The genus Bacillus consists of endospore forming rod-shaped bacteria that are members of the phylum firmicutes. Because species within the genus are largely common inhabitants of soil and aquatic sediments, they are widespread in nature and are found in virtually any environment. (Rooney et al. 2009). The reason Bacillus exploited for various biotechnological applications, because of their genetic variation lead to many of which were used for biotechnological and industrial applications (Price et al. 2007). The importance of Bacillus species in medicine, environment and industrial applications is reflected in the large number of Genome sequences deposited in the GenBank database 2017 (Anbu, et al. 2015).

Hernandez-Gonzalez et al, (2018) reported that by November 2017, more than 2000 complete and draft genome sequences of this genus had been deposited in the GenBank database. Most of these Bacillus sequences are either medically relevant strains, or are isolated from terrestrial environments. The number of Bacillus genome sequences publicly available isolated from aquatic environments has recently increased.

The change is a preliminary tool for the confirmation that the particles in nano size as well as the ability of bacterial extract in nanoparticle biogenic synthesis. Turn the culture to brown color in the reaction mixture (silver nitrate and bacterial culture) confirm the formation of silver nanoparticles (Fig.13 and 14). The reaction mixture turned into brown color after 48 hr  of incubation. The occurrences and turning to brown color indicate the formation of silver nanoparticles in the culture of reaction mixture and the efficacy of the broth media as reducing agent the Ag ions as in Fig. 10 and that with agreement with previous studies (Skiba and Vorobyova, 2019).

The information about the formation of silver nanoparticles using Bacillus species is confirmed in many previous studies. Reduction of silver ions and formation of silver may be due to the existence of bioactive molecules for examples polyphenolic compounds such as phenolic and flavonoids (Kaurinovic and Vastag, 2019). Other bioactive molecules were also confirmed to be excellent reducing agent in nanoparticles formation such as protein molecules, enzyme for examples nitrate reeducates which reported as good regulating agent in the nanoparticles biosynthesis and particularly for silver nitrate (Lee and Jun 2019).

The present study revealed a GC ratio of the bacillus martiums of 56.5% (Table 2). This finding is in line with previous studies confirming that firmicutes have GC content of up to 55 percent (e.g. Geobacillus thermocatenulatus). Some firmicutes make resistant progeny called endospores, while others can reproduce by binary fission only. It’s obvious that firmicutes are as varied as important. Different strains were isolated from Nile Tilapia in the present study, among which one strain named Asmo2020 (Fig. 1) showed excellent antimicrobial activity. The percentage identity is to estimate how closely the strain profile matches the taxon relative to the Bacillus Maritimus. The strain’s 16S rRNA sequence showed that the alignment similarity to Bacillus maritimus strain KS16-9 and Bacillus sp. was > 99 per cent. H-264 16S (Fig. 1).These species are considered to be members of the Bacillus group, and their similarity has been improved based on the 16S rRNA sequence. Chun and Bae (2000) reported that if the identity of the sequence showed similarity above 99 %. It could definitely conclude that the two strains belong to the same species in the current study it was identified as Bacillus maritimus. In addition, they reported that strains are classified as the same genus or the same family if the identity is higher than 97 %. Therefore the isolated strain was confirmed as Bacillus maritimus based on the morphological, physiological, biochemical characteristics, phylogenetic position, and 16S rRNA gene sequences.. The antioxidant capacity for the Bacillus Maritimus culture was investigated using two antioxidant model DDPH and ABTS; respectively (Figs 6 & 7). In the present study, Bacillus maritimus culture showed high antioxidant activity compared to vitamin C in agreement with other previous study (Elshaghabee et al. 2017). This is may be due to the presence of different bioactive compounds such as phenolic, flavonoids, carotenoids, fatty acids and lipids, carbohydrates and sulfated polysaccharide, amino acids and proteins (Figs 2 & 4) (Abd El Moneam et al. 2017a-e & 2018; Nabil-Adam et al. 2019 & 2020; Shreadah et al. 2017 & 2018a-c). Moreover, the current study revealed high anti-inflammatory activity of Bacillus maritimus compared to vitamin C (Fig. 5). This can be attributed to the presence also of over mentioned bioactive secondary metabolites (Abd El Moneam et al. 2017a-e & 2018; Nabil-Adam et al. 2019 &2020; Shreadah et al. 2017 & 2018a-c). It has been reported also that polysaccharides and sulfated polysaccharides particularly derived from Bacillus induce anti-inflammatory cytokine leading to suppressing T cell-dependent immune responses by inhibiting T cell activation (Xia et al. 2019). One reason behind the anti-inflammatory effect of Bacillus species is the production of the bioflocculant QZ-7 (Abu Tawila et al. 2018).

