Introduction

Silver nanoparticles (Ag° nanoparticles) are the most used nanomaterials due to their potent antimicrobial activity. Biogenic synthesis of Ag° nanoparticles is from a long time known for many researchers, and several excellent reviews were published in the last 6 years (Abbasi et al. 2014; Faramarzi and Sadighi, 2013; de Lima et al. 2012; Durán et al. 2009, 2010, 2011, 2014a; 2015a; Ingal and Chaudhari, 2013; Keat et al. 2015; Mashwani et al. 2015; Mittal et al. 2014; Moghaddam et al. 2015; Natsuki et al. 2015; Rai et al. 2009). Biogenic synthetic routes have several advantages over traditional chemical or physical ones, since they are environmentally friendly technology (Seabra and Durán, 2015). However, the great limitation of biogenic synthesis is the relatively poor reproducibility process. Therefore, the adequate characterization of biogenic Ag° nanoparticles is a crucial step prior to the evaluation of their antimicrobial activity. It is clear from the literature that the basic analysis of the structure of any metal-based nanomaterials is the x-ray diffraction (XRD). Although other techniques including UV–Vis, Fourier transform infrared (FTIR) spectroscopy, x-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) are important and complementary, XRD is a crucial technique to the correct identification of the silver-based nanoparticles.

In view of this statement, it is important to compare the silver derivative nanoparticles through XRD, especially in the case of biogenic synthesized Ag° nanoparticles. For this discussion, Fig. 1 represents the XRD pattern from inorganic crystal structure database (ICSD) standards of Ag° nanoparticles (Fig. 1a) and silver chloride nanoparticles (AgCl nanoparticles) (Fig. 1b), which are the most typical compounds found in the final product of biogenic synthesized silver-based nanoparticles. Figure 1a shows the XRD pattern 2θ peak values of around 38.10o, 44.47o, 64.63o, 77.44o, and 81.33o which correspond to the XRD pattern of indexed [111], [200], [220], [311], and [222] facets of Ag° nanoparticles. In the case of AgCl nanoparticles (Fig. 1b), the XRD pattern in 2θ peak values is 27.9o, 32.3o, 46.3o, 55.0o, 57.6o, 67.6o, 74.6o, 76.9o, and 85.7o corresponding to the indexed (111), (200), (220), (311), (222), (400), (331), (420), and (422) facets of AgCl nanoparticles.

Fig. 1
figure 1

XRD from International Crystal Structure Database (ICSD) standards: a Cubic Ago nanoparticles Code 64,994. b Cubic AgCl nanoparticles Code 64734 (modified from http://bdec.dotlib.com.br/cliente/login)

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Besides typical XRD patterns of silver-based nanoparticles, another important aspect in the characterization of the nanoparticles is the absorption bands of different silver nanoparticle derivatives. Pure Ago nanoparticles exhibit a typical plasmon resonance absorption band at the region ~400–500 nm (Fig. 2a), depending on the morphology and size of the nanoparticles (Fig. 2b). In contrast, pure AgCl nanoparticle exhibited only UV band (~250 nm), and not the plasmonic band (~400–500 nm) (Dong et al. 2009). However, upon contamination of AgCl nanoparticles with Ag° nanoparticle clusters, the plasmonic band appeared (Fig. 3). As indicated in Fig. 3, AgCl nanoparticles are photosensitive, yielding Ag° nanoparticles upon ambient light exposition (Trinh et al. 2015). The color of the AgCl nanoparticles suspension was initially opalescent, and upon irradiation, it changed to violet, and finally to yellow-brown (~420 nm). The presence of Ag° layer, produced by the chemical reduction of AgCl, might lead to the appearance of the visible light band absorption. The authors suggested that the direct and indirect band gaps of AgCl nanoparticles were 5.15 eV (240 nm) and 3.25 eV (380 nm), respectively, which avoids AgCl from absorbing light with the wavelength above 380 nm. Furthermore, it is reported that Ag° deposited on AgCl particles exhibited the plasmonic absorption of visible light (Trinh et al. 2015). Therefore, precaution must be taken during the synthesis process of AgCl nanoparticles, since Ag° nanoparticles might be simultaneously produced, as in the case of biogenic routes.

