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Ex-situ Biodegradation of petroleum hydrocarbons using Alcanivorax borkumensis enzymes
Bioremediation for degradation of hydrocarbons is a widely used alternative for the recovery of contaminated sites. The current study aimed to use Alcanivorax borkumensis crude enzymes preparation as an agent for enhanced microbial hydrocarbons biodegradation in contaminated water and soil. The inoculum and hydrocarbons concentration have a remarkable effect on the biodegradation with the crude enzymes. The use of 10% (v/v) of inoculum concentration increased the enzyme activity and thus biodegradation efficiency of hexadecane, BTEX, motor oil and contaminated soil. However, in the presence of higher concentration of hydrocarbons, the enzymes efficiency increased or decreased depending on the concentration. The study suggested that Alcanivorax borkumensis is a potential hydrocarbon-degrading bacterium with higher enzymatic capacities for bioremediation of hydrocarbon-polluted environment.
Keywords: Biodegradation;Petroleum Hydrocarbons; Alcanivorax borkumensis; Enzymes Production
Petroleum is the most cost-effective energy source in the current global economy. Its widespread and extensive use has led to serious ecological problems mainly due to improper waste management, polluting industries, maritime spills, and leaked oil from transport ships along with leaching landfills. Petroleum pollutants can cause severe disruption of the ecological balance (Marchut-Mikolajczyk et al., 2015).
Physical, chemical and biological methods have been expanded over the years to remediate petroleum spills. Bioremediation, either as a spontaneous or as a controlled process, is the application of biological methods for the treatment of unsafe chemicals present in the environment. This approach has been proven to be successful in the mineralization and removal of petroleum hydrocarbon contaminants in different habitats, such as water and soil (Nievas et al., 2008; McGenity, 2014; Ron and Rosenberg, 2014). Different microorganisms, such as bacteria, filamentous fungi and yeasts, can degrade alkanes ((Van Beilen et al., 2004; Rojo, 2009). Notably, some recently characterized bacterial species are highly specialized for hydrocarbon degradation. These species are called hydrocarbonoclastic bacteria (HCB), and they play a key role in the removal of hydrocarbons from polluted and non-polluted environments (Hara et al., 2003; Yakimov et al., 2007; L. Wang et al., 2010; W. Wang et al., 2010). Of particular importance is Alcanivorax, a marine bacterium that can assimilate various linear or branched alkanes (Liu et al., 2011; Wu et al., 2008; Yakimov et al., 2007). Alcanivorax bacteria are present in non-polluted seawater in low numbers; however, the number of Alcanivorax can increase as a result of an oil spill, and they are believed to play an important role in the natural bioremediation of oil spills worldwide (Golyshin et al., 2003; Hara et al., 2003; Naether et al., 2013; W. Wang et al., 2010; Yakimov et al., 2007; Bookstaver et al., 2015). In fact, chemotaxis facilitates the movement of microorganisms toward or away from chemical gradients in the environment, and this process plays a role in biodegradation by bringing cells into contact with degradation substrates (Parales et al., 2000; Parales and Harwood, 2002). Moreover, Alcanivorax borkumensis produces an anionic glucose lipid biosurfactant (Abraham et al., 1998; Yakimov et al., 1998) which may enhance the bioavailability of hydrocarbons by either increasing apparent hydrocarbon solubility in the aqueous phase or by expanding the contact surface area due to emulsification (Morán et al., 2000).
Enzymes produced from these microorganisms play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds (Das and Chandran, 2010; Van Beilen and Funhoff, 2007). The use of enzymes is advantageous because they can perform the same function as many chemicals, but under less harsh conditions (neutral pH and moderate temperature) (Ruggaber and Talley, 2006). Moreover, enzymatic treatment of soil and groundwater contaminated with hydrocarbons can be a replacement to conventional bioremediation (Gianfreda and Rao, 2004; Ruggaber and Talley, 2006). Their advantage over microbial treatment includes their important reaction activity, lower sensitivity towards the concentrations of recalcitrant, coverage of a wide range of physicochemical gradients in the environmental matrix and simple management of field application. Moreover, enzymes are biodegradable and have advanced chemo-, regio-, and stereo-selectivity, which results in small amounts or non-generation of by-product, all of which allows their progressive performance. Enzymes have the capability to mineralize a recalcitrant compound and also transform it to a state in which it is more biodegradable. Despite the cost of enzyme extraction and purification, they can be cost-effective due to negligible waste disposal and heating requirements (Gianfreda and Rao, 2004; Wu et al., 2008).
