Electrochemical performance and microbial community analysis in air cathode microbial fuel cells fuelled with pyroligneous liquor.

Microbial fuels cells (MFCs) have been applied for the degradation of pyroligneous liquor (PL) derived from apple tree branches, at different concentrations. The substrate removal, electrochemical properties, and microbial community characteristics were analysed to evaluate the performance of MFCs. Maximum current density (1.94 A/m2), coulombic efficiency (28%), and phenol removal rate (84%) were achieved with MFCs fed with PL at the optimal concentration of 1 g chemical oxygen demand (COD)/L. The polarisation test, cyclic voltammetry, and electrochemical impedance of the electrode redox reaction further explained how the addition of PL could stimulate formation of the electrochemically active biofilm, at the optimal concentration of 1 g COD/L. The microbial community of the anodic biofilm demonstrated that MFCs fed with 1 g COD/L had the highest relative abundance of the typical electrogenic bacteria Geobacter (33%), followed by Sphaerochaeta (6%) and Clostridium (4%). The results revealed that syntrophic interaction of these functional microorganisms contributed significantly to the PL degradation and electrical current generation.

Chemical signals stimulate Geobacter soli biofilm formation and electroactivity.

Biofilm formation and maturation have been demonstrated to be regulated by distinct forms of cell-cell communication factors such as chemical and physical signals. However, whether the Geobacter sp. biofilms, which are typical electroactive biofilms, are affected by chemical signals is poorly understood. This research investigated the effects and corresponding mechanisms of endogenous and exogenous chemical signals (i.e., N-acylhomoserine lactones, AHLs) on the Geobacter soli biofilm. The results showed that Geobacter soli GSS01 secreted detectable endogenous AHLs to facilitate the formation and electrochemical activity of the biofilm, and that exogenous AHLs could further promoted these activities. Analyses of surface proteins revealed that the mechanisms promoted by endogenous and exogenous AHLs were somewhat different. Endogenous AHLs improved the relative abundance of external membrane proteins, while exogenous AHLs further facilitated the formation of amide II and a stronger H-bond between the carbonyl group and the amide. Furthermore, the proteomics analysis indicated that endogenous AHLs enhanced extracellular polymeric substance production by up-regulating the expression of key enzymes participating in EPS production, and simultaneously affected the physiological performance of individual cells. These results demonstrate, for the first time, the importance of chemical signals in Geobacter sp. and provide a comprehensive understanding of the chemical signals involved in biofilm formation and electrochemical activity of Geobacter sp..

Abundance and diversity of iron reducing bacteria communities in the sediments of a heavily polluted freshwater lake.

Iron reduction mediated by Fe(III)-reducing bacteria (FeRB) occurs in aqueous environments and plays an essential role in removing contaminates in polluted freshwater lakes. Two model FeRB species, Shewanella and Geobacter, have been intensively studied because of their functions in bioremediation, iron reduction, and bioelectricity production. However, the abundance and community diversity of Shewanella and Geobacter in eutrophic freshwater lakes remain largely unknown. In this work, the distribution, abundance and biodiversity of Shewanella, Geobacter and other FeRB in the sediments of a heavily polluted lake, Chaohu Lake, China, across four successive seasons were investigated. Shewanella, Geobacter, and other FeRB were found to be widely distributed in the sediment of this heavily eutrophic lake. Geobacter was abundant with at least one order of magnitude more than Shewanella in cold seasons. Three Shewanella-related operational taxonomic units were detected and sixty one Geobacter-related operational taxonomic units were grouped into three phylogenetic clades. Thiobacillus, Desulfuromonas and Geobacter were identified as the main members of FeRB in the lake sediments. Interestingly, nutrients like carbon, nitrogen, and phosphorus were found to be the key factors governing the abundance and diversity of FeRB. Total FeRB, as well as Geobacter and Shewanella, were more abundant in the heavily eutrophic zone than those in the lightly eutrophic zone. The abundance and diversity of FeRB in the sediments of freshwater lakes were highly related with the degree of eutrophication, which imply that FeRB might have a great potential in alleviating the eutrophication and contamination in aqueous environments.

