Alfred M. Spormann - US grants

9723312 Spormann This research will investigate novel enzymes catalyzing the first step in anaerobic degradation of aromatic hydrocarbons. Recent work showed that the inital step in degradation of the methyl-benzenes toluene and m-xylene is the addition of the methyl carbon to the double bond of fumarate. Benzylsuccinate synthase, which catalyzes the addition of toluene to fumarate, will be studied as the prototype for this reaction. This enzymatic addition represents a novel biochemical reaction to activate aromatic hydrocarbons. Further, the activity may also define a novel class of enzymes that catalyze the formation of carbon-carbon bonds by a heretofore unknown reaction mechanism. Benzylsuccinate synthases may be of primary importance in anaerobic mineralization pathways in a range of different bacterial species. Research in this proposals directed towards gaining a better understanding of these unusual enzymatic reactions. This project will include purification of benzylsuccinate synthase and investigation of the enzyme's reaction mechanism. In addition to characterizing a novel class of enzymes, the results of this research will have important implications for our understanding of anaerobic pathways used by petroleum hydrocarbon-mineralizing bacteria. Because of the absence of molecular oxygen under typical conditions in fuel-contaminated aquifers, anaerobic bacteria capable of degrading petroleum hydrocarbons, such as toluene and the xylene isomers, are of key importance. Investigations of the key enzymes of these pathways may lead to the development of molecular tools that may assist in monitoring file abundance and activity of relevant microbes in situ. This research will investigate novel enzymes catalyzing the first step in anaerobic degradation of aromatic hydrocarbons. These compounds are significant environmental pollutants. Until recently it was thought that degradation of such compounds required oxygen. Recent work showed that the inital step in degradation of toluene and m-xylene, novel. In addition to characterizing this novel class of enzymes, the results of this research will have important implications for our understanding of anaerobic pathways used by petroleum hydrocarbon-mineralizing bacteria. Because of the absence of molecular oxygen under typical conditions in fuel-contaminated aquifers, anaerobic bacteria capable of degrading petroleum hydrocarbons, such as toluene and the xylene isomers, are of key importance. Investigations of the key enzymes of these pathways may lead to the development of molecular tools that may assist in monitoring the abundance and activity of relevant microbes in situ, and aid in bioremediation.

9733535 Spormann This project will investigate the biochemical and molecular mechanisms of aromatic hydrocarbon degradation by anaerobic bacteria. Current work in this research group has revealed several novel biochemical reactions involved in the initial steps of anaerobic degradation of toluene, xylenes, and ethylbenzene. Catabolic pathways for these compounds will be established and key enzymes of the novel pathways will be characterized. A genetic analysis of the pathways will be initiated to understand how these catabolic genes are regulated and which environmental factors control their expression. Further, it is proposed to isolate novel prokaryotes that degrade aromatic hydrocarbons under sulfidogenic and methanogenic conditions. BTEX (benzene, toluene, ethylbenzene, xylenes) compounds are among the most water-soluble components of gasoline and are frequently released in the environment by spills and improper handling. Contaminated sites often become anaerobic. Investigating anaerobic BTEX degradation in terms of the biochemistry involved, the molecular mechanisms that regulate expression of catabolic genes, and the microbial ecology of the hydrocarbon-degrading organisms will advance our fundamental knowledge of microbial metabolism, and will also be of practical use for evaluating and enhancing intrinsic bioremediation. The teaching activities are integrated with the research activities and have related focus. An Environmental Microbiology curriculum is currently being established to convey a comprehensive understanding of microbial activities in nature. An emphasis will be the mutual interactions between microorganisms and their environment, how microbial activities control the physical-chemical properties of an environment, and in turn, how this affects microbial activities, ranging from the elemental cycles of carbon, nitrogen, sulfur, oxygen and iron to microbial metabolism of compounds of geochemical and of anthropogenic origin.

