Bioremediation & Biodegradation
Research
Article
Research
Article
Saha et al., J Bioremed Biodeg 2013, 5:1
http://dx.doi.org/10.4172/2155-6199.1000211
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OpenAccess
Access
Development of Molecular Identification of Nitrifying Bacteria in Water
Bodies of East Kolkata Wetland, West Bengal
Mousumi Saha1*, Agniswar Sarkar2 and Bidyut Bandhophadhyay1
1
2
Department of Biotechnology, Vidyasagar University, West Bengal, India
Department of Biotechnology (Recognized by DBT, Govt. of India), The University of Burdwan, Burdwan, West Bengal, India
Abstract
Nitrifying bacteria plays a major role in converting the waste water to valuable renewable resources for the
society. Ammonia is the toxic excretory product of most aquatic organisms and nitrite formed by the oxidation of
ammonia is also toxic. Ammonia oxidizing bacteria converts ammonia to nitrite and Nitrite oxidizing bacteria convert
nitrite to nitrate. Among them, Nitrosomonas sp. and Nitrobacter sp. has been the most widely studied organisms. An
advanced molecular technique made it possible to explore the nitrifying bacteria in the environment and to enhance
our knowledge of its functioning. In view of this it would be of prime importance for rapid detection of these strains/
isolates using molecular techniques. The present study was undertaken to develop and validate 16S rDNA sequence
analysis of Nitrosomonas sp. and Nitrobacter sp. collected from different bheries in East Kolkata Wetland, West
Bengal.
Keywords: Ammonia oxidizing bacteria; Nitrite oxidizing bacteria;
16S rDNA; East Kolkata Wetland
Introduction
East Kolkata Wetland (EKW) is the world’s largest sewage-fed
aquaculture and natural water recycling system and contains a series
of wetland. It is renowned not simply as the sensitive ecosystem of
biodiversity value, but more specifically as an example of best practice
in the ‘wise use’ of wetlands (from 1879 onwards) for garbage farming
(2500 metric ton of solid waste is dumped every day), sewage-treated
fisheries (bheris) and sewage farming [1]. EKW located in the eastern
part of the city (22024/- 22036/N to 88023/-88032/E). In addition,
organic fertilizer produced by processing the solid waste from this area
is found to be commercially important [2-4]. The wetland also supports
the livelihoods of around 60,000 residents through the fisheries and
other socio-economic activities.
It serves as a vast area for research purpose and environment
protection through bioremediation or phytoremediation [3]. EKW
declared as a Ramsar site on November 2002 as per “Ramsar Guidelines”.
Wastewater in EKW flows through various channels into the fish ponds
covering about 4000ha, where a wide range of physical, biological and
chemical processes take place which helps in improving the quality of
the water [4,5]. Consequently this wetland system is popularly known
as the “kidney of the city”.
Various bacterial species distributed in different site or bheries in
EKW and involved in bioremediation of heavy metals. These diverse
microbial resources also produce some commercially important
enzymes like protease, oxidase etc. Streptococcus macedonicus produces
extracellular protease and food grade bacteriocine [6]. Pseudomonas sp.
are involved in the production of extracellular protease [7]. Klebsiella
sp. can help to overcome cadmium toxicity [8]. Actinobacteria and
Fermicutes participate in the degradation of nitrophenol, nitroaromatic
compound, pesticide and herbicide [3]. The metal accumulating or
removal property of these microbes can be exploited for treatment
of contaminants rich in metals in various sites. Along with the above
microbes, proteobacteria like Nitrosomonas sp. present in EKW and
enable to oxidize ammonia into nitrite [3].
Ammonia is the principal excretory product of most aquatic
organisms, but is toxic, acute and chronic to fish. Nitrite is formed
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
either by the oxidation of ammonia (Nitrification) or the reduction of
nitrate. Nitrite is also toxic to fish and another critical water quality
factor. Ammonia affects on the central nervous system by causing
“acute ammonia intoxication” which includes convulsions and death
[9]. Acute concentrations of ammonia may cause loss of equilibrium,
hyperexcitability, increased breathing, cardiac output, and oxygen
uptake; in extreme cases convulsions, coma, and death. Lower
concentrations of ammonia can cause a reduction in hatching success;
reduction in growth rate and morphological development; pathologic
changes in tissues of gills, livers, and kidneys [10]. Brown Blood
Disease occurs in fish when water contains high nitrite concentrations.
