Hydrocarbon degradation by a newly isolated thermophilic Anoxybacillus sp. with bioemulsifier production and new alkB genes

Abstract

Interest in biodegradation of petroleum derived pollutants by thermophilic bacteria has been steadily increasing in recent decades. In this work, a newly isolated thermophilic bacterial strain was isolated from a deep petroleum reservoir and identified as Anoxybacillus sp. WJ-4 based on an analysis of its physiological characteristics, 16S rRNA sequencing, GC content and cellular fatty acids. It is the first report that strain WJ-4 can degrade a wide range of hydrocarbons (C8–C22) at 67 °C. The production of an oligosaccharide–lipid–peptide bioemulsifier was detected. It exhibited an excellent emulsification activity with various oil phases (EI₂₄ > 60%), and the ability to increase cell surface lipophilicity during degradation, but has no significant impact on surface tension, with a reduction from 72.22 mN m⁻¹ to 52.45 mN m⁻¹. Four alkane monooxygenase genes showed a high phylogenetic relationship (>95%) with alkB genes from Geobacillus. These results indicate that this newly isolated bacterial strain and its bioemulsifier have great potential for environmental remediation and petroleum recovery under thermophilic conditions.

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1. Introduction

Petroleum is a predominant energy resource and raw material for the chemical industry in modern society. Spills, leaks, and other releases of petroleum, however, often result in soil and groundwater contamination.¹ The main sources of petroleum contamination include petrochemical industry processes, oil field installations, petroleum plants, liquid fuel distribution and storage devices and transportation equipment for petroleum products.² Pollution from such sources has significant environmental impact and presents hazards to human health. Petroleum is a complex mixture of hydrocarbons and related compounds. It is generally classified into four fractions: saturated alkanes, aromatics, resins, and asphaltenes, the latter two of which consist of polar molecules containing N, S, and O. Alkanes can constitute 50% to 95% of crude oil, depending on the source. As the main component of fuels and oils, their relative inertness poses ecological problems upon release to the environment. Due to their lack of functional groups as well as poor water solubility, alkanes exhibit both low chemical reactivity and low bioavailability for microorganisms.^3,4 Various linear, branched and cycloalkanes are known to cause respiratory, renal, or central nervous system disorders. As a result, considerable attention has been paid to the treatment of these pollutants.

Microbial bioremediation has been evaluated as one option in various polluted environments and is claimed to represent an efficient, economical, and versatile alternative to physicochemical treatments.^5,6 Hydrocarbon-degrading bacteria are widely distributed in nature. They include members of at least 60 genera of aerobic and five genera of anaerobic bacteria,^3,7–9 particularly Acinetobacter, Rhodococcus, Alcanivorax, Bacillus, Mycobacterium, Pseudomonas and Dietzia, which are among the most well known and studied.^10–13 However these bacteria generally exhibit good performance only under mesophilic conditions. Few thermophilic hydrocarbon-degrading species have been reported, although many thermophiles have been described.

The degradation of petroleum hydrocarbons by thermophilic bacteria has advantages over that by mesophilic or psychrophilic organisms, especially when they are incorporated into biotechnological applications.^14,15 Increasing attention has been paid directly to this field in recent decades.^16,17 Thus it is necessary to isolate more thermophilic strains with the capability to degrade these water-insoluble molecules. The majority of thermophilic strains are obtained from hot springs and oil reservoirs. High temperature petroleum reservoirs with temperatures exceeding 50 °C are one type of biotope attracting great interest as sites for the collection and screening of new thermophilic hydrocarbon-degrading bacteria.^4,14,17,18 Genera collected from these sites have been limited to Bacillus, Geobacillus, Thermoactinomyces, and Brevibacillus.^19,20 Only four studies on hydrocarbon degradation by Anoxybacillus have been reported,^21–24 where the degraded compounds were aromatic. As a new thermophilic genus, Anoxybacillus could be utilized in a large number of applications as previously described,²⁵ however these applications are currently limited to the hydrolysis of starch and lignocellulosic biomasses, not the degradation of alkanes.

Recognized bacterial mechanisms for enhancing hydrocarbon substrate availability and utilization can for simplicity generally be divided into (a) those related to the uptake of soluble fractions and (b) those related to the production of surfactants for physical modification of substrates and cell surface lipophilicity;^26–28 the mechanisms however overlap. Extremely limited water solubility is the most serious hurdle for the uptake of alkanes as a metabolic substrate, although higher temperatures can contribute to improved mass transfer rates. The uptake of hydrophobic components therefore commonly and frequently involves the production of microbial surfactant molecules as emulsifying agents or cell surface lipophilicity (CSL) altering agents to facilitate ultimate biodegradation.

