CN101041832A - Method for producing hydrogen by using marsh red pseudomonas - Google Patents
Method for producing hydrogen by using marsh red pseudomonas Download PDFInfo
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- CN101041832A CN101041832A CN 200710078405 CN200710078405A CN101041832A CN 101041832 A CN101041832 A CN 101041832A CN 200710078405 CN200710078405 CN 200710078405 CN 200710078405 A CN200710078405 A CN 200710078405A CN 101041832 A CN101041832 A CN 101041832A
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Abstract
The invention discloses a hydrogen preparing method with marsh red pseudomonas, which comprises the following steps: (1) putting fluid medium into autoclave sterilizing pot; sterilizing at 12-15 min; fetching out of the culture medium; cooling to normal temperature; embedding the fluid medium into bioreactor and photo biological hydrogen production reactor separately; setting the initial pH range at 4.5-8.8 and density range of organic subtract at 1.98g/l-45g/l; (2) fetching marsh red pseudomonas colony on solid double layer plate culture medium with inoculating needle; seeding in the cooling culture medium; charging into argon gas to remove air in the biological reactor of fluid medium; forming anaerobic environment; placing the seeding liquid medium at 25-35 deg.c; stewing and culturing at 36-96 h in constant-temperature culture box with illuminance at 1500lx-8000lx; getting the enlargement culture liquid in the subsequent hydrogen producing course.
Description
Technical field
The present invention relates to a kind of production by biological hydrogen methods, be specifically related to a kind of method of utilizing Rhodopseudomonas palustris hydrogen manufacturing.
Background technology
The annual production of present hydrogen in the world is more than 3,600 ten thousand tons, mainly from fossil oil, biomass and water, mainly be to adopt the partial oxidation etc. of traditional physico-chemical process such as water electrolysis, Sweet natural gas catalytic reforming and thermo-cracking, gasification of coal, naphtha to produce hydrogen, it is that cost exchanges Hydrogen Energy for that traditional hydrogen production process comes down to consume a large amount of fossil energies, have a net increase of can value low, economic serviceability is not strong, environment is produced pollute simultaneously.Utilization photoelectric technology and electrolysis tech bonded method also can be produced hydrogen, can produce higher transformation efficiency with solar-energy photo-voltaic cell or solar thermal collector, but because earth surface solar radiation rate only is 1kWm
-2Therefore need to build huge area to collect abundant energy, this significant limitation its commercial utilization, simultaneously the least favorable condition of this method be need be very high cost of investment and hi-tech requirement, thereby hindered its utilization to sunlight at technical elements in less developed country and area.Have many advantages and utilize microbial method to produce hydrogen: use abundant renewable resources microorganism and water to be raw material, directly or indirectly utilize the most economical and energy sunlight that cleans most on the earth, can curb environmental pollution; the protection environment; economic serviceability is strong, production safety, and equipment is simple; easy to operate; need not consume lot of energy, low, the reduced investment of cost, the photosynthetic hydrogen production microbe species is various; utilization has a extensive future, and is easy to generally apply.Therefore utilize microbial method to produce the research focus that hydrogen has become renewable energy utilization and development field.
Utilize the method for solar hydrogen making that solar heat hydrogen production by water decomposition, solar electrical energy generation water electrolysis hydrogen production, sunlight catalysis photolysis water hydrogen, sun power biological hydrogen production or the like are arranged at present, wherein have wide prospect with the sun power biological hydrogen production.
In the solar energy bio-hydrogen production technology photosynthetic bacterium because of can decomposing organic matter matter, transform and utilize solar energy to have special advantages for Hydrogen Energy, under the driving of luminous energy, be raw material promptly with the organism, decomposing organic matter obtains hydrogen, belongs to the photoheterotrophy type.In the production by biological hydrogen system, photosynthetic bacteria hydrogen production is considered to the most promising production by biological hydrogen methods, this be because: (1) photosynthetic bacteria hydrogen production has high theoretical light hydrogen inversion quantity; (2) do not produce O
2, avoided O
2The microorganism deactivated problem that causes; (3) spectral range in the light-use is wide; (4) have the ability that consumes the organic substrates that discharges in the wastewater treatment process, realized the purpose of the comprehensive utilization and the environment protection of organic waste, so this will be up-and-coming treatment technology to the exploitation of renewable energy source and the realization of the strategy of sustainable development.Foreign literature report hydrogen production with photosynthetic bacteria speed is generally: 0.00518-1.58mgh
-1L
-1, this class production with photosynthetic bacteria hydrogen efficiency is still lower.
