Enhanced Anaerobic Benzene Degradationwith the Addition of Sulfate

Jonathan M. Weiner,1 Terry S. Lauck,2 and Derek R. Lovley1*1 Department of Microbiology, University of Massachusetts, Amherst, MA 01003;

2Environmental Group, Conoco, Inc., Commerce City, CO 80022

Corresponding author. (Phone: 413-545-9651; Fax: 413-545-1578; E-mail: dlovley@rnicrobio.umass.edu)

Abstract: Potential mechanisms for stimulating anaerobic benzene degradation in methanogenic sediments froma petroleum-contaminated aquifer were evaluated. In short-term «2 weeks) incubations, addition of sulfateslightly stimulated benzene degradation and caused a small decrease in the ratio of methane to carbon dioxideproduction from benzene. However, in longer-term (>100 days) incubations, sulfate significantly stimulatedbenzene degradation with a complete shift to carbon dioxide as the end product of benzene degradation. Theaddition of Fe(III) and humic substances had short-term and long-term effects that were similar to the effects ofthe sulfate amendments. In studies in which anaerobic groundwater was pumped through columns of aquifersediments, addition of sulfate to the groundwater significantly enhanced the removal of -benzene from thegroundwater. The stoichiometry of sulfate and benzene removal from the groundwater passing through thesediment columns was consistent with benzene oxidation to carbon dioxide with sulfate serving as the primaryelectron acceptor. These results demonstrate for the first time that addition of sulfate may be an effective strategyfor enhancing anaerobic benzene removal in some petroleum-contaminated aquifers.

Keywords: bioremediation, petroleum, groundwater, sulfate reduction, Fe(III) reduction, chelator, humic substances.

Introduction aquatic sediments (Anderson et aI., 1998; Coates et aI.,1996; Kazumi et aI., 1997; Lovley, Coates, et aI.,1996; Lovley et al., 1995). However, in most instancesthe sediments had to be adapted for anaerobic benzenedegradation and/or the availability of electron accep-tors, and/or microbial populations had to be manipu-lated to stimulate anaerobic benzene degradation. Thus,there was little evidence for anaerobic benzene degra-dation in situ.

The flISt reported instance of anaerobic benzenedegradation under in situ conditions in sediments frompetroleum-contaminated aquifers was the finding thatbenzene was degraded in sediments from the Fe(ill)reduction zone of a petroleum-contaminated aquiferlocated in Bemidji, Minnesota (Anderson et aI., 1998).When tracer quantities of [l4C]-benzene were added tosediments incubated under anaerobic conditions, the

It has been widely reported that benzene is resistant tomicrobial degradation under anaerobic conditions inpetroleum-contaminated aquifers (Acton and Barker,1992; Anid et al., 1993; Barbaro et aI., 1992; Barker etal., 1987; Flyvbjerg et al., 1993; Hutchins et al., 1991;Kuhn et al., 1988; Lee et al., 1988; Patterson et aI.,1993; Thierrin et al., 1993). Benzene's recalcitrance inthis situation is of concern because (1) anaerobic con-ditions are common in heavily contaminated aquifers,and (2) benzene is carcinogenic and relatively mobilein groundwater (Anderson and Lovley, 1997; Lovley,

1997).Anaerobic benzene degradation has been observed

under anaerobic conditions in sediments from a fewpetroleum-contaminated aquifers as well as from

159

1058-8337/98/$.50@ 1998 by Battelle Memorial InstituteBioremediation Journal 2(3&4):159-173 (1998)

reduction by serving as an electron shuttle between thesurface of the Fe(lll)-reducing microorganisms and

the insoluble Fe(ill), again eliminating the need for

direct contact between the microorganisms and the

Fe(lll) oxides (Lovley, Coates, et al., 1996; Lovley et

al., 1998).Several studies have suggested that the addition of

nitrate might stimulate anaerobic benzene degradation

in petroleum-contaminated aquifers (Major et al., 1988;

Morgan et al., 1993). However, the vast majority ofstudies have found that nitrate additions do not en-

hance anaerobic benzene degradation (Lovley, 1997).

Sulfate is another potential electron acceptor for anaero-bic benzene degradation (Kazumi et al., 1997; Lovleyet al., 1995; Phelps et al., 1996; Weiner and Lovley,

1998a), but additions of sulfate have not been shown

previously to stimulate anaerobic benzene degradationin petroleum-contaminated aquifer sediments.

Benzene degradation coupled to sulfate reduction

has been stimulated in anaerobic aquifer sediments,

but only after inoculating the sediments with benzene-

degrading, sulfate-reducing microorganisms obtainedfrom aquatic sediments (Weiner and Lovley, 1998a).

The microorganisms were added as part of a mixed

microbial consortium, and the organisms directly re-

sponsible for benzene degradation were not identified.However, it was demonstrated that the benzene-de-

grading microorganisms in the inoculum were capable

of directly oxidizing benzene to carbon dioxide and

reducing sulfate without the production of commonlyconsidered extracellular intermediates.

The purpose of reviewing these studies was todetermine potential strategies for stimulating anaero-bic benzene degradation in the methanogenic, petro-

leum-contaminated aquifer at Ponca City. The results

indicate that several strategies resulted in stimulationof anaerobic benzene degradation and demonstrate forthe first time that, in some instances, anaerobic ben-

zene degradation may be accelerated merely by the

addition of sulfate.