Electrode plate-culture methods for colony isolation of exoelectrogens from anode microbiomes.

Exoelectrogens play central roles in microbial fuel cells and other bioelectrochemical systems (BESs), yet their physiological diversity remains largely elusive due to the lack of efficient methods for the isolation from naturally occurring microbiomes. The present study developed an electrode plate-culture (EPC) method that facilitates selective colony formation by exoelectrogens and used it for isolating them from an exoelectrogenic microbiome enriched from paddy-field soil. In an EPC device, the surface of solidified agarose medium was spread with a suspension of a microbiome and covered with a transparent fluorine doped tin oxide (FTO) electrode (poised at 0 V vs. the standard hydrogen electrode) that served as the sole electron acceptor. The medium contained acetate as the major growth substrate and Coomassie Brilliant Blue as a dye for visualizing colonies under FTO. It was shown that colonies successfully appeared under FTO in association with current generation. Analyses of 16S rRNA gene sequences of colonies indicated that they were affiliated with genera Citrobacter, Geobacter and others. Among them, Citrobacter and Geobacter isolates were found to be exoelectrogenic in pure-culture BESs. These results demonstrate the utility of the EPC method for colony isolation of exoelectrogens.

Dehalococcoides and general bacterial ecology of differentially trichloroethene dechlorinating flow-through columns.

The diversity of Dehalococcoides mccartyi (Dhc) and/or other organohalide respiring or associated microorganisms in parallel, partial, or complete trichloroethene (TCE) dehalogenating systems has not been well described. The composition of Dhc populations and the associated bacterial community that developed over 7.5 years in the top layer (0-10 cm) of eight TCE-fed columns were examined using pyrosequencing. Columns biostimulated with one of three carbon sources, along with non-stimulated controls, developed into complete (ethene production, whey amended), partial (cis-dichloroethene (DCE) and VC, an emulsified oil with nonionic surfactant), limited (<5 % cis-DCE and 95 % TCE, an emulsified oil), and non- (controls) TCE dehalogenating systems. Bioaugmentation of one column of each treatment with Bachman Road enrichment culture did not change Dhc populations nor the eventual degree of TCE dehalogenation. Pyrosequencing revealed high diversity among Dhc strains. There were 13 OTUs that were represented by more than 1000 sequences each. Cornell group-related populations dominated in complete TCE dehalogenating columns, while Pinellas group related Dhc dominated in all other treatments. General microbial communities varied with biostimulation, and three distinct microbial communities were established: one each for whey, oils, and control treatments. Bacterial genera, including Dehalobacter, Desulfitobacterium, Sulfurospirillum, Desulfuromonas, and Geobacter, all capable of partial TCE dehalogenation, were abundant in the limited and partial TCE dehalogenating systems. Dhc strain diversity was wider than previously reported and their composition within the community varied significantly depending on the nature of the carbon source applied and/or changes in the Dhc associated partners that fostered different biogeochemical conditions across the columns.

Sodium chloride concentration determines exoelectrogens in anode biofilms occurring from mangrove-grown brackish sediment.

Single-chamber microbial fuel cells (MFCs) were inoculated with mangrove-grown brackish sediment (MBS) and continuously supplied with an acetate medium containing different concentrations of NaCl (0-1.8M). Different from MFCs inoculated with paddy-field soil (high power outputs were observed between 0.05 and 0.1M), power outputs from MBS-MFCs were high at NaCl concentrations from 0 to 0.6M. Amplicon-sequence analyses of anode biofilms suggest that different exoelectrogens occurred from MBS depending on NaCl concentrations; Geobacter occurred abundantly below 0.1M, whereas Desulfuromonas was abundant from 0.3M to 0.6M. These results suggest that NaCl concentration is the major determinant of exoelectrogens that occur in anode biofilms from MBS. It is also suggested that MBS is a potent source of microbes for MFCs to be operated in a wide range of NaCl concentrations.

Comparison of electrochemical and microbiological characterization of microbial fuel cells equipped with SPEEK and Nafion membrane electrode assemblies.

