CN101805761A - Bio-detoxification application of Issatchenkia orientalis and method for synchronously detoxifying and fermenting hemicellulose hydrolysate by Iissatchenkia orientalis - Google Patents
Bio-detoxification application of Issatchenkia orientalis and method for synchronously detoxifying and fermenting hemicellulose hydrolysate by Iissatchenkia orientalis Download PDFInfo
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Abstract
The invention relates to a microbial strain of Issatchenkia orientalis (S-7) CCTCC NO:M 206098 and the application of domestic strain of the Issatchenkia orientalis. The invention is characterized in that the pure culture of the microbial strain of the Issatchenkia orientalis is inoculated to hemicellulose hydrolysate to culture for degrading the microbial metabolic inhibitor in the hemicellulose hydrolysate, thereby the bio-detoxification of the hemicellulose hydrolysate is realized. The invention further discloses a method for preparing xylitol and ethanol by using the strain. Compared with the traditional technology, the invention has higher production efficiency and higher quality of products.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of biological detoxication purposes of Issatchenkia orientalis, and it is to the method for the synchronous detoxification fermentation of hemicellulose hydrolysate.
Background technology
Hemicellulose is the glycan class except that Mierocrystalline cellulose in the plant cell wall, and content is difference to some extent with different plants, roughly accounts for the 20-35% of plant materials weight, be on the earth except that Mierocrystalline cellulose the abundantest polysaccharide.
Mierocrystalline cellulose is the homogeneity glycan that is aggregated into by glucose unit fully, hemicellulose then mainly be by wood sugar so that β-(1-4) glycosidic link constitutes main chain, the heterogencity glycan of Ah 's sugar, seminose and semi-lactosi formation branched structure.The xylan backbone of hemicellulose generally contains a 50-150 wood sugar unit, and no crystalline texture is combined in the surface of cellulose micro-fibers.
Hemicellulose than the easy hydrolysis of Mierocrystalline cellulose many,, under 100-130 ℃ of condition, just be easy to diluted acid hemicellulose, it is the hemicellulose hydrolysate of main component that hydrolysis generates with the five-carbon sugar.All kinds of Chemicals such as the Xylitol that fermentation hemicellulose hydrolysate production market capacity is big, ethanol are to utilize hemicellulose resources effective approach on a large scale.
Though with diluted acid hydrolysis of hemicellulose being generated with monose is that the process of hydrolyzate of main component is not difficult, but, the dilute acid hydrolysis process can be supervened a series of microbial metabolism inhibitions, improve the leavening property of hemicellulose hydrolysate, must remove microbial metabolism inhibition wherein, i.e. detoxification process is absolutely necessary.
The poisonous substance composition of having identified in hemicellulose hydrolysate has at present had tens kinds, and wherein representative poisonous substance comprises three major types, that is: furfural compounds, and as furfural, 5 hydroxymethyl furfural; Fatty acid compound, as formic acid, acetate, propionic acid; A series of compounds that contain phenyl ring that lignin degradation generates, as methyl catechol, phenyl aldehyde, forulic acid etc.
Reported vacuum-evaporation, solvent extraction, charcoal absorption, macroporous resin adsorption, ion exchange resin treatment can be sloughed some poisonous substance, improves the leavening property of hydrolyzate.Yet a kind of physics or chemical process can only be removed a certain class toxic substance, need unite usually and adopt multiple physics and chemistry measure to handle, and hemicellulose hydrolysate just can reach reasonable detoxification efficiency.But detoxification is a process that expends cost, uses multiple physics and chemistry measure that hemicellulose hydrolysate is carried out detoxification treatment simultaneously, must increase substantially the cost of product fermentation.
