Desalination 405 (2017) 1–9
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Biomineralization of calcium and magnesium crystals from seawater by halotolerant bacteria isolated from Atacama Salar (Chile) Dayana Arias a,b, Luis A. Cisternas b,c, Mariella Rivas a,c,⁎ a b c
Laboratory of Algal Biotechnology & Sustainability, Faculty of Marine Sciences and Biological Resources, University of Antofagasta, Av. Angamos 601, Antofagasta, Chile Department of Chemical Engineering and Mineral Process, University of Antofagasta, Av. Angamos 601, Antofagasta, Chile Science and Technology Research Center for Mining (CICITEM), General Velázquez 890 Of. 604, Antofagasta, Chile
H I G H L I G H T S • A new application for biomineralization is removal of calcium and magnesium ions from seawater as selective biodesalination • Twelve bacterial strains were identiﬁed from Atacama Desert with ureolytic activity precipitating calcite in seawater. • Rhodococcus erythropolis TN24F removed in just 7 days 95% of soluble calcium and in 14 days of bioassay the 8% magnesium ion.
a r t i c l e
i n f o
Article history: Received 13 September 2016 Received in revised form 26 November 2016 Accepted 30 November 2016 Available online xxxx Keywords: Biomineralization Seawater Ureolytic bacteria Calcite Struvite SW pretreatment
a b s t r a c t Antofagasta (Chile) is an arid region, and the climate is strongly inﬂuenced by the Atacama Desert, with few sources of fresh water. The pressure to use non-conventional water sources has boosted the construction of numerous desalination plants. High concentrations of secondary ions as calcium and magnesium cause problems in reverse osmosis plants and in other industries such as copper mining and cooling system. Biomineralization process based on hydrolysis of urea has been described in a wide variety of bacterial species with diverse applications. The selection of ureolytic halotolerant bacteria from Atacama Salar and their ability to precipitate calcium and magnesium crystals in seawater is described. Besides crystal structure and morphology were determined by electron microscopy analysis and X-ray diffraction. When assessing the mineral precipitate ability, Rhodococcus erythropolis precipitates a ~ 95% soluble calcium and 8% magnesium. The analysis of crystals showed that correspond to ~12.69% monohydrocalcite, ~30.72% struvite and ~56.59% halite. These results demonstrate that the biomineralization by ureolytic bacteria in seawater has great potential for its application as a pretreatment to improve water quality for industrial processes. © 2016 Published by Elsevier B.V.
1. Introduction In Chile, most of the mining activities take place in the North, a desert area located in the Atacama Desert. Where there is a great scarcity of fresh water (FW) sources, which, in recent years, has boosted the use of seawater (SW) as alternative water for mining processes and others industrial application. The industry uses both desalinated SW or directly SW [1,2]. Also, for human consumption desalinated SW is used, and in Abbreviations: SEM-EDX, Scanning electron microscopy with energy dispersive X-ray; XRD, X-ray diffraction; UA, Urease enzyme; Cu-Mo ores, Copper-molybdenum ores; NaHS, Sodium hydrosulﬁde; BIM, Biologically induced mineralization; BCM, Biologically controlled mineralization; CFU, Colony forming units; UAM, Urea agar medium; FW, Fresh water; SW, Seawater. ⁎ Corresponding author at: Laboratory of Algal Biotechnology & Sustainability, Faculty of Marine Sciences and Biological Resources, University of Antofagasta, Av. Angamos 601, Antofagasta, Chile. E-mail addresses: [email protected]
, [email protected]
http://dx.doi.org/10.1016/j.desal.2016.11.027 0011-9164/© 2016 Published by Elsevier B.V.
