了解深部微生物群落组成和功能对探索早期生命起源和利用其独特功能均有重要意义.地热区深部碳酸盐岩岩溶-裂隙热储环境中微生物群落组成和功能特性仍然不清.为了探测冀中地热区深部热水微生物群落组成与功能特性,以浅层水和土壤为参照,利用冀中地热区地热科学钻探孔抽水试验,采集深部热水样,同时采集每个钻孔上方附近的大田农灌井浅层水和土壤样品,用于16S rRNA基因高通量测序,并使用PICRUSt软件进行功能预测.结果显示冀中地热区深部热水含有38个菌门,541个菌属,以细菌为主(97.5％),古菌很少(2.5％),特征性菌群主要是厚壁菌门Firmicutes、硝化螺旋菌门Nitrospirae、热袍菌门Thermotogae和广古菌门Euryarchaeota,优势菌属包括硫酸盐还原菌,固碳作用、发酵作用和硫酸盐还原作用可能较强,而产甲烷作用和反硝化作用可能很弱.深部热水出现好氧嗜温菌,说明在大流量抽水条件下,深部热水受到富含变形菌门Proteobacteria浅层水的补给.本次研究揭示了冀中地热区深部碳酸盐岩岩溶-裂隙热储层含有种类丰富、功能多样的微生物群落,微生物群落特征可作为深部热水与浅层水水力联系的指示剂.

[背景]地下深部存在一个生物圈,深部沉积岩、玄武岩、花岗岩和变质岩等岩性环境的微生物群落已被调查,而地下深部碳酸盐岩岩溶-裂隙热储层微生物群落特征仍然不清.硫酸盐还原菌(sulfate-reducing bacteria,SRB)是地下深部频繁检出的微生物.[目的]建立快速准确定量深部热水硫酸盐还原菌的微滴数字PCR (droplet digital PCR,ddPCR)技术.[方法]以SRB的功能基因dsrB为检测目标,优化SRB ddPCR技术的退火温度,考察其线性范围、敏感性、重复性和特异性,并将该技术用于实际样品的检测. [结果]SRB ddPCR技术的最佳退火温度为54℃,检测的线性范围为1.1×100-1.1×105 copies/μL-DNA,相关系数R2为0.996,检出限为1 copy/μL-DNA,重复性的相对标准差优于9％,对3种非SRB人工构建的质粒均没有扩增,显示该技术具有很好的线性关系、敏感性、重复性和特异性.利用该技术对冀中地热区深部热水、浅层水和土壤样品进行了检测,平均含量分别为(4.0±8.4)× 103 copies/mL、(1.6±3.5)× 102 copies/mL和(1.5±1.2)× 103 copies/g-dw.与浅层水和土壤相比,深部热水富含SRB菌.[结论]为了提高地下深部生物圈认识和合理开发利用深部热水,建立了一种快速、灵敏、准确的SRB ddPCR检测技术,同时为其他指示菌检测技术的建立提供了参考.

The present work is designed to simulate the dissolution of sulphate rock under various conditions of different bacterial cell numbers, temperatures and reaction times both in water-rock system and water-rock-bacteria systems by laboratory experiment. The rate of sulphate reduction were estimated using the experimental data. The results suggested that the sulphate-reducing bacteria promote the sulphate rock dissolution and increase the amount of dissolved sulphate. The dissolution of sulphate rock driven by bacterial sulphate reduction results in the formation of sulphate rock karst. The research is an insight into biokarst, which provides a new perspective for the field of petroleum geology.

To reveal the evolution mechanism of groundwater quality in Liulin karst system in northern China,we used the major ions and saturation indices to analyze the hydrochemical characteristics and geochemical processes.Meanwhile,we established a reverse geochemical model to quantitatively simulate the water-rock interactions in karst aquifers.The results showed that the hydrochemical type changed from HCO3-Ca·Mg in the recharge area to HCO3·SO4-Ca·Mg in the runoff area and to HCO3·SO4-Ca·Na in the discharge area.The ge-ochemical processes of hydrochemical evolution in karst water were the dissolution of carbonate minerals and gypsum.Along the flow path, the processes were evolved from the simultaneous dissolution of calcite and dolomite in the recharge area to the dissolution of dolomite and gypsum in the runoff area,and the dissolution of halite was added in the discharge area.Besides,the dedolomitization and cation exchange between Na+and Ca2+in the runoff and discharge areas affected the hydrochemical types of karst groundwater in Liulin.

