Manganese peroxidase production from cassava residue by Phanerochaete chrysosporium in solid state fermentation and its decolorization of indigo carmine☆

Huixing Li,Ruijing Zhang ,Lei Tang ,*,Jianhua Zhang ,Zhonggui Mao ,*

1 The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China

2 School of Biological and Chemical Engineering,Nanyang Institute of Technology,Nanyang 473004,China

Keywords:Cassava residue Manganese peroxidase Phanerochaete chrysosporium Solid state fermentation Indigo carmine

ABSTRACT Bioconversion of lignocellulosic wastes to higher value products through fungal fermentation has economic and ecological benefits.In this study,to develop an effective strategy for production of manganese peroxidase(MnP)from cassava residue by Phanerochaete chrysosporium in solid state fermentation,the stimulators of MnP production were screened and their concentrations were optimized by one-at-a-time experiment and Box-Behnken design.The maximum MnP activity of 186.38 nk at·g?1 dry mass of the sample was achieved after 6 days of fermentation with the supplement of 79.5 mmol·L?1·kg?1 acetic acid,3.21 ml·kg?1 soybean oil,and 28.5 g·kg?1 alkaline lignin,indicating that cassava residue is a promising substrate for MnP production in solid state fermentation.Meanwhile,in vitro decolorization of indigo carmine by the crude MnP was also carried out,attaining the ratio of90.18%after 6 h of incubation.An oxidative mechanism of indigo carmine decolorization by MnP was proposed based on the analysis of intermediate metabolites with ultra-high performance liquid chromatography and gas chromatography tandem mass spectrometry.Using the crude MnP produced from cassava residue for indigo carmine decolorization gives an effective approach to treat dyeing effluents.

1.Introduction

Manganese peroxidase(MnP;EC 1.11.1.13)is one of ligninolytic enzymes including MnP,lignin peroxidase and laccase.It was discovered in Phanerochaete chrysosporium in 1983[1],with Mn2+as the preferred substrate to form Mn3+.The formed Mn3+is stabilized by chelators and acts in turn as a highly reactive low-molecular mass,diffusible redoxmediator that attacks organic molecules nonspecifically.Thus MnP is able to oxidize and depolymerize lignin as well as recalcitrant xenobiotics,such as nitroaminotoluenes and synthetic dyes[2].The remarkable degradation potential of MnP makes this enzyme attractive for biotechnological applications,e.g.cellulose pulping and bleaching,and hazardous waste removal[1-3].

These applications depend on the production of MnP at low cost and large scale.Solid state fermentation(SSF)is the most promising process for production of ligninolytic enzymes with fungi[4].Firstly,the SSF process can reduce the cost of enzyme production since substrates are omnipresence,including cereal raw material,agricultural or industrial waste,and wood.Secondly,SSF is environment friendly since less effort is needed in downstream treatment.Finally,the production of ligninolytic enzymes in SSF is usually higher than that in submerged fermentation,because SSF resembles the natural habitat for fungi[5,6].

Different lignocellulosic wastes have been used as raw materials for production of ligninolytic enzymes with SSF[4,7].Cassava residue is a lignocellulosic waste,generated in the distillation step of cassava based ethanol production.About 300000 metric tons(dry mass)of cassava residue is generated per year in China,which leads to disposal problems and may be more serious in the future with the increase of industrial production of ethanol[8].However,to our best knowledge,the production of ligninolytic enzymes from cassava residue with SSF has not been reported.

It is reported that at least 80000 metric tons of synthetic dyes is discharged in industrial effluent every year in the world,creating serious polluting problems since they are recalcitrant,toxic,mutagenic and carcinogenic[9-11].MnP has attracted increasing attention in decolorization and detoxification of synthetic dyes,as it is highly oxidative and substrate nonspecific.Many studies on dye decolorization involving MnP use submerged fermentation by white rot fungi[2,12],but a main obstacle for its development is low efficiency,requiring several days for its application[13].The mechanism of decolorization is also important,which is related to effective applications.Among the decolorization of synthetic dyes by ligninolytic enzymes,laccase-catalyzed reactions of decolorization have been investigated in depth[14,15].However,the decolorization mechanism of MnP is scarcely reported.

