Agrobacterial Transformation Enhancement by Improved Competent Cell Preparation and Optimized Electroporation.

The introduction of plasmids into Agrobacterium cells is one of the key steps in the Agrobacterium-mediated transformation of plants for gene editing applications. Depending on chromosomal background, some Agrobacterium strains exhibit a very low transformation efficiency, which results in a low number of colonies for subsequent screening and thus limits the potential for automated high-throughput transformation processes, especially with low copy or large plasmids. This study demonstrates improvements of transformation frequency by modifying the competent cell preparation process and optimizing electroporation parameters for two Agrobacterium strains. The competent cell preparation process was modified by prolonging bacterial growth in the log phase and optimizing the endpoint cell density for cell harvest which resulted in a significant cell yield increase and transformation frequency improvement. Optimization of electroporation by fine-tuning the parameters not only resulted in a 30-fold transformation frequency increase but also revealed a strain-dependent requirement for field strength and electric pulse length. To further improve transformation of a recalcitrant strain, different concentrations of dimethyl sulfoxide (DMSO) in recovery medium were examined. The study revealed an important role of DMSO in transformed cell recovery, with 5% DMSO resulting in the highest transformation frequency. The significant improvements in Agrobacterium transformation frequency addressed a critical bottleneck towards establishing a high throughput process.

Shifting the balance of fermentation products between hydrogen and volatile fatty acids: microbial community structure and function.

Fermentation is a key process in many anaerobic environments. Varying the concentration of electron donor fed to a fermenting community is known to shift the distribution of products between hydrogen, fatty acids and alcohols. Work to date has focused mainly on the fermentation of glucose, and how the microbial community structure is affected has not been explored. We fed ethanol, lactate, glucose, sucrose or molasses at 100 me- eq. L-1, 200 me- eq. L-1 or 400 me- eq. L-1 to batch-fed cultures with fermenting, methanogenic communities. In communities fed high concentrations of electron donor, the fraction of electrons channeled to methane decreased, from 34% to 6%, while the fraction of electrons channeled to short chain fatty acids increased, from 52% to 82%, averaged across all electron donors. Ethanol-fed cultures did not produce propionate, but did show an increase in electrons directed to acetate as initial ethanol concentration increased. In glucose, sucrose, molasses and lactate-fed cultures, propionate accumulation co-occurred with known propionate producing organisms. Overall, microbial communities were determined by the substrate provided, rather than its initial concentration, indicating that a change in community function, rather than community structure, is responsible for shifts in the fermentation products produced.

Combining microbial cultures for efficient production of electricity from butyrate in a microbial electrochemical cell.

Butyrate is an important product of anaerobic fermentation; however, it is not directly used by characterized strains of the highly efficient anode respiring bacteria (ARB) Geobacter sulfurreducens in microbial electrochemical cells. By combining a butyrate-oxidizing community with a Geobacter rich culture, we generated a microbial community which outperformed many naturally derived communities found in the literature for current production from butyrate and rivaled the highest performing natural cultures in terms of current density (∼ 11A/m(2)) and Coulombic efficiency (∼ 70%). Microbial community analyses support the shift in the microbial community from one lacking efficient ARB in the marine hydrothermal vent community to a community consisting of ∼ 80% Geobacter in the anode biofilm. This demonstrates the successful production and adaptation of a novel microbial culture for generating electrical current from butyrate with high current density and high Coulombic efficiency, by combining two mixed microbial cultures containing complementing biochemical pathways.

Selective enrichment yields robust ethene-producing dechlorinating cultures from microcosms stalled at cis-dichloroethene.

Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 10(9) Dehalococcoides mccartyi cells mL(-1). Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H2. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H2 for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.

Enrichment and analysis of anode-respiring bacteria from diverse anaerobic inocula.

One of the limitations currently faced by microbial electrochemical cell (MXC) technologies lies in the shortage of different organisms capable of forming a biofilm and channeling electrons from substrates to the anode at high current densities. Using a poised anode (-0.30 V vs Ag/AgCl) and acetate as the electron donor in a MXC, we demonstrated the presence of highly efficient anode-respiring bacteria (ARB) able to produce high current densities (>1.5 A/m(2) anode) in seven out of thirteen environmental samples. These included marshes, lake sediments, saline microbial mats, and anaerobic soils obtained from geographically diverse locations. Our microbial ecology analysis, using pyrosequencing, shows that bacteria related to the genus Geobacter, a known and commonly found ARB, dominate only two of the biofilm communities producing high current; other biofilm communities contained different known and/or novel ARB. The presence of ARB in geographically diverse locations indicates that ARB thrive in a wide range of ecosystems. Studying ARB from different environmental conditions will allow us to better understand the ubiquity of anode respiration, compare the capabilities of different ARB consortia, and find ARB with useful metabolic capacities for future applications.