Sustainable metal-free leather manufacture via synergistic effects of triazine derivative and vegetable tannins

Restrictions on heavy metals, especially chromium, have encouraged alternative tanning systems that can reduce environmental and human health risks from conventional chrome-based tanning. In this work, metal-free combination tanning was developed by using vegetable tannins and a triazine-based syntan containing active chlorine groups (SACC). Specifically, the relationship between leather performance (e.g., hydrothermal stability and organoleptic properties) and technical protocols (e.g., types and dose of tannins) was systematically established. The optimized protocol involving a unique procedure (i.e., 10% SACC pre-tanning, shaving, and 25% wattle tanning) endowed the leather with high shrinkage temperature (~ 92 °C) and met the Chinese standards for shoe upper leather (QB/T 1873-2010). Our method not only produces zero chrome-containing solid wastes, but also uses ~ 75% less tannin for leather manufacture. The excellent leather performance was ascribed to the synergistic effects, where SACC and wattle diffused into collagen fibrils and may bind to collagen via covalent, hydrogen and ionic bonding, locking the hierarchical structure of collagen from microfibrils to fiber bundles. Moreover, we summarized these findings and proposed a diffusion-binding-locking mechanism, providing new insights for current tanning theory. Together with the biodegradable spent tanning liquor, this approach will underpin the development of sustainable leather manufacture.


Introduction
Tanning is the critical process in leather industry through which animal hides and skins are converted into leathers that are resistant to heat and microbial attack. Chrome tanned leather has been dominant in the tanning industry because of their excellent comprehensive properties, such as good hydrothermal stability, reliable mechanical behavior, etc. [1]. However, conventional chrome tanning generates substantial non-renewable chrome-containing wastewater [2] and solid wastes [3,4]. These unfixed Cr(III) in leather and wastewater may be oxidized to carcinogenic Cr(VI) [5,6], which seriously threatens human health and environment. Therefore, various countries or regions have released stringent restrictions regarding the Cr discharge. In 2008, chrome-containing solid wastes have been listed in the "National Hazardous Waste Inventory" in China, which limits the production and circulation of chrome tanned leather. More recently, the European Union has limited the content of Cr(VI) in leather products to 3 mg/kg in 2014 (Annex XVII of REACH regulation) [7,8].
The utilization of non-chrome metal materials (e.g., Al 3+ , Zr 4+ and Ti 4+ ) as tanning agents has been reported to coordinate and crosslink collagen fibers, but their binding strength is weaker than chrome, leading to poor leather performance [9]. Moreover, many countries also have restricted the content of metals in leather and textiles, such as Leather Standard by Oeko-Tex (2021 version) [10]. To this end, the development of metal-free tanning materials and cleaner tanning systems is imperative [11]. This also requires a deeper understanding of the physicochemical interactions between tanning agents and collagen at the molecular and structural levels-the crosslinking effect of tanning agents is not only related to their reactivity [12], but also relevant to the collagen structure, size of tanning agents, and their diffusion capacity in leather matrix [13,14].
The tanning system using environmentally friendly organic tanning agents is considered as an alternative tanning approach [15]. One typical bio-based material is vegetable tannins from plant leaves, fruits, seeds and barks, which have molecular weights of 500-3000 Da and are classified into ester derived hydrolysable tannins (e.g., chestnut) and flavonoid derived condensed tannins (e.g., mimosa) [16,17]. They can penetrate and bind with collagen in multiple levels [18,19] via hydrogen and hydrophobic bonds [20] to endow the leather with good fullness, human skin compatibility and environmental friendliness. More recently, triazine derivatives as tanning agents for chrome-free leather has attracted immense interest [7,11,21,22]. It mainly reacts with the side-chain amino groups of collagens to form strong covalent bonds, thus enhancing hydrothermal stability of leather [22]. Compared with other organic tanning agents (e.g., aldehydes and syntan), triazine derivatives can effectively mitigate the health risks due to their biocompatibility [23,24]. Meanwhile, the released H + in tanning process (Additional file 1: Fig. S1) can simultaneously reduce the tanning liquor pH from ~ 7.8 to ~ 5.0, which facilitates the tanning process (e.g., dispense with pickling) and reduces the discharge of neutral salts [25]. In our previous work, the combination tanning system using tannic acid and triazine derivative (i.e., Granofin ® Easy F-90) has been studied to overcome the poor thermal stability and storage stability of solo tanned leather [26]. However, the restricted leather performance of such combination tannages and relatively expensive tannic acid limited their practical application. Moreover, the tanning mechanism underneath this approach lacks clear understanding from molecular level to collagen structure level.
Here we reported a sustainable metal-free leather processing for eco-leather manufacture involving triazine derivative pre-tanning, shaving and vegetable tannins tanning. The types of tannins including hydrolysable tannins (i.e., chestnut, valonea and tara extracts) and flavonoid derived condensed tannins (i.e., wattle, quebracho and bayberry extracts) in this method were systematically optimized (Additional file 1: Fig. S2). The characteristics and environmental performance of the tanning process were also investigated. This work not only revealed the synergistic effects of triazine derivative and tannins on leather matrix, but also established a reliable novel tannage to endow the leather with required organoleptic properties and physical performance. We envisaged this method would produce zero chrome-containing solid wastes (i.e., eliminating the risks of heavy metals to human health and environment) and emerge as a cost-effective tanning strategy, which contributes to the cleaner production towards a sustainable development of leather manufacture.

