Pedro J.J. Alvarez, Ph.D., P.E., DEE

Pedro J.J. Alvarez, Ph.D., P.E., DEE
Principles & Applications of BTEX Bioremediation Pedro J.J. Alvarez, Ph.D., P.E., DEE University of Iowa Rio de Janerio, July 29, 2002 As the title of this workshop suggests, I would like to give you a panoramic view of current BTEX biodegradation and bioremediation approaches, and discuss also their scientific basis.

Ya la fitorremediación se esta usando en mas de 200 lugares en los EEUU

Prospectus What are BTEX and why care about them?
What is needed to biodegrade them? How to exploit biodegradation for site cleanup? What are the more serious technical and political challenges related to BTEX bioremediation? What is epistemology and how can it help us address some of these challenges?

Let me begin by reminding you that groundwater contamination by petroleum hydrocarbons is one of the most common environmental problems that we face today. To put the magnitude of this problem in perspective, the EPA estimates that there are 2 million underground tanks storing gasoline in the U.S., and hundreds of thousands of these tanks are leaking. Analysts estimate that 40 million liters are spilled each year.

“Water, water everywhere, nor any drop to drink” The Rime of the Ancient Mariner, Samuel Taylor Coleridge These leaks contaminate potential drinking water sources with toxic and carcinogenic compounds. Since one-half of the U.S. population drinks groundwater, this is of concern to public health.

Contaminants of Concern: BTEX
CH3 CH2CH3 Benzene m-Xylene p-Xylene o-Xylene Toluene Ethylbenzene When such leaks occur, the contaminants of greatest concern are the monoaromatic hydrocarbons: benzene, toluene, ethylbenzene, and the 3 xylene isomers, ortho-, meta,- and para-xylenes, which are collectively known as BTEX. All of these compounds are powerful depressants of the central nervous system, and B can cause leukemia. Therefore, benzene is often the target compound that determines the need for corrective action. These compounds are volatile, so they can be removed by air strippers; they are moderately hydrophobic, so they can be removed with activated carbon filters, and they are also biodegradable under the right conditions, which makes bioremediation often the most feasible cleanup approach. But what are these right conditions? Importance: • Relatively high solubility = High migration potential • Toxicity: Benzene can cause leukemia at 5 µg/l • Volatile, hydrophobic, biodegradable

Requirements for Biodegradation
1. Existence of organism(s) with required catabolic potential. Xenobiotic will be degraded to an appreciable extent only if the organism has enzymes that catalyze its conversion to a product that is an intermediate or a substrate for common metabolic pathways. The greater the differences in structure between the xenobiotic and the constituents of living organisms (or the less common the xenobiotic building blocks are in living matter), the less likelihood of extensive transformation or the slower the transformation. Note that after glycosyl residues, the benzene ring is the second most common building block in the biosphere, and microorganisms have been in contact with such aromatic compounds throughout evolutionary periods of time. Therefore, it is not surprising that so many different microorganisms exist that can feed on BTEX This requirement is not an issue for BTEX

Requirements for Biodegradation (contd)
2. Presence of organism(s) in the environment. BTEX degraders are commonly found, but differences in relative abundance of dissimilar phenotypes may lead to apparent discrepancies in the biodegradability of a given BTEX compound at different sites. Depending on the relative abundance of different strains, B could degrade earlier than T at one site, but the opposite may be observed at other sites. Although BTEX degraders are fairly common, different strains may exhibit different idiosyncrasies and preferences for different compounds. B could degrade earlier than T at one site, but the opposite may be observed at other sites. Often, these apparent discrepancies are the result of differences in the relative abundance of different phenotypes.

Frequency Analysis of Biodegradation Capabilities of 55 Hydrocarbon Degraders
100 90 80 70 60 % Strains that degraded compound 50 40 30 Incidentally, we conducted a survey of the biodegradation capabilities of 55 aerobic strains, Toluene was the most frequently degraded compound (almost 90%) Less than 50% of the strains degraded o-Xylene or Benzene when fed alone. Thus, B and o-X were relatively recalcitrant. Note that the TOL plasmid does not code for enzymes that can degrade B or o-X Interestingly, the strains that could degrade the less frequently degraded compounds (B and o-X) exhibited broader biodegradation capabilities. 20 10 B T E p-X m-X o-X N

3. Compound must be accessible to organism: a) Physicochemical aspects (bioavailability). Desorption, dissolution, diffusion, and mass transport b) Biochemical aspects. Membrane permeability (important for intracellular enzymes), uptake regulation. Bioavailability is rarely a problem for BTEX, but some of these mass transfer processes could control biodegradation rates, such as desorption from aquifer material, dissolution from NAPL or diffusion of nutrients. Note that compounds get in the cell through selective gates, and the proteins that make these channels must be present. We do not understand the regulation of BTEX uptake very well yet.

