1. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 1 Applied Environmental Microbiology Chapter 35

2. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 2 Water Purification and Sanitary Analysis water purification –critical link in controlling waterborne disease

3. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 3 Insert Table 35.1

4. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 4 Water Purification water with high levels of suspended material  sedimentation basin –large particles settle out partially clarified water mixed with chemicals such as alum and lime and moved to a settling basin –more material precipitates out in coagulation or flocculation process removes microbes, organic matter, toxic contaminants and suspended fine particles

5. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 5 Water Purification… water  rapid sand filters –physically traps fine particles and flocs water treated with disinfectant –chlorine concern about the creation of disinfection by-products such as trihalomethanes which may be carcinogens –ozone

6. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 6 Water purification sedimentation basin settling basins

7. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 7 Problem Microbes not consistently removed by coagulation, rapid sand filtration, and disinfection processes –Giardia lamblia “backpackers disease” slow sand filters effectively remove Giardia cysts –Cryptosporidium small protozoan with oocysts that escape usual purification schemes –Cyclospora protozoan that causes diarrhea –viruses up to 99.9% are removed by usual purification schemes, but this not considered sufficient protection

8. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 8 Sanitary Analysis of Waters based on detecting indicator organisms –indicate fecal contamination of water supplies –indicate possible contamination by human pathogens

9. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 9 “Ideal” indicator organism suitable for analysis of all types of water present whenever enteric pathogens are present survives longer than hardiest enteric pathogen does not reproduce in contaminated water detected by highly specific test –test easy to do and sensitive harmless to humans its level in water reflects degree of fecal pollution

10. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 10 Two commonly used indicators Coliforms e.g. E. coli, Enterobacter aerogenes, Klebsiella pneumoniae fecal streptococci –increasingly used to test brackish and marine water

11. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 11 Coliforms facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hours at 35°C e.g. Escherichia coli, Enterobacter aerogenes, Klebsiella pneuminiae traditional method of detection is multiple-tube fermentation test

12. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 12 presumptive test confirmed test completed test Multiple-tube fermentation test

13. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 13 Other Tests for Indicator Organisms membrane filtration technique presence-absence (P-A) test defined substrate tests

14. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 14 Membrane filtration technique water passed through filter  filter placed on surface of growth medium  incubate  count colonies Used to detect total coliforms, fecal streptococci and fecal coliforms - from intestines of warm blooded animals - detected by incubation at 44.5°C

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16. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 16 Presence-absence test modification of MPN uses larger water sample (100 ml) sample added to lactose containing medium –contains pH indicator to detect acid production based on assumption that no indicator organisms should be present in 100 ml of water detects total coliforms and fecal coliforms

17. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 17 Defined substrate tests e.g., Colilert detects total coliforms and fecal coliforms 100 ml sample added to medium containing ONPG and MUG * ONPG: o-nitrophenyl-β-D-galactopyranoside * MUG: 4-methylumbelliferyl-β-D-glucuronide (a)Uninoculate control (b)Yellow color (o-nitrophenyl) due to the presence of coliforms (c)Fluorescent reaction due to the presence of fecal coliforms (E. coli modifies MUG to a fluorescent product)

18. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 18 Wastewater Treatment decreases organic matter and number of microorganisms in human waste-impacted water has lead to major reduction in spread of pathogens

19. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 19 Measuring Water Quality three major approaches –total organic carbon (TOC) –chemical oxygen demand (COD) –biochemical oxygen demand (BOD)

20. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 20 Total organic carbon quantifies all carbon organic matter oxidized at high temperature amount of CO 2 produced is measured fastest, but less informative

21. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 21 Chemical oxygen demand quantifies organic matter (except lignin) organic matter oxidized with strong acid (permanganate) slower test high cost for chemical waste disposal

22. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 22 Biochemical oxygen demand measures amount of dissolved oxygen needed for microbial degradation of organic matter –indirect measure –5 days at 20 o C can be affected by presence of ammonia –nitrogen oxygen demand use of oxygen during nitrification process inhibited by addition of chemicals to sample

23. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 23 Wastewater Treatment Processes similar to self-purification processes observed in rivers and streams except are controlled and intensified

24. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 24 An aerial view of a modern conventional sewage treatment plant in New Jersey

25. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 25

26. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 26 Activated sludge system

27. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 27 normal floc – settles well bulking sludge - does not settle properly

28. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 28 Trickling filter system

29. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 29 Extended aeration process Extended aeration period Reduces amount of sludge produced

30. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 30 Anaerobic digestion often sludges from aerobic sewage treatment, together with materials settled out in primary treatment are further treated by anaerobic digestion reduces the amount of sludge for disposal produces methane

31. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 31

32. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 32 Constructed wetlands Employed in treatment of liquid wastes and for bioremediation

33. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 33 Home Treatment Systems groundwater –water in gravel beds and fractured rocks below surface soil microbiological processes in groundwater are not well understood

34. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 34 Home septic tank system Frequently fail to work properly, contributing to groundwater contamination - this can lead to nutrient enrichment of other water bodies that groundwater flows into

35. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 35 Biodegradation and Bioremediation by Natural Communities metabolic activities of microbes can be exploited in natural environments –where physical and nutritional conditions for growth cannot be controlled –a largely unknown microbial community is present

36. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 36 examples –use of microbial communities to carry out biodegradation, bioremediation and environmental maintenance processes –addition of microbes to soils or plants for the improvement of crop production Biodegradation and Bioremediation by Natural Communities

37. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 37 biodegradation has at least three definitions –minor changes in an organic molecule leaving the main structure still intact –fragmentation of a complex organic molecule in such a way that the fragments could be reassembled to yield the original structure –complete mineralization Biodegradation Using Natural Microbial Communities

38. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 38

39. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 39 Bioremediation the use of microbes to transform toxic molecules to nontoxic degradation products the degradation of toxic molecules requires several stages, usually performed by different microbes reductive dehalogenation –removal of a halogen substituent while at the same time adding electrons to the molecule –usually occurs under anaerobic conditions

40. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 40 Fate of a chemical in Nature structure and stereochemistry play critical role in predicting the fate of specific chemical –meta effect occurs when constituent is in meta, as opposed to ortho position, the compound will be degraded at a slower rate many compounds added to environments are chiral –possess asymmetry and handedness enantiomer, stereoisomer microbes often can degrade only one isomer of a substance; the other isomer will remain in the environment

41. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 41 Meta effect ortho position

42. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 42 microbial communities change their characteristics in response to addition of inorganic or organic chemicals acclimation –occurs if chemical is repeatedly added, the community adapts and faster rates of degradation occur Fate of a chemical in Nature

43. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 43 Repeated exposure and degradation rate

44. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 44 Downside of Biodegradation some biodegradation processes can lead to the formation of degradation products with similar, or increased, levels of toxicity –e.g., conversion of DDT to DDE –e.g., conversion of TCE to vinyl chloride can lead to widespread damages and financial losses if occurs in inappropriate situation or in an uncontrolled manner e.g., corrosion of metals, especially iron pipes

45. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 45 Toxic degradation products

46. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 46 Microbial-mediated metal corrosion (e.g. Desulfovibrio)

47. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 47 Stimulating Biodegradation bioremediation usually involves stimulating degradative activities of microbes already present at contaminated sites it is necessary to determine the limiting factors at the site (e.g., nitrogen or phosphorus or other nutrients) and supply them or modify the environment

48. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 48 Changing Environmental Conditions to Stimulate Biodegradation engineered bioremediation –addition of O 2 or nutrients to soils or water in order to stimulate biodegradative activities of microbes –addition of easily metabolized organic matter increases degradation of recalcitrant compounds that are not usually used as carbon or energy sources an example of cometabolism

49. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 49 Some Examples stimulation of hydrocarbon degradation in aquatic environments phytoremediation –use of plants in bioremediation stimulation of bioleaching of metals from minerals –important in recovery of metals

50. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 50 Bioleaching Leptospirillum ferrooxidans Thiobacillus thiooxidans (Fe 2+ ) (Fe 3+ )

51. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 51 Bioaugmentation addition of natural (or genetically engineered) microorganisms to complex microbial communities generally has resulted in only short-term increases in desired degradative activity outcome can be improved by providing protective microhabitats –living microhabitats (e.g., surface of a seed, root, or leaf) –inert microhabitats (e.g., microporous glass) natural attenuation –use of natural microbial communities to carry out biodegradative process