentation, can utilize the cellulose raw material with huge capacity at present, and aims to realize low cost and high conversion of lactic acid fermentationThe scale industrial production of the rate provides reference.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for preparing lactic acid by fermentation.
Background
Polylactic acid is a biodegradable bio-based material obtained by polymerizing lactic acid as a main raw material, and is more and more concerned under the global plastic inhibition background, and the annual consumption is continuously increased. At present, the industrial production mode of lactic acid is mainly a microbial fermentation method, wherein the theoretical saccharic acid conversion rate of homotypic fermentation using lactic acid bacteria as strains can reach 100 percent, the fermentation process belongs to a facultative anaerobic or micro-oxygen consumption state, the process stirring and ventilation level is mild, and the application is wide.
When lactic acid is produced in a large scale by a microbial fermentation method, systematic optimization of middle and downstream of bioengineering, such as process improvement of strain transformation, cheap raw material substitution and separation and extraction, is required to realize the aims of yield increase, purity improvement, cost reduction, benefit improvement, thallus tolerance enhancement and the like, so that the potential is continuously mined from multiple links of a lactic acid production process, and the production efficiency is improved. At present, starch is mainly used as a raw material in the lactic acid fermentation process, glucose is obtained through liquefaction and saccharification, and the obtained glucose is used as the most commonly used lactic acid fermentation carbon source.
However, cellulose, which is a polysaccharide with the widest distribution and the largest content in nature, accounts for more than 50% of the carbon content in the plant world, and the annual output is as high as more than 1500 hundred million tons, but the cellulose is difficult to be directly utilized in lactic acid fermentation production. Therefore, it is urgently needed to develop a lactic acid fermentation process using cellulose as a raw material, which utilizes the cellulose with wide distribution and low price to achieve the purpose of low cost and high conversion rate of lactic acid production.
Disclosure of Invention
The invention provides a method for preparing lactic acid by fermentation based on the huge capacity of cellulose and aims to realize the purposes of low cost and high conversion rate of lactic acid production. By adopting the method provided by the invention, the total sugar acid conversion rate can reach 79 +/-2% in the lactic acid fermentation production process.
In order to achieve the above object, the present invention provides a method for producing lactic acid by fermentation, comprising: inoculating Pediococcus acidilactici to a fermentation culture medium, introducing oxygen-containing gas into a fermentation system for pre-fermentation until the content of Pediococcus acidilactici in the fermentation system reaches OD6008-13; and then stopping introducing oxygen-containing gas, and continuing to ferment until the consumption of the pentose in the fermentation system reaches 45-55 wt%, wherein the fermentation medium contains 30-100g/L glucose and 15-45g/L pentose before inoculation, and the ventilation amount of the oxygen-containing gas is 0.005-0.05vvm calculated by oxygen.
By adopting the technical scheme, the cellulose with low price and huge capacity can be used as a raw material to be applied to lactic acid fermentation production, so that the cost of lactic acid fermentation production is reduced, and the method provided by the invention can realize low-cost and high-conversion lactic acid fermentation at the level of small tests and medium tests, and provides reference for industrial scale lactic acid production process.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for preparing lactic acid by fermentation, which comprises the following steps: inoculating Pediococcus acidilactici to a fermentation medium, introducing oxygen-containing gas into a fermentation system for pre-fermentation after inoculation until the concentration of Pediococcus acidilactici in the fermentation system reaches OD6008-13; and then stopping introducing oxygen-containing gas, and continuing to ferment until the consumption of the pentose in the fermentation system reaches 45-55 wt%, wherein the fermentation medium contains 30-100g/L glucose and 15-45g/L pentose before inoculation, and the ventilation amount of the oxygen-containing gas is 0.005-0.05vvm calculated by oxygen. The unit "vvm" means L/(L.min).
According to a preferred embodiment of the present invention, the pediococcus acidilactici may be selected from pediococcus acidilactici with a collection number of CGMCCNo.16833. This Pediococcus acidilactici has been disclosed in CN 109536409A.
The fermentation medium before inoculation can contain 40-100g/L glucose besides pentose.
According to a preferred embodiment of the present invention, wherein the carbon source in the fermentation medium is provided by a cellulase hydrolysate.
Preferably, the addition amount of the cellulase hydrolysate in the fermentation medium is 10-30 vol% based on the fermentation system.
More preferably, the cellulose hydrolysate provides 25-50g/L of carbon element for the fermentation medium.
According to a preferred embodiment of the invention, wherein the five-carbon sugar comprises xylose and/or arabinose. The pentoses may be obtained by any conventional means in the art, for example by direct purchase of commercial products, or by processing any of the pentose-containing feedstocks.
According to a preferred embodiment of the present invention, the pentose is provided by a cellulose hydrolysate which is obtained by sequentially subjecting a cellulose raw material to acid hydrolysis, steam explosion, enzymatic hydrolysis and solid-liquid separation.
