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Accepted Manuscript

Title: ISOLATION AND BIOELECTROCHEMICALCHARACTERIZATION OF NOVEL FUNGAL SOURCES

WITH OXIDASIC ACTIVITY APPLIEDIN-SITUFOR THE

CATHODIC OXYGEN REDUCTION IN MICROBIAL

FUEL CELLS

Author: Kyriale Vasconcelos Morant Paulo Henrique da Silva

Galba Maria de Campos-Takaki Camilo Enrique La Rotta

Hernandez

PII: S0141-0229(14)00137-9

DOI: http://dx.doi.org/doi:10.1016/j.enzmictec.2014.07.007Reference: EMT 8665

To appear in: Enzyme and Microbial Technology

Received date: 28-5-2014

Revised date: 14-7-2014

Accepted date: 25-7-2014

Please cite this article as: Morant KV, Silva PH, Campos-Takaki GM, Hernandez CELR,

ISOLATION AND BIOELECTROCHEMICAL CHARACTERIZATION OF NOVEL

FUNGAL SOURCES WITH OXIDASIC ACTIVITY APPLIED IN-SITUFOR THECATHODIC OXYGEN REDUCTION IN MICROBIAL FUEL CELLS., Enzyme and

Microbial Technology(2014),http://dx.doi.org/10.1016/j.enzmictec.2014.07.007

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http://dx.doi.org/doi:10.1016/j.enzmictec.2014.07.007http://dx.doi.org/10.1016/j.enzmictec.2014.07.007http://dx.doi.org/10.1016/j.enzmictec.2014.07.007http://dx.doi.org/doi:10.1016/j.enzmictec.2014.07.007

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ISOLATION AND BIOELECTROCHEMICAL CHARACTERIZATION OF1

NOVEL FUNGAL SOURCES WITH OXIDASIC ACTIVITY APPLIED IN-SITU2

FOR THE CATHODIC OXYGEN REDUCTION IN MICROBIAL FUEL3

CELLS.4

Kyriale Vasconcelos Morant, Paulo Henrique da Silva, Galba Maria de Campos-5

Takaki and *Camilo Enrique La Rotta Hernndez.6

NPCIAMB - Ncleo de Pesquisas em Cincias Ambientais e Biotecnologia Centro de7

Cincias e Tecnologia (CCT) - Universidade Catlica de Pernambuco UNICAP,8

Recife PE, Brasil. Rua Nunes Machado, 42, Bloco J, Trreo, Boa Vista, 50050-590,9

Recife Brasil. Phone: +55 (81) 9927161210

*Corresponding author: [email protected]

ABSTRACT:12

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and13

Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatingas soils and14

applied during the in-situ cathodic oxygen reduction in fuel cells. All strains were15

cultivated in submerged cultures using and optimized saline medium enriched with 10 g16

L-1

of glucose, 3.0 g L-1

of peptone and 0.0005 g L-1

of CuSO4 as enzyme inducer.17

Parameters of oxidase activity, glucose consumption and microbial growth were18

evaluated. In-cell experiments evaluated by chronoamperometry were performed and19

two different electrode compositions were also compared. Maxima current densities of20

125.7, 98.7 and 11.5 A cm-2were observed before 24 h and coulombic efficiencies of21

56.5, 46.5 and 23.8% were obtained for SIS 31, SIS 21 and SIS 18, respectively.22

Conversely, maxima power outputs of 328.73, 288.80 and 197.77 mW m-3

, were23

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observed for SIS 18, SIS 21 and SIS31, respectively. This work provide the primary24

experimental evidences that fungi isolated from the Caatinga region in Brazil can serve25

as efficient biocatalysts during the oxygen reduction in air-cathodes to improve26

electricity generation in MFCs.27

28

Keywords: Oxidases,Filamentous fungi, Biocathodes, Biofuel cells, Cathodic Oxygen29

reduction.30

1. INTRODUCTION31

The gradual depletion of fossil fuels and the environmental concerns for their32

consumption have driven an intensive search for alternative sources for energy33

production. BioFuel Cells (BFC) are considered a promising alternative for clean energy34

generation and also obey general sustainability requirements (Karatay and Donmez,35

2011). However, the high cost of noble metals such as Au, Pt, Rh and Os, commonly36

used in coated electrodes as catalysts is still considered one of the limiting factors for37

scaled-up applications of microbial fuel cells (MFC) and conventional fuel cells. Even38

though, abiotic cathodes that use oxygen as electron acceptor are frequently adopted for39

BFC (Logan et al., 2006; Luo et al., 2010). Enzymes as biocathodes can potentially40

eliminate limiting factors such as: decreased efficiency due to the accumulation of41

metabolites, work under mild reaction condition such as temperature and pressure.42

Additionally, due to their high substrate specificity they are able to perform the electron43

transfer throughout suitable mediated systems and employing co-substrates (Da Silva et44

al., 2014; Farneth and DAmore, 2005). These types of enzymatic cathodes have been45

investigated in small scale enzymatic biofuel cells (Farneth and DAmore, 2005). On46

