Biodegradation of Benzene by Halophilic and Halotolerant Bacteria under Aerobic Conditions

A highly enriched halophilic culture was established with benzeneas the sole carbon source by using a brine soil obtained froman oil production facility in Oklahoma. The enrichment completelydegraded benzene, toluene, ethylbenzene, and xylenes within1 to 2 weeks. Also, [¹⁴C]benzene was converted to ¹⁴CO₂, suggestingthe culture's ability to mineralize benzene. Community structureanalysis revealed that Marinobacter spp. were the dominant membersof the enrichment.

INTRODUCTION

Top

Introduction

Large volumes of oily wastewater are generated during oil explorationand production activities. Produced waters display a wide rangeof salinities, ranging from low to high. Spilled brine inhibitsplant growth, leading to increased erosion and loss of topsoiland contamination of groundwater by both salt and hydrocarbons.Because conventional microbiological treatment processes donot function at high salt concentrations, bioremediation ofoilfield brine can only be accomplished by using indigenousbacteria capable of degrading petroleum compounds or throughbioaugmentation of halophiles or halotolerants. Unfortunately,information on the ability of such organisms to degrade petroleumcompounds is limited. Only recently have there been indicationsthat the halophilic bacteria may have a greater potential inthe degradation of pollutants than was previously assumed. Ahalophilic archaea (strain EH4) was found to be capable of degradinga wide range of n-alkanes and aromatic hydrocarbons in the presenceof high salt (1, 12, 15). Marinobacter hydrocarbonoclasticusdegraded a variety of aliphatic and aromatic hydrocarbons (7).A halotolerant Streptomyces sp., isolated from an oil fieldin Russia, degraded crude petroleum (9). In addition, bacteriaisolated from salt-impacted material degraded polycyclic aromatichydrocarbons (PAHs) (13). While these observations provide evidencefor the degradation of n-alkanes, PAHs, and other simple aromaticcompounds, little is known about the degradation of benzene,toluene, ethylbenzene, and xylene (BTEX) compounds. The primaryobjective of this work was to evaluate the ability of halophilicand halotolerant bacteria present in oilfield brine to degradeBTEX compounds. BTEX are most problematic, because they arehighly water soluble and are toxic. Among BTEX, benzene is ofmajor concern, because it is one of the most stable aromaticcompounds and is a known carcinogen.

Enrichment culture.

Top

Enrichment culture.

A stable and highly enriched aerobic microbial consortium thatdegraded benzene as the sole carbon and energy source was developedfrom a soil sample obtained from Seminole County in Oklahoma.Hereafter this culture is referred to as the Sem-2 enrichmentculture. The enrichment was initiated by adding 10 g of soil(wet weight) to duplicate 1-liter-capacity bottles containing500 ml of mineral salts medium (MSM). MSM contained (in grams/liter):NaCl, 145; MgCl₂, 0.5; KH₂PO₄, 0.45; K₂HPO₄, 0.9; NH₄Cl, 0.3;KCl, 0.3. Air in the headspace served as the source of oxygen.The bottles were closed with a black rubber stopper with a holein the middle that fit a cut 3-in. Hungate tube. The tubes wereclosed with Teflon-coated septa and aluminum caps. A 100-µlgas-tight glass syringe was used to introduce 22 µl ofundiluted benzene ( $~$ 245 µmol) to each bottle. The bottleswere incubated static in the dark at room temperature. After7 to 8 months of continuous enrichment, the culture consistentlydegraded benzene within 18 days at a rate of approximately 12µmol/day.

Biodegradation assay.

Top

Biodegradation assay.

Unless otherwise mentioned, all experiments involving the Sem-2enrichment culture were performed with 160-ml-capacity serumbottles filled with 45 ml of MSM containing 2.5 M NaCl. Bottleswere inoculated with 5 ml of Sem-2 enrichment culture and werespiked with 2 µl (17 to 22 µmol) of undiluted benzene,toluene, ethylbenzene, or xylenes. The bottles were then closedwith Teflon-coated septa and aluminum caps and were incubatedunder static conditions in the dark at 30°C. The headspacegas was withdrawn periodically, and degradation of BTEX wasmonitored by gas chromatography (GC).

