Kinetics and scale up of oxygen reducing cathodic biofilms.

The goals of this work were to study the kinetics and investigate the factors controlling the scale up of oxygen reducing mixed culture cathodic biofilms. Cathodic biofilms were enriched on different electrode sizes (14.5 cm2, 40.3 cm2, 131 cm2 and 466 cm2). Biofilm enrichment shifted the oxygen reduction onset potential from -0.1 VAg/AgCl to 0.3 VAg/AgCl, indicating the biofilm catalyzed oxygen reduction. The kinetics of oxygen reduction were studied by varying the bulk dissolved oxygen concentration. Oxygen reduction followed a Michaelis-Menten kinetics on all electrode sizes. The maximum current density decreased with increasing electrode surface area (-97.0 ± 10.6 µA/cm2, -76.0 ± 8.2 µA/cm2, -66.3 ± 3.0 µA/cm2 and -43.5 ± 10.5 µA/cm2, respectively). Cyclic voltammograms suggest that scale up was limited by ohmic resistance, likely due to the low ionic conductivity in the wastewater medium. Mathematical modeling using combined Michaelis-Menten and Butler-Volmer model supports that the decrease in current density with increasing electrode surface area is caused by ohmic losses. Analysis of the microbial community structure in different size electrodes and in multiple regions on the same electrode showed low variability, suggesting that the microbial community does not control the scale up of cathodic biofilms.

Large-scale switchable potentiostatically controlled/microbial fuel cell bioelectrochemical wastewater treatment system.

The treatment of municipal wastewater is an energy-intensive process, with the delivery of oxygen as an electron acceptor accounting for a significant share of the total energy consumption. Microbial communities growing on polarized electrodes can facilitate wastewater treatment processes by exchanging electrons with the electrodes. As a new approach, we combined the use of polarized electrodes with microbial fuel cells (MFCs) to develop a switchable dual-mode bioelectrochemical wastewater treatment system. In this system, we first enriched microbial communities on polarized anodes and cathodes. After enrichment, the system was switched to either a self-powered MFC (SP-MFC) mode or a potentiostatically controlled (PC) mode. The system was evaluated at the laboratory scale (260 L, 4 anode and cathode pairs) and the pilot scale (1200 L, 16 anode and cathode pairs). PC and SP-MFC systems showed improved COD removal relative to control (41.6 ± 3.5 and 38.4 ± 3.1 vs 28.8 ± 2.1 mg L-1 d-1, respectively). The laboratory-scale system was operated without biological or electrical interruption for one year. Finally, specific enrichment of active microbial communities was observed on PC anodes in comparison to mixed-operation and non-polarized control anodes. The combined PC and SP-MFC systems allowed us to develop a sustainable and failure-free bioelectrochemical wastewater treatment system.

Growth of 'Candidatus Liberibacter asiaticus' in a host-free microbial culture is associated with microbial community composition.

'Candidatus Liberibacter asiaticus' ('Ca. L. asiaticus'), the suspected causative agent of citrus greening disease, is one of many phloem-restricted plant pathogens that have not been isolated and grown in an axenic culture. In this study, infected Asian citrus psyllids were used to prepare a host-free source of 'Ca. L. asiaticus'. Host-free mixed microbial cultures of 'Ca. L. asiaticus' were grown in the presence of various antibiotic treatments to alter the composition of the microbial communities. Our hypothesis was that the presence of selected antibiotics would enhance or reduce the presence of 'Ca. L. asiaticus' in a host-free culture composed of a mixed bacterial population through changes in the microbial community structure. We determined how 'Ca. L. asiaticus' growth changed with the various treatments. Treatment with vancomycin (50 µg/mL), streptomycin (0.02 µg/mL), or polymyxin B (4 µg/mL) was associated with an increased abundance of 'Ca. L. asiaticus' of 7.35 ±â€¯0.27, 5.56 ±â€¯0.15, or 4.54 ±â€¯0.83 fold, respectively, compared to untreated mixed microbial cultures, while treatment with 100 µg/mL vancomycin; 0.5, 1, or 2 µg/mL streptomycin; or 0.5 µg/mL of polymyxin B was associated with reduced growth. In addition, the growth of 'Ca. L. asiaticus' was associated with the microbial community composition of the mixed microbial cultures. A positive relationship between the presence of the Pseudomonadaceae family and 'Ca. L. asiaticus' growth was observed, while the presence of 'Ca. L. asiaticus' was below the detection limit in cultures that displayed high abundances of Bacillus cereus. Our findings offer strategies for developing effective axenic culture conditions and suggest that enrichment of the Bacillaceae family could serve as a paratransgenic approach to controlling citrus greening disease.

Electron donor availability controls scale up of anodic biofilms.

The scale up of bioelectrochemical systems (BESs) is a challenging problem that limits the advancement and practical implementation of the technology. The goal of this work is to acquire an understanding of the limitations on scaling up anodic biofilms in BESs. We hypothesized that scaling up is dependent on the availability of electron donors. We tested this hypothesis by enriching anodic biofilms on electrodes of multiple sizes (15 cm2 to 466 cm2) and quantified the anodic current densities while varying the electron donor concentrations. The anodic biofilms were enriched on electrodes under two conditions: (1) in raw wastewater and (2) in wastewater supplemented with 20 mM acetate. Following anodic biofilm enrichment, the current density for each electrode was quantified in artificial wastewater medium with variable COD loadings using acetate as an electron donor. Current generated using anodic biofilms scaled up at a high COD loading (1500 mg/L), while current density decreased with increasing electrode size at lower COD loadings. Further, microbial community analysis revealed that the microbial community was independent of the electrode size but dependent on the medium composition during the enrichment phase. These results provide a practical framework for the design of large-scale BESs based on laboratory-scale measurements.

