Journal of Environmental Management 144 (2014) 197e202
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Microbial degradation of crude oil hydrocarbons on organoclay minerals Uzochukwu C. Ugochukwu*, David. A.C. Manning, Claire I. Fialips School of Civil Engineering and Geosciences, University of Newcastle Upon Tyne, Drummond Building, Newcastle Upon Tyne NE1 7RU, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 29 April 2014 Accepted 2 June 2014 Available online
The role of organoclays in hydrocarbon removal during biodegradation was investigated in aqueous clay/ oil microcosm experiments with a hydrocarbon degrading microorganism community. The clays used for this study were Na-montmorillonite and saponite. These two clays were treated with didecyldimethylammonium bromide to produce organoclays which were used in this study. The study indicated that clays with high cation exchange capacity (CEC) such as Na-montmorillonite produced an organomontmorillonite that was inhibitory to biodegradation of the crude oil hydrocarbons. Extensive hydrophobic interaction between the organic phase of the organoclay and the crude oil hydrocarbons is suggested to render the hydrocarbons unavailable for biodegradation. However, untreated Namontmorillonite was stimulatory to biodegradation of the hydrocarbons and is believed to have done so because of its high surface area for the accumulation of microbes and nutrients making it easy for the microbes to access the nutrients. This study indicates that unlike unmodified montmorillonites, organomontmorillonite may not serve any useful purpose in the bioremediation of crude oil spill sites where hydrocarbon removal by biodegradation is desired within a rapid time period. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Microbial cells Organoclay Unmodified clay Total petroleum hydrocarbon Adsorption Biodegradation
1. Introduction Clay minerals provide surfaces that may be inhibitory or stimulatory to biodegradation of organic compounds (Filip, 1973; Guerin and Boyd, 1992; Scow and Alexander, 1992; Knaebel et al., 1994; Van Loosdrecht et al., 1990; Chaerun and Tazaki, 2005; Warr et al., 2009). Several studies have demonstrated that some solid surfaces such as clay minerals are able to stimulate microbial growth enhancing biodegradation of organic compounds including hydrocarbons (Stotzky and Rem, 1966; Van Loosdrecht et al., 1990; Chaerun and Tazaki, 2005; Tazaki and Chaerun, 2008; Warr et al., 2009). Clay minerals are ubiquitous in the natural environment, non-toxic and available economically as commercial products (Tazaki and Chaerun, 2008). They play a vital role in terrestrial biogeochemical cycles and containment of toxic waste materials (Bergaya et al., 2006). Clay minerals are believed to have significant impacts on the environmental fate of pollutants such as oil spills (Chaerun and Tazaki, 2005). In addition to being able to stimulate microbial growth, some clay minerals are believed to have the ability to sorb organic and inorganic chemicals and microorganisms
* Corresponding author. E-mail address: [email protected] (U.C. Ugochukwu). http://dx.doi.org/10.1016/j.jenvman.2014.06.002 0301-4797/© 2014 Elsevier Ltd. All rights reserved.
which might be beneficial in some remediation processes (Lipson and Stotzky, 1983; 1984; Lee et al., 1990; van Loosdrecht et al., 1990; Guerin and Boyd, 1992; Khanna and Stotzky, 1992; Tapp and Stotzky, 1995; Vettori et al., 1999; Sposito et al., 1999; Lin and Puls, 2000; Murray, 2000; Churchman et al., 2006; Warr et al., 2009). For a few decades now, organoclays (especially those produced from alkyl ammonium cations) have been used as adsorbent for organic compounds. The increased environmental awareness and the need to remediate sites polluted with organic compounds (especially pesticides and crude oil hydrocarbons) have necessitated research advances in this area (Cornejo et al., 2008). Organoclay is quite common naturally in soils where the presence of organic compounds always occurs with the potential of generating organic cations. It is therefore important to understand how the organoclay interacts with hydrocarbon during its biodegradation. Producing organoclays in practice requires the replacement of the interlayer exchangeable inorganic cations with organic cations through ion-exchange reactions. The resultant organoclay modifies the surface of the original clay mineral from being hydrophilic to being hydrophobic (Hermosin et al., 1992; Groisman et al., 2004a). However, the role of organoclays during the microbial degradation of crude oil hydrocarbons has not been reported. During bioremediation of oil spill sites, it is desirable to effect biodegradation of hydrocarbons within a rapid time period. This study would enable
198
U.C. Ugochukwu et al. / Journal of Environmental Management 144 (2014) 197e202
the understanding of the usefulness of organoclays in this regard in comparison to the unmodified clay forms. This paper reports the result of a laboratory study conducted to investigate the potential ability of organoclays and their unmodified counterparts to enhance or inhibit the biodegradation of crude oil hydrocarbons. The report therefore addressed the following questions. (i) Do organoclays stimulate or inhibit the biodegradation of crude oil hydrocarbons in comparison with their unmodified counterparts? (ii) What are the factor(s) responsible for the behaviour observed in (i) above? (iii) Does adsorption of hydrocarbons occur alongside biodegradation with the clay samples?
