Valorization of tannery wastes: Lipoamino acid surfactant mixtures from the protein fraction of process wastewater

Valorization of tannery wastes: lipoamino acid surfactant mixtures from the protein fraction of process wastewater Mª Elena Bautista, Lourdes Pérez, M. Teresa García, Sara Cuadros, Agustín Marsal* Instituto de Química Avanzada de Cataluña, IQAC, CSIC, c/ Jordi Girona, 18-26, 08034 Barcelona, Spain Abstract The first stages of the transformation process of hides into leather (beamhouse process) generate an important waste in the tanning industry, since a considerable fraction of solubilised proteins ends up in waste water with the corresponding increase in contamination parameters, especially when the process is carried out without hair recovery (hair-pulping process). The objective of this work was the valorisation of this waste (the separated protein fraction) which conveniently hydrolyzed to amino acid level constituted the starting material for the production of biodegradable surfactants. The lipoamino acid surfactants were obtained by acylation of the amino acids from the protein hydrolisate. These surfactants were characterized and their physico-chemical and biological properties evaluated. They exhibit very low cmc values (about 40 mg/L). These surfactants are readily biodegradable and present an aquatic toxicity significantly lower than many common commercial surfactants derived whether from renewable or petrochemical feedstock. The mixtures of surfactants obtained are able to form oil/water emulsions that remain stable for at least one year. The results obtained in this work confirmed that it is possible the production of biodegradable and efficient lipoamino acid surfactant mixtures from the protein fraction present in beamhouse process wastewaters. This study constitutes a promising approach for the reduction of the pollution load from industrial tannery wastes and its valorisation as raw material for the production of surfactants with excellent environmental properties and good technical properties. Keywords: tannery waste; valorization; lipoamino acid surfactants; physico-chemical properties; biodegradation; ecotoxicity; *Corresponding author. Fax: +34 93 204 59 04 E-mail address: agusti.mars[email protected].es (A. Marsal) 1. Introduction Any industrial activity generates wastes to a greater o lesser extent and the growing demands of environmental respect in the production processes force companies to reuse the most of their wastes. Besides the respect for the environment, the reuse of wastes is of great importance and interest also from an economical point of view since the lower the amount of wastes generated, the lower the management cost in specialized plants. Moreover, the reuse of wastes can represent a sustainable solution to the lack of raw material to be used for the production of energy, fuel and chemicals than can be integrated again in the industry. The production of high added value materials as bioproducts, nanomaterials and bio-polymers starting from wastes underlines, even more, the interest for their reutilization [1-5]. The leather industry generates a considerable amount of wastes. However, the studies dealing with its potential valorization are scarce [6,7]. According to Sykes and Corning [8], each tonne of raw hide yields 200 kg of finished leather, 50 m3 of contaminated wastewater and the rest are solid wastes. Therefore, only 20% is transformed into useful material. According to Aloy [9], the main pollution load is produced in the beamhouse operation (stages before tanning process): 83% of BOD5 (five-day biochemical oxygen demand), 73% of COD (chemical oxygen demand) and 76% of toxicity. In another study, Portavella [10], states that for every 100 kg of dry raw sheepskin from Catalonia, 15 kg of solubilised proteins that are more or less structured (keratins, albumins, globulins, glycoproteins, etc) and that contain nearly 18% nitrogen will end up in waste water following beamhouse operations. Portavella [11] found that 70% of COD from the beamhouse operations is due to the skins themselves and that only 30% comes from added chemical products. Although the values of the parameters may vary as a function of the type of raw material treated, there is no doubt that the beamhouse operations produced most contamination, much of which is due to the solubilised components (proteins) of hides or skins. Therefore, separation of the dissolved protein fraction