[14C]-benzene was oxidized to 14CO2 without a lag,

suggesting that the microorganisms were adapted forbenzene degradation in situ. The capacity for benzene

degradation was associated with increased numbers of

microorganisms in the genera Geobacter (Anderson et

al., 1998), which are known to oxidize organic com-

pounds to carbon dioxide with the reduction of Fe(Ill)

(Lovley et al., 1989; Lovley and Lonergan, 1990).

Anaerobic benzene degradation also was observedunder methanogenic conditions in sediments from a

petroleum-contaminated aquifer in Ponca City, Okla-homa (Weiner and Lovley, 1998b). Benzene was con-

verted to methane and carbon dioxide with a stoichi-

ometry that was consistent with methanogenesis servingas the predominant terminal electron-accepting pro-

cess (TEAP). Benzene metabolism proceeded through

the extracellular intermediates phenol, propionate, and

acetate, suggesting that a cooperative consortium of

microorganisms was responsible for benzene mineral-ization.

Although there was evidence for anaerobic ben-zene degradation in the Ponca City aquifer, high con-

centrations of benzene were still found in the ground-

water. Therefore, a mechanism for stimulating benzene

degradation was desired. One strategy for stimulating

benzene degradation in such instances is to add oxy-

gen to the system. However, this approach can be

technically difficult and expensive, and it often is not

effective in heavily contaminated aquifers because of

the large chemical oxygen demand and plugging prob-

lems (Lee et aI., 1988; Major et al., 1988; Morgan and

Watkinson, 1992; Thomas and Ward, 1989). In such

instances it may be preferable to stimulate anaerobicbenzene degradation.

Previous studies have indicated that anaerobicbenzene degradation might be stimulated in aquifer

sediments by making alternative electron acceptorsavailable for microbial reduction. For example, it was

demonstrated in aquifers containing significant amounts

of Fe(lll) that anaerobic benzene degradation can be

stimulated with the addition of Fe(lll) chelators or

humic substances (Lovley et aI., 1994; Lovley, Wood-ward et al., 1996). Chelators stimulate the activity ofFe(Ill)-reducing microorganisms by solubilizing in-

soluble Fe(lll) (Lovley and Woodward, 1996). Solubi-lization makes the Fe(lll) more available for microbial

reduction by alleviating the need for Fe(lll)-reducing

microorganisms to come into direct physical contact

with the insoluble Fe(lll) oxides. In instances in which

Fe(Ill) is not present in the sediments, a similar stimu-lation of benzene degradation can be achieved by add-

ing chelated Fe(Ill) (Lovley et al., 1994; Lovley,

Woodward, et al., 1996). Humic acids stimulate Fe(lll)

Materials and Methods

Site Description and Sediment Collection

Sediments and associated groundwater were collected

from a sand and gravel, water table aquifer near an oil

refinery in Ponca City, Oklahoma. Refinery opera-tions, dating back to perhaps the mid-1940s, resultedin extensive subsurface petroleum contamination inthe area sampled. The aquifer was approximately 3 mbelow ground level at the sampling site. Sediments

were collected with a hand auger and placed into I-quart

Weiner, Lauck, and Lovley160

glass canning jars. The jars were filled as completelyas possible with sediment. Any remaining space in thebottles was filled with anoxic groundwater deliveredfrom a nearby monitoring well with a peristaltic pump.

The bottles were bubbled with N2 and sealed.Anoxic groundwater from the monitoring well wascollected in 50- and 160-mL serum bottles. The bottleswere sealed with thick butyl rubber stoppers that werepierced temporarily with a needle during closure tocreate a seal without trapping any air. The 50-mLbottles contained a drop of hydrochloric acid to pre-serve the samples for dissolved metal analysis. Thesediment and groundwater samples were packed incoolers containing ice packs and shipped via overnightcarrier to the laboratory for analyses.

the humic material served as the source of humic acids.For long-term studies with Fe(Ill)-EDTA or humicacids, additional Fe(lll) was added to the sediments aspoorly crystalline Fe(ill) oxide when the Fe(lll) be-came depleted. Benzene was added from concentratedstock solutions prepared in sterile, anoxic, deionizedwater. When the sediments became adapted to highlevels of benzene, pure benzene was added with amicroliter syringe.

For studies on the production of extracellular in-termediates, unlabeled phenol (40 ~M) or acetate(5 roM) was added from anoxic stock solutions toprovide an isotope trap as described by Lovley et al.(1995). The phenol and acetate were added immedi-ately before the addition of [14C]-benzene.

Column StudiesSediment Incubations

Glass columns (Figure I) were filled with aquifer sedi-ment under N2 in a glove bag to monitor the effects ofsulfate addition in a flowthrough system that mightmore closely simulate the physical conditions in theaquifer. Groundwater from the site was collected in55-gal (208-L) plastic drums. Prior to use, the ground-water was bubbled with N2-CO2 to remove dissolvedoxygen, and sodium sulfate (5 mM) was added to onealiquot. The groundwater was anaerobically dispensedinto l00-mL gastight glass syringes with TefionTM-coated plungers. Benzene was added anaerobically tothe groundwater in the syringes from anoxic, sterilestock solutions, and the syringes were connected to thecolumns with stainless steel tubing and fittings(Figure 1).

Groundwater in the syringes was pumped throughthe columns in an upflow direction with a variable-flow syringe pump (Harvard Apparatus, Inc., Holliston,MA). The influent and effluent groundwater wassampled via three-way stainless steel valves that per-mitted groundwater to be withdrawn from the systemunder positive pressure with a 5-mL luer lock glasssyringe. Studies in which Fe(Il)-containing solutionswere pumped through the columns found no oxidationof the Fe(Il), indicating that there was no oxygenleakage into the columns. Three columns each of sedi-ments receiving sulfate-amended groundwater andsediments receiving unamended groundwater wereincubated in the dark at 20°C.