4. If catabolic enzymes involved are not constitutive, they must be induced Inducer(s) must be present above specific treshold (e.g., [T] > 50 mg/L) The inducer must be present in sufficient quantities. T is usually the best inducer Diagram is a simplistic reminder that the inducer must be able to get into the cell and that metabolites or other substrates can repress enzyme synthesis (feedback inhibition) Thus, it is desirable to have other species that can remove such compounds if they accumulate In microbial communities, we often see such synthropic comensalistic interactions

Benzene Degradation by Pseudomonas CFS-215: Toluene enhanced enzyme induction
10 20 30 40 50 Control T = 0 Benzene Concentration (mg/L) T = 0.1 mg/L The presence of T usually has a beneficial effect on the degradation of other BTEX In this example, CFS-215 was capable of degrading benzene when present alone (2 wks). However, the presence of toluene significantly enhanced benzene degradation (2 days). Two separate mechanisms may explain this behavior. One, is that toluene acted as growth substrate in the proliferation of these microorganisms, which led to a greater number of microbes capable of attacking B and thus a faster degradation rate. However, B degradation was significantly enhanced even when only traces of toluene were initially present. The amount of T fed (0.1 mg/l) could not yield a significant relative increase in biomass. Thus, the observed enhancement must be due to another mechanism: toluene enhanced enzyme induction. This was confirmed by enzyme activity assays. T = 50 mg/L 15 10 5 Time (days) Alvarez and Vogel (1991) Appl. Environ. Microbiol., 57:

Cometabolic Degradation of o-Xylene by Denitrifying Toluene Degraders
2 4 6 8 10 TOLUENE mg/l Active Controls 50 40 30 20 10 days 0.0 0.5 1.0 1.5 2.0 Here we see another mechanism how T can enhance the degradation of another BTEX compound (o-X). In this case, T is serving as a primary substrate for the cometabolism of o-X. This sort of substrate interaction appears to be rather common under denitrifying conditions. In general, the presence of T enhances the catabolic range of the consortium o-XYLENE mg/l Active Controls 50 40 30 20 10 days Alvarez and Vogel (1995) Wat. Sci. Technol., 31: 15-28

5. Environment conducive to growth of desirable phenotypes and functioning of their enzymes: a) Presence of “recognizable” substrate(s) that can serve as energy and carbon source(s) (e.g., the BTEX) and limiting nutrients (N and P, trace metals, etc.). b) Moisture (80% of soil field capacity, or 15% H2O on a weight basis, is optimum for vadose zone remediation. Need at least 40% of field capacity). c) Availability of e- acceptors (e.g., O2 for oxidative reactions) or e- donors (e.g., H2 for reductive transformations). The e- acceptor establishes metabolism mode and specific reactions. Seven conditions or sub-requirements a) No need to add nutrients if [BTEX] < 1 mg/L b) Contamination can occur in different domains. Need water in the unsaturated zone

The electron tower concept
Half Reaction E °´ Reduction Potential Hierarchy H volts Reduced Oxidized -0.50 H H + 2 Benzene degradation to CO2 and CH4 under methanogenic conditions C H + 4.5 H O 2.25 CO CH benzene CO 2 6 6 2 2 4 D Go ’ = -(30 e-/ mol ) (96.63 kJ/volt) ( (-0.29) volts) -0.25 CH CO D Go ’ = -133 kJ/ mol of benzene, or - 4.5 kJ/e - equiv transferred 4 2 (barely feasible) HS - SO 2- 4 Benzene degradation to CO2 under aerobic conditions C H + 7.5 O 6 CO + 3 H O 6 6 2 2 2 D Go ’ = -(30 e-/ mol ) (96.63 kJ/volt) ( (-0.29) volts) D Go ’ = -3,200 kJ/ mol of benzene, or - 107 kJ/e - equiv transferred 0.25 Electron Tower (24 x more feasible) BTEX degradation is a redox reaction where electrons are transferred from the fuel molecule or electron donor to the electron acceptor. Falling ball analogy: potential energy = mgh The greater the potential difference between the donor and the acceptor, the greater the amount of energy potentially available to drive life functions Aerobic is much more feasible thermodynamically. Not surprisingly, B degrades much faster aerobically. 0.50 N NO - 0.75 2 3 H O O 2 2

Aerobic BTEX Degradation
BTEX are hydrocarbons (highly reduced) so their Oxidation to CO2 is highly feasible thermodynamically (fuel) Aerobic BTEX biodegradation is fast (O2 diffusion is often rate-limiting) Aerobic BTEX degraders are ubiquitous (e.g., Pseudomonas) Need oxygenase enzymes (i.e., enzymes that “activate” O2 and add it to carbon atoms in the BTEX molecule) The ring must be dihydroxylated before ring fission. Once the ring is opened, the resulting fatty acids are readily metabolized further to CO2. Often, BTEX degrade as fast as the O2 diffuses into the plume. Aerobic ring cleavage is also oxidative, needs O2

Anaerobic BTEX Degradation
Rates are much slower because anaerobic electron acceptors (e.g., NO3-, Fe+3, SO4-2, and CO2) are not as strong oxidants as O2. Benzene, the most toxic of the BTEX, is recalcitrant under anaerobic conditions (i.e., it degrades very slowly – after TEX, or not at all) Anaerobic degradation mechanisms are not fully understood Benzoyl-CoA is a common intermediate, and it is reduced prior to ring fission by hydrolysis. The oxygen in the evolved CO2 is from water. Anaerobic BTEX degradation processes (e.g., denitrifying, iron- reducing, sulfidogenic, and methanogenic) are important natural attenuation mechanisms. Also, humic acids, Mn, perchlorate can serve as electron acceptors benzyl succinate synthase is a key enzyme Martin Reinhard showed recently that you can enhance in situ BTEX degradation by adding nitrate + sulfate