According to a preferred embodiment of the present invention, the cellulose raw material may be any cellulose raw material commonly used in the art.
Preferably, the cellulosic feedstock is selected from at least one of straw, corncob, hardwood, softwood, husk, grass, paper, leaves, cottonseed fibre, willow branches and oat hulls.
More preferably, the cellulosic feedstock is selected from at least one of straw and oat hulls.
Wherein the straw can comprise any crop straw containing cellulose. Preferably at least one of corn stover, wheat straw, cotton straw, sorghum straw, and rice straw.
According to a preferred embodiment of the present invention, the acid hydrolysis method may be any acid hydrolysis method in the art, as long as the purpose of loosening the structure of the lignocellulose and increasing the exposure degree can be achieved.
Preferably, the acidolysis is performed in the following manner: the crushed cellulose raw material is contacted with a dilute sulfuric acid solution for a period of time, and then the obtained material is dehydrated to obtain a wet material.
According to a preferred embodiment of the present invention, wherein the particle size of the pulverized cellulose raw material may be 10 to 100 mm. The concentration of the dilute sulfuric acid solution is 1-2 wt%. The weight ratio of the crushed cellulose raw material to the dilute sulfuric acid solution is 0.03-0.08: 1. the contact time is more than 0.5h, preferably 0.6-0.8 h.
According to the preferred embodiment of the present invention, the dehydration mode can be any dehydration mode commonly used in the field, as long as the purpose of removing the moisture in the contacted materials is achieved.
Preferably, the dewatering manner may be extrusion dewatering. Its advantages are simple equipment, low energy consumption and thorough dewatering treatment.
According to a preferred embodiment of the invention, wherein the purpose of said steam explosion is to achieve a separation of the components of cellulose, hemicellulose and lignin. Any steam explosion method commonly used in the art can be applied to the method provided by the present invention as long as the above-mentioned objects can be achieved.
Preferably, the steam explosion mode is as follows: and (3) placing the wet material in a closed device for steam explosion treatment to obtain a steam explosion product. The steam explosion conditions include: the temperature of the saturated steam explosion treatment is 160-170 ℃, and the time is 40-60 min.
According to a preferred embodiment of the present invention, the enzymatic hydrolysis is performed by: contacting the steam explosion product with an enzyme.
Preferably, the enzyme is selected from at least one of cellulase, hemicellulase, amylase, protease, glucoamylase and lipase.
More preferably, the amylase may include at least one of α -amylase, β -amylase, and isoamylase.
More preferably, the protease may include at least one of papain, pepsin, and trypsin.
Further preferably, in order to ensure the enzymolysis effect, the cellulase may be a complex system consisting of cellobiase, hemicellulase and endoglucanase. The ratio of each enzyme can be adjusted differently according to the characteristics of the cellulose raw material. For example, Cellic CTec3 cellulose complex enzyme available from Novixin may be used.
Further preferably, in order to ensure the enzymolysis effect, the amylase can be a composite system consisting of α -amylase and β -amylase, wherein the proportion of each enzyme can be adjusted differently according to different characteristics of the cellulose raw material, for example, a composite enzyme system prepared by mixing food grade α -amylase in summer and β -amylase in a weight ratio of 1: 0.15-0.25 can be selected.
Further preferably, in order to ensure the enzymolysis effect, the protease may be a mixture of papain, pepsin and trypsin. The ratio of each enzyme can be adjusted differently according to the characteristics of the cellulose raw material. For example, Protamax complex protease available from Novitin may be used.
According to a preferred embodiment of the present invention, among others, the solid-liquid separation is because the supernatant is used as a fermentation medium raw material, which can reduce the burden of post-treatment, thereby reducing the post-treatment cost. Any solid-liquid separation means conventional in the art may be suitable for use in the process provided herein.
Preferably, the solid-liquid separation mode comprises the following steps: at least one of plate-and-frame filtration, centrifugal separation or natural sedimentation.
According to a preferred embodiment of the present invention, wherein the nitrogen source in the fermentation medium is provided by corn steep liquor.
Preferably, the corn steep liquor is added in an amount of 3-8 vol% based on the total volume of the fermentation medium.
More preferably, the corn steep liquor provides 0.8-2.3g/L of nitrogen to the fermentation medium.
According to a preferred embodiment of the present invention, the fermentation medium may further comprise an inorganic salt, if necessary. The inorganic salt functions to maintain osmotic pressure, act as an activating substance for functional protease, and the like.
Preferably, the inorganic salt may include: phosphates, soluble magnesium salts and soluble manganese salts. Such as KH2PO4、NaH2PO4、MgSO4、MgCl2、MnSO4And MnCl2And the like.
More preferably, the inorganic salt is added in an amount of 0.25 to 1.0 g/L.
