CN105087687A - Method for preparing microbial oil from paper pulp produced by using ammonium sulfite process - Google Patents
Method for preparing microbial oil from paper pulp produced by using ammonium sulfite process Download PDFInfo
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Abstract
The invention provides a method for preparing microbial oil from paper pulp produced by using an ammonium sulfite process to overcome the problems of great difficulty in pretreatment of a substrate, high investment cost and incapability of scaling-up of preparation of microbial oil from lignocellulose and the problems of oversupply of paper pulp, vacancy of equipment and the like in ammonium sulfite pulping. The novel method for preparing microbial oil from lignocellulose is obtained by integration of ammonium sulfite pulping and fermentation technology. The method comprises the following concrete steps: pretreating lignocellulose by using ammonium sulfite pulping; subjecting the pretreated lignocellulose to enzymatic hydrolysis to obtain monosaccharide and oligosaccharides; and with obtained sugar solution as a carbon source, carrying out fermentation by oleaginous microorganisms so as to obtain the microbial oil. The method for producing the microbial oil is beneficial for reduction in investment of production cost for the microbial oil, broadens the application scope of paper pulp of the traditional papermaking industry, increases profits of paper pulp papermaking enterprises, produces substantial economic benefits and facilitates scaling-up and mass production of the microbial oil.
Description
Technical field
The invention belongs to technical field of biochemical industry, particularly a kind of paper pulp obtained with ammonium salt process pre-treatment natural wooden fiber element is for raw material, by the method for oleaginous microorganism fermentation for microbial oil.The present invention can more effectively utilize natural lignocellulose material to produce microbial oil, and the production cost reducing microbial oil drops into, and is convenient to produce and amplifies.
Background technology
Some microorganisms can exceed the grease of its dry cell weight 20% at intracellular accumulation, main component is triglyceride level.Its oil fatty acid composition and plant oil seemingly, are mainly C
16and C
18lipid acid.Because the production of microbial oil is by the impact of time, season, place and climate change, grease mass-producing continuous seepage can be realized.Microbial oil production technology can be biofuel and oil prodution industry provides new raw material, has important application prospect.
Biomass are the fourth-largest energy on the earth, and are unique renewable energy sources.Lignocellulose-like biomass is formed primarily of Mierocrystalline cellulose, hemicellulose and xylogen three parts.Wherein content of cellulose 40-50%, hemicellulose 25-35%, xylogen 15-20%.Lignocellulosic material mainly comprises agricultural and forestry waste.Due to its wide material sources, cheap.Therefore, utilize lignocellulosic material to produce microbial oil, grease cost can not only be reduced, all right utilization of waste material, environmental protect problem.In plant tissue, xylogen is combined with covalent linkage form with hemicellulose, and by cellulosic molecule embedding wherein, forms a kind of firm natural cover for defense, makes general microorganism be difficult to enter and make it degrade.Therefore, the method needing some special destroys the structure of lignocellulose, thus utilizes lignocellulose.
