Microbial community structure in a dual chamber

Environ Sci Pollut Res DOI 10.1007/s11356-015-4582-8

RESEARCH ARTICLE

Microbial community structure in a dual chamber microbial fuel cell fed with brewery waste for azo dye degradation and electricity generation Waheed Miran 1 & Mohsin Nawaz 1 & Avinash Kadam 1 & Seolhye Shin 1 & Jun Heo 1 & Jiseon Jang 1 & Dae Sung Lee 1

Received: 28 January 2015 / Accepted: 21 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The expansion in knowledge of the microbial community structure can play a vital role in the electrochemical features and operation of microbial fuel cells (MFCs). In this study, bacterial community composition in a dual chamber MFC fed with brewery waste was investigated for simultaneous electricity generation and azo dye degradation. A stable voltage was generated with a maximum power density of 305 and 269 mW m−2 for brewery waste alone (2000 mg L−1) and after the azo dye (200 mg L−1) addition, respectively. Azo dye degradation was confirmed by Fourier transform infrared spectroscopy (FT-IR) as peak corresponding to –N=N– (azo) bond disappeared in the dye metabolites. Microbial communities attached to the anode were analyzed by high-throughput 454 pyrosequencing of the 16S rRNA gene. Microbial community composition analysis revealed that Proteobacteria (67.3 %), Betaproteobacteria (30.8 %), and Desulfovibrio (18.3 %) were the most dominant communities at phylum, class, and genus level, respectively. Among the classified genera, Desulfovibrio most likely plays a major role in electron transfer to the anode since its outer membrane contains c-type cytochromes. At the genus level, 62.3 % of all sequences belonged to the unclassified category indicating a high level Responsible editor: Bingcai Pan Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4582-8) contains supplementary material, which is available to authorized users. * Dae Sung Lee [email protected] 1

Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Republic of Korea

of diversity of microbial groups in MFCs fed with brewery waste and azo dye. Highlights • Azo dye degradation and stable bioelectricity generation was achieved in the MFC. • Anodic biofilm was analyzed by high-throughput pyrosequencing of the 16S rRNA gene. • Desulfovibrio (18.3 %) was the dominant genus in the classified genera. • Of the genus, 62.3 % were unclassified, thereby indicating highly diverse microbes. Keywords Microbial fuel cell . Brewery wastewater . Dye degradation . Bioelectricity . Microbial community analysis . Pyrosequencing

Introduction The increasing concern regarding shrinking fossil fuel supplies and their deteriorating effect on environmental change has stimulated the search for unconventional and clean energy sources (Sharma et al. 2011; Jafary et al. 2013). One of the emerging technologies of interest in this regard is microbial fuel cells (MFCs). Massive attention is being given to this technology as a novel approach aimed at bioelectricity production along with simultaneous wastewater treatment (Watanabe 2008; Sevda et al. 2013; Majumder et al. 2014). In recent years, the concept of combining recalcitrant wastewaters (like azo dyes) and easily biodegradable organics for simultaneous wastewater treatment (including dye degradation in the case of azo dyes) and electricity generation for making the technology cost effective has given a new

