The brominated aromatic compounds have been procured from Sisco Research Laboratories, India and Himedia Chemicals, India, while the solvents and media components were procured from Merck India, Ltd. All the chemicals were of AR grade, with an approximate 98% purity.

Two strains of Y. lipolytica, NCIM 3589 and NCIM 3590 were used in this study. Stock cultures of the strains were maintained on MGYP plates (%, w/v) (0.3, malt extract; 1.0, glucose; 0.3, yeast extract; 0.5, peptone; 2.5, agar) and the yeast has been sub-cultured every month.

Flasks containing liquid minimal medium (MMY, %, pH 7.4) [0.54, Na₂HPO₄.12H₂O; 0.14, KH₂PO₄; 0.05, (NH₄)₂SO₄; 0.02, MgSO₄. 7H₂O; 0.001, yeast extract; 0.5% (v/v) salts solution and 0.5% (v/v) filter-sterilized vitamin solution] (Janssen et al., 1984; Vatsal et al., 2015) were used. For growth kinetics, Y. lipolytica NCIM 3589 cells (2 × 10⁹) were inoculated in 250 mL bottles containing MMY medium (50 mL) along with varying BrB concentrations (0.1–2.0%, v/v; 9.5–190 mM) and were sealed with Teflon-lined screw caps. These cells were allowed to grow aerobically at 30°C with a shaking speed of 150 rpm for a time period of 72 h. After growth, cells were centrifuged; pellet was washed with autoclaved distilled water, vortexed for separating the cells and absorbance measured at A₆₀₀, wherein 1 OD was equivalent to 2 × 10⁹ cells/mL (Vatsal et al., 2011). The growth kinetic parameters were determined based on the relationship between the initial concentration of BrB and the specific growth rate and calculated by non-linear regression using Microcal Origin 6.0.

To investigate the effect of BrB concentration on growth of the cells, batch experiments were carried out. Initially, the yeast cells were grown on 1% MMYG [MMY containing 1% (w/v) of glucose] for a time period of 24 h to obtain biomass. The yeast cells were centrifuged at 4°C for 10 min at a speed of 7000 rpm. Then, the pellet was washed three times with sterile distilled water, vortexed, and absorbance measured at 600 nm. The cells were then collected and dried at 60°C to obtain a stable cell dry weight, which was estimated and a calibration curve for the optical density vs. dry weight plotted. 1 OD cells were inoculated with MMY medium (50 mL) in 250-mL Erlenmeyer flasks, containing BrB as the sole carbon source at a 0.5% v/v (47.6 mM) concentration till 96 h. Thereafter, the inoculated flasks were sealed for decreasing the abiotic loss of the volatile compounds. At a time interval of 24 h, one flask was collected and the cells were centrifuged at 7000 rpm at 4°C for 10 min. The cell free supernatant (CFS) obtained was used for estimating bromide ions and dehalogenase activity as described below. The residual concentration of BrB was determined by collecting the sample at the end of every time interval and the cell-free broth extracted as mentioned below. For determining the cell growth, the cells were vortexed for separating them from the hydrophobic droplets of BrB, then centrifuged and the pellet obtained was washed thrice with distilled water (sterile) and dried, and the dry weight was determined (Vatsal et al., 2011).

The cell growth on the metabolites of phenol and catechol (0.5, 1, 2.5, and 5 mM) in the MMY media was also determined after growing the yeast cells in aerobic conditions for 48 h and then dry weight determined.

For light microscopy studies, wet mounts of the washed yeast cells were prepared and carried out using a Zeiss microscope (Axioskope A1) with a camera and images were acquired, as described in Vatsal et al. (2011). The surface area was determined as πd² after assuming that the yeast cells were spherical. Though it is recognized that the individual yeast cells are not exactly spherical, the estimated surface area provides a relative index if there has been a change in the cell surface area after growing them on BrB.

The sedimentation velocity of the yeast cells was estimated using the Stokes law:

where, D is the cell diameter, g (gravity) is 9.807 m/s², Δρ (cell density minus water density) is 100 kg/m³ and η (viscosity of water) is 9.04 × 10^-4 kg/ms (Mortensen et al., 2005).

