CN103642773B - A kind of manioc waste anaerobic fermentation residue cellulase-producing and method for saccharifying thereof - Google Patents
A kind of manioc waste anaerobic fermentation residue cellulase-producing and method for saccharifying thereof Download PDFInfo
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- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The cellulase-producing and the method for saccharifying that the present invention relates to a kind of manioc waste anaerobic fermentation residue, belong to microbial technology field. Relate to and utilize manioc waste anaerobic fermentation residue to produce high activity as geotrichum sp carbon source<i>Galactomyces</i>The method of sp.CCZU11-1 cellulase, has efficiently utilized manioc waste anaerobic fermentation residue. The cellulase of producing can efficiently saccharifying 1-ethyl-1-methyl-imidazoles-diethyl phosphate ([Emim] [DEP]) pretreated manioc waste anaerobic fermentation of-phosphoric acid-aqueous systems residue, always conversion coefficient >40%. Technique of the present invention is simple, the advantage such as have that efficiency is high, process economy, the three wastes are few.
Description
Technical field
The invention discloses one utilizes manioc waste anaerobic fermentation residue for carbon source grown place mould GalactomycesSp. the method for tieing up plain enzyme and saccharification thereof, belongs to microbial technology field.
Background technology
Solid waste-manioc waste that tapioca produces in deep-processing process, contains abundant wooden in its buttCrude fibre, a small amount of starch and albumen. Crude fibre stability is higher, and is difficult for decomposing difficult treatment. According to statistics, every processing1000000 tons of cassava raw materials, will produce the manioc waste discarded object of approximately 200,000 tons. If direct this discarded object discharge or burning can be madeBecome the huge waste of environmental pollution and lignocellulosic resource. Utilizing manioc waste anaerobic fermentation to prepare biogas is a kind of important wayFootpath, but the biogas residue of its generation still contains a large amount of lignocellulosics. If rationally do not utilized, equally also canCause the problem such as environmental pollution and the lignocellulosic wasting of resources. Therefore, the efficient utilization of manioc waste anaerobic fermentation residue is valueThe problem that must study.
It should be noted that efficiently saccharifying manioc waste biogas residue, the production that can be bio-fuel provides the former of cheapnessMaterial. But, due to lignin, the crystalline texture of hemicellulose to cellulosic protective effect and cellulose itself, celluloseWhen raw material is directly hydrolyzed, its hydrolysis degree is very low. Therefore, must carry out effective pretreatment to cellulosic material.The cellulose preconditioning techniques such as Physical, chemical method, physical-chemical process and the bioanalysis occurring at present exist generally energy consumption andThe problem such as cost is high, efficiency is low and scale is difficult. Wherein, though the scale of diluted acid method exists that productive rate is low, in productHarmful side product affects further bio-transformation utilization. Bioanalysis is environmentally friendly, and energy consumption is low and accessory substance is few, but transformation efficiencyLow, cellulase activity is poor and cost is high. Adopt current conventional method to be difficult to cellulose Efficient Conversion to become carbohydrate chemical combinationThing, this has become the bottleneck that restriction cellulose Efficient Conversion utilizes. The expense of the quick-fried pretreating process of vapour is lower, but processes quality notStable. In recent years research shows that ionic liquid has good cellulose dissolution and disposal ability, wherein room temperature type ionic liquidShow more outstanding advantage. They are different from typical organic solvent, there is no electroneutral molecule in ionic liquid, withTraditional organic solvent is compared, that ionic liquid possesses is non-volatile, low melting point, high stability, incombustibility, good conduction andThe features such as thermal conductivity selective dissolution power, are called as " green solvent ". Utilize " green solvent " ionic liquid pretreatment cellulose,The crystalline texture that can destroy cellulose height itself, makes it loose, is easier to enzymolysis, has greatly improved its conversion coefficient.
Our seminar finds to utilize ionic liquid 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim][DEP]) aqueous solution as pretreating reagent, can improve the conversion coefficient of cellulosic material with the mixed liquor of a small amount of phosphoric acid greatly, subtractsThe consumption of few ionic liquid 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP]). Simultaneously. This pretreatment sideMethod not only can reduce the viscosity of ionic liquid 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP]), also canDissolve so that cellulose fully expands more complete, pretreatment of fiber element raw material more efficiently. The present invention has invented manioc wasteAnaerobic fermentation residue is the side that carbon source is produced high activity Galactomycessp.CCZU11-1 cellulase and efficiently saccharifying thereofMethod, has realized the efficient utilization that manioc waste anaerobic fermentation is prepared biogas residue, and this both can meet resource requirement, alleviates environment and pressesPower, can create again economic worth, has realized manioc waste anaerobic fermentation residue the like waste resource maximum using, has hugeDevelopment prospect.
Summary of the invention
The technical issues that need to address of the present invention are that open a kind of manioc waste anaerobic fermentation residue is carbon source grown place mouldThe method of cellulase and saccharification thereof, to realize manioc waste anaerobic fermentation residue as resources and comprehensive utilization.
