CN102041274A - Method for producing hydrogen by fermenting special anaerobic clostridium butyricum - Google Patents
Method for producing hydrogen by fermenting special anaerobic clostridium butyricum Download PDFInfo
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Abstract
The invention provides a method for producing hydrogen by fermenting special anaerobic clostridium butyricum. The screened special anaerobic bacterium is clostridium butyricum JCM1391. Proven by tests, the hydrogen is produced by fermenting by batch by using anaerobic bacteria under the sterile anaerobic condition, wherein the mixture of 10.0-15.0g of peptone, 2.0-3.0g of beef extract, 5.0-10.0g of yeast extract, 3.0-4.0g of Na2HPO4, 0.2-0.5g of L-cysteine, 0.2-0.5g of L-cysteine hydrochloride monohydrate, 1 ml of resazurin indicator which accounts for 0.2 percent by weight and 1 ml of distilled water is used as culture media, and glucose is used as fermentation matrix. The method has the advantage of high hydrogen production efficiency and low requirement on environmental condition for fermentation.
Description
Technical field
The present invention relates to a kind of method of utilizing the microbiological anaerobic ferment for hydrogen production, especially a kind of method of utilizing the hydrogen manufacturing of efficient anaerobic bacterium.
Background technology
The combustion heat value height of hydrogen is 3 times of gasoline, and unique products of combustion is a water, environmentally safe, so Hydrogen Energy is cleaning, efficient, reproducible green energy resource.
Hydrogen Energy is not a primary energy source, need produce from hydrogenous compound.The hydrogen of the whole world more than 90% is from fossil-fueled at present, and all the other are water electrolysis hydrogen producing.No matter be which kind of hydrogen production process all will directly or indirectly consume a large amount of fossil energies.The fossil oil reserves are limited, and are tending towards exhausted, simultaneously, the greenhouse gases that generate during combustion of fossil fuel, other organic compound such as toxic gas have caused the serious environmental pollution and have made global climate change.Biological hydrogen production is abundant with its raw material sources, lower-price characteristic is more and more received people's attention, in case large-scale production will be the most potential a kind of hydrogen production process.
The biological hydrogen production idea was proposed by lewis in 1966 at first, and the oil crisis that experiences the seventies in 20th century makes the whole world recognize the practicality and the urgency of biological hydrogen production then.Up to the nineties, when the world was faced with the dual-pressure of energy and environment, biological hydrogen production had been put on agenda once again.Biological hydrogen production comprises photosynthetic organism hydrogen generation and two kinds of approach of anaerobically fermenting hydrogen manufacturing.The latter have produce hydrogen rate height, hydrogen-producing speed fast, produce that hydrogen is continual and steady, the design operation of reaction unit is simple, raw material sources are extensive and characteristics such as cost is low, be easier to accomplish scale production, thereby become the main direction of biological hydrogen production research.Anaerobically fermenting hydrogen manufacturing bacterial classification comprises obligatory anaerobic bacteria and facultative anaerobe, wherein, the cultivation of obligatory anaerobic bacteria, transportation and industrialized condition harshness, and facultative anaerobe has adaptability widely, but the facultative anaerobe hydrogen generation efficiency is less than the obligatory anaerobic bacteria height, rationally cultivate under proper condition, can overcome its shortcoming, obligatory anaerobic bacteria will be the better bacterium of biological hydrogen production industrialization.At present, the obligatory anaerobic bacteria kind is fewer, lacks new mushroom-seed culturing, and hydrogen generation efficiency is on the low side, and the hydrogen manufacturing condition is treated further optimization.
