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Removal of crude oil polycyclic aromatic hydrocarbons via organoclay-microbe-oil interactions
Chemosphere 174 (2017) 28e38
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Removal of crude oil polycyclic aromatic hydrocarbons via organoclay-microbe-oil interactions Uzochukwu C. Ugochukwu a, *, Claire I. Fialips b a b
SHELL Centre for Environmental Management & Control, University of Nigeria, Enugu Campus, Enugu State, Nigeria Total SA, CSTJF, Avenue Larribau, 64018 PAU, France
h i g h l i g h t s Organo-montmorillonite and organo-saponite inhibit the biodegradation of dimethylnaphthalenes. Organo-saponite unlike organo-montmorillonite enhances the biodegradation of crude oil phenanthrenes. Organoclays sorb dimethylnaphthalenes extensively. Clays with relatively high CEC produces more hydrophobic organoclays and inhibit the biodegradation of PAHs. Unmodified montmorillonites enhances the biodegradation of PAHs except the LMWPAHs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2016 Received in revised form 31 December 2016 Accepted 14 January 2017 Available online 23 January 2017
Clay minerals are quite vital in biogeochemical processes but the effect of organo-clays in the microbial degradation of crude oil polycyclic aromatic hydrocarbons is not well understood. The role of organosaponite and organo-montmorillonite in comparison with the unmodified clays in crude oil polycyclic aromatic hydrocarbons (PAHs) removal via adsorption and biodegradation was studied by carrying out microcosm experiments in aqueous clay/oil systems with a hydrocarbon degrading microbial community that is predominantly alcanivorax spp. Montmorillonite and saponite samples were treated with didecyldimethylammonium bromide to produce organo-montmorillonite and organo-saponite used in this study. Obtained results indicate that clays with high cation exchange capacity (CEC) such as montmorillonite produced organo-clay (organomontmorillonite) that was not stimulatory to biodegradation of crude oil polycyclic aromatic compounds, especially the low molecular weight (LMW) ones, such as dimethylnaphthalenes. It is suggested that interaction between the organic phase of the organo-clay and the crude oil PAHs which is hydrophobic in nature must have reduced the availability of the polycyclic aromatic hydrocarbons for biodegradation. Organo-saponite did not enhance the microbial degradation of dimethylnaphthalenes but enhanced the biodegradation of some other PAHs such as phenanthrene. The unmodified montmorillonite enhanced the microbial degradation of the PAHs and is most likely to have done so as a result of its high surface area that allows the accumulation of microbes and nutrients enhancing their contact. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: Caroline Gaus Keywords: Polycyclic aromatic hydrocarbons Biodegradation Adsorption Organo-clays, Montmorillonite Saponite
1. Introduction So long as petroleum remains the main fossil fuel used as source of energy, spill incidents will likely remain inevitable. The spillage of crude oil and its fractions in the environment is responsible for many hydrocarbon polluted sites (Environmental agency, 2006). Soil contamination by hydrocarbons can cause extensive damage to
* Corresponding author. E-mail address: [email protected] (U.C. Ugochukwu). http://dx.doi.org/10.1016/j.chemosphere.2017.01.080 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
the local ecosystems affecting vegetation and wildlife adversely. Such contamination can also present a serious public health threat and in extreme case, can render the contaminated area uninhabitable for humans (Sunggyu, 1995). Broadly speaking, there are three key sources of hydrocarbons through which releases of PAHs to the environment can occur, namely, petrogenic, pyrogenic and biogenic sources. PAHs in the environment as a result of oil spill and natural oil seeps are petrogenic whereas those arising from burning wood, volcanoes, petroleum products and forest fires could be regarded as pyrogenic. Biogenic sources include PAHs from biologic or diagenetic processes. Simpson et al. (1998) demonstrated that
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the source of PAH input to the environment in the event of hydrocarbon contamination could be identified. Higher concentrations of retene and perylene in the environment are indicative of terrestrial organic inputs of biogenic origin while the predominant existence of lower molecular weight PAHs such as fluorene and acenaphthene indicates petrogenic origin. Higher concentrations of higher molecular weight PAHs (4e6 membered ring PAHs) such as fluoranthene, pyrene and benzo (a) pyrene in the environment are indicative of pyrogenic origin (Helfrich and Armstrong, 1986; Bence et al., 2007). Groundwater could also be at risk of contamination with PAHs from incidents of agricultural activities, improper waste disposal, underground oil storage tanks etc. Both natural and anthropogenic activities are responsible for the release of PAHs to the environment. Forest fires, volcanoes, biologic/diagenic processes and natural oil seepages etc represent the main natural sources of PAH in the environment while crude oil spills, municipal waste, and combustion of fossil fuels represent anthropogenic sources. PAHs are more recalcitrant to biodegradation than saturated hydrocarbons especially n-alkanes of equal molecular weight. The ability of PAHs to resist biodegradation in addition to their lipophilic nature account for why they persist in the environment and can penetrate tissues to bioaccumulate in organisms in the environment (Tuvikene, 1995; Boehm et al., 1981). Biomagnification of the PAHs takes place as they (the PAHs) are transferred to higher trophic levels in the food chain (Boehm et al., 1981). Very many PAHs have been reported to be mutagenic and carcinogenic making their control in the environment extremely important (ASTDR, 1995). The solubility of monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene and xylene (BTEX) is higher than that of PAHs. Generally speaking, the solubility of aromatic hydrocarbons in water (though poor) decreases with increase in molecular weight and is the reason why the concentration of PAHs in water column is relatively low. Organo-clays are mainly produced by the modification of 2:1 clay minerals such as smectites especially montmorillonites. The modification of clay minerals to produce organo-clays involves the replacement of the clay's interlayer exchangeable inorganic cation with a desired organic cation via ion-exchange reactions. This modification to produce organo-clay leads to a transformation from surface hydrophilcity to hydrophobicity (Hermosin et al., 1992; Groisman et al., 2004). Long chain alkyl ammonium cation such as didecyldimethylammonium (DDDMA) is used in the preparation of organoclay (Cornejo et al., 2008). Organoclays have been reported to be useful in the remediation of organic pollutants such as pesticides (Cornejo et al., 2008). However, it is not yet clear what role organoclays would play during the biodegradation of crude oil PAHs. Previous organo-clay mediated biodegradation studies reported by different authors (Malakul et al., 1998; Biswas et al., 2015) involved only single PAHs as substrate such as naphthalene and phenanthrene respectively. It was demonstrated in these studies that organoclay enhanced the biodegradation of the above substrates. There are scarcely any known studies on the effects of organo-montmorillonites or organo-saponites on the biodegradation of mixed PAHs as found in crude oil. When studying the effect of organo-clays, or any other material, on the microbial degradation and adsorption of PAHs present in petroleum, it is essential to do so in the presence of numerous PAHs rather than individual PAH fractions so as to understand how the biodegradation and adsorption of the individual PAHs proceed in the presence of other PAHs. Hence, this study would seek to understand how the presence of other PAHs affects the removal of the individual PAHs via organo-clay mediated adsorption and biodegradation. The effect of organoclays on the biodegradation of crude oil saturated fraction has been reported (Ugochukwu et al., 2013) however, our knowledge of the effect of organo-clays during
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biodegradation of crude oil PAHs is grossly limited. The result of the microcosm experiments carried out to investigate the ability of organo-clays in comparison with the unmodified clays to adsorb and stimulate or hinder the microbial degradation of crude oil PAHs is reported in this paper. The following objectives were pursued: (i) To produce organo-clay with hydrophobic surface by having the DDDMA successfully intercalated (ii) To investigate how the individual PAHs in crude oil respond to microbial degradation and adsorption on the organo-clays in the presence of other PAHs compared to unmodified clays?
2. Materials and methods Bentonite (Berkbent 163) was supplied by Steetley Bentonite & Absorbent Ltd (now Tolsa UK Ltd; www.tolsa.com) while natural saponite was collected from the Orrock Basalt Quarry Burntisland, Scotland (National Grid Reference NT 218887; Cowking et al., 1983). Clay mineral samples used in this study were a saponite and a montmorillonite separated by fractionation from the altered basalt rock samples and from the reference bentonite Berkbent 163, respectively, following the method of Cowking et al. (1983) modified as follows: Approximately 1000 g of basalt rock was crushed to particle size less than 10 mm and soaked in a 5 L beaker filled with water to 4.5 L mark. This mixture was allowed to stand overnight and the supernatant liquid decanted and sonicated for 3 min. Gravity sedimentation was employed for the settling of the crushed Orrock Basalt rock so as to separate particle size less than 2 mm by progressive size fractionation applying Stoke's Law. The same procedure was repeated for Berkbent 163 Bentonite except that there was no crushing and overnight standing as this reference material is provided as a powdery material. The microbial communities that were involved in the microbial degradation of crude oil PAHs were isolated from beach sediment samples that were collected in sterilised glass bottle from a site at St Mary's Lighthouse near Whitley Bay, Newcastle Upon Tyne, United Kingdom (National Grid Reference: NZ 352 754) and stored at 4 C in cold room until the commencement of the experiment. The microbial community has been reported to contain mainly Alcanivorax spp. as the dominant genera in another study carried out by Singh et al. (2009) conducted with exactly the same beach sediments as source of microbial cells. The Bushnel-Haas (BH) broth as the nutrient source, nutrient agar and all other chemicals were supplied by Sigma Aldrich. The undegraded North Sea crude oil sample for the experiments was supplied by British Petroleum (BP). 2.1. Organo-clay preparation The organo-clay minerals were prepared following the procedure reported in Ugochukwu et al., 2014a. Very succinctly, this procedure consisted in adding sufficient amounts of didecyldiammonium (DDDMA) bromide to the montmorillonite and saponite suspensions to reach 35% of their respective CEC followed by centrifugation, repeated washing of the clay with de-ionized water, drying and storing in a desiccator. 2.2. Characterization of the clay samples 2.2.1. XRD, FTIR, CEC, EGME-Surface area and TOC The untreated saponite and montmorillonite and the prepared organo-clays were characterized in detail using various techniques. The method employed for the X-Ray Diffraction (XRD), Fourier
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Transform Infra-Red (FTIR), Cation Exchange Capacity (CEC), Ethylene Glycol Monoethyl Ether (EGME)- Surface Area, and Total Organic Carbon (TOC) data are as reported in Ugochukwu et al. (2014a). The FTIR and XRD diffraction patterns of these clay samples having not been reported in any of the earlier published work is presented in the results section of this manuscript.
spectrophotometer. The obtained growth curve enabled determination of the exponential and stationary phase and hence an estimate of when there were active cells in the system. Thus active cells were harvested for preparing the subcultures. In addition to monitoring cell growth by absorbance method, cell growth was also monitored via standard cell plating.
