Powerpoint

ORIGINAL PAPER

Role of carbohydrases in minimizing use of harmful substances: leather as a case study

Jayanthi Durga1 • Ramakrishnan Ramesh3 • Chellan Rose2 • Chellappa Muralidharan3

Received: 10 August 2016 / Accepted: 10 December 2016 / Published online: 9 January 2017

� Springer-Verlag Berlin Heidelberg 2017

Abstract Leather processing is an important industrial

activity. Globally about 2.0 billon sqmt of leather is pro-

duced annually. Conventional cleansing operations carried

out prior to tanning generate large amounts of waste.

Among them dehairing and fibre opening process (relim-

ing) generate large amount of effluent containing haz-

ardous substances and alkaline sludge, resulting in high

negative impact on the environment. In this study, both

these pre-tanning process steps have been combined using

a cocktail of carbohydrases along with optimum quantity of

chemicals to minimize the environmental concerns. Car-

bohydrate and proteoglycan removal were chosen as the

parameters of study for efficacy of unhairing and fibre

opening. The morphology features of skins were analysed

using scanning electron microscopy and histology. Pollu-

tion load of the enzyme aided process effluent was deter-

mined and compared with conventional process. Findings

of the study indicate complete elimination of reliming

process step is possible when both unhairing and fibre

opening is carried out simultaneously using carbohydrases

as an adjunct. Reduction in use of harmful sulphide and

lime up to 40% apart from substantial saving in time and

water input is the major outcome of the present work.

Keywords Carbohydrases � Single-step processing � Leather making � Pollution reduction

Introduction

Tanneries are among the oldest manufacturing industries.

Tanneries are engaged in transforming the raw hides and

skins into leather through several unit operations. During

the last few decades, many new materials and technologies

are being studied and applied in manufacturing in order to

reduce the ecological impact of leather production (Jian

et al. 2011). Leather manufacturing has been, very often,

identified as one of the environmentally unfavourable

industrial activities. The non-substantive chemicals used in

the pre-tanning and tanning operations are predominantly

source for large amount of the harmful substances in tan-

ning effluents (Ludvik 1996).

Conventional pre-tanning involves use of chemicals

such as lime and sulphide, aimed at the removal of non-

leather making substances, which accounts for almost

80–90% of the total pollution load (Sivasubramanian et al.

2008). Besides this, sulphide in the effluent may librate

hydrogen sulphide under specific conditions, a toxic gas

that poses serious hazard for tannery workers. Many fatal

accidents have been reported due to generation of hydrogen

sulphide at high concentration, particularly at tannery

effluent treatment plants (Vijayaraghavan et al. 2015).

However, use of large amounts of lime (Ca(OH)2) and

sulphide (Na2S) for processing has remained unchanged

due to non-availability of viable cost-effective alternatives.

Unutilized lime contributes to significant quantity of sludge

Electronic supplementary material The online version of this article (doi:10.1007/s10098-016-1321-x) contains supplementary material, which is available to authorized users.

& Chellappa Muralidharan [email protected]

1 Academy of Scientific and Innovative Research (AcSIR),

AnusandhanBhawan, 2 Rafi Marg, New Delhi, 110 001, India

2 Department of Biotechnology, CSIR - Central Leather

Research Institute, Adyar, Chennai 600020, India

3 Leather Processing Division, CSIR - Central Leather

Research Institute, Adyar, Chennai 600020, India

123

Clean Techn Environ Policy (2017) 19:1567–1575

DOI 10.1007/s10098-016-1321-x

generation which under specific conditions can become

hazardous (Schlosser et al. 1986). Several lime- and sul-

phide-free pre-tanning methods have been studied exten-

sively in last few decades (Rose et al. 2007). These include

unhairing methods based on proteolytic enzymes, ionic

liquids and lactobacillus to replace sulphide (Seggiani et al.

2014; Sandhya et al. 2005). In leather making, many

enzyme-based processes have been reported to be cost-

ineffective (Ludvik 2000).

Enzymes have been widely used in leather manufacture

in soaking, unhairing, bating and degreasing processes

(Kandasamy et al. 2012). Currently enzyme-assisted

dehairing is being used in many industries due to its better

environmental performance (Senthilvelan et al. 2012).

