Waste Biomass Valor DOI 10.1007/s12649-017-9912-2
ORIGINAL PAPER
Characterization and Strain Improvement of Aerobic Denitrifying EPS Producing Bacterium Bacillus cereus PB88 for Shrimp Water Quality Management Prasenjit Barman1 · Partha Bandyopadhyay2 · Ahmet Kati3 · Tanmay Paul1 · Amit Kumar Mandal4 · Keshab Chandra Mondal1 · Pradeep Kumar Das Mohapatra1
Received: 9 August 2016 / Accepted: 20 March 2017 © Springer Science+Business Media Dordrecht 2017
Abstract A bacterial strain PB88 was isolated from intensive shrimp culture pond to study the denitrification process. On the basis of 16S rDNA analysis, strain PB88 was identified as Bacillus cereus PB88. It has the potential to remove 82.33 ± 3.24% NO−2 −Nin synthetic medium. The optimum pH, temperature and dissolved oxygen for the highest denitrification process of the PB88 were 8.0, 30 °C and 5.21 mg/l (150 rpm) respectively. PB88 harbour the genetic sequence of nitrite reductase (nirS) enzyme which is essential to complete aerobic denitrification process. One remarkable finding is that the experimental organism produced exopolysaccharide (EPS) during the denitrification process and EPS has the antibacterial property against shrimp pathogen Vibrio harveyi MTCC 7954 (inhibition zone of 5.21 mm) and Vibrio vulnificus MTCC 1145 (inhibition zone of 7.11 mm). Removal of NO−2 −N in open base shrimp wastewater system were recorded as 98.51% by B. cereus PB88 and average shrimp body weight gained in treated system as 6 ± 0.54 to 8 ± 0.74 g within 7 days. Overall result indicated that B. cereus PB88 has the immense
* Pradeep Kumar Das Mohapatra
[email protected] 1
Department of Microbiology, Vidyasagar University, Midnapore, West Bengal 721102, India
2
Biostadt India Limited, Poonam Chambers ‘A’ Wing, 6th Floor, Dr. A.B. Road, Worli, Mumbai, Maharashtra 400018, India
3
Department of Detergent and Chemical Technologies, Research and Development Center, Hayat Kimya, 41275 Gölcük, Kocaeli, Turkey
4
Chemical Biology Labortory, Department of Sericulture, Raiganj University, Raiganj, Uttar Dinajpur, West Bengal 733134, India
potential for the application in commercial shrimp culture as denitrifying agent. Keywords Bacillus cereus PB88 · Aerobic denitrification · Exopolysaccharide · Antimicrobial activity · Water quality management
Introduction Globally penaeid shrimp culture ranks 6th in terms of quantity and second in terms of value amongst all taxonomic groups of aquatic animal cultivated. Over the past three decades, shrimp farming in Asia has been expanding rapidly to a vibrant export industry currently valued to more than US $ 8 billion [1]. An intensive culture system is most commonly used for Black Tiger Shrimp (Penaeus monodon) because it produces higher yields than other systems [2]. Ammonia removal by nitrification, sludge removal by sedimentation or mechanical filtration and water exchange are the main criteria to maintain a good quality of water in recirculating aquaculture system [3]. Accumulated nitrogenous pollutant in form of black organic sludge (due to uneaten feed, dead plankton, soil minerals, air borne debris, shrimp feces and pathogenic microorganisms) at the bottom level in shrimp pond is a major environmental concern for shrimp cultivators [4, 5]. In an ecosystem, nitrite is a common compound of the nitrogen cycle but at present it is very crucial matter for intensive aquaculture industry because uneaten high protein feed and feces are generated in the process which causes rapid accumulation of the nitrite in to the intensive aquaculture pond [6, 7]. Denitrification is a respiratory process, in which heterotrophic bacteria converted nitrate and nitrite to gaseous nitrogen form with intermediates nitric oxide (NO), nitrous oxide
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(N2O) and lastly nitrogen. There are three processes to remove NO−2 −N in aquaculture system, i.e. physical, chemical and biological process [8]. Among them both physical and chemical process are not able to completely remove NO−2 −N whereas biological process is the best approaches to completely remove NO−2 −N without any secondary pollutant fermentation [9]. Denitrification process is occurs strictly under the anaerobic condition [10]. But in intensive aquaculture system like shrimp culture pond, need adequate dissolved oxygen (DO) for the aquatic animal [11]. As the culture environment need to be highly aerobic condition, aerobic denitrifying bacteria is important for NO−2 −N removal instant of anaerobic characteristic. Due to additional oxygen supply in intensive shrimp cultivation scientists are trying to find out aerobic denitrifying bacteria to remove completely NO−2 −N in aquatic system. Now a day’s scientific approaches have been showed that very few microorganisms are capable of performing aerobic denitrification. Some microorganisms are able to produce exopolysaccharide (EPS) in their environment [12]. EPS are high molecular weight polymers composed of saccharides subunits. The EPS is normally composed of 40–95% polysaccharides, 1–60% proteins, 1–10% nucleic acids and 1–40% lipids [13]. The composition of the EPS varies with the composition of the microbial consortia and the environmental conditions [14]. The EPS is one of the important compounds for the formation of biofilm. The biofilm matrix generally consist of up to 97% water, 2–5% microbial cells, 3–6% EPS and ions [15]. In the year of 2012, Orsod et al. [16] reported that EPS also showed antimicrobial activities against pathogenic bacteria. Vibrio harveyi, Vibrio vulnificus, Vibrio alginoliticus, Vibrio splendidus and Vibrio parahaemolyticus are the major pathogens in the shrimp culture pond [17]. They are responsible for several types of diseases and mortalities of up to 100% [18]. Barman et al. [19] reported that Bacillus strain can also inhibit certain Vibrio strain which can play a vital role to maintain good water quality in shrimp culture pond. So, this is the time to find out the aerobic denitrifying bacteria which can show a dual role like aerobic denitrification and also positive impact to reduce the growth of pathogenic Vibrio sp. by the formation of EPS. Therefore, the objective of the present study was to isolate the aerobic denitrifying bacteria which reduce the Vibrio sp. and investigate denitrification characteristics for the nitrite removal in the intensive culture ponds.