The yeast cell surface properties were measured with the help of the microbial adhesion to solvents (MATS) test and the contact angle measurements (Vatsal et al., 2011). MATS compares the microbial cell affinity to the polar and non-polar solvents (Bellon-Fontaine et al., 1996). The polar solvents can act as electron acceptors or as electron donor. For this, both solvents must possess similar components of the van der Waals surface tension. In our study, the electron donor character (EDC) was measured after determining the cell adhesion to chloroform, which is an electron acceptor solvent, and hexadecane, which is a non-polar solvent; while, the electron acceptor character (EAC) represents the cell adhesion to diethyl ether, which is a good electron donor solvent, and hexane, i.e., non-polar solvent (Bellon-Fontaine et al., 1996; Vatsal et al., 2011). Due to similar van der Waals properties displayed by these solvent pairs, the difference between the values obtained with chloroform and hexadecane and the values measured using diethyl ether and decane indicated the electron donor–electron acceptor interactions occurring at the yeast cell surface and reveals its hydrophilic and hydrophobic properties. The solvent affinity (i.e., percent adherence to every solvent), could be estimated as the proportion of cells which were suspended in these solvents in comparison to the initial cell concentration, with the help of the following equation:

where, A₀ and A refer to the A₅₇₀ values of the yeast cell suspension prior and after mixing with the solvent, respectively. The cell surface hydrophobicity (CSH) can be expressed as cells (%) adhered to hexadecane. For eachsolvent, affinity measurements were carried out in triplicates. All solvents were of AR grade and 99% pure according to the manufacturer.

Determination of water contact angle values was carried out with the sessile drop technique with the help of the DigiDrop (GBX, Surface Science technology, France). For this experiment, the cells grown for 72 h on 0.5% (v/v, 47.6 mM) BrB and glucose were mounted on the slides and the surface adhesion energy was determined with the help of the Young’s equation:

where, S is the surface energy, γ_l the surface tension of the liquid and 𝜃 is the contact angle (de Gennes, 1985).

Furthermore, the CFS obtained after growing the cells on BrB (0.5%, v/v; 47.6 mM) and glucose (0.5%, w/v) for 72 h was used for determining the surface tension and the emulsification activity by the method described earlier (Vatsal et al., 2011). The absorbance was measured at 570 nm of the aqueous phase and the absorbance value of 1.0 was defined as 1 U of emulsifying activity (EU/mL).

Concentrations of residual BrB were determined quantitatively by capillary gas chromatography (GC). The CFS collected (20 mL) at the end of every time point (0–96 h) was extracted with 2 volumes of diethyl ether, the sample was concentrated and then analyzed by capillary GC. 1 μl sample (split injection ration; 30:1) was injected on the RTX-5 column (30 m × 0.25 mm × 0.25 μm, Restek, United States), on a Shimadzu 2014 GC (Shimadzu, Japan) with a flame ionization detector, with nitrogen as the carrier gas. The initial temperature of 40°C, was held for 5 min, and then increased to 220°C at the rate of 10°C/min and then held further for 1 min. We also plotted calibration graphs for different BrB concentrations and the residual BrB levels ascertained. The retention time for standard BrB compound was 11.16 min, with the peak being well-resolved under the above conditions. The percent BrB degraded was determined as:

where C_control is the concentration of BrB in the media flasks without yeast (i.e., abiotic controls), while C_sample is the concentration of BrB in the media flasks with the culture.

For BrB, the degradation rate followed the first order growth kinetics and was determined as follows:

where, C is the final BrB concentration, C₀ is the initial BrB concentration, T is the time (per day) while the degradation rate constant can be represented by k (Okpokwasili and Nweke, 2005).

In this study, the CFS obtained after growing the cells on 0.5% BrB (47.6 mM), as stated above, was used for determining the bromide ions and the dehalogenase activity. Liberation of bromide at 460 nm was measured colorimetrically according to the method described by Iwasaki et al. (1952). Dehalogenase activity was determined by the method by Jesenska et al. (2000) with minor changes. The CFS (100 μL) was incubated with the substrate, i.e., BrB (5 mM) in 50 mM Na-acetate buffer (pH 4.5, 1.8 mL) at 30°C for 30 min and terminated by adding 25 μL of 3 M H₂SO₄. One unit (U) of dehalogenase activity could be defined as the amount of enzyme that catalyzed the formation of 1 nmole of bromide per min (Vatsal et al., 2015). All experiments were repeated thrice and the values expressed here are the mean of the three experiments.

