Lignosulfonate Research Articles

Application of lignosulfonate as the growth promotor for freshwater microalga Euglena gracilis to increase productivity of biomass and lipids

Biomass derived supplements have been recognized as environment-friendly and cost-effective sources of stimulants which can promote growth and valuable metabolites biosynthesis of microalgae. In this research, effect of lignosulfonates (LIGNs) on the growth, cell morphological changes, and valuable products accumulation of microalga Euglena gracilis was explored. At the optimal concentration of 5000 mg·L−1, LIGNs could promote the growth of E. gracilis up to 1.95 folds, and increase the yield of chlorophyll a, chlorophyll b and carotenoids by 3.50, 1.59 and 3.48 times, respectively. Cell morphological changes in aspect ratio and size caused by LIGNs were also observed. Fourier transform infrared spectroscopy measurements followed by multivariate analysis revealed difference in metabolic patterns and prominent metabolites with LIGNs treatment. LIGNs improved the lipid yield since both cell density and lipid content increased, which could contribute to the biofuel production in the future.

Acid hydrolysis of the waste newspaper: Comparison of process variables for finding the best condition to produce quality fermentable sugars

Environmentally hazardous solid waste is renewed to the feedstock of bio-fuel through the recovery of good quality fermentable sugars by acid hydrolysis of the waste newspapers. Conflicting effects of the variables of acid hydrolysis on the yield of fermentable sugars; and that on the generation of undesired inhibitors to subsequent enzymatic hydrolysis for the production of bio-fuel are compared to select the best condition of hydrolysis. The roles of temperature, acid concentration, the ratio of inorganic to organic acids on the yield of desired total sugars (TS) and lignosulfonate (LS), and that on the reduction of inhibitors such as 5-hydroxymethyl furfural (HMF) and acetic acid with time are studied. The hydrolysis experiments are performed at two different temperatures (80 °C and 121 °C), in the acid concentration range of 0.25–1 % (w/v) and with different ratios of two types of acids. The maximum recovery of TS is obtained as 17 % (w/w) at an acid concentration of 1% (w/v), the temperature of 121⁰C at a time of 60 min. Both of HMF and acetic acid are produced only at 121⁰C. The best condition of acid hydrolysis is found as the temperature of 121⁰C, a mixture of sulphuric acid to citric acid of the ratio 3:1 at a hydrolysis time of 60 min for higher values of desired TS and LS with a manageable production of inhibitors. The finer tuning of the hydrolysis variables to produce quality fermentable sugars is promising for the economical production of bio-fuel.

In-situ growth of graphene on carbon nanofiber from lignin

Design and synthesis of heterostructured carbon nanofibers is significant for applications in the field of optoelectronics, conductors and catalytic devices. Although lignin has been widely used as building blocks for fabricating nanofibers, it is still very challenging to synthesize conducting heterostructured carbon nanofibers without using additional catalysts. Herein, we first in-situ grew graphene grafted carbon nanofibers (G/CNFs) using calcium lignin sulfonate as starting materials without addition of catalysts. Through the electrospinning, pyrolysis and washing procedure, the as-spun nanofibers are converted into the flexible G/CNFs films. The in-situ formed CaS nanoparticles serve as catalysts for the growth of graphene on the surface of the CNFs, when the carbonization temperature is over 1100 °C. The as-prepared G/CNFs films exhibit superior electronic conductivity of 2377.9 S m−1, which is comparable to the values reported in the literature. As a proof-of-concept, the as-fabricated lithium-sulfur battery exhibits a high discharge of 808.7 mAh·g−1 after 600 cycles at 0.2 C by using the G/CNFs membrane as stand-alone and flexible sulfur cathode scaffolds. This work represents a promising new approach for designing heterostructured graphene/CNFs using low-cost lignin precursors, and provides significant insights for the sustainable development of CNFs in numerous applications.

Very Strong, Super-Tough, Antibacterial, and Biodegradable Polymeric Materials with Excellent UV-Blocking Performance.

