CN105624212A - Method for preparing 2,3-butanediol (BDO) by adopting microalgae as raw material - Google Patents
Method for preparing 2,3-butanediol (BDO) by adopting microalgae as raw material Download PDFInfo
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Abstract
The invention discloses a method for preparing 2,3-butanediol (BDO) by adopting microalgae as a raw material. The method comprises the following steps: (1) microalgae culture, namely, inoculating microalgae seed solution into a microalgae culture medium rich in N element, culturing till reaching the logarithm growth period, standing for sedimentation, discharging the supernate, adding with the microalgae culture medium containing trace amount of the N element, and further culturing till reaching the stable phase, thus obtaining a microalgae culture solution; (2) hydrolysis treatment, namely, adding helicase into the microalgae culture solution, carrying out wall-breaking treatment, adding inorganic strong acid into the solution according to the standard that the acid concentration in a final hydrolysis system is 0.5wt%-5.0wt%, heating to 100-150 DEG C, and carrying out hydrolysis, thus obtaining microalgae hydrolysate; and (3) fermentation culture, namely, preparing a fermentation culture medium by taking the obtained microalgae hydrolysate as a carbon source, and fermenting by adopting klebsiella variicola to prepare 2,3-BDO. According to method, through the regulation on the microalgae culture process and the hydrolysis process, the content of starch in the microalgae and the content of fermentable sugars in the hydrolysate are improved, finally, 2,3-BDO with high concentration is obtained, and the method has the characteristics of simple treatment process, low cost and high yield.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of method with microalgae for raw material production 2,3-butanediol.
Background technology
Due to the exhaustion of world petroleum resource and price gradually high, environmental issue is increasingly serious, and Global climate change causes a series of impact, since 20 century 70s, the development of many countries pay attention to day by day bioenergy, bio-based chemicals and biomaterial. 2,3-butanediol (2,3-Butanediol, 2,3-BDO), is a kind of very important industrial chemicals and liquid fuel, is widely used in the fields such as food, medicine, building materials, fuel and Aero-Space. The method synthesizing at present 2,3-butanediol has two kinds, chemical method and bioanalysis. Chemical method synthesis 2, four hydrocarbons produced when 3-butanediol is mainly with petroleum cracking are hydrolyzed under high temperature, high pressure and obtain, comparing with biological fermentation process, chemosynthesis cost is high, process is loaded down with trivial details, not easily operate, and is difficult to large-scale industrial production always. Utilize bioanalysis to produce 2,3-BDO, namely meet the industrial requirement of green chemical industry, national energy development strategy can be met by alleviating energy crisis again, become the new focus of biological field in recent years.
It is more with glucose for carbon source through fermentation that current bioanalysis produces 2,3-BDO reports, strives grain with people and problem that people strives ground if existed with traditional corn for raw material. The open one of CN1710086A utilizes the method that molasses, starch or Caulis Sacchari sinensis juice produce 2,3-BDO for carbon source through fermentation, but end product 2,3-BDO concentration is not high. CN101225408A provides a kind of method that fibrous matter produces alcohol fuel and 2,3-butanediol, and maximum bottleneck is exactly that cellulase cost is high. Therefore, seek efficiently, low cost 2,3-butanediol Biotechnology is always up the target of people's research, cheap biomass material is particularly efficiently utilized to produce 2,3-butanediol, not only facilitate the application expanding 2,3-butanediol further, additionally aid the general character bottleneck problem solved in bio-based chemicals Biotechnology.
In recent years, microalgae can carry out photosynthesis because of its simple in construction, whole organism, there is photosynthetic efficiency height, growth cycle feature short, fireballing, can accumulate the high value added products such as a large amount of oils and fats, albumen, starch in cell by regulation and control simultaneously, rapidly become study hotspot. But current research focuses primarily upon and utilizes microalgae to produce biodiesel, as CN103571612A discloses a kind of method utilizing microalgae to prepare biodiesel, CN103451101A discloses a kind of method producing high quality microalgae biodiesel, and CN102433362A discloses a kind of method utilizing microalgae to process biogas slurry producing biodiesel while. And the application in other biological product rarely has report.
