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A rapid methodology using fatty acid methyl esters to prole bacterial community structures in microbial fuel cells Kristina

Y. Nelson, Behrooz Razban, Dena W. McMartin, D. Roy Cullimore, Takaya Ono, Patrick D. Kiely PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S1567-5394(09)00175-3 doi: 10.1016/j.bioelechem.2009.09.005 BIOJEC 6409 Bioelectrochemistry 15 January 2009 6 September 2009 8 September 2009

Please cite this article as: Kristina Y. Nelson, Behrooz Razban, Dena W. McMartin, D. Roy Cullimore, Takaya Ono, Patrick D. Kiely, A rapid methodology using fatty acid methyl esters to prole bacterial community structures in microbial fuel cells, Bioelectrochemistry (2009), doi: 10.1016/j.bioelechem.2009.09.005

This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could aect the content, and all legal disclaimers that apply to the journal pertain.

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A rapid methodology using fatty acid methyl esters to profile bacterial community structures in microbial fuel cells Kristina Y. Nelsona*, Behrooz Razbana, Dena W. McMartina, D. Roy Cullimoreb, Takaya Onoc and Patrick D. Kielyd University of Regina, Faculty of Engineering, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada. b Droycon Bioconcepts Inc., 315 Dewdney Avenue, Regina, Saskatchewan, S4N 0E7 Canada. c Safe Water and LED Lighting Operatives Worldwide Inc., Regina, Saskatchewan, S4V 0C2, Canada. d Department of Civil and Environmental Engineering, 131 Sackett Building, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. kristina_nelson78@yahoo.com, behroozrazban@gmail.com, dena.mcmartin@uregina.ca, drc@dbi.ca, takaya.ono@swallowinc.org, patkiel01@yahoo.com Abstract

A new methodology is presented here as an effective, preliminary technique for the identification of indigenous aerobic and facultatively anaerobic bacterial communities found within microbial fuel cells (MFCs). The dual-phased method, named Rapid Agitation Static Incubation Microbial Identification, or RASI-MIDI, is comprised of rapidly agitating the sample within a SLYM-BART tester followed by stationary incubation produces a biomass that is subjected to extraction of methyl ester fatty acids. These distinctive fatty acid profiles represent a bacterial community fingerprint unique to the MFC, and are stored in a library for analysis. A total of 84 samples were analyzed for bacterial community structures from seven different groups of MFCs. Results showed that comparisons of replicate MFCs comprising the same bacterial communities generated high similarity index (SI) numbers (SI values ranging from 0.77 to 0.97), indicating highly correlated fatty acid profiles. In contrast, comparisons of MFCs having known dissimilar community structures did not consistently generate SI values in the analysis considered to be a significant match. It was found that this protocol described herein uniquely and accurately produced MFC fatty acid profiles contained in bacterial communities and thus provides a potential method for routinely studying MFC bacterial community fingerprints. Key words: Microbial fuel cells; Bacterial community structure; Fatty acid; FAME analysis; bacterial communities * Corresponding author. University of Regina, Faculty of Engineering, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada. Tel: +1 306 751 0775; Fax: +1 306 585 3000. Email address: kristina_nelson78@yahoo.com (Kristina Nelson).

