Accepted Manuscript

Title: ISOLATION AND BIOELECTROCHEMICAL

CHARACTERIZATION OF NOVEL FUNGAL SOURCES
WITH OXIDASIC ACTIVITY APPLIED IN-SITU FOR 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
Hern andez
PII: S0141-0229(14)00137-9
DOI: http://dx.doi.org/doi:10.1016/j.enzmictec.2014.07.007
Reference: 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, Hern andez CELR,
ISOLATION AND BIOELECTROCHEMICAL CHARACTERIZATION OF NOVEL
FUNGAL SOURCES WITH OXIDASIC ACTIVITY APPLIED IN-SITU FOR THE
CATHODIC 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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ISOLATION AND BIOELECTROCHEMICAL CHARACTERIZATION OF 1
NOVEL FUNGAL SOURCES WITH OXIDASIC ACTIVITY APPLIED I N-SI TU 2
FOR THE CATHODIC OXYGEN REDUCTION IN MICROBIAL FUEL 3
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 de 7
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) 99271612

10
*Corresponding author: camilo.larotta@pq.cnpq.br 11
ABSTRACT: 12
Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and 13
Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatingas soils and 14
applied during the in-situ cathodic oxygen reduction in fuel cells. All strains were 15
cultivated in submerged cultures using and optimized saline medium enriched with 10 g 16
L
-1
of glucose, 3.0 g L
-1
of peptone and 0.0005 g L
-1
of CuSO
4
as enzyme inducer. 17
Parameters of oxidase activity, glucose consumption and microbial growth were 18
evaluated. In-cell experiments evaluated by chronoamperometry were performed and 19
two different electrode compositions were also compared. Maxima current densities of 20
125.7, 98.7 and 11.5 A cm
-2
were observed before 24 h and coulombic efficiencies of 21
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
, were 23
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observed for SIS 18, SIS 21 and SIS 31, respectively. This work provide the primary 24
experimental evidences that fungi isolated from the Caatinga region in Brazil can serve 25
as efficient biocatalysts during the oxygen reduction in air-cathodes to improve 26
electricity generation in MFCs. 27
28
Keywords: Oxidases, Filamentous fungi, Biocathodes, Biofuel cells, Cathodic Oxygen 29
reduction. 30
1. INTRODUCTION 31
The gradual depletion of fossil fuels and the environmental concerns for their 32
consumption have driven an intensive search for alternative sources for energy 33
production. BioFuel Cells (BFC) are considered a promising alternative for clean energy 34
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, commonly 36
used in coated electrodes as catalysts is still considered one of the limiting factors for 37
scaled-up applications of microbial fuel cells (MFC) and conventional fuel cells. Even 38
though, abiotic cathodes that use oxygen as electron acceptor are frequently adopted for 39
BFC (Logan et al., 2006; Luo et al., 2010). Enzymes as biocathodes can potentially 40
eliminate limiting factors such as: decreased efficiency due to the accumulation of 41
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 electron 43
transfer throughout suitable mediated systems and employing co-substrates (Da Silva et 44
al., 2014; Farneth and DAmore, 2005). These types of enzymatic cathodes have been 45
investigated in small scale enzymatic biofuel cells (Farneth and DAmore, 2005). On 46
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the other hand, energy production obtained from the BFC is not yet satisfactory and 47
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 transfer 49
from the biocatalysts to the electrodic materials. Studies on enzymes for electron 50
interactions are being mainly focused on copper-containing oxidoreductases (Figure 1), 51
which can catalyze the direct reduction of oxygen while perform the simultaneous 52
oxidation of many organic compounds such as phenols. Mono and poly-phenol oxidases 53
from fungal species such as: Agaricus bisporus (Shervedani and Amini, 2012), Coriolus 54
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 Streptomyces 57
coelicolor, were already studied and applied to these bioelectrodes (Shleev et al., 58
2005). Others less electrochemically explored, but highly promising corresponds to the 59
fungal bilirubin oxidase (BOD) from Myrothecium verrucaria (Ivnitski et al. 2008; 60
Mano et al., 2002) and bacterial BOD from Bacillus pumilus (Durand et al. 2012). 61
EC 1.10.3.1 : Tyrosinase: polyphenol oxidase: 62
2 catechol+O
2
2 1,2-benzoquinone+2 H
2
O Eq. 1 63
EC 1.10.3.1: Laccase : Urishinol Oxidase 64
4 benzenediol+O
2
4 benzosemiquinone+2 H
2
O Eq. 2 65
EC 1.3.3.5: Bilirubin oxidase 66
2 bilirubin +O
2
2 biliverdin +2 H
2
O Eq.3 67
68
Laccase (LAC) and tyrosinase (TYR) are able to oxidize phenolic compounds and to 69
reduce simultaneously oxygen into water (Eq. 1 and 2). Depending the microbial 70
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source from which these enzyme were extracted, the redox potential of the T
1
site, may 71
vary from 430 mV up to 780 mV vs. NHE (Palmore and Kim, 1999). Laccase from 72
Trametes versicolor is the most attractive one since redox potential of its T
1
site is 73
ca.780 mVvs.NHE (Shleev et al., 2005). Nowadays, the best performances with laccase 74
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
at 76
only -70 mVvs. O
2
/H
2
O at pH 5.0. Under the same conditions, an identical current 77
density is obtained at -400 mVvs.O
2
/H
2
O with a platinum wire as catalyst. 78
Nevertheless, performances of laccase from Pleurotus Ostreatus electrodes drop 79
drastically in the presence of chloride ions what constitutes both a major problem and a 80
great challenge for its use in biofuel cells (Barton et al., 2002). On the other hand, BOD 81
(Eq. 3) is naturally capable of catalyzing the oxidation of bilirubin into biliverdin and to 82
simultaneously reduce dioxygen (Li et al., 2004). BOD is very similar to laccase. BOD 83
electrodes are greatly related to the amino-acids sequence around T
1
site of the enzyme 84
(Shimizu et al. 1999). It is clearly reported that the most efficient BOD enzyme comes 85
from Myrothecium verrucaria. Redox potential of its T
1
site is included between 650 86
and 750 mV vs. NHE, and its future application in BFC its close related with the 87
observed thermal stability up to 60 C (Mano et al., 2002). These biocatalysts have 88
been extensively used in cathodes for enzymatic fuel cells and electrochemical 89
biosensors due to their high redox potential, however the almost mandatory use of 90
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 been 92