Recently, a cocktail of carbohydrases has been successfully

employed to facilitate rapid fibre opening of skins in about

30 min compared to 72-h duration required in conventional

process replacing lime (Durga et al. 2015). In the present

study, an attempt has been made to carry out unhairing and

fibre opening process in a single step with a view to opti-

mize chemicals and time and to facilitate cleaner leather

production. The results of the study indicate substantial

benefits to leather making could be achieved by combining

process steps through use of the carbohydrases in pre-

tanning operation.

Materials and methods

Cocktail of carbohydrase enzymes produced by solid-state

fermentation (SSF) of Aspergillus terreus was used for

integrated dehairing and fibre opening along with optimum

quantities of sodium sulphide and lime. The enzyme

activity of carbohydrase was found to be 40,000 U/g of

substrate using a method of Dey and Pridham (1972) and

Miller (1972). The stability of the enzyme used in this

study was in the temperature range of 25–40 �C and pH range of 6–13. All the chemicals used for leather making

were of commercial grade.

Experimental

Goat skins were used as substrate in this study. Wet salted

goat skins were vertically cut into left (L) and right

(R) pieces, and were marked accordingly. The left pieces

were processed by conventional unhairing and fibre open-

ing (reliming) process by pasting method using 10% lime

20% water and 2.5% sodium sulphide. The paste was

applied on flesh side of the skin and left over night. Next d

the skins were unhaired and relimed with 5% lime and

100% water for a d in drum.

The respective right pieces were processed by employ-

ing different concentrations of sodium sulphide and lime in

the form of paste containing 20% water and 1% fibre

opening enzyme (carbohydrase). Next d the skins were

washed with 100% water for a period of 30 min. The pelts

thus obtained from both control and experiments were

assessed at this stage for fibre opening.

Optimization of sulphide concentration

Experiments were carried out to optimize the quantity of

sulphide for complete removal of hair in the presence of

carbohydrase and reduced lime quantity. The right pieces

were pasted on the flesh side with varying concentrations

Conventional process Experimental process

Soaking

Liming(unhairing)

Lime 10%; Sodium sulfide 2.5%; Water 10%

Re-liming (Fiber opening)

Lime 5% ; Water 100%

Tanning after flesh removal

Liming(unhairing) & Re-liming (Fiber opening)

Lime 5% ;Sodium sulfide 1.5% Water 20%; carbohydrase 1.0%

Soaking

Re-liming (Fiber opening)

Tanning after flesh removal

1568 J. Durga et al.

123

of sodium sulphide, i.e. 0.5, 1.0, 1.5, 2.0 and 2.5% with 5%

lime, 1% cocktail of carbohydrases and 20% water and left

overnight. Skins were unhaired the next d and assessed

visually. Removal of sugar and glycosaminoglycan were

assayed by using standard procedures.

Optimization of lime concentration

Second set of experiments was carried out to determine the

optimum concentration of lime required for this combined

process employing reduced sulphide in the presence of

carbohydrase. The sulphide of optimized concentration

along with 1% cocktail of carbohydrases and 20% water

was used for different lime concentrations, viz. 1.0, 2.0,

3.0, 4.0, 5.0, 6.0 and 7.0 without changing other parame-

ters. The skins were assessed visually and the carbohydrate

and proteoglycan levels in the pelt samples were quantified

spectrophotometrically to determine the optimum lime

concentration required for maximum removal of interfib-

rillarly materials. All the skins were converted into wet

blue leathers using standard chrome tanning process (given

as ‘‘Appendix 1’’). Chromium content of both the leathers

was analysed adopting IUC method.

Tanned leathers (wet blue) were then shaved to a uni-

form thickness and were converted into crust leather as per

the process given in ‘‘Appendix 2’’. The crust leathers were

assessed and evaluated as per standard test methods and

assessment procedures.

