CN101402973B - Integrated method for cyclic utilization with microorganism biomass in production process of 2,3-butanediol - Google Patents
Integrated method for cyclic utilization with microorganism biomass in production process of 2,3-butanediol Download PDFInfo
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- CN101402973B CN101402973B CN2008102028082A CN200810202808A CN101402973B CN 101402973 B CN101402973 B CN 101402973B CN 2008102028082 A CN2008102028082 A CN 2008102028082A CN 200810202808 A CN200810202808 A CN 200810202808A CN 101402973 B CN101402973 B CN 101402973B
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- butyleneglycol
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Abstract
The invention relates to an integration method of the production process of 2,3-butanediol and the recycling of microbial biomass, and the integration method comprises: the 2,3-butanediol is produced by the fermentation of the serratia marcescens seed bacteria; the recovered serratia marcescens is added in a fermentation culture medium of the second fermentation for carrying out the further fermentation culture. The serratia marcescens in fermentation products can be repeatedly utilized. The use of the method can not only improve the yield, the conversion rate and the production ability of the 2, 3-butanediol, but also effectively solve the thallus pollution and the recycling problem of resources, save the energy and reduce the emission.
Description
Technical field
The invention belongs to the microbiology field, be specifically related to a kind of serratia marcescens fermentative prodn 2 of utilizing recovery, the method for 3-butyleneglycol.
Background technology
2, and the 3-butyleneglycol (2,3-butanediol; Be called for short BD; Be widely used in every field such as chemical industry, food and aerospace down together), its calorific value 27200kJ/kg, approaching with alcoholic acid calorific value (29100kJ/kg); Can be used as fuel, also can be used to chiral support for preparing polymkeric substance, printing ink, perfume, frostproofer, perfumed incense, moistening agent, explosive and medicine etc.It is dehydrated into methylethylketone, can be used as the solvent of resin, paint; Be dehydrated into divinyl, can be used for viton; It can also replace 1, and the 4-butyleneglycol is used for the synthetic of polyester and Polyurethanes; With the methylethylketone condensation and carry out hydrogenation and form octane, can be used to produce senior aviation with oil.2,3-butyleneglycol and acetic acidreaction generate 2,3-butyleneglycol diacetate esters, and this ester class can be added to improves local flavor in the cream; 2, the 3-butyleneglycol also can be added in liquor, to improve the local flavor of liquor.
The fermentation volume of present biological technical field related industries has occupied preceding 3 of the world; The annual waste thallus that produces has reached 5,000,000 tons; The waste thallus of producing flood tide like this need consume mass energy (electricity, heat, water etc.) and resource (grain resource, organic and inorganic nitrogen etc.); And being limited to the current industry integral level of China, the recycling of these waste thallus and environmental protection treatment level are low, consequently cause the significant wastage of the energy and resource and serious environmental to pollute.
The target of China's Economic development is to promote economic Sustainable development, and therefore, the recycling of resource is very important.Shortage of resources, environmental degradation and population increase, and three factor superpositions have become stable, the fast-developing maximum restrained force of restriction China economic society.Thereby country's proposition, the Eleventh Five-Year Plan period target for energy-saving and emission-reduction is that energy consumption per unit reduction about 20%, total emissions of major pollutants reduce 10%, this is the inevitable choice of building a resource-conserving and environment-friendly society; Be to promote economic restructuring, change the only way of growth pattern.
Reclaim the thalline of the serratia marcescens in the fermented liquid, not only can significantly improve fermentative prodn 2, the transformation efficiency and the product output of the productivity of 3-butyleneglycol, main biomass carbon source can also significantly reduce the pollution problem of thalline discharging to environment.
A large amount of microbial cells are used to be used as fertilizer behind inactivation, can directly form fertilizer through liquid, also can be the accumulation fertilizer of solid substrate; Also some thalline often is used as waste treatment, in sewage work, decomposes, or is burnt.
The method of recycling thalline at present mainly contains: broken wall extracts the thalline soluble components, as the medium component of microorganism culturing, comprising mechanical crushing method and thalline autolysis method; Utilize the viable bacteria body, promptly production process is directly recycled the viable bacteria body.
