CN100575480C - One plant height is imitated Rhodopseudomonas palustris and the application thereof that utilizes nitrite nitrogen - Google Patents
One plant height is imitated Rhodopseudomonas palustris and the application thereof that utilizes nitrite nitrogen Download PDFInfo
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- CN100575480C CN100575480C CN200710089906A CN200710089906A CN100575480C CN 100575480 C CN100575480 C CN 100575480C CN 200710089906 A CN200710089906 A CN 200710089906A CN 200710089906 A CN200710089906 A CN 200710089906A CN 100575480 C CN100575480 C CN 100575480C
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Abstract
The present invention relates to a kind ofly has the photosynthetic bacterium of high assimilation utilising efficiency to nitrite nitrogen, specifically provides a kind of Rhodopseudomonas palustris bacterial strain and rejuvenation, preservation, biological assay and utilisation technology of assimilating nitrite nitrogen.The invention belongs to the category of biologic water cleaner and bio-feritlizer, the photosynthetic bacterium that provides can be used for controlling aquaculture system nitrite nitrogen content, improves environmental pollution, eliminates cinnamic acid and phenylformic acid, diazotroph fertilizer, continuous cropping cucumber volume increase growth promoter etc.
Description
Technical field
The present invention relates to a kind ofly can efficiently utilize nitrite nitrogen (with NO
2 -And the nitrogenous compound of its esters form existence) photosynthetic bacterium specifically provides a kind of Rhodopseudomonas palustris 2-8 (Rhodopseudomonas palustris 2-8) bacterial strain (CCTCC M 205147) of efficiently assimilatory reduction nitrite nitrogen and the bioassay method that industrialization cultural method, culture collection process, bacterial strain rejuvenation method and nitrite nitrogen are eliminated thereof.It belongs to the category of biologic water cleaner and bio-feritlizer.
Background technology
The biological residual body of the residual bait in the aquaculture system, fishes and shrimps movement, death and medicine, fertilizer etc. are in water bottom accumulation, decomposition, conversion, form hydrogen sulfide, ammonia nitrogen, nitrite nitrogen and other oxynitride etc. to the virulent compound of fishes and shrimps, make water quality deterioration, the existence of serious threat cultivated animals and normal growth.Under high-density, intensive culture condition, a large amount of bait throwing in often make the nitrite nitrogen content severe overweight in the water body, NO
2 -Be combined into protoferriheme with protoheme in the aquatic animal body, methemoglobin can not deliver oxygen, and aquatic animal just dies from anoxic.Non-ion state ammon nitrogen and nitrite nitrogen content increase with explosive fish disease in the breed pond substantial connection, so the elimination of the biology of aquaculture system nitrite nitrogen becomes outstanding problem demanding prompt solution in the aquatic products healthy aquaculture.
Nitrite nitrogen is eliminated by assimilation and two kinds of denitrification approach of alienation metabolism at nature.Assimilation is under the nitrite assimilatory reduction enzyme effect of microorganism or plant, and nitrite nitrogen is reduced into NH
4 +, NH then
4 +In cell, participate in the synthetic of material such as amino acid and be converted into protein and biological intravital other nitrogenous substances at last; The alienation metabolism is under the effect of nitrite alienation reductase enzyme, is reduced into N
2O further transforms and generates N
2Be discharged in the environment.Rhodopseudomonas palustris 2-8 bacterial strain provided by the invention is eliminated for assimilation the elimination of nitrite nitrogen: its anaerobic growth in containing the substratum of Sodium Nitrite, though NO in the substratum
2 -Gradually reduce, but NH do not occur
4 +And NO
3 -Increase, do not have gas yet and produce.Nitrite reductase extensively is present in microorganism and the plant materials, plays an important role in Nitrogen Cycling.Though extensively there are various types of denitrifying bacteriums in nature, can be finally NO
2 -Be converted into NO, N
2O and N
2But the distinctive eutrophic environment of aquaculture water, make in the water body own microorganism system that the self-purification capacity of nitrite nitrogen is not enough to nitrite nitrogen concentration is reduced to the level of cultivated animals tolerance, therefore usually need by other approach such as interpolation have the metabolism nitrite nitrogen microorganism, add the content that chemical reagent or dual mode bonded method reduce nitrite nitrogen in the water body.Chemical oxidization method and microorganism treatment are mainly used in the removal of nitrite nitrogen in the aquaculture water at present.Chemical oxidization method instant effect, but the weak point of holding time will constantly drop into chemicals with its oxidation, and cost is high and have a secondary pollution problem.Microniological proudcts based on various denitrifying bacteriums also can be eliminated water nitrite attitude nitrogen to a certain extent, but be subjected to the influence of microorganisms in water monoid, be difficult to effectively bring into play effect, chance of success is little in the actual production, has seriously restricted the use of biotechnological formulation in the fishery products healthy aquaculture.