widely recognized as an effective way to avoid the loss of current during the 93
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 the 97
ones commonly found in the surface of electrodic materials of BFC. In this regard, 98
biocathodes inoculated with the fungi for the in-situ oxidase production may offer a 99
potential solution (Wu et al., 2012; Rachinski et al., 2010). Also the in- situ secretion 100
of oxidases by the filamentous fungi in air cathodes might be a more attractive way to 101
achieve sustainable and cost-efficient electricity generation, especially for three main 102
reasons: longer life-time of the biocatalysts since these are being produced under more 103
compatible biological conditions; the use of low cost substrates such as residua or 104
contaminated effluents; and the possibility of concomitant production of other natural 105
occurring electro active molecules as microbial by-products like: azaphylones, quinone- 106
like pigments as melanins, terpene as carotenoids, etc. The simultaneous utilization of 107
such molecules could improve even more the couloumbic efficiencies by reducing the 108
charge and mass transportation problems previously observed for these systems. 109
PLEASE INSERTT FIGURE 1 HERE 110
Currently, the Brazilian North and Northeast Network of Filamentous Fungi 111
(RENNORFUN) aims to describe the biodiversity of filamentous fungi in soils from 112
the Caatinga and the Amazon regions of Brazil throughout poly-phasic and molecular 113
taxonomy as well as to demonstrate the applicability in industrial processes of the 114
isolated micro-organisms and their by-products. In this context, this study aimed the 115
isolation and identification of novel fungal species capable to produce biocatalysts with 116
high oxidasic activity that can be applied to the cathodic reduction of oxygen in 117
electrodes for biosensors and BFCs. 118
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119
120
2. METODOLOGY 121
2.1. Fungal Strains, media and cultivation conditions 122
All strains belong to the RENNORFUN Culture Collection from the Catholic University 123
of Pernambuco Brazil, stocked in slant tubes containing Sabouraud agar solid medium 124
under refrigeration at 4
o
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 these 127
enzymes (Koroljova-Skorobogat'ko et al., 1998; Saparrat et al., 2002). Table 1. Shows 128
the culture media used for selection of fungal strains with oxidasic activity. Twelve 129
fungal strains were originally chosen: two Rhizopus spp.; three Aspergillus spp.; three 130
Penicillium spp.; two Eupenicillium spp and two Talaromyces spp. The microorganisms 131
were visual evaluated in terms of substrate degradation and color formation, by 132
cultivation in solid plates incubated for 48 at 28
o
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 28
o
C and 180 rpm for 48 hours. After this 136
period of time, tubes were used for the inoculation of flasks containing 150 to 200 mL 137
or microbial cathodic compartiments of 100 mL of capacity at the bicompartmented 138
BFC. All cultures were incubated under the same controlled lab conditions. 139
140
2.2. Microbial growth parameters and sample post-treatment 141
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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 were 143
performed. First, samples and culture broths were separated from mycelia by filtration 144
through nylon cloth and centrifugation at 4500 rpm for 20 minutes at 4
o
C. Mycelia 145
were dried on paper filters at 60
o
C until constant weight. Finally, cell free supernatants 146
were used for the quantification of glycerol or glucose using specific enzymatic kits 147
purchased from BIOCLIN. Enzyme activities were determined using the below 148
described methodologies. 149
150
2.3. Oxidasic activity assays 151
Enzymatic crude extracts and fermentation samples were evaluated in terms of oxidase 152
activity using the methods summarized in Table 2. Oxidase and peroxidase activities 153
were distinguished throughout similar methods; however for peroxidase activity 5.0 154
mmol L
-1
hydrogen peroxide was added instead a saturated oxygen buffer solution as 155
oxidizing agent (assays 1 and 5, respectively). Mono and polyphenol oxidases were 156
differentiated using assays 2 and 3 both in saturated oxygen buffer solutions. And 157
finally the routine assay 4 was used for oxidase activity. All enzyme activities were 158
expressed as international units per mL (UI mL
-1
), defined as the amount of enzyme 159
required to produce 1 mol mL
-1
of the specific oxidized product, according to the 160
specific molar extinction coefficients, per minute under the reaction conditions used in 161
each assay. The increase in absorbance was monitored in a UV-Vis spectrophotometer 162
BioChrome Libra S32 . 163
PLEASE INSERT TABLE 1 HERE 164
PLEASE INSERT TABLE 2 HERE 165
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2.4. Electrochemical analyses 166
Chronoamperometric analysis: A bicompartmented BFC was employed (Scheme 1). A 167
100 mL cathodic compartment was composed by the fungal culture using the selected 168
medium, and two electrodic materials were evaluated. Immersed carbon felt electrodes 169
coated with carbon Black Vulcan plus 0.5% Pt (w\w) in PTFE or a Pt-free Black 170
Vulcan coated carbon felts; As anodes, immersed plates of exploded graphite in 20 171
mmol L
-1
potassium ferrocyanide were used. All electrodes had 19.6 cm
-2
of surface 172
area. As cation exchange system a saline bridge of agar in saturated KCl was used. The 173
potential (E) vs. time (min) was recorded using a multimeter Fluke 8080 with data 174
acquisition software. All experiments were allowed to stabilize for about 30 minutes 175
before each measurement. Chronovoltammetric data were converted into I
d
data using 176
the Ohms Law expression (Equation 4) since an external load resistance of 1 K was 177
employed. 178
I
d
= E * R Eq. 4 179
The Coulombic efficiency (%) in Equation 5, was calculated from the following 180
expressions according to previous studies (Logan et al., 2006; Dantas et al., 2013). 181
C
E
= (C
R
/C
T
) Eq. 5 182
where, C
T
corresponds to the theoretical amount to be obtained from each substrate and 183
C
R
corresponds to the practical coulombs recovery from the substrate. C
T
can be 184
calculated at any time using the expression: 185
C
T
= nzF Eq. 6 186
where n is the moles of substrate, z are the moles of electrons per mol of substrate (O
2
= 187
4e
-
, Glucose = 6 e
-
or Glycerol

