Accepted Manuscript Title: Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis Author: Dirk Dobritzsch Petra Ganz Michael Rother James Ehrman Renate Baumbach J¨urgen Miersch PII: DOI: Reference:
S0946-672X(15)00036-X http://dx.doi.org/doi:10.1016/j.jtemb.2015.03.006 JTEMB 25671
To appear in: Received date: Revised date: Accepted date:
23-10-2014 18-3-2015 27-3-2015
Please cite this article as: Dobritzsch D, Ganz P, Rother M, Ehrman J, Baumbach R, Miersch J, Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis, Journal of Trace Elements in Medicine and Biology (2015), http://dx.doi.org/10.1016/j.jtemb.2015.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Cadmium-induced formation of sulphide and cadmium sulphide particles
Dirk Dobritzsch1*, Petra Ganz1, Michael Rother1, James Ehrman2,
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Renate Baumbach1, Jürgen Miersch1
Institute of Biochemistry and Biotechnology, Division of Ecological and Plant Biochemistry,
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in the aquatic hyphomycete Heliscus lugdunensis
Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale),
*e-mail: [email protected] 2
Digital Microscopy Facility, Mount Allison University, Sackville N.B., E4L 1G7, Canada,
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Germany,
Key words: Cadmium, CdS crystallites, sulphide, glutathione, EDS, aquatic fungi, Heliscus
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e-mail: [email protected]
lugdunensis
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____________________ *) corresponding author Page 1 of 22
2 Abstract Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd2+ in the medium) was measured in the aquatic
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hyphomycete Heliscus lugdunensis. Up to 25 µmol g-1 dry mass the fungus formed sulphide
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in an exponentially Cd2+-concentration-dependent manner. Using light microscopy,
precipitates were observed outside of the hyphae which could be determined as amorphous
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particles by x-ray diffraction (XRD). Energy dispersive x-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1,
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but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd2+ accumulated 12 to 28 µmol metal g-1 dry mass over a period of 7 to 28 days.
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The results may indicate that sulphide could sequester excess Cd2+ under oxygen deprived
fungus.
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Abbreviations
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conditions and thereby reduce its toxicity via an additional avoidance mechanism of this
AAS, atomic absorption spectrometry; AQH, aquatic hyphomycetes, BB, Boss Brook; DMPD, N,N-dimethyl-p-phenylenediamine-dihydrochloride; d.m., dry mass; DTE, 45
dithioerythritol; DTNB, 5,5’-dithio-bis-(2-nitrobenzoic acid); EDS, energy dispersive x-ray spectroscopy; f.m., fresh mass; -EC, -glutamylcysteine; GSH, glutathione; Hl, acronym of Heliscus lugdunensis; HMW-complex, high molecular weight-complex; HPLC, high performance liquid chromatography; Hsp, heat shock protein; MT, metallothionein; PC, phytochelatin; SEM, scanning electron microscopy; TAP, time after pressure; TFA,
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trifluoroacetic acid; TTC, 2,3,5-Triphenyltetrazoliumchloride; XRD, x-ray diffraction
Introduction Page 2 of 22
3 Freshwater fungi play an important role in both, aquatic ecosystem integrity and health. They depend on decomposition of energy-rich organic compounds, produced by other organisms 55
and act as highly efficient contributors to aquatic ecosystems by transfer of nutrients between
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different trophic levels [1, 2]. Toxic metals and high concentrations of essential metals provoke major environmental and
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human health problems due to toxic impacts in different biota and food networks. Freshwater fungi that survive and thrive under harsh conditions, such as high metal and xenobiotics pollution have become a focus of increasing scientific attention [1].
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In freshwater fungi fluctuating environmental metal concentration are balanced to tolerable
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intracellular concentrations by a sophisticated metal homeostasis comprising various
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mechanisms [1], including biosorption to cell wall [3, 4], regulated cellular transport systems [5], heat shock proteins [6], modulation of reactive oxygen species [3, 5, 7], and contribution by thiolpeptides [3, 4, 8-10]. The thioltripeptide glutathione is a crucial compound in fungal
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cells, because it mediates redox reactions (by alteration between oxidized dithiol and reduced disulphide forms) as well it acts as starter compound for the biosynthesis of metal chelating
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phytochelatins.
The aquatic hyphomycete Heliscus lugdunensis is one of the most abundant fungal species 70
inhabiting highly heavy metal polluted sites (water [11], groundwater [12], sediments [13]). Several H. lugdunensis strains have already been reported to respond to Cd2+ exposition by cellular thiol peptide response, such as increase of glutathione level [3, 4, 8, 10] accompanied by rised levels of cysteine and γ-glutamyl-cysteine [3] as well as synthesis of phytochelatin 2 and a small metallothionein, MT1_HL [10]. As known from the aquatic hyphomycete
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Tetracladium marchalianum Cd2+ exposure raised intracellular soluble sulphide content [9]. Up to now, nothing is known about extracellular precipitation of CdS crystallites by aquatic hyphomycetes.
Page 3 of 22
4 In the present study, Cd2+-induced sulphide formation and extracellular precipitation of Cd2+ was determined using a H. ludgdunensis strain, isolated from an unpolluted habitat with 80
attention on possible involvement of sulphide in Cd2+ detoxification.
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Material and Methods Fungal strain and cultivation
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The strain Hl-BB of Heliscus lugdunensis Sacc. & Thérry was isolated from single conidia
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collected from a non-polluted stream Boss Brook near Amherst, Nova Scotia [14]. The strain is available from German Resource Centre for Biological Material (DSMZ Braunschweig, Hl-
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BB: DSM 18483).
Fungi were maintained on malt extract agar (0.5% malt extract, 0.1% peptone, 1.5% agar
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containing 270 µM sulphate). To examine heavy metals stress, the mycelia were cultivated in 150 mL medium in 200 mL Erlenmeyer flasks under stationary (non-shaking) conditions with limited oxygen supply (ca. 50% oxygenation of ca. 8 mg/L). The medium contained 0.5%
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malt extract, 0.1% peptone and 270 µM sulphate (sulphide free). In general, the cultures were
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inoculated with homogenized agar slices overgrown with 21-28 day old mycelia cultivated at 14±1 °C, the total fungal biomass introduced per flask was 3.4 mg d.m. Stock Cd2+ solutions (as CdCl2, filtrated through 0.2 µm membrane filters) were added on the fourth day of 95
cultivation to adjust the final metal concentration at 80 µM in the medium. Cultures were incubated in three replicates with and without Cd2+ at a final concentration of 80 µM and were harvested by filtration (Merck, filter 0869) after different times of cultivation. To isolate crystal-like particles in a higher yield for elemental analysis and HPLC, mycelia were cultivated in a ten-fold volume of medium inoculated with 82 mg d.m. of a
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liquid fungal preculture. Determination of dry mass
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5 Mycelia were rinsed twice with distilled water, blotted dry between filter paper and weighed (fresh weight). To determine wet to dry weight ratio, preweighed fresh mycelia were exposed to 80 °C for at least 2 h. Measurement of total dehydrogenase and oxygen
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Dehydrogenase activity was used as a general activity parameter and determined according to
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Fahmy and Walsh [15] using TTC reduction 2 h at 37 °C. The reaction mix contained 50 mg
f.m. in 5 mL 50 mM phosphate buffer (pH 7.5) containing 1 M sorbitol, 70 µL 1 M DTE and
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100 µL lyticase (Molzym). After centrifugation at 3600 × g (Hettich centrifuge type 30 RF) the pellet was resuspended in 2 mL 50 mM phosphate buffer (pH 7.5), mixed with 0.75 mL
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0.5% TTC (w/v) and incubated for 6 h at room temperature. The resulting formazane was
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extracted using 1 mL acetic acid and 2 mL toluol. Extinction was measured at 380 nm (n = 4). Oxygen in the culture medium was measured using an amperometric electrode (Mikro-
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Sauerstoffsensor 200, UMS GmbH, n = 5) and pH using a pH-meter (WTW 538; n = 5). Preparation and imaging of precipitates
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Culture liquid and mycelia were separated using a 1 × 1 mm 2 mesh-size sieve and subsequent
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centrifugation (10 min, 4 oC, 4000 × g). All following steps were carried out at 4 oC. The precipitate was resuspended in 20 mL Percoll medium (Pharmacia-Biotech, d = 1.130 g mL1
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), stirred for 10 min and centrifuged (30 min, 12000 × g). Hyphae without particles were
found at the top of the gradient while particles and hyphae attached particles precipitated at the bottom. This procedure was repeated with a 30 min stirring time. After decantation of Percoll, the precipitate was diluted with water (1:2) and centrifuged (4000 × g, 10 min). A portion of the particles was suspended in a drop of water and observed with a light microscope (Zeiss Axiolab) equipped with a digital camera (Sony DSC-S75). The particles
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were dried in vacuum and stored under N2 at 4 oC. Measurement of thiols and HPLC
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6 Non-protein thiols were extracted according to Grill et al. [16], modified by Miersch et al. [8]. Total thiols (TSH) were determined with Ellman’s reagent [17], glutathione was measured enzymatically [18], and non-protein thiols were analyzed using HPLC (Merck-Hitachi) [19]. Each sample consisting of 200 mg f.m. of hyphae or approximately 50 mg of wet particles
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was dissolved in 400 µL NaOH (1 N, added 1 mg mL-1 NaBH4) and centrifuged (5 min, 7000
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× g). After cooling on ice, 140 µL 3.6 N HCl was added and centrifuged (5 min, 10000 × g, 4 o
C). Samples of particles were diluted 1:4 with 1 N NaOH. 50 µL was separated on a RP 18
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column (SuperPac Sephasil-C-18, 5 µm, 4.6 × 250 mm). Absorbance was measured at 410 nm after post-column derivatization with DTNB as described earlier [20]. The thiols were
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eluted applying a linear gradient of 2-20% acetonitrile in TFA (0.1% v/v) over 25 min at 0.8
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mL min-1. In order to exclude the detection of non-thiolic compounds absorbing at 410 nm, the post column derivatization was repeated with KH2PO4 without Ellman’s reagent as a
Sulphide measurement
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negative control [21].