In fact, more natural microorganisms have degrading activity to some poisonous substance in the hemicellulose hydrolysate.For example, for example whiterot fungi (vast stretch of wooded country Lu Gang Zhang Qingna, the Screening and Identification and the application of degraded paper waste xylogen bacterial classification, University of Science ﹠ Technology, Beijing's journal of lignin degrading and a series of phenolic compounds of generating by lignin degradation.2007,29 (6): 569-573; Tao Yang Liao Jun and Luo Xuegang, bio-pulping technology more recent application progress.The Mierocrystalline cellulose science and technology.2007,15 (1): 70-74, degraded furfural, 5 hydroxymethyl furfural bacterium, yeast (tinkling of pieces of jade of writing legal document for others, Zhang Lujia, furfural and derivative microbiological deterioration (conversion) progress thereof.Amino acid and Biological resources 2007,29 (4): 41-45; Liu Aiping is permitted to learn book Fan Hangxue, utilizes yeast bio to transform vegetable fibre and is the alcoholic acid progress.The biotechnology communication, 2004,15 (2): 193--196), (what firm stifled state becomes Liu Liming to the ascomycetes of degraded short chain fatty acid, and thermophilic ascomycete utilizes short chain organic acid production at.The biotechnology journal, 2008,24 (5): 821-828)
In recent years the someone begins to attempt to remove poisonous substance in the hydrolyzate with biological enzyme.Human laccase and peroxidase combination treatment timber acid hydrolysis things such as Jonsson can have been removed most monocycle phenol thing (monoaromatic phenolic copounds).Hydrolyzate after the processing is used for ethanol fermentation, glucose consumption speed and ethanol generating rate have improved nearly 5 times (Jonsson J L as a result, Palmqvist E, Nilvebrant N O., Detoxification of wood hydrolysates with laccase andperoxidase from the white-rot fungus Trametes versicolor.Appl.Microbiol Biotechnol.1998, (49): 691-697)).But complicated poisonous substance composition needs the combined degradation of complicated enzyme system, and this has increased the cost of detoxification undoubtedly greatly.
People such as Lopez are with microorganism Conichaeta ligniaria NRRL 30616 fermentative processing wood fibre dilute acid hydrolysis things, and the effect of not only removing furfural, 5 hydroxymethyl furfural is remarkable, also obviously reduced the aldehydes matter in the hydrolyzate simultaneously.Use the producing and ethanol yeast fermentation again through the hydrolyzate that this bacterial strain is handled, 80h produces 1.66% ° of ethanol, and untreatedly do not have ethanol to produce (Lopez J M, Nichol s N N, Dien B Set al.Isolation of microorganisms for biological detoxification oflignocellulosec hydrolysates.Appl Microbiol Biotechnol.2004,64 (1): 125-131).
Above-mentioned studies show that utilizes that complicated toxic substance is in the cards in the microbiological deterioration wood fibre hydrolysis thing.Especially, if can obtain a kind of metabolic system that possesses three big representative poisonous substances in the degraded hemicellulose hydrolysate simultaneously, promptly can degrade simultaneously furfural, lipid acid and contain the microorganism of benzene ring structure compound, and this microorganism can not utilize wood sugar again, so directly utilize this microbial fermentation hemicellulose hydrolysate to carry out the detoxification mass treatment, just can effectively overcome the existing shortcoming that physics and chemistry poison-removing method technology is loaded down with trivial details, cost is high, and have eco-friendly advantage.
Summary of the invention
The objective of the invention is in order to solve existing hemicellulose hydrolysate physics and chemistry detoxification worker. the existing process of planting is loaded down with trivial details, the problem that cost is high.
Content of the present invention comprises: the contriver is from the paper mill, wood sugar factory, and furfural mill surrounding soil mud sample is rich in cultivation, separation, screening, and acquisition can effectively improve two isolate: the S-7 and the Lj-3 of wood fibre hydrolysis thing leavening property.The contriver identifies that isolate S-7 belongs to Issatchenkia orientalis (Issatchenkiaorientalis), it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on September 18th, 2006, preserving number is CCTCC NO:M206098, can not utilize wood sugar; Isolate Lj-3 belongs to her Sa yeast (Issatchenkia occidentalis) of west, it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on September 18th, 2006, preserving number is CCTCC NO:M206097, utilize the ability of wood sugar atomic a little less than.The contriver has set up the biological detoxication method of hemicellulose hydrolysate with the active new bacterial strain of this detoxification; Detoxification active bacterial strain and xylose-fermenting are generated the combined inoculation of Xylitol microorganism, realize the synchronous detoxification xylitol zymolysis production of hemicellulose hydrolysate; Detoxification active bacterial strain and xylose-fermenting are generated the combined inoculation of alcoholic acid microorganism, realize the synchronous detoxification fermentative production of ethanol of hemicellulose hydrolysate.The preservation information of above-mentioned two primary yeasts is accepted letter of information referring to two parts of appended preservations of present patent application.
Belong to two biological detoxication active bacterial strain S-7 of the present invention (CCTCC NO:M206098) and Lj-3 (CCTCC NO:M206097), its separation screening, and the process identified of classification as follows:
With the bagasse hemicellulose hydrolyzate is the basal component of isolation medium, suitably regulates pH 5-6 with alkali after the vacuum.If make flat board, add agar 20g/L as solid agent.
Soil, the mud of the environment collection of polluting from pulp mill wastewater are made sample separation.250ml triangular flask loading amount 25ml inserts waste water or the about 1g of mud sample separation, and 30 ℃, 200rpm shakes a bottle enrichment culture 72h.The separation of ruling on same culture medium flat plate of the nutrient solution of getting thalli growth, the type that selection can be grown fast forwards the inclined-plane to and preserves.
