细菌碳酸酐酶促进形成的碳酸盐矿物

刘 璐; 李福春; 李 磊; 张宠宏; 吕杰杰

doi:10.11932/karst20170402

细菌碳酸酐酶促进形成的碳酸盐矿物

doi: 10.11932/karst20170402

南京农业大学资源与环境科学学院

基金项目: 国家自然科学基金项目（41172308,41673083）;中国科学院黄土与第四纪地质国家重点实验室开放课题（SKLLQG1309）

计量
- 文章访问数: 1656
- HTML浏览量: 295
- PDF下载量: 942
- 被引次数: 0
出版历程
- 发布日期: 2017-08-25

Carbonic anhydrase excreted by bacteria induces the formation of carbonate minerals

College of Resources and Environmental Sciences, Nanjing Agricultural University

摘要

摘要: 微生物促进碳酸盐矿化机理的研究对于全球碳循环和土壤的形成与演化等科学问题具有重要的意义。为了探究细菌胞外碳酸酐酶（CA）在碳酸盐矿化过程中所起的作用，本文采用硫酸铵析出蛋白质和Tris-H2SO4缓冲液溶解蛋白质的方法提取MF-2菌株分泌的胞外CA，将其添加于一水乙酸钙—胰蛋白胨（不含碳酸根）体系中并进行了120 h的化学实验。同时设置一组不含CA的对照实验。实验结束后用离心法将固相和液相分离。利用X-射线衍射仪（XRD）和扫描电子显微镜（SEM/EDS）对沉淀物的矿物成分、元素组成和形态进行了研究，利用电感耦合等离子体发射光谱仪（ICP-OES）测定了溶液中的Ca2+浓度。实验结果表明：（1）在节杆菌属MF-2菌株胞外CA作用下形成了肉眼可见的沉淀物，其重量随着反应时间的延长而逐渐增大，而溶液中钙离子浓度随着反应时间的延长而逐渐降低。用双氧水（30%）处理的结果显示，碳酸盐矿物是沉淀物的主要组分。对照实验形成的沉淀物明显少于CA实验，而且其主要由有机物质组成。这充分地说明，MF-2菌株胞外CA可以显著地促进碳酸盐矿物沉淀。（2）在有胞外CA参与的实验早期（第48 h之前），未形成任何结晶态物质；在实验的中后期，形成的矿物以方解石为主，含少量或不含球霰石。这明显不同于MF-2菌株的培养实验。在后一种情况下仅形成球霰石一种矿物。（3）胞外CA作用下形成的矿物形态包括菱面体形、球形和半球形，其中菱面体形矿物占主导地位。这也有别于MF-2菌株的培养实验（以球状和碗状为主）。对多张SEM照片进行的统计结果显示，菱面体形矿物所占比例有随着反应时间的延长而逐渐降低的趋势（即球形和半球形所占比例逐渐升高），从第48 h的接近100%降低至第120 h的84%。（4）MF-2菌株胞外CA促进碳酸钙沉淀的主要机制是其加速CO2的水化反应，参与反应的CO2可能主要源于空气。
- 碳酸酐酶 /
- 节杆菌 /
- 方解石 /
- 球霰石 /
- 二氧化碳水化
Abstract: Study on the mechanism of carbonate mineralization in the presence of microorganisms is of great significance for the global carbon cycle, soil formation and evolution and other related issues. In order to clarify the role of extracellular carbonic anhydrase (CA) excreted by bacteria in the process of carbonate mineralization, a series of chemical experiments were carried out in the CA-calcium acetate-tryptone (without carbonate) system for 120 hours. The extracellular CA from MF-2 strains was extracted through precipitation of protein by ammonium sulfate and dissolution of protein by Tris-H2SO4 buffer solution. Synchronously, a series of control experiments without CA were performed. After the experiments, the solid and the liquid phases were separated using the centrifugation method. The mineral species, its chemical composition and morphology were characterized by X-ray diffractometer (XRD) and scanning electron microscope accompanied by energy-dispersive spectrometry (SEM/EDS). The concentration of calcium ion in the solution was determined by the inductively coupled plasma optical emission spectrometer (ICP-OES). The results show that (1) the precipitate with visible amount formed in the system with extracellular CA. With the prolongation of the reaction time, the precipitate weight increased gradually, while the Ca2+ concentration in the solution decreased. The treatment results with 30% hydrogen peroxide show that carbonate mineral was the dominant component in the precipitate. The precipitate formed in the control experiments was significantly less than that in the CA experiments, which was mainly composed of organic substances. This adequately demonstrates that the extracellular CA excreted by MF-2 strain could significantly promote precipitation of carbonate minerals. (2) No crystalline formed in the initial stage of experiments (before 48th h) with CA, and calcite was dominant mineral in the precipitate formed in the middle and late stages. This was obviously different from the incubation experiments with MF-2 strain. Only vaterite formed in the latter case. (3) The mineral morphologies formed by the action of extracellular CA were rhombohedral, spherical and hemi-spherical, in which then rhombohedral mineral dominated. This was also different from the incubation experiments with MF-2 strain (spherical and bowl-shaped in the latter case). The statistical results based on several SEM photographs show that the proportion of rhombohedral-shaped minerals decreased gradually with time, from nearly 100% at 48th h to 84% at 120th h, i.e. spherical and hemi-spherical minerals gradually increased. (4) CA accelerated hydration reaction of carbon dioxide and promoted formation of carbonate minerals. The CO2 participated in the reaction might largely come from air.
- Carbonic anhydrase /
- Arthrobacter sp. /
- calcite /
- vaterite /
- carbon dioxide hydration

