Salt tolerance mechanism of a hydrocarbon-degrading strain: Salt tolerance mediated by accumulated betaine in cells

Journal Pre-proof Salt tolerance mechanism of a hydrocarbon-degrading strain: salt tolerance mediated by accumulated betaine in cells Xin Hu, Dahui Li, Yue Qiao, Qianqian Song, Zhiguo Guan, Kaixuan Qiu, Jiachang Cao, Lei Huang

PII:

S0304-3894(20)30314-9

DOI:

https://doi.org/10.1016/j.jhazmat.2020.122326

Reference:

HAZMAT 122326

To appear in:

Journal of Hazardous Materials

Received Date:

18 December 2019

Revised Date:

15 February 2020

Accepted Date:

15 February 2020

Please cite this article as: Hu X, Li D, Qiao Y, Song Q, Guan Z, Qiu K, Cao J, Huang L, Salt tolerance mechanism of a hydrocarbon-degrading strain: salt tolerance mediated by accumulated betaine in cells, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122326

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Salt tolerance mechanism of a hydrocarbon-degrading strain: salt tolerance mediated by accumulated betaine in cells

Xin Hu, Dahui Li, Yue Qiao, Qianqian Song, Zhiguo Guan, Kaixuan Qiu, Jiachang Cao, Lei Huang*

Affiliations:

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College of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, Tianjin Key Laboratory of Drug Targeting and Bioimaging, Tianjin University of Technology, Tianjin 300384, China.

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*Corresponding Author: College of Chemistry and Chemical Engineering, Tianjin University

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Graphical abstract

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address: [email protected] (L. Huang)

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of Technology, Binshui West Road 391, Tianjin 300384, China. Tel.: 86-22-60214259; E-mail

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Highlights

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•Rhodococcus sp. HX-2 was found to be able to tolerate 1%–10% NaCl.

concentrations.

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•HX-2 can produce intracellular compatible substances (betaine) to survive in high NaCl

•Exogenously added betaine increased the production of exopolysaccharides and was

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transported into HX-2 cells.

•Genomic analysis identified the presence of betaine-producing and transport genes and their

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expression was induced by NaCl.

Abstract

Rhodococcus sp. HX-2 could degrade diesel oil in the presence of 1%–10% NaCl. The compatible solute betaine accumulated in cells with increasing NaCl concentration, and this was found to be the main mechanism of resistance of HX-2 to high salt concentration. Exogenously added betaine can be transported into cells, which improved cell growth and the 2

percentage degradation of diesel oil in the presence of high [NaCl] in solution and in soil. Scanning electron microscopy data suggested that addition of exogenous betaine facilitated salt tolerance by stimulating exopolysaccharide production. Fourier-transform infrared analysis suggested that surface hydroxyl, amide and phosphate groups may be related to tolerance of high-salt environments. Four betaine transporter-encoding genes (H0, H1, H3, H5) and the betaine producer gene betB were induced in Rhodococcus sp. HX-2 by NaCl stress. The maximal induction of H0, H1, H3 and H5 transcription depended on high salinity plus the presence of betaine. These results demonstrate that salt tolerance is mediated by

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accumulated betaine in Rhodococcus sp. HX-2 cells, and the potential of this strain for application in bioremediation of hydrocarbon pollution in saline environments.

Keywords: Hydrocarbon-degrading, Rhodococcus sp., Betaine, Salt tolerance mechanism,

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Transcriptional analysis

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1. Introduction

Pollution of soils by petroleum compounds is of great concern, mainly because of the

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solubilities of various molecules in water, which can endanger aquifers in contact with polluted zones [1]. Large-scale use of petroleum hydrocarbons and leakage during transportation can cause serious damage to the environment [2]. Diesel oil is extracted from

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crude oil. It is a more complex chemical mixture than gasoline, including many different isomers from about C10 to C22 [3]. Diesel oil poses environmental threats and is toxic to

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animals and humans. Physical and chemical remediation methods are costly and do not completely remove contaminants [4-5]. Biological methods offer an advantageous alternative, attributed to their low-energy design, ease of use, and high removal efficiency at low cost to

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restore hydrocarbon-contaminated soil sites [6-11].

Different microorganisms have varying abilities to utilize diesel oil hydrocarbons. There have been many reports on the ability of Rhodococcus spp. to degrade hydrocarbon pollutants. Auffret et al. isolated two strains, R. wratislaviensis IFP 2016 and R. aetherivorans IFP 2017, that can degrade and mineralize 11 compounds or degrade methyl tert-butyl ether and ethyl tert-butyl ether [1]. Huang et al. isolated a novel bacterium, R. erythropolis T7-2. They used a 3

five-factor central composite design to optimize the nutrition supplied to improve the degradation of diesel oil in seawater [12]. R. erythropolis strain NTU-1 degraded C10-C32 n-alkanes in diesel oil or crude oil [13]. Using fed-batch cultivation in mineral medium with phenol as the sole carbon source, large amounts of phenol were degraded by Rhodococcus spp. at 10 °C [14]. Quek et al. reported that both free and immobilized Rhodococcus sp. F92 were able to degrade approximately 90% of the total n-alkanes in the petroleum products tested within one week at 30 °C [15]. The soil-isolated strain R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complex model sulfur compound present in crude oil [16]. Laczi et al. found that R. erythropolis PR4 was able to degrade diesel oil, normal-, iso-

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and cycloparaffins, and aromatic compounds [17]. Lee isolated Rhodococcus sp. EC1 and showed it to have excellent cyclohexane-degrading ability [18]. Yang et al. investigated Rhodococcus sp. strain p52 and found that it could degrade dioxin or petroleum hydrocarbons [19]. Lee et al. showed that Rhodococcus sp. EH831, isolated from an enriched hexane-degrading consortium, was able to degrade hexane and various hydrocarbons,

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including alcohols, chlorinated hydrocarbons, cyclic alkanes, ethers, ketones, monoaromatic and polyaromatic hydrocarbons, and petroleum hydrocarbons [20]. Rhodococcus sp. P14 was

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capable of using three to five ring polycyclic aromatic hydrocarbons including phenanthrene (Phe), pyrene, and benzopyrene as sole carbon and energy sources [21]. Arif et al. reported the

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isolation of a phenol-degrading Rhodococcus sp. with a high tolerance toward phenol [22]. Rhodococcus sp. CMGCZ was capable of degrading 13.2% of the naphthalene supplied, 13.1% of the Phe, and 99.3% of the fluoranthene in 1 week, and 11% of the aliphatic fraction of

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Iranian light crude oil in 2 weeks when used as sole carbon and energy sources [23]. Bajaj et al. reported that in aqueous culture conditions, Rhodococcus sp. IITR03 degraded 282 μM dichlorodiphenyltrichloroethane (DDT) and could also use 10 mM of each of 4-chlorobenzoic

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acid, 3-chlorobenzoic acid and benzoic acid as sole carbon and energy sources [24]. Li et al.