Carbohydrate assay

Total carbohydrate content of the pelt samples, both the

experimental and control, was determined by phenol–sul-

phuric acid method using D-glucose as standard (Dubolis

et al. 1956). Soaked skin sample was used as blank to

compare the remaining sugar content of the both conven-

tional and experimental samples. Sample for assay was

prepared by hydrolysing 100 mg each of lyophilized

sample with 0.5 N sulphuric acid solution at 100 �C in sealed tube for 4 h. Assay was carried out by using 1 ml of

hydrolysed aliquot of pelt sample mixed with 5% (v/v)

phenol. And then the tubes were cooled in ice for 10 min

and 5 ml of concentrated sulphuric acid was added through

the sides of tubes. The contents were thoroughly mixed,

and the tubes were heated in a water bath at 80 �C for 20 min. After cooling the tubes to room temperature, the

absorbance was noted at 490 nm using a spectrophotome-

ter. A reagent blank was prepared in the same manner

using distilled water. The amount of carbohydrate

remained in the pelt samples was calculated as glucose

from the standard curve drawn using glucose solution of

known concentration.

Estimation of proteoglycan

In order to estimate the amount of proteoglycan in the pelt

sample, both the experimental and conventional samples

were assayed by Schiff’s colorimetric method (Mantle and

Allen 1978). Initially, 100 mg of sample was hydrolysed

using 0.5 N sulphuric acid solution at 100 �C in sealed tube for 16–18 h and allowed to cool to room temperature. To

1 ml of hydrolysed sample, 100 ll of decolorized Schiff reagent was added and incubated at 37 �C for 2 h. After- wards the reaction mixture was allowed to remain at room

temperature for 30 min for colour development. Absor-

bance of the reaction product was measured at 555 nm

using UV–Vis spectrophotometer and the total amount of

proteoglycan present in the sample was calculated using

mucin as standard.

Scanning electron microscopic analysis

Samples from conventional and experimental pelts were

cut, washed and fixed in formalin solution. Then the

samples were dehydrated using a graded ethanol series and

were finally freeze-dried. The dried samples were cut into

approximately 5 mm thickness and examined by scanning

electron microscopy. The samples were mounted both

vertically and horizontally on aluminium stubs. The stubs

were coated with gold using an Edwards E-306 sputter

coater and introduced into the specimen chamber of a FEI-

Quanta 200 scanning electron microscope. The micro-

graphs for the cross section were obtained by operating the

microscope at higher voltage.

Histological studies

Conventional and experimental limed skins were cut and

preserved in 10% formalin for 48 h. The samples of both

experimental and conventional trials were fixed using for-

malin (10%) in phosphate-buffered saline (PBS), cassetted

and blocked in paraffin wax. Sample sections of 4–5 lm thickness were cut using microtome (Leica) and mounted

on glass slide. The tissue specimens thus obtained were

dehydrated using series of alcohol (30, 60 and 100%) and

stained using haematoxylin and eosin and visualized in

bright-field microscope, to assess the extent of removal of

epidermis and opening of fibre bundles of collagen and

distribution in the sample.

Analysis of chrome content

Chromium content of leathers was determined by follow-

ing the official procedure (IUP 2 2000). A known quantity

(*1 g) of the sample was weighed, and the percentage of chromium was estimated as per standard procedures.

Role of carbohydrases in minimizing use of harmful substances: leather as a case study 1569

123

Initially the samples were analysed for moisture content;

chrome content was expressed on dry weight basis of

leather.

Evaluation of strength characteristics and visual

assessment of leathers

Various physical properties such as tensile strength, per-

centage elongation at break, tear strength and grain crack

strength of leather samples of experimental and conven-

tional processes were examined as per the standard pro-

cedure (IUP 6 2000; IUP 8 2000). Samples were

conditioned to the required relative humidity of 60 ± 4%

at 20 ± 2 �C for 48 h as per standard procedures. The crust leathers were assessed for softness, grain tightness and

general appearance by hand and visual examination.

Analysis of spent liquor

Spent liquor from both conventional and experimental

processes were collected and analysed for pollution

parameters such as biochemical oxygen demand (BOD),

chemical oxygen demand (COD) and total dissolved solids

(TDS) according to the method followed by Thangam et al.

(2001) and Eaton et al. (1995). The results are expressed in

parts per million (ppm).

