CN101333504A - Method for enhancing hydrogen-producing speed and yield of photosynthetic bacteria - Google Patents
Method for enhancing hydrogen-producing speed and yield of photosynthetic bacteria Download PDFInfo
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- CN101333504A CN101333504A CNA2007100430283A CN200710043028A CN101333504A CN 101333504 A CN101333504 A CN 101333504A CN A2007100430283 A CNA2007100430283 A CN A2007100430283A CN 200710043028 A CN200710043028 A CN 200710043028A CN 101333504 A CN101333504 A CN 101333504A
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Abstract
The invention discloses a method for increasing the hydrogen production rate and output of photosynthetic bacteria, which comprises the steps of: a) selecting strains: Rhodobacter sphearoides SH-2 strains are selected; b) cell culture: an RCVB culture medium is composed of a carbon source, a nitrogen source, basic salt, a phosphate buffer and vitamin in appropriate amount. The pH value is adjusted to 7.00; and a seed liquid is added and cultivated on a shaking table for one to two days at a speed of 160r/min at the culture temperature of 30+-2 DEG C; c) adding of microelements: an appropriate amount of molybdenum ions and iron ions are added into the culture medium; and d) waste water treatment: the domestic sludge, deteriorated milk or brewing waste water is added with basic salt, vitamin, phosphate buffer and L-glutamic acid after preprocessing for adjusting the pH value to 7.00 and sterilization. Experiments show that the concentration of iron and molybdenum ions in the culture medium obviously influences the hydrogen production rate of Rhodobacter sphearoides.
Description
Technical field
The energy and environment are the human two large problems that faces at present.Hydrogen is following optimal green energy resource.At occurring in nature, many microorganisms can be converted into hydrogen with the hydrogen proton in water or the carbohydrate in its metabolic process, and the biology product hydrogen technology that forms on this basis might make the mankind break away from oil crisis and take leave of the fuel-engined vehicle exhaust emission.Produce hydrogen transformation efficiency and product purification difficulty or ease from hydrogen-producing speed, carbohydrate, photosynthetic bacterium is that one of monoid of application prospect is arranged most.The photosynthetic hydrogen of putting can utilize multiple organic acid, and it produces the hydrogen process acquisition of clean energy, the utilization and the improvement organic pollutant three of luminous energy are organically combined.
But the photosynthetic hydrogen production technology to be applied to industrial production and satisfy human to the energy growing demand, still need further to improve the efficient of hydrogen production with photosynthetic bacteria, and seek suitable waste water as cheap carbon source.
Summary of the invention
Goal of the invention of the present invention is for a kind of method that improves hydrogen production with photosynthetic bacteria speed, output is provided, and the substratum by changing hydrogen production with photosynthetic bacteria, introduces suitable iron, molybdenum ion to improve hydrogen generation efficiency and to reduce cost.
Purpose of the present invention can be achieved through the following technical solutions.
A kind of method that improves hydrogen production with photosynthetic bacteria speed, output, its step is as follows:
A) select bacterial classification
The red bacterium of class ball (Rhodobacter sphearoides) the SH-2 bacterial strain of selecting for use; Provide by Shanghai school of life and health sciences plant physiology ecological Studies institute of Chinese Academy of Sciences molecule microorganism open laboratory.It can utilize oxysuccinic acid, acetate, butyric acid etc. to carry out anaerobism illumination to put hydrogen.
B) cell cultures
The RCVB substratum is by an amount of carbon source, nitrogenous source, and basic salt, phosphoric acid buffer, VITAMIN is formed.Adjust pH to 7.00; Insert seed liquor, 30 ± 2 degrees centigrade of culture temperature were cultivated 1-2 days with 160r/min on shaking table.
C) Wei Liangyuansu adding
In substratum, add an amount of molybdenum ion and iron ion.
D) wastewater treatment
To add basic salt, VITAMIN, phosphoric acid buffer, L-L-glutamic acid, adjust pH to 7.00, sterilization after domestic sludge, rotten milk or the pre-treatment of malting effluent process.
Studies show that the nitrogenase of photosynthetic bacterium is an iron-sulphur protein, is a unsettled bimolecular of oxygen, and molecule is very big very complicated.Mo-Fe active centre cluster is also contained in the active centre except containing Fe-S.
Experiment shows that iron, molybdenum ion concentration obviously influence the hydrogen-producing speed of the red bacterium of class ball in the substratum.