Slant culture is transferred in the half fiber dilute acid hydrolysis thing and was cultivated 24 hours shaking under bottle condition, the centrifugal thalline of removing, insert the yeast strain that can assimilate wood sugar then and ferment, selecting and remain, those can effectively improve the detoxification active bacterial strain of hemicellulose dilute acid hydrolysis thing leavening property.So through surplus ten batches sample separation, two the strongest isolates of the hemicellulose hydrolysate leavening property activity that is improved, numbering is respectively S-7 and Lj-3.
The detected result of high performance liquid chromatography shows, S-7 and Lj-3 are to the representative poisonous substance in the hemicellulose dilute acid hydrolysis thing: acetic acid, and furfural and phenol thing all have good degrading activity, and wherein S-7 does not utilize wood sugar, and Lj-3 can only faintly utilize wood sugar.
Aforesaid method separates two bacterial strains that obtain and refrigerates or the lyophilization preservation in 4 ℃.
According to the method that " yeast identification handbook " (press of Qingdao Marine University) book provides, identified cell, bacterium colony and the physiological and biochemical property (table 1, table 2) of S-7 and Lj-3.Issatchenkia orientalis (Issatchenkiaorientalis) described in the physiology of isolate S-7, biochemical character and " the yeast identification handbook " is identical; Her Sa yeast (Issatchenkia occidentalis) of west described in the physiology of isolate Lj-3, biochemical character and the same book is identical.
With primer 5 '-GCATATCAAAAGCGGAGGAAAAG-3 ' and 5 '-GGTCCGTGTTTCAAGACGG-3 ', (method is with reference to Kurtzman C P for the 26S rDNA D1/D2 zone nucleotide sequence of polymerase chain reaction (PCR) amplification S-7 and Lj-3, Four new Candida species from geographically diverselocations.Antonie van Leeuwenhoek, 2001,79:353-361).Measure the nucleotide sequence (table 3, table 4) of amplified production, and in GenBank nucleic acid sequence data storehouse, carry out homologous sequence search (Nucleotide-nucleotide BLAST) with this sequence.Search Results shows, the nucleotide sequence in the 26S rDNA D1/D2 zone of S-7 all reaches 100% with the existing Issatchenkia orientalis the same area nucleic acid sequence homology of GenBank; Lj-3 with wherein reach 100% with the homology of Issatchenkia occidentalis the same area nucleotide sequence.
According to can determining of above-mentioned cell, colonial morphology, Physiology and biochemistry and molecular biological characteristics, the classification position of S-7 belongs to Issatchenkia orientalis (Issatchenkia orientalis); Her Sa yeast (Issatchenkia occidentalis) of the west that the classification position of Lj-3 belongs to.
The preserving number of Issatchenkia orientalis (Issatchenkia orientalis) S-7 at China typical culture collection center (CCTCC) is CCTCC NO:M206098, and the registration number that the nucleotides sequence in its 26S rDNA D1/D2 zone is listed in GenBank is EF030708
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nucleotide&val＝116834296；
She Sa yeast (Issatchenkia occidentalis) Lj-3 of west is CCTCC NO:M206097 at the preserving number of CCTCC, and the GenBank registration number of the nucleotide sequence in its 26S rDNA D1/D2 zone is EF030710
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nucleotide&val＝116834298。
Cell and the colony morphology characteristic of table 1 isolate S-7, Lj-3
| Colonial morphology | Cell characteristic | |
| S-7 | Surface drying, the fringe radiation shape. | Multiterminal sprout, and the cell mean length is: between 10～20um. |
| Lj-3 | Glossy, surperficial thickness, neat in edge is level and smooth. | Multiterminal sprout, and the cell mean length is: 5～15um. |
The physiological and biochemical property of table 2 isolate S-7, Lj-3 (+, utilize;-, do not utilize)