Results and discussion

Initially, trials were performed to optimize concentrations

of sulphide and lime matching the requirements of the

conventional process. Trials with different concentrations

of sodium sulphide (0.5–2.5%), lime (1.0–7.0%) along

with 1% carbohydrases were carried out in the study. The

experimental skin was white in colour; it had cleaned grain

surface compared to its control (processed by traditional

method). Enzymatic fibre opening assisted the depilation of

hair at its roots. On the contrary, the hairs in the control

were removed by solubilization. The hair roots were still

present in the deep dermis regions, leading to unclean

appearance.

Breaking of O-glycosidic linkages of the lysyl residue of

collagen enables the loosening of the collagenous fibrillar

bundles which in turn facilitated the depilation of hair that

has been already discussed in our earlier report (Durga

et al. 2016).

Sodium sulphide optimization

Skins subjected to combined unhairing and fibre opening

process using 1% (v/w) enzyme dosage varying at varied

concentrations of sodium sulphide (0.5–2.5% w/w)

exhibited different degrees of unhairing. At 0.5–1.0%

sodium sulphide levels, unhairing was found not satisfac-

tory. Sulphide concentration of 1.5% along with 1%

enzyme was found to be optimum requirement for com-

plete unhairing. Visual assessment of unhaired skins indi-

cated that experimental pelts were comparable or

marginally better than the conventional pelts, and the data

obtained are presented in Table 1 on a 10-point scale. The

carbohydrase enzyme also was found to exhibit better

functionality at this sulphide concentration (1.5%), as

observed from the results of carbohydrate and proteoglycan

removal given in Table 2. The removal of sugars and

glycosaminoglycan at this optimum quantity is remarkably

high compared to other concentrations employed. While a

minimum of 1.5% sodium sulphide was found necessary

for dehairing, increasing sodium sulphide concentration

beyond was found not to be useful apart from adding to

harmful pollution. Higher concentration of sodium sul-

phide beyond 1.5% also was found to adversely affect the

fibre opening efficiency of carbohydrases.

Table 1 Visual assessment of unhaired pelt

Properties Conventional process Enzyme-assisted process

Sulphide 2.5% (w/w),

Lime 10%(w/w)

Optimized concentration

Sulphide 1.5% (w/w),

Lime 5% (w/w) ?

Enzyme 0.5%

Unhairing

efficiency

9.5 ± 0.2 9.5 ± 0.2

Grain pattern 9.6 ± 0.2 9.7 ± 0.2

substance 9.4 ± 0.2 9.8 ± 0.1

Smoothness 9.3 ± 0.2 9.5 ± 0.2

Pelt colour 9.2 ± 0.2 9.7 ± 0.2

Scale of 1–10; 1—poor; 10—best

Average value of 3 experts

Table 2 Extent of carbohydrate and proteoglycan removal

Sample Sugar removal (%)* GAG removal (%)*

Control 67.0 ± 0.5 71.6 ± 1.0

0.5% sulphide ? EL 73.5 ± 1.0 77.0 ± 1.0

1.0% sulphide ? EL 74.0 ± 0.9 83.0 ± 1.0

1.5% sulphide ? EL 88.0 ± 1.0 86.4 ± 1.5

2.0% sulphide ? EL 78.0 ± 1.0 84.0 ± 1.5

2.5% sulphide ? EL 79.5 ± 0.5 85.0 ± 0.5

EL enzyme 1%; Lime 5.0%, GAG Glycosaminoglycan

* Average value of 3 determinations

1570 J. Durga et al.

123

Optimization of lime

The process was carried out with different concentrations

[1.0–7.0% (w/w)] of lime along with standardized con-

centration of 1.5% sodium sulphide and 1% carbohydrase.

Compared to conventional processes employing 10% lime,

lime quantity of 5% was found sufficient for combined

process of unhairing and fibre opening. Visual assessment

of fibre opened experimental pelts indicated comparable or

marginally better features as presented in Table 3. The

results of carbohydrate and proteoglycan removal are pre-

sented in Table 4. This provides clear understanding that

the enzyme-assisted combined process is superior in

function compared to that of the conventional chemical

only process. Higher concentrations of lime beyond 7%

were found to be adversely affecting the enzyme activity.

