111Equation Chapter 1 Section 1Received 00th January 20xx,Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Experimental and geochemical simulation of nickel carbonate mineral precipitation by carbonate-laden ureolytic fungal culture supernatantsQianwei Li*a,b,#, Daoqing Liuc,#, Chunmao Chen*b, Zhiguo Shaoa, Huazhen Wangb, Jicheng Liub, Qiangbin Zhangb, Geoffrey Michael Gaddd
Ureolytic microorganisms have attracted much interest for the bioprecipitation, removal and biorecovery of toxic metals from aqueous solution but little is known about the distribution of cationic/anionic components or quantification of metal species in a fungal system, or the contribution of extracellular protein structure and adsorption during precipitation. In this research, carbonate-rich fungal growth supernatants were applied for bioprecipitation of Ni-containing minerals. The solubility and stability of relevant nickel species was calculated and simulated using Geochemists’ Workbench (GWB) and the structure and adsorption of extracellular protein were determined using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The results showed that hydrated nickel carbonate and trace nickel phosphate minerals, with a proportion being of nanoscale dimensions, were precipitated after mixing fungal growth supernatant and NiCl2. The solubility diagrams calculated by GWB showed that under the same condition nickel phosphates were the only precipitate in the simulated system. X-ray photoelectron spectroscopy (XPS) results showed the existence of protein N in the minerals and a protein-nanoparticle bond in C 1s spectra, which showed that extracellular protein might be an important factor in the difference between experimental results and the geochemical simulation. ATR-FTIR showed that during the biomineralization process, the conformation of extracellular proteins changed andstructures increased at the expense of highly ordered-helixes and other disordered structures, which might provide more nucleation sites to promote the crystallization of nickel carbonate especially under saturated conditions of CO3
2-. These results demonstrate that the combination of geochemical modelling and experimental analysis provide more complete scientific data to the explain differences between experimental results and theoretical outcomes. This can therefore enable reasonable predictions as to the effect of metal-containing nanoparticles on protein structure, and vice versa, during the process of metal bioprecipitation mediated by a carbonate-rich fungal growth supernatant system.
Introduction
Biomineralization is the process of forming minerals by organisms and the final complex materials may contain both
inorganic and organic components. Much research has been carried out on the precipitation of calcium carbonate by
ureolytic microorganisms1-7. Production of carbonate through the reactions of the nitrogen cycle can be established in three ways that include urea or uric acid degradation (ureolysis), ammonification of amino acids, and dissimilatory nitrate reduction8-10. Urease-positive microorganisms involved in the nitrogen cycle can produce carbonate through urea hydrolysis: one mole of urea (CO(NH2)2) is degraded to one mole carbamic acid (NH2COOH) and one mole ammonia (NH3)10, 11. The subsequent hydrolysis of carbamic acid releases one mole ammonia and one mole carbonic acid (H2CO3). Carbonate (CO3
2-) is then produced by the reaction of carbonic acid with hydroxide ions that are released by the reaction of ammonia with water. Free metal ions can interact with carbonate whether in solution or associated with cell surfaces. These metabolic activities therefore provide appropriate conditions for the
a. State Key Laboratory of Petroleum Pollution Control, Beijing, 102206, People’s Republic of China, E-mail: [email protected]
b. State Key Laboratory of Heavy Oil Processing, Beijing Key Laboratory of Oil and Gas Pollution Control, China University of Petroleum, Beijing, 18 Fuxue Road, Changping District, Beijing 102249, People’s Republic of China
c. Department of Environmental Engineering, Peking University, Beijing 100871, China
d. Geomicrobiology Group, School of Life Sciences, University of Dundee, DD1 5EH, Scotland, UK
# Co-first author.
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ARTICLE Journal Name
precipitation of metal carbonates, including those of nanoscale dimensions12-14.
