CN101812482A - Method for preparing PHBV (polyhydroxybutyrate-hydroxyvalerate) by using lignocellulose raw material - Google Patents
Method for preparing PHBV (polyhydroxybutyrate-hydroxyvalerate) by using lignocellulose raw material Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 53
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 title claims abstract description 50
- 239000002994 raw material Substances 0.000 title claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 229940106157 cellulase Drugs 0.000 claims abstract description 24
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 18
- 241000252867 Cupriavidus metallidurans Species 0.000 claims abstract description 18
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 15
- 238000001784 detoxification Methods 0.000 claims abstract description 15
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing PHBV by utilizing a lignocellulose raw material, which is technically characterized in that the lignocellulose raw material is pretreated and then is hydrolyzed by cellulase by a fed-batch method to obtain higher sugar concentration, activated carbon prepared by activating enzymolysis liquid by zinc chloride or phosphoric acid is collected for detoxification and decoloration, and the detoxified enzymolysis sugar liquid is used as a unique carbon source for synthesizing PHBV by fermenting alcaligenes eutrophus. The invention has important significance for developing and utilizing abundant crop straw resources in China, protecting the environment better and reducing white pollution, and has the characteristics of abundant raw materials, low cost and suitability for industrial application.
Description
Technical field
The invention belongs to technical field of biochemical industry, relate to a kind of method of utilizing straw lignocellulose for primary industry production PHBV.
Background technology
In recent years because global petroleum resources reduce and a large amount of plasthetics use the serious white pollution problems that brings later, havoc existent environment of people.Because in order to protect existent environment of people, the research of biodegradable material and production have been subjected to people and have paid close attention to widely.Wherein, a kind of new bio polymer 3-hydroxybutyric acid-co-3-hydroxyl pentanoate copolymer (being PHBV) is considered to the instead of chemical synthetic plastics, captures the new thermoplasticity polyester of the tool potentiality of white pollution, and the molecular structural formula of PHBV is as follows:
PHBV is to be not raw material with oil, Sweet natural gas, but utilizes starch and glucose as raw material, the biomaterial of producing by microbial fermentation technology.It is easy to by bacterial digestion, is decomposed into carbonic acid gas and water fully under soil or composting condition, does not have harmful toxic matter to generate, and environment is not polluted.Because PHBV has many excellent characteristic such as biodegradability, biocompatibility, piezoelectricity, optical activity, can be used widely at aspects such as various fields such as biodegradable wrapping material, tissue engineering material, slow-release material and electricity materials.Along with the aggravation of energy dilemma in the world wide and the enhancing of environmental protection consciousness, the exploitation of PHBV class material and utilization have caused the extensive interest of scientific research field and industry member.
The suitability for industrialized production of PHBV and the greatest problem that widespread use faced thereof are the too high problems of its production cost at present, and therefore it mainly still is applied in medical science and other demanding fields at present.The general technology flow process of producing PHBV with fermentation using bacteria is as follows:
Spawn culture → fermentation → Pasteur's method sterilization → precipitation → separation → throw out → extracting → drying
Because the selection problem of operational path only has enterprise of several family to produce at present in the world,, also all fail to realize scale operation because of production cost is very high.Institute of Micro-biology of China Chinese Academy of Sciences has developed at present with Dian Fentang and propionic fermentation and has produced the PHBV technology, and is equipped with the production equipment that limit company has set up an annual output 1kt PHBV cooperatively with the sky, Hangzhou.Along with succeed in developing, China has reached international most advanced level in the research and the production in this field, indicates that also China is stepping a new step aspect the production of biodegradable plastic.
Natural plant fibre is the renewable resources that enriches that obtains easily, such as, the annual agricultural crop straw output of China reaches 1,000,000,000 tons at present.Therefore, can produce PHBV to reduce the raw materials cost of fermentation, will get more and more people's extensive concerning by utilizing cheap natural cellulosic feedstocks.
At present, lignocellulose raw material hydrolysis and saccharification technology commonly used has two kinds to be respectively acid hydrolysis and enzymic hydrolysis.