Produced by a wide range of hydrocarbon-degrading microorganisms, microbial surfactants can be grossly classified into two major categories based on molecular weight (MW).^29,30 Examples of low-MW biosurfactants include glycolipids and lipopeptides, whose dominant function is the lowering of surface/interfacial tension. Examples of high-MW biosurfactants include emulsan, alasan, biodispersan, and extracellular or cell membrane-bound bioemulsifiers (such as exopolysaccharide and lipopolysaccharides), whose main function is emulsion stabilization.^30,31 For many hydrocarbon-degrading microorganisms, extensive changes of the cell surface lipophilicity were detected during growth on hydrocarbons.³² Several reports in the literature find correlation among CSL, microbial surfactant production, and hydrocarbon degrading capability.^26,30–32

In the present report, a newly isolated thermophilic hydrocarbon-degrading strain WJ-4 was isolated and investigated. This is the first report that Anoxybacillus strain can utilize alkanes (C8–C22) as a sole source of carbon for growth. With a goal of designing future applications for remediation of crude oil contaminated environments, the alkane degradation kinetics of strain WJ-4 was characterized. A bioemulsifier was detected during biodegradation and its structure was analyzed. Emulsification stability was evaluated as well. A tentative comparison of the alkB gene from this newly thermophilic strain was also carried out. This paper highlights an important potential use of a novel thermophilic strain for the cleanup of alkane or petroleum polluted environments.

2. Experimental

2.1. Isolation of microorganisms

Brine and oil samples were collected from a reservoir formation located at a depth of 1300 to 1600 meters underground with a temperature of 58–68 °C in Longhupao block (latitude 46.798383, longitude 124.350554) of Daqing oilfield. 50 ml of the brine and 5 g of the crude oil were transferred to a 250 ml flask filled with 50.0 ml of autoclaved minimal medium supplemented with a trace-element solution (0.1%, v/v), and then incubated at 65 °C with 180 rpm stirring for 20 days. The minimal medium contains (g L⁻¹): NH₄NO₃ 3.4, K₂HPO₄ 1.5, NaH₂PO₄ 1.5, MgSO₄ 0.3, yeast extract 0.3, glucose 0.3, pH 6.8–7.2; and the composition of the trace-element solution is (g L⁻¹): MnCl₂·4H₂O 0.1, CoCl₂·6H₂O 0.17, CaCl₂·2H₂O 0.02, FeCl₂ 0.4, H₂BO₃ 0.019, ZnCl₂ 0.1, NaMoO₄·2H₂O 0.1, vitamin B₁₂ 0.01, vitamin C 0.01, and NiCl₂·6H₂O 0.05. After 20 days incubation, 50 ml of the cultured sample was transferred into 50 ml of the above fresh sterilized medium again for a second 20 day incubation under the same conditions. Then 100 μl of the final cultured sample was spread onto LB agar plates and incubated at 65 °C for 48 h. Pure cultures of each morphologically distinct colony were selected by repetitive streaking onto solid LB agar medium. In order to isolate the strains with the ability to degrade hydrocarbons and produce biosurfactants, the selected pure strains were cultured for 10 days in the above sterilized minimal medium with crude oil as the carbon source, then the cell-oil-free supernatant of each cultured sample was spread onto an agar plate and a crude oil emulsification test was conducted following a reported method.³³

2.2. Characterization of strain

The final selected bacterial strain was characterized according to standard biochemical tests (morphology and biochemistry) following Bergey’s Manual of Systematic Bacteriology. Growth was determined by measuring the OD₆₀₀ value at different temperatures (20–80 °C), pHs (4–11) and NaCl concentrations (0–25%, g L⁻¹). The ability to utilize various carbon sources was examined in minimal medium. Carbon sources were added at a final concentration of 0.05% (w/v) with: fructose, glucose, L-rhamnose, sucrose, lactose, molasses, starch, xanthan, n-dodecane and n-hexadecane.

G + C content was determined according to Mesbah³⁴ by using HPLC. Non-methylated Lambda-DNA (Sigma) with a GC-content of 49.858 mol% served as an external standard.

DNA was extracted from the isolated strain following the instructions of an extraction kit (MoBio, USA) for phylogenetic analysis. The methods for 16S rRNA gene amplification and sequencing have been reported previously.²³ Phylogenetic analysis was performed by BLAST of the National Center for Biotechnology Information.³⁵ Multiple sequence alignments were carried out using ClustalX 1.8 and a neighbor-joining tree was constructed using MEGA Software Version 6.0.