2.2.2. Gas Chromatography-flame ionization detector and Gas Chromatography-Mass Spectrometer 2.2.2.1. Gas Chromatography-flame ionization detector (GC-FID). HP 5890 series II gas chromatograph equipped with a split/splitless injector and flame ionization detector (FID) was the Gas Chromatography (GC) instrument used. The sample was injected using an HP 7673 autosampler. The separation of the crude oil hydrocarbon compounds was carried out on an Agilent HP-5 capillary column (30 m 0.25 mm) coated with 5% phenylmethyl-polysiloxane (0.25 mm thick) stationary phase. The GC oven temperature was programmed from 50 C for 2 min and then ramped at 4 C/min up to 300 C where it was held for 20 min. The carrier gas used was hydrogen at a flow rate of about 2 mL/min at initial pressure of 100 kPa. The GC data was acquired using Atlas software on HP computer desktop.
2.3.2. Microbial degradation experiment in the presence of organoclay minerals The detailed procedure of the microbial degradation experiments in the presence of organosaponite, organomontmorillonite, and the unmodified clays has been reported in a previous study (Ugochukwu et al., 2014a). The procedure involved microcosm experiments consisting 0.25 g of clay and 0.050 g of crude oil in 10 mL of Bushnel-Haas medium with the microbial cells including control experiments to account for abiotic processes due to volatilization and adsorption. The experimental set up was such that the following test and control experiments were in place: ➢ Test experiment consisted of 0.25 g (organo-clay mineral sample or unmodified clay sample) þ 0.05 g crude oil þ 10 mL BH medium þ microbial cells
2.2.2.2. Gas Chromatography-Mass Spectrometer (GC-MS). The Gas Chromatography-Mass Spectrometer (GC-MS) used was Agilent 6890 Gas Chromatography instrument with split/splitless injector (280 ), connected to a 5975 MSD mass spectrometer. The stationary phase of the GC was a fused silica capillary column (30 m 0.25 mm) coated with 5% phenyl polysiloxane (HP-5). The GC oven was programmed from 40 C for 5 min and then ramped at 4 C/min, up to 300 C where it was held for 20 min. The carrier gas used was helium at a flow rate of about 1 mL/min and initial pressure of 50 kPa while split at 30 mL/min. The sample was injected by an HP7683 autosampler and the split opened after 1 min to vent the solvent. The mass spectrometer used electron ionization energy of 70 eV. The following operating conditions were used: Source temperature of 230 C; Quadrupole temperature of 150 C; Multiplier voltage of 2000 V; Interface temperature of 310 C. Selected ion monitoring (SIM) mode was used to monitor ions of m/z values: 156, 166, 170, 178, 180, 184, 192, 198, 206, 212, and 236 for various peaks used for identifying the PAHs namely: dimethylnaphthalenes, fluorene, trimethylnaphthalene, phenanthrene, methylfluorene, dibenzothiophene, methylphenanthrenes, methyldibenzothiophenes, dimethylphenanthrenes, dimethyldibenzothiophenes, and triaromatic steroids respectively.
The test experiment and Control-1 experiment are the only experiments that are biotic as they contain microbial cells as opposed to Control-2 and Control-3 experiments that do not contain microbial cells. Whereas Control-1 helps in the evaluation of biodegradation (a biotic process) as mediated by clays, Control-2 helps in the evaluation of adsorption (abiotic process) mediated by clays. See section 2.5. The Test experiment and Control-3 were used to evaluate biodegradation in the presence of organo-clay minerals or unmodified clay minerals whereas Control-1 and Control-2 were used to estimate biodegradation taking place in the absence of clay. Control-2 and Control -3 were used for estimating adsorption.
2.3. Biodegradation experiments
2.4. Extraction of hydrocarbons and analysis
The microorganisms indigenous to the collected sediments were proliferated subsequent to isolation and thereafter employed for microcosm microbial degradation experiments.
Extraction of the residual oil after incubation was carried out as follows: Three stages of extraction with 30 mL of dichloromethane (DCM) for each stage were employed to extract the residual oil subsequent to spiking with squalane as surrogate standard. Following the procedure of Bennett et al. (2002), the extract was further extracted on solid phase extraction (SPE) columns to separate the hydrocarbons from the polars. Prior to GC-FID and GCMS analysis, the samples were spiked with internal standards namely, heptadecylcyclohexane and 1,1-binaphthyl. The relative response factor (RRF) of the surrogate standard varied between 0.78 and 0.8 which is acceptable. However, for computing the percentage recovery of the surrogate standard, RRF was corrected to 1.0. The percentage recovery of the surrogate standard lied between 70% and 120% which is within acceptable range (USEPA method 8270). The selected aromatic compounds studied were: isomers of dimethyl naphthalene, isomers of trimethyl naphthalene, fluorene, isomers of methyl fluorene, phenanthrene, isomers
2.3.1. Enrichment culture preparation for microbial cell proliferation To prepare the initial enrichment culture, 0.5 g of crude oil, 20 g of the Whitley Bay beach sediment and 100 mL of BH medium were mixed in a 250 mL conical flask under a sterile atmosphere provided by bunsen burner flame. The flask was then closed with a cotton wool and continuously shaken while being incubated. Several weights (0.5 ge2.0 g) of the crude oil were used as carbon source in several subcultures after the initial culture so as to proliferate and purify the cells as they go through several serial transfers. The growth of the microbial cells were monitored in the subculture for each case by collecting 1 mL of cell suspension every two days and measuring absorbance at 600 nm using a UVeVisible
The control experiments were given as follows: ➢ Control-1: 10 mL BH medium þ microbial cells þ 0.05 g crude oil without clay ➢ Control-2: 10 mL BH medium þ 0.05 g crude oil without clay ➢ Control-3: Clay sample (organo-clay mineral sample or unmodified clay mineral sample) þ 10 mL BH medium þ 0.05 g crude oil.
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of methylphenanthrene, isomers of dimethylphenanthrene, dibenzothiophene, isomers of ethyldibenzothiophene, isomers of dimethyldibenzothiophene and triaromatic steroids.
Aromatic compound ð%Þ as adsorbed ¼
31
Xc2 Xcy 100 Xc2
(6)
2.5. Quantifying the biodegradation and adsorption of the PAHs 3. Results/discussion The mass and percentage of the aromatic compounds removed by biodegradation and adsorption are as described in the following equations:
Aromatic compounds as biodegraded ðmassÞ ¼ Xcy Xr Aromatic compound as biodegraded ð%Þ ¼
Xcy Xr Xcy
(1)
100 (2)
Xcy ¼ mass ðgÞ of the residual aromatic compound for the clay control sample Xr ¼ mass ðgÞ of the residual aromatic compound for the test sample The clay control experiment consisted of clay, Bushnel Haas medium, and crude oil with no microorganism added (abiotic experiment) whereas the test sample experiment consisted of clay, Bushnel Haas medium, crude oil, and microorganisms. Two other control experiments were conducted: Control 1 consisted of Bushnel-Haas medium, crude oil, and microorganisms but no clay whereas Control 2 consisted of Bushnel-Haas medium and crude oil only with neither clay nor microorganisms. For control-1,
Aromatic compound as biodegraded ðmassÞ ¼ Xc2 Xcx Aromatic compound as biodegraded ð%Þ ¼
(3)
Xc2 Xcx 100 Xc2 (4)
Xc2 ¼ mass ðgÞof the residual aromatic compound for control 2 Xcx ¼ mass ðgÞ of residual aromatic compound for control 1 Aromatic compound ðmassÞ as adsorbed ¼ Xc2 Xcy
(5)
3.1. Characterization of the clay samples 3.1.1. XRD patterns and FTIR bands of the organoclay samples The XRD patterns of the clay samples for both the organo-clay samples and the unmodified clays are presented in Figs. 1e3 showing the d001 reflections of the clay samples under air-dry, heat treated (300 C) and glycolated conditions. The FTIR spectra of the clay samples for both the organo-clay and unmodified clay samples are presented in Figs. 4e7. Characteristic FTIR bands for the saponites are as shown in Figs. 4 and 5 whereas the bands characterizing montmorillonites are as shown in Figs. 6 and 7. The basal air-dry d001 spacing for unmodified montmorillonite (BU), unmodified saponite (SU), organo-montmorillonite (BO), and organosaponite (SO) as deduced from Fig. 1 are 12.5 Å, 14.5 Å, 14.2 Å and 14.0 Å respectively. The observed reflections at 202q and 29.52q for BU and BO correspond to 4.27 Å (quartz) and 3.03 Å (calcite) respectively present in the montmorillonite clay mineral. The basal glycolated d001 spacing for BU, SU, BO, and SO as deduced from Fig. 2 are 17.1 Å, 16.2 Å, 16.8 Å, 14.7 Å whereas the heat treated d001 spacing as deduced from Fig. 3 are 10.6 Å, 10.7 Å, 13.2 Å and 13.8 Å respectively. Hence, the layer collapse of the organo-clays on heat treatment at 300 C is lower in comparison with the unmodified clays and is due to the intercalation of didecyldimethylammonium (DDDMA) in the interlayer of the clay samples. The two IR bands at 3390 cm1 and 3573 cm1 (Fig. 4) reflect octahedral character. The absorption band at 3570 cm1 is due to Mg/Fe2þ-OH stretch vibration of Fe-rich saponite. The absorption bands at 2861 cm1 and 2935 cm1 (Fig. 5) observed with organosaponite sample (SO) are assigned to symmetrical and assymetrical vibration stretch of C-H2 respectively from the hydrocarbon moiety of DDDMA used in the preparation of the organoclay sample. The broad IR absorption band at 3623 cm1 in the spectra for unmodified and organo-montmorillonite as shown in Fig. 6 is assigned to OH-stretching of AlAlOH which is typical of dioctahedral smectites such as montmorillonites (Wilson, 1987). Also, the absorption bands at 2861 cm1 and 2935 cm1 observed in Fig. 7 is due to symmetrical and assymetrical vibration stretch of C-H2 respectively from the hydrocarbon moiety of DDDMA (Gunzler and
Fig. 1. XRD patterns of the clay samples under air-dry conditions. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organosaponite.