Intracellular water-soluble sulphides were measured according to King and Morris [22] using
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a modified procedure. 50 mg of fungal sample were crushed in liquid nitrogen and extracted for 5 min by stirring in 1 mL oxygen-free water. The mixture was homogenized and 300 µL were transferred in an Eppendorf tube (n = 3) containing 1240 µL 2.6% Zn-acetate-solution 145
(w/v) and 240 µL 6% NaOH (w/v). After stirring for 15 min at 4 oC, 200 µL of 0.2% DMPD (Fluka) in 20% H2SO4 and 20 µL of 10% FeNH4(SO4) × 12 H2O (Fluka) in 2% H2SO4 were added to this mixture and stored in the dark for 30 min. The mixture was centrifuged (23000 × g, 10 min, 4 oC). The absorbance was determined at 665 nm from the clear supernatant. For calibration, solutions of 0.2-100 µM Na2S in distilled water were used The detection limit was
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0.25 µmol sulphide g-1 d.m. (with an SD of 25%). To estimate sulphide in medium, 1780 µL clear filtrate and 200 µL of 0.2% DMPD (Fluka) in 20% H2SO4 and 20 µL of 10%
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7 FeNH4(SO4) × 12 H2O (Fluka) in 2% H2SO4 were prepared as outlined above (LOD = 0.4 µM. Measurement of adsorbed and intracellular cadmium After 7, 14 and 28 d, the mycelia were harvested on a filter (Whatman No.3), washed for 5
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min with 100 mL distilled water, 3 times with 100 mL 20 mM NiCl2 solution, which causes
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the extracellular Cd2+ ions to be replaced by Ni2+ [23]. This washing did not cause any leakage of intracellular Cd²+ as it was tested by several washings using different Ni
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concentrations and washing time. The mycelia were then washed with 100 mL water, removed from the filter and dried 24 h at 80 °C. 50 mg dried fungal mass were dissolved with
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4 mL 65% (v/v) HNO3 and 2 mL 30% (v/v) H2O2 in a microwave (step 1: 130 bar, 10 min,
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TAP 5 min; step 2: 275 bar, 20 min, TAP 10 min, 180 oC; step 3: 558 bar, 10 min, 180 oC; CEM MDS 2100, Kamp-Lintfort).
Cadmium concentrations in the NiCl2 washing steps (adsorbed Cd2+) and in the mycelium digests (accumulated Cd2+) were determined by AAS at not interfering wavelength (ATI
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0.05 mg × L-1 Cd2+).
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Unicam). Standard solutions of 0.0625 to 2.0 mg L-1 Cd2+ were used for calibration (LOD =
SEM and EDS of particles
Particles were analyzed using scanning electron microscopy and energy dispersive x-ray 170
spectroscopy (Fig. 3). Specimens for SEM/EDS were handled inside a dry nitrogen flushed bag whenever possible. Transfer to coating equipment and the microscope was performed as quickly as possible to reduce exposure to atmosphere. Specimens residing on their collecting substrates (filter paper or cellophane) were attached to 10 mm aluminum supports using double-sided tape and rimmed with either colloidal carbon (ca. 15 nm using a Denton DV-
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502A vacuum evaporator, Denton Vacuum, Moorestown, NJ) or gold (ca. 15 nm gold using a Hummer 6.2 sputtering system, Anatech Ltd., Hayward, CA). Carbon coating allowed collection of EDS spectra without interfering Au M-shell x-ray lines that would obscure Page 7 of 22
8 elements of interest. Gold coating was required for high-resolution SEM imaging as well as analyses that were aimed at the detection of the presence of carbon in particles. All specimens 180
were examined using a JEOL JSM-5600 SEM (JEOL USA, Peabody MA) equipped with an
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Oxford Inca 200 EDS system (Oxford Instruments, High Wycombe, UK). SEM/EDS images and data were acquired using a 20 mm working distance and 15 kV accelerating voltage.
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Statistical analysis
Determination of standard deviation (SD) for dry weight, sulphide, cadmium, and thiols was performed with MS Excel 6.0 or Graph Pad Instat 3.05 (ANOVA). Significance of differences
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was calculated using Student’s t-test. P values < 0.05 were considered significant. Results
affecting growth and fungal activity
Heliscus lugdunensis strains BB growing under facultative anaerobic conditions showed a
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Cd2+ causes the formation of sulphide together with crystall-like precipitates while differently
reduced growth while crystal-like particles were formed in the medium accompanied by
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sulphide generation in cultures containing Cd2+ (Fig. 1). The formation of sulphide started
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immediately after addition of Cd2+ (Fig. 2) and within 28 days a concentration of 25±1.562 µmoles g-1 d.m. was reached. No sulphide was measured in controls (without Cd2+). 195
Cadmium addition at 80 µM decreased fungal growth by 18% compared to controls after 6 days (Fig. 1).
