BACKGROUND
wastewaters contain dissolved organics that require removal before discharge into the environment.
however, wastewaters are being recognized as a renewable resource for the production of electricity, fuels and chemicals.
bioelectrochemical wastewater treatment has, therefore, emerged as a potentially interesting technology for the production of energy  <cit> .
reducing the cost of the materials used in mfcs is essential for practical applications.
the cathode accounts for the greatest percentage of the total capital cost, and cathode surface area and materials generally limit higher power production in mfcs.
therefore, it is important to identify low-cost materials and efficient cathode architectures in order to improve mfc cost effectiveness and performance  <cit> .
the bioanode, a crucial component in bioelectrochemical systems , is composed of an anode biofilm and a conductive electrode.
the main catalytic components of interest in anode biofilms are exoelectrogens, microorganisms that are capable of exocellular electron transfer  <cit> .
in mixed-culture systems, exoelectrogens compete for electron donors with other functional groups such as fermenters, acetogens and methanogens  <cit> .
the complexity of anode biofilms makes it hard to elucidate electrochemical mechanisms at the bioanode, but a precise understanding of exoelectrogenesis and competition in anode biofilms will aid in improving the performance of bess.
several reviews provide insightful summaries and perspectives regarding bioanodes  <cit> .
electrochemical impedance spectroscopy  was used as a non-intrusive tool to identify and elucidate the electrochemical properties of redox mediators produced by microbes.
eis enabled the study of the individual contributions from different resistances.
these include ohmic resistance , charge transfer and concentration  resistance on the anode and cathode behavior of mediators and their impact on mfc impedance without the need to interrupt mfc operation  <cit> .
with the help of equivalent electrical circuit fitting analysis, eis can also provide quantitative estimates of the kinetic rate constants for the anodic and cathodic reactions, double layer capacitance at the electrode surface, and the diffusion coefficients of electro-active species in the bulk electrolyte  <cit> .
the aim of this study was to characterize community growth on the anode and in the anolyte and the impact of electrochemical impedance in a microbial fuel cell with an initial mixed inoculum and its selection process over time.
RESULTS
chemical oxygen demand
cod removed in the first week was 93%.
meanwhile, in week  <dig> it was  <dig> % due to increased planktonic cell concentration in the anolyte increasing the organic load and decreasing cod over time.
this is similar to what biffinger et al.
<cit>  observed.
the cod removed is found in the medium-high range  of results obtained by other authors in the literature  <cit> .
vfa concentrations at week  <dig> were  <dig>  mm,  <dig>  mm and  <dig>  mm for butyric, propionic and acetic acid, respectively.
these values were insignificant with respect to the initial concentration of the electron donor at  <dig>  mm of sodium acetate.
the ease with which acetate was metabolized by exoelectrogenic microorganisms was also observed by velasquez-orta et al.
<cit> , who obtained carboxylic acid concentrations of less than  <dig>  mm after  <dig> weeks.
the percentages of h <dig> and ch <dig> in the samples analyzed at week  <dig> of this study were less than  <dig> %.
this rules out a methanogenic biochemistry with a coulombic efficiency of  <dig> %.
these values were lower than those reported by jung and reagan  <cit>  in a pem-type mfc with pt catalyst at the cathode and acetate as the carbon source.
they obtained 6% h <dig> and ch <dig> with a coulombic efficiency of  <dig> %.
composition by pyrosequencing
the shannon index of diversity  was determined for all samples.
the h’ value was higher for “initial inoculum”  than “biofilm”  and “anolyte” .
this indicates, that the initial inoculum was higher diverse than the other samples.
the bacterial richness by chao estimate indicated that initial inoculum showed a higher number of species  than the biofilm  and anolyte  samples, as was indicated by shannon index.
the rarefaction curves  indicated higher otu  number on initial inoculum sample  than in biofilm  and anolyte .
in comparison to initial inoculum curve, the curve of biofilm and anolyte samples is flatter with increasing sampling effort, and therefore possesses lower bacterial species.figure  <dig>
rarefaction curves for each sample from mfc.
number of reads is shown on the x axis and number of otus at 95% sequence identity on the y axis.
initial inoculum
a total of  <dig>  bacteria were detected in this sample.
the phylogenetic spectrum  was dominated by proteobacteria , methylobacter sp.
and syntrophus sp.
as the predominant species.
within bacteroidetes , cytophagia order  was dominant with firmicutes  and clostridiales family .
proteobacteria phylum has been widely detected in microbial fuel cell studies.
for example, in floating microbial fuel cell , a modification of mfc the bacterial community was dominated by γ- and β-proteobacteria   <cit> .
bacterioidetes  <cit>  such as firmicutes  <cit>  have been frequently detected as dominant in mfc reactors.
primary clarifier effluents sampled from mfc showed phylotypes relatively abundant in proteobacteria, bacteroidetes and firmicutes phyla  <cit> .figure  <dig>
taxonomic classification of pyrosequences from predominant bacterial communities of initial inoculum, biofilm, anolyte of mfc at the phylum level.
