CN100371437C - Process for preparing lichem bacillus strain for producing composite amino acid and culture amino acid liquid fertilizer - Google Patents
Process for preparing lichem bacillus strain for producing composite amino acid and culture amino acid liquid fertilizer Download PDFInfo
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Abstract
The present invention relates to a bacterial strain of bacillus licheniformis for producing composite amino acid, and a preparation method of amino acid liquid fertilizer for cultivation. In the method, the bacterial strain of bacillus licheniformis, obtained by mutagenesis sieving is utilized at a normal temperature and in an anaerobic environment to obtain composite amino acid liquid under the condition of inorganic salt without carbon sources and with high concentration; ammonium salt and phosphate are compounded to form the amino acid liquid fertilizer. When 1 to 10kg of amino acid liquid fertilizer is put into 1 mou of water body with 1m of water depth for prawn cultivation, algae growth in the water body is effectively promoted, and the amino acid liquid fertilizer is also suitable for other occasions which need composite amino acid as a substance for promoting growth. Compared with similar methods, the present invention has the advantages of low price and convenient source of used raw materials, simple product technology, no need of large-sized fermentation equipment, great cost reduction and mass production adoption. The product contains no ingredients which are not beneficial to the water body for cultivation and products, and is particularly suitable for prawn cultivation.
Description
Technical field
The invention belongs to a kind of lichem bacillus strain and culture amino acid liquid fertilizer preparation method who produces aminoacids complex.
Background technology
The key of ammino acid liquid fertilizer production is to make a large amount of aminoacids complexs.Making aminoacids complex has multiple technologies, and the one, chemical process hydrolysis Biological resources, as utilize strong acid and strong base that keratoprotein (hair, feather, hoof tips), beans, blood meal, cottonseed cake and rapeseed cake are handled; The 2nd, utilize the thalline behind the fermentation production of citric acid; The 3rd, utilize fermentation process to produce ammino acid liquid fertilizer.Utilize waste material hydrolysis behind keratoprotein or the industrial fermentation, making processes is to utilize high strength hydrochloric acid or sulfuric acid decomposing protein, the environmental problem of utilizing strong acid and strong base to bring in the treating processes, and also raw materials cost and equipment are unfavorable for that scale operation serves agricultural.The someone studies by aerobic fermentation and produces aminoacids complex.As patent 95119430: add at the acid treatment keratoprotein and to produce the M-Zyme aspergillus oryzae, make aqueous soluble protein be converted into amino acid, allocate the trace element of plant and needed by human at last into, can produce has the growth promoting effect nutrition agent to plant.Patent 92110718 is cultivated and is produced one or more amino acid whose brevibacterium sps and corynebacterium sp. strain fermentative preparation of amino acids.It is that amino acid technology is produced in the main raw material fermentation that patent is produced the tankage vinasse with brewery for 94,102,211 1 kinds.Patent 96109736 is to adopt mixed strains to prepare the manufacture method of phosphoric ammino acid liquid fertilizer.Patent 95197497 is bacterial strain generation organic acid and the amino acid whose methods with Microbacterium.Above-mentioned patent has proposed the technology that effective aminoacids complex is produced, and can use in agricultural after being made as ammino acid liquid fertilizer.Agricultural amino acid fertilizer major part is used for foliage fertilizer and remunerative crop, and these ammino acid liquid fertilizers are not suitable for the water body in large of aquaculture because cost limits.
Summary of the invention
The purpose of this invention is to provide a kind of lichem bacillus strain and culture amino acid liquid fertilizer preparation method who produces aminoacids complex, the Bacillus licheniformis bacterial classification that utilizes mutagenesis screening to obtain, the normal temperature anaerobism obtains Moriamin S under no carbon source high density inorganic salt condition, composite again ammonium salt and phosphoric acid salt form ammino acid liquid fertilizer, be invested in the prawn culturing water body according to every mu every meter depth of water 1-10 kilogram, can effectively promote the algae and water growth, also be fit to the occasion of other needs aminoacids complex as short surplus matter.
