Chemistry and Physics of Lipids 208 (2017) 31–42

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Lipids of Dietzia sp. A14101. Part II: A study of the dynamics of the release of surface active compounds by Dietzia sp. A14101 into the medium

MARK

⁎

Ina Hvidstena, , Svein Are. Mjøsa, Gunhild Bødtkerb, Tanja Bartha a b

Department of Chemistry, University of Bergen, Allégaten 41, 5007 Bergen, Norway Uni Research CIPR, Uni Research, P.O. Box 7810, 5020 Bergen, Norway

A R T I C L E I N F O

A B S T R A C T

Keywords: Genus Dietzia Bacterial lipids Bio-surfactants Hydrocarbon-degradation Cell-bound Bio-surfactant-producing bacterial strain Gram-positive bacteria

Dietzia sp. A14101 isolated from an oil reservoir model column was found to induce a strong decrease of the interfacial tension (IFT) in hydrocarbon-water mixtures in the presence of the intact bacterial cells (Kowalewski et al., 2005). The strain was shown to be able to degrade a wide range of hydrocarbon substrates (Bødtker et al., 2009). Further studies showed that the surface-active compounds tentatively identified as glycolipids were produced by Dietzia sp. A14101 on non- and water-immiscible –hydrocarbon substrates, Part I (Hvidsten et al., 2017). The results suggested that biosurfactant (BS) was a mixture of several isomers. The study presented here is aimed to investigate whether BS are secreted into the aqueous medium, and if so, then at which phase of the culture growth and in which amounts – the dynamics of the BS release in incubations on water-immiscible hydrocarbons. Two methods of BS extraction from the medium were attempted and compared: a liquid–liquid extraction (LLE) and precipitation by acid. For qualitative and semi-quantitative assessment, gas chromatographymass spectrometry (GC/MS), thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC–MS), surface tension measurements (SFT), emulsification (E24) and oil-spreading tests were employed. The results indicated that BS only partially were secreted into the medium. Detectable amounts of glycolipids in media were first identified during the exponential growth phase. However, only a slight decrease of SFT was observed in the cell-free medium. The emulsification index values of the sampled material were lower than those reported for related strains. The results suggested that most of the BS produced by Dietzia sp. A14101 remains cell-bound during the culture development in a batch mode and only a narrow range of the BS isomers can be detected in small amounts in media.

1. Introduction The domain of prokaryotes, or bacteria, is a well-known source of a diversified range of novel biochemical compounds, including bio-surfactants (BS) (Banat et al. (2010), Rosenberg and Ron (1999)). BS exhibit a range of advantageous traits compared with synthetic surfactants: environmentally friendly both in terms of high biodegradability, low toxicity and low critical micelle concentration (CMC) and better specific activity at wider temperature-, pH and salinity ranges (Banat et al. (2000), Desai and Banat (1997), Kretschmer et al. (1982)). Further, several BS have been indicated to have biological activity (e.g. Omura (1992), Kurtböke (2012), Solanki et al. (2008)). Therefore, BS are of great industrial interest.

However, the main obstacles of the employing of bacteria for the large-industrial-scale production of a selected target compound is the necessity of the adjustment and a close monitoring of the incubation conditions and the subsequent isolation and purification steps, especially in the case of a large scale production (Mukherjee et al., 2006). Further, several BS-producing strains are strongly implicated as opportunistic pathogens (Van Hamme et al. (2003), Kuyukina et al. (2015), Koerner et al. (2009)). It seems that genetic engineering focused on modification of metabolic processes of a given bacterium could be a solution to design “generally regarded as safe” (G.R.A.S.) strains for industrial application (Wang et al., 2007). Thus, it is of interest to identify BS-synthesising bacteria that could be used either directly in industrial applications, e.g. microbial enhanced oil recovery

Abbreviations: BS, biosurfactant; BE, bioemulsifier; CFA, cyclopropane FA; ECL, equivalent chain length index; EOR, enhanced oil recovery; Fame, fatty acid methyl ester; FA, fatty acid; Gly, glycolipid; G.R.A.S., generally regarded as safe; HC, hydrocarbon; I, incubation; LLE, liquid–liquid extraction; LCFA, long-chain FA; MUFA, mono-unsaturated FA; L, lyophilised; PL, lhospholipid; SAT-FA, saturated; SCMA, short-chain mycolic acid; S, sampling ⁎ Corresponding author at: Department of Chemistry, University of Bergen, Allégaten 41, 5007 Bergen, Norway. E-mail address: [email protected] (I. Hvidsten). http://dx.doi.org/10.1016/j.chemphyslip.2017.08.007 Received 27 March 2017; Received in revised form 6 August 2017; Accepted 14 August 2017 Available online 31 August 2017 0009-3084/ © 2017 Elsevier B.V. All rights reserved.

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classical books on microbiology (e.g. Madigan et al. (2012)). Batch incubations were used in the project presented here, thus, the discussion and assumptions outlined in this paper are limited to closed incubation systems. Generally, batch incubations have a growth limited character, that is, allow a better control of growth factors, and follow the generally accepted growth pattern, the so-called growth curve. The general development pattern of a bacterial culture can be roughly split into several distinct phases: the initial lag phase, followed by the exponential or logarithmic growth; the stationary phase characterized by the high level of depletion of nutrients, and the subsequent transition of the culture into the autolysis (decline or death) phase. The duration of the phases can be strain-, genus- or family-specific. As mentioned above, the production of BS/BE is perceived to be necessary either for successful colonisation of a given ecological niche and/or due to competitive reasons. Thus, the biosynthesis of BS/BE is often trigged when the bacterium is exposed to some outer stress (e.g. temperature, toxic substrates). The stress is assumed to launch a cascade of secondary metabolic reactions that are dormant/non-active under otherwise favourable conditions. Such induced metabolism is often referred to as secondary metabolism; the compounds involved are, therefore, secondary metabolites. The onset of secondary metabolism including the biosynthesis of BS/BE is strongly associated with the late exponential and stationary growth phases when the active growth is in decline (Hogg, 2005). The production of BS/BE type compounds can be endogenous to a given strain, that is produced independent of stress factor and was e.g. reported for an actinomycete Corynebacterium lepus by (Duvnjak and Kosarik (1985). In such instances, BS/BE are rendered as essential primary metabolites vital for the normal cell functioning. There are also cases when a given strain is reported to co-produce both BS and BE that are structurally different, and the latter is secreted from the cellular membrane into the medium. Such cases of co-production are quite rare, e.g. one of the few reports on the subject concerns several strains of the actinomycete genus Gordonia (Franzetti et al., 2008). The mechanisms behind such co-production as well the evolutionary advantages of such energy-consuming biosynthesis are still poorly understood. Another important aspect of the production of BS/BE is whether the surface active compound is secreted/released from the cellular surface either as it is being produced or after a certain residence time during a specific growth phase, or whether BS/BE remains closely associated with the bacterial membrane. The understanding of production and release dynamics of BS/BE can have important implications for the eventual large scale production and/or whether the bacterium can be modified to increase yields, productions rates or to attain G.R.A.S.status. The order of Actinomycetes is taxonomically diverse and several members were reported to produce surface-active compounds (Kügler et al., 2015). Previously, a bacterial strain designated Dietzia sp. A14101 was identified as a Gram-positive actinomycete with high G + C content of DNA (Bødtker et al., 2009). The study has also shown that this bacterial strain is able of degrading a wide range of hydrocarbons. The initial investigations by Kowalewski and his team determined that the interfacial tension (IFT) value at the water-hydrocarbon interface in the presence of the intact bacterial cells was very low – 0.006 mN/m (Kowalewski et al. (2004), Kowalewski et al. (2005), Kowalewski et al. (2006)). Further, the results indicated that Dietzia sp. A14101 seemed to employ a range of adaptation strategies in order to survive and thrive on toxic HC-substrates (Hvidsten et al., 2015). First, the regulation of both fluidity (thus of permeability) and hydrophobicity of the outer cellular surface appeared to be, at least partially, related to/governed by modifications of the content of fatty acids (FA). Second, the exposure to a water–immiscible substrate trigged the development of the negative cellular surface charge and enhanced the production of carotenoidtype pigments. Third, the investigation of the cellular content of the