Late exponential phase cells were harvested by centrifugation (10 000 rpm for 10 min) and washed three times with distilled water for the analysis of cellular fatty acids. Lipid extraction and cellular fatty acid analyses were performed according to the method of Siristova.³⁶ The relative percentages of the fatty acids were determined from the peak areas of methyl esters by a gas chromatograph (Agilent 5890) equipped with a flame-ionization detector. The running method was as follows: injector temperature, 240 °C; detector temperature, 240 °C; carrier gas (He) flow rate, 10 ml min⁻¹. The oven temperature was programmed from 50 to 300 °C at 8 °C min⁻¹. The results are means of two independent experiments.

2.3. Kinetics of alkane degradation

Cells harvested from 50 ml of LB medium of the selected strain were washed with distilled water triply and transferred to a 250 ml flask containing 2 g of ultrafilter-sterile alkane mixture (n-alkane, C8–C22, purity > 99%, Sigma, USA) and 90 ml of minimal medium, then incubated at 70 °C with 180 rpm stirring for 40 days; cell-free sterile medium was used as a control. 4 parallel samples were prepared as interval samples for analyzing the biodegradation kinetics including cell growth, residual alkane, cell surface lipophilicity, surface tension and emulsification.

Cell growth was monitored by measuring the dry cell weight. Cells were collected by centrifugation (10 000 × g, 10 min) of a 100 ml culture broth and washed with distilled water twice, and then dried by heating at 40 °C until a constant weight was attained. The surface tension of cell-free samples was measured by a digital tensiometer (KRUESS klot, Germany) using a ring detachment method. Emulsification activity was determined by the addition of 5 ml of alkane mixture to 5 ml of the cell-alkane-free supernatant in a 15 ml graduated tube according to a previously described method.³⁷ Adherence of bacteria to hydrocarbons was used as a measurement of cell surface lipophilicity according to a reported method.³²

Alkanes were extracted with dichloromethane in triplicate (>99%, Sigma, USA), the organic phase collected and the solvent removed using a rotary evaporator at 30 °C, and then the residual alkanes were weighed. In order to profile the degradation characterization, a gas chromatograph equipped with an FID detector (Agilent 5890, USA) was applied to detect the change of each fraction in the alkane mixture. The GC running program was as follows: injector temperature, 240 °C; detector temperature, 240 °C; carrier gas (He) flow rate, 10 ml min⁻¹. The oven temperature was programmed from 50 to 300 °C at 8 °C min⁻¹. The relative percentages of the n-alkanes were determined from the peak areas; and the weight of each alkane was calculated by the relative percentage and the total weight of the residual alkane mixture. The results are means of two independent experiments.

2.4. Analysis of the bioemulsifier

Purification of the bioemulsifier was performed by solvent extraction and alcohol precipitation.^37,38 The measurement of surface tension and emulsification was applied to testify the surface/interface activities of the obtained materials, and then the chemical characteristics of these materials were further analyzed with or without hydrolysis. The carbohydrate moiety was detected by a phenol–sulfuric acid method using glucose as a standard.³⁹ The protein moiety was detected by the Lowry method using bovine serum albumin (BSA) as a calibration standard.⁴⁰ The lipid moiety was detected using a dichloromethane–methanol method.⁴¹ The number-average molecular weight (M_n), weight-average molecular weight (M_w) and polydispersity index (PDI) of the biosurfactant were measured by gel permeation chromatography (GPC) using Pullulan standards as described previously.^42,43

Monosaccharide composition was determined according to the method reported by Carrion.³⁸ The hydrolysates were used to identify and quantify the constituent monosaccharides by high-performance liquid chromatography (HPLC) using Aminex HPLC Carbohydrate Analysis Columns HPX-87P (300 × 7.8 mm) and HPX-87C (300 × 7.8 mm) (BioRad) with commercial sugars as standards for monosaccharide identification. Amino acids were analyzed following the methods of Xia³⁷ with an automatic amino acid analyzer (1100 series, Hewlett Packard, USA). Fatty acid compositions were extracted from the hydrolyzates with ether in triplicate and esterified with methanol at 100 °C for 1 h, then subjected to gas chromatograph mass spectrometer analysis.⁴⁴

The effect of environmental factors (salinity, temperature, pH, and metallic ions) on the performance of emulsification against the crude oil was determined. The concentration of the purified active bioemulsifier tested in emulsification tests was 2000 mg L⁻¹.

2.5. Analysis of alkane hydroxylase genes

DNA isolated from the Anoxybacillus strain was used as a template for PCR. Amplification of alkB fragments of the thermophilic bacteria was carried out with various degenerate oligonucleotide primers as described in the literature.^45,46 The purified PCR products of alkB genes were cloned using a pGEM-T vector system (Promega). Clones were sequenced with universal M13 primers in an ABI 3700XL genetic analyzer (Applied Biosystems). A preliminary analysis of the new sequences was done with the BLAST program of NCBI. The nucleotide sequences were aligned with homologous sequences retrieved from GenBank with ClustalX 1.8, and a neighbor-joining tree was constructed using MEGA Software Version 6.0.