The micro algae growth cycle is short, can effectively couple photosynthesis and saccharide produces, and photosynthetic efficiency is apparently higher than common plant, and starch is accumulative up to dry cell weight more than 30%, and part carbon also can be converted into other chemicals simultaneously; The cultivation of microalgae does not strive water with striving with crops, it is possible to use the nutrition such as N, P in waste water, thus reducing the eutrophication of water body, and saving water resource and nutritive salt cost. Therefore, utilize non-grain and reproducible microalgae to produce 2,3-BDO for fermenting raw materials, compare with other biomass 2,3-BDO, cost can be reduced to a certain extent, be also that the exploitation of microalgae downstream product provides a new approach simultaneously.
CN200810228100.4 discloses a kind of with rich in starch Chinese crude drug for fermenting raw materials production 2, the method of 3-butanediol, is with rich in starch Chinese crude drug for raw material, obtains saccharified liquid through liquefaction, saccharifying, with saccharified liquid for carbon source, mix with the mineral nutrition composition after sterilizing, access the strain producing 2,3-butanediol, carry out shake flask fermentation, batch fermentation or pass through to add solid glucose, carry out fedbatch culture fermentation, obtain higher concentration 2,3-butanediol. The pretreatment of raw material of this invention is complicated, and needs stream to add glucose not have economy.
CN200910015400.9 discloses one and utilizes starch materials to produce 2, the method of 3-butanediol, it is that starch materials is processed through ��-amylase and saccharifying enzyme, using saccharified liquid or liquefier as fermentation substrate, with Klebsiella pneumonia as fermentation strain, aseptically produce 2 by substep diastatic fermentation or simultaneous saccharification and fermentation, 3-butanediol, final 2, the concentration of 3-butanediol reaches 43��112g/L, the ultimate density of acetoin and 2,3-butanediol reaches 45��121g/L. Starch materials described in this patent is the grain class raw material such as Semen Maydis powder, corn starch, the problem that there is " striving grain with people ".
Summary of the invention
Problem low for microalgae content of starch, that hydrolysis sugar amount is low and other raw material production 2,3-butanediol costs are high, the invention provides a kind of method with microalgae for raw material production 2,3-butanediol. The present invention, by the regulation and control to microdisk electrode process and hydrolytic process, improves fermentable sugars content in microalgae content of starch and hydrolyzed solution, is finally obtained the 2,3-BDO of high concentration, has and processes the feature that technique is simple, cost is low, productivity is high.
The present invention method with microalgae for raw material production 2,3-butanediol, including following content:
(1) microdisk electrode: microalgae seed liquor is accessed rich in, in the micro-algae culture medium of N element, cultivating exponential phase and stop; Discharge supernatant after standing sedimentation, add the micro-algae culture medium containing trace N element and continue to cultivate, be cultured to stable phase and obtain micro algae culturing liquid;
(2) hydrolysis process: in above-mentioned micro algae culturing liquid add Snailase carry out broken wall treatment, subsequently according to acid concentration in final hydrolyzation system be 0.5wt%-5.0wt% add inorganic acid, be warming up to 100-150 DEG C and be hydrolyzed, it is thus achieved that microalgae hydrolyzed solution;
(3) fermentation culture: prepare fermentation medium with the microalgae hydrolyzed solution obtained for carbon source, and adopt klebsiella fermenting and producing 2,3-butanediol.
