Final Flashcards
(339 cards)
1
Q
What causes organic pollutant degradation?
A
Abiotic and biological mechanisms
2
Q
What are abiotic mechanisms of organic pollutant degradation?
A
Nonbiological mechanisms The types are: - photochemical - chemical (oxidation, reduction) - mechanical (wind, water, mixing, dilution)
3
Q
What are biological mechanisms of organic pollutant degradation?
A
Types:
- Plants and animals
- Microorganisms (most important)
4
Q
What are the different plant and animal biological mechanisms?
A
Direct consumption
Indirect degradation via:
- Compounds secreted by organisms
- Associated microbes biodegrade contaminants
- These microbes include rhizosphere and biofilm on roots
5
Q
What are the different microorganism biological mechanisms?
A
Mineralization
- Conversion of organic compounds to CO2
Modification or transformation
- End product may be more or less of a pollution problem after
6
Q
What are microbes?
A
Microscopic organisms of:
- Plants and animals (called protozoa)
- Bacteria (even visible bacteria)
- Archae
- Viruses & prions
7
Q
What is microbial hegemony?
A
The significant roles of microorganisms on earth
Dominance in global biomass with over 50% as microbial
Predominant influence over global biogeochemical cycling
Supremacy in extraordinary metabolic capacity
8
Q
What is the importance of microbial evolution in biodegradation?
A
Have been exposed to every imaginable organic compound and environment over the last 3-4 billion years of their existence
Have complex metabolic processes that evolved for biodegradation
Failures:
- Only ~1% of microbial species have been cultured
- we don’t know much about metabolic pathways
9
Q
Why are only ~1% of microbes cultured?
A
The vast majority of bacteria and archaea can’t be grown in culture
Some can be viable-but-not-culturable (Called VBNC)
10
Q
Tests to determine microbes that can’t be grown in culture
A
Microscopy: direct microscopic counts can exceed viable counts by several orders of magnitude
Respiration tests
Molecular biology (types: 16S rRNA sequences, the “molecular clock”, the position on tree of life)
11
Q
What is the central dogma of life?
A
An explanation of the flow of genetic information within a biological system
DNA -> RNA -> protein
12
Q
What is the tree of life?
A
The study of phylogenetic relationships between all cells: done by comparing the 16S rRNA sequences
- Proves that the greatest diversity in the living world is within the microbial world
- Was able to split the two branches of microbes: bacteria and archaea
13
Q
Bacteria on the tree of life
A
Include some well-known species
Majority of species have never been characterized
Found in every niche on the planet
14
Q
Archaea on the tree of life
A
Organisms previously thought to be limited to extreme environments (such as anaerobic sediments, hot springs, etc)
Dominate extreme environment niches, but are found everywhere (are ubiquitous)
Have unique physiological properties
15
Q
What happened with the Last Common Ancestor?
A
Gave rise to two branches: bacteria and archaea/eukarya
16
Q
What is the evolution of metabolic capabilities in bacteria?
A
Genetics -> physiology -> ecological niche -> genetics
Ecological niche consists of:
- competition
- change in conditions
These factors are part of the selective pressure that stimulate the evolution of metabolic capacities in microbes
17
Q
Importance of the evolution of metabolic capabilities in bacteria for biodegradation:
A
Microbes have been around since an anaerobic atmosphere
They have evolved many different/complex metabolic strategies
Can be used to degrade many toxic pollutants
High probability of finding a species that can bioremediate a particular pollution
- May have to use culture-based and/or molecular methods to find useful microbes
18
Q
Where do you look for microbial pollution biodegraders?
A
Soil & water in contaminated sites b/c
- biodegrades should be enriched and selected for in these environments
19
Q
What occurred 2.5 billion years ago?
A
Great oxidation event
When the amount of oxygen in the air increased so the atmosphere became aerobic
Due to oxygenic photosynthesis from microbes
20
Q
What are the key properties of prokaryotes?
A
Small size: 1-2 um High surface to volume ratio Developed biochemical pathways (favors chemistry) Metabolically diverse - Alternate energy sources - Light, organics, inorganics, alternate oxidants - O2, metals, CO2 Rigid cell wall
21
Q
What are the key properties of Eukaryotes?
A
Larger cell size (10-25um) Complex structures (multicellular) Flexible cell walls Metabolic specialization - O2 respiration - Organic C as fuel
22
Q
Microbial bioenergetics consists of:
A
Metabolism and thermodynamics
23
Q
How do cells grow?
A
Living cells are dynamic open systems in constant interaction with the immediate environment, where they obtain the raw material for the production of ATP and building blocks
They need to synthesize the building blocks (monomers) and harvest energy so biochemical reactions can occur, and the cell can grow
24
Q
What are the two sets of reactions of metabolism?
A
Catabolic
Anabolic
25
What is the electron donor of eukaryotes?
Organic carbon
26
What is the electron acceptors of eukaryotes?
Oxygen
27
Do many bacteria use carbon as an electron donor? If yes, what happens?
True
| Organic C is electron donor, oxygen is electron acceptor
28
What does lithoautotrophic prokaryotes mean?
Litho- -autotrophic = nonorganic eating
29
What are some electron donors of lithoautotrophic prokaryotes?
Hydrogen gas, hydrogen sulfur...
30
What are some electron acceptors of lithoautotrophic prokaryotes?
Oxygen
| Ferric hydroxide to iron (called iron respiration)
31
True/false different microbes can use different types of metabolisms to produce energy.
True
| Via redox reactions (also called oxidation reduction reactions)
32
What is oxidation?
The removal of electrons from an atom or molecule
| Can only occur with reduction (electron must be accepted by another atom or molecules)
33
What is reduction?
The addition of electrons to an atom or molecule
34
What happens to the energy released during redox reactions?
Captured in the form of energy-rich chemical bonds (becomes ATP)
35
What is NADH?
A reducing power
| Can take in the electron released by oxidation
36
What are ATP and NADH required for?
Cellular metabolism
37
What is anabolism?
Biochemical reactions involved in the synthesis of compounds and macromolecules such as proteins and nucleic acids
Biosynthesis
38
What is catabolism?
Biochemical reactions that break down compounds (mostly to allow cells to generate chemical energy)
39
What is an example of catabolism?
The oxidation of carbohydrates (sugars) during respiration (aerobic or anaerobic) or fermentation
40
What happens if glucose is the main nutrient for a bacterium?
Will be transported across the membrane
Then it will be oxidized to CO2 in 3 steps (IMPORTANT)
1. Glycolysis: Glucose (6 C) will be broken into 2 molecules of pyruvate (3 C) (produces 2ATP)
2. Krebs cycle or tricarboxylic acids cycle: Complete combustion of pyruvate to CO2 through a cyclic set of reactions (produces 2 ATP)
3. Oxidative phosphorylation: some reactions lead to the reduction of coenzymes (NADH, FADH) that will be further oxidized in the respiratory chain (electron transport system) which produces the proton motive force (produces the most ATP molecules: 34 ATP)
41
Why do microbes biodegrade pollutants?
They can get energy
42
What is atrazine?
Pesticide
43
What is toluene and xylene?
Hydrocarbons
44
What is dichloroethane (DCE)?
A solvent (a chlorinated hydrocarbon)
45
Process of the electron transfer system:
Electrons flow from the reduced coenzymes to a terminal electron acceptor (TEA) such as O2 (microorganisms can use different TEAs) via the electron transport chain (ETC)
Flow of electrons down ETC causes ETC to pump protons (H+) out of the cell, resulting in the proton motive force (PMF)
The PMF is used for different work (membrane transport, flagellar rotation...) and allows synthesis of ATP during respiration
46
What is aerobic respiration?
Overall process called oxidative phosphorlation
The use of O2 as the TEA
Reducing power (NADH...) generated by oxidation of energy source
Electrons transferred to ETC then to TEA (O2 -> H2O)
Results in (H+)/pH gradient
H+ gradient fuels processes like ATP synthesis
47
In what condition are organic pollutants better degraded (aerobic or anaerobic respiration)?
Aerobic
It is faster and more efficient
It results in more complete oxidation to CO2
48
Why is anaerobic respiration important in biodegradation?
Many contaminated environments quickly become anaerobic
| With anaerobic respiration of microbes, biodegradation is not limited by O2 as long as there are alternative TEAs
49
What are the O2 concentrations within soil aggregates?
Aerobic zones (outside) ->
Microaerophilic zones ->
Anaerobic zones (center)
Percent of oxygen decreases as it moves farther from external surface
The soil environment is a heterogeneous microbial habitat
50
What is a heterogeneous microbial habitat?
