Bioaccumulation of Arsenic by Fungi

Problem statement: Arsenic is a known toxic element and its presence and toxicity in nature is a worldwide environmental problem. The us of microorganisms in bioremediation is a potential method to reduce as concentration in cont aminated areas. Approach: In order to explore the possible bioremediation of this element, three fila mentous fungiAspergillus niger, Serpula himantioides and Trametes versicolor were investigated for their potential abilities to accumulate (and possibly solubilize) arsenic from an agar environment consis ting of non buffered mineral salts media amended with 0.2, 0.4, 0.6 and 0.8% (w/v) arsenopyrite (FeA sS). Growth rates, dry weights, arsenic accumulation and oxalate production by the fungi as well as the pH of the growth media were all assessed during this study. Results: There was no visible solubilization of FeAsS parti cles underneath any of the growing fungal colonies or elsewhere in the respective agar plates. No specific patterns of growth changes were observed from the growth ratios of the fungi on agar amended with different amounts of FeAsS although growth of all fungi was s timulated by the incorporation of varying amounts of FeAsS into the agar with the exce ption of A. niger on 0.4% (w/v) amended agar and T. versicolor on 0.8% (w/v) amended agar. The amounts of dry wei ghts obtained for all three fungi also did not follow any specific patterns with diff erent amounts of FeAsS and the quantities obtained were in the order A. niger > S. himantioides > T. versicolor. All fungi accumulated as in their biomasses with all amounts of FeAsS although to var ying levels and T. versicolor was the most effective with all amounts of FeAsS while A. niger was the least effective. Conclusion: The accumulation of arsenic in the biomasses of the tes t fungi as shown in this study may suggested a role for fungi through their bioaccumulating capabilitie s as agents in the possible bioremediation of arsen ic contaminated environments.


INTRODUCTION
Arsenic is a metalloid that is widely distributed in the earth's crust and is present at an average concentration of 2 mg kg −1 [1] . It is generally regarded as a toxic element and its toxicity to living organisms has long been acknowledged. For instance, in historical times in rural Germany, it was used as a means to dispose of the old owner of a house, farm or land thereby speeding up the inheritance process [2] . More recently, there has been an increased awareness of its risk to plants, animals and human health [3][4][5][6][7][8] .
In the environment, arsenic is present in more than 200 mineral species, the most common of which is arsenopyrite (FeAsS), an arsenic sulfide. Inorganic arsenic of geological origin is found in groundwater used for drinking purposes in several parts of the world such as Bangladesh and West Bengal, India [1,6] . Chronic exposure to high levels of arsenic and its compounds, especially in drinking water can have several carcinogenic, mutagenic and teratogenic implications [1,7,9] .
According to estimates, about a third of the atmospheric flux of arsenic is of natural origin, volcanic action being the most important natural source, followed by low-temperature volatilization [1] . Common anthropogenic sources such as mining, metal smelting and the use of arsenic containing agricultural chemicals, as well as the weathering of arsenic-containing rocks, e.g., arsenopyrite, lead to the contamination of air, water and soil [4,[9][10][11][12] . Of total world arsenic production, it has been estimated that about 70% is used in timber treatment as copper chrome arsenate, 22% in agricultural chemicals and the remainder in glass, pharmaceuticals and non-ferrous alloys [1] .
In nature, the responses of microorganisms to toxic metal (loid)s can dramatically alter metal(loid) abundances and elemental speciation, leading to a range of transformations such as mobilization, immobilization and mineral neogenesis [13] . In the course of these transformations, fungi in particular may be able to accumulate these metal(loid)s into their cells. The intracellular uptake of metal ions from a substrate into living cells, otherwise known as bioaccumulation may lead to the biological removal of metals by fungi [14] . Such accumulation of heavy metals by fungal biomass may be particularly relevant because of its potential low cost application in bioremediation and recovery of metals. Furthermore, as fungi play fundamental roles in the natural environment especially regarding decomposition, transformation and nutrient cycling, a knowledge of their responses in high metal concentration states may be particularly relevant, in this case, to the detoxification of arsenic polluted habitats. Prompted by the combined abilities of fungi to render metals soluble from insoluble compounds and accumulate them from the dissolved state, this study sought to evaluate the accumulation and possible solubilization of arsenopyrite at different concentrations in solid media by free-living filamentous fungi.