After cultivating in 30 ℃, 24 hours, to choose and wash bacterium colony, colony characteristics is irregular shape.With the conventional nutrient broth medium of making (1000 milliliters of peptone 5 grams, extractum carnis 3 grams, yeast extract paste 1 gram, sodium-chlor 5 grams, distilled water, adjusting medium pH is 7.4, conventional sterilization), at 30 ℃, per minute 140 changes shaking tables and cultivated 24 hours again.The same Temperature Treatment is used the dull and stereotyped purifying bacterial classification of nutrient broth agar again.Select bacterial classification, the same liquid culture is cultivated liquid and is added 20% glycerine, is stored in-20 ℃ as the purifying bacterial classification.
In Marine University Of Zhanjiang aquatic products disease research department the bacterial classification that adopts has been carried out determining again, determine that feature is as follows: bacterium elongated rod shape or fibrous, size is 1-10 micron * 0.3-0.8 micron, motion, Gram-positive, aerobic and amphimicrobian, produce gemma, gemma is rod-short, and is single, and V-P measures positive.Utilize propionic salt, glucose, pectinose, wood sugar or N.F,USP MANNITOL to produce acid, starch-splitting and gelatin, do not grow, growth is arranged at 55 ℃ at 5 ℃ and 10 ℃.Think B α cilluslicheniforms according to above-mentioned physiological and biochemical index.
Mutagenesis and seed selection: utilize sterilization (0.15MPa, 25 minutes, 250 milliliters of bottled 50 milliliters of nutrient solutions) nutrient broth medium, change shaking table at 30 ℃ of per minutes 180 and cultivated this bacterial classification 28 hours, getting culture inoculates on the nutrient broth agar, wash lawn in the bacterium logarithmic phase with stroke-physiological saline solution, add the aseptic glass strain of people and disperse lawn, leave the heart with per minute 3000 again, abandon supernatant, throw out adds stroke-physiological saline solution and makes bacteria suspension, the blood counting chamber counting, and adjusting concentration is 10
8/ milliliter.Get 2-3ml and place aseptic 5 centimetres of culture dish, be positioned over middle part, remove lid, shine and stopped 2 seconds in 10 seconds, shine again and stopped 2 seconds in 10 seconds, cumulative exposure 60 seconds-180 seconds through the household microwave oven (2450MHZ, 700 watts) of aseptically process.
Get peptone 2 grams, female extracting solution 0.1 gram of enzyme, N
aCl 0.3 gram, K
2HPO
40.5 gram, KH
2PO
40.1 gram, MgSO
47H
2O 0.3 gram, CaCl
20.1 800 milliliters of gram, distilled water, adjusting pH is 7.4, with pressure 0.15MPa, and sterilization in 15 minutes.Make 12 parts respectively, every part of difference increases by 10 grams again, 15 grams, 20 grams, 25 grams, 30 grams, 35 grams, 40 grams, 45 grams, 5 grams, 55 grams, 60 grams, 65 gram bicarbonate of ammonia.
Wherein every kind of concentration bicarbonate of ammonia is dissolved in respectively in 200 ml distilled waters, through the membrane filtration degerming of 0.25 micron pore size, joins in the nutrient solution of cool to room temperature of sterilized above-mentioned making, obtains the gradient seed selection substratum of bicarbonate of ammonia.Each irradiation back bacteria suspension is coated the gradient seed selection substratum of bicarbonate of ammonia, cultivates the mutagenesis bacterial classification 36-48 hour under 30 ℃ of conditions of lucifuge anaerobism.After each cultivation result is repeated once irradiating, progressively improve seed selection substratum ammonium salt concentration, step below the Bacillus licheniformis that selection can be grown continues in the above gradient seed selection of 40 gram bicarbonate of ammonia substratum.
Get peptone 2 grams, female extracting solution 0.1 gram of enzyme, N again
aCl 0.3 gram, MgSO
47H
2O0.3 gram, CaCl
20.1 800 milliliters of gram, distilled water are made 5 parts respectively, every part of difference increases by 4 grams again, 8 grams, and 12 grams, 16 grams, 20 gram phosphoric acid salt (potassium primary phosphate or calcium superphosphate) obtain phosphatic gradient seed selection substratum.Adjust pH7.4, with pressure 0.15MPa, sterilization in 15 minutes.Each part increases the bicarbonate of ammonia that is dissolved in the same membrane filtration degerming of the gram of 45 in 200 ml distilled waters again, inoculate the mutagenesis bacterial classification of seed selection, inoculum size 5% leaves standstill 3-7 day, and the microscope blood counting chamber measures bacterial biomass and ply of paper is analysed amino acid in the triketohydrindene hydrate dyeing mensuration supernatant liquor.
