CN101735966A

CN101735966A - Strong bacillus G25-1-2 and applications thereof

Info

Publication number: CN101735966A
Application number: CN200910192615A
Authority: CN
Inventors: 董俊德; 凌娟; 张燕英; 李丽璇; 王友绍; 张偲
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2009-09-25
Filing date: 2009-09-25
Publication date: 2010-06-16
Anticipated expiration: 2029-09-25
Also published as: CN101735966B

Abstract

The invention discloses a strong bacillus G25-1-2 and applications thereof. The bacillus has been preserved by China Center for Type Culture Collection in Wuhan University (short for CCTCC) on 4th, June, 2009, and the preservation number is CCTCC NO:M209122. The bacillus is a pure bacillus with high-efficiency nitrogen-fixing activity, and is separated and sieved from a seaweed ecological system. When the strong bacillus G25-1-2 is used for processing seaweed, compared with a test control group, the processed seaweed has higher plant height, stem diameter average value, carotene content, carotenoid content and soluble saccharide content, thereby the result shows that the bacillus has good growth promotion effect on the seaweed. The strong bacillus G25-1-2 can be used for seaweed nursing and restoration of the whole seaweed ecological system, and has good promotion functions on the nitrogen circulation of the coral reef-seaweed bed composite ecological system and the maintenance of high production level.

Description

Strong bacillus G 25-1-2 and application thereof

Technical field

The present invention relates to microbial technology field, be specifically related to microorganism fixed nitrogen, relate in particular to a kind of strong bacillus G 25-1-2 and application thereof.

Background technology

Wetlands ecosystems are a kind of special ecosystems, produce protein 9g/m every year on average ²Be 3.5 times of TERRESTRIAL ECOSYSTEMS, but also have the water source of to regulate and store, stablize the strand water front, regulate the climate, purify water and fix nutraceutical function, preserve species, provide basic ecological benefits such as wildlife habitat, for industry, agricultural, the energy, hospitality industry etc. provide the mass production raw material.

Karang and sea grass bed are the important component parts of tropical ocean Wetlands ecosystems, the higher level of the productive forces and important ecological significance are their basic features, but the vulnerability and the volatility that also possess Wetlands ecosystems simultaneously are particularly owing to variation and the effect of human activity aggravation along with global climate in recent years caused species diversity reduction, ecological functions degradation phenomena.In order to protect and to recover karang and sea grass bed Wetlands ecosystems, global various countries have launched a series of action in succession.

Discover, biological nitrogen fixation, promptly be meant the process of the biology of tool nitrogen fixing capacity with the reduction of the nitrogen in atmosphere ammonification, at karang and be that the sea grass bed ecosystem plays important effect, show airborne nitrogen is introduced the ecosystem, by nitrification, denitrification, ammonification diazotroph synthetic organonitrogen is carried out Transfer Mechanism to the ecosystem trophic structure of sea grass bed then, and then promoted the flow of matter and the Nutrient cycle of whole ecological system.

Be used for keeping of the ecosystem and recovery aspect about bio-azotobacter fertilizer, the mangrove ecosystem exploration that taken the lead in having launched, the scientist of country such as Mexico and India utilizes plant-growth to urge living bacterium (PGPB, plant growth-promoting bacteria) mangrove forest is urged living certain effect that obtained.

In the paddy rice of grass, also there be existing the expansion to use, Chile scientist Iris Pereira (2009, Development of a biofertilizer based on filamentous nitrogen-fixing cyanobacteriafor rice crops in Chile, Journal of Applied Phycology, 21 (1), 135-144) by the fixed nitrogen cyanobacteria being used for the big area test in rice field, found that with traditional nitrogenous fertilizer and compare, after using bio-bacterial manure, the amount of nitrogenous fertilizer of using can be reduced half, i.e. (50kg N ha ^-1), but still can obtain higher output (7.4t ha ^-1).

The bacterium of the existing strong class bacillus of discovering (Paenibacillus validus), has PAHs degradation function (Daane LL, 2002, PAH-degradation by Paenibacillus spp.anddescription of Paenibacillus naphthalenovorans sp nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants, International journal of systematic and evolutionary microbiology, 2002,52 (1), 131-139) with short function (the Ulrich H ildebrandt that gives birth to, 2006, The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation of fertile spores, FEMS Microbiology Letters, 254 (2), 258-267) etc., still find to have the fixed nitrogen function as yet.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, a kind of strong bacillus G 25-1-2 with higher nitrogenase activity is provided.

