time and cost efficient biodegradation of diesel in a continuous-upflow packed bed biofilm reactor...

Research ArticleReceived: 24 September 2011 Revised: 18 December 2011 Accepted: 21 December 2011 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.3736

Time and cost efficient biodegradation ofdiesel in a continuous-upflow packed bedbiofilm reactor and effect of surfactant GAELEMixtli Campos-Pineda,a,b Karim Acuna-Askar,a∗Jesus Alberto Martinez-Guel,a Marcela Mas-Trevino,a

Rolando Tijerina-Menchaca,a Luz Maria Martinez,b Marcelo Videab

and Roberto Parra-Saldivarc

Abstract

BACKGROUND: Biodegradation of diesel hydrocarbons using bioreactors has been proposed as an alternative for dieselcontaminated sites remediation. To make this alternative feasible, several factors must be optimized or improved: reducinghydraulic retention times (HRT) and applying design methods to enhance the access of the microorganisms to low solubleand recalcitrant compounds like hydrocarbon fuels. In the present work a time and cost efficient continuous-flow packed bedbioreactor at low HRT was designed and evaluated. The effect of non-previously studied anionic surfactant GAELE (glycolic acidethoxylate lauryl ether) was also investigated.

RESULTS: A continuous-upflow packed bed bioreactor (CPR) was built using an inexpensive support made of volcanic andalluvial stones. The biodegradation experiments conducted with a 12-month-old biofilm at a fixed HRT of 0.5 h, recordedremoval of up to 97.9% at a diesel concentration of 1120 mg L−1 with GAELE. A first-order rate constant of 0.10 h−1 wascalculated. Kinetic analysis using Arvin’s model, which introduces mass transfer to the biofilm, showed statistical differencesin the kinetic rate parameters (P < 0.001). Moreover, GAELE significantly increased biodegradability at high concentrations,with BOD5 and COD removals up to 90.8 and 80.7%, respectively. Putative hydrocarbon degrading bacteria responsible for thedegradation under nitrate-reducing conditions were positively identified.

CONCLUSIONS: The continuous-upflow packed bed reactor was capable of high percentage diesel biodegradation at shortHRTs. The use of GAELE increased diesel availability and thus enhanced hydrocarbon removal. Therefore, CPR packed withvolcanic and alluvial stones combined with GAELE showed potential for the remediation of diesel-impacted sites.c© 2012 Society of Chemical Industry

Keywords: biodegradation; diesel; fuels; GAELE; industrial residuals

INTRODUCTIONAutomotive diesel is a complex mixture of volatile and semi-volatile chemical compounds that include linear and branchedhydrocarbons often reported as recalcitrant contaminants of soiland water bodies. Therefore, remediation of diesel-impacted sitesrequires an in-depth assessment of the physical and chemicalproperties of these contaminants as well as characterization ofthe surrounding environment site to be treated. This includesevaluation of the degradation potential of indigenous biologicalconstituents to promote intrinsic remediation on the impactedsite and the implementation of technologies to accelerate cleanupprocesses through exogenous mechanisms involving the use ofbiological reactors. These reactors can operate under aerobicor anaerobic conditions and are widely known to enhance theremoval of contaminants by microbial populations in batchcultures, while growing before reaching the stationary phase,where they become capable of cleaving chemical bonds, andconsequently, modifying chemical structures.

Aerobic treatments for bioremediation of diesel-impacted siteshave been reported by several authors. Arrar et al.1 treated dieselfuel-contaminated soil with a jet-fluidized bed reactor throughthe use of particulate biofilm and obtained biodegradation

∗ Correspondence to: Karim Acuna-Askar, Laboratorios de BiorremediacionAmbiental y Microbiologia Sanitaria, Centro Regional de Control de En-fermedades Infecciosas, Depto. de Microbiologia, Facultad de Medicina,UANL, Madero Pte. y Dr. Aguirre-Pequeno, 64460 Monterrey, N.L., Mexico.E-mail: [email protected]

a Laboratorios de Biorremediacion Ambiental y Microbiologia Sanitaria, CentroRegional de Control de Enfermedades Infecciosas, Depto. de Microbiologia,Facultad de Medicina, UANL, 64460 Monterrey, N.L., Mexico

b Department of Chemistry, Tecnologico de Monterrey, Av. Eugenio Garza Sada2501 Sur, 64849, Monterrey, N.L., Mexico

c Centro del Agua para America Latina y el Caribe, Tecnologico de Monterrey,Av. Eugenio Garza Sada 2501 Sur, 64849, Monterrey, N.L., Mexico

J Chem Technol Biotechnol (2012) www.soci.org c© 2012 Society of Chemical Industry

www.soci.org M Campos-Pineda et al.

percentages up to 84% in 15 days at minimum fluidization. Afteroperating the reactor under different aeration and jet velocities,they concluded that jet velocities increase particle mixing, andthus enhance mass transfer to the biofilm, while aeration increasesabiotic losses. Lohi et al.2 used an aerobic fluidized bed reactor forbiodegrading diesel fuel in wastewater with a biofilm supportedon lava rock particles. The reactor operated at a hydraulicretention time (HRT) of 4 h and achieved 100% removal of dieselfuel hydrocarbon chains ranging from C10 to C20, at influentconcentrations of up to 200 mg L−1. In the case of anaerobictreatments, a less expensive alternative for bioremediation isattainable, although with longer residence times. Boopathy3

conducted a soil column study for diesel fuel biodegradationunder nitrate- and sulphate-reducing conditions where 81% dieselbiodegradation was reported within 310 days. On the other hand,Alvarez-Cuenca et al.4 used an anaerobic fluidized bed reactor forthe treatment of contaminated wastewater. At 300 mg L−1 dieselfuel influent, the biodegradation percentages obtained rangedfrom 86.7–96.5% at HRTs ranging from 6–96 h, respectively.The biomass support consisted of granulated carbon particles,providing high surface area and low diesel adsorption, althoughwith increased operational costs. Inexpensive alternative biomasssupports were explored by Morgan-Sagastume et al.5 for sewagewastewater. They used volcanic scoria as fixed support for anaerobic submerged filter and obtained COD removals of up to 90%at a HRT of 8.3 h. The use of volcanic scoria as biomass supportallowed an optimal surface area while reducing support washoutand avoiding the necessity for fluidization or filter backwash.