The present work aims to study the capacity of crude enzymes produced by Alcanivorax borkumensis, which is a hydrocarbonoclastic bacterium, to degrade petroleum hydrocarbons represented by hexadecane with a concentration varying from 5000 mg/L to 7000 mg/L, BTEX with a concentration of the six compounds varying from 30 mg/L to 70 mg/L (1:1:1:1:1:1), motor oil with a concentration varying from 500 mg/L to 1000 mg/L and contaminated soil with a concentration of total petroleum hydrocarbons varying from 2000 mg/L to 6000 mg/L. these concentrations were chosen depending on soil characterization and PAHs, BTEX and C10-C50 content. Also we varied the inoculum concentration from 3% (v/v) to 10% (v/v) and the biodegradation was carried out for 7 days.
2. Materials and methods
All chemical reagents of highest purity, such as hexadecane, BTEX (Benzene, Toluene, Ethylene, Xylene), NADPH (nicotinamide adenine dinucleotide phosphate) and DMSO (Dimethyl sulfoxide) among others, were procured from Sigma-Aldrich, Fisher scientific or VWR (Mississauga, Ontario, Canada). The strain, Alcanivorax borkumensis was purchased from DSMZ (Braunschweig, Germany).
2.1 Soil and motor oil characteristics
The contaminated soil used in this study was provided by TechnoRem Inc from a confidential site in Quebec. This soil was chosen due to its intense contamination with total petroleum hydrocarbons. The soil comprised 59% of 1 mm sized particles, 38% of particles with a size range between 250 µm and 500 µm and 3% of very fine particles with a size less than 250 µm.
The chemical characteristics of the heavily contaminated soil used during this study are presented in Tables 1, 2 and 3.
Total solids (TS) and moisture content were measured using the protocol 2540B of (Federation et al., 2005) which consists of heating the clean dish to 103±1 to 105±1 °C for 1 h. Later, it was stored and cooled in the desiccator. And finally, it was weighed immediately before use. To determine the elemental concentration of carbon and nitrogen, the sample was first dried at 60±1°C for 8 h and placed into a glass vial. Two sub-samples (2-3 mg each) were analyzed using a Leco-932 CHNS Analyzer in CHN mode. In CHN mode, samples are combusted in the presence of pure O2 and the combustion gases are measured to determine initial elemental concentrations of C, H and N.
Oil content was determined by adding n-hexane to the soil (1:1 w/v) and then centrifuging at 10,000 x g for 30 minutes to recover the pellet. Once again, n-hexane was added to the pellet (1:1 w/v) and then centrifuged at the same speed and time to recover the pellet. Finally, n-hexane was dried at room temperature and the oil content (supernatant) was weighed. Metals content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Samples digestion was fulfilled following the method MENVIQ.89.12/213.Mét 1.3. All the analyses were performed in triplicates.
The method used to determine the PAHs is MA. 400 – HAP 1.1, which consists of: extracting PAHs using dichloromethane or hexane after adding recovery standards (“surrogates”). Later, the extract was concentrated to a small volume under a nitrogen stream and purified on a silica gel-alumina column. The final volume of purified extract was concentrated to meet the target detection limits. Finally, the extract was concentrated and then analyzed by chromatography gas phase coupled to a mass spectrometer (GC-MS) operating in the mode selective ion acquisition (“SIM”) was used. The method used to determine the C10-C50 was MA. 400 – HYD. 1.1, which consists of extracting aqueous samples with hexane using a mechanical stirrer. Solid samples are first dried with acetone, and then extracted with hexane with the aid of an extraction system “mixer painting”. As for organic liquids, they are directly diluted in hexane. Thereafter, silica gel is added to the extract to adsorb polar substances and then the supernatant was analyzed by gas chromatography coupled to flame ionization detector.
The composition of motor oil in this study comprised: 69.8 mg/L of C10-C50, 1.83 mg/L of naphthalene, ≤ 44 mg/L of benzene, ≤30 mg/L of toluene, ≤ 44 mg/L of ethyl-benzene and ≤ 84 mg/L of xylene.
