Biomineralization of Metal Carbonates by Neurospora crassa

Article pubs.acs.org/est

Biomineralization of Metal Carbonates by Neurospora crassa Qianwei Li,† Laszlo Csetenyi,‡ and Geoffrey Michael Gadd*,†,§ †

Geomicrobiology Group, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom Concrete Technology Group, Department of Civil Engineering, University of Dundee, Dundee, DD1 4HN, Scotland, United Kingdom § Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China ‡

ABSTRACT: In this research, the urease-positive fungus Neurospora crassa was investigated for the biomineralization of calcium carbonate and its potential application in metal biorecovery and/ or bioremediation. After 12 d incubation at 25 °C in urea and calcium-containing medium, extensive biomineralization of fungal filaments was observed. Energy dispersive X-ray analysis of crystalline precipitates on the hyphae of N. crassa showed that the main elements present in the crystals were Ca, C, and O. X-ray diffraction (XRD) of the precipitates showed they were composed solely of calcite (CaCO3) and over 90% Ca could be removed from the media by the fungal biomass and associated calcite precipitation. To further investigate biologically induced metal carbonate biomineralization, CdCl2 was contacted with supernatants of N. crassa obtained after growth in urea-containing medium. XRD showed that the Cd2+ was precipitated as pure otavite (CdCO3) with a particle size range of 55 to 870 nm, and approximately 1.5% having nanoscale dimensions. These results provide direct experimental evidence for the precipitation of metal carbonates such as calcite and otavite based on biologically induced mineralization, and suggest that urease-positive fungi may play a potential role in the synthesis of novel biominerals and in metal bioremediation or biorecovery.

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INTRODUCTION Biomineralization is the process of forming minerals by organisms, the products of which are complex materials that may contain both minerals and organic components. Many microorganisms show the ability to precipitate a variety of minerals such as carbonates, phosphates, sulfides, oxides, and oxalates.1−9 The process of biomineralization is usually grouped into biologically controlled mineralization (BCM) and biologically induced mineralization (BIM) according to their variable degrees of biological control.6,10−13 BCM is where the organism exerts a great degree of control over the biomineralization process, and it is a process of molecular recognition and self-assembly, which may also be directed by an organic matrix.14,15 The cellular activities of organisms not only direct the nucleation, growth, morphology of the minerals deposited in BCM but also control the final location of minerals, with examples including algal coccoliths and frustules, and bacterial magnetosomes.6 BIM occurs when the organism modifies its local microenvironment creating conditions favorable for the extracellular precipitation of mineral phases.6 Most microbial biomineralization examples are BIM, and this can result from metal oxidation or reduction and metabolite excretion, with cell surfaces and outer layers often acting as a nucleation site, substrate or matrix for subsequent mineral precipitation.6,16 Some researchers have also used the term biologically influenced mineralization, which has been taken to © XXXX American Chemical Society

mean passive mineral precipitation on cell surfaces or extracellular polymeric substances (EPS).11,13,17 Several of these biomineralization processes may occur simultaneously in certain circumstances.13,18 Calcium carbonate (CaCO3) is the most common biomineral which can be found in soils, marine and fresh waters, and its formation can be mediated by a variety of organisms including bacteria, cyanobacteria, algae, fungi, and protista.6,19,20 Among the mechanisms of biologically induced calcium carbonate biomineralization, one mechanism is associated with urea degradation. Burbank et al.8 used urea-hydrolyzing microorganisms grown in a urea and calcium-rich medium in order to produce ammonium (NH4+) and dissolved carbonate which together with increasing medium pH, resulted in calcite precipitation (eqs 1 and 2): urease

CO(NH 2)2 (aq) + 2H 2O(aq) ⎯⎯⎯⎯⎯→ 2NH4 +(aq) + CO32 −(aq)

CO32 −(aq) + Ca 2 +(aq) → CaCO3(s)

(1) (2)

