3.1 Identification of fungi:
3.1.1 Phenotypic characterization:
Identification and Characterization of collected fungal strain was performed at NCMR by analyzing obtained results and species was identified as Penicilium rubens with 99% similarity.
Microscopic Observation were performed to study phenotypic expression. Branched, septate, smooth, hyaline hyphae was observed with width 37.66 x 5.82 μm. Conidiophore is un-branched and cylindrical. Size of Metulla is 8.67 x 3.06 μm and Phiallides is 7.43 x 2.09. Conidia is single chains, brown, and smooth, oval with size 1.56 μm.
3.2 Synthesis and Characterization:
In this experimental study, with a reduction of aqueous Ag+, silver nanoparticles were synthesized, by mixing ell filtrate with silver nitrate solution in 9:1 ratio. After an incubation, color chance was observed from pale yellow to dark brown color in 24 hrs. , indicating formation of silver nanoparticles. No tangible aggregate were observed, which indicate that the particle were well dispersed in the solution. Also the color intensity of mixture of AgNO3 and cell filtrate was suspended for 2 hrs. periodic monitoring of reaction mixture were recorded at regular time interval by UV visible spectroscopy.
The UV spectra of mixture of cell filtrate of AgNO3 shown a maximum absorption at 411 nm, which indicates the presence of silver nanoparticles. The silver nanoparticles formed were highly stabled for about 160 hours. Similar results were obtained by shivraj et al. 2013  using Aspergillus flavus. Moreover, Afreen et al. 2011  reported peak at 422 nm with Rhizopus stolonifer. The result were also consistent with maliszewska etal. 2009  since they obtained similar absorption spectrum of AGgNPs produced by penicillium with a maximum peak between 420-450 nm. FTIR analysis measurement accounts for identification of specific functional group which are responsible for synthesis and stabilization of silver nanoparticles. This FTIR spectrum revealed that, the synthesis of silver nanoparticles showed us a strong absorption peak at 3292.45 cm-1 which indicates the presence of carboxylic group. Similarly, the broad absorption found was observed at 3425 and 2927 cm-1 due to O-H stretching and H bounded alcohol and phenol group. A weak bond was observed at 1635 cm-1 corresponding to N-H bending primary amino. A small peak was formed at 773 cm -1 due to occurrence of alkalide halides. This analysis showed the functional biomolecule are hydroxyl, carboxyl, phenol and amine groups that strongly supported the formation of silver nanoparticles by fungi Penicillium rubens to stabilize the silver nanoparticle.
3.3 Optimization study:
The growth and metabolism of fungi is intensely altered by environmental conditions, similarly culture condition and other parameter also greatly influence the productivity and growth of fungi respectively. Various parameter like concentration, pH and temperature can directly affect the rate of which the silver nanoparticles are synthesized.
3.3.1 Effect of AgNO3 concentration:
Silver nanoparticles biosynthesis with different concentration of silver nitrate solution of 0.5 mM to 5 mM was studied with fungal filtrate. The optimize substrate concentrate was obtained as 1 mM by change in color and detected by UV-visible spectra at maximum absorbance of 411 nm. Our result correspond to Banu et al. 2011  as they too found 1 mM AgNO3 concentration as optimum concentration for production of silver nanoparticles using Rhizopus stolonifer.
3.3.2 Effect of pH:
To monitor certain features of the nanoparticles, modifying the pH range is preferable. The pH that is required for silver nanoparticles biosynthesis has a strong influence on growth and the production of enzymes. To research the effect of pH on the development of silver nanoparticles from fungi Penicillium rubens, a different pH ranging from 6 to 8 with a difference of 1 was used. At acidic ranges, small and broad peaks were observed whereas at neutral or slightly alkaline pH, studies have reported successful formation of narrower and sharper peaks indicating successful biosynthesis of silver nanoparticles. From the results obtained, the graph indicates maximum growth at the pH 7 at 410nm which indicates the presence of synthesized nanoparticles. The protein structure is impaired at low pH and the protein is denatured and loses its activity; hence, nanoparticles are aggregated at low pH or in acidic ranges.
It can be concluded that the AgNP formed is stable at pH 7 but not at acidic ph. Our results correspond with Banu et al. 2011 which shows maximum absorbance peak at 422 nm at pH 7.0 with Jain et al. 2001  using Aspergillus flavus at pH 7.
3.3.3 Effect of temperature:
In all reactions, temperature plays an important role. Temperature optimization experiments were carried out at temperatures of 28 °C, 35 °C and 40 °C respectively. UV-Visible spectroscopy was used to analyze the sample, and further effects of temperature on nanoparticles were studied. The temperature used in the synthesis of fungal mediated silver nanoparticles can affect parameters such as the synthesis, speed, size and stability of the nanoparticles. Earlier studies shows that synthesis rate increases as the temperature increases and the maximum rate of synthesis was observed at 35 °C and 40 °C and is considered to be the ideal temperature. While the majority of studies have recorded faster rates of synthesis at higher temperatures, the quality of nanoparticles should be taken into account. The temperature can affect the size and stability of the nanoparticle, in addition to affecting the synthesis rate. Comparing with the ideal conditions of temperature we have obtained optimum temperature of our research to be 35o C. At low temperature, Broad peaks are observed whereas at high temperature narrow and sharp peaks exist. When compared with other temperature range detected by UV spectrum, the maximum absorption spectra was observed at 425 nm which indicated the production of silver nanoparticles. The peak was somewhat symmetric at 35o C. On the other side, at greater temperature like 400 C, the enzyme activity gets lowered, hence the synthesis of silver nanoparticles is affected and peak formed is unsymmetrical.
3.4 Mechanism of dye reduction:
During a chemical reaction bond dissociation energy (BDE) plays a very important role in breaking and formation of new bond. During the reaction, methylene blue dye act as electron acceptor and NaBH4 acts as electron donor. The addition of silver nanoparticle in reaction lowers the BDE and wake the electron transfer more efficient. The percentage of degradation efficiency of silver nanoparticles was calculated as 97.7 % in 90 minutes. This increases the rate of reduction of methylene blue dye by NaBH4.Also, the degradation of the dye is known from gradual decrease of absorbance value of dye. Hence the silver nanoparticles obtained from fungi Penicillium rubens revealed favorable result in reduction of methylene blue dye.
The addition of silver nanoparticle in reaction mixture performed as potential intermediate between methylene blue dye & BH4- ions. At first, it lowered the BDE and made the electron transfer between them more efficient. Thus, the rate of reduction of MB by NaBH4 was increased in the presence of silver nanoparticles Xu L et.al 2008 .
Various application such as antimicrobial activity for multidrug resistant microorganisms as well as reduction of harmful industrial dyes, artificial food coloring, toxic food dyes are noted.