本文選題：銀納米粒子 + 生物合成　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：銀納米粒子(silver nanoparticles,AgNPs)具有許多獨(dú)特的物理化學(xué)特性,使其在光學(xué)、電子、催化以及醫(yī)藥領(lǐng)域等有著非常廣泛的應(yīng)用。過(guò)去的數(shù)十年,許多制備技術(shù)已經(jīng)能夠控制合成的銀納米粒子的粒徑、形狀和表面形貌。與傳統(tǒng)的物理化學(xué)方法相比,生物合成法利用細(xì)菌、真菌和植物等合成銀納米粒子,避免有毒化學(xué)試劑的使用,具有對(duì)環(huán)境無(wú)污染、成本低等優(yōu)點(diǎn),吸引了眾多研究者的關(guān)注。本文以海洋沉積物樣品作為實(shí)驗(yàn)對(duì)象,通過(guò)傳統(tǒng)的富集方法結(jié)合點(diǎn)種法分離拮抗菌,并篩選出一株拮抗菌,利用其產(chǎn)生的抑菌物質(zhì)合成銀納米粒子。利用紫外-可見分光光度法(UV-Visible spectrometry,UV-Vis)、掃描電子顯微鏡(Scanning electron microscopy,SEM)和X射線衍射分析(X-ray diffiraction,XRD)的方法對(duì)銀納米粒子進(jìn)行表征;以大腸桿菌和金黃色葡萄球菌作為抑菌對(duì)象,對(duì)銀納米粒子的抑菌性能進(jìn)行檢測(cè)。本實(shí)驗(yàn)以威海小石島自然海域(37° 31'2"N 122° 1'19" E)采集的底泥樣品作為實(shí)驗(yàn)對(duì)象,通過(guò)富集、稀釋涂布平板后,以大腸桿菌(Escherichia coli ATCC 25922)、金黃色葡萄球菌(Staphylococcus aureus ATCC 29213)、鮑曼不動(dòng)桿菌(Acinetobacter baumannii ATCC 19606)、副溶血弧菌(Vibrio parahaemolyticus ATCC 17802T)四株病原菌作為指示菌株,以點(diǎn)種法篩選出242株細(xì)菌,其中有177株細(xì)菌具有抑菌性�；�16SrDNA基因測(cè)序?qū)卓咕M(jìn)行鑒定,發(fā)現(xiàn)絕大多數(shù)菌株屬于厚壁菌門。從分離到的拮抗菌中,挑選出一株拮抗性強(qiáng)的細(xì)菌DXFD1,經(jīng)克隆測(cè)序得到其完整的16SrDNA序列(1474bp),經(jīng)比對(duì)分析,發(fā)現(xiàn)菌株DXFD1與枯草芽孢桿菌枯草亞種(sucillus subtilis subsp.subtilis NCIB 3610T)的 16SrDNA相似性最高(99.59%)。利用菌株DXFD1產(chǎn)生的抗菌物質(zhì)溶液與AgNO3溶液混合,置于磁力攪拌器上合成銀納米粒子,并對(duì)合成的銀納米粒子進(jìn)行UV-Vis光譜、SEM和XRD表征。結(jié)果顯示:銀納米粒子多為球形或近球形,粒徑分布在30-70 nm,含銀納米粒子的混合液的UV-Vis光譜顯示在480 nm處有吸收峰。以大腸桿菌和金黃色葡萄球菌為檢測(cè)菌株,對(duì)銀納米粒子的抑菌性能進(jìn)行檢測(cè)。結(jié)果表明:銀納米粒子在實(shí)驗(yàn)中表現(xiàn)出明顯的抑菌效果,對(duì)大腸桿菌和金黃色葡萄球菌的最小抑菌濃度均為5 μg/L,最小殺菌濃度為10 μg/mL。檢測(cè)去除表面附著物質(zhì)的銀納米粒子的抑菌活性,發(fā)現(xiàn)抑菌效果降低,結(jié)果表明抑菌物質(zhì)在合成銀納米粒子的過(guò)程中結(jié)合到粒子表面,提高銀納米粒子的抑菌性能。本文對(duì)從威海附近海域、文登鹽場(chǎng)、肥城精致鹽場(chǎng)樣品中分離到的編號(hào)為P131T、N62T、X7T、WDS2C4T與JZ3A21五株新菌進(jìn)行了多相分類學(xué)鑒定。菌株P(guān)131T分離自威海近海沉積物,為革蘭氏陰性菌、好氧、無(wú)滑動(dòng)性、細(xì)胞呈桿狀。菌株P(guān)131T的最適生長(zhǎng)溫度為28-30℃,最適pH為7.0-7.5,可耐受的鹽度最為2.0-3.0%(w/v)NaCl�；�16S rDNA序列的分析表明,菌株P(guān)131T屬于比齊奧氏菌屬,與屬內(nèi)物種的16SrDNA相似性范圍為94.6-97.0%。菌株P(guān)131T 主要脂肪酸組分為 iso-C15:0、iso-C15:0G、iso-C17:03-OH 和 iso-C171 ω9c。主要的極性脂成分包括磷脂酰乙醇胺、兩種氨脂質(zhì)、氨磷脂、磷脂和其他的脂質(zhì)。主要呼吸醌類型為MK-6�；蚪MDNA的G+C含量是36.7mol%。根據(jù)遺傳學(xué)和表型特征證據(jù),菌株P(guān)131T(=KCTC42587T=MCCC1H00124T)代表比齊奧氏菌屬的一個(gè)新物種,命名為沉積物比齊奧氏菌(Bizionia sediminis sp.nov.)。菌株N62T分離自自威海近海沉積物,為革蘭氏陰性、兼性厭氧、無(wú)運(yùn)動(dòng)性和細(xì)胞短桿狀。2216E固體培養(yǎng)基28℃培養(yǎng)2天,形成圓形、光滑、橘色和直徑約為0.6-1.5mm的單菌落。菌株N62T最適生長(zhǎng)溫度為28-30℃,最適pH為7.0-7.5和最適鹽度2.0-3.0%(w/v)NaCl。菌株N62T主要的脂肪酸組分是iso-C15:0、iso-C15:0G、iso-C17:03-OH 和 C17:13-OH。主要的呼吸醌類型是 MK-6。菌株N62T基因組DNA的G+C含量是35.3 mol%。主要的極性脂是磷脂酰乙醇胺、兩種氨脂質(zhì)、糖脂和三種其他的脂質(zhì)。基于16S rDNA序列的分析表明,菌株N62T屬于藏紅花黃色線菌科(Crocinitomicaceae),與寒冷微菌屬的物種的16SrDNA序列相似性范圍為95.8-97.2%。根據(jù)遺傳學(xué)和表型特征證據(jù),菌株N62T(=KCTC 42589T=MCCC1H00117T),代表寒冷微菌屬的一個(gè)新物種,命名為橙色寒冷微菌(Brumimicrobium aurantiacum sp.nov.)。菌株X7T分離自自威海近海沉積物,細(xì)胞革蘭氏陰性、無(wú)滑動(dòng)性、兼性厭氧和短桿狀。最適生長(zhǎng)溫度為28-30℃,最適pH為7.0和最適鹽度2.0-3.0%(w/v)NaCl。基于16S rDNA序列的分析表明,菌株X7T屬于鹽坑微菌屬,與Salinimicrobium gaetbulicola有著最高的 16SrDNA 序列相似性(96.3%)。主要脂肪酸為 anteiso-Ci5:0、iso-C15:0、anteiso-C17:1cω9c、iso-C17:1ω9c、C17;02-OH 和iso-C17;03-OH。主要極性脂是一種磷脂酰乙醇胺、一種磷脂、兩種氨脂質(zhì)和五種其他未確定的脂質(zhì)。