Description of drawings
Fig. 1 is the influence broken line graph of molybdenum ion concentration among the present invention to product hydrogen total amount;
Fig. 2 is the influence broken line graph of molybdenum ion concentration among the present invention to maximum hydrogen-producing speed;
Fig. 3 is the OD value graph of a relation behind molybdenum ion concentration of the present invention and the product hydrogen;
Fig. 4 is the influence broken line graph of iron concentration among the present invention to product hydrogen total amount;
Fig. 5 is the influence broken line graph of iron concentration among the present invention to maximum hydrogen-producing speed;
Fig. 6 is the OD value graph of a relation behind iron concentration of the present invention and the product hydrogen;
Embodiment
Further specify technical characterstic of the present invention below in conjunction with specific embodiment.
Experimental data of the present invention is tested by the following method:
A) select bacterial classification
The red bacterium of class ball (Rhodobacter sphearoides) the SH-2 bacterial strain of selecting for use; Provide by Shanghai school of life and health sciences plant physiology ecological Studies institute of Chinese Academy of Sciences molecule microorganism open laboratory.It can utilize oxysuccinic acid, acetate, butyric acid etc. to carry out anaerobism illumination to put hydrogen.
B) cell cultures
Every 1LRCVB substratum is by carbon source (30mM oxysuccinic acid), nitrogenous source (1g L-L-glutamic acid), and basic salt 50ml, phosphoric acid buffer 7.5ml, VITAMIN 1ml forms.Adjust pH to 7.00; Insert seed liquor, 30 ± 2 degrees centigrade of culture temperature were cultivated 1-2 days with 160r/min on shaking table.
C) Wei Liangyuansu adding
The molybdenum ion and the iron ion that in substratum, add different concns.Its concentration is respectively:
Mo
6+(μM)：0，5，10，15，20，30，60，100，3.5
Fe
2+(μM)：0，20，60，80，100，150，200，300，42
D) wastewater treatment
To add basic salt, VITAMIN, phosphoric acid buffer, L-L-glutamic acid, adjust pH to 7.00, sterilization after domestic sludge, rotten milk or the pre-treatment of malting effluent process.
E) organic acid is measured in the waste water
With island functional activities of the body fluid phase chromatogram (Shimadzu, Kyoto, Japan (FID-hydrogen flame detector) (capillary column), sample size 10 μ L, the sample introduction temperature is 250 ℃, 80 ℃ of column temperatures, detector temperature are 260 ℃.
F) gas production rate is measured
Be reflected in the test tube of 35mL and carry out, collect gas with the syringe of 60mL.Record aerogenesis volume in the gas generation process, hydrogen content by the U.S. gas-chromatography in sky (Techcomp GC7900, Shanghai) (TCD-thermal conductivity detector) (packed column) detects, sample size 0.1mL, 80 ℃ of injection ports, 80 ℃ of column temperatures, detector temperature are 130 ℃.
G) mensuration of OD value
With spectrophotometer (Unic UV2808, Shanghai) absorption value of detection 660nm.
Mo
6+To producing the influence of hydrogen
Mo in the substratum
6+Concentration all influential to product hydrogen total amount, substrate conversion efficiency, the maximum hydrogen-producing speed of the red Hydrogen Production by Bacteria of class ball, and have a tangible peak value, concrete data are as follows:
| Mo 6+Concentration (μ M) | H 2(ml/33m 1 substratum) | Maximum hydrogen-producing speed (ml H 2/L·h) | Mean rate (ml H 2/L·h) | Substrate conversion efficiency | Initial pH value | The final pH value | The |
| 0 | 29±4 | 45 | 15 | 19.84％ | 7.00 | 6.79 | 2.77 |
| 3.5 | 105±1 | 138 | 47 | 71.22％ | 7.00 | 7.08 | 2.28 |
| 5 | 94±3 | 89 | 42 | 63.90％ | 7.00 | 7.06 | 1.98 |
| 10 | 74±4 | 58 | 33 | 50.25％ | 7.00 | 7.07 | 2.01 |
| 15 | 87±6 | 73 | 35 | 59.49％ | 7.00 | 7.11 | 2.08 |
| 20 | 76±4 | 79 | 31 | 51.67％ | 7.00 | 7.11 | 1.93 |
| 30 | 75±4 | 84 | 29 | 50.79％ | 7.00 | 7.04 | 2.13 |
| 60 | 72±3 | 63 | 26 | 49.24％ | 7.00 | 7.03 | 1.98 |
| 100 | 71±5 | 65 | 25 | 48.52％ | 7.00 | 7.06 | 2.00 |
From table, see, work as Mo
6+Concentration is produced hydrogen total amount, maximum hydrogen-producing speed and substrate conversion efficiency and has all been reached the peak when 3.5 μ M, is respectively 105 ± 1ml/33ml substratum, 138 (ml H
2/ Lh), 71.22%; Work as Mo
6+When concentration surpassed 3.5 μ M, the product hydrogen effect of the red bacterium of class ball began to descend; Work as Mo
6+When concentration surpassed 10 μ M, producing hydrogen total amount, maximum hydrogen-producing speed and substrate conversion efficiency did not have big fluctuation, all maintains close level, was respectively about the 70-80ml/33ml substratum, 60-70ml H
2About/Lh, about 50%.
