CN101063098A - Culture method for photosynthetic bacterium strain having hydrogen-generation specificity - Google Patents
Culture method for photosynthetic bacterium strain having hydrogen-generation specificity Download PDFInfo
- Publication number
- CN101063098A CN101063098A CN 200710078404 CN200710078404A CN101063098A CN 101063098 A CN101063098 A CN 101063098A CN 200710078404 CN200710078404 CN 200710078404 CN 200710078404 A CN200710078404 A CN 200710078404A CN 101063098 A CN101063098 A CN 101063098A
- Authority
- CN
- China
- Prior art keywords
- culture
- bacterium
- red
- liquid
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture method of marsh red pseudomonas strain with generating hydrogen property, which comprises the following steps: (1) collecting upper superficial coat mud in farmland and sewer as bacterial source; filtering or stewing sample; collecting filter liquor or supernatant; (2) putting the filter liquid or supernatant into aseptic culture bottle; adding into fresh germicidal medium with proportion at 1 : 7-7 : 7; charging into argon gas to remove air; stewing; proceeding anaerobic culture; choosing daylight lamp as light source with illuminance at 2000 lux-5000 lux; setting the culture temperature at 25 deg. c-35 deg. c for 6-12 days; turning the culture liquid to purplish red; absorbing red bacterium on the wall of culture bottle; (3) fetching middle and lower layer liquid; adding into clean culture bottle.
Description
Technical field
The present invention relates to a kind of cultural method with photosynthetic bacterium bacterial strain of hydrogen-producing characteristic, belong to the bioenergy field, being specifically related to organic waste in the environment is nutritive substance, cultivates a kind of method with the photosynthetic bacterium bacterial strain that produces the hydrogen behavior.
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: 0.058016-17.696mlh
-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 enrichment culture 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, the purpose of its screening photosynthetic bacterium only is 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
One of purpose of the present invention provides a kind of cultural method with Rhodopseudomonas palustris bacterial strain of hydrogen-producing characteristic.
Two of purpose of the present invention provides a kind of cultural method with gluey red long life bacteria strain of hydrogen-producing characteristic.
Technical scheme one of the present invention is: a kind of cultural method with Rhodopseudomonas palustris bacterial strain of hydrogen-producing characteristic, adopt following steps:
(1) gather the bacterium source: the upper epidermis mud of gathering farmland, side bilge etc. is the bacterium source, filters or leave standstill sample, collects filtrate or supernatant liquor;
(2) multiplication culture of Rhodopseudomonas palustris bacterial strain: get above-mentioned filtrate or supernatant liquor and put into aseptic culturing bottle, add the substratum of fresh sterilization in culturing bottle in 1 to 7～7 to 7 ratio with it simultaneously, 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 2000 luxs～5000 luxs (lx), culture temperature is 25 ℃～35 ℃, cultivated 6 days～12 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of Rhodopseudomonas palustris bacterial strain: the middle lower floor liquid of getting above-mentioned red nutrient solution adds in the clean culturing bottle of sterilization, add sterilized fresh culture in 1 to 7～7 to 7 ratio with it, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with fluorescent lamp or the LED lamp that glows is light source, illuminance is 2000 luxs～5000 luxs (lx), culture temperature is 25 ℃～35 ℃, illumination is left standstill to transform and was cultivated 3 days～10 days once more, further eliminate non-photosynthetic bacterium, strengthen growth and the adaptive faculty of photosynthetic bacterium, the red nutrient solution of lower floor repeats this step more than 2 times in getting again, till observing a large amount of dominant bacterias under the opticmicroscope and being rod or oval bacterium;
(4) preparation of the gradient dilution liquid of Rhodopseudomonas palustris bacterial strain enrichment culture liquid: the enrichment culture liquid of getting the photosynthetic bacterium bacterial strain 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) separation and purification of Rhodopseudomonas palustris bacterial strain: 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 Rhodopseudomonas palustris bacterial strain enrichment culture liquid
-3-10
-10Gradient dilution liquid, the solid plate media surface was placed 5 minutes ~ 20 minutes, pour into 35 ℃～45 ℃ only contain agar and content is the upper strata covering liquid of 1.5%-2.0%, cover culture dish ware lid, placement is cooled to the top-layer agar covering liquid and solidifies fully, culture dish is placed 25 ℃～35 ℃, illuminance is an illumination cultivation 4 days～10 days in the illumination constant incubator of 2000lx～7000lx again; After waiting to grow bacterium colony, it is fast to select growth, and the single and color of colonial morphology is a red-purple, and cellular form is respectively oval person and 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) is carried out the preparation and the inoculation of substratum more set by step, repeat the separation and purification more than 3 times, up to the colonial morphology unanimity of observing growth, colony colour is to observe cellular form under red-purple and the opticmicroscope to be respectively ellipse and promptly to judge and obtain corresponding pure strain.