According to a preferred embodiment of the present invention, the inoculation method of pediococcus acidilactici may include: the pediococcus acidilactici is prepared into a seed solution and then inoculated into a culture medium.
Preferably, the preparation method of the seed liquid comprises the following steps: inoculating the low-temperature frozen lactococcus lactis strain into an activation culture medium for activation culture, and preparing a fresh seed solution after overnight culture. Wherein the activation medium can be any medium which can recover the low-temperature frozen pediococcus acidilactici strain. For example, it may be any one of MRS liquid medium and M17 medium. The conditions of the activation culture may include: the temperature is 33-48 ℃, and the rotation speed is 150-200 rpm.
More preferably, the concentration of Pediococcus acidilactici in the seed solution is OD600=7-12。
Further preferably, the inoculum size of the seed solution is 1-10% by volume based on the volume of the culture medium.
The inventor of the invention finds that proper amount of oxygen is introduced in the process of pre-fermentation, so that the amplification speed of the strain quantity can be accelerated, and the lactic acid fermentation period can be shortened.
According to a preferred embodiment of the present invention, the oxygen-containing gas may be any gas containing oxygen.
Preferably, the oxygen-containing gas is air for the purpose of cost reduction and ease of operation.
More preferably, the amount of the oxygen-containing gas introduced is 0.005 to 0.05vvm in terms of oxygen.
According to a preferred embodiment of the present invention, wherein the conditions of the pre-fermentation comprise: the temperature is 35-48 ℃, the pH value is 5.3-6.5, the time is 10-30h, and the stirring speed is 50-200 rpm.
More preferably, the conditions of the pre-fermentation comprise: the temperature is 37-45 ℃, the pH value is 5.5-6.2, the time is 15-25h, and the stirring speed is 70-150 rpm.
The inventors of the present invention have found in the course of research that when the consumption of five-carbon sugar in the medium reaches a specific range, the yield of lactic acid in a certain period reaches the maximum, and at this time, the yield and production intensity of lactic acid are not improved by continuing fermentation. Therefore, in order to avoid the unintentional extension of the fermentation time, the fermentation can be stopped when the amount of pentose consumed in the medium reaches a specific range, thereby shortening the fermentation period and improving the production efficiency.
According to a preferred embodiment of the present invention, the detection mode of the pentose content in the fermentation medium is high performance liquid chromatography.
According to a preferred embodiment of the present invention, when the five-carbon sugar consumption in the fermentation system is 45 to 55% by weight and the lactic acid fermentation yield is maximized, the fermentation may be stopped.
According to a preferred embodiment of the present invention, the conditions for the further fermentation and the conditions for the pre-fermentation may be the same or different. The conditions for continuing the fermentation include: the temperature is 37-45 ℃, the pH value is 5.3-6.5, the time is 30-50h, and the stirring speed is 50-200 rpm.
More preferably, the conditions for continuing the fermentation include: the temperature is 37-45 ℃, the pH value is 5.7-6.2, the time is 35-35h, and the stirring speed is 70-150 rpm.
According to a preferred embodiment of the present invention, wherein the pH during the pre-fermentation and/or fermentation process is adjusted by means of the addition of a neutralizing agent. According to a preferred embodiment of the present invention, the neutralizing agent comprises: at least one of lime milk, sodium hydroxide, calcium carbonate, ammonia water, potassium citrate and sodium citrate.
Preferably, the neutralizing agent is potassium citrate and/or sodium citrate. The inventors of the present invention found in the course of their research that when potassium citrate and/or sodium citrate is used as a neutralizing agent, it is possible to achieve the advantage of supplementing a carbon source while adjusting pH, and thus to further improve the yield of lactic acid.
According to a preferred embodiment of the present invention, wherein for the purpose of maximizing cost reduction and enhancing sugar acid conversion rate, the method may comprise: inoculating Pediococcus acidilactici to a fermentation medium, introducing oxygen-containing gas into a fermentation system for pre-fermentation after inoculation until the concentration of Pediococcus acidilactici in the fermentation system reaches OD600= 10-13; then stopping introducing the oxygen-containing gas, and continuing fermentation until the consumption of the pentose in the fermentation system reaches 48-53 wt%. Wherein the fermentation medium before inoculation contains 60-80g/L glucose and 25-35 g/L pentose, and the aeration quantity of the oxygen-containing gas is 0.008-0.05vvm calculated by oxygen.
According to a preferred embodiment of the present invention, the method may achieve a conversion rate of the sugar acid of 80% or more at a pilot scale. Wherein the pilot scale level is the level of producing lactic acid by fermenting with a fermentation tank of more than 100L.
Preferably, the fermentor has low shear, high mass transfer, and high mixing characteristics.
The invention will be further explained and illustrated by means of specific examples. It should be understood that the following examples are only for illustrating and explaining the present invention and are not to be construed as limiting the present invention.
