Bioaumentacion_TRATABILIDAD_Simon y Col (2004)

Ecological Engineering 22 (2004) 263–277

Evaluation of two commercial bioaugmentation products forenhanced removal of petroleum from a wetland

Mark A. Simona, James S. Bonnerb,∗, Cheryl A. Pageb, R. Todd Townsenda,Danica C. Muellera, Chris B. Fullera, Robin L. Autenrietha

a Civil Engineering, Texas A&M University, College Station, TX, USAb Conrad Blucher Institute for Surveying and Science, Texas A&M University-Corpus Christi,

6300 Ocean Drive, Corpus Christi, TX 78412, USA

Received 16 October 2003; received in revised form 26 May 2004; accepted 3 June 2004

Abstract

This research evaluated the performance of two commercial bioaugmentation products to enhance petroleum bioremediationin a wetland. The 152-day experiment was conducted at a research facility on the San Jacinto River near Houston, TX, USA,using a controlled oil application to reduce heterogeneity normally associated with spilled petroleum. Additional treatmentsincluded inorganic nutrients and an oiled control (intrinsic). Sediment samples were analyzed for petroleum chemistry, nutrients,microbial population numbers (most-probable-number), and toxicity (Microtox® 100% Test and amphipod bioassay). TheGC–MS results for “total target saturate hydrocarbons” and “total target aromatic hydrocarbons” were hopane-normalized forp o statisticald icrobialp ic nutrientst r all threeb this is duet( ronmentalc©

K

f

en-and

0

etroleum biodegradation interpretation. When comparing the enhanced treatments to the oiled control, there were nifferences in the first-order biodegradation rate coefficients. Similarly, there were no statistical differences in the mopulation numbers. The nutrient analyses indicated that there were higher ammonium concentrations for the inorgan

reatment and one of the bioaugmentation product treatments. There was statistically-higher amphipod mortality foioremediation treatments as well as the oiled control when compared to the unoiled control plots. However, whether

o the amendments or the oil is uncertain. There was no statistical correlation between oil concentrations and Microtox® toxicityr2 < 0.01). Overall, none of the bioremediation treatments appeared to benefit the wetland recovery in these envionditions.2004 Elsevier B.V. All rights reserved.

eywords:Bioremediation; Petroleum; Bioaugmentation; Biodegradation; Wetland

∗ Corresponding author. Tel.: +1 361-825-2717;ax: +1 361 825 2715.

E-mail address:[email protected] (J.S. Bonner).

1. Introduction

Hydrocarbon contamination is a major environmtal problem due to the manufacture, transportationdistribution of petroleum (Atlas and Cerniglia, 1995).

925-8574/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.ecoleng.2004.06.005

264 M.A. Simon et al. / Ecological Engineering 22 (2004) 263–277

The Gulf of Mexico is one of the most intensive oil-producing areas in the world and experiences the largestpercentage of oil spills in the United States (NationalResearch Council, 2003). Moreover, it has been es-timated that nearly 6 million acres of tidal marshesexist on the U.S. Gulf Coast (Alexander and Webb,1985). The proximity of the petroleum production andprocessing facilities to coastal environments results inchronic contamination and spill-related impacts to sen-sitive ecosystems. A report by the Office of TechnologyAssessment states bioremediation may be applicablein sensitive ecosystems in which traditional remedia-tion technologies may do more harm than good (OTA,1991).

Bioaugmentation, or “bacterial seeding”, is the ad-dition of microorganisms to stimulate biodegrada-tion (OTA, 1991). Commercial bioaugmentation prod-ucts are the result of large-scale fermentation of sin-gle or mixed cultures with desirable oil-degradingcapabilities (Forsyth et al., 1995). It has been welldocumented that hydrocarbon-degrading microorgan-isms are ubiquitous in the environment (Bartha andAtlas, 1977; Cerniglia, 1984; Leahy and Colwell, 1990;Prince, 1993; Bonner et al., 2002). In a previous phaseof research at SJWRF,Townsend et al. (2000)foundexponential number increases of both saturate- andPAH-degrading indigenous microbes subsequent topetroleum application in the wetland. Some bioaug-mentation researchers contend that adding exogenousmicroorganisms to a contaminated site may augmentte ticc hasb h,1 ndA

thec yade ndV n-d lurrys ofe yr ng ab noto iatedw trat-e ing

the transport of viable organisms to the site and the ap-plication of the microbes onto the contaminated areas(Atlas, 1991). Although past performance of bioaug-mentation products in field trials have produced mixedresults (Rosenberg et al., 1992; Venosa et al., 1992;Nadeau et al., 1993), few of these studies used a ro-bust experimental design with proper replication andrandomization that allow for a more statistical analy-sis of the results. One exception was a well-designedsand beach experiment that introduced an enriched cul-ture of the indigenous oil-degrading bacteria (Venosaet al., 1996). The researchers concluded that the micro-bial treatment did not provide any further enhancementover the nutrient treatment. It should be noted that thisstudy did not utilize a commercial product.