Described substratum composed as follows: K
2HPO
43H
2O:0.786g/l-1.5g/l; MgSO
47H
2O:0.03g/l-0.6g/l; KH
2PO
4: 0.272g/l-0.836g/l; FeSO
47H
2O:0.01g/l-0.09g/l; C
6H
12O
6H
2O:2.012g/l-8.048g/l; (NH
4)
6Mo
7O
244H
2O:0.0001g/l-0.0008g/l; NaCl:1g/l-2g/l; CaCl
2: 0.005g/l-0.03g/l; NH
4Cl:0.8g/l-2.6g/l; Growth factor solution: 3ml/l; ZnSO
47H
2O:0.0001g/l-0.004g/l.
Technical scheme two of the present invention is: a kind of cultural method with gluey red long life bacteria strain of hydrogen-producing characteristic, adopt following steps:
(1) gather the bacterium source: the upper epidermis mud of gathering farmland, side bilge etc. is the bacterium source, filters or leave standstill sample, collects filtrate or supernatant liquor;
(2) multiplication culture of gluey red long life bacteria strain: get above-mentioned filtrate or supernatant liquor and put into aseptic culturing bottle, add the substratum of fresh sterilization in culturing bottle in 1 to 7～7 to 7 ratio with it simultaneously, 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 2000 luxs～5000 luxs (lx), culture temperature is 25～35 ℃, cultivated 6～12 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of gluey red long life bacteria strain: the middle lower floor liquid of getting above-mentioned red nutrient solution adds in the clean culturing bottle of sterilization, add sterilized fresh culture in 1 to 7～7 to 7 ratio with it, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with fluorescent lamp or red LED lamp is light source, illuminance is 2000 luxs～5000 luxs (lx), culture temperature is 25 ℃～35 ℃, illumination is left standstill to transform and was cultivated 3 days～10 days once more, further eliminate non-photosynthetic bacterium, strengthen growth and the adaptive faculty of photosynthetic bacterium, the red nutrient solution of lower floor repeats this step more than 2 times in getting again, till observing a large amount of dominant bacterias under the opticmicroscope and being rod or oval bacterium;
(4) preparation of the gradient dilution liquid of gluey red long life bacteria strain enrichment culture liquid: the enrichment culture liquid of getting the photosynthetic bacterium bacterial strain 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) separation and purification of gluey red long life bacteria strain: 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 gluey red long life bacteria strain enrichment culture liquid
-3-10
-10Gradient dilution liquid, the solid plate media surface was placed 5 minutes-20 minutes, pour into 35 ℃～45 ℃ only contain agar and content is the upper strata covering liquid of 1.5%-2.0%, cover culture dish ware lid, placement is cooled to the top-layer agar covering liquid and solidifies fully, culture dish is placed 25 ℃～35 ℃, the fluorescent lamp illuminance is an illumination cultivation 4 days～10 days in the illumination constant incubator of 2000lx～7000lx again; After waiting to grow bacterium colony, it is fast to select growth, and the single and color of colonial morphology is orange-yellow, and cellular form is respectively quarter butt shape person and 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) is carried out the preparation and the inoculation of substratum more set by step, repeat the separation and purification more than 3 times, up to the colonial morphology unanimity of observing growth, colony colour is to observe cellular form under orange-yellow and the opticmicroscope to be respectively quarter butt shape and promptly to judge and obtain corresponding pure strain.
Described substratum composed as follows: K
2HPO
43H
2O:0.786g/l-1.5g/l; MgSO
47H
2O:0.03g/l-0.6g/l; KH
2PO
4: 0.272g/l-0.836g/l; FeSO
47H
2O:0.01g/l-0.09g/l; C
6H
12O
6H
2O:2.012g/l-8.048g/l; (NH
4)
6Mo
7O
244H
2O:0.0001g/l-0.0008g/l; NaCl:1g/l-2g/l; CaCl
2: 0.005g/l-0.03g/l; NH
4Cl:0.8g/l-2.6g/l; Growth factor solution: 3ml/l; ZnSO
47H
2O:0.0001g/l-0.004g/l.
Rhodopseudomonas palustris that technical scheme of the present invention obtains and gluey red long life bacteria strain have following characteristic:
(1) described bacterial classification nontoxicity, the hydrogen production potential height can absorb and conversion solar luminous energy, effectively decomposes and utilizes various organic substances;
(2) bacterial classification that obtains of the present invention is an amphimicrobian mode of life, but does not produce hydrogen under aerobic condition, can decompose various organic substances and produce hydrogen under the anaerobism illumination condition.
