Calix[n]arene Carboxylic Acid Derivatives as Regulators of Enzymatic Reactions: Enhanced Enantioselectivity in Lipase-Catalyzed Hydrolysis of (R/S)-Naproxen Methyl Ester

Candida rugosa lipase was immobilized with a sol–gel encapsulation procedure in the presence and absence of a calix[n]arene carboxylic acid derivative grafted onto magnetic nanoparticles or in the presence of the calix[n]arene carboxylic acid derivative with Fe₃O₄ magnetic nanoparticles as an additive. Through the enantioselective hydrolysis of racemic naproxen methyl ester and the hydrolysis of p-nitrophenylpalmitate, the relative enzyme activity was evaluated and tested. These results show that the encapsulated lipase without supports has lower conversion and enantioselectivity compared to the Calix[n]COOH-based encapsulated lipase. It has also been observed that the Calix[4]COOH-based encapsulated lipase has excellent enantioselectivity (enantiomeric ratio (E)?>?400) as compared to encapsulated-free lipase enantioselectivity (E?=?137), and it also has an enantiomeric excess value of ~98 % for S-naproxen.     

Layer-by-Layer Self-Assembly Immobilization of Catalases on Wool Fabrics

A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V _max values were found to be 2,500?±?238 U/mg protein for the free catalase and 1,000?±?102 U/mg protein for the immobilized catalase. The K _m value of free catalase (11.25?±?2.3 mM) was found to be lower than that of the immobilized catalase (222.2?±?36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.     

Extract from Eugenia punicifolia is an Antioxidant and Inhibits Enzymes Related to Metabolic Syndrome

The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS^·+, O₂ ^·?, and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using α-amylase, α-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS^·+, DPPH·, and O₂ ^·? scavenger (IC₅₀?=?10.5?±?1.2, 28.84?±?0.54, and 38.12?±?2.6 μg/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of α-amylase, α-glucosidase, and xanthine oxidase activities in vitro assays (IC₅₀?=?122.8?±?6.3; 2.9?±?0.1; 23.5?±?2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as α-amylase, α-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome.     

Immobilization and Kinetics of Catalase on Calcium Carbonate Nanoparticles Attached Epoxy Support

A novel hybrid epoxy/nano CaCO₃ composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO₃ nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO₃ support with a conjugation yield of 0.67?±?0.01 mg/cm² and 92.63?±?0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K _m for H₂O₂ was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V _max value from 1,500 to 421.10 μmol (min mg protein)^?1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.     

Immobilization of Lecitase? Ultra onto a Novel Polystyrene DA-201 Resin: Characterization and Biochemical Properties

A simple, rapid, and economic method of enzyme immobilization was developed for phospholipase Lecitase? ultra (LU) via interfacial adsorption. The effect of nature of the polystyrene supports and the kinetic behavior and stability of immobilized lecitase? ultra (IM-LU) were evaluated. Six macroporous resins (AB-8, X-5, DA-201, NKA-9, D101, D4006) and two anion resins (D318 and D201) were studied as the supports. DA-201 resin was selected because of its best immobilization effect for LU. Immobilization conditions were investigated, including immobilization time, pH, and enzyme concentration. IM-LU with a lipase activity of 1,652.4?±?8.6?U/g was obtained. The adsorption process was modeled by Langmuir and Freundlich equations, and the experimental data were better fit for the former one. The kinetic constant (K _m) values were found to be 192.7?±?2.2?mM for the free LU and 249.3?±?5.4?mM for the IM-LU, respectively. The V _max value of free LU (169.5?±?4.3?mM/min) was higher than that of the IM-LU (53.8?±?1.5?mM/min). Combined strategies of scanning electron micrograph, thermogravimetric analysis, and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the IM-LU. FTIR spectroscopy showed that the secondary conformation of the enzyme had changed after immobilization, through which a decrease of ??-helix content and an increase of ??-sheet content were observed. The IM-LU possessed an improved thermal stability as well as metal ionic tolerance when compared with its free form. The reusability of IM-LU was also evaluated through catalyzing esterification reaction between oleic acid and glycerol. It exhibited approximately 70?% of relative esterification efficiency after six successive cycles. This immobilized enzyme on hydrophobic support may well be used for the synthesis of structural lipids in lipid area.     