At present, produce microbial oil to lignocellulosic material both at home and abroad to conduct extensive research.Ion liquid dissolving lignocellulose is a kind of new pretreatment process that developed recently gets up, and it can effectively destroy lignocellulose structure, and removes xylogen, improves enzymolysis efficiency significantly.Use ionic liquid pretreatment lignocellulose, and then carry out oil fermentation and have bibliographical information (Xieetal.EnzymatichydrolysatesofcornstoverpretreatedbyaN-methylpyrrolidone-ionicliquidsolutionformicrobiallipidpr oduction.Greenchemistry.2012,14,1202-1210).But ionic liquid in use still exists some problems: ionic liquid cost is high, reuses efficiency low, viscosity is unfavorable for greatly amplifying.Dilute acid pretreatment uses method comparatively widely in lignocellulose pre-treatment.Diluted acid can destroy the structure of hemicellulose in lignocellulose, thus be beneficial to follow-up enzymic hydrolysis (Runetal.Co-HydrolysisofLignocellulosicBiomassforMicrobia lLipidAccumulation.Biotechnologyandbioengineering.2013,110,1039-1049).Dilute acid pretreatment effectively can destroy the structure of hemicellulose, but the by products such as furfural, hydroxymethylfurfural, acetic acid can be produced, affect follow-up oil fermentation (Huangetal.Biologicalremovalofinhibitorsleadstotheimprove dlipidproductioninthelipidfermentationofcornstoverhydrol ysatebyTrichosporoncutaneum.BioresourceTechnology.2011,102,9705-9709; Feng Chong etc. in Trichosporon fermentans Lipid-producing, cellulosic saccharified liquid detoxification process is to when optimizing research. Beijing University of Chemical Technology's journal (natural science edition) .2011,38,87-91), increase oil fermentation cost.In addition, also have gas explosion pre-treatment (Chen Shang Xing etc. steam explosion pre-treatment is on the impact of maize straw chemical constitution and characteristic fibrous structure. chemistry of forest product and industrial .2009, 29, 33-38), oxygenation pretreatment (Wangetal.Fastandefficientnanoshearhybridalkalinepretreat mentofcornstoverforbiofuelandmaterialsproduction.Biomass andBioenergy.2013, 51, 35-42), Biological Pretreatment (Yuetal.Theeffectofbiologicalpretreatmentwiththeselective white-rotfungusEchinodontiumtaxodiionenzymatichydrolysis ofsoftwoodsandhardwoods.Bioresourcetechnology.2009, 100, 5170-5175), the quick-fried pre-treatment of ammonia (Lauetal.Theimpactsofpretreatmentonthefermentabilityofpre treatedlignocellulosicbiomass:acomparativeevaluationbetw eenammoniafiberexpansionanddiluteacidpretreatment.Biotec hnologyforbiofuel.2009, 2, the pretreatment process such as 1-11).Although these methods can effectively destroy lignocellulose structure; but because the reason such as process is complicated, cost is high, by product is many; the complicacy of oil production and the input of capital will be increased; thus raise the production cost of microbial oil, and be unfavorable for microbe oil fermentation Product management model and large-scale production.
Slurrying is the important step of paper technology, is take vegetable fibre as raw material, through the fibrous material that different processing methods is obtained.Technical process comprises plant fiber material pulverizing, boiling, washing, screening, bleaching, purification, oven dry, paper pulp.At present, because the universal of electronic product produces huge impact to paper product market, the supply of paper is caused to be greater than demand, and the cost of some emerging pulping and paper-makings is lower, and then cause paper pulp sales volume and prices, cause papermaking enterprise profit to reduce, loss increases (MahdiMachanietal.Amathematically-basedframeworkforevalua tingthetechnicalandeconomicpotentialofintegratingbioener gyproductionwithinpulpandpapermills.http: //dx.doi.org/10.1016/j.biombioe.2014.02.024).Cause some enterprises to stop production thus to close down.
Summary of the invention
Microbial oil cost is prepared high for lignocellulosic material, should not amplify, and the problem of enterprises in pulp and paper industry production capacity surplus, present inventor has performed a large amount of scientific researches and creationary work, propose the novel method integrated pulping with ammonium sulfite method and fermentation technique and prepare microbial oil.Easy handling of the present invention and amplification, effectively can improve the economy of microbe oil fermentation, reduces microbial oil production cost.Expand traditional paper industry pulp applications scope, increase pulping and paper-making industrial profit.
The present invention is achieved by following technical proposals:
1, the preparation of paper pulp: by natural wooden fiber element raw material according to following pulping with ammonium sulfite method technique (Hou Yongfa etc. Pinus massoniana Lamb pulping with ammonium sulfite method. chemistry of forest product with industry, 1981,2) carry out pre-treatment.Reconstituted wood Mierocrystalline cellulose after washing and filtering.