biofilm
a total of  <dig>  bacteria were detected in the sample collected from the electrode.
the most abundant were proteobacteria , followed by bacteroidetes  and firmicutes .
bacteroidete identification in biofilm is consistent with many reports indicating its presence .
this suggests it may play a critical role in electricity generation or efficient anode function .
it is very interesting to note that of the 60% of proteobacteria found 51% of these bacteria corresponded to rhodopseudomonas palustris species.
this bacterium is a member of the phototrophic purple non-sulfur bacteria which proliferates in different environments due to its versatile metabolism.
it is photoautotrophic, photoheterotrophic, chemoheterotrophic and chemoautotrophic  <cit> .
its capability in electricity generation has been reported before in r. palustris dx- <dig> by xing et al.
<cit> .
this dx- <dig> strain can produce higher power densities when isolated than as mixed cultures in the same mfc.
the genome completes of cga <dig> strain of r. palustris have been sequenced  <cit> .
u.s. department of energy has anticipated that genome sequence comparisons between dx- <dig> and strains of r. palustris will probably reveal key biochemical characteristics of strain dx- <dig> that are critical for its ability to generate power .
anolyte
a total of  <dig>  bacteria were detected in the sample taken from electrode.
firmicutes  predominated in this sample, followed by bacteroidetes , proteobacteria  and lentispharaerae  .
predominance of firmicutes at week  <dig>  is in accordance with studies by ishii et al.
<cit> .
the dominance of firmicutes phylum was been reported in mfc at 79% in current production with acetate as the electron donor  <cit> .
thermincola sp.
was the predominant species that generated current independent of an electron shuttle with acetate as an electron donor.
in this study, bacillus species for firmicutes phylum were represented at 80%.
in this respect, nimjea et al.
<cit>  demonstrated that the aerobic gram-positive species bacillus subtilis was able to grow anaerobically and produce a biofilm in a microbial fuel cell which generated a long-term power output.
the electrochemical activity and the electron transfer mechanism were mainly due to excreted redox mediators in the broth solution and not to the membrane-bound proteins which were affected by physiological status.
numerous reports indicate that firmicutes are integral members of the mfc bacterial community, indicating their exocellular electron transfer .
thus, they dominated in the anolyte sample because of dependent electrode respiration .
power and current density
the maximum power and current densities were 100 ±  <dig> mw/m <dig> and 347 ±  <dig> ma/m <dig>  respectively, at week  <dig>  values reported in the literature range from  <dig> to  <dig>  mw/m <dig> and from  <dig>  ma/m <dig> to  <dig> ma/m <dig> in mfcs with no catalyst at the cathode.
the open circuit potential stabilized at  <dig> mv   <cit> .
electrochemical impedance spectroscopy
the eis measurement of an individual electrode provides information that permits the analysis of electrochemical reactions on electrodes and bacterial metabolism, as well as surface and material properties of electrodes.
these are critical to understanding the electricity-generating process and improving the power output of mfcs  <cit> .
anode chamber
from figure  <dig>  it is clear that at both week  <dig> and week  <dig> an ohmic resistance  is presented, followed by a semicircle at high frequencies, which corresponds to charge transfer processes on the surface of the electrode.
the semicircle is characteristic of a single “time constant”  <cit> .
subsequently, a predominantly capacitive linear segment is observed  with a slight inclination.
it is more evident in the diagram corresponding to week  <dig> due to the presence of other associated processes such as diffusion and/or charge transfer corresponding to the substrate.
generally, the bioelectrochemical substrate oxidation processes are slow, offer high impedance and are exhibited in the mid-to-low frequency domains  <cit> .
for week  <dig>  the charge transfer process of substrate oxidation is the rate limiting step since it is significantly slower than the mediator charge transfer process and oxygen reduction steps.
the microbial growth on the anode has a beneficial effect on the kinetics of the bio-electrochemical reaction as it decreases the anode activation losses due to increased biocatalyst density  <cit> .
likewise, it is clear that a lower impedance response was present at week  <dig>  if we consider impedance to be a vector with a specific magnitude  <cit> , then from figure  <dig> there is an improved performance in the anode chamber at week  <dig>  given that the impedance values are lower than week  <dig>  due to the presence of a biofilm.
this development is similar to the report by borole et al.
<cit>  used a consortium enriched in a compact, flow-through porous electrode chamber coupled to an air-cathode.
anode impedance initially decreased from  <dig>  Ω on day  <dig>  to  <dig>  Ω after  <dig> months .
ramasamy et al.
<cit>  reported that the anode impedance from two-chamber mfc on day  <dig> and week  <dig> were estimated to be  <dig> Ω and  <dig> Ω, respectively, indicating that the growth of the microbial biofilm was found to decrease the anode polarization resistance and facilitate the kinetics of the electrochemical reactions.
for this study, the following elements were considered due to the similarity of the behavior of the nyquist diagrams in accordance with what bisquert et al.
