A preliminary groundwater incubation study dataset to test isotope methods for distinction of various N transformation processes in different pH conditions

pH experiment 2 groundwater samples with high nitrate concentrations ('PZ4' and 'oxytree') were selected and incubated under oxic and anoxic conditions to investigate the effect of pH on nitrogen transformation. pH is a key factor influencing denitrification, as it regulates microbial enzyme activity and N₂O production; pH (7–8) is known to enhance complete denitrification and reduce N₂O emissions. Samples were flushed with N₂ gas for 30 minutes (anoxic) or with synthetic air (oxic), then adjusted to pH 5.0, 7.0, or 9.0 using 0.1 N NaOH or HCl. Each treatment had four replicates. Two types of sterile controls were prepared: one by filtration with sterile filters and addition of 2 mL 0.1 N HgCl₂, and another by sterile filtration followed by autoclaving. All samples were incubated at 16 °C, 100 rpm in the dark for 2 weeks. Glucose was added after one week to stimulate microbial denitrification. Headspace gas samples were analysed for GC and CRDS measurements. Further, Inorganic nitrogen species (NO₃⁻, NO₂⁻, NH₄⁺) were analyzed colorimetrically, and isotopic signatures determined using Pseudomonas aureofaciens for NO₃⁻ and Stenotrophomonas nitritireducens for NO₂⁻ isotopes.pH值影响实验 选取2份高硝酸盐浓度地下水样品（编号"PZ4"与"oxytree"），分别在有氧与厌氧条件下进行培养，以探究pH值对氮转化过程的影响。pH是影响反硝化作用的关键因子，其可通过调控微生物酶活性与一氧化二氮（N₂O）的生成过程；已有研究表明，pH值处于7~8区间时可促进完全反硝化进程并降低N₂O排放。实验前，将样品分别以氮气吹扫30分钟以营造厌氧环境，或以合成空气吹扫以营造有氧环境，随后使用0.1当量的氢氧化钠（NaOH）与盐酸（HCl）将样品pH值分别调节至5.0、7.0或9.0。每组处理设置4个重复样本。同时设置两类无菌对照：其一为采用无菌滤膜过滤后添加2 mL 0.1当量氯化汞（HgCl₂），其二为经无菌过滤后再进行高压蒸汽灭菌。所有样品均于16℃、100 rpm转速的黑暗环境中培养2周。培养至第1周时添加葡萄糖以刺激微生物反硝化作用。采集顶空气体样品，用于气相色谱（Gas Chromatography, GC）与腔环衰荡光谱（Cavity Ring-Down Spectroscopy, CRDS）检测。此外，采用比色法对无机氮形态（硝酸盐NO₃⁻、亚硝酸盐NO₂⁻、铵盐NH₄⁺）进行定量分析，并分别利用产金色假单胞菌（Pseudomonas aureofaciens）与亚硝酸盐还原寡养单胞菌（Stenotrophomonas nitritireducens）测定硝酸盐与亚硝酸盐的同位素特征。