Photosynthetic bacterium (Photosynthetic Bacteria, be called for short photosynthetic bacterium) be to carry out a photosynthetic bacterioid, be the prokaryotic organism that occur the earliest on the earth with original luminous energy synthetic system, it has constituted the original producer in the nature ecosystem with other photosynthetic organism, and the nature material transform and energy cycle in play an important role.Wherein some section, genus, the small organic molecule of under the illumination anaerobic condition, degrading, and utilize organism to decompose the basic substance of the organic acid, hydrogen sulfide and the synthetic thalline of ammonia nitrogen conduct that produce through heterotrophic bacterium, thereby COD in the reduction water body and ammonia nitrogen, improve the dissolved oxygen amount of water, thereby purification of organic waste water effectively, improve the breeding ecological environment of seawater, fresh water, improve cultured output.Some section, genus can also reduce the content of heavy metal ion in the water.Studies show that photosynthetic bacterium can make ammonia nitrogen, hydrogen sulfide in the aquaculture pond water body descend more than 50%, dissolved oxygen rate improves 14%～85%, so photosynthetic bacteria is a kind of effective biological weapon that improve environmental pollution.In addition, the protein content of photosynthetic bacterial thallus is up to more than 65%, be rich in vitamin B group and several physiological active substances such as coenzyme etc., and the thalline that generates is also to various biological nontoxics, harmless such as people, animal, fowl, therefore, all obtained application in every field such as aquaculture, livestock and poultry cultivation, agriculture production, health-care products, biological medicines.
Purple nonsulfur bacteria in the photosynthetic bacterium is the most changeable biology of finding on the earth of metabolic mechanism, and becomes the model animals of exploring many important vital processes.These bacteriums can utilize five kinds of main patterns of microorganism to carry out metabolism, comprise aerobic and anaerobic chemical heterotroph, aerobic chemosynthetic autotroph, anaerobism photosynthetic autotrophs and photoheterotrophy type.Rhodopseudomonas palustris belongs to purple nonsulfur bacteria, extensively is present in soil and the water body, and purple nonsulfur bacteria or any existing biophase ratio with other have its uniqueness, can be in unicellular the more biological process of catalysis.Rhodopseudomonas palustris has life condition extremely widely, and very strong viability can be grown and breeds with any in top listed all metabolic patterns; And it when photoheterotrophy is grown to the utilization of organic carbon source (comprising aromatic series carbohydrate and lignin monomer) and with the carbonic acid gas being sole carbon source when carrying out photoautotrophy, in the utilization to electron donor (sulphur compound and hydrogen) great handiness is arranged all.As far back as 1981, just in the pod membrane Rhodopseudomonas, found nitrite assimilatory reduction enzyme, and the domestic report of also eliminating nitrite nitrogen relevant for Rhodopseudomonas palustris.Lei Aiying etc. (2005) report screens the Rhodopseudomonas palustris of strain energy degrading nitrite, this bacterium can be reduced to 0.0309mg/L to the nitrite nitrogen of 0.0461mg/L at the 9th day, nitrite concentration then rises to 0.0825mg/L from initial 0.0418mg/L in the control group at this moment, actual this bacterial strain can be reduced to 0.0309mg/L to the nitrite of 0.0825mg/L at the 9th day, and its degradation rate is about 62.54%.Xu Shannan etc. (2004) separate from river and samples such as fish pond bed mud 18 strain swamp Rhodopseudomonas in the 28 strain Purple Non-sulfur photosynthetic bacteriums that obtain, wherein have 3 strain swamp Rhodopseudomonas to have the elimination ability of nitrite nitrogen, they are cultivated in the aquaculture water of sterilization, and the degradation capability to the 2.9mg/L nitrite nitrogen is respectively 68.96%, 81.03%, 93.79% behind the 72h.The result of document shows to exist really in the Rhodopseudomonas palustris to have the bacterial strain that nitrite nitrogen is eliminated ability, by simulation document (Xu Shannan etc., 2004) described condition, find can in 24h, will the sterilize nitrite nitrogen reduction by 99.68% of 2.9mg/L in the water of the shrimp pool of this patent bacterial strain, and can about 30h, reduce by 98.45% to same nitrite in the unsterilised shrimp pool water simultaneously, obviously under equal conditions the nitrite elimination ability than bibliographical information bacterial strain is stronger, faster for this patent bacterial strain.It should be noted that, needed bacterial strain in the aquaculture production practice, not only need it to possess the potentiality of eliminating nitrite nitrogen, also need and in water body, to breed fast, otherwise be difficult to production practice,, also contain various types of microorganisms because aquaculture water not only contains various abundant organism, especially comprising a large amount of is the planktonic organism of food with the bacterium, and the existence of these microorganisms can have influence on the effect of application.The Rhodopseudomonas palustris that patent of the present invention provides, at the problems referred to above, having passed through the purification, rejuvenation and a large amount of field tests that reach surplus 2 years, 30 time confirms, this bacterial strain not only has the efficient ability that reduces nature aquaculture water nitrite attitude nitrogen fast, also has to utilize the various organism ability of growth fast in aquaculture water.
Summary of the invention
The object of the present invention is to provide the industrialization substratum and the cultural method of a kind of Rhodopseudomonas palustris bacterial strain with assimilatory reduction nitrite nitrogen ability fast and efficiently and this bacterial strain, the purposes of utilizing the product that this method produces and bioassay method and the purposes that using method, culture collection process, bacterial strain rejuvenation method, nitrite nitrogen are eliminated.