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found that Rhodococcus sp. JZX-01 decomposed 65.27 ± 5.63% of crude oil in 9 days [25].

The members of the genus Rhodococcus are aerobic, nonsporulating bacteria, found in a wide diversity of environments, including tropical and arctic soils, deserts, and marine and deep-sea sediments [26-29]. Cappelletti et al. revealed the response of R. aetherivorans BCP1 and R. opacus R7 to various stress conditions and several antimicrobials [30]. The accumulation of different storage compounds by Rhodococcus probably permits cells to respond rapidly to changes in nutritional state and to balance metabolism in different environmental conditions [31]. Triacylglycerols (TAGs) are the main reserve material in 4

eukaryotes for energy and fatty acids required for membrane biosynthesis [32]. Bioremediation of soil contaminated by petroleum hydrocarbons and high salinity (3%-30% NaCl, w/v) is challenging because microbial activity is inhibited by salinity, and higher salinity reduces the oxygen level and the water solubility of hydrocarbons [33]. The presence of high salt in the process of degrading diesel oil not only inhibits the metabolic function of heterotrophic bacteria, but also reduces the efficiency of the repair process of the environment [34]. Isolation and enrichment of salt-tolerant bacteria has previously been used as a method to treat saline wastewaters [35-37]. de Carvalho et al. revealed the responses of R. erythropolis cells to osmotic stress, showing that the cells changed their total lipid fatty acid

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composition and net surface charge [38]. Due to inhibition of diesel oil degradation by salt, it is essential to screen for diesel-degrading bacteria with adaptability to high-salt environments.

Bacteria use different strategies to deal with excessive salt in the external environment [39]. The first is to accumulate K+/Cl− ions to maintain the internal osmotic balance of the cells in

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external high salt conditions [40]. This adaptation is common in extreme and anaerobic halophiles [41-42]. However, most salt-tolerant and moderately halophilic bacteria exhibit the

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second strategy—accumulating compatible solutes. These compatible solutes (amino acids, carbohydrates or their derivatives, sugars and polyols) can be absorbed from medium or be

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synthesized de novo [43]. In the cell, these solutes accumulate at a high concentration to maintain the balance of intracellular and extracellular osmosis [44]. This is the most widely used salt tolerance strategy in bacteria, eukaryotes and some methanogenic archaea [45-46].

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Betaine is an alkaloid that plays a significant role in maintaining cellular osmotic pressure and alleviating salt stress. Betaine has the advantages of no static charge, high solubility, and it has no effect on many enzymes and other biomacromolecules, even when present in high

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concentrations [47]. It is the most common osmoprotectant in prokaryotes [48]. Moreover, many bacteria, including Rhodococcus spp., produce extracellular polymeric substances (EPS)

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as a growth strategy, enabling adherence to solid surfaces and survival in adverse conditions [49-52]. EPS form a layer around the cell, providing protection against high or low temperatures, salinity, and possible predators. They are essential in the formation of aggregates, adhesion to surfaces and other organisms, biofilm formation, and nutrient uptake [53-55].

The lipids present in animals and plants are composed of choline or 2-hydroxyethyl trimethylammonium chloride and these two substances are ubiquitous in the environment [56]. 5

These substances are absorbed into cells by choline dehydrogenase (BetA, betA) or glycine betaine aldehyde dehydrogenase (BetB, betB) and oxidized by glycine betaine aldehyde enzyme to glycine betaine [57-59]. There are many types of betaine transporter, the most important of which are the betaine-choline-carnitine-transporter (BCCT) family [60], ABC transporters [61] and the major facilitator superfamily (MFS) [62]. Transporters of the BCCT family are ubiquitous in bacteria and their transport processes are driven by sodium ions or proton gradients [60]. The ABC transporter family is ubiquitous in all organisms [63]. Among secondary transporters, the MFS represents the most ubiquitous group and the largest

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transporter proteins [64].

Previous reports have only explored the ability of Rhodococcus sp. to degrade pollutants or its behavior under stress conditions. However, the interaction between the salt tolerance mechanism of Rhodococcus spp. and their ability to degrade hydrocarbons has not been revealed. In addition, previous research mainly focused on the ability of exogenous

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substances to improve strain salt tolerance; the related salt-tolerance genes in Rhodococcus sp. remain unconfirmed. This study aimed to determine the salt tolerance mechanism of

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Rhodococcus sp. HX-2: the principal salt-tolerance mechanism was found to be accumulation of the compatible solute betaine in cells. Meanwhile, transcriptional analysis of a

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betaine-producing gene and betaine transporter-encoding genes in different conditions revealed their potential roles in the response of the cells to salinity and exogenously added

environments.

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betaine. Our data provide a theoretical basis for bioremediation using strain HX-2 in polluted

2. Materials and methods

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2.1 Chemical Reagents and Media

Diesel oil (0#) was purchased from China Petrochemical Corporation and other reagents were

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analytically pure. Molecular biology reagents were purchased from Beijing Tiangen Biochemical Technology Company. LB medium contained (g L−1): peptone 10, yeast powder 5, NaCl 10, pH 7.0–7.2; 20 g L−1 agar were added to produce solid medium. Minimal salts medium contained (g L−1): Na2HPO4 1.5, KH2PO4 3.48, (NH4)2SO4 4, MgSO4 0.7, yeast powder 0.01, pH 7.2 [65]. Hydrocarbon degradation medium was minimal salts medium supplemented with 0.4% (v/v) 0# diesel oil. 6

The strain was a Rhodococcus sp. isolated from the laboratory [66].

