African Journal of Microbiology Research Vol. 6(10), pp. 2258-2264, 16 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.814 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Isolation and characterization of a denitrifying Acinetobacter baumannii H1 using NO2

--N as nitrogen

source from shrimp farming ponds

Haipeng Cao1, Huicong Wang1, Shan He2, Renjian Ou1, Sanling Hou1 and Xianle Yang1*

1Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources of Ministry of Education, Aquatic Pathogen

Collection Center of Ministry of Agriculture, Shanghai ocean university, Shanghai 201306, P. R. China. 2College of Education, Shanghai Normal University, Shanghai 200235, P. R. China.

Accepted 8 September, 2011

Recent studies have revealed that the use of denitrifying bacteria is an alternative to eliminate nitrite accumulation in the wastewaters. However, no information is available about Acinetobacter species as a denitrifying agent for reducing nitrite in aquaculture. In the present study, a potential denitrifying strain H1 was isolated from shrimp farming ponds using nitrite as nitrogen source, which displayed almost complete nitrite removal. It was initially identified as Acinetobacter baumannii using API identification kits, and confirmed to be A. baumannii strain (GenBank accession no. JF751054) by phylogenetic analysis. Three key denitrification genes nirS, norB and nirK were detected from strain H1 by PCR amplification. In addition, it was demonstrated to be safe for mammals and giant freshwater prawns, and showed in vitro high nitrite removal activities at a wide range of initial pH 5-9 and 15 to 35°C with a final cell density of 105-107 cfu/ml. To the best of our knowledge, this is the first report on a promising denitrifying A. baumannii strain from shrimp farming ponds. Key words: Characterization, nitrite removal, Acinetobacter baumannii, shrimp farming.

INTRODUCTION The shrimp farming industry is an important economic sector in many countries, including China, Thailand, Vietnam, Indonesia, India, etc. (Grǎslund et al., 2003; Anh et al., 2010). However, the intensive shrimp farming has led to serious water pollutions (Shang et al., 1998). In particular, nitrite has become the most common pollutant in intensified shrimp aquaculture because it is an intermediate product either during bacterial denitrification of nitrate or bacterial nitrification of ammonia (Xian et al., 2011). It has been reported that increased concentrations of nitrite can be formed directly within the culture period, reaching up to 20 mg/L in grow-out shrimp ponds (Tacon et al., 2002), and affect the immune response as well as oxygen consumption, ammonia excretion, haemolymph and haemocyanin protein levels of shrimps (Cheng and *Corresponding author. E-mail: xlyang@shou.edu.cn. Tel: +862161900453. Fax: +862161900452.

Chen, 1998; Tseng and Chen, 2004; Xian et al., 2011). For example, as low as 0.06 mg N/L nitrite could result in the significant reduction of total protein content and haemagglutinin levels in haemolymph of Macrobrachium malcolmsonii (Chand and Sahoo, 2006). Thus, besides the alternative control strategies such as feed input reduction, lower stocking densities, the use of denitrifying microorganisms is also widely expected to become an alternative method for the prevention and control of nitrite accumulation.

Microbial denitrification is a common phenomenon in nature and plays a major role in reducing or eliminating the incidence of nitrite in the wastewaters (Kariminiaae-Hamedaani et al., 2004). Recently, the application of Bacillus sp., Paracoccus sp. Bradyrhizobium sp., Pseudomonas sp. as denitrifying bacteria for removing nitrite shows promise (Denariaz et al., 1991; Polcyn and Luciński, 2003; Diep et al., 2009). For example, Wan et al. (2011) isolated a novel denitrifying Pseudomonas sp. strain yy7 that had a nitrite removal rate of 99.7%.

However, no information is available about Acinetobacter sp. as a denitrifying agent for shrimp farming pond treatment.