Nitrite enters the bloodstream through the gills and turns the blood
to a chocolate-brown color. Hemoglobin, which transports oxygen
in the blood, combines with nitrite to form Methemoglobin, which
is incapable of oxygen transport [11]. Warm water fishes like Carps,
Catfishes, and Tilapia are fairly sensitive to nitrite, and trout and other
cool water fish are sensitive to extremely small amounts of nitrite.
Nitrites also react directly with hemoglobin in human blood and other
warm-blooded animals to produce methemoglobin.
Thus, it is very important to understand the dynamics of nitrification,
where may be some microbe like ammonia oxidizing bacteria (AOBNitrosomonas sp.) and nitrite oxiding bacteria (NOB-Nitrobacter
sp.) plays the major role in converting the waste water to valuable
renewable resources for the society [12-14]. The genera Nitrosomonas
and Nitrobacter both are chemolithoautotrophic and members of the
Proteobacteria class [15]. Genus Nitrosomonas form a distinct group
within the β subdivision where as nitrite-oxidizing bacteria like
Nitrobacter occurring in α, δ, and γ subdivisions. Therefore, to maintain
*Corresponding author: Mousumi Saha, Department of Biotechnology,
Vidyasagar University, West Bengal, India, Tel: +91 9830736189; E-mail:
[email protected]
Received September 17, 2013; Accepted October 30, 2013; Published
November 07, 2013
Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of Molecular
Identification of Nitrifying Bacteria in Water Bodies of East Kolkata Wetland, West
Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/2155-6199.1000211
Copyright: © 2013 Saha M, et al. This is an open-a ccess article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Volume 5 • Issue 1 • 1000211
Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of Molecular Identification of Nitrifying Bacteria in Water Bodies of East Kolkata
Wetland, West Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/2155-6199.1000211
Page 2 of 5
the water quality and fish health it’s become very important to keep
the ammonia and nitrite within the safe tolerance limit and these can
be done some nitrifying bacteria. Various researchers described that
only about 1 to 4% of the entire microbial community of EKW has
been screened following the traditional and the conventional methods
and about 96 to 99% are yet to be isolated and identified [16,17].
EKW, which can prove to be important from point of bioremediation
and biotechnology and in this respect, it is necessary to develop the
rapid and accurate technology for isolation and identification of these
microbes using modern molecular techniques. Therefore, to gather
more information about these diverse microbial communities, it is
very important to use molecular technologies like 16S rRNA gene
sequence, phylogenetic analysis etc [18,19]. Study of AOB and NOB
by conventional cultivation techniques is also very difficult because
of their long generation times and low growth rates [20]. Therefore,
a rapid, culture-independent detection technique for nitrifiers would
be useful to provide a record about the natural availability and role
of these microbes in bioremediation of various bheries located in
the East Kolkata Wetland. In view of this economic importance to
aquatic animals, human and animal health hazard problems, it would
be of prime importance for rapid detection of these strains/isolates
using molecular techniques. With this in view, the present study was
undertaken with following objective: To develop and validate different
genotyping techniques viz 16S rDNA sequence analysis, Ribotyping and
bioinformatics analysis for Nitrosomonas and Nitrobacter sp. collected
from different bheries in West Bengal. To develop and standardize PCR
technique, for accurate and prompt diagnosis of the species, to create a
genomic fingerprint database for surveillance and monitoring genetic
variability of isolates and also to establish a biosecurity protocol for the
Bheries located in different areas of West Bengal.
Materials and Methods
Site selection
In West Bengal both sewage fed fisheries and floodplain wetlands
plays an important fishery resources. The commonly cultivated fish
includes Catla (Catla catla), Rohu (Labeo rohita), Mrigal (Cirrhinus
mrigala), Silver carp (Hypothalmichthys molitrix), Grass carp
(Ctenopharyngodon idella) and Common carp (Cyprinus carpio) are
considered to be the best culturable species.
Flood plain wetland in West Bengal covers an area of about 42000
ha. Within the West Bengal, water bodies for fish culture are present in
North and South 24 Praganas and Nadia Districts. Waste water fed fish
ponds in East Kolkata Wetland, West Bengal, India (called Bheris) have
a distinctly different architecture resulting in extensive purification of
waste along with integrated resource recovery [3]. Bheri, the shallow
(50-150 cm), flat bottom, waste water fed fish pond. Kolkata, Nadia,
South 24 Parganas and Burdwan were selected for the present study
because; they are important districts of West Bengal with respect to
both aquaculture and agriculture. East Kolkata Wetland in Kolkata;
Kulia, Bhomra in Nadia; Haruabhanga, Lakshmipur in Burdwan and
Malancha, Jaynagar in South 24 parganas were identified and surveyed
for our study. But for this present study EKW was chosen as it is a waste
water fishery. Preliminary physico-chemical and biological survey
indicates the presence of nitrifying bacteria in EKW as compared
to other sites of West Bengal. Among which research work was
initiated under East Kolkata Wetland at Nalban Fisheries (owned by
Government of West Bengal and runed by State Fisheries Development
Co-operation) and Jhagrasisa bhery (runed by private organization).