Leather tanning process
The solo SACC tanning process (Additional file 1: Table S1, above the dotted line) was conducted according to a previously reported method [26]. Specifically, the bated cattle pelt samples from back part (30 cm × 30 cm) were first immersed in 70 wt% water at 25 °C and tanned with 10 wt% SACC for 2 h at 25 rpm/min in a stainlesssteel temperature-controlled drum (GSD, Wuxi Xinda Light Industrial Machinery Co., LTD, China) to give a complete penetration. Then 50 wt% hot water (55-65 °C) was added to the drum twice, and the drum temperature was raised to 40 °C for 2 h and 45 °C for 4 h, respectively, to facilitate the binding between SACC and collagen. Meantime, the pH of the tanning system spontaneously reduced from ~ 7.8 (bating pH) to ~ 5.0. The obtained SACC-tanned leather was washed and piled overnight.
As for the combination tanning for metal-free leather (Additional file 1: Table S1), the above obtained SACCtanned leather was shaved, weighed (baseline of material dosage) and washed. Then, 0.5 wt% oxalic acid and 0.3 wt% disodium EDTA were used for rewetting and deferrization, preventing the tannin extract from darkening with iron. Subsequently, the leather was neutralized to pH 4.5-4.7 with 0.5 wt% NaHCO 3 solution and tanned with 10-30 wt% different tannin extracts (i.e., wattle, quebracho, bayberry, chestnut, tara, valonea). After tanning for 3 h, pH of the floats was adjusted to ~ 3.5 with 0.5 wt% HCOOH solution. The resulting SACC-tannins combination tanned leather was washed and piled for 24 h.
Post-tanning processing (Additional file 1: Table S2) was performed according to common procedures including retanning (e.g., acrylic resins, amino resins, syntan), dyeing and fatiliquoring. The obtained leather was termed as crust leather.

Measurement of shrinkage temperature
The shrinkage temperature (T s ) of SACC-tanned leather and SACC-tannins combination tanned leather were measured by a shrinkage tester (Sunshine electronic institute, Shaanxi University of Science & Technology) according to the ASTM method [15]. The leather samples (10 mm × 60 mm) were suspended vertically in water and the heating rate was maintained at 2 °C/min. The temperature at which samples shrink was recorded as T s . Each reported value was an average of three experiments.

Determination of the uptake of tannin extract
The maximum absorbance and standard curves of six tannin extracts were measured by an UV-visible spectrophotometer (UV3600, Shimadzu, Japan) according to the previously reported method [27]. Specifically, 10 mL of tanning spent liquor was collected and centrifuged at 5000 rpm for 30 min to remove suspended solids. The UV spectra of different tanning spent liquors were determined to obtain their maximum absorption peak (Wattle and Bayberry: 276 nm, Quebracho: 274 nm, Valonea: 270 nm, Chestnut: 278 nm, Tara: 265 nm) (Additional file 1: Fig. S3). Then 1.00 g/L standard solutions of different tannins were prepared and diluted to concentrations of 5, 10, 15, 20, 25, 30 and 35 mg/L. The absorbance values were measured with water as blank at maximum absorption peak and plotted against the concentration values as standard curves (Additional file 1: Fig. S3). Finally, the concentration of tannin extract in the spent liquor was acquired by the standard curves, and the absorptivity of tannin extract was calculated by the following formula: where C o and C t are the concentration of tannin extracts in tanning spent liquor before and after the tanning process, respectively.