MATERIALS AND METHODS
Organisms, media and culture conditions: The following fungi-Aspergillus niger (ATCC No. 201373), Serpula himantioides (from Dr N. White, University of Abertay Dundee, UK) and Trametes versicolor (from Prof. C. Evans, University of Westminster, UK) were used for the experiment. They were maintained on the same medium used for the experiment, a non-buffered mineral salts medium comprising (g L −1 distilled water) (NH 4

Mineral preparation and experimental set up:
Ground (i.e., powdered) arsenopyrite with particle sizes between 38-106 µm were obtained from R.G. Widdowson (Scarborough, UK) and used for the experiment. Amounts of arsenopyrite which would give 0.2, 0.4, 0.6 and 0.8% (w/v) in the final agar plates were weighed out into aluminium foil packets and oven sterilized at 70°C for at least 24 h. The agar was prepared using 15 g L −1 Lab M Agar No. 1 (IDG, Lancashire, UK) and autoclaved at 121°C for 15 min.
After cooling (to about 55°C) in a water bath, indicated quantities of individually autoclaved salts listed above from stock solutions were added to the molten agar. One pre-weight foil packet of each amount was added to the agar preparation and mixed well to ensure a homogenous distribution of the mineral particles. About 20 mL per plate of agar was poured into 90 mm diameter Petri dishes with or without the addition of arsenopyrite. After solidification of the agar, sterile dialysis membranes prepared by boiling twice for 10 min in ddH 2 O and rinsed thoroughly on each occasion prior to autoclaving at 121°C for 15 min were placed under aseptic conditions onto the surface of the agar in each Petri dish. The dialysis membrane allowed passage of nutrients and/or metabolites between the agar and the fungi and provided a convenient means of removing the mycelia from the agar.
All the plates (FeAsS-containing and FeAsS-free) were inoculated with 7 mm diameter discs of A. niger, S. himantioides and T. versicolor cut from the margin of a three day old grown mycelia on the mineral salts medium described above. The inoculated plates were incubated at 25°C over an eight-day period. Daily measurements of the growth radius of the hyphae and that of any clear zones under or around the colonies were taken either until the mycelia reached the edge of the Petri dish or the experimental period was over. Petri dishes were also examined under a light microscope for any transformations such as formation of clear zones, indicative of mineral dissolution and crystal formation in the agar. Growth rates were then calculated from this data. After eight days, colonies were harvested into aluminium foil cups by peeling the biomass from the dialysis membranes. These were oven-dried at 70°C until reaching a constant weight. The dry weights of the mycelia were then measured on a Mettler-Toledo PR203 weighing balance.
In order to obtain a pH profile of the agar surface under fungal colonies, pH measurements were made across the Petri dish at 20 mm intervals at the end of the experiment using an Orion model 720A pH meter.
All statistical analyses were carried out using Minitab for Windows 12.1 (Minitab Inc., USA). Correlation analysis was completed using the Pearson correlation method.

Arsenic content determination:
For arsenic content analysis of the biomasses, 2 mL concentrated HNO 3 was added to each biomass sample in a fume hood and incubated for at least 6 h after which they were transferred to a heating block at 90°C for 20 h. Arsenic was then determined using the methods described in Agrawal et al. [9] . Briefly, 1 mg mL −1 arsenic stock solution was prepared by dissolving 173.34 mg of sodium arsenite in 100 ml ddH 2 0. To a 1 mL aliquot of nitric acid-digested biomass, 0.4 mL of 1% potassium iodate was added followed by the addition of 0.2 mL of 0.5 M HCl. The reaction mixture was gently shaken upon the addition of 0.2 mL leucocrystal violet solution and 1-2 drops of 2 M NaOH solution. Leucocrystal violet solution was prepared by adding 25 mg of leucocrystal violet, 20 mL ddH 2 0 and 0.3 mL of 85% (v/v) phosphoric acid into a 250 mL Erlenmeyer flask, gently shaking until the dye dissolved and then diluted to give a final volume of 100 mL. Each aliquot of the nitric acid-digested biomass reaction mixture was kept in a heating block at 40°C for 5 min. The solution was diluted to 5 mL with ddH 2 0 and the absorbance was measured at 592 nm using a VERSAmax TM turnable microplate reader spectrophotometer with SOFTmax ® PRO software (Molecular Devices, California, USA).