Chromium content

The percentage of chromium oxide content in wet blue lea-

ther from conventional and experimental processes were

found to be 4.0 ± 0.3 and 4.9 ± 0.2 (w/w), respectively.

Experimental leather showed a marginal increase in chro-

mium uptake compared to conventionally processed leather.

The increased level of chromium uptake in the enzyme-

treated wet blue sample may be attributed to possible

availability of chromium binding functional groups on the

microfibrillar surface, due to the carbohydrase mediated

deglycosylating fibre opening process, although not con-

firmed experimentally.

Physical testing and visual assessment data

The strength properties such as tensile, tear and grain crack

strength values were determined by following standard

procedures. After dehairing, visual assessment of enzymatic

pelt from goat skin revealed that there was complete and

uniform removal of hair showing white clean pelt with the

complete absence of hair root. The strength properties of

control and experimental crust leathers are given in Table 5.

It is obvious from the results that the strength properties of

experimental crust leathers are comparable to that of con-

ventionally processed crust leathers. The visual assessment

and the hand evaluation of crust leathers revealed that the

enzyme-assisted process led to improve the organoleptic

properties such as grain pattern appearance, smoothness and

fullness (Fig. 1). Both the visual and feel tests suggested that

the crust leathers made out of enzyme-treated skins were

fuller probably due to improved diffusion of tanning and

post-tanning chemicals in to the skins because of better

opening up and removal of interfibrillary materials.

Environmental benefits

The spent liquors have been collected from conventional

and experimental processes, and analysed for pollution

parameters such as biochemical oxygen demand, chemical

oxygen demand and total dissolved solids. The values are

presented in Table 6. It is seen that the BOD, COD, TDS in

the experimental process is much lower than the control

process. This is mainly due to the partial replacement of

lime and sulphide with the help of fibre opening enzyme.

This method appreciably decreases the usage of chemicals,

reduces pollution load and eliminates intermediate process,

reliming. Moreover, the remarkable decrease in the BOD

level indicates the decreased solubilization of hair due to

judicious usage of sulphide (1.5% w/w) in the combined

process as against conventional two-stage chemical process

where sulphide is used to the extent of 2.5% (w/w) along

with lime 5% in reliming.

Economic benefits of integrated process

The combination of unhairing–fibre opening by using a

cocktail of carbohydrase has been developed to enhance

economic benefits of leather processing. The total cost

consumption of chemicals and enzyme used in

Table 3 Visual assessment of defleshed pelt

Properties Conventional process Enzymatic process

Lime 5.0% (w/w) with

100% water (v/w)

Unhaired pelt with 100%

water (v/w)

Grain

pattern

9.6 ± 0.1 9.7 ± 0.1

substance 9.4 ± 0.1 9.8 ± 0.1

Smoothness 9.3 ± 0.1 9.5 ± 0.1

Pelt colour 9.2 ± 0.1 9.7 ± 0.1

Scale of 1–10; 1—poor; 10—best

Average value of 3 experts

Table 4 Extent of carbohydrate and proteoglycan removal with conventional and experiment

Sample Sugar removal (%)* GAG removal (%)*

Control 65.0 ± 0.5 73.5 ± 0.5

1.0% lime ? ES 72.4 ± 1.0 76.2 ± 0.5

2.0% lime ? ES 75.0 ± 1.0 77.0 ± 0.5

3.0% lime ? ES 81.0 ± 1.0 84.2 ± 1.0

4.0% lime ? ES 84.0 ± 1.0 86.0 ± 1.5

5.0% lime ? ES 87.0 ± 1.5 87.5 ± 1.5

6.0% lime ? ES 85.2 ± 1.5 86.3 ± 1.5

7.0% lime ? ES 84.1 ± 1.5 85.4 ± 1.0

ES enzyme 1%; sulphide 1.5%, GAG glycosaminoglycan

* Average value of 3 determinations

Role of carbohydrases in minimizing use of harmful substances: leather as a case study 1571

123

conventional and experimental method are given in

Table 7. The enzymatic formulation of carbohydrase has

effectively used to remove the hair and interfibrillary

materials such as glycosaminoglycan and proteoglycan.

