CN104673712A - Bacterial strain for producing alcohol fuels by synchronously utilizing glucose and xylose and application of bacterial strain - Google Patents
Bacterial strain for producing alcohol fuels by synchronously utilizing glucose and xylose and application of bacterial strain Download PDFInfo
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Abstract
The invention discloses a bacterial strain for producing alcohol fuels by synchronously utilizing glucose and xylose and application of the bacterial strain. The bacterial strain is clostridium beijerinckii SE-2 with a culture preservation number of CCTCC No.M2014384. The bacterial strain capable of synchronously utilizing glucose and xylose is obtained by synchronously utilizing glucose and xylose in a natural environment to produce butyl alcohol and screening an alcohol microorganism, is a Gram-positive gemma-bearing bacterium, and the glucose and xylose in corncob hydrolysate can be synchronously utilized to produce the butyl alcohol and alcohol. A secondary growth phenomenon in a fermentation process can be eliminated by synchronously utilizing glucose and xylose in lignocelluloses hydrolysate, the fermentation period is shortened, the substrate utilization efficiency in the production process and the production strength of equipment are improved, and the economical efficiency of the industry of producing the butyl alcohol and the alcohol by the lignocelluloses are improved.
Description
Technical field
The present invention relates to and belong to technical field of bioengineering, particularly a strain synchronously utilizes bacterial strain and the application thereof of glucose and xylose production Aalcohols fuel in lignocellulosic material hydrolyzed solution.
Background technology
Due to the continuous aggravation of Greenhouse effect and environmental stress, the especially continuous exhaustion of fossil resource, the bioenergy being raw material with renewable biomass resource and chemicals (as the alcohols such as biological butanol, bio-ethanol) are subject to various countries and more and more pay attention to.In addition, greatly develop biomass energy more job opportunity can also be provided and reduce CO
2discharge.The alcohols such as butanols and ethanol is produced and is mainly contained chemical method and biological process, and wherein biological process is, by microorganism, carbohydrate fermentation is changed into butanols and ethanol, is the future directions of industry development.Lignocellulosic material with its abundance and reproducible feature, becomes the topmost raw material sources of Bioenergy development.Lignocellulosic material transforms and manufactures biological butanol, the operational path of ethanol may be summarized to be: raw materials pretreatment and be hydrolyzed to monose; Liquid glucose fermentation generates butanols, ethanol; Product Distillation recovery etc.Although above-mentioned operational path possesses feasibility, many technical bottlenecks are still had to need to overcome.Only with regard to bacterial classification, in fiber hydrolysate, the synchronous utilization of five-carbon sugar and hexose is one of difficult point in the urgent need to address.Wood sugar generates X 5P by xylose isomerase and the reaction of xylulokinase two-step catalysis after being transferred in born of the same parents, enters phosphopentose pathway subsequently.Microorganism is utilizing ubiquity catabolite repression effect in complex carbon source procedure, namely easily utilizes the quick utilization of carbon source (as glucose) to produce the metabolism of other carbon source (as wood sugar) and checks effect.Traditional biological butanol fermenting process mainly with the glucosyl group such as molasses and starch raw material for carbon source, therefore without the need to considering the problem that the five-carbon sugar such as wood sugar utilizes.Although product butanol acetone clostridium conventional in production efficiently can produce butanols by metabolizable glucose, and can directly utilize the five-carbon sugars such as wood sugar to produce butanols, but when there is glucose and wood sugar in fermented liquid simultaneously, it is on the impact of the metabolic process of wood sugar by glucose, namely there is " resistance to glucose ", fermenting process only has after glucose exhausts substantially, just starts xylose metabolism.Traditional butanols, ethanol fermentation mainly utilize molasses, starch etc. to be rich in glucose feed, and therefore whether bacterial strain exists resistance to glucose, for production not impact.But, with the development of Mierocrystalline cellulose butanols, ethanol industry, be that raw material carries out butanols, alcohol fermentation processes must in the face of resistance to glucose is on the impact of fermenting process with ligno-cellulose hydrolysate.Because, in lignocellulosic material hydrolyzed solution, not only containing glucose, and containing the five-carbon sugar such as wood sugar and pectinose.Therefore screening synchronously can utilize product butanols, the ethanol bacterial strain of five hexoses, for promoting that the development of Mierocrystalline cellulose butanols, ethanol is significant.
