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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: mohamedhatha@gmail.com
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