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Phenanthrene mineralization insoil in the presence of nonionicsurfactantsKauser Jahan a , Tariq Ahmed b & Walter J. Maier ca School of Engineering , Rowan University , 201 Mullica HillRoad, Glassboro, NJ, 08028–1701, USAb School of Environmental Science, Engg. and Policy , DrexelUniversity , Philadelphia, PA, 19104c Department of Civil Engineering , University of Minnesota ,Minneapolis, MN, 55455, USAPublished online: 19 Sep 2008.

To cite this article: Kauser Jahan , Tariq Ahmed & Walter J. Maier (1997) Phenanthrenemineralization in soil in the presence of nonionic surfactants, Toxicological & EnvironmentalChemistry, 64:1-4, 127-143, DOI: 10.1080/02772249709358544

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Toxicological and Environmental Chemistry, Vol. 64, pp. 127-143 © 1997 OPA (Overseas Publishers Association)Reprints available directly from the publisher Amsterdam B.V. Published under licensePhotocopying permitted by license only under the Gordon and Breach Science

Publishers imprint.Printed in India.

PHENANTHRENE MINERALIZATION IN SOILIN THE PRESENCE OF NONIONIC

SURFACTANTS

KAUSER JAHANa, TARIQ AHMEDb and WALTER J. MAIERC

aSchool of Engineering, Rowan University, 201 Mullica Hill Road, Glassboro, NJ

08028-1701 USA; bSchool of Environmental Science, Engg. and Policy, DrexelUniversity, Philadelphia, PA 19104; cDepartment of Civil Engineering, University of

Minnesota, Minneapolis, MN 55455 USA

(Received 12 February 1997; Revised 21 April 1997)

This research addresses the effect of surfactant addition on the microbial degradation of slightlysoluble organic compounds in soil. Biodegradation of phenanthrene, coated on sand with a lowf o c , was studied in the presence of nonionic surfactants. Phenanthrene coated sand was designedto simulate soil contaminated with excess phenanthrene which remains after evaporation ofthe lighter hydrocarbon solvents. A mixed culture acclimated to phenanthrene was used as theinoculum. Four nonionic surfactants were used in this study: Triton X-114, Brij 35, Tween 40 andCorexit 0600. Continuous flow columns were employed to simulate groundwater flow throughaquifers. The addition of Corexit 0600 and Tween 40 surfactants enhanced the biodegradation rateof phenanthrene while there was no enhancement by the other two surfactants. No appreciablelag period for mineralization was observed in these experiments. Additional tests are required toassess surfactant bacteria interactions to determine why certain surfactants perform better thanothers.

Keywords: Biodegradation; phenanthrene; nonionic surfactants; microorganism; soilcontamination; microbial degradation

INTRODUCTION

Many environmental pollutants, such as polynuclear aromatic hydrocarbons

(PAHs), polychlorinated biphenyls (PCBs) and dioxins are characterized by

their low aqueous solubility and hydrophobicity. These properties lead to

sorption of these compounds to the soil matrices and slow migration through

aquifer systems. Thus these compounds are typically found in soil at concen-

trations much higher than in the aqueous phase. Sorption of these compounds

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128 K. JAHAN et al.

onto soil is highly dependent on the soil organic fraction (Karickhoff, 1984).Results from several studies have indicated that the removal of such com-pounds from aquifers is difficult and site remediation based on pump andtreat technologies are lengthy and expensive (Sims, 1994). Cleanup of suchcontaminated sites is costly and current technologies include excavation anddisposal to a hazardous waste facility or excavation followed by incineration.Thus more cost effective insitu treatment methods are sought for the cleanupof soils contaminated with low solubility, hydrophobic compounds.

In recent years, there has been growing interest in the use of surfactants forinsitu aquifer restoration. Surfactants are known to increase the dispersion ofinsoluble hydrocarbons and oils and have been used in the petroleum industryto increase hydrocarbon mobility (Swisher, 1987). Several researchers haveinvestigated the effect of surfactants on the solubilization of hydrocarbons(Ellis et al, 1985; Vigon and Rubin, 1989; Kile and Chiou, 1989; Liu et al,1991). The results indicated that surfactant solutions in the range of 1 to 4%are promising for soil washing. Their presence can also affect the availabilityof insoluble hydrocarbons for microbial degradation.

