Estimation of hydrocarbon biodegradation rates in gasoline-contaminated sediment from measured respiration rates

Journal of Contaminant Hydrology 41 Ž2000. 175–192 www.elsevier.comrlocaterjconhyd

Estimation of hydrocarbon biodegradation rates in gasoline-contaminated sediment from measured respiration rates Ronald J. Baker ) , Arthur L. Baehr 1, Matthew A. Lahvis

2

U.S. Geological SurÕey, 810 Bear TaÕern Road, West Trenton, NJ 08628, USA Received 21 October 1997; received in revised form 15 June 1999; accepted 15 June 1999

Abstract An open microcosm method for quantifying microbial respiration and estimating biodegradation rates of hydrocarbons in gasoline-contaminated sediment samples has been developed and validated. Stainless-steel bioreactors are filled with soil or sediment samples, and the vapor-phase composition Žconcentrations of oxygen ŽO 2 ., nitrogen ŽN2 ., carbon dioxide ŽCO 2 ., and selected hydrocarbons. is monitored over time. Replacement gas is added as the vapor sample is taken, and selection of the replacement gas composition facilitates real-time decision-making regarding environmental conditions within the bioreactor. This capability allows for maintenance of field conditions over time, which is not possible in closed microcosms. Reaction rates of CO 2 and O 2 are calculated from the vapor-phase composition time series. Rates of hydrocarbon biodegradation are either measured directly from the hydrocarbon mass balance, or estimated from CO 2 and O 2 reaction rates and assumed reaction stoichiometries. Open microcosm experiments using sediments spiked with toluene and p-xylene were conducted to validate the stoichiometric assumptions. Respiration rates calculated from O 2 consumption and from CO 2 production provide estimates of toluene and p-xylene degradation rates within about "50% of measured values when complete mineralization stoichiometry is assumed. Measured values ranged from 851.1 to 965.1 g my3 yeary1 for toluene, and 407.2–942.3 g my3 yeary1 for p-xylene. Contaminated sediment samples from a gasoline-spill site were used in a second set of microcosm experiments. Here, reaction rates of O 2 and CO 2 were measured and used to estimate hydrocarbon respiration rates. Total hydrocarbon reaction rates ranged from 49.0 g my3 yeary1 in uncontaminated Žbackground. to 1040.4 g my3 yeary1 for highly contaminated sediment, based on CO 2 production data. These

)

Corresponding author. Tel.: q1-609-771-3900r3923; fax: q1-609-771-3915; E-mail: [email protected] Tel.: q1-609-771-3900; E-mail: [email protected]. 2 Tel.: q1-609-771-3900; E-mail: [email protected]. 1

0169-7722r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 7 7 2 2 Ž 9 9 . 0 0 0 6 3 - 7

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rate estimates were similar to those obtained independently from in situ CO 2 vertical gradient and flux determinations at the field site. In these experiments, aerobic conditions were maintained in the microcosms by using air as the replacement gas, thus preserving the ambient aerobic environment of the subsurface near the capillary zone. This would not be possible with closed microcosms. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Biodegradation; Hydrocarbon; Microcosm; Respiration; Porous media

1. Introduction Contamination of ground water by petroleum products is widely recognized as a serious water quality problem. There are an estimated 100,000 to 400,000 leaking gasoline storage tanks in the United States ŽAtlas and Cerniglia, 1995., as well as frequent spills from leaking pipelines and transportation accidents involving containers of gasoline and other petroleum products. Many gasoline hydrocarbons introduced to the subsurface are biodegradable in a wide variety of environments ŽAtlas, 1984; Chapelle, 1993.. Cleanup options that rely on natural biodegradation to contain or limit the spread of contaminants can be broadly categorized as Ž1. passive bioremediation requiring monitoring but no action, and Ž2. engineered solutions requiring physical andror chemical alteration of the site to enhance or stimulate the natural biodegradation process. Regardless of the bioremediation approach used, biodegradation rate information is essential for preliminary site assessment, progress-monitoring, and decision-making during remediation. The objectives of this paper are to describe a laboratory method for biodegradation rate determination, and to present experimental results that demonstrate the applicability of the method to measuring degradation rates in hydrocarbon-contaminated sediments. Several methods of measuring biodegradation rates have been described in recent literature, and most involve monitoring the depletion of the compoundŽs. of interest or change in a surrogate parameter, such as oxygen ŽO 2 ., carbon dioxide ŽCO 2 . or cellular metabolite concentrations. These can be categorized as closed microcosm, open microcosm, and column methods. Some reported methods are briefly described here to provide a context for presenting the attributes, applications and limitations of our method relative to other options. A closed microcosm can be described as a container filled with some combination of solid, liquid and vapor-phase ingredients and may be augmented with substrate, nutrients, microbial cultures or other amendments to achieve a desired initial condition. Typically, a large number of microcosms are prepared and subsets are periodically ‘‘sacrificed’’ for analysis, yielding a time-dependent record of microbial activity which can be used to calculate reaction rates or kinetic rate constants. Recent examples of closed-microcosm studies include Bregnard et al. Ž1997. who demonstrated the degradation of pristane, formerly thought to be recalcitrant, under nitrate-reducing conditions; Kao and Borden Ž1997. who investigated the site-specificity of benzene, toluene, ethylbenzene and xylene ŽBTEX. degradation in microcosms under denitrifying and aerobic conditions; and Bradley and Chapelle Ž1995. who monitored the mineralization