Environ Sci Pollut Res

dimension to research in this field (Sun et al. 2009; Solanki et al. 2013). The brewery industry discharges large quantities of wastewater containing high concentrations of degradable organic pollutants. Conventional methods used for the treatment of brewery wastewater demand large amounts of energy (Wang et al. 2008; Wen et al. 2010). Wastewater from breweries has been preferred among researchers as an appropriate substrate for MFCs for electricity generation, primarily due to the foodderived nature of the organic matter and the lack of high concentrations of inhibitory constituents. Azo dyes are the most extensively used dyes and one of the most problematic contaminants in the textile industry (Chengalroyen and Dabbs 2013). Azo dyes are considered to be very stable dyes that are toxic, recalcitrant, and potentially carcinogenic; their discharge into the environment raises very serious aesthetical, health, and environmental problems (Stolz 2001; Ong et al. 2009). Therefore, azo dye effluents are a major concern and require to be effectively treated before being discharged into the environment in order to remove their potential risks. A variety of physical, chemical, and biological processes have been used to treat dye effluents. Biological processes are more cost-effective, generate less sludge, and are more environmentally benign compared to the other two processes (Chen et al. 2003). Azo bonds can be cleaved successfully by some anaerobes in biological processes. However, cleavage without an external carbon source is very difficult. MFC anodic chambers with an anaerobic environment and brewery waste as an economical carbon source can be effectively used to achieve concurrent azo dye degradation and bioelectricity generation. Among the many factors which have an impact on the electrochemical features of the anode and operation of the MFC, a very prominent factor is the composition or structure of the biofilm (Cord-ruwisch 2008). Hence, it is crucial to identify the character, structure, and composition of the biofilm to achieve better performance of MFCs and increase our understanding of the electron transfer mechanisms (Yang et al. 2012). Denaturing gradient gel electrophoresis (DGGE) has been most widely used for microbial community profiling in MFCs. However, it is not very sensitive and cannot detect communities with less than 1 % representation which results in the underestimation of strains (Kan et al. 2011). Recently, pyrosequencing has been used as a next generation technique for the more detailed investigation of microbial communities in MFCs. It can detect rare microbial components which may affect the performance in terms of bioelectricity production (Zhi et al. 2014). However, the microbial communities in complex MFC systems that use brewery waste for simultaneous azo dye degradation and bioelectricity generation have not been investigated using this technique. In this study, pyrosequencing was used for a detailed microbial analysis of MFCs. Understanding the role of different microbes on the current generation and MFC performance

will help improve MFCs for future applications. The overall performance of the MFCs was determined by the maximum power density, dye degradation, chemical oxygen demand (COD) removal, internal resistance of the cell, and cyclic voltammetry (CV). In addition, the morphology of the bacterial community was assessed using field emission scanning electron microscopy (FE-SEM).

Materials and methods Dye and brewery waste The azo dye, Direct Red 80 (DR80), was purchased from Sigma-Aldrich, South Korea. DR80 had a purity of 25 % and a molar mass of 1373.07 g mol−1. Brewery waste was collected from a local barley brewery plant in Daegu, South Korea (having the composition of (in mg L−1): COD 61000, TSS 7500, chlorides 420, nitrates 380, sulfates 115, and phosphates 885). MFC reactor configuration, operation, and evaluation A dual chamber MFC (rectangular shape) with 0.2-L capacity for each anodic and cathodic chamber was used for simultaneous electricity production and dye degradation using brewery wastewater. The anode was comprised of graphite felt with 3.18-mm thickness and 25-cm2 projected surface area, and the cathode was comprised of graphite cloth, coated with platinum (20 wt.% Pt.), and a projected surface area of 25 cm2. Electrodes were separated by a proton exchange membrane (PEM) (Nafion® 117) to avoid the intermixing of solutions while allowing the transfer of protons from anodic chamber to cathodic chamber for the completion of the reduction reaction. The ethanol for acclimation (and startup) and later on brewery waste (with and without the added dye) was used with the defined medium (having a composition of [in mg L−1]: (NH4)2SO4 560, NaHCO3 420, MgSO4·7H2O 200, MnSO4·H2O 20, CaCl2 15, and other trace minerals and buffer solution) to aid the growth of exoelectrogens in the anodic chamber. The ethanol concentration of 1000 mg L−1 (as a readily degradable carbon source) was used for the startup and acclimation in the anode chamber using anaerobic culture. There was a lag time of about 10 h before the start of voltage generation, and stable state of voltage generation after three cycles of operation was achieved (indicating the development of electroactive biofilm on anode) (Fig. S1). The feed was purged with pure nitrogen for 15 min before use and a nitrogen gas bag was attached to the anodic chamber to ensure anaerobic conditions. An air pump was used to continuously supply air (oxygen) to the cathodic chamber as electron acceptor. A temperature of 30±2 °C was maintained in the MFC chamber with the help of a water bath.