Received: August 29, 2014 Revised: November 5, 2014 Accepted: November 12, 2014

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dx.doi.org/10.1021/es5042546 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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contained a final concentration of 40 or 330 mM urea instead of (NH4)2SO4 and 50 mM CaCl2·6H2O, while the control medium contained 40 mM urea and 0.2 mM CaCl2. Urea stock solutions were sterilized by filtration through a sterile 0.2 μm pore size cellulose acetate membrane filter (Sartorius Stedim Biotech, Göttingen, Germany). The initial pH of AP1 medium was adjusted to pH 5.5 using 1 M HCl after autoclaving. For the toxic metal-removal bioprecipitation experiments, 0.5 M CdCl2 was filtered using 0.2 μm pore size cellulose acetate membrane filters before being added to supernatants from the N. crassa carbonate-forming growth medium. pH and Ca Concentration Measurement and Metal Precipitation. After 12 days incubation at 25 °C in the dark, the pH of the media was measured using an Orion 3 Star benchtop pH meter (Thermo Fisher Scientific, Waltham, MA, U.S.A.) fitted with a flat tip electrode (VWR, U.S.A.). Fungal biomass was collected by centrifugation (4770g × 20 min, 4 °C), resuspended in 100 mL Milli-Q water and recentrifuged. Fungal biomass was dried at 105 °C for several days until the weight was constant, and then 50 mg dry biomass was dissolved in 3 mL 16 M HNO3 at 90 °C until the solution became clear: the liquor and supernatant were then filtered using 0.2 μm pore size cellulose acetate membrane filters and diluted to an appropriate concentration using 0.2 M HNO3. The concentration of calcium in the digests was measured using an AAnalyst 400 atomic absorption spectrophotometer (PerkinElmer Instruments, Waltham, MA, U.S.A.) using appropriate lamps and standards. N. crassa was grown in AP1 media amended with 40 mM urea and 0.2 mM CaCl2. After 12 d growth, fungal biomass was removed by centrifugation (4770g × 20 min, 4 °C) and the supernatant was collected. 200 mL CO32− containing supernatant was mixed with 50 mL 0.5 M CdCl2, and the precipitate was collected by centrifugation (4770g × 20 min, 4 °C), washed using Milli-Q water and dried in a desiccator for at least 3 weeks prior to X-ray diffraction (XRD) analysis. The pH of the supernatant was measured before and after centrifugation using an Orion 3 Star benchtop pH meter (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Scanning Electron Microscopy and XRD of Biominerals Produced by Fungi. Fungal biomass was fixed using 2.5% (v/vaq) glutaraldehyde in 5 mM PIPES buffer (pH was adjusted to 6.5 using 1 M NaOH) overnight at room temperature, and then washed twice using 5 mM PIPES (pH 6.5). Dehydration was performed by a 30−100% (v/vaq) ascending series of ethanol in Milli-Q water (15 min per step) and then dried using a CO2 critical point dryer (BAL-TEC company, Los Angeles, CA, U.S.A.). Samples were mounted on double-sided carbon adhesive tape on 10 mm diameter aluminum stubs prior to examination by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA). For SEM, particulate materials mounted on stubs were sputter coated for 5 min with gold and platinum (30 nm) using a Cressington 208HR sputter coater (Ted Pella, Inc., Redding, CA, U.S.A.). Specimens were examined using an environmental scanning electron microscope (ESEM) (Philips XL30 ESEM FEG) operating at an accelerating voltage of 15 kV. For energy dispersive X-ray analysis (EDXA), uncoated samples were used, and operation was at an accelerating voltage of 20 kV for at least 100 s. The mineralogy of the test materials was determined using a Hiltonbrooks X-ray Diffractometer (XRD) (Hiltonbrooks Ltd., Crewe, U.K.) with a monochromatic CuKα source and curved

