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Abstract
The treatment of chromium-containing leather waste (CCLW), the major solid waste generated at the post-tanning operations of
leather processing, has the potential to generate value-added leather chemicals. Various alkali and enzymatic hydrolysis were
compared, and calcium oxide was found to be important for effective (but still incomplete) hydrolysis. Three possible reasons are
given for the incomplete hydrolysis under alkaline conditions. Data for 19 amino acids are presented for four different treatment
products. On the basis of the results, a novel three-step CCLW treatment process is proposed. The gelatin extracted in the first step
is chemically modified to produce leather finishing agents. The collagen hydrolysates isolated in the second step are used as pro-
teinic retanning agents by chemical modification. The remaining chrome cake is further hydrolyzed with acids in the third step, and
the obtained chromium-containing protein hydrolysates could be used for the preparation of chromium-containing retanning
agents for leather industry. The proposed three-step process provides a feasible zero discharge process for the treatment of CCLW.
# 2003 Elsevier Ltd. All rights reserved.
addition, the method of making paper was introduced reaction with formaldehyde after chromium hydroxide
to produce both leather and paper substitutes. Between separation, showed very good tanning capability. They
1970 and 1993, a lot of publications and patents con- also studied the possibility of utilizing the hydrolysate
centrated on hydrolyzing CCLW to recycle amino acids or its copolymers with methyl methacrylate or acrylo-
and peptides for use in feeds and fertilizers (Alves Dos nitrile in leather finishing (Manzo et al., 1989). Unfor-
Reis and Beleza, 1991a,b; Ohtsuka, 1973; Taylor et al., tunately, they only utilized the collagen hydrolysate of
1992b, 1993a). Various treatment methods have been CCLW, the ‘‘chrome cake’’ was not investigated.
developed during this period. Basic hydrolysis, such as Research over several years in our laboratory has
using Ca(OH)2 with steam (Guardini, 1983; Holloway, been designed to fully utilize CCLW and convert them
1978) or NaOH (Galatik et al., 1988) at elevated tem- into value-added products, avoiding the production of
perature and/or pressure (Maire and Lipsett, 1980), acid any other waste. In the present study, we focus on
hydrolysis (Wojciech, 1998), and enzymatic hydrolysis establishing an appropriate hydrolysis method to yield
(Sivaparvathi et al., 1986a,b) were used for chrome the desired recyclable products. We also consider the
recovery and the isolation of protein fractions. Peroxide feasibility of completely utilizing CCLW with zero dis-
treatments (Cot et al., 1991; Cot and Aramon, 1986) can charge.
get collagen fiber and Cr(VI) by oxidization of leather
scraps. Wet air oxidation and incineration of CCLW
(Imai and Okamura, 1991) were used only to recycle 2. Experimental
chromium(VI). Unfortunately, strong toxic Cr(VI) by-
products were generated in these reactions, thus requir- 2.1. Analysis of chrome-containing leather waste
ing an additional reductive step. The removal of chro-
mium is, however, never complete and needs many CCLW obtained from a commercial pigskin tannery
repetitions, which is very expensive. Moreover, the more were kept at room temperature and analyzed for pH,
often the procedure is repeated, the more the decom- moisture, ash, Total Kjeldahl Nitrogen (TKN), chro-
position of the collagen proceeds and collagen goes into mium and fat by normal methods. Moisture was deter-
solution, resulting in a mixture of Cr(VI) with protein. mined by heating the sample at 110 C for 12 h. Ash in
Brown et al. did systematical laboratory (Cabeza et the dried products was determined by heating the sam-
al., 1999a; Taylor et al., 1993b, 1997, 1998a) and pilot ple at 600 C for 4 hours. TKN was determined by the
scale (Cabeza et al., 1998d, 1999b; Taylor et al., 1998b) semi-micro Kjeldahl method. Chromium was deter-
studies on the treatment of CCLW in the past 10 years. mined using a Perkin-Elmer model AA7003 atomic
The initial one-step process developed by them involved absorption spectrophotometer. Fat was extracted with
the use of alkaline proteolytic enzymes to isolate a chloroform.
chrome-free, hydrolysate product that can be used as
feed or fertilizer (Taylor et al., 1990, 1992b). A newer 2.2. Studies on hydrolysis methods
two-step process was to obtain a gelable protein, with
potential uses in adhesives, cosmetics, films, encapsula- 2.2.1. Alkali hydrolysis
tion or emulsifying, etc. in the first step, and a hydro- One hundred grams of CCLW were shaken in 1 l of
lysate in the second step. In this process the CCLW water, and added 10 g CaO, or 10 g MgO, or 7 g NaOH
were digested with an initial alkali step and with an at 98 C for 3, 6 and 24 h, respectively. Then the sam-
alkaline protease second step (Taylor et al., 1991, 1992a, ples were filtered warm through Büchner porcelain fun-
1993a, 1994), or using two consecutive enzymes (Cabeza nels with Whatman No. 1 filter paper under vacuum
et al., 1997, 1998b, 1999c) in the two-step process. condition. The filtered protein solutions were stored at
However, a chrome cake, consisting of residue protein 4 C, and the protein yield was calculated based on the
crosslinked with chromium, remained as a by-product dry weight of the waste. The remained chrome sludge
in both treatment processes (Cabeza et al., 1998c). was kept at room temperature for further analysis and
Therefore, a complicated multi-step chemical operation, treatment.
