Effect of fungal isolates from different samples upon radionuclide behavior and environmental hazard indices during bioleaching process in Gabal Um Hamd, Um Bogma area, southwestern Sinai, Egypt

Two fungal species were isolated from the studied rock samples and identified morphologically as Aspergillus hollandicus and Penicillium citrinum. Bioleaching process was applied to W1, W2 and W3. The average concentration activity of 238U, 226Ra, 232Th, and 40K are 5134.03, 5708.64, 189.51, and 1456.8 BqKg−1, respectively. Radionuclide’s distribution in leach liquor, residual, and fungal adsorption were followed and environmental hazard indices (Raeq, Iγ, Hex, Hin, and ECLR) were calculated. From the observed outcomes, the isolated fungal strains have the potential to reduce the harmful effect up to 50% compared to the original. As a result, application of these fungal strains offers a potential strategy for environmental remediation of radionuclides.


Introduction
Radionuclide contamination has become a part of the surrounding environment in the form of fallout from nuclear weapons, waste from nuclear energy-generating industries, and from medical uses of radioisotopes.Fungi play a significant role in providing ecosystem services in terrestrial ecosystems [1], the importance of organic soil horizons and their microbial communities (fungi and bacteria) highlighted as potential accumulators of nutrient elements and radionuclides within terrestrial ecosystems [2].According to Steinera et al. [3], fungi are one of the most crucial elements of forest ecosystems because they greatly influence the transit pathways and fate of radionuclides.The immobilization process of radionuclides achieved by saprotrophic fungi, which present in the surrounding environment depends on its ability to increases enormous surface area of hyphae that fitting them for absorbing a large amount of nutrient elements and radionuclides from the environment [4,5].
One of the main issues regarding ecotoxicology and human health is radionuclides in the environment.Consequently, the use of fungi in bioremediation of radioactively contaminated sites and cleanup of an industrial effluent is one of the most important requirements [6], Although heavy metals are naturally present in the soil, geologic and anthropogenic activities increase the concentration of these elements to amounts that are harmful to both plants and animals.Among there are the mining and smelting of metals, the burning of fossil fuels, the use of pesticides and fertilizers in agriculture, the manufacturing of batteries and other metal products in the industrial sector, and the disposal of sewage sludge [7].
Bioremediation happens spontaneously and is enhanced by providing live substances and fertilizers where the main principle behind biodegradation technology is bioremediation.It means total elimination of hazardous organic pollutants into nontoxic, naturally existing, or innocuous substances like carbon dioxide, water, and inorganic substances that are secure for use by people, animals, plants, and aquatic life [8] as well as the degradation of the contaminants is mediated by physical or enzymatic-regulated processes such as bioaccumulation, biosorption, biomineralization, and biotransformation [9].Qayyum et al. [10] showed that the industrial soil contaminated with heavy metals may be considered a valuable natural source of resistant fungal strains like Rhizomucor pusillus, Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, and Neosartorya hiratsukae, which can be widely used as a bioleaching or bioremediation tool.There are numerous studies that used microorganism like fungi in bioremediation process such as El Dabour et al. [11] were applied Aspragillus species on the waste samples that collected from Allouga locality southwestern Sinai, Egypt and demonstrated that A. lentulus had a higher bioleaching efficiency of uranium (68%) than A.niger (54%).Attia et al. [12] utilizing a natural fungus strain, Aspergillus nidulans, as a green technology, the uranium bioleaching efficiency was established at 80% under the ideal conditions of 3 pH, 3% pulp density, 7 days of incubation, and 30 °C incubation temperatures.On the other hand, the microorganism was utilized in the wastewater treatments as biosorption and/or bioaccumulation of contamination through its mycelium, which applied in different fields as pharmaceutical industry, dyes removal, agriculture as pesticides degradation, and metal removal (Th, Ra, U, Cu, Zn, Cd, Pb, Fe, Ni, and Ag) [13] by the mechanism of producing two main types of extracellular chemicals in response to metal stress known as microbial extracellular polymeric substances (EPS) and soluble microbial products (SMP), which affecting the metal binding properties to their active sites [14] as well as the biodegradation of a wide range of organic molecules has been explained by several mechanisms and pathways; for instance, it can occur both with and without oxygen [15].
The natural decay of uranium ( 238 U and 235 U) series, thorium ( 232 Th) series, and singly occurring isotopes like potassium ( 40 K) Khandaker et al. [16] contributed that the most of these invisible radiation sources cause environmental pollution.
The aim of this work is to study the relation between the bioleaching process and radionuclides behavior using two fungal isolates; A.hollandicus and P. citrinum, from the rock samples that collected from Gabal Um Hamd, Sinai, Egypt in addition to calculate their effects on the health hazard indices.