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of radio-labeled BTEX in soil at low temperatures in sealed 30-ml serum vials. Little or no maintenance is required between sacrifice events in closed microcosms. The principle shortcoming is that environmental conditions change during incubation, and adjusting the contents to maintain desired conditions is not possible. Open microcosms allow for continuous or intermittent addition or removal of substrates, nutrients, electron acceptors or other constituents, and sampling of one or more phases. Recent examples of open microcosms include Fish and Principe Ž1994. who quantified the reaction rate of a polychlorinated biphenyl ŽPCB. cogener in Hudson River sediment. 120-ml test tubes were filled with sediment, left open, and submerged in a container of ultra-pure water which was constantly oxygenated by recirculation. This design created an upper aerobic and a lower anaerobic zone. Godsy et al. Ž1992. studied the anaerobic degradation of creosote in 4-l microcosms that were open in that vapor and liquid samples were obtainable. Periodic quantitation of CO 2 , methane, organic acids and microbial substrates showed that methanogenic fermentation is a significant biodegradation pathway for selected organic contaminants at this site. The open microcosms of Alvey and Crowley Ž1995. for measuring atrazine biodegradation rates in soil had air constantly flowing through the headspace of 175-ml jars containing 100 g of soil, and exiting through a NaOH trap to capture CO 2 . 14 C-atrazine was used, and the fraction mineralized was quantified by scintillation counting of NaOH taken from the traps. Nielsen et al. Ž1996. buried ‘‘in situ microcosms’’, stainless-steel cylinders driven into the ground to enclose a volume of undisturbed aquifer material. Phenolic compounds were pumped into the enclosed porous media and sampled periodically to determine rated of aerobic biodegradation. This experimental design is essentially a buried open microcosm. As these examples show, the design and scale of open microcosms vary greatly and depend upon the research objectives. This paper presents an open-microcosm method for determining respiration rates and hydrocarbon biodegradation rates in unsaturated soils or sediments. Gas-tight stainlesssteel bioreactors developed by Nolan et al. Ž1997. are filled with soil or sediment contaminated with hydrocarbons. The bioreactor atmosphere is analyzed periodically to determine concentrations of hydrocarbons, O 2 , N2 , and CO 2 in the vapor phase, and Henry’s Law and carbonate system relations are applied to calculate the pore water composition. The vapor sample withdrawal and gas replacement system is a unique feature of the bioreactor design. Replacement gas composition can be selected and varied to control the environment within the bioreactor, such as aerobic or anaerobic conditions and volatile organic substrate levels. Column studies are usually designed to study concentration gradients or constituent transport under transient or steady-state conditions. The column can be an intact core obtained from the site of interest, or a container packed with solid material taken from the site. Moyer et al. Ž1996. used intact unsaturated soil cores with upflow air to simulate bioventing in soil with and without LNAPL present. Dolan and McCarty Ž1995. developed a small-column method in which ground water samples contaminated with vinyl chloride were passed through test tubes filled with aquifer porous media. Significant biotransformation of vinyl chloride by methanotrophic microbes was demonstrated. The greatest advantages of columns over microcosms are that steady-state conditions can be maintained, and sampling is usually not disruptive to the experimental environ-

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ment. Disadvantages include more complex and expensive hardware and more intensive maintenance requirements. The open microcosm method described in this paper was validated by filling bioreactors with sediments known to contain hydrocarbon-degrading microbes, adding hydrocarbons and oxygen periodically, and monitoring the reaction rates of hydrocarbons, O 2 and CO 2 . This set of experiments tested the hypothesis that hydrocarbon biodegradation rates can be calculated from O 2 and CO 2 reaction rates if it is assumed that complete hydrocarbon mineralization is the predominant biochemical pathway for hydrocarbon utilization. The bioreactors were then used in a second set of experiments to determine CO 2 and O 2 reaction rates in subsurface sediment samples from a gasoline spill site. Hydrocarbon degradation rates were estimated from these fixed-gas reaction rates and compared to rates determined from field measurements of hydrocarbon gradients in the unsaturated zone.