In order to increase diesel bioavailability, with a consequentreduction of residence times, surfactants and biosurfactantshave been used to enhance diesel biodegradation in water andsoil. Franzetti et al.6 used sorbitan derivatives (Tween) and alkylpolyethoxylates (Brij) in liquid systems; using a batch reactor theyreported a decrease in residual hydrocarbons from 37% to 36% and9% with the addition of 1 g l−1 of Tween80 and Brij56, respectively.Conversely, in soil column assays, addition of 0.5 and 1.0 g kg−1

of Brij56 and Tween80, respectively, resulted in an increase inthe percentage residual hydrocarbons. The authors attributed thiseffect to Tween80 having higher affinity to soil than for diesel,and to Brij56 lowering bioavailability. Nikakhtari et al.7 studiedthe effect of sodium dodecyl sulfate on diesel biodegradation in abaffled roller bioreactor and found no enhancement. Furthermore,Whang et al.8 explored the use of the biosurfactants surfactin andrhamnolipid on diesel biodegradation in batch reactors with aresidence time of 200 h. They found an increase in biodegradationfrom 40 to 100% with the addition of up to 160 mg L−1 ofrhamnolipid and an increase to 94% when using 40 mg L−1 ofsurfactin. However, an inhibition in biomass growth was observedwhen the addition of surfactin was higher than 40 mg L−1.

In order to contribute to the current need for more timeand cost efficient bioremediation technologies for diesel removalfrom water, a study of diesel biodegradation under nitrate-reducing conditions was conducted in this work using a CPRwith an inexpensive biomass support consisting of volcanic andalluvial stones. This support allowed for low HRTs ranging from3 to 0.5 h and influent diesel fuel concentrations ranging from50–1120 mg L−1. In order to broaden the available informationon inexpensive, non-toxic surfactants used in the biodegradationof diesel, the anionic surfactant glycolic acid ethoxylate laurylether (GAELE), not previously reported, was studied both inbatch and continuous-upflow reactors. For the analysis of dieselbiodegradation, the purgeable hydrocarbon fraction in the range

of C10 to C22 was considered. Furthermore, a first-order biphasicbiofilm model was compared with a kinetic model developedby Arvin9 and used to evaluate both diesel fuel biodegradationkinetics and the effect of GAELE on the kinetic rates. Substratekinetic rates provide helpful information for upscale designof reactors intended to remove biodegradable hydrocarbons.The distribution of 16S DNA-identified Gram-negative bacterialcommunities and biofilm thickness were estimated at all reactorsegment units.

MATERIALS AND METHODSChemicals and culture preparationChemicals, including glycolic acid ethoxylate lauryl ether (GAELE,an anionic surfactant), diesel range organics (DRO) calibrationstandard, and any other chemicals, were purchased from Sigma-Aldrich and were above 98% purity. Diesel was purchased from alocal PEMEX diesel station. Mineral medium I (MMI) was preparedaccording to Acuna-Askar et al.10 and used to maintain the growthof a bacterial consortium fed with diesel. Mineral medium II (MMII)had the following composition (in g L−1): KH2PO4, 2; NH4NO3, 0.5;MgCl2, 0.5; NaC2H3O2, 1.5; NaNO3, 1; glycerol, 0.5; K2HPO4, 3.5; tracenutrient stock solution11 (in mL L−1), 1. MMII was used for batch andcontinuous-flow biofilm experimental assays. The bacterial seedwas originally obtained from a petrochemical refinery wastewatertreatment plant and has been fed with 200 mg L−1 of diesel threetimes a week for 5 years. The enrichment reached 1360 mg L−1 ofvolatile suspended solids (VSS) and was further concentrated to3370 mg L−1. Nitrate reducing conditions were tested by the FlukaNitrate Reduction Test Kit (Sigma-Aldrich Chemie GmbH).

Design and operation of the continuous-flow packed bedreactor (CPR)An upflow glass-column reactor with an inner diameter of 5 cm anda height of 53.34 cm divided by eight sampling points distributedupwardly by seven intervals of 7.62 cm biofilm segment units wasused. This reactor was filled with biofilm support of volcanic andalluvial stones. Individual cost of volcanic and alluvial stone are$0.20/kg and $0.15 kg−1, respectively. A vapor chamber connectedat the upper end allowed an OrboTM(Supelco, Bellefonte, PA)activated carbon tube to monitor volatilization losses in theoff-gas. A peristaltic pump at flow rates of nearly 3.2, 6.5 and20 mL min−1 was used to feed the influent into the reactorat HRTs of 3, 1.5 and 0.5 h, respectively. Diesel concentrationswere monitored at the eight sampling points (as seen in Fig. 1)for each bioassay. The CPR bioassays were evaluated followingbiofilm growth at 6 and 12 months. For the 6-month study,two sets of influent streams were prepared: (a) 1.5 h HRT and3 h HRT with 50 mg L−1 of diesel without GAELE, and (b) 1.5 hHRT with 70 mg L−1 of diesel plus 25 mg L−1 of GAELE, slightlybelow the critical micelle concentration (CMC). For the 12-month,two sets of influent streams were prepared. One set containedthree diesel concentration levels without GAELE, namely: 50 (low),120 (medium) and 600 (high) mg L−1. The other set contained50 mg L−1 GAELE (slightly above the CMC) and the followingthree diesel concentration levels: 80 (low), 500 (medium) and 1120(high) mg L−1. Since addition of GAELE increased the solubility ofmost diesel hydrocarbons the concentration of total diesel wasincreased in those GAELE-containing influent streams. The HRT ofthe 12-month CPR study was fixed at 0.5 h for all influent streams.At the end of the study, the biofilm cell mass was determined asVSS for all seven degradation reactor segment units.

