Protonema of the moss Funaria hygrometrica can function as a lead (Pb) adsorbent

Water contamination by heavy metals from industrial activities is a serious environmental concern. To mitigate heavy metal toxicity and to recover heavy metals for recycling, biomaterials used in phytoremediation and bio-sorbent filtration have recently drawn renewed attention. The filamentous protonemal cells of the moss Funaria hygrometrica can hyperaccumulate lead (Pb) up to 74% of their dry weight when exposed to solutions containing divalent Pb. Energy-dispersive X-ray spectroscopy revealed that Pb is localized to the cell walls, endoplasmic reticulum-like membrane structures, and chloroplast thylakoids, suggesting that multiple Pb retention mechanisms are operating in living F. hygrometrica. The main Pb-accumulating compartment was the cell wall, and prepared cell-wall fractions could also adsorb Pb. Nuclear magnetic resonance analysis showed that polysaccharides composed of polygalacturonic acid and cellulose probably serve as the most effective Pb-binding components. The adsorption abilities were retained throughout a wide range of pH values, and bound Pb was not desorbed under conditions of high ionic strength. In addition, the moss is highly tolerant to Pb. These results suggest that the moss F. hygrometrica could be a useful tool for the mitigation of Pb-toxicity in wastewater.


Introduction
Water is essential for all living organisms on earth. Humans do not only ingest water, but use it also for agriculture and industrial activities. Water contamination with heavy metals from human industrial activities is a serious environmental concern. If polluted water enters drinking and agricultural water systems, heavy metals may cause serious toxicity to organisms [1]. To remove heavy metals from contaminated water, various methods and remediation materials have been developed and are widely used in current industrial procedures (e.g. chemical sedimentation, electro deposition, activated charcoal, ion-exchange resins, chelating resins) PLOS

Materials and methods
Moss sampling and spore sowing

Establishment of protonemal suspension cultures
Approximately 50 mg fresh weight of F. hygrometrica protonemal cells grown on agar were collected and suspended in modified Knop's liquid medium using a Polytron homogenizer (PT-MR2100, Kinematica, Switzerland) operated at minimum speed for 15 sec. The suspension cells were inoculated into 0.5 L liquid medium in a culture bottle, and cultured with aeration at 1000 mL min -1 under fluorescent lights. The growth rate of the culture was examined by measuring gain of the dry weight of cells at 5 and 8 days after inoculation.

Morphological observation of filamentous protonemal cells
Cultured F. hygrometrica protonemal cells were collected by filtration through a suction funnel fitted with a glass fiber filter (GS25, Advantec). The cells were freeze-dried and observed with a scanning electron microscope (SEM) (TM3000, Hitachi).

Adsorption test for 15 metal elements
Two-week-old cultured protonemal cells of F. hygrometrica were used as adsorbent materials. Twenty mL of the suspension culture was poured into a glass column (Fh-column; 10 mm inner diameter × 100 mm length). The Fh-column was washed and equilibrated with MQW using a peristaltic pump at a flow rate of ca. 12

Metal determination and quantification
To analyze the Fh-column filtrates, each solution was directly injected into an inductively coupled plasma mass spectrometer (ICP-MS; Perkin Elmer Elan6100DRC). For analysis of moss plant material, the protonemal cells were dried for 3 days at 60˚C and predigested with 5 mL aqua regia (HNO 3 :HCl = 1:3) overnight at room temperature. Thereafter, the organic compounds were totally decomposed by wet-ashing using a microwave sample preparation system (Perkin Elmer MultiWave-3000). The volume of the digested samples was adjusted to 50 mL with MQW, and the solution was filtered through 5B filter paper (Advantec, Tokyo, Japan). For ICP-MS analysis, a portion of the filtered samples was diluted appropriately with MQW.
Electron microscopic analysis For metal identification, F. hygrometrica cells used for Pb-adsorption tests were examined using a TEM-EDX spectroscopy system [JEM-1230 electron microscope with a MiniCup/EX-14033JTP energy dispersive X-ray (EDX) microanalyzer (JEOL, Tokyo, Japan)] [21]. Section thickness was increased to150 nm for EDX to get as much of signal as possible, and uranium staining was omitted to cut X-ray signal off originated from heavy metals except for Pb and to find dense particle more easily.

