Screening of white rot fungi for the treatment of olive mill waste-waters

Several white rot fungi were evaluated for their ability to decolorize olive mill waste-waters (OMW). Among these, Phanerochaete chrysosporium showed the highest potential for the biological depollution of OMW. Approximately 65% of the color and 73% of the chemical oxygen demand (COD) were removed by P. chrysosporium strain HD. Phlebia radiata, Dichomitus squalens, Polyporus frondosus and Coriolus versicolor could also decolorize the OMW but to a lesser extent. The different abilities of these white rot fungi to decolorize OMW correlated with their rates of depolymerization of high molecular weight aromatics and the degradation of the low molecular weight aromatic compounds. Fourteen strains of P. chrysosporium were compared on the basis of their lignin peroxidase (LiP) production, OMW decolorization and residual material dry weight. High lignin peroxidase producer strains, such as strains HD and BKM-F-1767, showed the highest rates of OMW decolorization, P. chrysosporium strains 79–36 and FP 104297 (low LiP producer strains) did not exhibit any decolorization activity.     

Decolourisation and treatment of pulp and paper mill effluent by lignin‐degrading Bacillus sp.

Three lignin‐degrading bacterial strains, identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been examined for the treatment of pulp and paper mill effluent. The results of this study revealed that all three bacterial strains effectively reduced colour (39–61%), lignin (28–53%), biochemical oxygen demand (BOD) (65–82%), chemical oxygen demand (COD) (52–78%) and total phenol (64–77%) within six days of incubation. However, the highest reduction in colour (61%), lignin (53%), BOD (82%) and COD (78%) was recorded by Bacillus sp. while, maximum reduction in total phenol (77%) was recorded with Paenibacillus sp. treatment. Significant reduction in colour and lignin content by these bacterial strains was observed after two days of incubation, indicating that bacterium initially utilized growth supportive substrates and subsequently chromophoric compounds thereby reducing lignin content and colour in the effluent. The total ion chromatograph (TIC) of compounds present in the ethyl acetate extract of control and bacterial treated samples revealed the formation of several lignin‐related aromatic compounds. The compounds identified in extracts of treated samples by Paenibacillus sp were t‐cinnamic acid and ferulic acid, while 3‐hydroxy‐4‐methoxyphenol, vanillic acid and vanillin acid by A. aneurinilyticus and gallic acid and ferulic acid by Bacillus sp. respectively indicating the degradation of lignin present in the effluent. The identified compounds obtained after different bacterial treatments were found to be strain‐specific. Among these identified compounds, ferulic acid, vanillic acid and vanillin could have immense value for their use in preservatives and in the food flavour industry. Copyright © 2007 Society of Chemical Industry     

Kraft lignin degradation products for tanning and dyeing of leather

Kraft lignin degradation by a biomimetic system was investigated, using hemin as a catalyst and hydrogen peroxide as an oxidising agent, which mimics the catalytic mechanism of lignin peroxidase (LiP) to produce phenolic compounds. The degradation products were identified using spectroscopy and gas chromatography–mass spectrometry (GC–MS): 2‐methoxyphenol, 4‐hydroxybenzaldehyde, vanillin and vanillic acid were produced and their formaldehyde polymerisation products used for tanning hide powders. The denaturation (shrinkage) temperature of hide powder was raised to 80 °C through hydrogen‐bonding interactions between the polymers and the collagenic hide powder. For dyeing of hide powder, the lignin degradation products were reacted with laccase (a polyphenol oxidase): 2‐methoxyphenol gave the darkest colour. These products have potential to be used as raw materials for tanning and dyeing of animal skins. Therefore, value can be added to this industrial byproduct and reduce its environmental impact. Copyright © 2004 Society of Chemical Industry     

Production and characterization of laccase and manganese peroxidase from the ligninolytic fungus Fomes sclerodermeus

Fomes sclerodermeus was grown on semi‐defined media based on yeast extract, peptone and glucose (YPG). The fungus produced a minimum basal level of laccase activity irrespective of culture medium. The highest laccase production (20 U cm^?3) was obtained in cultures supplemented with CuSO₄. Manganese peroxidase (MnP) could only be detected when MnSO₄ was added to the medium. None of the aromatic compounds tested stimulated further laccase or MnP production. Laccase and MnP stimulated by Cu²⁺ or Mn²⁺ respectively were purified. Two different laccase isoenzymes with the same molecular mass (67 kDa) and N‐linked carbohydrate content (3%) and a slight difference in their pI values (3.41 and 3.48) were characterized. In addition, two different MnP isoenzymes with the same molecular mass (47 kDa) and N‐linked carbohydrate content (4%) and different pI values (3.35 and 3.45) were characterized. Both enzymes showed good stability at 25 °C and over a wide range of pH. Both laccases oxidize ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) more efficiently than 2,6‐dimethoxyphenol (DMP) with similar efficiency values (K_cat/K_m) while the MnP I, the major peroxidase isoenzyme in the studied conditions, oxidizes the Mn²⁺ and Mn‐mediated activity on DMP more efficiently than MnP II. Copyright © 2006 Society of Chemical Industry     