With the development of nanotechnology, interactions between nanomaterials and biological systems have received much interest. Previous research has demonstrated that the formation of biominerals in the nanoscale resulted in a decrease of extracellular protein in a biological system15. However less is known about the specific contribution of protein, including structural and adsorption properties during mineral precipitation15. Some researchers have pointed out that in a protein-containing system, a “protein corona” is formed when the nanomineral appears which will modify the surface of the nanomaterials and change their interaction mode as well as biological and environmental fate16-21. Givens et al.17 investigated the conformation and adsorption of bovine serum albumin (BSA) on two nanoparticle surfaces including SiO2 and TiO2 at physiological and acidic pH values. These results showed that the secondary structure of BSA changed on adsorption to the nanoparticle surface. The functions of proteins are highly related to structure which can be unfolded and denatured under stressful conditions induced by, e. g. pH, temperature, ion concentration and surface energy17, 22-24. It is therefore important to understand interactions between proteins and biominerals due to their numerous applications and significance in biotechnology, geomicrobiology, biochemical engineering and medical sciences.
Ureolytic fungi have attracted considerable interest for carbonate biomineralization for removal of toxic metals from aqueous solution. Many potentially toxic metals, such as Co, Pb, Ni and Cd, can impair nerves, liver and other organs in humans, and block functional groups of enzymes25-27. For example, nickel is grouped as a possible human carcinogen, and is associated with birth defects and reproductive problems28. However, for the process of mineral precipitation, less attention has been paid to the distribution of cationic/anionic components or quantification of metal ions in a fungal system, as well as the contribution of extracellular protein related to structural and adsorption abilities. In this research, Neurospora crassa, a ureolytic fungus which shows various biomineralization and biosorption capacities for toxic metals14, 15, 29-31 was used to investigate the solubility and stability of nickel species during Ni biomineralization. The geochemical modelling programme, Geochemists’ Workbench (GWB), was applied for activity calculations of Ni2+, relevant Ni-containing minerals, and the predominance of aqueous Ni species in a simulated aqueous system. The structure and adsorption of extracellular protein was determined using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR).
Experimental
Organism, media and metals
The experimental fungus used was Neurospora crassa (WT ACCC #32256, Agricultural Culture Collection of China (ACCC), Beijing, China). It was routinely kept on malt extract agar (MEA) medium in 90-mm diameter Petri dishes and
grown at 25oC in the dark. N. crassa was grown for 1-2 d prior to subculture and inoculation plugs were taken from the margins of growing colonies using a sterilized cork borer ( 5 mm diameter). All experiments were conducted in triplicate32. A modified medium (AP1) was chosen for liquid experiments which contained (l-1, Milli-Q water): 111 mM D-glucose, 330 mM urea, 4 mM K2HPO4∙3H2O, 0.8 mM MgSO4∙7H2O, 0.2 mM CaCl2∙6H2O, 2 mM NaCl, 9 × 10-3 mM FeCl3∙6H2O and trace metals 1.4 × 10-2 mM ZnSO4∙7H2O, 1.8 × 10-2
mM MnSO4∙4H2O, and 1.6 × 10-3 mM CuSO4∙5H2O. All chemicals were purchased from Aladdin (Aladdin, Shanghai, China). All mineral salt, trace element solutions and urea solutions were prepared according to Li et al.33. The initial pH of AP1 medium was adjusted to pH 5.5 using 1 M HCl after autoclaving. Concentrated NiCl2·6H2O (0.5 M) was filtered using 0.2 m pore size membrane filters (Sartorius Stedim Biotech, Göttingen, Germany) prior to use in experiments.
Cultivation of N. crassa in modified AP1 media and mineral precipitation
N. crassa was grown in MEA medium for 1-2 d until the growing colonies occupied 2/3 of the medium surface. Then, different numbers of 5 mm-diameter fungal plugs were taken and inoculated into AP1 medium (1% (3 plugs per 100 ml) or 10% (30 plugs per 100 ml) inoculum) amended with 330 mM urea and incubated in a shaking incubator in the dark (125 rpm, 25°C). After 12 d incubation, fungal biomass was removed by centrifugation (4770 g × 20 min, 4°C) and the supernatant was collected.
