Assessment of the Pb2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions

Assessment of the Pb2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of the Pb 2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions Alessio Leins, Danae Bregnard, Ilona Schäpan, Wart Zonneveld, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4836282/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Geothermal Energy → Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract One solution for reducing the scaling risk of lead (Pb)-containing phases consists of removing the aqueous Pb 2+ ions from the brine by sorption before oversaturation of Pb 2+ phases at unwanted locations within the geothermal fluid loop. Hence, this study investigated the known capacity of fungal biomass to biosorb Pb 2+ ions to remove Pb 2+ from the brine. So far, biosorption studies have neither been done at high temperatures or salinity, nor under high pressure, three conditions that have to be considered within geothermal power plants. Thus, the overall goal of this study was to assess the Pb 2+ biosorption potential of dead biomass of the fungus Penicillium citrinum strain HEK1 under conditions mimicking those of natural highly saline geothermal fluids. This specific strain was isolated from geothermal brine circulating in a plant in which Pb 2+ scaling occurs. To assess biosorption, dead biomass of P. citrinum was added to synthetic solutions containing 260 g/L NaCl, 1g/L Pb, and (in half of the treatments) 60 mg/L acetic acid. These synthetic solutions, including the dead biomass, were then incubated at high pressure (8 bar), at different temperatures (25°C, 60°C, 98°C), and for different time intervals (1 h, 2 h, 3 h). Results showed that the structure of the biomass was stable in such conditions, at all temperatures tested, but small amounts of organic compounds, with a wide variety of low molecular weight (< 350 Da to 10,000 Da) have been released into the fluids from the biomass. In general, increased temperature resulted in an increase of dissolved organic carbon (DOC) concentration. The biosorption potential of P . citrinum HEK1 biomass was overall low (0.72% of total Pb 2+ ). While it was not affected by changes in temperature, time of exposure or by the presence of organic acids within the fluids, salinity showed to be influential as biosorption increased up to 19.22% of Pb 2+ removal in non-saline conditions. Therefore, the high salinity of the fluids was the factor limiting the biosorption to the highest extent, highlighting that working with highly saline geothermal fluids might be limiting for biosorption processes to happen efficiently. Geothermal energy Biosorption Fungal Biomass Pb2+ scaling Penicillium citrinum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Geothermal energy operators often encounter operational challenges such as mineral precipitation (scaling) that can clog the pipes and surface installations, leading to reduced flow rates and decreased injectivity of the reservoir (Demir et al., 2014 ; Regenspurg et al., 2015 ). Mineral precipitates can also insulate the pipes by creating a mineral layer, thus reducing the efficacy of heat exchange (Nitschke et al., 2014 ). Mineral scaling within geothermal power plants can include silicate, carbonate, hydroxide, sulfate, or sulfide scaling. Sulfide scales often incorporate heavy metals, such as copper, lead, iron, zinc or vanadium (Lebedev, 1972 ; Nitschke et al., 2014 ; Wolfgramm et al., 2009 ). In the case of Pb 2+ , scalings such as PbSO 4 , PbS, PbCO 3 , Pb(OH)Cl, PbOI or native Pb can be considered (Regenspurg et al., 2016 ). These different scales form depending on the Pb 2+ concentration present in the fluids, but in all cases, impact the power plants efficacy (Mouchot et al., 2018 ; Regenspurg et al., 2015 ; Scheiber et al., 2013 ; Zotzmann et al., 2023 ). These scalings are not only toxic but also typically contain the natural radioactive isotope 210 Pb, which presents an additional hazard for geothermal operators during maintenance or for the disposal of the radioactive Pb- containing filter residues (Regenspurg et al., 2014 ). The use of chemical scaling inhibitors is the most frequent practice to mitigate the scaling problem. However, inhibitors do not always present an optimal solution due to high costs and unknown long-term effect of the inhibitors to the reservoirs. Alternatively, metals can be removed from the fluids before precipitation using cation selective filters. For instance, the biopolymer chitosan has been shown to efficiently remove Cu and Pb from fluids (Zotzmann et al., 2023 ). Another alternative strategy could be to use the biosorption capacity of microorganisms. Indeed, the remediation of heavy metals from different substrates by biosorption is well known and fungi are efficient heavy metals biosorbents for instance (Congeevaram et al., 2007 ; Iram et al., 2015 ). As biosorbents, fungi can be used both as living - (Noormohamadi et al., 2019 ) or dead biomass (Pang et al., 2011 ; Verma et al., 2013 ), resting cells (i.e., spores) (Martins et al., 2016 ), or immobilized biomass (dead biomass entrapped in calcium alginate beads, for instance) (Verma et al., 2013 ). All of these technologies have different biosorption capacities depending on the targeted metals, the type of fungus and type of biomass preparation and the experimental conditions used during biosorption. More precisely, Pb 2+ biosorption has been broadly studied in fungi, especially in mitosporic fungi. Among other molds, biosorption has been demonstrated in Aspergillus niger (Amini et al., 2008 ), Aspergillus parasiticus (Akar et al., 2007 ), Penicillium citrinum (Pang et al., 2011 ; Verma et al., 2013 ), and Aspergillus flavus (Iram et al., 2015 ). For some of these fungi, metal removal ranged from 10–96.21% depending on the chemical conditions, the fungal species, and the target metal assessed (Amini et al., 2008 ; Akar et al., 2007 ; Verma et al., 2013 ) However, to the best of our knowledge, all previous studies have not been conducted under conditions that correspond to those found in geothermal power plants, i.e., high pressure and salinity, temperatures > 50°C. This represents a significant shortcoming as the environmental characteristics of geothermal fluids, such as higher temperatures, are known to affect the biosorption capacities of fungi. Moreover, within the same fungal strain, optimal biosorption temperatures can vary depending on the targeted metal (Iram et al., 2015 ). For instance, in the case of the fungus Phanerochaete chrysosporium , biosorption of (Pb 2+ ) and (Cd 2+ ) by living cells was equal at 25°C and 35°C, and even slightly better at 45°C for Pb 2+ (Lu et al., 2020 ). Additionally, salinity is another factor that can reduce the biosorption capacity and the high salinity of some geothermal fluids (i.e., 160 g/L of Cl − in the Heemskerk geothermal power plant (NL)) adds to the chemical complexity of these fluids. For example, the biosorption capacity for a dye of the fungus Rhizopus arrizhus dropped by 28.8% when the salinity was increased from 0 to 50 g/L (Aksu and Balibek, 2010 ) and the Cr 2+ biosorption capacity of the same fungus dropped by 17% with the same salinity change (Aksu and Balibek, 2007 ). In brewer yeasts ( Saccharomyces cerevisiae ), biosorption of Pb 2+ and Cu 2+ also decreased with higher concentrations of NaCl and CaCl 2 (Han et al., 2006 ). In addition, the impact of DOC, which can be present in high quantities in geothermal fluids (Leins et al., 2023 , 2022 ), on metal biosorption capacity has not been assessed systematically. Copper ions precipitated less with higher concentrations of DOC, while the increase of DOC concentrations resulted in higher abiotic Cd 2+ precipitation (Laurent et al., 2010 ). These examples demonstrate the need to test biosorption under geothermal conditions and investigate the influence of temperature, salinity, and DOC content to assess the feasibility of biosorption to prevent scaling in geothermal systems. Indeed, even if biosorption processes of heavy metals were investigated in many different systems and with many different types of metals, to the best of our knowledge, conditions similar to geothermal systems (pressure, temperature and salinity) have not been assessed yet. Adapting such biosorption processes to geothermal conditions could reduce the impact of scaling in geothermal power plants without the need to add chemical scaling inhibitors in the fluids. Moreover, utilizing the biosorption capacities of microorganisms to remove metal ions from geothermal fluids would not only help mitigate the impact of scaling but would also facilitate the recovery of these metals. Indeed, biorecovery, which is defined as the recovery of various metal ions through biosorption or bioaccumulation, is promising for fully exploiting geothermal fluids (Lo et al., 2014 ). For instance, the co-extraction of lithium from geothermal fluids is one of the processes explored to enhance the viability of geothermal power plants, which then do not only produce heat or electricity, but also a valuable metal for the industry (Weinand et al., 2023 ). The specific aims of the present study were to assess the stability of the structure of dead biomass of the fungus Penicillium citrinum HEK1 strain as well as its Pb 2+ biosorption capacity under conditions simulating the physico-chemical parameters (pressure, temperature, salinity) of the geothermal power plant from which this fungus was isolated. 2. Methods 2.1 Site description The Heemskerk geothermal power plant is situated north-west of Amsterdam (Netherlands) and produces primarily heat for greenhouses. It operates as a geothermal doublet, targeting a Permian sandstone (Rotliegend Slochteren) formation (TNO-GDN, 2023) in a depth of 2600 m. Fluids reach temperatures of 100–102°C in the production well and are reinjected at approximately 43.2°C. The pressure is at 9–11 bar before the injection pump, while it is around 11–15 bar after it. However, near the injection well, the pressure goes down to 2–6 bar. These fluids are characterized by high chloride concentrations of up to 160 g/L (Leins et al., 2023 ). The DOC concentrations in the produced fluids were found to be 26 mg C/L, containing high amounts of acetate (77 mg/L) and propionate (3 mg/L) (Leins et al., 2023 ). Lead is present in the Heemskerk fluids at an average concentration of 2.53 mg Pb/L (Kovács et al., 2023 ). 2.2 Scaling analysis In order to confirm the composition of the problematic scaling in the Heemskerk power plant, a scaling sample was collected from the heat exchanger of the power plant. It was powdered and analyzed by a Carl Zeiss Ultra Plus Gemini 55 field emission scanning electron microscope (FE-SEM) equipped with a tungsten-zircone field emission cathode. Images were taken at 10 kV acceleration voltage and apertures of 120 mm and 13 mm. Additionally, energy dispersive X-ray analysis (EDX) was conducted on the same sample with an UltraDry SDD detector (Thermo Fisher Scientific) on several points of the surface to obtain relative element concentrations. The same procedure was applied to the dried biomass samples. 