Contains both aerobic and anaerobic zones (O2 conc. decreases with depth into soil due to diffusion and utilization of O2 on the surface)
- O2 can be depleted within 1 mm below surface
Varies greatly (even within a soil aggregate)
Different micro-habitats and food sources
51
How do terminal electron acceptors work in energy generation?
Different TEAs available in different niches
Availability of TEAs differs with depth
Key parts of major biogeochemical cycles (C, N, and S)
Many organisms use metallic terminal electron acceptors that vary with depth
Some organisms able to use only one compound, some can use multiple (mostly from adjacent zones)
52
What is anaerobic respiration?
Electrons transferred to compounds other than oxygen
Examples:
- Denitrification (electron acceptor = NO3-)
- Sulfate reduction (electron acceptor = SO4-)
- Fermentation (electron acceptor = fumarate)
- Methanogenesis (electron acceptor = CO2)
53
Denitrification
An anaerobic respiration biodegradation
Use NO3- as TEA. Reduce it to gaseous N2O and N2
Most abundant bacteria: Pseudomonas and Alcaligenes
Reductions catalyzed by reductases located in membrane or periplasmic space that are part of the ETC
Important process in the nitrogen cycle
Ex. Thauera aromatica, Azoarcus tolutytics are both toluene degrading bacteria
54
Iron-reducing and manganese-reducing bacteria
An anaerobic respiration biodegradation
Use iron or manganese as TEAs
Ex.
Geobacter metallireducens GS15 degrade toluene under iron-reducing conditions
55
Sulfate-reducing bacteria
An anaerobic respiration biodegradation
Use sulfate as the TEA
Taxonomic groups = Desulfovibrio, Desulforomonas, Desulfosarcina
Desulfobacula toluolica degrade toluene under sulfate-reducing conditions
56
Methanogenic archaea
An anaerobic respiration biodegradation
Use CO2 as TEA
Use H2 as energy and electron source OR ferment acetate
Important bacteria for atmospheric trace gases
IMPORTANT in anaerobic degradation of toluene in sediment or activated sludge reactors
57
Fermentation in biodegradation
Use organic molecules as TEAs
Products in soil/sediment are acetate, formate, butyrate, lactate, succinate, caproate...
Clostridium is a fermenting bacteria
58
What is species Thauera aromatica?
A toluene degrading denitrifiers (anaerobic)
59
What is the species Azoarcus tolulyticus?
A toluene degrading denitrifiers (anaerobic)
60
What is species Geobacter metallireducens GS15?
An Fe-reducing bacterium degrading toluene under anaerobic conditions
61
What is species Desulfobacula toluolica?
Degrading toluene bacteria under sulfate-reducing conditions
62
What is genus Clostridium?
Fermenting bacteria in soil
63
What is the most metabolically efficient redox reaction?
Aerobic iron oxidation
64
What is the best reductant?
CH2O (organic carbon) (oxidized)
65
What is the worst reductant?
H2O (water) (oxidized)
66
What is the best oxidant?
O2 (oxygen) (reduced)
67
What is the worst oxidant?
CO2 (carbon dioxide) (reduced)
68
What is Shewanella oneidensis?
The super microbe
Extremely versatile with its electron acceptors (strains reduce multiple electron acceptors)
Donors: formate, lactate, pyruvate, amino acids, H2
Acceptors: O2, NO3-, NO2-, Mn (IV,III), Fe (III), Fumarate, DMSO, TMAO, S^0, S2O3^2-, U(VI), Cr (VI)
69
What is Geobacter spp.?
A cousin of Shewanella oneidensis
Energy source = organic carbon (acetate)
Reduces U (vi) to U (iv) and Fe3+ to Fe2+
U (vi) is highly soluble in water
U (iv) is highly insoluble and will precipitate out of water
70
How would you increase Geobacter numbers and activity in a Uranium contaminated aquifer?
Increase the amount of organic carbon and increase the number of Geobacter spp.
71
What is the bioremediation process of arsenic contaminated groundwater?
Using aerobic lithoautotrophic bacteria
Water cycles through oxygenation tanks containing bacteria that oxidize arsenite, iron, and manganese
Produces oxidized form of arsenic (arsenate)
- it chemically precipitates with iron and manganese for convenient removal
Electron donor = Fe2+, Mn2+, and arsenite
Electron acceptor = oxygen (O2)
72
How does mercury biodegradation occur?
Mercury concentrates in living tissues and is highly toxic
Mercury in atmosphere = elemental mercury (Hg^0) which is volatile
- Oxidized to mercuric ion (Hg2+) (how it enters aquatic environments
Hg2+ is metabolized by microorganisms which form methylmercury (CH3Hg+) (extremely soluble and neurotoxic compound)
Bacteria can transform methyl mercury into nontoxic form
73
How to define microbial nutritional categories?
Determine:
- Source of energy (photo- or chemo-)
- Source of electrons
- Source of carbon (-autotroph or -heterotroph)
74
Where do the two-part nutritional category names photoautotrophs and chemoheterotrophs come from?
Consideration of energy and carbon sources
75
Photoautotroph:
```
Photo- = light energy
-autotroph = CO2 carbon source
```
76
Chemoheterotroph/heterotrophs:
```
Chemo- = organic compounds for energy
-heterotroph = organic carbon source (plants/animals)
```
77
Where does the three-part nutritional category name chemolithoautotroph come from?
Energy, electron, and carbon sources
78
Chemolithoautroph:
Chemo- = chemical energy
- litho- = uses inorganic electron molecules
- autotroph = CO2 carbon source
79
Heterotrophic carbon utilization:
Heterotrophs assimilate organic compounds
| Take up organic compounds and then use them as a source of carbon in own biosynthetic reactions
80
Types of chemolithoautotrophs:
```
Able to oxidize reduced inorganic compounds to synthesize ATP for biosynthesis and fix CO2
Ammonium-oxidizing nitrifying bacteria
Nitrite-oxidizing nitrifying bacteria
Sulfur-oxidizing bacteria
H2-oxidizing bacteria
```
81
Ammonium-oxidizing nitrifying bacteria
Chemilithoautotroph
Uses inorganic compound NH4+ as energy source
Oxidize NH4+ to NO2-
Have a monooxygenase (ammonia monooxygenase, AMO) which may attack some pollutants (trichloroethylene, TCE)
Genuses Nitrosomonas & Nitrovibrio
Between ammonium-oxidizing and nitrite-oxidizing nitrifying bacteria, convert NH4+ to NO3- (a rate-limiting step)
82
Nitrite-oxidizing nitrifying bacteria
Chemolithoautotroph
Use inorganic compound NO2- as energy source
Oxidize NO2- to NO3-
Genus Nitrobacter
Between ammonium-oxidizing and nitrite-oxidizing nitrifying bacteria, convert NH4+ to NO3- (a rate-limiting step)
83
Sulfur-oxidizing bacteria
Chemolithoautotroph
Genus Thiobacillus
Use a variety of inorganic reduced sulfur compounds as energy source
- Such as S, H2S, S2O3
Oxidize reduced S compounds to SO4^2- using O2
Key enzymes: sulfide-, sulfur-, and sulphite-oxidases
Play critical role in S cycle by regenerating SO4 ^2- (main source of S for assimilation)
Exception is Thiobacillus denirificans
84
What is species Thiobacillus denitrificans?
Exception to sulfur-oxidizing bacteria
| Uses NO3- as an electron acceptor in the absence of oxygen
85
H2-oxidizing bacteria
Chemolithoautotroph
Uses H2 as energy and electron source
Considered a facultative chemolithoautotroph because they can use organic compounds instead of H2
Species are Paracoccus denitrificans and Desulfovibrio vulgaris
86
The course of biodegradation TEAs through sediment:
```
Based on energy per molecule
Begins with oxygen respiration (O2 -> H2O)
Then denitrification (NO3- -> N2))
Then iron reduction
Then sulfate reduction (SO4-2 -> H2S)
Then methanogenesis (CO2 -> CH4)
```
87
What happens when TEAs are used up through sediment?
SO4- is used = leads to sulfide accumulation
| CO2 is used = leads to CH4 accumulation
88
What does a purple gram stain indicate?
Gram-positive membrane
Found in Firmicutes and Actinobacteria (including Bacillus sp.)
Have thick cell wall outside of cytoplasmic membrane and have no outer membrane
89
What does a pink gram stain indicate?
Gram-negative membrane
Have thin cell wall in periplasmic space between the cytoplasmic and outer membranes
Have LPS and porins on outer wall not found in gram-positive membranes
90
What membranes do most of the bacterial phyla involved in biodegradation have?
