Your browser does not support the PubReader view.
Go to this page to see a list of supporting browsers.
The Korean Society Fishries And Sciences Education
[ Article ]
The Journal of the Korean Society for Fisheries and Marine Sciences Education - Vol. 32, No. 4, pp.891-898
ISSN: 1229-8999 (Print) 2288-2049 (Online)
Print publication date 31 Aug 2020
Received 01 Apr 2020 Revised 16 Jun 2020 Accepted 26 Jun 2020
DOI: https://doi.org/10.13000/JFMSE.2020.8.32.4.891

Oxidation of Hydrogen sulfide, Ammonia nitrogen and Nitrite nitrogen by Bacillus sp. Isolated from West Coast of Korea

Hong-Kook KIM ; Geun-Seop KIM ; Chae-Rin PARK ; Byeong-Soo KIM†
Kongju National University(student)
†Kongju National University(professor)
서해안에서 분리한 Bacillus sp.의 황화수소, 암모니아 질소, 아질산 질소에 대한 산화능
김홍국 ; 김근섭 ; 박채린 ; 김병수†
공주대학교(학생)
†공주대학교(교수)

Correspondence to: †041-330-1523, bskim@kongju.ac.kr

Abstract

In the aquaculture industry, the biological treatment using probiotics for eliminating hydrogen sulfide, ammonia nitrogen and nitrite nitrogen is receiving attention. In this study, Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 were isolated from the west coast of Korea and oxidation ability of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen were analyzed. Bacillus sp. TO-10 had high oxidation toward hydrogen sulfide by oxidizing 100% and Bacillus sp. TO-3 and TO-12 each had hydrogen sulfide oxidation of 49.53% and 69.03%. Bacillus sp. TO-10 and Bacillus sp. TO-12 had ammonia nitrogen oxidation approximately of 32.7 mg/L, while Bacillus sp. TO-3 had 11.4 mg/L of ammonia nitrogen oxidation. Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 all had high oxidation toward nitrite nitrogen by oxidizing 100% of 137.46 mg/L nitrite nitrogen. Therefore, Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 are possible probiotics used as water quality improvement of eliminating hydrogen sulfide, ammonia nitrogen and nitrite nitrogen.

Keywords:

Bacillus, Oxidation, Hydrogen sulfide, Ammonia nitrogen, Nitrite nitrogen

Ⅰ. Introduction

The modern aquaculture high rearing density for mass produce and low intensity flow tank system structure causes high concentration of nutrients such as nitrogen (N), phosphorus (P) and specific organic, resulting poor water quality in aquaculture environment (Kang and Yoon, 2004; Piedrahita, 2003). The sediments of metabolic wastes from fish and uneaten feed in the tank cause consumption of dissolved oxygen (DO) and the nutrient anaerobic decomposition by the bacteria occur, hydrogen sulfide (H2S), ammonia nitrogen (NH3-N) and nitrite nitrogen (NO2-N) (Handy and Poxton, 1993; Kioussis et al., 2000).

Hydrogen sulfide occurs when various bacteria decompose nutrient sediments in anaerobic environment using electron acceptor such as sulfate (SO2-4) (Kim et al., 2011). Hydrogen sulfide is toxic to cultured fish by decreasing hatching rates and killing fry causing an impact on productivity (Smith et al., 1976; Beauchamp et al., 1984).

Ammonia nitrogen (NH3-N) exists as dissolved ammonia (NH3) and ammoniumion (NH4+) in water (Zhou and Boyd, 2016). Ammonia (NH3) and ammoniumion (NH4+) are toxic to cultured fish, especially high concentration of un-ionized ammonia damage the fish gill, decreasing oxygen transportability in blood and decreasing osmoregulation by damaging the erythrocyte and tissue (Tomasso, 1994; Romano and Zeng, 2013).