Surfactants or surface active agents are substances which, when present inlow concentrations, have the property of adsorbing onto surfaces or at inter-faces and of reducing the surface or interfacial energy. When added to water,the surfactant molecule may dissolve as a monomer, adsorb at an interfacewith its hydrophobic end pointing away from the water or aggregate with othermolecules into clusters to form micelles. The first two forms are prominentbelow the sub-critical micelle concentrations (CMC) while the latter dominatesabove the CMC. Surfactants enhance the apparent solubility of a solute by atleast two mechanisms (Rosen, 1989). The first mechanism is dominant belowthe CMC and involves the association of the hydrophobic compound withthe hydrophobic groups of the surfactant molecules. The other mechanism isimportant above the CMC and involves the partitioning of the hydrophobiccompound into the hydrophobic nonpolar centers of the surfactant micelles.

Researchers have indicated that the microbial degradation of PAH com-pounds is technically feasible and has potential as a remedial technology(Gibson, 1984; Weissenfels et al, 1992; Sims, 1994). However, the low aque-ous solubility and high sorptive properties of these compounds result in slowbiodegradation rates. If the rate of biodegradation of PAHs in soil-watersystems could be enhanced, it could be a significant pathway for PAH removal.

Microorganisms are capable of producing their own emulsifying or solubi-lizing agents to enhance degradation of insoluble hydrocarbons (Zhang andMiller, 1992). The increase in apparent solubility in the presence of surfactantshas been reported in literature (Kile and Chiou, 1989; Edwards et al. 1991). A

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PHENANTHRENE MINERALIZATION IN SOIL 129

number of researchers have demonstrated that the increase in solubility leadsto greater substrate availability, thereby enhancing mineralization (Wodzinski,1974). A stimulation of microbial activity of lubricating oil in soil was obser-ved in the presence of a dispersant (Rittman, 1989). Aronstein et al. (1991)have also reported that low concentration of surfactants promoted the miner-alization rate of sorbed aromatic compounds. Laha and Luthy (1991) haveobserved complete inhibition of phenanthrene mineralization in the presenceof selected nonionic surfactants in soil-water systems at concentrations abovethe CMC. However, inhibition was found to be completely reversible afterdiluting the surfactant to a concentration below the CMC values. The diversityof results that have been reported in the literature on the effects of surfactantson biodegradation of hydrocarbons leads to the conclusion that there is a needfor further research in this area.

This research addresses the effect of nonionic surfactants at low concentra-tions on the biodegradation of phenanthrene from a contaminated soil. Phenan-threne coated sand was prepared to simulate the presence of excess phenan-threne in a contaminated soil. It has been known that PAHs are commonlyintroduced into the soil as a solute in a carrier solvent such as creosote.Examination of creosote contaminated soils suggest that the lighter hydrocar-bons evaporate quickly leaving residual PAHs, including phenanthrene as aquasi-solid/high viscosity liquid phase that covers the sand surfaces. Nonionicsurfactants were selected as they are known to have greater hydrocarbon solu-bilizing power, less toxicity to microbes, no adsorption to charged surfacesand low foaming properties than their ionic counterparts. This research eval-uates the effect of selected nonionic surfactants on the biodegradation ofphenanthrene in soil at sub-CMCs. Phenanthrene has a relatively low aqueoussolubility and high sorptive properties. Its physical characteristics are repre-sentative of PAHs and it has been identified as a pollutant at numerous sites.Phenanthrene coated sand was prepared to simulate the excess phase phenan-threne in contaminated soil. Low concentrations of surfactants were usedto investigate whether their presence would promote the mineralization ofphenanthrene without inhibiting microbial activity.

EXPERIMENTAL METHODS

Materials

The model aquifer material used in this study was obtained from theJ. L. Shiely Company, Minnesota, which mines Jordan sandstone near Jordan,Minnesota. This sand was almost pure quartz (% silt = 0, % clay = 0

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130 K. JAHAN«a/.

and % sand = 99.62) characterized by a very low (/o c = 0.01%) organiccarbon content (Chen and Maier, 1992). The sand was washed, oven-driedand sieved and the size fraction used was that passing the U.S. Standard sieve#30 and retained on #100. The sand was sterilized by autoclaving prior toall experiments. Autoclaving was carried out at a temperature of 121 °C and15 psi pressure.