Fig. 2
figure 2

a UV–Vis absorption spectra for the Ag° nanoparticles synthesized by citrate at different pH conditions, as indicated in the figure. b TEM images of the Ag° nanoparticles synthesized by different pHs, as indicated in the figure. (Modified from reference Dong et al. 2009 with permission from the American Chemical Society)

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Fig. 3
figure 3

UV–Vis absorption spectra of AgCl nanoparticle suspension before (solid line) and after 5 h of UV irradiation with λ = 254 nm irradiation (dashed line). The irradiation of AgCl nanoparticles led to the formation of the plasmonic band (~420 nm) due to the presence of Ag° nanoparticles. (Reproduced from reference Trinh et al. 2015 with permission from Vietnam Academy of Science and Technology, under Creative Commons Attribution license of an open access article)

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Upon these clarifications, regarding to the characterization of Ag° nanoparticles and AgCl nanoparticles and the differences among them, the next sections of this review present and discuss the most common misunderstanding related to the characterization of the nanostructure of biogenically synthesized Ag° nanoparticle, and consequently, the erroneous conclusion about their antimicrobial activities. Although several reviews reported the importance of biogenically synthesized Ag° nanoparticles for several applications (Abbasi et al. 2014; Faramarzi and Sadighi, 2013; de Lima et al. 2012; Durán et al. 2009, 2010, 2011, 2014a; 2015a; Ingal and Chaudhari, 2013; Keat et al. 2015; Mashwani et al. 2015; Mittal et al. 2014; Moghaddam et al. 2015; Natsuki et al. 2015; Rai et al. 2009) to our best knowledge, this is the first review to critically discuss the common problems on the erroneous characterization of Ag° nanoparticles obtained by biogenic routes. This review does not intend to cover all the literature; however, the aim of this paper is to alert that there are many published papers with confusing characterization of biogenic synthesized Ag° nanoparticles and their antibacterial effects, and it aims to be an alert for new developments in this field. In order to obtain relevant results on the antimicrobial effects of biogenic silver-based nanoparticles, it is necessary a complete and adequate characterization of these nanostructures. Although several previous studies reported the antibacterial effects of biogenic Ag° nanoparticles towards different bacteria, this review highlights the importance to report the antimicrobial effects of silver-based nanoparticles towards at least one standard strain in the microbiology protocols, to allow appropriated comparison among the antibacterial efficacy of different silver nanostructures.

Biogenic synthesis of Ag° nanoparticles

The bacterium Aeromonas sp. SH10 rapidly reduced silver ions, [Ag(NH3)2]+, to Ag° in the solution at basic pH, and the XRD pattern obtained clearly showed the Bragg reflections corresponding to (111), (200), (220), (311), and (222) sets of lattice planes of Ago nanoparticles (Fu et al. 2006). The same results was reported for the reduction of silver ions to Ag° (Kalishwaralal et al. 2008) in the presence of Bacillus licheniformis, from Morganella sp. (Parikh et al. 2008, 2011), from Streptomyces hygroscopicus (Sadhasivam et al. 2010), from Streptomyces albogriseolus (Samundeeswari et al. 2012), from Shewanella oneidensis MR-1 (Suresh et al. 2010), from Bacillus thuringiensis IS1 strain (Jain et al. 2010), and from Pseudomonas aeruginosa (Jeevan et al. 2012).

In the case of biogenic synthesis of Ag° nanoparticles by fungi, Phoma glomerata (MTCC-2210) (Gade et al. 2014) showed XRD pattern of 2θ peaks related to Ago nanoparticles (Fig. 4a), identical to that from Aspergillus oryzae (MTCC 1846) (Phanjom and Ahmed, 2015), similar to those Ago nanoparticles produced by Vitex negundo L. leaf extract (Zargar et al. 2011) (Fig. 4b). The same pattern were also published for Ag° nanoparticles synthesized from Phanerochaete chrysosporium (MTCC 787/ATCC 24725) culture (Vigneshwaran et al. 2006), Schizophyllum radiatum HE 863742.1 (Metuku et al. 2014), or from Aspergillus fumigatus (Bhainsa and D’Souza, 2006) following the Joint Committee on Powder Diffraction Standards (JCPDS; silver file No. 04-0783 or to the ICSD Code 64994).

Fig. 4
figure 4

XRD graphs of biogenic Ag° nanoparticles synthesized from a Phoma glomerata (MTCC-2210) and from b from Vitex negundo L. extract. (Reproduced with modification from reference Gade et al. 2014 with permission from Elsevier and reproduced from reference Zargar et al. 2011 with permission from MDPI, Basel, Switzerland, under Creative Commons Attribution license of an open access article)

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Plant sources such as Annona squamosa leaf broth (Arjunan et al. 2012) produced only Ago nanoparticles, the same was observed in the case of biogenic synthesis of Ag° nanoparticles from aqueous extract of Gloriosa superba Linn. (Glory Lily) (Ashokkumar et al. 2013), Dioscorea bulbifera (Ghosh et al. 2012), Sinapis arvensis seeds (Khatami et al. 2015), gum tragacanth exudate of the bark from Astragalus gummifer (Fabaceae family) (Kora and Arunachalam, 2012), Gum ghatti from Anogeissus latifolia (Kora et al. 2012), Medicago sativa seed exudates (Lukman et al. 2011), from Cinnamomum zeylanicum bark extract (Sathishkumar et al. 2009), from extract of Lawsonia inermis (Henna) (Gupta et al. 2014), and from latex of Jatropha curcas (Bar et al. 2009).