In the following examples, the strain used is pediococcus acidilactici with a collection number of CGMCC No.16833, see in particular CN109536409A, unless otherwise specified. Storing in a refrigerator at-80 deg.C, activating glycerol tube every 1-3 months, and subculturing.
In the following examples, the preparation method of the fresh seed liquid of pediococcus acidilactici comprises: inoculating the low-temperature frozen pediococcus acidilactici strain in MRS liquid culture medium, wherein the inoculation amount of the strain is 1-10 vol%. Culturing overnight at 40 + -2 deg.C and 170 + -30 rpm to obtain fresh seed liquid.
The preparation method of the MRS liquid culture medium comprises the following steps: 200 g/L glucose, 5g/L yeast extract, 10ml/L corn steep liquor with solid content of 40%, 2 g/L sodium acetate and KH2PO40.5 g/L，MgSO4·7H2O 0.5 g/L，MnSO4·H2O0.25 g/L and Tween 1 mL/L.
In the following examples, the preparation of the cellulase hydrolysate comprises: the cellulose is prepared by crushing cellulose raw materials, carrying out acidolysis by sulfuric acid (the concentration is 1.6 weight percent), carrying out steam explosion (the condition is 160 ℃, 40 min) and carrying out enzymolysis by cellulase (CellicCTec 3 cellulose complex enzyme of Novitin), and then carrying out filter cloth extrusion. The preparation method comprises the following steps:
1. crushing the lignocellulose raw material to the granularity of 50 +/-10 mm, putting the crushed lignocellulose raw material into a screw feeder, extruding and dehydrating the crushed lignocellulose raw material to the dry matter content of 40 +/-5 weight percent to form a compact material plug, and preventing the steam in a container from leaking out while ensuring that the cellulose raw material continuously enters a treatment container;
2. treating the material plug from a screw feeder, mixing sulfuric acid into the material plug, wherein the adding amount of the sulfuric acid is 2 +/-1 wt% based on the weight of the material plug, and mixing the material plug and the sulfuric acid in an acid mixing tank to obtain an acidic cellulose raw material;
3. contacting the acidic cellulose raw material with steam in a cooking treatment container under the condition of 0.6MPa for 50 min. Then discharging the container in a continuous discharge manner;
4. and (3) washing the obtained product with water, adjusting the pH to 5 +/-0.1, heating to 50 ℃, and adding cellulase, wherein the addition amount of the cellulase is 0.15g based on the dry weight of each gram of the product. Stirring for 72h at 50 deg.C to obtain cellulose hydrolysate.
In the following examples, the corn steep liquor is a corn thick stock product from elm, a staple grain chemical industry. KH (Perkin Elmer)2PO4、MgSO4•7H2O、MnSO4•H2O was purchased from national pharmaceutical agents. The cellulase is a CellicCTec3 cellulose complex enzyme product purchased from Novoxil. When not specifically described, the fermenter used was a 2-5L fermenter model Bio-Stat from Sadolis.
In the following examples, the conversion of sugar acids was calculated as: total weight of lactic acid/total weight of initial glucose and pentose at the end of fermentation. The five-carbon sugar comprises: xylose and arabinose. The glucose content was determined in the following manner: a biosensor analyzer (model SBA-40D biosensor analyzer of biological research institute of academy of sciences of Shandong province). The high temperature sterilization is carried out at 120 deg.C for 20 min.
In the following examples, the method for measuring the content of the five-carbon sugar is high performance liquid chromatography, and the conditions refer to Liujianwei, HPLC method for measuring the composition [ J ] of the xylose mother liquor, Anhui agricultural science, v.37(05): 1881-.
In the following examples, the lactic acid was measured by high performance liquid chromatography under the following conditions:
the instrument equipment comprises: agilent Technologies 1260 Infinity II;
a detector: RID;
separating Column, Aminex HPX-87H Column 300 × 7.8.8 mm;
column temperature: 55 deg.C
Mobile phase: 0.005M sulfuric acid;
flow rate: 0.5 mL/min;
sample introduction amount: 20 mu L of the solution;
the retention time of lactic acid is about 14 min.
Example 1
A2L fermenter was used for the pilot scale experiments.
Preparing a culture medium: the cellulose raw material is straw. According to the method, 30 volume percent of cellulose hydrolysate is used as a carbon source, 8 volume percent of corn slurry is used as a nitrogen source, and inorganic salt KH is further added2PO41 g/L、MgSO4·7H2O 0.5 g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 40 ℃ for later use. Wherein, the content of the pentose is 44 g/L, and the content of the glucose is 100 g/L.
