Contribution of ethylamine degrading bacteria to atrazine degradation in soils

Bacterial communities that cooperatively degrade atrazine commonly consist of diverse species in which the genes for atrazine dechlorination and dealkylation are variously distributed among different species. Normally, the first step in degradation of atrazine involves dechlorination mediated by atzA, followed by stepwise dealkylation to yield either N-ethylammelide or N-isopropylammelide. As the liberated alkylamine moieties are constituents of many organic molecules other than atrazine, it is possible that a large number of alkylamine-degrading bacteria other than those previously described might contribute to this key step in atrazine degradation. To examine this hypothesis, we isolated 82 bacterial strains from soil by plating soil water extracts on agar media with ethylamine as a sole carbon source. Among the relatively large number of isolates, only 3 were able to degrade N-ethylammelide, and in each case were shown to carry the atzB gene and atzC genes. The isolates, identified as Rhizobium leguminosarum, Flavobacterium sp., and Arthrobacter sp., were all readily substituted into an atrazine-degrading consortium to carry out N-ethylammelide degradation. The distribution of these genes among many different species in the soil microbial population suggests that these genes are highly mobile and over time may lead to generation of various atrazine-degrading consortia.     

Interactions of earthworms with Atrazine-degrading bacteria in an agricultural soil

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.     

Enzyme immunoassay determination of atrazine degradation in soil: Moisture,sterilization, and storage effects

Enzyme immunoassay (EIA) microtiter plate analysis was used to quantify atrazine (2‐chloro‐4‐ethylamino‐6‐isopropylamino‐1,3,5 triazine), fortified at 0, 50, and 500 or 549 ng/g, to Baxter and Maury silt loam soil sampled in 1965 and 1991. In the first experiment, aged soils (sampled in 1965 and stored air‐dried) were fortified with atrazine and then incubated in the dark at 0, 75, 150, 225 and 300 g/kg moisture for 15, 80, 154, and 289 d. In a second experiment, fresh soils were fortified with atrazine and incubated in the dark at 0, 150, and 300 g/kg moisture for 9, 15, 35, 55, 83, and 145 d. One half of the treatments in the second experiment were sterilized with 497 ng/g HgCl₂. Twenty milliliters of acetonitrile: water (9: 1) was used to extract 4 or 5 g of soil by vortex mixing at each sampling date. The soil extract was diluted, 80 μl incubated with antibody‐coated wells, and color development read using a microtiter plate reader. Recovery of atrazine from soil was 98% 5 d after fortification. Pesticide recoveries and first‐order degradation rates were dependent on the freshness and moisture content of the soil sample. Pesticide degradation was slower and recoveries higher in soil that had been air dried and stored since 1965, prior to fortification. More atrazine was extracted from soil maintained at 0 g/kg moisture than from soil maintained at 300 g/kg moisture over time.     

Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene

Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p-atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p-atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p-atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium-mediated transformation. Our work suggests that the in planta expression of p-atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome.     

Microbial aspects of atrazine degradation in natural environments

The potential toxicity of thes-triazine herbicide atrazine motivates continuous bioremediation-directed research. Several indigenous soilatrazine-catabolizing microbialassociations and monocultures have been enriched/isolated from compromised sites. Of these, Pseudomonas sp. strain ADP has become a reference strain and has been used to elucidate sequences of the catabolic enzymes atzA, atzB, atzCand atzD involvedin one aerobic degradation pathway and develop probes for the genes which encode these enzymes. Despite this, hitherto unknown or novel microorganisms, with unique sequences and different enzyme-mediated operative pathways, warrant continued investigations for effective site bioremediation. Also, the sustained effectiveness of natural attenuation must be demonstrated continually so regular site evaluations and results analyses, despite the limitations of chemical extraction methodologies, are crucial practices. For both directed and intrinsic bioremediation monitoring, traditional microbial association studies must be complemented by more advanced physiological and molecular approaches. The occurrence of catabolic plasmids, in particular, should be probed with DNA hybridization techniques. Also, PCR-DGGEand subsequent new sequenceelucidation should be used prior to developing new primers for DNA sequences encoding novel catabolic enzymes, and for hybridization probe development, to establish the degradative potential of a compromised site, or adoption of FISH to, for example, monitor bioaugmented remediation.     

