Chapter 21 Microorganisms and Metal Pollutants

1. Chapter 21Microorganisms and Metal Pollutants

2. Metals Defined • Metals • Gold, silver, copper, • Metaloids • Arsenic, boron, germanium tellurium • Heavy metals (environmental toxicity) • Arsenic, cadmium, copper, chromium, mercury, lead, zinc

3. Oligodynamic Effect of Metals • Many metals are required for normal biological functions • Iron, cobalt, nickel, copper, zinc • Many of the metals with known biological function can be toxic at high concentrations • Toxic metals exert their toxicity in a number of ways • Displacement of other essential metals from their binding site on biological molecules • Arsenic and cadmium compete with phosphate and zinc, respectively • Bioavailability: A metal that can be taken up by an organisms is considered to be bioavailable

4. Total versus soluble metal • In the environment, the soluble form of a metal is usually a small fraction of the total metal. Add 100 mg/L of a metal to a water sample and you retain only 20 mg/L Cd and 0.12 mg/L Cu in solution

5. Summary of various toxic influences of metals on the microbial cell

6. Mechanisms of metal resistance and detoxification Most common way microbes prevent metal toxicity is to pump the metal back out of the cell

7. Mercury • Why are we concerned with mercury? • High toxicity due to the affinity of Hg to sulfur – disruption of protein structure and function • Resistance in eukaryotes – biosynthesis of sulfhydryl-rich compounds (metallothioneins, phytochelatins). They contain –SH groups • Prokaryotes detoxify by reduction of Hg(II) to Hg(0) and subsequent volatilization

8. Anthropogenic Emission of Hg into the environment • Burning of fossil fuels • Coal fired power plants contributes 65% of anthropogenic emissions • Metal mining operations • gold and silver • Metal smelting and refining • Cement manufacture • Chemical manufacture • Production of goods • Disposal of Hg-waste • Municipal landfills

9. Volcanoes Deep-sea vents Natural Emission of Hg into the Biosphere Terrestrial hot springs

10. Hg(II) (via precipitation) Hg(0) →Hg(II) photochemical Microbes

11. Biomagnification of methyl-Hg Bacteria Algae Copepod HgCl2 CH3HgCl 0.010 ng/L 5 ng/g 30 ng/g Small fish Large fish Humans 5000 ng/g 180 ng/g 1000 ng/g

12. Bioaccumulation of methyl-Hg • Accumulates in tissues over time • Concentrates in the muscle tissue of fish • Accumulates in the envelopes of nerve cells • 100x more toxic than Hg0 and Hg2+ • Destroys muscle proteins and enzymes essential to cell function

13. The mercury resistance (mer) system in microbes CH3Hg(I) Lin et al., In: Environmental Chemistry and Toxicology of Mercury, in press • Common among bacteria in soils and natural waters • Applications in bioremediation and in monitoring of mercury in the environment

14. Radionuclides • Radioactive elements contribute to environmental contamination • Department of Energy has been releasing radionuclides produced during nuclear bomb manufacturing into the environment since the 1940s • Plutonium, uranium, cesium, technicium • These elements have a long half-life • Their concentration in environment is typically low, but the radiation produced by low concentrations is still toxic to higher life forms

15. Dissimilatory Metal Reduction • Some microorganisms can use metals and radionuclides as terminal electron acceptors. This is an enzymatic process and is termed dissimilatory metal reduction. It is also sometimes called direct metal reduction • Other microorganisms can reduce metals and rads indirectly through non-enzymatic mechanisms, usually involving a reaction between a microbial end product and the metal

16. e- e- e- e- Glucose 2 Pyruvate e- O2, Fe(III), NO3-, SO4 2-, etc. e- NADH Krebs Cycle FADH Cytochrome System e- H2O, Fe(II), N2, S2-, etc. Schematic of Dissimilatory (Direct) Reduction 2 Acetyl CoA

17. Bacterial Fe(III) Reduction • Not all Fe(III) reducing bacteria generate usable cellular energy via Fe(III) reduction. However, under certain conditions, iron reduction can potentially generate substantial energy. For example, when acetate is oxidized the standard free energy change at pH 7 is: -193.4 kcal/mol for Fe(III) -201 kcal/mol for O2 -5.5 kcal/mol for Fe(OH)3 This shows that insoluble iron oxides and hydroxides are less favored electron acceptors at neutral or alkaline pHs