Microbial fuel cells equipped with SPEEK-MEA (SPEEK-MFC) and Nafion-MEA (Nafion-MFC) were constructed with organic waste as electron donor and lake sediment as inoculum and were then evaluated comprehensively by electrochemical and microbial analyses. The proton conductivity of SPEEK was several hundreds-fold lower than that of Nafion 117, whereas the oxygen mass and diffusion transfer coefficients of SPEEK were 10-fold lower than those of Nafion 117. It was difficult to predict which was better membrane for MFC based on the feature of membrane. Analyses of polarization curves indicated that the potential of electricity production was similar in both MFCs, as the SPEEK-MFC produced 50-80% of the practical current density generated by the Nafion-MFC. Chronopotentiometry analyses indicated that the Nafion-MEA kept the performance longer than the SPEEK-MEA for long period, whereas performance of both anodes improved on time. Multidimensional scaling analyses based on DGGE profiles revealed the anolytic and biofilm communities of the SPEEK-MFC had developed differently from those of the Nafion-MFC. Clone library analyses indicated that Geobacter spp. represented 6.3% of the biofilm bacterial community in the Nafion-MFC but not detected in the SPEEK-MFC. Interestingly, the clone closely related to Acetobacterium malicum strain HAAP-1, belonging to the homoacetogens, became dominant in both anolytic and biofilm communities of the SPEEK-MFC. It was suggested that the lower proton conductivity of SPEEK-MEA allowed the bacteria closely related to strain HAAP-1 to be dominant specifically in SPEEK-MFC. These results indicated that Nafion-MFC ranked with SPEEK-MFC and that MEAs had strong selective pressure for electricity-producing bacterial community.

Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions.

This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen.

Size effects of potato waste on its treatment by microbial fuel cell.

The performance of microbial fuel cell (MFC) in treating potato cubes with different sizes (the edge size of 3, 5 and 7 mm) was investigated. Current density was found lower as the size of potato cubes increased, even if the differences in their removal were less apparent. At the end of MFC operation for 81 days, both total and soluble chemical oxygen demand reached nearly identical values, irrespective of the potato sizes; and citrate and isobutyrate were two major organic acids remaining in the solutions. Bacterial community analysis using polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing indicated that bacterial species on the anode and in the anodic solution were similar and did not change obviously with potato sizes, and that, in similarity with previous studies on potato-processing wastewater treatment, Proteobacteria and Firmicutes were two dominating phyla. Geobacter was found richer on the anode than in the anodic solutions.

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

Office paper platform for bioelectrochromic detection of electrochemically active bacteria using tungsten trioxide nanoprobes.

Electrochemically active bacteria (EAB) have the capability to transfer electrons to cell exterior, a feature that is currently explored for important applications in bioremediation and biotechnology fields. However, the number of isolated and characterized EAB species is still very limited regarding their abundance in nature. Colorimetric detection has emerged recently as an attractive mean for fast identification and characterization of analytes based on the use of electrochromic materials. In this work, WO3 nanoparticles were synthesized by microwave assisted hydrothermal synthesis and used to impregnate non-treated regular office paper substrates. This allowed the production of a paper-based colorimetric sensor able to detect EAB in a simple, rapid, reliable, inexpensive and eco-friendly method. The developed platform was then tested with Geobacter sulfurreducens, as a proof of concept. G. sulfurreducens cells were detected at latent phase with an RGB ratio of 1.10 ± 0.04, and a response time of two hours.

The impact of gamma radiation on sediment microbial processes.

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h(-1) for 8 weeks all displayed NO3 (-) and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h(-1) treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h(-1) treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO3 (-), Fe(III), or SO4 (2-) reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h(-1)-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h(-1) and 30-Gy h(-1) treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.

Geobacter, Anaeromyxobacter and Anaerolineae populations are enriched on anodes of root exudate-driven microbial fuel cells in rice field soil.

Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC.

Evidence of Geobacter-associated phage in a uranium-contaminated aquifer.

Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site.

Microbial community analysis in rice paddy soils irrigated by acid mine drainage contaminated water.

