Microbial Community Structure,Carbon And Nitrogen Mineralization And Nitrification In The Drawdown Zone Of The Three Gorges Reservoir Region

The challenge of linking pedology and hydrology has been identified in the recent past, semi-terrestrial ecosystems, particularly drawdown zone soils, their properties and ecological processes have been little studied compared to terrestrial soils. Drawdown zone soils are subject to large fluctuations in the water table and therefore to diverse soil moisture conditions. Often they have an oxidized zone that allows aerobic processes to occur. However, they can become reduced when submerged, which has a devastating effects on soil microbial community. Drawdown zone soils are frequently characterized by high carbon and nitrogen contents due to high rate of organic matter accumulation. Notwithstanding, the composition and diversity of soil microbial community largely determine biogeochemical cycles, the turnover processes of organic matter, and the fertility and quality of soils. Therefore, the quantitative description of microbial diversity in soils has become a topic of profound interest but remains one of the most difficult tasks facing microbial researchers.Many studies have focused on the factors influencing plant diversity in the riparian zone, like the drawdown zone, and found that the presence and abundance of species are, to a great extent, determined by a wide variety of environmental factors(i.e., flooding, soil contamination, and nutrient supply). Therefore, it is critical to understand the relation between the environmental factors and plant diversity for reconstructing floristically diverse riparian zone. It is now clear that soil communities are amongst the most species-rich components of terrestrial ecosystems and have been described as the ‘poor man’s rain forest. Soils have a key role in the biosphere with most of the annual carbon and nutrient fluxes mediated by soil organisms and occurring in the top 5–10 cm of the soil profile.Additionally, microbial properties respond rapidly to soil changes caused by both natural and anthropogenic factors. Soil microbial community structure is an important and sensitive indicator of soil quality. Soil microbial biomass is considered to be a source or sink of available nutrients and plays a critical role in nutrient transformation. Additionally, microbial biomass can be used for evaluating soil quality as well as for assessing soil disturbance and restoration.All wetlands, like water level drawdown zones, have in common that they are periodically or permanently water-saturated soil environments with characteristic vegetation and a water table at or close to the soil surface. As a consequence, they are characterized by steep gradients in soil redox conditions that sustain a complex pattern of biogeochemical cycling of elements.However, it is difficult to fully understand and predict the changes of complicated soil properties and functions based on physical and chemical parameters. Microorganisms inhabit soil with immense abundance and diversity and play key roles in decomposition of organic matter and cycling of nutrients. There are great variations between functions of different microbial groups. For instance, bacterial and fungal-based energy channels are considered as relating to different ecosystem functions. Besides the important functions of microbial community, microbial composition is sensitive to various environmental factors. Therefore, microbial composition can be used as a good indicator to evaluate the influence of human activities including soil management practices on soil properties and functions.A changing environment creates conditions that can be stressful for microorganisms. Soil microorganisms have a variety of evolutionary adaptations and physiological acclimation mechanisms that allow them to survive and remain active in the face of environmental stress. However, an extreme stress will force them into dormancy or cause death. In particular, since hydrology is considered to be the single most influential factor determining wetland characteristics, altered hydrologic regime could be responsible for drastic changes in structures and functions of wetlands. Several studies have reported the effect of hydrologic changes on wetlands in terms of their structure and ecological function. In general, enhanced mineralization rates and substantial nutrient leaching are well known consequences of successive drying and flooding.A disturbance such as flooding affects both above-ground and belowground ecosystem processes, although often ignored. In as much shifts in soil microbial community structure are expected when anaerobic conditions develop from flooding, changes in the belowground ecosystem may subsequently affect above-ground components of the ecosystem due to the critical roles that bacteria and fungi play in decomposition and nutrient cycling. With the