represent not only a way to significantly reduce the contamination of beamhouse wastewaters [12], with the economic savings this entails, but also provides a residue, the separated protein fraction, which conveniently hydrolyzed to amino acid level, could be used as the raw material for the production of surfactants. Protein hydrolysates from different sources have been used with the same aim [13, 14]. Other possible applications of the separated protein fraction from the beamhouse tannery process could be the production of liquid leaf fertilizer, solid fertilizer for soils or retanning agents for tannery. However, these possible applications will be the subject of future works. The use of this liquid waste of the leather industry as starting material for the production of surfactants is of great interest for several reasons: i) to reduce water pollution load, ii) to reduce the cost of waste disposal and iii) to provide an appropriate source of renewable raw materials not derived from petrochemical feedstocks. Amino acids are a very interesting raw material for the chemical preparation of environmentally friendly surfactants [15]. Twenty different α-amino acids are commonly present in proteins. Amino acid based surfactants are biodegradable and biocompatible compounds that can be prepared using natural compounds as fatty acids and amino acids as starting material [16,17]. Also, several commercial firms offer the possibility of finding amino acid surfactants on the market (series Amilite® from Ajinomoto; series Aminofoam™ from Croda; series Perlastan® from Schill&Seilacher, series Lamepon® from Basf; Nα-Lauroyl ethyl ester (LAE) from Lamirsa S.A [18], etc). One interesting strategy to reduce the tannery industrial pollution load as well as to obtain environmentally friendly compounds is to synthesize surfactants using the amino acids obtained by acid hydrolysis of the protein fraction recovered from the wastewater of soaking, unhairing-liming and conditioning operations of a hair-pulping (with hair destruction) beamhouse process. In the present work lipoamino acid surfactant mixtures using as raw material the protein fraction were obtained and their physico-chemical and biological properties were investigated. This study constitutes a promising approach to the reduction of the pollution load of industrial wastes and its possible reutilisation as starting material for surfactant production. 2. Experimental 2.1 Materials Dodecanoyl chloride, decanoyl chloride, dodecanoic acid, decanoic acid, pyreno, and squalane were purchase from Fluka. L-lysine, L-serine, L-proline, L-glutamic acid, glycine, L-leucine, L-arginine and sodium dodecyl sulfate were purchase from Sigma. Acetonitrile was purchase from Fisher Chemical. Acetone, n-hexane and ethanol absolute were purchase from Panreac. Trifluoroacetic acid, ndecane and hydrochloric acid were purchase from Merck. Sodium hydroxide was purchase from Carlo Erba. 2.2 Methods 2.2.1 Preparation of the amino acid mixture from the protein fraction. The protein fraction used in this work as starting material was obtained by acid precipitation (adding 2 M sulphuric acid solution up to the isoelectric point) [19] of the effluents of the unhairing-liming process and subsequent washings in a hair-pulping beamhouse process of hides. This protein fraction was subjected to a degreasing process with dichloromethane during 5 hours and subsequently hydrolyzed with 6 N HCl during 24 hours. The amino acid mixture was quantitatively determined in accordance with the AccQ-Tag Waters method [20] with previous derivatization with 6-AQC. A Waters 600 model with a 2487 UV detector was used for HPLC analyses. 2.2.2 Synthesis of surfactants from the amino acid mixture Surfactants were obtained by the introduction of a fatty acid residue, as an acid chloride, to the amino acids obtained from the protein hydrolisate in a strong alkaline aqueous medium. The mixture of amino acids obtained was dissolved in acetone/water (34/66) and NaOH was added until a pH of 10. This solution was filtered to remove the insoluble residue present in the medium. The residue was analyzed by thin-layer chromatography in order to check the absence of amino acids. Next, dodecanoyl chloride or decanoyl chloride were added dropwise maintaining the pH at 10 with NaOH. After adding the acid chloride, the reaction mixture was kept at -10 ºC for 3 hours. The progress of the reaction was checked by HPLC. Solvent was eliminated using a rotary evaporator and the sample was freeze-dried. Acylation reactions were carried out for different amino acid/acid chloride molar ratios. In some cases, salt formed in the reaction was removed with dry ethanol and subsequent filtration. In other cases, this purification process was not carried out (see Table 2). 