2. Experimental 2.1. Bioreactor design and preparation The reactor design consists of a 3.81-cm inside diameter Ži.d.. = 30.48 cm long Schedule 40 304 stainless-steel pipe, threaded end caps, and two vapor-sampling ports ŽFig. 1.. The sampling ports, made of 18-gauge stainless-steel syringe needles fitted with syringe valves, are threaded into the pipe near each end. Stainless-steel compression fittings Ž1r16 in. Ž0.1587 cm.. are used to attach the sampling port hardware to the reactor. The syringe needles extend through the reactor interior, are crimped at the point, and have several penetrating grooves along their length for obtaining a composite vapor sample from a cross-section of the reactor. To initiate a bioreactor experiment, one end-cap is removed, the reactor is partially or completely filled with the soil or sediment of interest, and the end-cap is replaced. Any amount of sample can be used, as long as the total reactor volume and characteristics of the sample Žmass, bulk density, porosity and moisture content. are known. The reactor is

Fig. 1. Bioreactor design.

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kept submerged in a water bath maintained at a desired experimental temperature Žusually that of the subsurface from which the sample was collected.. 2.2. Bioreactor Õapor sampling and analysis Reactors are removed from the water bath for sampling. Vapor sampling requires two gas-tight syringes. A vapor sample is withdrawn from the reactor into the empty syringe while an equal volume of replacement gas is injected into the port at the opposite end of the reactor. Rapid attainment of complete mixing of the vapor phase was confirmed by injecting toluene-saturated air into one port and sampling from the other port of a bioreactor filled with sterile sediment. Complete mixing occurred within 4 h. Hydrocarbons and fixed gases are quantified separately by gas chromatography ŽVarian 3600.. 3 Vapor-sample injection ports are equipped with Valco valves ŽModel A-60, Valco Instruments. and 0.5-ml sample loops. Hydrocarbons are separated in a 0.75-mm i.d.= 60-m long Vocol column ŽSupelco. and detected and quantified with a flame-ionization detector ŽFID.. A 0.32-cm = 5.5-m long stainless-steel Carbopack column ŽAlltech Associates. and a thermal-conductivity detector ŽTCD. are used to separate and detect the fixed gases ŽO 2 , N2 , and CO 2 .. A PC-based Turbochrom software system ŽPE-Nelson. is used to identify and quantify hydrocarbons and fixed gases. Linear standard curves with a minimum of three external standards are used for both methods. Relative standard deviations are less than "6% for all analyses. Additional analytical details are given in Baker et al. Ž1991.. 2.3. Mass-balance and cumulatiÕe reaction calculations Time-series gas composition data and a mathematical model are used to quantify reaction rates in the bioreactors. The conservation-of-mass principle applied to the reactor is expressed as follows: dG k ak s Vpm R k q d Ž t y t i . Ž A k Ž t i . y Bk Ž t i . Ž 1. dt where G k is the gaseous-phase concentration of kth constituent Žg cmy3 ., t is time Žs., Vpm is the volume occupied by porous media Žcm3 ., R k is the reaction rate of k th constituent per unit volume of porous media Žg cmy3 sy1 ., d Ž t y t i . is the Kroneker delta Žsy1 ., A k Ž t i . is the mass of k th constituent put into the reactor at sampling time t i Žg., Bk Ž t i . is the mass of k th constituent removed from the reactor at sampling time t i Žg., and a k is the storage coefficient for the k th constituent Žcm3 . and is defined as follows:

a k s Vw rHk q Va

Ž 2. 3.

where Vw is the reactor volume occupied by the aqueous phase Žcm , Va is the reactor volume occupied by the gaseous phase Žcm3 ., and Hk is Henry’s law coefficient for the y1 . . Ž k th constituent, dimensionless Žmg ly1 air r mg l water . 3

Use of brand, firm, or trade names in this paper is for identification purposes only and does not constitute endorsement by the US Geological Survey.

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This application does not consider solid-phase partitioning, however, Eq. Ž2. could be modified if significant sorption is expected, e.g., for a sediment with high organic content. Partitioning of O 2 heavily favors the vapor phase; therefore, aqueous O 2 was omitted from the mass-balance calculations. Vapor- and aqueous-phase CO 2 and all three dissolved carbonate species must be considered in CO 2 mass-balance calculations. Here and in Eq. Ž2., we define Hk for CO 2 to be the concentration of CO 2 in the air phase divided by C T , where C T is the sum of the concentrations of all dissolved carbonate x w 2y x.. species Ž C T s wCO 2 x q wH 2 CO 3 x q wHCOy 3 q CO 3 All of the aqueous-phase volume in the reactor is associated with the porous media; therefore, Vw s uw Vpm

Ž 3.

where uw is the volumetric content of the aqueous phase of the porous media Ždimensionless.. Va consists of the vapor-phase volume of the porous media plus any unoccupied volume in the reactor, and can be expressed as follows: Va s uaVpm q Vh

Ž 4.

where ua is the volumetric content of the gaseous phase of the porous media and Vh is the volume of the reactor unoccupied by porous media Žcm3 .. Integrating Eq. Ž1. over time yields the following:

a k G k Ž t n . y G k Ž t 1 . s Vpm

tn

Ht

1

ny1

R k d t q Ý A k Ž t i . y Bk Ž t i .