In this study, we isolated a potential denitrifying A. baumannii stain H1 using nitrite as nitrogen source, determined its taxonomic position, detected denitrification genes using PCR amplification, evaluated its in vitro nitrite removal effects under different initial pH, temperatures and final cell densities. The data could establish its potential as an environmentally friendly probiotic for shrimp farming pond treatment. MATERIALS AND METHODS Sample collection and isolation of denitrifying bacteria

Shrimp farming pond samples were collected from Qingpu Modern Agricultural Development Co., LTD. located in Shanghai, China. The samples were kept in a refrigerator until use. Isolation of

denitrifying bacteria from these samples was done with serial dilution technique on bromothymol blue (BTB) agar medium (Sinopharm Chemical Reagent Co. Ltd, Shanghai) as described by Li et al. (2005) and Mahmood et al. (2009a). Purification of denitrifying bacteria was done using repeated streaking and single colony culture at 30°C. Bacterial isolates were sub-cultured and transferred to basal medium containing (per liter) 0.05 g NaNO2, 4.72 g sodium succinate, 1.50 g KH2PO4, 1.00 g MgSO4·7H2O and 0.42 g Na2HPO4. Their ability to remove nitrite was examined according to Wan et al. (2011). The isolate with highest nitrite removal efficiency was suspended in 25% glycerol solution at -80°C for long-term storage as described by Das et al. (2006). Phenotypic characterization and identification The isolate was grown on nutrient agar (NA) (Sinopharm Chemical

Reagent Co. Ltd, Shanghai) plates at 30°C for 24 h, and then the bacterial suspension was used to inoculate the ID32GN

API strip

(Bio-Merieux, SA) following the manufacturer’s instruction. The strip was incubated at 30°C and observed after 48 h for checking against the API identification index and database.

Molecular identification

DNA extract, PCR and sequencing The genomic DNA was extracted from a pure culture of the isolate using a genomic DNA extraction kit following instructions of the manufacturer (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.). The 16S rRNA gene fragment was amplified by PCR using a pair of universal bacterial 16S rRNA gene primers (27f): 5’-AGAGTTTGATCCTGGCTCAG-3’ and

(1492r): 5’-TACGGCTACCTTGTTACGACTT-3’. The PCR was carried out according to Cao et al. (2010). The PCR product was electrophoresed on 1% agarose gel and visualized via ultraviolet trans-illumination. Sequencing was performed by a fluorescent labeled dideoxynucleotide termination method (with BigDye terminator) on ABI 3730 automated DNA Sequencer.

Phylogenetic analysis The partial 16S rRNA sequence was assembled using MegAlign,

Cao et al. 2259 Editseq and Seqman software with a Macintosh computer. Searches were done against the National Centre for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) program. The phylogenetic tree based on the 16S rRNA gene sequence of the isolate was constructed using the neighbor-joining method. PCR amplification of denitrification genes

The genomic DNA was extracted from a pure culture of the isolate using a genomic DNA extraction kit following instructions of the manufacturer (Shanghai Sangon Biological Engineering

Technology and Services Co., Ltd.). The denitrification genes nirS, norB and nirK were respectively amplified by PCR using specific nirS gene primers, norB gene primers and NirK gene primers recommended by Throback et al. (2004) and Garbeva et al. (2007). The PCR products were electrophoresed on 1% agarose gel and visualized via ultraviolet trans-illumination. In vitro nitrite removal efficiency assay

Influence of initial pH The isolate was inoculated into basal medium with the initial pH of 3, 5, 7, 9, 11 at a final cell density of 1.0×10

6 cfu/ml, and cultured at

30°C for 72 h. The test was carried out in triplicate. Samples of cultures were analyzed for nitrite at 12 h intervals using N-(1-naphthalene)-diaminoethane photometry method and the nitrite removal rate was calculated according to Yang et al. (2011).