Effects of biochar amendment on the sorption and degradation of atrazine in different soils

ABSTRACT

Sugarcane top-derived biochar was added to an alluvial soil, a moist soil and a paddy soil at the rate of 0.2% and 0.5% (w/w). After the addition of 0.2% and 0.5% biochar, the sorption coefficients (K_d) of atrazine (Ce = 10 mg L^?1) were increased by 26.97% and 79.58%, respectively, in the moist soil with a low level of total organic carbon (TOC), while it increased by 31.43% and 60.06%, respectively, in the paddy soil with a high TOC content. The half-time persistence values of atrazine in the alluvial soil, moist soil and paddy soil were 28.18, 23.74 and 39.84 d, respectively. In the 0.2% biochar amended soils, the corresponding half-times of atrazine for the alluvial soil, moist soil and paddy soil were extended by 10.33, 11.81 and 1.42 d, and they were prolonged by 16.83, 17.52 and 14.74 d, respectively, in the 0.5% biochar amended soils. Atrazine degradation products (deisopropylatrazine and desethylatrazine) decreased after they accumulated to 3.2 and 1 mg kg^?1, respectively. Generally, increasing sorption was accompanied by decreasing degradation of atrazine which is found in biochar-amended soils.     

Effects of dissolved organic matter from sewage sludge on the atrazine sorption by soils

The effects of dissolved organic matter (DOM), water soluble organic matter derived from sewage sludge, on the sorption of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-trazine) by soils were studied using a batch equilibrium technique. Six paddy soils, chosen so as to have different organic carbon contents, were experimented in this investigation. Atrazine sorption isotherms on soils were described by the linear equation, and the distribution coefficients without DOM (Kd) or with DOM (Kd*) were obtained. Generally, the values of Kd*/Kd initially increased and decreased thereafter with increasing DOM concentrations of 0-60 mg DOC ·L-1 in soil-solution system form. Critical concentrations of DOM (DOMnp) were obtained where the value of Kd* was equal to Kd. The presence of DOM with concentrations lower than DOMnp promoted atrazine sorption on soils (Kd* > Kd), whereas the presence of DOM with concentrations higher than DOMnp tended to inhibit atrazine sorption (Kd* < Kd). Interestingly, DOMnp for     

Effects of dissolved organic matter from sewage sludge on the atrazine sorption by soils

The effects of dissolved organic matter (DOM), water soluble organic matter derived from sewage sludge, on the sorption of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-trazine) by soils were studied using a batch equilibrium technique. Six paddy soils, chosen so as to have different organic carbon contents, were experimented in this investigation. Atrazine sorption isotherms on soils were described by the linear equation, and the distribution coefficients without DOM (Kd) or with DOM (Kd*) were obtained. Generally, the values of Kd*/Kd initially insoil-solution system form. Critical concentrations of DOM (DOMnp) were obtained where the value of Kd* was equal to Kd. The presence of DOM with concentrations lower than DOMnp promoted atrazine sorption on soils (Kd* ＞ Kd), whereas the presence of DOM with concentrations higher than DOMnp tended to inhibit atrazine sorption (Kd* ＜ Kd). Interestingly, DOMnp for tested soils was negatively correlated to the soil organic carbon content, and the maximum of Kd*/Kd (i.e.Kmax) correlated positively with the maximum of DOM sorption on soil (Xmax). Further investigations showed that the presence of hydrophobic fraction of DOM evidently promoted the atrazine sorption on soils, whereas the presence of hydrophilic DOM fraction obviously tended to inhibit the atrazine sorption. Interactions of soil surfaces with DOM and its fractions were suggested to be the major processes determining atrazine sorption on soils. The results of this work provide a reference to the agricultural use of organic amendment such as sewage sludge for improving the availability of atrazine in soils.     

The effect of some terrestrial oligochaeta on nitrogenase activity in the soil

Nitrogenase activity, assayed by the acetylene reduction technique, was estimated on control soils and soils which contained several species of earthworms. The worms and casts were separately assayed for nitrogenase activity. The results indicated that nitrogenase activity was associated with gut content or body surface of worms and also their casts. Nitrogen fixation was also enhanced in soils enriched with enchytracids in the presence of leaf litter.     

不同林型土壤微生物有机碳降解基因的多样性

应用寡聚核苷酸基因芯片,分析了米亚罗林区冷杉原始林（M—Y）和20世纪60年代云杉人工林（M-60）土壤微生物的功能基因多样性。该功能基因芯片含有与有机碳降解、碳固定、氮、磷、硫循环和金属抗性相关的1961个基因探针。在M—Y和M-60样地中分别检测到39和62个具有较强杂交信号（SNR≥2）的功能基因,其基因多样性水平指数分别为3．59和4．04,杂交信号强度总值分别为480280和630560。M—Y和M-60样地中分别检测到32个和37个有机碳降解基因,占总基因的82％和60％,这些基因分属于22个不同的基因类群,分别参与木质素、木聚糖、几丁质等有机碳的降解过程。有机碳降解基因在两个样地中存在较大的多样性和丰度差异。这些结果说明了大多数的土壤微生物直接参与了土壤有机碳的降解,同时,林型不同显著影响了土壤微生物群落结构和有机碳降解微生物的多样性。     