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Fig. 2. XRD patterns of the clay samples as glycolated. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organo-saponite.
Fig. 3. XRD patterns of the clay samples as heat treated at 300 C. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organosaponite.
Fig. 4. Infrared spectra of unmodified saponite (SU) and organosaponite (SO) (3000e4000 cm1).
Gremlich, 2002). The XRD patterns and FTIR bands suggest that the DDDMA was successfully intercalated in the interlayer of the montmorilonite and saponte samples.
3.1.2. EGME-surface area, CEC and TOC The EGME-surface area of the clay samples as reported in Ugochukwu et al., 2014a indicate that the specific surface area of the unmodified clays [unmodified montmorillonite (645 m2/g) and unmodified saponite (473 m2/g)] are higher than those of the organoclays (organomontmorillonite (471 m2/g) and
organosaponite (330 m2/g)). The TOC for the unmodified clay samples did not contain organic carbon while the TOC values for the organoclay were 3.4 and 7.3% for organo-saponite and organomontmorillonite respectively indicating that the organmontmorillonite contained more organic phase as was expected given the higher CEC value of montmorillonite (83.3 meq/100 g) than saponite (35.4 me/100 g).
3.1.3. Hydrophobicity In this study, the XRD and FTIR data indicate that the DDDMA
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Fig. 5. Infrared spectra of unmodified saponite (SU) and organo-saponite (SO) (2600e3400 cm1).
Fig. 6. Infrared spectra of unmodified montmorillonite (BU) and organomontmorillonite (BO) (3500e4000 cm1).
Fig. 7. Infrared spectra of unmodified montmorillonite (BU) and organo-montmorillonite (BO) (2700e3100 cm1).
was successfully intercalated in the organoclay samples. Furthermore, the TOC data confirms that the intercalation of the DDDMA was such that the organic phase of organomontmorillonite was more extensive than that of organosaponite. This derives from the fact that the CEC of montmorillonite is higher than that of saponite. 3.2. Biodegradation of crude oil PAHs on organomontmorillonite and organosaponite in comparison with the unmodified clays The preservation of n-C17/Pristane and n-C18/Phythane ratios at 2.0 and 2.1 respectively in Control-2 and all the clay control samples (where there was no addition of microbe) is an indication that there was no microbial activity in these control experiments (Fig. 8b, d, f,
h, and j). The disappearance of the hydrocarbon peaks in the test experiments indicates microbial activity (Fig. 8a, c, e, g, and i). The disappearance of the peaks appears to be more with unmodified clays especially unmodified montmorillonite hence providing evidence of enhanced biodegradation of the selected crude oil aromatic compounds (Fig. 9). In addition to relatively higher EGMEsurface area compared to organomontmorillonite, the unmodified montmorillonite seems to have the ability to promote contacts between nutrients and microbes to a degree that is sufficient enough to stimulate biodegradation of crude oil PAHs (Warr et al., 2009). There is usually a sequence of events that take place prior to cell colonization of clay surface. This sequence of events begins with transportation of cells to clay surface by means of diffusive
Fig. 8. (a) Chromatogram of Control-1 (no clay). (b) Chromatogram of Control-2 (no clay and no microbe). (c) Chromatogram of clay test sample containing unmodified montmorillonite. (d) Chromatogram of clay control sample containing unmodified montmorillonite. (e) Chromatogram of clay test sample containing organomontmorillonite. (f) Chromatogram of clay control sample containing organomontmorillonite (g) Chromatogram of test sample containing unmodified saponite (h) Chromatogram of clay control sample containing unmodified saponite. (i) Chromatogram of test sample containing organosaponite (j) Chromatogram of clay control sample containing organosaponite. The standards are labeled as: squ ¼ squalane; hdch ¼ heptadecylcyclohexane; 1,1-binaph ¼ 1,1-binaphthyl; andro ¼ 5a-androstane. Clay test samples contain both oil and microbes in a BH medium while clay control samples contain only oil in a BH medium but no microbes.
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35
Fig. 8. (continued).
transport, convective transport, cell motility and sedimentation under quiescent conditions (Van Loosdrecht et al., 1990). However, for there to be contact resulting from these modes of transport, the energy barrier imposed by the repulsive forces of interaction between the clay surface and the microbial cell must be overcome. Once it is overcome, contact is initiated and the cells adhere to the surface. The adhesion is reversible in suspensions with secondary minimum of total Gibb's free energy of interaction and irreversible in the primary minimum (Busscher et al., 1984; Van Loosdrecht et al., 1989). Subsequent to adhesion is firm attachment and surface colonization by the cells (Van Loosdrecht et al., 1990). Replacing the montmorillonite interlayer cations with considerable amount of DDDMA has the tendency of increasing repulsive energy of interaction thereby lowering the chances of adhesion. Also, the presence of DDDMA for the organomontmorillonite makes it have a lower specific surface area than the unmodified montmorillonite thereby making cell colonization on organomontmorillonite to be relatively less extensive. The nutrients employed in this study are hydrophilic and have a higher tendency to accumulate on clay surfaces that are hydrophilic than those that are not hence, nutrient accumulation on unmodified montmorillonite was expected to be higher than for organomontmorillonite.
The biodegradation of low molecular weight (LMW) PAHs such as dimethylnaphthalenes (DMNs) appears to be neither stimulated nor inhibited by the unmodified clays as the observed percentages of total dimethylnaphthalenes (SDMN) biodegradation are, for both the saponite and montmorillonite, very similar to that obtained in Control-1, in the absence of clay (88e95%). The biodegradation of triaromatic steroids is relatively enhanced by the unmodified clays in comparison with phenanthrenes. Though the extent of biodegradation of the aromatic compounds tends to decrease as the fused rings increase, stimulation of biodegradation of aromatic compounds by unmodified montmorillonite appears to relatively increase as the molecular weight of fused rings increase. The organoclay samples, especially organo-montmorillonite (BO), were inhibitory to the biodegradation of the low molecular PAH (LWMPAH) such as SDMN, as evidenced by the lower % biodegradation than in Control-1 (Fig. 9). These results suggest that these LMW PAHs like DMNs may have reached the adsorption sites of the organo-clay (before the other analytes) and interacted strongly enough with the organic phase of the organo-clay via hydrophobic interaction to render them partly unavailable for biodegradation. This hydrophobic interaction is expected to increase with the amount of organic phase of the organo-clay, thereby explaining
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Fig. 9. Biodegradation of the selected PAHs. SDMN ¼ sum of all isomers of dimethylnaphthalenes, STMN ¼ sum of all isomers of trimethylnaphthalenes, SF ¼ sum of fluorene and isomers of methyl fluorene, SP ¼ sum of phenanthrene and isomers of methyl- and dimethylphenanthrene, SDBT ¼ sum of dibenzothiophene and isomers of methyl- and dimethyldibenzothiophene, STAS ¼ sum of the isomers of triaromatic steroids. BO ¼ organomontmorillonite; BU ¼ unmodified montmorillonite; SO ¼ organosaponite; SU ¼ unmodified saponite; Control-1 ¼ BH þ oil þ cells (no clay). Values are reported as mean ± one standard error.
