Final Report Summary - AQUATIC N IN INDIA (Fate of anthropogenic nitrogen in aquatic systems of India)
The Indian Subcontinent occupies <3% of the total land area of the world; however, as much as 22% of the world’s human population lives here, in rough proportion to which the region accounts for ~19% (17 million tonnes of nitrogen per year) of the global synthetic nitrogen fertilizer consumption. Fossil fuel combustion is the other major source of new nitrogen introduced to the environment. The fate of the enormous nitrogen loading, which has increased by a factor of 50 over the past 4 decades, is largely unknown. Less than 5% of the anthropogenic nitrogen appears to reach the sea by river runoff; the rest presumably accumulates in the terrestrial aquatic systems where an unknown fraction may be removed as N2 or N2O through redox transformations, especially in anaerobic environments of the subsurface aquifers and hypolimnia of stratified reservoirs and lakes. Given the serious health hazards of high nitrate levels in drinking water and the high greenhouse potential of N2O, both nitrate accumulation in natural waters and conversion of fixed nitrogen to N2O are of immense socio-economic significance. The award of a Marie Curie International Incoming Fellowship to Dr. S.W.A. Naqvi to Max Planck Institute of Marine Microbiology, Bremen, Germany and its Return Phase at the National Institute of Oceanography (NIO), Goa, India, facilitated the first systematic study of nitrogen cycle processes in groundwaters and lakes/reservoirs in India, providing new insights into fates of anthropogenic reactive nitrogen.
Three man-made reservoirs and one natural lake, and groundwater aquifers of three different types were originally proposed to be studied during the Return Phase of the fellowship. The four major components of the proposed work were: (a) to study the spatial and temporal variations of various dissolved nitrogen species (organic nitrogen, nitrate, nitrite, ammonium, N2O and N2) in relation to organic matter loading and ambient dissolved oxygen levels; (b) to determine rates of redox transformations [(denitrification and anaerobic ammonium oxidation (anammox)] and assess their relative importance in N2 production; (c) to identify sources of nitrogen and understand mechanisms of its transformations through natural isotope abundance measurements; and (d) to characterize through molecular analyses the microbial community involved in redox transformations. However, based on the results of the Incoming Phase, the work plan was slightly modified. Since it had already been established during the Incoming Phase that anammox was not a major pathway of nitrogen loss in any system examined, objective (b) was not pursued in the Return Phase. Also, denitrification was convincingly established to be the predominant pathway of nitrogen loss in groundwaters; so, the groundwater work was excluded. Instead, the main focus during the Return Phase was on dam-reservoirs. A total of 9 such reservoirs were sampled spread over a large geographical area, 5 of them being studied for the first time. Repeat sampling was carried out in Selaulim (4 times), and in Tillari (9 times), the latter enabling construction of a year-long time series. The Lonar Lake, sampled for the Incoming Phase was sampled during the Reverse Phase as well.
All reservoirs examined experienced strong stratification during summer. This led to severe depletion of dissolved oxygen (DO) in the bottom layer (hypolimnion) in all cases with the exception of the Tungabhadra Reservoir, which was too shallow. The Markandeya, Koyna and Sardar Sarovar reservoirs were found to be suboxic but not sulfidic (i.e. these systems did not experience complete loss of nitrate). The other reservoirs (Tillari, Selaulim, Nagarjuna Sagar, Srisailam and Ukai) turned completely anoxic, with the highest H2S concentration of 80 µM occurring in the Srisailam Reservoir. The surface layer (epilimnion) had generally low concentration of nitrate (<2 µM), except in the case of Markandeya where it was ~10 µM. Accordingly, in general the chlorophyll a concentration was not very high (< 6 mg m-3), although in the case of Nagarjuna Sagar and Srisailam reservoirs it exceeded 20 mg m-3. Nitrate build-up within the thermocline (to a maximum of 46 µM) occurred in some but not all cases, and a decrease (even complete removal) was conspicuous in the hypolimnia of all systems barring the shallow Tungabhadra Reservoir. Ammonium (NH4+) accumulated in many reservoirs, especially those experiencing sulfate reduction, to a maximum concentration of 70 µM in Srisailam Reservoir. Two noteworthy features, also observed previously, are: (a) nitrite concentration was <1 µM, often close to the detection limit, except in rare cases (e.g. close to the bottom on 9 July, 2013 in the Tillari Reservoir), (b) large N2O buildup (>40 nM) rarely occurred except in Markandeya and Koyna Reservoirs. The results from the Return Phase of the Marie-Curie fellowship are thus consistent with those obtained during the Incoming Fellowship, where the presence of methane was found to greatly stimulate production of N2 from NO2- through a process that seems to bypass N2O.
