Program
Element 2:
Community Dynamics
and Microbial
Ecology
Fundamental research in ecological processes and interactions of biotic and abiotic components
of ecosystems to understand their influence on the degradation, persistence, and toxicity of mixed
contaminants.
PROGRAM OBJECTIVE
To further the understanding of the structure and function of the microbial and plant
community and interactions of components in natural and amended soil and
subsurface habitats containing mixed contaminants and to elucidate their role in
bioremediation.
BACKGROUND
Fundamental research is needed in community dynamics and microbial
ecology at both the molecular and the organismal level to better understand
bioremediation processes.
Fundamental research in community dynamics and microbial and plant ecology at
both the molecular and the organismal level is needed to better understand the
natural intrinsic processes of bioremediation in mixed contaminant sites. The
taxonomic scope of soil ecology includes viruses, prokaryotes, eukaryotic
microorganisms (such as fungi, protozoa, and microalgae), and metazoans including
nematodes, earthworms, and other microinvertebrates and their interactions with
plant roots. Carbon and electron flow through biological communities facilitates
degradation and transformation of organic and inorganic wastes. Our understanding
of these reactions must transcend taxonomic characterization and single-species
study from microorganisms through lower invertebrate communities populating
subsurface systems. A more complete understanding of energetics and
biogeochemical transformation at the community level may ultimately provide the
ability to control or stimulate communities capable of transformation and to channel
carbon flow (particularly of polluting organic compounds) through these
communities or populations. It is essential to understand the roles and
interactions of diverse communities in order to understand how and to
what extent the structure of the biological community influences the
course of bioremediation and to what extent the environmental factors
influence community dynamics in sites containing mixed contaminants.
As this issue is addressed, then the Acceleration program element can
determine how to best augment and sustain the microbial activity
involved in remediation.
Traditional methods for identifying microorganisms and
assessing their activity in soil have limited applicability for
bioremediation.
Genetic and phenotypic information is needed to better assess
community members. Traditional methods for identifying
microorganisms and assessing their activity in soil, such as plating and
most probable number (MPN) techniques, have limited applicability for
bioremediation (Metting, 1992). DNA and RNA molecular probes, polymerase
chain reaction (PCR) amplification of DNA, and genomic sequencing of ribosomal
RNA are being used to identify microorganisms or populations. In addition,
fluorescence stains and antibodies, microautoradiography, enzyme-linked
immunosorbent assay (ELISA), and microscopy are employed to monitor changes
in population types and numbers. Although these techniques can identify a larger
number of phenotypes present in a soil sample than direct plating, they do not
account for biodiversity with respect to function of the organisms under
environmental stress, competition, or seasonal changes. Both radioactive and stable
isotope technologies are being applied to measuring soil community respiration
activity along with methods to measure enzymatic activity. Specific patterns of
phospholipid ester-linked fatty acids (PLFA) and formation of polyhydroxy butyrate
(PHB) can indicate physiological stress within the microbial community (Tunlid and
White, 1992). However, we are still far from understanding or having the tools and
methods to provide cost-effective, real-time evaluation of metabolic activities and
physiology associated with bioremediation or to examine in detail changes in soil
populations over time to assess bioremediation activity. Even the modeling used to
predict microbial activity has been developed more for bioreactors that function as
closed (batch) and open (continuous) systems than for applications in soils (Lynch
and Hobbie, 1988).
APPROACH
In open systems, such as those found at mixed-contaminant sites or in continuous-flow bioreactors, organisms selectively take in substances, usually in solution,
process these substances, and return products to the surrounding environment.
Often the focus on bioremediation has been from an engineering point of view, i.e.,
to determine the mass balance (CISB, 1993). However, in nature this continuous
process of "substrates in" and "end products out" is dynamic, and members of the
community are continuously changing in numbers and types. Current approaches
to bioremediation focus on mass balance, providing quantitative descriptions of
initial and final concentrations as well as intermediates in selected cases. This gives
rise to the "black box" perception of bioremediation (Miller and Poindexter, 1994).
Basic research into community dynamics will provide a more complete
understanding of the path of carbon and electron flow through degrading and
transforming populations. This information will identify and quantify the signals and
switches at the molecular, cellular, and community level that lead to successional
changes required to induce and control bioremediation processes. The approach of
this program element is to build upon the previous laboratory studies on bioremedial
capabilities of organisms and transfer this knowledge to study communities in field
research centers where mixed contaminants exist in a variety of types and
concentrations. In the field research center it can be determined whether organisms
capable of bioremediation are widely dispersed, concentrated where the contaminant
levels are not toxic and diluted, or present where the contaminants are concentrated.