Summary of the invention
The object of this invention is to provide bacterial strain and application thereof that a strain synchronously utilizes glucose and xylose production Aalcohols fuel, this bacterial strain is that separation screening is out from physical environment, production for butanols and ethanol provides a kind of fermentation host, produces bacterial strain at present synchronously cannot utilize five limitation of hexose of glucose and wood sugar etc. in ligno-cellulose hydrolysate to overcome.
The present invention achieves the above object by the following technical programs: a strain synchronously utilizes the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that this bacterial strain Bai Shi clostridium SE-2, Latin name Clostridium beijerinckii SE-2; This bacterial strain is preserved in China typical culture collection center (CCTCC), address on August 19th, 2014: Wuhan, China Wuhan University, postcode 430072, preserving number CCTCC No.M2014384.
One strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, the Bai Shi clostridium SE-2 that to it is characterized in that with lignocellulosic material hydrolyzed solution be prepared using preserving number CCTCC No.M2014384 ferments, synchronously utilize glucose in hydrolyzed solution and xylose production butanols and ethanol, realize the full sugar of ligno-cellulose hydrolysate and utilize.
Concrete feature of the present invention also has, and lignocellulosic material comprises one or more in corn cob, maize straw, wheat stalk and branch.
One strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that it comprises the steps: (1) and configures seed culture medium, is accessed by Bai Shi clostridium SE-2 in seed culture medium and carries out first order seed cultivation and secondary seed cultivation; (2) in fermentor tank, anaerobically fermenting cultivation is carried out.
One strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that it comprises the steps: the first step, and preparation substratum, comprises seed culture medium (TYA substratum), butanols basal fermentation medium (MP2).Second step: first order seed is cultivated; Accessed by bacterial classification (500 mL inoculation bottles, liquid amount 400mL) in seed culture medium, before seed access, logical nitrogen flooding is except the oxygen in tank, and culture temperature 37 DEG C, quiescent culture 16 h obtains first order seed nutrient solution.3rd step: the first order seed nutrient solution obtained by second step is transferred to (3L stainless steel inoculation bottle in secondary seed medium by 3% volume ratio, liquid amount 2.5L), before seed access, logical nitrogen flooding is except the oxygen in tank, and culture temperature 30 DEG C, quiescent culture 20 h obtains secondary seed nutrient solution.4th step: the secondary seed nutrient solution the 3rd step obtained is by 3% volume ratio access containing in the fermention medium of Corncob hydrolysate, and fermenting process adopts anaerobism Batch fermentation, and before seed access, logical nitrogen flooding is except the oxygen in tank.Fermentation culture adopts 30L stirred-tank fermenter, and liquid amount 25L, culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5.5th step: fermenting process 72h, glucose, wood sugar and product butanols and ethanol content in sampling analysis fermented liquid.
The concrete feature of this programme also has, Corncob hydrolysate: total sugar content is 49.2 g/L, wherein glucose 33.5 g/L and wood sugar 15.8 g/L.
Seed culture medium (TYA substratum): glucose 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, CH
3cOONH
43 g, KH
2pO
40.5 g, MgSO
47H
2o 0.2 g, FeSO
47H
2o 0.01 g, distilled water 1000 mL, pH 6.5,121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
Butanols basal fermentation medium (MP2): Corncob hydrolysate, K
2hPO
40.5 g, KH
2pO4 0.5 g, CH
3cOONH
42.2 g, MgSO
47H
2o 0.2 g, MnSO
4h
2o 0.01 g, NaCl 0.01 g, FeSO4 7H
2o 0.01 g, thiamine (thiamines, vitamins B
1) 0.001g, biotin (vitamin H) 0.00001g, p-aminobenzoic acid (p-benzaminic acid) 0.001g, add water to 1000 mL;
During actual preparation, preparation mother liquor 1(Solution 1, S1): p-aminobenzoic acid 100 mg/L, Thiamine 100 mg/L, Biotin 1 mg/L, use the filtering with microporous membrane in aseptic 0.22 μm of aperture degerming, then save backup in 4 DEG C.
One strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that it comprises the steps: the first step, and preparation substratum, comprises seed culture medium (TYA substratum), butanols basal fermentation medium (MP2).Second step: first order seed is cultivated; Accessed by bacterial classification (500 mL inoculation bottles, liquid amount 400mL) in seed culture medium, before seed access, logical nitrogen flooding is except the oxygen in tank, and culture temperature 37 DEG C, quiescent culture 16 h obtains first order seed nutrient solution.3rd step: the first order seed nutrient solution obtained by second step is transferred to (3L stainless steel inoculation bottle in secondary seed medium by 3% volume ratio, liquid amount 2.5L), before seed access, logical nitrogen flooding is except the oxygen in tank, and culture temperature 30 DEG C, quiescent culture 20 h obtains secondary seed nutrient solution.4th step: contain in the fermention medium of Corncob hydrolysate by secondary seed nutrient solution by 3% volume ratio access, fermenting process adopts anaerobically fermenting, and before seed access, logical nitrogen flooding removes the oxygen in tank.Fermentation culture adopts 30L stirred-tank fermenter, and liquid amount 25L, culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5.Fermentation employing is continuously fermented mode, and in whole fermenting process, thinning ratio is 0.9 h
-1, fermentation time is 240 h, glucose, wood sugar and product butanols and ethanol content in sampling analysis fermented liquid.
The concrete feature of this programme also has, Corncob hydrolysate: total sugar content is 49.2 g/L, wherein glucose 33.5 g/L and wood sugar 15.8 g/L.
Seed culture medium (TYA substratum): glucose 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, CH
3cOONH
43 g, KH
2pO
40.5 g, MgSO
47H
2o 0.2 g, FeSO
47H
2o 0.01 g, distilled water 1000 mL, pH 6.5,121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
Butanols basal fermentation medium (MP2): Corncob hydrolysate, K
2hPO
40.5 g, KH
2pO4 0.5 g, CH
3cOONH
42.2 g, MgSO
47H
2o 0.2 g, MnSO
4h
2o 0.01 g, NaCl 0.01 g, FeSO4 7H
2o 0.01 g, thiamine (thiamines, vitamins B
1) 0.001g, biotin (vitamin H) 0.00001g, p-aminobenzoic acid (p-benzaminic acid) 0.001g, add water to 1000 mL;
During actual preparation, preparation mother liquor 1(Solution 1, S1): p-aminobenzoic acid 100 mg/L, Thiamine 100 mg/L, Biotin 1 mg/L, use the filtering with microporous membrane in aseptic 0.22 μm of aperture degerming, then save backup in 4 DEG C.
Applicant obtains a strain from the mud separation screening being arranged in Jinan first sewage work and can synchronously utilize glucose and wood sugar to produce the bacterial strain of butanols; Identify through morphologic observation and 16S rDNA, bacterial strain belongs to Bai Shi clostridium on taxonomy, called after Bai Shi clostridium SE-2, this bacterial strain contained in China typical culture collection center (being called for short CCTCC) on August 19th, 2014 by applicant, and its deposit number is: CCTCC No. M2014384.
Bai Shi clostridium SE-2 bacterial strain is that Gram-positive produces genus bacillus, and grow into about 18 h at anaerobic culture medium and start to form gemma, two ends are painted, and gemma diameter is suitable with thalline diameter, and thalline does not expand.Bai Shi clostridium SE-2 bacterial strain 16S rDNA sequence and Bai Shi clostridium NCIMB 8052 homology are more than 99%.
The invention has the beneficial effects as follows: 1, the present invention has successfully separated a strain from occurring in nature and can synchronously utilize glucose and wood sugar to produce the Bai Shi clostridium SE-2 of butanols, this bacterial strain exists in the fermenting process of glucose and wood sugar at the same time, with substantially identical speed metabolizable glucose and wood sugar.
2, traditional product butanols bacterial strain, owing to can not synchronously utilize glucose and wood sugar, the utilization when glucose consumption totally could start wood sugar is only had in fermenting process, and in the middle of two processes, have one section of bacterial strain to the growth caused because of the conversion of sugared source and damp production phase, in this process, the production of product butanols, ethanol stops substantially, need within 3-5 hour, just restart butanols, alcohol production afterwards, this phenomenon is called " diauxie ".This patent bacterial strain synchronously utilizes glucose and wood sugar characteristic can avoid the appearance of " diauxie ", ensure that whole fermenting process steadily carries out, therefore can shorten fermentation time, improve the utilization ratio of equipment, minimizing process energy consumption etc., and then improve the economy of producing.
3, pattern of continuously fermenting is the serialization being realized production process by fermenting process continuously feeding and continuous discharge, relative to Batch fermentation pattern, can the consumption such as artificial, the energy because reducing batch seed culture, in every batch fermentation time waste that thalli growth " lag phase " and fermentation unit cleaning etc. cause when starting and respective process, be therefore the first-selection of commercial process.Tradition bacterial strain is with glucose, wood sugar is that carbon source is when fermenting, after glucose exhausts, total sugar concentration in fermented liquid is very low, according to the feature of continuously fermenting, need further supplementary carbon source (ligno-cellulose hydrolysate, comprise glucose and wood sugar), if but now fill into hydrolyzed solution, because bacterial strain is to the existence of the preferential utilization level of glucose, bacterial strain will inevitably be caused to restart glucose utilization, further delay is to the utilization of wood sugar, wood sugar can accumulate further in fermented liquid, final wood sugar can only be discharged with fermented liquid, cause the waste of raw material.