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the other hand, energy production obtained from the BFC is not yet satisfactory and47

their performance and power output generation can be affected by a number of factors,48

such as cellular activity, substrate biotransformation and the inefficient electron transfer49

from the biocatalysts to the electrodic materials. Studies on enzymes for electron50

interactions are being mainly focused on copper-containing oxidoreductases (Figure 1),51

which can catalyze the direct reduction of oxygen while perform the simultaneous52

oxidation of many organic compounds such as phenols. Mono and poly-phenol oxidases53

from fungal species such as:Agaricus bisporus (Shervedani and Amini, 2012), Coriolus54

hirsutus (Farneth and DAmore, 2005), Trametes versicolor (Lou et al., 2010)55

Coriolopsis gallica(Tinoco et al., 2001) and Pleurotus ostreatus(Barton et al., 2002);56

plant laccase from Rhus vernicifera; and bacterial laccase from Streptomyces57

coelicolor, were already studied and applied to these bioelectrodes (Shleev et al.,58

2005). Others less electrochemically explored, but highly promising corresponds to the59

fungal bilirubin oxidase (BOD) from Myrothecium verrucaria (Ivnitski et al. 2008;60

Mano et al., 2002) and bacterial BOD fromBacillus pumilus (Durand et al. 2012).61

EC 1.10.3.1 : Tyrosinase: polyphenol oxidase:62

2 catechol+O22 1,2-benzoquinone+2 H2O Eq. 163

EC 1.10.3.1: Laccase : Urishinol Oxidase64

4 benzenediol+O24 benzosemiquinone+2 H2O Eq. 265

EC 1.3.3.5: Bilirubin oxidase66

2 bilirubin +O22 biliverdin +2 H2O Eq.367

68

Laccase (LAC) and tyrosinase (TYR) are able to oxidize phenolic compounds and to69

reduce simultaneously oxygen into water (Eq. 1 and 2). Depending the microbial70

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source from which these enzyme were extracted, the redox potential of the T1site, may71

vary from 430 mV up to 780 mV vs.NHE (Palmore and Kim, 1999). Laccase from72

Trametes versicolor is the most attractive one since redox potential of its T1 site is73

ca.780 mVvs.NHE (Shleev et al., 2005). Nowadays, the best performances with laccase74

electrodes are obtained with osmium based polymers as redox mediators (Mano et al.,75

2002) Actually these electrodes are able to deliver a current density of 860 A cm-2

at76

only -70 mVvs. O2/H2O at pH 5.0. Under the same conditions, an identical current77

density is obtained at -400 mVvs.O2/H2O with a platinum wire as catalyst.78

Nevertheless, performances of laccase from Pleurotus Ostreatus electrodes drop79

drastically in the presence of chloride ions what constitutes both a major problem and a80

great challenge for its use in biofuel cells (Barton et al., 2002). On the other hand, BOD81

(Eq. 3) is naturally capable of catalyzing the oxidation of bilirubin into biliverdin and to82

simultaneously reduce dioxygen (Li et al., 2004). BOD is very similar to laccase. BOD83

electrodes are greatly related to the amino-acids sequence around T1 site of the enzyme84

(Shimizu et al. 1999). It is clearly reported that the most efficient BOD enzyme comes85

from Myrothecium verrucaria. Redox potential of its T1 site is included between 65086

and 750 mV vs. NHE, and its future application in BFC its close related with the87

observed thermal stability up to 60 C (Mano et al., 2002). These biocatalysts have88

been extensively used in cathodes for enzymatic fuel cells and electrochemical89

biosensors due to their high redox potential, however the almost mandatory use of90

electron shuttles such as 2,2 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)91

and other suitable molecules more recently studied as triphenylmethane dyes has been92

widely recognized as an effective way to avoid the loss of current during the93

bioelectrochemical process (Bach et al., 2013; Smolander et al. 2008; La Rotta et al.,94

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2011). However, the application of such enzymatic fuel bioelectrodes has been limited,95

specially attributed to the high costs of production and purification and the short half-96

life time associated with the enzyme inactivation in non-biological environments as the97

ones commonly found in the surface of electrodic materials of BFC. In this regard,98

biocathodes inoculated with the fungi for the in-situoxidase production may offer a99

potential solution (Wu et al., 2012; Rachinski et al., 2010). Also the in- situsecretion100

of oxidases by the filamentous fungi in air cathodes might be a more attractive way to101

achieve sustainable and cost-efficient electricity generation, especially for three main102

reasons: longer life-time of the biocatalysts since these are being produced under more103

compatible biological conditions; the use of low cost substrates such as residua or104

contaminated effluents; and the possibility of concomitant production of other natural105

occurring electro active molecules as microbial by-products like: azaphylones, quinone-106

like pigments as melanins, terpene as carotenoids, etc. The simultaneous utilization of107

such molecules could improve even more the couloumbic efficiencies by reducing the108

charge and mass transportation problems previously observed for these systems.109

PLEASE INSERTT FIGURE 1 HERE110

Currently, the Brazilian North and Northeast Network of Filamentous Fungi111

(RENNORFUN) aims to describe the biodiversity of filamentous fungi in soils from112

the Caatinga and the Amazon regions of Brazil throughout poly-phasic and molecular113

taxonomy as well as to demonstrate the applicability in industrial processes of the114

isolated micro-organisms and their by-products. In this context, this study aimed the115

isolation and identification of novel fungal species capable to produce biocatalysts with116

high oxidasic activity that can be applied to the cathodic reduction of oxygen in117

electrodes for biosensors and BFCs.118

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119

120

2. METODOLOGY121

2.1. Fungal Strains, media and cultivation conditions122

All strains belong to the RENNORFUN Culture Collection from the Catholic University123

of Pernambuco Brazil, stocked in slant tubes containing Sabouraud agar solid medium124

under refrigeration at 4o

C until their use. Initial selection was based on previous in-125

plate observations associated with pigment production and oxidase or tannase activities,126

since pigment production can be by-products of the reactions catalyzed by these127

enzymes (Koroljova-Skorobogat'ko et al., 1998; Saparrat et al., 2002). Table 1.Shows128

the culture media used for selection of fungal strains with oxidasic activity. Twelve129

fungal strains were originally chosen: two Rhizopus spp.; threeAspergillus spp.; three130