主要呼吸醌類型是MK-6�；蚪MDNA的G+C含量是46.7 mol%。遺傳學(xué)、表型特征以及化學(xué)組分結(jié)果表明,菌株X7T(=KCTC 42585T=MCCC1H00115T)代表鹽坑微菌屬的一個(gè)新物種,命名為黃色鹽坑微菌(Salinimicrobium flavum sp.nov.)。菌株WDS2C4T和JZ3A21分別從文登鹽場(chǎng)、肥城精致鹽場(chǎng)的樣品分離到,細(xì)胞為革蘭氏陰性、長(zhǎng)桿狀和無(wú)滑動(dòng)性。生長(zhǎng)范圍:可耐受鹽度1.0-20.0%(w/v)NaCl(最適 6.0-10.0%),溫度 20-50℃(最適 37-40℃)和 pH6.5-9.5(最適 7.0-8.0)。菌株WDS2C4T和菌株JZ3A21主要脂肪酸組分均是cyclo-C19;0ω8c、summed features 8(C181 ω7cand/or C18:1 ω6c)、C18:0、C17:0、C16:0 和 11-methyl C18:1 ω7c。菌株WDS2C4T和JZ3A21主要的呼吸醌類型是Q-10。菌株WDS2C4T的基因組DNA的G+C含量為67.7 mol%,菌株JZ3A21的基因組DNA的G+C含量為67.3 mol%。菌株WDS2C4T主要極性脂成分是兩種磷脂、氨脂質(zhì)、磷脂酰甘油、磷脂酰膽堿和兩種糖脂。菌株 WDS2C4T與Rhodovulum marinum JCM 13300T 的 16S rDNA 序列相似性最高為 95.11%。與物種 Albidovulum xiamenense、Paracoccus aestuarii、Rhodobacter vinaykumarii 和Roseivivax pacificus 的模式菌株的相似性分別為94.75%、94.00%、93.88%和93.85%。根據(jù)16SrDNA序列建立的三個(gè)系統(tǒng)發(fā)育樹(Neighbour-joining,maximum-likelihood 和 maximurn-parsimony)表明,菌株WDS2C4T和JZ3A21位于Rhodobacteraceae科的單獨(dú)分支上。根據(jù)遺傳學(xué)和表型特征證據(jù),菌株 WDS2C4T(=KCTC 52227T=NBRC 112331T=MCCC 1H00148T)代表紅細(xì)菌科的一個(gè)新屬新種,命名為嗜鹽白色鹽桿菌(Albihalobacter halophilus gen.nov.sp.nov.)。
[Abstract]:Silver nanoparticles (AgNPs) has many unique physical and chemical properties, which makes it very widely used in the field of optics, electron, catalysis and medicine. In the past few decades, many preparation techniques have been able to control the size, shape and surface morphology of the synthesized silver nanoparticles. Compared with the method, biosynthesis method uses bacteria, fungi and plants to synthesize silver nanoparticles, avoids the use of toxic chemical reagents, has the advantages of no pollution to the environment and low cost, and attracts the attention of many researchers. A strain of antagonist was screened and silver nanoparticles were synthesized by the antibacterial substances produced by them. The silver nanoparticles were characterized by UV visible spectrophotometry (UV-Visible spectrometry, UV-Vis), scanning electron microscope (Scanning electron microscopy, SEM) and X ray diffraction analysis (X-ray diffiraction, XRD). The Bacteriostasis of silver nanoparticles was detected by Bacillus and Staphylococcus aureus as a bacteriostasis object. This experiment took the sediment samples collected in the Weihai small Shidao natural sea area (37 31'2 "N 122 1'19" E) as the experimental object, and by the enrichment and dilution coating plate, the Escherichia coli (Escherichia coli ATCC 25922) and golden yellow grapevine ball were used. Bacteria (Staphylococcus aureus ATCC 29213), Acinetobacter Bauman (Acinetobacter baumannii ATCC 19606), Vibrio parahaemolyticus (Vibrio parahaemolyticus ATCC 17802T) four strains of pathogenic bacteria as indicative strains, 242 strains of bacteria were screened by seed seed method, of which 177 bacteria were bacteriostasis. Based on the sequencing of 16SrDNA gene, the antagonistic bacteria were identified. It was found that most of the strains belonged to the phylum asiasis. From the isolated bacteria, a strain of resistant bacteria DXFD1 was selected and the complete 16SrDNA sequence (1474bp) was