CN101906458A - Detection method of saccharification capability of straw anaerobic microbe - Google Patents
Detection method of saccharification capability of straw anaerobic microbe Download PDFInfo
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- CN101906458A CN101906458A CN 201010235131 CN201010235131A CN101906458A CN 101906458 A CN101906458 A CN 101906458A CN 201010235131 CN201010235131 CN 201010235131 CN 201010235131 A CN201010235131 A CN 201010235131A CN 101906458 A CN101906458 A CN 101906458A
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Abstract
The invention provides a detection method of saccharification capability of a straw anaerobic microbe, and relates to the detection method of the straw saccharification capability, which solves the problem that the existing detection method of the saccharification capability of the straw microbe fails to truly reflect the saccharification capability of the microbe in real time to cause inaccurate detection. The detection method comprises the following steps: 1. fitting a regression equation; 2. adding no-carbon source culture solution to a double-chamber reactor, adding straw sections and inoculating bacterial liquid of an anaerobic straw saccharified composite microbial system or an anaerobic straw saccharified composite strain into a chamber A of the reactor, inoculating YUAN-3 bacterial liquid into a chamber B of the reactor, culturing and collecting hydrogen in the chamber B of the reactor, and measuring hydrogen content by high performance gas chromatography; and 3. substituting hydrogen production quantity in the chamber B of the reactor into the regression equation, and calculating quantity of reducing sugar produced by straw degradation via the anaerobic straw saccharified composite microbial system or the anaerobic straw saccharified strain in the chamber A of the reactor to finally obtain the straw saccharification capability. The detection method of the invention has good continuity and good effectiveness, and can help truly reflect the saccharification capability and quantize saccharification effect in real time.
Description
Technical field
The present invention relates to a kind of detection method of stalk saccharification capability.
Background technology
At present, in the stalk microbe saccharifying, mainly by measure fermentation system inner cellulose material be degraded the reducing sugar amount that produces how much, detect the microorganism saccharification capability.But owing to reducing sugar in glucose effect and extraction fermented liquid and the storage process can be by reasons such as sustainable utilizations, only extract fermented liquid and measure wherein residual reducing sugar amount, reaction microorganism saccharification capability that can not be real, real-time causes the detection of microorganism saccharification capability to lack continuity with effective.
Summary of the invention
The present invention seeks in order to solve the detection of existing stalk microbe saccharification capability, reaction microorganism saccharification capability that can not be real, real-time, cause detecting the problem that lacks continuity and actual effect, and a kind of detection method of saccharification capability of straw anaerobic microbe is provided.
The detection method of saccharification capability of straw anaerobic microbe realizes according to the following steps:
One, be to add 21.6ml in the anaerobism polycarbonate single chamber reactor of 30ml not have carbon source nutrient solution and 0.12g glucose to volume as fermented liquid, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 then, cultivate 32h down at 30 ℃, collect hydrogen every 4h during this time, the hydrogen utilization high resolution gas chromatography carries out assay, utilizing glucose consumption amount in the high-performance liquid chromatogram determination fermented liquid simultaneously, is Y=-0.00007x with the glucose consumption amount to hydrogen generating quantity The regression equation
2+ 0.0064x+0.0013 (R
2=0.9996);
Two, in volume is two chambers reactor of anaerobism polycarbonate of 60ml, add 43.2ml and do not have the carbon source nutrient solution, in the reactor A chamber, add stalk section and inoculate 2.4ml anaerobism stalk saccharification composite microbial system or the bacterium liquid of anaerobism stalk saccharification bacterial strain, in the reactor B chamber, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 simultaneously, cultivate down at 30 ℃ then, collect reactor B chamber hydrogen every 4h, and utilize high resolution gas chromatography to carry out hydrogen content and measure;