(3) bacterial strain of the present invention is extensive to the use pattern of substrate, can effectively decompose and utilize organic substances such as glucose, sodium tartrate, sodium acetate, Sodium Propionate, show that this bacterial strain utilizes and the ability of this type organic matter of metabolism is stronger, wherein special hobby utilizes glucose and multiple organic acid, and this helps the application in environmental pollution improvement.
(4) because the bacterial strain that the present invention obtains is to be carbon source with the organic substrates; nitrogenous source is provided simultaneously; inorganic salt and somatomedin etc. and cultivate to obtain; except that aspect producing Hydrogen Energy, having the special advantages; while energy decomposing organic matter; reduce the COD value of organic waste water; this bacterial classification can reach more than 98% the decomposition and the utilization ratio of organic substance; therefore this bacterial classification has very big advantage aspect the organic substrates transforming and utilize; simultaneously this bacterial classification is particularly suitable for decomposing and utilizes organic waste water behind the anaerobically fermenting; can further reduce the organic content in the waste liquid; obtain good purifying property; made full use of biomass energy; realized changing evil for precious, the purpose of protection environment.Therefore having broad application prospects and social value aspect environment protection and the realization Sustainable utilization of resources.
Technical solutions according to the invention compared with prior art, owing to adopt double-deck solid plate substratum to provide anaerobic environment for the growth and breeding of Rhodopseudomonas palustris and gluey red long life bacterium, simultaneously because solid medium is a printing opacity, can guarantee Rhodopseudomonas palustris and gluey red long life bacteria growing and produce in the hydrogen metabolic process demand luminous energy, guaranteed that the Rhodopseudomonas palustris and the gluey red long life bacterium bacterial classification that obtain have higher hydrogen production potential, can absorb and conversion solar luminous energy, effectively decompose and utilize various organic substances.
Embodiment
Embodiment 1:
(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) multiplication culture of Rhodopseudomonas palustris bacterial strain: get above-mentioned supernatant liquor 70ml and pour 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, leave standstill anaerobism and cultivate, be light source with the fluorescent lamp in the spawn culture, illuminance is 3000 luxs (lx), and culture temperature is 30 ℃, cultivates 10 days, nutrient solution color purpling gradually is red, and red bacterium absorption growth is arranged on the culturing bottle wall;
(3) enrichment culture of Rhodopseudomonas palustris bacterial strain: 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 are shaken up, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with the fluorescent lamp is light source, illuminance is 3000 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) preparation of the gradient dilution liquid of Rhodopseudomonas palustris bacterial strain enrichment culture liquid: 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) separation and purification of Rhodopseudomonas palustris bacterial strain: 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, after the method for (5) repeats 4 separation and purification more set by step, the colonial morphology unanimity of 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:1.2g/l; MgSO
47H
2O:0.5g/l; KH
2PO
4: 0.6g/l; FeSO
47H
2O:0.05g/l; C
6H
12O
6H
2O:6.012g/l; (NH
4)
6Mo
7O
244H
2O:0.0005g/l; NaCl:1.5g/l; CaCl
2: 0.015g/l; NH
4Cl1.5g/l; Growth factor solution concentration 3ml/l; ZnSO
47H
2O:0.002g/l;
Embodiment 2:
(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) multiplication culture of Rhodopseudomonas palustris bacterial strain: get above-mentioned supernatant liquor 70ml and pour in the triangular flask of 250ml, the fresh culture that adds the 60ml sterilization simultaneously is based in the bottle, supernatant liquor and fresh culture are shaken up, and charge into the argon gas excluding air, leave standstill anaerobism and cultivate, be light source with the fluorescent lamp in the spawn culture, illuminance is 5000 luxs (lx), and culture temperature is 35 ℃, cultivates 10 days, nutrient solution color purpling gradually is red, and red bacterium absorption growth is arranged on the culturing bottle wall;
(3) enrichment culture of Rhodopseudomonas palustris bacterial strain: the middle liquid 50ml of lower floor that gets above-mentioned nutrient solution is in the clean culturing bottle of sterilization, the sterilized fresh culture that adds 30ml again, nutrient solution and fresh culture are shaken up, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with the LED lamp that glows is light source, illuminance is 5000 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 4 times in getting again, and observing a large amount of dominant bacterias under the opticmicroscope is quarter butt shape or red oval bacterium;
(4) preparation of the gradient dilution liquid of Rhodopseudomonas palustris bacterial strain enrichment culture liquid: 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) separation and purification of Rhodopseudomonas palustris bacterial strain: 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 1.5% 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 10 days in the illumination constant incubator of 3000lx; 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, after the method for (5) repeats 4 separation and purification more set by step, the colonial morphology unanimity of growth, colony colour is a red-purple, observe cellular form under the opticmicroscope and be ellipse, promptly judge to obtain pure strain;
Wherein, substratum is composed as follows: K
2HPO
43H
2O:0.8g/l; MgSO
47H
2O:0.1g/l; KH
2PO
4: 0.3g/l; FeSO
47H
2O:0.02g/l; C
6H
12O
6H
2O:3.012g/l; (NH
4)
6Mo
7O
244H
2O:0.0002g/l; NaCl:1.8g/l; CaCl
2: 0.025g/l; NH
4Cl2.3g/l; Growth factor solution concentration 3ml/l; ZnSO
47H
2O:0.0002g/l;
The photosynthetic bacterium bacterial strain that obtains according to embodiment 1 and embodiment 2 is in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms culture presevation administrative center (CGMCC) preservation, and preservation date is on March 1st, 2007, preserving number CGMCC No.1947; Particular content is: Rhodopseudomonas palustris CQK 01, Rhodoseudomonas palustris CQK 01, CGMCC No.1947.