It is many that the former studies; Mainly contain: the fermentation of klebsiella has been studied by (1) German University Bielefeld, recombinant strain production of L-threonine by fermentation and the thalline in Bacillus licheniformis DSM 13 fermentative prodn subtilisins and the basic protein enzyme process of intestinal bacteria B-3996 recycled; Degrade and the dissolution of bacteria somatic cells with high-pressure homogenization method and protease treatment method, the thalline after the processing is put back in the former process with the form of substratum and is utilized; (2) R.Radmer of U.S. Martek Corporation etc. has studied carbon source and the energy of hydrolysis algae thalline as algal grown, and the result finds to improve the capacity usage ratio of algae culture in the loop system; (3) the partially recycled utilization of zymic in the beer prodn: behind the beer fermentation, a large amount of muddy yeasts can be made into dry yeast, both can be pressed into the sheet hyoscine, also can improve the nutrition of feed as the additive of feed.
The instance that utilizes of viable bacteria body mainly contains: the yeast in (1) beer prodn, except the broken wall utilization, can also recycle its viable bacteria body, and continue beer brewing; (2) in the environment protection, when disposing of sewage, floc sedimentation absorption, decomposing organic matter or the toxin with fixed attention of live bacterium, protozoon and other mikrobe commonly used, this active sludge often is recycled; (3) Minier Michel etc. has studied the recycling of producing the clostridium acetobutylicum thalline of butanols and acetone: this fermenting process can be cultured continuously and batch culture; Fermented liquid is put into the ultrafiltration district; The residue that then the comprises thalline entering fermentation zone that partly circulates, thus carry out recycle.
The applicant disclosed a kind of fermentative prodn 2 in the past, the method for 3-butyleneglycol, and Chinese publication number CN1884560 is inoculated in the serratia marcescens seed liquor in the substratum, and bubbling air stirs, and RQ is controlled between 0.9～1.2; Mend product then and promote the factor, replenish aqueous sucrose solution and amino acid solution, RQ is controlled between 1.5～1.8; Mend aqueous sucrose solution at last RQ is controlled between 1.9～2.2, fermentation culture promptly obtains to contain 2, the fermentation culture of 3-butyleneglycol.Utilize serratia marcescens fermentative prodn 2; The 3-butyleneglycol has remarkable progress; But a serious environmental problem is not only in the discharging of a large amount of waste thallus that produce in the fermentation production process, but also causes the loss of biomass resources such as a large amount of carbon source nitrogenous sources, and energy-saving and emission-reduction are brought immense pressure.
The method of existing recycling thalline soluble components is the effective ways of resource recycling; But; This process often comprises the indispensable stages such as broken wall dissolving, filtration, separation even drying; Therefore also can cause the waste of energy resource, can not realize the final purpose of energy-saving and emission-reduction; And present study fewer of the instance of recycling the viable bacteria body, therefore, recycling viable bacteria body improves product output, improves the biomass resource utilization rate and reduce energy consumption is the novel method that this area presses for researchdevelopment.
Summary of the invention
The object of the present invention is to provide the recycle serratia marcescens to produce 2, the method for 3-butyleneglycol.
In one aspect of the invention, provide a kind of serratia marcescens that utilizes to produce 2, the method for 3-butyleneglycol, said method comprises:
(1) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-15% (preferred 4-10%) inserted in the fermention medium, fermentation culture 30-50 hour, obtain containing 2, the fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid;
(2) from fermented liquid, reclaim serratia marcescens;
(3) serratia marcescens that reclaims is joined in the fermention medium, make the OD value of thalline in substratum be 20-80, fermentation culture 30-50 hour, from fermented liquid, separate results 2,3-butyleneglycol.
In another preference, in step (3), make the OD value of serratia marcescens in substratum be 30-70.
In another preference, in step (1), described recovery serratia marcescens is from fermented liquid, to separate acquisition through centrifugation method.
In another preference, the centrifugal condition is following: 4 ± 2 ℃, and 10000 ± 3000rpm, 15-20min.
In another preference, also comprise in step (3) back:
Repeating step (2)-(3) 1-5 time (preferred 1-3 time).
In another preference, described fermention medium contains:
Sucrose 90 ± 20g/L, peptone 20 ± 5g/L, yeast powder 5 ± 2g/L, Hydrocerol A 10 ± 3g/L, MnSO
40.1 ± 0.05g/L, KH
2PO
40.5 ± 0.2g/L, FeSO
40.02 ± 0.005g/L, MgSO
40.5 ± 0.2g/L.
In another preference, described fermention medium also contains: KAc6 ± 2g/L; NaNO
32 ± 0.5g/L.
