Elicitation of lignin peroxidase inStreptomyces lividans

Using a novel starch-based medium (DJMM) which elicits high expression of lignin peroxidase (ALiP-P3) fromStreptomyces viridosporus T7A, significant levels of ALiP-P3 (between 1135 and 1784 nmol/g cell-min) were excreted byS. lividans TK23, TK24, and TK64 with the supernatants capable of degrading dichlorophenol (these strains were previously reported to produce low levels of LiP). TheS. lividans wildtype strains produced 1/9 to 1/6 the cell-specific LiP activity previously detected inS. viridosporus T7A cultures grown in the same starch-based medium; however, by using DJMM to increase the cell density, the volumetric activity of wild-typeS. lividans TK23, TK24, and TK64 strains was increased 11 to 20-fold compared to cultivations in a yeast-extract-based medium. Consequently, this increase of LiP production allows the direct analysis of LiP activity in the supernatants of these strains without the need for enzyme concentration through ultrafiltration. Immunoblot analysis verified that a single 56.5 kDa band, secreted by all three strains, was extremely similar in size and immunologic reactivity to the 59.5 kDa ALiP-P3 isoform of S.viridosporus T7A. In addition, Western blot analysis was used to show that a previously cloned 4.1 kb chromosomal fragment ofS. viridosporus T7A DNA did not contain the ALiP-P3 structural genes.     

Modeling the Biomass Growth and Enzyme Secretion by the White Rot Fungus Phanerochaete chrysosporium: a Stochastic-Based Approach

The white rot fungus Phanerochaete chrysosporium has been identified to be an environmentally useful microorganism for the degradation of various hazardous pollutants, mainly because of its ligninolytic enzyme system, particularly the lignin peroxidase (LiP) secreted by the fungus. In the present work, the behavior of the fungus in liquid medium due to variation in physico-chemical parameters, i.e., glucose concentration, nitrogen concentration, agitation, etc., was studied. Increment of the initial concentration of glucose in the medium increases the biomass growth and LiP activity, when cultured under controlled conditions. The biomass growth and LiP activity by the fungus was modeled following stochastic approach. The behavior of growth and enzyme activity of the fungus observed from the model were found to be in agreement with the experiments qualitatively.     

Physiological aspects of the regulation of extracellular enzymes ofphanerochaete chrysosporiwn

The formation and decay of lignolytic enzymes, along with the generation of other extracellular metabolites in submerged cultures ofPhanerochaete chrysosporium, were studied under different physiological conditions. Whereas lignin peroxidase (LiP) was detectable only in a narrow range of O₂ tension and nitrogen concentration, manganese peroxidase (MnP) reached considerable levels over a broad range. The decay of LiP and MnP activities under lignolytic conditions paralleled that of heme and total proteins. The conditions that decrease or suppress LiP or MnP activities resulted in high levels of extracellular protease activity and/or polysaccharides.     

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.     

Olive mill wastewater treatment by a pilot-scale subsurface horizontal flow (SSF-h) constructed wetland

Performances of a pilot-scale reed bed for the olive mill wastewater (OMW) treatment were investigated, by monitoring influent and effluent pH, total suspended solids (TSS), chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), total phosphorus and polyphenols. In order to reduce the suspended matter concentration and to avoid clogging, OMW was pre-treated by adding lime putty, calcium hydroxide and hydraulic lime. The best results were obtained with 2 g/L of hydraulic lime. Pre-treated OMW was dosed in the reed bed at dilution ratios of 1/3 and 1/10 (v/v), pointing up that the latter only did not give rise to reed suffering and allowed to obtain good and durable removal efficiencies, above all for COD (74.1+/-17.6%) and polyphenols (83.4+/-17.8%). Recycling of the effluent was quite effective for the improvement of the wastewater quality, allowing a further removal of 26-70%, depending on the parameter taken into account. A post-dosage study, carried out by feeding the reed bed with the effluent of an activated sludge plant, pointed up a rapid decreasing of the outlet concentrations of the investigated parameters to values compatible with Italian regulations concerning wastewater discharge in surface water. Polyphenols were the exception, being their outlet concentration at the end of post-dosage study around 2 mg/L.     