Ž 5.

is1

where t 1 , t 2 , . . . , t n are the sampling times. Fk Ž t n ., the cumulative reaction of the k th constituent at time t n , is defined as follows: Fk Ž t n . s

tn

Ht

Rkdt

Ž 6.

1

Fk Ž t n . is calculated from Eq. Ž5., and the average reaction rates of O 2 , CO 2 , CH 4 , and hydrocarbons can then be calculated from changes in vapor composition over time as Fk Ž t n .rt n . 2.4. Determination of hydrocarbon biodegradation rates from O2 and CO2 reaction rates (respirometric method) Total aerobic hydrocarbon biodegradation rates can be estimated from rates of CO 2 production or O 2 utilization if certain stoichiometry assumptions are made. Aerobic microbial utilization of hydrocarbon can be expressed as the consumption of hydrocarbon, O 2 and a nitrogen source to yield biomass ŽC 18 H 20 N1O5 . or CO 2 and H 2 O: Biomass production: C x H y q Ž yr4 y 5 xr24 . O 2 Ž xr18 . Hqq Ž xr18 . NOy 3 ™ Ž xr18 . C 18 H 20 N1O5 q Ž yr2 y 19 xr36 . H 2 O CO 2 production: C x H y q Ž x q yr4 . O 2 ™ xCO 2 q Ž yr2 . H 2 O

Ž 7. Ž 8.

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181

Biomass can be oxidized to CO 2 , H 2 O and NOy 3 through aerobic endogenous respiration: q 4C 18 H 20 N1O5 q 87O 2 ™ 72CO 2 q 4NOy 3 q 38H 2 O q 4H

Ž 9.

Therefore, biomass can be considered an intermediate in the overall aerobic mineralization of hydrocarbons to CO 2 and H 2 O, and the sum of Eqs. Ž7. and Ž9., normalized to xr18 mol of biomass, produces Eq. Ž8.. Mineralization stoichiometry is a simplified summation of microbial biochemical reactions and does not consider metabolic intermediates, refractory organic by-products, microbial yield ŽEq. Ž7.., or endogenous microbial respiration ŽEq. Ž9... Eq. Ž8. can be used to calculate hydrocarbon mineralization rates from either O 2 consumption or CO 2 production. Note that O 2 and CO 2 concentrations are both measured; therefore, two independent estimates of hydrocarbon mineralization are possible. Complete mineralization stoichiometry is shown in Table 1 for several hydrocarbons. If the hydrocarbon composition at a contaminated site is complex or unknown, an estimated or averaged hydrocarbon formula may be used. For example, a weathered gasoline described by Johnson et al. Ž1990. has an averaged composition of C 8.30 H 14.43 yielding a molar HC:O 2 :CO 2 reaction ratio of 1:8.30:7.21. Little variation in this ratio is present among the entire spectrum of gasoline hydrocarbons. 2.5. Method Õalidation using hydrocarbon-spiked sediment The purpose of these experiments was to validate the method under controlled conditions, where mass balances of hydrocarbons, O 2 and CO 2 could be precisely maintained. The complete mineralization assumption could then be evaluated as a source of error when hydrocarbon reaction rates are calculated from O 2 or CO 2 reaction rates. Pertinent physical and chemical properties of fixed gases and selected hydrocarbons are listed in Table 2. 2.5.1. Source of the sediment used Unsaturated-zone sediment samples from a gasoline spill site were used in column experiments in which a method of quantifying hydrocarbon biodegradation rates based on reactive transport was developed ŽBaehr and Baker, 1995; Baker and Baehr, 1997.. The sediments were exposed to toluene or p-xylene for periods exceeding 1 year, and a microbial population was acclimated to utilizing the hydrocarbon as its sole carbon and energy source. Sediment samples from two completed column experiments were used in

Table 1 Aerobic mineralization stoichiometry of hydrocarbons Generic hydrocarbon: Benzene: Toluene: Ethylbenzene, xylenes: Weathered gasoline:

1C x H y qŽ x q yr4.O 2 ™ xCO 2 qŽ yr2.H 2 O 1C 6 H 6 q7.5O 2 ™6CO 2 q3H 2 O 1C 7 H 8 q9O 2 ™ 7CO 2 q4H 2 O 1C 8 H 10 q10.5O 2 ™8CO 2 q5H 2 O 1C 8.30 H 14.43 q11.91O 2 ™8.30CO 2 q7.21H 2 O

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Table 2 Properties of atmospheric fixed gases and selected gasoline constituents Species

Molecular weight

Density

Boiling point Ž8C.

k Ž Hwa 208C. b

Benzene Toluene Ethylbenzene m-Xylene p-Xylene o-Xylene N2 O2 CH 4 CO CO 2 pH4 a CO 2 pH5 CO 2 pH6 CO 2 pH7 CO 2 pH8

78.11 128.26 106.20 106.20 106.20 106.20 28.01 32.00 16.04 28.01 44.01 44.01 44.01 44.01 44.01