In another preference, the initial p H value of fermention medium is 7 ± 0.5.
In another preference, during the fermentation, also stream adds sucrose solution (sucrose solution of preferred 45-55%), and competent carbon source is provided.
In another preference, ferment after 6-12 hour, PH is maintained 6 ± 0.5.
In another preference, leavening temperature is 28-32 ℃.
In another aspect of this invention, provide a kind of serratia marcescens that utilizes to produce 2, the method for 3-butyleneglycol, said method comprises:
(a) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-15% (preferred 4-10%) inserted in the fermention medium, fermentation culture 30-50 hour, obtain containing 2, the fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid;
(b) from fermented liquid, reclaim serratia marcescens;
(c) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-10% (preferred 4-6%) inserted in the fermention medium; Fermentation culture was to 15-26 hour; The serratia marcescens that (b) reclaimed joins in the fermention medium, makes the OD value of thalline in substratum be 20-80 (preferred 20-40; More preferably 20-30), continued fermentation culture 4-25 hour, obtain containing 2, the fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid.
In another preference, in step (c), after fermentation culture 20-25 hour, the serratia marcescens that reclaims is joined in the fermention medium.
In another preference, also comprise in step (c) back:
Repeating step (b)-(c) 1-5 time.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
After Fig. 1 has shown the seed liquor (0 group) that in shaking bottle, inserts recovery viable bacteria body (1 group, 2 groups or 3 groups) or 5%, each group OD value changing conditions during the fermentation.
After Fig. 2 has shown the seed liquor (0 group) that in shaking bottle, inserts recovery viable bacteria body (1 group, 2 groups or 3 groups) or 5%, each group BD rate history during the fermentation.
After Fig. 3 has shown the seed liquor (0 group) that in shaking bottle, inserts recovery viable bacteria body (1 group, 2 groups or 3 groups) or 5%, the productive rate changing conditions of each group BD product during the fermentation.
After Fig. 4 has shown the seed liquor (0 group) that in shaking bottle, inserts recovery viable bacteria body (1 group, 2 groups or 3 groups) or 5%, each group sucrose utilization ratio comparable situation during the fermentation.
(4 groups) inserted and reclaim thalline, the OD value changing conditions during the fermentation that each is organized when (3 groups) were with 24h when (2 groups), 12h when Fig. 5 had shown respectively when fermentation 0h (1 group), 6h.
(4 groups) inserted and reclaim thalline, the sugar degree comparable situation during the fermentation that each is organized when (3 groups) were with 24h when (2 groups), 12h when Fig. 6 had shown respectively when fermentation 0h (1 group), 6h.
(4 groups) inserted and reclaim thalline, the BD rate history during the fermentation that each is organized when (3 groups) were with 24h when (2 groups), 12h when Fig. 7 had shown respectively when fermentation 0h (1 group), 6h.
(4 groups) inserted and reclaim thalline, the productive rate changing conditions of the BD product during the fermentation that each is organized when (3 groups) were with 24h when (2 groups), 12h when Fig. 8 had shown respectively when fermentation 0h (1 group), 6h.
(4 groups) inserted and reclaim thalline, the sucrose utilization ratio comparable situation during the fermentation that each is organized when (3 groups) were with 24h when (2 groups), 12h when Fig. 9 had shown respectively when fermentation 0h (1 group), 6h.
Embodiment
In order to reach the purpose that biomass resource effectively utilizes, the inventor is through deep research, develops first and optimized a kind of recycle serratia marcescens fermentative prodn 2, the method for 3-butyleneglycol.Described method compares 2 with inoculation kind of a daughter bacteria fermentation, and 3-butyleneglycol productive rate is significantly improved, and sugared transformation efficiency is high.Therefore, method of the present invention has improved the biomass resource utilization rate effectively, has reduced energy consumption, has realized cleaner production.
Among the present invention, utilize serratia marcescens to produce 2, the concrete reaction mechanism of 3-butyleneglycol is following: serratia marcescens resolves into glucose to sucrose (suc); Glucose is as unique carbon source and energy substance, and through glycolysis, glucose metabolism produces pyruvic acid; Pyruvic acid is a kind of important mesostate; A part pyruvic acid and diphosphothiamine (TPP) decarboxylation produces acetyl TPP, and this compound and another molecule pyruvic acid condensation form acetylactis, and acetylactis forms acetoin under the effect of acetylactis carboxylase; At last in reduced coenzyme (NADH) and 2; Obtained 2 under the effect of 3-butanediol dehydrogenation enzyme, the 3-butyleneglycol, final step is a reversible.
