BACKGROUND
microbial fuel cells  use bacteria as catalysts to oxidise organic and inorganic matter and generate electrical current.
the most widespread proposed use of mfcs, and now the broader term bioelectrochemical systems   <cit> , is for electricity generation during wastewater treatment  <cit> .
irrespective of the goal, the cornerstone of bess is the capacity of microorganisms to perform or participate in extracellular electron transfer .
in this process, microorganisms effectively pump electrons outside the cell, using direct or indirect mechanisms, towards the electron acceptor, i.e.
the anode, which is insoluble and exterior to the cell.
they also provide us with a platform to perform more fundamental research such as that presented in this paper.
direct eet occurs via electron flow through outer membrane proteins  <cit>  or potentially through electrically conductive bacterial appendages such as nanowires  <cit>  that make physical contact with the anode or other bacteria in the vicinity.
indirect eet involves exogenous   <cit>  or endogenous   <cit>  soluble molecules  that act to shuttle electrons through the extracellular aqueous matrix from the cells to the anode  <cit> .
although there is some evidence that increased current production in gram-positive bacteria in an mfc is achieved through redox shuttles  <cit> , other information pertaining to their role in eet is limited  <cit> .
generally, gram-positive bacteria on their own make limited current in comparison to the gram-negative  <cit> .
in most cases, the bacteria in the bes grow onto the electrode forming a biofilm, however, to understand the eet process fully we must also investigate the role of the biofilm.
a biofilm is an extracellular polymeric substance  encased, surface adhering microbial community  <cit> .
conventional theory categorizes biofilm structure around three basic stages of development, initial attachment, maturation and detachment  <cit> .
the eps physically immobilize the bacteria while at the same time provide them opportunity for cell to cell contact and communication.
moreover, electron transfer is constrained by the distance over which electrons need to travel to the electron acceptor and therefore, having a greater understanding of biofilm structure and development in bess may provide us with more of an insight in this area.
therefore this study aimed  to investigate the viability, structure and current production of gram-positive and -negative pure culture biofilms when growing on a closed circuit  and open circuit  anode  to investigate whether bacteria in co-culture generate different levels of current than pure cultures and  to investigate biofilm structure and development between pure and co-cultures on the anode.
for this, we used bacteria which had been isolated or used earlier in mfcs:  <dig> gram-negatives  pseudomonas aeruginosa pao <dig>   <cit> , geobacter sulfurreducens   <cit> , shewanella oneidensis  mr- <dig>  <cit> , and  <dig> gram-positive  organisms, clostridium acetobutylicum   <cit>  and enterococcus faecium   <cit> .
RESULTS
viability of pure culture anode biofilms
using the five pure cultures, closed circuit  and open circuit  batch experiments were run for three days each in an mfc .
during the closed circuit experiments, live/dead staining of the biofilm anode blocks indicated that for all species investigated the viability was higher adjacent to the electrode relative to the top of the biofilm.
the viability gradually decreased further away from the anode.
additional file  <dig> demonstrates the higher magnification  highlight the staining of the cells and not the matrix which can occur sometimes when using the live/dead stain.
as shown in figure  <dig>  the viability of p. aeruginosa was  <dig> ± 4% and  <dig> ± 6% at the top and the bottom of the biofilm respectively .
in contrast, the open circuit experiments showed greater viability on top of the biofilm, further away from the electrode, while more non-viable areas were detected closer to the electrode.
for example, when p. aeruginosa was using a soluble electron acceptor the viabilities were  <dig>  ±  <dig> % and  <dig>  ±  <dig> % top and bottom respectively .
development and current generation of pure and co-culture anode biofilms
during the pure culture closed circuit experiments the heights of the biofilms were less than that of the open circuit experiments .
for example, the biofilm height of p. aeruginosa was  <dig> ±  <dig> μm for the closed circuit experiment and  <dig> ±  <dig> μm for the open circuit experiment, as calculated with comstat.
all g- cultures developed an ample coverage of the electrode within the three ay period both in closed and open circuit.
for example, the s. oneidensis biofilm formed large towers of  <dig> μm high and up to ~ <dig> μm in diameter while the g+ species developed smaller microcolonies with the odd tower up to  <dig> μm high and 10- <dig> μm in diameter .
the latter was also reflected in the higher roughness coefficient between the g- and g+ biofilms indicating that during batch mode the g+ are flatter and more uniform than the g- .
