Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors

Microbial fuel cells (MFCs) harness the electrochemical activity of certain microbes for the production of electricity from reduced compounds. Characterizations of MFC anode biofilms have collectively shown very diverse microbial communities, raising ecological questions about competition and community succession within these anode-reducing communities. Three sets of triplicate, two-chamber MFCs inoculated with anaerobic sludge and differing in energy sources (acetate, lactate, and glucose) were operated to explore these questions. Based on 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE), all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone in the Bacteroidetes class (99% similarity). Various other Geobacter-like sequences were also enriched in most of the anode biofilms. While the anode communities in replicate reactors for each substrate generally converged to a reproducible community, there were some variations in the relative distribution of these putative anode-reducing Geobacter-like strains. Firmicutes were found only in glucose-fed MFCs, presumably serving the roles of converting complex carbon into simple molecules and scavenging oxygen. The maximum current density in these systems was negatively correlated with internal resistance variations among replicate reactors and, likely, was only minimally affected by anode community differences in these two-chamber MFCs with high internal resistance.     

Effect of enrichment procedures on performance and microbial diversity of microbial fuel cell for Congo red decolorization and electricity generation

The purpose of this study was to determine the effect of enrichment procedure on the performance and microbial diversity of an air-cathode microbial fuel cell (MFC) which was explored for simultaneous azo dye decolorization and electricity generation. Two different enrichment procedures in which glucose and Congo red were added into the MFCs sequentially (EP1) or simultaneously (EP2) were tested by operating parallel MFCs independently for more than 6 months. The power density, electrode potential, Congo red decolorization, biofilm morphology, and bacterial diversity of the MFCs under the two enrichment procedures were compared and investigated. The results showed that the enrichment procedures have a negligible effect on the dye decolorization, but significantly affected the electricity generation. More than 90% decolorization at dye concentration of 300 mg/L was achieved within 170 h for the two tested enrichment procedures. However, the MFC with EP2 achieved a maximum power density of 192 mW/m², which was 75% higher than that of the MFC with EP1 (110 mW/m²). The depressed surfaces of the bacteria in the MFC with EP1 indicated the allergic response caused by the subsequent addition of Congo red. 16S rRNA sequencing analysis demonstrated a phylogenetic diversity in the communities of the anode biofilm and showed clear differences between the anode-attached populations in the MFCs with a different enrichment procedure. This study suggests that the enrichment procedure is important for the MFC explored for simultaneous dye decolorization and electricity generation.     

Change in microbial communities in acetate- and glucose-fed microbial fuel cells in the presence of light

Power densities produced by microbial fuel cells (MFCs) in natural systems are changed by exposure to light through the enrichment of photosynthetic microorganisms. When MFCs with brush anodes were exposed to light (4000 lx), power densities increased by 8–10% for glucose-fed reactors, and 34% for acetate-fed reactors. Denaturing gradient gel electrophoresis (DGGE) profiles based on the 16S rRNA gene showed that exposure to high light levels changed the microbial communities on the anodes. Based on 16S rRNA gene clone libraries of light-exposed systems the anode communities using glucose were also significantly different than those fed acetate. Dominant bacteria that are known exoelectrogens were identified in the anode biofilm, including a purple nonsulfur (PNS) photosynthetic bacterium, Rhodopseudomonas palustris, and a dissimilatory iron-reducing bacterium, Geobacter sulfurreducens. Pure culture tests confirmed that PNS photosynthetic bacteria increased power production when exposed to high light intensities (4000 lx). These results demonstrate that power production and community composition are affected by light conditions as well as electron donors in single-chamber air-cathode MFCs.     

Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

The impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected, Burkholderiales and Rhodocyclales of the Betaproteobacteria class were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.     

微生物燃料电池利用乳酸产电性能与微生物群落分布特征

【目的】为探讨以乳酸为基质的微生物燃料电池(Microbial fuel cell,MFC)产电性能以及微生物群落在阳极膜、悬浮液、阳极沉淀污泥中的分布特征,【方法】试验建立了双室MFC,以乳酸为阳极主要碳源,研究了反应器的启动过程及产电效能,同时以电镜和PCR-变性梯度凝胶电泳(Denaturing gradient gelelectrophoresis,DGGE)技术解析了微生物群落的空间分布特征。【结果】结果表明,反应器启动第7天时外电压达到0.56 V,当外阻为80Ω时,电流密度为415 mA/m2,MFC的功率密度达到最大值82 mW/m2。电镜观察发现大量杆菌附着在阳极表面,结合较为紧密;DGGE图谱显示阳极膜表面微生物与种泥最为相似,与阳极悬浮液、底部沉淀污泥中的主要菌群一致,条带序列与睾丸酮丛毛单胞菌(Comamonas testosteroni)和布氏弓形菌(Arcobacter butzleri)等最为相似。【结论】本研究表明以乳酸为基质MFC可产生较高的功率密度,阳极附着的优势菌与接种污泥来源密切相关。     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