why BO (7.3% TOC) is more inhibitory to the biodegradation of the DMNs than SO (only 3.4% TOC). The organo-montmorillonite was also inhibitory to the biodegradation of the trimethylnaphthalenes (TMNs) as reflected by % biodegradation of total trimethylnaphthalenes (STMN) of 24% compared to 51% in Control-1 while the only inhibitory behavior of organo-saponite was with respect to DMNs. Considerably, enhanced biodegradation occurred in the presence of organo-saponite for all other groups of analytes compared to Control-1. The higher molecular weight of these analytes may have limited their diffusion to the organic phase of the organo-saponite, as transport to the organic phase is most likely diffusion controlled. In addition, the organic phase of the organosaponite is not as extensive as that of organo-montmorillonite. Most of the adsorptive sites of the organo-saponite were possibly saturated with DMNs, leaving limited spare adsorption sites for other PAHs, which were hereby more readily available for biodegradation. The % biodegradation of total triaromatic steroids (STAS) in the presence of organo-saponite and unmodified saponite are similar while the % biodegradation of total trimethylnaphthalenes (STMN), total fluorenes (SF), total phenanthrenes (SP) and total dibenzothiophenes (SDBT) are slightly higher in the presence of organo-saponite compared to that reached in the presence of the unmodified saponite (Fig. 9). The observed results indicate that the organo-saponite stimulate the biodegradation of those PAHs that are of higher molecular weight than DMNs by means of preference to adsorbing the DMNs and making the higher molecular weight PAHs available for biodegradation while rendering the adsorbed lower molecular weight compounds (DMNs) more difficult to biodegrade than usual. Hence, whether or not an organo-clay is going to be inhibitory to a given analyte will depend on whether the organo-clay would have any chance of holding on to the analyte by hydrophobic interaction. For the organo-clay to be able to inhibit the biodegradation of the various groups of the PAHs found in the crude oil, it must have sufficient organic phase with correspondingly extensive adsorptive sites that cannot be fully occupied by just a few LMW PAHs. Organo-saponite appears to stimulate the biodegradation of most PAHs even more than the unmodified saponite indicating that organoclays under certain conditions could enhance biodegradation of some PAHs. This ability of organosaponites to enhance the biodegradation of STMN, SF, SP, and SDBT nearly matches that of unmodified montmorillonite (Fig. 9). These findings lead to the suggestion that organoclays can actually be manipulated to either inhibit or enhance biodegradation of PAHs of choice in the crude oil. Previous study by Malakul et al. (1998)
and Biswas et al. (2015) demonstrated biodegradation enhancement of naphthalenes and phenanthrenes as single PAH substrates in the presence of organoclays but this work has studied the biodegradation of these substrates in the presence of other PAH substrates found in crude oil. Whereas this study in which organosaponite enhanced the biodegradation of phenanthrenes is in agreement with the study by Biswas et al. (2015), it is important to point out that Biswas et al. (2015) employed bentonite as clay and also did not study other PAHs alongside the phenanthrenes. The organosaponite and organomontmorillonite having inhibited the biodegradation of LMW PAHs such as dimethylnaphthalenes contrasts the study of Malakul et al. (1998) that enhanced the biodegradation of LMW PAHs such as naphthalenes. It is therefore possible that the different result obtained in this study is as a result of the presence of other PAHs in the crude oil among other possible reasons. 3.3. Adsorption of the crude oil PAHs on organo-montmorillonite and organo-saponite in comparison with the unmodified clays Adsorption of the selected crude oil aromatic compounds on organo-clays is significantly negative for the LMW PAHs, such as DMNs (Fig. 10). The implication of the negative adsorption is that the organoclay control samples have retained the analytes more than the volatility abiotic control, Control-2 where the DMNs as LMW PAH compounds must have been lost to volatilization than the HMW PAH compounds. With the organoclay, as shown in Fig. 9, this negative adsorption tends to be higher with LMW of the crude oil aromatic compounds. This corroborates the fact that the organoclays interact with the aromatic compounds hydrophobically and the interaction is strong enough to reduce the availability of the aromatic compounds for both biodegradation and volatilization. The higher negative adsorption of the LMW compounds such as DMNs indicates that these compounds reach the adsorptive sites of the organo-clay before the other analytes and are therefore largely adsorbed. Hydrophobicity of the organo-clays is due to the presence of organic phase in the interlayer of the clay minerals, in agreement with other reported studies (Hermosin et al., 1992; Groisman et al., 2004). The negative adsorption values reported with organo-clay controls (Fig. 10) is due to the fact that during extraction, the dichloromethane (DCM) used for extraction was able to overcome the hydrophobic interaction between the aromatic compounds and the organic phase of the organo-clay. Hence, the DCM desorbed and extracted the aromatic compounds,
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37
Fig. 10. Adsorption of the selected PAHs. CBU, CBO, CSU and CSO represent clay controls (in the absence of microbial cells) for unmodified montmorillonite, organomontmorillonite, unmodified saponite and organosaponite respectively. Values are reported as mean ± one standard error.
especially the LMW PAHs, which are expected to be more abundant in the organic phase of the organoclays. This therefore makes the concentration of the LMW PAHs in the organoclay controls outstrip that in Control-2 where there must have been losses due to volatilization. It follows therefore that hydrophobic interaction that holds the LMW PAHs in the organic phase of the organoclay is strong enough to prevent the LMW PAHs from volatilizing. Unmodified clay samples demonstrated moderate positive adsorption of the aromatic compounds, likely due to another kind of interaction (other than the hydrophobic interaction encountered with the organo-clay) known as p-cation interaction. Aromatic hydrocarbon compounds are donors of p electrons and therefore lend themselves to adsorption via p-cation interaction with the interlayer cations of the unmodified clay minerals. This p-cation interaction can be reasonably strong (in the order of that of hydrogen bonding) as indicated by recent molecular modelling and spectroscopic studies of Zhu et al. (2004). This type of interaction is generally stronger than the hydrophobic interaction discussed earlier. Hence, the clay controls of the unmodified clays showed positive adsorption of the aromatic compounds due to the relative strength of the p-cation interaction which cannot be overcome by DCM during extraction. The adsorption of the selected PAHs on the organo-clay and unmodified clay samples during the biodegradation process is as presented in Fig. 10.
Organo-montmorillonite with extensive organic phase was inhibitory to the biodegradation of all the PAHs whereas organosaponite, with less extensive organic phase, was stimulatory to the biodegradation of some of the PAHs such as phenanthrene. The biodegradation of triaromatic steroids was relatively enhanced by the unmodified clays in comparison with phenanthrenes. Though the extent of biodegradation of the aromatic compounds decreased as the fused rings increased, stimulation of biodegradation of aromatic compounds by montmorillonite relatively increased as the molecular weight of fused rings increased. This study therefore indicates that organoclays can actually be tailored to achieve biodegradation of some PAHs by carefully selecting the appropriate clay mineral for the production of organo-clays under certain conditions. Also, the inevitability of poor biodegradation of LMW PAHs in the presence of other PAHs on organo-clays and the reasons for that have been identified in this study. Acknowledgements We thank Petroleum Technology Development Fund (PTDF) of the Federal Republic of Nigeria for funding this project. Generally, we are grateful to the School of Civil Engineering and Geosciences of the University of Newcastle Upon Tyne, United Kingdom for providing the facilities used in this study. We appreciate Berny Bowler, Paul Donohue, Phil Green and Ian Harrison for the laboratory support received from them.
4. Conclusion Organo-montmorillonite and organo-saponite were both inhibitory to biodegradation of LMW PAHs such as dimethylnaphthalenes and the inhibition increased with increase in the amount of organic phase in the organo-clay. Organo-montmorillonite was therefore more inhibitory than organo-saponite to the biodegradation of LMW PAHs. Obtained results suggest that the organo-clay inhibited biodegradation of the LMW PAHs as a result of the hydrophobic interaction between the organic phase of the organoclay and the aromatic hydrocarbons. This hydrophobic interaction, though weak, was strong enough to render the aromatic compounds unavailable for degradation by the microorganisms. This interaction also made the LMW aromatic compounds, such as the dimethylnaphthalenes, resist volatilization. Hence, the organoclays retained and contained more of the LMW PAHs such as the dimethylnaphthalenes, trimethylnaphthalenes and fluorenes than the abiotic Control. Generally, the biodegradation of LMW aromatic compounds such as dimethylnaphthalenes was not enhanced by clay minerals.
References Agency for Toxic Substances and Disease Registry (ASTDR), 1995. Public Health Statement for Polycyclic Aromatic Hydrocarbons (PAHs) ATSDR. US Dept of Health and Human Services, Public Health Services, Atlanta, GA. Bence, A.E., Page, D.S., Boehm, P.D., 2007. Advances in forensic techniques for petroleum hydrocarbons: the Exxon Valdez experience. In: Wang, Z., Stout, S.A. (Eds.), Oil Spill Environmental Forensics. Academic Press, San Diego, California, USA, pp. 451e453. Bennett, B., Chen, M., Brincat, D., Gelin, F.J.P., Larter, S.R., 2002. Fractionation of benzocarbazoles between source rocks and petroleums. Org. Geochem. 33, 545e559. Biswas, B., Sarkar, B., Mandal, A., Naidu, R., 2015. Heavy metal eimmobilizing organoclay facilitates polyaromatic hydrocarbon biodegradation in mixedcontaminated soil. J. Hazard Mater. 298, 129e137. Boehm, P.D., Fiest, D.L., Elskus, A., 1981. Comparative weathering patterns of hydrocarbons from Amoco Cadiz oil spill observed at a variety of coastal environment. In: International Symposium on the Fate and Effects of Oil Spill. Brest, France, pp. 159e173. Busscher, H.J., Weerkamp, A.H., van Der mei, H.C., van Pelt, W.J., de Jong, H.P., Arends, J., 1984. Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion. Appl. Environ. Microbiol. 48, 980e983. Cornejo, J., Celis, R., Pavlovic, I., Ulibarri, M.A., 2008. Interactions of pesticides with clays and layered double hydroxides: a review. Clay Min. 43, 155e175.