2.3 Fungal strain and biomass production The fungal strain (strain HEK1) was previously isolated from the fluids of the Heemskerk geothermal power plant taken after the heat exchanger (Bregnard et al., 2024 ). Briefly, the fungal strain was isolated at 22°C and identified as a Penicillium citrinum through Sanger sequencing of the ribosomal Internal Transcriber Sequence (ITS). Strain HEK1 was routinely kept on inclined malt-agar (MA; malt: 12g/L, SIOS Homebrewing, Switzerland, agar: 15g/L, Biolife, Italiana, IT) agar tubes at 4°C. To produce biomass, the fungus was grown on MA plates at 30°C with an agar plug of 0.5 cm of diameter as a starting inoculum. Once the fungus had colonized the entire Petri dish, the agar and the mycelium were cut into pieces and put in a Falcon tube with 25 ml of malt-broth (MB; malt: 12g/L, SIOS Homebrewing, Switzerland). Biomass was reduced to small pieces and homogenized using an ULTRA-TURRAX® stem system (IKA, Germany). The mixture was poured into 250 ml of MB and incubated for 10 days at room temperature and under agitation of 110 rpm. The culture was then autoclaved and filtered through a plastic sieve to remove the supernatant. The supernatant was kept separately for exudates analysis, later labelled as “filtered exudates”. The biomass was rinsed with deionized water to remove the remaining growth medium. The biomass was then autoclaved once more, frozen at -80°C for 20 minutes and then dried for 32 h in a freeze-dryer (LyoQuest, Telstar®, condenser at -50°C, shelves with samples at room temperature). The dry biomass was powdered with a sterile pestle and mortar and kept at room temperature until use. 2.4 Assessment of the biomass stability at extreme conditions and the biosorption capacity of the biomass 2.4.1 General preparation Synthetic fluid solutions were prepared in 30 ml glass vials (Rotalibo screw neck ND24 EPA). To prepare the synthetic fluids, 260 g/L sodium chloride (NaCl, 99.8%, Cellpure, Merck, DE) and 1 g/L Pb 2+ , in form of lead nitrate (Pb(NO 3 ) 2 , Merck, DE) were added to ultrapure water. To assess the impact of acetic acid on Pb 2+ biosorption, acetic acid was added to half of the solutions, resulting in a final concentration of 60 mg/L. The pH value was stabilized to pH 5 in all solutions by adding NaOH. Finally, the amount of biomass used was standarized as the weight of dry biomass, as done in (Wahab et al., 2017 ; Khodabakhshi et al., 2019 ; Lacerda et al., 2019 ). In this study, biomass was added at a concentration of 4 g/L as done in (Wahab et al., 2017 ). The fluids were incubated in an autoclave at a pressure of 8 bar on a rotative shaker. The temperature was set at 25°C, 60°C or 98°C with three run times (1, 2 and 3 h). All treatments (combination of temperature, time and presence/absence of acetic acid) were performed in triplicates. For each treatment, two controls without biomass were done. Controls without the addition of NaCl were done in duplicate, at 25°C and for 2 h (Table 1). After incubation, the fluids were filtered through a 0.22 µm nitrocellulose filter (Sartorius Stedim Biotech, FR) to separate the biomass from the liquid. 2.4.2 Evaluation of the biomass effect on the fluid To assess whether organic matter from the biomass dissolved and contributed to the existing DOC, the DOC content was characterized in the liquid phase by size-exclusion-chromatography (SEC) with subsequent UV (λ = 254 nm) and IR detection by a liquid chromatography organic carbon detection (LC-OCD) system. Phosphate buffer (pH 6.85; 2.7 g/L KH 2 PO 4 , 1.6 g/L Na 2 HPO 4 ) was used as a mobile phase set to a flow of 1.1 mL/min (Huber et al., 2011 ). The fluids passed a 0.45 µm membrane syringe filter before entering the chromatographic column. With the LC-OCD the organic matter can be separated into different fractions according to their molecular mass: Makro 1 (Biopolymers) (> 10,000 Da), Makro 2 (Humic Substances) (~ 1000 Da), Makro 3 (Building Blocks) (350–500 Da), low molecular weight acids (LMWA) (< 350 Da), and low molecular weight neutrals (LMWN) (< 350 Da; Zhu et al., 2015). The hydrophobic organic carbon (HOC) is calculated by subtracting all DOC eluting through the column (chromatographic DOC) by the DOC as quantified by bypassing the SEC column. The DOC was quantified by IR-detection of the released CO 2 after UV-oxidation (λ = 185 nm) in a Gräntzel thin-film reactor. Humic and fulvic acid standards of the Suwannee River, provided by the International Humic Substances Society (IHSS), were used for molecular mass calibration. Fluids with chloride concentrations exceeding 1 g/L were diluted prior to the measurement to avoid disturbances during chromatographic separation. 2.4.2 Assessment of the biosorption capacity of the biomass The biomass on the filters was dried for 24 h at 45°C before being scraped from the filter, weighted on a precision scale, and kept in a Falcon tube at room temperature. The dried biomass (quantities available in the data repository Bregnard and Leins et al. ( 2024 )) was digested with 10 mL of nitric acid (HNO 3 ) (Suprapur® 65%, Merck KGaA, DE). The biomass used for scanning electron microscopy (SEM) was directly removed from the filters and dried on SEM stubs for 24 h at 45°C. The HNO 3 -digested solutions were further diluted at a 1:40 ratio. The amount of Pb 2+ was measured by atomic absorption spectrometry (contra 800G High-End AAS). In addition, the presence of Pb 2+ in the original biomass prepared for biosorption experiment was assessed. This biomass, not exposed to Pb 2+ , was re-hydrated (4 g/L) in ultrapure water (2 h, at room temperature), filtered (at 0.22 µm) and digested and processed as described before. 2.5 Statistical analyses A two-way Anova followed by a post-hoc Tukey test was performed on the Pb 2+ content of the biomass to determine significant differences among treatments temperature (25°C, 60°C, 98°C), exposure time (1, 2 or 3 h, with/without acid, with/without salt). 2.6 Data availability All results of the batch experiments with synthetic brines and the dead biomass are available as a data publication (Bregnard and Leins et al., 2024 ) using the following link: https://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807 . 3 Results 3.1 Characterization of the scales recovered from the geothermal power plant SEM microscopy images of the precipitate collected from the Heemskerk power plant showed dodecahedral crystal structures being the main constituent of the precipitate (Fig. 1 A). The EDX analysis revealed that these crystals correspond to the elements Pb and S, suggesting that these precipitates correspond to galena (PbS) crystals (Fig. 1 B). Some other elements present in the surroundings of the PbS crystals corresponded to probably amorphous phases containing iron (Fe), magnesium (Mg), aluminum (Al), sulfur (S), chloride (Cl) calcium (Ca). 3.2 Effect of the biomass on the fluid: DOC and bulk fractions LC-OCD analyses were conducted in order to assess the stability of the biomass under the tested experimental conditions. This was done through the assessment of the impact of the biomass on the fluid DOC and whether or not the presence of acetic acid has an additional effect on the release of organic compounds. In the LC-OCD data, the dissolved organic matter in the treated fluids and in the untreated fluids (controls) were separated into different fractions according to their molecular mass. The different DOC fractions correspond to peaks at specific retention times. The Makro 1 fraction peak is shown at 20 to 30 min, Makro 2 at 30 to 40 min, Makro 3 at 40 to 43 min, LMWA at 43 to 58 min, and LMWN at 58 to 140 min (Bregnard and Leins et al., 2024 ). The blank biomass (biomass of P . citrinum HEK1 in ultrapure water) showed a plateau in the Makro 1 to Makro 3 area, a small peak in the LMWA section, followed by a dominant peak in the LMWN area (Fig. 2 A). The chromatogram of the filtered exudates (culture supernatant) of P. citrinum HEK1 showed the same peak distribution as the blank biomass but with more distinct peaks in the Makro 1, Makro 3, LMWA, and LMWN area (Fig. 2 B). The strongest difference in the chromatograms is visible when comparing fluids with and without addition of acetic acid, as the added acetic acid results in a strong increase in the peak intensities of the LMWA fraction. All replicates showed a plateau in the Makro 1 to Makro 3 area and a peak in the LMWN area (Fig. 3 ). Generally, all peaks and plateaus seem to increase with increasing temperature. The Makro 1 fraction seems also to increase from 1 h to 2 h time of exposure and decrease after 3 h in the fluids without acetic acid. In the fluids where acetic acid was added, the increase of the Makro 1 peak continues to 3 h of exposure. Noticeable is also the Makro 2–3 peak in samples with 1 h run-time with and without acetic acid. It is stronger at 60°C compared to the other temperatures and decreases gradually at all temperatures with increasing time of exposure. The DOC concentrations in the samples, including the standard deviation, range between 4 and 81 mg C/L (Fig. 4 ). The DOC concentrations have been corrected for the acetate (60 mg/L = 24.4 mg C/L) that has been added in the "acetate experiments". At 25°C, the average DOC increases within the first 2 h from 30 — 40 mg C/L (no acetic acid) and 27 — 42 mg C/L (with acetic acid), respectively. While the samples without acetic acid remain relatively constant until the 3 h mark, the samples with acetic acid show a decrease to approximately 15 mg C/L. At 60°C, the average DOC in both with and without acetic acid samples decreases over time from 57 — 36 mg C/L (no acetic acid) and significantly from 61 — 15 mg C/L (with acetic acid, p value 0.01*), respectively. At 98°C, the samples with acetic acid show a constant decrease of DOC from approximately 67 — 43 mg C/L over time. In the samples without acetic acid, an initial increase of DOC from 47 — 67 mg C/L, is followed by a decrease to 38 mg C/L after 3 h run-time. Independent from the time of exposure, significantly more DOC was released into the fluids at 60°C (p value 0.0247) and 98°C (p-value 0.0005***) compared to a temperature of 25°C. However, within these temperature steps, the DOC appears to decrease with increasing time of exposure. 