Long-term nitrogen fertilization alters microbial community structure and denitrifier abundance in the deep vadose zone

Purpose

The excessive use of nitrogen (N) fertilizer in intensive agriculture has increased nitrate leaching into groundwater, but its impacts on N transformation processes and the associated microbial communities in the deep vadose zone remain unclear.

Materials and methods

Soil samples from 0–1050 cm depth were collected from a 20-year field experiment with two N fertilization treatments: 0 (N0) and 600 kg N ha^?1 year^?1 (N600). Amplicon sequencing and quantitative PCR analyses were performed to profile the vertical distribution of soil microbial communities and denitrification genes.

Results and discussion

The soil microbial community structure and diversity were strongly influenced by soil depth and N fertilization. The 250 cm depth was identified as a threshold depth, as dramatically different microbial communities were found below and above this depth. Quantitative PCR results showed that the absolute abundance of denitrification genes decreased with increasing soil depth.

Conclusion

This study elucidated the profound effects of long-term N input on the composition and diversity of the microbial communities and the abundance of denitrifiers in the deep vadose zone. Our results provide basic information for use in mitigating nitrate leaching by enhancing microbial denitrification in deep vadose zones in intensive agricultural areas.

     

基于开放式培养的微藻生物柴油生命周期环境影响评价

为定量评价以微藻为原料生产的生物柴油生命周期系统产生的各种潜在环境影响,以基于开放式培养的微藻生物柴油为研究对象,应用生命周期分析方法,以1 MJ能量的柴油产品为功能单位,对生产、使用微藻生物柴油产生的环境影响进行了分析。结果表明：微藻生物柴油生命周期最显著的环境影响类型为不可更新资源消耗,其次为光化学烟雾形成,生命周期总环境影响指数为4.63×10-4人当量,较石化柴油降低19.34%,以油菜籽为原料的生物柴油生命周期总环境影响指数是微藻生物柴油的7.19倍,微藻培养与生物柴油制取对生命周期总环境影响指数的贡献分别为27.95%与46.24%。基于开放式培养的微藻生物柴油相对于石化柴油与以油菜籽为原料的生物柴油具有较好的生命周期环境效益,控制微藻生物柴油生命周期环境影响的要点在于减少微藻培养与生物柴油制取阶段的动力消耗。     

应用新引物DGGE分析土壤中硝酸盐还原细菌群落

The narG gene is frequently used as a molecular marker for bacterial nitrate-reducing community analysis. In this study, a new set of primers targeting the narG gene was designed and applied to semi-nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assay. The potential of the new primers was verified on DNA directly extracted from soils from five different experimental sites distributed in Central and Southern Italy. Specificity of the primers was determined by excision, amplification, and sequencing of bands resolved by DGGE. A phylogenetic analysis showed the correlation between the sequences retrieved from the soils studied and the narG sequences from β and γ-Proteobacteria. These primers expanded the existing molecular tools for ecological study on the size and diversity of nitrate-reducing bacterial community in soil.     

Nitrate removal, denitrification and nitrous oxide production in the riparian zone of an ephemeral stream

