本文關(guān)鍵詞：真菌誘導碳酸鈣沉淀及其在土壤重金屬污染修復中的作用　出處：《華東師范大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 重金屬 鉻 鉛 Penicillium chrysogenum 生物修復 脲酶 方解石

【摘要】：鉻和鉛是現(xiàn)代工業(yè)生產(chǎn)活動中使用廣泛的金屬材料,隨著大量的原礦開采、冶煉、生產(chǎn)和排放,鉻和鉛最終在土壤中逐漸累積并造成土壤重金屬污染。傳統(tǒng)治理土壤鉻/鉛污染的方法不同程度地存在高能耗、高投入、二次污染等問題。這些治理技術(shù)的固有缺點使得人們不斷追求一種安全性更高、費用更低、對土壤環(huán)境擾動更小的修復技術(shù)。而生物修復技術(shù),包括微生物修復、植物修復、動物修復或聯(lián)合修復技術(shù),因具有良好的社會、生態(tài)綜合效益,正逐漸成為目前重金屬污染土壤修復的研究重點。其中,基于微生物誘導碳酸鈣沉淀(MICP)的新型重金屬修復技術(shù)是近年來最為受到關(guān)注的生物修復技術(shù)之一,被認為具有廣闊的發(fā)展前景和研究價值。作為一種微生物土壤重金屬原位修復新技術(shù),MICP作用的基本原理是:在微生物脲酶作用下,底物尿素被水解為NH4+和CO_3~(2-),其中NH4+有助于提升環(huán)境介質(zhì)pH,生成的CO_3~(2-)則能借助堿性環(huán)境沉淀介質(zhì)中的某些游離態(tài)重金屬離子。另一方面,Ca~(2+)的與CO_3~(2-)結(jié)合并析出的過程中也可將其他共存的重金屬離子共沉淀。相對于游離態(tài)重金屬,碳酸鹽形式的重金屬的生物可利用性和可遷移性大大降低,其生物毒性也隨之降低。大量實驗室內(nèi)研究證明,MICP能夠通過將目標污染物轉(zhuǎn)化為生物可利用性低且化學性質(zhì)更加穩(wěn)定的形態(tài),高效修復包括鉛、銅、鎘、鎳等金屬以及Sr、U等放射性核素在內(nèi)的污染物。但是,目前對于該技術(shù)的理論和實驗研究普遍關(guān)注原核生物以及其在去除單原子結(jié)構(gòu)金屬污染物的可行性,而真菌誘導的碳酸鹽沉淀機制及其對含氧形態(tài)復雜重金屬污染物的修復機制則缺乏相應(yīng)的研究。與細菌相比,真菌通常具有更強的環(huán)境耐受性、更快的生長速度、更大的生物量、更高的遺傳多樣性等優(yōu)勢,然而關(guān)于真菌參與的碳酸鹽礦化固定重金屬方面的研究卻十分有限。此外在重金屬污染修復領(lǐng)域,現(xiàn)有研究只證明了 MICP技術(shù)對于單原子形式的簡單重金屬離子,如Pb~(2+),Cu~(2+),Sr~(2+),Ni~(2+)等有顯著的去除效果,而對于另一類含氧形態(tài)污染物如CrO42-等的修復潛能以及修復機制相關(guān)研究尚為空白。因此,揭示真菌誘導碳酸鈣礦化過程在修復環(huán)境重金屬污染方面的可行性和機制,對于未來研發(fā)、設(shè)計和完善實地修復方案均具有重要的理論指導意義和參考價值。本研究將重點關(guān)注一株污泥中分離得到的耐鉻/鉛、具有脲酶活性的土著絲狀真菌,經(jīng)過分子鑒定種屬并初步優(yōu)化產(chǎn)脲酶特性后,分別在搖瓶實驗和土壤實驗中研究該菌誘導碳酸鹽沉淀過程對Cr(Ⅵ)和Pb的修復效果以及修復機理。本研究的主要內(nèi)容和結(jié)論如下:自污泥中篩選分離得到的一株產(chǎn)脲酶絲狀真菌CS1。經(jīng)MIC測定,該菌株分別對高達150mg/L和400mg/L的Cr(Ⅵ)和Pb有耐受能力。