2.2 Determination of percentage diesel oil degradation HX-2 was inoculated into hydrocarbon degradation medium and the biomass was determined as OD600 using a spectrophotometer.

A 30-mL culture of Rhodococcus sp. HX-2 (cultured in 100-mL flasks) in hydrocarbon degradation medium was mixed with 30 mL n-hexane in a separating funnel [12]. The organic

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phase was concentrated to 0.2 mL by Na2SO4 for gas chromatography (GC) analysis (Bruker 456-GC with Electron Capture Detector (ECD) and Flame Ionization Detector (FID), Headspace SHS-40) [65]. The column (PONA) length was 50 m with internal diameter 250 μm, coated film 0.25 μm. The conditions were: inlet temperature 280 °C and detector temperature 300 °C. The program was: initial temperature 50 °C, hold for 1 min, heat at 15°C

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min−1 to 280 °C, hold for 4 min. Carrier gas N2 10 mL min−1, H2 30 mL min−1, air 300 mL

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min−1. Split ratio 1:50; sample volume 10 μL.

2.3 Salt stress response

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2.3.1 Effects of NaCl concentration on growth and diesel oil degradation The diesel oil percentage degradation detection method was as described above. HX-2 (2% inoculum) was inoculated into hydrocarbon degradation medium containing 0%–10% NaCl.

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The growth and diesel oil degradation ability of the strain were investigated.

2.3.1 Effect of medium composition and inoculum size on diesel oil degradation

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First, HX-2 was inoculated at 108 colony-forming units (CFU)/mL (2%) into LB medium and hydrocarbon degradation medium containing different NaCl concentrations (2%–10%) at

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25 °C for 48 h. Then, different inoculum amounts (2%, 4%, 6%, 8% and 10%) were used in hydrocarbon degradation medium containing 6% NaCl at 25 °C for 5 d and the growth and diesel oil degradation ability of strain HX-2 were investigated.

2.4 Analysis of salt tolerance mechanism 2.4.1 Intracellular ion concentration and permeability The permeability of HX-2 and Staphylococcus aureus ATCC25923 was measured before and after heating at 100 °C for 20 min using a DDS-307A conductivity meter (REX, China), as 7

previously described [67]. Membrane penetration was calculated as:

Membrane penetration (%) =

β−α α

where  is the conductivity at room temperature and 𝛽 is the conductivity after heating at 100 °C for 20 min.

Cations are critical to maintaining cellular osmotic balance. Therefore, the concentrations of different cations within HX-2 were investigated for cells grown in medium with and without 6%

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NaCl. Culture broth was centrifuged at 10,000 × g for 10 min at 4 °C to collect cells, and the cells were treated with an ultrasonic cell analyzer (JYD-1200, China) at low temperature. The supernatant was collected for subsequent analysis. The concentrations of Na+, K+, Mg2+ and

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Ca2+ were determined by atomic absorption spectrometry (PerkinElmer Analyst 700).

2.4.2 Isolation and extraction of EPS

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Single colonies of HX-2 were inoculated into 250-mL flasks containing 100 mL of LB medium and cultured for 48 h at 25 °C. The OD600 value of the culture solution was adjusted to 1.0 with sterile pure water. An aliquot (5 mL) of the bacterial suspension was then added to

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a 250-mL flask containing 100 mL of LB medium with or without 6% NaCl (designated LB + 6.0% NaCl and LB, respectively). The procedure for extracting EPS according to Zhang et al. [68] was slightly modified. Briefly, all centrifugations were carried out at 12,000 × g for 15

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min at 4 °C unless otherwise stated. The extraction process consisted of the following steps: centrifuge the suspension and retain the supernatant; add three volumes of pre-cooled 95%

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ethanol to 250 mL of the supernatant, precipitate overnight, and centrifuge for 40 min to collect the precipitate; dissolve the polysaccharide precipitate in 250 mL ultrapure water at

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30–40 °C, add 250 mL of 10% trichloroacetic acid (to remove protein), and stir well at 4 °C for 10 h; centrifuge for 40 min, add three volumes of pre-cooled 95% ethanol to the supernatant, precipitate overnight, centrifuge for 40 min to collect the polysaccharide precipitate and redissolve in ultrapure water; place in a dialysis bag (molecular weight cutoff 14,000 Da) and dialyze into ultrapure water at 4 °C for 2 d. The EPS obtained after dialysis was freeze-dried.

2.4.3 Determination of intracellular compatible substances at different salinities 8

HX-2 was inoculated into hydrocarbon degradation medium containing different NaCl concentrations (2%, 4%, 6%, 8% and 10%). Three typical compatible solutes were selected—ectoine, betaine and glutamic acid—which were extracted by the modified Bligh-Dyer method [69]. The strain was cultured to OD600 of about 0.5 and the cells were harvested by centrifugation (10,000 × g, 20 min). Cells were dissolved using a solution of methanol, chloroform and distilled water (10:5:4, v:v:v). The extract was shaken vigorously for 30 min, an equal volume of chloroform and water were added (each approximately 130 μL), and the mixture was shaken for another 10 min. The mixture was then centrifuged (12000 × g, 20 min) and the aqueous phase was collected and dried. If ectoine was to be measured, the dry powder was dissolved in 500 μL of 80% acetonitrile. If glutamic acid and

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betaine were to be measured, the dry powder was dissolved in 500 μL distilled water. Determination of ectoine, betaine and glutamic acid was by high-performance liquid chromatography (Shimadzu-LC-2030) [70-72].

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Determination of ectoine: RP-18 column (Merck, Darmstadt, Germany). Chromatography was performed isocratically at a flow rate of 1 mL min−1 with acetonitrile/trifluoroacetate [4/1,

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v/v] (pH = 2.5) as the mobile phase. Detection wavelength 210 nm; column temperature 23 °C; injection volume 10 μL; detector: UV/vis. Determination of glutamic acid: Agilent

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TC-18 column; mobile phase—A: 50 mmol L−1 pH 6.8 sodium acetate buffer solution (containing 1% triethylamine), B: 50% aqueous acetonitrile solution. The above mobile phases were filtered through an organic filter and ultrasonically degassed for 20 min; flow

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rate 1 mL min−1; detection wavelength 360 nm; column temperature 23 °C; injection volume 10 μL; detection: UV-Waters 2487. Determination of betaine: Agilent TC-18 column; mobile phase—A: 0.01 mol L−1 ammonium formate–methanol, B: ultrapure water, mixing ratio 85:15.