2. Materials and methods Clay mineral samples used in this study were saponite and montmorillonite separated by fractionation from an altered basalt rock samples and from Berkbent 163 Bentonite respectively. 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). Microbial communities responsible for the biodegradation of crude oil hydrocarbons were isolated from beach sediment sample consisting of fine sand particles collected in a sterilised glass bottle (Duran) from a site at St Mary's Lighthouse near Whitley Bay, Newcastle Upon Tyne (National Grid Reference NZ 352 754), United Kingdom and stored at 4 C in cold room until start of the experiment. The BushneleHaas (BH) broth as the nutrient source, nutrient agar and all other chemicals were supplied by Sigma Aldrich. The crude oil was an undegraded North Sea crude oil originally supplied by British Petroleum (BP). 2.1. Characterization of the clay samples The basal spacings of the clay samples were measured by X-Ray Diffraction (XRD). Preferred orientation of the clay particles was performed by deposition of clay suspensions on a glass slide following standard procedure. The slides were air dried and placed in a desiccator containing silica gel to prevent rehydration. Glycolated and heat treated (at 300 C) samples were also prepared following standard procedure. The XRD patterns were acquired using Cu-Ka generated at 40 kV and 40 mA on a PANalytical X'Pert Pro MPD diffractometer fitted with an X'Celerator system. The data were collected over a range of 2e70 2q with a nominal step size of 0.0167 2q and nominal time per step of 1.00 s. Data were interpreted by reference to X'Pert accompanying software program High Score Plus in conjunction with the ICDD Powder Diffraction File 2 database (1999) and the Crystallography Open Database (October 2010; www.crystallography.net). To measure the Fourier-Transform Infrared spectroscopy (FTIR), the samples were prepared using KBr pellets at sample concentration of about 1% and the FTIR spectra were recorded on a Thermo Nicolet Nexus 870 spectrometer fitted with a transmission accessory and equipped with a DTGS detector. The spectra were obtained by collecting 100 scans for each sample over a wavenumber range of 400e4000 cm1 at 4 cm1 resolution. The specific surface area of the clay samples was determined by the EGME method following the method of Carter et al., (1965). The CEC of the clay mineral samples was determined following the ammonium acetate method (Lewis, 1949).
Total organic carbon (TOC) was measured to characterize the organoclay produced in this study. Approximately 100 mg of the organoclay and unmodified clay sample in a porous crucible was treated with sufficient (about 1 mL) hydrochloric acid at a concentration of 4 M in order to remove any carbonates that may be present. After the acid has drained from the crucible for about 4 h, the crucible and sample were dried overnight at 65 C. Residual (organic) carbon was then determined using a Leco CS244 Carbon analyser. This instrument uses a flow of oxygen that enables the sample to be ignited in an induction furnace so as to convert all residual carbon in the sample to carbon dioxide. The infrared detector in the instrument measures the carbon dioxide. Prior to running the sample, a blank was run to detect any background residual carbon. The organic carbon content is calculated as follows:
Total organic carbon; % ¼ Cs Cbl
(1)
where Cs and Cbl are organic carbon content of sample and blank, respectively.
2.2. Organoclay preparation Sufficient amounts of didecyldiammonium (DDDMA) bromide were added to the montmorillonite and saponite suspensions to reach 35% of their respective CEC. The mixture was stirred for 24 h and then centrifuged, after which the supernatant was discarded and the clay washed repeatedly with deionized water followed by drying at 48 C and storing in a dessicator.
2.3. Laboratory biodegradation 2.3.1. Microbial growth Indigenous microbial cells of Whitley Bay sediments were isolated and proliferated via several subcultures prior to use for laboratory biodegradation studies. Microbial growth during culture enrichment was monitored by measuring absorbance of the cell suspension at 600 nm using a UV-Visible spectrophotometer. Microbial growth during the experiments with clay samples was measured via standard plate count. The procedure used in carrying out the cell plate count was as follows: 1 mL of suspension from the clay/oil/microbe suspension was used in preparing 101e107 fold dilution. Then 0.1 mL of each of the above stated dilutions was spotted on the plate and carefully spread. The plates were incubated for 24 h at 28 C and enumerated.
2.3.2. Total petroleum hydrocarbon (TPH) The effect of the clay minerals on biodegradation of the crude oil hydrocarbons was determined by measuring the residual TPH after incubation of the aqueous clay/oil microcosm experiments with hydrocarbon degrading microorganism community for 60 days. Clay (dried)/oil ratio (w/w) of 5:1 was used in all the experiments. Hence, the microcosm consisted of 250 mg of clay mineral and 50 mg of oil in 10 mL of BushneleHaas medium with the microbial cells. Several control experiments were conducted to account for abiotic processes due to volatilization and adsorption, in addition to a positive control. Descriptions of those control experiments are as follows: Control-1 is a positive (biotic) control containing BH medium, oil and microbial cells (but no clay). Control 2 is a negative (abiotic) control containing BH medium and oil (but no clay and no microbial cells).