Biodegradation of BTEX compounds were assayed by using a HewlettPackard 6890 GC equipped with a flame ionization detector anda DB-1 capillary column (30 m by 0.320 mm by 1 µm; J&WScientific, Inc.). Helium served as both carrier and makeupgas at flow rates of 10 and 40 ml/min, respectively. The flowrates of hydrogen and air were set at 40 and 450 ml/min, respectively.The operating GC conditions were the following: oven temperature,70°C for 7 min; inlet temperature, 150°C; and detectortemperature, 220°C. Approximately 200 µl of headspacegas from microcosms was injected into the GC for quantification.The GC response for each compound tested was calibrated to givethe total mass in that bottle. Standards were prepared in 160-mlbottles filled with 50 ml of NaCl solution (0 to 4 M) and wereclosed with Teflon-faced septa and aluminum caps. After equilibrationat room temperature the GC response for a range of mass (inmicromoles/bottle) of each compound tested was plotted, andthe slopes were used to quantify the unknown. The quantificationof benzene in enrichment bottles was accomplished as describedabove by using a calibration curve prepared with 1-liter bottlescontaining 500 ml of 2.5 M NaCl solution. The GC detection limitfor benzene using our method was <1.0 µmol/bottle.

Microbial community structure.

Top

Microbial community structure.

Community structure of the Sem-2 culture grown on benzene inthe presence or absence of salt was analyzed by using denaturinggradient gel electrophoresis (DGGE) by Microbial Insights, Inc.(Rockford, Tenn.). PCR-amplified eubacterial 16S ribosomal DNA(rDNA) fragments were obtained by nested PCR using primer setscorresponding to Escherichia coli base pair positions 27 to1492 and 341 to 534 (the forward primer contained a 40-bp GCclamp). PCR products were separated on a DGGE gel, and eachprominent band was excised, purified, and sequenced.

Degradation BTEX by halophilic and halotolerant bacteria.

Top

Degradation btex by halophilic...

The Sem-2 enrichment consistently degraded all added benzene(250 µmol/bottle) in 2.5 weeks at room temperature (Fig.1). Experiments also evaluated the ability of the Sem-2 enrichmentto mineralize [¹⁴C]benzene to ¹⁴CO₂. Roughly 46% of the initiallyadded radiolabeled benzene was converted to ¹⁴CO₂ in 4 weeks(data not shown)_. In addition, the enrichment also degradedtoluene, ethylbenzene, or o-m-p-xylenes as the sole carbon sourcewhen grown in MSM with 2.5 M NaCl at 30°C (Fig. 2). Amongthe tested compounds, toluene was degraded fastest. Approximately20 µmol of toluene was completely degraded in less than1 week, while benzene, ethylbenzene, or xylenes required roughly2 to 3 weeks. Autoclaved control bottles showed no evidenceof degradation. Although a few reports have documented the abilityof halophiles or halotolerants to degrade hydrocarbons, includingphenol (16), nitrophenols (12), benzoate (5), pesticides (2),herbicides (11), n-alkanes (1), and PAHs (1, 13), the authorsare unaware of any report demonstrating the aerobic degradationof BTEX by halophiles or halotolerant bacteria. The abilityof the enrichment to degrade BTEX is significant, because thesecompounds are highly soluble and can contaminate large volumesof soil or aquifer material at exploration and production sites.Also, these compounds are toxic and carcinogenic and are amongthe U.S. Environmental Protection Agency's priority pollutants.

View larger version (20K):
[in this window]
[in a new window]
FIG. 1. Repeated use of benzene ( ${blacktriangleup}$ ) as the sole carbon and energy source in the presence of 2.5 M NaCl by the Sem-2 enrichment. The enrichments were maintained in 1-liter-capacity bottles containing 500 ml of MSM at room temperature. After an initial lag period, the enrichment degraded 200 to 300 µmol of added benzene/bottle consistently in 2.5 weeks. Results for only one bottle are shown; duplicate enrichments behaved similarly.