The anode and cathode were connected via an external resistor. The MFC voltage was recorded over an external resistance of 220Ω by using a Keithley 2700 digital multimeter (Keithley Instruments, USA). This voltage was used to calculate the current and power generation. By varying the external resistance from 10Ω to 10 kΩ, power density and polarization curves were generated to evaluate the maximum power production capacity of the cell. CV was carried out by using an Ivium CompactStat potentiostat (Ivium Technologies, Netherlands), where the anode and cathode of the MFC were serving as the working and counter electrode, respectively, and an Ag/AgCl electrode was used as the reference electrode. CV was performed at a scan rate of 50 mV s−1 in the potential range of −1.0 to 1.0 V while continuously monitoring the current responses. The COD concentration was measured using COD Hach kits and a spectrophotometer. Dye degradation and color removal were observed with the help of Fourier transform infrared spectroscopy (FT-IR) and an ultraviolet visible (UV-vis) spectrophotometer (Agilent 8453, USA), respectively. Anions were analyzed by using the DX ICS-1000 ion chromatography unit (Dionex, USA) equipped with a conductivity detector and self-regenerating suppressor. FE-SEM (Hitachi S-4300, Japan) was carried out to observe the morphology and surface structure of new carbon felt and carbon felt after biofilm attachment.

Korea). Pyrosequencing was carried out by Macrogen (Korea) with a 454 GS-FLX titanium platform and the sequence reads were processed to eliminate sequencing noise. The resulting sequencing data were evaluated using the ribosomal database project (RDP) pyrosequencing pipeline (http://pyro.cme.msu. edu/) (Cole et al. 2009). The reads that contained a barcode were used for evaluation, and the bar codes were later removed. Unanticipated or unspecific reads were eliminated manually with the help of the RDP classifier. Reads having a dissimilarity of 3 % were classified into one operational taxonomic unit (OTU) by setting a 0.05 distance limit using the MOTHUR program, and assignment of bacterial high quality reads was carried out with the help of the RDP naïve Bayesian rRNA classifier at 80 % confidence thresholds. OTUs and rarefaction curves were produced using the RDP pyrosequencing pipeline at a 3 % dissimilarity level. Diversity parameters like the Shannon-Weaver and Chao1 biodiversity indices and evenness were calculated by the RDP pyrosequencing pipeline after aligning and complete linkage clustering of the initial FASTA files.

PCR amplification, high-throughput pyrosequencing, and data analysis

The MFC was inoculated with anaerobic bacteria and a carbon source (ethanol with defined medium) was added in a fedbatch manner to facilitate the growth of exoelectrogens and encourage bioenrichment in the MFC. After achieving stable voltage generation, the chamber was operated with brewery waste as carbon source at a COD of approx. 1000 mg L−1 and at later stages (after dye addition) this was increased to 2000 mg L−1. Azo dye concentration was increased gradually from 25 to 200 mg L−1 and its effect on voltage generation and maximum power production (and other concerning parameters) was monitored (Fig. S2). To evaluate the maximum power, power density curves were generated by measuring the voltage change with the change of external resistance, starting from 10,000Ω and stepwise decreasing it to 10Ω. Voltage generation of around 0.41 V was achieved with brewery waste as the carbon source (2000 mg L−1) which decreased to about 0.38 V after addition of the azo dye (200 mg L−1). At these initial concentrations of brewery waste and azo dye, maximum power densities of 305 and 269 mW m−2 were obtained on the basis of anode surface area, corresponding to current densities of 745 and 700 mA m−2, for brewery waste alone and brewery waste with azo dye, respectively (Fig. 1). This decrease in power density after dye addition was most likely due to competition for electrons between the azo dye for its degradation and transfer of electrons to anode. Also, electrons may be consumed for the reduction of inorganic ions present in brewery waste, e.g., sulfate (which ultimately helps in