At present there is not to cultivate specially the bibliographical information of hydrogen yield photosynthetic bacterium method, concentrate on multiplication culture and the enlarged culturing of photosynthetic bacterium simultaneously in more documents and materials aspect the photosynthetic bacterium screening, what obtain is the photosynthetic bacterium nutrient solution that contains assorted bacterium, and the photosynthetic bacterium that screening obtains more is only to be used for feed or proteinic production or sewage disposal, organic waste is not handled with energy and be closely linked, the part information report is to adopt the anaerobism incubator to the screening of photosynthetic bacterium, but this method only provides anaerobic condition, and luminous energy is not provided simultaneously, therefore can not guarantee that the photosynthetic bacterium that obtains has higher hydrogen production potential.
Summary of the invention
The purpose of this invention is to provide a kind of hydrogen generation efficiency height, utilize Rhodopseudomonas palustris decomposing organic matter matter under anaerobism and illumination condition to produce the method for hydrogen.
A kind of method of utilizing Rhodopseudomonas palustris hydrogen manufacturing, adopt following steps to carry out:
(1) preparation liquid nutrient medium is put into high-pressure sterilizing pot sterilization 12-15 minute with liquid nutrient medium, takes out substratum and is cooled to normal temperature, and be respectively charged in seed enlarged culturing reactor and the light biological hydrogen producing reactor liquid nutrient medium stand-by; Wherein, the initial pH scope of liquid nutrient medium is: 4.5-8.8, and the concentration range of organic substrates is: 1.98g/l-45g/l;
(2) enlarged culturing of Rhodopseudomonas palustris bacterial classification: fall to being inoculated in the cooled liquid nutrient medium of sterilization with the Rhodopseudomonas palustris of growing on the inoculating needle picking solid double-layer plate culture medium; In the seed enlarged culturing reactor of this liquid nutrient medium of splendid attire, charge into the argon gas excluding air, form anaerobic environment, and postvaccinal liquid nutrient medium placed 25 ℃-35 ℃, fluorescent lamp is a light source, illuminance is to leave standstill in the illumination constant incubator of 1500lx-8000lx to cultivate 36-96 hour, obtains used enlarged culturing liquid in the follow-up product hydrogen process;
(3) light biological hydrogen producing of Rhodopseudomonas palustris: Rhodopseudomonas palustris enlarged culturing liquid is inoculated in the liquid nutrient medium of light biological hydrogen producing reactor splendid attire by the 8%-18% that cultivates base unit weight, and in light biological hydrogen producing reactor, charge into argon gas and obtain anaerobic environment, with fluorescent lamp or LED lamp is light source, illuminance is: 2000lx-5000lx, culture temperature is 20 ℃-35 ℃, leave standstill the constant temperature illumination cultivation after 10 hours-15 hours Rhodopseudomonas palustris begin to produce hydrogen.
Wherein, the best initial pH scope of described liquid nutrient medium is got: 6.0-8.5.
Wherein, the concentration range of the best organic substrates of described liquid nutrient medium is 11.880g/l-27.72g/l.
Wherein, described liquid culture based component is:
Organic substrates: 11.880g/l-27.72g/l; K
2HPO
43H
2O:0.3g/l-1.56g/l;
KH
2PO
4：0.22g/l-10.89g/l；NH
4Cl：0.677g/l-2.35g/l；
CaCl
2：0.002g/l-0.26g/l；FeSO
4·7H
2O：0.005g/l-0.417g/l
ZnSO
4：0.0001g/l-0.02g/l；MgSO
4·7H
2O：0.02g/l-0.5g/l；
NaCl：0.1g/l-1.5g/l；CuSO
4：0.001g/l-0.04g/l
(NH
4)
6Mo
4O
24·4H
2O：0.0001g/l-0.002g/l；
V
B2：0.00001g/l -0.00025g/l；
Nicotinic acid: 0.00001g/l-0.0002g/l; Extractum carnis: 0.5g/l-4.0g/l;
Water: 1000ml; PH value: 6.0-8.5
In mentioned component, some iron, calcium and magnesium etc. have bigger promoter action to hydrogen production with photosynthetic bacteria, while concentration is that the calcium salt of 0.002g/l-0.26g/l can improve the energy for growth of this bacterial classification to a certain extent and keep the cytoactive existence, but this class inorganic salt concentration must be followed the strict restriction of basal culture medium prescription, otherwise will influence the growth and the hydrogen production potential of hydrogen yield photosynthetic bacterium.In addition, because therefore the ability of this bacterial classification shortage self synthetically grown factor needs suitably to replenish somatomedin such as vitamin H, nicotinic acid and extractum carnis etc. in substratum.