The level of oxygen changed from 9±0.09 mg L-1 to 4.93±0.29 mg L-1 (control) and 4.35±0.17 mg L-1 (Cd2+ exposure) within 6 d, and had only slightly further decreased in Cd2+treated cultures and controls after 14 (16) d: 4.57±0.12 (3.93±0.12) mg L-1 in control and 200
4.10±0.33 (4.03±0.17) mg L-1 in Cd2+ exposed cultures. This may indicate that Cd2+ did not substantially affect fungal oxygen consumption. The pH of the medium (n = 4) decreased within 6 d from 5.63±0.04 to 4.99±0.11 and 4.84±0. in control and after Cd2+ exposure, respectively (p > 0.5). Afterwards, the pH was Page 8 of 22
9 nearly constant during 16 d reaching a final level of 4.96±0.05 in control and 5.03±0.04 in 205
Cd2+ cultures (p > 0.7). Cd-containing crystall-like particles show two types of elemental composition
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Initially, the cultures were investigated using light microscopy for their response to Cd2+. Yellow, crystal-like particles could be found from the middle of the log-phase of fungal
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growth in Cd2+-containing media (Fig. 1 insert) in both 150 mL as well as 1.5 L cultures. The yellow particles from the large culture were investigated using EDS and revealed two types of
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elemental composition. Samples of the first type contained a large number of relatively illdefined, flattened particles (data not shown) with lengths and widths in the 30-60 µm range,
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but only approximately 1 µm in thickness (Fig. 3A, insert). EDS analyses indicated that these
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particles contained a varying amount of Cd and S, with atomic% Cd:S between 2.39:2.09 and 30.52:25.62 (mean 9.15±8.82:8.31±7.30; n = 11). However, the extreme thinness of the
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specimens means that all spectra collected on such particles were actually composites of the
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particles and the substrate. Cd:S ratios were nevertheless reasonably close to 1:1 (1.05 ± 0.12; n =11). Thus, EDS alone could not rule out the possibility that fine, microcrystalline CdS was
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present in an amorphous matrix containing other elements from the culture broth, such as P and Cl (Fig. 3A). XRD analysis of this material does not support this hypothesis, however, as no prominent peaks for any crystalline material were obtained (data not shown). The second type of particles was well defined, ranging in size from 1 to 15 µm (Fig. 3 C, insert). All particles analyzed with EDS contained almost exclusively Cd and S (Fig. 3 C). EDS spectra collected from particles > 15 µm with flat top surfaces and minimum fracturing 225
yielded atom% Cd:S of 50.5±0.9:49.5±0.9 (n = 10) with only trace amounts of oxygen that could be attributed to surface oxidation or contaminants from solution. XRD analysis of this material produced prominent x-ray scattering peaks but the peaks did not correspond to known crystalline structures for either CdS or other cadmium compounds (data not shown).
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10 When hyphae of Cd2+-containing cultures or particles were exposed to acid a characteristic 230
smell of hydrogen sulphide could be noticed. In addition, purified particles were mixed with a saturated solution of lead acetate, producing the typical black needle-shaped crystals of PbS
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visible in the light microscope (not shown). Such samples contained a large number of elongated, typically cylindrical-shaped particles ranging in size from 5 × 2.5 µm to 12 × 4 µm
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(Fig. 3 B, insert). Lead and sulphur x-ray peaks are strongly overlapped in EDS spectra (Fig. 3 B), but peak deconvolution and subsequent semi-quantitative analyses indicated that Pb:S
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ratios for these particles were approximately 1:1. However all particles examined contained large concentrations of oxygen, approximately four times that of Pb and S, indicating that if
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PbS was formed, it was possibly rapidly oxidized to sulphate during sample processing.
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Characteristics of thiols derived from hyphae and particles
In control hyphae the eluted thiols were cysteine, GSH (coeluted with -EC, Fig. 4 A)
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whereas in Cd2+-stressed hyphae a remarkable peak at 7 min retention time (possibly
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sulphide) was observed (Fig. 4 B). In contrast to these results the elution profile of prepared enriched particles was dominated by only one peak at 7 min retention time, which is probably
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sulphide (Fig. 4 C) because i) sulphidic sulphur is indicated in most cases by post column derivatization by DTNB of particles, ii) paltry presence of oxygen (which imply the absence of sulphate) iii) complete absence of other elements in EDS (Fig. 3 C) and iv) during acidification hydrogen sulphide was released proved by precipitation of Pb-sulphide after addition of Pb-acetate (Fig. 3 B).
Small amounts of cysteine and GSH could not be excluded (Fig. 4 C). Other anions like citric, 250
malic and oxalic acid were not found by HPLC (data not shown). Apoplastic Cd2+-exposure leads to symplastic Cd2+-accumulation H. lugdunensis exposed to Cd2+ adsorbed metal ions externally by the apoplast and accumulated internally by the symplast. During 7 days mycelial biosorption increased rapidly to 80.1±5.9 µmoles g-1 d.m. and decreased initially (7-14 d) fast, then slows down to 54.3±3.4 Page 10 of 22
11 255
µmoles g-1 d.m. on 28 days (p < 0.05). Distinct levels of Cd2+ were accumulated by mycelia to 12.5±0.8 µmoles g-1 d.m. and reached 22.5±3.4 µmoles g-1 d.m. (p < 0.05) from 7 to 28 days (Fig. 5).
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Discussion Aquatic hyphomycetes are heterotrophic organisms of aerobic natural aquatic environments [1]. Their survival in metal polluted sites depends on avoidance/tolerance mechanisms.
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Different strains of the aquatic hyphomycete Heliscus lugdunensis showed distinct responses
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to metals, such as metal sorption to the cell wall, changes in plasma membrane integrity, intracellular changes of macromolecular complexes and reactive oxygen species as well as
265
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changes in thiol peptide biosynthesis [1]. To avoid interference with surplus of sulphur, in this study, CdCl2 was used without any negative impact of Cl- ions as tested with NaCl. However,
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Babich & Stotzky[24] suggested a decreased Cd2+ toxicity to fungi by formation of cadmium
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chloride complexes.
As known from other fungi the formation of cadmium sulphide could be another mechanism
Schizosaccharomyces pombe [27, 28], Saccharomyces cerevisiae [29]. In the aquatic
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to detoxify Cd2+, e.g. Candida glabrata [25], Fusarium oxysporum [26],
hyphomycete Tetracladium marchalianum Cd2+ exposure under aerobic conditions raised the sulphide level in the hyphae [9]. Agitated cultures of Heliscus lugdunensis strains were able to excrete sulphide without formation of extracellular CdS. Crystallites were formed only under static conditions providing a reduced oxygen supply, as also applied to H. lugdunenesis 275
strain Hl-BB in this study. Here, exposure to 80 µM Cd2+ inhibited fungal growth to 33% within the first 4 days (Fig. 1) as assayed by dehydrogenase activity as viability marker. Obviously, inhibition rate depends on strain origins. While strain Hl-BB from a non-polluted site [8] and strain H8-2-1 from a moderately polluted water [3, 11] showed lower Cd2+ sensitivity, strain H4-2-4 from a highly metal polluted habitat showed a much higher
280
tolerance [11]. Page 11 of 22
12 As shown in Fig. 1 (insert) extracellular particles were found in facultative anaerobic Cd2+ exposed cultures of H. lugdunensis while the strain was also able to generate sulphide up to 25 µmoles g-1 d.m. (Fig. 2). We assume an exclusively de-novo-biosynthetical origin of
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sulphide up to day 6. While a participation of thiol peptides in several intracellular detoxification of metal by
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aquatic fungi is undisputed up to now, the involvement of these peptides in formation of extracellular particles is still under discussion [1].
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The atom% Cd:S ratio is dependent on ion concentration in the nutrient medium [30]. In our study, EDS of amorphous particles from cultures of H. lugdunensis revealed two kinds of particles with different elemental composition: i) well defined particles containing almost
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exclusively Cd and S (Fig. 3 C) and ii) particles containing varying amounts of Cd and S and other elements/anions (Fig. 3 A). We assume, the aggregation started with small particles
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(Fig. 3 C) partially at the surface of the cell wall in the form of small clusters (Fig. 1 insert).
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Then, further elements of the medium were inserted and larger particles developed (Fig. 3 A), such as phosphorous and chlor. Since chloride were detectable only after longer period of
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time as well as not in the backround, no interference occured. No organic anions (citric-, oxalic-, malic acid) could be measured (Martienssen, personal communication). In terrestrial fungi, phosphates, polyphosphates or oxalates are common response to toxic heavy metal concentrations resulting in the formation of metal salts deposited intra- or extracellularly [31, 300
32].