Gram-negative membranes
91
What is biogeochemical cycling?
Organic matter decomposes into small inorganic molecules, which are immobilized by growing cells
Microbes play a great role in maintaining equilibrium between organic matter reservoir and mineralized reservoir
There is aerobic and anaerobic environment in each cycle
Important cycles: Nitrogen, Carbon, Sulfur
Cycles are altered by human activity
92
The carbon cycle is fixed into organic matter via:
Can be anaerobic or aerobic
Anaerobic: fixed by anoxygenic photosynthetic bacteria (Rhodospirillum, Chlorobium)
Aerobic: fixed by oxygenic photosynthetic organisms (cyanobacteria, algae, plants) and chemolithoautotrophic bacteria (nitrifying bacteria, sulfur-oxidizing bacteria)
93
What is merB
Breaks carbon-mercury bonds so methylmercury becomes mercuric ion
94
The carbon cycle:
- CO2 is fixed into organic matter (CH2O) under aerobic or anaerobic conditions
- Organic matter is oxidized back to CO2 via aerobic respiration or anaerobic respiration & fermentation
- Some organic matter and CO2 in anaerobic respiration can become CH4 by methanogens (diverse group of Archaea)
- CH4 is oxidized (aerobic) to CO2 by methanotrophs (group of bacteria: Methylosinus & Methylococcus)
95
What is carbon in organic matter?
-In soil/sediment, carbon in the organic matter can be active (living biomass) or inactive (dead)
96
What is the mineral reservoir of carbon?
Atmospheric or dissolved CO2 & calcareous rocks and coral
97
How do methanotrophs oxidize CH4
They possess a key enzyme (methane monooxygenase: MMO) that oxidized CH4 to methanol
Can be present on complex membrane structures
MMO can also oxidize trichloroethylene (TCE)
98
What is there a lot of in permafrost environments?
Organic carbon
The active layer of permafrost defrosts in the summer
- The active layer is becoming deeper so more organic carbon is being activated (and methane)
99
How much CO2 will be in atmosphere from permafrosts in 2100?
an extra 50ppm
100
How much CO2 is there in the environment today?
417ppm
101
What is the importance of sulfur?
Oxidized forms of S can be used as electron acceptors for anaerobic respiration (dissimilatory sulfate reduction)
Reduced forms of S are good energy sources
Sulfur is the most important element in the cell for amino acids
102
The Sulfur cycle (aerobic)
S^0 oxidized to sulfate (SO4-) (aerobic)
Dissimilatory sulfate reduction from SO4- to hydrogen sulfide (H2S) (anaerobic)
Sulfide oxidation from H2S to S^0 (aerobic)
103
The sulfur cycle (anaerobic)
Phototrophic oxidation of S^0 to H2S or SO4-
SO4- goes through dissimilatory sulfate reduction to H2S
H2S goes through sulfur respiration to S^0
104
What is an example of dissimilatory sulfate reduction?
sulfate-reducing bacteria (SRBs): Desulfovibrio
105
What does the dissimilatory sulfate reduction do?
Utilizes sulfate as a terminal electron acceptor
Uses H2 and/or organic carbon as an energy source
Therefore, SO4- becomes H2S
106
What is the importance of nitrogen?
Important element in cells for proteins and nucleic acids
Growth of organisms usually limited by nitrogen availability
Bacteria can use nitrate as a TEA (called dissimilatory nitrate reduction)
In bioremediation, adding N-fertilizer stimulates mineralization of organic matter by decreasing C/N ratio
107
What does nitrification do the ionic charge?
It alters the ionic charge of the fixed forms of nitrogen so that leaching occurs in soils
108
What happens in anaerobic denitrification?
Anaerobic respiration in soils and sediments returns molecular nitrogen to the atmosphere
109
The nitrate cycle (aerobic)
Nitrate oxidation: from nitrite (NO2-) to nitrate (NO3-)
Assimilation: Assimilatory NO3- reduction to R-NH2 ammonium assimilation to NH4+
Nitrification: Ammonium oxidation from NH4+ to NO2- using nitrifying bacteria with enzyme ammonium monooxygenase (AMO) or NO3-
Alternate root (from NO3- to NO2- through dissimilatory NO3- reduction (anaerobic)
110
The nitrate cycle (anaerobic)
Either:
- Nitrite ammonification: from NO2- to NH4+
- NH4+ to aerobic respiration
- Nitrite reduction: from NO2- to NO
- Denitrification: NO to N2O
- N2O to N2
- Nitrogen fixation: N2 to NH4+ (NH4+ to aerobic respiration)
Denitrification of NO3-: NO3- assimilated by microbes becoming organic matter or reduced to N2
111
How much of the atmosphere is N2 gas?
79%
112
What is the problem with N2 gas in the atmosphere?
Only diazotrophs (a small proportion of bacterial and archaeal species) can fix N2
113
What does the enzyme nitrogenase do?
An N2-fixing bacteria that reduces N2 to NH4+
| Ex. Rhizobium, Azotobacter, Azoarcus
114
What happens to NH4+ with assimilation?
Most microbes and plants can assimilate NH4+
| There is a dynamic equilibrium between assimilation and mineralization (the ammonification of N from amino acids)
115
What happens with NH4+ with oxidization?
NH4+ can be oxidized to NO2- and NO3- by bacterial. nitrification under aerobic conditions
Bacterial examples are Nitrosomonas (for NO2-) and Nitrobacter (for NO3-)
- Both have complex membrane infoldings to facilitate nitrification
116
What is Nitrosomonas sp. for?
Oxidize NH4+ to NO2-
| Possess ammonium monooxygenase (AMO) enzyme
117
What is Nitrobacter sp. for?
Oxidize NO2- to NO3-
118
Nitrate cycle in aquariums:
```
Urea/NH3 becomes nitrate (NO3-)
Uses denitrifying bacteria to filter out nitrate
Bacteria create biofilms on plastic
Uses methanol as the electron donor
Uses NO3 as electron acceptor
```
119
When does NO3 toxicity start manifesting?
Above 30mg NO3
| Leads to infections, parasites, mortality rates
120
How many die per year due to pollution?
~9million
| 1 in 6 deaths
121
Main human sources of organic pollutants:
Domestic waste (plastics, antibiotics...)
Pulp and paper (Cellulosics...)
Agriculture (lignin, chloro-organics...)
Food processing (proteins, fats, carbs...)
Mining (metals...)
Textile industry (fluorocarbons...)
Chemical, pharmaceutical industries (dyes, solvents, paints, resins...)
Internal combustion engines (hydrocarbons...)
122
What is the fate of industrial contaminants when they are released into the environment?
If volatile = air pollution
If non-volatile & soluble = water and groundwater pollution
If non-volatile & non-soluble = soil pollution, mineralization (degradation to CO2), persistence in food chain
123
Examples of air pollution:
Carbon tetrachloride
| CFCs
124
Examples of water pollution:
Pesticides
125
Examples of groundwater pollution:
Petrochemicals
| Pesticides
126
Examples of mineralization:
PAHs
| Petrochemicals
127
Examples of persistence in food chain:
PCBs
| DDT
128
What is biomagnification?
The increase in a pollutant in tissues of organisms at successive levels of a food chain
Results in bioaccumulation at higher trophic levels
129
What is bioaccumulation?
The increase in concentration of a compound within an organism compared to the level found in the environment
Accumulates in tissue if not metabolized or excreted
Has negative health/reproductive effects
130
What is biodegradation?
Degradation of a pollutant through a living organism (usually a microbe)
131
What is bioremediation?
Remediation of a contaminated site by using biodegradative capacity of biology (usually microbiology)
132
What interactions need to happen for biodegradation and bioremediation to occur?
1. The contaminant must be biodegradable
2. The environmental physical/chemical parameters must allow biodegradation
3. Biodegradative microorganisms must be present and active in the contaminated environment
133
What are xenobiotic compounds?
Compound alien to existing enzyme systems: man-made organic compounds with uncommon structures/properties
Not naturally occurring
Organic xenobiotics are often pollution problems due to:
- Toxicity
- Carcinogenicity (cancer causing)
- Recalcitrance (complexity)
134
What does recalcitrance mean?