The invention provides a kind of recycle serratia marcescens and produce 2; The method of 3-butyleneglycol comprises: (1) is inserted in the fermention medium fermentation culture 30-50 hour with serratia marcescens kind daughter bacteria according to the inoculum size of volume ratio 3-15% (preferred 4-10%); Obtain containing 2; The fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid; (2) from fermented liquid, reclaim serratia marcescens; (3) serratia marcescens that reclaims is joined in the fermention medium, make the OD value of thalline in substratum be 20-80, fermentation culture 30-50 hour, from fermented liquid, separate results 2,3-butyleneglycol.Serratia marcescens in the fermented liquid also can reuse.
The serratia marcescens of described recovery is meant that described serratia marcescens has lived through at least once the fermentative prodn 2 of (production cycle), the reaction of 3-butyleneglycol, and it is different from kind of a daughter bacteria.As optimal way of the present invention, the serratia marcescens of described recovery lives through 1-5 time fermentative prodn 2, the reaction of 3-butyleneglycol; Better, the serratia marcescens of described recovery lives through 1-3 time fermentative prodn 2, the reaction of 3-butyleneglycol.
The method that reclaims serratia marcescens the fermented liquid after fermentation stops is the known technology of those skilled in the art, preferably through method Separation and Recovery serratia marcescens from fermented liquid centrifugal and the collection lower sediment.The method of centrifugal recovery thalline is that those skilled in the art are known, preferred centrifugal as follows: 4 ± 2 ℃, 10000 ± 3000rpm, 15-20min.The thalline of centrifugal acquisition can directly be used for fermentation reaction next time, perhaps can after drying, preserve subsequent use through conventional thalline store method.
The inventor finds under study for action; Reclaim the consumption of thalline when fermentation and be different from kind of a daughter bacteria; Be appropriate to fermentative prodn 2, under the culture condition of 3-butyleneglycol, the add-on that the serratia marcescens of recovery joins in the fermentation system is a comparatively The key factor; Be directly connected in the follow-up fermenting process 2, the transformation efficiency of the output of 3-butyleneglycol and sugar.Add-on is very few to cause 2, and the output of 3-butyleneglycol is not high, and add-on too much not only can not play purpose, the waste biomass that improve output, but also has influence on the sugared transformation efficiency in the reaction process.Therefore, when adding the recovery thalline to fermented liquid (or being called substratum), being preferably among the present invention and making the OD value of recovery thalline in fermented liquid is 20-80; More preferably be to make the OD value of thalline in substratum be 20-70; Further better is to make the OD value be 20-50.The biomass that adds can obtain preferably 2 in this described OD value scope, 3-butyleneglycol output, and higher sugared transformation efficiency, thus reach purpose energy-conservation, High-efficient Production.
The serratia marcescens that reclaims can add in the starting stage of fermentation, also can carry out replenishing in the process in fermentation adding.Also be; 2 of the routine that can adopt earlier, 3-butyleneglycol working method are fermented (for example adopting kind of a daughter bacteria to inoculate in the fermented liquid ferments), the serratia marcescens that reclaims are joined in the fermented liquid during the fermentation again; To improve 2, the output of 3-butyleneglycol or sugared transformation efficiency.
The inventor is surprised to find that, after fermentation culture has been carried out 15-26 hour, the serratia marcescens that reclaims is joined in the fermented liquid, can improve 2, the output of 3-butyleneglycol and sugared transformation efficiency.Preferably after fermentation culture has been carried out 20-26 hour, the serratia marcescens that reclaims is joined in the fermented liquid.Therefore; As optimal way of the present invention; Described 2, the working method of 3-butyleneglycol is following: (a) the kind daughter bacteria with serratia marcescens is inoculated in the fermention medium with the inoculum size according to volume ratio 3-10% (preferably according to volume ratio 4-6%), carries out fermentation culture; (b) after fermentation culture 15-26 hour, the serratia marcescens that reclaims is joined in the fermention medium, make the OD value of thalline in substratum be 20-80, continue fermentation culture.