Baker’s yeast clearly produced Ago nanoparticles, as revealed by the XRD pattern of the obtained nanoparticle (Jha et al. 2008). The same results were reported from yeast strain from an acid mine drainage in Portugal (Mourato et al. 2011), and from yeast species (strain MKY3), which were isolated from garden soil (Kowshik et al. 2003).

Biogenic synthesis of AgCl nanoparticles

Apparently, the formation of AgCl nanoparticles is associated with the biosynthesis of Ago nanoparticles, thus it is important to correctly distinguish the XRD patterns between those two nanoparticles. In this direction, this section presents relevant reports on the biogenic synthesis of AgCl nanoparticles and their XRD characterization.

Using the Bacillus subtilis MTCC 3053, Paulkumar et al. (2013) demonstrated the biogenic synthesis of AgCl nanoparticles, in the total absence of Ago nanoparticles (Fig.5). The XRD spectrum of silver chloride nanoparticles synthesized by B. subtilis exhibits strong peaks at 2휃 values corresponding to (111), (200), (220), (311), (222), (400), (331), (420), and (422) planes, respectively (Fig. 5A). The XRD spectrum was in accordance with the standard AgCl nanoparticle from Joint Committee on Powder Diffraction Standards (JCPDS file no. 85–1355). Figure 5B shows the energy dispersive x-ray analysis (EDAX) of the AgCl nanoparticles demonstrating the presence of silver and chloride. Indeed, the quantitative percentage of silver and chloride atoms is represented in Fig. 5c. AgCl nanoparticles showed spherical-shaped morphology with average size in the range of 20–60 nm, as demonstrated by SEM analysis (Fig. 5d). Figure 5e shows the UV–Vis spectra recorded during the formation of AgCl nanoparticles from 6 to 48 h of monitoring (Paulkumar et al. 2013). Curiously, Fig. 5e clearly shows the presence of absorption band at around 400 nm, the authors attributed this band due to the presence of AgCl nanoparticles. However, the presence of absorption band at ~400 nm is not expected for AgCl nanoparticles, in contrast, this band indicates the formation of Ag° nanoparticles (plasmonic band) (Fig. 3). It can be assumed that for UV–Vis analysis, the author measured the real-time biogenic formation of AgCl nanoparticles (from 6 to 48 h), and Ag° nanoparticles were also yielded, leading to the appearance of the absorption band at ~400 nm. It can be assumed that for UV–Vis analysis, the samples were not washed. In contrast, for XRD analysis, Ag° nanoparticles were removed by washing/purification processes; therefore, the XRD pattern corresponded solely to the formation of AgCl nanoparticles, with no indication of the presence of Ag° nanoparticles. Hence, biogenic synthesis of AgCl nanoparticles by B. subtilis led to the formation of Ag° nanoparticles (as indicated by UV–Vis analysis, Fig. 5e), and upon AgCl purification, the Ag° nanoparticles were efficiently removed, as indicated by the XRD analysis (Fig. 5a). Therefore, caution must be taken with the methodology of biogenic synthesis of AgCl nanoparticles, in particular with the purification process and adequate characterization and discussion of the results in order to avoid confusing between the characterization of AgCl and Ag° nanoparticles.

Fig. 5
figure 5

Characterization of biogenic AgCl nanoparticles synthesized by B. subtilis. a XRD pattern. b, c EDAX analysis. d SEM image showing spherical-shaped AgCl nanoparticles. The particle size was characterized in the range of 20–60 nm for B. subtilis (scale bar = 500 nm). e UV–Vis spectra recorded during the formation of AgCl nanoparticle. Silver nitrate at 1 mM was added to B. subtilis and incubated at different growth periods. (Reproduced with modifications from reference Paulkumar et al. 2013 with permission from Hindawi Publishing Corporation, under Creative Commons Attribution license of an open-access article)

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Mokhtari et al. (2009) reported that Klebsiella pneumoniae synthesized AgCl nanoparticles as the final sole biogenic product. To justify their findings, the authors cited a reference that correctly demonstrated the characterization of chemically synthesized AgCl nanoparticles (Potiyaraj et al. 2007). Unfortunately, it was erroneously designated that the 2θ scale for the XRD pattern published by Mokhtari et al. (2009). In this case, the UV–Vis of as-prepared AgCl nanoparticles showed clearly no plasmon band before light irradiation, as shown in Fig. 3.

Synthesis of AgCl nanoparticles by using Rosa macdub petal extract was also reported by Shete et al. (2014), in which the XRD pattern clearly showed the presence of AgCl nanoparticles and the absence of Ag° nanoparticles. However, the UV–Vis spectra showed the presence of the plasmon absorption band, which is assigned to the presence of Ag° nanoparticles (probably the concentration of Ag° nanoparticles was very low, and not detectable by XRD analysis, or AgCl nanoparticles were purified for XRD analysis and not for UV–Vis analysis), as reported by Wang et al. (2008). Therefore, attention should be observed in the adequate purification process of the biogenically obtained nanoparticles, and in the correct form to analyze the different techniques for nanoparticle characterization. Adequate discrimination between AgCl and Ag° is required.