(MEOR) (Desouky et al., 1996), (Li et al., 2002) and bio-remediation (Hamme et al. (2009), Mulligan (2005), Ron and Rosenberg (2002)); or the bacterial strains that could be modified in order to enhance the production of the target compounds and, in the case of pathogenic BS/ BE producing strains, to render these safe for use in food, cosmetic and biomedical industries (Van Hamme et al. (2006), Lee et al. (2007)). 1.1. Background Biosurfactant (BS) is the contraction used to designate surface active agents produced by a living (biota) organism. BS are amphiphilic compounds that are comprise of a hydrophobic and a hydrophilic moiety; the latter is often employed for the classification of BS. BS that exhibit strong de- and emulsification behaviour are called bio-emulsifiers (BE), and are also referred to as biopolymers. BE are usually structurally complex compounds with high molecular weight. The domains – Eukarya, Archaea and Prokarya – are known to produce such compounds either as primary or secondary metabolites. However, one can reasonably suggest that the unicellular prokaryotic organisms are probably the most prolific domain in terms of BS/BE production (Rosenberg, 2006). The fact is further re-enforced by the evidence that the Prokaryotes are detected in all environmental niches. The range of the environmental habitats include, among others, hot springs, cold climatic zones, salt lakes with high pH, the deep subsurface, the stratosphere, and even on the internal surfaces of the nuclear reactors (Madigan et al., 2012). Since the cellular membrane of a Prokaryote is the ultimate barrier against and the only interaction site with the surroundings, the adaptation strategies cannot but include certain species-specific modifications of the cellular membrane (e.g. Goldfine (1984), Zhang and Rock, (2008)). The bacterial cellular membrane is generally perceived as the site where the biosynthesis of BS/BE takes place. It has been indicated that understanding of prokaryotic metabolism and metabolic products could be useful for application within the fields of pharmacology, agriculture, food industries or bioremediation (Banat et al. (2000), Solanki et al. (2008), Sen (2008), Beloqui et al. (2008), Hamme et al. (2009)). Bacterial metabolism appears to be quite diverse even within a single genus, the lowest taxonomical classification unit. (Maier, 2003) suggested that BS/BE are examples of metabolic products that coevolved among diverse genera that are functionally important, or convergent in function, but are not related genetically or on the level of molecular structure. Such metabolic divergence within a given genus can be partially explained in terms of the horizontal gene transfer, a phenomenon observed only in Prokaryotes. Whatever the driving cause, the scientific community recognizes the fact that strains of the same genus can be reported to produce a diverse range of BS, e.g. the recently established genus Dietzia, while another prokaryotic genus is consistently reported to produce only one type of BS/BE albeit as strain-specific isomers, e.g. the genus Pseudomonas. Thus, the molecular structure of the BS/BE produced most probably should be treated as strain-specific rather than making generalisations for a single genus, and even greater caution should be shown when attempting to make generalisations concerning higher taxonomical units. Several excellent reviews are available that provide detailed overviews of BS/BE-producing bacteria and address the classification of BS/BE according to the molecular structure, the molecular weight, and some other selected parameters (e.g. Lang (2002), Banat (1995), Banat et al. (2010), Hommel (1990), a series of articles by Dembitsky V.: e.g. Dembitsky (2004), and Rahman and Gakpe (2008)). The dynamics of the biosynthesis of BS/BE is an important aspect in terms of the onset of the production and the amounts of BS/BE produced. The latter has important implication for the large-scale production and extraction of BS. Closed batch incubations are employed for the laboratory studies of laboratory-cultivatable bacterial strains and differ in a number of aspects from continuous incubation systems. Both systems have their pros and cons and are covered in great detail in 32

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Poretics, Polycarbonate) and 0.2 μm (GE Water & Process Technologies, GE Polycarbonate). The methylation reagent, dry HCl in methanol (2.5 M), was prepared by dissolving HCl (g) in dry methanol (Meier et al., 2006). Pyrexglass tubes with Teflon lined screw caps were used for derivatisation. Two standard FAME mixtures (GLC 461 and 793, Nu-Check Prep., MN, USA) were employed. TLC plates (MACHEREY_NAGEL, DC-Fertgfolien ALUGRAM® SIL G/ UV254, silica gel 60 with fluorescent indicator; layer thickness 0.20 mm), TLC saturation pads (Analtech 8124, size 20 × 20 cm, pkg of 100 pieces), micro-pipettes for the application of samples (accuracy ± 1%, a dispenser bulb included, Drummond, Alltech), an adsorbent scraper (13 mm) with replacement blades and a nebuliser (10 mL) were bought from Sigma-Aldrich.

strain, described in Part I (Hvidsten et al., 2017), indicated that Dietzia sp. A14101 produced non-trehalose glycolipids on all the selected range of media and substrates – glucose, the complex medium and on the water-immiscible hydrocarbon substrates. Two trehalose-containing lipids were also detected in the cellular material obtained from the incubations on the selected range of substrates and media. Thus, the biosynthesis of these two trehalose-containing congeners was not related to the hydrocarbon uptake and can be, therefore, classified as primary metabolites endogenous to Dietzia sp. A14101. The exposure to the toxic water-immiscible HC substrate trigged the production of several trehalose-containing glycolipids during the exponential growth phase. The biosynthesis of these particular glycolipid species can be related directly to hydrocarbon uptake and, thus, can be assumed to be a secondary metabolic process. Interestingly, only low amounts of HC substrate were incorporated directly into the glycolipids produced. Most of the substrate seemed to be employed for the biosynthesis of the neutral lipids.