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Figure 1. Schematic diagram of the CPR: (a) influent solution, (b) peristalticpump, (c) check valve, (d) activated carbon tube. Numbers 1–8 representthe sampling points, 1 being the influent and 8 the effluent sampling point.R1-R7 are the reactor segment units between sampling points.

Since two different types of packed bed materials (volcanicand alluvial stones) were used to support bacterial growth persegment unit in the reactor, calculations on the overall biofilmthickness were computed based on the statistical weighted mean.Thus, Lf = XavLv + XaaLa, where Lf is the weighted mean of thebiofilm thickness of any segment unit in the reactor, Lv and La

are the biofilm thickness of the volcanic and alluvial stones persegment unit in the reactor, Xav and Xaa are the partial surface areafractions of volcanic and alluvial stones per segment unit whichwere calculated assuming spherical stones with an average radiusof 0.27 and 0.43 cm for alluvial and volcanic stones, respectively.

Diesel analysisDiesel was analyzed by a Varian 3400 GC/FID following USEPA’smethod 8015B. A PetrocolTM(Supelco, Bellefonte, PA) 100 m ×0.25 mm ID x 0.5 µm film DH fused silica GC capillary column wasused. The GC oven temperature rates allowed the separation ofdiesel peaks and avoidance of chromatogram bump formation.Injector and detector temperatures were kept isothermal at 220and 300 ◦C, respectively. A volume of 5 mL of liquid and vapor(headspace) of either standards or samples was injected intothe purge vessel of a Tekmar (Cincinati, OH) model LSC 2000purge and trap concentrator. Samples were purged at 80 ◦C for10 min. A method’s validation protocol was followed accordingto Bliesner.12 Substrate calibration curve was based on total peakareas of purgeable hydrocarbons from fresh commercial diesel.Two standard calibration curves were plotted for (a) medium-to-high diesel concentrations and (b) low diesel concentrations.The extrapolation of the y-intercept was taken as the MDL of theliquid-phase standard curve and rendered 3.8 mg L−1. Semivolatilehydrocarbons above C22 could not be readily extracted by thesample concentrator system at 80 ◦C. Based on the purgeablefraction of the DRO standard kit, the C10 –C22 hydrocarbon chainrange met the analytical quality control parameters followingUSEPA’s method 8015B

Design of batch bioassaysA batch study was also conducted for comparison purposes.The substrate mineral medium (SMM) of the batch experimentalbioassays was made up by mixing 50 mL of MMII with 10 µL ofdiesel into 122 mL amber-glass crimp-sealed bottles (WheatonScience Products, Millville, NJ). A 2 mL inoculum from theconcentrated bacterial cells was added to SMM to reach

130 mg L−1 VSS. An amount of nearly 30 mg L−1 in the liquidphase was the initial diesel concentration in the batch kineticstudies. Controls having only SMM and a group of three sets ofsamples were evaluated. Set 1 contained SMM and 130 mg L−1 VSSof microbial inoculum. Set 2 contained SMM, 130 mg L−1 VSS ofinoculum and 5.5% sterilized soil (SS). Sterilized soil was preparedby sieving through 3 mm mesh and wrapping 3 g samples inaluminum foil for a 3-cycle sterilization. Set 3 contained SMM,130 mg L−1 VSS of inoculum and 25 mg L−1 GAELE. Samples andcontrols were shaken at 7g at 28 ◦C, using a Lab-line oscillatingincubator shaker (Barnstead International, Dubuque, IA). Dieselmass was monitored in the liquid phase for 288 hours at differenttimes: 0, 24, 48, 72, 144 and 288 h. Diesel mass in the vapor phasewas obtained from the vapor-liquid phase coefficient partitioncurve.

Vapor-liquid phase partition coefficient of dieselThe vapor–liquid phase partition coefficient was obtained basedon diesel mass analysis both in vapor and liquid phases using twostandard sets of eleven 122-mL amber-glass crimp-sealed bottlescontaining known amounts of diesel. A practical method detectionlimit of diesel in the vapor phase (MDLdiesel

V ) was calculatedaccording to [An

V/(AnV + An

L)](Mn/VnV ), where An

V is the totalarea of GC peaks of the 5-mL vapor phase standards, An

L is thetotal area of GC peaks of the 5 mL liquid phase standards, n is anysample of either of the two sets of 11 standards with a knowndiesel mass analyzed for either the vapor or liquid phases, Mn isthe total mass of diesel added to each standard and Vn

V is theheadspace volume. An

V versus AnL was plotted and extrapolation

of the y-intercept was taken as the practical (MDLdieselV ) for mass

balance calculations.

Evaluation of BPF and Arvin’s modelsFor batch and CPR studies, the overall and specific dieselbiodegradation rate constants, K and k, respectively, were obtainedby the biphasic first-order (BPF) model according to Lee et al.14

and Acuna-Askar et al.10. For the 12-month steady-state CPR study,Arvin’s model9 was evaluated based on the influent, Si , andeffluent, Se, concentrations of diesel according to:

Se = Si/(1 + K(A/Q)) = Si/

(1 + kmaxXf

KSLε(A/Q)

)

where A is the cumulative area of biofilm (cm2), and Q is theflow rate (cm3 h−1) and K is the overall reaction rate (cm h−1),which is proportional to the maximum substrate utilization rateconstant (h−1), kmax, the active biomass concentration in thebiofilm (mg L−1), Xf , and the thickness of the biofilm (cm), L. KS

is Monod’s half-saturation constant and ε is the efficiency factor,considered as the ratio of the substrate diffusivities in the biofilmand the bulk liquid.

