Vol. 15(26), pp. 1413-1416, 29 June, 2016
Article Number: D49F1FD59232
Copyright © 2016
Author(s) retain the copyright of this article
African Journal of Biotechnology
Full Length Research Paper Recovery of silver from used X-ray film using alkaline
protease from Bacillus subtilis sub sp. subtilis
Amira Hassan Al-Abdalall* and Eida Marshid Al-Khaldi
Department of Biology, Faculty of Science, University of Dammam, El-Dammam, Kingdom of Saudi Arabia.
Received 14 March 2016; Accepted 9 June, 2016
Silver is an important industrial metal used in several areas such as photographic and x-ray films,
jewelries, silver wares and electronic objects. Silver is used for photographic film/x-ray film because of
its matchless quality as a light-sensitive material for making a photographic image. Silver is not
destroyed in the photographic process and it can be reused and recovered. Results have proven that,
bacterial alkaline protease can be used to extract silver in 30 min, but its activity decreases with
increasing incubation period. Gelatin hydrolysis was monitored by measuring the increase in turbidity
of the hydrolysate, which was accompanied by release of protein and hydroxyproline. The protease of
the culture filtrate used was 97 U/ml after 30 min, but it decreased to 86.5U/ml after 60 min. After 90 min,
it reached 85 U/ml. A great inactivation was recorded after 120 min; it got to 39.5 and 36.5% (U/ml) after
180 min. Gelatin layer was stripped completely within 30 min with 97 U ml-1 protease at 50°C and pH 8.
At the end of the treatment, gelatin layer was completely removed and the polyester film was left clean.
In addition, silver was recovered in the hydrolysate, both of which can be reused.
Key words: Silver recovery, x-ray films, gelatin, alkaline protease, Bacillus subtilis.
Alkaline protease is one of the most important enzymes
in the commercial field and it occupies a large area in the
field of enzyme production. It is widely used in leather
industry, diagnosis process, extraction of silver, animal
diet production and food processing. For these wide
applications, it is now commercially produced (Singhal et
al., 2012). Silver is a valuable metal used in
photographic and X-ray film, which is considered as an
important source of silver metal after recycling of used
films compared to other types of films. X-ray films contain
about 1.5 to 2% ratio of silver in gelatin-coated film made
from polyester layer. And it can restore this quantity of
silver by dissolving gelatin layer in alkaline protease to be
used for other purposes (Nakiboglu et al., 2003). X-ray
film is a rich source of silver, which is distributed in the
gelatin layer. Burning is a traditional way of extracting
silver. Silver oxidation is followed by electrolysis or
chemical treatment of the gelatin layers of X-ray films. All
these traditional ways are environmentally unsafe, so
enzymatic analysis of the gelatin layer is preferable. For
this reason, the considered methods of analysis for
enzymatic gelatin are the best alternatives to reduce
*Corresponding author. E-mail: firstname.lastname@example.org.
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1414 Afr. J. Biotechnol.
Table 1. Silver recovery from waste of X-ray films by alkaline protease.
Optical density (OD660 nm)
Protease activity (U/ml)
their harmful effects on the environment (Nakiboglu et al.,
2003). Gelatin is a protein from animal collagen, which
contains a large number of glycine, proline and 4-
hydroxyproline residues. Since the emulsion layer on X-
ray film contains silver and gelatin, it is possible to break
down the gelatin layer using proteases and to release the
silver (Nakiboglu et al., 2001). X-ray films are made of
polyester which cannot be recycled through traditional
methods of silver extraction. Enzyme hydrolysis does not
only extract silver from proteins, but also yields the
polyester base to be recycled (Gupta et al., 2002).
Nakiboglu et al. (2001) and Ahmed et al. (2008) used
alkaline protease from Bacillus subillis, Conidiobolus
coronatus and Streptomyces avermectinus to extract
silver. Kumaram et al. (2013) found that alkaline protease
from Bacillus grown on fish ruminants had a high activity
in silver extraction. The aim of this work was to detect the
use of alkaline protease to extract silver from X-ray films.
MATERIALS AND METHODS
The bacterial isolation
Bacillus subtilis isolated from soil in November 2009 at the Eastern
Province of Saudi Arabia was used in this research. Isolation was
done in Plant Protection Department, Faculty of Science and
Agriculture in King Saud University by Biolog Systems (Al-Yahya et
al., 2007). B. subtilis sub sp. subtilis was the highest isolate from
which Bacillus alkaline protease was obtained. Identified isolations
were evaluated for their ability to produce Bacillus alkaline protease
Cultural conditions and production of enzyme
The isolate was grown for enzyme production. This was done by
incubating it at 37°C for five days in media containing fructose (10
g), potassium nitrate (5 g), NaCl (150 g), dipotassium mono
hydrogen phosphate (5 g), magnesium sulfate (0.4 g), CaCl2 (0.2 g)
and Tween 80 (10 g) in one liter of sterilized water (AL-Khaldi,
2014). The enzyme was separated by centrifuging at 10,000 rpm.