Propane-oxidizing bacteria in surface soils are often used to indicate the position of oil and gas reservoirs. As a potential replacement for the laborious traditional culture-dependent counting method, we applied real-time fluorescent quantitative polymerase chain reaction detection as a quick and accurate technology for quantification of propane-oxidizing bacteria. The propane monooxygenase gene was set as the target and the assay is based on SYBR Green I dye. The detection range was from 9.75 × 10 8 to 9.75 × 10 1 gene copies/µl, with the lowest detected concentration of 9.75 copies/µl. All coefficient of variation values of the threshold cycle in the reproducibility test were better than 1%. The technique showed good sensitivity, specificity, and reproducibility. We also quantified the propane-oxidizing bacteria in soils from three vertical 250 cm profiles collected from an oil field, a gas field, and a nonoil gas field using the established technique. The results indicated that the presence of propane monooxygenase A genes in soils can indicate an oil or gas reservoir. Therefore, this technique can satisfy the requirements for microbial exploration of oil and gas.

In order to test the application effect of Diver automatic water level recorder in the study of hydrogeological parameters from pumping test data,a pumping test was carried out in the Liuli River area in Fangshan,Beijing.The characteristics of the original data from Diver and the manual measurement in the pumping test were compared,and the hydrogeological parameters were calculated based on Diver data.An average difference of the main well data between the Diver and the manual measurement was 0.17m,and the average difference of the auxiliary well data was 0.02m in the whole process of the pumping test.Finally,it was calculated that the value of the k was 91.51m/d,the value of the T was 1555.72m2/d and the value of the S was 0.0314 based on the Diver data.Diver has the advantages of large amounts of continuous and accurate data to ensure that the hydrogeological parameters are performed correctly.

The long-term oil exploitation in oil fields has led to pollution of surrounding soil, creating a serious ecological problem. In order to promote and improve the application of microbial remediation in oil contaminated soil, experiment is carried out in polluted area in Zhongyuan Oilfield. In the experiment, indigenous microorganisms and other physical and chemical methods are employed, ryegrass is grown, and environmental factors in soil are regulated to degrade the oil and treat the polluted soil. Results show that when the average oil content in the soil is about 523.08 mg/kg, 65 days' remediation through plants and microorganisms could help bring the oil content down to 74.61 mg/kg, achieving a degradation rate of 85.74%; through salinity treatment, salt content in soil is reduced by 62.93-82.03% to 399-823 mg/kg from previous 2.22 g/kg. Through this experiment, the bioremediation method is improved and its effectiveness and feasibility are testified. The result has been applied in Zhongyuan Oilfield and has brought fair ecological and economic benefits, providing technical support to the treatment of contaminated soil of the same kind, and offering some insights to the treatment of soil contaminated by other organic pollutants.

To evaluate the application of a DNA-based analysis of a microbial technique for oil and gas prospecting, the hydrocarbon potential predicted using the methanotrophic pmoA and propanotrophic prmA genes as targets was investigated in soil above a known oil reservoir. In total, 82 soil samples were collected at 50-cm depths above the Xiliu oil reservoir for quantitative real-time polymerase chain reaction (RT-PCR) analysis. The pmoA gene content ranged from 0 to 2.7x10(6) copies/g dw, whereas that of prmA varied from 0 to 1.6 x 10(6) copies/g dw. Microbial anomaly distribution maps of normalized values for the prmA gene as a main indicator suggested that the area with the highest potential of prospecting was an undeveloped location, and an area with a relatively large prmA background was identified at this oil-producing site. The areas of microbial anomaly exhibited different prmA/pmoA ratios (20, 1-5, 0.0n-0.25 and 0), and this ratio may be used as an indicator for predicting the properties of oil and gas reservoirs and biogenic methane or the influence of oil-producing activities. Our results suggest that a DNA-based analysis of a microbial technique is a powerful tool for oil and gas prospecting. This study provides a new microbial technique for the prospecting of oil and gas.