2, oil-containing thalline obtains by the following method:
(1) substep saccharification oil fermentation:
A, paper pulp enzymolysis: the paper pulp of gained after screening is carried out enzymolysis 12-72h, and cellulase addition is 10-30FPU/g dry pulp, and zytase addition is 0.1-10mg/g dry pulp, and enzymatic hydrolysis condition is pH4-6, temperature 40-60 DEG C.
In b, adjustment enzymolysis hydrolyzed solution, total reducing sugars concentration is at 30-150g/L, add yeast powder 0.1-2g/L wherein, ammonium sulfate 0.5-5g/L, potassium primary phosphate 0.1-1g/L, magnesium sulfate heptahydrate 0.5-3g/L, accesses oleaginous microorganism after sterilizing, at temperature 25-35 DEG C, pH4.5-6, dissolved oxygen carries out batch fermentation under being greater than 20% condition, obtains oil-containing thalline; Or
In c, adjustment enzymolysis hydrolyzed solution, the concentration of total reducing sugars is at 30-300g/L, add yeast powder 0.1-10g/L wherein, ammonium sulfate 0.5-10g/L, potassium primary phosphate 0.1-10g/L, magnesium sulfate heptahydrate 0.5-10g/L, accesses oleaginous microorganism after sterilizing, at temperature 25-35 DEG C, pH4.5-6, dissolved oxygen carries out cultured continuously under being greater than 20% condition, and control thinning ratio is 0.01-0.12h
-1, obtain oil-containing thalline; Or
In d, adjustment enzymolysis hydrolyzed solution, the concentration of total reducing sugars is at 30-300g/L, add yeast powder 0.1-10g/L wherein, ammonium sulfate 0.5-10g/L, potassium primary phosphate 0.1-10g/L, magnesium sulfate heptahydrate 0.5-10g/L, oleaginous microorganism is accessed, at temperature 25-35 DEG C, pH4.5-6 after sterilizing, dissolved oxygen carries out batch experiments under being greater than 20% condition, cultivate the logarithmic phase later stage and carry out feed supplement, add and carry out fed batch fermentation containing total reducing sugars 150-300g/L hydrolyzed solution, obtain oil-containing thalline.
(2) simultaneous saccharification and fermentation.
With water, paper pulp being adjusted to solid-to-liquid ratio is 3%-30%, add yeast powder 0.1-2g/L, ammonium sulfate 0.5-5g/L, potassium primary phosphate 0.1-1g/L, magnesium sulfate heptahydrate 0.5-3g/L, cellulase 5-20FPU/g (dry pulp) is added after sterilizing, zytase consumption is 0.05-10mg/g (dry pulp), according to the inoculum size access oleaginous microorganism of 0.1-5g/L stem cell, at temperature 25-35 DEG C, pH4.5-6, dissolved oxygen carries out synchronous saccharification oil fermentation under being greater than 20% condition, obtains oil-containing thalline;
3, microbial oil extracts: reference (LiYH of the present invention, ZhaoZB, BaiFW.High-densitycultivationofoleaginousyeastRhodospori diumtoruloidesY4infed-batchculture.EnzymeMicrobTechnol.2 007,41, method 312-317), after fermentation ends, through centrifugal, washing, collect bacterial sediment.Thalline is dried to constant weight at 105 DEG C, obtains dry mycelium.Reference literature (Li Zhifeng, Zhang Ling, Shen Xiaojing, Deng. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, and 2001,28 (6), 72-75) use acid heat-organic solvent method extracting to obtain grease, calculate thalline fat content.