Standard liquid chromatography columns are normally constructed withglass and Teflon end fittings. Lion et al. (1990) have indicated that theseTeflon components interfere with the sorption behavior of hydrophobic PAHsonto low-carbon sorbents and experiments should not be carried out in thepresence of competing hydrophobic materials such as PTFE septa. In thiscontext and due to the low organic carbon content of Jordan sand, an allglass column was assembled. The column (4.8 cm in length) was preparedby fusing two 25 mm filter assemblies (Millipore Corp., Bedford, MA). Thefilters had stainless steel mesh supports for the sand. Stainless steel tubes wereused to connect the column to the sample collection bottle. Thus the columnwas entirely constructed of borosilicate glass with stainless steel fittings. Therewere no Teflon components except two PTFE gaskets beneath the steel meshes.Reagent grade radiolabeled phenanthrene-9-14C with a specific activity of13.1 uCi/mmol (purity > 98%) was obtained from Sigma Chemical Co.,St. Louis, MO. Nonradiolabeled zone-refined phenanthrene was also obtainedfrom the same distributor. A phenanthrene stock solution consisting of a prede-termined mass ratio of radiolabeled to nonlabeled phenanthrene was preparedin HPLC grade methylene chloride (Fisher Scientific Co., Pittsburg, PA).

Four commercial nonionic surfactants were used in this study. Their chem-ical structures and other relevant physical properties are given in Table I. TheTriton X-114, Brij 35 and Tween 40 surfactants were obtained from SigmaChemical Co., St. Louis, MO. The Corexit 0600 surfactant was obtained from

TABLE I Structure, molecular weight and CMC of selected commercial surfactants

Surfactant Structure MW CMC(mg/L)

Triton X-114 Octylphenylethoxylate 536 110C8H17-C6H4-O-(CH2CH2O)nH

n = 7.5Corexit 0600 Blend of Surfactant Esters * 40Tween 40 Monopalmitate Polyoxyethylene 1277 30

SorbitanBrij 35 23 Lauryl Ether 1200 190

C,2H25(OCH2CH2)23OH

* Not available

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PHENANTHRENE MINERALIZATION IN SOIL 131

Exxon Chemical Co., Houston, TX. The surfactants were selected on the basisof their favorable solubility at groundwater temperatures and lack of toxicityto bacteria. They were used at a concentration of 25 mg/L in all experimentswithout further purification.

Acclimated enrichment cultures capable of degrading phenanthrene weredeveloped from contaminated soil. Petroleum contaminated soil samples werecollected from a Bemidji (Minnesota) oil spill site. Enrichment cultures weredeveloped in batch reactors incubated at room temperature (22 ±1° ) withparticulate phenanthrene as the sole carbon source. The culture was acclimatedto phenanthrene through repeated transfers into fresh autoclaved nutrient-buffer media. Nutrient buffer solution was prepared in distilled water similar incomposition to the Watanabe media (Watanabe, 1973). The media consisted of(per liter of distilled water) 0.5 g NaNO3, 0.65 g K2HPO4, 0.17 g KH2PO4,0.1 g MgSO4 • 1H2O, 0.03 g CaCl2 and 0.00375 g FeSO4 • 1H2O. The pHwas maintained at 7.2 ± 0 . 1 .

Sand Preparation

A phenanthrene solution was prepared in methylene chloride and added tosand in a glass beaker. The sand was then continuously stirred with a glassrod to uniformly distribute the phenanthrene within the sand while the methy-lene chloride was evaporated in a vented fume hood. Ten 1.0 g samples ofdried coated sand were tested by extraction with 30 mL of methylene chlo-ride and shaken for 24 hours. The supernatant was analyzed for phenanthreneconcentration by counting the samples on a liquid scintillation counter (ModelLS1801, Beckman Instruments, Inc., Palo Alto, CA). This procedure resultedin 97% recovery of phenanthrene.

