HW6 - MGP Site Bioremediation
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1 HW6 - MGP Site Bioremediation 1. Given the following group of compounds commonly found in coal tars at MGP sites, determine the relative concentration and total mass of each in the air, water, and soil of the following equilibrated system: 1-L closed vessel to which 500 g air dry, sterilized soil and 200 ml water added mg coal tar MIX added Remaining volume is air. Assume MIX composition is the following (in mg/kg each): 6,000 benzene; 20,000 toluene; 700,000 naphthalene; 200,000 phenanthrene; 30,000 benzo(a) anthracene ; 44,000 pyrene Soil specific gravity 2.5; and 2% organic carbon content. Ignore potential mixture effects on the solubility and sorption of the compounds. Make other assumptions as needed (such as temperature of system). assume the system is at 25 C. see Table for solution. -> calculate the initial total mass of each compound in the system -> pure phase (NAPL) of all PAHs since water solubility exceeded -> most mass of all compounds associated with the NAPL pure phase (PAHs) or soil (BTEX; also soil PAH mass greater than water or air) -> check to be sure that the final total mass equates to the total mass added to the system 2. Characterize the total risk to an unassuming janitor who finds the lab experiment from problem 1, opens the system, and then proceeds to dump the water down the sink and the soil into the trash. (consider potential exposure routes, and list any other assumptions used) RISK from inhalation of compounds, primarily benzene, toluene, and naphthalene from dermal contact with liquid, soil, and NAPL (soluble, sorbed, and pure phase) carcinogenic risk factors (CPF) for benzene (inhal and dermal), benz(a)anthracene (no data, but class B2 carcinogen = some evidence of carcinogen) non-carcinogenic RfD for toluene (inhalation and dermal), naphthalene (dermal pending), pyrene (dermal pending) (no data for phenanthrene) RISK, non-carcinogen = intake / RfD carcinogen = intake * CPF benzene only inhalation carcinogen; add benzene and BaA dermal risk add dermal non-carcinogen risks for toluene, naph, and pyrene; tol inhal non-carcinogen was the janitor wearing gloves? did he or did he not spill any on himself? for all, short-term exposure and concentrations below the level where acute noncarcinogenic effects should occur. 3. If, alternatively, you are a laboratory worker who wishes to use a biological process to clean-up the experiment from problem 1 prior to disposal: * what general procedure would you use? * where would you get the bacteria innoculum for the biosystem? what characteristics would you desire the innoculum to have? * what methods would you use to monitor the effectiveness of your remedial efforts? * at what point would you determine that the system is clean enough to dispose of in a routine manner (sink/trash)?
2 - keep the current closed vessel as the bioreactor, so that none of the toxics leave the system, and no further contamination of glassware, etc. occurs - add a bacterial innoculum into the 1-L vessel; potential source: soil from an MGP site where bioventing was being conducted this soil would contain an acclimated mixture of aerobic bacteria hopefully, this would contain mono-aromatic degrading bacteria possessing mono or dioxygenase enzymes for BTEX degradation (such as TOD pathway); also bacteria with the nah plasmid for naphthalene degradation; these bacteria could potentially cometabolically degrade the higher PAH compounds - mix the system periodically by some method, to facilitate gas transfer (O2) into the water and soils and drive partitioning - potentially increase the temperature of the system somewhat (maintain at 30 C) in order to encourage the most rapid biotransformation rates and increase the solubility of the compounds so that more is bioavailable for degradation. - initially, the system would contain oxygen and nutrients (N,P,etc) from the air and soil for the bacteria. However, additional nutrients, ph buffer, and oxygen may need to be added to the system to sustain biodegradation. Headspace monitoring of oxygen and CO2 levels is particularly critical. (analyze 0.1 ml sample with thermal conductivity detector) estimated oxygen required: SR B,T, and PAHs approx mg HC/mg O2 - total HC mass = 3 g => 9 g oxygen - initial oxygen in system: assume 21% oxygen in 600 ml air and 8 mg/l DO in 200 ml water = (0.21 L O2/L air * 0.6 L air * 1.3 g O2/L O2) + (8 mg O2/L * 0.2 L) = 164 mg O mg O2 ~ 166 mg O2 => therefore, need to add lots of O2! to add oxygen to system and prevent build-up of CO2 which could drive the system to acidic ph conditions (due to bicarbonate/carbonic acid): circulate air from 1 L flask through to secondary system which would contain a base trap ( NaOH) which will sorb the CO2 out of the air, creating a pressure drop which could be replaced with pure oxygen. Total amount of pure oxygen gas needed over time: (9 g g) * (1 L O2/1.3 g O2) = 6.8 L - Periodically measure headspace concentrations of VOCs and SVOCs (primarily benzene, toluene, and naphthalene); this could typically be done using a very small gas volume (approx. 