If do not increase ammonia nitrogen and inorganic phosphorus, when leaving standstill anaerobically fermenting, the quantity of experimental bacteria increases few, and the Keratin sulfate hydrolysis time is long, and the amino acid quantity of generation is few, and needs increase temperature (as 50 ℃) just can reach certain decomposition rate.In the present invention, this bacterium greater than, equal in the presence of the 4% ammonia salt, bacterial number increases rapidly, materials such as amino acid increase sharply, (28 ℃-30 ℃) can reach 14 grams per liters in the time of the 7th day at normal temperatures.Experimental results show that in this course because substratum does not have other carbohydrate as carbon source, bacterium has been strengthened keratic decomposition, under this ammonium salt and phosphate concn, produce justacrine amino acid simultaneously.
With clorox-potassium iodide method and hydrochloric acid-ethanol titration measuring amine, abandon producing amine and make the smelly bacterial strain of nutrient solution.Bacterial strain by above mutagenic and breeding obtains adds 20% glycerine, in-20 ℃ of preservations in bacterium liquid.
This bacterial classification is Bacillus licheniformis (Bacillus licheniforms), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (China GeneralMicrobiological Culture Collection Center on 01 04th, 2005, CGMCC), preservation and survival certificate number are CGMCCN
o.1285.
The bacterial classification safety experiment: the bacterial classification of seed selection is cultivated with nutrient broth, utilizes microscope inspection and 100 times of countings of dilution after the cultivation, and making bacterial concentration is 10
9/ milliliter.Culture about 10 centimetres healthy Environment of Litopenaeus vannamei Low of 6 tails, every group of 3 culturing jars, totally 3 groups with 32 liter of 15 ‰ clean seawater inflation in the culturing jar.Throw in 0.5 milliliter every day respectively, 1 milliliter, 2 milliliters of nutrient broths are cultivated bacterium liquid, and continuous 25 days, exchange 5-6 rose clean seawater in per 2 days.Observe the ingesting of Environment of Litopenaeus vannamei Low, cast off a skin, growth, activity be normally.
Determined amino acid:, adopt Paper Chromatography to carry out conventional semiquantitative determination because the interference of ammonia nitrogen in the culture can not be measured its amino acid with the amino acid whose method of conventional determining.28 * 28 centimetres of Xinhua's chromatography filter paper, applied sample amount 10-20 microlitre, exhibition layer liquid is propyl carbinol: glacial acetic acid: ethanol: water=4: 1: 1: 2 (V/V), developer are 0.5% triketohydrindene hydrate acetone soln.Elutriant be 75% ethanol and 0.1% copper-bath (38: 2, V/V).Use different standard amino acid (biochemical pure) to make various amino acid whose typical curves and slope respectively.Tomographic results subtracts down according to spot, and wash-out is measured absorbance value at OD520.Distinguish amino acid according to different Rf values, adopt Different Slope to calculate content.
HP LC quantitative assay amino acid: sample liquid is centrifugal through supercentrifuge 10000g, gets supernatant liquor, and 0.25 micron membrane filtration through pre-treatment, is measured supernatant liquor amino acid with high performance liquid chromatograph.
Measure protein content with the forint phenol method.Measure the content of phosphorus with the phosphorus molybdenum blue method.Soluble sugar detects with the sulfuric acid phynol method.
The making of ammino acid liquid fertilizer:
Utilize shaking table and 500 milliliters of bottles to carry out seed culture.Bacterium culture medium consists of peptone 5 grams, extractum carnis 3 grams, yeast extract paste 1 gram, NaC 15 grams, 1000 milliliters in water.Adjusting pH is 7.4, at 121 ℃, sterilizes 25-30 minute.The inoculation back is at 28-32 ℃, and 125 rev/mins of shaking tables were cultivated 24-48 hour.
