Environmental Microbiology and Biotechnology
The following is a list of currently active externally funded research projects:

Biochemistry and Genetics of Anaerobic Oxidation of Aromatic Hydrocarbons - Alfred Spormann (NSF)
Oxidation of aromatic hydrocarbons, such as benzene, toluene, ethylbenzene, and xylenes (BTEX) by anaerobic bacteria is of considerable biochemical and environmental interest. The enzymes involved in the initial degradation reactions of these substrates are dramatically different from the well-characterized aerobic oxidation reactions: In all aerobic pathways, the initial reactions involve mono- or dioxygenases that require molecular oxygen as a substrate. Anaerobic bacteria face the biochemical problem of how to perform the first oxidation step in the absence of molecular oxygen.

From an environmental point of view, degradation of these aromatic hydrocarbons is important, because they represent major components of the water soluble fraction of gasoline fuel. Leaking underground fuel storage tanks or surface spills often release these compounds into aquifers. Through research in the recent years, two fundamentally different modes to initiate anaerobic mineralization of alkylbenzenes are emerging: Methyl benzenes are activated by addition of the methyl groyup to fumarate, and benzylsuccinate synthase represents the prototype of this reaction. Alkylbenzenes with side chain lengths of * 2 carbons are anaerobically oxidized at the methylene carbon for which ethylbenzene dehydrogenase serves as the prototype.

One focus of our research is the novel enzyme benzylsuccinate synthase which catalyzes as the initial reaction in anaerobic activation of toluene the addition of the toluene methyl group to fumarate to form benzylsuccinate:
http://www-ce.Stanford.EDU:80/~spormann/Bss%20reaction.JPG

This is a unique non-redox reaction to activate methyl benzenes and also a novel mode to form a new carbon-carbon bond. This enzyme is of considerable interest because it carries an organic free glycyl radical. Biochemical and molecular studies are directed to elucidate the reaction mechanism. A similar reaction is involved in anaerobic activation of m-xylene.

The other focus is on anaerobic ethylbenzene dehydrogenase which catalyzes the first reaction in anaerobic ethylbenzene mineralization. Ethlbenzene dehydrogenase is an unusual enzyme because it catalyzes the oxidation of a hydrocarbon in the absence of molecular oxygen and converts ethylbenzene to 1-phenylethanol with p-benzoquinone as electron acceptor. http://www-ce.Stanford.EDU:80/~spormann/EBreactions.JPG

Biochemical studies are conducted with purified enzymes, and gas chromatography (GC), gas chromatography/ mass spectrometry (GC/MS), HPLC, and spectrophotometry assist in analyzing these unusual reactions.

A parallel genetic approach is directed to identify the structural genes involved and to understand the molecular mechanisms of their control. The current focus of the genetic approach is to generate mutants defective in aromatic hydrocarbon metabolism, and to isolate and to clone the genes involved by genetic complementation.

Anaerobic trnasformation of chloroethenes and evolution of pathways for transformation of "unnatural" compounds. - Alfred Spormann (SERDP)
Next to the BTEX compounds, chlorinated alkenes, such as tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE), and vinyl chloride (VC) are another important class of groundwater contaminants in the U.S.. These chlorinated aliphatic compounds are exclusively of anthropogenic origin and have been introduced involuntarily into the environment only within the last decades. Chlorinated aliphatic solvents are frequently used for the degreasing of engines and in dry-cleaning processes. PCE and TCE, but more importantly VC, are highly toxic compounds, and their concentrations in drinking water are regulated. Interestingly, under anaerobic conditions PCE and TCE are metabolized to VC, and, thus, are converted into a product that is more toxic than the parent compound. From a fundamental point of view, the microbial transformation of these compounds is very interesting because these unnatural chemicals were introduced into the environment only a few decades ago. Nevertheless, pathways that metabolize these compounds have evolved in some organisms. Thus, research on these novel microbes that can completely degrade these unnatural compounds allows us to address the more fundamental question of how novel pathways for the degradation of unnatural compounds evolve in nature.
Recently, we discovered a novel mode of anaerobic reductive vinyl chloride transformation to ethylene. Previously known reductive dehalogenation of PCE, TCE, or chlorobenzoates were shown to involve corrinoids as the catalytically active reagent. Through inhibitor studies with cell-free extracts from an anaerobic, VC-dehalogenating mixed culture, we discovered that VC reduction to ethene does not involve a corrinoid. Current, work investigates the microbial and molecular mechanism of this novel enzymatic activity. Understanding the fundamentals of natural dehalogenation of unnatural compounds will provide crucial knowledge that will allow also the development of novel in situ strategies to specifically target biodegradation of VC to this microbial mechanism. Furthermore, understanding this mechanism may also provide tools for engineering a chemical remediation scheme, as has been done using vitamin B12-based chemical reductive dehalogenation of PCE and TCE that was first discovered as a prokaryotic transformation reaction.

In addition, a novel research approach explores the in vitro engineering of enzymes with novel catalytic properties for the transformation of recalcitrant chemicals by use of directed evolution.

Functional Genomics and Physiology of Microbes in Biofilms - Alfred Spormann, Gordon E. Brown, Jr. (NSF)
Microbial biofilms are structures where microbes are attached to a surface and embedded in an extracellular polysaccharide (EPS) matrix. Microbial biofilms form on virtually all aqueous environmental interfaces, including the surfaces of ships, pipes, and sewers, and on soil mineral particles. Biofilms are of enormous economic, medical, and biogeochemical importance. Our work is at the interface of molecular microbiology, molecular microbial ecology, and biogeochemistry.

