硝酸盐氮氧同位素反硝化细菌法测试研究

进入21世纪,硝酸盐氮氧同位素测试技术的显著进展是反硝化细菌法测试技术的建立。反硝化细菌法具有可同时分析浓度低至μg/L的硝酸盐氮氧同位素组成、免去复杂的样品预处理、分析时间大大缩短以及从复杂溶液中只转化NO3-为N2O等优点。实验过程由细菌挑选和培养、样品NO3-转化为N2O、N2O提取纯化、同位素测试以及同位素测试结果校正等几个关键步骤组成。国外研究结果显示该技术不仅可用于海水和淡水中硝酸盐同位素测试,而且还可用于土壤样品,并得到不断的完善和应用;国内建立了这一技术,在包气带和地下水硝酸盐污染研究中取得重要进展。由于该技术诸多优点,建议大力促进这一技术在我国的研究和应用,希望有关部门加大支持力度,进一步完善该技术并推广应用。     

细菌反硝化法测定10–6级硝酸盐的氮和三氧同位素

硝酸盐的氮和三氧同位素(δ15N, δ17O和δ18O)及氧同位素非质量分馏(△17O)综合研究, 可以更有效地示踪硝酸盐的来源和形成过程、制约硝酸盐的形成条件。本文详细描述了细菌反硝化法测定10–6级硝酸盐氮和三氧同位素的分析测试方法和实验要点。综合优化改良的细菌反硝化前处理方法、全自动气体预浓缩富集纯化系统和测试流程, 实现了实验室长期测定数据的稳定性, 以及多批次标准样品测定的良好重现性。10 nmol NO– 3标准样品的δ18O和δ15N测试精度分别是0.25‰(1σ)和0.40‰(1σ)。80 nmol NO– 3标准样品的δ18O、δ17O和δ15N的测试精度分别是0.5‰(1σ)、0.4‰(1σ)和0.1‰(1σ), 据此计算出的Δ17O精度为0.46‰(1σ)。     

Distribution of nitrogenous nutrients and denitrifiers strains in estuarine sediment profiles of the Tanshui River, northern Taiwan

Chemical profiles of both oxidized (nitrate and sulfate) and reduced (ammonium, sulfide, acid-volatile sulfide [AVS], and pyrite) materials and the corresponding distribution of denitrifier microbial communities were measured at low tide in sediments at Guandu in the estuary of the Tanshui River, northern Taiwan in August 2002. Denitrifier strains were isolated for physiological and phylogenic analyses. Based on the distribution of nitrogenous compounds and denitrifier abundances, the vertical profile of Guandu sediments could be separated into four layers: a mixed layer (the top 1 cm of depth, respectively containing 0.82–2.37 and 535.9–475.0 μM of nitrate and ammonium), a nitrate-concentrated layer (1–5 cm in depth, 2.37–0.53 and 475.0–1192.1 μM, respectively), a denitrifier-aggregation layer (5–7 cm in depth, 0.53–0.72 and 1192.1–1430.1 μM, respectively), and an ammonium-enriched layer (7–12 cm in depth, 0.72–0.78 and 1430.1–2196.6 μM, respectively). Denitrifier strains were detected in all layers except for the mixed layer. A variety of metabolic processes by these strains may occur in different layers. Bacillus jeotgali-, Bacillus sphaericus-, and Bacillus firmus-related strains isolated from the nitrate-concentrated layer may be involved in the nitrification-denitrification coupling process due to the relatively low nitrate concentrations (maximum = 2.37 μM), and may contribute to denitrification not nitrification. Bacillus bataviensis- and B. jeotgali-related strains isolated from the denitrifier-aggregation layer comprised the predominant denitrifier population (3.64 × 10⁴ cells/g of denitrifier abundance). They possess the ability of dissimilatory nitrate reduction to ammonium (DNRA). Bacillus jeotgali-related strains and two newly identified strains of GD0705 and GD0706 isolated from the ammonium-enriched layer possibly use fermentative processes as the main metabolic pathway instead of denitrification when nitrate is scarce, and this further supports the high ammonium concentrations (up to 2.20 mM) found in the Guandu sediments. In addition, spore formation also enhances the chance of survival of these strains in the face with such a nitrate-deficient environment.     

湛江湾沉积物中反硝化和厌氧氨氧化细菌的丰度、多样性及分布特征

采用实时荧光定量PCR、高通量测序等方法对湛江湾沉积物中四个月份的反硝化细菌与厌氧氨氧化细菌的多样性和丰度进行了分析。结果表明:湛江湾沉积物中反硝化细菌和厌氧氨氧化细菌丰度在四个月份的变化和空间分布趋势为:nirS型反硝化细菌在二月份最高,四月份最低,且其平均丰度有从湛江湾湾内向湾口附近呈现先升高再降低的趋势;nirK型反硝化细菌丰度在九月份最高,十一月份最低;nosZ型反硝化细菌在四月份最高,其余月份变化不大;厌氧氨氧化细菌丰度在九月份最高,二月份最低。通过相关性分析结果表明,亚硝酸盐、铵盐等共同调控着湛江湾沉积物中反硝化和厌氧氨氧化细菌丰度变化。系统发育分析表明:湛江湾中存在着一些广泛分布的反硝化细菌,但也生活着一些新奇的nirK型和nosZ型反硝化细菌。对于厌氧氨氧化细菌而言,其主要属于浮霉菌门及Candidatus Scalindua属,具有较高的耐盐性,另外湛江湾海区的厌氧氨氧化细菌也生活着一类在其他地方没有的新分支。典范对应分析分析结果表明:硝酸盐显著影响湛江湾反硝化细菌和厌氧氨氧化细菌的群落结构。湛江湾沉积物中反硝化细菌和厌氧氨氧化细菌存在特殊的竞争与共存的关系,且由亚硝...