- Email: [email protected]
Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium
Journal Pre-proof Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium
C.M. Martínez, M. Rivera-Hernández, Luis H. Álvarez, Ismael Acosta-Rodríguez, F. Ruíz, V.D. Compeán-García PII:
S0254-0584(20)30176-0
DOI:
https://doi.org/10.1016/j.matchemphys.2020.122797
Reference:
MAC 122797
To appear in:
Materials Chemistry and Physics
Received Date:
16 December 2019
Accepted Date:
12 February 2020
Please cite this article as: C.M. Martínez, M. Rivera-Hernández, Luis H. Álvarez, Ismael AcostaRodríguez, F. Ruíz, V.D. Compeán-García, Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium, Materials Chemistry and Physics (2020), https://doi.org/10.1016/j.matchemphys.2020.122797
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium CM Martínez*, M. Rivera-Hernández, Luis H. Álvarez, Ismael Acosta-Rodríguez, Ruíz F, VD Compeán-García**
M. Rivera-Hernández Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E.mail: [email protected] Luis H. Álvarez Departamento de Ciencias Agronómicas y Veterinarias, Instituto Tecnológico de Sonora, 5 de Febrero 818 Sur, Ciudad Obregón, Sonora, México 85000. Email: [email protected] Ismael Acosta-Rodríguez Facultas de Ciencias Químicas, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México. ZP. 78290. E.mail: [email protected] Ruíz F. Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E.mail: [email protected] Corresponding authors: CM Martínez Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E-mail: *[email protected]. Tel.: +52 (444) 3813330 VD. Compeán-García CONACyT-Coordinación para la Innovación y Aplicación de la Ciencia y la Tecnología. Universidad Autónoma de San Luis Potosí. Sierra Leona #550, Lomas de San Luis, 78210 San Luis Potosí E-mail: **[email protected]
Journal Pre-proof Abstract Microbially induced carbonate precipitation (MICP) has been exploited as an efficient strategy to immobilize toxic metals in the form of carbonate salts. The present study investigated the cadmium carbonate (CdCO3) precipitation induced by an anaerobic granular sludge. The results revealed that anaerobic sludge showed high Cd utilization efficiencies (97.4% ± 1.1) after 18 hours of incubation at 30°C, and no differences were observed neither cadmium source nor substrate. According to SEM and X-ray diffraction results, the anaerobic sludge was able to precipitate Cd2+ as CdCO3 rhombohedral crystals in shape from 100 to 700 nm in size as a function of the source of cadmium and substrate used. FTIR results showed that 3 extra bands (1370, 848 and 688cm-1) corresponding to CO32- anion appeared once CdCO3 was synthesized. TEM revealed the bioaccumulation of CdCO3 on the bacterial cell wall and the synthesis of crystals smaller than 100 nm. The synthesis of CdCO3 crystals was associated with K. pneumoniae and E. coli, which had a synergetic effect during CdCO3 precipitation. Furthermore, the pH increase around 8.9 ± 2.5 in all the cases suggested that the precipitation of CdCO3 was through ureolysis.
Keywords: cadmium precipitation; CdCO3 crystals characterization; anaerobic sludge; K. pneumoniae, E. coli
1
Journal Pre-proof 1. Introduction In recently decades, the synthesis of materials with chemical formula CdXO3 (X = Ge, C, Si, Sn, and Pb) has attracted great interest due to their technological applications as anode material for Li-ion battery, chemical sensing, catalysis, luminescence, magnetic materials and precursor for preparing CdO [1,2]. In particular, cadmium carbonate (CdCO3) has been used as core material for fabrication of hollow polyelectrolyte capsules, solid phase reactor for indirect determination of cyanide, and a precursor for the preparation of technologically significant CdO nanostructured [1]. Likewise, Cd is used in the electroplating industry, pigments nickel–Cd batteries, dyes, plastics, pesticides and textile operations [3]. Different methods have been introduced to prepare cadmium carbonate, including the hydrothermal method [2,4,5], chemical bath [6,7], sonochemical method [8,9], and biomimetic synthesis using doublehydrophilic block copolymers [10]. Among these physico-chemical methods, the hydrothermal method is the most used due to induce the formation of high-crystallized powders with narrow particle size distribution and high purity. Adjusting the synthesis conditions as temperature, reaction time, pH and composition of the solution, the properties such as particle shape and size of the products can be controlled [4]. Well-defined superficial morphologies such as, nanobelts, nanowires, nanorolls and hierarchical structures are obtained by hydrothermal method [4,5]. In some cases, the use of organicinorganic ligands during the synthesis of CdCO3 has played an important role in determining not only the shape of the compounds but also the size [7,10]. Despite these promising results to synthesis CdCO3, all these synthesis procedures are energy-consuming and use chemicals that are toxic to the environment and are biologic hazards. [11]. In this context, environmentally friendly methods have been explored as feasible options to conventional methods with this purpose. Biological processes that bind metals and form minerals represent a fundamental part of key biogeochemical cycles [12]. In particular, the microbially induced carbonate precipitation is the process by which microorganisms form and precipitate minerals in the form of carbonates salts [13]. Some bacterial species specifically pure cultures have been used for microbially induced cadmium precipitation (MICP). Different studies have reported that pure cultures can mediate the precipitation of 2
Journal Pre-proof CdCO3. For example, Terrabacter tumescens, a urease-producing bacterium removed more than 90% Cd efficiently within 72h. The Cd in solution precipitated as spherical crystals of cadmium carbonate smaller than 50 µm in size [12]. Similar results were observed in the presence of Lysinibacillus sphaericus CH-5, which removed 99.95 % of Cd at 2 g/L in 48 h. The precipitated Cd was mostly spherical with a diameter of approximately 10–40 μm [14]. The precipitation of CdCO3 was also observed at low temperature (10°C) in the presence of Exiguobacterium undae YR10 [15], suggesting all these results, the enormous potential of pure cultures to precipitate Cd2+ and recover it as CdCO3, a material of great interest due to their technological applications. In contrast to pure cultures, the use of mixed microbial consortia offers attractive advantages since aseptic conditions are not necessary. Different studies have demonstrated that the metabolic versatility and synergistic interactions among bacteria favor the tolerance against toxic compounds present in the medium [16]. Thanks to these interactions, mixed microbial consortia, either aerobic and anaerobic granular sludges, have been able to remove recalcitrant pollutants from contaminated effluents [17–19]. However, certain aerobically recalcitrant contaminants are biodegraded under strictly anaerobic conditions and low removal efficiencies are achieved because oxygen is a more effective electron acceptor, therefore having more preference for reducing equivalents than those recalcitrant contaminants [20]. Many emerging organic and inorganic pollutants have been reduced under anaerobic conditions, such as mono- and polycyclic aromatic compounds, saturated aliphatic and cyclic hydrocarbons (alkanes), polyhalogenated hydrocarbons, alcohols, aldehydes and phenols, aromatic compounds with amine and nitro groups, azo groups, ethers, pharmaceuticals, personal care products and metalloids [19,20]. Thus, the ability of anaerobic consortia to remove recalcitrant pollutants in oxygen-free habitats, represent a bioremediation strategy of environmental matrices, such as soils, sediments, aquifers and wastewater contaminated. During the last decade, evidence has showed the great potential of pure cultures to form insoluble-metal carbonates as an important mechanism to decontaminate soils, sediments and contaminated effluents with As, Zn, Cu, Sr, Cr, Pb, and Cd [12]. Despite the advantage of using mixed microbial consortia in contrast to pure cultures, the microbially induced carbonate precipitation in the presence of an anaerobic granular sludge has not been 3
Journal Pre-proof documented. In this context, it is essential to explore the ability of an anaerobic granular sludge to precipitate Cd2+ from effluents contaminated and recover it as CdCO3. Furthermore, it is crucial to demonstrate that under this bioremediation strategy, it will be possible to recover a material of technological interest capable of competing with those synthesized by physico-chemical methods. Therefore, the objectives of this work were to investigate microbially induced cadmium precipitation using an anaerobic granular sludge and to characterize the crystals formed. Firstly, cadmium ion (Cd2+) utilization and pH changes in a liquid medium were evaluated to verify the removal of cadmium by the anaerobic granular sludge. Then, the biosynthesis of CdCO3 was proved by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), transmission electron microscope (TEM), Xray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Finally, the bacteria associated with CdCO3 precipitation were identified to propose the mechanisms of synthesis. To the best of our knowledge, this is the first work to evaluate the synthesis of CdCO3 by an anaerobic granular sludge as a strategy to remove this metal of contaminated effluents. 2. Experimental procedure 2.1.