Pre-fermentation: after adding 1.2L of the medium to the fermenter, the fresh seed solution of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 5 vol% (OD concentration)600= 0.45), controlling the temperature at 42 ℃, controlling the fermentation speed at 150 r/min, starting micro-aeration and introducing air, and recording the aeration quantity as 0.05vvm by oxygen. Sodium hydroxide solution (concentration 4 wt%) was used as a neutralizer, and the pH was controlled to 6 during the fermentation.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the concentration is not less than 10, the aeration is stopped, the fermentation is continued at 42 ℃ and the fermentation rate is 150 r/min, and lactic acid fermentation is carried out while controlling the pH to 6 by adding a sodium hydroxide solution (concentration: 4% by weight) as a neutralizing agent during the fermentation.
As a result: the total fermentation time is 75h, wherein the pre-fermentation time is 15h, and the continuous fermentation time is 60 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 48%. The total conversion rate of the saccharic acid reaches 78 percent, and the yield of the lactic acid can reach 95 g/L.
Example 2
A2L fermenter was used for the pilot scale experiments.
Preparing a culture medium: the cellulose raw material is straw. According to the method, 10 volume percent of cellulose hydrolysate is used as a carbon source, 3 volume percent of corn slurry is used as a nitrogen source, and inorganic salt KH is further added2PO41 g/L、MgSO4·7H2O 0.5g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 40 ℃ for later use. Wherein the content of the pentose is 15 g/L, and the content of the glucose is 36 g/L.
Pre-fermentation: after adding 1.2L of the medium to the fermenter, the fresh seed solution of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 1 vol% (OD concentration)600= 0.09), controlling the temperature at 37 ℃, controlling the fermentation speed at 150 r/min, starting micro-aeration and introducing air, and recording the aeration quantity as 0.05vvm by oxygen. Sodium hydroxide solution (concentration 4 wt%) was used as a neutralizer, and the pH was fed and controlled to 6.2.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the pH value is not less than 10, the aeration is stopped, the fermentation is continued at 37 ℃ and the fermentation speed is 150 r/min, and lactic acid fermentation is carried out while adding sodium hydroxide solution (concentration: 4 wt%) as a neutralizer and controlling the pH value to 6.2.
As a result: the total fermentation time is 48h, wherein the pre-fermentation time is 8h, and the continuous fermentation time is 40 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 53%. The total conversion rate of the saccharic acid reaches 80 percent, and the yield of the lactic acid can reach 35 g/L.
Example 3
A2L fermenter was used for the pilot scale experiments.
Preparing a culture medium: the cellulose raw material is straw. According to 20 volume percent of cellulose hydrolysate as a carbon source, 5 volume percent of corn slurry as a nitrogen source, and further adding inorganic salt KH2PO41 g/L、MgSO4·7H2O 0.5 g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 40 ℃ for later use. Wherein the content of the pentose is 30g/L, and the content of the glucose is 70 g/L.
Pre-fermentation: after adding 1.2L of the medium to the fermenter, the fresh seed solution of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 3 vol% (OD concentration)600= 0.27), controlling the temperature at 39 ℃, controlling the fermentation speed at 150 r/min, starting micro-aeration and introducing air, and recording the aeration quantity as 0.05vvm by oxygen. Sodium hydroxide solution (concentration 4 wt%) was used as a neutralizer, and the pH was fed and controlled to 6.5.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the pH value is not less than 10, the aeration is stopped, the fermentation is continued at 39 ℃ and the fermentation speed is 100 r/min, and lactic acid fermentation is carried out while adding sodium hydroxide solution (concentration: 4 wt%) as a neutralizer and controlling the pH value to 6.5.
The experimental results are as follows: the total fermentation time is 66h, wherein the pre-fermentation time is 15h, and the continuous fermentation time is 51 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 47%. The total conversion rate of the saccharic acid reaches 79 percent, and the yield of the lactic acid can reach 66 g/L.
Example 4
A small-scale experiment was carried out using a 5L fermenter.
Preparing a culture medium: the cellulose raw material is oat hulls. According to 15 volume percent of cellulose hydrolysate serving as a carbon source and 4 volume percent of corn slurry serving as a nitrogen source, and further adding an inorganic salt KH2PO41 g/L、MgSO4·7H2O 0.5 g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 45 ℃ for later use. Wherein the content of the pentose is 20 g/L, and the content of the glucose is 55 g/L.
Pre-fermentation: after 3.0L of the medium was added to the fermenter, the fresh seed liquid of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 7 vol% (OD concentration)600= 0.63), controlling the temperature at 45 ℃, controlling the fermentation speed at 50r/min, starting micro ventilation and introducing air, and recording the ventilation quantity as 0.008vvm by oxygen. Sodium hydroxide solution (concentration 4 wt%) was used as a neutralizer, and the pH was fed and controlled to 5.3.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600When the ratio is not less than 10,stopping introducing gas, continuing to perform lactic acid fermentation at 45 deg.C and fermentation speed of 70 r/min with ammonia water solution (concentration of 14 wt%) as neutralizer under the condition of controlling pH to 5.3.