Embodiment 3:
(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;
(2) multiplication culture of gluey red long life bacteria strain: get above-mentioned filtrate 70ml and pour in the triangular flask of 250ml, the fresh culture that adds the 30ml sterilization simultaneously is based in the bottle, filtrate and fresh culture are shaken up, and charge into the argon gas excluding air, leave standstill anaerobism and cultivate, be light source with the fluorescent lamp in the spawn culture, illuminance is 3000 luxs (lx), and culture temperature is 30 ℃, cultivates 10 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of gluey red long life bacteria strain: 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 are shaken up, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with the fluorescent lamp is light source, illuminance is 3000 luxs (lx), 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 quarter butt 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; After the 5th day, observe a large amount of have mobility, cell is quarter butt shape, the negative bacterium of gramstaining occurs, 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 opticmicroscope is quarter butt shape or red oval bacterium;
(4) preparation of the gradient dilution liquid of gluey red long life bacteria strain enrichment culture liquid: 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) separation and purification of gluey red long life bacterium: 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 quarter butt shape bacterium 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, the choosing colony growth is fast, and orange-yellow bacterium colony 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) is carried out the preparation and the inoculation of substratum more set by step, after repeating 4 separation and purification, observe the colonial morphology unanimity of growth, colony colour is orange-yellow, and observes cellular form under the opticmicroscope and be stock shape, promptly judges to obtain pure strain; This bacterial classification bacterium colony surface wettability, picking very easily, cell are difficult for disperseing, and gramstaining is negative, and cell mobility is strong, as seen breeds in the bipartition mode, and the many cells after the division connect into stock shape; The secretion of visible cell surface has a large amount of mucus under the Electronic Speculum; Flagellum is few and grow, and is extremely living, is about about 2.1-1.2 μ m, about wide about 0.5-0.9 μ m; This bacterial classification identifies that through physiological and biochemical analysis and 16SrRNA comparison this bacterial classification belongs to Rhodospirillales, Rhodospirillaceae, long life Pseudomonas, gluey red long life bacterium (Rubrivivax gelatinosus), and called after Rubrivivax gelatinosus CJK 02.