Penicillium spp.; twoEupenicillium spp and two Talaromycesspp. The microorganisms131

were visual evaluated in terms of substrate degradation and color formation, by132

cultivation in solid plates incubated for 48 at 28o C. For submerge cultures, a pre-133

inoculum of young mycelium disks of 0.8 cm of diameter were obtained from 2 days-134

old colonies in solid medium. Disks were disrupted in tubes and mycelium was re-135

suspended in fresh medium and incubated at 28o

C and 180 rpm for 48 hours. After this136

period of time, tubes were used for the inoculation of flasks containing 150 to 200 mL137

or microbial cathodic compartiments of 100 mL of capacity at the bicompartmented138

BFC. All cultures were incubated under the same controlled lab conditions.139

140

2.2. Microbial growth parameters and sample post-treatment141

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Comparisons between media composition and the effect over microbial parameters of:142

oxidase enzyme activity, final biomass (dried weight) and substrate consumption were143

performed. First, samples and culture brothswere separated from mycelia by filtration144

through nylon cloth and centrifugation at 4500 rpm for 20 minutes at 4o C. Mycelia145

were dried on paper filters at 60o C until constant weight. Finally, cell free supernatants146

were used for the quantification of glycerol or glucose using specific enzymatic kits147

purchased from BIOCLIN. Enzyme activities were determined using the below148

described methodologies.149

150

2.3. Oxidasic activity assays151

Enzymatic crude extracts and fermentation samples were evaluated in terms of oxidase152

activity using the methods summarized in Table 2. Oxidase and peroxidase activities153

were distinguished throughout similar methods; however for peroxidase activity 5.0154

mmol L-1

hydrogen peroxide was added instead a saturated oxygen buffer solution as155

oxidizing agent (assays 1 and 5, respectively). Mono and polyphenol oxidases were156

differentiated using assays 2 and 3 both in saturated oxygen buffer solutions. And157

finally the routine assay 4 was used for oxidase activity. All enzyme activities were158

expressed as international units per mL (UI mL-1

), defined as the amount of enzyme159

required to produce 1 mol mL-1 of the specific oxidized product, according to the160

specific molar extinction coefficients, per minute under the reaction conditions used in161

each assay. The increase in absorbance was monitored in a UV-Vis spectrophotometer162

BioChrome Libra S32 .163

PLEASE INSERT TABLE 1 HERE164


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2.4. Electrochemical analyses166

Chronoamperometric analysis:A bicompartmented BFC was employed (Scheme 1). A167

100 mL cathodic compartment was composed by the fungal culture using the selected168

medium, and two electrodic materials were evaluated. Immersed carbon felt electrodes169

coated with carbon Black Vulcan plus 0.5% Pt (w\w) in PTFE or a Pt-free Black170

Vulcan coated carbon felts; As anodes, immersed plates of exploded graphite in 20171

mmol L-1

potassium ferrocyanide were used. All electrodes had 19.6 cm-2

of surface172

area. As cation exchange system a saline bridge of agar in saturated KCl was used. The173

potential (E) vs. time (min) was recorded using a multimeter Fluke 8080 with data174

acquisition software. All experiments were allowed to stabilize for about 30 minutes175

before each measurement. Chronovoltammetric data were converted into Iddata using176

the Ohms Law expression (Equation 4) since an external load resistance of 1 K was177

employed.178

Id= E * R Eq. 4179

The Coulombic efficiency (%) in Equation 5, was calculated from the following180

expressions according to previous studies (Logan et al., 2006; Dantas et al., 2013).181

CE= (CR/CT) Eq. 5182

where, CTcorresponds to the theoretical amount to be obtained from each substrate and183

CR corresponds to the practical coulombs recovery from the substrate. CT can be184

calculated at any time using the expression:185

CT = nzF Eq. 6186

wherenis the moles of substrate, zare the moles of electrons per mol of substrate (O2=187

4e-, Glucose = 6 e

-or Glycerol= 12 e

-); and F is the Faradays constant 96485.4 C188

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mol-1. Applying Equation 6 to an integrated model of Id vs time for a microbial fuel189

cell we obtain Equation 7190

CE= M / F z VAC/CC S Eq. 7191

where M is the substrate molarity, A the electrode surface area, V is the total volume192

circulated inside the cathodic compartment and Sis the final substrate concentration.193

PLEASE INSERT SCHEME 1 HERE194

Cyclicvoltammetric analysis: The electrochemical system was composed by a glass 15195

mL glass cell, a 0.5 cm diameter glassy carbon as working electrode, a Pt wire of 1.0 cm196

as counter-electrode and Ag|AgCl2in saturated KCl as reference electrode. As enzyme197

substrate 1.0 mmol L-1

pyrogallol was used. And as support electrolyte 100 mmol L-1

198

Sodium acetate buffer solution pH 5.0 was employed. The cell was coupled to a199

PalmSens potentiostat/galvanostat with data acquisition software PS-Trace 4.4.200

Cyclic voltammetry parameters included: Pre-treatment of -1.2 V, 10 s; E deposition: -201