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1. Introduction One of the most significant current global challenges is to find methods to economically exploit the known alternative energy sources. The production of energy without the ongoing release of greenhouse gas emissions is of particular concern. The use of alternative energy remains to become widespread due to high capital costs, and the lack of ease in which they are implemented and reliably maintained. Thus, the need for renewable, cost-effective energy sources without a net carbon dioxide emission remains critical. Microbial fuel cells have only recently received any significant attention as a means to produce environmentally friendly electricity from organic substrates [1]. Microbial fuel cell (MFC) technology is a potentially rewarding energy production method whereby electrical energy is released through the conversion of energy during normal microbiological metabolic activities [2]. While it has been established that microbial fuel cells use bacteria to convert organic matter into electricity, it has not been completely exploited [3]. Evaluation of the microbiological generation of electrical potential began in the 1970s when MFCs were used to study the ability of microorganisms to be used as catalysts in fuel cells [4,5]. Research focus shifted in the 1990s to involve bacteria treating wastewater a fuel cell environment [6]. However, only recently has it been demonstrated that MFCs can produce power levels that make it possible to use them in practical applications, such as powering electronic sensors or treating wastewater [7,8]. In monitoring microbial fuel cells, attention must be paid to bacterial community structure and activity [9]. Fuel cell performance may be restricted due to insufficient knowledge of the bacterial ecosystem found in MFCs, and how they change and are manipulated [10,11]. Many studies have attempted to characterize microbial populations and activities to elucidate MFC behaviour and ecology [9,12-15]. To date, the examination of bacterial community structures and changes within MFCs have been discussed primarily in terms of specific bacterial strains, rather than examining the entire community and its associated fingerprint. The purpose of this study is to develop a preliminary methodology through which the differences and dynamics of bacterial communities in mixed culture MFCs may be assessed and compared. This was achieved through examining and identifying the aerobic and facultative anaerobic bacterial community fingerprint, or profile, rather than identifying individual bacterial species present in the community. This method is still being optimized, but the initial findings demonstrate that fatty acid profiling of bacterial communities provides a promising, new approach to examine and monitor changes in microbial structures, which warrants further research. To examine the bacterial community structures, fatty acid methyl ester (FAME) profiles of the overall community structures within the mixed culture MFCs were produced. This technique has been widely used for pure bacterial culture studies; however, this study developed a protocol for the application of FAME analysis of the bacterial community structures in MFC units. The method developed uses a two-phased culturing method (Rapid Agitation, Static Incubation or RASI) in a commercially available SLYM-BART tester using proprietary media (Droycon Bioconcepts, Inc., Regina, Canada) followed by Microbial Identification analysis (MIDI Inc, Newark, DE). The resulting FAME profiles of the bacterial communities were stored in libraries using the MIDI software. These profiles could later be accessed, analyzed, and compared to

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other profiles generated from other MFCs where the fuel cell performance has been documented. It was demonstrated that bacterial communities housed within a porous, structured, water-saturated natural organic matrix can be uniquely profiled using the described protocol. The significance of this methodology is that bacterial community profiles in MFCs can be analyzed with this technique rapidly and with precision. It was found that the type of community structure did relate to voltage outputs. Thus, the primary focus of this research is to develop and assess the robustness of this preliminary technique as grounds for further research and potential use in studying and profiling bacterial communities in microbial fuel cells. 2. Background It has been established that microbial fuel cells may be comprised of several strains of bacteria which are dynamic in nature and thus function together as a collective group [3,16]. Typically, individual bacterial strains generally are not observed in the environment without the direct interaction with other bacterial strains. Bacterial species generally co-exist interdependently within a coherent community to form a bacterial consortium. Because various bacterial communities dominate within a functioning MFC, identification of the entire bacterial community is more significant than that of any specific culturable strain [17]. As the environment changes, the dominating community within the bacterial consortium of the fuel cell will shift and the dynamics of the community should reflect this. Thus, a determination of the fuel cell bacterial consortial structure and its relationship to external factors, such as temperature and applied loads, improves our understanding of the nature, management and performance of microbial fuel cells. Typically for the identification of bacterial communities, there are two general methods accepted. The first method relies on utilizing molecular techniques to analyze DNA, while the second method identifies only the culturable bacteria present in the sample. Several molecular methods involving the extraction and analysis of DNA from entire bacterial communities are used to identify genetic fingerprints of bacteria. These methods, including automated ribosomal intergenic spacer analysis (ARISA), polymerase chain reaction (PCR) amplification of nucleic acid fragments, fluorescent in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE), generate profiles of bacterial community structures. The profiled structures are then analyzed through principal component analysis (PCA) or computational analysis (e.g. cluster and dendrogram analysis). While DNA fingerprinting is culture-independent, and can rapidly assess complex communities from various environments [18], the results may be affected from using PCR-based sequencing methods which may bias the results of molecularbased analysis of microbial community structure, as described by Jones and Thies, 2007 [19]. The second identification procedure employs traditional cultivation processes using selective nutrients to promote the growth of different types of bacteria within the sample. The community structure can then be assessed by identifying the isolates from the dominant colonies that were cultured. This can often be costly and laborsome as each isolate is further studied by examining its physiology, taxonomy and reactivity to stains [20]. Extensive databases comprised of numerous isolates have been developed to assist in the study of culture-dependent microbial communities [21]. The efficacy of this