Materials and Methods All the reagents, solvents and kits were used in the experiment purchased from Hi-media laboratories, India and Sigma–Aldrich, USA.
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Sampling Area Water and soil samples were collected from different black tiger shrimp (Penaeus monodon) culture pond located on Contai (latitude 24° and longitude 87°45′), Bay of Bengal region, India, to isolate aerobic denitrifying bacteria. Isolation of Aerobic Denitrifying Bacteria Luria–Bertani (LB) medium was used for the isolation of denitrifying bacteria from the collected samples with the composition of tryptone 10 g, Yeast extract 5 g and sodium chloride 5 g. Collected water (10 ml) and soil sediment (10 g) were transferred to the different 250 ml conical flasks that contained 90 ml of sterile LB liquid medium and incubated at 30 °C with shaking condition (200 rotations per min). After 24 h incubation, the sample of 0.1 ml was taken from each conical flask and spread on solid LB medium. The single and well grown colonies were picked out from the plates and streaked several times to obtained pure plate. For the confirmation of denitrifying bacteria isolated colonies were incubated on Bromothymol Blue (BTB) medium [ingredients of BTB medium were: l-asparagine 1 g, KNO3 1 g, KH2PO4 1 g, FeCl2–6H2O 0.05 g, CaCl2–2H2O 0.02 g, MgSO4-7H2O 1 g, BTB 1 g, agar 20 g, distilled water 1000 ml], pH 7.0–7.2 at 30 °C. After 3 days incubation, blue colour producing colonies were selected as the denitrifying bacteria [20]. Total seven different morphological colonies were selected and grown in to the liquid denitrification medium (DM) [ingredients of DM were: sodium succinate 4.72 g, N aNO2 0.05 g, K H2PO4 1.5 g, N a2HPO4 0.42 g, MgSO4-7H2O 1 g, distilled water 1000 ml, pH 7.2 (NaNO2 were used as N source for denitrification)] and incubated at 30 °C with shaking condition and periodically measured NO−2 −N removal and changes of dissolved oxygen and pH. Among them the highest NO−2 −N removal efficient bacteria was find out and designated as PB88 as well as preserved at 4 °C for further experiment. DNA Extraction, PCR Amplification and Analysis of 16S rDNA Gene Sequence of the Isolate The genomic DNA from pure culture was extracted according to the kit’s protocols using the Genomic DNA Isolation Kit (Invitrogen, USA). 16S rDNA genes were amplified by PCR using 27F (5′-AGAGTTTGATCCTGG CTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACT T-3′) primers. PCR was done in Biorad T100 thermal cycler and used i-Master Mix (Intron) which contains i-TaqDNA polymerase 2.5U, dNTPs 250 µM, Tris–HCl 10 mM, KCl 50 mM, MgCl2 1.5 mM and chemical stabilizer 1x. PCR reaction mixture (50 µl) was composed of 1 µl of DNA template, 25 µl PCR Master Mix, 1 µl of
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each primer (20 µM/l) and sterile water to volume. PCR conditions were set in a thermal cycler for an initial denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min and a final extension at 72 °C for 8 min. The PCR products were sequenced by Illumina HiSeq 2500 sequencer, at Macrogen, The Netherlands. The forward and reverse sequence data were analysed using CLC Main Workbench Tool. The consensus sequencing was studied using Basic Local Alignment Search Tool (BLAST) to find out the best match. FAME Analysis of the Isolated Strain The fatty acid methyl ester analysis (FAME) was performed by harvesting the pure cells from culture media, saponification by the liberation of fatty acids from the cellular lipids (NaOH and methanol), methylation by formation of fatty acid methyl esters (FAMEs, HCl and methanol), extraction by transfer of the FAMEs from the aqueous phase to organic phase (hexane and Methyl tert-butyl ether), and base (NaOH) wash of organic extract prior to chromatographic analysis [21]. Amplification of the Nitrite Reductase Gene of the Isolated Strain Fragments of the nirK and nirS gene of Bacillus strain YX-6 were amplified using primer pairs nirK1F-nirK5R for nirK and nirS1F-nirS6R for nirS, developed by Braker et al. (1998) [22]. The primers pairs were nirK1F: 5′GG(A/C) ATGGT(G/T)CC(C/G)TGGCA3′, nirK5R:5′ GCCTCG ATCAG(A/G)TT(A/G)TGG3′; nirS1F: 5′CCTA(C/T) TGGCCGCC (A/G)CA(A/G)T3′, nirS6R:5′CGTTGA ACTT(A/G)CCGGT3′ individually. The PCR conditions were according to the report described by Braker et al. [23]. Aliquots of 10 μl of the reactions were analyzed by 1% (w/v) agarose gels electrophoresis and bands were visualized by UV excitation. Measurement of Denitrification Rate of the Isolated Strain Strain PB88 was cultivated for 24 h in LB medium at 30 °C with shaking at 200 rpm. The bacterial sample were collected by centrifugation (4 °C, 15 min, 3000×g) and washed with sterile water for three times. During incubation, the cultures were examined periodically to determine optical density (OD at 620), removal efficiency of NO−2 −N, changes of pH and dissolved oxygen (DO).