Inoculum and basal medium
Anaerobic granular sludge samples were collected from a full-scale upflow anaerobic sludge bed (UASB) reactors treating effluents from a tequila factory (Jalisco, Mexico). The content of volatile solids (VS) in these anaerobic consortia was 10% (wt/wt). The basal medium used in all batch experiments contained (g l-1): NaHCO3 (5); NH4Cl (0.03); K2HPO4 (0.02); MgCl2*6H2O (0.012); CaCl2*2H2O (0.005); Na2S (0.013); and 1 ml l-1 of both trace elements and vitamins [21]. The basal medium was flushed with N2/CO2 (80/20) by passing this gas mixture through the liquid bulk for 5 min.
2.2.
Bioassays of Cd precipitation by anaerobic granular sludge
Incubations were conducted in batch mode by duplicate in glass serum bottles with a liquid volume of 30 mL. Acetate and glucose were evaluated as carbon sources. The basal medium was transferred directly to the vials, and then, 2 g of volatile suspended solids (VSS) l-1 and 2 g l-1 of glucose or acetate 4
Journal Pre-proof as carbon sources were added. Two sources of cadmium were evaluated, CdSO4·H2O and CdCl2·H2O at a final concentration of 33 mM. Vials were then flushed with N2/CO2 (80/20) for 5 min and sealed with butyl rubber stoppers and aluminum. All bioassays were incubated at 30 °C for 20 hours. Sterile controls without inoculum were also included in the experimental protocol in order to identify potential physicochemical processes (e.g. adsorption) involved during cadmium precipitation.
2.3.
Isolation and identification of bacteria
The bacterial suspensions from different treatments previously homogenized were incubated in 3 mL of liquid brain heart infusion (BHI) medium at 36°C for 24 h. Later, the cultures were incubated at 28°C for three days. The colonies obtained were purified by a successive spread in the same culture medium for their subsequent identification in the following selective media: Iron Agar Kligler, SIM (Sulfide Indol-Mobility) and OF (oxidation-fermentation) for Pseudomonas. All biochemical tests also performed for the identification of Enterobacteriaceae [22].
2.4.
Analytical techniques
Once finish the bioassays, the supernatant was separated and the precipitate was dried in an oven at 60°C for one day. The Cd concentrations and pH of liquid samples were analyzed by ICP-MS (ELANDRC II, PerkinElmer) and by a 530 pH meter (Corning Pinnacle). The precipitated was washed with chloroform-ethanol (1:1) and then dried with acetone, and subsequently dried at 60°C for one day. The structure and composition of the precipitated were characterized by X-ray powder diffraction (Empyream X-Ray diffractometer) equipment with a Cu-target at 45 kV and 40 mA and the IR spectra were recorded in the 400 – 4000 cm-1 range with a resolution of 4 cm-1, using Nicolet IS10 (Thermo Scientific) spectrometer. The morphological characteristics and size of precipitated were observed with a scanning electron microscope Inspect F50 operated at 30kV. The precipitated was resuspended in acetone (1mL) and collected on Si grid. TEM images were obtained with a Transmission Electron Microscope (TEM JEM-JEOL-2100) operated at 80kV. Samples were collected on nickel grids (mesh size, 200 μm) covered with a carbon-coated Formvar film, and stained with 2 % uranyl acetate. 5
Journal Pre-proof 2.5.
Statistical analysis
All the experiments were performed in triplicates. Error bars on graphs show the standard deviations of measurements. The data were analyzed by analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) version 20.0.
3. Results and discussions 3.1.
Cadmium ion utilization by anaerobic granular sludge
Cadmium ion (Cd2+) utilization in a liquid medium (1a and b) and pH changes (1c and d) were evaluated to verify the precipitation of cadmium by the anaerobic granular sludge under anaerobic conditions with CdSO4 and CdCl2 as precursors glucose and acetate as substrates. As shows the Fig. 1, complete cadmium utilization was achieved after 18 hours (97.4 ± 1.1) of continuous anaerobic incubation, and not differences were observed neither cadmium source nor substrate. In the sterile controls, not utilization was observed. With the decrease of cadmium in a liquid medium, the pH increased around 8.9 ± 2.5 in all the cases. This increase of pH is due to the formation of some intermediate products during the cadmium precipitation via ureolysis, such as bicarbonate, ammonium and hydroxyl ions [12, 15]. Although different metabolic pathways have been proposed for carbonate precipitation, the precipitation through ureolysis is the most straightforward and easily controlled mechanism to induce carbonate precipitation [23]. Through this pathway some pure cultures as Lysinibacillus sphaericus CH5 [14], Terrabacter tumescens [12] and Exiguobacterium undae YR10 [15] precipitated cadmium as CdCO3. For example, Lysinibacillus sphaericus CH-5 a bacterium isolated from an abandoned mine site with high urease activity (2.41 µmol min-1), removed 99.8% of Cd after 48 hours. However, once 2 g l1
Cd was added to the medium, the decrease of the growth of this bacterium was evident [14].
Exiguobacterium undae YR10 also precipitated Cd from a soil artificially contaminated with 100 mg CdSO4 kg-1 soil after 2 weeks [15]. In that last investigation, the cadmium precipitated as CdCO3 and co-precipitated with calcite (CaCO3). Although CdCO3 is sparsely soluble in the medium, it may combine with CaCO3 and remain immobilized. However, in the present investigation the concentration of calcium in the medium is very low to favor the co-precipitation with cadmium. 6
Journal Pre-proof 3.2.
Cadmium carbonate crystals produced by anaerobic granular sludge
In order to evaluate the precipitation of Cd2+ as CdCO3 by anaerobic granular sludge, all the samples were evaluated by FTIR. FTIR spectra of CdCO3 in presence of CdSO4 and CdCl2 are showed in Fig. 2a and 2b respectively. The absorption peak (Fig. 2a) around 1132 cm-1 appear due to SO42- groups present in CdSO4 as expected [24]. On the other hand, the FTIR spectrum of CdCO3 crystals is shown in the same Figure (bottom) where changes are observed. Once the anaerobic granular sludge precipitated the cadmium in presence of glucose, the adsorption peak of SO42- decreased considerably and extra bands correspond to CdCO3 appeared. The absorption band located at 1370 cm-1 corresponds to the asymmetric stretching vibration of CO32- anion, while bands located at 848 cm-1 and 688 cm-1 are assigned to the bending out of plane vibration and in-plane vibrations, respectively [1]. As shown in Fig. 2b, in the presence of acetate, CdCO3 crystals were synthesized and not differences in the FTIR spectrum were observed. Similar results were obtained when CdCl2 was used as a precursor (Fig. 2b), bands correspond to CdCO3 appeared after the incubation with both substrates. An structural analysis was carried out with XRD to confirmed the CdCO3 synthesis from CdSO4 and CdCl2 precursors. Fig. 3 a and 3b shows the obtained XRD patterns of CdCO3 in the presence of CdSO4 and CdCl2, respectively. In all cases evaluated, the obtained patterns were well indexed to the hexagonal crystal of CdCO3 [JCPDS. 72- 1939] with an R-3c space group (rhombohedral lattice). The average crystallite sizes of the CdCO3 crystals were calculated from X-ray line broadening of the (012) and (104) reflections, respectively, by using the Scherer’s equation: kλ/(dcosθ). In that equation λ is the wavelength of the X-ray radiation, k is a constant taken as∼0.9, θ is the diffraction angle, and d is the full width at half-maximum and (FWHM) for the CdCO3 crystals. The crystallite sizes of the CdCO3 obtained under all conditions evaluated were similar and no significant differences were observed (Table 1). The XRD patterns also confirmed that under conditions evaluated in this work, there had not peaks associated with precipitation of additional metals such as calcium. The corresponding SEM micrographs of the samples in the presence of CdSO4 and CdCl2 are shown in Fig. 4. The sample with CdSO4 precursor showed that crystals produced during anaerobic incubation with glucose (Fig. 4a) were rhombohedral and generally 500 nm in size. Similar results were observed with glucose in the presence of CdCl2 as a precursor (Fig. 4c). In contrast to these results, rhombohedral 7
Journal Pre-proof crystals around from 300 to 500 nm were observed in the presence of CdSO4 and acetate as substrate (Fig. 4b), while in the presence of CdCl2 rhombohedral crystals from 100 to 700 nm were observed (Fig. 4d). EDS patterns of CdCO3 crystals is depicted in Table 2. EDS results showed that Cd, O, and C were the main elements presents in media amended with CdSO4 and CdCl2 (Table 2). The additional peak of Si obtained was due to Si grid used for the sampling. In contrast to the results obtained in this investigation, different morphologies have been reported during cadmium precipitation in the presence of pure culture. For example, with T. tumescens and L. sphaericus the precipitated obtained were spheres in shape approximately of 10 and 40 µm respectively with CdCl2 as the precursor [12,14]. Irregular shape around 100 nm was also observed in the presence of Proteus mirabilis and Cd-acetate as Cd source [3]. In this sense, the source of cadmium and substrate may favor the synthesis of crystals of different morphologies and sizes. The metabolic versatility and synergistic interactions among bacteria of anaerobic sludge favored the synthesis of crystals of different sizes, which could represent an advantage due to broadly technological and environmental applications of these materials. TEM micrographs also confirmed the precipitation of CdCO3 on the bacterial cell wall and the synthesis of crystals smaller than 100 nm in the presence of both precursors with acetate as substrate (Fig. 5a and b). 3.3.