Mineralization Tests

The columns were operated in a downfiow mode under saturated flow condi-tions. Phenanthrene was coated on the sand as described above. The coatedsand was packed into the columns by pouring a continuous thin stream withgentle tapping to achieve a uniform packing. A peristaltic pump at a flowrateof 0.125 mL/min was used to pump nutrient buffer with or without surfactants.The buffer feed bottles were continuously sparged with filtered air to main-tain saturation dissolved oxygen concentrations. The effluent dissolved oxygen(DO) and the pH were also monitored. Each column received a total of 997 ugof phenanthrene (labeled and nonlabeled). The activity of 14C phenanthrenein these tests was 0.5 uCi. As indicated above, duplicate mineralization tests

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132 K. JAHANcfa/.

were carried out for each test condition and average values are reported inthe results. Error bars represent either ±1 standard deviation around the meanof triplicate measurements or the ranges of duplicate determinations in allfigures. Samples were collected in glass vials with crimp top caps havingTeflon-lined septa. The septa were covered with aluminum foil to preventlosses to the hydrophobic septa. Sodium azide (0.2%, w/w) was added toprevent any microbial activity in the sample bottles (Chen and Maier, 1992).Sample bottles were acidified with 1 mL concentrated sulfuric acid to lowerthe pH to about 1.09. 14CC>2 released from the mineralization of phenanthrenewas then trapped in an apparatus described below.

Mineralization of 14C-labeled phenanthrene was detected by trapping andanalyzing the liberated 14CC>2 in a CO2 trap. A similar system was devised byMarinucci and Bartha (1979) for monitoring the mineralization of volatile Re-labeled compounds. The apparatus consisted of three 10 mL glass scintillationvials in series. All vials contained 10 mL Permaflour (Packard Instrument Co.,Downer Grove, IL) while the second and third vials also contained 1 mL ofCarbo-Sorb E (Packard Instrument Co., Downer Grove, IL) for absorbing CO2.The first vial was designed to trap any volatilized phenanthrene and the secondand third vials were designed for 14CC>2 collection. Each vial had an inlet (22gauge X 7.5 cm stainless steel needle) and an outlet (22 gauge X 1.27 cmstainless steel needle). All vials were connected in series with stainless steeltubing. After acidification, the air was sucked through the three vials to purgeCO2 from the sampling bottle. The trapping efficiency of the system waschecked by evolving 14CO2 from acidified radiolabeled NaHCO3. An overall99% recovery of CO2 was achieved in 8 minutes of flushing. The scintillationvials were stored overnight in the dark to minimize chemiluminescence. Afterthe 14CC>2 had been purged from the serum bottles, 0.5 mL of each sample wasadded to 10 mL of scintillation cocktail and counted on the liquid scintillationcounter to measure total counts. One mL of the same sample was centrifuged at10,000 rpm on a centrifuge (Eppendorf Model 5415C, Brinkmann Instruments,Inc., Westbury, NY) for 8 minutes. One-half mL supernatant of the centrifugedsample was counted to measure the soluble 14C. The difference in the totaland supernatant counts was ascribed to pellet cell mass associated 14C.

Fifty ml of the harvested phenanthrene acclimated cell suspension waspumped through each column. Cell mass was estimated as protein by themethod of Lowry et al. (1951) on a spectrophotometer (Spectronic 1001,Bausch and Lomb, Rochester, NY). Bovine serum albumin (Sigma Chemicals,St. Louis, MO) was used as the protein standard.

At the end of each experiment the sand was removed from the columnsin three equal portions, representing the top, mid and bottom third of the

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PHENANTHRENE MINERALIZATION IN SOIL 133

column. Each portion was analyzed for residual protein and 14C remaining.For the sand, 0.2 g sand portions in triplicates were heated with 0.5 mL NaOHat 90 CC for 15 minutes in a water bath. One mL of water was added to thesamples and centrifuged at 14,000 rpm in a microcentrifuge for 8 minutes. Thesupernatant was then analyzed for protein by the above mentioned method.Duplicate 1.0 g air dried sand samples from the above mentioned three sandfractions were added to 30 mL of methylene chloride in a crimp top vialand put on a shaker for 24 hours. One-half mL of the supernatant from thevial was added to 10 mL scintillation cocktail for counting. The sand wassubjected to sequential extractions with methylene chloride until the countsfell to background levels.