For mineral precipitation experiments, different volumes of fungal growth supernatant (5 ml, 7.5 ml and 10 ml) were mixed with 0.5 ml 50 mM NiCl2 (diluted from the concentrated stock NiCl2 solution) and kept in a shaking incubator (125 rpm, 25oC) for 24 h. The resulting precipitate was collected by centrifugation (4770 g × 20 min, 4°C) and then washed using Milli-Q water and dried in a desiccator at room temperature to constant weight prior to further investigations. The pH of the supernatant was measured before and after mixing using a PHS-3C bench top pH meter (Mettler-Toledo, Zurich, Switzerland).
Quantification of extracellular proteins produced by N. crassa
The concentration of extracellular protein produced by N. crassa before and after mixture with NiCl2 was determined using a Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China). A series of bovine serum albumin (BSA) standards were prepared according to the protocol provided. 1 ml fungal growth supernatant was well mixed with 1 ml Milli-Q water, to which was then added 1 ml Bradford reagent and then left for 15 min. The absorbance was measured at 595 nm using a UV-2100 spectrophotometer (Beifeng, Beijing, China).
Identification of minerals precipitated by fungal growth supernatants
Washed and dried mineral particles were mounted on double-sided carbon adhesive tape on 20 mm diameter aluminium electron microscopy stubs prior to observation by scanning electron
microscopy (SEM) (ZEISS Gemini 300, Oberkochen, Germany) and elemental analysis using energy dispersive X-ray analysis (EDXA). X-ray diffraction (XRD) (Bruker D8 Advance, Karlsruhe, Germany), and Fourier transform infrared spectroscopy (FTIR) (Thermo Fisher 6700, Waltham, USA) were used for the identification of biominerals: sample preparation was conducted according to the procedures described in Li et al.34.
X-ray Photoelectron Spectroscopy (XPS) of fungal growth supernatants and precipitated minerals
Precipitated minerals were collected by centrifugation (4770 g, 15 min, 4oC), washed with Milli-Q water twice, and then suspended in the Milli-Q water. A drop of the fungal growth supernatant or the solution containing minerals was placed on the top of a monocrystalline silicon chip (5 x 5 mm) and dried in a desiccator for XPS analysis. XPS spectra were collected on a K-Alpha instrument (Thermo Fisher, Waltham, MA, USA) fitted with a conventional non-monochromatic Al-Kα X-ray source and a multichannel video detector. The spectra collected were analyzed using CasaXPS V2.3.16.PR1.6 software (Casa Software Ltd, Teignmouth, Devon, UK) and a 30% Gaussian-Lorentzian function was used for the curve fitting procedure.
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)
Extracellular protein adsorbed on biominerals and changes in protein secondary structure during the bioprecipitation process was assessed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) (Bruker Optics Vertex 70, Karlsruhe, Germany). The spectra for water and air were subtracted from spectra obtained in the presence of the fungal growth supernatant. After addition of NiCl2 (to a final concentration of 50 mM), the mixture was shaken gently to provide a uniform suspension and then exposed to the uncoated reflectance element to collect the spectra. The resulting spectra were fitted to a Gaussian-Lorentzian shape containing five components for the amide I region, which indicated the secondary structure of extracellular protein in the fungal supernatant.
Geochemical modelling of mineral precipitation using Geochemist’s Workbench (GWB)
The Geochemist’s Workbench (GWB, 11.0.6) (Aqueous Solutions LLC, Urbana-Champaign, USA) is designed for simulation of chemical reactions, calculation of stability diagrams as well as determination of the chemical equilibrium states of natural waters. GWB contains three versions depending on different requirements, including GWB Professional, GWB Standard and GWB Essentials. Here, GWB Essentials was used which contains six programs, GSS, Rxn, Act2, Tact, SpecE8 and Gtplot with Act2 generating stability diagrams for activity, Eh, pe, pH and fugacity axes (more details can be found at https://www.gwb.com/essentials.php). In this experiment, the simulated fungal growth supernatant contained the same concentrations of ions as present in AP1 medium. Without fungal incubation, urea would not be degraded and the concentration of urea in the control system was set as 330 mM. To simulate fungal growth activity, the concentration of carbonate (degraded from urea
by fungal urease) in the system was set as 33 mM (1% inoculation) and 330 mM (completely degraded from an initial concentration of 330 mM urea by a 10% inoculation according to previous experiments).