In experiment, also observe, work as Mo
6+When concentration was lower than 30 μ M, about about the 18 hours red bacteriums of class ball in inoculation back began aerogenesis, reach the aerogenesis peak in the little period of 20-30; Work as Mo
6+When concentration was higher than 30 μ M, about about the 27 hours red bacteriums of class ball in inoculation back began aerogenesis, reach the aerogenesis peak in the little period of 30-40.
By detection, find to work as Mo to substratum OD value behind the aerogenesis
6+When concentration is lower than 5 μ M, Mo
6+Concentration is big more, and the OD value is more little; Work as Mo
6+When concentration surpasses 5 μ M, Mo
6+The variation of concentration and OD value there is no obviously gets in touch.Mo is described
6+Concentration has certain influence to the growth of the red bacterium of class ball, but this influence is little, and the red bacterium of class ball is at Mo
6+Concentration is to grow preferably (except 0 μ M) in the substratum of 3.5 μ M.
Therefore, control Mo
6+Concentration can strengthen the product hydrogen effect of the red bacterium of class ball greatly at 3.5 μ M, improves and produces hydrogen total amount, maximum hydrogen-producing speed and substrate conversion efficiency.
Molybdenum ion concentration can intuitively be expressed by Fig. 1-Fig. 3 the influence of producing the hydrogen effect.
Fe
2+To producing the influence of hydrogen
Fe in the substratum
2+Concentration all influential to product hydrogen total amount, substrate conversion efficiency, the maximum hydrogen-producing speed of the red Hydrogen Production by Bacteria of class ball, concrete data are as follows:
| Fe 2+Concentration (μ M) | H 2(ml/33ml substratum) | Maximum hydrogen-producing speed (ml H 2/L·h) | Mean rate (ml H 2/L·h) | Substrate conversion efficiency | Initial pH value | The final pH value | The |
| 0 | 22±1 | 50 | 21 | 14.97％ | 7.00 | 7.45 | 1.78 |
| 20 | 70±2 | 70 | 24 | 47.62％ | 7.00 | 7.28 | 1.82 |
| 42 | 115±2 | 105 | 37 | 78.23％ | 7.00 | 7.05 | 2.21 |
| 60 | 102±3 | 100 | 38 | 69.39％ | 7.00 | 7.07 | 1.93 |
| 80 | 98±5 | 91 | 29 | 66.67％ | 7.00 | 7.14 | 1.89 |
| 100 | 108±3 | 98 | 31 | 73.47％ | 7.00 | 7.11 | 1.93 |
| 150 | 122±4 | 136 | 42 | 82.99％ | 7.00 | 7.21 | 1.88 |
| 200 | 117±2 | 135 | 37 | 79.59％ | 7.00 | 7.26 | 2.13 |
| 300 | 115±3 | 134 | 35 | 78.23％ | 7.00 | 7.16 | 1.90 |
Can see from last table, along with Fe
2+The increase of concentration, the product hydrogen total amount of the red bacterium of class ball, maximum hydrogen generation efficiency, substrate conversion efficiency all become greatly, work as Fe
2+When concentration reached 42 μ M and 150 μ M, product hydrogen total amount, maximum hydrogen generation efficiency, substrate conversion efficiency peaked, and Fe
2+Concentration is that the substratum effect of 150 μ M is best, and product hydrogen total amount, maximum hydrogen generation efficiency, substrate conversion efficiency are respectively 122ml/33ml substratum, 136ml H
2/ Lh, 82.99%, Fe
2+Concentration is that the substratum effect of 42 μ M is slightly poor, and product hydrogen total amount, maximum hydrogen generation efficiency, substrate conversion efficiency are respectively 115ml/33ml substratum, 105ml H
2/ Lh, 78.23%; Work as Fe
2+Concentration is between 42 μ M and 150 μ M the time, Fe
2+The variation of concentration is little to producing the hydrogen influential effect, produces hydrogen total amount, maximum hydrogen generation efficiency, substrate conversion efficiency and be respectively about the 100-110ml/33ml substratum, 90-100ml H
2About/Lh, about 65%-75%; Work as Fe
2+When concentration surpassed 150 μ M, product hydrogen effect was slightly poor than 150 μ M's, but remains basically stable, and does not have great fluctuation process.