2.3. Incubation conditions, sampling and pre-treatment The details concerning incubation, sampling and pre-treatment have been described in detail previously (Hvidsten et al. (2015), Part I (Hvidsten et al., 2017)). In short, a pure culture of Dietzia sp. A14101 was cultivated at 30 °C without agitation. Six incubations on liquid media were performed. D20 medium (Bødtker et al., 2009) was used for incubation on water-immiscible HC-substrate, and for non-HC source incubations, a complete (“nutrient-rich”) medium Gym Streptomyces nr.65 (DSMZ, Braunschweig, Germany) was used. The sampling points were designated as the lag phase (S1); the transitory I (lag-to-exponential, S2); the exponential phase (S3); the transitory II (exponential-to-stationary, S4); the stationary phase (S5), and the advanced stationary phase (S6). The medium generated by the incubation I-1 was sampled at S6; the medium recovered from the incubations I-5 and I-6 was sampled as “time series” at sampling points S1-S6. BHT, a phenolic antioxidant that has been shown to inhibit lipid peroxidation, was added as 0,005% (w/v, in chloroform) to protect samples against autoxidation during the sample work-up and storage (−80 or −20 °C). The selected aspects of sampling and cultivations are summarized in Table 1. Since the aim was to elucidate whether BS is released into the medium during incubation on hydrocarbons, it was important to remove bacterial cells. Microfiltration, as opposed to centrifugation, produced cell-free media. The metabolites released into the media were sampled as follows. The sampled cell-free media was divided into two parts. One portion was extracted by liquid–liquid extraction (LLE) “as it is” with equal volume of methyl tert-butyl methyl ether (MTBE) according to Kuyukina et al. (2001) and the extract was designated EXNA. The other

1.2. Objectives This paper describes the assessment of media and the secreted BS obtained from the incubations of Dietzia sp. A14101 on a water-immiscible HC substrate. The objective is to investigate whether the trehalose-containing glycolipid isomers, associated with the uptake of the water-immiscible HC-substrate, were secreted by Dietzia sp. A14101 into the medium or remained associated with the cellular membrane. Two other aspects of the production dynamics, time of release and the released relative amounts, are also addressed in a semi-quantitative manner. 2. Experimental and methods 2.1. Bacterial strain The aerobic chemoorganotrophic strain Dietzia sp. A14101 was isolated from an oil reservoir model column (Bødtker et al., 2009) and affiliated to the genus Dietzia with 99.9% sequence similarity to type species D. maris. The genus Dietzia has been established only recently (Rainey et al., 1995). It is further affiliated to the Family Dietziaceae (the Suborder Corynebacterineae, the Order of Actinomycetes (Goodfellow et al., 2006)) and is characterized by having a high G + C content (guanineplus-cytosine content of DNA). The range was identified as being from 66.1 to 73.0 mol% for the recognized members of the genus Dietzia (Li et al., 2008a). 2.2. Materials

Table 1 A short summary of cultivation conditions for Dietzia sp. A14101 on non- and HC-substrate. I = incubation; HC = hydrocarbon; C = material sampled by centrifugation; F = sampling by micro-filtration (0.2 μm); BP = bacterial cells/pellet; L = lyophilised/ freeze-dried/desiccated; EXNA = extract obtained by LLE from the cell-free media; EXA = extract obtained by LLE from the after the precipitate; AcP = precipitate obtained by acidification of the cell-free media followed by microfiltration; n.d. = not detected; — = not collected; + = collected. * Cell-free medium obtained by centrifugation or micro-filtration (0.2 μm) of the crude broth was designated as “pure”.

Solvents, all HPLC-grade and p.a. grade, and solid chemicals (sodium azide; butylated hydroxytoluene, (BHT)), reference lipid standards (TAG, DAG; PLs: PC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), PE (1,2-diacyl-sn-glycero-3phosphoethanolamine from E. coli); MA - mycolic acid(s) (Mycobacterium tuberculosis, bovine strain, MA as a mixture of homologues Mw range 110–1300 g/mol); LP (surfactin)); reagents for detection of lipids on thin-layer chromatography (TLC) Molybdenum Blue spray- reagent (1.3%); 2,2-dixydroxy-1,3-indanedione (ninhydrin); 1-hydroxynaphthalene (α-naphthol); 4-methoxybenzaldehyde (p-anisaldehyde); 5-methylbenzene-1,3-diol (orcinol); the universal detection reagents (iodine; primuline, ethanolic phosphomolybdic acid (PMA)) were purchased from Sigma-Aldrich. Standard reference glycolipid compounds, mono-glycolipid n-octyl-β-Dglucoside (mono-glycolipid) and di-glycolipid n-dodecyl- β-D-maltoside (di-glycolipid) were purchased from Avanti Polar Lipids, USA. The filtration system Millipore® was purchased from Sigma-Aldrich. Filters had i.d. Ø 47 mm, the pore size was 1.2 μm (Whatman, Schleicher & Schuell, Glass Microfibre GF/C); 0.8 μm (OSMONICS Inc.,

Incubation

I-1

I-2 I-4 I-3 I-5 I-6

33

Medium, V (L)

Substrate

Gym Streptomyces nr.65/1 L (complex nutrient rich medium) D20/1 L Simple HC, D20/5 L n-C12 D20/0.5 L

Sampling method

Type of sampled material EXNA

EXA

AcP

C

+

+

n.d.

C F C F F

– – + + +

– – – + +

– +(L) – + +

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2.6. LC–MS of selected extracellular AcP type sample

portion of medium was acidified to pH 2.0 with concentrated hydrochloric acid according to the method by Deziel et al. (1999). The acidified medium was kept overnight at the 4 °C, and the precipitate was collected by filtration on 0.2 μm filters. The precipitated material recovered from medium by acidification was designated AcP. The AcP was dried on filter prior to analyses. The AcP collected from I-4, was lyophilised and designated AcP(L). The AcP material had light orange coloration. After the removal of AcP, the medium was adjusted to the original pH 7.5, re-extracted with MTBE and the extract was designated as EXA. In liquid–liquid extractions the solvent was removed on a rotary evaporator at the reduced pressure. The obtained viscous light-yellow residue was re-extracted with MTBE and subjected to further analysis.