= 12 e
-
); and F is the Faradays constant 96485.4 C 188
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mol
-1
. Applying Equation 6 to an integrated model of Id vs time for a microbial fuel 189
cell we obtain Equation 7 190
C
E
= M / F z V
AC/CC
S Eq. 7 191
where M is the substrate molarity, A the electrode surface area, V is the total volume 192
circulated inside the cathodic compartment and S is the final substrate concentration. 193
PLEASE INSERT SCHEME 1 HERE 194
Cyclicvoltammetric analysis: The electrochemical system was composed by a glass 15 195
mL glass cell, a 0.5 cm diameter glassy carbon as working electrode, a Pt wire of 1.0 cm 196
as counter-electrode and Ag|AgCl
2
in saturated KCl as reference electrode. As enzyme 197
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 a 199
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: 0.005 202
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 electrochemical 205
system for the MFC, but placing the reference electrode at the cathodic compartment 206
and same apparatus described above, samples with the highest recorded activity were 207
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; Linear 209
sweep: 8 s, E
o
= 0.8 V, E
f
=-0.5 V, E
step
= 0.002 V, Scan rate 0.01 V s
-1
. The polarization 210
curves of I
d
vs E obtained were used to the determination of the Power-output curves (P
d
211
vs E) and maxima P
d
values according to Equation 8 212
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P
d
= E * I
d
Eq. 8 213
214
215
3. RESULTS AND DISCUSSION 216
3.1. Screening of novel fungal oxidases 217
Initially the solid culture medium A (MA) was supplemented with gallic acid or tannic 218
acid as carbon sources, to visualize the production of oxidase or tannase in plates by the 219
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 screening 221
in MA supplemented with those substrates can be observed in Table 3. Six of twelve 222
strains presented the availability to degrade tannic acid and only four were able to 223
oxidize efficiently gallic acid. Unexpectedly, only one strain (SIS-21) showed both 224
pigments production and exhibited high oxidase and tannase activities, while the other 225
pigment producers strains showed low to moderate tannase activity and none oxidase 226
activity. Since one of the goals of this study was also to find fungal strains able to 227
biotransform alternative carbon sources, different than glucose (Ex. Glycerol), with the 228
retention of the oxidasic activity, the use of a modified medium A or (MMA), enriched 229
with glycerol was also tested. Nevertheless, only strains SIS-18, 21, 31 and 39 retained 230
the availability of performing the oxidization of gallic acid in plates while grew very 231
well in a medium containing glycerol. 232
PLEASE INSERT TABLE 3 HERE 233
This also was confirmed when the pre-selected strains according to the halo formation 234
in plates containing (MA) and (MMA) media, were cultivated in liquid medium (B) or 235
modified medium B (MMB) where glycerol was added instead glucose as can be seen in 236
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Table 4. As such, no oxidase activity was observed at all in MMB. At this point the best 237
oxidase producers corresponded to strains SIS-21, 31 and 18, with 5, 1.7 and 1.6 KU 238
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 of 240
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 it 242
was expected a significant increase on oxidase activity was observed in all cases. This 243
increase represents almost 1.65 fold-times in the case of the oxidasic activities observed 244
for SIS 18 and SIS-21, an almost 2.4 fold-times in the case of SIS-31. In contrast, SIS 245
21 showed the highest values for fungal growth in terms of dried weight of 1.02 g, and 246
lower fungal growths were observed for SIS 31 followed by SIS 18, with 0.43 and 0.95 247
g, respectively. In terms of glucose consumption, all three strains caused the complete 248
depletion of this substrate in 120 h. 249
PLEASE INSERT TABLE 4 HERE 250
Since the oxidation of pyrogallol can be unspecific in terms of which oxidasic activity is 251
present in the fungal cultures, specific assays were performed using cell free samples 252
obtained from submerged cultures in CuB medium, in a way to identify which one of 253
the oxidase activities (monophenol oxidase - LAC, polyphenol oxidase -TYR or 254
bilirubin oxidase - BOD) or eventually if peroxidase activity (POD) was present. Table 255
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, since 257
no oxidation products were observed in the absence of oxygen and the presence of 258
hydrogen peroxide. Likewise, when BOD was evaluated using bilirubin as specific 259
substrate in the presence of oxygen, no oxidized products were observed either. On the 260
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other hand, when samples were tested in terms of oxidase activity using specific 261
substrates in the presence of oxygen, interesting results were found. Firstly, only two 262
strains presented LAC activity, corresponding to Penicillium sp. (SIS-18) and Rhizopus 263
sp (SIS-31). Additionally, both fungi showed almost half of the oxidase activity 264
observed when the unspecific assay of pyrogallol was used. At that point, when TYR 265
activity was tested only Aspergilllus sp. and Rhizopus sp. showed a fraction of the 266
observed activity with the unspecific assay. These discrepancies among oxidase 267
activities can be related with the specificity that each enzyme has for each substrate 268
used during the assays. According to the available enzyme data bases, no records 269
relating mono or polyphenol oxidases have been yet found for Rhizopus sp, but 270
polyphenol oxidases was already reported for two Aspergillus spp.: A. niger (Sutay et 271
al., 2008) and A. oryzae (Gasparetti et al., 2009); On the other hand, several laccase-like 272
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 promising 274
since a novel fungal species as Rhizopus is now being discovered as source of not one, 275
but two different oxidase activities. 276
PLEASE INSERT TABLE 5 HERE 277
3.2. Electrochemical studies 278
Fig 2. shows the cyclic voltammograms (CV) obtained for cell free culture media 279
samples containing the highest oxidasic activity observed for the selected fungi. At 280
first, it can be observed that profiles showed to be very similar for all strains, but 281