Identification of bacteria associated to synthesis of cadmium carbonate crystals
The identification assays revealed the presence of Klebsiella pneumoniae and Escherichia coli in all the samples evaluated. K. pneumoniae has urease activity [25,26]. Through this pathway, the urease produced by bacterium breakdowns the urea to produce NH4+ and CO32 -, which react with cadmium to form CdCO3. The changes in the pH support that the precipitation of CdCO3 was through ureolysis. In contrast to K. pneumoniae, E. coli, a non-urease producing bacterium cannot precipitate CdCO3 on its own because it does not decompose urea. Recently, it was reported that non-ureolityc bacteria were able to precipitate calcium as calcium carbonate under aerobic conditions [27–29]. For example, during the precipitation of calcium in presence of Lysinibacillus sp. YS11, this bacterium interacted with
8
Journal Pre-proof calcium during its exponential growth phase by binding positively charged calcium ions on net negatively charged cell walls and then gradually releasing them. During that same period, there was more biofilm and EPS formation, suggesting this results that calcium ions might alter bacterial surface properties and EPS-mediated bacteria calcium carbonate composites [27]. Similar results were reported in the presence of Sporosarcina pasteurii a urease-producing bacterium and Bacillus thuringiensis a non-urease producing bacterium [28]. The cocultivation of S. pasteurii and B. thuringiensis (8:2) had a synergetic effect on CaCO3 precipitation attributed to the presence of more nucleation sites by the presence of B. thuringiensis. The surface of S. pasteurii is saturated due to the precipitation of calcium with carbonate formed upon urea degradation by the release of urease by this bacterium. In contrast, B. thuringiensis enhances binding capacity for calcium and by carbonate formed by S. pasteurii, providing additional nucleation sites outside of those offered by S. pasteurii embedded in CaCO3 and improve the precipitation of CaCO3 [28]. Thus, considering these previous reports, probably K. pneumoniae and E. coli had a synergetic effect on CdCO3 precipitation. Urease produced by K. pneumoniae catalyzed the hydrolysis of urea to generate CO2 and ammonia that increased pH to produce supersaturation of Cd2+ and CO32- around of its cell membrane. Escherichia coli with its exopolymers attracted the Cd2+ near its own cell wall, which reacted with CO32- produced by K. pneumoniae to precipitate as CdCO3. Possible biochemical reactions to precipitate CdCO3 at the cell surface in the presence of both bacteria can be summarized as follows: 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐶𝑑2 + → 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 ― 𝐶𝑑2 + 𝐻2𝑁 ― 𝐶𝑂 ― 𝑁𝐻2 ↔2𝑁𝐻4+ + 𝐶𝑂23 ― 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐸. 𝑐𝑜𝑙𝑖 ― 𝐶𝑑2 + + 𝐶𝑂23 ― →𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐸. 𝑐𝑜𝑙𝑖 + 𝐶𝑑𝐶𝑂3 ↓ However, are necessarily more studies to understand the role of E. coli to precipitate CdCO3 under anaerobic conditions. Although the metal precipitation via bacterial ureolysis is an efficient strategy for metal bioremediation, the urea is not ubiquitous in natural environments [27]. Thus, more studies are necessary to comprehend the mechanisms of non-urease producing bacteria to precipitate metal carbonates. 4. Conclusions 9
Journal Pre-proof This study showed that Cd2+ can be precipitated under anaerobic conditions in the presence of a ureaseproducing bacterium and a non-urease producing bacterium. Although the precipitation of CdCO3 was through ureolysis, the synergistic interactions between both bacteria favored the cadmium precipitation under all conditions evaluated. In this sense, with the use of mixed microbial consortia for cadmium bioremediation, it will be possible to precipitate cadmium from soils, sediments and wastewater contaminated. Microbial synthesized CdCO3 could be used as a precursor of CdO nanoparticles used for solar cells, transparent electrode, and photodiodes. Furthermore, this mechanism of bioremediation could represent an important strategy to CO2 fixation in natural environments.
Acknowledgements
This study was financially supported by the Council of Science and Technology of Mexico (Postdoctoral fellowship number 206474).
References [1]
S. Ashoka, G. Nagaraju, K. V Thipperudraiah, G.T. Chandrappa, Controlled synthesis of cadmium carbonate nanowires, nanoribbons, nanorings and sphere like architectures via hydrothermal method Mater. Res. Bull., 45(11) (2010) 1736–1740. doi:10.1016/j.materresbull.2010.06.055.
[2]
P. Huang, X. Zhang, J. Wei, J. Pan, Y. Sheng, B. Feng, The preparation , characterization and optical properties of Cd2V2O7 and CdCO3 compounds Mater. Chem. Phys., 147(3) (2014) 996–1002. doi:10.1016/j.matchemphys.2014.06.050.
[3]
S. Kang, Y. Kim, Y. Lee, Y. Roh, Microbially Mediated-Precipitation of Cadmium Carbonate Nanoparticles J. Nanosci. Nanotechnol., 15(1) (2015) 418–420. doi:10.1166/jnn.2015.8379.
[4]
S. Ashoka, P. Chithaiah, G.T. Chandrappa, Studies on the synthesis of CdCO3 nanowires and porous CdO powder Mater. Lett., 64(2) (2010) 173–176. doi:10.1016/j.matlet.2009.10.036.
[5]
Z. Jia, Y. Tang, L. Luo, B. Li, Shape-Controlled Synthesis of Single-Crystalline CdCO3 and Corresponding Porous CdO Nanostructures Cryst. Growth Des., 8(7) (2008) 2116–2120.
[6]
G.E. Moreno Morales, M.E. Araiza Garcia, S. Cruz Cruz, B. Rebollo Plata, O. Portillo Moreno, R. Gutiérrez Pérez, CdCO3 nanocrystalline thin fi lm grown by chemical bath and its transition to porous CdO by thermal annealing treatment Optik (Stuttg)., 171 (2018) 347–355. doi:10.1016/j.ijleo.2018.06.068. 10
Journal Pre-proof [7]
O. Portillo Moreno, R. Gutiérrez Pérez, M. Chávez Portillo, M.E. Araiza García, G.E. Moreno, S. Cruz Cruz, E. Rubio Rosas, M. Hernandez Lascano, Morphological, optical and structural analysis in CdS, CdS-CdCO3 and CdCO3 thin solid films grown by chemical bath Optik (Stuttg)., 157 (2018) 388–399. doi:10.1016/j.ijleo.2017.11.036.
[8]
A. Askarinejad, A. Morsali, Syntheses and characterization of CdCO3 and CdO nanoparticles by using a sonochemical method Mater. Lett., 62(3) (2008) 478–482. doi:10.1016/j.matlet.2007.05.082.
[9]
A. Askarinejad, A. Morsali, Synthesis of cadmium (II) hydroxide, cadmium (II) carbonate and cadmium (II) oxide nanoparticles; investigation of intermediate products Chem. Eng. J. 150(23) (2009) 569–571., doi:10.1016/j.cej.2009.03.005.
[10] S.H. Yu, H. Colfen, M. Antonietti, Polymer-Controlled Morphosynthesis and Mineralization of Metal Carbonate Superstructures J. Phys. Chem. B., 107(30) (2003) 7396–7405. doi.org/10.1021/jp034009+. [11]
D. Baruah, M. Goswami, R.N.S. Yadav, A. Yadav, A.M. Das, Biogenic synthesis of gold nanoparticles and their application in photocatalytic degradation of toxic dyes J. Photochem. Photobiol. B., 186 (2018) 51–58. doi.org/10.1016/j.jphotobiol.2018.07.002.
[12]
M. Li, X. Cheng, H. Guo, Heavy metal removal by biomineralization of urease producing bacteria isolated from soil Int. Biodeterior. Biodegradation., 76 (2013) 81–85. doi:10.1016/j.ibiod.2012.06.016.