Information is also needed to assess what organisms can degrade the
contaminants irrespective of their ability to be isolated or counted
(Miller and Poindexter, 1994). Other factors that may influence
community bioremedial activities also need to be elucidated at both the
molecular and the organismal level, such as rates and mechanisms of
molecular evolution of expressed bioremedial genes and environmental
factors influencing bioavailability and rates of biotransformation and
biodegradation and the partitioning of contaminants within the geologic
medium.
Understanding the complex relationships and interactions
of all the organisms involved will improve prediction, assessment, and
optimization of bioremedial processes.
Research in the Community Dynamics and Microbial Ecology program
element will be integrated with research in several other elements. With
respect to Biomolecular Science and Engineering, Community Dynamics
will identify important prokaryote and eukaryote bioremedial
populations present within one or more damaged sites that may have
greater potential for bioremedial activity and genes for cloning or
biological molecules for overexpression. Similarly, for the
Biotransformation and Biodegradation element, Community Dynamics
will provide organisms to examine for poorly understood or novel
enzymatic pathways. In collaboration with the Assessment element,
Community Dynamics will provide the basic data that need to be
monitored and assessed for soil community bioremedial activity of the
consortia as a whole in situ and ex situ and for the
maintenance of the community in relation to structure and function of the
consortia. Finally, for the System Integration, Prediction, and
Optimization element, Community Dynamics will provide information
on bioremedially active members of the community to design and
calibrate the models.
Community Dynamics and Microbial Ecology will use molecular and
macro approaches to identify the individual species of the community
and detect variations within the species and differences in species
composition at different sites. It will characterize the consortia which
may, through molecular evolution, become a robust community that
utilizes and/or detoxifies mixed contaminants. It will also explore
interrelationships between organisms in the community, evaluate the
community as a whole system in remediating the individual components
of the mixed contaminants in the presence of geologic and hydrogeologic
factors, and correlate the presence of metabolic genes with effective
bioremedial processes. By understanding the complex multi-organismal
relationships of each of the populations involved and their interactions, the overall
goal to improve bioremedial activity in the field research center should be achieved.
Community Dynamics and Microbial Ecology will focus on four subelements to
meet the overall goals described above. These subelements are:
Subelement 2.1: Community Identification Using
Molecular and Biochemical Techniques.
Fundamental research in using molecular and biochemical techniques to identify members of
the community existing in sites containing mixed contaminants.
Objectives
Use molecular biology tools and biochemical techniques to characterize pristine and
damaged-site biological communities and to establish with high reliability the spatial
and temporal diversity of populations that make up natural communities of
microbiota.
Goals
Three-Year
Develop strategies to identify important transient and permanent bioremedial
members of the community and monitor their natural changes or shifts with respect
to the impact of various contaminants on the community structure, and compare to
similar unpolluted sites by use of molecular biology, genomic sequencing, and
biochemical analysis of metabolites.
Five-Year
Characterize populations to identify isolates that are resistant to or are utilizers of
toxic components.
Identify and verify presence and activity of specific genes responsible for
biodegradation and biotransformation activity on mixed contaminants and integrate
information with Biomolecular Science and Engineering and Biotransformation and
Biodegradation program elements.
Based on information obtained from molecular biology and biochemistry techniques,
develop new isolation procedures that will enable the non-culturables to be cultured
for kinetics studies on biochemical capabilities as defined in the program element
Biotransformation and Biodegradation.
Determine the fate of introduced organisms that have demonstrated superior
bioremedial activity and compare their numbers and types to those of competitive
intrinsic populations.
Ten-Year
Compare populations throughout a ten-year period for changes due to environmental
parameters including concentrations of contaminants that may either optimize or
enhance the bioremedial activity of the community as a whole.
Subelement 2.2: Molecular Evolution and Gene
Transfer within the Community
Fundamental research in identifying how genes are transferred in mixed-waste contaminated
habitats to understand how the community evolves to utilize the organic contaminants as substrates
and/or tolerate high concentrations of toxic compounds.
Objectives
To genetically and molecularly evaluate how the microbial community as a whole
deals with contaminants by using the experimental field research center to examine
consortia that have through molecular evolution become a robust community that
utilizes and/or detoxifies mixed-waste contaminants.
Goals
Three-Year
Determine whether the frequency of genetic transfer of various elements such as
phage and plasmids present in prokaryotic and eukaryotic members of the
community at sites containing mixed contaminants is greater or lesser due to
environmental stress than at clean sites of similar geological characteristics.
Identify the regulatory elements of genes responsible for bioremedial activity of
various populations within the community.
Within sites containing mixed contaminants, begin to identify signaling mechanisms
of plant-microorganism communication such as the chemical signal in root exudates
to induce microbial metabolic response and determine whether this relationship
could favor bioremedial activity between the two groups of organisms.
Five-Year
Use a consortium isolated from one or more sites containing mixed contaminants as
a nursery and inoculate other similar mixed-contaminant sites to see how
information is transferred and/or used for bioremedial activities.
Understand how different members of the community, including rhizosphere
populations, work together in the presence of mixed contaminants to handle the
individual components, and link this information to the Biotransformation and
Biodegradation program element.
Identify key species and metabolic dynamics, including stress recognition and
response, and interactions that are critical to the remediation capabilities of the
communities.
Determine the minimal amount of redundancy of similar, identical, or different genes
of degradative enzymatic pathways needed within the community for bioremedial
activity of mixed contaminants.
Determine whether microbial bioremedial genes and species boundaries are
maintained in a highly contaminated mixed-waste site.
Determine how genes and plasmids are being transferred (horizontal gene transfer,
transduction, conjugation, or transformation), selected (cryptic or spontaneous
mutations), or rearranged to produce naturally occurring variants in the field
research center and/or generate novel genotypes, and determine whether this transfer
is retained in the autochthonous population even when the original organism (natural
or genetically engineered) can no longer be detected.
Ten-Year
Begin to determine the long-term effect of molecular evolution on the stability of
community bioremediation of mixed wastes.
Evaluate the consortium under different environmental conditions such as diverse
geologic and hydrologic conditions and geochemical matrices, different
concentrations of mixed contaminants, and temporal as well as spatial
variations.
Identify and utilize the robust populations for applications in bioremediation.
Subelement 2.3: Community Structure and Function
Relationships
Fundamental research in examining principles and dogmas of ecology that may influence the
overall ability of the community to remediate mixed contaminants.
Objectives
To examine and understand ecological theories with respect to bioremedial structure
and function activities of the community within one or more soil and subsurface
samples containing mixed contaminants.
Goals
Three-Year
Determine range of changes in the community structure and function of populations
in response to different environmental parameters, including different morphological
types exhibited in the presence of mixed contaminants.
Examine the role of and tendency toward biofilm formation by community members
and its influence on bioremedial activity.
Examine and compare community structure and function of biodegradation and
biotransformation activities found in contaminated sites containing mixed wastes to
other, similar samples collected from more than one site and correlate this data with
the Biodegradation and Biotransformation and Biogeochemical Dynamics program
elements.
In collaboration with the Biotransformation and Biodegradation program element,
examine the rate and degree of utilization of the mixed contaminants as substrates
supporting microbial growth to that of co-metabolism where the substrate is only
partially metabolized and does not support growth.
Five-Year
Determine the ecological significance of biological behavior in mixed-contaminant
sites in terms of cell replication, nutrition, and response; e.g., bacterial chemotaxis
and chemotropism in eukaryotic microorganisms, phototaxis and phototropism,
orientation to gravity and magnetism, responses to contact, pressure, heat, and other
factors.
Examine the effects of bioremediation activity on microbial developmental
processes; e.g., differentiation; secondary metabolism; sporulation of bacteria,
actinomycetes, and fungi; heterocyst formation of cyanobacteria; cell starvation; and
survival of non-sporulating eubacteria and archaebacteria.
Apply the understanding of successional cues to induce and control biodegradation
activity in field research center experiments.
Ten-Year
Determine the direct or indirect effects bioremediation activity has on interactions
with other cells, such as parasitism, predation, amensalism, mutualism, and
commensalism.
Apply tested and validated ecological principles to further enhance bioremedial
activities of communities present in sites containing mixed contaminants.
Subelement 2.4: Influence of Environmental Factors
Fundamental research on the response of microbial and rhizosphere communities to in situ
physical and chemical factors that affect the survival, growth, and active bioremediation of
contaminant mixtures.
Objectives
To understand the relative importance and interactive influences of in situ
physicochemical parameters such as pH, Eh, ionic strength, temperature, water
activity, and other factors on properties of microbial populations and communities
responsible for biodegradation and biotransformation activity in soil material
containing mixed contaminants.
Goals
Three-Year
Identify and evaluate key physicochemical parameters that influence microbial
community structure and function in environments containing mixed contaminants.
Five-Year
Understand and demonstrate those physicochemical parameters that support or
interfere with biodegradation and biotransformation under controlled conditions
in situ and ex situ.
Develop a knowledge base sufficient for the design and implementation of
laboratory and field experiments to understand how physicochemical processes, in
the presence of mixed contaminants, interact at the soil community level, including
plants, under hydrological flow regimes and in natural heterogeneous
environments.
Ten-Year
Understand the interactions and relative importance of key physical and chemical
variables that influence or control microbial community dynamics and how these can
be manipulated to optimize biodegradation and biotransformation activities of the
community.
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