The rate-limiting diffusion across the biofilm layers wasapproached by portraying diesel as a sole hypothetical structurebased on the concept of the molecular weight of the diesel centroidmolecule reported by Lee et al.15 Thus, the molar volume of diesel16

was computed as 301.2 cm3 mol−1. The diffusion coefficient inthe bulk liquid was calculated, based on the molar volume ofthe diesel centroid, to be 3.54 × 10−6 cm2 s−1.17 The diffusioncoefficient of the diesel centroid in the biofilm was estimated18 tobe 2.83 × 10−6 cm2 s−1.

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Bacterial identification and predominancePetri plates were prepared with bacteriological agar and spikedwith known concentrations of diesel to grow and isolate bacteriapresent in batch and CPR assays. For batch studies, samples weretaken at each monitoring time throughout 288 h. For the CPRstudies, bacterial samples were withdrawn from all seven biofilmsegment units and grown on diesel-spiked agar plates. All plateswere incubated at 35 ◦C for 72 h. All isolates were then distributedinto either API 20NE or API 20E test strips (Marcy L’Etoile, France)and the resulting numerical profiles were interpreted with theAPIWEB database V7.0. DNA samples were extracted from isolatesaccording to Sambrook and Russel19 and submitted to MIDI Labs,Inc. (Newark, DE) for 16S DNA identification. Bacterial populationspresent at each CPR sampling point were grown separately onsoy-tripticase agar (STA) plates. Percentage predominance ratio inthe 12-month steady-state CPR study was obtained by countingthe total number of identified colonies and dividing by the totalnumber of colonies grown on STA plates.

RESULTS AND DISCUSSIONContinuous-flow kinetics after 6 months of biofilm growthBioassays were conducted with a 6-month-old biofilm at HRTsof 3 and 1.5 h without GAELE and at a HRT of 1.5 h with GAELE.Due to limitations concerning the MDL of diesel (3.8 mg L−1), theremoval of diesel was slightly lower (91–92%) for the bioassayswith 50 mg L−1 of diesel at the influent. As shown in Fig. 2(b),the 6 month continuous-flow bioassays showed that the higherbiodegradation rate of C10 –C22 occurred at the HRT of 3 h,whereas the HRT of 1.5 h showed lower biodegradation rate.The aforementioned can be confirmed as shown in Table 1, wherethe 3 h HRT had a first-phase biodegradation kinetic rate 1.8-foldhigher than the 1.5 h HRT kinetic rate run without GAELE. The3 h HRT also had a first-phase kinetic rate 2.5-fold higher thanthe 1.5 h HRT kinetic rate run with GAELE. It can also be noticedthat all second-phase kinetic rates were in the range 5- to 13-foldlower than the first-phase kinetic rate constants. Interestingly,the highest C10 –C22 biodegradation percentage (95.8%) occurredat the lower HRT of 1.5 h with GAELE, although the addition ofGAELE resulted in a first-phase kinetic rate 2.5-fold lower thanthe 3 h HRT kinetics. This confirms that higher biodegradationkinetic rates do not always guarantee higher biodegradationpercentages, because some intermediate metabolites could beproduced throughout the kinetics and slow down or inhibit thetransformation of substrates at any time. Furthermore, the highestHRT had the highest ORP shift due to higher oxygen consumption.

Packing materials may play a significant role on increasingcontact area between microbial populations and substrates in thebulk liquid. Arvin9 used a Plexiglas cylinder with a surface area of0.16 m2 in a reactor volume of 0.95 L for an HRT of 4.8 h, whereaswe used a mixture of alluvial stones and previously characterizedsmall volcanic rocks5 that provided 0.39 m2 in a reactor volumeof 0.55 L for HRTs of 1.5–3 h. The use of glass beads has beenreported20 in the biodegradation of chlorinated hydrocarbonswith HRTs of 24–96 h, yielding kinetic rates 4- to 10-fold lowerthan those reported here.

Continuous-flow kinetics after 12 months of biofilm growthA two-way statistical analysis of variance (ANOVA) was performedto test the effect of surfactant, influent diesel concentration, andthe interaction between surfactant and total diesel concentration

Figure 2. Biodegradation kinetics of the purgeable fraction of diesel in(a) batch throughout 288 h, (b) at 6-month period of biofilm growth withHRTs of 1.5 and 3 h, and (c) at 12-month steady-state maturation period atHRT of 0.5 h.

on BPF and Arvin’s kinetic models using MINITAB 15. Statisticaltests were analyzed on BPF and Arvin’s kinetic rates of C10 –C22,C10 –C18 and C20 –C22 hydrocarbon chain ranges. For the BPFoverall kinetic rate of C10 –C22, only influent diesel concentrationwas statistically significant (P < 0.025) with the highest valuerecorded at the medium-range diesel concentration (Table 2).Likewise, for the kinetic rates of C10 –C18, significant factorswere the interaction (P < 0.002) and surfactant concentration(P < 0.007) with the largest shift on kinetic rates occurring alsoat the medium-range diesel concentration. As for the kinetic ratesof C20 –C22, significant factors were influent diesel concentration(P < 0.002) and surfactant (P < 0.03) with the highest fluctuationsoccurring at the medium-range diesel concentration as well.

For Arvin’s overall kinetic rate of C10 –C22, the most significantfactor was the enhancing effect of the surfactant (P < 0.001),followed by the interaction (P < 0.01), at all influent dieselconcentration ranges. For the kinetic rates of C10 –C18 and



Table 1. Diesel removal conditions in the biofilm reactor after 6 months of biofilm growth for various HRTs

Substrate Diesel (50 mg L−1) Diesel (50 mg L−1) Diesel (70 mg L−1) + GAELE

HRT (h) 3 1.5 1.5

DO (mg L−1), Mean, (S.D.)

Influent 6.2 (0.15) 6.5 (0.2) 6.6 (0.25)

Effluent 1.1 (0.04) 1.7 (0.06) 1.9 (0.06)

ORP (mV), Mean, (S.D.)