By patent retrieval, at present existing patent mainly is the research to fermentation substrate, fermentation unit and raising hydrogen generation efficiency, method (application number: 200710032658.0 weeks are strange less etc.) as a kind of mud and the hydrogen manufacturing of organic waste mixed biologic, photosynthetic microorganism hydrogen-manufacturing reactor (application number: 200620032282.4), the fermentative hydrogen production device (application number: the 00231651.X power of building in Shen), a kind of CO that utilizes
2Make the algae fast breeding be directly used in the method (200710010804.X) and the efficient fermentation method biological hydrogen production expanded bed equipment (application number: 03260012.7 Nan Qi etc.) etc. of biological hydrogen production, and for fermented bacterium particularly a little less than the screening study relative thin of highly effective hydrogen yield bacterial classification, a kind of biological hydrogen production method and special bacterium thereof (application number: 200910088408.8 Xing Xin can wait) are only arranged, but the used bacterial classification of this method is the reorganization bacterium after importing by gene, operation is complicated, and the strict anaerobism of fermentation condition is difficult for promoting.So, very necessary for the exploration optimization of the screening of novel obligate anaerobic new mushroom-seed culturing and process for making hydrogen condition.
Summary of the invention
The present invention mainly comprises a kind of method of utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production, this method is under the strictly anaerobic condition, use clostridium butylicum LMG1217(Clostridium butyricum) JCM1391, with glucose is fermentation substrate, adopt mode of batch fermentation, can begin to produce the hydrogen process after 24 hours.
Technical scheme of the present invention is: a kind of method of utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production, under aseptic anaerobic condition, utilize anerobe, with peptone, extractum carnis, yeast extract, Na
2HPO
4, the mixture formed of L-halfcystine, L-cysteine hydrochloride one water, resazurin indication and distilled water is substratum, with glucose is fermentation substrate, adopt mode of batch fermentation to carry out hydrogen manufacturing, described anerobe is clostridium butylicum (Clostridium butyricum) JCM1391.
Consisting of of substratum: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2HPO
43.0 resazurin indicator 1mL and the distilled water 1L of～4.0g, L-halfcystine 0.2～0.5g, L-cysteine hydrochloride one water 0.2～0.5g, 0.2%wt.
The concentration of described fermentation substrate glucose is 5.0～10.0g/L
Concrete steps are: under the aseptic anaerobic environment, it is 7.0～7.5 that substratum is transferred pH with phosphate buffered saline buffer, substratum in the heating in water bath bottle, simultaneously from bottleneck inflated with nitrogen 5 minutes, treat that liquid phase becomes colorless, seal up bottle cap at last, 115 ℃ of high-temperature sterilizations, after the oven dry with the inoculation of clostridium butylicum to the bottle that substratum is housed, leave standstill under 37～40 ℃ of conditions be cultured to dry cell weight to reach 0.658g/L after as inoculum, then with inoculum and substratum by volume the ratio of 1:10 be inoculated in the substratum in the fermentation flask and obtain fermented liquid; Under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, produce hydrogen continuously.
The obligatory anaerobic bacteria that the present invention screened belongs to fusobacterium (Clostridium Prazmowski in the bacteriology classification, 1880), be the Gram-positive anaerobic bacillus(cillus anaerobicus), title is clostridium butylicum (Clostridium butyricum) JCM1391, available from Japanese biotechnology research microbial strains preservation center (the Marine Biotechnology Institute Culture Collection of institute, Marine Biotechnology Institution, MBIC).
Clostridium butylicum (Clostridium butyricum) is an obligatory anaerobic bacteria, culture environment oxygen is required relatively stricter.The present invention finds optimum process condition to make and reaches the best hydrogen output of this obligatory anaerobic bacteria through to the test of fermentation condition combining and configuring.The bacterium enlarged culturing, the fermentation inoculation is all operated in anaerobic box, and adopts mode of batch fermentation; Hydrogen generation efficiency height under this condition, low to the yeasting conditional request, have broad application prospects.
