technology as an alternative to conventional chemicals in leather industry
N. R. Kamini, C. Hemachander, J. Geraldine Sandana
Mala and R. Puvanakrishnan*
Department of Biotechnology, Central Leather
Research Institute, Adyar, Chennai 600 020, India
Leather industry contributes
to one of the major industrial pollution problems facing the country, and the pollution
causing chemicals, viz. lime, sodium sulphide, salt, solvents, etc. arise mainly from the
pre-tanning processes of leather processing. In order to overcome the hazards caused by
the tannery effluents, use of enzymes as a viable alternative has been resorted to in
pre-tanning operations such as soaking, dehairing, bating, degreasing and offal treatment.
This review focuses on the use of microbial enzymes as an alternate technology to the
conventional methods, and highlights the importance of these enzymes in minimizing the
Environmental pollution has been a major irritant to
industrial development. Chemical and chemical-based industries are the prime targets of
the environmentalists for their crusade against pollution, and leather industry has also
not been left out of the reckoning. The generation of pollution is significantly high in
the pre-tanning operations compared to the post-tanning operations1. The
chemicals mainly responsible for pollution in pre-tanning processes are lime, sodium
sulphide, and caustic soda apart from common salt and degreasing chemicals. In fact, one
third of the pollution caused by the leather industries results from the wastes generated
during dehairing operations2. The wastes from the tanneries are let out into
the drains which in turn empty into the main sewerage causing hazard to those who use this
water. Many tanneries have been forced to close down because of their noncompliance with
the standards laid down. In a short span of time, Indian leather industry has faced
serious challenges such as German ban on pentachlorophenate, certain azo dyes,
formaldehyde, etc. on one hand, and court order for compliance with environmental
regula-tions on the other3. The attention of tanners is
focused towards revamping the processing methods, recovery systems, and effluent treatment
techniques to make leather processing eco-friendly. Intensive efforts are being directed
towards using a viable alternative technology for pre-tanning processes using enzymes4,5.
This could be one of the ways of solving the industrial pollution problems resulting
from tannery effluents.
Conventional leather processing
The raw hide has to undergo a series of chemical
treatments before it turns into a flattering leather. This includes soaking, liming,
dehairing, deliming, bating, degreasing, and pickling1. For all these steps,
the chemicals used are quite toxic. Thus due to these pretanning operations, the leather
processing industry is one of the worst offenders of the environment.
The principal leather making protein, collagen,
exists in hides and skins in association with various globular proteins, viz. albumin,
globulin, mucoids; and fibrous proteins such as elastin, keratin, and reticulin. During
leather manufacture, the noncollagenous constituents are removed partially or completely
in the various pre-tanning operations; the extent of removal of these constituents decides
the characteristics of the final leather. Besides chemical treatment, certain enzymatic
treatments are also necessary to get optimum results. One such treatment, bating, is the
only step in leather processing where enzymatic process cannot be substituted by chemical
processes. The process of bating gives certain desired characteristics to the finished
leather. Earlier, the process was carried out using dog dung or manure6. The
use of this was not only unhygienic but fermentation could also not be controlled.
In pre-tanning operations, the hides and skins are
first subjected to a water soak. For loosening the hair7, the oldest method is
the sweating process a natural autolysis or breakdown
process. It is a mild putrefaction process induced at random. Since the type and quantity
of the putrefying bacteria cannot be controlled, the process itself eludes control.
Moreover, since the sensitivity to attack the epidermal proteins and the fibrous proteins
of the corium by the proteolytic enzymes is more or less the same, the sweating may result
in serious damage to the hide surface. Dehairing is used to be followed by opening up of
fibre structure in liming. The dehaired hide is transferred to an alkaline
solution of lime milk where swelling occurs and the nonfibrillar proteins are dissolved.
After mechanical removal of the subcutaneous tissue, deliming is performed in order to
remove the adsorbed lime from the hide and to eliminate the lime swell.
The fat present in the hide skins is removed either
as soluble lime soap or hydrolysis products like fatty acids. Kerosene, chlorinated
hydrocarbons, and white spirit are used in the degreasing system which add to the toxicity
of the environment and effluents1. The various steps of the pre-tanning
processes of leather manufacture are shown in Figure 1.
