CN103773714B - The application of a kind of photosynthetic bacterium in sea farming water correction - Google Patents
The application of a kind of photosynthetic bacterium in sea farming water correction Download PDFInfo
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- CN103773714B CN103773714B CN201310730681.2A CN201310730681A CN103773714B CN 103773714 B CN103773714 B CN 103773714B CN 201310730681 A CN201310730681 A CN 201310730681A CN 103773714 B CN103773714 B CN 103773714B
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Abstract
The present invention relates to the Application Areas of microorganism in sea farming water correction, specifically a kind of photosynthetic bacterium SC01 and application thereof.Does is photosynthetic bacterium hydrogenlike silicon ion (Rhodobacter? sphaeroides), preserve center preservation on September 25th, 2013 at China Microbiological, does is deposit number CGMCC? No.8248.Described photosynthetic bacterium can be applicable to process the pollutent produced in sea farming process.Bacterial strain of the present invention has simple and feasible screening process, produces and accumulating convenience, the features such as result of use is obvious.Have a good application prospect.
Description
Technical field
The present invention relates to the Application Areas of microorganism in sea farming water correction, specifically a kind of photosynthetic bacterium SC01 and application thereof.
Background technology
The mariculture industry of China is through the fast development of many decades, play a part very important in coastal economy and even the national economic development, with the fast development of mariculture industry, the process problem of the disease in breeding process and breeding wastewater becomes increasingly conspicuous, and becomes one of bottleneck of restriction industry sustainable health development.Particularly breeding wastewater can not get effectively process for a long time and directly arranges sea.Due in the seawater after cultivation, the rich in nutrition content such as the Ammonia In Sea Water that bait remains, the ight soil of aquaculture organism etc. causes, nitrite nitrogen, phosphoric acid salt, arbitrarily row sea easily causes growing of the harmful organisms such as algae.When envrionment conditions is suitable, some breed the outburst of red tide algae rapidly, usually cause the sharply decline of extra large water oxygen level, cause the aquaculture organism mortality of mariculture area, simultaneously, owing to containing the objectionable impuritiess such as microbiotic, sanitising agent, sterilant in breeding wastewater, arbitrarily the offshore even minimizing of neritic organism diversity or disappearance is likely caused in row sea, causes a series of serious ecological problems such as aqueous desert.
Therefore, develop some based on microbial technique and can be used in sea farming, be both conducive to aquaculture organism health, and had the probiotics that can carry out cultivation water improvement, greatly can alleviate generation and the development of the problems referred to above.Thus be conducive to reducing cultivation consumption, reduce row's sea wind danger, increase farmers' income, promote long-term stability and the Sustainable development of industry.
Photosynthetic bacterium one class can utilize the organism such as the sulfide of occurring in nature, ammonia to carry out photosynthetic microorganism as hydrogen donor carbon source of holding concurrently using light as the energy, under anaerobism illumination or aerobic dark condition.Photosynthetic bacterium is the prokaryotic organism earth occurring the earliest have original luminous energy synthetic system.In long-term evolutionary process, photosynthetic bacterium defines the metabolic system of oneself uniqueness, in newtype drug, function enzyme and environmental improvement, have potential scientific research and using value.
In aquaculture, people have started to utilize photosynthetic bacterium to carry out the improvement of cultivation water and the practice of Disease management aspect, but lack the theoretical direction of system.Bacterial classification is selected, the usage quantity of photosynthetic bacterium, the aspects such as use opportunity still do not have systematic theoretical direction at present, in production and use procedure, still there is larger blindness.
Summary of the invention
The invention provides photosynthetic bacterium SC01 and the application in sea farming water correction thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of photosynthetic bacterium SC01, photosynthetic bacterium taxonomy called after hydrogenlike silicon ion (Rhodobactersphaeroides), on September 25th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8248.Depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Described strain culturing mode is:
1) sampling and process: get mud 5-10g residual at the bottom of plant area of turbot fry breeding factory waterways bottom seawater, pool wall dirt settling, pool bottom sludge, nursery and culturing pool bottom seawater, dirt settling, pool wall and pond respectively, add the sterilizing seawater of 50-100ml, be placed in the aseptic triangular flask of 150-300ml, abundant concussion 10-15min, remove the large particulate matter such as weeds, rock, be prepared into sample suspension liquid.
