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Geobacter biofilms synthesize an electroactive exopolysaccharide matrix with conductive pili and c-cytochromes that spatially organizes cells optimally for growth and electron transport to iron oxide substrates, soluble metal contaminants, and current-harvesting electrodes. Despite its relevance to bioremediation and bioenergy applications, little is known about the developmental stages leading to the formation of mature (20 μm thick) electroactive biofilms. Thus, we developed a transposon mutagenesis method and a high-throughput screening assay and identified mutants of Geobacter sulfurreducens PCA interrupted in the initial stages of surface colonization (attachment and monolayer formation) and the vertical growth and maturation of multilayered biofilms. The molecular dissection of biofilm formation demonstrated that cells undergo a regulated developmental program to first colonize the surface to saturation and then synthesize an electroactive matrix to support optimal cell growth within structured communities. Transitioning from a monolayer to a multilayered, mature biofilm required the expression of conductive pili, consistent with the essential role of these extracellular protein appendages as electronic conduits across all layers of the biofilms. The genetic screening also identified cell envelope processes, regulatory pathways, and electron transport components not previously linked to biofilm formation. These genes provide much-needed understanding of the cellular reprogramming needed to build electroactive biofilms. Importantly, they serve as predictive markers of the physiology and reductive capacity of Geobacter biofilms during the bioremediation of toxic metals and radionuclides and current harvesting in bioelectrochemical systems.

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. Here, we investigated how the commonly-implemented practice of continued microbial community transfer from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa var. crispa) growth in nitrogen-limited aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Lettuce above- and below-ground growth were significantly reduced across replicates treated with a pre-existing aquaponic system inoculum when compared to replicates treated with a commercial inoculum. Reduced productivity was associated with enrichment in specific bacterial genera in plant roots, including Pseudomonas, following inoculum transfer from pre-existing systems. Increased productivity was associated with enrichment of nitrogen-fixing Rahnella in roots of plants treated with the commercial inoculum. Thus, we show that inoculation from a pre-existing system, rather than from a commercial inoculum, is associated with lower yields. Further work will be necessary to test the putative mechanisms involved.

Through complex interspecies interactions, microbial processes drive nutrient cycling and biogeochemistry. However, we still struggle to predict specifically which organisms, communities and biotic and abiotic processes are determining ecosystem function and how environmental changes will alter their roles and stability. While the tools to create such a predictive microbial ecology capability exist, cross-disciplinary integration of high-resolution field measurements, detailed laboratory studies and computation is essential. In this perspective, we emphasize the importance of pursuing a multiscale, systems approach to iteratively link ecological processes measured in the field to testable hypotheses that drive high-throughput laboratory experimentation. Mechanistic understanding of microbial processes gained in controlled lab systems will lead to the development of theory that can be tested back in the field. Using N2 O production as an example, we review the current status of field and laboratory research and layout a plausible path to the kind of integration that is needed to enable prediction of how N-cycling microbial communities will respond to environmental changes. We advocate for the development of realistic and predictive gene regulatory network models for environmental responses that extend from single-cell resolution to ecosystems, which is essential to understand how microbial communities involved in N2 O production and consumption will respond to future environmental conditions.

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. Efforts to increase conventional agricultural output come with significant environmental impacts stemming from deforestation and excessive use of chemicals, including soil salinization, erosion, and nutrient runoffs. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. For instance, much of our knowledge of beneficial and detrimental microbial communities vis-à-vis crop productivity comes from studies on plant-microbiome interactions in soil. Here, we investigated how the practice of continued transfer of microbial communities from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa) growth in aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Strikingly, L. sativa plant and root growth was significantly reduced across all replicates inoculated with the established microbiome. Further analyses revealed the reduced productivity was potentially a consequence of plant-specific pathogen enrichment, including Pseudomonas, through transfer of microbiomes from pre-existing systems – a phenomenon consistent with negative feedbacks in soil ecology. These findings underscore the need for diagnostic tools to monitor microbiome composition, detect negative feedbacks early, and minimize pathogen accumulation in aquaponic systems.

We established Fe(III)-reducing co-cultures of two species of metal-reducing bacteria, the Gram-positive Desulfotomaculum reducens MI-1 and the Gram-negative Geobacter sulfurreducens PCA. Co-cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co-culture. Co-cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co-cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co-cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co-culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c-type cytochromes and type IV pili proteins, were significantly increased in abundance in co-cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co-cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co-culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co-cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.

The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.