Green Nano Actinobacteriology – An Interdisciplinary Study

Green nano actinobacteriology has been considered as a novel field of study in order to develop least expensive and highly qualitative strategies for production of eco-friendly beneficial products with diverse applications. The uniqueness of bioactive ac‐ tinomycetes has turned the attention of scientists worldwide in order to explore its potentiality as effective “micronanofactories”. This chapter provides a brief overview of the synthesis, characterization, and application of actinobacterial nanoparticles with an added note to actinobacterial detoxification.


Introduction
Nanotechnology is a rapid, upcoming, multidisciplinary promising area that has an influence in medical, agricultural, and industrial fields, where they manufacture materials at the nano scale (one billionth of a meter or 10 -9 in size). These nanomaterials are synthesized by physical, chemical, and biological methodologies. Physical processes include mechanical smashing, solid phase reaction, laser ablation, melt mixing, high-energy ball milling, physical vapor deposition, freeze drying, spread drying, ion sputtering, solvothermal synthesis, sol-gel technique, and precipitation [16,17]. Chemical processes include aqueous and nonaqueous chemical reduction, electrochemical reduction, template method, ultrasonic-assisted reduction, photocatalytic reduction, microwave-assisted synthesis, biochemical reduction, irradiation reduction, and micro-emulsion method [32]. Both these physical and chemical processes need high temperature and high pressure to execute. These processes were costlier and produced highly toxic compounds along with consumption of high energy. Therefore, scientists employ inexpensive, eco-friendly, easier scale-up biological synthesis methodology by utilizing both plant extracts [6,10,21,26,28] and microbes. The phytochemicals and enzymes present in plants and microbes reduce the metal compounds into corresponding nanoparticles [1]. Initially, among microbes, bacteria was utilized for green synthesis, which was later overcome by the use of yeast, algae [7,25] and fungi [5,8,20,27]. But the use of actinomycetes (filamentous bacteria) was comparatively less reported, though they act as valuable resource for numerous diverse bioactive metabolites [2]. Compared to bacteria, fungi and actinobacteria secrete more proteins which in turn increase biosynthesis production. Compared to fungi, actinobacteria comes under prokaryotes group. Hence, they can be manipulated genetically in order to achieve better control over size and polydispersity of the nanoparticles [14,36]. This biogenic process takes place at ambient temperature and pressure conditions and no toxic chemical is involved [37].
In this chapter, green synthesis of nanoparticles by actinobacteria and their application in varied fields were studied under green nano actinobacteriology, which acts as the interface between "nanotechnology" and "actinobacteriology".

Biosynthesis of Nanoparticles
Actinomycetes are helpful in the biosynthesis of nanoparticles with good surface and size characteristics showing wide range of bioproperties. Actinobacterial production of metallic nanomaterials was mediated by extracellular and intracellular methodologies. Extracellular synthesis has got more commercial advantages compared to intracellular synthesis since polydispersity plays a significant role. In the case of intracellular synthesis, the accumulated nanoparticles are of particular dimension with less polydispersity [4]. In extracellular synthesis, steps such as fermentation, filtration, followed by enzyme substrate complex formation in dark condition were involved. Whereas in intracellular synthesis, additional steps like ultrasound treatment of enzyme substrate complex or its reaction with suitable detergents were required [4]. Hence due to the above-mentioned drawbacks, unlike bacteria and fungi, only less number of intracellular actinobacterial nanoparticle production was reported.
The basic principle is actinobacteria, when exposed to metal ions, releases enzymes that reduce the metal ions to yield highly stable nanoparticles. Usually, silver ions in the form of silver nitrate (AgNO 3 ) solution [9] or gold ions in the form of chloroauric acid (AuCl 4 ) solution were used as substrate to the enzyme secreted from microbes. Both gold and silver are nonallergic, biocompatible with slow oxidation rate, and effective antimicrobial agents. Several researches are conducted by using other metals like zinc, manganese, and copper for nanoparticle formation.
Most of the nanoparticle biosynthesis work was carried out in Streptomyces sp., except the first novel work in Rhodococcus sp where Ahmed et al. [38] reported the intracellular synthesis of gold nanoparticle of the dimension 5-15 nm. They were concentrated more on cytoplasmic membrane than on cell wall. Using Streptomyces sp., intracellular synthesis of gold nanoparti-cles [39,40], silver nanoparticles [41,42], zinc and manganese nanoparticles [33] were reported till date.

