CN102212491A - Simple culture method of photosynthetic bacteria with high cell concentration - Google Patents
Simple culture method of photosynthetic bacteria with high cell concentration Download PDFInfo
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Abstract
The invention provides a simple culture method of photosynthetic bacteria with a high cell concentration. The components of the culture medium of photosynthetic bacteria are simplified. The main components of the culture medium comprise the following steps: 0.3% of sodium acetate, 0.6% of sodium bicarbonate, 0.1% of NH4Cl, 0.1% of NaCl, 0.02% of MgSO4.7H2O, 0.05% of K2HPO4, 0.02% of KH2PO4, 0.005% of CaCl2 and 0.05% of yeast extract. The production process of photosynthetic bacteria is also simplified. The invention has the following advantages: the composition and components of the culture medium are optimized, and the production cost can be reduced under the premise of ensuring the concentration of a bacteria solution; the culture process of photosynthetic bacteria is simplified to ensure that the scale production of photosynthetic bacteria has high operability; and the required equipment is simple and culturists can produce photosynthetic bacteria at home so as to greatly reduce the use cost of photosynthetic bacteria. The photosynthetic bacteria produced by the method can be used to adjust the algal phase of a water body, effectively reduce the ammonia-nitrogen content and increase the content of dissolved oxygen in the water body.
Description
Technical field
The present invention relates to biotechnology and microorganism Application Areas, the simple and easy cultural method and the fermented liquid that relate to a kind of high cell concentration photosynthetic bacteria are specifically regulated the application of water quality.
The production method of concrete detailed a kind of simple and practical high cell concentration photosynthetic bacteria of saying so and fermented liquid are regulated application on the water quality in aquaculture.
Background technology
In recent years, along with culture fishery cultured area and production-scale continuous expansion, and the rapid increase of cultured output and culture improving constantly of intensification degree, breeding production itself has produced pollution to a certain degree to the breed water environment; In addition, the breeding water water source has further been polluted in discharging such as the excess of human lives's sewage, various trade effluent, agriculture water-break etc., causes cultivation water to descend, and breeding environment worsens.Culture water pollution and caused enormous economic loss not only for the aquatic products aquaculture, and, cause very big harm to people ' s health because objectionable impuritiess such as the interior microbiotic of cultivated animals body are residual.The general purpose that adopts probiotics to reach the breeding water recycle or reduce harmful substance contents in the waste discharge in the at present external high-density intensive culture.Use commonplace probiotics that photosynthetic bacterium, (instead) nitrobacteria are arranged in the aquaculture, based on the compound formulation of genus bacillus, EM composite fungus agent etc.Wherein photosynthetic bacterium (photosynthesis bacteria is called for short PSB) can utilize organic acid, ammonia, hydrogen sulfide, alkane and low molecule organic matter to carry out photosynthesis as carbon source and hydrogen donor in the growth and breeding process, degrade simultaneously and remove excessive organism and objectionable impurities in the water body environment, prevent body eutrophication, improve the dissolved oxygen amount of water body, thereby purify water, improve the growing environment of aquatic animal.Therefore, photosynthetic bacterium is to use relatively early also to be to use commonplace a kind of probiotics in aquaculture.
