Accepted Manuscript Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells Chongyang Gao, Aijie Wang, Wei-Min Wu, Yalin Yin, Yang-Guo Zhao PII: DOI: Reference:

S0960-8524(14)00838-4 http://dx.doi.org/10.1016/j.biortech.2014.05.120 BITE 13528

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

13 April 2014 29 May 2014 31 May 2014

Please cite this article as: Gao, C., Wang, A., Wu, W-M., Yin, Y., Zhao, Y-G., Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.120

This is a PDF file 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 final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells

Authors: Chongyang Gaoa, Aijie Wanga*, Wei-Min Wub, Yalin Yinc, Yang-Guo Zhaoc**

Affiliations: a. School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China b. Department of Civil and Environmental Engineering, Codiga Resource Recovery Center, Center for Sustainable Development & Global Competitiveness, Stanford University, Stanford, CA 94305-4020, USA c. Key Laboratory of Marine Environment and Ecology (Ocean University of China), Ministry of Education, Qingdao 266100, China

Corresponding authors: E-mail address: [email protected] (A. Wang); [email protected] (Y.-G. Zhao).

1

Abstract Aerobic sludge after anaerobic pretreatment and anaerobic sludge were separately used as inoculum to start up air-cathode single-chamber MFCs. Aerobic sludge-inoculated MFCs arrived at 0.27 V with a maximum power density of 5.79 W m-3, while anaerobic sludge-inoculated MFCs reached 0.21 V with 3.66 W m-3. Microbial analysis with DGGE profiling and high-throughput sequencing indicated that aerobic sludge contained more diverse bacterial populations than anaerobic sludge. Nitrospira species dominated in aerobic sludge, while anaerobic sludge was dominated by Desulfurella and Acidithiobacillus species. Microbial community structure and composition in anodic biofilms enriched respectively from aerobic and anaerobic sludges tended gradually to be similar. Potentially exoelectrogenic Geobacter and Anaeromusa species, biofilm-forming Zoogloea and Acinetobacter species were abundant in both anodic biofilms. This study indicated that aerobic sludge performed better for MFCs startup, and the enrichment of anodic microbial consortium with different inocula but same substrate resulted in uniformity of functional microbial communities. Keywords microbial fuel cell; startup; inoculated sludge; anodic microbial community

2

1. Introduction Biocatalyzed electrolysis systems (BESs) including microbial fuel cell (MFC) can generate electrical power from organic waste and wastewater. Recently, more researches have been focused on wastewater treatment, bioremediation of contaminants and stabilization of recalcitrant compounds using BESs to improve cost-effectiveness and sustainability (Logan & Rabaey, 2012). The reactor configuration, anodic and cathodic materials, type of substrates, internal resistance, buffer solutions and anodophilic populations have been proved to be the important factors affecting the operational efficiency of MFCs (Logan, 2012; Pant et al., 2010). In general, MFCs are inoculated with either aerobic activated sludge or anaerobic digestion sludge from municipal wastewater treatment plant. However, a few studies have addressed the impact of sludge types in anodic chamber on the performance of MFCs as well as microbial community. Rodrigo et al. (2007) reported that after short-term anaerobic pretreatment of aerobic sludge, MFC was started up efficiently within 10 d. Lobato et al. (2012) found that seeding an MFC with anaerobic acclimated sludge from anaerobic digestor led to more rapid startup of electricity production than with aerobic sludge for 20 d. The apparently contradictory statements, therefore, indicate that the influence of the type and source of inoculum sludge on the startup and performance of MFC should be further investigated. A short startup time and highly negative anode potential are favorable to improve single-chamber air-cathode MFCs. Many researchers have tried to reduce startup time by the methods from changing the configurations to altering the operational parameters. Zhang et al. (2013) used the glass fiber separator to reduce the startup time from 10 d to 3

8 d, and the separator also reduced the anode potentials. Boghani et al (2013) reduced significantly startup time from 42 d to 22 d, along with 3.5-fold increase in biocatalytic activity after startup by applying the strategy of controlled electrogenic film at an electrode. Lobato et al. (2012) observed that the MFC achieved the steady-state operation conditions inoculated with anaerobic sludge in 10 d while the aerobic sludge-seeded MFC required more than 20 d to achieve this regime. In order to quickly startup MFC, some researchers inoculated MFC with mixture of aerobic activated sludge and anaerobic sludge, which is well known to contain a greater diversity of electrochemical active bacteria (Luo et al., 2009). Other than sludge type incubated, the startup time depends on the successful growth and attachment of exoelectrogenic biofilm on the electrode. The microorganisms grown on the MFCs’ anode surface are the key factors because they are responsible for degradation of contaminants and production of electricity simultaneously (Logan & Regan, 2006). Thus the populations and activity of the bacteria on the anode are very important for the electricity production (Logan, 2009). During the startup of MFCs, a seed sludge containing more exoelectrogens and biofilm-forming bacteria will be capable of accelerating the formation of biofilm on the anode. However, there are few literatures reporting the electricity-producing and biofilm-forming flora during MFC startup. In this study, the experiments were designed to investigate the impact of sludge type on performance of MFCs and characterize the morphology, microbial composition and community structure of the biofilm on MFC anode using scanning electron 4