Distribution of Pb between cell walls and other components
After F. hygrometrica protonemal cells adsorbed Pb, the cells were freeze-dried and divided into two parts: one portion was used to prepare a cell-wall fraction (CWF) and the other portion served as the total cell fraction (TC). To prepare the CWF, the cells were suspended in aqueous 80% (v/v) ethanol and homogenized using a pestle and a mixer (Pellet Pestle 1 Cordless Motor, Kimble Chase, USA). After centrifugation at 1,200 × g for 5 min, the pellet was washed in a sequence of 80% ethanol, 95% ethanol, 99.5% ethanol, chloroform:methanol (1:1), and acetone. The alcohol-insoluble residues were dried in air and analyzed as the cell-wall fraction as previously described by Matsunaga et al. [22]. The Pb contents of the CWF ([Pb] CWF ) and TC ([Pb] TC ) preparations were determined by ICP-MS. To determine the cell-wall proportion in F. hygrometrica protonemal cells (CWF/TC), F. hygrometrica protonemal suspension cells were washed with MQW, freeze-dried, and weighed. Then, the cell-wall fraction was prepared as described above and weighed. Distributions of Pb in the cell walls were estimated according to: Pb content in cell walls ð½Pb CW Þ ¼ ½Pb CWF Â CWF=TC Distribution ð%Þ of Pb in cell walls ¼ ½Pb CW =½Pb TC Â 100 Pb-adsorption to the prepared cell-wall fraction F. hygrometrica protonemal cells were cultured for 10 days on the modified Knop's-agar medium, harvested and freeze-dried. Cell walls were extracted by homogenizing the dried samples in 50 mM Na-phosphate buffer (pH 6.5), collecting the cell walls by centrifugation, followed by washing several times with 50 mM Na-phosphate buffer (pH 6.5) until the wash solution was visibly colorless. Finally, cell walls were sequentially washed with acetone, a water/chloroform/methanol mixture (5:6:3, v/v/v) and freeze-dried. The prepared cell-wall fractions were left to stand for 4 h in glass bottles filled with 1 mM PbCl 2 , Pb(NO 3 ) 2 , or MQW (Mock). After washing with MQW, bound Pb was detected as a red lead-rhodizonate complex visualized by staining with 3 mM sodium rhodizonic acid and 100 mM tartaric acid (pH 2.8) on a slide glass or by X-ray analysis with an X-ray analyzer (XGT-5000, Horiba, Japan).

Effects of pH on metal adsorption
The test solutions were prepared by diluting the commercial standard solutions (Wako Pure Chemical Industries) with MQW to concentrations of 5 mg L -1 (Au and Pt-group metals) or 10 mg L -1 (other metals). Five g (wet weight) of F. hygrometrica protonemal cells were suspended in 250 mL of the test solutions containing the tested metals at various pH values, and the suspensions were incubated for 5 h with shaking at 100 r.p.m. During the incubation, the pH was occasionally checked and adjusted within ±0.15 of the initial value with diluted HCl or NaOH. After incubation, the filtrate was recovered and analyzed using an inductively coupled plasma atomic emission spectrometer (ICP-AES; SPS5100, SII NanoTechnology, Chiba, Japan).

Effects of ionic strength on Pb desorption
Pb-loaded F. hygrometrica protonemal cells were prepared by incubation with 100 mg L -1 Pb (pH 5.0) for 24 h, followed by washing with MQW. The resulting Pb concentration of the protonemal cells was 4.02 mg g -1 wet weight. Five g (wet weight) of the Pb-adsorbed cells was suspended in 250 mL of MQW, the pH was adjusted to 5.0 with HCl, and the ionic strengths of the suspensions were adjusted by adding an aqueous solution of NaCl. The suspensions were incubated for 5 h with shaking at 100 r.p.m. After incubation, the samples were filtered and the recovered filtrate was subjected to ICP-MS (Agilent 7500, Agilent Technologies).

F. hygrometrica protonemal suspension cultures
We collected F. hygrometrica Hedw. on a landfill area in Omuta City, Japan, on Apr. 16,2003 ( Fig 1A). To evaluate the ability of F. hygrometrica to adsorb various metal ions, it was essential to work with uniform material. Thus, we collected sporophytes, allowed the spores to germinate on agar plates (Fig 1B), and established a suspension culture of protonemal cells derived from a single spore (Fig 1C). When the protonemal cells were cultured in modified Knop's liquid medium, the constant growth rate in the exponential phase (μ e ) was 0.144 g dry weight L -1 d -1 and the final yield was ca. 0.57 g dry weight L -1 . The collected protonemal cells at the late stationary phase (i.e. 2 weeks) were used as metal adsorbents. Observation of the fine structure of collected F. hygrometrica protonemal cells with SEM showed that the filamentous protonemal cells were intertangled (Fig 1D).