Photosynthetic bacterium provided by the invention is Rhodopseudomonas palustris 2-8 (Rhodopseudomonas palustris 2-8), is deposited in Chinese typical culture collection center (being abbreviated as CCTCC) on December 16th, 2005, and preserving number is CCTCC M 205147.This strains separation is from the pool bottom sludge in fish pond, Shaoguan, Guangdong, and this bacterium is a gram negative bacterium, and thalline is little, is shaft-like to avette, and older culture forms rosette and boundling shape, asymmetric division.Bacterial strain is circular in acidic culture, and thalline is circular to oval in the alkaline medium, and part becomes shaft-like.Grow into little water white transparency small colonies in adding the solid medium of sodium sulphite, the later stage bacterium colony is begun to become reddish brown by the center, constantly to external diffusion.Bacterium liquid is pink in the early stage, and the later stage becomes scarlet.Centrifugal collection thalline, and record thalline after suspending again with 40% sucrose solution and absorption peak is arranged at 750～900nm and 790～1030nm place, can not in containing the substratum of 3%NaCl, grow, can in the substratum that contains L-Ala, Sodium Propionate, sodium acetate, Trisodium Citrate, para-amino benzoic acid, grow, can not utilize tartrate to do the sole carbon source growth, has the gelatine liquefication ability, can hydrolyzed starch, can on sodium cellulosate or pectin are the substratum of sole carbon source, grow, have nitrogen fixing capacity.
Indoor bioassay is found, bacterial strain provided by the invention can be under inoculum size be 0.14 ‰ situation, eliminate the nitrite nitrogen of 1.89mg/L more than 99.2% in the 12h, through domestication, this bacterial strain is under the situation of same inoculum size, in 24h, can eliminate the nitrite nitrogen of 35mg/L more than 99.98%.It has fast, efficient, consumption is few, eliminate advantage completely, and the ability of eliminating nitrite nitrogen can be improved by domestication; In containing the substratum of Sodium Nitrite, cultivate this bacterium,, NH do not occur along with gradually reducing of nitrite nitrogen in the substratum
4 +And NO
3 -Increase, do not have gas yet and produce, in addition, this bacterial strain also has metabolism cinnamic acid and benzoic ability and nitrogen fixing capacity simultaneously.
The present invention includes following several respects content:
(1) swamp Rhodopseudomonas 2-8 belongs to the Rhodopseudomonas of purple nonsulfur bacteria, has assimilation NO
2 -Ability (be after anaerobism cultivate to be eliminated nitrite nitrogen, in substratum, to detect less than NH
4 +And NO
3 -Rising, do not have gas yet and produce), metabolism NO under the condition that it can be aerobic in dark and illumination is aerobic
2 -
(2) be applicable to the culture medium prescription and the cultural method of photosynthetic bacterium industry cultivation in (1).The present invention on the basis of known substratum according to the characteristic of this patent bacterial strain, optimum medium to this bacterial strain has carried out optimizing improvement, filter out three substratum that are applicable to that industrialization is cultivated, they are respectively: 1. sodium acetate 0.2%, ammonium chloride 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid; 2. Sodium Propionate 0.2%, ammonium chloride 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid; 3. sodium succinate 0.2%, ammonium chloride 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid.Adopt this optimization substratum can be the bacterial concentration of nutrient solution from 10
8Cfu/ml brings up to 1.8 * 10
11More than the cfu/ml.
(3) Rhodopseudomonas palustris of mentioning in above-mentioned (1), by any prescription large-scale production of mentioning in above-mentioned (2), the fermented liquid of acquisition, its viable count reaches 10
11More than the cfu/ml, this fermented liquid is used by 4 kilograms/mu, this bacterium can be in 24h be reduced to the water nitrite attitude nitrogen of the severe overweight more than the 0.3mg/L in the Penaeus vannamei aquaculture water below the 0.05mg/L, and in the 10d afterwards, continues to keep nitrite nitrogen content about 0.05mg/L.After this, threw in once, can control cultivation water nitrite fully and not exceed standard every 15 days.
(4) bacterial strain and the cultured products of mentioning in above-mentioned (1)～(3) thereof is used for the elimination of aquaculture system nitrite nitrogen.
(5) any one bacterial strain of mentioning of a kind of being applicable to (1)～(4) has the photosynthetic bacterium bacterial strain rejuvenation method of nitrite nitrogen assimilative capacity with other.Concrete operations: take by weighing the fertile fresh bed mud 200～300g in the shrimp pool or fish pond, place the 500ml saline bottle, in saline bottle, add 10g Penaeus vannamei feed then, fill it up with saline bottle with fish pond or shrimp pool water, after the autoclaving cooling, inoculum size by 3% inserts treats the rejuvenation bacterial strain, sealing back illumination cultivation 2～6 days, the bacterial strain that both can obtain to have high reactivity nitrite nitrogen reducing power.If necessary, can repeat aforesaid operations more than three times.
(6) any one bacterial strain of mentioning and other photosynthetic bacterium with nitrite nitrogen assimilative capacity keep the culture collection process of its efficient nitrite nitrogen reducing power in a kind of being applicable to (1)～(5).Concrete operations are: the inoculum size inoculation by 3% treats that preservation bacterium liquid (promptly takes by weighing the fertile fresh bed mud 200～300g in the shrimp pool or fish pond to aseptic prawn feed bed mud substratum, place the 500ml saline bottle, in saline bottle, add 10g Penaeus vannamei feed then, fill it up with saline bottle with fish pond or shrimp pool water, standby after the autoclaving cooling) in, after the illumination cultivation 3 days, the low light level is according to the room temperature preservation.
