一株高效氯嘧磺隆降解菌的分离鉴定及其降解机理初探

消除农业生产中除草剂残留是现代农业绿色发展的关键,为发掘更多磺酰脲类除草剂高效降解微生物资源,系统阐述氯嘧磺隆的微生物降解途径。以氯嘧磺隆为唯一氮源,从某磺酰脲类除草剂生产厂废水处理活性污泥中分离出的高效氯嘧磺隆降解菌LAM2021。生理生化特性和16S rRNA基因序列比对分析结果表明,该菌株属小坂菌属(Kosakonia sp.)。单因素试验结果经RSM优化后,菌株LAM2021在无机盐培养基中对50 mg/L氯嘧磺隆的最佳降解条件为:接种量5%、培养温度30℃、pH 6.0,11 h后降解率可达94.6%。利用高效液相色谱(HPLC)对接菌后降解体系内代谢产生的酸类物质进行分离与鉴定,结果表明产物主要为柠檬酸。推测菌株LAM2021受高浓度氯嘧磺隆胁迫,利用葡萄糖产生柠檬酸,通过降低环境pH使氯嘧磺隆发生水解,从而解除其胁迫。从菌株全基因组信息中发现有参与氯嘧磺隆降解的酯酶编码基因。采用液相色谱-质谱联用(LC-MS)对菌株LAM2021与氯嘧磺隆混合培养后的代谢产物进行检测与鉴定,共有5种主要物质被检测到。根据氯嘧磺隆的化学结构式和中间代谢产物特征,推测菌株LAM2021降解氯嘧磺隆的代谢途径为:氯嘧磺隆中的脲桥先水解断裂成2-氨基-4-氯-6-甲基嘧啶和邻甲酸乙酯苯磺酰胺,随后酯键被脱酯断裂成邻甲酸乙酯苯磺酰胺,最后环化为N-醛基糖精。     

土壤中结合残留态甲磺隆的微生物降解研究

主要进行了优选菌株青霉 (Penicilliumsp .)对土壤中结合残留态甲磺隆的降解研究 ,结果表明 ,优选菌株的引入对土壤中可提态甲磺隆的影响不大 ,但对结合残留态甲磺隆的降解和矿化有较大影响。在结合残留态甲磺隆中 ,优选菌株青霉的引入有利于松结态甲磺隆尤其是松结态富里酸甲磺隆的降解。     

4株苯磺隆降解菌的分离鉴定及其生长特性

从长期受农药苯磺隆污染的土壤中通过采用富集培养分离技术得到4株以苯磺隆为唯一碳源生长的细菌,分别将其命名为B1、B2、B3和B4。通过观察这4种菌株的形态学特征,研究其生理生化特性以及分析其16S rDNA序列,初步鉴定菌株B1为铜绿假单胞菌(Pseudomonas aeruginosa),B2为戴尔福特菌(Delftia sp.),B3为微杆菌(Microbacterium sp.),B4为产碱杆菌(Alcaligenes sp.)。并通过研究温度、初始pH值、接种量、苯磺隆初始浓度、培养基体积、氮源、碳源、Mg2+浓度等因素对4种菌株生长情况的影响,确定了菌株的最佳生长条件。结果显示,B1菌株的最适温度为35℃,其他3株菌株均为30℃。菌株B3最适pH为8.0,其余3株菌株均为pH7.0。B1和B3菌株最适接种量为15%,B2和B4最适接种量为10%。菌株B3最适苯磺隆初始浓度为100mg·L-1,其余菌株最适苯磺隆初始浓度均为200mg·L-1。4株菌株最适培养基体积均为75mL,最适氮源均为硝酸铵,最适碳源均为葡萄糖。B2菌株最适Mg2+浓度为100mg·L-1,其余3株菌株均为200mg·L-1。B1和B4菌株最适NaCl浓度为20g·L-1,B2菌株NaCl浓度为5~30g·L-1,B3菌株最适NaCl浓度为50g·L-1。该结果为利用微生物对农药苯磺隆污染的土壤进行原位生物修复提供理论依据。     

