Iron (Fe) has long been a recognized physiological requirement for life, yet for many microorganisms that persist in water, soils and sediments, its role extends well beyond that of a nutritional necessity. Fe(II) can function as an electron source for iron-oxidizing microorganisms under both oxic and anoxic conditions and Fe(III) can function as a terminal electron acceptor under anoxic conditions for iron-reducing microorganisms. Given that iron is the fourth most abundant element in the Earth's crust, iron redox reactions have the potential to support substantial microbial populations in soil and sedimentary environments. As such, biological iron apportionment has been described as one of the most ancient forms of microbial metabolism on Earth, and as a conceivable extraterrestrial metabolism on other iron-mineral-rich planets such as Mars. Furthermore, the metabolic versatility of the microorganisms involved in these reactions has resulted in the development of biotechnological applications to remediate contaminated environments and harvest energy.

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Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction

Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times ( 2 S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Recent Advances in Petroleum Microbiology

Understanding the dynamics of methane (CH4) emissions is of paramount importance because CH4 has 25 times the global warming potential of carbon dioxide (CO2) and is currently the second most important anthropogenic greenhouse gas. Wetlands are the single largest natural CH4 source with median emissions from published studies of 164 Tg yr 1 , which is about a third of total global emissions. We provide a perspective on important new frontiers in obtaining a better understanding of CH4 dynamics in natural systems, with a focus on wetlands. One of the most exciting recent developments in this field is the attempt to integrate the different methodologies and spatial scales of biogeochemistry, molecular microbiology, and modeling, and thus this is a major focus of this review. Our specific objectives are to provide an up-to-date synthesis of estimates of global CH4 emissions from wetlands and other freshwater aquatic ecosystems, briefly summarize major biogeophysical controls over CH4 emissions from wetlands, suggest new frontiers in CH4 biogeochemistry, examine relationships between methanogen community structure and CH4 dynamics in situ, and to review the current generation of CH4 models. We highlight throughout some of the most pressing issues concerning global change and feedbacks on CH4 emissions from natural ecosystems. Major uncertainties in estimating current and future CH4 emissions from natural ecosystems include the following: (i) A number of important controls over CH4 production, consumption, and transport have not been, or are inadequately, incorporated into existing CH4 biogeochemistry models. (ii) Significant errors in regional and global emission estimates are derived from large spatial-scale extrapolations from highly heterogeneous and often poorly mapped wetland complexes. (iii) The limited number of observations of CH4 fluxes and their associated environmental variables loosely constrains the parameterization of process-based biogeochemistry models.

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Methane emissions from wetlands: biogeochemical, microbial, and modeling perspectives from local to global scales

Salinization, a widespread threat to the structure and ecological functioning of inland and coastal wetlands, is currently occurring at an unprecedented rate and geographic scale. The causes of salinization are diverse and include alterations to freshwater flows, land-clearance, irrigation, disposal of wastewater effluent, sea level rise, storm surges, and applications of de-icing salts. Climate change and anthropogenic modifications to the hydrologic cycle are expected to further increase the extent and severity of wetland salinization. Salinization alters the fundamental physicochemical nature of the soil-water environment, increasing ionic concentrations and altering chemical equilibria and mineral solubility. Increased concentrations of solutes, especially sulfate, alter the biogeochemical cycling of major elements including carbon, nitrogen, phosphorus, sulfur, iron, and silica. The effects of salinization on wetland biogeochemistry typically include decreased inorganic nitrogen removal (with implica...

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A global perspective on wetland salinization: ecological consequences of a growing threat to freshwater wetlands

Bioremediation has the potential to restore contaminated environments inexpensively yet effectively, but a lack of information about the factors controlling the growth and metabolism of microorganisms in polluted environments often limits its implementation. However, rapid advances in the understanding of bioremediation are on the horizon. Researchers now have the ability to culture microorganisms that are important in bioremediation and can evaluate their physiology using a combination of genome-enabled experimental and modelling techniques. In addition, new environmental genomic techniques offer the possibility for similar studies on as-yet-uncultured organisms. Combining models that can predict the activity of microorganisms that are involved in bioremediation with existing geochemical and hydrological models should transform bioremediation from a largely empirical practice into a science.

Cleaning up with genomics: applying molecular biology to bioremediation

Benzene contamination is a significant problem It is used in a wide range of manufacturing processes and is a primary component of petroleum-based fuels Benzene is a hydrocarbon that is soluble, mobile, toxic and stable, especially in ground and surface waters It is poorly biodegraded in the absence of oxygen However, anaerobic benzene biodegradation has been documented under various conditions Although benzene biomineralization has been demonstrated with nitrate1, Fe(III)2,3,4,5, sulphate6,7 or CO28,9 as alternative electron acceptors, these studies were based on sediments or microbial enrichments Until now there were no organisms in pure culture that degraded benzene anaerobically Here we report two Dechloromonas strains, RCB and JJ, that can completely mineralize various mono-aromatic compounds including benzene to CO2 in the absence of O2 with nitrate as the electron acceptor This is the first example, to our knowledge, of an organism of any type that can oxidize benzene anaerobically, and we demonstrate the potential applicability of these organisms to the treatment of contaminated environments

Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas

Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 × 103 to 1.5 × 104 cells g−1 wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to Chromobacterium violaceum, a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and C. violaceum incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N2 and N2O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO2 for growth as well as the assimilation of 14C-labeled CO2 into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.