slightly higher cathodic currents were observed for SIS 18 and 31. Also a wide 282
reduction peak, probably corresponding to a process of two close reduction events was 283
observed for SIS-31 between 500 and 800 mV. On the other hand, a higher redox span 284
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can be expected for Tyrosinase, since not one but two oxidations can occur over the 285
same molecule, or one -OH insertion on a phenolic ring following by a dual oxidation 286
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 in 288
accordance to previous studies where, a lower redox span was observed for LAC 289
(Fernndez-Snchez et al., 2002). In general, control experiments with all enzymatic 290
extracts in the absence of substrate confirmed the enzymatic oxygen reduction, but in all 291
cases low electrochemical activity ranging only 2 to 4 A, were observed. 292
PLEASE INSERT FIGURE 2 HERE 293
When CVs were repeated in the presence of the unspecific substrate pyrogallol, more 294
asymmetric anodic and cathodic peak shapes were detected in potentials ranging 295
between -0.2 to 1.0 V vs. Ag|AgCl sat. KCl. This response was especially evident in the 296
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 lower 298
levels, being only of 7 A and 3 A for SIS-31 and SIS-18, respectively. The 299
substantial shift of currents demonstrates the active catalysis of the enzymatic oxygen 300
reduction at the surface of the working electrode, and the simultaneous oxidation of the 301
substrate, indicating that an active oxidase was being detected in all cases. Since most of 302
oxidase enzymes are multicenter enzymes, intermediate redox states are expected. We 303
assume that the pair of anodic and cathodic redox peaks in the evaluated samples can be 304
attributed to the process of direct electrical transfer between the T2/T3 redox copper 305
center of the oxidase and the glassy carbon electrode, being mediated by the oxidized 306
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 derivative 309
called purpurogallin. Finally, voltammetries were repeated in the presence of a specific 310
substrate for TYR or poly-phenoloxidase. Cresol corresponds to a methyl derivative of 311
phenol, that can be easily oxidized to quinone by the insertion of an oxygen atom 312
depending on the activation caused by the -CH3 position respect the OH group. As 313
such, this substrate can be oxidized not once but twice, and under strong oxidizing 314
conditions cycled derivatives are not formed, instead of them poly-quinone-like 315
derivatives can be appeared as dark precipitates since they are usually insoluble in water 316
(Ramsden and Riley, 2014). As we expected, the results pointed the main polyphenol 317
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 I
a
= 2 A 320
and I
c
= 1 A, were observed at the same potentials for SIS-18. In contrast, no 321
significant response was observed for SIS-21, confirming the absence of tyrosinase 322
activity for this strain. The chronoamperometric analyses were used to determine 323
electrochemical parameters of maxima current intensities during the cultivation and in- 324
situ simultaneous oxygen reduction. These profiles allow us to determine the amount of 325
electrons that effectively were starved from the substrate, and expressed them as 326
coulombic efficiencies. The profiles obtained for the evaluated fungal strains cultivated 327
at air-cathodes using medium CuB , during 120 h can be observed in Fig. 3. Since no 328
significant changes in current densities were observed from the 72 h up to end of the 329
experiment at 120 h, only these data are shown. In-situ culture experiments were 330
compared with the response of a simulated behavior observed for the addition of 900 UI 331
mL
-1
of Laccase from Trametes versicolor (LAC Tv) to a volume of 100 mL of fresh 332
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CuB medium. The chronoamperometric profile for control LAC Tv was very flat 333
compared with the highest density current peaks observed when all three strains 334
evaluated in-situ. 335
PLEASE INSERT FIGURE 3 HERE 336
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 experiment 339
containing pure LAC Tv the lowest value of 11.47 mA cm-
2
,
-
was observed. Even when 340
we tried to mimic the same conditions present during the fermentations, including 341
enzyme concentration, many factors could affect the activity of pure LAC Tv causing 342
such low response, including: enzyme inactivation, electrode deposition and passivation 343
or lack of a suitable electron transfer mechanism, that can be present while the fungi are 344
growing. These observations are in concordance with previous studies about MFC 345
using fungus-based biocathodes, where longer and stable performances were achieved 346
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 densities 348
(in the case of SIS 31) than the control using carbon electrode and free LAC from 349
Trametes versicolor. This clearly shows that the oxygen reduction in the air biocathodes 350
can be efficiently performed and enhanced by the used of in-situ fungal cultures. For the 351
coulumbic efficiency, it was observed that all strains have, from moderate to very good 352
levels of electron starvation from the substrate (in terms of glucose biotransformation). 353
Especially, SIS-31 achieved a very high value of C
E
of 56.5%, which means that this 354
fungus is able to remove almost 50% of the electrons available in the substrate to the 355
biocatalyst during its biosynthesis and reducing the available oxygen present inside the 356
Page 16 of 35
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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 the 359
evaluated strains, only SIS-31 was more efficient transforming the substrate during the 360
fermentation into tyrosinase and laccase and subsequently this conducted to a higher 361
and more stable production of energy in-situ. 362
PLEASE INSERT TABLE 6 HERE 363
The power outputs observed for the strains and the enzyme control of LAC-Tv were 364
compared using two different electrode compositions. At first, all experiments were 365
evaluated with free-platinum carbon air cathodes. As such, Figure 4, exemplifies the 366
polarization curves and power output profiles that were obtained for this approach. The 367
highest power outs were observed for SIS-18 and SIS-21, with close values of 328 and 368
288 mW m
-3
, respectively. In contrast, with previous observations, SIS-31 showed only 369