The nucleotide sequence (GenBank registration number EF030708) in Issatchenkia orientalis (Issatchenkia orientalis) S-7 (CCTCC NO:M206098) 26SrDNA D1/D2 zone:
1??taagcggagg?aaaagaaacc?aacagggatt?gcctcagtag?cggcgagtga?agcggcaaga
61?gctcagattt?gaaatcgtgc?tttgcggcac?gagttgtaga?ttgcaggttg?gagtctgtgt
121ggaaggcggt?gtccaagtcc?cttggaacag?ggcgcccagg?agggtgagag?ccccgtggga
181tgccggcgga?agcagtgagg?cccttctgac?gagtcgagtt?gtttgggaat?gcagctccaa
241gcgggtggta?aattccatct?aaggctaaat?actggcgaga?gaccgatagc?gaacaagtac
301tgtgaaggaa?agatgaaaag?cactttgaaa?agagagtgaa?acagcacgtg?aaattgttga
361aagggaaggg?tattgcgccc?gacatgggga?ttgcgcaccg?ctgcctctcg?tgggcggcgc
421tctgggcttt?ccctgggcca?gcatcggttc?ttgctgcagg?agaaggggtt?ctggaacgtg
481gctcttcgga?gtgttatagc?cagggccaga?tgctgcgtgc?ggggaccgag?gactgcggcc
541gtgtaggtca?cggatgctgg?cagaacggcg?caacaccgcc?cgtcttgaaa?cacgga
The 26S rDNA D1/D2 zone nucleotide sequence (GenBank registration number EF030710) of she Sa yeast (Issatchenkia occidentalis) Lj-3 (CCTCC NO:M206097) of west:
1??tatcaataag?cggaggaaaa?gaaaccaaca?gggattgcct?cagtagcggc?gagtgaagcg
61?gcaaaagctc?agatttgaaa?tcgtgtttcg?gcacgagttg?tagattgcag?gttggagtct
121ttgtggaagc?gtgtgtctaa?gtcccttgga?acagggtgcc?attgagggtg?agagccccgt
181gagacgcgtg?cggaagctgt?aaggcccttc?tgacgagtcg?agttgtttgg?gaatgcagct
241ctaagtgggt?ggtaaattcc?atctaaggct?aaatattggc?gagagaccga?tagcgaacaa
301gtactgtgaa?ggaaagatga?aaagcacttt?gaaaagagag?tgaaacagca?cgtgaaattg
361ttgaaaggga?agggtattgg?gctcgacatg?ggatttgcgc?accgctgctc?cttgtgggcg
421gcgctctgtg?cttttcctgg?gccagcatcg?gtttttgccg?caggagaagg?cgtgctggaa
481tgtggctctt?cggagtgtta?tagccagtgc?gagatgctgc?gtgcggggac?cgaggactgc
541gacatctgtc?tcggatgctg?gcacaacggc?gcaataccgc?ccgtcttgta?a
The present invention prepares needed hemicellulose hydrolysate as follows:
Bagasse, corn cob, utilizing rice straw or other crops ' stalk are crushed to suitable granularity, the dilute sulphuric acid or the dilute hydrochloric acid solution that add 0.3～3% (w/w) by solid-to-liquid ratio 1: 6～7, stir,, kept 0.5～3 hour acid proof pressurized vessel internal heating to 100～130 ℃.Hydrolysis finishes back filtering residue, and filtrate is hemicellulose hydrolysate.With solid carbonic acid calcium or calcium hydroxide hemicellulose dilute acid hydrolysis liquid is transferred to pH value 3～8, can directly or after concentrating carry out biological detoxication again.Usually, can use lower hydrolysis temperature than the condition of high acid concentration, or corresponding shortening hydrolysis time, lower acid concentration then must improve hydrolysis temperature or prolong hydrolysis time.
The present invention cultivates as follows and is used for the hemicellulose hydrolysate biological detoxication, xylitol fermentation, and the microbial strains of ethanol fermentation:
Detoxification bacterial strain culture of seed liquid: the slant culture of CCTCC NO:M206098 or CCTCC NO:M206097 is inserted liquid seed culture medium, liquid amount is to shake 20% of bottle capacity, at 27--30 ℃, shaking table was cultivated 12～18 hours under the rotating speed 200rmp condition, can obtain can be used for the kind liquid of detoxification.Seed culture medium consists of: glucose 50g/L, MgSO
4.7H
2O 2g/L, K
2HPO
44g/L, KH
2PO
46g/L, yeast extract paste 5g/L.
Xylose-fermenting generates the bacterial strain seed liquor of Xylitol and cultivates: used bacterial strain is candida tropicalis (Candida tropicalis), it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on June 15th, 2005, preserving number is CCTCC NO:M205067, classification called after candida tropicalis 1-18Candida tropicalis 1-18, (see patent of invention " separation of candida tropicalis bacterial strain and be used for the method that Xylitol is produced ", application for a patent for invention numbers 200510037580.2), contain glucose 20g/L, wood sugar 20g/L, all the other compositions and culture condition are with detoxification bacterial strain seed culture.