3. Results and discussion

3.1. Characterization of bacteria

The well-characterized strain WJ-4 is a facultative aerobic, Gram-positive, motile, spore-forming, rod-shaped bacterium with a length of 3.9 to 5.6 μm and a width of 0.6 to 0.9 μm (Table 1). Colonies were gray white, with an umbonate surface and undulate edge. The sporangium was not swollen, whereas the spores were oval and terminally positioned. Growth was observed both on carbohydrates (arabinose, fructose, glucose, L-rhamnose, mannose, sucrose, lactose, molasses, starch, and xanthan) and hydrocarbons (n-dodecane, n-hexadecane and xylene). Indole was not produced; nitrate was not reduced; the Voges–Proskauer reaction and methyl-red test were negative. Like most thermophilic bacilli, the catalase reaction was positive for this tested strain. It can survive at a wide range of temperatures, from 45 °C to 80 °C, and the maximum optical density was achieved at 68–72 °C. Tolerance to NaCl levels was weakened with an increase of salt concentration, the growth almost ceased when salinity reached 12%. The optimum pH for the growth was 6.0–8.0.

Table 1 Comparison of the phenotypic characteristics of Anoxybacillus sp. WJ-4 and related species^a

Characteristic	WJ-4	AB04	R270	MR3C	GS5-97
a +positive; −negative; w weak response; N.D. not determined. Anoxybacillus ayderensis AB04T;^47 Anoxybacillus amylolyticus MR3C;^48 Geobacillus tepidamans GS5-97;^49 Anoxybacillus rupiensis R270.^50
Cell length (μm)	3.9–5.6	4.6	3.3–7.0	2.0–2.5	3.9–4.7
Oxygen	Facultative	Aerobic	Aerobic	Facultative	Aerobic
Motility	+	+	+	+	+
Spore shape	Spherical	Spherical	Spherical	Spherical	Oval
Optimal temperature (°C)	68–72	50	55	61	55
Optimal pH	6.5–8.0	7.5–8.5	6.0–6.5	5.6	7.0
Catalase	+	+	+	+
Oxidase	+	+	+	+
Arabinose	+	+	+	+	ND
Ribose	ND	ND	+	+	ND
Xylose	+	+	+	+	+
Fructose	+	+	+	+	+
Galactose	ND	ND	−	+	+
Mannose	+	+	ND	+	+
Rhamnose	+	−	+	+	−
Sucrose	+	+	−	+	+
Lactose	−	−	−	ND	w
Starch	+	+	+	+	ND
G + C (mol%)	44.3	54	41.7	43.5	42.4

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The phylogenetic tree (Fig. 1) showed that strain WJ-4 was almost positioned between the Anoxybacillus genus on one side and the Geobacillus genus on the other side. The closest (more than 98%) sequence relatives found by a BLAST search were Anoxybacillus species (KJ842629.1, EU710556.1, KF266689.1) and Geobacillus species (FJ823100.2, EU087702.1). Although Anoxybacillus is phylogenetically close to Geobacillus species,^49,51 strain WJ-4 was related to the genus Anoxybacillus on the basis of phylogenetic similarity with Anoxybacillus species combined with the morphological, physiological, and biochemical properties. The sequence of the 16S rRNA gene of the strain WJ-4 is deposited and available under the GenBank accession number JX673944.1.

Fig. 1 Phylogenetic relationship based on the 16S rRNA gene sequences between strain WJ-4 and species in the Anoxybacillus and Geobacillus genera as determined by the neighbor-joining algorithm and evaluated by the maximum-likelihood and maximum parsimony algorithms. The bar represents 2 nucleotide substitutions per 1000 nucleotides.

The G + C content of the genomic DNA for the strain WJ-4 was 44.3 mol%, which was significantly lower than those of the genus Geobacillus (48.2–58 mol%),⁵¹ but similar to the closest Anoxybacillus relatives (42–57 mol%, Anoxybacillus amylolyticus 43.5 mol%; Anoxybacillus voinovskiensis 43.9 mol%; Anoxybacillus contaminans 44.3 mol%). Although the G + C content of DNA was 43.2 mol% for Geobacillus tepidamans,⁴⁹ it currently has been reclassified as Anoxybacillus tepidamans. Therefore, based on the similar percentage ranges of G + C content between Geobacillus and Anoxybacillus, it is possible that an intimate phylogenetic relationship existed between them.