2.2 Bacterial strain
Aerobic bacterial strain, Alcanivorax borkumensis SK2 (DSM 11573) which has the capability to degrade petroleum hydrocarbons was used in all the experiments in this study. The strain was stored at 4 °C on agar plates coated with a film of hexadecane.
2.3 Culture conditions
Initial culture of A. borkumensis strain was grown aerobically in batch culture in 250-mL Erlenmeyer flasks for 72h at 30±1 °C and 150 rpm in synthetic sea water medium SM1 in order to mimic the conditions of an oil spill in the environment (high carbon concentration and nitrogen limitation). This medium was supplemented with 3% (v/v) hexadecaneas the sole carbon and energy source, and all major cations and anions present in concentrations higher than 1 mg/L in sea water. SM1 contained (per liter of distilled water): 23 g NaC1, 0.75 g KCl, 1.47 g CaCl2 . 2H2O, 5.08 g MgCl2 . 6H2O, 6.16 g MgSO4. 7H2O, 0.89 g Na2HPO4. 2H2O, 5.0 g NaNO3 and 0.03 g FeSO4. 7H2O. To prevent precipitation, four separate solutions were prepared and later mixed together after autoclaving when the solutions had cooled to room temperature; the first solution contained Na2HPO4 and NaNO3 (the pH value of medium was adjusted to 7.5 by the addition of a 10%solution of NaOH), the second solution contained NaCl, KCl and CaCl2, the third solution contained MgCl2 and MgSO4,and the fourth solution contained FeSO4. Bacto agar (Difco, Fisher Scientific, Mississauga, Ontario) (15 g/L) was added to the first solution for the preparation of solid media (Yakimov et al., 1998). About 3% (v/v) and 10% of sub-culture was used to inoculate the medium which contained 3% (v/v) of hexadecane as a sole source of carbon and energy.
For enzymes recovery, the bacterial culture was centrifuged at 12,000 x g for 30 minutes at 4 °C, and the supernatant containing the extracellular enzymes was recovered and stored at -20 °C for further biodegradation experiments. A. borkumensis cell pellet (1 g) frozen at -20 °C was re-suspended in phosphate buffer (1 ml, 0.1 M, pH 8.0). The mixture was sonicated by using two frequencies of ultrasounds (22 kHz and 30 kHz) for 6 min at 4 °C and centrifuged at 12 000 × g for 30 min. The supernatant was used as a crude intracellular enzyme extract.
2.4 Biodegradation using the crude enzymes mixture
The performance of the extracellular and intracellular crude enzymes from A. borkumensis was evaluated in batch tests in Milli-Q water. The test solutions contained 50 mL Milli-Q water, 10 mg/mL of an equal mixture of intracellular and extracellular crude enzyme and the different petroleum hydrocarbons concentrations: 500, 600 and 700 mg/L of hexadecane, 30 mg/L, 50 mg/L and 70 mg/L of BTEX compounds mixture (1:1:1:1:1:1), 500, 750 and 1000 mg/L of motor oil, and 2000, 4000 and 6000 mg/mL of contaminated soil. The solutions were incubated at 30 °C for 7 days with shaking at 100 rpm in an incubator shaker. All experiments were carried out in triplicates.
2.5 Sampling strategy and parameters assayed
At the beginning (T0) and at the end (T7) of the experimental period, sub-samples of enzymatic degradation were taken. Measures of enzymes activities (alkane hydroxylase, lipase and esterase) and concentrations of different petroleum hydrocarbons (hexadecane, BTEX and motor oil) were carried out. All experiments were performed in triplicates.
2.6 Proteins and enzymatic assays
2.6.1 Total protein assay
Total protein concentration was determined according to the Bradford method. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex (Bradford, 1976).
2.6.2 Alkane hydroxylase assay
Alkane hydroxylase activity was measured using a cofactor (NADPH) depletion assay to determine relative activities. The intracellular enzyme was diluted into phosphate buffer (0.1 M, pH 8), alkane substrate (0.5-1 mM), and dimethyl sulfoxide (DMSO; 1%, vol/vol). Alkanes were added to the buffer using alkane stock solutions in DMSO. The reaction was initiated by addition of NADPH (200 µM), and the oxidation of NADPH was monitored at 340 nm (Glieder et al., 2002).The alkane substrate used in this study was hexadecane.
