Using Bacillus amyloliquefaciens for remediation of aquaculture water
© Xie et al.; licensee Springer. 2013
Received: 29 November 2012
Accepted: 27 February 2013
Published: 20 March 2013
Remediation of aquaculture water using microorganisms like Bacillus species is a burgeoning trend for the sustainable development of aquaculture industries. In this work, a Bacillus amyloliquefaciens strain (namely B. amyloliquefaciens HN), isolated from activated sludge of a polluted river, was evaluated for its potential in water remediation using simulated aquaculture water. B. amyloliquefaciens HN exhibited high tolerance towards 80 mg l-1 of nitrite-N and ammonia-N. It could effectively remove 20 mg l-1 of nitrite-N, but was inefficient in eliminating ammonia-N when the ammonia-N concentration was below 20 mg l-1. Further studies showed that the ammonia-N removal by B. amyloliquefaciens HN was more efficient at 30°C and 35°C than 25°C, and that maximum nitrite-N removal rate was achieved at pH 8.
KeywordsBacillus amyloliquefaciens Aquaculture water remediation Nitrogenous compound Nitrite removal
The rapid expansion of intensive aquaculture industries, are often companied by rotted uneaten feed, sedimentation of feces and organic residues. The water quality rapidly deteriorates as a result. In particular, nitrogenous compounds such as ammonia and nitrite quickly build up, which are both harmful to fish and shrimp even at low concentrations (Crab et al. 2007; Mohapatra et al. 2012). Water exchange can be applied to maintain good water quality. However, frequent water exchange is not only laborious and costly, but also may incur disease causing agents and pollute nearby rivers and coastal areas (Mohapatra et al. 2012). Therefore, there is an urgent demand for cost-effective and environment-friendly approaches for remediation of aquaculture water.
In recent years, the use of microorganisms to improve water quality becomes a burgeoning trend (Ninawe and Selvin 2009; Verschuere et al. 2000). Bacillus species are widely used for water remediation because they are stable for long period due to spore formation, easily prepared by fermentation and possess antagonistic effects on pathogens (Hong et al. 2005). Strains belonging to several Bacillus species, such as Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus were isolated and evaluated for their potential as biological agents for water quality enhancement. Several strains with good nitrogen removal properties were thus found. To date, screening strains with good remediation characteristics still remains a fundamental step towards developing commercial microbial agents.
Previously, we isolated a Bacillus amyloliquefaciens strain, named as B. amyloliquefaciens HN from the activated sludge of a polluted river. This strain was shown to effectively remove nitrogenous compounds and grow in broad temperature, pH, and salts concentration in preliminary studies. Moreover, no previous studied has characterized the nitrogen removal ability of B. amyloliquefaciens. Therefore, the aim of this study is to evaluate B. amyloliquefaciens HN for its remediation properties using simulated aquaculture water.
Materials and methods
Strains and culture media
B. amyloliquefaciens HN was isolated from a polluted pool in Xiqing District, Tianjin. The strain was identified by Agricultural Culture Collection of China, and deposited at China General Microbiological Culture Collection Center (CGMCC No. 3261).
The culture medium (also referred as basal medium) for B. amyloliquefaciens HN contained (per liter): peptone 8 g, beef extract 3 g, soluble starch 5 g, NaCl 5 g (plus agar 15 g for solid medium). The simulated aquaculture water was prepared referring to Chen (2005) and LuzE. De-Bashanet (2002), which contained (per liter): beef extract 0.5 g, sucrose 0.5 g, NaCl 0.25 g, KH2PO4 0.075 g.
Nitrite and ammonia-N tolerance tests
Sodium nitrite and ammonium sulfate were added to the culture medium to make the final concentrations of nitrite-N 1, 5, 10, 20, 40, 80, and 100 mg l-1, respectively, and those of ammonia-N 10, 20, 40, 80, and 160 mg l-1, respectively. All the experiments were repeated thrice with the basal medium as control. Three milliliters of strain culture were inoculated into 250 ml shake flask with 100 ml culture media and cultured at 37°C.
Removal tests of nitrite-N and ammonia-N
The simulated polluted water was prepared by making the final concentrations of nitrite-N 10, 20, 40, and 80 mg l-1, respectively, and the concentrations of ammonia-N 5, 10, 20, and 40 mg l-1, respectively. Three milliliters of strain culture were inoculated into 250 ml shake flask with 100 ml culture media and cultured at 37°C.
