真菌诱导碳酸钙沉淀及其在土壤重金属污染修复中的作用

发布时间：2018-01-16 18:40

本文关键词：真菌诱导碳酸钙沉淀及其在土壤重金属污染修复中的作用　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：铬和铅是现代工业生产活动中使用广泛的金属材料,随着大量的原矿开采、冶炼、生产和排放,铬和铅最终在土壤中逐渐累积并造成土壤重金属污染。传统治理土壤铬/铅污染的方法不同程度地存在高能耗、高投入、二次污染等问题。这些治理技术的固有缺点使得人们不断追求一种安全性更高、费用更低、对土壤环境扰动更小的修复技术。而生物修复技术,包括微生物修复、植物修复、动物修复或联合修复技术,因具有良好的社会、生态综合效益,正逐渐成为目前重金属污染土壤修复的研究重点。其中,基于微生物诱导碳酸钙沉淀(MICP)的新型重金属修复技术是近年来最为受到关注的生物修复技术之一,被认为具有广阔的发展前景和研究价值。作为一种微生物土壤重金属原位修复新技术,MICP作用的基本原理是:在微生物脲酶作用下,底物尿素被水解为NH4+和CO_3~(2-),其中NH4+有助于提升环境介质pH,生成的CO_3~(2-)则能借助碱性环境沉淀介质中的某些游离态重金属离子。另一方面,Ca~(2+)的与CO_3~(2-)结合并析出的过程中也可将其他共存的重金属离子共沉淀。相对于游离态重金属,碳酸盐形式的重金属的生物可利用性和可迁移性大大降低,其生物毒性也随之降低。大量实验室内研究证明,MICP能够通过将目标污染物转化为生物可利用性低且化学性质更加稳定的形态,高效修复包括铅、铜、镉、镍等金属以及Sr、U等放射性核素在内的污染物。但是,目前对于该技术的理论和实验研究普遍关注原核生物以及其在去除单原子结构金属污染物的可行性,而真菌诱导的碳酸盐沉淀机制及其对含氧形态复杂重金属污染物的修复机制则缺乏相应的研究。与细菌相比,真菌通常具有更强的环境耐受性、更快的生长速度、更大的生物量、更高的遗传多样性等优势,然而关于真菌参与的碳酸盐矿化固定重金属方面的研究却十分有限。此外在重金属污染修复领域,现有研究只证明了 MICP技术对于单原子形式的简单重金属离子,如Pb~(2+),Cu~(2+),Sr~(2+),Ni~(2+)等有显著的去除效果,而对于另一类含氧形态污染物如CrO42-等的修复潜能以及修复机制相关研究尚为空白。因此,揭示真菌诱导碳酸钙矿化过程在修复环境重金属污染方面的可行性和机制,对于未来研发、设计和完善实地修复方案均具有重要的理论指导意义和参考价值。本研究将重点关注一株污泥中分离得到的耐铬/铅、具有脲酶活性的土著丝状真菌,经过分子鉴定种属并初步优化产脲酶特性后,分别在摇瓶实验和土壤实验中研究该菌诱导碳酸盐沉淀过程对Cr(Ⅵ)和Pb的修复效果以及修复机理。本研究的主要内容和结论如下:自污泥中筛选分离得到的一株产脲酶丝状真菌CS1。经MIC测定,该菌株分别对高达150mg/L和400mg/L的Cr(Ⅵ)和Pb有耐受能力。对该菌株提取核DNA并扩增其rRNA的5'片段,并进行基因测序,并将测序结果与GeneBank中的数据进行比对和计算遗传差异,然后以最大简约法建立系统发育树对其归属。最终结果显示该菌株属于Penicillium chrysogenum.Penicillium chrysogenum是自然界中广泛存在的真菌之一,且对极端环境有着较好的耐性。在污染修复、产酶工程等多个领域中,Penicillium chrysogenum均有丰富的应用。由于脲酶是MICP过程中的核心酶,在获取实验菌株后设计了条件优化实验以初步确定P.chrysogenumCS1最适生长和最优产酶条件。其中单因素实验用于确定最佳产酶碳源和氮源,正交实验用于确定各因素(碳源和氮源、初始pH、温度、Ni添加量)的最适水平。结果显示,最适宜P.chrysogenum CS1生长和脲酶产量的碳源和氮源分别为蔗糖和酵母浸膏。正交实验结果表明,当蔗糖添加量为20g/L,酵母浸膏添加量为10g/L,初始pH为6.5-7.5,培养温度为30℃,且添加Ni浓度为10 mg/L时,真菌能达到的最大生长量和脲酶产量,分别为6.54 g 和 36.91 U/ml。在进行正式真菌MICP去除重金属实验之前,进行了以真菌脲酶水解尿素后的富含CO_3~(2-)上清液去除Cr(Ⅵ)和Pb的预实验。预实验结果表明,Pb~(2+)能迅速与上清液中的CO_3~(2-)离子结合并生成白色不溶物沉淀,而Cr(Ⅵ)不能经此途径去除。随后,开展摇瓶实验以研究产脲酶丝状真菌P.chrysogenumCS1诱导下的碳酸钙矿化过程对Cr(Ⅵ)和Pb的去除效果和机理。研究发现,由于重金属对真菌P.chrysogenum CS1的毒害作用,在低浓度组别中,pH、脲酶活性以及重金属去除率均高于高浓度组,但是MICP处理可削弱重金属的生物毒性从而获得更高的脲酶产量。同时,经过MICP处理的产脲酶丝状真菌P.chrysogenum CS1对Cr(VI)和Pb的去除率均明显高于对照组。此外,在MICP实验组中P.chrysogenum CS1菌丝对Cr和Pb的富集量分别提高了 32%-42.2%和29.9%-61.4%。进一步进行显微镜和扫描电镜观察发现,在MICP处理组中有大量形态丰富的晶体形成,大部分为紧紧粘附于菌丝的小颗粒型晶体并且大部分菌丝被这种坚硬的矿物质外壳所覆盖,同时也有少数为较大颗粒的散落的斜六方晶型矿物,显示了与已有细菌MICP研究结果不同的现象。同时也说明,真菌的菌丝结构在矿物生成过程中能为其提供结构支持、粘结和包裹作用,明显优于单细胞细菌,这极大地方便了水体修复中后续的分离步骤。能谱分析显示,在空白组样品中矿物的元素组成为C、O、Ca,以及微量的C1;而在MICP组样品中同时还分别检测出Pb(3.59%)和Cr(1.29%)。该结果说明了含重金属碳酸钙矿物的生成是重金属去除效率提高的主要原因。同时,还有N、Na、P、S、K等微量元素检出,说明细胞表面大分子物质参与了含重金属碳酸钙矿物的形成。在Pb-MICP样品中,还观察到一些球形的、大小均一的矿物颗粒,经能谱分析其为组成元素仅仅为C、O、Pb的碳酸铅。而在Cr-MICP样品中,并未发现仅由C、O、Cr元素组成的矿化产物,这进一步佐证了 Cr(Ⅵ)不同于Pb的矿化途径。为了进一步探究Cr(Ⅵ)的矿化途径,对菌丝及矿物晶体进行矿物学特性分析。傅里叶红外光谱表明,在控制组中重金属主要与真菌细胞表面的大分子物质如多聚糖、蛋白质、糖蛋白、磷脂中的官能团(氨基、羧基、磷酸酯类)进行配位耦合,这属于生物吸附效应。而在MICP组样品中,这些官能团的吸附效应依然存在但是强度减弱,同时还发现了大量碳酸盐的生成。该结果进一步佐证了在MICP处理下,生物矿化作用生成碳酸盐沉淀是重金属去除效率提升的主要原因。此外,Pb-MICP光谱还指出Pb_3(OH)_4C0_3的存在;而在Cr-MICP光谱中,归属于CrO_4~(2-)离子团的特征峰表明Cr(Ⅵ)是以CrO_4~(2-)形式存在于矿物中。粉末X射线衍射谱比对结果表明,在MICP处理下方解石为主要矿物成分,同时还有少量球霰石、草酸钙。在Pb-MICP样品中,Pb以水合碳酸铅矿物形式存在,进一步佐证了 Pb以碳酸铅矿物沉淀方式从水体中去除。而在Cr-MICP样品中,发现了铬氧碳酸钙盐矿物所属特征峰以及明显的方解石晶格的变形,说明CrO42-通过取代方解石中的部分C和O位置而整合进入方解石晶格中,形成稳定形态的含铬碳酸钙矿物。据此,本研究提出了在真菌诱导碳酸钙沉淀过程中Cr(Ⅵ)矿化的可能机制。