<cit>  reported.
both a warburg element  and anomaly diffusion  were used to evaluate diffusion resistance  in terms of two parameters: y <dig> and b. y <dig> is the magnitude of the admittance at ω =  <dig> rad/s while b characterizes the time it takes for a reactant to diffuse through a thin fi w. the ratio b/y <dig> indicates the magnitude of diffusion resistance  <cit> .figure  <dig>
nyquist diagrams corresponding to anode chamber at different times of growth: a) week  <dig> and b) week  <dig>
on analyzing the values obtained, the ohmic resistance  was not observed to present significant changes between week  <dig>  and week  <dig> .
this contrasted with the resistance values corresponding to the charge transfer phenomena in which a reduction was observed due to the presence of the biofilm .
given its high conductivity, it facilitated the mobility of electrons through the electrode/biofilm interface.
this behavior is similar to what manohar et al.
<cit>  found, as well as other authors such as malvankar et al.
<cit>  and borole et al.
<cit> .
cpe values for week  <dig> and  <dig> were  <dig>  e-03 ±  <dig>  e- <dig> Ω−1sα and  <dig>  ±  <dig> Ω−1sα with α =  <dig> , respectively.
in both systems, the incorporated cpe is considered to be a non-ideal capacitor.
for determination, it was considered a faradaic system with time-constant interfacial capacitance rc  <cit> .
<dig> c=q1r1+1r2α−11/α
q  is the admittance when ω= <dig> j=−1; α is an ideal constant and r <dig> and r <dig> are the ohmic and charge transfer resistances, respectively.
on comparing the capacitance values between week  <dig> cdl =  <dig>  e-04 ±  <dig>  e- <dig> f and week  <dig> cdl =  <dig>  e-02 ±  <dig>  e- <dig> f, an increase in capacitance was observed by two orders of magnitude due to the fact that the biofilm facilitates charge accumulation at the electrode interface.
although the values may vary based on the experimental conditions   <cit> , the behavior is similar to that observed in previous works.
for example, borole et al.
<cit>  reported their values of cdl in the anode of  <dig>  f during the first two months .
also, it increases by two orders of magnitude from  <dig> to  <dig> days , obtaining a value of  <dig>  ±  <dig>  f. also, ramasamy et al.
<cit>  reported values for double layer capacitance from  <dig>  mf  to  <dig>  mf  for a surface of  <dig> cm <dig> in anode, which is expected capacitance or carbon electrode.
this confirmed that cdl increased due to the stabilization of the system.
as for the diffusion processes, these can occur when the solution species are diffused through the biofilm to the surface of the electrode  <cit> .
at week  <dig>  they are represented as a warburg element   with linear, semi-infinite diffusion behavior  equivalent to rd =  <dig>  ±  <dig>  Ωcm <dig>  at week  <dig>  meanwhile, this element is replaced by a modified anomalous diffusion element   equivalent to a linear transmission model reported by bisquert et al.
<cit>  with rd =  <dig>  ±  <dig>  Ωcm <dig>  this decrease in resistance is caused by proton exchange due to the presence of the biofilm.figure  <dig>
equivalent circuits used for the analysis of impedance data for the three systems: anode chamber, biofilm and anolyte at a) week  <dig> and b) week  <dig>
the model includes these elements, given that the impedance in the diffusion processes is defined as  <cit> :  <dig> ziω∝iω−β/20<β< <dig> where, in the majority of cases, it is a warburg-type impedance .
anomalous diffusion  is characterized by a mean squared displacement of the diffusing particles that does not follow the ordinary linear law r2 ∝ t but, more generally, has a power law dependence on time: r2 ∝ tβ at low frequencies.
the reason for this is the frequency transient time for a diffusing particle injected at x =  <dig> to cover a distance l. for ω > > ωd the particles will not sense the boundary at x = l so that the system will behave as semi-infinite.
the presence of the biofilm promotes anomalous diffusion processes, i.e.
protonic diffusion which can be spatially restricted in either planar, cylindrical or spherical forms, producing variation in the limits of the diffusion region 0 < x < l present at low frequencies.
many different mechanisms give rise to this anomalous behavior, including complex flows, structural complexity in the substrate of diffusion and in the diffusing tracers.
thus, no single theory of anomalous diffusion can account for all possible phenomenologies as in this case for the presence of biofilm.
biofilm
a more common “coating layer” on the electrodes of mfcs is a biofilm.
figure  <dig> shows the nyquist diagram of the biofilm at weeks  <dig> and  <dig>  first of all, there is a difference between the two systems at the same frequencies with week  <dig> presenting lower impedance, once again considering impedance to be a magnitude vector.