Micrococcus luteus (Vimalanathan et al. 2013) was able to produced Ago nanoparticles; however, the XRD pattern exhibited only the formation of AgCl nanoparticles, in the absence of Ago nanoparticles. Similar erroneous interpretation was reported by Roy et al. (2015), which used Saccharomyces cerevisiae to biogenically produced AgCl nanoparticles, as revealed by XRD analysis; however, the authors assumed the formation of Ag° nanoparticles (Roy et al. 2015). In a similar manner, Streptomyces sp. LK3 (Karthik et al. 2014) apparently produced AgCl nanoparticles, and not Ago nanoparticles, as suggested by the authors. Therefore, from the revised literature, it is clear that several papers published erroneous interpretation regarding to the characterization of biogenic synthesized Ag° and AgCl nanoparticles.

Biogenic synthesis of Ago nanoparticles/AgCl nanoparticles

The next section discusses some relevant publications of biogenic synthesis of both Ago nanoparticles and AgCl nanoparticles, probably as Ag/AgCl nanocomposite.

Fungi

In the case of Penicillium brevicompactum WA 2315, it was suggested the exclusively formation of Ago nanoparticles by XRD pattern (Shaligram et al. 2009). However, the pattern does not correspond solely to Ago nanoparticles; probably, there is a mixture of both Ag° and AgCl nanoparticles, as following the JCPDS or ICSD standards. Dhanasekaran and Thangaraj (2013) reported the biogenic synthesis of Ago nanoparticles from Agaricus bisporus acting on Culex mosquito larvae, however, the XRD pattern revealed the presence of both Ago nanoparticles and AgCl nanoparticles. Therefore, it is not possible to assign if the effective antimicrobial action of the nanomaterial against larvae was due to Ag° nanoparticles or to AgCl nanoparticles, or the combination of both nanoparticles.

Li et al. (2012) reported the biogenic synthesis of Ago nanoparticles from Aspergillus terreus (BC0603), and for the crystalline nature of the Ago nanoparticles intense XRD peaks were observed. However, the authors pointed out that some of the intense diffraction peaks observed were related to the presence of AgCl nanoparticles. The authors attributed the presence of AgCl nanoparticles to the addition of chloride ions involved during the preparation of the cell filtrate. Similarly, Vanaja et al. (2015) demonstrated XDR patterns of both Ag° and AgCl nanoparticles upon biogenic synthesis by using Aspergillus niger. Aspergillus pseudodeflectus, and the synthesized nanoparticles were found to be effective against Malassezia furfur (dandruff diseases). The authors attributed the formation of exclusively Ag° nanoparticles, although XRD analysis showed mainly the formation of AgCl nanoparticles. Therefore, AgCl nanoparticles were effective against Malassezia, not Ag° nanoparticles, as concluded by the authors (Mathew et al. 2015). Vigneshwaran et al. (2007) produced both nanoparticles, Ago and AgCl nanoparticles from Aspergillus flavus (NCIM 650). Gade et al. (2011) reported that Phoma sorghina was capable of synthesizing the Ago nanorods extracellularly. However, negligible amounts of AgCl were observed in the XRD pattern.

Patil et al. (2011) reported that A. flavus challenged with silver nitrate solution accumulated Ago nanoparticles on the surface of its cell wall. However, the XRD pattern revealed that Ago nanoparticles are in minor proportion in comparison with AgCl nanoparticles, and other impurities. Cochliobolus lunatus produced Ago nanoparticles; however, AgCl nanoparticles and probably silver oxides were also produced, as demonstrated by the XRD analysis (Salunkhe et al. 2011). The formation of AgCl nanoparticles and silver oxides was not discussed in the publication (Salunkhe et al. 2011). Fusarium oxysporum, isolated from mangrove soil samples, showed the presence of only Ago nanoparticles although no complete pattern was analyzed (Selvi and Sivakumar, 2012). However, Gaikwad et al. (2013) showed that F. oxysporum, from infected plant materials such as fruits, vegetable, and food collected from different places of India, exhibited by its XRD analysis mainly Ago nanoparticles, and a very small amount of AgCl nanoparticles. Similarly, Husseiny et al. (2015) produced Ago nanoparticles from F. oxysporum f. sp. lycopersici; however, XRD pattern showed also the presence of AgCl nanoparticles. Soni and Prakash (2011) demonstrated the presence of Ago nanoparticles together with silver oxides mixtures and other impurities from F. oxysporum Schltdl 2480 and from Chrysosporium tropicum Carmichael-2828 strains. Penicillium atramentosum KM was suggested to be able to produce Ago nanoparticles; however, the XRD pattern represented a mixture of silver oxide nanoparticles and not Ago nanoparticles (Sarsar et al. 2015). Aspergillus foetidus (Roy and Das 2015) demonstrated mainly the presence of Ago nanoparticles; however, traces of AgCl nanoparticles were also observed in the XRD pattern and not discussed in the publication.