Results and discussion
Geochemical simulation of nickel-containing mineral bioprecipitation using GWB
Previous experiments have demonstrated that growth supernatants of urease-positive fungi provide a promising system for the precipitation and biorecovery of toxic and valuable metal ions14, 33-35. The effectiveness of mineral precipitation depends strongly on the concentration of metal ions, dissolved carbonate, pH, alkalinity, temperature, medium composition, availability of nucleation sites and the Hartree energy (Ha)10, 34, 36, 37. Geochemist’s workbench (GWB) provides a geochemical modelling system for calculation of the equilibrium state of natural fluids, activity/temperature diagrams, theoretical systems as well as reaction path modelling 15, 38, 39. Here, GWB and a thermodynamic database included with the package were applied for the geochemical simulation of Ni-containing mineral precipitation in a fungal growth supernatant system. In the modelling system, the same concentrations of the different salts incorporated in the fungal growth media were applied into the system for simulation, and the concentration of urea was set as 20 g/L (~ 330 mM). However, fungal growth and metabolism cannot be simulated using the software, and the release of CO3
2- due to urea degradation by the fungus was set as an indicator for fungal growth. According to previous experiments, 330 mM urea was degraded completely by ureolytic fungi (at a 10% inoculation) giving a theoretical release of 330 mM CO3
2- (eq. 1). For a 1% fungal inoculation, urea would only be partially degraded and the concentration of CO3
2- released was therefore set at 33 mM.
When all these related data was uploaded into the simulation system, the solubility and stability of Ni2+, Ni-containing species or minerals were calculated individually using GWB Act2 (Fig. 1,2). The results showed that the precipitation of Ni-containing minerals occurred at certain metal ion concentrations and pH range, and at given physico-chemical conditions, different Ni-containing species occurred in the aqueous system.
In the control medium without fungal inoculation, there was no CO3
2- release and Ni3(PO4)2 and NiO were the two dominant minerals over a particular concentration of Ni2+ and pH range. Here, the PO4
3- originated from the medium, being necessary for fungal growth. The lowest concentration of Ni2+ for nickel phosphate precipitation was around 0.32M (here, a (Ni2+) was equal to Ni2+ concentration, log a (Ni2+) ≈ -6.5, c (Ni2+) ≈ a (Ni2+) ≈ 0.32M) and the system was at pH 8 in this condition (Fig. 1). When inoculated with N. crassa, NiCO3
became another dominant mineral, occurring because of the degradation of urea by this urease-positive fungus (Fig. 2). In the system containing 33 mM CO3
2- (1% fungal inoculation) (Fig. 2a), NiCO3 could be precipitated when the concentration of Ni2+ was ＞ 3.2 M (log a (Ni2+) ≈ -5.5) and the pH of the system was
below 7, while Ni3(PO4)2 could be precipitated when the Ni2+
concentration was ＞ 0.5 M (log a (Ni2+) ≈ -6.3) (Fig. 2a). With lower Ksp values (Ksp (NiCO3) = 1.4 x 10-7, Ksp (Ni3(PO4)2) = 4.7 x 10-32)40, 41, nickel phosphate is less soluble than nickel carbonate, and therefore precipitates before nickel carbonate. With more fungal inoculation (10%) (Fig. 2b), urea would be degraded completely and would release 330 mM CO3
2- into the
system, and so less Ni2+ was needed for precipitation of carbonate minerals (0.32M, log a (Ni2+) ≈ -6.5) while more Ni2+ (32M, log a (Ni2+) ≈ -4.5) was needed for the precipitation of phosphate minerals (Fig. 2b). The formation of minerals depends on the solution supersaturation, pH, temperature, ionic strength and inhibitors in the system42.
