Continuous fermentation method and continuous fermentation system of lactic acid

Abstract

The invention provides a continuous fermentation method and a continuous fermentation system of lactic acid, and the method comprises the following steps: inoculating strains in an aerobic fermentation tank, and carrying out fermentation culture in an aerobic environment; conveying the thalli subjected to fermentation culture into an anaerobic fermentation tank for anaerobic continuous fermentation to generate fermentation liquor containing lactic acid, centrifuging one part of the fermentation liquor in a centrifugal machine after sugar consumption of the fermentation liquor in a saccharification tank, discharging aged thalli, and recovering supernate after fermentation; the other part of fermentation liquor is filtered through a ceramic membrane, thalli are recycled to an anaerobic fermentation tank, supernate obtained through centrifugation and ceramic membrane separation is dissociated through an electrodialysis bipolar membrane to obtain a sodium hydroxide solution and a lactic acid solution, and the sodium hydroxide solution serves as a neutralizing agent and is returned to the anaerobic fermentation procedure. According to the method disclosed by the invention, sodium hydroxide is used as a neutralizing agent to generate sodium lactate, and a continuous fermentation mode is adopted, so that inhibition of sodium lactate on thallus activity is relieved, and the production efficiency of lactic acid is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid fermentation, and in particular to a continuous fermentation method and a continuous fermentation system using sodium salt as a neutralizer in the lactic acid fermentation process. Background Art

[0002] Lactic acid is a naturally occurring organic acid with numerous applications, particularly as a monomer for the biodegradable material polylactic acid. However, since lactic acid production primarily relies on microbial fermentation, the traditional calcium salt method is the primary method. This method is energy-intensive, consumes large amounts of sulfuric acid, and produces phosphorus-containing calcium sulfate waste. Improvements are urgently needed.

[0003] The sodium salt method produces lactic acid with sodium hydroxide as the neutralizing agent, and the sodium ions can be recycled during the fermentation process, without waste residues such as calcium sulfate. However, sodium lactate has a higher osmotic pressure than calcium lactate, which means that the accumulation of sodium lactate in the fermentation broth will gradually reduce the activity of the strain and cause a decrease in the specific lactic acid production rate, resulting in a rapid decrease in yield.

[0004] Therefore, there is an urgent need for new methods and related systems for continuous lactic acid fermentation that can effectively reduce the inhibitory effect of sodium lactate, enhance bacterial activity, thereby increasing the acid production rate and improving the purity of lactic acid. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a continuous lactic acid fermentation method and a continuous lactic acid fermentation system.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a continuous fermentation method for lactic acid, comprising:

[0008] The strain is inoculated into an aerobic fermentation tank and fermented under an aerobic environment;

[0009] The bacterial cells after the fermentation culture are transported to an anaerobic fermentation tank for anaerobic continuous fermentation to produce a fermentation liquid containing lactic acid. After the fermentation liquid consumes sugar in a saccharolysis tank, a portion of the fermentation liquid is centrifuged in a centrifuge to remove aged bacterial cells and recover the fermented supernatant. The other portion of the fermentation liquid is filtered through a ceramic membrane and the bacterial cells are recovered to the anaerobic fermentation tank. The supernatant obtained by centrifugation and ceramic membrane separation is dissociated through an electrodialysis bipolar membrane to obtain a sodium hydroxide solution and a lactic acid solution. The sodium hydroxide solution is returned to the anaerobic fermentation process as a neutralizer.

[0010] Wherein, in the anaerobic continuous fermentation, the carbon source addition rate Q of the control flow to the anaerobic fermentation tank is controlled. g and bacterial flow rate Q cTo control the residual sugar concentration in the anaerobic fermentation tank to 1-2g / L, OD 600 25 to 27;

[0011] In the ceramic membrane separation process, the output of the supernatant is controlled o To maintain the concentration of lactic acid in the anaerobic fermentation tank at 60-72 g / L, preferably 60-62 g / L.

[0012] The continuous fermentation method of the present invention controls the residual sugar concentration in the anaerobic fermentation tank to 1-2 g / L to avoid insufficient substrate, which leads to a decrease in lactic acid yield, and also prevents excessive concentration of miscellaneous acids in the fermentation broth. As is well known to those skilled in the art, sodium lactate has an inhibitory effect on bacterial growth. The lower the concentration of lactic acid in the anaerobic fermentation tank, the lower the inhibitory effect and the higher the lactic acid yield. However, if the lactic acid concentration is too low, the cost of lactic acid separation will increase significantly. Researchers of the present invention have found that controlling the lactic acid concentration in the anaerobic fermentation tank to 60-72 g / L can maximize the lactic acid yield from an economic perspective.