Taken together, from the revise literature, there is a misunderstanding in the XRD analysis of biogenically synthesized Ag° and AgCl nanoparticles. It is important to correctly attribute the presence of Ag° and/or AgCl nanoparticles in order to investigate the antimicrobial action of the obtained nanoparticles in a proper manner.

Bacteria

Chauhan et al. (2013) reported that the culture supernatant Streptomyces sp. JAR1 was able to synthesize Ago nanoparticles; however, the XRD pattern clearly revealed the presence of AgCl nanoparticles that was not discussed by the authors. In similar manner, Streptomyces sp. ERI-3 produced both Ago nanoparticles and AgCl nanoparticles, as assayed by XRD analysis, and this fact was not discussed by the authors in their publication (Zonooz and Salouti 2011). The same mistake was observed for the biogenic formation of both AgCl and Ag° nanoparticles by Brevibacterium frigoritolerans DC2, and not appropriated discussed in the publication (Singh et al. 2015). Therefore, bacteria may lead to the synthesis of both Ag° and AgCl nanoparticles, and appropriate XRD interpretation is necessary.

Plants

As in the case of fungi and bacteria, plants might result in the formation of both Ag° and AgCl nanoparticles; therefore, caution must be taken to the correct interpretation of the nanomaterial characterization. Gopinath et al. (2013) reported the synthesis of AgCl nanoparticles by leaf extract of Cissus quadrangularis Linn, and the crystalline nature of the synthesized AgCl nanoparticles was confirmed using XRD analysis showing the typical peaks at 2θ planes of the cubic AgCl nanoparticles (ICDD file no. 00-001-1013; ICSD Code 64734). However, the authors demonstrated a minor production of Ago nanoparticles, as shown by the peaks that correspond to (111), (200), and (220) planes for cubic Ago nanoparticles (ICDD file no. 00-087-0718 or ICSD Code 64,994). Similarly, Satureja hortensis L produced Ago nanoparticles together with AgCl nanoparticles; however, this issue was not discussed by the authors (Afshar P, Sedaghat 2016). Awwad et al. (2015a,b) reported the formation of Ag/AgCl nanoparticles by Albizia julibrissin flowers extract. Murraya koenigii leaves extract produced mainly Ago nanoparticles; however, the presence of AgCl was also evident from XRD pattern of the final silver nanostructured material, and not discussed in the paper (Bonde et al. 2012).

Ibrahim (2015) reported the production of Ago nanoparticles from banana (Musa paradisiaca) peels, as suggested by XRD analysis. However, the XRD pattern demonstrated the presence of extra peaks, which the authors attributed to organic compounds, found in the extract, and responsible for the reduction of silver ions as well as to the stabilization of resultant nanoparticles. Unfortunately, the XRD pattern was clearly due to the presence of AgCl nanoparticles. Similarly, Johnson and Prabu (2015) reported that Cycas circinalis, Ficus amplissima, Commelina benghalensis, and Lippia nodiflora produced Ago nanoparticles; however, in the case of F. amplissima, AgCl nanoparticles were clearly produced.

Parthenium hyterophorus extract yielded Ago nanoparticles; however, AgCl nanoparticles were also produced as assayed by XRD and not discussed in the paper (Kumar 2012), and similar mistake was also reported by Prosopis farcta extract (Miri et al. 2015) or Pongamia pinnata (L) Pierre (Fabaceae family) plant leaves extract (Naik et al. 2014) or from Curcuma longa tuber powder and extract (Sathishkumar et al. 2010). Leela and Vivekanandan (2008) suggested the presence of Ago nanoparticles in their publication; however, the XRD pattern does not correspond to the Ag° nanoparticle. In the presence of Asiatic Pennywort and Bryophyllum leaf extracts, the formation of Ag° nanoparticles was wrongly assigned by XRD technique, and the presence of AgCl nanoparticles was ignored (Saikia et al. 2015). Subarani et al. (2013) reported the formation of Ago nanoparticles from Vinca rosea (L.) (Catharanthus roseus); however, some silver oxides probably contaminated nanostructure, as revealed by XRD analysis. The Opuntia ficus-indica produced both Ago nanoparticles and AgCl nanoparticles (Gade et al. 2010).

Ago nanoparticles were suggested to be obtained by using Rhinacanthus nasutus leaf extract; however, the XRD pattern did not correspond to Ag° nanoparticles. Probably, traces of Ago nanoparticles and some AgCl nanoparticles were presented in the low-quality XRD diffractions pattern reported (Pasupuleti et al. 2013). Clitoria ternatea and Solanum nigrum extracts were suggested to produce Ago nanoparticles: however, from the XRD pattern, it is clear the presence of AgCl nanoparticles (Krithiga et al. 2015). Therefore, inadequate XRD analysis occurs during characterization of biogenic synthesized silver-based nanoparticles.

