Genus-Wide Physicochemical Evidence of Extracellular Crystalline Silver Nanoparticles Biosynthesis by Morganella spp

This study was performed to determine whether extracellular silver nanoparticles (AgNPs) production is a genus-wide phenotype associated with all the members of genus Morganella, or only Morganella morganii RP-42 isolate is able to synthesize extracellular Ag nanoparticles. To undertake this study, all the available Morganella isolates were exposed to Ag+ ions, and the obtained nanoproducts were thoroughly analyzed using physico-chemical characterization tools such as transmission electron microscopy (TEM), UV-visible spectrophotometry (UV-vis), and X-ray diffraction (XRD) analysis. It was identified that extracellular biosynthesis of crystalline silver nanoparticles is a unique biochemical character of all the members of genus Morganella, which was found independent of environmental changes. Significantly, the inability of other closely related members of the family Enterobacteriaceae towards AgNPs synthesis strongly suggests that AgNPs synthesis in the presence of Ag+ ions is a phenotypic character that is uniquely associated with genus Morganella.

Recently, in an attempt to understand the biomolecular mechanism of extracellular AgNPs synthesis, we demonstrated that Morganella morganii strain RP-42 isolate [10] was capable of synthesizing AgNPs extracellularly, and explored the phenotypic and genotypic characters of putative silver resistant machinery in Morganella sp. RP-42 [10]. It, however, remains an established fact that microbial physiology tends to evolve rapidly to the environmental changes to increase the chances of survival. To understand AgNPs synthesis by Morganella in a more clear way, it was found significantly important to evaluate whether the AgNPs synthesis was an adaptive physiology of Morganella sp. strain RP-42 as a result of environmental factors or it was independent to that [12]. In the present study, we aim to further strengthen the understanding of AgNPs synthesis in Morganella, and validate whether extracellular AgNPs synthesis in the presence of Ag + ions is a genus-wide phenotype in Morganella spp. This has been achieved by performing a detailed time-dependent UV-visible absorption spectroscopy study with respect to AgNPs synthesis capability of all the known Morganella biogroups till date. We have also investigated the presence of gene homologue of putative gene of the silver binding protein (silE) from all strains of Morganella spp. that were tested for AgNPs synthesis. To achieve this, we have carried out a genus-wide characterization of AgNPs synthesis using all the members of genus Morganella isolated from different environments.