0.879 0.867 0.867 0.864 0.861 0.880 – – – – – – – – –

80.1 110.6 136.2 139.1 138.3 144.4 y195.8 y183.0 y164 y191.5 y78.5 y78.5 y78.5 y78.5 y78.5

0.227 0.270 0.323 0.283 0.287 0.202 58.82 30.30 27.78 41.67 1.06 1.02 0.750 0.205 0.0248

a b

Henry’s law constant, dimensionless Žmg ly1 .air rŽmg ly1 . water . k x w 2y x. Hwa s wCO 2 xair r C T , where C T saqueous wCO 2 xqwH 2 CO 3 xqwHCOy 3 q CO 2

the open microcosm experiments presented here. These sediments are coastal plain sands with less than 1% organic content. No loss to adsorption was observed when 30 g of sediment was mixed in 1 l of D.I. water containing 4.3 mgrl of toluene and p-xylene for 20 h. Therefore, adsorption and desorption are not expected to be significant sinks or sources for hydrocarbons. 2.5.2. Preparation of open microcosms Bioreactors were filled with sediment from the completed column experiments and toluene or p-xylene was provided as a component of the replacement gas Žair saturated with hydrocarbon vapor.. One bioreactor was sterile Žautoclaved. and another Žcontrol. received no hydrocarbon in the replacement gas Ž100% dry air.. Five bioreactors were filled with sediment taken from different elevations in the column to represent various moisture conditions Žunsaturated, water-table, or saturated zone of the column experiment.. Mass balances of O 2 , CO 2 and hydrocarbon were maintained for at least 28 days. Eqs. Ž5. and Ž6. were applied to the vapor-composition data to determine total amounts reacted of O 2 , CO 2 and hydrocarbon as a function of time during the experiments Ž Fk Ž t n ... 2.6. Preparation of bioreactors using sediments from a gasoline-contaminated site Sediment samples were obtained from a gasoline-spill research site in Beaufort, SC. The site is characterized by highly permeable sandy Coastal Plain sediment with very low background organic content and low hydrocarbon adsorption coefficient Ž K d . as determined by adsorption isotherm partitioning experiments ŽLandmeyer et al., 1996.. These sediments contained complex mixtures of hydrocarbons, and small amounts of weathered gasoline product in some cases. Therefore, maintaining hydrocarbon mass

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183

balances was not possible, and total hydrocarbon biodegradation rates were calculated from CO 2 production and O 2 utilization as described in Section 2.4. Samples were collected from uncontaminated Žcontrol. and hydrocarbon-contaminated locations. For each sampling location, three reactors were filled, one with unsaturated-zone sediment, one with capillary-zone sediment and one with saturated-zone sediment. Initial experimental conditions are shown in Table 3. Gas samples were

Table 3 Initial experimental conditions for bioreactor experiments, Beaufort, SC site Ž –, not applicable. Site and sample date:

Beaufort site Ž2r09r95.

Beaufort background Ž2r10r95.

Sampling location: Depth below land surface: Moisture condition Žzone.:

NJVW4 NJVW4 NJVW4 6–8 ft 8–10 ft 10–12 ft Unsaturated Capillary Saturated

NJVWB NJVWB NJVWB 3–5 ft 5–7 ft 7–9 ft Unsaturated Capillary Saturated

Sample properties (model Õariable) Temperature, 8C ŽT. 15.9 Contamination level moderate Oxygen condition aerobic

16.7 high aerobic

18.5 13.0 saturated None anaerobic aerobic

15.0 low aerobic

16.5 low aerobic

Volume (ml) Sediment volume Ž Vpm . Unoccupied volume Ž V h . Water volume Ž uw Vpm . Gas-filled volume Ž Va . Total reactor volume Ž Vpm qV h .

245.79 99.71 20.62 158.21 345.5

335.36 3.34 102.33 67.70 338.7

350.20 0 120.21 57.58 350.2

282.24 78.06 30.75 166.4 360.3

293.57 63.83 102.10 117.26 357.4

356.1 0 136.17 55.96 356.1

Sediment properties Mass Ž Mpm . Bulk density Ž r b . Air-filled porosity Ž ua . Water-filled porosity Ž uw . Total viable microbes Total petroleum H.C., mg gy1

462.3 1.77 0.238 0.142 2.2=10 5 0.27

549.3 1.88 0.192 0.188 1.7=10 4 11.22

577.1 1.78 0.175 0.205 6.4=10 2 55.76

463.1 1.65 0.313 0.067 1.7=10 5 0.00

467.9 1.82 0.182 0.198 7.9=10 5 0.00

570.7 1.56 0.181 0.199 7.9=10 3 0.00

Sample properties (model Õariable) temperature, shallow ground water properties pH – – 5.9 – Alkalinity, mg ly1 as CaCO 3 – – 21.0 –

– –

5.1 2.5

Initial major fixed gas concentrations (%Õr Õ) N2 Ž G N2 . 82.77 O 2 Ž GO2 . 20.09 CO 2 Ž GCO2 . 0.23