Sediment samples (40 g) were transferred into anaero-bic pressure tubes in a glove bag under an N2 atmo-sphere. After the tubes had been sealed with thickbutyl rubber stoppers, they were removed from theglove bag. The heads pace of the tubes was flushedwith N2:CO2 (93:7) that had been passed over heatedcopper turnings to eliminate traces of oxygen. Alladditions to sediments were made under strict anaero-bic conditions with syringes and needles. The sedi-ments were amended with sodium sulfate, ferroussulfate, sodium nitrate, or Fe(llI)-sodium ethylene-diarninetetraacetate [Fe(llI)-EDTA] from concentrated,anaerobic, sterile stock solutions. Poorly crystallineFe(ill) oxide was synthesized as described previously(!..oyley and Phillips, 1986) and was added as an anaero-bic slurry.

Additions of Fe(ill) oxide were accompanied byadditions of either the chelator nitrilotriacetic acid(NTA), commercially available humic acids (AldrichChemical Co., Inc., Milwaukee, WI), or the sodium orammonium salt of a humic material derived as a wastefrom the processing of titanium dioxide ore. Whennoted, the sediments were inoculated with the previ-ously described (Weiner and Lovley, 1998a) benzene-oxidizing, sulfate-reducing enrichment along with 20mM sodium sulfate. Then [i4C]-benzene (0. 75 ~Ci)was added from an anaerobic sterile stock (58.2 mCi/mmole diluted in sterile, anoxic, deionized water to aconcentration of approximately 3 ~Ci/rnL). The tubeswere inverted and incubated at 20°C in the dark.

The long-term sediment incubations were con-ducted in a manner similar to the short-term incuba-tions, with the exception that the sediments were incu-bated in 30-mL serum bottles rather than pressuretubes. For the long-term studies, the ammonium salt of

Analytical Methods

Sulfate and nitrate concentrations in the groundwaterwere analyzed on a Dionex DX-IOO ion chromato-graph using an IonPac AG4A-SC column. Methane

Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate 161

to waste

3- sampling port (Iuer adapter)

~" 10 0.125" Swagelock reducer60 11m glass fritted disc ~

I" to ~"glass reducer

I" Swagelock union

'"glass colunm end tapered to I"

S cmI.D.

aquifer sediment

-60 I'm glass flitted disc

""

A

""""

"- -/' "- glass column end tapered to ~"

",,--.t: ~"to 0.125" Swagelock reducer

sampling port (Ioer adapter)~ ~3-way valve

r "L_!"~.~g!

Ti'II!lllllllllllln! i

~1astight syringe

1/8" to 1/16" Swagelock reducer

1/16" stainless steel tubing

r syringe pump

Figure 1. Schematic of flowthrough columns.

concentrations in the groundwater and loss of benzenein the long-term sediment incubations were determinedvia headspace analysis with a capillary column gaschromatograph equipped with a flame ionization de-tector. Concentrations of dissolved benzene in theflowthrough column studies were measured with apurge-and-trap apparatus connected to a gas chromato-graph with a flame ionization detector. HCI-extract-able Fe(ll) and Fe(ffi) (Lovley and Phillips, 1987b)and dissolved sulfide (Cline, 1969) were measured asdescribed previously.

Acid-extractable sulfate concentrations in the Site2 sediments were analyzed after 120 g of sediment wasmixed with 100 rnL of 20% hydrochloric acid whilebeing stirred on a hot plate. The acid extract was thenfiltered through a biichner funnel, diluted in deionizedwater, and analyzed for sulfate by ion chromatographyas described above.

Concentrations of 14CH4 and 14COZ were deter-mined by gas proportional counting as described byLovley et al. (1994). Production of [l4C]-labeled extra-cellular intermediates in the sediments was analyzed

162 Weiner, Lauck, and Lovle}

by adding I mL of water to the sediment, centrifugingthe sediments within the pressure tubes and collectingthe supernatant. Aliquots of the supernatant wereinjected into a liquid chromatograph. Fractions sepa-rated on Supelcosil LC-18 (phenol analysis) or AminexHPX-87H (acetate analysis) high-performance liquidchromatography (HPLC) columns were collected andanalyzed for radioactivity by liquid scintillation count-ing as described by Lovley et al. (1995).

consumed in the sediments containing humics and Fe(III)in the same amount of time it took for one benzeneaddition to be consumed in the control sediments.

Lack of Stimulation of BenzeneDegradation with Nitrate

Addition of nitrate completely inhibited benzene deg-radation (Figure 2). This result is consistent with thefinding in most studies that benzene is not degradedwith nitrate as an electron acceptor (Lovley, 1997).Results and Discussion

Stimulation of BenzeneDegradation with Fe(III)

As observed previously by Weiner and Lovley (1998b),significant amounts of [l4C]-benzene were convertedto 14CH4 and 14CO2 in the Ponca City sediments incu-bated under in situ conditions without amendments(Figure 2). Addition of the Fe(IlI) chelator, NTA, orhumic substances along with Fe(lII) oxide resulted inthe short-term stimulation of benzene degradation (Fig-ure 2). Of the combinations evaluated, no consistentdifferences were found in the degree of stimulationbetween NT A and the different types and concentra-tions of humic substances added.