completion of the Three Gorges Dam project in 2008, the water level of the reservoir fluctuates from 145 m in summer(May to September) to 175 m in winter(October to April), resulting in formation of a drawdown zone with a total area of ~350 km2 along the reservoir..The reversal of flooding time, creating prolonged flooding duration and a 30 m in elevation of water fluctuation, has dramatically altered environmental conditions in the drawdown zone. The Three Gorges Reservoir is exposed to annual redox fluctuation due to changes in the water table in the drawdown zone of 350 km2 with elevation of 30 m receives intermittent seasonal flooding. As a result of the tremendous construction effort, much of the native plant community along the reservoir slopes was cleared with considerable erosion. The remaining scattered native plants were incapable to adapt to prolonged redox disturbance due to flooding and have been subjected to a wide range of water stresses annually. The alteration in the submergence time and duration in the drawdown zone of the Three Gorges Reservoir has tremendously left most part of the drawdown zone bald of native plants due to prolonged redox disturbance.Despite huge efforts are channeled to restore the riparian ecosystems of the Three Gorges Reservoir Region, little is known about the effects of redox disturbance on the microbial community structure, carbon and nitrogen mineralization, nitrification potential and net nitrification in the drawdown zone. Knowing these data could strengthen the restoration strategy in the drawdown zone, this study conducted a field survey in June 2014 in three areas including Wanzhou-upstream(N 30.712576°,E 108.416496°), Changshou-middlestream(N 29.799030°, E 107.082710°) and Fengdu-downstream(N 29.904140°, E 107.754242°) based on the geographical characteristics of the drawdown zone from upstream to downstream in the Reservoir. The study areas are typical of the southeast subtropical monsoon with a mean annual temperature of 5.3°C in January and 29°C in July. Mean annual precipitation is about 1,100 mm with 80% falling between April and October. Soils studied are Cambisols, including purplish soil in Genetic Soil Classification of China(GSCC)(47.8%; Haplic Cambisols in World Reference Base for soil resources(WRB). From 2006 to 2014, seasonal inundation caused the development of a redox gradient with elevation, as elevations from 150-156 m are submerged annually for 8 months, 157- 170 m are submerged for 6 months, and 171-175 m are submerged for 3 monthsSoil samples were collected from three sections of elevation from 150 m to 182 m representing redox regimes(sites) due to repeated flooding from each of the three different sampling areas(Wanzhou from the upper portion of the reservoir, Changshou from the middle portion, and Fengdu from the lower portion) in the Three Gorges Reservoir Region.. The elevation range of 150 m to 156 m was flooded 8 times(8-cycle) from 2006 to 2014, the range of 161 m to 170 m flooded 5 times(5-cycle) since 2009, and 80 m to 82 m was never flooded(0-cycle) which consisted of native vegetation. For Wanzhou, Fengdu, and Changshou, 3 plots were established at each site(with the exception of Fengdu having 4 plots) and were randomly selected along the elevation range from 150 to 182 m. For each sampling plot(1×1 m), five soil samples of the 0–20-cm soil depth interval were collected and then well mixed to form 1 composite sample per each of the 3 plots per site. Samples were sealed in plastic bags and stored at 4°C for soil physiochemical analyses including soil p H, TOC, TN and nitrate, and at-20°C for soil microbial community structure analyses using the Phospholipid Fatty Acid(PLFA) analysis.Air dried soil samples were used to investigate C and N mineraliation, and nitrification kinetics in the drawdown zone. The soil nitrogen mineralization incubation was performed with two treatments: i) control without nitrogen amendment and ii) arginine containing nitrogen amendment of 0.34 mg arginine g-1 soil(88.4mg N arginine Kg-1 soil) in order to determine the changes in the inorganic N between the two treatments. Whereas, soil carbon mineralization incubation also consisted of two treatments: i) control without carbon amendment and ii) glucose containing carbon amendment of 1% glucose to determine changes in CO2-C between the two treatments. For the nitrification kinetics, we performed two treatments wherein half of the flasks were amended with 0.518 mg(NH4)2SO4 g-1(0.0884 mg N g-1) dry soil to study nitrification and the other half considered as control. The three soil samples were pre-incubated for 7 days at 28°C and 70% water-holding capacity(WHC). All the treatments were incubated in 250 ml flash with the replicates using 30 g soil maintaining 70% WHC throughout 15-day incubation of 28 oC in the dark. Samples were collected on day-0, day-5, day-10 and day-15. Moreover, at incubation days 