Pollution induced community tolerance (PICT) and analysis of 16S rRNA genes to evaluate the long-term effects of herbicides on methanotrophic communities in soil

There is an increasing interest in agricultural systems in which the use of herbicides is forbidden. Therefore, soils treated with herbicides atrazine and metolachlor for the last 20 years were compared with soil samples from the same field that had never been treated (control soil). We determined the pollution induced community tolerance (PICT) by evaluating the methane oxidation capacity of soil samples after adding increasing amounts of a methane oxidation inhibitor, 2,4‐dichlorophenoxyacetic acid (2,4‐D). Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes assessed whether the soil methanotrophic community differed between the two treatments. Addition of 60 µg 2,4‐D per g soil clearly inhibited methane oxidation in both soils but increased the time needed to oxidize 5% methane in the headspace by 250% for the control soil compared with 175% for the herbicide‐treated soil. This indicates that the soil with a long‐term herbicide history had a greater tolerance to the methane oxidation inhibitor than did the control soil. The DGGE of 16S rRNA genes amplified directly from soil community DNA could also distinguish the two treatments. The banding patterns of the Type I methanotrophs contained fewer bands in the herbicide‐treated soil. It seems that both the PICT approach and DGGE analysis are effective assays to distinguish a long‐term herbicide‐treated soil from an untreated soil.     

A phylogenetic microarray targeting 16S rRNA genes from the bacterial division Acidobacteria reveals a lineage-specific distribution in a soil clay fraction

We designed an oligonucleotide microarray using probe sequences based upon a phylogenetic analysis of 16S rRNA genes recovered from members of the bacterial division Acidobacteria. A total of 42,194 oligonucleotide probes targeting members of the Acidobacteria division at multiple phylogenetic levels were included on a high-density microarray. Positive control hybridizations revealed a linear relationship between hybridization signal and template concentration, and a substantial decrease in non-specific hybridization was achieved through the addition of 2.5 M betaine to the hybridization buffer. A mean hybridization signal value was calculated for each Acidobacteria lineage, with the resultant lineage-specific hybridization data revealing strong predictive value for the positive control hybridizations. The Acidobacteria phylochip was then used to evaluate Acidobacteria rRNA genes from a Wisconsin soil and within a soil clay fraction. The Acidobacteria hybridization profile revealed the predominance of Acidobacteria subdivisions four and six, and also suggested a decrease in the abundance of subdivision six relative to subdivision four in the soil clay fraction. The change in relative abundance of these subdivisions in a soil clay fraction was supported by data from quantitative PCR. These results support the utility of a phylogenetic microarray in revealing changes in microbial population-level distributions in a complex soil microbial assemblage.     

Characterization of redox-related soil microbial communities along a river floodplain continuum by fatty acid methyl ester (FAME) and 16S rRNA genes

Redox states affect substrate availability and energy transformation, and, thus, play a crucial role in regulating soil microbial abundance, diversity, and community structure. We evaluated microbial communities in soils under oxic, intermittent, and anoxic conditions along a river floodplain continuum using fatty acid methyl ester (FAME) and 16S rRNA genes-based terminal-restriction fragment length polymorphism (T-RFLP) bacterial fingerprints. In all the soils tested, microbial communities clustered according to soil redox state by both evaluation techniques. Bacteria were dominant components of soil microbial communities, while mycorrhizal fungi composed about 12% of the microbial community in the oxic soils. Gram-positive bacteria consisted >10% of the community in all soils tested and their abundance increased with increasing soil depth when shifting from oxic to anoxic conditions. In the anoxic soils, Gram-positive bacteria composed about 16% of the total community, suggesting that their growth and maintenance were not as sensitive to oxygen supply as for other microbes. In general, microorganisms were more abundant and diverse, and distributed more evenly in the oxic layers than the anoxic layers. The decrease in abundance with increasing oxygen and substrate limitation, however, was considerably more drastic than the decrease in diversity, suggesting that growth of soil microorganisms is more energy demanding than maintenance. The lower diversity in the anoxic than the oxic soils was attributed primarily to the differences in oxygen availability in these soils.     