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protocol is restricted to identifying only culturable strains. Such selective techniques may bias the results by favoring only cultivatable strains able to grow in typical laboratory conditions, while ignoring those species unable to be cultured. According to Hattori [22], populations of bacteria taken from natural samples may not be accurately represented when grown on agar media. The principal focus of classical methods to culture bacteria was for the study of pathogenic bacteria, thus ignoring many bacterial species and their communities [23]. Another option is to identify different isolates through fatty acid analysis of the cell membrane. By analyzing fatty acid methyl esters (FAMEs) for each bacterial species, a fatty acid profile is produced that is unique to that particular species. FAME analysis has become a common identification tool and has been used to identify bacterial communities through clustering isolate profiles into conspecific or similar groups [20]. Commercial libraries have been created to identify FAME profiles of bacterial strains. However, because the libraries are generally developed based on stock cultures used in public health clinical labs for identifying pathogenic strains, they frequently do not include naturally-occurring environmental strains. As well, newly discovered isolates may not have been sufficiently studied such that they are included in commercial libraries. This study investigates the use of FAME analysis to examine dominating bacterial community fingerprints in microbial fuel cells. Bobbie and White, 1980 [24] noted that FAME analysis of bacteria within a mixed culture had sufficiently diverse fatty acids such that they could be separated and identified. This is advantageous over having to identify each individual isolate. Instead of distinguishing the species or genus of each bacterial isolate and examining each physiological profile, FAME analysis can be used to compare microbial community profiles, because several categories of microorganisms are comprised of unique fatty acid structures [25]. FAME analysis of bacterial communities allows a profile to be quickly developed of bacteria that can be grown in nutrients [26]. Thus, the precision of FAME analysis is subjected the applied cultural techniques. There are several ways to interpret fatty acid profiles from bacterial strains. Generally, assumptions are made that phospholipids from which the fatty acids are derived comprise a relatively constant fraction of the cell. According to Zelles [27], not all bacterial constituents of the community can be identified because different proportions of some fatty acids may be present in the generated profiles due to blending that may lead to possible bias when examining complex community structures. As such, there is concern surrounding the ability to accurately interpret bacterial community fatty acid profiles [28] because profiles can vary as a result of different environmental effects [29,30]. As well, in order to prevent biased results, if selective media is used in preparing the sample for FAME analysis, attention must be paid to choosing the appropriate media to promote the growth of both dominant and non-dominant strains. One detailed study examined FAME profiles of model bacterial communities using soil bacterial isolates where the taxonomic structure had been previously identified [31]. Two model communities were constructed from a combination of the isolates in known proportions, and the resulting whole-community profile was examined using MIDI and their commercially available gas chromatograph software package. Reproducibility of the profiles and the accuracy of the profile representing the underlying taxonomic structure were examined. Using mathematical analysis, the differences

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between the two communities were analyzed. Haack et al. demonstrated that comparisons of microbial communities to subsequently determine if they are similar or not can be performed using MIDI-FAME analysis. The study also confirmed that each fatty acid peak was correlated with at least one isolate; there was no uncertainty in the peaks that comprised the profile. Classical methods to analyze bacterial communities were performed either by culturing techniques or by light microscopic inspection, generally time consuming and costly [32,33]. However, analysis of fatty acid methyl esters provides a promising means by which to study microbial community profiles. Bacterial communities represent the prime component of microbial fuel cells and are responsible for the generation of electricity [16] and can also be used for wastewater treatment. This paper discusses the application and improvements in the use of FAME analysis applied to microbial fuel cells in the analysis of bacterial community structure. The scope of this research does not include identifying specific strains within the community, but rather it involves developing an analytical technique that can be used to assess and monitor overall bacterial community structures, or profiles, and how they change in response to fuel cell lifetime, optimization, power production, nutrient loading, etc. The purpose of this paper is to identify a novel approach to studying the dynamic nature of bacterial communities found in microbial fuel cells by examining the overall community fatty acid profile. Because sediment and other mixed culture MFCs are living reactors and are thus affected by various factors, such as temperature, available nutrients, external loads applied to the electrodes, current flow, etc., it is valuable to have a technique to study and assess the evolution and changes in the bacterial communities in response to these factors. Hence, the focus of this paper was to develop and evaluate a preliminary methodology to be further researched and used in microbial fuel cell analysis. Ultimately, it is possible that MFCs will be used as power sources in remote applications requiring small amounts of electricity, such as powering deep-ocean research stations or for use in wastewater treatment plants to reduce biochemical oxygen demand levels and improve wastewater quality. Both applications involve MFCs that comprise several bacterial species. While molecular methods can be used to define the bacterial community, it can become expensive and time consuming. And, in order to determine how the bacterial community can change within an MFC due to external factors or over time, it may be required to successively profile the community several times, thus substantially increasing the cost. With an analytical methodology available such as the one presented here, MFCs can quickly be analyzed for overall changes in response to treating wastewater or producing power. This technique can be used to profile the MFC bacterial community, compare the profile to other MFC community profiles stored in the library software where the performance levels are already known, and then assessed for performance and functionality. With further research, the profiles can be used to assess MFC performance, to detect deviations in bacterial communities in MFCs, to determine the presence of certain bacterial species based on the fatty acid composition, and to optimize bacterial community structures in MFCs for different MFC applications. 3. Methodology and materials