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Cowking, A., Wilson, M.J., Tait, J.M., Robertson, R.H.S., 1983. Structure and swelling of fibrous and granular saponitic clay from Orrock Quarry Fife, Scotland. Clay Min. 18, 49e64. Environmental agency, (2006) http://www.environment-agency.gov.uk/ commondata/103601/poll_incidents_2005_1438766.xls Accessed Nov, 2007. Groisman, L., Rav-Acha, C., Gerstl, Z., Mingelgrin, U., 2004. Sorption of organic compounds of varying hydrophobicities from water and industrial waste-water by long- and short- chain organoclays. Appl. Clay Sci. 24, 159e166. Gunzler, H., Gremlich, H., 2002. IR Spectroscopy: an Introduction. Wiley VCH Verlag GmbH, Weinheim, Germany. Helfrich, J., Armstrong, D.E., 1986. Polycyclic aromatic hydrocarbons in sediments of the Southern basin of lake Michigan. J. Gt. Lakes. R 12 (3), 192e199. Hermosin, M.C., Ulibarri, M.A., Mansour, M., Cornejo, J., 1992. Assaying sorbents for 2,4-dichlorophenoxyacetic acid from water. Fresen Environ. Bull. 1, 472e481. Malakul, P., Srinivasan, K.R., Wang, H.Y., 1998. Metal toxicity reduction in naphthalene biodegradation by use of metal-chelating adsorbents. Appl. Environ. Microbiol. 64, 4610e4613. Simpson, C.D., Harrington, C.F., Cullen, W.R., Bright, D.A., Reimer, K.J., 1998. Polycyclic aromatic hydrocarbon contamination in marine sediments near Kitimat, British Columbia. Environ. Sci. Technol. 32 (21), 3266e3272. Singh, A.K., Sherry, A., Gray, N.D., Jones, M.D., Rolling, W.F.M., Head, I.M., 2009. How specific microbial communities benefit oil industry: dynamics of Alcanivorax spp. in oil contaminated intertidal beach sediments undergoing bioremediation. In: Proceedings of International Symposium. Applied Microbiology and
Molecular Biology in Oilfield Systems, pp. 199e210. Sunggyu, L., 1995. Bioremediation of polycyclic aromatic hydrocarboncontaminated soil. J. Clean. Prod. 3, 255. Tuvikene, A., 1995. Response of fish to polycyclic aromatic hydrocarbons (PAHs). Ann. Zool. Fenn. 32, 295e309. Ugochukwu, U.C., Jones, M.D., Head, I.M., Manning, D.A.C., Fialips, C.I., 2013. Biodegradation of crude oil saturated fraction supported on clays. Biodegradation 24 (3). Ugochukwu, U.C., Manning, D.A.C., Fialips, C.I., 2014a. Microbial degradation of crude oil hydrocarbons on organoclay minerals. J. Environ. Manag. 144, 197e202. Van Loosdrecht, M.C.M., Lyklema, J., Norde, W., Zehnder, J.B., 1990. Influence of interfaces on microbial activity. Mirobiol. Rev. 54, 75e87. Van Loosdrecht, M.C.M., Lyklema, J., Norde, W., Zehnder, J.B., 1989. Bacterial adhesion: a physicochemical approach. Microb. Ecol. 17, 1e15. Warr, L.N., Perdrial, J.N., Lett, M., Heinrich-Salmeron, A., Khodja, M., 2009. Clay mineral-enhanced bioremediation of marine oil pollution. Appl. Clay Sci. 46, 337e345. Wilson, M.J., 1987. Infrared methods-smectites. In: A Handbook of Determinative Methods in Clay Mineralogy. Blackie, Glasgow and London, UK, p. 154. Zhu, D., Herbert, B.E., Schlautman, M.A., Carraway, E.R., Hur, J., 2004. Cation-p bonding: a new perspective on the sorption of polycyclic aromatic hydrocarbons to mineral surfaces. J. Environ. Qual. 33, 1322e1330.
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Removal of crude oil polycyclic aromatic hydrocarbons via organoclay-microbe-oil interactions Uzochukwu C. Ugochukwu a, *, Claire I. Fialips b a b
SHELL Centre for Environmental Management & Control, University of Nigeria, Enugu Campus, Enugu State, Nigeria Total SA, CSTJF, Avenue Larribau, 64018 PAU, France
h i g h l i g h t s Organo-montmorillonite and organo-saponite inhibit the biodegradation of dimethylnaphthalenes. Organo-saponite unlike organo-montmorillonite enhances the biodegradation of crude oil phenanthrenes. Organoclays sorb dimethylnaphthalenes extensively. Clays with relatively high CEC produces more hydrophobic organoclays and inhibit the biodegradation of PAHs. Unmodified montmorillonites enhances the biodegradation of PAHs except the LMWPAHs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2016 Received in revised form 31 December 2016 Accepted 14 January 2017 Available online 23 January 2017
Clay minerals are quite vital in biogeochemical processes but the effect of organo-clays in the microbial degradation of crude oil polycyclic aromatic hydrocarbons is not well understood. The role of organosaponite and organo-montmorillonite in comparison with the unmodified clays in crude oil polycyclic aromatic hydrocarbons (PAHs) removal via adsorption and biodegradation was studied by carrying out microcosm experiments in aqueous clay/oil systems with a hydrocarbon degrading microbial community that is predominantly alcanivorax spp. Montmorillonite and saponite samples were treated with didecyldimethylammonium bromide to produce organo-montmorillonite and organo-saponite used in this study. Obtained results indicate that clays with high cation exchange capacity (CEC) such as montmorillonite produced organo-clay (organomontmorillonite) that was not stimulatory to biodegradation of crude oil polycyclic aromatic compounds, especially the low molecular weight (LMW) ones, such as dimethylnaphthalenes. It is suggested that interaction between the organic phase of the organo-clay and the crude oil PAHs which is hydrophobic in nature must have reduced the availability of the polycyclic aromatic hydrocarbons for biodegradation. Organo-saponite did not enhance the microbial degradation of dimethylnaphthalenes but enhanced the biodegradation of some other PAHs such as phenanthrene. The unmodified montmorillonite enhanced the microbial degradation of the PAHs and is most likely to have done so as a result of its high surface area that allows the accumulation of microbes and nutrients enhancing their contact. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: Caroline Gaus Keywords: Polycyclic aromatic hydrocarbons Biodegradation Adsorption Organo-clays, Montmorillonite Saponite
1. Introduction So long as petroleum remains the main fossil fuel used as source of energy, spill incidents will likely remain inevitable. The spillage of crude oil and its fractions in the environment is responsible for many hydrocarbon polluted sites (Environmental agency, 2006). Soil contamination by hydrocarbons can cause extensive damage to
* Corresponding author. E-mail address: [email protected] (U.C. Ugochukwu). http://dx.doi.org/10.1016/j.chemosphere.2017.01.080 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
the local ecosystems affecting vegetation and wildlife adversely. Such contamination can also present a serious public health threat and in extreme case, can render the contaminated area uninhabitable for humans (Sunggyu, 1995). Broadly speaking, there are three key sources of hydrocarbons through which releases of PAHs to the environment can occur, namely, petrogenic, pyrogenic and biogenic sources. PAHs in the environment as a result of oil spill and natural oil seeps are petrogenic whereas those arising from burning wood, volcanoes, petroleum products and forest fires could be regarded as pyrogenic. Biogenic sources include PAHs from biologic or diagenetic processes. Simpson et al. (1998) demonstrated that
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the source of PAH input to the environment in the event of hydrocarbon contamination could be identified. Higher concentrations of retene and perylene in the environment are indicative of terrestrial organic inputs of biogenic origin while the predominant existence of lower molecular weight PAHs such as fluorene and acenaphthene indicates petrogenic origin. Higher concentrations of higher molecular weight PAHs (4e6 membered ring PAHs) such as fluoranthene, pyrene and benzo (a) pyrene in the environment are indicative of pyrogenic origin (Helfrich and Armstrong, 1986; Bence et al., 2007). Groundwater could also be at risk of contamination with PAHs from incidents of agricultural activities, improper waste disposal, underground oil storage tanks etc. Both natural and anthropogenic activities are responsible for the release of PAHs to the environment. Forest fires, volcanoes, biologic/diagenic processes and natural oil seepages etc represent the main natural sources of PAH in the environment while crude oil spills, municipal waste, and combustion of fossil fuels represent anthropogenic sources. PAHs are more recalcitrant to biodegradation than saturated hydrocarbons especially n-alkanes of equal molecular weight. The ability of PAHs to resist biodegradation in addition to their lipophilic nature account for why they persist in the environment and can penetrate tissues to bioaccumulate in organisms in the environment (Tuvikene, 1995; Boehm et al., 1981). Biomagnification of the PAHs takes place as they (the PAHs) are transferred to higher trophic levels in the food chain (Boehm et al., 1981). Very many PAHs have been reported to be mutagenic and carcinogenic making their control in the environment extremely important (ASTDR, 1995). The solubility of monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene and xylene (BTEX) is higher than that of PAHs. Generally speaking, the solubility of aromatic hydrocarbons in water (though poor) decreases with increase in molecular weight and is the reason why the concentration of PAHs in water column is relatively low. Organo-clays are mainly produced by the modification of 2:1 clay minerals such as smectites especially montmorillonites. The modification of clay minerals to produce organo-clays involves the replacement of the clay's interlayer exchangeable inorganic cation with a desired organic cation via ion-exchange reactions. This modification to produce organo-clay leads to a transformation from surface hydrophilcity to hydrophobicity (Hermosin et al., 1992; Groisman et al., 2004). Long chain alkyl ammonium cation such as didecyldimethylammonium (DDDMA) is used in the preparation of organoclay (Cornejo et al., 2008). Organoclays have been reported to be useful in the remediation of organic pollutants such as pesticides (Cornejo et al., 2008). However, it is not yet clear what role organoclays would play during the biodegradation of crude oil PAHs. Previous organo-clay mediated biodegradation studies reported by different authors (Malakul et al., 1998; Biswas et al., 2015) involved only single PAHs as substrate such as naphthalene and phenanthrene respectively. It was demonstrated in these studies that organoclay enhanced the biodegradation of the above substrates. There are scarcely any known studies on the effects of organo-montmorillonites or organo-saponites on the biodegradation of mixed PAHs as found in crude oil. When studying the effect of organo-clays, or any other material, on the microbial degradation and adsorption of PAHs present in petroleum, it is essential to do so in the presence of numerous PAHs rather than individual PAH fractions so as to understand how the biodegradation and adsorption of the individual PAHs proceed in the presence of other PAHs. Hence, this study would seek to understand how the presence of other PAHs affects the removal of the individual PAHs via organo-clay mediated adsorption and biodegradation. The effect of organoclays on the biodegradation of crude oil saturated fraction has been reported (Ugochukwu et al., 2013) however, our knowledge of the effect of organo-clays during
29
biodegradation of crude oil PAHs is grossly limited. The result of the microcosm experiments carried out to investigate the ability of organo-clays in comparison with the unmodified clays to adsorb and stimulate or hinder the microbial degradation of crude oil PAHs is reported in this paper. The following objectives were pursued: (i) To produce organo-clay with hydrophobic surface by having the DDDMA successfully intercalated (ii) To investigate how the individual PAHs in crude oil respond to microbial degradation and adsorption on the organo-clays in the presence of other PAHs compared to unmodified clays?
