1. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 1 Applied and Industrial Microbiology Chapter 41

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 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

4. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 4 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

5. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 5 Water purification sedimentation basin settling basins

6. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 6 Problem microbes not consistently removed by coagulation, rapid sand filtration, and disinfection processes –Giardia intestinalis “backpackers disease” (diarrhea) 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

7. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 7 Sanitary Analysis of Waters based on detecting indicator organisms –indicate fecal contamination of water supplies –indicate possible contamination by human pathogens

8. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 8 “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

9. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 9 Two commonly used indicators coliforms fecal streptococci –increasingly used to test brackish and marine water

10. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 10 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 for presumptive test

11. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 11 Multiple-tube fermentation test presumptive test confirmed test completed test

12. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 12 Other tests for indicator organisms membrane filtration technique presence-absence (P-A) test defined substrate tests

13. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 13 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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15. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 15 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

16. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 16 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)

17. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 17 Potable water – suitable for human consumption

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 reacted 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

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24. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 24 Wastewater Treatment Processes similar to self-purification processes observed in rivers and streams except are controlled and intensified

25. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 25 An aerial view of a modern conventional sewage treatment plant in New Jersey

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27. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 27 Activated sludge system

28. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 28 normal floc – settles well bulking sludge - does not settle properly

29. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 29 Trickling filter system

30. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 30 Extended aeration process Reduces amount of sludge produced Extended aeration period

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

32. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 32 A sequential process during anaerobic digestion

33. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 33 Constructed wetlands Employed in treatment of liquid wastes and for bioremediation

34. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 34 Home Treatment Systems groundwater –water in gravel beds and fractured rocks below surface soil microbiological processes in groundwater are not well understood

35. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 35 Home septic tank system Frequently fail to work properly, contributing to groundwater contamination.

36. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 36 Choosing Microorganisms for Industrial Microbiology must first find suitable microbes –genetically stable –easy to maintain and grow –well suited for extraction or separation of desire product

37. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 37 Finding Microorganisms in Nature bioprospecting –hunting for new microorganisms most major sources of microbes for use in industrial microbiology have been natural materials hunting for industrially useful microbes in nature continues because < 1% of microbes have been cultured from nature

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39. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 39 Genetic Manipulation of Microorganisms Used to produce microorganisms with new and desirable characteristics –mutation –protoplast fusion widely used with fungi can fuse protoplasts of different species –insertion of short DNA sequences –transfer of genetic information between different organisms –modification of gene expression –natural genetic engineering

40. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 40 mutations used to increase yield of penicillin initial yield Final yield Mutation of Penicillium chrysogenum to increase penicillin yield X-ray (X) UV (UV) Mustard gas (N)

41. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 41 Insertion of short DNA sequences site-directed mutagenesis –insertion of chemically synthesized DNA to create small genetic changes –used in protein engineering deliberately making alterations in amino acid sequence of a protein in order to generate enzymes with new or improved properties

42. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 42 Transfer of Genetic Information Between Different Organisms the transfer and expression of genes between different organisms can give rise to novel metabolic processes and products part of field of combinatorial biology heterologous gene expression –the insertion of functional genes from one organism into another e.g., creation of patented “superbug” with enhanced ability to degrade hydrocarbons

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44. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 44 e.g., recombinant vaccine production

45. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 45 Modification of Gene Expression allows for overproduction of a product, production of more than one product by the same organism, or synthesis of modified products pathway architecture –analysis, design, and modification of biochemical pathways to increase process efficiency –metabolic pathway engineering intentional alteration of metabolic pathway by inactivation of specific genes –metabolic control engineering alteration of control mechanisms of specific genes

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47. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 47 Metabolic engineering to create modified antibiotics 6-deoxyerythronilide

48. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 48 Protein Evolution one of the newest approaches for creating new metabolic capabilities in a given microorganism employs forced evolution and adaptive mutations –involve use of specific environmental stresses to “force” microbes to mutate and adapt, thus creating microbes with new biological capabilities in vitro evolution –starts with purified nucleic acids rather than whole organisms high-throughput screening enables the rapid selection of a single desirable mutant or molecule from tens of thousands of new constructed stains, molecules, or compounds

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50. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 50 Preservation of Microorganisms once microbe or virus has been selected or created, it must be preserved in its original form for further use and study periodic transfers can lead to mutations and phenotypic changes –to prevent this problem, a variety of culture preservation techniques can be used

51. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 51 Preservation of Microorganisms

52. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 52 Microbial Growth in Controlled Environments - Fermentation

53. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 53 Medium Development to maintain economic competitiveness, low-cost crude materials are frequently used levels of minerals (especially iron) and growth factors may be critical

54. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 54

55. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 55 Growth of Microorganisms in an Industrial Setting requires precise control of agitation, temperature, pH changes, and oxygenation non-Newtonian broth –viscous, plastic medium resulting from growth of filamentous fungi and actinomycetes –resists stirring and aeration, making maintenance of appropriate levels of O 2 difficult scaleup –modifications of growth process so that it works efficiently in large fermenters

56. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 56 Filamentous growth during fermentation Initial cultureAfter 18 h growth

57. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 57 A typical industrial stirred fermentation unit

58. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 58 Supplying nutrients can supply a critical nutrient in limiting amounts –can cause shift from growth to production of desired metabolites continuous feed –continual addition of a critical nutrient so that microbes will not have excess substrate available at any given time –prevents production and accumulation of undesirable metabolic waste products

59. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 59 Alternate methods for mass culture

60. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 60 Types of microbial products primary metabolites –related to synthesis of microbial cells in the growth phase –include amino acids, nucleotides, fermentation end products, and enzymes secondary metabolites –accumulate following active growth –have no direct relationship to synthesis of cell material and natural growth –include antibiotics and mycotoxins

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62. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 62 Major Products of Industrial Microbiology industrial and agricultural products food additives products for human and animal health biofuels

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64. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 64 Penicillin production requires precise control of nutrients Final product can be modified to yield a variety of semisynthetic penicillins

65. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 65 Streptomycin production

66. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 66 Amino Acids typically produced using regulatory mutants, which have reduced ability to limit synthesis of an end product

67. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 67 Glutamic acid production by Corynebacterium glutamicum

68. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 68 Organic Acids

69. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 69 Specialty Products for Use in Medicine and Health

70. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 70 Biopolymers used to modify flow characteristics of liquids and to serve as gelling agents include: –dextrans and other polysaccharides –cyclodextrins

71. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 71 (from Thermoanaerobacter sp.)

72. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 72 Biosurfactants used for emulsification, increasing detergency, wetting and phase dispersion, and solubilization important in bioremediation, oil spill dispersion, and enhancing oil recovery many have antibacterial and antifungal activity; some inactivate enveloped viruses

73. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 73 Bioconversion Processes microbial transformations or biotransformations –minor changes in molecules carried out by nongrowing microbes –microbes act directly or indirectly as biocatalysts can be carried out by free enzymes or cells or by immobilized enzymes or cells advantages –produce appropriate stereoisomer –very specific –done using mild conditions

74. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 74 e.g., Biotransformation to modify a steroid progesterone 11α-hydroxyprogesterone

75. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 75 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

76. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 76 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

77. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 77 Biodegradation Using Natural Microbial Communities biodegradation has at least three definitions –minor changes –fragmentation –mineralization

78. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 78

79. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 79 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

80. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 80 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 microbes often can degrade only one isomer of a substance; the other isomer will remain in the environment

81. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 81 Meta effect

82. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 82 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

83. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 83 Repeated exposure and degradation rate

84. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 84 Downside of biodegradation 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

85. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 85 Microbial-mediated metal corrosion (e.g. Desulfovibrio)

86. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 86 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

87. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 87 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

88. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 88 Some examples stimulation of hydrocarbon degradation phytoremediation –use of plants stimulation of bioleaching of metals from minerals

89. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 89 Subsurface engineered bioremediation system M: monitoring wells

90. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 90 Phytoremediation Hexachlorobenzene is cometabolized by microbes in the presence of organic matters relased from plant roots.

91. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 91 Types of phytoremediation

92. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 92 Bioleaching (Fe 2+ ) (Fe 3+ ) Leptospirillum ferrooxidans Thiobacillus thiooxidans

93. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 93 Bioaugmentation addition of 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

94. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 94 Microbes as Products nanotechnology –e.g., use of diatom 3-D structures as templates –e.g., use magnetosomes from magnetotactic bacteria in magnetic resonance tomography (MRI) applications and as probes to detect cancer

95. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 95 Marine diatom surface features

96. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 96 Biosensors living microbes, enzymes or organelles are linked to electrodes to detect specific substances –detection is done by converting biological reaction products into electrical currents have a broad range of applications

97. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 97 Biosensor design

98. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 98

99. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 99 Streptavidin-Biotin binding and biotechnology Streptavidin: from Streptomyces avidini

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101. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 101 Biopesticides biological agents, such as bacteria, fungi, viruses, or their components, which can be used to kill a susceptible insect

102. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 102

103. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 103 Bacteria as biopesticides Bacillus thuringiensis –parasporal body produced during sporulation as an intracellular protein toxin crystal acts as microbial insecticide for specific groups of insects –insecticide (Bt) used for over 40 years unlike chemical insecticides, does not accumulate in environment

104. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 104 Mode of action of Bt toxin Release of prototoxin from the parasporal body Modification of the prototoxin by proteases

105. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 105 68 kDa active toxins

106. Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. 106 Impacts of Microbial Biotechnology industrial ecology –concerned with tracking flow of elements and compounds through natural world (biosphere) and social world (anthrosphere) microbiologists must: –understand potential impacts of new products and processes on the broader society as well as on microbiology –communicate effectively with the various “societal stakeholders” about the immediate and longer-term potential impacts of biotechnologies