U.C. Ugochukwu et al. / Journal of Environmental Management 144 (2014) 197e202
Control-3 (clay controls) represents a negative abiotic set of controls containing BH medium, clay and oil but no microbial cells. Control-2 accounts for volatilization and is also used (in combination with the clay controls) to estimate the extent of adsorption of the hydrocarbons (see Section 2.5). Clay controls are important in estimating biodegraded and adsorbed hydrocarbons. All experiments were carried out in triplicate to ensure reproducibility and amenability to statistical analysis. 2.3.3. Effect of spent water from ogranoclay on microbial growth/ activity The bactericidal effect of the spent water from organoclay was tested by collecting the spent water (10 mL) arising from the final washings of the organoclay (organosaponite and organomontmorillonite) and using it to prepare the BH medium by dispersing the appropriate quantity (32.7 mg) of BH broth into it and adding 50 mg of crude oil. The control experiment contained deionized water and is in this study known as Control-3*. Both the test and control experiments were inoculated with the same cells that use crude oil hydrocarbons as carbon source. 2.3.4. Extraction of hydrocarbons and analysis Three stages of extraction (with 302.3.2mL of DCM for each stage) were employed to extract the residual oil (i.e. extractable organic matter-EOM) subsequent to spiking with squalane as a surrogate and TPH quantitation standard. The EOM was extracted on solid phase extraction (SPE) columns following the procedure of Bennett et al., (2002) to separate the hydrocarbons from the residual oil (EOM). Prior to GC-FID analysis, the samples were spiked with internal standards (heptadecylcyclohexane, 5a-androstane 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, a RRF of unity was assumed. The percentage recovery of the surrogate standard was between 70% and 120% which is within acceptable range (USEPA method 8270). 2.4. Analytical Instrumentation for the Hydrocarbons
Table 2 EGME-surface area, total organic carbon (TOC) and cation exchange capacity (CEC) of the clay samples. Sample
Surface area (m2/g)
CEC (meq/100 g)
TOC (%)
BU BO SU SO
645 471 473 330
83.3 e 35.4 e
e 7.3 e 3.4
100 kPa. The GC data was acquired using Atlas software on a HP desktop computer. 2.5. Determination of total petroleum hydrocarbon (TPH) The basis for the assessment of biodegradation in this study is the determination of TPH using GC. This TPH value as determined by GC is the residue of the original TPH after incubation. The determination of the TPH concentration was done by measuring the total GC area between 10 and 70 min followed by quantitation with squalane. 2.5.1. Biodegraded versus adsorbed TPH The following equations were used to determine the weight and percentage of hydrocarbons removed by biodegradation and adsorption.
TPH biodegradedðmgÞ ¼ TPHcy TPHr TPHbiodegradedð%Þ ¼
(2)
TPHcy TPHr 100 TPHcy
(3)
where TPHcy is TPH(mg) of the clay control ( clay þ oil þ BH, but without cells); and TPHr is TPH(mg) of clay test sample ( clay þ oil þ cells þ BH). For control-1:
TPH biodegradedðmgÞ ¼ TPHc2 TPHcx TPH biodegradedð%Þ ¼
2.4.1. GC-FID The GC instrument used was an HP 5890 series II gas chromatograph equipped with a split/splitless injector and flame ionization detector (FID). The sample was injected using a 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% phenylmethylpolysiloxane (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
199
(4)
TPHc2 TPHcx 100 TPHc2
(5)
where TPHc2 is TPH(mg) of Control-2 (BH þ oil: with neither clay nor cells); and TPHcx is TPH(mg) of Control-1 (BH þ oil þ cells: but without clay). 3. Results/discussion 3.1. Characterization of the clay samples 3.1.1. XRD and FTIR The XRD and FTIR data are shown in Table 1 below.
Table 1 Basal spacing of 001 reflections and selected FTIR absorption bands of the clay samples Sample
XRD
FTIR Absorption band (cm1)
d-spacing (Å)
BU BO SO SU
Ethylene glycolated
Air dried
Heat treatment (300 C)
OH-stretch
CeH
Carbonate
17.1 16.8 14.7 16.2
12.5 14.2 14.0 14.5
10.6 13.2 13.8 10.7
3623 3623 3570 3570
e 2861, 2935 2861, 2935 e
1430 1430 1430 1430
BU ¼ unmodified montmorillonite, SU ¼ unmodified saponite, SO ¼ organosaponite, and BO ¼ Organomontmorillonite.
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The d001 reflections at 17.1 Å, 12.5 Å and 10.6 Å (Table 1) following ethylene glycolation, air drying and heat treatment, respectively, are indicative of montmorillonite with mainly sodium in the interlayer whereas the corresponding d001 reflections at 16.2 Å, 14.5 Å and 10.7 Å (Table 1) are indicative of trioctahedral smectite such as saponite with a divalent metal in the interlayer. The FTIR absorption band at 3623 cm1 (Table 1) is due to the OHstretch of AlAlOH typical of montmorillonites while the band at 3570 cm1 indicates the occurrence of Mg/Fe2þ within the trioctahedral smectite (Fe-rich saponite; Shayan et al., 1988). The layer collapse on heat treatment of the organoclay samples is lower than that for the unmodified clay samples and is due to the intercalation of the didecyldimethylammonium (DDDMA) ion in the interlayer of the organoclay. The FTIR absorption bands at 2861 cm1 and 2935 cm1 observed with the organoclay samples were due to CH2 symmetrical and asymmetrical vibration stretch from alkyl chain moiety of the DDDMA. Fig. 1. Effect of organoclay minerals on growth of hydrocarbon degrading bacteria. BO250 ¼ organo montmorillonite; BU-250 ¼ unmodified montmorillonite; SO250 ¼ organosaponite; SU-250 ¼ unmodified saponite; Control-1 ¼ BH þ oil þ cells (no clay). Control-2 ¼ BH þ oil (no clay and no cells). CBU-250, CSU-250, CBO-250 and CSO250 are clay controls where there was no growth (not shown on the chart). Values are reported as mean ± one standard error.
3.1.2. Surface area, TOC and CEC of the clay samples The EGME-surface area, total organic carbon (TOC) and cation exchange capacity (CEC) of the clay samples are shown in Table 2 below. The TOC values confirm that the organoclay contains organic phase unlike the unmodified clay samples. The organoclay samples have lower EGME-surface area in comparison with their unmodified clay counterparts. This lower EGME-surface area may be as a
Fig. 2. (a) Chromatogram (of TPH fraction) showing no biodegradation-sample Control-2. Control-2 ¼ negative control (BH þ oil) no clay and no cells. The nC17/pristane and nC18/ phytane ratios for this sample are 2.0 and 2.1, respectively. The standards are labelled as: Squ ¼ squalane; hdch ¼ heptadecylcyclohexane; 1,1-binaph ¼ 1,1-binaphthyl; andro ¼ 5aandrostane. (b) Chromatogram (of TPH fraction) showing moderate to heavy biodegradation-sample BO-250. BO-250 ¼ organomontmorillonite. (c) Chromatogram (of TPH fraction) showing heavy biodegradation-sample BU-250. BU-250 ¼ unmodified montmorillonite.