2. Materials and methods Bentonite (Berkbent 163) was supplied by Steetley Bentonite & Absorbent Ltd (now Tolsa UK Ltd; www.tolsa.com) while natural saponite was collected from the Orrock Basalt Quarry Burntisland, Scotland (National Grid Reference NT 218887; Cowking et al., 1983). Clay mineral samples used in this study were a saponite and a montmorillonite separated by fractionation from the altered basalt rock samples and from the reference bentonite Berkbent 163, respectively, following the method of Cowking et al. (1983) modified as follows: Approximately 1000 g of basalt rock was crushed to particle size less than 10 mm and soaked in a 5 L beaker filled with water to 4.5 L mark. This mixture was allowed to stand overnight and the supernatant liquid decanted and sonicated for 3 min. Gravity sedimentation was employed for the settling of the crushed Orrock Basalt rock so as to separate particle size less than 2 mm by progressive size fractionation applying Stoke's Law. The same procedure was repeated for Berkbent 163 Bentonite except that there was no crushing and overnight standing as this reference material is provided as a powdery material. The microbial communities that were involved in the microbial degradation of crude oil PAHs were isolated from beach sediment samples that were collected in sterilised glass bottle from a site at St Mary's Lighthouse near Whitley Bay, Newcastle Upon Tyne, United Kingdom (National Grid Reference: NZ 352 754) and stored at 4 C in cold room until the commencement of the experiment. The microbial community has been reported to contain mainly Alcanivorax spp. as the dominant genera in another study carried out by Singh et al. (2009) conducted with exactly the same beach sediments as source of microbial cells. The Bushnel-Haas (BH) broth as the nutrient source, nutrient agar and all other chemicals were supplied by Sigma Aldrich. The undegraded North Sea crude oil sample for the experiments was supplied by British Petroleum (BP). 2.1. Organo-clay preparation The organo-clay minerals were prepared following the procedure reported in Ugochukwu et al., 2014a. Very succinctly, this procedure consisted in adding sufficient amounts of didecyldiammonium (DDDMA) bromide to the montmorillonite and saponite suspensions to reach 35% of their respective CEC followed by centrifugation, repeated washing of the clay with de-ionized water, drying and storing in a desiccator. 2.2. Characterization of the clay samples 2.2.1. XRD, FTIR, CEC, EGME-Surface area and TOC The untreated saponite and montmorillonite and the prepared organo-clays were characterized in detail using various techniques. The method employed for the X-Ray Diffraction (XRD), Fourier
30
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Transform Infra-Red (FTIR), Cation Exchange Capacity (CEC), Ethylene Glycol Monoethyl Ether (EGME)- Surface Area, and Total Organic Carbon (TOC) data are as reported in Ugochukwu et al. (2014a). The FTIR and XRD diffraction patterns of these clay samples having not been reported in any of the earlier published work is presented in the results section of this manuscript.
spectrophotometer. The obtained growth curve enabled determination of the exponential and stationary phase and hence an estimate of when there were active cells in the system. Thus active cells were harvested for preparing the subcultures. In addition to monitoring cell growth by absorbance method, cell growth was also monitored via standard cell plating.
2.2.2. Gas Chromatography-flame ionization detector and Gas Chromatography-Mass Spectrometer 2.2.2.1. Gas Chromatography-flame ionization detector (GC-FID). HP 5890 series II gas chromatograph equipped with a split/splitless injector and flame ionization detector (FID) was the Gas Chromatography (GC) instrument used. The sample was injected using an HP 7673 autosampler. The separation of the crude oil hydrocarbon compounds was carried out on an Agilent HP-5 capillary column (30 m 0.25 mm) coated with 5% phenylmethyl-polysiloxane (0.25 mm thick) stationary phase. The GC oven temperature was programmed from 50 C for 2 min and then ramped at 4 C/min up to 300 C where it was held for 20 min. The carrier gas used was hydrogen at a flow rate of about 2 mL/min at initial pressure of 100 kPa. The GC data was acquired using Atlas software on HP computer desktop.
2.3.2. Microbial degradation experiment in the presence of organoclay minerals The detailed procedure of the microbial degradation experiments in the presence of organosaponite, organomontmorillonite, and the unmodified clays has been reported in a previous study (Ugochukwu et al., 2014a). The procedure involved microcosm experiments consisting 0.25 g of clay and 0.050 g of crude oil in 10 mL of Bushnel-Haas medium with the microbial cells including control experiments to account for abiotic processes due to volatilization and adsorption. The experimental set up was such that the following test and control experiments were in place: ➢ Test experiment consisted of 0.25 g (organo-clay mineral sample or unmodified clay sample) þ 0.05 g crude oil þ 10 mL BH medium þ microbial cells
2.2.2.2. Gas Chromatography-Mass Spectrometer (GC-MS). The Gas Chromatography-Mass Spectrometer (GC-MS) used was Agilent 6890 Gas Chromatography instrument with split/splitless injector (280 ), connected to a 5975 MSD mass spectrometer. The stationary phase of the GC was a fused silica capillary column (30 m 0.25 mm) coated with 5% phenyl polysiloxane (HP-5). The GC oven was programmed from 40 C for 5 min and then ramped at 4 C/min, up to 300 C where it was held for 20 min. The carrier gas used was helium at a flow rate of about 1 mL/min and initial pressure of 50 kPa while split at 30 mL/min. The sample was injected by an HP7683 autosampler and the split opened after 1 min to vent the solvent. The mass spectrometer used electron ionization energy of 70 eV. The following operating conditions were used: Source temperature of 230 C; Quadrupole temperature of 150 C; Multiplier voltage of 2000 V; Interface temperature of 310 C. Selected ion monitoring (SIM) mode was used to monitor ions of m/z values: 156, 166, 170, 178, 180, 184, 192, 198, 206, 212, and 236 for various peaks used for identifying the PAHs namely: dimethylnaphthalenes, fluorene, trimethylnaphthalene, phenanthrene, methylfluorene, dibenzothiophene, methylphenanthrenes, methyldibenzothiophenes, dimethylphenanthrenes, dimethyldibenzothiophenes, and triaromatic steroids respectively.
The test experiment and Control-1 experiment are the only experiments that are biotic as they contain microbial cells as opposed to Control-2 and Control-3 experiments that do not contain microbial cells. Whereas Control-1 helps in the evaluation of biodegradation (a biotic process) as mediated by clays, Control-2 helps in the evaluation of adsorption (abiotic process) mediated by clays. See section 2.5. The Test experiment and Control-3 were used to evaluate biodegradation in the presence of organo-clay minerals or unmodified clay minerals whereas Control-1 and Control-2 were used to estimate biodegradation taking place in the absence of clay. Control-2 and Control -3 were used for estimating adsorption.
2.3. Biodegradation experiments
2.4. Extraction of hydrocarbons and analysis
The microorganisms indigenous to the collected sediments were proliferated subsequent to isolation and thereafter employed for microcosm microbial degradation experiments.
Extraction of the residual oil after incubation was carried out as follows: Three stages of extraction with 30 mL of dichloromethane (DCM) for each stage were employed to extract the residual oil subsequent to spiking with squalane as surrogate standard. Following the procedure of Bennett et al. (2002), the extract was further extracted on solid phase extraction (SPE) columns to separate the hydrocarbons from the polars. Prior to GC-FID and GCMS analysis, the samples were spiked with internal standards namely, heptadecylcyclohexane and 1,1-binaphthyl. The relative response factor (RRF) of the surrogate standard varied between 0.78 and 0.8 which is acceptable. However, for computing the percentage recovery of the surrogate standard, RRF was corrected to 1.0. The percentage recovery of the surrogate standard lied between 70% and 120% which is within acceptable range (USEPA method 8270). The selected aromatic compounds studied were: isomers of dimethyl naphthalene, isomers of trimethyl naphthalene, fluorene, isomers of methyl fluorene, phenanthrene, isomers
2.3.1. Enrichment culture preparation for microbial cell proliferation To prepare the initial enrichment culture, 0.5 g of crude oil, 20 g of the Whitley Bay beach sediment and 100 mL of BH medium were mixed in a 250 mL conical flask under a sterile atmosphere provided by bunsen burner flame. The flask was then closed with a cotton wool and continuously shaken while being incubated. Several weights (0.5 ge2.0 g) of the crude oil were used as carbon source in several subcultures after the initial culture so as to proliferate and purify the cells as they go through several serial transfers. The growth of the microbial cells were monitored in the subculture for each case by collecting 1 mL of cell suspension every two days and measuring absorbance at 600 nm using a UVeVisible
The control experiments were given as follows: ➢ Control-1: 10 mL BH medium þ microbial cells þ 0.05 g crude oil without clay ➢ Control-2: 10 mL BH medium þ 0.05 g crude oil without clay ➢ Control-3: Clay sample (organo-clay mineral sample or unmodified clay mineral sample) þ 10 mL BH medium þ 0.05 g crude oil.
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of methylphenanthrene, isomers of dimethylphenanthrene, dibenzothiophene, isomers of ethyldibenzothiophene, isomers of dimethyldibenzothiophene and triaromatic steroids.