Chemical resistance stability measurements
The SACC tanned leather and SACC-tannins combination tanned leather samples (5 cm × 5 cm) were incubated with distilled water, 10% urea solution, 0.5% Na 2 CO 3 solution, 10% n-propanol solution or HCOOH solution (pH 2.5) in conical flask, respectively. The incubation was carried out at room temperature for 12 h in a water bath thermostatic oscillator. Each of them was piled overnight. The T s of the leather samples was then measured before and after washing.

Amino acids analysis
The bated pelt and SACC-tanned leather samples were first crushed into powder. Then, 0.2 g samples were hydrolyzed in 20 mL of 6 mol/L HCl at 110 °C for 10 h. The hydrolysates were dried to remove HCl, the obtained residues were dissolved and diluted with deionized water. After filtered through a 0.22 μm filter syringe, the solution was measured by using an amino acid analyzer (A300, MembraPure GmbH, Germany) to analyze the content of amino acids [28]. The detection wavelength was 570 nm and the injection volume was 20 μL.

Fourier transform infrared spectroscopy (FTIR) studies
The SACC tanned leather and SACC-tannins combination tanned leather samples after freeze drying for 24 h were tested by a Fourier transform infrared spectroscopy (FTIR, Nicolet iS10, Thermo Fisher Scientific Inc., USA) in the range of 400-4000 cm −1 .

Raman spectral measurements
The SACC tanned leather and SACC-tannins combination tanned leather samples after freeze drying were measured on a confocal Raman spectrometer (Horiba Labra HR, France) excited by a laser at 785 nm. The (1) Raman shifts were carefully calibrated using Si plate with an uncertainty of 0.5 cm −1 . The scanning was in the range of 700-1800 cm −1 .

Scanning electron microscopy (SEM) observation
After freeze drying at − 45 °C in a freeze dryer (Alpha 1-2 LD, Christ, Germany) for 24 h, the bated pelt, SACC tanned leather and SACC-wattle combination tanned leather samples were cut into defined specimens with uniform thickness (~ 1 cm × 1 cm) by a freezing microtome (CM1900, Leica, Germany) at − 20 °C. Then the samples were sputter-coated with Au before SEM observation. The SEM images for the cross section were obtained by operating a scanning electron microscope (JSM-7500F, JEOL, Japan) at an accelerating voltage of 15 kV.

Pore structure measurement
The bated pelt, SACC tanned leather and SACC-wattle combination tanned leather samples were freeze dried at − 45 °C for 24 h. Then the pore structures of leather samples were measured using a mercury intrusion porosimetry (MIP, AutoPore IV 9500, Micromeritics, USA) [29]. The porosity, total pore area and average pore diameter were given by this instrument.

Organoleptic property assessment
The quality and grade of the leathers largely depend on its organoleptic properties, such as softness, fullness, grain smoothness, grain compactness and color shade. Thereinto, softness is defined as an integration of the thickness, compressibility, smooth handle, bend-ability, and extensibility of leather. Fullness is defined as a feeling of compressibility and smoothness to the touch when the leather is deformed under an external force [30]. Grain smoothness and compactness are related to the area of leather, and grain smoothness will reduce and compactness will increase as the leather shrinks [31]. Color shade of leather is influenced by the color of tannins. These organoleptic properties were assessed by hand and visual examination. The crust leathers were evaluated on score from 1 to 10 points for each property by three experienced tanners [32].

Determination of physical properties
The crust leather samples were first conditioned for 48 h at 20 ± 2 °C with a relative humidity of 65 ± 2%. Then physical properties of the samples were examined using the standard IULTCS methods [33]. Specifically, tear load, elongation at 10 N, bursting strength and softness were measured as per standard procedures. Each reported value was an average of four samples (2 along the backbone, 2 across the backbone).

The environmental impact assessment (EIA)
of metal-free tanning process

Extractable metal content measurement of crust leather
In order to simulate the scenario where leather products contact human skin, the crust leather samples were first extracted with acidic artificial sweat, filtered and acidified using international standard ISO 17072-1:2019 method [34]. Then the common metal concentration (i.e., Cr, Al, Zr, Ti, Fe) in the extracts were measured by inductively coupled plasma-optical emission spectrometer (ICP-OES, IRIS Intrepid II, Thermofisher, USA).

