Dr. Lisa A Waidner

Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H2 production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H2 production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H2 production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.
•Chimeric algal hydrogenase gene mutants were created from three genes of two microalgal species
•Swapping gene segments resulted in some constructs with improved hydrogen production
•Sequence and electrostatic potential models may help predict improved function with mutation

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are water- and foodborne bacteria that can cause several distinct human diseases, collectively called vibriosis. The success of oyster aquaculture is negatively impacted by high Vibrio abundances. Myriad environmental factors affect the distribution of pathogenic Vibrio, including temperature, salinity, eutrophication, extreme weather events, and plankton loads, including harmful algal blooms. In this paper, we synthesize the current understanding of ecological drivers of Vv and Vp and provide a summary of various tools used to enumerate Vv and Vp in a variety of environments and environmental samples. We also highlight the limitations and benefits of each of the measurement tools and propose example alternative tools for more specific enumeration of pathogenic Vv and Vp. Improvement of molecular methods can tighten better predictive models that are potentially important for mitigation in more controlled environments such as aquaculture.

The increased potential for contamination of seawater by crude oils requires studies of bacterial biodegradation potential, but little is known of the differential negative impacts of oils on bacterial growth. No two wells generate chemically identical oils; and importantly, solar exposure of crude oil may differentially affect the bacterial response. Elucidating the role that sunlight plays on the potential toxicity of spilled crude oils is imperative to understanding how oil spills might affect microbes in the tropical and subtropical waters of Florida. This study examined light exposure of six different crude oils, and subsequent microbial responses to altered oils. Marine bacterioplankton heterotrophic activities were measured via3H-leucine incorporation after the addition of oils’ water accommodated fractions (WAFs) that were created under varied solar conditions. Inhibition of production increased with higher concentrations of WAFs, but dose-response trends varied among the oils. Increased solar exposure during WAF preparation generally led to more inhibition, but trends varied among oils. WAFs were also prepared under different parts of the solar spectrum. Solar-irradiated WAFs resulted in significant but variable acute toxicity vs. dark counterparts. Solar-induced toxicity was primarily a result of visible and not ultraviolet light exposure. Results indicate responses to oil spills are highly dependent on the source of the oil and solar conditions at the time and location of the spill. The data presented here demonstrate the importance of photochemical changes and oil source in modulating microbial activity and bioremediation potential.

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component
nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to β-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.

Vibriosis is the general term for human illnesses caused by infection of pathogenic Vibrio species. Vibrio vulnificus (Vv) and parahaemolyticus (Vp) are two problematic waterborne pathogens that have yet to be enumerated in northwest Florida coastal Gulf of Mexico estuaries. In this regionally novel study, we surveyed 43 locations in two subtropical estuarine systems, Perdido Bay and Pensacola Bay, over seven dates in winter 2020. Sampling included three substrate types: surface waters, sediments, and invertebrate biofilms. We determined baseline abundances of presumptive viable Vv and Vp appearing as colonies on CHROMagar (Vv, blue; Vp, purple). Vv was detected in 37 out of 43 water samples, with maximum levels of 3,556 CFU/mL. Vp was only detected in 15 water samples, with a maximum concentration of 8,919 CFU/mL. Sediments contained Vv in all but one sample, with concentrations ranging from 121 to 607,222 CFU/mL. In contrast, Vp were only detected in 33 sediment samples, where concentrations ranged from 28 to 77,333 CFU/mL. Opportunistically-sampled surface swabs (biofilms), collected from shells (either oyster or barnacle) and polychaete worms found in sediment samples, contained on average 7,735 and 1,490 CFU/mL of Vv and Vp, respectively. Surface water Vv abundances covaried with bottom water pH, maximum prior cumulative wind speeds, and tidal coefficient on the day of sampling. Vp surface water abundances negatively correlated with surface water salinity, surface water pH, and bottom water pH and positively correlated with total surface dissolved inorganic and total Kjeldahl nitrogen concentrations, and wind. Spatially, there was large variation in Vibrio densities in surface waters; abundances of both species were strongly correlated with wind, suggesting resuspension was important. Sedimentary abundances of both putative Vv and Vp shared a correlation with one parameter: salinity stratification. Due to the length of this study, temperature was not considered a major factor. This short-term (one month) study was designed not to enumerate pathogenic Vv or Vp, but rather to establish the first winter baseline of Vibrio abundances for this region. Determination of these baseline winter cultivable putative Vibrio abundances will be valuable in predicting relative risk factors in each waterbody of interest.