82.79 15.07 2.23

Initial Õapor-phase hydrocarbon concentration ( m g l y 1) Total hydrocarbons ŽG HC . 603 22 363 BTEX Ž G BT EX . 320 3787 Benzene Ž G ben . 227 6738 Toluene Ž Gtol . 198 1210 Ethylbenzene Ž Getb . 12 511 m, p-Xylene Ž Gm, p . 101 1654 o-Xylene Ž Goy x . 0 188

75.50 16.82 1.95

83.70 20.22 0.31

82.61 20.46 0.46

85.95 17.82 0.72

99 751 15 671 0 9666 622 1991 113

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

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Fig. 2. Cumulative reaction of toluene calculated from toluene, O 2 and CO 2 data, in bioreactors filled with toluene-acclimated sediment.

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185

collected from the microcosms at t s 0, 0.5, 2, 4, and 6 days, then at less frequent intervals. The microbial origin of the CO 2 produced was verified experimentally by autoclaving a reactor filled with hydrocarbon-contaminated sediment at 1208C. and monitoring the vapor phase for changes in hydrocarbon, O 2 and CO 2 concentrations. 3. Results and discussion 3.1. Open microcosm experiments using spiked sediments Time series of the bioreactor experiments which received toluene and p-xylene in the replacement gas are shown if Figs. 2 and 3, respectively. Units of g, my3 and daysy1 Žor yearsy1 . are used for a more intuitive presentation of the data, especially when discussing site-wide amounts and rates of hydrocarbon biodegradation. Cumulative hydrocarbon utilization Ž FHC Ž t n .. is determined three ways: from direct monitoring of

Fig. 3. Cumulative reaction of p-xylene calculated from p-xylene, O 2 and CO 2 data, in bioreactors filled with p-xylene-acclimated sediment.

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the hydrocarbon mass balance, and by calculating hydrocarbon utilization from O 2 consumption data or CO 2 production data and assuming complete mineralization stoichiometry ŽEq. Ž8... A close match of the three lines would support the use of CO 2 and O 2 data to estimate hydrocarbon utilization. These data ŽTable 4. show an error of about "50% in estimating hydrocarbon consumption from O 2 and CO 2 data. This error is a measure of the inherent limitations in applying the mineralization assumption in this method, and several measurement and reaction characteristics contribute to this error. These include accuracy in sampling and analysis of the vapor phase, pH of the pore water Žwhich may change over time and effects the C T quantitation., net production or endogenous degradation of biomass, and microbial production of refractory organic compounds. These sources of error are commonly present in respiration-based studies. Figs. 2 and 3 show that both O 2 and CO 2 reaction rates and the stoichiometric hypothesis over-predicted hydrocarbon degradation rates of toluene and p-xylene in sediment from unsaturated zone. The reverse was true for water-table and saturated-zone sediments. Microbiological data could explain some of this error in four of the five experiments. Total viable microbes increased by about three orders of magnitude in the water-table and saturated-zone sediments during the bioreactor experiments. The anticipated net increase in biomass necessitates a greater consumption of substrate Žtoluene or p-xylene. than would be evidenced by CO 2 or O 2 reaction rates. This is consistent with the data, as shown in Fig. 2ŽB,C.Fig. 3ŽB., where hydrocarbon is utilized at a higher rate than would be predicted by respiration rates. There was no change in total viable microbial count in the bioreactor filled with unsaturated-zone sediment with p-xylene as the substrate, and both O 2 and CO 2 data closely predicted hydrocarbon utilization ŽFig. 3ŽA... The high respiration rate relative to toluene utilization in unsaturated-zone sediment ŽFig. 2ŽA.. is not consistent with microbial data, where total viable count remained unchanged during the experiment. There was apparently a microbial substrate source, such as nonviable biomass, that contributed to the measured respiration rates in this experiment. If significant biochemical or abiotic CO 2 production or O 2 consumption unrelated to hydrocarbon degradation are present, the use of O 2 and CO 2 data may not be valid for estimating hydrocarbon biodegradation rates. This suggest a lower hydrocarbon Table 4 Mean reaction rates of constituents in bioreactors, spiked experiments Experiment

Unsaturated zone, toluene Water table, toluene Saturated zone, toluene Unsaturated zone, p-xylene Water table, p-xylene

Measured reaction rates Žg my3 yeary1 .

Calculated hydrocarbon reaction rates Žg my3 yeary1 .

O2

CO 2

Hydrocarbon

From O 2

% error

From CO 2

% error

4488.0

5079.6

965.9

1445.1

q49

1521.2

q57

1472.6 1531.1

1784.3 2202.2

851.5 868.3

471.2 491.3

y45 y44

531.74 656.3

y37 y25

3471.2

4436.6

942.3

952.41

q1

885.1

y6

9607.2

8214.2

407.2

263.5

y35

216.4

y47

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reaction-rate measurement threshold for respiration-based experiments. For this reason, control experiments using uncontaminated sediment samples are an essential component of applying this method. Respiration observed in control experiments, attributable to

Fig. 4. Cumulative reactions of O 2 in bioreactors filled with gasoline-contaminated sediment from the Beaufort, SC gasoline spill site.