Characterization of Nanoparticles
Techniques used for characterizing nanoparticles and their respective applications are as follows.

a. Colour change test
The formation of nanoparticles was detected by the color change within 72 h. Change of color from pale yellow to brownish color shows formation of silver nanoparticles [19]. Change of color from pale yellow to pinkish color indicates formation of gold nanoparticles. Formation of whitish yellow to yellow color shows formation of manganese and zinc nanoparticles [33].

b. UV--Visible Spectroscopy
The reduction of metal ions was estimated by measuring the absorption level using UV-visible spectroscopy. Light wavelengths of 200-800 nm are considered for nanoparticle characterization. Absorption measurements in the wavelength range of 400-450 nm and 500-550 nm are used in characterizing silver and gold nanoparticles, respectively [4].

c. X-ray Diffraction (XRD)
The phase identification and characterization of nanoparticle crystal structure were studied using XRD. X-rays penetrate into the freeze-dried and powdered nanoparticle at the scan speed of 0.02/min and the resultant diffraction is compared with the standard in order to derive its structural information [2,4].

d. Fourier Transform Infrared Spectrometer (FT-IR) Analysis
Dried nanoparticle sample was mixed with KBr in 1:100 ratio and scanned using IR rays of around 4000-400 cm -1 with reflectance mode at 4 cm -1 resolution. The chemistry and the variation in functional group attached to the nanoparticle surface are analyzed using FT-IR [4,12,30].

e. Dynamic Light Scattering (DLS)
DLS was performed to determine the size, surface charge, and distribution of nanoparticles suspended in a liquid [17].

f. Energy Dispersive X-ray (EDX)
EDX was performed to determine the elemental composition of metal nanoparticles [17].

g. Atomic Force Microscopy (AFM)
AFM was performed to determine the size and topological appearance of metal nanomaterials. Porosity, roughness, and fractal dimension are also analyzed with the help of AFM images. For AFM study, the sonicated metal nanoparticles were formed as a thin film on slide [29,32].

h. Scanning Eelectron Microscope (SEM)
SEM was performed to determine the size and surface morphology of biosynthesized metal nanomaterials. For SEM study at an operating voltage of 15 kV, the nanoparticle solution was sonicated, centrifuged, and the resultant dried powdered nanoparticles were used as sample [29].

i. Transmission Electron Microscope (TEM)
Compared to SEM, TEM gives 1,000 fold higher morphological resolution including both size and shape. The ultrasonicated nanomaterial sample was placed on copper grid coated with 300 mesh palladium and carbon for TEM study at 80 kV [33].

Applications
Nowadays, biosynthesized nanoparticles, especially gold and silver, were used in diverse applications especially in diagnostic field due to its antibacterial, antifungal, larvicidal, antifouling, anticancerous, antioxidant properties. Green synthesis of nanoparticles using dextran as ligand was explored since dextran was considered as cheaper, nontoxic and biocompatible agent [31].

a. Antibacterial activity
Using Streptomyces viridogens, gold nanoparticles of spherical shape with 18-20 nm was synthesized through intracellular mode [39]. A considerable number of silver nanoparticles using Streptomyces sp. were reported to show antibacterial activity against varied human pathogens through extracellular mode [14,22,24,29,51,[53][54][55][56][57]. Unnamed actinomycetes mediated silver nanoparticles were also reported to produce antibacterial activity [13,18,44]. Rare actinobacteria such as Thermoactinomyces sp. produce spherical shaped 20-40 nm sized silver nanoparticles were reported by Deepa et al. [30]. Similarly, Usha et al. [43] reported the production of copper and zinc nanoparticles sized 100-150 nm using Streptomyces sp. showing antimicrobial activity. The mechanism behind this bactericidal effect is silver nanoparticles attach to the bacterial cell membrane and disturb both respiration and permeability. Then by penetrating deep into the DNA part, it causes further damage by disrupting DNA replication due to release of silver ions, which leads to target cell destruction [32,34].

