This is the accepted version of the following article: Villagrasa E., Ballesteros B., Obiol A., Millach L., Esteve I., Solé A.. Multi-approach analysis to assess the chromium(III) immobilization by Ochrobactrum anthropi DE2010. Chemosphere, (2020). 238. 124663: - . 10.1016/j.chemosphere.2019.124663, which has been published in final form at https://dx.doi.org/10.1016/j.chemosphere.2019.124663 © https://dx.doi.org/10.1016/j.chemosphere.2019.124663. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 1 Title: Multi-approach analysis to assess the chromium(III) immobilization 2 by Ochrobactrum anthropi DE2010 3 4 Abstract 5 Ochrobactrum anthropi DE2010 is a microorganism isolated from Ebro 6 Delta microbial mats and able to resist high doses of chromium(III) due to its 7 capacity to tolerate, absorb and accumulate this metal. The effect of this 8 pollutant on O. anthropi DE2010 has been studied assessing changes in 9 viability and biomass, sorption yields and removal efficiencies. Furthermore, 10 and for the first time, its capacity for immobilizing Cr(III) from culture media was 11 tested by a combination of High Angle Annular Dark Field (HAADF) Scanning 12 Transmission Electron Microscopy (STEM) imaging coupled to Energy 13 Dispersive X-ray spectroscopy (EDX). 14 The results showed that O. anthropi DE2010 was grown optimally at 0-2 15 mM Cr(III). On the other hand, from 2 to 10 mM Cr(III) microbial plate counts, 16 growth rates, cell viability, and biomass decreased while extracellular polymeric 17 substances (EPS) production increases. Furthermore, this bacterium had a 18 great ability to remove Cr(III) at 10 mM (q= 950.00 mg g-1) immobilizing it mostly 19 in bright polyphosphate inclusions and secondarily on the cellular surface at the 20 EPS level. Based on these results, O. anthropi DE2010 could be considered as 21 a potential agent for bioremediation in Cr(III) contaminated environments. 22 Keywords: Ochrobactrum anthropi DE2010; CLSM; ICP-OES; HAADF-STEM 23 EDX; polyphosphate inclusions 24 1. Introduction 1 25 The increase in industrial and anthropogenic activities has largely 26 contributed towards the introduction of heavy metals and other pollutants into 27 the environment (Gupta et al., 2014; Bhattacharya et al., 2015). Pollution by 28 heavy metals has become a serious threat to the environment and public 29 health, since, in general, they are highly toxic and accumulate throughout the 30 food chain (Feng et al., 2012; Xu et al., 2014). 31 Chromium is one of the earth crust’s most abundant elements (Mohana 32 and Pittman, 2006) and it is a redox active 3d transition metal with different 33 oxidation states ranging from 2+ to 6+ (Greenwood and Earnshaw, 1998; 34 Srivastava and Thakur, 2007). The trivalent (Cr(III)) and hexavalent (Cr(VI)) 35 forms, from industrial by-products or by being part of phosphate fertilizers used 36 in agriculture (Nziguheba and Smolders, 2008; Manahan, 2009) among other 37 chromium sources, are stable in the majority of terrestrial surface and aquatic 38 environments (Kimbrough et al., 1999; Ihsanullah et al., 2016). Cr(III) is 39 considered to be an essential element (micronutrient) with known cellular 40 biological functions (Balk et al., 2007; Evert et al., 2013). However, with long- 41 term exposure it can produce toxic effects in the cells, but at a much lower 42 degree than Cr(VI), which is more toxic and unhealthy for living beings and the 43 environment (Shanker et al., 2005; Francisco et al., 2011; Ihsanullah et al., 44 2016). Even so, Cr(III) is involved in some human diseases such as: structural 45 perturbation in erythrocyte membrane and cancer (Kusiak et al., 1993; 46 Suwalsky et al., 2008; Figgit et al., 2010). 47 In the last decade, our research group has isolated different 48 heterotrophic and phototrophic (cyanobacteria and algae) microorganisms from 49 the Ebro Delta microbial mats (Tarragona, Spain). One of these 2 50 microorganisms, Ochrobactrum anthropi DE2010, was isolated from the 51 microalgae Scenedesmus sp. DE2009 consortium from the same ecosystems, 52 and recently was characterized and identified by means of microbiological, 53 biochemical and molecular methods. Furthermore, the analysis of the effect of 54 deprived nitrogen source conditions on this heterotrophic bacterium 55 demonstrate that it is able to overcome this limiting condition through a still 56 unknown nifH–independent mechanism, although there were changes in shape, 57 size, and abundance of pleomorphic cells (Villagrasa et al., 2019). 