Bacterial strain of the present invention is owing to can synchronously utilize glucose and wood sugar, in Continuous Fermentation Processes when carbon source in fermented liquid declines, that glucose and wood sugar are being consumed simultaneously, now fill into new carbon source (ligno-cellulose hydrolysate, comprise glucose and wood sugar), glucose inside bacterial strain can continuously, synchronously utilize and wood sugar, can not cause wood sugar accumulation in fermented liquid, ensure that the steady running of Continuous Fermentation Processes and raw material make full use of.
4, bacterial strain of the present invention is laid a good foundation for the fermentation of ligno-cellulose hydrolysate full sugar produced in butanols process synchronously utilizes on the one hand, simultaneously for butanols bacterial strain glucose and xylose metabolism mechanism are produced in further investigation, and then carry out genetic engineering modifiedly providing material to high yield industrialization bacterial strain.
Accompanying drawing explanation
Fig. 1 be in the present invention bacterial strain with glucose and wood sugar mixing sugar for sugar during carbon source through fermentation utilizes graphic representation.
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is described further.
Embodiment 1: one strain can synchronously utilize glucose and wood sugar to produce being separated of the Bai Shi clostridium of butanols and ethanol, selection systems.
The first step: sampling
Collected specimens from the sludge of sewage treatment plant of Jinan first.
Second step: enrichment culture
The blue lid bottles of 100 mL enrichment mediums put into by the sample getting 1 g collection, at 80 DEG C of heat shock 10 min, 37 DEG C of constant incubator enrichment culture 3 d are placed on water cooling, select the nutrient solution of a large amount of bubble formation, enrichment is carried out further in enrichment medium, go down to posterity after 5 times, gas chromatographic analysis detection is carried out in sampling.The fermented liquid generated there being butanols is transferred in isolation medium and is continued fermentation culture.
Enrichment medium (for anaerobic bacteria culture): wood sugar 5 g, peptone 10 g, beef powder 10 g, yeast powder 3 g, Zulkovsky starch 1 g, sodium-chlor 5 g, sodium-acetate 3 g, L-cysteine hydrochloride 0.5 g, agar 0.5 g, resazurin 0.3 g, distilled water 1000 mL.Fully mixed by above composition during preparation, pH value 6.5, be sub-packed in the indigo plant lid bottle of 100 mL, every bottled liquid measure is 50 mL, and 121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
3rd step: separation and purification
The fermented liquid getting enrichment culture in anaerobic culture box gradient dilution to 10
-8, utilize rolling tube technique method to be separated single bacterium colony.Picking list bacterium colony is in the indigo plant lid bottle of TYA substratum, and place 37 DEG C of constant incubators and cultivate 2 d, 25 % glycerine are stored in-80 DEG C of refrigerators.
Isolation medium (being separated single bacterium colony for rolling pipe): D-wood sugar 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, ammonium acetate 3 g, KH2PO4 0.5 g, MgSO47H2O 0.2 g, FeSO47H2O 0.01g, agar 20 g, distilled water 1000 mL, pH 6.5, be sub-packed in the indigo plant lid bottle of 100 mL, every bottled liquid measure is 50 mL, 121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
This research obtains with enrichment and separation purification method the bacterial strain that butanols is produced in 10 strains from 60 samples, and because wood sugar in isolation medium is sole carbon source, 10 therefore obtained strain bacterium all can produce butanols by xylose-fermenting.Bacterial strain is carried out culture presevation, and the mixing sugar fermentation carrying out next step sieves experiment again.
4th step: isolated strains produces solvent research
The primary dcreening operation getting glycerine preservation produces 80 DEG C of heat shock 10 min in butanols bacterial strain access TYA substratum, cools rearmounted 37 DEG C of constant incubators and cultivates activation 20 h, be then transferred in TYA substratum to cultivate and arrive OD
600be about 1 as seed liquor.By seed liquor in 5% inoculum size be inoculated into (cubic capacity 100 mL in the indigo plant lid bottle of the substratum that 60 g/L sugar (wherein the ratio of glucose and wood sugar is 2:1) are housed, liquid amount 50 mL), in 37 DEG C of thermostat containers, carry out solvent mensuration after standing for fermentation 72 h.