Nitrite nitrogen (NO2-N) occurs as an intermediate product of ammonia and nitric acid when ammonia is oxidized by bacteria. Nitrite nitrogen is toxic to cultured fish by combining with the fish erythrocyte, reducing the oxygen transport ability. Therefore, death by asphyxia happen even when air supply is enough in the aquaculture tank. Nitrite nitrogen poisoning doesn’t have external symptoms while progressing into chronic. (Russoet al., 1974; Brown and McLeay, 1975).

Aquaculture farms use chemical and physical treatment to eliminate hydrogen sulfide, ammonia nitrogen and nitrite nitrogen. However the chemical treatment occurs secondary containment and the physical treatment is expensive (Gadre, 1989; Mook et al., 2012). Therefore, biological treatment using probiotics is receiving attention in aquaculture for the purpose of water quality improvement. Probiotics are beneficial microorganism to organism. Especially a gram positive strain Bacillus sp. is an effective probiotics for water quality improvement (Verschuere et al., 2000; Sahu et al., 2008).

In this study, Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 strains were isolated from the west coast of Korea and oxidation ability of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen were analyzed for development purpose of water quality improvement probiotics.

Ⅱ. Material and methods

1. Strains isolation and identification

TO-3, TO-10 and TO-12 strains were isolated from the west coast of Korea. The water samples were spread in a mineral salt medium appeared in Table 1 and cultured at 35℃ for 48 hours. Strains which have high oxidation of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen were selected and isolated. The selected strains were identified by 16s rRNA gene from Solgent Co., Ltd (Daejeon, Korea).

Mineral salt medium component

2. Hydrogen sulfide oxidation analysis

The component of the mineral salts medium used in the hydrogen sulfide oxidation analysis is in <Table 1>. The mineral salts medium was made into anaerobic condition by injecting nitrogen (N) gas for 30 minutes and was autoclaved at 121℃ for 20 minutes. Sodium thiosulfatepentahydrate (Na2S2O3·5H2O) 8 g/L was separately made and after making into anaerobic condition and autoclaved, it was combined with the mineral salts medium. The isolated strains TO-3, TO-10 and TO-12 were anaerobic cultured in the mineral salts medium at 35℃ for 24 hours. The cultured medium was collected at 0 hour and 24 hour and was centrifuged at 10000 rpm for 5 minutes. The supernatant was used as sample. The hydrogen sulfide concentration was analyzed by the methylene blue method from marine environment official test methods (Ministry of Oceans and Fisheries, 2013). Sample 500 μL was mixed with diamine solution 10 μL and left at room temperature for 20 minutes. Absorbance was measured at 670 nm using microplate spectrophotometer (BioTek Instruments, USA). Sodium sulfide nonahydrate (NaS·9H2O) was used as a standard and the sample concentration of hydrogen sulfide (mg S/L) was measured using a standard curve.

3. Ammonia nitrogen oxidation analysis

LB broth containing ammonia sulfate ((NH4)2SO4) 0.1 g/L was used as medium for ammonia nitrogen oxidation analysis. Isolated strains TO-3, TO-10 and TO-12 were cultured in the medium at 35℃ for 24 hours. The cultured medium was collected at 0, 6, 12, 24 hour and was centrifuged at 10000 rpm for 5 minutes. The supernatant was separated and used as sample. The ammonia nitrogen concentration was analyzed by indophenol method from marine environment official test methods (Ministry of Oceans and Fisheries, 2013). Sample 2.5 mL was mixed with phenol solution 100 μL and nitroprussidesodium solution 100 μL. Oxidation solution 250 μL was added and was developed at 35℃ water bath for 30 minutes. Absorbance was measured at 640 nm using microplate spectrophotometer (BioTek Instruments, USA). Ammonia sulfate ((NH4)2SO4) was used as a standard and the sample concentration of ammonia nitrogen (mg/L) was measured using a standard curve.