Substratum is produced in preparation, can adopt following two kinds of substratum:
(1) feather meal is with 2-2.5 times of 30%-40% mixed in hydrochloric acid, at 121 ℃, and 0.15MPa sterilization 30-60 minute, perhaps 126 ℃ of oil removals of oil bath were handled 60-120 minute, were 7 with the NaOH accent pH of 4M, obtained handling feather meal;
Get NH
4Cl 0.5 gram-1 gram, N
aCl 0.3 gram-1 gram, K
2HPO
40.5 gram-1 gram, KH
2PO
40.1 gram-1 gram, MgSO
47H
2O 0.1g, CaCl
20.3 gram, yeast extract 0.1 gram-0.5 gram, 1000 milliliters in feather albumen powder 5 grams-15 grams, water, accent pH is 7.4-8.0, enlarges consumption according to preparing with equal proportion.
(2) NH
4Cl 0.5 gram-1 gram, NaCl 0.3 gram-1 gram, K
2HPO
40.5 gram-1 gram, KH
2PO
40.1 gram-1 gram, MgSO
47H
2O 0.1-1 gram, CaCl
20.1 gram-0.3 restrains, yeast extract 0.1 gram-0.5 restrains, feather albumen powder 5 restrains-10 grams, peptone 3 restrains 1000 milliliters in-5 grams, water, accent pH is 7.4-8.0.Enlarge consumption according to preparing with equal proportion.
In 100 liters of fermentor tanks, add medium component and water, 121 ℃ of sterilizations 60 minutes, be cooled to 30 ℃, inoculation bacterial classification, inoculum size 1-5%.30 ℃-35 ℃ of temperature aerobic fermentation 24-36 hour.
Add 2%-6.5% bicarbonate of ammonia (W/V) in above-mentioned nutrient solution, the sterilization phosphoric acid dioxy potassium of 0.4%-2% or calcium superphosphate (W/V) fully stir, and divide to install in 10 liters of containers.Seal, in 28 ℃-35 ℃ of temperature or room temperature, or higher temperature, placed 5-10 days, obtain aminoacids solution.Product has the certain esters flavor, is thick liquid attitude, and black or tawny, pH value are 8.
In the aminoacids solution that obtains, add bicarbonate of ammonia and 0.7%-2% potassium primary phosphate or the calcium superphosphate of 7%-10%, obtain ammino acid liquid fertilizer, can preserve 6 months as product.Potassium primary phosphate that uses or calcium superphosphate were 121 ℃ of sterilizations 30 minutes, and the carbonic acid gas ammonium requires to select quality good, does not have other impurity person, directly uses
Utilize the aminoacids content of high performance liquid phase spectrum quantitative check Moriamin S.
Amino acid anaerobism 7 days, mg/ml amino acid anaerobism 7 days, mg/ml
asp 0.035 cys 0.732
ghu 0.274 val 1.427
ser 0.308 met 6.327
his 0.067 phe 0.462
gly 0.785 ile 0.423
thr 0.192 leu 0.805
arg 0.261 lys 0.425
ala 0.638 pro 1.008
tyr 0.279
Amount to 14.446
According to the analysis met of a plurality of samples, val, pro is higher amount always, gly, ser, leu, ala have higher amount as a rule, phe, cys and lys have moderate, and arg and ile have a certain amount of under a few cases.The minimum number of his and asp wherein.
The safety experiment of ammino acid liquid fertilizer: culture about 10 centimetres healthy Environment of Litopenaeus vannamei Low of 6 tails, totally 3 groups, every group of 3 culturing jars with 32 liter of 15 ‰ clean seawater inflation in the culturing jar.Throw in the ammino acid liquid fertilizer of 1 milliliter/liter making every day respectively, continuous 25 days, exchange 5-6 rose clean seawater in per 2 days.The ingesting of Environment of Litopenaeus vannamei Low, cast off a skin, growth, activity be normally.
