What is bioremediation?

1. What is bioremediation? The use of bacteria and fungi and plants to break down or degrade toxic chemical compounds that have accumulated in the environment

2. Site of contaminants • Water • Air • Soil • Indoor environments

3. Sources 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

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. Non-point source: Agricultural land receiving fertilizers & pesticides Point source Point source aquifer Subsurface contamination

6. Teknik Bioremediasi • Optimasi kontak antara mikroorganisme dengan pencemar yang dimanfaatkan sebagai sumber makanan • Lebih ditujukan pada materi organik • Teknik bioremediasi tanah tercemar: • In-situ: pengolahan setempat • Ex-situ: pengolahan di tempat lain

7. CO2 CO2 NH4 PO4 The importance of microorganisms You could probably remove all higher organisms without significantly altering the Earth’s biogeochemistry or homeostatic properties. Microbial Systems responsible for decomposition of dead organic material (detritus) under aerobic and anaerobic conditions Higher Trophic Levels Metabolically Simple Animals Microbial Systems Detritus Plants As well as being consumed by higher trophic levels, microbial systems also recycle many inorganic nutrients: N, P, S, Trace metals. William B. Whitman, David C. Coleman, and William J. Wiebe. Prokaryotes: The unseen majority. PNAS 95 (12):6578-6583, 1998.

8. Rumen Microbial Ecosystem Complex anaerobic microbial system found in the rumen Feed (grasses or grain) Cellulose and Starch Glucose Fermentation Lactate Succinate H2 + CO2 Formate Methanogens Acetate Propionate Butyrate CH4 CO2 Protein Greenhouse gases Digested Reactions mediated by dozens of bacterial species, including protozoan grazers such as ciliates. Similar systems found in termites

9. Protein Biomass Cellulose Lipids Glucose AA Glyc LCFA Acetate CO2 H2 Diversity of Anaerobic Methanogenic Pathways EtOH Lact Prop Buty CH4 Biogas: CH4 + CO2 “Distributed Metabolic Network”

10. H2S oxidation by NO3- Anammox NH4+ + NO2- = N2 + 2H2O CH4 oxidation by SO42- Chemical Potential Exploitation Boetius et al. 2000: Schulz et al. 1999: Thiomargarita namibiensis Strous et al. 1999: Planctomycete 1 mm CH4 oxidation by NO3-(Raghoebarsing et al. 2006) 5CH4 + 8NO3- + 8H+ 5CO2 + 4N2 + 14H2O

11. Other examples, Microbially-coupled Systems Symbiosis and Endosymbiosis Lichen: Fungi+Algae Dinoflagellates in flatworm Sulfur bacteria in Riftia Mycorrhizae Caldwell et al. 1997: Biological systems develop with multiple levels of organization and multiple levels of proliferation.

12. Geoengineering and Biofuels Iron Fertilization? Biodiesel from Algae Oceanus, 46 (1) 2008 http://www.whoi.edu/oceanus/

13. Other Important Microbial Processes • Nutrient cycling under aerobic and anaerobic conditions. • Removal of excess nitrogen via nitrification and denitrification in eutrophic systems. • NH4 NO3 N2 • Fixation of N2 gas into organic N, especially in root nodules via symbiosis with bacteria, such as Rhizobium. • N2 Amino Acids • Remediation of toxic substances (bioremediation or natural attenuation). • Almost all biomass and processes in the oceans are are dominated by microbes. • Largest organism on Earth is a fungus (Armillaria ostoyae; honey mushroom). • Major sources and sinks for atmospheric trace greenhouse gasses (CH4, N2O). • Cycling of iron (Fe3+ Fe2+), Manganese (Mn4+ Mn2+) and other metals. • Cause of many diseases, especially in 3rd world countries. • Very high species abundance, current estimate of 107 species in 10 g of soil.