the main city of northern Chile (Antofagasta) 80% of the water used is desalinated water. The technology utilized for desalination is reverse osmosis. Due to the high energy demand of desalination techniques there is a great need for alternatives to reduce the salinity from SW . In addition, secondary ions as calcium and magnesium in the SW cause scale problems in reverse osmosis plants, mining, and others industries such as cooling system. These problems cause increased costs and reduce the efﬁciency of these processes. SW has a broad corrosive power which depends on many factors including material composition, SW chemistry, pH, dissolved oxygen content, salinity, galvanic interactions, temperature and ﬂuid velocity characteristics . Speciﬁcally ions in solution present various problems, for example, the chloride ions in saline water are one of the most aggressive substances in SW because ions can react with dissolved ferrous ions to create ferrous chloride which then can react with dissolved oxygen and produce ferric oxide (Fe2O3) and ferric chloride (FeCl3), which are considered an oxidizing agent that can enhance the
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general corrosion rate and pitting corrosion . On the other hand, calcium concentrations in SW lead to clogging of pipelines through scaling as carbonate, sulfate or phosphate precipitates . Speciﬁcally, in reverse osmosis there are problems of scale deposition on membranes due to concentration polarization. In cooling systems there are scale deposits on heat transfer surface due to temperature change and pressure drops. Through mineral ﬂotation gold, copper and molybdenum are extracted among other ores; the latter is produced as the main byproduct of copper mining in Chile, becoming the fourth export mining product. When untreated SW and lime are used to raise the pH and depress pyrite in copper, molybdenum and gold ﬂotation processes (Cu-Mo-Au) molybdenite MoS2 is also depressed . The reason is that the calcium and magnesium ions which are present in high concentration (Ca2+ 400 mg/L; Mg2+ 1576 mg/L) [8,9] precipitate at alkaline pH as hydroxy-complex species of Ca(OH)+ and Mg(OH)+ type, which are adsorbed on the anionic sites of the surface of MoS2 and the surface of bubbles giving positive charge thereby inhibiting its buoyancy [8,10]. The concept of biomineralization is deﬁned as the process that involves the formation of minerals by living organisms as a result of cellular activity that promotes the physic-chemical conditions required to carried out the formation and growth of biominerals [11,12,13]. Biomineralization processes are based on two types according to the degree of existing biological control, divided in biologically controlled mineralization (BCM) and biologically induced mineralization (BIM) [13,14]. Within this last point, we have identiﬁed several biological processes that create the necessary conditions for the mineral precipitation [15,16]. These include metabolic pathways of the denitrifying microorganisms, iron reducers, sulfate reducers and action of carbonic anhydrase and urease enzymes [17,18,19]. Speciﬁcally, the urease enzyme action mechanism (urea amidohydrolase; EC 126.96.36.199) is based on catalyzing the hydrolysis of urea generating ammonia and carbamate. Carbamate spontaneously decomposes to produce another molecule of ammonia and carbonic acid. Two ammonia molecules and carbonic acid subsequently equilibrate in water with their deprotonated and protonated forms, resulting in an increase in the pH [11,19,20,21]. This increased pH in the microenvironment surrounding the bacteria induces the precipitation of crystals around acting as a nucleation site . The biomineralization processes mediated by urea hydrolysis are widely described in the literature in various biotechnology applications such as soil stabilization, particulate matter control  and cement remediation [24,25], soil and water [11,13,26,27]. Under this last point, the main research is related to the treatment of wastewater and groundwater [6,28,29]. There are few studies on biomineralization in SW. Drew  ﬁrst described involving marine bacteria in the precipitation of calcium carbonate, and then McCallum and Guhathakurta  described the precipitation of aragonite from SW isolated from marine origin in various culture mediums. Regarding the calcium and magnesium biomineralization in SW, Greenﬁeld  and, Malone and Towe  detailed the struvite and monohydrocalcite precipitation from SW culture, emphasizing the importance of the metabolism of nitrogen compounds and the consequent increase in pH on its precipitation. However, some microbial species have been associated with precipitation of biominerals in many different