Laccase improvement in submerged cultivation: induced production and kinetic modelling

Improvement of laccase production by Trametes versicolor was made by employing different operational strategies. In the cell growth medium, various glucose concentrations were compared for improving laccase production. A clear and significant stimulation of enzyme production under carbon limitation was obtained. Copper, 2,5‐xylidine, and a phenolic mixture were also used as laccase inducers. A cooperative effect between the inducers on laccase production was identified. Mixtures of inducers produced higher laccase activities, reaching values of 5500 U dm^?3. Further productivity enhancement can be obtained using the inducers along with the carbon limitation strategy. It is shown that low laccase concentrations are obtained by a primary metabolism of T versicolor, and that phenolic compounds and carbon limitation induce a secondary metabolism, providing higher laccase concentrations. A mathematical model for laccase production based on a direct experimental measure of biomass, along with substrate consumption and enzymatic activity over time is proposed for non‐homogeneous fermentations of T versicolor. Copyright © 2005 Society of Chemical Industry     

曝气式反应器中木质素过氧化物酶的合成及对印染废水配制液的脱色处理

比较了黄孢原毛平革菌在3种生物反应器(搅拌式反应器、鼓泡式反应器、曝气式反应器)中合成木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)的差异. 结果表明，曝气式反应器对酶的合成(尤其是LiP)最为有利. 考察了曝气式反应器中半连续培养和连续培养两种方式下酶的合成和橙I脱色情况，发现半连续培养可使培养体系长时间保持较高酶活力，置换比例为1/2时染料废水可连续脱色5批，脱色率达到90%以上，比脱色率在46.7 g/(g×d)以上. 连续培养条件下酶很快失活，废水的脱色率迅速下降. 在曝气式反应器中用半连续培养的方式(置换比例1/2)对实际印染废水进行处理，可处理废水4批，前3批脱色率达到90%以上，第4批有明显下降.     

Decolorization of Direct Blue GLL with enhanced lignin peroxidase enzyme production in Comamonas sp UVS

BACKGROUND: The present work aims to study the production of lignin peroxidase (LiP) enzyme by Comamonas sp UVS using various media, and lignocellulosic waste materials, and its effect on decolorization of Direct Blue GLL (DBGLL). RESULTS: Yeast extract medium was found to be more effective for the production of LiP and also for the decolorization of DBGLL. The bagasse powder along with yeast extract induced LiP activity. Comamonas sp UVS decolorized DBGLL dye (50 mg L^?1) within 13 h at static condition in YE broth. It could degrade up to 300 mg L^?1 of dye within 55 h. The maximum rate (Vmax) of decolorization was 12.41 ± 0.55 mg dye g cell^?1 h^?1 with the Michaelis constant (Km) value as 6.20 ± 0.27 mg L^?1. The biodegradation was monitored by UV‐Vis, GC‐MS and HPLC. CONCLUSION: The use of agricultural by‐products for the activity enhancement of the ligninolytic enzymes is a cost effective process. It also resolves the problem of the disposal of agro‐residues. This system can be applied for the degradation of different recalcitrant compounds. Copyright © 2008 Society of Chemical Industry     

木质素降解酶的酶活测试方法的评价与分析

作为最丰富的可再生有机芳香族聚合物之一,木质素的生物降解和利用具有较好的环境、经济和社会效益,但繁多的木质素降解酶活测试方法为生物降解木质素的相关研究带来一定困难。本文围绕已发现的5种重要木质素降解酶（漆酶、锰过氧化物酶、木质素过氧化物酶、多功能过氧化物酶和染料脱色过氧化物酶）,针对其来源、特性和酶与底物的作用机理,深入地比较和分析了多种木质素降解酶酶活测试方法。通过对ABTS法、 2,6-DMP法、藜芦醇法、天青B法和其他特色方法从原理到应用的综合讨论,全面评估了上述方法的底物多样性、测试条件及适用范围。此外,本文还针对木质素降解酶酶活测试研究中所存在的主要挑战提出了解决思路,旨在为生物法降解木质素及其高值化利用的研究提供有效参考和策略。     

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-β- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s⁻¹·mM⁻¹ for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1–3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28–1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.     