3.2.1 Impact of temperature and acetic acid on the structure of the biomass itself SEM images combined with EDX analysis were done on the biomass exposed to lead, presence or absence of acetic acid and salt at the different temperatures and exposure times tested. The structure of the biomass was visible at all temperatures, with and without acetic acid (Fig. 5 , Supplementary Fig. 1 for all treatments). Moreover, the precipitation of salt (NaCl) on the biomass was observed at all temperatures and on all EDX analyses performed on the biomass while no peaks indicating the presence of Pb 2+ in the biomass were found. The presence of acetic acid visually seemed to reduce the NaCl quadratic structures, but these structures were nevertheless present in all treatments and NaCl was detected through EDX. 3.3 Lead biosorption capacity of the biomass at extreme conditions 3.3.1 Impact of temperature, acetic acid and incubation time on lead biosorption Overall, the average amount of Pb 2+ associated with the biomass treated with 260 g/L NaCl was 1.81 ± 0.51 mg of Pb 2+ per g of biomass. A blank biomass showed a concentration of 0.01 mg of Pb 2+ per g of biomass. Therefore, the Pb 2+ natively sorbed to the biomass was considered as negligible compared to the values measured in the biomass after biosorption. The Pb 2+ content in the biomass ranged from 1.43 mg Pb 2+ per g of biomass to 2.72 mg Pb 2+ per g of biomass. The lowest mean amount of Pb 2+ in the biomass was detected in the experiments at 25°C, for 1 h and without acid acetic (mean 1.43 ± 0.37 mg Pb 2+ per g of biomass), closely followed by the biomass exposed to 98°C, for 3 h and without acetic acid (mean 1.44 ± 0.73 mg Pb 2+ per g of biomass) (Fig. 6 ). The highest mean amount of Pb 2+ in the biomass was found in the experiments at 25°C, for 2 h and without acetic acid (mean 2.72 ± 0.8 mg Pb 2+ per g of biomass), followed by the same conditions but with 3 h contact (mean 2.26 ± 0.4 mg Pb 2+ per g of biomass). The temperature had a significant impact on the amount of lead present in the biomass (p value 0.0159*), while acetic acid (p value 0.1990) and contact time (p value 0.5650) did not (Supplementary Table 2). Differences between sorption with and without acetic acid at 25°C are still noticeable, but not significant (Fig. 6 A). However, there was a significant difference between 25°C and 98°C (p value 0.0178) but only between the biomass at 98°C with acetic acid compared to the biomass at 25°C without acetic acid. Within the 25°C treatment, there was a significant difference only between the biomass exposed to Pb 2+ for 1 and 2 h without acetic acid (p value 0.0393). Within the two other temperatures (60°C and 98°C), the contact time and presence or absence of acetic acid did not have an influence on the amount of Pb 2+ present in the biomass. Overall, the biosorption capacity was low, with only 0.72% of the total Pb 2+ in solution sorbed in the biomass. 3.3.2 Impact of high salinity in the presence or absence of salt on the lead removal capacity of the biomass The amount of lead detected in the biomass without salt ranged from 13.04 mg Pb 2+ per g of biomass to 79.76 mg Pb 2+ per g of biomass while the amount of lead detected in the biomass in the presence of salt ranged from 1.43 mg Pb 2+ per g of biomass to 3.35 mg per g of biomass (Fig. 7 ). This showed that the presence of salt had a significant impact on the lead sorbed by the biomass (p value 0.0209*). Again, the addition of acetic acid had no impact (p value 0.9163) on this process. The pH value stayed constant before and after the experiments at around pH 5. 4 Discussion Many geothermal sites such as Heemskerk in the Netherlands have to deal with Pb 2+ scales. The presence of Pb 2+ within the scaling precipitates was confirmed in this study by SEM microscopy. These analyses suggest galena (PbS) as a major constituent of the scales. The main aim of this study was to test the feasibility of biosorption for Pb 2+ removal from the fluids. The biosorption potential for Pb 2+ by dead biomass of the fungus P . citrinum HEK1, isolated from the Heemskerk geothermal power plant, was investigated under physicochemical conditions mimicking those in the plant. First, the stability of the biomass under those experimental conditions was tested through the assessment of the release of organic compounds within the fluids as well as the assessment of the structure of the biomass itself after exposure to lead, pressure, different temperatures, salinity and the presence or absence of acetic acid. Biosorption was then assessed at different temperatures (25°C, 60°C and 98°C), contact times (1 h, 2 h, 3 h), and the presence or absence of acetic acid. Additionally, the impact of salinity (NaCl) on the biosorption potential of the biomass was further investigated. 4.1 DOC composition and impact of the biomass at elevated temperatures As expected, the introduction of biomass affected the DOC composition of the fluids as the Makro 1 to Makro 3 and the LMWN fraction were shown to derive from the biomass. This was shown independently from the temperature, time of exposure and presence of acetic acid. This was further supported by the chromatograms of the blank biomass compared to the filtered exudates of P. citrinum HEK1 where the same peak signals were found. The leaching of organic carbon into DOC pools is already a well-known process in soils or inland water networks for instance (Kindler et al., 2011 ; Nakhavali et al., 2021 ). Polysaccharides, proteins and amino sugars are characteristic compounds for the Makro 1 fraction that is also known as the biopolymer fraction (Huber et al., 2011 ). In this study, polysaccharides deriving from remains of the dead biomass could be the cause of this peak, as the fungal cell wall is rich in polysaccharides (Barbosa and Carvalho Junior, 2020). The Makro 2 and Makro 3 are also known as humic substances and building blocks, respectively (Huber et al., 2011 ). Humic substances and their building blocks are generally known as complex heterogeneous mixtures of organic compounds of biotic origin that have undergone extensive transformation (Filella et al., 2005 ; Tranvik, 2009 ). Polysaccharides, proteins, lipids, and nucleic acids deriving from the biomass are likely the precursors of the compounds forming these fractions. The LMWN fraction refers to low molecular weight and low ion density compounds such as low molecular weight alcohols, aldehydes, ketones, sugars, but also amino acids (Huber et al., 2011 ). These compounds could be metabolic byproducts of P. citrinum HEK1 during the growth of biomass which were still present in the dead biomass or even though hyphal structures were still recognizable, cell walls may have become porous, thus leaching cytoplasmic compounds in the liquid phase. Another explanation for the strong presence of these compounds could be the remains of the malt extract where the fungus was grown in, despite washing the biomass. The malt extract typically contains sugars which could be the source for the LMWN signal in this case. The stronger LMWA peaks in the acetic acid fluids were very likely caused by the artificial addition of acetic acid. Nonetheless, fluids without acetic acid also showed a small LWMA peak. The acids in these fluids likely derived from the biomass as shown by the slight peak in the LMWA area in the blank biomass and exudate samples. The DOC concentration of the fluids ranged from an average of 15 mg C/L at 25°C with acetic acid and a contact time of 3 h to 81 mg C/L at 98°C in the absence of acetic acid and a contact time of 2 h. The absence or presence of acetic acid did not have a significant effect on the amount of DOC that was released from the biomass. The temperature had a significant impact on the release of DOC into the fluids from the biomass at both 60°C and 98°C compared to 25°C. This suggests that organic compounds derived from the biomass are more easily dissolved into the fluid at elevated temperatures. Cell wall polymers may be more denatured, allowing cell contents to leach into the fluid. However, at 60°C and 98°C, respectively, the DOC appeared to decrease after a run-time of 3 h. At temperatures above 80°C, thermal degradation has been described as an increasingly important factor in the amount of organic acid anions found in oilfield waters (Kharaka and Hanor, 2003 ). A similar observation of decreasing concentrations was made for DOC data from fluids in the temperature range of 30–200°C (Leins et al., 2022 ). The decrease in DOC at 60°C can be explained by the high concentration of NaCl in the experiments, which may alter the solubility or stability of certain organic compounds, potentially promoting aggregation or precipitation of organic material over time (Kho et al., 2022). This would result in a net reduction of measurable DOC in the liquid phase. Interestingly, this trend is less clear at 98°C, possibly because the higher temperature promotes greater thermal degradation of the biomass. This process could release new DOC, counteracting the decrease caused by thermal degradation of DOC, aggregation or precipitation. In addition, the aggregation effect induced by high NaCl concentrations may itself be temperature dependent, with stronger aggregation occurring at 60°C than at 98°C. While elevated temperatures appear to favor the release of organic compounds into the fluid, a prolonged exposure appears to lead to the decrease of DOC. 4.2 Effect of temperature, contact time and the presence or absence of NaCl and acetic acid on biosorption In the biomass exposed to both lead (Pb 2+ ) and salt (NaCl), the amount of Pb 2+ measured varied greatly (from 0.85 mg to 3.35 mg of Pb 2+ per g of biomass), with the highest values reached at 25°C in the absence of acetic acid and with a contact time of 2 h (mean of 2.72 ± 0.8 mg Pb 2+ per g of biomass). The presence or absence of acetic acid did not have a significant impact on the amount of lead sorbed on the biomass, even though differences were more marked within the biomass at 25°C. Temperature during biosorption had a significant impact on the biosorption capacity of the biomass. However, a statistically significant difference was noticed only between the biomass at 25°C without acetic acid and the biomass exposed at 98°C with acetic acid. Indeed, these two combinations of variables are those that resulted in, respectively, the highest and lowest mean amount of lead present within the biomass. In another study, the optimization of biosorption processes using P. citrinum biomass (Wahab et al., 2017 ) resulted in an average of 329 ± 33.4 mg of Pb 2+ per mg of biomass at 30°C, 150 rpm, 60 minutes contact time, 4 g/L of biomass, and a biomass aged of 5 days. However, in this study, the amount of Pb 2+ in the biomass was calculated using the biosorption capacity of the biomass (q) that considers the initial and final concentration of metal ions in the liquid cultures, alongside the initial dry weight of biomass used. Here the amount of Pb 2+ in the biomass was measured directly by considering the dry weight of the biomass after Pb 2+ exposure and the consecutive Pb 2+ measure. The values of both studies should thus be compared with caution. Moreover, the biosorption experiment conducted by (Wahab et al., 2017 ) did not conduct experiments under saline conditions. If considering the biosorption performed without salt, the maximal biosorption values from (Wahab et al., 2017 ) were roughly 6.85 times higher than in the present study (329 mg Pb 2+ per g of biomass for (Wahab et al., 2017 ) compared to a maximal mean of 48.05 ± 28.96 mg Pb 2+ /g at 25°C, 2 h contact time, no acetic acid, and no salt in the present study). This suggests that the conditions used here are not optimal for Pb 2+ biosorption and should be adapted to increase the biosorption yield. Nevertheless, a striking difference in the Pb 2+ content of the biomass was noticed between the biomass treated with the salt-containing fluid compared to the one treated without salt. Furthermore, considering geothermal fluids in general, the composition of these fluids will add another layer of complexity as many other ions may be present within the fluids (Kovács et al., 2023 ). These ions may compete with the Pb 2+ ions, for instance by competing for binding sites of the dead biomass and thus interfering with the Pb 2+ biosorption capacity of the biosorbent (Michalak et al., 2013 ; Sağ, 2001 ), and this may explain why salinity is an issue for biosorption processes. It was indeed assessed that when biosorption is done with different heavy metals, including Pb 2+ , and different types of biosorbents, the adsorption capacity of the biosorbent was reduced compared to systems where only one metal was present (Mahamadi, 2019 ). In the latter case, lead ions tended to outcompete the other metals assessed and lead was the only metal being successfully sorbed during most of the tested multi-metals biosorption experiments (Mahamadi, 2019 ). It however highlights the fact that multi-metals interactions must be considered (Mahamadi, 2019 ; Sağ, 2001 ) before upscaling any biosorption method to an industrial scale, in our case, the geothermal power plant scale. Indeed, the amount of Pb 2+ sorbed by the biomass without salt was drastically higher as compared to the biomass with a high concentration of salt. This highlights the fact that working with highly saline fluids may be challenging for biosorption, decreasing the absolute quantity of metals that can be biosorbed by the dead biomass. This is consistent with a chromium biosorption study using the fungus Rhizopus arrhizus , where increasing salt (NaCl) concentrations gradually reduced the biosorption capacity, leading to a maximum of 28% biosorption decrease (Aksu and Balibek, 2007 ). In our case, the addition of 260 g/L of NaCl induced a 96.25% decrease of biosorption capacity compared to the no salt condition. Moreover, SEM microscopy images coupled to EDX analysis of the biomass showed the presence of NaCl crystals associated with fungal biomass. This can easily explain why no Pb 2+ was detected with EDX analysis, as Pb was probably not sorbed due to competition with sodium ions and NaCl oversaturation. Nonetheless, SEM analysis showed that the biomass structure was not destroyed by heat and pressure, as hyphal structures were still observable after exposure, which confirms its stability at high temperature and pressure conditions. Still, several limitations of the current study should be indicated. First of all, for biosorption experiments at 60°C and 98°C, it needs to be taken into consideration that the fluids and biomass were not directly at the right temperature, as the preparation took place at room temperature (25°C) and as the autoclaves took around 30 minutes to reach the desired temperature. It can therefore not be excluded that the biosorption observed had happened during that time. However, even if biosorption occurred during that time frame, our results showed that it did not seem to be reversed by exposure to high temperature or high pressure as the results at higher temperatures were not significantly different from those at room temperature in most cases. Desorption processes, used to recover the metal ions sorbed by the biomass, usually involve the use of solvents (Sen and Dastidar, 2010 ; Şenol et al., 2021 ) rather than temperature treatment of the biomass. This suggests that biosorption processes may not be reversed by higher temperature treatments. Moreover, it is also important to consider that the maximum temperature tested in this study was 98°C, which is higher than temperatures usually tested in biosorption studies. However, it is consistent with the temperature of fluids used for heat production in the geothermal context, but it is still low when considering geothermal fluids used for electricity production. In fact, geothermal fluids used for electricity production are usually fluids above 150°C, with some exceptions. Nevertheless, a biosorption process at lower temperature could be applicable after the heat exchange for instance. Secondly, the manipulation steps of the biomass after exposure, such as the separation of the liquid phase and the biomass by filtration and the subsequent biomass removal from the filters, can induce experimental biases as some variable portions of lead could desorb and sorb again on the filter material, therefore induce losses during the process. This is especially true while working with highly saline fluids, which could lead to salt precipitation on filters. These biases were attenuated by using triplicates. However, in future the experimental design should be improved and better adapted to highly saline fluids such as by using decantation processes to separate the biomass from the liquid phase or by keeping the biomass on the filters for lead measurements. Moreover, the filtration processes, here done on 20 mL samples, proved to be impractical and should be adapted to scale experiments up to industrial processes for biosorption to be used in geothermal plants settings. This participates in the challenge to adapt biosorption processes to industrial scale processes in general (de Freitas et al., 2019 ). Finally, in this study, only dead biomass of P . citrinum was used as a biosorbent. Nevertheless, testing dead biomass from other fungi in the context of geothermal fluids could allow to find optimal organisms for different fluid conditions. Testing other types of biomass use, such as immobilized biomass, for instance in calcium alginate beads, could also help to improve the uptake capacity of the biomass (Verma et al., 2013 ), which would be a necessary step to advance toward an application of such processes at a larger scale. Conclusion The current study investigated the potential of dead biomass of the fungus P. citrinum to be used as a lead biosorbent at conditions simulating geothermal fluids. To the best of our knowledge, this is the first study assessing the biosorption potential of a fungus under geothermal conditions. Such preliminary study is a necessary step to assess the feasibility of using biosorption to reduce scaling in geothermal plants. Maximum Pb 2+ biosorption by P. citrinum in highly saline fluids was 3.35 mg/g and the average across all treatments was 1.84 ± 0.51 mg Pb 2+ /g. The process was slightly impacted by temperature but was not influenced by changes in contact time and presence or absence of acetic acid. However, at 25°C and 2 h of contact time, biomass treated with the fluid without salt had a mean biosorption of 48.05 ± 28.96 mg Pb 2+ /g biomass, thus indicating that the main limiting factor under these tested conditions is the high salinity of the fluids. DOC ranging from LMWN’s (< 350 Da) to Makro 1 fraction compounds (10,000 Da) were released from the biomass into the fluids. This increases with temperature but does not appear to affect the stability of the biomass with regard to its biosorption potential. It can be assumed that lead extraction using P. citrinum biomass on a larger scale (e.g., in geothermal facilities) would be more effective with low salinity fluids. Enhancement of biosorption processes is needed even for low salinity waters in order to test competition of Pb 2+ with other ions in geothermal brines and to improve the overall biosorption capacity. Moreover, large amounts of biomass would be needed for an effective solution for geothermal operators, thus requiring an optimization of the fungal culture conditions to ensure a larger biomass production. Further research in conditions simulating different geothermal fluids, combined with other types of biomass, other organisms, and other metals is required to enhance our knowledge on biosorption processes under extreme environmental conditions. In addition, to upscale our finding, on-site test pipes at selected geothermal facilities would be crucial to assess the feasibility to integrate biosorption systems into geothermal facilities. Despite the limited potential use of P. citrinum biomass, this study highlights that microbial strains coming directly from the targeted geothermal fluids might be part of the solution to deal with scaling issues within geothermal power plants. The isolation of more strains from these extreme environments could then not only be beneficial for the biosorption in various contexts, but also within the geothermal industry itself. Abbreviations DOC: Dissolved organic carbon EDX: energy dispersive X-ray FE-SEM: Field emission scanning electron microscope HOC: Hydrophobic organic carbon IHSS: International Humic Substances Society LC-OCD: Liquid chromatography organic carbon detection LMWA: Low molecular weight acids LMWN: Low molecular weight neutrals MA: Malt-agar MB: Malt-broth SEC: Size-exclusion-chromatography SEM: scanning electron microscopy Declarations Acknowledgements We wish to thank Florian Lüdicke (GFZ Potsdam) for the technical assistance and Kristin Günther (GFZ Potsdam) for the laboratory analyses of the samples. Availability of data and materials All results of the batch experiments with synthetic brines and the dead biomass are available as a data publication (Bregnard and Leins et al., 2024) using the following link: https://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807. Funding Supported within the funding programme "Open Access Publikationskosten" Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project Number 491075472 and the project CRM geothermal EU HORIZON program under grant number 101058163. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable Contributions AVH, PJ and SR designed the project. AL and DB designed and conducted the experiment. IS performed the SEM microscopy and EDX analysis. AL and DB analyzed the data. AL and DB wrote the manuscript (equal contribution). AVH, PJ, SB, WZ and SR reviewed and edited the manuscript. All authors read and approved the final manuscript. References Akar, T., Tunali, S., Çabuk, A., 2007. Study on the characterization of lead (II) biosorption by fungus Aspergillus parasiticus. Appl. Biochem. Biotechnol. 136, 389–405. https://doi.org/10.1007/s12010-007-9032-8 Aksu, Z., Balibek, E., 2010. 