O2 > NO3- > Mn+4 > Fe+3 > SO4-2 > CO2
In aquifers, electron acceptors are used in sequence. Those of higher oxidation potential are used preferentially: O2 > NO3- > Mn+4 > Fe+3 > SO4-2 > CO2 Source: Wiedemeier et al., 1999 Electron acceptors are often used in sequence… Thus, contamination by gasoline often results in geochemical transitions such as shown here. Typically, you have strong anaerobic conditions near the source, where the biochemical oxygen demand exerted by the pollutants is very high and the preferred electron acceptors are depleted, and you transition into aerobic processes near the fringes of the plume, where the BTEX concentration is low.

5. Favorable environment (continued): d) Adequate temperature (rates double for ∆T = +10°C). e) Adequate pH (6-9). f) Absence/control of toxic substances (e.g., precipitation of heavy metals, dilution of toxic conc.). g) Absence of easily degradable, non-target substrates that could be preferentially metabolized (ethanol?). 6. Time. Without engineered enhancement, benzene half-lives on the order of 100 days are common in aquifers. Want degradation rate > migration rate Groundwater temperature is typically within 1C of the mean annual air temperature In Alaska, use steam pipes to heat groundwater pH is important for getting the correct degree of protonation of enzymes, which affects 3-D structure and activity. Landfill leachates have organic acids that lower the pH to inhibitory levels Sulfide production by SRB enhances metal precipitation, but too much (200 mg/L) can be toxic Ethanol in gasoline can hinder BTEX degradation

What is Bioremediation?
It is a managed or spontaneous process in which biological, especially microbiological, catalysis acts on pollutants, thereby remedying or eliminating environmental contamination present in water, wastewater, sludge, soil, aquifer material, or gas streams. (a.k.a. biorestoration). Ex Situ (Above ground) In Situ (In its original place, below ground) Engineered Systems (biostimulation vs. bioaugmentation) Natural Attenuation (intrinsic/passive) Now that we have a basic understanding of biodegradation, I would like to discuss bioremediation, which I would like to define it as the use of microorganisms, or the enzymes they produce, to degrade environmental pollutants. There are several strategies to exploit such natural degradative processes. For example, you can use conventional reactors similar to those used to treat industrial and domestic wastewater to treat contaminated groundwater and soil above-ground. However, most people think of bioremediation as an in situ process that works with or without human intervention to stimulate microbial activity.

Why Use Bioremediation?
Can be faster and cheaper (at least 10x less expensive than removal & incineration, or pump and treat) Minimum land and environmental disturbance (e.g., generation of lesser volume of remediation wastes) Can attack hard-to-withdraw hydrophobic pollutants Done on site, eliminates transportation cost & liability Environmentally sound (natural pathways) Does not dewater the aquifer Bioremediation and natural attenuation are the technologies of choice for delaing with BTEX contamination, and these are some of the main reasons why Bioremediation, however, is not always applicable...

When is engineered bioremediation feasible?
Feasibility depends on: 1) Kh  distribution of nutrients and e- acceptors (Kh > 10-5 m/s) 2) Adsorption  bioavailability (depends on Kow and foc, problem for PAHs) 3) Potential degradation rate (half life < 10 days) 2 Feasible 1 with Feasible Enhancement log k; (per day) -1 For example there are three variables that determine the feasibility of converting a contaminated aquifer into a biological reactor Lets review 4 common engineered bioremediation approaches Not feasible -2 -3 - log K (cm/s) h

Bioventing Used to bioremediate BTEX trapped above water table
Vacuum pumps pull air through unsaturated soil Need to infiltrate water (with nutrients) to prevent desiccation The general idea of engineered bioremediation is to convert the contaminated zone into a bioreactor. Source: MacDonald and Rittmanm (1993) ES&T, 27(10)

Water Circulation Systems
Used to bioremediate BTEX in saturated zone (Raymond) Contaminated water is extracted, treated (air-stripping, activated carbon, or biodegradation), and recycled. Some is amended with nutrients and reinjected (pulsing is better). Clogging near injection well screens and infiltration galleries can be a problem (bacterial growth, mineral precipitation) but pulsing reduces clogging (may need occasional Cl2, H2O2) This was actually the first method, which was patented by Raymond in the 1970’s Clogging near injection well screens and infiltration galleries can be a problem (bacterial growth, mineral precipitation) pulsing reduces clogging (may need occasional Cl2, H2O2).

Air Sparging Injection of compressed air directly into contaminated zone stimulates aerobic degradation, strips BTEX into unsaturated zone to be removed by vapor-capture system Not effective when low-permeability soil traps or diverts airflow Air sparging is commonly used by the Air Force to stimulate the aerobic biodegradation of jet fuel spills. The idea is to add nutrients and moisture through an infiltration gallery and inject oxygen to stimulate microbial activity. In this case, the air we inject also strips volatile contaminants, which are captured by vacuum pumps. Thus, this a hybrid system that exploits both physical and a biological processes.

