Biorecovery of cobalt and nickel using biomass-free culture supernatants from Aspergillus niger
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In this research, the capabilities of culture supernatants generated by the oxalate-producing fungus Aspergillus niger for the bioprecipitation and biorecovery of cobalt and nickel were investigated, as was the influence of extracellular polymeric substances (EPS) on these processes. The removal of cobalt from solution was >90% for all tested Co concentrations: maximal nickel recovery was >80%. Energy-dispersive X-ray analysis (EDXA) and X-ray diffraction (XRD) confirmed the formation of cobalt and nickel oxalate. In a mixture of cobalt and nickel, cobalt oxalate appeared to predominate precipitation and was dependent on the mixture ratios of the two metals. The presence of EPS together with oxalate in solution decreased the recovery of nickel but did not influence the recovery of cobalt. Concentrations of extracellular protein showed a significant decrease after precipitation while no significant difference was found for extracellular polysaccharide concentrations before and after oxalate precipitation. These results showed that extracellular protein rather than extracellular polysaccharide played a more important role in influencing the biorecovery of metal oxalates from solution. Excitation–emission matrix (EEM) fluorescence spectroscopy showed that aromatic protein-like and hydrophobic acid-like substances from the EPS complexed with cobalt but did not for nickel. The humic acid-like substances from the EPS showed a higher affinity for cobalt than for nickel.
KeywordsAspergillus niger Biorecovery Extracellular polymeric substances Fluorescence quenching Cobalt Nickel Oxalate
Microorganisms can play an important role in both the remediation and biorecovery of metals (Gadd 2010; Liang and Gadd 2017). Although metals cannot be degraded into harmless compounds, their chemical form, mobility, toxicity, and bioavailability can be changed via the growth, metabolism, and metabolic products of microorganisms (Peng et al. 2018). Metal immobilization or recovery from solution can be achieved by bioprecipitation where metals are transformed from soluble species to insoluble compounds, such as oxides, carbonates, phosphates, oxalates, and sulfides (Haferburg and Kothe 2007; Tsezos 2009; Gadd et al. 2014; Liang and Gadd 2017). For example, Fe, Zn, and Cd in wastewater showed more than 99% precipitation rates in downflow fluidized bed reactors containing sulfate-reducing bacteria (SRB) (Gallegos-Garcia et al. 2009). Biological sulfide precipitation combined with solvent extraction can even result in nanosized metal sulfides for biorecovery (Nanusha et al. 2019). Microbially induced calcite precipitation (MICP) is also a promising biotechnology for recovery or immobilization of metals from wastewater or groundwater (Li et al. 2014; Li and Gadd 2017a, b; Kumari et al. 2016; Zhu and Dittrich 2016; Torres-Aravena et al. 2018). A total of 61% of calcium and 56% of strontium precipitation rates were obtained in porous media reactors via MICP mediated by the bacterium Sporosarcina pasteurii (Lauchnor et al. 2013). Calcium recovery of more than 90% was achieved from a calcium-rich industrial wastewater using bacterial MICP (Hammes et al. 2003). Supernatants obtained from ureolytic fungi are also very efficient in forming copper and other metal carbonate nanoparticles for biorecovery (Li et al. 2014, 2015, 2019; Li and Gadd, 2017a, b; Liu et al. 2019).
Organic acids, e.g. oxalic acid produced by fungi, can also play an important role in the immobilization or biorecovery of metals (Clarholm et al. 2015; Gadd 1999; Gadd et al. 2014; Mishra et al. 2017; Yang et al. 2019). Wood-rotting fungi can immobilize toxic metals in a metal-amended substrate by precipitation as metal oxalates (Kaewdoung et al. 2016). Lead immobilization via oxalates was also found with fluorapatite and Aspergillus niger (Li et al. 2016). In previous studies, it was also found that metabolites of the geoactive fungi Aspergillus niger and Beauveria caledonica could immobilize rare earth elements and toxic metals as oxalates (Fomina et al. 2005; Kang et al. 2019). Chemical oxalate precipitation is also widely used for the recovery of actinides (Abraham et al. 2014). High purity magnesium oxalate was obtained from Uyuni salar brine via chemical oxalate precipitation (Tran et al. 2013). Nickel is a primary co-existing element in Co minerals (Hazen et al. 2017), while industrially, cobalt, and nickel are also normal elements used in several kinds of batteries (Lupi et al. 2005; Rodrigues and Mansur 2010; Chen et al. 2011). In this research, we have used fungal products for biorecovery of cobalt and nickel from solution to (1) investigate the efficiency of culture supernatants from Aspergillus niger for cobalt and nickel biorecovery, (2) identify the bioprecipitation products formed, (3) determine the possible influence of extracellular polymeric substances (EPS) on the biorecovery process. This study will provide insights into the roles of fungal metabolites in metal–mineral interactions and their potential for metal biorecovery from solution.