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biodegradation of background organic substances, can be subtracted from the total respiration observed in contaminated samples, yielding a respiration value for biodegradation of the contaminants of interest. This background subtraction should be used whenever reaction rates of respiration gases ŽO 2 or CO 2 . are used to estimate reaction rates of contaminants, and is demonstrated with the next data set. All experiments conducted to date have used highly permeable sandy sediments, and rapid mixing of the vapor phase was observed. Mixing may be problematic if silty or clayey sediments are used, and confirming rapid mixing is always advisable with this method. 3.2. Open microcosm experiments sediments from a gasoline-spill site in Beaufort, SC Cumulative reactions of O 2 and CO 2 , Fk Ž t n ., are shown in Fig. 4 for sediment taken from uncontaminated Žcontrol. and contaminated areas of the Beaufort, SC gasoline-spill site. Fk Ž t n .rt n for the time increment of t s 0–15 days was used to quantify average O 2 and CO 2 reaction rates ŽTable 5.. CO 2 and O 2 reaction rates are significantly above background in the saturated zone and capillary zone at the site and slightly above background in the unsaturated zone. Direct mass-balance calculations for hydrocarbons cannot be accomplished with the model because separate-phase product is present. Therefore, hydrocarbon reaction rates are estimated from O 2 and CO 2 reaction rates, assuming complete mineralization. No CO 2 production or O 2 utilization were observed in sterilized Žautoclaved. experiments over a 2-week period, but Fig. 4 reveals that CO 2 production and O 2 consumption were significant in the control experiments. This is attributed to microbial respiration unrelated to biodegradation of hydrocarbons from the gasoline spill. These respiration rates define the lower limit of sensitivity for this open microcosm method. Hydrocarbon: O 2 : CO 2 mass ratios of 1: 3.41: 3.27, reflecting mineralization stoichiometry of weathered gasoline ŽJohnson et al., 1990. were used to convert O 2 and CO 2 reaction rates to hydrocarbon biodegradation rates. Hydrocarbon reaction rates calculated from control experiment CO 2 and O 2 data have no physical meaning, as they reflect respiration of background organic matter and are not related to the gasoline spill;

Table 5 Mean reaction rates of constituents in bioreactors, Beaufort, SC gasoline-spill site sediment Sample location

Reaction rate Žg my3 yeary1 . O2

Control (no hydrocarbons) Unsaturated NJVW-B 3–5 ft Capillary zone NJVW-B 5–7 ft Saturated NJVW-B 7–9 ft

283.7 341.8 128.3

HeaÕily contaminated (near source) Unsaturated NJVW-4 6–8 ft Capillary zone NJVW-4 8–10 ft Saturated NJVW-4 10–12 ft

752.7 1539.6 no data

CO 2 252.6 413.8 156.5

468.4 3329.3 1568.6

HC Žfrom O 2 . 84.9 102.2 38.4

225.4 460.9 no data

HC Žfrom CO 2 . 79.1 129.2 49.0

146.4 1040.4 490.2

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however, the CO 2 produced and O 2 consumed expressed as hydrocarbon degraded indicates a lower threshold of measurable hydrocarbon biodegradation rates. Hydrocarbon degradation rates calculated from O 2 and CO 2 data for the unsaturated zone at NJVW-4 were 225.4 g my3 yeary1 and 146.4 g my3 yeary1 . These values are marginally above background rates Ž84.9 and 79.1 g my3 yeary1 ., and caution should be used in attributing this respiration to hydrocarbon utilization. Hydrocarbon concentrations in this sediment were low compared to those in deeper sediment samples. The capillary zone exhibited the highest calculated hydrocarbon reaction rates of the three NJVW-4 samples Ž460.9 and 1040.4. g my3 yeary1 for O 2 and CO 2 data, respectively.. Ideally, the rates of CO 2 production and O 2 utilization would yield independent but similar estimates of the hydrocarbon reaction rate; however, in the examples given here the CO 2 method yields more consistent reaction rates when different experimental time increments are compared and may be a more reliable estimator of hydrocarbon reaction rates ŽLahvis and Baehr, 1996.. Oxygen data would provide more consistent rate estimates if lower initial O 2 concentrations were used, so that a greater fraction of the total O 2 would be consumed between sampling times. The risk here is that an O 2-limiting environment could be created. The experiment in which water-table sediment from NJVW-4 was used may be an example of this. At about t s 10 days the O 2 concentration was approaching 0%, and CO 2 production rates had decreased ŽFig. 4.. At that time the volume of sample collected and replacement gas Ždry air with no CO 2 . injected was increased from 5 to 11 ml, and O 2 and CO 2 reaction rates increased immediately. An advantage of using rates of O 2 consumption rather than CO 2 production to determine hydrocarbon reaction rates is that the O 2 mass-balance calculations do not significantly depend on aqueous-phase partitioning. Additionally, abiotic CO 2 production from dissolution could be significant in sediment containing carbonate minerals. 3.3. Hydrocarbon mass-flux determinations For site-wide determination of aerobic hydrocarbon biodegradation a three-dimensional perspective is required. Reaction rates are depth-dependent, because hydrocarbon concentrations, O 2 availability, moisture content, microbial populations, and other conditions vary with depth. If multiple bioreactor experiments are conducted for sediments from various depths at a given location, then integration of depth-dependent reaction rates gives a total reaction rate per unit area, or CO 2 flux Žg my2 yeary1 .. If vertical reaction rate profiles are determined at several locations at the site, a three-dimensional view of reaction rate distribution can be constructed. Fig. 5 depicts the CO 2 reaction rates as a function of depth at NJVW-4. The CO 2 production attributable to degradation of background organic material at each depth interval can be subtracted from the total to determine the CO 2 production from hydrocarbon biodegradation. The depth-integrated CO 2 production rate at NJVW-4 is 5166 g my2 yeary1 , or 4866 g my2 yeary1 above background. On the basis of mineralization stoichiometry, after subtracting background CO 2 production, these values imply hydrocarbon degradation rates at NJVW-4 of 1522 g my2 yeary1 .