2.6.3 Lipase and esterase assay
Extracellular lipase and esterase activities were performed bytitrimetric method according to Lopes et al., (2011) by using olive oil as a substrate for both enzymes and arabic gum as emulsifier for lipase assay. One unit of enzyme activity was defined as the amount of enzyme which liberated 1 µmol of fatty acids per minute.
- Gas chromatography
GC analysis of petroleum hydrocarbons biodegradation was performed using Hewlett-Packard 6890/5973 with flame ionization detector (FID). Analyses were carried out with helium as the carrier gas at a flow rate of 2 mL/min on a DB-1 column (30 m, 0.53 mm i.d., 1.0 mm film thickness). Oven temperature was programmed from 60 °C 260 °C at a rate of 4 °C/min. Split/Splitless injector and detector (FID) temperatures were 260 °C and 260 °C, respectively, and 1 mL of the sample was injected.
- Statistical analysis
All the experiments were performed in replicates and an average of 3 replicates was calculated along with the standard deviation. Database was subjected to an analysis of variance (ANOVA) and the results which have P < 0.05 were considered as significant.
- Results and discussion
- Dynamics of petroleum hydrocarbons degradation with crude enzymes
The present study deals with the enzymatic degradation of A. borkumensis which was grown in the presence of xenobiotic petroleum hydrocarbons being reported to be toxic substances, such as hexadecane, motor oil and BTEX. Overall, degrading capacities of crude enzymes produced were also tested in the presence of contaminated soil which contains a range of contaminants as described in Table 1. Accordingly, aerobic enzymatic degradation can be affected by many physical, chemical and biological conditions (e.g. pollutant concentration, availability of inorganic nutrients and enzymes adaptation. ect) that influence the pollutant degradation efficiency (Singh and Celin, 2010). Herein, the variation of operational factors (inoculum percentage, substrate concentrations and degradation time) was carried out to judge the efficiency of biodegradation of the selected strain. In fact, biodegradation progression is affected by the bacterial strain used for bioremediation and the capacity of each bacterial isolate to use efficiently the available nutrients and produce enzymes. Fig. 2 and Fig. 3 show the efficiency of the removal capacities of hexadecane, motor oil, BTEX and hydrocarbons in soil by A. borkumensis crude enzymes as a function of different parameters. A. borkumensis was able to use various tested substrates as sole carbon source and energy for its growth and proliferation which was confirmed by cell count. The cell number reached around 108 CFU/mL (Colony Forming Units per mL) in the case of hexadecane, BTEX, motor oil, as well as petroleum hydrocarbons contaminated soil. Other than the bacterial strain and the enzymes produced by this strain, other parameters were demonstrated previously in other studies to influence the degradation process which include the hydrocarbons present in the contaminated site, and the environmental compartment in which the process is being carried out (Bamard et al., 2011). In the current study the influence of substrate concentration on the degradation rate of A. borkumensis, was very representative. Herein, the percentage of removal was correlated with initial concentration of hydrocarbons.