1.0 V, 10 s; T eq.: 8 s; E start: 0 V; E vertex 1: -0.2 V; E vertex 2: 1.0 V; E step: 0.005202

V and Scan rate: 0.05 V s-1

. All readings used at least 5 scans.203

204

Polarization analysis and power output determination: Using the same electrochemical205

system for the MFC, but placing the reference electrode at the cathodic compartment206

and same apparatus described above, samples with the highest recorded activity were207

analyzed in terms of cathodic current densities within potential range of: 0.80 to -0.5 V.208

The following parameters were used: OCV of 0.85 V vs Ag|AgCl in sat. KCl; Linear209

sweep: 8 s, Eo = 0.8 V, Ef =-0.5 V, Estep= 0.002 V, Scan rate 0.01 V s-1

. The polarization210

curves of Id vs E obtained were used to the determination of the Power-output curves (Pd211

vs E) and maxima Pdvalues according to Equation 8212

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Pd= E * Id Eq. 8213

214

215

3. RESULTS AND DISCUSSION216

3.1. Screening of novel fungal oxidases217

Initially the solid culture medium A (MA) was supplemented with gallic acid or tannic218

acid as carbon sources, to visualize the production of oxidase or tannase in plates by the219

formation of green to black halos according to previous studies (Leite et al., 2012;220

Koroljova-Skorobogat'ko et al., 1998; Saparrat et al., 2002). Results from the screening221

in MA supplemented with those substrates can be observed in Table 3. Six of twelve222

strains presented the availability to degrade tannic acid and only four were able to223

oxidize efficiently gallic acid. Unexpectedly, only one strain (SIS-21) showed both224

pigments production and exhibited high oxidase and tannase activities, while the other225

pigment producers strains showed low to moderate tannase activity and none oxidase226

activity. Since one of the goals of this study was also to find fungal strains able to227

biotransform alternative carbon sources, different than glucose (Ex. Glycerol), with the228

retention of the oxidasic activity, the use of a modified medium A or (MMA), enriched229

with glycerol was also tested. Nevertheless, only strains SIS-18, 21, 31 and 39 retained230

the availability of performing the oxidization of gallic acid in plates while grew very231

well in a medium containing glycerol.232


This also was confirmed when the pre-selected strains according to the halo formation234

in plates containing (MA) and (MMA) media, were cultivated in liquid medium (B) or235

modified medium B (MMB) where glycerol was added instead glucose as can be seen in236

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Table 4. As such, no oxidase activity was observed at all in MMB. At this point the best237

oxidase producers corresponded to strains SIS-21, 31 and 18, with 5, 1.7 and 1.6 KU238

mL-1, respectively. For the next experiments only these three strains were evaluated.239

Finally, since the presence of copper was already mentioned as a beneficial inducer of240

oxidase enzymes as blue-oxidases (Ex. LAC, TYR and BOD) (Fonseca et al., 2010),241

this was added to the medium B (MB), now called Copper-medium B or (CuB). As it242

was expected a significant increase on oxidase activity was observed in all cases. This243

increase represents almost 1.65 fold-times in the case of the oxidasic activities observed244

for SIS 18 and SIS-21, an almost 2.4 fold-times in the case of SIS-31. In contrast, SIS245

21 showed the highest values for fungal growth in terms of dried weight of 1.02 g, and246

lower fungal growths were observed for SIS 31 followed by SIS 18, with 0.43 and 0.95247

g, respectively. In terms of glucose consumption, all three strains caused the complete248

depletion of this substrate in 120 h.249


Since the oxidation of pyrogallol can be unspecific in terms of which oxidasic activity is251

present in the fungal cultures, specific assays were performed using cell free samples252

obtained from submerged cultures in CuB medium, in a way to identify which one of253

the oxidase activities (monophenol oxidase - LAC, polyphenol oxidase -TYR or254

bilirubin oxidase - BOD) or eventually if peroxidase activity (POD) was present. Table255

5 shows this activity characterization performed for the three selected fungal strains.256

Thus, as clearly appeared, none of the evaluated strains showed any POD activity, since257

no oxidation products were observed in the absence of oxygen and the presence of258

hydrogen peroxide. Likewise, when BOD was evaluated using bilirubin as specific259

substrate in the presence of oxygen, no oxidized products were observed either. On the260

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other hand, when samples were tested in terms of oxidase activity using specific261

substrates in the presence of oxygen, interesting results were found. Firstly, only two262

strains presented LAC activity, corresponding toPenicilliumsp. (SIS-18) andRhizopus263

sp (SIS-31). Additionally, both fungi showed almost half of the oxidase activity264

observed when the unspecific assay of pyrogallol was used. At that point, when TYR265

activity was tested only Aspergilllus sp. and Rhizopus sp. showed a fraction of the266

observed activity with the unspecific assay. These discrepancies among oxidase267

activities can be related with the specificity that each enzyme has for each substrate268

used during the assays. According to the available enzyme data bases, no records269

relating mono or polyphenol oxidases have been yet found for Rhizopus sp, but270

polyphenol oxidases was already reported for two Aspergillusspp.: A. niger (Sutay et271

al., 2008) andA. oryzae (Gasparetti et al., 2009); On the other hand, several laccase-like272

enzymes were already reported for several Penicillium spp., such as P. acuelatum, P.273

cyclopium and P. digitatum (El-Shora et al., 2008). These results are very promising274

since a novel fungal species asRhizopusis now being discovered as source of not one,275

but two different oxidase activities.276


3.2. Electrochemical studies278

Fig 2. shows the cyclic voltammograms (CV) obtained for cell free culture media279

samples containing the highest oxidasic activity observed for the selected fungi. At280

first, it can be observed that profiles showed to be very similar for all strains, but281

slightly higher cathodic currents were observed for SIS 18 and 31. Also a wide282

reduction peak, probably corresponding to a process of two close reduction events was283

observed for SIS-31 between 500 and 800 mV. On the other hand, a higher redox span284