Oxidation of crocein orange G by lignin peroxidase isoenzymes

The ligninolytic enzyme system ofPhanerochaete chrysosporiun is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium ofP. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from theN-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H₂O₂ concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 μM COG was obtained when the H₂O₂ concentration was 150 μM. This was also the lowest H₂O₂ concentration for maximal decolorization of 100 μM COG, regardless of the amount of lignin peroxidase used in the reaction.     

Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Bleacing of olive mill wastewater by clay in the presence of hydrogen peroxide

Treatment of olive mill wastewater (OMW) with clayey soils in the presence of hydrogen peroxide (H₂O₂) allows the elimination of phenolic compounds responsible for the black-brownish color of this industrial effluent. The aim of this research was to define optimal physicochemical parameters for the bleaching of OMW with clay in the presence of hydrogen peroxide. Two clayey soil powders were tested (A and B) and the results obtained indicate that high bleaching could be reached after 24 hours exposure of OMW to 7 % (W/V) clay material A in the presence of 0.5 % (V/V) hydrogen peroxide. Under these conditions, the bleaching led to about 87 % decrease of polyphenols (PF) and a 66 % decrease of the Chemical Oxygen Demand (COD). The structure of clay and its concentration in iron salts have an effective adsorbent and catalytic effect on the removal of the majority of polyphenols.     

Mineralization and Kinetics of Reactive Brilliant Red X-3B by a Combined Anaerobic–Aerobic Bioprocess Inoculated with the Coculture of Fungus and Bacterium

Mineralization of Reactive Brilliant Red X-3B by a combined anaerobic–aerobic process which was inoculated with the co-culture of Penicillium sp. QQ and Exiguobacterium sp. TL was studied. The optimal conditions of decolorization were investigated by response surface methodology as follows: 132.67 g/L of strain QQ wet spores, 1.09 g/L of strain TL wet cells, 2.25 g/L of glucose, 2.10 g/L of yeast extract, the initial dye concentration of 235.14 mg/L, pH 6.5, and 33 °C. The maximal decolorization rate was about 96 % within 12 h under the above conditions. According to the Haldane kinetic equation, the maximal specific decolorization rate was 89.629 mg/g˙h. It was suggested that in the anaerobic–aerobic combined process, decolorization occurred in the anaerobic unit and chemical oxygen demand (COD) was mainly removed in the aerobic one. Inoculation of fungus QQ in the anaerobic unit was important for mineralization of X-3B. Besides, the divided anaerobic–aerobic process showed better performance of COD removal than the integrated one. It was suggested that the combined anaerobic–aerobic process which was inoculated with co-culture was potentially useful for the field application.     

Comparison of the performances of Ti/SnO2-Sb, Ti/SnO2-Sb/PbO2, and Nb/BDD anodes on electrochemical degradation of azo dye

In this paper, the preparation conditions of antimony-doped SnO₂ and PbO₂ electrode were optimized for the degradation activity of AO7 dye solution. The results showed that when the doping content of Sb is 8 mol %(SnO₂-Sb(0.08)), the SnO₂ electrode exhibited best activities for the decolorization and mineralization of AO7. The concentration of NaF in electroplating solution had a noticeable effect on PbO₂ electrode for the decolorization of AO7 solution, but little influence on the COD removal rate. The anodic stability tests showed that the electrode prepared in the solution containing 0.10 g l⁻¹ NaF (PbO₂-F(0.10)) was best for environmental application. The comparison of SnO₂-Sb(0.08), PbO₂-F(0.10) and Boron-doped Diamond (BDD) electrodes revealed that a more rapid decolorization rate was obtained on SnO₂-Sb(0.08) and PbO₂-F(0.10) electrodes in dilute AO7 solutions, while higher COD removal rate of concentrated AO7 solutions was on BDD and SnO₂-Sb(0.08) electrodes. The effect of concentration of Na₂SO₄ on the degradation rate of AO7 was very notable on BDD electrode for its highest overpotential of oxygen evolution reaction. In the chloride-containing medium, the decolorization was accelerated greatly but the completed mineralization of AO7 was inhibited with the increasing of chloride ions concentration when these high-overvoltage anodes were used Published in Russian in Elektrokhimiya, 2008, vol. 44, No. 7, pp. 865–875. The text was submitted by the authors in English.     

