Bioremediation Flashcards
(20 cards)
Bioremediation: definition, why bioremediate
Use of biotechnology to remove or reduce pollution (in water, soil, food, ecosystems, etc) to acceptable levels
Microbes have evolved to use pollutants as energy source and nutrition source so we can utilise to reduce pollution via converting to something else
Why:
Industrial activities had low levels of regulation of dangerous compounds and accidents (chemical spills, etc)
Increased demand for development of new properties so now Brownfield sites (previously used for industry) want to be used for building houses on, but doesn’t meet regulation
Additionally, digging through soils from unsaturated zone (gaps in soil are air) to saturated zone (gaps in soil are water) then the soluble pollutants will move in water underground to sea, or a public well of water supply, or into field growing crops
Soil and water samples are tested to identify level of pollution
Pollution: definition, classes, examples, persistence,
Pollution is the introduction by man into the environment of substances liable to cause hazards to human health, harm to living resources and ecological damage
Pollution can be point source (factory smoke) or a non-point source (fumes from cars, pesticides on farm)
Natural: compounds that occur naturally somewhere in the environment but human activity (mining industrial sites, metal smelting) leads to unnaturally high concentrations.
Ex. crude oil, heavy metals (lead, mercury, etc), carbon dioxide, phosphates
Xenobiotics: chemically synthesised compounds that have never occurred in nature
Ex. pesticides, herbicides, plastics
Persistance:
Pollutants can be classified as:
Degradable: Not stable in the environment
Degraded by biological and non-biological processes
Ex. simple hydrocarbons and petroleum fuels (degradability decrease as MW and degree of branching increase)
Persistent: Stable in the environment (at least in some conditions) for long periods of time.
Ex. typically fat soluble molecules, chlorinated hydrocarbons
Recalcitrant: Intrinsically resistant to any degradation
When may using microbes be inefficient/difficult in bioremediation
Xenobiotics are likely to be more persistent/recalcitrant because microbes have not had time to evolve pathways to degrade them. e.g. halogenated hydrocarbons
Insoluble compounds such as nylon, polyethylene and other plastics are recalcitrant because of low biological availability (not in a form that can be taken up by microbe, too large). Microbes can’t access them to degrade them since insoluble in water
Heavy metals can’t be enzymatically degraded by microbes so instead are made less harmful via oxidation, precipitated (therefore less bioavailable), purified and reused, or concentrated and stored safely
Case study of bioremediation project; reason, process, how pollutants were cleared
Clean-up of a 350-hectare area of Brownfield site for London 2012 Olympic Park.
Started with >3,500 sampling locations, creating more than 5 million chemical test results to check which pollutants were there (PAH, heavy metals, chlorinated solvents, etc)
Soil was excavated, and was treated with on and off site by soil washing, chemical stabilisation, bioremediation (biopiles for PAH and petroleum hydrocarbon degradation) or sorting. Or was removed
Free product (LNAPL hydrocarbon) was removed and contaminated groundwater pumped and treated.
Free products: the pollutant in it’s original form (not diluted in water, etc) sitting underground
DDT: structure, what it is, use, downside
DDT is a very effective insecticide (Paul Muller won the Nobel Prize in 1948 for developing)
Benefits
Sprayed over crops, in houses, etc
Controlled spread of vector borne diseases (malaria) which provided crop protection
Downside
DDT is stable and slow to degrade (half-life in soil up to 30 years) so remains in ecosystem
Accumulates in insect eating crops, and when bird eats insect has higher concentration. Process repeats up the food chain (biomagnification)
Accumulates and persists in fatty tissue
DDT accumulated in bird eggs (ex. eagle and falcons), thinned eggs so more fragile and breeding success declined
In humans over time it has carcinogenic potential
DDT ban increased breeding success
[See structure]
Mercury case study: how it occurred, effect, solution
In 1951, Chisso factory producing acetaldehyde via a Mercury Sulphate catalyst, producing inorganic mercury biproduct. After being dumped into harbour, aquatic microbes converted into methylmercury (neurotoxin)
Local cats going crazy: “dancing cat” disease and families in coastal villages eating local fish and shellfish were affected from mercury poisoning
1958/1959 cause identified as organic mercury compounds
* Up to 2kg mercury per ton sediment at mouth of plant outflow
* Company change waste-water route to pollute river instead of harbour and installed “water treatment plant” that was ineffective
* Continued to pollute until 1968 when production of acetaldehyde with mercury stopped and another catalyst was used
* Over 2000 certified victims and 10,000 other claimants have been paid $100s millions in compensation
Pollution cycle
Pollutants introduced via chemical spills, vehicle emissions, toxic chemical (pesticides), etc
Enters air, water (drinking water, groundwater), rain, soil
Transports long distances via air currents, water flow, and soil erosion
Acid rain dissolving monuments, bioaccumulation in our bodies from eating in food, etc
Underground:
Top layer soil
Unsaturated zone; soil is full of air
Saturated zone; soil is full of water (wells for drinking water)
Petrol from leak at petrol station goes through gaps of air in soil and puddles on top of water table as a free product (aka LNAPL).