Previous studies have demonstrated that carbonand electron flow can be shifted from methane produc-tion to Fe(ill) reduction when Fe(ill) is added tomethanogenic sediments (Lovley and Phillips, 1987a).However, there was only a slight decrease in the pro-portion of 14CH4 produced from [l4C]-benzene in theseshort-term studies with Fe(IlI) oxide treatments. Incontrast, the addition of Fe(ill)-EDT A completely in-hibited production of 14CH4 while stimulating benzenedegradation to a greater extent than any of the otherFe(lII) amendments (Figure 2).

To investigate the potential for Fe(IlI) amend-ments to stimulate benzene degradation over the longterm, sediments amended with Fe(IlI)- EDT A or Fe(IlI)oxide and humics were incubated under strict anaero-bic conditions, and benzene consumption was moni-tored. More benzene was added to the sediments whenmost of the benzene had been depleted from the sedi-ments.

Although Fe(IlI)-EDT A stimulated benzene degra-dation in the short-term study with [l4C]-benzene, inlonger-term incubations the rate of benzene degradationin the presence of Fe(lll)-EDTA was comparable to thatobserved in controls without added Fe(IlI) (Figure 3).Addition of a combination of Fe(IlI) oxide and humicacids did stimulate benzene degradation (Figure 4). Thiswas most apparent in the second 100 days of incubationduring which several additions of benzene were

Stimulation of BenzeneDegradation with Sulfate

Based on the results of previous studies with sedi-

ments from a different aquifer (Weiner and Lovley,

1998a), it was expected that stimulation of benzene

degradation with sulfate would require that the sedi-ments also be inoculated with benzene-degrading sul-fate reducers. In the short-term studies, addition of

sulfate and the benzene-degrading inoculum did stimu-late benzene degradation (Figure 2). However, addi-

tion of sulfate alone also stimulated benzene degrada-tion and resulted in an increase in the proportion of

I4CO2 produced from p4C]-benzene. The additions of

Fe(ll) sulfate stimulated benzene degradation morethan did the additions of sodium sulfate. However,

further studies were conducted with sodium sulfate

because the Fe(ll) sulfate was contaminated with

Fe(llI), and it was important to ascertain whether the

stimulation effect of the sulfate addition could be at-tributed to sulfate alone.

The long-term static incubations also demonstratedthat sulfate additions could stimulate benzene degra-

dation (Figure 5). For approximately the first 2 months,the rates of benzene degradation in the sulfate-amended

sediments were comparable to those in the unamended

sediments. However, with extended incubation, ben-zene degradation in the sulfate-amended sedimentsbegan to become significantly faster than in unamended

sediments (Figure 5). For example, on day 83 both

sediments received the same level of benzene (ca. 200

~). However, benzene was completely consumedwithin 9 days in the sulfate-amended sediments but

persisted for more than 3 weeks in the unamended

sediments.

Aquifer sediments were incubated in glass col-

umns under anaerobic conditions (Figure 6) to simu-late sulfate additions to groundwater moving through

the aquifer. Groundwater from the site was bubbled

with N2 to remove oxygen, amended with benzene,and pumped through the sediment columns. Initially,

benzene concentrations were relatively low in the

Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate 163

4mMNTA

.33 mg NaHum

6.67 mg NaHum

13.3 mgNaHum

1.33 mg NH4Hum

6.67 mg NH4Hum

13.3 mg NH4Hum

1.33 mg Aldrich

6.67 mg Aldrich

N ~ 0\ 00 <=><=> <=> <=> <=> 0 0

No additions~~:~~~//;////// //~//////~///J;i, :'10 rnM FeEDTAI

W//////////////////M I

W///////////////////J ,W // // // // // // // // // // ~ -c'

I

W//////////////////M

W////////////////////////MI W////////////////J

W//////////////////////////J.

(fj'::=ff//J, !.W/ // // // // // // // ///A :

~~(j'--'~~g-

13.3 mg Aldrich

500 J.1M NO3

2 mM Na2S04

10 rnM Na2S04

20 mM Na2S04

2 mM FeSO4

10 roM FeSO4

20 mM FeSO4

10% Inoculum

W//////////AW//////////////~

I W////////////////A

W // // // // // // // //A S\J S\JI :"~~~ff~ Cf) Cf)

~ ~n n0 ~ I

t-.) I W///~//////~//////~//////~ I ,

0 N ~ 0\ 00 -0 0 0 0 0

0

Figure 2. Effects of various amendments on the conversion of [14C]-benzene to 14CH4and 14CO2 over a 13-day incubation period.The results are the means of duplicate incubations. Abbreviations for amendments: FeEDT A, Fe(III)-EDT A; NT A, nitrilotriacetic acidplus Fe(lll) oxide; NaHum, sodium salt of humics plus Fe(lll) oxide; NH4Hum, ammonium salt of humics plus Fe(lll) oxide; Aldrich,Aldrich humic acids plus Fe(lll) oxide; 10% Inoculum, inoculum of benzene-oxidizing, sulfate-reducing enrichment along with 20

mM sodium sulfate.

Weiner, Lauck, and Lovlev164

0,.-,

';nI--'

~

<<

'00\0

<

oj~ro15-"

~-

uic0~.c:J0.~Q)

~~(/)casQ

)

E

-0IN--/

Q)

c:Q)

Nc:OJ

.c

Q)

55~

'0<

000<\D<<<

0<~

<<<0<N

<Q

)

:5'0

~0

000In

165E

nhanced A

naerobic B

enzene D

egradation w

ith the Addition

of Sulfate

CD

:E~(\1

.'!!"5C

/)

~CD

.cI-

<<

'0u-u"Q.

u'0

~

gQ

)~+~~

I-~~~0

Weiner, Lauck, and Lovley

166

cIic:0"ia

.c:Ju.~aJ

~"Q.

