CN103773722A - Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis - Google Patents
Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis Download PDFInfo
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Abstract
The invention relates to the technical field of biology and particularly relates to salt-tolerance bacillus subtilis with a low-temperature biological deamination function and an application of the bacillus subtilis. The bacillus subtilis DA-1 provided by the invention is classified and named as Bacillus subtilis and is preserved in China General Microbiological Culture Collection Center (CGMCC); the preservation number is CGMCC No.8292 and the preservation time is October 8th, 2013. The bacillus subtilis provided by the invention is low in cost, simple and convenient to operate and high in viable organism rate. A prepared bacillus subtilis micro-ecological preparation can be used for removing ammonia nitrogen in a eutrophic water body under a low-temperature condition, and is particularly suitable for fishery cultivation, and can be used for rapidly and effectively reducing the content of the ammonia nitrogen in the water body so as to improve the environment of an aquatic water body and bring obvious social benefits.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the Bei ﹑ processed of Fang Fa ﹑ that a kind of Xi Jun ﹑ under cold condition with high deamination activity cultivates this bacterium low-temperature biological deamidization take this bacterium as chief component composition and use the application of said preparation in the degraded of water body nitrogen.
Background technology
Nitrogen Cycling is one of composition of the of paramount importance ecosystem material cycle of occurring in nature.But the use of nitrogenous fertilizer and the development of high-density aquiculture have also caused serious environmental problem in bringing great economic benefit.In aquaculture, the eutrophication situation causing because nitrogen phosphorus element content is too high frequently occurs, except making water quality deterioration and destroying biotic balance wherein, ammonia nitrogen wherein and nitrite nitrogen have stronger toxicity to cultivated animals, particularly ammonia nitrogen (NH
4 +-N), the Chang ﹑ of Sheng that it can directly or indirectly affect fish breeds and even death, and it can directly be absorbed by plant on the one hand, increases planktonic organism amount, for fish provide abundanter nutrition bait.It is toxic to fish and other hydrocoles on the other hand, and it can destroy gill tissue, and infilters in blood, reduces blood oxygen carrying capability, and breathing function is declined.
Studies have reported that and shown that candiyeast, Nocardia bacteria, photosynthetic bacterium, nitrobacteria etc. have stronger removal ability to the ammonia nitrogen in aquaculture water, but there is the shortcomings such as inoculum size is large, application cost is high, and genus bacillus is easily separated, easily cultivation, this quasi-microorganism not only can produce the outer lytic enzyme of multiple born of the same parents, residual organism in water of decomposition, also can produce statoblast, can resist severe environment, facilitate preparation processing and accumulating, aspect reduction water body ammonia-nitrogen content and regulating water quality, having good application prospect.
Subtilis, except ammonia nitrogen is had and removed preferably ability, can also produce a large amount of extracellular enzyme classes, thereby consumes in a large number the macromole organic matter such as residual bait and animal excrements in pond.Macromole organic matter is broken down into the material such as small molecular organic acid, amino acid, can provide nutrition for photosynthetic bacterium, algae and other planktonic organism in water body, has objectively promoted the stable of micro-ecological environment; Genus bacillus also can make the enteron aisle pH of hydrocoles reduce, and produces strong active proteolytic enzyme and amylase, promotes the digestion of hydrocoles; In addition, genus bacillus field planting, in target animal enteron aisle, is taken oxygen effect by force through biology, can suppress growing and improving the immunizing power of animal of pathogenic bacteria, and final prophylactic generation finally promotes target animal growth and improves food conversion ratio.
The subtilis of having reported at present exists removing of ammonia nitrogen indifferent mostly; envrionment temperature is required to high (25 ℃ of following ammonia nitrogen removals are indifferent); bacterial strain survival rate is not high; production cost is large; bacterial strain is the problems such as denitrification activity is low in high salinity water body, have affected the application of subtilis in environment protection.
Summary of the invention
The invention discloses a strain and there is the bioactive bacterium of low temperature deamination: subtilis DA-1, this bacterium has higher ammonia nitrogen removal ability.This class bacterial strain is aerobic bacteria, can be effectively by NH in biological denitrificaion
4 +-N directly changes into self component of thalline, and more specifically, this bacterioid is a bacterioid for bacillus, in the soil being gathered, screens and obtains by contriver in Chinese lute lakeside, Tourism Resource in Zhongshan Scenic Area, Nanjing.Bacterium disclosed by the invention is subtilis DA-1, Classification And Nomenclature: subtilis, Bacillus subtilis.Its preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.8292.The preservation time: on October 08th, 2013, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese science microorganisms institute.