Aromatic compound ð%Þ as adsorbed ¼
31
Xc2 Xcy 100 Xc2
(6)
2.5. Quantifying the biodegradation and adsorption of the PAHs 3. Results/discussion The mass and percentage of the aromatic compounds removed by biodegradation and adsorption are as described in the following equations:
Aromatic compounds as biodegraded ðmassÞ ¼ Xcy Xr Aromatic compound as biodegraded ð%Þ ¼
Xcy Xr Xcy
(1)
100 (2)
Xcy ¼ mass ðgÞ of the residual aromatic compound for the clay control sample Xr ¼ mass ðgÞ of the residual aromatic compound for the test sample The clay control experiment consisted of clay, Bushnel Haas medium, and crude oil with no microorganism added (abiotic experiment) whereas the test sample experiment consisted of clay, Bushnel Haas medium, crude oil, and microorganisms. Two other control experiments were conducted: Control 1 consisted of Bushnel-Haas medium, crude oil, and microorganisms but no clay whereas Control 2 consisted of Bushnel-Haas medium and crude oil only with neither clay nor microorganisms. For control-1,
Aromatic compound as biodegraded ðmassÞ ¼ Xc2 Xcx Aromatic compound as biodegraded ð%Þ ¼
(3)
Xc2 Xcx 100 Xc2 (4)
Xc2 ¼ mass ðgÞof the residual aromatic compound for control 2 Xcx ¼ mass ðgÞ of residual aromatic compound for control 1 Aromatic compound ðmassÞ as adsorbed ¼ Xc2 Xcy
(5)
3.1. Characterization of the clay samples 3.1.1. XRD patterns and FTIR bands of the organoclay samples The XRD patterns of the clay samples for both the organo-clay samples and the unmodified clays are presented in Figs. 1e3 showing the d001 reflections of the clay samples under air-dry, heat treated (300 C) and glycolated conditions. The FTIR spectra of the clay samples for both the organo-clay and unmodified clay samples are presented in Figs. 4e7. Characteristic FTIR bands for the saponites are as shown in Figs. 4 and 5 whereas the bands characterizing montmorillonites are as shown in Figs. 6 and 7. The basal air-dry d001 spacing for unmodified montmorillonite (BU), unmodified saponite (SU), organo-montmorillonite (BO), and organosaponite (SO) as deduced from Fig. 1 are 12.5 Å, 14.5 Å, 14.2 Å and 14.0 Å respectively. The observed reflections at 202q and 29.52q for BU and BO correspond to 4.27 Å (quartz) and 3.03 Å (calcite) respectively present in the montmorillonite clay mineral. The basal glycolated d001 spacing for BU, SU, BO, and SO as deduced from Fig. 2 are 17.1 Å, 16.2 Å, 16.8 Å, 14.7 Å whereas the heat treated d001 spacing as deduced from Fig. 3 are 10.6 Å, 10.7 Å, 13.2 Å and 13.8 Å respectively. Hence, the layer collapse of the organo-clays on heat treatment at 300 C is lower in comparison with the unmodified clays and is due to the intercalation of didecyldimethylammonium (DDDMA) in the interlayer of the clay samples. The two IR bands at 3390 cm1 and 3573 cm1 (Fig. 4) reflect octahedral character. The absorption band at 3570 cm1 is due to Mg/Fe2þ-OH stretch vibration of Fe-rich saponite. The absorption bands at 2861 cm1 and 2935 cm1 (Fig. 5) observed with organosaponite sample (SO) are assigned to symmetrical and assymetrical vibration stretch of C-H2 respectively from the hydrocarbon moiety of DDDMA used in the preparation of the organoclay sample. The broad IR absorption band at 3623 cm1 in the spectra for unmodified and organo-montmorillonite as shown in Fig. 6 is assigned to OH-stretching of AlAlOH which is typical of dioctahedral smectites such as montmorillonites (Wilson, 1987). Also, the absorption bands at 2861 cm1 and 2935 cm1 observed in Fig. 7 is due to symmetrical and assymetrical vibration stretch of C-H2 respectively from the hydrocarbon moiety of DDDMA (Gunzler and
Fig. 1. XRD patterns of the clay samples under air-dry conditions. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organosaponite.
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Fig. 2. XRD patterns of the clay samples as glycolated. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organo-saponite.
Fig. 3. XRD patterns of the clay samples as heat treated at 300 C. BU ¼ unmodified montmorillonite; BO ¼ organo-montmorillonite; SU ¼ unmodified saponite and SO ¼ organosaponite.
Fig. 4. Infrared spectra of unmodified saponite (SU) and organosaponite (SO) (3000e4000 cm1).
Gremlich, 2002). The XRD patterns and FTIR bands suggest that the DDDMA was successfully intercalated in the interlayer of the montmorilonite and saponte samples.
3.1.2. EGME-surface area, CEC and TOC The EGME-surface area of the clay samples as reported in Ugochukwu et al., 2014a indicate that the specific surface area of the unmodified clays [unmodified montmorillonite (645 m2/g) and unmodified saponite (473 m2/g)] are higher than those of the organoclays (organomontmorillonite (471 m2/g) and
organosaponite (330 m2/g)). The TOC for the unmodified clay samples did not contain organic carbon while the TOC values for the organoclay were 3.4 and 7.3% for organo-saponite and organomontmorillonite respectively indicating that the organmontmorillonite contained more organic phase as was expected given the higher CEC value of montmorillonite (83.3 meq/100 g) than saponite (35.4 me/100 g).
3.1.3. Hydrophobicity In this study, the XRD and FTIR data indicate that the DDDMA
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33
Fig. 5. Infrared spectra of unmodified saponite (SU) and organo-saponite (SO) (2600e3400 cm1).
Fig. 6. Infrared spectra of unmodified montmorillonite (BU) and organomontmorillonite (BO) (3500e4000 cm1).
Fig. 7. Infrared spectra of unmodified montmorillonite (BU) and organo-montmorillonite (BO) (2700e3100 cm1).
was successfully intercalated in the organoclay samples. Furthermore, the TOC data confirms that the intercalation of the DDDMA was such that the organic phase of organomontmorillonite was more extensive than that of organosaponite. This derives from the fact that the CEC of montmorillonite is higher than that of saponite. 3.2. Biodegradation of crude oil PAHs on organomontmorillonite and organosaponite in comparison with the unmodified clays The preservation of n-C17/Pristane and n-C18/Phythane ratios at 2.0 and 2.1 respectively in Control-2 and all the clay control samples (where there was no addition of microbe) is an indication that there was no microbial activity in these control experiments (Fig. 8b, d, f,
h, and j). The disappearance of the hydrocarbon peaks in the test experiments indicates microbial activity (Fig. 8a, c, e, g, and i). The disappearance of the peaks appears to be more with unmodified clays especially unmodified montmorillonite hence providing evidence of enhanced biodegradation of the selected crude oil aromatic compounds (Fig. 9). In addition to relatively higher EGMEsurface area compared to organomontmorillonite, the unmodified montmorillonite seems to have the ability to promote contacts between nutrients and microbes to a degree that is sufficient enough to stimulate biodegradation of crude oil PAHs (Warr et al., 2009). There is usually a sequence of events that take place prior to cell colonization of clay surface. This sequence of events begins with transportation of cells to clay surface by means of diffusive
Fig. 8. (a) Chromatogram of Control-1 (no clay). (b) Chromatogram of Control-2 (no clay and no microbe). (c) Chromatogram of clay test sample containing unmodified montmorillonite. (d) Chromatogram of clay control sample containing unmodified montmorillonite. (e) Chromatogram of clay test sample containing organomontmorillonite. (f) Chromatogram of clay control sample containing organomontmorillonite (g) Chromatogram of test sample containing unmodified saponite (h) Chromatogram of clay control sample containing unmodified saponite. (i) Chromatogram of test sample containing organosaponite (j) Chromatogram of clay control sample containing organosaponite. The standards are labeled as: squ ¼ squalane; hdch ¼ heptadecylcyclohexane; 1,1-binaph ¼ 1,1-binaphthyl; andro ¼ 5a-androstane. Clay test samples contain both oil and microbes in a BH medium while clay control samples contain only oil in a BH medium but no microbes.
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35
Fig. 8. (continued).
transport, convective transport, cell motility and sedimentation under quiescent conditions (Van Loosdrecht et al., 1990). However, for there to be contact resulting from these modes of transport, the energy barrier imposed by the repulsive forces of interaction between the clay surface and the microbial cell must be overcome. Once it is overcome, contact is initiated and the cells adhere to the surface. The adhesion is reversible in suspensions with secondary minimum of total Gibb's free energy of interaction and irreversible in the primary minimum (Busscher et al., 1984; Van Loosdrecht et al., 1989). Subsequent to adhesion is firm attachment and surface colonization by the cells (Van Loosdrecht et al., 1990). Replacing the montmorillonite interlayer cations with considerable amount of DDDMA has the tendency of increasing repulsive energy of interaction thereby lowering the chances of adhesion. Also, the presence of DDDMA for the organomontmorillonite makes it have a lower specific surface area than the unmodified montmorillonite thereby making cell colonization on organomontmorillonite to be relatively less extensive. The nutrients employed in this study are hydrophilic and have a higher tendency to accumulate on clay surfaces that are hydrophilic than those that are not hence, nutrient accumulation on unmodified montmorillonite was expected to be higher than for organomontmorillonite.