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Collection of samples and process
Water samples (25 ml) were collected from Nalban Fisheries and
Jhagrasisa bhery. A total 6 fish ponds or tanks were selected for the
study and marked as N1, N2, N3 for Nalban Fishery and J1, J2, J3
for Jhagrasisa bhery in East Kolkata Wetland. Samples (two aliquots
from each place) were also collected from Topsia (Sewage Pumping
Station) and from different channels like Ambedkar Bridge, Bantala,
Bamunghata, Ghosher Khal which is connected to the fish pond and
Ghusighata (outlet). Samples from each site was collected at around
6.30-7.00 in the morning in sterile plastic bottle (Tarson) from a depth
of 1-2.0cm below the surface of the water, kept in ice box (Milton, India)
and then taken in laboratory for immediate culture. Water samples
were collected in different seasons as winter, summer, post monsoon
and monsoon. In each seasons two aliquots were collected from each
tanks and a total of 96 aliquots were collected and studied.
Samples were collected and processed on Winogradskyi medium
for the isolation of nitrifying bacteria. Nitrosomonas sp. was isolated
using Winogradsky phase 1 medium, which contains (NH4)2SO4, 2.0
g; K2HPO4, 1.0 g; MgSO4, 7H2O, O.5 g; NaCl, 2.0 g; FeSO4,7H2O, 0.4 g;
CaCO3, 0.01g, and miliQ water upto 1000 ml, pH 7.8). Nitrobacter sp.
was isolated using Winogradsky phase 2 medium (KNO2, 0.1 g; Na2CO3,
1.0 g; NaCl, 0.5 g; FeSO4, 7H2O, 0.4 g, and miliQ water upto 1000 ml,
pH 7.8) [21-23]. Tubes were incubated at 28°C for 90 hrs aerobically
[21,24,25]. Cultures were initially identified by gram bacteria [26].
Suspected cultures were evaluated for presence of ammonia and nitrite
and vice versa for production of nitrite and nitrate. These tests were
performed and confirmed by a series of reaction. Selected cultures were
preserved for further studies at -80°C with 30-40% glycerol stock.
Isolation of genomic DNA
Bacterial biomass from 10 ml of culture was collected by
centrifugation for 5 min at 10000×g and resuspended in a 2 ml
polypropylene tube (Eppendrof) containing glass beads with 500 ml
buffer containing 20 mM sodium acetate, 1 mM EDTA (pH 5.5), 50 ml
25% SDS and 600 ml phenol: chloroform: isoamyl alcohol (25: 24: 1).
Cells were lysed by the addition of 200 μl of 10 mg ml-1 lysozyme and
incubation at 37°C for 60 min. 20% Sodium dodecyl sulfate was added to
a final concentration of 1% and incubated at 37°C for 60 min. Proteinase
K solution was added (10 mg/ml) to a final concentration of 2 mg/ml
and incubated at 50°C for 35 min and the mixture was then centrifuged
at 10,000 g×10 min at 4°C. The aqueous phase was transferred carefully
to a fresh tube, mixed with 600 ml chloroform: isoamyl alcohol (24: 1)
and centrifuged 10000 g×10 min. The aqueous phase was transferred to
a fresh tube and, after the addition of 0.1 vol. 3 M sodium acetate. DNA
was precipitated by incubation with 0.6 volume of isopropanol and 5 ml
glycogen (5 mg/ml) for 1 h and subsequently pelleted by centrifugation
at 10 000 g×20 min at 4°C. Pellets were washed with 1 ml ice-cold 70%
alcohol and dried it by Maxi Vacuum Dryer and resuspended in 500μl
TE buffer (pH 8.0). The DNA concentration was estimated by visual
comparison with the standard DNA size markers after electrophoresis
through 0.8% agarose gel, stained with 0.5 mg ml-l ethidium bromide
(Sigma Chemicals Co.).