Thermo scientific (LTQ-Orbitrap XL) with high resolution (HRMS, > 1000 at m/z 400) with an electrospray ion source ION MAX was used for global profiling of the isolates. Accela HPLC Thermo Fisher Scientific (USA) with an Supelco Acquity UPLC BEH C18 reverse phase column (2.1 mm × 50 mm, 1.7 μm) was used employed for chromatographic separation of lipid species. The analysis was performed according to the method by Nygren et al. (2013). In short, the gradient elution was run from 65% A (ultrapure water (1% 1 M ammonium acetate, 0.1% formic acid)) and 35% B (LC–MS-grade acetonitrile–isopropanol (1:1, 1% 1 M ammonium acetate, 0.1% formic acid)), reached 80% B in 2 min, 100% B in 7 min, and remained there for 7 min. The column temperature was 50 °C. The flow rate was 0.500 mL/min. The following modifications were found to be necessary: the injection volume was set to 1 μL. Electron ionisation (ESI) both in positive and negative mode were employed for lipid profiling. The lyophilised AcP type samples were-dissolved in chloroformmethanol (2:1 by vol.), and further diluted with isopropanol-acetonitrile (1:1 by vol.) so that the final concentration was in the range 20–60 μm/ml prior to injection.

2.4. Fatty acids in EX and AcP samples by GC–MS The total fatty-acid content was determined by methylation and the subsequent GC–MS analysis. The method was adopted from Meier et al. (2006). Minor modifications to the method, the instrument specifications and the program were as described previously (Hvidsten et al., 2015). FA were identified by the evaluation of both “equivalent chain length” (ECL) index value of the FA species identified and mass spectral data. FA are reported by using the IUPAC nomenclature. The yields are presented as the relative percent amounts of the total crude sample, which are either LLE extracts taken to dryness or the precipitate obtained by acidification of the aqueous incubation medium.

2.7. Structure determination by ATR-FTIR spectroscopy The infrared analyses of the material obtained from AcP type sample by preparative TLC was carried on Nicolet Protege 460 FTIR spectrometer equipped with a diamond attenuated total reflection (ATR) sampler cell (Dura, SensIR). The recorded spectrum range was 700–4000 cm−1, 64 scans and a resolution of 8 cm−1. The spectrum of the solvent mixture was measured and used for background correction. The cell surface was checked prior to each analysis by measuring of the baseline after removing/washing away the previous sample residue. Samples were dissolved in dichloromethane-methanol (93:7, by vol.) and applied as a small drop onto the cell surface. The solvent was allowed to evaporate before the spectrum was recorded. Samples were analysed as triplicates.

2.5. Thin-layer chromatography (TLC) Crude bacterial mass samples collected at different stage of the culture development, TLE of the corresponding crude samples and the SPE fractions obtained from TLE samples were subjected to one-dimensional (1D) and/or two-dimensional (2D) TLC. Plates, pre-developed with the respective solvent system (Subsections 2.5.1–2.5.2), dried, and were activated at 110 °C for an hour. The material was applied as 4 superimposed spots, so that the final amount applied was in the range 10–15 μL from 30 mg/ml solutions, each application was allowed to dry before a new one. Spacing between spots was 1 cm for 1D TLC. The plates were allowed to dry for 30 min at room temperature. Application and drying at room temperature in-between development steps and/or prior to visualisation were performed under a stream of nitrogen (g). In addition to the detection reagents (Section 2.2) aqueous sulphuric acid (50%) followed by heating at 100 °C for 10 min was used as a universal reagent.

2.8. Physico-chemical properties of extracellular material Selected tests were employed to assess the performance potential the secreted lipid metabolite fraction. Each test was performed in triplicate. The oil-spreading test (Morikawa et al., 2000) allowed quick assessment whether the isolated lipid species are surface active. In short, 20 μL of non-biodegraded crude oil were spread over the water surface (50 mL of distilled water, Petri dish, 20 cm diameter). 10 μL of the 0.2 μm filtrated medium were carefully applied onto the oil-film. The diameter of the clear zone, that corresponds to the amount and activity of the BS added, was measured after 2 min. This was also investigated under acidic condition, pH 3.0 (Ishigami et al., 1987). Surface tension (SFT) was determined by the DU NOÜY RING method with Sigma 700 Tensiometer, 240VAC, 50 Hz (KSV, Biolin Scientific) equipped with a motorised sample stage, an automatic calibration and locking. Distilled water and freshly prepared D20 medium were used as standards. The emulsification potential for crude cell-free medium, AcP, EXNA and EXA samples was expressed as the emulsification index (E24) and determined according to the method by Mnif et al. (2011). Briefly, samples (crude medium broth or the sampled material dissolved in distilled water) were added as 4 mL to the equal amount of the hydrocarbon phase (decane, n-hexadecane and kerosene). The obtained bi-phasic solution was mixed by vortex for 2 min and left for 24 h. All the samples were analysed as triplicate. The E24 index was calculated as the ratio of the height of the emulsion layer to the total height of the liquid column. Temperature tolerance and pH stability were evaluated (Qiao and

2.5.1. Screening by TLC 2D TLC was used to separate the complex mixture obtained as AcP type sample. The method was developed by Matsuyama et al. (1987), and was employed previously for screening of lipid fraction cellular material of Dietzia sp. A14101 (Hvidsten et al., 2015). The screening of lipopeptides by 1D TLC was performed according to Li et al. (2008b). To investigate the dynamics of the release of BS/BE into the medium by Dietzia sp. A14101 a version of the neutral eluent system chloroform-methanol-water (65:15:2, by vol.) developed by Paściak et al. (2003) was employed.