Influence of temperature The isolate was inoculated into basal medium with the initial pH of 7 at a final cell density of 1.0×10

6 cfu/ml, and cultured at 15, 20, 25,

30, 35, 40°C for 72 h, respectively. The test was carried out in triplicate. Samples of cultures were analyzed for nitrite at 12 h

intervals using N-(1-naphthalene)-diaminoethane photometry method and the nitrite removal rate was calculated according to Yang et al. (2011). Influence of cell density The isolate was inoculated into basal medium with the initial pH of 7 at a final cell density of 1.0×10

3, 1.0×10

4, 1.0×10

5, 1.0×10

6, 1.0×10

7

cfu/ml, and cultured at 30°C for 72 h. The test was carried out in triplicate. Samples of cultures were analyzed for nitrite at 12 h intervals using N-(1-naphthalene)-diaminoethane photometry method and the nitrite removal rate was calculated according to Yang et al. (2011). Virulence assay

Hemolytic activity assay of the isolate was carried out with rabbit blood agar (RBA) plates (Sinopharm Chemical Reagent Co., Ltd) at 30°C for 2 days. Virulence was further assayed in four-week-old female BALB/c mice (20 g in weight) and giant freshwater prawns (20 g in weight) as described by Vijayan et al. (2006) and Zheng et al. (2008). Animals were challenged with the isolate’s pure cells prepared as mentioned above. The isolate’s suspension was oral administered at the final cell density of 10

5, 10

6, 10

7, 10

8 and 10

9

cfu/g through a micropipette fitted with a fine micropipette tip and thin flexible tube. The control animals were oral administered with the autoclaved physiological saline solution (0.85%). Ten mice and

2260 Afr. J. Microbiol. Res.

0

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A1 B1 C1 D1 E1 F1 G1 H1 J1 K1 L1 M1

Strain

Nitri

te r

em

oval ra

te (

%))

Figure 1. Nitrite removal activity of the isolates.

twenty giant freshwater prawns were tested in each dilution. The animals were bred at 20 to 25°C, fed with the pellet feed. Animals were examined daily and any signs of disease and mortality were recorded up to 14 days. The 50% lethal dose (LD50) was determined according to Mittal et al. (1980).

Statistical analysis

Data were presented as the mean ± the standard deviation (S.D.) for the indicated number of independently performed experiments. P<0.05 was considered statistically significant using one-way

analysis of variance. RESULTS Isolation of denitrifying strains A total of 12 denitrifying bacteria were isolated from the shrimp farming ponds. Only one isolate, named H1, was found to exhibit strong nitrite removal activity, displaying almost complete nitrite removal (Figure 1). Thus, strain H1 was chosen for further study as recommended by Zhou et al. (2011). Characterization and identification The API identification kits identified strain H1 as A. baumannii (Table 1), and it showed an identity of 96.9% with the type strain ATCC19606 in phenotypic characteri-zation. Strain H1 and the type strain ATCC19606 were found both positive for glucose, ribose, arabinose, suberate, propionate, malonate, decanoate, acetate, valerate, 3-oxhydryl-butyrate, lactate, citrate, 4-oxhydryl-benzoate, alanine, histidine, serine, proline, and negative for rhamnose, melibiose, sucrose, fucose, maltose, itaconic sugar, glycogen, mannitol, N-acetyl- glucosamine,

salicin, inositol, sorbierite, 5-keto-gluconate,

3-oxhydryl-benzoate. These data indicated that strain H1 was a A. baumannii strain.