氯嘧磺隆降解菌筛选及基于16S rDNA序列的系统学分析

氯嘧磺隆是长残留除草剂,污染土壤后影响土壤肥力和后茬作物生长。筛选氯嘧磺隆降解菌21株,纯培养条件下,7 d对初始浓度50 mg/L氯嘧磺隆的降解达到0.75%~80.77%。对筛选到的氯嘧磺隆降解菌进行了16S rDNA基因扩增、序列测定和系统学分析,结果显示,所选菌株在系统发育地位上分别属于肠杆菌、短杆菌、柠檬酸杆菌、志贺氏菌、寡养单胞菌、无色杆菌、假单胞菌等7个属。     

除草剂降解真菌对氯嘧磺隆的降解作用

利用自行筛选和保存的3种高效降解真菌菌株,对氯嘧磺隆除草剂降解作用的影响进行了系统的研究,采用高效液相色谱法测定其降解率。真菌黑曲霉、黄曲霉、F8酿酒酵母均可以很好的降解氯嘧磺隆,其中黑曲霉的降解率最高,为96.52%;其次是黄曲霉为88.21%。混合菌株对氯嘧磺隆的降解效率均高于其单一菌株,真菌黑曲霉与黄曲霉混合菌株降解率最高,为98.74%;其次是真菌黑曲霉与F8混合菌株为98.22%。在培养基中随着氯嘧磺隆降解率的增加,反应液的pH逐渐降低。     

降解菌S113对甲磺隆污染土壤生物修复作用的研究

在室内条件下,研究了降解菌S113(Methylopila sp.)对甲磺隆污染土壤的修复作用。S113能够以甲磺隆为唯一碳源生长,72h对50mgL-1甲磺隆的降解率达98.38%。投加降解菌S113可显著提高土壤中甲磺隆的降解速率。当甲磺隆浓度为10mgkg-1干土,S113接种量为108个g-1土时,第30天土壤中甲磺隆降解率为76.9%,对照土壤中甲磺隆降解率仅为11.9%。S113降解甲磺隆的速率和接种量呈正相关,当接种量减少为105个g-1干土时,降解菌对甲磺隆的降解作用微弱。在土壤中甲磺隆浓度较低的条件下,S113的降解效果显著,而当土壤中甲磺隆浓度达到50mgkg-1时,甲磺隆降解率仅为39.6%。S113降解土壤中甲磺隆的最适温度为30℃,第30天的降解率可达75.9%。当温度为25℃、20℃时,第30天甲磺隆降解率仅为53.5%和23.9%。S113菌剂灌根,能不同程度地缓解土壤中浓度为40、80μgkg-1的甲磺隆对玉米生长的抑制作用,但当甲磺隆浓度增加到120μgkg-1时,接种S113对药害解除作用不显著。结果表明,人工接种降解菌S113,能有效去除土壤中甲磺隆残留。     

生物肥对氯嘧磺隆残留的降解及对水稻生长的影响

为筛选具有降解氯嘧磺隆残留和作物增产效果的优质生物肥, 采用田间小区模拟试验, 研究了不同浓度氯嘧磺隆施用下, 3种生物肥对氯嘧磺隆残留的降解及对水稻生长的影响。结果表明, 供试的农大生物菌剂、世绿环保生物肥和农大生物肥对施用量分别为3 μg·kg^-1和6 μg·kg^-1的氯嘧磺隆残留均有降解作用, 对施用量为6 μg·kg^-1的氯嘧磺隆残留的降解率分别为61.5%、58.3%和62.7%, 比自然降解率分别高17.8%、14.6%和19.0%。土壤中氯嘧磺隆施用量为0.03 μg·kg^-1时即对水稻株高、穗长、结实率和产量性状产生明显影响, 氯嘧磺隆施用量越大药害影响越显著。3种供试生物肥均可解除0.03 μg·kg^-1氯嘧磺隆的残留对水稻产量的影响, 但随氯嘧磺隆施用量的增加, 供试生物肥虽可减轻药害影响, 但不能完全解除残留药害。农大生物菌剂和农大生物肥解除氯嘧磺隆残留药害能力高于世绿环保生物肥。田间氯嘧磺隆的施用量小于0.03 μg·kg^-1时, 施用供试的3种生物肥均可保证水稻生产安全。     