During exposition of alder leaves in a high heavy metal polluted water in Mansfeld area a metal-rich layer (zincowoodwardite) was deposited on the leave surfaces with significant amounts of aluminium, copper and zinc but also sulphur [33]. HPLC profiles show peaks at 7 min (sulphide, Fig. 4 B, C), which most likely could be 305
sulphide (Fig. 4 C). Sulphide was bound and precipitated as Pb-sulphide after addition of water-dissolved Pb-acetate and could be analyzed by EDS (Fig. 3 B). Page 12 of 22
13 Sulphide is probably generated in the symplast but nothing is known about fungal efflux mechanisms of sulphide It can be hypothesized, that the regulation of sulphate assimilation in H. lugdunensis changed under facultative anaerobic conditions and Cd2+ exposure. Sulphite-reductase activity and its
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transcript were enhanced in H. lugdunensis strain H4-2-4 under Cd2+ influence, too (Nathan,
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unpublished results). Increased sulphur assimilation was found in earlier experiments showing that copper uptake by copper-tolerant strains of S. cerevisiae induced an intensive cysteine
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metabolism, resulting in black deposits of copper sulphide on the cell wall [34]. Hydrogen sulphide was found to act as a population sychronizer during aerobic continuous culture of the
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same yeast [35]. In response to Cd2+ the yeasts Schizosaccharomyces pombe and Candida
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glabrata synthesized intracellularly acid-labile sulphide- and PC-containing CdS quantum semiconductor crystallites [36] in which sulphide increased both stability of the HMW-
activity, required methionine or cysteine for growth and could not synthesize PC-coated CdS-
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complex and the Cd2+ amount [37]. Mutants of C. glabrata, devoid in cysteine-synthetase
particles. However, these cells were able to accumulate high levels of sulphide as well as to
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precipitate CdS outside the cell [38] as found similary in aquatic hyphomycete Heliscus by our experiments. Extracellular sulphide-containing deposits (sometimes as crystallites) were also produced by S. pombe [27, 28] and C. glabrata [25]. 325
All in all, these results may indicate that under conditions of diminished intracellular phytochelatine synthesis [10] sulphide could sequester excess Cd2+ and thereby reduce its toxicity via an additional avoidance mechanism.
Acknowledgements 330
The authors thank Dr. R. Bruening (Physics Dept., Mount Allison University, Sackville) for helping in XRD-analyses, Dr. M. Martienssen (Helmholtz Centre for Environmental Research
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14 – UFZ, Dept. of Hydrogeology, Halle) for the analysis of organic acids, Mrs. E. Püschel and E. Funke for technical assistance.
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Krauss GJ, Solé M, Krauss G, Schlosser D, Wesenberg D, Bärlocher F. Fungi in freshwaters: ecology, physiology and biochemical potential. FEMS microbiology reviews 2011;35:620-51. Schneider T, Gerrits B, Gassmann R, Schmid E, Gessner MO, Richter A, et al. Proteome analysis of fungal and bacterial involvement in leaf litter decomposition. Proteomics 2010;10:1819-30. Braha B, Tintemann H, Krauss G, Ehrman J, Barlocher F, Krauss GJ. Stress response in two strains of the aquatic hyphomycete Heliscus lugdunensis after exposure to cadmium and copper ions. Biometals 2007;20:93-105. Jaeckel P, Krauss GJ, Krauss G. Cadmium and zinc response of the fungi Heliscus lugdunensis and Verticillium cf. alboatrum isolated from highly polluted water. Sci Total Environ 2005;346:274-9. Azevedo MM, Carvalho A, Pascoal C, Rodrigues F, Cassio F. Responses of antioxidant defenses to Cu and Zn stress in two aquatic fungi. Sci Total Environ 2007;377:233-43. Miersch J, Grancharov K. Cadmium and heat response of the fungus Heliscus lugdunensis isolated from highly polluted and unpolluted areas. Amino acids 2008;34:271-7. Azevedo MM, Almeida B, Ludovico P, Cassio F. Metal stress induces programmed cell death in aquatic fungi. Aquat Toxicol 2009;92:264-70. Miersch J, Bärlocher F, Bruns I. Effects of cadmium, copper, and zinc on growth and thiol content of aquatic hyphomycetes. Hydrobiologia 1997;346:77-84. Miersch J, Neumann D, Menge S, Bärlocher F, Baumbach R, Lichtenberger O. Heavy metals and thiol pool in three strains of Tetracladium marchalianum. Mycol Progress 2005;4:185-94. Jaeckel P, Krauss G, Menge S, Schierhorn A, Rucknagel P, Krauss GJ. Cadmium induces a novel metallothionein and phytochelatin 2 in an aquatic fungus. Biochemical and biophysical research communications 2005;333:150-5. Krauss G, Bärlocher F, Schreck P, Wennrich R, Glasser W, Krauss GJ. Aquatic hyphomycetes occur in hyperpolluted waters in Central Germany. Nova Hedwigia 2001;72:419-28. Krauss G, Sridhar KR, Jung K, Wennrich R, Ehrman J, Barlocher F. Aquatic hyphomycetes in polluted groundwater habitats of central Germany. Microbial ecology 2003;45:329-39. Sridhar KR, Bärlocher F, Wennrich R, Krauss GJ, Krauss G. Fungal biomass and diversity in sediments and on leaf litter in heavy metal contaminated waters of Central Germany. Fund Appl Limnol 2008;171:63-74. Bärlocher F. Aquatic hyphomycete spora in 10 streams of New Brunswick and Nova Scotia. Canadian Journal of Botany 1987;65:76-9. Fahmy AR, Walsh EO. The quantitative determination of dehydrogenase activity in cell suspensions. The Biochemical journal 1952;51:55-6. Grill E, Winnacker EL, Zenk MH. Phytochelatins. Methods Enzymol 1991;205:33341.
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Ellman GL. Tissue sulfhydryl groups. Archives of biochemistry and biophysics 1959;82:70-7. Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Method Enzymol 1985;113:548-55. Bräutigam A, Schaumlöffel D, Krauss GJ, Wesenberg D. Analytical approach for characterization of cadmium-induced thiol peptides-a case study using Chlamydomonas reinhardtii. Anal Bioanal Chem 2009;395:1737-47. Bruns I, Siebert A, Baumbach R, Miersch J, Gunther D, Markert B, et al. Analysis of heavy-metals and sulfur-rich compounds in the water moss Fontinalis Antipyretica L. ex Hedw. Fresen J Anal Chem 1995;353:101-4. Berlich M, Menge S, Bruns I, Schmidt J, Schneider B, Krauss GJ. Coumarins give misleading absorbance with Ellman's reagent suggestive of thiol conjugates. The Analyst 2002;127:333-6. King TE, Morris RO. Determination of acid-labile sulfide and sulfhydryl groups. In: Ronald W. Estabrook MEP, editor. Method Enzymol: Academic Press, 1967. p. 63441. Brown D, Wells J. Sequention elution technique for determing the cellular location of cations. In: Glime J, editor. Methods in Bryology Proc bryol methods Workshop, Mainz Hattori bot lab, Nichigan, 1988. p. 227-33. Babich H, Stotzky G. Influence of chloride-ions on the toxicity of cadmium to fungi. Zbl Bakt Mik Hyg I C 1982;3:421-6. Mehra RK, Mulchandani P, Hunter TC. Role of CdS quantum crystallites in cadmium resistance in Candida glabrata. Biochemical and biophysical research communications 1994;200:1193-200. Ahmad A, Mukherjee P, Mandal D, Senapati S, Khan MI, Kumar R, et al. Enzyme mediated extracellular synthesis of CdS nanoparticles by the fungus, Fusarium oxysporum. Journal of the American Chemical Society 2002;124:12108-9. Murasugi A, Wada Nakagawa C, Hayashi Y. Formation of cadmium-binding peptide allomorphs in fission yeast. Journal of biochemistry 1984;96:1375-9. Gade A, Ingle A, Whiteley C, Rai M. Mycogenic metal nanoparticles: progress and applications. Biotechnology letters 2010;32:593-600. Prasad K, Jha AK. Biosynthesis of CdS nanoparticles: An improved green and rapid procedure. Journal of colloid and interface science 2010;342:68-72. Pal U, Loaiza-Gonzáles G, Bautista-Hernández A, Vázquez-Cuchillo O. Synthesis of CdS nanoparticles through colloidal rout. Superfic Vacio 2000;11. Gadd GM. Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycological research 2007;111:3-49. Gadd GM. Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology 2010;156:609-43. Ehrman JM, Bärlocher F, Wennrich R, Krauss GJ, Krauss G. Fungi in a heavy metal precipitating stream in the Mansfeld mining district, Germany. Sci Total Environ 2008;389:486-96. Kosman DJ. Transition metal ion uptake in yeasts and filamentous fungi. In: Winkelmann G, Winge DR, editors. Metal ions in fungi. New York: Marcel Dekker, 1994. p. 1–38. Sohn H, Kuriyama H. Ultradian metabolic oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide, a population synchronizer, is produced by sulphite reductase. Yeast 2001;18:125-35.