Enzymes in pre-tanning
An important enzyme used in pre-tanning processes
belongs to the group of proteolytic enzymes, proteases. Obtained by microbial
fermentation, the proteases are meant for use in the leather industry for dehairing,
bating and soaking processes, and in the detergent industry for breaking down
proteinaceous matter caused by body secretions, food stuffs, and blood8. The
main advantages of the use of enzymes are specificity, stereospecificity, activity under
mild conditions, possibility of producing natural products, nonpollutants, and
Although enzymes from plants, animals, and microbial
sources have been used for decades, large-scale use of microbial enzymes received a boost
only in 1960s following the introduction of fermentation technology. The enzymes or
enzymatic formulations need not be pure but must be cheap compared to that of commercial
chemicals used in leather industry.
Animal proteases and microbial proteases from
bacteria and fungi are used in the pre-tanning processes of leather manufacture. The most
important criteria for their selection are their specificity, pH activity range as well as
pH and thermal stability. If an enzyme is to act uniformly, it must be able to diffuse
into the hide and this is obviously acheived with skins rather than with hides. In the
latter case, an accumulation of enzyme at the surface of the grain occurs. A pronounced
difference between the pH value of the solution and that of the hide is also possible10.
The animal proteases are mixtures of trypsin,
chymotrypsin, and various peptidases which may contain amylase or lipase as secondary
enzymes. Mainly for economic reasons, enzymes from microorganisms have come to play a
significant role in recent years and enzyme products of microbial origin are already being
produced on a wide scale11.
Since microorganisms can be made to propagate
rapidly and profusely, they are an ideal source for enzymes12. Mainly, neutral
and alkaline proteases are obtained from bacteria, which differ in their pH activity
range. Fungal proteases are also classified according to the pH activity range13 : fungal
acid proteases act between pH 2.5 and 6.0 and can be derived from A. satoi. These
are used for bating prior to pickling and serve to open up the fibre structure. Fungal
alkaline proteases14 belong to the same group of serine proteases as alkaline
bacterial proteases. However, these are more heat sensitive and are quickly deactivated
above 60° C. Fungal neutral proteases are mainly obtained from Aspergillus or Penicillium
Table 1 shows the various enzymes produced by various microorganisms used in the leather
Apart from bacterial and fungal proteases, specific
proteases like keratinases15 are known. Keratinases which hydrolyse keratins,
are obtained from Streptomyces fradiae and can be used for dehairing10.
Some of the important lipase-producing microorganisms used in degreasing are shown in
Table 1. Lipases are used (i) in the oil and fat industry to modify fats for use in foods;
(ii) in detergent compositions; (iii) for fatty acid production, lipid synthesis via
reversal of hydrolysis and lipid modification by interesterification, and (iv) in
degreasing of hides and skins16.
Enzymes in soaking
Soaking is the first operation in the tannery
wherein the hides and skins are cleaned and softened with water17. Wet-salted
or freshly slaughtered hides and skins do not require any chemical agent for their proper
soaking4. Soaking is necessary for solubilization and elimination of salts and
globular proteins contained within the fibrous structure of hides and skins. It is carried
out under alkaline conditions at low temperature between 10° C and 20° C in water
treated with antiseptics such as sodium hypochlorite, sodium pentachlorophenate, formic
acid, etc.1. It is accelerated by some of the nonionic detergents and additives
such as sodium sulphide or sodium tetrasulphide.
The advantages of enzymatic soaking include
loosening of the scud, initiation of the opening of the fibre structure, and production of
leather with less wrinkled grain when used at an alkaline pH of less than 10.5 (ref. 6).
Use of enzyme preparation in soaking of rabbit skins improves the softness and elasticity,
and increases the area yield of the fur by 3.3% while reducing the processing time by
1020 h (ref. 18).
Grimm18 has described a soaking method
using proteolytic enzymes and carbohydrases in the pH range of 5.5 to 10.0. Enzymes from Aspergillus
parasiticus, A. flavus, A. oryzae, and Bacillus subtilis have
been used alone or in mixtures. Rokhvarger and Zubin20 suggested the use of
carbohydrase from the mold culture A. awamori in soaking. Botev et al.21
have reported the use of bacterial amylase for soaking dried wool lamb skins. Alkaline
proteases of bacterial and fungal origin have been used for soaking which reduces the need
for the liming chemicals by 3060% (refs 22,23). Soaking of dried furs in an aqueous
bath containing 1% acid proteinase from Rhizopus rhizopodiformis and sodium
bisulphite at 25° C for about 20 h has been reported by Asbeck et al.24.