Exceed eucaryon or the prokaryotic micro-organisms of its biological total amount 20% (w/w) at intracellular accumulation grease after the present invention uses oleaginous microorganism to refer to fermentation culture.They include but not limited to, produce oil bacterium, and as Arthrobacter genus, Rhodococcus belongs to or Mycobacterium belongs to; Oleaginous yeast, as Candida genus, Cryptococcus belongs to, Hansenula belongs to, Lipomyces belongs to, Rhodosporidium belongs to, Rhodotorula genus, Endomyces belong to, Schwanniomye belongs to, Trichosporon belongs to, Sporobolomyces belongs to, and Trigonopsis genus or Yarrowia belong to; Produce oil mould, as Mortierella genus, Cunninghamella belongs to, Mucor belongs to or Aspergillus belongs to; Oil-producing microalgae, as Crypthecodinium genus, Botryococcus belongs to, Chlorella belongs to, Nannochloropsis genus, Haliphthoros genus or Schizochytrium genus etc.These bacterial strains can directly be bought from comprising the Spawn preservation organizations such as China General Microbiological culture presevation administrative center (CGMCC), American type culture collection (ATCC) and Chinese industrial Microbiological Culture Collection administrative center (CICC) or be separated from occurring in nature, also can use the artificial or spontaneous mutation bacterial strain different from original bacterial strain proterties.
Advantage of the present invention and beneficial effect: compared with producing microbial oil with existing natural wooden fiber element, the present invention utilizes the paper pulp of ammonium salt process process natural wooden fiber element acquisition to be raw material production microbial oil.It can utilize existing pulping equipment, saves capital contribution, reduces grease production cost.Improve pulping equipment utilization ratio, increase pulping and paper-making industrial profit.And slurrying and fermentation technique are ripe, are convenient to lignocellulose and produce microbial oil Product management model and large-scale production.
Accompanying drawing explanation
Fig. 1 prepares microbial oil general flow chart for utilizing ammonium salt process paper pulp for raw material.
Embodiment
Following examples have chosen some paper pulp and prepare microbial oil process, illustrate and utilize ammonium salt process paper pulp to be prepared the method for microbial oil by fermentable for raw material.This will contribute to understanding this patent, but not limit other produce oil bacterial strain in any form apply the present invention on other paper pulp.
Embodiment 1
(1) by the ammonium sulphite of stalk by mass concentration 12%, with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 120 DEG C, and insulation 30min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 71%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 48h according to the regeneration stalk of 30FPU/g drying and the regeneration stalk of 3mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast RhodosporidiumtoruloidesAS2.1389 (purchased from China General Microbiological culture presevation administrative center) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 30%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 28.6g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 66.0%.As calculated, oil yield coefficient is 0.12g oil/g stalk.
The present embodiment result shows: stalk, by after pulping with ammonium sulfite method art breading, can be changed into grease by oleaginous microorganism through enzymolysis.Same disclosed application number is a kind of CN201210208132 " method utilizing corn stalk hydrolysis fermentative production microbial oil ", CN200810114616 " a kind of method being prepared microbial oil by wood fibre raw material " compares with CN200910040550 " taking agricultural wastes as the method that fermenting raw materials produces microbial oil " patent, by integrating slurrying and fermentation technique production microbial oil, the input of the lignocellulose preprocessing process equipment such as stalk can be saved, to produce the microbial oil of 10000 tons per year, pre-processing device can be reduced and drop into more than 5,000,000 yuan.And ammonium sulphite is neutral salt, pretreated lignocellulose does not need to adjust back pH and detoxification treatment with acid or alkali.Same disclosed application number is that CN201180066061.2 " integrating process system " patent is compared, the paper pulp that this patent is prepared with imonium pulping method is for raw material, and greatly remain hemicellulose components in paper pulp, this patent implements the range of application can expanding traditional paper pulp, solves the problem of paper pulp production capacity surplus.
Embodiment 2
(1) by the ammonium sulphite of stalk by mass concentration 12%, with the solid-to-liquid ratio of 1:8 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 120 DEG C, and insulation 30min, carries out secondary temperature elevation to 160 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 68%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 48h according to the regeneration stalk of 25FPU/g drying and the regeneration stalk of 5mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast RhodosporidiumtoruloidesAS2.1389 (purchased from China General Microbiological culture presevation administrative center) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 50%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 28.1g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 65.0%.As calculated, oil yield coefficient is 0.11g oil/g stalk.