Five rice paddy soils located in southwest China were selected for geochemical and microbial community analysis. These rice fields were irrigated with river water which was contaminated by Fe-S-rich acid mine drainage. Microbial communities were characterized by high-throughput sequencing, which showed 39 different phyla/groups in these samples. Among these phyla/groups, Proteobacteria was the most abundant phylum in all samples. Chloroflexi, Acidobacteria, Nitrospirae, and Bacteroidetes exhibited higher relative abundances than other phyla. A number of rare and candidate phyla were also detected. Moreover, canonical correspondence analysis suggested that pH, sulfate, and nitrate were significant factors that shaped the microbial community structure. In addition, a wide diversity of Fe- and S-related bacteria, such as GOUTA19, Shewanella, Geobacter, Desulfobacca, Thiobacillus, Desulfobacterium, and Anaeromyxobacter, might be responsible for biogeochemical Fe and S cycles in the tested rice paddy soils. Among the dominant genera, GOUTA19 and Shewanella were seldom detected in rice paddy soils.

Long-term nitrogen fertilization of paddy soil shifts iron-reducing microbial community revealed by RNA-(13)C-acetate probing coupled with pyrosequencing.

Iron reduction is an important biogeochemical process in paddy soils, yet little is known about the microbial coupling between nitrogen and iron reduction. Here, we investigated the shift of acetate-metabolizing iron-reducers under long-term nitrogen fertilization using (13)C-acetate-based ribosomal RNA (rRNA)-stable isotope probing (SIP) and pyrosequencing in an incubation experiment, and the shift of putative iron-reducers in original field samples were investigated by 16S rRNA gene-based pyrosequencing. During SIP incubations, in the presence of iron(III) oxyhydroxides, more iron(II) formation and less methane production were detected in nitrogen-fertilized (N) compared with non-fertilized (NF) soil. In (13)C-rRNA from microcosms amended with ferrihydrite (FER), Geobacter spp. were the important active iron-reducers in both soils, and labeled to a greater extent in N (31% of the bacterial classified sequences) than NF soils (11%). Pyrosequencing of the total 16S rRNA transcripts from microcosms at the whole community level further revealed hitherto unknown metabolisms of potential FER reduction by microorganisms including Pseudomonas and Solibacillus spp. in N soil, Dechloromonas, Clostridium, Bacillus and Solibacillus spp. in NF soil. Goethite (GOE) amendment stimulated Geobacter spp. to a lesser extent in both soils compared with FER treatment. Pseudomonas spp. in the N soil and Clostridium spp. in the NF soil may also be involved in GOE reduction. Pyrosequencing results from field samples showed that Geobacter spp. were the most abundant putative iron-reducers in both soils, and significantly stimulated by long-term nitrogen fertilization. Overall, for the first time, we demonstrate that long-term nitrogen fertilization promotes iron(III) reduction and modulates iron-reducing bacterial community in paddy soils.

Geobacter soli sp. nov., a dissimilatory Fe(III)-reducing bacterium isolated from forest soil.

A novel Fe(III)-reducing bacterium, designated GSS01(T), was isolated from a forest soil sample using a liquid medium containing acetate and ferrihydrite as electron donor and electron acceptor, respectively. Cells of strain GSS01(T) were strictly anaerobic, Gram-stain-negative, motile, non-spore-forming and slightly curved rod-shaped. Growth occurred at 16-40 °C and optimally at 30 °C. The DNA G+C content was 60.9 mol%. The major respiratory quinone was MK-8. The major fatty acids were C(16:0), C(18:0) and C(16:1)ω7c/C(16:1)ω6c. Strain GSS01(T) was able to grow with ferrihydrite, Fe(III) citrate, Mn(IV), sulfur, nitrate or anthraquinone-2,6-disulfonate, but not with fumarate, as sole electron acceptor when acetate was the sole electron donor. The isolate was able to utilize acetate, ethanol, glucose, lactate, butyrate, pyruvate, benzoate, benzaldehyde, m-cresol and phenol but not toluene, p-cresol, propionate, malate or succinate as sole electron donor when ferrihydrite was the sole electron acceptor. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain GSS01(T) was most closely related to Geobacter sulfurreducens PCA(T) (98.3% sequence similarity) and exhibited low similarities (94.9-91.8%) to the type strains of other species of the genus Geobacter. The DNA-DNA relatedness between strain GSS01(T) and G. sulfurreducens PCA(T) was 41.4 ± 1.1%. On the basis of phylogenetic analysis, phenotypic characterization and physiological tests, strain GSS01(T) is believed to represent a novel species of the genus Geobacter, and the name Geobacter soli sp. nov. is proposed. The type strain is GSS01(T) ( =KCTC 4545(T) =MCCC 1K00269(T)).