In the present invention, the microalgae described in step (1) selects the chlorella that content of starch is higher, it is preferable that chlorella (Chlorella), chlamydomonas (Chlamydomonas), scenedesmus (Scenedesmus) etc. in one or more. Micro-algae culture medium is the culture medium of cellar culture microalgae, as adopted SE, TAP or BG11 culture medium. The referring to of microalgae seed liquor is cultured to the algae solution that exponential phase obtains after microalgae is accessed culture medium; During microdisk electrode, the inoculum concentration controlling microalgae seed liquor is 2v%-10v% for (accounting for culture volume, lower same). The actual conditions of microalgae seed liquor and microdisk electrode is as follows: temperature 25-30 �� of C, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, speed of agitator 50-200rpm, pH6.0-8.0, Light To Dark Ratio 10:14-14:10.
In the present invention, the micro-algae culture medium rich in N element described in step (1) refers to and adds excessive inorganic nitrogen in conventional micro-algae culture medium, as being NaNO3��NH4NO3��NH4Cl etc., addition is 2.0-5.0g/L; The described micro-algae culture medium containing trace N element refers to and reduces the content of nitrogenous source in conventional micro-algae culture medium, and addition is 0-0.5g/L.
In the present invention, the microdisk electrode described in step (1) can adopt the training method of routine, as passed into CO2Content is 1v%-3v%, carries out the microdisk electrode rich in N element and the microdisk electrode containing trace N element when Light To Dark Ratio is 10:14-14:10. Present invention preferably employs in the following manner to cultivate: when cultivating in rich in the micro-algae culture medium of N element, the CO passed into2Content is 1v%-3v%, and Light To Dark Ratio is 10:14-14:10, cultivates exponential phase and stops; Supernatant is discharged, simultaneously by CO after standing sedimentation2Content bring up to 3v%-5v%, pH regulator is 8.0-9.0, and Light To Dark Ratio is 18:6-24:0, is cultured to stable phase. By above-mentioned cultivation, help speed up microalgae to the adaptability of environment and fast breeding, thus being favorably improved in algae solution content of starch.
In the present invention, microdisk electrode to stable phase being obtained the algae solution that content of starch is higher, add Snailase according to 5-50mg/g algae solution and cell carries out broken wall treatment, the pH value controlling broken wall treatment is 5.0-8.0, and temperature is 30-45 �� of C. Frustule is carried out broken wall treatment, is possible not only to by the starch release within frustule out, improve amylolytic accessibility, and can hydrolyzable moiety starch and cell-wall component, significantly reduce sequential hydrolysis difficulty. Subsequently according to acid concentration in final hydrolyzation system be 0.5wt%-5.0wt% add inorganic acid, be warming up to 100-120 DEG C, be incubated 10-60min. Inorganic acid selects sulphuric acid or hydrochloric acid. Microalgae is except born of the same parents are contained within much starch, possibly together with the material such as cellulose, hemicellulose in cell wall. Adopt Snailase combination dilute acid hydrolysis namely Starch Hydrolysis in born of the same parents can be become fermentable sugars, moreover it is possible to cellulose, hemicellulose etc. in cell wall to be together hydrolyzed into fermentable sugars, improves the fermentable sugars content in hydrolyzed solution.
In the present invention, prepare fermentation medium with microalgae hydrolyzed solution prepared by step (2) for carbon source, also include: nitrogenous source (NH simultaneously4)2HPO42.0-6.0g/L, (NH4)2SO44.0-8.0g/L; Inorganic salt K2HPO4��3H2O10.0-20.0g/L, KH2PO41.0-5.0g/L; Trace element MgSO4��7H2O0-1.0g/L, FeSO4��7H2O0-1.0g/L, ZnSO4��7H2O0-0.5g/L, MnSO4��H2O0-0.5g/L, CaCl20-1.0g/L, EDTA0-1.0g/L, pH value is 6.0-8.0.
