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Wastewater Microbiology & Bioaugmentation
November 17, 2016
WAISA – San Salvador
Christopher Flannery, Technical Service Manager
Agenda
• Novozymes background
• What are microorganisms?
• Wastewater microbiology
• Principles of bioaugmentation
Introducing nature's problem solvers
Everywhere you need us
Our biological answers, near you
What is a microorganism?
What is a microorganism?
Organism - an individual living thing that can
• react to stimuli
• grow
• reproduce (sexually or asexually)
• maintain homeostasis (maintain an internal equilibrium with variables such as temperature, pH, salinity)
What makes them “micro-”?
BASIC LAYOUT Use: This is the basic slide with no extra Novozymes graphics added. Edit Layout: Click Layout in the top menu Home. And choose between +30 different layouts. Edit Header and footer: In the top left corner you find Slide no., Date and Header. Change settings in the menu: > Insert > Header and Footer
guide
Size of microorganisms
Typically 0.01 µm to 1 mm
Bacterial morphology
BASIC LAYOUT Use: This is the basic slide with no extra Novozymes graphics added. Edit Layout: Click Layout in the top menu Home. And choose between +30 different layouts. Edit Header and footer: In the top left corner you find Slide no., Date and Header. Change settings in the menu: > Insert > Header and Footer
guide
7200X Magnification
FISH Microscopy
Durban (2016) Water Science & Technology - IWA
Cellular Composition and Nutrition
Composition of a Bacterial Cell
Element Percentage by Weight (%)
Carbon 50
Oxygen 20
Nitrogen 14
Hydrogen 8
Phosphorus 3
Sulfur 1
Potassium 1
Sodium 1
Calcium 0.5
Magnesium 0.5
Chlorine 0.5
Iron 0.2
All others ~0.3
Nutrition
Requirements are essentially the same as the elemental composition
Wastewater applications focus on C, N, O, and P
Needed for:
Respiration Cell structure, growth Energy, electron acceptor Enzymes, cofactors
Enzymes Fast starting biochemicals Very specific for a certain reaction (“lock and key”) Proteins that can be degraded by other enzymes Do not reproduce
Bacteria Take a bit longer to awaken and show activity Produce enzymes on demand, in response to compounds present (in situ enzyme factories) Can produce suite of enzymes to completely degrade a compound Can use the components and energy from the enzymatic action to produce more bacteria.
Comparison of Bacteria and Enzymes
Enzymes
Bacteria
Enzymes feed the bacteria
Only ~3% of enzymes used by microorganisms are secreted; the rest are accessible for useful applications only by using the whole cell
Microbial Growth
new cells
CO2
reduced acceptor
end products
carbon source
energy source
electron acceptor
nutrients
respiration
Nutritional Types
Microorganisms can be further classified by where they get their nutrition from:
• carbon source • electron donor • electron acceptor
Wastewater Process Classification Wastewater System C Source e- donor e- acceptor Byproducts
Aerobic Oxidation Heterotrophic Aerobic OM OM O2 CO2/H2O/NH3
Nitrification Autotrophic Aerobic CO2/HCO3 NH3/NO2 O2 NO2/NO3
Sulfur Oxidation Autotrophic Aerobic CO2/HCO3 H2S/S/ S2O3 O2 SO4
Denitrification Heterotrophic Anoxic/Facultative OM OM NO2/NO3 N2/CO2/H2O
Acidification Heterotrophic Anaerobic OM OM OM VFA
Acetogenesis Heterotrophic Anaerobic CO2/HCO3 H2/CO VFA CH3COOH
Sulfur Reduction Heterotrophic Anaerobic OM OM SO4 H2S/CO2/H2O
Methanogenesis Heterotrophic Anaerobic OM H2/CH3COOH CO2 CH4
Notes: OM = organic material VFA = volatile fatty acids
Biomass Growth Pressures
Temperature pH
Substrate/ concentration
Dissolved Oxygen
Nutrients
Oxygen Considered the most important electron acceptor in wastewater treatment (aerobic processes).
But not all organisms use oxygen; to some it’s toxic:
• Aerobic – Can exist only when there is a supply of molecular oxygen. • Anaerobic – Can exist only in an environment where there is no oxygen. • Facultative – Can exist in an environment with or without molecular oxygen.
Temperature
Every microorganism has a temperature range.
In general, we say that warmer environments have more microbial activity (catalysis).
• In wastewater, we mostly focus on the mesophiles. • Some anaerobic treatment can be thermophilic.
Mesophilic bacteria have the ability adapt to a wide range, however they must have time to acclimate.
Temperature (°C)
Group Minimum Optimum Maximum
Thermophiles 40 - 50 55 - 75 60 - 80
Mesophiles 10 - 15 30 - 45 35 - 47
Psychrophiles -5 – 5 15 - 18 19 – 22
Each microorganism has a pH optimum and a pH range for growth.
• Natural habitats usually are between 5 and 9. • Few organisms are able to grow at a pH below 2 and above 10.
pH changes can be tolerated, but need acclimation.
• Effects the physiology of microbial cells/reactions.
pH
Group pH Range
Acidophiles <1 to 4.5
Neutrophiles 5.5 to 8.5
Alkalophiles 7.5 to 11.5
Microbial Growth
Lag phase
Lag Phase: • Acclimatization to the substrate • Long generation times and slower growth rates. • Nutrients taken into the cells • Both the size and the mass of the bacteria increase • Amount of enzymes and nucleic acid increases Exponential Growth (“Log”) Phase: • Cells rapidly divide • Discernible increase in the growth rate
Stationary Phase: • Substrate becomes limiting • Growth rate = death rate (aka “endogenous decay”)
Death Phase: • Microbial growth rapidly declines • Generation time increases • Loss in cell mass due to oxidation of internal storage
products for energy for cell maintenance • Cell death • Predation by organisms higher in the food chain
kd µmax
Wastewater Biology
Wastewater Microorganisms
The goal of biological wastewater treatment is to engineer an environment in which microbes consume the maximum amount of organic substrate and produce clear effluent water.