U.C. Ugochukwu et al. / Journal of Environmental Management 144 (2014) 197e202
201
Table 3 Maximum cell yield, specific growth rate, % TPH biodegraded and % TPH adsorbed due to the effect of unmodified clay samples and organoclay samples. Values are reported as mean ± one standard error of the mean. Sample
Maximum cell yield (CFU/ml)/108
Specific growth rate (h1) 103
%TPH biodegraded
%TPH adsorbed
SU-250 BU-250 Control-1 BO-250 SO-250 BO-250* SO-250* Control-3*
8.3 ± 0.3 11.2 ± 0.4 8.1 ± 0.3 7.0 ± 0.3 8.0 ± 0.2 8.1 ± 0.2 7.9 ± 0.2 8.0 ± 0.2
9.1 ± 0.4 10.2 ± 0.4 9.1 ± 0.2 8.4 ± 0.2 8.8 ± 0.2 9.1 ± 0.2 9.0 ± 0.2 9.1 ± 0.3
59 ± 3 72 ± 2 50 ± 1 38 ± 3 50 ± 1 50 ± 1 49 ± 1 49 ± 2
27 ± 2.4 27 ± 3.7 e 8.5 ± 0.9 8.5 ± 0.6 e e e
BO-250* ¼ BH in spent water from the organomontmorillonite, SO-250* ¼ BH in spent water from the organosaponite, Control-3* ¼ BH in deionized water. There is no growth in Control 2, hence it (Control 2) has not been entered in this table.
result of EGME being polar and therefore not able to interact with the non-polar portions of the organoclay. 3.2. Microbial growth and biodegradation of crude oil hydrocarbons in the presence of organoclay samples. The effect of organoclay minerals on microbial growth with crude oil hydrocarbons as the only carbon source is presented in Fig. 1. The growth of the hydrocarbon degrading bacteria is affected by the clay mineral samples to varying extents depending on the type and form of the clay mineral sample (Fig. 1). The gas chromatograms of selected samples showing extent of biodegradation are shown in Fig. 2aec. Fig. 2b and c showd that the nC17/pristane and nC18/phytane ratios for the organomontmorillonite (BO-250) and the untreated montmorillonite (BU-250) are not measurable following biodegradation. The effect of the clay mineral samples on microbial growth and biodegradation of crude oil hydrocarbons assessed by the maximum cell yield, specific growth rate, % TPH biodegraded and % TPH adsorbed is presented in Table 3. The maximum cell yield, specific growth rate constant and percentage of biodegradation of the hydrocarbons were highest in sample BU-250 (Table 3). The 2-sample t-test at 95% confidence interval (CI) with respect to maximum cell yield, specific growth rate constant and percentage biodegradation of TPH indicates that unmodified montmorillonite (BU-250) is significantly different from Control-1. Results for the unmodified saponite (SU-250) are not significantly different from Control-1 and therefore this clay is not stimulatory to biodegradation of crude oil hydrocarbons. To some extent, results of the stimulatory role of the montmorillonite presented in this study appear to be consistent with those reported by Warr et al. (2009) although the later did not specifically cover the effect of saponites on microbial growth and biodegradation of crude oil hydrocarbons. However, with respect to adsorption, the study reported here is inconsistent with the study of Warr et al. (2009) which assumed that adsorption of hydrocarbons is not significant during biodegradation of the oil. As shown in Table 3, this is not always true. The absence of a clay control (i.e. an experiment with only clay and oil) in the study of Warr et al. (2009) made it impossible for the authors to estimate losses due to adsorption. The microbial growth and percentage of biodegradation of crude oil hydrocarbons in the presence of unmodified clay minerals appears to increase with the surface area of the clay mineral samples (Fig. 1 and Table 3). Interlayer cations are believed to cause ‘local bridging effect’ (effective delivery of nutrients to cells due to reduction of zeta potential or electrical double layer repulsion) hence clays with interlayer cations and high surface area would tend to stimulate biodegradation of crude oil hydrocarbons (Bright and
Fletcher, 1983; Fletcher and Marshal, 1982;; Liu et al., 1991; Warr et al., 2009). Unmodified montmorillonite has both high surface area (645 m2/g) and interlayer cations which led to the accumulation and delivery of the nutrients to the cells, enhancing their growth and effecting biodegradation of crude oil hydrocarbons (Bright and Fletcher, 1983; Fletcher and Marshal, 1982; Warr et al., 2009). The modification of the clay samples to produce organoclay seemed to lead to inhibition of biodegradation and this is quite noticeable by comparing organomontmorillonite, BO-250 and unmodified montmorillonite, BU-250 (Table 3). Also, on comparing the organoclay samples with Control-1, microbial growth and biodegradation of the hydrocarbons seemed to be depressed in sample BO-250 as the maximum cell yield, specific growth rate constant and percentage TPH biodegraded are lower with this sample than either Control-1 or the unmodified saponite (SO-250). The 2-sample t-test with respect to maximum cell yield, specific growth rate and percentage TPH biodegraded indicates that there is no statistical significant difference between Control-1 and either of the organoclays, BO-250 or SO-250 (P-values >0.05). Therefore BO250 and SO-250 do not stimulate the microbial growth and biodegradation of crude oil hydrocarbons although SO-250 does not depress it either. There is significant statistical difference between BU-250 and BO-250 with respect to maximum cell yield, specific growth rate constant and percentage TPH biodegraded (Pvalues