Understanding the groundwater evolution has always been a challenging work for geochemists and hydrogeologists. The purpose of this study is to reveal the evolution mechanism of groundwater quality in karst groundwater system from Liulin, northern China. Multiple hydrochemical indicators were used to differentiate the dominant geochemical processes of hydrogeochemical evolution. The results suggested that the dissolution of calcite and dolomite widely occurred in Liulin karst water system, especially in the recharge area, and the dissolution of halite, gypsum and mirabilite locally occurred in the runoff and discharge areas of the system. Dedolomitization driven by gypsum dissolution was found in the runoff areas of karst aquifers on the north and south of Liulin springs. Cation exchange between Ca2+, Mg2+ and Na+ was proved to take place in the runoff and discharge areas. Besides, the springs in the north of Sanchuan River were more saline than those in the south, which could be attributed to the common effect of geologic background and recharge sources. Eventually, an updated conceptual model of the geochemical processes in Liulin karst water system was developed, which illustrated the chemical evolution and geochemical processes of karst groundwater along the flow paths.

Methanotrophs have long been used as an important biological indicator for prospecting of oil and gas, while the indication of propanotrophs in hydrocarbon micro-seep systems is still poorly investigated. In this study, the abundance and diversity of the methanotrophic pmoA gene and the propanotrophic prmA gene as target genes were investigated in soils above Yangxin oil reservoir and Beiguan non-petroliferous area using molecular biological techniques. A total of 14 soil samples were collected at different depths (5, 20, 50, 100, 150, 200 and 250 cm) of two 2.5-m soil profiles located separately within the oil field and the non-petroliferous area for analysis of fluorescent quantitative real-time polymerase chain reaction (RTPCR) (14 samples) and clone libraries (4 samples). The results demonstrated high presence of the propanotrophic prmA gene ranging from 7.68 x 10(5) to 2.29 x 10(7) copies/g dw (gene copies per gram soil of dry weight) in soil from the oil field relative to the non-petroliferous area for which the same measurements yielded results all below detection limit except for the 5-cm sample. On the other hand, oil field soil yielded much lower content of the methanotrophic pmoA gene (below detection limit to 5.6 x 10(2) copies/g dw) than the non-petroliferous area (1.14 x 10(3) copies/g dw to 1.26 x 10(5) copies/g dw) below 20-cm depth due to influence of biogenic methane, implying that propanotrophs may be better indicator bacteria for prospecting of oil and gas. Almost all pmoA clones of two 50-cm soil samples phylogenetically belonged to Gamma-Proteobacteria and the predominant pmoA OTUs were all uncultured bacteria. All prmA clones of two 5-cm soil samples were derived predominantly from Actinobacteridae (25.7%) and Alpha-Proteobacteria (74.3%), and all dominant prmA OTUs were also clustered with uncultured bacteria. Our results confirm that propanotrophs may be better indicator bacteria for prospecting of oil and gas and enrich the knowledge on diversity of methanotrophs and propanotrophs in the oil field and the non-petroliferous area.

Experimental study of enhanced in-situ micro-ecological remediation of petroleum contaminated loess soil was carried out in Zhongyuan oil production areas, and the enhanced in-situ micro-ecological remediation technique includes optimistic in-situ microbial communities, physical chemistry methods, alfalfa planting and regulation of soil environmental elements. Experiments showed that the oil content in the contaminated soil with oil content about 2 898.25 mg/kg can be reduced about 98.61% after in-situ micro-ecological remediation for 99 days, which demonstrated the effectiveness of in-situ micro-ecological remediation methods for petroleum contaminated soil in central plains of China, and explored the practical and feasible application of these methods.

mineral facies included dolomite, calcite, gypsum and halite.Dolomite is the most important dissolved mineral, followed by calcite and gypsum in recharge area, as well as calcite, gypsum and halite in runoff and discharge areas.