Technical solutions according to the invention are compared with existing photosynthetic bacteria used for light bio-hydrogen production technology has special advantages; the hydrogen production efficiency height; cost is low; with domestic and international similar photosynthetic bacterium bacterial classification relatively this bacterial classification hydrogen production potential under given conditions be in the prostatitis; secondly; Rhodopseudomonas palustris has wide application potential aspect environment protection; various organic acids in Rhodopseudomonas palustris energy decomposing organic matter of the present invention and the anaerobic fermented liquid; discover that this bacterial classification can reach 100% to the decomposition and the utilization ratio of organic substance; be particularly suitable for decomposing and utilize organic waste water behind the anaerobically fermenting; further reduce the organic content in the waste liquid; made full use of biomass energy; realized changing evil for precious; therefore the purpose of protection environment is having broad application prospects and social value aspect environment protection and the realization Sustainable utilization of resources.
Description of drawings
Fig. 1 is the influence curve of concentration of substrate to the Rhodopseudomonas palustris hydrogen output
Fig. 2. be initial pH value of medium is produced the hydrogen behavior to Rhodopseudomonas palustris influence curve
Fig. 3 substratum Fe
2+Rhodopseudomonas palustris is produced the influence curve of hydrogen behavior
Fig. 4 temperature variation is produced the influence curve of hydrogen behavior to Rhodopseudomonas palustris
Fig. 5 intensity of illumination is produced the influence curve of hydrogen behavior to Rhodopseudomonas palustris
Embodiment
Embodiment 1: the cultivation of Rhodopseudomonas palustris:
(1) gather the bacterium source: the upper epidermis mud of gathering farmland, side bilge etc. is the bacterium source, leaves standstill 5 hours, collects supernatant liquor; Kind and the distributed number of the preliminary microscopy microorganism of microscopically, finding has other a large amount of assorted bacterium, and as beads phycomycete, green alga etc., photosynthetic bacterium quantity is seldom;
(2) has the multiplication culture of the Rhodopseudomonas palustris bacterial strain that produces the hydrogen behavior: get above-mentioned supernatant liquor 70ml and pour into respectively in the triangular flask of 250ml, the fresh culture that adds the 30ml sterilization simultaneously is based in the bottle, supernatant liquor and fresh culture are shaken up, and charge into the argon gas excluding air, leaving standstill anaerobism cultivates, be light source with the fluorescent lamp in the spawn culture, illuminance is 3000 luxs (lx), culture temperature is 30 ℃, cultivated 10 days, nutrient solution color purpling gradually is red, and red bacterium absorption growth is arranged on the culturing bottle wall;
(3) have the enrichment culture of the Rhodopseudomonas palustris bacterial strain that produces the hydrogen behavior: the middle liquid 40ml of lower floor that gets above-mentioned nutrient solution is in the clean culturing bottle of sterilization, the sterilized fresh culture that adds 40ml again, nutrient solution and fresh culture shaken up charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with fluorescent lamp or LED lamp is light source, illuminance is 4000 luxs, culture temperature is 30 ℃, illumination is left standstill to transform and is cultivated once more, cultivate and observe solution becomes muddiness in the culturing bottle after 1 day, cultivate after 3 days microscopic examination after the dyeing of viable bacteria alkalescence methylene blue, carry out sediments microscope inspection, find that the strain of beads phycomycete disappears, contain the bacterium (Gram-positive) that the many cells of non-mobility bacterium of a large amount of stock shapes (gramstaining is not obvious) and part long filament shape are connected, observe a spot of mobility, cell is the bacterium of ellipse garden shape, also contain the part sphere, the bacterium that cellular form is very little (gramstaining is negative) also has the bacterium of the quick flexural deformation campaign of a spot of energy; Observed a large amount of have mobility, cell ovalizes after the 7th day, the negative bacterium of gramstaining occurs, and the quantity of other bacterium obviously reduces.The red nutrient solution of lower floor repeats this step 3 times in getting again, and observing a large amount of dominant bacterias under the opticmicroscope is quarter butt shape or red oval bacterium;