Under cold condition, DA-1 of the present invention still has higher ammonia nitrogen removal ability.
DA-1 of the present invention can be at 15～30 ℃, the shaking flask rotating speed of 120～250r/min, under the condition of pH5～10, in every liter of substratum containing 10～25g glucose, 1～3g Zulkovsky starch, 10～40g soybean cake powder, 20～40mg manganous sulfate, 0.1～0.4g yeast extract paste, 0.1～0.3g iron trichloride and 0.1～0.3g calcium carbonate, cultivate.
Preferred cultural method is: DA-1 of the present invention is at 30 ℃, the shaking flask rotating speed of 200r/min, under the condition of pH7.0, in every liter of substratum containing 15g glucose, 2g Zulkovsky starch, 30g soybean cake powder, 30mg manganous sulfate, 0.2g yeast extract paste, 0.1g iron trichloride, 0.1g calcium carbonate.
In above-mentioned substratum, sterilising conditions is preferred: 121 ℃ of sterilizing 20min, add in aseptic culture medium after the independent 115 ℃ of sterilizing 15min of glucose.In solid medium, need to add 1.5% agar powder.Per-cent of the present invention is all weight percentage.
The invention also discloses the fermentation process in high density of DA-1, by liquid submerged fermentation, this bacterium can form a large amount of gemma, in ammonia and nitrogen pollution water body, use separately this biological denitrifier can effective elimination water body in ammonia nitrogen.
The invention discloses a kind of large scale and high density and cultivate the method for low temperature deamination genus bacillus DA-1, comprise: at 30 ℃, mixing speed 200～300r/min, air flow 0.3～0.8vvm, connect bacterium amount 3～6%, under the condition of initial pH7.0, contain 10～25g glucose at every liter, 1～3g Zulkovsky starch, 10～40g soybean cake powder, 20～40mg manganous sulfate, 1～3g dipotassium hydrogen phosphate, 1～2g potassium primary phosphate, 0.2～1.0g magnesium sulfate, 0.1～0.5g yeast extract paste, in the nutrient solution of 0.1～0.3g iron trichloride and 0.1～0.3g calcium carbonate, carry out fermentor cultivation bacterium.
Preferred method is included in 30 ℃, mixing speed 250r/min, air flow 0.5vvm, connects bacterium amount 5%, under the condition of initial pH7.0, in every liter of nutrient solution containing 15g glucose, 2g Zulkovsky starch, 30g soybean cake powder, 30mg manganous sulfate, 3g dipotassium hydrogen phosphate, 1.5g potassium primary phosphate, 0.5g magnesium sulfate, 0.2g yeast extract paste, 0.1g iron trichloride and 0.1g calcium carbonate, carry out fermentor cultivation bacterium.
DA-1 of the present invention is the bacterium with outstanding ammonia nitrogen removal ability, after fermentation culture, can obtain the bacterial strain that viable count is many, gemma rate is high, just can show ammonia nitrogen removal activity under cold condition.
In use, generally DA-1 of the present invention is carried out after large scale and high density cultivation, low-temperature centrifugation results thalline, thalline is resuspended in 12～15% skimmed milk, is lyophilized into thalline lyophilized powder.The preferred activity of lyophilized powder is 1.0～2.7 × 10
11cFU/g.When use, directly lyophilized powder is dropped into the water body for the treatment of ammonia nitrogen removal.
The invention also discloses a kind of method of water body being carried out to biological deamination, comprising: thalline lyophilized powder is directly dropped into freshwater aquiculture water body, and making its final concentration is 10
7～10
8cFU/m
3.Best Combination of Methods comprises low temperature deamination genus bacillus is carried out after large scale and high density cultivation, by 8000r/min, 4 ℃ of centrifugal 10min results thalline, is resuspended in 15% skimmed milk, freeze-drying.Thalline lyophilized powder directly drops into freshwater aquiculture water body, and making its final concentration is 10
7～10
8cFU/m
3.Low temperature deamination genus bacillus DA-1 of the present invention just has outstanding deamination activity under cold condition, and under the combination culture condition such as suitable Wen Du ﹑ pH, sole carbon source, C/N compare, DA-1 can show stronger ammonia nitrogen removal ability.
