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As dangerous and uninviting as the conditions may seem based on the quote above, the solutions draining fom Iron Mountain mines are not sterile. In fact, flourishing communities of microorganisms populate this habitat. These microbial communities, and their role in the generation of acid mine drainage, are the focus of DOE (Microbial Genome Program) and NSF (Biocomplexity Program)-funded work at Iron Mountain. Previous studies have demonstrated that microorganisms promote acid mine drainage (AMD) formation by oxidizing ferrous to ferric iron. Ferric iron is a much more effective oxidant of pyrite surface sulfide groups than O2. Thus, understanding of AMD formation requires understanding of microbial activity.
The goal of these projects is to obtain a molecular-level understanding of the metabolism of organisms involved in AMD formation. Because so few organisms can be cultivated for detailed study, our approach has been to develop cultivation-independent methods to identify the organisms present and determine their roles. This web page provides basic project information and describes genomic and genomically-enabled studies of microbial biofilms that populate the Richmond Mine, the source of much of the acid mine drainage (AMD) at Iron Mountain. The genome sequencing is being conducted at the Joint Genome Institute under sequencing awards from the DOE's Microbial Genome Program.
Iron Mountain Mine is located approximately 9 miles northwest of Redding, CA in the foothills of the Kalamath Mountains. Mining operations at Iron Mountain began in the late 1800's and continued intermittently until 1962. The 4,400 arce property contains many miles of underground tunnels and workings, an open-pit mining area, waste-rock dumps, and tailings piles. The ore body is a massive hydrothermal sulfide deposit in rhyolite host-rock and was mined for iron, silver, gold, copper, and zinc. Blasting and tunneling have made the sulfide ore permeable to air and water which results in the generation of vast quantities of metal-contaminated sulfuric acid solutions. It has been estimated that there is enough remaining ore in the disused mines at Iron Mountain to allow the acid mine drainage process to continue for about 3,000 years. The biofilms under investigation are growing in regions within the ore body where acid mine drainage is currently forming. The sampling sites are accessed by a recently renovated horizontal entrance tunnel that is maintained as part of the Superfund site remediation effort. The ore deposit is encountered about 460 meters (1,500 feet) inside the mountain. The entrance tunnel meets with 4 other tunnels in a region referred to as the five-way (see figure below). In the first years of our work at Iron Mt. (starting in 1994), our sampling was conducted within ~20 m of the five-way. Recently, we have been working in three areas in the A drift, AB-B drift junction area (AB muck dam) and C drift. Here, temperatures are high due to the exothermic nature of pyrite oxidation, varying between 35-50 degrees C (95-120 F). Drainage waters which are collecting and flowing through the tunnels have a pH between 0-1, which is indeed as acidic as battery acid. Map of the field site used in this study.
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Typically, it had been assumed that the microbiology of acid mine drainage environments is well represented by a few readily cultured bacteria species (Acicithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans and related species). However, results from our early studies showed that extreme pH and temperature conditions at acid generating sites are dominated by other unknown or little studied organisms.
We have documented the microbial species involved in metal and sulfur cycling at our field site using an array of molecular techniques. These include DNA sequence analysis and fluorescence in-situ hybridizations (FISH). A FISH image of a biofilm is shown below.
FISH image of a biofilm. Cells shown in yellow are Leptospirillum group II, those in white are Leptospirillum group III, blue cells are a variety of archaea.
DNA extractions and sequence analyses can answer "who's out there" but offer limited information about the abundance, in-situ metabolic activity, and ecological niche of individual organisms. Therefore, we have used FISH to label cells on mineral surfaces and in suspension to quantify cell type distribution and correlate the distribution of organisms (species level) with geochemical conditions (Edwards et al. 1999).
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We are continuing to enrich for and isolate bacteria and archaea from within an extreme acid mine drainage environment. Microbial samples are collected from different environments, including sediments, pore fluids, free-flowing waters, and subaerial biofilms. These field samples are used as innoculum in a variety of different media which are incubated under aerobic, microaerophillic, and anaerobic conditions.
As isolates are obtained, metabolic and biochemical characterizations are being
carried out, including assessments of their growth rates, optimal growth conditi
ons (pH, temperature, ionic strength) and their range of metabolic capability.
New and unknown isolates are being identified through DNA sequence analyses.
Although these isolates are important for some aspects of our work, our primary goal is to investigate entire natural communiites in situ, without cultivation biases and with the ability to understand the roles of closely related organism types (i.e., without assuming that isolates are representatives of their species or that laboratory conditions adequately represent those in the field).