Xylose-fermenting generates alcoholic acid bacterial strain seed liquor and cultivates: used bacterial strain is had a liking for tannin pipe capsule yeast (Pachysolen tannophilus) CICC 1770 for what be preserved in Chinese industrial microbial strains preservation administrative center (CICC), and medium component and culture condition generate the spawn culture of Xylitol with xylose-fermenting.
The present invention carries out the hemicellulose hydrolysate biological detoxication as follows:
The hemicellulose hydrolysate total sugar content is 4～20% (w/w), and wherein reductive monosaccharide accounts for total sugar content＞more than 90%, be principal constituent with the wood sugar.With alkali lye (calcium hydroxide, ammoniacal liquor etc.) be adjusted to pH3-7, insert cultured CCTCCNO:M206098 or CCTCC NO:M206097 seed liquor by 10% (v/v) inoculum size, at 25～35 ℃, aeration condition bottom fermentation detoxification 5～20 hours, most of microorganism toxic ingredient wherein can be degraded and remove.The thalline of collecting centrifugation is recycled and reused for the fresh hemicellulose hydrolysate detoxification of next batch, and centrifuged supernatant can directly or after concentrating be used for xylitol fermentation, or ethanol fermentation.
The hemicellulose hydrolysate that the present invention is fermented after the detoxification is as follows produced Xylitol:
Passed through CCTCC NO:M206098, or the hemicellulose hydrolysate of CCTCC NO:M206097 fermentation detoxification, vacuum concentration is to the about 150g/L of wood sugar, and ammoniacal liquor is adjusted to pH=6, other adds yeast extract paste 5g/L, obtains being used for the hemicellulose hydrolysate substratum of xylitol fermentation.Xylitol fermentation is carried out with shaking bottle, shakes bottled amount 10%, inserts candida tropicalis (Candida tropicalis) CCTCCNO:M205067 seed liquor by 5% (v/v) inoculum size, 200rmp, and 33 ℃ ferment to wood sugar and run out of.Collect the thalline of centrifugation, put into the xylitol fermentation that continues a new round in the fresh hemicellulose hydrolysate substratum, centrifuged supernatant is used to prepare Xylitol.
The present invention utilizes hemicellulose hydrolysate to carry out synchronous detoxification xylitol zymolysis production as follows:
The vacuum concentration hemicellulose hydrolysate is to the about 100--150g/L of wood sugar content, transfers to pH 3--pH7 with alkali (calcium hydroxide, or ammoniacal liquor),, centrifugal or remove by filter precipitation, add yeast extract paste 5g/L, the hemicellulose hydrolysate substratum, be sub-packed in triangular flask; Get cultured strain CCTCC NO:M206098 seed liquor, the perhaps seed liquor of CCTCC NO:M206097, and mix with isopyknic CCTCC NO:M205067 seed liquor, by in 5% (v/v) and the sowing quantity access hemicellulose hydrolysate, shake-flask culture to wood sugar runs out of then.The yeast cell of centrifugal collecting precipitation also is used for fresh substratum, continues synchronous detoxification fermentation.So constantly circulation can not utilize until yeast cell.
The present invention utilizes hemicellulose hydrolysate to carry out synchronous detoxification fermentative production of ethanol as follows:
The substratum of the hemicellulose hydrolysate that synchronous detoxification fermentative production of ethanol is used is identical with the substratum of synchronous detoxification xylitol zymolysis production.Get cultured strain CCTCC NO:M206098 seed liquor, the perhaps seed liquor of CCTCCNO:M206097, mix with isopyknic CICC 1770 seed liquor, insert in the hemicellulose hydrolysate culture media shaking vase by 5% (v/v) and sowing quantity then, be cultured to wood sugar and run out of.The yeast cell circulation of centrifugal collecting precipitation is used to the fresh substratum that ferments, and continues synchronous detoxification fermentation, and so constantly circulation can not utilize until yeast cell.The ethanol in the centrifuged supernatant is reclaimed in distillation.
The present invention to the representative poisonous substance matter in the hemicellulose hydrolysate, reaches main aldehydes matter degrading activity with high performance liquid chromatography detection of biological detoxification bacterial strain.
Select the representative of acetate, furfural and methyl catechol as dissimilar inhibitions in the hemicellulose hydrolysate, these three kinds of compounds are joined in the common yeast culture base together, and access detoxification active bacterial strain (CCTCCNO:M206097, or CCTCC NO:M206098) ferments.Utilize these three kinds of compounds that the characteristics of stronger uv-absorbing are all arranged at the 198nm place, at following chromatographic condition: the Waters486 high performance liquid chromatograph, ZORBAX XDB-C18 chromatographic column, mobile phase methanol: phosphoric acid (0.2%, w/w)=75: 25 (v/v), the content of these three kinds of compounds of comparison and detection before and after fermentation judges that these two bacterial strains all have degrading activity (Fig. 1) to these three kinds of representative toxic ingredients.