this is due to the fact that at week  <dig> the biofilm had already achieved growth and stabilization, favoring processes which reduce the total resistance of the system  <cit> .figure  <dig>
nyquist diagrams corresponding to biofilms at different times of growth: a) week  <dig> and b) week  <dig>
likewise, at both weeks  <dig> and  <dig>  the final part of a semicircle is observed, corresponding to charge transfer processes on the surface of the electrode  followed by a linear segment with an incline angle that corresponds to diffusion processes, and finally a predominantly capacitive linear portion similar to the behavior presented by the anode chamber.
therefore, the equivalent circuits proposed for the anode chamber at weeks  <dig> and  <dig> were used .
on analyzing the data obtained, ohmic resistance was not found to present significant changes between week  <dig>  and week  <dig> .
with respect to the cpe, as in the case of the anode chamber, the behavior is mainly non-ideal capacitance.
the values obtained  at weeks  <dig> and  <dig> were  <dig> e-01 ±  <dig> e- <dig> f and  <dig>  e-04 ±  <dig>  e- <dig> f, respectively.
this allows for the inference that the capacitance contribution in the anode chamber can be attributed to the behavior of the biofilm as has been reported previously by other research groups  <cit> .
the charge transfer resistance for week  <dig> presented higher values  compared to those at week  <dig>  due to the interaction of the biofilm with the anolyte.
diffusion processes at weeks  <dig> and  <dig> were equivalent to rd  <dig>  ±  <dig>  Ωcm <dig> and  <dig>  ±  <dig>  Ωcm <dig>  these values were similar to those for the anode chamber at week  <dig>  suggesting that the anolyte presents diffusion processes from the outset.
anolyte
figure  <dig> shows the nyquist diagram corresponding to the anolyte at weeks  <dig> and  <dig>  in both cases, it was not possible to observe a variation in the impedance value, considering it to be a vector.
the ohmic resistance is followed by an incomplete semicircle which indicates the presence of charge transfer processes.
it is unlikely that any biochemically derived redox compounds, synthesized mediators, yield a complete faradaic response to an ac signal faster than  <dig> hz.
hence, the reaction in the high frequency region depicts a fast electrochemical process such as oxidation of soluble metal ions in the growth medium.figure  <dig>
nyquist diagrams corresponding to anolyte at different times of growth: a) week  <dig> and b) week 20).
the ohmic resistance values  do not present significant changes due to the fact that they correspond to the resistance of the components of the system itself .
the charge transfer resistance and diffusion resistance for week  <dig>  r2 = 199 ±  <dig>  Ωcm <dig> and rd =  <dig>  ±  <dig>  Ωcm <dig>  compared to week  <dig>  r2 = 179 ±  <dig>  Ωcm <dig> and rd =  <dig> e + 03 ±  <dig> e- <dig> Ωcm <dig>  show that the r <dig> values are similar, meaning that there is no contribution from the anolyte to the charge transfer processes.
the anolyte without biofilm on the electrode is not redox-active toward acetate under these conditions.
eis of bare electrodes showed a very high charge transfer resistance  <cit> .
when comparing the results of rd obtained for the anolyte with those for the anode chamber and the biofilm, the anolyte at week  <dig> presents similar values to those for the anode chamber.
however, at week  <dig> the value increases by an order of magnitude, whilst in the anode chamber it decreases by an order of magnitude because the diffusion resistance decreased considerably due to the presence of the biofilm.
cpe values for weeks  <dig> and  <dig> were  <dig> e-03 ±  <dig> e- <dig>  and  <dig> e-03 ±  <dig>  e- <dig> , corresponding to the capacitances   <dig> e-05 ±  <dig> e- <dig> f and  <dig> e-05 ±  <dig> e- <dig>  respectively.
although there is evidence in the literature for the presence of diffusion processes in this type of bioelectrochemical system indicating that the mass transfer limitations were insignificant and masked by the dominant kinetic limitations for the anode bio-electrochemical reaction, this study proved that under the experimental conditions used there is an important contribution from the interaction of the biofilm and the anolyte on cell performance.
table  <dig> presents a summary of the specific influence of each element of the system under study .table  <dig>
influence of each of the elements corresponding to the phenomena in the mfc
configuration
system
charge transfer processes
diffusion processes
r1
r2
cdl
rd
anode chamber
c
a
a
b
anolyte
c
c
c
a
biofilm
c
a
a
b
CONCLUSIONS
the microbial fuel cell with no catalyst  and mixed inoculum in this study showed certain members of the bacterial electrode community increased in relative abundance from the initial inoculum.
for example, proteobacteria species are important for electricity generation in the anode biofilms and firmicutes phylum was predominant on anolyte to transfer electron.
likewise, the biofilm can act as a barrier which reduces diffusion of the anolyte towards the electrode, all the while behaving like a porous material.
the anolyte and its interaction with the biofilm exert a considerable influence on diffusion processes, given that it presents the highest values for rd.
