ENVR 890/296Microbe/Pathogen Survival/Reduction in the

Environment, Excreta and Excreta Treatment Processes

Mark D. SobseyDept. of Environmental Sciences and Engineering

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

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Microorganism survival periods in faeces, sludge and soil

Some Physical Factors Influencing Microbe Survival in the Environment

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.)

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

Microbes are most stable in the environment and will grow in some media (e.g., foods) in the mid pH range

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

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

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

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

Dispersed: least protected

Embedded: most protected

Adsorbed: partially protected

Clumped: interior microbes protected

: Antimicrobial agent

Some Chemical Factors Influencing Microbe Survival in the Environment

Effects

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

Some Biological Factors Influencing Microbe Survival in the Environment

Effects

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

Factors Affecting Survival in Liquid

• Temperature

• Ionic Strength

• Chemical Constituents/Composition of Medium

• Microbial Antagonism

• Sorption Status

• Type of Microbe

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

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

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

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

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.

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

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

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

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

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

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

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)

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

Microbe Survival on Surfaces

• Adsorption State– Air Water Interface– Triple Phase Boundary

• Physical State– Dispersed– Aggregation– Solids associated

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

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)

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

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.

Least

Most

High

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

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

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

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.

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

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.

Aerobic microbes utililize carbon and other nutrients to form a healthy activated sludge AS biomass (floc)

The biomass floc is allowed to settle out in the next reactor; some of the AS is recycled

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.)

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

“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

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

Facultative Oxidation (Waste Stabilization) Pond

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.

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

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.

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

Log10 Reduction of Pathogens by Wastewater Rx Processes

Log10 Reduction of Pathogens by Wastewater Rx Processes

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%

Entamoeba histolytica Reduction by Sewage Treatment

ORGANISM PROCESS % REMOVALE. 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, 87E. histolytica " 100E. histolytica Aerated lagoon (no settling) 84%

Microbial Reductions by Wastewater Treatment

% Reduction

Microbe 1o&2o Filt. Disinfect. Store Total Rx.Tot. colif. 98 69 99.99 75 99.99999

Fec. colif. 99 10 99.998 57 99.999996

Coliphage 82 99.98 90 90 99.99997

Entero-virus

98 84 96 91 99.999

Giardia 93 99 78 50 99.9993

Crypto-sporidium

93 98 61 <10 99.95

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

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

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

Indicator Microbe Levels in Raw and Treated Municipal Sewage: Sewage Treatment Efficacy

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F. col. E. coli Ent. C. p. F+ phg.

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RawTreated (geom. mean values of 24 biweekly samples)

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