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[35]
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430 [37] [38] 435
Dameron CT, Reese RN, Mehra RK, Kortan AR, Carroll PJ, Steigerwald ML, et al. Biosynthesis of cadmium-sulfide quantum semiconductor crystallites. Nature 1989;338:596-7. Reese RN, Winge DR. Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe. The Journal of biological chemistry 1988;263:12832-5. Barbas J, Santhanagopalan V, Blaszczynski M, Ellis WR, Winge DR. Conversion in the peptides coating dadmium-sulfide crystallites in Candida glabrata. J Inorg Biochem 1992;48:95-105.
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[36]
Fig. 1: Growth of H. lugdunensis shown as mg d.m. in 150 mL culture medium. Control -
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Legends to figures
white rhomb, 80 µM Cd - black rhomb, * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 3).
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Dehydrogenase activity was used as a general parameter of cell vividness (data not shown)
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Inserts show crystal-like formations aggregated at a hypha after 14 d of this culture. Fig. 2: Generation of sulphide by hyphae of H. lugdunensis in response to cadmium. The hyphae were cultivated in 150 mL medium without (control, white square) or with Cd (80
d
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µM, black square). Cd(II) was added at fourth day of cultivation. *** p < 0.001 (n = 3). Fig. 3: Scanning electron microscopy (insert) and energy dispersive x-ray spectroscopy of
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particles isolated from a 21 d old culture of H. lugdunensis. EDS spectra from areas analyzed on particles (bold line) and background substrate (thin line) are indicated by boxes labeled on 450
SEM micrograph (insert in A – C). (A) Amorphous particles containing Cd and S but also other elements from culture broth. (B) PbS derived by acidification of particles and reaction of hydrogen sulphide with lead acetate. (C) Crystal-like particles primarily containing approximately equal atomic% values for Cd and S (n = 7). Fig. 4: HPLC of sulphur-containing compounds in hyphae and particles isolated after 14 d of
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cultivation of H. lugdunensis in 1.5 L medium; A, control; B, hyphae exposed to 80 µM Cd; C, partial purified crystal-like material (All samples were assayed by HPLC in three technical replicates).
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17 Fig. 5: Measurement of adsorbed and intracellular Cd2+ by AAS as well as sulphide according to King and Morris [22] in Cd2+-containing cultures of H. lugdunensis up to 28 days (Three
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biological replicates were assayed in three technical replicates).
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S0946-672X(15)00036-X http://dx.doi.org/doi:10.1016/j.jtemb.2015.03.006 JTEMB 25671
To appear in: Received date: Revised date: Accepted date:
23-10-2014 18-3-2015 27-3-2015
Please cite this article as: Dobritzsch D, Ganz P, Rother M, Ehrman J, Baumbach R, Miersch J, Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis, Journal of Trace Elements in Medicine and Biology (2015), http://dx.doi.org/10.1016/j.jtemb.2015.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
Cadmium-induced formation of sulphide and cadmium sulphide particles
Dirk Dobritzsch1*, Petra Ganz1, Michael Rother1, James Ehrman2,
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Renate Baumbach1, Jürgen Miersch1
Institute of Biochemistry and Biotechnology, Division of Ecological and Plant Biochemistry,
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in the aquatic hyphomycete Heliscus lugdunensis
Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale),
*e-mail: [email protected] 2
Digital Microscopy Facility, Mount Allison University, Sackville N.B., E4L 1G7, Canada,
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Germany,
Key words: Cadmium, CdS crystallites, sulphide, glutathione, EDS, aquatic fungi, Heliscus
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e-mail: [email protected]
lugdunensis
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____________________ *) corresponding author Page 1 of 22
2 Abstract Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd2+ in the medium) was measured in the aquatic
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hyphomycete Heliscus lugdunensis. Up to 25 µmol g-1 dry mass the fungus formed sulphide
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in an exponentially Cd2+-concentration-dependent manner. Using light microscopy,
precipitates were observed outside of the hyphae which could be determined as amorphous
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particles by x-ray diffraction (XRD). Energy dispersive x-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1,
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but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd2+ accumulated 12 to 28 µmol metal g-1 dry mass over a period of 7 to 28 days.
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The results may indicate that sulphide could sequester excess Cd2+ under oxygen deprived
fungus.
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Abbreviations
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conditions and thereby reduce its toxicity via an additional avoidance mechanism of this
AAS, atomic absorption spectrometry; AQH, aquatic hyphomycetes, BB, Boss Brook; DMPD, N,N-dimethyl-p-phenylenediamine-dihydrochloride; d.m., dry mass; DTE, 45
dithioerythritol; DTNB, 5,5’-dithio-bis-(2-nitrobenzoic acid); EDS, energy dispersive x-ray spectroscopy; f.m., fresh mass; -EC, -glutamylcysteine; GSH, glutathione; Hl, acronym of Heliscus lugdunensis; HMW-complex, high molecular weight-complex; HPLC, high performance liquid chromatography; Hsp, heat shock protein; MT, metallothionein; PC, phytochelatin; SEM, scanning electron microscopy; TAP, time after pressure; TFA,
50
trifluoroacetic acid; TTC, 2,3,5-Triphenyltetrazoliumchloride; XRD, x-ray diffraction
Introduction Page 2 of 22
3 Freshwater fungi play an important role in both, aquatic ecosystem integrity and health. They depend on decomposition of energy-rich organic compounds, produced by other organisms 55
and act as highly efficient contributors to aquatic ecosystems by transfer of nutrients between
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different trophic levels [1, 2]. Toxic metals and high concentrations of essential metals provoke major environmental and
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human health problems due to toxic impacts in different biota and food networks. Freshwater fungi that survive and thrive under harsh conditions, such as high metal and xenobiotics pollution have become a focus of increasing scientific attention [1].
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In freshwater fungi fluctuating environmental metal concentration are balanced to tolerable
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intracellular concentrations by a sophisticated metal homeostasis comprising various
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mechanisms [1], including biosorption to cell wall [3, 4], regulated cellular transport systems [5], heat shock proteins [6], modulation of reactive oxygen species [3, 5, 7], and contribution by thiolpeptides [3, 4, 8-10]. The thioltripeptide glutathione is a crucial compound in fungal
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cells, because it mediates redox reactions (by alteration between oxidized dithiol and reduced disulphide forms) as well it acts as starter compound for the biosynthesis of metal chelating
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phytochelatins.
The aquatic hyphomycete Heliscus lugdunensis is one of the most abundant fungal species 70
inhabiting highly heavy metal polluted sites (water [11], groundwater [12], sediments [13]). Several H. lugdunensis strains have already been reported to respond to Cd2+ exposition by cellular thiol peptide response, such as increase of glutathione level [3, 4, 8, 10] accompanied by rised levels of cysteine and γ-glutamyl-cysteine [3] as well as synthesis of phytochelatin 2 and a small metallothionein, MT1_HL [10]. As known from the aquatic hyphomycete
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Tetracladium marchalianum Cd2+ exposure raised intracellular soluble sulphide content [9]. Up to now, nothing is known about extracellular precipitation of CdS crystallites by aquatic hyphomycetes.