Electricity-producing bacterial communities in microbial fuel cells

Microbial fuel cells (MFCs) are not yet commercialized but they show great promise as a method of water treatment and as power sources for environmental sensors. The power produced by these systems is currently limited, primarily by high internal (ohmic) resistance. However, improvements in the system architecture will soon result in power generation that is dependent on the capabilities of the microorganisms. The bacterial communities that develop in these systems show great diversity, ranging from primarily delta-Proteobacteria that predominate in sediment MFCs to communities composed of alpha-, beta-, gamma- or delta-Proteobacteria, Firmicutes and uncharacterized clones in other types of MFCs. Much remains to be discovered about the physiology of these bacteria capable of exocellular electron transfer, collectively defined as a community of "exoelectrogens". Here, we review the microbial communities found in MFCs and the prospects for this emerging bioenergy technology.     

Electricity generation from cellulose by rumen microorganisms in microbial fuel cells

In microbial fuel cells (MFCs) bacteria generate electricity by mediating the oxidation of organic compounds and transferring the resulting electrons to an anode electrode. The objective of this study was to test the possibility of generating electricity with rumen microorganisms as biocatalysts and cellulose as the electron donor in two-compartment MFCs. The anode and cathode chambers were separated by a proton exchange membrane and graphite plates were used as electrodes. The medium in the anode chamber was inoculated with rumen microorganisms, and the catholyte in the cathode compartment was ferricyanide solution. Maximum power density reached 55 mW/m(2) (1.5 mA, 313 mV) with cellulose as the electron donor. Cellulose hydrolysis and electrode reduction were shown to support the production of current. The electrical current was sustained for over 2 months with periodic cellulose addition. Clarified rumen fluid and a soluble carbohydrate mixture, serving as the electron donors, could also sustain power output. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA genes revealed that the microbial communities differed when different substrates were used in the MFCs. The anode-attached and the suspended consortia were shown to be different within the same MFC. Cloning and sequencing analysis of 16S rRNA genes indicated that the most predominant bacteria in the anode-attached consortia were related to Clostridium spp., while Comamonas spp. abounded in the suspended consortia. The results demonstrated that electricity can be generated from cellulose by exploiting rumen microorganisms as biocatalysts, but both technical and biological optimization is needed to maximize power output.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

Alteration of bacterial communities and organic matter in microbial fuel cells (MFCs) supplied with soil and organic fertilizer

The alteration of the organic matter (OM) and the composition of bacterial community in microbial fuel cells (MFCs) supplied with soil (S) and a composted organic fertilizer (A) was examined at the beginning and at the end of 3 weeks of incubation under current-producing as well as no-current-producing conditions. Denaturing gradient gel electrophoresis revealed a significant alteration of the microbial community structure in MFCs generating electricity as compared with no-current-producing MFCs. The genetic diversity of cultivable bacterial communities was assessed by random amplified polymorphic DNA (RAPD) analysis of 106 bacterial isolates obtained by using both generic and elective media. Sequencing of the 16S rRNA genes of the more representative RAPD groups indicated that over 50.4% of the isolates from MFCs fed with S were Proteobacteria, 25.1% Firmicutes, and 24.5% Actinobacteria, whereas in MFCs supplied with A 100% of the dominant species belonged to γ-Proteobacteria. The chemical analysis performed by fractioning the OM and using thermal analysis showed that the amount of total organic carbon contained in the soluble phase of the electrochemically active chambers significantly decreased as compared to the no-current-producing systems, whereas the OM of the solid phase became more humified and aromatic along with electricity generation, suggesting a significant stimulation of a humification process of the OM. These findings demonstrated that electroactive bacteria are commonly present in aerobic organic substrates such as soil or a fertilizer and that MFCs could represent a powerful tool for exploring the mineralization and humification processes of the soil OM.     

Microbial fuel cells meet with external resistance

The influence of external load on the composition of the anodic biofilm microbial community and biomass yield was investigated in a microbial fuel cell fed with glucose and domestic wastewater was used as source of electrogens. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments revealed distinct differences in anodic bacterial communities formed at the anode of each MFC operated under a different external load. These results implied that in an MFC, electrogenic bacteria were enriched under higher current densities, i.e., low external load, and were able to sustain better current and effluent quality. The influence of the external resistance applied to the MFCs during formation of the bacterial communities from sewage wastewater was shown to have no significant effect on power performance of the MFCs nor to have a significant influence on their anodic activity with both glucose and brewery wastewater as fuel. As expected, current generation, COD removal and the biomass yield were all directly influenced by the external load. Significantly, when operated under lower external load, the biomass yield in the MFC was less than that in conventional anaerobic digestion (i.e., control).     