4. Nitrite nitrogen oxidation analysis

LB broth containing sodium nitrite (NaNO2) 0.1 g/L was used as medium for nitrite nitrogen oxidation analysis. Isolated strains TO-3, TO-10 and TO-12 were cultured in the medium at 35℃ for 24 hours. The cultured medium was collected at 0, 6, 9, 12 hour and was centrifuge at 10000 rpm for 5 minutes. The supernatant was separated and used as sample. The nitrite nitrogen concentration was analyzed by diazotization method from marine environment official test methods (Ministry of Oceans and Fisheries, 2013). Sample 5 mL was mixed with sulfanilamide solution 100 μL and left at room temperature for 5 minutes. Napeutiretillendiamin solution 100 μL was mixed and left at room temperature for 20 minutes. Absorbance was measured at 540 nm using microplate spectrophotometer (BioTek Instruments, USA). Sodium nitrite (NaNO2) was used as standard and the sample concentration of nitrite nitrogen (mg/L) was measured using a standard curve.

5. Heat resistant analysis

Isolated strains TO-3, TO-10 and TO-12 were each cultured in LB broth at 35℃ for 24 hours. Each cultured mediums were incubated in 50℃, 60℃, 70℃ water bath for 15 minutes and were spread at LB agar and incubate at 35℃ for 18 hours. The strains survival rate was analyzed by calculating the CFU/mL.

Ⅲ. Result and Discussion

1. Isolated strains identification

The identification result of isolated strains from the west coast of Korea is shown in <Table 2>. Nineteen strains were isolated from the water samples and three strains which have high oxidation of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen were selected. The selected three strains TO-3, TO-10 and TO-12 were each identified as Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis. In this study, the three isolated strains would be named as Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12.

Identification of Bacillus sp. strains using 16s rRNA sequencing

2. Hydrogen sulfide oxidation ability

The result of Bacillus sp. strains analyzed of hydrogen sulfide oxidation ability is shown in figure 1. Bacillus sp. TO-3 oxidized 49.53% of the hydrogen sulfide by reducing 1.07±0.02 mg S/L hydrogen sulfide to 0.54±0.03 mg S/L for 24 hours. Bacillus sp. TO-12 oxidized 69.03% of hydrogen sulfide by reducing 1.13±0.04 mg S/L hydrogen sulfide to 0.35±0.03 mg S/L for 24 hours. Bacillus sp. TO-10 oxidized 100% of the 1.13±0.02 mg S/L hydrogen sulfide for 24 hours, which stands out to have the most effective hydrogen sulfide oxidation ability between the three Bacillus sp. strains.

[Fig. 1]

Hydrogen sulfide oxidation of Bacillus sp. strains.

Hydrogen sulfide have high toxicity toward cultured fish even in small concentration. Exposure of 0.5 mg/L hydrogen sulfide resulted hyperpnea, apnea and finally respiratory arrest (Torrans and Clemens, 1982). Therefore eliminating hydrogen sulfide is important in aquaculture industry. The result of Bacillus sp. oxidation ability of hydrogen sulfide also appeared in Nakada and Ohta(1999); Choi et al.(2018). The Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 appeared to have similar oxidation ability of hydrogen sulfide comparing with Choi et al.(2018).

3. Ammonia nitrogen oxidation ability

The result of Bacillus sp. strains analyzed of ammonia nitrogen oxidation ability is shown in figure 2. Bacillus sp. TO-3 oxidized 11.4 mg/L ammonia nitrogen by reducing 276±3.67 mg/L ammonia nitrogen to 264±6.33 mg S/L for 12 hours. However after 12 hours ammonia nitrogen concentration didn’t appeared reducing. Bacillus sp. TO-10 appeared to have oxidation ability after 12 hour. Bacillus sp. TO-10 oxidized 32.7 mg/L ammonia nitrogen by reducing 275±1.22 mg/L ammonia nitrogen to 242±4.11 mg/L. Bacillus sp. TO-12 oxidized 31 mg/L ammonia nitrogen by reducing 271±5.11 mg/L ammonia nitrogen to 240±1.44 mg/L for 24 hour, which appeared to have similar ammonia nitrogen oxidation ability with Bacillus sp. TO-10.