In the presentwork,higher value MnP is produced by P.chrysosporium with cassava residue as substrate in SSF.To enhance the MnP production,inexpensive stimulators of MnP are screened and their concentrations are optimized.In addition,in vitro decolorization of staple and toxic dye indigo carmine[16]by the crude MnP is evaluated for its potential in effective treatment of dyeing effluent.Furthermore,an oxidative mechanism of indigo carmine decolorization by MnP is proposed based on the analysis of decolorizing intermediates with mass spectrometry.

2.Materials and Methods

2.1.Raw materials

Cassava residue was provided by Yong Xiang Ethanol Co.,Ltd.,Wujiang,China.The cassava residue was dried at105°C till the constant mass was obtained.Components of cassava residue were as follows:total organic carbon,33.85%±0.17%;total nitrogen,1.53%±0.08%;lignin,20.19%±0.22%;cellulose,31.46%±0.63%;and hemicellulose,10.49%±0.20%.

Soybean oil was obtained from China Oil&Foodstuffs Co.,Ltd.,Beijing,China.Alkaline lignin was obtained from Tokyo Chemical Industry Co.,Ltd.,Tokyo,Japan.Indigo carmine and HPLC grade acetone were obtained from Sinopharm Chemical Reagent Co.,Ltd.,Shanghai,China.All other reagents were of analytical grade.

2.2.Fungal strain and cultivation

P.chrysosporium CICC 40719 obtained from Beina Chuanglian Biological Research Institute was used in this study.Fungus was maintained on potato dextrose agar(PDA)plates at 4°C and periodically subcultured.The conidia were harvested from 7 day old fungal cultures grown on PDA plates.Incubation was carried out in 250 ml Erlenmeyer flasks containing cassava residue and distilled water(1:2,mass ratio)and four 10 mmagarplugs removed from the PDAplate at30°C and humidity 70%.

2.3.Effect of stimulators and mass of cassava residue on MnP activity

Effects of concentration of stimulators and mass of cassava residue were investigated by one-at-a-time experiment.Five gram cassava residue supplemented with different stimulators or different mass of cassava residue was incubated for four days.

2.4.Optimization of stimulator concentration with Box–Behnken design

Based on the experimental results,a Box-Behnken design[17]with three settings for each of three stimulators(acetic acid,soybean oil,and alkaline lignin)were applied to optimize MnP activity in solid state fermentation and to explore the interactions between these variables.The experimental plan and levels of independent variables are shown in Table 1,where X1presents that concentration of acetic acid has a lower limit of 0 mmol·L?1· kg?1and a higher limit of 100 mmol·L?1· kg?1,and X2(concentration of soybean oil)varies between 0 and 6 ml·kg?1.The lower and higher limits of X3(concentration of alkaline lignin)are 0 and 60 g·kg?1,respectively.The design of matrices and analysis of variance for the variables were conducted using the SAS program(Version8.0,SAS Institute,Cary,NC).The p-value was calculated by the program with F-test and considered significant for p-value＜0.05 and very significant for p-value＜0.01.Moreover,under the optimized and un-optimized conditions,cultivations were incubated for 10 days and the time courses of MnP activity were recorded and compared.

Table 1 Box-Behnken design to optimize concentration of stimulators

2.5.Enzyme extraction and determination

Enzyme activities were analyzed from extracted samples.One gram of sample was taken from each flask and mixed with 50 mmol·L?1sodiumtartrate buffer(1/10)at pH 4.5.The mixture was placed in a shaker at150 r·min?1for 1 h and then centrifuged at4500 g for 10 min.The supernatants collected were analyzed for MnP activity.The activity of MnP was measured at 270 nm by quantifying the formation of Mn(III)-malonate complex after incubating reaction mixtures containing 1 mmol·L?1MnSO4and 0.1 mmol·L?1H2O2at 37 °C at certain time intervals.The dry mass of the sample was determined by weighing the sample dried at105°C till constantmass.The MnP activity was calculated as nkat·g?1dry mass of the sample[7].