Brown/Sporman
9905755
Microbial biofilms are common coatings on mineral surfaces in many environments and are known to sorb metal ions such as Pb(II). However, little is currently known about the chemical form(s) of these metal ions at a molecular level, their spatial distribution in the biofilm, and the mechanisms by which the biofilm affects metal ion sorption on mineral surfaces. This multidisciplinary project will address these and related issues through laboratory studies of Pb(II) sorption on biofilm-coated surfaces of aluminum and iron (hydr)oxides and quartz as a function of several solution variables. The molecular-scale speciation and spatial distribution of Pb(II) in biofilms grown on these surfaces will be studied using synchrotron-based x-ray and electron microscopy methods. In addition, we will use infrared spectroscopy to study the effects on Pb(II) uptake of the common inorganic ligand PO43- and several organic ligands that are dominant functional groups on bacterial surfaces and biofilm exopolysaccharides. Burkholderia cepacia, an aerobic gram-negative bacterium that readily forms biofilms under nutrient-limited conditions, was chosen for the initial phases of this work. Our choice of system variables will be guided by the results of our ongoing studies of Pb speciation in several Pb-contaminated tailings at Leadville, CO and soils in northern France. The role of extracellular phosphate and of intracellular ortho- and polyphosphate metabolism in the biofilm in producing Pb-phosphates will be explored. Our approach starts with relatively simplified model systems and systematically adds some of the complexities of natural systems. The ultimate goal of this work is to develop a fundamental understanding of some of the factors that control the geochemical cycling (and potential bioavailability) of Pb and other metals in aquatic environments.

This proposal was submitted in response to the Environmental Geochemistry and Biogeochemistry solicitation NSF 99-9, and is being funded jointly by the Divisions of Chemistry, Earth Sciences, Ocean Sciences, and MPS Office of Multidisciplinary Activities.

Microbial enzymes, specifically those of anaerobic microbes, are receiving significant attention because they provide an invaluable source for diverse natural catalysts. Of particular interest are those enzymes that catalyze the transformation of relatively unreactive organic compounds. Methyl-substituted benzenes, such as toluene, xylenes, and trimethylbenzenes, are relatively stable and represent important drinking water contaminants that are released into the environment from leaking underground gasoline storage tanks and surface spills. Several diverse, anaerobic, toluene--mineralizing microbes have been isolated and studied in more detail to gain insight into the mechanism of anaerobic activation of toluene. These studies showed that the initial reaction of anaerobic toluene metabolism is the addition of the toluene methyl group to fumarate to form benzylsuccinate. Benzylsuccinate synthase (BSS), which catalyzes this reaction, is emerging as a prototype of a new class of enzymes for activation of methylbenzenes. Recent studies strongly suggest that BSS is a radical enzyme, which carries an essential glycyl radical. Using spectroscopic as well as biochemical and genetic studies, this project will investigate the involvement of a radical in the BSS reaction and examine the mode of BSS activation.

This project will further our understanding of the pathways used by microbes to degrade highly stable organic compounds that represent important contaminants of drinking water. This information should help in developing effective biological means of removing these contaminants from water.

This Collaborative Research Activities in Environmental Molecular Science (CRAEMS) Award to Stanford University is supported by the Special Projects Office in the Chemistry Division. Gordon Brown, Scott Fendorf, and Alfred Spormann supported by this award will study the chemical and microbial interactions at environmental interfaces among solids, aqueous solutions, natural organic and plant matter, microorganisms and atmospheric gases. Support is also provided to Satish Myneni at Princeton University through a subaward. Model systems of increasing complexity will be studied with molecular-level probes to explore (1) geometric and electronic surface structures of environmentally relevant hydrated solids; (2) the structure of water at solid-aqueous interfaces and in the vicinity of nonpolar organic molecules absorbed on solid surfaces; (3) the mode of interaction of metal ion cations and oxoanions with these surfaces and with organic ligands and microbial organisms; (4) the structure and bonding of aqueous and surface complexes of these ions; (5) the rates of abiotic and biotic reaction pathways of redox-sensitive metalloids such as arsenic, selenium and uranium; (6) the hydrophobic interactions of polycyclic aromatic hydrocarbons with environmental solids at the molecular level; (7) the effects of microbial biofilm coatings on solids on the adsorption and transformation reactions of heavy metal and organic pollutants; and, (8) genomic-level interactions of microorganisms with mineral surfaces and organic and inorganic pollutants. Model studies will be coupled with laboratory studies of natural contaminated systems. Advanced spectroscopic methods, particularly those based on tunable synchrotron radiation, computational chemistry and molecular genomic technologies will be utilized.

This fundamental interdisciplinary research will advance our understanding of the role of sorption/precipitation/transformation at environmental interfaces in sequestering heavy metal and organic pollutants and will lead to the development of new remediation methodology. The affiliation with the Stanford Synchrotron Radiation Laboratory will also introduce environmental scientists and students to synchrotron-based studies in environmental chemistry.

