Jonathan P. Badalamenti

Generation of High Current Densities by Pure Cultures of Anode-Respiring Geoalkalibacter spp. under Alkaline and Saline Conditions in Microbial Electrochemical Cells

ABSTRACT Anode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity of Geobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for the Geobacteraceae family of the Deltaproteobacteria. Here we report high current densities generated by haloalkaliphilic Geoalkalibacter spp., thus broadening the capability for high anode respiration rates by including other genera within the Geobacteraceae. In this study, acetate-fed pure cultures of two related Geoalkalibacter spp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m−2 under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilic Glk. subterraneus DSM 23483 and alkaliphilic Glk. ferrihydriticus DSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles. Glk. ferrihydriticus also oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m−2 and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiring Gb. sulfurreducens biofilms producing similar current densities (~5 to 9 A m−2). IMPORTANCE This study establishes the highest current densities ever achieved by pure cultures of anode-respiring bacteria (ARB) under alkaline and saline conditions in microbial electrochemical cells (MXCs) and provides the first electrochemical characterization of the genus Geoalkalibacter. Production of high current densities among the Geobacteraceae is no longer exclusive to Geobacter sulfurreducens, suggesting greater versatility for this family in fundamental and applied microbial electrochemical cell (MXC) research than previously considered. Additionally, this work raises the possibility that different members of the Geobacteraceae have conserved molecular mechanisms governing respiratory extracellular electron transfer to electrodes. Thus, the capacity for high current generation may exist in other uncultivated members of this family. Advancement of MXC technology for practical uses must rely on an expanded suite of ARB capable of using different electron donors and producing high current densities under various conditions. Geoalkalibacter spp. can potentially broaden the practical capabilities of MXCs to include energy generation and waste treatment under expanded ranges of salinity and pH. This study establishes the highest current densities ever achieved by pure cultures of anode-respiring bacteria (ARB) under alkaline and saline conditions in microbial electrochemical cells (MXCs) and provides the first electrochemical characterization of the genus Geoalkalibacter. Production of high current densities among the Geobacteraceae is no longer exclusive to Geobacter sulfurreducens, suggesting greater versatility for this family in fundamental and applied microbial electrochemical cell (MXC) research than previously considered. Additionally, this work raises the possibility that different members of the Geobacteraceae have conserved molecular mechanisms governing respiratory extracellular electron transfer to electrodes. Thus, the capacity for high current generation may exist in other uncultivated members of this family. Advancement of MXC technology for practical uses must rely on an expanded suite of ARB capable of using different electron donors and producing high current densities under various conditions. Geoalkalibacter spp. can potentially broaden the practical capabilities of MXCs to include energy generation and waste treatment under expanded ranges of salinity and pH.

EFFECTS OF TEMPERATURE SHIFTS ON GROWTH RATE AND LIPID CHARACTERISTICS OF SYNECHOCYSTIS SP. PCC6803 IN A BENCH-TOP PHOTOBIOREACTOR

Synechocystis sp. PCC6803 exhibited a high degree of variation in biomass and lipid production rates in response to temperature changes in a photobioreactor. Compared with an optimal temperature of 30-33°C, a higher temperature of 44°C and lower temperatures of 22°C and 18°C severely inhibited the specific growth rate (up to a 66% decrease), biomass production rate (up to a 71% decrease), nutrient utilization rates (up to a 77% decrease), and lipid production rate (up to a 80% decrease). Temperature stress triggered changes in the relative percentage of individual fatty acids (mainly for C16:0 and C18:3), and degree of unsaturation significantly changed: 0.87 at 30°C, 0.62 at 44°C, and 1.29 at 18°C. Although PCC6803 survived temperature stress and maintained its predominate position in the culture, it could not fully recover from long-term temperature stress. Thus, avoiding prolonged exposure to extreme temperature is crucial for using PCC6803 as feedstock for biofuel production.

Integrating High-Throughput Pyrosequencing and Quantitative Real-Time PCR to Analyze Complex Microbial Communities

New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, we discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. We explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. We also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. We provide three examples in which we used pyrosequencing and QPCR together to define and quantify members of microbial communities: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. We highlight our key findings in these systems and how both methods were used in concert to achieve those findings. Finally, we supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation.

Engineering motility as a phenotypic response to LuxI/R-dependent quorum sensing in Escherichia coli.

The repertoire of functional outputs interfaced with the LuxI/LuxR quorum sensing system in engineered Escherichia coli has been expanded to include motility via inducible expression of motB. Appropriate choice of ribosome binding site controlling MotB translation was crucial to achieving control over motility.

Light‐responsive current generation by phototrophically enriched anode biofilms dominated by green sulfur bacteria

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co‐enrichment of non‐phototrophic anode‐respiring bacteria (ARB). This approach resulted in light‐responsive electricity generation from fresh‐ and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non‐mediated electron transfer, but kinetic parameters differed from values previously reported for non‐phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria. Biotechnol. Bioeng. 2013; 110: 1020–1027.

Coupling dark metabolism to electricity generation using photosynthetic cocultures

We investigated the role of green sulfur bacteria inlight‐responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode‐respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light‐responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S0 as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14‐fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real‐time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current. Biotechnol. Bioeng. 2014;111: 223–231.

Isolation and genomic characterization of 'Desulfuromonas soudanensis WTL', a metal- and electrode-respiring bacterium from anoxic deep subsurface brine

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, ‘Desulfuromonas soudanensis’ strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that ‘D. soudanensis’ releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. ‘D. soudanensis’ contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of ‘D. soudanensis’ underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens

ABSTRACT Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.

Complete Genome Sequence of Achromobacter xylosoxidans MN001, a Cystic Fibrosis Airway Isolate

ABSTRACT The genome of Achromobacter xylosoxidans MN001, a strain isolated from sputum derived from an adult cystic fibrosis patient, was sequenced using combined single-molecule real-time and Illumina sequencing. Assembly of the complete genome resulted in a 5,876,039-bp chromosome, representing the smallest A. xylosoxidans genome sequenced to date.

Draft Genome Sequence of the Gram-Positive Thermophilic Iron Reducer Thermincola ferriacetica Strain Z-0001T

ABSTRACT A 3.19-Mbp draft genome of the Gram-positive thermophilic iron-reducing Firmicutes isolate from the Peptococcaceae family, Thermincola ferriacetica Z-0001, was assembled at ~100× coverage from 100-bp paired-end Illumina reads. The draft genome contains 3,274 predicted genes (3,187 protein coding genes) and putative multiheme c-type cytochromes.