Another object of the present invention is to provide the application of above-mentioned strong bacillus G 25-1-2 in the biological nitrogen fixation of sea grass-karang complex ecosystem.

Another object of the present invention is to provide above-mentioned strong bacillus G 25-1-2 in the short application of giving birth in the preparation of preparation sea grass.

Above-mentioned purpose of the present invention is achieved by following scheme:

Strong bacillus G 25-1-2 of the present invention belongs to Paenibacillus and belongs to (class Bacillus), its English name is Paenibacillus validus G25-1-2, be preserved in Wuhan University China typical culture collection center on June 4th, 2009, it abbreviates CCTCC as, and deposit number is CCTCCNO:M209122.

Strong bacillus G 25-1-2 of the present invention is that the Paenibacillus of the reported first isolated nifH of having gene from sea grass belongs to bacterium, this strong bacillus G 25-1-2 is the bacterium direct rod shape, Gram-positive, move with peritrichous, bacterium size 0.2-0.4 * 3.1-5.2 μ m, cell is single, also has less paired.

Strong bacillus G 25-1-2 of the present invention is grown on nitrogen-free agar and is canescence, and is translucent, smooth, and circle is cultivated down for 30 ℃, colony diameter 1～1.5mm after three days, and the optimum growth temperature scope is 25～37 ℃, maximum growth temperature is 42 ℃; Oval-shaped brood cell is arranged in the cyst that expands, endways or near terminal place, both sides to outer process or or form at a side and thalline and must angle to expand projection, on nutrient agar medium, there is not soluble pigment, strict aerobic, catalase feminine gender, oxidase negative, the V-P feminine gender, edwardsiella hoshinae.

Strong bacillus G 25-1-2 of the present invention detects through acetylene reduction method (ARA), finds that this bacterium can utilize N ₂As nitrogenous source, have higher biological nitrogen fixation activity, under the pure culture condition, its nitrogenase activity 30～60umolC ₂H ₂h ^-1Mg ^-1Fresh weight.

Use the specific primer PolF/PolR of nif (with reference to Poly et al., 2001, Comparison ofnifH Gene Pools in Soils and Soil Microenvironments with Contrasting Properties, Applied and Environmental Microbiology, 2001,67 (5) 2255-2262,) and bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (with reference to Weisenburg et al., 1991,16Sribosomal DNA amplification for phylogenetic study.Journal of bacteriology, 1991173 (2): 697-703) strong bacillus G 25-1-2 of the present invention is carried out pcr amplification, all amplification obtains the purpose fragment, proves that bacterial strain of the present invention has nif.

Nif nifH sequence and 16S rDNA sequence that above-mentioned amplification obtains are carried out sequencing analysis, by the BLAST software among the GenBank correlated series in nif nifH sequence and 16S rDNA sequence and the GenBank database is compared, on all four sequence is not found in discovery in database, show the new gene order of these 2 gene orders, successfully submit new gene order to Genebank and obtain accession number and be: EU884567 (16S rDNA sequence) and FJ875968 (nif nifH sequence) for finding first

Adopt next sea grass with strong bacillus G 25-1-2 processing of the present invention from San Yawan; found that; its plant height of edible seaweed after treatment and the thick mean value of stem are all apparently higher than untreated sea grass; and carotene carotene content, carotenoid content and soluble sugar content also all are higher than untreated sea grass; explanation thus; strong bacillus G 25-1-2 of the present invention comes into force fruit better for sea grass short, has huge potential significance for the protection and the recovery of the sea grass bed ecosystem.

Strong bacillus G 25-1-2 of the present invention can be used for the biological nitrogen fixation of sea grass-karang complex ecosystem, also can be used for preparing the short preparation of giving birth to of sea grass.