Biodegradation of aged residues of atrazine and alachlor in a mix-load site soil

A selected microbial consortium (SMC) capable of degrading two specific herbicides, alachlor (2-chloro-2′,6′-diethyl-N-[methoxymethyl]-acetanilide; AL) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-S-triazine; AT) was isolated from a pesticide-contaminated mix-load site soil. Evaluation of bioaugmentation as a feasible bioremediation strategy for this mix-load site soil (Site 5A) was initiated in standard laboratory biometer flasks utilizing the isolated SMC. The biometer flasks were monitored for CO₂ evolution and pesticide degradation. The total amount of CO₂ evolved from the treated biometer flasks was significantly different from the control flasks. The rate of CO₂ evolution was 2.6 times faster in the treated soil (0.0123 mM CO₂ d⁻¹ vs. 0.0048 mM CO₂ d⁻¹). The total net CO₂ produced in the treated biometer flasks was 0.9481 mM, representing mineralization of approximately 10% of the AT and AL initially present. Forty-eight percent of AT and 70% of AL was degraded in the inoculated biometer flasks. The first-order rate constants were 0.0064 d⁻¹ and 0.1331 d⁻¹ for AT and AL, respectively. The calculated half-life of AT was 108 d while a 50% decrease in AL occurred by Day 5. In just 2 d, 20% of the AT was degraded while only 10% of the AL disappeared. The initial fast degradation rate of AT was followed by a much slower, more gradual degradation rate period that lasted about 35 d. Alternatively, the rate of AL degradation increased after the second day resulting in 60% of the AL being transformed by the end of the first week. Alachlor degradation appeared to be dependent upon AT degradation especially during the first several days of the incubation period. Complete disappearance of the herbicides over the study time was not achieved.     

Bacterial Communities Responsible for the Decomposition of Rice Straw Compost in a Japanese Rice Paddy Field Estimated by DGGE Analysis of Amplified 16S rDNA and 16S rRNA Fragments

To estimate the succession and phylogenetic composition of the bacterial communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analyses targeting 16S rDNA and 16S rRNA, followed by sequencing were conducted in a Japanese paddy field. The DGGE bands of the bacterial communities in the rice straw compost were significantly more numerous in the DNA samples than in the RNA samples. Although the band number of the DNA samples was almost constant throughout the period, RNA samples showed fewer DGGE bands after mid-season drainage than before it. Thus, about 81% of the bacteria present in rice straw compost were considered to be metabolically "active" before mid-season drainage and about 62% after it. The changes in the DGGE patterns of bacterial DNA and RNA before and after mid-season drainage, respectively, were also revealed by cluster analysis and principal component analysis of the DGGE patterns. These results indicated that the bacterial communities of rice straw compost incorporated into flooded paddy fields changed gradually along with the decomposition, except for the period of mid-season drainage, but that they were influenced by mid-season drainage. Members of β-, γ- and δ-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Chlorobia, Verrucomicrobia, Chloroflexi, Spirochaetes, Firmicutes (clostridia) and Actinobacteria were present during the decomposition of rice straw compost. Characteristic "active" bacteria among them were as follows: Clostridium, Acinetobacter (γ-Proteobacteria) and β-Proteobacteria before mid-season drainage, Flavobacterium, Chondromyces , Chlorflexi and δ-Proteobacteria after mid-season drainage, and Spirochaeta and myxobacteria throughout the period.     

Intragenomic variation in the internal transcribed spacer regions between 16S–23S rRNA genes among the three copies of Sinorhizobium fredii strains

The soybean-nodulating Sinorhizobium fredii strain has been reported to possess three copies of rRNA gene operons. In the present study, we investigated the diversity of the 16S–23S rDNA internal transcribed spacer (ITS) regions of S. fredii strains. Based on the sequences of the ITS regions, we divided the sequences of the S.   fredii strains into two groups, type A and type B. A dot-matrix analysis indicated that the region flanked by tRNA-Ile and tRNA-Ala is longer in type A than in type B, whereas type B sequences possess longer regions upstream of tRNA-Ile and downstream of tRNA-Ala than those of the type A sequence. Restriction fragment length polymorphism of polymerase chain reaction product (PCR-RFLP) of the ITS region in the cloned plasmids as templates could reconstruct the PCR-RFLP pattern from the total DNA as a template. The results of Southern hybridization using the insert sequence between tRNA-Ile and tRNA-Ala in type A as a probe indicated differences in the copy numbers of the type A ITS regions among the strains tested. These results indicated that S. fredii strains possess the type A and type B sequences of the ITS regions at ratios of 3:0, 2:1, 1:2 or 0:3. These S. fredii strains may be useful biological materials for the study of intraspecific variations.     