58 The aims of this work are: (i) to evaluate the cytotoxic effect of chromium 59 and the capacity of Ochrobactrum anthropi DE2010, growing in axenic cultures, 60 to remove it; (ii) to determine changes in total biomass and cellular viability and 61 in composition and production of EPS in cultures exposed to this metal and (iii) 62 to determine the Cr(III) uptake efficiency of Ochrobactrum anthropi DE2010 and 63 its ability to capture chromium(III) extra and/or intracellularly. With this in mind, 64 an analytical multi-approach combining classical microbial plate counts, growth 65 methods, optical microscope techniques, and biochemical and chemical 66 analysis besides high resolution microscopy techniques have been applied. 67 2. Materials and methods 68 2.1 Microorganism, chromium(III) stock solutions and culture conditions 69 O. anthropi DE2010 (accession number DDBJ/ENA/GenBank, 70 KY575285) isolated from a Scenedesmus sp. DE2009 consortium from Ebro 71 Delta microbial mats (Villagrasa et al., 2019) was grown at 27 °C in Luria– 72 Bertani (LB) agar medium, containing tryptone (10.0 g L-1), yeast extract (5.0 g 3 73 L-1), NaCl (10.0 g L-1) and agar (15.0 g L-1), and preserved in Cryoinstant® vials 74 (Thermo Fisher Scientific) at -80 ºC. 75 76 Chromium(III) stock solution was prepared as Cr(NO3)3· 9H2O salt 77 (Sigma-Aldrich, Bellefonte, PA, USA). The 2,600 mg L-1 (50 mM) Cr(III) stock 78 solution was made by dissolving the exact quantities of Cr(NO3)3 in double 79 deionized water and sterilized by filtration through a 0.2 μm filter (Millipore, 80 Merck Millipore). The Cr(III) working concentrations of 0 (control experiment), 81 0.5, 2, 5, 7, and 10 mM (equivalent to 0, 26, 104, 260, 364, and 520 mg L-1, 82 respectively) were obtained by serial dilution. The Cr(III) stock and serial 83 working solutions were prepared just before use and its pH were adjusted at 84 6.5. 85 86 Unpolluted (0 mM) and polluted (0.5, 2, 5, 7 and 10 mM) cultures were 87 prepared at the same conditions in the following manner: 2 mL of a 24 h culture 88 of O. anthropi DE2010 grown in LB (OD600 between 1.4-1.6, approximately 1010 89 cfu mL-1) was inoculated into 18 mL of LB liquid medium with the different 90 tested Cr(III) concentrations. These cultures were used for all experiments and 91 were incubated in an orbital shaker (Infors HT, Ecotron) (150 rpm) at 27 ºC for 92 24 h. Triplicate cultures were grown for each heavy metal concentration. 93 94 2.2 Bacterial plate counts and growth curves 95 To determine the concentration of bacterial cells, 0.1 mL from 24 h 96 cultures grown at different Cr(III) concentrations were spread onto LB agar 97 plates also supplemented with the same final chromium working concentrations: 4 98 0, 0.5, 2, 5, 7, and 10 mM. In addition, in order to evaluate the number of 99 dormant O. anthropi DE2010 cells, 0.1 mL from each 24 h culture polluted with 100 Cr(III) was spread onto new LB agar plates without Cr(III). The plates were 101 incubated at 27 ºC for 2 d in darkness. The viability counts were expressed in 102 cfu/mL, and 8 replicates were performed for each experiment. The dormant cell 103 counts were determined using the following formula (Sachidanandham et al., 104 2009): CC 105 DC  [100  ( x100)] CC  RC 106 where DC is the dormant cells relative ratio; CC is the cultivable cells grown 107 with Cr (III) count (cfu mL-1) and RC is the resurrected cells count (cfu mL-1) and 108 corresponds to the difference between cfu mL-1 obtained without and with Cr 109 (III). 110 111 For growth curve measurements, Ochrobactrum anthropi DE2010 culture 112 was grown in LB medium at 27 °C for 24 h in the dark. After this incubation 113 time, a 96-well microplate was prepared by inoculating 20 μL of the above- 114 mentioned over-night culture in microplate columns 2, 4, 6, 8, 10, and 12 filling 115 all the wells. Working Cr(III) concentrations 0, 0.5, 2, 5, 7, and 10 mM at a final 116 volume of 200 µL were used. Blank media samples were considerate by adding 117 bacteria-free LB medium uncontaminated and contaminated with Cr(III) using 118 the same working concentrations in microplate columns 1, 3, 5, 7, 9, and 11, 119 respectively. OD (λ = 600 nm) was measured every 30 min during 24 h with a 120 microtiter plate photometer (Thermo Scientific Variokan® Flash, Waltham, 121 USA). Before each measurement, microtiter plate was shaken for 5 s at 120 122 rpm. Eight replicates were analyzed for each heavy metal concentration. 