Multi-walled carbon nanotubes used as support for lipase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

Commercial lipase from Burkholderia cepacia is immobilized on functionalized multi-walled carbon nanotubes (MWNT-COOH and MWNT-OH) provided by a physical adsorption. The immobilization processes for the carbon nanotubes are defined using immobilization time (0–30 min) and distinct adsorbent:adsorbate ratios (1:4, 1:7, and 1:10) with lipase loading of 100, 175, and 250 mg, respectively. The characterization of the immobilized preparations, the free lipase, and the pure nanotubes (MWNT-COOH and MWNT-OH) indicate that the lipase adsorption is increased. Thermogravimetric analysis, differential scanning calorimetry, and scanning electron microscopy are used. The specific surface area, pore volumes, and average pore diameters are determined by nitrogen adsorption–desorption isotherms. For the pure lipase, in the range between 40 and 300 °C, the micrograph is acquired. Experimental results clearly show an effective lipase adsorption in a lower period of time (5 min) in MWNT-COOH and MWNT-OH as well as a decrease in the surface area (98.30–45.9(86)?±?2.5 and 97.61–37.71?±?3.3(7) m² g^?1) and the pore volume (0.48–0.25?±?0.01 and 0.39–0.24?±?0.05 cm³ g^?1), indicating that functionalized multi-walled carbon nanotubes can be successfully used as enzyme support.     

Production of Metabolites with Antioxidant and Emulsifying Properties by Antarctic Strain Sporobolomyces salmonicolor AL1

The Sporobolomyces salmonicolor AL₁ Antarctic strain was cultivated and two bioproducts were obtained: exopolysaccharide and biomass. The biologically active substances ergosterol, torularhodin, torulene, β-carotene and CoQ₁₀ were extracted from the biomass and were quantified as follows: ergosterol 5.2?±?0.2 mg/g, torularhodin 458.3?±?24.5 μg/g, torulene 273.7?±?14.5 μg/g, β-carotene 129.2?±?7.3 μg/g and coenzyme Q₁₀ (CoQ₁₀) 236.1?±?12.1 μg/g. Their antioxidant activity was estimated according to the cathode voltammetry method. The most pronounced antioxidant activity (according to trolox) was exhibited by β-carotene 3.78, followed by CoQ₁₀ 3.60, both of them being the main contributors to the total extract activity of 3.19. The biologically active metabolites in combination with exoglucomannan as emulsifier were used for the creation of model emulsion systems characterised by great stability. The absorption of UVA rays by the model emulsions was studied.     

α-Glucosidase Inhibition and Antioxidant Properties of Streptomyces sp.: In Vitro

Streptomyces strain isolated from the soil sediment was studied for its in vitro α-glucosidase and antioxidant properties. Morphological characterization and 16S rRNA partial gene sequencing were carried out to confirm that the strain Loyola AR1 belongs to genus Streptomyces sp. Modified nutrient glucose broth was used as the basal medium for growth and metabolites production. Ethyl acetate extract of Loyola AR1 (EA-Loyola AR1) showed 50 % α-glucosidase inhibition at the concentration of 860.50?±?2.68 μg/ml. Antioxidant properties such as total phenolic content of EA-Loyola AR1 was 176.83?±?1.17 mg of catechol equivalents/g extracts. EA-Loyola AR1 showed significant scavenging activity on 2,2-diphenyl-picrylhydrazyl (50 % inhibition (IC₅₀), 750.50?±?1.61 μg/ml), hydroxyl (IC₅₀, 690.20?±?2.38 μg/ml), nitric oxide (IC₅₀, 850.50?±?1.77 μg/ml), and superoxide (IC₅₀, 880.08?±?1.80 μg/ml) radicals, as well as reducing power. EA-Loyola AR1 showed strong suppressive effect on lipid peroxidation (IC₅₀, 670.50?±?2.52 μg/ml). Antioxidants of β-carotene linoleate model system reveals significantly lower than butylated hydroxyanisole.     

CO2-expanded bio-based liquids as novel solvents for enantioselective biocatalysis

For the first time, CO₂-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO₂, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO₂-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.     

改性Ultrastable-Y分子筛固定化对非水相中脂肪酶拆分(R,S)-2-辛醇的影响

采用改性Ultrastable-Y分子筛固定Penicillium expansum PED-03 脂肪酶(PEL), 利用固定化PEL在非水相中对(R,S)-2-辛醇进行手性拆分, 考察了改性Ultrastable-Y分子筛固定化处理对PEL催化性能的影响. 结果表明, 与游离PEL及经其它载体固定化的PEL相比, 改性Ultrastable-Y分子筛固定的PEL所催化的拆分反应的转化率(c)和对映体过量值(ee)以及对映体选择性(E)均得到了较大提高. 经固定化处理后, PEL的最适反应温度明显升高, 适宜反应温度范围变宽, 其稳定性也得到了明显改善, 而适宜反应pH值则具有“记忆”性. 在间歇式反应器中利用Ultrastable-Y分子筛固定化PEL对(R,S)-2-辛醇进行手性拆分, 50 ℃反应24 h转化率(c)可达理论值的97.68%, 对映体过量值(ee)可达98.75%. 连续8批拆分反应的结果表明: 改性Ultrastable-Y分子筛固定化脂肪酶催化效率高、立体选择性强(平均E 值＞460), 且催化性能稳定, 显示了该固定化酶在(R,S)-2-辛醇的手性拆分方面具有良好的应用前景.     