Reduction in usage of lime and sodium sulphide in

experimental process provides cost reduction to an extent

of US$ 40/ton of skins. The experimental process would

lead to saving in chemical cost due to 40–50% reduction in

lime and sulphide process. In this work, a single-step

process leads way to a cleaner technology. This new

technology will result in increased productivity with the

existing methods due to substantial time saving.

Evaluation using scanning electron microscopy

The pelt sample processed by both conventional and

experimental (enzyme-assisted) methods was examined

through scanning electron microscope, and the details are

shown in Fig. 2. According to the micrographs, the cross-

sectional study of the experimental sample indicated

smooth and opened fibre bundles compared to that of

conventionally produced pelt specimen. The micrographs

confirm that the enzyme was able to bring out a conspic-

uous complete removal of hair and opening up of fibre

structure compared to conventional process. Efficacy of

enzyme-assisted and chemical-driven unhairing and fibre

opening process was corroborated with the results of his-

tological studies. Harish et al. (2015) also reported

enzyme-dehaired skins exhibit better characteristics, com-

pared to conventional process.

Histology

Haematoxylin- and eosin-stained sections of both the

samples of chemical- and enzyme-assisted processes were

analysed for extent of removal of epidermis, glandular

structures and hair root. The absence of the keratinous

structural features was observed in pelts obtained by

enzyme-assisted single-stage treatment, whereas removal

of such components was incomplete in the conventional

pelts. Haematoxylin and eosin staining clearly distin-

guished the removal of residues of interfibrillary materials

through histological studies of both conventional and

experimental pelts as shown in Fig. 3. It is seen that cross

section of control samples exhibited moderate opening of

collagen fibre bundles, while the experimental pelts

showed distinct fibres with good orientation. Complete

removal of epidermal layer from skin was observed when

dehairing was performed using carbohydrase.

Conclusion

The present work deals with combining the unhairing and

fibre opening by using a cocktail of enzyme in the presence of

judicious concentration of lime, and sulphide has been

developed to enhance economic and environmental benefits

of leather making. Only 1.5% sulphide and 5% lime were

found to be sufficient for unhairing when simultaneously

treated with 1.0% carbohydrases for fibre opening leading to

saving in 40–50% chemicals usage. Graphical representation

of the process is given in Supplementary Figure. This present

8.4

8.6

8.8

9

9.2

9.4

9.6

9.8

10

10.2

A ss

es sm

en t

R at

in g

Control

Experiment

Fig. 1 Visual assessment of wet blue leathers

Table 6 Environmental benefits

Process BOD (ppm)* COD (ppm)* TDS (ppm)*

C 5780 ± 10 7560 ± 10 15,320 ± 10

E 2264 ± 10 5040 ± 10 8330 ± 10

* Average value of 3 determinations

Table 5 Physical testing results of conventional and experimental leathers

Experiment Tensile strength (Kg/cm 2 )* % Elongation at break* Tear strength (Kg/cm)* Grain crack strength*

Load (Kg) Distension (mm)

C 242.8 ± 0.2 68.8 ± 0.5 45.0 ± 0.2 41.6 ± 0.2 9.5 ± 0.2

E 248.0 ± 0.2 57.8 ± 0.5 41.9 ± 0.2 46.6 ± 0.2 8.9 ± 0.2

* Average value of 3 determinations

1572 J. Durga et al.

123

Fig. 2 Scanning electron microscopy images of control and experimental goat skins. a Control, b experiment

Table 7 Cost of chemicals for processing one ton of raw skins to tanned leather

Chemicals Conventional method Experimental method

Quantity required (%) Cost (US $/ton) Quantity required (%) Cost (US $/ton)

Lime (Ca(OH)2) 10 15.04 5.0 7.52

Sodium sulphide (Na2S) 2.5 13.54 1.5 7.9

Enzyme – – 1.0 3.01

Lime (Ca(OH)2) 10 15.04 – –

Ammonium chloride (NH4Cl) 3.0 2.71 2.1 1.89

Alkali Bate 1.0 13.54 1.0 –

Salt (NaCl) 10 6.02 10 6.02

Formic acid (HCOOH) 0.5 0.23 0.5 0.23

Sulphuric acid (H2SO4) 1.0 0.3 1.0 0.3

Basic chromium sulphate (BCS) 4.0 42.11 4.0 42.11

Basic chromium sulphate (BCS) 4.0 42.11 4.0 42.11

Sodium formate 0.7 4.74 0.7 4.74

Sodium bicarbonate 1.0 13.54 1.0 13.54

Total 47.7 168.92 31.8 129.37

Fig. 3 Photomicrographs of H- and E-stained control- and

experiment-treated goat skins.