LC-MS/MS法研究甲磺隆和氯磺隆在不同条件土壤中的残留行为

在建立液相色谱三重串联四极杆质谱（LC-MS/MS）检测技术的基础上,采用PVC材料的圆柱形盆钵栽培方法,研究了不同土壤环境条件下甲磺隆和氯磺隆的残留特性。结果表明,甲磺隆和氯磺隆在水稻根际和非根际土壤中的残留量均呈不断下降趋势。处理后15d,根际和非根际土壤中两种农药残留量分别下降了44.7%和41.5%（甲磺隆）及38.7%和40.1%（氯磺隆）,根际和非根际的残留差异不显著（P〉0.05）;处理后31d,残留量分别下降了77.7%和64.8%（甲磺隆）及62.7%和50.1%（氯磺隆）,差异达极显著水平（P〈0.01）;处理后63d,残留量分别下降了96.4%和85.1%（甲磺隆）及90.0%和79.4%（氯磺隆）,残留差异达极显著水平（P〈0.01）。甲磺隆的降解趋势和氯磺隆基本一致,但下降幅度比氯磺隆大。二者在水稻根际和非根际土壤中的残留量均符合一级动力学方程式C=C0e-λ（tC代表浓度;C0代表初始浓度;t时间）,决定系数范围在0.9342～0.9957之间。在种植水稻的条件下,下层土的农药残留量呈先上升后下降的趋势,处理后数日内达最高点,之后不断下降,122d后低于检测限。水稻下层土残留的从无到有说明农药在土壤中的淋溶可能是水田环境农药残留降解的原因之一。干旱土壤条件下,土壤的淋溶作用不明显。说明水旱轮作有利于农药残留在土壤环境中的降解。     

等高草篱对坡耕地土壤苯磺隆残留迁移的影响

在北京市昌平区冬小麦田采用模拟降雨的方法,对不同坡位（坡中和坡底）的土壤进行分层（0~5、5~10、10~15 cm）取样测定,研究了草木樨和狼尾草两种等高草篱在不同雨强（15、20 mm·h-1）和坡度（15%和20%）条件下对土壤苯磺隆残留迁移及分布特征的影响。结果表明,前期干旱条件下,降雨后土壤苯磺隆残留纵向迁移特征明显,无明显的水平移动,亦未产生残留径流输出。草篱能够促进土壤中苯磺隆残留向下迁移,导致草篱带内和无草篱区域土壤中的残留纵向分布特征有较大差异：草篱带内3个土层残留水平相近,而对应坡位无草篱区域残留主要滞留于土壤表层,并随土层加深而显著降低。雨强、草篱、坡度均对土壤苯磺隆残留量有显著影响,草篱的贡献超过雨强和坡度,成为影响坡耕地农药残留纵向迁移行为的首要因素。     

戊菌隆在棉花和土壤中残留规律研究

采用田间试验方法，研究了戊菌隆农药在棉花和土壤中的残留规律。结果表明，每100kg棉种用戊菌隆75-112.5g(有效成分）拌种，防治棉花苗期病害，戊菌隆在土壤中的残留降解较慢，半衰期7.8-10.1d，施药后35d，降解达90%。收获期土壤、棉叶、棉籽中均未检出戊菌隆。     