Samples for microbial community analysis were collected at the end of the experiments by scraping the anode surface and centrifuged at 10,000 rpm for 10 min. Samples were preserved at −80 °C until further processing to avoid any changes in microbial composition. DNA extraction from the sludge for bar-coded pyrosequencing was carried out with the help of a Fast-DNA kit (MPbio Solon, OH) according to the manufacturer’s instructions. Bacterial 16S rRNA genes containing variable regions (V1 to V3) were amplified using primers Bac541R (5′-adaptor A-X-AC-WTT ACC GCG GCT GCT GG-3′, where BX^ represents 7 to 11 barcode sequences for sample identification when multiple samples were inserted in parallel on one 454 life sciences adaptor A sequence and the common linkers, AC and GA) and Bac9F (5′-adaptor B-ACGAG TTT GAT CMT GGC TCA G-3′) (Chun et al. 2010). For polymerase chain reaction (PCR), genomic deoxyribonucleic acid (DNA) template (1 μL), primers (20 pmol each), and a Taq polymerase mixture were mixed (50 μL final volume) and amplified in a C1000 thermal cycler (Bio-Rad, USA) using a cycling regime as described earlier (Lee et al. 2012), i.e., 1 cycle of 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 60 °C (bacteria) or 58 °C (archaea) for 30 s, and 72 °C for 1 min 20 s, and 1 final cycle of 72 °C for 10 min. PCR products were purified using a PCR purification kit (Solgent,

Results and discussion Bioelectricity and power density


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Fig. 1 Power density and polarization curve for brewery waste and brewery waste with azo dye

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growth and activity of sulfate reducing cultures). Internal resistance, calculated by the slope method from voltage and current (Logan et al. 2006), was about 200Ω (R2 =0.988) in the case of brewery waste and increased up to about 226Ω (R2 =0.986) in the case of azo dye addition. The regression linear equations can be found in supplementary data (Fig. S3). The internal resistance of an MFC is mainly influenced by the anode, cathode (major factor), membrane, and solutions, which ultimately affects the power output (Fan et al. 2008). The change in internal resistance observed here was probably caused by dye addition. However, overall performance of the MFC in terms of stability of voltage generation was not much altered even after the addition of the azo dye. Dye and COD removal Bacterial degradation of azo dyes was investigated thoroughly. It is generally recognized that reductive cleavage of azo groups is the first step towards the decolorization of azo dyes (dos Santos et al. 2007). FT-IR (Fig. 2a.) showed a peak around 1640 cm−1 in the control azo dye which mainly corresponded to azo bonds, and this peak was absent in the degraded dye metabolites. This is a clear indication of azo dye cleavage during dye decolorization. Dye removal was also analyzed by measuring the absorbance using UV-vis spectra (200–800 nm) during the batch experiments. Figure 2b clearly shows that there was an obvious decrease in absorbance in the visible region around 540 nm at the end of the experiments. There were also changes in peaks in the ultraviolet region suggesting the destruction of primary chromophores (Qu et al. 2012). Peaks in the visible region did not completely disappear indicating that color removal was not complete; color removal of approximately 75 % was achieved at the

initial brewery waste and dye concentrations of 2000 and 200 mg L−1, respectively. A very important feature of MFCs along with bioelectricity generation is their application as wastewater treatment chambers. As COD is a very common measure of wastewater treatment efficiency, COD concentrations after azo dye addition were obtained from brewery waste fed batches. About 80 % of the initial COD (2000 mg L−1) was removed from brewery waste, and there was a decrease in COD removal of up to 62 % after the dye (200 mg L−1) addition (the dye itself has a COD of 337.4 mg L−1 at a concentration of 200 mg L−1). In terms of the COD removed per unit time, the value was 34.4 and 30.7 mg L−1 h−1 for brewery waste before and after the dye addition, respectively. This is probably due to the well-known fact that most azo dye intermediates have a higher COD than their parent dye. As no pure carbon source was used, combining brewery waste and azo dye wastewater for simultaneous power generation and waste water treatment could be accomplished at pilot to industrial scales (Duteanu et al. 2010; Logan. 2010). Cyclic voltammetry CV is an electroanalytical technique which provides information about an electrochemical system and helps to interpret the electrochemical reactions occurring at the electrodes (Zhao et al. 2012). In this study, voltammograms were used to evaluate the electrochemical behavior of the biofilms at different phases of formation as evidenced by significant variations in electron discharge and energy generation. Voltammograms were recorded for three different batches, anode (a) without biofilm, after the attachment of biofilm using brewery waste (b) without dye, and (c) with dye during the steady states of