during these pure culture batch experiments g+ species delivered low current throughout while the g- produced a much higher current as shown in table  <dig>
co-culture experiments with
during the pure culture continuous experiments, g. sulfurreducens and s. oneidensis initially showed very similar development, although slower than p. aeruginosa, with small towers averaging a height of  <dig> μm and diameters between 10- <dig> μm.
moreover, the biofilms became less dense with higher towers developing while prolonged biofilm development revealed less coverage of the electrode giving way to the formation of channels and loss of biofilm mass, similar to that observed in the p. aeruginosa biofilm .
additionally, a few towers reaching  <dig> μm in height were observed in the g. sulfurreducens biofilm while the s. oneidensis biofilm revealed an occasional tower structure up to  <dig> μm dispersed throughout the biofilm.
these results also correlated with the high level of roughness coefficient measurement from comstat  again indicating the non-uniformity of these biofilms throughout the duration of the continuous pure culture experiment.
during continuous mode the g+ c. acetobutylicum and e. faecium biofilms started out slowly and similarly with only small  aggregates of biofilm growth on the electrode.
these biofilms did not increase in height like the g- and as time progressed the heights of these biofilms remained low .
by the end of  <dig> hours the biofilms highest point reached  <dig> μm, with colony diameters of less than  <dig> μm.
a more detailed description of the pure culture continuous experiments can be seen in additional file  <dig>  roughness coefficients for g+ during continuous experiments were higher than those of the batch experiments  indicating more non-uniformity during the continuous experiments.
the continuously fed mfcs revealed the g- consistently generating more current than the g+ .
p. aeruginosa reached its peak in current production  between 24- <dig> hours, however, by  <dig> hours it had decreased to  <dig>  ±  <dig>  ma.
g. sulfurreducens and s. oneidensis, on the other hand, both increased current generation later in the experiment while the g+ e. faecium and c. acetobutylicum maintained a low current throughout.
during the continuous co-culture experiments, e. faecium remained in the close vicinity of the electrode while the g- colonized the top of the biofilm.
as time progressed they separated with the g- forming towers and e. faecium developed a lawn over the electrode surrounding the g-.
confocal microscopy revealed large towers of p. aeruginosa  surrounded by a lawn of e. faecium .
initially, these towers of p. aeruginosa were very sparse and the growth of the two together was patchy although covering more of the electrode than any of the pure cultures.
similarly, s. oneidensis and e. faecium  and g. sulfurreducens and e. faecium co-culture  biofilms also separated during development with g. sulfurreducens and s. oneidensis forming smaller towers.
a more detailed description of the co-culture experiments is presented in additional file  <dig>  roughness coefficients from the co-culture continuous experiments were lower than those of the pure cultures indicating a more uniform and even biofilm .
co-culture continuous experiment with e. faecium and a g- all produced more current compared to the pure cultures .
for example, s. oneidensis and e. faecium separately generated  <dig>  ±  <dig>  and  <dig>  ±  <dig>  ma respectively while together the highest current generated was  <dig>  ±  <dig>  ma.
this co-culture generated more current initially than the geobacter and pseudomonas ones, but levelled off between 24- <dig> hours after which it began to decrease.
this same behaviour was observed across the triplicate experiments.
contrary to e. faecium, none of the co-culture experiments with c. acetobutylicum showed any difference in performance relative to the pure culture experiments .
discussion
in this study, we observed quite low current densities relative to a number of dedicated pure culture studies  <cit> .
to accommodate the growth of five different species, we created a joint medium which may have caused suboptimal growth conditions for each culture.
however, it eliminated any discrepancies caused by differing constituents within the media when analyzing biofilms.
to observe the viability of the anodic biofilms, live/dead staining was employed.
this stain is an assay for membrane integrity and does not exclusively separate live from dead cells or unequivocally confirms metabolic inactivity  <cit> , nevertheless, it has been successfully used in many studies to indicate viability of the bacteria  <cit> .
in this study, this method was thought to be the best option compared to other viability indicators which have to be incubated for a considerable time period or have redox activity by themselves.
viability, structure and current of pure culture anode biofilms
during the closed circuit batch experiments viability was maintained in the proximity of the electrode, with slight variations between cultures .
this may suggest that the most active part of the biofilm, playing a major role in the eet process, is within 10- <dig> μm of the electrode.