environments including saline habitats (SW, brine), bioﬁlms and soils [22,34]. Rivadeneyra et al.  described halotolerant and halophilic bacteria adapted to a high salinity range environment that can be used for mineralization of calcium and magnesium by forming calcite and struvite. Also González-Muñoz et al.  demonstrated that strains from the Idiomarina genus precipitate struvite similar to SW salinity. Therefore, the main goal of this study was to isolate and select bacterial strains able to live in SW and whose metabolism allows selective precipitation of ions therefrom. For this purpose, the ability of halotolerant ureolytic bacteria isolated from the Jere Valley, Tara Salar and the National Reserve “Los Flamencos” in the Atacama Desert (Chile) to biomineralize calcium, magnesium and sodium chloride crystals present in high concentration in SW was evaluated in order to propose for the ﬁrst time
the application of biomineralization bacterially mediated as biotechnological tool to improve the quality of water for industrial mining processes through the selective removal of these ions. 2. Materials and methods 2.1. Description and physico-chemical parameters from the sampling site Environmental samples of water and sediment were collected from three locations inland from San Pedro de Atacama in Northern Chile in June 2014. Table 1 shows the geographical location, altitude and physico-chemical parameters of the selected locations. At the National Reserve “Los Flamencos” (located 135 Km from San Pedro de Atacama) 5 environmental samples were collected, from Tara Salar 3 samples and from Jere Valley 2 samples. The samples were placed in sterile tubes and stored at 4 °C until use. Physical-chemical parameters (Table 1) were measured using a multiparameter probe (Multi 340i Set, WTW Germany). Calcium and magnesium concentrations were measured using a portable photometer HI-96752 and HI-96821 (Hanna Instruments). 2.2. Enrichment cultures and isolation of halotolerant bacteria Original samples were enriched with culture mediums prepared in SW at Antofagasta shore (Chile) and distilled water supplemented with 35 g/L NaCl during 7 days at ~ 30 °C. The culture mediums used were designated as TN (Tryptone Soya Broth, TSB; DIFCO™) supplemented with NaCl, TM (TSB in SW), LN (Luria broth, LB; MoBio Lab., Inc.) supplemented with NaCl and LM (LB in SW). SW and distilled water were sterilized by ﬁltration 0.2 μm. To isolate culturable halophilic bacteria, the described culture mediums were prepared in plates with 15 g/L agar (Oxoid) inoculating 10 μL of each enriched sample and incubated at ~30 °C until growing up. The colony forming units (CFU) were selected according to their morphology and were subcultured in order to identify the presence of urease activity. 2.3. Urease activity qualitative assay To determine the presence of urease activity bacterial isolates were grown up in tubes with urea agar medium (UAM) modiﬁed from Christensen's  (20 g/L urea,1 g/L peptone, 5 g/L sodium chloride, 2 g/L potassium dihydrogen phosphate, 20 g/L agar, 1 g/L glucose and 0.012 g/L phenol red; pH 6.5). This selective medium allows the rapid detection of urease enzyme activity and additionally 35 g/L NaCl allowed the detection of halophilic bacteria . Bacterial strain were streaked on the surface of the media and incubated at 37 °C for six days (limit of chemical hydrolysis of urea) and observed the change of color in the medium from yellow to pink. The assay was performed in triplicate. Proteus mirabilis was used as positive control and Escherichia coli JM109 as a negative control. 2.4. Bacterial identiﬁcation by rDNA 16S, phylogenetic analysis and nucleotide sequence accession numbers To identify isolates, strains were grown up overnight and centrifuged at 3000 × g for 5 min. Total DNA was extracted from the cell pellet by the DNA Extraction Power Soil kit (Mobio Cat. No. 12888-100) following the manufacturer's instructions. Using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′TACGGYTACCTTGTTACGACTT-3′) was ampliﬁed 16S rDNA gene by PCR  and afterwards sequenced in Macrogen Inc. (South Korea). Sequences were edited using the program FinchTV version 1.4 (http://www.geospiza.com). The assembling sequences were performed using version Chromas Pro 1.6 (2012) and then analyzed by BLASTN database (http://www.ncbi.nlm.nih.gov) using the non-redundant GenBank nucleotide collection. Evolutionary analyses were conducted in MEGA 6.0 software using Maximum Likelihood with a bootstrap of 1000. The model used was General Time Reversible while the branch swap ﬁlter analysis setting used was weak.
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Table 1 Geographical location physicochemical parameters of sampling sites.