Eh0 142 (2.5) 137 (4.0) 46 (3.5)

Ehf −56.3 (5.3) −22.2 (9.2) 7.1 (6.2)

pH

Influent 7.4 7.5 7.1

Effluent 7.9 7.8 7.2

Volatilization (%) <3.3 <3.3 <3.3

Biodegradation (%) 91.9 91.4 95.8

Overall rate constants (K)

First-phase k1 (h−1), (R2) 0.412 (0.997) 0.224 (0.989) 0.175 (0.978)

Second-phase k2 (h−1), (R2) 0.030 (0.997) 0.018 (0.989) 0.036 (0.978)

C20 –C22, the effects of surfactant, influent diesel concentrationand the interaction were significant (P < 0.001). Kinetic ratesof C10 –C18 and C20 –C22 with and without GAELE fluctuatedstrongly depending on influent diesel concentration. However,statistical data on both BPF and Arvin’s models confirmed thatthe kinetic rates of C20 –C22 became restrained at the high-range diesel concentration when compared with medium- andlow-range diesel concentrations. A possible explanation is thatlonger-length hydrocarbon molecules may exert inhibition at thehigh-range diesel concentration.

Statistical analysis for Arvin’s maximum utilization rate ofC10 –C22 showed GAELE had a significant enhancing effect(P < 0.001). As for Arvin’s maximum utilization rates of C10 –C18

and C20 –C22, the effects of surfactant, influent diesel concen-tration and the interaction were also significant (p < 0.001 andP < 0.005). Maximum utilization rates of C10 –C18 without GAELEshowed the highest rate occurring at the high-range dieselconcentration among all concentration ranges. This could be ex-plained as if microbial substrate uptake may have not yet reachedthe saturation point among all diesel concentrations and thereforemicroorganisms can continue metabolizing larger amounts ofC10 –C18 substrates. In contrast, maximum utilization rates ofC20 –C22 without GAELE may have promptly reached a saturationpoint at the medium-range diesel concentration, suggesting thatany further concentration increase would not bring forth a highermaximum utilization rate. Based on these findings, it could beassumed that the concentration of GAELE on the bioavailability ef-fect is different on C10 –C18 compared with C20 –C22. Nevertheless,a larger set of diesel concentration ranges with different concen-trations of GAELE would be required to prove this assumption.

The addition of GAELE helped increase the maximum utilizationrates of C10 –C22 at all three concentration ranges tested(Fig. 3(a)). This confirms that as the solubility increased, so did thebioavailability and thus, higher maximum utilization rates wereobtained. The addition of GAELE on the maximum utilization ratesof C10 –C18 (Fig. 3(b)) showed the highest positive impact on thehigh-range diesel concentration, followed by the medium- andlow-range diesel concentrations. The steady-state of the maximumutilization rate being reached by the addition of GAELE to thehigh-range diesel concentration is also clearly noticeable. This

suggests that microbial substrate uptake had the potentialto increase between both medium- and low-range dieselconcentrations following the addition of surfactant. In contrast,the addition of GAELE on the maximum utilization rates ofC20 –C22 (Fig. 3(c)) recorded the highest positive impact on thelow-range diesel concentration, followed by a poor increase in themedium- and high-range diesel concentrations. This may lead toassume that longer-length hydrocarbons would reach saturationpoints at concentrations lower than short-length chains and thus,potentially impacting more significantly on substrate uptake.

Fluidized bed reactors can achieve relatively high removal effi-ciencies in the biodegradation of diesel hydrocarbons. However,massive biofilm washout would be a concern if high flow rateswere applied. In a study, using an anaerobic fluidized bed reactorfor wastewater treatment, where no kinetic rates were reported,biodegradation efficiencies of diesel up to 70–96.5% with influ-ent concentrations varying from 100–300 mg L−1 achieved CODremovals of 61.9–84.1%.4 That study involved HRTs of 6–96 h,which proved to be longer than those HRTs utilized in our study.Another jet-fluidized bed reactor study for the aerobic treatmentof diesel-contaminated soils (4%) obtained first-order kinetic ratesin the range 0.10–0.22 day−1 with biodegradation efficiencies upto 84% following 15 days of treatment.1

One study using continuous-flow systems for the bioremedia-tion of diesel-contaminated soil with removal efficiencies of upto 80% has reported a maximum substrate utilization rate k of0.113 day−1, at a solids retention time of 97 days.13 Although ourstudy reached higher maximum utilization rates, it is well knownthat the treatment of soil involves different complexities otherthan those found in aqueous samples. Other study designs, whereno kinetic rates were reported, achieved up to 81% biodegrada-tion of diesel, as a primary substrate, on the bioremediation of soilcontaminated with 550 mg kg−1 following 310 days of treatment.3

The aerobic batch biodegradation of diesel in aqueous samplesfor specific n-alkanes (C14, C18 and C25) previously eluted frompolarity-based open column chromatography revealed thehighest biodegradation efficiency up to 65% for n-alkanes in therange C14–C18 in 8 days.21 In that study, the authors reporteddecreasing first-order kinetic rates as the molecular weight of spe-cific n-alkanes increased, i.e. k = 0.173 d−1 (n-C14), k = 0.144 d−1



Table 2. Diesel removal conditions in the biofilm reactor at 12-month steady-state maturation period at a fixed HRT of 0.5 h

Low-range Medium-range High-range

SubstrateDiesel

(50 mg L−1)Diesel

(80 mg L−1) + GAELEDiesel

(120 mg L−1)Diesel



(1120 mg L−1) + GAELE

DO (mg L−1, Mean, (S.D.)

Influent 6.2 (0.15) 6.5 (0.2) 6.6 (0.25) 6.2 (0.2) 6.1 (0.4) 6.1 (0.2)

Effluent 0.75 (0.12) 1.19 (0.25) 0.74 (0.15) 0.61 (0.4) 0.98 (0.2) 0.70 (0.14)

ORP (mV), Mean, (S.D.)