Convergent development of anodic bacterial communities in microbial fuel cells

Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470±20 mV) and maximum power densities (590±170 mW m⁻²). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.     

The significance of the initiation process parameters and reactor design for maximizing the efficiency of microbial fuel cells

Microbial fuel cells (MFCs) can be used for electricity generation via bioconversion of wastewater and organic waste substrates. MFCs also hold potential for production of certain chemicals, such as H₂ and H₂O₂. The studies of electricity generation in MFCs have mainly focused on the microbial community formation, substrate effect on the anode reaction, and the cathode’s catalytic properties. To improve the performance of MFCs, the initiation process requires more investigation because of its significant effect on the anodic biofilm formation. This review explores the factors which affect the initiation process, including inoculum, substrate, and reactor configuration. The key messages are that optimal performance of MFCs for electricity production requires (1) understanding of the electrogenic bacterial biofilm formation, (2) proper substrates at the initiation stage, (3) focus on operational conditions affecting initial biofilm formation, and (4) attention to the reactor configuration.     

Microbial communities in contrasting freshwater marsh microhabitats

Heterotrophic microorganisms are widely recognized as crucial components of ecosystems; yet information on their community structure and dynamics in benthic freshwater habitats is notably scarce. Using denaturing gradient gel electrophoresis (DGGE), we determined the composition of bacterial and fungal communities in a freshwater marsh over four seasons. DGGE revealed diverse bacterial communities in four contrasting microhabitats. The greatest compositional differences emerged between water-column and surface-associated bacteria, although communities associated with sediment also differed from those on plant litter and epiphytic biofilms. Sequences of bacterial clones derived from DGGE bands belonged to the Alphaproteobacteria (31%), Actinobacteria (19%) and Bacteriodetes (19%). Betaproteobacteria were notably absent. Fungal clones obtained from leaf litter were mainly Ascomycota , but two members of the Basidiomycota were also identified. Overall, habitat type was the most important factor explaining variation in bacterial communities among samples, whereas temporal patterns in community composition were less pronounced in spite of large seasonal variation in environmental conditions such as temperature. The observed differences among bacterial communities in different microhabitats were not caused by random variation, but rather appeared to be determined by habitat characteristics, as evidenced by largely congruent community profiles of replicate samples taken at 10–100 m distances within the marsh.     

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.     

Site-specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.     

Analysis of microbial diversity in oligotrophic microbial fuel cells using 16S rDNA sequences

Molecular ecological techniques were applied to analyze the bacterial diversity of two oligotrophic microbial fuel cells (MFCs) enriched using river water or artificial wastewater (AWW) as fuel. Denaturing gradient gel electrophoresis (DGGE) of the PCR amplified 16S rDNA showed that different microbial communities were present in the two MFCs and these were different from the river sediment used to initiate the enrichment. Nearly complete 16S rDNA was amplified and sequenced. Over 80% of the clones were Proteobacteria. Betaproteobacteria were the dominant clones (46.2%) in MFCs fed with river water, and about 64.4% of the clones in MFCs fed with AWW were Alphaproteobacteria. Actinobacteria were found only in the MFC fed with AWW, and Deltaproteobacteria, Acidobacteria, Chloroflexi and Verrucomicrobia in the MFC fed with river water. Many clones were related to uncultured bacteria, some with homology less than 95%, indicating that many novel bacteria were enriched in the oligotrophic MFCs.     

Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA profiles were objectively digitized using an image analyzer; the individual microbial species in a community can thus be precisely quantified. The similarity between various microbial communities was compared to the digitized DGGE profiles using the cluster analyses technique. The microbial community in a biofilm was considerably different from that in suspended sludge obtained from the same system.     

Microbial community differences between propionate-fed microbial fuel cell systems under open and closed circuit conditions

We report the electrochemical characterization and microbial community analysis of closed circuit microbial fuel cells (CC-MFCs) and open circuit (OC) cells continuously fed with propionate as substrate. Differences in power output between MFCs correlated with their polarization behavior, which is related to the maturation of the anodophilic communities. The microbial communities residing in the biofilm growing on the electrode, biofouled cation-exchange membrane and anodic chamber liquor of OC-and CC-MFCs were characterized by restriction fragment length polymorphism screening of 16S rRNA gene clone libraries. The results show that the CC-MFC anode was enriched in several microorganisms related to known electrochemically active and dissimilatory Fe(III) reducing bacteria, mostly from the Geobacter spp., to the detriment of Bacteroidetes abundant in the OC-MFC anode. The results also evidenced the lack of a specific pelagic community in the liquor sample. The biofilm growing on the cation-exchange membrane of the CC-MFC was found to be composed of a low-diversity community dominated by two microaerophilic species of the Achromobacter and Azovibrio genus.