Biobarriers Containment method that prevents further transport (hydraulic or physical controls on groundwater movement may be required to ensure that BTEX pass through barrier Biologically active zone is created in the path of the plume by injecting nutrients and electron acceptors (could use oxygen-releasing compounds, or inject compressed air and form an air curtain) Recently, there has been a lot of interest placed on biobarriers, especially for dealing with shallow and narrow plumes that could flow along buried river channels. This is a containment method... Treated Water Air Curtain

Benzoate addition as auxiliary substrate (1 mg/L) stimulated benzene attenuation through 1-D “biobarrier” 200 Sterile control 150 Effluent Benzene (µg/L) Not amended 100 The efficiency of biobarriers could be enhanced by adding non-toxic structural analogues, such as benzoate (1000 mg/l in Cola). This graph shows the effluent benzene concentration that breaks through from different aquifer columns that are mimicking a 1-D barrier. It takes time for microbes to acclimate and begin to grow. When this happens, B degradation rates increase and the effluent concentration decreases. Acclimation was enhanced by adding small amount of benzoate (1 mg/l). This enhanced aerobic B degradation and significantly attenuated its breakthrough. No enzyme induction. Apparently, the aromatic nature of benzoate enhanced the proliferation of BTX degraders, which resulted in faster rates. But care should be taken not to add too much benzoate, which could exert a significant BOD and consume the available O2 - C O O 50 with benzoate 1 2 3 4 5 6 7 8 9 10 Time (days) Alvarez P.J.J., L. Cronkhite, and C.S. Hunt (1988). Environ. Sci. Technol. 1998; 32(5)

Bioremediation Market
According to the Organization for Economic Cooperation & Development), the global market potential for environmental biotechnology doubled in the past 10 years to $75 billions in the year 2000 In USA, we have 400,000 highly contaminated sites, and NRC estimates the cleanup cost to be on the order of $1,000 billions In USA, the current bioremediation market is only about $0.5 billions These past slides illustrate that numerous bioremediation approaches exist that can be very effective, and environmental biotechnologies are becoming very popular, doubling their share of the market in the past 10 years. But considering the relatively small share of bioremediation in the site remediation market, we must conclude that it is generally an underutilized technology. Part of the reason for this is that bioremediation is surrounded by a lot of controversy, which dates back to its origins.

Bioremediation experienced many up- and downturns
1950’s: Microbial infallibility hypothesis (Gayle, 1952) 1970’s: Regulatory pressure stimulates development. Adding bacteria to contaminated sites becomes common practice. Failure to meet expectations (e.g., DDT accumulation) prompts a major downturn. 1980’s: It becomes clear that fundamental processes need to be understood before a successful technology can be designed. This realization, along with the fear of liability and Superfund, stimulates the blending of science and engineering to tackle environmental problems. 1990’s: Many bioremediation and hybrid technologies are developed. However, decision makers insist on pump and treat, and Superfund is depleted. Poor cleanup record and high costs stimulate paradigm shift towards natural attenuation and RBCA. Bioremediation has experienced many upturns and downturns as a result of political and economic forces. (Chinese, 2k ago) Gayle proposed that for any conceivable organic compound, there exists a microorganism that can degrade it under the right conditions. If not, evolution and adaptation would produce such a strain. This hypothesis cannot be proven wrong, because failure to degrade a contaminant can be attributed to your failure to use the right strain under the right conditions. In other words, as Carl Seagan says, The absence of evidence is not in itself evidence of absence In the 70’s, environmental statutes of unprecedented scope passed, and the interest in bioremediation grew. Unfortunately, bioremediation became attractive for snake-oil salesmen who claimed to solve all problems of contamination by adding bacteria to contaminated sites (bioaugmentation). They did not recognize that the indigenous bacteria already present were probably better suited to do the job, and if they were not degrading the contaminants is probably because they lacked an essential ingredient, like oxygen, or the contaminant was not bioavailable, or it was thermodynamically unfeasible to oxidize it and the only way to degraded it was to reduce it. Their failures created an bandwagon effect. Bioremediation research boomed in the 1980’s, and numerous successes occurred, primarily with cleaning up petroleum product releases. However, the majority of decision makers viewed it as a risky technology and sticked with pump and treat. Eventually, Superfund ($10 billion) dried up. This caused an interesting change in site remediation philosophy towards RBCA and NA.