Manioc waste anaerobic fermentation residue of the present invention is the method for carbon source grown place mould cellulase and saccharification thereof, according toFollowing steps are carried out:
(1) cultivation of bacterial strain and product enzyme process thereof:
In 5L fermentation tank, pack 3L culture medium into, at 30 DEG C, mixing speed (400rpm) and throughput (2.4L/Min) under condition, cultivate geotrichum sp Galactomycessp.3 days; The cellulase fermentations liquid obtaining is for saccharification manioc wasteAnaerobic fermentation residue.
(2) enzymolysis pretreatment manioc waste anaerobic fermentation residue:
At 110-130 DEG C, manioc waste anaerobic fermentation residue is placed in to 1-ethyl-3-methyl-imidazoles-diethyl phosphateIn ([Emim] [DEP])-phosphoric acid-water pretreatment system, dissolve after pretreatment 30-120min, add deionized water washingRegeneration obtains cellulosic material; The cellulosic material deionized water of regeneration is washed after three times, adds acetic acid-sodium acetate bufferThe mixed system that (50mM, pH4.8) and cellulase fermentations liquid form, wherein manioc waste anaerobic fermentation residue cellulose is formerMaterial final mass concentration is 3%, and oscillating reactions on the constant-temperature table of 50 DEG C and 180rpm, can reach enzymolysis manioc waste and detestThe object of aerobe fermentation residue.
Wherein step (1) culture medium is composed as follows: NH4Cl5g/L，KH2PO42g/L，MgSO40.3g/L，CaCl20.6g/L, Macrogol 6000 4g/L, manioc waste anaerobic fermentation residue 10g/L, pH6.5.
Wherein in step (2), 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP])-phosphoric acid-water is located in advanceThe mass ratio of managing total system and manioc waste anaerobic fermentation residue is 15:1.
Wherein in step (2), 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP])-phosphoric acid-water is located in advanceIn reason system, mass fraction is composed as follows:
1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP]) 70-85 part,
Phosphoric acid 0.5-5 part,
Water 14.5-25 part.
Wherein in step (2), the volume ratio of acetic acid-sodium acetate buffer and cellulase fermentations liquid is 8:2.
Beneficial effect of the present invention:
The present invention utilizes manioc waste anaerobic fermentation residue for carbon source production Galactomycessp.CCZU11-1 celluloseEnzyme, cassava that can efficiently saccharifying 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP])-phosphoric acid-water pretreatmentSlag anaerobic fermentation residue, can alleviate environmental pressure, can create again economic worth. Whole technique has process economy, the three wastes are fewEtc. advantage.
Brief description of the drawings
SEM (SEM) figure before and after the pretreatment of Fig. 1 manioc waste anaerobic fermentation residue, a is untreated residueSEM figure, b is pretreatment residue SEM figure.
Detailed description of the invention
The cellulase producing bacteria strain of the present invention mould Galactomycessp.CCZU11-1 in ground, belongs to Geotrichum,This bacterial strain was declared Chinese invention patent on 04 16th, 2012, and application number is 201210109397.9, publication numberCN102604844A. (detailed description is shown in above-mentioned patent)
Cellulase unit of activity definition of the present invention: a Ge Meihuo unit (U) is defined as at 50 DEG C, pH=4.8 acetic acid-1min hydrolysis CMC or filter paper (1.0 × 6.0cm) generation 1 μ mol reduced sugar under sodium acetate buffer solution (50mM) condition (withGlucose is as the criterion) required enzyme amount. 3,5-dinitrosalicylic acid for the content of reduced sugar (DNS) is analyzed.
Reducing sugar yield=concentration of reduced sugar × 100%/manioc waste anaerobic fermentation sludge concentration.
Embodiment 1 fermenting and producing geotrichum sp Galactomycessp.CCZU11-1 cellulase
In 5-L fermentation tank, pack 3L culture medium (NH into4Cl5g/L，KH2PO42g/L，MgSO40.3g/L，CaCl20.6g/L, Macrogol 6000 4g/L, manioc waste anaerobic fermentation residue 10g/L, pH6.5), 30 DEG C, mixing speed(400rpm) and under throughput (2.4L/min) condition cultivate Galactomycessp.CCZU11-13d. The fibre obtainingFilter paper enzyme activity (FPA), restriction endonuclease (CMCase) alive and the total protein concentration of tieing up plain enzyme fermentation liquid are respectively 29.3U/mL, 59.9U/ML and 1.6g/L.
The auxiliary 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] of phosphoric acid of embodiment 2 quality final concentrations 0.5%[DEP]) pretreatment manioc waste anaerobic fermentation residue
In pretreatment system, the manioc waste anaerobic fermentation residue that 3g is dry utilizes 1-ethyl-3-first of 85% (w/w)The water mixed liquid of the phosphoric acid and 14.5% (w/w) of base-imidazoles-diethyl phosphate ([Emim] [DEP]), 0.5% (w/w) existsAt 130 DEG C, dissolve after pretreatment 30min, add deionized water regenerated from washing, obtain regenerated cellulose raw material. The fiber of regenerationAfter cellulosic material deionized water washing three times, add 80mL acetic acid-sodium acetate buffer (50mM, pH4.8) and 20mL fiberElement enzyme fermentation liquid forms 100mL mixed system, and manioc waste anaerobic fermentation residue cellulosic material final mass concentration is 3%. ?Oscillating reactions 72h on the constant-temperature table of 50 DEG C and 180rpm, reducing sugar yield is 40.5%.