Artificial reefs have been deployed throughout US coastal waters since the late 1970s, primarily to enhance fisheries. Although numerous studies have examined their effects on fish communities, few have examined interactions between artificial reefs and primary producers or their effects on biogeochemistry of the surrounding water column. Understanding how reefs may alter biogeochemistry and primary producers is key to understanding overall reef productivity. In this study, we examined the relationships among epifauna, algae, and biogeochemical processes on artificial reefs located on the shallow Florida shelf in the Northeast Gulf of Mexico over a year following their deployment. We measured oxygen and nutrient fluxes, attached chlorophyll a, and invertebrate macrofauna. Temporal differences in biomass and chlorophyll a production occurred due to changes in in situ conditions including fluctuations in bottom-water temperature over the year as well as decreasing bottomwater oxygen and increasing chlorophyll a fluorescence during the summer. Invertebrate biomass was greater than micro- or macroalgal biomass. Biomass of the invertebrate epifaunal community increased exponentially during the first 5 months of this study. The reef was net heterotrophic with few differences between oxygen or nutrient fluxes in the light and dark. Positive nitrate
and nitrite fluxes and abundances of amoA genes in the microbiomes of benthic invertebrates indicate significant nitrification associated with the epifaunal community. Reef biogeochemistry was directly related to the composition and biomass of the epifaunal community at the reef sites.

3-Nitro-1,2,4-triazol-5-one (NTO) is one of the main ingredients of many insensitive munitions, which are being used as replacements for conventional explosives. As its use becomes widespread, more research is needed to assess its environmental fate. Previous studies have shown that NTO is biologically reduced to 3-amino-1,2,4-triazol-5-one (ATO). However, the final degradation products of ATO are still unknown. We have studied the aerobic degradation of ATO by enrichment cultures derived from the soil. After multiple transfers, ATO degradation was monitored in closed bottles through measurements of inorganic carbon and nitrogen species. The results indicate that the members of the enrichment culture utilize ATO as the sole source of carbon and nitrogen. As ATO was mineralized to CO₂, N₂, and NH₄⁺, microbial growth was observed in the culture. Co-substrates addition did not increase the ATO degradation rate. Quantitative polymerase chain reaction analysis revealed that the organisms that enriched using ATO as carbon and nitrogen source were Terrimonas spp., Ramlibacter-related spp., Mesorhizobium spp., Hydrogenophaga spp., Ralstonia spp., Pseudomonas spp., Ectothiorhodospiraceae, and Sphingopyxis. This is the first study to report the complete mineralization of ATO by soil microorganisms, expanding our understanding of natural attenuation and bioremediation of the explosive NTO.

Dichloronitrobenzenes (DCNB) are intermediates in the production of dichloroanilines, which are key feedstocks for synthesis of diuron and other herbicides. Although DCNB is a major contaminant at certain chemical manufacturing sites, aerobic DCNB biodegradation is poorly understood and such sites have not been candidates for bioremediation. When a bench-scale aerobic fluidized- bed bioreactor was inoculated with samples from a DCNB contaminated site in Brazil 2,3-DCNB, 3,4-DCNB, 1,2-dichlorobenzene (o-DCB), and chlorobenzene (CB) were biodegraded simultaneously. Biodegradation of the mixture was complete even when the reactor was operated at high flow rates (1.6 h hydraulic residence time), and bacteria able to degrade the individual contaminants were isolated from the reactor by selective enrichment. The enrichments yielded 2 strains of bacteria able to degrade 3,4-DCNB and one able to degrade 2,3-DCNB. The isolates released nitrite during growth on the respective DCNB isomers under aerobic conditions. The draft genome sequence of Diaphorobacter sp. JS3050, which grew on 3,4- DCNB, revealed the presence of putative nitroarene dioxygenase genes, which is consistent with initial attack by a dioxygenase analogous to the initial steps in degradation of nitrobenzene and dinitrotoluenes. The results indicate clearly that the DCNB isomers are biodegradable under aerobic conditions and thus are candidates for natural attenuation/bioremediation.