Design of different bioreactor configurations: application to ligninolytic enzyme production in semi‐solid‐state cultivation

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) in laboratory‐scale bioreactors was studied. The cultivations were carried out in semi‐solid‐state conditions, employing corncob as carrier, which functioned both as a place of attachment and as a source of nutrients. Several bioreactor configurations were investigated in order to determine the most suitable one for ligninolytic enzyme production: a 1‐dm³‐static‐bed bioreactor, a 1‐dm³‐static‐bed bioreactor with air diffusers into the bed, a 0.5‐dm³‐static‐bed bioreactor with air diffusers into the bed and a tray bioreactor. Although the static‐bed configurations produced maximum individual lignin peroxidase (LiP) activities about 400 U dm⁻³ (1.0‐dm³ bioreactor) and about 700 U dm⁻³ (0.5‐dm³ bioreactor), manganese‐dependent peroxidase (MnP) was not detected throughout the cultures. Nevertheless, the tray configuration led to maximum individual MnP and LiP activities of about 200 U dm⁻³ and 300 U dm⁻³, respectively. Therefore, this configuration is the most adequate of the different bioreactor configurations tested in the present work, since the ligninolytic complex formed by MnP and LiP is more efficient for its application to bio‐processing systems. In addition, the results indicated the influence of the oxygen in ligninolytic enzyme production. © 2001 Society of Chemical Industry     

仿生酶菌协同体系预处理木质素机理及特性

白蚁-真菌自然共生体系可有效转化木质纤维素类生物质,其本质在于对木质素物理结构的破坏和官能团的修饰,减少木质素对酶的非生产性吸附,从而提升酶解糖化效率,为生物质高效能源化利用提供新思路。本文基于白蚁肠道中存在的分解木质素酚类单元的漆酶（La）和蚁巢内降解木质纤维素的蚁巢伞菌（Te）,构建La和Te协同预处理木质素体系,比较La和典型的木质素降解菌黄孢原毛平革菌（PC）对木质素模型化合物碱木素的预处理特性。结果表明,在降解La预处理的碱木素过程中,Te产生的漆酶（La）和木质素过氧化物酶（LiP）活性最大值较未处理的碱木素样品分别提升43.3%、58.5%,PC产生的漆酶（La）和锰过氧化物酶（MnP）活性最大值较未处理的碱木素样品分别提升35.9%、31.6%。漆酶预处理强化了Te、PC对碱木素官能团的修饰和物理结构的破坏。傅里叶红外转换光谱分析（FTIR）表明酶菌协同体系处理后碱木素特征官能团的吸收峰显著降低。扫描电镜（SEM）和压汞测试结果表明酶菌协同体系对碱木素表面结构破坏严重,La和Te协同（La+Te）体系预处理后的碱木素平均孔径比单一La和单一Te分别显著提升31.1%、45.6%。经La+Te体系预处理后的碱木素最大酶吸附量较未处理的碱木素减少了51.5%,由于非生产性吸附显著减少,后续纤维素酶的转化率较未处理的碱木素样品提高了71.5%。本文证明了通过漆酶与真菌协同作用可有效改变碱木素物化特性,从而有效促进后续纤维素的酶解糖化,为木质纤维素类生物质高效利用提供指导。     

Improvement of the catalytic performance of lignin peroxidase in reversed micelles

BACKGROUND: Anionic surfactant sodium bis (2‐ethylhexyl) sulfosuccinate (AOT) had an inhibiting effect on lignin peroxidase (LiP). To improve the catalytic activity of LiP in an AOT reversed micelle in isooctane, nonionic surfactant polyoxyethylene lauryl ether (Brij30) was incorporated into the interfacial membrane. H₂O₂ played dual roles in the LiP‐catalyzed oxidation of substrates. To obtain a sustainable high activity of LiP, a coupled enzymatic reaction, i.e. the glucose oxidase (GOD)‐catalyzed oxidation of glucose was used as an H₂O₂ source. RESULTS: Owing to modification of the charge density of the interfacial membrane, the activity of LiP in an optimized AOT/Brij30 reversed micellar medium (χ_B (the molar percentage of Brij30) = 0.53, ω₀ ([H₂O]/([AOT] + [Brij30]) = 23, pH = 4.8) was 40 times that in a single AOT reversed micelle. Due to the controlled release of H₂O₂, the concentration of H₂O₂ in the mixed reversed micellar medium was maintained at a moderately high level throughout, which made the LiP‐catalyzed oxidation of substrates proceed at a higher conversion rate than counterparts in which H₂O₂ was supplied externally in one batch at the beginning of the reaction. Decolourization of two waterless‐soluble aromatic dyes (pyrogallol red and bromopyrogallol red) using LiP coupled with GOD in the medium also demonstrated that a higher decolourization percentage was obtained if H₂O₂ was supplied enzymatically. CONCLUSION: The proposed measures (both physicochemical and biochemical) were very effective, giving significant improvement in the catalytic performance of LiP in a single AOT reversed micelle in isooctane, which helped to degrade or transform hydrophobic aromatic compounds with LiP in reversed micelles more efficiently. Copyright © 2007 Society of Chemical Industry     

