In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) fromPhanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Comparative study on using carbon or nitrogen limited medium to culture white rot fungi for reactive brilliant red dye K-2BP decolotization under non-sterile conditions

In order to explore ways for the application of white rot fungus in dye effluent treatment under non-sterile conditions, experiment on decolorization of reactive brilliant red was carried out, employing nitrogen-limited and carbon-limited medium with C/N ratio of 56/2.2 and 28/44 (in mmol/L), respectively. The results showed that the decolorization rate reached 92% while culturing white rot fungus with ni- trogen-limited medium; however, the decolorization process ended in carbon-limited medium (n(C)/n(N) = 28/44) because of bacterial contamination. In addition, pH rose up to 9.31 after 4 d of decolorization, which was caused by bacterial contamination in the carbon-limited system. Therefore, it is concluded that nitrogen-limited medium can inhibit bacterial growth to some extent while carbon-limited medium is more easily contaminated by bacteria. Nitrogen-limited medium is more suitable in culture of white rot fungus for decolorization of reactive dye. Medium with the ability of inhibiting yeast growth should be developed by adjusting other components of nitrogen-limited medium.     

Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp

The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO₃ completely inhibited the enzyme synthesis while (NH₄)₂SO₄ at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu²⁺ had the most inhibiting effect while K⁺ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation.     

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.     

Biodegradation of poly (vinylalcohol) with enzymatic extracts of phanerochaete chrysosporium

Commercial poly(vinylalcohol) samples (PVOH) of average molecular mass (Mw) 120,000 g/mol, were subjected to biodegradation with enzymatic extracts of the Phanerochaete chrysosporium fungus, in which the lignine peroxidase activity (LiP) was detected. The results of differential refractive index and ultraviolet (UV) absorption Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography coupled to a Mass Detector (GC-MS) allow to deduce that in 15 days, under the conditions of this study, the average molar mass of the polymer decreases in 79.3 %. The benzaldehyde was detected as the main degradation product.     

液相色谱-串联质谱法测定偶氮染料橙黄G的Fenton法降解氧化产物苯胺

采用液相色谱-串联质谱法测定偶氮染料橙黄G的Fenton法降解氧化产物苯胺的含量。采用OnGuard II H固相萃取柱去除Fenton反应液中的亚铁离子,然后在XR-ODS II色谱柱上,采用电喷雾电离正离子扫描多反应监测模式测定。苯胺的线性范围为2.0~100.0μg·L-1,方法的检出限(3S/N)为0.6μg·L-1。加标回收率在94.0%~98.4%之间,日内相对标准偏差在2.8%~6.4%之间,日间相对标准偏差在5.2%~8.6%之间。     

磷钨酸均相光催化还原降解水中偶氮染料酸性大红3R

以磷钨酸(H3PW12O4记为PW12)作为光催化剂,在异丙醇作为电子给体的条件下对偶氮染料酸性大红3R（记为AR3R）进行光催化均相还原脱色研究。循环伏安法、暗反应、O2竞争抑制等实验表明杂多蓝（PW12O404-）对AR3R 具有明显的还原脱色作用。实验研究了催化剂PW12用量、异丙醇浓度、染料浓度、盐浓度对PW12/异丙醇光催化还原降解酸性大红3R的影响。结果表明：AR3R的光催化脱色速率随催化剂PW12、异丙醇浓度的增加而增加,最后趋于恒定;随染料初始浓度增加,初始光解速率增大,且符合Langmuir-Hinshelwood动力学方程;随盐浓度增加,染料脱色速率减小,表现为负的盐效应。由此推测AR3R与光反应生成的杂多蓝预先进行复合,然后发生电子转移引起偶氮染料还原脱色,杂多蓝氧化复原。本研究结果表明磷钨酸/异丙醇/UV绿色光催化还原体系能够有效用于偶氮染料废水的还原脱色处理。     

The Biodegradation of Indigo Carmine by Bacillus safensis HL3 Spore and Toxicity Analysis of the Degradation Products

The aims of this article were to investigate Bacillus safensis HL3 spore for its capacity to degrade and detoxify indigo carmine and to provide an effective biological agent for the treatment of isatin dye wastewater. Bacillus safensis HL3 spore was found to decolorize indigo carmine by 97% in the presence of acetosyringone within 2 h. Significantly increased activities of spore laccase, intracellular tyrosinase, and lignin peroxidase upon exposure to indigo carmine were observed. The results of RT–qPCR also showed that the expression of laccase gene was significantly increased. The spore has the ability to degrade indigo carmine through oxidization. Furthermore, the pathway by which indigo carmine is degraded was investigated using liquid chromatography–mass spectrometry analysis to identify the biodegradation products. A detailed pathway of indigo carmine degradation by bacterial spores was proposed for the first time. Toxicity tests indicated that the biodegradation products of indigo carmine are non-toxic to Nicotiana tabacum seeds and are less hazardous to human erythrocytes than the original dye. Indigo carmine is a typical recalcitrant dye and severely jeopardizes human health. The results demonstrate the utility of the spore from Bacillus safensis HL3 for the degradation of indigo carmine and simultaneous reduction of its toxicity.     