Analytical Methods During cultivation, the experimental culture and culture environment was examined periodically for determinations of cell density, chemical analyses, and measurements of pH and DO. The optical density of the culture broth was measured at 620 nm (OD 620) using a spectrophotometer (UV–VIS, Model no. UV2300II, Techcomp, Chaina). Total nitrite nitrogen was measured by the N-(1-naphthalene)diaminoethane photometry method and OD was taken at 543 nm [24]. Extraction of Exopolysaccharide and Study of Antimicrobial Activities of the Isolate After the cultivation of PB88 in DM medium, culture was centrifuged at 10,000×g for 15 min at 4 °C for the extraction of exopolysaccharide. Supernatant taken after centrifugation and ethanol precipitation was done by 1:4 ratios (v/v). Then again centrifuge the solution at 10,000×g at 4 °C for 15 min and collect the pallet. The EPS was made dialysis for the partial purification over night at 10 kDa cut off dialysis bag (Hi-media, India). Finally, freeze–dried technique was used for further experiment of the EPS. The pathogenic strains, V. harveyi MTCC 7954 and Vibrio vulnificus MTCC 1145 were diluted 10− 3 times using sterile Normal Saline Solution (NSS) to reach the concentration of 106 cfu/ml. The diluted cultures of pathogenic strains were spread over the Nutrient Agar (NA) plates. The freeze dried crude EPS mixed with distilled water as a concentration of 2 mg/ml and poured (0.1 ml) on the plates spread with different pathogenic bacteria. After overnight incubation at 30 °C, the EPS producing clear inhibition zones on the pathogenic strains were recorded [25]. Effects of Different pH, DO, Temperature and Salinity for Denitrification The degradation rates of nitrite nitrogen by the isolated strain PB88 was determined under different pH, temperature, DO (Shaking condition set as different rotation per minute) and salinity. The pH was set at 5–11; the temperature was set at 15, 20, 25, 30, 35 and 40 °C; RPM was set at 50, 100, 150, 200, 250 and 300 respectively; the salinity of the artificial seawater was set at 5, 10, 15, 20, 25, 30, 35 and 40 ppt respectively. Influence of Carbon Source on Aerobic Denitrification Isolated PB88 was cultured in DM medium with the addition of the following carbon sources: glucose, citrate (trisodium citrate) and acetate (sodium acetate). Medium containing glucose was autoclaved for 15 min at 110 °C;
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all other media were autoclaved for 30 min at 121 °C. The amount of N aNO2 (N source) was fixed and provided 12 ± 0.049 mg/l NO−2 −N. The culture conditions were as described above. All experiment was carried out in triplicate. Denitrifying Efficiency of PB88 with an Open Aquarium Base System Experimental Design The nitrogen removal capacity of the isolated bacteria was performed in six glass aquarium system having height 30 cm, length 60 cm and width 37 cm. Where total water holding capacity was approximately 70 l and working volume of each aquarium was 61 l. Among them three were with the PB88 and others were control without the bacterial strain. The activated sludge used as nitrogen source was collected from sludge outlet main drain of Black Tiger Shrimp (Penaeus monodon) culture pond, Contai (21.68°N and 87.55°E), Bay of Bengal region, India, applied in each experimental opened aquarium system as a concentration of 0.216 ± 0.004 mg/l NO−2 −N. Average body weight of shrimps was 6 ± 0.15 g and day of culture was 50 and average salinity of the pond was 15–17 ppt. Every aquarium was built with additional air flow to maintain the DO concentration (4–6 mg/l) with six shrimp. CP feed (Charoen Pokhpond aquaculture India Pvt. Ltd., Chennai, India) were used for the shrimp fed. Feed were applied in to four times in a day. The ratio of applied feed sample was 25, 20, 30 and 25% (according to CP chart) in the morning (5.00 AM), noon (11.00 AM), evening (5.00 PM) and night (10.00 PM) respectively. The open aquarium base experimental design was performed at room temperature (30 ± 3 °C) with 12 h day night condition. Removal capacity of NO−2 −N, growth of the bacteria, total dissolved solids (TDS), changes of pH, DO and average body weight of shrimp during the experiment were measured every 12 h interval. Statistical Analysis All the experiments were performed in triplicate, variation was expressed as SD. Analysis of variance (ANOVA) and the Duncan’s multiple range tests were used to differentiate each parameter of all the experiments and the value P ≤ 0.05 was indicated that the results were statistically significance. The data in these experiments were analysed by Microsoft Excel 2007 and SPSS 13.0 software.