Algae

Azziz et al. (2015) reported the synthesis of Ag° nanoparticles from Chlorella pyrenoidosa (NCIM 2738), and the authors cited that the characterization of Ago nanoparticles was in accordance to the results reported by Yang et al. (2011), in which the peaks at 2θ values were correctly ascribed to the reflection of (111), (200), (220), (311) planes of the face-centered cubic silver, respectively (matched well with the patterns of silver) (JCPDS file No. 04-0783 or ICSD Code 64994). However, Azziz et al. (2015) showed XRD peaks at 2θ values that clearly correspond to AgCl nanoparticles, not to Ago nanoparticles. Vivek et al. (2011) clearly obtained, from Gelidiella acerosa, AgCl nanoparticles and probably traces of Ago nanoparticles, as stated by the authors, and as shown by XRD patterns and UV–Vis spectra. Aqueous extract of Sargassum plagiophyllum produced mainly AgCl nanoparticles, as stated by the authors (Dhas et al. 2014); however, traces of Ago nanoparticles were also shown by UV–Vis spectra, since pure AgCl nanoparticles did not exhibit plasmon absorption spectra. Gracilaria crassa was able to produce Ago nanoparticles as stated by the authors (Lavakumar et al. 2015); however, presence of AgCl nanoparticles can be observed from the XRD pattern. The authors ignored the presence of AgCl nanoparticles.

The formation of synthesized Ago nanoparticles by Spirulina platensis was suggested by XRD measurements (Namasivayam et al. 2015). The authors stated that the distinct high diffraction peaks at 21.6°, 35.5°, and 43.5° erroneously corresponded to the face-centered cubic structure of crystalline silver nanoparticles. Probably (since the published XRD graphic has low quality), there is the appearance of Ago nanoparticles at 38o, 44o, and 64o; however, there is mainly the presence of AgCl nanoparticles (monoclinic) (ICSD Code 56543). Therefore, erroneous interpretation of XRD results can be observed for biogenic synthesis of Ag° and AgCl nanoparticles from algae.

Yeasts

Candida diversa strain JA1 was reported to synthesize Ago nanoparticles together with some AgCl nanoparticles (Chauhan et al. 2014), in a similar manner as described by Candida lusitaniae (strain TERM 73 and TERM77) (Eugenio et al. 2016). Indeed, Candida albicans produced AgCl nanoparticles as shown by XRD pattern, together with Ago nanoparticles, and the authors unfortunately did not discuss this pattern (Saminathan 2015).

Enzymes

Kumar et al. (2007) reported the synthesis of Ag° nanoparticles from lysozyme, although the XRD analysis confirmed the formation of the Ag° nanoparticles, the authors ignored the presence of AgCl nanoparticles, as revealed by XRD. Das et al. (2009) and Kumar et al. (2012) reported the lysozyme-mediated synthesis of Ago nanoparticles in aqueous solution under controlled pH and light conditions. XRD pattern showed that the reaction of laccase, obtained from Trametes versicolor, with silver ions was compatible with the cubic phase of Ag° nanoparticles with diffractions points at planes of the facet centered cubic (FCC) structure (JCPDS file: 65-2871 or ICSD Code 64994) that coexisted with the cubic phase of AgCl planes (JCPDS file: 31-1238 or ICSD Code 64734) (Durán et al. 2014b).

Similarly, Zhao et al. (2015) generated both Ago and AgCl nanoparticles by Trichoderma hamatum extract as shown by its XRD pattern. Plasmonic silver-embedded silver chloride (Ag/AgCl) nano-photocatalysts were prepared in a microbe-free aqueous lysogeny broth (Miller) solution, with sunlight exposure and at room temperature, as shown by XRD analysis. Probably, in situ photo-reduction and photo-accelerated ripening of AgCl colloids were promoted by the attached biomolecular ligands (Shen et al. 2015).

Antibacterial activities of biogenic Ago nanoparticles, AgCl nanoparticles, and Ag/AgCl nanocomposite

From the revised literature, there is a confusion regarding to the characterization of biogenic synthesized Ag° nanoparticles, AgCl nanoparticles, and Ag/AgCl nanocomposites. This misunderstanding affects the appropriate interpretation of the antimicrobial effects of biogenic silver-based nanomaterials and impairs the establishment of regulatory nanotechnological platform in countries that are using silver nanoparticles for the manufacturing of medical or technical products. All silver-based biogenic nanomaterials (Ag° nanoparticles, AgCl nanoparticles, and Ag°/AgCl) are reported to have antimicrobial activity. Tables 1, 2, and 3 summarize the antibacterial activity of biogenic synthesized Ag° nanoparticles, AgCl nanoparticles and Ag°/AgCl nanoparticles, respectively. The tables present the conclusion from XRD pattern analysis, the biogenic organism used for the synthesis, the size and zeta potential of the nanoparticles, the bacteria strain employed for the microbiological assay, the minimum inhibitory concentration (MIC) values, and the corresponding reference number.