Industrial indigo dyeing wastewater purification: Effective COD removal with peroxi-AC electrocoagulation system

Direct current (DC) electrocoagulation has obvious decolorization effect on indigo dyeing wastewater produced in actual production, while the removal rate of chemical oxygen demand (COD) was less than 40%, accompanied by serious electrode corrosion and high energy consumption. In order to enhance the COD removal rate, reduce the electrode loss and power consumption, a peroxi-alternating current (AC) electrocoagulation (EC) system was constructed. With COD as the main evaluation index, the effects of power type, electrode combination and process parameters on COD removal rate and decolorization rate were focused on and explored, as well as revealing the degradation mechanism of COD. The results showed that AC electrocoagulation enhanced the floc adsorption capacity by improving the floc structure, and the COD removal rate after treatment was 51.19%. The coupled system of peroxi-AC electrocoagulation further removed sulfite and residual dyes in the wastewater, and the COD removal and decolorization rate of the treated wastewater reached 78.09% and 98.47%, respectively. In addition, the specific energy consumption analysis of COD removal showed that the coupled system was far less energetic than the DC electrocoagulation process, with only 30% of its energy consumption. The uniform corrosion of the electrode under the action of AC weakened the passivation, which reduced the electrode loss and power consumption by 22.73% and 43.75%, respectively. Results from the present work indicated that the peroxi-AC EC system could be an effective method for reducing the concentration of COD in indigo dyeing wastewater.     

Octyl glucoside inhibits [14C]DHP mineralization whereas peroxidase activity is stimulated inPhanerochaete Chrysosporium

Octyl glucoside stimulated peroxidase formation inPhanerochaete chrysosporium ME-446 cultivated in cellulose-based media. Addition of 0.1% of the nonionic surfactant resulted in a ninefold (143 U/L) and sixfold (119 U/L) increase in LiP formation under conditions of N limitation and N excess, respectively. Octyl glucoside also stimulated MnP formation, but to a lesser extent than observed with LiP. The cellobiose-oxidizing enzymes (cellobiose dehydrogenase and cellobiose:quinone oxidoreductase) were stimulated by octyl glucoside when used at a concentration of up to 0.05%, but higher concentrations gave values similar to those for the controls. Little proteolytic activity was detected in the presence of the surfactant. In general, activities of the enzymes studied were of the same order as those seen using Tween-80. In contrast with Tween-80, octyl glucoside markedly inhibited [¹⁴C]DHP mineralization. Attempts to account for the observed inhibition of synthetic lignin degradation by P.chrysosporium in the presence of octyl glucoside are discussed.     

Determination of pKa values of some hydroxylated benzoic acids in methanol-water binary mixtures by LC methodology and potentiometry

An accurate estimation of pK_a values in methanol-water binary mixtures is very important for several separation techniques such as liquid chromatography and capillary electrophoresis that use these solvent mixtures. In this study, the pK_a values of 11 polyphenolic acids have been determined in methanol-water binary mixtures (10%, 20% and 30% (v/v)) by potentiometry, liquid chromatography (LC) and LC-DAD methodology.The results show a similar trend for the pK_a values of all the studied compounds, as they increase with increasing concentration of organic modifier, which allows a linear relationship between pK_a values and mole fraction of methanol to be obtained. The pK_a values obtained in aqueous medium have been compared with those given in the literature, and also with the values predicted by the SPARC on-line pK_a calculator. The data obtained have been used to test the feasibility of an estimation of dissociation constants in a methanol-water medium from the relationship between pK_a values and the organic cosolvent fraction in the mixtures.     

Aerobic Biodegradation of a Sulfonated Phenylazonaphthol Dye by a Bacterial Community Immobilized in a Multistage Packed-Bed BAC Reactor

A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (η _COD ) and 4-ABS (η _ABS ) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a–c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.     

Variations in some extracellular enzyme activities during degradation of lignocellulose byPhanerochaete chrysosporium

The white-rot fungusPhanerochaete chrysosporium is able to degrade lignin only when its primary growth phase is completed. We have recently shown that the organism is able to establish new growth at 10–15 d intervals by recycling its own nitrogen (2). We have now further characterized this growth-rest cycle by measuring changes in extracellular protease, cellulase, and xylanase activities together with total extracellular protein during growth on different carbon sources.