a Control, b experiment

Role of carbohydrases in minimizing use of harmful substances: leather as a case study 1573

123

invention also resulted in significant removal of interfibrillar

substances without damage to collagen structure. The partial

reduction in sulphide and lime lowers sulphide toxicity, hair

solubilization, BOD and sludge formation. This study

therefore provides an important solution to one of the long-

pending problems of leather processing.

Acknowledgements The authors gratefully acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi, for funding

this research. Authors thank ‘‘Science and Technology Revolution in

Leather with a Green Touch’’ (STRAIT)—1190.

Appendix 1

The pelts were washed with 200% water for 10 min.

Subsequently, the pelts were delimed by adding 100%

water and 1% (w/w) ammonium chloride for 45 min in a

drum. Deliming was ascertained by checking the cross

section of the delimed pelts for colourlessness due to

phenolphthalein indicator. After deliming, bating process

were carried out in the same bath for 30 min by the addi-

tion of bating enzyme. The pelts were washed with 100%

water for 10 min. Pickling was carried out. 1% sulphuric

acid (w/w) was added in 4 feeds at 10-min interval and

tumbled in a drum for 60 min to obtain pickled skin at pH

of 2.8. The pickled skins were tanned using 8% (w/w) basic

chromium sulphate (BCS) in 50% pickle water for 90 min.

Then 50% (w/w) water was added and the drum was run

further for 30 min. To the running drum, 1% (v/w) sodium

formate (mixed with 10% w/v water) was added. After

30 min, 1% (w/w) sodium bicarbonate (mixed with 10%

w/v water) was added in 3 feeds at 10-min interval and

continued the tumbling for 60 more min to bring the pH to

3.8.

Appendix 2

Post-tanning operations comprise of rechroming of semi-

finished wet blue leather, neutralization, dyeing, fat

liquoring and finishing. The wet blue leathers obtained by

the procedure under Appendix 1 to were shaved to 1.0 mm

thickness. All the samples were washed in 100% (w/v)

water in a drum for 10 min. After draining, the wet blue

leathers were treated with 1.0% (w/w) neutralizing syntan

with 100% water for 20 min. Sodium formate 0.5% (w/w)

and sodium bicarbonate were then added to the drum in 3

feeds at 10-min interval, while the drum was in running

mode. After ensuring the pH of the cross sections at 5.0, the

leather samples were washing twice with 200% (w/v) water

for 10 min. The neutralized skins were washed with water

followed by treatment with resin syntan (3% w/w) and

allowed to run in the drum for 20 min. After this, dying

(2% w/w acid dye) and fat liquoring (4% w/w synthetic fat

liquor) were carried out by drumming for 30 min. Subse-

quently melamine- and naphthalene-based retanning syn-

tans 4% (w/w) was added and run for 40 min followed by

the addition of synthetic fat liquor 4% (w/w), polymeric fat

liquor 3% (w/w) and natural fat liquor oil 4% (w/w) and

further running the drum for 40 min. Finally the auxiliaries

were fixed using 2% (v/w) formic acid diluted with 20% (v/

w) water and added at 3 feeds at every 10-min interval and

the drum was further run for 30 min and piled overnight.

The leathers were set, conditioned, again set with rever-

sible setting machine and hooked for drying. After drying,

leather was staked and buffed using 400-grit emery paper.