2) enrichment culture: sample thief suspension liquid 5-8mL, adds the aseptic enrichment culture liquid (KH containing 35-50mL
2pO
40.025-0.5g/L, yeast extract paste 0.5-1.5g/L, Tryptones 0.02-0.05g, CaCl0.03-0.08g, NaAc0.5-1.5g/L, NH
4cl0.1-0.3g/L, MnSO
40.025-0.05g, MgSO
40.025-0.05g, FeSO
40.002-0.005g, pH value 6.8-7.4, be settled to 1000mL with distilled water) 100-150ml triangular flask in, add 10-15ml paraffin oil, sealed membrane seal.28-30 DEG C, 1500-3000lux illumination cultivation.Every 48h observes nutrient solution colour-change.This enrichment culture process continues to 30-60 days.
3) separation and Culture: when enrichment culture liquid obviously presents redness, get 2-5ml, adds the liquid separation culture medium (K of 30-50ml
2hPO
40.025-0.5g/L, KH
2pO
40.025-0.5g/L, yeast extract paste 0.5-1.5g/L, NaAc0.5-1.5g/L, NH
4cl0.1-0.3g/L, Glu L-glutamic acid 0.002-0.005g, pH value 6.8-7.4, is settled to 1000mL with distilled water), 28-30 DEG C, 1500-3000lux illumination, 3-5d is cultivated in 150-300rpm concussion.Now nutrient solution becomes scarlet by the naked eye, gets this nutrient solution of 50-200ul and is coated on solid separation culture medium flat board.Select red colonies, repeat line and be separated, to obtaining the consistent pure growth of colonial morphology, called after SC01 also preserves.Analyze through Physiology and biochemistry and 16srDNA, determine that SC01 is a strain class ball photosynthetic bacterium.
This photosynthetic bacterium is a kind of hydrogenlike silicon ion (Rhodobactersphaeroides).Gramstaining is negative, without gemma, without pod membrane.On LB flat board, bacterium colony becomes red, circular, and intermediate projections is smooth moistening.
Photosynthetic bacterium of the present invention is separated from turbot fry breeding factory and obtains.This photosynthetic bacterium gramstaining is negative, without gemma, without pod membrane.On LB flat board, bacterium colony becomes red, circular, and intermediate projections is smooth moistening.16SrDNA sequencing is carried out to the bacterial strain obtained, bacterial universal primers 27F and 1492R is adopted to carry out pcr amplification, to get PCR primer 2 ~ 5 μ l and carry out 1wt% agarose gel electrophoresis, ultraviolet detection after EB dyeing. the PCR primer of cutting glue recovery 1.5kb size with agarose gel purification test kit (purchased from the precious biotech firm in Dalian) carries out sequencing analysis.The 16SrDNA sequence result of bacterial strain logs in GenBank, and carry out tetraploid rice by the 16SrDNA sequence in BLAST and GenBank, by the 16SrDNA of similar strain obtained, utilize MEGA4.0 software, phylogenetic tree construction, analyzes the evolutionary degree of each isolated strains.Through qualification: be a kind of be hydrogenlike silicon ion (Rhodobactersphaeroides).On September 25th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8248.The GenBank number of logging in of this bacterial strain 16SrDNA is: KF791043.
The application of photosynthetic bacterium SC01, described photosynthetic bacterium can be applicable to process the pollutent produced in sea farming process.
Described photosynthetic bacterium put in breeding seawater and then improves water inlet and the discharge water of used in mariculture water, improving the survival rate of sea farming biology simultaneously.
The advantage that the present invention has: photosynthetic bacterium of the present invention is separated from the turbot fry breeding factory of Yantai City and obtains.This photosynthetic bacterium in the Main Economic animal cultivation processes such as turbot because the process after bait remains, ight soil etc. causes water quality deterioration has good effect.To low aeration rate or the existence without cultivated animals main under aeration significant.Can be used for the improvement of water quality and the process of breeding wastewater in aquaculture process, have a good application prospect.