2.2.3 Synthesis of pure Nα-acyl amino acid surfactants A series of standard Nα–acyl amino acid surfactants was prepared for the characterization of the surfactants synthesized in this work. The standard surfactants were prepared with those amino acids that were present at the highest percentage in the starting protein fraction (glycine, leucine, proline, arginine, glutamic acid, lysine and serine). Dodecanoyl chloride was selected as the acylating agent. The reaction was carried out under the above mentioned conditions. 0.5 g of pure amino acid were taken and the reaction was carried out at the 1:1 ratio. Once obtained, the surfactants were purified by repetitive washings with n-hexane. For identification purposes, the purified standard surfactants prepared for each amino acid were added one by one to the final product of the acylation reaction. HPLC calibration curves were prepared for every pure Nα–acyl amino acid surfactant and then, the concentration of the major components in the mixture was calculated. 2.2.4 High Performance Liquid Chromatography (HPLC) To check the progress of the acylation reaction and for identification purposes, HPLC analyses were performed on a VWRHitachi ELITE LaChrom system which consisted of an injection valve fitted with a 20 µl loop, and pump L-2200 and a UV-Vis detector L-2400 at 215 nm wavelength. A Lichrocart 2504, lichrospher 100 CN (5μm) column was used at room temperature. The flow-rate through the HPLC column was 1.0 ml/min. Elution was performed in a gradient system of water/acetonitrile. Eluent A was 0.1% (v/v) trifluoroacetic (TFA) in water, and eluent B was 0.085% TFA in water/acetonitrile 1:4. The initial composition A/B of the gradient was 75/25 (v/v), changing over 24 min to a final composition of 5/95. 2.2.5 Surface tension measurements The critical micelle concentrations (cmc) of the obtained surfactant mixtures were determined by surface tension measurements. Surface tension measurements were carried out at 25 ºC in accordance with the Wilhelmy plate method [21] using a K12 Krüss tensiometer. A stock solution of 1 mg/ml in water (pH = 6) was prepared from which different solutions were obtained for cmc determination. The cmc of decanoic and dodecanoic acids were also determined for the sake of comparison. 2.2.6 Spectrofluorimetry Fluorescence measurements were carried out with a Shidmadzu RF 540 spectrofluorometer. The fluorescence emission spectra of pyrene dissolved in surfactant aqueous solutions were recorded from 340 to 450 nm after excitation at 332 nm. Pyrene exhibits fine structure in 370–400 nm regions of the steady-state fluorescence emission spectra. The nature and the intensity are extremely dependent on the polarity of the environment. The ratio of the first to the third vibronic peaks, i.e., I1/I3, shows the greatest solvent dependency, and can be used to obtain the cmc of the surfactant solutions [21]. A pyrene aqueous solution of 10-6 M (pH = 6) was used. The different surfactant concentrations were prepared with this pyrene aqueous solution. All measurements were carried out at 25 ºC. 2.2.7 Qualitative phase behaviour Optical microscopy was used to study the qualitative phase behaviour of binary water/L-3 and water/C-2 systems as a function of temperature. Optical observations were performed according to the “flooding” (penetration) method of Lawrence [22]. A polarising microscope Reichert Polyvar® 2 Leica equipped with a hot stage was employed. A videocamera and a PC with Leica IM 500 software were used for the image capture. In the flooding experiment, water was allowed to diffuse into anhydrous surfactants placed between a slide and a cover slip. After a short time, gradients in composition were produced and different separated mesophases developed around the crystalline surfactant [23]. 