References

Dey PM, Pridham JB (1972) Biochemistry of a-galactosidase. Adv Enzymol 36:911–930

Dubolis M, Gillis KA, Hamilton JK, Rebers PA, Smith F (1956)

Colorimetric method for determination of sugars and related

substances. Anal Chem 28:350–356

Durga J, Ranjithkumar A, Ramesh R, Rose C, Muralidharan C (2015)

Rapid fiber opening process for skins: an approach for fail-safe

chemical free process. J Am Leather Chem Assoc 110:07–12

Durga J, Ranjithkumar A, Ramesh R, Girivasan KTP, Rose C,

Muralidharan C (2016) Replacement of lime with carbohy-

drases—a successful cleaner process for leather making. J Clean

Prod 112:1122–1127

Eaton AD, Clesceri LS, Greenberg AE (1995) Standard methods of

the examination of water and waste water. The American Public

Health Association (APHA), Washington

Harish B, KhandelwalS MoreV, Kalal KM, Seeta Laxman R (2015)

Eco-friendly enzymatic dehairing of skins and hides by C.

brefeldianus protease. Clean Technol Environ Policy

17:393–405

IUP 2 (2000) Sampling. J Soc Leather Technol Chem 84:303

IUP 6 (2000) Measurement of tensile strength and percentage

elongation. J Soc Leather Technol Chem 84:317–321

IUP 8 (2000) Measurement of tear load—double edge tear. J Soc

Leather Technol Chem 84:327–329

Jian S, Wenyi T, Wuyong C (2011) Kinetic of enzymatic unhairing by

protease in leather industry. J Clean Prod 19:325–331

Kandasamy N, Velmurugan P, Sundarvel A, Jonnalagadda RR,

Bangaru C, Palanisamy T (2012) Eco-benign enzymatic dehair-

ing of goat skins utilizing a protease from Pseudomonas

fluorescens species isolated from fish visceral waste. J Clean

Prod 25:27–33

Ludvik J (1996) Cleaner tanning technologies, UNIDO report,

pp 18–25

Ludvik J (2000) The Scope of decrease of pollution load in leather

processing (US/RAS/92/120/11-51). United Nation Industrial

Development Organsiation-Regional programme for pollution

control in the Tanning industry in south-East Asia, 2000-08-09

Mantle M, Allen A (1978) Periodic acid/Schiff assay for glycopro-

teins and Mucin. Bio Chem Soc Trans 6:607–609

Miller J (1972) Experiments in molecular genetics. Cold Spring

Harbor Laboratory, NY, pp 352–355

Rose C, Suguna L, Samivelu N, Rathinasamy V, Ramalingam S,

Iyappan K, Parvathaleswara T, Ramasami T (2007) Process for

1574 J. Durga et al.

123

lime and sulfide free unhairing of skins or hides using animal/or

plant enzymes. US Patent 7,198,647

Sandhya C, Sumantha A, Szakacs G, Pandey A (2005) Comparative

evaluation of neutral protease production by Aspergillusoryzae

in submerged and solid-state fermentation. ProcBiochem

40:2689–2694

Schlosser L, Keller W, Hein A, Heidemann E (1986) Utilisation of a

Lactobacillus culture in the beamhouse. J Soc Leather Technol

Chem 70:163–168

Seggiani M, Puccini M, Vitolo S, Chiappe C, Pomeli CS, Castiello D

(2014) Eco-friendly titanium tanning for the manufacture of

bovine upper leathers: pilot scale studies. Clean Technol Environ

Policy 16:1795–1803

Senthilvelan T, Kanagaraj J, Mandal AB (2012) Application of

enzymes for dehairing of skins: cleaner leather processing. Clean

Technol Environ Policy 14:889–897

Sivasubramanian S, Manohar BM, Rajaram A, Puvanakrishnan R

(2008) Ecofriendly lime and sulfide free enzymatic dehairing of

skins and hides using a bacterial alkaline protease. Chemosphere

70:1015–1024

Thangam EB, Nagarajan T, Suseela RG, Chandrababu NK (2001)

Application of alkaline protease isolated from Alcaligenesfae-

calis for enzymatic unhairing in tanneries. J Ind Leather

37:215–222

Vijayaraghavan R, Vedaraman N, Muralidharan C, Mandal AB,

MacFarlane DR (2015) Aqueous ionic liquid solutions as

alternatives for sulphide-free leather processing. Green Chem

17:1001–1007

Role of carbohydrases in minimizing use of harmful substances: leather as a case study 1575

123

Reproduced with permission of copyright owner. Further reproduction prohibited without permission.