The research reported in the current study focuseson bioaugmentation in a wetland setting, using the SanJacinto Wetland Research Facility (SJWRF). The capa-bilities of two bioaugmentation products and a fertilizertreatment to enhance petroleum biodegradation werestudied, using replication and random interspersion oftreatments. This paper presents the temporal results ofpetroleum concentrations in the sediments, along withsupporting microbial and toxicological results. The re-sults of the nutrient analyses are also presented. Otherresearch efforts at the SJWRF are detailed elsewhere(Mills et al., 1997, 2003; Wood et al., 1997; Harris et al.,1999; Mueller et al., 1999, 2003; Bizzell et al., 1999;Townsend et al., 2000; Page et al., 2002; LaRiviere etal., 2003).

2

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he capabilities of the indigenous populations (Forsytht al., 1995). Enhanced biodegradation of xenobioompounds through the use of bioaugmentationeen documented for many years (Daughton and Hsie977; Edgehill and Finn, 1983; Finn, 1983; Barr aust, 1994).Some bioaugmentation products have shown

apability to degrade petroleum in the lab (Fat al., 1992; Aldrett et al., 1997; Raghaven aivekanandan, 1999) and under some controlled coitions such as wastewater treatment and soil systems (Forsyth et al., 1995). In a comparative setxperiments,Lee et al. (1997)found that laboratoresults indicated enhanced oil biodegradation usiioaugmentation product, but similar results werebtained in the field. There are problems associth implementing a bioaugmentation treatment sgy, especially in a terrestrial environment, includ

. Methods and materials

.1. The San Jacinto Wetland Research FacilitySJWRF)

The SJWRF was established in 1994 followingil spill on the San Jacinto River near Houston Tetails of this 21-plot facility are discussed inHarrist al. (1999)andMills et al. (2003).

.2. Experimental design

A controlled application of petroleum in the wetlaas used to test four treatment strategies for enh

ng bioremediation. The strategies were intrinsic reiation (oiled control), biostimulation with inorgan

M.A. Simon et al. / Ecological Engineering 22 (2004) 263–277 265

fertilizer (diammonium phosphate), and two commer-cial bioaugmentation products. A randomized com-plete block design with repeated measures (as detailedin Venosa et al., 1996) was altered in favor of an unbal-anced, complete block design. This modified designwas incorporated to allow testing of three treatmentsand an oiled control without the need for additional plotstructures. Collaborating with the Texas A&M Univer-sity Statistics Department, data from previous studieswere used to calculate (and post validate) the statisticalpower for the SJWRF. It was determined that there wasadequate replication for the proposed experiment.

Three blocks were established with six plots as-signed per block. Plot elevation and location relativeto the San Jacinto River served as blocking criteria.Treatments were assigned randomly to plots within ablock such that each block would have all the four po-tential treatments, but not necessarily in equal numberswithin the block. Unoiled plots (located within one ofthe blocks) were monitored for physical transport ofthe bulk oil phase, but not included in the evaluationof treatment performance. The location of these un-oiled plots was based on two criteria: (a) relative plotelevation (for more consistent plot inundation due totidal effects) and (b) site history (locations that havenot been oiled during this multi-year project). Each en-hanced treatment was employed in replicates of five,while oiled and unoiled controls were each conductedin triplicate. A schematic of the plot assignments ispresented inFig. 1.

2

usei rudei ulfC umw ath-e ore-l tioni duc-t ann tiesf ianm

lya ray-i n air-

Fig. 1. San Jacinto Wetland Research Facility Site map (Houston,TX, USA) showing experimental plot layout and treatment assign-ments.

less spray gun, a six-foot wand, and a fan-type spraynozzle) using a timed spraying technique. The appli-cation procedure is detailed inTownsend et al. (2000).A controlled application of weathered petroleum mini-mizes heterogeneity typically associated with the depo-sition of spilled petroleum, allowing for a more quan-titative analysis of the data. Prior to oil application,existing plant cover was sprayed with water. A pre-vious field trial demonstrated the water-washed plantstended to repel the oil, allowing the sediments to receivea more even application of petroleum. Moreover, basedon observations of marine wetland oil spills, plants areoften wetted and then oiled by receding tides and sothis exposure is thought to be more realistic.