The raw material (like carbon source, phosphorus source, organic nitrogen source, inorganic salt, trace element) and the usage quantity thereof that are used to prepare fermention medium can be utilized serratia marcescens fermentative prodn 2 according to this area routine; Used raw material and usage quantity during the 3-butyleneglycol and decide; As long as described fermention medium can improve enough nutritive ingredients for serratia marcescens growth, metabolism and production, and do not bring detrimentally affect (as checking effect) or spinoff (or producing by product).
As optimal way of the present invention, cultivate serratia marcescens fermentative prodn 2, the used initial medium of 3-butyleneglycol contains: sucrose 90 ± 20g/L, peptone 20 ± 5g/L, yeast powder 5 ± 2g/L, Hydrocerol A 10 ± 3g/L, MnSO
40.1 ± 0.05g/L, KH
2PO
40.5 ± 0.2g/L, FeSO
40.02 ± 0.005g/L, MgSO
40.5 ± 0.2g/L.More preferably, described fermention medium also contains: KAc6 ± 2g/L; NaNO
32 ± 0.5g/L.The initial p H value of the fermention medium of preferably, stating is 7 ± 0.5.After fermenting, PH is maintained 6 ± 0.5 to 6-12 hour.The method of pH value is that those skilled in the art are known in the control substratum, preferably can adopt H
2SO
4Solution and NaOH solution are regulated pH value in the substratum.
As optimal way of the present invention, during the fermentation, also stream adds or batch adds sucrose solution in fermentation system, makes that the sucrose amount can enough satisfy the required carbon source of fermenting in the fermented liquid.The preparation of sucrose solution can preferably be used the sucrose solution of 45-55% according to the known technology of those skilled in the art.
As optimal way of the present invention, leavening temperature is 28-32 ℃.
Utilize serratia marcescens to produce 2, other condition of 3-butyleneglycol can adopt condition known in the art.For conditions such as air flow, stirring velocitys, those skilled in the art also can be rule of thumb and the scale of fermentation carry out suitable accommodation.
Among the present invention, the thalline that fermentation is adopted can be a multiple serratia marcescens known in the art.As the preferred embodiments of the present invention, described serratia marcescens (Serratia marcescens) is the serratia marcescens that is numbered CICC10187.
After fermentative prodn finished, also can comprise step: isolated or purified 2 from fermention medium, the 3-butyleneglycol.Isolated or purified 2 from cultured products, and the 3-butyleneglycol can adopt separating and purifying technology well known to those skilled in the art.
The positively effect of method of the present invention is:
(1) the present invention is through reusing serratia marcescens during the fermentation, and optimized the add-on that reclaims thalline and adding opportunity, thereby improved 2 effectively, the output of 3-butyleneglycol, transformation efficiency and throughput.
(2) recycle method of the present invention is in fermented liquid, to add the serratia marcescens viable bacteria body of an amount of recovery; This method is compared with the method for traditional recycling thalline, has reduced the stages such as broken wall dissolving, filtration, separation and drying of waste energy resource.
(3) the present invention has reasonably utilized serratia marcescens to reclaim thalline, has solved the recycling problem of fouled by microzyme and resource effectively, has also reduced energy consumption, has reached the purpose of energy-saving and emission-reduction, thereby realizes cleaner production.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The cultivation of embodiment 1. serratia marcescens
Described serratia marcescens (Serratia marcescens) is provided by Chinese industrial microbial strains preservation administrative center, is numbered serratia marcescens CICC10187.
(1) seed culture
Glycerine is guaranteed the seed shaking table cultivation of Tibetan, and incubation time is 24 hours, transfers once in the time of 12 hours, and culture temperature is 30 ℃, 190r/min.
The component of substratum and content are (content in every liter): glucose 10g; Yeast powder 1g; Peptone 2g; (NH
4)
2SO
46g; K
2HPO
410g; NaCl 0.5g; MgSO
40.5g; Transfer pH6.6 with NaOH solution before the inoculation of sterilization back.Surplus is a water.
(2) fermentation culture
3.7L bioreactor culture: with cultivating in the inoculum size access fermention medium of seed liquor with 5% (volume ratio), fermention medium is (content in every liter):
Sucrose 90g; Peptone 20g; Yeast powder 5g; Hydrocerol A 10g; MnSO
40.1g; KH
2PO
40.5g; MgSO
40.5g; FeSO
40.02g; KAc6g; NaNO
32g; And the water of surplus; Transferring pH with NaOH solution before the sterilization is 7.2.