Although the Chinese people have, through continuous efforts, built the constantly improving Yellow River flood control system, and created a miracle which has been tranquil for over 50 years, the tendency for the downstream watercourse of the Yellow River to uplift every year has not been fundamentally curbed, and the aboveground river is still the "scourge" of the sons and daughters of the Yellow River. By the use of a variety of modern investigation and survey methods, the geological environment characteristics of the downstream of the Yellow River have been identified basically, including the environmental and geological factors affecting the stability of aboveground rivercourse of the lower Yellow River such as the active fracture of the lower Yellow River, crustal uplift, land subsidence, seismic activity, geological conditions of dike foundation engineering, hydrodynamic conditions of rivers, and geomorphology of watercourses. After a comprehensive analysis of the inability mode of aboveground river on the downstream of the Yellow River and its corresponding impact factors, by using the fuzzy comprehensive evaluation method, we have evaluated the crustal stability of the aboveground river, dike foundation stability, watercourse landform stability and overall stability. The results of comprehensive results show that the stability of downstream aboveground rivercourse of the Yellow River can be divided into four grades and 11 sections, ie. "basically stable, unstable, very unstable and extremely unstable". On the basis of the stability segmentation, we consider the influence of integrally the future structural faults, earthquakes, the difference of watercourse between forward and backward heights of dikes, river regime and river type, historical crevasses, foundation soil liquefaction and seepage deformation, and find out 17 most unstable danger points. Finally, from 17 danger points, we select 7 danger points which are most prone to instability including Wuzhi, Zhongmou, Kaifeng, Fengqiu, Dongming, Changyuan and Dong'e. The calculation and analysis of the range and area inundated by 7 danger points, area, and number of people threatened, possible economic loss, and environmental damage, the inability caused by any one of 7 points could bring disastrous consequences to the downstream.

[目的]建立快速检测甲烷氧化菌含量的SYBR Green Ⅰ实时荧光定量PCR技术,用于油气微生物勘探.[方法]以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测.[结果]该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976％,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3％,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性.利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值.[结论]为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考.

Thanks to the increasingly-improved flood-control engineering projects in the lower reaches of the Yellow River safety has been maintained over the past 50 years. Nonetheless, the rising trend of the riverway bed of the lower reaches has not been reversed. This has been a potential hazard to the local people. Using various investigation methods we basically ascertain the geological environment of the lower reaches including active faults, crust uplift, ground subsidence, seismic activities, engineering conditions of dike foundation, hydraulic conditions and fluvial geomorphology, etc. Based on the comprehensive analysis of the instability modes and corresponding influencing factors we choose fuzzy comprehensive assessing method to assess the ground stability, dike-foundation stability and riverway stability and general stability of the suspended river. The results show that the stability of the suspended river of the Yellow River could be classified into four grades, i.e. “basically stable, unstable, very unstable and extremely unstable” and 11 segments. Taking into account future tectonic faults, earthquake, difference between riverway and ground outside riverway, riverway shapes, historical breaches, ground fluidization and seepage deformation, etc., we further find out 17 the most unstable sites, among of which 7 are the most possible breaching sites, i.e. Wuzhi, Zhongmou, Kaifeng, Fengqiu, Dongming, Changyuan and Dong’e. Calculations of the possible inundation scope and area, number of affected people, economic losses and environment damage etc. Any of them, if breached, could bring about disaster results in the lower reaches of the Yellow River.

以某废弃采油井井场做为研究对象,对油井不同方位、距离和深度剖面土壤石油含量和物理化学性质进行测试,并利用实时定量PCR技术对场地细菌和真菌生物量展开调查,以期能够为修复工作提供数据基础.结果显示,该场地遭受了严重石油污染和盐碱污染.土壤细菌16S rDNA和真菌ITS rDNA定量调查结果显示,细菌和真菌的生物量均高于普通土壤,尤其石油含量与细菌16S rDNA基因具有一定的正相关性,土壤的生态系统的健康和稳定性变弱.通过土壤三氮的测定,结合石油和微生物量的对比得出,嗜油微生物大量生长导致了土壤硝化作用的减弱和NH4+的积累、土壤N素的缺失和土壤健康及稳定性的减弱.因此,有必要对该污染场地进行治理.基于分析数据,提出了一些该污染场地的修复建议.