[Fig. 2]

Ammonia nitrogen oxidation of Bacillus sp. strains.

Ammonia nitrogen damage the fish gill, decrease oxygen transportability in blood. Exposure to 40 mg/L ammonia nitrogen caused mortality in cultured fish (Chen and Lei, 1990). Therefore eliminating ammonia nitrogen is important in aquaculture industry. The result of Bacillus sp. having oxidation ability of ammonia nitrogen also appeared in Lee et al.(2003). Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 had a similar ammonia nitrogen oxidation for 24 hours and it is considered the three Bacillus sp. strains would continue to oxidize ammonia nitrogen for 48 hours as in Lee et al.(2003).

4. Nitrite nitrogen oxidation ability

The result of Bacillus sp. strains analyzed of nitrite nitrogen oxidation ability is shown in figure 3. Bacillus sp. TO-3 oxidized 133.22±1.36 mg/L nitrite nitrogen to 78.05±0.82 mg/L at 9 hour and oxidized 100% of nitrite nitrogen at 12 hour. Bacillus sp. TO-10 oxidized 100% of 137.46±0.27 mg/L nitrite nitrogen at 9 hours, resulting the fastest nitrite nitrogen oxidation ability between the three Bacillus sp. strains. Bacillus sp. TO-12 oxidized 100% of the 134.18±2.18 mg/L nitrite nitrogen at 12 hours.

[Fig. 3]

Nitrite nitrogen oxidation of Bacillus sp. strains.

Nitrite nitrogen is toxic to cultured fish by reducing oxygen transport ability and causing asphyxia. Exposure to 20 mg/L nitrite nitrogen caused mortality in cultured fish (Chen and Lei, 1990). Therefore eliminating nitrite nitrogen is important in aquaculture industry. The result of Bacillus sp. having oxidation ability of nitrite nitrogen also appeared in Lee et al.(2003). In this research Bacillus sp. oxidized nitrite nitrogen for 48 hours, however nitrite nitrogen still remained. Unlike Lee et al.(2003), Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 strains had completely oxidized nitrite nitrogen before 12 hours, resulting higher oxidation ability of nitrite nitrogen.

5. Heat resistance

The result of Bacillus sp. strains analyzed of heat resistance is shown in figure 4. At 50℃, the three Bacillus sp. strains appeared to maintain the same CFU/mL compare with the result at 35℃ which indicates the three Bacillus sp. strains have strong heat resistance at 50℃. Bacillus sp. TO-3 appeared to have 5 log10 CFU/mL of cell survivability at 70℃. Bacillus sp. TO-10 and Bacillus sp. TO-12 appeared to have 4 log10 CFU/mL of cell survivability at 70℃. All three Bacillus sp. strains appeared to have high heat resistance at 50℃ and also survive at 70℃. Therefore, the three Bacillus sp. strains are able to survive the occurred heat during the process of water quality improvement product.

[Fig. 4]

Heat resistance of Bacillus sp. strains.

Ⅳ. Conclusion

Three Bacillus sp. strains were isolated from the west coast of Korea and oxidation ability of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen were analyzed for development purpose of water quality improvement probiotics. Bacillus sp. TO-3, Bacillus sp. TO-10 and Bacillus sp. TO-12 were each identified as Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis. Bacillus strains are appropriate and safe probiotics used in aquaculture, known to have effect of increasing fry body weight, digestion ability and reducing the severity of cellular stress (Acella et al., 2010).