Acid hydrolysis speed is finished soon and easily, and the mineral acid of acid hydrolysis employing is H usually
2SO
4Or HCl, 160～240 ℃ of temperature.Cellulosic material not only contains fermentable sugar in pyroprocessing posthydrolysis liquid, and contain fermentation inhibitors such as furfural and 5 hydroxymethyl furfural, produced by the sugar degraded, these compounds have suppressed the fermentation of microorganism to sugar, therefore need fermentation inhibitors such as alditol and 5-hydroxymethylfurfural are removed before fermentation.The method that removes is a lot, as lime neutralisation, method of evaporation, sulphite pre-treatment, ion-exchange, enzyme, activated carbon treatment etc., can remove the part fermentation inhibitor, but processing cost is too high, is difficult in the production realize.
Therefore, what cellulosic hydrolysis was mainly adopted now is cellulase hydrolysis, and fibrous material must could obtain higher enzymolysis transformation efficiency through pre-treatment.Pretreatment process can reduce the consumption of enzyme preferably.The factor that influences cellulose hydrolysis comprises the degree of crystallinity of porousness, cellulose bundle and the content of xylogen and hemicellulose.It is very difficult that the existence of xylogen and hemicellulose makes that cellulase contacts Mierocrystalline cellulose, therefore reduced hydrolysis efficiency.Xylogen and hemicellulose are removed in pre-treatment, reduce cellulosic degree of crystallinity and increase porousness, can significantly improve hydrolysis efficiency.A lot of pretreatment process purposes of fibrous material are to remove hemicellulose and xylogen, but the contact area of increased fiber element, and then improve hydrolysis efficiency.Because xylogen, hemicellulose are to the crystalline texture of cellulosic provide protection and Mierocrystalline cellulose itself, when natural cellulose raw material directly was hydrolyzed, its hydrolysis degree was very low, so must carry out certain pre-treatment to Mierocrystalline cellulose.
At present, lignocellulose enzyme digestion reaction pretreatment process is a lot, comprises physical method, chemical process and biological method etc.Fibrous material physics pretreatment process commonly used is a mechanical crushing method, by cutting, grind and technology such as mill diminishing the granularity of fibrous material, increases the surface-area that substrate contacts with enzyme, reduces cellulosic degree of crystallinity.Comprise pulverizing, ball milling, the compression technology such as pulverizing, ultrasonic wave of milling.Steam explosion is one of more effective pretreatment process of research both at home and abroad at present.Used steam explosion is with steam raw material to be heated to 160～260 ℃ at present, keeping 20s～30min causes lignocellulose softening under High Temperature High Pressure, make the raw material decompression cause Mierocrystalline cellulose crystal and cellulosic breaking then rapidly, xylogen separates with Mierocrystalline cellulose, and hemicellulose at high temperature takes place can dissolve from hydrolytic action, and the part degraded also takes place xylogen.
Above preprocessing process is handled in conjunction with diluted acid, diluted alkaline method can significantly improve enzymolysis efficiency.But high temperature or diluted acid diluted alkaline treating processes can cause cellulose degradation to generate fermentation inhibitor, so the removal effect of fermentation inhibitor has directly influenced the application of follow-up fermentation process.
Gac is a kind of brute force sorbent material and discoloring agent efficiently, is widely used in water purification, adsorbs obnoxious flavour and various flowing products and beverage are carried out many aspects such as decolorization and purification.Gac is very strong to the decolouring usefulness of liquid glucose, external many sugar refinery of making refined sugar, will be with gac through the liquid glucose decolorization and purification after the clarifying treatment, " gac-liquid glucose discoloring agent " efficiently (Guangxi light industry, a 2003) literary composition in Huo Han town is studied the sugar decolouring theory of gac.It is raw material that gac is normally used various crushed aggregate of plant (wood chip, coconut husk and bagasse etc.) or suitable coal or charcoal, makes through special processing treatment.There are a lot of atomic thin holes the inside of gac.The diameter of hole is very little, so total surface area is very big, and very strong adsorptive power is arranged.Different because of raw material and manufacture method, the kind of gac is quite a lot of, is applicable to different purposes respectively.