Wherein, substratum is composed as follows: K
2HPO
43H
2O:1.2g/l; MgSO
47H
2O:0.5g/l; KH
2PO
4: 0.6g/l; FeSO
47H
2O:0.05g/l; C
6H
12O
6H
2O:6.012g/l; (NH
4)
6Mo
7O
244H
2O:0.0005g/l; NaCl:1.5g/l; CaCl
2: 0.015g/l; NH
4Cl1.5g/l; Growth factor solution concentration 3ml/l; ZnSO
47H
2O:0.002g/l;
Embodiment 4:
(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;
(2) multiplication culture of gluey red long life bacteria strain: get above-mentioned filtrate 70ml and pour in the triangular flask of 250ml, the fresh culture that adds the 60ml sterilization simultaneously is based in the bottle, filtrate and fresh culture are shaken up, and charge into the argon gas excluding air, leave standstill anaerobism and cultivate, be light source with the fluorescent lamp in the spawn culture, illuminance is 5000 luxs (lx), and culture temperature is 35 ℃, cultivates 10 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of gluey red long life bacteria strain: the middle liquid 50ml of lower floor that gets above-mentioned nutrient solution is in the clean culturing bottle of sterilization, the sterilized fresh culture that adds 30ml again, nutrient solution and fresh culture are shaken up, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with the fluorescent lamp is light source, illuminance is 5000 luxs (lx), 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 quarter butt 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; After the 5th day, observe a large amount of have mobility, cell is quarter butt shape, the negative bacterium of gramstaining occurs, the quantity of other bacterium obviously reduces.The red nutrient solution of lower floor repeats this step 4 times in getting again, and observing a large amount of dominant bacterias under opticmicroscope is quarter butt shape or red oval bacterium;
(4) preparation of the gradient dilution liquid of gluey red long life bacteria strain enrichment culture liquid: 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) separation and purification of gluey red long life bacterium: 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 quarter butt shape bacterium 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 1.5% 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 10 days in the illumination constant incubator of 3000lx; After growing bacterium colony, the choosing colony growth is fast, and orange-yellow bacterium colony 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) is carried out the preparation and the inoculation of substratum more set by step, after repeating 4 separation and purification, observe the colonial morphology unanimity of growth, colony colour is orange-yellow, and observes cellular form under the opticmicroscope and be stock shape, promptly judges to obtain pure strain;
Wherein, substratum is composed as follows: K
2HPO
43H
2O:0.8g/l; MgSO
47H
2O:0.1g/l; KH
2PO
4: 0.3g/l; FeSO
47H
2O:0.02g/l; C
6H
12O
6H
2O:3.012g/l; (NH
4)
6Mo
7O
244H
2O:0.0002g/l; NaCl:1.8g/l; CaCl
2: 0.025g/l; NH
4Cl2.3g/l; Growth factor solution concentration 3ml/l; ZnSO
47H
2O:0.0002g/l;
The photosynthetic bacterium bacterial strain that obtains according to embodiment 3 and embodiment 4 is in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms culture presevation administrative center (CGMCC) preservation, and preservation date is on March 1st, 2007, preserving number CGMCC No.1948; Particular content is: gluey red long life bacterium CJK 02, Rubrivivax gelatinosus CJK 02, CGMCC No.1948.
Embodiment 5:
The hydrogen test is produced in Rhodopseudomonas palustris Rhodopseudomonaspalustris CQK 01 strain that embodiment 1 or embodiment 2 obtain, and concrete experimental technique is:
Bacteria suspension is made in Rhodopseudomonas palustris Rhodopseudomonas palustris CQK 01 strain, bacteria suspension is added in the substratum in the bio-reactor that the transparent material of rectangular build makes in 10% ratio, reactor volume is 500ml, and the reaction solution volume is 300ml, and the upper space volume is 200ml, charge into argon gas and obtain anaerobic environment, with the LED lamp that glows is light source, and illuminance is 3000lx, and temperature is 30 ℃, leave standstill constant temperature culture, the culture media composition composition of employing is as follows:
Medium component and content
Glucose: 19.8g/l; NH
4Cl:1.99g/l; K
2HPO
43H
2O:0.6g/l; KH
2PO
4: 0.38g/l; CaCl
2: 0.1g/l; FeSO
47H
2O:0.0834g/l;
ZnSO
4·7H
2O：0.0005g/l；MgSO
4·7H
2O：0.05g/l；
CuSO
4：0.03g/l；(NH
4)
6Mo
7O
24·4H
2O：0.00075g/l；
Growth factor solution: 2ml g/l; V
B2: 0.00002g/l;
Nicotinic acid: 0.000015g/l; Extractum carnis: 3g/l; Water: 1000ml;
PH value: 6.2
With postvaccinal nutrient solution by above-mentioned condition cultured continuously 5 days, during regularly detect the hydrogen situation of producing, find: the 12h photosynthetic bacterium 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
Embodiment 6:
This bacterial strain that embodiment 3 or embodiment 4 are obtained is gluey red long life bacterium (Rubrivivaxgelatinosus) CJK 02 strain, produces the hydrogen test, and concrete experimental technique is:
Bacteria suspension is made in the red long life bacterium of glue (Rubrivivax gelatinosus) CJK 02 strain, bacteria suspension is added in the substratum in the bio-reactor that the transparent material of rectangular build makes in 10% ratio, reactor volume is 500ml, and the reaction solution volume is 300ml, and the upper space volume is 200ml, charge into argon gas and obtain anaerobic environment, with the fluorescent lamp is light source, and illuminance is 3000lx, and temperature is 30 ℃, leave standstill constant temperature culture, the culture media composition composition of employing is as follows:
Glucose: 19.8g/l; NaCl:1.5g/l; NH
4Cl:1.99g/l;
K
2HPO
4·3H
2O：0.6g/l；KH
2PO
4：0.3g/l；
CaCl
2：0.1g/l；FeSO
4·7H
2O：0.0834g/l；
ZnSO
4·7H
2O：0.0005g/l；MgSO
4·7H
2O：0.005g/l；
CuSO
4：0.03g/l；(NH
4)
6Mo
7O
24·4H
2O：0.00075g/l；
V
B2: 0.00002g/l; Nicotinic acid: 0.000015g/l;
Extractum carnis: 2.5g/l; Water: 1000ml; PH value: 6.5
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 0.0103mgh
-1L
-1, biomass this moment (dry weight) is 0.083gl
-1, when being cultured to 28h, hydrogen-producing speed reaches maximum, is 1.35mgh
-1L
-1, this moment, biomass was 0.49gl
-1After this maintain 0.98-1.07mgh substantially to the 72h hydrogen-producing speed
-1L
-1Between, hydrogen-producing speed descends gradually afterwards, when being cultured to 120h, producing hydrogen and still continues as 0.025mgh
-1L
-1, biomass slightly is reduced to 0.37gl
-1
By embodiment 5 and embodiment 6 as can be seen, cultivate the photosynthetic bacterium bacterial classification and domestic and international similar photosynthetic bacterium bacterial classification comparison that obtains with technical solution of the present invention, hydrogen production potential is in the prostatitis.