Aerobic Unsaturated Zone
Volatilization Oxygen Exchange To explain what natural attenuation and risk-based corrective action are, consider a common contamination scenario When contamination occurs, a dissolved plume develops and expands downgradient. Fortunately, there are numerous processes that reduce contaminant concentrations and attenuate their migration, including volatilization (10%) sorption, dilution, and biodegradation (aerobic in the fringes, anaerobic in the core, where BOD > DO). The combined effect of these mechanisms is known as natural attenuation. Dissolution Anaerobic core Advection Aerobic uncontaminated groundwater Aerobic Processes Mixing, Dilution

Atenuação Natural Fluxo da água subterrânea PE
Numerous field investigations have recently shown that BTEX plumes do not expand forever. They reach a maximum size, typically smaller than 100 m, and stabilize. Let me illustrate this --- This “steady-state” is achieved when any additional contamination released from the source is degraded and “naturally attenuated” at the same rate as it is introduced. Eventually, as the source disappears, the rate of introduction becomes smaller than the rate of degradation and the plume shrinks and disappears, completing the life cycle. Fluxo da água subterrânea

Atenuação Natural PE Fluxo da água subterrânea

Plume Source A candle might be a good analogy of how a plume behaves. The flame, which symbolizes the plume, does not grow forever, but reaches a constant size and then goes out when the wax is used up. Note that the size of the candle does not affect the size of the flame, but it determines how long the flame stays on Similarly, the mass of the source of a plume does not affect the plume length, which is controlled by the flux of pollutants out of the source zone and the flux of electron acceptors like oxygen from the surrounding uncontaminated groundwater. The source mass, however, influences how long it would take for the plume to disappear.

What is Monitored Natural Attenuation?
MNA is the combination of natural biological, chemical and physical processes that act without human intervention to reduce the mass, toxicity, mobility, volume, or concentration of the contaminants (e.g., biodegradation, dispersion, dilution, sorption, and volatilization). Success depends on adequate site characterization, a long-term monitoring plan consistent with the level of knowledge regarding subsurface conditions at the site, control of the contaminant source, and a reasonable time frame to achieve the objectives. MNA should not be a default technology or presumptive remedy. The burden of proof (e.g., loss of contaminants at field scale, and geochemical foot-prints) should be on proponent, and evidence of its effectiveness should emphasize biodegradation. So now that you have a feeling for what natural attenuation is, let us define it formally as… Typically MNA is used in conjunction with active remediation measures (e.g., source control) or as a follow-up to such measures, and its success depends... “Reasonable time frame” is a site-specific decision, generally meaning not excessive compared to other remedies. Reasonableness depends on current and future potential uses of the aquifer. MNA can be controversial since it seems that officials are walking away from contaminated sites. That is why it is important to get public participation early in the decision making process and to ensure that MNA is not a default...

Plume Dimensions Reflect Natural Attenuation MEDIAN PLUME DIMENSIONS
BTEX Plumes (604 Sites) 132 ft 1000 ft TCE Plumes (88 Sites) Other chlorinated solvent plumes (29 Sites) 500 ft A survey of plume dimensions shows that BTEX plumes are relatively small compared to chlorinated solvent plumes. This reflects that BTEX are relatively easy to degrade and are attenuated to a greater extent than chlorinated solvents. Salt Water Plumes (chloride) (25 Sites) 700 ft 200 400 600 800 1000 Feet

Because of its low cost (10%) and the eagerness of some regulators to “close the books” on low-risk sites, natural attenuation has become the technology of choice in the past 5 years for dealing with contamination by leaking USTs. Used successfully in more than 15,000 sites, but there is a tendency to over-prescribe it. Natural attenuation is often used in conjunction with risk-based corrective action, and I would like to discuss what this entails.

A few years ago, the law required that all contamination everywhere had to be removed below a stipulated level, such as the drinking water standard, even if contamination occurred on the middle of nowhere and there were no people at risk. Unfortunately, the cost of environmental compliance was too high, and there was a concern that cleaning up every site to meet DW standards could only happen at the expense of other important social and educational programs. Sometimes, it was technically impossible, if no unfeasible, to meet DW standards. In a 1994 NRC study, conventional P&T technologies restored contaminated groundwater to regulatory standards at only 8 of 77 sites (10%). Therefore, resource allocation problems, coupled with the realization that plume expansion is limited, motivated a paradigm shift in site remediation practices towards natural attenuation and risk-based corrective action. Concentration “safe”

What is Risk-Based Corrective Action?
Clean source only to a level that will result in an acceptable risk at the potential receptor’s location (e.g., property boundary) Need a mathematical model to calculate the required Co receptor Co =? Specifically, remediation laws were changed so that contamination now has to be removed only to a level that results in an acceptable risk at the point where potential receptors are located. This is known as risk-based corrective action. For example, the objective would be to ensure that benzene concentrations are below the DW standard of 5 mg/L here, which means that we only have to clean the source area to this level, and rely on NA to decrease the concentration to a safe level at the receptor’s location. Also, if potential receptors are beyond the reach of the plume, there would be no need for active cleanup. The new law would only require monitoring (sentinel wells). This paradigm shift has stimulated a lot of debate. Sometimes, it is not clear RBCA is a solution to the resource allocation problem, or an excuse to do nothing. Sometimes, it seems that the question has changed from How clean is clean to How safe is dirty. One thing is clear, RBCA relies heavily of mathematical fate and transport models to determine how far will a plume get and how to set site-specific cleanup goals. Thus, it is important that we understand the capabilities and limitations of such models. Concentration “safe”