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Microbial degradation of crude oil hydrocarbons on organoclay minerals Uzochukwu C. Ugochukwu*, David. A.C. Manning, Claire I. Fialips School of Civil Engineering and Geosciences, University of Newcastle Upon Tyne, Drummond Building, Newcastle Upon Tyne NE1 7RU, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 29 April 2014 Accepted 2 June 2014 Available online
The role of organoclays in hydrocarbon removal during biodegradation was investigated in aqueous clay/ oil microcosm experiments with a hydrocarbon degrading microorganism community. The clays used for this study were Na-montmorillonite and saponite. These two clays were treated with didecyldimethylammonium bromide to produce organoclays which were used in this study. The study indicated that clays with high cation exchange capacity (CEC) such as Na-montmorillonite produced an organomontmorillonite that was inhibitory to biodegradation of the crude oil hydrocarbons. Extensive hydrophobic interaction between the organic phase of the organoclay and the crude oil hydrocarbons is suggested to render the hydrocarbons unavailable for biodegradation. However, untreated Namontmorillonite was stimulatory to biodegradation of the hydrocarbons and is believed to have done so because of its high surface area for the accumulation of microbes and nutrients making it easy for the microbes to access the nutrients. This study indicates that unlike unmodified montmorillonites, organomontmorillonite may not serve any useful purpose in the bioremediation of crude oil spill sites where hydrocarbon removal by biodegradation is desired within a rapid time period. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Microbial cells Organoclay Unmodified clay Total petroleum hydrocarbon Adsorption Biodegradation
1. Introduction Clay minerals provide surfaces that may be inhibitory or stimulatory to biodegradation of organic compounds (Filip, 1973; Guerin and Boyd, 1992; Scow and Alexander, 1992; Knaebel et al., 1994; Van Loosdrecht et al., 1990; Chaerun and Tazaki, 2005; Warr et al., 2009). Several studies have demonstrated that some solid surfaces such as clay minerals are able to stimulate microbial growth enhancing biodegradation of organic compounds including hydrocarbons (Stotzky and Rem, 1966; Van Loosdrecht et al., 1990; Chaerun and Tazaki, 2005; Tazaki and Chaerun, 2008; Warr et al., 2009). Clay minerals are ubiquitous in the natural environment, non-toxic and available economically as commercial products (Tazaki and Chaerun, 2008). They play a vital role in terrestrial biogeochemical cycles and containment of toxic waste materials (Bergaya et al., 2006). Clay minerals are believed to have significant impacts on the environmental fate of pollutants such as oil spills (Chaerun and Tazaki, 2005). In addition to being able to stimulate microbial growth, some clay minerals are believed to have the ability to sorb organic and inorganic chemicals and microorganisms
* Corresponding author. E-mail address: [email protected] (U.C. Ugochukwu). http://dx.doi.org/10.1016/j.jenvman.2014.06.002 0301-4797/© 2014 Elsevier Ltd. All rights reserved.
which might be beneficial in some remediation processes (Lipson and Stotzky, 1983; 1984; Lee et al., 1990; van Loosdrecht et al., 1990; Guerin and Boyd, 1992; Khanna and Stotzky, 1992; Tapp and Stotzky, 1995; Vettori et al., 1999; Sposito et al., 1999; Lin and Puls, 2000; Murray, 2000; Churchman et al., 2006; Warr et al., 2009). For a few decades now, organoclays (especially those produced from alkyl ammonium cations) have been used as adsorbent for organic compounds. The increased environmental awareness and the need to remediate sites polluted with organic compounds (especially pesticides and crude oil hydrocarbons) have necessitated research advances in this area (Cornejo et al., 2008). Organoclay is quite common naturally in soils where the presence of organic compounds always occurs with the potential of generating organic cations. It is therefore important to understand how the organoclay interacts with hydrocarbon during its biodegradation. Producing organoclays in practice requires the replacement of the interlayer exchangeable inorganic cations with organic cations through ion-exchange reactions. The resultant organoclay modifies the surface of the original clay mineral from being hydrophilic to being hydrophobic (Hermosin et al., 1992; Groisman et al., 2004a). However, the role of organoclays during the microbial degradation of crude oil hydrocarbons has not been reported. During bioremediation of oil spill sites, it is desirable to effect biodegradation of hydrocarbons within a rapid time period. This study would enable
198
U.C. Ugochukwu et al. / Journal of Environmental Management 144 (2014) 197e202
the understanding of the usefulness of organoclays in this regard in comparison to the unmodified clay forms. This paper reports the result of a laboratory study conducted to investigate the potential ability of organoclays and their unmodified counterparts to enhance or inhibit the biodegradation of crude oil hydrocarbons. The report therefore addressed the following questions. (i) Do organoclays stimulate or inhibit the biodegradation of crude oil hydrocarbons in comparison with their unmodified counterparts? (ii) What are the factor(s) responsible for the behaviour observed in (i) above? (iii) Does adsorption of hydrocarbons occur alongside biodegradation with the clay samples?