山药根际土壤微生物16S rRNA多样性及影响因素

通过采集山药连作2年的植株根际土壤及根茎,采用高通量测序技术研究根际土壤细菌多样性,分析7个样本中菌群的组成、丰度、α多样性、β多样性、菌群差异性。采用典型相关分析(Canonical Correlation Analysis,CCA)分析优势菌群与土壤化学因子、根茎糖类物质的关系,探讨根际土壤中细菌群落与土壤化学指标、根茎糖类物质的相关性。结果表明:山药根际土壤细菌优势群落为变形菌、绿弯菌、酸杆菌、放线菌;土壤pH是影响细菌群落结构及分布的重要因子;酸杆菌和硝化螺旋菌与土壤中蔗糖酶、脲酶活性及总有机碳、碱解氮呈正相关,奇古菌和绿弯菌与土壤总有机碳、碱解氮呈正相关,土壤中碳、氮养分又与山药根茎中可溶性糖、粗多糖及淀粉呈正相关关系,说明这些微生态环境因子可能对山药根茎糖类物质积累有促进作用。     

巢式PCR检测无乳链球菌16SrRNA方法的建立及应用

无乳链球菌（Streptococcus agalactiae）引起的奶牛乳房炎主要表现为隐性型,需要通过实验室检查才能确诊。研究建立了一种特异、敏感和快速的巢式PCR方法用于检测奶样中的无乳链球菌。利用链球菌属（Sgeptococcus）16S rRNA基因的保守区设计通用引物作为链球菌属的阳性控制,在保守区内的可变区设计无乳链球菌特异的引物。研究结果表明,通过奶样中无乳链球菌的增菌培养,简便、快速提取DNA和特异性PCR反应,可以检测到奶样中1CFU／mL的无乳链球菌,可以用作牛群中无乳链球菌感染的早期流行病学调查。     

PCR结合表型鉴定对超高压处理后的冷藏带鱼细菌菌相分析

为了研究超高压处理对带鱼微生物菌群组成的影响,该文通过形态学特征、生理生化鉴定、16S r RNA序列分析鉴定及系统发育树的建立,分别分析290 MPa、6 min超高压处理前后于4℃冷藏12 d内的带鱼菌相变化,最终分离筛选到24株不同特征的纯化菌株。结果显示,带鱼初始菌相中出现的布式假单胞菌(Pseudomonas brenneri)、黄褐假单胞菌(Pseudomonas fulva)、粪嗜冷杆菌(Psychrobacter faecalis)菌株,经超高压处理后的样品中未能筛选到;波罗的海希瓦氏菌(Shewanella baltica)、隆德假单胞菌(Pseudomonas lundensis)、嗜根寡养单胞菌(Stenotrophomonas rhizophila)、表皮葡萄球菌(Staphylococcus epidermidis)、氧化微杆菌(Microbacterium oxydans)等微生物在超高压处理后的贮藏期间数量逐渐减少至消失;另有一些微生物在贮藏期间逐渐恢复生长,如Rhizobium larrymoorei、Microbacterium halimionae、溶酪大球菌(Macrococcus caseolyticus),而奥斯陆莫拉菌(Moraxella osloensis)、藤黄微球菌(Kocuria rhizophila)、产乳酸菌素的肉杆菌(Carnobacterium maltaromaticum)、西宫皮肤球菌(Dermacoccus nishinomiyaensis)等受超高压的影响较小,尤其是产乳酸菌素的肉杆菌(Carnobacterium maltaromaticum)占好氧菌和厌氧菌菌落总数的比例均较高;Leucobacter aerolatus、成团泛菌(Pantoea agglomerans)、结肠炎耶尔森杆菌palearctica亚种(Yersinia enterocolitica subsp.palearctica)、Chryseobacterium vrystaatense、鼠李糖短杆菌(Brachybacterium rhamnosum)在贮藏末期出现。从带鱼冷藏过程中细菌的组成与变化分析可见,超高压处理对革兰氏阴性菌的抑菌效果较好,而革兰氏阳性菌对超高压处理的耐受性较强。在超高压技术的影响下,致腐败能力较强的微生物被抑制,腐败能力稍弱的微生物成为优势菌,这可能是超高压技术能够有效延长带鱼货架期的因素之一。     