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can be expected for Tyrosinase, since not one but two oxidations can occur over the285

same molecule, or one -OH insertion on a phenolic ring following by a dual oxidation286

process causing the formation of quinone-like derivatives (La Rotta et al., 2011).287

Monophenol oxidase activity observed for SIS-21 and its voltammetric profile was in288

accordance to previous studies where, a lower redox span was observed for LAC289

(Fernndez-Snchez et al., 2002). In general, control experiments with all enzymatic290

extracts in the absence of substrate confirmed the enzymatic oxygen reduction, but in all291

cases low electrochemical activity ranging only 2 to 4 A, were observed.292

PLEASE INSERT FIGURE 2 HERE293

When CVs were repeated in the presence of the unspecific substrate pyrogallol, more294

asymmetric anodic and cathodic peak shapes were detected in potentials ranging295

between -0.2 to 1.0 V vs. Ag|AgCl sat. KCl. This response was especially evident in the296

case of SIS 21 between 0.6 and 0.8 V, where an increase on anodic current of 10 A,297

was achieved. The same response was observed for the other two strains but at lower298

levels, being only of 7 A and 3 A for SIS-31 and SIS-18, respectively. The299

substantial shift of currents demonstrates the active catalysis of the enzymatic oxygen300

reduction at the surface of the working electrode, and the simultaneous oxidation of the301

substrate, indicating that an active oxidase was being detected in all cases. Since most of302

oxidase enzymes are multicenter enzymes, intermediate redox states are expected. We303

assume that the pair of anodic and cathodic redox peaks in the evaluated samples can be304

attributed to the process of direct electrical transfer between the T2/T3 redox copper305

center of the oxidase and the glassy carbon electrode, being mediated by the oxidized306

forms of pyrogallol. Also, oxidation of the pyrogallol can easily occur on any of the 307

OH groups, and its hardly expected to occur a simultaneous oxygen insertion.308

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Pyrogallol oxidation is mostly followed by the formation of a cycled oxidized derivative309

called purpurogallin. Finally, voltammetries were repeated in the presence of a specific310

substrate for TYR or poly-phenoloxidase. Cresol corresponds to a methyl derivative of311

phenol, that can be easily oxidized to quinone by the insertion of an oxygen atom312

depending on the activation caused by the -CH3 position respect the OH group. As313

such, this substrate can be oxidized not once but twice, and under strong oxidizing314

conditions cycled derivatives are not formed, instead of them poly-quinone-like315

derivatives can be appeared as dark precipitates since they are usually insoluble in water316

(Ramsden and Riley, 2014). As we expected, the results pointed the main polyphenol317

oxidase to be present in SIS-31, confirming our previous biochemical observations.318

Clearly, SIS-31 possess a strong oxidation peak of 4 A was observed at 100 mV,319

followed by a 2 A reduction peak at 200 mV. Similar, but lower currents of Ia= 2 A320

and Ic = 1 A, were observed at the same potentials for SIS-18. In contrast, no321

significant response was observed for SIS-21, confirming the absence of tyrosinase322

activity for this strain. The chronoamperometric analyses were used to determine323

electrochemical parameters of maxima current intensities during the cultivation and in-324

situ simultaneous oxygen reduction. These profiles allow us to determine the amount of325

electrons that effectively were starved from the substrate, and expressed them as326

coulombic efficiencies. The profiles obtained for the evaluated fungal strains cultivated327

at air-cathodes using medium CuB , during 120 h can be observed in Fig. 3. Since no328

significant changes in current densities were observed from the 72 h up to end of the329

experiment at 120 h, only these data are shown. In-situ culture experiments were330

compared with the response of a simulated behavior observed for the addition of 900 UI331

mL-1

of Laccase from Trametes versicolor (LAC Tv) to a volume of 100 mL of fresh332

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CuB medium. The chronoamperometric profile for control LAC Tv was very flat333

compared with the highest density current peaks observed when all three strains334

evaluated in-situ.335


The summarized results achieved with the chronoamperometries are shown in Table 6.337

Maximum current densities were observed for SIS 31 followed by SIS 21 and SIS 18,338

with 125.75, 98.68 and 29.75 mA cm-2, respectively. While for the experiment339

containing pure LAC Tv the lowest value of 11.47 mA cm-2, -was observed. Even when340

we tried to mimic the same conditions present during the fermentations, including341

enzyme concentration, many factors could affect the activity of pure LAC Tv causing342

such low response, including: enzyme inactivation, electrode deposition and passivation343

or lack of a suitable electron transfer mechanism, that can be present while the fungi are344

growing. These observations are in concordance with previous studies about MFC345

using fungus-based biocathodes, where longer and stable performances were achieved346

for the fungal cultures than with the pure laccase-based controls (Wu et al., 2012).347

Moreover, the fungi inoculated into the MFC had about 12-time higher current densities348

(in the case of SIS 31) than the control using carbon electrode and free LAC from349

Trametes versicolor. This clearly shows that the oxygen reduction in the air biocathodes350

can be efficiently performed and enhanced by the used of in-situ fungal cultures. For the351

coulumbic efficiency, it was observed that all strains have, from moderate to very good352

levels of electron starvation from the substrate (in terms of glucose biotransformation).353