首先,Cr0_4~(2-)被菌丝细胞表面的大分子有机物如多聚糖、蛋白质、糖蛋白、磷脂中的配位官能团(氨基、羧基、磷酸酯类)吸附并逐步积累。随着真菌脲酶降解尿素产生大量的C032-和pH提升后,不断释放的Ca2+和C032-在细胞表面附近形成局部过饱和并开始沉淀,逐步将Cr0_4~(2-)包裹在碳酸钙晶体中。另一方面,本研究认为真菌诱导产生的生物碳酸钙是一种典型的多孔材料,并对重金属有着优良的吸附作用。被生物碳酸钙吸附后的Cr0_4~(2-)能随着晶体的不断生长而逐步被包裹、固定。最后,进行土壤重金属修复实验,初步评价了真菌P.chrysogenum CS1诱导碳酸钙沉淀在土壤介质中对重金属污染物的固定作用。结果表明,MICP处理下的大部分可交换态Pb和Cr转化为化学性质更加稳定、生物可利用性更小的碳酸盐结合态,其生物可利用性和可移动性大幅度降低。同时发现在土壤的不同深度(0-20cm),矿化修复效果有所降低,但是幅度变化不大。针对MICP处理后的土壤孔隙率研究结果表明,MICP处理会在一定程度上降低土壤孔隙率,尤其在土壤表层。这可能是由生物矿化形成的碳酸盐颗粒填充了土壤颗粒间的空隙导致的。但是总体上,MICP处理过后土壤孔隙率依旧保持在40%以上,且并未导致土壤钙化板结,该条件依旧适合大部分微生物和植物生长。以上结果说明,丝状真菌P.chrysogenum CS1诱导的碳酸钙矿化沉淀过程是一种有效的土壤重金属污染修复技术。
[Abstract]:Lead and chromium is the use of metal materials of the modern industrial production activities, with a large number of ore mining, smelting, production and emissions of lead and chromium in the soil gradually accumulated and eventually caused pollution of soil heavy metals. The traditional management method of soil Cr / lead pollution in different degree in high energy consumption, high investment, two pollution problems. The shortcomings inherent in these governance technology makes people continue to pursue a higher security, lower cost, smaller disturbance restoration technology on soil environment. Bioremediation technology, including microbial remediation, phytoremediation, animal repair or joint repair technology, because of its good social and comprehensive benefits of ecology, is gradually become the focus of research in remediation of heavy metal contaminated soil. The microbial induced carbonate precipitation (MICP) based on the model of heavy metal remediation technology in recent years is the most concern for bioremediation One of the techniques is thought to have a broad prospect and study value. As a kind of microorganism of soil heavy metals in situ remediation technology, the basic principle of MICP effect is: in microbial urease under the action of urea is hydrolyzed into NH4+ substrate and CO_3~ (2-) NH4+, which helps to improve the environment of medium pH, the generated CO_3~ (2-) can use alkaline precipitation medium some free heavy metal ions. On the other hand, Ca~ (2+) and CO_3~ (2-) and the combination of precipitation can also be other coexisting metal ions coprecipitation. Compared with the free state of heavy metal, heavy metal carbonate form and bioavailability mobility is greatly reduced, the biological toxicity is reduced. A large number of studies prove that the laboratory, MICP can be transformed into the form of target pollutants can be more stable with low biological and chemical properties, including efficient repair Lead, copper, cadmium, nickel and other metals as well as Sr, U, radionuclide pollutants. However, the current theoretical and experimental studies on the widespread concern of the prokaryotes and its feasibility in removal of single atomic structure of metallic pollutants, but the fungi induced carbonate precipitation mechanism and repair mechanism of oxygen containing complex forms of heavy metal pollutants the lack of corresponding research. Compared with bacteria, environmental tolerance fungi usually has a stronger, faster growth, greater biomass, genetic diversity advantage more, but the research for the carbonate