Geological setting
Gabal Um Hamd in southwest Sinai is where the rock samples were collected.Primary and secondary minerals that are allogenic and authigenic make up the research region in Um Hamd, as seen in Fig. 1 radioactive (U, Th), non-radioactive REEs, and base metal-bearing minerals [17].A lower siltstone-Fe, Mn ore member, a middle siltstone-mudstoneshale member, and an upper dolostone-dolomitic limestone member might all be classified as parts of the Um Bogma Formation in Gabal Um Hamd.In the western region of Gabal Um Hamd, where the middle and upper members are the thinnest, otherwise the bottom member, which exhibits an increase in thickness.The three members' rocks vary in their levels of Fe oxide and to a lesser extent of Mn-bearing deposit richness [18].The Um Bogma Formation's sediments underwent a variety of diagenetic processes, including both iso-chemical and allo-chemical processes.The lower member indicates a maritime habitat that is subtidal to intertidal.The shallow subtidal marine habitat follows the open ocean shelf environment in the middle member.The upper member has shallow subtidal to lower intertidal environments, which are followed by clastic rocks that suggested a regressive sea within a tidal flat [19].

Samples preparation
Three geologically gripped radioactive rock samples were collected from Gabal Um Hamd in southwest Sinai.Each sample is a representation of the location where it was gathered.The samples were sealed in sterile polyethylene packets before being crushed, quartered, and ground for analysis.The specification of the studied samples was as follows: i) The first sample (W1) is composed of compact siltstone, which is medium hard and highly feriginous, ii) The second sample (W2) is made up of grey shale, which is fissile ferruginous and gypsiferrous, and iii) The third sample (W3) is made up of sandy dolostone, which is jointed and fractured and is dark grey.

Microbiological studies
The microbial studies upon the collected samples were explained in three steps as follows: Step1: Fungal isolation Fine ore powder was spread directly upon the surface of sabouraud dextrose agar plate, this agar media is particularly useful for fungal regrowth [20,21], in which under septic conditions, sealed and incubated at 30 ºC until the fungal colonies grew [22,23].
Step2: Purification and identification of isolated fungi Hyphal tips of each colony were removed and plated upon the surface of sabouraud agar plates.The developed colonies were examined under a microscope to detect contamination.The pure isolated colonies were identified at the regional center for mycology and biotechnology (RCMB) Al-Azhar-University, Cairo, Egypt [23][24][25].The microbiological experiments included the fungal strains isolation from the studied rock samples are followed by the bioleaching process.

Step3: Impact of fungi on the test for Bioleaching Ability
Studying the behaviors of isolated fungal strains from studied rock samples (W1, W2 and W3), which contain different grades (high, moderate, and low) of uranium and thorium concentrations.Using 100 g of ore from each sample was placed in a1000 mL Erlenmeyer flask then, adding three hundred ml of sabouraud dextrose media to be autoclaved.Inoculation of 10% (v/v) of 1 × 10 8 spore/mL from each fungus separately with each sample in a separate flask.bioleaching factors investigated included: ore concentration 3%, 1/3 solid liquid ratio, incubation period 7 days at temperature between 30 and 35 °C and pH value equals 3, which applied to the determined optimum conditions.
The radionuclides activity concentrations for the original samples (BqKg −1 ), bioleach liquor (BqL −1 ), fungal biosorption (BqKg −1 ), and residual (BqKg −1 ) because of bioleaching process phases will be measured using a high-purity germanium (HPGe) detector as a non-destructive examination.The relative efficiency of this detector, which has a resolution of 1.90 keV and a relative efficiency of approximately 50% of the 3′′ 3′′ NaI (Tl) crystal efficiency, was used to determine the activity concentrations of various radionuclides, including 238 U, 235 U, 234 Th, 234 Pa, 232 Th, 226 Ra, and 40 K.The peak/Compton ratio is 69.9 at 1.33 MeV based on 60 Co is changing [26].