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Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Geothermal Energy → Version 2 posted Editorial decision: Revision requested 12 Oct, 2025 Reviews received at journal 14 Sep, 2025 Reviews received at journal 29 Aug, 2025 Reviews received at journal 28 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviewers agreed at journal 24 Aug, 2025 Reviewers invited by journal 01 May, 2025 Editor assigned by journal 12 Apr, 2025 Submission checks completed at journal 12 Apr, 2025 First submitted to journal 02 Apr, 2025 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4836282","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2024-09-23 11:32:03","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444944386,"identity":"417617ac-182e-4db5-9f67-974c8cad166c","order_by":0,"name":"Alessio Leins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie2QsQrCMBCGLxTikrbrFYc+gaAIOhTxWcTBB3Bx0BIQ4kuI7+CSOSXQLvoOiuDk4KjQwbQ6iEPQzSEfJOTgPv67ADgcf0gAhAPMIHzVXnW1rQqtlR1EvKrUd4qBiJ+UxlIc75sFxtzPTtcyiVvc2x6sCstWXV8WSHgwbmdi0pGKTq0xFEeiSWSeevGlhxnXRCrWQ6sSH0V0X+dIwXSqUg+N0r/ZU4hAn8+R1QrVoyrFZphdzGAsV4hgdtmLyVhqOrUOFjaKc3Sbp/WPHWZlMpDFcnu1xjzR5rDX2/ui35C+KQ6Hw+H44AEY3UZeAcQ0SAAAAABJRU5ErkJggg==","orcid":"","institution":"GFZ Helmholtz Centre for Geosciences","correspondingAuthor":true,"prefix":"","firstName":"Alessio","middleName":"","lastName":"Leins","suffix":""},{"id":444944387,"identity":"ccb0a775-9dde-4e47-b9bb-acac4d8946a0","order_by":1,"name":"Danae Bregnard","email":"","orcid":"","institution":"University of Neuchâtel","correspondingAuthor":false,"prefix":"","firstName":"Danae","middleName":"","lastName":"Bregnard","suffix":""},{"id":444944388,"identity":"b6c788e6-7e94-48ad-ac49-50a0676e0c61","order_by":2,"name":"Ilona Schäpan","email":"","orcid":"","institution":"GFZ Helmholtz Centre for Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Ilona","middleName":"","lastName":"Schäpan","suffix":""},{"id":444944389,"identity":"96ede47c-8d1c-4705-a7cf-5bed15331e51","order_by":3,"name":"Wart Zonneveld","email":"","orcid":"","institution":"Floricultura b.v.","correspondingAuthor":false,"prefix":"","firstName":"Wart","middleName":"","lastName":"Zonneveld","suffix":""},{"id":444944390,"identity":"7eea6fc4-c304-4867-a3a4-c364a21c33ef","order_by":4,"name":"Saskia Bindschedler","email":"","orcid":"","institution":"University of Neuchâtel","correspondingAuthor":false,"prefix":"","firstName":"Saskia","middleName":"","lastName":"Bindschedler","suffix":""},{"id":444944391,"identity":"e6902615-3298-44b5-8403-87f114eddbae","order_by":5,"name":"Andrea Vieth-Hillebrand","email":"","orcid":"","institution":"GFZ Helmholtz Centre for Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Vieth-Hillebrand","suffix":""},{"id":444944392,"identity":"1904afa9-9587-4d48-987b-20f77ffdab54","order_by":6,"name":"Pilar Junier","email":"","orcid":"","institution":"University of Neuchâtel","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Junier","suffix":""},{"id":444944393,"identity":"3e7b1865-5869-4704-9c3b-b0270cbe8884","order_by":7,"name":"Simona Regenspurg","email":"","orcid":"","institution":"GFZ Helmholtz Centre for Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Simona","middleName":"","lastName":"Regenspurg","suffix":""}],"badges":[],"createdAt":"2024-07-31 14:18:00","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-4836282/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-4836282/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40517-025-00368-z","type":"published","date":"2025-12-02T15:58:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81614865,"identity":"e2adc62d-9c78-451d-8e31-c1bdb7a905a5","added_by":"auto","created_at":"2025-04-29 08:00:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359284,"visible":true,"origin":"","legend":"\u003cp\u003eSEM microscopy image of the precipitate obtained from the heat exchanger of the Heemskerk geothermal power plant, coupled with EDX analysis. A) SEM microscopy image of the precipitate with the location of the EDX measure shown (red star). Scale bar represents 100 µm. B) Graph of the EDX measure.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/62f7234fcb704f42c5bbb8d3.png"},{"id":81613638,"identity":"c4eee103-0dd6-4f10-a7f7-79d5c0db11c1","added_by":"auto","created_at":"2025-04-29 07:52:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103154,"visible":true,"origin":"","legend":"\u003cp\u003eSize exclusion chromatograms of A) the biomass blank (filtered fluids after 80 mg biomass were added to ultra-pure water at room temperature for two hours) and B) the filtered \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecitrinum \u003c/em\u003eHEK1\u003cem\u003e \u003c/em\u003eexudates released in malt broth (MB).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/a3258af5b7aada36c8848885.png"},{"id":81613637,"identity":"2dee362a-50b6-4559-b21b-9bb5755c7d73","added_by":"auto","created_at":"2025-04-29 07:52:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246203,"visible":true,"origin":"","legend":"\u003cp\u003eSize exclusion chromatograms of the fluids at 22 °C, 60 °C and 98 °C for 1, 2 and 3 h, with and without acetic acid, NaCl, and biomass.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/9b6587ea4c35ab13389231ce.png"},{"id":81614862,"identity":"d454cb8f-a432-48b8-8de4-db351d51b18d","added_by":"auto","created_at":"2025-04-29 08:00:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260352,"visible":true,"origin":"","legend":"\u003cp\u003eDOC fluid concentrations (mg C/L) with standard error bars after reaction at different temperatures (25 °C, 60 °C, 98 °C), contact times (1 h, 2 h, 3 h) and presence [60 mg/L] or absence [0 mg/L] of acetic acid. The DOC concentrations have been corrected for the acetate (60 mg/L = 24.4 mg C/L) that has been added in the \"acetate experiments\". A) DOC concentration in the fluid exposed to 25 °C. B) DOC concentration in the fluid exposed to 60 °C. C) DOC concentration in the fluid exposed to 98 °C.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/f0924c9ab81f037d3dc8c88a.png"},{"id":81613641,"identity":"11800a71-10b5-4789-b669-c43e43811ff5","added_by":"auto","created_at":"2025-04-29 07:52:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":676688,"visible":true,"origin":"","legend":"\u003cp\u003eSEM microscopy images of biomass exposed to different treatments of temperature, salinity, pressure, and acetic acid and corresponding EDX analysis: NaCl [260 g/L], Pb\u003csup\u003e2+\u003c/sup\u003e [1 g/L], Contact time: 2 h. Temperature: 98 °C, presence [60 mg/L] of acetic acid. Scale: present on each image. Red arrows highlight typical structures of fungal hyphaes as examples. One EDX measure point is shown for each condition (red circle).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/93b1b90c33edbb46c8f1e436.png"},{"id":81614868,"identity":"d9d51df8-118a-4d5a-99c7-12fa2ad1cbdb","added_by":"auto","created_at":"2025-04-29 08:00:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":403588,"visible":true,"origin":"","legend":"\u003cp\u003eLead (Pb\u003csup\u003e2+\u003c/sup\u003e) content in the biomass (mg of Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass) with standard error bars after reaction at different temperatures (25 °C, 60 °C, 98 °C), contact times (1 h, 2 h, 3 h) and presence [60 mg/L] or absence [0 mg/L] of acetic acid. A) Lead concentration in the biomass exposed to 25 °C. B) Lead concentration in the biomass exposed to 60 °C. C) Lead concentration in the biomass exposed to 98 °C.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/df99f24329957c0cc0e73543.png"},{"id":81613633,"identity":"f97c769d-e16f-41f7-9668-275b4ba39268","added_by":"auto","created_at":"2025-04-29 07:52:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":59635,"visible":true,"origin":"","legend":"\u003cp\u003eLead (Pb\u003csup\u003e2+\u003c/sup\u003e) concentration (mg of Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass) with standard error bars in the biomass in the presence [260 g/L] and absence [0 g/L] of salt (NaCl) and in the presence [60 mg/L, blue bars] or absence [0 mg/L, orange bars] of acetic acid. Temperature: 25\u0026nbsp;°C. Exposure time: 2 h. As only duplicates were done for each treatment, error bars of the standard deviation represent the minimal and maximal values reached.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/fe2806e3350a8dc080ab4fef.png"},{"id":97724838,"identity":"d64083b1-5a62-43bf-8e57-09f71ddb6e71","added_by":"auto","created_at":"2025-12-08 16:13:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3195348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/b6fdb8fd-5726-41f7-8e15-553cfb149117.pdf"},{"id":81613674,"identity":"b7c2d59c-34d2-429a-9a72-aa59c716c92e","added_by":"auto","created_at":"2025-04-29 07:52:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5385409,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4836282/v2/e04952931a6e5488b9deca25.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAssessment of the Pb\u003csup\u003e2+ \u003c/sup\u003ebiosorption potential of the fungus Penicillium citrinum under geothermal conditions\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGeothermal energy operators often encounter operational challenges such as mineral precipitation (scaling) that can clog the pipes and surface installations, leading to reduced flow rates and decreased injectivity of the reservoir (Demir et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Regenspurg et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Mineral precipitates can also insulate the pipes by creating a mineral layer, thus reducing the efficacy of heat exchange (Nitschke et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Mineral scaling within geothermal power plants can include silicate, carbonate, hydroxide, sulfate, or sulfide scaling. Sulfide scales often incorporate heavy metals, such as copper, lead, iron, zinc or vanadium (Lebedev, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Nitschke et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wolfgramm et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the case of Pb\u003csup\u003e2+\u003c/sup\u003e, scalings such as PbSO\u003csub\u003e4\u003c/sub\u003e, PbS, PbCO\u003csub\u003e3\u003c/sub\u003e, Pb(OH)Cl, PbOI or native Pb can be considered (Regenspurg et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These different scales form depending on the Pb\u003csup\u003e2+\u003c/sup\u003e concentration present in the fluids, but in all cases, impact the power plants efficacy (Mouchot et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Regenspurg et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Scheiber et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zotzmann et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These scalings are not only toxic but also typically contain the natural radioactive isotope \u003csup\u003e210\u003c/sup\u003ePb, which presents an additional hazard for geothermal operators during maintenance or for the disposal of the radioactive Pb- containing filter residues (Regenspurg et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of chemical scaling inhibitors is the most frequent practice to mitigate the scaling problem. However, inhibitors do not always present an optimal solution due to high costs and unknown long-term effect of the inhibitors to the reservoirs. Alternatively, metals can be removed from the fluids before precipitation using cation selective filters. For instance, the biopolymer chitosan has been shown to efficiently remove Cu and Pb from fluids (Zotzmann et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Another alternative strategy could be to use the biosorption capacity of microorganisms. Indeed, the remediation of heavy metals from different substrates by biosorption is well known and fungi are efficient heavy metals biosorbents for instance (Congeevaram et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Iram et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As biosorbents, fungi can be used both as living - (Noormohamadi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or dead biomass (Pang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), resting cells (i.e., spores) (Martins et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), or immobilized biomass (dead biomass entrapped in calcium alginate beads, for instance) (Verma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). All of these technologies have different biosorption capacities depending on the targeted metals, the type of fungus and type of biomass preparation and the experimental conditions used during biosorption.