mineralization of heavy metals in fixed fungi is very limited. In addition in the field of remediation of heavy metal pollution, existing research only the MICP technique for simple single atom forms of heavy metal ions, such as Pb~ (2+), Cu~ (2+), Sr~ (2+), Ni~ (2+), the removal effect is remarkable, and for another Study on repairing potential and repair mechanisms related with oxygen form of pollutants such as CrO42- is still blank. Therefore, the feasibility and mechanism of fungal induced calcium carbonate mineralization process in heavy metal polluted environment remediation, for future research and development, design and improvement of field rehabilitation program has important theoretical significance and reference value in this study. Will focus on the separation of chromium / lead resistant strain in sludge, filamentous fungi with native urease activity, the molecular identification of species and preliminary optimization of urease producing characteristics, respectively in shake flask experiments and experimental study of the soil bacteria induced carbonate precipitation process of Cr (VI) and Pb repair and repair effect the mechanism. The main contents and conclusions of this study are as follows: since the sludge was isolated a strain of urease producing filamentous fungi CS1. measured by MIC, the strain of up to 150mg/L and 400mg/ respectively L Cr (VI) with tolerance and Pb. The strain DNA and its rRNA nuclear extract amplified 5'fragments were sequenced, and the sequencing results and the GeneBank data were compared and calculated genetic differences, and then to the maximum parsimony phylogenetic tree was constructed on the final results show that the attribution. Penicillium chrysogenum.Penicillium chrysogenum is a fungal strain belongs to one of the existing widely in nature, and has a good tolerance to extreme environment. In many areas of pollution remediation, enzyme engineering, application of Penicillium chrysogenum are abundant. Because the urease is core enzyme of MICP, in order to obtain experimental strains after optimized experimental design in order to determine the optimum growth initial P.chrysogenumCS1 and the optimal conditions of enzyme production. For the single factor experiment to determine the best enzyme production of carbon source and nitrogen source, the orthogonal experiment was used to determine the various factors (carbon The source and the nitrogen source, initial pH, temperature, dosage of Ni) optimum level. The results showed that the optimum carbon source and nitrogen source P.chrysogenum CS1 growth and the yield of urease were sucrose and yeast extract. The experimental results showed that when sucrose 20g/L, yeast extract was added into 10g/L, the initial pH 6.5-7.5, culture temperature was 30 C, and the added Ni concentration was 10 mg/L, the fungus can achieve maximum growth amount and urease yield were 6.54 g and 36.91 U/ml. before the fungal MICP was heavy metal removal experiment, the fungus urease hydrolysis of urea containing CO_3~ (2-) Cr (supernatant removal VI) pre experiment and Pb. The results of pre experiment, Pb~ (2+) can rapidly and the supernatant of CO_3~ (2-) ion binding and generate white insoluble