In the present invention, prepare 2,3-butanediol with klebsiella for fermented bacterium, it is preferable that klebsiella and acid-producing Klebsiella bacterium. The preparation method of zymocyte seed liquor is as follows: take the klebsiella of activation, is inoculated into containing glucose 5-50g/L, yeast extract 2-6g/L, peptone 5-15g/L, in the culture medium of sodium chloride 5-10g/L, at temperature 35-40 DEG C, rotating speed 150-250rpm, pH are cultivation 12-24h under 6.0-7.5.
In the present invention, klebsiella seed liquor is inoculated in described fermentation medium with the inoculum concentration of 2v%-10v%, at temperature 30-40 DEG C, rotating speed 150-300rpm, carry out fermentation when ventilation 0.1-1.0vvm, pH are 6.0-8.0 and produce 2,3-butanediol.
In the inventive method, 2,3 butyleneglycol fermentation can adopt Batch fermentation or fedbatch culture fermentation. Start to add microalgae hydrolyzed solution when remaining sugar concentration to 10-20g/L, fermentation 48-96h terminates. The microalgae hydrolyzed solution that wherein stream adds is the algae solution that microalgae is repeatedly cultivated, and discharges supernatant after staticly settling, and the algae mud obtained obtains fermentable sugars liquid after utilizing Snailase and diluted acid combined hydrolysis.
Compared with prior art, the method have the advantages that
1, by the Discrete control of nitrogen content in microdisk electrode process, contributing to propagation and the starch accumulation of microalgae cell, the frustule of high-content of starch can be obtained; Dilute acid hydrolysis process is combined, it is possible to significantly improve fermentable sugars content in hydrolyzed solution, thus being favorably improved final Fermentation Substance Concentration by Snailase. The invention solves and utilize microalgae to produce, for substrate, the problem that 2,3-BDO productivity is low, cost is high.
2, during microdisk electrode, low content CO is passed at initial period2, control Light To Dark Ratio is 10:14-14:10, cultivates exponential phase and stops; Discharge supernatant after standing sedimentation, then improve CO2Content and pH value to improve Light To Dark Ratio be 18:6-24:0, be cultured to stable phase. By above-mentioned cultivation, help speed up microalgae to the adaptability of environment and fast breeding, be favorably improved the content of starch of frustule.
3, the present invention raw materials used be absent from striving grain with people and grain strive etc. problem, it is possible to effectively lowering bioconversion produces the cost of 2,3-butanediol; Directly adopt the fermentation of algae mud band slag, save microalgae and be centrifuged and dry run, energy-conservation energy consumption, the protein in microalgae can also be made full use of as 2,3-butanediol fermentation N source simultaneously. Present invention process is that microalgae downstream industry opens a kind of new way.
Detailed description of the invention
The present invention utilizes starch-containing microalgae for the method for raw material production 2,3-butanediol, first microalgae seed liquor is accessed rich in, in the micro-algae culture medium of N element, cultivating exponential phase and stop, making proliferation of microalgae papidly; Discharge supernatant after standing sedimentation, add the micro-algae culture medium containing trace N element and continue to cultivate, contribute to the stable accumulation of starch in microalgae born of the same parents, be cultured to stable phase and obtain amyloid algae solution. In above-mentioned cultured algae solution, add Snailase carry out broken wall treatment, subsequently according to acid concentration in final hydrolyzation system be 0.5wt%-5.0wt% add inorganic acid, be warming up to 100-150 DEG C and be hydrolyzed, it is thus achieved that microalgae hydrolyzed solution. Finally preparing fermentation medium using the microalgae hydrolyzed solution obtained as carbon source, remainder, as the stream liquid feeding of subsequent fermentation, adopts klebsiella fermenting and producing 2,3-butanediol.
In the present invention, microdisk electrode reactor is ventilating/stirring closed reactor, with controllable light lighting system, it is possible to pass into carbon dioxide, and arranges the determinator of carbon dioxide and dissolved oxygen, adjusts ventilation as required, to obtain good culture effect. Stirring paddle has contractility, packs up stirring paddle when algae precipitate, does not affect effect of settling. Microdisk electrode reactor can accurately control pH and agitation revolution, it is ensured that stablizing of microdisk electrode and 2,3-butanediol sweat.