Embodiment 3
(1) by the ammonium sulphite of stalk by mass concentration 8% (w/v), with the solid-to-liquid ratio of 1:10 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 120 DEG C, and insulation 30min, carries out secondary temperature elevation to 160 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 65%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 48h according to the regeneration stalk of 20FPU/g drying and the regeneration stalk of 5mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast RhodotorulaglutinisAS2.499 (purchased from China General Microbiological culture presevation administrative center) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 25%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 26.9g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 60.0%.As calculated, oil yield coefficient is 0.09g oil/g stalk.
Embodiment 4
(1) by the ammonium sulphite of stalk by mass concentration 12%, with the solid-to-liquid ratio of 1:8 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 120 DEG C, and insulation 30min, carries out secondary temperature elevation to 160 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 68%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 60h according to the regeneration stalk of 15FPU/g drying and the regeneration stalk of 15mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 4g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast TrichosporoncutaneumAS2.571 (purchased from China General Microbiological culture presevation administrative center) of 0.3g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 20%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 29.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 69.0%.As calculated, be 0.12g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 5
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:8 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 105 DEG C, and insulation 30min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 71%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 72h according to the regeneration stalk of 20FPU/g drying and the regeneration stalk of 15mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 4g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.3g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 40%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 30.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 68.0%.As calculated, oil yield coefficient is 0.13g oil/g stalk.
Embodiment 6
((1), by the ammonium sulphite of stalk by mass concentration 18% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, carries out twice intensification pre-treatment.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 30min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 66%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 72h according to the regeneration stalk of 10FPU/g drying and the regeneration stalk of 15mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.4g/L, at 33 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 35%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 28.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 61.0%.As calculated, oil yield coefficient is 0.10g oil/g stalk.
Embodiment 7
(1) by the ammonium sulphite of stalk by mass concentration 20% (w/v), with the solid-to-liquid ratio of 1:5 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 30min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 60min.Must regenerate stalk after filtration washing, yield is 64%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 72h according to the regeneration stalk of 10FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.3g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 24%, cultivate residual sugar in fermented liquid and be less than 5g/L, terminate fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 31.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 54.0%.As calculated, oil yield coefficient is 0.10g oil/g stalk.
Embodiment 8
(1) by the ammonium sulphite of stalk by mass concentration 6% (w/v), with the solid-to-liquid ratio of 1:10 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 71%.
(2) enzymolysis of pretreated straw: the regeneration stalk 200g that ammonium salt process is obtained, add water and be modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 72h according to the regeneration stalk of 10FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.3g/L, at 30 DEG C, pH5.6 cultivates, and by regulating air flow and stirring, controls dissolved oxygen 32%.It is the hydrolyzed solution of 100g/L that stream adds concentrated rear concentration of reduced sugar, and thinning ratio is 0.03h
-1.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 29.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 56.0%.As calculated, be 0.10g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 9
(1) by the ammonium sulphite of stalk by mass concentration 13% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 67%.
(2) enzymolysis of pretreated straw: the regeneration stalk that ammonium salt process obtains is added water and is modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 72h according to the regeneration stalk of 10FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.3g/L, at 30 DEG C, pH5.6 cultivates, and by regulating air flow and stirring, controls dissolved oxygen 20%.It is the hydrolyzed solution of 300g/L that stream adds concentrated rear concentration of reduced sugar, and thinning ratio is 0.01h
-1.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 83.2g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 56.0%.As calculated, oil yield coefficient is 0.12g oil/g stalk.