(7) a kind of bacterial strain nitrite nitrogen that is applicable to that any one bacterial strain of mentioning in evaluation (1)～(6) and other have a similar functions is eliminated the bioassay method of ability.This method adopts two evaluation systems.Evaluation system 1: substratum 1 (ammonium chloride 0.1%, sodium hydrogen phosphate 0.05%, sal epsom or magnesium chloride 0.02%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, above composition is water-soluble, 121 ℃ of steam sterilizing 20min.Prepare following solution and filtration sterilization respectively: 1. 10% sodium bicarbonate, 2. 3. 0.1N phosphoric acid of ethanol.Add sodium hydrogen carbonate solution 50ml in above-mentioned basic medium, ethanol 1.5～2.0ml transfers to pH7.0 with 0.1N phosphoric acid) the middle Sodium Nitrite that adds, according to 10
6The concentration of cfu/ml adds microorganism to be measured, and the anaerobism illumination cultivation is measured the content that remains nitrite in the substratum every 4～6h, and METHOD FOR CONTINUOUS DETERMINATION 3～4 times, data can be used for estimating the nitrite nitrogen elimination ability difference of different strains or different treatment mode; Evaluation system 2: gather and to contain the pool water of nitrite and the bed mud on this pool, supernatant is got in the mixed that adds 200～400g bed mud by the every liter of pool water clarification of spending the night.The concentration of pressing 2g/L before measuring adds sodium acetate, presses the inoculum size inoculation in the evaluation system 1 then, and illumination is aerobic or keep natural lighting and room temperature, and per 4～6h measures the content of a nitrite, is used for the difference that the comparison nitrite nitrogen is eliminated ability.Evaluation system 1 and evaluation system 2 are united use, can reflect the difference of bacterial strain nitrite nitrogen elimination ability preferably, specifically see embodiment 5.But in the process of biological assay, should be noted that: 1. employed nitrite nitrogen colouring reagents can be to the NO of 0～1.9mg/L among the present invention
2 -Normal colour developing, and at the OD at 520nm place value and NO
2 -Concentration have linear relationship, the colouring reagents prescription is: the 1.0g alpha-naphthylamine, the 10g Sulphanilic Acid, 89g tartrate is ground into powder in mortar, mixes then, is stored in the brown bottle standby.2. each mensuration adds excessive slightly reagent, about 0.01～0.1g (, needing behind double-deck filter paper filtering, to add again colouring reagents) if pool water is too muddy, and mixing leaves standstill colour developing 10min, measures the OD value of colour developing back liquid under 520nm with spectrophotometer.If 3. the nitrite nitrogen content of pool water is too high, precipitation appears during colour developing, need dilute in order to avoid the mensuration that precipitation influences the OD value occurs pool water.4. before the color reaction, need to use the centrifugal 15min of speed of 15000rpm to remove thalline, especially photosynthetic bacterium, its pigment that contains has the certain absorption peak at the 520nm place, as not centrifugal removal thalline, then can influence the result of mensuration.
(8) any one photosynthetic bacterium of mentioning is cultivated through domestication in above-mentioned (1)～(7), can reach 35mg/L to the highest tolerance of Sodium Nitrite, and the nitrite nitrogen of this concentration can be reduced by 99.98%.
(9) any one photosynthetic bacterium of mentioning has the ability of fixed nitrogen in above-mentioned (1)～(8), can be used as diazotroph fertilizer.
(10) any one photosynthetic bacterium of mentioning has production-increasing function to cucumber in above-mentioned (1)～(9), can be used as continuous cropping cucumber volume increase, promotes growth microbial inoculum.
(11) any one photosynthetic bacterium of mentioning can utilize cinnamic acid and phenylformic acid to do the sole carbon source growth in above-mentioned (1)～(10), can be used for the biological elimination of cinnamic acid and phenylformic acid.
Embodiment
Enumerate embodiments of the invention explanation embodiment of the present invention and effect below, but the present invention is not limited to these embodiment.
The preparation of embodiment 1 bacterial classification
Getting Rhodopseudomonas palustris 2-8 bacterium liquid 100 μ l coating is inoculated on the flat board that contains substratum 2, place anaerobic jar, in intensity of illumination is 2000lux, temperature is to cultivate under 28 ℃～30 ℃ conditions after 3 days, from medium slant picking 1 transfering loop bacterial classification inoculation to liquid nutrient medium 1, anaerobism was cultivated 3 days under the same temperature light condition, by increasing 2 times in 3% the inoculum density liquid medium within 1, promptly obtained can be used for the strain cultured solution of scale operation then again.
The prescription of A, substratum 1 is as follows: ammonium chloride 0.1%, sodium hydrogen phosphate 0.05%, sal epsom or magnesium chloride 0.02%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, and above composition is water-soluble, 121 ℃ of steam sterilizing 20min.Prepare following solution and filtration sterilization respectively: 1. 2. 3. 0.1N phosphoric acid of ethanol of 10% sodium bicarbonate.Add sodium hydrogen carbonate solution 50ml (annotating: add the 50ml sodium hydrogen carbonate solution in the above-mentioned substratum of 1L) in above-mentioned basic medium, ethanol 1.5～2.0ml transfers to pH7.0 with 0.1N phosphoric acid.