Direct electrochemistry and electrochemical biosensing of glucose oxidase based on CdSe@CdS quantum dots and MWNT-modified electrode

The direct electron transfer of glucose oxidase (GOx) was achieved based on the immobilization of CdSe@CdS quantum dots on glassy carbon electrode by multi-wall carbon nanotubes (MWNTs)-chitosan (Chit) film. The immobilized GOx displayed a pair of well-defined and reversible redox peaks with a formal potential (E ^θ’) of ?0.459 V (versus Ag/AgCl) in 0.1 M pH 7.0 phosphate buffer solution. The apparent heterogeneous electron transfer rate constants (k _s) of GOx confined in MWNTs-Chit/CdSe@CdS membrane were evaluated as 1.56 s^?1 according to Laviron's equation. The surface concentration (Γ*) of the electroactive GOx in the MWNTs-Chit film was estimated to be (6.52?±?0.01)?×?10^?11?mol?cm^?2. Meanwhile, the catalytic ability of GOx toward the oxidation of glucose was studied. Its apparent Michaelis–Menten constant for glucose was 0.46?±?0.01 mM, showing a good affinity. The linear range for glucose determination was from 1.6?×?10^?4 to 5.6?×?10^?3?M with a relatively high sensitivity of 31.13?±?0.02 μA?mM^?1?cm^?2 and a detection limit of 2.5?×?10^?5?M (S/N=3).     

Improvement of catalytic activity of lipase in the presence of wide rim substituted calix[4]arene carboxylic acid-grafted magnetic nanoparticles

Candida rugosa lipase immobilized on calix[4]arene carboxylic acid-grafted magnetic nanoparticles using a sol–gel encapsulation technique was tested for activity, which was assessed both in the enantioselective hydrolysis of racemic Naproxen methyl ester and that of p-nitrophenylpalmitate. It has also been noticed that, compared to the free enzyme (E = 137) with an ee value of >98 %, S-Naproxen calix[4]arene carboxylic acid-grafted magnetic nanoparticles based on encapsulated lipase (Calix-1-MN and Calix-2-MN) offer excellent enantioselectivity (E = 373 and E = 381). Moreover, the results indicated that after the fifth reuse in the enantioselective reaction, the encapsulated lipase (Calix-2-MN) still retained about 43 % of its conversion power.     

Isolation and Identification of Antioxidative Peptides from Frog (Hylarana guentheri) Protein Hydrolysate by Consecutive Chromatography and Electrospray Ionization Mass Spectrometry

Frog (Hylarana guentheri) proteins were hydrolyzed by papain and Flavourzyme to obtain antioxidative peptides. The antioxidant activities of the frog protein hydrolysates (FPHs) were measured, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀?=?9.94?±?0.13 mg/mL), reducing power (0.39?±?0.01 at 5.0 mg/mL), and oxygen radical absorbance capacity (ORAC) value (789.15?±?75.10 μmol Trolox equivalents/g). The hydrolysates were purified by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of ESI-MS/MS, two dipeptides were identified as Leu/Ile-Lys (259.1607 Da) and Phe-Lys (293.1446 Da), respectively.     

Purification,Partial Characterization,and CNBr-Sepharose Immobilization of a Vasorelaxant Glucose/Mannose Lectin from Canavalia virosa Seeds

A novel mannose/glucose-binding lectin from Canavalia virosa (designated as ConV) has been purified from seeds of C. virosa by affinity chromatography on a mannose-Sepharose 4B column. ConV strongly agglutinates rabbit erythrocytes and was inhibited by monosaccharides (D-mannose, D-glucose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). SDS-PAGE revealed three bands corresponding to three subunits (α, β, and γ) confirmed by ESI mass spectrometry with exact mass of 25,480?±?2 Da, 12,864?±?1 Da, and 12,633?±?1 Da, respectively. The purified lectin was more stable in pH ranging from 7.0 to 9.0, supported up to 80?ºC without any loss in activity and unaffected by EDTA. ConV showed no toxicity against Artemia sp. nauplii and relaxed endothelized rat aorta, with the participation of the lectin domain. In our tests, the lectin immobilized on CNBr-Sepharose was capable of binding 0.8 mg of ovalbumin per chromatography, allowing the use of ConV as a tool for capture and purification of glycoproteins.     