Page 3 of 22
4 In the present study, Cd2+-induced sulphide formation and extracellular precipitation of Cd2+ was determined using a H. ludgdunensis strain, isolated from an unpolluted habitat with 80
attention on possible involvement of sulphide in Cd2+ detoxification.
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Material and Methods Fungal strain and cultivation
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The strain Hl-BB of Heliscus lugdunensis Sacc. & Thérry was isolated from single conidia
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collected from a non-polluted stream Boss Brook near Amherst, Nova Scotia [14]. The strain is available from German Resource Centre for Biological Material (DSMZ Braunschweig, Hl-
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BB: DSM 18483).
Fungi were maintained on malt extract agar (0.5% malt extract, 0.1% peptone, 1.5% agar
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containing 270 µM sulphate). To examine heavy metals stress, the mycelia were cultivated in 150 mL medium in 200 mL Erlenmeyer flasks under stationary (non-shaking) conditions with limited oxygen supply (ca. 50% oxygenation of ca. 8 mg/L). The medium contained 0.5%
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malt extract, 0.1% peptone and 270 µM sulphate (sulphide free). In general, the cultures were
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inoculated with homogenized agar slices overgrown with 21-28 day old mycelia cultivated at 14±1 °C, the total fungal biomass introduced per flask was 3.4 mg d.m. Stock Cd2+ solutions (as CdCl2, filtrated through 0.2 µm membrane filters) were added on the fourth day of 95
cultivation to adjust the final metal concentration at 80 µM in the medium. Cultures were incubated in three replicates with and without Cd2+ at a final concentration of 80 µM and were harvested by filtration (Merck, filter 0869) after different times of cultivation. To isolate crystal-like particles in a higher yield for elemental analysis and HPLC, mycelia were cultivated in a ten-fold volume of medium inoculated with 82 mg d.m. of a
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liquid fungal preculture. Determination of dry mass
Page 4 of 22
5 Mycelia were rinsed twice with distilled water, blotted dry between filter paper and weighed (fresh weight). To determine wet to dry weight ratio, preweighed fresh mycelia were exposed to 80 °C for at least 2 h. Measurement of total dehydrogenase and oxygen
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105
Dehydrogenase activity was used as a general activity parameter and determined according to
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Fahmy and Walsh [15] using TTC reduction 2 h at 37 °C. The reaction mix contained 50 mg
f.m. in 5 mL 50 mM phosphate buffer (pH 7.5) containing 1 M sorbitol, 70 µL 1 M DTE and
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100 µL lyticase (Molzym). After centrifugation at 3600 × g (Hettich centrifuge type 30 RF) the pellet was resuspended in 2 mL 50 mM phosphate buffer (pH 7.5), mixed with 0.75 mL
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0.5% TTC (w/v) and incubated for 6 h at room temperature. The resulting formazane was
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extracted using 1 mL acetic acid and 2 mL toluol. Extinction was measured at 380 nm (n = 4). Oxygen in the culture medium was measured using an amperometric electrode (Mikro-
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Sauerstoffsensor 200, UMS GmbH, n = 5) and pH using a pH-meter (WTW 538; n = 5). Preparation and imaging of precipitates
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Culture liquid and mycelia were separated using a 1 × 1 mm 2 mesh-size sieve and subsequent
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centrifugation (10 min, 4 oC, 4000 × g). All following steps were carried out at 4 oC. The precipitate was resuspended in 20 mL Percoll medium (Pharmacia-Biotech, d = 1.130 g mL1
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), stirred for 10 min and centrifuged (30 min, 12000 × g). Hyphae without particles were
found at the top of the gradient while particles and hyphae attached particles precipitated at the bottom. This procedure was repeated with a 30 min stirring time. After decantation of Percoll, the precipitate was diluted with water (1:2) and centrifuged (4000 × g, 10 min). A portion of the particles was suspended in a drop of water and observed with a light microscope (Zeiss Axiolab) equipped with a digital camera (Sony DSC-S75). The particles
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were dried in vacuum and stored under N2 at 4 oC. Measurement of thiols and HPLC
Page 5 of 22
6 Non-protein thiols were extracted according to Grill et al. [16], modified by Miersch et al. [8]. Total thiols (TSH) were determined with Ellman’s reagent [17], glutathione was measured enzymatically [18], and non-protein thiols were analyzed using HPLC (Merck-Hitachi) [19]. Each sample consisting of 200 mg f.m. of hyphae or approximately 50 mg of wet particles
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130
was dissolved in 400 µL NaOH (1 N, added 1 mg mL-1 NaBH4) and centrifuged (5 min, 7000
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× g). After cooling on ice, 140 µL 3.6 N HCl was added and centrifuged (5 min, 10000 × g, 4 o
C). Samples of particles were diluted 1:4 with 1 N NaOH. 50 µL was separated on a RP 18
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column (SuperPac Sephasil-C-18, 5 µm, 4.6 × 250 mm). Absorbance was measured at 410 nm after post-column derivatization with DTNB as described earlier [20]. The thiols were
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eluted applying a linear gradient of 2-20% acetonitrile in TFA (0.1% v/v) over 25 min at 0.8
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mL min-1. In order to exclude the detection of non-thiolic compounds absorbing at 410 nm, the post column derivatization was repeated with KH2PO4 without Ellman’s reagent as a
Sulphide measurement
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negative control [21].
Intracellular water-soluble sulphides were measured according to King and Morris [22] using
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a modified procedure. 50 mg of fungal sample were crushed in liquid nitrogen and extracted for 5 min by stirring in 1 mL oxygen-free water. The mixture was homogenized and 300 µL were transferred in an Eppendorf tube (n = 3) containing 1240 µL 2.6% Zn-acetate-solution 145
(w/v) and 240 µL 6% NaOH (w/v). After stirring for 15 min at 4 oC, 200 µL of 0.2% DMPD (Fluka) in 20% H2SO4 and 20 µL of 10% FeNH4(SO4) × 12 H2O (Fluka) in 2% H2SO4 were added to this mixture and stored in the dark for 30 min. The mixture was centrifuged (23000 × g, 10 min, 4 oC). The absorbance was determined at 665 nm from the clear supernatant. For calibration, solutions of 0.2-100 µM Na2S in distilled water were used The detection limit was
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0.25 µmol sulphide g-1 d.m. (with an SD of 25%). To estimate sulphide in medium, 1780 µL clear filtrate and 200 µL of 0.2% DMPD (Fluka) in 20% H2SO4 and 20 µL of 10%
Page 6 of 22
7 FeNH4(SO4) × 12 H2O (Fluka) in 2% H2SO4 were prepared as outlined above (LOD = 0.4 µM. Measurement of adsorbed and intracellular cadmium After 7, 14 and 28 d, the mycelia were harvested on a filter (Whatman No.3), washed for 5
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min with 100 mL distilled water, 3 times with 100 mL 20 mM NiCl2 solution, which causes
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the extracellular Cd2+ ions to be replaced by Ni2+ [23]. This washing did not cause any leakage of intracellular Cd²+ as it was tested by several washings using different Ni
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concentrations and washing time. The mycelia were then washed with 100 mL water, removed from the filter and dried 24 h at 80 °C. 50 mg dried fungal mass were dissolved with
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4 mL 65% (v/v) HNO3 and 2 mL 30% (v/v) H2O2 in a microwave (step 1: 130 bar, 10 min,
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TAP 5 min; step 2: 275 bar, 20 min, TAP 10 min, 180 oC; step 3: 558 bar, 10 min, 180 oC; CEM MDS 2100, Kamp-Lintfort).
Cadmium concentrations in the NiCl2 washing steps (adsorbed Cd2+) and in the mycelium digests (accumulated Cd2+) were determined by AAS at not interfering wavelength (ATI
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0.05 mg × L-1 Cd2+).