對該菌株提取核DNA并擴增其rRNA的5'片段,并進行基因測序,并將測序結(jié)果與GeneBank中的數(shù)據(jù)進行比對和計算遺傳差異,然后以最大簡約法建立系統(tǒng)發(fā)育樹對其歸屬。最終結(jié)果顯示該菌株屬于Penicillium chrysogenum.Penicillium chrysogenum是自然界中廣泛存在的真菌之一,且對極端環(huán)境有著較好的耐性。在污染修復、產(chǎn)酶工程等多個領(lǐng)域中,Penicillium chrysogenum均有豐富的應(yīng)用。由于脲酶是MICP過程中的核心酶,在獲取實驗菌株后設(shè)計了條件優(yōu)化實驗以初步確定P.chrysogenumCS1最適生長和最優(yōu)產(chǎn)酶條件。其中單因素實驗用于確定最佳產(chǎn)酶碳源和氮源,正交實驗用于確定各因素(碳源和氮源、初始pH、溫度、Ni添加量)的最適水平。結(jié)果顯示,最適宜P.chrysogenum CS1生長和脲酶產(chǎn)量的碳源和氮源分別為蔗糖和酵母浸膏。正交實驗結(jié)果表明,當蔗糖添加量為20g/L,酵母浸膏添加量為10g/L,初始pH為6.5-7.5,培養(yǎng)溫度為30℃,且添加Ni濃度為10 mg/L時,真菌能達到的最大生長量和脲酶產(chǎn)量,分別為6.54 g 和 36.91 U/ml。在進行正式真菌MICP去除重金屬實驗之前,進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的預實驗。預實驗結(jié)果表明,Pb~(2+)能迅速與上清液中的CO_3~(2-)離子結(jié)合并生成白色不溶物沉淀,而Cr(Ⅵ)不能經(jīng)此途徑去除。隨后,開展搖瓶實驗以研究產(chǎn)脲酶絲狀真菌P.chrysogenumCS1誘導下的碳酸鈣礦化過程對Cr(Ⅵ)和Pb的去除效果和機理。研究發(fā)現(xiàn),由于重金屬對真菌P.chrysogenum CS1的毒害作用,在低濃度組別中,pH、脲酶活性以及重金屬去除率均高于高濃度組,但是MICP處理可削弱重金屬的生物毒性從而獲得更高的脲酶產(chǎn)量。同時,經(jīng)過MICP處理的產(chǎn)脲酶絲狀真菌P.chrysogenum CS1對Cr(VI)和Pb的去除率均明顯高于對照組。此外,在MICP實驗組中P.chrysogenum CS1菌絲對Cr和Pb的富集量分別提高了 32%-42.2%和29.9%-61.4%。進一步進行顯微鏡和掃描電鏡觀察發(fā)現(xiàn),在MICP處理組中有大量形態(tài)豐富的晶體形成,大部分為緊緊粘附于菌絲的小顆粒型晶體并且大部分菌絲被這種堅硬的礦物質(zhì)外殼所覆蓋,同時也有少數(shù)為較大顆粒的散落的斜六方晶型礦物,顯示了與已有細菌MICP研究結(jié)果不同的現(xiàn)象。同時也說明,真菌的菌絲結(jié)構(gòu)在礦物生成過程中能為其提供結(jié)構(gòu)支持、粘結(jié)和包裹作用,明顯優(yōu)于單細胞細菌,這極大地方便了水體修復中后續(xù)的分離步驟。能譜分析顯示,在空白組樣品中礦物的元素組成為C、O、Ca,以及微量的C1;而在MICP組樣品中同時還分別檢測出Pb(3.59%)和Cr(1.29%)。該結(jié)果說明了含重金屬碳酸鈣礦物的生成是重金屬去除效率提高的主要原因。同時,還有N、Na、P、S、K等微量元素檢出,說明細胞表面大分子物質(zhì)參與了含重金屬碳酸鈣礦物的形成。在Pb-MICP樣品中,還觀察到一些球形的、大小均一的礦物顆粒,經(jīng)能譜分析其為組成元素僅僅為C、O、Pb的碳酸鉛。