microscope (SEM), PCR-denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing techniques. The results demonstrated that the sludge type did impact the MFC performance but the final dominant microbial community was less influenced by the type of sludge inoculated.

2. Materials and Methods 2.1 MFC configuration The air-cathode single-chambered MFCs were constructed by Plexiglas vessel with a working volume of 28 mL. Carbon brush with 1.5 cm in radius and 2 cm in length was used as MFC anode. Carbon cloth (Ruibang Carbon Material Co., Shanghai, China) with a working area of 7 cm2 was used as air-cathode. The cathode containing Pt catalyst (0.5 mg Pt cm-2) was pretreated as described by Cheng et al. (2006). Carbon brush anode and air-cathode carbon cloth were installed in the cylindrical chamber with 1 cm apart. Titanium wires were used to connect the circuit and all wire interfaces were sealed with epoxy resin. Unless otherwise specified, the external resistance used in this study was 1 kΩ. 2.2 MFC setup and operation Six identical MFCs were divided into two groups in this study. One group received aerobic activated sludge as inoculum while another received anaerobic sludge. The anaerobic sludge was obtained from an anaerobic baffled reactor (ABR) used to treat high sulfate containing wastewater in our lab in 2010 (Li et al., 2010). Aerobic activated sludge was collected from the aeration tank of Licunhe Sewage Treatment Plant of 5

Qingdao, China and kept in a closed tank without aeration for one week as anaerobic pretreatment in order to obtain a mixed culture of aerobic and anaerobic microorganisms at the room temperature of 20°C (Rodrigo et al., 2007). The anolyte contained 500 mg L-1 lactate (as COD), phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4•2H2O, 2 mM KH2PO4, pH 7.4), trace mineral and vitamin solutions. The influent conductivity and pH were finally maintained at 8 ± 1 mS cm-1 and 8.5 ± 0.5, respectively. The anolyte was replaced regularly with new solution once the voltage declined below 0.01 V. The MFCs were operated in fed-batch mode at room temperature (20-25°C). 2.3 Electrochemical measurements Voltage (U) across the external resistance (R) of the MFC was measured using a data acquisition system (PISO-813, Hongge Taiwan) connected to a computer. Current (I) was calculated from I =U/R, and cell power output (P) was calculated from P =U×I. The power density (PV) was calculated using the equation: PV(W m-3)= P/V, where P is cell power output, V is the volume of the anodic chamber. The polarization curves were obtained by changing external circuit resistances from 1000 Ω to 20 Ω during the steady state. The data were recorded at a time interval of 10 min. 2.4 Microbial analysis 2.4.1 Scanning electron microscopy (SEM) observation At the end of each current production, a small piece of anode electrode was taken to examine the bacterial morphology on the anode by using SEM (S-4800, Hitachi Japan). The sample preparation was performed as described by Chung and Okabe (2009). 6

2.4.2 Microbial community analysis Attached biofilm samples were collected from the carbon anode surface at the end of each test stage. Genomic DNA was extracted directly with the soil DNA extraction kit (Mobio, CA USA) according to manufacturer's instructions. Bacterial universal primers BA101F (5’- TGGCGGACGGGTGAGTAA -3’) and BA534R (5′-ATTACCGCGGCTGCTGG-3′) were used to amplify the V2~V3 region of bacterial 16S rDNA. A GC clamp was attached to the 5’ terminal of primer BA534. PCR amplification of 16S rDNA, DGGE profiling and 16S rDNA sequencing for community analysis were conducted as previously described (Zhao et al., 2014). After DGGE profiles were digitized, SPSS software (SPSS Inc., Chicago IL) was used to perform cluster analysis for the DGGE profiles by Ward's method. Partial 16S rDNA based high-throughput sequencing was used to determine the diversity and composition of the bacterial communities in each sample. PCR amplifications were conducted in triplicate with the primer set 515F (5'-GTGCCAGCAGCCGCGGTAA-3') and 806R (5'GGACTACCAGGGTATCTAAT-3') that amplifies the V4 region of the 16S rDNA. The reverse primer contained a 6-bp error-correcting barcode unique to each sample. DNA was amplified following the protocol described previously (Qin et al., 2010). Sequencing was subsequently determined on an Illumina MiSeq platform by Novogene (Beijing, China). Pairs of reads from the original DNA fragments were merged by using FLASH (Magoč & Salzberg, 2011). Sequencing reads was assigned to each sample according to 7