It is little to the growth effect of the red bacterium of class ball that the detection of OD value has reflected that iron concentration changes, and works as Fe
2+Concentration is when 42 μ M, and the red bacterial growth of class ball gets best, differs very little but compare with other concentration.
Therefore, Fe in the control substratum
2+Concentration is when 42 μ M or 150 μ M, and the red Hydrogen Production by Bacteria effect of class ball is best, certainly, if Fe
2+Concentration is 150 μ M, though hydrogen-producing speed is bigger, but also is accompanied by simultaneously the rising of cost.
Iron concentration can intuitively be expressed by Fig. 4-Fig. 6 the influence of producing the hydrogen effect.
Mo
6+With Fe
2+Comparison
Two groups that aerogenesis effect in the experimental data of above-mentioned two kinds of particulates is best compare, and find that the red bacterium of class ball is at 150 μ MFe
2+Substratum in hydrogen-producing speed a little less than at 3.5 μ M Mo
6+Substratum, but produce the hydrogen total amount and substrate conversion efficiency bigger.
As shown in the table:
| Concentration (μ M) | H 2(ml/33 ml substratum) | Maximum hydrogen-producing speed (ml H 2/L·h) | Mean rate (ml H 2/L·h) | Substrate conversion efficiency | Initial p H value | Final pH value | The OD value |
| 3.5(Mo 6+) | 105±1 | 138 | 47 | 71.22％ | 7.00 | 7.08 | 2.28 |
| 150(Fe 2+) | 122±4 | 136 | 42 | 82.99％ | 7.00 | 7.21 | 1.88 |
The product hydrogen situation of the red bacterium of class ball in malting effluent
Vinegar is a kind of polytrophic acid condiment that China people like very much, studies show that, acetic acid is the main component in making vinegar, and sugar, amino acid, ester, aldehyde, alcohol, phenol, ketone etc. are the microchemistry composition.This problem has been tested the product hydrogen effect of the red bacterium of class ball in the waste water of brewery's system vinegar.
Carry out organic acid by the waste water to brewery's system vinegar and detect, find wherein to contain certain density acetate, concrete data are as follows:
| Acetate (mM) | Propionic acid (mM) | Butyric acid (mM) | Initial p H value | |
| Vinegar | 92.8 | / | / | 2.00 |
Modulate behind 1.5 times of this wastewater dilutions, add basic salt, VITAMIN, phosphoric acid buffer, L-L-glutamic acid, adjust pH to 7.00 is as nutrient solution.Adding the 33mL nutrient solution in the 35mL test tube, is the light source irradiation 7 days of 4000-5000Lux with illumination intensity.
Product hydrogen situation such as the following table of the red bacterium of class ball in this waste water:
| Malting effluent | Acetic acid content in the raw wastewater (mM) | H 2(ml/33ml substratum) | Maximum hydrogen-producing speed (ml H 2/L·h) | Substrate conversion efficiency | Initial ph value | Final pH value | The OD value |
| 92.8 | 31±3 | 93 | 65.61％ | 7.00 | 10.26 | 4.21 |
Above data declaration, the red bacterium of class ball can be put hydrogen in malting effluent, and effect is better.The product hydrogen situation of the red bacterium of class ball in domestic sludge, rotten milk
Contain lot of organic acids in the domestic sludge, and certain density butyric acid arranged in the putrid milk, domestic sludge and corrupt milk through with handle after organic acid content see the following form:
| Acetate (mM) | Propionic acid (mM) | Butyric acid (mM) | Initial p H value | Final pH value | The OD value | |
| Mud | 25 | 13 | 6.9 | 6.35 | 9.25 | 4.14 |
| Milk | 33 | / | 65.8 | 5.73 | 8.85 | 6.30 |
Domestic sludge and corrupt milk is centrifugal, in the corrupt milk of domestic sludge and 2.7 times of dilutions, add basic salt respectively, VITAMIN, phosphoric acid buffer, L-L-glutamic acid transfers pH value to 7.00 as nutrient solution.Adding the 33mL nutrient solution in the 35mL test tube, is the light source irradiation 7 days of 4000-5000Lux with illumination intensity.