5 123 124 2.3 Cell viability and biomass analysis by Confocal Laser Microscopy (CLSM) 125 Biomass and viability of O. anthropi DE2010 cells at different Cr(III) 126 concentrations were determined according to the fluorochromes-confocal laser 127 scanning-image analysis method (FLU-CLSM-IA) described by Puyen et al. 128 (2012a) with some modifications introduced for this particular study. This 129 method combined the use of specific fluorochromes, CLSM, and image analysis 130 using the ImageJ/FIJI v.1.46r software (Schneider et al., 2012). 131 132 For this experiment, 1 mL from each 24 h culture contaminated with a 133 different Cr(III) working concentration was centrifuged at 2000xg for 10 min at 4 134 ºC (Eppendorf 5804R refrigerated centrifuge). Then, the obtained pellets were 135 stained with a mixture of 100 μL of two fluorochromes, SYTOX® Green (5 μM) 136 and Hoechst 33342 (64.9 μM) for 30 min at room temperature in the dark. 137 Subsequently, 25 μL of these stained pellets were smeared onto Polylysine® 138 slides (ThermoFisher Scientific, USA), producing a sticky surface and 139 preventing cell movement, mounted in BacLight mounting oil (ThermoFisher 140 Scientific, USA) and immediately covered and sealed with coverslips (24x24 141 mm). Stained cells were observed with a confocal microscope (Leica TCS SP5; 142 Leica Microsystems CMS GmbH, Mannheim, Germany) using a HCX PL APO 143 lambda blue 63.0x1.40 OIL UV at 2 zoom. In this study, xyz CLSM images were 144 taken in a sequential scan in two channels to distinguish the fluorescence 145 emitted by Hoechst 33342 (414-485 nm) and SYTOX Green (515-580 nm), 146 respectively. In order to differentiate between living and dead cells, they were 147 stained with Hoechst 333429 or SYTOX Green respectively, blue (live cells) and 6 148 green (dead cells) pseudo-colors were used. In each bacterial culture, once the 149 20 blue and green CLSM images (1024x1024 pixels) were acquired, the 150 corresponding CLSM binary images (black/white) were generated by means of 151 the ImageJ/FIJI v.1.46r program. In addition, Voxel counter plugin of this 152 software was applied to assess the cellular viability and biomass at each Cr(III) 153 concentration (Puyen et al., 2012a). 154 155 2.4 Extraction and Biochemical analysis of the total EPS 156 Total EPS of O. anthropi DE2010 from all analyzed cultures were 157 extracted using the physico-chemical method described by Adav and Lee 158 (2008), with some modifications. For this analysis, 20 mL from each 159 contaminated (0.5, 2, 5, 7, and 10 mM Cr(III)), and non-contaminated (control) 160 culture were centrifuged at 2000xg for 10 min at 4 ºC (Eppendorf 5804R 161 refrigerated centrifuge), and pellets were resuspended in 10 mL of sterile 162 double deionized water. Cellular suspensions were mixed with 60 μL of ultra- 163 pure formamide (ThermoFisher Scientific, USA) and incubated on ice for 60 164 min. Then, 4 mL of 1N NaOH was added and the resulting mixtures were 165 incubated for 40 min on ice, and afterward were subjected to ultrasound at 120 166 W for 5 min on ice in an ultrasonic bath (Sonorex, Bandelin). All sonicated 167 suspensions were centrifuged at 10000xg for 20 min at 4 ºC. The supernatants 168 obtained were filtered, to avoid the presence of cells, through 0.22 μm pore size 169 filters (Millipore, Merck Millipore) to collect the total EPS. Aliquots of 5 mL of 170 each of the EPS extracts were stored at -20 ºC until further analysis. 171 Biochemical composition of each EPS extract was determined using various 172 colorimetric methods as follows: total carbohydrate content with the phenol– 7 173 sulphuric acid method (Dubois et al., 1956), using glucose (1 mg mL-1, Merck) 174 as standard; protein content by the Bradford method (Bradford, 1976), with 175 bovine serum albumin (2 mg mL-1, Pierce) as standard, and the uronic acids 176 content by the m-hydroxyphenyl method (Kintner and Van Buren, 1982), using 177 galacturonic acid (1 mg mL-1, Fluka) as standard. The cell lysis caused by EPS 178 extraction was evaluated by quantification of the DNA present in the extracts by 179 the diphenylamine colorimetric method (Burton, 1956) using salmon sperm DNA 180 (1 mg mL-1, Sigma) as standard. Low content of DNA (1-1.2%) indicates that 181 the EPS extracted are not contaminated by intracellular substances (Liu and 182 Fang, 2002). A total of 15 replicates for each EPS extract were analyzed, with 183 the aid of a Beckman Coulter DU® 730 spectrophotometer (Beckman Coulter, 184 Harbor Blvd., Fullerton CA, USA). All calculations were expressed in ppm (parts 185 per million) corresponding to 1 microgram of EPS component (protein, total 186 carbohydrates or uronic acid) per milliliter of total EPS extract (µg mL-1). 