[13] D. Kumari, X.Y. Qian, X. Pan, V. Achal, Q. Li, G.M. Gadd, Microbially-induced Carbonate Precipitation for Immobilization of Toxic Metals Adv. Appl. Microbiol., 94(2016) 79-108. doi:10.1016/bs.aambs.2015.12.002. [14]
C.H. Kang, S.H. Han, Y. Shin, Y. Shin, S.J. Oh, J.S. So, Bioremediation of Cd by Microbially Induced Calcite Precipitation Appl Biochem Biotechnol., 172(4) (2014) 1929–1937. doi:10.1007/s12010-014-0737-1.
[15]
D. Kumari, X. Pan, D.J. Lee, V. Achal, Immobilization of cadmium in soil by microbially induced carbonate precipitation with Exiguobacterium undae at low temperature Int. Biodeterior., Biodegradation. 94 (2014) 98–102. doi:10.1016/j.ibiod.2014.07.007.
[16] A.M. Pat-Espadas, E. Razo-Flores, J.R. Rangel-Mendez, J.A. Ascacio Valdes, C.N. Aguila, F.J. Cervantes, Immobilization of biogenic Pd (0) in anaerobic granular sludge for the biotransformation of recalcitrant halogenated pollutants in UASB reactors Appl Microbiol Biotechnol., 100(3) (2016) 1427–1436. doi:10.1007/s00253-015-7055-6. [17] G. Yang, N. Zhang, J. Yang, Q. Fu, Y. Wang, D. Wang, L. Tang, J. Xia, X. Liu, X. Li, Q. Yang, Y. Liu, Q. Wang, B. Ni, Interaction between perfluorooctanoic acid and aerobic granular sludge Water Res. (2019) 115249. doi:10.1016/j.watres.2019.115249. [18]
N. Schmidt, D. Page, A. Tiehm, Biodegradation of pharmaceuticals and endocrine disruptors with oxygen , nitrate , manganese ( IV ), iron ( III ) and sulfate as electron acceptors J. Contam. 11
Journal Pre-proof Hydrol. 203 (2017) 62–69. doi:10.1016/j.jconhyd.2017.06.007. [19]
A.-K. Gattas, F. Fischer, A. Wick, T.A. Ternes, Anaerobic biodegradation of (emerging) organic contaminants in the aquatic environment Water Res. 116 (2017) 268–295. doi.org/10.1016/j.watres.2017.02.001.
[20]
F.P. Van der Zee, F.J. Cervantes, Impact and application of electron shuttles on the redox (bio)transformation of contaminants: A review Biotechnol. Adv. 27 (2009) 256–277. doi:10.1016/j.biotechadv.2009.01.004.
[21]
F.J. Cervantes, S. van der Velde, G. Lettinga, J.A. Field, Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia FEMS Microbilogy Ecol., 34 (2000) 161–171. doi:https://doi.org/10.1111/j.15746941.2000.tb00766.x.
[22]
M. de G. Moctezuma Zárate, J.F. Cárdenas González, A.S. Rodríguez Pérez, E.E. Domínguez, J. Tovar Oviedo, V.M. Martínez Juárez, I. Acosta Rodríguez, Isolation and Identification of Bacteria and Fungi Resistant to crude oil J. Multidiscip. Eng. Sci. Technol., 3 (2016) 5273– 5278. doi:ninive.uaslp.mx/jspui/handle/i/4265.
[23]
A.I. Omoregie, R.H. Ginjom, P.M. Nissom, Microbially Induced Carbonate Precipitation Via Ureolysis Process: A Mini-Review Trans. Sci. Technol., 5(4) (2018) 245–256. doi.org/10.1016/bs.aambs.2015.12.002.
[24]
G.R. Khayati, H. Dalvand, E. Darezereshki, A. Irannejad, A facile method to synthesis of CdO nanoparticles from spent Ni – Cd batteries Mater. Lett., 115 (2014) 272–274. doi:10.1016/j.matlet.2013.10.078.
[25]
A.L. Barry, K.L. Bernsohn, L.D. Thrupp, Four-hour urease test for distinguishing between Klebsiella and Enterobacter Appl. Microbiol., 18(2) (1969) 156–158. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=377935&tool=pmcentrez&renderty pe=abstract.
[26]
M. Umar, K.A. Kassim, Effect of Cementation Reagents Concentrations on Microbial Calcite Precipitation in Residual Soil Malaysian J. Civ., Eng. 29 Spec. Issue. (1) (2017) 79–90. doi:https://doi.org/10.11113/mjce.v29.134..
[27]
Y.S. Lee, H.J. Kim, W. Park, Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus J. Microbiol., 55(6) (2017) 440–447. doi:10.1007/s12275-017-7086-z.
[28]
H.M. Son, H.Y. Kim, S.M. Park, H.K. Lee, Ureolytic/Non-ureolytic bacteria co-cultured selfhealing agent for cementitious materials crack repair Materials (Basel)., 11(5) (2018) 782–795. doi:10.3390/ma11050782.
[29]
J. Xu, W. Yao, Z. Jiang, Non-Ureolytic Bacterial Carbonate Precipitation as a Surface Treatment Strategy on Cementitious Materials J. Mater. Civ., Eng. 26(5) (2014) 983–991. doi:10.1061/(ASCE)MT. 12
Journal Pre-proof
Table 1. Crystallite size using Sherrer equation.
CdCl2-a CdCl2-g CdSO4-a CdSO4-g
Plane (012) (104) (012) (104) (012) (104) (012) (104)
Grain size (nm) 24.5 ± 3.5 25.8 ± 5.3 25.3 ± 4.8 25.9 ± 3.7 25.8 ± 5.4 26.5 ± 6.1 25.0 ± 4.1 24.8 ± 3.2
a, acetate; g, glucose
13
Journal Pre-proof
Table 2. Results of quantitative EDS analyses of the samples. Precursor Carbon source
CdSO4 Glucose
CdCl2
Acetate
Glucose
acetate
Weight (%) C
15.81 ± 2.4
14.35 ± 4.2
19.5 ± 2.1
22.07 ± 2.1
O
7.71 ± 1.7
7.53 ± 1.6
4.11 ± 3.5
7.54 ± 1.8
Cd
4.59 ± 3.3
6.90 ± 2.5
1.50 ± 5.1
4.65 ± 1.9
14
Journal Pre-proof
Figure captions Fig. 1. Time in course of Cd2+ utilization (close figures) and pH change (open figures) in the presence of anaerobic granular sludge in liquid medium with (a) CdSO4 and (b) CdCl2 as precursors. Fig. 2. FT-IR spectrum of CdCO3 crystals by anaerobic granular sludge in presence of CdSO4 (a) and CdCl2 (b) as precursors and glucose and acetate as substrate.
Fig. 3. XRD patterns of CdCO3 crystals by anaerobic granular sludge in presence of CdSO4 (a) and CdCl2 (b) as precursors and glucose and acetate as substrate. Fig. 4. SEM images of CdCO3 crystals in presence of CdSO4 with (a) glucose, (b) acetate and CdCl2 with (c) glucose, (d) acetate as substrates by anaerobic granular sludge. Fig. 5. TEM images of CdCO3 crystals in presence of (a) CdSO4 (b) CdCl2 with acetate as substrate. The arrows indicate the formation of precipitated smaller than 100 nm. c) Precipitation of CdCO3 on the bacterial cell wall.
15
Journal Pre-proof
Fig. 1
16
Journal Pre-proof
Fig. 2
17
Journal Pre-proof
Fig. 3
18
Journal Pre-proof
Fig. 4
19
Journal Pre-proof
Fig. 5
20
Journal Pre-proof CrediT author statement
CM Martinez Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft and Writing - Review & Editing.
M. Rivera-Hernández Investigation
Luis H-Alvarez Investigation
Ismael Acosta-Rodriguez Funding acquisition and Supervision
F. Ruiz Funding acquisition
V.D. Compeán-García Conceptualization, Formal analysis, Resources and Writing - Review & Editing
Journal Pre-proof Highlights
An anaerobic granular sludge was able to precipitation Cd2+ as CdCO3 crystals.
No differences were observed neither cadmium source nor substrate.
Cd2+ precipitated as CdCO3 rhombohedral crystals from 100 to 700 nm.
Synthesis was associated to K. pneumoniae and E. coli.
The co-precipitation of cadmium with other metals was not observed.