including sulfuric acid dissolving, sodium hydroxide
precipitation, filtering and washing, were used to purify 2.2.2. Enzymatic hydrolysis
the chrome cake for the chrome recovery (Cabeza et al., One hundred grams of CCLW were shaken in 1 L of
1998d, 1999b). This process produces wastewater with water and 3 g MgO at room temperature for 4 h. This
salts and protein residues, requiring further treatment. pretreatment step is necessary to obtain the optimal pH
The collagen hydrolysate of CCLW once separated for the enzymatic digestion. Then 2 g alkaline protease,
from the chromium might have potential use as a lea- such as 2709 enzyme (Wenjiang enzyme Inc., China), or
ther tanning or finishing agent. Manzo and Fedele 1398 enzyme (Wenjiang enzyme Inc., China), or Trypsin
(1994, 1996) not only demonstrated that the con- (Wenjiang enzyme Inc., China), or Alcalase 2.5 L
densates, produced by the hydrolysis of CCLW and (Novozymes Inc., China), was added at their individual
C. Mu et al. / Waste Management 23 (2003) 835–843 837
optimal temperature for 3, 6 and 24 h, respectively. for 3–5 h. The resultant chromium-containing protein
Then the solution was filtered warm through Whatman hydrolysates solution was slowly adjusted to the appro-
No. 1 filter paper in porcelain funnels under vacuum priate pH value with sodium bicarbonate for prepara-
condition. The filtered protein solutions and remained tion of retanning agents.
chrome sludge were analyzed and treated as before.
2.6. Analysis of recycled products from three-step
2.3. Multi-step alkali hydrolysis process
One hundred grams of CCLW were shaken in 1 l of The recovered samples from three-step process were
water and 10 g CaO at 98 C for 3 h. The protein solu- analyzed for pH, ash, protein as TKN, chromium, cal-
tion was filtered and separated, stored at 4 C. Then, the cium or magnesium. The molecular weight ranges of
chrome sludge was continuously hydrolyzed four times protein fractions were estimated by SDS–PAGE (poly-
at 98 C for 3 h, with 2 g CaO and 100 ml water added acrylamide gel electrophoresis in sodium sulfate) and
each time. All of hydrolysates filtered were calculated ÄKTATM explorer (Amershampharmacia, Inc.) GFC
into the total protein yield. The mixed hydrolyzed pro- (gel filtration chromatograph).
tein solutions and the final chrome cake (containing
protein residues and chromium) were stored at 4 C for
the analysis of amino acid composition. 3. Results
2.4. Analysis of amino acid composition 3.1. Analyses of chrome leather waste
The amino acid compositions of the hydrolyzed pro- Table 1 shows the analysis results of the CCLW used
tein products and the residue protein in chrome cake in our experiments. It indicates that the waste contains
were determined using a Hitachi model 835-50 analyzer. about 4% chromic oxide and about 80% protein (the
The results were compared with natural type I collagen nitrogen content of collagen is 18%).
and pickled pigskin obtained from the same pigskin
tannery. 3.2. Comparison of alkali and enzymatic hydrolysis
2.5. Three-step hydrolysis process Table 2 presents the protein yields via various alkali
hydrolysis or enzymatic treatment assisted by magne-
In the first step, 100 g of the CCLW were suspended sium oxide.