Class Bacilli organisms are capable of producing larger amounts of polysscharide compared to lactic acid bacteria (Ruiz Rodriguez, et al. 2019). In an earlier study, a bioflocculant-producing thermotolerant species Bacillus subtilis F9 was isolated from wastewater sludge (Giri et al. 2015). NO is a signaling molecule which has important functions in inflammatory pathogenesis. It appears to be an anti-inflammatory in normal condition, but NO functions as a pro-inflammatory mediator thus promoting inflammation in abnormal physiological states through its overproduction (Chen, et al. 2017). Thus it has been shown that NO inhibitors play a vital role in controlling inflammatory diseases. The present study showed that, in agreement with that of Paynich et al. (2017), different concentrations of Bacillus maritimus (mg / ml) led to maximum inhibition of NO production. Bacillus’ anti-inflammatory effect was investigated against pretreated Bacillus Caco-2 cells, which resulted in the release of IL-8 to various pro-inflammatory signals IL-1β, deoxynivalenol, or flagellin. In all stressed conditions tested, Bacillus was able to decrease IL-8 production considerably. Mechanistically, during IL-1β exposure Bacillus maritimus appeared to limit the nuclear translocation of NF-kB by pre-exposure by preventing degradation of IκB (Rhayat et al. 2019). The effects of Bacillus 29784 were observed independently with supernatant and cells, but to a lesser extent than with the combination, indicating that both secreted metabolites and cell-associated compounds are thus likely to be attributed to them. In addition, Bacillus 29784 significantly reduced the upregulation of the iNOS protein levels under inflammatory conditions further underlining its anti-inflammatory intestinal potential. Previous studies have reported an immunomodulatory effect of soluble secondary metabolites of Bacillus, inhibiting the signaling pathway of compounds associated with NF-yB, or / and bacterial membranes (Valdivieso-Ugarte et al. 2019). As antibiotics have been known for decades, the potential of Bacillus species to produce a broad diversity of bioactive secondary metabolites acts. It is well known that at least 4–5 per cent of the genome of Bacillus acts as antimicrobial compounds. The nature of the mainly antimicrobial peptides in these compounds. Their structures are hydrophobic cyclic, and for example intermolecular thioether bonds and/or D-amino acids (AA) properly contain peculiar moieties. In addition, volatile compounds also have a large family of antimicrobials that exhibit variety of functional and metabolic roles in antimicrobial compounds. the main biosynthesis pathway and the secondary metabolites bioactivity clades of antimicrobial compounds within the Bacillus group: i.e., the polyketides (PKs), (Enzymes and bacteriocins),the ribosomal peptides (RPs) the non-ribosomal peptides (NRPs) and the volatiles (Caulier, et al. 2019).

The growing antimicrobial resistances in modern clinical pathology and medicine interest and focus on bioactive compounds from natural products in order to generate new and novel antimicrobials in general and particular antibiotics (Fiedler et al. 2019). Bacilli have capability able to synthesis more than two dozen of antibiotics with variety in function and structures as they well known for the production of a vast array of structurally unrelated antimicrobial compounds. The formation of these antibiotics is depending on the environmental characteristics, the metabolic pathways including lipopeptides such as surfactin, iturin, bacteriocins, bacteriocin and fengycins (Ramachandran, et al. 2014). one of known antimicrobial mechanisms against pathogen is that lipopeptides allows certain Bacillus to change their outer surface which form biofilm through allow them to regroup together to proliferate and distributed in the area.

Formation of silver nanoparticles is easily detected by spectroscopy because the colored nanoparticle solution shows a peak ~400 nm. In the current study, the optical density of the solution was measured by using a Jenway spectrophotometer and a transmission electron microscopy was applied. The result of micrograph of TEM analyzed of biogenic silver nanoparticles using Bacillus maritimus. as reducing agent showed that nanoparticles is essentially spherical furthermore they are well-disperse and size ranging from 18.68-26.9nm. The Ag nanoparticles have particle size in the range of 18.68- 26.96 nm. However, in the current study we investigate the possible mechanism of metal and microbes interaction which may be due to structural specificity of the cell of microbes and how metal availability influences microbia. In this paper we report the culture form bacteria Bacillus for synthesis of silver nanoparticles using the culture filtrate figure. the Bacillus maritimus able to biogenic synthesis of silver nanoparticles which confirmed using UV, FT-IR, SEM and TEM (Fig. s 11-14 & table 4). Image showed that the nano-silver particles shape and crystal structure of silver nanoparticles as well as size of the particles. Furthermore, the EDX analysis confirm this finding with presence of other element such as sodium, chlorine, magnesium Sulfur and potassium in addition to carbon and oxygen and that due to culture of bacillus that contain all that also the metabolite of the Bacillus maritimus. as in Fig. 13 The UV-Vis absorption spectra (Fig. 11). The control samples were subjected to the solution of silver nitrate have been shown the wide spectrum range around 400 nm where in case of nanoparticles formation of silver nitrate a broad resonance confirm the aggregation of the silver nanoparticles in the solution (Adebayo-Tayo et al.2019). Many studies have confirmed the advantages of biogenic synthesis over physical, chemical, since these methods attach to toxic substances on the surface of nanoparticles, in addition to the instability of nanoparticles, in addition to producing hazardous as by-products (Singh et al. 2018). In addition, Igiri et al. (2018) investigated the microorganism mechanism that reduces hazard and toxic substance by accumulating metallic ions to reduce its toxicity. Reduction of nanoparticles occurs intracellularly with different reduction of bioactive compounds that have been extracellularly produced by different metabolites, inside the cell and/or on the cell wall. The biogenic synthesis of bioactive compounds such as polyphenolic (flavonoids, phenolic and tannic) carotenoids and water-soluble biomolecules produces stable nanoparticles, additional active surface area without formation for interaction in the biological environment, hazardous by-product (Singh, et al. 2018; Siddiqi et al. 2018).

