Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials &

p; Metallurgy

RECOVERY OF SILVER FROM EXPOSED X-RAY FILM

USING BROMELAIN
ARUN.C*. , PRAVEEN K.C*., AND Dr.RADHA .K.V#
* M.TECH, ENVIRONMENTAL SCIENCE AND TECHNOLOGY, A.C .TECH, CHENNAI-25.
# ASSISTANT PROFESSOR, DEPARTEMENT OF CHEMICAL ENGINEERING, A.C.TECH, CHENNAI-25.

ircarun@gmail.com
ABSTRACT
In the present work an environmental friendly recycling system which peels away the base coat from the
X-ray film by using an enzyme was developed. Bromelain, a proteolytic enzyme extracted from waste apex
of pineapple was used for stripping the silver layer on the exposed film by hydrolysis of gelatin. The silver
recovery conditions were optimized and the slurry obtained from treatment of X-ray with Bromelain was
analyzed showed 30% of silver and the slurry underwent smelting in the presence of flux (Na2CO3) in a
furnace to recover 99% of pure silver. Thus it helps in recycling of X ray film and recovery of silver
without any harm to Environment.

Key words: X-ray films, Pineapple Apex, Bromelain, silver, Gelatin.

1. INTRODUCTION
Silver is a rare, precious metal and naturally occurring metal, often found deposited as a
mineral ore in association with other elements. Fifty percent of silver produced is used in
photographic and imaging materials. Normally X-ray is discarded by incineration and
chemical processing. However, the standard silver loss in an incineration process is about
25%-30% and the smoke produced is very toxic and illegal. Any chemical process is very
difficult, expensive, inefficient, and sometimes a dangerous process, especially when
working in bulk. Now days various studies have been carried out using Proteases
(Akihiko Masui et al.,2008 ) which play a crucial role in the recycling of exposed X-ray
film .

X-rays films used in medical applications are composed of a plastic sheet coated with a
thin coating of gelatin (protein) impregnated with silver grain. Bromelain breaks down
the gelatin protein that serves as the support medium for the emulsion by hydrolysis
reaction. If the gelatin is destroyed the black silver grains will slough off, leaving a clear
Plastic backing of the x-ray film. The obtained slurry undergoes drying and smelting in
the presence of flux in a furnace to obtain pure silver.

The objective of the present study was Extraction and purification (Krzysztof N.
Waliszewski, et al., 2009) of Bromelain enzymes from waste apex of pineapple and to
study the effect of optimal environmental parameters (khan R H, 2003). Treatment of
exposed X-ray films by using enzyme solution , recycling of X ray films and
simultaneously recovery of silver without any harm to environment (Nuri Nakiboglu et
al., 2001).

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

2 EXPERIMENTS
2.1 EXTRACTION OF BROMELAIN
Waste Pineapple apex was collected from a market in Chennai, India, used for extraction
of Bromelain a Proteolytic enzyme. Pineapple apex was cut into pieces and blended using
juice extractor. The blended tissues were homogenized using homogenization buffer
(Phosphate buffer) .The crude extract was passed through Buckner funnel and the filtrate
was centrifuged at 7000 rpm for 15 minutes. Save the supernatant and this was used as
the crude homogenate.

2.2 CONFIRMATION TEST USING GELATION METHOD

The test tube was labeled as crude homogenate, control and gelatin. The test tube labeled
as crude homogenate was taken and to it 1ml of crude homogenate and 1ml of 1% gelatin
was added. The test tube labeled as gelatin was taken and to it 1ml of gelatin and 1ml of
distilled water was added. The test tube labeled as control was taken and to it 2ml water
was added. All test tubes are kept in a plastic beaker containing ice cubes for 30 minutes.
After 30 min all the tubes was removed from the plastic beaker. The consistency of the
gelatin in each tube was noted and tabulated. This procedure was explained by Glider
et al., (2002).

2.3 EFFECT OF TEMPERATURE AND pH ON ENZYME ACTIVITY

The effect of temperature and pH of the crude enzyme was determined by incubating the
reaction mixture contains Bromelain solution and 1% gelatin. Reaction was allowed for
15 minutes at different temperature from 30C to 60C at interval of 5 and with different
pH from 4.5 to 8 at interval of 0.5. The reaction stopped with 3 ml of 5% Trichloro acetic
acid. An appropriate blank solution was also prepared and absorbance values at 620 nm
were measured with reference to the blank. The absorbance values obtained could be
correlated with enzyme activity.