cloned and sequenced. The strain DXFD1 was similar to the 16SrDNA of Bacillus subtilis subspecies (sucillus subtilis subsp.subtilis NCIB 3610T). The highest (99.59%). The antibacterial substance produced by the strain DXFD1 is mixed with the AgNO3 solution, and the silver nanoparticles are synthesized on the magnetic stirrer, and the silver nanoparticles are characterized by UV-Vis, SEM and XRD. The results show that the silver nanoparticles are mostly spherical or near the spherical shape, the particle size distribution is 30-70 nm, and the mixing of silver nanoparticles is mixed. The UV-Vis spectrum of the liquid showed a peak of absorption at 480 nm. The Bacteriostasis of silver nanoparticles was detected with Escherichia coli and Staphylococcus aureus. The results showed that silver nanoparticles showed obvious bacteriostasis effect in the experiment, and the minimum inhibitory concentration for Escherichia coli and Staphylococcus aureus were 5 g/L. The bacteriostasis activity of the silver nanoparticles which removed the surface attachment material was detected by the small bactericidal concentration of 10 g/mL.. The results showed that the bacteriostatic effect was reduced. The results showed that the bacteriostatic substance was combined with the surface of the particles in the process of synthesizing silver nanoparticles and improved the antibacterial properties of the silver nanoparticles. This paper is a refined salt field from the sea area near Weihai, Wendeng salt field and Feicheng. Five strains of P131T, N62T, X7T, WDS2C4T and JZ3A21 were identified by multiphase taxonomy. The strain P131T was isolated from the coastal sediments of Weihai. It was Gram-negative bacteria, aerobic, non gliding, and rod-shaped. The optimum growth temperature of the strain P131T was 28-30, the optimum pH was 7.0-7.5, and the tolerable salinity was the most 2.0-3.0%. (w/v) NaCl. based on the analysis of 16S rDNA sequence, the strain P131T belongs to the genus of Bacillus, and the 16SrDNA resemblance to the internal species is the 94.6-97.0%. strain P131T main fatty acids in iso-C15:0, iso-C15:0G, iso-C17:03-OH, and iso-C171 Omega respectively, including phosphatidyl ethanolamine, two kinds of ammonia lipid, ammonia and phosphorus. Lipid, phospholipids and other lipids. The G+C content of the main respiratory quinone type is the MK-6. genome DNA, which is 36.7mol%. based on genetic and phenotypic characteristics. The strain P131T (=KCTC42587T=MCCC1H00124T) represents a new species of the genus Qi, which is named as the sediment than the Bizionia sediminis sp.nov.. The strain N62T is isolated from the self power. The marine sediments were gram-negative, facultative, facultative anaerobic, no motion and cell short rod like.2216E solid medium at 28 C for 2 days, forming a single colony of round, smooth, orange and 0.6-1.5MM diameter. The optimum growth temperature of strain N62T was 28-30, and the optimum pH was 7.0-7.5 and 2.0-3.0% (w/v) NaCl. strain N62T main fat. The acid components are iso-C15:0, iso-C15:0G, iso-C17:03-OH and C17:13-OH., the main type of respiratory quinone is the G+C