Absorbed dose rate (DR)
The absorbed dose rate (nGy/h) for the current study has been caculated using Eq.(1) where A U , A Th , and A K are the average specific activities of 226 Ra, 232 Th, and 40 K respectively in Bq kg −1 , respectively [27,28].

Annual effective dose equivalent (AEDE)
Annual effective dose due to radionuclides activities for different studied samples are calculated for outdoor or indoor using Eq. 2, the conversion factor from absorbed dose in air to effective dose must be taken in unit of mSv per year, is calculated from the following Eq.( 2) [27,28].
where mSvy −1 is the effective dose rate, (n Gy h −1 ) is the dose rate and SvGy −1 is the conversion coefficient.

Excess lifetime cancer risk (ELCR)
Excess lifetime cancer risk gives the probability of developing cancer over a lifetime at a given exposure level, considering 70 years as the average duration of life for human beings, as displayed in Eq. ( 3) [29].
where AEDE is the Annual Effective Dose Equivalent, DL is the average Duration of Life (estimated to be 70 years) and RF is the Risk Factor (Sv) represents fatal cancer risk per Sievert.For stochastic effects, ICRP uses RF as 0.05 for the public.

Radium equivalent activity (Ra eq )
The combined specific activity of 226 Ra, 232 Th, and 40 K develop a numerical indicator of an external dose to public [23,30].Suggested Eq. ( 4), to calculate radium equivalent and stated the value of 370 Bq kg −1 as the maximum allowed dose public.
where A Ra , A Th and A K are the specific activities of 226 Ra, 232 Th and 40 K, in Bq Kg −1 , respectively.

External hazard (H ex )
External hazard index (H ex ) are used to measure the external hazard due to the emitted gamma radiation, which has been calculated for the present study using Eq. ( 5) [27].
where H ex is the external hazrd index and A Ra , A Th and A K are the specific activity of 226 Ra, 232 Th and 40 K in Bq Kg −1 , respectively.

Internal hazard (H in )
The internal radon and its daughter products are quantified by the internal hazard index H in , which is caculated for the studied samples in the present study using Eq. ( 6): It is also possible to use an activity utilization index proposed by EC [31] and UNSCEAR [27] due to the raw materials used in building and covering materials, that facilitates the derivation of dose rates in air from different combinations of these three radionuclides has been caculated for the different studied samples using Eq. ( 7) where C K , C U and C Th are the specific activities of 226 Ra, 232 Th and 40 K respectively in Bqkg −1 due to building materials.The safty vlue of this index is ≤ 1 [32].

Results and discussion
The microbiological experiments included the fungal strains isolation from the studied rock samples are followed by the bioleaching ability assay.

Microorganisms isolation and identification
Two fungal species were isolated from the tested samples.The most dominant fungal strains belong to two species, known as Asperigillus and Penicillium, identified according to their morphological features, (RCMB), meanwhile, the first one of Aspergillus species was identified as Aspergillus hollandicus (A.hollandicus) and the second of Penicillium species as Penicillium citrinum (P.citrinum).They were grown in the presence of sabouraud agar medium at 30−35°C for 7 days.