Time series observations in Tillari Reservoir reveal that the water column is highly stratified during summer with H2S accumulation in the hypolimnion. Vertical mixing occurs during the southwest monsoon season when the region is subjected to strong winds and intense rainfall. Reoxygenation of hypolimetic waters also occurs through lateral advection of oxygen rich waters. At the end of the southwest monsoon, stratification is re-established and the hypolimnion again loses oxygen, but it does not become anoxic. Convective mixing finally causes the development of well-oxygenated conditions during winter. Stable isotopic compositions (δ13C and δ15N) of suspended particulate organic matter (SPOM) and δ15N of NH4+ and nitrate have been measured in the Tillari Reservoir. The δ13C of SPOM showed higher values ranging between -23.3 and -27‰ when the water column was well mixed in September, 2013. Much lower values (-26 to -34‰) were recorded during period of stratification and anoxia in May, 2013. This could be due to the contribution of chemoautotrophic and/or methanotrophic biomass in the anoxic hypolimnion. The δ15N of SPOM ranged between 2 and 4.3‰ in September, but much lower values (-4.3 to 2.3‰) were observed during the anoxic period. Highly depleted values (~-4‰) close to the bottom during May, 2013 coincide with high [NH4+] ( ~18 μM) and [H2S] ( ~5 μM), thus confirming degradation of organic matter under anoxic conditions and subsequent microbial assimilation of NH4+ , which is depleted in 15N (1.25-2.66 ‰), by chemoautotrophic bacteria present in the water column. The δ15N of nitrate varied greatly in this reservoir (from -0.01 to 23.99‰), reflecting diverse sources and cycling in the reservoir.
Observations in Lonar Lake, a highly alkaline (pH ~10) and shallow (depth 4 m) brackish water (salinity ~10) system, carried out in March 2013 were consistent with previously collected data. The water column was vertically stratified below 1 m. Sharp decreases in temperature and DO occurred below the epilimnion. The water column was strongly sulfidic below 1m and H2S concentration reached up to 316 µM close to the bottom. Both NO3- (< 3 µM) and NO2- (<0.4 µM) were low as was N2O (<8 nM) throughout the water column.
Water and sediment samples were collected from Lonar Lake and Koyna, Nagarjuna Sagar and Srisailam reservoirs. DNA was isolated from water and sediment samples of Lonar Lake. Also 16S rDNA was amplified and subjected to amplicon sequencing. In addition, amplicon sequencing of metagenomes obtained from Nagarjuna Sagar, Srisailam and Koyna dams is being carried out.
Three man-made reservoirs and one natural lake, and groundwater aquifers of three different types were originally proposed to be studied during the Return Phase of the fellowship. The four major components of the proposed work were: (a) to study the spatial and temporal variations of various dissolved nitrogen species (organic nitrogen, nitrate, nitrite, ammonium, N2O and N2) in relation to organic matter loading and ambient dissolved oxygen levels; (b) to determine rates of redox transformations [(denitrification and anaerobic ammonium oxidation (anammox)] and assess their relative importance in N2 production; (c) to identify sources of nitrogen and understand mechanisms of its transformations through natural isotope abundance measurements; and (d) to characterize through molecular analyses the microbial community involved in redox transformations. However, based on the results of the Incoming Phase, the work plan was slightly modified. Since it had already been established during the Incoming Phase that anammox was not a major pathway of nitrogen loss in any system examined, objective (b) was not pursued in the Return Phase. Also, denitrification was convincingly established to be the predominant pathway of nitrogen loss in groundwaters; so, the groundwater work was excluded. Instead, the main focus during the Return Phase was on dam-reservoirs. A total of 9 such reservoirs were sampled spread over a large geographical area, 5 of them being studied for the first time. Repeat sampling was carried out in Selaulim (4 times), and in Tillari (9 times), the latter enabling construction of a year-long time series. The Lonar Lake, sampled for the Incoming Phase was sampled during the Reverse Phase as well.