Fig. 1. Solubility diagram of Ni2+ versus pH at 25oC in simulated control media without fungal inoculation. The simulated system was set at 20 g urea (330 mM), 4 mM KH2PO4, 0.8 mM MgSO4·7H2O, 1.7 mM NaCl, 0.2 mM CaCl2·6H2O, 9 μM FeCl3·6H2O, 0.01 mM ZnSO4·7H2O and 0.02 mM MnSO4·4H2O. The symbol a on the y-axis represents the effective concentration of a given species in a mixture.
Compared with the system containing 33 mM CO32- (Fig. 2a),
a higher concentration of CO32-
(330 mM) might be supersaturated and affect the precipitation of phosphates, either by blocking phosphate nucleation sites or inducing carbonate precipitation43. The simulation by the GWB modelling system was based on thermodynamic and kinetic data, and did not include the contribution of other possible extracellular metabolites in the fungal culture supernatant such as proteins, amino acids, organic acids and polysaccharides.
Mineral precipitation using fungal growth supernatant
To further compare the GWB simulation with experimental results, different volumes (5 ml, 7.5 ml and 10 ml) of fungal growth supernatants from 1% and 10% fungal inoculations were mixed with 0.5 ml 50 mM NiCl2 for mineral precipitation. Obvious mineral precipitates appeared after mixing and the test tube containing the mixture was left in a shaking incubator for 24 h until the pH of the mixture became constant. After this, the supernatant was collected by centrifugation (4770 g × 20 min, 4°C). The pH, removal of Ni2+, extracellular protein and polysaccharides after mixture were measured, respectively.
After mixture, the pH of the supernatant decreased to different levels (Table 1, 2). The removal of Ni2+ and extracellular protein increased significantly when more fungal growth supernatant was mixed with the same amount of NiCl2, while only 5-8% of polysaccharides was removed after mixture (Table S1). When mixed with the supernatant that was obtained from media inoculated with more fungal biomass, more Ni2+
and extracellular protein was removed. With a higher fungal inoculation, the same amount of urea would be degraded completely, and more carbonate ions would be released for mineral precipitation. Similarly, more extracellular proteins would be secreted and contribute to the biomineralization process. Previous studies have demonstrated that extracellular
Fig. 2. Solubility diagram of Ni2+ versus pH at 25oC in simulated media modified with 20 g urea (330 mM) with (a) 1% , or (b) 10% inoculation of N. crassa. The simulated system was set at (a) 33 mM CO3
2-, 66 mM NH4+ (b) 330 mM CO3
2-, 660 mM NH4
+, 4 mM KH2PO4, 0.8 mM MgSO4·7H2O, 1.7 mM NaCl, 0.2 mM CaCl2·6H2O, 9 μM FeCl3·6H2O, 0.01 mM ZnSO4·7H2O and 0.02 mM MnSO4·4H2O. The symbol a on the y-axis represents the effective concentration of a given species in a mixture.
proteins may act as a template for mineral precipitation and take part in the formation of specific nanominerals14, 15.
The precipitates produced were collected by centrifugation (4770 g × 20 min, 4°C) and dried in a desiccator to constant weight prior to morphological observations and elemental analysis. The minerals were found to be spherical with a proportion being of nanoscale dimensions (Fig. 3). Minerals precipitated with the higher amount of fungal inoculation were smaller (~60 nm) (Fig. 3c,d). EDXA results showed that the main elements in both minerals were the same, consisting of Ni, C, O and P (Fig. 3b). Besides the minerals precipitated with fungal growth supernatant, minerals formed by the mixture of NiCl2 and AP1 medium containing different concentrations of (NH4)2CO3 were examined as a control group (Fig. S1). In the control group, granular and block minerals (indicated by arrows) with rough or smooth surfaces were formed and showed various sizes ranging from ～ 300 nm to 1m (Fig. S1). XRD results showed that the minerals precipitated were amorphous (Fig. 4a) and FTIR was therefore applied for further investigation of their composition (Fig. 4b). The spectra showed broad bands in the high energy region which were probably water related (around 3400 cm-1) which meant that the amorphous minerals were hydrated (Fig. 4b). According to previous experiments, peaks in the region of 1619 and 1418 cm -
1 are attributable to the 3 vibrational mode of the carbonate ion, while the peaks at 835 cm-1 are due to the 2 vibrational mode of CO3
2- (Fig. 4b) 33, 44.This data therefore confirms the existence of carbonates and
the production of NiCO3∙xH2O. The peaks in the region of 1044 cm-1 (Fig. 4b) may be due to the1 vibrational mode of CO3
2-, or the3 vibrational mode (~1029 cm-1) of PO4
3- which was the composition of AP1 medium. Compared with the EDXA results, it can be concluded that the minerals precipitated were hydrated nickel carbonate with trace amounts of nickel phosphates.