Characterization,Analysis, and Application of Fabricated Fe3O4-Chitosan-Pectinase Nanobiocatalyst

The investigation on fabrication of Fe₃O₄-chitosan-pectinase nanobiocatalyst was performed by covalently binding the pectinase onto carboxyl group activated chitosan-coated magnetic nanoparticles (CMNPs). The morphological and size distribution analysis of the different magnetic nanoparticles (MNPs) was done using transmission electron microscopy (TEM), and the average diameter was 11.07?±?3.04, 11.55?±?3.16, and 11.59?±?3.16 nm for MNPs, CMNPs, and fabricated nanobiocatalyst, respectively, suggesting that there was no significant change in the size of MNPs after coating and binding. The characteristic peaks occurred at 2θ of 30.39, 35.43, 43.37, 57.22, and 62.9, and their corresponding indices 220, 311, 400, 520, and 441 for different MNPs from the X-ray diffraction (XRD) studies confirmed the presence of Fe₃O₄ with the spinel structure, and there was no phase change even after coating and binding. The various required characteristic absorption peaks (575, 585, 1,563, 1,614, 1,651, and 1,653 cm^?1) from Fourier transform infrared (FT-IR) spectroscopy confirmed the surface modifications and binding of pectinase onto the MNPs. At the weight ratio of about 19.8?×?10^?3 mg bound pectinase/mg activated CMNPs, the activity of fabricated nanobiocatalyst was found to be maximum. In order to monitor their improved activity, the pH, temperature, reusability, storage ability, and kinetic studies were established.     

New Biosilified Pd-lipase hybrid biocatalysts for dynamic resolution of amines

In this work lipase CaLB was immobilized on functionalized Pd-SiO₂ nanoparticles in order to simplify the DKR of α-methylbenzylamine. Hybrid biocatalysts showed immobilization efficiencies of 82%, 80% and 76% when containing 1, 5 and 10% of Pd respectively. On DKR reaction values of ee?>?99% and conversion of 82% were found with only 1% of Pd, generating a productivity of 2.21?mg of product h^?1?mg of support^?1 against 0.76 found by N435®. Compared to commercial N435®, the novel biocatalysts showed protein loads about 15-fold lower and higher activity, demonstrating competitive performances and high industrial applications.     

Effect of lipase immobilization on resolution of (R, S)-2-octanol in nonaqueous media using modified ultrastable-Y molecular sieve as support

The lipase from Penicillium expansum PED-03 (PEL) was immobilized onto modified ultrastable-Y (USY) molecular sieve and the resolution of (R, S)-2-octanol was carried out in a bioreactor in nonaqueous media by the immobilized lipase. It was found that the conversion rate, enantiomeric excess (ee) value, and enantioselectivity (E) value of the resolution catalyzed by PEL immobilized on modified USY molecular sieve were much higher than those of the reaction catalyzed by free PEL and PEL immobilized on other supports. Immobilized on modified USY molecular sieve, the PEL exhibited obvious activity within a wider pH range and at a much higher temperature and showed a markedly enhanced stability against thermal inactivation, by which the suitable pH of the buffer used for immobilization could be “memorized.” The conversion rate of the reaction catalyzed by PEL immobilized on modified USY molecular sieve reached 48.84%, with excellent enantio-selectivity (avarege E value of eight batches >460) in nonaqueous media at “memorial” pH 9.5, 50°C for 24 h, demonstrating a good application potential in the production of optically pure (R, S)-2-octanol.     

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76?U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080?±?0.256 and 0.0003?±?0.018?mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010?±?0.173?mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003?mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178?mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.     

Synthesis of SBA-15 from low cost silica precursor obtained from sugarcane leaf ash and its application as a support matrix for lipase in biodiesel production

Ordered mesoporous silica material was synthesized from a low-cost precursor, sugarcane leaf ash, was used as a support matrix for lipase for the production of biodiesel. The mesoporous samples were characterized using Fourier transform infra red spectroscopy. The surface topography and morphology of the mesoporous materials were studied using scanning electron microscope. The pore diameter, pore volume, Brunauer Emmett and Teller surface area of the mesoporous material were determined by N₂ gas adsorption technique. Different pore size Santa Barbara Acid-15 (SBA-15) samples were synthesized and their lipase immobilization capacity and specific enzyme activity of immobilization lipase were determined and compared. Lipase from Candida Antarctica immobilized on SBA-15 (C) had shown maximum percentage immobilization and specific enzyme activity. The immobilized lipase mesoporous matrix was used for biodiesel production from crude non-edible Calophyllum inophyllum oil. The percentage yield of fatty acid methyl ester, 97.6 % was obtained under optimized conditions: 100 mg of lipase immobilized on SBA-15, 6:1 methanol to oil molar ratio, the reaction of 2 g C. inophyllum oil with methanol.     

Green Synthesized Iron Oxide Nanoparticles Effect on Fermentative Hydrogen Production by Clostridium acetobutylicum

A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV–vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm^?1 is assigned to Fe–O of γ-Fe₂O₃. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be ～59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74?±?0.08 mol H₂/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33?±?0.09 mol H₂/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34?±?0.8 to 52?±?0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO₄ on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO₄. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.