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The above mobile phases were filtered through an organic filter and ultrasonically degassed for 20 min; flow rate 1 mL min−1; detection wavelength 195 nm; column temperature 30 °C;

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injection volume 10 μL; detection: UV-Waters 2487. A standard curve was drawn using the relationship between different concentrations of the standard and the peak area, and then the sample content was calculated from peak areas.

2.5 Effect of exogenous addition of betaine 2.5.1 Effect of exogenous addition of betaine on diesel oil degradation Strain HX-2 was inoculated (2%) into hydrocarbon degradation medium containing 0-10% NaCl with or without 150 mg L−1 betaine. After cultivation for 5 d, the intracellular betaine 9

content was determined.

Strain HX-2 was inoculated (2%) into hydrocarbon degradation medium containing 6%, 7%, 8%, 9% or 10% NaCl and different concentrations of betaine (50–250 mg L−1). The diesel oil degradation ability of HX-2 was observed at 25 °C for 5 d.

2.5.2 Effect of exogenous addition of betaine on surface groups of HX-2 Cells were inoculated (2%) into hydrocarbon degradation medium containing 6% NaCl with or without 150 mg L−1 betaine. Cells were collected by centrifugation and dried at 30 °C for

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24 h. The dried cells were mixed with KBr (100 mg) and then pressed into pellets for Fourier-transform infrared spectrum determination (FTIR; NICOLET iS10, Thermo Scientific, USA) in the frequency range 4000–400 cm−1 with resolution 4 cm−1.

2.5.3 Effect of exogenous addition of betaine on surface morphology of HX-2

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Cells were inoculated (2%) into hydrocarbon degradation medium containing 6% NaCl with or without 150 mg L−1 betaine. Cells were collected by centrifugation (10,000 × g, 20 min)

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and washed three times with deionized water. Cells were fixed using glutaraldehyde, and then dehydrated with 70%, 90% and 100% ethanol. The dried cells were glued onto scanning

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electron microscopy (SEM; FEI-Verios 460L, USA) stubs and gold-coated, and then SEM images were obtained at an accelerating voltage of 3.0 kV.

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2.5.4 Soil experiments

The tested soil was from Tianjin University of Technology, China. The soil is used to plant trees, and had physical and chemical properties: pH 7.2-7.4, moisture content 12.35%-15.28%,

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organic matter content 102-122 g kg−1, total nitrogen content 12-20 g kg−1, and effective phosphorus content 10.0-19.6 g kg−1. Plant residue and gravel were removed from the soil by

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passage through a 3-mm sieve, then it was dried for 7 d. Diesel oil and n-hexane (1:1) were added into test soil so that the diesel oil content reached 0.4% (v g−1). After waiting for the n-hexane to evaporate, HX-2 was inoculated into the soil (10%, v g−1, 108 CFU per mL). The experimental soil was placed in a constant-temperature incubator (25 °C) for cultivation. Minimal salts medium was added to the soil regularly to ensure that it contained sufficient moisture and nutrients to allow HX-2 to grow. Experimental soil contained 2%, 4% or 6% NaCl (NaCl was dissolved in 50 mL of sterile water and added to the soil; soil was used in the experiment after drying for 7 d). Betaine (150 mg g−1) was added to the above soil (negative 10

control, soil without betaine).

Samples were taken periodically for GC-mass spectrometry (GC-MS) analysis. Soil (5 g) was mixed with 50 mL of n-hexane in a separating funnel (the collected soil was dried at 50 °C for 48 h). The organic phase was passed through Na2SO4 and concentrated to 0.2 mL. Three samples for each group were analyzed by GC-MS.

2.6 RNA isolation and qPCR RNA extraction and gene transcription analysis of betB, H0, H1, H3 and H5 was performed

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on Rhodococcus sp. HX-2 grown in different conditions (hydrocarbon degradation medium containing 0%, 2%, 4%, 6%, 8% and 10% NaCl, with or without supplementation of 150 mg L−1 betaine). RNA extraction and fluorescence-based quantitative real-time PCR (qPCR) were slightly modified from the method of Tao et al. [73]. Cultures were collected during the logarithmic growth phase and total RNA was extracted according to the instructions of the

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Tiangen RNAprep Pure Cell/Bacteria Kit (containing DNase). The total RNA concentration of the separated material was measured using a Qubit spectrophotometer. Next, according to the

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TOYOBO One-step qPCR Kit specification, synthesis of first strand cDNA was carried out using 500 ng RNA as the template in a 20-μL reaction system. Transcriptional analysis of the

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genes was performed by qPCR; gene-specific primer pairs for each gene are listed in Table S1. Each qPCR reaction mixture contained 10 μL of IQTM SYBR® Green Supermix, 2.0 μL of cDNA, 10 mM primer-F and primer-R (each 0.4 μL), and diethyl pyrocarbonate-treated water

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to 20 μL. The qPCR reaction procedure was: 95 °C for 3 min, 95 °C for 30 s, 61 °C for 20 s, 95 °C for 30 s, 95 °C for 5 s, 55 °C for 10 s, 74 °C for 15 s, the next 43 cycles, 72 °C for 5 min, and cooling to 20 °C. The experimental data were analyzed according to the 2−ΔΔCt

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method [74] and standardized by the 16S rRNA gene as an internal reference control.

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2.7 Statistical analysis

The SPSS statistical software 26.0 was used for all data analyses. Results are expressed as the mean ± SD. Single factor analysis for variance was performed using the analysis of variance program, followed by the least significant difference test. P<0.05 indicates a significant value.

3. Results 3.1 Salt tolerance 11

3.1.1 Effect of salt concentration on diesel oil percentage degradation Salt concentration may limit the effective biodegradation of contaminated hydrocarbons by natural microbial populations [75]. Figure 1 indicates that HX-2 can grow in the range 0%-10% NaCl. The diesel oil percentage degradation was >80% in the range 0%–2% NaCl, declining as [NaCl] increased. This data indicates that HX-2 can be applied to bioremediation in moderately saline environments.

