2017 2nd International Conference on Environmental Science and Energy Engineering (ICESEE 2017) ISBN: 978-1-60595-417-2

Isolation and Identification of Denitrifying and Its Growth and Characteristics

Xin-ran JIANG, Dian JIAO, Lei ZHANG and Li-na ZHENG College of Marine Technology and Environmental, Dalian Ocean University, Dalian 116023, China

Keywords: , , Isolation and identification, Activity research.

Abstract. A denitrifying bacterium DN20 was isolated from the biofilm after centrifugation in the aquaculture purification system of a aquaculture base, and the denitrification characteristics of the strain were further studied. The strain was identified by 16S rDNA sequence analysis, the effects of temperature, salinity, pH, substrate concentration and C/N ratio on denitrification activity were studied.

Introduction Denitrifying bacteria are a kind of bacteria that can cause denitrification. Biological denitrification is the use of denitrifying bacteria, nitrate into gaseous products to remove, is considered to be the most economical and effective way of nitrogen removal [1]-[2]. The application of denitrifying bacteria and the removal of nitrate in water have become the research focus[3]-[5], many methods have been reported [6]-[8]. In recent years, some studies on salt tolerant and halophilic denitrifying bacteria have been reported abroad [9]-[10]. In this study, the sludge from the culture pond was taken as the main separation source, and the enrichment, separation and purification were carried out.To obtain a highly efficient nitrate removal ability of denitrifying bacteria, named strain DN20. The aim of this study is to provide effective bacteria source for practical application of nitrate-N treatment in aquaculture water.

Materials and methods

Experimental Materials

Source of Sample. Samples from Dalian City in Liaoning Province, a aquaculture base in Ganjingzi, the collecting point is the biofilm after centrifugation on the carrier of the culture purification system. -1 -1 Culture Medium. Enrichment culture liquid medium: KNaC4H4O6 ∙4H2O 20 g·L , KNO3 2 g·L , -1 -1 K2HPO4 0.5 g·L , MgSO4∙7H2O 0.2 g·L , pH 7.2-7.6. Purification medium: In the above-mentioned enrichment culture liquid medium adding agar 18 g·L-1, for bacterial purification. Enrichment, Separation and Purification of Denitrifying Bacteria

Enrichment of Bacteria. Bacterial samples were derived from water treatment unit of biological film on soft filler in circulating water culture system of sea cucumber. 10 g of the biofilm after centrifugation, was added to 100 ml of denatured bacteria enrichment culture liquid-based serum bottle, adding sterilized glass beads, in the low temperature on the shaker at 20℃ after a number of - - days of culture, the concentration of NO3 -N in the medium was determined. When the NO3 -N transformation reached more than 90%, the bacterial solution could be used for bacterial isolation. Isolation and Purification of Bacteria. Denitrifying bacteria were isolated from the enriched bacteria by serial dilution and plate streak, and the strain was purified several times. During the bacterial culture, the plates were placed in an anaerobic tank, and joined in the tank to ensure the

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hypoxic environment of pyrogallic acid. Finally, the anaerobic tank was placed in a low-temperature incubator at 20℃for several days. Identification of Strains

External Morphological Characteristics of Strains. The colonies were pale yellow, round, smooth and moist. It was observed by electron microscope that the shape of the strain was rod-shaped, the size is（0.5-0.7）µm×（1.1-1.3）µm, gram stain was negative, Fig. 1 is an optical micrograph of the stained cells.

Figure 1. Pattern of strain DN20 under microscopy.

Internal Structure Characteristics of Strains. In order to observe the internal structure of DN20 strain, the strain was cultured for ultrathin section and re-staining, observation under transmission electron microscope

Figure 2. Transmission electron micrograph of strain DN20.

Effects of Various Factors on Growth and Metabolism of Bacteria

Temperature - The control temperature is 4℃, 20℃, 30℃, 37℃, 41℃, 45℃.Determination of NO3 -N and protein - initiation and termination concentrations by 3h at 20℃ in a serum bottle, the removal rate of NO3 -N was calculated (Formula 1).

C   C  3  hNNO )3( 3  hNNO )0( q   (1) 3 NNO 3h  protein h )0(

75 3.0

2.5

2.0 -N (ugN/mg Protein/h) (ugN/mg -N - 3 1.5

1.0

0.5

0.0

Specific removal rate of NO 0 1020304050 Tem perature ( °C)

- Figure 3. Relationship between temperature and NO3 -N ratio removal rate.

Salinity - Control salinity by NaCl solution is 2%, 5%, 7%, 10%. Determination of NO3 -N and protein - initiation and termination concentrations by 3h at 20℃ in a serum bottle, the removal rate of NO3 -N was calculated (Formula 1).

7

6

5

4

3

2

1

0 024681012 specific removal rate of NO3--N(ugN/mg Protein/h) NO3--N(ugN/mg of rate removal specific Salinity(%)

- Figure 4. Relationship between salinity and NO3 -N ratio removal rate. pH The pH value of the medium was adjusted with MES (pH 5.0-6.0), PIPES (pH 6.5-7.0), Tricine (pH -1 - 7.5-8.5), CHES (pH 9-10) at a concentration of 50 mmol·L . Determination of NO3 -N and protein - initiation and termination concentrations by 3h at 20℃ in a serum bottle, the removal rate of NO3 -N was calculated (Formula 1).