Results and Discussion
On exposure to 5 mM colourless AgNO 3 solutions, all Morganella biogroups formed dark brown coloured solutions within 20 h of reaction, except for M. psychrotolerans that formed greenish brown colloidal solution, indicating formation of extracellular AgNPs by all the biogroups. The color of the solutions did not significantly change from that point onward (except in intensity), even after continuing the reaction for up to 5 days. The AgNPs solutions remained stable for at least 8 weeks without any visible aggregation or precipitation. To understand the nature of nanoparticles, detailed physico-chemical characterization of ex-tracellular AgNPs formed by all Morganella strains was carried out using UV-Vis absorbance spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD) studies as described in the materials and methods section [10]. Figure 1 shows the time-dependent UV-vis absorbance spectra of colloidal solutions obtained after reaction of all Morganella biogroups with 5 mM AgNO 3 for 1, 3,8,16,20,48 and 120 h. The presence of a characteristic Ag surface plasmon resonance (SPR) between 400 and 500 nm is clearly evident in all the samples, thus confirming the formation of extracellular AgNPs by all Morganella biogroups [10]. The differences in the position of absorbance maxima of SPR features of AgNPs synthesized by different biogroups is notable, which is most likely due to the difference in the size and/or shape of Ag nanocrystals synthesized by these biogroups [12,31,]. It is also interesting to note that most of the Morganella biogroups started synthesizing AgNPs as early as within 1 h of reaction and the yield of AgNPs by different biogroups increased as the reaction progresses over a period of time. However, the amount of AgNPs produced by different biogroups reached to a saturation state somewhere between 20 h and 120 h of reaction, which varied from one biogroup to another. This suggests that although all Morganella biogroups have the capability to reduce Ag + ions to form AgNPs (Ag 0 ), the rate of AgNPs formation may vary among them. To compare the rate of AgNPs formation by different biogroups, the maximum absorbance intensity (A max ) of the Ag SPR feature of different biogroups was plotted with respect to different time points of the biosynthesis reaction ( Figure 2). It is clearly evident from Figure 2 that different biogroups indeed followed different reaction kinetics in terms of AgNPs formation, among which M. psychrotolerans showed the fastest activity towards AgNPs biosynthesis, followed by M. morganii strain RP-42 (compare absorbance intensities at 1 h). However, after 8 h of reaction, AgNPs production by M. morganii strain RP-42 superseded that from M. psychrotolerans, thus RP-42 strain showing largest overall AgNPs production capability within 120 h time frame. In a control experiment, SPR signatures corresponding to Ag nanoparticles were found absent in the media control wherein no bacteria were inoculated, thus ruling out the possibility of direct role of media components on AgNPs synthesis, and affirming that AgNPs synthesis resulted as a whole function of extracellular micro-environment created by different strains of Morganella.
It is also interesting to note that AgNPs production by most of the Morganella biogroups (except biogroups RP-42 and A) reached to a saturation state within 20 h of reaction, after which no further increase in AgNPs synthesis was observed. Therefore, in this study, although UV-Vis analysis was performed up to 5 days to follow the reaction kinetic, we performed TEM and XRD analysis on AgNPs obtained after 20 h of reaction. In our opinion, 20 h time point provides a better representation than 120 h time point for comparison between AgNPs synthesized by different biogroups, predominantly because at 20 h time point AgNPs biosynthesis is in its log (growth) phase, which enables to capture the state of asformed particles, rather than a possibility of their further modification while AgNPs stay in the bacterial growth media up to 120 h. It should also be noted that when we previously performed a detailed precursor concentration-dependent experiment on M. morganii strain RP-42, the rate of AgNPs formation was found to be maximum at 5 mM AgNO 3 concentration, and was reduced by increasing the precursor concentration [10]. This motivated us to perform all the experiments reported in the current study at 5 mM AgNO 3 concentration. However, considering the differences in the rate of AgNPs biosynthesis by different Morganella biogroups, it is likely that optimal precursor concentration for maximum AgNPs synthesis rate may vary from one biogroup to another. This will require separate detailed investigations concerning each of the biogroups in the future, wherein influence of various parameters such as precursor concentration, solution pH, reaction time and temperature should be studied in detail to obtain more insights about each system.
To understand the morphology of AgNPs formed by different biogroups of Morganella, the TEM analysis of AgNPs synthesized by all biogroups of Morganella was performed after 20 h of biosynthesis ( Figure 3). It is evident from TEM images that AgNPs formed by all biogroups were quasi-spherical in shape, ranging 10-50 nm in diameter (Figure 3a-r). The particle size distribution of AgNPs formed by different Morganella biogroups was assessed using TEM micrographs for at least 200 particles in each biogroup, which revealed that the average particle diameters with the standard error of mean of quasi-spherical AgNPs synthesized by different biogroups were 39. The crystallography of AgNPs formed by different biogroups of Morganella after 20 h of reaction was investigated by XRD. As is evident from XRD patterns in Figure 4, extracellular AgNPs synthesized by all the biogroups are highly crystalline in nature, that could be perfectly indexed to the {111}, {200}, {220} and {311} Bragg reflections of the face centered cubic (fcc) form of crystalline silver [12,24,[31][32]. XRD analysis thus provided a clear indication of formation of high quality crystalline AgNPs using a Morganella mediated biosynthesis process by all its type strains. Additionally, the crystallinity of AgNPs was further confirmed by performing SAED analysis of AgNPs formed by M. morganii strain RP-42 and M. psychrotolerans during TEM imaging ( Figure 5). The SAED patterns from both the samples revealed well-defined diffraction spots in the form of rings, which are indicative of polycrystalline silver.
The UV-vis, TEM, XRD and SAED results presented in this study clearly demonstrate that formation of AgNPs is a genus-wide characteristic phenotype of all reported type strains of Morganella to date. Further experiments were performed to explore whether AgNPs formation is a characteristic phenotype restricted to genus Morganella, or whether other taxonomically related genera of Enterobacteriaceae family also show this feature. To obtain this insight, when comparative analysis of AgNPs synthesis using laboratory strains of Escherichia coli, Salmonella typhimurium, Kelebisella pneumoniae and Serratia marcescens was performed in the presence of 5 mM AgNO 3 , no AgNPs formation was observed in any of these closely related organisms. Additionally, distant taxonomic relatives of Morganella such as Firmicutes and Actinobacter did not lead to formation of detectable AgNPs in solutions. This strongly suggests that AgNPs synthesis in the presence of Ag + ions is a phenotypic character that is uniquely associated with genus Morganella.
It has been previously established that in silver resistant bacteria, silver resistance mechanism involves the gene (SilE) which encodes a periplasmic silver binding protein (silE). This macromolecule plays a major role in highly specific uptake of Ag + ions from surrounding environment by providing histidine sites as primary candidate for Ag + ions binding [33]. Similarly, in our previous study, we established that silver resistance machinery in Morganella morganii RP-42 is associated with AgNPs synthesis capability of this particular strain [10]. Since, in the current study, all reported strains of genus Morganella were found to exhibit phenotype of AgNPs synthesis in the presence of Ag + ions, we found it important to associate the presence of silver resistance (SilE gene) in all these strains with their AgNPs synthesis capability [10]. Therefore, to determine whether all strains of Morganella spp. exhibit silver resistance, further efforts were made to identify gene homologue of SilE in all the members of genus Morganella. As can be seen from Figure 6, all Morganella strains showed the presence of SilE gene homologue. This observation further strengthens the probable role of silver resistance genes and gene products in AgNPs synthesis in Morganella. Detailed investigation of functional role of silver resistance genes in AgNPs synthesis is currently being pursued.
In the present study, we have demonstrated that the phenotype of extracellular AgNPs synthesis in Morganella is not just restricted to an isolate pertaining to one environment, but it is indeed a unique biochemical character associated with all the members of this genus isolated from different environment [34][35][36][37]. This clearly establishes that AgNPs synthesis by genus Morganella is a phenotype independent of environmental influence. Although AgNPs synthesis has been previously reported by other microor- ganisms, this is for the first time that extracellular synthesis of AgNPs by all the members a particular Genus (Morganella) has been established, and their AgNPs synthesis capability has been followed in a time-dependent manner. The observation that members of other genera of the same Enterobacteriacea family are incapable of AgNPs synthesis, establishes AgNPs synthesis as a unique phenotypic character of genus Morganella. These observations might, in future, not only provide a complementary tool for easy detection and purification of Morganella in the presence of other members of Enterobacteriaceae family, but might also establish new evolutionary links between different microorganisms by comparing their metal ion reducing capabilities.