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Unicam). Standard solutions of 0.0625 to 2.0 mg L-1 Cd2+ were used for calibration (LOD =
SEM and EDS of particles
Particles were analyzed using scanning electron microscopy and energy dispersive x-ray 170
spectroscopy (Fig. 3). Specimens for SEM/EDS were handled inside a dry nitrogen flushed bag whenever possible. Transfer to coating equipment and the microscope was performed as quickly as possible to reduce exposure to atmosphere. Specimens residing on their collecting substrates (filter paper or cellophane) were attached to 10 mm aluminum supports using double-sided tape and rimmed with either colloidal carbon (ca. 15 nm using a Denton DV-
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502A vacuum evaporator, Denton Vacuum, Moorestown, NJ) or gold (ca. 15 nm gold using a Hummer 6.2 sputtering system, Anatech Ltd., Hayward, CA). Carbon coating allowed collection of EDS spectra without interfering Au M-shell x-ray lines that would obscure Page 7 of 22
8 elements of interest. Gold coating was required for high-resolution SEM imaging as well as analyses that were aimed at the detection of the presence of carbon in particles. All specimens 180
were examined using a JEOL JSM-5600 SEM (JEOL USA, Peabody MA) equipped with an
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Oxford Inca 200 EDS system (Oxford Instruments, High Wycombe, UK). SEM/EDS images and data were acquired using a 20 mm working distance and 15 kV accelerating voltage.
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Statistical analysis
Determination of standard deviation (SD) for dry weight, sulphide, cadmium, and thiols was performed with MS Excel 6.0 or Graph Pad Instat 3.05 (ANOVA). Significance of differences
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was calculated using Student’s t-test. P values < 0.05 were considered significant. Results
affecting growth and fungal activity
Heliscus lugdunensis strains BB growing under facultative anaerobic conditions showed a
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Cd2+ causes the formation of sulphide together with crystall-like precipitates while differently
reduced growth while crystal-like particles were formed in the medium accompanied by
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sulphide generation in cultures containing Cd2+ (Fig. 1). The formation of sulphide started
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immediately after addition of Cd2+ (Fig. 2) and within 28 days a concentration of 25±1.562 µmoles g-1 d.m. was reached. No sulphide was measured in controls (without Cd2+). 195
Cadmium addition at 80 µM decreased fungal growth by 18% compared to controls after 6 days (Fig. 1).
The level of oxygen changed from 9±0.09 mg L-1 to 4.93±0.29 mg L-1 (control) and 4.35±0.17 mg L-1 (Cd2+ exposure) within 6 d, and had only slightly further decreased in Cd2+treated cultures and controls after 14 (16) d: 4.57±0.12 (3.93±0.12) mg L-1 in control and 200
4.10±0.33 (4.03±0.17) mg L-1 in Cd2+ exposed cultures. This may indicate that Cd2+ did not substantially affect fungal oxygen consumption. The pH of the medium (n = 4) decreased within 6 d from 5.63±0.04 to 4.99±0.11 and 4.84±0. in control and after Cd2+ exposure, respectively (p > 0.5). Afterwards, the pH was Page 8 of 22
9 nearly constant during 16 d reaching a final level of 4.96±0.05 in control and 5.03±0.04 in 205
Cd2+ cultures (p > 0.7). Cd-containing crystall-like particles show two types of elemental composition
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Initially, the cultures were investigated using light microscopy for their response to Cd2+. Yellow, crystal-like particles could be found from the middle of the log-phase of fungal
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growth in Cd2+-containing media (Fig. 1 insert) in both 150 mL as well as 1.5 L cultures. The yellow particles from the large culture were investigated using EDS and revealed two types of
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elemental composition. Samples of the first type contained a large number of relatively illdefined, flattened particles (data not shown) with lengths and widths in the 30-60 µm range,
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but only approximately 1 µm in thickness (Fig. 3A, insert). EDS analyses indicated that these
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particles contained a varying amount of Cd and S, with atomic% Cd:S between 2.39:2.09 and 30.52:25.62 (mean 9.15±8.82:8.31±7.30; n = 11). However, the extreme thinness of the
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specimens means that all spectra collected on such particles were actually composites of the
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particles and the substrate. Cd:S ratios were nevertheless reasonably close to 1:1 (1.05 ± 0.12; n =11). Thus, EDS alone could not rule out the possibility that fine, microcrystalline CdS was
220
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present in an amorphous matrix containing other elements from the culture broth, such as P and Cl (Fig. 3A). XRD analysis of this material does not support this hypothesis, however, as no prominent peaks for any crystalline material were obtained (data not shown). The second type of particles was well defined, ranging in size from 1 to 15 µm (Fig. 3 C, insert). All particles analyzed with EDS contained almost exclusively Cd and S (Fig. 3 C). EDS spectra collected from particles > 15 µm with flat top surfaces and minimum fracturing 225
yielded atom% Cd:S of 50.5±0.9:49.5±0.9 (n = 10) with only trace amounts of oxygen that could be attributed to surface oxidation or contaminants from solution. XRD analysis of this material produced prominent x-ray scattering peaks but the peaks did not correspond to known crystalline structures for either CdS or other cadmium compounds (data not shown).
Page 9 of 22
10 When hyphae of Cd2+-containing cultures or particles were exposed to acid a characteristic 230
smell of hydrogen sulphide could be noticed. In addition, purified particles were mixed with a saturated solution of lead acetate, producing the typical black needle-shaped crystals of PbS
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visible in the light microscope (not shown). Such samples contained a large number of elongated, typically cylindrical-shaped particles ranging in size from 5 × 2.5 µm to 12 × 4 µm
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(Fig. 3 B, insert). Lead and sulphur x-ray peaks are strongly overlapped in EDS spectra (Fig. 3 B), but peak deconvolution and subsequent semi-quantitative analyses indicated that Pb:S
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ratios for these particles were approximately 1:1. However all particles examined contained large concentrations of oxygen, approximately four times that of Pb and S, indicating that if
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PbS was formed, it was possibly rapidly oxidized to sulphate during sample processing.
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Characteristics of thiols derived from hyphae and particles
In control hyphae the eluted thiols were cysteine, GSH (coeluted with -EC, Fig. 4 A)
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whereas in Cd2+-stressed hyphae a remarkable peak at 7 min retention time (possibly
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sulphide) was observed (Fig. 4 B). In contrast to these results the elution profile of prepared enriched particles was dominated by only one peak at 7 min retention time, which is probably
245
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sulphide (Fig. 4 C) because i) sulphidic sulphur is indicated in most cases by post column derivatization by DTNB of particles, ii) paltry presence of oxygen (which imply the absence of sulphate) iii) complete absence of other elements in EDS (Fig. 3 C) and iv) during acidification hydrogen sulphide was released proved by precipitation of Pb-sulphide after addition of Pb-acetate (Fig. 3 B).
Small amounts of cysteine and GSH could not be excluded (Fig. 4 C). Other anions like citric, 250
malic and oxalic acid were not found by HPLC (data not shown). Apoplastic Cd2+-exposure leads to symplastic Cd2+-accumulation H. lugdunensis exposed to Cd2+ adsorbed metal ions externally by the apoplast and accumulated internally by the symplast. During 7 days mycelial biosorption increased rapidly to 80.1±5.9 µmoles g-1 d.m. and decreased initially (7-14 d) fast, then slows down to 54.3±3.4 Page 10 of 22
11 255
µmoles g-1 d.m. on 28 days (p < 0.05). Distinct levels of Cd2+ were accumulated by mycelia to 12.5±0.8 µmoles g-1 d.m. and reached 22.5±3.4 µmoles g-1 d.m. (p < 0.05) from 7 to 28 days (Fig. 5).
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Discussion Aquatic hyphomycetes are heterotrophic organisms of aerobic natural aquatic environments [1]. Their survival in metal polluted sites depends on avoidance/tolerance mechanisms.