0, 5, 10 and 15, 3 replicate bottles of each treatment were randomly selected to extract DNA and amo A genes was performed by quantitative PCR(q PCR). The DNA was extracted for three sub-samples from 0.50 g of soil with the Fast DNA Spin Kit for soil(MP Biomedicals, United States), according to the protocol of the manufacturer. The quality and quantity of the DNA extracts were determined with a spectrophotometer(Nanodrop, Peq Lab, Germany), and were pooled and stored at-20 °C until use. Quantitative PCR of amo A genes was performed to estimate the abundance of the ammonia-oxidizing bacterial and archaeal communities, respectively.The results showed that a total of 31 different PLFA biomarkers were identified in the drawdown zone and native soils. The prolonged redox disturbance caused shifts in the PLFA functional groups. The straight chain saturated PLFAs showed a ranged of 18%- 22% and 12%- 15% increase in comparison with the 0-cycle sites in the 8-cycle and 5-cycle sites respectively in Changshou, Wanzhou and Fengdu, while the branch chain saturated PLFAs were 24%- 26% higher in the 5-cycle plots and 10%- 12% higher in the 8-cycle sites than in the 0-cycle sites. The cyclopropyl PLFAs experienced a 30%- 32% and 47%- 50% increase in the 8-cycle and 5-cycle sites, respectively, compared to 0-cycle sites. Both linear monounsaturated PLFAs and polyunsaturated PLFAs decreased in the drawdown zone compared to the 0-cycle sites of the native community. The linear monounsaturated PLFAs decreased by 41%- 43% and 19%-20% in the 8-cycle and 5-cycle sites, respectively; and the Polyunsaturated PLFAs was not found in the 8-cycle sites but had 80%- 84% reduction in the 5-cycle sites compared to the 0-cycle sites of Changshou, Wanzhou and Fengdu. General bacteria, gram-positive bacteria, sulfate-reducing bacteria(SRB) and anaerobic bacteria were predominant in the 8-cycle and 5-cycle sites, whereas fungi, gram-negative bacteria, actinomycetes, methanotrophs, aerobic bacteria and protozoa enriched in the 0-cycle sites in Changshou, Wanzhou and Fengdu. Significant statistical variation existed between the drawdown zone(8-cycle and 5-cycle plots) and the native soils(0-cycle plots)(P < 0.002), which also explained the disadvantage for survival for certain microorganisms such as fungi, protozoa, gram-negative bacteria, actinomycetes, and aerobic bacteria in the drawdown zone. Protozoa was not found in drawdown zone of Changshou but was only found in the 5-cycle in the drawdown zone of both Wanzhou and Fengdu. Soil p H, TOC, and TN were positively correlated to G+/G- ratio, but negatively correlated to Tot PLFA, F/B, A/B and Mono/Branch ratios. While the C:N ratio was positively correlated to Tot PLFA and G+/G- ratio, it was negatively correlated to F/B, A/B and mono/branch ratios.The p H dynamic in both drawdown zone(8-cycle and 5-cycle) and native community(0-cycle) soils showed declining patterns, although the drawdown zone p H was higher compared to the native soil throughout the 15-day incubation in all the three areas. Interestingly, amongst the amendments arginine caused an increase in p H while glucose caused a decrease in p H. The 0-cycle sites of the native soils witnessed the highest decline in p H compared to the drawdown zone(0-cycle > 5-cycle > 8-cycle), though not significant different amongst plot in the drawdown zone(p < 0.05) in all the three areas. NH4+ and N2 O decrease with increasing incubation time while NO3-increasing with increasing incubation time during 15-day incubation in both the drawdown zone(8-cycle and 5-cycle) and native community(0-cycle) soils. All the redox regimes(sites) maintained significant declining patterns throughout the 15-day incubation, although the 8-cycle and 5-cycle of the drawdown zone had significant higher N2O- N and NH4+- N concentration than the 0-cycle of the native soils at day-15; Whereby, all the redox regimes(sites) maintained significant increasing patterns throughout the 15-day incubation, although the 8-cycle and 5-cycle of the drawdown zone had significant lower NO3-- N concentration than the 0-cycle of the native soils at day-15. The net nitrogen mineralization was significantly higher for 0-cycle than 8-cycle and 5-cycle throughout the 15-day incubation, but at the end of incubation the opposite trends were observed for net nitrogen mineralization rates.. The result showed that net nitrogen mineralization process was reduced or immobilized during day-5 in the 8-cycle and 5-cycle of the stressed soils in the drawdown zone of only Fengdu. But the 0-cycle of the unstressed soils in the native plant community witnessed no immobilization of the net nitrogen mineralization throughout incubation in all the three areas. After the addition of glucose all the commutative carbon dioxide concentrations increased in all the different redox regimes significantly to 401.74, 451.58 and 568.90 mg CO2-C Kg-1 soil for 8-cycle, 