LU501700B1 - Marine bacterium aerococcus urinaeequi hz and polysaccharide produced by the same - Google Patents

Abstract

Disclosed is a marine bacterium Aerococcus urinaeequi HZ.

Description

MARINE BACTERIUM AEROCOCCUS URINAEEQUI HZ AND LUS01700

POLYSACCHARIDE PRODUCED BY THE SAME TECHNICAL FIELD

[01] The present invention relates to a marine bacterium Aerococcus urinaeequi HZ as well as a polysaccharide produced by the same and an application thereof.

BACKGROUND ART

[02] With the development of industrial and agricultural production and the improvement of people’s living standards, the discharge of wastewater increases rapidly, and wastewater treatment has become one of the current social problems. The use of bacteria to treat wastewater is a research hotspot now, but no ideal strain is available.

SUMMARY

[03] In view of the above-mentioned prior art, the present invention provides a marine bacterium Aerococcus urinaeequi HZ used for wastewater treatment as well as a polysaccharide produced by the same and an application thereof.

[04] The present invention is realized by the following technical solution:

[05] a marine bacterium Aerococcus urinaeequi HZ is characterized in that Aerococcus urinaeequi HZ is a rod-like Gram-negative bacterium, and the bacterial colony is large (a diameter of 2 mm) in a Zobell 2216E solid medium, milk-white, round, raised, non-transparent and migration-free with rounded edges.

[06] A polysaccharide is derived by culturing the marine bacterium Aerococcus urinaeequi HZ.

[07] The experiment results show that the polysaccharide also features an excellent clearance efficiency for free radicals and an excellent oxidation resistance. The polysaccharide also has an excellent flocculation ability for muddy wastewater. It can be seen that in wastewater treatment, the strain and polysaccharide of the present invention have exceptional advantages, such as being made into additives. It is believed that the strain and polysaccharide of the present invention will occupy a place in the field of wastewater treatment in the near future.

BRIEF DESCRIPTION OF THE DRAWINGS

[08] FIG 1: A cell growth curve and a polysaccharide fermentation curve.

[09] FIG 2: A schematic diagram of ultraviolet scanning data.

[10] FIG 3: A schematic diagram of a main peak collected by gradient elution.

[11] FIG 4: A schematic diagram of an elution peak obtained by elution.

[12] FIG 5-1: A linear regression equation for a polysaccharide molecular weight, with an abscissa: elution volume, and an ordinate: molar mass. The figure is obtained by a machine.

[13] FIG 5-2: A schematic diagram of a polysaccharide molecular weight and a molecular weight distribution, with an abscissa: elution volume, and an ordinate: detector response. The figure is a scanned picture.

[14] FIG 6: An infrared spectrum of EPS-2. 1

[15] FIG 7: A'H NMR spectrum (30°C) of EPS-2. LU501700

[16] FIG 8: AC NMR spectrum (30°C) of EPS-2.

[17] FIG 9: A schematic diagram of a flocculation effect of exopolysaccharide EPS2 on wastewater.

[18] FIG 10: A schematic diagram of a clearance rate of exopolysaccharide and Vc for -OH.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[19] The present invention will be further described below in combination with embodiments.

[20] Example 1 Acquisition of strain

[21] A sample was collected from a sea area of Rongcheng, Shandong Province, at a time when a larva (abalone) at a seedling stage was stripped from a substratum (mid-May). The sample, attachments of a 10 cm’ area scraped from a plate, was preserved in a 2 ml sterilized freezing tube, sealed in an ice box, and returned to a laboratory within 24 h. The sample was subject to heterotrophic bacterium separation and DNA extraction. The sample was diluted by sterile seawater to four concentrations, i.e. 10-3, 10-4, 10-5 and 10-6, then evenly applied to a 2216E solid medium plate by a sterilized spreader, and cultured at a constant temperature of 25°C for 48 h; then a bacterial colony with a significant morphological difference was selected. The bacterial colony was purified by a method of scoring. Whether the resulting strains produced a polysaccharide was detected by a phenol-sulfuric acid method; a strain with a polysaccharide output of more than 0.5 g/L was screened, and a species relation thereof was determined by a DNA sequence analysis, and the strain belonged to aerococcus.