Riparian zones are important features of the landscape that can buffer waterways from non-point sources of nitrogen pollution. Studies of perennial streams have identified denitrification as one of the dominant mechanisms by which this can occur. This study aimed to assess nitrate removal within the riparian zone of an ephemeral stream and characterise the processes responsible, particularly denitrification, using both in-situ and laboratory techniques. To quantify rates of groundwater nitrate removal and denitrification in-situ, nitrate was added to two separate injection-capture well networks in a perched riparian aquifer of a low order ephemeral stream in South East Queensland, Australia. Both networks also received bromide as a conservative tracer and one received acetylene to inhibit the last step of denitrification. An average of 77 ± 2% and 98 ± 1% of the added nitrate was removed within a distance of 40 cm from the injection wells (networks with acetylene and without, respectively). Based on rates of N₂O production in the network with added acetylene, denitrification was not a major mechanism of nitrate loss, accounting for only 3% of removal. Reduction of nitrate to ammonium was also not a major pathway in either network, contributing <4%. Relatively high concentrations of oxygen in the aquifer following recent filling by stream water may have reduced the importance of these two anaerobic pathways. Alternatively, denitrification may have been underestimated using the in-situ acetylene block technique. In the laboratory, soils taken from two depths at each well network were incubated with four nitrate-N treatments (ranging from ambient concentration to an addition of 15 mg N l⁻¹), with and without added acetylene. Potential rates of denitrification, N₂O production and N₂O:N₂ ratios increased with nitrate additions, particularly in shallow soils. Potential rates of denitrification observed in the laboratory were equivalent in magnitude to nitrate removal measured in the field (mean 0.26 ± 0.12 mg N kg of dry soil⁻¹ d⁻¹), but were two orders of magnitude greater than denitrification measured in the field with added acetylene. The relative importance of assimilatory vs. dissimilatory processes of nitrate removal depends on environmental conditions in the aquifer, particularly hydrology and its effects on dissolved oxygen concentrations. Depending on seasonal conditions, aquifers of ephemeral streams like the study site are likely to fluctuate between oxic and anoxic conditions; nevertheless they may still function as effective buffers. While denitrification to N₂ is a desirable outcome from a management perspective, assimilation into biomass can provide a rapid sink for nitrate, thus helping to reduce short-term delivery of nitrate downstream. Longer-term studies are needed to determine the overall effectiveness of riparian buffers associated with ephemeral streams in mitigating nitrate loads reaching downstream ecosystems.     

Denitrification activity in the vadose zone beneath a sludge‐amended semi‐arid soil

Abstract

Soil cores were collected to a depth of 14 m from a Southwest semi‐arid soil amended with either anaerobically digested sludge or inorganic fertilizer. Twenty sections partitioned from each core were characterized for their physical and chemical properties. Denitrification potential was estimated in each core section in the laboratory using the acetylene reduction method. The sludge‐amended soil had significantly higher denitrification rates within and below the root zone than the fertilizer‐amended soil. Additionally, significant correlation values were obtained in both cores between denitrification rates and particle size distribution, moisture, and total organic carbon (C). Sludge applications in semi‐desert soils may add much needed organic C in the soil profile. This additional soluble organic C may help control nitrate (NO₃) ground water pollution by providing substrate C for denitrifying bacteria below the root zone.     

Effects of microbial inoculants on phosphorus and potassium availability,bacterial community composition,and chili pepper growth in a calcareous soil: a greenhouse study

Purpose

Phosphorus (P) and potassium (K) are two important essential nutrient elements for plant growth and development but their availability is often limited in calcareous soils. The objective of this study was to determine the effects of applying microbial inoculants (MI, containing effective strains of Bacillus megaterium and Bacillus mucilaginous) on the availability of P and K, plant growth, and the bacterial community in calcareous soil.

Materials and methods

A greenhouse experiment was conducted to explore the effects of the addition of MI (control: without MI addition; treatment: with MI addition at the rate of 60 L ha^?1) on the concentrations of P and K in soil and plant, soil bacterial community diversity and composition, and chili pepper (Capsicum annuum L.) growth.

Results and discussion

The results showed that MI inoculation significantly increased the fruit yields by 28.5% (p?<?0.01), available P and K in the rhizosphere soil by 32.1% and 28.1% (p?<?0.05), and P and K accumulation in the whole plants by 40.9% and 40.2%, respectively (p?<?0.05). Moreover, high-throughput sequencing revealed that Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, and Gemmatimonadetes were the dominant phyla of soil bacteria. MI application did not significantly impact the diversity and composition of soil bacterial communities, but increased relative abundances of bacterial genera Flavobacterium responsible for promoting root development across growing stages (p?<?0.05), and changed the soil bacterial community structure associated closely with soil properties of available P, K, and pH in soil.

Conclusions

The application of MI improved the bioavailability of P and K and plant growth due to its impact on the soil bacterial community structure.