As a result: the total fermentation time is 55 h, wherein the pre-fermentation time is 12h, and the continuous fermentation time is 43 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 51%. The total conversion rate of the saccharic acid reaches 78 percent, and the yield of the lactic acid can reach 51 g/L.
Example 5
A2L fermenter was used for the pilot scale experiments.
Preparing a culture medium: the cellulose raw material is corncob. According to the method, 25 volume percent of cellulose hydrolysate is used as a carbon source, 7 volume percent of corn slurry is used as a nitrogen source, and inorganic salt KH is further added2PO41 g/L、MgSO4·7H2O 0.5 g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 40 ℃ for later use. Wherein, the content of the pentose is 35g/L, and the content of the glucose is 85 g/L.
Pre-fermentation: after adding 1.2L of the medium to the fermenter, the fresh seed solution of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 5 vol% (OD concentration)600= 0.45), controlling the temperature at 40 ℃, controlling the fermentation speed at 120 r/min, starting micro-aeration and introducing air, and recording the aeration quantity as 0.01vvm by oxygen. An aqueous ammonia solution (concentration 12% by weight) was used as a neutralizer, and the pH was controlled to 6.5 by feeding during the process.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the pH value is not less than 10, the aeration is stopped, the fermentation is continued at 40 ℃ and the fermentation speed is 120 r/min, and lactic acid fermentation is carried out while adding sodium hydroxide solution (concentration: 4 wt%) as a neutralizer and controlling the pH value to 6.5.
As a result: the total fermentation time is 75h, wherein the pre-fermentation time is 18h, and the continuous fermentation time is 57 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 52%. The total saccharic acid conversion rate reaches 81 percent, and the yield of the lactic acid can reach 84 g/L.
Example 6
A pilot-scale horizontal experiment was performed using a 100L fermenter, which was a 100L fermenter of the BIOTECH-100JS model of Shanghai Baoxing bioengineering, Inc.
Preparing a culture medium: the cellulose raw material is straw. According to 20 volume percent of cellulose hydrolysate serving as a carbon source and 6 volume percent of corn slurry serving as a nitrogen source, and further adding an inorganic salt KH2PO41 g/L、MgSO4·7H2O 0.5 g/L、MnSO4·H20.25g/L of O and the balance of water, sterilizing at high temperature, and cooling to 45 ℃ for later use. Wherein the content of the pentose is 30g/L, and the content of the glucose is 70 g/L.
Pre-fermentation: after adding 57L of the medium to the fermenter, the fresh seed liquid of Pediococcus acidilactici was inoculated into the fermenter at an inoculum size of 5 vol% (OD concentration)600= 9), controlling the temperature at 42 ℃, controlling the fermentation speed at 80 r/min, starting micro-aeration and introducing air, and recording the aeration quantity as 0.01vvm by oxygen. An ammonia solution (concentration 14%) is used as a neutralizer, and the pH is controlled to be 6.0 during the process.
And (3) continuing fermentation: when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the pH value is not less than 10, the aeration is stopped, the fermentation is continued at 42 ℃ and the fermentation speed is 50r/min, and lactic acid fermentation is carried out while feeding an aqueous ammonia solution (concentration: 14 wt%) as a neutralizer and controlling the pH value to 6.
As a result: the total fermentation time is 62 h, wherein the pre-fermentation time is 14h, and the continuous fermentation time is 48 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 46%. The total conversion rate of the saccharic acid reaches 78 percent, and the yield of the lactic acid can reach 65 g/L.
Example 7
The procedure of example 1 was followed except that a sodium citrate solution (concentration of 11% by weight) was used as the neutralizer.
As a result: the total fermentation time is 80 h, wherein the pre-fermentation time is 20 h, and the continuous fermentation time is 60 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 49%. The total conversion rate of the saccharic acid reaches 80 percent, and the yield of the lactic acid can reach 97 g/L.
Example 8
The procedure of example 1 is followed, except that the fermentation is continued in the following manner:
when the content of Pediococcus acidilactici in the fermentation system reaches OD600If the concentration is not less than 8, the aeration is stopped, the fermentation is continued at 42 ℃ and the fermentation rate is 150 r/min, and lactic acid fermentation is carried out while controlling the pH to 6 by adding a sodium hydroxide solution (concentration: 4% by weight) as a neutralizing agent during the fermentation.
As a result: the total fermentation time is 70h, wherein the pre-fermentation time is 12h, and the continuous fermentation time is 58 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 48%. The total conversion rate of the saccharic acid reaches 72 percent, and the yield of the lactic acid can reach 90 g/L.