Some is soluble and goes into water table, carrying pollutants, which can end up in water for public or agriculture
Oil pollution: source, localisation, bioremediation strategy
Oil has been in environment for millions of years
Cracks in sediment allows oil to seep out naturally and microbes have evolved to use as carbon and energy source
Humans drill underwater to access deposits of oil underground
Around 2/3rds of oil in sea is from industrial waste
10% from natural sources
Localisation depends on weight:
Oil seeps out of reservoir underground
Some deposit as heavy oil seep on seabed
The rest as oil droplets migrate up through water column to become oil slick on water surface
Light petroleum hydrocarbons evaporate into atmosphere
Over time some deposits back down to seabed as fallout plume of heavy petroleum hydrocarbons
Methane from reservoir bubbles up and releases into atmosphere or dissolves in water column
Bioremediation:
Microbes degrade as all this happens
Make more accessible/speed process by spraying dispersant (detergent) onto oil spill
Could cause oil spill to become more toxic and sink down into water
Also injected into well to form oil clouds underwater
Bag oil
Biostimulation with fertiliser
Oxygen and phosphate levels decreased around clouds suggesting microbial activity
16s sequencing to identify microbe and microarray showed genes involved in oil degradation increased
Oil contamination case study, prevention example
Exon valdez - oil spill
Ship with oil tanker ran into rocks and spilt a fifth of oil
Wind wasn’t high so oil wasn’t broken up and dispersed, instead blew ashore onto beaches killing wildlife
Took ~10 years for bird and mammal populations to recover
MTBE added to petrol to make petrol burn better by delivering O2 in too in car engine (used to be lead until realised it was toxic)
Reduces harmful emissions
Components of crude oil, petrol, extraction
Crude oil:
Aliphatics - pentane, etc
Aromatics - benzene, etc (a little more water soluble than straight chain)
Cyclic hydrocarbons - cyclohexane, etc
Non-hydrocarbons
Sulphurs
Nitrogens
Oxygens
Metallics
Petrol:
~20% is the BTEXs: benzene, toluene, ethylbenzene and xylene
Very dangerous, benzene is a carcinogen
Aromatic so resistant to degradation
Slightly water soluble
~80% is other hydrocarbons (cyclic, branched, straight chain)
Fractional distillation: Heat crude oil and filter based on temperature it heats at. Take 3-10 C in length
Light non-aqueous phase liquids: definition, effect, removal
Non-aqueous phase liquid - Most hydrocarbons don’t mix well with water so crude oil and petrol float to top after a while
Underground petrol storage tank leakage into soil below then onto top of ground water of light non aqueous phase liquid
Moves down to unsaturated zone then settles on impermeable rock or travels to saturated zone
Lighter than water so floats on top of water table and is carried downstream
Slightly soluble bits (ex. BTEX) dissolve into water and carried with water flow in pollution plume and can end up in water used for wells
Residual phase binds soil particles
Removal:
Drill well into ground and pump up oil (groundwater comes up too)
Separate water and oil to recover
Energy intensive
Biosparging
Biostimulation with fertiliser
Bioaugmentation
Microbes to degrade hydrocarbons: how to improve, requirements for bioremediation
Degraded by specific enzymatic pathways, and different microbes degrade different pollutants
Complete degradation - mineralisation into CO2, water and salts
Since degradative enzymes are encoded on genomic DNA or plasmids, can engineer to degrade new pollutants or move plasmid from one to another
Requirements:
Microorganisms capable of producing enzymes to degrade or immobilise target (bioaugmentation; add microbes that will reduce pollution)
Biostimulation: For growth, require energy source (hydrocarbon oxidation) and electron acceptor (O, N, etc reduction) is required for growth
Biostimulation: Appropriate conditions for cell growth (pH, moisture, nutrients (N, P, K), temperature)
Absence of toxicity (pollutant doesn’t kill microbes)
Removal of metabolites (complete conversion of pollutant, not into another toxic compound)
Absence of competitive organisms
Redox reaction: importance, use in bioremediation
Microbes can use hydrocarbon pollutants as an energy source (electron donor/acceptor in redox reaction) and nutrition source (carbon and nitrogen source)
In aerobic conditions, hydrocarbons are used as electron donors and O2 as electron acceptor, breaking down the pollutant to generate ATP
Very important:
Macronutrients - C, H, N, O, P (comprises most of microbe)
Micronutrients - S, K, Na, Mg, Fe, etc
Humans oxidise carbohydrates/acetyl CoA to get energy
Microbes can use multiple as electron acceptors:
Reduce oxygen (predominantly), sulphur, nitrogen (and sometimes) carbon.