3.1.2 Effect of medium components and inoculum size on salt tolerance

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The growth of the strain was examined after 48 h by measuring OD600 in LB and minimal salts media containing different NaCl concentrations (Figure S1A). Up to 6% salinity, the growth of HX-2 in LB medium was significantly better than that in minimal salts medium. Above 6% NaCl, the growth of HX-2 was inhibited in both media. Thus, 6% NaCl was

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implemented for subsequent experiments.

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Figure S1B indicates that diesel oil degradation has minimal dependence on inoculum amount in hydrocarbon degradation medium containing 6% NaCl. Compared with a 2% inoculum, the

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use of 8% inoculum only increased the percentage degradation by 12.4%. This experiment suggested that changing the inoculum size at 6% NaCl would not significantly improve diesel

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oil percentage degradation.

3.2 Salt Tolerance Mechanism

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3.2.1 Cell membrane permeability and intracellular cations We compared the permeability of strain HX-2 with that of S. aureus ATCC25923 to explore

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the salt tolerance mechanism of HX-2. As shown in Figure 2A, the membrane permeability of HX-2 increased substantially with increasing NaCl concentration. This suggests that extra ions were expelled into the medium and cell disruption occurred at high salt concentrations. S. aureus ATCC25923 (which can withstand 10% NaCl) [76], exhibited a different response; the membrane permeability of some halophilic strains does not increase sharply with increasing salt concentration [77]. 12

We also detected intracellular ions for further study of the salt tolerance mechanism of strain HX-2. Cations are critical to maintaining cellular osmotic balance [78]. Therefore, the concentrations of different cations within HX-2 were investigated for cells grown in medium with and without 6% NaCl. Figure 2B displays the amounts of Ca2+, Mg2+, K+ and Na+ in cells grown in LB medium and in LB + 6.0% NaCl (the inset in Figure 2 shows the concentrations of Ca2+ and Mg2+). The cation content in cells grown in LB + 6.0% NaCl was similar to that in LB medium. This indicates that in a high-salt environment, excessive cations do not accumulate intracellularly in strain HX-2 to maintain the osmotic pressure balance

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across the cell membrane. The salt-tolerant hydrocarbon degrading strain Enterobacter cloacae MU-1 rapidly accumulates K+ to cope with high-salt environments [77]. However, it can be seen that the accumulation of intracellular ions is not the salt tolerance mechanism of

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HX-2.

3.2.2 EPS secretion

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Zeng found that microbes secrete more EPS at high salinity to protect themselves [79]. In this study, we researched the potential role of EPS in the salt tolerance of HX-2. Figure S2

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illustrates the amount of EPS secreted by HX-2 at varying salinities. The EPS yield increased with increasing salt concentration (0%-6% NaCl), reaching a maximum at 6% NaCl (222.67

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mg g−1). The EPS yield declined at higher NaCl concentrations. When the salinity exceeded 6%, the bacteria could not withstand the high-salt environment, resulting in cell death and hence a reduction in EPS synthesis. Hua reported that the EPS yield of mutant strains

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(Enterobacter cloacae MU-1) increased from 1350 mg g−1 dry cell weight to 1825 mg g−1 in high-salt conditions (LB + 9% NaCl) [77]. Compared with the above report, the level of EPS

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produced by HX-2 was relatively low in high-salt conditions. We suspect that EPS production may not be the only mechanism by which HX-2 cells resist high-salt environments.

3.2.3 Accumulation of intracellular compatible substances at different salinities It was reported that many salt-tolerant strains accumulate compatible substances in cells to resist the negative effects caused by the high-salt environment [80]. To determine whether the type and content of compatible substances in HX-2 was related to NaCl concentration, the 13

accumulation of ectoine, betaine and glutamic acid was investigated at different NaCl concentrations (2%–10%). It can be seen from Figure 3 that betaine increased significantly with increasing NaCl concentration. When the NaCl concentration reached 10%, the betaine and glutamic acid content reached 1.5 and 0.6 mmol L−1, respectively, while the content of ectoine was almost unchanged compared with growth in 2% NaCl. These results indicated that the accumulation of betaine was the main protective mechanism of HX-2 against high salt concentration. Betaine was chosen for subsequent exogenous addition experiments.

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3.3 Effect of exogenous addition of betaine 3.3.1 Conversion of exogenous betaine and its effect on diesel degradation

To investigate the accumulation of exogenously added betaine in cells, the same concentration of betaine (150 mg L−1) was added to hydrocarbon degradation medium containing 0%, 2%,

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4%, 6%, 8% and 10% NaCl. In the control, no betaine was added. Intracellular betaine was extracted and determined after 5 d. Figure 4A shows that the amount of betaine synthesized

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increased with the NaCl concentration. The reason for this may be that salinity induces the expression of betaine-producing genes, and as the salt concentration increases, the gene

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expression level continues to increase. The exogenous betaine absorbed by the cells first rose (0%–6% NaCl) and then decreased (6%–10% NaCl), which indicates that the betaine

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transporter genes have different responses to external salinity; the maximum response was to 4%–6% NaCl. The amount of exogenously absorbed betaine in the cells was 3.58, 12.85,

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44.48, 45.78, 23.54 and 10.65 mg L−1 at 0%, 2%, 4%, 6%, 8% and 10% NaCl, respectively.

The addition of compatible substances can improve the adaptability of many microorganisms

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to osmotic pressure (Beblo-Vranesevic et al., 2017). These substances can accumulate in large amounts in cells to maintain the balance of internal and external osmotic pressure without affecting the normal physiological functions of the cells. Betaine (0–250 mg L−1) was added exogenously to hydrocarbon degradation medium containing 6%–10% NaCl to investigate the effect on diesel oil degradation (Figure 4B). When 150 mg L−1 betaine were added, the degradation of diesel oil by HX-2 was obviously promoted. The percentage diesel oil degradations increased by 33.8%, 14.8%, 12.1%, 10.1% and 9.0% compared with cultures 14

with no added betaine at 6%, 7%, 8%, 9% and 10% NaCl, respectively. However, addition of further betaine did not further improve the degradation; therefore, the betaine concentration used in subsequent experiments was 150 mg L−1.

3.3.2 Effect of exogenous addition of betaine on surface morphology of HX-2 cells SEM analysis showed a slightly roughened cell surface of cells grown in hydrocarbon degradation medium (Figure 5A). Figure 5B illustrates that the morphology of cells under the dual stress of diesel oil and 6% NaCl changed greatly; the grew longer and become damaged.