Phytotoxicity of cobalt,vanadium, titanium,silver, and chromium

Abstract

The phytotoxicity of five nonessential elements (Co, V, Ti, Ag, Cr) to higher plants was studied in solution culture experiments with bush beans (Phaseolus vulgaris L. C.V. Improved Tendergreen). All, but in varying degrees, tended to concentrate in roots with a decreasing gradient to stems and leaves. Cobalt was one of the more mobile of the five trace metals. Its toxicity was expressed as severe chlorosis; 43 (with 10^‐5 M) and 142 (with 10^‐4 M) μg Co/g dry weight in leaves resulted in severe chlorosis. Vanadium as 10^‐4 M vanadate resulted in smaller plants but not in chlorosis. Leaf, stem, and root V, respectively, were 13, 8, and 881 μg/g dry weight. Titanium was somewhat mobile with considerable yield decrease at 10^‐4 M; leaf, stem, and root Ti concentrations, respectively, were 202, 48, and 2420 μg/g. Symptoms were chlorosis, necrotic spots on leaves, and stunting. Silver was very lethal at 10^‐4 M AgNO₃; at 10^‐5 M yields were greatly decreased, but plants were grown without symptoms. Leaf, stem, and root concentrations of Ag for this treatment, respectively, were 5.8, 5.1, and 1760 μg/g dry weight. Plants grown with 10^‐5 N Cr₂O₇ were decreased in yield by about 25% with or without EDTA (ethylenediamine tetraacetic acid) while the same level of Cr₂(SO₄)₃ was essentially without effect. For the two salts, the leaf, stem, root concentrations for Cr, respectively were 2.2 and 1.3, 0.7 and 0. 7, and 140 and 104 μg/g. Most of the trace metals studied here had interactions in the uptake and/or distribution of other elements.     

Variation of N,P, K,Ca, Mg,Mn, Zn,and al in slash pine foliage

Abstract

The seasonal patterns of foliage nutrient concentrations and contents were monitored for two growing seasons in an 11‐year—old Pinus el1iottii stand. In the first growing season after needle initiation, N, P, K, Mg, and Zn concentrations decreased, but this was followed by an increase in the fall and winter months. Another drop in concentration of all elements, except P, occurred in the second growing season. Decreases in total contents indicated that this drop was a result of translocation to other tissues. In contrast to the mobile elements, the concentration and fascicle contents of Ca, Mn, and Al increased with aging of the needles.

Between‐tree variability was least for N, P, and Zn and the N, K, Mg, Mn, and Zn in the current foliage had consistently lower variation than that in the 1‐year‐old foliage. Between‐tree variation for K was lower in the winter than the spring.

For pine foliage, recommended sampling period for N, P, Mg, and Zn is mid to late summer and for the other elements it is late fall to late winter.

There are several sources of variation that influence the level of nutrients in tree foliage. The most important of these, apart from the tree nutrient status, are seasonal fluctuations, variation between trees, and age of needles . Smaller sources of variation are associated with position of the needles within the crown, diurnal changes, year to year variation, and analytical errors^1,2. These variables must be studied in order to develop suitable sampling techniques and in Pinus this has been undertaken for P. banksiana ¹, P. taeda ³, P. strobus ⁴, P. resinosa ⁴, P. sylvestris ⁵, and P. radiata ^6,7. However, foliage sampling has not been studied in detail for slash pine (Pinus elliottii Englem var. elliottii) and earlier studies with other pines have been largely confined to temperate or cool climates.

This study reports the variation in elemental concentrations with season, age of foliage, and between slash pine trees growing in a subtropical climate in Florida.     

Bioavailability of As,Cd, Co,Cr, Cu,Hg, Mo,Ni, Pb,Se, and Zn from Biosolids Amended Compost

Appropriate compost standards are being considered in Canada. Five aspects of compost safety and quality are being evaluated; probably the most controversial aspect is the standards for metals in compost. In order to assist in the development of appropriate standards, the authors began an extensive research project in October, 1993 to determine the bioavailability of metals from compost and compost-metal mixtures. Swiss chard was grown in compost-amended soils or compost in a growth room using five treatments of increasing percentages of compost in the media (0, 25 percent, 50 percent, 75 percent, 100 percent compost (v/v)). A Truro loamy sand and a race-track manure-biosolids compost (RTM-biosolids) supplemented with a high metal biosolids were used in a completely randomized design with five replicates. Dry matter yield, metal content in plant tissue, and total metal uptake were evaluated as well as the total and DTPA-extractable metal content in the compost-soil mixes. The results of this and five other experiments conducted by the authors will help determine whether the suggested limits for As, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Se and Zn in composts are appropriate.     