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Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, strain 2002.

Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter, Geothrix, and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 x 10(1) in aquifer sediments to a high of 9.33 x 10(6) in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N(2). Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the PROTEOBACTERIA: Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the PROTEOBACTERIA: The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

Diversity and Ubiquity of Bacteria Capable of Utilizing Humic Substances as Electron Donors for Anaerobic Respiration

Swine production is an important industry in the United States, with an annual value of more than 12 billion dollars. In 1997, the annual nationwide production was more than 100 million pigs, with a resultant production of 8.5 × 106 tons (dry weight) of manure per year requiring treatment and disposal (21, 28a). The low profit margins associated with swine production and the difficulties associated with poor public acceptance of hog facilities in many regions have resulted in a significant increase in the number of high-density-confinement rearing facilities in an attempt to improve efficiency based on economies of scale. Although hog wastes can be used as a good nutritional source of nitrogen and phosphorous for agronomic crops, the practice of land spreading has come under intensive scrutiny in the last decade because of runoff contamination of water resources and the odor of the untreated slurry. In addition, swine manures contain high concentrations of metals such as iron, copper, zinc, cobalt, and cadmium that are present as both soluble organic complexes and insoluble oxides and can accumulate in a soil environment as a result of land spreading (34). Thus, major air, soil, and water pollution problems are associated with storage and disposal of swine waste.

Odor management has become a crucial issue for the swine industry, and the sustainability, productivity, and profitability of a producer depend on the extent to which odor emissions can be controlled (62). Malodorous components of swine waste can be divided into four classes: volatile fatty acids (VFAs), indoles and phenols, ammonia and volatile amines, and volatile sulfur compounds (63). Each of these components is primarily microbially formed through the activity of fermentative bacteria that degrade the complex organics present in the waste (56, 62). These compounds accumulate in storage facilities, where the mixtures of feces and urine collected from under-floor collection pits decompose under the prevailing anaerobic conditions. Although these pits are left exposed to the atmosphere, the high organic content of swine waste ensures that oxygen diffusing from the atmosphere is rapidly removed biologically (10). As such, although the malodorous components are readily biodegraded by many respiratory microbial species, the activity of these organisms is inhibited by the limiting availability of suitable electron acceptors or by the organisms' low rate of metabolism under methanogenic conditions (29, 48).

Swine waste can be treated microbially in aerobic activated sludge systems; however, these systems are energy intensive, and there is a large production of microbial biomass (1.0 to 1.5 mol mol−1 waste treated) (33) that also requires treatment and disposal. Alternatively, anaerobic treatment processes, such as the use of methanogenic bioreactors, can be applied for the removal of odor and the generation of combustible biogases. This type of treatment has the advantages over aerobic systems of lower energy input and of much lower yield of biomass per liter of treated waste (0.032 mol mol−1 waste treated) (25). However, traditional methanogenic systems are slow due to the long doubling times of the fatty acid-degrading, syntrophic bacteria whose activity is central to the process (46). Alternative treatment systems based on sulfate- or nitrate-reducing bacteria could potentially be faster, due to the favorable thermodynamics of these metabolisms (46, 58). However, both of these systems can produce noxious and toxic products (e.g., sulfide, nitrite, and nitrogen oxides).

Here, we describe an alternative approach for treatment of the malodorous compounds associated with hog waste by stimulating their removal through Fe(III) supplementation and bioaugmentation with a novel dissimilatory Fe(III)-reducing organism. This process can be used to treat hog waste directly in primary and secondary lagoons without the need to construct dedicated bioreactors. Microbial Fe(III) reduction is an energetically favorable process, and in the natural environment, Fe(III)-reducing bacteria (FeRB) can outcompete and inhibit both sulfate-reducing and methanogenic bacteria (10, 38, 44, 60). FeRB also have diverse metabolisms, and many pure-culture examples exist that can completely oxidize straight- and branched-chain fatty acids and aromatic organics without the need for the activity of the rate-limiting syntrophic bacteria (14, 37). The respiratory end product of microbial Fe(III) reduction, Fe(II), is nontoxic and can be recycled after abiotic reoxidation through its reaction with O2. In addition, added iron will abiotically react with malodorous HS− ions, forming non-odor-causing metal sulfide precipitates. This approach has the potential for the long-term and sustainable removal of the major odor-causing components of hog manure.

Kimberly A. Cole

Papers

Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas

Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, strain 2002.

Diversity and Ubiquity of Bacteria Capable of Utilizing Humic Substances as Electron Donors for Anaerobic Respiration

Biological Control of Hog Waste Odor through Stimulated Microbial Fe(III) Reduction

Pesticide residues in water, sediment, and fish at the SpartaI, IL, USA, National Guard Armory.