2.4 EFFECT OF SUBSTRATE CONCENTRATION ON PRECENTAGE

REMOVAL OF SILVER

100ml of enzyme solution was taken in conical flask and to it different grams of waste X
ray film(gelatine) was added at 55c with pH 6.5 for 1hour to strip out the silver
materials from the Xray films. The precipitated was dried and weight was taken and this
could used to calculate removal % of silver.

2.5 EFFECT OF INITIAL ENZYME CONCENTRATION ON PRECENTAGE

REMOVAL OF SILVER

20 grams of waste X- ray film (gelatine) was taken in conical flask and to it 5-35ml at
equal interval of enzyme solution was added and the reaction was taken at 55c with pH 6
for 1hour to strip out the silver materials from the Xray films. Solution was centrifuged,
the precipitated are dried and weight was taken and this could used to correlate with
removal % of silver.

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

2.6 EFFECT OF METAL IONS ON ENZYME ACTIVITY

The effect of metal ion (ferrous, copper, silver, lead, cobalt, chromium) was determined
by reacting
1% gelatin solution in presence of 1gram of different metal ions with enzyme solution.
Reaction allowed for 15 minutes at temperature 550C and the reaction stopped with 3 ml
of 5% trichloroacetic acid. An appropriate blank solution was prepared and absorbance
values at 620 nm were measured with reference to the blank. The absorbance values
obtained could be correlated with enzyme activity.

2.7 TREATMENT OF X-RAY FILM USING BROMELAIN

X-ray film, coated on both sides with gelatin first washed with distilled water and wiped
with cotton impregnated with ethanol and they are cut into pieces. Before treatment it
was heated in an oven at 50 C for 30 minutes. The film was immersed in Bromelain
solution for at optimal condition to form a tough, hard, black solid consisting of silver
and products of silver with the gelatine breakdown products. Essentially, all the silver
had been removed from the film and the treated X-ray films are separated from the
solution and solution was centrifuged. The precipitated was dried and mixed with sodium
carbonate and smelted for 30 min at 750C. The smelted silver was then quenched to
form silver grain. The grain was dried and analyzed.

3. RESULTS AND DISCUSSION

3.1 Extraction of Bromelain

500 g of waste pineapple apex taken and cut into smaller pieces. Using juices extractor
and cell debris removed using Buckner funnel filter. The solution centrifuged to obtain
crude homogenate stored at 4 C. Similarly Frattini Fileti et al (2005) extracted the fruit
juice of pineapple and filtered it to get crude homogenate and stored at 4 C, this solution
were used to determine the optimizing conditions for Bromelain extraction by using
reversed micelles .

3.2 CONFIRMATION TEST USING GELATION METHOD

The extracted sample undergone a confirmatory test by using Gelation method .In this
method powdered gelatin is mixed with water and warmed, it dissolve and while cool,
they form semi solid. But when they reacted with Proteolytic enzymes, gelatin will not
solidify when cooled because peptide bonds between the amino acids of the gelatin are
broken. (Table 1).

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

S.No NO
TREATMENT GELATION
. GELATION

1 Enzyme + gelatin

2 Gelatin + water -
3 Water(control) -

Table 1 Confirmation Test for the presence of protease

3.3 EFFECT OF PH ON ENZYME ACTIVITY

Protease enzymes generally can be categorized into two groups with respect to their
optimal pH (acidic or alkaline). The effect of pH on the activity of the enzyme was
investigated using gelatin as substrate. The maximum operating pH for Bromelain from
pineapple apex was found to be pH 6.5(Fig 1). From the above result, Bromelain
extracted from pineapple apex was confirmed as acidic protease. Similar result was
reported by Waliszewski et al (2004) showed that fruit Bromelain has optimum pH 6.

3.4 EFFECT OF TEMPERATURE ON ENZYME ACTIVITY

The effect of temperature on enzyme activity with varies temperature ranges were
studied. Increase in temperature will increase the reaction rate until the enzyme becomes
denatured and the maximum operating temperature was determined as 55C using gelatin
as substrate (Fig 2) .similar work has been conducted by Joonseok Koh et al (2006)
showed that the optimum temperature was found to be 60C for fruit extract from
pineapple using azo casein as substrate.

3.5 EFFECT OF SUBSTRATE CONCENTRATION AND INITIAL ENZYME

CONCENTRATION ON % REMOVAL OF SILVER

Removal percentage of silver was higher for a substrate concentration (Fig 4) of 35g of
X-ray film in 50 ml of enzyme solution. The Effect of Initial enzyme concentration (Fig
5) shows the linear increment in removal % of silver and it was higher at a value of 20
grams of X-ray film in 30 ml of enzyme solution.