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Results Isolation and Identification of Strain PB88 In the present study, ten morphologically different purified blue colonies were selected according to BTB plate’s reaction. The blue colonies were appeared when pH increased in the BTB plate medium. Among them strain PB88 was considered for the further experiment due to the better denitrifying efficiency. Colony morphology of the PB88 was large, irregular and flat an undulate margin (Fig. 1a). It was found that strain PB88 is an aerobic, Gram positive, rod shaped (Fig. 1b), spore forming, motile bacterium. The catalase reaction, gelatin liquefaction, starch hydrolysis, casein hydrolysis, sugar fermentation, and Voges–Proskauer tests were all positive, while the oxidase reaction, indole test were negative and other biochemical test of PB88 by VITEK2 was illustrate in Table 1. A 1434 bp fragment of partial 16 S rDNA obtained from PCR was sequenced and homology searches in NCBI data base using BLAST shown similarity of 99% sequence with Bacillus cereus SBTBC-008 (accession number KF601957.1) and phylogenetic tree was constructed on the basis of 16 S rDNA sequence (Fig. 1c). The nucleotide sequence of PB88 was submitted to the GenBank (accession number KP843556). According to fatty acid profile, PB88 possessed iso-C15:0 (32.00%), iso-C17:0 (15.61%), isoC13:0 (6.05%), summed feature 3 (C16:w17c/16:w16c; 6.68%), iso C14:0 (3.30%), iso-C16:0 (6.65%), isoC17:1w 5c (5.64%) as the major cellular fatty acids. The FAME analysis of the isolate PB88 supported the 16 S rDNA sequence homology and confirmed that PB88 was B. cereus PB88. Denitrifying Efficiency of Bacillus cereus PB88 The denitrifying capacity of Bacillus cereus PB88 was studied in denitrification medium (DM) under aerobic circumstances at 30 °C, where sodium nitrite was the N source. The experimental organism able to removed 82.33 ± 3.24% NO−2 −N in 39 h (12 mg/l initial NO−2 −N concentration) and the denitrification rate was about 0.25 mg/l/h (Fig. 2). During the shaking cultivation time growth (OD at 620 nm) of the PB88 also measured. The stationary phase of the bacterium was begins at 18 h of cultivation and retained up to 27 h with a long duration of the phase of 9 h (Fig. 2). pH and Dissolved oxygen (DO) were also calculated during the cultivation period and observed that the lowest and highest pH and DO of the culture medium varied from 7.2 ± 0.119 to 7.8 ± 0.129
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Fig. 1 Isolated PB88 was identified based on a Colony characteristic, b SEM micrograph showing shape of the cell and c Phylogenetic tree of strain PB88 and related bacteria based on partial 16S rDNA sequence. The tree was constructed using MEGA 4.0
and 6.64 ± 0.029 to 6.84 ± 0.030 mg/l respectively (Fig. 3). Effect of DO, pH, Temperature and Salinity for Denitrifying Capacity of PB88 The maximum NO−2 −N removal efficiency (82 ± 2.38) of B. cereus PB88 was observed in the denitrification media with the shaking speed of 150 rpm and DO was 5.21 ± 0.09 mg/l
(Fig. 4). In this study a wide range of pH was set to find out the optimum Nitrite nitrogen degradation rate and observed that maximum 82 ± 3.23% removal establish at pH 8.0 (Fig. 5). Temperature played an important function on the denitrification reductase activity with pH value. Nitrite nitrogen removal by PB88 occurred at a wide range of temperature profile. At the 30 °C, PB88 showed its highest 82 ± 3.23% NO−2 −Nremoval efficiency (Fig. 6) then decreased slowly with increase in temperature (at 40 °C
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Table 1 Biochemical test of B. cereus PB88 by VITEK 2 Sl No.
VITEK 2 Systems GP card
Result
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
d-Amygdalin
– (−) – – – + + – – – – + + – – + – – – – – – – + – – + – + – – – – – – – + – – + – + –
Phosphophatidyllinositol Phosphate d-Xylose Arginine dihydrolase 1 β-Galactosidase α-Glucosidase Ala-phe-pro arylamidase Clyclodextrin l-Aspartate arylamidase β-Galactopyranosidase α-Mannosidase Phosphatase LeucineArylamidase l-ProlineArylamidase β-Glucuronidase α-Galactosidase l-Pyrrolidonyl- arylamidase β-Glucoronidase Alanine arylamidase Tyrosine arylamidase d-Sorbitol Urease Polymixin B resistance d-galactose d-Ribose l-Lactate alkalization Lactose N-acetyl-d-glucosamine d-Maltose Bacitracin resistance Novobiocin resistance Growth in 6.5% NaCl d-Mannitol d-Mannose Methyl-β-d-Glucopyranose Pullulan d-Rafinose O/129 rsistance (Comp. Vibrio) Salicin Sucrose d-Trehalose Arginine dihydrolase Optochin resistance Phosphatidylinositol phaspholopase C
52 ± 2.05%). The performance of nitrite nitrogen removal by PB88 was also observed in different salinity. From the experiment it was found that the maximum nitrite nitrogen
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removal was occurred at 20 ppt of salinity (Fig. 7) then decreased slowly with increase in salinity of the medium. Model Equation for Quantitative Interrelationship Study The experimental organism was able to denitrify the nitrogenous waste of shrimp cultivation medium significantly. Denitrification process by the organism Bacillus cereus PB88 was mainly regulated by growth of the organism, pH of the medium, dissolved oxygen and cultivation period. An effort has been undertaken to express the quantitative interrelationship of the most influencing factors in denitrification by B. cereus PB88 through following equation.