Though acetate, butyric acid are arranged as carbon source in domestic sludge and the corrupt milk, and the red bacterial growth of class ball is all right, produces hydrogen hardly.Suspect and contain the material that suppresses the red Hydrogen Production by Bacteria of class ball in above-mentioned two kinds of waste water.Because NH
4 +Can suppress hydrogen production with photosynthetic bacteria,, be [NH so checked ammonia nitrogen value wherein
4 +]=0.7523mM not in suppress producing the scope of hydrogen, does not lie in and is subjected to " ammonium inhibitions " so photosynthetic bacterium does not produce the reason of hydrogen, and concrete reason still needs further analysis.
(1), in substratum, adds molybdenum, iron ion can improve the hydrogen generation efficiency of the red bacterium of class ball from above-mentioned experimental data.Work as Mo
6+Concentration produces hydrogen total amount, maximum hydrogen-producing speed and substrate conversion efficiency and all reached the peak, and upgrowth situation is best when 3.5 μ M.
Work as Fe
2+When concentration reached 42 μ M or 150 μ M, product hydrogen total amount, maximum hydrogen generation efficiency, substrate conversion efficiency peaked, wherein again with Fe
2+Concentration is that the substratum effect of 150 μ M is best, the hydrogen-producing speed maximum, but cost is also bigger.
Both compare, and the red bacterium of class ball is at 150 μ M Fe
2+Substratum in hydrogen-producing speed a little less than at 3.5 μ M Mo
6+Substratum, but produce the hydrogen total amount and substrate conversion efficiency bigger.
(2) can draw from above-mentioned data: malting effluent can be used as the cheap carbon source of the red bacterium photosynthetic hydrogen production of class ball.Although but it does not produce hydrogen in corrupt milk and domestic sludge--all contain abundant small molecular organic acid in this two classes waste water.Got rid of and suppressed by ammonium, concrete reason remains further to be inquired into.
Claims (6)
1, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output, its step is as follows:
A) select bacterial classification
The red bacterium of class ball (Rhodobacter sphearoides) the SH-2 bacterial strain of selecting for use; Provide by Shanghai school of life and health sciences plant physiology ecological Studies institute of Chinese Academy of Sciences molecule microorganism open laboratory.It can utilize oxysuccinic acid, acetate, butyric acid etc. to carry out anaerobism illumination to put hydrogen;
B) cell cultures
The RCVB substratum is by an amount of carbon source, nitrogenous source, and basic salt, phosphoric acid buffer, VITAMIN is formed.Adjust pH to 7.00; Insert seed liquor, 30 ± 2 degrees centigrade of culture temperature were cultivated 1-2 days with 160r/min on shaking table;
C) Wei Liangyuansu adding
In substratum, add an amount of molybdenum ion and iron ion;
D) wastewater treatment
To add basic salt, VITAMIN, phosphoric acid buffer, L-L-glutamic acid, adjust pH to 7.00, sterilization after domestic sludge, rotten milk or the pre-treatment of malting effluent process.
2, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output according to claim 1 is characterized in that: described Fe
2+Ion is selected 0 μ M～300 μ M for use.
3, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output according to claim 2 is characterized in that: described Fe
2+Ion is selected 42 μ M～150 μ M for use.
4, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output according to claim 3 is characterized in that: described Fe
2+Ion is selected 150 μ M for use.
5, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output according to claim 1 is characterized in that: described Fe
2+Ionic concn is 0 μ M～100 μ M.
6, a kind of method that improves hydrogen production with photosynthetic bacteria speed, output according to claim 5 is characterized in that: described Fe
2+Ionic concn is 3.5 μ M.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343643A (en) * | 2019-08-07 | 2019-10-18 | 陕西科技大学 | One plant of hydrogenlike silicon ion and its fermentation and hydrogen production methods and applications |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343643A (en) * | 2019-08-07 | 2019-10-18 | 陕西科技大学 | One plant of hydrogenlike silicon ion and its fermentation and hydrogen production methods and applications |
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Open date: 20081231 |