2.2.8 Foaming properties: foaming capacity (FC) and foaming stability (FS) Foaming properties were measured using a modified Padmashree’s method [24-26]. Different amounts of surfactants (10, 20 and 30 mg) were mixed with 2 ml of distilled water (pH = 6) at 25 ºC in a graduated test tube. Purified and unpurified (with salts) surfactant samples were tested. Sodium dodecyl sulfate was used as control. Given its higher foaming capacity a lower amount (3 and 4 mg) was taken for the test. Solutions were continuously shaken by hand during 1 min. After 30 s shaking, the volume was measured. The foaming capacity (FC) was expressed as the percentage of volume according to the following formula: FC= ((Volume after stirring – Volume before stirring) /Volume before stirring)) *100 (1) The foam volume was recorded at 5, 30, 60, 120 and 180 min after shaking. Foaming stability (FS) was calculated using the following formula: FS= (Foam volume after a time “t” / Initial foam volume) *100 (2) 2.2.9 Emulsifying capacity To check the emulsifying capacity of the surfactant mixtures, they were dissolved in water and then oil was added, thus favouring the formation of Oil in Water (O/W) emulsions. Emulsions were prepared by adding dropwise 1 g of oil (decane or squalane) to aqueous solutions containing 10 or 20 mg of surfactant at 25 ºC. The volume of water was 0.2, 0.4 or 1 ml. During the addition of the oil, the samples were stirred with a Heidolph Reax 2000. When the addition of the oil was finished, the samples were left to stand. 2.2.10 Biodegradability assessment The biodegradability of the surfactants under aerobic conditions was evaluated according to the ISO-14593 CO2 headspace test [27]. This method allows the evaluation of the ultimate aerobic biodegradation (mineralization to carbon dioxide) of an organic compound in aqueous medium by measuring the increase in total inorganic carbon over time with respect to a blank without the addition of the test substance. The surfactants were tested at a concentration of 20 mg C/L. Samples were inoculated with activated sludge (10 mg dry solids/L) collected from a municipal wastewater treatment plant (Manresa, Barcelona) and then incubated in the dark at 22 ± 1 ºC in 250 mL sealed vessels (air headspace/liquid volume ratio, 1:2). Sodium n-dodecyl sulphate was used as reference substance. Three replicates of the surfactants, blank and reference substance were measured for each sampling day. The test ran for 28 days. Each sampling day, after injecting a sodium hydroxide solution to the vessels, shaking for 1 h and allowing settling, appropriate volumes were withdrawn by syringe from the liquid phase of each vessel and kept in small beakers carefully filled to the brim and covered with a cap to prevent CO2 exchange with the air. The concentration of inorganic carbon was determined in a carbon analyzer (Shimadzu TOC-5050). The biodegradation level was expressed as a percentage of the theoretical amount of inorganic carbon based on the initial amount of the test compound. 2.2.11 Aquatic toxicity assessment The aquatic toxicity determination of the obtained surfactants was carried out in accordance with the Daphnia magna method where the swimming incapability is the end point [28]. The pH of the medium was 8.0 and the total water hardness was 250 mg/L (expressed as CaCO3), with a Ca/Mg ratio of 4/1. Tests were performed in the dark at 20 ºC. Twenty daphnia, divided into four batches of five animals each, were used at each test concentration. The concentration range was first established in a preliminary test and 10 concentrations in a geometric series were tested for each surfactant. The percentage immobility at 48 h was plotted against concentration on a logarithmic-probability scale and a linear relationship was obtained. The Probit method was employed as statistical procedure to determine the IC50 (the estimated concentration to immobilise 50% of the daphnia after 48 h exposure) and the corresponding 95% confidence interval (CI). 3. Results and discussion 3.1 Amino acid composition of the protein fraction The amino acid composition obtained after the hydrolysis of the protein fraction separated by acid precipitation from the effluents of the unhairing-liming operation in a hair-pulping beamhouse process of hides is shown in Table 1. Amino acid percentages were determined in accordance with the AccQ-Tag method [20]. As observed, the major amino acids in the protein fraction were: glutamic acid + glutamine (14.12%), serine (9.46%), arginine (9.00%) and proline (7.08%). Notice that the fraction of neutral amino acids was the most abundant (64.21%) in the protein fraction. 