CN101988075B - Method for preparing hydrogen by fermentation through using special anaerobic clostridium pasteurianum - Google Patents
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Abstract
The invention provides a method for preparing hydrogen by fermentation through using special anaerobic clostridium pasteurianum. Under the sterile and anaerobic condition, anaerobic bacteria are used, a mixture consisting of peptone, beef extracts, yeast extracts, Na2HPO4, L-cysteine, L-cysteine hydrochloride monohydrate, diazoresorcinal indicators and distilled water is used as a culture medium, glucose is used as a fermentation substrate, a batch fermentation method is adopted for preparing the hydrogen, and the special anaerobic bacteria are clostridium pasteurianum JCM1408T. The culture medium consists of 10.0 to 15.0g of peptone, 2.0 to 3.0g of beef extracts, 5.0 to 10.0g of yeast extracts, 3.0 to 4.0g of Na2HPO4, 0.2 to 0.5g of L-cysteine, 0.2 to 0.5g of L-cysteine hydrochloride monohydrate, 1ml of 0.2 percent wt diazoresorcinal indicators, 40 to 60ml of horse serum and 1L of distilled water. The hydrogen can be continuously prepared.
Description
Technical field
The present invention relates to a kind of method of utilizing microbiological anaerobic ferment for hydrogen production, especially a kind of method of utilizing the hydrogen manufacturing of efficient anaerobic bacterium.
Background technology
Hydrogen, as the new generation of green energy, has the features such as clean, efficient, renewable, if can industrialization produce, must replace the fossil energy such as oil, Sweet natural gas future, alleviates the pressure to environment.
In addition, Hydrogen Energy is not primary energy source, need to from hydrogeneous compound, produce.More than 90% hydrogen in the whole world 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.Fossil oil reserves are limited, and are tending towards exhausted, meanwhile, the greenhouse gases that generate when combustion of fossil fuel, other organic compound such as toxic gas, have caused serious environmental pollution and global climate are changed.Biological hydrogen production is with the concern that its raw material sources are abundant, lower-price characteristic is more and more received people, once large-scale production will be most potential a kind of hydrogen production process.
Biological hydrogen production was proposed by lewis in 1966 at first, and then the oil crisis of 20 century 70 experience makes the whole world recognize practicality and the urgency of biological hydrogen production.Until the nineties, in the time that the world is faced with the dual-pressure of energy and environment, biological hydrogen production has 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 has and produces that hydrogen rate is high, hydrogen-producing speed fast, produces that hydrogen is continual and steady, the design operation of reaction unit is simple, raw material sources are extensive and the feature such as cost is low, is easier to accomplish scale production, thereby becomes the main direction of biological hydrogen production research.Anaerobically fermenting hydrogen manufacturing bacterial classification comprises obligatory anaerobic bacteria and facultative anaerobe, wherein, cultivation, transportation and the industrialized condition harshness of obligatory anaerobic bacteria, and facultative anaerobe has adaptability widely, but facultative anaerobe hydrogen generation efficiency is high less than obligatory anaerobic bacteria, rationally cultivate under proper condition, can overcome its shortcoming, obligatory anaerobic bacteria will be more preferably bacterium of biological hydrogen production industrialization.At present, obligatory anaerobic bacteria kind is fewer, lacks new mushroom-seed culturing, and hydrogen generation efficiency is on the low side, and hydrogen manufacturing condition is treated further optimization.
By patent retrieval, at present existing patent is mainly the research to fermentation substrate, fermentation unit and raising hydrogen generation efficiency, as a kind of method of mud and the hydrogen manufacturing of organic waste mixed biologic (application number: 200710032658.0 weeks few strange etc.), photosynthetic microorganism hydrogen-manufacturing reactor (application number: 200620032282.4), Biofermentation hydrogen producer (application number: the 00231651.X power of building in Shen), one is utilized CO
2make algae fast breeding be directly used in method (200710010804.X) and the high-efficiency fermenting 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, only there are a kind of biological hydrogen production method and special bacterium thereof (application number: 200910088408.8 Xing Xin can wait), but the method bacterial classification used is the recombinant bacterium after importing by gene, operate more complicated, and fermentation condition requires strictly anaerobic, be difficult for promoting.So, very necessary for the screening of novel obligate anaerobic new mushroom-seed culturing and the exploration optimization of process for making hydrogen condition.