View larger version (17K):
[in this window]
[in a new window]
FIG. 2. Biodegradation of benzene ( ${circ}$ ), toluene ( ${blacklozenge}$ ), ethylbenzene ( ${square}$ ), and xylenes (•) in 160-ml-capacity bottles containing 45 ml of MSM containing 2.5 M NaCl and inoculated with 5 ml of Sem-2 enrichment. The microcosms were incubated at 30°C. The data are means ± standard deviations for triplicate active microcosms and average of duplicate autoclaved control bottles. Since all controls behaved similarly, only the control for xylenes (asterisks) is shown.

Our study also evaluated the effect of low concentrations ofyeast extract (YE), vitamins, or trace elements on benzene degradationrate. Benzene was completely removed within 8 days in microcosmsamended with 0.02% YE, 1 µl of vitamin solution/ml, or1 µl of trace elements/ml (10) compared to 15 days inthe absence of the stimulants (Fig. 3). It was suggested thathalophiles have more demanding nutritional requirements at highsalt concentrations, and complex media may help stimulate growthof halophilic bacteria at high salt concentrations (14). Anarchaea (strain EH4) isolated from a salt marsh was able todegrade a higher percentage of eicosane (C₂₀H₄₂) in the presenceof YE, peptone, and Casamino Acids (1).

View larger version (19K):
[in this window]
[in a new window]
FIG. 3. Biostimulation of benzene degradation by YE, vitamin, or trace elements. Microcosms (160 ml) were established with 45 ml of MSM containing 2.5 M NaCl and were inoculated with 5 ml of Sem-2 enrichment. Biodegradation of benzene (22 µmol/bottle) was evaluated in the presence or absence of growth stimulants. Symbols: ${blacksquare}$ , YE; ${triangleup}$ , vitamins; ${circ}$ , trace elements; ${square}$ , no amendments. The autoclaved microcosms were also amended with filter-sterilized 0.02% YE, 1 µl of vitamin solution/ml, or 1 µl of trace elements/ml. The results are means ± standard deviations for triplicate active microcosms and the average of two autoclaved bottles. Because all controls behaved similarly, only control data for YE ( ${blacktriangleup}$ ) are shown.

Biodegradation of benzene was evaluated at various salt concentrations,ranging from 0 to 4 M (Fig. 4). The Sem-2 degraded 25 to 30µmol of benzene in 7 to 14 days with no apparent lag timein bottles containing 0.5, 1.0, 2.0, or 2.5 M NaCl. No degradationoccurred in bottles containing 0, 3.0, or 4.0 M NaCl, even after4 weeks of incubation (data not shown). Degradation of benzenerequired the addition of at least 0.5 M NaCl in MSM while nodegradation occurred in its absence, indicating that the enrichmentharbored true halophiles. Also, the ability of the culture todegrade benzene over a wide range of NaCl (0.5 M to 2.5 M) suggeststhat this culture is well suited for field bioremediation applications,because many produced waters or oilfield brines display a widerange of temporal and spatial salinity flux. The reason forthe lack of benzene degradation at 3.0 and 4.0 M NaCl is notknown. Few studies have dealt with the effect of salinity onmicrobial degradation of hydrocarbons. These studies (3, 4,15) have found that biodegradation of hydrocarbons declinedwith increasing NaCl concentrations. In contrast, Fernandez-Linareset al. (6) showed that increasing salt level had no effect oneicosane degradation by a Marinobacter sp.