Uptake by rice seedlings and in-plant degradation of atrazine as influenced by the oxidative stress induced by added arsenic or phosphate deficiency

The uptake and degradation of atrazine (ATR) by rice seedlings (Oryza sativa L.) was investigated with and without arsenate and phosphate nutrient in the cultured solution over a period of 48 h. The hydrogen peroxide (H₂O₂) contents in plants under different treatments were measured to evaluate the oxidative stress of the plant cell and its influence on the plant uptake and degradation of ATR. Results indicated that the ATR levels and main degradation products, deethylatrazine (DEA) and deisopropylatrazine (DIA), in plants varied significantly in different treatments. Added arsenate in solution increased the level of DEA and the ratios of DEA to the total (ATR, DEA, and DIA) in roots, while it either increased or decreased the H₂O₂ content in roots. Added arsenate increased the ratios of degradation products to the total in shoots, which corresponded to the 110%–285% increase of the H₂O₂ content. In phosphate-deficient systems, the H₂O₂ contents in shoots increased significantly, especially when exposed to a low level of ATR while the ratios of DIA and DEA to the total in shoots increased. The oxidative stress in rice seedlings induced by arsenic coexisting with ATR and by phosphate deficiency affected the plant uptake and degradation of ATR.     

Subcellular aspects of the protective effect of spermine against atrazine in pea plants

The action of the exogenously applied tetraamine spermine in reversing the effect of atrazine stress on intact pea plants (Pisum sativum L., cv. Koray) was investigated at the ultrastructural level. The results indicate that atrazine increases cell senescence by lipid peroxidation and loss of unsaturated fatty acids from thylakoid membranes of pea plant chloroplasts. Spermine acts as a polyfunctional effector. It stabilises the molecular composition of the membranes by preventing lipid peroxidation and as a consequence protects the conformation of the thylakoid system and structural integrity of the chloroplasts. Spermine treatment also contributes to the process of neutralisation of the free radicals by peroxisomes.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

高寒草甸退化对祁连山土壤微生物生物量和氮矿化速率的影响

为深入了解高寒草甸退化对草原生态系统中土壤微生物碳氮量、土壤氮矿化及土壤微生物相关酶的变化特征,以祁连山东缘4个不同退化程度(未退化、轻度退化、中度退化和极度退化)的高寒草甸为研究对象,采集了深度为0—10 cm的土壤样品,并对不同退化程度高寒草甸中植物因子、土壤理化性质、土壤氨化速率、土壤硝化速率、土壤净氮矿化速率以及转化氮素的相关酶和微生物进行了相关研究。结果表明:(1)随退化程度的加剧,高寒草甸土壤中氨化速率和净氮矿化速率逐渐降低,硝化速率逐渐升高;(2)高寒草甸的退化降低了有关氮素转化相关酶,如土壤蛋白酶、脲酶、亮氨酸氨基肽酶的活性,而β-乙酰葡糖胺糖苷酶的活性呈先下降后上升趋势,且在极度退化草地活性最高;(3)随退化程度的加剧,高寒草甸土壤中微生物生物量碳和氮的含量逐渐降低,同时土壤基础呼吸、土壤微生物熵和代谢熵的指数也呈下降趋势。RDA分析表明,高寒草甸中氨化速率和净氮矿化速率与微生物生物量碳、微生物生物量氮、土壤基础呼吸、植物高度、植被盖度、地上生物量、蛋白酶、脲酶以及亮氨酸氨基肽酶呈显著正相关,而硝化速率则表现为负相关性。因此,高寒草甸退化对土壤微生物特性以及氮素转化和...     

Production of aromatic compounds during methanogenic degradation of straw in rice field soil

Production of CH(4) in anoxic rice field soil is stimulated by the addition of rice straw. Previous experiments showed that acetate and propionate are the most important intermediates of the carbon flow to CH(4), and accumulate if CH(4) production is inhibited by 2-bromoethanesulfonate (BES). However, some unidentified compounds were found to accumulate in addition. We now identified them as benzoate, phenylpropionate, and phenylacetate by comparison of the retention times in HPLC chromatograms with authentic standards and by mass spectrometry. These aromatic compounds accumulated only to concentrations <100 microM, especially in soil amended with rice straw (stem, sheath or blade straw). Phenylpropionate and benzoate were the most abundant aromatic intermediates contributing up to 4% to total CH(4) production. Phenylacetate, on the other hand, contributed very little (<0.3%). Gibbs free energies (DeltaG) were calculated for different anaerobic degradation pathways of the aromatic compounds at the actual incubation conditions. Conversion of benzoate to acetate, CO(2) and H(2) was strongly exergonic (DeltaG = -86 kJ mol(-1)) under methanogenic conditions, but became less exergonic (DeltaG = -30 kJ mol(-1)) when CH(4) production was inhibited. The primary oxidation of phenylpropionate was only exergonic for alpha-oxidation (i.e. phenylacetate as product) but not for beta-oxidation (i.e. benzoate as product). However, the DeltaG values for the complete degradation of phenylpropionate to acetate, CO(2) and H(2) were similar for both pathways and were also similar to those of benzoate degradation. Collectively, the results suggest that aromatic compounds are minor intermediates of anaerobic degradation of organic matter in rice field soil, and are syntrophically degraded by coupling to methanogenesis.     