The characterization and quantification of methanotrophic bacterial populations in constructed wetland sediments using PCR targeting 16S rRNA gene fragments

Three mesocosms were studied to evaluate the effect of wetland plants on the methanotrophic bacterial populations in the sediments of a full-scale constructed wetland. Cores were collected from two vegetated mesocosms and one unvegetated mesocosm from fall 2002 through summer 2003. Competitive quantitative PCR revealed no significant differences in the quantities of either Type I or Type II methanotrophic bacteria between the vegetated and unvegetated mesocosms. Type I methanotroph-biased nested PCR-DGGE resulted in the detection of 23 different populations related to Methylococcus, Methylomonas, Methylobacter, Methylocaldum, and Methylosarcina spp. Type II methanotroph-biased nested PCR-DGGE resulted in the detection of 5 different populations, more than 90% of which were related to previously uncultivated Type II methanotrophs. While wetland vegetation did not affect the structure of either the Type I or Type II methanotrophic communities, the Type I methanotrophic community structure was observed to vary seasonally. This work suggests that wetland plants neither enhanced nor adversely affected the size or structure of methanotrophic communities in our constructed wetland. Substantial quantities of both Type I and Type II methanotrophic populations were detected in both planted and unplanted mesocosms, suggesting that the constructed wetland had substantial potential for xenobiotic bioremediation whether or not plants were present.     

不同植被对晋陕蒙矿区排土场土壤养分16 a恢复程度的影响

为评价晋陕蒙矿区排土场土地复垦过程中不同植被措施对土壤肥力的恢复作用及程度,在内蒙古准格尔旗黑岱沟煤矿东排土场,分别选取已恢复16 a的7种不同人工植被(沙打旺、长芒草、紫穗槐+长芒草、刺槐林、沙棘林、杨树林、樟子松),并以周边原地貌条件下两种植被(退耕梯田长芒草、自然坡地沙棘林)为对照,分析了不同植被条件下0~10、10~20、20~40 cm土层土壤全氮、有机碳、硝态氮以及铵态氮的含量及其差异。结果表明:1)9种植被配置中除樟子松地和沙打旺草地外,土壤养分含量均随深度增加而递减。2)经过近16 a植被修复后,7种人工植被不同程度地改善了土壤肥力状况,其中刺槐纯林配置的恢复效果最佳,土壤肥力恢复程度接近或超过原地貌条件下植被的50%,沙棘次之。杨树和樟子松两种配置对土壤全氮、有机碳、硝态氮恢复效果相对较差,恢复程度均不超过40%。7种不同植被措施对土壤铵态氮的恢复程度相对较好,分别恢复到自然植被长芒草样地和自然植被沙棘样地的79.50%~93.77%,89.47%~105.70%。土壤全氮、有机碳、硝态氮恢复程度则相对较低,分别恢复到自然植被长芒草样地的24.70%~44.15%、25.09%~44.65%、47.25%~85.33%;分别恢复到自然植被沙棘样地的33.52%~56.85%、32.83%~54.82%、47.18%~84.66%。总之,尽管经过近16 a的复垦,7种人工植被下的土壤养分状况尚未完全恢复到该地区自然水平,但仍可认为该地区进行生态修复时可优先选择以刺槐为主的豆科乔木、以沙棘为主的灌木、以及长芒草等草本植物,并进行适当的套种、间种,以增强复垦效果。     

Investigation of heavy metal concentrations on urban soils,dust and vegetables nearby a former smelter site in Mortagne du Nord,Northern France

Environmental situations near former industrial sites like Mortagne du Nord (Northern France) in which the soil, dust and homegrown vegetables have been contaminated by heavy metals can represent a sanitary risk to the surrounding population. In order to evaluate the contamination level in different exposition media in this site, 26 urban topsoils, 17 dust and 38 vegetable samples were taken near the former zinc smelter site. All the observed levels on urban soil samples (except one) are over regional agricultural reference values for Cd and Pb. 45 % of the vegetable samples are over the European foodstuff limits and one dust sample taken in the school playground outdoor tarmac area exceeds the French limit in dust set up at 1,000 μg m^?2 for Pb. The Cd and Pb levels fluctuate depending on the use of the ground (kitchen garden, lawn, courtyard) and its location. The lack of simple correlations between the distance from the former smelter site and the Pb concentrations on sampled soils let us think that human activities contributed in the local urban contamination. In this context, who is now responsible for the soil contamination and the human exposure?     