Exploiting stored TiO2 electrons for multi-electron reduction of an azo dye methyl orange in aqueous suspension

The mechanism of multi-electron reduction of methyl orange (MO) azo dye on TiO₂ nanoparticles has been studied performing stopped flow technique. A multi-electron reduction of azo dye has been investigated. It was found that a multistep reduction of the dye takes place: the stored electrons reduce the conjugative system of the azo group resulting in the decolorization of the dye and leading to the formation of hydrazine derivative followed by further 2 electron transfer step leading to the cleavage of the N–N bond and the formation of aromatic amines. The FTIR analysis of the products confirms the proposed mechanism of the dye reduction. The kinetic parameters and of the multi-electrons reduction of the MO have been determined. The rate of MO reduction was found to be dependent on both the TiO₂ electrons and the dye concentrations.     

Biosorption of mercury by carboxymethylcellulose and immobilized Phanerochaete chrysosporium

Phanerochaete chrysosporium basidiospores immobilized onto carboxymethylcellulose were used for the removal of mercury ions from aqueous solutions. The biosorption of Hg(II) ions onto carboxymethylcellulose and both immobilized live and heat-inactivated fungal mycelia of Phanerochaete chrysosporium was studied using aqueous solutions in the concentration range 30-700 mg l⁻¹. The biosorption of Hg(II) ions by the carboxymethylcellulose and both live and heat-inactivated immobilized preparations increased as the initial concentration of mercury ions increased in the medium. Maximum biosorption capacity for immobilized live and heat-inactivated fungal mycelia of Phanerochaete chrysosporium was found to be 83.10 and 102.15 mg Hg(II) g⁻¹, respectively, whereas the amount of Hg(II) ions adsorbed onto the plain carboxymethylcellulose beads was 39.42 mg g⁻¹. Biosorption equilibria were established in approximately 1 h and the correlation regression coefficients show that the adsorption process can be well defined by a Langmuir equation. Temperature changes between 15 and 45 °C did not affect the biosorption capacity. The effect of pH was also investigated and the maximum adsorption of Hg(II) ions onto the carboxymethylcellulose and both live and heat-inactivated immobilized fungal mycelia was observed at pH 6.0. The carboxymethylcellulose-fungus beads could be regenerated using 10 mM HCl, with up to 95% recovery. The biosorbents were used in three biosorption-desorption cycles and no significant loss in the biosorption capacity was observed.     

Sorption of certain heavy-metal ions by hydrolyzed lignin and its derivatives

The ability to form intermolecular coordination of lignin macromolecules and metal ions through both ionic and coordinative metal—ligand bonds was shown by studying the sorption activity of hydrolyzed lignin from cotton seed husks (HLCSH) and its aminated derivatives. The studied lignins exhibited high sorption activity for metal ions over the whole range of concentrations used. The heavy-metal ions fell in the following order of decreasing lignin sorption activity: Fe > Pb > Cu > Cd > Zn. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 386–388, July–August, 2006.     

Elucidation of lignin structure by quantitative 2D NMR

Quick quantitative HSQC (QQ‐HSQC) was applied to quantitative evaluation of different inter‐unit linkages in an array of milled softwood and hardwood and technical lignins by using the guaiacyl C2 and syringyl C2–C6 signals as internal standards. The results were found to be highly reproducible and comparable with earlier literature reports. The advantage of QQ‐HSQC NMR analysis of lignin is contemporary detection and quantification of lignin inter‐unit linkages with a direct, non‐destructive method requiring short acquisition times.     

Production and characterization ofPhanerochaete chrysosporium lignin peroxidases for pulp bleaching

The production of lignin peroxidase fromPhanerochaete chrysosporium was studied using immobilized mycelia in nylon-web cubes in semicontinuous fermentation using glucose pulses or ammonium tartrate pulses. Consistent enzyme production was achieved when glucose pulses were used, leading to an average activity of 253 U/L. The crude enzyme was added to eucalyptus kraft pulp before conventional and ECF bleaching sequences. Optimization of the enzymatic pretreatment led to the following operational conditions: enzyme load of 2 U/g of pulp, hydrogen peroxide addition rate of 10 ppm/h, and reaction time of 60 min. Pulp final characteristics were dependent on the chemical treatment sequence that followed enzymatic pretreatment. The chief advantage of enzymatic pretreatment was pulp viscosity preservation, which was observed in most of the experiments carried out with seven different chemical treatment sequences     