Furthermore, intracellular catechol 1,2 dioxygenase and the catechol 2,3 dioxygenase activities were measured according to Hupert-Kocurek et al. (2012).

For detection of the metabolites formed due to BrB degradation, initially the glucose-grown Y. lipolytica cells were harvested after 24 h of growth. Thereafter, the cells were centrifuged to collect the cell pellet (8000 × g at 4°C for 10 min). This pellet was then re-inoculated in 100 mL flasks containing 20 mL of MMY, with 0.5% BrB (47.6 mM) as the substrate. Control flasks containing MMY and 0.5% BrB and no cells were also included. These flasks were then incubated for 48 h; the CFS was collected, concentrated with lyophilisation and then subjected to HPLC. The HPLC analysis was carried out on a C18 column, namely, Poroshell 120 EC (50 mm × 4.6 mm × 2.7 μm) connected to 1260-Quat pumps and a1260 VWD (1260 Infinity, Agilent Technologies, Germany) at 254 nm. Compounds and standards were eluted at the flow rate of 0.7 mL/min with a mobile phase of water–acetic acid (99.5:0.5%, v/v) as solvent A and solvent B as solvent A: acetonitrile (20:80%, v/v).

LCMS was carried out on a Thermo Scientific Q Exactive^TM quadrupole-Orbitrap mass spectrometer associated with Accela 1250 pump and Accela open AS. The samples were loaded on a Thermo Scientific Hypersil Gold column (50 mm × 2.1 mm) with a particle size of 1.9 mm. MS and MS/MS experiments were carried out in an ESI-negative ion mode with the following method: sheath gas flow rate 45, auxiliary gas flow rate 10, sweep gas flow rate 2, spray voltage (|KV|) 3.60, spray current (μA) 3.70, capillary temperature (°C) 320, s-lens RF level 50, heater temperature (°C) 350. The ESI–MS data was recorded in a full scan mode for the mass range, m/z of 66–1000. The samples were diluted 1:20 and 2 μL samples were injected. The solvents used were methanol and water with 0.1% formic acid at the flow rate of 0.3 mL/min.

The presence of phenol was also confirmed by extracting 1 mL of the sample with a mixture of acetic acid and dichloromethane (1:10, v/v) and GCMS analysis done on a Trace-Ultra GC (Thermo Scientific, United States) with a DB-5 column (30 m × 0.25 mm × 0.25 μm) (Agilent Technologies, United States) and a mass detector (ITQ 1100, Thermo Scientific, United States) with helium as the carrier gas. The column temperature at 60–160°C was increased at the rate of 15°C/min to 200°C at the rate of 3°C/min and held for 1 min. The peaks were identified based on both the m/z values and comparison of retention times with standards.

For detecting carbon dioxide, cells were inoculated in sealed serum bottles (60 mL) containing 15 mL MMY with BrB and incubated at 30°C for 120 h at 150 rpm. The headspace in the bottles (inoculated and uninoculated control) was checked for CO₂ using a GC (Chemito 3800, India) equipped with thermal conductivity detector (TCD) and packed Porapak Q column (stainless steel, 80/100 mesh, 6 feet × 1/8 inch, Sigma-Aldrich, United States) with argon as the carrier gas (flow rate of 40 mL/min). The temperature of the column was 40°C, with the injector and detector temperatures at 70 and 100°C, respectively (Bhadbhade et al., 2002). As a reference standard, carbon dioxide from the gas cylinder containing 80:20 hydrogen:carbon dioxide (mixed gas cylinder, BOC, India) was used.

For confirming that the phenol (Metabolite 1) formed was as a result of the hydrolytic dehalogenation reaction, experiments using labeled water (H₂O¹⁸) were carried out. For this, resting cells as well as CFS (100 μL) from above as enzyme source were incubated with BrB (5 mM) in 100 mM Na-acetate buffer (pH 4.5, 1 ml) and an equal volume of labeled water (H₂O¹⁸) at 30°C for 2 h. The samples were then centrifuged and supernatant subjected to UPLC–MS or extracted and subjected to GCMS as mentioned above.

