Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia Sp NL1, Isolated from an Algerian Oilfield

Geomicrobiology Journal

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Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield

Nesrine Lenchi, Salima Kebbouche-Gana, Pierre Servais, Mohamed Lamine Gana & Marc Llirós

To cite this article: Nesrine Lenchi, Salima Kebbouche-Gana, Pierre Servais, Mohamed Lamine Gana & Marc Llirós (2020): Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield, Geomicrobiology Journal, DOI: 10.1080/01490451.2020.1722769 To link to this article: https://doi.org/10.1080/01490451.2020.1722769

Published online: 05 Feb 2020.

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Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield

Nesrine Lenchia, Salima Kebbouche-Ganab, Pierre Servaisc, Mohamed Lamine Ganad, and Marc Lliros e,f,g aDepartment of Natural and Life Sciences, Faculty of Sciences, Benyoucef Benkhedda University (Algiers 1), Algiers, Algeria; bFaculty of sciences, M’Hamed Bougara University of Boumerdes, Boumerdes, Algeria; cEcology of Aquatic Systems, Universite Libre de Bruxelles, Brussels, Belgium; dCenter of research and development, SONATRACH, Boumerdes, Algeria; eDepartment of Genetics and Microbiology, Universitat Autonoma de Barcelona, Bellatera, Catalunya, Spain; fFaculty of Sciences and Technology, Universitat de Vic – Universitat Central de Catalunya, Vic, Catalunya, Spain; gInstitut d’Investigacio Biomedica de Girona – Dr Josep Trueta, Salt, Catalunya, Spain

ABSTRACT ARTICLE HISTORY In this study, a diesel oil-degrading bacterium was isolated from an oilfield water injection (water- Received 14 March 2019 bearing formations, 1,205 m depth) in Algeria. The bacterial strain, designated NL1, was cultivated Accepted 22 January 2020 on diesel oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene Delftia KEYWORDS sequence (KY397882) placed NL1 strain closely related to distinct cultivated species of the Delftia genus. Optimal diesel oil biodegradation by Delftia sp NL1 strain occurred at pH 11, 40 C, 2 M Biosurfactant; sp v/v NL1; diesel; oilfield; NaCl and initial hydrocarbon concentration of 5% ( ) as sole carbon source. GC-MS analyses evi- pH; salinity denced that strain Delftia sp NL1 was able to degrade more than 66.76% of diesel oil within only 7 days. On the other hand, and in the same conditions, biosurfactant production by Delftia sp NL1 was also evaluated evidencing high emulsifying capacity (E24 ¼ 81%), ability to lower the surface tension of growing media (with the value of 25.7 mN m21), and production of glycolipids (8.7 g L1) as biosurfactants. This research presents indigenous strain Delftia sp NL1 for diesel degradation and synthesis of biosurfactant in extreme conditions. In this sense, strain NL1 is a good candidate for possible in situ oil recovery and in wastewater treatment in refineries and oil terminals in petroleum industry.

Introduction of lowering costs, and environmental safety with absence of secondary pollutants (Philip et al. 2005; Sun et al. 2019). The extensive use of petroleum components or hydrocar- Biodegradation of diesel oil by microorganisms had bons as fuels has as negative drawback their release into the been widely demonstrated by the use of several species of environment in form of spills and subproducts of their con- the following genera, to cite some, Bacillus, Pseudomonas, sumption (Peixoto et al. 2011). Diesel oil is a common prod- Sphingomonas, Acinetobacter, Serratia, Citrobacter, uct of crude oil distillation with a very complex Raoultella, Stenotrophomonas mainly isolated from a wide composition, mainly consisting of alkanes and polycyclic diversity of oil-contaminated soils (Leahy and Colwell 1990; aromatic hydrocarbons (PAHs). In fact, PAHs can cause Morales-Guzman et al. 2017; Palanisamy et al. 2014; Zhang carcinogenic and mutagenic effects and are potent immune- et al. 2014). suppressants (Abdel-Shafy and Mansour 2016; Handley et al. However, many oilfields are situated in regions with arid 2017). Diesel compounds in the environment may be a great and semi-arid climates, where salinity of soil and therefore human health problem and exert a severe impact on the of wells is high. Thus, in such environments, it is necessary environment and dependent economies (Barron 2012). In that the potential bioremediation agents are able to cope case of an uncontrolled industrial leakage, diesel oil and its with high salinity. Various salt-tolerant hydrocarbon- constituents might act as a persistent water and soil pollu- degrading bacteria from different genera (either saline tant (Anjana et al. 2014). tolerant ones (e.g., Marinobacter, Halomonas, Alcanivorax, A variety of methods have been developed to treat diesel Haloferax, Haloarcula, or Ochrobactrum anthropi)or contamination. Physico-chemical treatments to the remedi- generalist ones (e.g., Bacillus thuringiensis, Bordetella bron- ation of hydrocarbons are usually laborious, expensive, and chialis and Pseudomonas sp. CQ2), have been isolated from use hazardous solvents. However, bioremediation is hypersaline oil reservoirs, saline oilfield-produced water, recognized as an effective method for the treatment of oil and high-salinity hydrocarbon impacted environments contaminated areas (Zhang et al. 2014). Bioremediation (Fathepure 2014; Kebbouche-Gana et al. 2009; Sun et al. technology utilizes microorganisms and their activities to 2018; Sun et al. 2019). In oil industry, the isolation of degrade toxic pollutants to harmless products with the aim microorganisms that degrades hydrocarbons in highly saline