2.6.In vitro decolorization of indigo carmine by crude MnP

Crude MnP solution was obtained from the supernatants and stored at 4°C.The decolorization of indigo carmine by crude MnP was carried out at30°C.The reaction mixture contained 3.3 mlsodium tartrate buffer(50 mmol·L?1,pH 4.5),0.5 ml crude MnP(activity 5.3 nkat·ml?1),0.1 ml 40 mmol·L?1MnSO4,0.1 ml 4 mmol·L?1H2O2,and indigo carmine(30 mg·L?1).Indigo carmine was added to this mixture in order to get an initial absorbance of approximately 1.0 at its maximum wavelengths(615 nm).Decolorization ratio(%)was calculated by(A0?A)/A0×100%,where A0is the initial absorbance in maximum wavelengths and A is the corresponding final absorbance.A control test containing the same amount of heat-denatured MnP was performed in parallel,by heating MnP in a boiling water bath for 30 min.

2.7.Analysis of indigo carmine and its intermediate metabolites of decolorization

The reaction mixture of indigo carmine and the crude MnP were mixed with alcohol(1:3,volume ratio)for protein precipitation,and centrifuged at 6000 g for 10 min.The supernatants were evaporated to dryness.The obtained crystals were dissolved in small volume HPLC grade acetone for analysis by ultra-high performance liquid chromatography(UPLC)and gas chromatography(GC)tandem mass spectrometry.The UPLC(Waters,USA)was carried out on BEH C18(2.1 mm×100 mm,1.7 μm)column by using acetonitrile/water(10:90,volume ratio)as mobile phase,and the water contained 0.1%formic acid.The flow rate of mobile phase was 0.3 ml·min?1.The GC-MS was applied using a TSQ Quantum XLS(Thermo Scientific,USA).The ionization voltage was 70 eV.GC was conducted in the temperature programming mode with a Rtx-5MS column(0.25 mm,30 m).The initial column temperature was 80 °C for 2 min,then increased linearly at 10 °C·min?1to 280°C and held for 7 min.The temperature of the injection port was 280 °C,and the GC-MS interface was maintained at 290 °C.The helium carrier gas flow rate was 1.0 ml·min?1.

3.Results

3.1.Effect of stimulators and cassava residue mass on MnP activity

Fig.1.Effects of stimulators and cassava residue mass on MnP activity.

The activity of MnP is generally enhanced by the addition of exogenous stimulators because MnP derived from fungi is an inducible enzyme.However,stimulators such as veratryl alcohol,syringic acid,and cyclic adenosine monophosphate are expensive and restricted their application in industry[18-20].Therefore,it is necessary to screen inexpensive stimulators for enhancing MnP production.As shown in Fig.1,alkaline lignin is beneficial for MnP production(a),while hydroquinone and o-phthalic acid inhibit the production of MnP(b,c).For the effects of chelators of MnP including acetic acid(d),methanoic acid(e),and lactic acid(f),85 mmol·L?1·kg?1acetic acid and 55 mmol·L?1·kg?1methanoic acid increase MnP activity to 143.02 and 111.91 nkat·g?1,respectively.Fig.1 also shows the effects of mediator on MnP activity,including oleic acid(g),soybean oil(h)and Tween 80(i).Soybean oil is highly polyunsaturated and could act as a mediator for MnP because it contains carbon-carbon double bond(C=C)[21,22].Fig.1(h)shows that it is efficient and leads to the MnP activity of 93.75 nkat·g?1with the addition of 4 ml·kg?1,while more soybean oil decreases the MnP activity.Fig.1(j)shows that MnP activity increases with the addition of cassava residue,reaching the maximum of 65.87 nkat·g?1with 12.5 g cassava residue added,and then decreases with the cassava residue increased to 15.0 g.It demonstrates that too much cassava residue will limit oxygen diffusion in the flask,resulting in cell growth and inhibiting the metabolism of P.chrysosporium as it is an aerobic process.

3.2.Optimization of stimulator concentration for MnP activity

3.2.1.Analysis of variance

Based on the above experiments,the mass of cassava residue was set to 12.5 g.Acetic acid,methanoic acid,soybean oil and alkaline lignin have been proven to stimulate MnP production significantly.Acetic acid and methanoic acid are similar in chemical properties and structure,and acetic acid is better than methanoic acid for improving MnP activity.Therefore,the concentrations of acetic acid,soybean oil and alkaline lignin are further investigated with Box-Behnken design.Fifteen trials are used to determine optimum concentrations for maximum MnP activity.The results are shown in Table 2.