The little algae usage quantity of laboratory culture: eutrophy requires planktonic algae such as marine chlorella: 1% (sample/water, V/V); The moderate nutritional requirement as: egg capsule algae, Skeletonema Greville 1 ‰ (sample/water, V/V); Poor nutritional requirement such as part diatom: 0.1 ‰.
The prawn culturing water body uses: 2-15ppm.Splash.
The effect that produces: measure the effect of ammino acid liquid fertilizer for algal grown
1, cultivates little algae of prawn feed and breed water planktonic microalgae.
Clean seawater boils postcooling, adds an amount of little algae of people respectively, 1 grams per liter ammino acid liquid fertilizer, and the algae with same initial concentration adds the inorganic salt nutrient solution in addition, in contrast.Cultivation results is counted at microscopically with blood counting chamber.
Table 1 ammino acid liquid fertilizer is to the cultivation of chlorella
| Marine chlorella quantity | Initial | 24 hours | 48 hours | 192 hours | 216 hours | 240 hours |
| ×10 6/ milliliter | ||||||
| Ammino acid liquid fertilizer inorganic salt nutrient solution | 0.61 0.61 | 5.68 5.40 | 7.90 6.13 | 20.92 14.80 | 37.20 16.80 | 55.12 19.24 |
Chlorella inorganic salt nutrient solution composition: NaNO
380 milligrams, K
2HPO
48 milligrams, FeC
6H
5O
7(1% solution) 0.2 milliliter, VB
1200 micrograms, VB
120.2 microgram, NaHCO
3(0.5 gram.1000 milliliters in seawater.Cultivating liquid pH is 7-8, and culture temperature 25-28 ℃, illumination is 2000-8000LUX, salinity 10.
The cultivation of table 2 ammino acid liquid fertilizer centering Skeletonemacostatum
| Middle Skeletonemacostatum quantity * 10 6/ milliliter | Initial | 24 hours | 48 hours | 192 hours | 216 hours | 240 hours |
| Ammino acid liquid fertilizer inorganic salt nutrient solution | 23.41 23.41 | 38.9 32.4 | 76.4 44.2 | 245 106 | 288 102 | 321 88 |
Middle Skeletonemacostatum inorganic salt nutrient solution composition: KNO
360 milligrams, Na
2HPO
412H
2The O10 milligram, Na
2 SiO
310 milligrams, 1000 milliliters in seawater.Cultivating liquid pH is 8.4, culture temperature 25-28 ℃, and illumination 2000-8000LUX, salinity 25.
Table 3 ammino acid liquid fertilizer is to the cultivation of Zhanjiang Isochrysis galbana quantity
| Zhanjiang Isochrysis galbana quantity * 10 6/ milliliter | Initial | 24 hours | 48 hours | 192 hours | 216 hours | 240 hours |
| Ammino acid liquid fertilizer inorganic |
15 | 56 34 | 98 68 | 548 344 | 680 276 | 732 198 |
Zhanjiang Isochrysis galbana inorganic salt nutrient solution composition: NaNO
330 milligrams, 15 milligrams in urea, KH
2PO
46 milligrams, FeC
6H
5O
75H
20.5 milligram of O, 1 milligram of water glass, VB
1200 micrograms, VB
120.2 microgram, 1000 milliliters in seawater.Cultivating liquid pH is 8.4, culture temperature 25-28 ℃, and illumination 2000-8000LUX, salinity 25.
In addition, this product has similar effect to egg capsule algae, Nannochloropsis oceanica, spirulina, flat algae, Dunaliella salina.
2, big area behaviour in service
In Zhanjiang region, in water temperature 26-27 ℃ of April, sunshine is abundant.Before the prawn mature stage is put seedling, 2.5 kilograms of the ammino acid liquid fertilizers of every mu every meter depth of water input making, plant plankton began to increase sharply after 24 hours, and 48 hours make water colour is light green or Sandy.Algae and water reached the 104-105/ milliliter in 72 hours.After throwing seedling, this water colour can be kept 15-20 day.