N=k
DX tpH
6 ≤ t ≤ 18
(1)
where N = Nitrite nitrogen concentration, D = Dissolved oxygen, X = Cell mass, t = Cultivation time (h), pH = pH of the medium, k = Constant). Antimicrobial Activity of EPS V. harveyi and Vibrio vulnificus, the most common pathogens in shrimp cultivation were selected for the antimicrobial activities test. The in-vitro antimicrobial activities of the EPS produced by PB88 exposed that it was more effective against V. vulnificus (MTCC 1145) than V. harveyi (MTCC 7954) and the clear zone of inhibition were 7.11 mm and 5.21 mm respectively (Table 2). Denitrifying Nitrite Reductase Genetic Study For identification of the possible pathway of the aerobic denitrification process, the key gene Nitrite reductase (nirS) was searched. Denitrifying nitrite reductase gene nirS was amplified by PCR for the confirmation of nitrite reductase gene (Fig. 8) of PB88. Amplification result of nirS gene was positive and 480 bp. Effect of Carbon Source in Aerobic Denitrification Three different carbon sources such as glucose, acetate and citrate were added to the denitrification media to influence of B. cereus PB88 for NO−2 −Nremoval. When glucose, acetate and citrate were provided to the denitrification media, the percentages of NO−2 −N removal were 85.85 ± 3.00%, 78.38 ± 2.19 and 69.39 ± 2.22 respectively (Table 3). In normal denitrification media, the stationary phase of B. cereus PB88 reached 0.04 to 1.58 as the OD620 in 18 h but when supplementary carbon sources such as glucose, acetate and citrate used in denitrification medium the stationary phases reached 0.05–1.94,
Waste Biomass Valor Fig. 2 Denitrifying potentiality of B. cereus PB88 in denitrification medium. Nitrite nitrogen removal rate, percentage of nitrite nitrogen removal and growth pattern of B. cereus PB88
Fig. 3 The changes of pH and DO during the denitrification process of B. cereus PB88 in denitrification medium
Fig. 4 Optimisation of DO for nitrite nitrogen removal of B. cereus PB88 in denitrification medium
0.04–1.34 and 0.03–1.22 respectively (Table 3) at the same hour. The maximum pH of glucose, acetate and citrate containing media were 7.8 ± 0.13, 7.8 ± 0.12, 7.7 ± 0.13 respectively. Bacillus cereus PB88 showed 60% denitrification activity up to pH 9.0. Simultaneously
Fig. 5 Optimisation of pH for nitrite nitrogen removal of B. cereus PB88 in denitrification medium
Fig. 6 Optimisation of temperature for nitrite nitrogen removal of B. cereus PB88 in denitrification medium
the lowest DO of the media was 5.8 ± 0.038, 5.8 ± 0.043 and 5.7 ± 0.041 mg/l when glucose, acetate and citrate respectively supplemented as carbon sources in the denitrification medium (Table 3).
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Study of Denitrification in an Open Aquarium Base Wastewater System by Bacillus cereus PB88
Fig. 7 Optimisation of salinity for nitrite nitrogen removal of B. cereus PB88 in denitrification medium Table 2 Antimicrobial activity of EPS against shrimp pathogen Name of the pathogen
Diameter of inhibition zone (mm)
Vibrio harveyi MTCC No. 7954 Vibrio vulnificus MTCC No. 1145
5.21 7.11
The experiment was designed considering aquaculture wastewater sludge as the source of nitrogen waste in an open aquarium base system with strain PB88 (treatment) and without strain PB88 (control) to study NO−2 −N removal. After 7days it has been observed that the amount of NO−2 −N was 0.003 ± 0.002 mg/l in treatment aquarium and 0.176 ± 0.004 mg/l in control. The removal of NO−2 −N in the treated aquarium and control aquarium were 98.51 and 19.26% respectively (Table 4). Initial pH in the treatment was 8.3 ± 0.04 and control was 8.3 ± 0.03, which were in an alkaline condition but after 7 days treatment pH still in an alkaline condition (8.0 ± 0.02) in control system but mostly in neutral 7.3 ± 0.03 in treatment system (Table 4). Table 3 indicates that in treatment system DO dropped 4.91 ± 0.04 to 3.92 ± 0.03 due to oxygen consumption by PB88 and in control system DO change 4.92 ± 0.03 to 4.62 ± 0.03. In this study aquaculture wastewater treated with PB88 showed decrease of TDS up to 74.32% after 7 days treatment and in control system TDS changes only 6.74% (Table 4). During the 7 days experiment trial, the cell density also measured. It was also been observed that the highest cell density were 98 × 109 cfu/ml on 3rd day and after 7 days the cell density remain 92 × 107 cfu/ml. It is clearly indicated that the PB88 plays a major roll for elimination of nitrite nitrogen with high salinity. The average shrimp body weight of treated tank gained 6 ± 0.15 g to 7.5 ± 0.27 g within 7 days, where in control 6 ± 0.18 g to 7 ± 0.14 g, which indicates nitrogenous stress affect shrimp growth.