[0013] In a specific embodiment of the continuous fermentation method of the present invention, at the initial stage of fermentation, Escherichia coli is inoculated into the culture medium in the aerobic fermentation tank, and sodium hydroxide solution is added, and the culture is cultured under an aerobic environment until the OD 600 Between 20 and 35; in the initial accumulation stage of lactic acid, maintain OD 600 The temperature is 20 to 35 and the material is continuously added to the anaerobic fermentation tank. When the liquid level of the anaerobic fermentation tank is higher than 20%, stirring is turned on, and alkali solution and carbon source are continuously added to the anaerobic fermentation tank through the alkali buffer tank and the sugar feeding tank. In some specific embodiments, when the lactic acid concentration in the anaerobic fermentation tank is higher than 40g / L, the fermentation liquid containing lactic acid is added to the saccharolysis tank; in some specific embodiments, when the liquid level in the saccharolysis tank is higher than 40% and the lactic acid concentration in the anaerobic fermentation tank is greater than 60g / L, steady-state continuous fermentation is entered. In the steady-state continuous fermentation stage of lactic acid, after the bacteria are generated by the aerobic fermentation tank, they are continuously transported to the anaerobic fermentation tank, and the fermentation liquid containing lactic acid is continuously produced by fermentation in the anaerobic continuous fermentation tank.

[0014] In some specific embodiments, in the anaerobic continuous fermentation, the carbon source feed rate Q in the anaerobic fermentation tank is g =3.75K g ×C a ×Q a , where C a is the mass content of sodium hydroxide in the anaerobic fermentation tank, Q a is the flow rate of sodium hydroxide in the anaerobic fermentation tank, K g is the mixed acid coefficient in the anaerobic fermentation tank, K g =1+C Sa / C La1 , CSa is the concentration of miscellaneous acids in the anaerobic fermentation tank, C La1 is the lactic acid concentration in the anaerobic fermentation tank. In some specific embodiments, the operating parameters in the anaerobic fermentation tank can be measured by instruments to obtain real-time data, for example, the flow rate Q of sodium hydroxide in the anaerobic fermentation tank a It can be measured using a flow meter installed at the discharge port of the alkali buffer tank.

[0015] In the specific embodiment of the continuous fermentation method of the present invention, during the anaerobic continuous fermentation process, the cell flow rate in the anaerobic fermentation tank is Qc=V×Kc×exp(1-62 / C La1 ), where V is the weight of the fermentation liquid in the anaerobic fermentation tank, K c is the dilution rate of bacteria supplementation in the anaerobic fermentation tank, ranging from 0.08 to 0.12, preferably from 0.10 to 0.11, and exp is the natural index.

[0016] In the ceramic membrane separation process, the extraction amount of supernatant Q o =K o ×(Q g +Q a ), K o is the extraction ratio coefficient, K o =exp(1-62 / C La2 ), where C La2 is the lactic acid concentration in the ceramic membrane, Q g is the carbon source replenishment rate in the anaerobic fermentation tank, Q a is the flow rate of sodium hydroxide in the anaerobic fermentation tank. In some specific embodiments, the lactic acid concentration C in the anaerobic fermentation tank La1 and the lactic acid concentration C in the ceramic membrane La2 are numerically equal.

[0017] In some specific embodiments, the various operating parameters in the ceramic membrane separation process can be measured by instruments to obtain real-time data, for example, the concentration of lactic acid in the ceramic membrane C La2 Online near-infrared instrument can be used for real-time measurement.

[0018] The bacterial strain in the aerobic fermentation tank is selected from Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris;

[0019] The culture medium in the aerobic fermentation tank is a nitrogen source culture medium, which includes: 10g / L yeast powder, 2-5g / L urea, 2-3g / L KCl, 0.01-0.2g / L CaCl2·2H2O, 2-8g / L KH2PO4, 2-8g / L Na2HPO4·12H2O, 0.2-2g / L MgSO4 and 1-5mL / L trace elements;

[0020] Wherein, the trace elements include 0.8g / L vitamin B1, 0.1g / L FeCl3·6H2O, 0.8g / L ZnSO4·7H2O, 0.1g / L CoCl2·6H2O, 0.075g / L CuSO4·5H2O, 0.75g / LMnSO4·H2O, 0.15g / L H3BO3 and 0.05g / L Na2MoO4·2H2O; in some preferred embodiments, the OD in the aerobic fermentation tank is 600 Maintain between 20 and 35, for example, 22, 25, 28, 30, 32.

[0021] In some specific embodiments, the anaerobic continuous fermentation is carried out at 42-45° C., pH 6.8-7.1, and a stirring rate of 250-350 rpm;

[0022] The carbon source added to the anaerobic fermentation tank is selected from at least one of glucose, starch or sucrose; the concentration of the nitrogen source in the anaerobic fermentation tank is 1.0-1.2 g / L, for example, 1.1 g / L.