Example 9
The procedure of example 1 is followed, except that the fermentation is continued in the following manner:
when the content of Pediococcus acidilactici in the fermentation system reaches OD600If not more than 13, the aeration was stopped, and the fermentation was continued at 42 ℃ and a fermentation rate of 150 r/min, while adding a sodium hydroxide solution (concentration: 4% by weight) as a neutralizer, and carrying out the lactic acid fermentation while controlling the pH to 6.
As a result: the total fermentation time is 72h, wherein the pre-fermentation time is 16h, and the continuous fermentation time is 56 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of pentose is 50%. The total conversion rate of the saccharic acid reaches 77 percent, and the yield of the lactic acid can reach 93 g/L.
Comparative example 1
The method of example 1 was used, except that a whole course anaerobic fermentation was used, i.e., no air was introduced during the pre-fermentation and the continuous fermentation.
As a result: the total fermentation time was 90 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 42%. The total conversion rate of the saccharic acid reaches 73 percent, and the yield of the lactic acid can reach 86 g/L.
Comparative example 2
The procedure of example 1 was followed, except that the aeration during the pre-fermentation was 0.1vvm in terms of oxygen.
As a result: the total fermentation time is 72h, wherein the pre-fermentation time is 12h, and the continuous fermentation time is 60 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 52%. The total conversion rate of the saccharic acid reaches 66 percent, and the yield of the lactic acid can reach 81 g/L.
Comparative example 3
The procedure of example 1 was followed except that the inoculum size of the inoculated pediococcus acidilactici seed solution was 1% by volume.
As a result: the total fermentation time is 90 h, wherein the pre-fermentation time is 20 h, and the continuous fermentation time is 70 h. After the fermentation is finished, no glucose residue exists, and the consumption of pentose is 33%. The total conversion rate of the saccharic acid reaches 66 percent, and the yield of the lactic acid can reach 76 g/L.
Comparative example 4
The procedure of example 1 was followed except that the fermentation was stopped when the medium had a five carbon sugar consumption of 60%.
As a result: the total fermentation time is 120 h, wherein the pre-fermentation time is 15h, and the continuous fermentation time is 105 h. After the fermentation is finished, no glucose residue exists, and the consumption of pentose is 60%. The total conversion rate of the saccharic acid reaches 69 percent, and the yield of the lactic acid can reach 87 g/L.
Comparative example 5
The procedure of example 1 was followed, except that, after the pre-fermentation, Pediococcus acidilactici biomass OD was obtained in the fermentation system600≥20 。
As a result: the total fermentation time is 68 h, wherein the pre-fermentation time is 18h, and the continuous fermentation time is 50 h. After the fermentation is finished, no glucose residue exists, and the consumption of the pentose is 49%. The total conversion rate of the saccharic acid reaches 66 percent, and the yield of the lactic acid can reach 80 g/L.
Comparative example 6
The method of example 1 was used, except that air was introduced during both the pre-fermentation and the further fermentation, i.e. air was introduced by whole aeration.
As a result: the total fermentation time is 66h, wherein the pre-fermentation time is 14h, and the continuous fermentation time is 52 h. After the fermentation is finished, no obvious glucose residue exists, and the consumption of the pentose is 56%. The total conversion rate of the saccharic acid reaches 54 percent, and the yield of the lactic acid can reach 67 g/L.
As can be seen from the comparison of the results of example 1 with comparative example 1, the preferred embodiment provided by the present invention, i.e., the manner of introducing air during the pre-fermentation stage, results in less total fermentation time and time cost savings in lactic acid production applications. Meanwhile, the consumption of pentose, the conversion rate of saccharic acid and the yield of lactic acid in the fermentation medium are all increased, and the production efficiency is improved.
As can be seen from comparison of the results of example 1 with comparative example 2, although an increase in the amount of oxygen introduced may slightly decrease the total fermentation time and the consumption of pentose in the medium, the sugar acid conversion rate and lactic acid production rate thereof are significantly decreased. From this result, it can be seen that the increased consumption of five carbon sugars was not used for lactic acid fermentation, but more of the microbial cells were supplied for growth, which was not beneficial to the production efficiency of lactic acid fermentation production.
As can be seen from the comparison of the results of example 1 and comparative example 3, when the inoculation amount of Pediococcus acidilactici is lower than the preferable range of the inoculation amount of the present invention, the total fermentation time during the lactic acid fermentation production process is increased, and the consumption amount of pentose, the sugar-acid conversion rate and the lactic acid production amount in the medium are all reduced. The overall production efficiency is reduced, which is not beneficial to industrial scale production and application.
As can be seen from the comparison of the results of example 1 with comparative example 4, when the consumption of pentose in the medium exceeds the preferable consumption range of the present invention, the sugar acid conversion rate and the lactic acid production rate are not increased correspondingly as the total fermentation time is increased. And the overall production efficiency is low, which is not beneficial to industrial scale production and application.
