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Recovery of silver from used X-ray film using alkaline protease from Bacillus subtilis sub sp. subtilis

  • Imam Abdulrahman Bin Faisal University

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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.
Content may be subject to copyright.
Vol. 15(26), pp. 1413-1416, 29 June, 2016
DOI: 10.5897/AJB2016.15340
Article Number: D49F1FD59232
ISSN 1684-5315
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
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1414 Afr. J. Biotechnol.
Table 1. Silver recovery from waste of X-ray films by alkaline protease.
Time (min)
Optical density (OD660 nm)
Std. error
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.
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
(AL-Khaldi, 2014).
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).
Statistical analysis
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
polyester layer.
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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... This result indicated that, the alkaline protease produced from M. luteus has facilitated the recovery of silver from X-ray films. In an earlier study Al-Abdalall and Al-Khaldi, [14] recovered silver from alkaline protease of B. subtilis sub sp. Hakim et al., [30] observed the capable exclusion of silver from partially purified protease synthesized by B. subtilis. ...
The study aims at the characterization of purified alkaline protease Micrococcus luteus strain isolated from marine sediments collected at Tuticorin, Tamilnadu, Southeast coast of India.The16S rRNA sequencing and biochemical characterization exposed that the strain resembles Micrococcus luteus. The protease purification produced 2.5 fold purification with 2490 U/mg and 48.6% yield. The molecular weight of the enzyme was determined as 41.1kDa by SDS PAGE and exhibited activity and stability of pH (2-12) and temperature (20-80ºC). Metal activity of protease was studied which revealed highest activity with Mg2+ (90.2%), Ca2+ (85.3%) and reduced activity with metals such as Cu2+, Fe2+, Zn2+and Co2+. The partially purified alkaline protease was used to test various applications such as removal of hair from goat skin (activity at 10 h), breakdown of egg albumin (activity at 6 h), haemoglobin hydrolysis (activity at 12 h) wash performance of blood stain (9 h) and decomposition of gelatinous layer from used X-ray film (4 h). All the experiments have shown excellent results and hence it can be concluded that protease production from marine bacterium can be used in industrial and several biotechnological applications
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... With alkaline protease of Bacillus subtilis NCIM 2724 complete stripping of gelatin occur within 4 to 6 days with crude protease at 37 °C and pH 8 used for 0.5012 g recovery of silver (Parpalliwar et al., 2015) [28] . Al-Abdalall and Al-Khaldi (2016) employed B. subtilis protease for recovery of silver within 30 min at 50°C and pH 8. Hamza et al. (2017) [29] demonstrated recovery of gelatin and silver from waste X-ray film at pH 9, 40°C in 40 min by Bacillus sp. THZ14. ...
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The waste X-ray/ photographic films contain 1.5 - 2 % (w/w) black metallic silver which is recovered and reused. Around 18-20% of the world's silver needs are supplied by recycling photographic waste. Since silver is linked to gelatin in the emulsion layer, it is possible to break the same and release the silver using proteolytic enzymes. Alkaline protease from Conidiobolus coronatus was investigated for enzymatic hydrolysis of gelatin from waste X-ray films. At the end of the treatment, gelatin layer was completely removed leaving the polyester film clean and silver was recovered in the hydrolysate, both of which can be reused. Various parameters such as pH, temperature, enzyme concentration, time etc on silver removal from the film were studied. Gelatin hydrolysis was monitored by measuring increase in turbidity in the hydrolysate, which was accompanied by release of protein and hydroxyproline. Gelatin layer was stripped completely within 6 min with 1.35 U ml -1 of protease at 40°C, pH 10. Rate of gelatin hydrolysis increased with increased in protease concentration. The enzyme could be effectively reused for four cycles of gelatin hydrolysis. Silver in hydolysate was around 3.87% (w/w) based on total weight of sludge. Key words: Silver recovery; X-ray films; gelatin hydrolysis; alkaline protease; Conidiobolus coronatus DOI: 10.3126/kuset.v6i1.3311 Kathmandu University Journal of Science, Engineering and Technology Vol.6(1) 2010, pp60-69