Freeze‐drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability

The effect of different lyoprotectants (sucrose, dimethyl‐succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran, mw 60 kDa) on the stability of the enzyme lignin peroxidase (LiP, EC number: 1.11.1.‐), both during the freeze‐drying process and storage were investigated. The shelf stability tests were performed at 4 °C and 27 °C. Both DMS buffer and sucrose showed a good protective action: the former was particularly effective during the process, while the latter improved the stability during storage. In contrast, mannitol and dextran had negative effects, reducing the activity also in the lignin peroxidase solution. BSA was discarded because, in the range of compatible concentrations with LiP, it does not confer a consistent structure to the freeze‐dried product. © 2002 Society of Chemical Industry     

Removal of lignin in a liquid system by an isolated fungus

The screening of a strain which could perform lignin removal was carried out. Based on taxonomic study the isolated strain (LM‐2) was identified as Penicillium sp. LM‐2 could decolorize 0.6 g dm^?3 lignin within 4 days in a shaking culture at 25 °C. The efficiency of decolorization of the lignin was over 80% in the pH range of 4.0–6.0, but was low above pH 6.2. The rise of temperature had a slight adverse effect on the lignin decolorization in the range of 25–35 °C. Lignolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase were not detected in the culture broth or within the fungal cells. The lignin was removed from the high molecular weight fraction mainly by adsorption and accumulation inside the cells. © 2001 Society of Chemical Industry     

白腐菌木素过氧化物酶发酵条件及其酶液对稻草降解的研究

研究营养条件对白腐菌合成木素过氧化物酶(ligninperoxidase,LiP)的影响。在最适培养条件下,第6天获得7560U/L的酶活。利用LiP粗酶液在体外直接降解稻草,3天后Klason木素的降解率为8.7%,经红外光谱分析其降解产物,推断LiP粗酶液在体外降解稻草的反应主要是氧化反应。     

Alkaline fungal degradation of oxidized polyethylene in black liquor: Studies on the effect of lignin peroxidases and manganese peroxidases

High‐molecular‐weight polyethylene is resistant to natural environmental degradation for its crystalline, hydrophobic structure. In this study, waste polyethylene bags are chemically oxidized at 80°C for 5 days by potassium dichromate solutions of various concentrations along with sulfuric acid. Absorbance peaks of carbonyl and carboxylate ions in the Fourier transform infrared spectroscopy spectra and formation of amorphous phase from crystalline one as indicated in X ray diffraction studies of oxidized polyethylenes indicate the formation of a polar hydrophilic and low‐molecular‐weight material after oxidation. From the scanning electron microscopy studies, it is observed that reacted polyethylene surface is disintegrated and numerous fissures are formed throughout the surface. The respective weight loss of incubated oxidized polyethylene with Phanerochaete chrysosporium (MTCC‐787) after 15 days of incubation is 70%, respectively, in black liquor–glucose–malt extract medium. As both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected in this media, further degradation of oxidized polyethylene is carried out in four different media with varying amount of N and Mn. The weight loss is observed only in media with excess nitrogen (N) and limited manganese (Mn), the condition which enhances the presence of LiP and MnP. This indicates that these enzymes are essential for degradation of lignin as well as oxidized polyethylene. UV spectroscopic studies indicate 40% decrease in the lignin concentration. This process of fungal degradation of chemically oxidized polyethylene using black liquor is very quick compared to the other related studies, leading to the simultaneous degradation of two waste materials, polyethylene and black liquor. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40738.     