Whiffin et al.3 used a five meter sand column treated with bacteria, urea, and calcium to mimic field conditions. In this research, they found that the column treated with bacteria and reagents showed a significant improvement of strength and stiffness due to microbial CaCO3 precipitation, and proposed that this soil treatment method could be applied in the subsurface to improve soil strength. Toxic metal and radionuclide pollution is a very important environmental problem, and some of these substances may pass through the food chain from soil or water and lead to serious ecological and human health effects.21−23 It is well-known that potentially toxic metals, such as Co, Pb, and Cd, can damage nerves, liver and other organs, and block some functional enzymes.21,24,25 However, many microbes can grow and flourish in metal-contaminated environments and metal resistance may be due to a variety of active and incidental mechanisms that affect metal mobility and toxicity.6 Both living and dead microorganisms, including fungi, bacteria and algae, can effectively remove toxic metals from solution.6,26−30 Biologically induced calcium carbonate precipitation has been suggested as a promising method for toxic metal remediation of contaminated environments.24,31,32 Since urease-positive microorganisms show the ability to precipitate Ca as CaCO3, this indicates they could also be utilized to trap other toxic metals and form toxic metal-containing carbonates.24,32−34 However, most research to date has concentrated on prokaryotic systems, and the possible significance of ureasepositive fungi should not be neglected. Ammonia fungi can be defined as an abundant chemo-ecological group of fungi that increase growth in response to the addition of nitrogenous substances to soil, including urea, the degradation of which is accompanied by soil alkalinization to pH 9−10.35 Strong urease activity is exhibited by most saprotrophic ammonia fungi as well as nonammonia saprotrophs, and ectomycorhizal fungi.36,37 The overall aim of this research was therefore to examine fungal biomineralization of CaCO3 using a urease-positive model organism, Neurospora crassa, to provide further understanding of the mechanisms involved and also to indicate possible applications for microbially mediated production of novel biomineral products, including those at nanoscale dimensions, and in metal bioremediation or biorecovery by fungal systems.

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METHODS Organism, Media, and Metals. The experimental fungus used was Neurospora crassa (WT FGSC # 2489, Fungal Genetics Stock Centre (FGSC), Kansas, U.S.A.). It was routinely maintained on malt extract agar (MEA, Lab M limited, Bury, U.K.) in 90 mm diameter Petri dishes and grown at 25 °C in the dark. All experiments were conducted at least in triplicate. A modified medium AP1 was prepared of the following composition: 111 mM D-glucose (Merck, Readington Township, NJ, U.S.A.), 38 mM (NH4)2SO4 (Sigma-Aldrich, St. Louis, MO, U.S.A.), 4 mM K2HPO4·3H2O (Sigma-Aldrich, U.S.A.), 0.8 mM MgSO4·7H2O (Sigma-Aldrich, U.S.A.), 0.2 mM CaCl2· 6H2O (Sigma-Aldrich, U.S.A.), 2 mM NaCl (Sigma-Aldrich, U.S.A.), 9 × 10−3 mM FeCl3·6H2O (Sigma-Aldrich, U.S.A.)and trace metals 1.4 × 10−2 mM ZnSO4·7H2O (VWR, Radnor, PA, U.S.A.), 1.8 × 10−2 mM MnSO4·4H2O (Sigma-Aldrich, U.S.A.), and 1.6 × 10−3 mM CuSO4·5H2O (VWR, U.S.A.). All the salt and trace element solutions were sterilized separately by autoclaving (121 °C, 15 min) and mixed with sterile D-glucose solution when cool. The calcium carbonate-forming medium B



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Table 1. pH of Uninoculated or Inoculated Liquid Media Amended with Different Concentrations of Urea and CaCl2a uninoculated inoculated

AP1 + 40 mM urea + 0.2 mM CaCl2

AP1 + 40 mM urea + 50 mM CaCl2

AP1 + 330 mM urea + 50 mM CaCl2

5.50 ± 0.01 9.22 ± 0.01

5.50 ± 0.04 7.29 ± 0.10

5.50 ± 0.04 8.68 ± 0.14

Measurements from at least three replicates were taken after removal of the fungal biomass by centrifugation (4770 g × 20 min, 4 °C) after 12 days growth at 25 °C. The values indicate the standard error of the mean. The medium amended with 40 mM urea and 0.2 mM CaCl2 was used as the control while the other two media amended with 40 or 330 mM urea and 50 mM CaCl2 were the carbonate-forming media.