Each has different oxidation states
Carbon in CO2 = +4
Carbon in methane = -4
Carbon in sugar = 0
Means it takes different number of electrons to convert into CO2
In respiration:
1 O2 can be reduced into 2 water
Can couple oxidation of O2 to one carbon in a carbohydrate to turn into CO2
Nitrate can be used a acceptor into nitrogen and carbohydrate as donor
Sulphate to hydrogen sulphide (smells)
Most energy efficient is O2 but used up early on so microbes that utilise nitrate (2nd most energy releasing) dominates, then the next when that runs out
Methanogenesis is least efficient (reduction of CO2 to methane) and is slow to degrade pollutant
Metabolism of straight chain alkanes: toxicity
Short (C5-C10) alkanes can disrupt plasma membrane (toxicity)
Medium length are slightly soluble and less toxic
Higher length can be solid (depends on temperature)
and therefore have reduced bioavailability
Bioavailability / toxicity
Bacteria can transport hydrocarbons across their membrane
Some bacteria produce biossurfactant on surface or secrete to help adhere to oil droplets, increasing the surface area for hydrocarbon uptake
Degradation of aromatics: example, pathway, draw, enzymes
Aromatics (ex. benzene) have a stable pi electron ring structure, making them chemically inert and resistant to degradation
A few species of microbes are known to degrade and pseudomonas is most studied that degrades to catechol/catechol-like compounds (upper pathway) before fully oxidising as energy source to produce CO2 (lower pathway)
[draw]
Contain di- and monooxygenases to initiate ring cleavage of BTEX compounds
Ring hydroxylating dioxygenases is used to degrade polyaromatic hydrocarbons (multiple benzenes fused together)
Pathways:
Degradation pathways are often on plasmids and mobile elements
An organism usually has one upper and one lower pathway
Can move plasmid from cell to cell by conjugation
Ex. Toluene and xylene metabolism - TOL plasmid
Whole Tol region can move from plasmid to plasmid via DNA transposition
Degradation via reduction
In anaerobic conditions, compounds like trichloroethylene; TCE (industrial solvent) are degraded via reductive dechlorination
Organohalide respiring bacteria (OHRB) use H or organic compounds (ex. lactate) as e donor and TCE organohalide as e acceptor.
Produces dichloroethene (DCE) and vinyl chloride (VC), more toxic than TCE
Only a few strains of dehalococcoides can completely dechlorinate TCE, DCE and VC to nontoxic ethene
Injection of molasses into contaminated site first leads to development of anaerobic environment then development of a dehalogenating microbial community. Can be followed by microbiome sequencing.
Identification of bacteria that can degrade a pollutant
Soil and water samples taken
Metagenome sequencing - extract and sequence all DNA from environmental sample without isolating microbes (comprehensive overview of which microbes present, genes they carry, etc) since some can’t be cultured in lab
Selective enrichments to isolate strains that are actively degrading pollutants
Bacteria identified:
PCR amplification and sequencing 16S rRNA genes (conserved in bacteria and variable between species, identify with database)
Whole genome sequencing to explore degradation pathways, resistance traits, mobile genetic elements (ex. TOL plasmid)
Community structure characterised - within the sample quantify % of each species, then compare with contaminated/pristine or before/after bioremediation
Selective enrichment:
Inoculate culture with soil into liquid culture of medium with pollutant as only C/N source
Grow for weeks, subculture, repeat
Plate onto agar; only bacteria which can use pollutant survive
Characterization of Microorganisms
Demonstrate that isolated organisms can degrade pollutant
Growth rate on pollutant
Identification of metabolic intermediates
Identification of enzymes responsible for degradation and
characterization of their mechanisms
Identification and cloning of genes and operons responsible
for degradation
Elucidation of gene regulation
In-situ Bioremediation strategies, pros/cons
Natural attenuation
Includes intrinsic bioremediation: chemical (redox reactions, reductive dechlorination, etc) and physical processes (dilution in water, vapourisation, etc)
Leave nature to get on with it (slow)
Monitored natural attenuation involves regularly sampling area to ensure process is occuring
Bioventing (petrol spills) - supply air and nutrients through wells to unsaturated zone to stimulate indigenous bacteria. Deliver minimum air required for biodegradation while minimising volatilisation and release of contaminants into atmosphere
Biosparging (petrol contamination in water table and below) - Inject air and nutrients under pressure into saturated zone. Drives volatiles out of water. Can recover vapour coming out of soil
In situ Landfarming - mix soil above ground with nutrients and
bulking agents and plough into ground
Enhance bioremediation - Biostimulation (addition of nutrients)
Microbes need nitrogen (150g), phosphates (30g), etc
Providing as fertiliser, allows them to uptake with more hydrocarbon from spill (1kg) to increase growth
Bioaugmentation (addition of microbes)
Less Costly
Slower
Ex-situ –Bioremediation:
strategies, pros/cons
Dig up the contaminated material and treat it elsewhere
Slurry-phase:
Soil in tank combined with
water, additives, fertilisers, microorganisms, nutrients,
oxygen
Solid-phase:
Land-farming (hydrocarbons): spread waste in a field on top of impermeable soil/clay/etc with raised barrier or specially constructed site.
Keep moist and turn over with farm equipment to aerate.
Biostimulate/Bioaugment
Collect leachate and treat
Soil biopiles: contaminated soil heaped, air & nutrients pumped in via pipes
Collect leachate (run-off) and any vapour
Maintain correct levels of moisture, pH, temp. for microbial growth
Biostimulation/Bioaugmentation
Pros/Cons
Easier to control
Used to treat wider range of contaminants and soil types
Costly
Faster