Methods and materials
Microorganism and media
The experimental fungus used in this study was Aspergillus niger (ATCC 1015), which was incubated on malt extract agar slants (Lab M Limited, Heywood, Lancashire, UK) at 25 °C in the dark for 7 days to prepare spore suspensions for inoculation of liquid media according to a previous study (Kang et al. 2019). Modified Czapek-Dox (MCD) medium consisted of (g L−1 Milli-Q water): d-glucose, 30; NaNO3, 3; Na2HPO4, 1; MgSO4·7H2O, 0.5; KCl, 0.5; and FeSO4·7H2O, 0.01. All components were individually prepared as 100 mL stock solutions at the appropriate concentration and sterilized at 115 °C for 20 min prior to experiments. The initial pH of MCD media was adjusted to pH 5.5 using 1 M HCl before autoclaving. The initial spore concentration in the medium was 5 × 105 mL−1. A. niger was grown in 100-mL liquid medium in 250-mL Erlenmeyer flasks incubated on a shaker at 150 rpm at 25 °C in the dark. Biomass-free culture supernatants after 1- or 2-week incubation were obtained by filtering the medium through 0.45-μm cellulose nitrate membrane filters (Minisart syringe filters, Sartorius, Göttingen, Germany). All chemicals were obtained from Sigma-Aldrich Ltd., St. Louis, MO, USA unless stated otherwise.
Recovery of cobalt and nickel via bioprecipitation by fungal supernatants
Different concentrations of cobalt, nickel, and their mixture were used to examine their recovery from solution as biogenic minerals. Aliquots from stock solutions of 100 mM CoCl2∙4H2O and/or NiCl2∙6H2O were added to 27 mL biomass-free supernatants to reach a total volume of 30 mL, with or without the addition of Milli-Q H2O, in 50-mL tubes and mixed at 60 rpm for 24 h on a tube rotator. The resulting precipitate and supernatants from the tubes were collected after centrifugation (2012 g, 30 min). The concentrations of cobalt and nickel in the supernatants were measured by atomic absorption spectrophotometry (AAS) (AAnalyst 400 Atomic Absorption Spectrophotometer, PerkinElmer Ltd., Beaconsfield, UK). All experiments were carried out at least in triplicate.
Environmental scanning electron microscopy (ESEM) and X-ray diffraction (XRD) analysis of the collected precipitates were used to examine the morphology and mineralogical composition of the precipitated minerals. Detailed experimental procedures can be found in previous publications (Li et al. 2014; Li and Gadd 2017a). Precipitates were dried in a desiccator at ambient temperature for at least 5 days, mounted on aluminum stubs using carbon adhesive tape and coated with 10 nm Au/Pd using a Cressington 208HR sputter coater (Cressington, Watford, UK) and examined using a Philips XL30 ESEM (Philips XL 30 ESEM FEG) operating at an accelerating voltage of 15 kV. The mineral phases of the precipitates were identified using a Hiltonbrooks X-ray diffractometer (HiltonBrooks Ltd., Crewe, UK) equipped with a single graphite crystal monochromatic CuKα chronometer (30 mA, 40 kV).