The cellular fatty acids (FAs) of the strain WJ-4 are largely composed of branched saturated aliphatic acids, and contain a similar percentage of anteiso-fatty acids as minor components to other thermophilic bacilli (Table 2). Iso-branched fatty acids constitute 83.22% of total WJ-4 fatty acids and greatly predominate over anteiso-branched members, and contain iso-branched saturated fatty acids (iso-C15:0 and iso-C17:0) as major fatty acids, especially iso-C15:0 which accounts for 48.79%. The presence of branched FAs is considered to be a means of maintaining membrane fluidity; iso-branched FAs generally have higher melting points, while anteiso-branched FAs typically have lower melting points. This is a possible explanation for the thermophilic property of strain WJ-4. The percentages of iso-C15:0 and iso-C17:0 for the other genus representatives were: 48.30% for Geobacillus stearothermophilus 5965, 68.59% for Anoxybacillus contaminans, 72.8% for Anoxybacillus amylolyticus and 86.36% for Anoxybacillus rupiensis R270.⁵⁰

Table 2 Cellular fatty acid compositions (%, w/w) of strain WJ-4 and some of the most closely related strains in the phylogenetic tree shown in Fig. 1^a

Fatty acid	WJ-4	5965	GS5-97	MR3C	R270	LGM	3721
a Anoxybacillus amylolyticus MR3C;^48 Geobacillus tepidamans GS5-97;^49 Anoxybacillus rupiensis R270, Anoxybacillus contaminans LGM, Geobacillus stearothermophilus 5965, Geobacillus stearothermophilus 3721.^42
13:0 iso	—	—	0.4	—	—	—	—
14:0 iso	—	0.20	0.60	—	—	—	0.30
14:0	2.87	0.20	4.10	—	0.30	3.00	1.10
15:0 iso	48.79	22.90	44.33	41.2	52.81	52.00	16.90
15:0 ai	1.32	5.10	6.60	2.13	1.64	7.00	2.40
15:0	0.32	1.70	—	0.10	0.31	—	2.90
16:0 iso	5.68	7.30	3.20	7.00	2.01	5.00	15.20
16:0 ai	—	—	—	0.12	—	—	—
16:0	12.65	14.0	15.10	6.3	5.44	11.00	18.40
17:0 iso	28.45	25.40	15.00	31.60	33.55	12.00	29.90
17:0 ai	4.02	8.10	6.10	0.70	3.94	7.00	6.40
17:0	—	6.30	—	—	—	—	2.50
18:0 iso	0.30	0.7	0.60	1.30	—	—	1.30
18:1	—	—	—	0.70	—	—	0.10
18:0	—	2.2	—	1.90	—	—	1.40
19:0 iso		0.3					0.3
19:0		—					0.1

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The similar physiological characteristics, G + C content of DNA, fatty acid profile and phylogenetic similarity (98.0–99.0% to the closest relatives) with representatives of the genus Anoxybacillus allow us to place the strain WJ-4 in the genus Anoxybacillus as the type strain for the novel species. Particularly, it was possible that Anoxybacillus sp. WJ-4 has some similar characteristics with Geobacillus species.

3.2. Degradation kinetics

Due to a lack of functional groups as well as low water solubility, alkanes exhibit low chemical reactivity and bioavailability for microorganisms. However, some microorganisms possess the metabolic capacity to use these compounds as carbon and energy sources for their growth. In this study, the isolated themophilic and halotolerant strain WJ-4 was evaluated for the degradation of alkanes (C8–C22). The results of the hydrocarbon degradation kinetics shown in Fig. 2A demonstrate the excellent ability for alkane degradation of this novel strain with maximum growth reached at the 20^th day. The degradation rate became flattened after 20 days incubation but in total 58.75% of alkanes were decomposed after 40 days at 70 °C under aerobic conditions.

Fig. 2 Degradation of an alkane mixture by Anoxybacillus strain WJ-4 under aerobic conditions at 70 °C. (A) Biodegradation kinetics, including the changing of cell surface lipophilicity, surface tension, emulsification index, residual alkane mixture and cell weights during the degradation; (B) the degradation kinetics of the individual alkanes in the mixture. The values are means ± standard deviation (n = 3).

The thermophilic bacterial genera Anoxybacillus and Geobacillus were nearly described in the radiation from the Gram-positive genus Bacillus,^51,52 and Bacillus and Geobacillus have presented the ability of alkane degradation.⁵³ Compared with Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000.⁵² Little research on hydrocarbon degradation by Anoxybacillus genus has been reported, so the application of this new genus for bioremediation and enhanced oil recovery was rare. The Anoxybacillus strain was able to efficiently degrade synthetic aromatic hydrocarbons.^21–24 Al-Jailawi et al. found that Anoxybacillus was the most predominant genus of thermophilic bacteria with aromatic hydrocarbon degradation activity for hydrocarbon contaminated soil in Iraq.²² However, none of the literature has investigated or explored the possibility and characteristics of alkane degradation by this relatively new genus; this study therefore is the first report about alkane biodegradation by the Anoxybacillus genus.