Claims (4)
1, a kind of cultural method with Rhodopseudomonas palustris bacterial strain of hydrogen-producing characteristic is characterized in that adopting following steps:
(1) gather the bacterium source: the upper epidermis mud of gathering farmland, side bilge etc. is the bacterium source, filters or leave standstill sample, collects filtrate or supernatant liquor;
(2) multiplication culture of Rhodopseudomonas palustris bacterial strain: get above-mentioned filtrate or supernatant liquor and put into aseptic culturing bottle, add the substratum of fresh sterilization in culturing bottle in 1 to 7～7 to 7 ratio with it simultaneously, 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 2000 luxs～5000 luxs (lx), culture temperature is 25～35 ℃, cultivated 6～12 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of Rhodopseudomonas palustris bacterial strain: the middle lower floor liquid of getting above-mentioned red nutrient solution adds in the clean culturing bottle of sterilization, add sterilized fresh culture in 1 to 7～7 to 7 ratio with it, charge into argon gas and obtain anaerobic environment, culturing bottle is placed constant incubator, with fluorescent lamp or the LED lamp that glows is light source, illuminance is 2000 luxs～5000 luxs (lx), culture temperature is 25～35 ℃, illumination is left standstill to transform and was cultivated 3～10 days once more, further eliminate non-photosynthetic bacterium, strengthen growth and the adaptive faculty of photosynthetic bacterium, the red nutrient solution of lower floor repeats this step more than 2 times in getting again, till observing a large amount of dominant bacterias under the opticmicroscope and being rod or oval bacterium;
(4) preparation of the gradient dilution liquid of Rhodopseudomonas palustris bacterial strain enrichment culture liquid: get Rhodopseudomonas palustris 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) separation and purification of Rhodopseudomonas palustris bacterial strain: 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 Rhodopseudomonas palustris bacterial strain enrichment culture liquid
-3-10
-10Gradient dilution liquid, the solid plate media surface was placed 5-20 minute, pour into 40～45 ℃ only contain agar and content is the upper strata covering liquid of 1.5%-2.0%, cover culture dish ware lid, placement is cooled to the top-layer agar covering liquid and solidifies fully, culture dish is placed 25 ℃～35 ℃, illuminance is an illumination cultivation 4～10 days in the illumination constant incubator of 2000lx～7000lx again; After waiting to grow bacterium colony, it is fast to select growth, and colonial morphology is single, and colony colour be redness, and cellular form is that oval 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) is carried out the preparation and the inoculation of substratum more set by step, repeat the separation and purification more than 3 times, up to the colonial morphology unanimity of observing growth, colony colour is to observe cellular form under red-purple and the opticmicroscope to be respectively ellipse and promptly to judge and obtain corresponding pure strain.
2, the cultural method with Rhodopseudomonas palustris bacterial strain of hydrogen-producing characteristic according to claim 1, described substratum composed as follows: K
2HPO
43H
2O:0.786g/l-1.5g/l; MgSO
47H
2O:0.03g/l-0.6g/l; KH
2PO
4: 0.272g/l-0.836g/l; FeSO
47H
2O:0.01g/l-0.09g/l; C
6H
12O
6H
2O:2.012g/l-8.048g/l; (NH
4)
6Mo
7O
244H
2O:0.0001g/l-0.0008g/l; NaCl:1g/l-2g/l; CaCl
2: 0.005g/l-0.03g/l; NH
4Cl:0.8g/l-2.6g/l; Growth factor solution: 3ml/l; ZnSO
47H
2O:0.0001g/l-0.004g/l.