In the present invention, pH controls acid neutralizing agent is HCl, H2SO4��HNO3Deng conventional mineral acid, alkali nertralizer is NaOH, NaHCO3��KOH��Ca(OH)2, inorganic base that ammonia etc. is conventional.
Below in conjunction with embodiment, the present invention program is described further. In the present invention, wt% is mass fraction, and v% is volume fraction.
Embodiment 1
The preparation of micro-algae culture medium: adopt BG11 culture medium (in every liter): NaNO31.5g, K2HPO4��3H2O0.04g, MgSO4��7H2O0.075g, CaCl2��2H2O0.036g, citric acid 0.006g, ferric ammonium citrate 0.006g, EDTA0.001g, NaCO30.02g, A5+Co solution 1mL. Wherein the formula of A5+Co solution is: H3BO32.86g, MnCl2��H2O1.81g, ZnSO4��7H2O0.222g, CuSO4��5H2O0.079g, Na2MoO4��2H2O0.390g, Co (NO3)2��6H2O0.049g��
The preparation of microalgae seed liquor: by chlorella (Chlorellavulgaris) the seed above-mentioned micro-algae culture medium of access, at temperature 30 DEG C, ventilation 0.25vvm, CO2Content is 1v%, and speed of agitator 100rpm, pH are 7.0, are cultured to exponential phase when Light To Dark Ratio 10:14.
(1) microdisk electrode: microalgae seed liquor is accessed rich in, in the micro-algae culture medium of N element, referring to rich in N element and add NaNO according to 2.5g/L in the medium according to inoculum concentration 5v%3, cultivate exponential phase and stop; Discharging supernatant after standing sedimentation, proceed to the culture medium containing trace N element and continue to cultivate, the N element containing trace refers to and adds NaNO according to 0.05g/L in the medium3, it is cultured to stable phase. Above-mentioned microdisk electrode is at temperature 30 DEG C, ventilation 0.25vvm, CO2Content is 1v%, and speed of agitator 100rpm, pH are 7.0, is cultured to stable phase when Light To Dark Ratio 10:14, gathers in the crops algae solution, and wherein content of starch reaches 48%. Content of starch detection method: take two parts of 10mL (V) algae solutions, centrifugal deionization is washed 2 times, adds 10mL deionized water resuspended. A copy of it 80 DEG C drying obtains algae dry weight W to constant weight, another part of 600W ultrasonication, ultrasonic time 1s, intermittent time 2s, ultrasonic 60 times, the algae solution crushed completely. Regulate pH to 6.5, be separately added into 0.20��lLiquozymeSupra85-95 DEG C of insulation liquefaction 1h; Regulate pH to 4.5, be separately added into 0.4��lDextrozymeDX60 DEG C of insulation liquefaction 10h, centrifugal precipitation of abandoning, supernatant constant volume 100mL, glucose content C in detection hydrolyzed solution after hydrolysis completely. Add equivalent amounts of enzyme with deionized water and be hydrolyzed to comparison. Content of starch (g/g)=C (g/L) �� V (L) �� 0.9/W (g).
(2) algae solution processes: in the algae solution of step (1), adds Snailase according to 20mg Snailase/g algae solution and carries out broken wall treatment, controls the pH6.0 of broken wall treatment, and temperature is 40 DEG C; Subsequently according to acid concentration in final hydrolyzation system be 2.5wt% add sulphuric acid, be warming up to 115 DEG C and be hydrolyzed, be incubated 10-60min, it is thus achieved that microalgae hydrolyzed solution. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 75g/L.
(3) fermentation culture: prepare fermentation medium for carbon source with microalgae hydrolyzed solution, adopts klebsiella fermenting and producing 2,3-butanediol.