Making gac generally will be through charing and two step of activation.Especially activation, this is a committed step of making gac, and two class methods are arranged: the one, the gas activation method is an activator with water vapour or carbonic acid gas etc.; Another is the chemical agent activation method, is activator with zinc chloride or phosphoric acid etc.The gas activation method is that charcoal is at high temperature reacted with gas, and carbide surface is corroded, and forms the structure of micropore prosperity, and the shape of product is based on particulate state, and internal microcellular structure is flourishing, and surface-area is big, the loading capacity height.Be suitable for adsorbing the less material of molecular weight in the liquid and gas, but should not be used for absorption macromolecular substance.Chemical activation zinc chloride commonly used or phosphoric acid are made activator, the gac that utilizes zinc chloride or phosphoric acid activation to make, and the size of hole is big slightly usually, and this class gac is applicable to the macromolecular substance in the adsorbent solution, for example decolouring of liquid glucose.Gac has the structure of fragrant ring type, is good at adsorbing aromatic series organism (the colored substance major part in the syrup belongs to this class), and is good at adsorbing other organism that contain more than three carbon atoms.It is stronger to the adsorptive power of neutral species, and a little less than the absorption to charge species (as negatively charged ion).Absorption to the latter is relevant with the pH value of solution value: in the acidic solution absorption stronger, in the basic solution a little less than.The adsorption of gac is relevant with temperature.For the physisorption of majority, can reach bigger adsorptive capacity at low temperatures, but the speed of absorption is slower.Use in most cases in sugar refinery, the time that gac contact with liquid glucose is not long, so require absorption to carry out comparatively fast, just uses higher temperature always, 70～85 ℃ under this temperature, general adsorption through 15～30 minutes gacs is just near its maximum value.
Chinese patent CN1186461C discloses a kind of method for preparing lower polyxylose, use xylanase hydrolysis after utilizing the corn cob alkaline purification, hydrolyzed solution is prepared xylan with gac and ion-exchange resin decolorization, this patent has been introduced the method for xylan decolouring, relative complex operation is not suitable for the fermentation system of subsequent request decolouring detoxification.Chinese patent CN1020042C discloses a kind of preparation method of crystalline xylose, with bagasse is that raw material obtains dilute acid hydrolysis liquid, decolour together with Zeo-karb and gac, processes such as this process need concentrating and precipitating, operate more loaded down with trivial detailsly, be not suitable for the fermentation system that subsequent request decolouring detoxification is arranged.It is the method that raw material application enzyme and membrane technique prepare high-purity oligoxylose with the stalk that Chinese patent CN1020042C discloses a kind of, the enzymolysis liquid glucose is utilized the processing of decolouring of nanofiltration, gac, ion exchange resin, obtain xylo-oligosaccharide, be not suitable for the fermentation system that subsequent request decolouring detoxification is arranged.More than for cellulosic material hydrolyzate decolorizing process, be not suitable for the fermenting process that the detoxification requirement is arranged.
Produce PHBV technology at alcaligenes eutrophus (Alcaligenes eutrophus), in flat (the culture condition optimization of alcaligenes eutrophus fermentative production PHBV, the Chinese food journal, 2007), stifled state becomes (synthetic 3-hydroxybutyric acid of alcaligenes eutrophus and 3-hydroxyl pentanoate copolymer Fermentation Conditions, the microorganism journal, 1999), Yang Ming (the research of the poly-B-hydroxybutyric acid (PHBV) of alcaligenes eutrophus fermentative production, food and fermentation industries, 1999) etc. usefulness glucose is that carbon source is studied the condition that alcaligenes eutrophus produces PHBV, and the extensive supply of raw material can be restricted comparatively speaking.Therefore, for alcaligenes eutrophus is produced PHBV technology, the problem that prior art exists can be summed up as: (1) microbial fermentation raw material mainly is starch or grape glucide, and raw material production cost height is if scale operation meeting existence and people strive the problem of grain; (2) the cellulosic material pretreatment technology need use the high temperature high pressure process method mostly; Diluted acid or diluted alkaline that pretreatment stage uses can form degraded product, and these degraded products can produce toxic action to the fermentation of follow-up microorganism, must carry out the detoxification decolouring to the enzymolysis liquid glucose and handle, and could be utilized by microorganism; (3) the decolouring detoxification process of enzymolysis solution is used chemical reagent and material is more, even can introduce new impurity atom, and complicated operation has increased raw materials cost.
Summary of the invention
Technical purpose of the present invention has been to provide the new lignocellulosic material that utilizes of a kind of usefulness to produce the method for PHBV, makes this method have the characteristics of abundant raw material, with low cost, suitable industrial applications.