Embodiment
Be described in detail around bacterial strain of the present invention and embodiment, but embodiments of the present invention are not limited thereto.
The screening of photosynthetic bacterium
The screening of embodiment 1 photosynthetic bacterium and cultivation
1) sampling and process: get mud 8g residual at the bottom of plant area of turbot fry breeding factory waterways bottom seawater, pool wall dirt settling, pool bottom sludge, nursery and culturing pool bottom seawater, dirt settling, pool wall and pond respectively, add the sterilizing seawater of 100ml, be placed in the aseptic triangular flask of 300ml, abundant concussion 15min, remove the large particulate matter such as weeds, rock, be prepared into sample suspension liquid.
2) enrichment culture: sample thief suspension liquid 5mL, adds the aseptic enrichment medium (KH containing 50mL
2pO
40.5g/L, yeast extract paste 1.5g/L, Tryptones 0.05g, CaCl0.05g, NaAc1.0g/L, NH
4cl0.2g/L, MnSO
40.05g, MgSO
40.025g, FeSO
40.002g, pH value 7.2, is settled to 1000mL with distilled water) 150ml triangular flask in, add 10ml paraffin oil, sealed membrane seals.28 DEG C, 2000lux illumination cultivation.Every 48h observes nutrient solution colour-change.This enrichment culture process continues to 30 days.
3) isolation identification: when enrichment culture liquid obviously presents redness, get 2ml, adds the liquid separation culture medium (K of 50ml
2hPO
40.5g/L, KH
2pO
40.5g/L, yeast extract paste 1.5g/L, NaAc1.0g/L, NH
4cl0.2g/L, L-glutamic acid 0.005g, pH value 7.4,1000mL is settled to distilled water), 28 DEG C, 2000lux illumination, 300rpm shaking culture 3d, now nutrient solution becomes scarlet by the naked eye, gets this nutrient solution of 100ul and is coated on solid separation culture medium flat board (K
2hPO
40.5g/L, KH
2pO
40.5g/L, yeast extract paste 1.5g/L, NaAc1.0g/L, NH
4cl0.2g/L, L-glutamic acid 0.005g, agar powder 10g, pH value 7.4, is settled to 1000mL with distilled water.) on, 28 DEG C, 2000lux illumination, is inverted and cultivates 7d, selects red colonies, and repeat line on above-mentioned solid medium and be separated, to obtaining the consistent pure growth of colonial morphology, called after SC01 also preserves.Analyze through Physiology and biochemistry and 16srDNA, determine that SC01 is hydrogenlike silicon ion (Rhodobactersphaeroides).On September 25th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8248.
4) fast culture: get 50ul-80 DEG C of frozen SC01 and be seeded to 10ml fast culture media (K
2hPO
40.25g/L, KH
2pO
40.25g/L, yeast extract paste 3.0g/L, NaAc2.0g/L, NH
4cl1.0g/L, pH value 6.8, is settled to 1000mL with Chen Haishui (removing throw out after staticly settling 14 days).) in, 28 DEG C, 300rpm, cultivates 120h to OD under natural lighting
600to 1.0, in this, as seed liquor, according to 5%(volume ratio) seed liquor is inoculated in fast culture media by inoculum size, and 28 DEG C, 150rpm, cultivates 120h under natural lighting.
Embodiment 2:SC01 is on the impact of the objectionable impuritiess such as ammonia nitrogen in breeding seawater
Get 0 respectively, 0.1,0.5,2,5,10mlOD
600to the SC01 nutrient solution of 1.0, be added in the storage pond, holding pond of 50L seawater respectively, wherein, think and add SC01 nutrient solution group non-control group, in each culturing pool, put into the healthy turbot that 10 health total lengths are 6-8cm.Indoor normal illumination, every day, conventional granulates feed addition was by every bar fish 0.5-0.8g, divided and added for 3 times.Respectively at add the 4th after SC01,18,28,40h water sampling, pH in test water, the change (see table 1) of ammonia nitrogen, nitrite nitrogen, dissolved oxygen, activared carbon sulfur salts contg, by visible at each tested time point in table 1, the pH of each culturing pool and dissolved oxygen change not obvious.But along with the carrying out of breeding process, ammonia nitrogen in aquaculture water, nitrite nitrogen, the content of reactive phosphate raises gradually.