     

Effectiveness of acetylene inhibition of N2O reduction for measuring denitrification in soils of varying wetness

Abstract

The use of acetylene (C₂H₂) in the inhibition of N₂O to N₂ is widely used for measuring denitrification. The objective of this study was to determine the effectiveness of acetylene inhibition of N₂O reduction for short‐term and prolonged incubation studies in soils of varying water saturation, and to find out the possible reasons for lower N₂O recovery in continuously sealed incubations. Two experiments carried out in the laboratory reconfirmed that acetylene was very effective in inhibiting the reduction of N₂O in denitrification even for the prolonged incubation period (up to 96 h) under moist to saturated soil water contents. With 90 and 120% water‐filled pore space (WFPS), the accumulated N₂O in containers kept sealed throughout the study period, was 28 to 41% less than total headspace N₂O produced in containers that were opened, flushed and fresh C₂H₂ added every 24 h. Interpretation of our results suggest the lower N₂O amount recovered from continuously sealed containers at high WFPS, as compared to short‐term incubations (flushed containers), resulted primarily from delayed N₂O release from soil and not greater N₂O dissolved in soil solution, lower rates of denitrification, or decomposition/loss of C₂H₂ during prolonged incubation. Reduction of N₂O diffusion from soil cores showed direct relationship with head space concentration‐of N₂O and soil WFPS. From these results it is concluded that to obtain quantitative recovery of N₂O produced via denitrification, especially from soil with high WFPS soil cores should be vigorously shaken before head‐space N₂O analysis.     

Effectiveness of acetylene inhibition of N2O reduction for measuring denitrification in soils of varying wetness

Abstract

The use of acetylene (C₂H₂) in the inhibition of N₂O to N₂ is widely used for measuring denitrification. The objective of this study was to determine the effectiveness of acetylene inhibition of N₂O reduction for short‐term and prolonged incubation studies in soils of varying water saturation, and to find out the possible reasons for lower N₂O recovery in continuously sealed incubations. Two experiments carried out in the laboratory reconfirmed that acetylene was very effective in inhibiting the reduction of N₂O in denitrification even for the prolonged incubation period (up to 96 h) under moist to saturated soil water contents. With 90 and 120% water‐filled pore space (WFPS), the accumulated N₂O in containers kept sealed throughout the study period, was 28 to 41% less than total headspace N₂O produced in containers that were opened, flushed and fresh C₂H₂ added every 24 h. Interpretation of our results suggest the lower N₂O amount recovered from continuously sealed containers at high WFPS, as compared to short‐term incubations (flushed containers), resulted primarily from delayed N₂O release from soil and not greater N₂O dissolved in soil solution, lower rates of denitrification, or decomposition/loss of C₂H₂ during prolonged incubation. Reduction of N₂O diffusion from soil cores showed direct relationship with headspace concentration of N₂O and soil WFPS. From these results it is concluded that to obtain quantitative recovery of N₂O produced via denitrification, especially from soil with high WFPS soil cores should be vigorously shaken before head‐space N₂O analysis.     

Succession of bacterial community and methanotrophy during lake shrinkage

Purpose

The shrinkage of vast inland lakes affects microbially mediated soil biogeochemical processes, which are critical for maintaining ecosystem sustainability, such as microbial diversity and a balanced CH₄ budget. Here we aimed to elucidate shifts in the bacterial community and methanotrophy during the shrinkage of a saline lake.

Materials and methods

Sediments and soils along a gradient transecting a saline lake, saline riparian land, and grassland were collected. The succession of microbial communities was characterized by high-throughput sequencing of the V4-V5 region of 16S rRNA genes coupled to non-metric multidimensional scaling (NMDS), linear discriminant effect size (LEfSe), community assembly, and co-occurrence network analyses. We further incubated these samples under a 10% CH₄ (v/v) atmospheric condition to determine the response of methane oxidation potentials and of methanotrophs to lake shrinkage by using pmoA-based qPCR and amplicon sequencing.

Results and discussion

LEfSe and NMDS analyses showed significant differences in bacterial communities among 3 stages of lake shrinkage. The microbial taxa with the highest increase were phylogenetically affiliated with unclassified Rhizobiales, Panacagrimonas, and Pseudomonas in saline and grassland soils when compared with sediments. Microbial community assembly was largely determined by deterministic rather than stochastic processes (NTI?>?2). The drastic increase of Methylocystis-like (type II) methanotrophs was observed during lake shrinkage, while type I methanotrophs showed a decreasing trend. However, upon consuming high-concentration methane of about 10%, type I methanotrophs dominated methane-oxidizing communities in lake sediment (Methylomonas), riparian saline soil (Methylomicrobium), and grassland soil (Methylobacter). Structural equation model identified soil pH, C/N ratio, and soil texture as key factors affecting methane oxidation rates and the methanotrophic community.