Analysis for phenanthrene metabolic intermediates was carried out becausethere was visual evidence of orange color at certain stages during the devel-opment of enrichment cultures. Biochemical studies indicate that many ofthe phenanthrene degradation products are hyroxylated aromatic compounds(Evans et al. 1965; Kiyohara and Nagao, 1978; Guerin and Jones, 1988a,b). 1H2NA (l-hydroxy-2-naphthoic acid) which often accumulates in the growthmedium and turns it an orange color has been identified as the major inter-mediate produced during phenanthrene degradation. Thus the column effluentwas analyzed for the presence of phenolic compounds by a modification of theFolin-Ciocalteau reaction by Box (1983). Resorcinol was used as the standardand the absorbance was measured in a spectrophotometer (Bausch and Lomb,Spectronic 1001) at 750 nm.

RESULTS AND DISCUSSION

Washing of Phenanthrene Coated Sand

Mineralization of phenanthrene was monitored by measuring 14CO2 concen-tration in the column effluent as a function of time. Six columns with sandcoated with a mixture of labeled and nonlabeled phenanthrene were tested.The operating conditions are listed in Table II. The first column was dosedwith nutrient buffer without any cells and was designed to measure the extentand rate of phenanthrene washout from the sand by dissolution and flushingalone. The other columns were pretreated with 50 mL of acclimated inoculumwith initial protein concentrations of 275 mg/L. All surfactant solutions werepumped at a concentration of 25 mg/L.

Figure 1 shows the data on phenanthrene washout from the column whichreceived no cells. Effluent phenanthrene concentrations were measured by

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Column #

12 .3456

TABLE II

Phenanthrene(tig)

Runl

994994994994994994

Run2

997997997997997997

Initial

K. JAHAN et at.

parameters of soil

Mass of sand(8)

Runl

18.518.618.518.618.618.5

Run2

18.518.618.518.618.618.5

column

Bulk density(g/mL)

Runl

1.761.761.761.761.761.76

Run2

1.761.771.761.771.771.76

experiments

Pore volume(mL)

3.593.593.593.593.593.59

Nutrient buffersolution

BufferBuffer (Cells)Corexit 0600

Tween 40Triton XI14

Brij35

Column Length: 4.8 cm, Diameter: 1.668 cm, Flowrate: 0.125 mL/min, Temperature: 22±0 .5°C

100

80

Control column (No cells)

Phenanthrene concentration (ug/ml) J

% of initial 14C0.8 |

0.6

3goo3

0.4 |53

0.2 jT

50 100 150Time (hrs)

200 250

FIGURE 1 Phenanthrene concentration and percent 14C in column effluent versus time.

collecting samples every 10 hours and measuring 14C concentration. The dataare presented as percent cumulative phenanthrene and as concentration washedout of the column versus time. The % cumulative phenanthrene indicates thecumulative 14C in the effluent expressed as percent of initial dpm. Fiftynine %of the total counts were recovered from the column effluent in 220 hours whichcorresponds to 460 pore volumes. The figure indicates that there is a fairlyrapid removal of phenanthrene (50%) in the first 130 hours. After 200 hoursthe rate of removal is less than 0.5% per 10 hours whereas the initial rates wereapproximately 6% per 10 hours. There is only an additional 9% removal inthe following 90 hours. This slower removal towards the later stages indicatethat the residual phenanthrene is not readily available for dissolution from

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PHENANTHRENE MINERALIZATION IN SOIL 135

the soil. This may be due to the presence of pore spaces in which thereis restricted contact with the flowing water. Another possible explanation isthat phenanthrene is initially present as a separate phase on the sand as acoating and is thus readily available for dissolution. During the later stages,phenanthrene may become sorbed to the sand and thus not be as readilyavailable for dissolution. The finding that washing to remove slightly solubleorganic chemicals from soils is a very slow process is consistent with otherlaboratory and field studies (Edwards, 1991; Pritchard, 1994). It is recognizedthat desorption may be a much more slower process than adsorption thuscontributing to the slow rate of removal. There was no mineralization in thiscolumn as indicated by the absence of 14CC>2 production.

Phenanthrene Mineralization Tests

All the effluent from the columns were collected for ten hours and analyzed for14C concentration. Each sample was acidified and purged for trapping I4CC"2and then measured for total counts and counts remaining after centrifugation.14CC>2 production is a direct indicator of the extent of mineralization and hencea quantitative descriptor of the rate of biodegradation. Volatilization lossesduring 14CO2 trapping were negligible. The total 14C counts in the columneffluents comprised of undegraded soluble phenanthrene, soluble byproductsand cell mass. The effluent from the columns was thus analyzed to measuresoluble phenanthrene and byproducts and cell mass by measuring the 14Ccounts before (total) and after centrifugation. The difference between the totaland the supernatant counts is the 14C associated with cell mass or byproducts(termed as pellet counts). The total counts in the centrifuged supernatant werea measure of soluble phenanthrene and its byproducts.

