Michael Hyman

Many critical processes depend on metalloenzymes, and scarcity of the trace metals required for these enzymes may limit their activity, thus causing potential bottlenecks biogeochemical cycles. A recent revision to the microbial tree of life has revealed widespread and abundant soil bacteria that produce lanthanum-dependent methanol dehydrogenase, an enzyme potentially important in their metabolism and the cycling of carbon in soil. This exciting discovery further expands the periodic table of life and raises many questions about the biogeochemistry of lanthanum and other rare earth elements (REYs). Our research project���s central assertion is that microbes���specifically those utilizing REY-dependent methanol dehydrogenase���will require a specific REY uptake strategy, akin to other biologically necessary trace metals. We propose to utilize cutting-edge coordination, soil, and analytical chemistry approaches to identify and characterize ligands that promote solubilization and binding of REYs from soils. The successful completion of the project will result in a transformative new paradigm for the transport of REYs in biological systems, and may provide significant advance in other related fields.

This project has two aims: First, we propose to develop a molecular biological tool (MBT) using activity based labeling (ABL) that can associate biomarkers such as monooxygenases with the biotransformation rate of key per��� and polyfluoroalkyl substances (PFAS) precursors under conditions relevant to the aqueous film��� forming foam (AFFF)���impacted sites. Second, we propose to assess the extent of sequestration of end products from precursors biotransformation into biomass and better understand the environmental fate of PFAS precursors.

The overall aim of this project is to evaluate the use of aerobic alkane-oxidizing bacteria for the in situ cometabolic degradation of 1,4-dioxane (14D). The project will involve testing field samples for the stimulation of either indigenous gaseous alkane and alkyne-metabolizing bacteria, testing the potential for bioaugmentation of field samples and detecting the presence of active alkane-oxidizing bacteria in field samples using activity-based labeling of catalytically active monooxygenases.

The alkane-oxidizing bacterium Rhodococcus rhodochrous ATCC 21198 can degrade 1,4-dioxane (14D) at high rates for over 300 days when it is co-encapsulated in gellan gum with orthosilicate slow release compounds (SRCs) that hydrolyze to produce alcohols. The overall goal of this project is to further develop this co-encapsulation technology for passive and sustainable aerobic cometabolic systems for the treatment of emerging contaminants such as N-nitrosodimethylamine (NDMA), 1,2,3-trichloropropane (TCP), as well as important contaminant mixtures such as 14D and its common co-contaminants, 1,1,1 trichloroethane and cis-dichloroethene.The contaminant-degrading activity of ATCC 21198 is due to non-specific, alkane-inducible monooxygenases that normally function to initiate alkane catabolism. In the absence of alkanes, the factors that control expression of these monooxygenases and enable sustained contaminant degradation are unknown but are key to further developing the co-encapsulation technology. Activity measurements, activity-based monooxygenase labeling and whole cell proteome analyses will be used to separately characterize the effects of alcohols, SRCs, starvation, encapsulation, and non-growth supporting contaminants on expression of monooxygenases and other enzymes in strain ATCC 21198. The physiological and enzymatic changes that occur the 300-day life cycle of this strain when co-encapsulated with model SRCs will also be determined. Similar genome-enabled approaches will also be used to identify other pure cultures with alternative monooxygenase complements that can sustainably degrade chlorinated ethenes (trichloroethene, vinyl chloride and 1,1-dichloroethene), and emerging contaminants (NDMA, and TCP) when co-encapsulated with SRCs. The activities of the co-encapsulated strains will be tested in batch reactors containing beads with single cultures and SRC as well as bead mixtures with different cultures and SRCs.

The project objective is to use apply a recently-developed activity-based labeling (ABL) technique to detect, and identify contaminant-degrading monooxygenase enzymes expressed by native or augmented microorganisms. The 2-step technique involves an initial inactivation of target enzymes using diyne probes with subsequent use of copper-catalyzed alkyne/Azide cycloaddition (CuAAC) reaction which generates a fluorescent protein adduct. This adduct can be detected and quantified by a number of different analytical approaches such as SDS-PAGE, microscopy, and flow cytometry. The technical objective of the proposed work will be to use ABL approaches to support a concurrent but separately funded study that aims to demonstrate that in situ aerobic cometabolic treatment of dilute plumes of chlorinated volatile organic compounds (CVOC)s can be achieved using bacteria grown on substrates including 2-butanol and benzyl alcohol. The ABL technique is expected to provide data that will enable the accompanying project to confirm the role of specific monooxygenases in in situ contaminant biodegradation and to determine the abundance of bacteria expressing these specific monooxygenases.