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The abundance and diversity of microbial life at pH < 1.0 (extreme acidophiles) raises interesting questions about microbial adaptions to the environmental extremes of acidity and dissolved metal concentrations. Microoragnsims thriving in acid mine drainage must have systems for protection from their environment . Of particular interest is the ability of microorganisms to maintain internal pH homeostasis against enormous pH gradients. The only well studied acidophilic chemolithotroph is Acidithiobacillus ferrooxidans. Optimal growth of A. ferrooxidans occurs between pH 1.5 to 3.5, yet cytoplasmic pH remains near neutral. Internal pH maintenance in T. ferrooxidans has been attributed to a reversed membrane potential. However, the mechanisms of pH homeostasis operating in extreme acidophiles are unknown. It is likely that the system(s) differ from those previously identified in A. ferrooxidans and the well-studied neutrophiles, such as Escherichia coli and Salmonella.
Our investigations of pH tolerance have focused on the archaeal inhabitants. We have analyzed the lipids of F. acidarmanus and shown that the cells are bounded by predominantly tetraether-linked membranes that prevent proton penetration so that the cytoplasm can be maintained at ~ pH 5.3 (Macalady et al. Extremophiles, 2004).
More recently, our focus has been on protein stability at low pH. The results of this work are noted below.
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Despite the diversity of iron oxidizing organisms, the vast majority of research has been on the respiratory chain of A. ferrooxidans. However, studies have shown that many different types of iron oxidizing pathways exist throughout the Bacterial and Archeal domains. Further characterization of the functional diversity of iron oxidation in these two domains and groups within these domains may reveal evolutionary relationships between them.
Our team is trying to determine the mechanisms for iron oxidation in microbial communities at very low pH. Our primary approach has been to use genomic sequence information to identify candidate proteins that may be involved in the electron transport chains of the different organisms. This information can be used to identify proteins that occur abundantly in the biofilm (e.g., that are extracted from the biofilm, purified, and N-terminal sequenced). More recently, our focus has been on using proteomic methods to identify those candidate molecules that exist in high concentration and occur in the membrane of periplasm (see below).
The pink color of many biofilms is due to a high concentration of a red heme-based cytochrome.
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In prior work we conducted experiments to determine the rates of oxidation and the surface morphologies of different iron sulfide minerals that are oxidized abiotically and in the presence of bacteria and archaea. The correlation between attached cells and surface "pitting" has been evaluated. Results indicate that although many pits form inorganically, some cell-mediated local pitting can occur (e.g., F. acidarmanus on arsenopyrite and Leptospirillum cells on pyrite).
Dissolution rates for attached and unattached microorganisms have been measured for a number of species with different metabolic capabilities (
Edwards et al. 1998, 1999, 2000).
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The central goal of this part of our research is to understand the chemical, structural, and microbial factors that control the speciation and release of arsenic into the environment. In prior work we conducted experiments to understand the reactions that result in arsenic release from sulfide minerals and evaluated the processes that determine its form and distribution in water, secondary minerals, and organic materials. The ways in which both biological and inorganic processes control the rates of dissolution of arsenic-bearing minerals in acid mine drainage conditions has also been studied. In addition, we are determining which microorganisms in acid mine drainage environments have the ability to utilize or transform arsenic and what the mechanisms are. Most recently, this work has invovled testing of a very novel archaeal gene with similarity to arsenate reductase by inserting the gene in E.coli cells that lacked this capacity (Flanagan et al. in prep.).
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Table 1 provides geochemical information from a series of sampling time points to illustrate the range in pH, temperature, redox potential, and metal concentrations.
TABLE 1 : Geochemical data (from Druschel, Baker et al. 2004).
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Isolate genomics
The genome of Ferroplasma acidarmanus, an iron-oxidizing extreme acidophile from the site, was sequenced at the Joint Genome Institute and at UW Madison. The genome (with manual annotation) is available at:
To access this site, login as guest, password banfield
The extraordinary simplicity of AMD microbial communities (compared to other communities in less extreme environments) makes it possible to go beyond use of analysis of isolates for genomic characterization.
Our approach is to sample the genomes of an entire microbial community, without cultivation.
Community genomics for study of lateral gene transfer (LGT)
Our approach has been to simultaneously characterize the genomes of organisms that represent a natural microbial community. This is feasible because prior work has demonstrated that the communities have low species-level diversity.
An advantage of acidophilic communities for study of LGT is that the microbes have been largely isolated from most organismal diversity over geological-evolutionary time scales due to their extreme habitat. A hierarchy of LGT probabilities may exist, based on the likelihood of interactions with organisms in other environmental niches. Our goal is to identify the genes that have been transferred, to determine whether these are peripheral or central to the survival of the host organism, and to assess the predicted relationship between LGT frequency and the probability of inter-species interaction.
AMD community genomics
Samples for the first community genomic study were collected in March 2002. Several 50 ml tubes of biofilm were collected from six of the seven previously sampled locations (pH ~ 0.6- 1.2, 45-50 °C). Water samples were also collected for geochemical analysis. A pink biofilm was selected for detailed study. Macroscopically, the community formed a mm thick biofilm that grew at the air-AMD solution interface in a small stream running through fine-grained pyrite sediment.