Hydrogen sulfide was oxidized each by Bacillus sp. TO-3 49.53%, Bacillus sp. TO-10 100% and Bacillus sp. TO-12 69.03% for 24 hours. Ammonia nitrogen was oxidized each by Bacillus sp. TO-3 11.4 mg/L, Bacillus sp. TO-10 32.7 mg/L and Bacillus sp. TO-12 31 mg/L for 24 hours. All three Bacillus sp. strains have oxidized 100% nitrite nitrogen for 12 hours and Bacillus sp. TO-10 had the fastest nitrite nitrogen oxidation ability. Due to these results, all three Bacillus sp. strains had the oxidation ability toward hydrogen sulfide, ammonia nitrogen and nitrite nitrogen. Above all, Bacillus sp. TO-10 had the most effective oxidation ability between the three isolated Bacillus strains. All three Bacillus sp. strains had strong heat resistance at 50℃ and survived at 70℃. Therefore the three Bacillus sp. strains are possible to be used as probiotics for water quality improvement of hydrogen sulfide, ammonia nitrogen and nitrite nitrogen.

References

  • Avella MA, Gioacchini, G, Decamp O, Makridis P, Bracciatelli C and Carnevali O(2010). Application of multi-species of Bacillus in sea bream larviculture. Aquaculture 305(1-4), 12~19. [https://doi.org/10.1016/j.aquaculture.2010.03.029]
  • Beauchamp RO, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA and Leber P(1984). A critical review of the literature on hydrogen sulfide toxicity. CRC Critical Reviews in Toxicology 13(1), 25~97. [https://doi.org/10.3109/10408448409029321]
  • Brown DA and McLeay DJ(1975). Effect of nitrite on methemoglobin and total hemoglobin of juvenile rainbow trout. The Progressive Fish-Culturist 37(1), 36~38. [https://doi.org/10.1577/1548-8659(1975)37[36:EONOMA]2.0.CO;2]
  • Chen JC and Lei SC(1990). Toxicity of ammonia and nitrite to Penueus monodon juveniles. Journal of the World Aquaculture Society 21(4), 300~306. [https://doi.org/10.1111/j.1749-7345.1990.tb00543.x]
  • Choi JH, Lee JH, Park JJ, Lee MS, Bae JS, Shin DH and Park KH(2018). Reduction of dissolved hydrogen sulfide and mortality of white leg shrimp, Litopenaeus vannamei by Bacillus spp. Microorganisms. The Korean Society of Fish pathology 31(1), 41~48. [https://doi.org/10.7847/jfp.2018.31.1.041]
  • Gadre RV(1989). Removal of hydrogen sulfide from biogas by chemoautotrophic fixed-film bioreactor. Biotechnology and bioengineering 34(3), 410~414. [https://doi.org/10.1002/bit.260340317]
  • Handy RD and Poxton MG(1993). Nitrogen pollution in mariculture: toxicity and excretion of nitrogenous compounds by marine fish. Reviews in Fish Biology and Fisheries 3(3), 205~241. [https://doi.org/10.1007/BF00043929]
  • Kang YH and Yoon YH(2004). Nitrogen Dynamics and Growing of Shrimp (Fenneropenaeus chinensis) in the High Density Aquaculture Ponds. Korean Journal of Fisheries and Aquatic Sciences 37(1), 24~32. [https://doi.org/10.5657/kfas.2004.37.1.024]
  • Kim BMN, Choi A, An SU, Kim HC, Jung RH, Lee WC and Hyun JH(2011). Rates of sulfate reduction and iron reduction in the sediment associated with abalone aquaculture in the southern coastal waters of Korea. Ocean and Polar Research 33(4), 435~445. [https://doi.org/10.4217/OPR.2011.33.4.435]
  • Kioussis DR, Wheaton FW and Kofinas P(2000). Reactive nitrogen and phosphorus removal from aquaculture wastewater effluents using polymer hydrogels. Aquacultural Engineering 23(4), 315~332. [https://doi.org/10.1016/S0144-8609(00)00058-3]
  • Lee YS, Yoo JS, Chung SY, Park CS and Choi TL(2003). Biochemisty, molercular biology : microbial immobilization, characterization and isolation of nitrogen oxidizing bacteria. Journal of the Korean Society for Applied Biological Chemisty 46(1), 1~6.