Wen Du ﹑ pH, the impact of salinity on DA-1 degradation of ammonia nitrogen:
Subtilis DA-1 is inoculated into every liter and contains 5g extractum carnis, 10g peptone, and 5g sodium-chlor, cultivates 24h in shaking table in the basic medium of pH7.0, makes biomass reach 10
9more than CFU/mL.Adopt viable bacteria counting method to measure genus bacillus bacterium dense.Thalline is after centrifugal physiological saline rinsing three times, abandon supernatant, add isopyknic physiological saline suspension thalline, bacteria suspension is seeded to the screening culture medium of ammonia nitrogen concentration as 50mg/L take 1% inoculum size (volume ratio), screening culture medium composition: 5.0g glucose, 0.25g (NH
4)
2sO
4, 1.0g NaCl, 0.5g K
2hPO
4, 0.25g MgSO
4, 1000mL water, pH7.0.By culture temperature (℃) be made as respectively 10 ﹑ 15 ﹑ 20,25,30, pH7.0,200r/min shaking table are cultivated; PH is made as respectively to 5.0,6.0,7.0,8.0,9.0, and 30 ℃ of culture temperature, 200r/min shaking table are cultivated; Salinity (%) is made as respectively to 0,1,2,3,4,30 ℃ of culture temperature, pH7.0,200r/min shaking table is cultivated, and measures ammonia nitrogen degradation rate after 48h.
As can be seen from Figure 1, in the temperature range from 15-30 ℃, the ammonia nitrogen degradation rate of DA-1 can remain on more than 95%, and when temperature is too low while reaching 10 ℃, ammonia nitrogen degradation rate can be affected, and drops to 60%.
As seen from Figure 2, temperature is 30 ℃, and in the time that pH is 5.0, DA-1 is to the degradation rate of ammonia nitrogen lower than 10%, and in the time of pH > 6.0, mineralized nitrogen rate is higher, and in the time that pH is 7.0, ammonia nitrogen degradation rate is the highest.DA-1 is adapted at growth metabolism under neutral and slight alkalinity environment.This is because hydrogen ion concentration can change the electric charge of microorganism and plasmalemma, and then affect enzymic activity and the growth metabolism of microorganism, pH can also affect the degree of ionization of nutritive substance in substratum, thereby affect the absorption of microorganism to nutritive substance, affect toxicity on microorganism of objectionable impurities in environment and affect activity of various enzymes in metabolic reaction etc.
DA-1 is adaptable to pH, all shows very strong ammonia nitrogen degradation ability in pH7～9, can meet the degraded of ammonia nitrogen in aquaculture water completely.
As can be seen from Figure 3, be 30 ℃ in temperature, under the condition of pH7.0, while cultivation with the shaking speed of 200r/min, salinity does not have a significant effect to the reducing activity of bacterial strain.In the water body of aquaculture, have certain salt concn, fresh water salt concn is generally no more than 0.5%, and seawater average salt concentration is 3.5%.To sum up, bacterial strain can adapt to the high salt concentration of seawater, can remove fast the ammonia nitrogen in water body.
Low temperature deamination subtilis DA-1 of the present invention is the aerobic heterotrophic microorganism of a strain, it is (15～25 ℃) under cold condition, can be to eutrophication water, particularly the water body of ammonium salt-containing carries out the processing of nitrogen harmless biological, reaches the comparatively safe level of fishes and shrimps through the freshwater aquiculture water body of low temperature deamination subtilis biological treatment.The water body harmless treatment of low temperature deamination subtilis is carried out under aerobic conditions; do not need special equipment; ammonia nitrogen is converted into the component of thalline self in a large number; thereby bacterium can be used as the food of fish and enters food chain; finally realize environment protection, and really accomplished harmless, the reasonable recirculation of the energy and material simultaneously.
The invention discloses a kind of method of nitrogen-containing wastewater being carried out biological deamination under cold condition, the subtilis DA-1 in the present invention with low temperature ammonia nitrogen removal ability can join in nitrogen-containing wastewater, 15～25 ℃ of physical environments under static or whipped state, after 3～4 days, can remove 80～95% of water body ammonia nitrogen.
