ENVR 890

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Microbial Survival/Inactivation
ENVR 890

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1. ENVR 890/296 Microbe/Pathogen Survival/Reduction in the Environment, Excreta and Excreta Treatment Processes Mark D. Sobsey Dept. of Environmental Sciences and Engineering

2. Microbial Survival/Inactivation ? A Kinetic Phenomenon Microbe inactivation is best described by the rate(s) or kinetic of inactivation Survival depends on reduction rate and time (duration of exposure) Expressing survival in absolute terms based on time only such as days or weeks is misleading Depends on initial and final microbe concentrations Express extent of inactivation per unit of time at specified conditions of exposure

3. Microorganism survival periods in faeces, sludge and soil

4. Some Physical Factors Influencing Microbe Survival in the Environment

5. TEMPERATURE Greater Inactivation/death rates at higher temperatures Lower survival rates at higher temperatures But, some microbes will grow or grow better at higher temperatures Many microbes survive better at lower temperature Some bacteria experience ?cold injury? or?cold shock? and cold inactivation Thermal inactivation differs between dry heat and moist heat Dry heat is much less efficient than moist heat in inactivating microbes Some microbes survive very long times when frozen Other microbes are destroyed by freezing Ice crystals impale them Increased environmental temperatures can promotes pathogen spread by insect vectors (mosquitoes, flies, etc.)

6. pH Relative acidity or alkalinity A measure of hydrogen ion (H+) concentration Scale: 1 (most acidic) to 14 (most alkaline or basic) pH 7 is neutral Moving toward pH 1 the substance is more acidic Moving toward pH 14, the substance is more alkaline. Extreme pH inactivates microbes Chemically alters macromolecules Disrupts enzyme and transport functions Some enteric pathogens survive pH 3.0 (tolerate stomach acidity) Some pathogens survive pH 11 and fewer survive pH 12

7. Moisture Content ? Drying and Dessiccation Drying or low moisture inactivates/kills some microbes Survival depends on moisture content or ?water activity?But, removing water content of some foods can preserve them Most viruses rapidly inactivated in soil at <1% moisture; Some inactivated rapidly at a few % moisture Time for 90% reduction in days-weeks-months; depends on moisture level & temperature Some protozoan parasites (Cryptosporidium parvum) are rapidly inactivated when dried (>90% reduction within hours at room temp.) Some helminth ova (Entamoeba hystolytica) are very persistent dry Bacteria persistence to drying and desiccation is highly variable Most bacteria can survive for extended periods of time Days to weeks for >90% reduction Bacteria and fungi spores are very persistent when dry

8. Physical Factors Influencing Survival, Continued Ultraviolet radiation: about 330 to 200 nm Primary effects nucleic acids; absorbs the UV energy and is damaged Sunlight: Ultraviolet radiation in sunlight inactivates microbes Visible light is antimicrobial to some microbes Promotes growth of photosynthetic microbes Ionizing radiation X-rays, gamma rays, beta-rays, alpha rays Generally antimicrobial; bacterial spores relatively resistant Main target of activity is nucleic acid Effect is proportional to the size of the ?target? Bigger targets easier to inactivate; a generalization; exceptions Environmental activity of ionizing radiation in the biosphere is not highly antimicrobial Ionizing radiation is used in food preservation and sterilization

9. Atmospheric and Hydrostatic Pressure Most microbes survive typical atmospheric pressure Some pathogens in the deep ocean are adapted to high pressure levels (hydrostatic pressures): barophiles Survive less well at low atmospheric pressures Spores and (oo)cysts survive pressure extremes High hydrostatic pressure is being developed as a process to inactivate microbes in certain foods, such as shellfish Several 100s of MPa of pressure for several minutes inactivates viruses and bacteria in a time- and pressure-dependent manner

10. Role of Solids-Association in Microbial Survival Microbes can be on or in other, usually larger particles or they can be aggregated (clumped together) Association of microbes with solids or particles and microbial aggregation is generally protective Microbes are shielded from environmental agents by association with solids Protection depends on type of solids-association See diagrams, right Protection varies with particle composition Organic particles: often highly protective Biofilms protect microbes in them React with/consume antimicrobial chemicals Inorganic particles vary in protection Opaque particles protect from UV/visible light Inorganic particles do not always protect well against chemical agents Some inorganic particles are antimicrobial Silver, copper, other heavy metals/their oxides