Summary of the invention
The present invention mainly comprises a kind of method of utilizing obligate anaerobic Clostridium baratii ferment for hydrogen production, and the method is under strictly anaerobic condition, uses Clostridium baratii (Clostridium pasteurianum) JCM1408
t, take glucose as fermentation substrate, adopt the mode of batch fermentation, after 24 hours, can start to produce hydrogen process.
The obligatory anaerobic bacteria that the present invention screens is specially Clostridium baratii (Clostridium pasteurianum) JCM1408 of fusobacterium (Clostridium Winogradsky, 1895)
t, purchased from Japanese biotechnology research institute's microbial strains preservation center (Marine Biotechnology Institute Culture Collection, Marine Biotechnology Institution, MBIC).
Technical scheme of the present invention is: a kind of method of utilizing obligate anaerobic Clostridium baratii ferment for hydrogen production, under aseptic anaerobic condition, utilize anerobe, with peptone, extractum carnis, yeast extract, Na
2hPO
4, Cys, Cys hydrochloride one water, resazurin indication and distilled water composition mixture be substratum, take glucose as fermentation substrate, adopt the mode of batch fermentation to carry out hydrogen manufacturing, described obligatory anaerobic bacteria is Clostridium baratii JCM1408
t.
Consisting of of substratum: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2hPO
4resazurin indicator 1mL, horse serum 40 ~ 60ml and the distilled water 1L of 3.0～4.0g, Cys 0.2～0.5g, Cys hydrochloride one water 0.2～0.5g, 0.2%wt.
The concentration of described fermentation substrate glucose is 5.0～20.0g/L
Concrete steps are: under aseptic anaerobic environment, it is 5.5～6.5 that substratum is adjusted to pH with phosphate buffered saline buffer, substratum in heating in water bath bottle, simultaneously from bottleneck inflated with nitrogen 5 minutes, treat that liquid phase becomes colorless, finally seal up bottle cap, 115 ℃ of high-temperature sterilizations, after oven dry by the inoculation of Clostridium baratii to being equipped with in the bottle of substratum, under 37～40 ℃ of conditions, leave standstill and be cultured to OD
600value is 1.402 rear as inoculum, then by inoculum and substratum by volume the ratio of 1:10 ~ 40 be inoculated in the substratum in fermentation flask and obtain fermented liquid; Under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, produce continuously hydrogen.Under this condition, hydrogen generation efficiency is high, low to yeasting conditional request, has broad application prospects.
beneficial effect:
First, filter out a kind of novel fermentation hydrogen manufacturing bacterial classification, this bacterial classification is obligatory anaerobic bacteria, has growth rapidly, hydrogen generation efficiency advantages of higher;
Secondly, determined technological condition for fermentation, determined that fermentation substrate is that concentration is the glucose of 5.0～20.0g/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, Cys 0.2～0.5g, Cys hydrochloride one water 0.2～0.5g, resazurin indicator (0.2%) 1mL, horse serum 40～60 mL/L, distilled water 1L; It is 5.5～6.5 that substratum is adjusted pH with phosphate buffered saline buffer, and in heating in water bath bottle, liquid inflated with nitrogen 5 minutes, in vitro keeps anaerobic environment, and fermentation and inoculation all operate in anaerobic box, adopt the mode of batch fermentation; Under 37～40 ℃ of conditions, leave standstill and cultivate 24 hours as inoculum, then by being connected in fermentation flask, under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, can produce continuously hydrogen.In addition, Clostridium baratii is Batch Culture in fermentor tank, and hydrogen output is obviously high to be cultivated with sample, produces hydrogen ratio and reaches 1.7mol H
2/ mol glucose.