in our set-up, the electron donor was generally provided in excess concentrations.
as a result, the decreasing viability away from the anode can rather be attributed to limitations for the electron transfer towards the electrode than substrate limitation.
at the current densities observed, it appears unlikely that proton accumulation limited the biofilm performance, as observed previously  <cit> .
during these batch experiments the g- biofilms remained viable while the thinner g+ biofilms rapidly lost viability.
a very insightful study using g. sulfurreducens reported no loss of viability as biofilm thickness and current increased  <cit> , while our study revealed a notable increase of the non-viable cells over the duration of the study, with decreasing viability away from the anode.
although this experiment uses the same strain of g. sulfurreducens as the reguera et al.
study, the media and fuel cells are not the same which may explain the variations between these two studies.
for all bacteria in the batch experiments, the closed circuit biofilms were thinner than the open circuit biofilms .
this could be related to the larger thermodynamic gain  and availability of the soluble electron acceptors relative to the electrode , which can lead to higher bacterial growth yield  <cit> .
while the biofilm structure of the g- batch experiments was different to the continuous experiments , the clsm images indicate that they developed in the typical stages conceptualized in other biofilm studies  <cit> , initially as small clusters of biofilm and later as larger towers.
the roughness coefficients also differed, suggesting that the biofilms grown in batch mode were more uniform and flatter than those of the continuous experiments, which had higher roughness coefficients.
the g+ biofilms were very similar in development during batch and continuous experiments, also in this case the supply of soluble electron acceptors produced thicker biofilms.
the pure cultures of g. sulfurreducens and s. oneidensis used in this study did not produce as much current as previous studies  <cit> .
this may be due to the compromised medium used to grow all five cultures, as well as the suboptimal configuration of the mfc in terms of internal resistance.
during all experiments g+ bacteria generated limited current by themselves, while g- bacteria generated much higher currents .
this was expected as, unlike the g- bacteria, most g+ on their own have limited eet competence  <cit> .
some are electrochemically active to a certain extent such as a clostridium butyricum strain isolated from a mediatorless mfc  <cit>  and enterococcus sp.
<cit> .
in previous work g+ generally require either bacterially produced redox shuttles or humics  <cit>  to generate significant current.
one exception to this so far is a thermophilic isolate, thermincola sp.
strain jr  <cit> .
in some instances g+ have been seen to dominate populations in mixed culture mfcs  <cit> .
hence, while g+ have some capacity for electron transfer, it is apparent that the g- used here generated much greater current in our mfc conditions.
interestingly, the current generated by p. aeruginosa in batch mode was larger than in continuous mode which may be concomitant with the gradual loss of redox shuttles previously implicated in electron transfer by p. aeruginosa  <cit> .
p. aeruginosa as a pure culture decreased its current production after the  <dig> hour timepoint  in continuous mode, however, in batch mode it continued to increase current.
potentially, a gradual wash-out of redox shuttles, which can be produced by p. aeruginosa, explains the lower performance in continuous mode  <cit> .
a comprehensive, non-mfc based study using pa <dig> to investigate phenotypic differentiation and seeding dispersal also noted a halt in biofilm height after about  <dig> hours  <cit> .
during that study microcolonies of  <dig> μm diameter became differentiated, leaving the microcolony hollow by day  <dig>  similarly to our current study, by  <dig> hours pao <dig> had formed  <dig> ±  <dig> μm thick biofilms, which did not increase throughout the duration of the experiment.
although the aforementioned study used different parameters, the growth and retardation of the pa <dig> biofilms coincided with the timing of the assumed decreased eet activity in our mfc.
co-culture versus pure culture current generation
the three co-cultures  used in this study all generated more current together then when grown as pure cultures.
although this has not yet been investigated at a deeper level, several studies have noted the coexistence between g+ and g- within the mfc environment.
for example, the role of a phenazine electron shuttle has been verified in an earlier mfc study where it was observed to increase current generation in co-cultures of brevibacillus sp.
and enterococcus sp.
with pseudomonas sp.
these studies determined that the g+ were able to use electron shuttles  produced by pseudomonas sp  <cit> , the combination of both bacteria being the more successful one.
whether other mechanisms such as quorum regulation or the establishment of a syntrophic association is in play is yet to be investigated.
in a recent study, nevin et al.,  <cit>  described how pure culture biofilms of g. sulfurreducens were able to reach current densities of the same order of magnitude as mixed population current densities.