Discussion
Fig. 8 PCR amplification of nitrite reductase (nirS) and (nirK) genes in B. cereus PB88 (M- 100 base pair ladder)
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The present study aimed to isolate and characterise aerobic denitrifying bacteria from shrimp culture pond. Earlier concept was that denitrification process is occurs strictly under the anaerobic condition [10]. But in intensive aquaculture system like shrimp culture pond, which need adequate dissolved oxygen (DO) supply to maintain a certain concentration of DO into the culture pond for survival of aquatic animal [11]. So aerobic denitrifying bacteria is only the alternative way to reduce the NO−2 −N in shrimp culture pond. In this context the newly isolated B. cereus PB88 was able to remove nitrogenous compound up to 82.33 ± 3.24% by denitrification. Though aerobic denitrification process by bacteria was studied by some of the researchers [26–29] but the experimental organism has an additional advantage of the production of EPS, which also has the antimicrobial effect. Antimicrobial activities of the EPS showed against Vibrio
Citrate
Acetate
(mg/L)
(mg/L)
7.2 ± 0.129
6.3 ± 0.045
DO (mg/L)
0.03 ± 0.001
0 ± 0
6.2 ± 0.045
7.2 ± 0.129
0.03 ± 0.001
0 ± 0
6.3 ± 0.045
7.3 ± 0.131
0.06 ± 0.002
2.42 ± 0.077
14.06 ± 0.264
7.4 ± 0.118
14.41 ± 0.270 14.41 ± 0.270
pH
Cell growth (OD)
removal (%)
NO−2 −N
NO−2 −N
7.2 ± 0.115
0.07 ± 0.003
4.14 ± 0.115
13.17 ± 0.251
6.3 ± 0.042
7.3 ± 0.124
0.11 ± 0.004
4.28 ± 0.149
13.4 ± 0.363
6.2 ± 0.044
6.3 ± 0.045
0.04 ± 0.001
0.14 ± 0.003
6
6.3 ± 0.045
7.2 ± 0.115
DO (mg/L)
0.04 ± 0.001
0 ± 0
pH
Cell growth (OD)
removal (%)
NO−2 −N
6.4 ± 0.042
7.2 ± 0.122
13.74 ± 0.262 13.72 ± 0.262
6.4 ± 0.042
DO (mg/L)
NO−2 −N
7.2 ± 0.122
pH
Cell growth (OD)
removal (%) 0.06 ± 0.002
0.71 ± 0.024
0 ± 0
NO−2 −N
0.05 ± 0.002
13.9 ± 0.376
14 ± 0.379
(mg/L)
3
Glucose
0
NO−2 −N
Cultivation time (h)
Parameter
Carbon source 9
6.2 ± 0.045
7.4 ± 0.133
0.14 ± 0.005
5.75 ± 0.184
13.58 ± 0.255
6.1 ± 0.043
7.3 ± 0.116
0.14 ± 0.006
8.0 ± 0.224
12.64 ± 0.241
6.2 ± 0.041
7.5 ± 0.127
0.24 ± 0.010
11.85 ± 0.414
12.34 ± 0.334
6.0 ± 0.043
7.3 ± 0.131
0.36 ± 0.015
11.48 ± 0.367
12.74 ± 0.239
5.9 ± 0.042
7.6 ± 0.121
0.38 ± 0.016
14.33 ± 0.401
11.77 ± 0.224
6.3 ± 0.042
7.4 ± 0.125
0.68 ± 0.029
22.35 ± 0.782
10.87 ± 0.294
12
5.8 ± 0.038
7.6 ± 0.129
1.21 ± 0.052
42.0 ± 1.47
8.12 ± 0.220
5.9 ± 0.043
7.5 ± 0.135
0.66 ± 0.028
19.63 ± 0.628
11.58 ± 0.217
5.8 ± 0.041
7.5 ± 0.120
0.72 ± 0.031
25.69 ± 0.719
10.21 ± 0.195
15
18
5.7 ± 0.041
7.4 ± 0.133
1.22 ± 0.052
34.14 ± 1.092
9.49 ± 0.178
6.1 ± 0.043
7.7 ± 0.123
1.34 ± 0.059
36.89 ± 1.032
8.67 ± 0.165
5.9 ± 0.039
7.8 ± 0.132
1.94 ± 0.083
58.42 ± 2.044
5.82 ± 0.157
21
5.9 ± 0.043
7.7 ± 0.138
1.21 ± 0.051
45.59 ± 1.458
7.84 ± 0.147
6.0 ± 0.043
7.9 ± 0.126
1.36 ± 0.066
60.4 ± 1.691
5.44 ± 0.103
5.8 ± 0.038
8.1 ± 0.137
1.92 ± 0.082
71.64 ± 2.507
3.97 ± 0.107
Table 3 Characteristics of denitrifying efficacy in denitrifying medium with additional carbon source (glucose, acetate and citrate) 24
4.66 ± 0.087
5.9 ± 0.042
7.7 ± 0.123
1.35 ± 0.058
76.56 ± 2.143
3.22 ± 0.061
6.2 ± 0.041
7.9 ± 0.134
1.94 ± 0.083
85.5 ± 2.992
2.03 ± 0.055
6.2 ± 0.045
7.5 ± 0.135
1.22 ± 0.052
6.0 ± 0.043
7.6 ± 0.136
1.23 ± 0.052
56.14 ± 1.796 67.66 ± 2.165
6.32 ± 0.118
5.8 ± 0.041
7.8 ± 0.124
1.35 ± 0.059
71.68 ± 2.00
3.89 ± 0.074
6.1 ± 0.040
7.9 ± 0.134
1.93 ± 0.083
79.42 ± 2.779
2.88 ± 0.078
27
30
6.1 ± 0.044
7.9 ± 0.142
1.22 ± 0.052
69.39 ± 2.220
4.41 ± 0.082
6.1 ± 0.043
7.8 ± 0.124
1.33 ± 0.058
78.38 ± 2.194
2.97 ± 0.056
6.3 ± 0.042
8.0 ± 0.136
1.94 ± 0.083
85.85 ± 3.00
1.98 ± 0.053
33
5.9 ± 0.043
7.7 ± 0.138
1.06 ± 0.045
69.39 ± 2.220
4.41 ± 0.082
6.1 ± 0.043
7.8 ± 0.124
1.12 ± 0.049
78.38 ± 2.194
2.97 ± 0.056
6.3 ± 0.042
7.8 ± 0.132
1.67 ± 0.071
85.85 ± 3.00
1.98 ± 0.053
Waste Biomass Valor
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Waste Biomass Valor
Table 4 Water quality management of shrimp cultivation by Bacillus cereusPB88 in ex-situ model Parameters
Nitritenitrogen (ppm)
Aquarium Days
Treatment
Initial
1
2
3
4
5
6
7
0.216 ± 0.004
0.0058 ± 0.003
0.0042 ± 0.005
0.0034 ± 0.004