Convert soluble organic pollutants (BOD) in to insoluble biomass (microorganisms) which can be separated.
Bacteria and Floc Formation
The goal is to achieve the growth of a biological floc which is a microscopic size grouping of bacteria in a semi-gelatinous mass which is heavy enough to settle.
A nice dense floc, as seen under a microscope, is shown below. A good floc is usually light brown and settles at a uniform rate leaving a clear liquid.
Why do we care about floculation?
Biomass Growth Pressures
Inert material
Operator
Temperature
SRT
pH
Substrate/ concentration Competition & Cooperation
Toxins
Dissolved Oxygen
Nutrients
Filamentous Bacteria
String or threadlike bacteria
Chains of cells which can extend from the floc, grow within the floc, or even free in the bulk water.
Prevent effective settling by interfering with floc formation (bulking).
Can be helpful in small quantities, acting as a backbone for floc to form.
Filamentous Scale
Examples of Filamentous Bacteria
Beggiatoa Microthrix S. natans
H. hydrossis Nocardia Thiothrix
Filaments and associated causes Manual on the Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems 3rd edition Jenkins, Richard, Daigger Lewis Publishers
Cause Filaments
Low Dissolved oxygen Sphaerotilus natans, Type 1701, Haliscomenobacter hydrosis
Low F/M Type 0041, Type 0675, Type 1851, Type 0803
Septicity Type 021N, Thiothrix I and II, Type 0914, Type 0411, Type 0961, Type 0581, Type 0092, Nostocoida limicola I, II, and III
Oil and grease Nocardia sp., Microthrix parvicella, Type 1863
Nutrient deficiency Type 021N, Thiothrix I and II, S. natans, N. limicola III, H. hydrosis
Low pH Fungi
Higher Life Forms
BOD Bacteria Protozoa Metazoa
Protozoa
Amoeba Flagellates Free-Swimming Ciliates Carnivorous Ciliates
Crawling Ciliates Stalked Ciliates Suctoria
Metazoa
Rotifers Gastrotrichs Aeolosoma Worms
Tardigrades Nematodes
Having a wide range of microorganisms is important
• Demonstrates stability of the ecosystem
• No upsets (spills impacting pH, Temperature, organic loading, toxins).
Useful as an operational parameter
• Monitoring sludge age and F/M
Diversity
Biotechnology is an emerging innovation platform in the wastewater industry
Biological treatment will remain a go-to option due to its cost-effectiveness, sustainability, flexibility, and capabilities
Operational innovations: Changes growth characteristics and selects for different microorganisms.
Chemical innovations: Flocculants, for example, change solids settling and dewatering characteristics.
Equipment innovations: Membranes, for example, change the way solids are removed from water.
Process innovations: Creates an environment to select for desired microorganisms and activities.
Biotechnology innovations:
Addition of specific microorganisms or enzymes to a process to enable desired activities.
Bioaugmentation is the practice of assisting the bacterial population of a wastewater treatment system by the addition of specialized cultures developed to provide increased rates of organic reduction or degrade compounds that are hard to break down. The goal is not to replace the existing biological population, but to supplement it for improved treatment.
Microbials Development
Collect microbial
strains from nature
Isolate and identify
organisms
Evaluate the strains for their
capabilities and tolerances
Ferment key organisms
Develop and test formulations
of strains
Solve customer problems
The four main applications for bioaugmentation
Bioaugmentation addresses many wastewater problems
COD Removal Odors Sludge Bulking
Foaming Cold Weather Biogas
Bioaugmentation In Action
Sludge reduction in paper mill lagoons
• Pulp and paper operations, 8.5 MGD
• Facultative sludge pond, 1.3 acres
• Fed 1.15 MGD of primary sludge
• ~ 7 days HRT (design)
• Pond was very full, constantly off-gassing methane and hydrogen sulfide
• Odor complaints from employees who had to work near the pond
• Poor solids settling, bypassing of flow
Treatment goal: Decrease the volume of settled sludge in the lagoon using BioSpikes 5000
Results - View 1
Day 0
Day 60
Results – View 2
Day 0
Day 60
Summary
7-10% sludge volume reduction
0.43 MG of capacity captured.
9 hour increase in hydraulic retention time.
$40,000 per acre sludge dredging estimate
Saved $20,000
Biogas enhancement at a pork processor
• 8,700 m3/d (2.3 MGD) • High proteins and fats • Up to 60,000 mg/L COD • Anaerobic follow by activated sludge • Unheated covered lagoons
System Information
Treatment goal: Increase the quantity of biogas produced by using BG Max 3000
• Low yield system at 0.34m3/kg of COD • High solids buildup • High FOG scum layer • Poor hydrolysis of proteins and lipids
Diagnosis
Biogas enhancement at a pork processor
• 39% increase in the quantity of biogas produced
• $170K/year worth of additional biogas produced
• Methane content stayed constant at 60-70%
• Addition benefits were low COD loading to the aerobic system (energy and sludge volume savings)
Odor Control
ManholeSterilization
Open SumpSite Sewer Site SewerDischarge to POTW
Represents confirmed points of odor Represents where NZ products are applied
Odor Control
57% reduction in volatile organic acids
76% reduction in reduced sulfurs
81% reduction in skatole
0
10
20
30
40
50
60
70
Total VOAs Total Reduced S Skatole
Day 0
Day 15
Day 36
• No odor complaints from neighbors, even in the warmest months
• CAPEX and OPEX cost avoidance related to odor scrubbing