The prescription of B, substratum 2 is to add 1.5% agar powder on the basis of substratum 1.
The scale operation of embodiment 2 thalline is cultivated
Sterilize with the chlorine bleach liquor earlier and cultivate, neutralize with Sulfothiorine then with container and tap water.In culture vessel, press any culture medium prescription described in the summary of the invention (2) then and add reagent, be mixed with substratum, insert 10% three-class strain, at last with sterilization after in the neutralizing agent and the tap water of crossing fill it up with entire container, place 28 ℃～30 ℃, intensity of illumination is that 2000lux cultivated 5～7 days down, get final product product.
Disinfect reagent and time such as table 1:
Disinfection technology parameter when table 1, this patent bacterial strain 2-8 scale operation
The rejuvenation of embodiment 3 bacterial strains
Gather the shrimp pool or the fertile fresh bed mud 200～300g in fish pond, place the 500ml saline bottle, and adding 10g Penaeus vannamei feed, fill it up with saline bottle with fish pond or shrimp pool water then, after the autoclaving cooling, inoculum size by 3% inserts treats rejuvenation bacterial strain, anaerobism illumination cultivation 2～6 days, the bacterial strain that can obtain to have high reactivity nitrite nitrogen reducing power.If necessary, can repeat aforesaid operations more than three times.The evaluation method of introducing according to embodiment 6 after the bacterial strain rejuvenation to rejuvenation after the nitrite nitrogen assimilative capacity of bacterial strain measure.
Behind the rejuvenation this patent bacterial strain 2-8, measure result such as following table according to the method described above through the evaluation method of embodiment 6:
Table 2, rejuvenation bacterial strain and the original strain elimination ability to nitrite nitrogen in evaluation system 1 compares
Table 3, rejuvenation bacterial strain and the original strain elimination ability to nitrite nitrogen in evaluation system 2 compares
Purpose to the bacterial strain rejuvenation is to utilize various organism to breed and eliminate the ability of nitrite nitrogen fast in natural water in order to improve bacterial strain, from evaluation result, though original strain and rejuvenation bacterial strain all can effectively be eliminated nitrite nitrogen in evaluation system 1, but original strain can not be eliminated the nitrite in the water of the shrimp pool in evaluation system 2, the rejuvenation bacterial strain then can effectively eliminate 97.50% at 30h with the nitrite nitrogen in the water of the shrimp pool, obviously by above-mentioned rejuvenation operation, improve the viability of bacterial strain in natural water, thereby reached the stable purpose of eliminating nitrite nitrogen.
Embodiment 4 high density nitrite nitrogens are to the domestication of bacterial strain tolerance
The fermented liquid of this patent bacterial strain inserted by 3% inoculum size contain in the substratum 1 of 15mg/L Sodium Nitrite, place illumination box, illuminance is controlled in the 1200-2000lux scope, carrying out the illumination anaerobism under 28 ℃ cultivates, measure remaining nitrite nitrogen concentration in the substratum every 12h, cultivate after 7 days, be inoculated in the substratum 1 that nitrite concentration is 25mg/L taming the inoculum size of nutrient solution by 3%, place similarity condition to cultivate, measure residue nitrite nitrogen concentration in the substratum every 12h simultaneously, cultivate after seven days, again nutrient solution is inoculated in the substratum 1 that nitrite concentration is 35mg/L by 3% inoculum size.The result shows, in above-mentioned tolerance gradient domestication, bacterial strain all in 24h respectively the nitrite of 25mg/L, 35mg/L eliminate 99.86%, more than 99.98%.The mensuration of maximum nitrite tolerance concentration is not done in this research.
The preservation of embodiment 5 bacterial strains
Take by weighing the fertile fresh bed mud 200～300g in the shrimp pool or fish pond, place the 500ml saline bottle, in saline bottle, add 10g Penaeus vannamei feed then, fill it up with saline bottle with fish pond or shrimp pool water, after the autoclaving cooling, the inoculum density by 3% inserts treats preservation strain, after the illumination cultivation 6 days, the low light level is according to the room temperature preservation, and transfer once every half a year, and measures its nitrite nitrogen assimilative capacity and the cultivation proterties has no change.Such method can keep the function of bacterial strain and growth performance not to degenerate.
Bacterial strain after the aforesaid method preservation, after the activation, as shown in the table by embodiment 6 described evaluation system evaluation results.
Table 4, preservation activation bacterial strain elimination ability to nitrite nitrogen in evaluation system 1 are measured
As can be known from Table 4, compare with original strain by the bacterial strain that above-mentioned culture collection process activates out, nitrite nitrogen is eliminated ability and is still kept higher level.