The 14CO2 production in columns 2 to 6 are shown as the cumulativemass of 14C versus time in Figure 2. The inoculated control column (#2)received nutrient buffer only whereas columns 3 to 6 were dosed with nutrientbuffer media containing 25 mg/L of surfactants. Biodegradation was enhancedin the presence of the Tween 40 and Corexit 0600 surfactants. There wasno appreciable enhancement nor inhibition in the presence of the other twosurfactants when compared to the inoculated control column. The rapid initialrelease of CO2 indicates that the culture was well acclimated to phenanthrene.The rate decreased after the first 100 hours. Approximately 55% of the initial14C was converted to 14CC>2 in the presence of Tween 40 surfactant in the first130 hours that corresponds to 272 pore volume displacements. Cumulative14CO2 production for Corexit 0600, Triton X-114 and Brij 35 for the sametime interval were 50.5%, 41.7% and 41.6%, respectively, compared to 41.3%

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136 K. JAHAN et al.

ou

I>u

U

I

U

50 100 150Time (hrs)

200 250

FIGURE 2 Percent I 4C evolved as 14CO2 versus time.

100

v 80

60

20

Control (With cells)Tween 40Corexit 0600Triton X-l 14Brij 35Control (No cells)

50 100 150Time (hrs)

200 250

FIGURE 3 Comparison of total I 4C in column effluent in inoculated and uninoculated columns.

conversion in the control column. During the period beyond 130 hours, anadditional 2 to 5% of 14C has been converted to 14CO2.

The 14C content of the column supernatant after purging represents solublephenanthrene and its soluble intermediates and is shown in Figure 3. It is

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PHENANTHRENE MINERALIZATION IN SOIL 137

50

c 4 0u

i| 30

3

6

Control SupernatantControl pelletCorexit 0600 supernatantCorexit 0600 pellet

50 100 150Time (hrs)

200 250

FIGURE 4 Percent 14C in column effluent associated with supernatant and cell pellet.

evident that the concentrations from the inoculated and inoculated/surfactantadded columns are lower than that of uninoculated columns. The effluentconcentration in the presence of the surfactants is slightly higher for Tween40 and Corexit 0600 as compared to the inoculated control.

The total counts in the supernatant and in the cell pellet recovered in thecolumn effluent for the control column and for one of the surfactants (Corexit0600) are presented in Figure 4. All surfactants showed similar trends in thesupernatant and the pellet counts. Higher counts were eluted in the presenceof the Tween 40 and Corexit 0600 surfactants as compared to the controland the other two surfactants. An average of 22% of the total counts wererecovered as soluble dpm in the column effluents, indicating that removal bybiodegradation is higher than loss of solubilized phenanthrene and cell mass.In all five columns, pellet associated counts were considerable during the first20 hours. After 20 hours, pellet associated dpm were relatively small for atotal of 7-9% at the end of 230 hours. The pellets obtained after centrifugationwere also analyzed for protein concentrations.

Analysis of Protein

Accumulated cell mass in the soil at the end of the column tests was measuredas residual protein. Protein measurements yield a measure of total cell mass

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138 K. JAHAN et al.

100

jo1sI

8 0

60

I 40

20

Buffer

Triton XI14

Corexit 0600

Brij35

Tween 40

0.0-1.3 1.3-3.2

Column Depth (cm)

FIGURE 5 Percent protein distribution along column depth.

3.2-4.8

which includes that retained in the column from the initial inoculum as wellas new cell mass produced by biodegradation of coated phenanthrene. Theprotein distribution along the column depth at the end of the experiment isshown in Figure 5. All columns showed similar trends in cell retention, withmore cells being retained in the upper 1.6 cm. Protein concentrations werehigher in the presence of the Tween 40 and Corexit 0600 surfactants. Thesesurfactants also had the highest mineralization rates. The figure indicates thatan average of 58% of the residual protein in the columns was present in thetop 1.6 cm, 29% in the middle and 12.78% in the lower third portions.

