The trajectory of microbial single-cell sequencing.

Over the past decade, it has become nearly routine to sequence genomes of individual microbial cells directly isolated from environmental samples ranging from deep-sea hydrothermal vents to insect guts, providing a powerful complement to shotgun metagenomics in microbial community studies. In this review, we address the technical aspects and challenges of single-cell genome sequencing and discuss some of the scientific endeavors that it has enabled. Specifically, we highlight newly added leaves and branches in the genomic tree of bacterial and archaeal life and illustrate the unique and exciting advantages that single-cell genomics offers over metagenomics, both now and in the near future.

Optimal intensity and biomass density for biofuel production in a thin-light-path photobioreactor.

Production of competitive microalgal biofuels requires development of high volumetric productivity photobioreactors (PBRs) capable of supporting high-density cultures. Maximal biomass density supported by the current PBRs is limited by nonuniform distribution of light as a result of self-shading effects. We recently developed a thin-light-path stacked photobioreactor with integrated slab waveguides that distributed light uniformly across the volume of the PBR. Here, we enhance the performance of the stacked waveguide photobioreactor (SW-PBR) by determining the optimal wavelength and intensity regime of the incident light. This enabled the SW-PBR to support high-density cultures, achieving a carrying capacity of OD730 20. Using a genetically modified algal strain capable of secreting ethylene, we improved ethylene production rates to 937 µg L(-1) h(-1). This represents a 4-fold improvement over a conventional flat-plate PBR. These results demonstrate the advantages of the SW-PBR design and provide the optimal operational parameters to maximize volumetric production.

In situ UV disinfection of a waveguide-based photobioreactor.

Compact waveguide-based photobioreactors with high surface area-to-volume ratios and optimum light-management strategies have been developed to achieve high volumetric productivities within algal cultures. The light-managing strategies have focused on optimizing sunlight collection, sunlight filtration, and light delivery throughout the entire bioreactor volume by using light-directing waveguides. In addition to delivering broad-spectrum or monochromatic light for algal growth, these systems present an opportunity for advances in photobioreactor disinfection by using germicidal ultraviolet (UV) light. Here, we investigated the efficacy of in situ, nonchemical UV treatment to disinfect a heterotrophic contaminant in a compact photobioreactor. We maintained a >99% pure culture of Synechocystis sp. PCC 6803 for an operating period exceeding 3 weeks following UV treatment of an intentionally contaminated waveguide photobioreactor. Without UV treatment, the culture became contaminated within only a few days (control). We developed a theoretical model to predict disinfection efficiency based on operational parameters and bioreactor geometry, and we verified it with experimental results to predict the disinfection efficiency of a Bacillus subtilis spore culture.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium.

BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

Nucleopolyhedrovirus detection and distribution in terrestrial, freshwater, and marine habitats of Appledore Island, Gulf of Maine.

Viruses in aquatic ecosystems comprise those produced by both autochthonous and allochthonous host taxa. However, there is little information on the diversity and abundance of viruses of allochthonous origin, particularly from non-anthropogenic sources, in freshwater and marine ecosystems. We investigated the presence of nucleopolyhedroviruses (NPV) (Baculovirus), which commonly infect terrestrial lepidopteran taxa, across the landscape of Appledore Island, Gulf of Maine. PCR and qPCR primers were developed around a 294-bp fragment of the polyhedrin (polH) gene, which is the major constituent protein of NPV multivirion polyhedral occlusion bodies. polH was successfully amplified from several aquatic habitats, and recovered polH sequences were most similar to known lepidopteran NPV. Using quantitative PCR designed around a cluster of detected sequences, we detected polH in Appledore Island soils, supratidal freshwater ponds, nearshore sediments, near- and offshore plankton, and in floatsam. This diverse set of locations suggests that NPVs are widely dispersed along the terrestrial--marine continuum and that free polyhedra may be washed into ponds and eventually to sea. The putative hosts of detected NPVs were webworms (Hyphantria sp.) which form dense nests in late summer on the dominant Appledore Island vegetation (Prunus virginiana). Our data indicate that viruses of terrestrial origin (i.e., allochthonous viruses) may be dispersed widely in coastal marine habitats. The dispersal of NPV polH and detection within offshore net plankton (>64 µm) demonstrates that terrestrial viruses may interact with larger particles and plankton of coastal marine ecosystem, which further suggests that viral genomic information may be transported between biomes.

Function-driven single-cell genomics uncovers cellulose-degrading bacteria from the rare biosphere.

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus 'Cellulosimonas argentiregionis'. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

Novel approaches in function-driven single-cell genomics.

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.

Analysis of single-cell genome sequences of bacteria and archaea.

Single-cell genome sequencing of individual archaeal and bacterial cells is a vital approach to decipher the genetic makeup of uncultured microorganisms. With this review, we describe single-cell genome analysis with a focus on the unique properties of single-cell sequence data and with emphasis on quality assessment and assurance.

Metabolic engineering of Rhodopseudomonas palustris for the obligate reduction of n-butyrate to n-butanol.