197 mW m
-3
, being the lowest value among strains. This could indicate that even when 370
this microorganism starves efficiently electrons from the substrates, the produced 371
enzyme did not perform very well the cathodic oxygen reduction or that some 372
unidentified problems related with charge or mass transportations were present. The 373
control experiment using LAC Tv showed the lowest value of 43.4 mW m
-3
. This 374
proved again that the fungal metabolites present inside the cultures and the crude 375
extracts contributed indeed to improve the energy generation inside the biocathodes. 376
Since similar enzyme concentrations were used, this cannot be consider as the direct 377
responsible for the power loss. 378
PLEASE INSERT FIGURE 4 HERE 379
PLEASE INSERT FIGURE 5 HERE 380
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The second electrode approach used a Pt load of 0.5% (w/w), and intended to identify if 381
a real improvement can be achieved in electrodes with no metallic catalysts and with the 382
addition of fungal oxidases. These results were put together in figure 5. Using as 383
control experiment a submerge electrode in free-enzyme medium, a very low level of 384
cathodic oxygen reduction of just 5.46 mW m
-3
was observed. When the electrode 385
contained Pt, an increase of almost 75 mW m
-3
was observed. Similar increases were 386
observed in all cases, following the same patter observed for Free-Pt electrodes. As 387
such, were observed increases of 100, 60 and 20 mW m
-3
, when we used crude extracts 388
of SIS-18, SIS-21 and SIS-31, respectively. Comparing both systems it can be observed 389
that the differences are not quite significant between an oxidase biocathode plus free-pt 390
electrode and when the Pt-loaded electrode was used. These differences were really 391
evident when no catalysts were used and between pure enzyme electrodes and the ones 392
obtained by in-situ cultures. 393
394
4. CONCLUSIONS 395
Three strains were selected as the best producers of oxidasic activity and then can be 396
used as potential biocatalysts for oxygen reduction in MFCs air-cathodes. The efficient 397
utilization and biotransformation of glucose was observed specially for Aspergillus sp 398
SIS 18. and Rhizopus sp. SIS 31. However, all strains showed low oxidasic activity in 399
the presence of glycerol; Penicillium sp. - SIS 21 was responsible for the highest 400
generation of current density, while SIS 31 showed the best transformation of glucose 401
into energy according to the highest coulombic efficiency observed. This fact, turn the 402
oxidases from these microorganisms into potential targets for the isolation, 403
characterization and use of their biocatalysts applied to the oxygen reduction in biotic 404
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cathodes. The results thus far suggest the observed electrochemical activity is due to the 405
oxidase enzymes. This family of enzymes can catalyze the four-electron reduction of 406
O
2
to H
2
O coupled to the one-electron oxidation of different substrates. In nature these 407
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 coupled 409
to the degradation of some interesting substrates including pollutants as phenolic 410
compounds and dyes present in waste-waters. In a MFC, electrons were replenished to 411
the cathode from the anode, which accepts electrons from anode-respiring and other 412
bioentities with cross-membrane electron transfer capabilities. The results above 413
provide the primary experimental evidences that fungi isolated from the Caatinga region 414
in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes 415
to improve electricity generation in MFCs. Such biosystem confers many advantages 416
over conventional abiotic or pure enzyme cathodes, such as low costs, good pH 417
buffering capability and the possibility for sustainable MFC operation. 418
419
Acknowledgments 420
The authors wish to thank the Regional Scientific Development Research Grant 421
Program (DCR No.0008-1.06/11) and Scientific Initiation Grant Program given by the 422
Brazilian Research Council - CNPq and The Foundation for Support of Science and 423
Technology from the State of Pernambuco - FACEPE, Brazil. Special thanks are given 424
to the NPCIAMB from the Catholic University of Pernambuco for the facilities and 425
infrastructure used during the execution of this research. And the invaluable help given 426
by the group of Electrochemistry from the Institute of Chemistry University of So 427
Paulo, Brazil. 428
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429
430
REFERENCES 431
1. Bach, C.E., Warnock, D.D., Van Horn, D.J., Weintraub, M.N., Sinsabaugh, R.L., Allison, 432
S.D., German, D.P. 2013. Measuring phenol oxidase and peroxidase activities with 433
pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type. Soil Biol 434
Biochem. 67. 183-191 435
2. Barton, S.C., Pickard, M., Vazquez-Duhalt, R., Heller, A. 436
2002. Electroreduction of O
2
to water at 0.6 V (SHE) at pH 7 on the wired Pleurotus 437
ostreatus laccase cathode Biosens. Bioelectron. 17. 10711074 438
3. Da Silva, P.H., Morant, K. V., Takaki, G. M. C., La Rotta, H.C.E. 2014. Production of 439
electrogenic pigments from new fungal sources applied as electron shuttles in biofuel cells. 440
In: Industrial, medical and environmental applications of microorganisms: current status and 441
trend. S/N. Ed. Wageningen Academic Publishers, In press. Wageningen, The Netherlands. 442
4. Dantas, P.V., Peres S., Takaki, G.M.C, La Rotta, H.C.E. 2013. Utilization of Raw Glycerol 443
for Pyocyanin Production from Pseudomonas Aeruginosa in Half-Microbial Fuel Cells: 444
Evaluation of Two Electrochemical Approaches. J. Electrochem. Soc. 160. G1-G7. 445
5. Durand, F., Hauge-Kjaergaard, C., Suraniti, E., Gounel, S. Hadt, R.G., Solomon, E.I., 446
Mano, N. 2012 Bilirubin oxidase from Bacillus pumilus: A promising enzyme for the 447
elaboration of efficient cathodes in biofuel cells. Biosens. Bioelectron. 35, 1. 140-146 448
6. El-Shora, H., Youssef, M., Khalaf, S., 2008. Inducers and inhibitors of laccase from 449
Penicillium, Biotechnol. 7. 35-42. 450
7. Espin, J.C., Tudela, J., Garcia-Canovas, F. 1998. 4-Hydroxyanisole: the mostsuitable 451
monophenolic substrate for determining spectrophotometrically the monophenolase activity 452
of polyphenol oxidase from fruits and vegetables. Anal. Biochem. 259. 11826. 453
Page 20 of 35
A
c
c
e
p
t
e
d