Compared with prior art, the present invention has following beneficial effect:

The present invention from sea grass separation screening to pure bacterial strain, and by this pure bacterial strain is carried out triple screenings: nitrogen-free agar growth situation, nitrogenase activity determination result, nifH gene amplification have nothing, thereby determine fast whether bacterial classification is vinelandii, finally obtain having new bacterial strain---the strong bacillus G 25-1-2 of efficient nitrogen fixation activity, strong bacillus G 25-1-2 has that adaptive faculty is wide, the characteristic of strong stress resistance and efficient nitrogen fixation activity, and its nitrogenase activity can reach 30～60umolC ₂H ₂h ^-1Mg ^-1Fresh weight;

2. strong bacillus G 25-1-2 of the present invention has nitrogen fixation effect preferably, can be prepared into the azotogen of nitrogenase activity height, anti-drying, strong stress resistance, and be convenient to standing storage and transportation after the microbial inoculum drying can being made dry powder, be suitable for making powdery or particulate state microbial fertilizer, be the best bacterial classification that Azobacter updates, therefore have broad application prospects;

3. strong bacillus G 25-1-2 of the present invention has significant growth-promoting functions to sea grass, can be used for the child care of sea grass and the recovery of the ecosystem, and to karang-sea grass bed complex ecosystem the circulation and the keeping of the higher level of the productive forces of nitrogen promoter action is preferably all arranged.

Description of drawings

Fig. 1 is the PCR electrophoresis result figure of the pure bacterial strain of embodiment 1 separation and purification gained;

Wherein, 1 is the genomic dna of bacterial strain, the 2 16S rDNA that obtain for pcr amplification, and the 3 nif nifH that obtain for pcr amplification, M1 is a λ DNA/Hind III standard molecular weight object of reference, M2 is Marker DL2000;

Fig. 2 is the phylogenetic tree of strong bacillus G 25-1-2 in 16S rDNA.

Embodiment

Below in conjunction with specific embodiment the present invention is done description further, but specific embodiment is not done any qualification to the present invention.

The separation and purification of embodiment 1 bacterial strain

The sampling position of present embodiment is sea grass (18 ° of 15 ' 08.6 " N of growth after karang protective belt, gulf, Sanya, Sanya, Chinese Hainan Province coral is degenerated; 109 ° 30 ' 51.1 " E), main sea grass kind is Old Taylor algae (Thalassia hemprichii), behind the sample collecting, each sample is classified respectively, is numbered, sealing in the polyethylene bag of the sterilization of packing into places and takes back the laboratory in the ice chest ,-20 ℃ of preservations.

With the sea grass sample of above-mentioned collection, get rhizosphere (being leaf sheath and root stock), be cut into fritter about 2cm with the scissors of sterilization, (this substratum contains NaCl 25g, KCl 0.56g, MgSO to put into selectivity fixed nitrogen liquid nutrient medium ₄-7H ₂O 4.8g, MgCl ₂-6H ₂O 4.0g, K ₂HPO ₄0.01g, FeSO ₄-7H ₂00.001g, Tris 0.48g, peptone 4.0g, yeast extract 2.0g, glycerine 2.0ml and distilled water 1L, whole pH is 8.2) add and richly cultivate (30 ℃, 180rpm, constant temperature 48h), then nutrient solution is diluted to the concentration of a series of gradients, the concentration of each gradient is taken out 200 μ L and is inoculated with coating method, three repetitions of each gradient, 30 ℃, 48h cultivates the back and selects single bacterium colony to be further purified.

With the above-mentioned single bacterium colony of inoculating needle picking, through smear staining, under opticmicroscope, observe thalli morphology, impure as the isolating bacterium colony of institute, should repeat once more to separate with spread plate once more after the series concentration dilution, until obtaining pure bacterial strain, then with pure inoculation to slant medium (test tube behind the potato agar medium sterilization of conventional formulation is inclined to the inclined-plane to be solidified preparation and gets), be used for the late detection nitrogenase activity.