C.M. Martínez, M. Rivera-Hernández, Luis H. Álvarez, Ismael Acosta-Rodríguez, F. Ruíz, V.D. Compeán-García PII:
S0254-0584(20)30176-0
DOI:
https://doi.org/10.1016/j.matchemphys.2020.122797
Reference:
MAC 122797
To appear in:
Materials Chemistry and Physics
Received Date:
16 December 2019
Accepted Date:
12 February 2020
Please cite this article as: C.M. Martínez, M. Rivera-Hernández, Luis H. Álvarez, Ismael AcostaRodríguez, F. Ruíz, V.D. Compeán-García, Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium, Materials Chemistry and Physics (2020), https://doi.org/10.1016/j.matchemphys.2020.122797
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Biosynthesis and characterization of cadmium carbonate crystals by anaerobic granular sludge capable of precipitate cadmium CM Martínez*, M. Rivera-Hernández, Luis H. Álvarez, Ismael Acosta-Rodríguez, Ruíz F, VD Compeán-García**
M. Rivera-Hernández Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E.mail: [email protected] Luis H. Álvarez Departamento de Ciencias Agronómicas y Veterinarias, Instituto Tecnológico de Sonora, 5 de Febrero 818 Sur, Ciudad Obregón, Sonora, México 85000. Email: [email protected] Ismael Acosta-Rodríguez Facultas de Ciencias Químicas, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México. ZP. 78290. E.mail: [email protected] Ruíz F. Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E.mail: [email protected] Corresponding authors: CM Martínez Facultad de Ciencias, Universidad Autónoma de San Luis Potosí. Lateral Av. Salvador Nava Martínez S/N, Zona Universitaria, San Luis Potosí, SLP México 78290. E-mail: *[email protected]. Tel.: +52 (444) 3813330 VD. Compeán-García CONACyT-Coordinación para la Innovación y Aplicación de la Ciencia y la Tecnología. Universidad Autónoma de San Luis Potosí. Sierra Leona #550, Lomas de San Luis, 78210 San Luis Potosí E-mail: **[email protected]
Journal Pre-proof Abstract Microbially induced carbonate precipitation (MICP) has been exploited as an efficient strategy to immobilize toxic metals in the form of carbonate salts. The present study investigated the cadmium carbonate (CdCO3) precipitation induced by an anaerobic granular sludge. The results revealed that anaerobic sludge showed high Cd utilization efficiencies (97.4% ± 1.1) after 18 hours of incubation at 30°C, and no differences were observed neither cadmium source nor substrate. According to SEM and X-ray diffraction results, the anaerobic sludge was able to precipitate Cd2+ as CdCO3 rhombohedral crystals in shape from 100 to 700 nm in size as a function of the source of cadmium and substrate used. FTIR results showed that 3 extra bands (1370, 848 and 688cm-1) corresponding to CO32- anion appeared once CdCO3 was synthesized. TEM revealed the bioaccumulation of CdCO3 on the bacterial cell wall and the synthesis of crystals smaller than 100 nm. The synthesis of CdCO3 crystals was associated with K. pneumoniae and E. coli, which had a synergetic effect during CdCO3 precipitation. Furthermore, the pH increase around 8.9 ± 2.5 in all the cases suggested that the precipitation of CdCO3 was through ureolysis.
Keywords: cadmium precipitation; CdCO3 crystals characterization; anaerobic sludge; K. pneumoniae, E. coli
1
Journal Pre-proof 1. Introduction In recently decades, the synthesis of materials with chemical formula CdXO3 (X = Ge, C, Si, Sn, and Pb) has attracted great interest due to their technological applications as anode material for Li-ion battery, chemical sensing, catalysis, luminescence, magnetic materials and precursor for preparing CdO [1,2]. In particular, cadmium carbonate (CdCO3) has been used as core material for fabrication of hollow polyelectrolyte capsules, solid phase reactor for indirect determination of cyanide, and a precursor for the preparation of technologically significant CdO nanostructured [1]. Likewise, Cd is used in the electroplating industry, pigments nickel–Cd batteries, dyes, plastics, pesticides and textile operations [3]. Different methods have been introduced to prepare cadmium carbonate, including the hydrothermal method [2,4,5], chemical bath [6,7], sonochemical method [8,9], and biomimetic synthesis using doublehydrophilic block copolymers [10]. Among these physico-chemical methods, the hydrothermal method is the most used due to induce the formation of high-crystallized powders with narrow particle size distribution and high purity. Adjusting the synthesis conditions as temperature, reaction time, pH and composition of the solution, the properties such as particle shape and size of the products can be controlled [4]. Well-defined superficial morphologies such as, nanobelts, nanowires, nanorolls and hierarchical structures are obtained by hydrothermal method [4,5]. In some cases, the use of organicinorganic ligands during the synthesis of CdCO3 has played an important role in determining not only the shape of the compounds but also the size [7,10]. Despite these promising results to synthesis CdCO3, all these synthesis procedures are energy-consuming and use chemicals that are toxic to the environment and are biologic hazards. [11]. In this context, environmentally friendly methods have been explored as feasible options to conventional methods with this purpose. Biological processes that bind metals and form minerals represent a fundamental part of key biogeochemical cycles [12]. In particular, the microbially induced carbonate precipitation is the process by which microorganisms form and precipitate minerals in the form of carbonates salts [13]. Some bacterial species specifically pure cultures have been used for microbially induced cadmium precipitation (MICP). Different studies have reported that pure cultures can mediate the precipitation of 2
Journal Pre-proof CdCO3. For example, Terrabacter tumescens, a urease-producing bacterium removed more than 90% Cd efficiently within 72h. The Cd in solution precipitated as spherical crystals of cadmium carbonate smaller than 50 µm in size [12]. Similar results were observed in the presence of Lysinibacillus sphaericus CH-5, which removed 99.95 % of Cd at 2 g/L in 48 h. The precipitated Cd was mostly spherical with a diameter of approximately 10–40 μm [14]. The precipitation of CdCO3 was also observed at low temperature (10°C) in the presence of Exiguobacterium undae YR10 [15], suggesting all these results, the enormous potential of pure cultures to precipitate Cd2+ and recover it as CdCO3, a material of great interest due to their technological applications. In contrast to pure cultures, the use of mixed microbial consortia offers attractive advantages since aseptic conditions are not necessary. Different studies have demonstrated that the metabolic versatility and synergistic interactions among bacteria favor the tolerance against toxic compounds present in the medium [16]. Thanks to these interactions, mixed microbial consortia, either aerobic and anaerobic granular sludges, have been able to remove recalcitrant pollutants from contaminated effluents [17–19]. However, certain aerobically recalcitrant contaminants are biodegraded under strictly anaerobic conditions and low removal efficiencies are achieved because oxygen is a more effective electron acceptor, therefore having more preference for reducing equivalents than those recalcitrant contaminants [20]. Many emerging organic and inorganic pollutants have been reduced under anaerobic conditions, such as mono- and polycyclic aromatic compounds, saturated aliphatic and cyclic hydrocarbons (alkanes), polyhalogenated hydrocarbons, alcohols, aldehydes and phenols, aromatic compounds with amine and nitro groups, azo groups, ethers, pharmaceuticals, personal care products and metalloids [19,20]. Thus, the ability of anaerobic consortia to remove recalcitrant pollutants in oxygen-free habitats, represent a bioremediation strategy of environmental matrices, such as soils, sediments, aquifers and wastewater contaminated. During the last decade, evidence has showed the great potential of pure cultures to form insoluble-metal carbonates as an important mechanism to decontaminate soils, sediments and contaminated effluents with As, Zn, Cu, Sr, Cr, Pb, and Cd [12]. Despite the advantage of using mixed microbial consortia in contrast to pure cultures, the microbially induced carbonate precipitation in the presence of an anaerobic granular sludge has not been 3
Journal Pre-proof documented. In this context, it is essential to explore the ability of an anaerobic granular sludge to precipitate Cd2+ from effluents contaminated and recover it as CdCO3. Furthermore, it is crucial to demonstrate that under this bioremediation strategy, it will be possible to recover a material of technological interest capable of competing with those synthesized by physico-chemical methods. Therefore, the objectives of this work were to investigate microbially induced cadmium precipitation using an anaerobic granular sludge and to characterize the crystals formed. Firstly, cadmium ion (Cd2+) utilization and pH changes in a liquid medium were evaluated to verify the removal of cadmium by the anaerobic granular sludge. Then, the biosynthesis of CdCO3 was proved by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), transmission electron microscope (TEM), Xray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Finally, the bacteria associated with CdCO3 precipitation were identified to propose the mechanisms of synthesis. To the best of our knowledge, this is the first work to evaluate the synthesis of CdCO3 by an anaerobic granular sludge as a strategy to remove this metal of contaminated effluents. 2. Experimental procedure 2.1.
