| As an essential nutrient element for all organisms,nitrogen is a limiting factor in controlling primary production of terrestrial and aquatic ecosystems.Human activities such as combustion of fossil fuel and application of artificial nitrogen fertilizers have greatly increased the level of reactive nitrogen in the Earth surface ecosystem over the past several decades.A large quantity of the reactive nitrogen has been transported into estuarine and coastal seas through river,groundwater,and atmospheric deposition,consequently causing serious ecological and environmental issues such as eutrophication,harmful algal blooms,oxygen depletion and acidification.It is thus important to understand the nitrogen loss processes and associated microbial mechanisms controlling the availability of reactive nitrogen in these aquatic ecosystems.Denitrification,converting nitrate(NO3-)to nitrite?NO?2-)),nitric oxide?NO?,nitrous oxide?N2O?and finally to nitrogen gas?N2?,is an important pathway for permanent removal of reactive nitrogen from estuarine and coastal ecosystems.This pathway is catalyzed by various enzymes,including nitrate reductase?Nar?,nitrite reductase?Nir?,nitric oxide reductase?Nor?and nitrous oxide reductase?Nos?.In addition to denitrification,the anaerobic ammonium oxidation?anammox?is an innovative discovery for biogeochemical cycling of nitrogen,which is also involved in the emission of nitrogen gas?N2?from estuarine and coastal environments through ammonium oxidation accompanied by nitrate/nitrite reduction.Estuarine tidal flat wetland is widely distributed in the world,and it is an important part of estuarine ecosystems.It can provide shelter for coastal cities to resist storm and provide habitat and reproduction for many fish,birds and invertebrates.At the same time,estuarine tidal flat wetland is also a natural filter of land-based pollutants,which has the functions of interception,storage and purification of pollutants,and plays an important role in reducing the nitrogen level of estuarine and offshore ecosystems,and can effectively alleviate water eutrophication.The estuarine tidal flats are subject to the interaction of tidal current and runoff,and the physical,chemical and biological factors in these aquatic systems are highly dynamic.Thus,the associations between the nitrogen transformation processes and associated microbial dynamics remain largely uncertain in these aquatic environments.The Indus River is an important river in South Asia.It originates near Lake Manasarovar in the Tibetan Plateau,with a total length of 3200 km.Among the world's rivers,the Indus River Delta area ranks seventh?3×104 km2?and the drainage area ranks twelfth?9.7×107 km2?.The annual rainfall is very low?about 180-220 mm?,resulting in dry environmental conditions.The tide in the Indus estuary is semidiurnal with an amplitude of about 2.7 m.The Indus River is considered the backbone and lifeline of Pakistan.Pakistan is a big agricultural country,and most people are closely related to agriculture.More than 70%of the population is rural.Nowdays,although the economy has been developed,more than 3×106 tons of nitrogen enter the Indus River Basin every year due to human activities such as excessive use of chemical fertilizers in agriculture,improper wastewater treatment,sewage discharge and fossil fuel combustion.The excessive emission of anthropogenic reactive nitrogen also led to the sharp decrease of biodiversity and serious environmental problems,such as eutrophication,hypoxia and harmful algal blooms.The ecosystem of the Indus River Estuary has been seriously damaged by the increasing artificial nitrogen input.However,little is known about the denitrification and anammox processes and associated microbial community dynamics in the Indus estuary.Therefore,it is of great ecological and environmental significance to study these nitrogen removal process and related microbial driving mechanism in the Indus River estuary ecosystem,which can deepen the understanding of the process and mechanism of nitrogen transformation in estuarine wetland,and provide scientific basis for the ecological and environmental protection of estuarine wetland.In this study,taking the Indus River Estuary as a typical study area,the denitrification rate,anammox rate and related functional microbial community dynamics were detected along the salinity gradient of the estuary by using 15N isotope tracer and molecular biology technology.The main objectives of this study are:?1?to determine the