14. Microbial Systems as Model Ecosystems Closed Systems (such as Earth) Energy P Primary producers Self organization R Resources C Heat (Entropy) Consumers

15. Metabolic Classification of Life

16. Winogradsky Column • Sediment supplements: • CaCO3 • CaSO4 • Carbon source H2S O2 Cyanobacteria Algae Water Sulfur bacteria Purple nonsulfur bacteria • All five major metabolic groups will develop • Sulfate reduction • S oxidation • Fermentation • Photosynthesis PS II • Photosynthesis PS I • Methanogenesis • Nitrification • Denitrification? Purple S bacteria Green S bacteria Sediment Desulfovibrio Clostridium

17. Contaminants Potentially Amenable to Bioremediation____________________________________________

18. Pollutants may exist at high, toxic concentrations degradation may depend on another nutrient that is in limiting supply Xenobiotics microbes may not yet have evolved biochemical pathways to degrade compounds may require a consortium of microbial populations What challenges exist for bioremediation of pollutants and xenobiotics?

19. Fundamentals of cleanup reactions • Aerobic metabolism • Microbes use O2 in their metabolism to degrade contaminants • Anaerobic metabolism • Microbes substitute another chemical for O2 to degrade contaminants • Nitrate, iron, sulfate, carbon dioxide, uranium, technicium, perchlorate

20. Metabolism of a Pollutant-degrading 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

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

22. Volatile organic compounds (VOC) • These are major contributors to air pollution • Paint industry • Pharmaceutical industry • bakeries • printers • dry cleaners • auto body shops

23. Cometabolism • Bacterium uses some other carbon and energy source to partially degrade contaminant (organic aromatic ring compound) degradation products contaminant bacterium corn starch CO2 + H2O

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

25. Degradation of chlorinated hydrocarbons • Degradation of organic toxins requires the participation of entire biochemical pathways involving many enzymes coded for by many genes. • Some of the genes exist on the chromosome while other genes reside on plasmids.

26. CO2 + H2O • Phenol-degrading dmp operon is regulated by DmpR, a NtrC-like positive regulator.

27. Genetic engineering of bacteria to remove toxic metals from the environment E. coli bacterium New gene/transport proteins Hg2+-metallothein Hg2+→Hgo Hg2+ New gene/enzyme Hgo (less toxic form of metal)

28. Questions to answer before starting bioremediation

29. What is limiting nutrient at site of contaminant? • nitrogen • add as ammonium chloride • phosphate • add as polyphosphate, tri-metaphosphate • electron donor • cheap source of energy • electron acceptor • oxygen as air, or nitrate, sulfate

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

32. 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

33. 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-

34. 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

35. 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

36. 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

37. 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

38. Examples of brand-name products on the market • Oil Spill Eater • Alpha Biosea • Marine D • Bioblend • A mixture of microbes is on the market that will deodorize pig manure.

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

40. 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

41. Most important factors controlling contaminant biodegradation

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

43. Degradation of chlorinated hydrocarbons Degradation of organic toxins requires the participation of entire biochemical pathways involving many enzymes coded for by many genes. Some of the genes exist on the chromosome while other genes reside on plasmids.

44. Groundwater contamination • Groundwater constitutes 96% of available freshwater in U.S. • 95% of potable water in rural areas of U.S. comes from groundwater • In 1988, EPA confirmed that 26 states had various amounts of 44 different pesticides in their groundwater • Cost of cleanup is in the $ trillions • Issues that are still hotly debated • How clean is clean?

46. Specific Technologies • Established Technologies • Incineration- ex situ; destructive • Excavation- ex situ; isolation

47. Bioremediation • in situ; destructive • Uses microorganisms such as bacteria in engineered processes to break down organic contaminants into harmless substances

48. In - Situ • Kelebihan: • Mengurangi gangguan thd lokasi • Pengolahan pencemaran yang lebih dalam • Kontak dengan pencemar minimal terutama pencemar volatil • Mengurangi biaya transportasi

49. In - situ • Kekurangan: • Data geohidrologi yang lengkap • Pengendalian kondisi reaksi dan hasiol akhir sulit • Monitoring yang lebih hati-hati • Perlu rekayasa lebih lanjut untuk suply oksigen dan nutrien

50. In - Situ • Contoh: • Soil-venting: kontaminan yang volatil dan di evakuasi untuk diolah lebih lanjut • Bio-venting: kontaminan semi dan non-volatil dengan suplai oksigen dan nutrien