Gamma counting
The obtained results for the activity concentrations of 238 U, 226 Ra, 235 U, 232 Th, and 40 K as well as their averages are reported in Table 1 respectively (Bq Kg −1 ) for the studied rock samples (W1, W2, And W3) by HPGe detector, the radionuclides distribution for different studied samples shown in (Fig. 2).For the sample W1 the activity concentration of 238 U, 226 Ra, 235 U, 232 Th and 40 K was 2982, 3011,136, 102.6, and 744 Bq Kg −1 , respectively.The activity concentration of W2 was 4765.73,5358.12,216.09, 198.1, and 1723 Bq Kg −1 , respectively.Finally, for the sample W3 the activity concentration was 7654.3, 8756, 347.91, 267, and 1903 Bq Kg −1 , respectively.There are respective world-wide concentration limitations of 33, 32, 45, and 420 Bq Kg −1 for 238 U, 226 Ra, 232 Th, and 40 K [27].The obtained data demonstrates that it is obvious that Sandy dolostone, jointed and fractured (W3), have the highest concentration of uranium, (7) thorium, radium, and potassium.The obtained results of ore samples for the activity concentrations showed that all the studied radionuclides in the representative samples have higher values relative to that recorded in the UNSCEAR [27].As a result, it has higher concentrations of uranium and thorium than sediments described in the IAEA's [33] publication, but lower 40 K levels.The Fe oxide cement is also attached to and/or adsorbed with some uranium content.

Fractional dissolution of radionuclides during bioleaching process
Results of specific activities of various isotopes have been examined for each different phase of the bioleaching process: (a) bioleach liquor phase (Bq L −1 ), (b) fungal biosorption phase (Bq Kg −1 ) and (c) residual phase (Bq Kg −1 ) shown in Fig. 3. Leachability percentage for each radionuclide was adjusted varied using A. hollandicus, as listed in a group of Tables 2, 3, 4, the investigated measurements with the following characteristics:    On the other hand, the calculated leachability is varied for each radionuclide using P. citrinum as follows:   From the obtained results of the radionuclides activity concentrations of the studied samples and different bioleaching phases, numerous useful ratios as 238 U/ 235 U, 226 Ra/ 238 U, and 238 U/ 232 Th were calculated. 238U/ 235 U ratios demonstrated that the predominant normality was achieved for each phase around the natural value (21.7) deducing the same behavior of the two radioactive elements [34][35][36].Also, the 226 Ra/ 238 U ratio represents a meaningful ratio in the geological systems whereas its normal value equals unity, if any deviation is recorded whether greater or lesser pointing to deviation from the secular equilibrium, may be that due to 226 Ra in excess of equilibrium with 238 U enhanced leaching of 224 U according to the occurring of alpha decay-recoil phenomenon causing nucleus damage to the crystal structure of the ore [37].Finally, the obtained ratio 238 U/ 232 Th are compared with the ratio in the upper crust is typically 0.8 [38] indicating a relatively enhancement of 238 U to 232 Th.Therefore, the slight enhancement of uranium relative to thorium in some samples may be due to dissolution and readsorption of uranium.

Radiological hazards (EhzIs)
From Table 8 The absorbed dose rate was ranged from (2666.83-4286 n Gy/h), and the annual effective dose equivalent calculated for outdoor from 1.82 to 5.25 mSv/y.Since the results of absorbed dose rate (D R ) and the annual effective dose rate (AEDE) are higher than the international recommended values, which are 60 n Gy/h and 1 mSv/y, respectively [27,28].
The Level index I-gamma calculated values were ranged from 21.59 to 62.3, which found to be higher than the world level index I-gamma (I γ ≤ 6) [27].The obtained values for calculated H ex ranges from 8.96 to 25.09 are higher than the world external hazard (unity) [27], likewise, the values of internal hazard index (H in ) ranges from 16.44 to 25.09 are higher than the world internal hazard unity, as well as the radium equivalent activity (Ra eq ) ranged from ( 3728.86 Bq Kg −1 ) to (10,603.12Bq Kg −1 ), which is higher than the recommended maximum value (370 Bq Kg −1 ) [26].The value of the excess life cancer risk (ELCR) varied from (6.37 × 10 -3 to 18.39 × 10 -3 ), which is greater than the standard ELCR that is given as (0.29 × 10 -3) [29].From the previous results can be concluded that A. hollandicus decreased the AEDE value in the three studied samples as in Table 9.For sample W2 and W3 the value of D R , AEDE, I γ , H ex , H in , Ra eq , and ECLR in residual phase was higher than that in the leach liquor represented low leachability percentage.On the other hand, the calculated hazard indices using P. citrinum, exhibited an effective role in decreasing the D R , AEDE, Iγ, H ex , H in , Ra eq in the residual phase R1 as (497.84n Gy/h), (0.61 mSv/y), (7.28), (5.49), (2.92), (1278.9BqKg−1 ), and (2.13 × 10 -3 ), respectively.and R3 as (957.76n Gy/h), (1.17 mSv/y), (14.19), (9.95), (5.60), (3265.53BqKg −1 ), and (4.11 × 10 -3 ).As well as in fungal biosorption phase whereas it has high values of D R , AEDE, H ex , H in , Ra eq , also in B2 as (461.11nGy/h),(0.56 mSv/y), (5.11), (2.71), and (1179.83BqKg−1 ), respectively.These results agreed with Maity et al. [39], showed that high doses of gamma radiation cause dose-dependent inhibitory effects in the fungi that make soil fungal community structure appeared to shift toward species that may be more radiation resistant, and melanin-containing fungi ascended to dominate the soil fungal communities with increase in radionuclide pollution [40].