This Institute will create fundamental molecular-level understanding of environmental interfaces and the important chemical and biological processes that occur at these interfaces. The Institute's efforts will focus on understanding the structure of water at the surfaces of common metal oxides, the interaction of water with these solids, the effect of hydration on the structure of solid surfaces, the nature of the electrical double layer at solid-water interfaces, the sorption of heavy metal and metalloid ions onto solid surfaces, and how microbial biofilms influence all of these features or processes. Accurate models of the processes occurring at these interfaces are key to understanding solute-substrate interactions. The multidisciplinary team will use a combination of state-of-the-art in situ synchrotron radiation-based spectroscopy and scattering, computational chemistry, and molecular genomic methods to examine the interaction of water and selected metal and metalloid contaminant ions with environmentally important solid substrates with and without microbial biofilm coatings in simplified model systems. In addition, parallel molecular-level studies of more complex natural systems containing contaminant species, such as arsenic and lead, as well as bacteria will be carried out, building on these fundamental studies.

Physicochemical and microbiological processes taking place at environmental interfaces influence many natural processes, such as the weathering of earth materials and the formation of solils, as well as the transport and fate of environmental contaminants. A team of scientists and engineers has been assembled to develop and apply new experimental and computational techniques to studies of environmental interfaces. The Institute includes chemists, geochemists, microbiologists, physicists, and soil chemists to ensure that the basic research will also inform more applied research. Some of the important applications of this research include studies that will lead to predictions of the transport behavior and potential bioavailability of heavy metal contaminants in natural aquatic systems and environmental remediation of toxic metals. A cohort of talented and diverse graduate and undergraduate students and post-docs will be trained to work on these complex problems in an interdisciplinary setting. The researchers will also help train middle and high school science teachers in summer institutes on environmental science and will interact with science writers to disseminate their discoveries to the larger public.

Environmental Molecular Science Institute (EMSI) awards are given to interdisciplinary teams of university, industrial, and/or national laboratory scientists working on problems aimed at increasing fundamental understanding of natural processes and processes resulting from human activities in the environment at the molecular level. The emphasis in these awards is on collaborative research among teams with complementary research interests and the creation of broad educational experiences for students. The Stanford EMSI team is a partnership among eight faculty at Stanford University, Princeton University, and the University of Alaska-Fairbanks (funded by the National Science Foundation Divisions of Chemistry and Earth Sciences), three researchers drawn from Lawrence Berkeley National Laboratory and the Pacific Northwest National Laboratory (funded by the Department of Energy Division of Environmental Remediation Sciences), and one researcher from the National Institute of Standards and Technology. Additional collaborators include a researcher from the U.S. Geological Survey, researchers from several French universities, and four industrial partners (Corning, Inc., DuPont, Skeletal Kinetics, and Zyomyx). The NSF EMSI award incorporates a Collaborative Research in Environmental Molecular Science award, CHE-0089215.

Intellectual Merit
Most microbes in nature are believed to exist in biofilms that develop on biotic or abiotic surfaces at almost any aqueous interface. These surface-associated, structured microbial communities are of medical and environmental importance. Stability and resilience of mature biofilms is controlled by two interrelated cellular processes: attachment and detachment. However, the underlying molecular mechanism(s) of detachment and attachment are largely unknown. This project explores the first steps towards a physiological, genetic and biochemical understanding of the detachment/attachment processes. Shewanella oneidensis MR-1, a geochemically important microbe that metabolically interacts with Fe- and Mn-containing soil minerals in biofilms serves as model microbe for these studies. The multi-tiered approach involves physiological studies on detachment including energetic and cell mixing experiments. The results expected from this research should provide fundamental insights into the molecular mechanism(s) of microbial detachment from biofilms. While the initial processes of biofilm formation have been investigated in the past, the important questions of the control of stability of established biofilms and their long-term survival have not been addressed. These studies will reveal some important physiological, mechanistic, and genetic principles of determining biofilm stability.

Broader Impact
This research will generate fundamental knowledge on the biology of microbial biofilms and educate graduate and undergraduate students and the broader community. This laboratory has a record of attracting and retaining minorities (70% of the lab are female, 33% ethnic minority). The PI teaches on an annual basis three courses in Environmental Microbiology including a new sophomore seminar on "Environment and Human Health." The broader impact of the activities of the entire lab is on several levels. The PI served for the last four years as co-director of the MBL "Microbial Diversity Course" in Woods Hole, MA; served 2003/04 as a member on the NRC/NAS committee on "Alternatives and Strategies for Future Use and Production of Hydrogen"; the PI has been on the scientific advisory board of Sound Vision for the production of "The DNA Files"; has served since 2000 as the director of the Stanford Biofilm Research Center, since 2002 as co-organizer of the annual "West Coast Bacterial Physiologists' Meeting", Asilomar, CA; and since 2003 as the program coordinator of Stanford's Environmental Engineering and Science Program. In addition, since the fall of 2004, this laboratory has been working with the San Jose Museum of Technology to establish an exhibition module on "Microbes in Nature and their Role in Water Treatment". Members of this laboratory have been active as judges in the "Synopsys Silicon Valley Science and Technology Championship", in "Girls for a Change", and in tutoring East Palo Alto High School students. Activities such as these will continue throughout the current project.