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Fig. 2 a FT-IR and b UV-vis spectroscopy for control dye and dye after degradation

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electricity generation (Fig. 3). No significant electrochemical activity was observed in the anode medium without bacterial culture, indicating that the medium used had no effect on the CV measurement. Cyclic voltammograms of batches (b) and (c) showed noticeable peaks indicating electrochemical activity. Significant oxidation peaks in the forward scan were observed at 0.25 and 0.45 V for brewery waste with and without the addition of dye, respectively. This difference in oxidation peak at different locations was most probably due to the dye addition. Earlier, similar results were found, when the oxidation peak (potential) of the bioanode with Congo red-fed MFC was shifted by 0.25 V toward negative potential, in comparison to the bioanode which was not used for Congo red decolorization (Sun et al. 2011). The reduction peak was found in the reverse scan at −0.6 V both for brewery waste with and

without dye. A noticeable improvement in electrochemical activity was observed with the formation of the biofilm. We have shown here that under long-term electrochemical tension, bacteria attached to the anode form a biofilm and develop a capability for electricity generation. Scanning electron microscopy Surface morphologies of carbon felt anode and electrode in the MFC anodic chamber was examined by SEM after completing the MFC experiments. Figure 4c–e clearly show the attachment of a biofilm consisting of a variety of bacteria on electrodes in comparison to plain carbon felt in Fig. 4a, b. The bacteria grown on the anodic electrode exhibited a predominantly rod-shaped structure, similar to what has previously

Environ Sci Pollut Res Fig. 3 Cyclic voltammetry curves of the anode before biofilm formation (a) and after biofilm formation with brewery waste (b) and brewery waste with azo dye (c)

15 Fresh anolyte without biofilm Brewery waste with biofilm Brewery waste with dye and biofilm

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been reported (Qiao et al. 2008). In MFCs, rod-shaped bacteria participate in direct electron transfer either by forming a pilus connection which functions as a route for electron transfer from the inside of the microbe to the anodic electrode or by transferring electrons through the outer membrane via cytochrome c (Reguera et al. 2005). The conductance of electrons by both pilus (nano wires) and cytochromes after degradation of substrates by microorganisms promotes electrogenesis. Bacterial community analysis could provide further insights Fig. 4 SEM images: a, b new carbon felt and c–e biofilms firmly attached on the surface of the anode electrode

into the significance of morphologies and elucidate their exact function. Microbial community analysis Microbial community analysis of enriched sludge biomass from MFC is very important for understanding the performance of MFCs in terms of current generation and waste degradation. The PCR primer trimmed 16S rRNA gene

a)

b)

c)

d)

e)

Environ Sci Pollut Res Fig. 5 Bacterial taxonomic compositions at a phylum, b class, and c genus level. d Rarefaction curve of bacterial 16S rRNA gene sequence from anodeattached microbial communities. Sequences were classified using the RDP naive Bayesian rRNA Classifier with an 80 % confidence threshold

sequences derived from sludge were classified at phylum, class, and genus levels using the RDP Classifier (Fig. 5a–c). A total of 1606 reads were found, which after trimming were reduced to 1177 high-quality reads. The rarefaction curve was obtained by plotting the OTUs (OTU with 97 % similarity) against the number of sequences to assess the extent to which sampling covered species richness (Fig. 5d). The sharp slope of the curve showed that the rarefaction curve did not reach saturation, indicating the presence of highly diverse bacterial communities. Several key analytical diversity indices, such as the Shannon–Weaver Index, Chao1, and evenness were analyzed using the RDP pyrosequencing pipeline (Table 1). At the phylum level, Proteobacteria (67.3 %), Bacteroidetes (15.8 %), and Firmicutes (6.3 %) covered the majority of the bacterial community as more than 85 % of it was composed by these three phyla. Other phyla present in small quantities were Chloroflexi (2 %), Chlorobi (1.7 %), Verrucomicrobia (0.2 %), Acinobacteria (0.2 %), and Armatimonadetes (0.1 %). Of the total community at phylum

Table 1 Summary of pyrosequencing data and statistical analysis of bacterial communities from the anode of the MFC fed with brewery waste and azo dye Parameter