A compound that is attacked poorly or not at all by microbial enzyme systems due to molecular complexity
- Oligomerization: converts monomers to macromolecular complexes
- Halogen substitutions: replacing H with chlorine, fluorine, or bromine
- Other substitutions: replacing H with nitro- or sulfo- groups
- Branching
- Large size: molecules are too big to fit into enzyme pockets with catalytic sites; large molecular organic contaminants are more hydrophobic so less bioavailable
135
Examples of xenobiotic compounds:
```
DDT
Malathion
2,4-D
Atrazine
Monuron
Chlorinated biphenyl (PCB)
Trichloroethylene
Mirex (KNOW)
Kepone (KNOW)
Benzaanthracene (KNOW)
Benzoapyrene (KNOW)
```
136
Naturally made xenobiotic compounds:
Some can be made naturally in minute quantities
- Ex. chloroorganics in forest fires
Conc. of man-made compounds causes them to be "foreign to life"
If man-made compounds are similar to existing compounds, microbes might be able to easily switch to metabolism of them
137
Biodegradation of xenobiotic compounds:
Might take a long time
Look in environments where previous contaminations occurred
Not all compounds can be biodegraded
Determine how much needs to be degraded before environment is safe AND if the contaminant is bioavailable
138
What does bioavailable mean?
Available to biological systems for utilization as energy and C sources or to be biotransformed
139
Most to least bioavailable organic pollutants
Physical is greater than chemical
Highest (extractable): particulate pollutant, liquid form, absorbed in soil
Middle (extractable): aged compounds (absorbed in soil, water phase in soil pores, a separate phase in soil pores)
Lowest (non-extractable): chemically bound to soil
140
To optimize bioremediation, the following needs to be optimal:
```
Soil moisture
Soil type
Aeration
Redox potential
pH
Temperature
```
141
Aeration of soil:
Oxygen
Aerobes: require oxygen as an electron acceptor
Facultative anaerobes: grow with or without oxygen
Strict anaerobes: oxygen in inhibitory to growth
142
Impact of aeration on biodegradation & bioremediation:
Aerobic conditions are essential for petroleum hydrocarbons
O2 is required as TEA and substrate in oxygenase-catalyzed biodegradative reactions
O2 is often limited in soil & aqueous systems
- The rate-limiting variable in petroleum degradation in soil & gasoline in groundwater
143
How to increase aeration?
Tilling
Adding bulking agents in polluted soil systems
Venting aquifers
...
144
Importance of anaerobic conditions in biodegradation/bioremediation:
Needed for BTEX, PAHS, halogenated organic compounds (TCE, PCBs,...)
In anoxic conditions, uses denitrifying, methanogenic, sulphate-reducing, and iron-reducing conditions
Allows for reductive dechlorination (substitute Cl with H) rxns
Halogenated hydrocarbons are the TEA
145
How to inc. anaerobic degradation:
Supplying the appropriate electron acceptors
146
What is the pH of bacteria?
~6-9/log[H+]
147
What is the pH of yeast?
~5-9/log[H+]
148
What is the pH of acidophiles?
Grow as low as 1/log[H+]
149
What is the normal pH of soil?
~2.5-11/log[H+]
150
What is the optimal pH for petroleum hydrocarbon degradation?
pH = 7-8
151
How to increase pH for bioremediation?
Add lime to acid soils to inc. pH
152
Why is pH important?
It can effect water solubility and sorption of contaminants to soil/sediment
Ex. Inc. acidity (lower pH) = inc. solubility of heavy metals
153
What is the optimal temperature for psychrophiles?
Less than 20C
| Can survive from 0-15/20C
154
What is the optimal temperature for psychotrophs?
Over 20C
| Can survive between 0-30/35C
155
What is the optimal temperature for mesophiles?
About 40C
| Can survive between 10-45/50C
156
What is the optimal temperature for thermophiles?
Less than 65-105C
| Can survive from 50C-80/110C
157
Temperature change of 10C:
A change of 10C will generally inc. or dec. an enzyme's activity by 2x
158
Importance of temperature in bioremediation:
Raising temp of contaminated soils/water can inc the rate of degradation by increasing microbial activity & solubility of contaminants
- Difficult to manipulate in the filed (except for biopiles/bioreactors)
Compost materials = thermophilic biodegradation
159
Soil moisture content (%)/water content is:
Amount of water present in soil
| Expressed as the ratio of dry weight/wet weight
160
Water activity (Aw):
Microbes require available water for growth and metabolism
Measures water actually available for microbial use
Distilled water = 0.0-1.0
Most microbes need about 0.9-1.0 (optimally 0.96)
161
Water holding capacity (WHC)
Amount of water a soil can hold before becoming saturated
| Dry soil soaks up water to excess -> excess is drained -> increase in weight of soil = WHC
162
Importance of soil moisture content in bioremediation:
```
Optimal soil moisture content for aerobic microbial activity = 60-80% WHC
Optimal soil moisture content for hydrocarbon degradation = 30-90% WHC
Low moisture content = low Aw => microbial activity decrease
Waterlogged soils (where WHC is greater than 100%) are anoxic
```
163
How to decrease soil moisture content:
Amending soil with:
- Agents that bind to free water (gypsum)
- Bulking agents (alfalfa)
164
Nutrient supply:
Heterotrophic microbes require growth factors (such as amino acids, B vitamins, fat-soluble vitamins, other organic molecules)
Limiting nutrients for heterotrophs in normal sites can be carbon
Limiting nutrients in contaminated environments can be nitrogen and phosphorous
There are usually adequate amounts of K, S, Mg, Ca, Fe...
165
Impact of nutrient supply on bioremediation:
Treating contaminated sites with additions of nitrogen and phosphorous = biostimulation
In bacterial cells: C/N = 20/1 & C/P = 50/1
In soils = C/N = range from 20/1 - 50/1 by adding nitrogen compounds
Increases biomass & degradative activity
Biostimulation caused by oleophilic fertilizers (contain N & P)
166
How is biodegradation achieved?
Using concentrated efforts of a number of enzymes in a regulated manner
167
How is complete mineralization achieved?
A consortium of microorganisms present in the contaminated matrix
168
Bacteria with De-:
Removes ...
| Ex. Dehalococcoides (removes halogens)
169
What is mineralization?
Complete breakdown or degradation by a microorganisms of organic compounds into inorganic compounds
170
What is biotransformation?
Transformation by a microorganism of an organic or inorganic compound into another organic compound or inorganic compound, respectively
171
What is cometabolism?
The gratuitous metabolic transformation of a substance by a microbe growing on another substrate
The substrate is not incorporated into the microorganism's biomass and the microorganisms does not derive energy from the transformation of the substrate
172
Example of cometabolism:
Cyclohexane uses Mycobacterium vaccae to become cyclohexanol
Mycobacterium vaccae can only gain energy if it has the substrate propane attached when degrading cyclohexane
Therefore: to get cyclohexanol, just add propane so M. vaccae can gain energy from degradation
173
Difficult to biodegrade hydrocarbons:
Depends upon molecular complexity (recalcitrant compounds)
Compounds quite inaccessible to microbes due to low solubility with water
Remain at spill sites for years
The lightest alkanes are toxic, but tend to volatilize quickly
174
Hydrocarbon compound structures:
Know -enes or cyclo- are usually rings of carbon
| Know -anes are usually branches of carbon
175
Mineralization of alkanes:
Initiated by oxidation of either terminal or subterminal carbon which form either a primary or secondary alcohol
176
What are the major steps of mineralization of an alkane via oxidation?
1. Oxidation of alkane to primary alcohol via monooxygenase or dioxygenase (requires O2)
2. Formation of a fatty acid (requires O2)
3. Beta-oxidation of fatty acids to acetyl-CoA
4. Beta-oxidation of acetyl-CoA via the TCA cycle & glyoxylate shunt
177
Biodegradation of complex aromatic compounds (phenolic or aromatic rings):
1. Aromatic compounds are first oxidized to catechol (under aerobic conditions)
- Enzymes are monooxygenase or dioxygenase
2. Then the catechol phenolic ring is cleaved
- Two main cleavage pathways (ortho cleavage and meta cleavage)
- The pathway is defined by the position of the ring cleavage site in an aromatic compound
178
Ortho cleavage:
Between the OH groups
| Better cleavage pathway
179
Meta cleavage:
Beside one of the OH groups
| Worse cleavage pathway
180
What are monooxygenases?
When one oxygen atom is transferred to the substrate and the other is reduced, yielding water
Ex. Methane monooxygenase (MMO) oxidized CH4 to CH3OH
Ex. Alkane monooxygenase oxidizes alkanes to alcohols
181
What are dioxygenases?
When both oxygen atoms are transferred to the substrate
| Ex. Catechol dioxygenases oxidize catechol (NB two O) to cis, cis-muconic acid (NB four O)
182
What does BTEX stand for?