Water temperature (°C)
Redox potential (mV)
Jere Valley (VJ) Tara Salar (ST) Los Flamencos Reserve site 1 (RF1) Los Flamencos Reserve site 2 (RF2)
23°11′18′ 23°8′31′ 23°6′47′ 23°4′56′
67°59′33′′ 67°25′23′′ 67°32′42′′ 67°35′55′′
2295 4200 4553 4600
10.50 32.60 0.50 0.20
8.30 7.22 9.17 9.80
8.10 1.94 10.30 2.95
−78 −15 −27 −172
0.20 15,000 2000 5500
30 210 55 560
30 690 205 5720
meters above sea level.
of urea solution due to production of ammonium. The amount of ammonia released from urea was measured according to the phenol-hypochlorite assay method described by Achal et al.  with the followings modiﬁcations: 50 μL of bacterial ﬁltrates were added to a mixture containing 700 μL of 0.1 M potassium phosphate buffer (pH 8.0). Two hundred μL of phenol nitroprusside and alkaline hypochlorite were added to the mixture and incubated at room temperature for 25 min. Optical density was measured at 630 nm against the blank (250 μL of 0.1 M potassium phosphate buffer pH 8.0, 200 μL phenol nitroprusside sodium, 200 μL sodium hypochlorite in a total volume of 1150 μL) in a spectrophotometer and compared to a standard curve prepared with NH4Cl (range 50–1000 μM).
The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The 16S rRNA gene sequences that we obtained for the strains isolated from Atacama Desert were upload to GenBank with the following accession numbers: KX185697.1 (strain LM19A), KX185698.1 (strain LM20KC), KX018272.1 (strain TN21G), KX185699.1 (strain LM22A), KX185700.1 (strain LM24AB), KX018273.1 (strain LM24AC), KX185704.1 (strain TN24F), KX185701.1 (strain LM25A), KX018274.1 (strain TN25G), KX185705.1 (strain TN26I), KX185703.1 (strain LN27F), KX018275.1 (strain TM27IT) and KX018276.1 (strain TN27K). 2.5. Biomineralization tests Bacterial strains with urease activity were inoculated in triplicate in 10% LN medium supplemented with 20 g/L urea and CaCl2 × 2H2O to evaluate the bacterial capacity to precipitate calcium crystals in distilled water. Then to evaluate the biomineralization in SW, bacterial strains were inoculated in LM medium with 20 g/L urea. For both assays the cultures were performed in triplicate and incubated for a week at 30 °C and 120 rpm. pH of the assays were measured daily while calcium, magnesium ions, total carbonates and bicarbonates were measured at the beginning and end of both trials as described below. The precipitates obtained were washed three times with distilled water and dried at 60 °C for 24 h for SEM-EDX and DRX.
2.8. Scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) spectroscopy and XRD analysis The morphology of the dried precipitates produced in bacterial biomineralization tests were examined at accelerating voltages ranging from 10 to 20 kV by a SEM (JEOL JSM 6360LV), which was equipped with energy dispersive X-ray analyzer (Oxford, INCA) for elemental analysis. Samples were arranged in a carbon tape and covered with gold via sputtering (108 Auto Sputter Coater, 115VAC) prior to examination. The XRD analyses were obtained using a diffractometer (Siemens D5000) with a graphite secondary monochromator and CuKα radiation. Data were collected for a 1.0 s integration time in 0.02° 2θ steps at 40 kV and 30 mA and scanning from 3° to 70° 2θ. The sample components were identiﬁed by comparing them with standards established by the International Center for Diffraction Data.
2.6. Chemical analysis Calcium and magnesium ions were analyzed by atomic absorption spectrophotometry (220FS, Varian) with direct suction. Total carbonates and bicarbonates were measured through acid-base titration . NaCl concentration was measured by a digital refractometer.
3. Results and discussion Biomineralization or MICP has been proposed as a new strategy for pretreatment of SW by selective removal of ions present in high concentration, at this point the key is hydrolysis of urea to ammonia through the enzymatic urease activity with subsequent precipitation of calcium as calcium carbonate and magnesium as struvite. This new application
2.7. Measurement of urease activity Urease activity can be measured through the production of CO2, ammonium ions, changes of pH, or by the changes of electric conductivity
Table 2 Phylogenetic identiﬁcation and calcium carbonate production of halotolerant ureolytic bacterial strains isolated from Atacama Salar.
Species with sequence homologya
Homolog GenBank accession no.
