Research

Current Investigation of the Bacterial Microflora of the Human Intestinal Tract

The composition of the human large intestinal microflora has a profound influence on health and disease through its involvement in nutrition, pathogenesis, and immunology of the host. A proper comprehension of the diversity of species present and their numerical preponderance is therefore of utmost importance. Although this microflora has been the subject of intensive investigation, using anaerobic culturing techniques combined with phenotypic methods of characterization, there is now a universal recognition that these approaches have provided a vastly incomplete picture of the predominant gut flora diversity and as much as 80% may have eluded scientific description. Molecular phylogenetic studies are now being employed to provide as complete as possible inventory of the microbial biodiversity present.

Indigenous Communities & Human Microbiome Research

This work is a collaboration between our laboratory and Dr. Cecil Lewis (Department of Anthropology, OU) investing the GI microflora of indigenous populations of Peru. Studies using novel state-of-the-art pyrosequencing techniques have provided unprecedented insights into the microbial diversity of the human gut and also suggest previous estimates of species have been significantly underestimated. At present, little is known about the distinctiveness of intestinal microbes and what influences their diversity. Three hypotheses are of interest: 1) human microbiomes are geographically structured; 2) the modern global economy has disrupted the microbial ecologies that co-adapted with our ancestors; 3) unique species of microbes, with unique functional potentials, can be found within different world populations. To test these hypotheses, samples from remote communities Peruvian communities, less affected by the global economy, are ideal.

Three communities will be sampled that include remote villages within different environments: dry desert, high mountain, and jungle. The fourth community is prehistoric, providing fecal samples extracted directly from mummies. Microbes from these communities are analyzed using state-of-the-art, high-throughput molecular methods and culture-specific methods. Non-mutually exclusive factors include diet, human genome-microbiome interactions, and the shared history with their host population. Much of our understanding of human microbiomes is a result of model organism studies and clinical samples from individuals in industrialized nations. Because of global subsistence catchment of dietary components, human microbial ecosystems in industrialized nations are likely too heterogeneous for systematic studies of their co-evolutionary history with humans. Therefore, samples from remote communities, less affected by the global economy, are ideal for investigations into the structure and function of the GI tract microbiota. Unlike our modern industrialized communities where family and communities are subject to constant change due the individual’s ability to move away from family; within indigenous peoples, family groups and the village way of life may be more favorable conditions to the retention of stable microbiota profiles from generation to generation.

This preservation of close-knit, family units could, potentially contain species or groups of organisms that are exclusive to certain populations within Peru. Most of the gastrointestinal microbiota appears to be anaerobic and are obligate host-specific microbes that do not grow outside of their host; therefore it is likely that they rely on the close contact of parents and offspring for transmission. A number of organisms known to be associated with both commensal and pathogenic mechanisms will be targeted for enrichment and isolation to allow studies on the comparison of inter-stain variability. Genome plasticity represented by genetic diversity within strains of a species allows these species to respond to a wide variety of conditions. Thus “phylotypes” do not necessarily reflect “ecotypes” in that those strains of a species from one geographical location, or within an individual’s gastrointestinal tract, do not necessarily have identical biochemical or physiological properties to those of a second location. Because of the number of as yet-uncultured organisms, it is highly probable that we will find novel bacteria and some may be exclusive to certain Peruvian populations.

Previous Work With Biofuels

Search for Novel Microorganisms Capable Of Producing Ethanol and Other End Products World energy consumption is projected to increase by 71% from 2003-2030 contributing to a potential crippling energy crisis. Combined with the CO2 emissions and climate change issues now becoming ever more prevalent, there is now a great need to use and develop alternative fuels. One such example is biofuels whereby the chemical energy in biomass can be converted to chemical energy in the form of ethanol, hydrogen or other microbial end products. In order to capitalize on these developments and manage the projects in an organized manner, the Oklahoma Center for Bioenergy (OCB) was established. I have become involved in two OCB sponsored projects.

 

  • Isolation and characterization of novel microbial catalysts for direct fermentation of lignocellulose to ethanol.
    One of the key problems hindering efficient utilization of lignocellulosic biomass and conversion to ethanol is the low susceptibility of lignocellulose to hydrolysis, a critical step for generating sugars for microbial fermentation. Pretreatment of cellulosic biomass to break down the cellulose structure using various mechanical, physical and chemical approaches is required prior to the subsequent steps of hydrolysis and fermentation. Furthermore, due to the inhibitory effect of end products and/or inhibitive reagents used in pretreatment processes, the final ethanol yield, generally is low, and does not meet the industrial requirements. An ideal solution is to develop biological processes that do not require pretreatment of plant materials. In addition, the current technology for conversion of cellulosic biomass to ethanol is not efficient or economically viable – a key bottleneck in using cellulosic biomass as an energy source. Thus, novel technologies are urgently needed for efficient ethanol production from cellulosic biomass. Hydrolysis of lignocellulosic materials by fungi generates a mix of sugars including both 5- and 6-carbon sugars. An objective of this project will be the isolation of bacteria capable of ethanol production from fungi-degraded plant biomass.
    Some unexpected outcomes of biofuels research are coming into the public arena. For example, the competition of energy companies and food companies for the same starting materials, such as of corn, is leading to vastly increased food prices. Secondly, the destruction of the South American rainforests to plant sugar-beets as a starting material for biofuels is also causing much concern. Moreover, in response to the above problems, in July 2008, the UK government and other European countries announced a slowing down in the adoption of biofuels in preference for other alternative fuels. However there are alternative starting materials for biofuels for which there is no competition with foodstuffs. 
  • Indirect fermentation of biomass to ethanol and other products.
    Biomass can be first burned to produce a gas called Syngas composed of carbon monoxide, hydrogen and carbon dioxide that is then passed into a bioreactor where bacteria ferment the Syngas and produce ethanol. The advantage of this process is that any biomass that can be burned can be used as starting material. For example, switch grass, a plant that can be grown on marginal land where normal agricultural practices are not possible, city garbage recycling plants, and paper and pulp mills are all potential sources for the generation of Syngas for renewable energy purposes. Dr. Ralph Tanner has long been at the forefront of this technology and we are currently involved into the search for new microbial catalysts that may be more efficient in the above process and may in turn be enhanced by molecular genetic manipulate techniques.