Hyper-accumulation of Pb and other metal ions by F. hygrometrica
To examine the capacity of F. hygrometrica to adsorb industrially important metal ions, we prepared protonema-packed small columns and loaded 15 metal or metalloid ion solutions onto the columns (S1A Fig). To establish the maximum adsorption capacities, thirty-five-column volumes of metal solutions were loaded. After extensive washing, the metal contents were analyzed using an inductively coupled plasma mass spectrometer (ICP-MS) ( Table 1). The protonemal cells accumulated remarkably high levels of some metals: Pb was efficiently adsorbed to the breakthrough point (S1B Fig) and accumulated up to 74.1% on a dry weight base. Au was also highly accumulated up to 11.3% (Table 1, S1B Fig), but Li did not accumulate. The order of maximum adsorption capacity was Pb>Au>Cr>Tl>Pt>Co>Mn>Mo>Ni>Al>Ag>Zn>Y>Se>Li. Since the capacity for Pb accumulation was remarkably high, we focused on Pb adsorption by F. hygrometrica in our further studies.

Cellular localization of accumulated Pb
To obtain insights into the mechanism for Pb accumulation, we fixed the F. hygrometrica protonema used in the column tests, and analyzed these materials with a transmission electron microscope-linked energy-dispersive X-ray (TEM-EDX) microanalysis unit. When the cells were treated with Pb, the cell walls became significantly thicker than the non-treated control (S2 Fig), and high-density particles were detected in cell walls, endoplasmic reticulum (ER)like membrane structures, and chloroplast thylakoids (Fig 2A-2E). X-ray microanalysis showed that typical Pb signals originated from the high-density particles (Fig 2F-2H). These results suggested that living F. hygrometrica accumulated Pb at multiple sites.
To examine the distribution of Pb between the cell wall and other compartments, we prepared cell-wall fractions from the Pb-treated F. hygrometrica protonemal cells and analyzed their Pb content. We found that most of the Pb (88.7%) was present in the cell-wall fraction (S1 Table). These results suggest that the main Pb-accumulating cellular compartment is the cell wall.

Adsorption of Pb to a prepared cell-wall fraction of F. hygrometrica
We next examined the adsorption of Pb to a prepared cell-wall fraction of F. hygrometrica. A Pb-free cell-wall fraction was prepared from F. hygrometrica protonema, and aliquots of the preparation were treated with 1 mM PbCl 2 or Pb(NO 3 ) 2 . After washing, the bound Pb was detected by staining with rhodizonic acid followed by X-ray analysis. In both Pb-treatments,  the cell-wall fraction was strongly stained with rhodizonic acid, and typical Pb signals were detected in the X-ray analysis (Fig 3B-3D), indicating that the F. hygrometrica cell-wall fraction can adsorb Pb. Notably, Pb-treated cell-wall fractions were swollen compared to those receiving the mock treatment as observed by TEM analysis (Fig 3A and S2 Fig).

Characterization of cell-wall components by two-dimensional nuclear magnetic resonance
To obtain information about the chemical components of F. hygrometrica cell walls, the cellwall components were characterized by two-dimensional nuclear magnetic resonance (NMR). We also prepared cell-wall fractions of two mosses, Physcomitrella patens and Ceratodon purpureus, for reference. Typical hetero-nuclear single quantum coherence (HSQC) spectra are shown in Fig 4A. In all three moss species, polysaccharide signals predominated in the spectra, including correlations in the chemical shift range of δC/δH 60-80/3.0-4.4 ppm, whereas the anomeric correlations in the range of δC/δH 90-110/4.5-5.5 ppm were well-resolved. Among the species, the spectral patterns were similar, but not identical. Initially, we attempted to identify or annotate using our chemical shift database of cell-wall components [30,31]; however, it was difficult to find matched chemical shifts due to the limited cell-wall component data from land plants. Therefore, we compared chemical shifts for several individual commercial reagents, 1,3-beta-glucan, pectin, polygalacturonic acid (PGA) and cellooligosaccharides, that could be assumed to be cell-wall components of bryophytes. A large part of the observed signals from the bryophytes overlapped with PGA and cellooligosaccharides, suggesting that the major cell-wall components adsorbing Pb are PGA and cellulose. To find the characteristic feature for Pb adsorption by F. hygrometrica cell walls, we compared our data with previously reported spectral data prepared from land plants and seaweeds [24,26] and bryophytes P. patens and C. purpureus. Hierarchical clustering analysis of normalized HSQC spectra showed that land plants and seaweeds were clustered by separation at the first branch, and bryophytes including F. hygrometrica were clustered in another branch ( Fig  4B). Among members of the clade that includes seaweeds, bryophytes grouped at the edge of the cluster, and F. hygrometrica was separate from the other two mosses. Since the major factor for the edge location was a predominant cell-wall composition of PGA, our results suggested that the chemical component in bryophyte cell wall is a PGA-enriched material.