a

Figure 1. Scanning electron microscopy of mineral deposition by N. crassa grown in different media. Control medium: (A) AP1 media amended with 40 mM urea and 0.2 mM CaCl2, scale bar = 50 μm; Test media: (B) AP1 media amended with 40 mM urea and 50 mM CaCl2, scale bar = 100 μm, (C, D) AP1 media amended with 330 mM urea and 50 mM CaCl2, scale bars: C = 100 μm, D = 50 μm. Inset in D is a higher magnification image of the area indicated by the square, scale bar = 10 μm. All samples were incubated for 12 days at 25 °C in the dark. Typical images are shown from many similar examples.

Formation of Calcium Carbonate. Biomass of N. crassa grown in media amended with urea and CaCl2 was observed using scanning electron microscopy (SEM) (Figure 1). The minerals induced by the fungus were analyzed by energydispersive X-ray microanalysis (EDXA) (Figure 2). In media amended with 40 mM urea and a low concentration of CaCl2 (0.2 mM), there was little mineral deposition around the hyphae (Figure 1A), while at a high CaCl2 concentration (50 mM), abundant minerals of varying size and morphology appeared (Figure 1B). Moreover, in media amended with a higher concentration of urea (330 mM) and CaCl2 (50 mM), the whole surface of the fungal hyphae became rough and enveloped with mineral deposits (Figure 1C). Imprints of fungal hyphae were observed on the surface of larger mineral structures (Figure 1D) which indicated that the fungal hyphae were providing nucleation sites for the precipitation of these minerals. EDXA results showed that calcium, carbon and oxygen were the main elements present in precipitates from media amended with 50 mM CaCl2 and different concentrations of urea (Figure 2A). Some common Ca-containing minerals produced by fungi with this elemental profile are calcite (CaCO3) or calcium oxalate (CaC2O4·nH2O).39 X-ray diffraction (XRD) analysis revealed that the mineral that appeared in media with CaCl2 and 40/330 mM urea was calcite (CaCO3) (Figure 2B). The sample pattern fitted very well with standard calcite which indicated a very fine size or cryptocrystalline nature of the phase.

graphite, single crystal chrometer (30 mA, 40 kV). The hand ground samples obtained were firmly compacted on the reverse side of an aluminum specimen holder (15 × 20 × 2 mm3), held against a glass side. After compaction, the back cover was snapped into place and the glass side removed from the holder. Duplicate samples were analyzed over the range 3−60° 2θ at a scan rate of one degree/min in 0.1 degree increments.

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RESULTS pH Profile of Media after Growth of Neurospora crassa. N. crassa is a fast-growing urea-hydrolyzing fungus38 and was cultured on media amended with different concentrations of urea and CaCl2 for deposition of calcium carbonate. After inoculation and growth of N. crassa, the media became alkaline and the pH increased (Table 1). For media amended with 40 mM urea and 0.2 mM CaCl2, the pH increased from pH 5.5 to 9.2 while media amended with the same concentration of urea but a higher concentration of CaCl2 (50 mM), reached pH 7.3. This might be because as soon as the urea was hydrolyzed by the fungus, the high concentrations of Ca 2+ precipitated formed CO 3 2− as CaCO 3 . With consumption of available CO32− in the media, the resulting final pH reached a lower value than in media with 0.2 mM CaCl2. When the concentration of urea was increased to 330 mM, the medium rose to pH 8.7 providing suitable conditions for the precipitation of calcium carbonate. C



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Figure 3. Scanning electron microscopy of the Cd-containing precipitate produced by mixture of the supernatant of N. crassa grown in urea-containing medium with 0.5 M CdCl2. Scale bar = 2 μm. A typical image is shown from many similar examples.

Figure 2. (A) Energy dispersive X-ray analysis (EDXA) and (B) X-ray diffraction of minerals formed in AP1 media amended with 40 mM urea and 50 mM CaCl2 after incubation with N. crassa. Typical data are shown from one of several determinations.