Gas chromatography was applied to detect the change of each component in the alkane mixture during degradation, and the residual weight of each alkane was calculated as well. The results in Fig. 3 show that this strain can utilize different chain alkanes (C8–C22) with varying efficiency, and especially that C8 and C9 were almost depleted after the first 10 days of degradation. The degradation kinetics of each alkane (in Fig. 2B) demonstrated that the degradation rate of the alkanes can be classified into three types based on the rate calculated for the first 10 days of degradation. The first type, with a degradation rate higher than 10 mg d⁻¹, is limited to C8–C10 with 14.49 mg d⁻¹, 14.33 mg d⁻¹ and 11.92 mg d⁻¹ respectively; for the second type, the degradation rate is between 5 mg d⁻¹ and 9 mg d⁻¹ which includes C11–C12 with 8.13 mg d⁻¹ and 6.47 mg d⁻¹ respectively; for the third type, the degradation rate is less than 3 mg d⁻¹ for the long chain alkanes (C13–C22). Combining the results in Fig. 2A and B, it was obvious that the degradation of C13–C22 between the 10^th and 30^th day was facilitated along with cell surface lipophilicity increasing and reaching a CSL of 65% at the 30^th day, while the cell surface lipophilicity of the strain before the 10^th day was relatively lower than 20%. Two possible mechanisms of alkane uptake have been proposed and well proven: direct uptake of small chain alkanes, and the changing of physiochemical properties of the cell to the alkane.²⁶ In addition, suggested mechanisms for the uptake of hydrophobic contaminants by degrading bacteria include direct contact of substrates with microorganisms having a high CSL and biosurfactant-mediated uptake by microorganisms capable of producing biosurfactants (and bioemulsifiers).²⁸ Therefore, the possible mechanism of degradation of Anoxybacillus WJ-4 when fed with different alkane molecules could be explained by the above proposed mechanisms based on the results in Fig. 2 and 3. It was hypothesized that this bacterial strain assimilated the small chain alkanes (<C10) following the first mechanism (because of the depletion of C8 and C9 in Fig. 2B and 3); and the degradation of the long chain alkanes (>C10) conformed to the second mechanism (because of the increasing of cell surface lipophilicity in Fig. 2A).

Fig. 3 Gas chromatography analysis of the degradation of the alkane mixture by Anoxybacillus strain WJ-4 under aerobic conditions at 70 °C.

Most alkane-degrading bacteria secrete diverse surfactants that facilitate emulsification of hydrocarbons. Zhao et al. elaborated a relationship between cell surface lipophilicity and the level of biosurfactant production.²⁶ Hassanshahian and Emtiazi reported a correlation between emulsification activity, cell adherence to hydrocarbons and growth rate of the crude oil degrading bacteria in crude oil media.⁵⁴ Kundu described the effect of biosurfactant production on cell surface hydrophobicity and the degradation of heteroaromatic hydrocarbons.²⁸ In this study, the cell surface lipophilicity of Anoxybacillus cells and the emulsification index were increased from 5.9% to 70.4% and from 0 to 72.2% respectively along with a decrease in the amount of residual alkanes (Fig. 2A). Therefore, it could be concluded that this novel thermophilic and halotolerant Anoxybacillus WJ-4 could synthesize a biosurfactant or bioemulsifier to facilitate the degradation of the alkanes.