LCMS was carried out on a Waters XEVO^TM TQD model (Waters, United States) associated with the quadrupole analysers (MS2). The standard and samples were diluted with methanol (1:1) and 10 μL injected on a C18 column of dimensions 100 mm × 2.1 mm × 1.7 μm (Acquity, Waters, United States). Compounds and standards were eluted at the flow rate of 0.7 mL/min, where the mobile phase was water–acetic acid (99.5:0.5%, v/v) as solvent A and solvent B was solvent A: acetonitrile (20:80%, v/v) as mentioned above. MS/MS experiments were carried out in an ESI-positive ion mode using the cone voltage 5 V and collision energy of 3 V. ESI–MS data were recorded in full scan mode. For GCMS, analysis was carried out on a GC 7890 with a HP5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, United States) and MS 7000 (Agilent technologies, United States) as mass detector. All other conditions were similar to those mentioned above.

All the experiments have been carried out in three separate sets where every set has been replicated thrice. The data is expressed as average ± SEM (standard error of mean). A statistical analysis technique of ANOVA (analysis of variance) was also used along with the F-test with the help of Mathstat. All values for p that were ≤ 0.05 were considered to be significant.

To study the degradation and growth on brominated aromatics, two marine strains of Y. lipolytica NCIM 3589 and 3590 were selected. Y. lipolytica 3589 is a tropical marine strain isolated from the oil polluted regions off Mumbai high with an optimal growth temperature of 30°C while Y. lipolytica 3590 (NCYC 789) is a psychotropic marine isolate obtained from Scottish seawaters and has an optimal growth temperature of 20°C (Zinjarde and Pant, 2002). As seen from Table 1, Y. lipolytica 3589 could grow to differing degrees on MMY containing BrB, 1,2 and 1,3-dibromobenzene and 1,3,5-tribromobenzene with maximal growth on BrB (1.47 g/L). Growth was low on1,2-dibromobenzene, 1,3-dibromobenzeneand 1,3,5-tribromobenzene at 0.1 g/L in 72 h. No growth was seen on 1,4-dibromobenzene and epibromohydrin. Y. lipolytica 3590 exhibited a low growth on BrB (0.1 g/L) but failed to grow on any of the other di- or tri-bromobenzenes in 72 h. Thus, it is to be noted that an increase in a number of bromide groups, as well as their position on the ring molecule, affected growth of the yeast. Based on the above studies, Y. lipolytica 3589 was selected for further studies. Both these strains have been maintained in the laboratory for over 2 years.

To ascertain the probable route in the utilization of the BrB, studies with Y. lipolytica were performed where the cells were grown on 0.5% (v/v, 47.6 mM) of BrB. The increase in cell mass resulted in the decrease of the BrB concentration with a simultaneous formation of biomass and release of inorganic bromide ions.

The time course of cells grown on 0.5% v/v (7.48 g/L, 47.6 mM) BrB concentration is shown in Figure 3. Growth, as seen by an increase in cell dry weight, resulted in the decrease in concentration of BrB and a concomitant release of bromide in the medium. A maximum cell mass of 1.86 g/L was seen within 48 h after which there was a decline in the cell growth. The concentration of bromide released in the medium was determined to be 12.87 mM in 24 h which increased to 15.89 mM in 96 h. The residual BrB concentration was seen to decrease from the initial concentration of 47.6 mM to 31.3 mM at the end of 96 h, i.e., attaining a degradation of 34% with a degradation rate of 0.106 per day. Very less BrB degradation was noted in the abiotic controls lacking yeast cells. The abiotic BrB degradation ranged between 0.3 and 4.1 mM for the time points ranging between 0 and 96 h. The values for the biotic degradation, reported here, refer to the values taken into account after deducting the abiotic degradation for every time point.

A metabolite, which we have identified as phenol (see below) was detected in the media with a maximal concentration of 14.34 mM at 24 h. This metabolite declined thereafter to 2.51 mM in 72 h (Figure 3). Thus, the metabolite was being formed as an intermediate during degradation of BrB. The increase in the metabolite concentration in the culture broth coincided with the decrease in BrB concentration, increase in bromide release and higher dehalogenase activity.