abundance,diversity,distribution,and activity of denitrifying bacteria and anammox bacteria in the sediments of the Indus River Estuary;?2?to analyze the dynamics of denitrification and anammox bacteria and their potential relationships with activities and environmental factors;?3?to evaluate potential contribution of denitrification and anammox to total nitrogen removal from the sediments of Indus River Estuary.The results of this study can enrich the study of nitrogen biogeochemical cycle in estuarine and coastal ecosystems,provide new insights into the relationship between nitrogen transformation and microbial dynamics in estuarine and coastal ecosystems,and can help to control the nitrogen pollution and to maintain the eco-environmental health in the estuarine and coastal ecosystems.The main findings are as follows:?1?The results of nir S gene labeled denitrifying bacteria showed that the abundance of denitrifying bacteria varied widely from 5.3 × 106 copies g-1 to 2.5 × 108 copies g-1.The abundance of denitrifying bacteria(average: 2.2 × 108 copies g-1)was higher than that of low salinity area(average: 9.3 × 107 copies g-1);the abundance of denitrifying bacteria in winter(average: 1.1 × 108 copies g-1)was slightly higher than that in summer(average: 9.4 × 107 copies g-1).Based on 97% similarity of nir S gene,it was divided into 1625 OTUs.The results of OTU diversity showed that there was a significant seasonal variation in denitrifying bacteria community diversity?ANOVA,P < 0.05?,but there was no significant difference between high and low salinity regions?ANOVA,P > 0.05?.The results of phylogenetic analysis showed that there was significant spatial difference in the structure of denitrifying bacteria along the salinity gradient.The gene sequences of nir S type denitrifying bacteria obtained in this study are similar to those reported in NCBI database,but most of them belong to unclassified denitrifying bacteria.Based on the results of phylogenetic analysis,the community structure was divided into 9 groups?cluster? cluster IX?,and cluster?and cluster? were widely distributed denitrification groups,in which cluster?was the dominant population in high salinity area,while cluster ? was dominant in fresh water area;the genetic relationship of cluster?X was similar to Pseudomonas xanthomarina,mainly in summer sediment samples.Based on the community structure,principal component analysis of nir S type denitrifying bacteria was conducted at all sampling sites.The results showed that there were obvious spatial differences in the structure of denitrifying bacteria,indicating that salinity may be the key environmental factor regulating the geographical distribution of denitrifying bacteria in the Indus River Estuary.At the same time,the community structure of denitrifying bacteria was quite different from that of sediments in the estuary of China,suggesting that there was a unique geographical distribution of denitrifying bacteria in the Indus River Estuary.The results showed that the diversity of denitrifying bacteria was significantly correlated with temperature?R =-0.532,P = 0.007?,while the abundance of denitrifying bacteria was significantly correlated with nitrate concentration?R = 0.421,P = 0.040?,suggesting that temperature and nitrate concentration might have significant effects on denitrifying bacteria.Canonical correspondence analysis showed that nitrate,water content,Fe???,total organic carbon?TOC?and salinity were the key environmental factors affecting denitrifying bacteria community structure.?2?Based on specific 16 S r RNA gene fragments,anammox bacteria in sediments of the Indus River Estuary were studied.The results showed that the abundance of anammox bacteria ranged from 1.64 × 106 copies g-1 to 8.21 × 108 copies g-1 along the salinity gradient.The abundance of anammox bacteria in high salinity area(1.99 × 108 copies g-1)was higher than that in low salinity area(8.07 × 107 copies g-1)and fresh water area(1.17 × 108 copies g-1);the abundance of anammox bacteria in summer was higher than that in winter(1.75 × 108 copies g-1 and 6.14 × 108 copies g-1,respectively).Based on 97% similarity,113 OTUs were obtained from high-throughput sequencing of anammox 16 S r RNA gene.Only 5 OTUs were shared by sediments in different salinity regions,indicating that anammox bacteria had large spatial variability in the Indus River Estuary.Phylogenetic analysis showed that the sequences of anammox bacteria were identified as Brocadia,Kuenenia,Jettenia and Scalindua.Among