BACKGROUND: Rhodopseudomonas palustris is a versatile microbe that encounters an innate redox imbalance while growing photoheterotrophically with reduced substrates. The resulting excess in reducing equivalents, together with ATP from photosynthesis, could be utilized to drive a wide range of bioconversions. The objective of this study was to genetically modify R. palustris to provide a pathway to reduce n-butyrate into n-butanol for maintaining redox balance. RESULTS: Here, we constructed and expressed a plasmid-based pathway for n-butanol production from Clostridium acetobutylicum ATCC 824 in R. palustris. We maintained the environmental conditions in such a way that this pathway functioned as the obligate route to re-oxidize excess reducing equivalents, resulting in an innate selection pressure. The engineered strain of R. palustris grew under otherwise restrictive redox conditions and achieved concentrations of 1.5 mM n-butanol at a production rate of 0.03 g L-1 day-1 and a selectivity (i.e., products compared to the consumed substrate) of close to 40%. Since the theoretical maximum selectivity is 45%, the engineered strain converted close to its maximum selectivity. CONCLUSIONS: The innate redox imbalance of R. palustris can be used to drive the reduction of n-butyrate into n-butanol after expression of a plasmid-based enzyme from a butanol-producing Clostridium strain.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Single-Genotype Syntrophy by Rhodopseudomonas palustris Is Not a Strategy to Aid Redox Balance during Anaerobic Degradation of Lignin Monomers.

Rhodopseudomonas palustris has emerged as a model microbe for the anaerobic metabolism of p-coumarate, which is an aromatic compound and a primary component of lignin. However, under anaerobic conditions, R. palustris must actively eliminate excess reducing equivalents through a number of known strategies (e.g., CO2 fixation, H2 evolution) to avoid lethal redox imbalance. Others had hypothesized that to ease the burden of this redox imbalance, a clonal population of R. palustris could functionally differentiate into a pseudo-consortium. Within this pseudo-consortium, one sub-population would perform the aromatic moiety degradation into acetate, while the other sub-population would oxidize acetate, resulting in a single-genotype syntrophy through acetate sharing. Here, the objective was to test this hypothesis by utilizing microbial electrochemistry as a research tool with the extracellular-electron-transferring bacterium Geobacter sulfurreducens as a reporter strain replacing the hypothesized acetate-oxidizing sub-population. We used a 2 × 4 experimental design with pure cultures of R. palustris in serum bottles and co-cultures of R. palustris and G. sulfurreducens in bioelectrochemical systems. This experimental design included growth medium with and without bicarbonate to induce non-lethal and lethal redox imbalance conditions, respectively, in R. palustris. Finally, the design also included a mutant strain (NifA(*)) of R. palustris, which constitutively produces H2, to serve both as a positive control for metabolite secretion (H2) to G. sulfurreducens, and as a non-lethal redox control for without bicarbonate conditions. Our results demonstrate that acetate sharing between different sub-populations of R. palustris does not occur while degrading p-coumarate under either non-lethal or lethal redox imbalance conditions. This work highlights the strength of microbial electrochemistry as a tool for studying microbial syntrophy.

Stacked optical waveguide photobioreactor for high density algal cultures.

In this work, an ultracompact algal photobioreactor that alleviates the problem of non-optimal light distribution in current algae photobioreactor systems, by incorporating stacked layers of slab waveguides with embedded light scatterers, is presented. Poor light distribution in traditional photobioreactor systems, due to self-shading effects, is responsible for relatively low volumetric productivity. The optimal conditions for operating a 10-layer bioreactor are outlined. The bioreactor exhibits the ability to sustain uniform biomass growth throughout the bioreactor for 3 weeks, and demonstrates an 8-fold increase in biomass productivity. Using a genetically engineered algal strain, constant secreted ethylene production for over 45 days is also demonstrated. Since the stacked architecture leads to improved light distribution throughout the volume of the bioreactor, it reduces the need for culture mixing for optimum light distribution, and thereby potentially reducing operational costs.

An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system.

Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 µM arsenite with a linear range up to 100 µM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.

Slab waveguide photobioreactors for microalgae based biofuel production.

Microalgae are a promising feedstock for sustainable biofuel production. At present, however, there are a number of challenges that limit the economic viability of the process. Two of the major challenges are the non-uniform distribution of light in photobioreactors and the inefficiencies associated with traditional biomass processing. To address the latter limitation, a number of studies have demonstrated organisms that directly secrete fuels without requiring organism harvesting. In this paper, we demonstrate a novel optofluidic photobioreactor that can help address the light distribution challenge while being compatible with these chemical secreting organisms. Our approach is based on light delivery to surface bound photosynthetic organisms through the evanescent field of an optically excited slab waveguide. In addition to characterizing organism growth-rates in the system, we also show here, for the first time, that the photon usage efficiency of evanescent field illumination is comparable to the direct illumination used in traditional photobioreactors. We also show that the stackable nature of the slab waveguide approach could yield a 12-fold improvement in the volumetric productivity.

Quantitative correlation of absolute hydroxyl radical rate constants with non-isolated effluent organic matter bulk properties in water.

Absolute second-order rate constants for the reaction between the hydroxyl radical (*OH) and eight water samples containing non-isolated effluent organic matter (EfOM) collected at different wastewater and reclamation sites were measured by electron pulse radiolysis. The measured rate constants ranged from 0.27 to 1.21 x 10(9) Mc(-1) s(-1), with an average value of 0.86 (+/-0.35) x 10(9) Mc(-1) s(-1). These absolute values were 3-5 times faster than previously reported values using natural organic matter and wastewater isolates. The obtained rate constants were correlated (R2 > 0.99) to bulk EfOM properties through an empirical equation that included terms relating to the polarity, apparent molecular weight, and fluorescence index of the effluent organic matter. The obtained data were used to model steady state *OH concentrations during UV advanced oxidation. The steady-state *OH concentration was lower than that obtained using previously reported values for the reaction with dissolved organic matter, indicating that accurate measurement of reaction rate constants at specific sites would greatly improve the design and prediction of the removal of organic contaminants. These results will improve the ability of researchers to accurately model scavenging capacities during the advanced oxidation processtreatment of wastewaters.