precipitates, while Cr (VI) not removed via this way. Then, carry out the shake flask experiments to study urease producing filamentous fungi P. Calcium carbonate mineralization induced by chrysogenumCS1 on Cr (VI) removal effect and mechanism and Pb. The study found that the toxic effect of heavy metal on the fungus P.chrysogenum CS1, in the low concentration group, pH, the removal rate of urease activity and heavy metals were higher than that of the high concentration group, but MICP treatment can weaken the biological toxicity of heavy metals to to obtain higher yield of urease. At the same time, after MICP treatment of CS1 urease producing fungus P.chrysogenum Cr (VI) and the removal rate of Pb were significantly higher than control group. In addition, the MICP in the experimental group the concentration of P.chrysogenum CS1 and Pb Cr of hyphae were enhanced by 32%-42.2% and 29.9%-61.4%. further microscope and scanning electron microscope observation shows that a large number of rich crystal formation in the MICP group, most closely adhered to the hyphae of small particles of crystal and most of this kind of hard mycelium Covered with mineral shell, also a few scattered oblique six party crystal mineral larger particles, shows different from the existing research results of bacterial MICP phenomenon. At the same time that the hyphal structure of fungi can provide support for the structure in the mineral formation process, bonding and inclusion effect, significantly better than the single cell the bacteria, which greatly facilitates the subsequent separation step water remediation. Energy spectrum analysis showed that the mineral elements in the samples in the control group consisting of C, O, Ca, and trace C1; while the MICP group samples were also detected in Pb (3.59%) and Cr (1.29%) of the results. The generation of heavy metal containing calcium carbonate minerals is the main reason for the heavy metal removal efficiency. At the same time, there are N, Na, P, S, K and other trace elements detected that cell surface molecules involved in the formation of heavy metal containing calcium carbonate minerals. In the sample of Pb-MICP, Also observed some spherical minerals, uniform particle size, by energy spectrum analysis for the elements of only C, O, Pb and lead carbonate. In Cr-MICP samples, found only by C, O, Cr mineralization product elements, this is further evidence of Cr (VI) mineralization in different ways in Pb. In order to further explore Cr (VI) mineralization pathways, mineralogical characteristics analysis of hyphae and mineral crystals. Fourier transform infrared spectroscopy showed that the heavy metals in the control group with fungal cell surface macromolecules such as polysaccharides, proteins, glycoproteins, functional groups in phospholipid (amino, carboxyl and phosphate esters) with ligand coupling, which belongs to the biological adsorption effect. While in the MICP group in the sample, the adsorption effect of these functional groups still exist but weakened, also found to generate a lot of carbonate. The results further indicated in the treatment of MICP, biological Mineralization formation of carbonate precipitation is the main reason for heavy