Especially, SIS-31 achieved a very high value of CEof 56.5%, which means that this354

fungus is able to remove almost 50% of the electrons available in the substrate to the355

biocatalyst during its biosynthesis and reducing the available oxygen present inside the356

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media. Good results of 46.5 and 23.8%, were also achieved for SIS-21 and SIS-18,357

respectively. Even when SIS-21 produced almost 2 fold-times more biomass than SIS-358

31 and achieved the maximum unspecific oxidase activity of 5000 UI mL-1 among the359

evaluated strains, only SIS-31 was more efficient transforming the substrate during the360

fermentation into tyrosinase and laccase and subsequently this conducted to a higher361

and more stable production of energy in-situ.362


The power outputs observed for the strains and the enzyme control of LAC-Tv were364

compared using two different electrode compositions. At first, all experiments were365

evaluated with free-platinum carbon air cathodes. As such, Figure 4, exemplifies the366

polarization curves and power output profiles that were obtained for this approach. The367

highest power outs were observed for SIS-18 and SIS-21, with close values of 328 and368

288 mW m-3

, respectively. In contrast, with previous observations, SIS-31 showed only369

197 mW m-3, being the lowest value among strains. This could indicate that even when370

this microorganism starves efficiently electrons from the substrates, the produced371

enzyme did not perform very well the cathodic oxygen reduction or that some372

unidentified problems related with charge or mass transportations were present. The373

control experiment using LAC Tv showed the lowest value of 43.4 mW m-3

. This374

proved again that the fungal metabolites present inside the cultures and the crude375

extracts contributed indeed to improve the energy generation inside the biocathodes.376

Since similar enzyme concentrations were used, this cannot be consider as the direct377

responsible for the power loss.378



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The second electrode approach used a Pt load of 0.5% (w/w), and intended to identify if381

a real improvement can be achieved in electrodes with no metallic catalysts and with the382

addition of fungal oxidases. These results were put together in figure 5. Using as383

control experiment a submerge electrode in free-enzyme medium, a very low level of384

cathodic oxygen reduction of just 5.46 mW m-3 was observed. When the electrode385

contained Pt, an increase of almost 75 mW m-3

was observed. Similar increases were386

observed in all cases, following the same patter observed for Free-Pt electrodes. As387

such, were observed increases of 100, 60 and 20 mW m-3, when we used crude extracts388

of SIS-18, SIS-21 and SIS-31, respectively. Comparing both systems it can be observed389

that the differences are not quite significant between an oxidase biocathode plus free-pt390

electrode and when the Pt-loaded electrode was used. These differences were really391

evident when no catalysts were used and between pure enzyme electrodes and the ones392

obtained by in-situ cultures.393

394

4. CONCLUSIONS395

Three strains were selected as the best producers of oxidasic activity and then can be396

used as potential biocatalysts for oxygen reduction in MFCs air-cathodes. The efficient397

utilization and biotransformation of glucose was observed specially for Aspergillus sp398

SIS 18. and Rhizopus sp. SIS 31. However, all strains showed low oxidasic activity in399

the presence of glycerol; Penicillium sp. - SIS 21 was responsible for the highest400

generation of current density, while SIS 31 showed the best transformation of glucose401

into energy according to the highest coulombic efficiency observed. This fact, turn the402

oxidases from these microorganisms into potential targets for the isolation,403

characterization and use of their biocatalysts applied to the oxygen reduction in biotic404

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cathodes. The results thus far suggest the observed electrochemical activity is due to the405

oxidase enzymes. This family of enzymes can catalyze the four-electron reduction of406

O2to H2O coupled to the one-electron oxidation of different substrates. In nature these407

electrons are supplied by several phenols, amines, and lignins, as well as inorganic ions.408

Thus, the production of these biocatalysts in air-cathodes for MFC can be also coupled409

to the degradation of some interesting substrates including pollutants as phenolic410

compounds and dyes present in waste-waters. In a MFC, electrons were replenished to411

the cathode from the anode, which accepts electrons from anode-respiring and other412

bioentities with cross-membrane electron transfer capabilities. The results above413

provide the primary experimental evidences that fungi isolated from the Caatinga region414

in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes415

to improve electricity generation in MFCs. Such biosystem confers many advantages416

over conventional abiotic or pure enzyme cathodes, such as low costs, good pH417

buffering capability and the possibility for sustainable MFC operation.418

419

Acknowledgments420

The authors wish to thank the Regional Scientific Development Research Grant421

Program (DCR No.0008-1.06/11) and Scientific Initiation Grant Program given by the422

Brazilian Research Council - CNPq and The Foundation for Support of Science and423

Technology from the State of Pernambuco - FACEPE, Brazil. Special thanks are given424

to the NPCIAMB from the Catholic University of Pernambuco for the facilities and425

infrastructure used during the execution of this research. And the invaluable help given426

by the group of Electrochemistry from the Institute of Chemistry University of So427

Paulo, Brazil.428

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429

430

REFERENCES431

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Coriolopsis rigida. Appl. Environ. Microbiol. 68. 153440.507

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Samejima, T. 1999Myrothecium verrucariaBilirubin Oxidase and Its Mutants for Potential512

Copper Ligands. Biochem. 9, 38. 3034-42.513

30. Shleev, S., Tkac, J., Christenson, A., Ruzgas, T., Yaropolov, A.I. , Whittaker, J.W., Gorton,514L. 2005. Direct electron transfer between copper-containing proteins and electrodes515