Expected significance of proposed research
This research seeks to provide a physiological and molecular understanding of microbial attachment, detachment, and the transitioning between both states using S. oneidensis as a model system. In addition, the results will also serve as a useful basis for the applied environmental and medical fields to manage microbial biofilms. For example, the dissolution of biofilms beneficial for wastewater and groundwater treatment often needs to be prevented or controlled, whereas corrosion of pipes, ships, as well as formation of medically detrimental biofilms on teeth (caries), subgingival pockets, and catheters could be mitigated (or prevented) by engineered manipulation of the microbial detachment physiology. Therefore, this research is not only of fundamental but also applied relevance.

The Hopkins Microbiology Course is a new, four-week, intensive summer course in microbiology focusing on the intricate interplay between physiological, ecological, evolutionary and geochemical processes that constitute, cause, and maintain microbial diversity. The course is taught at the Hopkins Marine Station, Pacific Grove, CA, and consists of lectures and an extensive laboratory component. This course builds on the inquiry-driven teaching tradition introduced by C. B. van Niel in his Hopkins Microbiology Course taught in the 1950s and 1960s. The objectives of the course are to enable students (i) to isolate key microorganisms driving the biological and geochemical diversity in marine environments, and to conduct and interpret culture-independent molecular characterization of microbial species and their activities; (ii) to assess, evaluate, and recognize physiological and metabolic diversity; (iii) to evaluate and experimentally test ecological and evolutionary factors causing microbial diversification, and (iv) to understand and predict the causes as well as the biological and geochemical consequences of microbial diversity. The distinguishing aspect of this Hopkins Microbiology Course is the unique integration and application of the concepts and experimentation of microbial physiology, ecology, population genetics and experimental evolution in a cohesive study of the environment. The broader impact of this teaching activity is the training of an integrated, multi-level understanding of microbial biology to the next generation of academic and industrial leaders.

Evaluation of biological data often needs statistical insight to detect whether apparent treatment effects are real and useful. Typical applications are to personalized medicine and drug resistance. Standard statistical methods rely on unrealistic assumptions (data is supposed to be independent and identically distributed). This project will provide biologists with new tools for detecting, quantifying and leveraging hierarchical dependencies in areas of microbiology currently revolutionized by the emergence of new sequencing technologies. The PIs propose to tailor new ``treeness'' and ``clustering'' indices incorporating relevant distance and structural information computed from sequence and contingent information. The investigators will also use the treeness indices to provide improved multiple testing programs that improve the power of corrected multiple testing procedures in the case of hierarchical dependencies between variables. This will enhance the power in detecting significant functional differences between different conditions. The methods will first be developed and calibrated on data simulated according to known tree structures. Calibration will evaluate the indices under various types of perturbations and thresholding. The methods will then be used on real data generated, as part of the proposed work by directed evolution experiments in microbial ecology.


This work is an application-driven project for providing useful multiple testing correction under hierarchical dependencies. The goal is to tailor statistical methods to the exact needs of biologists working in bacterial ecology and in HIV/HCV drug resistance. This project provides the integration of a broad range of cutting edge mathematics, probability and statistics with computational advances that cater to the realities of data collection and analyses in the fields of phylogenetics, metatranscriptomics and metagenomics. Advances in the study of evolution, in microbial ecosystems (the human gut, sewage treatment plants) or virus evolution (HCV/HIV in a human host) would have repercussions on overall health practices at both the individual and epidemiological levels. Quantitative estimates of confidence in `entero-types' or other inferred clusters would be important in the cost analysis of personalized medicine. Students and Postdoctoral fellows will be trained both in biology and statistics, so they can understand the biologist's requests and constraints. Consulting workshops will be organized regularly where the effectiveness of planned experiments and applied statistics can be discussed. During the academic year, classes targeted to molecular biologists and microbiologists teach multivariate visualization and geometrical statistics methods using R. These will be open source and available from the class web pages. The PIs will offer several Summer schools in Microbiology and Metagenomics where they teach both multivariate statistics, phylogenetic analyses, metagenomic analysis, metatranscriptomics as well as experimental techniques for studying evolution in action.