16S rDNA-PCR amplification
The 16S rRNA genes were amplified by PCR using the universal
primers: forward primer 27F 5’-AGA GTT TGA TCC TGG CTC AG3’ and reverse primer 1492R 5’- GGT TAC CTT GTT ACG ACT T-3’
(Chromous Biotech Ltd., Bengaluru, India) [27]. PCR reactions has
been carried out by following the series of standardizing experimental
Volume 5 • Issue 1 • 1000211
Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of Molecular Identification of Nitrifying Bacteria in Water Bodies of East Kolkata
Wetland, West Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/2155-6199.1000211
Page 3 of 5
protocol as annealing temperature, concentration of MgCl2, template
DNA, Taq DNA polymerase, dNTP’s and primers. The PCR reaction
components consists of 200 mm dNTP, 20 pico moles of primer, 2
units of Taq DNA polymerase enzyme, assay buffer with working
concentration of 1.5 mM MgCl2, 20-30 ng template DNA in an assay
volume of 25 µl. These concentrations were determined by a series of
preliminary standardizing experiments.
A cycling program was performed using a Thermal Cycler (Thermo
Scientific) with an initial denaturation step at 95°C×3 min, 35 cycles of
denaturation at 94°C×1 min, annealing at 55°C×1 min, and extension
at 72°C×2 min, and final extension at 72°C×3 min. The presence of the
PCR products were examined and visualized by electrophoresis in 1.5%
agarose gel in TBE buffer. The gel was stained with EtBr (Sigma) and
viewed in Gel Doc System (Biorad Gel Doc. 2000 system).
Sequencing of 16S rDNA amplicons
The PCR products were purified by the Exosap treatment in the
Departmental Molecular Biology Laboratory. For the validation and
identification, chosen strains were identified by 16S rDNA PCR pattern
analysis on 1.5% agarose gel electrophoresis and those were selected
for sequencing. In total, the PCR amplified products were sequenced
by Ion torrent Sequencer from the Xcelris Lab Ltd, Ahmedabad, India.
Computer assisted pattern analysis
Chromatogram of sequence data confirmed the peak, reproducibility,
quality and gene sequences of 16S rRNA amplicons. Raw sequence data
are arranged as complementary and consensus through online ReverseComplement tools (www.bioinformatics.org/sms/rev_comp.html) and
Genefisher 2, the BiBiServ (Bielefeld BioInformatics Service) is a unit
of the Institute for Bioinformatics (bibiserv.techfak.uni-bielefeld.de/
genefisher2/submission.html). Identification of species and similarity
calculations were performed, comparing sequences of approximately
1500 bases with sequences available in GenBank using BLAST network
services (blast.ncbi.nlm.nih.gov/). Multiple Sequence Alignments
(MSA) are carried out by Clustal W following all the sequence pairs
were aligned separately for the calculation of distance matrix and
constructed a guide tree and sequences are aligned according to the
hiearchy.
Phylogenetic analysis between Ammonia Oxidizing and
Nitrite Oxidizing Bacteria
Phylogenetic relationship, similarity index, distance between
all isolates and maximum likelihood pattern analysis outlined and
phylogenetic tree generated by Mega v5.05. The 16S rDNA sequences
in this study were submitted to the GenBank database by using Sequin
software (www.ncbi.nlm.nih.gov/projects/Sequin/) under nucleotide
accession number. Analysis of the 16S rRNA gene proved as useful
taxonomic tools with discriminatory properties for identification up to
the species level.
another two Nitrosomonas includes eutrtopha and euroraea species; on
the other hand, two strains were found as Nitrobacter sp. and another
single strain was identified as Nitrobacter vulgaris (Figures 1 and 2).
Phylogenetic analysis
Grimont and Grimont (1986) described the importance of the 16S
rRNA-based probes as taxonomic tools [29]. In the present study, we
approached the use of the properties of the 16S rDNA for phylogenetic
identification from a different perspective and results apparently covered
the majority of NOBs and AOBs can be discriminated by 16s rDNA
targeted primers therefore need to take into account the intragenomic
diversity of the 16S rRNA gene. Nitrosomonas europaea cell are capable
of doing nitrification in wastewater with high efficiency. A recent study
Strains
Collection Site
code
Name of isolates
NCBI-GenBank
Acc. No.
NSW1
Bantala
Nitrosomonas eutropha
KF618623
NSW2
Nalban fishery tank 2
Nitrosomonas europaea
KF618624
NSW3
Jhagrasisa bhery tank 1 Nitrosomonas sp.
KF618625
NSW4
Ambedkar bridge
KF618626
Nitrosomonas eutropha
NBW1
Bantala
Nitrobacter sp.
KF618620
NBW2
Nalban fishery tank 2
Nitrobacter sp.
KF618621
NBW3
Jhagrasisa bhery tank 1 Nitrobacter vulgaris
KF618622
Table 1: Details of samples and their source(s).
Figure 1: 1.5% Agarose gels showing atypical 16S rDNA-PCR amplicons of
about 1.5 kb in size.