CONTACT Nesrine Lenchi [email protected], [email protected] Department of Natural and Life Sciences, Faculty of Sciences, University of Algiers 1 Benyoucef Benkhedda, Algiers, Algeria. ß 2020 Informa UK Limited, trading as Taylor & Francis Group 2 N. LENCHI ET AL. milieus is of great importance for effective wastewater treat- Tabankort; TFT). Site description has been published else- ment strategies in refineries, oil terminals, and depots (Riis where (Lenchi et al. 2013). Water was collected in sterile et al. 2003). jerry cans directly from the wellhead, completely filled after One of the main factors affecting the biodegradation effi- three overflows and sealed directly with screw caps to avoid ciency of complex hydrocarbon compounds is the low avail- contamination and oxygen intrusion. The samples were ability of contaminants to microbial attack. An alternative to immediately transported at ambient temperature to the expand the bioavailability and the contaminant metabolism laboratory and stored at 4 C until analyses. Samples were is increasing substrate solubilization by using biosurfactants treated within 24 h after collection. Temperature, pH, and (Cerqueira et al. 2011). Biosurfactants are amphiphilic com- salinity were measured in situ using a multi-parameter probe pounds (i.e. both hydrophobic and hydrophilic moieties are (Hanna Instruments, Smithfield, RI, USA). In fact, injection present) produced by many living organisms within the abil- water collected from this oilfield was from 1205 m depth ity to reduce interfacial tension between different fluid and had a temperature of 98 C, pH of 7.11 and a salinity of phases (Banat 1995; Santos et al. 2016). In this sense, biosur- 7.30 g L 1. factants have a wide variety of industrial (e.g. petroleum, pharmaceutics, food, cosmetic, detergent, textiles, paints and agriculture, … ) and environmental (e.g. wastewater treat- Isolation of NL1 strain ment and bioremediation of sites contaminated with hydro- In order to isolate viable bacterial species from injection carbons) applications (Banat et al. 2000; Akbari et al. 2018). waters, 100 mL were spread on the surface of Pseudomonas Currently, the petroleum industry is the main market of bio- agar media (PA, Merck) and incubated at 37 C for 7 days surfactant production (Akbari et al. 2018; Santos et al. under aerobic conditions. The obtained colonies were fur- 2016). In fact, Microbial enhanced oil recovery (MEOR) is ther sub-cultured in the same media and culture conditions an important and one of the most promising tertiary proc- to obtain pure colonies. Isolates were further screened for esses that uses microorganisms or their metabolites (biosur- the ability to degrade diesel oil on mineral salt medium factants, biopolymers, biomass, acids, solvents, gases and 1 1 1 (MSM; 3.0 g L NH4 NO3, 0.5 g L KH2 PO4, 0.5 g L also enzymes) to increase oil recovery from depleted oil res- K2HPO4 3H2O and trace amounts of MgSO4 7H2O (0.008 g ervoirs after primary (mechanical) and secondary (physical) 1 1 1 L ), CuSO4 4H2O (0.002 g L ), MnSO4 H2O (0.002 g L ), recovery procedures (Pacwa-Płociniczak et al. 2011). 1 1 FeSO4 7H2O (0.002 g L ) and CaCl2 2H2O (0.002 g L ); Biosurfactants, by reducing interfacial tension between oil/ Palanisamy et al. 2014) supplemented with diesel oil water and oil/rock, reduces the capillary forces preventing (commercially available in Algerian local petrol stations and oil displacement from rock pores. Biosurfactants can also filtered through 0.22 mm pore-size filters (syringe filters, tightly bind to the oil-water interface and form emulsions. DuraporeTM)) as the sole carbon source. Cultures were Such processes stabilize the desorbed oil in water and allow incubated for 7 days at 37 C in an orbital shaker at removal of oil driven by the injection of water (Pacwa- ł 120 rpm. Bacterial growth was monitored at regular intervals P ociniczak et al. 2011; Hosseininoosheri et al. 2016; Suthar by measuring the optical density at 600 nm using a UV/vis et al. 2008). spectrophotometer (Zuzi). A recent metagenomic study in Algerian oilfields (Lenchi et al. 2013) evidenced the presence of a wide variety of hydrocarbon degrading microbes with putative interest as Morphological, biochemical and physiological bioremediation agents. In line with these results, we cultured characterization and tested several bacterial isolates from an Algerian oilfield For morphological and physiological characterization, strain water injection well (1,205 m depth) for the biodegradation of diesel oil and possible production of biosurfactants of NL1 was cultured in PA media at 37 C for 48 h. Cell motil- interest. Among them, NL1 (KY397882) isolate was charac- ity and shape were examined by phase contrast microscopy. terized on the basis of its culture parameters, its efficient Gram staining was determined using common Gram color- diesel oil degradation and, finally, its biosurfactant charac- ation procedures. Tests for catalase and oxidase activities, teristics and was placed within the Delftia genus of the starch, gelatin, casein, and Tween 80 hydrolysis, and nitrate Comamonadaceae family. Even also being described as crude reduction were performed by using standard procedures oil degraders on a contaminated soil (Wedulo et al. 2014), (Gerhardt et al. 1981). Growth response to NaCl was exam- this is the first report on the description of a Delftia isolate ined in liquid medium (mineral salt medium -MSM- supple- from an oilfield water injection well with high degradation mented with diesel oil) using serial NaCl concentrations efficiency of diesel oil and production of biosurfactant under ranging from 0,5 to 35% and to pH by testing growth at pH – extreme conditions of salinity and pH. 4 10. The growth response to temperature was examined by testing growth in liquid medium up to 55 C. Moreover, to obtain a broader description of strain NL1 Materials and methods capacities, the selected strain was screened for phenotypic resistance to antibiotics by using the disk diffusion method Water sample 1 against Ampicillin (10 mgmL, Thermo-FisherVR ), VR Injection water samples were collected from an oilfield Clindamycin (2 mgmL1, Thermo-Fisher ), Fusidic acid VR located in the southern Algerian Sahara (Tin Fuin (10 mgmL1, Thermo-Fisher ), Cefoxitine (10 mgmL1, GEOMICROBIOLOGY JOURNAL 3