1. WhenP. chrysosporium is grown on a N-limited glucose medium, the cessation of primary growth is closely connected to the increase in extracellular proteolytic activity. When the culture is not O₂-limited (2) it becomes ligninolytically active after about 2 d with a simultaneous decrease in proteolytic activity and an increase in extracellular protein. In O₂-limited cultures, the proteolytic activity remains on a high level for up to 6–7 d. During the second growth phase, the proteolytic activity again increases.
2. When P.chrysosporium is grown on a N-limited glucose medium supplemented with lignocellulosic materials the cellulase and xylanase activities are suppressed and the growth is again connected to an increase in extracellular proteolytic activity. Lignin is not degraded during the growth phases.
3. When P.chrysosporium is grown on a N-limited medium with lignocellulose as the only energy source, the growth phases are connected with increased cellulolytic, xylanolytic, and proteolytic activities. Again during the growth phase, lignin is not degraded. During the ligninolytic phase the level of measured extracellular enzyme activities decreases. A simultaneous increase in total extracellular protein seems to indicate that these enzymes are partly reused for synthesis of the ligninolytic system. Proteins associated with the ligninolytic system appear to be partly reused to synthesize the hydrolytic enzymes for the next growth phase.

     

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.     

Efficient photocatalytic treatment of sugar mill wastewater with 2%Ag3PO4/Fe/GTiP nanocomposite

The large amount of colored substances exist in the sugar mills wastewater that give higher organic load to the effluent. Therefore, a novel study of sugar mill wastewater treatment was carried out under photocatalysis by using a nanocomposite of silver phosphate-iron-graphene oxide-titanium phosphate (Ag₃PO₄/Fe/GTiP). The catalyst was prepared by simple chemical process with 2% content of Ag₃PO₄ to Fe/GTiP. The light, catalyst dosage, pH, and scavenger impacts on the decolorization and chemical oxygen demand (COD) removal from the sugar mill wastewater were observed. The highest decolorization and COD removal of 85.02% and 80.3% was achieved under pH-1 by using 50 W visible halogen light at catalyst dosage of 100 mg/75 ml in 200 min. The excellent recycled results were observed up to four cycles. The obtained results proves that this catalyst has high photocatalytic efficiency to treat the sugar mill wastewater.     

Manganese Peroxidase H4 Isozyme Mediated Degradation and Detoxification of Triarylmethane Dye Malachite Green: Optimization of Decolorization by Response Surface Methodology

A cDNA encoding for manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of alcohol oxidase I promoter. The recombinant MnPH4 was efficiently secreted onto media supplemented with hemin at a maximum concentration of 500 U/L, after which purified rMnPH4 was used to decolorize the triarylmethane dye malachite green (MG). Response surface methodology (RSM) was employed to optimize three different operational parameters for the decolorization of MG. RSM showed that the optimized variables of enzyme (0.662 U), MnSO₄ (448 μM), and hydrogen peroxide (159 μM) decolorized 100 mg/L of MG completely at 3 h. Additionally, UV–VIS spectra, high-performance liquid chromatography, gas chromatography–mass spectrometry, and liquid chromatography–electrospray ionization/mass spectrometry analysis confirmed the degradation of MG by the formation of main metabolites 4-dimethylamino-benzophenone hydrate, N, N-dimethylaniline (N,N-dimethyl-benzenamine), and methylbenzaldehyde. Interestingly, it was found that rMnPH4 mediates hydroxyl radical attack on the central carbon of MG. Finally, rMnPH4 degraded MG resulted in the complete removal of its toxicity, which was checked under in vitro conditions.     

Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion

The anaerobic biodegradability and toxicity of olive mill waste-waters (OMW) were studied in batch anaerobic digestion experiments. Anaerobic digestion of OMW or the supernatant of its centrifugation, the methane production was achieved at up to 5–15% (V/V) dilution corresponding to only 5–20 g/L COD. The washed suspended solids of OMW were toxic at up to 80 g/L COD; however, the kinetic of biodegradability of OMW or the supernatant was faster than for suspended solids, which are constituted meanly of cellulose and lignin. The darkly colored polyphenols induce the problem of biodegradation of OMW, whereas the long chain fatty acids (LCFA), tannins and simple phenolic compounds are responsible its toxicity for methanogenic bacteria.