Beneficial effect:
At first, filter out a kind of novel fermentation hydrogen manufacturing bacterial classification, this bacterial classification is an obligatory anaerobic bacteria, has growth rapidly, the hydrogen generation efficiency advantages of higher;
Secondly, determined technological condition for fermentation, determined that fermentation substrate is that concentration is the glucose of 5.0～10.0g/L, substratum is formulated as: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2HPO
43.0～4.0g, L-halfcystine 0.2～0.5g, L-cysteine hydrochloride one water 0.2～0.5g, resazurin indicator (0.2%) 1mL, distilled water 1L; It is 7.0～7.5 that substratum is transferred pH with phosphate buffered saline buffer, and liquid and inflated with nitrogen are 5 minutes in the heating in water bath bottle, in vitro keep anaerobic environment, and fermentation and inoculation are all operated in anaerobic box, adopt mode of batch fermentation; Under 37～40 ℃ of conditions, leave standstill and cultivate 24 hours, by being connected in the fermentation flask, carry out constant-temperature shaking culture under 37～40 ℃ of conditions then, can produce hydrogen continuously as inoculum.
Description of drawings
Fig. 1 is a device of the present invention.
Wherein, 1 is nitrogen wash mouth and liquid sampling mouth, and 2 are the gas sampling mouth, and 3 is the collection and confinement of gases pipe, and 4 is leveling bottle.
Fig. 2 is a bacterium liquid OD value change curve in time.
Fig. 3 is a hydrogen output change curve in time.
Fig. 4 is the hydrogen output change curve of scheme one and scheme two.
Embodiment
A kind of method of utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production under aseptic anaerobic condition, is utilized anerobe, with peptone, extractum carnis, yeast extract, Na
2HPO
4, the mixture formed of L-halfcystine, L-cysteine hydrochloride one water, resazurin indication and distilled water is substratum, with glucose is fermentation substrate, adopt mode of batch fermentation to carry out hydrogen manufacturing, described anerobe is clostridium butylicum (Clostridium butyricum) JCM1391.
Consisting of of substratum: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2HPO
43.0 resazurin indicator 1mL and the distilled water 1L of～4.0g, L-halfcystine 0.2～0.5g, L-cysteine hydrochloride one water 0.2～0.5g, 0.2%wt.
The concentration of described fermentation substrate glucose is 5.0～10.0g/L
Concrete steps are: under the aseptic anaerobic environment, it is 7.0～7.5 that substratum is transferred pH with phosphate buffered saline buffer, substratum in the heating in water bath bottle, simultaneously from bottleneck inflated with nitrogen 5 minutes, treat that liquid phase becomes colorless, seal up bottle cap at last, 115 ℃ of high-temperature sterilizations, after the oven dry with the inoculation of clostridium butylicum to the bottle that substratum is housed, leave standstill under 37～40 ℃ of conditions be cultured to dry cell weight to reach 0.658g/L after as inoculum, then with inoculum and substratum by volume the ratio of 1:10 be inoculated in the substratum in the fermentation flask and obtain fermented liquid; Under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, produce hydrogen continuously.
?
The enlarged culturing of bacterial classification:
Embodiment 1:Bacterial classification anaerobic operation Technology Selection is determined
The preparation basic medium:
(1) scheme one: the nutrient solution component is: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2HPO
44g/L, L-halfcystine 0.2 g/L, L-cysteine hydrochloride one water 0.5 g/L, distilled water 1000mL.Regulate pH=7.0, every 100mL nutrient solution 250mL Erlenmeyer flask of packing into, internal layer wraps up with gauze, and outer bag newspaper seals.120 ℃ of high-temperature sterilization 25min after the oven dry, are seeded to bacterial strain Clostridium butyricum in the bottle that substratum is housed (aseptic anaerobic operation) in 60 ℃ of baking ovens, leave standstill under 37 ℃ and cultivate 24～48h.
(2) scheme two: basic identical with above-mentioned nutrient solution component, in addition, other adds resazurin indicator (0.2%) 1mL/L.Regulate pH=7.0, every 100mL nutrient solution 250mL saline bottle (the band bottle stopper can be airtight) of packing into, liquid in the heating in water bath bottle is blown into nitrogen from bottleneck simultaneously, treats that liquid phase becomes colorless (being oxygen-free environment in the bottle), seals up bottle cap.120 ℃ of high-temperature sterilization 25min after the oven dry, are seeded to bacterial strain Clostridium butyricum in the bottle that substratum is housed (aseptic anaerobic operation) in 60 ℃ of baking ovens, leave standstill under 37 ℃ and cultivate 24～48h.