Value

No. of reads

1606

No. of high-quality reads Average read length (bp) Standard deviation of sequence length OTUs Shannon-Weaver index (H′) Chao1 index Evenness No. of phyla No. of genera

1177 402 69.22 287 4.73 445.20 0.84 9 31

OTUs were determined using the RDP pipeline with a 97 % OTU cutoff of the 16S rRNA gene sequences. All unclassified phyla or genera were counted as one phylum or genus


level, 6.5 % was unclassified. At the level of class, 60 % of the total community was composed of Betaproteobacteria (30.8 %) and Deltaproteobacteria (29.2 %). Other dominant classes covering more than 15 % of the composition were mainly Flavobacteria, Bacteroidia, Alphahotobacteria, and Bacilli, while 15.4 % of the total community was assigned to the unclassified category. At the genus level, the community was mainly composed of Desulfovibrio (18.2 %), Petrimonas (3.0 %), Trichocccus (3.0 %), Sphingopyxis (2.3 %), Thiobacillus (2.0 %), and Geobacter (0.6 %). Interestingly, 62.4 % of the total composition was unclassified at the genus level, which implied the presence of very complicated and diverse microbial communities in the MFC. The bacterial community at the anode is mainly affected by the type of substrates used and ultimately influences the current generation (Zhang et al. 2011). Also, it is important to know that there are a number of bacteria in MFCs which may not be directly involved in the current production. Instead, they are involved in the degradation of complex organic substrates to simpler ones which are then used by exoelectrogens (Patil et al. 2009). In MFCs with food derived substrates, exoelectrogens, fermentative bacteria, and other bacteria with specific functions could play a collective role in determining the overall performance of the MFC (Jia et al. 2013). In the current study, Desulfovibrio was the dominant genus among the classified genera and was most likely responsible for electron transfer to the anode via outer membrane cytochrome c. The percentage of well recognized exoelectrogens, i.e., Geobacter, Shewanella, Pseudomonas, etc. was relatively low, which was in line with the community composition reported for alcohol fed MFCs (Kim et al. 2007). The role of Desulfovibrio in the enhancement of current generation in a mediator-less MFC was recently studied. It was shown that cytochrome c of Desulfovibrio was attached to the anodic surface leading to a more efficient electron transfer than that observed for Geobacter (Kang et al. 2014). However, in many studies, overall performance of MFCs was much better with mixed cultures than pure cultures probably due to synergistic interactions of communities in the MFC and many unknown mechanisms (Jung and Regan. 2007; Yates et al. 2012). Desulfovibrio is a very important member of the sulfate-reducing bacteria (SRB). Desulfobulbus and Desulfovibrio, two other members of the SRB group, were among the dominant communities in rice straw hydrolysate fed MFC anodic biofilm (Wang et al. 2014) and it was suggested that they take part in electron transfers to the anode. SRB have also been successfully used for the azo dye degradation and mineralization of intermediates under anaerobic, sulfate-reducing conditions (Rasool et al. 2013). Based on these findings, SRB cultures could also be used in MFCs in the future to simultaneously generate currents and degrade azo dyes.

Conclusions In this study, it was demonstrated that MFCs were effectively enriched with exoelectrogens for simultaneous electricity generation and azo dye degradation using brewery waste as a cheap external carbon source. CV and SEM showed that bacteria were attached to the anaerobic electrode (anode) forming a biofilm and as a result, capability for electricity generation was developed by specific communities. Pyrosequencing was employed for bacterial community analysis and provided insight into the dominant communities at phylum, class, and genus level. A large percentage of the bacterial composition could not be determined at the genus level, which shows the huge diversity of microbes. An important member of the SRB class, Desulfovibrio, was the dominant genus present among the classified genera and which suggested that it is responsible for electron transfer to the anode. Acknowledgments This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation (NRF) of Korea (NRF-2014H1C1A1066929). This study was also supported by grants (NRF-2013R1A1A4A01008000 and NRF-2009-0093819) through the ME and NRF of Korea. This research was also supported by the NRF grant by the Korea government (MSIP) (NRF-2015M2A7A1000194).

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