Semi qualitative urease activityb
Total carbonates (mg/L)c
LM19A LM20KC TN21G LM22A LM24AB LM24AC TN24F LM25A TN25G TN26I LN27F TM27IT TN27K
VJ VJ ST ST RF1 RF1 RF1 RF1 RF1 RF2 RF2 RF2 RF2
Pseudomonas sp. Shewanella putrefaciens LHC79 Bacillus subtilis NZ2-4-2 Shewanella sp. AC-WP-R Pseudomonas gessardii N13 Bacillus subtilis CICR-NG Rhodococcus erythropolis LZ1312-1-23 Pseudomonas ﬂuorescens FLM05-2 Bacillus subtilis H184 Marinilactibacillus psychrotolerans Halomonas sp. GOS-3a Bacillus subtilis CRB115 Bacillus subtilis CRB115
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
100 100 100 100 100 99 100 100 99 100 100 100 100
99 99 100 99 99 98 100 99 100 99 99 99 99
KF544922.1 KC951912.1 KR999922.1 FJ231175.1 KT883843.1 KC438378.1 KT597543.1 DQ084460.1 JX515570.1 AB681123.1 JQ246431.1 GQ161967.1 GQ161967.1
+++ +++ +++ + +++ +++ +++ +++ +++ ++ +++ +++ +++
6881 ± 0.001 2970 ± 0.211 6853 ± 0.001 1965 ± 0.016 889 ± 0.015 695 ± 0.022 6826 ± 0.003 3258 ± 0.224 4115 ± 0.192 6863 ± 0.001 1292 ± 0.009 1604 ± 0.003 6844 ± 0.003
Underline corresponds to the bacterial strains that possess the highest urease activity and were selected for the accomplishment of the biomineralization tests. a Sequence homology was determined with BLASTN. b Activity expression in UAM plates at 24 h. High: +++; Medium: ++; Low: +. c Biomineralization test of calcium carbonate precipitates on distillated water-LB broth.
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requires the use of bacteria able to biomineralize carbonate in SW tolerating high salinities.
3.1. Selection, isolation and identiﬁcation of halotolerant ureolytic bacterial strains The enrichment of each sample in the 4 culture mediums described generated a total of 40 enrichment cultures from which a number of culturable heterotrophic bacteria were obtained (~97 CFU) selected by colony morphology, color and appearance. Only 13.4% demonstrated ureolytic enzyme activity. The activity of E. coli DH5-α and P. mirabilis were used as control. The speciﬁc activity of P. mirabilis was measured by Prywer and Torzewska . E. coli strain DH5-α has a low-level of urease activity . According to the results, all strains have a high urease activity, except for strains LM22A and TN26I which have low urease activity (Table 2). All isolates with ureolytic activity were identiﬁed phylogenetically based on 16S rRNA gene sequences through BLASTN (Table 2). The isolates identiﬁed positive urease belong to phylum Firmicutes, Proteobacteria and Actinobacteria according to phylogenetic analysis (Fig. 1). The highest bacterial diversity occurred at Los Flamencos Reserve with nine isolates, strain LM24AB and LM25A correspond to Pseudomonas gessardii with identity 99% and P. ﬂuorescens with 99%, respectively; strains LM24AC, TN25G, TM27IT and TN27K correspond to Bacillus subtilis with identity between 98 and 100%; strain TN24F identiﬁed as Rhodococcus erythropolis with identity 100%; strain TN26I as Marinilactibacillus psychrotolerans with identity 99% and the strain LN27F as Halomonas sp. with identity 99%. From the Jere Valley only two strains were isolated which correspond to Shewanella putrefaciens with identity 99% and Pseudomonas sp. with identity also 99%. From Tara Salar it was also possible to identify two strains with ureolytic activity, TN21G as Bacillus subtilis with identity 100% and strain LM22A as Shewanella sp. with identity 99% (Table 2). Unlike other studies on isolation of culturable bacteria with ureolytic activity from land, we do not identify any strain belonging to Sporosarcina genus . The 38.46% of strains with ureolytic activity belong to the Bacillus genus, previously described due to its ability to precipitate calcium carbonate crystals in saline environments, conﬁrming our study , indicating also that most microorganisms belonging to Firmicutes phylum are the dominant phylotypes involved in the precipitation of carbonates in extreme environments . In general, species from Bacillus group are able to precipitate calcite in their microenvironment around their cells through converting urea to ammonia and carbon dioxide [20,44].