The pure bacterial strain that present embodiment is separated to is the bacterium direct rod shape, Gram-positive, and with the peritrichous motion, bacterium size 0.2-0.4 * 3.1-5.2 μ m, cell is single, also has less paired; Growth is canescence on nitrogen-free agar, and is translucent, smooth, and circle is cultivated down for 30 ℃, colony diameter 1-1.5mm after three days, and the optimum growth temperature scope is 25～37 ℃, maximum growth temperature is 42 ℃; Oval-shaped brood cell is arranged in the cyst that expands, endways or near terminal place, both sides to outer process or or form at a side and thalline and must angle to expand projection, on nutrient agar medium, there is not soluble pigment, strict aerobic, catalase feminine gender, oxidase negative, the V-P feminine gender, edwardsiella hoshinae.

The nitrogenase vitality test of embodiment 2 bacterial classifications

The nitrogenase activity of present embodiment bacterial classification adopts gas chromatograph to survey acetyiene reduction activity (the Acetylene-reducing Activity of vinelandii, ARA) (with reference to Capone 1993, Capone DG (1993) Determination of nitrogenase activity in aquatic samples using the acetylenereduction procedure.Aquatic Microbial Ecology, 621-631).

Get above-mentioned 40 μ L reaction back gas and go up mensuration ethene peak value at GC-17A type gas chromatograph (Tianjin, island), the concentration of standard gas ethene is 138ppm.The working conditions of gas chromatograph is set to: 100 ℃ of sensing chamber's temperature, and 60 ℃ of column temperatures, injection port 60, column length 1m, gauge outfit nebulizer gas pressure nitrogen is 0.95kgcm ^-2, hydrogen is 0.8kgcm ^-2, air is 0.6kgcm ^-2The acetyiene reduction activity method of calculation are:

ARA(nmol?C?H·H ^-1·Culture ^-1)＝Vst×Cst×Asa×Vtu/Vsa/Ast/H/22.4

Wherein, Vst is the volume (mL) of injection standard gas, and Cst is the concentration of standard gas, and Vtu is a used test tube volume (mL), and Asa is the area (cm) at sample ethene peak, and Vsa is that sample injects volume (mL), and Ast is a standard gas peak area (cm), and H is an incubation time.

Present embodiment divides experimental group and blank group.

Experimental group: embodiment 1 separation and purification obtains pure bacterial strain, and be kept on the slant medium, should pure inoculation in the 10mlL selected liq does not have nitrogen liquid improved culture medium, in 30 ℃ of shaking table shaking culture 72h, get 1mL in the 5mL bottle of sterilization, cover soft rubber ball, the bottle cap edge is with paraffin sealing, take 5% air from container away with syringe, add the acetylene gas of equal volume then.

Blank group: do not add acetylene gas in the control container.

All samples is in 30 ℃ of reaction 12h, with the growing amount of SQ-204 gas chromatographic detection ethene.

Detected result shows that the pure bacterial strain that embodiment 1 separation and purification obtains can utilize N ₂As nitrogenous source, and have higher biological nitrogen fixation activity, average nitrogenase activity is 30～60umolC under the pure culture condition ₂H ₂h ^-1Mg ^-1Fresh weight.

The selected liq of present embodiment does not have nitrogen liquid improved culture medium, and its prescription is: NaCl 25g, KCl0.56g, MgSO ₄-7H ₂O 4.8g, MgCl ₂-6H ₂O 4.0g, K ₂HPO ₄0.01g, FeSO ₄7H ₂O0.001g, Tris 0.48g, peptone 4.0g, 2.0g yeast extract 2.0g, glycerine 2.0mL, distilled water 1L, whole PH are 8.2; The agar of adding 2% in the solid medium; Liquid nutrient medium adopts the sterilization of 0.22um suction method, and solid medium adopts autoclave sterilization.

The Sequence Identification of embodiment 3 bacterial strains

The template preparation: the pure bacterial strain of selecting embodiment 1 separation and purification to obtain carries out DNA extraction and purifying, the extraction and purification of described DNA, and its working method is with reference to the elegant pearl in east " common bacteria system identification handbook " P ₄₀₉, very the follow-up pcr amplification of present embodiment is template used for the DNA that the extraction purifying obtains.

1, the amplification of nifH gene

Adopt the nifH gene universal primer PolF/PolR (with reference to Poly et al., 2001) of nitrogen-fixing bacteria to carry out pcr amplification:

The PCR reaction system: totally be 25uL, 1 * PCR Buffer, 200umol L ^-1DNTPs, 2.0mmol L ^-1Mg ²⁺, 1umol L ^-1Primer, the Taq archaeal dna polymerase of the BSA of 4ug and 2.0U, template 1ul.