[22] Example 2 Polysaccharide extraction

[23] A polysaccharide was derived by culturing the marine bacterium Aerococcus urinaeequi HZ, specifically as follows:

[24] (1) Culturing: A seed medium was obtained from the marine bacterium Aerococcus urinaeequi HZ strain by a slant culture and a seed culture, inoculated to a fermentation medium with an inoculum size of 5% (volume percentage), and subject to shake cultivation at 230 rpm at 26°C for 35 h to obtain a fermentation broth. A cell growth curve and a polysaccharide fermentation curve are as shown in FIG 1, with a polysaccharide output of up to 2.34 g/L.

[25] (2) Extraction: The fermentation broth was centrifuged at 5,000 rpm for 20 min, the resulting thallus was removed by filtration, then a 3-fold volume of 95% ethanol (volume percentage) was added to the supernate, the solution was kept static for 10 h, and centrifuged to obtain a precipitate, i.e. a mixture of protein and polysaccharide;

[26] (3) Impure protein removal: The mixture of protein and polysaccharide was dissolved in water, and a 1/4 volume of Sevag solvent (chloroform:n-butanol = 4:1, volume ratio) was added, and the solution was fully vibrated for 10 min and centrifuged for removal of organic phase; the steps were repeated seven times to obtain a crude polysaccharide solution; an ultraviolet scanning was adopted to detect whether all the impure protein was removed (if no absorption peak appeared at 260 nm and 280 nm wavelengths, it indicated that no nucleic acid or protein residue was available), wherein 2 the ultraviolet scanning data were as shown in FIG. 2. LU501700

[27] (4) Separation and purification: The crude polysaccharide was graded by DEAE-52 and then gradient-eluted by 0-2 M NaCl Tris-HCI (0.01 M), and a main peak (as shown in FIG 3) was collected; the main peak collected was concentration-desalted and freeze-dried, dissolved in a 0.1 M NaCl solution, subject to chromatography by Sephadex G100 and eluted by a 0.1 M NaCl solution, and a first elution peak was collected and freeze-dried to obtain a polysaccharide, named EPS-2, as shown in FIG 4.

[28] A slant medium used in the culture was: Zobell 2216E medium: 5.0 g of peptone, 1.0 g of yeast extract, 0.01 g of iron phosphate, 20.0 g of agar, and 1,000 ml of aged seawater, with a pH value adjusted to 7.6-7.8.

[29] A seed medium used in the culture was: Zobell 2216E medium: 5.0 g of peptone,

1.0 g of yeast extract, 0.01 g of iron phosphate, and 1,000 ml of aged seawater, with a pH value adjusted to 7.6-7.8.

[30] The fermentation medium was: 0.25% of beef extract, 3% of sucrose, sea salt (0.00016% of ammonium nitrate, 0.0022% of boric acid, 0.18% of calcium chloride,

0.0008% of sodium hydrogen phosphate, 0.01% of ferric citrate, 0.88% of magnesium chloride, 0.008% of potassium bromide, 0.055% of potassium chloride, 0.016% of sodium bicarbonate, 1.945% of sodium chloride, 0.0004% of sodium silicate, 0.0324% of sodium sulfate, 0.0034% of strontium chloride, and 0.00024% of sodium fluoride), and a remaining amount of water, with a pH value of 7.6-7.8. The percentages were all mass percent.

[31] Determination of polysaccharide purity and molecular weight:

[32] A molecular weight of the polysaccharide EPS-2 was determined by a high performance gel permeation chromatography (HPGPC), with a chromatographic column: SHODEX SB-806HQ (8.0 mx300 mm); a mobile phase: 0.2 M NaCl (17.85 g of sodium chloride and 0.15 g of sodium azide were taken, dissolved by pure water and diluted to 1,500 mL) solution; and a control sample: 0.1 mg/mL of polystyrene sulphate sodium salt series control sample; 100 pL of control sample was injected into a liquid chromatograph respectively at a flow velocity of 0.5 mL/min at a column temperature of 35°C, a chromatogram was recorded, and universal calibration was performed and the linear regression equation was calculated by means of GPC software, as shown in FIG 5-1. AK value of the control sample was 0.0006, and an a value was 0.75, then 100 uL of test sample solution was collected and determined in the same method, and the molecular weight and a molecular weight distribution of the sample were calculated by means of GPC software, as shown in FIG. 5-2.