2. Materials and methods Clay mineral samples used in this study were saponite and montmorillonite separated by fractionation from an altered basalt rock samples and from Berkbent 163 Bentonite respectively. 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). Microbial communities responsible for the biodegradation of crude oil hydrocarbons were isolated from beach sediment sample consisting of fine sand particles collected in a sterilised glass bottle (Duran) from a site at St Mary's Lighthouse near Whitley Bay, Newcastle Upon Tyne (National Grid Reference NZ 352 754), United Kingdom and stored at 4 C in cold room until start of the experiment. The BushneleHaas (BH) broth as the nutrient source, nutrient agar and all other chemicals were supplied by Sigma Aldrich. The crude oil was an undegraded North Sea crude oil originally supplied by British Petroleum (BP). 2.1. Characterization of the clay samples The basal spacings of the clay samples were measured by X-Ray Diffraction (XRD). Preferred orientation of the clay particles was performed by deposition of clay suspensions on a glass slide following standard procedure. The slides were air dried and placed in a desiccator containing silica gel to prevent rehydration. Glycolated and heat treated (at 300 C) samples were also prepared following standard procedure. The XRD patterns were acquired using Cu-Ka generated at 40 kV and 40 mA on a PANalytical X'Pert Pro MPD diffractometer fitted with an X'Celerator system. The data were collected over a range of 2e70 2q with a nominal step size of 0.0167 2q and nominal time per step of 1.00 s. Data were interpreted by reference to X'Pert accompanying software program High Score Plus in conjunction with the ICDD Powder Diffraction File 2 database (1999) and the Crystallography Open Database (October 2010; www.crystallography.net). To measure the Fourier-Transform Infrared spectroscopy (FTIR), the samples were prepared using KBr pellets at sample concentration of about 1% and the FTIR spectra were recorded on a Thermo Nicolet Nexus 870 spectrometer fitted with a transmission accessory and equipped with a DTGS detector. The spectra were obtained by collecting 100 scans for each sample over a wavenumber range of 400e4000 cm1 at 4 cm1 resolution. The specific surface area of the clay samples was determined by the EGME method following the method of Carter et al., (1965). The CEC of the clay mineral samples was determined following the ammonium acetate method (Lewis, 1949).
Total organic carbon (TOC) was measured to characterize the organoclay produced in this study. Approximately 100 mg of the organoclay and unmodified clay sample in a porous crucible was treated with sufficient (about 1 mL) hydrochloric acid at a concentration of 4 M in order to remove any carbonates that may be present. After the acid has drained from the crucible for about 4 h, the crucible and sample were dried overnight at 65 C. Residual (organic) carbon was then determined using a Leco CS244 Carbon analyser. This instrument uses a flow of oxygen that enables the sample to be ignited in an induction furnace so as to convert all residual carbon in the sample to carbon dioxide. The infrared detector in the instrument measures the carbon dioxide. Prior to running the sample, a blank was run to detect any background residual carbon. The organic carbon content is calculated as follows:
Total organic carbon; % ¼ Cs Cbl
(1)
where Cs and Cbl are organic carbon content of sample and blank, respectively.
2.2. Organoclay preparation Sufficient amounts of didecyldiammonium (DDDMA) bromide were added to the montmorillonite and saponite suspensions to reach 35% of their respective CEC. The mixture was stirred for 24 h and then centrifuged, after which the supernatant was discarded and the clay washed repeatedly with deionized water followed by drying at 48 C and storing in a dessicator.
2.3. Laboratory biodegradation 2.3.1. Microbial growth Indigenous microbial cells of Whitley Bay sediments were isolated and proliferated via several subcultures prior to use for laboratory biodegradation studies. Microbial growth during culture enrichment was monitored by measuring absorbance of the cell suspension at 600 nm using a UV-Visible spectrophotometer. Microbial growth during the experiments with clay samples was measured via standard plate count. The procedure used in carrying out the cell plate count was as follows: 1 mL of suspension from the clay/oil/microbe suspension was used in preparing 101e107 fold dilution. Then 0.1 mL of each of the above stated dilutions was spotted on the plate and carefully spread. The plates were incubated for 24 h at 28 C and enumerated.
2.3.2. Total petroleum hydrocarbon (TPH) The effect of the clay minerals on biodegradation of the crude oil hydrocarbons was determined by measuring the residual TPH after incubation of the aqueous clay/oil microcosm experiments with hydrocarbon degrading microorganism community for 60 days. Clay (dried)/oil ratio (w/w) of 5:1 was used in all the experiments. Hence, the microcosm consisted of 250 mg of clay mineral and 50 mg of oil in 10 mL of BushneleHaas medium with the microbial cells. Several control experiments were conducted to account for abiotic processes due to volatilization and adsorption, in addition to a positive control. Descriptions of those control experiments are as follows: Control-1 is a positive (biotic) control containing BH medium, oil and microbial cells (but no clay). Control 2 is a negative (abiotic) control containing BH medium and oil (but no clay and no microbial cells).