(4) have the preparation of the gradient dilution liquid of the Rhodopseudomonas palustris bacterial strain enrichment culture liquid that produces the hydrogen behavior: get enrichment culture liquid that step (3) obtains with the distilled water of sterilization by 10
-1-10
-9Progression dilutes, and obtaining extension rate respectively is 10
-1, 10
-2... 10
-10Gradient dilution liquid;
(5) has the separation and purification of the Rhodopseudomonas palustris bacterial strain that produces the hydrogen behavior: adopt gradient dilution flat-plate bacterial colony partition method, utilize the double-layer plate solid medium to provide anaerobic environment for the growth and breeding of bacterial classification; At first in aseptic culture dish, make subfoundation nutritive solid plate culture medium, treat 10 of inoculation step (4) obtains on each solid plate substratum respectively after the cooling of subfoundation nutritive solid plate culture medium enrichment culture liquid
-3-10
-10Gradient dilution liquid, the solid plate media surface was placed 15 minutes, only contain agar and the content poured into about 40 ℃ are 1.8% upper strata covering liquid, cover culture dish ware lid, placement is cooled to the top-layer agar covering liquid and solidifies fully, culture dish is placed 30 ℃ again, illuminance is an illumination cultivation 8 days in the illumination constant incubator of 5000lx; After growing bacterium colony, it is fast to select growth, and colonial morphology is single and big, colony colour is that red person is the purpose bacterial classification,
(6) the purpose bacterial classification that obtains of picking step (5), the method of (4) is diluted set by step, the method of (5) repeats 3 separation and purification more set by step, up to colonial morphology unanimity to growth, colony colour is a red-purple, observe cellular form under the opticmicroscope and be ellipse, promptly judge to obtain pure strain; This bacterial classification bacterium colony surface wettability, picking very easily, cell easily disperses, and gramstaining is negative, and cell mobility is strong, and the secretion of visible cell surface has a large amount of mucus under the Electronic Speculum; Flagellum is many and grow, Zhousheng, and diameter is 100-200 μ m, is about about 4.0 μ m, about wide about 2.5 μ m; This bacterial classification is through physiological and biochemical analysis and 16SrRNA comparison, identify that this bacterial classification belongs to Rhodospirillales, Rhodospirillaceae, Rhodopseudomonas, Rhodopseudomonas palustris (Rhodopseudomonas palustris), and called after Rhodopseudomonas palustris CQK 01.
Wherein, substratum is composed as follows: K
2HPO
43H
2O concentration 1.2g/l; MgSO
47H
2O: concentration 0.5g/l; KH
2PO
4: concentration 0.6g/l; FeSO
47H
2O: concentration 0.05g/l; C
6H
12O
6H
2O: concentration 6.012g/l; (NH
4)
6Mo
7O
244H
2O: concentration 0.0005g/l; NaCl: concentration 1.5g/l; CaCl
2Concentration 0.015g/l; NH
4Cl concentration 1.5g/l; Growth factor solution concentration 3ml/l; ZnSO
47H
2O concentration 0.002g/l;
Embodiment 2:
(1) manufacturing of light biological hydrogen producing reactor: selecting transparent synthetic glass for use is the light biological hydrogen producing reactor material, thickness of slab 0.8cm, the long 10cm of reactor, wide 5cm, high 10cm, reactor is a rectangular structure, reactor cubic capacity 500ml, each side open a hole in reactor head, be used to charge into argon gas and discharged air, open a hole near lower portion at reactor, be used for liquid sampling and discharge opeing, each hole is connected with outside with adapter, and wherein venting hole is connected with the extraneous gas collection device and is used to collect hydrogen, and reactor is answered air tight and leakage except that necessary perforate;
(2) preparation of product hydrogen liquid culture based formulas:
Glucose: 7.92g/l; K
2HPO
43H
2O:0.6g/l;
KH
2PO
4：0.38g/l；NH
4Cl：1.99g/l；
CaCl
2：0.1g/l；FeSO
4·7H
2O：0.0834g/l；
ZnSO
4：0.0005g/l；MgSO
4·7H
2O：0.05g/l；
NaCl：1.0g/l；CuSO
4：0.03g/l
(NH
4)
6Mo
4O
24·4H
2O：0.00075g/l；V
B2：0.00002g/l；
Nicotinic acid: 0.00002g/l; Extractum carnis: 3g/l;
Water: 1000ml; Growth factor solution: 2ml;
The initial pH value of regulating liquid nutrient medium is 8.0