Embodiment 10
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) enzymolysis of pretreated straw: the regeneration stalk that ammonium salt process obtains is added water and is modulated into the slurries that solid content is 8% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 12h according to the regeneration stalk of 30FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Load in 3L fermentor tank, be added into final concentration: ammonium sulfate 0.5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, and by regulating air flow and stirring, controls dissolved oxygen 32%.It is the hydrolyzed solution of 30g/L that stream adds concentrated rear concentration of reduced sugar, and thinning ratio is 0.12h
-1.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 9.6g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 32.0%.As calculated, be 0.08g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 11
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) enzymolysis of pretreated straw: the regeneration stalk that ammonium salt process obtains is added water and is modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 60h according to the regeneration stalk of 30FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Get 1800mL supernatant liquor to load in 3L fermentor tank, be added into final concentration: ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, and by regulating air flow and stirring, controls dissolved oxygen 38%.Adding concentrated rear concentration of reduced sugar is the hydrolyzed solution of 300g/L, and cultivate 160h, residual sugar is less than 5g/L, terminates fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 64.6g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 71.0%.As calculated, be 0.12g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 12
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) enzymolysis of pretreated straw: the regeneration stalk that ammonium salt process obtains is added water and is modulated into the slurries that solid content is 15% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 60h according to the regeneration stalk of 30FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Get 1800mL supernatant liquor to load in 3L fermentor tank, be added into final concentration: ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast CryptococcusalbidusATCC56298 (purchased from American Type Tissue Collection) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, and by regulating air flow and stirring, controls dissolved oxygen 70%.Adding concentrated rear concentration of reduced sugar is the hydrolyzed solution of 150g/L, and cultivate 160h, residual sugar is less than 5g/L, terminates fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 44.6g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 57.0%.As calculated, be 0.11g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 13
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) enzymolysis of pretreated straw: the regeneration stalk that ammonium salt process obtains is added water and is modulated into the slurries that solid content is 10% (w/v), cellulase and zytase is added, at 50 DEG C and pH4.8 Water Under solution 60h according to the regeneration stalk of 30FPU/g drying and the regeneration stalk of 10mg zytase/g drying.
(3) oil fermentation: the enzymolysis solution of above-mentioned gained is collected supernatant liquor at the centrifugal 5min of 3000g.Get 1800mL supernatant liquor to load in 3L fermentor tank, be added into final concentration: ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1.5g/L, at 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L.After cooling, according to inoculum size access oleaginous yeast RhodotorulaglutinisAS2.704 (purchased from China General Microbiological culture presevation administrative center) of 0.5g/L, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 46%.Adding concentrated rear concentration of reduced sugar is the hydrolyzed solution of 300g/L, and cultivate 180h, residual sugar is less than 5g/L, terminates fermentation.
(4) extraction of grease: step (3) gained fermented liquid is centrifugal, collects thalline, dries to constant weight, determine that dry mycelium content is 91.6g/L at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease, determine that fat content is 65.0%.As calculated, be 0.14g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 14
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) the synchronous saccharification oil fermentation of stalk after pre-treatment: just ammonium salt process pretreated straw 200g, add water and be modulated into the slurries that pulp content is 10%, add ammonium sulfate 3g/L, magnesium sulfate heptahydrate 1g/L, 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L, after cooling, regenerating stalk according to 7FPU/g regeneration stalk and 0.5mg/g will add cellulase and zytase respectively.According to inoculum size access oleaginous yeast TrichosporoncutaneumAS2.571 (purchased from China General Microbiological culture presevation administrative center) of 10%, at 30 DEG C, pH5.6 cultivates, by regulating air flow and stirring, control dissolved oxygen 33%, cultivate 84h, terminate fermentation.
(3) extraction of grease: step (2) gained fermented liquid is centrifugal, collects thalline, dries to constant weight at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease.As calculated, be 0.10g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 15
(1) by the ammonium sulphite of stalk by mass concentration 15% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 65%.