Alkaline protease used for silver extraction (hydrolysis of
gelatin and release of silver)
After washing the X-ray film, it was rubbed over with ethanol. The X
- ray film was cut into small pieces (4 × 4 cm), and dried at 40°C for
30 min. Each piece was soaked in a solution containing 500 µl
enzyme and 1000.0 µl buffer solution (0.2 M, pH 8). They were
incubated at 50°C in a water bath and were shaken (90 rpm) at
different periods (30, 60, 90, 120, 150 and 180 min). The turbidity of
the reaction mixture (hydrolysate) increased with time and no
further increase in turbidity was observed when the hydrolysis was
complete. Hence, progress of hydrolysis, that is, turbidity was
monitored by measuring the absorbance at 660 nm. Samples were
removed at 1 min interval and time required for complete removal of
gelatin layer was noted. The resultant color was determined
spectrophotometrically at 660 nm (shankal et al., 2010).
The statistical analyses were performed in a complete randomized
design of three replicates for each treatment. The results were
analyzed and compared at 0.05 level of probability using the least
significant difference (LSD) and SPSS 16 version of program
according to the method of Norusis (1999).
Alkaline protease produced by B. subtilis was used in this
study. The protease activity of the culture filtrate used
was 97 U/ml after 30 min, then it decreased to 86.5%
U/ml after 60 min, while after 90 min, it got to 85 U/ml. A
great inactivation was recorded after 120 min; it got to
39.5 and 36.5% (U/ml) after 180 min. It was noticed that it
took 30 min to decompose the gelatin layer completely at
the given experimental conditions (50°C and pH 8). Table
1 and Figures 1 and 2 show the enzyme alkaline
protease activity in extracting silver from X-ray films. At
the end of the treatment, gelatin layer was completely
removed leaving the polyester film clean, and the silver
was recovered in the hydrolysate, both of which can be
From the results obtained in this study, it was noticed that
alkaline enzyme protease was effective in recovering the
silver layer during the first 30 min. Subsequently, it went
down with increase in the period of incubation. The
reason for this decline is due to exposure of the enzyme
Al-Abdalall and Al-Khaldi 1415
Figure 1. X-ray film used in this study as control
which contains silver in gelatin-coated film made from
Figure 2. The extract silver layer from treated films by crude alkaline enzyme
after: (a) 30 min, (b) 60 min, (c) 90 min, (d) 120 min, (e) 150 min, (f) 180 min,
where gradual removal of gelatinous layer was noted.
to a temperature of 50°C, which is the optimum
temperature in the different periods of time. This led to
breakage of weak peptide bonds, making the enzyme to
lose its activity (Bholay et al., 2012). The time factor is
important for the stability of the temperature. Alkaline
protease proved its activity in extracting silver from used
X-ray films. Seid (2011) proved that silver could be
extracted after 3 min treatment with alkaline protease at
55°C and pH 10.5, while Shankar et al. (2010) mentioned
complete silver extraction after 6 min of using alkaline
protease extracted from C. coronatus. Nakiboglu et al.
(2003) could extract silver in 15 min after using the
protease enzyme extracted from B. subtilis ATCC 6633;
while using enzyme extracted from Aspergillus versicolor,
Choudhary et al. (2013) extracted silver in 15 min. Also,
Foda et al. (2013) extracted silver after 1 h of incubation
with alkaline protease.
Silver extraction was also tested at 40°C for 20 min or
incubation at 24 h with alkaline protease (Sangeetha et
al., 2011). Furthermore, Pathaka and Deshmukh (2012)
could extract silver after 24 h. Kumaran et al. (2013)
mentioned high activity of the enzyme extracted from
Bacillus grown on fish remains in silver extraction
Recycle of natural mineral resources especially silver
metal remains the most practical option to slow down the
exhaustion caused by their diminution. This study shows
that the alkaline protease of B. subtilis sub sp. subtilis
1416 Afr. J. Biotechnol.
has the potential of being reused for extracting silver from
used X-ray films in an eco-friendly way.
Conflict of Interests
The authors have not declared any conflict of interests.
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