187 On the other hand, the production of EPS was expressed as a percentage of 188 the total concentration of EPS in relation to cell concentration. 189 190 2.5 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) 191 Cr (III) removal capacity of O. anthropi DE2010 was analyzed by ICP- 192 OES and the specific metal removal (q), expressed as mg of chromium/g of dry 193 weight, was calculated using the following formula (Chakravarty and Banerjee, 194 2012): V(Ci  Cf) 195 q m 196 where V is the sample volume (L); Ci and Cf are the initial and final metal 197 concentrations (mg L-1), and m is the dry culture biomass (g). 8 198 199 After 24 h, O. anthropi DE2010 cultures growing at each tested 200 concentration were centrifuged at 2000xg for 10 min at 4 ºC (Eppendorf 5804R 201 refrigerated centrifuge). Samples from the resulting supernatants and liquid 202 samples from the initial Cr(III) working concentrations (without microorganism) 203 were analyzed by ICP-OES. Pellets were resuspended in 10 mL of sterile 204 double deionized water and the corresponding EPS extracts were obtained 205 using the same method as that described above (Adav and Lee, 2008). These 206 extracts and samples from the same cellular suspensions but without EPS were 207 also analyzed by ICP-OES, in order to detect the presence of chromium 208 immobilized into the cells. Before chemical assays, all samples were digested 209 with HNO3 in a 600 W microwave digestion system to remove all organic 210 matter. Chromium analysis at 267.716 nm was carried out on the resultant 211 digestions using an ICP-OES spectrometer Optima 4300DV (Perkin Elmer LLC, 212 761 Main Avenue, Norwalk, USA), in quadruplicate assays. 213 214 2.6 Energy dispersive X-ray spectroscopy (EDX) analysis coupled to Scanning 215 transmission electron microscope (STEM) imaging 216 Metal sorption capability of O. anthropi DE2010 was confirmed by STEM 217 EDX. Samples were prepared following the TEM-EDX protocol described by 218 Maldonado et al. (2010). To this end, a sample from each 24 h culture 219 contaminated with a Cr(III) working concentration was centrifuged at 4000xg for 220 5 min at 4 ºC (Eppendorf 5804R refrigerated centrifuge). The resulting pellets 221 were included in soft agar and subsequently fixed in 2.5% glutaraldehyde 222 diluted in Millonig phosphate buffer (Millonig, 1961) for 2 h and washed four 9 223 times (15 min) in the same buffer. Then samples were post-fixed in 1% OsO4 at 224 4 °C for 2 h and washed again in the same buffer. They were then dehydrated 225 in a graded series of acetone (50, 70, 90, 95, and 100%), and embedded in 226 Spurr's resin. Semi-fine sections (200 nm thick) were mounted on a titanium 227 grid covered with a thin carbon layer without additional staining (lead citrate and 228 uranyl acetate). High angle annular dark field scanning transmission electron 229 microscopy (HAADF-STEM) images and STEM EDX profiles were acquired 230 using a FEI Tecnai G2 F20 microscope operated at 200 kV and equipped with 231 an EDAX super ultra-thin window (SUTW) X-ray detector (FEI, Hillsboro, 232 Oregon, USA). 233 234 2.7 Data analysis 235 Statistical analysis was carried out by one-way analysis of variance 236 (ANOVA) and Tukey multiple comparison post-hoc test. Significant differences 237 in ANOVA and Tukey’s test were accepted at p ≤0.05. The analyses were 238 performed using SPSS software (version 20.0 for Windows 7). 