14 C Mass Balance

Table III shows the mass balance on the total counts in each column. The totalcounts in each column can be divided as follows:

14C (total) = 14CO2 + 14C(effluent) + 14C(sand)

14C material balances show recovery efficiencies ranging from 91 to 95% withan average of ~93%. The measurements of 14C in the soil phase is subjectto sampling errors; small losses may also be introduced due to volatilization.Comparison of these recoveries indicate that greater mineralization can be

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PHENANTHRENE MINERALIZATION IN SOIL 139

TABLE III Mass Balance on Total I4C Counts

Column Type

Buffer OnlyBuffer (Cells)Triton X-l 14Corexit 0600Brij 35Tween 40

% I 4C as CO2

.—43.89 (1.9)47.45 (1.3)52.98 (2.9)45.89 (1.3)60.21 (1.3)

% 14C Eluted inwater

59.13 (2.7)18.03 (1.5)19.15 (2.0)24.79 (1.7)18.98 (1.2)27.86 (1.5)

% 14C Recoveredfrom sand

36.16 (2.3)33.07 (2.0)25.09 (2.2)14.02 (2.3)27.42 (2.3)5.72 (1.2)

Total %recovered

95.395.091.791.892.393.8

The number in the paranthesis is the standard deviation of the data

achieved in the presence of surfactants. Elution of phenanthrene in the waterphase accounted for 59% in the control compared to 18-27% in the inoculatedcolumns. However, it is interesting to note that there are substantial differencesin the presence of surfactants. Tween 40 and the Corexit 0600 showed highermineralization and elution losses as compared to the other surfactants and theinoculated control. The performance of the surfactants can be rated as:

Tween 40 > Corexit 0600 > Triton X - 114 > Brij 35 > Control

Residual 14C on the sand at the end of the experiments were different,varying from 5.7 to 36.2%. 14C residuals on the sand were measured by extrac-tion of the soil and therefore include residual phenanthrene and cell mass. Itis evident that Tween 40 is more effective followed by Corexit 0600, Triton X114 and Brij 35. The absence of surfactant resulted in much higher residual 14C(33%) which is essentially the same as flushing without biodegradation (36%).

Assessments of the possible sources of lower mass balances indicates thatthe soil measurement are the least reliable primarily due to sampling problems.If the deviations from 100% recovery are assigned to % 14C recovered fromsand, the relative efficiencies of removal still remain unchanged.

During the time course of these experiments there was no DO limitation andthe system was well buffered. The average DO concentration and pH valuesin the column effluent were 8.2 mg/L and 7.2, respectively. The acclimatedculture used in these experiments were obtained from earlier batch biodegrada-tion tests (Jahan, 1992), and, none of the selected surfactants were biodegradedduring the duration (10 days) of the batch experiments. The fact that the mixedculture was preacclimated to phenanthrene and earlier tests did not demonstratethe biodegradation of the surfactants in preference to phenanthrene, supportthe contention that the surfactants were not biodegraded in these experiments.Breuil and Kushner (1980) investigated the effects of Triton X-100 and Brij35 on the bacterial utilization of hexadecane. These surfactants were not used

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for growth but stimulated bacterial growth on hexadecane. Guerin and Jones(1989) used five Tween surfactants to stimulate phenanthrene biodegradation.None of the Tween surfactants were utilized when they were present as thesole carbon source. Laha and Luthy (1991) have also indicated that noneof the surfactants selected (Brij 30, Triton X-100 and Tergitol NP-10) werebiodegraded when used in phenanthrene biodegradation studies.

The effluent samples were analyzed for intermediate concentrationsexpressed as resorcinol equivalents. Intermediate concentrations could notbe detected in the column effluents indicating the absence of metaboliteaccumulation. Guerin and Jones (1988a,b) have indicated that the intermediateconcentrations are insignificant when the phenanthrene concentration is low.These authors reported that at low phenanthrene concentrations, 1H2NAaccumulates to a lesser extent or is utilized concurrently with phenanthrene.