Table 1 ． pH changes of the supernatant obtained from media with a 1% fungal inoculation after mixing with 50 mM NiCl2, and the proportion of Ni2+, extracellular protein removed by precipitation.
Table 2. pH changes of the supernatant obtained from media with 10% fungal inoculation after mixing with 50 mM NiCl2, and the proportion of Ni2+, extracellular protein removed by precipitation.
In this experiment, when 0.5 ml 50 mM NiCl2 was mixed with 10 ml fungal growth supernatant, the final concentration of Ni2+
in the system was equal to 2.4 mM. However, in the GWB
solubility diagrams , Ni3(PO4)2 was the only mineral appearing in the simulated system with a 1% fungal inoculation (pH = 8.7, log a (Ni2+) = log (2.4 x 10-3) = -2.6) (Fig. 2a) . Similarly, with a 10% fungal inoculation (pH = 8.9, log a (Ni2+) = log (2.4 x 10-3) = -2.6), Ni3(PO4)2 was the only dominant mineral in the solubility diagram (Fig. 2b). Carbonate minerals were not precipitated in the simulated system which was not in accordance with our experimental results.
The only difference between the GWB simulation system and actual experimentation was that GWB cannot take fungal metabolites into account. According to Table 1 and 2, most of the extracellular protein was removed after mixture, which may provide more nucleation or adsorption sites for nickel carbonate precipitation15, 45, 46, which could then inhibited the formation of nickel phosphate to some extent. To further investigate the role of extracellular protein in the formation of nickel-containing minerals, XPS was applied.
Fig. 3. Scanning electron microscopy and elemental analysis of precipitated minerals. Minerals were precipitated by mixture of 0.5 ml 50 mM NiCl 2 and growth supernatant obtained from media with (a, b) 1%, (c, d) 10% fungal inoculation. Different inoculum amounts of N. crassa were inoculated in urea-amended AP1 media and incubated in a shaking incubator for 12 d: the growth supernatant was collected by centrifugation (4770 g × 20 min, 4°C). Inset shows EDXA analysis of the precipitated minerals. Typical spectra and images are shown from one of several examples. Scale bars: (a, c) = 500 nm, (b, d) = 300 nm.
XPS analysis of minerals precipitated by mixture of fungal growth supernatant and NiCl2
XPS was further applied to explore the interaction between the organic functional groups present in the fungal supernatant and the precipitated bio minerals (Fig. 5). The general spectrum showed the presence of C 1s, O 1s, N 1s core levels in precipitated minerals and the fungal growth supernatant, respectively. Ni 2p core levels were also detected in minerals with a binding energy of 848~886 eV (Fig. 5). No P 2p spectra were detected in the biominerals which may be because the amount was too low to be detected. The amount of N in the minerals was 17%, higher than that in the growth supernatant (11%), which may be due to the coprecipitation of extracellular protein with the minerals (Table S2). C 1s spectra of the fungal
supernatant and precipitated minerals are shown in Fig.6a. In the fungal growth supernatant, C 1s spectra were fitted with four major peaks with binding energies of 283.4 eV, 284.9 eV (C-C，C=C or C-H), 286.4 eV (C-O) and 287.7 eV (C=O)47, 48. The component appearing at 283.4 eV can be attributed to a metal-C bond49, 50, which could be Ni2+, or other metal ions present in the fungal growth medium. The peak in the 287.8 eV region was attributed to electron emission from carbon atoms in carbonyl groups (ketonic or aldehydic carbon) that are present in proteins. For the precipitated minerals, except for these four major peaks, another peak appeared with the binding energy of 286.7 eV, which was attributed to the presence of aromatic carbon atoms present in amino acids from proteins that are bound to the surface of nanoparticles51, 52, such as nickel carbonate51. In the O 1s region (Fig. 6b), the peaks at 529.9 ± 0.1 eV, 530.6 ± 0.1 eV, 531.2 ± 0.1 eV and 531.7 ± 0.2 eV were attributed to –OH and carbonate related groups (i.e. C=O or COOH) due to the hydrolysis of urea53. Peaks for N 1s centred at 397.5 eV and 398.2 eV were assigned to neutral amine and protonated amine groups in the protein moiety, while the peak at 398.8 ± 0.1 eV contributed to the ammonium arising from the hydrolysis of urea52, 54, 55 (Fig. 6c). The binding energy of 854.6 and 872.2 eV in the Ni 2p region was allocated to Ni 2p3/2 and Ni 2p1/2 (Fig. 6d), indicating the existence of NiCO3