rd increased at week  <dig>

 wastewaters contain dissolved organics that require removal before discharge into the environment. however, wastewaters are being recognized as a renewable resource for the production of electricity, fuels and chemicals. bioelectrochemical wastewater treatment has, therefore, emerged as a potentially interesting technology for the production of energy. reducing the cost of the materials used in mfcs is essential for practical applications. the cathode accounts for the greatest percentage of the total capital cost, and cathode surface area and materials generally limit higher power production in mfcs. therefore, it is important to identify low-cost materials and efficient cathode architectures in order to improve mfc cost effectiveness and performance. the bioanode, a crucial component in bioelectrochemical systems, is composed of an anode biofilm and a conductive electrode. the main catalytic components of interest in anode biofilms are exoelectrogens, microorganisms that are capable of exocellular electron transfer. in mixed-culture systems, exoelectrogens compete for electron donors with other functional groups such as fermenters, acetogens and methanogens. the complexity of anode biofilms makes it hard to elucidate electrochemical mechanisms at the bioanode, but a precise understanding of exoelectrogenesis and competition in anode biofilms will aid in improving the performance of bess. several reviews provide insightful summaries and perspectives regarding bioanodes. electrochemical impedance spectroscopy was used as a non-intrusive tool to identify and elucidate the electrochemical properties of redox mediators produced by microbes. eis enabled the study of the individual contributions from different resistances. these include ohmic resistance, charge transfer and concentration resistance on the anode and cathode behavior of mediators and their impact on mfc impedance without the need to interrupt mfc operation. with the help of equivalent electrical circuit fitting analysis, eis can also provide quantitative estimates of the kinetic rate constants for the anodic and cathodic reactions, double layer capacitance at the electrode surface, and the diffusion coefficients of electro-active species in the bulk electrolyte. the aim of this study was to characterize community growth on the anode and in the anolyte and the impact of electrochemical impedance in a microbial fuel cell with an initial mixed inoculum and its selection process over time.  chemical oxygen demand cod removed in the first week was 93%. meanwhile, in week it was % due to increased planktonic cell concentration in the anolyte increasing the organic load and decreasing cod over time. this is similar to what biffinger et al. observed. the cod removed is found in the medium-high range of results obtained by other authors in the literature. vfa concentrations at week were mm, mm and mm for butyric, propionic and acetic acid, respectively. these values were insignificant with respect to the initial concentration of the electron donor at mm of sodium acetate. the ease with which acetate was metabolized by exoelectrogenic microorganisms was also observed by velasquez-orta et al., who obtained carboxylic acid concentrations of less than mm after weeks. the percentages of h and ch in the samples analyzed at week of this study were less than %. this rules out a methanogenic biochemistry with a coulombic efficiency of %. these values were lower than those reported by jung and reagan in a pem-type mfc with pt catalyst at the cathode and acetate as the carbon source. they obtained 6% h and ch with a coulombic efficiency of %. composition by pyrosequencing the shannon index of diversity was determined for all samples. the h’ value was higher for “initial inoculum” than “biofilm” and “anolyte”. this indicates, that the initial inoculum was higher diverse than the other samples. the bacterial richness by chao estimate indicated that initial inoculum showed a higher number of species than the biofilm and anolyte samples, as was indicated by shannon index. the rarefaction curves indicated higher otu number on initial inoculum sample than in biofilm and anolyte. in comparison to initial inoculum curve, the curve of biofilm and anolyte samples is flatter with increasing sampling effort, and therefore possesses lower bacterial species.figure rarefaction curves for each sample from mfc. number of reads is shown on the x axis and number of otus at 95% sequence identity on the y axis. initial inoculum a total of bacteria were detected in this sample. the phylogenetic spectrum was dominated by proteobacteria, methylobacter sp. and syntrophus sp. as the predominant species. within bacteroidetes, cytophagia order was dominant with firmicutes and clostridiales family. proteobacteria phylum has been widely detected in microbial fuel cell studies. for example, in floating microbial fuel cell, a modification of mfc the bacterial community was dominated by γ- and β-proteobacteria. bacterioidetes such as firmicutes have been frequently detected as dominant in mfc reactors. primary clarifier effluents sampled from mfc showed phylotypes relatively abundant in proteobacteria, bacteroidetes and firmicutes phyla.figure taxonomic classification of pyrosequences from predominant bacterial communities of initial inoculum, biofilm, anolyte of mfc at the phylum level. biofilm a total of bacteria were detected in the sample collected from the electrode. the most abundant were proteobacteria, followed by bacteroidetes and firmicutes. bacteroidete identification in biofilm is consistent with many reports indicating its presence. this suggests it may play a critical role in electricity generation or efficient anode function. it is very interesting to note that of the 60% of proteobacteria found 51% of these bacteria corresponded to rhodopseudomonas palustris species. this bacterium is a member of the phototrophic purple non-sulfur bacteria which proliferates in different environments due to its versatile metabolism. it is