Prepare fermentation medium with microalgae hydrolyzed solution prepared by step (2) for carbon source, and add following material: nitrogenous source (NH4)2HPO43.3g/L, (NH4)2SO46.6g/L; Inorganic salt K2HPO4��3H2O13.7g/L, KH2PO42.0g/L; Trace element MgSO4��7H2O0.25g/L, FeSO4��7H2O0.05g/L, ZnSO4��7H2O0.002g/L, MnSO4��H2O0.001g/L, CaCl20.01g/L, EDTA0.05g/L, pH control 7.0, Initial sugar concentration 75g/L.
Preparing 2,3-butanediol with klebsiella for fermented bacterium, the preparation method of zymocyte seed liquor is as follows: take the klebsiella of activation, Chinese industrial Microbiological Culture Collection administrative center provide, be numbered klebsiella CICC10011. Being inoculated into containing glucose 20g/L, yeast extract 3g/L, peptone 8g/L, in the culture medium of sodium chloride 5g/L, temperature 37 DEG C, rotating speed 200rpm, pH are cultivation 12h, dry cell weight about 3.0g/L under 7.0 conditions.
Above-mentioned klebsiella seed liquor is inoculated in fermentation medium with the inoculum concentration of 5v%, at temperature 35 DEG C, rotating speed 150rpm, carries out fermentation when ventilation 0.25vvm, pH7.0 and produce 2,3-butanediol. After fermentation 16h, remaining sugar concentration is lower than 20g/L, and stream adds microalgae hydrolyzed solution makes fermentation system sugar concentration be 200g/L, and fermentation carries out 48h altogether, and in fermentation liquid, 2,3-butanediol and acetoin ultimate density sum are 70.21g/L.
Embodiment 2
Process technique and operating condition with embodiment 1, be different in that: (1) microalgae employing Chlamydomonas reinhardtii (Chlamydomonasreinhardtii), micro-algae culture medium adopts TAP culture medium; (2) N source is NH4Cl, when cultivating rich in N, addition is 3g/L, and when cultivating containing trace N, addition is 0.02g/L; (3) adding Snailase according to 30mg Snailase/g algae solution and carry out broken wall treatment, control the pH8.0 of broken wall treatment, temperature is 30 DEG C.
After microdisk electrode terminates, gathering in the crops algae solution, wherein content of starch reaches 47%. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 72g/L. Fermentation 14h residual sugar is lower than 20g/L, and stream adds microalgae hydrolyzed solution makes the sugared concentration of fermentation system be 200g/L, and fermentation carries out 48h altogether, and in fermentation liquid, 2,3-butanediol and acetoin ultimate density sum are 68.9g/L.
TAP culture medium prescription is (in every liter): H2NC(CH2OH)32.42g, TAP salt 25mL (NH4Cl15g/L, MgSO4��7H2O4g/L, CaCl 2H2O2g/L), phosphoric acid solution 1mL (K2HPO428.8g/100mL, KH2PO414.4g/100mL), trace element 1mL (Na2EDTA��2H2O5.00g/100mL, ZnSO4��7H2O2.20g/100mL, H3BO31.14g/100mL, MnCl2��4H2O0.50g/100mL, FeSO4��7H2O0.50g/100mL, CoCl2��6H2O0.16g/100mL, CuSO4��5H2O0.16g/100mL, (NH4)6Mo7O24��4H2O0.11g/100mL), acetic acid 1mL.
Embodiment 3
Process technique and operating condition with embodiment 1, be different in that: (1) microalgae adopt four tail coral algaes (Scenedesmusquadricanda); (2) N source is NH4NO3, addition is 4g/L; (3) adding Snailase according to 25mg Snailase/g algae solution and carry out broken wall treatment, control the pH7.0 of broken wall treatment, temperature is 35 DEG C.