\u003c/p\u003e \u003cp\u003eMore precisely, Pb\u003csup\u003e2+\u003c/sup\u003e biosorption has been broadly studied in fungi, especially in mitosporic fungi. Among other molds, biosorption has been demonstrated in \u003cem\u003eAspergillus niger\u003c/em\u003e (Amini et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003eAspergillus parasiticus\u003c/em\u003e (Akar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), \u003cem\u003ePenicillium citrinum\u003c/em\u003e (Pang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u003cem\u003eAspergillus flavus\u003c/em\u003e (Iram et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For some of these fungi, metal removal ranged from 10\u0026ndash;96.21% depending on the chemical conditions, the fungal species, and the target metal assessed (Amini et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Akar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) However, to the best of our knowledge, all previous studies have not been conducted under conditions that correspond to those found in geothermal power plants, i.e., high pressure and salinity, temperatures\u0026thinsp;\u0026gt;\u0026thinsp;50\u0026deg;C. This represents a significant shortcoming as the environmental characteristics of geothermal fluids, such as higher temperatures, are known to affect the biosorption capacities of fungi. Moreover, within the same fungal strain, optimal biosorption temperatures can vary depending on the targeted metal (Iram et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, in the case of the fungus \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e, biosorption of (Pb\u003csup\u003e2+\u003c/sup\u003e) and (Cd\u003csup\u003e2+\u003c/sup\u003e) by living cells was equal at 25\u0026deg;C and 35\u0026deg;C, and even slightly better at 45\u0026deg;C for Pb\u003csup\u003e2+\u003c/sup\u003e (Lu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, salinity is another factor that can reduce the biosorption capacity and the high salinity of some geothermal fluids (i.e., 160 g/L of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in the Heemskerk geothermal power plant (NL)) adds to the chemical complexity of these fluids. For example, the biosorption capacity for a dye of the fungus \u003cem\u003eRhizopus arrizhus\u003c/em\u003e dropped by 28.8% when the salinity was increased from 0 to 50 g/L (Aksu and Balibek, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and the Cr\u003csup\u003e2+\u003c/sup\u003e biosorption capacity of the same fungus dropped by 17% with the same salinity change (Aksu and Balibek, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In brewer yeasts (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e), biosorption of Pb\u003csup\u003e2+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e also decreased with higher concentrations of NaCl and CaCl\u003csub\u003e2\u003c/sub\u003e (Han et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition, the impact of DOC, which can be present in high quantities in geothermal fluids (Leins et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), on metal biosorption capacity has not been assessed systematically. Copper ions precipitated less with higher concentrations of DOC, while the increase of DOC concentrations resulted in higher abiotic Cd\u003csup\u003e2+\u003c/sup\u003e precipitation (Laurent et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese examples demonstrate the need to test biosorption under geothermal conditions and investigate the influence of temperature, salinity, and DOC content to assess the feasibility of biosorption to prevent scaling in geothermal systems. Indeed, even if biosorption processes of heavy metals were investigated in many different systems and with many different types of metals, to the best of our knowledge, conditions similar to geothermal systems (pressure, temperature and salinity) have not been assessed yet. Adapting such biosorption processes to geothermal conditions could reduce the impact of scaling in geothermal power plants without the need to add chemical scaling inhibitors in the fluids. Moreover, utilizing the biosorption capacities of microorganisms to remove metal ions from geothermal fluids would not only help mitigate the impact of scaling but would also facilitate the recovery of these metals. Indeed, biorecovery, which is defined as the recovery of various metal ions through biosorption or bioaccumulation, is promising for fully exploiting geothermal fluids (Lo et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For instance, the co-extraction of lithium from geothermal fluids is one of the processes explored to enhance the viability of geothermal power plants, which then do not only produce heat or electricity, but also a valuable metal for the industry (Weinand et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe specific aims of the present study were to assess the stability of the structure of dead biomass of the fungus \u003cem\u003ePenicillium citrinum\u003c/em\u003e HEK1 strain as well as its Pb\u003csup\u003e2+\u003c/sup\u003e biosorption capacity under conditions simulating the physico-chemical parameters (pressure, temperature, salinity) of the geothermal power plant from which this fungus was isolated.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Site description\u003c/h2\u003e \u003cp\u003eThe Heemskerk geothermal power plant is situated north-west of Amsterdam (Netherlands) and produces primarily heat for greenhouses. It operates as a geothermal doublet, targeting a Permian sandstone (Rotliegend Slochteren) formation (TNO-GDN, 2023) in a depth of 2600 m. Fluids reach temperatures of 100\u0026ndash;102\u0026deg;C in the production well and are reinjected at approximately 43.2\u0026deg;C. The pressure is at 9\u0026ndash;11 bar before the injection pump, while it is around 11\u0026ndash;15 bar after it. However, near the injection well, the pressure goes down to 2\u0026ndash;6 bar. These fluids are characterized by high chloride concentrations of up to 160 g/L (Leins et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The DOC concentrations in the produced fluids were found to be 26 mg C/L, containing high amounts of acetate (77 mg/L) and propionate (3 mg/L) (Leins et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Lead is present in the Heemskerk fluids at an average concentration of 2.53 mg Pb/L (Kov\u0026aacute;cs et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Scaling analysis\u003c/h2\u003e \u003cp\u003eIn order to confirm the composition of the problematic scaling in the Heemskerk power plant, a scaling sample was collected from the heat exchanger of the power plant. It was powdered and analyzed by a Carl Zeiss Ultra Plus Gemini 55 field emission scanning electron microscope (FE-SEM) equipped with a tungsten-zircone field emission cathode. Images were taken at 10 kV acceleration voltage and apertures of 120 mm and 13 mm. Additionally, energy dispersive X-ray analysis (EDX) was conducted on the same sample with an UltraDry SDD detector (Thermo Fisher Scientific) on several points of the surface to obtain relative element concentrations. The same procedure was applied to the dried biomass samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fungal strain and biomass production\u003c/h2\u003e \u003cp\u003eThe fungal strain (strain HEK1) was previously isolated from the fluids of the Heemskerk geothermal power plant taken after the heat exchanger (Bregnard et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, the fungal strain was isolated at 22\u0026deg;C and identified as a \u003cem\u003ePenicillium citrinum\u003c/em\u003e through Sanger sequencing of the ribosomal Internal Transcriber Sequence (ITS). Strain HEK1 was routinely kept on inclined malt-agar (MA; malt: 12g/L, SIOS Homebrewing, Switzerland, agar: 15g/L, Biolife, Italiana, IT) agar tubes at 4\u0026deg;C. To produce biomass, the fungus was grown on MA plates at 30\u0026deg;C with an agar plug of 0.5 cm of diameter as a starting inoculum. Once the fungus had colonized the entire Petri dish, the agar and the mycelium were cut into pieces and put in a Falcon tube with 25 ml of malt-broth (MB; malt: 12g/L, SIOS Homebrewing, Switzerland). Biomass was reduced to small pieces and homogenized using an ULTRA-TURRAX\u0026reg; stem system (IKA, Germany). The mixture was poured into 250 ml of MB and incubated for 10 days at room temperature and under agitation of 110 rpm. The culture was then autoclaved and filtered through a plastic sieve to remove the supernatant. The supernatant was kept separately for exudates analysis, later labelled as \u0026ldquo;filtered exudates\u0026rdquo;. The biomass was rinsed with deionized water to remove the remaining growth medium. The biomass was then autoclaved once more, frozen at -80\u0026deg;C for 20 minutes and then dried for 32 h in a freeze-dryer (LyoQuest, Telstar\u0026reg;, condenser at -50\u0026deg;C, shelves with samples at room temperature). The dry biomass was powdered with a sterile pestle and mortar and kept at room temperature until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Assessment of the biomass stability at extreme conditions and the biosorption capacity of the biomass\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 General preparation\u003c/h2\u003e \u003cp\u003eSynthetic fluid solutions were prepared in 30 ml glass vials (Rotalibo screw neck ND24 EPA). To prepare the synthetic fluids, 260 g/L sodium chloride (NaCl, 99.8%, Cellpure, Merck, DE) and 1 g/L Pb\u003csup\u003e2+\u003c/sup\u003e, in form of lead nitrate (Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, Merck, DE) were added to ultrapure water. To assess the impact of acetic acid on Pb\u003csup\u003e2+\u003c/sup\u003e biosorption, acetic acid was added to half of the solutions, resulting in a final concentration of 60 mg/L. The pH value was stabilized to pH 5 in all solutions by adding NaOH. Finally, the amount of biomass used was standarized as the weight of dry biomass, as done in (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khodabakhshi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lacerda et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, biomass was added at a concentration of 4 g/L as done in (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The fluids were incubated in