Analytical Solution of the Advection-Dispersion-Sorption Equation with First-Order Decay, for Constant Rectangular Source (Domenico, 1987) ï þ ý ü î í ì ú û ù ê ë é - + ÷ ø ö ç è æ = ) ( 2 / 2) 4 1 8 , x Z erf Y y vt v erfc C t z o a l [ ] exp k s Models are useful analytical tools, and can be used to demonstrate that natural attenuation is occurring Limited predictive capability (order-of-magnitude accuracy): groundwater flow and microbial behavior rarely follow simplifying assumptions. This is a typical model used in risk-based corrective action to describe the concentration of a contaminant at different times and locations. This widely used model is based on many simplifying and influential assumptions that often do not account for the complexity and heterogeneity of the site. Consequently, such models are good screening tools to evaluate if natural attenuation is occurring, but their predictive capabilities are limited to order-of -magnitude accuracy. It seems ironic that the main advantage of these models, which is their simplicity, is also their main disadvantage. This model has lots of parameters, but which ones are most important?

Variable Baseline Value Lp (%)
Sensitivity Analysis: Effect of Doubling a Variable on Plume Length (Lp) Variable Baseline Value Lp (%)  (day-1) Co (ppb) , Z (m) Y (m) x (m) foc n b (g/cm3) Vw (m/day) A sensitivity analysis of this model shows that the parameters having a greater influence on model predictions of maximum plume length are the biodegradation rate and the groundwater flow velocity. Thus, one needs to pay special attention to these parameters Vw can usually be measured accurately. Lambda, however, is a volume-weighted average parameter that is subject to considerable variability both in time and space, since it depends on electron acceptor availability and microbial community structure. Therefore, we are going to focus on this parameter. Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez (1999). Proc. 6th International Petroleum Environmental Conference, Houston, TX.

Frequency Distribution for  (n=79)
How variable are biodegradation rates in the field, and What are “reasonable” parameters for RBCA models? Frequency Distribution for  (n=79) Mean = day -1 Median = day-1 (t1/2 = 139 days) 100 Density 50 We conducted a survey of 79 sites and estimated l values by model fitting. The purpose of this exercise was to characterize the variability and central tendencies of this parameter. Lambda varied widely (3 to 4 orders of magnitude). The median value corresponds to a half life of 139 days 0.00 0.05 0.10  (day-1) Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez (1999). Proc. 6th International Petroleum Environmental Conference, Houston, TX.

Current Status of Bioremediation
We have made significant advances towards understanding the biochemical and genetic basis for biodegradation. However, bioremediation is still an underutilized technology. Bioremediation is not universally understood, or trusted by those who must approve it. To take full advantage of its potential, we need to communicate better the capabilities and limitations of bioremediation, and answer: What is being done in the subsurface, Why, How, and Who is doing what? How fast is it being done, and can we control it and make it go faster? When can we meet cleanup standards in a cost-effective manner? Can we reasonably predict that what we want to happen, will happen? So we have come a long way in the past 10 years and made significant advances… We need to communicate better the capabilities and limitations of bioremediation, and answer several questions... Some questions related to microbial ecology have a direct bearing on the feasibility of natural attenuation, such as: Which attenuation processes are responsible for the loss of contaminants? Why are some processes occurring at some sites but not others? How are different microbes degrading different contaminants? Others are more practical questions related to process engineering, such as… So in the last few minutes I have left, I would like to discuss research philosophies to address these questions.

EPISTEMOLOGY OF BIOREMEDIATION
episteme = knowledge Theory of the method and basis we use to acquire knowledge, including the possibility and opportunity to advance fundamental understanding, sphere of action, and the philosophy of the scientific disciplines that we rely upon. Reductionism: System analysis through separation of its components (eliminates complexity to enhance interpretation). Based on the premise that a system can be known by studying its components, and that an idea can be understood if we understand its concepts separately. Used increasingly in bioremediation research to investigate mechanisms. Holism: The totality of a system is greater than the sum of its parts (synergism & antagonism) Specifically, I would like to finish with some thoughts on epistemology of bioremediation. For those of you not familiar with this term, let me give you some definitions. There are two extreme research styles or philosophies ..synergistic and antagonistic combinations of system components.

Epistemology’s Uncertainty Principle
Reductionism simplifies the system, enhances hypothesis testing, and interpretation It also augments lab artifacts and hinders the relevance of the information we obtain High Low High Low Expt. control, Lab artifacts Complexity, Relevance Both approaches have their advantages and disadvantages. As we unfold each layer of reductionism (L to R), we simplify the the system, enhance experimental control, hypothesis testing, and interpretation of the results. Unfortunately, reductionism augments lab artifacts and hinders the relevance of the information that we obtain with regards to site cleanup These contrasting philosophies are, in a way, SCALE DEPENDENT (RIGHT) Most microbiologists work at the in vitro and in vivo scale, and rely on reductionist disciplines like genetics and physiology. They are great at taking apart the components of a cell and learning how they work. (LEFT) In contrast, geologists traditionally work in situ, and rely more on field-scale observation than in experimentation, because they cannot reproduce in the lab the spatial and time scales associated with geologic phenomena such as continental drift. The enormous temporal and spatial scale of earth’s phenomena cannot be reproduced in the lab, so Geology has traditionally been an observational science. For example, the most important unifying concept in geology is perhaps the plate tectonics model. This model, which was proposed by Wegener and Holmes, explains continental drift, oceanic trenches, geosynclines, and other phenomena, was based entirely on observational evidence. Experimental evidence, indicating that rocks were insufficiently deformable for continents to move at all, largely muddied the issues involved. Microbiology, on the other hand, is an experimental science. It used to be an observational hobby until Pasteur and Koch introduced rigorous experimental procedure that transformed it into a highly prolific science. Since microbes are difficult to observe in their natural habitats, the notion that advancement of knowledge must rely on hypothesis-driven experimentation is deeply rooted in microbiologists. Holism Reductionism Scale: Field Microcosms Cells Extracts Genes Disciplines: Ecology Biogeochemistry Physiology Biochemistry Genetics Molecular Biology