Ca Removal by Fungal Biomass. The inclusion of urea in the media provided a crucial condition for the precipitation of calcite. More crystals were precipitated by N. crassa when the concentration of urea rose to 330 mM. Furthermore, compared with the fungal hyphae shown in Figure 1A,B, the hyphae in Figure 1C showed an obvious calcareous character due to the high concentration of urea and CaCl2. After 12 days incubation, the concentration of calcium in the supernatant and accumulated by the biomass was measured by atomic absorption spectroscopy (AAS) (Table 2). The results showed that after inoculation with N. crassa, the concentration of Ca in the supernatant of media amended with 40 mM urea containing 0.2 mM CaCl2 was 0.17 mM, and only around 16% of Ca was therefore removed from solution. However, for media amended with 40 mM urea and 50 mM CaCl2, about 53% of Ca was

Figure 4. Particle size distribution of Cd-containing minerals obtained by mixture of N. crassa supernatant with 0.5 M CdCl2 measured by Nano Measurer 1.2.5. The vertical line indicates the mean diameter of the particles.

removed while for media containing the highest concentration of urea, almost 93% of Ca was removed by the fungal biomass and the concentration of Ca in the supernatant was reduced from 50 to 4.5 mM (Table 2).

Table 2. Amount of Ca Remaining in the Supernatant and Removed by the Fungal Biomass after Incubation of N. crassa in Different Mediaa media AP1 + 40 mM urea + 0.2 mM CaCl2 AP1 + 40 mM urea + 50 mM CaCl2 AP1 + 330 mM urea + 50 mM CaCl2

concentration of Ca remaining in the supernatant (mM)

Ca removed by the fungal biomass (μmol mL−1)

proportion of Ca removed from solution (%)

0.17 ± 0.01

0.03 ± 0.01

16.4

23.64 ± 2.01

26.35 ± 6.01

52.7

4.47 ± 1.71

46.62 ± 2.57

93.2

a

Measurements were taken from at least three replicates and the values indicate the standard error of the mean. N. crassa was grown for 12 days at 25 °C in the dark. The medium amended with 40 mM urea and 0.2 mM CaCl2 was used as the control while the other two media amended with 40 or 330 mM urea and 50 mM CaCl2 were the carbonate-forming media. D



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Table 3. pH Change of the Supernatant after Mixing with 0.5 M CdCl2 and the Concentration of Cd Remaining in the Supernatant and the Proportion Removed by CdCO3 Precipitationa pH of the supernatant

pH of the mixture

concentration of Cd remaining in the supernatant after mixing (M)

proportion of Cd removed from solution (%)

9.18 ± 0.02

6.09 ± 0.03

0.24 ± 0.01

51.2

a

At least three replicates were measured and the values indicate the standard error of the mean. The fungal growth supernatant was collected after removal of the N. crassa biomass by centrifugation (4770g × 20 min, 4 °C) after 12 days growth in medium amended with 40 mM urea at 25 °C. The concentration of Cd remaining in the supernatant after mixing with CdCl2 was measured after removal of the CdCO3 precipitate by centrifugation.

Figure 5. Frequency histograms for particle sizes and the probability curve of a Gaussian distribution (f(x, μ, σ2), μ = 280.5, σ = 111.3). The random number sampled was 785.

Cd-Precipitation Mediated by Growth Supernatants of N.crassa after Growth with Urea. The calcite precipitation induced by N. crassa after growth in urea-modified medium provides a model system for metal carbonate precipitation. We hypothesize that this system could be used for precipitation of other toxic metals in a bioremediation or biorecovery context. The crystals formed by reaction of N. crassa supernatant and CdCl2 were approximately capsular in shape (cylinder with hemispherical ends) but aggregated together in various forms and displayed differing sizes (Figures 3 and 4). After the reaction, the pH of the solution had decreased from pH 9.2 to 6.1, and the concentration of Cd in the supernatant decreased to 0.24 M which indicated that 51% of the initially supplied Cd had been precipitated (Table 3). To further characterize the precipitated mineral, particle size analysis was carried out so that important parameters such as size and surface area, that are important in determining chemical reactivity, could be measured. Such parameters are