Orlita and Beseda25 have tested three commercial bacterial alkaline protease
preparations for the soaking of salted cow hides. Thus, use of enzyme preparations results
in a decrease in soaking time.
Soaking is usually carried out using a combination
of proteolytic enzymes that are optimally active in the neutral or alkaline pH range. For
enzymatic soaking, the average soaking period for salted raw stock is about 4 h and
for dried raw stock is about 810 h (ref. 26). A water soak without auxiliary
agents takes 24 h for salted hides, and 3648 h for dried hides.
Enzymes in dehairing
Dehairing is one of the main operations in the
beamhouse. Five methods of dehairing are generally adopted, viz. (i) clipping process,
(ii) scalding process, (iii) chemical process, (iv) sweating process, and (v) enzymatic
process1. Of these, the most commonly practiced method of dehairing of hides
and skins is the chemical process using lime and sodium sulphide. However, the use of high
concentrations of lime and sodium sulphide creates an extremely alkaline environment
resulting in the pulping of hair and its subsequent removal. While one cannot question the
efficacy of this process, its inherent disadvantages have to be taken note of. Significant
amongst these are:
(i) It contributes in no small measure to the
pollution load. Beamhouse processes generally account for 7080% of the total COD of
effluent from all leather making processes. About 75% of the organic waste from a tannery
is from the beamhouse and 70% of this waste is from hair which is rich in nitrogen. These
figures clearly illustrate the contribution made by the lime and sulphide process towards
(ii) Sulphide is highly toxic with obnoxious odor.
If left untreated, it can cause major problems in the sewers.
(iii) The severe alkaline condition is a health
hazard for the workers.
Enzymatic dehairing is suggested as an
environmentally friendly alternative to the conventional chemical process6. The
enzyme digests the basal cells of the hair bulb and
the cells of the malphigian layer. This is followed by loosening of hair with an attack on
the outermost sheath and subsequent swelling and breakdown of the inner root sheath and
parts of the hair that are not keratinized28. Advantages of enzymatic dehairing
(i) Significant reduction or even complete
elimination of the use of sodium sulphide.
(ii) Recovery of hair of good quality and strength
with a good saleable value.
(iii) Creation of an ecologically conducive
atmosphere for the workers.
(iv) Enzymatically dehaired leathers have shown
better strength properties and greater surface area.
(v) Simplification of pre-tanning processes by
cutting down one step, viz. bating.
(vi) A significant nature of the enzymatic dehairing
process is the time factor involved. The lime-sulphide process takes about 16 h,
whereas the enzymatic dehairing would be also completed between 12 and 20 h (ref.
Proteolytic enzymes are of great commercial
importance, contributing to more than 40% of the worlds commercially produced
enzymes30. Approximately 50% of the enzymes used as industrial process aids are
proteolytic enzymes31. Proteolytic enzymes are more efficient in enzymatic
dehairing than amylolytic enzymes1.
Microbial proteases are derived from a wide variety
of yeasts, molds, and bacteria32. Yeast proteases are mainly intracellular in
nature and therefore these enzymes have not gained significant commercial interest. The
protease from A. flavus was earlier being used for dehairing, and later it was
reported that simultaneous dehairing and bating is possible with the protease of A.
flavus33. Gillespie34 has observed that the enzyme preparation
from cultures of A. oryzae, A. parasiticus, A. fumigatus, A.
effusus, A. ochraceus, A. wentii, and P. griseofulvum exhibit
marked depilatory activity on sheep skins.
CLRI has developed Clarizyme, an alkaline serine
protease, produced by A. flavus used for the dehairing of skins and hides35.
A. flavus grows rapidly on wheat bran and produces large amounts of extracellular
proteases. Extensive trials carried out in CLRI tannery have confirmed the successful use
of this enzyme as a depilatory agent. The use of this enzymatic depilation process
completely eliminates the use of sulphide, a toxic pollutant.