them,Kuenenia was the dominant genus in the Indus River Estuary accounting for 60.2% of all bacterial groups,Brocadia was the main anammox genus in fresh water sediment samples,and Scalindua was obviously enriched in the lower reaches of the Indus River Estuary.The diversity analysis of anammox bacteria showed that there was no significant spatial and temporal difference in anammox bacterial diversity in the Indus River Estuary.Correlation analysis showed that the diversity of anammox bacteria was significantly correlated with nitrate?R =-0.452,P = 0.027?,while the abundance of anammox bacteria was significantly correlated with Fe????R = 0.587,P = 0.003?.Canonical correspondence analysis showed that temperature and sulfide were the key environmental factors affecting the anammox bacterial community structure,indicating that the key environmental factors had significant effects on the abundance and diversity of anammox bacteria in the Indus River Estuary.?3?Potential rates of denitrification in the sediments of the Indus Estuary were determined with nitrogen isotope tracing technique.The measured rates were in the range of 0.01 ?mol N kg-1 h-1 to 6.27 ?mol N kg-1 h-1.The average denitrifying bacterial activity inclined to be higher in winter than in summer,with average values of 1.9 ?mol N kg-1 h-1and 1.2 ?mol N kg-1 h-1,respectively.However,the analysis of variance showed that the seasonal variation of denitrifying bacteria activity was not significant?P > 0.05?,which might be attributed to the relatively weak seasonal temperature difference.Along the Indus River Estuary,there was no significant spatial variation of denitrification rate in winter?ANOVA,P > 0.05?;however,in summer,the denitrification rate in sediments from high salinity habitat was significantly higher than that in freshwater habitat?ANOVA,P < 0.05?,with average values of 3.9 ?mol N kg-1 h-1and 0.1 ?mol N kg-1 h-1,respectively.Correlation analysis showed that there was a significant positive correlation between the potential denitrification rate and the gene abundance of denitrifying bacteria?P < 0.05?,reflecting that the abundance of denitrifying bacteria can indicate the denitrification potential of sediments.Moreover,the denitrification rate was significantly correlated with TOC?R = 0.438,P = 0.032?,which may depend on the fact that high organic matter content is conducive to promoting heterotrophic denitrification rate.The correlation between denitrification rate and Fe????R = 0.523,P = 0.009?might suggest that ferrous driven denitrification process may also exist in the sediments of the study area.It was estimated that approximately 7.2 × 105 ton nitrogen was annually removed from the Indus Estuary by the denitrifiers,which accounted for about 20% of the total amount of the inorganic nitrogen delivered to the Indus River Basin.?4?Nitrogen isotope tracing experiments showed that the measured anammox activity was in the range of 0.01 ?mol N kg-1 h-1 to 0.32 ?mol N kg-1 h-1.Anammox bacteria were more active in saline habitats than in freshwater habitats,with average rates of 0.14 ?mol N kg-1 h-1 and 0.06 ?mol N kg-1 h-1,respectively?ANOVA,P<0.05?.Althoug there was no significant seasonality of anammox rates?ANOVA,P>0.05?,anammox bacteria were more active in winter than in summer,with average rates of 0.11 ?mol N kg-1 h-1 and 0.06 ?mol N kg-1 h-1,respectively.Statistical analysis showed that there was a significant positive correlation between the activity of anammox bacteria and total organic carbon?R = 0.527,P = 0.008?,which indicated that organic matter could promote the activity of denitrifying bacteria and thus provide nitrite reaction substrate for anammox bacteria.This conclusion was also confirmed by the significant positive correlation between the activity of anammox bacteria and denitrifying bacteria?R = 0.127,P = 0.049?.The positive correlation between the activity of anammox bacteria and Fe????R = 0.422,P = 0.040?suggested the occurrence of ferrous driven ammonia oxidation in the study area.Compared with denitrification,anammox bacteria on average contributed 21.9% to the total loss of reactive nitrogen in the sediments of the Indus Estuary.If the estimated potential rates of anammox were extrapolated to the whole delta region,approximately 4.1 × 104 ton nitrogen was annually removed by anammox bacteria.Therefore,the anammox process played an important role in relieving the environmental stress of nitrogen overload in the estuarine and coastal environments. |
PDF Full Text Request