The mode of cultivating photosynthetic bacterium at present both at home and abroad generally has the dynamic cultured continuously of the airtight photoreactor of employing and adopts the container of printing opacity to leave standstill airtight cultivation.Chinese invention patent application number 200910039524.0 " a kind of production method of additive of bait for photosynthetic bacteria aquaculture " is though can guarantee the photosynthetic bacterium fermented liquid steady quality of production, but need full-automatic photosynthetic microorganism fermentor tank, and output also is subjected to the restriction of fermentor tank tank body, and production cost is higher will to limit applying of this technology.The patent No. is that 03220904.5 " vertical photosynthetic bacterium culture apparatus " is though saved certain place, also can utilize source of artificial light, but this device needs specific customization, maneuvering ability is poor, as utilize sunlight then to need the strong glass room place of similar light transmission, utilize artificial lighting fully as utilizing, then energy consumption is more.Analyze from substratum; carbon source accounts for more than 50% of dry weight of photosynthetic bacterium; the research of therefore relevant photosynthetic bacterium carbon source is also more; general with the main organic carbon source of sodium acetate as substratum; replace sodium acetate with ethanol among the patent No. 200410009803.x " a kind of method of cultivating the superelevation cell concentration photosynthetic bacteria "; the photosynthetic bacterium mycetocyte concentration of preparation can reach every milliliter more than 4,500,000,000; but the alcoholic acid price is higher comparatively speaking; and along with the aggravation of energy dilemma, the alcoholic acid price can be soaring day by day.Therefore, how simplifying culture apparatus and production process, reduce production costs, make the raiser " afford to use " photosynthetic bacterium, is the key of photosynthetic bacterium energy large scale application.
Summary of the invention:
The object of the present invention is to provide a kind of simple and easy cultural method of high cell concentration photosynthetic bacteria, guaranteeing to reduce the production cost of photosynthetic bacterium under the photosynthetic bacterium product quality premise.
The technical scheme that the present invention takes is:
A kind of simple and easy cultural method of high cell concentration photosynthetic bacteria is characterized in that: the inorganic carbon source of interpolation--sodium bicarbonate as photosynthetic bacterium main carbon source, the medium component and the content of simplification are as follows: sodium acetate 0.3%, sodium bicarbonate 0.8%, NH
4Cl 0.1%, and NaCl 0.1%, MgSO
47H
2O 0.02%, K
2HPO
40.05%, KH
2PO
40.02%, CaCl
20.005%, yeast extract paste 0.05%.
Described culture medium prescription, developing medium is tap water or cleaning underground water, the tower culture apparatus of indoor employing multicolumn, the bacterial classification of this laboratory preservation of the bacterium amount that connects inoculation by 20%, temperature is controlled at about 30 ℃, when the intensity of illumination of source of artificial light was about 3000lux, the bacterial concentration of cultivating 7d can reach every milliliter more than 2,000,000,000.
Described culture medium prescription, can be directly with tap water or cleaning underground water configuration substratum, outdoor employing white plastic bucket, the bacterial classification of this laboratory preservation of the bacterium amount that connects inoculation by 20%, daily mean temperature is more than 25 ℃, the bacterial concentration of cultivating 7d can reach every milliliter more than 4,000,000,000, and bacterium liquid can be preserved more than 1 year.
Utilize the production method of described high cell concentration photosynthetic bacteria, the application of the photosynthetic bacterium of production on the aquaculture water quality regulation.
The invention has the advantages that: optimized the composition and the composition of substratum, under the prerequisite that guarantees bacterial concentration, reduced production cost; Simplified the culturing process of photosynthetic bacterium, made the large-scale production manipulative capability of photosynthetic bacterium strong, required equipment is simple, and the raiser can produce at home voluntarily, thereby reduces the use cost of photosynthetic bacterium greatly.Algae phase in the adjustable water saving body of photosynthetic bacterium that the present invention produces effectively reduces ammonia-nitrogen content, improves the content of dissolved oxygen in the water body.
Description of drawings:
Fig. 1 be in the embodiment of the invention 4 photosynthetic bacterium to the influence of oxygen in water content.Wherein X-coordinate is the time, unit: day; Ordinate zou is the content of dissolved oxygen in the water body, the mg/L of unit.The demonstration dissolved oxygen content is passed in time to present and is fallen after rising among Fig. 1, again trend of rising.Dissolved oxygen content reaches maximum value at test 7d in the water body, and reducing subsequently is because 7d has closed light source evening, the new oxygen that produces seldom, and original oxygen is discharged in the air along with the stirring of sampling.The rising that dissolved oxygen content in the water body is described is carried out photosynthesis with a large amount of breedings of algae in the water body, and to discharge oxygen relevant, can find a large amount of bubbles at the water surface in the process of the test.