Simultaneously the various objectionable impurities of passing content in the seawater in time raises gradually as shown in Table 1, especially obvious with ammonia nitrogen.Dosage be 0.1,0.5, in the culturing pool of 2.0mlSC01 nutrient solution, every Testing index of seawater is all less than the control group not adding SC01 nutrient solution, illustrates that appropriate SC01's has the control being beneficial to ammonia nitrogen in aquaculture water, nitrite nitrogen and reactive phosphate.But when the dosage of SC01 increases to 5-10ml, each monitoring index in seawater goes up not down, even higher than the control group not adding SC01 nutrient solution.The visible bacterial number owing to adding is too many, and the metabolite of bacterium and thalline itself make water body be secondary polluted on the contrary.
Table 1SC01 is on the impact of objectionable impurities in breeding seawater
NH
4-N（μg/L）
NO
2-N（μg/L）
PO
4-P（μg/L）
Embodiment 3: the impact on turbot mortality ratio under without aeration condition SC01
Get 0,0.1,0.5,2,5,10mlOD
600to the SC01 nutrient solution of 1.0, be added in the storage pond, holding pond containing 50L seawater respectively, not add SC01 nutrient solution group for control group, putting into 10 bodies in each culturing pool respectively long is 6-9cm, and mean body weight is the healthy turbot seedling of 6-8g.Indoor normal illumination, each culturing pool per minute air flow is 6-8L, the dosage of conventional granulates feed according to every fish 0.8-1.0g every day, in every day 7:00,13:00,19:00 divide and add for three times.After adding SC00148h, aeration 5-8h is stopped to all experimental group and control group, observe the death condition (see table 2) of cultured fishes.Under stuffiness condition, the dissolved oxygen content in culturing pool declines rapidly.After stopping aeration in 5h, the turbot seedling do not added in the culturing pool of SC01 is very fast all dead, and in the culturing pool adding SC01 nutrient solution the death time of turbot seedling obviously postpone; The survival rate of turbot seedling exists directly related with SC01 add-on: in the 8h after stopping aeration, and add-on is none death of turbot seedling in the storage pond, holding pond of 1%; Add-on is the mortality ratio of turbot seedling in the storage pond, holding pond of 4% is 20%.And when the addition of SC01 be less than 0.2% or be greater than 10% time, good protected effect could not be played to turbot seedling.Therefore, under suitable concentration, the existence of SC01 can consume the organism such as swill, ight soil in culturing pool, avoids the too high death causing aquaculture organism of the concentration of narmful substances such as ammonia nitrogen.But too much the existence of SC01 may cause again gathering of opsonigenous substances or by product, unfavorable to aquaculture organism.Therefore to pay special attention to working concentration in actual use procedure, avoid having too much of a good thing.
Table 2 without turbot seedling under aeration condition in the mortality ratio of 8h
Claims (3)
1. a photosynthetic bacterium SC01, is characterized in that: photosynthetic bacterium be hydrogenlike silicon ion (
rhodobactersphaeroides), preserve center preservation on September 25th, 2013 at China Microbiological, deposit number is CGMCCNo.8248.
2. an application of photosynthetic bacterium SC01 according to claim 1, is characterized in that: described photosynthetic bacterium is applied to the pollutent produced in process sea farming process.
3. by the application of photosynthetic bacterium SC01 according to claim 2, it is characterized in that: described photosynthetic bacterium put in breeding seawater and then improves water inlet and the discharge water of used in mariculture water, improving the survival rate of sea farming biology simultaneously.
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| CN106676050A (en) * | 2017-03-17 | 2017-05-17 | 烟台寒武纪生物科技有限公司 | Method and application for preparing photosynthetic bacterium through photonic fermentation |
| CN115386521B (en) * | 2022-09-20 | 2024-01-05 | 广东海洋大学 | Photosynthetic bacteria and application thereof in prawn culture |
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