The fungal culture, Conidiobolus sp.,
isolated at NCL, produces high yields of extracellular alkaline protease36. The
enzyme is active at pH 10.0 and is being tried for many industrial applications. Enzymes
derived from bacteria have gained much commercial interest37,38 because of
their easy production capabilities by submerged cultivation, high yield of enzyme, short
duration for production, and easy recovery of the enzyme.
Proteolytic enzymes derived from a large number of Bacillus
sp. and Streptomyces sp. have been used in dehairing of hides and skins39,40.
A lime and sulphide-free process of dehairing has been developed for the manufacture of
suede from sheep skins using protease from B. subtilis41. Schlosser et al.42
have reported a method of depilation in an acid medium containing Lactobacillus
In dehairing, the hair loosening is effected at pH
10.0 using fungal or bacterial enzymes; the treatment period being approximately
1216 h, followed by hair removal using mechanical means10. The
treatment period can be substantially reduced if the enzyme solution is fed in from the
flesh side under pressure43. Enzymatic hair loosening processes play a role
wherever high-quality hair, wool or bristles are to be recovered.
Three methods of application are commonly used in
the enzymatic dehairing process: (i) paint method, (ii) dip method, and (iii) spray
method. In the paint method, the enzyme solution is mixed with an inert material like
kaolin, made into a thin paste, adjusted to the required pH, applied on the flesh side of
hides and skins, piled flesh to flesh, covered with polythene sheets and kept till
dehairing takes place. In the dip method of enzymatic unhairing, the hides or skins are
kept immersed in the enzyme solution at the required pH in a pit or tub. The disadvantage
encountered in this method is the unavoidable dilution of the enzyme solution. Even though
enzyme penetration is observed to be uniform, dehairing at backbone and neck is not up to
the mark. A novel spraying technique has been adopted for the application of multienzyme
concentrate in depilation44. The advantages of this method over the painting
and dip methods are that (i) even concentrated solutions can be sprayed, (ii) when the
enzyme solution is sprayed on the flesh side with force, entry becomes easier, (iii)
backbone and neck can be sprayed with more amount of enzyme, thereby making the process
quicker, (iv) there is no effluent arising out of this method, and (v) after depilation,
hair will be almost free from all the adhering skin tissues. Of late, dehairing by
drumming is being practiced, and industrially this should be feasible.
Enzymes in bating
Bating is a very important process in which enzymes
have been successfully employed for centuries. The concept of softening hides by treating
them in a warm infusion of animal dung has been termed as bating and the
product used for such process is known as a bate. The main object of bating is to remove
some of the nonleather-forming proteinous materials like albumins, globulin, and mucoids
from hides and skins, and to allow splitting up of collagen fibres to facilitate the
penetration of tanning materials and other processing chemicals, thereby giving the
finished leather the desired characteristic properties like feel, softness, pliability,
Deliming and bating, the subsequent steps in the
processing of the pelts after liming, are really two separate operations although they are
usually carried out in one step and often overlap each other. The principal materials
which a bate contains are a proteolytic enzyme, a carrier for the enzyme like wood flour,
and a suitable deliming agent like ammonium chloride or sulphate or both. The deliming
agents are used for the removal of lime salts which are used during the dehairing process.
The comparatively richer source for the proteolytic
enzyme is the pancreas from bovine and pig. The proteolytic enzymes in the pancreas are
present in inactive forms; chymotrypsin as chymotrypsinogen, trypsin as trypsinogen, and
carboxypeptidase as procarboxypeptidase. A process has been patented for the activation of
pancreatic enzymes by the use of acid protease from A. fumigatus45.
Underkofler and Hickey46 have described a
process for the manufacture of enzyme bate from mold source. Trabitzch47 have
reported the use of enzymes from Aspergillus species in bating and dehairing. A
procedure has been developed for bating pig skins, using an enzyme preparation from B.
subtilis, and bated skins exhibit good physicochemical properties18.
Bacterial preparation from S. rimosus and B. licheniformis have been tested
for their bating action and it is found that solubilization of collagen has been less
pronounced under the influence of microbial proteases than under the influence of
pancreatic protease48. A combination of both mold and pancreatic enzymes in
suitable proportions will be an ideal bate for different types of leather.
In bating, pancreatic enzymes are used in
combination with neutral and alkaline bacterial or fungal proteases. After loading the
drum with the pelts, the float is fed in at 3537° C and, then, the bating agent
containing enzyme, ammonium salts and carrier material is added.