In this work, inspired by the dynamic sacrificial hydrogen bonds in biological materials, a very strong, super-tough, antibacterial, and cost-effective biodegradable poly(vinyl alcohol) (PVA) nanocomposite material was developed by incorporating the nanoscale antibacterial agent TA@LS-Ag. TA@LS-Ag was prepared from the green biomass tannic acid (TA) and sodium lignosulfonate (LS), and was facilely incorporated into the PVA matrix with a homogeneously interspersed nanoparticle size of about 20 nm. The PVA nanocomposite film with 2 wt % addition of TA@LS-Ag achieved the highest specific toughness of 262 J g-1 among the PVA-based films to date, which is far higher than that of natural spider silk (150-190 J g-1 ), as well as a very high tensile strength of 131.6 MPa. The excellent tensile strength and superior toughness were attributed to synergy of the nanophase separation structure and the intense hydrogen-bonding interactions between the nanoparticles and PVA matrix. The PVA/TA@LS-Ag nanocomposite films exhibited good antibacterial properties, despite the extremely low silver content (0.032-0.32 wt ‰). TA@LS-Ag also endowed the PVA films with excellent antioxidant and UV-shielding performance. As the biomass-derived LS and TA and the PVA matrix are all biodegradable, this work offers a facile strategy for preparing high-performance antibacterial and biodegradable polymeric materials.

Novel and eco-friendly flame-retardant cotton fabrics with lignosulfonate and chitosan through LbL: Flame retardancy, smoke suppression and flame-retardant mechanism

Bio-based materials have been noticed for the continuous environmental pollution and resource shortage. In this paper, lignosulfonate (LS) and chitosan (CS) were selected to be flame-retardants. In the vertical flame test (VFT), LS/cotton-17.1 wt%, CS/LS/cotton-17.0 wt%, and CS/LS/cotton-25.2 wt% obtained lower afterflame times than that of uncoated cotton fabrics, while LS/cotton-17.1 wt% had serious afterglow time of 70 s. However, the afterglow time of CS/LS/cotton-25.2 wt% was 11 s, which decreased owing to the increased amount of CS. LS/cotton-17.1 wt%, CS/LS/cotton-17.0 wt%, and CS/LS/cotton-25.2 wt% presented limiting oxygen index values of 24.7%, 25.0%, and 26.0%, respectively. The scanning electron microscope images of char residues after the VFT showed that CS and LS formed an intumescent flame-retardant system, and cotton fibers remained intact structure. Meanwhile, the results of thermogravimetric analysis suggested that flame-retardant cotton fabrics can generate stable char residues. In N2 atmosphere, CS/LS/cotton-25.2 wt% generated 27.1% stable char residues. Moreover, the addition of CS/LS decreased the heat release rate and total heat release values and this system performed well in smoke suppression. The flame-retardant mechanism of the system might belong to gas-phased because more non-combustible products were generated in the thermal degradation process.

Adsorption behavior of two glucanases on three lignins and the effect by adding sulfonated lignin

The adsorption behaviors of two glucanases, TvEG and TrCel7A, on three lignins were investigated. Three lignins were isolated from raw aspen and its pretreated solid residue. The isolated lignins were labeled as Asp-MWL, DA-MWL (pretreated by dilute acid), and GL-MWL (pretreated by green liquor), respectively. The surface properties of lignins and spin-coated lignin films were characterized by zeta potential, atomic force microscope (AFM) and contact angle. The enzyme adsorption behavior was monitored by quartz crystal microbalance (QCM) and fluorescence spectrometer. TlCel7A had similar adsorption capacities on the three lignin films but were higher than those of TvEG. The TrCel7A adsorptions on the three lignin films were affected by synergistic effect of electrostatic and hydrophobic interaction while the TvEG adsorptions on the three lignin films were mainly dominated by hydrophobic action. The adsorption capacities of TlCel7A and TvEG on the three lignin films were decreased by adding SL. Plausible explanation was that the SL and glucanase formed a complex with more negative charges, which suppressed the adsorption of glucannase on lignin through electrostatic repulsion. It also explained the improved enzymatic hydrolysis efficiency of lignocellulose upon adding SL.

Lignin derivatives stabilizing oil-in-water emulsions: Technological aspects, interfacial rheology and cytotoxicity

The emulsifier ability to stabilize oil-in-water emulsions of two industrial lignins (calcium lignosulfonate and kraft lignin) were evaluated and compared with whey protein isolate that is a widely used emulsifier, as well as their cytotoxic effect in Caco-2 and HT-29 cell lines. The surface activity of calcium lignosulfonate (LG) and kraft lignin (LA) was evidenced by the reduction of interfacial tension between oil-water phases, and interfacial activity was lower and presented a faster decrease than those obtained for whey protein. This behavior was associated to the great driving force promoted by the high number of hydrophobic groups on the lignins. The emulsions with lignins presented smaller diameter (0.717 μm) and higher zeta potential than emulsion produced with WPI (1.065 μm). In addition, WPI emulsions were the most unstable after 28 days of storage. Among lignins, emulsions with kraft lignin were the most stable due to differences in the functional groups on these lignins. The cytotoxicity of lignins towards HT-29 and Caco-2 cells showed a dose-response relationship, determining non-cytotoxic concentrations for emulsifiers that can produce stable emulsions. In order to encourage the industries to use these lignins in polymeric form, these results present the safety level for both lignins capable of producing a stable emulsion, in addition to presenting characteristics of these two biopolymers that will influence the different applications.