Three, will produce amounts of hydrogen in the reactor B chamber, substitution The regression equation Y=-0.00007x
2+ 0.0064x+0.0013 (R
2=0.9996),, promptly finishes the detection of saccharification capability of straw anaerobic microbe by calculating the amount of the reducing sugar that anaerobism stalk saccharification composite microbial system in the reactor A chamber or anaerobism stalk saccharification bacterial strain degrading straw produced;
Wherein in the step 1 no carbon source substratum by the NH of 1.0g
4The K of Cl, 3.5g
2HPO
4, 1.5g KH
2PO
4, 0.5g MgCl
2, the VITAMIN liquid of yeast powder, 5ml of peptone, 2.0g of halfcystine, 2.0g of KCl, 0.5g of NaCl, 0.2g of 1.0g and 1ml trace element solution be dissolved in the distilled water of 1000mL and form, described VITAMIN liquor is by the Thioctic Acid of 50.0mg, the vitamin H of 20.0mg, the nicotinic acid of 0.35g, the vitamin of 5.0mg, the para-amino benzoic acid of 50.0mg, the folic acid of 20.0mg, the calcium pantothenate of 50.0mg, the vitamins B of 1.0mg
12Be dissolved in the distilled water of 1000ml with the pyridoxine hydrochloride of 100.0mg and form, the pH value is 6.8～7.0, and described trace element solution is dissolved in by the five water sodium selenates of the sodium wolframate of the water Sodium orthomolybdate of the six water nickelous chlorides of the CoCL2 of the copper chloride dihydrate of the tetrahydrate manganese chloride of the boric acid of the zinc chloride of the iron protochloride of 1.5g, 70mg, 6mg, 0.1g, 2mg, 0.19g, 24mg, 36mg, 15mg and 15mg in the distilled water of 1000mL to be formed; Y is the glucose consumption amount in the single chamber reactor in the step 1, and unit is gram, and x is a hydrogen volume, and unit is a milliliter; Y is the glucose consumption amount in the reactor B chamber in the step 3, is the amount of the reducing sugar that produces in the reactor A chamber, and unit is gram, and x is a hydrogen volume, and unit is a milliliter.
The present invention is with anaerobism stalk saccharification composite microbial system or anaerobism stalk saccharification bacterial strain and utilize reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 to carry out the locellus simultaneous saccharification and fermentation, according to utilizing reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 hydrogen generating quantity under optimum conditions, be certain proportionlity with the reducing sugar amount of the glucose form of its consumption, by measuring the instant hydrogen output of YUAN-3, calculate generation unit volume H
2The time the reducing sugar consumption, detect thereby anaerobism stalk saccharification composite microbial system under the locellus synchronized culture condition or anaerobism stalk saccharification bacterial strain are separated the carbohydrogenic ability of stalk.The present invention compares with reducing sugar residual quantity in the direct mensuration anaerobism stalk fermentation system, more can actual response anaerobism stalk saccharification composite microbial system or the stalk saccharification capability and the saccharification potentiality of anaerobism stalk saccharification bacterial strain, the highly effective hydrogen yield bacterial strain is connected with the diastatic fermentation system, can be by fixed its hydrogen output of time actual measurement, detect the saccharification capability of stalk amylomyces timely, the reaction stalk is converted into the effect of reducing sugar, this detection method has possessed good continuity and actual effect, quantitation of glycated effect that can be real-time, method is easy and simple to handle, the accuracy height, synchronism is good.
Description of drawings
Fig. 1 is the device synoptic diagram of two chambers reactor of used anaerobism polycarbonate in the step 2 of embodiment one; Fig. 2 utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 hydrogen output to glucose consumption amount matched curve figure in the embodiment three; Fig. 3 is a hydrogen producing change dynamics graphic representation in the reactor B chamber in the embodiment three; Fig. 4 is that reactor produces and the graphic representation of residual reducing sugar amount in the embodiment three.