白腐菌降解焦化废水的研究

介绍了白腐菌在焦化废水中的驯化生长和产酶条件优化,考察了白腐菌及其酶类对焦化废水的降解能力,结果表明:白腐菌在培养条件下能够产生白腐菌漆酶,是降解焦化废水中的多环芳香化合物的主要活性成分。经过白腐菌和活性污泥混合,得到较好的处理效果:实验时间内COD去除率达到89%,NH3-N达到88%,较单纯活性污泥效率高。     

Tyrosinase and peroxidase production by Rhizopus oryzae strain ENHE obtained from pentachlorophenol‐contaminated soil

BACKGROUND: The aim of this study was to investigate the ability of a zygomycete isolated from pentachlorophenol (PCP)‐contaminated soil to produce peroxidase and phenoloxidase enzymes, and to determine the effect of tyrosine and PCP on the enzyme activities. The ability of the isolate to tolerate and remove PCP was also studied. RESULTS: A zygomycete capable of tolerating and removing PCP was isolated from contaminated soil and identified by molecular techniques as Rhizopus oryzae strain ENHE. This fungus produced extra‐ and intracellular tyrosinase and extracellular lignin peroxidase. Tyrosinase activity increased with 0.1 g tyrosine L^?1 added to the culture medium. PCP had no effect on tyrosinase activity but increased lignin peroxidase activity. It was shown that R. oryzae ENHE grew until 100 mg PCP L^?1 and removed 90% of the initial PCP concentration of 12.5 mg L^?1 in 24 h and that the enzymes tyrosinase and lignin peroxidase were probably involved in the PCP removal process. CONCLUSION: The results indicate that R. oryzae ENHE has the potential to be used to produce tyrosinase and lignin peroxidase enzymes. In the few studies that report the production of peroxidase and extracellular tyrosinase by fungi, these enzymes are produced mainly by basidiomycetes. Copyright © 2008 Society of Chemical Industry     

Decolorization of textile dyes by Bjerkandera sp. BOL 13 using waste biomass as carbon source

BACKGROUND: Phanerochaete chrysosporium, Trametes versicolor and Bjerkandera sp BOL13 were compared for decolorization of azo dyes supplied individually or as a mixture. The dye decolorization was also evaluated during continuous treatment under non‐sterile conditions using a lignocellulosic growth substrate. RESULTS: Bjerkandera sp BOL13 showed the highest dye decolorization potential. This fungus was also found to support high decolorization of Remazol Red RR at an initial pH of 4‐6 and when using straw as co‐substrate. The fungus was evaluated for Remazol Red RR decolorization in a continuously fed packed‐bed bioreactor operated under non‐sterile conditions with 3 days of hydraulic retention time. When glucose was supplied as growth‐substrate, decolorization efficiencies of 65‐90% were maintained for 12 days in a bioreactor packed with wooden material. The decolorization efficiency was lower when glucose was not fed to the fungus or when a plastic material was used as packing. Higher manganese peroxidase and laccase activities were also recorded when the wood packing was used. Contamination caused a drop in decolorization efficiency after 17‐19 days operation. CONCLUSIONS: The potential of Bjerkandera sp BOL13 for decolorization of azo dyes under non‐sterile conditions using lignocellulosic growth substrates was demonstrated. Research is needed to reduce contamination under non‐sterile conditions. © 2012 Society of Chemical Industry     

Genes Identification,Molecular Docking and Dynamics Simulation Analysis of Laccases from Amylostereum areolatum Provides Molecular Basis of Laccase Bound to Lignin

An obligate mutualistic relationship exists between the fungus Amylostereum areolatum and woodwasp Sirex noctilio. The fungus digests lignin in the host pine, providing essential nutrients for the growing woodwasp larvae. However, the functional properties of this symbiosis are poorly described. In this study, we identified, cloned, and characterized 14 laccase genes from A. areolatum. These genes encoded proteins of 508 to 529 amino acids and contained three typical copper-oxidase domains, necessary to confer laccase activity. Besides, we performed molecular docking and dynamics simulation of the laccase proteins in complex with lignin compounds (monomers, dimers, trimers, and tetramers). AaLac2, AaLac3, AaLac6, AaLac8, and AaLac10 were found that had low binding energies with all lignin model compounds tested and three of them could maintain stability when binding to these compounds. Among these complexes, amino acid residues ALA, GLN, LEU, PHE, PRO, and SER were commonly present. Our study reveals the molecular basis of A. areolatum laccases interacting with lignin, which is essential for understanding how the fungus provides nutrients to S. noctilio. These findings might also provide guidance for the control of S. noctilio by informing the design of enzyme mutants that could reduce the efficiency of lignin degradation.