(3) liquid nutrient medium is put into high-pressure sterilizing pot sterilization 15 minutes, taken out substratum and be cooled to normal temperature, and be respectively charged in seed enlarged culturing reactor and the light biological hydrogen producing reactor liquid nutrient medium stand-by;
(4) enlarged culturing of Rhodopseudomonas palustris bacterial classification: the Rhodopseudomonas palustris of growing on the solid double-layer plate culture medium that obtains with inoculating needle picking embodiment 1 falls to being inoculated in the cooled liquid nutrient medium of sterilization; In the seed enlarged culturing reactor of this liquid nutrient medium of splendid attire, charge into the argon gas excluding air, form anaerobic environment, and postvaccinal substratum placed 25 ℃-35 ℃, fluorescent lamp is a light source, illuminance is to leave standstill in the illumination constant incubator of 1500lx-8000lx to cultivate 36-96 hour, obtains used enlarged culturing liquid in the follow-up product hydrogen process;
(5) photobiological hydrogen production of Rhodopseudomonas palustris: be inoculated into Rhodopseudomonas palustris enlarged culturing liquid in the cooled liquid nutrient medium of sterilization of using the light biological hydrogen producing reactor splendid attire by the 8%-18% that cultivates base unit weight, and in light biological hydrogen producing reactor, charge into argon gas and obtain anaerobic environment, with fluorescent lamp or LED lamp is light source, illuminance is 5000lx, culture temperature is 30 ℃, leaves standstill the constant temperature illumination cultivation;
Regularly detect the hydrogen situation of producing between incubation period, find that the 12h photosynthetic bacterium begins to produce hydrogen after inoculation culture, hydrogen-producing speed is 0.6731mlh
-1L
-1, biomass this moment (dry weight) is 0.019gl
-1, when being cultured to 44h, hydrogen-producing speed reaches maximum, is 5.432mlh
-1L
-1, this moment 0.23gl
-1After this hydrogen-producing speed descends gradually, when being cultured to 105h, producing hydrogen and stops substantially, and biomass is reduced to 0.14gl
-1, obtaining 364mlH altogether in the 105h cultivation continuously
2
(1) liquid nutrient medium preparation: get glucose 19.8g/l; K
2HPO
43H
2O:0.6g/l;
KH
2PO
4：0.38g/l；NH
4Cl：1.99g/l；
CaCl
2：0.1g/l；FeSO
4·7H
2O：0.0834g/l；
ZnSO
4：0.0005g/l；MgSO
4·7H
2O：0.05g/l；
NaCl：1.0g/l；CuSO
4：0.03g/l
(NH
4)
6Mo
4O
24·4H
2O：0.00075g/l；V
B2：0.00002g/l；
Nicotinic acid: 0.00002g/l; Extractum carnis: 3g/l;
Water: 1000ml; Growth factor solution: 2ml;
The initial pH value of regulating liquid nutrient medium is 8.0
Other conditions are identical with embodiment 2 with step, find that the 12h Rhodopseudomonas palustris begins to produce hydrogen after inoculation culture, and hydrogen-producing speed is 0.5376mlh
-1L
-1, biomass this moment (dry weight) is 0.037gl
-1, when being cultured to 23h, hydrogen-producing speed reaches maximum, is 14.0672mlh
-1L
-1, this moment, biomass was 0.28gl
-1After this maintain 10.976-14.224mlh substantially to the 64h hydrogen-producing speed
-1L
-1Between, hydrogen-producing speed descends gradually afterwards, when being cultured to 120h, producing hydrogen and still continues as 0.645mlh
-1L
-1, biomass slightly is reduced to 0.12gl
-1, in the cultivation of continuous 120h, obtain 626.08mlH altogether
2
Comparative example 2 and 3 finds that concentration of substrate is very big to the product hydrogen influence of this bacterial classification, and suitable high concentration of substrate helps to improve the product hydrogen performance of this bacterial classification.Found through experiments Rhodopseudomonas palustris can utilize the concentration range of organic substrates to be: 1.98-35.64g/l (is example with glucose), wherein suitable concentration of substrate scope is: 11.880g/l-27.72g/l.Fig. 1 is illustrated under the Incubation Condition concentration of substrate Rhodopseudomonas palustris is produced the hydrogen Effect on Performance.When concentration of substrate was lower than 5.94g/l, the hydrogen production with photosynthetic bacteria amount was very low, and behind cultured continuously 62h, nutrient solution promptly becomes redness, produced hydrogen and stopped fully.When concentration of substrate is higher than 23.76g/l, to produce the hydrogen process and be suppressed, the high more restraining effect of concentration is strong more.