In culturing process, with the absorbance of 756 ultraviolet-visible pectrophotometers, draw bacterium liquid OD value change curve in time at 600 nm places timing working sample, bacterial growth OD value changes in time sees accompanying drawing 2.
Experimental result shows: the total growth time of scheme two (strictly anaerobic condition) bacterium is 40h, final OD
600Value is 1.425, and bacterial reproduction is rapid, and it is good that anaerobic condition is controlled, and is fit to bacterial growth.Scheme one (non-strictly anaerobic condition) the total growth time of bacterium is 48h, final OD
600Value is 1.109.Though scheme one is not in strict accordance with the anaerobic condition culturing bacterium, bacterium still can grow.Obviously, two pairs of bacterial growths of scheme more have superiority.
The fermentation and hydrogen production of bacterial classification:
Embodiment 2:The selection of strain fermentation environment is determined
The fermention medium component is: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2HPO
44g/L, L-halfcystine 0.2g/L, L-cysteine hydrochloride one water 0.5g/L, resazurin (0.2%) 1mL/L, glucose 10g/L; Regulate initial pH=7.0; In the preparation substratum process, utilize conventional heated and boiled expulsion liquid phase dissolved oxygen and the mode of utilizing gas phase air in the nitrogen stripping bottle, guarantee that strictly anaerobic is in 115 ℃ of high-temperature sterilization 25min; Aseptic anaerobic operation is inoculated in Clostridium butyricum high-concentration bacterial liquid (dry cell weight reaches 0.658g/L) in the substratum of 200ml and obtains fermented liquid with volume ratio 1:10 ratio, leave standstill cultivation in 37 ℃ anaerobic box:
(1) scheme one: will inoculate one bottle of good fermented liquid, total amount is 220mL, and anaerobic box is taken out in the sealing back, is 37 ℃, the shaking table shaking culture of 170r/min and connects hydrogen and receive acquisition means in temperature.
(3) scheme two: anaerobic box is taken out in another bottle 220mL fermented liquid sealing back, leave standstill cultivation in temperature is 37 ℃ water-bath, and connects the hydrogen collection device.
In culturing process, the gas and the real time record gas yield of collection scheme one (constant temperature oscillation and fermentation) and scheme two (constant temperature standing for fermentation) are regularly got fermented liquid sample mensuration bacterium OD in the scheme one
600Indexs such as value and breakdown of glucose rate; Bacterium OD
600Value is measured at 600 nm places with 756 ultraviolet-visible pectrophotometers; Hydrogen content is measured and is adopted gas chromatograph TCD thermal conductance device to detect; The breakdown of glucose rate adopts film colorimetric method for determining, and hydrogen output changes in time sees accompanying drawing 3.
Experimental result shows: about 23 hours of scheme one aerogenesis total duration, be initially breeding stage of bacterial classification, and gas production rate is low, the highest at 10 ~ 15 hours gas production rates, final gas production rate 267mL, amounts of hydrogen 169.67mL, hydrogen ratios 63.5%, inversion rate of glucose are 50.87%; About 35 hours of scheme two aerogenesis total durations are initially breeding stage of bacterial classification, and gas production rate is low, and gas production rate is stable, final gas production rate 153mL, and amounts of hydrogen 32.19mL, hydrogen ratios 21.04%, inversion rate of glucose is 63.04%.Contrast finds that it is fast that scheme one is produced hydrogen speed, and hydrogen percentages height, and glucose utilization rate height are with the obvious advantage, are suitable for using in the fermenting process.