11. Some Chemical Factors Influencing Microbe Survival in the Environment

12. Chemicals and Nutrients Influence Microbial Survival Antimicrobial chemicals Strong oxidants and acids Strong bases Ammonia: antimicrobial at higher pH (>8.0) Sulfur dioxide and sulfites: used as food preservatives Nitrates and nitrites: used as food preservatives Enzymes: Proteases Nucleases Amylases (degrade carbohydrates) Ionic strength/dissolved solids/salts High (or low) ionic strength can be anti-microbial Many microbes survive less in seawater than in freshwater High salt (NaCl) and sugars are used to preserve foods Has a drying effect; cells shrink and die Heavy metals: Mercury, lead, silver, cadmium, etc. are antimicrobial Nutrients for growth and proliferation Carbon, nitrogen, sulfur and other essential nutrients

13. Some Biological Factors Influencing Microbe Survival in the Environment

14. Biological Factors Influence Microbial Survival Chemical antagonistic activity by other microorganisms: Proteolytic enzymes/proteases Nucleases Amylases Antibiotics/antimicrobials: many produced naturally by microbes Oxidants/oxides Fatty acids and esters; organic acids (acetic, lactic, etc.) Predation Vectors Reservoir animals

15. Factors Affecting Survival in Liquid Temperature Ionic Strength Chemical Constituents/Composition of Medium Microbial Antagonism Sorption Status Type of Microbe

16. Factors Affecting Survival in Aerosols Temperature Relative Humidity Moisture Content of Aerosol Particle Composition of Suspending Medium Sunlight Exposure Air Quality (esp. ?open air? factor) Size of Aerosol Particle Type of Microbe

17. Factors Affecting Survival on Surfaces Type of Microbe Type of Surface Relative Humidity Moisture Content (Water Activity) Temperature Composition of Suspending Medium Light Exposure Presence of Antiviral Chemical or Biological Agents

18. Microbe Survival in Liquid Media Temperature Increased inactivation with increasing temperature Most are inactivated rapidly (minutes) above 50oC Some microbes are more thermotolerant than others (e.g. Hepatitis A virus, bacterial/fungal spores, some helminth ova (ascarids) Most are inactivation more at higher temperatures Chemical composition of media influences survival Protein/other organics & Mg&Ca ions protect Generally very stable at ultra-cold temperatures, Some loss of infectivity occurs with freezing and thawing

19. Survival in Liquid Media pH Direct effects on conformation of proteins and other biomolecules Indirect effects on adsorption and elution from particles pH range of stability is microbe-dependent Polio: 3.8 to 8.5 for maximum stability Salt Content Variable effects on microbe survival Affects microbe physiology (isotonic conditions), adsorption and stability of biomolecules Divalent cations (Mg2+) can increase thermo-stability of viruses and bacteria E,g., MHV, enteroviruses, HAV

20. Microbe Survival in Liquid Media Microbial Antagonism Microflora influences microbe survival Metabolites: enzymes, VFAs, NH3 are antiviral Use of pathogen as a nutrient source Greater microbe survival documented in sterilized or pasteurized matrices, as compared to non-sterile matrices Phenomena demonstrated in sewage, fresh, estuarine, and marine waters, soils and sediments.

21. Microbe Survival in Liquid Media Adsorption Several possible mechanisms: Ionic attractions and repulsions covalent reactions (with active chemicals) hydrogen bonding hydrophobic interactions double layer interactions van der Waal?s forces Adsorption status greatly influences survival adsorbed microbes generally survive longer than unadsorbed microbes Protection and accumulation in sediments and soils

22. MicrobeSurvival in Liquid Media Organic Matter In liquid media, organic matter increases microbe survival Increased oxidant demand protects from oxidation If an enzyme substrate, protects from enzymatic attack Can coat to protect microbe particles In soils, organic matter has variable effects on microbes Possible competition for adsorption sites May coat or protect microbe particles Bacteria may grow of organics are nutrients

23. Microbe Survival in Liquid Media Antimicrobial Chemicals Ionic and non-ionic detergents, particularly for enveloped viruses and some bacteria Ammonia is virucidal; ammonium ion is not Germicides (chlorine, ozone, etc.) Light Direct microbicidal activity below wavelengths of 370 nm Indirect antimicrobial activity: stimulation of microflora growth triggering formation of reactive oxidants activation of photoreactive chemicals

24. Microbe (Virus) Survival in Aerosols Relative Humidity and Moisture Content Viruses with lipid survive better at lower relative humidity Viruses with little or no lipid content survive better at higher relative humidity Viral inactivation or retention of infectivity may be a function of stabilization (drying of aerosol) and of rehumidification of aerosol particle upon collection Effect of relative humidity on virus survival may be influenced by temperature effects