E"6(/)c:(IjaJEaJ~aJ@f!."5(/)

~aJ.c:I-m~!:JaJc:aJNc:aJ

.cc:0!?.!Q"5(/)

"6f!.~ffitriaJ~C

I~

rn~0

00In

'=>

'=>

~0

00

0('f")

N

CW

rl) ~u~zu~

g

00.-0

167E

nhanced A

naerobic B

enzene D

egradation w

ith the Addition

of Sulfate

168

G)

G)-

~=

,==

0

G)"t:!"t:!

G)~

G)G

)~

~"t:!"t:!

.==

0

53 53

::g::g~~

~

~

I I

G)G

)G)G

)

§§cEcE

:5:5~~

~+

~

~00V

\

00~

....A

~.,

..J-

/

.

00NtI

<:>

0r'"I

(wrl)

;}U;}Z

U;}1

~

'tB

~

m

~;t:j-I~~

0

0Ir) or)r- <=

><

=>

Ir}N 0In I/')r---- 00N<

=>

'rIN

<<

~Q

>.(/)(/)

"OC

t1C::. ~

.2.oc;;(/)rnQ

)Q).c

.I:: ~

::.

-0 u

-u.5o~

Q)

.oc;;Q)rn

0E

uE

=.-c."0 -

~

Q)

.~o(/)-u-Q

)o.r

(/)

$.~c

C

C

ct1Q

) .-Q

)

E~

E.-I-"O

O:Q

)~

:I:£~

~Q

)Q

)Q)~

'§E~

0".- -C

t1-"5C

)Q)(/)

CU

Q)

.-C

~

cQ)Q

).-"0

.I::ct1 .-

I--(/)C

Q

) .

0 ~

(/)

uu>'

(/).- ct1

C"5"O

E~

CD

::'"0>.

"0 >

'm

U.l::rn

.l::Q)E

C).I::

.-::'-x0

-0~

~~

.I:: (/)

c. .

.-c. C

~~

ct1.g0=

)(

!Q

.~

oQ)~

~_.1::

1-"0

c-Q)C

)O:"E

::'c:I:Ct1

5::oQ)"O

Q)""(ij£ffi

"O(/)

--C

"O(/)(/)

Ct1C

t1~Q

)-Q

)Q)C

Q).I::~

o-~

~.5

0 ~

~

C

~

0 ct1

.-Co

t: c

(/) ct1

C)

C

Q)

Q)

'm

o.l::.£::Q)

:0:;-""'"0~

cQ)(/)

-Q

)"o~

C

.-ct1

Q)Q

)$..cU

~

::.

c"'"O

Q)

OQ

) .I::

u.cuil-Q

)"O>

..C

Q)C

t1-Q

)-"O

cct1

Q)

NU

Lt)Ec'5>

.-Q

)c-Ct1

m.- ~

~(/)ct1-

.ct1 E

.I::

CD

~.-

U

4I"O~

Ct1

"o~Q

)::.

'C

c. ~

ClQ

)c.oii:c.C

t1-

Lauck, and

Lovley

Effect of Sulfate and Fe(III) onPathway for Benzene Degradation

Although there was only a slight change in the relativeimportance of methane and carbon dioxide as endproducts of benzene degradation in the short-term in-cubations, in long-term incubations the addition ofsulfate (Figure 7) and Fe(III) (Figure 8) resulted in theproduction of carbon dioxide as the sole end product.Benzene degradation showed similar results in othersulfate-reducing (Lovley et al., 1995; Weiner andLovley, 1998a) and Fe(III)-reducing (Anderson et al.,1998; Lovley et al., 1994; Lovley, Woodward, et al.,1996) environments.

In other sulfate-reducing environments, benzenedegradation was found to proceed directly to carbondioxide without the production of extracellular inter-mediates such as aromatic compounds or fatty acids(Lovley et al., 1995; Weiner and Lovley, 1998a).However, in methanogenic Ponca City sediments, phe-nol, acetate, and propionate were important extracellu-lar intermediates in benzene metabolism (Weiner and

Lovley, 1998b).Isotope-trapping experiments were conducted to

determine whether long-term exposure to sulfate hadhad an effect on the production of extracellular inter-mediates from benzene. These experiments were simi-lar to those used by Lovley et al. (1995) and Weinerand Lovley (1998b) to evaluate the pathway of ben-zene degradation in sulfate-reducing and methanogenicsediments. Addition of acetate as an isotope trap hadno effect on the production of I4CO2 from [l4C]-ben-zene (Figure 7). However, the addition of phenol in-hibited 14CO2 production, suggesting the phenol wasan extracellular intermediate in benzene degradation.This was confirmed by the finding that, when thesediments were sampled at 9 h, radioactivity equiva-lent to 21% of the amount of I4CO2 produced wasrecovered in the phenol pool. In contrast, no radioac-tivity was recovered from the acetate pool.

The findings that phenol was an extracellular in-termediate, but that acetate was not, indicate that ben-zene metabolism in these sediments was different thanfor previously described methanogenic or sulfate-re-ducing sediments. A potential interpretation of theseresults is that, although benzene continues to be me-tabolized first to phenol in the sulfate-amended sedi-ments as previously found for unamended methano-genic sediments (Weiner and Lovley, 1998b), incontrast to the unamended sediments, the phenol isthen directly oxidized to carbon dioxide by phenol-oxidizing sulfate-reducers.

groundwater being pumped through the columns. Af-ter 18 days, no benzene was detected in the outflow ofany of the columns. These observations were truewhether the groundwater was amended with sulfate orno sulfate was added to the groundwater. When thebenzene concentrations in the groundwater and thegroundwater flow were increased, a significant differ-ence was noted in the extent of benzene degradationbetween the two treatments.