Pollution load assessment of metal-free tanning system
The total dissolved solids (TDS) in the tanning spent liquor was tested according to the standard method of the American Wastewater Association (AWWA) [35]. The tanning effluent after vacuum filtration was dried in a drying oven at 102 °C until reaching a constant residual weight, and the ratio of the dried solid mass to the volume of the spent liquor was calculated as the TDS content (g/L). The chloride ion (Cl − ) content, chemical oxygen demand (COD Cr ) and biological oxygen demand (BOD 5 ) in spent tanning liquor were measured by ion chromatograph (CIC-D160, Shenghan Chromatograph, Qingdao), COD analyzer (DR1010, Hach, USA) and BOD analyzer (TrakII, Hach, USA), respectively. COD Cr refers to chemical oxygen demand measured with potassium dichromate as the oxidant. BOD 5 represents the amount of oxygen required for 5 days of microbial biodegradation.

Optimizations of metal-free combination leather tannage
We first investigated the effect of various tannin extracts and SACC on the T s of tanned leathers in a solo tanning method. The pickled pelts were tanned by using conventional vegetable tannage and the dosages of chemicals were applied based on the weight of limed pelts (a piece of limed pelt ~ 30 kg) (Fig. 1a). Six types of tannins including wattle, quebracho, bayberry, tara, chestnut, and valonea were chosen as the representative tannins, covering both condensed and hydrolysable tannins (Additional file 1: Fig. S2). The dosage of tannin extracts was kept at 25% based on the weight of pickled pelts. The T s of leather tanned by condensed tannins (80-85 °C) was much higher than that of hydrolysable tannins (75-80 °C), which is mainly due to the stronger astringency of condensed tannins (i.e., possible covalent reaction between the basic amino groups of collagen and aromatic carbon in the condensed tannin molecules via quinoid structures) than hydrolysable tannins (Fig. 1b) [19,36]. In contrast, solo SACC tanning gave the leather an increased T s (~ 78 °C) compared to the bated pelts (~ 60 °C), owing to the covalent bonding between the active chlorines of SACC and amino groups of collagen [26]. Notably, the T s of SACC tanned leather can meet the requirement of shaving (T s > 75 °C).
The unique combination tanning strategy by using SACC and tannins was then studied for better performance of the leather products. Specifically, bated pelts were first treated by 10% SACC for 8 h, then these pretanned leathers were shaved before the tanning with different tannins (Note: the dosages of the tanning chemicals applied were based on the weight of shaved leather; a piece of shaved leather ~ 7 kg) (Fig. 1a). This method produces no chrome-containing solid waste and reduces the dosages of tannins by ~ 75% compared to conventional vegetable tanning (Additional file 1: Table S3). It is notable that SACC-condensed tannin system conferred the leather an increased T s (> 87 °C), much higher than solo SACC or tannins tanning (Fig. 1c), implying the presence of synergistic interactions of SACC and tannins. For example, the T s of combination tanning leather (10 wt% SACC and 25 wt% wattle) can reach ~ 92 °C. Moreover, although the introduction of hydrolysable tannins (even up to 30 wt%) into combination tanning system gave the leather a limited increase (4-6 °C) in T s , which is still above the critical T s standard (80 °C) of the qualified shoe upper leather products (QB/T 1873-2010).
The uptake efficiency of tannins in the different combination protocols was then determined. Generally, the spent liquor after tanning with condensed tannins was clearer than that of the hydrolysable tannins (Fig. 2). For example, the spent liquor from 20-25 wt% wattle was clear, while the spent liquor from 10-15 wt% tara was still turbid. The uptake efficiency of condensed tannins is significantly higher than that of hydrolysable Fig. 2 The photos of tanning spent liquor and uptake efficiency of tannins in different SACC-tannin combination tanning: a wattle, b bayberry, c quebracho, d valonea, e chestnut, and f tara. The concentration of SACC was fixed at 10% and the concentration of tannins varied from 10 to 30 wt% tannins (85-90% vs. < 85%) based on absorbance measured by UV-vis spectroscopy. Especially, the uptake of wattle tannin could reach ~ 92% (Fig. 2a). The uptake of tannins in combination tanning is as follows: Wattle > Bayberry > Quebracho > Chestnut > Valonea > Tara. Therefore, the combination tanning with 20-25 wt% wattle tannins was applied in the following assessment based on tanning effects and economic benefits.