Conclusions

Lake shrinkage showed profound impacts on the overall bacterial communities and methane oxidizers. Soil physico-chemical properties likely shaped the bacterial community and phylogenetically distinct methanotrophs during lake shrinkage.

     

不同原料制备生物柴油生命周期能耗和排放评价

建立了大豆、油菜籽、光皮树和麻疯树4种原料制生物柴油生命周期能源消耗和排放评价模型，并对其进行了生命周期能源消耗和排放评价。结果表明：与石化柴油比较，大豆和油菜籽制生物柴油生命周期整体能源消耗与石化柴油基本相当；光皮树和麻疯树制生物柴油的生命周期整体能源消耗比石化柴油低约10%；所有原料制生物柴油生命周期化石能源消耗显著降低，生命周期HC、CO、PM₁₀、SOx和CO₂排放降低，NOx排放升高。     

Verification of an in‐situ method for measuring denitr1f1cation

Abstract

The most common direct in‐situ method for the measurement of soil denitrification requires many acetylene (C₂H₂) supply probes and airflow lines to measure nitrous oxide (N₂O) flux from the soil under a sealed cover. A modification to this method simplified C₂H₂ supply by placing a single acetylene supply probe 30 cm deep into the soil and measured soil N₂O emission flux over a 0.11 m² area. Acetylene concentrations ranging from 0.1–10.0% were readily and predictably established by radial diffusion from the supply probe. Over 94% of the N₂O released into the enclosed air space of the soil cover was recovered at an air flow rate of 21 L/h. Recovery decreased rapidly with increased flow rates of 31 and 37 L/h.     

Denitrification rate relationships with soil parameters in the field

Abstract

It has been recently shown that there is a large spatial variability in denltrification rates measured in the field. The objective of this study was to assign this variability to twelve measurable or determined soil parameters known or suspected to be Important to denitrification during the early part of the growing season. Relationships were sought with 16 cores at a grid spacing of 25 cm on three sites (dates) within a 0.07 ha area of a cultivated silt loam soil. The denitrification rate was estimated from the N₂O production rate with the acetylene blockage technique.

Only few statistical significant relationships were found with simple and multiple regression analyses and there was a lack of consistency from site to site. Plotting the data revealed a tentative negative relationship between the N₂O production rate and percent air‐filled porosity. A few cores at each site showed a much greater N₂O production rate for no discernable reason, but these rates were also negatively related to percent air‐filled porosity. Tentative positive relationships between N₂O production rate and total organic carbon or water‐soluble carbon were similarly found.     

Characterization of Groundwater Microbial Communities, Dechlorinating Bacteria, and In Situ Biodegradation of Chloroethenes Along a Vertical Gradient

The variability of hydrogeochemical conditions can affect groundwater microbial communities and the natural attenuation of organic chemicals in contaminated aquifers. It is suspected that in situ biodegradation in anoxic plumes of chloroethenes depends on the spatial location of the contaminants and the electron donors and acceptors, as well as the patchiness of bacterial populations capable of reductive dechlorination. However, knowledge about the spatial variability of bacterial communities and in situ biodegradation of chloroethenes in aquifers is limited. Here, we show that changes of the bacterial communities, the distribution of putative dechlorinating bacteria and in situ biodegradation at the border of a chloroethenes plume (Bitterfeld, Germany) are related to local hydrogeochemical conditions. Biotic reductive dechlorination occurred along a 50 m vertical gradient, although significant changes of the hydrogeochemistry and contaminant concentrations, bacterial communities and distribution of putative dechlorinating bacteria (Dehalobacter spp., Desulfitobacterium spp., Dehalococcoides spp., and Geobacter spp.) were observed. The occurrence and variability of in situ biodegradation of chloroethenes were revealed by shifts in the isotope compositions of the chloroethenes along the vertical gradient (??¹³C ranging from ?14.4?? to ?4.4??). Our results indicate that habitat characteristics were compartmentalized along the vertical gradient and in situ biodegradation occurred with specific reaction conditions at discrete depth. The polyphasic approach that combined geochemical and biomolecular methods with compound-specific analysis enabled to characterize the spatial variability of hydrochemistry, bacterial communities and in situ biodegradation of chloroethenes in a heterogeneous aquifer.     