Above purpose the present invention is achieved through the following technical solutions in order to reach:
A kind of method of utilizing lignocellulosic material to prepare PHBV, it is characterized in that lignocellulosic material is through after the pre-treatment, use cellulase hydrolysis, collect enzymolysis solution and be prepared into fermention medium, and carry out fermentative production PHBV with the gac detoxification decolouring sole carbon source that PHBV is synthesized in fermentation as Ralstonia eutropha.
Method for preprocessing lignocellulose raw material of the present invention is the prior art of any lignocellulose pretreatment method, for example steam explosion, high-temperature water facture, ball milled, screw extrusion method etc.;
Can add before the feed pretreatment step of the present invention 0～1.0% diluted acid or diluted alkaline as auxiliary to improve follow-up enzymolysis efficiency;
Wherein, described diluted acid comprises HCl or H
2SO
4Diluted alkaline comprises NaOH, KOH;
Enzymatic saccharification step of the present invention: pretreated raw material adds substrate according to initial solid-to-liquid ratio at 1: 10, regulating the pH value is 4.5～5.0, interval 24h adds cellulosic material 1～4 time in the enzymolysis process, keeping the enzymatic hydrolysis system solid content is 10%, according to the amount adding cellulase of 5.0～30.0FPU/g raw material, 50 ℃ of temperature of reaction, stir enzymolysis 36～120h, after enzymolysis finished, the solids removed by filtration residue obtained the enzymolysis liquid glucose;
Gac of the present invention is the gac that makes with zinc chloride activation or phosphoric acid activation;
The decolorization condition of gac of the present invention is pH2.5～8.0,40 ℃～100 ℃ of temperature, powdered carbon dosage 0.1～5%, 0.1～3h;
The culture medium culturing condition of Ralstonia eutropha fermentative production PHBV of the present invention is: carbon source is that glucose concn is 2.0～10.0% cellulase hydrolysis liquid, ammonium sulfate 0.1～0.5%; Regulate pH7.0～8.0 with ammoniacal liquor, inoculum size is cultivated 35～50h for 10%, 32～38 ℃;
Lignocellulose of the present invention comprises stalk, sorghum stalk, bagasse or careless class.
Beneficial effect of the present invention is:
1, directly add the gac that zinc chloride or phosphoric acid activation make in the enzymolysis liquid glucose, not only have good decolorizing effect, and can remove various fermentation inhibitors, and this process do not have the introducing of impurity atom, simple to operate.
2, utilize lignocellulosic material to produce PHBV, thereby polymerizable obtains final product PHBV, have the characteristics of abundant raw material, with low cost, suitable industrial applications.
3, the popularization of biomaterial PHBV and use have good biodegradable, have reduced the white pollution in the environment.
Embodiment
The calculation of correlation formula and the measuring method that use in the embodiment of the invention are as follows:
The overall enzyme activity determination of 1, the mensuration of filter paper enzyme activity---cellulase:
[Ghose, T.K., et al.Pure﹠amp are measured in the standard method of adopting international theory and applied chemistry association (IUPAC) to recommend; Appl.Chem., 1987,59,257～268], the international unit of a filter paper enzyme activity (FPIU) equals the enzyme amount that under standard reaction condition per minute generates 1 μ mol glucose amount.Measuring method is as follows:
Get 3 25mL scale test tubes and add 50mg respectively therein and be ground into pulverous filter paper, the plain enzyme of the anchoring fiber of certain mass, 0.05M citrate buffer solution 5.0mL.
Above-mentioned test tube is placed thermostatical water bath, remain on amplitude 100r/min and temperature and take out adding 5.0mL DNS reagent immediately behind the insulation 60min down for 50 ℃, in boiling water, react 5min, add water to 25mL after the cooling and shake up, measure the absorbance A value down in the 550nm wavelength.The amount of the glucose that reaction generates is tried to achieve according to the glucose typical curve; Calculate and generate the relative enzyme amount of 2mg glucose, be calculated as follows the filter paper enzyme activity (FPU) of sample, net result is averaged:
Wherein, a filter paper enzyme activity unit definition is that per minute generates the required enzyme amount of 1 μ mol glucose, unit: IU/mL.
The filter paper enzyme activity 93FPIU/mL of cellulase Accellerase 1000 used in the present invention (available from international corporation of outstanding person's energy section).