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Thirty available bacterial cultures belonging to Bacillus thuringiensis (B.t.) obtained from various culture collections were grown on two media, mainly NYSM & Soybean, and screened with respect to their ability to produce alkaline protease (AP) under shaking culture condition; they all were able to produce AP, except B.t. subsp. subtoxicus NRC16a. Growing high AP-Producers under solid-state fermentation (SSF) condition on an inexpensive substrate, wheat bran, as the main medium substrate, the highest AP yield was detected in culture of B.t. subspecies dendrolimus IP 4A/4B. Thus, it was selected for further studies. Optimum initial moisture content was 67% (v/w), inoculum size 60% (v/w), Incubation period 3 days, and incubation temperature 30°C for the production of AP from B.t. subspecies dendrolimus IP 4A/4B, under SSF condition. Investigating some agro-wastes as sole medium, wheat bran was the promising one for AP production. On the other hand, addition of different carbon sources as a supplement of SSF medium-substrate (wheat bran) induced AP production by different ratios; maximum increases in the AP levels (about 220%) were obtained using mannitol and fructose. Only soluble starch caused slight inhibition of enzyme production, as compared with the original medium control. The highest enzymatic level was obtained with the lowest value tested of particle size of medium substrate (<1mm). Hydrolyzing the gelatin of used X-ray film with AP enzyme, the gelatin layer was completely removed within 1 hr.
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A bacterium producing an alkaline protease was isolated from the Lonar soda lake, Buldhana district (19 degrees 58' N; 76 degrees 31' E), Maharashtra, India. The most appropriate medium for the growth and protease production was composed of (g/L): casein 10; yeast extract 4; KH2PO4 0.5, K2HPO4 0.5 and CaCl2 0.5. The enzyme showed maximum activity with and without 5 mM Ca2+ at 70 and 60 degrees C, respectively. The enzyme retained 40 and 82% of its initial activity after heating for 60 min at 60 degrees C, in absence and presence of 5 mM CaCl2 respectively. The enzyme remained active and stable at pH 8-12, with an optimum at pH 10. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. It also showed excellent stability and compatibility with commonly used laundry detergents. Wash performance analysis revealed that enzyme could effectively remove blood stains. It also showed decomposition of gelatinous coating on X- ray film.
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The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.
A different enzymatic method was developed for stripping the silver from waste X-ray photographic films. The silver having purity of > 99% was recovered by smelting the obtained slurry in the presence of borax. Enzyme extract, obtained from Bacillus subtilis ATCC 6633 by modifying Horikoshi medium, was used for stripping the silver layer. The metal impurities (Al, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb and Sn) in the recovered silver were determined using the ICP-MS method. The results were compared those in with literature for high purity silver using the same method.
A novel, simple, fast, cheap and pollution-free method was developed for recovering the silver from waste X-ray photographic films with NaOH stripping. The method has a number of advantages because it obviates the need for burning, oxidizing, electrolysis or purifying steps. Moreover, all experiments were carried out in the same flask, unlike other techniques. Silver recovery conditions were optimized and silver a purity level of 99% was recovered. The metal impurities (Al, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb and Sn) in the recovered silver were determined using the ICP-MS method. The results were compared with results in the literature for high-purity silver using the same method.
With the modern world focusing on eco-friendly products and product output, more and more chemical processes are being replaced by enzymatic methods. Alkaline proteases are one of the most important group of microbial enzymes that find varied uses in various industrial sectors such as leather, detergents, textile, food and feed etc. Industrially important alkaline proteases from bacterial sources have been studied extensively, of which Bacillus sp. is most reported. Most of the alkaline proteases that play a role in industries are thermostable as their optimal activity lies between 500C to 700C. The recently used statistical methods have given way to a more rapid optimization process for alkaline protease production. Other than traditional industrial uses, alkaline proteases have promising application in feather degradation and feather meal production for animal feed. This review highlights the alkaline proteases production, optimization of process parameters, characterization as well as their different applicability.
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.
Recovery of silver from used X-ray films by Aspergillus versicolor protease
  • V Choudhary
Choudhary V (2013). Recovery of silver from used X-ray films by Aspergillus versicolor protease. J. Acad. Indus. Res. 2(1):39-41.