  • Ministry of Oceans and Fisheries(2013). Marine environment official test methods. Ministry of Oceans and Fisheries, Sejong, Republic of Korea.
  • Mook WT, Chakrabarti MH, Aroua MK, Khan GMA, Ali BS, Islam MS and Hassan MA(2012). Removal of total ammonia nitrogen (TAN), nitrate and total organic carbon (TOC) from aquaculture wastewater using electrochemical technology: a review. Desalination 285, 1~13. [https://doi.org/10.1016/j.desal.2011.09.029]
  • Nakada Y and Ohta Y(1999). Purification and properties of hydrogen sulfide oxidase from Bacillus sp. BN53-1. Journal of bioscience and bioengineering 87(4), 452~455. [https://doi.org/10.1016/S1389-1723(99)80093-0]
  • Piedrahita RH(2003). Reducing the potential environmental impact of tank aquaculture effluents through intensification and recirculation. Aquaculture 226(1~4), 35~44. [https://doi.org/10.1016/S0044-8486(03)00465-4]
  • Romano N and Zeng C(2013). Toxic effects of ammonia, nitrite, and nitrate to decapod crustaceans: a review on factors influencing their toxicity, physiological consequences, and coping mechanisms. Reviews in Fisheries Science 21(1), 1~21. [https://doi.org/10.1080/10641262.2012.753404]
  • Russo RC, Smith CE and Thurston RV. (1974). Acute toxicity of nitrite to rainbow trout (Salmo gairdneri). Journal of the Fisheries Board of Canada 31(10), 1653~1655. [https://doi.org/10.1139/f74-208]
  • Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T and L. Kannan(2008). Probiotics in aquaculture: importance and future perspectives. Indian journal of microbiology 48(3), 299~308. [https://doi.org/10.1007/s12088-008-0024-3]
  • Smith Jr LL, Oseid DM, Kimball GL and Andelgy SM(1976). Toxicity of hydrogen sulfide to various life history stages of bluegill (Lepomis macrochirus). Transactions of the American Fisheries Society 5(3), 442~449. [https://doi.org/10.1577/1548-8659(1976)105<442:TOHSTV>2.0.CO;2]
  • Tomasso JR(1994). Toxicity of nitrogenous wastes to aquaculture animals. Reviews in Fisheries Science 2(4), 291-314. [https://doi.org/10.1080/10641269409388560]
  • Torrans EL and Clemens HP(1982). Physiological and biochemical effects of acute exposure of fish to hydrogen sulfide. Comparative biochemistry and physiology. C: Comparative pharmacology 71(2), 183~190. [https://doi.org/10.1016/0306-4492(82)90034-X]
  • Verschuere L, Rombaut G, Sorgeloos P and Verstraete W(2000). Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev 64(4), 655~671. [https://doi.org/10.1128/MMBR.64.4.655-671.2000]
  • Zhou L and Boyd CE(2016). Comparison of Nessler, phenate, salicylate and ion selective electrode procedures for determination of total ammonia nitrogen in aquaculture. Aquaculture 450, 187~193. [https://doi.org/10.1016/j.aquaculture.2015.07.022]

[Fig. 1]

[Fig. 1]
Hydrogen sulfide oxidation of Bacillus sp. strains.

[Fig. 2]

[Fig. 2]
Ammonia nitrogen oxidation of Bacillus sp. strains.

[Fig. 3]

[Fig. 3]
Nitrite nitrogen oxidation of Bacillus sp. strains.

[Fig. 4]

[Fig. 4]
Heat resistance of Bacillus sp. strains.

<Table 1>

Mineral salt medium component

Component Concentration (g/L)
(NH4)2SO4 3.0
KH2PO4 0.1
MgSO4·7H2O 0.5
CaCl2·2H2O 0.3
FeSO4·7H2O 0.01
NaCl 19.45

<Table 2>

Identification of Bacillus sp. strains using 16s rRNA sequencing

Microorganism Identified strains Identity
Match Total Pct(%)
TO-3 B. amyloliquefaciens 1476 1480 99
TO-10 B. licheniformis 1476 1480 99
TO-12 B. subtilis 1452 1456 99