而在Cr-MICP樣品中,并未發(fā)現(xiàn)僅由C、O、Cr元素組成的礦化產(chǎn)物,這進一步佐證了 Cr(Ⅵ)不同于Pb的礦化途徑。為了進一步探究Cr(Ⅵ)的礦化途徑,對菌絲及礦物晶體進行礦物學特性分析。傅里葉紅外光譜表明,在控制組中重金屬主要與真菌細胞表面的大分子物質(zhì)如多聚糖、蛋白質(zhì)、糖蛋白、磷脂中的官能團(氨基、羧基、磷酸酯類)進行配位耦合,這屬于生物吸附效應(yīng)。而在MICP組樣品中,這些官能團的吸附效應(yīng)依然存在但是強度減弱,同時還發(fā)現(xiàn)了大量碳酸鹽的生成。該結(jié)果進一步佐證了在MICP處理下,生物礦化作用生成碳酸鹽沉淀是重金屬去除效率提升的主要原因。此外,Pb-MICP光譜還指出Pb_3(OH)_4C0_3的存在;而在Cr-MICP光譜中,歸屬于CrO_4~(2-)離子團的特征峰表明Cr(Ⅵ)是以CrO_4~(2-)形式存在于礦物中。粉末X射線衍射譜比對結(jié)果表明,在MICP處理下方解石為主要礦物成分,同時還有少量球霰石、草酸鈣。在Pb-MICP樣品中,Pb以水合碳酸鉛礦物形式存在,進一步佐證了 Pb以碳酸鉛礦物沉淀方式從水體中去除。而在Cr-MICP樣品中,發(fā)現(xiàn)了鉻氧碳酸鈣鹽礦物所屬特征峰以及明顯的方解石晶格的變形,說明CrO42-通過取代方解石中的部分C和O位置而整合進入方解石晶格中,形成穩(wěn)定形態(tài)的含鉻碳酸鈣礦物。據(jù)此,本研究提出了在真菌誘導碳酸鈣沉淀過程中Cr(Ⅵ)礦化的可能機制。首先,Cr0_4~(2-)被菌絲細胞表面的大分子有機物如多聚糖、蛋白質(zhì)、糖蛋白、磷脂中的配位官能團(氨基、羧基、磷酸酯類)吸附并逐步積累。隨著真菌脲酶降解尿素產(chǎn)生大量的C032-和pH提升后,不斷釋放的Ca2+和C032-在細胞表面附近形成局部過飽和并開始沉淀,逐步將Cr0_4~(2-)包裹在碳酸鈣晶體中。另一方面,本研究認為真菌誘導產(chǎn)生的生物碳酸鈣是一種典型的多孔材料,并對重金屬有著優(yōu)良的吸附作用。被生物碳酸鈣吸附后的Cr0_4~(2-)能隨著晶體的不斷生長而逐步被包裹、固定。最后,進行土壤重金屬修復實驗,初步評價了真菌P.chrysogenum CS1誘導碳酸鈣沉淀在土壤介質(zhì)中對重金屬污染物的固定作用。結(jié)果表明,MICP處理下的大部分可交換態(tài)Pb和Cr轉(zhuǎn)化為化學性質(zhì)更加穩(wěn)定、生物可利用性更小的碳酸鹽結(jié)合態(tài),其生物可利用性和可移動性大幅度降低。同時發(fā)現(xiàn)在土壤的不同深度(0-20cm),礦化修復效果有所降低,但是幅度變化不大。針對MICP處理后的土壤孔隙率研究結(jié)果表明,MICP處理會在一定程度上降低土壤孔隙率,尤其在土壤表層。這可能是由生物礦化形成的碳酸鹽顆粒填充了土壤顆粒間的空隙導致的。但是總體上,MICP處理過后土壤孔隙率依舊保持在40%以上,且并未導致土壤鈣化板結(jié),該條件依舊適合大部分微生物和植物生長。以上結(jié)果說明,絲狀真菌P.chrysogenum CS1誘導的碳酸鈣礦化沉淀過程是一種有效的土壤重金屬污染修復技術(shù)。
[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.

【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X53

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本文編號：1434355