The JCM1391 clostridium butylicum is the obligate anaerobic hydrogenogens:
Embodiment 3: novel clostridium butylicum fermentation and hydrogen production
The fermention medium component is: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2HPO
44g/L, L-halfcystine 0.2g/L, L-cysteine hydrochloride one water 0.5g/L, resazurin (0.2%) 1mL/L, glucose 10g/L; Regulate initial pH=7.0; In the preparation substratum process, utilize heated and boiled expulsion liquid phase dissolved oxygen and the mode of utilizing gas phase air in the nitrogen stripping bottle, guarantee that strictly anaerobic is in 115 ℃ of high-temperature sterilization 25min; Clostridium butyricum high-concentration bacterial liquid is inoculated in the 200ml fermention medium with the 1:10 ratio, aseptic anaerobic operation, leave standstill cultivation in 37 ℃ anaerobic box:
(1) scheme one: clostridium butylicum JCM1391 is seeded to one bottle of fermented liquid, and total amount is 220mL, and anaerobic box is taken out in sealing back, is 37 ℃, the shaking table shaking culture of 170r/min and connects hydrogen and receive acquisition means in temperature.
(2) scheme two: clostridium butylicum As1. 209 (purchase in Chinese common micro-organisms DSMZ) is seeded to one bottle of fermented liquid, total amount is 220mL, anaerobic box is taken out in sealing back, is 37 ℃, the shaking table shaking culture of 170r/min and connects hydrogen and receive acquisition means in temperature.
Under the culture condition condition identical with producing hydrogen technology, be the bacterium difference, the gas of collection scheme one and scheme two is drawn final hydrogen output such as Fig. 4.Final plan one hydrogen ultimate production is 168mL, and no gas produces in the scheme two.Therefore the result proves that the generic clostridium butylicum is not can both fermentation and hydrogen production, and the clostridium butylicum JCM1391 that is screened can be used to ferment for hydrogen production.
Under anaerobic, it is consistent that microbial fermentation generates being reflected in the different bacterial classifications of pyruvic acid, but the by product of different microbial strains anaerobic metabolism is not quite similar, and promptly pyruvic acid has a plurality of pathways metabolisms.In clostridium butylicum, cell generates acetyl-CoA under the effect of ferritin oxydo-reductase, and discharges CO
2And hydrogen, final main acetate and the butyric acid of generating of the further metabolism of acetyl-CoA; Sometimes also can be attended by the generation of formic acid and propyl carbinol, and not have hydrogen to generate.
In sum, clostridium butylicum (Clostridium butyricum) JCM1391 is as obligate anaerobic hydrogenogens kind, can be used for fermentation substrate is in the ferment for hydrogen production process of glucose, find by test, the general technology condition of its ferment for hydrogen production is that the best anaerobic operation technology of 1. bacterium is: utilize heated and boiled expulsion liquid phase dissolved oxygen and the mode of utilizing gas phase air in the nitrogen stripping bottle, guarantee strictly anaerobic; When 2. best bacterial classification inoculation is basic medium and fermentation substrate coexistence opportunity; 3. substratum is formulated as: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2HPO
43.0～4.0g, L-halfcystine 0.2～0.5g, L-cysteine hydrochloride one water 0.2～0.5g, resazurin indicator (0.2%) 1mL, distilled water 1L; 4. the concentration of fermentation substrate glucose is 5.0～10.0g/L; 5. leavening temperature is 37～40 ℃; 6. the initial pH that ferments is 7.0～7.5; 7. earthquake condition bottom fermentation liquid hydrogen generation efficiency is higher.
Claims (3)
1. a method of utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production is characterized in that, under aseptic anaerobic condition, utilizes anerobe, with peptone, extractum carnis, yeast extract, Na
2HPO
4, the mixture formed of L-halfcystine, L-cysteine hydrochloride one water, resazurin indication and distilled water is substratum, with glucose is fermentation substrate, adopt mode of batch fermentation to carry out hydrogen manufacturing, described anerobe is clostridium butylicum (Clostridium butyricum) JCM1391.
2. the method for utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production as claimed in claim 1 is characterized in that, the consisting of of substratum: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2HPO
43.0 resazurin indicator 1mL and the distilled water 1L of～4.0g, L-halfcystine 0.2～0.5g, L-cysteine hydrochloride one water 0.2～0.5g, 0.2%wt.