2.5.2. Preparative TLC The eluent system by Paściak et al. (2003) was employed for the isolation of the compounds of interest from the AcP type-samples. Ten pre-treated TLC Plates 10 × 10 cm were used to collect enough of the material. The spots were scraped from plate, re-dissolved in chloroformmethanol (2:1, by vol.), and silica removed by filtration. The samples were taken to dryness, the weight was determined, and the stock solution was prepared. The spots were applied under a stream of nitrogen. 34

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stable through S2-S5, but increased at S6. Such an increase can be partially explained by the increasing rate of the decline of the batch culture characterized by the high rates of autolysis of the cell, which means the release of the cellular material into the media. However, the same pattern was not observed for C18:0, the secretion of which seemed to fall drastically at S3. Thus, the sudden increase of C16:0 cannot be explained by autolysis alone. C20:1 showed consistent and steady increase along the growth curve, thus its production was certainly growth associated. The same growth and HC-uptake associated pattern and also high relative amounts of C20:1 FA, identified in the trehalose-containing fraction of the cellular lipid content, were detected previously in cell material (Part I (Hvidsten et al., 2017)). Another interesting feature of the EXNA type-sample was the presence of the fatty acid C12:0 at small constants amounts even in the lag and the transitory I growth phases, S1 and S2, respectively. The C12:0 amounts increased substantially during the log phase, S3. However, it is interesting to note that the decrease in the amount of the C12:0 from S4 to S6 sampling points coincided with the increase in C14:0, Fig. 2. In EX NA type-sample C20:1n-7 was present in high amounts, Fig. 3. The amounts of this FA decreased from S1 to S6 sampling points, that is the development profile along the time line was found to be the opposite to that identified for EXA samples. A branched isomer of the saturated C16:0 and a long-chain saturated C25:0 were next-to-dominant species in the profile. Low amounts, less than 10%, of C12:0 were also observed. It was clear that the removal of some secreted metabolic products by precipitation with acid had a pronounce effect on the profile of metabolites that could not be extracted as precipitate under acidic conditions. The third sample type, AcP, isolated by precipitation with an acid, could only be recovered when the bacterial culture entered the exponential growth phase, corresponding roughly to the sampling point S3. The analysis of the hydrophobic content of the isolated secreted material indicated that two FA, a saturated C16:0 and a monounsaturated C20:1, dominated the profile, as illustrated in Fig. 4. The profile of C16:0 showed a steady increase along the time line, S3-S6 sampling points. C12:0 and C14:0 were detected at minor amounts. No C25:0 was identified in this type of the samples. Fig. 5 illustrates the fatty acid profiles for all three sample types for the incubation I-4 at the sampling point S4, corresponding to the transitory II growth phase. The comparison gives the following observations. First, the acidification of the media and the subsequent removal of the precipitate by micro-filtration allowed isolating most of the lipid material with the hydrophobic moiety (-ies) comprised of the following fatty acids: three saturated fatty acids, C16:0 (a dominant FA) and C12:0 and C14:0 (present at lower amounts), and one monosaturated C20:1n-7 fatty acid. The lipid species with C25:0 could not be removed from media by the precipitation with an acid, though it seemed that the previous treatment of the media by acidification and the subsequent return to pH 7 had a positive effect on the extraction yield of the lipid species with C25:0.

Shao, 2009). In short, to assess the temperature tolerance an aliquot of samples (4 mL) was kept in a Pyrex tube for 1 h at 50, 70, 100 °C and 15 min at 121 °C and the emulsification test was repeated for the heattreated sample. pH tolerance was assessed by adjusting an aliquot of a sample to pH 3 with hydrochloric acid (100 mmol l−1) and 10 with sodium hydroxide (mmol l−1) and the oil-spreading test was repeated. 3. Results 3.1. Extraction of lipid species from media The isolation of the metabolites secreted into the medium was attempted by both LLE extraction and precipitation with an acid. The obtained material gave rise to 3 types of samples EXNA, EXA and AcP, Section 2.3 gave the designation of the samples names. LLE with MTBE was employed to determine which neutral lipid species were secreted. Generally, neutral glycolipids, both non-ionic trehalose-containing and lactone forms of glycolipids could be extracted from the non-acidified medium. The LLE extraction of the media produced relatively variable yields and could not be used for conclusive and rigid qualitative assessment of the metabolites. Thus, the yields were assessed only semiqualitatively. Some examples of the yields are provided in Supplemental Material, Section S-1. The extraction by precipitation with a concentrated acid of the cellfree-medium was applied to investigate the presence of acidic lipid compounds. The extraction yields of the weakly ionic forms of glycolipids, including trehalose-containing variants, was improved by acidification of the solution, since they become protonated and, thus, less soluble in an aqueous phase. Glycolipids exist under the acidic conditions pH 3.0 in their protonated form, e.g. rhamnolipids have pKa 5.6, (Ishigami et al., 1987). Later Schenk et al. (1995) confirmed that water solubility of glycolipids is affected severely by changing pH. The AcPtype sample was sampled only from the liquid medium incubations on water-immiscible HC substrate. Fig. 1 presents a graphical summary of the production dynamics of metabolites that were related to the hydrocarbon uptake and were secreted into the media, and which could be recovered by precipitation. The AcP-type material could not be recovered from the incubations on the HC substrate during the lag and Transitory 1 phases, S1 and S2 sampling points, respectively. The deviation between the yields of the AcP-type material isolated from the I-5 and I-6 incubation media was more pronounced in the exponential growth phase, sampling points S3 and S4. 3.2. Fatty acids in secreted material The hydrophobic content of the lipids isolated by LLE from crude untreated cell-free media was, as expected, less varied than the corresponding content of the cellular material described previously (Hvidsten et al., 2015). Two fatty acids, a saturated C16:0 and a monounsaturated C20:1 dominated the profile of the EXNA type-sample, Fig. 2. However, the fatty acids exhibited different time profile in terms of the determined relative amounts. The amount of C16:0 was relatively

3.3. TLC All sample types were assessed by TLC. The qualitative assessment of the samples by analytical TLC did not indicate the presence of any surface-active compounds with a lipopeptide structure. No glycolipids were detected in medium obtained from I-1 incubation either at S1 or S6 sampling points. The 1D TLC results, Fig. 6 and Supplemental Material, Section S-2, Fig. S-2.1, suggested that both ACP-L and ACP type-samples, Lanes 3 and 4 respectively, contained several glycolipid species. Interestingly, the non-lyophilized sample performed better, which could indicated that the lyophilised sampled required a somewhat longer than 20 min rehydration. Some glycolipid-reagent positive spots were elongated/ tailing in spite of the modifications of the eluent solvent mix and other

Fig. 1. Yields of the AcP type material recovered from the incubations on dodecane substrate. AcP = precipitate obtained by microfiltration.

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Fig. 2. Fatty acids profiles for the sampling points S1-S6, in the EX NA type-sample for I-6.