photoautotrophic, photoheterotrophic, chemoheterotrophic and chemoautotrophic. its capability in electricity generation has been reported before in r. palustris dx- by xing et al.. this dx- strain can produce higher power densities when isolated than as mixed cultures in the same mfc. the genome completes of cga strain of r. palustris have been sequenced. u.s. department of energy has anticipated that genome sequence comparisons between dx- and strains of r. palustris will probably reveal key biochemical characteristics of strain dx- that are critical for its ability to generate power. anolyte a total of bacteria were detected in the sample taken from electrode. firmicutes predominated in this sample, followed by bacteroidetes, proteobacteria and lentispharaerae. predominance of firmicutes at week is in accordance with studies by ishii et al.. the dominance of firmicutes phylum was been reported in mfc at 79% in current production with acetate as the electron donor. thermincola sp. was the predominant species that generated current independent of an electron shuttle with acetate as an electron donor. in this study, bacillus species for firmicutes phylum were represented at 80%. in this respect, nimjea et al. demonstrated that the aerobic gram-positive species bacillus subtilis was able to grow anaerobically and produce a biofilm in a microbial fuel cell which generated a long-term power output. the electrochemical activity and the electron transfer mechanism were mainly due to excreted redox mediators in the broth solution and not to the membrane-bound proteins which were affected by physiological status. numerous reports indicate that firmicutes are integral members of the mfc bacterial community, indicating their exocellular electron transfer. thus, they dominated in the anolyte sample because of dependent electrode respiration. power and current density the maximum power and current densities were 100 ±  mw/m and 347 ±  ma/m respectively, at week values reported in the literature range from to mw/m and from ma/m to ma/m in mfcs with no catalyst at the cathode. the open circuit potential stabilized at mv. electrochemical impedance spectroscopy the eis measurement of an individual electrode provides information that permits the analysis of electrochemical reactions on electrodes and bacterial metabolism, as well as surface and material properties of electrodes. these are critical to understanding the electricity-generating process and improving the power output of mfcs. anode chamber from figure  it is clear that at both week and week an ohmic resistance is presented, followed by a semicircle at high frequencies, which corresponds to charge transfer processes on the surface of the electrode. the semicircle is characteristic of a single “time constant”. subsequently, a predominantly capacitive linear segment is observed with a slight inclination. it is more evident in the diagram corresponding to week due to the presence of other associated processes such as diffusion and/or charge transfer corresponding to the substrate. generally, the bioelectrochemical substrate oxidation processes are slow, offer high impedance and are exhibited in the mid-to-low frequency domains. for week the charge transfer process of substrate oxidation is the rate limiting step since it is significantly slower than the mediator charge transfer process and oxygen reduction steps. the microbial growth on the anode has a beneficial effect on the kinetics of the bio-electrochemical reaction as it decreases the anode activation losses due to increased biocatalyst density. likewise, it is clear that a lower impedance response was present at week if we consider impedance to be a vector with a specific magnitude, then from figure  there is an improved performance in the anode chamber at week given that the impedance values are lower than week due to the presence of a biofilm. this development is similar to the report by borole et al. used a consortium enriched in a compact, flow-through porous electrode chamber coupled to an air-cathode. anode impedance initially decreased from Ω on day to Ω after months. ramasamy et al. reported that the anode impedance from two-chamber mfc on day and week were estimated to be Ω and Ω, respectively, indicating that the growth of the microbial biofilm was found to decrease the anode polarization resistance and facilitate the kinetics of the electrochemical reactions. for this study, the following elements were considered due to the similarity of the behavior of the nyquist diagrams in accordance with what bisquert et al. reported. both a warburg element and anomaly diffusion were used to evaluate diffusion resistance in terms of two parameters: y and b. y is the magnitude of the admittance at ω =  rad/s while b characterizes the time it takes for a reactant to diffuse through a thin fi w. the ratio b/y indicates the magnitude of diffusion resistance.figure nyquist diagrams corresponding to anode chamber at different times of growth: a) week and b) week on analyzing the values obtained, the ohmic resistance was not observed to present significant changes between week and week. this contrasted with the resistance values corresponding to the charge transfer phenomena in which a reduction was observed due to the presence of the biofilm. given its high conductivity, it facilitated the mobility of electrons through the electrode/biofilm interface. this behavior is similar to what manohar et al. found, as well as other authors such as malvankar et al. and borole et al.. cpe values for week and were e-03 ±  e- Ω−1sα and  ±  Ω−1sα with α = , respectively. in both systems, the incorporated cpe is considered to be a non-ideal capacitor. for determination, it was considered a faradaic system with time-constant interfacial capacitance rc. c=q1r1+1r2α−11/α q is the admittance when ω= j=−1; α is an ideal constant and r and r are the ohmic and charge transfer resistances, respectively. on comparing the capacitance values between week cdl =  e-04 ±  e- f and week cdl =  e-02 ±  e- f, an