3, a kind of cultural method with gluey red long life bacteria strain of hydrogen-producing characteristic is characterized in that adopting following steps:
(1) gather the bacterium source: the upper epidermis mud of gathering farmland, side bilge etc. is the bacterium source, filters or leave standstill sample, collects filtrate or supernatant liquor;
(2) multiplication culture of gluey red long life bacteria strain: get above-mentioned filtrate or supernatant liquor and put into aseptic culturing bottle, add the substratum of fresh sterilization in culturing bottle in 1 to 7～7 to 7 ratio with it simultaneously, and charge into the argon gas excluding air, leaving standstill anaerobism cultivates, be light source with fluorescent lamp or the LED lamp that glows in the spawn culture, illuminance is 2000 luxs～5000 luxs (lx), culture temperature is 25～35 ℃, cultivated 6～12 days, purpling is red gradually up to the nutrient solution color, ends when having red bacterium absorption to grow on the culturing bottle wall;
(3) enrichment culture of gluey red long life bacteria strain: the middle lower floor liquid of getting above-mentioned red nutrient solution adds in the clean culturing bottle of sterilization, add sterilized fresh culture in 1 to 7～7 to 7 ratio with it, charge into argon gas and create anaerobic environment, culturing bottle is placed constant incubator, with fluorescent lamp or red LED lamp is light source, illuminance is 2000 luxs～5000 luxs (lx), culture temperature is 25～35 ℃, illumination is left standstill to transform and was cultivated 3～10 days once more, further eliminate non-photosynthetic bacterium, strengthen growth and the adaptive faculty of photosynthetic bacterium, the red nutrient solution of lower floor repeats this step more than 2 times in getting again, till observing a large amount of dominant bacterias under the opticmicroscope and being rod or oval bacterium;
(4) preparation of the gradient dilution liquid of gluey red long life bacteria strain enrichment culture liquid: get gluey red long life bacterium 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) separation and purification of gluey red long life bacteria strain: 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 gluey red long life bacteria strain enrichment culture liquid
-3-10
-10Gradient dilution liquid, the solid plate media surface was placed 5-20 minute, pour into 40～45 ℃ only contain agar and content is the upper strata covering liquid of 1.5%-2.0%, cover culture dish ware lid, placement is cooled to the top-layer agar covering liquid and solidifies fully, culture dish is placed 25 ℃～35 ℃, the fluorescent lamp illuminance is an illumination cultivation 4～10 days in the illumination constant incubator of 2000lx～7000lx again; After waiting to grow bacterium colony, it is fast to select growth, and colonial morphology is single, and colony colour is orange-yellow, and cellular form is that quarter butt shape 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) is carried out the preparation and the inoculation of substratum more set by step, repeat the separation and purification more than 3 times, up to the colonial morphology unanimity of observing growth, colony colour is that to observe cellular form under orange-yellow and the opticmicroscope be that quarter butt shape is promptly judged and obtained corresponding pure strain.
4, the cultural method with gluey red long life bacteria strain of hydrogen-producing characteristic according to claim 3, described substratum composed as follows: K
2HPO
43H
2O:0.786g/l-1.5g/l; MgSO
47H
2O:0.03g/l-0.6g/l; KH
2PO
4: 0.272g/l-0.836g/l; FeSO
47H
2O:0.01g/l-0.09g/l; C
6H
12O
6H
2O:2.012g/l-8.048g/l; (NH
4)
6Mo
7O
244H
2O:0.0001g/l-0.0008g/l; NaCl:1g/l-2g/l; CaCl
2: 0.005g/l-0.03g/l; NH
4Cl:0.8g/l-2.6g/l; Growth factor solution: 3ml/l; ZnSO
47H
2O:0.0001g/l-0.004g/l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100784042A CN101063098B (en) | 2007-04-20 | 2007-04-20 | Culture method for photosynthetic bacterium strain having hydrogen-generation specificity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100784042A CN101063098B (en) | 2007-04-20 | 2007-04-20 | Culture method for photosynthetic bacterium strain having hydrogen-generation specificity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101063098A true CN101063098A (en) | 2007-10-31 |
| CN101063098B CN101063098B (en) | 2010-12-29 |
Family
ID=38964385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100784042A Expired - Fee Related CN101063098B (en) | 2007-04-20 | 2007-04-20 | Culture method for photosynthetic bacterium strain having hydrogen-generation specificity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101063098B (en) |
Cited By (6)