239 3. Results and discussion 240 In this study, several analytical methods were applied to batch cultures in 241 order to evaluate the cytotoxic effect of Cr(III) on O. anthropi DE2010, and the 242 capacity of this bacterium to remove it. 243 244 3.1 Changes in plate counts, cell viability and biomass 245 The changes in plate counts, growth curves, cell viability, and biomass in 246 the presence of chromium are shown in Figure 1 (Log10 cfu mL-1 of total and 10 247 cultivable cells, and growth curves), and Figure 2 (FLU-CLSM-IA imaging, live 248 and dead cells%, mg C (cm3)-1, and DC%). The effect of Cr(III) on O. anthropi 249 DE2010 varied significantly depending on the concentration, although is more 250 evident at the highest metal concentrations. This is evidenced by a reduction in 251 the number of cfu mL-1, cell viability and biomass, and an increase in DC% and 252 duplication time. 253 254 The maximum values of the four biological parameters were obtained at 255 0 mM Cr(III) (9.26x109 cfu mL-1 of cultivable cells, 4.02 h duplication time, 256 99.82% of live cells and 46.76 mg C (cm3)-1 of biomass) and the minimum were 257 recovered out for 7 mM (<10 cfu mL-1 of cultivable cells, 21 h duplication time, 258 62.72% of live cells and 3.30 mg C (cm3)-1 of biomass) (Fig 1, and Fig 2). 259 Statistically significant differences were obtained, ranging from 0 mM (control) 260 to 7 mM Cr(III) for viability (F= 213.434) (p <0.05) and biomass (F= 212.253) (p 261 <0.05) results. Using the Tukey multiple comparisons post-hoc test, statistically 262 significant differences (p <0.05) were observed among all the metal 263 concentrations tested for cell viability and biomass (Fig 2). On one hand, 264 decreasing values from 0 mM to 7 mM Cr (III) corresponding to reductions in cfu 265 mL-1 of 99.99%, cell viability of 37.1% and biomass of 92.94%, were also noted. 266 On the other hand, results of cell viability and biomass at 10 mM Cr(III) could 267 not be determined from FLU-CLSM images due to the formation of cellular 268 aggregates (Fig 2), but a minimal cfu mL-1 value was achieved at this 269 concentration (Fig 1). Comparing these results with those obtained by different 270 phototrophic microorganisms, Scenedesmus sp. DE2009 and Geitlerinema sp. 271 DE2011 (Millach et al., 2015) and Chroococcus sp. PCC 9106 (Puyen et al., 11 272 2017) and the yeast Pichia stipitis (Yilmazer and Saracoglu, 2008), it can be 273 demonstrated that O. anthropi DE2010 shows a great tolerance and resistance 274 to this metal at higher concentrations, even at 10 mM. 275 276 It is important to highlight that at 7 mM Cr(III) a high percentage of cells 277 remain alive (FLU-CLSM-IA method) despite minimal viable cells being 278 detected in plate counts. A possible explanation for this fact is the presence of 279 dormant cells (DC) in cultures contaminated with Cr(III) that are unable to grow 280 in plates. Lewis (2010) defined DC as a small sub-population of cells that 281 spontaneously enter a dormant, non-dividing, and resistant state. In this paper, 282 to study the role of DC, 24 h cultures of O. anthropi DE2010 grown at the 283 different tested Cr(III) concentrations were also spread onto LB agar without 284 metal (Fig 1A, cfu mL-1 of total cells). These results are shown in Figure 2. The 285 number of DC increases as the metal concentration increases. Results were 286 significant at 5 mM Cr(III), with a relative ratio of dormant cells of 96.68%. This 287 can be seen even more clearly at 7 mM and 10 mM Cr(III), where the values 288 were 99.98% and 99.89%, respectively. 289 290 3.2 Changes in the EPS composition and production 291 The results concerning the biochemical analysis of EPS extracts from O. 292 anthropi DE2010 cultures grown with Cr(III) at different concentrations are 293 shown in Table 1. Changes in the EPS composition were observed as a 294 response to an increase in Cr(III) concentration. At lower concentrations of 295 Cr(III) (0-2 mM) the major EPS component is carbohydrates (88-89%), followed 296 by proteins (5-7%) and uronic acids (3-5%). However, at 5 mM Cr(III) the most 12 297 drastic metal effect was assessed. At this concentration, a considerable 298 increase of 37.88% in proteins was observed in contrast to a significant 299 reduction in 35.45% in carbohydrates, with as well as a slight decrease in uronic 300 acid content. Guibaud et al. (2003) demonstrated that the protein played a 301 major role in the complexation of metal ions and the number of binding sites 302 (alcohol, carboxyl and amino groups) for heavy metals. This change in the EPS 303 composition coincide with that described by Seng et al. (2005), who indicate 304 that the content of extracellular proteins increased when bacteria were grown in 305 adverse environmental conditions, and Yin et