[33] The molecular weight information obtained by standard substance and standard curve integral is as shown in Table 1.

[34] Table 1 Number-Average Weight-Average Molecular Lu ; Molecular Weight/(g/mol) Weight/(g/mol) Distribution Coefficient

2.22x10° 2.84x10° 1.28

[35] The commonly used average molecular weights include a number-average molecular weight (Mn) with number as a statistical weight and a weight-average molecular weight (Mw) with weight as a statistical weight. A distribution coefficient of 3 the polysaccharide was 1.28, indicating that a distribution degree of the molecular LU501700 weight of the bacterial polysaccharide was low.

[36] Monosaccharide composition analysis of EPS-2:

[37] The mass spectrum analysis results of EPS-2 hydrolysate are as shown in Table

2. Upon a contrastive analysis of standard mass spectrum library searching, seven kinds of monosaccharides were detected, mainly including two kinds of pentose (arabinose and xylose), two kinds of hexose (mannose and glucose), as well as a little glucoside, galactose and glyceraldehyde.

[38] Table 2 Monosaccharide Composition Analysis of EPS-2 Retention | Monosaccharide Molecular Formula Monosaccharide o Time Derivative CAS of Monosaccharide Name Peak Area (%) Derivative

[39] Infrared spectroscopic analysis of EPS-2:

[40] From the infrared spectroscopy (as shown in FIG 6) of EPS-2, it can be seen that the EPS-2 had specific absorption peaks of polysaccharide substances, wherein broad peaks within 3,200 cm-1-3,600 cm-1 were O-H stretching vibration peaks on the EPS-2; 2935.6 cm-1 and 2,887.4 cm-1 were C-H stretching vibration of -CH2 and -CH3, and 1,319.3 cm-1-1,454.33 cm-1 were C-H variable angle vibration absorption peaks; 1,651.07 cm-1 was a stretching vibration peak of C=O, or a N-H variable angle vibration peak, which needs to be further verified, about 1,140 cm-1 was a C-O highly-absorptive stretching vibration peak, or a pyranose ring characteristic absorption peak; 927.7 cm-1 was a pyranoid ring asymmetrical stretching vibration peak, or a characteristic absorption peak of furan derivative, and 812 cm-1 may be a C-H variable angle vibration of furan ring, which needs to be further verified due to possible deviations as a result of instruments, operation techniques, sample treatment, etc.

[41] Nuclear magnetic resonance spectroscopy analysis of EPS-2:

[42] In order to further determine a structure of EPS-2, the EPS-2 was subject to "H-NMR (FIG. 7) and ’C-NMR (FIG. 8) analysis. In the "H-NMR spectrum, the 5 value of a proton on anomeric carbon C-1 was near 4.7 ppm, not exceeding 5 ppm, indicating that these glucose residues were all B configurations, which was consistent with the infrared spectroscopic analysis. In the *C-NMR spectrum, no carbonyl carbon signal appeared between 160-180 ppm, indicating that the polysaccharide contained no uronic acid and glycoprotein, while seven groups of signal peaks appeared between 95-105 ppm and all were greater than 102, and thus it could be determined according to literatures that the polysaccharide had seven different monosaccharide residues, all of which were B configurations; no absorption peak appeared between 67-70 ppm, indicating that no C6 was substituted, while three absorption appeared between 78-85 4 ppm, indicating that three carbon atoms were substituted in various monosaccharide LU501700 residues 2, 3 and 4, wherein the specific locations need to be further determined; no carbon signal appeared at 6<20 ppm, indicating that the EPS-2 contained no methyl sugar.

[43] Example 3 Applications of polysaccharide

[44] I. Flocculation of EPS-2 on wastewater

[45] (1) Acquisition of domestic wastewater

[46] Wastewater was collected from Domestic Wastewater Treatment Plant of Qilu University of Technology, and an A value of the wastewater was detected as 1.626.