After microdisk electrode terminates, gathering in the crops algae solution, wherein content of starch reaches 45.6%. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 78g/L. Fermentation 17h residual sugar is lower than 20g/L, and stream adds microalgae hydrolyzed solution makes the sugared concentration of fermentation system be 200g/L, and fermentation carries out 48h altogether, and in fermentation liquid, 2,3-butanediol and acetoin ultimate density sum are 69.5g/L.
Embodiment 4
Process technique and operating condition with embodiment 1, be different in that: when microdisk electrode is cultivated in rich in the micro-algae culture medium of N element, the CO passed first into2Content is 2v%, and Light To Dark Ratio is 10:14, cultivates exponential phase and stops; Supernatant is discharged, simultaneously by CO after standing sedimentation2Content bring up to 5v%, pH and be increased to 8.0, Light To Dark Ratio is 24:0, is cultured to stable phase. After cultivation terminates, gathering in the crops algae solution, wherein content of starch reaches 53%. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 84g/L.
Embodiment 5
Process technique and operating condition with embodiment 2, be different in that: when microdisk electrode is cultivated in rich in the micro-algae culture medium of N element, the CO passed first into2Content is 1v%, and Light To Dark Ratio is 10:14, cultivates exponential phase and stops; Supernatant is discharged, simultaneously by CO after standing sedimentation2Content bring up to 3v%, pH and be increased to 7.5, Light To Dark Ratio is 18:6, is cultured to stable phase. After cultivation terminates, gathering in the crops algae solution, wherein content of starch reaches 51%. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 80g/L.
Comparative example 1
The treatment process condition of fermentation part, with embodiment 1, is different in that: do not adopt microalgae hydrolyzed solution to ferment, and directly adopts glucose as carbon source, and glucose initial concentration is 80g/L. Adopting fedbatch culture fermentation mode, when remaining sugar concentration is lower than 20g/L, adds Glucose Liquid and make the sugared concentration of fermentation system be 200g/L, fermentation carries out 48h altogether, and in fermentation liquid, 2,3-butanediol and acetoin ultimate density sum are 73.2g/L.
Compared with embodiment 1, the present invention utilizes Microalgae fermentation to produce the level of 2,3-butanediol with directly employing glucose fermentation level disparity is little, and the present invention alternative grain raw material of amyloid microalgae or the supplementary production 2,3-butanediol as grain raw material are described.
Comparative example 2
Processing technique and operating condition with embodiment 1, be different in that: during microdisk electrode, not adopting nitrogenous source content Discrete control, whole process adds nitrogenous source in a conventional manner, namely adds NaNO31.5g/L. After cultivation terminates, gathering in the crops algae solution, wherein content of starch reaches about 30%.
Comparative example 3
Process technique and operating condition with embodiment 1, be different in that: during microdisk electrode, only obtain fermentable sugars by dilute acid hydrolysis process. Sugared concentration 40g/L after hydrolysis process, in hydrolyzed solution.
Comparative example 4
Process technique and operating condition with embodiment 1, be different in that: during microdisk electrode, adopt the mode described in CN200910015400.9 to be hydrolyzed. After hydrolysis process, the sugared concentration in hydrolyzed solution is about 60g/L.
Claims (11)
1. the method with microalgae for raw material production 2,3-butanediol, it is characterised in that include following content:
(1) microdisk electrode: microalgae seed liquor is accessed rich in, in the micro-algae culture medium of N element, cultivating exponential phase and stop; Discharge supernatant after standing sedimentation, add the micro-algae culture medium containing trace N element and continue to cultivate, be cultured to stable phase and obtain micro algae culturing liquid;
(2) hydrolysis process: in above-mentioned micro algae culturing liquid add Snailase carry out broken wall treatment, subsequently according to acid concentration in final hydrolyzation system be 0.5wt%-5.0wt% add inorganic acid, be warming up to 100-150 DEG C and be hydrolyzed, it is thus achieved that microalgae hydrolyzed solution;
(3) fermentation culture: prepare fermentation medium with the microalgae hydrolyzed solution obtained for carbon source, and adopt klebsiella fermenting and producing 2,3-butanediol.