Benzene, toluene, ethylbenzene, xylene
183
What does ppm stand for?
parts per million = mg pollutant/kg or liter of sample
184
What does ppb stand for?
parts per billion = ug pollutant/kg or liter of sample
185
What does TPH stand for?
Total Petroleum Hydrocarbons
| It is a common chemical procedure used to quantify the amount of hydrocarbons in a contaminated sample
186
What are the common modes of biodegradation of organic compounds?
Cellular metabolism
Detoxifying enzymatic reactions
Non-enzymatic reactions
Cometabolism
187
Cellular metabolism:
Either catabolism or anabolism
Catabolism: carbon used as source of energy, CO2 released
Anabolism: carbon converted to biomass
Pollutants are converted to: cells (biomass), residual organics, or inorganics (CO2...)
188
Detoxifying enzymatic reactions:
Antibiotic degradation
Metal transformations
Ex. CH4Hg+ -> Hg2+ -> Hg^0
Ex. U6+ -> U4+
189
Non-enzymatic reactions:
By-products of microbial metabolism can change environmental conditions via:
- deplete O2
- change pH
- Produce H2O2 -> strong oxidant
- SO4 -> H2S (via SRB) (H2S reacts with heavy metals -> insoluble metal sulfides
190
Cometabolism:
Compound is modified but not used for generation of energy or biomass
Enzymes with low specificity
- Often via excreted enzymes (extracellular enzymes)
191
What are the basic bioremediation steps?
1. Bacterium identifies contaminant
2. Bacteria ingests contaminant
3. Bacteria uses multiple enzymes to break contaminant into something it can digest
- Each enzyme step releases carbon & ATP
- Enzymes are found in the operons in plasmids of bacteria
4. Bacteria excrete CO2 and H2O
192
What are the parameters for successful bioremediation?
Each site is unique
Need multidisciplinary expertise
Determine waste characteristics, then find out:
- Waste characteristics (composition, properties)
- Optimal microbiology (nutrients, moisture, aeration, inoculum)
- Remediation technology (land treatment, bioslurry, compositing, bioventing)
- Analytical methods (correct method, QA/QC)
- Statistical sampling (statistic procedures)
- Regulatory approval (cleanup standards, closure requirements, permitting)
193
What is the most cost effective remediation technology?
Bioremediation (between $40m and $150m)
| It is sustainable
194
What is the most expensive remediation technology?
Incineration (between $350m to $1,600m)
195
What is the problem with bioremediation?
Unpredictable outcomes
Changes with every contaminant
Sometimes slower
196
What are the conventional remediation technologies?
Based on pollutant removal
Excavation is often a necessary first step
Then either:
- Incineration
- Containment (landfill, land farming, solidification/stabilization)
- Chemical addition and soil washing (chelating agents, hydrogen peroxide addition)
- Pollutant removed/neutralized based on physical/chemical properties
197
What are the Sydney Tar Pits?
Located in Nova Scotia
Contains 1.2m metric tons of contaminated sediments
Tar pits -> solidification/stabilization with cement (no movement of pollutants)
$400m remediation program
198
What are coke ovens?
Spread of contaminated soil through land farming
Till to aerate and add fertilizer
Works with hydrocarbons
199
Weaknesses of conventional remediation technologies:
High cost
Don't destroy pollutant
- Pollutants can be converted to another form but still pollutants
- Pollutants can be moved to another environment
200
Why is bioremediation usually better than conventional technologies?
Pollutants are usually completely destroyed
| Cost is generally lower
201
Types of bioremediation:
1. Following excavation
2. In situ bioremediation
2. a. intrinsic bioremediation
2. b. enhanced bioremediation
202
Intrinsic in situ bioremediation:
No intervention
Rely on existing microbes, nutrients, and other environmental parameters
Inexpensive but slow/may never be complete
Requires a comprehensive monitoring program contaminant has limited toxicity, not travelling, and concentrations are reducing
203
Enhanced in situ bioremediation:
Enhanced by additions but no excavation
| Can speed up degradation time & percent reduction of pollutant
204
What is bioaugmentation:
Addition of microbes (natural or genetically engineered) known to break down the pollutant
Not commonly used because isn't optimal for particular environment needed in (every environment is different)
205
What is biostimulation?
Addition of O2 or another electron acceptor
Addition of fertilizers to optimize C:N:P ratio (so growth is not limited by a nutrient)
Addition of inducers (of gene expression)
- Ex. CH4 stimulates production of MMO
Alteration of any other important environmental parameter
206
The bioremediation assessment study determines:
1. if contaminants are biodegradable
2. If biodegradable microbes are present at site
3. If the contaminated environment parameters are optimal for biodegradation
4. If any parameters are limiting the biodegradation activity
207
Using the bioremediation assessment study, a bioremediation treatment strategy is developed to:
Optimize biostimulation parameters
| Apply the optimized parameters to the field for in situ bioremediation
208
Bioremediation assessment steps:
Lab -> pilot-scale -> field-scale
1.
- Use of controls and methods for detection of pollutants or biodegradation end-products, detecting and quantifying pollutant-degrading microorganisms
- Lab testing and determining optimal bioremediation treatments
2.
- Utilized in bioremediation strategy once basic processes are understood
209
Examples of in situ bioremediation:
Bioventing
Biosparging
Stimulation
Phytoremediation
210
Examples of excavated bioremediation:
```
On- & off-site:
- Land farming
- Composting
- Biopile
- Bioreactor
- Pump and treat
Off-site:
- Phytoremediation
```
211
Pros of phytoremediation:
```
One of the few methods for removing heavy metals from soil & shallow aquifers
Inexpensive
Can promote soil regeneration
Additional uses for plant material
- Can produce biomass for fuel
- Pioneer species
Can lead to effective stimulation of petroleum breakdown (organic biodegradation)
Energy efficient
Occurs in situ
```
212
Cons of phytoremediation:
Can be slow (15-100 years)
Difficult to predict
How would you treat multi-contaminated sites?
What is the contaminant concentration threshold?
May give a false image of site restoration
213
What is phytoremediation?
Uses plants to remove elemental pollutants or lower their bioavailability in soil
214
Eureka, Ellesmere Island
Military Base as far north as possible
Eureka station = runway strip and communication area
Fuel line broke causing 37k L of diesel fuel to leak: contaminated soils
215
Bioremediation of Eureka
Far away
Extremely cold, very dry
Did bioremediation assessment: moisture content, WHC, soil pH, total petroleum hydrocarbons (TPH), microbial count
Used fertilizer + peat moss for best results
216
Colomac Hydrocarbon Tank Farm
Tank farms begin to leak: migrated into river (hydrocarbons)
| Use biopile remediation treatment
217
Steps of biopile:
1. Contaminated soil is placed in pile & inspected for foreign material
2. Blend of chemicals and organisms added (fertilizer)
3. Synthetic cover is applied to control emissions and humidity
4. Aeration system added
4a. Perforated pressure piping in biopile adds air (via blower assembly)
4b. Perforated vacuum piping in biopile removes air (via blower assembly
5. Recirculated air is monitored to determine rate of treatment
6. Demister (knock out drum) collects condensation
7. Monitor pressure and temp of the pile core
218
Treating contaminated aquifers:
```
Contaminated from a leaking fuel storage tank
Occurs in situ
Types:
Bioventing
Biosparging
Permeable reactive barrier
```
219
Bioventing:
Bioventing Steps:
1. Pump air into soil (promotes aerobic conditions of soil)
2. Vacuum applied to draw out volatile contaminants (bind to activated carbon)
220
Biosparging:
Biosparging steps:
1. Pumps air into aquifer (inc aerobic activity)
- Requires porous environments
221
Permeable reactive barrier:
```
For shallow aquifers
Permeable reactive barriers steps:
1. Add barrier with reactant to react to pollutant
- Allows treated water through while capturing pollutant
2. Removes or breaks down contaminants
- Removal methods are:
a. sorption & precipitation
b. chemical reaction
c. biodegradation mechanisms
```
222
Pros and cons of tar sands:
Pros:
- Generate jobs, wealth
Cons:
- Generate greenhouse gasses
- Tailing sands & tailing water (byproducts)
- Have inc. ion content, alkaline pH, nutrient depletion, residual hydrocarbons
- Generate soil & water pollution (fix with phytoremediation)
223
What is phytoremediation?
Planting of plants to:
- add nutrients that support bacteria
- Bacteria then protect roots & encourage growth
- Plant root enzymes (from bacteria) degrade PAH
224
How is most oil released into the ocean?