3.2 Synthesis of lipoamino acid surfactant mixtures The main purpose of this work was the exploration of novel and advanced routes for the valorisation of a waste from the leather industry. The chemical procedure used to prepare surfactants from the mixture of amino acids was easy and very efficient. It consisted of the N-acylation of the amino groups of the amino acids with two different acid chlorides, decanoyl chloride and dodecanoyl chloride. This is a traditional synthetic method in which the reaction is carried out in a mixture of water/acetone, no organic waste is generated and it is not necessary to increase the temperature. The N-acylation reaction was carried out with different amino acid/acid chloride ratios (Table 2). A pondered molecular weight was calculated for the amino acid mixtures taking into account the amino acids and their percentages. Using the 1/0.5 amino acid/acid chloride ratio it was observed that part of the amino acids remained without reacting (Figure 1 B). When the percentage of acid chloride was increased up to a 1/0.75 ratio all the amino acids present in the mixture reacted (Figure 1 C). Because of that, the proportion of acid chloride was not further increased and the ratio 1/1 was not used. This behaviour indicates that the amino acid mixture also contained some salts from the hydrolysis process. It is important to emphasize that for each amino acid/ acid chloride molar ratio three replicates were performed and the chromatograms obtained presented always the same profile. Chromatograms for lipoamino acid surfactants with C12 alkyl chains are similar to those for lipoamino acid mixtures containing C10 alkyl chains being the retention time the only difference. Because of the enhanced hydrophobicity of the compounds with C12 alkyl chains regarding compounds containing C10 alkyl chains, the HPLC of the mixture obtained with dodecanoyl chloride contained peaks with retention times higher than those obtained with decanoyl chloride. The retention times of the surfactants obtained ranged from 9 to 22 minutes. Given that different amino acids were present in the starting material (Table 1), the obtained mixture contained surfactants with different retention times. The retention time of the surfactant depends on the hydrophobicity of the amino acids that form the polar head. In the case of basic amino acids with two amino groups, such as lysine or arginine, it is possible to obtain amphiphilic molecules with two alkyl chains. It is also possible that the acyl chloride reacts in some cases with the hydroxyl group present on serine and threonine. Because of that, peaks of the HPLC chromatograms at high retention times (>18 minutes) are expected to correspond to the surfactants containing two alkyl chains. The mixtures of lipoamino acid surfactants were not further purified because the aim of the work was to obtain surfactants with easy and clean technologies. The isolation of pure surfactants from these heterogeneous mixtures would require different purification methods with the use of huge quantities of solvents. The only additional process carried out in this study after surfactant synthesis was the removal of some inorganic salts. The acylation reaction produces salts, most of them can be removed with dry ethanol and subsequent filtration. With this objective, the sample was dissolved in dried ethanol and filtered through a 0.22 µm porous membranes. As expected, the chromatographic peaks of the surfactant mixtures after removing salts were sharper than those corresponding to the lipoamino acid surfactants containing salts. 3.3 Critical micelle concentration of the surfactant mixtures In the lipoamino acid surfactant mixtures, the alkyl chain is linked to the amino group of the amino acid through an amide bond. It means that the polar groups of the surfactant mixture contain a carboxylic group that can be neutral or negatively charged. 