Inoculum and basal medium
Anaerobic granular sludge samples were collected from a full-scale upflow anaerobic sludge bed (UASB) reactors treating effluents from a tequila factory (Jalisco, Mexico). The content of volatile solids (VS) in these anaerobic consortia was 10% (wt/wt). The basal medium used in all batch experiments contained (g l-1): NaHCO3 (5); NH4Cl (0.03); K2HPO4 (0.02); MgCl2*6H2O (0.012); CaCl2*2H2O (0.005); Na2S (0.013); and 1 ml l-1 of both trace elements and vitamins [21]. The basal medium was flushed with N2/CO2 (80/20) by passing this gas mixture through the liquid bulk for 5 min.
2.2.
Bioassays of Cd precipitation by anaerobic granular sludge
Incubations were conducted in batch mode by duplicate in glass serum bottles with a liquid volume of 30 mL. Acetate and glucose were evaluated as carbon sources. The basal medium was transferred directly to the vials, and then, 2 g of volatile suspended solids (VSS) l-1 and 2 g l-1 of glucose or acetate 4
Journal Pre-proof as carbon sources were added. Two sources of cadmium were evaluated, CdSO4·H2O and CdCl2·H2O at a final concentration of 33 mM. Vials were then flushed with N2/CO2 (80/20) for 5 min and sealed with butyl rubber stoppers and aluminum. All bioassays were incubated at 30 °C for 20 hours. Sterile controls without inoculum were also included in the experimental protocol in order to identify potential physicochemical processes (e.g. adsorption) involved during cadmium precipitation.
2.3.
Isolation and identification of bacteria
The bacterial suspensions from different treatments previously homogenized were incubated in 3 mL of liquid brain heart infusion (BHI) medium at 36°C for 24 h. Later, the cultures were incubated at 28°C for three days. The colonies obtained were purified by a successive spread in the same culture medium for their subsequent identification in the following selective media: Iron Agar Kligler, SIM (Sulfide Indol-Mobility) and OF (oxidation-fermentation) for Pseudomonas. All biochemical tests also performed for the identification of Enterobacteriaceae [22].
2.4.
Analytical techniques
Once finish the bioassays, the supernatant was separated and the precipitate was dried in an oven at 60°C for one day. The Cd concentrations and pH of liquid samples were analyzed by ICP-MS (ELANDRC II, PerkinElmer) and by a 530 pH meter (Corning Pinnacle). The precipitated was washed with chloroform-ethanol (1:1) and then dried with acetone, and subsequently dried at 60°C for one day. The structure and composition of the precipitated were characterized by X-ray powder diffraction (Empyream X-Ray diffractometer) equipment with a Cu-target at 45 kV and 40 mA and the IR spectra were recorded in the 400 – 4000 cm-1 range with a resolution of 4 cm-1, using Nicolet IS10 (Thermo Scientific) spectrometer. The morphological characteristics and size of precipitated were observed with a scanning electron microscope Inspect F50 operated at 30kV. The precipitated was resuspended in acetone (1mL) and collected on Si grid. TEM images were obtained with a Transmission Electron Microscope (TEM JEM-JEOL-2100) operated at 80kV. Samples were collected on nickel grids (mesh size, 200 μm) covered with a carbon-coated Formvar film, and stained with 2 % uranyl acetate. 5
Journal Pre-proof 2.5.
Statistical analysis
All the experiments were performed in triplicates. Error bars on graphs show the standard deviations of measurements. The data were analyzed by analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) version 20.0.
3. Results and discussions 3.1.
Cadmium ion utilization by anaerobic granular sludge
Cadmium ion (Cd2+) utilization in a liquid medium (1a and b) and pH changes (1c and d) were evaluated to verify the precipitation of cadmium by the anaerobic granular sludge under anaerobic conditions with CdSO4 and CdCl2 as precursors glucose and acetate as substrates. As shows the Fig. 1, complete cadmium utilization was achieved after 18 hours (97.4 ± 1.1) of continuous anaerobic incubation, and not differences were observed neither cadmium source nor substrate. In the sterile controls, not utilization was observed. With the decrease of cadmium in a liquid medium, the pH increased around 8.9 ± 2.5 in all the cases. This increase of pH is due to the formation of some intermediate products during the cadmium precipitation via ureolysis, such as bicarbonate, ammonium and hydroxyl ions [12, 15]. Although different metabolic pathways have been proposed for carbonate precipitation, the precipitation through ureolysis is the most straightforward and easily controlled mechanism to induce carbonate precipitation [23]. Through this pathway some pure cultures as Lysinibacillus sphaericus CH5 [14], Terrabacter tumescens [12] and Exiguobacterium undae YR10 [15] precipitated cadmium as CdCO3. For example, Lysinibacillus sphaericus CH-5 a bacterium isolated from an abandoned mine site with high urease activity (2.41 µmol min-1), removed 99.8% of Cd after 48 hours. However, once 2 g l1
Cd was added to the medium, the decrease of the growth of this bacterium was evident [14].
Exiguobacterium undae YR10 also precipitated Cd from a soil artificially contaminated with 100 mg CdSO4 kg-1 soil after 2 weeks [15]. In that last investigation, the cadmium precipitated as CdCO3 and co-precipitated with calcite (CaCO3). Although CdCO3 is sparsely soluble in the medium, it may combine with CaCO3 and remain immobilized. However, in the present investigation the concentration of calcium in the medium is very low to favor the co-precipitation with cadmium. 6
Journal Pre-proof 3.2.
Cadmium carbonate crystals produced by anaerobic granular sludge
In order to evaluate the precipitation of Cd2+ as CdCO3 by anaerobic granular sludge, all the samples were evaluated by FTIR. FTIR spectra of CdCO3 in presence of CdSO4 and CdCl2 are showed in Fig. 2a and 2b respectively. The absorption peak (Fig. 2a) around 1132 cm-1 appear due to SO42- groups present in CdSO4 as expected [24]. On the other hand, the FTIR spectrum of CdCO3 crystals is shown in the same Figure (bottom) where changes are observed. Once the anaerobic granular sludge precipitated the cadmium in presence of glucose, the adsorption peak of SO42- decreased considerably and extra bands correspond to CdCO3 appeared. The absorption band located at 1370 cm-1 corresponds to the asymmetric stretching vibration of CO32- anion, while bands located at 848 cm-1 and 688 cm-1 are assigned to the bending out of plane vibration and in-plane vibrations, respectively [1]. As shown in Fig. 2b, in the presence of acetate, CdCO3 crystals were synthesized and not differences in the FTIR spectrum were observed. Similar results were obtained when CdCl2 was used as a precursor (Fig. 2b), bands correspond to CdCO3 appeared after the incubation with both substrates. An structural analysis was carried out with XRD to confirmed the CdCO3 synthesis from CdSO4 and CdCl2 precursors. Fig. 3 a and 3b shows the obtained XRD patterns of CdCO3 in the presence of CdSO4 and CdCl2, respectively. In all cases evaluated, the obtained patterns were well indexed to the hexagonal crystal of CdCO3 [JCPDS. 72- 1939] with an R-3c space group (rhombohedral lattice). The average crystallite sizes of the CdCO3 crystals were calculated from X-ray line broadening of the (012) and (104) reflections, respectively, by using the Scherer’s equation: kλ/(dcosθ). In that equation λ is the wavelength of the X-ray radiation, k is a constant taken as∼0.9, θ is the diffraction angle, and d is the full width at half-maximum and (FWHM) for the CdCO3 crystals. The crystallite sizes of the CdCO3 obtained under all conditions evaluated were similar and no significant differences were observed (Table 1). The XRD patterns also confirmed that under conditions evaluated in this work, there had not peaks associated with precipitation of additional metals such as calcium. The corresponding SEM micrographs of the samples in the presence of CdSO4 and CdCl2 are shown in Fig. 4. The sample with CdSO4 precursor showed that crystals produced during anaerobic incubation with glucose (Fig. 4a) were rhombohedral and generally 500 nm in size. Similar results were observed with glucose in the presence of CdCl2 as a precursor (Fig. 4c). In contrast to these results, rhombohedral 7
Journal Pre-proof crystals around from 300 to 500 nm were observed in the presence of CdSO4 and acetate as substrate (Fig. 4b), while in the presence of CdCl2 rhombohedral crystals from 100 to 700 nm were observed (Fig. 4d). EDS patterns of CdCO3 crystals is depicted in Table 2. EDS results showed that Cd, O, and C were the main elements presents in media amended with CdSO4 and CdCl2 (Table 2). The additional peak of Si obtained was due to Si grid used for the sampling. In contrast to the results obtained in this investigation, different morphologies have been reported during cadmium precipitation in the presence of pure culture. For example, with T. tumescens and L. sphaericus the precipitated obtained were spheres in shape approximately of 10 and 40 µm respectively with CdCl2 as the precursor [12,14]. Irregular shape around 100 nm was also observed in the presence of Proteus mirabilis and Cd-acetate as Cd source [3]. In this sense, the source of cadmium and substrate may favor the synthesis of crystals of different morphologies and sizes. The metabolic versatility and synergistic interactions among bacteria of anaerobic sludge favored the synthesis of crystals of different sizes, which could represent an advantage due to broadly technological and environmental applications of these materials. TEM micrographs also confirmed the precipitation of CdCO3 on the bacterial cell wall and the synthesis of crystals smaller than 100 nm in the presence of both precursors with acetate as substrate (Fig. 5a and b). 3.3.