Growth and identification of Morganella strains
The genus Morganella comprises of two species viz. Morganella morganii with two sub-species morganii and sibonii, and Morganella psychrotolerans. On the basis of biochemical and taxonomic profiling, two sub-species morganii and sibonii have been further divided into a total of eight biogroups (Table 1) [34][35][36][37]. All strains of Morganella were routinely maintained on LB agar slants and preserved at 280uC. The identity of all strains was confirmed by 16S rRNA gene sequencing as shown earlier [10], and sequences were submitted to GenBank with accession numbers HM122047, HM122048, HM122049, HM122050, HM122051, HM122052, HM122053, HM122054 and HM122055.

Extracellular biosynthesis of silver nanoparticles by Morganella spp. and nanoparticles characterization
All reported type strains of Morganella were used for AgNPs synthesis in the present study (Table 1). All strains of Morganella were initially grown at 37uC for 24 h in a 500-mL Erlenmeyer flask that contained LB broth (100 mL) without added NaCl in a shaker incubator set at 200 rpm, as reported previously for M. morganii strain RP-42 [10], except for M. psychrotolerans, wherein the bacteria were grown at 20uC, while maintaining all other synthesis conditions similar to those for other Morganella strains. For M. psychrotolerans, 20uC was used as the growth temperature, because this being a psychrotolerant species, 15-20uC is its optimum growth temperature and M. psychrotolerans could not be grown successfully at 37uC even after 5 days of incubation [12]. For all other Morganella biogroups, the optimum growth temperature being 37uC, they were grown at 37uC. Following bacterial growth, all the culture suspensions were incubated with aqueous 5 mM solutions of AgNO 3 at 37uC in a shaker incubator at 200 rpm in the dark, and the reactions were carried out in a time-dependent manner for up to 120 h (5 days). The M. morganii strain RP-42 that was earlier reported by us to synthesize extracellular AgNPs was considered as a positive control in all the experiments [10]. The  extracellular synthesis of AgNPs was initially detected by visual inspection of the culture flask for a change in color of culture medium from clear light-yellow to brown/green. Extracellular AgNPs were separated from bacterial cells by centrifuging aliquots of culture supernatants (1.5 mL) at 3000 rpm for 6 min at 25uC. The supernatants thus obtained were clear brown/green homogenous suspensions of AgNPs, which were analyzed in a timedependent fashion using UV-vis spectroscopy (Cary 50 Biospectrophotometer) at a spectral resolution of 2 nm, transmission electron microscopy (Jeol 1010 TEM) at an accelerating voltage of 100 keV, selected area electron diffraction (SAED) coupled with TEM instrument, and X-ray diffraction (XRD -Bruker AXS D8 Discover) using Cu K a radiation and a General Area Detector Diffraction System (GADDS). For UV-vis analysis, the AgNPs suspensions were diluted 10 times using MilliQ deionized water at every time point and UV-vis spectra were obtained. For TEM analysis, AgNPs samples obtained after 20 h of reaction were prepared by drop casting the colloidal suspensions of AgNPs onto carbon-coated Cu grids followed by drying under air for 24 hours.
For XRD analysis, the samples were prepared by precipitating AgNPs obtained after 20 h of biosynthesis at 10,000 rpm for 15 min, followed by three washings with MilliQ deionised water, and drop casting the samples onto a glass substrate.