The 1.0 kb 16S rRNA sequence of strain H1 was submitted to GenBank database with the accession no. JF751056. Similarities between the 16S rRNA sequence of strain H1 and those of A. baumannii strains in the GenBank database were 99.0%, which proved the initial identification. The constructed phylogenetic tree using neighbor-joining method further demonstrated that strain H1 was closely related to the A. baumannii strain (GenBank accession no. JF751054) (Figure 2). The identification result from phylogenetic analysis was consistent with that found through API identification kits. Denitrification genes The three denitrification genes’ fragments were obtained using the specific nirS gene primers, norB gene primers and nirK gene primers, respectively (Figure 3). The PCR amplification result suggested the presence of the three key denitrification genes nirS, norB and nirK in strain H1. Nitrite removal efficiency under different initial pH The influence of initial pH on the nitrite removal efficiency of strain H1 was shown in Figure 4. Strain H1 displayed high nitrite removal rates at an initial pH range of 5-9. Only nitrite removal rates of 7% and 9.1% were found after incubation for 72h at an initial pH of 3 and 11, respectively. The nitrite removal of strain H1 was optimal at an initial pH of 7, because a nitrite removal rate of 94.3% was obtained after incubation for 12 h, significantly higher than those at other tested pH (P<0.05). Nitrite removal efficiency under different temperatures The influence of temperature on the nitrite removal efficiency of strain H1 was shown in Figure 5. Strain H1 removed nitrite well from 15 to 35°C, showing the high nitrite removal rate of 98 to 100% after incubation for 72 h. However, strain H1 showed only a nitrite removal rate of 14.8% at the temperature of 40°C, significantly lower than those at other tested temperatures (P<0.05). Nitrite removal efficiency under different final cell densities

The influence of cell density on the nitrite removal efficiency of strain H1 was shown in Figure 6. Strain H1 had good nitrite removal activity at a final cell density of 10

5-10

7 cfu/ml, and the optimal nitrite removal was found

at a final cell density of 106-10

7 cfu/ml, showing

approximately complete nitrite removal. Only a nitrite

Cao et al. 2261

Table 1. Phenotypic characterization comparison of isolate H1 and the type strain using API identification kits.

Tested items Reaction

Isolate H1 ATCC19606

Rhamnose - -

Mannitol - -

N-acetyl-glucosamine - -

Glucose + +

Ribose + +

Salicin - -

Inositol - -

Melibiose - -

Sucrose - -

Fucose - -

Maltose - -

Sorbierite - -

Itaconic sugar - -

Arabinose + +

Suberate + +

Propionate + +

Malonate + +

Decanoate + +

Acetate + +

Valerate + +

Glycogen - -

3-Oxhydryl-butyrate + +

Lactate + +

Citrate + +

Alanine + +

Histidine + +

5-Keto-gluconate - -

2-Keto-gluconate + -

3-Oxhydryl-benzoate - -

4-Oxhydryl-benzoate + +

Serine + +

Proline + +

“+”: Positive; “-”: Negative.

Safety No hemolytic activity was detected with strain H1, with no zones of hemolysis being formed on the RBA plates (data not shown). In addition, no acute mortality or any visible disease signs were observed in the test mice and giant freshwater prawns treated with 10

5 to 10

9 cfu/g of strain

H1’s suspension (data not shown). It was concluded that the LD50 value of strain H1 was estimated to exceed 10

9

cfu/g according to Mittal et al. (1980). DISCUSSION The use of denitrifying bacteria is widely expected to

become an alternative method for the control of nitrite accumulation in aquaculture systems (Kim et al., 1999; Shan and Obbard, 2001). Nitrite has been well-documented to reach a high concentration toxic to aquatic organisms in a short period because of high stocking densities of aquatic animals (Jensen, 2003; Ren et al., 2008). Thus, potential denitrifying bacteria should be screened to reduce the length of time for nitrite elimination in aquaculture. The present study reported a promising denitrifying A. baumannii H1 from shrimp farming ponds, which showed almost fast complete removal property towards 10 mg N/L nitrite. Our data indicated that the isolate could be a suitable candidate probiotic for shrimp farming: (1) a significant in vitro nitrite removal effect at an initial pH of 5-9, a temperature range

2262 Afr. J. Microbiol. Res.

Acinetobacter baumannii strain H-s-1 [FR774581]

Acinetobacter sp. strain PND-4 [EF494199]

Acinetobacter baumannii strain SRMC 27 [EU760630]

Acinetobacter baumannii strain KK14 [GQ200824]

Acinetobacter sp. strain R3 [EU236731]

Acinetobacter sp. strain X1 [EU236725]

Acinetobacter sp. strain TW [FJ753401]

Acinetobacter baumannii strain 14 [FJ907197]

Acinetobacter baumannii strain AQ-3 [JF751054]

strain H1

Bacillus subtilis strain QD517 [EF472261]

93

54

99

66

100

81

66

77

0.02

Figure 2. The constructed phylogenetic tree for isolate H1 using neighbor-joining method.