Influence of extracellular polymeric substances on recovery of cobalt and nickel
Supernatants were collected from MCD media after growth of A. niger and then dialyzed using dialysis membrane to obtain an extracellular polymeric substances (EPS) solution. Twenty-five millimolar of sodium oxalate was dissolved in the EPS solution, and the pH was adjusted to pH 2.2 using 1 M HCl, the same value as in oxalate-free supernatants. The EPS solution containing oxalate was then used to precipitate cobalt and nickel from single and mixed solutions and using a 25-mM sodium oxalate solution treatment as a control. The content of extracellular polysaccharide and protein before and after precipitation was determined using the phenol-sulfuric acid method (DuBois et al. 1956) and Bradford method (Bradford 1976), respectively.
Excitation emission matrix fluorescence spectroscopy and quenching titrations
A Hitachi F-7000 fluorescence spectroscope (Hitachi, Tokyo, Japan), equipped with a 1.0-cm quartz cell and thermostatic bath, was used to determine the excitation emission matrix (EEM) spectra of EPS obtained from the A. niger culture supernatants. EEM spectra were obtained according to published methods (Song et al. 2012; Wang et al. 2018). For the quenching titration, the EPS solution was titrated with incremental microliter additions of Co(II) and Ni(II) solution at 308 K (35 °C). After each addition of metal salt, the solution was mixed using a magnetic stirrer for 15 min and EEM spectra were recorded during this process. Fifteen minutes was set as the equilibrium time because the fluorescence intensities showed almost no change after 15-min reaction.
Recovery of cobalt and nickel as oxalate minerals using A. niger biomass-free culture supernatants
Influence of extracellular polymeric substances on recovery of cobalt and nickel
Characterization of cobalt and nickel oxalate minerals
Fluorescence properties of EPS from A. niger
The fluorescence position (Ex/Em, nm) and intensity (arbitrary units) of EPS in the absence and presence of cobalt and nickel at concentrations of 3.67 mM
Stern–Volmer fluorescence quenching constant, Ksv, and quenching rate constant, Kq, of EPS in the presence of different metals (cobalt concentration range 0–5.67 mM; nickel concentration range 0–3.67 mM; R2: determination coefficients)
Quenching constants, Ksv (× 103/M)
Quenching rate constants, Kq (× 1011/M/s)
The quenching rate constant (Kq) values were one order of magnitude larger than the maximum diffusion collision quenching rate constant (2.0 × 1010/M/s), indicating that the fluorescence quenching process was mainly governed by static quenching by formation of complexes. However, if the Kq value was smaller than 2.0 × 1010/M/s, this indicated the fluorescence quenching process was dominated by dynamic quenching by intermolecular collisions. According to the quenching constant rates shown in Table 2, it was observed that the fluorescence quenching of EPS by cobalt was due to complexation. The fluorescence quenching of EPS by Ni was both a dynamic (peak A and peak D) and static process (peak C). Furthermore, the quenching strength of cobalt to fluorescence of EPS was larger than that for nickel.
Binding constants and binding sites
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively; Kb is the binding constant; n is the number of binding sites; and [Q] is the concentration of metal. The binding constant (Kb) reflects the interactive intensity between EPS and the metal.