2001-2003, the ammino acid liquid fertilizer of making has been used in record prawn intensive culture pond under Suixi, Zhanjiang, and the prawn culturing success ratio is bordering on 100% for several years running, and the contrast pond success ratio of same shrimp seedling is lower than 50%.At whole prawn culturing early metaphase, the ammino acid liquid fertilizer that input in per 15 days-20 days is made once can keep water colour stable in whole breed period.Its advantage has: 1, when prawn culturing is thrown in inorganic nutrient salt to water body in early days, increase sharply ammonia nitrogen or nitrite nitrogen concentration, and make the shrimp seedling produce disease easily, the ammino acid liquid fertilizer of use has been avoided this problem.2, can in 15 days-20 days, stablize water colour, keep planktonic algae quantity.3,, throw in the back and do not pollute because the ammino acid liquid fertilizer of making belongs to fermented product.Render to shrimp culture pond with the ammino acid liquid fertilizer that chemical process is made, make the water body blackening easily.In June, 2003-September, seedling 13-15 ten thousand tails are put in this prawn intensive culture pond, and early metaphase is all cultivated water colour with the ammino acid liquid fertilizer of making, and use altogether 4 times in preceding 65 days, keep water colour good.Culturing time is 70 days-90 days, results prawn 64-76 tail/kilogram, and every mu of 11500-1250 kilogram of output, every mu every batch has profit about 12000.
The experimental result of the ammino acid liquid fertilizer of prawn intensive culture pond use in record and East Sea Island, Zhanjiang Buddhist nunnery under the Suixi, Zhanjiang in 2004 is good, and breed is succeeded.Gather in the crops prawn every mu 1250-1777 kilogram in the East Sea Island Buddhist nunnery in July, 2004, and size is 90 tail/kilograms.
The ammino acid liquid fertilizer of experiment use in prawn intensive culture pond in East Sea Island, Zhanjiang Buddhist nunnery on May 31,13 days to 2004 February in 2004 does not re-use other and cultures articles for use and medicine except that bait throwing in.Before the breed, throw in the ammino acid liquid fertilizer of making, each 2.5 kilograms every mu, put seedling and strengthened once in 3 days later on, threw in once, and threw in altogether 10 times in later per 15 days.Can see that from diagram early stage water temperature 18-25 ℃, high-density breeding algae and water chlorophyll changes steadily in the 10-20 micrograms per litre.Be elevated to 25-30 ℃ in breed middle and later periods temperature, chlorophyll increases, in experiment 3# pond (5 mu of areas) algae chlorophyll 30-60 micrograms per litre, in experiment 2# pond (5 mu of areas) algae chlorophyll 35-70 micrograms per litre.Whole breeding process does not have " pouring " phenomenon of the algae sudden death of overrich to take place, and has guaranteed the stable of prawn growing environment, has effectively suppressed the quantity of water body pathogenic bacteria simultaneously.
13 days to 2004 Mays of February in 2004 experiment pool algae on the 31st chlorophyll amounts figure, as shown in Figure 1, 2.
On May 31st, 2004 was measured the water-quality guideline of experiment pool, and the water quality that data declaration is cultured is good, and data are as follows:
The lichem bacillus strain of mutagenic and breeding of the present invention can be grown under 4-6.5% bicarbonate of ammonia .0.4-2% phosphoric acid salt and no carbohydrate, FAF and organic acid condition; At 4-6.5% bicarbonate of ammonia, 0.4-2% phosphoric acid salt with do not have carbohydrate, FAF and organic acid and anaerobism, temperature are to produce the amino acid justacrine to the extracellular under 25 ℃-35 ℃ the condition, do not produce the amine substance that makes nutrient solution smelly.
The method that the present invention utilizes this Bacillus licheniformis bacterial classification to produce ammino acid liquid fertilizer comprises the steps:
(1) the Bacillus licheniformis bacterial classification utilized conventional nutrient broth to cultivate, and adjustment pH is 7.0-8.0,121 ℃ of sterilizations 25-30 minute, cultivated 24-48 hour at 28 ℃ of-32 ℃, 125 rev/mins shaking tables the inoculation back, culture adds 20% glycerine as preserving bacterial classification, in-20 ℃ of preservations, every half a year transferred species once.