25. Microbe Survival in Aerosols Temperature Survival decreases with increased temperature Suspending Media composition influences microbe stability effect is microbe dependent Salts stabilize some viruses (e.g. Poliovirus) Removal of salts stabilize other viruses (e.g. Langat, Semiliki Forest virus) Proteinacious material and organic matter may have similar mixed effects, depending on microbe type Polyhydroxy compounds stabilize some virus types (e.g. Influenza) but have no effect on other viruses

26. Microbes Survival in Aerosols Oxygen and Air Ions Oxygen has little direct effect on most viruses but may influence bacteria But, oxygenation may be synergistic with higher temperature and sunlight to inactivate microbes The ?Open Air Factor? has been shown to have virucidal activity Poorly characterized chemical agents in open air that reduce virus survival compared to clean laboratory air May be reaction products of ozone and olefins

27. Microbe Survival in Aerosols Light Virucidal activity of UV light is a greater in air than in liquid media Photosensitivity is virus type-dependent and may be related to the envelope Non-enveloped viruses (Poliovirus, Adenoviruses and FMDV) are more resistant to UV light than enveloped viruses (vaccinia, herpes simplex, influenza, and Newcastle disease virus) (Jenson, 1964; Donaldson 1975; Applyard, 1967)

28. Microbe Survival in Aerosols Aerosol Particle Size Airborne microbes may be more rapidly inactivated in smaller aerosol particles than larger ones (some studies) Other studies observed no effect of particle size on virus survival Aerosol Collection Method Abrupt rehydration of virus particles and other microbes upon collection may lead to their inactivation Prehumidification may improve recovery of infectious virus Effect is virus type-dependent

29. Microbe Survival on Surfaces Adsorption State Air Water Interface Triple Phase Boundary Physical State Dispersed Aggregation Solids associated

30. Microbe Survival on Surfaces Relative humidity Similar effects as seen in aerosols; effects are microbe type dependent Moisture Content In soils moisture content directly related to microbe survival Dessication Enhanced predation

31. Microbe Survival on Surfaces Temperature Effects as observed in liquid media and aerosols Interaction between relative humidity and temperature pronounced on surfaces for certain virus types (e.g. Polio, Herpes Simplex), less important for others (e.g. Vaccinia) (Edward, 1941)

32. Microbe Survival on Surfaces Suspending Media Effects similar to effects on survival in aerosols Presence of fecal material Presence of salts Type of Surface Little effect by non-porous surfaces on most viruses important for some virus types (Herpes simplex) Effects more pronounce for porous surfaces (e.g. fabrics: cotton, synthetics and wool Light Effects similar to those in aerosols and liquids

33. Microbe type: Resistance to chemical disinfectants: Vegetative bacteria: Salmonella, coliforms, etc.: low Enteric viruses: coliphages, HAV, Noroviruses: moderate Bacterial Spores Fungal Spores Protozoan (oo)cysts, spores, helminth ova, etc. Cryptosporidium parvum oocysts Giardia lamblia cysts Ascaris lumbricoides ova Acid-fast bacteria: Mycobacterium spp.

34. Factors Influencing Microbial Reductions by Wastewater Treatment Processes Solids association: microbes embedded in larger particles or aggregated are: more likely to sediment (settle) protected from disinfection and other antagonists possibly different in their surface properties due to the other material present

35. Factors Influencing Microbial Reductions by Wastewater Treatment Processes Temperature produces more microbial rapid inactivation: at higher temp. by thermal effects (denaturation) in biological processes by more rapid biological metabolism and enzymatic activity in chemical processes by faster reaction rates

36. Factors Influencing Microbial Reductions by Wastewater Treatment Processes Temperature elevation for some pathogens may promote growth: Naegleria fowlerii and other amebas Legionella species Mycobacteria species Aeromonas species Vibrio species

37. Factors Influencing Microbial Reductions by Wastewater Treatment Processes Biological activity can decrease pathogens by: Grazing, phagocytosis and other predation mechanisms Increased enzymatic activity by bacteria and other treatment microbes: proteases, amylases, nucleases, etc. Increased adsorption to and accumulation in microbial biomass complexes: floc particles, biofilms, etc.