VR Thermo-Fisher ), Gentamicin (10 mgmL1, Thermo- effect of different carbon and nitrogen sources in the grow- VR VR Fisher ) and Kanamycin (30 mgmL 1, Thermo-Fisher ). ing characteristics of the different isolates, MSM cultures were supplemented with 5% (v/v) diesel oil and 0.1% carbon (e.g. fructose and glucose) and 0.1% nitrogen (e.g. peptone 16S rRNA gene sequencing of NL1 strain and yeast extract) sources. For all experimental conditions, Two mL exponential phase of NL1 culture were used for growth was monitored by regular optical density measure- V nucleic acid extraction by means of Gentra Puregene R ments using a UV/visible spectrophotometer at 600 nm V Yeast/Bact extraction Kit (Qiagen R , UK) according to manu- (Zuzi). In parallel, control experiments (i.e. inoculated MSM facturer instructions. Nearly full-length (27f–1492r; Lane Erlenmeyer flasks without diesel supplement or diesel sup- 1991) of 16S rRNA gene was sequenced at external facilities plemented MSM Erlenmeyer flasks without inoculum) were (Macrogene Company (Seoul, Korea)). Phylogenetic assign- subjected to the same incubation conditions and followed in ment of consensus sequences was conducted by means of parallel as the experimental setup. Each experiment was con- ARB software package (http://www.arb-home.de/; Ludwig ducted in triplicate. et al. 2004) loaded with a 16S rRNA ARB-compatible database (SSUREF132, Dec 2017) available at SILVA webpage Gas chromatography–mass spectrometry analyses (www.silva-arb.de). Nearly full-length 16S rRNA gene sequences available in the non-redundant 132 SSURef In order to detect the degradation effect of diesel oil by the SILVA database closely related to strain NL1 (KY397882) isolate, NL1, under optimal growing conditions, Gas were selected to construct a maximum likelihood phylogen- Chromatography–Mass Spectrometry (GC–MS) analyses etic tree using ARB. The tree was constructed using were conducted. Cultures were grown for 7 days under opti- AxML þ FastdnaML algorithm implemented in ARB using mal conditions (i.e. pH 11, 40 C, 2 M NaCl and 5% hydro- AxML as program and filters termini and ssuref:bacteria. carbon) and 5 mL of the cultures were extracted in two Once constructed, the tree was exported form ARB and volumes (20 mL) of n-hexane (Sigma-Aldrich, purity >97%) inferred by PHYML (Guindon et al. 2010) using HKY þ as a solvent by using separating funnels to remove cellular 4G þ I model of nucleotide evolution where G distribution, material. The residues were transferred to tarred vials and invariable sites proportion, transition/transversion ratio the volume of each extract was adjusted to 100 mL by add- parameters were kept as default. The confidence of each ing n-hexane. The vials were kept at 4 C until gas chroma- node was determined by assembling a consensus tree of 100 tographic analyses. Culture media without inoculum were bootstrap replicates. used as negative controls to monitor abiotic losses of the substrate. Diesel oil degradation was detected by GC–MS using an Abiotic growth factors’ optimization for maximum Agilent 7890B apparatus equipped with a DB 50-MS capil- degradation of diesel oil lary standard non-polar column (30 m 250 mm 0.25 mm) In order to determine the best growing conditions for and split-split less injector (split ratios 100:1). One mL of the obtained isolates in front of diesel oil degradation, a range organic phase was analyzed by GC–MS. The oven tempera- of pH, temperature, NaCl concentration, carbon and nitro- ture was initially at 60 C and then programed to 325 Cat 1 gen sources, hydrocarbon concentration, and chemical sur- a rate of 8 C min where it was held for 5 min. The tem- factants role were tested. Accordingly, 250 mL Erlenmeyer peratures of injector, transfer line and ionization source conical flasks containing 100 mL MSM media supplemented were all 250 C. The electron impact ionization was tuned at with 1% (v/v) diesel oil were inoculated with culture. Flasks 70 eV and Helium was used as carrier gas with an average 1 were incubated under agitation (120 rpm) and at different linear velocity of 1.0 mL min . The quantification of diesel conditions: for pH (range from 4 to 11 units using either oil degradation was done by the calculation of peak HCl or NaOH), temperature (25 C, 30 C, 37 C, 40 C and area decrease. 45 C), and NaCl concentration (1 mM, 100 mM, 500 mM, 1 M, 2 M, and 5 M). For temperature and pH growth range Biosurfactant production tests, cultures were amended with 1% diesel oil, whereas for the other experiments they were supplemented with 5% die- To evaluate the capacity of biosurfactant production by our sel oil (optimal value). In the case of hydrocarbon concen- isolate, bacteria were grown in 500 mL Erlenmeyer flasks tration, MSM cultures were supplemented with a set of with 100 mL of MSM supplemented with NaCl (2 M) and concentrations spanning from 1 to 10% (v/v) diesel oil and diesel oil (5%). Culture flasks were maintained in a shaker incubated at 40 C and under agitation (120 rpm). In order for 7 days at 120 rpm, pH 11 and 40 C. After reaching to test the effect of chemical surfactants, MSM cultures were production period, bacterial cells were removed by centrifu- supplemented with 5% (v/v) diesel oil and 0.02% of different gation (12,000 g at 4C for 20 min). The cell-free culture surfactants (e.g. sodium dodecyl sulfate (SDS, Sigma- broth suspected to contain biosurfactants was used for fur- Aldrich), Tween 80 (Sigma-Aldrich) and Cetyl Trimethyl ther assays (see below). Ammonium Bromide (CTAB, Sigma-Aldrich) to enhance A qualitative measurement of biosurfactant production by diesel oil degradation and cultures were incubated at 40 C NL1 isolate was carried out using a 100-fold dilution of the and under agitation (120 rpm). In order to evaluate the cell-free culture broth using a TS90000 surface tensiometer 4 N. LENCHI ET AL.