Using FISH, it was determined that the 5-way community contained approximately 75% Leptospirillum group II, 10% Leptospirillum group III, and 10% Ferroplasma-like archaea (specifically 3 distinct types within this group, including the Ferroplasma acidarmanus population), and 5% eukaryotes (not sampled genomically). Two PCR-clone libraries were constructed using Archaea- and Bacteria-specific 16S rDNA primers applied to DNA extracted from the target community to verify the simplicity of the sample.
Small insert libraries were constructed from DNA extracted from the biofilm sample, and ~ 100 Mb of sequence obtained. We obtained >95% coverage of the genomes of all 5 dominant organism types. The results of this study were reported in an Article in Nature by Tyson et al. (2004).
New sequencing of four additional biofilm samples is planned in 2005.
Post-genomic studies
Ongoing work is focusing on using the existing genomic data to evaluate microbial function in the environment. The first approach has been to combine genomics and mass spectrometry-based proteomics to identify the abundant proteins in a biofilm. The paper reporting results of this work is currently in review (Ram et al.). The second phase of this work will involve microarray-based experiments.
Visit Jill Banfield's Home Pages
(Initial web site version written by Tom Gihring, updated by Jill Banfield)
Of special importance to understanding acid mine drainage and for the development of predictive models, remediation strategies, etc., is an assessment of the importance of known and new microbial species and the determination of their role in the generation of AMD.
Cultivation-based methods have been used in order to obtain organisms for physiological characterization and for experiments designed to evaluate the impact of microbial activity on pyrite dissolution. To date, we have isolates of Leptospirillum group II, Leptospirillum group III (obtained only after genomic data provided clues to an isolation strategy), and Ferroplasma acidarmanus.
A very important and fundamental question is "How do microorganisms survive (and thrive) in highly acidic and metal-rich solutions?"
Biologically-mediated pyrite oxidation has probably exerted a fundamental geochemical control on the global iron and sulfur cycles for a significant fraction of geologic time. Iron oxidation occurs in the bacterial and archaeal domains, and in both of the two, early diverging archaeal kingdoms. An important challenge is to relate metabolic innovations to the geological record, and place these on an absolute time scale. Evolution of iron oxidizing metabolisms may have been coupled to the build up of atmospheric oxygen. This capability may be distributed through the prokaryotes because it is a characteristic of an ancester to both domains, or it may have multiple evolutionary origins. Diversity or conservation of metabolic pathways provides key information to constrain these possibilities.
The debate surrounding the exact mechanisms and rates of microbial sulfide mineral dissolution continues. Attempts to clarify the role and relative importance of the two proposed mechanisms, "direct" and "indirect" attack, have not been conclusive. A new model for biofilm function is currently being developed. This may change our view of how organisms function in communities and how energy metabolism is structured.
Large doses of arsenic can be fatal and chronic, repeated exposure results in body-wide effects including skin lesions, cancer of the internal organs, heart and blood vessel damage, and liver and/or kidney failure. Water supplies in many areas of the world are contaminated with high levels of arsenic. While some arsenic contamination can be attributed to industrial and agricultural sources (e.g. tanneries and pesticides), a significant fraction is liberated through the alteration of arsenic-bearing minerals. In general, the mineralogical, biological, and geochemical factors leading to arsenic contanimation are not well understood.
Waters from the Richmond 5-way at Iron Mountain Mine are being collected in parallel with microbial studies at the site. The interactions between microbial organisms and the minerals and waters they are associated with is being investigated. Complete chemical analyses of these waters using a variety of techniques (including - ion chromatography, atomic absorption spectrophotometry, colorimetric spectrometry, and inductively-coupled plasma mass spectrometry) are coupled with field measurements (pH, Eh, T, cond, D.O.) to rigorously define the aqueous chemistry at the site both spatially and temporally. Waters at the Iron mountain Mine contain extremely high concentrations of Fe (15,000 - 80,000 mg/l), SO4 (100,000 - 500,000 mg/l), Zn (1,000 - 6,000 mg/l), and other metals (including As and Cu). Very low acidities (pH -3 to 1) and high concentration of metals and sulfur give these waters ionic strengths from over 1 to up to approximately 7 m. Speciation of the waters is accomplished in part by using the PHRQPITZ model, but there are limitations in the utilization of that model with respect to redox chemistry due to the lack of information on Fe3+ specific interaction parameters in the current PHRQPITZ database. Aqueous geochemical changes correlated to changes in the microbiological community are being coupled with both forward and inverse modeling codes (PHRQPITZ, PHREEQC, CCBATCH, and an in-house mass balance spreadsheet) to understand the role of microbes in the overall generation and maintenance of a highly acidic environment. Mass balance modeling will be coupled with several laboratory column experiments, a set of in-situ field experiments at Iron Mountain, and finally on observed geochemical changes in acid mine drainage waters to assess relative reaction rates of sulfide minerals in the field and the effect of microbial catalysis and oxygen infiltration.
Organism and Community Genomics: Ecology and Lateral Gene Transfer