in the latter case, the anode surface was minimized in order to ensure that the anode became the limiting factor.
it is important to distinguish here that while the electron flux achievable through a defined surface area can indeed be similar for a pure culture and a mixed population, in conditions where the surface area is not limiting  mixed populations  consistently perform better than pure cultures by apparently generating larger reducing power.
it is likely that, similar to earlier biofilm studies, metabolic cooperation leads to increased performance  <cit> , further research on this is warranted.
the tower development by the g- organisms in coculture may be an ecological strategy to gain greater access to the carbon source, while maintaining contact with the electrode via a superior electron transfer mechanism.
the competition for substrate does not exclude a simultaneous metabolic cooperation for electron transfer.
hansen et al.,  <cit>  studied the evolution of species within a co-culture and described a symbiotic relationship which in a short duration apparently stabilized species interactions and affected community function.
spatial structure was the key environmental factor provided in our current study as well as in the hansen study mentioned above.
given suitable conditions to establish a community, the co-cultures used in this study have been allowed to evolve and form their own structure and interactions, which have produced a more productive community.
CONCLUSIONS
this study has shown that biofilms of pure culture g- and g+ remain viable closest to the electrode while becoming non-viable on top or the further away from the electrode.
this result was also reiterated by the reverse experiment, where a soluble electron acceptor was offered, with the top of the biofilm remaining viable and the bottom of the biofilm becoming non-viable.
the g- cultures developed thicker biofilms, higher towers and produced higher current while the g+ produced thinner biofilms, smaller towers and lower current.
co-culture experiments between e. faecium and g- bacteria evidenced a significant increase in current generation when grown together in the mfc, indicating a synergistic or mutualistic relationship between e. faecium and g- bacteria within this system which warrants further investigation.

 microbial fuel cells use bacteria as catalysts to oxidise organic and inorganic matter and generate electrical current. the most widespread proposed use of mfcs, and now the broader term bioelectrochemical systems, is for electricity generation during wastewater treatment. irrespective of the goal, the cornerstone of bess is the capacity of microorganisms to perform or participate in extracellular electron transfer. in this process, microorganisms effectively pump electrons outside the cell, using direct or indirect mechanisms, towards the electron acceptor, i.e. the anode, which is insoluble and exterior to the cell. they also provide us with a platform to perform more fundamental research such as that presented in this paper. direct eet occurs via electron flow through outer membrane proteins or potentially through electrically conductive bacterial appendages such as nanowires that make physical contact with the anode or other bacteria in the vicinity. indirect eet involves exogenous or endogenous soluble molecules that act to shuttle electrons through the extracellular aqueous matrix from the cells to the anode. although there is some evidence that increased current production in gram-positive bacteria in an mfc is achieved through redox shuttles, other information pertaining to their role in eet is limited. generally, gram-positive bacteria on their own make limited current in comparison to the gram-negative. in most cases, the bacteria in the bes grow onto the electrode forming a biofilm, however, to understand the eet process fully we must also investigate the role of the biofilm. a biofilm is an extracellular polymeric substance encased, surface adhering microbial community. conventional theory categorizes biofilm structure around three basic stages of development, initial attachment, maturation and detachment. the eps physically immobilize the bacteria while at the same time provide them opportunity for cell to cell contact and communication. moreover, electron transfer is constrained by the distance over which electrons need to travel to the electron acceptor and therefore, having a greater understanding of biofilm structure and development in bess may provide us with more of an insight in this area. therefore this study aimed to investigate the viability, structure and current production of gram-positive and -negative pure culture biofilms when growing on a closed circuit and open circuit anode to investigate whether bacteria in co-culture generate different levels of current than pure cultures and to investigate biofilm structure and development between pure and co-cultures on the anode. for this, we used bacteria which had been isolated or used earlier in mfcs: gram-negatives pseudomonas aeruginosa pao, geobacter sulfurreducens, shewanella oneidensis mr-, and gram-positive organisms, clostridium acetobutylicum and enterococcus faecium.  