Because phenolic compound all has strong uv-absorbing near 270nm, so with 270nm is to detect wavelength, with the bagasse hemicellulose hydrolyzate of high performance liquid phase comparison and detection operation 1, operation 3 through Issatchenkia orientalis CCTCC NO:M206098 biological detoxication, the bagasse hemicellulose hydrolyzate (Fig. 2) of her Sa yeast CCTCC NO:M206097 biological detoxication through the west.And analyze the information (table 4) of some characteristic peaks under this detection wavelength condition, and hemicellulose hydrolysate is after biological detoxication is handled as can be known, and furfural wherein and many deleterious aldehydes matters are degraded.
The present invention's detoxification efficiency of the method detection of biological detoxification bacterial strain of contrast biological fermentation to hemicellulose hydrolysate:
Ferment through CCTCC NO:M206097 with CCTCC NO:M205067 contrast, or, detect the tunning Xylitol through CCTCCNO:M206098 detoxification treatment front and back hemicellulose hydrolysate; Or with CICC1770 contrast fermentation through CCTCC NO:M206097, or before and after CCTCC NO:M206098 detoxification treatment hemicellulose hydrolysate, detect tunning ethanol.Can find no matter the hemicellulose hydrolysate through after biological bacterial strain provided by the present invention and the poison-removing method processing is used for xylitol fermentation or ethanol fermentation, output, the generating rate of fermented product all are improved largely.Can affirm that thus biological detoxication bacterial strain provided by the invention and biological detoxification method can improve the leavening property of hemicellulose hydrolysate effectively.
Substantive distinguishing features and marked improvement that the present invention gives prominence to are:
1. the present invention Issatchenkia orientalis CCTCC NO:M 206097 of finding and providing, her Sa yeast CCTCC NO:M 206098 these two bacterial strains of west, to three big representative microorganism metabolic antagonists in the hemicellulose hydrolysate: with acetic acid is the organic acid of representative, with the furfural is the carbohydrate degraded product of representative, reaching with the methyl catechol is the phenolic compound of representative, all have degrading activity simultaneously, they all have outstanding biological detoxication activity to hemicellulose hydrolysate.
2. the present invention utilizes Issatchenkia orientalis CCTCC NO:M 206097, or the multiple toxic ingredient in her the Sa yeast CCTCCNO:M 206098 biological degradation hemicellulose hydrolysates of west, and the fact has reduced the impurity in the matrix.Compare with traditional physics and chemistry detoxification process, hemicellulose hydrolysate is carried out detoxification, obviously have simple and direct, low-cost, and eco-friendly outstanding advantage with the present invention.
3. two detoxification active bacterial strains provided by the invention, it is also very faint that wherein CCTCC NO:M 206097 can not assimilate the metabolic capacity of wood sugar, 206098 pairs of wood sugars of CCTCC NO:M fully, utilize them to the hemicellulose hydrolysate detoxification, and can't cause wood sugar loss wherein.Because these characteristics, they and xylose-fermenting are generated Xylitol, or xylose-fermenting generation alcoholic acid microorganism places same fermentation system, can realize hemicellulose hydrolysate detoxification process and xylitol fermentation, or with ethanol fermentation coupling mutually, greatly simplify fermentation hemicellulose hydrolysate production xylitol fermentation, or produce alcoholic acid technology.
Description of drawings
Fig. 1 is that the high performance liquid chromatography of inhibition after the degraded of detoxification active bacterial strain changes.Wherein parameter is: (A) inhibition, and (B) substratum (C) has added the substratum of inhibition, and the substratum that (D) contains inhibition is through CCTCC NO:M206097 fermentation detoxification, and E contains inhibition and cultivates it through CCTCC NO:M206098 fermentation detoxification.Peak number and compound: 1 acetate, 2 furfurals, 1,3 methyl catechol, 4,5 medium components, 6 furfural degraded products, 7 methyl catechol degraded products.
Fig. 2 is the HPLC collection of illustrative plates (detecting wavelength 270nm) before and after the bagasse hemicellulose hydrolysate biological detoxication.Wherein parameter is: A, furfural standard specimen; B, the bagasse hemicellulose hydrolysate; C is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206098 detoxification; D is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206097 detoxification.