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3.1 Methodology to profile MFC bacterial communities The methodology to profile bacterial community structures was performed on 7 sets, or groups, comprised of replicate microbial fuel cells. Each group consisted of three to four replicates of mixed culture, sediment MFCs, where it was assumed that the bacterial communities in each replicate were the same [34,35]. Each group of cells comprised a different natural organic matrix, thus having different initial bacterial communities. The bacterial community structures in the MFCs were analyzed approximately three weeks after the cells were built and had reached a steady-state open circuit voltage, with no load ever being applied to the cells. The procedure for bacterial community analysis can be completed in 26 hours. Each fuel cell was sampled four times and subjected to the proposed methodology. Using a pipette, the 1.5 ml semi-solid sample was drawn near the anode and placed in a SLYM-BART tester using standard aseptic techniques [3]. Cullimore and Alford [36] patented broad spectrum biodetection systems using proprietary selective media that became known as the biological activity reaction tests (BART). The SLYM-BART employs a formulation of tryptone and proteose peptone which triggers rapid generation of bacterial growth [37], with the major bacterial genera identified by Cullimore [38]. A modified version of the SLYM-BART is the commercially available HAB-BART tester, where methylene blue has been added as the oxidation reduction potential indicator to determine the presence of aerobic or anaerobic bacteria and can be further used to determine active bacterial population numbers in water samples [39]. Sterile water was then added to each tester to generate the required 15 ml sample for cultural testing. To generate the methyl ester fatty acid fingerprint of the microbial community in the fuel cells, the SLYM-BART tester undergoes a two-phased culturing technique (RASI): 1) The sample contained in the tester is rapidly agitated, or rotated, at room temperature (22 2 C) for four hours using a LabQuake Tube Shaker (Thermo Scientific, Dubuque, IA) at 8rpm, causing turbulence and oxidative conditions. This allows the media contained in the SLYM-BART to be well dispersed and mixed with the MFC sample. 2) After rotating the sample, the sample is statically incubated in an upright, passive manner for 20 hours at 30 1 C. Incubation was completed at 30 C since this is well within the accepted range for typical mesophiles and is also representative of the sample growing conditions throughout the experiments [40,41]. The formation of foam may occur during the agitation in phase one due to a highly active bacterial population, but generally subsides during the sedentary second phase of incubation. The combined RASI conditions create a selective stimulative culture of aerobic and facultative anaerobic bacteria, followed by an incubation phase, to allow for a growth period for the bacteria. In this series of experiments the objective was to test our hypothesis that bacterial community fingerprints can be repeatedly detected and differentiated from one another within microbial fuel cells. To achieve this, we chose to first focus on profiling aerobic and facultative anaerobic bacteria as a baseline to develop this preliminary methodology. This was achieved through oxygenating the sample to encourage the growth of aerobic and facultative anaerobic bacteria, and through using the selective, proprietary media found in the SLYM-BART [37]. Future research activities