Unlike biosurfactants (that can significantly decrease surface/interfacial tension), bioemulsifiers are always high molecular weight biosurfactants that are able to form stable emulsions with hydrophobic materials (usually oil-in-water and less commonly water-in-oil), but hardly reduce the surface or interfacial tension. Fig. 2A presents the surface tension change of the cell-alkane-free supernatant and indicates that the microbial surfactant produced by the novel strain WJ-4 has no significant influence on the surface tension, which decreases from 72.22 mN m⁻¹ to 52.45 mN m⁻¹. A 890 Da-biosurfactant from Rhodococcus could decrease the surface tension from 71 to 29 mN m⁻¹.²⁸ Hazra found that a 1044 Da surfactin from Bacillus could reduce surface tension from 69.07 mN m⁻¹ to 30 mN m⁻¹.⁵⁵ Bao et al.⁵⁶ reported that a small molecule lipopeptide biosurfactant has a lowest surface tension of 26.30 mN m⁻¹ when using crude oil as a carbon source. Bioemulsifiers are higher in molecular weight than biosurfactants as they are complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins.^30,57–59 They are also known as high molecular weight biopolymers or exopolysaccharides, which possess only emulsifying activity and not surface activity. Similar to biosurfactants, these molecules can efficiently emulsify two immiscible liquids such as hydrocarbons or other hydrophobic substrates even at low concentrations, but in contrast are less effective at surface tension reduction.³⁰ In addition, the number-average molecular weight (M_n), weight-average molecular weight (M_w) and polydispersity index (PDI) of the bioemulsifier were measured by gel permeation chromatography (GPC) using Pullulan standards. The results show that the number-average molecular weight (M_n), and weight-average molecular weight (M_w) of the obtained bioemulsifier were between 129 485 ± 1827 Da and 210 000 ± 1827 Da, with PDI average values of 1.212, respectively. Therefore, we can infer that the strain WJ-4 produces a relatively high molecular weight bioemulsifier which was not able to reduce the surface tension obviously, but has the ability to change the cell surface lipophilicity and emulsify the alkanes as evidenced in Fig. 2A. A large amount of bacterial strains can produce high molecular weight bioemulsifiers which can stabilize the various kinds of emulsion formed with immiscible phases but cannot decrease surface tension significantly. The bioemulsifier of the Anoxybacillus strain WJ-4 has a similar characteristic to these reported bioemulsifiers.^31,42 Although the numerous literature reports on CSL, emulsification and biodegradation are limited to small molecule biosurfactants (generally glycolipids and lipopeptides), it is not ignorable and increasingly significant that bioemulsifiers (high molecule weight biosurfactants) could be applied efficiently in many fields, especially in hydrocarbon bioremediation.

3.3. Analysis of the biosurfactant

A chromogenic reaction was developed to analyze the composition of the bioemulsifier from Anoxybacillus sp. WJ-4. The results in Fig. 4 show that there was a blue-green spot, purplish-red spot and yellow brown spot on silica gel plates when separately using phenol, ninhydrin and iodine vapor as a color developing reagent, indicating that peptides, glycosides and lipids were present with approximate percentages of 6.25%, 53.12% and 40.63% respectively in this bioemulsifier. Thus the bioemulsifier produced by Anoxybacillus sp. WJ-4 was a high molecular weight oligosaccharide–lipid–peptide complex in which these moieties could be structurally associated involving either covalent or non-covalent bonds. The oligosaccharide composition of the hydrolysed bioemulsifier consisted of D-glucose, D-galactose, D-mannose and L-rhamnose in an approximate molar ratio of 2 : 1 : 2 : 1. GC–MS analysis of lipid fractions showed that hexadecanoic acid and octadecanoic acid were the major fatty acids that account for 89.85% of total fatty acids. Other fatty acids determined at lower levels were decanoic acid (2.38%), dodecanoic acid (4.25%) and tetradecanoic acid (3.52%). The amino acids of the peptides of the bioemulsifier were analyzed and are tabulated in Table 3.

Fig. 4 Chromogenic reaction of the bioemulsifier obtained from Anoxybacillus sp. WJ-4 when utilizing alkane hydrocarbons. Points A, B and C were developed with 0.2% phenol–sulfatic acid solution, 0.1% ninhydrin solution and iodine vapor to detect glycoside, peptides and lipids respectively.

Table 3 Protein composition of the bioemulsifier from Anoxybacillus strain

Amino acid	Concentration (mg ml^−1)	Amino acid	Concentration (mg ml^−1)
Asp	6.126	Gly	3.236
Thr	2.781	Ala	6.519
Ser	2.031	Leu	2.773
Glu	7.231	Phe	1.121
Pro	1.142	Lys	2.452
Val	2.828	Ile	1.859

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3.4. Emulsification activity

Emulsification activity is critical for a bioemulsifier to be promising in different environmental and industrial applications. The emulsification activity of the Anoxybacillus bioemulsifier against various oil phases was investigated at room temperature for 24 hours. The results in Fig. 5A show that the Anoxybacillus bioemulsifier can effectively emulsify different hydrocarbons and form stable emulsions with pure alkanes (C6–C10, C12 and C16), as well as mixtures (crude oil, kerosene, paraffin). Furthermore, it had a better emulsification activity with hydrocarbon mixtures than with pure alkanes, with crude oil as the best oil phase, and weaker emulsions were formed with an increase of alkane chain number.

Fig. 5 Emulsification activity evaluation of the bioemulsifier produced from the novel thermophilic Anoxybacillus strain when utilizing different hydrocarbons. (a) The bioemulsifier solution against pure alkanes and hydrocarbon mixtures; (b–d) the bioemulsifier solution against the crude oil from Daqing oilfield. The values are means ± standard deviations (n = 3).