Embodiment
Embodiment one: in conjunction with Fig. 1 present embodiment is described, the detection method of present embodiment saccharification capability of straw anaerobic microbe realizes according to the following steps:
One, be to add 21.6ml in the anaerobism polycarbonate single chamber reactor of 30ml not have carbon source nutrient solution and 0.12g glucose to volume as fermented liquid, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 then, cultivate 32h down at 30 ℃, collect hydrogen every 4h during this time, the hydrogen utilization high resolution gas chromatography carries out assay, utilizing glucose consumption amount in the high-performance liquid chromatogram determination fermented liquid simultaneously, is Y=-0.00007x with the glucose consumption amount to hydrogen generating quantity The regression equation
2+ 0.0064x+0.0013 (R
2=0.9996);
Two, in volume is two chambers reactor 1 of anaerobism polycarbonate of 60ml, add 43.2ml and do not have the carbon source nutrient solution, in the 1-1 of reactor A chamber, add stalk section and inoculate 2.4ml anaerobism stalk saccharification composite microbial system or the bacterium liquid of anaerobism stalk saccharification bacterial strain, in the 1-2 of reactor B chamber, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 simultaneously, cultivate down at 30 ℃ then, collect reactor B chamber 1-2 hydrogen every 4h, and utilize high resolution gas chromatography to carry out hydrogen content and measure;
Three, will produce amounts of hydrogen among the 1-2 of reactor B chamber, substitution The regression equation Y=-0.00007x
2+ 0.0064x+0.0013 (R
2=0.9996),, promptly finishes the detection of saccharification capability of straw anaerobic microbe by calculating the amount of the reducing sugar that anaerobism stalk saccharification composite microbial system among the 1-1 of reactor A chamber or anaerobism stalk saccharification bacterial strain degrading straw produced;
Wherein in the step 1 no carbon source substratum by the NH of 1.0g
4The K of Cl, 3.5g
2HPO
4, 1.5g KH
2PO
4, 0.5g MgCl
2, the VITAMIN liquid of yeast powder, 5ml of peptone, 2.0g of halfcystine, 2.0g of KCl, 0.5g of NaCl, 0.2g of 1.0g and 1ml trace element solution be dissolved in the distilled water of 1000mL and form, described VITAMIN liquor is by the Thioctic Acid of 50.0mg, the vitamin H of 20.0mg, the nicotinic acid of 0.35g, the vitamin of 5.0mg, the para-amino benzoic acid of 50.0mg, the folic acid of 20.0mg, the calcium pantothenate of 50.0mg, the vitamins B of 1.0mg
12Be dissolved in the distilled water of 1000ml with the pyridoxine hydrochloride of 100.0mg and form, the pH value is 6.8～7.0, and described trace element solution is dissolved in by the five water sodium selenates of the sodium wolframate of the water Sodium orthomolybdate of the six water nickelous chlorides of the CoCL2 of the copper chloride dihydrate of the tetrahydrate manganese chloride of the boric acid of the zinc chloride of the iron protochloride of 1.5g, 70mg, 6mg, 0.1g, 2mg, 0.19g, 24mg, 36mg, 15mg and 15mg in the distilled water of 1000mL to be formed; Y is the glucose consumption amount in the single chamber reactor in the step 1, and unit is gram, and x is a hydrogen volume, and unit is a milliliter; Y is the glucose consumption amount among the 1-2 of reactor B chamber in the step 3, is the amount of the reducing sugar that produces among the 1-1 of reactor A chamber, and unit is gram, and x is a hydrogen volume, and unit is a milliliter.
In the present embodiment step 1 in the single chamber reactor glucose as sole carbon source.
Utilize in the present embodiment step 1 reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 (
Ethanoligenens harbinense) be Harbin producing and ethanol bacillus, be the highly effective hydrogen yield bacterium, for purchase obtains.
R represents degree of fitting in the present embodiment step 1.
Anaerobism stalk saccharification composite microbial system or anaerobism stalk saccharification bacterial strain obtain for buying in the present embodiment step 2.