Enzymes in degreasing
Degreasing is an essential step in the production of
glove and clothing leather. In this process there is removal of excess natural fats from
greasy skins. The presence of natural grease in raw hides and skins, especially woolly
sheep skins, results in various defects, viz. fatty spues, uneven dyeing and finishing,
waxy patches in alum-tanned leathers, and pink stain on wet blues1. During the
degreasing operation in the pretanning process, the fat or grease is removed from the
interfibrillary spaces of the skins to facilitate the even penetration of tanning
materials, fat liquors, and dyes, etc. Degreasing helps to obtain soft and pliable leather
for garment manufacture.
Degreasing is carried out after pickling, using
aqueous emulsification with detergents, or by solvent extraction. It is well known that
organic solvents like kerosene, petrol, perchloroethylene and trichloroethylene are highly
unsafe and hazardous to the workers and heavily pollute the environment. The detergents,
though not hazardous while handling and storing, cause serious pollution problems. These
detergents and solvents add to the BOD load of the pickling effluent, and the chlorinated
hydrocarbons and solvents add to the toxicity of the effluent49.
Enzymatic degreasing is suggested as a viable
alternative to combat the pollution problems caused by the use of solvents and detergents.
Lipases which are projected as alternatives for solvents and detergents, catalyze the
breakdown of fats and can be obtained from animal, microbial and plant sources. The
advantages of using enzymes for degreasing are the elimination of solvents, reduction in
surfactants, and possible recovery of valuable by-products. The disadvantages are that the
lipases do not remove all types of fats in the same way that solvents do, and they add
cost to the process.
In 1966, Trabitzsch47 described the
potential for lipases in degreasing skins. Baldano and Shestakova50 compared
the enzymatic and solvent degreasing of pig skin and have shown that both these methods
remove approximately 50% of the grease. Yeshoda et al.51 used a
fungal lipase for the degreasing of woolly sheep skins, pH range of 3.23.6 at 37° C
for 1 h. Subsequently, Yeshoda et al.52 observed that
degreasing and bating could be carried out simultaneously in the pH range of 7.88.0.
An acid lipase from Rhizopus nodosus has been noticed to be very effective in the
degreasing of sheep skins49. Zhang reported use of alkaline lipase in
combination with the proteinase and pancreatin in softening pig skin to improve the
degreasing effect53. Pfleiderer et al.54 carried out
degreasing of hides by soaking in an acidic bath containing a proteolytic enzyme
(0.013.0%), and a nonionic surfactant (0.21.5%) or its mixture with anionic
emulsifiers. A combination of proteolytic enzymes and emulsifiers gives optimum results in
wet degreasing of sheep skins1.
CLRI has developed a potent fungal lipase from A.
niger55 and a potent bacterial lipase56. Comparative studies on
degreasing of sheep skins using the bacterial lipase and commercial detergent-based
degreasing agent Gelon-PK have been carried out. Improved degreasing results with the
bacterial lipase, with added advantages of better softness, smoothness, and improvement in
other physical properties57. Furthermore, the lipase without detergent is
observed to show 70% degreasing in 2 h, with the effluent showing minimal pollution
Enzymatic degreasing can be carried out with acidic
or alkaline lipases of fungal or bacterial origin. For degreasing, pickled pelts are kept
immersed in an enzyme bath containing microbial lipase and water pH of 3.6, and left in
the same bath overnight at a temperature of 2832° C. The degreased pelts are then
removed from the bath and subjected to salt wash twice with water and common salt for
40 min. The washed pelts are repickled, chrome tanned and taken for further
processing50. The use of an alkaline lipase at a pH of 9.0 to 9.3 in the
degreasing of pig skin results in short degreasing time and high degreasing efficiency58.
Enzymes for by-products utilization and effluent treatment
Enzymes could be used in the treatment of fleshings
and effluent from tannery processes. A combination of hydrolytic enzymes, viz. proteases,
carbohydrases, and lipases would be required. The advantages to be realised include a
protein by-product suitable for animal feed as well as energy conservation and fat
recovery. Again, the major disadvantage would be the cost6.