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0 5678910 specific removal rate of NO3--N(ugN/mg Protein/h) NO3--N(ugN/mg of rate removal specific pH

- Figure 5. Relationship between pH and NO3 -N ratio removal rate.

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NO3 Concentration - -1 -1 -1 The concentrations of NO3 -N were controlled respectively 10 mg·L , 50 mg·L , 100 mg·L , 200 -1 -1 mg·L , 400 mg·L , according to the actual reaction of KNaC4H4O6 ∙4H2O and KNO3 molar ratio - (0.47:1), the experimental culture solution was prepared (Table 1). Determination of NO3 -N and protein initiation and termination concentrations by 3h at 20℃ in a serum bottle, the removal rate of - NO3 -N was calculated (Formula 1). Table 1. Substrate concentration in medium for matrix concentration test.

-1 KNaC4H4O6 ∙4H2O（g·L 0.10 0.50 1.00 2.00 4.00 ） -1 KNO3（g·L ） 0.072 0.36 0.72 1.44 2.88

20

18

16

14

12

10

8

6

4

2

0 0 100 200 300 400 500 specific removal rate of NO3--N(ugN/mg Protein/h) NO3--N(ugN/mg of rate removal specific NO concentration (m g/L) 3

- Figure 6. Relationship between NO3 concentration and NO3 -N ratio removal rate.

COD:N - -1 The concentrations of NO3 -N were controlled respectively 20 mmol·L , adjust with COD:N value - respectively were 1, 4, 8, 16, 32 (Table 2). Determination of NO3 -N and protein initiation and - termination concentrations by 3h at 20℃ in a serum bottle, the removal rate of NO3 -N was calculated (Formula 1). Table 2. Matrix concentration in culture medium of COD: N test. COD:N 2:1 4:1 8:1 16:1 32:1 -1 KNO3（g·L ） 2 2 2 2 2 COD 560 1120 2240 4480 8960 -1 KNaC4H4O6 ∙4H2O（g·L 1.23 2.46 4.92 9.84 19.68 ）

9

8

7

6

5

4

3

2

1

0 2:1 4:1 8:1 16:1 32:1 specific removal rate of NO3--N(ugN/mg Protein/h) NO3--N(ugN/mg of rate removal specific C:N

- Figure 7. Relationship between C:N and NO3 -N ratio removal rate.

77 Conclusion DN20 strain is a typical anaerobic salt tolerant denitrifying bacteria, which has strong denitrification activity, the best electron acceptor is nitrate nitrogen. Under the conditions of temperature 41℃, salinity is 10%, pH value of CHES (pH 9-10), substrate concentration of 400 mg·L-1 and C / N ratio of 4: 1, the denitrifying activity of the strain was the strongest.

Acknowledgement This research was financially supported by the National Marine public welfare project of sea cucumber.

References [1] Angelopoulos K., Spiliopoulos I.C., Mandoulaki A., et al.Groundwater nitrate pollution in northern part of Achaia Prefecture. Desalination, 2009, 248(1- 3): 852- 858. [2] Gomez M.A., Hontoria E., Gonzalez- Lopez J. Effect of dissolved oxygen concentration on nitrate removal from groundwater using a denitrifying submerged filter. Journal of Hazardous Materials, 2002, 90(3): 267- 278. [3] Bandpi M.A., Elliott D.J., Memeny-Mazdek A. Denitrification of groundwater using acetic acid as a carbon source [J]. Water Science and Technology, 1999, 40 (2): 53-59. [4] Green M., Schnizer M., Tarre S., et al. Groundwater denitrification using an upflow sludge blanket reactor [J]. Water Research, 1994, 28 (3): 631-637. [5] Lin Y.F., Jing S.R., Wang T.W., et al. Effects of macrophytes and external carbon sources on nitrate removal from groundwater in constructed [J]. Environmental Pollution, 2001, 119: 413-420. [6] Hu J.Y., Ong S.L., Ng W.J., et al. A new method for characterizing denitrifying phosphorus removal bacteria by using three different types of electron acceptors [J].Water Research, 2003, 37: 3463-3471. [7] Kariminiaae-Hamedaani H., Kanda K., Kato F. Denitrification activity of the bacterium sp.ASM-2-3 isolated from the Ariake Sea Tideland [J]. Journal of Bioengineering, 2004, 97(1): 39-44. [8] Rocca C.D., Belgiorno V., Meric S. Heterotrophic/autotrophic denitrification (HAD) of drinking water: Prospective use for permeable reactive barrier [J]. Desalination, 2007, 210: 194-204. [9] T.M. Caton, L.R. Witte, HD Ngyuen, et al. Halotolerant aerobic heterotrophic bacteria from the greatsalt plains of oklahoma. Microbial Ecology, 2004, 48: 449−462. [10] Charles Glass, Joann Silverstein. Denitrification of high-nitrate, high-salinity wastewater. Wat Res, 1999, 33(1): 223−229.

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