(2) aerobic fermentation increases the bacterial classification amount: get NH
4Cl 0.5 gram-1 gram, N
aCl 0.3 gram-1 gram, K
2HPO
40.5 gram-1 gram, KH
2PO
40.1 gram-1 gram, MgSO
47H
2O 0.1 gram-1 gram, CaCl
20.1 gram-0.3 gram, yeast extract 0.1 gram-0.5 gram, feather albumen powder 5 restrain 1000 milliliters in-15 grams, water, accent pH is 7.4-8.0; Enlarge consumption according to preparing with equal proportion; According to routine sterilization, the cooling back is by 1% inoculation bacterial classification, 30 ℃-35 ℃ aerobic fermentation 24-48 hour, obtain nutrient solution; Feather albumen powder 2-2.5 times of 30%-40% mixed in hydrochloric acid, at 121 ℃, steam sterilizer sterilization 30-60 minute, perhaps 126 ℃ of oil removals of oil bath were handled 60-120 minute, were 7 with the NaOH accent pH of 4M, obtained handling feather meal;
(3) add 2%-6.5% (W/V) bicarbonate of ammonia in nutrient solution, sterilization potassium primary phosphate or the calcium superphosphate of 0.4-2% (W/V) dissolve and stir, with 10 liters of-20 liters of container packing, after the sealing, placed 5-10 days for 28 ℃-35 ℃, get Moriamin S in temperature;
(4) bicarbonate of ammonia and 0.7%-2% potassium primary phosphate or the calcium superphosphate of adding 7%-10% in the Moriamin S that obtains obtain ammino acid liquid fertilizer, can preserve 6 months as product.
The present invention and relatively with class methods, the cost of material that the present invention uses is cheap, convenient sources, product technology is simple, does not need large-scale fermentation equipment, reduces the cost significantly, is suitable for big production; Product does not contain any composition that is unfavorable for aquaculture water and product, is particularly suitable for prawn culturing.
Description of drawings
Fig. 1 is a high-density prawn culturing water body chlorophyll variation diagram of the present invention;
Fig. 2 is an aquaculture water temperature variation of the present invention.
Embodiment
Bacterial classification of the present invention comes from Chinese common micro-organisms culture presevation administrative center (Beijing, bacterial classification is Bacillus licheniformis B α cillus licheniforms (Weigmann) Chester, strain number 1.813), fetch behind the laboratory with 95 ℃ of water bath processing 40 minutes, coat 2216E seawater plate culture medium at once, grams per liter, chemical oxygen demand 22.36 mg/litre, nitrate nitrogen 0.008 mg/litre, nitrous acid nitrogen 0.021 mg/litre, ammonia nitrogen 0.805 mg/litre, basicity 2.891, hydrogen sulfide is less than 0.01 mg/litre.
33000/milliliter of the aerobic autotrophic bacterias of protein type, 15000/milliliter of anaerobism autotrophic bacterias, 1475/milliliter in vibrios, 2000/milliliter on fermented type bacterium, 2000/milliliter of nitrobacterias, chlorophyll 44.65 mg/litre.
10000/milliliter of the good type oxygen of albumen autotrophic bacterias, 10000/milliliter of anaerobism autotrophic bacterias, 1010/milliliter in vibrios, 300/milliliter on fermented type bacterium, 2300/milliliter of nitrobacterias, chlorophyll 41.86 mg/litre.
Claims (2)
1. a lichem bacillus strain of producing aminoacids complex is characterized in that it is the lichem bacillus strain of mutagenic and breeding, can and not have under carbohydrate, FAF and the organic acid condition and grow at 4-6.5% bicarbonate of ammonia, 0.4-2% phosphoric acid salt; At 4-6.5% bicarbonate of ammonia, 0.4-2% phosphoric acid salt with do not have carbohydrate, FAF and organic acid and anaerobism, temperature are to produce the amino acid justacrine to the extracellular under 25 ℃-35 ℃ the condition, do not produce the amine substance that makes nutrient solution smelly; This bacterial classification is Bacillus licheniformis (Bacilluslicheniforms), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (China General Microbiological Culture Collection Center on 01 04th, 2005, CGMCC), preservation and survival certificate number are CGMCC No.1285.