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Control-3 (clay controls) represents a negative abiotic set of controls containing BH medium, clay and oil but no microbial cells. Control-2 accounts for volatilization and is also used (in combination with the clay controls) to estimate the extent of adsorption of the hydrocarbons (see Section 2.5). Clay controls are important in estimating biodegraded and adsorbed hydrocarbons. All experiments were carried out in triplicate to ensure reproducibility and amenability to statistical analysis. 2.3.3. Effect of spent water from ogranoclay on microbial growth/ activity The bactericidal effect of the spent water from organoclay was tested by collecting the spent water (10 mL) arising from the final washings of the organoclay (organosaponite and organomontmorillonite) and using it to prepare the BH medium by dispersing the appropriate quantity (32.7 mg) of BH broth into it and adding 50 mg of crude oil. The control experiment contained deionized water and is in this study known as Control-3*. Both the test and control experiments were inoculated with the same cells that use crude oil hydrocarbons as carbon source. 2.3.4. Extraction of hydrocarbons and analysis Three stages of extraction (with 302.3.2mL of DCM for each stage) were employed to extract the residual oil (i.e. extractable organic matter-EOM) subsequent to spiking with squalane as a surrogate and TPH quantitation standard. The EOM was extracted on solid phase extraction (SPE) columns following the procedure of Bennett et al., (2002) to separate the hydrocarbons from the residual oil (EOM). Prior to GC-FID analysis, the samples were spiked with internal standards (heptadecylcyclohexane, 5a-androstane 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, a RRF of unity was assumed. The percentage recovery of the surrogate standard was between 70% and 120% which is within acceptable range (USEPA method 8270). 2.4. Analytical Instrumentation for the Hydrocarbons
Table 2 EGME-surface area, total organic carbon (TOC) and cation exchange capacity (CEC) of the clay samples. Sample
Surface area (m2/g)
CEC (meq/100 g)
TOC (%)
BU BO SU SO
645 471 473 330
83.3 e 35.4 e
e 7.3 e 3.4
100 kPa. The GC data was acquired using Atlas software on a HP desktop computer. 2.5. Determination of total petroleum hydrocarbon (TPH) The basis for the assessment of biodegradation in this study is the determination of TPH using GC. This TPH value as determined by GC is the residue of the original TPH after incubation. The determination of the TPH concentration was done by measuring the total GC area between 10 and 70 min followed by quantitation with squalane. 2.5.1. Biodegraded versus adsorbed TPH The following equations were used to determine the weight and percentage of hydrocarbons removed by biodegradation and adsorption.
TPH biodegradedðmgÞ ¼ TPHcy TPHr TPHbiodegradedð%Þ ¼
(2)
TPHcy TPHr 100 TPHcy
(3)
where TPHcy is TPH(mg) of the clay control ( clay þ oil þ BH, but without cells); and TPHr is TPH(mg) of clay test sample ( clay þ oil þ cells þ BH). For control-1:
TPH biodegradedðmgÞ ¼ TPHc2 TPHcx TPH biodegradedð%Þ ¼
2.4.1. GC-FID The GC instrument used was an HP 5890 series II gas chromatograph equipped with a split/splitless injector and flame ionization detector (FID). The sample was injected using a 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% phenylmethylpolysiloxane (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
199
(4)
TPHc2 TPHcx 100 TPHc2
(5)
where TPHc2 is TPH(mg) of Control-2 (BH þ oil: with neither clay nor cells); and TPHcx is TPH(mg) of Control-1 (BH þ oil þ cells: but without clay). 3. Results/discussion 3.1. Characterization of the clay samples 3.1.1. XRD and FTIR The XRD and FTIR data are shown in Table 1 below.
Table 1 Basal spacing of 001 reflections and selected FTIR absorption bands of the clay samples Sample
XRD
FTIR Absorption band (cm1)
d-spacing (Å)
BU BO SO SU
Ethylene glycolated
Air dried
Heat treatment (300 C)
OH-stretch
CeH
Carbonate
17.1 16.8 14.7 16.2
12.5 14.2 14.0 14.5
10.6 13.2 13.8 10.7
3623 3623 3570 3570
e 2861, 2935 2861, 2935 e
1430 1430 1430 1430
BU ¼ unmodified montmorillonite, SU ¼ unmodified saponite, SO ¼ organosaponite, and BO ¼ Organomontmorillonite.
200
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The d001 reflections at 17.1 Å, 12.5 Å and 10.6 Å (Table 1) following ethylene glycolation, air drying and heat treatment, respectively, are indicative of montmorillonite with mainly sodium in the interlayer whereas the corresponding d001 reflections at 16.2 Å, 14.5 Å and 10.7 Å (Table 1) are indicative of trioctahedral smectite such as saponite with a divalent metal in the interlayer. The FTIR absorption band at 3623 cm1 (Table 1) is due to the OHstretch of AlAlOH typical of montmorillonites while the band at 3570 cm1 indicates the occurrence of Mg/Fe2þ within the trioctahedral smectite (Fe-rich saponite; Shayan et al., 1988). The layer collapse on heat treatment of the organoclay samples is lower than that for the unmodified clay samples and is due to the intercalation of the didecyldimethylammonium (DDDMA) ion in the interlayer of the organoclay. The FTIR absorption bands at 2861 cm1 and 2935 cm1 observed with the organoclay samples were due to CH2 symmetrical and asymmetrical vibration stretch from alkyl chain moiety of the DDDMA. Fig. 1. Effect of organoclay minerals on growth of hydrocarbon degrading bacteria. BO250 ¼ organo montmorillonite; BU-250 ¼ unmodified montmorillonite; SO250 ¼ organosaponite; SU-250 ¼ unmodified saponite; Control-1 ¼ BH þ oil þ cells (no clay). Control-2 ¼ BH þ oil (no clay and no cells). CBU-250, CSU-250, CBO-250 and CSO250 are clay controls where there was no growth (not shown on the chart). Values are reported as mean ± one standard error.
3.1.2. Surface area, TOC and CEC of the clay samples The EGME-surface area, total organic carbon (TOC) and cation exchange capacity (CEC) of the clay samples are shown in Table 2 below. The TOC values confirm that the organoclay contains organic phase unlike the unmodified clay samples. The organoclay samples have lower EGME-surface area in comparison with their unmodified clay counterparts. This lower EGME-surface area may be as a
Fig. 2. (a) Chromatogram (of TPH fraction) showing no biodegradation-sample Control-2. Control-2 ¼ negative control (BH þ oil) no clay and no cells. The nC17/pristane and nC18/ phytane ratios for this sample are 2.0 and 2.1, respectively. The standards are labelled as: Squ ¼ squalane; hdch ¼ heptadecylcyclohexane; 1,1-binaph ¼ 1,1-binaphthyl; andro ¼ 5aandrostane. (b) Chromatogram (of TPH fraction) showing moderate to heavy biodegradation-sample BO-250. BO-250 ¼ organomontmorillonite. (c) Chromatogram (of TPH fraction) showing heavy biodegradation-sample BU-250. BU-250 ¼ unmodified montmorillonite.