One possible mechanism behind the stability of nanoparticles is the bioactive compounds that make capping layers that usually affect the biological activity of nanoparticles with particular and highly stable long-term stability during biosynthesis. Previous study confirmed that both nucleation and surface growth undergoes the biosynthesis of nanoparticles from microorganisms. In both positive gram negatives such as Streptomyces and gram negative such as Bacillus, antimicrobial metabolites are widely produced among genus bacteria in many bacterial genera (Chen et al. 2019).

Bacillus strains in particular are widely distributed in the environment, and have also been isolated from various environmental sources. Most species of Bacillus members consider good sources of biologically active compounds, in particular enzyme inhibitors, therapeutic proteins, antibiotics, and bioactive pharmacological agents (Perez et al. 2017). They produce bioactive metabolites with vast numbers of secondary antimicrobial metabolites, as well as bio-peptides with varied diversity and chemical structures (Andryukov et al. 2019).). Although most of these compounds are bioactive against Gram-positive bacteria, some of them have a wide range of bioactivity towards Gram-negative bacteria and filamentous fungi, such as bacteriocins, bacteriocin-like substances and lipopeptides (Silva et al. 2018).

Although several studies have been conducted on the isolation and characterization of antimicrobial metabolite producing Bacillus from different origins, few or no studies were evaluations of their anti-microbial performance from Bacillus maritimus. Seven peaks were shown in FT-IR profile (Fig. 5A & 5B). The position were at 3356.94, 2102.67, 1635.81, 1076.61, 582.94, 497.15 and 420.23 cm⁻¹ ; respectively for Bacillus maritimus culture whereas the Bacterial culture AgNPs reaction mixture at the end of 30  hours showed 23 spectral bands (Fig. 10 B) at the following wave numbers 3891.64, 3860.27, 3741.97, 3281.64, 2962.15, 2919.01, 2360.62, 219018, 2107.47, 1988.82, 1790.83, 1627.81, 1539.13, 1453.60, 1406.37, 1221.83, 1062.53, 778.32, 691.03 and 442.32 cm⁻¹. With respect to the AgNPs reaction mixture FT-IR analysis after 30 hours (Fig. 10B), the absorbance bands located between 3000–3500 cm⁻¹ were assigned to the stretching vibrations of amine groups and hydroxyl (Geana et al. 2019). Illustrated distinctive broad spectral bands around 3356.94 -3891.64 cm⁻¹ are characteristic of the O-H stretching vibration type of the hydroxyl functional group in polyphenols and N-H stretching vibrations of amino acids , peptides, and proteins in primary and secondary amines. The peak change in the spectral profile of AgNPs could be attributed to the interactions between these chemical functional groups and AgNPs. Protein molecules have been documented collaborating with AgNPs via free amide groups (Hamouda et al. 2019). FT-IR data showed that the amide protein linkage possessed a higher potential for joining silver and thus forming protein covering around AgNPs to prevent agglomeration and thus stabilize the medium (Elamawi et al. 2018).The involvement of hydroxyl groups in the bioreduction process could be confirmed in this study by shifting the deformation vibration of O-H positioned at about wave number 1100 cm⁻¹ in the AgNP preparations as well as the observed shift from 1100 to 1132 cm⁻¹ which did not appear in the crude extract of Bacillus maritimus. The FTIR showed spectral peaks at 2962.15, 2919.01, 2360.62, 2190.18, and 2107.47 cm⁻¹ may refer to N-H bending vibration and hydrocarbon chains stretching C-H vibration (Coates, 2000).The spectral vibration of the aldehyde group (C = O) was illustrated by a wave length of 1740 cm⁻¹, whereas the peak at a range of 1648–1621 cm⁻¹ could be attributed to amides (N-H) stretching in addition to peptide bond and C = C stretching which may involve the stabilization of nanoparticles by proteins as explained by Castro et al. (2013), who investigated the biosynthesis of AgNPs using aqueous extract of either t. The spectral band at 1384 cm⁻¹ could be designated to the residual amount of AgNO₃, which appeared only in the three AgNPs preparations. The data revealed the presence of 1221,83cm⁻¹ absorption bands that may have been attributed to vibration of proteins as a stabilizing agent is attributed to free amine groups or cysteine groups (Coates, 2000). The peaks in the Bacillus maritimus extract IR profile were positioned at wave numbers 1551 and 1415 cm⁻¹ and disappeared in the AgNPs preparations. The bending region of the aliphatic chain was indicated by spectral peaks of 616–672, 522–575 & 441–493 cm⁻¹ In addition, bands assigned at 1381, 1269, 1132 & 1034 cm⁻¹ can be attributed to either sulfur or phosphorus function groups that may attach silver and perform both nanoparticles capping and stabilisation processes (Castro et al. 2013).