| 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 |
| CN107410493A (en) * | 2017-05-08 | 2017-12-01 | 田丙申 | A kind of preparation method of selenium-rich milk |
| CN109666611A (en) * | 2019-01-28 | 2019-04-23 | 南通龙洋水产有限公司 | A kind of preparation of photosynthetic bacteria used for aquiculture and purification process |
| 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 |
| CN109929898A (en) * | 2019-03-28 | 2019-06-25 | 河南农业大学 | A method of promoting the energy-efficient production hydrogen of HAU-M1 photosynthetic bacteria group |
| CN112877242A (en) * | 2021-02-05 | 2021-06-01 | 广东工业大学 | Bacterial source photosensitizer for wastewater treatment and preparation method and application thereof |
-
2007
- 2007-04-20 CN CN2007100784042A patent/CN101063098B/en not_active Expired - Fee Related
Cited By (7)
| 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 |
| CN107410493A (en) * | 2017-05-08 | 2017-12-01 | 田丙申 | A kind of preparation method of selenium-rich milk |
| CN109666611A (en) * | 2019-01-28 | 2019-04-23 | 南通龙洋水产有限公司 | A kind of preparation of photosynthetic bacteria used for aquiculture and purification process |
| CN109929898A (en) * | 2019-03-28 | 2019-06-25 | 河南农业大学 | A method of promoting the energy-efficient production hydrogen of HAU-M1 photosynthetic bacteria group |
| CN109929898B (en) * | 2019-03-28 | 2022-08-30 | 河南农业大学 | Method for promoting HAU-M1 photosynthetic bacteria 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 |
| CN112877242A (en) * | 2021-02-05 | 2021-06-01 | 广东工业大学 | Bacterial source photosensitizer for wastewater treatment and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101063098B (en) | 2010-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tiang et al. | Recent advanced biotechnological strategies to enhance photo-fermentative biohydrogen production by purple non-sulphur bacteria: an overview | |
| CN101285075B (en) | Coupling method for biogas fermentation and autotrophic freshwater microalgae culture | |
| CN101063098B (en) | Culture method for photosynthetic bacterium strain having hydrogen-generation specificity | |
| Unpaprom et al. | A newly isolated green alga, Scenedesmus acuminatus, from Thailand with efficient hydrogen production | |
| CN102443542B (en) | High-density culture process of autotrophic oil-producing microalgae | |
| CN101041832A (en) | Method for producing hydrogen by using marsh red pseudomonas | |
| CN104611227B (en) | Scenedesmus obliquus with tolerance to high pH and breeding method thereof | |
| Zhang et al. | Removal of pollutants from biogas slurry and CO 2 capture in biogas by microalgae-based technology: a systematic review | |
| CN110684667B (en) | Microalgae biofilm culture method capable of simultaneously improving biomass and grease yield | |
| Liu et al. | Material biosynthesis, mechanism regulation and resource recycling of biomass and high-value substances from wastewater treatment by photosynthetic bacteria: a review | |
| CN113233587B (en) | Microalgae culture pond-artificial wetland coupling system and method for deep purification of sewage | |
| Rameshprabu et al. | A newly isolated green alga, Pediastrum duplex Meyen, from Thailand with efficient hydrogen production | |
| Hamouda et al. | Removal of heavy metals and production of bioethanol by green alga Scenedesmus obliquus grown in different concentrations of wastewater | |
| CN107760586A (en) | A kind of microalgae biofilm system of Immobilized culture | |
| CN101045902A (en) | Biophotosynthetic reactor apparatus and process for producing micro algae | |
| Rai et al. | Hydrogen economy and storage by nanoporous microalgae diatom: special emphasis on designing photobioreactors | |
| CN110479079B (en) | Membrane bioreaction system for coprocessing greenhouse gas by bacteria and algae | |
| CN113105991B (en) | Micro-nano bubble culture and recovery integrated microalgae biofilm reactor and microalgae culture and recovery method thereof | |
| CN106906270A (en) | Waste water and the method for producing carotenoid, gaseous fuel and organic fertilizer are processed using microalgae | |
| CN117568178A (en) | High-carbon-fixation microalgae and method for treating sewage by using same | |
| CN101280273A (en) | Industrial culture method and device for photosynthetic autotrophic microorganisms | |
| CN1796268A (en) | Hydrogen production plant by photosynthetic organism of solar energy | |
| CN107043693A (en) | It is a kind of from oxygen uptake formula tubular type bioreactor | |
| CN101275117B (en) | Method for fast algae proliferation directly used for bio-hydrogen production by using CO2 | |
| CN1280405C (en) | Method of increasing hydrogen releasing efficient of chlamydomonas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20101229 Termination date: 20130420 |