(2) the synchronous saccharification oil fermentation of stalk after pre-treatment: just ammonium salt process pretreated straw 200g, add water and be modulated into the slurries that pulp content is 10%, add ammonium sulfate 0.5g/L, magnesium sulfate heptahydrate 1g/L, 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L, after cooling, regenerating stalk according to 7FPU/g regeneration stalk and 1mg/g will add cellulase and zytase respectively.According to inoculum size access oleaginous yeast RhodosporidiumtoruloidesAS2.1389 (purchased from China General Microbiological culture presevation administrative center) of 10%, at 32 DEG C, pH5.2 cultivates, by regulating air flow and stirring, control dissolved oxygen 40%, cultivate 72h, terminate fermentation.
(3) extraction of grease: step (2) gained fermented liquid is centrifugal, collects thalline, dries to constant weight at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease.As calculated, be 0.13g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Embodiment 16
(1) by the ammonium sulphite of stalk by mass concentration 13% (w/v), with the solid-to-liquid ratio of 1:6 (g/ml), in encloses container, twice intensification pre-treatment is carried out.Rotary spherical digester idle running 5min before heating up, first time is warming up to 115 DEG C, and insulation 50min, carries out secondary temperature elevation to 150 DEG C after little steam bleeding 5min, insulation 90min.Must regenerate stalk after filtration washing, yield is 66%.
(2) the synchronous saccharification oil fermentation of stalk after pre-treatment: just ammonium salt process pretreated straw 200g, add water and be modulated into the slurries that pulp content is 10%, add ammonium sulfate 5g/L, magnesium sulfate heptahydrate 1g/L, 121 DEG C of sterilizing 18min after potassium primary phosphate 0.4g/L, after cooling, regenerating stalk according to 10FPU/g and 1mg/g will add cellulase and zytase respectively.According to inoculum size access oleaginous yeast RhodosporidiumtoruloidesAS2.1389 (purchased from China General Microbiological culture presevation administrative center) of 10%, at 32 DEG C, pH5.2 cultivates, by regulating air flow and stirring, control dissolved oxygen 25%, cultivate 72h, terminate fermentation.
(3) extraction of grease: step (2) gained fermented liquid is centrifugal, collects thalline, dries to constant weight at 105 DEG C.By document (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. the comparative studies of four kinds of fungal oil extracting method. microbiology is circulated a notice of, 2001,28 (6), 72-75) use acid heat-organic solvent method to extract grease.As calculated, be 0.10g oil/g stalk at oil and fat accumulation stage oil yield coefficient.
Superior combination of the present invention is commented:
The present invention utilizes the pretreated lignocellulose of ammonium salt process to be raw material, adopts different fermentation modes, and efficient lignocellulose changes into microbial oil.Utilize existing pulping with ammonium sulfite method technique, decrease the cost input that lignocellulose prepares microbial oil, reduce the production cost of grease.Also improve the utilization ratio of pulping and papermaking equipments simultaneously, widened the range of application of paper pulp, effectively increase pulp and paper industry profit.And be convenient to lignocellulose and prepare microbial oil Product management model and large-scale production.