Oxalate determination in agar:
In order to determine the amounts of oxalate formed in the agar, agar from each plate (mineral-containing and mineral-free) was mashed and put into 250 mL Erlenmeyer conical flasks. 40 mL of 1 M HCl was added to each flask and the flasks were shaken overnight in a rotating incubator at 120 rpm. A 1 mL aliquot of the resulting liquid solution for each sample was collected in an eppendorf tube, to which cation exchange resin (Bio-Rad AG 50W-X4; BioRad Laboratories, Richmond, CA, USA) was added. The oxalate content of the samples was then determined enzymatically according to the methods described in the Sigma diagnostics oxalate kit (Procedure no. 591) [15] . In summary, Sigma sample diluents and oxalate reagents A and B were reconstituted according to the reagent preparation instructions contained in the kit. A known volume of sample diluent was added to an equal volume of agar sample for analysis. The pH was measured intermittently and adjusted using either 1 M HCl or 1 M KOH until the pH was between 5 and 7. 1 mL of oxalate reagent A was added to each sample after which 0.1 mL of oxalate reagent B was added. Upon addition, each sample was mixed by gentle inversion and incubated at room temperature (between 18-37°C) for 5 min. Standards were prepared in the following concentrations 0 mM (blank), 0.125 mM, 0.25 mM, 0.5 mM and 1 mM. Absorbances of standards and samples derived from the agar were measured at 590 nm using a VERSAmax TM turnable microplate reader spectrophotometer with SOFTmax ® PRO software (Molecular Devices, California, USA).

Growth rates:
The growth rates of the fungi grown on agar containing different amounts of arsenopyrite are shown in Table 1 and are expressed in terms of ratios. The growth ratio is the colony growth rate in the presence of the mineral compound (R m ) relative to the colony control growth rate (R c ) [16] . There was no visible solubilization of FeAsS particles underneath any of the growing fungal colonies or elsewhere in the respective agar plates. As a result, solubilization rates could not be obtained nor ratios calculated. A growth ratio greater than 1.0 indicates that growth of the fungus was stimulated by the addition of the metal compound. On the other hand, a growth ratio less than 1.0 indicates growth inhibition. No specific patterns of growth changes with different amounts of FeAsS were observed from the ratios. The incorporation of 0.2% (w/v) FeAsS into the agar medium stimulated the growth of A. niger as the growth ratio was greater than 1.0. When 0.4% (w/v) FeAsS was present, the growth ratio was 0.95 indicating growth inhibition with this amount. Growth at higher amounts of 0.6 and 0.8% (w/v) was stimulated with ratios of 1.05 and 1.07 respectively. The growth of S. himantioides was stimulated in the agar medium with all amounts of FeAsS since the derived growth ratios were all higher than 1.0. With T. viride, growth was stimulated with all amounts except when 0.8% (w/v) was present in the agar medium. The ratio when this amount was present in the agar was 0.95, thus indicating inhibition.

Dry weights of fungal biomass and pH of agar:
The dry weight values recorded for A. niger, S. himantioides and T. versicolor are shown in Fig. 1. Increasing amounts of FeAsS had differing influences on the biomasses of all three fungi. With A. niger, the highest amount of biomass was obtained when 0.2% (w/v) FeAsS was present in the agar. There were subsequent decreases in biomasses with increasing FeAsS amounts from 0.4-0.8% (w/v). Dry weights of S. himantioides were increased by the presence of FeAsS in the growth medium with all amounts although it followed no specific pattern. Biomass was highest in the 0.6% (w/v) FeAsS-amended medium, followed by 0.8%, then 0.2 and 0.4% (w/v) FeAsS-amended media.    The values under the biotic control column represent the pH underneath growing colonies of A. niger, S. himantioides and T. versicolor on FeAsS-free agar (i.e., 0% FeAsS) with the exception of the abiotic control. *: The values in the abiotic control row represent the pH of agar with the addition of the respective amounts of FeAsS, without fungal growth (i.e., agar only)     Table 2 show a gradual increase in the pH of the experimental medium (without any fungal growth, i.e., abiotic control) from 4.68±0.03 (mean value±standard error of mean; three replicates each) in the FeAsS-free medium to 5.94±0.01 with 0.8% (w/v) FeAsS-amended medium. Fungal growth on FeAsS-free media (control) revealed A. niger was the most acidic with a pH of 2.01, followed by T. versicolor with a pH of 2.96, then S. himantioides with a pH of 3.00±0.01. Also, all three fungi reduced the pH of varying amounts of FeAsS-amended media and in all cases, there was reduced acidity with higher amounts. In other words, as the amounts of FeAsS in the media increased, pH as a result of fungal growth also increased with all three fungi and this is indicated in the positive correlation between both variables (r = 0.997, 0.987 and 0.990, for A. niger, S. himantioides and T. versicolor respectively).