Table 1. Differential characteristics of strain NL1 and two other strains of Delftia species. Characteristicsa Delftia sp NL1 Delftia tsuruhatensis T7T Delftia lacustris 332T Morphology Rod Rod Rod Gram –– – Motility – þþ Pigmentation Creamish White NA NaCl (% (w/v; optimum) 0.5–35 (10) – 0–0.6 (0.1) pH (optimum) 5–9 (8.0) 5–9 (7.0) 5–10 (6.0–7.0) Temperature (C; optimum) 20–50 (30) 10 to 40 (35) 3–37 (25) Catalase – þþ Oxidase þþ þ Hydrolysis of: Starch –– NA Gelatin – NA NA Casein – NA NA Esterase activity against Tween 80 þþ NA H2S production – NA NA Nitrite reduction – þþ Production of acid from: Glucose þ – NA Maltose – NA NA Lactose – NA NA aþ: positive reaction; –: negative reaction; NA: no data available. Data for Delftia tsuruhatensis T7T and Delftia lacustris 332 T were taken respectively from Shigematsu et al. (2003) and Jørgensen et al. (2009).

Delftia lacustris 332T (EU888308) T 97 Delftia tsuruhatensis T7 (AB075017) 92 NL1 isolate (KY397882) T T 81 Delftia acidovorans ACM 489 (ATCC15668 , AF078774) T 100 Delftia litopenaei wsw−7 (GU721027) 78 Delftia sp. TS40 (EU073106) Acidovorax valerianellae DSM 16619T (KF931150) Comamonas denitrificans 123T (AF233877) Rubrivivax gelatinosus ATCC 17011T (D16213)

0.10 Figure 1. Maximum likelihood phylogenetic tree computed using nearly full-length 16S rRNA gene sequences available in the non-redundant 132 SSURef SILVA database belonging to and closely related to Delftia species. The tree was constructed using AxML þ FastdnaML algorithm and termini and ssuref:bacteria filters implemented in ARB. PHYML using HKY þ 4G þ I model of nucleotide evolution was used to infer the final confident tree by assembling a consensus tree of 100 bootstrap replicate.

(Nima, Coventry, England) at room temperature. At the In order to obtain, extract and analyze crude biosurfactants same time, a more quantitative method was also performed. free of other culture media components on the cell-free cul- In this sense, the oil-spread technique was carried out as ture broth of NL1 experiments, we applied a solvent extrac- previously reported (Morikawa et al. 2000; Youssef et al. tion based methodology recently described (Shah et al. 2016). 2004) to evaluate the biosurfactant production of NL1. Accordingly, the cell-free culture broth was centrifuged at Briefly, 30 mL of distilled water were added into a Petri dish 12,000 rpm for 20 min and extracted with chloroform/metha- and 50 mL of diesel oil (commercially available in Algerian nol (2:1 v/v)at4C overnight for biosurfactant isolation. The local petrol stations) were dropped on the water surface solvents were then removed by rotary evaporation. The pre- allowing the formation of a thin oil film. Afterwards, 5 mLof cipitated biosurfactants were lyophilized and then weighted a 10-fold dilution (using deionized water) of cell-free culture (Chandran and Das 2010) using the following formula: broth was carefully added to the middle of the oil film. If Wb ¼ Wpb Wp biosurfactants are present in the cell-free culture broth, the oil will be displaced and a clear zone will be generated, indi- where Wb stands for the dry weight of the biosurfactant; cative of biosurfactant presence. Diameters of the oil expel- Wpb refers to the weight of the Petri dish and biosurfactant ling circles were measured by means of a slide caliper residue after drying; and finally, Wp stands for the weight of (accuracy degree 0.02 mm; Zhang et al. 2012). the empty Petri dish. The weights of the biosurfactants were The emulsifying capacity of products generate by NL1 expressed in terms of grams per liter (dry weight). Primary growth under the experimental conditions tested, was eval- characterization of the biosurfactants present in the cell-free uated by means of the Emulsification index value (E24). culture broth was carried out using ninhydrin, anthrone, The E24 index of the culture samples was determined by and saponification tests, following standardized methodology adding 2 mL of diesel oil to the same amount of culture, (Patowary et al. 2015, 2016). The ninhydrin test will deter- mixed for 2 min with a vortex, and allowed to stand for mine the presence of amino acids and their polymer pro- 24 h. The E24 index is defined as the percentage of height of teins, whereas the anthrone test will determine the presence emulsifion layer (mm) divided by the total height of the of carbohydrate moieties; finally, saponification will estimate liquid column (mm) multiplied by 100 (Iqbal et al. 1995). the lipid content in the analyzed samples. The biosurfactants GEOMICROBIOLOGY JOURNAL 5