Total = 5x10^6 tons/year
1. Drains
2. Maintenance
3. Smoke
4. Natural
5. Big spills
6. Offshore drilling
225
What is the ocean hydrocarbon cycle?
The use of CO2 to alkanes, which are used as energy for hydrocarbon degrading bacteria
Two cycles:
- Short term hydrocarbon cycle occurs over days
- Long term hydrocarbon cycle occurs over thousands of years
226
Ocean hydrocarbon cycle: how are alkanes produced?
Cyanobacteria convert CO2 to alkanes with photosynthesis
CO2 -> sugars -> acetyl-ACPS -> alkanes
About 500m tons of alkanes are produced each year
227
Ocean hydrocarbon cycle: how are alkanes essential to oil spills?
Alkanes metabolize for hydrocarbon-degrading bacteria
| Alkanes -> fatty acids -> TCA cycle -> respiration
228
What is the species Alcanivorax borkumensis?
Obligate hydrocarbonoclastic bacterium
It plays a significant role in the biological removal of petroleum hydrocarbons from polluted oceans
It secretes natural emulsifiers to break down oil droplets
229
What are the remediation treatment options for marine oil spills?
```
Takes Hours:
- Dispersion (underwater)
- Evaporation aerosolization (surface)
- Dissolution (underwater)
- Photooxidation (surface)
- Physical recovery (surface)
Takes days:
- In-situ burning (surface)
- Emulsification (surface)
- Oil-article aggregation (underwater)
- Dispersants (surface)
Takes weeks to years:
- Sedimentation (underwater)
- Biodegradation (underwater)
```
230
Major steps to clean beaches covered in crude oil:
1. Clean up bulk of oil by physical means (ex. physical washing)
2. Clean up remaining oil with in-situ bioremediation
- This was done as a trial with the Exxon Valdez oil spill (ex. applied fertilizer)
231
What compounds are added to stimulate biodegradation of crude oil on beaches?
```
Inipol EAP 22
- 360g/m^2
Customblen
- 17g/m^2
To determine if they worked:
- Quantify residual oil contamination per area & by type of hydrocarbon over time
- Look for enhanced hydrocarbon degrader conc.
- Look for inc. rate of biodegradation
```
232
What is Inipol EAP 22?
```
Contains:
- Oleic acid: surfactant
- Urea: N & C source
- Triphosphate: detergent, P source
- 2-butoxyethanol: solvent
Oleophilic, slow release fertilizer (sticks around)
```
233
What is Customblen?
Contains:
- Ammonium nitrate: N source (NO3 acts as TEA in anaerobic zones)
- Ammonium phosphate: N & P source
- Calcium phosphate: P source
234
How to test for biodegradation rate:
Mix treated and untreated sediment slurry with C-labeled hexadecane or phenanthrene
- Hexadecane is a straight-chain alkane often used as a model alkane in biodegradation studies
- Phenanthrene is a 3-ring PAH often used as a model PAH in biodegradation studies
Compare rate of CO2 produced over time and in treated & untreated sediments
235
Hydrocarbon-degrading bacteria at Exxon Valdez Oil Spill:
Significicant proportion of total population of environment were hydrocarbon degraders
Had important hydrocarbon degrading genes:
- xylE gene (encodes catechol 2,3-dioxygenase)
- alkB gene (encodes alkane hydroxylase)
236
xylE gene:
Encodes catechol 2,3-dioxygenase protein
| Used as a probe to detect bacteria that have the capacity to grow on aromatic compounds
237
alkB gene:
Encodes alkane hydroxylase protein
| Used as a probe to detect bacteria that have the capacity to grow on C6-C12 alkane ocmpounds
238
What is metagenomics in bioremediation?
Sequencing of all DNA from polluted sample and looking for biodegradative genes & pathways of interest
Sometimes paralleled with metatranscriptomics (which genes are coming from active microbes) and metaproteomics (which genes are active themselves)
239
What are the three garbage patches in the pacific?
Western garbage patch (by Asia)
Subtropical convergence zone (north pacific/in between)
Eastern Garbage Patch (by N.A.)
240
How does mineralization of plastics work?
In oceans (VERY slow process)
1. Initial attachment of microbes on plastic surface
2. Microbial biofilm formation
3. Biodeteriation (secretion of extracellular enzymes & EPS)
4. Biofragmentation (formation of oligomers, dimers, monomers)
5. Mineralization (microbial biomass, CO2, H2O)
241
What provides a point of attack for hydrolytic enzymes in plastics?
Ester bond (C-O)
Not all plastics have ester bond
Ex w/: PET, PEF
242
What does PET stand for?
Polyethylene terephthalate
243
What does PEF stand for?
Polyehtheylene-2,5-furandicarboxylate
244
What are plastics made of?
Hydrocarbons
245
What are the oil-derive non-biodegradable and bio-based non-biodegradable plastics?
```
Oil-derived:
Polyethylene tereaphthalate (PET): biotransforms using cutinases & lipases
Polyethylene (PE)
Polyporpylene (PP)
Polystyrene (PS)
Polyvinyl chloride (PVC)
Bio-based:
polyethylene-2,5-furandicarboxylate (PEF)
```
246
What are oil-based biodegradable plastics?
Polybutylene adipate terephthalate (PBAT)
| Polycaprolactone (PCLA)
247
What are the bio-based biodegradable plastics?
```
Polyhydroxyalkanoate (PHA): uses Amycolatopsis serine proteases & thermophilic lipases -> lactic acid -> biomass, H2O, CO2
Polylactic acid (PLA): uses PHA depolymerases -> R-3-HAA -> biomass, H2O, CO2
Thermoplastic starch (TPS): uses cutinases -> caproic acid -> biomass, H2O, CO2
```
248
What are halogenated compounds?
Organic compounds containing Cl, Fl, Br
More difficult to degrade
Because Cl-C bonds are relatively strong and difficult to break
249
What determines the biodegradability of halogenated phenolic compounds?
Also called haloaromatic compounds
| Molecules with greater degree of halogenation are more recalcitrant + the general complexity of hte molecule
250
What are the major steps in biodegradation of halogenated organic compounds?
Begins with modified halogenated aromatic compounds (ex. halogenated pheonxyacetate, organophosphate)
1. Modified halogenated aromatic compounds are debranched/ring-cleavage
- Creates simple halogenated aromatic compounds (ex. halogenated benze, halogenated phenol)
2. Simple HACs are dehalogenated (key step) & hydroxylated
- Creates central metabolites (ex. catechol, hydroquinone)
3. Central metabolites go through ring cleavage & redox
- Creates common metabolites (ex. acetyl Coa, pyruvate)
4. Mineralization to CO2
251
How can dehalogenation occur?
Aerobically or anaerobically usually
| Sometimes must be aerobically
252
What type of compound is PCP?
Haloaromatic compound
Biodegradation occurs aerobically (sometimes) or anaerobically (usually: via reductive dechlorination)
Uses H2 as electron donor (called hydrogenolytic reductive dechlorination)
253
What type of compounds are PCBs?
Haloaromatic compounds
Resistance to biodegradation inc with inc number of Cl substitutions
Can use anaerobic or aerobic degradation
Aerobic results in CBAs (can't keep biodegrading)
254
What type of compound is 2,4-D?
```
Haloaromatic compound
One of the most widely used herbicides
Highly toxic
Readily biodegraded in soil/water
- Half life in soil = 2-16 days
```
255
Biodegradation of nitroaromatic compounds:
Explosive
Anaerobic or aerobic biodegradation
First reductive states can use aerobic respiration; as it goes down, use strict anaerobes
Anaerobic: nitro groups used as TEAs
- Ex. TNT to TAT using Clostridium, Desulfovibrio
Aerobic: nitro groups are cleaved off using monooxygenase or dioxygenase activity
256
Best parts of bacteria:
```
Penetration power
Voracious appetite
Tough surface
Multi-functional respiration
Biosurfactant producing
Nutrient reserves
HIghly motile
```
257
Genetically engineer bacteria to:
Add genes from pollutants into bacteria to biodegrade them
258
How to genetically engineer bacteria:
Take multiple plasmids and splice until wanted genes are in new plasmid
259
Example of genetically modified bacteria:
Mineralize PCB
| Modified strain Cupriavidus nacator JMS34
260
Aerobic cometabolisms of trichloroethene:
Trichloroethene (TCE)
Uses methanotrophic microorganisms
TCE + oxygen TEA = TCE epoxide
Methanotrophs don't utilize the products (other microbes complete the degradation)
261
What are the water-bourne microorganisms?