Arsenic accumulation:
The data presented in Table 3 shows the amounts of arsenic (As) accumulated in the biomasses of Aspergillus niger, Serpula himantioides and Trametes versicolor. The values are expressed in nmol mg −1 dry weight and they show that T. versicolor had the highest levels of As accumulation per mg dry weight of biomass with all amounts of FeAsS in the agar.  Oxalate production: The amounts of oxalate produced by the fungi as shown in Table 4 indicate with A. niger, the incorporation of 0.2% (w/v) FeAsS into the agar increased oxalate production from 0.056±0.003 mM (mean values±standard error of mean; three replicates each) to 0.070±0.003 mM followed by a decrease with increasing FeAsS amounts. A similar pattern was followed by T. versicolor as there was an increase from 0.063±0.029 mM in the control to 0.103±0.019 mM when 0.2% (w/v) FeAsS was present in the medium.
No specific pattern was detected with S. himantioides although the presence of 0.6% (w/v) FeAsS led to an increase in oxalate production to 0.306±0.052 mM when compared to the control where 0.279±0.031 mM oxalate was produced. The amounts of oxalate produced by S. himantioides were greater than those of either A. niger or T. versicolor with all amounts of FeAsS. When subjected to statistical analysis, these results showed negative correlations between the amounts of FeAsS in the media and oxalate production by A. niger, S. himantioides and T. versicolor (r = -0.731,-0.071 and -0.544 respectively).

DISCUSSION
The three fungi tested in this study accumulated arsenic from arsenopyrite in their biomasses to varying degrees. T. versicolor was the most efficient in accumulation with all amounts, accumulating up to 15 times the amounts accumulated by A. niger which was the least effective in accumulation. In environments or media laden with high toxic-metal concentrations, the general expectation is usually a reduced microbial growth response as a result of the toxicity exerted by the pollutants on microbial cells.
This study has demonstrated this may not always be the case. The increased growth which resulted when FeAsS was present in the media at most amounts, relative to the control indicate these fungi show some resistance to As toxicity although the precise mechanism(s) by which toxic effects are overcome remain unclear. A plausible explanation for this is the possible utilization of Fe present in FeAsS for fungal growth. One mechanism however, by which fungi may be able to survive in and tolerate high toxic metal-containing environments is through the formation of oxalates which results from the oxalic acid they secrete [17] . Despite the negative correlation of increasing amounts of arsenopyrite to oxalate production in all three fungi shown by statistical analysis (r = -0.731 for A. niger, -0.071 for S. himantioides and-0.544 for T. versicolor), there was an increase in growth by the fungi when compared to the control in the majority of cases. With the exception of growth by A. niger on 0.4% (w/v) and T. versicolor on 0.8% (w/v) FeAsS amended media, fungal growth was enhanced with the addition of arsenopyrite to the media. Biomasses produced by the fungi with the varying amounts of FeAsS were either equal to or greater than that produced by the control in each case except for T. versicolor grown on 0.8% (w/v) FeAsS amended medium.
Oxalate, organic acid production and acidification through proton exudation by fungi may result in the solubilization of insoluble metal-bearing minerals by fungal species. Often times especially with organic acids, this is usually evident through crystal formation in the solubilized zones, underneath growing colonies and elsewhere in the agar. In contrast to previous studies in which such solubilization was demonstrated [18][19][20][21][22] , no visible solubilization of arsenopyrite by these fungi occurred throughout the duration of this study. Also, upon examination under a light microscope, no crystals (usually indicative of cation immobilization as oxalates) were observed underneath growing colonies of the fungi or elsewhere in the agar plates with all amounts of FeAsS. Thus, there was no biological immobilization of arsenic in this study and although possible arsenic immobilization as crystals has implications for disposal and bioavailability, it has only been demonstrated by physico-chemical processes [7,[23][24][25] and there is yet to be any evidence that microorganisms can play a role in achieving this.
Environmental factors such as pH influence the abundance of different arsenic forms in natural environments [26] . For instance, a decrease in pH is known to increase heavy metal availability [27] . In this study, the addition of increasing amounts of FeAsS to the experimental agar generally increased the pH of the media in all cases. However, fungal growth with the varying FeAsS amounts reduced the pH of the media when compared with the abiotic controls (i.e., agar with the addition of the respective amounts of FeAsS, without fungal growth). With all three fungi, acidity generally reduced with higher amounts of FeAsS and the order of acidification of growth media by the fungi was A. niger > S. himantioides > T. versicolor. In addition, the differences in the pH of the agar with the different amounts by all three fungi were statistically significant (p<0.05). Fungal acidification of media could have arisen through organic acid secretion during growth, proton extrusion via the proton translocating ATPase, absorption of nutrients in exchange for protons or carbonic acid formation through respiratory CO 2 production [28] .

CONCLUSION
In summary, while the immobilization of arsenic as a result of fungal interaction with arsenopyrite did not occur in this study, the accumulation of arsenic in the biomasses of the test fungi may suggest a role for fungi through their bioaccumulating capabilities as agents in the possible bioremediation of arsenic contaminated environments.