30 ℃ of leavening temperatures, ventilation flow rate 1vvm, air pressure 0.1-0.2; 6h is controlled at 450rpm before the rotating speed, after per hour increase by 50 and change, reach 650rpm to 10h, be maintained to 18h, every 5h reduces 50rpm behind the 18h, is maintained until fermentation ends when being reduced to 400rpm.Sucrose concentration adds to maintain through stream and enough satisfies the required carbon source of bacterial strain; Mend during 15h into an amount of bottle thalline that shakes, pH transfers to 6.0-6.5 when 9h, through H
3PO
4Solution and NaOH solution are kept pH at 6.0-6.5; The initial liquid amount of fermentor tank is the 3.7L canned fermented liquid 1.8L that ferments.The end in 40 hours of fermenting.
(3) reclaim thalline
After fermentation ends, from fermented liquid, separating 2, behind the 3-butyleneglycol product, from fermented liquid, reclaim serratia marcescens through centrifugation method.Centrifugal condition is following: 4 ℃, and 10000rpm, 15-20min.
The optimization that the amount of thalline is reclaimed in feasibility analysis that embodiment 2. thalline are recycled and adding fermentation
Pass through fermenting experiment; Insert the viable bacteria body (promptly reclaiming thalline) in fermentation latter stage of 3.7L bioreactor culture in the blank fermented liquid; Insert between the recovery thalline of different amounts in the research fermenting process; And and control group (do not add reclaim thalline) between output (g/l), the utilization ratio (g/g) of sucrose and the productive rate (g/l*h) of product of tunning whether notable difference is arranged, and examination adds the influence of different biomasses to each index.
Experiment material:
(1) seed culture medium (content in every liter): glucose 10g, yeast powder 1g, peptone 2g, (NH
4)
2SO
46g, KH
2PO
410g, NaCl0.5g, MgSO
40.5g.Surplus is a water.
(2) shake flask fermentation substratum (content in every liter): sucrose 90g, peptone 20g, yeast powder 5g, Hydrocerol A 10g, MnSO
40.1g, KH
2PO
40.5g, FeSO
40.02g, MgSO
40.5g.Surplus is a water.
Experimental technique:
Shake in the bottle and to insert 40h3.7L bio-reactor fermentation latter stage through the centrifugal recovery viable bacteria body that obtains, control group be the seed liquor of twice of the activation of normal access 5%, initial OD values table 1 specific as follows:
Table 1
| 0 group (control group) | 1 |
2 |
3 groups | |
| OD | 1.28 | 35.5 | 75.2 | 165.1 |
Fermentation condition is following:
30 ℃ of leavening temperatures, 190r/min, the fermenting process middle and later periods is with 50% aqueous sucrose solution feed supplement; Concentration of sucrose is controlled at enough satisfy bacterial strain required; Fermentation initially according on the said adding of table reclaim the viable bacteria body or plant daughter bacteria, pH transfers to 6.0-6.5 when 9h, through H
2SO
4Solution and NaOH solution are kept pH at 6.0-6.5; The liquid amount that shakes bottle is the bottled 50ml fermented liquid of 500ml triangle.
Experimental data and analysis:
1.OD analyze
The OD value that control group is 0 group meets the rule of microorganism growth, and the OD value is respectively 35.5 and 75.2 behind 1 group, the 2 groups thalline that add, and its OD descends slowly, and 3 groups of OD values that add behind the thalline are 165.1, and its OD descends apparent in view, like Fig. 1.
2.BD rate ratio
Added 1 group, 2 groups of thalline and 3 groups BD yield increased group and be significantly improved for 0 group, but 1 group, 2 groups and 3 groups are when fermentation 36h in latter stage, BD output is respectively 108.1g/l, 118.3g/l and 115.1g/l, and its difference is not obvious; And these the three groups OD value gradients that add behind the viable bacteria body are very big, are respectively 35.5,75.2 and 165.1, and this explanation is not that the thalline of adding is many more, and the output of BD is good more, and is as shown in Figure 2.
3. the productive rate of product (g/l*h) relatively
BD/T (g/l*h)=BD output (g/l)/time (h).
Three groups of product productive rates than control group that add centrifugal viable bacteria body are significantly improved, and three groups of product productive rate differences are little, as shown in Figure 3 when still fermenting latter stage.