Autocthonous: indigenous water column organisms
Allocthonous: transient; either harmless or pathogens
262
Common pathogens found in waste water/drinking water:
Salmonella sp. = typhoid fever, GI problems
Shigella sp. = dysentery
E. coli = mostly harmless, some cause Gi problems
V. cholerae = cholera
263
How many people lack safe drinking water?
1.1 billion
264
How much of diarrheal disease/year come from contaminated water?
88%
| 1.8 mill people die/year from diarrheal diseases
265
What is the water quality like vs dissolved oxygen (DO) content?
```
Good = 8-9ppm
Slightly polluted = 6.7-8ppm
Moderately polluted = 4.5-6.7ppm
Heavily polluted = 4-4.5ppm
Gravely polluted = below 4ppm
```
266
How much dissolved solids are in a typical municipal wastewater?
500mg/l
267
How much suspended solids are in typical municipal wastewater?
200mg/l
268
How much ultimate biochemical oxygen demand (BOD) is in typical municipal wastewater?
300mg/l
269
How much chemical oxygen demand is in typical municipal wastewater?
400mg/l
270
How much free ammonia (NH3) is in typical municipal wastewater?
25mg/l
271
How much phosphorous is in typical municipal wastewater?
10mg/l
272
What is the biochemical oxygen demand (BOD)?
Measures the amount of O2 (in mg/l) required for aerobic degradation of organic material in a water sample
- An indirect measure of biologically-utilizable organic material via determination of dissolved O2 (DO) conc.
273
BOD5:
Test
Gives an index of the pollution potential of an organic pollutant
Higher the BOD5 = more polluted the water
274
How to test BOD?
```
BOD = D1-D2/P
D1 = initial DO measure
D2 = final DO measure after 5 days incubated at 20C
```
275
BOD analyzer determines:
O2 required for inorganic oxidation
O2 required by nitrifiers (BODn)
Useful for:
- Estimation of waste loading to treatment plants (needed to properly design treatment plant)
- Evaluate the efficiency of a treatment plant
- Predict the effect of effluent release on DO in receiving stream
276
Chemical Oxygen Demand (COD):
Amount of O2 consumed in complete oxidation of organic matter
Reaction occurs in acidic conditions & uses strong oxidizing agents to oxidize organic compounds -> CO2
277
When will the COB be higher than the BOD?
If biologically recalcitrant organic compounds are present
278
What is the purpose of municipal wastewater treatment?
Remove/reduce nutrients
Remove/inactivate pathogenic microbes
Reduce organic C content (leads to reduced BOD)
Protects receiving ecosystems from nutrient overload and humans from wastewater pathogens
279
Which wastewater treatments use physical and chemical processes?
Primary & tertiary
| Only ones used in Montreal
280
Which wastewater treatment use biological processes?
Secondary treatments
281
Primary treatment:
```
Physical/chemical process
Removes up to 90% of organic matter
Steps:
- Bar screen (large debris removed to landfill)
- Grit chamber
- Add floculant to aid solids & colloid settling as well as some phosphate-removal
- Floculants = alum, FeCl3,...
- Settling (primary clarifier)
```
282
What is a wastewater clarifier?
Clarifier/sedimentation tank (slow water flow)
- Skin off grease/foam from surface
- Settled material (sludge is removed from bottom (sent to landfill/anaerobic digester)
- Clear effluent (raw sewage) flow over the top edge of weir and goes to secondary treatment
283
Secondary treatment:
```
Microbial process
Steps:
- Activated slug (aerobic)
- Trickling filter (aerobic)
- Sludge digestor (anaerobic)
Microbial processes that occur (during aerobic steps):
1. Nitrification (NH4 -> NO2 -> NO3)
2. Removal of pathogens
3. Removal of nutrients (BOD) as biomass
```
284
Activated sludge process:
Aerobic (uses aeration)
Part of secondary treatment
Can reduce organics (BOD) 90% in 4-8hrs
- Requires lots of O2 quickly
N, P, C are converted to microbial biomass (called flocs)
- Go from dissolved to solid form
- After aeration, flocs settle out of solution, removing BOD
- Key element is reycling of a portion of the settle floc (this is the "activated" sludge)
285
Sequence batch reactor (SBR):
Part of secondary treatment activated sludge
Has four tanks because its continuous flow (don't want new water mixing with settling water)
Steps:
1. Fill
2 . React (mix for about 1-2 hrs)
4. Settle
5. Decant
286
Main things that happen in activated sludge:
1. Decreasing the BOD of wastewater so add oxygen through aeration to encourage multiplication of aerobic bacteria that consume the nutrients
2. NH4+ is toxic at high conc. so nitrification occurs
- NH4+ + O2 -> NO2-
- NO2- + O2 -> NO3-
- NO3- assimilated into biomass or Denitrificationof NO3- -> N2 at anaerobic zones at bottom of tank
3. Removal of pathogens through floc/biofilm, consumed by predators
4. Removal of nutrients (BOD) as biomass
- Settling of floc (sludge) leaves cleaner water to flow out
287
Poorly settled flocs:
Due to open or porous structures
Causes organic material is released with the treated water
Mostly due to over-abundance of filamentous organisms in sewage population (called bulking)
- Caused by changes in: nutrients, flooding, seasonal changes, toxic chemical influx, pH changes
Increases BOD (high BOD -> pollution of receiving waters
288
How to control bulking:
FInd causes & reverse if possible
By predation via ciliated protozoans on filamentous bacteria
Chemical amendments
Tricky to control
289
Trickling filter:
Part of secondary treatment
Aerobic
Relies on formation of biofilm on surface of 2m deep loose gravel
Apply highly aerated sewage spray (think sprinkler)
Requires periodic backwash
Same principles as activated sludge (removal of pollutants as biomass, nitrification, trapped pathogens)
As wastewater flows through filter, nutrients are absorbed by microbes in biofilms
Cleaner effluent flows out of bottom
290
Biofilms in trickling filters:
```
Mostly algae & fungi; can be bacteria and protozoa
Stuck together by polysaccharides
Foodweb
Micro-habitats/environments
Micro-channels
```
291
Sludge digestors:
```
Part of secondary treatment
Anearobic
Slow (similar to septic tank)
Done in batches in large tanks
Expensive and large
How to speed up the process:
- Mix tanks & add heat
- Recycle "ripe" sludge
- Burn natural gas produced to head & power system
```
292
Types of sludge digestor tanks:
- Unheated, unmixed sewage digester
- Conventional heated and mixed digester
- Anaerobic contact process
- Fastest, flow-through (still slow)
293
What are the microbial processes in anaerobic digestion of secondary treatment:
Denitrification:
- NO3- -> N2
Fermentation & methanogenesis:
- Biomass/organic matter converted to gases CO2, CH4, H2S (vented)
- Fermentation yields heat
- Effluent contains organic acids & recalcitrant organic compounds
- Effluent can be released, returned to aerobic secondary treatment or to tertiary treatment
294
Anaerobic digestion pathway of secondary treatment:
Fermentation: organic polymers -> butyrate, propionate, lactate, succinate, ethanol, acetate... ->
Acetogenic reactions: butyrate, proprionate, lactate... -> acetate, H2, CO2 ->
Methanogenic reactions: Acetation, H2, HCO3- -> CH4 + CO2, CH4
295
What happens to the sludge from the sludge digesters:
Digester breaks down input sludge to simple components & residual sludge
Kills/destorys pathogens
Residual sludge is stabilized (not pathogenic, not smelly)
Good fertilizer (except for heavy metal content)
Residual sludge de-watered & usually land-filled
Effluent (high BOD) goes back into sewage treatment system
296
What happens to the effluent from secondary treatment?
```
Release with or without disinfection
OR
Second roud of secondary treatment
OR
Send to tertiary treatment
```
297
Settled sewage viable bacteria:
```
Number/ml = 1.4x10^7
Percent = 2.0%
```
298
Secondary effluents viable bacteria:
```
number/ml = 5.7x10^5
Percent = 1.1%
```
299
Tertiary effluents viable bacteria:
```
number/ml = 4.1x10^4
Percent = 0.12%
```
300
Tertiary treatment:
Physical/chemical process
Removal of specific compounds
Not always necessary
Removes PO4 via precipitation if P is too high
- primary and secondary treatments only remove 30% P
Final clarifier
Charcoal filters
301
Charcoal filters:
In big tanks
Remove organic compounds recalcitrant to biodegradation
Often in specific industrial applications
Re-use charcoal after burning to destroy organics
302
What does primary treatment remove?