As can be seen by comparing the results of example 1 with those of comparative example 5, when there were too many Pediococcus acidilactici in the fermentation system at the end of the pre-fermentation stage, the total fermentation time decreased, but the pre-fermentation time increased accordingly, and the total conversion of sugar and acid and the yield of lactic acid decreased, and the overall production efficiency decreased. In combination with the increased consumption of pentose, it is likely that the nutrients in the fermentation system provide more for growth of the bacteria than for lactic acid production.
As can be seen by comparing the results of example 1 with those of comparative example 6, the total conversion of sugar and lactic acid production were reduced although the fermentation time was reduced. According to the comparison of the consumption of pentose, under the condition of whole ventilation, more nutrient components in a fermentation system supply thallus growth instead of lactic acid production, so that the production efficiency is reduced, and the industrial scale production application is not facilitated.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention. Including the combination of specific features in any suitable manner, the invention is not described in detail in order to avoid unnecessary repetition. Such simple modifications and combinations should be considered within the scope of the present disclosure as well.
Claims (11)
1. A method for preparing lactic acid by fermentation, the method comprising: lactic acid Pediococcus (A), (B), (C)Pediococcus acidilactici) Inoculating into fermentation medium, introducing oxygen-containing gas into the fermentation system for pre-fermentation until the content of Pediococcus acidilactici in the fermentation system reaches OD6008-13; then stopping introducing oxygen-containing gas, and continuing fermenting until the consumption of pentose in the fermentation system is 45-55 wt%;
wherein the fermentation medium before inoculation contains 30-100g/L glucose and 15-45g/L pentose, and the aeration quantity of the oxygen-containing gas is 0.005-0.05vvm in terms of oxygen.
2. The method according to claim 1, wherein the Pediococcus acidilactici has a accession number of CGMCC No. 16833;
and/or, the five-carbon sugar comprises xylose and/or arabinose.
3. The method according to claim 1 or 2, wherein the pentose is provided by a cellulose hydrolysate obtained by subjecting a cellulose raw material to acid hydrolysis, steam explosion, enzymatic hydrolysis and solid-liquid separation in this order.
4. The method of claim 3, wherein the cellulosic feedstock is selected from at least one of straw, corncobs, hardwoods, softwoods, husks, grasses, paper, leaves, cottonseed, willows, and oat hulls.
5. The method as claimed in claim 3, wherein the acid hydrolysis is carried out by: contacting the crushed cellulose raw material with a dilute sulfuric acid solution, and then dehydrating the obtained material to obtain a wet material;
and/or the steam explosion mode is as follows: placing the wet material in a closed device for steam explosion treatment, wherein the conditions comprise that: the temperature of the saturated steam explosion treatment is 160-170 ℃, and the time is 40-60min, so as to obtain a steam explosion product;
and/or the enzymolysis mode is as follows: contacting the steam exploded product with an enzyme selected from at least one of cellulase, hemicellulase, amylase, protease, glucoamylase, and lipase.
6. The method according to claim 5, wherein the particle size of the comminuted cellulosic feedstock is 10-100mm, the concentration of the dilute sulfuric acid solution is 1-2 wt.%, and the weight ratio of the comminuted cellulosic feedstock to the dilute sulfuric acid solution is 0.03-0.08: 1, the contact time is more than 0.5 h.
7. The method according to claim 1 or 2, wherein the pediococcus acidilactici is inoculated in such an amount that its final concentration is OD relative to the fermentation system600=0.07-1.2；
And/or, the pediococcus acidilactici is inoculated in the form of seed liquid, and the concentration of the pediococcus acidilactici in the seed liquid is OD600(ii) =7-12, and the inoculation amount of the seed liquid is 1-10 vol% relative to the fermentation system.
8. The method of claim 1, wherein the oxygen-containing gas is air.
9. The method of claim 1, wherein the conditions of the pre-fermentation comprise: the temperature is 35-48 ℃, the pH value is 5.3-6.5, the time is 10-30h, and the stirring speed is 50-200 rpm.
10. The process according to claim 1 or 9, wherein the conditions for the continued fermentation are the same or different from the conditions for the pre-fermentation, comprising: the temperature is 35-48 ℃, the pH value is 5.3-6.5, the time is 30-50h, and the stirring speed is 50-200 rpm.
11. The method of claim 1, wherein the method comprises: inoculating Pediococcus acidilactici to a fermentation medium, introducing oxygen-containing gas into a fermentation system for pre-fermentation after inoculation until the concentration of Pediococcus acidilactici in the fermentation system reaches OD600= 10-13; then stopping introducing oxygen-containing gas, and continuing fermenting until the consumption of pentose in the fermentation system reaches 48-53 wt%;
wherein the fermentation medium before inoculation contains 60-80g/L glucose and 25-35 g/L pentose, and the aeration quantity of the oxygen-containing gas is 0.008-0.05vvm calculated by oxygen.
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