[0023] In the continuous fermentation method of the present invention, the lactic acid fermentation broth consumes sugar in the fermentation broth in the saccharolysis tank. In some specific embodiments, the residence time of the fermentation broth in the saccharolysis tank is 0.5 to 1 hour, for example, 0.7 hours, 0.8 hours; the carbon source concentration at the discharge port of the saccharolysis tank is less than 1 g / L, for example, 0.5 g / L, 0.2 g / L; the nitrogen source concentration is less than 0.2 g / L, for example, 0.10 g / L, 0.05 g / L.

[0024] In some specific embodiments, the residence time of the fermentation liquid in the centrifuge is 1 to 10 s, for example, 3 s, 5 s, 8 s; the feed amount of the centrifuge is equal to the amount of bacterial flow added in the anaerobic fermentation tank; in some specific embodiments, the solid content at the discharge port of the centrifuge is 1 to 5 wt%, for example, 2 wt%, 3 wt%.

[0025] In the continuous fermentation method of the present invention, the supernatant obtained by centrifugation and ceramic membrane separation is subjected to dissociation in an electrodialysis bipolar membrane device; specifically, the electrodialysis bipolar membrane device is a dual-chamber electrodialysis device in which cationic homogeneous membranes and anionic bipolar membranes are alternately arranged. In some specific embodiments, the membrane stack of the electrodialysis bipolar membrane device is composed of five alternating anionic bipolar membranes and six cationic homogeneous membranes.

[0026] In a second aspect, the present invention provides a continuous fermentation system for lactic acid, which is applied to the above-mentioned continuous fermentation method and includes an aerobic fermentation tank, a sugar supplement tank, an alkali buffer tank, an anaerobic fermentation tank, a saccharolysis tank, a ceramic membrane, a centrifuge, and an electrodialysis bipolar membrane;

[0027] Among them, the discharge port of the aerobic fermentation tank, the discharge port of the sugar supplement tank and the discharge port of the alkali buffer tank are respectively controllably connected to the feed port of the anaerobic fermentation tank, the discharge port of the anaerobic fermentation tank is connected to the feed port of the saccharolysis tank, the discharge port of the saccharolysis tank is respectively controllably connected to the feed port of the ceramic membrane and the feed port of the centrifuge, the supernatant discharge port of the ceramic membrane and the supernatant discharge port of the centrifuge are controllably connected to the feed port of the electrodialysis bipolar membrane, and the bacterial cell outlet of the ceramic membrane is connected to the feed port of the anaerobic fermentation tank.

[0028] The above technical solution has the following technical effects:

[0029] The fermentation method of the invention uses sodium hydroxide as a neutralizer to generate sodium lactate, and adopts a continuous fermentation mode to eliminate the inhibition of bacterial activity by sodium lactate, thereby improving the production efficiency of lactic acid.

[0030] The fermentation method of the present invention adopts a mathematical model to control key parameters of the anaerobic continuous fermentation process, such as residual sugar, OD and lactic acid concentration in the anaerobic fermentation process, thereby further enabling the continuous fermentation process to operate stably and efficiently for a long time.

[0031] In the lactic acid continuous fermentation system of the present invention, sodium ions are recycled and no phosphorus, calcium sulfate or other waste residue is generated. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : A specific embodiment of a continuous lactic acid fermentation system provided by the present invention;

[0033] Figure 2 : Average production rate diagram of the continuous lactic acid fermentation system in Example 3 of the present invention;

[0034] Among them, 10. aerobic fermentation tank, 11. sugar supplement tank, 12. alkali buffer tank, 13. anaerobic fermentation tank, 14. saccharolysis tank, 15. ceramic membrane, 16. centrifuge, 17. electrodialysis bipolar membrane, 18. pump. DETAILED DESCRIPTION

[0035] The biological experimental methods not specifically described in the following examples were all performed according to conventional methods or in accordance with the kits and product instructions. The reagents and biological materials used in the specific examples were all commercially available unless otherwise specified.

[0036] In the following embodiments, the Figure 1The continuous lactic acid fermentation system shown includes: an aerobic fermentation tank 10, a sugar supplement tank 11, an alkali buffer tank 12, an anaerobic fermentation tank 13, a saccharolysis tank 14, a ceramic membrane 15, a centrifuge 16, and an electrodialysis bipolar membrane 17. The discharge ports of the aerobic fermentation tank 10, the sugar supplement tank 11, and the alkali buffer tank 12 are controllably connected to the feed port of the anaerobic fermentation tank 13, respectively. The discharge port of the anaerobic fermentation tank 13 is connected to the feed port of the saccharolysis tank 14, and the discharge port of the saccharolysis tank 14 is controllably connected to the feed port of the ceramic membrane 15 and the feed port of the centrifuge 16, respectively. The supernatant discharge ports of the ceramic membrane 15 and the supernatant discharge ports of the centrifuge 16 are controllably connected to the feed port of the electrodialysis bipolar membrane 17, and the bacterial cell outlet of the ceramic membrane 15 is connected to the feed port of the anaerobic fermentation tank 13. Pumps 18 are provided between the discharge port of the anaerobic fermentation tank 13 and the feed port of the saccharolysis tank 14, between the discharge port of the saccharolysis tank 14 and the feed port of the ceramic membrane 15, and between the discharge port of the saccharolysis tank 14 and the feed port of the centrifuge 16 to control the material flow rate in each pipeline.