important when considering possible biotechnological applications of microbial biominerals. Approximately 800 particles were randomly chosen for size measurement using the software Nano Measurer 1.2.5 (Figure 4). To clarify the particle size distribution, particles were divided into 31 groups according to their size range, and the frequency of the different groups was counted: the probability of a single random particle to be in a particular size group was also calculated by the Gaussian Distribution equation:40 f (x , μ , σ ) = 1/[σ(2π )1/2 ]exp[ − (x − μ)2 /2σ 2]

Here, x is the length of the particles, μ is the mean diameter of sampled particles, and σ is the standard deviation based on the sample (Figures 5 and 6). According to the frequency histogram, particles within the range 224−252 nm were the most common while the highest probability ( f(x) ≈ 0.0036, x = 252−280 nm) in the Gaussian Distribution curve confirmed that particles around 252−280 nm represented the largest E



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Figure 7. (A) Energy dispersive X-ray analysis and (B) X-ray diffraction of crystals produced by mixture of growth supernatant of N. crassa and 0.5 M CdCl2. Typical data are shown from one of several determinations.

Figure 6. Capsular morphology assumption for the Cd-containing minerals. h is the height of the assumed cylinder, and r is the radius of the hemisphere.

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DISCUSSION Biomineralization of calcium carbonate has been widely studied and shows promising applications in various contexts, especially in construction and toxic metal remediation.3,8,24,32−34,41,42 There is little knowledge of fungal roles in this context however. The aim of this research was therefore to examine fungal-mediated calcium carbonate precipitation to provide more understanding of its environmental importance and possible biotechnological applications in novel biomaterial synthesis, and metal bioremediation or biorecovery. Burford et al.43 demonstrated that fungal hyphae became biomineralized with calcite (CaCO3) and calcium oxalate (CaC2O4·nH2O) crystals in simulated limestone microcosms. It has also been shown that calcite can be surrounded with oxalate crystals.44 Calcite precipitation phenomena occur in alkaline conditions and Stocks-Fischer et al.18 showed that ureasepositive Bacillus pasteurii favored the precipitation of calcite in an alkaline environment. To obtain an appropriate pH system for the precipitation of CaCO3 therefore, urea was used as the sole nitrogen source for the cultivation of urea-hydrolyzing fungi. The degradation of urea releases ammonium (NH4+)

proportion. To further understand some physical differences between particles of differing size, a simple model was used to calculate particle surface area and volume assuming a capsular geometry (Figure 6, Table 4). Table 4 shows that the mean diameter of the Cd-containing crystals was 280.5 nm, the maxmium diameter was 870 nm while the minimum diameter was about 55 nm which indicated that a proportion (∼1.5%) of the crystals were nanoscale. The surface area of particles with the maximum diameter was almost 250 times that of those with minimum diameter, while for the same volume of particles, the total surface area of the smallest particles was about 17 times that of the largest particles (Table 4). The precipitate produced by metal addition to the supernatant of N. crassa culture medium was analyzed by EDXA and XRD (Figure 7A,B). EDXA showed that the main elements in the crystals were Cd, C, and O (Figure 7A). According to the XRD pattern (Figure 7B) the crystals were identified as pure otavite (CdCO3). This indicates that the supernatant of ureagrown N. crassa could be a potentially useful reagent for precipitation of metal-containing carbonate minerals.