Intellectual Merit. The Hopkins Microbiology Course (http://hmc.stanford.edu) is an intensive, 4-week course, targeting microbiology students, who seek to obtain an integrated view of microbial life. The course also attracts non-microbiologists, such as physicists or engineers who want to understand microbial biology in order to begin molecular and population level studies. As a "big picture" course, the Hopkins Microbiology Course requires students to have already acquired a significant background in microbiology. While details are important and stressed abundantly as required, the emphasis is on providing the intellectual framework to weave together multiple disciplines into an integrated perspective. This integration is further intensified by providing students with an introduction and hands-on use of biocomputational tools and methods to exploit large biological data sets in order to obtain new biological understandings. Such a broad view is important for students entering not only the academic environment, but also professional fields that involve microbes (chemical, pharmaceutical, or environmental and water industry).

Broader Impacts. This advanced level summer course seeks to provide students with an integrated conceptual framework and experimental tools necessary to understand and study how the interplay of physiology, ecology, evolution, and population genetics causes and maintains diversity of microbes in diverse environments. The course emphasizes a critical, cross-disciplinary integration and application of the knowledge gained and experimental skills learned.

1712411
Spormann

The International Society for Microbial Electrochemistry and Technology (ISMET) was formed in 2011 as a new international umbrella organization (private nonprofit) with the goal to link researchers, engineers, and scientists in the area of microbial electrochemistry and their derived technologies. This conference, was held at Stanford University on October 5-7, 2016 with the objective of linking together an interdisciplinary group of researchers to discuss recent advancements in microbial electrochemistry and technologies. This technology is one of many that is being investigated related to the food-energy-water nexus.

The topics that were covered at the North American-International Society for Microbial Electrochemistry and Technology conference was to integrate both fundamental and applied research, and cover topics were in the area of fundamental research: 1. Novel microbial pathways in microbial electrochemistry, 2. Fundamentals of extracellular electron transfer, 3. Synthetic biology approaches to optimize microbial electrochemical systems, and, 4. Microbial ecology of mixed culture systems. In the area of applied research the topics were: 1. Wastewater treatment and other bioremediation/bioenergy/biosensing applications, 2. Materials: electrodes and membranes for microbial electrochemistry and technologies, 3. Design towards applications and scale-up, and, 4. Modeling and optimization. The ISMET promotes the International and Continental meetings occurring every other year on alternating years. However, ISMET does not organize these meetings; the annual meetings are organized at the host level. The interaction among researchers from different fields created a unique environment to foster ideas and future collaborations. At the same time, these ideas could lead to novel technologies that can impact the future infrastructure at the water-energy nexus. The conference also had workshops to educate new researchers in techniques such as molecular biology and mathematical modeling. Through these workshops, the PI expected to train students that will become leaders in our field. The conference also serves to be a springboard for forging the thought processes of researchers from various backgrounds across the globe to enable the success of microbial electrochemical technologies.

Project summary In nature, bacteria often form complex communities that enable them to adapt to their environment and to carry out particular functions. For example, the community of bacteria in the human gut in uences a variety of aspects of health and disease, while some infections are characterized by the formation of lms of living cells (bio lms). These bio lms have been extensively investigated in medical and industrial contexts, but the biophysical rules underlying these living communities have remained unclear. To close this gap, the proposed investigations will develop genetic constructs, bacterial strains, visu- alization tools, and biophysical models in order to set the stage for the rational engineering of complex microbial communities that carry out de ned functions. Speci cally, the investigators propose to (1) develop quantitative experimental tools to optically pattern single-species and multispecies bio lms; and (2) through biophysical modeling and experimentation, investigate the structural development of ecolog- ically interacting consortia from initial seeding. Critically, the proposed investigations will establish and validate a new platform, bio lm lithography, that will enable major advances in the use of optogenetics and synthetic biology for bioengineering, research and therapeutic purposes. This broadly applicable platform will answer two pairs of crucial questions in the proposed inves- tigations. 1a) Can the light-activated expression of bio lm genes yield robust, high-resolution spatial patterning of bio lms on a 2D surface? 1b) Can the platform be used to control multiple genes and populations in parallel by using multichromatic light stimuli? 2a) Can these tools be used to generate, study, and understand stable, spatially patterned multispecies bio lm consortia? 2b) Are experimental measurements of bio lm dynamics supported by quantitative biophysical models? Taken together, the combination of theory and experiment proposed here will set the stage for the plug-and-play design of microbial communities with both complex structure and function. These advances will signi cantly lower access barriers to complex synthetic biology, driving innovation across elds as well as across socioeconomic divisions. When coupled with the future rational design of cellular genomes and structures, our platform for the construction and manipulation of light-patterned living communities has the potential to signi cantly advance medicine and also material sciences.