[0037] Example 1

[0038] Aerobic fermentation stage: Escherichia coli is inoculated into the culture medium in the aerobic fermentation tank, sodium hydroxide solution is added, and fermentation is carried out in an aerobic environment until OD 600 The temperature is 20-35°C, and the whole operation temperature is 37°C. The culture medium used is 10 g / L yeast powder, 5 g / L urea, 3 g / L KCl, 0.1 g / L CaCl2·2H2O, 5 g / L KH2PO4, 5 g / L Na2HPO4·12H2O, 1 g / LMgSO4 and 2 mL / L trace elements, which include 0.8 g / L vitamin B1, 0.1 g / L FeCl3·6H2O, 0.8 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.075 g / L CuSO4·5H2O, 0.75 g / L mnSO4·H2O, 0.15 g / L H3BO3 and 0.05 g / L Na2MoO4·2H2O.

[0039] Initial lactic acid accumulation stage: The bacteria after aerobic fermentation culture are transported to the anaerobic fermentation tank for anaerobic continuous fermentation. When the liquid level in the anaerobic fermentation tank is higher than 20%, stirring is started, and glucose is continuously added to the anaerobic fermentation tank through the sugar feeding tank, and sodium hydroxide solution is added to the anaerobic fermentation tank through the alkali buffer tank; when the lactic acid concentration is higher than 40g / L, materials are continuously transported to the saccharolysis tank. When the liquid level in the saccharolysis tank is higher than 40% and the lactic acid concentration in the anaerobic fermentation tank is greater than 60g / L, the steady-state continuous fermentation process is entered.

[0040] Steady-state continuous fermentation stage: The bacteria cultured by aerobic fermentation are continuously transported to the anaerobic fermentation tank, and anaerobic continuous fermentation is carried out under the conditions of 42°C, pH 7.0, and stirring rate of 250rpm. The glucose feed rate Q added to the anaerobic fermentation tank is controlled. g and bacterial flow rate Q c , so that the residual sugar concentration in the anaerobic fermentation tank is 1g / L, OD 600 Specifically, the glucose replenishment rate Q in the anaerobic fermentation tank is adjusted according to the following parameters: g , Q g =3.75K g ×C a ×Q a , where C a Q is the mass content of sodium hydroxide in the anaerobic fermentation tank, in g / kg; a is the flow rate of sodium hydroxide in the anaerobic fermentation tank, in kg / h; K g is the mixed acid coefficient in the anaerobic fermentation tank, K g =1+C Sa / C La1 , C Sa is the concentration of miscellaneous acids in the anaerobic fermentation tank, in g / kg, C La1 is the lactic acid concentration in the anaerobic fermentation tank, in g / kg. By collecting the real-time values ​​of the above parameters in the anaerobic fermentation tank in real time, the flow rate of sodium hydroxide in the anaerobic fermentation tank is adjusted to control the glucose feeding rate into the anaerobic fermentation tank. At the same time, the cell flow rate Q is adjusted according to the parameters in the following formula: c , Q c =V×K c ×exp(1-62 / C La1 ), where V is the weight of the fermentation liquid in the anaerobic fermentation tank, in kg, K c is the dilution rate of bacteria supplementation in the anaerobic fermentation tank, K c Taken from 0.08 to 0.12 hours -1 , exp is the natural exponential.

[0041] After anaerobic fermentation, the fermentation liquid containing lactic acid is retained in the saccharolysis tank for 5 hours to consume sugar. The carbon source concentration at the discharge port of the saccharolysis tank is 0.5 g / L, and the nitrogen source concentration is 2 g / L. A portion of the fermentation liquid is discharged into a butterfly centrifuge for centrifugal treatment to obtain the fermented supernatant and discharge the aged bacteria; the other portion enters the ceramic membrane filtration, and the filtered bacteria are recovered to the anaerobic fermentation tank. The supernatant of the ceramic membrane and the supernatant obtained after centrifugation enter the electrodialysis bipolar membrane device for dissociation; wherein, the supernatant extraction amount of the ceramic membrane is Q o =K o ×(Q g +Q a), K o is the extraction ratio coefficient, K o =exp(1-62 / C La2 ), where C La2 is the lactic acid concentration in the ceramic membrane, in g / kg, Q g is the glucose feeding rate in the anaerobic fermentation tank, in kg / h, Q a The flow rate of sodium hydroxide in the anaerobic fermentation tank is kg / h. The supernatant output Q of the ceramic membrane is controlled o , so that the concentration of lactic acid in the anaerobic fermentation tank is maintained at 67g / L.