Table 4. Size Measurements of the Cd-Containing Minerals Obtained by Mixture of N. crassa Growth Supernatant with 0.5 M CdCl2 minimum mean maximum

length (nm)

surface area (nm2)

volume (nm3)

55.64 280.51 870.86

4.86 × 10 1.43 × 105 1.19 × 106

2.82 × 10 3.61 × 106 1.08 × 108

3

4

S/V

number of particles in 1 cm3

total surface area of particles in 1 cm3 (nm2)

0.17 0.04 0.01

3.55 × 10 2.77 × 1014 9.25 × 1012

1.73 × 1020 3.96 × 1019 1.10 × 1019

16

a

Particles were randomly chosen and the length and width of the particles were measured by using Nano Measurer 1.2.5. The particle surface area and volume were calculated by the formula: S = 4πr2 + 2πrh; V = 4/3πr3 + πr2h; h = x − 2r. x is the length of the particle, r is the assumed hemisphere radius which is half the width of the particle, and h is the height of the remaining cylinder (see Figure 6). F



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into the medium resulting in the pH increasing. It was found that the crystals formed in the media or deposited around fungal hyphae were calcite (CaCO3) which confirmed that the addition of urea in media was a crucial requirement for the precipitation of calcium carbonate. The key factors which affect CaCO3 precipitation include the concentration of calcium, the concentration of dissolved carbonate, pH, alkalinity, the medium composition and the availability of nucleation sites.19,45 Scanning electron microscopy clearly showed that many crystals formed around the fungal hyphae providing them with a thick calcareous coating. Furthermore, cracks involving fungal hyphae were observed on the surface of some of the crystals which indicated that the fungal hyphae were acting as nucleation sites for the precipitation of calcite. This assumption agrees with Stocks-Fischer et al.18 who found that the cell surface of B. pasteurii, which was located in the middle of calcite crystals, acted as a nucleation site during the precipitation process. Compared to the simpler bacterial cell form, the fungal filamentous growth habit could provide more framework support and stability for the precipitation of calcite or other biomineral crystals. Fungi are highly effective biosorbents for diverse metals including Pb, Cu, Zn, Fe, Ni, Ag, and La.6,26−28,43 The capacity of fungal biomass for binding metals such as Cd, Zn, and Cu declines at low pH.46 There is now much more attention given to removal of toxic metals from contaminated soil or water through biological methods for bioremediation as well as for metal biorecovery. Achal et al.24,32−34,42 applied microbially induced calcite precipitation (MICP) system to the remediation of toxic metals (Cu2+, Sr2+, Pb2+, As3+, and Cr6+) by using various urease-producing bacteria and proposed that this technology may provide an effective and economical treatment for toxic metal contaminated sites. Li et al.47 demonstrated that 88−99% of supplied toxic metals (Ni, Cu, Pb, Co, Zn, and Cd) could be removed as carbonates by urease-producing bacteria isolated from soil. In our experiments, the incubation of ureasepositive N. crassa in urea-modified media provided a means for the formation of calcite, as well as carbonates containing other metals, such as those related to toxic metal remediation. When the supernatant was mixed with CdCl2, the toxic Cd ions were precipitated in the form of otavite (CdCO3) thus immobilizing the cadmium. The otavite was found to be of high purity, and a small proportion exhibited nanoscale dimensions, which may provide further advantages for industrial applications than larger size biominerals. According to the morphology of the otavite particles, we assumed that the physical structure of these minerals could be defined by two hemispheres and a cylinder. Unsurprisingly, particles exhibiting nanoscale dimensions had a much greater surface area to volume ratio than the larger particles. The properties of minerals are influenced by various physical, chemical, and biological processes, and especially for nanoscale biominerals, their atomic and electronic structure, and the surface area to volume ratio can show a significant difference to bulk minerals.48 The utilization of carbonate-laden supernatants therefore provides us with a promising system for removal of metals from aqueous solution as carbonates. It may also provide a means for selective removal of metals and/or preparation of pure metal carbonates. Thus, we have demonstrated that urea-containing media provides a biotechnologically promising system for calcite precipitation by N. crassa. It is concluded that urease-positive fungi could be used for the precipitation of metal-containing carbonates, which therefore provides a means of metal biorecovery and purification.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +44 (0)1382 384767; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the help of Martin Kierans (Central Imaging Facility, College of Life Sciences, University of Dundee, U.K.) for assistance with scanning electron microscopy. We also acknowledge financial support from the China Scholarship Council through a PhD scholarship to Q.L. (No. 201206120066).

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