The sediments receiving sulfate-amended ground-water adapted to completely remove the increasedbenzene input by day 50. In contrast, benzene contin-ued to pass through the columns receiving no sulfateadditions. When the benzene concentration in thegroundwater being pumped through the columns wasfurther increased on day 60, the capacity for thesediments receiving sulfate-amended groundwater toremove all of the benzene was exceeded. However,the amount of benzene removed from the sulfate-amended sediments remained considerably greaterthan the benzene removed from the sediments notreceiving sulfate throughout the remainder of the study

(Figure 6).Benzene removal in the sulfate-amended col-

umns was associated with the removal of sulfate fromthe groundwater. For example, on day 99 the sulfateconcentration in the inlets to the sulfate-amendedcolumns was 5.28 :!: 0.094 roM (mean:!: standarddeviation, n = 3), whereas the outlet concentrationwas 4.66 :!: 0.41 roM. The difference in benzeneconcentrations between the influent and the effluent(Figure 6) was 177.4 ~. Thus, the ratio of sulfateremoved to benzene removed was 3.5:1. This ratio isclose to the theoretical (Lovley et al., 1995) sulfate-to-benzene consumption ratio of 3.75 moles of sul-fate reduced per mole of benzene oxidized to carbondioxide, with sulfate serving as the sole electron ac-

ceptor.Despite the apparent reduction of significant

quantities of sulfate, there was little free sulfide in thegroundwater exiting the columns. The concentrationof dissolved sulfide in the effluent from the sulfate-amended columns was 8.5 ~ compared to 1.9 IlMin the effluent from the columns not receiving sulfateadditions. The sulfate-amended columns became pro-gressively darker over the course of the study, sug-gesting that much of the sulfide produced was se-questered in the sediments as iron sulfides. The factthat these sediments contained 19 mmoles of Fe(II)per kilogram of sedim~t indicates that there is sig-nificant potential for Fe(II) to sequester sulfides inthe sediments.

16QEnhanced Anaerobic Benzene Degradation with the Addition of Sulfate

~4)

~4)

~u...

-I--'

-04)

-0

~'+-.0

E

~e:-

M

0U~

Hours

Figure 7. Effects of phenol and acetate on the oxidation of [14C]-benzene in sediments from long-term incubation in the presence

of sulfate. There was no detectable production of 14CH4. The results are the means of duplicate incubations.

Similar studies on the fate of [l4C]-benzene withthe sediments incubated with a combination of Fe(llI)oxide and humic acids indicated that, as with the sul-fate-amended sediments, the addition of a phenol iso-tope trap inhibited production of 14CO2, but the addi-tion of acetate as the isotope trap did not. These resultssuggest that phenol was also an extracellular interme-diate in the Fe(Ill)/humics-amended sediments.

Why Sulfate Additions Stimulate BenzeneDegradation in Ponca City Sediments

Previous studies have indicated that increasing theavailability of Fe(Ill) for reduction with humic acidscan stimulate anaerobic benzene degradation in sedi-ments from a petroleum-contaminated aquifer (Lovley,Coates et al., 1996; Lovley, Woodward et al., 1996).However, the finding that sulfate could stimulateanaerobic benzene degradation at this site contrastswith results of studies from previous sites where ben-zene persisted in the presence of sulfate (Chapelle et

al., 1996; Lovley et al., 1994; Weiner and Lovley,

1998a).The chronic, long-term benzene contamination of

the Ponca City site may be an important factor leadingto the establishment of anaerobic benzene-degradingmicroorganisms at this site (Weiner and Lovley, 1998b).Furthermore, even though initial studies indicated thatmethanogenesis is the predominant TEAP at this site(Weiner and Lovley, 1998b), the data in that study aswell as the data presented here (Figure 2) indicate thatthe amount of methane produced from benzene wastoo low for methanogenesis to be the only TEAP in-volved in naturally occurring benzene degradation in

the aquifer.Small amounts of Fe(Ill) and sulfate are present in

the sediments and could permit maintenance of smallFe(ill)-reducing and sulfate-reducing communitieswhose activity could be stimulated when more Fe(lll)or sulfate is made available.

For example, the sediment contained 2 mmoles/kgof HCI-extractable Fe(ill), which is considered to be

Weiner, Lauck, and Lovley170

---G)

55~u

...~'0G)

'0

~0

5~b""

0u

~

Hours

Figure 8. Effects of phenol and acetate on the oxidation of ["C]-benzene in sediments from long-term incubation in the presenceof Fe{lll) oxide and humic acids. There was no detectable production of I'CH," The results are the means of duplicate incubations.

develop and persist in the aquifer. Once the availabil-ity of Fe(lll) and/or sulfate is increased artificially,further stimulation of these populations and rates ofbenzene degradation that are higher than those ob-served in unamended, methanogenic sediments couldresult.