0.0032 ± 0.002
0.0032 ± 0.003
0.0032 ± 0.003
0.0032 ± 0.002
Control
0.218 ± 0.005
0.218 ± 0.003
0.212 ± 0.004
0.205 ± 0.006
0.198 ± 0.004
0.191 ± 0.003
0.184 ± 0.004
0.176 ± 0.004
pH
Treatment
8.3 ± 0.04
7.5 ± 0.03
7.5 ± 0.02
7.4 ± 0.02
7.4 ± 0.02
7.3 ± 0.03
7.3 ± 0.02
7.3 ± 0.03
Control
8.3 ± 0.03
8.2 ± 0.02
8.2 ± 0.03
8.1 ± 0.03
8.2 ± 0.02
8.1 ± 0.04
8.1 ± 0.03
8.0 ± 0.02
DO (mg/l)
Treatment
4.91 ± 0.04
4.56 ± 0.03
4.48 ± 0.02
4.35 ± 0.04
4.14 ± 0.04
4.12 ± 0.03
4.08 ± 0.02
3.92 ± 0.03
Control TDS (ppm) Treatment
4.92 ± 0.03
4.91 ± 0.02
4.88 ± 0.03
4.86 ± 0.03
4.81 ± 0.04
4.77 ± 0.02
4.72 ± 0.03
4.62± 0.03
27 ± 1.02
24 ± 1.06
21 ± 1.11
18 ± 0.94
17 ± 0.85
17 ± 0.77
17 ± 0.81
17 ± 0.88
Control
27 ± 1.12
27 ± 1.04
28 ± 1.08
29 ± 1.21
29 ± 1.14
30 ± 1.07
30 ± 1.04
31 ± 1.06
Salinity (ppt)
Treatment
15.21 ± 0.012
15.20 ± 0.014
15.18 ± 0.017
15.16 ± 0.014
15.13± 0.013
15.11± 0.018
15.08± 0.013
15.05± 0.012
Control
15.22 ± 0.013
15.21± 0.011
15.19± 0.016
15.17 ± 0.012
15.13± 0.015
15.10± 0.011
15.09± 0.017
15.06± 0.014
Cell growth (cfu/ml)
Treatment
57 × 106
69 × 108
86 × 109
88 × 109
84 × 108
71 × 108
51 × 108
89 × 107
Average body weight
Treatment
6 ± 0.15
6 ± 0.13
6 ± 0.31
6 ± 0.28
7 ± 0.17
7 ± 0.13
7 ± 0.21
7.5 ± 0.27
Control
6 ± 0.18
6 ± 0.16
6 ± 0.29
6 ± 0.14
6 ± 0.17
7 ± 0.18
7 ± 0.11
7 ± 0.14
vulnificus MTCC No. 1145 (7.11 mm) and V. harveyi MTCC No. 7954 (5.21 mm) which are common shrimp pathogen (Table 2). For the aerobic denitrification, dissolved oxygen was a major factor. In the present study, the denitrification rate of PB88 increased with the increased of DO and then declined. So the result supported that PB88 able to remove nitrite nitrogen in an aerobic condition. pH is one of the another factor for the growth of any bacteria. The optimum pH of denitrifying bacteria was neutral or alkalescency and activity of denitrifying bacteria could be reduced when pH will be high [30, 31]. B. cereus PB88 removed NO−2 −N optimally (82 ± 3.23%) at pH 8.0 but it has also showed the removal efficiency up to 70% at pH 9.0. It has a clear indication of alkaline environment for NO−2 −N removal by the experimental organism. The same has been reflected in Bacillus sp. YX-6 [9]. The experimental bacterium B. cereus PB88 removed 82 ± 3.23% NO−2 −N optimally at 30 °C but it has also showed up to 50% removal efficacy at 40 °C. So the result clearly indicated that NO−2 −N removal can occurred by the experimental bacteria at high temperature and this result also reflected in P. stutzeri YZN001 [32]. B. cereus PB88 maximum removed 82 ± 3.23% NO−2 −N at 20 ppt salinity but it has also exhibited up to 55% removal efficiency at 30 ppt salinity. Denitrification properties of bacteria mostly dependent on nitrite reductase functional gene, and plenty of reports were found on composition and correlative gene of nitrite reductase [23, 33]. Philippot et al. [34] and Barman et al. [35] reported that expression of nirS gene is suggestive of
13
transformation of NO−2 −N to NO or N 2O. In the present study result indicated that nirS gene present in the PB88. The carbon source is one of the important subjects for the aerobic denitrification process. During this process, carbon source was used by the denitrifiers as an electron donor and increasingly reduce nitrate to N2, thus eliminate nitrate and organic matter concurrently. The molecular content and structure of the carbon source were clearly talented to power of the denitrification [36]. In this study, it has been observed that glucose was the most effective carbon source for PB88 for reduction of NO−2 −N level. This result was consistent with previous finding of Bacillus subtilis A1 [37] where glucose was the best carbon source for nitrogen removal. On the other hand, some scientific article reported that citrate was the best carbon source for nitrogen removal by P. stutzeri T1 [38] and P. stutzeri YG-24 [28]. According to Duan et al. [39] and Li et al. [28] different carbon source can influence nitrogen removal efficiency by different organisms. When additional carbon sources were used in the denitrifying medium, the stationary phases of B. cereus PB88 increased than the normal denitrifying medium (Table 3). As a result, the entire carbon source supported PB88 for growth and removal of NO−2 −N. Denitrifying capacity of the aerobic bacteria has been reported mainly on the basis of laboratory mode. Only few reports are available that denitrifying performance with an open base wastewater system [28, 37, 40, 41] but no report available where efficiency examined with shrimp. The effort has been taken through the present study to observed NO−2 −Nremoval efficiency of aquaculture wastewater
