Timberley M. Roane, Christopher Rensing, Ian L. Pepper, and Raina M. Maier 1. Address the structural differences between prokaryotic and eukaryotic cells and how these differences influence cell resistance or sensitivity to metal toxicity. Eukaryotic cells contain internal organelles such as mitochondria (site for ATP synthesis) which is a target for metal toxicity. In contrast, bacterial prokaryotic cells generate their ATP through the electron transport chain located in the cytoplasmic membrane. The exposed nature of the electron transport chain in prokaryotes would make this target more susceptible to metal influence than the more protected mitochondria in eukaryotes. Additionally, the thick cell wall of some eukaryotic cells, e.g., fungal cells, can confer some resistance when compared to the thinner cell walls of prokaryotes. Specifically within the prokaryotes, comparing gram positive and gram negative bacteria to each other, gram positive bacteria have a thick peptidoglycan cell wall where metals bind whereas reactive metals can often be found in the periplasm of gram negative bacteria.
2. In a metal-contaminated lake, discuss metal bioavailability throughout the water column including the sediment. Metals become less bioavailable with depth from surface to sediment in the water column. Metals are generally more soluble under oxidizing conditions existing as soluble metal salts and ionic species, such as CdCl2 and Cd2+. With depth, however, conditions become more reducing and a shift in the metal species to less soluble forms is observed. For example, in reducing sediments where conditions are anaerobic, sulfate-reducing bacteria predominate resulting in metal precipitation as metal sulfides, e.g., CdS, reducing metal bioavailability.
3. Is dredging a viable option for removal of metal contamination in an aquatic system? What are the potential problems? Depending on the volume and depth of the contaminated sediment, dredging is a viable option in that it will physically remove the metal-contaminated material from an aquatic system. However, in many systems, the contaminated sediments are anaerobic and the process of dredging introduces oxidizing conditions to the otherwise anoxic sediments. In particular, oxidizing conditions can solubilize cationic metals. Once soluble, the metals are more mobile and chemically reactive, increasing bioavailability.
4. What factors need to be considered when bioaugmenting a metal-contaminated site with a metal resistant microorganism? Factors influencing the survival of an introduced microorganism have to be considered. Is the metal-resistant microorganism an aerobe or an anaerobe? Will the organism carry out metal-detoxification under oxidizing or reducing conditions? Other factors for consideration include pH, nutrient availability, and even metal concentration, e.g., is the metal concentration appropriate for the remediative ability of the introduced organism?
In order for bioaugmentation to be successful, the introduced organism has to survive long enough to carry out the desired metal transformation. In some cases, the organism may become part of the indigenous microbial community. In other cases, the introduced microorganism may transfer its metal resistance genes to recipient indigenous microbial populations. In either case, resistance is established and remediation can occur.
5. Which chemical groups in proteins are most reactive with metals? In nucleic acids? In membranes? Metals are highly reactive with numerous chemical groups on proteins, nucleic acids and membranes. Specifically, the sulfhydryl groups in proteins are most susceptible to metal attack, while the phosphate groups in nucleic acids readily react with metals disrupting the phosphate backbone. Membranes are a more complicated system as any charged group, e.g., hydroxyl and phosphate groups, is vulnerable to metal binding and interference. For example, membranes contain proteins involved in nutrient transport, environmental chemotaxis, and energy generation. Due to their partial external nature, membrane-associated proteins are highly susceptible to metal interference. Membrane integrity can also be at risk when metals are present, as some metals can cross the membrane to enter the cell.
6. Metal resistant microorganisms are often isolated from noncontaminated environments (with no prior metal exposure). Discuss possible reasons why. Metal resistant microorganisms can be found in noncontaminated environments because of the development of early resistance mechanisms to the toxic metals present on early Earth. Additionally, metal resistance can occur through the use of and modification of existing mechanisms. For example, exopolymer production occurs in a number of microorganisms and not necessarily in response to metal exposure. However, an exopolymer producing microorganism is inherently resistant to metals due to the ionic interactions with the exopolymer. Likewise, a surfactant-producing microorganism and a sulfate-reducing microorganism both have mechanisms already in place to detoxify metals, e.g., biosurfactant-metal complexation and formation of metal-sulfides, respectively.