1 2 3 4

400

bp

700

bp

1000 bp

Figure 3. PCR amplification of isolate H1’s denitrification genes. 1:

1000 bp ladder; lane 2: nirS gene, lane 3: norB gene, lane 4: nirK gene.

of 15 to 35°C with a final cell density of 10

5-10

7 cfu/ml

within 72 h; (2) safety for mammalians and giant freshwater prawns.

0

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0 12 24 36 48 60 72Time (h)

Nitrite r

em

oval rate

(%

))

pH3 pH5 pH7

pH9 pH11

Figure 4. Influence of initial pH on the nitrite removal rate of isolate H1.

Reduction of nitrite to nitric oxide was catalysed by the products of two different nir genes: one product contained the copper (NirK) and the other contained cytochrome cd1 (NirS) (Braker et al., 1998), and nitric oxide was further catalysed by nitric oxide reductase (Nor) expressed by norB gene (Philippot et al., 2001). Thus, the detection of denitrification genes was useful for understanding of nitrite removal mechanisms. To date, some denitrifying bacteria in aquaculture had been

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Time (h)

Nitrite r

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oval ra

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%))

(%

))

15°C 20°C 25°C

30°C 35°C 40°C

Figure 5. Influence of temperature on the nitrite removal rate of isolate H1

demonstrated to have these functional genes (Krishnani, 2010). The present study also proved the presence of the nirS, norB and nirK genes from strain H1 using specific PCR amplification (Figure 3), further indicating the distribution and importance of these denitrification genes in the nitrite removal of denitrifying bacteria. For appli-cation of strain H1 as a probiotic for nitrite accumulation control in shrimp farming, the data on the nitrite removal efficiency were essential. The present study indicated that strain H1 could significantly reduce the nitrite concentration by 98 to 100% after incubation for 72 h at an initial pH of 5-9, a temperature range of 15 to 35°C with a final cell density of 10

5-10

7 cfu/ml (Figures 4 to 6).

In a related study conducted by Mahmood et al. (2009b), Ochrobactrum sp. strain QZ2 only exhibited high nitrite removal activity at a narrow range of initial pH6.5-7.0 and 25 to 30°C. Therefore, strain H1 might have more potential for the removal of nitrite in shrimp farming.

A. baumannii was considered as a pathogen for humans and fish (Bergogne-Berezin and Towner, 1996; Xia et al., 2008). Thus, in order to be considered as a probiotic for application, strain H1 had to be evaluated for its pathogenicity as recommended by Verchuere et al. (2000). The present study revealed that strain H1 could not form any hemolytic rings on the RBA plates (data not shown), and the LD50 value to BALB/c mice and giant freshwater prawns exceeded 10

9 cfu/g. As described by

Cutting (2010), a potential probiotic strainwas regarded as no infectivity or toxicity when its oral LD50 value was above 4.7×10

8 cfu/g. Thus, strain H1 was evaluated as a

safe strain. In conclusion, the present study for the first time

reported a denitrifying bacterium, identified as A.

Cao et al. 2263

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(%))

103 cfu/ml 104 cfu/ml

105 cfu/ml 106 cfu/ml 107 cfu/ml

Figure 6. Influence of final cell density on the nitrite removal rate

of isolate H1.

baumannii strain H1, that could use nitrite as a nitrogen. The high denitrifying activity of strain H1 at a wide pH and temperature range, as well as its safety to the mammalian system and giant freshwater prawns, supported this strain as a promising agent for the removal of nitrite in shrimp farming.

ACKNOWLEDGMENTS This work has been contributed equally by Huicong Wang and financially supported by the National 863 program (No. 2011AA10A216), the Special Fund for Agroscientific Research in the Public Interest (No. 201203085), the Earmarked Fund for China Agriculture Research System (No. CARS-46). REFERENCES

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