2, the PHBV Determination on content adopts vapor-phase chromatography.
Take by weighing the 50mg stem cell, adding 2mL chloroform, 2mL acidifying methyl alcohol (contain 3%H
2SO
4), place the cock test tube, 100 ℃ of esterification 4h, cooling adds 1mL distilled water, thermal agitation 2min, layering, the gas chromatographic analysis of chloroform phase calculates the percentage composition of PHBV in the sample.
PHBV mass concentration (g/L)=dry cell weight (g/L) * PHBV percentage composition
Embodiment 1
Raw materials pretreatment: maize straw is steamed quick-fried processing 5min in 210 ℃, regulating the pH value is 5.0,50 ℃ of temperature of reaction, add cellulase 15FPU/g raw material, solid-to-liquid ratio 1: 10, behind the 24h feed supplement once, keeping the enzymatic hydrolysis system solid content is 10%, stir enzymolysis 36h, the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: phosphoric acid activation gac (river, Liyang City Yang Huoxingtanchang) decolorization condition is pH2.5,40 ℃ of temperature, powdered carbon dosage 5%, 0.1h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 2.0%, ammonium sulfate 0.1% is regulated pH7.0 with ammoniacal liquor, 32 ℃, cultivates 35h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 0.5g/L.
Embodiment 2
Raw materials pretreatment: 180 ℃ of maize straws, 1.0% dilute sulphuric acid, 25min handles, and getting material adjusting pH value is 4.5, add cellulase 15FPU/g raw material, 50 ℃ of temperature of reaction, solid-to-liquid ratio 1: 10,24, enzymolysis 78h is stirred in feed supplement twice behind the 48h, and the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: zinc chloride activation gac (Shanxi Xinhua gac) decolorization condition is pH8.0,100 ℃ of temperature, powdered carbon dosage 0.1%, 3.0h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 10.0%, ammonium sulfate 0.1% is regulated pH8.0 with ammoniacal liquor, 38 ℃, cultivates 50h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 25g/L.
Embodiment 3
Raw materials pretreatment: bagasse adds the 1.0%NaOH ball milling, and 25min handles, and getting material adjusting pH value is 4.5, add cellulase 30FPU/g raw material, 50 ℃ of temperature of reaction, solid-to-liquid ratio 1: 10, feed supplement is four times behind 24h, 48h, 72h, the 96h, stirs enzymolysis 120h, and the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: phosphoric acid activation gac (river, Liyang City Yang Huoxingtanchang) decolorization condition is pH8.0,80 ℃ of temperature, powdered carbon dosage 2.1%, 1.0h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 9.0%, ammonium sulfate 0.1% is regulated pH7.8 with ammoniacal liquor, 38 ℃, cultivates 48h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 15g/L.
Embodiment 4
Raw materials pretreatment: 210 ℃ of jowar stalks, 0.5% dilute hydrochloric acid 5min steams quick-fried processing, and regulating the pH value is 5.0,50 ℃ of temperature of reaction add cellulase 15FPU/g raw material, solid-to-liquid ratio 1: 10, feed supplement twice behind 24h, the 48h, stirs enzymolysis 72h, and the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: phosphoric acid activation gac (Harbin gac factory) decolorization condition is pH2.5,40 ℃ of temperature, powdered carbon dosage 5%, 0.1h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 8.0%, ammonium sulfate 0.1% is regulated pH7.0 with ammoniacal liquor, 32 ℃, cultivates 48h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 19g/L.
Embodiment 5
Raw materials pretreatment: straw, 180 ℃ of screw extrusions of screw rod handle twice, and regulating the pH value is 4.5,50 ℃ of temperature of reaction add cellulase 5FPU/g raw material, solid-to-liquid ratio 1: 10, feed supplement twice behind 24h, the 48h, stirs enzymolysis 72h, and the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: phosphoric acid activation gac (river, Liyang City Yang Huoxingtanchang) decolorization condition is pH2.5,40 ℃ of temperature, powdered carbon dosage 5%, 0.1h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 6.0%, ammonium sulfate 0.5% is regulated pH7.0 with ammoniacal liquor, 38 ℃, cultivates 40h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 15g/L.