.3. Application of petroleum

An Arabian medium crude oil was selected forn this phase of the research. Arabian medium cs a common crude oil imported into the Texas Goast region. Artificial weathering of the petroleas conducted to more closely approximate the wered state of spilled oil once it strands on a sh

ine. The petroleum was weathered by recirculan an open atmosphere tank until either a 25% reion in volume or loss of all hydrocarbons lighter thonane (n-C9) was achieved. The physical proper

or the Arabian medium oil and the weathered Arabedium oil are presented inTable 1.Twenty-one liters of weathered oil was uniform

pplied to each assigned plot with a customized spng system (air compressor, pressurized canister, a


Table 1Physical properties of Arabian Medium and weathered Arabian Medium crude oil used in this study

Properties Units Weathered Arabianmedium

Arabian medium Method

Specific gravity 0.9129 0.8724API gravity API degree 23.5 30.7 ASTM D 287Reid vapor pressure kPa 2.1 2.5 ASTM 323

Viscosity@15 CST 102.4 21.45 ASTM D 445@20 CST 80.7 19.12 ASTM D 445

Pour point ◦C −14 −23 ASTM D 97Sulfur content wt.% 2.96 2.58 Dohrmann oxidative-

microcoulometry

2.4. The treatments

The two bioaugmentation products were selectedbased on specific scientific selection criteria, whichconsidered the performance enhancement in a studyconducted in our laboratory (Aldrett et al., 1997). Inthis lab study, 13 products listed on the National Con-tingency Plan (NCP) product schedule (CFR, 1995)were evaluated for effectiveness in the biodegradationof petroleum relative to a seawater control and sea-water with nutrient addition. The study was conductedunder blind testing conditions, where the products wererandomly coded (BP 1 through BP 13). The best per-forming bioaugmentation products (BP 8 and BP 10)were selected for this field study. The numbers of thesetwo products are used here to maintain continuity.

BP 8 is a commercially-available bioremediationagent described as a dry, wheat-bran-based pow-der containing a large consortium of hydrocarbon-degrading bacteria. It was applied five times during theexperiment (Days 4, 7, 11, 18, 28). Vendor-suppliednutrients were applied with a broadcast spreader priorto each application. BP 10 is also a commercially-available bioremediation agent and is a dry, wheat-bran-based (plus non-ionic surfactant) powder contain-ing large numbers of oil-degrading microorganisms.BP 10 was applied twice (Days 4 and 28). The appli-cation schedules were based on manufacturer recom-mendations. Plate counts of these products revealed thepresence of several bacterial genera includingAcine-t yb al-y onu

The vendor-recommended application methodswere modified to minimize differences between treat-ments and to provide more experimental control. Bothbioproducts were applied in slurry form. For each plot,the appropriate amount of dry product (as per manufac-turer instructions) was mixed overnight in 15 liters ofdistilled water buffered with 0.1% disodium pyrophos-phate. Prior to application, the slurry was transferred toa 20 liters container equipped with a submersible pumpto suspend the slurry. A siphon sprayer connected to anair compressor was used to apply the product to eachplot. The most-probable-number (MPN) analyses ofthe product suspensions before and during applicationrevealed no loss of microorganisms due to the applica-tion procedure.

The biostimulation treatment provided nutri-ents where lack of availability would otherwiselimit petroleum biodegradation. Commercial-grade di-ammonium phosphate ((NH3)2HPO4) fertilizer wasapplied by broadcast spreading of granular pellets fromthe elevated scaffolds. The application days were 4, 11,28, 42, 56, 70, 84, and 126. This treatment was utilizedin a previous phase of research at SJWRF (Mills et al.,1997; Mills, 1997). Prior research at the site establisheda target level of 40 mg available N/kg dry soil.