4. the utilization ratio of sucrose relatively
Consumption sugar amount (g/l)=original fermented solution sugar degree (g/l)+benefit sugar amount (g/l)-residual sugar amount (g/l);
BD/ sugar consumption (g/g)=BD output (g/l)/sugared consumption (g/l);
Three groups of utilization ratios than the sucrose of control group that add centrifugal viable bacteria body are significantly improved, and 1 group and 2 groups are the highest, as shown in Figure 4 in the utilization ratios of the sucrose in fermentation latter stage.
5. analysis-by-synthesis
Comprehensive above-mentioned analysis, 1 group, 2 groups and the 3 groups OD values that add behind the centrifugal viable bacteria body are respectively 35.5,75.2 and 165.1, are significantly improved than the maximum OD value (15.4) of 0 group of control group; When fermentation 36h in latter stage; BD output (g/l), product productive rate (g/l*h) difference of 1 group, 2 groups and 3 groups are not obvious, and the utilization ratio (g/g) of 1 group, 2 groups sucrose is higher than 3 groups, explanation thus; The recovery thalline that is not adding is The more the better, and adding OD value is preferable at 20-80.
Test through shaking table; Fermented liquid inserts the viable bacteria body period at different fermentations; Relatively whether the output (g/l) of tunning, the utilization ratio (g/g) of sucrose, the productive rate (g/l*h) of product have notable difference between each group, and among several groups, confirm to insert the best period of viable bacteria body.
Experiment material:
(1) seed culture medium (content in every liter): glucose 10g, yeast powder 1g, peptone 2g, (NH
4)
2SO
46g, KH
2PO
410g, NaCl 0.5g, MgSO
40.5g.Surplus is a water.
(2) shake flask fermentation substratum (content in every liter): sucrose 90g, peptone 20g, yeast powder 5g, Hydrocerol A 10g, MnSO
40.1g, KH
2PO
40.5g, FeSO
40.02g, MgSO
40.5g.Surplus is a water.
Experimental technique:
1 group, 2 groups, 3 groups, 4 groups are respectively fermention medium and insert when fermentation during 0h, 6h, during 12h and during 24h and reclaim thalline, detect output (g/l), the utilization ratio (g/g) of sucrose and the productive rate (g/l*h) of product of the tunning of each group.
Experimental procedure:
(1) insert thalline in the fermention medium, table 2 specific as follows:
Table 2
| 1 group (control group) | 2 |
3 |
4 groups |
| Fermented to insert in the fermention medium of 0h and reclaimed thalline | Fermented to insert in the fermention medium of 6h and reclaimed thalline | Fermented to insert in the fermention medium of 12h and reclaimed thalline | Fermented to insert in the fermention medium of 24h and reclaimed thalline |
(2) shaking table is cultivated: with cultivating in the inoculum size access fermention medium of seed liquor with 5% (volume ratio); The condition of shake-flask culture is 30 ℃ of leavening temperatures, 190r/min, and the fermenting process middle and later periods is with 50% aqueous sucrose solution feed supplement; Concentration of sucrose is controlled at enough satisfy bacterial strain required; Mend into an amount of recovery viable bacteria body in the said moment of last table, pH transfers to 6.0-6.5 when 9h, through H
2SO
4Solution and NaOH solution are kept pH at 6.0-6.5.
(3) respectively organize product output with the gas chromatograph survey.
(4) and calculate the output (g/l) of product, the utilization ratio (g/g) of sucrose and the productive rate (g/l*h) of product.
Experimental result and analysis:
1.OD relatively
1 group, 2 groups, 3 groups and the 4 groups fermented liquids that are respectively fermented 0h, 6h, 12h and 24h insert the recovery thalline, and the OD that inserts thalline is respectively 25.8,24.2,23.5 and 22.5, and it is close to mend biomass, therefore has comparability; 1 group and increase of 2 groups of OD values elder generations and then decline, 3 groups, 4 groups OD values have only downtrending, and are as shown in Figure 5.
2. sugar degree analysis
Sugar degree is relatively seen Fig. 6, and the zig-zag broken line is represented the sugar degree after each residual sugar amount of organizing of a certain moment and this are mended sugar constantly among the figure.
3.BD rate ratio
The output of 1 group of BD is all lower in each time period, and the rate ratio of 2 groups, 3 groups and 4 groups BD is more approaching, and 4 groups of output are higher, like Fig. 7.
4.BD the productivity ratio of product
BD/T (g/l*h)=BD output (g/l)/Time (h)
The productive rate of product (g/l*h) comparative result, 1 group of productive rate is minimum, 4 groups when 24h productive rate higher, as shown in Figure 8.