Figure 2. Effect of temperature on growth of NL1 isolate using diesel oil as sole carbon source. obtained from the cell-free culture broth were also submit- biochemical proprieties of NL1 strain with respect to other ted to Thin Layer Chromatography (TLC) analyses. Briefly, Delftia species are summarized in Table 1. biosurfactants were extracted by chloroform: methanol: Strain NL1 was resistant to ampicillin (10 mgmL 1) and water (65:15:2; v/v/v) solution as developing solvent and Clindamycin (2 mgmL 1), However, it was sensitive to presence of glycolipids was determined by brown dot blot Fusidic acid (10 mgmL 1), Cefoxitine (10 mgmL 1), (Zhang et al. 2010) by using a chromogenic reagent (mixture Gentamicin (10 mgmL 1) and Kanamycin (30 mgmL 1). of ethanol (95 mL), phenol (3 gr), and sulfuric acid (5 mL)). After the characterization at the physiological and biochemical level of the isolated strain, a molecular approach was performed in order to assign the isolate to a bacterial Results and discussion taxonomic group. In this sense, sequence analyses of the Phenotypic and phylogenetic characterization of the 16S rRNA gene of NL1 strain were conducted placing the NL1 isolate NL1 isolate within the genus Delftia (Figure 1), which belong to the Comamonadaceae family within the order During the characterization of microorganisms able to cope Burkholderiales of the class Betaproteobacteria. Sequence with crude oil present in injection waters of Algerian oil- similarity values (Table S1) with close cultivated species fields, strain NL1 was isolated and selected for detailed anal- within the Delftia genus evidenced that NL1 isolate was yses due to promising results. In this study, we describe the closely related to Delftia lacustris strain 332T (EU888308; morphological, biochemical and phylogenetic characteristics 99.92% 16S rRNA gene sequence similarity) and to D. tsur- of the isolate and propose it as novel species within the uhatensis strain T7 (AB075017; 99.85% similarity) and Delftia genus. more distantly to D. acidovorans strain ACM 489T Strain NL1 is a gram-negative and rod-shaped bacteria (AF078774) and D. litopenaei strain wsw-7 (GU721027) showing circular and cream pigmented colony phenotype (98.6% and 98.5%, respectively) at 16S rRNA gene level. As when growing on solid PA media. The strain NL1 was able per the information gathered in the present work, the to grow in liquid media containing 0.5–35% (w/v) NaCl, bacterial isolate retrieved from injection water well from and optimal growth was observed when growing at 10% an Algerian oilfield was named as Delftia sp. NaCl (w/v), 30 C and pH 8. The phenotypic and NL1 (KY397882). 6 N. LENCHI ET AL.

Figure 3. Effect of pH on growth of NL1 isolate using diesel oil as sole carbon source.

As isolation source directly correlates with crude oil pres- of microbial hydrocarbon-degrading activities by its effect on ence, detailed studies on NL1 strain capacity to cope with the physical nature and chemical composition of oil diesel oil were performed (see below). (Margesin and Schinner, 1997), which might be equivalent to the role of nutrients on rate degradation. As for the temperature test conditions, NL1 isolate evi- Optimization of abiotic factor for maximum denced a preference for higher pH (maximum growth at pH degradation of diesel oil 11.0; Figure 3), whereas minimal growth was observed at The optimization of environmental conditions (e.g. tempera- lower pH values (e.g. pH 5.0 and 6.0). Generally, pH is an ture, pH, NaCl concentration) is an important factor for the important parameter for microbial growth and development enhancement of bacterial growth and for effective bioremedi- of microbes in different environmental conditions, especially ation strategies (Davey 1994). In this sense, various abiotic for soil (Fierer and Jackson 2006; Lauber et al. 2009), and factors affecting the NL1 strain growth were optimized for for bioremediation strategies (Pawar 2012). In this sense, achieving maximum diesel oil degradation. Amongst the fac- many other diesel-degrading bacteria have growth require- tors controlling growth characteristics in microbes, tempera- ments on neutral or near neutrality for optimal growth con- ture is one of the most important ones thus affecting ditions (Kwapisz et al. 2008; Marquez-Rocha et al. 2005; bacterial diesel degradation potential and efficiency. Figure 2 Palanisamy et al. 2014; Rajasekar et al. 2007; Usharani and showed the growth of NL1 isolate at different temperatures in Lakshmanaperumalsamy 2016). To the best of our know- MSM media supplemented with 1% diesel oil. Optimal and ledge, this is the first study showing the requirement of alka- maximum growth were observed at 40 C after 72 h of incu- line pH (pH 11) for optimal bacterial growth on medium bation, whereas minimal growth was observed at 45 Cand containing diesel as sole carbon source. 25 C. Luo and coworkers (Luo et al. 2013) reported that the Furthermore, we evaluate the effect of different NaCl diesel oil degrading ability of Pseudomonas sp. strain F4 was concentrations on NL1 isolate growth capabilities on MSM reported to be at 37 C whereas, Pseudomonas aeruginosa media supplemented with 5% diesel oil as carbon source, dou-1 showed optimal growth at 35 C (Usharani and after results on growth with initial hydrocarbon concentra- Lakshmanaperumalsamy 2016). Temperature affects the rates tion (see below). The results showed that strain NL1 was GEOMICROBIOLOGY JOURNAL 7