Fig. 3. Fatty acids profiles for the sampling points S1-S6 in the EX A type-sample for I-6.

probable that the elongated spot with Rf 0.5 was a mixture of homologues, some of which can acquire an ionic site(s) at low pH values. The neutral homologues could not be removed by precipitation with an acid and seemed to be minor components in the mixture. The high Rf-value can be indicative of a higher hydrophobic nature of the secreted glycolipids. Several minor glycolipid-positive spots were detected close to the origin with Rf values 0.05 and 0.1, the marked field at the bottom of the chromatogram in Lanes 3 and 4, Fig. 6. These glycolipid compounds were totally removed from the media by precipitation with an acid and were absent from EXA-type samples, Lanes 2 and 6 in Fig. 6. All LLE extracts, EXA and EXNA, contained also fatty acids and a range of neutral lipids, identification of which was not pursued further. The material isolated from the cell-free media was screened by 2D TLC. The results indicated that the Dietzia sp. A14101 secreted a mixture of mycolic acids, fatty acids and glycolipids, Fig. 7 and Supplemental Material, Section S-2, Fig. S-2.2 (A). The four intense clearly defined spots and 3 less intense spots with much smaller relative areas were glycolipid-positive in the EXNA type material. Fig. 7 indicates the

analysis parameter. 1D TLC eluent systems usually cannot provide further separation, as efforts to achieve a higher resolution could be time-consuming. Four spots in the upper region of Lanes 3 and 4 were tentatively identified as MA, otherwise no pigments or neutral lipid species were detected in AcP-type samples, Fig. 6 Lanes 3 and 4. The elongated spots in the area marked with the hatched line had Rf 0.5, the Rf value was calculated for the centre of the spot. Such elongated glycolipid-reagent positive spots were detected in all three sample types, but were most prominent in the AcP-type samples. Such elongated form usually indicates that the spot is a mixture of several homologs. The chromatogram in Fig. 6 provides the evidence that the extraction by acidification enhanced the extraction yield of these glycolipid species, the upper marked field in Lanes 3 and 4. Still, weaker spot with the same elongated form and Rf 0.5 value were detected in all LLE extracts, both EXNA and EXA, Lanes 1, 2, 5, and 6 in Fig. 6. A weak glycolipid-reagent positive spot was present in both EXNA and EXA, and according to the Rf value corresponded to non-ionic trehalose glycolipid. Thus, it is very

Fig. 4. Fatty acids profiles of AcP type-sample for I6S3-S6.

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Fig. 5. A comparison of fatty acid profiles determined for the sampling point I-6S4 in the three sample types.

Fig. 7. Distribution of lipid species secreted into the medium and sampled as EXNA type material for I-6S5, see L4 I-6S5 AcP in Fig. 6. (1) MA; (2) neutral and simple lipids; and the marked areas tentatively-identified as (3) acidic trehalose-containing glycolipids; (4) neutral trehalose-containing glycolipid; (5) di-corynomycolate containing a sugar moiety. The areas marked by hatched lines denote spots that produced a glycolipid positive response. TLC system (Matsuyama et al., 1987). Detection as described in Fig. 6. Supplemental Material Section S-2 Fig. S-2.2 (A). Fig. 6. Distribution of lipid species secreted into the medium (L = lane). L1 I-4S5 EXNA; L2: I-4S5 EXA; L3 I-4S5 AcP(L); L4 I-6S5 AcP; L5 I-6S5 EXNA; L6 I-6S5 EXA; L7 standard reference glycolipid compounds. The most intensive spots are marked by a solid line. The areas marked by hatched lines denote spots that produced a glycolipid positive response. TLC system: CHCl3-MeOH-H2O (65:15:2, by vol.).The visualisation reagents used (three replicates): universal reagent (aqueous sulfuric acid, 50%), phospholipid-specific reagent (Molybdenum Blue spray-reagent, or 1.3% molydbenum oxide in 4.2 M sulfuric acid), and glycolipid specific reagent (4-methoxybenzaldehyde). Supplemental Material Section S-2 Fig. S-2.1.

presence glycolipid-positive compounds and allowed a decent resolution of these, which otherwise were eluted as one elongated spot in 1D TLC, Fig. 6. Further, the 2D method by Matsuyama et al. (1987) allowed a much better resolution of the mycolic acids compared to the 2D method by Mordarska and Paściak (1994), Supplemental Material, Section S-2, Fig. S2.2 (A versus B). According to the 2D TLC chromatogram the mixture consisted of four glycolipid species. EXna and EXa type-samples did not produce clear well defined glycolipid-positive spots. Further, TLC was employed for the assessment of the dynamics of the glycolipid release into the media. The production of the glycolipids in detectable amounts coincided with S3 sampling point as indicated by 1D TLC, Fig. 8 Lane 1. The results indicated that two polar glycolipid species (Rf values 0.05 and 0.11), and a homologous mixture of less polar glycolipids (Rf 0.5, Rf value calculated for the centre of the spot) were released by Dietzia sp. A 14101 into the medium during the exponential growth phase on the water-immiscible HC substrate (dodecane). The polar glycolipid species had the same Rf value as reported previously for trehalose-containing lipids detected in the cellular material of Dietzia sp. A14101 Part I (Hvidsten et al., 2017). The peak of release of latter appeared to occur at approximately S5, which

Fig. 8. Dynamics of the release into the medium of lipid species identified in AcP(L) typesampler. (L = lane); L1: I-4S3; L2: I-4S4; L3: I-4S5; L4: I-4S6; L5: standard reference glycolipid compounds. The most intensive spots are marked by a solid line. The areas marked by stippled lines denote spots that produced a glycolipid positive response. TLC system: CHCl3-MeOH-H2O (65:15:2, by vol.), Detection as described in Fig. 6, but also with 5-methylbenzene-1,3-diol (orcinol), which is shown here.

corresponded to the stationary phase of the culture development.

3.4. LC–MS of selected extracellular AcP type sample The initial results of the LC·MS analysis of the AcP type sample confirmed that several glycolipid species are secreted by Diertzia sp. 37

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Fig. 9. (A) Total ion chromatogram (TIC) LC/ESI negative ion mode of the AcP type sample (I-5S5); (B) MS (ESI, negative ion mode) spectrum of the glycolipids obtained from the AcP sample (I-5S5), the spectrum is divided into regions (1) mono-glycolipids and di-glycolipids with short fatty acids residues as the hydrophobic moiety; (2) di- glycolipids and (3) and tetraglycolipids.