4. Conclusions Lipoamino acid surfactant mixtures were obtained by acylation of the amino acids obtained from the acid hydrolysis of the protein fraction present in the waste waters of the tannery beamhouse processes. The surfactant mixtures from this type of waste show very low cmc values indicating that these surfactants form aggregates at very low concentrations. Moreover, they are very efficient in reducing the surface tension of water. The mixtures obtained form lamellar liquid crystal structure and very stable O/W emulsions and foams. In addition, these surfactants are readily biodegradable and result to be non-toxic or only slightly toxic to the aquatic environment. Consequently, bearing in mind their physical-chemical and environmental properties the surfactant mixtures could be used as green solubilizers, green emulsifiers or foaming agents in different industrial applications. Our results confirm that it is possible the valorisation of a waste of the tanning industry that entails the reduction of the pollution load from this sector. This approach also contributes to save fossil resources such as crude oil and natural gas. Acknowledgements The authors are grateful to the Spanish Ministry of Economy and Competitiveness for the financial support given through the CTQ2013-41514P, CTQ2013-43029P and MAT2012-38047-C02-02 Projects. References [1] A. 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Minutes 0 5 10 15 20 25 mAU -50 0 50 100 150 200 250 300 350 400 450 500 mAU -50 0 50 100 150 200 250 300 350 400 450 500 UV reaccion 6 C12 inicial Retention Time Minutes 0 5 10 15 20 25 mAU -50 0 50 100 150 200 250 300 350 400 450 500 mAU -50 0 50 100 150 200 250 300 350 400 450 500 UV reaccion 6 C12 FINAL Retention Time Minutes 0 5 10 15 20 25 mAU 0 50 100 150 200 250 300 350 mAU 0 50 100 150 200 250 300 350 UV R4 Retention Time Fig. 1. HPLC chromatogram corresponding to the initial mixture of amino acids (A), lipoamino acid mixture L-1 (B) and lipoamino acid mixture L-2 (C). 10 20 30 40 50 60 70 80 Surface tension (mN/m) Concentration (g/L) 0,01 0,1 1 1E-3 0,01 0,1 1 1,0 1,2 1,4 1,6 I1 / I 3 Concentration (g/L) Fig. 2. Influence of the amount of acid chloride in the acylation reaction on the cmc in water (pH 6) at 25ºC of the L-1(▲) and L-2 (■) lipoamino acid surfactant mixtures (A: Surface tension; B: Fluorescence). 1E-3 0,01 0,1 1 1,0 1,2 1,4 1,6 I1 /I3 Concentration (g/L) 20 30 40 50 60 70 80 1E-3 0,01 0,1 1 Surface tension (mN/m) Concentration (g/L) Fig. 3. Influence of the length of the hydrocarbon chain on the cmc in water (pH 6) at 25ºC of the L-2 (■) and C-2 (●) lipoamino acid surfactant mixtures (A: Surface tension; B: Fluorescence). 1E-3 0,01 0,1 1 20 25 30 35 40 45 50 55 60 65 70 Surface Tension (mN/m) Concentration (g/L) Fig. 4. Influence of the salt removal on the cmc in water (pH 6) at 25ºC of the L-2 (■) and L-3 (▲) lipoamino acid surfactant mixtures (A: Surface tension; B: Fluorescence). 0,01 0,1 1 1,0 1,2 1,4 1,6 I1 / I 3 Concentration (g/L) Table 2. Abbreviations used to name the obtained surfactant mixtures, N-acylation reaction conditions and critical micellar concentration (cmc) values of the lipoamino acid surfactant mixtures obtained by surface tension and fluorescence measurements Lipoamino acid surfactant mixture abbreviations Alkyl chain Amino acid/acid chloride molar ratio Salt Removal cmc (surface tension) (g/L) cmc (fluorescence) (g/L) L-1 C12 1/0.5 No 0.13 0.08 L-2 C12 1/0.75 No 0.09 0,06 L-3 C12 1/0.75 Yes 0.03 0.04 C-1 C10 1/0.5 No 0.24 0.24 C-2 C10 1/0.75 No 0.12 0.15 Table 3. Critical micellar concentration (cmc), surface tension at the cmc (γcmc) and the concentration necessary to reduce by 20 mN/m the surface tension of water (C20). Values of lipoamino acid surfactant mixtures obtained by surface tension measurements in water at 25ºC. Surfactant mixture cmc (g/l) γ cmc (mN/m) C 20 (g/l) L-1 0.135 28.4 0.038 L-2 0.094 29.2 0.015 L-3 0.034 28.1 0.004 C-1 0.24 28.3 0.022 C-2 0.12 32.8 0.021 Table 4. Foaming capacity (%) of L-2, L-3 and SDS in water at at 25ºC. Surfactant mixture Concentration (mg / ml) pH Foaming Capacity (%) L-2 5 7 275 L-2 10 7 300 L-2 15 7 325 L-3 10 7 325 L-3 15 7 475 SDS 1,5 7 300 SDS 2 7 400 Table 5. Composition of the emulsions investigated Emulsion Surfactant Mixture Water content (ml) Oil Name Content (mg) Type Content (g) E1 C-1 20 1 Decane 1 E2 C-1 10 0.4 Decane 1 E3 C-1 10 1 Squalane 1 E4 C-2 10 1 Squalane 1 E5 L-2 10 0.2 Decane 1 E6 L-2 10 0.4 Decane 1 E7 L-3 10 0.2 Decane 1 E8 L-3 10 0.4 Decane 1 Table 6. Acute toxicity of lipoamino acid surfactant mixtures on Daphnia magna after a 48 h exposure time expressed as the EC50 value and its corresponding 95% confidence interval (95 % CI). Surfactant mixture EC50 (mg/L) 95% CI (mg/L) C-2 245 196-326 L-2 69 43-99 Table 7. Ecotoxicity hazard classification of the surfactant mixture on the basis of the EC50 values of the Daphnia magna test Surfactant mixture Acute toxicity EC50 US fish and Wildlife services OECD C-2 >100 mg/L Practically non toxic --- L-2 10-100 mg/L Slightly toxic Acute Toxicity III (harmful to aquatic life)