Embodiment two: what present embodiment and embodiment one were different is that two chamber reactors (1) are formed by two onesize anaerobism polycarbonate single chamber combination of reactors described in the step 2, separate with 0.45 μ m bacterium filter membrane between reactor A chamber (1-1) and reactor B chamber (1-2), do not add carbon source in the reactor B chamber (1-2), and the reducing sugar that utilizes anaerobism stalk saccharification composite microbial system in the reactor A chamber (1-1) or anaerobism stalk saccharification bacterial strain degrading straw to be produced ferments and produces hydrogen.Wherein step and parameter are identical with embodiment one.
Embodiment three: in conjunction with Fig. 1,2 and 3 explanation present embodiments, the detection method of present embodiment present embodiment saccharification capability of straw anaerobic microbe realizes according to the following steps:
One, be to add 21.6ml in the anaerobism polycarbonate single chamber reactor of 30ml not have carbon source nutrient solution and 0.12g glucose to volume as fermented liquid, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 then, cultivate 32h down at 30 ℃, collect hydrogen every 4h during this time, the hydrogen utilization high resolution gas chromatography carries out assay, utilizing glucose consumption amount in the high-performance liquid chromatogram determination fermented liquid simultaneously, is Y=-0.00007x with the glucose consumption amount to hydrogen generating quantity The regression equation
2+ 0.0064x+0.0013 (R
2=0.9996);
Two, in volume is two chambers reactor 1 of anaerobism polycarbonate of 60ml, add 43.2ml and do not have the carbon source nutrient solution, in the 1-1 of reactor A chamber, add stalk section and inoculate 2.4ml anaerobism stalk saccharification composite microbial system or the bacterium liquid of anaerobism stalk saccharification bacterial strain, in the 1-2 of reactor B chamber, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 simultaneously, cultivate down at 30 ℃ then, collect reactor B chamber 1-2 hydrogen every 4h, and utilize high resolution gas chromatography to carry out hydrogen content and measure;
Three, will produce amounts of hydrogen among the 1-2 of reactor B chamber, substitution The regression equation Y=-0.00007x
2+ 0.0064x+0.0013 (R
2=0.9996),, promptly finishes the detection of saccharification capability of straw anaerobic microbe by calculating the amount of the reducing sugar that anaerobism stalk saccharification composite microbial system among the 1-1 of reactor A chamber or anaerobism stalk saccharification bacterial strain degrading straw produced;
Wherein in the step 1 no carbon source substratum by the NH of 1.0g
4The K of Cl, 3.5g
2HPO
4, 1.5g KH
2PO
4, 0.5g MgCl
2, the VITAMIN liquid of yeast powder, 5ml of peptone, 2.0g of halfcystine, 2.0g of KCl, 0.5g of NaCl, 0.2g of 1.0g and 1ml trace element solution be dissolved in the distilled water of 1000mL and form, described VITAMIN liquor is by the Thioctic Acid of 50.0mg, the vitamin H of 20.0mg, the nicotinic acid of 0.35g, the vitamin of 5.0mg, the para-amino benzoic acid of 50.0mg, the folic acid of 20.0mg, the calcium pantothenate of 50.0mg, the vitamins B of 1.0mg
12Be dissolved in the distilled water of 1000ml with the pyridoxine hydrochloride of 100.0mg and form, the pH value is 7.0, and described trace element solution is dissolved in by the five water sodium selenates of the sodium wolframate of the water Sodium orthomolybdate of the six water nickelous chlorides of the CoCL2 of the copper chloride dihydrate of the tetrahydrate manganese chloride of the boric acid of the zinc chloride of the iron protochloride of 1.5g, 70mg, 6mg, 0.1g, 2mg, 0.19g, 24mg, 36mg, 15mg and 15mg in the distilled water of 1000mL to be formed; Y is the glucose consumption amount in the single chamber reactor in the step 1, and unit is gram, and x is a hydrogen volume, and unit is a milliliter; Y is the glucose consumption amount among the 1-2 of reactor B chamber in the step 3, is the amount of the reducing sugar that produces among the 1-1 of reactor A chamber, and unit is gram, and x is a hydrogen volume, and unit is a milliliter.