生物柴油对能源和环境影响分析

生物柴油是从植物或动物脂肪酸通过酯化反应而得到,由于生物柴油无毒,可生物降解和可以再生,因此受到越来越多人的关注。生物柴油的性质和普通柴油非常相似,它能直接被用到发动机上而不需要改动发动机的结构。该文基于美国能源部对生物柴油的统计数据,利用生命循环分析法,对生物柴油从生产到消耗的生命循环中的能量消耗和产出、循环中的排放以及生物柴油汽车尾气排放等方面进行了分析。生命循环开始于普通柴油或生物柴油生产的原料提取,结束于成品油在发动机上的使用。只有分析生命循环中的所有过程,才能确定它对自然环境总量的影响。例如研究温室效应就要对整个生命循环中CO₂的排放进行分析。该文利用生命循环分析法分析了在生产生物柴油或柴油生命循环过程中的能量平衡、温室气体排放及对气体和固体污染物排放,提供了生物柴油生产过程和在发动机上使用的详细数据。分析结果表明∶生物柴油循环的石化能效比大大提高,大约是柴油的4倍;生物柴油循环中CO₂排放大大降低,大约降低了78.4%;发动机排气管有害物质的排放中,除NO_x排放增加8.89%外,CO、HC、PM等有害物质的排放大大降低（分别降低了46%、37%和68%）。     

Influx and concentration of triazine pesticides in the Amaterska cave system,Moravian Karst,Czech Republic

Purpose

The aim of this study was to detect three triazine pesticides and their metabolites in the drip water and the sediment of the Amaterska cave system. Diversity of the bacterial community in the sediment was also assessed, and the potential role of bacteria in degradation of these pesticides was evaluated.

Materials and methods

Triazines and their metabolites were analyzed in the soil, drip water, and sediment of the Amaterska cave system area in seven sampling sites (S1–S7) based on the above ground cover that included forest, permanent grassland, and agriculture cropland. The bacterial community in the cave sediments (S1–S6) was also analyzed using the Illumina sequencing of the V3 and V4 regions of 16S rDNA.

Results and discussion

Triazines were present in the soil and drip water in all sites below grassland and agricultural land but not under the forest area. Only atrazine metabolites were detected in the surface soil. In contrast, atrazine was detected in all cave sediments regardless of above ground cover, and this is likely due to the occasional alluvial influx. The overall prevalence of bacteria potentially capable of atrazine degradation in the cave sediment ranged from 13.4 to 64.0% of the entire bacterial community. The concentrations of atrazine in the cave sediment were 16 to 70 times higher than in those in drip water.

Conclusions

High concentrations of atrazine in the cave sediment indicate a slow degradation rate of triazines in the cave likely due to low temperatures and absence of photolysis. The main source of atrazine in the Amaterska cave system is likely not drip water but the alluvial influx. Bacteria potentially capable of triazine degradation in the cave sediment were detected; however, their role in this process remains to be investigated.

     

Differentiation of nitrogen and microbial community in the littoral and limnetic sediments of a large shallow eutrophic lake (Chaohu Lake,China)

Purpose

Nitrogen (N) is one of the major elements causing eutrophication in freshwater lakes, and the N cycle is mainly driven by microorganisms. Lake littoral zones are found to be “hotspots” for N removal from both the basin and receiving waters. However, the environmental factors that drive the distribution of microorganisms are diverse and unclear. Here, we examined the differentiation of nitrogen and microbial community between the littoral and limnetic sediments to explore their interactions.

Materials and methods

Sediment samples were collected in the littoral and limnetic zones of Chaohu Lake in winter (ca. 7 °C) and autumn (ca. 22 °C). Abundances of the bacterial and archaeal genes amoA (ammoxidation), nirS and nirK (denitrification), hzsB (anaerobic ammonium oxidation; anammox), and nrfA (dissimilatory nitrate reduction to ammonium; DNRA) were measured via quantitative real-time polymerase chain reaction (qPCR). Clone libraries were constructed for further phylogenetic analysis to study the community composition.