The biological assay of embodiment 6 bacterial strains assimilation nitrite nitrogen ability
(1) mensuration of content of sodium nitrite typical curve
Preparation 1L substratum 1 (ammonium chloride 0.1%, sodium hydrogen phosphate 0.05%, sal epsom or magnesium chloride 0.02%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, above composition is water-soluble, 121 ℃ of steam sterilizing 20min.Prepare following solution and filtration sterilization respectively: 1. 10% sodium bicarbonate, 2. 3. 0.1N phosphoric acid of ethanol.In above-mentioned basic medium, add sodium hydrogen carbonate solution 50ml, ethanol 1.5～2.0ml, transfer to pH7.0 with 0.1N phosphoric acid), behind the autoclaving, aseptic technique, other components after the adding filtration sterilization, and mix up the pH value, with the 500ml volumetric flask of sterilization, be solvent then with above-mentioned substratum 1, being made into final concentration is the Sodium Nitrite mother liquor of 5mg/L, weighs nitrite nitrogen content with the content of Sodium Nitrite.Get a certain amount of substratum that contains Sodium Nitrite 1 and a certain amount of substratum 1 solution of Sodium Nitrite that do not contain in the clean tube of sterilization, make that the final concentration of Sodium Nitrite is respectively 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8,1.9mg/L, each concentration repeats for three times.Colouring reagents (the 1.0g alpha-naphthylamine described in summary of the invention (5) that adds excessive slightly (nitrite nitrogen is developed the color fully) again, the 10g Sulphanilic Acid, 89g tartrate, in mortar, be ground into powder, mix then) about 0.1g, mixing, leave standstill colour developing 10min after, under 520nm, measures the colour developing OD value of liquid afterwards with spectrophotometer.At last according to Sodium Nitrite concentration and corresponding OD thereof
520nmValue is drawn substratum Central Asia sodium nitrate concentration typical curve.The equation that obtains between OD value and the Sodium Nitrite concentration is that (wherein y is OD to y=0.4526x+0.0356
520nm, X is substratum 1 Central Asia sodium nitrate concentration, the likeness coefficient of equation reaches R
2=0.9966), this equation is used for the evaluation system 1 described in the summary of the invention (7).
In like manner, measure process according to above-mentioned Sodium Nitrite concentration standard curve, change substratum 1 into distilled water, measure nitrite typical curve in the water body, the equation that gets between water-outlet body Central Asia sodium nitrate concentration and the OD value according to this typical curve is y=0.4633x+0.0313, and wherein y is OD
520nmValue, X is a Sodium Nitrite concentration, the likeness coefficient of equation reaches R
2=0.9954, this equation is used for the evaluation system 2 described in the summary of the invention (7).
(2) measure the elimination ability (in summary of the invention (7) application example of evaluation system 1) of this patent bacterial strain 2-8 to Sodium Nitrite in the substratum 1
In the ground Erlenmeyer flask of 50ml, add the aseptic substratum 1 of 50ml and the sodium nitrite solution of filtration sterilization, make the Sodium Nitrite final concentration reach 1.4mg/L, inoculate this patent bacterial classification 2-8 then, inoculum size is by 10
6Cfu/ml calculates, and is contrast with the substratum of not inoculating.30 ℃, anaerobism is cultivated under the 2000lux, measures remaining content of sodium nitrite in the substratum every 6h.
Measuring method: get the centrifugal 15min of 2ml bacterium liquid 15000rpm, take out supernatant liquor in test tube, add excessive slightly about 0.01～0.1g and (add a little about 0.01g earlier, shaking up colourless then need not of back liquid adds again, as there is redness to occur after shaking up, then need add a certain amount of developer again, but total amount need be controlled at 0.1g, excessive developer can influence the accuracy of measurement result) the colouring reagents described in summary of the invention (7), mixing, static colour developing 10min uses spectrophotometer to measure the OD value of colour developing back liquid then under 520nm.According to the equation that is used for evaluation system 1, draw remaining nitrite nitrogen content in the liquid, according to the nitrite nitrogen content of contrast, calculate the nitrite nitrogen residual value shi then, concrete outcome sees Table 5.
Table 5, this patent bacterial strain 2-8 in evaluation system 1 to the elimination bioassay results of nitrite nitrogen
Can see that from the data of table 5 inoculation this patent bacterial strain 24h eliminates the Sodium Nitrite in the evaluation system 1 100% in containing the nutrient solution of nitrite nitrogen.
(3) measure this patent bacterial strain 2-8 assimilative capacity (application example of evaluation system 2 in the summary of the invention (7)) to nitrite nitrogen in the water of the shrimp pool
In the Penaeus vannamei cultivating pool, adopt back the pool water and the bed mud that contain nitrite nitrogen, at first the basic condition to pool water writes down (as: salinity, pH value, color, turbidity, contain algae situation etc.), and add developer as the aforementioned, leave standstill 10min after, measure OD
520nmBe worth, calculate the initial nitrite nitrogen content of pool water according to typical curve y=0.4633x+0.0313.Then according to the method for evaluation system 2 in the summary of the invention (5), get 1000ml pool water, add the 200g bed mud, after the stirring, clarification is spent the night, about the morning on the secondth 8:00, pour out supernatant, in supernatant liquor, press 2g/L concentration and add sodium acetate, mix the back and install in the clean tool cover glass bottle, then by 10 by 50ml/ bottle branch
6The inoculum size of cfu/ml inserts this patent bacterial strain 2-8, is contrast with the sodium acetate pool water of not inoculating that adds.Mixture is in room temperature, and aerobic cultivation under the 2000lux illumination is measured remaining nitrite nitrogen content in the water of the pool every 6h, and measuring method the results are shown in Table 6 with evaluation system 1.
