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Is nitrogen fixing heterotrophic bacteria balancing the nutrient ratio (N:P) in a tropical eutrophic estuary (Cochin, India)?
Jabir T1., Jesmi Y1,3., Sudheesh V1., Prabhakaran, M P2., Aravind Singh4 and Mohamed Hatha A. A.1
1Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Kochi 682016, Kerala, India
2Kerala University of Fisheries and Ocean Studies, Panangad, Kochi, Kerala, 682506
3School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala
4Physical Research Laboratory, Ahmedabad, India
*Corresponding Author:
Biological nitrogen fixation contributes new nitrogen to the majority of ecosystems, but despite its importance in ecosystem functioning, ecological controls of N fixation are poorly understood in Indian waters. Studies on quantification of nitrogen (N) fixation rates in a tropical estuarine system undergoing anthropogenic disturbance are scant. Here, we report the first investigation on potential contribution of heterotrophic bacteria in N-fixation in a tropical eutrophic ecosystem, Cochin estuary (CE), along the west coast of India. The role of heterotrophic N-fixing bacteria in balancing N:P ratio in estuarine waters is also studied. Nitrogen fixation rate (NFR) was anti-correlated with N:P ratio (pre-monsoon; R2=0.62, p -=0.05;  monsoon=0.83, p=0.05; post-monsoon R2=0.57, p=0.05). It suggests that nitrogen fixation may be controlled by N:P ratio of CE. NFR ranged from 0.12-2.02 nmolN2L-1h-1. Abundance of nitrogen fixing heterotrophic bacteria (NFHB) in the estuary ranged from 5.01x102 to 7.1×104cfu/ml. NFR is significantly correlated with NFHB (Pre-monsoon - R2=0.62,p=0.05, Monsoon - R2=0.97, p=0.05, Post-monsoon, R2= 0.47, p=0.05) suggesting that heterotrophic bacteria have pivotal role in nitrogen fixation in CE. Metagenomic study of nitrogenase gene nifH revealed that major nitrogen fixing heterotrophic bacteria are under the class proteobacteria such as alpha-, beta-, gamma-, proteobacteria and firmicutes.
Key words
Nitrogen fixing heterotrophic bacteria, Nutrient stoichiometry, Eutrophic estuary, nifH gene
1.      Introduction
The microbial conversion of molecular nitrogen (N2) to ammonium, known as nitrogen fixation, is fundamental for biological productivity in many aquatic environments (Bentzon-Tilia et al., 2015). Nitrogen fixation appears important in making up deficits in nitrogen (N) availability relative to phosphorus in aquatic ecosystems, contributing to the phosphorus (P) limited status of these systems (Cerna et al., 2009; Voss et al., 2011). N limits biological production in aquatic environments, so it is essential to understand processes that control N availability. The ability of N2 fixation is a widely distributed trait among prokaryotes such as autotrophic and heterotrophic bacteria that accounts for an essential input of new N to aquatic ecosystems (Bentzon-Tilia et al., 2015).
            The biogeochemistry and microbial ecology of estuaries depends on nutrient discharges from various sources such as rivers, industrial, agricultural and sewage, etc. Excess nutrients enter the system as organic and inorganic N and P compounds and threaten the ecological stability of coastal ecosystems (Galloway et al., 2004). Human activity has accelerated the flow of nutrients into the estuaries (Nixon et al., 1996) over the past few decades and caused many estuarine systems to shift drastically from nutrient limitation to nutrient surplus leading to eutrophication (Bricker et al., 1999). Moreover, the heavy nutrient load of any one of the nutrients (N or P) can display either P limitation or N limitation, resulting from changes in nutrient ratio or co-limitation (Conley et al., 2009). In estuaries, the region of transition between fresh and saline water, P can often be the limiting nutrient (Conley, 1999; Blomqvist, 2004). The inventory of bioavailable (fixed) N is largely regulated by N2 fixation (Hamersley et al., 2009). From this perspective, P availability is a crucial factor in controlling the process of N2 fixation. The availability of dissolved nutrients in the estuaries are the major selective force for bacterial communities to do physiological activity (Langenheder et al., 2005). High P content in estuary induces the nitrogen fixation (Howarth and Marino, 2006) for maintaining the nutrient stoichiometry and heterotrophic bacteria play a major role in this process.