Accompanying drawing explanation
Fig. 1 is device for producing hydrogen schematic diagram of the present invention.
Wherein, 1 is nitrogen wash mouth and liquid sampling mouth, and 2 is gas sampling mouth, and 3 is gas-collecting pipe, and 4 is leveling bottle.
Fig. 2 is the impact on gas ultimate production of the mass concentration of glucose.
Fig. 3 is the impact that the mass concentration of glucose is grown up on cell.
Fig. 4 is the impact of initial pH on hydrogen ultimate production.
Fig. 5 is the impact that initial pH grows up on cell.
Embodiment
the enlarged culturing of bacterial classification:
embodiment 1:determine the basic medium of strain expanded culture
Substratum consists of: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2hPO
44g/L, Cys 0.2 g/L, Cys hydrochloride one water 0.5 g/L, horse serum 40～60 mL/L, distilled water 1000mL.In addition, separately add resazurin indicator (0.2%) 1mL/L.Regulate pH=6.5, every 100mL nutrient solution packs 250mL saline bottle (band bottle stopper, can be airtight) into, and liquid in heating in water bath bottle, is blown into nitrogen from bottleneck simultaneously, treats that liquid phase becomes colorless (in bottle, being oxygen-free environment), seals up bottle cap.120 ℃ of high-temperature sterilization 25min, after drying, are seeded to bacterial strain Clostridium pasteurianum in the bottle that substratum is housed (aseptic anaerobic operation) in 60 ℃ of baking ovens, leave standstill and cultivate 24～48h at 37 ℃.
In culturing process, the absorbance with 756 ultraviolet-visible pectrophotometers at 600 nm place timing working samples, 48h bacterial growth OD value is 1.402.
Experimental result shows: the total growth time of bacterium is 45h, final OD
600value is 1.402, and bacterial reproduction is rapid, and anaerobic condition control good is applicable to bacterial growth.
the fermentation and hydrogen production of bacterial classification:
embodiment 2:the selection of strain fermentation substrate concentration configuration is determined
Fermentation consisting of with substratum: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2hPO
44g/L, Cys 0.2g/L, Cys hydrochloride one water 0.5g/L, horse serum 40～60 mL/L, resazurin (0.2%) 1mL/L, glucose 5 ~ 20g/L; Regulate initial pH=5.5 ~ 6.5; In preparation substratum process, utilize heated and boiled expulsion liquid phase dissolved oxygen and the mode of utilizing gas phase air in nitrogen stripping bottle, guarantee that strictly anaerobic is in 115 ℃ of high-temperature sterilization 25min; Under aseptic anaerobic operation, by the Clostridium pasteurianum OD cultivating before
600value is that 1.402 inoculum high-concentration bacterial liquid and substratum are inoculated in the substratum in 200ml fermentation flask by the inoculum size in table 1, in the anaerobic box of 37 ℃, leaves standstill and cultivates, and concrete experimental factor and level are in table 1:
table 1
| PH value: | 5.0 | 5.5 | 6.0 | 6.5 |
| Concentration of substrate (g/L): | 5 | 10 | 15 | 20 |
| Inoculum size (V/V): | 1：40 | 1：20 | 1：10 | — |
| Level numbering: | 1 | 2 | 3 | 4 |
By the fermented liquid of having inoculated, total amount is 220mL, takes out anaerobic box after sealing, is 37 ℃, the shaking table shaking culture of 170r/min and connects hydrogen and receive acquisition means in temperature.In culturing process, collect gas real time record gas yield.
Experimental result shows: Clostridium baratii aerogenesis total duration approximately 24 hours, and concentration of substrate is shown in Fig. 2 to the impact of Clostridium baratii gas ultimate production and Growth of Cells, 3.Maximum gas production rate 480mL, amounts of hydrogen 302mL, hydrogen ratios 63%, is suitable for applying in fermenting process.The highly significant of glucose concn to Clostridium baratii gas ultimate production and Growth of Cells, in the time that glucose quality concentration is 20g/L, gas ultimate production is minimum, is only 255mL/L, and now the dry cell weight of Clostridium baratii also only has 0.38g.In the time that glucose quality concentration is 10g/L, gas ultimate production reaches maximum value 480mL/L, and dry cell weight reaches maximum value 0.73g/L, but lower than or during higher than this concentration, gas ultimate production and dry cell weight have all presented downtrending.