7. A metal-contaminated soil has been remediated using a metal-complexing microorganism. What factors need to be considered to ensure that the metal does not become “reavailable ” ? In order for microbially-induced metal-immobilization to be a long-term treatment of a metal-contaminated soil, the metal-complexing organism must become part of the soil microbial community. As part of the community, the metal-complexing microorganism can continuously immobilize the metal as soil conditions change or if there is new metal input. Contributing to the stabilization of the metal, however, is prevention of environmental changes that may result in either (i) kill the metal-complexing population or (ii) result in metal solubilization. Such changes include a dramatic pH fluctuation as may occur with chemical introduction; oxidation of the soil which can result from soil disturbance; or loss of organic materials which can result in decreased metal binding as well as decreased microbial activity.
8. Summarize the possible mechanisms of metal resistance in microorganisms and discuss which mechanism would be most effective in remediating a metal-contaminated surface soil, a metal contaminated soil in the vadose zone, a metal-contaminated stream, and a metal-contaminated groundwater. General mechanisms of metal resistance involve those mechanisms not specifically designed to detoxify metals but do so because of nonselectivity. General mechanisms include nonspecific metal reduction as metal sulfides; metal complexation due to ionic interactions with cell wall components and extracellular binding to exopolymers; and metal complexation with siderophores and biosurfactants, both produced for other purposes by the cell. Metal-dependent mechanisms of resistance involve mechanisms specifically designed to address metal toxicity. Examples include metal efflux systems, which actively remove metals from the cell; intracellular complexation by metal-binding metallothioneins; and methylation of certain metals, such as mercury.
Only certain metal resistance mechanisms are appropriate for remediation purposes. In remediating a contaminated surface soil, metal removal mechanisms, such as surfactant binding and solubilization or metal methylation would be most effective. These types of mechanisms facilitate metal removal through soil washing techniques. In remediating a metal-contaminated subsurface vadose zone soil, complexation mechanisms would be most effective. Due to the difficulty in removing subsurface soils for treatments, mechanisms to reduce metal mobility and toxicity in situ are ideal. This can be accomplished through modification of existing soil conditions to facilitate the growth of indigenous microorganisms or through bioaugmentation with a complexing microbial population. Metal reduction as metal sulfides is an efficient mechanism for treating metal-contaminated sediments in a contaminated stream. The existing anaerobic conditions of the sediment will encourage the growth of sulfate-reducing bacteria and the precipitation of metals as sulfides, which are less toxic and insoluble. To control metal content in the free flowing water in a stream, a microibally-based bioreactor system can be used to trap and complex metals for removal. Finally, a similar approach can be used to treat metal-contaminated groundwater. In this case, groundwater will need to be pumped to the surface for treatment with a metal-complexing microorganism.
9. Discuss the fate (both chemical and biological) of lead in (a) an acid soil, pH 4.0; (b) a neutral soil; (c) a basic soil, pH 8.5; (d) an anaerobic soil; and (e) an aerobic–anaerobic soil interface. (a) Lead in an acidic soil will be found in its free ionic species (Pb2+) and other readily soluble species, such as Pb(NO3)2. Lead in these forms are biologically available and toxicity effects will be observed.
(b) Lead in a neutral soil will be less available and less toxic. Lead will more likely exist as hydroxides (Pb(OH)2) and salts with limited chemical and biological reactivity, although some reactivity will still be present.
(c) Lead will be least soluble and least toxic in a basic soil where the predominate lead forms will be as insoluble carbonates (Pb(CO3)2).
(d) Under anaerobic conditions, lead will most likely exist as lead sulfide (PbS), an insoluble and non-toxic form of lead.
(e) At the aerobic-anaerobic interface, lead will exist as both soluble and insoluble forms, and will achieve equilibrium between the insoluble, anaerobic species and the more soluble, aerobic species. The anaerobic forms of lead can become soluble as the aerobic side of the interface is encountered and visa versa with soluble species becoming less soluble anaerobically.