Microorganisms and Organic Pollutants

1. Microorganisms and Organic Pollutants Chapter 20 Lecture 16

2. Legacy Waste • 430,000 confirmed cases of leaking underground storage tanks in U.S. as of 2003 • >90% of the monitored stream and >55% of shallow underground sites contaminated with pesticides • >1.4 million acres of chemical plumes in groundwater

3. Dealing with the problem • National Environmental Policy Act in 1970 • Environmental Impact Statements • Applicant required to take a hard look at the environmental consequences of the proposed action (development). • Environmental laws • Clean Air Act • Clean Water Act • Comprehensive Environmental Response, Compensation and Liability Act (Superfund) • Superfund Amendments and Reauthorization Act

4. Pollutants naturally-occurring compounds in the environment that are present in unnaturally high concentrations. Examples: crude oil refined oil phosphates heavy metals Xenobiotics chemically synthesized compounds that have never occurred in nature. Examples: pesticides herbicides plastics What are environmental contaminants?

5. Types of contamination • Point source contamination • contaminant emanating from a defined source • discharge pipe from an industrial operation • Non-point-source • source of contaminant emanating from a large area • fertilizers or pesticides applied to agricultural land

6. Overall process of biodegradation Electron donor Carbon source Organic C Carbon dioxide H2O Electron acceptor O2aerobic respiration NO3, Fe(III), Mn(IV), SO4, CO2anaerobic respiration

7. Complete (mineralization) Glucose→ CO2 + H2O TCA cycle Complete vs incomplete biodegradation Incomplete Glucose → pyruvate →→CO2 + H2O TCA cycle

8. 2e-, H+ 2e-, H+ 2e-, H+ H Cl Cl H Cl Cl Cl H Cl Cl H H Cl H H H H Cl Cl H Cl Cl Cl Biological Reductive Dechlorination Pathway PCE TCE cis-1,2-DCE vinyl chloride 2e-, H+ Cl Vinyl chloride intermediate is more toxic than PCE ethene

9. Biological Reductive Dechlorination Pathway Hydrogen is preferred electron donor VC ethene 93

11. Cometabolism • Bacterium uses some other carbon and energy source to partially degrade contaminant degradation products contaminant bacterium corn starch CO2 + H2O

12. Consortium interactions • Bacterium A uses some other carbon and energy source to partially degrade contaminant. • Bacterium B metabolizes contaminant degradation products to carbon dioxide and water. Bacterium B CO2 + H2O O2 contaminant degradation products Bacterium A O2 corn starch CO2 + H2O

13. Combining cometabolism and consortium interactions (TCE) CH4 Cl2C=CHCl Methanotrophic bacterium Methane Monooxygenase O CH3OH Cl2-CHCl Other populations of bacteria H2CO HCOOH CO2 CO2 + Cl-

14. Carrying Capacity • Although many chemical contaminants in the environment can be readily degraded because of their structural similarity to naturally occurring organic carbon, the amounts added may exceed the carrying capacity of the environment. • Carrying capacity is the maximum level of microbial activity that can occur under the existing environmental conditions

15. What limits carrying capacity? • Physical-chemical factors • pH, temperature, nutrients • types of microbes present and their biomass Low carrying capacity High carrying capacity Contaminant breakthrough No contaminant left

16. Determinants of extent and rate of contaminant biodegradation • Genetic potential of microbes to mutate key genes in such a way that gene product (enzyme) can catalyze step in contaminant degradation • This requires period of time for such adaptation to occur (weeks, months, years?)

17. Determinants of extent and rate of contaminant biodegradation • Bioavailability • First step in biodegradation process is the uptake of the contaminant compound by the cell in order for intracellular enzymes to access the contaminant • If contaminant is not water-soluble, it is difficult for cell to access and take up contaminant. Low-density, non-aqueous phase liquid (hydrocarbon, benzene H2O Dense, non-aqueous phase liquid (TCE, PCBs)

18. Determinants of extent and rate of contaminant biodegradation • Bioavailability • Production of surfactants • Attachment to liquid-liquid interface Low-density, non-aqueous phase liquid (hydrocarbon, benzene H2O Dense, non-aqueous phase liquid (TCE, PCBs)