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include analyses of anaerobic bacteria within the microbial fuel cells using a modified SLYM-BART method. The final step involves the recovery of bacterial cellular growth cultured in the SLYM-BART tester. Using a standard Millipore filtration apparatus with 0.45 m pore size membrane filters and a vacuum pump operating at 20 1 psi, the entire 15 ml contents of the SLYM-BART tester are poured onto the filter apparatus. Using the tester cap, the ball in the tester is prevented from rolling out and onto the membrane. Should any dense or gelatine-like mass form in the tester, the tester and contents are gently shaken to evenly mix the contents. The entire contents of the tester are allowed to pass through the membrane filter, thus trapping the bacterial cellular mass on the filters surface. Once the contents of the tester are effectively collected on the filter paper, a 1 l sterile, plastic disposable loop is used to scrape the cultured cellular material from the filter surface (equivalent to approximately 2.5-3 mg of material). This is done by slowly stroking the loop in all directions across the filter until there is a notable collection of material sticking to the loops center. This material is completely deposited into a 1 ml glass vial. The sample is then prepared according to the Sherlock Microbial Identification System 6.0 Operating Manual MIS Whole Cell Fatty Acid Analysis by Gas Chromatography. This method isolates the methyl ester fatty acids using the Instant FAME protocol. Three reagents are sequentially used to extract the fatty acids: 1) a solution of methanol and potassium hydroxide; 2) solvent extraction using hexane; and 3) application of hydrochloric acid with a red indicator dye. After the fatty acids are extracted from the original bacterial contents, the sample is now ready for FAME analysis using an Agilent GC 6850 gas chromatograph and Sherlock software. The Sherlock software quantifies and interprets the fatty acid peak profiles and compares the results to built-in or customised libraries based on specific culturing techniques using different standard culturing practices. For the FAME profiles generated by the proposed methodology applied to microbial fuel cells, a unique library was generated using the software tools included in the library generation system. New libraries using the described methodology are subsequently generated based on the fatty acid configurations of bacterial communities. Assessment of bacterial communities is evaluated through the methyl ester fatty acid configurations where each bacterial consortium produces a unique profile. The Sherlock library identification system uses probability percentages to express the closeness of any given match, or closeness of fit, between the sample and the fatty acid configurations stored in the library, given as a similarity index (SI) number. According to the softwares literature, to be considered as an acceptable match, an SI value of at least 0.50 must be produced, provided that the next closest identification is at least 0.10 unit of an SI below the highest generated SI number. Generally a three standard deviate window from the mean, giving an SI value of 0.70, signifies a minimal, but acceptable match. However SI values of greater than 0.80 indicates a significantly closer match, with an SI of 1.00 indicating a perfect complete match. In the following analysis, an SI value of 0.70 was chosen as an acceptable match between the test sample and the profiles stored in the library. A unique library was created which included only the MFC bacterial communities profiled using this methodology.

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3.2 Methodology to validate the protocol The purpose of this paper is to assess and validate the proposed protocol that profiles bacterial communities in microbial fuel cells. The method in which this was done was to compare known similar and different bacterial community structures formed in MFCs. Analysis of SI numbers of similar bacterial community structures compared to each other should yield high values, while SI numbers of different bacterial community profiles compared to each other should yield significantly lower values. For each of the two series of experiments conducted, libraries were produced from samples that were known to contain similar bacterial communities. Each library, or grouping of samples, was then compared to the samples that it was generated from as well as samples contained in different libraries. It is expected that samples compared to the libraries to which they belong should yield SI values that are significantly higher than libraries containing samples taken from MFCs with known different bacterial communities. It is also expected that a portion of the fatty acid profile in one bacterial community structure may be similar to the profile of a different bacterial community structure since some bacteria may be present in more than one type of media used. However, the incidence of this should not be frequent. Suspected outliers in SI date were identified using Grubbs tests and were excluded from the analysis [42]. 3.3 Microbial fuel cell designs Sixteen MFCs were built for the first series of experiments. The single chamber MFCs were housed in a plastic box with dimensions h = 4.5 cm, w = 6.5 cm, and l = 12.0cm. Grade 1 carbon rods, with a 0.6 cm diameter, were used as electrodes (Canemco Inc, Canton de Gore, QC). A cylindrical anode, 7.0 cm long, was horizontally placed 0.6 cm from the bottom of the MFC box. A 2.5 cm long cathode was vertically suspended from the lid of the box, providing a 0.8 cm gap between the anode and the bottom of the cathode. The top of the cathode protruded 0.5 cm through the lid of the box to allow for contact with air. A 22 gauge copper wire was attached to each electrode to provide a connection point for external circuitry. Each MFC was filled with approximately 140 cm 3 of media, including creek water for added moisture, such that the horizontal anode was embedded in the media but the bottom of the vertical cathode was not. The 16 MFCs were divided into four groups of four cells each. Each group, labeled alphabetically A-D, comprised different media, including soil from a dog park, soil from a creek area, and two different compost materials. This allowed each group of MFCs to contain a different community of bacteria. After assembly, the MFCs were allowed to mature for five weeks and reach an average steady-state open circuit voltage of 0.71 0.04 V. The proposed methodology for profiling bacterial communities was performed in triplicate on each cell. Three weeks later, the voltage of one MFC dropped to 0.42 V, and the bacterial communities were analyzed for this cell. All 16 MFCs were kept at room temperature (22 3C). For the second series of experiments, 12 larger single chamber MFCs were constructed. The MFCs were housed in 15.3 cm long cylindrical plastic containers, with a 10.2 cm diameter. Square 2.5 cm by 2.5 cm electrodes were made from carbon waste generated by Evraz Regina Steel (Regina, SK). The anode-cathode configuration was similar to the first set of MFCs; the 7.5 cm long anode was horizontally placed on the end cap, at the bottom of the MFC. The 14 cm long cathode was vertically suspended above