2. the method for a culture amino acid liquid fertilizer preparation is characterized in that comprising the steps:
(1) lichem bacillus strain of claim 1 utilizes conventional nutrient broth to cultivate, adjustment pH is 7.0-8.0,121 ℃ of sterilizations 25-30 minute, cultivated 24-48 hour at 28 ℃ of-32 ℃, 125 rev/mins shaking tables the inoculation back, culture adds 20% glycerine as preserving bacterial classification, in-20 ℃ of preservations, every half a year transferred species once;
(2) aerobic fermentation increases the bacterial classification amount: get NH
4Cl 0.5 gram-1 gram, NaCl 0.3 gram-1 gram, K
2HPO
40.5 gram-1 gram, KH
2PO
40.1 gram-1 gram, MgSO
47H
2O 0.1 gram-1 gram, CaCl
20.1 gram-0.3 gram. yeast extract 0.1 gram-0.5 restrains, handles 1000 milliliters in feather meal 5 grams-15 grams, water, and accent pH is 7.4-8.0; Enlarge consumption according to preparing with equal proportion; According to routine sterilization, the cooling back is by 1% inoculation bacterial classification, 30 ℃-35 ℃ aerobic fermentation 24-48 hour, obtain nutrient solution; Described processing feather meal is obtained by following mode: feather meal and 2-2.5 times of 30%-40% mixed in hydrochloric acid, 121 ℃ of steam sterilizer sterilizations 30-60 minute, perhaps 126 ℃ of oil removals of oil bath were handled 60-120 minute, were 7 with the NaOH accent pH of 4M, obtained handling feather meal;
(3) add 2%-6.5% (W/V) bicarbonate of ammonia in the nutrient solution of step (2), sterilization potassium primary phosphate or the calcium superphosphate of 0.4-2% (W/V) dissolve and stir, with 10 liters of-20 liters of container packing, after the sealing, placed 5-10 days for 28 ℃-35 ℃, get Moriamin S in temperature;
(4) bicarbonate of ammonia and 0.7%-2% potassium primary phosphate or the calcium superphosphate of adding 7%-10% in the Moriamin S that step (3) obtains obtain ammino acid liquid fertilizer, can preserve 6 months as product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100331113A CN100371437C (en) | 2005-02-01 | 2005-02-01 | Process for preparing lichem bacillus strain for producing composite amino acid and culture amino acid liquid fertilizer |
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| CNB2005100331113A CN100371437C (en) | 2005-02-01 | 2005-02-01 | Process for preparing lichem bacillus strain for producing composite amino acid and culture amino acid liquid fertilizer |
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| CN102718605B (en) * | 2012-07-19 | 2013-12-11 | 湖南省烟草公司郴州市公司 | Method for producing amino acid tobacco-specific foliar fertilizer by using abandoned fur |
| CN103121884B (en) * | 2013-02-04 | 2014-12-10 | 河北根力多生物科技股份有限公司 | Preparation method of multifunctional bioprotein controlled release fertilizer |
| CN105925484A (en) * | 2016-06-29 | 2016-09-07 | 广东海洋大学 | Normal temperature preservation method for oocystis |
| CN111733118B (en) * | 2020-08-17 | 2020-12-04 | 中国科学院烟台海岸带研究所 | Bacillus PL-2 and application thereof in aquaculture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5063161A (en) * | 1988-03-31 | 1991-11-05 | North Carolina State University | Method of degrading keratinaceous material and bacteria useful therefor |
| CN1070687A (en) * | 1991-09-17 | 1993-04-07 | 底古萨股份公司 | The method of fermentative preparation of amino acids |
| CN1133275A (en) * | 1995-12-29 | 1996-10-16 | 洛滨 | Znzyme transformed organic fertilizer |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5063161A (en) * | 1988-03-31 | 1991-11-05 | North Carolina State University | Method of degrading keratinaceous material and bacteria useful therefor |
| CN1070687A (en) * | 1991-09-17 | 1993-04-07 | 底古萨股份公司 | The method of fermentative preparation of amino acids |
| CN1133275A (en) * | 1995-12-29 | 1996-10-16 | 洛滨 | Znzyme transformed organic fertilizer |
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