2.5. Sampling strategy

Using a 5 cm-diameter coring device, sedimentsamples (0–5 cm depth) were collected on the fol-l 8,4 ap-p resw hen

obacter,Pseudomonas, andBacillusas indentured both fatty acid analysis (FA/MIS) and BIOLOG ansis. Product viability was verified prior to applicatising the most-probable-number (MPN) method.

owing days: −14 (baseline), 4, 7, 11, 18, 22, 56, 70, 84, 105, 126, and 152 (where oillication was Day 0). For each plot, three coere extruded (randomized sampling grid) and t


homogenized in pre-cleaned glass jars, stored onice, and transported to the laboratory for analy-sis. More details of the standard sampling proce-dures at SJWRF are outlined elsewhere (Mills, 1997;Harris et al., 1999; Townsend et al., 2000).

2.6. Petroleum analysis

Aliquots (65 g) of the sediment samples were frozenat −20◦C, freeze dried, ground, and homogenized.The dried samples were extracted by pressurized fluidextraction with dichloromethane (DCM) using anAccelerated Solvent Extractor (Dionex Corporation,Sunnyvale, CA) with conditions developed previously(Bauguss, 1997). The DCM extract was concentratedto 1 ml by evaporative concentration with a TurboVapII Concentration Workstation (Zymark Corporation,Hopkinton, MA) and then reconstituted to a finalvolume of 5 ml. A 1�l aliquot of the DCM extractwas injected into a Hewlett-Packard (HP) 5890 SeriesII gas chromatograph (GC) interfaced to a HP 5972mass selective detector (MS) and operated usingHP MS Chemstation software (Hewlett-PackardCorporation, Palo Alto, CA). The petroleum analysismethod yields data on 28 saturated hydrocarbon and37 polycyclic aromatic hydrocarbon (PAH) analytes.The saturated analytes weren-decane (C10) throughn-pentatriacontane (C35), while the PAH analyteswere two-ring through five-ring PAHs including naph-thalene, phenanthrene, fluorene and akyl-substituteda pa-r ures,e

2a

de-fi ep theb ,t eitya ath-e nantp egra-d(

where (A/H) is the hopane-normalized concentrationof the analyte at timet (day) and (A/H)0 is the ini-tial hopane-normalized concentration of the analyte.The first-order biodegradation rate coefficient is repre-sented byk (day−1).

Non-linear regression analysis using JMP StatisticalDiscovery Software (SAS Institute Inc., Cary, NC) wasperformed to obtain the first-order biodegradation ratecoefficient for each analyte. The rate coefficients forthe treatments were compared to the oiled control forstatistically significant differences. A significant differ-ence was determined if the 95% confidence intervalsfor the treatments were exclusive of the 95% confi-dence intervals for the oiled control. A more completeexplanation of the model is available inVenosa et al.(1996).

2.8. Nutrients

Nutrient concentrations were determined by themethods ofHarris et al. (1999). The analysis was com-pleted by flow injection analysis using an autoana-lyzer (Quikchem 8000, Lachat Instruments, Milwau-kee, WI). Nitrate and ammonium were extracted si-multaneously with a potassium chloride solution, whileavailable phosphorus was extracted with a Bray solu-tion (an ammonium fluoride hydrochloric acid solu-tion). The reported nitrate value includes nitrate andnitrite species, the reported ammonium value includesammonium and ammonia species, and the reporteda atesa cal-c hos-p ahlp d di-g

2

asc ble-n ed toae d eta wasu dingp n-t was

nalogs. The complete list of analytes, operatingameters of the GC–MS, standards, QAQC procedtc. are detailed inMills et al. (1999).

.7. First-order biodegradation model and datanalysis

A first-order biodegradation model, previouslyned byVenosa et al. (1996), was used to interpret thetroleum data. All analytes were normalized toioconservative compound 17�(H) 21�(H)-hopane

hus reducing the influence of sample heterogennd any physical losses of petroleum. For this wered oil, flushing was presumed to be the domihysical loss mechanism. The losses due to biodation can be described as follows:

A

H

)=

(A

H

)0e−kt (1)

vailable phosphorus value includes orthophosphnd easily acid-soluble forms of phosphate (i.e.ium phosphates, aluminum phosphates, and iron phates). The total Kjeldahl nitrogen and total Kjeldhosphorus were determined in one extraction anestion procedure.

.9. Microbial populations analysis

Enumeration of the microbial population wompleted using hydrocarbon-specific most-probaumber protocols. The MPN procedure was adapt96-well plate format as described previously (Hainest al., 1996; Wrenn and Venosa, 1996; Townsenl., 2000). For hydrocarbon sources, hexadecanesed to quantify the saturate-hydrocarbon-degraopulation, while a mix of four PAHs (phena

hrene, anthracene, fluorene, dibenzothiophene)


used for the aromatic degraders. After incubation, theplates were scored and the most-probable-number ofmicroorganisms per milliliter was calculated usingthe Most-Probable-Number Calculator (Version 4.00)computer program (Klee, 1993). An analysis of vari-ance (ANOVA) procedure was used to assess treatmentdifferences (P < 0.05) on individual sample days.