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Fig. 5. CO 2 production from biodegradation of background organic compounds and hydrocarbons at location NJVW-4, Beaufort, SC, site.

CO 2 reaction rates determined from the bioreactor experiments for the Beaufort gasoline-spill site were compared to CO 2 reaction rates obtained by using a method based on analyzing in situ gas concentration profiles to calculate the flux of CO 2 in the unsaturated zone. This method and its application are discussed by Lahvis and Baehr Ž1996.. Baehr and Baker Ž1995. applied the method with minor modifications to analyze the results of laboratory column experiments. At NJVW-4, the CO 2 mass-flux estimates are 1.64Ey8 g cmy2 sy1 Ž5166 g my2 yeary1 . and 1.76Ey8 g cmy2 sy1 Ž5554 g my2 yeary1 . for bioreactor and field calibration methods, respectively. The agreement in reaction rates verifies the ability of the open microcosm method to accurately measure CO 2 reaction rates. On the basis of this comparison, the bioreactor method can provide meaningful aerobic respiration rate information, and this can be used to estimate in situ hydrocarbon biodegradation rates.

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3.4. Application to assess passiÕe and engineered remediation Biodegradation rates can be used in conjunction with an understanding of the hydrogeology of a site to assess the effectiveness of passive remediation. The rates can be directly incorporated into contaminant transport models to predict and quantify the contaminant fate and transport. The transport simulations can be used to help decide whether passive remediation is an acceptable plan or whether engineered remediation is required. The passive-remediation option was selected for the Beaufort Site on the basis of closed microcosm studies and solute-transport modeling ŽLandmeyer et al., 1996.. The accuracy of simulated hydrocarbon migration rates is limited by the accuracy of available reaction rate data. The open microcosm method can be used to improve contaminant fate predictions by providing reaction rate data obtained under conditions more closely resembling those in the field. This method with minor modifications can be used to compare treatment alternatives at a contaminated site. Additional hydrocarbons, moisture, andror nutrients can be mixed into the soil before filling the reactor. Hydrocarbon vapors also can be added to the replacement gas to maintain a microbial carbon and energy source. A continuous stream of a gas mixture could be passed through the reactor, effectively simulating purging, vapor extraction, or bioventing, three remediation technologies used to remove hydrocarbons from contaminated sites. Respiration experiments under steady-state conditions would also be possible with a flow-through regimen, which could simulate field conditions more closely than either open or closed microcosms.

4. Summary and conclusions An open microcosm method of measuring respiration and hydrocarbon biodegradation in porous media has been developed and validated. Stainless steel bioreactors were filled with sandy sediments containing microbial populations acclimated to utilizing gasoline hydrocarbons. In the first set of experiments bioreactors were periodically spiked with hydrocarbons to verify stoichiometric hypotheses. A mass-balance model was applied to vapor-phase composition data collected incrementally to determine reaction rates of CO 2 , O 2 and hydrocarbons. Hydrocarbon reaction rates were estimated from O 2 and CO 2 data with an accuracy of about "50% of measured hydrocarbon reaction rates when complete mineralization was assumed. In a second set of experiments, bioreactors were filled with sandy sediment extracted from a gasoline-spill site. Time-series vapor-phase composition data were used to determine reaction rates of CO 2 and O 2 . Hydrocarbon biodegradation rates were calculated from CO 2 and O 2 reaction rates. Respiration rates and estimated hydrocarbon biodegradation rates were compared to those obtained using an in situ method based on measuring the transport of CO 2 in the unsaturated zone. Results of the two methods were similar, indicating that the bioreactor method can be used to produce meaningful and cost-effective estimates of in situ hydrocarbon degradation.

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