Embodiment 6
Raw materials pretreatment: straw, 210 ℃ of high-temperature water are handled, and getting material adjusting pH value is 4.7, and 50 ℃ of temperature of reaction add cellulase 5FPU/g raw material, solid-to-liquid ratio 1: 10, feed supplement twice behind 24h, the 48h, stirs enzymolysis 72h, and the enzymolysis after-filtration is got enzymolysis solution.
The detoxification decolouring: phosphoric acid activation gac (Gongyi City surmounts filtrate factory) decolorization condition is pH5,40 ℃ of temperature, powdered carbon dosage 3%, 0.1h.
Inoculation fermentation: activated liquid alcaligenes eutrophus ATCC 17699, inoculum size 10%, cellulase hydrolysis liquid glucose concn 9.0%, ammonium sulfate 1.5% is regulated pH7.0 with ammoniacal liquor, 38 ℃, cultivates 40h.
The product separation and Extraction: fermented liquid is centrifugal, gets thalline, after the drying, measures the PHBV mass concentration and can reach 25g/L.
Claims (9)
1. method of utilizing lignocellulosic material to prepare PHBV, it is characterized in that lignocellulosic material is through after the pre-treatment, use cellulase hydrolysis, collect enzymolysis solution and be prepared into fermention medium, and carry out fermentative production PHBV with the gac detoxification decolouring sole carbon source that PHBV is synthesized in fermentation as Ralstonia eutropha.
2. the method for utilizing lignocellulosic material to prepare PHBV according to claim 1 is characterized in that described lignocellulose pretreatment method comprises steam explosion, high-temperature water facture, ball milled, screw extrusion method.
3. the method for utilizing lignocellulosic material to prepare PHBV according to claim 2 is characterized in that can adding before the described lignocellulose pre-treatment 0～1.0% diluted acid or diluted alkaline as auxiliary.
4. the method for utilizing lignocellulosic material to prepare PHBV according to claim 3 is characterized in that described diluted acid comprises HCl or H
2SO
4
5. the method for utilizing lignocellulosic material to prepare PHBV according to claim 3 is characterized in that described diluted alkaline comprises NaOH or KOH.
6. the method for utilizing lignocellulosic material to prepare PHBV according to claim 1, the method that it is characterized in that described cellulase hydrolysis is: pretreated raw material adds substrate according to initial solid-to-liquid ratio at 1: 10, regulating the pH value is 4.5～5.0, interval 24h adds cellulosic material 1～4 time in the enzymolysis process, keeping the enzymatic hydrolysis system solid content is 10%, amount according to 5.0～30.0FPU/g raw material adds cellulase, 50 ℃ of temperature of reaction, stir enzymolysis 36～120h, after enzymolysis finishes, the solids removed by filtration residue obtains the enzymolysis liquid glucose.
7. the method for utilizing lignocellulosic material to prepare PHBV according to claim 1, the decolorization condition that it is characterized in that described gac is pH2.5～8.0,40 ℃～100 ℃ of temperature, powdered carbon dosage 0.1～5%, 0.1～3h.
8. the method for utilizing lignocellulosic material to prepare PHBV according to claim 1, the culture medium culturing condition that it is characterized in that described Ralstonia eutropha fermentative production PHBV is: carbon source is that glucose concn is 2.0～10.0% cellulase hydrolysis liquid, ammonium sulfate 0.1～0.5% is regulated pH7.0～8.0 with ammoniacal liquor; Inoculum size is cultivated 35～50h for 10%, 32～38 ℃.
9. according to the described method of utilizing lignocellulosic material to prepare PHBV of one of claim 1 to 9, it is characterized in that described lignocellulose comprises stalk, sorghum stalk, bagasse or careless class.
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| CN114149844A (en) * | 2021-12-07 | 2022-03-08 | 上海海事大学 | Preparation method of multifunctional modified molybdenum disulfide nano additive |
| CN114377660A (en) * | 2021-12-15 | 2022-04-22 | 绍兴市上虞区武汉理工大学高等研究院 | Preparation method of BC/PHB composite material for removing heavy metal ions |
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| CN114149844A (en) * | 2021-12-07 | 2022-03-08 | 上海海事大学 | Preparation method of multifunctional modified molybdenum disulfide nano additive |
| CN114377660A (en) * | 2021-12-15 | 2022-04-22 | 绍兴市上虞区武汉理工大学高等研究院 | Preparation method of BC/PHB composite material for removing heavy metal ions |
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