Previous Work With Biocorrosion

In 2006, I was very pleased to be approached by Dr. Joseph M. Suflita to become involved in some studies arising from the oil pipe breakage that occurred on the North Slope, Alaska. We initiated some preliminary studies into the role of microorganisms in microbially influenced corrosion (MIC). To explore the fundamental scientific issues that lead to new knowledge, understanding and technology for the diagnosis and mitigation of fuel biodeterioration and biocorrosion problems. This collaboration with a number of OU-base laboratories as led to formation of a Biodeterioration and Biocorrosion Center here at OU, sponsored by ConocoPhillips and the University. The University recognized this research with the prestigious designation of University Strategic Organization, and therefore ear-marking this as an important area of future research. This exciting development should lead to long-term funding with other energy-based companies being encouraged to take part and address the problems being encountered with MIC.

Previous Work With Molecular Identification & Phylogenetic Analysis of Unique Microorganisms From Swine Feces & Manure Storage Pits

Intensive farming practices have resulted in huge amounts of manure being produced. Odious compounds such as phenolics, indols, volatile amines and ammonia produced by microorganisms can be detrimental to the health of agricultural works, animals and the general population. ln collaboration with Drs. Cotta/ Whitehead, Agricultural Research Service, USDA, we have begun to unravel the complex ecosystems and organisms present in swine manure and the storage facilities of this waste material. This work has resulted in a number of publications describing novel bacterial taxa from manure sources. We propose to extend this work to specifically look at samples from several difference facilities here in Oklahoma. Initial studies will employ a culture-independent molecular approach to create a clone library to produce a comprehensive inventory of the representative organisms present. Organisms will be targeted for directed cultivation work involving the isolation of organisms that will be subjected to further analysis for more complete characterizations. Strategies can then be developed to decrease or eliminate organisms producing noxious compounds. In addition to detecting harmful compounds, samples will be analyzed for the presence of substances such as alcohols and solvents that may be beneficial to industry and renewable energy programs such as bioethanol production and effective waste recycling projects.
Investigations to Into Microbial Assemblages Associated With Hot Springs

This is a collaboration with Hot Springs National Park, Ar were a number of hot springs led to the growth of the town around a number of Bath-houses become a very popular vacation destination from the late 1800s. In addition to my laboratory Dr. Bradley Stevenson (Bot/Micro) and Dr. Mark Nanny (School of Civil Engineering and Environmental Science, College of Engineering Institute for Energy and the Environment, College of Earth and Energy, OU) are also collaborators on this project.

Thermal springs have generated much interest among scientists due to their immense microbial diversity and as an analog to early Earth environments. Volcanically super-heated springs as exemplified by those located in Yellowstone National Park have long been a favorite haunt for biologists and studied extensively. In addition to elevated temperatures, many of these locations also have extremes of pH and high salt concentrations. Molecular surveys of the microbial diversity in Obsidian Pool were pivotal in the discovery of many novel phyla. Examples of organisms resident to these environments are chemolithotrophic, thermophilic anaerobes that grow optimally at temperatures above 50oC and derive their energy from the oxidation of inorganic substances.

Non-volcanic geothermal springs such as those found at Hot Springs National Park, exhibit elevated but less-extreme temperatures (55-65oC) as well as unique geochemical parameters but have received much less attention from biologists. These springs therefore, provide a unique opportunity to discover underrepresented microbial diversity. Furthermore, the influence of temperature and geochemistry on extant microbial diversity in these non-volcanic hot springs can be more readily determined through comparisons with cold-temperature analogs. Could these organisms reveal intermediate physiologies between extreme temperatures akin to early earth environments and mesophilic conditions more prevalent on present day earth? These questions are also gaining increasing attention with respect to Astro- and Extrem- biology as we go in search of life in the Universe.
Identification of Novel Emerging Human & Animal Pathogens

An increasing number of novel organisms, that have previously eluded identification by traditional methods, including many potentially pathogenic bacteria, are now being revealed by modern molecular methodologies. Many of these organisms are anaerobes co-existing in complex community structures; some may act as opportunistic pathogens causing veterinary or clinical problems only when particular circumstances arise. Some organisms are present as a “reservoir” in the environment from where they may infect both man and animals when contact is made and/or the appropriate conditions arise. In my laboratory in collaboration with a number of international groups we are adding to the knowledge of base line microbial communities in man and animals and organisms that arise and cause disease processes.

Consultancy / Miscellaneous Projects

In addition to the studies outlined above, because of my long-standing collaborations with a network of national and international research groups, I am often approached for my opinion on unusual or difficult to identify microorganisms.

Center For Microbial Identification & Taxonomy

Unidentified bacteria that require a rapid identification are taken as pure cultures or isolated DNA. Samples are subjected to 16S rDNA sequence analysis. A report is then generated providing details of the analysis prior to further characterization if requested.