Present study was carried out in Cochin estuary (CE), which is the largest tropical eutrophic salt wedge estuary (Qasim, 2003) along south west coast of India. CE is facing serious pollution problems following the release of untreated effluents from industrial (0.104 × 106 m3d-1) and domestic sewage (0.26 ×103 m3d-1) leading to deleterious changes in the estuarine ecosystems substantially (Menon et al., 2000). These inputs through anthropogenic activities can alter the nutrient stoichiometry of the estuary leading to change in microbial activity and biogeochemistry (Martin et al., 2008). CE was highly autotrophic five decades ago (Sankaranarayanan and Qasim, 1969) but has evolved into a heterotrophic system due to anthropogenic activities (Shoji et al., 2008; Gupta et al., 2009).
There are several studies depicting the environmental changes of the CE caused by anthropogenic activities (Menon et al., 2000; Martin et al., 2008, Bhavya et al., 2016). Neverthless, there have been no published report on microbial nitrogen fixation in the Cochin estuary. A recent study by Bhavya et al. (2016) on nitrogen uptake and nitrogen fixation in the CE suggests that the nitrogen fixation occurs even in high DIN concentration. Due to relative abundance of P compared to N in the estuary cause imbalance in nutrient stoichiometry (N/P). In addition, hydrographic and environmental parameters also influence DIN uptake and nitrogen fixation under the nutrient replete condition of the estuary. The present study focused to find out (i) the role of diazotrophic heterotrophic bacteria in nitrogen fixation (ii) the importance of N:P stoichiometry in the microbial N-fixing process in the study area.
2. Materials and methods
2.1. Study Area
            Cochin estuary (between 9° 40' - 10° 12' N and 76° 10' - 76° 30' E) forms a complex micro tidal tropical estuary situated along the west coast of India having an area of about 256 Km2 (Martin et al., 2008). The depth of the estuary varies from 2 to 3 m, but the ship channel at the Cochin harbor region is continuously dredged and maintained at 10 to 13 m (Qasim, 2003). The estuary stretches parallel to the coastal line and connected to the Arabian Sea by two permanent opening viz. Cochin and Azheekode. Six major rivers with their tributaries and several canals discharge 20000×109 m3/year of fresh water in to backwaters annually (Sreenivas et al., 2003). The high fresh water flow into the estuary during summer monsoon reduces the tidal characteristics and increases the stratification (Quasim and Gopinathan, 1969).
2.2 Sampling and Analysis
            The samples were collected at seven different locations within the estuary based on the salinity gradient during summer monsoon (August) and post monsoon (December) period of 2014 and pre-monsoon (March) of the year 2015. Water samples were collected using 2L niskin sampler (General Oceanic, Miami, USA). Salinity was measured using Systronics water quality analyzer (Model No. 317; Accuracy ± 0.01) calibrated with standard sea water (APHA, 2005). Samples for dissolved oxygen (DO) were analyzed by Winkler method (Grasshoff et al., 1999). The detection limit is ~0.05 ml L-1(~2µM). Samples for nutrients (phosphate, nitrate, nitrite and ammonium) were analyzed spectrophotometrically (Hitachi - 2J1-0004, Tokyo, Japan) within few hours of collection following standard procedures (Grasshoff et al., 1999). The analytical precision, expressed as standard deviation, was ± 0.01, 0.01, 0.07 and 0.05 for phosphate, nitrite, ammonium and nitrate, respectively. The samples for microbiological studies were collected in sterile bottles. The samples were analyzed within 4 hours in the laboratory.