Accompanying drawing explanation
Accompanying drawing 2 is pH impacts on subtilis DA-1 ammonia nitrogen removal
Accompanying drawing 3 is salinity impacts on subtilis DA-1 ammonia nitrogen removal
Growth curve when accompanying drawing 4 is DA-1 shake flask fermentation
When accompanying drawing 5 is 10L fermentor cultivation, DA-1 gemma rate changes
Accompanying drawing 6 is DA-1 gemma forms (1000 ×)
Accompanying drawing 7 is DA-1 ammonia nitrogen removal curves in actual aquaculture water
Embodiment
DA-1 shake flask fermentation
1. seed activation substratum:
Extractum carnis 5g, peptone 10g, sodium-chlor 5g, distilled water is settled to 1L, pH7.0.Add 1.5% agar powder to make solid slant culture base.
2. fermention medium:
Glucose 15g, Zulkovsky starch 2g, soybean cake powder 30g, manganous sulfate 30mg, dipotassium hydrogen phosphate 3g, potassium primary phosphate 1.5g, magnesium sulfate 0.5g, yeast extract paste 0.2g, iron trichloride 0.1g, calcium carbonate 0.1g, distilled water is settled to 1L, initial pH regulator to 7.0.
Bacterial classification DA-1 is inoculated into seed activation substratum, 30 ℃ of constant temperature culture 18h, 5% inoculum size is to being equipped with in the 500mL Erlenmeyer flask of 100mL fermention medium, and 30 ℃, the concussion of 200r/min condition is cultivated.As shown in Figure 4, while cultivating 0～2h, DA-1 strain growth is in lag phase for result, and bacterial number is few; After 2h, bacterium enters logarithmic phase, and bacterial number growth is exceedingly fast; 12～20h is the growth stationary phase of DA-1; After 20h, bacterial count reduces gradually, and bacterium enters decline phase.
The fermentor tank large scale culturing of genus bacillus
1. seed culture medium:
Extractum carnis 5g, peptone 10g, sodium-chlor 5g, distilled water is settled to 1L, pH7.0.Add 1.5% agar powder to make solid slant culture base.
2. fermention medium:
Glucose 15g, Zulkovsky starch 2g, soybean cake powder 30g, manganous sulfate 30mg, dipotassium hydrogen phosphate 3g, potassium primary phosphate 1.5g, magnesium sulfate 0.5g, yeast extract paste 0.2g, iron trichloride 0.1g, calcium carbonate 0.1g, distilled water is settled to 1L, initial pH regulator to 7.0.
Meet DA-1 two from agar slant and encircle in the 500mL Erlenmeyer flask that lawn is incorporated with 100mL seed culture medium, at 30 ℃, 200r/min shaking table is cultivated 12h.By 5% inoculum size inoculation, 500mL seed culture fluid is transferred in the 10L fermentor tank that 9.5L fermention medium is housed.According to the result of study of shake flat experiment, determine the culture condition of best fermentor tank: 30 ℃, mixing speed 200r/min, air flow 0.5vvm, connect bacterium amount 5%, initial pH is 7.0, in every liter of nutrient solution containing 15g glucose, 2g Zulkovsky starch, 30g soybean cake powder, 30mg manganous sulfate, 3g dipotassium hydrogen phosphate, 1.5g potassium primary phosphate, 0.5g magnesium sulfate, 0.2g yeast extract paste, 0.1g iron trichloride and 0.1g calcium carbonate, carries out fermentation culture.In fermentor tank, DA-1 bacterial strain gemma rate changes as shown in Figure 5, through latent period, bacterium ramp breeding, when 24h, DA-1 bacterial strain starts to form gemma, and when 28h, DA-1 bacterial strain gemma rate reaches 40%, in the time that fermentation culture arrives 33h, as shown in Figure 6, gemma rate reaches 85%, now can stop fermentation, and fermented liquid is carried out to subsequent disposal by embodiment 3.
Lyophilized powder preparation and viable bacteria concentration determination
According to embodiment 2, by the fermentor cultivation subtilis DA-1 thalline of 24 hours, by 12,000g, at 4 ℃, centrifugal 15min collects, and is then suspended in the 400mL fermented liquid that contains 15% skim-milk, last freeze-drying.Freeze-drying programming is first paragraph :-30 ℃, and 240min(4h); Second segment :-10 ℃, 600min(10h); The 3rd section: 0 ℃, 960min(16h); The 4th section: 4 ℃, 360min(6h).
Thalline freeze-dried powder preparation viable bacteria concentration range is 2.7 × 10
11cFU/g, the about 96g of gross weight.