Table 6, this patent bacterial strain 2-8 in evaluation system 2 to the elimination bioassay results of nitrite nitrogen
Can see from the data of table 6, in containing the nutrient solution of nitrite nitrogen, behind the inoculation this patent bacterial strain 12h, the nitrite nitrogen in the evaluation system 2 is reduced to normal level.Though during from 6h to 12h, the nitrite nitrogen concentration of contrast has very significantly rising, the shrimp pool water nitrite nitrogen residue content that 2-8 handles still keeps stable.
Embodiment 7 this patent bacterial strains are eliminated the treatment process of nitrite nitrogen in the aquaculture system
At first according to the good bacterium liquid of embodiment 2 described method large scale culturing, concentration reaches 10 before experimentizing
11Cfu/ml is then according to using by 4 kilograms/mu amount.Earlier product is mixed in larger container before using, and shine in the sun about 2h, to activate this patent bacterial strain, the whole then water surface is evenly splashed.The nitrite nitrogen initial content on the prawn pool is measured before adding bacterium liquid, every 12h, gets the content that pool aquametry is cultured residue nitrite nitrogen in the water of the pool.In December, 2005 is field test in the winter in the smooth continent of Zhong Shan, Guangdong canopy Penaeus vannamei cultivating pool, in the 24h, uses in a manner described, and this patent bacterial strain drops to 0.05mg/L with the nitrite nitrogen of this aquaculture water from 0.3mg/L, and continues to have kept about 20 days.
Embodiment 8 bacterial strains utilize cinnamic acid and phenylformic acid
(1) mensuration of cinnamic acid and phenylformic acid typical curve
Prepare a series of certain density phenylformic acid and cinnamic acid standardized solution at first respectively, measure the pairing OD value of these standardized solution at 290nm and 272nm place respectively with spectrophotometer then, according to cinnamic acid and benzoic normal concentration and their pairing OD drawing standard curves, and draw calculation equation between OD value and cinnamic acid and OD value and concentration of benzoic acid and be respectively y=46.394x-0.0023 (wherein Y is OD
290nm, X is cinnamic acid concentration g/L, R
2=0.9997) and y=3.6171x+0.0038 (wherein Y is OD
272nm, X is cinnamic acid concentration g/L, R
2=0.9992).
(2) this patent bacterial strain utilizes the mensuration of cinnamic acid and phenylformic acid ability
At first, configuration photosynthetic bacterium minimum medium (no organic carbon source), this culture medium prescription are 1. 2. 3. 1ml, pH7.0 of 1ml, distilled water 100ml, trace element solution of 0.1g, potassium hydrogen phosphate 0.02g, bitter salt 0.02g, sodium-chlor 0.1g, growth cofactor of ammonium chloride 0.1g, sodium bicarbonate.
1. prepared beforehand 5% sodium bicarbonate aqueous solution, (sodium bicarbonate 5g, distilled water 100ml) after the degerming, are got 2ml and are mixed through autoclaved substratum with other after filtration.
2. the cofactor of growing: take by weighing beta-amino phenylformic acid 0.1mg, be dissolved in the 10ml distilled water filtration sterilization.
3. trace element solution: six Ferric Chloride Hydrated 5mg, Salzburg vitriol 0.05mg, boric acid 1mg, four hydration Manganous chloride tetrahydrate 0.05mg, Zinc vitriol 1mg, Cobaltous nitrate hexahydrate 0.5mg, be dissolved in above reagent in the distilled water respectively, and be settled to 1000ml, filtration sterilization then.
(above except that 1., 2., 3., each composition dissolves back 121 ℃ of steam sterilizing 20min, adds respectively 1., 2., 3. then, transferring pH again is 7.0)
Then, the cinnamic acid and the phenylformic acid that in above-mentioned minimum medium, add 1.0g respectively.And this patent bacterial strain 2-8 is inoculated in above-mentioned containing in cinnamic acid or the benzoic substratum by 3% concentration, each concentration is provided with three repetitions.(cultivating down with the minimum medium similarity condition is contrast) observes its growing state and record.Represent the degree of its growth with "+", "-" expression is not grown.Measure OD value (wavelength is respectively 290nm and 272nm) with ultraviolet spectrophotometer at last, the results are shown in Table 7.
The result shows the cinnamic acid that this bacterial strain can be degraded more than 50% at the 4th day, but para Toluic Acid's degradation rate is not very high, can only reach about 28%.
Table 7, this patent bacterial strain 2-8 are to cinnamic acid and benzoic degraded measurement result
Embodiment 9 bacterial strain nitrogen fixing capacities are measured
With 1.5ml cultured bacterial strain in above-mentioned substratum 1,12000rpm, centrifugal 5min cleans for several times with aseptic physiological saline then, and with the aseptic physiological saline resuspension of 1ml, is inoculated on Ah Xu the shellfish nitrogen-free agar.Every strain bacterium connects 3 plates, and other gets 1 plating 1ml stroke-physiological saline solution in contrast, behind 30 ℃ of cultivation 5d, observes its growing state.The result shows that this bacterium can grow on nitrogen-free agar, illustrate that it has the fixed nitrogen characteristic.