This be because: in the time that glucose concentration of substrate is low, it can not offer Clostridium baratii sufficient nutrient material, thereby its growth is restricted, and glucose concn is when higher, Clostridium baratii can not utilize cmpletely to it, and residual glucose is piled up in reaction flask, and the pH of fermented liquid is reduced, on the contrary Clostridium baratii is produced to restraining effect, made it produce hydrogen and be all subject to influence with growth.
embodiment 3:best fermentation pH determines
Initial pH value on the impact of Clostridium baratii gas ultimate production and Growth of Cells in table 4,5.Under the condition that is 10g/L at glucose concn, the gas ultimate production of Clostridium baratii is along with the variation of initial pH presents fluctuation status.When initial pH is in 5.0 ~ 6.0 scopes, gas ultimate production and dry cell weight all increase with the increase of initial pH, and in the time that initial pH is 6.0, gas ultimate production reaches maximum value 480mL/L, and dry cell weight reaches maximum value 0.72g/L; In the time that initial pH continues to increase, gas ultimate production and dry cell weight all reduce along with the increase of initial pH.Under this explanation condition that is 10g/L in glucose quality concentration, initial pH is 6.0 to be growth conditionss of the suitableeest aerogenesis of Clostridium baratii, all can produce restraining effect to Clostridium baratii below or above this value.
embodiment 4:determining of best inoculative proportion
Inoculative proportion on the impact of Clostridium baratii gas ultimate production and Growth of Cells in table 2:
table 2
| Inoculum size: | 1：40 | 1：20 | 1：10 | — |
| Gas production rate | 263 | 485 | 210 | — |
| Bacterium dry weight | 0.375 | 0.836 | 0.348 | — |
Under the condition that is 10g/L at glucose concn, initial pH is 6.0, and the gas ultimate production that Clostridium baratii inoculum size is 1:20 reaches maximum value 485mL/L, and dry cell weight reaches maximum value 0.836g/L; In the time that inoculum size increases or reduces, gas ultimate production and dry cell weight all reduce.This illustrates in the situation that other conditions are identical, the restraining effect minimum of inoculum size to Clostridium baratii growth and nutritive substance contention generation, the most applicable fermentation and hydrogen production.
jCM1408
t
clostridium baratii is obligate anaerobic hydrogenogens:
embodiment 5: novel Clostridium baratii fermentation and hydrogen production
Fermention medium component is: peptone 10g/L, extractum carnis 2.4 g/L, yeast extract 5.0g/L, Na
2hPO
44g/L, Cys 0.2g/L, Cys hydrochloride one water 0.5g/L, resazurin (0.2%) 1mL/L, horse serum 40 ~ 60ml, glucose 10g/L; Regulate initial pH=6.0; In fermentor tank, pack 8L substratum into, utilize heated and boiled expulsion liquid phase dissolved oxygen and the mode of utilizing gas phase air in nitrogen stripping bottle, guarantee that strictly anaerobic is in 115 ℃ of high-temperature sterilization 25min;
(1) scheme one: by JCM1408
tclostridium baratii high-concentration bacterial liquid is inoculated in 8L fermention medium (aseptic anaerobic operation) with 1:20 ratio, stir culture under the constant temperature of 37 ℃, and connect gas collector.
(2) scheme two: by LMG3285
tclostridium baratii (being purchased from Ghent, Belgium university microorganism hereditary laboratory) is inoculated in 8L fermention medium (aseptic anaerobic operation) with 1:20 ratio, stir culture under the constant temperature of 37 ℃, and connect gas collector.