To identify the intermediates or metabolites formed as a consequence of degradation of BrB, studies were carried out under the conditions mentioned in the experimental procedures and samples analyzed by HPLC, LC–MS, and GC–MS. During BrB degradation by the yeast cells, four metabolites (I to IV) appeared in the culture medium in 48 h (Figure 4A). Metabolite I, which co-eluted with the authentic phenol standard, was detected with the help of the HPLC (RT = 6.5 min) (Figure 4A) and GC (RT = 6.19 min). GC–MS analysis showed that it had a molecular mass of m/z = 94.38 and exhibited fragments at the m/z 94, 80, 79.6, 47 (Figure 4B) similar to that of standard phenol. Furthermore, we also identified the Metabolite II as catechol based on the co-chromatography using an authentic catechol standard by HPLC (RT = 3.83 min), and obtained a negative-mode mass spectra of m/z = 109.03 (M^-H⁺), with fragments at m/z 54.6, 65, 81, 91, 99, 109 (Figure 4C). Metabolite III, m/z = 127.1 (M^-H⁺) could not be identified. Metabolite IV was identified as cis, cis muconic acid by mass spectrometry with a m/z = 141.01 (M^-H⁺) and fragments at m/z 75, 79, 85, 93, 97, 115, 119, 128, 141 (Figure 4D).

Studies with labeled water (H₂O¹⁸) showed the presence of a single peak which eluted at RT = 0.99 min (Figure 5A) on LCMS in a positive mode. The standard phenol (m/z = 97) eluted at 1.07 min (Figure 5A) with m/z of daughter ions at 65, 47, and 33. The MRM scan of the test sample with labeled water exhibited two m/z values of 97 and 99 with a difference of 2 mass units (Figure 5B). MS–MS analysis of the peak with m/z = 97 exhibited daughter ions at m/z = 65, 47, and 33 (Figure 5B), similar to that of standard phenol. The peak with m/z = 99 was unable to ionize well under the conditions used and a proper fragmentation profile could not be obtained. The LCMS analysis thereby suggests that incorporation of O¹⁸ from H₂O¹⁸ into phenol is likely. To further confirm the same, analysis was also carried out using GCMS. A single peak eluted at RT = 4.712 min and no other peak could be detected under the experimental conditions used (Figure 5C). Ion fragmentation pattern of the peak showed the presence of fragments at m/z = 95.2, 94.2, 79.1, 77, 76.1, 75.3, 67.8, 66, 65, 63.7, 63, 55, 53.1, 52, 51.2, 50.5, 49.9, 41.9, 40.1 (Figure 5C, inset). These fragment ion pattern was similar to that of phenol formed from oxygen incorporated from H₂O while some ion fragments originated from H₂O¹⁸, suggesting the incorporation of O¹⁸ into phenol. Thus, the analysis by both LC–MS and GC–MS suggests that incorporation of O¹⁸ from the labeled water occurs into phenol by a hydrolytic mechanism.

After analyzing the gas in the headspace of the bottles, we observed that CO₂ could be detected at the Retention Time (RT) of 2.55 min (GC area = 15265.45) and it was co-eluted with the CO₂ standard. No significant carbon dioxide was detected in the uninoculated control. The presence of the carbon dioxide in the headspace showed that the BrB could be mineralized by the yeast. Thus, from the above studies, as the initial product formed was via an initial dehalogenation step, the CFS was tested for dehalogenase activity, if any.

The release of bromide and presence of phenol as an initial product occurring during cell growth in the above experiments indicates an attack on the ring carbon to which the bromide is attached and suggests that the bromide is being released by a dehalogenation reaction. The CFC collected after the growth of Y. lipolytica cells on 0.5% (v/v; 47.6 mM) of BrB was tested for the presence of the extra-cellular debrominating or dehalogenase activity of Y. lipolytica.

Maximal extracellular dehalogenase activity of 16.16 U/mL was noted on 0.5% (v/v) of BrB (47.6 mM) after 24 h; which decreased thereafter to 8.4 U/mL at the end of 72 h (Figure 3).

No extracellular dehalogenase activity could be detected when glucose was used as the carbon source for growth of the yeast. Similarly, no enzyme activity could be detected in the CFS of cells that were grown on the corresponding products such as phenol or catechol. This suggested that the extracellular dehalogenase enzyme from Y. lipolytica was induced in the presence of brominated compounds. A similar observation has been reported when the yeast was grown on bromoalkanes (Vatsal et al., 2015).