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After the addition of exogenous betaine, a membranous substances appeared on the cell surface (Figure 5C). Although the overall morphology of the cells was similar to that in Fig. 5B, the cell surface was now covered by EPS, which contributed to the resistance of the bacteria to the high-salt environment. Because of the low level of EPS normally produced by

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these cells in a high-salt environment in the absence of added betaine (Figure S2), they were unable to withstand the external osmotic pressure, therefore the percentage diesel oil

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degradation decreased with increasing salinity (Figure 5B). EPS better encapsulates cells and prevents them from being exposed to osmotic pressure. To prove this hypothesis, we

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determined EPS levels after addition of exogenous betaine, as shown in Figure S3. The yield of EPS increased on addition of 150 mg L−1 betaine at different NaCl concentrations, and

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increased by 223 mg g−1 (approximately doubling compared with experiments without added betaine) at 6% NaCl. These data indicate that betaine can improve diesel oil degradation

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efficiency (Figure 5B) by changing HX-2 cell surface morphology.

3.3.3 Effect of exogenous addition of betaine on surface groups of HX-2 cells

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The FT-IR spectrum of betaine is shown in Figure 6C. In accordance with the literature [82-84], a broad and strong peak at 3425 cm−1, indicating the presence of a hydroxyl (-OH) or amine (-NH) group. The peaks at 2991 cm−1 could be due to the stretching vibration of the -CH2 group, and those at 1622, 1446 and 1251 cm−1 could be attributed to the stretching vibration of the -NH and -CN groups, respectively. The absorption peak at 756 cm−1 was attributed to monosubstituted benzene, and the absorption peak at 574 cm−1 represented C–X stretching vibration in organic halides. 15

The functional groups on the surface of HX-2 cells were characterized via FTIR analysis (as shown in Figure 6). A broad stretching peak at about 3464 cm−1 suggested a large number of hydroxyl groups were present on HX-2 cells [85]. The absorption peak at 2933 cm−1 was due to C–H stretching vibration, and strong absorption at around 1631 cm−1 corresponds to the amide I C=O stretch and C–N bending of protein and peptide amines [86]. The absorption bands observed at 1041 cm−1 usually represent the O–P–O symmetric stretching of organic phosphate in P–O–C [87]. The absorption peak at 756 cm−1 was attributed to monosubstituted

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benzene, and the absorption peak at 574 cm−1 represented C–X stretching vibration in organic halides [88]. Figure 6B shown that after adding 150 mg L−1 betaine to the hydrocarbon

degradation medium, the absorption peaks at 3464 cm−1 and 1041 cm−1 broadened, and the peak at 1631 cm−1 became stronger. We speculated that betaine transferred into the cell or

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adhered to the cell surface caused changes in these groups (hydroxyl, amide and phosphate). And these changes are presumed to be related to increased salt tolerance of the strains and

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better degradation of diesel oil in high-salt environments.

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3.3.4 Effect of exogenous addition of betaine on diesel oil degradation in soil The degradation of diesel oil in saline soil by strain HX-2 was studied with or without

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addition of 150 mg g−1 betaine. As shown in Figure 7, GC analysis indicated that soil salinity had a significant effect on diesel oil degradation. Exogenous addition of 150 mg g−1 betaine promoted diesel oil degradation at 2% NaCl (percentage degradation increased about 16.8%

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in 15 days compared with experiments without added betaine). However, when 150 mg g−1 of betaine were added to soil containing 4% and 6% NaCl, its effect on the percentage

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degradation was less pronounced (increases of 8% and 5.5% respectively, compared with no betaine addition). The addition of betaine increased the salt tolerance of the bacteria, enabling the strain to more quickly adapt to the saline soil environment and degrade diesel oil faster. A longer lag period is required without added betaine.

3.5 Transporters and betaine production genes present in Rhodococcus sp. HX-2 Four transporter genes, H0, H1, H3 and H5, identified in Rhodococcus sp. HX-2 respectively 16

belong to the BCCT family, MFS, ABC transporter ATP-binding proteins, and ABC transporter permeases by genetic comparison with data in the GenBank database. Their GenBank accession numbers are MN567074, MN567075, MN567076 and MN567077, respectively. A betaine-producing gene was also identified and it encodes a betaine aldehyde dehydrogenase (GenBank accession numbers: MN567073). Figure S4 shows the PCR products on amplification from genomic DNA of betB, H0, H1, H3 and H5.

To investigate the transcription levels of the betB, H0, H1, H3 and H5 genes at different

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salinities, Rhodococcus sp. HX-2 was cultivated in hydrocarbon degradation medium containing various concentrations of NaCl (0%–10%). qPCR was used to analyze RNA isolated from bacteria in logarithmic growth phase. The transcription level of betB increased at 2–10% NaCl salinity, reaching a maximum of 30.42-times that in the control (0% NaCl) at

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10% NaCl (Figure 8A). This result supports the conclusion drawn from Figure 3 that the amount of betaine in the cells increases with NaCl concentration in the range 2%–10% NaCl.

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The transcriptional level of H0 and H1 increased with NaCl (2%–6%), and the maximum level was 1.74- and 4.18-fold that in the control (0% NaCl) at 6% NaCl (Figure 8B–C). The

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transcriptional level of H3 and H5 increased at 2%–4% NaCl, and the maximum was 10.21-

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and 6.73-fold that in the control (0% NaCl) at 4% NaCl (Fig. 8D–E).

3.6 Effects of betaine and NaCl on the transcriptional level of transporters H0, H1, H3 and H5

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To investigate the relationship between the relative expression of the four transporter genes (H0, H1, H3 and H5) and betaine or NaCl in Rhodococcus sp. HX-2, cells were first cultured

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to logarithmic growth phase in LB medium containing 2% NaCl. These cells were inoculated into hydrocarbon degradation medium containing 6% NaCl with or without 150 mg L−1 betaine (betaine-free as a control). Figure 9 shows that compared with HX-2 without NaCl and betaine, the expression levels of the four transporter-encoding genes were not significantly affected in the presence of NaCl or betaine alone. However, the transcriptional levels of H0, H1, H3 and H5 were upregulated 2.91-, 22.36-, 37.13- and 25.73-fold in the presence of betaine and NaCl. These data demonstrate that the maximal induction of H0, H1, 17

H3 and H5 transcription depends on high salinity plus the presence of betaine.