Biofilms are dynamic structures and undergo a developmental process consisting of (i) initial attachment, (ii) microcolony formation, (iii) maturation, and (iv) detachment. Because cells are constrained in a biofilm and because free diffusion of small molecules is affected within a biofilm, these bioflms exhibit a high degree of internal heterogeneity, where the overall biological activity of a biofilm is controlled by an intricate structure-function relationship. Therefore, individual cells, as well as the overall microbial community, respond to changes in the environment. Thus, the activity of a microbe within a biofilm is a function of its position in the biofilm.

We are studying the cell-cell and cell-substratum intractions using a new genomic approach in conjunction with confocal laser scanning microscopy (CLSM). Our goal is to understand and to predict the complex metabolic and signaling interactions that occur between biofilm microbes. We use two model microbes, Shewanella oneidensis MR-1 and Vibrio cholerae. The genome of both microorganisms has been sequenced, and we are using DNA microarrays to generate profiles of whole cell gene expression during cell-cell and cell-surface interactions. CLSM is a powerful technology to visualize individual microbial cells in biofilms and to visualize the expression of individual genes in biofilm cells. This allows us to observe directly metabolic and signaling cell-cell interactions in active biofilms. This CLSM work is conducted here at the new Stanford Biofilm Research Center.
S. oneidensisutilizes the surface of insoluble Fe(III) minerals as electron acceptor and forms biofilms on such minerals. The reduced Fe(II) minerals, in turn, are critically important for geochemical redox reactions involved in abiotic heavy metal mobilization and reductive degradation reactions. We are specifically interested in the molecular processes that control S. oneidensis biofilm formation and stability, and that are involved in redox reactions between biofilm cells and the mineral subtratum.

V. cholerae forms biofilms on inorganic and organic, including chitin, surfaces. It is believed that chitin provides a critical metabolizable substrate for this microbe to persistent in marine environments. Also here, we are using a genomic approach to uncover cell-cell and cell-substratum interactions of V. cholerae in marine environments and on chitin surfaces.

This project is part of an interdisciplinary NSF-funded research effort (CREAMS) on "Chemical and Microbial Interactions on Environmental Surfaces"

Reductive processes for the bioremediation of chlorinated solvent metal mixtures - Craig Criddle, Alfred Spormann (NIEHS).
Mixtures of chlorinated solvenets and metals pose a significant challenge for bioremediation. This work focuses on the transfer of the the carbon tetrachloride degradation genes encoding production of pyridine-2,6-bis(thiocarboxylate) (PDTC) from Pseudomonas stutzeri strain KC to Shewanella oneidensis MR1 to create an organism with increased metal tolerance and the ability to dechlorinate carbon tetrachloride.

Field-scale evaluation of biostimulation for remediation of uranium-contaminated groundwater - Craig Criddle, Peter Kitanidis, and Gary Hopkins (U.S. DOE).
Microbial reduction of uranium may prevent its migration to receptor streams. However, application of this technology to field sites is untested, and future site remediation will require improved understanding of basic processes and implementation strategies in heterogeneous environments. The goals of this work are (1) to develop a predictive capability for the rates and mechanisms controlling microbial reduction of U in heterogeneous field settings, and (2) to develop a system capable of delivering electron donor to a highly heterogeneous subsurface environment enabling spatially uniform in situ immobilization of U in groundwater upon passage through a subsurface biocurtain. This work involves a 3-phase field study in a near surface aquifer at Oak Ridge, TN. This aquifer contains very high levels of nitrate and part per million levels of U(VI). The nitrate must be removed because it prevents reduction of U, and, if the nitrate is reduced to N2 the resulting gas could reduce aquifer permeability. We will test in-situ concepts for nitrate removal: an in-well vacuum stripper; an in-well bioreactor; and ion selective resins. The most effective and least expensive system will be coupled to a system for in-situ uranium removal. By removing the nitrate, we will be able to impose hydrological and geochemical controls on the U source permitting reliable determination of U reduction rates within a downgradient biocurtain for U immobilization. We plan to use field-scale and companion bench-scale studies to evaluate hypotheses on dissimilatory metal-reducing activity, and we will be monitoring changes in microbial community dynamics using molecular methods.

Proof of gene expression during bioaugmentation - Craig Criddle (WRHSRC, EPA)
Experimental justification for bioaugmentation is typically obtained by comparing the bioremediation of inoculated and uninoculated samples. This approach is adequate for bench-scale studies. At full scale, however, design and operation of uninoculated controls is difficult and expensive. Inadvertent inoculation of "uninoculated" regions must be avoided, and the inoculated and uninoculated regions must initially be geologically, chemically, and biologically similar. Other methods, besides the use of uninoculated control regions, are needed to establish that added organisms are in fact mediating the desired transformations. A logical approach is to prove expression of the genes required for the desired transformation. Gene expression occurs at different levels as the synthesis of mRNA (transcription), the formation of polypeptides (translation), and the biochemical reaction itself. Proof of gene expression is best obtained at each level, because each piece of evidence strengthens the conclusion that gene expression is occurring as intended. This proposal explores each level of gene expression for the bioremediation of carbon tetrachloride by Pseudomonas stutzeri KC.

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