al. (2011), who report that the 306 presence of heavy metals resulted in a higher production of proteins than total 307 carbohydrates and uronic acids in the EPS. A decreasing value in 308 carbohydrates and proteins content is observed from 7 mM Cr(III), reaching a 309 similar value between both EPS components, although a slight increase was 310 observed at 10 mM, as result of cell aggregation. Regarding the uronic acids 311 content, in this study its role in O. anthropi DE2010 is less clear, although 312 different authors have been demonstrated the active role of this EPS 313 components in heavy metals chelation (Guibaud et al., 2005; Morillo Pérez et 314 al., 2008; Pal and Paul, 2008). The results showed in Table 1 demonstrated that 315 the concentration of uronic acid decreases from 0 to 5 mM and it is maintained 316 at higher Cr(III) concentrations, especially at 7 mM, when the carbohydrates 317 and proteins reach the lowest values. This data coincides with the formation of 318 cellular aggregates, which probably indicates that under these conditions, 319 uronic acid could contribute to chromium immobilization. Statistically significant 320 differences were found among all the metal concentrations assayed for 321 carbohydrates (F= 271.15) (p <0.05), proteins (F= 471.14) (p <0.05), and uronic 13 322 acids (F= 130.28) (p <0.05). Using the Tukey multiple comparisons post-hoc 323 test, statistically significant differences (p <0.05) were labelled in Table 1. 324 325 As regards the EPS production, a maximum increase in EPS 326 concentration in relation to cell concentration (7.82%) was obtained from 0 mM 327 (10.17%) to 5 mM (17.99%) Cr(III) coinciding with the increment in the protein 328 content. It is well known that the EPS generally contains high molecular weight 329 compounds with charged functional groups with adsorptive and adhesive 330 properties. These functional groups provide binding sites for heavy metals, as 331 demonstrated by Decho (1990) and more recently by other authors (More et al., 332 2014; Yue et al., 2015). These results could explain the increase in total amount 333 of EPS at high concentrations of Cr(III) in O. anthropi DE2010. 334 335 As previously mentioned the most important cytotoxic effect of Cr(III) on 336 O. anthropi DE2010 is produced at 5 mM Cr(III). At this concentration, the 337 percentage of dead cells increases, provoking a drop in viability and biomass. 338 This is linked to an enhancement in the cellular synthesis of EPS by O. anthropi 339 DE2010 to protect the cells themselves from chromium toxicity, increasing, in 340 turn, the protein content and reducing the amount of carbohydrate and uronic 341 acids. 342 343 3.3 Removal capacity for Cr(III) of O. anthropi DE2010 344 Quantitative chemical data for the ability of O. anthropi DE2010 cells to 345 capture Cr(III) and the corresponding cellular uptake efficiency (q) are shown in 346 Table 2. These results indicate that there is a strong correlation between the 14 347 initial concentration of metal and the total of chromium taken from the cells, 348 reaching maximum values of removal of 40.83% at 7 mM and of 38.38% at 10 349 mM Cr(III). In addition, practically all the chromium removed was detected 350 inside the cells (cytoplasm), with values ranging between 97% and 99%, 351 depending on the metal concentration, with the rest of the metal being found in 352 the EPS extracts (1-3%). These results indicate that O. anthropi DE2010 is able 353 to immobilize chromium in both the EPS and the cytoplasm of the cells. The q 354 values also increase by increasing the initial metal concentration, and a 355 maximum value of 950 mg g-1 was reached at 10 mM Cr(III). However, the most 356 notable increase in q values (590 mg g-1) was observed from 5 mM to 7 mM 357 Cr(III). The highest q value reported here could be related to the cellular 358 aggregation monitored by CLSM imaging at 10 mM. This cellular organization is 359 more efficient to capture Cr(III) and to protect the cells from the cytotoxic effect 360 of the metal than the individual cells observed at the other metal concentrations. 