3.2. Biomineralization of calcium carbonate crystals in FW All bacterial strains positive urease were able to induce the formation of crystals in FW only with the addition of NaCl, urea and CaCl2 as a calcium source conﬁrming that enzyme urease activity plays a key role in microbiologically-induced calcite precipitation . Quantiﬁcation of total carbonates produced for each of the strains at day seven of test determined that the highest concentrations are produced by the strains Pseudomonas sp. LM19A (6881 ± 0.001 mg/L), M. psychrotolerans TN26I (6863 ± 0.001 mg/L), B. subtilis TN21G (6853 ± 0.001 mg/L), B. subtilis TN27K (6844 ± 0.003 mg/L) and R. erythropolis TN24F (6826 ± 0.003 mg/L) (Table 2). No mineral formation was observed in any of the controls. M. psychrotolerans has been described so far as a slightly halophilic bacteria present in marine environments and in deep-sea sediments [45,46]. They produce lactacte, formate, acetate, ethanol and carbon dioxide [45,47]. Except
for the other species, this is the ﬁrst record of its ureolytic activity and its ability to produce carbonate crystals. The composition and morphology of the crystals from three bacterial strains on day 7 of the test were determined through analysis by EDX microanalytical data and SEM observations. Fig. 2 shows that crystals form spheres with the presence of bacteria (Fig. 2A and C). EDX data for Pseudomonas sp. LM19A, B. subtilis TN21G and R. erythropolis TN24F indicated that the precipitates are composed essentially of Ca, O and C. These strains were selected to assess their ability to biomineralize calcium and magnesium ions in SW. 3.3. Biomineralization of calcium and magnesium crystals in SW It has been widely described that Bacteria can serve as nuclei of carbonate precipitation upon adsorbing Ca2+ and Mg2+ cations onto the cell surface due to their possessing a negative surface charge [23,48]. Each bacterium has a different type of cell surface resulting in a different cell surface charge. During the bioassay of biomineralization in SW, the pH change induced by the ureolytic activity of bacteria, ammonium production from urea hydrolysis and the growth of the selected strains was evaluated. A directly proportional tendency was observed between produced ammonia concentration and the pH evolution in cultures. Regarding cell density in culture strains no relationship was observed between these parameters (Fig. 3A). From day 1 of the test, an increase in the cell number for most bacterial strains being Pseudomonas sp. LM19A which showed the highest cell density at day 6 (7.7 × 109 cells/mL). However, this density is similar to that achieved by strains of the Bacillus sp. on day 7 of culture (strains TN21G and TN27K, 7.7 × 109 cells/mL and 6.2 × 109 cells/mL, respectively). Unlike, on day 6 of culture M. psychrotolerans TN26I only reaches a cell density equal to 3.2 × 109 cells/mL. For R. erythropolis strain TN24F a low cell number was determined mainly because its growth occurs as aggregate making difﬁcult to count living cells. The greatest production of ammonia in all cultures was observed from day four of bioassay maintaining constant levels until day 7 of test. R. erythropolis TN24F produced the highest concentration of ammonium (43,349 ± 0.065 μM) higher by approximately 63 ± 4.03% compared with the other strains (Fig. 3B). When determining the evolution of pH, it was determined that in presence of R. erythropolis an increase in pH is induced from the ﬁrst day of the bioassay (from 7.44 to 8.3 ± 0.05) to reach its maximum on day 6 of test (pH 9.07 ± 0.02; Fig. 3C). This behavior is not observed in the other bacterial strains, in which was observed a pH increase sustained over time from day 1 of bioassay reaching a peak on day 7 of test close to pH 8.6 (B. subtilis TN21G), but below the maximum value of pH of R. erythropolis (Fig. 3C). In other bacteria isolated from saline environments Virgibacillus pantothenticus and Bacillus marisﬂavi it was observed that these strains initially decreased the pH of culture mediums at values 5.0 and 5.8, respectively, but then slowly these values were increased to a pH near 8.0 at the end of the experiments and after 30 days testing . On day 7 of the bioassay, the concentration of calcium, magnesium ions, total carbonates and total bicarbonates present with respect to control was determined (Fig. 4). The control corresponds to SW without adding bacteria, in this case the concentration of calcium present corresponds to 403 ± 2 mg/L. and of magnesium to 1301 ± 2 mg/L. No carbonates were present in the control but there were total bicarbonates (942 ± 4 mg/L). In the presence of all bacterial strains, concentrations of carbonates and total bicarbonates signiﬁcantly increase in regards to the control, except for M. psychrotolerans in which no presence of total carbonates was observed and concentration of calcium and
Fig. 1. Molecular Phylogenetic analysis on partial 16S rRNA sequences by Maximum Likelihood method. The analysis involved 13 nucleotide sequences (~1100 bp) of 16S rRNA gene of halotolerant ureolytic bacterial strains isolated from Atacama Salar indicated in Table 2. Filled circles indicate isolates from Jere Valley, squares show the isolates from Tara Salar, ﬁlled triangles indicate the isolates from Los Flamencos National Reserve site 1, therefore the diamonds indicated the isolates from Los Flamencos National Reserve site 2. Planctomyces maris was used as an outgroup.