an autoclave at a pressure of 8 bar on a rotative shaker. The temperature was set at 25\u0026deg;C, 60\u0026deg;C or 98\u0026deg;C with three run times (1, 2 and 3 h). All treatments (combination of temperature, time and presence/absence of acetic acid) were performed in triplicates. For each treatment, two controls without biomass were done. Controls without the addition of NaCl were done in duplicate, at 25\u0026deg;C and for 2 h (Table\u0026nbsp;1). After incubation, the fluids were filtered through a 0.22 \u0026micro;m nitrocellulose filter (Sartorius Stedim Biotech, FR) to separate the biomass from the liquid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Evaluation of the biomass effect on the fluid\u003c/h2\u003e \u003cp\u003eTo assess whether organic matter from the biomass dissolved and contributed to the existing DOC, the DOC content was characterized in the liquid phase by size-exclusion-chromatography (SEC) with subsequent UV (λ\u0026thinsp;=\u0026thinsp;254 nm) and IR detection by a liquid chromatography organic carbon detection (LC-OCD) system. Phosphate buffer (pH 6.85; 2.7 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.6 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) was used as a mobile phase set to a flow of 1.1 mL/min (Huber et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The fluids passed a 0.45 \u0026micro;m membrane syringe filter before entering the chromatographic column. With the LC-OCD the organic matter can be separated into different fractions according to their molecular mass: Makro 1 (Biopolymers) (\u0026gt;\u0026thinsp;10,000 Da), Makro 2 (Humic Substances) (~\u0026thinsp;1000 Da), Makro 3 (Building Blocks) (350\u0026ndash;500 Da), low molecular weight acids (LMWA) (\u0026lt;\u0026thinsp;350 Da), and low molecular weight neutrals (LMWN) (\u0026lt;\u0026thinsp;350 Da; Zhu et al., 2015). The hydrophobic organic carbon (HOC) is calculated by subtracting all DOC eluting through the column (chromatographic DOC) by the DOC as quantified by bypassing the SEC column. The DOC was quantified by IR-detection of the released CO\u003csub\u003e2\u003c/sub\u003e after UV-oxidation (λ\u0026thinsp;=\u0026thinsp;185 nm) in a Gr\u0026auml;ntzel thin-film reactor. Humic and fulvic acid standards of the Suwannee River, provided by the International Humic Substances Society (IHSS), were used for molecular mass calibration. Fluids with chloride concentrations exceeding 1 g/L were diluted prior to the measurement to avoid disturbances during chromatographic separation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Assessment of the biosorption capacity of the biomass\u003c/h2\u003e \u003cp\u003eThe biomass on the filters was dried for 24 h at 45\u0026deg;C before being scraped from the filter, weighted on a precision scale, and kept in a Falcon tube at room temperature. The dried biomass (quantities available in the data repository Bregnard and Leins et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)) was digested with 10 mL of nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e) (Suprapur\u0026reg; 65%, Merck KGaA, DE). The biomass used for scanning electron microscopy (SEM) was directly removed from the filters and dried on SEM stubs for 24 h at 45\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe HNO\u003csub\u003e3\u003c/sub\u003e-digested solutions were further diluted at a 1:40 ratio. The amount of Pb\u003csup\u003e2+\u003c/sup\u003e was measured by atomic absorption spectrometry (contra 800G High-End AAS). In addition, the presence of Pb\u003csup\u003e2+\u003c/sup\u003e in the original biomass prepared for biosorption experiment was assessed. This biomass, not exposed to Pb\u003csup\u003e2+\u003c/sup\u003e, was re-hydrated (4 g/L) in ultrapure water (2 h, at room temperature), filtered (at 0.22 \u0026micro;m) and digested and processed as described before.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analyses\u003c/h2\u003e \u003cp\u003eA two-way Anova followed by a post-hoc Tukey test was performed on the Pb\u003csup\u003e2+\u003c/sup\u003e content of the biomass to determine significant differences among treatments temperature (25\u0026deg;C, 60\u0026deg;C, 98\u0026deg;C), exposure time (1, 2 or 3 h, with/without acid, with/without salt).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data availability\u003c/h2\u003e \u003cp\u003eAll results of the batch experiments with synthetic brines and the dead biomass are available as a data publication (Bregnard and Leins et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the following link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807\u003c/span\u003e\u003cspan address=\"https://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of the scales recovered from the geothermal power plant\u003c/h2\u003e \u003cp\u003eSEM microscopy images of the precipitate collected from the Heemskerk power plant showed dodecahedral crystal structures being the main constituent of the precipitate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The EDX analysis revealed that these crystals correspond to the elements Pb and S, suggesting that these precipitates correspond to galena (PbS) crystals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Some other elements present in the surroundings of the PbS crystals corresponded to probably amorphous phases containing iron (Fe), magnesium (Mg), aluminum (Al), sulfur (S), chloride (Cl) calcium (Ca).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of the biomass on the fluid: DOC and bulk fractions\u003c/h2\u003e \u003cp\u003eLC-OCD analyses were conducted in order to assess the stability of the biomass under the tested experimental conditions. This was done through the assessment of the impact of the biomass on the fluid DOC and whether or not the presence of acetic acid has an additional effect on the release of organic compounds.\u003c/p\u003e \u003cp\u003eIn the LC-OCD data, the dissolved organic matter in the treated fluids and in the untreated fluids (controls) were separated into different fractions according to their molecular mass. The different DOC fractions correspond to peaks at specific retention times. The Makro 1 fraction peak is shown at 20 to 30 min, Makro 2 at 30 to 40 min, Makro 3 at 40 to 43 min, LMWA at 43 to 58 min, and LMWN at 58 to 140 min (Bregnard and Leins et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The blank biomass (biomass of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecitrinum\u003c/em\u003e HEK1 in ultrapure water) showed a plateau in the Makro 1 to Makro 3 area, a small peak in the LMWA section, followed by a dominant peak in the LMWN area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The chromatogram of the filtered exudates (culture supernatant) of \u003cem\u003eP. citrinum\u003c/em\u003e HEK1 showed the same peak distribution as the blank biomass but with more distinct peaks in the Makro 1, Makro 3, LMWA, and LMWN area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe strongest difference in the chromatograms is visible when comparing fluids with and without addition of acetic acid, as the added acetic acid results in a strong increase in the peak intensities of the LMWA fraction. All replicates showed a plateau in the Makro 1 to Makro 3 area and a peak in the LMWN area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Generally, all peaks and plateaus seem to increase with increasing temperature. The Makro 1 fraction seems also to increase from 1 h to 2 h time of exposure and decrease after 3 h in the fluids without acetic acid. In the fluids where acetic acid was added, the increase of the Makro 1 peak continues to 3 h of exposure. Noticeable is also the Makro 2\u0026ndash;3 peak in samples with 1 h run-time with and without acetic acid. It is stronger at 60\u0026deg;C compared to the other temperatures and decreases gradually at all temperatures with increasing time of exposure.\u003c/p\u003e \u003cp\u003eThe DOC concentrations in the samples, including the standard deviation, range between 4 and 81 mg C/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The DOC concentrations have been corrected for the acetate (60 mg/L\u0026thinsp;=\u0026thinsp;24.4 mg C/L) that has been added in the \"acetate experiments\". At 25\u0026deg;C, the average DOC increases within the first 2 h from 30 \u0026mdash; 40 mg C/L (no acetic acid) and 27 \u0026mdash; 42 mg C/L (with acetic acid), respectively. While the samples without acetic acid remain relatively constant until the 3 h mark, the samples with acetic acid show a decrease to approximately 15 mg C/L. At 60\u0026deg;C, the average DOC in both with and without acetic acid samples decreases over time from 57 \u0026mdash; 36 mg C/L (no acetic acid) and significantly from 61 \u0026mdash; 15 mg C/L (with acetic acid, p value 0.01*), respectively. At 98\u0026deg;C, the samples with acetic acid show a constant decrease of DOC from approximately 67 \u0026mdash; 43 mg C/L over time. In the samples without acetic acid, an initial increase of DOC from 47 \u0026mdash; 67 mg C/L, is followed by a decrease to 38 mg C/L after 3 h run-time. Independent from the time of exposure, significantly more DOC was released into the fluids at 60\u0026deg;C (p value 0.0247) and 98\u0026deg;C (p-value 0.0005***) compared to a temperature of 25\u0026deg;C. However, within these temperature steps, the DOC appears to decrease with increasing time of exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Impact of temperature and acetic acid on the structure of the biomass itself\u003c/h2\u003e \u003cp\u003eSEM images combined with EDX analysis were done on the biomass exposed to lead, presence or absence of acetic acid and salt at the different temperatures and exposure times tested. The structure of the biomass was visible at all temperatures, with and without acetic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Fig.\u0026nbsp;1 for all treatments). Moreover, the precipitation of salt (NaCl) on the biomass was observed at all temperatures and on all EDX analyses performed on the biomass while no peaks indicating the presence of Pb\u003csup\u003e2+\u003c/sup\u003e in the biomass were found. The presence of acetic acid visually seemed to reduce the NaCl quadratic structures, but these structures were nevertheless present in all treatments and NaCl was detected through EDX.