The biodegradation of low molecular weight (LMW) PAHs such as dimethylnaphthalenes (DMNs) appears to be neither stimulated nor inhibited by the unmodified clays as the observed percentages of total dimethylnaphthalenes (SDMN) biodegradation are, for both the saponite and montmorillonite, very similar to that obtained in Control-1, in the absence of clay (88e95%). The biodegradation of triaromatic steroids is relatively enhanced by the unmodified clays in comparison with phenanthrenes. Though the extent of biodegradation of the aromatic compounds tends to decrease as the fused rings increase, stimulation of biodegradation of aromatic compounds by unmodified montmorillonite appears to relatively increase as the molecular weight of fused rings increase. The organoclay samples, especially organo-montmorillonite (BO), were inhibitory to the biodegradation of the low molecular PAH (LWMPAH) such as SDMN, as evidenced by the lower % biodegradation than in Control-1 (Fig. 9). These results suggest that these LMW PAHs like DMNs may have reached the adsorption sites of the organo-clay (before the other analytes) and interacted strongly enough with the organic phase of the organo-clay via hydrophobic interaction to render them partly unavailable for biodegradation. This hydrophobic interaction is expected to increase with the amount of organic phase of the organo-clay, thereby explaining
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Fig. 9. Biodegradation of the selected PAHs. SDMN ¼ sum of all isomers of dimethylnaphthalenes, STMN ¼ sum of all isomers of trimethylnaphthalenes, SF ¼ sum of fluorene and isomers of methyl fluorene, SP ¼ sum of phenanthrene and isomers of methyl- and dimethylphenanthrene, SDBT ¼ sum of dibenzothiophene and isomers of methyl- and dimethyldibenzothiophene, STAS ¼ sum of the isomers of triaromatic steroids. BO ¼ organomontmorillonite; BU ¼ unmodified montmorillonite; SO ¼ organosaponite; SU ¼ unmodified saponite; Control-1 ¼ BH þ oil þ cells (no clay). Values are reported as mean ± one standard error.
why BO (7.3% TOC) is more inhibitory to the biodegradation of the DMNs than SO (only 3.4% TOC). The organo-montmorillonite was also inhibitory to the biodegradation of the trimethylnaphthalenes (TMNs) as reflected by % biodegradation of total trimethylnaphthalenes (STMN) of 24% compared to 51% in Control-1 while the only inhibitory behavior of organo-saponite was with respect to DMNs. Considerably, enhanced biodegradation occurred in the presence of organo-saponite for all other groups of analytes compared to Control-1. The higher molecular weight of these analytes may have limited their diffusion to the organic phase of the organo-saponite, as transport to the organic phase is most likely diffusion controlled. In addition, the organic phase of the organosaponite is not as extensive as that of organo-montmorillonite. Most of the adsorptive sites of the organo-saponite were possibly saturated with DMNs, leaving limited spare adsorption sites for other PAHs, which were hereby more readily available for biodegradation. The % biodegradation of total triaromatic steroids (STAS) in the presence of organo-saponite and unmodified saponite are similar while the % biodegradation of total trimethylnaphthalenes (STMN), total fluorenes (SF), total phenanthrenes (SP) and total dibenzothiophenes (SDBT) are slightly higher in the presence of organo-saponite compared to that reached in the presence of the unmodified saponite (Fig. 9). The observed results indicate that the organo-saponite stimulate the biodegradation of those PAHs that are of higher molecular weight than DMNs by means of preference to adsorbing the DMNs and making the higher molecular weight PAHs available for biodegradation while rendering the adsorbed lower molecular weight compounds (DMNs) more difficult to biodegrade than usual. Hence, whether or not an organo-clay is going to be inhibitory to a given analyte will depend on whether the organo-clay would have any chance of holding on to the analyte by hydrophobic interaction. For the organo-clay to be able to inhibit the biodegradation of the various groups of the PAHs found in the crude oil, it must have sufficient organic phase with correspondingly extensive adsorptive sites that cannot be fully occupied by just a few LMW PAHs. Organo-saponite appears to stimulate the biodegradation of most PAHs even more than the unmodified saponite indicating that organoclays under certain conditions could enhance biodegradation of some PAHs. This ability of organosaponites to enhance the biodegradation of STMN, SF, SP, and SDBT nearly matches that of unmodified montmorillonite (Fig. 9). These findings lead to the suggestion that organoclays can actually be manipulated to either inhibit or enhance biodegradation of PAHs of choice in the crude oil. Previous study by Malakul et al. (1998)
and Biswas et al. (2015) demonstrated biodegradation enhancement of naphthalenes and phenanthrenes as single PAH substrates in the presence of organoclays but this work has studied the biodegradation of these substrates in the presence of other PAH substrates found in crude oil. Whereas this study in which organosaponite enhanced the biodegradation of phenanthrenes is in agreement with the study by Biswas et al. (2015), it is important to point out that Biswas et al. (2015) employed bentonite as clay and also did not study other PAHs alongside the phenanthrenes. The organosaponite and organomontmorillonite having inhibited the biodegradation of LMW PAHs such as dimethylnaphthalenes contrasts the study of Malakul et al. (1998) that enhanced the biodegradation of LMW PAHs such as naphthalenes. It is therefore possible that the different result obtained in this study is as a result of the presence of other PAHs in the crude oil among other possible reasons. 3.3. Adsorption of the crude oil PAHs on organo-montmorillonite and organo-saponite in comparison with the unmodified clays Adsorption of the selected crude oil aromatic compounds on organo-clays is significantly negative for the LMW PAHs, such as DMNs (Fig. 10). The implication of the negative adsorption is that the organoclay control samples have retained the analytes more than the volatility abiotic control, Control-2 where the DMNs as LMW PAH compounds must have been lost to volatilization than the HMW PAH compounds. With the organoclay, as shown in Fig. 9, this negative adsorption tends to be higher with LMW of the crude oil aromatic compounds. This corroborates the fact that the organoclays interact with the aromatic compounds hydrophobically and the interaction is strong enough to reduce the availability of the aromatic compounds for both biodegradation and volatilization. The higher negative adsorption of the LMW compounds such as DMNs indicates that these compounds reach the adsorptive sites of the organo-clay before the other analytes and are therefore largely adsorbed. Hydrophobicity of the organo-clays is due to the presence of organic phase in the interlayer of the clay minerals, in agreement with other reported studies (Hermosin et al., 1992; Groisman et al., 2004). The negative adsorption values reported with organo-clay controls (Fig. 10) is due to the fact that during extraction, the dichloromethane (DCM) used for extraction was able to overcome the hydrophobic interaction between the aromatic compounds and the organic phase of the organo-clay. Hence, the DCM desorbed and extracted the aromatic compounds,
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37
Fig. 10. Adsorption of the selected PAHs. CBU, CBO, CSU and CSO represent clay controls (in the absence of microbial cells) for unmodified montmorillonite, organomontmorillonite, unmodified saponite and organosaponite respectively. Values are reported as mean ± one standard error.
especially the LMW PAHs, which are expected to be more abundant in the organic phase of the organoclays. This therefore makes the concentration of the LMW PAHs in the organoclay controls outstrip that in Control-2 where there must have been losses due to volatilization. It follows therefore that hydrophobic interaction that holds the LMW PAHs in the organic phase of the organoclay is strong enough to prevent the LMW PAHs from volatilizing. Unmodified clay samples demonstrated moderate positive adsorption of the aromatic compounds, likely due to another kind of interaction (other than the hydrophobic interaction encountered with the organo-clay) known as p-cation interaction. Aromatic hydrocarbon compounds are donors of p electrons and therefore lend themselves to adsorption via p-cation interaction with the interlayer cations of the unmodified clay minerals. This p-cation interaction can be reasonably strong (in the order of that of hydrogen bonding) as indicated by recent molecular modelling and spectroscopic studies of Zhu et al. (2004). This type of interaction is generally stronger than the hydrophobic interaction discussed earlier. Hence, the clay controls of the unmodified clays showed positive adsorption of the aromatic compounds due to the relative strength of the p-cation interaction which cannot be overcome by DCM during extraction. The adsorption of the selected PAHs on the organo-clay and unmodified clay samples during the biodegradation process is as presented in Fig. 10.
Organo-montmorillonite with extensive organic phase was inhibitory to the biodegradation of all the PAHs whereas organosaponite, with less extensive organic phase, was stimulatory to the biodegradation of some of the PAHs such as phenanthrene. The biodegradation of triaromatic steroids was relatively enhanced by the unmodified clays in comparison with phenanthrenes. Though the extent of biodegradation of the aromatic compounds decreased as the fused rings increased, stimulation of biodegradation of aromatic compounds by montmorillonite relatively increased as the molecular weight of fused rings increased. This study therefore indicates that organoclays can actually be tailored to achieve biodegradation of some PAHs by carefully selecting the appropriate clay mineral for the production of organo-clays under certain conditions. Also, the inevitability of poor biodegradation of LMW PAHs in the presence of other PAHs on organo-clays and the reasons for that have been identified in this study. Acknowledgements We thank Petroleum Technology Development Fund (PTDF) of the Federal Republic of Nigeria for funding this project. Generally, we are grateful to the School of Civil Engineering and Geosciences of the University of Newcastle Upon Tyne, United Kingdom for providing the facilities used in this study. We appreciate Berny Bowler, Paul Donohue, Phil Green and Ian Harrison for the laboratory support received from them.
4. Conclusion Organo-montmorillonite and organo-saponite were both inhibitory to biodegradation of LMW PAHs such as dimethylnaphthalenes and the inhibition increased with increase in the amount of organic phase in the organo-clay. Organo-montmorillonite was therefore more inhibitory than organo-saponite to the biodegradation of LMW PAHs. Obtained results suggest that the organo-clay inhibited biodegradation of the LMW PAHs as a result of the hydrophobic interaction between the organic phase of the organoclay and the aromatic hydrocarbons. This hydrophobic interaction, though weak, was strong enough to render the aromatic compounds unavailable for degradation by the microorganisms. This interaction also made the LMW aromatic compounds, such as the dimethylnaphthalenes, resist volatilization. Hence, the organoclays retained and contained more of the LMW PAHs such as the dimethylnaphthalenes, trimethylnaphthalenes and fluorenes than the abiotic Control. Generally, the biodegradation of LMW aromatic compounds such as dimethylnaphthalenes was not enhanced by clay minerals.
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