in water and CaO/NaOH was added to increase the
alkalinity to pH=11 0.5. The reaction mixture was 3.3. Amino acid composition of different products from
stirred at 70–80 C for 3.5 h, then filtered warm to multi-step alkali hydrolysis
separate the gelatin from chrome sludge. In the second
step, 100 g water and 2 g CaO were added to the chrome The chromed scraps, with alkali alone or alkali-assis-
sludge at 97–99 C for 3–4 h. Then the mixture was fil- tant protease treated, were difficult to completely
tered warm through the filter press to separate the col- hydrolyze, even when increasing dosage of alkali and
lagen hydrolysates from the chrome cake. During the enzymes, prolonging reaction time or enhancing hydro-
whole process, pH was controlled at 9–10 to maintain lysis temperature. Typically, in multi-step alkali hydro-
optimum alkalinity for avoiding chrome dissolution. In
the third step, the chrome cake was dissolved in con-
centrated sulfuric acid with a pH of 1.0–2.0, at 97–99 C Table 2
Efficiency of basifiers and enzymes on hydrolysis the waste
Table 3
Amino acid compositions of the hydrolysate and the residue protein
(mol%)
Table 4
Properties of the recovered products from a three-step process
ondly, the total ash of the resultant hydrolysate was hydroxy-amino-acid (Ser, Tyr and Thr) have much
minimized when using Calcium oxide, and can con- higher contents in protein residues than in the hydro-
sistently be maintained below 10% on a dry-basis. This lysates, while the hydrophilic acidic amino acids (Asp
agrees with results reported elsewhere (Stockman, and Glu) contents of protein residues are lower than
1996). Third, using NaOH in the treatment of CCLW that of the hydrolysates. Note that the alkaline amino
can lead to a rapid increase of alkali strength at the acids are hydrophobic under alkaline conditions.
initial stage, thus resulting in excessive digestion of col- Therefore, we can conclude that the protein residues in
lagen. Chromium hydroxide is an amphoteric hydroxide chrome cake were difficult to hydrolyze and dissolve in
and it will be dissolved when the liquor pH is below 5.5 alkaline solution.
or over 12 [as shown in Eq. (1)]. It seems that using We see three possible reasons for the incomplete
Ca(OH)2 resulted in steady hydrolysis due to its buffer- hydrolysis of CCLW in our experiments.
ing property.
OH OH 1. Hydrophobic interaction: Non-polar amino acids
Cr3þ *
) CrðOHÞ3 # *
) CrO
2 ð1Þ and alkaline amino acids are all hydrophobic in
Hþ Hþ
alkaline conditions, and because they are com-
As shown in Table 5, the 14 kinds of amino acid mon in the residue protein, they could form
contents differ greatly between the protein residues and hydrophobic domains and tight clusters inacces-
hydrolysates. Non-polar amino acids (Val, Phe, Leu, Ile sible to water, leading to the protein resisting
and Met), alkaline amino acid (Hyl, Arg and His) and hydrolysis.
Fig. 2. A schematic of oxolation chromium(III) complexes formed during basification and its complexation with protein carboxyl.
840 C. Mu et al. / Waste Management 23 (2003) 835–843
2. Covalent bridges: The products in the residue Cr(OH)3. However, oxolation chromium(III)
protein might have covalent cross-linking formed complexes formed during basification (as sche-
between alkaline amino acids and hydroxy amino matically shown in Fig. 2) have strong resistance
acids. These covalent bonds might break under to alkali. It could crosslink with the carboxyl
acidic conditions leading to the complete hydro- groups of a few Asp and Glu, or protein hydro-
lysis of protein residues with acids to obtain lysates, and form insoluble macromolecular
chromium-containing protein hydrolysates. metal complexes. When using peroxide to oxidize
3. Cross-linking with chromium(III) complex: the Cr(III) to Cr(IV), we found that most of the
Under alkaline condition (pH < 12), most of the protein residues were dissolved soon even in
coordinated chromium are divorced from the alkaline solution, which supports this possibility.
carboxyl groups on the side chain of aspartic acid In addition, a certain amount of cystine (Cys)
(Asp) and glutamic acid (Glu) and precipitated as appeared in the hydrolysates, but not in chrome
Fig. 3. Outline of three-step process for treatment and utilization of chromium-containing leather wastes.
C. Mu et al. / Waste Management 23 (2003) 835–843 841
cake, which indicates that the S–S crosslinking in A possible flow chart is given in Fig. 4. In this scheme,
cystine has nothing to do with the residue protein. the recovered collagen polypeptides (i.e., protein
hydrolysates) are chemically modified with certain
The results presented here are for the CCLW of one bifunctional monomers of acrylic acid series and mod-
specific tannery; however, we have found that the chro- ified wax to produce leather retanning agents. The
mic oxide and protein contents of CCLW vary within chromium containing hydrolysates are further modified
relatively narrow ranges. As a result, we believe the with acrylic acid series monomers and developed into
treatment procedures proposed could be easily adjusted retanning agent which can be recycled into tanning and
for other CCLW. retanning process. The detailed preparation and use of
The gelatin isolated by hydrolysis (see Fig. 3) could be the final products will be the subject of future work. A
chemically modified for the preparation of proteinic patent has been applied for (Mu et al. 2000) and pilot
leather finishing agents, a highly value-added product. scale studies are being carried out at present.
Fig. 4. Flow diagram of the procedure for gelatin modification to produce leather finishing.
842 C. Mu et al. / Waste Management 23 (2003) 835–843
Table 5
Comparison of some amino acids in hydrolysates and protein residues (mol%)
Amino acid Side chain group Performance of the Group Hydrolysates Protein residues
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