increase in capacitance was observed by two orders of magnitude due to the fact that the biofilm facilitates charge accumulation at the electrode interface. although the values may vary based on the experimental conditions, the behavior is similar to that observed in previous works. for example, borole et al. reported their values of cdl in the anode of f during the first two months. also, it increases by two orders of magnitude from to days, obtaining a value of  ±  f. also, ramasamy et al. reported values for double layer capacitance from mf to mf for a surface of cm in anode, which is expected capacitance or carbon electrode. this confirmed that cdl increased due to the stabilization of the system. as for the diffusion processes, these can occur when the solution species are diffused through the biofilm to the surface of the electrode. at week they are represented as a warburg element with linear, semi-infinite diffusion behavior equivalent to rd =   ±  Ωcm at week meanwhile, this element is replaced by a modified anomalous diffusion element equivalent to a linear transmission model reported by bisquert et al. with rd =   ±  Ωcm this decrease in resistance is caused by proton exchange due to the presence of the biofilm.figure equivalent circuits used for the analysis of impedance data for the three systems: anode chamber, biofilm and anolyte at a) week and b) week the model includes these elements, given that the impedance in the diffusion processes is defined as: ziω∝iω−β/20<β< where, in the majority of cases, it is a warburg-type impedance. anomalous diffusion is characterized by a mean squared displacement of the diffusing particles that does not follow the ordinary linear law r2 ∝ t but, more generally, has a power law dependence on time: r2 ∝ tβ at low frequencies. the reason for this is the frequency transient time for a diffusing particle injected at x =  to cover a distance l. for ω > > ωd the particles will not sense the boundary at x = l so that the system will behave as semi-infinite. the presence of the biofilm promotes anomalous diffusion processes, i.e. protonic diffusion which can be spatially restricted in either planar, cylindrical or spherical forms, producing variation in the limits of the diffusion region 0 < x < l present at low frequencies. many different mechanisms give rise to this anomalous behavior, including complex flows, structural complexity in the substrate of diffusion and in the diffusing tracers. thus, no single theory of anomalous diffusion can account for all possible phenomenologies as in this case for the presence of biofilm. biofilm a more common “coating layer” on the electrodes of mfcs is a biofilm. figure  shows the nyquist diagram of the biofilm at weeks and first of all, there is a difference between the two systems at the same frequencies with week presenting lower impedance, once again considering impedance to be a magnitude vector. this is due to the fact that at week the biofilm had already achieved growth and stabilization, favoring processes which reduce the total resistance of the system.figure nyquist diagrams corresponding to biofilms at different times of growth: a) week and b) week likewise, at both weeks and the final part of a semicircle is observed, corresponding to charge transfer processes on the surface of the electrode followed by a linear segment with an incline angle that corresponds to diffusion processes, and finally a predominantly capacitive linear portion similar to the behavior presented by the anode chamber. therefore, the equivalent circuits proposed for the anode chamber at weeks and were used. on analyzing the data obtained, ohmic resistance was not found to present significant changes between week and week. with respect to the cpe, as in the case of the anode chamber, the behavior is mainly non-ideal capacitance. the values obtained at weeks and were e-01 ±  e- f and e-04 ±  e- f, respectively. this allows for the inference that the capacitance contribution in the anode chamber can be attributed to the behavior of the biofilm as has been reported previously by other research groups. the charge transfer resistance for week presented higher values compared to those at week due to the interaction of the biofilm with the anolyte. diffusion processes at weeks and were equivalent to rd  ±  Ωcm and  ±  Ωcm these values were similar to those for the anode chamber at week suggesting that the anolyte presents diffusion processes from the outset. anolyte figure  shows the nyquist diagram corresponding to the anolyte at weeks and in both cases, it was not possible to observe a variation in the impedance value, considering it to be a vector. the ohmic resistance is followed by an incomplete semicircle which indicates the presence of charge transfer processes. it is unlikely that any biochemically derived redox compounds, synthesized mediators, yield a complete faradaic response to an ac signal faster than hz. hence, the reaction in the high frequency region depicts a fast electrochemical process such as oxidation of soluble metal ions in the growth medium.figure nyquist diagrams corresponding to anolyte at different times of growth: a) week and b) week 20). the ohmic resistance values do not present significant changes due to the fact that they correspond to the resistance of the components of the system itself. the charge transfer resistance and diffusion resistance for week r2 = 199 ±  Ωcm and rd =   ±  Ωcm compared to week r2 = 179 ±  Ωcm and rd =  e + 03 ±  e- Ωcm show that the r values are similar, meaning that there is no contribution from the anolyte to the charge transfer processes. the anolyte without biofilm on the electrode is not redox-active toward acetate under these conditions. eis of bare electrodes showed a very high charge transfer resistance. when comparing the results of rd obtained for the anolyte with those for the anode chamber and the biofilm, the anolyte at week presents similar values to those for the anode chamber. however, at week the value increases by an order of magnitude, whilst in