Conclusions

This study provides the first example of stimulation ofanaerobic benzene degradation in sediments from apetroleum-contaminated aquifer with the addition ofsulfate. Although it is clear from previous studies(Weiner and Lovley, 1998a) that addition of sulfatemay not stimulate benzene degradation in all pe~o-leum-contaminated aquifers, in aquifers such as thePonca City site, sulfate additions may have severaladvantages over other potential electron acceptors thatmight be added to stimulate benzene degradation. Sul-fate additions may be preferable to oxygen additionsfor the following reasons:

available for microbial reduction (Lovley and Phillips,1987b). Although the Fe(lll) in these sediments ac-counted for less than 10% of the total HCl-extractableiron, similar concentrations of Fe(IJI) have been foundto support Fe(llI) reduction in other aquifers. Concen-trations of sulfate in the groundwater were 30 ~ orless, but HCl extracts of the sediment indicated that itcontained 1.4 mmoles/kg of an acid-extractable sulfatemineral following 1 year of anaerobic incubation inthe laboratory. This particulate sulfate could provide alow, steady supply of dissolved sulfate to the sulfate-reducers in the sediment. Furthermore, because theaquifer in the present study is a water table aquifer,inputs of oxygenated surface recharge water may re-sult periodically in the oxidation of Fe(ll) to Fe(ill)and introduce sulfate into the aquifer (Vroblesky and

Chapelle, 1994).Thus, there may be the opportunity for small

communities of Fe(ill)-reducing and sulfate-reduc-ing microorganisms that can metabolize benzene (orproducts of benzene metabolism such as phenol) to

171Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate

4.

Unlike oxygen, sulfate is not consumed chemi-cally by reaction with reduced species such asFe(II) and sulfide.The addition of oxygen can cause plugging ofthe aquifer due to the formation of insolubleFe(ill) oxides, whereas sulfate will not reactwith Fe(II) to form Fe(III) oxides.The solubility of sulfate is much higher thanthat of oxygen, so it is easier to add higherconcentrations to groundwater.One mole of sulfate has twice the oxidizing

(electron accepting) capacity of one mole ofoxygen.

When sulfate additions are successful in stimulat-ing benzene degradation, addition of sulfate may bepreferable to the use of Fe(Ill) to stimulate anaerobicbenzene degradation because sulfate does not requirethe addition of other components such as humic acidsor synthetic chelators. In those aquifers in which theaddition of sulfate alone does not stimulate anaerobicbenzene degradation, it may still be possible to en-hance benzene degradation with sulfate if, as previ-ously described (Weiner and Lovley, 1998a), the aquiferis also seeded with benzene-degrading sulfate-reduc-ers. Although laboratory fIowthrough simulations ofthe aquifer have demonstrated that addition of sulfatehas the potential to be a useful bioremediation strat-egy, field trials are necessary to confirm that sulfateaddition is an effective treatment.

AcknowledgmentsThis research was supported by a grant fromConoco, Inc. and grant DEB-9523932 from theNational Science Foundation. We thank Jeff Meyersfor initiating this project; Robert Siebert for providingthe humic matrials; Robert Anderson for helpful sug-gestions; and Keith Coffman for continued support.

References

Acton, D.W., and J.F. Barker. 1992. "In situ biodegradationpotential of aromatic hydrocarbons in anaerobicgroundwaters." J. Contam. Hydrol. 9:325-352.

Anderson, R.T., and D.R. Lovley. 1997. "Ecology and bio-geochemistry of in situ groundwater bioremediation."Adv. Microbial Ecol. 15:289-350.

Anderson, R.T., J. Rooney-Varga, C.V. Gaw, and D.R.Lovley. 1998. "Anaerobic benzene oxidation in theFe(Ill)-reduction zone of petroleum-contaminated aqui-fers." Environ. Sci. Technol.32:1222-1229.

Anid, P.J., P.J.J. Alvarez, and T.M. Vogel. 1993. "Biodegra-dation of monoaromatic hydrocarbons in aquifer col-

umns amended with hydrogen peroxide and nitrate."Wat. Res. 27:685-691.

Barbaro, J.R., J.F. Barker, L.A. Lemon, and C.I. Mayfield.1992. "Biotransformation of BTEX under anaerobicdenitrifying conditions: Field and laboratory observa-tions." J. ContaIn. Hydrol. 11:245-272.

Barker, J.K., P. Major, and D. Major. 1987. "Natural attenu-ation of aromatic hydrocarbons in a shallow sand aqui-fer." Growzd Wat. Monitor. Rev. 7:64-71.

Chapelle, F.H., P.M. Bradley, D.A. Vroblesky, and D.R.Lovley. 1996. "Measuring rates of biodegradation in apetroleum hydrocarbon-contaminated aquifer:' Ground-water 34:691-698.

Cline, J.D. 1969. "Spectrophotometric determination of hy-drogen sulfide in natural waters." Limnol. Oceanogr.14:454-458.

Coates, J.D., R. T. Anderson, J.C. Woodward, E.J.P. Phillips,and D.R. Lovley. 1996. "Anaerobic hydrocarbon.deg-radation in petroleum-contaminated harbor sedimentsunder sulfate-reducing and artificially imposed iron-reducing conditions." Environ. Sci. Technol. 30:2784-2789.

Flyvbjerg, J., E. Arvin, B.K. Jensen, and S.K. Olsen. 1993."Microbial degradation of phenols and aromatic hydro-carbons in creosote-contaminated groundwater undernitrate-reducing conditions." J. ContaIn. Hydrol.12:l33-150.

Hutchins, S.R., G. W. Sewell, D.A. Kovacs, and G.A. Smith.1991. "Biodegradation of aromatic hydrocarbons byaquifer microorganisms under denitrifying conditions."Environ. Sci. Technol. 25:68-76.

Kazumi, J., M.E. Caldwell, J.M. Sutlita, D.R. Lovley, and L.Y.Young. 1997. "Anaerobic degradation of benzene in di-verse environments." Environ. Sci. Technol. 31:813-818.