5-cycle and 0-cycle respectively, significantly decreased throughout the period of incubation. Prior to incubation the C mineralization were 370.43, 408.21 and 522.77 mg CO2-C Kg-1 soil for 8-cycle, 5-cycle and 0-cycle respectively, which indicate significant difference amongst the different redox regimes. Despite the 0-cycle CO2-C mineralization was higher than 8-cycle and 5-cycle, the different sites(redox regimes) showed a significant declining pattern throughout the 15-day incubation.The NO3--N concentrations increase with increasing incubation time during 15-day incubation in the different redox regimes in all the three areas even with ammonia sulphate addition. Regression analysis showed that prolonged redox disturbance did not change the time dependent kinetics of potential nitrification pattern which was best fitted by zero order model for the all the redox regimes; however, the potential nitrification rate for 0-cycle of the native community was 20.2 mg Kg-1 day-1 which was 3.4 and 1.9 times higher compared to 8-cycle and 5-cycle respectively in the water fluctuation zone. The nitrification reaction rates(k0) of the stimulated parameters for potential nitrification revealed that 1.95 and 3.8 times higher in the 0-cycle compared to the 5-cycle and 8-cycle respectively. Meanwhile, the stimulated parameters for the time dependent kinetics of net nitrification was best fitted by a zero-order model for 8-cycle and 5-cycle, while the 0-cycle best fitted the first-order kinetic model. The Va and nitrification reaction rates(k0) did not show significant difference for 8-cycle and 5-cycle. Va was 6.39, 5.31 and 446 mg Kg-1 day-1 soil which reflected no statistical difference in the 0-cycle of the native community compared to the 5-cycle and 8-cycle of the water fluctuation zone. The initial potential nitrification was 12.5, 21.1 and 36.3 mg Kg-1 soil in 8-cycle, 5-cycle and 0-cycle. Although N0 was higher in the native soil compared to the water fluctuation zone, the prolonged redox disturbance caused a slight increase in the Va in the water fluctuation zone. AOB amo A gene copy numbers for the sites of different redox regimes with ammonium application were higher than those without N supply controls at the end of incubation. Whereby, AOA dynamics during incubation showed similar trend for all soils, with or without additional N supply, and the highest levels in AOA amo A gene copies were observed at the end of the 15-day incubation.Exposure to prolonged redox disturbance following recurrent flooding shifted microbial community structure in the drawdown zone. This phenomenon disadvantaged fungi, protozoa, gram-negative bacteria, actinomycetes, and aerobic bacteria. There was a complete extinction of protozoa owing to the 8-year prolonged redox disturbance in the 8-cycle sites of the drawdown zone in all the areas of the Three Gorges Reservoir Region. The significance of the shifts in microbial activity interacting with available substrate in the drawdown zone was critical in the reduction of carbon and nitrogen mineralization at different flooding regimes in the Three Gorges Reservoir Region. Apart from Fengdu wherein nitrogen immobilization was observed at day-5 in the 8-cycle and 5-cycle sites, Changshou and Wanzhou observed the immobilization of N mineral at day-5. All the 0-cycle sites in all the areas observed no N mineral immobilization during the 15-day incubation. The stimulated net nitrification kinetic model in the drawdown zone soils was affected by the prolonged redox disturbance, which reflects the abundance of substrate over microbial activity compared to the unstressed soils of the native plant community which were dominated by microbial activity. But the prolonged redox disturbance did not alter potential nitrification pattern though the 0-cycle sites had higher nitrate concentrations than the 8-cycle and 5-cycle sites of the drawdown zone. Moreover, the prolonged redox disturbance favors only the AOA community.Therefore, disturbances such as recurrent flooding in the drawdown zone created temporal and seasonal redox gradients due to reversal of submergence time and prolonged inundation duration which caused changes in the soil microbial community structure that made them different from the native microbial community. Change in the microbial community structure by repeated flooding events, or a change in the physiology of the extant population can reduce the ability of the microbial community to mineralize available substrate thus causing a reduction in carbon and nitrogen mineralization. Moreover, continuation of fluctuating redox condition in the drawdown zone might have a continuous devastating effect on the ammonia microbial-catalyzed nitrifiers community. This might cause further reduction in the metabolic mechanism of nitrification and in turn make the water fluctuation zone to be more and more substrate independence compared to catalyst.