Waste Biomass Valor
sludge in an open aquarium base system with B. cereus PB88. During the experiment different water parameters were monitored periodically on both treated and control system and also checked the growth performance of the B. cereus PB88 with high salinity (Table 4). The experimental result showed that B. cereus PB88 removed NO−2 −N 98.51% in high salinity (15 ppt) after 7 days. The result in this study clearly indicated that PB88 not only survive in high salinity but also maintain good NO−2 −N degradation rate. Therefore PB88 could be used for the removal of NO−2 −N in marine or shrimp culture where 16–35 ppt salinity was the ideal for P. monodon culture [42]. After 7 days surveillance pH of the treatment goes to 8.3 ± 0.04–7.3 ± 0.03 which was mostly neutral condition (Table 4). Although strain PB88 showed its best denitrifying capacity when DO was 5.21 ± 0.09 mg/l (Fig. 4) but in an open aquarium base system PB88 was active for denitrification at low DO concentration (4.14 ± 0.04 mg/l). It is another advantage of B. cereus PB88. Total Dissolved Solid (TDS) is a measurement of inorganic salts, organic matter and other dissolved materials in water. Total dissolved solids cause toxicity through increases in salinity, changes in the ionic composition of the water and toxicity of individual ions. Present study illustrated that the initial and final TDS of the treated aquarium were 27 ± 1.12 and 17 ± 0.88 respectively (Table 4). Where in the control aquarium, TDS increased slowly up to 31 ± 1.06 ppm. Strain PB88 has the capability to reduce NO−2 −N and maintain optimum water quality parameters. It was found that after 7 days the cell density remained 89 × 107 cfu/ml that means PB88 can survive with high salinity and have been completed the nitrite nitrogen removal role. The most important part of this experiment, average body weight of the shrimp was achieved 1.5 g within 7 days without showing any sign of disease (Table 4). On the other hand, in the control aquarium shrimp growth was slow it may be due to aquatic environmental stress like, nitrogenous compound, high TDS etc. This results denote that Bacillus cereus PB88 have the capability to organize eco-friendly aquatic environment by denitrifying activity.
Conclusion Bacillus cereus PB8 showed excellent aerobic denitrifying ability both in artificial media and real wastewater treatment. The NO−2 −N removal rate was 0.25 mg/l/h. The optimum pH, temperature, shaking speed and salinity for denitrification were 8.0, 30 °C, 150 rpm and 20 ppt respectively. Expression of nirS gene was the additional verification of B. cereus PB88 for the denitrification process. The most important finding of this study was EPS production during the denitrification process can play a major role to
inhibit the common shrimp pathogen Vibrio sp. The strain PB88 did not showed pathogenicity against shrimp. So it is confirmed that the bacterium Bacillus cereus PB88 belongs to aquaculture probiotic group’s bacteria and a novel aerobic denitrifying bacterium which can eliminate the pathogenic Vibrio sp. in shrimp pond. It also played a major role to maintain good water quality parameters in commercial shrimp cultivation. Acknowledgements The authors are thankful to Mr. D. K. Chopra group CEO of Biostadt India Limited, Mumbai, India for his kind support and Mr. Pravas Singh Assistant professor, Department of Computer Science, Vidyasagar University for development of equation. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest.
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