Cooking,Roasting, and Fermentation of Chickpeas,Lentils, Peas,and Soybeans for Fortification of Leavened Bread

The effects of cooking, roasting, and fermentation on the composition and protein properties of grain legumes and the characteristics of dough and bread incorporated with legume flours were determined to identify an appropriate pretreatment. Oligosaccharide content of legumes was reduced by 76.2–96.9% by fermentation, 44.0–64.0% by roasting, and 28.4–70.1% by cooking. Cooking and roasting decreased protein solubility but improved in vitro protein digestibility. Mixograph absorption of wheat and legume flour blends increased from 50–52% for raw legumes to 68–76, 62–64, and 74–80% for cooked, roasted, and fermented ones, respectively. Bread dough with cooked or roasted legume flour was less sticky than that with raw or fermented legume flour. Loaf volume of bread baked from wheat and raw or roasted legume flour blends with or without gluten addition was consistently highest for chickpeas, less for peas and lentils, and lowest for soybeans. Roasted legume flour exhibited more appealing aroma and greater loaf volume of bread than cooked legume flour, and it appears to be the most appropriate preprocessing method for incorporation into bread.     

Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A.

A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.     

Distribution of Zinc,Manganese, Copper,Cobalt, and Nickel in Andosols Profiles

The distribution of zinc, manganese, copper, cobalt, and nickel in Andosols was investigated. Sixty nine soil samples were collected from different horizons of an Andosols profile in Miyakonojo Basin in south Kyushu, Japan, The total contents of heavy metals were determined by digestion and four extraction solutions, 1 M NH₄Ac (ammonium acetate) pH 4.5, 0.1 M HCl, 0.01 M EDTA (ethylenediaminetetraacetic acid) pH 6.5, and 0.005 M DTPA (diethylenetri-aminepentaacetic acid) pH 7.3 were used to determine the contents of available Zn, Mn, Cu, Co, and Ni in Andosols in relation to the organic carbon content. The results of the extraction analysis showed that by the use of 0.1 M H Cl high value of extracted heavy metals in the upper layers of the humus horizons were obtained while EDTA extraction yielded a large amount of the above mentioned metals in the high humus horizons. The extractable heavy metals contents were high and these metals closely related to the organic carbon content mostly in the humus horizons in the profile. Where, biocycling process may play an important role in the concentration of heavy metals. Based on the study, it was found that the total content of Zn increased towards the C horizons or pumice layers in the soil profile. Such a trend was also found in the case of the Mn content. While the Cu content in the humus horizons was much higher in the upper part of each humus horizon. According to this study the distribution of heavy metals, Cu (organic matter complexes) in the Andosols profile was more stable than that of Zn (organic matter complexes) in soils. It was shown that Zn in the surface humus horizon was enriched but that some amount was leached under buried conditions. The same phenomenon was also observed in the distribution of Mn in the profile. The movement of Co and Ni in the soil profile was limited, as evidenced by the sharp reduction in the concentrations of these two metals in buried soils.

Hence, it is concluded that the distribution of Zn, Mn, Cu, Co, and Ni was considerably higher in the humus horizons of the Andosols profiles.     

Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, brussels sprouts, kale, and cabbage

The importance of dietary sulforaphane in helping maintain good health continues to gain support within the health-care community and awareness among U.S. consumers. In addition to the traditional avenue for obtaining sulforaphane, namely, the consumption of appropriate cruciferous vegetables, other consumer products containing added glucoraphanin, the natural precursor to sulforaphane, are now appearing in the United States. Crucifer seeds are a likely source for obtaining glucoraphanin, owing to a higher concentration of glucoraphanin and the relative ease of processing seeds as compared to vegetative parts. Seeds of several commonly consumed crucifers were analyzed not only for glucoraphanin but also for components that might have negative health implications, such as certain indole-containing glucosinolates and erucic acid-containing lipids. Glucoraphanin, 4-hydroxyglucobrassicin, other glucosinolates, and lipid erucic acid were quantified in seeds of 33 commercially available cultivars of broccoli, 4 cultivars each of kohlrabi, radish, cauliflower, Brussels sprouts, kale, and cabbage, and 2 cultivars of raab.     

Hydrophilic chromatographic determination of carnosine, anserine, balenine, creatine, and creatinine

A new HPLC procedure based on hydrophilic interaction chromatography (HILIC) has been developed for the simultaneous determination of carnosine, anserine, balenine, creatine, and creatinine in meat. This is the first time that HILIC has been directly applied to the study of meat components, having the advantage of not requiring complex cleanup and/or sample derivatization procedures. The chromatographic separation has been developed using a silica column (4.6 x 150 mm, 3 microm), and the proposed methodology is simple, reliable, and fast (<13 min per sample). The method has been validated in terms of linearity, repeatability, reproducibility, and recovery and represents an interesting alternative to methods currently in use for determining the mentioned compounds and other polar substances. The detection limits are 5.64, 8.23, 3.66, 3.99, and 0.06 microg/mL for carnosine, anserine, balenine, creatine, and creatinine, respectively.     

Estimation of Reference Values for Cadmium,Cobalt, Chromium,Copper, Nickel,Lead, and Zinc in Brazilian Soils

Abstract

The objectives of this study were 1) to recommend reference values (RVs) and tolerance limits (TLs) for representative Brazilian soils and 2) to propose a model to calculate natural contents of cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) in a soil from the silt, clay, manganese (Mn), iron (Fe), and cation exchange capacity (CEC) values. A set of 256 soil samples was classified by similarity in seven groups, and the concentrations corresponding to the upper quarter of data collected were then calculated. These concentrations are proposed as RVs for Brazilian soils. Additionally, TLs were obtained for each group from the antilog expression (m+2s), where m=mean value and s=standard deviation of data transformed in log₁₀. The classification functions of discriminant analysis proved to be suitable to allocate new samples in the established groups. Thus, it is possible to evaluate soils under anthropic activity and, by comparison with reference values, to be aware of pollution risks in a given area.     

Volatilization of trifluralin,atrazine, metolachlor,chlorpyrifos, alpha-endosulfan,and beta-endosulfan from freshly tilled soil

The volatile and soil loss profiles of six agricultural pesticides were measured for 20 days following treatment to freshly tilled soil at the Beltsville Agricultural Research Center. The volatile fluxes were determined using the Theoretical Profile Shape (TPS) method. Polyurethane foam plugs were used to collect the gas-phase levels of the pesticides at the TPS-defined critical height above a treated field. Surface-soil (0-8 cm) samples were collected on each day of air sampling. The order of the volatile flux losses was trifluralin > alpha-endosulfan > chlorpyrifos > metolachlor > atrazine > beta-endosulfan. The magnitude of the losses ranged from 14.1% of nominal applied amounts of trifluralin to 2.5% of beta-endosulfan. The daily loss profiles were typical of those observed by others for volatile flux of pesticides from moist soil. Even though heavy rains occurred from the first to third day after treatment, the majority of the losses took place within 4 days of treatment, that is, 59% of the total applied atrazine and metolachlor and >78% of the other pesticides. Soil losses generally followed pseudo-first-order kinetics; however, leaching due to heavy rainfall caused significant errors in these results. The portion of soil losses that were accounted for by the volatile fluxes was ordered as follows: alpha-endosulfan, 34.5%; trifluralin, 26.5%; chlorpyrifos, 23.3%; beta-endosulfan, 14.5%; metolachlor, 12.4%; and atrazine, 7.5%.