Tree species traits cause divergence in soil acidification during four decades of postagricultural forest development

A change in land use from agriculture to forest generally increases soil acidity. However, it remains unclear to what extent plant traits can enhance or mitigate soil acidification caused by atmospheric deposition. Soil acidification is detrimental for the survival of many species. An in‐depth understanding of tree species‐specific effects on soil acidification is therefore crucial, particularly in view of the predicted global increases in acidifying nitrogen (N) deposition. Here, we report soil acidification rates in a chronosequence of broadleaved deciduous forests planted on former arable land in Belgium. This region receives one of the highest loads of potentially acidifying atmospheric deposition in Europe, which allowed us to study a ‘worst case scenario’. We show that less than four decades of forest development caused significant soil acidification. Atmospheric deposition undoubtedly and unequivocally drives postagricultural forests towards more acidic conditions, but the rate of soil acidification is also determined by the tree species‐specific leaf litter quality and litter decomposition rates. We propose that the intrinsic differences in leaf litter quality among tree species create fundamentally different nutrient cycles within the ecosystem, both directly through the chemical composition of the litter and indirectly through its effects on the size and composition of earthworm communities. Poor leaf litter quality contributes to the absence of a burrowing earthworm community, which retards leaf litter decomposition and, consequently, results in forest‐floor build‐up and soil acidification. Also nutrient uptake and N₂ fixation are causing soil acidification, but were found to be less important. Our results highlight the fact that tree species‐specific traits significantly influence the magnitude of human pollution‐induced soil acidification.     

应用土壤质量退化指数计算松嫩盐碱草地土壤营养位

土壤质量退化指数是一种定量评价土壤质量的方法,以某一土壤类型为基准,计算其他土壤类型与基准土壤类型之间各土壤属性的差异,进而反应土壤质量退化或改善的程度.采用1999年的数据应用该方法评价土壤营养状况,计算土壤营养位,分析松嫩盐碱草地植被种群分布格局与土壤营养的关系,从而探讨应用土壤质量退化指数计算土壤营养位的可行性.应用土壤质量退化指数评价土壤营养位得到的结果与主成分分析法的基本一致,并且方法更为简便,更利于土壤营养位分析在生产实践中应用.     

若尔盖湿地高寒草甸退化过程中土壤有机碳含量变化及成因分析

土壤碳输入与输出之间的收支差决定土壤有机碳(SOC)含量。若尔盖湿地高寒草甸退化过程中, 土壤碳输入和输出哪个过程对SOC含量的影响占主导作用还不明确。该研究用空间序列代替时间序列的方法研究了若尔盖湿地高寒草甸不同退化阶段(高寒草甸(AM)、轻度退化高寒草甸(SD)和重度退化高寒草甸(HD)) SOC含量变化及原因。首先, 通过测定高寒草甸退化阶段上主要的土壤理化性状、微生物生物量、植物生物量和功能群组成的变化, 分析了退化阶段上土壤碳输入量的变化及原因; 其次, 结合室内土壤碳矿化培养实验结果和研究区的月平均气温以及土壤呼吸温度敏感性(Q₁₀)估算了该区域土壤碳输出, 并分析了其变化原因; 最后, 分析了造成SOC含量变化的主要原因和过程。结果表明: 在退化梯度上, 土壤含水量(SWC)、SOC和全氮(TN)含量、微生物生物量碳氮含量降低; 植物群落组成逐渐从莎草科、禾本科占优势过渡到杂类草占优势, 且植物生物量降低; SOC矿化量降低; 有机碳潜在积累量降低(与AM阶段相比, SD和HD阶段有机碳潜在输入量、输出量和积累量分别降低了16%、18%、15%和59%、63%、41%)。SWC降低引起土壤容重、SOC含量、TN含量、全磷含量、C:N的改变, 进而导致植物功能群分布模式和土壤微生物的变化, 最终引起SOC输入和输出量的降低。SWC降低导致的植物碳潜在输入量的降低是若尔盖湿地高寒草甸退化过程中SOC含量下降的主要原因。