To assess whether the anomalies of methanotrophic pmoA and propanotrophic prmA genes as target genes can reflect the existence of underlying oil and gas reservoirs, natural abundance and distribution of target genes were investigated in soil above known gas fields, oil fields and non-oil and gas fields, China. A total of 45 soil samples were collected from different depths of 2.5-m soil profiles in Xiaoquan gas field, Xiliu oil field and Beiguan non-oil and gas field and from 50cm depth of the old oil area, the new developed area, the oil producing area and the undeveloped area of Xiliu oil field, respectively for analysis of target genes by SRBR Green-based fluorescent quantitative real-time polymerase chain reaction (RT-PCR) technique. Distributions of target gene contents in 2.5-m soil profiles of the three fields show that 50cm was considered to be optimal sampling depth for microbial prospecting of oil and gas reservoirs. At 50cm depth of 2.5-m soil profiles, the gas field had very high contents of methanotrophic pmoA gene (3.8×105copies/gdw) and propanotrophic prmA gene (5.92×105copies/gdw), the oil field had medium contents of methanotrophic pmoA gene (2.8×104copies/gdw) and propanotrophic prmA gene (4.1×104copies/gdw), and the non-oil and gas field had low contents of propanotrophic prmA gene (below detection limit) and relatively high contents of methanotrophic pmoA gene (1.26×105copies/gdw) due to the influence of biogenic methane. The anomalies of methanotrophic pmoA gene and propanotrophic prmA gene can reflect the existence of underlying gas deposits. Different areas of the oil field had different target gene contents in the new developed area ((1.7–2.3)×106copies/gdw for the prmA gene, 1.0×103–1.2×104copies/gdw for the pmoA gene), the old oil area (below detection limit −2.0×106copies/gdw for the prmA gene, below detection limit −7.1×104copies/gdw for the pmoA gene), the undeveloped area (1.8×105–1.5×106copies/gdw for the prmA gene, 5.3×104−3.3×105copies/gdw for the pmoA gene) and the oil producing area (below detection limit −1.0×106copies/gdw for the prmA gene, 1.4×105–4.8×105copies/gdw for the pmoA gene). Comparisons of target gene contents in different areas of Xiliu oil field indicate that oil productive activities had a large impact on contents of target genes in soil above the oil field. The anomalies of propanotrophic prmA gene with low contents of methanotrophic pmoA gene can reflect the existence of underlying oil reservoirs. Analysis of the relationships between contents of target genes may be used for identification of gas fields, oil fields and the influence of biogenic methane. Our results suggest that quantitative RT-PCR assay-based methanotrophic pmoA gene and propanotrophic prmA gene anomalies can reflect the existence of underlying oil and gas reservoirs.

Bacterial sulfate reduction is significant for the karst development and pyrite formation within the Ordovician weathering crust in the Ordos Basin of China. Bacterial communities were studied to determine their potential geomicrobiological functioning by constructing 16S rRNA clone library for in situ samples. The results showed that 147 positive clones sequenced were divided into 23 operational taxonomic units (OTUs), 8 OTUs accouting for 80% of all the selected clones belonged to the genus Desulfosporosinus. Bacterial sulfate reduction has been demonstrated to take place in the Ordovician by the classical hydrogeological information together with the stable sulfur isotope analysis from both the pyrite in the weathering crust and the products of the laboratory experiments on the dissolution of sulfate rock. The H2S produced by bacterial sulfate reduction combined with iron to form pyrite, resulting in the development of hypogenic karst in the weathering crust. This process provided a reasonable interpretation for karst development in the vicinity of sulfur deposits in the Ordos Basin.

在现代油气勘探中,油气微生物勘探技术可以辅助地质与地球物理勘探方法进行油气勘探,以降低勘探风险.本文系统总结了油气微生物勘探的理论基础,着重介绍了现代微生物技术在该领域的应用,指出现代微生物技术从分子水平出发,有效避免了传统方法中的弊端,显示出分子生物技术的优越性.将现代分子生物学技术应用于油气勘探,有望为微生物油气勘探开创新的战略局面.

For understanding the effects of ambient temperature, water flow rate, and pH of water on the dissolution of sulfate rocks, the present work aims at studying the mechanism of sulfate rock dissolution through laboratory experiments. The results suggest that each experimental factor has a different effect on the dissolution of sulfate rock. High temperature caused the transformation of the sulfate minerals, resulting in the dissolution of sulfate rocks. At high water flow rate solutions remain under-saturated with respect to sulfate mineral phases resulting in an increase of dissolution rates. The sulfate karst formation is different from carbonate karst, i. e. the water is corrosive with regards to the sulfate rock system even in the absence of CO2 in the system. The results will give an insight into the mechanism of karst with respect to the coexistence of sulfate and carbonate rocks.