Implications Quantitative extrapolation from the lab to the field is taboo. (interpolate but do not extrapolate) Rely more on holistic disciplines (e.g., ecology, biogeochemistry) and iterate more between the field and the lab, between basic and applied research. Multidisciplinary Research (interstices) Aurea mediocridad (San Ignacio de Loyola) We should try to get the best of both approaches to advance our knowledge of bioremediation. To do so, we should keep in mind the following axioms 1) Be careful about extrapolating results from reductionist experiments to complex systems. Interpolation is OK, but Extrapolation can get you in trouble. For example, rates measured in the lab are often one order of magnitude slower than in the field, where you have nutrient and mass transfer limitations and lower temperatures. 2) Repeat bullet 3) I think it is important to collaborate in multidisciplinary teams because some of the most intellectually stimulating and technologically productive research lies at the interstices between disciplines. In this regard, it may be a mistake to try to be be all encompassing, like trying to be a microbiologist an a geologist at the same time. Rather, build on strength and think in systems while you act in your disciplines. This is analogous to thinking globally and acting locally. Finally, it is important to balance our scientific drive to understand nature with our engineering mission to solve problems, and to balance breadth with depth. This is why I wrote down this quotation from the founder of the Jesuit order: the gold is in the middle, and the virtue lies is in the equilibrium. (science should be humane in spirit, wise in its uses, and moral in purpose)

Pay attention to detail. You never know who is watching your
work, and where your next promotion or demotion will come from. Dreaming should be a part of your job (Mario Molina) Bioremediation is seldom a straight line to an imagined goal (many branching decision points requiring flexibility and versatility) Remedial technologies are rapidly evolving. Be committed to life-long learning, and be aware that imagination and creativity could more important than knowledge

Conclusions Indigenous microorganisms can often destroy BTEX and other common groundwater contaminants, making bioremediation (often) technically feasible. The pendulum recently swung towards natural attenuation. This can save money but take much longer to achieve cleanup and appear as if officials are walking away from contaminated sites. Early public involvement is critical to minimize such controversy. We should keep in mind that neither bioremediation not natural attenuation is a panacea that is universally applicable, and that some sites may require you to use physo-chemical approaches. Nevertheless, bioremediation is quickly achieving pedagogical maturity, and has earned an important place in the menu of alternatives from which we select solutions to our environmental pollution problems.

Lets Take a Break!

TYPES OF MICROBES USED A. Indigenous Microorganisms
Used in most applications (99%) Pseudomonas have wide catabolic capacity May need to enhance proliferation/enzyme induction B. Acclimated Strains Preselected naturally occurring bacteria Generally not needed for BTEX Often fail to function in situ; common reasons: - Conc. of target compound too low to support growth - Other substances and organisms inhibit growth - Microbe uses other food than target contaminant - Target compound not accessible to microbe C. Genetically Engineered Microbes (GEMs) Could combine desirable traits from different microbes: - Ability to withstand stress & degrade recalcitrant compounds - Not needed for BTEX, many technical & political constraints

Want converging lines of evidence, some of which could be circumstantial such as higher concentration of protozoa (feeding on bacteria) Inverse relation vetween DO and BTEX Higher H2 = more reducing environment, weaker electron accepting conditions

Now that we understand biodegradation better, let us discuss how to exploit it for site remediation

Análisis de varianza de las interacciones BTEXN
Las capacidades de degradación fueron mas amplias cuando los BTEXN fueron alimentados como mezcla que separadamente (particularmente cuando el T estaba presente) Las interacciones negativas (e.g., inhibición competitiva, toxicidad) fueron estadísticamente significativas cuando se alimentó 1 mg/L a cada una. Por estadística de Kappa se encontró una correlación significativa entre las habilidades para degradar T y E, p-X y m-X, y p-X y o-X. La falla de degradar B fue correlacionada con la inhabilidad para degradar o-X. We also conducted Analysis of Variance to identify general trends regarding how the presence or absence of a compound is likely to affect biodegradation capabilities. The interesting findings of a factorial analysis of variance were: (1. Particularly when T was present... possibly because of enhanced induction of catabolic enzymes of relaxed substrate specificity)