Embodiment
Operation 1
Claim to have washed the new dry bagasse 3kg that crosses that lays equal stress on, by solid-to-liquid ratio (w/v) is 1: 7 adding H2SO4 solution (2.5%, w/w), 120 ℃ of hydrolysis 2h, centrifugal collection filtrate, filter residue is washed once, merging filtrate, with lime carbonate solid adjust pH to 3, suction filtration is removed precipitation, gets the bagasse hemicellulose hydrolyzate.This hydrolyzate reducing sugar content 3%, wood sugar monose accounts for more than 80% of total reducing sugar.
Get already pulverised dried corn cob 2kg, add the H of 2% (w/w) by solid-to-liquid ratio at 1: 7
2SO
4Solution, 120 ℃ of hydrolysis 2h collect liquid portion, and lime carbonate is neutralized to pH value 3, and the suction filtration disgorging is able to corn cob hemicellulose dilute acid hydrolysis liquid.This hydrolyzed solution total sugar content 5%, wherein wood sugar accounts for 60% of total reducing sugar.
Issatchenkia orientalis CCTCC NO:M206098 inclined-plane is inoculated on the liquid seed culture medium, 200rmp, 30 ℃ of shaking tables are cultivated 12h, get activated seed liquid.By the inoculum size access operation 1 of 15% (v/v), in the bagasse hemicellulose dilute acid hydrolysis liquid of gained, 200rmp, 33 ℃ of shaker fermentation detoxification 45h, centrifuged supernatant is bagasse half hydrolyzate through Issatchenkia orientalis CCTCC NO:M206098 biological detoxication.
(,, obtain the bagasse hemicellulose hydrolyzate of her Sa yeast CCTCC NO:M206097 biological detoxication through the west with the bagasse hemicellulose hydrolyzate of her Sa yeast CCTCC NO:M206097 processing operation 1 of west perhaps by same condition.)
Operation 4
Operate the bagasse hemicellulose hydrolyzate through the biological detoxication processing of 3 gained, continue vacuum concentration to wood sugar 150g/L, ammoniacal liquor is adjusted to pH=6, other adds yeast extract paste 5g/L, insert candida tropicalis (Candidatropicalis) CCTCC NO:M205067 seed liquor, the access amount is 5% (v/v), 200rmp, and 33 ℃ ferment.Contrast HPLC detected result (table 3) is used for xylitol fermentation through the hemicellulose hydrolysate after the biological detoxication processing, and not only the product generating rate is more than doubled, and product yield is also near doubling.
Table 3. biological detoxication is handled the influence to xylitol fermentation
(initial xylose concentration, 150g/L; Fermentation time, 61.5h)
Hemicellulose hydrolysate with operation 1 gained, be evaporated to the about 120g/L of soluble solid wood sugar content, ammoniacal liquor is adjusted to pH5, one group of equivalent inserts the Issatchenkia orientalis CCTCCNO:M206098 and candida tropicalis CCTCC NO:M 205067 cells of centrifugal collection, and another is organized then equivalent and inserts her Sa yeast CCTCC NO:M206097 cell and candida tropicalis CCTCC NO:M 205067 cells of west.Their total inoculum size all is about stem cell 50g/L.33 ℃, shake bottle synchronous detoxification fermentation 30 hours under the 200rpm condition.Result's (table 4) shows that the detoxification fermentation energy obviously improves tunning concentration, product generating rate and product yield synchronously.
Comparison (initial wood sugar, the 120g/L of Xylitol performance produced in synchronous detoxification fermentation of table 4. hemicellulose hydrolysate and direct fermentation; Fermentation time, 30 hours)
Hemicellulose hydrolysate with operation 2 gained, be evaporated to the about 60g/L of soluble solid wood sugar content, ammoniacal liquor is adjusted to pH5, press Issatchenkia orientalis CCTCC NO:M206098 and candida tropicalis CCTCC NO:M 205067 combinations respectively, the mode of her Sa yeast CCTCC NO:M206097 of west and candida tropicalis CCTCC NO:M 205067 combinations, carry out the balanced mix cell inoculation, total inoculum size is about cultivates 5% of its volume, 33 ℃, shake bottle synchronous detoxification fermentation 80 hours under the 200rpm condition.Result's (table 5) shows that the detoxification fermentation energy obviously improves tunning alcoholic acid generating rate and alcohol concn synchronously.