Identification of bacteria associated to synthesis of cadmium carbonate crystals
The identification assays revealed the presence of Klebsiella pneumoniae and Escherichia coli in all the samples evaluated. K. pneumoniae has urease activity [25,26]. Through this pathway, the urease produced by bacterium breakdowns the urea to produce NH4+ and CO32 -, which react with cadmium to form CdCO3. The changes in the pH support that the precipitation of CdCO3 was through ureolysis. In contrast to K. pneumoniae, E. coli, a non-urease producing bacterium cannot precipitate CdCO3 on its own because it does not decompose urea. Recently, it was reported that non-ureolityc bacteria were able to precipitate calcium as calcium carbonate under aerobic conditions [27–29]. For example, during the precipitation of calcium in presence of Lysinibacillus sp. YS11, this bacterium interacted with
8
Journal Pre-proof calcium during its exponential growth phase by binding positively charged calcium ions on net negatively charged cell walls and then gradually releasing them. During that same period, there was more biofilm and EPS formation, suggesting this results that calcium ions might alter bacterial surface properties and EPS-mediated bacteria calcium carbonate composites [27]. Similar results were reported in the presence of Sporosarcina pasteurii a urease-producing bacterium and Bacillus thuringiensis a non-urease producing bacterium [28]. The cocultivation of S. pasteurii and B. thuringiensis (8:2) had a synergetic effect on CaCO3 precipitation attributed to the presence of more nucleation sites by the presence of B. thuringiensis. The surface of S. pasteurii is saturated due to the precipitation of calcium with carbonate formed upon urea degradation by the release of urease by this bacterium. In contrast, B. thuringiensis enhances binding capacity for calcium and by carbonate formed by S. pasteurii, providing additional nucleation sites outside of those offered by S. pasteurii embedded in CaCO3 and improve the precipitation of CaCO3 [28]. Thus, considering these previous reports, probably K. pneumoniae and E. coli had a synergetic effect on CdCO3 precipitation. Urease produced by K. pneumoniae catalyzed the hydrolysis of urea to generate CO2 and ammonia that increased pH to produce supersaturation of Cd2+ and CO32- around of its cell membrane. Escherichia coli with its exopolymers attracted the Cd2+ near its own cell wall, which reacted with CO32- produced by K. pneumoniae to precipitate as CdCO3. Possible biochemical reactions to precipitate CdCO3 at the cell surface in the presence of both bacteria can be summarized as follows: 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐶𝑑2 + → 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 ― 𝐶𝑑2 + 𝐻2𝑁 ― 𝐶𝑂 ― 𝑁𝐻2 ↔2𝑁𝐻4+ + 𝐶𝑂23 ― 𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐸. 𝑐𝑜𝑙𝑖 ― 𝐶𝑑2 + + 𝐶𝑂23 ― →𝐾. 𝑝𝑛𝑒𝑢𝑚𝑜𝑛𝑖𝑎𝑒 + 𝐸. 𝑐𝑜𝑙𝑖 + 𝐶𝑑𝐶𝑂3 ↓ However, are necessarily more studies to understand the role of E. coli to precipitate CdCO3 under anaerobic conditions. Although the metal precipitation via bacterial ureolysis is an efficient strategy for metal bioremediation, the urea is not ubiquitous in natural environments [27]. Thus, more studies are necessary to comprehend the mechanisms of non-urease producing bacteria to precipitate metal carbonates. 4. Conclusions 9
Journal Pre-proof This study showed that Cd2+ can be precipitated under anaerobic conditions in the presence of a ureaseproducing bacterium and a non-urease producing bacterium. Although the precipitation of CdCO3 was through ureolysis, the synergistic interactions between both bacteria favored the cadmium precipitation under all conditions evaluated. In this sense, with the use of mixed microbial consortia for cadmium bioremediation, it will be possible to precipitate cadmium from soils, sediments and wastewater contaminated. Microbial synthesized CdCO3 could be used as a precursor of CdO nanoparticles used for solar cells, transparent electrode, and photodiodes. Furthermore, this mechanism of bioremediation could represent an important strategy to CO2 fixation in natural environments.
Acknowledgements
This study was financially supported by the Council of Science and Technology of Mexico (Postdoctoral fellowship number 206474).
References [1]
S. Ashoka, G. Nagaraju, K. V Thipperudraiah, G.T. Chandrappa, Controlled synthesis of cadmium carbonate nanowires, nanoribbons, nanorings and sphere like architectures via hydrothermal method Mater. Res. Bull., 45(11) (2010) 1736–1740. doi:10.1016/j.materresbull.2010.06.055.
[2]
P. Huang, X. Zhang, J. Wei, J. Pan, Y. Sheng, B. Feng, The preparation , characterization and optical properties of Cd2V2O7 and CdCO3 compounds Mater. Chem. Phys., 147(3) (2014) 996–1002. doi:10.1016/j.matchemphys.2014.06.050.
[3]
S. Kang, Y. Kim, Y. Lee, Y. Roh, Microbially Mediated-Precipitation of Cadmium Carbonate Nanoparticles J. Nanosci. Nanotechnol., 15(1) (2015) 418–420. doi:10.1166/jnn.2015.8379.
[4]
S. Ashoka, P. Chithaiah, G.T. Chandrappa, Studies on the synthesis of CdCO3 nanowires and porous CdO powder Mater. Lett., 64(2) (2010) 173–176. doi:10.1016/j.matlet.2009.10.036.
[5]
Z. Jia, Y. Tang, L. Luo, B. Li, Shape-Controlled Synthesis of Single-Crystalline CdCO3 and Corresponding Porous CdO Nanostructures Cryst. Growth Des., 8(7) (2008) 2116–2120.
[6]
G.E. Moreno Morales, M.E. Araiza Garcia, S. Cruz Cruz, B. Rebollo Plata, O. Portillo Moreno, R. Gutiérrez Pérez, CdCO3 nanocrystalline thin fi lm grown by chemical bath and its transition to porous CdO by thermal annealing treatment Optik (Stuttg)., 171 (2018) 347–355. doi:10.1016/j.ijleo.2018.06.068. 10
Journal Pre-proof [7]
O. Portillo Moreno, R. Gutiérrez Pérez, M. Chávez Portillo, M.E. Araiza García, G.E. Moreno, S. Cruz Cruz, E. Rubio Rosas, M. Hernandez Lascano, Morphological, optical and structural analysis in CdS, CdS-CdCO3 and CdCO3 thin solid films grown by chemical bath Optik (Stuttg)., 157 (2018) 388–399. doi:10.1016/j.ijleo.2017.11.036.
[8]
A. Askarinejad, A. Morsali, Syntheses and characterization of CdCO3 and CdO nanoparticles by using a sonochemical method Mater. Lett., 62(3) (2008) 478–482. doi:10.1016/j.matlet.2007.05.082.
[9]
A. Askarinejad, A. Morsali, Synthesis of cadmium (II) hydroxide, cadmium (II) carbonate and cadmium (II) oxide nanoparticles; investigation of intermediate products Chem. Eng. J. 150(23) (2009) 569–571., doi:10.1016/j.cej.2009.03.005.
[10] S.H. Yu, H. Colfen, M. Antonietti, Polymer-Controlled Morphosynthesis and Mineralization of Metal Carbonate Superstructures J. Phys. Chem. B., 107(30) (2003) 7396–7405. doi.org/10.1021/jp034009+. [11]
D. Baruah, M. Goswami, R.N.S. Yadav, A. Yadav, A.M. Das, Biogenic synthesis of gold nanoparticles and their application in photocatalytic degradation of toxic dyes J. Photochem. Photobiol. B., 186 (2018) 51–58. doi.org/10.1016/j.jphotobiol.2018.07.002.