Results and discussion

We observed significant higher concentration values in terms of sedimentary NH₄⁺-N and NO₃^?-N in the limnetic zone than littoral zone (p?<?0.05; n?=?12). In general, abundance values of the above six genes in the littoral zone were all higher than those in the limnetic zone, while higher in winter (7 °C) than in autumn (22 °C) (p?<?0.05; n?=?6). The spatial heterogeneity had the most significant effect on the distribution of ammonia-oxidizing archaea (AOA) and anammox bacteria abundance. Both temporal (temperature) and spatial heterogeneity affected the abundance of ammonia-oxidizing bacteria (AOB). The variation in the abundance of denitrifying bacteria and DNRA bacteria mainly reflected the temporal (temperature) heterogeneity.

Conclusions

The six N-cycle-related microorganisms were affected by different environmental factors and presented different distribution patterns. The lower nitrogen content and the higher microbial abundance and diversity showed that the littoral zone was the “hotspot” of N-cycling-related microorganisms in a large, eutrophic, and turbid lake. It is suggested that increasing the area and restoring the ecological function of the littoral zone was effective and significant in eutrophic lake management.

     

棉籽油生物柴油和柴油混合燃料的润滑特性

为研究生物柴油的润滑特性,该研究用棉籽油与甲醇和乙醇分别进行酯交换反应,制备出甲酯生物柴油和乙酯生物柴油。然后将它们分别以不同体积比率与市售0#柴油混合,制备出不同比率的生物柴油/柴油混合物。通过四球摩擦磨损试验机考察了这些混合物的润滑特性。结果表明：不同碳醇与棉籽油制备的生物柴油对柴油的润滑性能均具有增效作用,且随着生物柴油添加量的增加,柴油的润滑性能得到提高;但市售0#柴油对不同碳醇制备的生物柴油感受性不同,分别添加20%的甲酯生物柴油和乙酯生物柴油到0#柴油时,发现混合柴油的最大无卡咬负荷（PB值）分别提升了94.1%和29.4%;同时,甲酯生物柴油对柴油的最大无卡咬负荷影响大,而乙酯生物柴油/柴油的减摩性和抗磨性都好于甲酯生物柴油/柴油;游离脂肪酸对生物柴油润滑性也有较大影响。该研究为生物柴油的应用打下基础。     

Fungal and bacterial denitrification are differently affected by long-term pH amendment and cultivation of arable soil

Bacteria are considered as playing a predominant role in the production of nitrous oxide (N₂O) in arable soil. Despite the knowledge that fungi are able to denitrify their contribution to denitrifier N₂O production from arable soil is uncertain. Here, we assess the capability of fungi and bacteria to contribute to N₂O emission from arable soil by measuring potential denitrification rates (PDR) as N₂O production, after application of selective inhibitors aimed at distinguishing between fungal and bacterial denitrification, and related PDR to characteristics of the soil microbial community. Soil was sampled from a long-term crop rotation maintained since 1961 at seven different pH levels, ranging in 0.5 increments from pH 4.5 to 7.5, and along a cultivation gradient from freshly ploughed soil to three years under ley grass. Over both pH and cultivation gradients, bacteria contributed up to 54% and fungi contributed to 18% of the PDR. Residual N₂O production that was not targeted by the selective inhibitors and hence could not be attributed to fungi or bacteria might be due to pre-synthesised enzymes or resistant organisms. The PDR of the bacterial community responded positively to increase in soil pH with the lowest PDR at pH 4.2 and the highest around pH 5.9. In contrast, fungal denitrification was not influenced by soil pH. Changes in ester linked fatty acids (ELFA) concentrations showed that whilst total bacterial biomass decreased with increasing pH fungal biomass was not significantly influenced by pH, driving an increase in the ratio of fungal–bacterial biomass. Both fungal biomass and bacterial biomass, and the PDR from the control treatment (no inhibitor application) across the pH gradient were greatest under long-term ley. Concentrations of fatty acids a15:0, 16:1ω7 and 17:1ω8 of microbial origin were positively correlated with the proportion of denitrification activity that was repressed by bacterial inhibitors. This suggests that there is a relationship between organisms that possess the fatty acids a15:0, 16:1ω7 and 17:1ω8, and the function of denitrification. Our results demonstrate that both fungal and bacterial denitrification were occurring in this arable soil. That management for pH and cultivation had differing effects on the potential contribution of fungal and bacterial denitrification to N₂O production has implications for the development of appropriate management practices for mitigation of this greenhouse gas.