View larger version (22K):
[in this window]
[in a new window]
FIG. 4. Biodegradation of benzene by the Sem-2 enrichment at different salt concentrations (NaCl). Microcosms were established with 160-ml-capacity serum bottles filled with 45 ml of MSM and were inoculated with 5 ml of Sem-2 culture. All microcosms were spiked with benzene (22 µmol/bottle) and various concentrations of NaCl, including 0 ( ${triangleup}$ ), 0.5 ( ${blacklozenge}$ ), 1 ( ${blacksquare}$ ), 2 ( ${blacktriangleup}$ ), 2.5 ( ${circ}$ ), 3 ( ${square}$ ), or 4 M (•). The results are means ± standard deviations for triplicate active microcosms. Although results for 0, 3.0, and 4.0 M NaCl bottles are shown for only 2 weeks of incubation, these bottles were incubated for more than 4 weeks. No benzene degradation occurred even after 4 weeks of incubation (data not shown).

The community structure of the Sem-2 grown in the presence orabsence of added NaCl was characterized by profiling the 16SrDNA gene by using DGGE (Fig. 5). Multiple bands were amplified,and sequences from each of these bands aligned well (>99%)with the Marinobacter spp. sequences (GenBank accession nos.AY136121, AF513448, and AF237685). From analysis, it appearsthat bands A and B were heteroduplexes of bands C and D (accessionnos. AJ294359, AF212213, AY136121, AY129889, and AF546961).It may be that bands C and D represent two different yet closelyrelated bacteria or that there are two ribosomal sequences forMarinobacter. Also, similar bands were missing in the DGGE obtainedfrom the enrichment devoid of NaCl. These results are consistentwith the degradation activity; benzene was not degraded in bottleswith no added salt, thus suggesting that perhaps the Marinobactersp. was responsible for the observed degradation of benzene.Marinobacter spp. have been isolated from geographically differentlocations, including the French Mediterranean coast, at themouth of a petroleum refinery outlet, from deep-sea sedimentsin the western Pacific, and from oil wells off the coasts ofVietnam and California (7, 8).

View larger version (22K):
[in this window]
[in a new window]
FIG. 5. DGGE gel image of amplimers of bacterial 16S rDNA extracted from microcosms amended with 0 and 2.5 M NaCl. Each microcosm was filled with 45 ml of MSM and 22 µmol of benzene and was inoculated with 5 ml of Sem-2 enrichment. When all the added benzene was degraded (in bottles with 2.5 M NaCl), the culture was analyzed for community composition by DGGE. Bottles without salt did not degrade benzene even after 4 weeks of incubation, and these bottles were analyzed after 4 weeks of incubation. The bands were excised and sequenced. The sequences were compared with closely related sequences in the database. Results show that the dominant members of the Sem-2 culture grown in the presence of 2.5 M NaCl had 97 to 100% sequence similarity to the members of the genus Marinobacter (bands A, B, C, and D). Similar bands were missing in DGGE gel from the Sem-2 culture that did not degrade added benzene (0 M NaCl). Bands labeled F failed to yield usable sequences.

Overall our research has demonstrated the ability of halophilicand halotolerant bacteria to rapidly degrade BTEX compoundsunder aerobic conditions, and such activity could be enhancedmarkedly by the addition of growth promoting nutrients suchas YE. Thus, these observations provide hope for the establishmentof cost-effective methods to remediate brine-impacted soil andaquifers.

ACKNOWLEDGMENTS

This research was funded through grants from the IntegratedPetroleum Environmental Consortium and the Environmental Institute'sCenter for Water Research at Oklahoma State University.

We gratefully acknowledge the assistance of Steve Sower, OklahomaEnvironmental Resources Board, for providing contaminated oilfieldsamples. We also thank Alok Bhandari, Greg Wilber, Vijai Elango,and Kanchan Joshi for their assistance with radioisotopic andGC analyses.

FOOTNOTES

* Corresponding author. Mailing address: Oklahoma State University, Department of Microbiology and Molecular Genetics, 307 Life Sciences East, Stillwater, OK 74078-3020. Phone: (405) 744-7764. Fax: (405) 744-6790. E-mail: fathepu{at}okstate.edu.

REFERENCES

Top