M
a
n
u
s
c
r
i
p
t
20
8. Farneth, W.E., DAmore, M.B. 2005 Encapsulated laccase electrodes for fuel cell cathodes. 454
J. Electroanal. Chem. 581. 197205. 455
9. Fernndez-Snchez, C., Tzanov, T., Gbitz, G.M., Cavaco-Paulo, A. 2002. Voltammetric 456
monitoring of laccase-catalysed mediated reactions. Bioelectrochem. 58. 149 156. 457
10. Fonseca, M.I, Shimizu, E., Zapata, P.D., Villalba, L.L. 2010. Copper inducing effect on 458
laccase production of white rot fungi native from Misiones (Argentina). Enz. Microb. 459
Technol. 46. 534539. 460
11. Gasparetti, C., Faccio, G., Arvas, M., Buchert, J., Saloheimo, M., Kruus, K., 2009. 461
Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed 462
domain: production and characterization of an Aspergillus oryzae catechol oxidase. Appl. 463
Microbiol. Biotechnol. 86. 213-226. 464
12. Ghadiri, M., Kariminia, H.R., Roosta, R.A. 2013. Spectrophotometric determination of 465
sulde based on peroxidase inhibition by detection of purpurogallin formation. Ecotoxicol. 466
Environ. Saf. 91. 117121. 467
13. Harkin, J., Obst, J., 1973. Syringaldazine, an effective reagent for detecting laccase and 468
peroxidase in fungi. Experientia 29. 387. 469
14. Ivnitski, D., Artyushkova, K., Atanassov, P. 2008 Surface characterization and direct 470
electrochemistry of redox copper centers of bilirubin oxidase from fungi Myrothecium 471
verrucaria. Bioelectrochem., 74, 1. 101-110 472
15. Karatay, S.E., Donmez, G., 2011. Microbial oil production from thermophile cyanobacteria 473
for biodiesel production. Appl Energy. 88, 36325. 474
16. Kimura, S., Iyama, S., Yamaguchi, Y., Hayashi., S., Yanagihara, T. 1999. Enzymatic assay 475
for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD). J. Clin. Lab. Anal.13, 476
5. 219-23. 477
17. Koroljova-Skorobogat'ko, O.V., Stepanova, E.V., Gavrilova, V.P., Morozova, O.V., 478
Lubimova, N.V., Dzchafarova, A.N., Jaropolov, A.I., Makower, A. 1998. Purification and 479
Page 21 of 35
A
c
c
e
p
t
e
d