[47] (2) Preparation of microbial flocculant exopolysaccharide

[48] A marine bacterium HZ strain was inoculated to 1 L of sterilized nitrogen-free medium and cultured at 28°C for 2 d until a flocculating substance was produced at a bottom of a bottle, the solution was centrifuged by a precipitation method and freeze-dried, and exopolysaccharide of marine bacterium Pseudoalteromonus isachenkonii HZ was obtained, then subject to deproteinization by a sevage method, and further separated and purified by DEAE-52 and Sephadex G100 to obtain EPS-2 (i.e.

the EPS-2 extracted by the method of example 2).

[49] (3) Treatment of domestic wastewater

[50] 100 mL of wastewater to be treated and 0.2 g of prepared EPS-2 of marine bacterium Pseudoalteromonus isachenkonii HZ were added to a 250 mL conical flask, mixed evenly, and kept static for | h, and a treatment effect of EPS on wastewater was detected by the following indicators.

[S1] (4) Measurement of flocculation rate [S2] A flocculation rate was measured in 550 nm wavelength by means of a 752 spectrophotometer, wherein, an upper phase liquid Dy value of the original wastewater was marked as A1; and an upper phase liquid absorbance value (Dy value) of the treated wastewater was marked as Bl. The flocculation effect was represented by the flocculation rate: Flocculation rate (%) = (A1-B1)/A1X 100% [S3] The analysis of experimental results of wastewater flocculation is as shown in FIG 9. After the solution was kept static for 1 h, the flocculation rate was measured as

79.90%. From the treatment results of domestic wastewater, the EPS-2 prepared from the marine bacterium Pseudoalteromonus isachenkonii HZ strain has achieved the treatment requirements for domestic wastewater, and the effect has been exactly as expected. Therefore, the EPS-2 can be applied as a flocculant.

[54] II Study on an application of EPS2 in clearing free radicals [S5] (1) Clearing of hydroxyl radical

[56] 1. AH,0,/Fe™ system was adopted, -OH was generated by Fenton reaction, and then a salicylic acid was added to the system to capture the -OH and generate a colored matter, which had a maximum absorption at 510 nm. The reaction system contained 1 mL of 9.8 mmol/LH202, 1 mL of 9 mmol/LFeS04, 1 mL of 9 mmol/L salicylic acid-ethanol, and 1 mL of marine bacterium Pseudoalteromonus isachenkonii HZ EPS2 solution of different concentrations, wherein the H202 was added at last and started the whole reaction. The absorbance at each concentration was measured at 510 nm, with distilled water as a reference, after reaction for 30 min at 37°C, and centrifugation at LU501700 5,000 rpm for 10 min. [S7] In view of different absorbance values of a solution to be measured, 1 mL of 9 mmol/L. FeSO,, 1 mL of 9 mmol/L salicylic acid-ethanol, 1 mL of polysaccharide solution of different concentrations and 1 mL of distilled water were served as background absorption values of the solution to be measured. A calculation formula of clearance rate was as follows:

[58] Clearance rate = [AO-(Ax-Ax0)]/AOX 100% [S9] where: AO represented the absorbance of a blank control solution, Ax represented the absorbance after the polysaccharide solution added, and AxO represented the background absorption value of the polysaccharide solution.

[60] 2. A 2/Fe” system was adopted, "OH was generated by Fenton reaction, then a salicylic acid was added to the system to capture the -OH and generate a colored matter, which had a maximum absorption at 510 nm. The reaction system contained 1 mL of

9.8 mmol/L H202, 1 mL of 9 mmol/L FeSO,, 1 mL of 9 mmol/L salicylic acid-ethanol, and 1 mL of Vc solution of different concentrations, wherein the H202 was added at last and started the whole reaction. The absorbance of each concentration was measured at 510 nm, with distilled water as a reference, after reaction for 30 min at 37°C, and centrifugation at 5,000 rpm for 10 min.