0.1 ml) analyzed on a gas chromatograph with flame ionization detector (GC- FID), in the analysis process the toxics are destroyed. However, this will only give an indication of BT and nap concentrations in the system. Analyze: once per week also liquid concentrations of the compounds could be measured, particularly for the nonvolatile PAH compounds. Similar analysis procedure (GC-FID) after extraction from liquid into solvent (such as pentane or cyclohexane) or high pressure liquid chromatography (HPLC) of sample. However, somewhat large sample volumes are needed (typically about 1 ml), so less frequent sampling would be used. - benzene and toluene will likely be completely degraded from the system first, due to most rapid biodegradation rates and bioavailability. Naphthalene will probably degrade the next most rapidly, but there is an extremely HIGH amount of naphthalene present (particularly in NAPL). approximate time: using aerobic degradation rates in Table and assuming that degradation from the liquid only with 200 mg/l bacteria in water:
3 ds/dt benzene = K * B * X / (KsI + B) assume re-equilibration due to mixing maintains liquid concentrations of benzene and toluene at near current levels ~10 mg/l each and benzene/toluene competitively inhibit each other : I = 1 + T/Ks-t assume: Benzene: K = 0.77 g/g-d, Ks = 0.13 mg/l (these are for mixed cultures, 20 C so Toluene: K = 0.94 g/g-d, Ks = 0.18 mg/l 30 C rates should be higher, conservative estimate) db/dt = 88.7 mg/l-d = 17.7 mg ben biodeg/d; dt/dt = 78.2 mg/l-d = 15.6 mg tol biodeg/d benzene biodegraded in approximately 1 day, toluene biodegraded in approx. 4 days (will take time to get 200 mg/l bacteria, but still short (WEEK) remediation time for B&T) naphthalene mass = 2100 mg if 200 mg/l bacteria, K = 0.45 g/g-d, Ks = 0.25 mg/l assume that benzene and toluene will inhibit naphthalene degradation, but most of degradation will occur after B&T already biodegraded out of the system assume all biodegradation of liquid phase, and that system mass transfer from solids/napl/air to water with biodegrad. can maintain liquid concs of naph approx. 5 mg/l (conservative estimate), then: dn/dt = 0.45 * 5 * 200 / ( ) = 86 mg/l-d = 17 mg nap/d => 124 days therefore, naphthalene biodegradation will take at least 4 months phenanthrene can also serve as a growth substrate, but less data available on rates. - during biodegradation of PAHs from the liquid, dissolution of these compounds from the pure phase NAPL will continue to maintain high liquid concentrations until all the pure phase is gone biodegradation of the other PAHs may be very slow, esp. with cometabolic degradation, but naph will be present for at least a few months to serve as a cosubstrate. After naphthalene is fully biodegraded from the system, no cometabolite compounds left. Will then need to add to system (salicylate, protocatechuate, or other) to get further biodegradation of high ring PAHs, particularly BaA. - cleaning of the soil will be the most difficult due to the high sorption and low water solubility of the compounds. After the easy to degrade compounds are gone, the system could be innoculated with special bacteria able to degrade the PAHs and/or biosurfactantproducing bacteria. - other methods: look for increase in turbidity of liquid which can indicate biomass growth, also genetically probe for specific bacteria (such as NAH plasmid, TOD pathway, etc), measure enzyme levels: monooxygenase and dioxygenase,... visually look for the pure phase NAPLs in the system (stop mixing of system and look for floating or sunk NAPLs and/or intra-soil blobs) - clean enough to dispose system? when liquid concentrations are below sewage discharge levels as set by the city municipal water treatment plant, the water could be dumped (standards may be concentration based or total mass). Must analytically measure to determine concentrations, and document discharge level on a sewer log. (in some municipalities) soil concentrations are somewhat more ambiguous. difficult to measure sorbed contaminant concentrations, particularly if sorbed into the soil organic matrix. if total mass is small, based on city standards can often dispose.