viability of pure culture anode biofilms using the five pure cultures, closed circuit and open circuit batch experiments were run for three days each in an mfc. during the closed circuit experiments, live/dead staining of the biofilm anode blocks indicated that for all species investigated the viability was higher adjacent to the electrode relative to the top of the biofilm. the viability gradually decreased further away from the anode. additional file demonstrates the higher magnification highlight the staining of the cells and not the matrix which can occur sometimes when using the live/dead stain. as shown in figure the viability of p. aeruginosa was ± 4% and ± 6% at the top and the bottom of the biofilm respectively. in contrast, the open circuit experiments showed greater viability on top of the biofilm, further away from the electrode, while more non-viable areas were detected closer to the electrode. for example, when p. aeruginosa was using a soluble electron acceptor the viabilities were ± % and ± % top and bottom respectively. development and current generation of pure and co-culture anode biofilms during the pure culture closed circuit experiments the heights of the biofilms were less than that of the open circuit experiments. for example, the biofilm height of p. aeruginosa was ± μm for the closed circuit experiment and ± μm for the open circuit experiment, as calculated with comstat. all g- cultures developed an ample coverage of the electrode within the three ay period both in closed and open circuit. for example, the s. oneidensis biofilm formed large towers of μm high and up to ~ μm in diameter while the g+ species developed smaller microcolonies with the odd tower up to μm high and 10- μm in diameter. the latter was also reflected in the higher roughness coefficient between the g- and g+ biofilms indicating that during batch mode the g+ are flatter and more uniform than the g-. during these pure culture batch experiments g+ species delivered low current throughout while the g- produced a much higher current as shown in table co-culture experiments with during the pure culture continuous experiments, g. sulfurreducens and s. oneidensis initially showed very similar development, although slower than p. aeruginosa, with small towers averaging a height of μm and diameters between 10- μm. moreover, the biofilms became less dense with higher towers developing while prolonged biofilm development revealed less coverage of the electrode giving way to the formation of channels and loss of biofilm mass, similar to that observed in the p. aeruginosa biofilm. additionally, a few towers reaching μm in height were observed in the g. sulfurreducens biofilm while the s. oneidensis biofilm revealed an occasional tower structure up to μm dispersed throughout the biofilm. these results also correlated with the high level of roughness coefficient measurement from comstat again indicating the non-uniformity of these biofilms throughout the duration of the continuous pure culture experiment. during continuous mode the g+ c. acetobutylicum and e. faecium biofilms started out slowly and similarly with only small aggregates of biofilm growth on the electrode. these biofilms did not increase in height like the g- and as time progressed the heights of these biofilms remained low. by the end of hours the biofilms highest point reached μm, with colony diameters of less than μm. a more detailed description of the pure culture continuous experiments can be seen in additional file roughness coefficients for g+ during continuous experiments were higher than those of the batch experiments indicating more non-uniformity during the continuous experiments. the continuously fed mfcs revealed the g- consistently generating more current than the g+. p. aeruginosa reached its peak in current production between 24- hours, however, by hours it had decreased to ± ma. g. sulfurreducens and s. oneidensis, on the other hand, both increased current generation later in the experiment while the g+ e. faecium and c. acetobutylicum maintained a low current throughout. during the continuous co-culture experiments, e. faecium remained in the close vicinity of the electrode while the g- colonized the top of the biofilm. as time progressed they separated with the g- forming towers and e. faecium developed a lawn over the electrode surrounding the g-. confocal microscopy revealed large towers of p. aeruginosa surrounded by a lawn of e. faecium. initially, these towers of p. aeruginosa were very sparse and the growth of the two together was patchy although covering more of the electrode than any of the pure cultures. similarly, s. oneidensis and e. faecium and g. sulfurreducens and e. faecium co-culture biofilms also separated during development with g. sulfurreducens and s. oneidensis forming smaller towers. a more detailed description of the co-culture experiments is presented in additional file roughness coefficients from the co-culture continuous experiments were lower than those of the pure cultures indicating a more uniform and even biofilm. co-culture continuous experiment with e. faecium and a g- all produced more current compared to the pure cultures. for example, s. oneidensis and e. faecium separately generated ± and ± ma respectively while together the highest current generated was ± ma. this co-culture generated more current initially than the geobacter and pseudomonas ones, but levelled off between 24- hours after which it began to decrease. this same behaviour was observed across the triplicate experiments. contrary to e. faecium, none of the co-culture experiments with c. acetobutylicum showed any difference in performance relative to the pure culture experiments. discussion in this study, we observed quite low current densities relative to a number of dedicated pure culture studies. to accommodate the growth of five different species, we created a joint medium which may have caused suboptimal growth conditions for each culture. however, it eliminated any discrepancies caused by differing constituents within the media