Alkaline sulfonation and thermomechanical pulping pretreatment of softwood chips and pellets to enhance enzymatic hydrolysis

To assess the impact of alkalinity on sulfonation and the enzyme-mediated hydrolysis of softwood cellulose, Lodgepole pine chips were impregnated with 8% sodium sulfite and increasing loadings of sodium carbonate before thermomechanical pulping. It was apparent that alkali addition enhanced lignin sulfonation with an additional 4% loading of sodium carbonate proving optimal. TEM indicated that sulfonation predominantly occurred within the secondary-cell-wall lignin, increasing cellulose accessibility to the cellulase enzymes. Although increasing alkalinity did not significantly enhance lignin sulfonation, likely due to the lower acetyl content of the softwood chips, it increases mannan solubilization. Despite their smaller particle size, softwood pellets were more poorly sulfonated, probably due to their higher lignin content and lower amount of acid groups. This more condensed lignin structure was confirmed by 2D-NMR and GPC analyses which indicated that the EMAL derived from softwood pellets contained less native β-O-4 linkages and had a higher molecular weight.

A novel strategy to prepare COFs based BN co-doped carbon nanosheet for enhancing mechanical performance and fire safety to PVA nanocomposite

N doped carbon nanosheet and B N co-doped carbon (BNC) nanosheet are first prepared through exfoliation of carbon of Schiff based covalent organic frameworks (COFs) under ultrasound using sodium lignin sulfonate as the intercalation agent and modifier. Then poly (vinyl alcohol) (PVA)/BNC nanocomposite with enhanced thermal oxidation stability, good mechanical performances and enhanced flame retardancy has been prepared through solution blending. The temperature at 5 wt% weight loss and peak heat release rate of PVA loaded with 4 wt% of BNC nanosheet increase by 31.9 °C and 57.3% compared with that of untreated PVA, respectively. A new fire hazard evaluation system based on analytic hierarchy process (AHP) is raised to comprehensively estimate fire risk of prepared sample. It is deduced from AHP that PVA loaded with 4 wt% BNC nanosheet shows the lowest fire risk (77.9 score). The effect of BNC nanosheet on pyrolysis mode of PVA in gaseous and condensed phase has also been investigated to figure out the reason for low fire risk of treated PVA.

Generation of sulfonated kraft lignin acrylic acid polymer and its use as a flocculant

ABSTRACT The main purpose of this work was to generate sulfonated lignin-acrylic acid polymer and to study its flocculation performance for aluminum oxide suspension. The sulfonation pretreatment enhanced the charge density and solubility of kraft lignin facilitating its subsequent polymerization with acrylic acid. The maximum anionic charge densities of 3.3 and 2.5 meq/g and maximum molecular weights of 898 and 330 kg/mol were obtained for purified sulfonated lignin – acrylic acid and purified kraft lignin–acrylic acid polymers, respectively. The elimination of polyacrylic acid (i.e., homopolymer) from the lignin derivatives improved the efficiency of lignin products as flocculants for aluminum oxide particles.

Preparation of semicokes by medium–low temperature pyrolysis of long flame coal and their adsorption performance on sodium lignin sulfonate dispersant for high concentration coal water slurry

To get a high concentration of coal water slurry (CWS) for gasification application, semicokes were prepared by pyrolysis of Dongrong long flame coal (DRC) at medium–low temperature. The obtained semicokes were characterized by thermogravimetry, Fourier transform infrared spectrometry, X-ray photoelectron spectroscopy, X-ray diffraction, scanning electron microscope, and nitrogen adsorption-desorption methods. After preparation, the adsorption experiment of sodium lignin sulfonate (NaLS) on these semicokes was also carried out. It was found that medium–low temperature pyrolysis have changed the pore structure, surface properties, and molecular structure of DRC, which increased the saturated adsorption capacity of semicokes for NaLS dispersant. The enhanced adsorption capacity for dispersant can significantly reduce the apparent viscosity of CWS and increase its concentration. Under the optimized pyrolysis conditions, the maximum solid concentration of CWS prepared with as-prepared semicokes reached up to 68.09% when the dosage of NaLS disperdant was 1%, which met the gasification application of CWS. Such medium–low temperature pyrolysis method is an effective way to large-scale development and utilization of low rank coal through modifying their physicochemical properties.