Seed culture medium (TYA substratum): glucose 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, ammonium acetate 3 g, KH
2pO
40.5 g, MgSO
47H
2o 0.2 g, FeSO
47H
2o 0.01 g, distilled water 1000 mL, pH 6.5,121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
Mixing sugar fermention medium: mixing sugar (glucose and wood sugar are mixed in proportion), yeast powder 5 g, calcium carbonate 3.0 g, calcium superphosphate 0.7 g, ammonium sulfate 3.0 g, with distilled water constant volume to 1000 mL, pH 6.5.Mixed by above composition, be sub-packed in the blue lid bottle of 100 mL, 115 DEG C of autoclaving 20 min, blow 5min with sterile nitrogen, drive most oxygen.
Shown by the multiple sieve result of table 1, bacterial strain S3, S7 and S8 are high relative to other bacterial strain to the fermentation capacity of mixing sugar, and in different batches fermentation stability, butanols average productivity is respectively 7.17g/L, 8.54 g/L and 6.04 g/L, and its carbohydrate metabolism and product butanols ability are a little less than bacterial strain SE-1.Because bacterial strain SE-1 is that a strain utilizes molasses to produce the clostridium acetobutylicum industrial strain of butanols, and the primary and foremost purpose of this test wants to obtain the product butanols bacterium that synchronously can utilize glucose and wood sugar, to carry out the object that fermentation of ligno-cellulose hydrolysate produces butanols.Relatively bacterial strain S3, S7, S8 and SE-1 find, in mixing sugar fermenting process, the xylose utilization rates of bacterial strain S3 faster than other three strains bacterium, will infer that bacterial strain S3 likely realizes the synchronous fermentation of glucose and wood sugar in mixing sugar fermenting process.Therefore, identification of strains is carried out to bacterial strain S3 and carbohydrate metabolism characteristic is furtherd investigate.
Table 1 isolated strains fermentation mixing sugar is produced acetone, butanols and ethanol and is compared
5th step: isolated strains glucose and xylose utilization rates are studied
The bacterial strain of production butanols screening obtained carries out glucose and wood-sugar fermentation is tested.By the bacterial classification of activation with 5% inoculum size be inoculated into (cubic capacity 100 mL in the indigo plant lid bottle of the mixing sugar substratum that 60 g/L sugar (wherein the ratio of glucose and wood sugar is for 2:1) are housed, liquid amount 50 mL), in 37 DEG C of thermostat containers, carry out solvent mensuration after standing for fermentation 72 h.
As shown in Figure 1, in bacterial strain S3 fermenting process, wood sugar is the same with glucose, along with the prolongation of fermentation time, is utilized gradually, illustrates that bacterial strain S3 synchronous consumption glucose and wood sugar can carry out production of butanol.
6th step: thalli morphology and molecular biology identification
1, identification of morphology utilizes gemma dye liquor to dye, microscopy.With reference to " common bacteria system identification handbook " (eastern elegant pearl, 2001).
2, the Molecular Identification of bacterial strain
Extract thalline STb gene, utilize bacterial 16 S rRNA gene universal primer to increase and obtain the 16S rRNA gene order of bacterial strain, sequencing result carries out Blast compare of analysis in GenBank database.
S3 bacterial strain is gram-positive product genus bacillus, and grow into about 18 h at anaerobic culture medium and start to form gemma, two ends are painted, and gemma diameter is suitable with thalline diameter, and thalline does not expand.The online software of 16S rRNA sequence result Blast of S3 bacterial strain carries out similarity analysis.As shown in Figure 1, this bacterial strain and Bai Shi clostridium NCIMB 8052 have the homology of 99 more than %, are initially identified as Bai Shi clostridium, called after Bai Shi clostridium SE-2.
Embodiment 2
Bai Shi clostridium SE-2 utilizes Corncob hydrolysate Batch fermentation to produce butanols
1, bacterial classification:
Bai Shi clostridium SE-2, this bacterial strain contained in China typical culture collection center (being called for short CCTCC) on August 19th, 2014 by applicant, and its deposit number is: CCTCC No. M2014384.