The auxiliary 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] of phosphoric acid of embodiment 3 quality final concentrations 5%[DEP]) pretreatment manioc waste anaerobic fermentation residue
In pretreatment system, the manioc waste anaerobic fermentation residue that 3g is dry utilizes 1-ethyl-3-first of 70% (w/w)The water mixed liquid of the phosphoric acid, 25% (w/w) of base-imidazoles-diethyl phosphate ([Emim] [DEP]), 5% (w/w) is at 110 DEG CAfter lower dissolving pretreatment 120min, add deionized water regenerated from washing; The cellulosic material deionized water washing of regeneration three timesAfter, add 80mL acetic acid-sodium acetate buffer (50mM, pH4.8) and 20mL cellulase fermentations liquid to form 100mL mixedFit system, manioc waste anaerobic fermentation residue cellulosic material final mass concentration is 3%. Shake with the constant temperature of 180rpm at 50 DEG COscillating reactions 72h on bed, reducing sugar yield is 42.4%. The manioc waste anaerobic fermentation residue that 3g is dry add 80mL acetic acid-Sodium acetate buffer (50mM, pH4.8) and 20mL cellulase fermentations liquid form 100mL mixed system, manioc waste anaerobismFermentation residue cellulosic material final mass concentration is 3%. Oscillating reactions 72h on the constant-temperature table of 50 DEG C and 180rpm,Reducing sugar yield is 29.3%. From Fig. 1 (a) and Fig. 1 (b), manioc waste anaerobic fermentation residue cellulosic structure is dredged and is become thinnerPine, possible its higher structure is destroyed, makes cellulose surface or inside have more contact site to come out, and is easier to fibreTie up absorption and the enzymolysis of plain enzyme, thereby improved the conversion coefficient of manioc waste anaerobic fermentation residue.
Claims (1)
1. manioc waste anaerobic fermentation residue is the method for carbon source grown place mould cellulase and saccharification thereof, it is characterized in that according toFollowing steps are carried out:
(1) cultivation of bacterial strain and product enzyme process thereof:
In fermentation tank, packing the culture medium that accounts for its volume 3/5 into, is that 400rpm and throughput are 2.4 at 30 DEG C, mixing speedUnder L/min condition, cultivate geotrichum sp CCZU11-1(Galactomycessp.) 3 days; The cellulase fermentations liquid obtaining is for sugarChange manioc waste anaerobic fermentation residue;
(2) enzymolysis pretreatment manioc waste anaerobic fermentation residue:
Manioc waste anaerobic fermentation residue is placed in to 1-ethyl-3-methyl-imidazoles-diethyl phosphate at 110-130 DEG CIn ([Emim] [DEP])-phosphoric acid-water pretreatment system, dissolve after pretreatment 30-120min, add deionized water washingRegeneration obtains cellulosic material; After the cellulosic material deionized water washing three times of regeneration, add acetic acid-sodium acetate buffer andThe cellulase fermentations liquid that step (1) obtains forms mixed system, and wherein manioc waste anaerobic fermentation residue cellulosic material is finalMass concentration is 3%, and oscillating reactions on the constant-temperature table of 50 DEG C and 180rpm, can reach enzymolysis manioc waste anaerobic fermentationThe object of residue;
Wherein step (1) culture medium is composed as follows: NH4Cl5g/L，KH2PO42g/L，MgSO40.3g/L，CaCl20.6G/L, Macrogol 6000 4g/L, manioc waste anaerobic fermentation residue 10g/L, pH6.5;
Wherein 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP])-phosphoric acid-water pretreatment body in step (2)System is 15:1 with the mass ratio of manioc waste anaerobic fermentation residue;
Wherein 1-ethyl-3-methyl-imidazoles-diethyl phosphate ([Emim] [DEP])-phosphoric acid-water pretreatment body in step (2)In system, mass fraction is composed as follows:
1-ethyl-3-methyl-imidazoles-diethyl phosphate 70-85 part,
Phosphoric acid 0.5-5 part,
Water 14.5-25 part;
Wherein in step (2), the volume ratio of acetic acid-sodium acetate buffer and cellulase fermentations liquid is 8:2, described acetic acid-acetic acidSodium buffer concentration is 50mM, and pH is 4.8.
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Effective date of registration: 20201201 Address after: S201-202, building 10, wushengdian commercial street, Zhongquan capital, Chengnan street, Linquan County, Fuyang City, Anhui Province Patentee after: Linquan Jiahong Decoration Engineering Co., Ltd Address before: Gehu Lake Road Wujin District 213164 Jiangsu city of Changzhou province No. 1 Patentee before: CHANGZHOU University |