Binding constants (logKb) and binding sites (n) of peaks A, B, and C from EPS complexation with different metals (cobalt concentration range 0–5.67 mM; nickel concentration range 0–3.67 mM; R2: determination coefficients)
Binding constant, log Kb
Binding sites n
Bioprecipitation is regarded as a potential technique for the removal and recovery of metals from solution (Gadd et al. 2014; Gadd and Pan 2016; Liang and Gadd 2017; Torres-Aravena et al. 2018). For example, carbonates, oxides, phosphates, and oxalates produced by microbial activities can transform metals from soluble species into the corresponding insoluble compounds via precipitation (Li and Gadd 2017a, b; Liang and Gadd 2017; Peng et al. 2018). Oxalic acid is produced by a wide variety of fungi, and this plays an important role in metal detoxification, weathering and cycling of metals, bioleaching, and biomineralization (Anjum et al. 2010; Fomina et al. 2008; Gadd 2007; Gadd et al. 2014; Kang et al. 2019; Nancharaiah et al. 2016; Yang et al. 2019). Metal oxalate crystal formation has been widely found using Aspergillus species, such as zinc oxalate (Sutjaritvorakul et al. 2016), lanthanum oxalate (Kang et al. 2019), manganese oxalate (Wei et al. 2012), and calcium oxalate (Pinzari et al. 2010). Chemically produced oxalate has also been used as a precipitant for recovery of valuable metals from a bioleaching solution of spent lithium-ion batteries (Sun and Qiu 2012) and as a leaching solution for monazite (de Vasconcellos et al. 2006). Copper recovery can reach 99.5% using chemical oxalate precipitation (Gyliene and Salkauskas 1995). Cobalt recovery from a spent lithium-ion batteries leachate could reach ~ 93% using chemical oxalate (Chen et al. 2011). In this work, approximately 94% and 98% cobalt recovery from a 10-mM solution were obtained using biomass-free A. niger culture supernatants and chemical oxalate, respectively, thus demonstrating that supernatants from A. niger were almost as efficient as chemical means to recover cobalt via bioprecipitation. Moreover, culture supernatants were more efficient for recovery of nickel compared with the chemical oxalate treatment. In a Co/Ni mixture, cobalt and nickel recoveries were increased compared to the single metal treatments. However, the apparent dominance of cobalt oxalate in the mixed Co/Ni precipitate could be explained by the solubility product constants (Ksp) of the two oxalate minerals. The Ksp of cobalt oxalate (2.70 × 10−9) is significantly smaller than that of nickel oxalate (1.2 × 10−3) (IUPAC-NIST Solubility Database).
Extracellular polymeric substances (proteins and/or polysaccharides) can play an important role in biomineralization, regulating and controlling nucleation, and growth of crystal structures (Ercole et al. 2012; Kawaguchi and Decho 2002; Li and Gadd 2017a, b; Perri et al. 2018; Tourney and Ngwenya 2014). In our study, it was found that the supernatants, or EPS obtained from supernatants, did not exert a significant influence on crystal morphology compared with chemical methods during the formation of cobalt and nickel oxalate. Only for the Co/Ni–mixed solution was there a difference in crystal morphology between those derived from the supernatants and the chemical system (Fig. 5). In other studies, fungal growth supernatants from Neurospora crassa were found to greatly influence the scale of crystal morphology for metal carbonates (Li and Gadd 2017a, b). Although EPS did not change the morphology of the nickel precipitate in the single metal solution, it did influence nickel recovery (Fig. 3).
In other studies, significant amounts of extracellular protein in fungal supernatants were removed by precipitation of copper carbonate (Li and Gadd 2017a, b; Liu et al. 2019). Here, extracellular protein was only partly removed during oxalate precipitation, while the extracellular polysaccharide concentration did not show much change compared to the control. Much more protein was removed with cobalt compared to nickel. It is well known that extracellular polymeric substances contain a variety of metal-binding groups (Wang et al. 2014; Liu et al. 2015; Song et al. 2016). Lower binding affinities between nickel and EPS could lead to a decrease in recovery of Ni in the presence of EPS in the oxalate solution compared to cobalt (Fig. 3). The aromatic protein-like, hydrophobic acid-like substances and humic acid-like substances (peak C) had a clear binding affinity for cobalt. Aromatic protein-like and humic acid-like substances have been found to easily trap copper (Wang et al. 2015; Wei et al. 2017). The differences in affinities of these substances for metals could explain the differences in the recovery efficiency for cobalt and nickel influenced by EPS. This work has demonstrated that fungal derived oxalate can be used for the recovery of cobalt from solution and provides insights into the role of other extracellular products during this process.
We gratefully thank Yongchang Fan (School of Science and Engineering, University of Dundee, Dundee, DD1 4HN, Scotland, UK) for assistance with scanning electron microscopy.
GMG received research funding from the Natural Environment Research Council (NE/M011275/1 (COG3—The geology, geometallurgy and geomicrobiology of cobalt resources leading to new product streams)). GMG also received additional research support of the Geomicrobiology Group from NERC (TeaSe project: NE/M010910/1). JF received receipt of a NERC PhD studentship as part of the COG3 award.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any data from studies on human participants or animals performed by any of the authors.
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