Devin F. R. Doud

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.

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.

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.

In situ hollow fiber membrane facilitated CO2 delivery to a cyanobacterium for enhanced productivity

Recently, cyanobacteria have been metabolically engineered to secrete valuable biofuel precursors eliminating the requirement to harvest and post-process algal biomass. However, development of new photobioreactors (PBRs) that can efficiently deliver light and address the mass transport challenges associated with maintaining high cyanobacteria productivity has been lagging. Hollow fiber membranes (HFMs) are a method for bubble-less gas exchange which has been shown to be effective at enhancing mass transfer. Previous applications of HFM technology to PBRs have been limited to exploring its ability to enhance CO2 delivery to the bulk liquid volume. To investigate potential strategies for novel PBR design configurations, we examined the growth pattern of Synechococcus elongatus around individual HFMs to determine the optimal spacing and conditions for maximizing photosynthetic activity. We have shown that a single fiber enabling passive transport from/to the atmosphere can provide enough gas exchange to increase biomass accumulation by >2.5 times with respect to a non-fiber control. This increased growth was found to decay in the radial direction with the enhanced growth area spanning between 1.2 mm and 1.7 mm depending on the initial inoculation concentration.

Hollow fibre membrane arrays for CO2 delivery in microalgae photobioreactors

Microalgae can serve as a carbon sink for CO2 sequestration and as a feedstock for liquid biofuel production. Methods for microalgal biomass and biofuel cultivation are progressing, but are still limited in the efficiency of light delivery and gas exchange within cultures. Specifically, current gas exchange methods are very energy intensive since they rely on mixing algal cultures at high flow rates. One method that can improve gas exchange within photobioreactors without excessive mixing is the use of hollow fibre membranes, which enable simultaneous transport of gases deep into the reactor and rapid exchange with the culture media. Here we demonstrate the optimal geometric and operational conditions for CO2 transport to planar cultures of Synechococcus elongatus via hollow fibre membrane arrays. Specifically, we investigated the effects of inter-fibre spacing and active/passive aeration on the growth rate, planar surface density, and total biomass accumulation. We show that spacing in excess of 3 times the fibre diameter lead to significant variations in the uniformity of the surface density and spatially resolved growth rate, whereas spacing of 3 times the fibre diameter supported culture surface densities nearing 90%, which were maintained for 17 days without decreasing. Active aeration with the fibres showed an increase in the specific growth rate and the average surface density with respect to passive aeration by approximately 15% and 35%, respectively, while also eliminating gradients in localized growth rates along the length of the fibres.

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.

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.

Novel approach in algae biofuel production using advanced photonics

Utilization of evanescent fields for photosynthetic bacteria to produce biofuel can lead to thousandfold decrease in the volume of photobioreactors. We demonstrate the growth of bacteria enabled by the evanescent excitation using planar waveguides.