Removes suspended solids
| About 30-40% of BOD
303
What does secondary treatment remove?
Removes dissolved organic substrates, pathogens, NH4
| Removes 80-90% of BOD (to 20-30mg/l)
304
What does tertiary treatment remove?
REduces recalcitrant organics PCBs, chlorophenols...
| Removes PO4
305
Sewage lagoons:
Requires plentiful land/sunshin
Low tech
Low costs
Takes days to weeks
306
Facultative sewage pond system:
```
1 to 2.5m depth
Aerobic & anaerobic zones
Reduces BOD by 75-95%
Requires a ~7 to 50 day retention time
Used in rural & small communities
```
307
Aerated sewage lagoon:
Used by small municipal sewage systems
Inexpensive
Range from 1.5-5 meters
Use motor-driven aerators floating on surface of wastewater that:
- transfer air into basins required by biological oxidation reactions
- Provide mixing required for dispersing the air and optimizing contact between reactants
Not as efficient as activated sludge systems but:
- 80-90% removed BOD
308
Septic tanks:
Anaerobic degradation of waste
Similar to sludge digestion
Effluent dispersed into well-drained soil for consumption by aerobic bacteria
Sludge is periodically removed
309
Wastewater treatment plants vs agriculture:
Impractical for purifying runoff from large agriclutural operations
Some use artificial wetlands to replace treatment plants
Not appropriate for large municipalities:
- Too much waste
310
Drinking water purification:
Secondary treatment. not yet potable or safe for human consumption
Requires further treatment to remove pathogens, eliminate taste/odor, reduce chemicals, & decrease turbidity
Typical drinking water treatment purifies untreated water
Steps:
- Sedimentation (removes particles)
- Coagulation & flocculation (form additional aggregates to settle out)
- Filtration (remove remaining particulates & organic/inorganic compounds)
- Disinfection (typically with Cl gas or UV radiation) (kill remaining microorganisms & prevent growth)
311
Viruses found in water:
Primarily enteric viruses (about 100 different types)
| Ex. hepatitis, polio
312
Protozoa and algae in water:
```
Entamoeba histolytica: dysentery
Giardia sp: diarrhea
Cryptosporidium: diarrhea
Algal blooms: toxins
Red tides: blooms of diatoms
Pfiesterica piscicida: dinoflagellates, fish kills, human illness
```
313
Coliform test:
Detects fecal contamination in water, wastewater
Coliforms: enteric rod-shaped gram-negative, non-spore forming, bacteria which can ferment lactose
Presence of coliforms = fecal contamination
Three stages:
- Presumptive test: gas production in lactose broth
- Confirmed test: gas production in brilliant green lactose bile broth
- Completed test: Coliform colonies on Levine's EMB agar; gas production
- Takes 4 days
314
Other methods to calculate bacteria in water samples:
```
Filter plate method
Direct count method
Other tests for:
- Fecal coliforms
- Fecal streptococci
- E. coli (microbiological quality determine by testing for this b/c indicates fecal contamination: 0/100ml acceptable)
```
315
Maximum acceptable concentration (MAC):
```
For E.coli = 0
For coliforms = 0
- Testing over 10 samples, no consecutive or over 10% of tests should show presence of coliforms
For Heterotrophic plate count (HPC) bacteria = no test (any inc is bad)
For protozoa = not possible (achieve 3log reduction)
For viruses = not possible (achieve 4log reducation or inactivation of virus)
For lead (in Montreal) = less than 5ppb
```
316
Filtration processes of drinking water:
```
Rapid filtration (used in US)
Slow sand filtration (used in Europe)
```
317
Rapid filtration:
Water moves vertically through sand
Often with activated carbon or anthracite coal that traps organic C
Fast filtration rates
Backwashing needed
318
Slow sand filtration:
Uses graded layers of sand (coarsest sand at bottom & finest at top)
Drains at base move treated water away for disinfection
Slow filtration
Removal of biological layer needed
Higher removal rates for all microorganisms
319
Disinfection of drinking water objectives:
Primary objective: kill, remove all pathogens
| Secondary objective: remove chemicals, contaminants, suspended solids & gases from drinking water
320
Main ways to disinfect drinking water:
Chlorine
Ozone (O3)
UV irradiation
321
Chlorine as a disinfectant for drinking water:
Very strong oxidant with:
Residual activity: minimal residual chlorine = .5ppm after 15mins
Common chlorine is sodium hydrochlorite
- Inexpensive, realtively safe
- When dissolved in water, slowly decomposes, releasing Cl, O2, & sodium & hydroxide ions
- disadvantage = organic molecules in drinking water become chlorinated, forming Trihalomethanes (THMs) which are carcinogenic
Alternative = monochloroamines (less THMs formed but less effective)
322
Ozone as a disinfectant for drinking water:
No THMs formed
Can produce bormate (is carcinogenic)
No residual activity
Must be made onsite
323
UV light as a disinfectant for drinking water:
Effective but no residual activity
Optimization difficult (effectiveness dec. as turbidity inc.)
Damages DNA of bacteria
Less effective against viruses
324
What are biosolids?
Sewage sludge that has been treated (removed pathogens & stabilize material)
Treatment includes digestion & usually additions of liming agents
Used in agricultural lands
- 80% of Ontario's municipalities spread sludge on agricultural land
325
Why are biosolids applied to arable lands?
```
High organic matter content
Improves soil structure
Improves water & nutrient holding capacities
Rich in N, P, S
Rich in micronutrients
Inexpensive amendment
```
326
Concerns of biosolids in agriculture?
```
Trace metals
Pathogens
Organic contaminants
PPCPs
Antibiotic resitant material
Odorous vapors
N & P (contributes to nutrient loading of water bodies)
```
327
Pathogens in biosolids:
Large conc. of microbes (relative to topsoil; similar to manure)
High levels of fecal coliforms (higher than manure)
Benefits of microbes = Inc organic molecule decomposition rates; inc. mineralization of N & P
Problems: some are pathogenic (very high fecal coliform count)
Treatments to reduce microbes: digestion, alkalinization, composting, heat-drying...
328
Organic contaminants in biosolids:
Conc. in biosolids are low (below levels for acut toxicity)
Most are volatile, removed during treatment process
Are biodegradable
329
What is NASM?
Non-agricultural source material
330
What are emerging concerns for biosolids?
PPCPs
Detect ppt
Don't pose risk to human health
In environment cause: chronic toxicity, endocrine disruption, behavior effects data
331
Growth promoting hormones:
GPHs
Used in farms
Concern for human health
Environmental effects are largely unknown
332
Antibiotics:
Majority are excreted in feces/urine
Three improtant groups in livestock: tylosin, tetracycline, sulfonamides
Arms race between bacteria and antibiotics cause bacteria to be better
333
Anthropogenic sources of N & P loading:
Agriculture: 293x10^3 tons/year of N; 55x10^3 tons/year of P
| Municipal wastewater treatment plants (MWTPs): 80.3x10^3 tons/year of N; 5.6x10^3 tons/year of P
334
Eutrophication is caused by:
Inc. P & N from erosion & run-ff from agriculture
Causes cyanobacteria growth (some are toxic)
- As phytoplankton dies, use up O2, water becomes increasingly anoxic leading to hypoxia
335
Remediation strategies of Eutrophic Lakes:
Chemical treatments
Dredging lake sediments
Ecological restoration
* Only work if N & P have been reduced
336
Chemical treatments for eutrophic lakes:
```
Addition of Al or Fe
Pros:
- Fast
- Cost effective
Cons:
- Doesn't nreduce microcystis sp.
- Can lower pH
- Al3+ is toxic
- Fe is less effective in anoxic conditions
```
337
Dredging lake sediments for eutrophic lakes:
```
Release of P stored in sediment
Pros:
- Is successful (depends on specific hydrologic characteristics of water body & sediment characteristics)
Cons:
- Time consuming
- Costly
```
338
Ecological restoration of eutrophic lakes:
```
Promote macrophyte populations
Pros:
- Release O2
- Reduce turbidity
- Compete with algae & cyanobacteria for nutrients & space
- Provide habitats
Cons:
- Difficult to establish
- Time consuming
- Can be undesirable for lakes
```
339
Nitrate sources from agriculture:
Non-point sources
- Leaching of chemical fertilizers
- leaching of animal manure
- groundwater pollution from septic & sewage discharges
Health concerns:
- Harm infants in large doses (reduce oxygen transport in blood)
- Cause blue-baby syndrome
N2O (nitrous oxide) builds up if too much NO3- is present
- Prevalent in hypoxic ocean waters