Eh0 105.7 (2.5) 103.2 (3.7) 56.4 (6.2) 112.6 (3.5) 74.7 (4.5) 85.3 (5.9)

Ehf −15.2 (5.3) −11.3 (3.4) −42.1 (9.0) −52.3 (8.4) −13.2(3.2) −67.3(5.6)

pH

Influent 7.4 7.5 7.1 7.3 7.3 7.5

Effluent 7.7 7.6 7.7 7.6 6.8 7.5

Biodegradation (%) 92.4 95.3 96.8 99.0 98.4 97.9

Overall BPF rate (K) 0.131 0.123 0.132 0.114 0.104 0.103

(h−1), (R2) (0.998) (0.989) (0.996) (0.994) (0.941) (0.997)

C10 –C18 BPF rate (K) 0.196 0.192 0.118 0.254 0.219 0.193

(h−1), (R2) (0.953) (0.974) (0.922) (0.989) (0.984) (0.996)

C20 –C22 BPF rate (K) 0.138 0.138 0.169 0.136 0.111 0.093

(h−1), (R2) (0.969) (0.986) (0.996) (0.987) (0.955) (0.866)

Overall Arvin’s rate K , 9.13 20.67 8.35 18.88 12.01 17.60

(R2) (0.948) (0.898) (0.932) (0.913) (0.955) (0.920)

C10 –C18 Arvin’s rate 7.37 8.82 3.53 11.67 9.38 24.00

K , (R2) (0.869) (0.950) (0.959) (0.970) (0.937) (0.820)

C20 –C22 Arvin’s rate 2.58 24.706 17.73 31.87 17.82 21.197

K , (R2) (0.796) (0.870) (0.932) (0.913) (0.963) (0.886)

Arvin’s Maximum 1.47 3.53 1.19 3.36 1.68 3.42

utilization rate kmax(R2)

(0.970) (0.950) (0.943) (0.948) (0.993) (0.997)

C10 –C18 Arvin’s 0.77 1.40 0.61 1.13 1.21 2.73

maximumutilization rate kmax

(R2)

(0.962) (0.960) (0.962) (0.934) (0.849) (0.845)

C20 –C22 Arvin’s 0.23 8.31 3.43 5.10 2.04 3.84

maximumutilization ratekmax (R2)

(0.959) (0.933) (0.969) (0.910) (0.964) (0.996)

GAELE concentration in the influent 50 mg L−1.

(n-C18), and k = 0.095 d−1 (n-C25), whereas the aromatic elutedfraction was more resistant over 165 days of incubation. In contrast,we found that in continuous flow assays, at the influent concentra-tion of 1120 mg L−1 of diesel, homologous series of paraffin chainsC10 –C18 and C20 –C22 can be degraded in the range 97–98% withkinetic rates 20- to 30-fold higher at an HRT of 0.5 h (Fig. 2(c)).

In column studies, short HRTs can be met at high flow rates.Therefore, it is necessary to combine high-surface area packingmaterials with balanced growth of effective biofilm layers toprevent massive detachment by erosion or sloughing, as usedin our study. As is known, low-energy demand is among thekey factors of using facultative over aerobic treatments andtherefore, our study offers some perspective for potential pilot-scale remediation design.

Estimation of volatilization lossesContinuous-flow reactors usually involve an open system design;consequently, mass balances for substrate mass volatilizationshould not be ignored, mainly because volatilization losses occur

at the influent and throughout the biological reactor. Based onthe solubility of diesel in water,22 0.2–5 mg L−1, the molar fractionof diesel at the saturation point would range from 1.54 × 10−8 to3.86 × 10−7. In this paper, the influent molar fractions of dieselranged 2.46 × 10−5 to 1.96 × 10−4 for the continuous-flow assays.Despite these molar fractions being higher than the saturationsolubility, dilute solution conditions may have been enhancedby both vigorous mixing in the influent and reduced surfacetension. Figure 4 shows the profile of the vapor pressure releasedat all diesel concentrations with and without surfactant by usingtheoretical calculations based on the Henry’s law constant of dieselfor dilute solutions.

Microbial characterization of biofilm segmentsPredominant communities forming the biofilm were characterizedin seven biofilm segment units (Table 3). Biofilm thickness basedon cell mass VSS, revealed that the first reactor biofilm segmentunit (R1), located between the influent and the second samplingpoint, recorded biofilm 1.4-fold thinner than R2. One explanation



Figure 3. Maximum utilization rates of (a) purgeable fraction of wholerange of hydrocarbons of diesel, (b) purgeable fraction of C10 –C18 hydro-carbon chain range, and (c) purgeable fraction of C20 –C22 hydrocarbonchain range.

is that R1 received the highest diesel concentration and thusthe highest toxicity, causing inhibitory growth. Biofilm thicknessdecreased by 2.3-fold from R2 to R7, inclusive, and all biofilmsegments became thinner as the cell mass decreased by 4.3-foldthrough the upper levels of the reactor. This suggests that, as dieselcompounds became degraded as secondary substrates, loweramounts were bioavailable through upper biofilm communities.

Figure 4. Partial vapor pressures of the purgeable fraction of dieselresulting from substrate concentrations at biofilm reactor sampling pointsaccording to Henry’s law constant of diesel in dilute solution at 12-monthof biofilm growth.

This is also proven for primary substrates. Rittmann and McCarty23

showed that as the concentration of acetate, supplied asprimary substrate, decreased along the length of the column,so did the biofilm thickness. Interestingly, only two bacterialspecies at segment R1 made up 83% of the total population,whereas at R2 they made up 28%. It could be possible that,as the higher diesel loads enter the reactor, the more resistantpopulations predominate. In contrast, as diesel components breakdown into smaller chains, the biofilm diversifies the quality ofbacterial species to mineralize hydrocarbons. From R3 to R7,inclusive, predominant species ranged from 50–80%, indicatingthat at all segments, hydrocarbon-degrading populations showedconsistent significant presence.