The pcr amplification condition: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 55 ℃ of renaturation 30s of annealing temperature, 72 ℃ are extended 30s, carry out 30 circulations and extend 10min for back 72 ℃.

Get 5 μ L amplified productions and carry out electrophoresis, voltage is 100V, sepharose is 1%, EB steeps glue, observe the swimming result and take pictures with gel imaging system, electrophorogram as shown in Figure 1, remaining PCR product directly gives Invitrogen Bioisystech Co., Ltd with the order-checking of ABI Prism 3730XL DNA analysis system.The nifH sequence that obtains is directly submitted the Genbank database, and sequence number is: FJ875968.

2, the amplification of 16S rDNA complete sequence

Adopt bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (Weisenburg et al., 1991,16S ribosomal DNA amplification for phylogenetic study., Journal of bacteriology, 1991173 (2): 697-703), carry out pcr amplification.

The PCR reaction system is as shown in table 1.

Table 1PCR reaction system

Template	??1μL
Template	??1μL	Upstream and downstream primer (1mM)	Each 1 μ L
10 * PCR damping fluid	??5μL	Upstream and downstream primer (1mM)	Each 1 μ L
10 * PCR damping fluid	??5μL	??MgCl ₂(25mM)	??4μL
DNTP mixed solution (each 10mM)	??1μL	??MgCl ₂(25mM)	??4μL
DNTP mixed solution (each 10mM)	??1μL	Bovine serum albumin (5mg mL ^-1)	??1μL
Ex-Taq enzyme (5U μ L ^-1)	??0.4μL	Bovine serum albumin (5mg mL ^-1)	??1μL
Ex-Taq enzyme (5U μ L ^-1)	??0.4μL	Sterilized water	??34μL
Amount to	??50μL	Sterilized water	??34μL

The PCR reaction conditions is: pre-95 ℃ of 5min of sex change; 95 ℃ of 30s of sex change, the 55 ℃ of 30s that anneal extend 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 10min are extended in the back.

After reaction finishes, getting 2 μ l PCR reaction product detects with 0.8% agarose gel electrophoresis, electrophoresis result as shown in Figure 1, all the other PCR products directly give Invitrogen Bioisystech Co., Ltd to check order with ABIPrism 3730XL DNA analysis system, the 16S rDNA sequence that obtains is directly submitted the Genbank database, and sequence number is: EU884567.

Among Fig. 1,1 is the genomic dna of embodiment 1 separation and purification obtained strains, the 2 16S rDNA that obtain for pcr amplification, the 3 nif nifH that obtain for pcr amplification, M1 is a λ DNA/Hind III standard molecular weight object of reference (Marker), and M2 is DL2000 (Marker).

Nif sequence nifH (the Genbank accession number is FJ875968) and 16S rDNA sequence (the Genbank accession number is EU884567) that above-mentioned sequencing analysis is obtained, compare by the sequence in BLAST among the GenBank and the GenBank database, in database, do not find on all four sequence, show the new gene order of these 2 gene orders for finding first.

By the analysis of embodiment 2 and 3 as can be seen, embodiment 1 separation and purification obtains a new bacterial strain, this bacterial strain belongs to Paenibacillus and belongs to (class Bacillus), its Chinese name is a strong bacillus G 25-1-2, English name is Paenibacillus validus G25-1-2, be preserved in Wuhan University China typical culture collection center on June 4th, 2009, it abbreviates CCTCC as, and deposit number is CCTCC NO:M209122.

Part and the higher representative gene order of measuring of 16S rDNA sequence similarity in the GenBank database, have been chosen, by ClustalW software and Mega software with Neibor-joining method constructing system evolutionary tree (as shown in Figure 2), carry out Phylogenetic Analysis, has the sequence of nearer sibship all from strong class bacillus with the 16S rDNA sequence of strong bacillus G 25-1-2 as can be seen from phylogenetic tree, and has 99% similarity with the 16S rDNA sequence of known bacterial strain Paenibacillus validus PR-P9 (AF353697) and Paenibacillus validus (AB073203), show that it is defined as strong class bacillus, but it has higher nitrogenase activity and nif nifH and has and shown that itself and general strong class bacillus have difference, and physiological and biochemical index as shown in table 2 has also confirmed this point.