HTML

参考文献(28)

[1]	?Lal R. Soil carbon sequestration impacts on global climate change and food security［J］. Science, 2004, 304 (5677): 1623-1626.
[2]	Aquilano D, Otálora F, Pastero L, et al. Three study cases of growth morphology in minerals: Halite, calcite and gypsum ［J］. Progress in Crystal Growth and Characterization of Materials, 2016, 62 (2): 227-251.
[3]	Hirmas D R, Amrhein C, Graham R C, et al. Spatial and processbased modeling of soil inorganic carbon storage in an arid piedmont ［J］. Geoderma, 2010, 154 (3-4): 486-494.
[4]	Lian B, Hu Q, Chen J, et al. Carbonate biomineralization induced by soil bacterium Bacillus megaterium ［J］. Geochimicaet CosmochimicaActa, 2006, 70 (22): 5522-5535.
[5]	Meldrum N U, Roughton F J W. Carbonic anhydrase. Its preparation and properties ［J］. Journal of Physiology, 1933, 80 (80): 113-142.
[6]	Lindskog S. Structure and mechanism of carbonic anhydrase ［J］. Pharmacology and Therapeutics, 1997, 74 (1): 1-20.
[7]	Liu Z, Bartlow P, Dilmore R M, et al. Production, purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum ［J］. Biotechnology Progress, 2009, 25 (1): 68-74.
[8]	Kim I G, Jo B H, Kang D G, et al. Biomineralizationbased conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase ［J］. Chemosphere, 2012, 87 (10): 1091-1096.
[9]	Kanth B K, Min K, Kumari S, et al. Expression and characterization of codonoptimized carbonic anhydrase from Dunaliella species for CO2 sequestration application ［J］. Applied biochemistry and biotechnology, 2012, 167 (8): 2341-2356.
[10]	Achal V, Pan X. Characterization of urease and carbonic anhydrase producing bacteria and their role in calcite precipitation ［J］. Current microbiology, 2011, 62 (3): 894-902.
[11]	Dhami N K, Reddy M S, Mukherjee A. Synergistic role of bacterial urease and carbonic anhydrase in carbonate mineralization ［J］. Applied biochemistry and biotechnology, 2014, 172 (5): 2552-2561.
[12]	李为, 曹龙, 周蓬蓬, 等. 温度对细菌碳酸酐酶催化碳酸钙沉积的影响［J］. 2013, 41(4): 371-377.
[13]	崔建东, 李莹, 姬晓元, 等. 静电纺丝制备中空纤维原位固定化碳酸酐酶用于二氧化碳的吸收［J］. 高等学校化学学报, 2014, 35(9): 1999-2006.
[14]	Xu Q L, Zhang C H, Li F C, et al. Arthrobacter sp. strain MF-2 induces high-mg calcite formation: Mechanism and implications for carbon fixation ［J］. Geomicrobiology Journal, 2017, 34 (2): 157-165.
[15]	Li W, Chen W S, Zhou P P, et al. Influence of initial pH on the precipitation and crystal morphology of calcium carbonate induced by microbial carbonic anhydrase ［J］. Colloids and Surfaces B: Biointerfaces, 2013, 102 (1):281-287.
[16]	鲍士旦. 土壤农化分析 (第2版)［M］. 农业出版社, 1981: 130-132.
[17]	Reinke L A, Moyer M J. p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol ［J］. Drug Metabolism and Disposition, 1985, 13 (5): 548-552.
[18]	张道勇, 潘响亮, 张京梅. 环境因子对Synechocystis sp.钙化动力学的影响［J］. 矿物岩石地球化学通报, 2008, 27 (2): 105-111.
[19]	Kontoyannis C G, Vagenas N V. Calcium carbonate phase analysis using XRD and FTRaman spectroscopy［J］. The Analyst. 2000,125(2):251-255.
[20]	Ridgwell A, Zeebe R E. The role of the global carbonate cycle in the regulation and evolution of the Earth system ［J］. Earth and Planetary Science Letters, 2005, 234 (3-4): 299-315.
[21]	Dupraz C, Reid R P, Braissant O, et al. Processes of carbonate precipitation in modern microbial mats ［J］. Earth-Science Reviews, 2009, 96 (3): 141-162.
[22]	Arp G, Reimer A, Reitner J. Photosynthesis-induced biofilm calcification and calcium concentrations in Phanerozoic oceans ［J］. Science, 2001, 292 (5522): 1701-1704.
[23]	Faridi S, Satyanarayana T. Characteristics of recombinant alpha-carbonic anhydrase of polyextremophilic bacterium Bacillus halodurans TSLV1 ［J］. International journal of biological macromolecules, 2016, 89: 659-668.
[24]	Elder I, Han S, Tu C K, et al. Activation of carbonic anhydrase II by active-site incorporation of histidine analogs ［J］. Archives of Biochemistry and Biophysics. 2004, 421 (2): 283-289.
[25]	Power I M, Harrison A L, Dipple G M, et al. Carbon sequestration via carbonic anhydrase facilitated magnesium carbonate precipitation ［J］. International Journal of Greenhouse Gas Control, 2013, 16: 145-155.
[26]	雷云. 球霰石型碳酸钙的研究进展［J］. 长江大学学报, 2014 (34): 35-39.
[27]	李福春, 郭文文. 三种好氧细菌诱导碳酸钙矿物的形成［J］. 南京大学学报(自然科学), 2013, 49 (6): 665-672.
[28]	马芳. 赖氨酸芽孢杆菌和节杆菌作用下碳酸盐矿物的形成［D］. 南京: 南京农业大学, 2014.