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

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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-”?

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guide

Size of microorganisms

Typically 0.01 µm to 1 mm

Bacterial morphology

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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