The catechol 1,2-dioxygenase and thcatechol 2,3-dioxygenase activities were determined and it was seen that the catechol 1,2-dioxygenase activity was 39.91 nmol/min/mg protein while the catechol 2, 3 dioxygenase activity was much lower at 1.3 nmol/min/mg protein. A higher catechol 1,2 dioxygenase activity provides further evidence that it is a likely route for catechol (Metabolite II) to be degraded via the ortho-cleavage pathway yielding cis, cis muconic acid (Metabolite III).

The tropical marine yeast Y. lipolytica NCIM 3589 was able to grow well on bromobenzene, an environmentally hazardous compound. Further, it was noted that an increase in a number of the substituent bromide groups, as well as their position on the benzene ring structure, affected the growth of the yeast on other bromoaromatics to differing degrees. The fate and the persistence of several water insoluble aromatic compounds are seen to be dependent on the capability of microbes to use and degrade them. Microbes can either solubilize these hydrophobic compounds using certain surface-active compounds or they could attach to them after altering some of their cell surface properties. Both these phenomena are crucial in understanding the biodegradation and utilization of hydrocarbons by cells (Rosenberg et al., 1980). In the case of Y. lipolytica, there has been evidence for both these mechanisms for the hydrophobic compounds (Fickers et al., 2005; Vatsal et al., 2011). To understand the probable mechanism by which Y. lipolytica 3589 would be utilizing BrB, both the hypotheses were tested.

When the Y. lipolytica cells were grown on 0.5% BrB (47.6 mM), the yeast cells were seen to adhere to the hydrophobic BrB droplet within a short time period (Figure 2). We have earlier reported such a phenomenon by the same yeast for n-dodecane (Bankar et al., 2009) and bromoalkanes (Vatsal et al., 2011). This fact was significant as it suggested that the bioavailability of the brominated substrate, i.e., BrB would occur due to the attachment of the cells to the hydrophobic droplets. We also observed that there was an increase in the surface area of the cells grown on BrB as compared to those on glucose. Hence, it could be possible that an increased surface area of the cells would lead to a higher attachment of the yeast cells to the hydrophobic BrB droplets; thus, improving its utilization and subsequent debromination. The cells grown on BrB had a higher ESV value as compared to cells grown on glucose. In fact, it was seen that an increasing cell size was directly related to the increased adhesion rate in the yeast Debaryomyces hansenii, where a larger cell diameter and a large ESV value resulted in a high adherence of the cells to the substrate (Mortensen et al., 2005). Hence, it could be said that such a similar correlation could exist between the cell size, which led to a high surface area, large ESV and a higher adherence of the Y. lipolytica 3589 cells to the hydrophobic substrate, i.e., BrB.

Furthermore, we also determined that there was no change in the surface tension and the emulsification activity, when the yeast cells were grown on the substrate. Thus, a decrease in the surface tension or higher emulsification activity due to production of the extracellular emulsifier is not the strategy employed by Y. lipolytica NCIM 3589 for utilizing BrB. Also, we studied the effect of the substrate on the CSH of the Y. lipolytica3589 cells with the help of the MATS test and the contact angle measurements. The yeast cells also showed a high CSH when grown on BrB and glucose. We have earlier shown that the strain itself possesses adhering capacity to the hydrophobic substrates (Bankar et al., 2009; Vatsal et al., 2011). Our results were similar to those noted for Y. lipolytica 180 where the cells that were grown on glucose and crude oil showed a CSH more than 90% (Kim et al., 1999). Also, the MATS analysis carried out on Y. lipolytica IMUFRJ 50682 showed a higher cell adhesion to the hydrophobic compounds (Amaral et al., 2006). The yeast cells grown on BrB exhibited an increase in EDC suggesting that the cell surface of the Y. lipolytica 3589 cells display an overall electron donating character. Our results based on the interactions between the cells and BrB show that when Y. lipolytica cells were grown on BrB there was no further increase in the CSH, as the cells displayed an inherent hydrophobic surface. However, a small increase in its EDC was noted which suggests that adsorption of the yeast cells to the BrB drop is also due to its electron donating capacity apart from the hydrophobic interactions. Similarly, Y. lipolytica W29 cell surface was determined by the MATS test to have much less hydrophobic character while possessing a higher EDC Aguedo et al. (2003).

Based on the light microscopy and cell surface properties of Y. lipolytica, when grown on BrB, we conclude that the overall increase in cell size, high CSH and EDC character of cell surface led to an increase in adherence to the BrB droplets enabling its degradation and utilization.