M
a
n
u
s
c
r
i
p
t
21
characterization of the constitutive form of laccase from the basidiomycete Coriolus 480
hirsutus and effect of inducers on laccase synthesis. Biotechnol. Appl. Biochem. 1. 47-54. 481
18. La Rotta, C.E.; Ciniciato, G.P; Gonzlez, E.R. 2011. Triphenylmethane dyes, an alternative 482
for mediated electronic transfer systems in glucose oxidase biofuel cells. Enz Microb. 483
Technol. 2011, 48. 487497. 484
19. Leite, M.V.; Morant, K.V.; Silva, H.L.; Luna, M.A.C.; Alves Da Silva, C.A.; Okada, K.; 485
Horie, Y.; Campos-Takaki, G.M. 2012. Phenotypic and molecular characterization of 486
Penicillium decubens and glabrum and polyphenol oxidase production. Biological 487
Resource Centres, Closing tha gap between science and society. Micoteca Univ. Minho. 94 488
20. Li, H., Webb, S.P., Ivanic, J., Jensen, J.H. 2004. Determinants of the Relative Reduction 489
Potentials of Type-1 Copper Sites in Proteins. J. Am. Chem. Soc. 30, 126. 8010-9. 490
21. Logan, B.E., Hamelers, B., Rozendal, R., Schroder, U., Freguia, K.J., Verstraete, A.P., 491
Rabaey, W.K. 2006. Environ. Sci. Technol. 40. 7. 5181. 492
22. Luo, H., Jing, S., Fallgren, P.H., Park, H., Johnson, P.A. 2010. A novel laccase-catalyzed 493
cathode for microbial fuel cells. Chem. Eng. J. 165. 524528. 494
23. Mano, N., Kim, H., Zhang, H.Y., Heller, A. 2002a. An Oxygen Cathode Operating in a 495
Physiological Solution. J. Am. Chem. Soc. 124, 22. 6480-6. 496
24. Palmore, G.T.R., Kim, H. 1999. Electro-enzymatic reduction of dioxygen to water in the 497
cathode compartment of a biofuel cell J. Electroanal. Chem. 464, 1. 110117 498
25. Rachinski, S., Carubelli, A., Mangoni, A.P., Mangrich, A.S. 2010. Pilhas de Combustveis 499
Microbianas Utilizadas na Produo de Eletricidade a Partir de Rejeitos Orgnicos: Uma 500
Perspectiva de Futuro. Quim. Nova. 33, 8. 1773-1778. 501
26. Ramsden, C,A. and Riley, P.A. 2014. Tyrosinase: The four oxidation states of the active 502
site and their relevance to enzymatic activation, oxidation and inactivation. Bioorgan. Med. 503
Chem. 22, 8. 2388-2395. 504
Page 22 of 35
A
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t
22
27. Saparrat, M.C., Guilln, F., Arambarri, A.M., Martnez, A.T., Martnez, M.J. 2002 505
Induction, isolation, and characterization of two laccases from the white rot basidiomycete 506
Coriolopsis rigida. Appl. Environ. Microbiol. 68. 153440. 507
28. Shervedani, R.K; Amini, A. 2012. Direct electrochemistry of dopamine on gold Agaricus 508
bisporus laccase enzyme electrode: characterization and quantitative detection. 509
Bioelectrochem. 84: 25-31, Apr. 510
29. Shimizu, A., Kwon, J.H., Sasaki, T., Satoh, T., Sakurai, N., Sakurai, T., Yamaguchi, S., 511
Samejima, T. 1999 Myrothecium verrucaria Bilirubin Oxidase and Its Mutants for Potential 512
Copper Ligands. Biochem. 9, 38. 3034-42. 513
30. Shleev, S., Tkac, J., Christenson, A., Ruzgas, T., Yaropolov, A.I. , Whittaker, J.W., Gorton, 514
L. 2005. Direct electron transfer between copper-containing proteins and electrodes 515
Biosens. Bioelectron. 20, 12. 25172554 516
31. Smolander, M., Boer, H., Valkiainen, M., Roozeman, R., Bergelin, M., Eriksson, J.E., 517
Zhang, X.E., Koivula, A., Viikari. L. 2008. Development of a printable laccase-based 518
biocathode for fuel cell application. Enz. Microbial Technol. 43. 2. 93-102 519
32. Sutay, K.D., Bakir, U., Phillips, S.E., McPherson, M.J., Ogel, Z.B. 2008 Purification, 520
characterization, and identification of a novel bifunctional catalase-phenol oxidase from 521
Scytalidium thermophilum Appl. Microbiol. Biotechnol. 79. 407-415. 522
33. Tinoco, R., Pickard, M.A., Vazquez-Duhalt, R., 2001. Kinetic differences of puried 523
laccases from six Pleurotus ostreatus strains. 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 production 526
of a microbial fuel cell. Applied Energy. 98. 594596. 527
35. Xican Li. 2012. Improved Pyrogallol Autoxidation Method: A Reliable and Cheap 528
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
H
2
O
O
2
+ H
+
e
-
e
-
536
Scheme 1. Diagram for the bicompartmented microbial fuel cell using an air-cathode 537
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
K
2
HPO
4