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Fig. 2. SEM-EDX analysis of precipitates generated in the biomineralization test in FW-LB broth. Precipitates from Pseudomonas sp. strain LM19A (a), R.erythropolis strain TN24F (b) and B. subtilis strain TN21G (c).
magnesium have no variation with respect to control. From all the strains tested, R. erythropolis TN24F precipitates the greatest concentration of carbonates and total biocarbonates. The increase on total carbonates increases from 0 to 1587 ± 152 mg/L and the increase of total biocarbonates from 924 ± 4 to 5257 ± 4 mg/L. For this same strain with respect to the calcium ion, a decrease of the soluble calcium concentration was determined in ~ 95%, while the magnesium concentration decreased only in ~4% on day 7 of bioassay. Similar results were determined in bacteria isolated from saline environments, V. pantothenticus S3 with decreased soluble calcium concentration around 69.5% and magnesium only 4.4%, while B. marisﬂavi M3 decreased concentrations in 72.3% and 9.1%, respectively, within 30 days of bioassay . One possible explanation is that the formation of the complex mediated by ammonium and Ca, together with the increase in pH favors binding to calcium cells over magnesium ion . In addition, on the negatively charged surface (as in the case of cell envelope of bacteria) Ca2+ adsorbs with greater ionic selectivity than Mg2+ . Castanier et al. , and McConnaughey and Whelan  suggest that ion transport across the cell membrane is related to the precipitation of crystals. For R. erythropolis TN24F calcium concentration decreases by 95% within only 7 days of bioassay justifying a greater ionic selectivity of Ca2+ over Mg2+ conﬁrming Silva-Castro et al. . No signiﬁcant differences were observed with respect to the decrease in calcium and magnesium in presence of other bacterial strains with respect to control (Fig. 4). The greatest difﬁculty for biomineralization in SW may be due to excess salts and high concentration of Mg2+ which would adversely affect
the precipitation of CO2− 3 mediated by bacteria [51,52]. However, all bacterial species analyzed precipitate crystals in saline conditions conﬁrming that the inhibitory effect of Mg2+ is not signiﬁcant in moderately halophilic bacteria [53,54]. Moreover, for R. erythropolis TN24F the evolution of the 95% decrease in soluble Ca2+ ion from day 7 was determined, which is maintained until day 14 (The concentration decreases from ~395 mg/L to ~18.36 mg/L) (Fig. 5). In the case of Mg2+ ion, the decrease on day 7 is close to 4% and on day 14 it reaches 8% (from ~1258 mg/L to ~1130 mg/L). The evolution of the pH for the SW in the presence of the bacterium increases from pH ~7 to pH ~8.5 as opposed to the control without bacteria with a constant pH equal to ~7.3. 3.4. Analysis of the precipitates produced by R. erythropolis TN24F in SW The crystals produced by R. erythropolis TN24F on day 14 of biomineralization bioassay were analyzed by SEM-EDX (Fig. 6). Irregular crystals were observed with a different morphology to crystals produced in FW. The EDX analysis in four random sites showed that the precipitates are mainly composed of Ca, P, Na, Cl and C (Fig. 6). Finally, the precipitates were characterized by XRD analysis determining that are mainly composed of monohydrocalcite (CaCO3 × H2O) in ~ 12.69%, struvite (MgNH4PO4 × 6H2O) in ~30.72% and halite (NaCl) in ~ 56.59% (Fig. 7; Table 3). The order in which minerals precipitate by R. erythropolis TN24F in SW indicates that there is predominance to precipitate calcium crystals ﬁrst, shaped like monohydrocalcite (day 7 of test; Fig. 4) then
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Fig. 4. Quantiﬁcation of calcium, magnesium, carbonate and bicarbonate ion concentration in the biomineralization test in seawater-LB broth at day 7 of cultures.