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the anode with nylon bolts, leaving a 2 cm gap between the anode and the bottom of the cathode. Copper wires were attached to the electrodes to monitor MFC performance. Approximately 295 cm3 of media were added to the bottom of the MFC, including creek water for added moisture, thus completely immersing the anode without touching the bottom of the anode. The 12 MFCs were divided into three groups of four cells each. Each group, termed E, F, and G, comprised media different from each other, as well as different from MFCs in groups A-D. The three types of media, including commercially available cow manure, sludge from the Regina Wastewater Treatment Plant (Regina, SK), and a mixture of organic material, potting soil and horse manure, provided three different initial bacterial communities. The MFCs were allowed to mature for three weeks prior to performing in triplicate the described methodology for profiling bacterial communities. All 12 MFCs were kept at 30 1C. The three different groups of MFCs had various steady-state open circuit potentials: the average voltage output of Group E measured 0.23 0.03 V, Group F average potential was 0.51 0.02 V, and Group G average voltage output was -0.06 0.04 V. 4. Results and discussion For the first series of experiments, 16 MFCs comprising four different initial bacterial communities, and each having four replicates, were subjected to the RASI-MIDI protocol to generate fatty acid profiles. A total of 47 comparisons were made between the four groups of MFCs (A, B, C, and D). This first series of tests was used to explore and develop the technique. Due to five failed samples that did not produce fatty acid profiles, the number of analyses varied between the MFCs. The failed samples occurred due to insufficient cellular material available for analysis. This problem was eventually resolved for the second series of experiments by taking the sample from deeper within the fuel cell, still alongside the anode, to capture more bacteria in the sample. For the four groups of bacterial communities, the highest SI values and highest correlations were obtained from comparisons between samples and the library generated from those samples: i.e. each cell in Group A matched the generated library A; B cells matched library B, with C and D grouped cells also showing matches to C and D libraries, respectively (Table 1). Here Table 1 Comparisons between the four bacterial community groups generated a lower incidence of correlations. Correlation was calculated by counting the number of times an SI value was generated between a sample and the library to which it is being compared to (indicating a match was made between the sample and library), and dividing it by the total number of comparisons made between the sample and library (indicating the total potential number of times an SI value could have been generated). This indicated that although a high SI value may be generated, the frequency of this occurring was low. For example, MFCs in Group B and the libraries generated from MFCs in groups A, C, and D gave no SI correlation of 25%, meaning that only 25% of the samples from Group B cells produced an SI value of 0.69 when compared to Group A cells. For Group A MFCs, seven out of the eight SI comparison yielded an average value of 0.77 with a variability of 12.7%. There were no significant SI correlations between the communities recovered from the MFC Group A and any other groups of