361 Statistically significant differences for q values (F= 1,596.45) (p <0.05) were 362 found among all the metal concentrations assayed. Using the Tukey multiple 363 comparisons post-hoc test, statistically significant differences (p <0.05) were 364 observed from 5 mM Cr(III) and were included in Table 2. The specific removal 365 efficiency (q) of O. anthropi DE2010 is higher with respect to those obtained by 366 other prokaryotic: 120 mg g-1 in Sphaerotilus natans (Solisio et al., 2000), 185 367 mg g-1 in Spirullina sp. (Chojnacka et al., 2005), and 14.28 mg g-1 in 368 Rhodococcus opacus (Calfa et al., 2008); and eukaryotic microorganisms: 369 11.30 mg g-1 in Saccharomyces cerevisiae (Ksheminska et al., 2005), and 370 41.18 mg g-1 in Chlorella miniata (Han et al., 2006). For this reason, it can be 371 considered that O. anthropi DE2010 show a higher capacity to sequestrate 15 372 chromium into the cells than other microorganisms and is, therefore, a great 373 candidate to bioremediate natural/artificial environments polluted with 374 chromium. 375 376 3.4 Cr(III) localization at cellular structure level 377 Electron microscopy imaging, SEM and TEM, have become a crucial tool 378 to visualize the ultrastructure of microorganisms (Rachel et al., 2010; Golding et 379 al., 2016; Solé et al., 2019) and coupled to an EDX detector to analyze the 380 elemental composition and its distribution in the samples (Maldonado et al., 381 2011, Burgos et al., 2013; Coreño-Alonso et al., 2014; Millach et al., 2015; 382 Povedano-Priego et al., 2017). Although, these techniques have been very 383 useful in this regard, we applied for the first time the HAADF-STEM EDX for 384 locating, at the nanoscale level, heavy metals immobilized on different cellular 385 structures (Fig 3) evidencing whether specific microorganisms have a capacity 386 to immobilize them inside and/or outside cells in the same semi-fine section. 387 388 HAADF-STEM images exhibited discernible changes (changes in cell 389 morphology, an increase in bright inclusions, and the appearance of cellular 390 pleomorphic forms) in cells (Fig 3 A1, B1, and C1) due to the Cr (III), mainly at 391 the highest concentration (10 mM Cr(III)). EDX microanalyses shows that 392 chromium was not detectable, either externally or internally, in control samples 393 (0 mM) and up to 2 mM Cr(III). However, results obtained from 5 mM to 10 mM 394 indicated that the chromium signal was detected in the cellular structure. 395 Figures 3 A2, B2, and C2 show the STEM EDX chromium and phosphorous 396 composition line profiles that were taken along a bright inclusion from a semi- 16 397 fine section micrograph of O. anthropi DE2010 grown at 0, 5, and 10 mM Cr(III). 398 When compared the image intensity profile, with an increased signal in the 399 bright inclusion, it is clear that the most part of chromium was located in the 400 bright inclusions, together with an important phosphorous signal, and 401 secondarily on the cellular surface at the EPS level. 402 403 It is known that microorganisms play an important role in heavy metal 404 immobilization processes (Cheng et al., 2010) either at extra-cellular 405 (biosorption) and/or intra-cellular (bioaccumulation) levels (Guibaud et al., 2005; 406 Guine et al., 2006; Kothe et al., 2010; Puyen et al., 2012b; Esteve et al., 2013; 407 Burgos et al., 2013; Gutiérrez-Corona et al., 2016). On the other hand, 408 Velásquez and Dussan, (2009) demonstrated the relation between external 409 capture and passive process (live or dead cells) and internal bioaccumulation 410 and active process (live cells). 411 412 The present STEM EDX results demonstrate that O. anthropi DE2010 413 accumulates Cr(III) externally in EPS, and internally mainly in cytoplasmic 414 polyphosphate inclusions. Therefore, the chromium removal via sorption and 415 accumulation mechanisms in this bacterium was demonstrated using STEM 416 EDX. 417 Conclusions 418 Our results indicate that O. anthropi DE2010 is able to (i) tolerate and 419 resist the presence of high Cr(III) concentrations, showing changes in 420 production and EPS composition and increasing the number of dormant cells, 17 421 (ii) capture chromium at higher removal efficiencies, and (iii) immobilize it mainly 422 inside the cell in polyphosphate inclusions (bioaccumulation process), as well 423 as externally in the EPS (biosorption process). 424 As a future perspective, O. anthropi DE2010 could be considered as a 425 potential agent for bioremediation in Cr(III) contaminated environments since it 426 shows a great ability to tolerate and efficiently remove Cr(III) from polluted 427 cultures. 