Embodiment 4:
(1) liquid nutrient medium preparation: get glucose 3.96g/l; K
2HPO
43H
2O:0.6g/l;
KH
2PO
4：0.38g/l；NH
4Cl：1.99g/l；
CaCl
2：0.1g/l；FeSO
4·7H
2O：0.00695g/l；
ZnSO
4：0.0005g/l；MgSO
4·7H
2O：0.05g/l；
NaCl：0.1g/l-1.5g/l；CuSO
4：0.03g/l
(NH
4)
6Mo
4O
24·4H
2O：0.00075g/l；CuSO
4：0.03g/l；
V
B2: 0.00002g/l; Nicotinic acid: 0.00002g/l; Extractum carnis: 3g/l;
Water: 1000ml; Growth factor solution: 2ml;
Respectively adorn 0.31 above-mentioned substratum in two identical light biological hydrogen producing reactors, the initial pH value of regulating liquid nutrient medium respectively is 8.0,5.0;
Following steps are identical with embodiment 2 with condition, find that initial pH value of medium is at 5.0 o'clock, and 12h hydrogen production with photosynthetic bacteria speed is 1.08mlh after inoculation culture
-1L
-1, when being cultured to 60h, producing hydrogen and stop fully, obtain 24.5ml H altogether
2, when initial pH value of medium is 8.0,12h hydrogen-producing speed 1.64mlh after inoculation
-1L
-1, obtaining 33.2ml H altogether in the 120h cultivation continuously
2
Think that thus medium pH value is bigger to the influence of the product hydrogen of this bacterial classification, when the pH value is 8.0 its hydrogen production potential than the pH value be 5.0 much higher.
Fig. 2 shows initial pH value of medium to producing the hydrogen Effect on Performance, as shown in Figure 2, when initial pH is 7,8,9 o'clock, the hydrogen production potential of Rhodopseudomonas palustris is stronger, particularly pH value summary meta-alkalescence is 8 o'clock, it is maximum that the hydrogen production potential of Rhodopseudomonas palustris reaches, therefore the appropriate pH scope that helps Rhodopseudomonas palustris product hydrogen and growth is: 6.0-8.5, in experiment, find to work as too meta-acid of pH simultaneously, occur a large amount of dead bacterium in the nutrient solution, when the pH value was 9, growth was very fast, but a large amount of dead bacterium of very fast appearance in the nutrient solution subsequently greatly influence the hydrogen production potential of this bacterial classification.
Embodiment 5:
(1) liquid nutrient medium preparation: get glucose 3.96g/l; K
2HPO
43H
2O:0.6g/l;
KH
2PO
4：0.38g/l；NH
4Cl：1.99g/l；
CaCl
2：0.1g/l；FeSO
4·7H
2O：0.0417g/l；
ZnSO
4：0.0005g/l；MgSO
4·7H
2O：0.05g/l；
NaCl：0.1g/l-1.5g/l；CuSO
4：0.03g/l
(NH
4)
6Mo
4O
24·4H
2O：0.00075g/l；CuSO
4：0.03g/l；
V
B2: 0.00002g/l; Nicotinic acid: 0.00002g/l; Extractum carnis: 3g/l;
Water: 1000ml; Growth factor solution: 2ml;
The initial pH value of regulating liquid nutrient medium is 8.0;
Following steps are identical with embodiment 2 with condition, with postvaccinal nutrient solution by above-mentioned condition cultured continuously 5 days, during regularly detect the hydrogen situation of producing, find that the 12h photosynthetic bacterium begins to produce hydrogen after inoculation culture, hydrogen-producing speed is 0.616mlh
-1L
-1, biomass this moment (dry weight) is 0.012gl
-1, when being cultured to 24h, hydrogen-producing speed reaches maximum, is 5.219mlh
-1L
-1, this moment, biomass was 0.15gl
-1After this hydrogen-producing speed descends gradually, when being cultured to 120h, producing hydrogen and stops substantially, obtains 37.3ml H altogether
2
Comparing embodiment 4 and 5 is found, iron concentration is very big to the product hydrogen influence of photosynthetic bacterium under other identical culture condition, when iron concentration was 0.0417g/l (being 0.15mmol/l), the obvious specific concentration of hydrogen production potential was the height of 0.00695g/l (being 0.025mmol/l).Biomass growth simultaneously is also higher.