Effects of different conditions on the removal of nitrite-N and ammonia-N
The model polluted water was prepared to make the initial concentration of nitrite-N was 10 mg l-1, and ammonia-N was 20 mg l-1. The inoculum volume was 3%, and cultured at different temperature (25°C, 30°C, and 35°C), and different initial pH (pH5.0, 6.0, 7.0, 8.0, and 9.0, adjusted by NaOH and HCl).
The Nesslar method was used for ammonia determination. The 1,2-ethanediamine, N-1-naphthalenyl-,dihydrochloride spectrophotometric method was used for the nitrite measurement (APHA, 2005). The nitrate concentration was measured using the salicylic (2-hydroxybenzoic) acid method (Cataldo et al. 1975). Cell concentration was determined by plate counting.
Nitrite and ammonia-N tolerance tests for of B. amyloliquefaciens HN
For ammonia-N tolerance test (Figure 1b), all cell concentrations with tested ammonia-N concentrations were higher than that of control, except the one with 160 mg l-1 ammonia-N. The ammonia-N concentration of 20 mg l-1 resulted in the highest cell growth, 79.9% higher than that of control. The results indicated that B. amyloliquefaciens HN can tolerate ammonia-N ranging from 10 mg l-1 to 80 mg l-1. An ammonia-N concentration of 160 mg l-1 was shown to inhibit the growth of the strain.
Nitrite-N and ammonia-N removal tests for B. amyloliquefaciens HN
With high initial ammonia-N concentrations (20 mg l-1 and 40 mg l-1) ,the removal rates of ammonia-N were significant, and maximum removal rates of 59.8% and 79.8% were reached at 48 h (Figure 2b). As for low initial ammonia-N concentrations (10 mg l-1 and 20 mg l-1), the removal efficiencies of ammonia-N were not efficient. During the time course, the residue ammonia-N levels fluctuated around 10 mg l-1, suggesting that B. amyloliquefaciens HN could not only utilize ammonia-N but also produce it.
Effects of temperature on the nitrite-N and ammonia-N removal abilities of B. amyloliquefaciens HN
Effects of pH on the nitrite-N and ammonia-N removal abilities of B. amyloliquefaciens HN
In this work, we investigated the potential of using B. amyloliquefaciens in the remediation of aquaculture water. Particularly, its remediation characteristics regarding nitrite and ammonia removal were evaluated. The accumulation of nitrite and ammonia is highly toxic to aquatic fauna. 0.1 ~ 10 mg l-1 of nitrite can cause 50% mortality (LC 50) of a number of fish and shrimp (Philips et al. 2002). Ammonia-N can be toxic to commercially cultured fish at concentrations above 1.5 mg l-1 (Crab et al. 2007).
Bacillus species are important candidates for developing commercial biological agents for nitrogen removal and water quality enhancement (Hong et al. 2005). Previously, a few studies reported that some strains of B. subtilis (Chen and Hu 2011; Meng et al. 2009), B. lichenformis (Meng et al. 2009) and B. cereus (Lalloo et al. 2007) exhibited strong nitrite removal ability. Physiological studies on Bacillus spp. also showed that Bacillus spp. could utilize nitrate and nitrite as alternative electron acceptors and nitrogen sources (Nakano et al. 1998; Hoffmann et al. 1998).
This study showed that an B. amyloliquefaciens strain, isolated from the activated sludge, was also a very efficient nitrite-N cleaner, which was able to completely remove 10 mg l-1 of nitrite-N present in the simulated aquaculture water within 24 h. In the past, B. amyloliquefaciens were applied in enzyme production (Wei et al. 2011), plant disease control (Alvindia and Natsuaki 2009; Arrebola et al. 2010) and food preservation (Wang et al. 2006). This is the first study on its potential application in improving water quality. It suggested that B. amyloliquefaciens might be an important alternative Bacillus species for nitrite removal.
The ammonia-N removal ability of B. amyloliquefaciens was not satisfying, because when the ammonia-N concentration was below 20 mg l-1, the removal rate was at best 39.4% and the ammonia-N concentration cannot be reduced below 10 mg l-1. Past literature seems to suggest that Bacillus species are not very efficient in ammonia removal, and no ammonia removal efficiency of a single Bacillus strain has been reported to exceed 90%. Therefore, to create a microbal agent which can simultaneously eliminate nitrite and ammonia, B. amyloliquefaciens NH can be formulated with efficient ammonia reducing bacteria, such as nitrifying bacteria (Meng et al. 2009).