the anode chamber it decreases by an order of magnitude because the diffusion resistance decreased considerably due to the presence of the biofilm. cpe values for weeks and were e-03 ±  e- and e-03 ±  e-, corresponding to the capacitances e-05 ±  e- f and e-05 ±  e- respectively. although there is evidence in the literature for the presence of diffusion processes in this type of bioelectrochemical system indicating that the mass transfer limitations were insignificant and masked by the dominant kinetic limitations for the anode bio-electrochemical reaction, this study proved that under the experimental conditions used there is an important contribution from the interaction of the biofilm and the anolyte on cell performance. table  presents a summary of the specific influence of each element of the system under study.table influence of each of the elements corresponding to the phenomena in the mfc configuration system charge transfer processes diffusion processes r1 r2 cdl rd anode chamber c a a b anolyte c c c a biofilm c a a b  the microbial fuel cell with no catalyst and mixed inoculum in this study showed certain members of the bacterial electrode community increased in relative abundance from the initial inoculum. for example, proteobacteria species are important for electricity generation in the anode biofilms and firmicutes phylum was predominant on anolyte to transfer electron. likewise, the biofilm can act as a barrier which reduces diffusion of the anolyte towards the electrode, all the while behaving like a porous material. the anolyte and its interaction with the biofilm exert a considerable influence on diffusion processes, given that it presents the highest values for rd. rd increased at week 
eis were performed on the anode chamber, biofilm and anolyte. the biofilm makes a significant contribution to the charge transfer processes at the electrode and, consequently, on the performance of the anode chamber. the results showed certain members of the bacterial electrode community increased in relative abundance from the initial inoculum. this study proved that under the experimental conditions used there is an important contribution from the interaction of the biofilm and the anolyte on cell performance. the results showed that the system tended to increase power density from 15 ±  to 100 ±  mw/m and current density 211 ±  to 347 ±  ma/m. electrochemical impedance spectroscopy was used to study the behavior of the mfc during initial biofilm and maximum power density. microbial fuel cellcommunity growth and electrochemical impedance spectroscopyissue-copyright-statement© the author 2014 the biofilm can act as a barrier which reduces diffusion of the anolyte towards the electrode, all the while behaving like a porous material. for example, proteobacterial species are important for electricity generation in the anode biofilms and firmicutes phylum was predominant on anolyte to transfer electron. the samples were identified by pyrosequencing of the 16s rrna gene and showed that initial inoculum was constituted by proteobacteria, bacteroidetes and firmicutes. at week proteobacterial species were predominant for electricity generation in the anode biofilm, being 51% rhodopseudomonas palustris. a laboratory-scale two-chamber microbial fuel cell employing an aerated cathode with no catalyst was inoculated with mixed inoculum and acetate as the carbon source. meanwhile on anolyte, firmicutes phylum was predominant with bacillus sp. table presents a summary of the specific influence of each element of the system under study. the anolyte and its interaction with the biofilm exert a considerable influence on diffusion processes, given that it presents the highest values for rd which increased at week 
 a laboratory-scale two-chamber microbial fuel cell employing an aerated cathode with no catalyst was inoculated with mixed inoculum and acetate as the carbon source. electrochemical impedance spectroscopy was used to study the behavior of the mfc during initial biofilm and maximum power density. eis were performed on the anode chamber, biofilm and anolyte. nyquist plots of the eis data were fitted with two equivalent electrical circuits to estimate the contributions of intrinsic resistances to the overall internal mfc impedance at weeks and respectively.  the results showed that the system tended to increase power density from 15 ±  to 100 ±  mw/m and current density 211 ±  to 347 ±  ma/m. the samples were identified by pyrosequencing of the 16s rrna gene and showed that initial inoculum was constituted by proteobacteria, bacteroidetes and firmicutes. at week proteobacterial species were predominant for electricity generation in the anode biofilm, being 51% rhodopseudomonas palustris. meanwhile on anolyte, firmicutes phylum was predominant with bacillus sp. this study proved that under the experimental conditions used there is an important contribution from the interaction of the biofilm and the anolyte on cell performance. table presents a summary of the specific influence of each element of the system under study.  the results showed certain members of the bacterial electrode community increased in relative abundance from the initial inoculum. for example, proteobacterial species are important for electricity generation in the anode biofilms and firmicutes phylum was predominant on anolyte to transfer electron. r is the same in the three systems and no variation is observed over time. the biofilm makes a significant contribution to the charge transfer processes at the electrode and, consequently, on the performance of the anode chamber. the biofilm can act as a barrier which reduces diffusion of the anolyte towards the electrode, all the while behaving like a porous material. the anolyte and its interaction with the biofilm exert a considerable influence on diffusion processes, given that it presents the highest values for rd which increased at week keywords microbial fuel cellcommunity growth and electrochemical impedance spectroscopyissue-copyright-statement© the author 2014 