Molecular mechanisms in the combination tanning system
We next explore the dominant stabilizing interactions in the leather matrix. The chemical interactions, e.g., ionic bonding, hydrogen bonding and hydrophobic bonding between the tanning agent and collagen can be disrupted by washing with certain chemical reagents, which in turn leads to the change of T s of the leather. This provides us a clue to infer the key interaction after the combination tanning [37]. For example, distilled water can wash out tannins that are free or weakly adsorbed in the leather; Urea and n-propanol are hydrogen bond and hydrophobic bond breaking reagents, respectively [38,39]. The solo SACC tanned leather showed no obvious change (< 1.0 °C) in T s after washing with water, urea, n-propanol and sodium carbonate solution (Fig. 3a). This is mainly due to the formation of robust covalent interaction between active chlorine and the side-chain amino groups of Lys, Arg, His and Hyl in collagen [26]. In contrast, a minor decrease (2.9 °C) in T s was observed after formic acid treatment. This may be attributed to the instability of the covalent interaction between SACC and collagen amino groups in acidic environment (i.e., formic acid) [40][41][42]. The ∆T s of SACC-tannins combination tanning leathers was further investigated (Fig. 3a). A slightly reduced T s was observed after washing with distilled water and formic acid solution (2.0-2.5 °C and 2.5-3.5 °C, respectively). This indicated that compared with solo SACC tanning, SACC-tannin combination tanning had a multi-modal tanning effect on collagen fibers. All the leather products remain a relatively high T s (> 80 °C), implying the good water and acid resistance for posttanning procedures (e.g., filling, dyeing and fatliquoring). It is notable that the urea solution and n-propyl alcohol brought about a decrease of T s (3.6-4.2 °C and 4.2-4.7 °C, respectively) via breaking hydrogen and hydrophobic bonds. Moreover, sodium carbonate solution generated an obvious decrease of T s (6.2-7.7 °C), which is probably because of the deprotonation of tannins (i.e., deflocculating) at high pH leading to the reduced multi-point interaction (e.g., hydrogen bonding) with collagen [43]. Overall, this suggested the SACC and tannin can form hydrogen bonds, hydrophobic bonds, and covalent bonds between collagen molecules.
To understand the collagen structure and tanning mechanism after this combination tanning process. Firstly, the amino acids content of bated pelt and SACCtanned leather were analyzed. The content of Lys, His and Arg in leather fiber is reduced by 27-30% after SACC tanning (Fig. 3b). This may be due to the covalent interaction of active chlorine on SACC with collagen amino groups. Then, the FT-IR spectra and Raman spectra of leather samples before and after different tannins tanning were collected. Type I collagen is known to possess a special triple-helix conformation which has a backbone structure with a high proportion of amide and imide groups. The absorption peaks of leather at 3410 cm −1 and 3084 cm −1 (N-H stretching of amides A and B), 1642 cm −1 (peptide chain C=O stretching of amide I), 1532 cm −1 (couple of N-H bending and C-N stretching of amide II) and 1240 cm −1 (-CH 2 wagging, C-N stretching and N-H bending of amide III) indicated the presence of collagen backbone (Fig. 3c) [44]. Moreover, no obvious shift of the amide I band absorption peak was observed, showing that the triple-helix structure of collagen largely remained after the introduction of SACC and tannins [45]. The increased intensity of amide A band can be attributed to the formation of hydrogen bond between phenolic hydroxyl groups of tannins and side-chain amino groups of collagen [46]. In addition, the appearance in absorption peaks of C-O-C and C-N in the Raman spectra (Fig. 3d) may be attributed to the covalent interaction of active chlorine with side-chain amino groups of collagens and phenolic hydroxyl groups of tannins [26].
Based on these results, the synergistic mechanism of SACC and tannin in the metal-free tanning system was summarized in Fig. 3e. Active chlorine groups on SACC molecules can not only interact with the amino groups on the collagen fibers via covalent bonding, but also bind to the phenolic hydroxyl groups on the tannin molecules, exerting robust crosslinking effects [47]. In addition, -SO 3 − of SACC molecules can form ionic bonding with -NH 3 + of collagen [48], and the phenolic hydroxyl groups on the tannin molecules can interact with amino, hydroxyl or carboxyl groups of collagen by hydrogen bonds and/or hydrophobic bonds [49]. Collectively, the synergistic interactions within collagen fibers are formed, which substantially contribute to the enhanced hydrothermal stability of the metal-free leather.