A pedological investigation of an archaeological site in Ontario,Canada, I. An examination of the soils in and adjacent to a former village

The Benson site is a former village which was inhabited by Huron Indians in the 1600's. It is located 90 km northeast of Toronto, Canada. The soils of a portion of the site were sampled on a 1-m grid 15 cm below the surface. These soils were then analyzed for magnesium, calcium, potassium, sodium, phosphorus, carbonates, pH and organic carbon. Three off-site soil profiles (Podzols) were sampled and analyzed for comparative purposes.Of the chemical attributes of soil materials studied at the Benson site amounts of magnesium, organic phosphorus, and inorganic phosphorus were sufficiently different to be useful in distinguishing formerly occupied soils from off-site soils. Such information may thus be an aid to archaeologists in determining the presence or absence of sites occupied in the past.     

Methodological development for combined analysis of S and 35S in a Scots pine forest study

Abstract

A study of sulphur movement through a Scots pine forest involved the analysis of S and ³⁵S in rainfall, throughfall and stem flow solutions, and aqueous extracts of needles and soil, together with measurement of total levels in soil and needles. Solution preparation procedures were developed so that the solutions could be used for liquid scintillation counting, ion chromatography and ICP/OES instrumental methods, ie., it was possible to analyse stable and radioactive sulphur fractions on the same sample solutions. Counting efficiency of the beta radiation from ³⁵S in the study solutions ranged from 10 to 80%, with the greatest quenching occurring in soil nitric acid digestion solutions. A single non‐linear mathematical relationship was applied to correct the beta counts for energy quenching in all solutions with minimal bias when compared to the values derived from using separate factors for each solution type.     

Anomalous contents of heavy metals in soils and vegetation of a mine area in S.W. Sardinia,Italy

Samples of soils and vegetation from the mining area of South-West Sardinia (Italy) were analyzed for Pb, Zn, Cd, and Cu content. The area (more than 100 km²) is inhabited by many thousands of people; land utilization includes mainly grapes on some small plains and permanent sheep pasture on the hills. The levels of Pb, Zn, and Cd were found to be exceptionally high in most samples. Lead concentration was up to 71000 μg g^?1 in the soils and 4000 jig g^?1 in vegetation; Cd concentration was found up to 665 μg g^?1 in soils and 26.5 μg g^?1 in vegetation. The heavy metal content of some soil samples was highly variable. Data show that Pb is easily absorbed by plant roots and translocated to foliage. In spite of the high heavy metal level, no signs of toxicity were apparent in vegetation.     

Long-term record and analysis of soil temperatures and soil heat fluxes in a grassland area, The Netherlands

A 27-year soil temperature record at five depths and soil heat flux record at one depth were analyzed for a grassland area in The Netherlands. The annual mean soil temperature of the last 23 years of soil measurements (no data gaps) showed a statistically significant increase of about 1.0 °C, consistent with the observed air temperature increase of about 1.3 °C for the same period. This positive trend correlates well with global brightening of 5.3 W m⁻² per decade. The 10-day mean soil temperature varied smoothly throughout the year with relatively small inter-annual variability. The deeper the measurement depth, the smoother the annual cycle and the smaller the variability. In February and at the end of the year the variability appears to be somewhat larger. A Fast Fourier Transform was applied to the measurements and revealed an annual and daily damping depth of 1.80 m and 0.10 m, respectively. An example of the usefulness of this data is provided for agriculture, where an aggressive root-knot nematode is affected by the mean soil temperature increase. It appears that the 600 degree day life-cycle threshold is reached 16 days earlier, which may lead to a potential serious increase in agriculture crop damage.     