Figure 4. Effect of NaCl concentration on growth of NL1 isolate using diesel oil as sole carbon source. able to grow and degrade diesel oil at high NaCl concentra- test for enhancement for diesel oil degradation by NL1 iso- tions (up to 2 M NaCl, Figure 4) with the exception of 5 M late (Figure 5). All tested carbon and nitrogen sources NaCl, thus evidencing an halotolerant behavior. Such char- exhibited none effect over the first 48 h of incubation. acteristic would be of interest in MEOR processes where sal- Glucose supplemented culture exhibited a significant inity of production wells on oilfields is high (Lenchi et al. enhancement of diesel oil degradation with respect to fruc- 2013) or for removal of stranded oil on beaches or intertidal tose, which never overpass glucose effects. Accordingly, areas where bacterial species would have to survive exposure glucose was selected as carbon source for further studies. to pollutants and sea level (or higher) saline concentrations. Ganesh and Lin (2009) reported that the addition of glu- Recently, Usharani and Lakshmanaperumalsamy (2016) cose to the liquid medium had a positive effect, with an reported that the amount of oil degraded by Pseudomonas increase in growth of the isolates thus leading to signifi- aeruginosa strain dou-1 increased to maximum levels when cantly (p < 0.05) higher percentages of diesel degradation growing at 3 mg L 1 of NaCl. Furthermore, Mnif and cow- and greater emulsification activity. On the other hand, nei- orkers (Mnif et al. 2014) also isolated an aromatic-degrading ther peptone nor yeast extract exhibited higher effect than bacterium (Aeribacillus pallidus VP3) from a geothermal oil control cultures (MSM supplemented with 5% diesel oil) field with 10 g L 1 as optimal NaCl concentration for deg- suggesting no effect of these nitrogen sources on NL1 radation of crude oil. In other study, Mnif et al. (2009) iso- growth capacity. lated an halotolerant bacterial strain Halomonas sp. C2SS100 from production water of an oilfield, which was able to Effect of initial hydrocarbon concentration degrade hydrocarbons efficiently at 100 g L 1 of NaCl. To the best of our knowledge, this is the first report on The isolated bacterial strain was screened based on the abil- bacteria, and especially Delftia sp, that could degrade diesel ity to utilize diesel oil. In order to evaluate the effect of ini- oil at such high salt concentration; 116 g L 1 (2 M) more tial hydrocarbon concentration (i.e. diesel oil) on the growth than three times salt concentration in seawater. of NL1 isolate, several incubations with a range of hydrocar- Various carbon and nitrogen sources such as fructose, bon concentrations spanning from 1 to 10% were used glucose, yeast extract and peptone (0.1%) were added to (Figure 6). Higher (10%) and lower (1%) diesel oil concen- the MSM medium containing 5% diesel oil as nutrient to trations reflected minimal growth enhancing effect, whereas 8 N. LENCHI ET AL.

Figure 5. Effect of different carbon and nitrogen sources on NL1 isolate growth using diesel oil as sole carbon source. middle range (3–5%) showed a slight enhancement. In this distinct chemical surfactants (i.e., SDS, Tween 80 and sense, the addition of 5% diesel oil as initial carbon source CTAB) on diesel oil degradation by our bacterial isolate is for NL1 isolate under MSM media showed maximum shown in Figure 7. Results evidenced a marked growth on growth effect (Figure 6). Biodegradation studies on diesel oil 0.02% SDS (an anionic surfactant) with respect to control have been carried out using diesel concentrations ranging experiment (no surfactant) all over the growing experiment from 0.5 to 1.5% (Lee et al. 2006; Mukherji et al. 2004; (144 h). By contrast, the addition of a nonionic surfactant Ueno et al. 2007). At high diesel oil concentration, diesel oil (Tween 80 at 0.02%) on the culture under optimal growth provided a better carbon source for bacterial growth (Luo conditions (MSM media supplemented with 5% of diesel oil) et al. 2012). Degradation at a much higher concentration did not show any marked difference with respect to the con- (6% v/v diesel) has been reported for some bacterial species, trol, so evidencing a negative effect on diesel oil degradation but with glucose (0.2%, w/v) and yeast extract (0.1%, w/v) by NL1. The addition of a cationic surfactant (CTAB) on supplements (Kwapisz et al. 2008). However, growth of the the culture showed a toxic effect on Delftia sp NL1 strain as bacterial species on higher amounts of diesel oil can be reflected by its marked growth reduction compared to the inhibited by recalcitrant effect of such compounds, as control incubation condition (see Figure 7). Thus, our occurred in our case at 10% diesel oil concentration results demonstrated a preferential utilization of anionic (Figure 6). In effect, the high concentration of diesel could SDS by Delftia sp NL1 under aerobic conditions. be harmful to the bacterial population in enclosed However, Tween 80 has been reported as the best systems due to its solvent effect that may damage the bacter- amongst nonionic surfactants in improving low molecular ial cell membrane (Putri Pranowo and Sulistiyaning weight PAHs (i.e. naphthalene, phenanthrene, and anthra- Titah 2016). cene) degradation by different soil bacterial species (named Pseudomonas sp., Enterobacter sp., and Stenotrophomonas sp.) (Bautista et al. 2009). Effect of chemical surfactants Even cationic surfactants has been reported in the litera- Surfactants facilitate the utilization of hydrocarbons at ture to promote microbial degradation of hydrocarbons in higher rates by enhancing their bioavailability. The effect of some cases and for some species (Anaukwu et al. 2016), our GEOMICROBIOLOGY JOURNAL 9