Fig. 2 be in the embodiment of the invention 4 photosynthetic bacterium to the influence of frustule concentration in the water.Wherein X-coordinate is for handling; Ordinate zou is a frustule concentration in the water body, unit/milliliter.Show among Fig. 2 that the frustule concentration of processing is near 2 times of contrast.
Fig. 3 be in the embodiment of the invention 4 photosynthetic bacterium to the influence of ammonia-nitrogen content in the water.Wherein X-coordinate is the time, unit: day; Ordinate zou is an ammonia nitrogen content in the water body, the mg/L of unit.Fig. 3 shows that As time goes on ammonia nitrogen sharply reduces, and the rapid degraded of ammonia nitrogen may be relevant with the algal bloom in the water body in the water body.Wherein the degradation rate of Chu Liing is very fast relatively, and final ammonia nitrogen concentration is also minimum, and the input that microbial inoculum is described has play a part certain to the degraded of ammonia nitrogen.
Specific embodiments:
Below in conjunction with the simple and easy cultural method of specific embodiments, be described further high cell concentration photosynthetic bacteria of the present invention.
A kind of production method of high cell concentration photosynthetic bacteria:
1. photosynthetic bacterium bacterial classification
The bacterial classification of production usefulness separates the Rhodopseudomonas palustris Rhodopseudomonas palustris of the Rhodospirillaceae Rhodopseudomonas of preserving for this laboratory.
2. photosynthetic bacteria culture medium and developing medium
Medium component that the present invention relates to and content: sodium acetate 0.3%, sodium bicarbonate 0.8%, NH
4Cl 0.1%, NaCl0.1%, MgSO
47H
2O 0.02%, K
2HPO
40.05%, KH
2PO
40.02%, CaCl
20.005%, yeast extract paste 0.05%.
Photosynthetic bacterium produces must be with cleaning fresh water, and the pH value 7.0～8.5 of water is advisable, and assorted bacterial content is few, is lower than 100/milliliter.Can be directly with tap water or underground water.Low best of ground water hardness.Chloride tap water will be placed 24～48h, or add in an amount of Sulfothiorine and after can use.
3. the device of the scale production of microbial inoculum
Indoor production can be used the tower culture apparatus of multicolumn, and outdoor production can be directly with the wide aperture cylinder of 25L or 50L, and the diluent with 20～30 times of antiformins before using sprays inwall, cleans with clear water then.
4. inoculum size
It is fermentation period that inoculum size size mainly influence the time that bacterium liquid reaches maximum biomass, and to final photosynthetic bacterium biomass influence not quite.Therefore take all factors into consideration each side factors such as cost in the scale operation, adopt 20% inoculum size more suitable.
5. culture condition
1. temperature: photosynthetic bacterium is well-grown in 25～35 ℃ temperature range, and optimum growth temperature is 30 ℃.
2. intensity of illumination: the suitableeest intensity of illumination of photosynthetic bacterium growth is 2500～5000lux, can select natural lighting to cultivate in order to save cost when scale operation.
3. pH value: photosynthetic bacterium all can normal growth between pH value 6.5～9.0, and growth is best when pH8.0.When scale production such as the pH of institute's water be between 6.5～8.5, can uncomfortable pH.
6. culture cycle
The tower photosynthetic bacterium reactor of indoor multicolumn, temperature be at 30 ℃, pH8.0～8.5, and inoculum size is 20%, and illumination cultivation (illumination is more than 2500lux) can reach fermentation termination at 7～9d, and bacterial concentration is generally 2.0 * 10
9Individual/milliliter.Outdoor cultivation, daily mean temperature is more than 25 ℃, and weather is fine, and general 5～7d can reach fermentation termination, and bacterial concentration is generally 4.0 * 10
9Individual/more than the milliliter, the culture effect of natural lighting is better than source of artificial light.
Application on the aquaculture system water quality regulation of photosynthetic bacterium.
Simulation laboratory test shows that the photosynthetic bacterium that produces among the present invention can make useful algae such as ball algae in the water body, grid algae be multiplied, along with a large amount of breeding ammonia-nitrogen contents of algae reduce more than 90%, and being elevated to more than the 10mg/L of dissolved oxygen.