51,
53. There are two satellites with binding energies of 859.8 and 878.0 eV arising from Ni2+ in the Ni 2p region53, 56, 57. This evidence, including the increase of N in the precipitated minerals and the appearance of the C1s peak with a binding energy of 286.7 eV, indicated that extracellular proteins played a crucial role in determining the difference between the experimental results and the GWB simulation.
Fig. 4. (a) X-ray diffraction and (b) Fourier transform infrared spectroscopy of minerals precipitated by mixture of NiCl2 and fungal supernatant obtained from medium with a 10% fungal inoculation. Ni-containing minerals were precipitated by mixture of growth supernatant of N. crassa (10% inoculation) with NiCl2; commercial nickel carbonate (NiCO3·2Ni(OH)2·xH2O) was used as a standard. N. crassa was incubated in 330 mM urea-amended AP1 media for 12d at 25oC. Typical pattern and spectra are shown from one of several determinations.
Fig. 5. XPS survey spectrum for minerals precipitated by mixture of fungal growth supernatant (black) with NiCl2 (red). Typical spectra are shown from one of several determinations.
Fig. 6. High resolution XPS spectra of (a) C 1s, (b) O 1s, (c) N 1s and (d) Ni 2p for minerals precipitated by mixture of fungal growth supernatant with NiCl2. Typical spectra are shown from one of several determinations.
ATR-FTIR spectroscopy of fungal growth supernatant after mixture with NiCl2
The key advantage of the ATR-FTIR technique is its capability to collect information about conformational changes in adsorbed proteins58. Givens et al.17 studied the interaction of bovine serum albumin (BSA) with two nanoparticles, TiO2 and SiO2. ATR-FTIR showed that the protein corona structure was significantly impacted by a protein-nanoparticle interaction17. In order to characterize the interaction between extracellular protein and minerals in this study , ATR-FTIR spectroscopy was used to analyse protein adsorption during mineral precipitation. After subtraction of the water contribution, adsorption of other solution-phase components could
be monitored spectroscopically (Fig. 7). The bands centred at 1700-1600 cm-1 refer to the protein backbone, and the 3 vibrational mode of carbonate was detected in the region 1500-1400 cm -1 33, 34. Bands in the range of 1155 and at 1080 cm−1 could be attributed to stretching of C–O–C and OH in polysaccharides, respectively59, while the small bands around 1000 cm-1 are due to the 3 vibrational mode of PO4
3-. There are nine characteristic IR absorption bands for protein, namely, amide A, B, and I-VII regions 60. The amide I band (1700-1600 cm-1), related to the C=O stretch vibration of the peptide linkage, is the most sensitive spectral region for protein secondary structure including-helical, sheets/turns and random-coil structures19, 22, 58, 60. To further investigate the changes in protein structure due to absorption, the spectra of the amide I band region for extracellular protein in the fungal supernatant (10% fungal inoculation) adsorbed to Ni-containing nanoparticles were curve-fitted using symmetric Gausisian curves (Fig. 7). The percentage content of the different secondary structures is shown in Table 3. Five contributions indicated the following secondary structures in the extracellular protein found in the fungal supernatant: β-sheets/turns (1663.2 cm-1, 25.7%),-helices (1650.4 cm-1, 2.56%), random chains (1641.5 cm-1, 6.87%), extended chains/β-sheets (1627.3 cm-1, 34.64%) and side chain moieties (1600.3 cm-1, 30.14%). After adsorption to surfaces of nanoparticles, the content of -helices, random chains and side chain moieties