Kuhn, E.P., J. Zeyer, P. Eicher, and R.P. Schwarzenbach.1988. "Anaerobic degradation of alkylated benzenes indenitrifying laboratory aquifer columns." Appl. Environ.Microbiol. 54:490-496.

Lee, M.D., J.M. Thomas, J.C. Borden, P.B. Bedient, C.H.Ward, and J.T. Wilson. 1988. "Biorestoration ofaqui-fers contaminated with organic compounds." CRC Grit.Rev. Environ. Control 18:29-89.

Lovley, D.R. 1997. "Potential for anaerobic bioremediationof BTEX in petroleum-contaminated aquifers." J.1ndustr. Microbial. 18:75-81.

Lovley, D.R.,M.J. Baedecker, D.J. Lonergan,I.M. Cozzarelli,E.J.P. Phillips, and D.I. Siegel. 1989. "Oxidation ofaromatic contaminants coupled to microbial iron reduc-tion." Nature 339:297-299.

Lovley, D.R., J.D. Coates, E.L. Blunt-Harris, E.J.P. Phillips,and J.C. Woodward. 1996. "Humic substances as elec-tron acceptors for microbial respiration." Nature382:445-448.

Lovley, D.R., J.D. Coates, J.C. Woodward,andE.J.P.Phillips.1995. "Benzene oxidation coupled to sulfate reduc-tion." Appl. Environ. Microbiol. 61:953-958.

Lovley, D.R., J.L. Fraga, E.L. Blunt-Harris, L.A. Hayes,E.J.P. Phillips, and J.D. Coates. 1998. "Humic

Weiner, Lauck, and Lovley

Morgan, P., and R.J. Watkinson. 1992. "Factors limiting thesupply and efficiency of nutrient and oxygen supple-ments for the in situ biotreatment of contaminated soiland groundwater." Wat. Res. 26:73-78.

Patterson, B.M., F. Pribac, C. Barber, G.B. Davis, and R.Gibbs. 1993. "Biodegradation and retardation of PCEand BTEX compounds in aquifer material from West-ern Australia using large-scale columns." J. Contam.HydroL 14:261-278.

Phelps, C.D., J. Kazumi, and L. Y. Young. 1996. "Anaero-bic degradation of benzene in BTX mixtures depen-dent on sulfate reduction." FEMS Microbiol. Lett.145:433-437.

Thierrin, J., G.B. Davis, C. Barber, B.M. Patterson, F. Pribac,T.R. Power, and M. Lambert. 1993. "Natural degrada-tion rates of BTEX compounds and naphthalene in asulphate reducing groundwater environment." Hydro-logical Sciences 38:309-322.

Thomas, J.M., and C.H. Ward. 1989. "In situ biorestorationof organic contaminants in the subsurface." Environ.Sci. TechnoL 23:760-766.

Vroblesky, D.A., and F.H. Chapelle. 1994. "Temporal andspatial changes of terminal electron accepting processesin a petroleum hydrocarbon contaminated aquifer andthe significance for contaminant biodegradation." Wat.Resour. Res. 30:1561-1570.

Weiner, J., and D.R. Lovley. 1998a. "Anaerobic benzenedegradation in petroleum-contaminated aquifersediments after inoculation with a benzene-oxidizingenrichment." AppL Environ. MicrobioL 64:775-778.

Weiner, J., and D.R. Lovley. 1998b. "Rapid benzene degra-dation in methanogenic sediments from a petroleum-contaminated aquifer." Appl. Environ. Microbiol.64:1937-1939.

substances as a mediator for microbially catalyzedmetal reduction." Acta Hydrochirn. Hydrobiol.26:152-157.

Lovley, D.R., and D.J. Lonergan. 1990. "Anaerobic oxida-tion of toluene, phenol, and p-cresol by the dissimila-tory iron-reducing organism, GS-15." Appl. Environ.Microbiol.56:1858-1864.

Lovley D.R., and E.J.P. Phillips. 1986. "Organic mattermineralization with reduction of ferric iron in anaerobicsediments." Appl. Environ. Microbiol. 51:683-689.

Lovley, D.R., and E.J.P. Phillips. 1987a. "Competitive mecha-nisms for inhibition of sulfate reduction and methaneproduction in the zone of ferric iron reduction in sedi-ments." Appl. Environ. Microbiol. 53:2636-2641.

Lovley, D.R., and E.J.P. Phillips. 1987b. "Rapid assay formicrobially reducible ferric iron in aquatic sediments."Appl. Environ. Microbiol. 53:1536-1540.

Lovley, D.R., and J.C. Woodward. 1996. "Mechanisms forchelator stimulation of microbial Fe(IlI)-oxide reduc-tion." Chern. Geol. 132:19-24.

Lovley, D.R., J.C. Woodward, and F.H. Chapelle. 1994."Stimulated anoxic biodegradation of aromatic hydro-carbons using Fe(IlI) ligands." Nature 370: 128-131.

Lovley, D.R., J.C. Woodward, and F.H. Chapelle. 1996."Rapid anaerobic benzene oxidation with a variety ofchelated Fe(IlI) forms." Appl. Environ. Microbiol.62:288-291.

Major, D.W.,C.I. Mayfield, andJ.F. Barker. 1988. "Biotrans-formation of benzene by denitrification in aquifer sand."Ground Water 26:8-14.

Morgan, P., S.T. Lewis, and R.J. Watkinson. 1993. "Biodeg-radation of benzene, toluene, ethylbenzene, and xylenesin gas-condensate-contaminated ground-water.»Environ. Pollu. 82:181-190.

173Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate