Objectives:
(1) Develop an ecological understanding of the interactions among
heavy metals (lead and chromium), the physico-chemical environment,
and microbes capable of remediating organic pollutants, to supplant
the current empirical approach; (2) use microbial community diversity
and heavy metal tolerance to determine the load of "bioavailable"
metal in contaminated sites; (3) use experimental microcosms to test
ecological conclusions derived from analysis of waste sites. The purpose
of these experiments is to optimize organic bioremediation, and provide
feedback to field site remediators.
Approach:
In this project, we examine the impact of lead and chromium contamination
upon microbial community diversity and activity. We propose to analyze
hazardous waste sites, via samples provided by the Waterways Experiment
Station, Army Corps of Engineers. Three experimental approaches will
be used. (1) Analyses of microbial community diversity and activity
in contaminated soils will be conducted. Analysis of extracted phospholipids
will be used to assess biomass and community structure. Community structure
will also be assessed by PCR amplification of a portion of the 16ssrRNA
genes, and separation of these products on a denaturing gradient gel.
Microbial activities will be assessed using catabolism of 14C
organic substrates. (2) Metal-resistant bacteria will be isolated from
these habitats. We will determine both the levels of metal resistance
via the inhibition of respiration (as a bioindicator of in situ available
metal) and the physiological mechanism(s) of metal resistance. The level
of metal resistance will also be determined in the natural microbial
community by assaying how metal concentration affects activity of the
extracted microbial population. (3) Experimental manipulations of soils
in microcosms will be made to test factors that (a) determine microbial
biomass levels, (b) lead to the dissemination of metal resistance, and
(c) impact the rates of organic bioremediation.
Expected
results: Long-term metal contamination selects for a metal-resistant
microbial population much like other "extreme" environments
(hot spring or acid pH) select for specific populations. We hypothesize
that in soil, a diverse metal-resistant population will develop and
conduct a broad set of microbial activities similar to an uncontaminated
soil. The resistance level of the microbial population will be related
to the level of "bioavailable" rather than total metal. Therefore,
physiological analyses can be used to indicate the level of metal stress
in the waste site. Physico-chemical conditions which reduce the level
of free, toxic metal ions (high pH, complexes with organic ligands)
will reduce metal toxicity and promote optimal activities of microbes
that catabolize organic pollutants.
We propose
to couple the introduction of bacteria over expressing phosphatase (OXP)
with the addition of organic phosphorus (OP) and examine the resulting
impact on the microbial community and the sequestration of uranium.
We will examine three critical hypotheses (1) The introduction of the
OXP bacteria will increase microbial activity and decrease community
diversity (2) The introduction of the bacteria and an organic phosphorus
source will enhance formation of phosphate minerals and immobilization
of uranium, and (3) The introduction of the OXP bacteria will increase
microbial heterogeneity. The introduced bacteria will make both naturally
occurring and added OP bioavailable by enzymatically transforming the
OP to inorganic form, potentially relieving nutrient limitation and
making PO43- available for contaminant precipitation.
This approach is superior to introduction of PO43-
because many of the organic forms are more mobile in the subsurface
due to their lack of charge. We will determine the effects of the organism
on the composition, activity, and heterogeneity of the indigenous microbial
community and examine factors (e.g., concentration of OP and organic
carbon) controlling ecosystem response. We will use uranium in experiments
with sediments from NABIR "analog" sites and will assess heterogeneity
issues impacting phosphorus availability and cycling at these sites.
In the initial studies, we will use genetically engineered organisms.
However, we will develop a natural variant that could be used in the
field. This research addresses the Community Dynamics and Microbial
Ecology and Microbial Ecology and the Biogeochemical Dynamics NABIR
Program Element.