Claims (5)
1. utilize ammonium salt process paper pulp to prepare a method for microbial oil for raw material, it is characterized in that: with the paper pulp of natural wooden fiber's element after the process of ammonium sulphite pulping method for raw material production microbial oil;
One, 1), first enzymolysis secondary fermentation:
A, paper pulp enzymolysis: lignocellulose obtains paper pulp enzymatic hydrolysis condition and is after ammonium sulphite pre-treatment: during enzymolysis, pulp slurry solid content is 5-30% (w/v), cellulase consumption is 5-30FPU/g (dry pulp), and zytase consumption is 0.1-10mg/g (dry pulp); Enzymatic hydrolysis condition is: pH3.5-7, temperature 30-60 DEG C, saccharification time 12-72h; FPU is that cellulosic filter paper enzyme is lived;
In b, adjustment enzymolysis hydrolyzed solution, total reducing sugars concentration is at 30-150g/L, final concentration is added to wherein: yeast powder 0.1-2g/L before fermentation, ammonium sulfate 0.5-5g/L, potassium primary phosphate 0.1-1g/L, magnesium sulfate heptahydrate 0.5-3g/L, access 0.1-5g/L oleaginous microorganism after sterilizing, at temperature 25-35 DEG C, carry out batch fermentation under pH4.5-6, dissolved oxygen 10-90% condition, obtain oil-containing thalline;
Or, in adjustment enzymolysis hydrolyzed solution the concentration of total reducing sugars at 30-300g/L, be added to final concentration wherein: yeast powder 0.1-10g/L, ammonium sulfate 0.5-10g/L, potassium primary phosphate 0.1-10g/L, magnesium sulfate heptahydrate 0.5-10g/L, access 0.1-5g/L oleaginous microorganism after sterilizing, at temperature 25-35 DEG C, carry out cultured continuously under pH4.5-6, dissolved oxygen 10-90% condition, control thinning ratio is 0.01-0.12h
-1, obtain oil-containing thalline;
Or, in adjustment enzymolysis hydrolyzed solution the concentration of total reducing sugars at 30-300g/L, be added to final concentration wherein: yeast powder 0.1-10g/L, ammonium sulfate 0.5-10g/L, potassium primary phosphate 0.1-10g/L, magnesium sulfate heptahydrate 0.5-10g/L, 0.1-5g/L oleaginous microorganism is accessed after sterilizing, at temperature 25-35 DEG C, pH4.5-6, dissolved oxygen carries out batch experiments under being greater than 10-90% condition, cultivate the logarithmic phase later stage and carry out feed supplement, add and carry out fed batch fermentation containing total reducing sugars 150-300g/L hydrolyzed solution, obtain oil-containing thalline;
Or 2) simultaneous saccharification and fermentation: with water, paper pulp being adjusted to solid-to-liquid ratio is 3%-30% (w/v), be added to final concentration: yeast powder 0.1-2g/L, ammonium sulfate 0.5-5g/L, potassium primary phosphate 0.1-1g/L, magnesium sulfate heptahydrate 0.5-3g/L, cellulase 5-20FPU/g (dry pulp) is added after sterilizing, zytase 0.05-10mg/g (dry pulp), be hydrolyzed after 0-8h, according to the inoculum size access oleaginous microorganism of 0.1-5g/L cell, at temperature 25-35 DEG C, pH4.5-6, synchronous saccharification oil fermentation is carried out under dissolved oxygen 10-90% condition, obtain oil-containing thalline,
Two, microbial oil extracts.
2. in accordance with the method for claim 1, it is characterized in that: described pulping with ammonium sulfite method, ammonium sulphite mass concentration is 10-20%; Natural wooden fiber's element is 1:5-10 (g/ml) with ammonium sulfite solution solid-to-liquid ratio; In encloses container, twice intensification is adopted to remove xylogen: first time is warming up to 100-150 DEG C, insulation 20-60min, steam bleeding 2-10min; Second time is warming up to 110-170 DEG C, and insulation 90-150min, obtains reconstituted wood Mierocrystalline cellulose, i.e. paper pulp after filtration washing.
3. according to the method described in claim 1 or 3, it is characterized in that: described natural wooden fiber element comprises:
Agricultural thing stalk, timber, a kind of or mixture of above-mentioned at least two kinds of crop seeds skin;
Comprise specifically: straw, wheat straw, reed straw, cotton stalk, peanut straw, Pericarppium arachidis hypogaeae, the mixture of maize straw, leaf of Semen Maydis, mealie skin, bagasse a kind of or at least two kinds.
4. method according to claim 1, is characterized in that: described oleaginous microorganism is eukaryotic microorganisms or the prokaryotic micro-organisms that can be greater than 20% (w/w) at thin intracellular accumulation neutral fat content.
5. method according to claim 1, is characterized in that: described oil fermentation is batch oil fermentation, batch feed supplement oil fermentation and continuous oil fermentation.
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