content of the N62T genome DNA of the MK-6. strain is 35.3 mol%., the main polar lipid is phosphatidyl ethanolamine, two kinds of lipid, sugar and three other lipids. Based on the rDNA sequence of 16S, the strain N62T belongs to the yellow line of saffron. The family of bacteria (Crocinitomicaceae), similar to the 16SrDNA sequence of the species of the cold microbacteria, is 95.8-97.2%. based on the evidence of genetic and phenotypic characteristics, strain N62T (=KCTC 42589T=MCCC1H00117T), representing a new species of cold microbacteria, named orange cold microbacteria (Brumimicrobium aurantiacum sp.nov.). The strain X7T is isolated from self self. The sediments of Weihai offshore were gram-negative, non gliding, facultative and short rod-shaped. The optimum growth temperature was 28-30, the optimum pH was 7 and the optimum salinity 2.0-3.0% (w/v) NaCl. based on 16S rDNA sequence analysis showed that the strain X7T belonged to the salt pit Micrococcus, and the highest 16SrDNA sequence resemblance with Salinimicrobium gaetbulicola (96) .3%). The main fatty acids are anteiso-Ci5:0, iso-C15:0, anteiso-C17:1c Omega 9C, iso-C17:1 Omega 9C, C17, 02-OH and iso-C17; 03-OH. major polar lipid is a phosphatidyl ethanolamine, a kind of phospholipid, two kinds of lipids, and five other undetermined lipids. The results showed that the strain X7T (=KCTC 42585T=MCCC1H00115T) represented a new species of salt pit microbacteria, named as the yellow salt pit microbacteria (Salinimicrobium flavum sp.nov.). The strain WDS2C4T and JZ3A21 were separated from the Wendeng salt field and the Feicheng delicate salt field, and the cells were gram-negative, long rod-shaped and non gliding. Growth range: tolerance to salinity 1.0-20.0% (w/v) NaCl (optimum 6.0-10.0%), temperature 20-50 C (optimum 37-40 C) and pH6.5-9.5 (optimum 7.0-8.0). The main fatty acid components of strain WDS2C4T and strain JZ3A21 are cyclo-C19, 0 Omega 8C, summed features 8 The main respiratory quinone type of S2C4T and JZ3A21 is the G+C content of the genomic DNA of Q-10. strain WDS2C4T is 67.7 mol%, the G+C content of the genomic DNA of the strain JZ3A21 is 67.3 mol%., and the main polar lipids are two kinds of phospholipids, the ammonia lipid, the phosphatidyl glycerol, the phosphatidylcholine and the two kinds of glycolipids. The similarity of the 16S rDNA sequence of 3300T was the highest of 95.11%. and the species Albidovulum xiamenense, Paracoccus aestuarii, Rhodobacter vinaykumarii and Roseivivax pacificus were 94.75%, 94%, 93.88% and three phylogenetic trees established according to the sequence. Elihood and maximurn-parsimony) showed that strain WDS2C4T and JZ3A21 were located in the separate branch of the family Rhodobacteraceae. According to the evidence of genetic and phenotypic characteristics, strain WDS2C4T (=KCTC 52227T=NBRC 112331T=MCCC 1H00148T) represented a new species of a new genus of the family red bacteria family, named halophilic white halophilus (Albihalobacter halophilus). V.sp.nov.).
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TB383.1;Q93

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