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MFC indicating that the bacterial community in Group A MFCs was distinctive and unique, apart from the single failed sample drawn from one cell (thus giving an 87% correlation value). Direct community comparisons between Group B MFCs included 10 analyses, all of which correlated with the other bacterial communities in Group B. An average SI value of 0.800.06 with a variability of 7.8% was produced. Fatty acid profiles of bacterial communities in Group B MFCs were the only profiles that had a portion of their fatty acid structure match profiles in each of the other groups. There were also nine SI values recorded between the MFC group B communities and other MFC including two SI with group A; five values with group C and two with group D. However, two of SI values were less than 0.70, indicating a weak match. Group C MFCs showed good SI correlation among the four cells in that group, with an average SI value of 0.89 and a 3.8% variability. One MFC in Group C dropped to 0.42 V and was found to contain the only bacterial community that now fell significantly out of the range observed for the remaining fifteen microbial fuel cells (A, B, C and D), according to the SI values. Clearly in this MFC, there was some perturbation in the community structures as it fell out of an acceptable SI range four out of the five times it was analyzed. Group D MFCs showed significant SI correlations 13 out of 14 times that the RASI-MIDI analysis was performed. For group D it was found that the SI average was 0.79 with a variability of 17.9%. According to the SI values, there were eleven incidents where there was cross detection with MFCs from other groups. In this case, there were four matches with Group B fatty acid profiles and seven matches with Group C profiles. However, these SI values were found to be lower than those generated within Group D. Due to the promising results generated by the first series of experiments, a second set of tests was performed, including a more detailed analysis. For the second series of experiments, 12 MFCs comprising three different initial bacterial community fatty acid profiles were analyzed using the RASI-MIDI protocol. To demonstrate repeatability, three samples were taken from each MFC. In this analysis, 15 different libraries were created from the MFC samples. One library was created from the three samples (replicates) drawn from each cell, to produce 12 libraries, with each library containing the profiles of the three samples. Also, a library was created from each group of MFCs (E, F, and G), to produce three more libraries. These three libraries, with similar initial bacterial communities, comprised 12 samples in total: the three samples from each of the four MFCs in that group. Thus, the three group libraries contained the four individual cell libraries from which the group was made. Each of the three samples taken from each MFC produced 36 samples available for analysis. The 36 samples were compared to each of the 15 libraries, producing a total number of 540 potential combinations of comparisons that were subsequently analyzed. The first level of comparison was between each of the three samples of a cell and the library created by those three samples. We expect a very high SI value since the library profiles contain one third of the sampled fatty acid structures. An average value and standard deviation was calculated from the three SI values generated for each of the 12 cells under analysis. The highest and lowest average SI values were 0.97 0.02 and 0.85 0.01, respectively, with 100% correlation. The mean SI values generated by

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comparing each sample to its own cell, averaged for each of the three groups and for all 12 groups, were significantly high, as reported in Table 2. Here Table 2 The second level of comparison was between each of the three samples of a cell and the group library which the individual cell (along with the three other cells containing similar bacterial communities) belongs to. Again, average SI values were calculated from comparisons of the three samples, producing a range of SI values from 0.78 0.11 to 0.96 0.01, with 100% correlation. Averaged SI values for comparisons within all three groups are found in Table 3. The high SI values indicate that the profiles from individual MFCs are uniquely identified as part of a group of MFCs with similar bacterial community structure. However, due to the group of MFCs samples from other MFCs, the SI values are lower than those produced from comparing fatty acid structures of samples directly to the cells from which they came. Here Table 3 In this series of experiments, the SI numbers generated from comparisons between a sample and the pool of samples from which the sample under analysis was taken from has yielded significantly high values. In fact, all but one of the SI values of the 36 samples compared to SI values of samples containing different bacterial communities were an average of 0.27 SI units higher, significantly higher than the required 0.1 SI unit criterion to demonstrate an acceptable match. As well, all SI values were correlated to the other bacterial community samples to which it was compared. To further validate that the protocol is effective in differentiating dissimilar fatty acid profiles, analyses of bacterial communities between different sets of samples were performed. Bacterial community structures in each cell were compared to other cells of the same group, excluding the cell from which the sample was taken from. It is expected that there will be notably similar fatty acid profiles since each group contains MFCs with the same media and initial bacterial communities. However, there should be a significantly lower incidence of correlation compared to cells from which the sample was directly drawn from. For Group E MFCs, the average SI value of fatty acid profiles compared to other MFCs in the same group was 0.71, which occurred 74% of the time. For Group F, the average SI value was 0.58, with a 53% correlation. And, for Group G, the average SI value was 0.72, correlating to 69% of the samples. Similar analysis was done by comparing MFC fatty acid profiles to cells in different MFC groups, with notably different bacterial communities. The average SI value for Group E MFC samples compared to Group F and G samples was 0.62, with a 21% correlation value. Group F samples had an average SI value of 0.48 when compared to Groups E and G, with 11% correlation. No SI values were given for Group G samples with respect to other groups. This signifies that the unique bacterial communities known to be formed in the different MFC groups were accurately identified and distinguished. As such, analysis of the application of the RASI-MIDI protocol verifies its potential use in routinely investigating MFC bacterial communities through examining the methyl ester fatty acid profiles. Use of the Sherlock MIDI system typically has been used to identify single strains of bacteria. There have been relatively few studies on the application of Sherlock MIDI analysis of bacterial communities. From our results, comparisons of samples to the library comprised of those samples generated the highest SI values, with at least 0.1 SI