The effectiveness of a bioemulsifier over wide ranges of temperature, pH and salinity can endow it with broad applications. Exposed to different temperature extremes (<10 °C and >80 °C) for 2 hours, our bioemulsifier exhibited a high activity (EI > 60%) stably over a wide temperature range (Fig. 5B), and a slight decrease was detected when the temperature was higher than 80 °C. In pH evaluation, the Anoxybacillus emulsifier showed relatively stable emulsification performance at a pH from 6 to 12 and was significantly inhibited under extreme acid conditions (Fig. 5C). The previous literature has reported that emulsifying activity was significantly inhibited at a NaCl concentration greater than 5%,⁴² whereas the bioemulsifier from Anoxybacillus sp. WJ-4 showed a better halotolerance, retaining high emulsification activity (EI > 60%) under salinity up to 20% (Fig. 5D). In contrast, the commercial chemical surfactants SDS (sodium dodecyl sulfate), Triton X-100 and Tween 80 have no emulsifying activity at NaCl concentrations of 100–120 g L⁻¹.⁶⁰ Although the production cost of bioemulsifiers/biosurfactants is still high, it is obvious that the trend of the replacement of chemical surfactants by bio-counterparts is being accelerated due to the unparalleled advantages over the commercial petro-derived chemical surfactants and the rapid development of biological methods and purification technology.

3.5. Alkane hydroxylase gene analysis

Because of the nearest phylogenetic relationship with the Geobacillus genus,^25,50 the total DNA of the strain WJ-4 grown on alkane containing medium was used to amplify alkB gene fragments with various degenerate primers targeting the most conserved region of the Geobacillus alkB gene.^45,46 The selected 50 clones were analyzed by DNA sequencing using plasmid primers. The obtained nucleotide sequences were compared to the known sequences of alkB geo homologs, as well as with alkB genes of various bacteria deposited in GenBank. Four homologs of the alkB gene were revealed in the Anoxybacillus strain WJ-4, namely, alkB-an1 (10 clones), alkB-an2 (11 clones), alkB-an3 (8 clones) and alkB-an4 (21 clones). The sequenced Anoxybacillus alkB fragments were blasted with the homologs available from GenBank. The corresponding phylogenetic trees differ from each other, as shown in Fig. 6. The GenBank access numbers of the four genes are from KR153280 to KR153283.

Fig. 6 Position of the Anoxybacillus alkB homologs on phylogenetic trees based on nucleotide sequences. The sequences obtained in our study are labeled with bold circles. Numerals indicate the statistical reliability of branching order as determined by a bootstrap analysis of 1000 alternative trees. Values exceeding 90% are considered significant.

The sequences of alkB-an1, alkB-an2, alkB-an3 and alkB-an4 belonging to the phylogenetic clusters were similar to Geobacillus alkB-geo1, Geobacillus alkB-geo2, Geobacillus alkB-geo3, and Geobacillus alkB-geo4 found in the Geobacillus genus.^45,46,53 The nucleotide sequence similarity to the corresponding Geobacillus alkB homologs is 97.0, 95.0, 97.0, and 98.0% for these four alkB genes respectively. Although PCR and sequencing of the alkane gene from Anoxybacillus were implemented in this study, it is obviously insufficient to explain the alkane degradation mechanism of this novel strain. In order to reveal the metabolic pathway of alkane degradation, the whole genome sequence, reverse transcription, functional analysis and structure of the alkB-an series, and the intermediate metabolites will be further investigated.

4. Conclusions

A newly isolated thermophilic Anoxybacillus strain from a high temperature petroleum reservoir was evaluated for degradation of hydrocarbon pollutants. The produced oligosaccharide–lipid–peptide bioemulsifier showed a good emulsification performance (EI₂₄ > 60%) with various oil phases under different conditions. This bioemulsifier reduced surface tension from 72.22 mN m⁻¹ to 52.45 mN m⁻¹, and increased cell surface lipophilicity to 65% during hydrocarbon degradation. Four hydrocarbon-hydrolysis genes were detected in this strain, and showed a high sequence similarity (>95%) to the corresponding Geobacillus alkB homologs. Although the mechanism of degradation and bioemulsifier production need more study, it has been shown that the bioemulsifier, cell surface lipophilicity and hydrocarbon monooxygenase have a significant effect on alkane degradation, even under thermophilic conditions.

Acknowledgements

We thank the 863 National High Technology Research and Development Program of China (2013AA064402) for financial support; the project was also supported by the National Natural Science Foundation of China (Grant No. D308/41573068).

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