Biosynthesis of Silver Nanoparticles Using Microorganism

미생물을 이용한 은 나노입자 생합성

Abstract

The aim of this study was to develop a simple, environmentally friendly synthesis of silver nanoparticles (SNPs) without the use of chemical reducing agents by exploiting the extracellular synthesis of SNPs in a culture supernatant of Bacillus thuringiensis CH3. Addition of 5 mM $AgNO_3$ to the culture supernatant at a ratio of 1:1 caused a change in the maximum absorbance at 418 nm corresponding to the surface plasmon resonance of the SNPs. Synthesis of SNPs occurred within 8 hr and reached a maximum at 40-48 hr. The structural characteristics of the synthesized SNPs were investigated by various instrumental analysis. FESEM observations showed the formation of well-dispersed spherical SNPs, and the presence of silver was confirmed by EDS analysis. The X-ray diffraction spectrum indicated that the SNPs had a face-centered cubic crystal lattice. The average SNP size, calculated using DLS, was about 51.3 nm and ranged from 19 to 110 nm. The synthesized SNPs exhibited a broad spectrum of antimicrobial activity against a variety of pathogenic Gram-positive and Gram-negative bacteria and yeasts. The highest antimicrobial activity was observed against C. albicans, a human pathogenic yeast. The FESEM observations determined that the antimicrobial activity of the SNPs was due to destruction of the cell surface, cytoplasmic leakage, and finally cell lysis. This study suggests that B. thuringiensis CH3 is a potential candidate for efficient synthesis of SNPs, and that these SNPs have potential uses in a variety of pharmaceutical applications.

본 연구에서는 간단하고, 환경친화적인 은 나노입자 합성법을 개발하기 위하여 화학적 환원제 사용없이 Bacillus thuringiensis CH3의 배양상등액만을 사용하여 은 나노입자의 세포외 합성을 조사하였다. 5 mM $AgNO_3$와 배양상등액을 1:1로 혼합하여 반응시켰을 때, 은 나노입자의 표면 플라스몬 공명에 해당하는 418 nm에서 최대 흡광도를 나타내었다. 은 나노입자의 합성은 8시간 내에 일어났고, 40-48시간에 최대가 되었다. 합성된 은 나노입자의 구조적 특성을 다양한 기기분석에 의하여 조사하였다. FESEM 관찰은 잘 분산된 구형의 은 나노입자가 합성되었음을 보여주었고, 은의 존재는 EDS 분석으로 확인되었다. X선 회절 스펙트럼은 은 나노입자가 면심 입방결정격자임을 나타내었다. DLS를 사용하여 계산된 은 나노입자의 평균 입자 크기는 약 51.3 nm이었고, 범위는 19-110 nm이었다. 합성된 은 나노입자는 다양한 병원성 그람양성 세균, 그람음성 세균 및 효모에 대해 항균활성을 나타내었다. 가장 높은 항균활성은 인체병원성 효모인 C. albicans에서 관찰되었다. FESEM 관찰 결과, 은 나노입자의 항균활성은 세포 표층구조의 파괴와 세포질 누출에 따른 세포 용해에 기인하는 것으로 판단되었다. 본 연구는 B. thuringiensis CH3는 은 나노입자의 효율적인 합성을 위한 잠재적인 후보이며, 합성된 은 나노입자는 다양한 제약 분야에서 잠재적 응용가능성이 있음을 시사한다.

Fig. 1. Color change (A) and UV—Vis spectrum (B) of culture supernatant of B. thuringiensis CH3 in a time-dependent manner.

Fig. 2. Influence of temperature (A) and pH (C) on synthesis of silver nanoparticles. Spectra (B) and (D) show the synthesis of silver nanoparticles at 40℃ and pH 8.5, respectively.

Fig. 3. FESEM (A), TEM (B) images and EDS spectrum of silver nanoparticle synthesized by culture supernatant of Bacillus thuringiensis CH3.

Fig. 4. X-ray diffraction pattern of silver nanoparticle synthesized by culture supernatant of Bacillus thuringiensis CH3.

Fig. 5. Particle-size distribution (A) and zeta potential (B) of silver nanoparticle synthesized by culture supernatant of Bacillus thuringiensis CH3.

Fig. 6. Antimicrobial activity of silver nanoparticle (SNP) against K. pneumoniae (A), L. monocytogenes (B) and C. albicans (C). FESEM images present intact cells and silver nanoparticle-treated cells.