Hexadecane used in this study is part of the aliphatic fraction of crude oil and it is one of the most important components of diesel (Chénier et al., 2003). This compound is present at many oil-contaminated sites and its biodegradability has been well characterized (Graham et al., 1999). For these reasons, hexadecane is used in this study as a model molecule to study aliphatic hydrocarbon biodegradation since it has been always considered as a model (Schoefs et al., 2004). The concentrations adopted in our research (5000 mg/L, 6000 mg/L and 7000 mg/L) were based on the concentration of C10-C50 that has been found in the characterized contaminated soil (Table 2). The removal percentage of C16H34 decreased from 73.75% to 59.74% after 7 days of enzymatic degradation, while increasing the concentration of hexadecane from 5000 to 7000 mg/L when using 10% (v/v) of inoculum concentration (Fig. 2). These findings are in agreement with Maletić et al., (2013) (Maletić et al., 2013) who reported that hydrocarbon degradation is ultimately dependent on their concentration. Most of the previous studies on hexadecane biodegradation have used an initial concentration lower than 1 g/L and have been carried out for up to 45 days (Haines and Alexander, 1974; Hanstveit, 1992). Setti et al., (1993) (Setti et al., 1993) reported 86.4% of hexadecane mineralization at an initial concentration of 12 g/L by Pseudomonas sp. after 31 days of biodegradation. Colombo et al., (1996) (Colombo et al., 1996) reported that several fungal strains were able to biodegrade up to 80% of aliphatic hydrocarbons after 90 days, using a contaminated soil with a concentration of 10% crude oil which contain 16.5 mg of aliphatic hydrocarbons per g of soil. Moreover, Volke-Sepulveda et al., (2003) (Volke-Sepulveda et al., 2003) found that an initial concentration of 45 g/L of hexadecane was totally mineralized after 31 days of culture using a solid state fermentation. In this study, the inoculum concentration had high effect on the enzymes activities which reached 0.93 U/mL of alkane hydroxylase, 57.8 U/mL of lipase and 60.1 U/mL of esterase (Fig. 1). Consequently, it had an effect on the degradation efficiency of hexadecane. This effect can be clearly noticed on the three different concentrations of hexadecane used. In fact, the degradation percentage increased almost twice (from 37.70±0.72 to 73.75±3.11) when using 5000 mg/L of hexadecane and from 32.40±1.46 to 38.50±2.13 when using 6000 mg/L of hexadecane and also almost two time degradation increase was observed when using 7000 mg/L of hexadecane (from 28.31±0.37 to 59.74±0.62), after 7 days of degradation with enzymes produced by the inoculated A. borkumensis (Fig. 2).
Peng et al., (2015) (Peng et al., 2015) has stated that BTEX mixture is the most toxic TPH component for living cells). The BTEX degradation abilities of the enzymes produced by A. borkumensis inoculated with 3% (v/v) and 10% (v/v), were evaluated during 7 days using a BTEX mixture containing 30 mg/L, 50 mg/L and 70 mg/L of each of the six compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene (1:1:1:1:1:1)) in the enzymatic preparation The removal percentage of benzene, ethylbenzene and o-xylene decreased from 72.22%, 74.8 %, 79.3% to 69.5%, 50.25% and 63.71%, respectively while increasing substrate concentration from 50 mg/L to 70 mg/L using 3% (v/v) of inoculum concentration. However, in the presence of higher inoculum concentration (10% (v/v)), the degradation rate decreased from 76.48%, 71.69%, 81.2% to 71.22%, 65.45%, 74.8% respectively as shown in Fig. 3. These observations were in agreement with Li et al., (2006) (Li et al., 2006) who showed an inhibitory effect of higher benzene concentration (more than 80 mg/L) in the presence of Planococcus sp. strain ZD22 Similarly, Hamed et al., (2003) (Hamed et al., 2003) reported that specific growth rate of P. putida in batch systems has been set up to be a decreasing function of benzene and toluene concentrations. BTEX compounds upon reaching certain concentrations can inhibit the microbes and their enzymatic activity due to complex micro- and macro-level interactions (Jo et al., 2008). Furthermore, Mathur and Majumber (2010) (Mathur and Majumder, 2010) claimed that at higher initial concentrations (>150 mg/L benzene and >200 mg/L toluene), degradation rate was lower. The removal efficiency could also be attributed to the simple structure and the molecular composition of BTEX (Fedorak and Westlake, 1981; Horowitz and Atlas, 1977). In the case of toluene about 84.22% of removal percentage was obtained at an initial concentration of 70 mg/L on day 7 (Fig. 3). Thus, toluene had been claimed as the most easily biodegradable among the six compounds of BTEX. This may be probably due to the existence of the substituent groups on the ring that offer an alternative path of attack on the side chain or oxidization of the aromatic ring (El-Naas et al., 2014). Accordingly, the enzymatic attack was presumed to affect the aromatic ring of hydrocarbons via hydroxylation reactions by the addition of molecular oxygen on one or more carbon (Haddock, 2010). In the current study, the mixture of different BTEX compounds (benzene, toluene, ethylbenzene and xylene) together is seen to affect one another. For example, they can interact synergistically or antagonistically