Pickering/Non-Pickering Emulsions of Nanostructured Sulfonated Lignin Derivatives.

Sulfoethylated lignin (SEKL) polymeric surfactant and sulfoethylated lignin nanoparticles (N-SEKL) with a size of 750±50 nm are produced by using a facile green process involving a solvent-free reaction and acidification-based fractionation. SEKL forms a liquid-like conventional emulsion with low viscosity that has temporary stability (5 h) at pH 7. However, N-SEKL forms a gel-like, motionless, and ultra-stable Pickering emulsion through a network of interactions between N-SEKL particles, which creates steric hindrance among the oil droplets at pH 3. The deposition of SEKL and N-SEKL on the oil surface is monitored by a using a quartz crystal microbalance. Experimentally, the formation of emulsions at pH 7 is found to be reversible owing to the low adsorption energy ΔE of SEKL on the oil droplet (ΔE≈15 kB T), which is determined with the help of three-phase contact-angle measurements. However, the high desorption energy (ΔE≈6.0×105 kB T) of N-SEKL makes it irreversibly adsorb on the oil droplets. SEKL is too hydrophilic to attach to the oil interface (ΔE≈0) and thus does not facilitate emulsion formation at pH 11. Therefore, it is feasible to apply SEKL for the formulation of Pickering or non-Pickering emulsions in the form of nanoparticles or polymeric surfactants, depending on the targeted application.

Effect of lignosulfonate on the adsorption performance of hematite for Cd(II)

Lignin is a precursor of humus in soil and sediment. Lignin can be separated from vascular plants in the form of lignosulfonate via pulping processes. On the other hand, composites of iron oxide and organic matter can adsorb heavy metals, and thus influence the migration of these heavy metals in the environment. In this paper, a hematite/lignosulfonate composite (HLS) was prepared via coprecipitation to compare the adsorption performance of hematite (α-Fe2O3) toward Cd(II) before and after the incorporation of lignosulfonate (LS). The HLS is found to exhibit a weakly crystalline structure and possess a large number of nanoscale particles. Specific surface area of HLS (291.97 m2/g) is about 11 times that of α-Fe2O3, and the pore volume of HLS (0.22 cm3/g) is twice that of α-Fe2O3. The adsorption of Cd(II) is well illustrated by the pseudo-second-order adsorption kinetics and the initial adsorption rate (h) of HLS is 13.83 times that of α-Fe2O3. The maximum adsorption capacities are significantly improved from 4.89–6.35 mg/g (α-Fe2O3) to 39.03–53.65 mg/g (HLS). A greater affinity and more favorable association between Cd(II) and HLS is observed via fitting models. The incorporation of LS provides HLS with significantly better adsorption properties toward Cd(II) than α-Fe2O3, as is further confirmed by FT-IR and XPS characterization. Fe-O-O-H and Fe-O-H structures as well as more hydroxyl groups are observed, which promote the adsorption performance since the process are mainly influenced by complexation via coordination bonds.

Carbonized lignosulfonate-based porous nanocomposites for adsorption of environmental contaminants

Carbon-based adsorbents possess exceptional adsorption capability, making them an ideal platform for the remediation of environmental contaminants. Here, we demonstrate carbonized lignosulfonate (LS)-based porous nanocomposites with excellent adsorption performance towards heavy metal ions and cationic dye pollutants. Through microwave-assisted hydrothermal carbonization, a green approach was employed to carbonize lignosulfonate to carbon spheres. The LS-derived carbon spheres were then oxidized into nanographene oxide (nGO) carbon dots. A facile two-step procedure that involved the self-assembly of nGO and gelatin into a hydrogel precursor coupled with freeze-drying enabled the construction of three-dimensional (3D) free-standing porous composites without the use of organic solvents or chemical crosslinking agents. The favorable pore structure and abundance of surface functional groups on the nGO/gelatin porous composite proved to substantially facilitate the adsorption of Cu(II) in comparison to conventionally-used activated carbon. Further enhancement of adsorption performance was achieved by introducing additional surface functional groups through a non-covalent functionalization of the porous composite with lignosulfonate. The presence of negatively-charged sulfonate groups increased the Cu(II) equilibrium adsorption capacity (66 mg/g) by 24% in comparison to the non-functionalized nGO/gelatin counterpart. Both functionalized and non-functionalized composites exhibited significantly faster adsorption rates (40 min) compared to many graphene- or GO-based adsorbents reported in literature. In addition to the adsorption of heavy metal ions, the composites also demonstrated good adsorption capacity towards cationic dyes such as methylene blue. This paves the way for a high value-added application of lignin in environmental remediation and opens up new possibilities for the development of sustainable materials for adsorption and water purification.