[0042] After dissociation by an electrodialysis bipolar membrane device, crude lactic acid product and sodium hydroxide solution are obtained, and the sodium hydroxide solution is returned to the alkali buffer tank, so that the continuous fermentation of lactic acid can run stably.

[0043] As shown in Table 1 below, during continuous fermentation for 72 hours, the dilution rate of bacteria in the anaerobic fermentation tank was K. c Take 0.10h -1 The conversion rate of glucose was 89.1%, and the average production rate of lactic acid was 8.81 g / L·h. The conversion rate and the average production rate of lactic acid were optimal.

[0044] Table 1 Different bacterial supplementation dilution rates K c Effect on the average production rate of lactic acid

[0045] <![CDATA[K c (h -1 )]]> Average lactic acid production rate (g / L·h) Glucose conversion rate (%) 0.08 7.86 89.8 0.10 8.81 89.1 0.12 9.02 86.1

[0046] Example 2

[0047] Aerobic fermentation stage: Escherichia coli is inoculated into the culture medium in the aerobic fermentation tank, sodium hydroxide solution is added, and fermentation is carried out in an aerobic environment until OD 600 The temperature is 20-35°C, and the whole operation temperature is 37°C. The culture medium consists of 10 g / L yeast powder, 5 g / L urea, 3 g / L KCl, 0.1 g / L CaCl2·2H2O, 5 g / L KH2PO4, 5 g / L Na2HPO4·12H2O, 1 g / LMgSO4 and 2 mL / L trace elements, which include 0.8 g / L vitamin B1, 0.1 g / L FeCl3·6H2O, 0.8 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.075 g / L CuSO4·5H2O, 0.75 g / L mnSO4·H2O, 0.15 g / L H3BO3 and 0.05 g / L Na2MoO4·2H2O.

[0048] Initial lactic acid accumulation stage: The bacteria after aerobic fermentation culture are transported to the anaerobic fermentation tank for anaerobic continuous fermentation. When the liquid level in the anaerobic fermentation tank is higher than 20%, stirring is started, and glucose is continuously added to the anaerobic fermentation tank through the sugar feeding tank, and sodium hydroxide solution is added to the anaerobic fermentation tank through the alkali buffer tank; when the lactic acid concentration is higher than 40g / L, materials are continuously transported to the saccharolysis tank. When the liquid level in the saccharolysis tank is higher than 40% and the lactic acid concentration in the anaerobic fermentation tank is greater than 60g / L, the steady-state continuous fermentation process is entered.

[0049] Steady-state continuous fermentation stage: The bacteria cultured by aerobic fermentation are continuously transported to the anaerobic fermentation tank, and anaerobic continuous fermentation is carried out at 42°C, pH 6.8-7.1, and a stirring rate of 250rpm. The glucose feed rate Q added to the anaerobic fermentation tank is controlled. g and bacterial flow rate Q c , so that the residual sugar concentration in the anaerobic fermentation tank is 2g / L, OD 600 Specifically, the glucose replenishment rate Q in the anaerobic fermentation tank is adjusted according to the following parameters: g , Q g =3.75K g ×C a ×Q a , where C a Q is the mass content of sodium hydroxide in the anaerobic fermentation tank, in g / kg, a is the flow rate of sodium hydroxide in the anaerobic fermentation tank, in kg / h, K g is the mixed acid coefficient in the anaerobic fermentation tank, K g =1+C Sa / C La1 , C Sa is the concentration of miscellaneous acids in the anaerobic fermentation tank, in g / kg, C La1 is the lactic acid concentration in the anaerobic fermentation tank, in g / kg. By collecting the above parameter values ​​in the anaerobic fermentation tank in real time, the flow rate of sodium hydroxide in the anaerobic fermentation tank is adjusted to control the glucose feeding rate into the anaerobic fermentation tank. At the same time, the cell flow rate Q is adjusted according to the parameters in the following formula: c , Q c =V×K c ×exp(1-62 / C La1 ), where V is the weight of the fermentation liquid in the anaerobic fermentation tank, in kg; C La1 is the lactic acid concentration in the anaerobic fermentation tank, in g / kg; K c is the dilution rate of bacteria supplementation in the anaerobic fermentation tank, K c Taken from 0.10h -1 , exp is the natural exponential.