2.10. Toxicity analysis

2.10.1. Microtox® 100% TestAcute toxicity was evaluated by performing the

Microtox® 100% Test (Model 500 Analyzer, Azur En-vironmental, Carlsbad, CA) on the water-extractablephase (or elutriate) of the sediment samples. Elutriatepreparation was conducted as detailed byMueller etal. (1999). Wet sediment (7 g) was vigorously stirredin Microtox® diluent (35 ml) and then centrifuged toseparate the aqueous phase (elutriate) from the solidsediment. An aliquot of the elutriate was then tested ac-cording to the Microtox® 100% Test protocol, whichinvolves inoculation of the sample concentration testseries with a bacterial reagent (Vibrio fisheri) and mea-suring the bioluminescence after a 5 min exposure. Theconcentration calculated to effect a 20% reduction inbioluminescence was reported as the EC20. Sampleswith non-detectable toxicity were assigned an EC20value of 99%. Details of the procedure are outlined inthe operator’s manual (Microbics Corp, 1992). Eachsample was tested in duplicate. For this study, toxicityv erebyr ingi eent ue.

2s

w y bothE de-sf est-e erep literb gan-i tests ater( ys-t test

beakers to the 850 ml mark. The beakers were placedin a water bath (15◦C) and received controlled aer-ation. The following day, acclimated test organismswere sifted from the control sediments using a 1 mmsieve and counted. Twenty organisms were randomlyadded to their respective test chamber according to apredetermined random order list. After 10 days, the testorganisms were sieved from the sediments. Both deadand live organisms were counted and recorded. Mortal-ity data was determined by subtracting the number ofrecorded live organisms from the number of organismsinitially exposed to the test sediments. The bioassaywas conducted on three sample sets (Days 4, 7, 28).

3. Results

3.1. Observations

Prior to oil application, the plots received frequentinundation because of an elevated river stage and 7 cmof rainfall over the 3 days preceding oil application.Application of the petroleum occurred during low tideand additional rainfall occurred within 12 h. Completecoverage of the plots with water occurred within 24 hafter oil application and continued for an additional30 h. Free phase oil was not observed floating in thewater, although a sheen was noted in the areas imme-diately surrounding the plots and almost continuouslyalong the western shoreline. Floating debris contami-n loy-m entb leumf tiona ut thec

3

val-u s as-s ntlys m-p sicall Ara-bo , ac ed

alues are presented as the inverse of the EC20, theflecting an increase in toxicity with a correspondncrease in EC value. Statistical differences betwreatments were calculated with the ANOVA techniq

.10.2. Amphipod bioassayThe burrowing amphipodEohaustaurius estuariu

as used as the test species as recommended bnvironment Canada and USEPA. Test guidelinescribed inEnvironment Canada (1992)were followedor all tests. Animals were purchased from Northwrn Aquatic Sciences (Newport, OR). All tests werformed under static conditions for 10 days in 1eakers. One day prior to exposing the test or

sms to the sediments, approximately 125 ml ofediment was added to each beaker. Control wsalinity-matched using Instant Ocean, Aquarium Sems, Mentor OH) was then gently added to the

ated with petroleum was floating in the water. Depent of two sorbent booms and a skirted containmoom at the mouth of the cove prevented any petro

rom entering the river channel. Some contaminand debris were observed on the sorbent booms, bontainment boom was free of contamination.

.2. Physical loss of petroleum

Physical loss of petroleum was determined by eating temporal hopane concentrations. Hopane iumed to biologically degrade at a rate significalower than other analytes of interest. Also, all oil coonents are assumed to experience similar phy

osses. The hopane concentration in the weatheredian medium crude oil was determined to be 260�g/g-il. Based on an application of 21 l of oil per plotoncentration of 5.2�g hopane/g dry soil was expect


in the top 5 cm of soil. The average measured back-ground hopane concentration for all oiled plots wasdetermined to be 1.77± 0.89�g/g dry soil while theaverage hopane concentration observed 4 days after oilapplication for all oiled plots was 6.96± 2.51�g/g drysoil. These values indicate close agreement betweenpredicted concentrations (based on hopane concentra-tions in the oil) and measured hopane concentrationsin the plots. Using a first-order model, a rate coeffi-cient was calculated for the hopane data for all oiledplots and was assumed to represent physical losses ofhopane. The results of this analysis indicate that physi-cal losses accounted for removal of approximately 30%of the bulk petroleum from the oiled plots over the du-ration of the study.

