Investigation Of The Surface Modification Of Gold Nanoparticles On Rhododospirillum Rubrum Surface

In nature,bacteria possess specific morphologies including sphere,rod,arc,and spiral.The in-depth study of bacterial morphology is important to explore various biological activities such as feeding,movement,disease-causing ability,and chemotaxis.In addition,the study will also provide theoretical basis for the development of nanoparticle-bacteria hybrid materials with unique biological functions.Rhododospirillum rubrum(R.rubrum)is one type of proteobacteria,alphaproteobacteria,Gram-negative bacteria.Because of the light-dependent self-feeding property and hydrogen production ability through nitrogen bio-fixation,as well as its unique spiral shape with 2-4 cycles,R.rubrum is an idea model system to explore nanoparticle-bacteria hybrid material for nitrogen bio-fixation and to prepare micrometer scale chiral plasmonic structure,attracting wide attention by researchers in the relevant fields.Although people have studied R.rubrum for decades,the biomolecular basis of its spiral morphology and the parameters regulating the bacteria growth process are still not clear.For example,spherical,rod-like,arc-like morphologies are occasionally found during the growth process.The cause of the change of bacterial morphology is still unknown.In addition,modification of R.rubrum surface with nanoparticles is rarely explored.In this thesis,we used the R.rubrum as the model bacteria.In order to address some scientific questions in synthesizing nanoparticle-bacteria hybrid materials,we first studied different experimental conditions on the regulation of R.rubrum morphologies,figuring out the optimized conditions to control the spiral morphology of the R.rubrum.Then,based on these conditions,we used in situ biosynthesis,direct deposition,and layer-by-layer deposition methods for the modification of R.rubrum surface with gold nanoparticles(Au NPs),preparing Au NP-bacteria hybrid materials.The major discoveries in this thesis are listed below:(1)We systematically studied the effects of different culture conditions of R.rubrum on bacterial morphology.Different culture conditions,such as medium composition,pH,and light,can have significant effects on bacterial morphologies.For example,bacteria showed spiral morphology in nutrient-rich SMN medium and rod-like morphology in nutrition-limit MG medium.Bacteria showed spiral morphology in neutral pH and showed rod-like morphology in weak acid(pH = 6,pH= 7)or weak basic(pH = 8)conditions.The absence of light will also change the morphology of bacteria from spiral to rod-like.Different culture temperatures(28 ?,30 ?,32 ?,37 ?)showed no effect on bacterial morphology.We hypothesized that only under culture conditions which are suitable for R.rubrum growth and metabolism,bacteria would then have excess energy for other biological activities other than survival,such as the assembly of Cres skeleton proteins on the cell membrane to form spiral shape.(2)We systematically studied three methods for R.rubrum surface modification of gold nanoparticles(Au NPs),including in situ bio-synthesis,direct deposition,and layer-by-layer deposition.UV-vis spectroscopy,dynamic light scattering(DLS),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and inductively coupled plasma mass spectrometry(ICP-MS)were used for the characterization of Au NP-modified bacteria.By using in situ bio-synthesis method,R.rubrum cultured in a medium containing chloroquine acid can generate a small amount of Au NPs on surface.However,the surface density Au NPs is low due to the weak reduction ability of R.rubrum.By using layer-by-layer deposition method,we deposited polyethylenimine(PEI)and poly(diallyldimethylamonium chloride)(PDDA)onto bacteria surface,changing the bacterial surface charge into positive,then modified bacteria with negatively-charged Au NPs through electrostatic adsorption.We found that 10% PDDA showed the maximized modification efficiency with low toxicity.We further explored the effects of Au NPs sizes and surface charges on the modification efficiency,and found that the 13 nm Au NPs showed best efficiency.Surfactants such as Triton-X and Tween 20 also improved modification efficiency,in which Triton-X showed the best efficiency.We tested two direct deposition methods,the electrostatic interaction and hydrogen bonding interaction.We used positively-charged PEI-Au NPs to modify negatively-charged bacteria through electrostatic adsorption.However,and the modification efficiency was not high due to the uneven charge distribution on the bacterial surface.We also explored the surface modification of R.rubrum with Au NPs synthesized from hydroxylamine hydrochloride(HAHC).The modification efficiency was high,probably due to the hydrogen bonding between protein and lipid polysaccharides amine groups on bacterial surface and hydroxyl groups on Au NPs.The pH value of the reaction system also played a vital role on the direct deposition of Au NPs on bacteria with the optimized pH value of 3.Under pH 3,the bacteria were homogeneously covered with a large number of Au NPs with the spiral morphology well preserved.The direct deposition method using HAHC-Au NPs was generalizable,which was successfully applied to both Gram-negative E.coli and Gram-positive S.aureus.To conclude,this thesis systematically studied the effects of culture conditions on the spiral morphology of R.rubrum,obtaining R.rubrum with well-defined spiral shapes.By using these bacteria as template,we systematically studied three methods for R.rubrum surface modification of Au NPs and achieved highly efficient Au NPs modification using direct deposition method.The prepared Au NP-bacteria hybrid material is expected to improve the photo-induced biocatalytic activity,to synthesize micron-scale chiral plasma materials for regulation of polarizing light,and to provide a new experimental tool to monitor the dynamic motion of microorganisms using nanoparticles.