1411 cm−1 corresponded to CeH/OeH deformation of the carbohydrate moieties. Signals between approximately 2935 and 2860 cm−1 and between 1470 and 720 cm−1 indicated presence of long linear aliphatic chains (symmetric stretching vibrations of CeH in aliphatic groups). The less pronounced signals at 1411 and 1386 cm−1 suggested stretching of CeO bonds between carbon atoms and hydroxyl groups of carbohydrate moieties. The compounds isolated by the means of the preparative TLC produces similar spectra with minor variations, which indicated glycolipid structures. The screening for lipopeptides by ATR FT-IR of the sampled material did not identify such compounds in any of the sample types that was supported by TLC results, Section 3.3.

A14101 into the medium, Fig. 9, the work on the identification of the exact structure of these is in progress. 3.5. ATR FT-IR of the secreted glycolipids The ATR FTIR of the compounds sampled by the means of preparative TLC (the two lowest spots, Fig. 8, were pooled together to produce enough material) confirmed the glycolipid structures, Fig. 10(A) and (B). The interpretation of the spectra were based on Willimas and Flemming (1995) and Coates (2000). A very weak peak at approximately 1731 cm−1, stretching band C]O is indicative of a carboxylic group and ester bonds. A second strong absorption between 1650–1600 cm−1, that is a pronounced peak at 1649.8 cm−1 below 1700 cm−1 (at the low end of the range [1850-1650] cm−1 the indicative area for a carbonyl group moiety) was related to C]O stretching vibrations of carbonyl moieties and suggested also that conjugation might be present. A range of weak signals was observed at

3.6. Surface activity of the AcP-type sample The total AcP-type samples produced clouded unclear aqueous solutions which indicated that the material was only partially dissolved in water. The material though dissolved completely on addition of low 38

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Fig. 10. An example of an ATR FTIR spectrum of (A) AcP(L) type-sample obtained by preparative TLC; (B) an overlapping > spectre of the (red) di-glycolipid standard reference compound (Avanti) and (violet) the total AcP-type sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the highest E24 value (59 ± 1.37%, determined for the sample EXNA at S5 sampling points) in incubations on the water-immiscible HC-substrate. The EXA type-material exhibited the poorest emulsification activity (29 ± 0.53%). The AcP type sample, despite of being only partially water-soluble, generated E24-index of 49 ± 0.44%. The values reported here are quite average when compared to otherwise reported emulsification index values (Vasconcellos et al., 2011). A time-series investigation of the development of the emulsification ability, E24, for the sampling points S1-S6 indicated that the release of the surface-active metabolite mixture should have occurred between S2 and S3. Though the detectable amounts of biosurfactants (by the TLC method) were identified only at the sampling point, S3, a slight emulsification was observed already for S2 (a 5 mm high emulsion layer, E24 6.3 ± 0.40%) and at S3, where a stable well-defined emulsion layer (the height 44 mm, E24 23.4 ± 0.5%) was formed.

amounts of methanol and ethanol. The results of the oil-spreading test indicated some surface activity of the total AcP-type material dissolved in water without any solvents being added, Fig. 11. The shape of the clear zone was highly asymmetrical with clear channels radiating from the clear zone. Thus, the determination of the exact area of the clear zone was impossible. Qualitatively the area of the clear zone increased from S3 and continued to increase through S4 and S5 sampling points. However, the relative area of the S6 clear zone was comparable to that of S3. The oilspreading test of the individual glycolipid isolates obtained by the preparative TLC showed smaller but well-defined clear areas. No inhibition of the emulsification activity by heat treatment was observed. The samples showed stable oil-spreading performance in the range pH 2–9. The results of the surface tension tests supported the results of the oil spreading test, Fig. 12. The decrease of the surface tension was not large, and the lowest value determined was 46.9 mN/m for EXNA-type samples. The partially dissolved AcP type-material produced a SFT value of 56.2 mN/m. E24-index test indicated a relatively strong emulsification ability of the material secreted into the medium. It seems that the lipid metabolites secreted into the medium had an additive emulsification ability. That is, only samples containing all the species, EXNA, were able to produce a reasonable emulsification effect, which was manifested by

4. Discussion Three glycolipid-reagent positive spots were identified by 1D TLC in the material secreted into the media. Two of these were tentatively identified as acidic glycolipid species with trehalose-disaccharide as the hydrophilic moiety. The pattern of elution and the Rf values suggested these two compounds are anionic ester forms of trehalose-glycolipids. The third glycolipid-reagent positive spot corresponded to a non39

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probably was a structural part of all the four congeners, while the second constituent was represented by a range of shorter fatty acid residues thus giving rise to several co-eluted congeners. This fatty acid residue was not identified in the AcP type samples, that is, it was a constituent of non-ionisable secreted metabolites. The investigation of the dynamics of the release shows that the surface-active metabolites were released already between S2 and S3, since the glycolipid metabolites were detected only at the sampling point S3 but the surface activity was observed for material sampled at S2. Probably, the biosurfactant mixture started to be secreted around S2, but at very low concentrations undetectable for the TLC chromatographic method. The partial solubility in the distilled water of the extracted glycolipid species prevented the unambiguous determination of CMC, while for the complex extracts EXNA and EXA-type samples the results were not reproducible − probably either due to the presence of some unidentified macrostructures or due to the complex composition of the extracts. Regarding the hydrophobic moiety of the detected surface-active compounds, the range of the fatty acids was too wide to be related to the narrow range of the glycolipids secreted and identified by TLC. The upper part of the chromatograms displayed in Figs. 6 and 7, suggests that some FA were released as free species along with mycolic acids. These lipids can be either a part of the overall mechanism to survive and thrive at the interface of water and the toxic hydrocarbon substrate or just a consequence of the general metabolic activity. A surprising aspect of the dynamics of the BS secretion by Dietzia sp. A14101 is that the amounts of trehalose-containing acidic species detected in the media were relatively constant and, thus, were not associated with growth. On the other side, the tentatively identified di-corynomycolate mixture of four congeners seemed to be growth associated. The E24 values reported here are average compared to other strains. This contrasts the results of the hydrophobicity test (determined to be 66%, which is quite high if compared to the reported values otherwise) of the intact cells Dietzia sp. A14101 by BATH (n-C16) reported previously (Hvidsten et al., 2015). Such discrepancy can be probably explained by assuming that most of the synthesised surface active compounds, both in terms of amounts and the diversity, continue to be cell-associated even as the bacterial culture entered the advanced stationary growth phase, S6, while only small amounts and a modest diversity range of biosurfactants are released into the media as confirmed by global LS–MS profiling. The discrepancy between the BATH test results for intact cells reported earlier and the E24 test results for secreted into the media material reported in this paper, probably exemplifies the phenomenon when emulsions can be stabilised in the presence of a particulate matter. From this perspective, the cellular membrane of Dietzia sp. A14101 can contain inclusions or sites with a high distribution density of amphiphilic glycolipids. Thus, the whole intact cell can be perceived as a kind of particulate matter able to produce a stabilizing effect on