[61] In view of different absorbance values of a solution to be measured, the 1 mL of 9 mmol/L FeSO4, 1 mL of 9 mmol/L salicylic acid-ethanol, 1 mL of Vc solution of different concentrations and 1 mL of distilled water were served as background absorption values of the solution to be measured. A calculation formula of clearance rate was as follows:

[62] Clearance rate = [AO-(Ax-Ax0)]/A0x100%

[63] where: AO represented the absorbance of a blank control solution, Ax represented the absorbance after the Vc solution added, and AxO represented the background absorption value of the Vc solution.

[64] The analysis of experimental results of hydroxyl radical (OH) clearance was as shown in FIG 10. EPS2 had a relatively obvious clearance effect on hydroxyl radical. With an increase of polysaccharide concentration and a presentation of obvious dose-effect relationship, the clearance rate reached 45.65% when the polysaccharide concentration was 100 ug/mL. Compared with the clearance rate of Vc on hydroxyl radical, the effect of the EPS2 was relatively ideal with a relatively low clearance rate.

[65] According to the previous experimental results, the marine bacterial polysaccharide EPS2 has many unique effects in various fields. The marine bacterial polysaccharide EPS2 plays a significant role in promoting wastewater flocculation. This effect not only enables us to have a better understanding of wastewater treatment but also opens up a way to use microbial rapid flocculation, which provides strong supports for future water purification and wastewater treatment research. In clearing free radicals, the marine bacterial polysaccharide EPS2 has a clearing effect comparable to Vc on hydroxyl radicals and superoxide radicals. Though the results are not as obvious as Vc, in the same order of magnitude, and Vc itself is an excellent antioxidant, the results are more satisfactory. Moreover, the marine bacterial EPS has an excellent flocculation 6 ability on muddy wastewater, and the Pseudoalteromonus isachenkonii HZ EPS2 has LU501700 exceptional advantages when being used as a wastewater treatment additive.

It is believed that the EPS2 will occupy a place in the field of wastewater treatment in the near future. 7

Claims (4)

WHAT IS CLAIMED IS: LUS01700

1. A polysaccharide, wherein the polysaccharide is derived by culturing a marine bacterium Aerococcus urinaeequi HZ.

2. The polysaccharide of claim 1, wherein the polysaccharide is obtained by the following method: (1) culturing: obtaining a seed medium from marine bacterium Aerococcus urinaeequi HZ strain by a slant culture and a seed culture, inoculating the seed medium to a fermentation medium with an inoculum size of 2%-5%, and performing shake cultivation at 150-250 rpm at 20-35°C for 30-40 h to obtain a fermentation broth; (2) extraction: centrifuging the fermentation broth at 5,000 rpm for 20 min, removing the resulting thallus by filtration, then adding a 3-fold volume of 95% ethanol to the supernate, keeping the solution static for 8-12 h, and centrifuging the solution to obtain a precipitate, i.e. a mixture of protein and polysaccharide; (3) impure protein removal: dissolving the mixture of protein and polysaccharide in water, and adding a 1/4 volume of Sevag solvent, fully vibrating the solution for 10 min, then centrifuging the solution for removal of organic phase; repeating the steps eight times to obtain a crude polysaccharide solution; and (4) separation and purification: grading the crude polysaccharide by DEAE-52, then gradient-eluting the crude polysaccharide by 0-2 M NaCl Tris-HCI, and collecting a main peak; performing concentration desalination and freeze drying for the main peak collected, dissolving the main peak in a 0.1 M NaCl solution, performing chromatography by Sephadex G100, and eluting the solution by a 0.1 M NaCl solution, collecting a first elution peak and performing freeze drying to obtain a polysaccharide.

3. The polysaccharide of claim 2, wherein a slant medium used in the slant culture is: Zobell 2216E medium: 5.0 g of peptone, 1.0 g of yeast extract, 0.01 g of iron phosphate, 20.0 g of agar, and 1,000 ml of aged seawater, with a pH value adjusted to

7.6-7.8.

4. The polysaccharide of claim 2, wherein a seed medium used in the seed culture was: Zobell 2216E medium: 5.0 g of peptone, 1.0 g of yeast extract, 0.01 g of iron phosphate, and 1,000 ml of aged seawater, with a pH value adjusted to 7.6-7.8.

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