The field application test shows that the photosynthetic bacterium that produces among the present invention can make useful algae such as ball algae in the water body, grid algae increase, and ammonia-nitrogen content reduces more than 40%, and the content rising of dissolved oxygen is more than 8.5%.
Determining of photosynthetic bacteria culture medium and culture condition
Photosynthetic bacteria culture medium (g/L): CH
3COONa 3.0, NaHCO
38.0, NH
4Cl 1.0, and NaCl 1.0, MgSO
47H
2O0.2, K
2HPO
40.5, KH
2PO
40.2, CaCl
20.05, yeast extract paste 0.5, pH nature.
Temperature: will close bacterium and be transferred in the substratum, and place 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ following anaerobism illumination (the about 3000lux of light intensity) to cultivate respectively, and test 7d statistics bacterium number, and the results are shown in Table 1 by 10% inoculum size.
Table 1 temperature is to the influence of photosynthetic bacterium growth
As shown in Table 1, well-grown in 25～35 ℃ temperature range, and difference is little, and the photosynthetic bacterium optimum growth temperature is 30 ℃, and photosynthetic bacterium growth is subjected to obvious inhibition when temperature reaches 40 ℃.Therefore when scale production at ordinary times temperature being controlled at 25～35 ℃ gets final product.
Intensity of illumination: will close bacterium and be transferred in the substratum, and place the following 30 ℃ of cultivations of different light intensity, and test 7d statistics bacterium number by 10% inoculum size.
Table 2 intensity of illumination is to the influence of photosynthetic bacterium growth
As shown in Table 2, the suitableeest intensity of illumination of photosynthetic bacterium growth is 2500～5000lx, and adjustment intensity of illumination that can be suitable according to the internal diameter of photosynthetic bacterium reactor can select natural lighting to cultivate in order to save cost when scale operation in process of production.
PH value: will close bacterium and be transferred in the different initial pH substratum, and place 30 ℃ of illumination anaerobism (light intensity is about 3000lux) to cultivate, and test 7d statistics bacterium number by 10% inoculum size.
Table 3 initial pH value is to the influence of photosynthetic bacterium growth
As shown in Table 3, photosynthetic bacterium is equal energy normal growth between pH value 6.5～9.0, and growth is best when pH8.0.When scale production is between 6.5～8.5 as the water pH of institute, can uncomfortable pH.
Indoor open culture test
The collocation method of photosynthetic bacteria culture medium is with embodiment 1, the tower photosynthetic bacterium culture apparatus of multicolumn is cleaned, with the bacterial classification that connects 20% in the post, add 80% nutrient solution then, incandescent light with 100w gives source of artificial light, the distance of adjusting bulb makes intensity of illumination be about 3000lux, and temperature is controlled at about 30 ℃, and every day, bacterial concentration was surveyed in sampling.
Table 4 photosynthetic bacterium concentration over time (10
8Individual/milliliter)
As shown in Table 4, under above-mentioned culture condition, the culture cycle of photosynthetic bacterium is 7d, and bacterial concentration can reach every milliliter more than 2,000,000,000, and bacterium liquid can be preserved more than 1 year.When actual production because temperature and light intensity different slightly variant.
Outdoor open culture test
The collocation method of photosynthetic bacteria culture medium is with embodiment 1,25L white drum is cleaned, as be new bucket, can directly use after the cleaning, as using, then the diluent with 20～30 times of antiformins sprays inwall, clean with clear water then, with connecing 20% bacterial classification in the bucket, add 80% nutrient solution then, lid is built to be placed under the outdoor sunlight cultivate.Daily mean temperature is more than 25 ℃, and weather is fine, and every day, bacterial concentration was surveyed in sampling.
Table 5 photosynthetic bacterium concentration over time (10
8Individual/milliliter)
Test-results shows, in summer, and general continuous 5 fine days or 7 interrupted cumulative fine days, photosynthetic bacterium can reach maximum bacterial concentration, and bacterial concentration generally can reach every milliliter more than 4,000,000,000, and bacterium liquid can be preserved more than 1 year.