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201
Table 3 Maximum cell yield, specific growth rate, % TPH biodegraded and % TPH adsorbed due to the effect of unmodified clay samples and organoclay samples. Values are reported as mean ± one standard error of the mean. Sample
Maximum cell yield (CFU/ml)/108
Specific growth rate (h1) 103
%TPH biodegraded
%TPH adsorbed
SU-250 BU-250 Control-1 BO-250 SO-250 BO-250* SO-250* Control-3*
8.3 ± 0.3 11.2 ± 0.4 8.1 ± 0.3 7.0 ± 0.3 8.0 ± 0.2 8.1 ± 0.2 7.9 ± 0.2 8.0 ± 0.2
9.1 ± 0.4 10.2 ± 0.4 9.1 ± 0.2 8.4 ± 0.2 8.8 ± 0.2 9.1 ± 0.2 9.0 ± 0.2 9.1 ± 0.3
59 ± 3 72 ± 2 50 ± 1 38 ± 3 50 ± 1 50 ± 1 49 ± 1 49 ± 2
27 ± 2.4 27 ± 3.7 e 8.5 ± 0.9 8.5 ± 0.6 e e e
BO-250* ¼ BH in spent water from the organomontmorillonite, SO-250* ¼ BH in spent water from the organosaponite, Control-3* ¼ BH in deionized water. There is no growth in Control 2, hence it (Control 2) has not been entered in this table.
result of EGME being polar and therefore not able to interact with the non-polar portions of the organoclay. 3.2. Microbial growth and biodegradation of crude oil hydrocarbons in the presence of organoclay samples. The effect of organoclay minerals on microbial growth with crude oil hydrocarbons as the only carbon source is presented in Fig. 1. The growth of the hydrocarbon degrading bacteria is affected by the clay mineral samples to varying extents depending on the type and form of the clay mineral sample (Fig. 1). The gas chromatograms of selected samples showing extent of biodegradation are shown in Fig. 2aec. Fig. 2b and c showd that the nC17/pristane and nC18/phytane ratios for the organomontmorillonite (BO-250) and the untreated montmorillonite (BU-250) are not measurable following biodegradation. The effect of the clay mineral samples on microbial growth and biodegradation of crude oil hydrocarbons assessed by the maximum cell yield, specific growth rate, % TPH biodegraded and % TPH adsorbed is presented in Table 3. The maximum cell yield, specific growth rate constant and percentage of biodegradation of the hydrocarbons were highest in sample BU-250 (Table 3). The 2-sample t-test at 95% confidence interval (CI) with respect to maximum cell yield, specific growth rate constant and percentage biodegradation of TPH indicates that unmodified montmorillonite (BU-250) is significantly different from Control-1. Results for the unmodified saponite (SU-250) are not significantly different from Control-1 and therefore this clay is not stimulatory to biodegradation of crude oil hydrocarbons. To some extent, results of the stimulatory role of the montmorillonite presented in this study appear to be consistent with those reported by Warr et al. (2009) although the later did not specifically cover the effect of saponites on microbial growth and biodegradation of crude oil hydrocarbons. However, with respect to adsorption, the study reported here is inconsistent with the study of Warr et al. (2009) which assumed that adsorption of hydrocarbons is not significant during biodegradation of the oil. As shown in Table 3, this is not always true. The absence of a clay control (i.e. an experiment with only clay and oil) in the study of Warr et al. (2009) made it impossible for the authors to estimate losses due to adsorption. The microbial growth and percentage of biodegradation of crude oil hydrocarbons in the presence of unmodified clay minerals appears to increase with the surface area of the clay mineral samples (Fig. 1 and Table 3). Interlayer cations are believed to cause ‘local bridging effect’ (effective delivery of nutrients to cells due to reduction of zeta potential or electrical double layer repulsion) hence clays with interlayer cations and high surface area would tend to stimulate biodegradation of crude oil hydrocarbons (Bright and
Fletcher, 1983; Fletcher and Marshal, 1982;; Liu et al., 1991; Warr et al., 2009). Unmodified montmorillonite has both high surface area (645 m2/g) and interlayer cations which led to the accumulation and delivery of the nutrients to the cells, enhancing their growth and effecting biodegradation of crude oil hydrocarbons (Bright and Fletcher, 1983; Fletcher and Marshal, 1982; Warr et al., 2009). The modification of the clay samples to produce organoclay seemed to lead to inhibition of biodegradation and this is quite noticeable by comparing organomontmorillonite, BO-250 and unmodified montmorillonite, BU-250 (Table 3). Also, on comparing the organoclay samples with Control-1, microbial growth and biodegradation of the hydrocarbons seemed to be depressed in sample BO-250 as the maximum cell yield, specific growth rate constant and percentage TPH biodegraded are lower with this sample than either Control-1 or the unmodified saponite (SO-250). The 2-sample t-test with respect to maximum cell yield, specific growth rate and percentage TPH biodegraded indicates that there is no statistical significant difference between Control-1 and either of the organoclays, BO-250 or SO-250 (P-values >0.05). Therefore BO250 and SO-250 do not stimulate the microbial growth and biodegradation of crude oil hydrocarbons although SO-250 does not depress it either. There is significant statistical difference between BU-250 and BO-250 with respect to maximum cell yield, specific growth rate constant and percentage TPH biodegraded (Pvalues