Fig. 11. An example of the clear zone formed, crude cell-free medium I-6 through the sampling points S-1-S6. 0 = freshly prepared D20 medium.

trehalose sugar-containing corynomycolate lipid structure. The spot had an elongated shape and consisted of 4 isomers/congeners as confirmed by the selected 2D TLC method. The extraction method using precipitation with an acid produced intense spots suggests that both the two trehalose secreted lipid and non-trehalose sugar-containing dicorynomycolate were secreted into and existed in the medium in their acidic forms. Rapp and Gabriel-Jürgens (2003) reported that trehalose mycolates and their derivatives are frequently non-ionic, however trehalose-containing lipids can be synthesised as anionic tetraesters by some strains. The hydrophobic moiety of trehalose-containing species was similar to that of the trehalose-containing glycolipids previously identified in the cellular material of Dietzia sp. A14101 and consisted of a monosaturated C20:1n-7 and a shorter saturated chain of 16 carbon atom and the isomer of this glycolipid where C16:0 was substituted with either C12:0 or C14:0 fatty acid residues. The mixture of non-trehalose glycolipid isomers, tentatively identified as sugar-containing di-corynomycolates, seemed to be more diversified in terms of the constituents of their hydrophobic moiety. The results suggested that the residue of C 25:0 fatty acid dominated the profile and is thus most

Fig. 12. Surface Tension in Pure D20 medium (—, solid line), crude cell free medium (- - -, dotted line), medium after removal of AcP material ….., hatched line).

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Fig. 13. (A) The appearance of the batch culture of Dietzia sp. A14101 at the water-hydrocarbon interface prior to sampling, the incubation I-5; (B) a cluster of cells I-6S3; (C) an enlarged fragment of the cluster in (B)-photo. The brightness of Fig. 13(A) was enhanced in order to show the tight colonisation by Dietzia sp. A14101 of the interface between the aqueous and the water-insoluble phases.

microemulsions at interfaces of the aqueous media – water-immiscible hydrocarbons. The fact that the Dietzia sp. A14101 culture was not more or less evenly distributed through the aqueous phase, which would be the case if a sufficient emulsification of the water-insoluble hydrocarbon substrate at a macro-level was created, but rather resided as a relatively tight compact amorphous formation/body at the interface between the two phases, Fig. 13(A), can also serve as additional qualitative evidence that the biosurfactant mixture produced is mostly cell-bound. Fig. 13(B) that depicts a cluster of cells of Dietzia sp. A14101 grown on HC and the magnification of a cluster section shown at Fig. 13(C), illustrate a halo of microemulsion in the immediate proximity of the cell. The observations outlined here agree well with hypothesis by Rosenberg and Ron (1999). They suggested that the emulsification caused by the bioactivity of the prokaryotic organisms is a “cell-densitydependent” phenomenon. Thus, the laboratory batch-based investigations of pure bacterial cultures achieve highly dense population in a relatively short strain specific time interval and do often exhibit extraordinary emulsification ability on a macro-level. However, Rosenberg and Ron (1999) have pointed out that such dense populations are rather atypical in the real open systems which get naturally diluted (due to i.e. the continuous flow of ground water, precipitation, or the flow of reservoir fluids). Therefore, formulated in a simplistic way, it is more advantageous for the bacteria to hold on its biosurfactant, and let it work for the given cell by creating small micro-droplets with food rather than using energy to produce the valuable compounds. Which, on being released, could and probably would “run away” from the bacterial cell with the water flow, dragging away the emulsified, that is pseudo-water-solubilized, “food-substrate”. That scenario would probably lead to the death of the bacterium due to starvation. The results outlined in this paper suggest that virtually most of the BS produced by Dietzia sp. A14101 remains cell-bound. The results of the SFT analysis were not promising for the production of biosurfactants that can be harvested and applied as isolates in EOR as the decrease of the surface tension was too small, Fig. 12, while the generally accepted SFT value required for the applications in EOR should at least 30 mN/m or lower. A similar poor effect on SFT, 43 mN/m, was previously reported for a mixture of trehalose lipids produced by Rhodococcus fascians (Yakimov et al., 1999). However, the previously reported by Kowalewski et al. (2005) IFT values for Dietzia sp. A14101 were one of the lowest IFT values ever reported for known biological systems peer publishing date. These findings, together with the results described here, suggest that it is rather the whole microorganism should be used in EOR applications. The fact that Dietzia sp. A14101 was isolated from an oil reservoir model column incubated with the sample of the real reservoir biota (Bødtker et al., 2009) agrees with the train of arguments from BS-specialised research groups quoted above and our results. That is, the bacterium

can survive and thrive on water-immiscible hydrocarbon substrates in a diluted environment of the subsurface if it preserves BS closely associated with the cellular membrane, albeit with rather low water flow rates. But the main concern regarding the usefulness of the isolated Dietzia sp. A14101 is that the strain is mesophilic and thrives at neutral pH. This is probably a limiting factor when considering Dietzia sp. A14101 for MEOR applications in the reservoirs with high temperature regime and/or salinity. However, MEOR is a tertiary methodology and as such is applied to well-developed field. This means that the original saline aqueous phase and/or brines associated with the hydrocarbon phase in the reservoir have already been diluted due to the application of the secondary EOR methods (e.g. water-, steam-injection) with simultaneous decrease of the temperature. It is possible that such diluted aqueous phase and the temperature regime can support mesophilic bacterial strains. In respect to bioremediation, Dietzia sp. A14101, being a potent degrader of a wide range of hydrocarbon compounds, should be definitely considered as member of bacterial consortia in bioremediation projects.

5. Conclusions and prospects The following conclusion can be made:

• Diezia sp. A14101 did secreted surface active compounds of glycolipid nature into the medium; • The range of the released BS was narrow with regard to chemical activity; • The two acidic trehalose-containing glycolipids were not growth • •

associated, neither were the free fatty acids and pigments; while the secretion of one non-ionic trehalo-lipid and a mixture of four dicorynomycolate congeners were growth associated; The secreted mixture did not exhibit a pronounced effect on SFT, but showed some moderate emulsification properties. The results reported here together with previously reported assessment of the cellular material suggest that for the large industrial applications the presence of intact active cells of this strain is necessary.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.2017.08. 007.

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