FeSO
4

MnSO4
MgSO4
10.0
3.0
0.6
0.001
0.4
0.0005
0.05
0.05
Glycerol
Peptone
KH2PO4
ZnSO4
K
2
HPO
4

FeSO
4

MnSO4
MgSO4
20.0
3.0
0.6
0.001
0.4
0.0005
0.05
0.05
Glucose
Peptone
KH2PO4
ZnSO4
K
2
HPO
4

FeSO
4

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 L

1
cm

1

at 420 nm
(Xican, 2012;
Ghadiri et al., 2013)
Laccase Syringaldazine |
0.05 mmol L
-1

O
2
20 mmol L
-1

Phosphate buffer,
pH 7.0 at 30C
Oxidized Syringaldazine
|
65,000 mol L
1
cm1
at 525 nm
(Harkin and Obst, 1973;
;Espin et al., 1998)
Tyrosinase 3-Methyl-2-
benzothiazolinone
hydrazone | 6.0
mmol L
-1

O
2
20 mmol L
-1

Phosphate buffer,
pH 7.0 at 30C
L-DOPA |
38,000 mol L
1
cm
1
at 505 nm
(Harkin and Obst, 1973;
Espin et al., 1998)
BOD Bilirubin | 0.002%
(w/v)
O2 200 mmol L
-1
Tris
HCl buffer, pH 8.4
at 37C
Biliverdin |
56,300 mol L

1
cm

1
at 440 nm

(Kimura et al., 1999)
Peroxidase Pyrogallol | 0.01
mmol L
-1

Syringaldazine |
0.05 mmol L
-1

H2O2

H
2
O
2

100 mmol L
-1

Acetate Buffer pH
5.0 at 25
o
C
20 mmol L
-1

Phosphate buffer,
pH 7.0 at 30C
Purpurogallyn |
24,700 mol L

1
cm

1

at 420 nm
Oxidized Syringaldazine
|
65,000 mol L

1
cm
1

at 525 nm
(Xican, 2012; Ghadiri
et al., 2013)

(Harkin and Obst, 1973;
Espin et al., 1998)
572
573
574
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575
576
Table 3. Preliminary selection of fungal strains with oxidase activity and pigment 577
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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595
596
597
Table 4. Results obtained for the screening in liquid media using the selected fungal 598
strains. 599
Liquid Media
B MB CuB CuB CuB CuB

Fungal Strain

*Oxidase
Activity
(UI mL
1
)
*Oxidase
Activity
(UI mL
-1
)
*Oxidase
Activity
(UI mL
-1
)
Maximum
Dried weight
(g)
Residual
Glucose
(g L
-1
)
X glucose
%
Aspergillus sp. SIS 18
1600 80.0 N.R.
2630
131.5
0.95 0.5 0.000 0.0005 100.0 1.0
Penicillium sp. SIS 21
5500 275..0 N.R.
8900
445.0
1.02 0.5 0.001 0.0005 99.99 1.0
Rhizopus sp. SIS 31
1800 90.0 N.R.
4180
209.0
0.43 0.5 0.003 0.0095 99.97 1.0
N.R.: Negative or no response; *Oxidase activity determined by pyrogallol assay (Xican, 2012; Ghadiri et al., 2013) 600
601
602
603
604
605
606
607
608
609
610
611
612
613
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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
Aspergillus sp. SIS
18
2630 131.5
N.R 1200 60.0 N.R. N.R.
Penicillium sp. SIS
21
8900 445.0
5600 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 response 617
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-cell 633
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

(%)
Aspergillus sp. 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
O
2
H
2
O
O
2
H
2
O
O
2
H
2
O
643
Figure 1. Reactions catalyzed by multi-copper oxidases applied to biocathodes 644
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
-1
657
cell free sample (blue), plus 0.01 mol L
-1
pyrogallol (red); plus 0.01 mol L
-1
cresol 658
(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
140
Control Lac Tv
SIS 31
SIS 21
SIS 18
I
d
,

m
A

c
m
-
2
Time, h
672
Fig. 3. Chronoamperometric profiles for the selected strains observed during 120 h at 673
25
o
C in medium BCu compared to a control of free cell medium dopped with Lacase 674
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
I
d
, A m
-3
E
,

V
0 1 2 3 4 5 6
0
50
100
150
200
250
300
350
400
SIS18
SIS21
SIS31
P
d
,

m
W

m
-
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-set 684
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 as 686
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
P
d
m
a
x
,

m
W

m
-
3
75.80
5.46
105.46
43.40
438.16
328.73
344.10
288.90
317.30
197.80
692
Figure 5. Maxima In-cell Power-outputs (vs Ag|AgCl2 sat. KCl) for crude extracts with 693
the highest oxidasic activity obtained from the evaluated strains applied in to air 694
biocathodes using two different electrode compositions: 0.5% (w/w) Pt-Black Carbon 695
PTFE carbon felt (grey bars) and Free Pt Carbon PTFE carbon felt (white bars). 696
Control experiments contain no enzyme. 697
698
699
700
701
702
703
Highlights 704
We isolated three novel filamentous fungi with high oxidasic activity from 705
soils of the Brazilian Scrubland (Caatinga). 706
We found interesting mono- and poly-phenol oxidasic activities 707
compared with other fungal sources. 708
These fungal strains were applied during the in-situ cathodic reduction of 709
oxygen in microbial fuel cells air-cathodes . 710
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In bioelectrochemical terms, we observed high current densities and 711
power out generations. 712
Also high levels of substrate biotransformation into energy according to 713
the coulombic efficiency were observed. 714
715
716
717