Among the bacteria identified in this research, the nitrate-reducing Citrobacter sp. and Stenotrophomonas sp. have beenreported to degrade diesel in a microbial fuel cell,24 whereasthe strain ZP2 of Pseudomonas stutzeri has been shown todegrade phenanthrene in an oil refinery waste.25 Moreover,Kim and Jaffe26 reported the capacity of facultative Ralstoniapicketii PKO1 to degrade toluene, whereas Juhas et al.27 showedthe ability of Pseudomonas aeruginosa and Burkholderia cepaciato form mixed biofilms and hold intercellular communication.Furthermore, the strain AR-46 of Acinetobacter haemolyticus hasbeen reported to degrade n-hexadecane28 and Achromobacterxylosoxidans was able to grow on crude oil under oxygen-limitingconditions.29

Influent and effluent BOD and CODIn Table 4 can be seen that as diesel concentration increased 12-fold in the influent, where no GAELE was added, BOD5 and CODpercentage removals decreased 1.3- and 2.2-fold, respectively. Incontrast, in those streams where GAELE was added and dieselconcentrations increased by 14-fold in the influent, BOD5 andCOD removals decreased by 1.1- and 1.2-fold, respectively. Themajor change observed in the decrease of COD removal in thepresence versus absence of GAELE proves the effect of GAELEon increasing the bioavailability of high diesel concentrations.Biofilm development provided efficient removals of organic matterconcentrations within the COD load range previously reported.5



Table 3. Microbial predominance per segment unit in the biofilm reactor

Biofilm reactor segment unit Identified bacteria Predominance (%) Weighted mean of biofilm thickness (cm) Cell mass (g VSS)

R1 Pseudomonas stutzeri 83 0.078 4.22

Pseudomonas aeruginosa


Pseudomonas aeruginosa

R3 Burkholderia cepacia 50 0.081 4.42

Stenotrophomonas spp.

R4 Pseudomonas aeruginosa 80 0.076 4.01

Achromobacter xylosoxidans

Citrobacter freundii

Acinetobacter haemolyticus

R5 Pseudomonas aeruginosa 60 0.072 3.71

Achromobacter xylosoxidans

Acinetobacter haemolyticus


Burkholderia cepacia

Escherichia coli 1

R7 Achromobacter xylosoxidans 80 0.047 2.42

Burkholderia cepacia

Table 4. Effect of GAELE on BOD5 and COD removals in CPR kinetic assays at 12-month biofilm maturation period at a fixed HRT of 0.5 h

RemovalsDiesel

(50 mg L−1)Diesel





(1120 mg L−1) + GAELE

% BOD5 96.1 89.4 73.1 98.1 92.3 90.8

% COD 92.9 82.9 41.6 94.7 85.6 80.7

GAELE concentration in the influent: 50 mg L−1.

For those streams where diesel concentrations were set at50, 120 and 600 mg L−1 with no GAELE added, with influentBOD5/COD ratios recording 0.85, 0.66 and 0.53, respectively,the effluent BOD5/COD ratios decreased to 0.47, 0.51 and 0.38,respectively. Based on the 12-month steady-state CPR study,effluent BOD5/COD ratios suggest that organic residuals attributedto semi-volatile diesel compounds, acetate or glycerol may nothave been entirely amenable to biological degradation. In contrast,12-month CPR effluent BOD5/COD ratios, where GAELE was added,with influent diesel concentrations of 80, 500 and 1120 mg L−1,decreased to 0.30, 0.34 and 0.37, respectively. This reduction ofBOD5/COD ratios, which translate into increased recalcitrance, canbe attributed to remnants from GAELE that may have contributedto organic residuals non-amenable to biodegradation. In a similar12-month CPR study, where GAELE was added to the culturemedium with no diesel, influent and effluent BOD5/COD ratiosrecorded 0.31 and 0.26, respectively, which indicates that organicresiduals other than diesel compounds remain in the effluentstream. This may not be a disadvantage since organics are neededin the bulk to provide carbon and energy source for biofilmmaintenance.

Batch kineticsTwo non-fermentative, oxidase-positive, Gram-negative rods,Pseudomonas stutzeri and Ralstonia picketii were predominantspecies in batch bioassays, and were identified with a likelihoodpercentage of 99.9 and 99.0%, respectively. As seen in Table 5,molecular oxygen was fully consumed as the redox potential

decreased substantially, suggesting that bacterial activity turnedfacultative. Set 1 and Set 2 samples showed that the first-phasekinetic rates were nearly eight-fold higher than the correspondingsecond-phase kinetic rates, suggesting that most diesel degrada-tion occurred within the first few days of incubation. Furthermore,the first-phase kinetic rate constant of Set 3 samples was nearly 70-fold higher than the corresponding second-phase kinetic rate con-stant, which confirms that the highest bacterial activity occurredwithin the first 3 days of the bioassays. This is relevant, since reportsindicate that first-order kinetic calculations can effectively be ap-plied to the first 4 days of incubation due to the rapid degradationof the most degradable n-alkanes of diesel oil.21 It is notewor-thy that, as the overall biodegradation percentage increased, thefirst-phase biodegradation rate constants also increased for each ofthe three sets run, accordingly. In addition, the oxidation-reductionpotential (ORP) values between the start and the end of the exper-iments shifted 256, 74 and 26 mV for Sets 1, 2 and 3, respectively.This suggests that soil and GAELE, on an individual basis, were ableto reduce the ORP span. However, no relationship was found be-tween the magnitude of change on ORP as measured between thestart and the end of the kinetics and the change recorded on thefirst-phase biodegradation rate constants. Set 1 samples showedthat the purgeable fraction of diesel hydrocarbons within the chainlengths between C10 and C18 were largely degraded within thefirst 48–72 h, whereas those chains in the C20 –C22 range mostlyremained at the end of the 288 h experiment. Set 2 samples con-firmed the degradation trend with the purgeable fraction of dieselhydrocarbons in the interval C10 –C18 being removed slightly faster