Photosynthetic bacterium water quality regulation simulation laboratory test
3.5L glass jar in fill it up with culturing pool after the husky filter, add NH in the water
4Cl, making ammonia nitrogen concentration is 80mg/L.Add the somatic cells after centrifugal in the cylinder, make bacteria containing amount in the water 10
9Individual/L, place the incubator temperature to be made as 25 ℃ in cylinder, give artificial lighting, light intensity is about 2500lux.Add behind the bacterium the 1st, 2,3,4,6,7,8,10 day and measure water dissolution oxygen and ammonia nitrogen content, and frustule concentration and the microscopy surveyed in the water in the 10th day in test.
By Fig. 1～Fig. 3 as can be known, photosynthetic bacterium can make dissolved oxygen content rising in the water body, and ammonia-nitrogen content reduces, and frustule concentration is increased, and microscopy is found to be mainly the ball algae, grid algae, useful algae such as little Nitzschia closterium minutissima.
Embodiment 5
Photosynthetic bacterium water quality regulation field test
Fishes and shrimps in fish pond, Xiqing District worker-peasant alliance farm, Tianjin overlap to support and carry out the photosynthetic bacterium test, in the whole culture-cycle, add the treating pond of photosynthetic bacterium and throw bacterium altogether 6 times, press 1～2 kilogram/mu amount full pool spilling head at every turn, do not use any microbiotic and sterilant, the contrast pond is by daily management.The result shows that behind the throwing bacterium 4d, the ammonia-nitrogen content in contrast pond has raise 4.0%, and dissolved oxygen content has reduced by 2.0%; And the treating pond ammonia-nitrogen content has reduced by 41.8% before adding bacterium, and dissolved oxygen content has raise 8.5%.Under the situation of identical water source and identical feed injected volume, use the pond water quality of photosynthetic bacterium salubrious, the transparency height, the algae lover is green algas such as chlorella in the pond, planktonic quantity such as useful algae and wheel animalcule obviously increase, and have improved feed coefficient.
Table 6 contrast pond and treating pond water-quality guideline
Claims (4)
1. the production method of a high cell concentration photosynthetic bacteria is characterized in that: the inorganic carbon source of interpolation--sodium bicarbonate as photosynthetic bacterium main carbon source, the medium component and the content of simplification are as follows: sodium acetate 0.3%, sodium bicarbonate 0.8%, NH
4Cl0.1%, NaCl 0.1%, MgSO
47H
2O 0.02%, K
2HPO
40.05%, KH
2PO
40.02%, CaCl
20.005%, yeast extract paste 0.05%.
2. the production method of high cell concentration photosynthetic bacteria according to claim 1, it is characterized in that: described culture medium prescription, developing medium is tap water or cleaning underground water, the tower culture apparatus of indoor employing multicolumn, the bacterial classification of this laboratory preservation of the bacterium amount that connects inoculation by 20%, temperature is controlled at about 30 ℃, and when the intensity of illumination of source of artificial light was about 3000lux, the bacterial concentration of cultivating 7d can reach every milliliter more than 2,000,000,000.
3. the production method of high cell concentration photosynthetic bacteria according to claim 1, it is characterized in that: described culture medium prescription, can be directly with tap water or cleaning underground water configuration substratum, outdoor employing white plastic bucket, the bacterial classification of this laboratory preservation of the bacterium amount that connects inoculation by 20%, daily mean temperature is more than 25 ℃, and the bacterial concentration of cultivating 7d can reach every milliliter more than 4,000,000,000, and bacterium liquid can be preserved more than 1 year.
4. utilize the production method of described high cell concentration photosynthetic bacteria, the application of the photosynthetic bacterium of production on the aquaculture water quality regulation.