Lane 1, Nitrosomonas eutropha (KF618623); 2, Nitrosomonas europaea
(KF618624); 3, Nitrosomonas sp. (KF618625); and 4, Nitrosomonas eutropha
(KF618626). Samples isolated from Wastewater of East Kolkata Wetland; Lane
M, 100bp DNA size marker (Promega).
Results and Discussion
Identification of strains
AOB and NOBs were initially identified by gram staining and
produced gram negative characteristics. The nitrite accumulation test
was performed to confirm AOBs and the nitrite consumption test was
performed to confirm NOBs by following the standard protocol of
APHA, (1998) [28]. A comparative 16S rRNA sequence analysis finally
confirmed the genus and species (Table 1). Out of total seven strains
were sequenced and tested, two were found as Nitrosomonas sp. and
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Figure 2: 1.5% Agarose gels showing atypical 16S rDNA-PCR amplicons of
about 1.5 kb in size.
Lane 1, Nitrobacter sp. (KF618620); 2, Nitrobacter sp. (KF618621) and 3,
Nitrobacter vulgaris (KF618622). Samples isolated from Wastewater of East
Kolkata Wetland; Lane M, 100bp DNA size marker (Promega).
Volume 5 • Issue 1 • 1000211
Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of Molecular Identification of Nitrifying Bacteria in Water Bodies of East Kolkata
Wetland, West Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/2155-6199.1000211
Page 4 of 5
was developed by Niranjan Uemoto and Saiki and Kumar Shrestha et
al. [12,30], where nitrogen removed using Nitrosomonas europaea and
Paracoccus denitrificans from waste water. Nitrobacter population can
be detected by using PCR- analysis [13].
As waste water is introduced into the bheries or the fish pond
with high load of organics or heavy metals, the bacterial populations
neutralizes or break down the organics or heavy metals to achieve
maximum benefit and also enhance fish production. Thus, the
application of probiotics in sewage fed bheries along with the details
molecular characterization of microbes present in bheries can enhance
the fish production and can reduce the level of ammonia, H2S stress.
Analysis of different samples of Nitrosomonas spp. found in freshwater
was done using 16S ribosomal DNA and PCR technique [18].
Classification of any bacterial group, taxonomically as well as molecular
characterization is required, thus for studying ammonia- oxidizing
bacteria polygenic and molecular analysis was done [19]. Along with
this molecular characterization of the bacteria is also important for
further investigation and to analyze these bacterial communities.
The isolation of samples collected from the distribution network in
this study could be attributed to the effective strategy employed in the
system, which may increase the survival of the organism as suspended
or as part of the biofilm community. The similarity of isolated 16S
ribosomal RNA gene, partial sequences were identical and ranging
between 94 to 97% (Figures 3 and 4). To draw a proper biosecurity
protocol, molecular diagnosis and phylogenetic relationships between
the species in EKW, the present technique used in this study can be able
to overcome threats and control the disease outbreaks. These data and
methodology has been proved in this study as advance molecular tool.
Our results emphasize the need to take into account the intragenomic
diversity of the 16S rRNA gene. This method has shown discriminatory
properties in the identification up to the species level. The study
provided a quick identification tool.
Conclusion
Views were recognition of the heterogeneity detected in the
sequences is important to avoid strain misidentification and faithful to
the acknowledgement of diversity. Our study aimed towards prompt
and accurate identification of members of the genus AOBs and NOBs as
Figure 4: Phylogenetic distance tree displaying the evolutionary origin among
different species of Nitrosomonas and Nitrobacter as reference, collected from
NCBI- GenBank with query sequences within a lineage shared by the different
species through Mega v5.05. Numbers indicate GenBank accession no. of
reference sequences query sequences shared existence with them is indicated.
indicators in environment and as the most prevalent bacterium in the
waterways of EKW in West Bengal, India. The method provided in this
work, apart from being a reliable identifier at reasonable cost.
Acknowledgements
The author acknowledges to Dr. Bidyut Bandhophadhyay for his intellectual
input. We also acknowledge Mr. S. D. Ghosh for his kind permission to work at
EKW.
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Volume 5 • Issue 1 • 1000211
Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of Molecular Identification of Nitrifying Bacteria in Water Bodies of East Kolkata
Wetland, West Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/2155-6199.1000211
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Citation: Saha M, Sarkar A, Bandhophadhyay B (2013) Development of
Molecular Identification of Nitrifying Bacteria in Water Bodies of East Kolkata
Wetland, West Bengal. J Bioremed Biodeg 5: 211. doi:10.4172/21556199.1000211
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