Figure 6. Effect of initial diesel oil concentration on growth of NL1 isolate using diesel oil as sole carbon source and mineral salt medium as basal media.

results are on line with primary works such the one of of SDS was also previously reported (Margesin and Volkering and coworkers (Volkering et al., 1997) statin that: Schinner 1998). ‘at a pH of 7 and higher, cationic surfactants are the most toxic ones, while anionic surfactants display the most toxic GC-MS analysis behaviour at lower pH’. In this sense and as result of growing conditions for Delftia sp. NL1 strain (pH 11), the use of In order to determine the biodegradation capabilities of isolated strain NL1 of diesel oil under the experimental set-up, CTAB surfactant to putatively increase hydrocarbon biodeg- GC–MS analyses were conducted. GC–MS chromatograms radation resulted on toxic effects for Delftia sp. NL1. Such evidenced a clear reduction in peak intensities of diesel oil toxicity might be related to either cellular membrane disrup- degradation products after incubation with NL1 isolate tion by interaction with membrane lipids or reaction with (Figure 8(b)) when compared with abiotic control experi- essential proteins for cell functioning as proposed Volkering ments (Figure 8(a)). In general, when bacterial isolate NL1 and coworkers (Volkering et al. 1997) based on early sugges- was grown with MSM media supplemented with 5% crude tions on previous reports (Helenius and Simons 1975) oil at 40 C, 2 M NaCl, and pH 11 for 7 days (168 h), a The bioavailability of the primary substrate can be reduction of 66.76% of the diesel oil was achieved (based on improved with biodegradation of the surfactant due to peak area for higher alkane molecules (i.e. C10 to C19)). To greater release of hydrocarbons from the micellar phase into the best of our knowledge, this is the first report on an iso- the aqueous phase, making the substrates more readily avail- lated bacteria which is able to degrade more than 66% of able to microorganisms (Mohanty et al. 2013). Study of diesel in few days (7) and on such high salt (2 M) and pH Zeng et al. (2007) on the degradability of different types of (11) conditions. These abilities may be due to the source of surfactants (CTAB, Triton X-100, SDS, and rhamnolipids) this strain. In fact, petroleum reservoirs are complex ecosys- by Pseudomonas aeruginosa, Bacillus subtilis, and a microbial tems located in deep geological formations where high tem- consortium obtained from solid waste compost reported that perature, pressure, and salinity exist. Accordingly, this type comparing to the other the anionic surfactant SDS was eas- of environments are excellent sources of fascinating micro- ily degraded by all three microbial strains. The degradation bial strains and metabolic processes. 10 N. LENCHI ET AL.

● 1.5 ●

●

● 1.0 ●

● Control

● CTAB ● ● ● SDS

● Tween80 OD (600nm) ●

● ● 0.5 ● ● ● ●

● ●

●

● ●●● ● 0.0 ●

0 50 100 150 200 250 time (min) Figure 7. Effect of chemical surfactants on growth of NL1 isolate using diesel oil as sole carbon source.