Table 1 Biogenic Ago nanoparticles characterized by XRD technique and their antibacterial activities
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Table 2 Biogenic AgCl nanoparticles characterized by XRD technique and their antibacterial activities
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Table 3 Biogenic Ag/AgCl nanoparticles characterized by XRD technique and antibacterial activities
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As can be seen in Tables 1, 2, and 3, the most important parameters to compare the antimicrobial efficacy of a compound are the MIC and/or minimal bactericidal concentration (MBC) (CLSI, 1999) values. However, most of studies reported the antimicrobial effect of silver-based nanomaterials in terms of zone of inhibition, which is a qualitative measurement, and in many cases, the concentration of silver-based nanomaterial is not reported.

While the antimicrobial efficacy of biogenic silver-based nanomaterials is increasing, irregular reporting of both nanomaterial characterization and microbiology parameters continue to limit the development of biogenic silver-based nanomaterials because comparison between reports are difficult. Microbiological results must be reported using standard conventions to allow for appropriate comparison among the therapeutic activity between the biogenic nanomaterials.

From Tables 1, 2, and 3, it can be conclude that MIC values of Escherichia coli from different strains were completely different, as shown in the strain BL-21 (Sathishkumar et al. 2009) vs ATCC 25922 (Suresh et al. 2010), in which the MIC values were found to be 59 and 2 μg/mL, respectively. Obviously, this discrepancy depends on the intermediate resistant of the strains. Therefore, this is an indication that it is necessary to use standard strains in order to compare the results in an adequate manner. Indeed, from the results presented in Table 2, it is not possible to compare the antibacterial activity, since the studies used different strains, and unfortunately, MIC values were not reported. Analyzing Tables 1, 2, and 3, it can be observed that very few studies reported MIC values and employed same bacterial strains for the biological tests.

Attempt to correlate the antibacterial activities among the silver-based nanoparticles

This section attempts to compare the microbiological results published in the literature regarding to the antimicrobial effects of biogenic synthesized silver-based nanomaterials (Ag° nanoparticles, AgCl nanoparticles, and Ag/AgCl). Table 4 presents the biogenic silver-based nanostructure material, the size of the nanoparticle, the MIC values towards E. coli, Staphylococcus aureus, C. albicans, B. subtilis, P. aeruginosa, and the corresponding references. As can be observed, comparison between the nanostructures are complicated since the size of the nanoparticles are reported from different techniques ranging from solid-state measurements (TEM and SEM) to aqueous suspension measurements (DLS). It should be noted that generally, DLS values are found to be higher in comparison with nanoparticle size obtained from TEM/SEM analyses, since in aqueous environment there is the presence of extra hydrate layers on the nanoparticle surface. Therefore, standardization of size measurements of nanoparticles is required. Moreover, in microbiological assays, different strains are selected for the antibacterial activities, and a few papers reported the results in terms of MIC or MBC, in contrast, zone of inhibition is still observed in several publications as the single microbiological assay.

Table 4 Comparison between MIC and size values of biogenically synthesized Ag° nanoparticles and Ag°/AgCl
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Despite these differences, Table 4 aims to highlight and summarize the published results regarding to antibacterial effects of biogenic silver-based nanomaterials against E. coli, S. aureus, C. albicans, B. subtilis, and P. aeruginosa. In the case of Ag° nanoparticles acting on E. coli, no correlation between the published papers was possible to make, since different strains and different size measurements were performed. For Ag/AgCl nanoparticles; generally, it can be conclude that MIC values of 2–7 μg/mL were obtained by treating E. coli with 20–70 nm nanoparticle sizes in different strains including ATCC 8739 and ATCC 25922. In the case of S. aureus, MIC values in the range of 1.9–3.4 μg/mL were found for ~20 nm of Ag/AgCl nanoparticles (ATCC 06538 and 05,923) (Marcato and Durán 2011; Longhi et al. 2016; Ballottin et al. 2016). Table 4 reveals that higher values of MIC are observed for nanoparticles with higher size values, as measured by DLS technique.

In the case of antimicrobial activity of silver-based biogenic nanoparticles towards different strains (ATCC 26790, ATCC 10231) of C. albicans, the nanocomposite Ag/AgCl showed MIC values in the range of 4–12 μg/mL and the size values range from 24–70 nm, as measured by DLS technique. Although, Ag/AgCl synthesized from banana peel no effect was observed against C. albicans. Probably, in this case, the protein corona effect (Durán et al. 2015b) (capped protein on the surface of Ag/AgCl nanoparticle) impaired the nanoparticle antimicrobial activity.