Conclusion
Bioremediation will be required as a highly effective way of pollutant removal and nutrient recycling in an ecosystem.Fungi, as decomposers have the unusual capacity to metabolize various organic and inorganic contaminants, which utilizing them as energy and carbon sources as well as reducing them to non-hazardous concentrations.Radiometric studies clearly show high values of uranium contents in the rock samples and bioleaching process phases (leach liquor, residual, and fungal adsorption) relative to those international recorded values.The EHzIs were calculated for the original samples cleared that (D R ), (AEDE) and (Ra eq ) were more than the international recommended limits.The effect of the two fungal isolates upon radionuclides were followed in the three phases of bioleaching process showed that A. hollandicus exhibited a high leaching efficiency with low uranium  contents samples as well as achieved a low leaching efficiency with high uranium content samples.Since P. citrinum can withstand the high uranium content samples, also it exhibited a high leachability of uranium whereas displays a better result to decrease the EHzIs in the studied samples.Consequently, can utilize the P. citrinum to remediate the environment by applying bioleaching process several times to reach the safe international limit values of EHzIs.The hazard indices in leach liquor phase are directly proportional to the leachability percentage otherwise in the case of residual phase are inversible.Therefore, it is recommended to use a combined leaching experiment and hazard index calculation as an indicator of fungal leaching efficiency.

Fig. 1
Fig. 1 Geological map showing the location of the studied area and samples location (Modified after Hamza et al., [17])

Table 2
[11]sample W1, which recorded radionuclides leachability percent are 69%, 72%, 46%, and 69% for 238 U, 226 R,232Th and 235 U, respectively.The calculated activity ratio of 238 U/ 235 U exhibited within the natural value between 20.00 and 21.92, which reflects a slight deviation from the natural ratio (21.7).Activity ratio of 226 Ra/ 238 U recorded 1.009, 0.92, 1.07, and 1.009 for original sample, residual, bioleach liquor, and fungal biosorption, respectively.It is clear from the activity ratio that the calculated values come close to the unity that attained to secular equilibrium while ratio of 238 U/ 232 Th ranged from 16.01 to 60.55 showed a deviation from the natural (magmatic) ratio 1:3 in the direction of U-migration-in[11].(ii)Theradionuclides leachability percent for sample W2 are recorded 49%, 53%, 28%, and 48% for 238 U, 226 R, 232 Th and 235 U, respectively are shown in Table3, as well as the calculated ratio of 238 U / 235 U exhibited slightly higher values than the natural value (21.7) between 22.05 and 24.01. the 226 Ra/ 238 U ratio recorded 1.12, 1.05, 1.18, and 1.51 for original sample, residual, bioleach liquor, and fungal biosorption, respectively, while the ratio of 238 U / 232 Th ranged from 15.26 to 51.58.

Table 1
Radioelements content in the collected soil samples from Gabal Um Hamd area

Table 7
Radionuclides distribution of original and bioleaching different phases by P. citrinum upon sample W3 including leachability percent *

Table 8
Radiological hazard indices (EHzIs) of the original samples

Table 9
Specific activity concentration and radiological hazard indices of different bioleaching phases by A. hollandicus Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/.