[0050] After anaerobic fermentation, the fermentation liquid containing lactic acid is retained in the saccharolysis tank for 5 hours to consume sugar. The carbon source concentration at the discharge port of the saccharolysis tank is 0.5 g / L, and the nitrogen source concentration is 2 g / L. A portion of the fermentation liquid is discharged into a butterfly centrifuge for centrifugal treatment to obtain the fermented supernatant and discharge the aged bacteria; the other portion is filtered through a ceramic membrane, and the filtered bacteria are recovered to the anaerobic fermentation tank. The supernatant of the ceramic membrane and the supernatant obtained after centrifugation are entered into an electrodialysis bipolar membrane device for dissociation; wherein, the supernatant extraction amount of the ceramic membrane is Q o =K o ×(Q g +Q a ), K o is the extraction ratio coefficient, K o =exp(1-62 / C La2 ), where C La2 is the lactic acid concentration in the ceramic membrane, in g / kg, Q g is the glucose feeding rate in the anaerobic fermentation tank, in kg / h, Q a The flow rate of sodium hydroxide in the anaerobic fermentation tank is kg / h. The supernatant output Q of the ceramic membrane is controlled o , thereby controlling the lactic acid concentration C in the ceramic membrane La2 , that is, the concentration of lactic acid in the anaerobic fermentation tank C La1 .

[0051] After dissociation by an electrodialysis bipolar membrane device, crude lactic acid product and sodium hydroxide solution are obtained, and the sodium hydroxide solution is returned to the alkali buffer tank, so that the continuous fermentation of lactic acid can run stably.

[0052] As shown in Table 2 below, after 72 hours of continuous fermentation, the amount of supernatant extracted through the ceramic membrane is Q o , so that the concentration of lactic acid in the anaerobic fermentation tank is maintained at 60-62g / L, the average production rate of lactic acid is the highest and the conversion rate of glucose is the highest.

[0053] Table 2 Different lactic acid concentrations C La1 Effect on the average production rate of lactic acid

[0054] Lactic acid concentration (g / L) Average lactic acid production rate (g / L·h) Glucose conversion rate (%) 60～62 8.92 89.3 65～67 8.05 89.1 70～72 5.06 78.2

[0055] Example 3

[0056] Aerobic fermentation stage: Escherichia coli is inoculated into the culture medium in the aerobic fermentation tank, sodium hydroxide solution is added, and fermentation is carried out in an aerobic environment until OD 600The temperature is 20-35°C, and the whole operation temperature is 37°C. The culture medium consists of 10 g / L yeast powder, 5 g / L urea, 3 g / L KCl, 0.1 g / L CaCl2·2H2O, 5 g / L KH2PO4, 5 g / L Na2HPO4·12H2O, 1 g / LMgSO4 and 2 mL / L trace elements, which include 0.8 g / L vitamin B1, 0.1 g / L FeCl3·6H2O, 0.8 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.075 g / L CuSO4·5H2O, 0.75 g / L mnSO4·H2O, 0.15 g / L H3BO3 and 0.05 g / L Na2MoO4·2H2O.

[0057] Initial accumulation stage of lactic acid: After fermentation and cultivation, the bacteria are transported to the anaerobic fermentation tank for anaerobic continuous fermentation. When the liquid level in the anaerobic fermentation tank is higher than 20%, stirring is started, and glucose and sodium hydroxide solution are continuously added to the anaerobic fermentation tank through the sugar feeding tank and the alkali buffer tank respectively. When the lactic acid concentration is higher than 40g / L, the saccharolysis tank is continuously added. When the liquid level in the saccharolysis tank is higher than 40% and the lactic acid concentration in the anaerobic fermentation tank is greater than 60g / L, the steady-state continuous fermentation process is entered.

[0058] Steady-state continuous fermentation stage: The bacteria cultured by aerobic fermentation are continuously transported to the anaerobic fermentation tank, and anaerobic continuous fermentation is carried out at 42°C, pH 6.8-7.1, and a stirring rate of 250rpm. The glucose feed rate Q added to the anaerobic fermentation tank is controlled. g and bacterial flow rate Q c , so that the residual sugar concentration in the anaerobic fermentation tank is 2g / L, OD 600 Specifically, the glucose replenishment rate Q in the anaerobic fermentation tank is adjusted according to the following parameters: g , Q g =3.75K g ×C a ×Q a , where C a Q is the mass content of sodium hydroxide in the anaerobic fermentation tank, in g / kg, a is the flow rate of sodium hydroxide in the anaerobic fermentation tank, in kg / h, K g is the mixed acid coefficient in the anaerobic fermentation tank, K g =1+C Sa / C La1 , C Sa is the concentration of miscellaneous acids in the anaerobic fermentation tank, in g / kg, C La1is the lactic acid concentration in the anaerobic fermentation tank, in g / kg. The glucose feeding rate in the anaerobic fermentation tank is controlled by adjusting the flow rate of sodium hydroxide in the anaerobic fermentation tank. At the same time, the cell flow rate Q is adjusted according to the parameters in the following formula: c , Q c =V×K c ×exp(1-62 / C La1 ), where V is the weight of the fermentation liquid in the anaerobic fermentation tank, in kg, K c is the dilution rate of bacteria supplementation in the anaerobic fermentation tank, K c Taken from 0.10h -1 , exp is the natural exponential.