Nanofiltration Membranes via Layer-by-layer Assembly and Cross-linking of Polyethyleneimine/Sodium Lignosulfonate for Heavy Metal Removal

Layer-by-layer (LbL) assembly technology is a facile method for constructing thin film composite membrane. Herein, a novel nanofiltration (NF) membrane was prepared by LbL assembly of polyethyleneimine (PEI) and sodium lignosulfonate (LS) followed by cross-linking. The surface composition, morphology, and property of PEI/LS bilayer were detailedly investigated by FTIR/ATR, XPS, SEM, AFM, and water contact angle test. The PEI/LS bilayer full of amino and hydroxyl groups presents increased roughness and improved hydrophilicity. Moreover, the NF performance of PEI/LS LbL assembly membranes can be modulated by bilayer number, polyelectrolyte concentration, and salt content. The water flux reduced while the salt rejection greatly improved as increasing the bilayer numbers, PEI concentration, or NaCI content. More than 95% MgSO4 and MgCI2, as well as 80% NaCI can be rejected by a NF membrane prepared by 6 PEI/LS bilayers, 1 wt% PEI, 0.5 wt% LS, and 1 mol/L NaCI. Furthermore, this NF membrane can be used to remove more than 95% heavy metal ions (Cd2+, Zn2+, Mn2+, Cr2+, Cu2+, and Ni2+). This work proposed a promising NF membrane by using PEI/LS as low cost polyelectrolytes and facile LbL assembly method, which should receive much attention in water purification.

Efficient Toluene Adsorption on Metal Salt-Activated Porous Carbons Derived from Low-Cost Biomass: A Discussion of Mechanism.

Porous carbons (PCS) derived from sodium lignin sulfonate were activated by four common metal salts. The samples exhibit distinct characteristics of irregular, sunflower-like, interconnected sheet, and tine block morphologies under the impact of NaCl, CaCl2, ZnCl2, and FeCl3, respectively (PCS-MClx). Surprisingly, the maximum and minimum specific surface areas are 1524 and 44 m2/g corresponding to PCS-ZnCl2 and PCS-NaCl. All of the samples have plentiful functional groups; herein, PCS-NaCl and PCS-FeCl3 are detected with the highest O and S contents (11.85, 1.08%), respectively, which signifies sufficient active sites for adsorption. These porous materials were applied in toluene adsorption from paraffin liquid and matched the Langmuir isotherm models well. Thus, the activation mechanism was discussed in detail. PCS-MClx has a completely different pyrolysis behavior according to thermogravimetry/derivative thermogravimetry (TG/DTG) analysis. It is speculated that H[ZnCl2(OH)] would have an etching effect on the carbon structure of PCS-ZnCl2, and HCl or H2SO4, resulting from FeCl3 hydrolysis and a reduction reaction, would be corrosive to the sodium lignin sulfonate (SLS) surface. Each metal salt plays a different role in activation. The devised method for the synthesis of porous carbons is green and economical, which is suited to mass production.

Preparation and characterization of biodegradable lignin-sulfonate nanoparticles using the microemulsion method to enhance the acetylation efficiency of lignin-sulfonate

In this study, a novel method is presented for producing lignin-sulfonate nanoparticles. Then, the effect of produced nanoparticles is investigated on enhancing the acetylation efficiency. For these purposes, lignin-sulfonate was isolated from black-liquor of pulp-and-paper mill wastewater. Next, lignin-sulfonate nanoparticles were obtained using the oil-in-water (O/W) microemulsion, followed by modification of micro/nano-lignin-sulfonate particles. The physical, chemical, and morphological properties of lignin sulfonate micro/nanoparticles and modified forms of both samples were analyzed using FTIR, DLS, FE-SEM, AFM, 1H NMR, and 13CNMR analyses. Surface morphology revealed that the nanoparticles were homogenized and spherical with an average diameter of 25.5 nm. The chemical structure of the nanoparticles was similar to that of the microparticles. On the other hand, the chemical structure of acetylated lignin-sulfonate was slightly different from that of unmodified samples. The results also showed that the production of nano-lignin-sulfonate increased the acetylation efficiency and reduced the time and temperature of acetylation.