when analyzing biofilms. to observe the viability of the anodic biofilms, live/dead staining was employed. this stain is an assay for membrane integrity and does not exclusively separate live from dead cells or unequivocally confirms metabolic inactivity, nevertheless, it has been successfully used in many studies to indicate viability of the bacteria. in this study, this method was thought to be the best option compared to other viability indicators which have to be incubated for a considerable time period or have redox activity by themselves. viability, structure and current of pure culture anode biofilms during the closed circuit batch experiments viability was maintained in the proximity of the electrode, with slight variations between cultures. this may suggest that the most active part of the biofilm, playing a major role in the eet process, is within 10- μm of the electrode. in our set-up, the electron donor was generally provided in excess concentrations. as a result, the decreasing viability away from the anode can rather be attributed to limitations for the electron transfer towards the electrode than substrate limitation. at the current densities observed, it appears unlikely that proton accumulation limited the biofilm performance, as observed previously. during these batch experiments the g- biofilms remained viable while the thinner g+ biofilms rapidly lost viability. a very insightful study using g. sulfurreducens reported no loss of viability as biofilm thickness and current increased, while our study revealed a notable increase of the non-viable cells over the duration of the study, with decreasing viability away from the anode. although this experiment uses the same strain of g. sulfurreducens as the reguera et al. study, the media and fuel cells are not the same which may explain the variations between these two studies. for all bacteria in the batch experiments, the closed circuit biofilms were thinner than the open circuit biofilms. this could be related to the larger thermodynamic gain and availability of the soluble electron acceptors relative to the electrode, which can lead to higher bacterial growth yield. while the biofilm structure of the g- batch experiments was different to the continuous experiments, the clsm images indicate that they developed in the typical stages conceptualized in other biofilm studies, initially as small clusters of biofilm and later as larger towers. the roughness coefficients also differed, suggesting that the biofilms grown in batch mode were more uniform and flatter than those of the continuous experiments, which had higher roughness coefficients. the g+ biofilms were very similar in development during batch and continuous experiments, also in this case the supply of soluble electron acceptors produced thicker biofilms. the pure cultures of g. sulfurreducens and s. oneidensis used in this study did not produce as much current as previous studies. this may be due to the compromised medium used to grow all five cultures, as well as the suboptimal configuration of the mfc in terms of internal resistance. during all experiments g+ bacteria generated limited current by themselves, while g- bacteria generated much higher currents. this was expected as, unlike the g- bacteria, most g+ on their own have limited eet competence. some are electrochemically active to a certain extent such as a clostridium butyricum strain isolated from a mediatorless mfc and enterococcus sp.. in previous work g+ generally require either bacterially produced redox shuttles or humics to generate significant current. one exception to this so far is a thermophilic isolate, thermincola sp. strain jr. in some instances g+ have been seen to dominate populations in mixed culture mfcs. hence, while g+ have some capacity for electron transfer, it is apparent that the g- used here generated much greater current in our mfc conditions. interestingly, the current generated by p. aeruginosa in batch mode was larger than in continuous mode which may be concomitant with the gradual loss of redox shuttles previously implicated in electron transfer by p. aeruginosa. p. aeruginosa as a pure culture decreased its current production after the hour timepoint in continuous mode, however, in batch mode it continued to increase current. potentially, a gradual wash-out of redox shuttles, which can be produced by p. aeruginosa, explains the lower performance in continuous mode. a comprehensive, non-mfc based study using pa to investigate phenotypic differentiation and seeding dispersal also noted a halt in biofilm height after about hours. during that study microcolonies of μm diameter became differentiated, leaving the microcolony hollow by day similarly to our current study, by hours pao had formed ± μm thick biofilms, which did not increase throughout the duration of the experiment. although the aforementioned study used different parameters, the growth and retardation of the pa biofilms coincided with the timing of the assumed decreased eet activity in our mfc. co-culture versus pure culture current generation the three co-cultures used in this study all generated more current together then when grown as pure cultures. although this has not yet been investigated at a deeper level, several studies have noted the coexistence between g+ and g- within the mfc environment. for example, the role of a phenazine electron shuttle has been verified in an earlier mfc study where it was observed to increase current generation in co-cultures of brevibacillus sp. and enterococcus sp. with pseudomonas sp. these studies determined that the g+ were able to use electron shuttles