428 Acknowledgments 429 We express our thanks for the assistance of the UAB staff of Servei de 430 Micròscopia (http://sct.uab.cat/microscopia/) in particular Dr Alejandro Sanchez- 431 Chardi, the UAB Servei de Anàlisi Química (http://sct.uab.cat/saq/) especially Dr 432 Ignacio Villarroya. We also appreciate the help of Cristina Sosa and the 433 valuable comments and suggestions of Diana Gutiérrez, Irene López-Gómez, 434 and Estefania Solsona. This research was supported by the following grants: 435 FONCICYT (Ref. 95887), Ministerio de Economía y Competitividad (Refs. 436 CTQ2014-54553-C3-2-R and CGL2008-01891) and UAB postgraduate 437 scholarship to EV. The ICN2 is supported by the Severo Ochoa (MINECO, 438 grant no. SEV-2017-0706) and CERCA programmes. 439 Conflicts of interest 440 The authors declare no conflict of interests. 441 References: 18 442 Adav, S.S., Lee, D.J., 2008. Extraction of extracellular polymeric substances 443 from aerobic granule with compact interior structure. J. Hazard. Mater. 154 444 (1-3), 1120-1126. https://doi.org/10.1016/j.jhazmat.2007.11.058. 445 Balk, E.M., Tatsioni, A., Lichtenstein, A.H., Lau, J., Pittas, A.G., 2007. 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Technol. 194, 399-402. 736 https://doi.org/10.1016/j.biortech.2015.07.042. 31 Table 1 Variations in the production and composition of the EPS of Ochrobactrum anthropi DE2010 Cr(III) [DNA] [Carbohydrates] [Proteins] [Uronic acids] [Cell co concentration (ppm) (ppm) (ppm) (ppm) (ppm) (mM) 0 0.28 ± 0.02 148.12 ± 4.94 9.98 ± 0.71 8.40 ± 0.71 1640 ± 8 0.5 0.58 ± 0.02** 150.50 ± 2.09 11.95 ± 0.52 6.30 ± 0.34** 1580 ± 1 2 0.91 ± 0.02**++ 133.28 ± 5.44 11.89 ± 0.88 6.02 ± 0.20** 1505 ± 1 5 1.20 ± 0.02**++$$ 45.36 ± 0.95**++$$ 39.37 ± 0.44**++ 1.35 ± 0.16**++$$ 485 ± 24 7 0.87 ± 0.03a**++&& 12.46 ± 0.15**++$$&& 10.72 ± 1.16&& 0.97 ± 0.07**++$$&& 255 ± 4.6 10 1.27 ± 0.03a**++$$^^ 17.36 ± 0.24**++$$&& 12.47 ± 4.36*&& 1.37 ± 0.26**++$$^^ 220 ± 4.7 a values out of range indicating cell lysis due to Cr(III) Data are expressed as mean ±SD (n= 15) *p <0.05; **p <0.01: 0.5, 2, 5, 7 and 10 mM treatments vs. control (0 mM) +p <0.05; ++p <0.01: 2, 5, 7, and 10 mM treatments vs. 0.5 mM treatment $$p <0.01: 5, 7, and 10 mM treatments vs. 2 mM treatment &&p <0.01: 7, and 10 mM treatments vs. 5 mM treatment ^^p <0.01: 7 treatment mM vs. 10 mM treatment Table 2 Removal Cr(III) capacity by Ochrobactrum anthropi DE2010 in the EPS and cytoplasm Culture Cr(III) Supernatant Total metal Metal in cells Dry we Medium Metal in EPS concentration (Cf) removed without EPS (m) (Ci) (mg L-1) (mM) (mg L-1) (mg L-1) (mg L-1) (g) (mg L-1) 0 0 0 0 0 0 0.033 ± 0.5 24 ± 1.15 16.50 ± 0.57 7.50 ± 1.73 0.20 ± 0.006 7.30 ± 1.71 0.031 ± 2 99 ± 2.30 83.50 ± 1.73 15.50 ± 4.04 0.21 ± 0.010 15.29 ± 4.03 0.030 ± 5 257 ± 2.31 199 ± 3.46 58 ± 1.15 0.52 ± 0.015 57.48 ± 1.14 0.010 ± 7 360 ± 3.46 212.50 ± 2.89 147.50 ± 0.58 1.85 ± 0.045 145.15 ± 0.54 0.005 ± 10 495 ± 9.24 305 ± 8.08 190 ± 8.99 5.10 ± 0.095 184.90 ± 0.90 0.004 ± Data are expressed as mean ±SD (n= 4) *p <0.05, **p <0.01: 0.5, 2, 5, 7 and 10 mM treatments vs. control (0 mM) ++p <0.01: 2, 5, 7, and 10 mM treatments vs. 0.5 mM treatment $$p <0.01: 5, 7, and 10 mM treatments vs. 2 mM treatment &&p <0.01: 7, and 10 mM treatments vs. 5 mM treatment ^^p <0.01: 7 treatment mM vs. 10 mM treatment 1 Figure captions 2 Figure 1 Growth parameters of O. anthropi DE2010 growing at different Cr(III) 3 concentrations. (A) Plate counts (cfu mL-1) from total (grown on LB agar without 4 Cr(III)) and cultivable cells (grown on LB agar with Cr(III)). p <0.01**: 0.5, 2, 5, 5 7, and 10 mM treatments vs. control (0 mM). (B) Growth curves at different 6 Cr(III) working concentrations. In both graphics, data are expressed as mean 7 ±SD (n= 8). 8 Figure 2 Cytotoxic effect of Cr(III) in the O. anthropi DE2010 cultures. *p <0.05, 9 **p <0.01: 0.5, 2, 5, and 7 mM treatments vs. control (0 mM). +p <0.05, ++p < 10 0.01: 2, 5, and 7 mM treatments vs. 0.5 mM treatment. $$p <0.01: 5, and 7 mM 11 treatments vs. 2 mM treatment. Data are expressed as mean ±SD (n= 20). 12 Figure 3 HAADF STEM analysis of O. anthropi DE2010 cells grown at 0 (A), 5 13 (B), and 10 mM (C) of Cr(III). (A1, B1, and C1) HAADF STEM imaging, and 14 (A2, B2, and C2) HAADF STEM-EDX analysis and line profiles (250 nm) along 15 the black line showing the intensity of the HAADF image (top), phosphorus 16 (middle) and chromium (bottom).