than those in the C20 –C22 range. The reduction in the degradationrate of those chains in the C20 –C22 range may be attributed to theparticular composition of the bacterial populations present in themicrobial consortium and possible toxicity associated with the hy-drocarbon chains in the C20 –C22 range. It has been reported thathydrocarbon fuel chain lengths can have different toxicity patternsdepending on the range of carbon chain length tested.30 Addition-ally, the decrease of the overall first-phase kinetic rate constant ofSet 2 samples, with respect to those of Set 1 samples, may havebeen influenced by the presence of soil particles. It has been re-ported that soil particles can slow down the biodegradation kineticrates not only for small oxygenated hydrocarbons,10 but also forlarge linear alkanes of diesel hydrocarbons.31 As the availability be-comes reduced due to chemical sorption and limited diffusion,32

sorption rates may be affected by substrate diffusion through thepores of soil aggregates.33 The presence of GAELE in Set 3 samplesstimulated the degradation of those hydrocarbon chains largerthan C22, and contributed to render the overall first-phase degra-dation rate of diesel hydrocarbons nearly three-fold and five-foldhigher than the corresponding rates of Set 1 and Set 2 samples,respectively (Fig. 2(a)). The observed enhancement of biodegra-dation through the use of surfactant is in accordance with otherwork. Whang et al.8 studied the effect of rhamnolipid and surfactinbiosurfactants on the biodegradation of diesel-contaminated wa-ter in an aerobic batch reactor. They found a five-fold increase inthe kinetic rate kbio from 4.24 to 20×10−6 h−1 mg VSS−1 followingthe addition of 80 mg L−1 of rhamnolipid and a three-fold increasefrom 5.7 to 17.75 × 10−6 h−1 mg VSS−1 following the addition of40 mg L−1 of surfactin.

CONCLUSIONSThe continuous-upflow packed bed bioreactor built with inexpen-sive support made of volcanic and alluvial stones was capableof degrading diesel hydrocarbons under nitrate-reducing condi-tions at low hydraulic retention times and high concentrations ofsubstrate. Biodegradation percentages of up to 98.4% of dieselfuel at influent concentrations of 600 mg L−1 without GAELE wereobtained. The addition of surfactant GAELE resulted in biodegra-dation percentages of nearly 98% of diesel fuel at influent con-centrations of 1120 mg L−1. Results showed high biodegradationpercentages with a cost-efficient anaerobic CPR. It is noteworthyto mention that the overall anaerobic biodegradation percentagesobtained under nitrate reducing conditions were near or abovethe biodegradation percentages of diesel degradation with aero-bic treatments.1,2 Moreover, although other anaerobic treatmentshave reached similar diesel biodegradation percentages,3,4 in thiswork this was achieved with a HRT of 0.5 h. The use of surfactantshowed a slight increase in percentage biodegradation for boththe 6 month and 12 month biolfilm studies. However, significantincreases on COD and BOD5 removals were observed when GAELEwas added to those bioassays containing high concentrations ofdiesel. Although the addition of surfactant can increase opera-tional costs of the bioreactor, the concentrations of GAELE usedwould not represent an excessive additional cost, especially be-cause GAELE can attain a low and effective CMC. Furthermore,GAELE showed a significant statistical influence on the transportof substrate into the biofilm. On the other hand, batch bioassaysresulted in diesel removals of up to 52% without GAELE, increas-ing to 82% with addition of the surfactant. While percentages ofdiesel removals in batch studies were lower than those obtainedby CPR assays, these studies confirm that GAELE helps increase

Table 5. Microaerophilic degradation of the purgeable fraction ofdiesel in batch assays

Set 1 ∗ Set 2 ∗∗ Set 3 ∗∗∗

Time (h) 288 288 288

DO (mg L−1, (SD)

Initial 6.6 (0.1) 6.9 (0.05) 6.9 (0.11)

Final 0.00 0.00 0.00

ORP (mV), (SD)

Eh0 126 (5.0) 62 (4.7) 29.5 (5.5)

Ehf −130 (2.6) −12.3 (2.5) 3.5 (3.1)

pH, Initial, Final 7.1, 8.0 7.2, 7.4 7.2, 7.3

Volatilization (%) <5.0 <5.5 <1.9

Overall biodegradation(%)

52.6 48.0 82.0

Overall rate constants (K)

First-phase (h−1) 0.074 0.039 0.200

(R2) (0.999) (0.989) (0.998)

Second-phase (h−1) 0.009 0.005 0.003

(R2) (0.999) (0.989) (0.998)

Specific rate constants (k)

First-phase[h−1(mg/L)−1]

5.70 × 10−4 3.00 × 10−4 1.53 × 10−3

(R2) (0.999) (0.989) (0.998)

Second-phase[h−1(mg/L)−1]

6.92 × 10−5 3.84 × 10−5 2.30 × 10−5

(R2) (0.999) (0.989) (0.998)

R2 = correlation coefficient.∗ SMM + 130 mg L−1 VSS.∗∗ SMM + 130 mg L−1 VSS + 5.5% SS.∗∗∗ SMM + 130 mg L−1 VSS + 25 mg L−1 GAELE.

the biodegradation of diesel. The effectiveness of nitrate-reducingbacteria of the genus Pseudomonas in both batch and continuousflow reactors proves the capability of this genus to adapt undervarying environmental conditions to break down small and largehydrocarbon molecules.

ACKNOWLEDGEMENTSThis research was supported under auspices of SEP-CONACyT-82761, CONACyT-SNI-LIC-101753, CONACyT-SNI-91360 researchprojects, and under the fund of the Research Chair of Nanoma-terials and Advanced Materials from Tecnologico de MonterreyCAT-120.

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