Morphologies and porous structures of leather
The microstructures of collagen after tanning processes and the distribution of SACC-tannin in leather matrix were then analyzed. SEM showed the morphologies of bated pelt, solo SACC tanned leather and SACC-tannin combination tanned leather (Fig. 4a-f ). Bating pelt exhibited sticky, tight and thick collagen fiber bundles (Fig. 4a). In contrast, the collagen fibers after the combination tanning became loose and isolated. Moreover, the larger SACC-tannin complexes with micron dimensions were well deposited between fiber strands to form the crosslinked network structure (Fig. 4c) [14]. Magnified SEM image confirmed that the collagen fibrils in all groups exhibited the quarter-stagger structure  Fig. 4d-f ), which was the typical collagen banding pattern of grooves and ridges [50]. Specifically, the collagen fibrils maintained native D-periodic banding patterns with a longitudinal repeat of ~ 64 nm with SACC and tannin dispersed between the fibrils [51]. The intact D-periodic pattern indicated that the collagen molecules were  (a and d), solo SACC tanned leather (b and e) and SACC-wattle combination tanned leather (c and f). Red dash frame in c indicates the SACC-tannin complexes. g Porosity and pore size distribution of tanned leathers. h Total pore area and average pore diameter of tanned leathers. i Schematic diagram of diffusion-binding-locking mechanism proposed in this combination tanning. The distribution of SACC-tannins in collagen was highlighted in multiple levels from collagen molecule to fiber bundle still stacked together in order, retaining their native conformation [52]. Therefore, we concluded that the complexes were incorporated into the fibrillar structure of collagen without disrupting their primary and conformational structures.
The pore size distribution of leather matrix including microfibrils (< 12 nm), fibrils (~ 100 nm), elementary fibers (1-3 µm) and fiber bundles (> 3 µm) was categorized by using a theoretical model [53]. Measurements based on the MIP confirmed that the pore size of bated pelt was mainly from 3.0 to 40 µm (Fig. 4g), which indicated that bating pelts were woven at the fiber bundle level. In contrast, after the introduction of SACC, the percentage of pore size (< 3.0 µm) increased from 1.6 to 12.2%, indicating that the SACC molecules were able to diffuse into the collagen fiber network and form bindings between the microfibril, fibril and elementary fiber. Whereas the pores in the range of 3.0-40 µm were almost unaffected. Thus, the SACC tanning led to the increased total porosity from 58.4 to 72.0%. Notably, SACC-tannin combination tanned leather exhibited a higher proportion in the range of 5.5 nm-3.0 µm and a lower proportion in the range of 3.0-40 µm. This gave rise to a decreased total porosity of leather (61.1%), and indicated the larger SACC-tannin complexes mainly distributed in space among collagen fiber bundles and bound to these bundles. Overall, SACC-tannin played a role in "locking" the multi-level structure of collagen to form a network structure. Moreover, the average pore diameters of leather decreased and the total pore areas increased after this combination tanning (Fig. 4h). We reasoned that the deposition of SACC and tannin in the collagen fibers can increase the small pore size (5.5 nm-3.0 µm) and form dense intertwining networks in the leather matrix. Therefore, we proposed a diffusion-binding-locking mechanism for this combination tanning (Fig. 4i), where SACC-tannins diffused into the hierarchical leather matrix and interacted with collagen molecules. The multiple levels of interaction prevented fibers from collapsing into the interstices, reduced the ability of collagen to shrink, and eventually improved the denaturation temperature [54].

Organoleptic properties and physical performance of the leathers
The organoleptic properties (i.e., softness, fullness, grain smoothness, grain compactness and color shade) determine the quality of leather products, which can be evaluated through haptic and visual feedback. The performance comparison was summarized in Fig. 5a, b. Condensed tannins outperformed hydrolyzed tannins in terms of fullness and grain compactness of tanned leathers. This is because condensed tannins have larger molecular weights and stronger astringency, and therefore are more effectively deposited in collagen fibrils to form hierarchical network structure [31]. Particularly, the flavanol polycondensation structure and strong tanning ability of wattle tannin endowed the leather with excellent softness and grain smoothness. Notably, the color shade of the leathers are as follows: Valonea > Bayberry > Quebracho > Chestnut > Wattle > Tara. Therefore, choosing tannins in this metal-free tanning systems can selectively meet the market requirements. For example, metal-free tanning system based on the combination of 10 wt% SACC and 25 wt% wattle can be optimized for manufacturing light-colored or bright-colored leather. We further evaluated the potential of the obtained leather for market requirements. As given in Table 1, the dispersing effect of wattle tannin on collagen fibers increased the softness of leather by ~ 1.1 mm. The combination tanned metal-free leather exhibited enhanced physical and mechanical properties (tearing load, elongation at 10 N, T s and bursting strength) compared to solo SACC tanned leather. For example, the introduction of wattle tannin increased the T s of leather from ~ 79 to ~ 92 °C, and the tearing load from ~ 102 to ~ 122 N. Furthermore, all the physical properties (elongation