4 4. [6 pts] Given an MGP site with BTEX and PAH (2 to 5 ring) contaminated groundwater, due to the low solubility of oxygen in water you wish to explore alternate electron acceptor addition as a method to enhance in situ bioremediation. a. What preliminary tests and site data would you collect to evaluate the feasibility of alternate electron acceptors for site remediation? (2 pts) look for the presence of alternate electron acceptor compounds naturally in the subsurface, such as nitrate, Fe+3, Mn+4, sulfates, etc. determine the hydraulic conductivity (permeability) of the aquifer in the saturated zone look at subsurface geochemistry - soil types, ph, etc. that might react with added substrates other subsurface characteristics: nutrients (N, P), heavy metals, organic content, hydraulic gradient, etc. are PAH/BTEX degrading bacteria from different redox potentials present? do lab scale studies with native aquifer samples and addition of each potential electron acceptor, look for evidence of degradation of the 2 to 5 ring PAHs; evalulate relative degradation rates under each condition and the amount of added substrate required per amount of PAH degraded. Also, look for production and accumulation of undesirable products, such as H2S or nitrite. b. Selecting one of the potential options, what methods would you use to add the selected electron acceptor and what monitoring approach would you use to verify bioremediation? What are the advantages of this system over the other options? (2 pts) - list which compounds you would expect to be more or less readily degradable under the conditions Select nitrate as the alternate electron acceptor. Add as dissolved nitrate in water solution, which is pumped into the subsurface via a well in the contaminated zone. Due to bioenergetics, nitrate is the next most favorable electron acceptor, after oxygen. Therefore, maintain fairly high biodegradation rates of BTEX compounds. Advantages over Other options: if nitrate is present, these bacteria will naturally outcompete bacterial consortia which would use other electron acceptors, due to energetic favorability. In terms of the BTEX and PAH compounds, there has been more evidence of degradation of these compounds under nitrate reducing conditions compared to the other alternate electron acceptors. Problems are also associated with negative by-products such as H2S from sulfate reducers, Fe which may plug the aquifer (due to precipitated forms of Fe+2), etc. Potential degradation of compounds: toluene > ethylbenzene > xylenes > benzene > naphthalene > other PAHs (may be cometabolic degradation) c. What other factors might limit clean-up in the saturated zone, and potential approaches to evaluate and overcome these limitations? (2 pts) NAPLs (LNAPLs and DNAPLs) serving as continuing sources of contaminants to dissolve into the groundwater - soln: find and physically REMOVE LNAPLs (bioslurping or draw-down, etc)
5 try to locate DNAPL zones and remove free phase if possible - also, very high concentrations of contaminants (esp. BTEX) near NAPLs could be toxic to denitrifying bacteria; therefore, may only have biodegradation away from the NAPLs in the lower concentration areas - NAPL degradation only occurs at interface or edge, so limited by bioavailability; may add surfactants or emulsifiers to increase NAPL solubilization compounds sorbed onto the soil solids in the saturated zone which could desorb into the groundwater, may be limited by bioavailability; add surfactants (may grow biosurfactant producing bacteria in the lab and add harvested biosurfactants, which have less change of biotoxicity compared to synthetic surfactants) getting the introduced nitrate into all of the contaminated areas - subsurface heterogeneity may yield preferred flow paths so that nitrate will not get to some pockets of contamination; soln: pulsed injection and/or variable pressure injection which may change preferred flow paths and create non-steady state conditions build-up of nitrite (produced from the added nitrate) to levels which are toxic to the bacteria - are nitrite -> N2 gas bacteria present? if so, how to increase activity if not, add? or encourage heavy metals in the subsurface which are toxic to denitrifying bacteria - if present, nothing can be done, must encourage other bacteria (perhaps sulfatereducers or methanogens) which are not as susceptible to the heavy metal toxicity excess biogrowth may plug the aquifer, increasing the difficulty of pumping in water - less of a problem than some of the other electron acceptors - if allow system to become somewhat nutrient limited (P) may limit biogrowth - may be a positive if attempting a biobarrier type of remediation
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