Under the culture condition condition identical with producing hydrogen technique, just bacterium difference, the gas gross of collection scheme one and scheme two.Final plan one hydrogen ultimate production is 16.8L, and in scheme two, hydrogen ultimate production is 13.4L.Although therefore result proves that the clostridium butylicum that belongs to together can fermentation and hydrogen production, the bacterial classification JCM1408 that this screens
tclostridium baratii ferment for hydrogen production efficiency is higher, and productive rate can reach 1.7mol H
2/ mol glucose.
In sum, Clostridium baratii (Clostridium pasteurianum) 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 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 nitrogen stripping bottle, guarantee strictly anaerobic; 2. bacterial classification inoculation is that basic medium and fermentation substrate are while coexisting 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, Cys 0.2～0.5g, Cys hydrochloride one water 0.2～0.5g, resazurin indicator (0.2%) 1mL, horse serum 40～60 mL/L, distilled water 1L; 4. the concentration of fermentation substrate glucose is 10.0g/L; 5. leavening temperature is 37～40 ℃; 6. the initial pH that ferments is 6.0～6.5; 7. under earthquake or agitation condition, make fermented liquid produce hydrogen.
Claims (3)
1. a method of utilizing obligatory anaerobic bacteria ferment for hydrogen production, is characterized in that, under aseptic anaerobic condition, utilizes anerobe, with peptone, extractum carnis, yeast extract, Na
2hPO
4, Cys, Cys hydrochloride one water, resazurin indication, horse serum and distilled water composition mixture be substratum, take glucose as fermentation substrate, adopt the mode of batch fermentation to carry out hydrogen manufacturing, described obligatory anaerobic bacteria is Clostridium baratii JCM1408
t; Consisting of of described substratum: peptone 10.0～15.0g, extractum carnis 2.0～3.0 g, yeast extract 5.0～10.0g, Na
2hPO
4resazurin indicator 1mL, horse serum 40 ~ 60ml and the distilled water 1L of 3.0～4.0g, Cys 0.2～0.5g, Cys hydrochloride one water 0.2～0.5g, 0.2%wt.
2. the method for utilizing obligatory anaerobic bacteria ferment for hydrogen production as claimed in claim 1, is characterized in that, the concentration of described fermentation substrate glucose is 5.0～20.0g/L.
3. the method for utilizing obligatory anaerobic bacteria ferment for hydrogen production as claimed in claim 1, it is characterized in that, concrete steps are: under aseptic anaerobic environment, it is 5.5～6.5 that substratum is adjusted to pH with phosphate buffered saline buffer, substratum in heating in water bath bottle, simultaneously from bottleneck inflated with nitrogen 5 minutes, treat that liquid phase becomes colorless, finally seal up bottle cap, 115 ℃ of high-temperature sterilizations, after oven dry by the inoculation of Clostridium baratii to being equipped with in the bottle of substratum, under 37～40 ℃ of conditions, leave standstill and be cultured to OD
600value is 1.402 rear as inoculum, then by inoculum and substratum by volume the ratio of 1:10 ~ 40 be inoculated in the substratum in fermentation flask and obtain fermented liquid; Under 37～40 ℃ of conditions, carry out constant-temperature shaking culture, produce continuously hydrogen.
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| CN106746436B (en) * | 2017-02-06 | 2020-06-26 | 同济大学 | Method for improving anaerobic degradation of L-glucose |
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| CN117965645A (en) * | 2024-03-29 | 2024-05-03 | 广东省科学院生态环境与土壤研究所 | Method for improving hydrogen production rate and hydrogen production amount of clostridium fermentation and application |
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| AGENCY OF IND SCI & TECHNOLOGY.JP特开平6-133785A的德温特专利文献摘要.《德温特数据库》.1994,德温特专利文献摘要. |
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| James D. Brosseau et al..Hydrogen-gas Production with Citrobacter intermedius and Clostridium pasteurianum.《J. Chem. Tech. Biotechnol》.1982,第32卷496-502. * |
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