38. Primary Treatment or Primary Sedimentation Settle solids for 2-3 hours in a static, unmixed tank or basin. ~75-90% of particles and 50-75% of organics settle out as ?primary sludge? enteric microbe levels in 1o sludge are sometimes ~10X higher than in raw sewage enriched by solids accumulation Overall, little removal of many enteric microbes: typically ~50% for viruses and bacteria >50% for parasites, depending on their size

39. Enteric Microbe/Pathogen Reductions in Secondary or Biological Treatment Aerobic biological treatment: typically, activated sludge (AS) or trickling filtration (TF) Then, settle out the biological solids produced (2o sludge) ~90-99% enteric microbe/pathogen reductions from the liquid phase Enteric microbe retention by the biologically active solids: accumulation in AS flocs or TF biofilms Biodegradation of enteric microbes by proteolytic enzymes and other degradative enzymes/chemicals Predation by treatment microbes/plankton (amoeba, ciliates, rotifers, etc.

40. Waste Solids (Sludge) Treatment Treatment of settled solids from 1o and 2o sewage treatment Biological ?digestion? to biologically stabilize the sludge solids Anaerobic digestion (anaerobic biodegradation) Aerobic digestion (aerobic biodegradation) Mesophilic digestion: ambient temp. to ~40oC; 3-6 weeks Thermophilic digestion: 40-60oC; 2-3 weeks Produce digested (biologically stabilized) sludge solids for further treatment and/or disposal (often by land application) ?Thickening? or ?dewatering? drying or ?curing? Waste liquids from sludge treatment are recycled through the sewage treatment plant Waste gases from sludge treatment are released (or burned if from anaerobic digestion: methane, hydrogen, etc.)

41. Enteric Microbe/Pathogen Reductions by Sludge Treatment Processes Anaerobic and aerobic digestion processes Moderate reductions (90-99%) by mesophilic processes High reductions (>99%) by thermophilic processes Thermal processes Reductions depend on temperature Greater reductions at higher temperatures Temperatures >55oC usually produce appreciable pathogen reductions. Alkaline processes: lime or other alkaline material Reductions depend on pH; greater reductions at higher pHs pH >11 produces extensive pathogen reductions Composting: high temperature, aerobic biological process Reductions extensive (>99.99%) when temperatures high and waste uniformly exposed to high temperature Drying and curing Variable and often only moderate pathogen reductions

42. ?Processes to Further Reduce Pathogens? ?PFRP?: Class A Sludge Class A sludge: <1 virus per 4 grams dried sludge solids <1 viable helminth ovum per 4 grams dried sludge solids <3 Salmonella per 4 grams of dried sludge solids <1,000 fecal coliforms per gram dry sludge solids PFRPs: Thermal (high temperature) processes (incl. thermophilic digestion); hold sludge at 50oC or more for specified times lime (alkaline) stabilization; raise pH 12for 2 or more hours composting: additional aerobic treatment at elevated temperature Class A sludge or ?biosolids? disposal by a variety of options or used as a soil conditioner Class A biosolids can be marketed/distributed as soil conditioner for use on non-edible plants

43. Alternative Biological Treatment of Wastewater: Alternatives for Small and Rural Communities Lagoons, Ponds and Ditches aerobic, anaerobic and facultative; for smaller communities and farms enteric microbes are reduced by ~90-99% per pond multiple ponds in series increases microbe reductions Constructed Wetlands aerobic systems containing biologically active, oxidizing microbes and emergent aquatic plants Lagoons and constructed wetlands are practical and economical sewage treatment alternatives when land is available at reasonable cost

44. Facultative Oxidation (Waste Stabilization) Pond

45. Stabilization Ponds or Lagoons Aerobic and Facultative Ponds: Biologically Rx by complementary activity of algae and bacteria. Used for raw sewage as well as primary- or secondary-Rx?d. effluent. Bacteria and other heterotrophs convert organic matter to carbon dioxide, inorganic nutrients, water and microbial biomass. Algae use CO2 and inorganic nutrients, primarily N and P, in photosynthesis to produce oxygen and algal biomass. Many different pond designs have been used to treat sewage: facultative ponds: upper, aerobic zone and a lower anaerobic zone. Aerobic heterotrophics and algae proliferate in the upper zone. Biomass from upper zone settles into the anaerobic, bottom zone. Bottom solids digested by anaerobic bacteria.

46. Enteric Microbe/Pathogen Reductions in Stabilization Ponds BOD and enteric microbe/pathogen reductions of 90%, esp. in warm, sunny climates. Even greater enteric microbe /pathogen reductions by using two or more ponds in series Better BOD and enteric microbe/pathogen reductions if detention (residence) times are sufficiently long (several weeks to months) Enteric microbes reduced by 90% in single ponds and by multiples of 90% for ponds in series. Microbe removal may be quite variable depending upon pond design, operating conditions and climate. Reduction efficiency lower in colder weather and shorter retention times

48. Constructed Wetlands and Enteric Microbe Reductions Surface flow (SF) wetlands reduce enteric microbes by ~90% Subsurface flow (SSF) wetlands reduce enteric microbes by ~99% Greater reduction in SSF may be due to greater biological activity in wetland bed media (porous gravel) and longer retention times Multiple wetlands in series incrementally increase microbial reductions, with 90-99% reduction per wetland cell.