Biosurfactant production and characterization increasing the cell hydrophobicity. Consequently, the interaction between biosurfactant, contaminant and cells are Production of biosurfactants by microbes can be inferred by optimized and biodegradation increased. (Meyer et al. 2012). the evaluation of the emulsification index (E ); a higher E 24 24 Chemical composition of biosurfactants produced by NL1 value evidence the capacity of emulsify those hydrocarbons isolate was evaluated by the ninhydrin (formation of present in a sample and render them more accessible for Ruhemann’s purple complex; Ruhemann 1910) and biological break down and thus the presence of biosurfactant anthrone (formation of blue-green coloring, Sawhney and substances (Abouseoud et al. 2008). Bacteria with E24 higher than 50% have been defined as potential biosurfactant pro- Singh 2000) tests. In the ninhydrin test, no purple complex ducers (Rodriguez-Rodriguez et al. 2012) and thus of bio- was observed thus biosurfactants present in the cell-free cul- technological interest. The assimilation of diesel as the sole ture broth of NL1 isolate were not protein/amino-acid carbon source by the isolated strain led to the production of based, whereas a bluish green color was noted in the biosurfactants as evidenced by the clearance zone in the oil- anthrone test, which denotes the existence of carbohydrates spreading method (up to 4.52 cm of diameter of oil expelling in the biosurfactants produced by NL1 isolate. Furthermore, ¼ saponification experiments evidenced the presence of a lipid circles), the emulsification index (E24 81%) and the lower- 2 ing of the surface tension (25.7 mN m 1 in front of 74.4 mN portion in the biosurfactants produced by NL1 by appear- m 1 for deionized water and 61.7 mN m 1 for culture ance of brown spots in the TLC plates. In conclusion, NL1 media without inocula) generated by the cell-free culture isolate could produce biosurfactants of glycolipidic nature by broth. The result indicated that the isolate NL1 was able to fermentation when grown on diesel oil as sole carbon source lower the surface tension of diesel liquid media, putatively (up to 8.7 g 1 under optimal growing conditions). via biosurfactant production. The results demonstrated that even at high salt concen- For hydrocarbons, the biosurfactants influence the tration (2 M) and alkaline media (pH 11), strain Delftia sp. biodegradation rate in two different ways: by increasing NL1 was able to produce biosurfactants, thus facilitating the non-polar molecule dissolution in the aqueous phase, and uptake of hydrocarbons and their biodegradation. In recent changing the affinity between cell and contaminant, thus decades the search for biosurfactant producing GEOMICROBIOLOGY JOURNAL 11

(a) (b)

Figure 8. GC–MS profiles of diesel remaining in basal medium after aerobic incubation, without (a) and with Delftia sp.NL1 (b) at 40 C, pH 11 and 2 M NaCl for 7 days. microorganisms isolated from saline environments is exten- saline-alkali sites, in wastewater treatment in refineries and sive (Kebbouche-Gana et al. 2009) but the use of indigenous oil terminals and in MEOR process in petroleum industry. microorganisms to such extreme environment like oilfield Delftia sp. NL1 provided promising evidence for the biodeg- must be encouraged. Thus, the indigenous strain Delftia sp. radation of diesel in extreme condition like high salinity NL1 could be a potential candidate for the production of and pH. glycolipidic biosurfactant which could be useful in a variety of biotechnological and environmental applications particu- larly in the main stages of the oil production chain, such as Acknowledgment extraction, transportation, and storage. We gratefully acknowledge the work of Adriana Anzil from the ESA In petroleum industry, indigenous species present in oil- unit at Universite Libre de Bruxelles. fields are the best candidate for the MEOR processes. In fact, the injection of microbes producing stable biosurfac- Author contributions tants at extreme condition of production wells causes the reduction of oil viscosity and interfacial tension between oil Conceived and designed experiments: NL. hydrocarbons and surface of rock matrix, which can facili- Performed experiments: NL. Analyzed data: NL, MLl. tate the mobilization of oil and further increment of oil Contributed reagents/materials/analysis tools: NL, SKG, PS, MLG, MLl. recovery. In fact, biosurfactants form an emulsion at the oil- Wrote the paper: NL, MLl, PS, SKG. water interface, which stabilizes the desorbed oil in water and allows oil removal along with the injection water (Pacwa-Plociniczak et al. 2011). Disclosure statement No potential conflict of interest was reported by the author(s). Conclusion References The present study focused on the degradation of diesel oil by a bacterial isolate retrieved from an Algerian oilfield- Abdel-Shafy HI, Mansour M.S.M. 2016. A review on polycyclic aro- water injection sample. The strain was identified as Delftia matic hydrocarbons: source, environmental impact, effect on human – sp. NL1, which exhibited the ability to degrade more than health and remediation. Egyptian J Petrol 25:107 123. Abouseoud M, Maachi R, Amrane A, Boudergua S, Nabi I. 2008. 66.76% of diesel oil within only 7 days of incubation at ini- Evaluation of different carbon and nitrogen sources in production tial pH 11 and 40 C and in the presence of 2 M NaCl. of biosurfactant by Pseudomonas fluorescens. Desalination 223: Furthermore, the described isolate (Delftia sp. NL1) pro- 143–151. duced glycolipids as biosurfactant at a concentration of 8.7 g Akbari S, Abdurahman NH, Yunus RM, Fayaz F, Alara OR. 2018. L 1. These biosurfactant will help to improve the accessibil- Biosurfactants: a new frontier for social and environmental safety: a ity and bioavailability of water-immiscible diesel to degrad- mini review. Biotechnol Res Innovat 2:81–90. ing bacteria and reducing environment contamination. Anaukwu CG, Ekwealor AI, Ezemba CC, Anakwenze VN, Agu KC, Nwankwegu AS, Okeke BC, Awah NS. 2016. Influence of anionic, Based on our observations, we concluded that the degrad- cationic and non-ionic surfactants on growth of hydrocarbon utilization of diesel hydrocarbon was significantly achieved by the ing bacteria. Amer J Curr Microbiol 4:10–16. indigenous strain Delftia sp. NL1 which can be further Anjana S, Poonam K, Meenal Budholia R. 2014. Biodegradation of die- exploited in in situ bioremediation of diesel contaminated sel hydrocarbon in soil by bioaugmentation of Pseudomonas 12 N. LENCHI ET AL.

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