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

50
40
E n z ym e activity in %

40

Enzyme Activity in %
30
30
20
20

10
10

0 0
25 30 35 40 45 50 55 60
3 4 5 6 7 8
Temperture in Degree celusies
pH

Fig 1 Effect of pH on enzyme activity Fig 1 Effect of temperature on enzyme activity

After Before

Fig 3. X-ray film before and after treatment

3.6 EFFECT OF METAL IONS ON ENZYME ACTIVITY

The effect of metal ion (ferrous, copper, silver, lead, cobalt, chromium) on enzyme
activity shows the cobalt and copper (Fig 6) seems to affect the Enzyme activity at large
extent. Silver have smaller effect on enzyme activity so this shows that silver present in
X-ray film will not affect the enzyme and substrate reaction.

3.7 TREATMENT OF X-RAY FILM USING BROMELAIN:

20g of waste X ray film was treated using 100ml of Bromelain crude extract to strip out
the silver materials from the Xray films at 55c with pH 6 for 35min and the cleared
polymeric (Fig 3) material can be reused. Similar Nuri Nakiboglu (2001) shows the
decomposes the gelatin layer at 50c for 30 min using protease from bacillus subtitles and
the film cleared by stripping out the silver grains on them but purification step is large
and maintain the microbe in its optimum is very important and difficult . But when using
Bromelain the process was very cheaper because enzyme was extracted from waste pine
apple apex only and crude enzyme was used for treatment and can be reused.

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

30

% Removal of silver
25
20
15
10
5
0
0 5 10 15 20 25 30 35 40 45 50 55
substrate concentration ,g/m l

Fig 4 Effect of Substrate Concentration on % removal of silver

25
% Removal of silver

20
15
10
5
0
0 5 10 15 20 25 30 35 40 45
Enz yme conce ntration ,mg/ml

Fig. 5 Effect of Enzyme Concentration on % removal of silver

E n z y m e a c t iv it y %

60
50
40
30
20
10
0
p o ta s s iu m d ic h r o
c o p p e r s u lp h a te
F errous
none

s u lp h a te

s ilv e r n itr a te

le a d n itr a te

c o b a lt s u lp h a te

m a te

Fig.6 Effects of Metal Ions on Enzyme Activity.

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Proceedings of National Symposium for Post Graduate Students (NSPGS 2010), 24 25 April, 2010: Vol 4 Civil, Materials & Metallurgy

4. CONCLUSIONS

The Bromelain enzyme was extracted by using juice extractor from pineapple apex.
Maximum operating pH and temperature for Bromelain from waste pineapple apex was
found to be 6.5 and 55C respectively. A waste X-ray film undergo treatment process
using 100ml of Bromelain crude extract to strip out the silver materials from the Xray
films at optimal condition. After 1 hour cleared X-ray film was collected and dried. The
collected film can be reused for manufacturing of X-ray film or other material production
like fabrics, packaging films, recording tapes, and soft-drink bottles. And all above
results shows that this process is cheaper pollution free and efficient. By this process 99
% of silver can be recovered form X-ray film and the silver can be reused for silver
solder, jewellery, the silver plating of utensils, electrical components and film
manufacture.

REFERENCE

1) Akihiko Masui, Masahiro Yasuda, Nobuaki Fujiwara, Haruo Ishikawa., Enzymatic Hydrolysis of
Gelatin Layers on Used Lith Film Using Thermostable Alkaline Protease for Recovery of Silver
and PET Film. Biotechnology Progress.20 (4), 1267 1269, 2004.
2) Frattini Fileti, A. M., Fischer, G. A. , Tambourgi, E. B. , Optimizing Bromelain Extraction by
Reversed Micelles from Pineapple Fruit, Brazilian Journal of Chemical Engineering, 17, 29-38,
2005 .
3) Glider, W. V., Hargrove, M. S., Using Bromelain in pineapple juice to investigate enzyme
function. Tested studies for laboratory teaching. 275-295, 2002.
4) Joonseok Koh, Sang-Mo Kang, Soo-Jin Kim, Min-Kyung Cha, Yoon-Jung Kwon. Effect of
pineapple protease on the characteristics of protein fibers.7,2,180-185, 2006
5) Khan, R .H., Rasheedi, S ., Haq S K ., Effect of pH, temperature and alcohols on the stability of
glycosylated and deglycosylated stem bromelain. J. Biosci. 28, 709714, 2003.
6) Krzysztof Waliszewski , N., Ofelia Mrquez , Violeta Pardio, T., Quantification and
characterisation of polyphenol oxidase from vanilla bean. Food Chemistry .117, (2) 196-203,
2009.
7) Nuri Nakiboglu, Duygu Toscali , Ihsan Yasa . Silver Recovery from Waste Photographic Films by
an Enzymatic Method. Turk J Chem. 349 -353, 2001.

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