Growth performance of different species in excess Pb
We examined the viability and growth potential of F. hygrometrica in the presence of Pb. Even in modified Knop's medium containing 0.5 mM PbCl 2 , F. hygrometrica protonemal cells grew (Fig 5A). To see whether other species are similarly tolerant, we compared the growth yields of F. hygrometrica and P. patens in the presence Pb concentrations varying from 0.001 to 1 mM (Fig 5B). Both species belong to the Funariaceae family, and P. patens is an established model species for studies in plant evolution and development [32]. At 0.5 mM PbCl 2 , F. hygrometrica maintained about 80% of its growth yield compared with that observed under Pb-free control conditions, whereas the growth yield of P. patens was significantly less in 0.05 mM PbCl 2 and almost completely inhibited at 0.5 mM PbCl 2 . In addition, when we compared the chlorophyll content of F. hygrometrica and P. patens grown under a range of Pb concentrations, the chlorophyll a and b levels of F. hygrometrica changed little with increasing Pb, whereas those of P.   Table). These results indicate that F. hygrometrica is a Pb-tolerant species.

Effect of pH on adsorption
To investigate the effects of chemical factors on metal adsorption and desorption, we prepared protonemal cells on a large scale and first examined the dependence of adsorption on pH. Protonemal cells growing in bottles on a shaking incubator were treated with 40 metals including Pb at various pH values, and metal adsorption in the recovered cells was analyzed. The protonemal cells showed various pH dependency patterns for metal adsorption (Fig 6). Among them, adsorption of Pb and Sn was high and stable from pH 3 to 9, whereas adsorption of some other metals, such as Cu, decreased gradually with lower pH, indicating that the Pb adsorption ability was retained in a wide range of pH values. In addition to Pb, the protonemal efficiently adsorbed platinum-group metals (Ru(III), Rh(II), Pd(II), Ir(III), Pt(III)) and Au(III) within a wide pH range.

Effect of ionic strength on desorption
We further examined the ability to retain Pb in the same system used for monitoring pH effects. When Pb-loaded F. hygrometrica protonemal cells were incubated in solutions of varying ionic strengths, Pb was stably retained in the cell fraction. More than 95% of the Pb remained adsorbed to protonemal cells, even at 0.5 mol kg -1 NaCl (S3 Fig). This result suggests that the bound Pb is not readily desorbed, even under high ionic strength conditions.