Ah Xu shellfish nitrogen-free agar prescription: glucose 10.0g, potassium primary phosphate 0.2g, sal epsom 0.2g, calcium chloride 0.2g, calcium sulfate 0.1g, lime carbonate 5.0g, distilled water 1000ml, pH7.0.
Embodiment 10 bacterial strains are to the effect of increasing production research of continuous cropping cucumber
In the greenhouse of Center of Agriculture Sciences ﹠ Research, Zhuhai City, carry out volume increase research between in May, 2006 to June to continuous cropping cucumber, cucumber variety is Sa Ruige (Israel's import kind), design sub-district simultaneous test, contrast is general planting, handles and then use this patent bacterial strain 2-8 bacterium liquid 50ml (10 when culturing and transplanting seedlings
6Cfu/ml) pouring root, each sub-district 30 young plant, 3 repetitions, and from June 1 to June when cucumber was gathered in the crops on 17th, write down the output of different districts, the average ultimate production of check plot in 17d is the 3525g/30 strain, and the average ultimate production of sub-district in 17d after handling with 2-8 is the 5850g/30 strain.With the cell production after the 2-8 processing is 1.7 times that contrast, and this patent strains expressed goes out tangible effect of increasing production.
Claims (8)
1. one kind has the photosynthetic bacterium of efficient elimination factor to nitrite nitrogen, and this bacterial strain is Rhodopseudomonas palustris 2-8 (Rhodopseudomonas palustris 2-8), and the bacterial strain preserving number is CCTCC M 205147.
2. with the described photosynthetic bacterium of claim 1 aquaculture pool water is eliminated the treatment process of nitrite nitrogen for one kind, it is characterized in that using the described photosynthetic bacterium of following culture medium prescription production claim 1, the fermented liquid viable cell concentrations reaches 10
11Cfu/ml in the Penaeus vannamei aquaculture water, splashes by the consumption of every mu of 4kg, throws in once in 10～15 days; Substratum be following any one:
1. sodium acetate 0.2%, ammonium chloride (NH
4Cl) 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid; 2. Sodium Propionate 0.2%, ammonium chloride (NH
4Cl) 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid; 3. sodium succinate 0.2%, ammonium chloride (NH
4Cl) 0.1%, Sodium phosphate dibasic 0.05%, magnesium chloride 0.001%, Iron trichloride hexahydrate 0.001%, Zinc Sulphate Heptahydrate 0.0022%, sodium-chlor 0.2%, yeast extract paste or peptone 0.1～0.2%, 10% sodium hydrogen carbonate solution 50ml/L, ethanol 1.5～2.0ml/L transfers to pH7.0 with 0.1N phosphoric acid.
3. the application of the described photosynthetic bacterium of claim 1 is characterized in that being used for the elimination of aquaculture system nitrite nitrogen.
4. the rejuvenation method of the described photosynthetic bacterium of claim 1, concrete steps are: directly adopt the fertile mud at the bottom of the shrimp pool, the fish pond, add pool water and prawn feed, after the autoclaving cooling, rejuvenation bacterial strain Rhodopseudomonas palustris 2-8 is treated in access, anaerobism illumination cultivation 2～6 days, the bacterial strain that can obtain to have high reactivity nitrite nitrogen reducing power.
5. the culture collection process of the described photosynthetic bacterium of claim 1, after it is characterized in that gathering the fertile fresh bed mud 200～300g in the shrimp pool or fish pond, place the 500ml saline bottle, in saline bottle, add 10g Penaeus vannamei feed then, fill it up with saline bottle, cool off through autoclaving with fish pond or shrimp pool water, preservation strain Rhodopseudomonas palustris 2-8 is treated in access, after the illumination cultivation 3 days, the low light level is according to the room temperature preservation, and transfer once every half a year.
6. a bioassay method that is applicable to the nitrite nitrogen elimination ability of estimating the described photosynthetic bacterium of claim 1 is characterized in that this method adopts two evaluation systems, form by evaluation system 1 and evaluation system 2, wherein evaluation system 1, adds Sodium Nitrite in substratum, according to 10
6The concentration of cfu/ml adds microorganism to be measured, and the anaerobism illumination cultivation is measured the content that remains Sodium Nitrite in the substratum every 4～6h, and METHOD FOR CONTINUOUS DETERMINATION 3-4 time, data can be used for estimating the nitrite nitrogen of bacterial strain under this processing mode and eliminate ability; Evaluation system 2 is gathered and to be contained the pool water of nitrite nitrogen and the bed mud on this pool, and the mixed that adds 200～400g bed mud by the every premium on currency clarification of spending the night is got supernatant and is used for measuring, and presses the concentration adding sodium acetate of 2g/L before measuring, according to 10
6The concentration of cfu/ml adds microorganism to be measured, and illumination is aerobic or keep natural lighting and room temperature, the content of nitrite in solution of per 4～6h mensuration, and data are used to estimate the nitrite nitrogen of bacterial strain under this processing mode and eliminate ability.
7. the application of the described photosynthetic bacterium of claim 1 is characterized in that being used for cinnamic acid and benzoic biological degradation.
8. the application of the described photosynthetic bacterium of claim 1 is characterized in that being used to prepare continuous cropping cucumber volume increase microbial fertilizer.
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