19. Determinants of extent and rate of contaminant biodegradation • Bioavailability • Production of surfactants • Attachment to liquid-liquid interface • Make cell surface more hydrophobic-nonpolar LPS or EPS Low-density, non-aqueous phase liquid (hydrocarbon, benzene H2O Dense, non-aqueous phase liquid (TCE, PCBs)

20. Contaminant Particle Determinants of extent and rate of contaminant biodegradation • Bioavailability • Sorption of contaminant to soil particles • Diffusion of contaminant into soil matrix Bacterial cell Soil particles

21. Determinants of extent and rate of contaminant biodegradation • Bioavailability • Sorption of contaminant to soil particles • Diffusion of contaminant into soil matrix Contaminant no longer available to microbes contaminant Soil particles

22. Determinants of extent and rate of contaminant biodegradation • Contaminant structure • Steric effects active site for enzyme blocked

23. Determinants of extent and rate of contaminant biodegradation • Contaminant structure • Electronic effects • as electronegativity of substituents increased, biodegradation rates decreased

24. Electonic Effects

25. Determinants of extent and rate of contaminant biodegradation • Environmental factors • organic matter (source of carbon and energy) • subsurface, unsaturated zones have low organic matter concentrations • oxygen availability • nutrient (N,S, P) availability • temperature • pH • Eh • salinity • water activity

26. Most important factors controlling contaminant biodegradation

28. Bioaugmentation • The addition of microorganisms with specific metabolic capabilities that are under-represented in the natural microbial populations that will promote degradation of the contaminant. • This can increase carrying capacity of the system to degrade a contaminant

29. Bioaugmentation • There are lots of companies around today that sell a variety of "formulations" to: • remove animal wastes • keep ponds free of algae • clean up gasoline leaked from underground storage tanks

30. Hard to degrade contaminants • Chlorinated hydrocarbons • solvents • lubricants • plasticizers • insulators • herbicides and pesticides.

31. Cl Cl Cln Cln Types of chlorinated compounds • Aromatics • Benzene • Poly chlorinated biphenyls

33. Meta-pathway for catechol degradation is often used for degradation of chlorinated aromatics Catechol is a common intermediate for metabolizing many aromatic compounds and utilizes enzymes encoded by the catA, catB, catC, and catD genes

34. CatR cis, cis-muclonate

36. p ORF1pheBpheA ORF2 Promoter forms 2 complexes with catR in absence of inducer and 1 complex with catR in presence of inducer catC catB p catR plasmid activation CatR chromosome phenol, cis, cis-muconate are inducers

37. The chlorocatechol degradative pathway is used to degrade these chlorinated compounds. • Similar to catechols, chlorocatechols are common intermediates of the degradation of chloroaromatics such as chlorobenzenes and chlorophenoxyacetates. • Chlorocatechol-degrading genes that have been isolated from bacteria: • clc for chlorocatechol • tcb for trichlorobenzene • tfd for 2,4-dichlorophenoxyacetate

38. The layout of the genes involved in chlorocatechol-degradation on the plasmid is similar to the layout of the catechol-degrading genes on the chromosome

39. The clcABD operon is positively regulated by the clcR gene product, just as the catBC operon is controlled by the catR gene product. • The clcA, tcbC and tfdC genes, all of which encode a similar chlorocatechol dioxygenase activity, have high nucleotide sequence identity. • The clcB, tcbD and tfdD genes, all of which encode a similar chloromuconate cyclosomerase activity, have high nucleotide sequence identity.

40. Each of these chlorocatechol-degrading genes closely resembles the corresponding catechol-degrading cat genes, implying they evolved from common ancestral genes. • CatR and ClcR cross-bind each other’s target promoter regions, indicating that the regulatory regions have considerable homology. • CatR can regulate the clcABD operon but ClcR cannot regulate the catBC operon.

41. Treatment strategies for subsurface contamination

42. In situ

43. Summary • Many factors control biodegradability of a contaminant in the environment • Before attempting to employ bioremediation technology, one needs to conduct a thorough characterization of the environment where the contaminant exists, including the microbiology, geochemistry, mineralogy, geophysics, and hydrology of the system