metal removal efficiency. In addition, it is pointed out that the Pb_3 spectrum of Pb-MICP (OH) _4C0_3; and in the Cr-MICP spectrum, belonging to CrO_4~ (2-) ion clusters show that the characteristic peaks of Cr (VI) to CrO_4~ (2-) in the form of mineral powder X. The X-ray diffraction spectra results showed that, in MICP calcite as the main mineral composition, and there is a small amount of vaterite, calcium oxalate. In the sample of Pb-MICP, Pb in hydrated lead carbonate mineral form, further evidence of the removal of Pb from water to lead carbonate mineral precipitation. In Cr-MICP samples, found chromium oxide calcium carbonate minerals are characteristic peaks and deformation of the calcite lattice obviously, indicating that CrO42- by replacing part of C and O in calcite position integrated into calcite. The chromium containing calcium carbonate minerals to form a stable form. Accordingly, this study proposes induced calcium carbonate precipitation process in fungi Cr (VI) the possible mechanism of mineralization. Firstly, Cr0_4~ (2-) are organic molecules such as mycelial cell surface polysaccharides, protein, glycoprotein, phospholipid ligand functional groups (carboxyl, amino, phosphorus esters) and stepwise adsorption with the accumulation of fungal degradation of urea. Urease produced large amounts of C032- and pH after the upgrade, the continuous release of Ca2+ and C032- on the cell surface is formed near the local supersaturation and precipitation, gradually Cr0_4~ (2-) wrapped in calcium carbonate crystals. On the other hand, this study suggests that fungi induced by calcium carbonate is a kind of biological the typical porous materials, and has a good adsorption effect on heavy metals. By biological calcium carbonate after adsorption of Cr0_4~ (2-) with the continuous growth of the crystal can gradually be wrapped, fixed. Finally, the soil heavy metal remediation experiment, preliminary evaluation Fungus P.chrysogenum CS1 induced calcium carbonate precipitation in soil medium to heavy metal contaminants fixation. The results showed that most of the exchangeable Pb and Cr into a more stable chemical properties under MICP treatment, the bioavailability of smaller carbonate bound, its bioavailability and mobility can be greatly reduced. At the same time found in different depth of soil (0-20cm), mineralization repair effect decreased, but the rate changes little. According to the results of soil porosity after MICP treatment showed that MICP treatment could reduce the soil porosity in a certain extent, especially in the surface soil. This may be the carbonate particles formed by biomineralization to fill the void of soil particles the lead. But on the whole, MICP treatment after the soil porosity still remained above 40%, and did not lead to the calcification of soil compaction, the condition is still suitable for the Department Microorganism and plant growth. The above results indicate that the P.chrysogenum CS1 induced mineralization of calcium carbonate in filamentous fungi is an effective remediation technology for heavy metal pollution in soil.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X53

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