indicating that the Mg2+/Ca2+ ratio is very important for crystals precipitation, coincidently with these authors the Mg2+/Ca2+ ratio present in SW is approximately 3.94, favoring the primary formation of monohydrocalcite crystals and then of struvite. 4. Conclusions Our results effectively demonstrate an active role for halotolerant bacteria selected in the precipitation of different minerals compound of 12.69% monohydrocalcite, 30.72% struvite and 56.59% halite from the ions present in SW. The formation of these minerals is directly related to the removal of the soluble calcium, with decreasing the calcium concentration by 95% and the magnesium concentration decreasing by ~4% on day 7 of bioassay. At day 14 the concentration of the calcium ion decrease from ~400 mg/L to 18.36 mg/L (~95%), and magnesium ion decrease from ~1258 mg/L to ~1130 mg/L (~8%). However, this study only describes the results obtained in 14 days of bioassay, in future studies it will be necessary to optimize biomineralization conditions in R. erythropolis TN24F for potential use in pretreatment processes at industrial scale. Acknowledgements This publication was supported by Anillo Project–Grant no ACT1201-Atacama Seawater, PhD Scholarship CONICYT no 21130712, CICITEM Project no R10C1004 and the Regional Government of Antofagasta.
Fig. 3. Analysis of the cellular density (a), ammonium production (b) and pH evolution (c) in the biomineralization test in seawater-LB broth.
precipitate magnesium crystals shaped like struvite (day 14 of test; Fig. 7, Table 3). The struvite formation (MgNH4PO4 × 6H2O) mediated by bacteria was ﬁrst described by Robinson , suggesting that microorganisms can precipitate struvite as a consequence of the release of NH4, the metabolism of organic nitrogenous substances and the presence of magnesium phosphate which is present in the medium. The results obtained for R. erythropolis TN24F differ from the above described by Rivadeneyra et al. [51,56], Raz et al.  and Delgado et al.  in which it was determined that the presence of magnesium ions inhibit the precipitation of calcium carbonate crystals, speciﬁcally calcite. These authors indicate that the removal of magnesium ions from the medium through struvite formation can generate favorable conditions for the formation of carbonates. One possible explanation may be that the inhibitory effect of magnesium affects mostly non-halotolerant bacteria that halotolerant bacteria [53,57,58]. Sánchez-Román et al.  indicate a clear difference between halophilic and non-halophilic bacteria
Fig. 5. Evolution of the concentration of calcium and magnesium ions and pH during biomineralization test in seawater-LB broth from day 0 until day 14.
D. Arias et al. / Desalination 405 (2017) 1–9
Fig. 6. SEM-EDX analysis of the precipitates produced by R. erythropolis strain TN24F. Calcium crystals (a), presence of bacteria between crystals (b) and panoramic view of crystals types in precipitates with EDX spectrum indicates that they are composed of Ca, Mg, Na, Cl, C and P (c).
Fig. 7. XRD analysis of the precipitates produced by R. erythropolis strain TN24F. Struvite was represented by (S), Monohydrocalcite by (M) and Halite by (H). The chemical composition of these biominerals and abundance percentage are represented in the Table 3.
Table 3 Quantitative analysis (%) of precipitates produced in SW as calcium and magnesium source by R. erythropolis TN24F. Crystaline phase
Halite Monohydrocalcite Struvite
NaCl CaCO3 × H2O MgNH4PO4 × 6H2O
56.59 12.69 30.72
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