Table 2 MnP activity at different levels of variables

Due to the complex nature ofMnP production,it is difficult to predict the effect of all stimulators,which may have multiple interactions.Therefore,response surface methodology is applied to obtain a second-order polynomial model[Eq.(1)]for MnP activity(Y)in terms of acetic acid concentration(X1),soybean oil concentration(X2)and alkaline lignin concentration(X3).

The analysis of variance(Table 3)indicates that Eq.(1)is statistically significant and adequate to represent the relationship between the response and variables,with the model p-value＜0.05,lack of fit p-value 0.36(＞0.1).The multiple correlation coefficient of 96.30%also indicates that this model is highly significant and only 3.70%of the total variations is not defined by the model.The coefficient of variance is 5.75%,implying the precision and reliability of the experimental data.The overalleffect of the three variables on the MnP activity is further analyzed by F-test.The results reveal that the quadratic term of soybean oil concentration(X2X2)is very significant,with p-value＜0.01,acetic acid concentration(X1)and its quadratic term(X1X1),quadratic term of alkaline lignin concentration(X3X3),and interactions of acetic acid concentration and alkaline lignin concentration(X1X3)are significant,with p-value＜0.05.

3.2.2.Production of MnP at the optimal concentration of stimulators

The optimal concentrations of stimulators were determined by the ridge max analysis.The method computes the estimated ridge of maximum response for increasing radii from the center of the original design[23].The ridge max analysis indicates that the maximum MnP activity of 163.69 nkat·g?1is obtained at concentrations of acetic acid 79.5 mmol·L?1·kg?1,soybean oil 3.21 ml·kg?1and alkaline lignin 28.5 g·kg?1at the distance of coded radius 0.6.Fig.2 demonstrates the time course of MnP activity under the optimized and un-optimized conditions.Under the optimized conditions,MnP activity attained 162.77 nkat·g?1after 4 days of fermentation and the maximum MnP activity reached 186.38 nkat·g?1on the 6th day.Under the un-optimized conditions,the maximum MnP activity of 93.89 nkat·g?1was also achieved on the 6th day,lower than that under the optimized conditions.The results indicate that addition of stimulators in an optimal concentration enhances the MnP production.

Fig.2.Time course of MnP activity.(? MnP activity under optimized conditions;● MnP activity under un-optimized conditions).

3.3.In vitro decolorization of indigo carmine using crude MnP and its intermediate metabolite analysis

To identify the effect of the crude MnP on the decolorization of synthetic dyes,the process of decolorization of indigo carmine is studied.As shown in Fig.3(a),the visible spectrum absorption of indigo carmine with crude MnP for 30 min(curve 2)decreases sharply compared with that of indigo carmine with crude MnP for 0 min(curve 1),suggesting that the crude MnP decolorizes indigo carmine efficiently.There is no decolorization effect when the MnPis inactivated by heating(curves 3 and 4).Fig.3(b)illustrates the time course of indigo carmine decolorization with the crude MnP and demonstrates that the decolorization mainly occurs in the first 30 min,and the maximum decolorization ratio reaches 90.18%after 6 h of incubation.

Fig.3.Indigo carmine decolorization by the crude MnP.(a)Visible absorbance spectra of reaction mixture before and after treatment(1—with addition ofMnP,0 min;2—with addition of MnP,30 min;3—with addition of heat-denatured MnP,0 min;4—with addition of heat-denatured MnP,30 min);(b)time course of indigo carmine decolorization.

Fig.4.Absorption peaks of indigo carmine decolorization at 615 nm(A)and 550 nm(B)by UPLC.(a)Standard sample;(b)decolorization 15 min;(c)decolorization 30 min;(d)decolorization 1 h;(e)decolorization 6 h.

To investigate the catalytic action of MnP,the intermediate metabolites of indigo carmine decolorization were analyzed by UPLC and GCMS.As shown in Fig.4(A),the retention time of indigo carmine in UPLC is approximately 3.0 min,and the peak area at 615 nm,which is the wavelength of maximum absorbance of indigo carmine,decreases from 9397 to 0 in the decolorization.The visible absorbance spectra of the reaction mixture[Fig.3(a)]show that decolorization metabolites have slight absorption at 550 nm,detected by UPLC.The retention time of the decolorization metabolites was approximately 0.7 min and the absorption peak area did not increase continuously between 830 and 360[Fig.4(B)],indicating that the products were intermediates rather than final products.The purification of the products is difficult and this is consistent with the previous report[24].Therefore,GC-MS was employed to identify the products,and isatin was found with the retention time of 14.95 min through database mining(Fig.5).The mass spectrum of isatin is shown in Fig.6.Isatin,an orange-red product,is the main product in oxidation of indigo carmine with electro-Fenton and photoelectro-Fenton.Oxidation of indigo carmine breaks its C=C double bond,forming two molecules of isatin[25].

