Course Topics
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Field trips. The course will begin with a discussion of and field trip to the shallow aquifer underlying the now closed landfill research site in Norman, OK. This will be the location where students can test their hand with various procedures to obtain samples (sediment and ground water) for their studies. Mr. Jason Masoner of the USGS will coordinate the field activities. He will assist the students as they take their samples. The students will be introduced to a variety of site-related issues such as safety, site characterization contaminant containment, sample transport, storage conditions, past history as well as the goals and objectives of the exercises. The experience will also serve as a primer for subsequent field work on the Tallgrass Prairie Preserve. The Nature Conservancy has granted access to the prairie, the largest protected remnant of tallgrass prairie left on earth. Oil and gas collection and separation facilities are common on the preserve. There is ongoing hydrocarbon biodegradation research on preserve property. The students will investigate oil field issues such as corroding pipelines and storage tanks or even historical environmental petroleum releases. The students will thus relate their findings to real environmental issues and at the same time, learn how energy is produced, transported, stored, used, and occasionally spilled in modern society. The students will use samples from these sites for use in the laboratory portion of the course. The students will employ a battery of techniques in order to provide a multi-faceted view of the microbial diversity present at these sites.
Anaerobic microbiology procedures. Anaerobes are now known to be far more diverse and to possess greater metabolic capabilities then was generally believed even a few years ago. Research over the past few decades has revealed a variety of potentially useful catabolic activities, including hydrocarbon metabolism, as well as many environmental impacts that were considered rather improbable under anoxic conditions. The development of this area of microbiological research was made possible by the application of anaerobic techniques that permit the manipulation and cultivation of oxygen and redox sensitive microorganisms. The methods of anaerobic microbiology will be taught in sufficient detail to permit students to appreciate and enter into this field of research.
Enrichments. Enrichment protocols will be used to obtain sulfate-reducing bacteria methanogens and hydrocarbon-degrading bacteria. These enrichments and eventual isolates will be used in other exercises associated with the course. To ensure student success, a positive control oil-degrading methanogenic enrichment will be used if required. Initially, enrichment cultures will be established using contaminated aquifer sediments and groundwater obtained from the Norman landfill research site. The same procedures will be used subsequently on tallgrass prairie samples that are hydrocarbon-impacted. For the purpose of this course, alkane biodegradability has been chosen as the model system for the reasons outlined above. Enrichments will be set up using a variety of environmental and selective conditions, including combinations of the following: (1) different concentrations of alkanes, including biphasic systems with the alkane present in excess of its aqueous solubility, (2) different chain length (likely C5-C8) alkanes, and (3) different terminal electron accepting conditions (sulfate-reducing and methanogenic). The existing methanogenic alkane-degrading enrichment will serve as a positive control. The progress of the enrichments will be monitored by having the students check for the increase in numbers of bacteria using direct microscopic observations as well as increase in optical density. This will be augmented by assays of both the loss of the alkane primary substrate(s) coupled with the utilization of a terminal electron acceptor in the case of sulfate or methane formation. Mass balance calculations will be attempted. Primary isolates obtained from these procedures will be purified and the students will then test the purified isolates for catabolic versatility by evaluating growth and activity of isolates on a range of different hydrocarbons, including different chain length alkanes and aromatic hydrocarbons such as benzene, ethylbenzene, the isomers of xylene, etc. A positive control culture, Desulfoglaeba alkanexedens, will be used as a positive control. Well characterized bacterial isolates will be chosen by the students for DNA isolation, which will be used to estimate abundance of alkane degraders as described in the Molecular Analysis of Isolated Strains objectives, below.
Molecular profiling of microbial populations. DNA will be extracted from various types of samples, including those considered difficult due to low microbial biomass and/or substances inhibitory to PCR. The DNA will be PCR amplified with standard small ribosomal subunit primers designed to amplify partial 16S rRNA gene sequences of bacteria and archaea. The students will construct clone libraries by direct cloning of the PCR products and will analyze sequence data (see Bioinformatics) to characterize bacterial community structure and dynamics in environmental samples. Such 16S sequence profiling will initially be used to observe changes in microbial population dynamics along previously established biogeochemical transects through the aquifer underlying the landfill research site. The analyses will target changes in bacterial and archaeal populations specifically. The same profiling techniques will then be used on other environmental samples as well as any alkane-degrading enrichments or to complement other techniques for assessing pure cultures.