5. the utilization ratio of sucrose relatively
Can calculate sugared consumption according to residual sugar and the sugared situation of benefit:
Consumption sugar amount (g/l)=original fermented solution sugar degree (g/l)+benefit sugar amount (g/l)-residual sugar amount (g/l).
BD/ sugar consumption (g/g)=BD output (g/l)/sugared consumption (g/l).
The comparison of the utilization ratio of sucrose (g/g):
4 groups of sucrose utilization ratios when 24h and 30h are obviously high, as shown in Figure 9 than other group.
6. analysis-by-synthesis
In sum, 1 group, 2 groups, 3 groups and 4 groups are mended respectively into aforementioned fermentation when the 0h that fermented, 6h, 12h and 24h respectively and are reclaimed thalline, mend the OD that goes into and are respectively 25.8,24.2,23.5 and 22.5, and it is close to mend the amount of going into, and therefore has comparability; According to analysis to the productive rate (g/l*h) of the utilization ratio (g/g) of the output (g/l) of product, sucrose and product; The situation of 4 groups (mending into the recovery thalline during 24h) is best; When 30h; The utilization ratio (g/g) of the output of product (g/l), sucrose and the productive rate (g/l*h) of product are respectively 58.6g/l, 0.42g/g and 1.95g/l*h, therefore, mend into recovery thalline situation preferable during 24h.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (11)
1. one kind is utilized serratia marcescens (Serratia marcescens) to produce 2, and the method for 3-butyleneglycol is characterized in that, said method comprises:
(1) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-15% inserted in the fermention medium, fermentation culture 30-50 hour, obtain containing 2, the fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid;
(2) from fermented liquid, reclaim serratia marcescens;
(3) serratia marcescens that reclaims is joined in the fermention medium, make the OD value of thalline in substratum be 20-80, fermentation culture 30-50 hour, from fermented liquid, separate results 2,3-butyleneglycol.
2. the method for claim 1 is characterized in that, in step (3), makes the OD value of serratia marcescens in substratum be 30-70.
3. the method for claim 1 is characterized in that, in step (1), described recovery serratia marcescens is from fermented liquid, to separate acquisition through centrifugation method.
4. method as claimed in claim 3 is characterized in that, the centrifugal condition is following: 4 ± 2 ℃, and 10000 ± 3000rpm, 15-20min.
5. the method for claim 1 is characterized in that, also comprises in step (3) back:
Repeating step (2)-(3) 1-5 time.
6. the method for claim 1 is characterized in that, described fermention medium contains:
Sucrose 90 ± 20g/L, peptone 20 ± 5g/L, yeast powder 5 ± 2g/L, Hydrocerol A 10 ± 3g/L, MnSO
40.1 ± 0.05g/L, KH
2PO
40.5 ± 0.2g/L, FeSO
40.02 ± 0.005g/L, MgSO
40.5 ± 0.2g/L.
7. method as claimed in claim 6 is characterized in that, described fermention medium also contains: KAc 6 ± 2g/L; NaNO
32 ± 0.5g/L.
8. the method for claim 1 is characterized in that, leavening temperature is 28-32 ℃.
9. one kind is utilized serratia marcescens to produce 2, and the method for 3-butyleneglycol is characterized in that, said method comprises:
(a) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-15% inserted in the fermention medium, fermentation culture 30-50 hour, obtain containing 2, the fermented liquid of 3-butyleneglycol separates results 2,3-butyleneglycol product from fermented liquid;
(b) from fermented liquid, reclaim serratia marcescens;
(c) inoculum size of serratia marcescens kind daughter bacteria according to volume ratio 3-10% inserted in the fermention medium, fermentation culture was to 15-26 hour, and the serratia marcescens that (b) reclaimed joins in the fermention medium; Make the OD value of thalline in substratum be 20-80; Continue fermentation culture 4-25 hour, and obtained containing 2, the fermented liquid of 3-butyleneglycol; From fermented liquid, separate results 2,3-butyleneglycol product.
10. method as claimed in claim 9 is characterized in that, in step (c), after fermentation culture 20-25 hour, the serratia marcescens that (b) reclaimed joins in the fermention medium.
11. method as claimed in claim 9 is characterized in that, also comprises in step (c) back:
Repeating step (b)-(c) 1-5 time.
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