The ammonia nitrogen removal ability of subtilis DA-1 in actual aquaculture water
The lyophilized powder of preparing according to embodiment 3, by thalline freeze-dried powder preparation (2.7 × 10
11cFU/g) directly drop into polluted seawater cultivating pool, the input amount of thalline is 10
8cFU/m
3, every day is with the ammonia-nitrogen content of indigo spectrphotometric method for measuring pond waters.
Result demonstration, under the water body environment of 17 ℃ of water temperatures, pH7.8, NH in water
4 +-N starting for the 2nd day from thalline is thrown in declines, during by the 5th day, drop to 0.17mg/L by 2.4mg/L, see Fig. 7, subtilis has the effect of the degradation of ammonia nitrogen of highly significant, can be in the situation that not taking additive method, by water body ammonia nitrogen fast degradation to the comparatively safe level of fishes and shrimps.In whole implementation process, pH fluctuates between 7.6～7.9 scope, and this scope is applicable to the growth of genus bacillus.
Claims (8)
1. a subtilis, its preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.8292.
2. cultivate the method for the subtilis of claim 1 for one kind, comprise: under 15～30 ℃, 120～250r/min shaking flask rotating speed, pH5～10 condition, in every liter of nutrient solution containing 10～25g glucose, 1～3g Zulkovsky starch, 10～40g soybean cake powder, 20～40mg manganous sulfate, 0.1～0.4g yeast extract paste, 0.1～0.3g iron trichloride and 0.1～0.3g calcium carbonate, cultivate.
3. the method for claim 2, comprise: at 30 ℃, 200r/min shaking flask rotating speed under pH7.0 condition, is cultivated in every liter of nutrient solution containing 15g glucose, 2g Zulkovsky starch, 30g soybean cake powder, 30mg manganous sulfate, 0.2g yeast extract paste, 0.1g iron trichloride and 0.1g calcium carbonate.
4. the method for the subtilis of a mass-producing fermentation culture claim 1, comprise: at 30 ℃, mixing speed 200～300r/min, air flow 0.3～0.8vvm, connect bacterium amount 3～6%, under the condition of initial pH7.0, contain 10～25g glucose at every liter, 1～3g Zulkovsky starch, 10～40g soybean cake powder, 20～40mg manganous sulfate, 1～3g dipotassium hydrogen phosphate, 1～2g potassium primary phosphate, 0.2～1.0g magnesium sulfate, 0.1～0.5g yeast extract paste, in the nutrient solution of 0.1～0.3g iron trichloride and 0.1～0.3g calcium carbonate, carry out fermentor cultivation bacterium, described per-cent is weight percentage.
5. the method for claim 4, comprise: at 30 ℃, the mixing speed of 250r/min, under the condition of the air flow of 0.5vvm, 5% the bacterium amount that connects, initial pH7.0, in every liter of nutrient solution containing 15g glucose, 2g Zulkovsky starch, 30g soybean cake powder, 30mg manganous sulfate, 3g dipotassium hydrogen phosphate, 1.5g potassium primary phosphate, 0.5g magnesium sulfate, 0.2g yeast extract paste, 0.1g iron trichloride and 0.1g calcium carbonate, carry out fermentation culture.
6. the application of the subtilis in claim 1 in aquaculture water deamination.
7. a method of marine culture water being carried out to biological deamination, comprising: after the subtilis of claim 1 is cultivated, be suspended in skimmed milk freeze-drying, lyophilized powder is directly dropped into water body, in physical environment pH7～9, cultivate after 3～4 days, can remove the ammonia nitrogen of water body.
8. the method for claim 7, comprising: after the subtilis of claim 1 is cultivated, be suspended in skimmed milk freeze-drying, lyophilized powder is directly dropped into water body, the input amount of thalline is 10
7～10
8cFU/m
3, in physical environment pH7～9,15～20 ℃ of temperature, cultivated after 3～4 days, can remove the ammonia nitrogen in water body.
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| CN113699066A (en) * | 2021-08-24 | 2021-11-26 | 山东京青农业科技有限公司 | Cold-resistant bacillus subtilis and application thereof |
| CN116621320A (en) * | 2023-04-03 | 2023-08-22 | 江苏金山环保科技有限公司 | Biological composite carbon source prepared from blue algae and preparation process thereof |
| CN116621320B (en) * | 2023-04-03 | 2023-11-07 | 江苏金山环保科技有限公司 | Biological composite carbon source prepared from blue algae and preparation process thereof |
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