2, substratum:
Corncob hydrolysate: total sugar content is 49.2 g/L (wherein glucose 33.5 g/L and wood sugar 15.8 g/L)
Seed culture medium (TYA substratum): glucose 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, CH
3cOONH
43 g, KH
2pO
40.5 g, MgSO
47H
2o 0.2 g, FeSO
47H
2o 0.01 g, distilled water 1000 mL, pH 6.5,121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
Butanols basal fermentation medium (MP2): Corncob hydrolysate, K
2hPO
40.5 g, KH
2pO4 0.5 g, CH
3cOONH
42.2 g, MgSO
47H
2o 0.2 g, MnSO
4h
2o 0.01 g, NaCl 0.01 g, FeSO4 7H
2o 0.01 g, thiamine (thiamines, vitamins B
1) 0.001g, biotin (vitamin H) 0.00001g, p-aminobenzoic acid (p-benzaminic acid) 0.001g, add water to 1000 mL.
During actual preparation, preparation mother liquor 1(Solution 1, S1): p-aminobenzoic acid 100 mg/L, Thiamine 100 mg/L, Biotin 1 mg/L, use the filtering with microporous membrane in aseptic 0.22 μm of aperture degerming, then save backup in 4 DEG C.
3, zymotechnique:
The first step: accessed by Bai Shi clostridium SE-2 in seed culture medium (500 mL inoculation bottles, liquid amount 400mL), culture temperature 37 DEG C, quiescent culture 16 h, before seed access, logical nitrogen flooding is except the oxygen in tank, obtains first order seed nutrient solution.
Second step: the first order seed nutrient solution the first step obtained is transferred to (3L stainless steel inoculation bottle in secondary seed medium by 3%, liquid amount 2.5L), culture temperature 30 DEG C, quiescent culture 20 h, before seed access, logical nitrogen flooding is except the oxygen in tank, obtains secondary seed nutrient solution.
3rd step: the secondary seed nutrient solution obtained by second step is by 3% access containing in the fermention medium of Corncob hydrolysate, and fermenting process adopts anaerobism Batch fermentation, before seed access, logical nitrogen flooding is except the oxygen in tank.Fermentation culture adopts 30L stirred-tank fermenter, and liquid amount 25L, culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5.
4th step: fermentation 72h, glucose, wood sugar and product butanols and ethanol content in sampling analysis fermented liquid.
4, fermentation ends, in fermented liquid, glucose and xylose concentration is respectively 5.3 g/L and 0.2 g/L, and butanols and alcohol concn are respectively 11.76 g/L and 2.11 g/L.
Embodiment 3:
Clostridium acetobutylicum ATCC824 utilizes Corncob hydrolysate Batch fermentation to produce butanols
1, repeat no more with embodiment 2 something in common, difference separately adds explanation.
2, bacterial classification: clostridium acetobutylicum ATCC824, this bacterial strain, for producing butanols type strain, can utilize glucose and wood sugar, but fermenting process exists " resistance to glucose ", only has and just starts to utilize wood sugar after glucose utilization completely.
2, fermentation ends, in fermented liquid, glucose and xylose concentration is respectively is 0.5 g/L and 4.2 g/L, and butanols and alcohol concn are respectively 11.03 g/L and 1.68 g/L.
Embodiment 4
Bai Shi clostridium SE-2 (CCTCC No. M2014384) utilizes Corncob hydrolysate to continuously ferment product butanols
1, the present embodiment and embodiment 2 something in common repeat no more, and difference separately adds explanation.
2, zymotechnique
By the access of secondary seed nutrient solution containing in the fermention medium of Corncob hydrolysate, fermenting process adopts anaerobically fermenting, and before seed access, logical nitrogen flooding is except the oxygen in tank.Fermentation culture adopts 30L stirred-tank fermenter, and liquid amount 25L, culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5.
Fermenting process adopts continuous feeding fermentation mode, and in whole fermenting process, thinning ratio is 0.9 h
-1, fermentation time is 240 h.
3, sampling analysis, in fermented liquid, glucose and xylose concentration are respectively 7.3 g/L and 3.1 g/L, wherein glucose and wood sugar ratio are 2.35, basic identical with wood sugar ratio (2.12) with glucose in Corncob hydrolysate, illustrate that glucose and wood sugar are being utilized with identical speed substantially in Continuous Fermentation Processes, and the accumulation of wood sugar does not occur.
Embodiment 5
Clostridium acetobutylicum ATCC 824 utilizes Corncob hydrolysate to continuously ferment product butanols
1, repeat no more with embodiment 3 something in common, difference separately adds explanation.