Table 5 biological detoxication is handled the influence to lignocellulose hydrolytic residue simultaneous saccharification and fermentation alcohol performance
Referring to Fig. 1. the high performance liquid chromatography of inhibition after the degraded of detoxification active bacterial strain changes.Wherein parameter is: (A) inhibition, and (B) substratum (C) has added the substratum of inhibition, and the substratum that (D) contains inhibition is through CCTCC NO:M206097 fermentation detoxification, and E contains inhibition and cultivates it through CCTCC NO:M206098 fermentation detoxification.Peak number and compound: 1 acetate, 2 furfurals, 1,3 methyl catechol, 4,5 medium components, 6 furfural degraded products, 7 methyl catechol degraded products.Fig. 2 is the HPLC collection of illustrative plates (detecting wavelength 270hm) before and after the bagasse hemicellulose hydrolysate biological detoxication.Wherein parameter is: A, furfural standard specimen; B, the bagasse hemicellulose hydrolysate; C is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206098 detoxification; D is through the bagasse hemicellulose hydrolysate of CCTCCNO:M206097 detoxification.
The content of some uv-absorbing thing (detecting wavelength 270nm) in the bagasse hemicellulose hydrolysate after table 6 biological detoxication is handled
Claims (10)
1. the purposes of a microorganism strains Issatchenkia orientalis Issatchenkia orientalis (S-7), it is characterized in that, be seeded to hemicellulose hydrolysate and cultivate with the pure growth of described microorganism strains Issatchenkia orientalis, be used for the microbial metabolism inhibition of described hemicellulose hydrolysate of degrading, and then be used to realize biological detoxication described hemicellulose hydrolysate.
2. purposes according to claim 1 is characterized in that, described microorganism strains Issatchenkia orientalis Issatchenkia orientalis is CCTCC NO:M 206098 and naturalized strain thereof.
3. one kind is used for the synchronous detoxification fermentation process of hemicellulose hydrolysate that Xylitol is produced, it is characterized in that, with microorganism strains Issatchenkia orientalis (Issatchenkia orientalis) S-7 with biological detoxication function, the pure growth of CCTCC NO:M 206098 is seeded to hemicellulose hydrolysate, and inoculate the microorganism that the five-carbon sugar that can ferment generates Xylitol to described hemicellulose hydrolysate simultaneously, the biological detoxication of described hemicellulose hydrolysate, xylitol fermentation are finished in same reactor simultaneously.
4. the synchronous detoxification fermentation process of hemicellulose hydrolysate according to claim 3 is characterized in that, the microorganism that the described five-carbon sugar that can ferment generates Xylitol is candida tropicalis (Candida tropicalis) CCTCC NO:M205067.
5. according to claim 3 or the synchronous detoxification fermentation process of 4 described hemicellulose hydrolysates, it is characterized in that described hemicellulose hydrolysate need add nitrogenous source, as inorganic nitrogen-sourced or organic nitrogen source.
6. the synchronous detoxification fermentation process of hemicellulose hydrolysate according to claim 5 is characterized in that, described inorganic nitrogen-sourced be urea or ammonium sulfate; Described organic nitrogen source is yeast or rice bran hot water extract.
7. synchronous detoxification fermentation process of hemicellulose hydrolysate that is used for alcohol production, it is characterized in that, with microorganism strains Issatchenkia orientalis (Issatchenkia orientalis) S-7 with biological detoxication function, the pure growth of CCTCC NO:M 206098, and inoculate the five-carbon sugar generation alcoholic acid microorganism of fermenting to described hemicellulose hydrolysate simultaneously, described hemicellulose hydrolysate detoxification and ethanol fermentation are finished in same reactor simultaneously.
8. the synchronous detoxification fermentation process of hemicellulose hydrolysate according to claim 7 is characterized in that, the described five-carbon sugar that can ferment generates the alcoholic acid microorganism for having a liking for tannin pipe capsule (Pachysolen tannophilus) CICC 1770.
9. as claim 7 or the synchronous detoxification fermentation process of 8 described hemicellulose hydrolysates, it is characterized in that described hemicellulose hydrolysate need add nitrogenous source, as inorganic nitrogen-sourced or organic nitrogen source.
10. the synchronous detoxification fermentation process of hemicellulose hydrolysate according to claim 9 is characterized in that, described inorganic nitrogen-sourced be urea or ammonium sulfate; Described organic nitrogen source is yeast or rice bran hot water extract.
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Non-Patent Citations (4)
| Title |
|---|
| ZHANG HOU-RUI ET AL: "Novel Isolates for Biological Detoxification of Lignocellulosic Hydrolysate", 《APPL BIOCHEM BIOTECHNOL》 * |
| 刘天霞等: "嗜单宁管囊酵母木糖酒精发酵的研究", 《纤维素科学与技术》 * |
| 刘天霞等: "稻壳粉水解条件优化及燃料酒精发酵的研究", 《宁夏农林科技》 * |
| 赵磊: "管囊酵母发酵木糖产乙醇", 《中国优秀硕士学位论文全文数据库》 * |
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| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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