Fig. 3 shows substratum Fe
2+To producing the hydrogen Effect on Performance, as shown in Figure 3, in the metabolism of this bacterial classification to Fe
2+Demand is big, works as Fe
2+Hydrogen output is the highest when reaching 0.15mmol/l.
In embodiment 2, embodiment 3, embodiment 4 and embodiment 5, glucose can be changed into sodium acetate or organism such as sodium tartrate or Sodium Propionate.
In addition the inventor also obtained by experiment the substratum of determining form and content the same terms under temperature variation shown in Figure 4 to producing metabolic influence curve of hydrogen and intensity of illumination shown in Figure 5 to producing hydrogen Effect on Performance curve; Temperature mainly is to influence enzymatic activity in the hydrogen production with photosynthetic bacteria metabolism.Be illustrated under the defined medium prescription temperature by Fig. 4 Rhodopseudomonas palustris is produced the hydrogen Effect on Performance, hence one can see that: when temperature is 25 ℃, this bacterial classification hydrogen production potential the best, when temperature is below or above 25 ℃, the hydrogen production with photosynthetic bacteria amount is all on a declining curve, but need consider also in the practical application that bacterial classification produces the hydrogen metabolic rate, the growth temperature range that shows this bacterial classification is 10 ℃-43 ℃, and producing hydrogen optimal temperature scope is 20 ℃-35 ℃.Rhodopseudomonas palustris produces in the hydrogen metabolism needs luminous energy as motivating force.Fig. 5 represents that intensity of illumination is to the influence of this bacterial classification product hydrogen behavior under the specified conditions, and studies show that required intensity of illumination is in this growth and the product hydrogen process: 0lx-9000lx, wherein Shi Yi intensity of illumination is: 2000lx-5000lx.Intensity of illumination shows as the influence that this bacterial classification produces the hydrogen behavior under the specified conditions: this bacterial classification is not grown or poor growth under dark anaerobic condition substantially, and when illuminance was 4000lx, it is maximum that the hydrogen output of acquisition reaches, and is 49.57mlH
2/ (100ml culture medium) for the illuminance of the best, when illuminance is below or above 4000lx, will limits or suppress the photosynthetic bacterium physiological activity and carry out to the direction of synthetic hydrogen.
Claims (5)
1, a kind of method of utilizing Rhodopseudomonas palustris hydrogen manufacturing is characterized in that adopting following steps to carry out:
(1) preparation liquid nutrient medium is put into high-pressure sterilizing pot sterilization 12-15 minute with liquid nutrient medium, takes out substratum and is cooled to normal temperature, and be respectively charged in seed enlarged culturing reactor and the light biological hydrogen producing reactor liquid nutrient medium stand-by; Wherein, the initial pH scope of liquid nutrient medium is: 4.5-8.8, and the concentration range of organic substrates is: 1.98g/l-45g/l;
(2) enlarged culturing of Rhodopseudomonas palustris bacterial classification: fall to being inoculated in the cooled liquid nutrient medium of sterilization with the Rhodopseudomonas palustris of growing on the inoculating needle picking solid double-layer plate culture medium; In the seed enlarged culturing reactor of this liquid nutrient medium of splendid attire, charge into the argon gas excluding air, form anaerobic environment, and postvaccinal liquid nutrient medium placed 25 ℃-35 ℃, fluorescent lamp is a light source, illuminance is to leave standstill in the illumination constant incubator of 1500lx-8000lx to cultivate 36-96 hour, obtains used enlarged culturing liquid in the follow-up product hydrogen process;
(3) light biological hydrogen producing of Rhodopseudomonas palustris: Rhodopseudomonas palustris enlarged culturing liquid is inoculated in the liquid nutrient medium of light biological hydrogen producing reactor splendid attire by the 8%-18% that cultivates base unit weight, and in light biological hydrogen producing reactor, charge into argon gas and obtain anaerobic environment, with fluorescent lamp or LED lamp is light source, illuminance is: 2000lx-5000lx, culture temperature is 20 ℃-35 ℃, leave standstill the constant temperature illumination cultivation after 10 hours-15 hours Rhodopseudomonas palustris begin to produce hydrogen.
2, the method for utilizing Rhodopseudomonas palustris hydrogen manufacturing according to claim 1, the best initial pH scope of described liquid nutrient medium is got: 6.0-8.5.