18. 2 Lactate + SO42- + H+  2 Acetate + 2CO2 + 2H2O + HS- Indirect (Non-enzymatic) Metal Reduction • Hydrogen sulfide produced by sulfate reducing bacteria may reduce ferric to ferrous iron via the following equation:

19. (at pH 7-9) 2HFeO2 + 3H2S 2FeSppt + So + 4H2O Indirect Metal Reduction goethite greigite mackinawite pyrrhotite pyrite Adapted from: Geomicrobiology by H. L. Erhlich

20. Microbial dissimilatory metal/rad reduction is a rapidly evolving area of study. In the past several years investigators have discovered that microbes are capable of directly reducing a wide variety of metals/rads. • These discoveries are of considerable importance because they provide: • Information on natural metal cycling and deposition in nature • Potential bioremediation options metals and radionuclides

21. Metabolism of a Fe(III)-Reducing Bacterium Fe(III) ACETATE *U(VI) *Co(III) *Cr(VI) *Se(VI) *Pb(II) *Tc(VII) *Benzoate *Toluene *Phenol *p-Cresol *Benzene ATP CO2 Fe(II) *CCl4 *Cl-ethenes *Cl-aromatics *Nitro-aromatics

22. Cr(VI) Fe(III) U(VI) Mn(IV) Se(VI), (IV), (0) Tc(VII) Hg(II) Cu(II) Co(III) Pd(II) Np(V) Pu(IV) Mo(VI) V(V) Au(III), (I) Ag(I) Metals Known to be Reduced via Dissimilatory Microbial Reduction

23. U6+sol U4+insol Uranium reduction leads to uranium precipitation and immobilization U6+sol U6+sol U4+insol

24. bacterium U6+sol U4+insol Mineral particle-associated metal reducing bacteria as catalysts of uranium reduction

25. Nutrients electron donors bacteria precipitated U4+ Biostimulation Goal: to promote uranium reduction and immobilization • increase microbial biomass to increase enzyme-mediated transformation • avoid excess biomass production that leads to formation plugging (impedes nutrient delivery to U reduction zone)

26. Bacterium Flow Flow Matrix Matrix Matrix Matrix Bacteria with exopolymer slime Mineral surface Avoid stimulation that leads to excess bacterial biomass accumulation in flow path of fluid delivering nutrients to stimulate metal reduction

27. NABIR Field Research Center • Located on the Oak Ridge Reservation • S-3 Ponds consist of 4 unlined ponds constructed in 1951 at the Y-12 Plant • Ponds received liquid wastes composed of nitric acid plating wastes containing nitrate and various metals and radionuclides (U, Tc) from 1951-1983.

28. Groundwater • Contains >40,000 mg/L total dissolved solids • S-3 Pond plume contains elevated levels of nitrate, bicarbonate, Al, U, Tc, and tetrachloroethylene • Plume is stratified • Mobile nitrate and Tc are extensively distributed • Nitrate has migrated approx. 1 km in the Nolichucky Shales since 1951 via preferential pathways. • Less mobile U and other metals are more restricted in distribution

29. Are indigenous bacteria capable of uranium reduction?

30. Evaluate ability of subsurface microbial communities at this site to reduce metals • Assess expression of genes known to be involved in metal reduction • Look for evidence of metal reduction during stimulation

31. AREA 3 Nitrate=8200mg/L pH=3.1 U6+=50 mg/L Tc-99=40,000 pCi/L Al=438 mg/L SO4=1,000mg/L TCE=3.1 mg/L Nitrate N pH Al U 2 Reactor effluent Chemical extraction Lactate or EtOH FBR surface Side-stream coupon reactor subsurface Down - hole U reduction zone coupon reactor in MLS wells

32. Summary • Metals can be used by microbes for essential metabolism or be toxic to cells, depending on availability and concentration in the environment. • Most common way for microbes to avoid toxicity is to pump metal back out of cell into the environment. • Some metals are becoming increasingly bioavailable in the environment and this increases exposure and health risks to organisms up the food chain. • Microbes can transform redox active metals from soluble toxic forms to insoluble less toxic forms.