This work is supported by Key Projects in Science and Technology Support Program of Tianjin, China (10ZCZDSY06100), Major Projects in Science and Technology Support Program of Tianjin, China (11ZCKFNC00500) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-15-03).
- Alvindia DG, Natsuaki KT: Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing pathogens. Crop Prot 2009, 28(3):236-242. 10.1016/j.cropro.2008.10.011View ArticleGoogle Scholar
- Apha A WPCF: Standard methods for the examination of water and wastewater. Washington, DC: Public Health Association; 2005.Google Scholar
- Arrebola E, Jacobs R, Korsten L: Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J Appl Microbiol 2010, 108(2):386-395. 10.1111/j.1365-2672.2009.04438.xView ArticleGoogle Scholar
- Cataldo D, Haroon M, Schrader L, Youngs V: Rapid colorimetric determination of nitrate in plant-tissue by nitration of salicylic-acid. 1975, 1(6):71-80.Google Scholar
- Chen GK, Chen G: Effect of synthetic wastewater on some physico-chemical properties of soil in Avicennia marina simulated wetland system. Mar Environ Sci 2005, 24(2):26-28.Google Scholar
- Chen S, Hu Y: Use of Bacillus subtilis in purification of slightly-polluted water. Acta Scienctiae Circumstantiae 2011, 31(8):1594-1601.Google Scholar
- Crab R, Avnimelech Y, Defoirdt T, Bossier P, Verstraete W: Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture 2007, 270(1–4):1-14.View ArticleGoogle Scholar
- De-Bashan LE, Moreno M, Hernandez JP: Removal of ammonium and phosphorus ions from synthetic wastewater by the microalgae Chlorella vulgaris coimmobilizedin alginate beads with the microalgae growth promoting bacterium Azospirilum brasilense . Water Res 2002, 36(12):2941-2948. 10.1016/S0043-1354(01)00522-XView ArticleGoogle Scholar
- Hoffmann T, Frankenberg N, Marino M, Jahn D: Ammonification in Bacillus subtilis utilizing dissimilatory nitrite reductase is dependent on resDE. J Bacteriol 1998, 180(1):186-189.Google Scholar
- Hong HA, Duc H, Cutting SM: The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 2005, 29(4):813-835. 10.1016/j.femsre.2004.12.001View ArticleGoogle Scholar
- Lalloo R, Ramchuran S, Ramduth D, Görgens J, Gardiner N: Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. J Appl Microbiol 2007, 103(5):1471-1479. 10.1111/j.1365-2672.2007.03360.xView ArticleGoogle Scholar
- Meng R, He LS, Xi BD, Hu X, Li YY: Experimental study on purifying aquaculture wastewater between Bacillus and nitrifying bacteria. Environ Sci Technol 2009, 32(11):28-31.Google Scholar
- Mohapatra S, Chakraborty T, Kumar V, Deboeck G, Mohanta KN: Aquaculture and stress management: a review of probiotic intervention. J Anim Physiol Anim Nutr 2012, 1-26. 10.1111/j.1439-0396.2012.01301.xGoogle Scholar
- Nakano MM, Hoffmann T, Zhu Y, Jahn D: Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE. J Bacteriol 1998, 180(20):5344-5350.Google Scholar
- Ninawe AS, Selvin J: Probiotics in shrimp aquaculture: avenues and challenges. Crit Rev Microbiol 2009, 35(1):43-66. 10.1080/10408410802667202View ArticleGoogle Scholar
- Philips S, Laanbroek HJ, Verstraete W: Origin, causes and effects of increased nitrite concentrations in aquatic environments. 2002, 1(2):115-141.Google Scholar
- Verschuere L, Rombaut G, Sorgeloos P, Verstraete W: Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 2000, 64(4):655-671. 10.1128/MMBR.64.4.655-671.2000View ArticleGoogle Scholar
- Wang J, Quan C, Xu H, Fan S: Antifungal characterization of Bacillus amyloliquefaciens Q-12. Food Ferment Ind 2006, 32(6):47-50.Google Scholar
- Wei X, Luo M, Xu L, Zhang Y, Lin X, Kong P, Liu H: Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. J Agric Food Chem 2011, 59(8):3957-3963. 10.1021/jf1049535View ArticleGoogle Scholar
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