3. the method for utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production as claimed in claim 1 is characterized in that, the concentration of described fermentation substrate glucose is 5.0～10.0g/L
The method of utilizing obligate anaerobic clostridium butylicum ferment for hydrogen production as claimed in claim 1, it is characterized in that, concrete steps are: under the aseptic anaerobic environment, it is 7.0～7.5 that substratum is transferred pH with phosphate buffered saline buffer, substratum in the heating in water bath bottle, simultaneously from bottleneck inflated with nitrogen 5 minutes, treat that liquid phase becomes colorless, seal up bottle cap at last, 115 ℃ of high-temperature sterilizations, after the oven dry with the inoculation of clostridium butylicum to the bottle that substratum is housed, leave standstill under 37～40 ℃ of conditions be cultured to dry cell weight to reach 0.658g/L after as inoculum, then with inoculum and substratum by volume the ratio of 1:10 be inoculated in the substratum in the fermentation flask and obtain fermented liquid; Under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, produce hydrogen continuously.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462874A (en) * | 2015-11-25 | 2016-04-06 | 清华大学 | Clostridium butyricum and application of clostridium butyricum in anaerobic fermentation hydrogen production |
| CN105695370A (en) * | 2016-04-13 | 2016-06-22 | 南京工业大学 | Clostridium butyricum and culture method and application thereof |
| CN115895982A (en) * | 2023-02-06 | 2023-04-04 | 大连理工大学 | Arsenic-resistant fermentation hydrogen-producing bacterium, screening and application |
| CN116042478A (en) * | 2023-02-06 | 2023-05-02 | 大连理工大学 | Fermentation hydrogen-producing bacteria, directional screening and enhanced hydrogen production method |
| CN116439315A (en) * | 2023-04-21 | 2023-07-18 | 南京中医药大学 | Clostridium butyricum fermentation product of astragalus membranaceus stem and leaf as well as preparation method and application thereof |
-
2010
- 2010-12-14 CN CN 201010586487 patent/CN102041274A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 《International Journal of Hydrogen Energy》 20050831 Wen-Ming Chen Fermentative hydrogen productionwithClostridiumbutyricum CGS5 isolated fromanaerobic sewage sludge 1-8 1-3 第30卷, 第10期 * |
| 《NSTL期刊》 20071231 Pei-Ying Lin Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation 摘要 1-3 第32卷, 第12期 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462874A (en) * | 2015-11-25 | 2016-04-06 | 清华大学 | Clostridium butyricum and application of clostridium butyricum in anaerobic fermentation hydrogen production |
| CN105462874B (en) * | 2015-11-25 | 2019-04-30 | 清华大学 | A kind of clostridium butyricum and its application in hydrogen production through anaerobic fermentation |
| CN105695370A (en) * | 2016-04-13 | 2016-06-22 | 南京工业大学 | Clostridium butyricum and culture method and application thereof |
| CN115895982A (en) * | 2023-02-06 | 2023-04-04 | 大连理工大学 | Arsenic-resistant fermentation hydrogen-producing bacterium, screening and application |
| CN116042478A (en) * | 2023-02-06 | 2023-05-02 | 大连理工大学 | Fermentation hydrogen-producing bacteria, directional screening and enhanced hydrogen production method |
| CN115895982B (en) * | 2023-02-06 | 2024-04-12 | 大连理工大学 | Arsenic-resistant fermentation hydrogen-producing bacterium, screening and application |
| CN116439315A (en) * | 2023-04-21 | 2023-07-18 | 南京中医药大学 | Clostridium butyricum fermentation product of astragalus membranaceus stem and leaf as well as preparation method and application thereof |
| CN116439315B (en) * | 2023-04-21 | 2024-04-26 | 南京中医药大学 | Clostridium butyricum fermentation product of astragalus membranaceus stem and leaf as well as preparation method and application thereof |
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Application publication date: 20110504 |