Table 2 physiological and biochemical property

In the above-mentioned table 2 ,+expression positive reaction;-expression negative reaction.

In the above-mentioned table 2, P.validus T reference data is from Paenibacillus (Formerly Bacillus) gordonae (Pichinoty et al.1986) Ash et al.1994Is a Later Subjective Synonym of Paenibacillus (Formerly Bacillus) validus (Nakamura 1984) Ash et al.1994:Emended Description of P.validus, M.heyndrickx?etl.Int.J.Syst.Evol.Microbiol.45(4)，661-669(1995)45，(4)。

The efficiency assay of embodiment 4 bacterial strain preparations in soil

The efficiency assay of present embodiment is chosen in Bioexperiment station, the torrid zone, the Chinese Academy of Sciences South Sea to carry out, experiment usefulness sea grass (Thalassia emperichii) be to gather ground San Yawan from raw sample, choose growing way identical (specific targets have plant height, stem is slightly identical, growing way health) 20 strain sea grass test, ten strains are one group, are respectively experimental group and control group.

The soil of the potted plant experiment of sea grass and growth water all come from sea grass sample collecting ground---San Yawan, and wherein the soil of sea grass vitellarium and seawater all pass through sterilising treatment.

The strong bacillus G 25-1-2 that embodiment 1 separation and purification is obtained is in LB liquid nutrient medium (the conventional substratum when those skilled in the art carry out spawn culture), 30 ℃, 180rpm, cultivated 18 hours, get gained growth bacterium liquid, the sea grass root and the root stock of experimental group all are soaked in 3～4h in this growth bacterium liquid, and plastic tub is gone in plantation then; Control group is left intact; Two groups all place under the outdoor same condition and cultivate.

Cultivate after 60 days, it (is plant height that experimental group and control group are carried out every index determining respectively, stem is thick, chlorophyll content, carotenoid, soluble sugar and soluble proteins etc.), observe this bacterium and whether the growth of sea grass is had growth-promoting functions, the measurement result of every index is as shown in table 3.

The influence that the inoculation of table 3 strong bacillus G 25-1-2 was grown for sea grass after 60 days

	Plant height (cm)	Stem thick (cm)	Chlorophyll (ug/gFW)	Carotenoid (ug/gFW)	Soluble sugar (ug/gDW)	Soluble proteins (ug/gFW)
	Plant height (cm)	Stem thick (cm)	Chlorophyll (ug/gFW)	Carotenoid (ug/gFW)	Soluble sugar (ug/gDW)	Soluble proteins (ug/gFW)	Experimental group	??12.6	??1.3	??972	??329	??4728	??156
Control group	??10.2	??0.8	??628	??162	??3172	??132	Experimental group	??12.6	??1.3	??972	??329	??4728	??156

In the above-mentioned table 3, all numerical value are 10 groups of parallel sample mean values.

As shown in table 3, the contrast of group and control group can be found by experiment, the thick mean value of the plant height of experimental group and stem is apparently higher than control group, carotene carotene content and carotenoid content also exceed 46.8% and 103% than control group respectively, soluble sugar content is 1.49 times of control group, and soluble proteins and control group do not have significant difference.These results show, strong bacillus G 25-1-2 of the present invention comes into force fruit better for the short of sea grass, have huge potential significance for the protection and the recovery of the sea grass bed ecosystem.

Claims

1. a strong bacillus G 25-1-2 is preserved in Wuhan University China typical culture collection center on June 4th, 2009, and it abbreviates CCTCC as, and deposit number is CCTCCNO:M209122.

2. according to the described strong bacillus G 25-1-2 of claim 1, it is characterized in that the growth temperature of this strong series bacillus G25-1-2 on nitrogen-free agar is 25～37 ℃.

3. the application of the described strong bacillus G 25-1-2 of claim 1 in the biological nitrogen fixation of sea grass-karang complex ecosystem.

4. the described strong bacillus G 25-1-2 of claim 1 is in the short again application of giving birth in the preparation of preparation sea grass-karang.