as reviewed by (Dou et al., 2008). Synergistic interactions enhance the degradation rates of individual contaminants by inducing the required catabolic enzyme. Meanwhile, antagonistic interactions inhibit the degradation rates through exertion of toxicity, diauxic behaviour, catabolite repression, competitive inhibition for enzymes, or depletion of electron acceptors. Herein, the interaction between BTEX components in this study seems to be synergistic since a high removal percentage (up to 60%) is reached while using the mixture of the above contaminants in a time period of 7 days. Other xenobiotic components were tested in the current study for their removal efficiency, such as motor oil which showed a potential degradation rate (Fig. 2). As mentioned earlier in this study, this compound was characterized and it is composed of (in mg/L): 69.8 C10-C50, 1.83 naphthalene, ≤ 44 benzene, ≤30 toluene, ≤44 ethyl-benzene and ≤84 xylene. Thus, A. burkumensis grew well on engine oil producing high crude enzymes activities and the higher removal was obtained within 7 days with about 83% disappearance of oil. The variation of the initial concentration of motor oil from 500 to 1000 mg/L with an inoculum size varying from 3% to 10 % (v/v) was accompanied by an increase of degradation efficiency reaching around 83% within day 7 with an inoculum size of 10% as well as an initial concentration of 1000 mg/L. As discussed earlier, the biodegradation carried out by A. burkumensis seemed to be concentration dependent as observed with BTEX. In the case of motor oil, this compound is recognized to have a complex structure since it is composed of aliphatics, monoaromatics and polyaromatics. But despite its complexity, A. burkumensis enzyme preparation was able to degrade it efficiently. These results are advantageous compared with Pseudomonas aeruginosa isolated from hydrocarbon-polluted environment which was able to utilize 81% of used engine oil within 4 weeks compared to 7 days in the current study (Thenmozhi et al., 2011). Likewise, Basuki et al., (2015) (Basuki et al., 2015) reported the removal of 35 out of 47 components of used oil by Acinetobacter junii TBC 1.2. Besides, Pseudomonas aeruginosa LP5 degraded more than 90% of all oil types within 21 days. Taken together, the selected strain is a potential candidate for the degradation of motor oil (known to be generally more resistant to degradation) in minimal time. Herein, the incomplete degradation obtained (between 59-83%) can be further improved by extending the fermentation time to more than 7 days.
In the current study the degradation efficiency varied largely between the different compounds, hexadecane, BTEX or motor oil reflecting a complexity in the structure and chain-length of the different studied substrates. These observations substantiated the findings of Das and Chandran (2010) (Das and Chandran, 2010) who stated that all the mechanisms of biodegradation and the degradative enzymes produced are dependent on physical and chemical properties of hydrocarbons.
In all the previous experiments, the inoculum concentration affects the biodegradation of hydrocarbons and the higher percentage of removal was obtained at 10% (v/v) of inoculum size. Thus, the bacterial growth and proliferation obtained due to higher inoculum is accompanied by the higher incidence of hydrolysing enzymes i.e lipase, esterase and alkane hydroxylase. These enzymes helped the biodegradation of hydrocarbons through aerobic pathways (Marchut-Mikolajczyk et al., 2015).
In order to determine the biodegradation, the application of crude enzyme extracted from Alcanivorax borkumensis was carried out. Fig. 1 presents the evolution of the enzyme activities in the presence of different substrates. As seen from Fig. 2 and 3 from 1st to 7th day of degradation for the different substrates , the enzyme activities of lipase, esterase and alkane hydroxylase were almost constant, which suggested higher stability of the enzymes even at higher concentrations of substrates. The higher activities ranged from 40 to 71 U/mL of lipase for the different studied substrates. These results suggested that the enzymatic activities implicated in the biodegradation processes can be preserved for 7 days which offer a wide range of application. Ruggaber and Talley (2006) (Ruggaber and Talley, 2006) and Wu et al., (2008) (Wu et al., 2008) have revealed that enzymes remained active during the process of biodegradation and even when changing the environmental conditions. The crude enzymatic consortium composed of lipase, esterase and alkane hydroxylase in the current study showed a potential efficiency which may suggest their crucial role in the biodegradation process. Thus, the use of enzymatic preparation from Alcanivorax borkumensis is effective in bioremediation of up to 80% and contributes to the acceleration of the process (7 days). In this regard, enzymatic activities, such as lipase, esterase and alkane hydroxylase were investigated. Higher enzymatic activities were obtained during the degradation of BTEX with 0.93 U/mL of alkane hydroxylase, 57.8 U/mL of lipase and 60.1 U/mL of esterase when using 10% (v/v) of inoculum concentration after 7 days of degradation (Fig. 1). These results confirm the high removal percentage obtained on the different BTEX components (up to 80%).