Near-infrared spectroscopy for analysis of chemical and microbiological properties of forest soil organic horizons in a heavy-metal-polluted area

In industrial areas, heavy metals may accumulate in forest soil organic horizons, affecting soil microorganisms and causing changes in the chemical composition of the accumulated organic matter. The objectives of this study were to test the ability of near-infrared spectroscopy (NIRS) to detect heavy metal effects on the chemical composition of forest soil O horizons and to test whether NIRS may be used to quantitatively determine total and exchangeable concentrations of Zn and Pb (Zn_t, Pb_t, Zn_ex, Pb_ex) and other chemical and microbial properties in forest soil O horizons polluted with heavy metals. The samples of O horizons (n = 79) were analyzed for organic C (C_org), total N and S (N_t, S_t), Zn_t, Pb_t, Zn_ex, Pb_ex, basal respiration (BR), microbial biomass (C_mic) and C_mic-to-C_org ratio. Spectra of the samples were recorded in the Vis-NIR range (400–2,500 nm). To detect heavy-metal-induced changes in the chemical composition of O horizons principal components (PC1–PC7) based on the spectral data were regressed against Zn_t + Pb_t values. A modified partial least squares method was used to develop calibration models for prediction of various chemical and microbial properties of the samples from their spectra. Regression analysis revealed a significant relationship between PC3 and PC5 (r = −0.27 and −0.34, respectively) and Zn_t + Pb_t values, indicating an effect of heavy metal pollution on the spectral properties of the O horizons and thus on their chemical composition. For quantitative estimations, the best calibration model was obtained for C_org-to-N_t ratio (r = 0.98). The models for C_org, N_t, and microbial properties were satisfactory but less accurate. NIRS failed to accurately predict S_t, C_org-to-S_t, Zn_t, Pb_t, Zn_ex, and Pb_ex.     

Assessing the risk of a 50-year-old dump site in the Baltic Sea by combining chemical analysis, bioaccumulation, and ecotoxicity

Purpose

During the late 1950s and early 1960s, industrial waste material highly enriched with various contaminants (e.g., heavy metals, polycyclic aromatic hydrocarbons (PAHs)) was dumped in the inner Bay of Mecklenburg, western Baltic Sea. Between 2002 and 2004, a research program was initiated using chemical analysis in combination with bioanalytical techniques to assess the extent and variability in contamination at this dump site (DS). The data were compared to a reference area (RS) with similar environmental conditions, which is representative of the western Baltic Sea.

Materials and methods

Twelve PAHs were investigated to assess their ecological hazard, as they were identified as major pollutants in the dumped material. In addition to analyzing the actual PAH contamination status in the sediments, PAHs measured in the soft tissue of Arctica islandica were also used as an indicator of contaminant bioaccumulation. A biotest battery was applied to determine the toxic effects of contaminants in the sediment.

Results and discussion

Significantly elevated PAH concentrations (sum of 12 PAHs) of ～3,000 ng g^?1 dw and higher bioaccumulation factors (BAFs) were determined in the soft body tissue of A. islandica collected at DS (t test, p?=?0.025). The results also showed that the sediment PAH contamination was significantly higher at DS (1,952–5,466 ng g^?1 dw) than at RS (1,384–2,315 ng g^?1 dw). The results revealed a major heterogeneity in the PAH concentration at DS due to an attempt to cover the toxic material with clean clay. This resulted in a more heterogeneous distribution of the dump material rather than covering it up completely. However, not all relevant contaminants were included in this study, not only because it is too costly to determine them all but also because unidentified contaminants present at concentrations below the limit of detection cannot be measured. Bioassays were used to fill this gap in the hazard assessment in a cost-effective way by investigating the possible effects of sediment contamination on benthic organisms. The results showed a high variability and magnitude of growth and luminescence inhibition. Bacterial contact tests with marine organisms showed a high toxicity response (>80 % inhibition) from DS sediments. In contrast, the luminescent bacteria test (Vibrio fischeri) showed equivalent effects of sediments from both DS and RS.

Conclusions

The spatial distribution of toxicity in DS, the bioaccumulation in mussels and the analytical evidence of PAH pollution clearly show that the dumped material still represents a potential risk even after 60 years.