3.3. Total target analytes

The 28 resolved saturate hydrocarbon analytes weresummed and the resulting value (TTsat, total target sat-urates) was normalized to hopane. Similarly, the 37 re-solved aromatic hydrocarbon analytes were summedand the resulting value (TTaro, total target aromatics)was normalized to hopane. The temporal values forTTsat and TTaro are presented inFig. 2. Each datapoint represents the mean of the replicates for that treat-ment and the errors bars indicate± one standard de-viation. The modeling results for TTsat and Ttaro arealso presented inFig. 2, including the initial concen-trations,(A/H)0, the biodegradation rate coefficients,k,ab fer-e tivet

3

izedfi edo ent.T y oft anyo im-i nos rel-a erno ho-m uted

homologues indicated decreasing first-order biodegra-dation rate constants with increasing molecular weightand increasing alkylation of the PAH. This pattern isconsistent with patterns of PAH degradation previouslyreported (Cerniglia, 1992; Venosa et al., 1996; Mills,1997; Mills et al., 2003). This trend was not observedfor the phenanthrene family of compounds; however,low sediment concentrations may have affected theseresults. More details of the PAH results are explainedin Simon et al. (1999).

3.5. Nutrients analysis

Although both nitrogen and phosphorus analyseswere conducted, a qualitative review of the data in-dicated that nitrogen was the more-limiting nutrient.Consequently, only those results are presented. The soilconcentrations for ammonium and nitrate are presentedin Fig. 3(mean± standard deviation). For ammonium(Fig. 3a), the nutrient treatment had statistically higherconcentrations for Days 4, 7, 28, 42 and 56 as comparedto the oiled control (P< 0.05), while the BP 8 treatmentshowed statistically higher ammonium concentrationsfor Days 7 and 14 when compared to the oiled control.For nitrate (Fig. 3b), none of the enhanced treatmentswere statistically different than the oiled control for anygiven sampling event.

3.6. Microbial populations

arept low-i ilarf nu-t ringt weren fora bersf ntlyg

lsoi a-t or-ds oveu them p-

nd the calculated biodegradation half-lives (t1/2). Foroth TTsat and TTaro, there were no significant difnces in the rate coefficients for any treatment rela

o the oiled control.

.4. Target analytes

Non-linear regression using the hopane-normalrst-order biodegradation model was also performn each individual target analyte for each treatmhere were no significant differences between an

he remediation treatments and the oiled control forf the individual saturate hydrocarbon analytes. S

larly for the individual aromatic target analytes,ignificant differences existed between treatmentstive to the oiled control. However, the overall pattf degradation for naphthalene and akyl-substitutedologues and dibenzothiophene and akyl-substit

The results of the microbial population countsresented inFig. 4. For the saturate degraders (Fig. 4a),

here was an exponential increase in numbers folng oil application. Population increases were simor all oiled plots, including those amended withrients or bioaugmentation products. When compahe enhanced treatments to the oiled control, thereo significant differences in population numbersny sampling event except Day 18 (where the num

or the nutrient and BP 8 treatments were significareater than the oiled control).

PAH-degrading populations in all oiled plots ancreased exponentially following the oil applicion (Fig. 4b). These numbers increased threeers of magnitude (from 104 to 107 microbes/g-dryoil) over initial populations and remained well abnoiled control populations for the duration ofonitoring. This dramatic increase following oil a


Fig. 2. Hopane-normalized concentrations vs. time for (a) total target saturates (TTsat) and (b) total target aromatics (TTaro) for each treatment(oiled control, nutrient, bioproduct 8, bioproduct 10).


Fig. 3. Nutrient concentrations for (a) ammonium and (b) nitrate for each treatment (oiled control, nutrient, bioproduct 8, bioproduct 10).

plication illustrates a classical growth response to anintroduced abundant carbon source. Statistical analy-sis revealed no significant differences between PAH-degrading populations in oiled control plots and biore-mediation treatment plots on any individual sampleday. More complete information on the microbial pop-ulation analyses is presented elsewhere (Townsend,1999; Townsend et al., 1999).