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Cited By (10)
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| CN102399735A (en) * | 2011-12-21 | 2012-04-04 | 大连洪家畜牧有限公司 | Method for culturing photosynthetic bacteria by biogas liquid |
| CN102690777A (en) * | 2012-05-29 | 2012-09-26 | 浙江荣怀环保科技有限公司 | Rapid culture method for high density of photosynthetic bacteria |
| CN102992894A (en) * | 2012-11-20 | 2013-03-27 | 蒋常德 | Preparation method for high-concentration photosynthesis bacterial manure |
| CN103087938A (en) * | 2011-11-04 | 2013-05-08 | 天津绿动植物营养技术开发有限公司 | Application of medium for rapid propagation of photosynthetic bacteria in slightly polluted water |
| CN104946569A (en) * | 2015-07-06 | 2015-09-30 | 天津市农科旺生物菌培育专业合作社 | Method and application for easily and rapidly culturing agricultural high-concentration photosynthetic bacteria |
| CN104962549A (en) * | 2015-07-27 | 2015-10-07 | 厦门市科环海洋生物科技有限公司 | Rhodopseudomonas palustris synchronized concentration method and application |
| CN106635928A (en) * | 2017-02-13 | 2017-05-10 | 天津市农业生物技术研究中心 | Culture method for continuous and scaled production of photosynthetic bacteria |
| CN109810915A (en) * | 2018-04-19 | 2019-05-28 | 湛江恒兴养殖技术服务有限公司 | A kind of photosynthetic bacteria culture simplicity spreads cultivation culture medium and preparation method thereof |
| CN112126591A (en) * | 2020-05-28 | 2020-12-25 | 安吉国千环境科技有限公司 | Culture medium formula for kitchen waste recycling composite flora culture and culture method thereof |
| CN112300967A (en) * | 2020-11-16 | 2021-02-02 | 佛山市顺德区活宝源生物科技有限公司 | Photosynthetic bacteria pond head expanding culture medium and method for preparing photosynthetic bacteria liquid by using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087938A (en) * | 2011-11-04 | 2013-05-08 | 天津绿动植物营养技术开发有限公司 | Application of medium for rapid propagation of photosynthetic bacteria in slightly polluted water |
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| CN102690777A (en) * | 2012-05-29 | 2012-09-26 | 浙江荣怀环保科技有限公司 | Rapid culture method for high density of photosynthetic bacteria |
| CN102992894A (en) * | 2012-11-20 | 2013-03-27 | 蒋常德 | Preparation method for high-concentration photosynthesis bacterial manure |
| CN102992894B (en) * | 2012-11-20 | 2014-08-06 | 北京市波普克生态科技有限公司 | Preparation method for high-concentration photosynthesis bacterial manure |
| CN104946569A (en) * | 2015-07-06 | 2015-09-30 | 天津市农科旺生物菌培育专业合作社 | Method and application for easily and rapidly culturing agricultural high-concentration photosynthetic bacteria |
| CN104962549A (en) * | 2015-07-27 | 2015-10-07 | 厦门市科环海洋生物科技有限公司 | Rhodopseudomonas palustris synchronized concentration method and application |
| CN104962549B (en) * | 2015-07-27 | 2018-10-19 | 厦门市科环海洋生物科技有限公司 | A kind of Rhodopseudomonas palustris synchronizes method and the application of concentration |
| CN106635928A (en) * | 2017-02-13 | 2017-05-10 | 天津市农业生物技术研究中心 | Culture method for continuous and scaled production of photosynthetic bacteria |
| CN109810915A (en) * | 2018-04-19 | 2019-05-28 | 湛江恒兴养殖技术服务有限公司 | A kind of photosynthetic bacteria culture simplicity spreads cultivation culture medium and preparation method thereof |
| CN112126591A (en) * | 2020-05-28 | 2020-12-25 | 安吉国千环境科技有限公司 | Culture medium formula for kitchen waste recycling composite flora culture and culture method thereof |
| CN112300967A (en) * | 2020-11-16 | 2021-02-02 | 佛山市顺德区活宝源生物科技有限公司 | Photosynthetic bacteria pond head expanding culture medium and method for preparing photosynthetic bacteria liquid by using same |
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Application publication date: 20111012 |