4. Discussion Due to sewage discharge and other problems lead to imbalance of water and salt in soil, inhibiting the growth and metabolism of microorganisms, and reducing the efficiency of traditional bioremediation of diesel oil pollution. Therefore, identification of excellent salt-tolerant bacteria is prerequisite for microbial remediation of petroleum-contaminated saline soils. Liu et al. reported that at an initial NaCl concentration of 1.2% and 2.4%,

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80%–90% removal of alkane was achieved within 56 h, and 65% removal was achieved at 3.6% NaCl within 68 h, by R. erythropolis NTU-1 [89]. A Rhodococcus sp. bacterium used benzene in the liquid phase as sole carbon source and grew at 6% NaCl at 0–37 °C [90]. Rhodococcus sp. RB1 was able to thrive in medium containing up to 0.9 M NaCl or KCl and in the

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presence of high concentrations of nitrate (up to 0.9 M) [91]. Here we show that Rhodococcus strain HX-2 can grow in 0%–10% NaCl. The diesel oil percentage degradation was >80% at

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0–2% NaCl, declining as [NaCl] increased, although when the NaCl concentration reached 6%, the percentage degradation was still 44.8%. Higher NaCl concentration (7%–10%)

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significantly inhibited the degradation of diesel oil. Importantly, none of the studies mentioned above investigated the salt tolerance mechanism of the hydrocarbon-degrading

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strain. Our experiments proved that the salt tolerance of strain HX-2 was principally due to accumulation of a compatible substance (betaine) in the cells.

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Salinity is an environmental factor that affects the growth of microorganisms. Within a certain salinity range, microorganisms balance the intracellular osmotic pressure and protect the

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protoplasts in the cells through various osmotic adjustment mechanisms in conjunction with regulating their metabolism to adapt the salinity perturbations [92]. The salinity range in which microorganisms are capable of normal metabolism is approximately 0.5% to 3% [93]. Bacteria use different strategies to deal with a high-salt environment. The first is to accumulate K+/Cl− ions to maintain the internal osmotic balance of the cells. The second is accumulation of intracellular compatible substances. These solutes reduce the difference in osmotic pressure between the inside and outside of the cell, thereby minimizing water loss 18

and helping to maintain cell expansion pressure. An adaptation period was not required by Rhodococcus sp. RB1 for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl [91]. Komarova et al. showed that R. erythropolis E-15 responded to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid [94]. Cappelletti et al. reported that the salt tolerance of Rhodococcus sp. R7 could be supported by the presence in the genome of genes encoding both a proline/glycine betaine transporter (ProP) and a multicomponent proline permease (ProU) [30]. Lu et al. found that at NaCl concentrations of 0%–16%, the content of betaine in cells

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(Tetragenococcus halophilus) increased with salinity [95]. Qian et al. reported that the moderately halophilic bacterium Virgibacillus halodenitrificans PDB-F2 could degrade phenol in high salinity environments, and responded to external salinity changes through biosynthesis or uptake of compatible solutes [96]. Han et al. showed that the betaine content

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in Lactobacillus delbrueckii cells when NaCl and betaine were both added to the medium was 1.5-times that when only NaCl was added [97]. The intracellular glutamic acid content in L.

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bulgaricus 34.5 was higher in the presence of both salt and glutamic acid compared with that in the presence of salt alone [97]. Canovas et al. found that the accumulation of betaine in

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Halomonas elongata was gradually increased at 0.75%–3.0% NaCl [98]. It has been reported that adding the proper amount of betaine can increase the growth rate of bacteria under salt

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stress [99]. In the present study, Rhodococcus sp. HX-2 accumulated the compatible substance betaine with increasing external salt concentration (2%–10%). The amount of exogenous betaine transported into cells at different NaCl concentrations was different. The

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amount of exogenously absorbed betaine (150 mg L−1) in the cells was 3.58, 12.85, 44.48, 45.78, 23.54 and 10.65 mg L−1 at 0%, 2%, 4%, 6%, 8% and 10% NaCl, respectively. When

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150 mg L−1 betaine were added, the degradation of diesel oil by HX-2 was obviously promoted. The percentage diesel oil degradation increased by 33.8%, 14.8%, 12.1%, 10.1% and 9.0% compared with cultures with no added betaine at 6%, 7%, 8%, 9% and 10% NaCl, respectively. Thus, although the bacteria were able to synthesize betaine by themselves, they could also absorb betaine and use it. We speculate that bacteria that can directly absorb betaine into cells can adapt to a high-salt environment faster than those that need to self-synthesize betaine, and that exogenous addition of betaine will help with resisting a 19

high-salt environment.

As an important component of activated sludge flocs, EPS play a significant role in the adsorption of contaminants, maintenance and formation of microbial aggregates, as well as protection of microbial organisms from external harsh environments [79]. The EPS produced in many salt-tolerant bacteria is a growth strategy in response to adverse conditions and it adheres to the surface of the cells to form a membranous structure that protects the cells from the external environment [100]. Kumari and Khanna reported that enhanced EPS production

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by rhizobacteria was initially observed with a rise in salinity, then it declined with a further increase in NaCl concentration [101]. The present study showed that HX-2 cells produced less EPS as the external salinity increased. However, the addition of exogenous betaine led to an increase in EPS production, resulting in a membrane structure forming outside the cells,

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which contributed to the resistance of the bacteria to high-salt environments. The results of SEM supported this (Fig. 5). FTIR analysis suggested that hydroxyl, amide and phosphate

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groups may be involved in the defense of the bacteria against high-salt environments.

