一株新的砷还原菌分离鉴定及其对天然矿物臭葱石的溶解作用

Isolation and identification of a novel As-reducing bacterium for the reductive dissolution of scorodite

摘要:

[目的] 探究江汉平原土著砷还原微生物如何介导臭葱石的溶解和释放过程，以及硝酸盐和硫酸盐对该过程的影响。[方法] 采集江汉平原高砷沉积物，利用多轮传代富集方法筛选出一株兼性厌氧砷还原菌；克隆其16S rRNA基因、砷还原酶基因（arsC）、硫代硫酸盐还原酶基因（phsA）、硝酸盐还原酶基因（nar）以获得其分类地位；分析该细菌的As（V）、NO₃^-、Fe（III）、S₂O₃^2-还原功能；利用microcosm技术分析该菌株催化臭葱石中不可溶砷和铁的溶解和释放作用及硝酸盐和硫酸盐对此过程的影响；采用X-射线衍射（XRD）和扫描电镜（SEM）等方法对细菌作用前后的矿物表面形貌进行分析。[结果] 16S rRNA基因测序结果表明该细菌为柠檬酸杆菌属（Citrobacter sp.），故命名为Citrobacter sp.A11；在Citrobacter sp.A11作用下，0.45 mmol/L As（V）在4 d内被还原成As（III），2.0 mmol/L S₂O₃^2-在6 d内被还原成S^2-，1.0 mmol/L Fe（III）在3 d内被还原成Fe（II），140.0 mg/L NO₃^-在28 h内被还原成NO₂^-；经过28 d该细菌的催化作用使得体系中不可溶砷和铁的释放量分别为33.68 μmol/L、51.93 μmol/L；硫酸根的加入使得砷和铁的释放量分别增长了41.04%和34.30%，硝酸根的加入则使砷和铁释放量分别降低了35.07%和53.46%。XRD、SEM-EDS分析表明，细菌作用后的臭葱石表面形貌发生明显改变，并出现细小且分散的溶解性颗粒。[结论] 本次研究从江汉平原高砷沉积物中富集分离得到一株兼性厌氧砷还原细菌Citrobacter sp.A11，能有效还原As（V）、S₂O₃^2-、NO₃^-、Fe（III）；砷还原细菌Citrobacter sp.A11能显著促进臭葱石中砷和铁的溶解和释放，硫酸根离子的存在会促进细菌介导臭葱石中固态砷、铁的释放，而硝酸根离子的存在则对此过程起明显抑制作用。

关键词:砷还原菌;硫酸根;硝酸根;臭葱石;江汉平原

Abstract:

[Objective] This study aimed to investigate the reductive dissolution of scorodite by an indigenous As-resistant bacterium isolated from the Jianghan plain and the influences of nitrate and sulfate on the mobilization in this microbiological process.[Methods] We used the microbial enrichment technique to isolate a facultative anaerobic arsenate-reducing strain (A11), amplified the 16S rRNA and functional genes (arsenate-reductase gene, thiosulfate reductase gene and nitrate reductase genes) from the bacterial cells, and analyzed arsenate-, ferric iron-, thiosulfate-, and nitrate-reducing activities of this strain. We also examined the abilities of A11 cells to catalyze the mobilization and release of As and Fe from scorodite under anaerobic conditions, and further explored how sulfate and nitrate affected this microbial process. The microbial reduction and dissolution of the scorodite mineral were verified by XRD and SEM-EDS analysis. [Results] Citrobacter sp. A11 completely reduced 0.45 mmol/L As(V), 2 mmol/L S₂O₃^2-, 1 mmol/L Fe(III) and 140 mg/L NO₃^- in 4 days, 6 days, 3 days, 28 hours, respectively. In the presence of A11 cells, approximately 33.68 μmol/L As(III) and 51.93 μmol/L Fe(II) were released from the scorodite slurries after 28 days of anaerobic incubations. The presence of sulfate caused 41.04% and 34.30% increases of the A11-catalyzed release of As(III) and Fe(II) over the course of 28 days. However, the addition of nitrate caused 35.07%, 53.46% decreases of the A11-catalyzed release of As(III) and Fe(II), respectively. XRD and SEM-EDS observations for the reduced scorodite revealed that the mineral particles were altered dramatically. The particles size of the treated scorodite was much smaller than the control one. [Conclusion] Citrobacter sp. A11 strain has been identified with the ability of arsenate, thiosulfate, ferric iron and nitrate reduction, also can efficiently catalyze the mobilization and release of As and Fe from scorodite under anaerobic conditions, and this process enhanced when sulfate was added to the reactions. However, this process was inhibited when nitrate was added.

Key words:As-resistant bacteria;sulfate;nitrate;scorodite;Jianghan plain