Environmental assessment and cost analysis of our method
Heavy metals in leather are harmful to the ecological environment, and the metal ions commonly involved in leather tanning include Cr 3+ , Al 3+ , Zr 4+ , Ti 4+ , and Fe 3+ . Table 2 revealed the extractable metals from the leather products in the scenario where leather products contact human skins. It showed that Cr 3+ , Al 3+ , Zr 4+ and Ti 4+ were not detected in the acidic artificial sweat. Trace amount of Fe 3+ (0.48 mg/kg) was detected in leather, which may be caused by the leached Fe 3+ from the stainless-steel drum. The conventional chrome tanning process not only has the risk of toxic Cr ions, but also produces substantial wastewater containing neutral salts, which harms the aquatic environment. Compared with the wastewater from chrome tanning process, the Cl − content of the waste liquor produced by the SACC-tannin combination tanning process was significantly lower by 71.7% (Table 3). This is because no NaCl was used for the pickling process in our metal-free tanning system. Moreover, the reduced amount of Cr 3+ and Cl − also led to a lower TDS content in the waste of combination system. For example, BOD 5 /COD Cr is the index of biochemical degradation of wastewater. When the ratio of BOD 5 /COD Cr is higher than 0.3, the biodegradability of organic pollutants in wastewater can be classified as good [55]. BOD 5 /COD Cr value of wastewater in our combination tanning system was 0.35, suggesting the improved biodegradability. Therefore, the SACC-tannin combination tanning process is environmentally friendly, and the leather products can meet the development demands of metal-free leather.
We then evaluate the market viability of this tanning process including commercial value, cost effectiveness and sustainability [22]. In a case of 1000 kg limed cowhide, the total cost of chemicals for the SACC-tannin acid combination tanning and this tanning method (i.e., SACC pre-tanning, shaving and wattle tanning) are given in Table 4. Our tanning method exhibited a total chemical cost of ~ 459.1 $, which is 46.3% lower than that of the SACC-tannin acid process (~ 854.5 $) [26]. Together with the environmental assessment, the significant improvement of cost suggests our reported method as an attractive option for sustainable leather manufacture.  Table 2 The content of extractable metals in the metal-free finished leather ND = not detected, Quantification limit: quantification limit possible with ICP-OES, from EN ISO 17072-1-2019

Metal species Detected concentration Quantification limit
Cr (mg/kg) ND 10 Al (mg/kg) ND 100 Zr (mg/kg) ND 10 Ti (mg/kg) ND 10 Fe (mg/kg) 0. 48 25 Table 4 The cost of main chemicals used in the SACC-tannin acid and SACC-wattle combination tanning (1) The amount was calculated according to the weight 1000 kg limed cowhide. (2) The amount was calculated according to the weight 250 kg shaving leather

Conclusion
We reported a sustainable metal-free leather processing based on SACC and vegetable tannins. The results demonstrated that a unique combination tanning (i.e., SACC pre-tanning, shaving and wattle tanning) can significantly enhance the hydrothermal stability of the leather (T s ~ 92 °C). We revealed that SACC and wattle can evenly diffuse into the collagen fibrils, and bind to the collagen via synergistic effects including covalent, hydrogen and ionic bonding. We also revealed that SACCwattle complexes can "lock" the multi-level structure of leather matrix without destabilizing the conformational structure of collagen. Therefore, we proposed a diffusion-binding-locking mechanism, which provided new insights of tanning mechanisms. This novel eco-friendly metal-free tanning system can not only improve the organoleptic properties and physical performance of the leather products, but also can reduce environmental risks of chrome-containing wastes and improve biodegradability of tanning spent liquor. Of particular interest is that these leather products meet current Chinese standard requirements for shoe upper leather (QB/T 1873-2010), validating it as an attractive translational technology. We envision that this work is beneficial to the sustainable development of eco-leather manufacturing.
The solo tanning process of the vegetable tannins.