Geobacter sulfurreducens subsp. ethanolicus, subsp. nov., an ethanol-utilizing dissimilatory Fe(III)-reducing bacterium from a lotus field.

An ethanol-utilizing Fe(III)-reducing bacterial strain, OSK2A(T), was isolated from a lotus field in Aichi, Japan. Phylogenetic analysis of the 16S rRNA gene sequences of OSK2A(T) and related strains placed it within Geobacter sulfurreducens PCA(T). Strain OSK2A(T) was shown to be a Gram-negative, motile, rod-shaped bacterium, strictly anaerobic, 0.76-1.65 µm long and 0.28-0.45 µm wide. Its growth occurred at 20-40℃, pH 6.0-8.1, and it tolerated up to 1% NaCl. The G+C content of the genomic DNA was 61.2 mol% and DNA-DNA hybridization value with Geobacter sulfurreducens PCA(T) was 60.7%. The major respiratory quinone was MK-8. The major fatty acids were 16：1 ω7c, 16：0, 14：0, 15：0 iso, 16：1 ω5c, and 18：1 ω7c. Strain OSK2A(T) could utilize H2, ethanol, acetate, lactate, pyruvate, and formate as substrates with Fe(III)-citrate as electron acceptor. Amorphous Fe(III) hydroxide, Fe(III)-NTA, fumarate, malate, and elemental sulfur were utilized as electron acceptors with either acetate or ethanol as substrates. Results obtained from physiological, DNA-DNA hybridization, and chemotaxonomic tests support genotypic and phenotypic differentiation of strain OSK2A(T) from its closest relative. The isolate is assigned as a novel subspecies with the name Geobacter sulfurreducens subsp. ethanolicus, subsp. nov. (type strain OSK2A(T)=DSMZ 26126(T)=JCM 18752(T)).

When is a microbial culture "pure"? Persistent cryptic contaminant escapes detection even with deep genome sequencing.

UNLABELLED: Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (-400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 10(5) copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at -400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode. IMPORTANCE: Repeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.

Geobacter luticola sp. nov., an Fe(III)-reducing bacterium isolated from lotus field mud.

A novel species of Fe(III)-reducing bacterium, designated strain OSK6(T), belonging to the genus Geobacter, was isolated from lotus field mud in Japan. Strain OSK6(T) was isolated using a solid medium containing acetate, Fe(III)-nitrilotriacetate (NTA) and gellan gum. The isolate is a strictly anaerobic, gram-negative, motile, straight rod-shaped bacterium, 0.6-1.9 µm long and 0.2-0.4 µm wide. The growth of the isolate occurred at 20-40 °C with optima of 30-37 °C and pH 6.5-7.5 in the presence of up to 0.5 g NaCl l(-1). The G+C content of the genomic DNA was determined by HPLC to be 59.7 mol%. The major respiratory quinone was MK-8. The major fatty acids were 16 : 1ω7c and 16 : 0. Strain OSK6(T) was able to grow with Fe(III)-NTA, ferric citrate, amorphous iron (III) hydroxide and nitrate, but not with fumarate, malate or sulfate as electron acceptors. Among examined substrates grown with Fe(III)-NTA, the isolate grew on acetate, lactate, pyruvate and succinate. Analysis of the near full-length 16S rRNA gene sequence revealed that strain OSK6(T) is closely related to Geobacter daltonii and Geobacter toluenoxydans with 95.6 % similarity to the type strains of these species. On the basis of phylogenetic analysis and physiological tests, strain OSK6(T) is described as a representative of a novel species, Geobacter luticola sp. nov.; the type strain is OSK6(T) ( = DSM 24905(T) = JCM 17780(T)).