Specific degradation rate
Monod’s Equation k Specific degradation rate dC/dt/X dC k C X k = - 2 dt K + C S Let me review some basic principles: Modod’s equation is a hyperbolic function, predicting that rates increase with C until a maximum rate dictated by the microbe’s metabolic capacity is reached. This asymptote is k (max. specific substrate utilization rate) The other hyperbolic coefficient coefficient that defines the rate law is Ks, the half-velocity coefficient. Ks is inversely related to enzyme affinity for the substrate. Monod’s equation also predicts faster rates with higher X Let us focus on the region when the contaminant conc. is relatively low (relative to Ks), as is often the case for B in aquifers. Here, the rate approaches a first-order law. (i.e., rate is proportional to C) KS Contaminant Concentration, C 7

Why First-Order Degradation Rates?
Monod’s Equation, When C << KS dC k X C k X = - - C dt K + C K S S dC = - lC dt This analysis shows that l can be explained in terms of Monod parameters. When C is relatively low, we can ignore C in the denominator and Monod’s equation reduces a linear equation. This theoretical analysis lambda (units of per time) depends on: k (which in turn depends on the type of microbe and e.a.- conditions). In fact, the single most important factor affecting the rate of B degradation is probably O2 availability. Ks (related to enzyme affinity and bioavailability) X (which is not constant, and depends on environmental conditions and aquifer chemistry, including available substrates). The bottom line is that lambda is not a constant, but a coefficient that varies in time and space due to microbial population shifts resulting from changes in aquifer chemistry. l = S K X k (not constant)

Also, Mass Transfer Limitations Are Conducive to First-Order Kinetics (even if C > Ks)
Sometimes, first-order kinetics is also observed even when the contaminant concentration is higher than Ks (say 10 mg/L). This can be explained by considering mass transfer limitations through the boundary layer that separates cells from the bulk liquid. Diffusion through this layer follows first-order kinetics. Thus, ff mass transfer is the rate-limiting step (i.e., slower than metabolism), diffusion rates control the process and contaminant removal rates are proportional to their bulk liquid concentration. (Otherwise, you get hyperbolic kinetics, as predicted by Monod’s equation) The bottom line is that the first-order kinetics assumption can be appropriate for natural attenuation of BTEX

Alta concentración microbiana = Taza más rápida
Simulaciones empleadas: k = 0.28 g-T/g-células/día KS = 8.6 mg-T/L Y = 0.6 g- células/g-T 80 60 107 células/mL TOLUENO (mg/L) 40 102 células/mL Another important factor affecting rates, is the type and concentration of prevailing microbial species. If mass transfer processes are not rate-limiting, the rate is proportional to the microbial concentration. In this example, with denitrifying bacteria, an enriched culture degraded T in 2 weeks, while the culture which with a lower microbial conc. required 20 weeks to do the job. This suggests that the lag period one commonly observes regarding contaminant degradation often reflects the time required for the specific degraders to grow to a critical mass capable of exerting measurable degradation rates. (e.g., 106 cells/g of soil or per ml) The take-home message is that effort to selectively increase the concentration of specific degraders may be well justified. Let me show you an example. 20 30 60 90 120 150 Tiempo (Días)

¿Por qué es tan difícil limpiar acuíferos?
¿Porque es tan dificil y costoso limpiar acuiferos? Generlamente los contaminates no estan a la vista y son dificiles de encontrar. En este ejemplo, los pozos de monitoreo estan muy profundos (izq.) o no penetran lo suficiente (der) para encontrar el centro de la pluma, y solo en 1 de 4 pozos se detecta. Es decir, un pozo es simplemente un hoyo en la tierra que no dice toda la verdad. … y cuando encontramos la contaminacion, es dificil removerla. Les voy a dar un ejemplo. Imaginense que ustedes meten su mano en aceite contaminado y toman un puno. Su reto es limpiarse la mano sin abrir el puno, para reflejar la dificultad de alcanzar los contaminates bajo tierra. Podrian enjuagarse el puno en un balde de agua. Una pequena parte de los contamimantes se saldrian y ensuciarian el agua, pero la mayor parte quedaria en el puno. Esto es analogo a “bombear y tratar” el agua contaminada en la superficie, pues es muy dificil extraer contaminantes hidrofobicos que se adhieren a la tierra y contaminan el agua continuamente. Por eso bombear y tratar raramente funciona. Podrian anadirle detergentes al balde, lo que seria analogo a lavar con tensoactivos para remover los contaminates hidrofobicos, pero esto no es 100% efectivo. Podrian tambier usar una secadora de pelo para volatizar algunos contaminantes, lo cual seria analogo a la extraccion con vacio, pero esto tambien dejaria mucha contaminacion residual. Finalmente, podrian excavar e incinerar la tierra contaminanda, pero seria como cortarse la mano por su alto costo. La biorremediacion ofrece una oportunidad de remover la contaminacion al degradarla donde esta, sin tener que abrir la mano. Detectar la contaminación en aguas subterráneas es como buscar una aguja en un pajar. Los puertos de muestreo pueden ser demasiado profundos, no muy profundos o en un lugar equivocado.

Pedro J.J. Alvarez, Ph.D., P.E., DEE

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