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Different strains of the aquatic hyphomycete Heliscus lugdunensis showed distinct responses
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to metals, such as metal sorption to the cell wall, changes in plasma membrane integrity, intracellular changes of macromolecular complexes and reactive oxygen species as well as
265
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changes in thiol peptide biosynthesis [1]. To avoid interference with surplus of sulphur, in this study, CdCl2 was used without any negative impact of Cl- ions as tested with NaCl. However,
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Babich & Stotzky[24] suggested a decreased Cd2+ toxicity to fungi by formation of cadmium
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chloride complexes.
As known from other fungi the formation of cadmium sulphide could be another mechanism
Schizosaccharomyces pombe [27, 28], Saccharomyces cerevisiae [29]. In the aquatic
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to detoxify Cd2+, e.g. Candida glabrata [25], Fusarium oxysporum [26],
hyphomycete Tetracladium marchalianum Cd2+ exposure under aerobic conditions raised the sulphide level in the hyphae [9]. Agitated cultures of Heliscus lugdunensis strains were able to excrete sulphide without formation of extracellular CdS. Crystallites were formed only under static conditions providing a reduced oxygen supply, as also applied to H. lugdunenesis 275
strain Hl-BB in this study. Here, exposure to 80 µM Cd2+ inhibited fungal growth to 33% within the first 4 days (Fig. 1) as assayed by dehydrogenase activity as viability marker. Obviously, inhibition rate depends on strain origins. While strain Hl-BB from a non-polluted site [8] and strain H8-2-1 from a moderately polluted water [3, 11] showed lower Cd2+ sensitivity, strain H4-2-4 from a highly metal polluted habitat showed a much higher
280
tolerance [11]. Page 11 of 22
12 As shown in Fig. 1 (insert) extracellular particles were found in facultative anaerobic Cd2+ exposed cultures of H. lugdunensis while the strain was also able to generate sulphide up to 25 µmoles g-1 d.m. (Fig. 2). We assume an exclusively de-novo-biosynthetical origin of
285
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sulphide up to day 6. While a participation of thiol peptides in several intracellular detoxification of metal by
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aquatic fungi is undisputed up to now, the involvement of these peptides in formation of extracellular particles is still under discussion [1].
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The atom% Cd:S ratio is dependent on ion concentration in the nutrient medium [30]. In our study, EDS of amorphous particles from cultures of H. lugdunensis revealed two kinds of particles with different elemental composition: i) well defined particles containing almost
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exclusively Cd and S (Fig. 3 C) and ii) particles containing varying amounts of Cd and S and other elements/anions (Fig. 3 A). We assume, the aggregation started with small particles
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(Fig. 3 C) partially at the surface of the cell wall in the form of small clusters (Fig. 1 insert).
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Then, further elements of the medium were inserted and larger particles developed (Fig. 3 A), such as phosphorous and chlor. Since chloride were detectable only after longer period of
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time as well as not in the backround, no interference occured. No organic anions (citric-, oxalic-, malic acid) could be measured (Martienssen, personal communication). In terrestrial fungi, phosphates, polyphosphates or oxalates are common response to toxic heavy metal concentrations resulting in the formation of metal salts deposited intra- or extracellularly [31, 300
32].
During exposition of alder leaves in a high heavy metal polluted water in Mansfeld area a metal-rich layer (zincowoodwardite) was deposited on the leave surfaces with significant amounts of aluminium, copper and zinc but also sulphur [33]. HPLC profiles show peaks at 7 min (sulphide, Fig. 4 B, C), which most likely could be 305
sulphide (Fig. 4 C). Sulphide was bound and precipitated as Pb-sulphide after addition of water-dissolved Pb-acetate and could be analyzed by EDS (Fig. 3 B). Page 12 of 22
13 Sulphide is probably generated in the symplast but nothing is known about fungal efflux mechanisms of sulphide It can be hypothesized, that the regulation of sulphate assimilation in H. lugdunensis changed under facultative anaerobic conditions and Cd2+ exposure. Sulphite-reductase activity and its
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310
transcript were enhanced in H. lugdunensis strain H4-2-4 under Cd2+ influence, too (Nathan,
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unpublished results). Increased sulphur assimilation was found in earlier experiments showing that copper uptake by copper-tolerant strains of S. cerevisiae induced an intensive cysteine
315
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metabolism, resulting in black deposits of copper sulphide on the cell wall [34]. Hydrogen sulphide was found to act as a population sychronizer during aerobic continuous culture of the
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same yeast [35]. In response to Cd2+ the yeasts Schizosaccharomyces pombe and Candida
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glabrata synthesized intracellularly acid-labile sulphide- and PC-containing CdS quantum semiconductor crystallites [36] in which sulphide increased both stability of the HMW-
activity, required methionine or cysteine for growth and could not synthesize PC-coated CdS-
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320
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complex and the Cd2+ amount [37]. Mutants of C. glabrata, devoid in cysteine-synthetase
particles. However, these cells were able to accumulate high levels of sulphide as well as to
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precipitate CdS outside the cell [38] as found similary in aquatic hyphomycete Heliscus by our experiments. Extracellular sulphide-containing deposits (sometimes as crystallites) were also produced by S. pombe [27, 28] and C. glabrata [25]. 325
All in all, these results may indicate that under conditions of diminished intracellular phytochelatine synthesis [10] sulphide could sequester excess Cd2+ and thereby reduce its toxicity via an additional avoidance mechanism.
Acknowledgements 330
The authors thank Dr. R. Bruening (Physics Dept., Mount Allison University, Sackville) for helping in XRD-analyses, Dr. M. Martienssen (Helmholtz Centre for Environmental Research
Page 13 of 22
14 – UFZ, Dept. of Hydrogeology, Halle) for the analysis of organic acids, Mrs. E. Püschel and E. Funke for technical assistance.
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Dameron CT, Reese RN, Mehra RK, Kortan AR, Carroll PJ, Steigerwald ML, et al. Biosynthesis of cadmium-sulfide quantum semiconductor crystallites. Nature 1989;338:596-7. Reese RN, Winge DR. Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe. The Journal of biological chemistry 1988;263:12832-5. Barbas J, Santhanagopalan V, Blaszczynski M, Ellis WR, Winge DR. Conversion in the peptides coating dadmium-sulfide crystallites in Candida glabrata. J Inorg Biochem 1992;48:95-105.
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[36]
Fig. 1: Growth of H. lugdunensis shown as mg d.m. in 150 mL culture medium. Control -
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Legends to figures
white rhomb, 80 µM Cd - black rhomb, * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 3).
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Dehydrogenase activity was used as a general parameter of cell vividness (data not shown)
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Inserts show crystal-like formations aggregated at a hypha after 14 d of this culture. Fig. 2: Generation of sulphide by hyphae of H. lugdunensis in response to cadmium. The hyphae were cultivated in 150 mL medium without (control, white square) or with Cd (80
d
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µM, black square). Cd(II) was added at fourth day of cultivation. *** p < 0.001 (n = 3). Fig. 3: Scanning electron microscopy (insert) and energy dispersive x-ray spectroscopy of
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particles isolated from a 21 d old culture of H. lugdunensis. EDS spectra from areas analyzed on particles (bold line) and background substrate (thin line) are indicated by boxes labeled on 450
SEM micrograph (insert in A – C). (A) Amorphous particles containing Cd and S but also other elements from culture broth. (B) PbS derived by acidification of particles and reaction of hydrogen sulphide with lead acetate. (C) Crystal-like particles primarily containing approximately equal atomic% values for Cd and S (n = 7). Fig. 4: HPLC of sulphur-containing compounds in hyphae and particles isolated after 14 d of
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cultivation of H. lugdunensis in 1.5 L medium; A, control; B, hyphae exposed to 80 µM Cd; C, partial purified crystal-like material (All samples were assayed by HPLC in three technical replicates).
Page 16 of 22
17 Fig. 5: Measurement of adsorbed and intracellular Cd2+ by AAS as well as sulphide according to King and Morris [22] in Cd2+-containing cultures of H. lugdunensis up to 28 days (Three
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biological replicates were assayed in three technical replicates).
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