Fig.5.GC elution profile of indigo carmine decolorization metabolites.

Fig.6.Mass spectrum of isatin.

4.Discussion

Some reports emphasize on the selection of stimulators for enhancing MnP production[19,26].In this study,three inexpensive stimulators,acetic acid,soybean oil and alkaline lignin,are screened out and their concentrations were optimized.The maximum MnP activity was achieved with the supplement of 79.5 mmol·L?1·kg?1acetic acid,3.21 ml·kg?1soybean oiland 28.5 g·kg?1alkaline lignin.Organic acids stimulate MnP production because of their chelator role in the presence of manganese[19].The unsaturated fatty acids and their derivatives can mediate MnP through peroxidation of the unsaturated C=C in the fatty acids,which enhance the production and oxidizing capacity of MnP[22].Lignin and phenolic compounds are substrates of MnP and actas stimulators,such as indulin AT,veratryl alcohol,3,4-dimethoxycinnamicacid,and 3,4,5-trimethoxycinnamic acid[18,26,27].Alkaline lignin is mainly utilized in the production of biodegradable materials as its renewable,degradable and low-cost particle[28].In this study,the experimental result shows that alkaline lignin enhances MnP production effectively,extending its application range.

Lignocellulosic wastes such as barley bran,wheat bran and wheat straw are extensively used for production of ligninolytic enzymes[4-6].In this study,cassava residue is used as substrate for MnPproduction.The maximum MnP activity achieved 186.38 nkat·g?1in SSF by P.chrysosporium after 6 days of fermentation.Table 4 lists severalsuccessful examples of MnP production from lignocellulosic wastes in SSF.MnP activity is usually lower without stimulators added[7,29,30]and higher with the addition of inorganic salt and organic nitrogen stimulators[30,31].Moreover,MnP activity is significantly affected by the variety of substrates and stimulators[32,33].It usually needs 14 days ofcultivation to reach the maximum MnP activity,while we obtain the maximum MnP activity in the same order of magnitude in 6 days of cultivation,suggesting that the productive efficiency of MnP in this study is superior.It demonstrates that cassava residue is a promising substrate for MnP production,which may be due to its biodegradability derived from the process of mechanical and biological treatment[8,34].

Table 4 MnP activity in solid state fermentation using lignocellulosic wastes

Fig.7.An oxidative mechanism for MnP-catalyzed decolorization of indigo carmine.

Indigo carmine is extensively employed in industries,such as paper,plastic and textile,generating dyeing effluent.This anionic dye is highly toxic[16].In this study,we use the crude MnP for the decolorization of indigo carmine,attaining the decolorization rate of 90.18%after 6 h of incubation.Furthermore,isatin is identified as an intermediate metabolite of indigo carmine decolorization.Based on identification of the isatin and catalysis mechanism of MnP[1,14,25,35],a pathway for decolorization of indigo carmine by MnP is proposed as depicted in Fig.7.Electrochemical oxidation of indigo carmine appears first since sulfonic acid groups and amino-aromatic groups are oxidized by MnP catalysis.Then the oxidative compound is easily attacked by nucleophiles such as water molecules.Finally,another oxidation step degrades the products to form isatin since phenolic compounds are oxidized by hydrogen abstraction.Isatin is mineralized further,with final products of indigo carmine decolorization separated and determined.The proposed mechanism will enrich the pool of heme peroxidases and help its application in industry.

5.Conclusions

Cassava residue could be effectively used to produce MnP by P.chrysosporium in solid state fermentation.Supplements of acetic acid,soybean oil and alkaline lignin stimulated MnP production significantly.In addition,indigo carmine was effectively decolorized by the crude MnP from the solid state fermentation.An oxidative mechanism for the decolorization of indigo carmine by MnP was proposed based on the analysis of intermediate metabolites by UPLC and GC-MS.