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Soil contaminated with salinized petroleum hydrocarbons is a major problem in environmental restoration. The diesel oil degradation observed in soil was significantly lower

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than that in liquid medium in the present study. Diesel oil biodegradation in soil is limited by factors such as adsorption to soil particles and the absence of enough oxygen to support the microbial degradation processes. Extracellular enzyme activity is a key step in degradation

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and use of organic polymers, since only compounds with molecular mass <600 Da can pass through cell pores [102]. Kebria et al. reported a Bacillus sp. strain that was capable of

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degrading 85.2% of diesel oil fuel (10,000 ppm) in 15 d in soil without added salt [103]. Wu et al. observed 20% degradation on inoculation of Serratia sp. BF40 and 59.1% on addition of biosurfactant in saline conditions [104]. Al-Kaabi et al. showed that Bacillus sorensis isolate D1 had the ability to remove 88% of the diesel oil (584 ± 7 ppm) and 63% of the polycyclic aromatic hydrocarbons from oil-contaminated soil within 2 weeks [105]. Here we show that Rhodococcus sp. HX-2 can repair soil contaminated with salinized diesel oil. Furthermore, we found that exogenous addition of betaine facilitated diesel oil degradation in saline soil. 20

B. subtilis possesses five osmotically regulated transporters (Opu) for the uptake of various compatible solutes for osmoprotective purposes [106]. The proU locus and the kdp operon encode a high-affinity betaine transport system, and are the principal osmoresponsive genes in Escherichia coli and Salmonella typhimurium [107]. Culham et al. revealed that transporters encoded in loci putP, proP and proU mediate proline and/or betaine accumulation by E. coli K-12, and defined relationships between the proP sequence and a transporter superfamily [108]. BetS protein displays significant sequence identity to the choline transporter BetT of E.

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coli (34%) and to the glycine betaine transporter OpuD of B. subtilis (30%). The BetS protein shows a common structure with BCCT systems [109]. Angelidis et al. reported three osmolyte transport systems in Listeria monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter (OpuC) [110]. The present paper explored the

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relative expression of a betaine synthesis gene (betB) and of betaine transporter genes (H0, H1, H3, H5) in Rhodococcus sp. HX-2 in high-salt conditions. The expression level of the

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betaine production gene was positively correlated with NaCl concentration, while the relative expression of the betaine transporter genes was affected by the external NaCl and betaine

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concentrations. Qian et al. reported the transcription level of the betaine transporter gene of V. halodenitrificans PDB-F2 at different NaCl concentrations [96]. However, such data has not

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previously been reported for the betaine transporter genes in Rhodococcus spp.; thus, our data lay a foundation for the discussion of the salt tolerance mechanisms of Rhodococcus spp.

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5. Conclusions

Analysis of the salt tolerance mechanism of Rhodococcus strain HX-2 revealed that it can

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resist an external high-salt environment (2%–10% NaCl) by accumulation of compatible substances (mainly betaine) in the cell. Exogenously added betaine can be transported into the cells and increase the degradation rate of diesel oil in high salt conditions. We also analyzed the effect of betaine addition on the surface morphology and surface groups of the cells. SEM analysis demonstrated that a membranous structure was produced outside the cell due to an increase in EPS production as a result of betaine supplementation. This membranous structure helps the bacteria resist the high-salt environment. FTIR analysis suggested that surface 21

hydroxyl, amide and phosphate groups may be related to tolerance of high-salt environments. Finally, we found that the betaine producer gene betB was induced by NaCl stress. NaCl also induces four transporter genes for betaine, and maximal transcription depends on the combination of a high salt concentration and the presence of external betaine.

Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or

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financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No.

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21777113), the Natural Science Foundation of Tianjin City (Grant No. 15JCQNJC08800), the National Undergraduate Training Programs for Innovation and Entrepreneurship (No.

Author contributions:

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Colleges and Universities in Tianjin.

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201810060147 and 201910060142) and the Training Project of the Innovation Team of

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Xin Hu contributed to the conception of the study.

Dahui Li and Yue Qiao contributed significantly to analysis and manuscript preparation;

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Qianqian Song, Zhiguo Guan Kaixuan Qiu and Jiachang Cao performed the data analyses and

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wrote the manuscript;

Lei Huang helped perform the analysis with constructive discussions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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List of figures and tables

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Figure 1 Degradation of diesel oil by strain HX-2 at different NaCl concentrations.

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Figure 2 Figure 2 (A) Permeability of the cell membrane of strain HX-2 and Staphylococcus aureus ATCC25923 at different NaCl concentrations. (B) Cation contents of HX-2 in different culture media (*P<0.05, **P<0.01,

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***P<0.001).

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Figure 3 Figure 3 Content of betaine, glutamic acid and ectoine in strain HX-2 at different concentrations of NaCl (*P <

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0.05, **P < 0.01, ***P < 0.001).

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Figure 4 Figure 4 Effect of exogenous addition of betaine on strain HX-2. (A) Accumulation of betaine in cells at different NaCl concentrations. (B) Effect of exogenous addition of betaine on diesel oil degradation (*P < 0.05,

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**P < 0.01, ***P < 0.001).

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Figure 5 Figure 5 Scanning electron micrographs of HX-2 cells. (A) Cells grown in hydrocarbon degradation medium; (B) Cells grown in hydrocarbon degradation medium containing 6% NaCl; (C) Cells grown in hydrocarbon

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degradation medium containing 6% NaCl and 150 mg L−1 betaine.

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Figure 6 Figure 6 Effect of betaine on surface groups of HX-2. Curve (A) shows the Fourier-transform infrared absorption spectrum of HX-2 after adding 0.4% diesel oil to 6% NaCl-containing hydrocarbon degradation medium; Curve (B) shows the absorption spectrum after addition of 0.4% diesel oil and 150 mg L−1 betaine to 6%

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NaCl-containing hydrocarbon degradation medium. Curve (C) shows the absorption spectrum of betaine.

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Figure 7 Figure 7 Effect of exogenous addition of betaine on the percentage degradation of diesel oil in soil at different

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salinities (*P < 0.05, **P < 0.01, ***P < 0.001).

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Figure 8 Figure 8 Relative expression of five genes in strain HX-2 at different NaCl concentrations (0%–10%) (*P < 0.05,

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Figure 9 Figure 9 Relative expression of genes H0, H1, H3 and H5 in Rhodococcus sp. HX-2 depending on NaCl and betaine. The strain was grown in hydrocarbon degradation medium in the presence or absence of NaCl or betaine to exponential growth phase and then subjected to RNA isolation and qPCR analysis. The relative expression was normalized to cells grown in control conditions (without NaCl or betaine) (*P < 0.05, **P < 0.01, ***P <

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