过氧化氢调控藜芦醇介导木素过氧化物酶催化氧化邻苯三酚红研究

木素过氧化物酶(LiP)催化H₂O₂氧化邻苯三酚红(PR)反应的氧化产物受H₂O₂与PR的摩尔比控制, H₂O₂与PR的摩尔比不同, 所得降解产物不一样. 分析表明, H₂O₂在LiP催化氧化PR过程中的双重作用(即低浓度的H₂O₂是LiP的激活剂, 高浓度的H₂O₂是LiP的抑制剂)是导致上述现象的根本原因. 藜芦醇(VA)对LiP催化氧化PR的反应有促进作用, 尤其是当H₂O₂与PR的摩尔比较高时这种促进作用更为明显; 然而PR对LiP催化氧化VA的反应却有抑制作用. 后者可以用来解释为什么在用白腐菌降解染料时在培养液中常常检测不到LiP的藜芦醇活力. 分析表明, VA的存在不但促进了LiP酶中间体LiP(II)和/或LiP(III)向LiP的转化, 使LiP的催化循环加速, VA生成的VA^＋·也间接氧化了染料PR, 从而使PR的氧化速率提高.     

Kinetic Studies on of Veratryl the Lignin Peroxidase Catalyzed Oxidation Alcohol by H2O2 in AOT/Isooctane/ Toluene/Water Reverse Micelles

The steady state kinetics of the lignin peroxidase （LIP） catalyzed oxidation of veratryl alcohol （VA） by H2O2 in a sodium bis（2-ethylhexyl） sulfosuccinate （AOT）/isooctane/toluene/water reverse micellar medium was studied and a comparison with the corresponding aqueous medium was made to understand the effect of the reverse micellar medium on the catalytic mechanism and kinetic parameters. Results indicated that the model reaction in the AOT reverse micelle followed the ping-pong mechanism with true kcat, Km,VA and KmH2O2 being 59.6min^-1, 13.9 mmol· L^-1 and 94.8 μmol·L^-1, respectively; inhibition of high level of H2O2 on LiP followed the reversible competitive pattern with Ki being 0.140 mmol·L^-1. The reaction mechanism and inhibition pattern in the AOT reverse micellar medium were the same as those in bulk aqueous medium, but the kinetic parameters except KmH2O2 were greatly different in the two media. The kcat and Ki values in the reverse micelle were approximately 2 and 20 times smaller than the corresponding values in the aqueous solution, but the Michaelis constant of VA was approximately 100 times greater than that in the aqueous solution. The above mentioned differences in the kinetic parameters were caused by the microheterogeneity and the interface of the AOT reverse micelle, which resulted in the partitioning of VA and H2O2, and by the changes of the conformation of LiP and the reactivity of the substrates.     

胺化木质素对水中硝基苯吸附性能的研究

含硝基苯的工业废水对环境造成了严重的污染.本文用环氧氯丙烷、乙二胺对碱木质素进行改性制备胺化木质素,详细研究了溶液pH、接触时间、温度、浓度等因素对胺化木质素吸附水溶液中硝基苯性能的影响.结果表明:溶液pH对吸附有较大的影响,吸附的最佳pH为8,胺化木质素对硝基苯吸附3h后达到平衡,与准一级吸附动力学模型有较好的拟合;...     

Kinetics of phenol oxidation by peroxidase

Studies of the kinetic behavior of horseradish peroxidase (HRP) at pH 8 and at room temperature indicate that the reaction of phenol with H₂O₂ catalyzed by HRP exhibits normal Michaelis-Menten saturation kinetics. An irreversible reaction mechanism for the steady-state kinetics of HRP, which is consistent with the experimental data, is considered. The second-order rate constants for the reactions of HRP with H₂O₂ and compound II with phenol are 4.14 × 10⁵ M^-1s^-1 and 5.54 × 10⁴M^-1s^-1, respectively.     

Addition of polyglycol to old culture of Phanerochaete chrysosporium

Polyglycols increased lignin peroxidase activity in shaken cultures of Phanerochaete chrysosporium even when they were added to an old fungus (5 or 10 d after inoculation). The effects depended on the polyglycol mol wts (10²–10⁶ Daltons) as well as on the backbone structure (i.e., poly[ethylene glycol], poly[butylene glycol], poly[propylene glycol]) and terminal groups (i.e., poly[ethylene glycol], poly[ethylene glycol] methyl ether, poly[ethylene glycol] dimenthyl ether). The residual quantity of polyglycol in the biomass and in the culture filtrate also varied among different polyglycols. The polyglycols act after being adsorbed to the cell membrane exaggerating the asymmetry of the membrane environment. In old fungus, the incompatibility of polyglycols and glycans decreases the adsorption and the effect of high mol wtpolyglycols.