decreased to different levels. In general, the decrease in -helices was expected to be balanced by an increase in β structures17, 61. The results indeed showed that the major increase of 8% was inβ-sheet structures. Therefore, the protein remained intact and close to its native form although there were some changes in protein folding on adsorption. It has been reported that bands of the amide I component centred at ~1610 cm-1 (side chain moieties) were sensitive to hydrogen bonding and reflect protein-protein interactions61. The decrease of side chain moieties was probably an effect of surface-induced deformation because adsorption on nanoparticles would reduce the interactive area between neighbouring adsorbed proteins61. With the addition of Ni2+, the secondary structure of proteins changed and might provide more nucleation sites to promote the nucleation and crystallization of nickel carbonate especially in a saturated condition of CO3
2-. The
changes in secondary structure indicated that the highly helical structure of extracellular protein unfolded on adsorption to Ni-containing nanoparticles. Compared with the extracellular protein in Ni2+-free supernatants, the spectral shifts and large changes in relative intensities were the result of strong hydrogen bonding with hydroxyl (-OH) groups on the surface17, 62.
Fig. 7. Absorbance spectra of fungal growth supernatant before and after mixture with 50 mM NiCl2. Typical spectra are shown from one of several determinations.
Fig. 8. Absorbance spectra of fungal growth supernatant before and after mixture with 50 mM NiCl2. Typical spectra are shown from one of several determinations. Fig. 8. Background subtracted and normalized extracellular protein amide I band for curve fitting for structural analysis. (a) fungal growth supernatant; (b) fungal growth supernatant (10% inoculation) mixed with NiCl2. Component bands are given for (1) side chain moieties, (2) sheets, (3) random chains,-helices,turns. Typical patterns are shown from one of several determinations.
Table 3. Secondary structure content (%) of extracellular proteins in fungal growth supernatant determined via curve fitting.
Sample Side chain moieties β-sheets Random
chains α-helices β- turns
Fungal growth
supernatant (S)
30.14% (1600.25)
34.64%(1627.3)
6.87%(1641.45)
2.65%(1650.35)
25.7%(1663.2)
S + 50 mM NiCl2
25.59%(1600.4)
42.71%(1627.7)
3.74%(1641.25)
0.51%(1650.4)
27.45%(1663.3)
Fungal growth supernatant was collected by centrifugation (4770 g × 20 min, 4°C) after 12 d incubation at 25oC in the dark.
ConclusionsIn the process of biomineralization, extracellular protein can
play an important role in regulating and controlling nucleation, growth, and morphology of the resulting biominerals. The application of GWB calculations and simulation has provided more scientific data that explains differences between experimental results and theoretical outcomes. The curve fitting method for the second derivative spectra confirmed conformational changes of the extracellular protein on adsorption during biomineralization. The combination of geochemical modelling and experimental analysis can therefore enable reasonable predictions as to the effect of metal-containing nanoparticles on protein structure, and vice versa, during biomineralization in a fungal-mediated carbonate precipitation system.
Conflicts of interestThere are no conflicts to declare.
AcknowledgementsFinancial support from the Open Project Program of the State Key Laboratory of Petroleum Pollution Control (Grant No. PPC2016011), CNPC Research Institute of Safety and Environmental Technology is gratefully acknowledged. We also acknowledge financial support from the National Natural Science Foundation of China (Grant No.41701352) and the Science Foundation of the China University of Petroleum, Beijing (No. 2462017YJRC010). GMG gratefully acknowledges support of the Geomicrobiology Group from the Natural Environment Research Council, UK (NE/M010910/1 (TeaSe); NE/M011275/1 (COG3)).
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