b. Antifungal activity
Biosynthesis of gold nanoparticles using Streptomyces sp. VITDDK3 showed antifungal activity against M. gypseum and T. rubrum by changing the membrane potential and inhibiting ATP synthase activity of the target cell [49].

c. Anti-biofouling property
Anti-biofouling is defined as the process of eliminating the microbes that aggregate on wetted surface forming biofilms leading to foul smell production. Shanmugasundaram et al. [42] reported the antibiofouling property of 5-50 nm spherical-shaped silver nanoparticles using Streptomyces naganishii MA7.

d. Antioxidant property
An antioxidant prevents the oxidation of other molecules by interfering with defense mechanism leading to ROS generation [46]. Antioxidant property of silver nanoparticles mediated by Streptomyces naganishii MA7 was reported by Shanmugasundaram et al. [42], showing positive DPPH scavenging activity.

e. Larvicidal property
Silver nanoparticles synthesized by using Streptomyces sp. GRD showed effective larvicidal activity against Culex quinquefasciatus and Aedes aegypti, which would be an effective bioprocess for mosquito control [35]. Nanoparticles penetrate through larval membrane into the intracellular space leading to denaturation of organelles and enzymes [35]. Karthik et al. reported the biosynthesis of gold nanoparticles with antimalarial activity using Streptomyces sp. LK3 [50].

f. Anticancerous property
Varied researches are going on in order to treat cancer and reduce its side effects worldwide. Manivasagan et al. reported the cytotoxic effect of silver nanoparticles mediated by Nocardiopsis sp. MBRC-1 [23]. Same way, an actinomycete PSBVIT-13 [18] and Streptomyces naganishii MA7 [42] were also reported to mediate silver nanoparticles with cytotoxic effect.

g. Others
Torres-Chavolla et al. [52] reported the production of gold nanoparticles using Thermomonospora sp. which can be utilized as biosensing enhancement analytical device meant for detection purpose in military field and pollution control field.

Drawbacks (Nanoparticle toxicity)
Exposure of cells to actinobacterial nanoparticles like silver nanoparticles alters mitochondrial functioning by collapsing proton-motive force across the cell membrane, which leads to increased membrane permeability. As a result, ROS (reactive oxygen species) will be generated, which seems to be the initiator for toxicity. Factors like breakdown of its unique surface plasmon resonance, magnetic, chemical, and optical properties are responsible for toxicity.

Actinobacterial detoxification
Actinomycetes are resistant to toxic heavy metals due to their chemical detoxification, solubility alteration, as well as energy-dependent ion efflux from the cell by membrane proteins that function either as ATPase or chemiosmotic cation or proton anti-transporters. Detoxification can be made by reduction, precipitation, biosorption, biomineralization, and bioaccumulation [4].

Future directions
Compared to physical and chemical approaches, biosynthesis of nanoparticles consumes extra time. Actinobacterial-mediated nanoparticle biosynthesis has got more advantages such as the possibility of scaling up the process, economic viability, and the possibility of covering a large surface area due to mycelial growth. [11]. Hence, several criteria like synthesis time, particle size, solubility, stability, monodispersity should be given utmost priority in order to obtain highly efficient actinobacterial nanoparticles. By optimizing parameters like type of microbe, growth stage, growth medium, synthesis conditions, source compound of target nanoparticle, etc., it is possible to obtain clean, nontoxic, and eco-friendly nanoparticles [15].

Conclusion
As discussed earlier, due to increased cognizance towards green chemistry approach, the need of the hour is to develop eco-friendly strategies for the synthesis of nontoxic nanoparticles. To serve that purpose, the least explored actinomycetes have to be further explored so that they can emerge as effective "micronanofactories" in future. Still, the exact mechanism of nanoparticle biosynthesis is not clear. By decrypting the never ending clues involved in its synthesis, actinomycetes would be considered as the efficient microbial group for harnessing nanoparticles.