2.3 DNA Extraction
Genomic DNA from water samples was extracted following Boström et al. (2004) with some modification. Briefly, 500 ml of water sample was filtered through 0.2 μm pore size polycarbonate membrane filter (Millipore; GTTP2500). Filtered samples were incubated at 37 °C for 1 h in a lysis buffer (NaCl 400 mM, sucrose 750 mM, EDTA 20 mM, and Tris-HCl 50 mM) containing 1 mg/ml lysozyme. Subsequently, SDS (1%) and 100 μgm/l proteinase K were added to the solution and continued incubation for overnight at 55 °C. Further, 60% volume of isopropanol was added and DNA was precipitated by keeping at −20 °C for 60 min. DNA pellet was washed copiously with 70 % ethanol, dissolved in TE buffer.
2.4 PCR amplification of the nifH gene fragment
The nifH sequences were amplified from the sample with high efficiency DNA polymerase (Takara) on a Thermal Cycler (Applied Biosystem Inc.). A nested reaction was used, as previously described (Zehr et al., 1998), with  modification: 50 µl PCRs were amplified for 30 cycles (1 min at 98 °C, 45 seconds at 57 °C, 1 min at 72 °C), first with the outer PCR primers (Zani et al., 2000), followed by amplification with the inner PCR primers (Zehr and McReynolds, 1989). To minimize the possibility of amplifying contaminants (Zehr et al., 2003), negative controls were run with every PCR reaction. To avoid potential sample biases and to obtain enough PCR products for cloning, three replicate amplifications were conducted for each sample.
PCR conditions consisted of 30 cycles at 94 °C, 55 °C (1 min) for the annealing step and 72 °C (2 min), with a 5 min extension at 72 °C for the last cycle. PCR products were analyzed by electrophoresis on 1.4% agarose gel to confirm the size of the bands. The amplification products were purified using the PCR clean-up system (Nucleospin PCR clean up kit, MN, Duren, Germany) and inserted into pGEM-T using the pGEM-T Easy Vector system kit (Promega, Madison, WI) according to the manufacturer's instructions. The colonies that exhibited vector inserts (white colour) were selected for insert detection by amplification with the M13F and M13R primers. The resulting PCR products with the inserts were isolated and plasmid isolated and sent for sequencing at Scigenome (Kochi, India). Using this methodology, 350 clones containing nifH gene inserts were obtained.
The partial nifH sequences of selected representative clones (Using Vec Screen tool from NCBI) obtained in this study were compared with the GenBank database using the algorithm BLAST-N to identify the most similar nifH sequences and then aligned with representative nitrogenase sequences obtained from the same database using the software package CLUSTAL X (Thompson et al., 1997). BIOEDIT v. 7.0.0 (http:// was used for manual editing of the sequences. Nucleotide sequences were also checked for chimeric sequences by performing BLAST searches with partial sequences. All clones that were affiliated with nifH sequences had their identity checked by phylogenetic analyses. The phylogeny-based analysis was made by comparing the obtained sequences to those from public databases (GenBank) in neighbor-joining trees constructed using MEGA 4.0 (Tamura et al., 2013).
2.5 Nucleotide Sequence and Accession Numbers: - The sequences reported in this study were deposited in GenBank under the accession numbers.
2.6 Nitrogen fixation rates
            Nitrogen fixation rates were estimated using acetylene reduction assay method (Stewart et al., 1967, Wilson et al., 2012., Fulweiler, et al., 2015). The water samples were transferred to acid washed 300 ml crimp sealed vials after rinsing with the sample with triplicates. Bottles were carefully closed using septum closure caps (Sigma-Aldrich) with no headspace and inverted 10-15 times. Subsequently, 30 ml C2H2 was injected into serum bottles using a gas-tight syringe (Hamilton) through the septum cap. Control samples were filtered through 0.2 µm filters and processed the same way as samples have done. Immediately after the C2H2 addition, the samples were incubated in situ for 4 hours, approximately symmetrical to the local noon. Post incubation samples were fixed using saturated HgCl2 and ethylene (C2