3, the method for utilizing Rhodopseudomonas palustris hydrogen manufacturing according to claim 1, the concentration range of the best organic substrates of described liquid nutrient medium is 11.880g/l-27.72g/l.
4, according to claim 1 or the 2 or 3 described methods of utilizing Rhodopseudomonas palustris hydrogen manufacturing, described liquid culture based component is:
Organic substrates: 11.880g/l-27.72g/l; K
2HPO
43H
2O:0.3g/l-1.56g/l;
KH
2PO
4：0.22g/l-10.89g/l；NH
4Cl：0.677g/l-2.35g/l；
CaCl
2：0.002g/l-0.26g/l；FeSO
4·7H
2O：0.005g/l-0.417g/l；
ZnSO
4：0.0001g/l-0.02g/l；MgSO
4·7H
2O：0.02g/l-0.5g/l；
NaCl：0.1g/l-1.5g/l；CuSO
4：0.001g/l-0.04g/l
(NH
4)
6Mo
4O
24·4H
2O：0.0001g/l-0.002g/l；
V
B2：0.00001g/l-0.00025g/l；
Nicotinic acid: 0.00001g/l-0.0002g/l; Extractum carnis: 0.5g/l-4.0g/l;
Water: 1000ml; PH value: 6.0-8.5.
5, the method for utilizing Rhodopseudomonas palustris hydrogen manufacturing according to claim 4, described organic substrates comprise glucose or sodium acetate or sodium tartrate or Sodium Propionate.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101760480B (en) * | 2008-12-25 | 2012-11-07 | 中国科学院上海生命科学研究院 | Bio-hydrogen production method by photosynthetic bacteria |
| WO2013082988A1 (en) * | 2011-12-08 | 2013-06-13 | 中国科学院华南植物园 | Biological reactor with full-wavelength controllable light sources |
| CN104498516A (en) * | 2014-11-14 | 2015-04-08 | 成都理工大学 | High-efficiency hydrogen-production functional gene carrier pETD-SL and construction and application thereof |
| CN105713948A (en) * | 2014-12-01 | 2016-06-29 | 中粮集团有限公司 | Method for fermenting starchy raw material with mixed strain to produce hydrogen |
| CN109929783A (en) * | 2019-04-22 | 2019-06-25 | 广东宏隆生物科技有限公司 | The culture medium and cultural method of Rhodopseudomonas palustris |
| CN109937839A (en) * | 2019-04-03 | 2019-06-28 | 青岛农业大学 | A kind of tobacco plant preparation method with anti-wildfire characteristic |
| CN107937327B (en) * | 2018-01-28 | 2021-05-04 | 杭州富阳优信科技有限公司 | Bacteria and algae composite preparation and application thereof in preparation of hydrogen |
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- 2007-04-20 CN CN 200710078405 patent/CN101041832A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760480B (en) * | 2008-12-25 | 2012-11-07 | 中国科学院上海生命科学研究院 | Bio-hydrogen production method by photosynthetic bacteria |
| WO2013082988A1 (en) * | 2011-12-08 | 2013-06-13 | 中国科学院华南植物园 | Biological reactor with full-wavelength controllable light sources |
| CN104498516A (en) * | 2014-11-14 | 2015-04-08 | 成都理工大学 | High-efficiency hydrogen-production functional gene carrier pETD-SL and construction and application thereof |
| CN104498516B (en) * | 2014-11-14 | 2018-05-15 | 成都理工大学 | Highly effective hydrogen yield functional gene carrier pETD-SL and its structure and application |
| CN105713948A (en) * | 2014-12-01 | 2016-06-29 | 中粮集团有限公司 | Method for fermenting starchy raw material with mixed strain to produce hydrogen |
| CN107937327B (en) * | 2018-01-28 | 2021-05-04 | 杭州富阳优信科技有限公司 | Bacteria and algae composite preparation and application thereof in preparation of hydrogen |
| CN109937839A (en) * | 2019-04-03 | 2019-06-28 | 青岛农业大学 | A kind of tobacco plant preparation method with anti-wildfire characteristic |
| CN109929783A (en) * | 2019-04-22 | 2019-06-25 | 广东宏隆生物科技有限公司 | The culture medium and cultural method of Rhodopseudomonas palustris |
| CN113943759A (en) * | 2021-11-04 | 2022-01-18 | 重庆大学 | Method for regulating and controlling ethanol production performance of synthesis gas fermentation by two-step method |
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