Eco-friendly remediation for lead-contaminated riverine sediment by sodium lignin sulfonate stabilized nano-chlorapatite

Achieving high immobilization performance to reduce the toxicity and bioavailability of heavy metals is critical for the remediation of heavy metals contaminated sediment. Herein, a novel chlorapatites (SNClAP) with high stability and dispersibility is successfully synthesized using sodium lignin sulfonate (SLS) as dispersant. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) characterization demonstrated that SNClAP possessed an internal layered structure, and exhibited better dispersibility, which was conducive to the immobilization of lead (Pb) in sediment. Besides, SNClAP showed excellent remediation performance towards Pb contaminated sediment, with the highest Pb immobilization efficiency of 80.29%, which was much higher than that of pristine chlorapatite (ClAP) (45.62%). The results of available phosphorus (AP) showed that the better dispersion of chlorapatites could make soluble phosphorus (P) released by chlorapatites more fully combine with Pb to form the extremely stable Pb10(PO4)6Cl2 through precipitation, thus reducing the retention of P in sediment to greatly lower the risk of eutrophication. Therefore, SLS, a by-product of pulping and papermaking industry, was used to synthesize SNClAP, which could not only improve the remediation efficiency of Pb-contaminated sediments, but also greatly promote the recycling of waste, thereby achieving harmony between materials and ecological environment.

Ultrafast gelling using sulfonated lignin-Fe3+ chelates to produce dynamic crosslinked hydrogel/coating with charming stretchable, conductive, self-healing, and ultraviolet-blocking properties

Developing an ecological and economical method for fabrication and application of polymer hydrogels is important for the hydrogel/coating electronics. Here, a dynamic oxidation and coordination system composed of sulfonated lignin (SL) and Fe3+ was introduced to assemble multifunctional Fe-SL-g-polyacrylic acid (PAA) hydrogel at 20 ℃ in a time scale of minutes instead of hours. The SL-Fe3+ system can rapidly activate the ammonium persulfate (APS, initiator), and then it instantaneously provides abundant semiquinone and hydroxyl radicals to accelerate the formation of polymer chains. Moreover, the functional SL-Fe3+ chelates can also reversibly crosslink the polymer chains to form multifunctional hydrogels. Surprisingly, the resultant hydrogel obtained charming extension ratio (up to 1680%), conductivity (up to 7.0 × 10-2 S·m−1), and adhesive strength (up to 36.4 kPa). This technology was also applied to rapidly fabricate a transparent (up to 81%, thickness: 2 mm), UV-blocking (up to 99.7%, thickness: 2 mm), adhesive, and conductive hydrogel coating on substrates. Besides, the dynamic coordination inside the hydrogel imparts desirable self-healing capabilities to damaged network, with up to 98.5% recovery in conductivity and 85.7% in extension ratio. In short, this technology based on the sustainable SL-Fe3+ system greatly facilitates the rapid and low-cost preparation of multifunctional hydrogel/coatings, which have promising industrial applications in human–machine electronics.

Stepwise extraction of gold and silver from refractory gold concentrate calcine by thiourea

Cyanide leaching, as a typical traditional method, hardly achieves the cleaner production of gold due to high consumption of auxiliary materials, inefficient production and heavy environmental pollution. In this work, stepwise thiourea leaching was used to extract gold and silver from refractory gold concentrate calcine. In the first stage, gold and silver extractions of 84.42% and 44.15% are obtained by thiourea leaching without any additive, respectively, which are further enhanced to 88.71% and 52.65% by adding the additives of lignin sulfonate sodium, urea and iron ion. The spent thiourea can be recycled to leach next batch of calcine after the treatment of replacing gold by iron powder. In the second stage with mechanical activation, higher gold and silver extractions are achieved 96.51% and 70.43%, respectively. Furthermore, a pilot-scale test is carried out to prove the reliability of this technology, which is hopefully adopted in industry to extract gold and silver efficiently and cleanly