Discussion
In this study, we found that F. hygrometrica has a remarkable ability to tolerate and accumulate Pb and characterized some physicochemical aspects of metal accumulation by this moss. Our findings led us to propose the use of F. hygrometrica as a biomaterial for the bio-sorbent filtration of metals. Extensive efforts are now being made to identify and characterize key genes involved in heavy metal uptake and resistance, mainly in vascular plants [33][34][35][36][37][38]. Our study suggests that bryophytes should be included as target species in these studies.
Our analysis indicated that the cell wall is the main compartment for Pb adsorption; over 80% of the total absorbed Pb was found in the wall (S1 Table). Furthermore, prepared cell-wall fractions adsorbed Pb (Fig 3), suggesting that the chemical constituents and structure of F. hygrometrica cell walls are responsible for the specificity and large capacity of Pb-accumulation.
Our NMR analyses suggest that the chemical constituents of F. hygrometrica cell walls are similar to other mosses but distinct from land plants and seaweeds and that PGA is a major component of bryophyte cell walls (Fig 4). At present, it is difficult to identify the chemical properties underlying the hyper-accumulation of Pb in F. hygrometrica. However, in our previous study, we found that commercially available PGA was as effective in adsorbing divalent Pb as a well-known metal adsorbent, chitosan [39]. Therefore, PGA in the cell walls of F. hygrometrica might be involved in Pb binding. Although PGA is known to be a major component of pectin as homogalacturonan, NMR analysis did not detect two other major components of Potential use of Funaria hygrometrica as a Pb adsorbent pectin, rhamnogalacturonan-I and II. These results suggest that (1) F. hygrometrica cell walls do not contain typical pectins and (2) this unusual cell-wall composition and structure might be involved in the ability of F. hygrometrica to adsorb Pb.
Although the mechanism underlying Pb hyper-accumulation in F. hygrometrica cell walls has not been elucidated, accumulation of heavy metals in cell walls has been reported for other moss species. In S. cataractae, Satake et al. found that Cu accumulated in the cell wall [40], and Konno et al. suggested that this Cu was bound mainly to pectin [41]. In general, pectin has a high potential for binding divalent ions [42]. For instance, the crosslinking of pectinates by Ca 2+ ions plays an important role in the organization of polysaccharides in plant cell walls, according to the so-called 'egg box model' of pectin structure [43]. Thus, Pb ions may bind to negatively charged polysaccharides such as those found in pectin in F. hygrometrica cell walls. Comparative biological and physicochemical studies using these mosses are necessary to elucidate the mechanisms for metal specificity and the large capacity for Pb adsorption.
Alternative Pb-binding components in F. hygrometrica cell walls are the neutral cellulose matrix and/or other constituents with negatively charged moieties such as phosphate groups, carboxyl groups, amines, and amides [44]. Plant cell walls are composed of a complex matrix, whose structure is very diverse. Perhaps F. hygrometrica has a specialized cell-wall structure for containment of specific metals, or multiple chemical and structural properties might be additively involved in F. hygrometrica's tolerance and accumulation of Pb. Riaz et al. reported that the main functional groups involved in Pb adsorption by waste biomass from Gossypium hirsutum (cotton) were carboxyl, carbonyl, amino, and alcoholic groups [45].
In our experimental conditions, the thickening of cell walls in the presence of high Pb concentrations was observed in vivo and in vitro (S2 Fig and Fig 3). Although we cannot provide an unequivocal explanation for the volume enlargement caused by capture of Pb, an extreme structural change could occur in the cell-wall matrix resulting from metal binding. It might be caused by replacement of cell-wall bound Ca 2+ by Pb 2+ as previously suggested [46,47].
In our TEM-EDX analysis, Pb was detected not only in cell walls but also in chloroplasts and ER-like structures (Fig 2). Given that F. hygrometrica can grow under high PbCl 2 concentrations, it is plausible that F. hygrometrica has a Pb detoxification mechanism. For example, the Pb detected in chloroplasts and ER might be detoxified by sequestration with chelating compounds or proteins and excretion to the extracellular space or vacuole. Elucidation of the detoxification mechanism will be important for understanding the specialized competence of this moss in metal tolerance and accumulation.
Our study also revealed the efficient adsorption of other metals, such as Sn, Au and Ptgroup metals, by F. hygrometrica protonema under a wide pH range (Fig 6). At present, it is not clear whether there is a common adsorption mechanism for these metals with that for Pb. Interestingly, under the same experimental conditions, the binding properties of the protonemal cells to Pb(II), Au(III), Ru(III), Rh(II), Pd(II) appear comparable or superior to that of a commercial chelating resin that immobilizes ethylenediaminetriacetic acid and iminodiacetic acid groups [48], tempting us to test the use of the moss for recovery of other metals.
Our results suggest that F. hygrometrica is a useful bio-material for the recovery of heavy metals, especially Pb from aqueous solutions. Currently, metal ions are recovered by chemical sedimentation, electro deposition, or ion-exchange adsorption, using materials and energy ultimately derived from fossil resources. Although current methodologies work well, alternative technologies based on plant-derived materials will become more important in the future. The application of mosses for Pb-recovery from industrial wastewaters should be valuable for creating a sustainable recycling system.   Table. Chlorophyll content of protonemal cells exposed to different PbCl 2 concentrations in F. hygrometrica and P. patens. F. hygrometrica and P. patens protonemal cells were cultured in modified Knop's liquid media containing the indicated concentrations of PbCl 2 for 10 days. Thirty mg of freeze-dried samples were used to measure the chlorophyll concentration as described by Arnon (1949). Chl a, chlorophyll a; Chl b, chlorophyll b; NI, no inhibition; ND, not determined. a , Relative Inhibition (RI, %) was calculated as RI = (1 -A/B) × 100, where A is the average value determined in the control (0 mM PbCl 2 ) and B is the average for the treatment. Ã , Significant difference as assessed by Welch's t-test at p <0.05 (n = 3). (TIFF)