Biosens. Bioelectron. 20, 12. 25172554516

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Zhang, X.E., Koivula, A., Viikari. L. 2008. Development of a printable laccase-based518

biocathode for fuel cell application. Enz. Microbial Technol. 43. 2. 93-102519

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characterization, and identification of a novel bifunctional catalase-phenol oxidase from521

Scytalidium thermophilumAppl. Microbiol. Biotechnol. 79. 407-415.522

33. Tinoco, R., Pickard, M.A., Vazquez-Duhalt, R., 2001. Kinetic differences of purified523

laccases from sixPleurotus ostreatusstrains. Lett. Appl. Microbiol. 32, 5. 331-335.524

34. Wu, C.; Liu, X.W., Li, W.W.; Sheng, G.P.; Zang, G.L.; Cheng, Y.Y.; Shen, N.; Yang, Y.P.;525

Yu, H.Q. 2012. A white-rot fungus is used as a biocathode to improve electricity production526

of a microbial fuel cell.Applied Energy. 98. 594596.527

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Superoxide-Scavenging Assay Suitable for All Antioxidants. J. Agricul. Food Chem. 60.529

6418-6424.530

531

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532

533

534

535

Anode

Air Cathode

Saline bridge

Fungal mycelium

V

50 mm

Cathodic

compartment

Anodic

compartment

Air

Inlet

Fe +2| Fe +3

Enzyme Ox

Enzyme Red

H2O

O2 + H+

e-

e-

536

Scheme 1. Diagram for the bicompartmented microbial fuel cell using an air-cathode537

and in-situ fungal growth.538

539

540

541

542

543

544

545

546

547

548

549

550

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551

552

553

554

Table 1.Culture media used for screening of fungal strains with oxidasic activity.555

Solid Media Liquid Media

MA* MA MMA MB [27,28] MMB [27,28] CuB

Component / g L-1 Component / g L-1

Peptone

Meat ext.

Tannic acid

Agar

6.0

4.0

4.0

4.0

Peptone

Meat ext.

Gallic acid

Agar

6.0

4.0

5.0

4.0

Glycerol

Peptone

Meat ext.

Gallic acid

Agar

20.0

6.0

4.0

5.0

4.0

Glucose

Peptone

KH2PO4

ZnSO4

K2HPO4

FeSO4

MnSO4

MgSO4

10.0

3.0

0.6

0.001

0.4

0.0005

0.05

0.05

Glycerol

Peptone

KH2PO4

ZnSO4

K2HPO4

FeSO4

MnSO4

MgSO4

20.0

3.0

0.6

0.001

0.4

0.0005

0.05

0.05

Glucose

Peptone

KH2PO4

ZnSO4

K2HPO4

FeSO4

MnSO4

MgSO4

CuSO4

10.0

3.0

0.6

0.001

0.4

0.0005

0.05

0.05

0.0005

*Media used for visualization of tannase activity in plates.556

557

558

559

560

561

562

563

564

565

566

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567

568

569

570

Table 2.Enzyme activity methods used for screening of oxidasic enzymes.571

Enzyme

Activity

for:

Substrate

Initial

concentration

Co-

substrate

Buffer, pH,

Temperature

Product | ABS Ref.

Oxidase Pyrogallol | 0.01

mmol L-1

O2 100 mmol L-1

Acetate Buffer pH

5.0 at 25

o

C

Purpurogallyn |

24,700 mol L1cm

1

at 420 nm

(Xican, 2012;

Ghadiri et al., 2013)

Laccase Syringaldazine |

0.05 mmol L-1

O2 20 mmol L-1

Phosphate buffer,

pH 7.0 at 30C

Oxidized Syringaldazine

|

65,000 mol L1cm1

at 525 nm

(Harkin and Obst, 1973;

;Espin et al., 1998)

Tyrosinase 3-Methyl-2-

benzothiazolinone

hydrazone | 6.0

mmol L-1

O2 20 mmol L-1

Phosphate buffer,

pH 7.0 at 30C

L-DOPA |

38,000 mol L1cm

1

at 505 nm


Espin et al., 1998)

BOD Bilirubin | 0.002%

(w/v)

O2 200 mmol L-1Tris

HCl buffer, pH 8.4

at 37C

Biliverdin |

56,300 mol L1cm

1

at 440 nm

(Kimura et al., 1999)

Peroxidase Pyrogallol | 0.01

mmol L-1

Syringaldazine |

0.05 mmol L-1

H2O2

H2O2

100 mmol L-1

Acetate Buffer pH

5.0 at 25oC

20 mmol L-1

Phosphate buffer,

pH 7.0 at 30C

Purpurogallyn |

24,700 mol L1cm

1

at 420 nm

Oxidized Syringaldazine

|

65,000 mol L1cm1

at 525 nm

(Xican, 2012; Ghadiri

et al., 2013)


Espin et al., 1998)

572

573

574

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575

576

Table 3. Preliminary selection of fungal strains with oxidase activity and pigment577

production based on observations made in plates.578

RENNORFUN

CODE

Microorganism

Pigment

production

Tannase assay Oxidase

Assay

SIS-39 Rhizopus sp. - ++ ++

SIS-31 Rhizopus sp. - +++ +++

SIS-18 Aspergillus sp. - +++ ++

SIS-27 Penicillium sp. - +++ +

SIS-4(E) Aspergillus sp. Red ++ -

SIS-7 Aspergillus sp. Dark + ++ -

SIS-21 Penicillium sp. Yellow ++ +++ +++

CP1 10-3 H Penicillium sp. Green ++ ++ -

N.C. Eupenicillium sp. Orange ++ - -

A2P1 10-3 Eupenicillium sp. Orange + - -

A2P1 10-4 G Talaromyces sp. Orange ++ + -

A2P1 10-3 Talaromyces sp. Orange + - -

NC = Not codified.579

Intensity of color = Negative or absence (-); Positive weak (+); Positive moderate (++); Positive strong (+++)580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