2, zymotechnique
Fermentation culture adopts 30L stirred-tank fermenter, and liquid amount 25L, culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5.By seed liquor access containing in the fermention medium of Corncob hydrolysate, fermenting process adopts anaerobically fermenting, and before seed access, logical nitrogen flooding is except the oxygen in tank.
In whole fermenting process, thinning ratio is 0.9 h
-1, fermentation time is 240 h.
3, fermentation ends, in fermented liquid, glucose and xylose concentration are respectively 1.6 g/L and 8.7 g/L, and wherein wood sugar ratio is apparently higher than the ratio of wood sugar in Corncob hydrolysate, illustrate that wood sugar accumulates during the fermentation.
Claims (8)
1. a strain synchronously utilizes the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that this bacterial strain is Bai Shi clostridium SE-2, Latin name Clostridium beijerinckii SE-2; This bacterial strain is preserved in China typical culture collection center on August 19th, 2014, preserving number CCTCC No.M2014384.
2. a strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, the Bai Shi clostridium SE-2 that to it is characterized in that with lignocellulosic material hydrolyzed solution be prepared using preserving number CCTCC No.M2014384 ferments, synchronously utilize glucose in hydrolyzed solution and xylose production butanols and ethanol, realize the full sugar of ligno-cellulose hydrolysate and utilize.
3. a strain according to claim 2 synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that lignocellulosic material comprises in corn cob, maize straw, wheat stalk and branch one or more.
4. a strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel according to claim 2, it is characterized in that it comprises the steps: (1) and configure seed culture medium, Bai Shi clostridium SE-2 is accessed in seed culture medium and carry out first order seed cultivation and secondary seed cultivation; (2) in fermentor tank, anaerobically fermenting cultivation is carried out.
5. a strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that it comprises the steps:
The first step, preparation substratum, comprises seed culture medium, butanols basal fermentation medium;
Second step: first order seed is cultivated; Accessed by bacterial classification in seed culture medium, before seed access, logical nitrogen flooding is except the oxygen in tank, culture temperature 37 DEG C, and quiescent culture 16 h obtains first order seed nutrient solution;
3rd step: the first order seed nutrient solution obtained by second step is transferred in secondary seed medium by 3% volume ratio, before seed access, logical nitrogen flooding is except the oxygen in tank, culture temperature 30 DEG C, and quiescent culture 20 h obtains secondary seed nutrient solution;
4th step: the secondary seed nutrient solution the 3rd step obtained is by 3% volume ratio access containing in the fermention medium of Corncob hydrolysate, and fermenting process adopts anaerobism Batch fermentation, and before seed access, logical nitrogen flooding is except the oxygen in tank; Fermentation culture adopts 30L stirred-tank fermenter, liquid amount 25L, and culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5;
5th step: fermenting process 72h.
6. a strain synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that it comprises the steps:
The first step, preparation substratum, comprises seed culture medium, butanols basal fermentation medium;
Second step: first order seed is cultivated; Accessed by bacterial classification in seed culture medium, before seed access, logical nitrogen flooding is except the oxygen in tank, culture temperature 37 DEG C, and quiescent culture 16 h obtains first order seed nutrient solution;
3rd step: the first order seed nutrient solution obtained by second step is transferred in secondary seed medium by 3% volume ratio, before seed access, logical nitrogen flooding is except the oxygen in tank, culture temperature 30 DEG C, and quiescent culture 20 h obtains secondary seed nutrient solution;
4th step: contain in the fermention medium of Corncob hydrolysate by secondary seed nutrient solution by 3% volume ratio access, fermenting process adopts anaerobically fermenting, and before seed access, logical nitrogen flooding removes the oxygen in tank; Fermentation culture adopts 30L stirred-tank fermenter, liquid amount 25L, and culture temperature 37 DEG C, by 10 M sodium hydroxide control ph 6.5;
Fermentation employing is continuously fermented mode, and in whole fermenting process, thinning ratio is 0.9 h
-1, fermentation time is 240 h.
7. the strain according to claim 5 or 6 synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that Corncob hydrolysate: total sugar content is 49.2 g/L, wherein glucose 33.5 g/L and wood sugar 15.8 g/L;
A strain according to claim 5 or 6 synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that seed culture medium: glucose 40 g, extractum carnis 2 g, yeast powder 2 g, peptone 6 g, CH
3cOONH
43 g, KH
2pO
40.5 g, MgSO
47H
2o 0.2 g, FeSO
47H
2o 0.01 g, distilled water 1000 mL, pH 6.5,121 DEG C of autoclaving 15 min, blow 5 min with sterile nitrogen, drive most oxygen.