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unit away from the next highest SI value for 97% of the samples. As well, MFC samples constructed of one type of media, with a corresponding bacterial community, compared to samples containing a notably different media, with a different corresponding bacterial community, did not produce any significant SI values. This demonstrates the ability of this protocol to be applied to identify and analyze fatty acid profiles of bacterial communities found in MFCs. The research presented here is of a preliminary method found to be repeatable in specific laboratory conditions for microbial community profiling. However, additional research is required to optimize the technique. To further validate the results, independent molecular based techniques, such as DGGE, are required. This would also provide a baseline for future work in identifying individual strains that comprise the fatty acid fingerprint. As well, refining the technique for use in profiling strict anaerobic bacteria is necessary for producing a more complete analysis of the bacterial communities found in microbial fuel cells. Because initial results show promise in examining and profiling bacterial community structures in a repeatable manner, further research is warranted to optimize the FAME technique for various types of MFCs. 4. Conclusion From these series of experiments, it is seen that the bacterial communities within MFCs comprised of known different bacterial communities maintain a distinct and definable fatty acid profile that can be accurately and repeatedly profiled. The communities within each group of MFC replicates, where each fuel cell was known to contain the same, original bacterial communities, were recognized as being statistically the same as each other, and uniquely different from communities in other MFCs. The co-application of this preliminary methodology for the enrichment of bacteria and the rapid Sherlock MIDI system is inherently different from standard methods of culturing bacteria in that 1) the objective is to identify bacterial communities within consortia rather than single species on the basis that fatty acid configurations remain constant even in groups of bacteria consisting of several strains; and 2) samples were taken directly from their source and analyzed using the RASI-MIDI protocol without any attempt to selectively isolate any specific strains. Analysis of fatty acids extracted from the bacterial consortia from seven distinct groups of MFCs were found to have different but relatively stable and identifiable fatty acid peaks as determined by the Sherlock Microbial Identification System. When MFC voltage output significantly changed in one MFC, analysis showed the presence of a distinctly different bacterial community. The RASI-MIDI protocol was found to be repeatable and appears to provide a functional method to rapidly and routinely identify bacterial communities in microbial fuel cells. 5. Acknowledgements The authors are grateful to Droycon Bioconcepts, Inc. (Regina, SK), Ahmed Sultan, DBI technologist, Mary S. Nelson, and Dr. Ioannis Ieropoulos of the University of Bristol.

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Table 1 Comparison of SI values between MFCs in each group and the libraries generated by each group

Library A

87%

25%

0%

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20% Library C 0 0% Library D 0 0%

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0.790.05 0.890.03 0.790.01 SI value 78% 50% SI correlation

0.670.13 0.790.01 0.790.14 SI value 14% 57% 100% SI correlation

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SI value

SI correlation

SI correlation

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Group B

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Table 2 SI values of individual samples compared to the cell from which the sample was drawn, averaged for each group. average SI value 0.90 0.93 0.95 0.93 standard deviation 0.04 0.03 0.02 0.04

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Group E Group F G roup G all groups

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Table 3 SI values of individual samples compared to the group containing the cell from which the sample was drawn, averaged for each group. average SI value 0.87 0.84 0.88 0.86 standard deviation 0.05 0.07 0.07 0.06

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Group E Group F G roup G all groups

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