produced by pseudomonas sp, the combination of both bacteria being the more successful one. whether other mechanisms such as quorum regulation or the establishment of a syntrophic association is in play is yet to be investigated. in a recent study, nevin et al., described how pure culture biofilms of g. sulfurreducens were able to reach current densities of the same order of magnitude as mixed population current densities. in the latter case, the anode surface was minimized in order to ensure that the anode became the limiting factor. it is important to distinguish here that while the electron flux achievable through a defined surface area can indeed be similar for a pure culture and a mixed population, in conditions where the surface area is not limiting mixed populations consistently perform better than pure cultures by apparently generating larger reducing power. it is likely that, similar to earlier biofilm studies, metabolic cooperation leads to increased performance, further research on this is warranted. the tower development by the g- organisms in coculture may be an ecological strategy to gain greater access to the carbon source, while maintaining contact with the electrode via a superior electron transfer mechanism. the competition for substrate does not exclude a simultaneous metabolic cooperation for electron transfer. hansen et al., studied the evolution of species within a co-culture and described a symbiotic relationship which in a short duration apparently stabilized species interactions and affected community function. spatial structure was the key environmental factor provided in our current study as well as in the hansen study mentioned above. given suitable conditions to establish a community, the co-cultures used in this study have been allowed to evolve and form their own structure and interactions, which have produced a more productive community.  this study has shown that biofilms of pure culture g- and g+ remain viable closest to the electrode while becoming non-viable on top or the further away from the electrode. this result was also reiterated by the reverse experiment, where a soluble electron acceptor was offered, with the top of the biofilm remaining viable and the bottom of the biofilm becoming non-viable. the g- cultures developed thicker biofilms, higher towers and produced higher current while the g+ produced thinner biofilms, smaller towers and lower current. co-culture experiments between e. faecium and g- bacteria evidenced a significant increase in current generation when grown together in the mfc, indicating a synergistic or mutualistic relationship between e. faecium and g- bacteria within this system which warrants further investigation. 
p. aeruginosa and e. faecium together generated a current of ± ma while alone they produced ± and ± ma respectively. microbial fuel cells rely on electrochemically active bacteria to capture the chemical energy contained in organics and convert it to electrical energy. closed circuit anode pseudomonas aeruginosa biofilms were considerably thinner compared to the open circuit anode, which is likely due to the higher energetic gain of soluble electron acceptors used. bacteria develop biofilms on the mfc electrodes, allowing considerable conversion capacity and opportunities for extracellular electron transfer. to understand how bacteria form biofilms within mfcs and how their development, structure and viability affects electron transfer, we performed pure and co-culture experiments. the two gram-positive bacteria used only provided a fraction of current produced by the gram-negative organisms. we postulate that this segregation may be an essential difference in strategy for electron transfer and substrate capture between the gram-negative and the gram-positive bacteria used here.
 microbial fuel cells rely on electrochemically active bacteria to capture the chemical energy contained in organics and convert it to electrical energy. bacteria develop biofilms on the mfc electrodes, allowing considerable conversion capacity and opportunities for extracellular electron transfer. the present knowledge on eet is centred around two gram-negative models, i.e. shewanella and geobacter species, as it is believed that gram-positives cannot perform eet by themselves as the gram-negatives can. to understand how bacteria form biofilms within mfcs and how their development, structure and viability affects electron transfer, we performed pure and co-culture experiments.  biofilm viability was maintained highest nearer the anode during closed circuit operation, in contrast to when the anode was in open circuit where viability was highest on top of the biofilm, furthest from the anode. closed circuit anode pseudomonas aeruginosa biofilms were considerably thinner compared to the open circuit anode, which is likely due to the higher energetic gain of soluble electron acceptors used. the two gram-positive bacteria used only provided a fraction of current produced by the gram-negative organisms. power output of co-cultures gram-positive enterococcus faecium and either gram-negative organisms, increased by 30-70% relative to the single cultures. over time the co-culture biofilms segregated, in particular, pseudomonas aeruginosa creating towers piercing through a thin, uniform layer of enterococcus faecium. p. aeruginosa and e. faecium together generated a current of ± ma while alone they produced ± and ± ma respectively.  we postulate that this segregation may be an essential difference in strategy for electron transfer and substrate capture between the gram-negative and the gram-positive bacteria used here. 