Molecular analysis of functional genes for sulfate reduction, methanogenesis, and anaerobic alkane metabolism. DNA will be extracted from various types of environmental matrices that may or may not have been exposed to n-alkanes. This DNA will be PCR amplified with primers designed to amplify genes associated with sulfate reduction, methanogenesis and anaerobic hydrocarbon-biodegradation. This assay will include the key gene of dissimilatory sulfate reduction, dsrAB, that codes for the alpha and beta subunits of the enzyme dissimilatory sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Similarly, the coenzyme M methyl reductase is the key enzyme of methanogenesis. This enzyme catalyzes the terminal step of the methanogenesis, the reduction and release of the coenzyme-M-bound methyl group as free methane. An important advance on the genes involved in the “fumarate addition” mechanism of anaerobic alkane metabolism has recently been described. In this reaction, alkanes are activated by an alkylsuccinate synthase (Ass), which adds the sub-terminal carbon of an alkane across the double bond of fumarate. Since the discovery of Ass, the ubiquity of assA genotypes in hydrocarbon-impacted environments has only begun to be explored. Degenerate PCR primers that allow amplification of partial assA genes in environmental communities have been developed and will be used. Sequencing of the PCR products (after cloning) will yield information on the diversity of these genes in various environmental matrices. The nucleotide sequences will be compared to other sequences in the database and phylogeny constructed. The abundance of various functional genes will be estimated by real-time PCR.
Molecular analysis of isolated strains and enrichment cultures. Pure strains obtained from the enrichment cultures will be subjected to PCR with primers as described in the previous paragraphs. In addition, DNA extracted from enrichment cultures will be subjected to the same analyses to see the difference between culture based and non-culture based data.
Microarray experiments. The students will use a comprehensive functional gene microarray system, termed GeoChip. Such technology represents a state-of-the-art molecular approach to detect and monitor microbial communities important to various biogeochemical, ecological and environmental processes. The use of the GeoChip on the environmental samples obtained by the students (as well as selected enrichments and isolates) will provide the opportunity to identify and track thousands of microbial genes simultaneously. The GeoChip contains 24,243 oligonucleotide (50 mer) probes and covers 410,000 genes in 4,150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, as well as genes associated with the biodegradation of hydrocarbons and other organic contaminants. The students will also learn about the requisite bioinformatics tools to analyze large amounts of microarray information. The resulting information will also be coupled with biogeochemical data collected by the students during other portions of the course. Lastly, genome-wide analyses will be performed to understand what genes are expressed during growth on alkanes by the model sulfate reducing bacterium AK-01. This is important to understand that there are a host of other factors in the cell that are involved in hydrocarbon degradation besides just the genes coding for the biodegradative pathway.
Environmental metabolomics. In order for the students to obtain information on the nature of the hydrocarbon contaminants in their environmental samples (aquifer or hydrocarbon-contaminated area), the nature of signature microbial metabolites produced as a result of anaerobic biodegradation of these substrates will be analyzed by GC-MS protocols. Initially, analyses will be performed on actual field samples. In addition, sub-samples of the environments from which the enrichments have been established will also be treated similarly. We will also have the students analyze their enrichment cultures at the time of subculturing for microbial isolation to determine the nature of the microbial metabolites produced during the enrichment protocol. An outcome of this investigation will be direct hands-on experience in the extraction of metabolites, comparison with control samples and analysis of mass spectral data.
Sequence analysis and bioinformatics. Students will learn how to analyze sequence data generated during the course and other sets of sequence data using suites of programs readily available on the web (RDP, ARB project, greengenes, DOTUR, and others).
Student presentations. Students are invited to bring with them a poster that illustrates their research (the poster can have been previously presented at a conference). The students will have the opportunity to introduce themselves and their research interests on the first day of the course. The posters will be displayed throughout the course in a readily accessible location to facilitate conversations among students, faculty, and guest speakers. Students will make group presentations on the next to the last day of the course summarizing their results and suggesting future research questions and approaches.