The rationale for testing the effects of low concentration of surfactants toenhance biodegradation of slightly soluble chemicals is based on several ideasthat relate to the physical-chemical-biological behavior of surfactants. It is wellknown that addition of surfactants below their CMC reduces surface tensionof water with some indications that apparent solubility of slightly solubleorganics is also enhanced (Kile and Chiou, 1989). It is not clear whetherthe enhancement represents an increase in molecular solution or a surfactantstabilization of micelles, colloids or particles of the slightly soluble chemical.Enhancement of apparent solubility of slightly soluble organic chemicals athigh surfactant concentrations above the CMC is ascribed to partitioning intosurfactant micelles. Microorganisms may not have access to the hydrocarbonwhen it partition into the micelles (Rosen, 1989). A number of commercialsurfactants have been reported to be inhibitory or toxic to microorganisms athigh concentrations (Whitworth et al., 1973; Thai, 1993). Therefore, surfac-tants were used at a concentration of 25 mg/L which is below the CMC forall surfactants.

Tween 40 and Corexit 0600 enhanced mineralization of phenanthrene whilethe other two neither inhibited nor enhanced mineralization. The enhancedmineralization may be attributed to the greater solubilization of phenanthrenein presence of the surfactants and in terns of their nonpolar contents. Kileand Chiou (1989) have correlated the increased solvency of hydrocarbons tothe inner nonpolar core of the surfactants. Tween 40 has the highest nonpolarcontent among the selected surfactants that could explain its better perfor-mance. The nonpolar content in increasing order for the selected surfactantsis as follows:

Tween 40 > Triton X - 114 > Brij 35

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The molecular structure for Corexit 0600 was unavailable from the manufac-turer.

Solubilizaition of hydrophobic compounds in surfactant solutions has alsobeen suggested to be related to the hydrophile-lipophile balance (HLB) number(Vigon and Rubin, 1989). The HLB number (which can range from 0 to 40)is related to the balance between the hydrphphilic and hydropphobic portionsof a molecule and is indicative of emulsification behavior of an emulsifyingagent (Rosen, 1989). The HLB numbers of the surfactants used in these exper-iments were obtained from the manufacturers and are presented in Table IV.Vigon and Rubin (1989) suggested that HLB values are useful for prelim-inary selection of surfactants for solubilization enhancement. Typically, thelower the HLB number the higher the surfactant solubilization efficiency.However, it is evident from Table IV that the HLB numbers and mineral-ization enhancements do not correlate. Guerin and Jones (1988a,b) also couldnot relate the order of enhancement of phenanthrene biodegradation by variousTween surfactants to their HLB numbes indicating that one single parametercannot be a determining factor in surfactant selection.

Surfactant performance can not be predicted by a single physical or chem-ical property. Most research on surfactant enhanced biodegradation suggestthat physiological as well as physicochemical properties are responsible forenhanced mineralization. This was also concluded by Vigon and Rubin (1989)where they evaluated performance parameters that included surface tensionminimization, CMC, HLB number and the partition coefficient. Another majorfactor that needs to be investigated is the surfactant interaction with cellmembranes. Nonionic surfactants can form complexes with cell membraneproteins which are significant in the transport of materials across the cell wall.Surfactant-protein complex formation occurs at nonionic surfactant concen-trations near or above the CMC (Swisher, 1897). The interaction of thePAH with surfactant monomers, the sorption of the PAH and surfactant tothe soil, and surfactant-bacteria interactions are the most important factorsgoverning decontamination and bioavailability of insoluble hydrocarbons insoils.

TABLE IV Surfactants and theirHLB numbers

Surfactant HLB

Triton X-l 14 12.9Corexit 0600 15.0

"Tween 40 15.6Brij 35 16.9

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CONCLUSIONS

This study indicates that the presence of low concentration of nonionic surfac-tants can increase the mineralization of low aqueous solubility compounds.Phenanthrene was converted to carbon dioxide and cell mass without accumu-lation of intermediates. The surfactants were applied at sub-CMC and neitherinhibition nor any toxic effects were observed. The mixed preacclimatedculture was capable of degrading phenanthrene in the columns without anyappreciable lag period. Additional research is required to address surfactant-bacteria interactions, why certain surfactants yield better results as comparedto others and optimum surfactant dosages.

Acknowledgments

This work was supported by Department of Civil Engineering, Universityof Minnesota, Minneapolis. The authors would like to thank Eric Tam foranalytical help. The results were presented at the 1994 Annual Conference ofthe Water Environment Federation, Chicago, IL.

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