3.7. Toxicity

The results of the toxicity analyses are presented inFig. 5. For the Microtox® 100% Test (Fig. 5a, mean

± standard error), some evidence of toxic responsesduring the first week of the experiment was indicated.However, there were no statistical differences at anytime point when the oiled control was compared anyof the three remediation treatments (P < 0.05). Theseresults were not as dramatic as similar analyses in theprevious controlled-spill study conducted at SJWRF(Mueller et al., 2003), where the average toxicity val-ues (1/EC20) for all oil treatments were higher than0.20 for the first time point following the oil applica-tion. Moreover, in the current study, there was no corre-lation between the oil concentrations in the sedimentsand the Microtox® results (r2 < 0.01). This lack of


Fig. 4. Population dynamics of (a) saturate-degrading microorganisms and (b) PAH-degrading microorganisms for each treatment using most-probable-number (MPN). Treatments included oiled control, nutrient, bioproduct 8, bioproduct 10. Oil application occurred on Day 0.

correlation may be due in part to the generally-insoluble nature of most of the petroleum compounds.In addition, the oil chemistry concentrations weredetermined from DCM sediment extracts while theMicrotox® results were determined from aqueous elu-triates. If both the toxicity and chemistry analyses wereconducted on the same elutriate, a correlation betweenthe two may exist.

To further test this hypothesis, elutriates were againprepared from oiled sediment samples by the methodpreviously discussed for the Microtox® 100% Test.

One aliquot was used to conduct the Microtox®

test when another aliquot was extracted with DCMand analyzed by GC–MS. The dominant petroleumconstituents were lightweight PAHs (parent/alkyl-substituted naphthalenes, phenanthrene, and diben-zothiophene). The concentrations of these dominantPAHs observed in elutriate extracts were summed,compared and correlated with Microtox® 100% Testresults (Fig. 5b). This linear regression produced anr2

value of 0.63, demonstrating a much stronger correla-tion between the Microtox® 100% Test toxicity and


Fig. 5. (a) Microtox® 100% Test toxicity results for all oil treatments (oiled control, nutrient, bioproduct 8, bioproduct 10); (b) Microtox®

toxicity (100% Test) vs. polycyclic aromatic hydrocarbon (PAH) concentrations for additional elutriate samples. The elutriates were preparedfrom sediments samples from the experimental study. Toxicity and GC–MS analyses were conducted on the elutriates.Note: EC20 is theconcentration calculated to effect a 20% reduction in bioluminescence.

select PAH concentrations. Total naphthalenes ap-peared to be the greatest contributors to toxicity (r2

= 0.73).The amphipod assay has demonstrated sensitivity to

petroleum hydrocarbons (Mearns et al., 1995; Muelleret al., 2003). The cumulative amphipod mortality foreach treatment is depicted inFig. 6 (mean± standarderror). Contrary to published literature, there were nostatistical differences between the oiled control treat-ment and the unoiled control (P< 0.05). However, whencompared to the unoiled plots, all the enhanced treat-ments were statistically different for at least two time

points (Day 4 for all three enhanced treatments, Day7 for the two bioaugmentation treatments, and Day28 for the fertilizer treatment). The amphipod assayhas demonstrated sensitivity to ammonia, a microbialby-product and potentially-toxic side effect of multiplenutrient additions (Ames et al., 1975; USEPA, 1994).Ammonia has been suspected as a sediment toxicantfor many years; ammonia presence in standard am-phipod sediment tests has been shown to contribute toincreased mortality (USEPA, 1994; Borgmann, 1994;Pinza et al., 1996; Mueller et al., 2003). For this exper-iment, both the nutrients treatment and BP 8 treatment


Fig. 6. Amphipod mortality for all treatments for Days 4, 7, and 28. Treatments are oiled control, nutrient, bioproduct 8, bioproduct 10.

had statistically higher mortality than the oiled con-trol for Day 4. The average ammonium concentrationswere consistently higher for these two treatments ascompared to the other treatments (Fig. 3a). There maybe a causal linkage between ammonium concentrationsand amphipod mortality.