[0059] After anaerobic fermentation, the fermentation liquid containing lactic acid is retained in the saccharolysis tank for 5 hours to consume sugar. The carbon source concentration at the discharge port of the saccharolysis tank is 0.5 g / L, and the nitrogen source concentration is 2 g / L. A portion of the fermentation liquid is discharged into a butterfly centrifuge for centrifugal treatment to obtain the fermented supernatant and discharge the aged bacteria. The other portion is filtered through a ceramic membrane. The filtered bacteria are recovered to the anaerobic fermentation tank. The supernatant of the ceramic membrane and the supernatant obtained after centrifugation are separated into an electrodialysis bipolar membrane device. The supernatant extraction amount of the ceramic membrane is Q o =K o ×(Q g +Q a ), K o is the extraction ratio coefficient, K o =exp(1-62 / C La2 ), where C La2 is the lactic acid concentration in the ceramic membrane, in g / kg, Q g is the glucose feeding rate in the anaerobic fermentation tank, in kg / h, Q a The flow rate of sodium hydroxide in the anaerobic fermentation tank is in kg / h. The amount of supernatant produced through the ceramic membrane is Q o , so that the concentration of lactic acid in the anaerobic fermentation tank is 67g / L.

[0060] After dissociation by an electrodialysis bipolar membrane device, crude lactic acid product and sodium hydroxide solution are obtained, and the sodium hydroxide solution is returned to the alkali buffer tank, so that the continuous fermentation of lactic acid can run stably.

[0061] As attached Figure 2 As shown, after 216 hours of continuous fermentation, the highest lactic acid yield was 9.09 g / L·h, the lowest yield was 7.59 g / L·h, the average production rate during the entire fermentation cycle was 8.29 g / L·h, and the glucose conversion rate was 87.2%, which had good economic effects.

[0062] Comparative Example 1

[0063] Aerobic fermentation stage: Escherichia coli is inoculated into the culture medium in the aerobic fermentation tank, sodium hydroxide solution is added, and fermentation is carried out in an aerobic environment until OD 600 The temperature is 20-35°C, and the whole operation temperature is 37°C. The culture medium is 10 g / L yeast powder, 5 g / L urea, 3 g / L KCl, 0.1 g / L CaCl2·2H2O, 5 g / L KH2PO4, 5 g / L Na2HPO4·12H2O, 1 g / LMgSO4 and 2 mL / L trace elements, which include 0.8 g / L vitamin B1, 0.1 g / L FeCl3·6H2O, 0.8 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.075 g / L CuSO4·5H2O, 0.75 g / L mnSO4·H2O, 0.15 g / L H3BO3 and 0.05 g / L Na2MoO4·2H2O.

[0064] Lactic acid accumulation stage: The bacteria after aerobic fermentation culture are transported to the anaerobic fermentation tank for anaerobic continuous fermentation. When the liquid level in the anaerobic fermentation tank is higher than 20%, stirring is started, and glucose is continuously added to the anaerobic fermentation tank through the sugar feeding tank, and sodium hydroxide solution is added to the anaerobic fermentation tank through the alkali buffer tank; when the lactic acid concentration is higher than 40g / L, the material is continuously transported to the saccharolysis tank. When the lactic acid concentration in the anaerobic fermentation tank is greater than 60g / L, the fermentation is stopped.

[0065] After 46 hours of continuous fermentation, the highest lactic acid yield was 8.8 g / L·h, the lowest yield was 7.1 g / L·h, the average lactic acid yield during the entire fermentation cycle was 7.9 g / L·h, the glucose conversion rate was 80%, the fermentation cycle was short, and the lactic acid yield was low.

Claims

1. A continuous fermentation method for lactic acid, characterized in that: include: The strain is inoculated into an aerobic fermentation tank and fermented under an aerobic environment; The bacterial cells after the fermentation culture are transported to an anaerobic fermentation tank for anaerobic continuous fermentation to produce a fermentation liquid containing lactic acid. After the fermentation liquid consumes sugar in a saccharolysis tank, a portion of the fermentation liquid is centrifuged in a centrifuge to remove aged bacterial cells and recover the fermented supernatant. The other portion of the fermentation liquid is filtered through a ceramic membrane and the bacterial cells are recovered to the anaerobic fermentation tank. The supernatant obtained by centrifugation and ceramic membrane separation is dissociated through an electrodialysis bipolar membrane to obtain a sodium hydroxide solution and a lactic acid solution. The sodium hydroxide solution is returned to the anaerobic fermentation process as a neutralizer. Wherein, in the anaerobic continuous fermentation, the carbon source addition rate Q of the control flow to the anaerobic fermentation tank is controlled. g and bacterial flow rate Q c To control the residual sugar concentration in the anaerobic fermentation tank to 1-2g / L, OD 600 25 to 27; In the ceramic membrane separation process, the output of the supernatant is controlled o To maintain the concentration of lactic acid in the anaerobic fermentation tank at 60-72 g / L, preferably 60-62 g / L.

2. The continuous fermentation method according to claim 1, characterized in that In the anaerobic continuous fermentation, the carbon source replenishment rate Q in the anaerobic fermentation tank is g =3.75K g ×C a ×Q a ; Among them, C a Q is the mass content of sodium hydroxide in the anaerobic fermentation tank; a is the flow rate of sodium hydroxide in the anaerobic fermentation tank; K g is the mixed acid coefficient in the anaerobic fermentation tank, K g =1+C Sa / C La1 , C Sa is the concentration of miscellaneous acids in the anaerobic fermentation tank, C La1 is the lactic acid concentration in the anaerobic fermentation tank.

3. The continuous fermentation method according to claim 1 or 2, characterized in that: In the anaerobic continuous fermentation, the cell flow rate Q in the anaerobic fermentation tank is c =V×K c ×exp(1-62 / C La1 ); Among them, V is the weight of the fermentation liquid in the anaerobic fermentation tank, K c is the dilution rate of bacteria supplementation in the anaerobic fermentation tank, ranging from 0.08 to 0.12, preferably from 0.10 to 0.11, and exp is the natural index.

4. The continuous fermentation method according to any one of claims 1 to 3, characterized in that In the ceramic membrane separation process, the extraction amount of supernatant Q o =K o ×(Q g +Q a ); Among them, K o is the extraction ratio coefficient, K o =exp(1-62 / C La2 ), C La2 is the lactic acid concentration in the ceramic membrane, Q g is the carbon source replenishment rate in the anaerobic fermentation tank, Q a is the flow rate of sodium hydroxide in the anaerobic fermentation tank.

5. The continuous fermentation method according to any one of claims 1 to 4, characterized in that: The bacterial strain in the aerobic fermentation tank is selected from Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris; The culture medium in the aerobic fermentation tank is a nitrogen source culture medium, which includes: 10g / L yeast powder, 2-5g / L urea, 2-3g / L KCl, 0.01-0.2g / L CaCl2·2H2O, 2-8g / L KH2PO4, 2-8g / L Na2HPO4·12H2O, 0.2-2g / L MgSO4 and 1-5mL / L trace elements; The trace elements include 0.8 g / L vitamin B1, 0.1 g / L FeCl3·6H2O, 0.8 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·6H2O, 0.075 g / L CuSO4·5H2O, 0.75 g / L MnSO4·H2O, 0.15 g / L H3BO3 and 0.05 g / L Na2MoO4·2H2O; Preferably, the OD in the aerobic fermentation tank is 600 Maintain between 20 and 35.

6. The continuous fermentation method according to claim 5, characterized in that The anaerobic continuous fermentation is carried out at 42-45° C., pH 6.8-7.1, and a stirring rate of 250-350 rpm; The carbon source added to the anaerobic fermentation tank is selected from at least one of glucose, starch or sucrose; The concentration of the nitrogen source in the anaerobic fermentation tank is 1.0-1.2 g / L.

7. The continuous fermentation method according to any one of claims 1 to 6, characterized in that: The residence time of the fermentation liquid in the saccharolysis tank is 0.5 to 1 hour, the carbon source concentration at the discharge port of the saccharolysis tank is less than 1 g / L, and the nitrogen source concentration is less than 0.2 g / L.

8. The continuous fermentation method according to any one of claims 1 to 7, characterized in that The residence time of the fermentation liquid in the centrifuge is 1 to 10 seconds, and the solid content at the slag discharge port of the centrifuge is 1 to 5 wt%.

9. The continuous fermentation method according to claim 8, characterized in that: The supernatant obtained by centrifugation and ceramic membrane separation is dissociated in an electrodialysis bipolar membrane device; The electrodialysis bipolar membrane device is a double-chamber electrodialysis device in which a cationic homogeneous membrane and an anionic bipolar membrane are alternately arranged.

10. A continuous fermentation system for lactic acid, characterized in that: The continuous fermentation system is applied to the continuous fermentation method according to any one of claims 1 to 9, comprising an aerobic fermentation tank, a sugar supplement tank, an alkali buffer tank, an anaerobic fermentation tank, a saccharolysis tank, a ceramic membrane, a centrifuge, and an electrodialysis bipolar membrane; Among them, the discharge port of the aerobic fermentation tank, the discharge port of the sugar supplement tank and the discharge port of the alkali buffer tank are respectively controllably connected to the feed port of the anaerobic fermentation tank, the discharge port of the anaerobic fermentation tank is connected to the feed port of the saccharolysis tank, the discharge port of the saccharolysis tank is respectively controllably connected to the feed port of the ceramic membrane and the feed port of the centrifuge, the supernatant discharge port of the ceramic membrane and the supernatant discharge port of the centrifuge are controllably connected to the feed port of the electrodialysis bipolar membrane, and the bacterial cell outlet of the ceramic membrane is connected to the feed port of the anaerobic fermentation tank.

Images