49. Septic Tank-Soil Absorption Systems for On-Site Sewage Rx Used where there are no sewers and community sewage treatment facilities: ex.: rural homes Septic tank: solids settle and are digested Septic tank effluent (STE) is similar to primary sewage effluent Distribute STE to soil via a sub-surface, porous pipe in a trench Absorption System: Distribution lines and drainfield Septic tank effluent flows through perforated pipes located 2-3 feet below the land surface in a trenches filled with gravel, preferably in the unsaturated (vadose) zone. Effluent discharges from perforated pipes into trench gravel and then into unsaturated soil, where it is biologically treated aerobically. Enteric microbes are removed and retained by the soil and biodegraded along with STE organic matter; extensive enteric microbe reductions are possible But, viruses and other pathogens can migrate through the soil and reach ground water if the soil is too porous (sand) and the water table is high

50. Log10 Reduction of Pathogens by Wastewater Rx Processes

51. Log10 Reduction of Pathogens by Wastewater Rx Processes

52. REMOVAL OF ENTERIC BACTERIA BY SEWAGE TREATMENT PROCESSES ORGANISM PROCESS % REMOVAL Fecal indicators Primary sed. 0-60% E. coli Primary sed. 32 and 50% Fecal indicators Trickling filt. 20-80% Fecal indicators Activated sludge 40-95% Fecal indicators Stab. ponds, 1 mo. >99.9999% @ high temp. Salmonellae Primary sed. 79%, 6-7 hrs. Salmonellae " 73%, 6-7 hrs. Salmenellae Trickling filt. 92% Salmonellae Activated sludge ca. 99%

53. Entamoeba histolytica Reduction by Sewage Treatment ORGANISM PROCESS % REMOVAL E. histolytica Primary Sed. 50% E. histolytica Primary Sed., 2 hr. 64% E. histolytica Primary sed., 1 hr. 27% E. histolytica Primary sed. + Trickl. Filt. 25% E. histolytica " 74% E. histolytica " 91% E. histolytica Primary sed. + Act. Sludge 83% E. histolytica Oxidation ditch + Sedimentation 91% E. histolytica Stabilization ponds + sedimentation 100% E. histolytica " 100, 94, 87 E. histolytica " 100 E. histolytica Aerated lagoon (no settling) 84%

54. Microbial Reductions by Wastewater Treatment

55. Disinfection of Wastewater Intended to reduce microbes in 1o or 2o treated effluent Typically chlorination Alternatives: UV radiation, ozone and chlorine dioxide Good enteric bacterial reductions: typically, 99.99+% Meet fecal coliform limits for effluent dicharge Often 200-1,000 per 100 ml geometric mean as permitted discharge limit Less effective for viruses and parasites: typically, 90-99% reduction Toxicity of chlorine and its by-products to aquatic life now limits wastewater chlorination; may have to: Dechlorinate Use an alternative, less toxic chemical disinfectant or Use an alternative treatment process to reduce enteric microbes granular medium (e.g., sand) filtration membrane filtration

56. When Wastewater Disinfection is Recommended or Required Discharge to surface waters: near water supply intakes used for primary contact recreation used for shellfish harvesting used for irrigation of crops and greenspace other direct and indirect reuse and reclamation purposes Discharge to ground waters waters: used as a water supply source used for irrigation of crops and greenspace other direct and indirect reuse and reclamation purposes

57. Wastewater Reuse Wastewater is sometimes reused for beneficial, non-potable purposes in arid and other water-short regions. Often uses advanced or additional treatment processes, sometimes referred to as ?reclamation? Biological treatment in ?polishing? ponds and constructed wetlands Physical-chemical treatment processes as used for drinking water: Coagulation-flocculation and sedimentation Filtration: granular medium filters; membrane filters Granular Activated Carbon adsorption Disinfection Details of these water treatment process will be presented in lectures on water treatment

59. Estimated Pathogen Reductions by Sewage Treatment Processes: An Example Sewage Treatment Rx: % Reduction Total % Reduction Primary settling 50 50 2o biological treatment 99 99.5 Granular medium filtration 90 99.95 Disinfection 99 99.9995

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