These results are advantageous compared to similar strains that presented higher degradation capacities. Acinetobacter baumannii isolated from crude oil exhibited 62.8% of TPH biodegradation after 7 days (Mishra et al., 2004). Cellulosimicrobium cellulans exhibited hydrocarbon degradability of 18.86% after 15 days (Nkem et al., 2016). Similarly, Ijah (1998) (Ijah, 1998) reported more than 52% obtained in 16 days.
3.2 Biodegradation kinetics of contaminated soil
The higher removal percentage of contaminated soil was observed after 7 days of culture with 64.23%, 79.59% and 88.52% of degradation calculated with an initial concentration of contaminated soil of 2000 mg/L, 4000 mg/L and 6000 mg/L of soil, respectively. In the 3rd day, 40.67%, 54.43% and 67.29% were observed with the high inoculum percentage (10% (v/v)) in the case of 2000 mg/L, 4000 mg/L and 6000 mg/L of contaminated soil, respectively (Fig. 2). This rapid degradation rate on the 3rd day is likely due to the consumption of the easily degradable compounds of low molecular weight found in the soil.
Based on a first order model, degradation constant (k) and half-life (t1/2) were determined. Table 4 presents the kinetic parameters calculated for the removal of TPH in the soil as well as the hydrocarbons consumption data (i.e. global consumption rate; maximum consumption rate). The half-life was around 5.12 days. This indicates that it would take about 10 days to achieve complete biodegradation of the carbon source by applying the tested inoculum and enzymes. According to the analysis, the estimated biodegradation in the soil reached around 64% of TPH removal. The calculated global consumption rate (GCR) was about 566.14±42.1 mg kg-1 d-1 for contaminated soil with 6170.7 mg-1 kg-1 of TPH while the maximum consumption was 363.4 mg-1 kg-1.
Further analysis of these results in contaminated soil showed that on the 7th day of treatment, there was a rapid decline in the concentration of hydrocarbons in the soil; higher enzyme activities were implicated in the biodegradation and about 21.5 µg/ml of proteins were used in the current experiments.
Herein, the degradation rate was very important compared to other reported strains. In fact, Diaz-Ramirez (2000) (Díaz-Ramírez, 2000) evaluated the biodegradation of hydrocarbons using a bacterial consortium and has found around 62% of removal within 30 days. In the contaminated soil, the biotransformation of hydrocarbons was probably due to degradation of short chain compounds (low molecular weight) and medium sized alkanes. Herein, the higher content of petroleum hydrocarbons C10-C50 (6020 mg kg-1) activated the capacity of biodegradation in the selected microorganism. The biodegradation pathway initially involved the degradation of short to medium chain aliphatics (C10) presented by methyl naphthalene up to 25 mg kg-1. Similarly, Marquez-Rocha et al. (2001), reported the degradation of medium sized hydrocarbons (> C12) contained in the diesel, together with the short-chain compounds. To make the process wholesome, ecotoxicological studies should be carried out and special attention should be put on the effects of hydrocarbons on the physicochemical properties of the soil.
Crude enzyme extracted from Alacanivorax borkumensis showed better efficiency in terms of high removal of hydrocarbons. This study confirms the possibility of using bacterial enzymes for the bioremediation of hydrocarbons. More than 89% of enzymatic removal was obtained in the presence of different compounds namely BTEX, motor oil, hexadecane and contaminated soil. The use of enzyme degrading activities in both liquid medium and soil assay are promising alternatives for the application of this approach as a part of active remediation strategies for contaminated sites. Alcanivorax borkumensis is an excellent bioremediation tool with the ability to use hydrocarbons as carbon source. Thus, Alacanivorax borkumesis derived enzymes may be used as a powerful approach for the clean-up of environments polluted with petroleum compounds in both aquatic and terrestrial ecosystem.
The authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (Discovery Grant 355254, CRD Grant and Strategic Grant 447075) and Techno-Rem Inc. for financial support. The views or opinions expressed in this article are those of the author.
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