When raw hides are processed to leather, a number of
by-products such as native hide material (claws, tails, necks, fleshings), pelt waste
(trimmings, machine fleshings, gluestock, pelt cuts), and tanned material (shavings,
leather cuts, buffing dust, chrome cuttings) are obtained10.
Braeumer et al.59 have
described the enzymatic conversion of glue stock and other hide offal to technically
useful byproducts by hydrolysing the pulverised hide wastes with an alkaline protease, pH
9.013.0, in the presence of urea, and then at pH 2.05.0 in the presence of a
strong acid. Bronowski et al.60 have shown that treating fleshings
with pancreatic enzymes instead of heat treatment for separating the fat from the
proteinaceous matter requires much less energy, and the yield is increased from
6065% to over 90%. Sauer61 has described a process for the utilization of
fleshings which consists of the enzymatic hydrolysis of the proteins, conditioning of the
resulting liquid, and separating the fats and solids present in the hydrolysate. The
outstanding feature of the process is a recovery of 91% of the fat in the fleshings and
the application of the hydrolysate directly to the soil, as a fertilizer. Iliskovic and
Mersed62 have described the separation of fats from the fleshy wastes from
cattle hide processing by treatment with enzymes.
The problem of waste treatment can be approached (i)
by getting rid of the pollution by proper effluent treatment, and (ii) by controlling
pollution occurring at different stages of leather manufacture2. Biotechnology
plays an important role in tannery effluent treatment. The secondary treatment of tannery
effluents, which relies on living organisms, is normally by anaerobic lagoons and aerobic
lagoons. Open waste-ponds or anaerobic lagoons are installed in few south Indian tanneries
where the atmospheric temperature (2040° C) is suitable for this operation. In
these ponds, microorganisms which thrive in oxygen-less environments are allowed to digest
the waste. Anaerobic lagoons can be used for cleaning wastes coming from both the
vegetable tanning and chrome tanning procedures. Closed type anaerobic systems are useful
for tanneries situated in cold temperatures (510° C). Aerobic lagoon is a shallow
water-tight pond of about 23 m depth. The wastes are kept for about a week.
Fixed or floating type surface aerators blow oxygen or air into these for helping growth
of organisms. This system requires less land and is economical for larger tanneries
located in urban areas.
The necessity for chromium removal in tannery waste
water is another area of waste management2. Microorganisms such as A.
fumigatus and species of Pseudomonas when grown on chrome waste can
leach out chromium. Pentachlorophenol, a preservative used for raw as well as
semi-processed skins, creates problems during handling and also during biological effluent
treatment. P. aeruginosa could be used successfully to degrade pentachlorophenol2.
Other potential techniques for reduction of pollution load are recycling of immobilized
enzymes to hydrolyse the solid waste, and recycling of immobilized whole cells to absorb
or detoxify toxic metals in the effluent.
Indian Leather Industry Foundation (ILIFO), a
nonprofit association of major Indian tanners, and UNIDOs Regional Programme for
Pollution Control in the Tanning Industry in SouthEast Asia (RePO) have recently
launched a research programme to find uses for treated tannery effluents in agriculture.
At present, the experiment is on in the North Arcot district of Tamil Nadu, where several
fruit, flower and vegetable plants are grown with irrigation from treated tannery
effluents. Like treated effluent, tannery sludge also contains some nutrients which could
be applied to agricultural fields. Disposal of sludge generated in the tannery effluent
treatment process is a major bottleneck in tackling tannery pollution63.
The tanneries in future will use a combination of
chemical and enzymatic processes. The potential for use of microbial enzymes in leather
processing lies mainly in areas in which pollution-causing chemicals, such as sodium
sulphide, lime and solvents, are being used and conversion of waste products into
potentially saleable by-products is possible. Future may witness ecolabelled
leather/leather products emerging as niche products, and the experience gained by the
Indian leather industry in this area might greatly help India to emerge as a global leader
in leather industry.
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ACKNOWLEDGEMENTS. We thank Dr T. Ramasami, Director,
Central Leather Research Institute, Madras, for his permission to publish this work. We
gratefully acknowledge the financial assistance extended to Mrs N. R. Kamini and Mr C.
Hemachander by the Department of Biotechnology, Government of India, and to Ms J.
Geraldine Sandana Mala by Council of Scientific and Industrial Research, Government of