Used stalk section is the rice straw section in the present embodiment;
In the present embodiment step 1 with the glucose consumption amount to hydrogen generating quantity The regression equation Y=-0.00007x
2+ 0.0064x+0.0013 (R
2=0.9996), press YUAN-3 hydrogen output shown in Figure 2 to the matched curve of glucose consumption amount, by shown in Figure 3 YUAN-3 hydrogen output substitution regression equation gained anaerobism stalk saccharification composite microbial system JY (abbreviating JY in the text as) is produced the reducing sugar amount as shown in Figure 4, the reducing sugar amount that YUAN-3 is consumed during the fermentation is in continuous rising, and present dynamic change trend, terminal point maximum value 164.80mg, calculating JY degrading straw generation reducing sugar amount is the 0.5150g/g stalk, and the straw degradative rate reaches 61.06%; And residual reducing sugar content is all lower in the reactor, and maximum also has only 3.31mg respectively, and does not present variation tendency; Simultaneously, utilize the DNS method to measure the residual reducing sugar amount in the reaction system, JY and YUAN-3 reducing sugar content are all lower in the reactor, and maximum also has only 3.310mg and 3.005mg respectively, and dynamic change and irregular; The result shows that in the process of straw degradative, possible reducing sugar is just in time utilized after generation, be difficult in reaction system and find the residual of sugar; This shows that to produce the glucose amount that certain volume hydrogen is consumed, the reducing sugar amount of explaining the generation of JY degrading straw is feasible and effective.
Claims (2)
1. the detection method of a saccharification capability of straw anaerobic microbe is characterized in that the detection method of saccharification capability of straw anaerobic microbe realizes according to the following steps:
One, be to add 21.6ml in the anaerobism polycarbonate single chamber reactor of 30ml not have carbon source nutrient solution and 0.12g glucose to volume as fermented liquid, inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 then, cultivate 32h down at 30 ℃, collect hydrogen every 4h during this time, the hydrogen utilization high resolution gas chromatography carries out assay, utilizing glucose consumption amount in the high-performance liquid chromatogram determination fermented liquid simultaneously, is Y=-0.00007x with the glucose consumption amount to hydrogen generating quantity The regression equation
2+ 0.0064x+0.0013;
Two, in volume is two chambers reactors (1) of anaerobism polycarbonate of 60ml, add 43.2ml and do not have the carbon source nutrient solution, in reactor A chamber (1-1), add stalk section and inoculate 2.4ml anaerobism stalk saccharification composite microbial system or the bacterium liquid of anaerobism stalk saccharification bacterial strain, in reactor B chamber (1-2), inoculate the bacterium liquid that 2.4ml utilizes reducing sugar highly effective hydrogen yield bacterial strain YUAN-3 simultaneously, cultivate down at 30 ℃ then, collect reactor B chamber (1-2) hydrogen every 4h, and utilize high resolution gas chromatography to carry out hydrogen content and measure;
Three, amounts of hydrogen, substitution The regression equation Y=-0.00007x will be produced in reactor B chamber (1-2)
2+ 0.0064x+0.0013 by calculating the amount of the reducing sugar that anaerobism stalk saccharification composite microbial system in the reactor A chamber (1-1) or anaerobism stalk saccharification bacterial strain degrading straw produced, promptly finishes the detection of saccharification capability of straw anaerobic microbe;
Wherein in the step 1 no carbon source substratum by the NH of 1.0g