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614

615

Table 5.Analyses for oxidasic activity present in the crude extracts.616

Strain Oxidase Activity Tested( UI mL-1)

Unespecific

Phenol Oxidase

Activity

Assay 1

Monophenol

oxidase Activity

LAC

Assay 2

Polypheno

oxidasel Activity

TYR

Assay 3

Bilirubin

oxidase Activity

BOD

Assay 4

Peroxidase

Activity

POD

Assay 5

Aspergillussp. SIS

18

2630 131.5

N.R 1200 60.0 N.R. N.R.

Penicillium sp.SIS

21

8900 445.05600 280.0 N.R. N.R. N.R.

Rhizopus sp. SIS 31 4180 209.0 2580 129.0 1400 70.0 N.R. N.R.

N.R.: Negative or no response617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

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632

Table 6. General results for the chronoamperometric analyses during in-cell633

experiments.634

635

636

637

638

639

640

641

642

Fungal Strain

Maximum Oxidase

Activity achieved in-situ

(UI mL-1) at 120h

Idmax

(

A cm-2)

Time of

Idmax

(h)

Residual

Glucose

(g L-1) at 120h

*CE

(%)

Aspergillussp. SIS 18 2800 140 29.75 1.0 13 0.000 0.0005 23.8 1.2

Penicillium sp. SIS 21 4600 230 98.68 1.0 23 0.001 0.0005 46.5 2.3

Rhizopus sp. SIS 31 2700 135 125.75 1.0 23 0.003 0.0095 56.5 2.8

Laccase from Trametes

versicolor 600 30 11.47 1.0 1 0.000 0.0005 --

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Laccase Tyrosinase

BOD

O2 H2O

O2 H2O

O2 H2O

643

Figure 1. Reactions catalyzed by multi-copper oxidases applied to biocathodes644

645

646

647

648

649

650

651

652

653

654

655

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-0.2 0.0 0.2 0.4 0.6 0.8 1.0-4

-2

0

2

4

6

8

10

12

I,A

E, V

Aspergillus sp. SIS-18

-0.2 0.0 0.2 0.4 0.6 0.8 1.0-4

-2

0

2

4

6

8

10

12

I,

A

E, V

Penicillium sp. SIS-21

-0.2 0.0 0.2 0.4 0.6 0.8 1.0-4

-2

0

2

4

6

8

10

12

I,A

E, V

Rhizopus sp. SIS-31

656

Fig. 2.Cyclic-voltammograms for the selected fungal strains. Control of 200 UI mL-1657

cell free sample (blue), plus 0.01 mol L-1 pyrogallol (red); plus 0.01 mol L-1 cresol658

(green); Potential was measured versus a Ag|AgCl in sat. KCl. Scan rate: 5 mV s-1

.659

660

661

662

663

664

665

666

667

668

669

670

671

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0 12 24 36 48 60 72

0

20

40

60

80

100

120

140Control Lac Tv

SIS 31

SIS 21

SIS 18

Id,mAc

m-2

Time, h

672

Fig. 3. Chronoamperometric profiles for the selected strains observed during 120 h at673

25oC in medium BCu compared to a control of free cell medium dopped with Lacase674

from T. versicolor.675

676

677

678

679

680

681

682

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-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0 1 2 3 4 5 6

Id, A m-3

E,V

0 1 2 3 4 5 6

0

50

100

150

200

250

300

350

400SIS18

SIS21

SIS31

Pd,mWm

-3

I, A m-3

328.73

288.80

197.77

43.40

683

Figure 4.Power density output profiles obtained from the polarization curves (in-set684

plot) for crude extracts obtained from cultures of SIS-21 (black), SIS-18 (red) and SIS-685

31 (blue). And free cell medium doped with 900 UI of Lac Tv (green) was used as686

control.687

688

689

690

691

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Control LAC TV SIS 18 SIS 21 SIS 31

0

100

200

300

400

500

Pdmax,mWm

-3

75.80

5.46

105.46

43.40

438.16

328.73

344.10

288.90317.30

197.80

692

Figure 5. Maxima In-cell Power-outputs (vs Ag|AgCl2 sat. KCl) for crude extracts with693

the highest oxidasic activity obtained from the evaluated strains applied in to air694

biocathodes using two different electrode compositions: 0.5% (w/w) Pt-Black Carbon695

PTFE carbon felt (grey bars) and Free Pt Carbon PTFE carbon felt (white bars).696

Control experiments contain no enzyme.697

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Highlights704

We isolated three novel filamentous fungi with high oxidasic activity from705

soils of the Brazilian Scrubland (Caatinga).706We found interesting mono- and poly-phenol oxidasic activities707

compared with other fungal sources.708

These fungal strains were applied during the in-situ cathodic reduction of709

oxygen in microbial fuel cells air-cathodes .710

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In bioelec trochemical terms, we observed high current densities and711

power out generations.712

Also high levels of substrate biotransformation into energy according to713

the coulombic efficiency were observed.714

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Morant 2014