In the case of antimicrobial activity against B. subtilis and P. aeruginosa, different MIC values and different particle sizes are reported. Therefore, these results highlight the importance of standardization of the characterization and the microbiological assays of silver-based biogenic nanoparticles.

According to Table 4, in the case of E. coli, few publications reported the MIC values for silver-based nanoparticles. Among them, Ago nanoparticles (size of 30–40 nm) obtained from the plant extract of C. zeylanicum demonstrated an MIC value of 10 μg/mL, whereas Ag° nanoparticles (size of 2–11 nm) obtained from S. oneidensis showed an MIC value of 2 μg/mL (Table 4). This is an indication that small-sized Ago nanoparticles are more active against the studied bacterium. Similar results were observed for Ag/AgCl nanoparticles, the decrease of MIC values for small-sized nanoparticles (Table 4). Unfortunately, in the case of AgCl nanoparticles, the literature does not report enough data to allow comparison, as indicated in Table 2. Therefore, more studies are necessary.

Final remarks

From the selected publications presented and discussed in this review, it can be observed that the biogenic synthesis of silver-based nanomaterials (Ag° nanoparticles, AgCl nanoparticles, and Ag/AgCl nanocomposites) has become an attractive field of research due to the combination of the biological/technological impact of silver nanoparticles and the use of environment-friendly routes in the place of traditional chemical/physical synthetic protocols. Although much progress in this area (Durán et al. 2016), the characterization of the obtained silver-based biogenic nanostructures contains errors, which are perpetuating in many recent publications. In special, the XRD and UV–Vis analyses of the nanoparticles, reported in several papers, demonstrate incorrect interpretations and conclusions.

Besides the lack of appropriate characterization of biogenic silver-based nanomaterials, the antimicrobial experiments, reported in the literature, make difficult comparisons among the reports. As demonstrated in this review, it is difficult to compare the results reported by different papers, since the measurements of nanoparticle size varies among the reports, as well as the authors used different microorganism strains, and in most of the cases, the results are not reported in terms of MIC values. In addition, an important point to be considered is the thermodynamic stabilization of Ag° and AgCl nanoparticles, which may interfere in their antibacterial activities (de Lima et al. 2012). In general, bared nanoparticles obtained by chemical/physical routes are unstable and tend to aggregate. To avoid this, the nanoparticle stabilization is achieved by the addition of capping agents via covalent bonds or chemical interactions. In contrast, biogenically synthesized nanoparticles have a superior stability due to the presence of capping agents. Indeed, biogenically synthesized nanoparticles are obtained from the biological resources, which act not only as a reducing agent but also as efficient capping agents (Seabra and Durán, 2015). The presence of capping agents is important to avoid the oxidation of biogenic Ag° nanoparticles to silver ions. During biogenic synthesis of Ag° nanoparticles, oxidation of the metallic nanoparticle is avoid by the presence of reducing/capping agents (Durán et al. 2015a,b).

This review highlights that irregular reporting of both nanomaterial characterization and microbiological parameters impairs the development of biogenic silver-based nanomaterials, since the comparison between the reports are difficult. In contrast, comparable results from different research groups are necessary for the development of regulatory issues in safety norms and procedures. Microbiological results need be reported using standard conventions allowing appropriate comparison among the therapeutic activity between the biogenic nanomaterials synthesized from different research groups.

For microbiological evaluations, this review highlights the importance to select the target microorganism and the protocol by following the international foundations, such as the Pharmaceutical Microbiology Manual (2014). ORA.007, Version 1.2 date: March 30, 2015 (http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM397228.pdf). According to this manual, the protocol for the evaluation of antimicrobial effectiveness of a certain compound should use cultures with no more than five passages removed from the original stock culture. Moreover, the manual suggests the following standard microorganisms: C. albicans (ATCC 10231), Aspergillus brasiliensis (ATCC 16404) (formerly A. niger), E. coli (ATCC 8739), P. aeruginosa (ATCC 9027), and S. aureus (ATCC 6538). There are other suggestions of regulatory manuals to be followed, including the US Pharmacopeia. US Pharmacopeia Convention, Rockville, MD, USA, 2009, pp. 87–93), and British Pharmacopeia. The stationary office, London, 2011, pp. A348–A355). Determination of MIC values by broth microdilution assays in 96-well microplates (Corning®) as suggested by the Clinical and Laboratory Standards Institute (CLSI, 2015) is the most common assay (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Approved Standards. Tenth Edition. M07-A10 Vol. 35 (2). Replaces M07-A9. Jan. 2015) (http://shop.clsi.org/c.1253739/site/Sample_pdf/M07A10_sample.pdf), and for MBC (CLSI, 1999) (http://shop.clsi.org/site/Sample_pdf/M26A_sample.pdf).

Finally, as stated in this review, it is necessary an exhaustive adequate characterization and interpretation of the final products obtained from biogenic synthesis of silver-based nanomaterials, followed by comparable reports of their antimicrobial activities.