4The K of Cl, 3.5g
2HPO
4, 1.5g KH
2PO
4, 0.5g MgCl
2, the VITAMIN liquid of yeast powder, 5ml of peptone, 2.0g of halfcystine, 2.0g of KCl, 0.5g of NaCl, 0.2g of 1.0g and 1ml trace element solution be dissolved in the distilled water of 1000mL and form, described VITAMIN liquor is by the Thioctic Acid of 50.0mg, the vitamin H of 20.0mg, the nicotinic acid of 0.35g, the vitamin of 5.0mg, the para-amino benzoic acid of 50.0mg, the folic acid of 20.0mg, the calcium pantothenate of 50.0mg, the vitamins B of 1.0mg
12Be dissolved in the distilled water of 1000ml with the pyridoxine hydrochloride of 100.0mg and form, the pH value is 6.8～7.0, and described trace element solution is dissolved in by the five water sodium selenates of the sodium wolframate of the water Sodium orthomolybdate of the six water nickelous chlorides of the CoCL2 of the copper chloride dihydrate of the tetrahydrate manganese chloride of the boric acid of the zinc chloride of the iron protochloride of 1.5g, 70mg, 6mg, 0.1g, 2mg, 0.19g, 24mg, 36mg, 15mg and 15mg in the distilled water of 1000mL to be formed; Y is the glucose consumption amount in the single chamber reactor in the step 1, and unit is gram, and x is a hydrogen volume, and unit is a milliliter; Y is the glucose consumption amount in the reactor B chamber (1-2) in the step 3, is the amount of the reducing sugar that produces in the reactor A chamber (1-1), and unit is gram, and x is a hydrogen volume, and unit is a milliliter.
2. the detection method of a kind of saccharification capability of straw anaerobic microbe according to claim 1, it is characterized in that two chamber reactors (1) are formed by two onesize anaerobism polycarbonate single chamber combination of reactors described in the step 2, separate with 0.45 μ m bacterium filter membrane between reactor A chamber (1-1) and reactor B chamber (1-2), do not add carbon source in the reactor B chamber (1-2), and the reducing sugar that utilizes anaerobism stalk saccharification composite microbial system in the reactor A chamber (1-1) or anaerobism stalk saccharification bacterial strain degrading straw to be produced ferments and produces hydrogen.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624109A (en) * | 2004-08-27 | 2005-06-08 | 哈尔滨工业大学 | Self-coagulating producing hydrogen bacteria and its screening process |
| CN1772877A (en) * | 2005-09-28 | 2006-05-17 | 哈尔滨工业大学 | Continuous flow culture method of industrial biological hydrogen preparing spawn and biological hydrogen preparing system reinforcing method |
| CN101402926A (en) * | 2008-11-18 | 2009-04-08 | 哈尔滨工业大学 | Biological reinforcing method of hydrogenogen compensating material cultivation and biological hydrogen production system |
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2010
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624109A (en) * | 2004-08-27 | 2005-06-08 | 哈尔滨工业大学 | Self-coagulating producing hydrogen bacteria and its screening process |
| CN1772877A (en) * | 2005-09-28 | 2006-05-17 | 哈尔滨工业大学 | Continuous flow culture method of industrial biological hydrogen preparing spawn and biological hydrogen preparing system reinforcing method |
| CN101402926A (en) * | 2008-11-18 | 2009-04-08 | 哈尔滨工业大学 | Biological reinforcing method of hydrogenogen compensating material cultivation and biological hydrogen production system |
Non-Patent Citations (3)
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| 《哈尔滨工业大学学报》 20100630 刘冰等 秸秆糖化复合菌系的筛选方法及糖化效果评价 1-2 第42卷, 第6期 2 * |
| 《太阳能学报》 20070430 冯玉杰等 混合菌群用于纤维素糖化和燃料酒精发酵的试验研究 第28卷, 第4期 2 * |
| 《沈阳建筑大学学报(自然科学版)》 20080531 包红旭等 新菌X_9协同B_(49)同步发酵纤维素产氢能力分析 第24卷, 第3期 2 * |
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Application publication date: 20101208 |