2. in accordance with the method for claim 1, it is characterised in that: microalgae described in step (1) select chlorella that content of starch is higher (Chlorella), chlamydomonas (Chlamydomonas), scenedesmus (Scenedesmus) in one or more, micro-algae culture medium adopt SE, TAP or BG11 culture medium.
3. in accordance with the method for claim 1, it is characterised in that: the microalgae seed liquor described in step (1) refers to and is cultured to the algae solution that exponential phase obtains after microalgae is accessed culture medium, and the inoculum concentration controlling microalgae seed liquor during microdisk electrode is 2v%-10v%.
4. in accordance with the method for claim 1, it is characterised in that: microalgae seed liquor and the actual conditions of microdisk electrode described in step (1) are as follows: temperature 25-30 DEG C, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, speed of agitator 50-200rpm, pH6.0-8.0, Light To Dark Ratio 10:14-14:10.
5. in accordance with the method for claim 1, it is characterised in that: the micro-algae culture medium rich in N element described in step (1) refers to and adds excessive NaNO in conventional micro-algae culture medium3��NH4NO3Or NH4Cl, addition is 2.0-5.0g/L; The described micro-algae culture medium containing trace N element refers to and reduces the content of nitrogenous source in conventional micro-algae culture medium, and addition is 0-0.5g/L.
6. in accordance with the method for claim 1, it is characterised in that: step (1) adopts following two step training methods: when cultivating in rich in the micro-algae culture medium of N element, the CO passed into2Content is 1v%-3v%, and Light To Dark Ratio is 10:14-14:10, cultivates exponential phase and stops; Supernatant is discharged, simultaneously by CO after standing sedimentation2Content bring up to 3v%-5v%, pH regulator is 8.0-9.0, and Light To Dark Ratio is 18:6-24:0, is cultured to stable phase.
7. in accordance with the method for claim 1, it is characterised in that: step (2) adds Snailase according to 5-50mg/g algae solution and cell carries out broken wall treatment, and the pH controlling broken wall treatment is 5.0-8.0, and temperature is 30-45 DEG C.
8. in accordance with the method for claim 1, it is characterised in that: step (2) is that 1.0wt%-2.5wt% adds inorganic acid according to acid concentration in final hydrolyzation system, is warming up to 100-120 DEG C, is incubated 10-60min.
9. in accordance with the method for claim 1, it is characterised in that: prepare fermentation medium with microalgae hydrolyzed solution prepared by step (2) for carbon source, also include: nitrogenous source (NH simultaneously4)2HPO42.0-6.0g/L, (NH4)2SO44.0-8.0g/L; Inorganic salt K2HPO4��3H2O10.0-20.0g/L, KH2PO41.0-5.0g/L; Trace element MgSO4��7H2O0-1.0g/L, FeSO4��7H2O0-1.0g/L, ZnSO4��7H2O0-0.5g/L, MnSO4��H2O0-0.5g/L, CaCl20-1.0g/L, EDTA0-1.0g/L, pH value is 6.0-8.0.
10. in accordance with the method for claim 1, it is characterized in that: step (3) prepares 2 with klebsiella for fermented bacterium, 3-butanediol, klebsiella seed liquor is inoculated in described fermentation medium with the inoculum concentration of 2v%-10v%, at temperature 30-40 DEG C, rotating speed 150-300rpm, ventilation 0.1-1.0vvm, carry out fermentation when pH is 6.0-8.0 and produce 2,3-butanediol.
11. in accordance with the method for claim 1, it is characterised in that: in step (3), 2,3-butanediol fermentation adopts fedbatch culture fermentation, starts to add microalgae hydrolyzed solution when remaining sugar concentration to 10-20g/L, and fermentation 48-96h terminates.
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