All reservoirs examined experienced strong stratification during summer. This led to severe depletion of dissolved oxygen (DO) in the bottom layer (hypolimnion) in all cases with the exception of the Tungabhadra Reservoir, which was too shallow. The Markandeya, Koyna and Sardar Sarovar reservoirs were found to be suboxic but not sulfidic (i.e. these systems did not experience complete loss of nitrate). The other reservoirs (Tillari, Selaulim, Nagarjuna Sagar, Srisailam and Ukai) turned completely anoxic, with the highest H2S concentration of 80 µM occurring in the Srisailam Reservoir. The surface layer (epilimnion) had generally low concentration of nitrate (<2 µM), except in the case of Markandeya where it was ~10 µM. Accordingly, in general the chlorophyll a concentration was not very high (< 6 mg m-3), although in the case of Nagarjuna Sagar and Srisailam reservoirs it exceeded 20 mg m-3. Nitrate build-up within the thermocline (to a maximum of 46 µM) occurred in some but not all cases, and a decrease (even complete removal) was conspicuous in the hypolimnia of all systems barring the shallow Tungabhadra Reservoir. Ammonium (NH4+) accumulated in many reservoirs, especially those experiencing sulfate reduction, to a maximum concentration of 70 µM in Srisailam Reservoir. Two noteworthy features, also observed previously, are: (a) nitrite concentration was <1 µM, often close to the detection limit, except in rare cases (e.g. close to the bottom on 9 July, 2013 in the Tillari Reservoir), (b) large N2O buildup (>40 nM) rarely occurred except in Markandeya and Koyna Reservoirs. The results from the Return Phase of the Marie-Curie fellowship are thus consistent with those obtained during the Incoming Fellowship, where the presence of methane was found to greatly stimulate production of N2 from NO2- through a process that seems to bypass N2O.
Time series observations in Tillari Reservoir reveal that the water column is highly stratified during summer with H2S accumulation in the hypolimnion. Vertical mixing occurs during the southwest monsoon season when the region is subjected to strong winds and intense rainfall. Reoxygenation of hypolimetic waters also occurs through lateral advection of oxygen rich waters. At the end of the southwest monsoon, stratification is re-established and the hypolimnion again loses oxygen, but it does not become anoxic. Convective mixing finally causes the development of well-oxygenated conditions during winter. Stable isotopic compositions (δ13C and δ15N) of suspended particulate organic matter (SPOM) and δ15N of NH4+ and nitrate have been measured in the Tillari Reservoir. The δ13C of SPOM showed higher values ranging between -23.3 and -27‰ when the water column was well mixed in September, 2013. Much lower values (-26 to -34‰) were recorded during period of stratification and anoxia in May, 2013. This could be due to the contribution of chemoautotrophic and/or methanotrophic biomass in the anoxic hypolimnion. The δ15N of SPOM ranged between 2 and 4.3‰ in September, but much lower values (-4.3 to 2.3‰) were observed during the anoxic period. Highly depleted values (~-4‰) close to the bottom during May, 2013 coincide with high [NH4+] ( ~18 μM) and [H2S] ( ~5 μM), thus confirming degradation of organic matter under anoxic conditions and subsequent microbial assimilation of NH4+ , which is depleted in 15N (1.25-2.66 ‰), by chemoautotrophic bacteria present in the water column. The δ15N of nitrate varied greatly in this reservoir (from -0.01 to 23.99‰), reflecting diverse sources and cycling in the reservoir.
Observations in Lonar Lake, a highly alkaline (pH ~10) and shallow (depth 4 m) brackish water (salinity ~10) system, carried out in March 2013 were consistent with previously collected data. The water column was vertically stratified below 1 m. Sharp decreases in temperature and DO occurred below the epilimnion. The water column was strongly sulfidic below 1m and H2S concentration reached up to 316 µM close to the bottom. Both NO3- (< 3 µM) and NO2- (<0.4 µM) were low as was N2O (<8 nM) throughout the water column.
Water and sediment samples were collected from Lonar Lake and Koyna, Nagarjuna Sagar and Srisailam reservoirs. DNA was isolated from water and sediment samples of Lonar Lake. Also 16S rDNA was amplified and subjected to amplicon sequencing. In addition, amplicon sequencing of metagenomes obtained from Nagarjuna Sagar, Srisailam and Koyna dams is being carried out.