8. the strain according to claim 5 or 6 synchronously utilizes the application of the bacterial strain of glucose and xylose production Aalcohols fuel, it is characterized in that butanols basal fermentation medium: Corncob hydrolysate, K
2hPO
40.5 g, KH
2pO4 0.5 g, CH
3cOONH
42.2 g, MgSO
47H
2o 0.2 g, MnSO
4h
2o 0.01 g, NaCl 0.01 g, FeSO4 7H
2o 0.01 g, thiamine 0.001g, vitamin H 0.00001g, p-benzaminic acid 0.001g, add water to 1000 mL;
During actual preparation, preparation mother liquor 1:p-aminobenzoic acid 100 mg/L, thiamine 100 mg/L, vitamin H 1 mg/L, use the filtering with microporous membrane in aseptic 0.22 μm of aperture degerming, then save backup in 4 DEG C.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106746436A (en) * | 2017-02-06 | 2017-05-31 | 同济大学 | A kind of method of raising L glucose anaerobic degradations |
| CN106754553A (en) * | 2017-01-23 | 2017-05-31 | 天津大学 | Clostridium beijerinckii and application using xylose production butanol |
| CN109504713A (en) * | 2018-12-04 | 2019-03-22 | 齐鲁工业大学 | The method and application of high ester bond humic acid preparation are prepared using Clostridium beijerinckii |
| CN111500485A (en) * | 2020-03-19 | 2020-08-07 | 南京工业大学 | Clostridium acetobutylicum capable of co-utilizing glucose and xylose and application thereof |
| CN112852891A (en) * | 2021-02-03 | 2021-05-28 | 天津大学 | Artificial dual-bacterium system for producing mcl-PHA and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1355318A (en) * | 2000-11-27 | 2002-06-26 | 中国科学院成都生物研究所 | Process for preparing natural active dormin |
| CN1629298A (en) * | 2003-12-19 | 2005-06-22 | 首都师范大学 | Process for preparing alcohol through utilizing xylose and glucose by microzyme |
-
2015
- 2015-01-16 CN CN201510021602.XA patent/CN104673712B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1355318A (en) * | 2000-11-27 | 2002-06-26 | 中国科学院成都生物研究所 | Process for preparing natural active dormin |
| CN1629298A (en) * | 2003-12-19 | 2005-06-22 | 首都师范大学 | Process for preparing alcohol through utilizing xylose and glucose by microzyme |
Non-Patent Citations (3)
| Title |
|---|
| 张杰: "以木质纤维素为原料发酵生产丙酮丁醇的过程以及科学机理研究", 《中国博士学位论文全文数据库》 * |
| 李岩等: "玉米秸秆烯酸水解与水解液发酵的实验研究", 《现代化工》 * |
| 潘微等: "玉米浆干粉在葡糖糖和木糖混合丙酮丁醇发酵中的应用", 《化工进展》 * |
Cited By (9)
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|---|---|---|---|---|
| CN106754553A (en) * | 2017-01-23 | 2017-05-31 | 天津大学 | Clostridium beijerinckii and application using xylose production butanol |
| CN106754553B (en) * | 2017-01-23 | 2020-04-03 | 天津大学 | Clostridium beijerinckii for producing butanol by using xylose and application thereof |
| CN106746436A (en) * | 2017-02-06 | 2017-05-31 | 同济大学 | A kind of method of raising L glucose anaerobic degradations |
| CN106746436B (en) * | 2017-02-06 | 2020-06-26 | 同济大学 | Method for improving anaerobic degradation of L-glucose |
| CN109504713A (en) * | 2018-12-04 | 2019-03-22 | 齐鲁工业大学 | The method and application of high ester bond humic acid preparation are prepared using Clostridium beijerinckii |
| CN109504713B (en) * | 2018-12-04 | 2022-05-17 | 齐鲁工业大学 | Method for preparing high-ester-bond humic acid preparation by using clostridium beijerinckii and application |
| CN111500485A (en) * | 2020-03-19 | 2020-08-07 | 南京工业大学 | Clostridium acetobutylicum capable of co-utilizing glucose and xylose and application thereof |
| CN111500485B (en) * | 2020-03-19 | 2022-04-15 | 南京工业大学 | Clostridium acetobutylicum capable of co-utilizing glucose and xylose and application thereof |
| CN112852891A (en) * | 2021-02-03 | 2021-05-28 | 天津大学 | Artificial dual-bacterium system for producing mcl-PHA and application thereof |
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