the unique barcode of each sample. QIIME software package (http://qiime.org/) and UPARSE pipeline (http://drive5.com/uparse/) were used to analyze the reads and pick operational taxonomic units (OTUs). Sequences were assigned to OTUs at 97% similarity. We picked a representative sequence for each OTU and used the RDP classifier (Wang et al., 2007) to assign taxonomic data to each representative sequence. In order to reveal Alpha diversity, rarefaction curves were generated based on these two metrics: the observed species metric is simply the count of unique OTUs found in the sample, and Shannon index. We used unweighted unifrac in QIIME to do principal coordinate analysis (PCoA). 2.5 Accession number of DNA sequence Sequences obtained in DGGE are deposited in GenBank, and the accession numbers were KJ875868 - KJ875898; 16S rDNA sequencing reads by high-throughput sequencing are deposited in MG-RAST with the IDs 4565696.3, 4565697.3, 4565698.3 and 4565699.3.

3 Results and discussion 3.1 Electricity production of MFCs Anaerobic pretreated aerobic sludge or anaerobic sludge was inoculated directly into the anodic chambers of the two groups of MFCs. The anolyte was replaced each day to acclimate the anodophilic populations in biofilm. The changes in voltage with time in the MFCs inoculated with aerobic activated sludge and anaerobic ABR sludge are presented in Fig. 1A and 1B, respectively. During 35 day operation, voltage of aerobic 8

sludge-seeded (Os) MFC varied between 0.2 and 0.3 V (Fig.1.A). It reached 0.22 V at 50 h after incubation, and showed the maximum voltage at 0.27 V at 170 h. On the other hand, the voltage of anaerobic sludge-seeded (As) MFC increased slowly and reached its maximum of 0.21 V at about 400 h (Fig.1B). At the end of operation, the polarization curves of the two MFCs were recorded (Fig. 2). The open circuit voltage was 0.7 and 0.6 V for Os and As MFCs, respectively. Maximum power density for Os MFC was 5.79 W m-3 with external resistance of 200 Ω, while the As MFC was 3.66 W m-3 with external resistance of 820 Ω. Above results indicated that Os MFC performed better than As MFC. The MFC inoculated with aerobic activated sludge started up more rapidly, reached higher potential and presented lower internal resistance. Besides wastewater type and operational parameters, the inoculum type is a key factor influencing the startup of bioreactors including MFCs (Lobato et al., 2012). For MFCs, anode chamber usually works under anaerobic or anoxic condition to avoid competition of oxygen for electrons. Anaerobic digestion sludge has been used by several investigators. Lobato et al. (2012) reported that seeding an MFC with anaerobic sludge led to rapid startup of electricity production and better performance of the MFC than aerobic sludge. Tao et al. (2011) used anaerobic digester sludge to start up MFCs for copper (II) reduction. However, contradictory results were also observed. Higher power density was observed in a two-chambered MFC inoculated with aerobic activated sludge (Patil et al., 2009). In fact, the startup process is the enrichment of exoelectrogens on the anodic biofilm of MFC (Sun et al., 2009). Exoelectrogens have massive diversity and distribute widely in classes Alpha-, Beta-, Gamma-, and 9

Delta-proteobacteria and phylum Firmicutes (Logan & Regan, 2006), including anaerobic and facultative anaerobes. Facultative anaerobes are more easily enriched from a source with more diverse of microorganisms than a source with relative low diverse and specific-function microbial community. Anaerobic digester in wastewater treatment plant commonly receives aerobic activated sludge with long-term (e.g. 20 to 30 d or longer) under anaerobic conditions, resulting in more strict anaerobes and less microbial diversity. In this study, it showed that the MFC with anaerobic pretreated aerobic sludge started up more quickly and performed better than anaerobic sludge. Anaerobic pretreatment of aerobic sludge with a short term (

Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells.

Aerobic sludge after anaerobic pretreatment and anaerobic sludge were separately used as inoculum to start up air-cathode single-chamber MFCs. Aerobic...
2MB Sizes 0 Downloads 3 Views