[12]
M. Li, X. Cheng, H. Guo, Heavy metal removal by biomineralization of urease producing bacteria isolated from soil Int. Biodeterior. Biodegradation., 76 (2013) 81–85. doi:10.1016/j.ibiod.2012.06.016.
[13] D. Kumari, X.Y. Qian, X. Pan, V. Achal, Q. Li, G.M. Gadd, Microbially-induced Carbonate Precipitation for Immobilization of Toxic Metals Adv. Appl. Microbiol., 94(2016) 79-108. doi:10.1016/bs.aambs.2015.12.002. [14]
C.H. Kang, S.H. Han, Y. Shin, Y. Shin, S.J. Oh, J.S. So, Bioremediation of Cd by Microbially Induced Calcite Precipitation Appl Biochem Biotechnol., 172(4) (2014) 1929–1937. doi:10.1007/s12010-014-0737-1.
[15]
D. Kumari, X. Pan, D.J. Lee, V. Achal, Immobilization of cadmium in soil by microbially induced carbonate precipitation with Exiguobacterium undae at low temperature Int. Biodeterior., Biodegradation. 94 (2014) 98–102. doi:10.1016/j.ibiod.2014.07.007.
[16] A.M. Pat-Espadas, E. Razo-Flores, J.R. Rangel-Mendez, J.A. Ascacio Valdes, C.N. Aguila, F.J. Cervantes, Immobilization of biogenic Pd (0) in anaerobic granular sludge for the biotransformation of recalcitrant halogenated pollutants in UASB reactors Appl Microbiol Biotechnol., 100(3) (2016) 1427–1436. doi:10.1007/s00253-015-7055-6. [17] G. Yang, N. Zhang, J. Yang, Q. Fu, Y. Wang, D. Wang, L. Tang, J. Xia, X. Liu, X. Li, Q. Yang, Y. Liu, Q. Wang, B. Ni, Interaction between perfluorooctanoic acid and aerobic granular sludge Water Res. (2019) 115249. doi:10.1016/j.watres.2019.115249. [18]
N. Schmidt, D. Page, A. Tiehm, Biodegradation of pharmaceuticals and endocrine disruptors with oxygen , nitrate , manganese ( IV ), iron ( III ) and sulfate as electron acceptors J. Contam. 11
Journal Pre-proof Hydrol. 203 (2017) 62–69. doi:10.1016/j.jconhyd.2017.06.007. [19]
A.-K. Gattas, F. Fischer, A. Wick, T.A. Ternes, Anaerobic biodegradation of (emerging) organic contaminants in the aquatic environment Water Res. 116 (2017) 268–295. doi.org/10.1016/j.watres.2017.02.001.
[20]
F.P. Van der Zee, F.J. Cervantes, Impact and application of electron shuttles on the redox (bio)transformation of contaminants: A review Biotechnol. Adv. 27 (2009) 256–277. doi:10.1016/j.biotechadv.2009.01.004.
[21]
F.J. Cervantes, S. van der Velde, G. Lettinga, J.A. Field, Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia FEMS Microbilogy Ecol., 34 (2000) 161–171. doi:https://doi.org/10.1111/j.15746941.2000.tb00766.x.
[22]
M. de G. Moctezuma Zárate, J.F. Cárdenas González, A.S. Rodríguez Pérez, E.E. Domínguez, J. Tovar Oviedo, V.M. Martínez Juárez, I. Acosta Rodríguez, Isolation and Identification of Bacteria and Fungi Resistant to crude oil J. Multidiscip. Eng. Sci. Technol., 3 (2016) 5273– 5278. doi:ninive.uaslp.mx/jspui/handle/i/4265.
[23]
A.I. Omoregie, R.H. Ginjom, P.M. Nissom, Microbially Induced Carbonate Precipitation Via Ureolysis Process: A Mini-Review Trans. Sci. Technol., 5(4) (2018) 245–256. doi.org/10.1016/bs.aambs.2015.12.002.
[24]
G.R. Khayati, H. Dalvand, E. Darezereshki, A. Irannejad, A facile method to synthesis of CdO nanoparticles from spent Ni – Cd batteries Mater. Lett., 115 (2014) 272–274. doi:10.1016/j.matlet.2013.10.078.
[25]
A.L. Barry, K.L. Bernsohn, L.D. Thrupp, Four-hour urease test for distinguishing between Klebsiella and Enterobacter Appl. Microbiol., 18(2) (1969) 156–158. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=377935&tool=pmcentrez&renderty pe=abstract.
[26]
M. Umar, K.A. Kassim, Effect of Cementation Reagents Concentrations on Microbial Calcite Precipitation in Residual Soil Malaysian J. Civ., Eng. 29 Spec. Issue. (1) (2017) 79–90. doi:https://doi.org/10.11113/mjce.v29.134..
[27]
Y.S. Lee, H.J. Kim, W. Park, Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus J. Microbiol., 55(6) (2017) 440–447. doi:10.1007/s12275-017-7086-z.
[28]
H.M. Son, H.Y. Kim, S.M. Park, H.K. Lee, Ureolytic/Non-ureolytic bacteria co-cultured selfhealing agent for cementitious materials crack repair Materials (Basel)., 11(5) (2018) 782–795. doi:10.3390/ma11050782.
[29]
J. Xu, W. Yao, Z. Jiang, Non-Ureolytic Bacterial Carbonate Precipitation as a Surface Treatment Strategy on Cementitious Materials J. Mater. Civ., Eng. 26(5) (2014) 983–991. doi:10.1061/(ASCE)MT. 12
Journal Pre-proof
Table 1. Crystallite size using Sherrer equation.
CdCl2-a CdCl2-g CdSO4-a CdSO4-g
Plane (012) (104) (012) (104) (012) (104) (012) (104)
Grain size (nm) 24.5 ± 3.5 25.8 ± 5.3 25.3 ± 4.8 25.9 ± 3.7 25.8 ± 5.4 26.5 ± 6.1 25.0 ± 4.1 24.8 ± 3.2
a, acetate; g, glucose
13
Journal Pre-proof
Table 2. Results of quantitative EDS analyses of the samples. Precursor Carbon source
CdSO4 Glucose
CdCl2
Acetate
Glucose
acetate
Weight (%) C
15.81 ± 2.4
14.35 ± 4.2
19.5 ± 2.1
22.07 ± 2.1
O
7.71 ± 1.7
7.53 ± 1.6
4.11 ± 3.5
7.54 ± 1.8
Cd
4.59 ± 3.3
6.90 ± 2.5
1.50 ± 5.1
4.65 ± 1.9
14
Journal Pre-proof
Figure captions Fig. 1. Time in course of Cd2+ utilization (close figures) and pH change (open figures) in the presence of anaerobic granular sludge in liquid medium with (a) CdSO4 and (b) CdCl2 as precursors. Fig. 2. FT-IR spectrum of CdCO3 crystals by anaerobic granular sludge in presence of CdSO4 (a) and CdCl2 (b) as precursors and glucose and acetate as substrate.
Fig. 3. XRD patterns of CdCO3 crystals by anaerobic granular sludge in presence of CdSO4 (a) and CdCl2 (b) as precursors and glucose and acetate as substrate. Fig. 4. SEM images of CdCO3 crystals in presence of CdSO4 with (a) glucose, (b) acetate and CdCl2 with (c) glucose, (d) acetate as substrates by anaerobic granular sludge. Fig. 5. TEM images of CdCO3 crystals in presence of (a) CdSO4 (b) CdCl2 with acetate as substrate. The arrows indicate the formation of precipitated smaller than 100 nm. c) Precipitation of CdCO3 on the bacterial cell wall.
15
Journal Pre-proof
Fig. 1
16
Journal Pre-proof
Fig. 2
17
Journal Pre-proof
Fig. 3
18
Journal Pre-proof
Fig. 4
19
Journal Pre-proof
Fig. 5
20
Journal Pre-proof CrediT author statement
CM Martinez Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft and Writing - Review & Editing.
M. Rivera-Hernández Investigation
Luis H-Alvarez Investigation
Ismael Acosta-Rodriguez Funding acquisition and Supervision
F. Ruiz Funding acquisition
V.D. Compeán-García Conceptualization, Formal analysis, Resources and Writing - Review & Editing
Journal Pre-proof Highlights
An anaerobic granular sludge was able to precipitation Cd2+ as CdCO3 crystals.
No differences were observed neither cadmium source nor substrate.
Cd2+ precipitated as CdCO3 rhombohedral crystals from 100 to 700 nm.
Synthesis was associated to K. pneumoniae and E. coli.
The co-precipitation of cadmium with other metals was not observed.