4. Discussion

The extent of biodegradation was quantitativelycompared to a “background” or baseline value for thesite. For all 18 oiled plots, the target analyte data ofthe baseline samples (Day−14) were compared to thesamples taken at experimental end (Day 152). For the“total target saturates”, the baseline value averaged 9± 6�g/g dry sediment, while the baseline value forthe “total target PAHs” was 3± 2�g/g dry sedimentfor the study site. By Day 152, the total target satu-rates averaged 15± 11�g/g dry sediment while thetotal target PAHs were 14± 9�g/g dry sediment. Fromthis perspective, the site conditions were approachingbackground levels by experimental end, regardless oftreatment.

Before further evaluating the efficacy of the bio-products in this wetland, the nutrient treatment wascompared to the same nutrient treatment from a pre-vious SJWRF experiment (Mills et al., 1997; Mills,1997). For the current experiment, the biodegradationrate did not exhibit the same enhancement reported for

the same nutrient treatment in the previous experimentnor in literature (Lee and Levy, 1989; Lee and Levy,1991; Lee et al., 1995; Venosa et al., 1996). Severalfactors may have contributed to the disparity in the re-sults. More rainfall was experienced during this experi-ment, especially early in the study, and several elevatedriver stage events occurred as compared to the previousexperiment. Sometimes, fertilizer application (and oc-casionally bioproduct application) occurred when thesurface of the plots were covered with water. This re-sulted in a dilution (or possible loss) of the treatmentdue to flushing of the system. When comparing nu-trient levels during the first 40 days (when the mostoil biodegradation was occurring), the available phos-phorus levels were similar, but the more-soluble am-monium levels averaged 52 mg-N/kg-dry soil for thisexperiment and 72 mg-N/kg-dry soil for the previousexperiment. Besides lowering nutrient levels, the fre-quent inundation may have also affected the microbialpopulations and their efficiency at degrading petroleumhydrocarbons. It has been established that oil can be de-graded in anaerobic or anoxic conditions, though therate of biodegradation is lower than in aerobic condi-tions (Hambrick et al., 1980; DeLaune et al., 1990).

The biodegradation rates obtained for the bioaug-mentation treatments did not show any significant dif-ferences as compared to the oiled control (Fig. 2), al-though the products demonstrated enhanced perfor-mance in the laboratory flask experiment (Aldrett etal., 1997). Whether the bioaugmentation microbes can


quickly acclimate to conditions at the site is unknown.The ability of exogenous organisms to adapt to the con-ditions present in the field has been cited as a possi-ble limitation for the use of seed organisms, especiallyin soil ecosystems (Atlas, 1977). In this wetland, asthe river stage increases/decreases, periodic inundationof the plots creates an alternating aerobic/anaerobicenvironment.LaRiviere et al. (2004)draws the cor-relation between the constantly-changing river stageheight at the SJWRF and the concomitant redox con-ditions. The redox potential (Eh) would decrease (i.e.,increased reduction) as the stage height rose and inun-dated the sediments. The indigenous microbes at thesite are well adapted to these alternating conditions.Harris et al. (2001)conducted laboratory slurry ex-periments using sediments from SJWRF. They com-pared the oil-degrading capabilities of the indigenousmicrobes under aerobic, FeIII-reducing and nitrate-reducing conditions and discovered that the oil wasbeing degraded in all three scenarios, though at dif-fering rates. The overall lack of significant differencesin microbial population numbers for the bioaugmenta-tion treatments as compared to the oiled control sug-gests that the allochthonous microbes did not augmentthe oil-degrading capabilities of the indigenous consor-tium of microorganisms. This is further supported bythe lack of statistical differences in the biodegradationrates of these treatments as compared to the control.

5

atet tionp umi iledc os-p n ofp iatedw msr thep r oila ureh tud-i mi-ce mt her,

suggesting that there was no additional response fromthose microbes as compared to the indigenous popula-tions. First-order biodegradation rates were also com-pared to evaluate the performance of the treatments.No significant differences in biodegradation rate coef-ficients for total target saturate and total target aromatichydrocarbons were observed between treatments.There was some evidence of higher amphipod mortal-ity for all three bioremediation treatments, but whetherthis is due to the amendments or the oil is uncertain.

Acknowledgements

The authors would like to thank the Texas Gen-eral Land Office for the funding and support providedthroughout the project. A special thanks is extended toour Project Officer, Robin Jamail. The authors wouldalso like to acknowledge the research group, in par-ticular Jason Leik and Frank Stephens. Permissionto release crude oil at the facility for research pur-poses was granted by the Texas Natural Resource Con-servation Commission (Hazardous Waste OperatingPermit #HW-50367-001). At the time of the experi-ment, the site was registered under EPA ID NumberTXR000009332.

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