Host-free biofilm culture of "Candidatus Liberibacter asiaticus," the bacterium associated with Huanglongbing.

Inability to culture the phloem-restricted alpha-proteobacterium "Candidatus Liberibacter asiaticus" ("Ca. L. asiaticus") or the closely related species ("Candidatus Liberibacter americanus" and "Candidatus Liberibacter africanus") that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free "Ca. L. asiaticus" cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of "Ca. L. asiaticus" and the use of genetics-based methods to understand and mitigate the spread of HLB.

Syntrophic anaerobic photosynthesis via direct interspecies electron transfer.

Microbial phototrophs, key primary producers on Earth, use H2O, H2, H2S and other reduced inorganic compounds as electron donors. Here we describe a form of metabolism linking anoxygenic photosynthesis to anaerobic respiration that we call 'syntrophic anaerobic photosynthesis'. We show that photoautotrophy in the green sulfur bacterium Prosthecochloris aestaurii can be driven by either electrons from a solid electrode or acetate oxidation via direct interspecies electron transfer from a heterotrophic partner bacterium, Geobacter sulfurreducens. Photosynthetic growth of P. aestuarii using reductant provided by either an electrode or syntrophy is robust and light-dependent. In contrast, P. aestuarii does not grow in co-culture with a G. sulfurreducens mutant lacking a trans-outer membrane porin-cytochrome protein complex required for direct intercellular electron transfer. Syntrophic anaerobic photosynthesis is therefore a carbon cycling process that could take place in anoxic environments. This process could be exploited for biotechnological applications, such as waste treatment and bioenergy production, using engineered phototrophic microbial communities.

Regulation of electron transfer processes affects phototrophic mat structure and activity.

Phototrophic microbial mats are among the most diverse ecosystems in nature. These systems undergo daily cycles in redox potential caused by variations in light energy input and metabolic interactions among the microbial species. In this work, solid electrodes with controlled potentials were placed under mats to study the electron transfer processes between the electrode and the microbial mat. The phototrophic microbial mat was harvested from Hot Lake, a hypersaline, epsomitic lake located near Oroville (Washington, USA). We operated two reactors: graphite electrodes were polarized at potentials of -700 mVAg/AgCl [cathodic (CAT) mat system] and +300 mVAg/AgCl [anodic (AN) mat system] and the electron transfer rates between the electrode and mat were monitored. We observed a diel cycle of electron transfer rates for both AN and CAT mat systems. Interestingly, the CAT mats generated the highest reducing current at the same time points that the AN mats showed the highest oxidizing current. To characterize the physicochemical factors influencing electron transfer processes, we measured depth profiles of dissolved oxygen (DO) and sulfide in the mats using microelectrodes. We further demonstrated that the mat-to-electrode and electrode-to-mat electron transfer rates were light- and temperature-dependent. Using nuclear magnetic resonance (NMR) imaging, we determined that the electrode potential regulated the diffusivity and porosity of the microbial mats. Both porosity and diffusivity were higher in the CAT mats than in the AN mats. We also used NMR spectroscopy for high-resolution quantitative metabolite analysis and found that the CAT mats had significantly higher concentrations of osmoprotectants such as betaine and trehalose. Subsequently, we performed amplicon sequencing across the V4 region of the 16S rRNA gene of incubated mats to understand the impact of electrode potential on microbial community structure. These data suggested that variation in the electrochemical conditions under which mats were generated significantly impacted the relative abundances of mat members and mat metabolism.

Localized electron transfer rates and microelectrode-based enrichment of microbial communities within a phototrophic microbial mat.

Phototrophic microbial mats frequently exhibit sharp, light-dependent redox gradients that regulate microbial respiration on specific electron acceptors as a function of depth. In this work, a benthic phototrophic microbial mat from Hot Lake, a hypersaline, epsomitic lake located near Oroville in north-central Washington, was used to develop a microscale electrochemical method to study local electron transfer processes within the mat. To characterize the physicochemical variables influencing electron transfer, we initially quantified redox potential, pH, and dissolved oxygen gradients by depth in the mat under photic and aphotic conditions. We further demonstrated that power output of a mat fuel cell was light-dependent. To study local electron transfer processes, we deployed a microscale electrode (microelectrode) with tip size ~20 µm. To enrich a subset of microorganisms capable of interacting with the microelectrode, we anodically polarized the microelectrode at depth in the mat. Subsequently, to characterize the microelectrode-associated community and compare it to the neighboring mat community, we performed amplicon sequencing of the V1-V3 region of the 16S gene. Differences in Bray-Curtis beta diversity, illustrated by large changes in relative abundance at the phylum level, suggested successful enrichment of specific mat community members on the microelectrode surface. The microelectrode-associated community exhibited substantially reduced alpha diversity and elevated relative abundances of Prosthecochloris, Loktanella, Catellibacterium, other unclassified members of Rhodobacteraceae, Thiomicrospira, and Limnobacter, compared with the community at an equivalent depth in the mat. Our results suggest that local electron transfer to an anodically polarized microelectrode selected for a specific microbial population, with substantially more abundance and diversity of sulfur-oxidizing phylotypes compared with the neighboring mat community.