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Lead biosorption capacity of the biomass at extreme conditions\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Impact of temperature, acetic acid and incubation time on lead biosorption\u003c/h2\u003e \u003cp\u003eOverall, the average amount of Pb\u003csup\u003e2+\u003c/sup\u003e associated with the biomass treated with 260 g/L NaCl was 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 mg of Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass. A blank biomass showed a concentration of 0.01 mg of Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass. Therefore, the Pb\u003csup\u003e2+\u003c/sup\u003e natively sorbed to the biomass was considered as negligible compared to the values measured in the biomass after biosorption. The Pb\u003csup\u003e2+\u003c/sup\u003e content in the biomass ranged from 1.43 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass to 2.72 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass. The lowest mean amount of Pb\u003csup\u003e2+\u003c/sup\u003e in the biomass was detected in the experiments at 25\u0026deg;C, for 1 h and without acid acetic (mean 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass), closely followed by the biomass exposed to 98\u0026deg;C, for 3 h and without acetic acid (mean 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The highest mean amount of Pb\u003csup\u003e2+\u003c/sup\u003e in the biomass was found in the experiments at 25\u0026deg;C, for 2 h and without acetic acid (mean 2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass), followed by the same conditions but with 3 h contact (mean 2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass).\u003c/p\u003e \u003cp\u003eThe temperature had a significant impact on the amount of lead present in the biomass (p value 0.0159*), while acetic acid (p value 0.1990) and contact time (p value 0.5650) did not (Supplementary Table\u0026nbsp;2). Differences between sorption with and without acetic acid at 25\u0026deg;C are still noticeable, but not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, there was a significant difference between 25\u0026deg;C and 98\u0026deg;C (p value 0.0178) but only between the biomass at 98\u0026deg;C with acetic acid compared to the biomass at 25\u0026deg;C without acetic acid. Within the 25\u0026deg;C treatment, there was a significant difference only between the biomass exposed to Pb\u003csup\u003e2+\u003c/sup\u003e for 1 and 2 h without acetic acid (p value 0.0393). Within the two other temperatures (60\u0026deg;C and 98\u0026deg;C), the contact time and presence or absence of acetic acid did not have an influence on the amount of Pb\u003csup\u003e2+\u003c/sup\u003e present in the biomass. Overall, the biosorption capacity was low, with only 0.72% of the total Pb\u003csup\u003e2+\u003c/sup\u003e in solution sorbed in the biomass.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3.2 Impact of high salinity in the presence or absence of salt on the lead removal capacity of the biomass\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe amount of lead detected in the biomass without salt ranged from 13.04 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass to 79.76 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass while the amount of lead detected in the biomass in the presence of salt ranged from 1.43 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass to 3.35 mg per g of biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This showed that the presence of salt had a significant impact on the lead sorbed by the biomass (p value 0.0209*). Again, the addition of acetic acid had no impact (p value 0.9163) on this process. The pH value stayed constant before and after the experiments at around pH 5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eMany geothermal sites such as Heemskerk in the Netherlands have to deal with Pb\u003csup\u003e2+\u003c/sup\u003e scales. The presence of Pb\u003csup\u003e2+\u003c/sup\u003e within the scaling precipitates was confirmed in this study by SEM microscopy. These analyses suggest galena (PbS) as a major constituent of the scales. The main aim of this study was to test the feasibility of biosorption for Pb\u003csup\u003e2+\u003c/sup\u003e removal from the fluids. The biosorption potential for Pb\u003csup\u003e2+\u003c/sup\u003e by dead biomass of the fungus \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecitrinum\u003c/em\u003e HEK1, isolated from the Heemskerk geothermal power plant, was investigated under physicochemical conditions mimicking those in the plant. First, the stability of the biomass under those experimental conditions was tested through the assessment of the release of organic compounds within the fluids as well as the assessment of the structure of the biomass itself after exposure to lead, pressure, different temperatures, salinity and the presence or absence of acetic acid. Biosorption was then assessed at different temperatures (25°C, 60°C and 98°C), contact times (1 h, 2 h, 3 h), and the presence or absence of acetic acid. Additionally, the impact of salinity (NaCl) on the biosorption potential of the biomass was further investigated.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 DOC composition and impact of the biomass at elevated temperatures\u003c/h2\u003e \u003cp\u003eAs expected, the introduction of biomass affected the DOC composition of the fluids as the Makro 1 to Makro 3 and the LMWN fraction were shown to derive from the biomass. This was shown independently from the temperature, time of exposure and presence of acetic acid. This was further supported by the chromatograms of the blank biomass compared to the filtered exudates of \u003cem\u003eP. citrinum\u003c/em\u003e HEK1 where the same peak signals were found. The leaching of organic carbon into DOC pools is already a well-known process in soils or inland water networks for instance (Kindler et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nakhavali et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePolysaccharides, proteins and amino sugars are characteristic compounds for the Makro 1 fraction that is also known as the biopolymer fraction (Huber et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this study, polysaccharides deriving from remains of the dead biomass could be the cause of this peak, as the fungal cell wall is rich in polysaccharides (Barbosa and Carvalho Junior, 2020). The Makro 2 and Makro 3 are also known as humic substances and building blocks, respectively (Huber et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Humic substances and their building blocks are generally known as complex heterogeneous mixtures of organic compounds of biotic origin that have undergone extensive transformation (Filella et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tranvik, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Polysaccharides, proteins, lipids, and nucleic acids deriving from the biomass are likely the precursors of the compounds forming these fractions. The LMWN fraction refers to low molecular weight and low ion density compounds such as low molecular weight alcohols, aldehydes, ketones, sugars, but also amino acids (Huber et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These compounds could be metabolic byproducts of \u003cem\u003eP. citrinum\u003c/em\u003e HEK1 during the growth of biomass which were still present in the dead biomass or even though hyphal structures were still recognizable, cell walls may have become porous, thus leaching cytoplasmic compounds in the liquid phase. Another explanation for the strong presence of these compounds could be the remains of the malt extract where the fungus was grown in, despite washing the biomass. The malt extract typically contains sugars which could be the source for the LMWN signal in this case. The stronger LMWA peaks in the acetic acid fluids were very likely caused by the artificial addition of acetic acid. Nonetheless, fluids without acetic acid also showed a small LWMA peak. The acids in these fluids likely derived from the biomass as shown by the slight peak in the LMWA area in the blank biomass and exudate samples.\u003c/p\u003e \u003cp\u003eThe DOC concentration of the fluids ranged from an average of 15 mg C/L at 25°C with acetic acid and a contact time of 3 h to 81 mg C/L at 98°C in the absence of acetic acid and a contact time of 2 h. The absence or presence of acetic acid did not have a significant effect on the amount of DOC that was released from the biomass. The temperature had a significant impact on the release of DOC into the fluids from the biomass at both 60°C and 98°C compared to 25°C. This suggests that organic compounds derived from the biomass are more easily dissolved into the fluid at elevated temperatures. Cell wall polymers may be more denatured, allowing cell contents to leach into the fluid. However, at 60°C and 98°C, respectively, the DOC appeared to decrease after a run-time of 3 h. At temperatures above 80°C, thermal degradation has been described as an increasingly important factor in the amount of organic acid anions found in oilfield waters (Kharaka and Hanor, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A similar observation of decreasing concentrations was made for DOC data from fluids in the temperature range of 30–200°C (Leins et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The decrease in DOC at 60°C can be explained by the high concentration of NaCl in the experiments, which may alter the solubility or stability of certain organic compounds, potentially promoting aggregation or precipitation of organic material over time (Kho et al., 2022). This would result in a net reduction of measurable DOC in the liquid phase. Interestingly, this trend is less clear at 98°C, possibly because the higher temperature promotes greater thermal degradation of the biomass. This process could release new DOC, counteracting the decrease caused by thermal degradation of DOC, aggregation or precipitation. In addition, the aggregation effect induced by high NaCl concentrations may itself be temperature dependent, with stronger aggregation occurring at 60°C than at 98°C. While elevated temperatures appear to favor the release of organic compounds into the fluid, a prolonged exposure appears to lead to the decrease of DOC.\u003c/p\u003e \u003cp\u003e4.2 Effect of temperature, contact time and the presence or absence of NaCl and acetic acid on biosorption\u003c/p\u003e \u003cp\u003eIn the biomass exposed to both lead (Pb\u003csup\u003e2+\u003c/sup\u003e) and salt (NaCl), the amount of Pb\u003csup\u003e2+\u003c/sup\u003e measured varied greatly (from 0.85 mg to 3.35 mg of Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass), with the highest values reached at 25°C in the absence of acetic acid and with a contact time of 2 h (mean of 2.72 ± 0.8 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass). The presence or absence of acetic acid did not have a significant impact on the amount of lead sorbed on the biomass, even though differences were more marked within the biomass at 25°C. Temperature during biosorption had a significant impact on the biosorption capacity of the biomass. However, a statistically significant difference was noticed only between the biomass at 25°C without acetic acid and the biomass exposed at 98°C with acetic acid. Indeed, these two combinations of variables are those that resulted in, respectively, the highest and lowest mean amount of lead present within the biomass.\u003c/p\u003e \u003cp\u003eIn another study, the optimization of biosorption processes using \u003cem\u003eP. citrinum\u003c/em\u003e biomass (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) resulted in an average of 329 ± 33.4 mg of Pb\u003csup\u003e2+\u003c/sup\u003e per mg of biomass at 30°C, 150 rpm, 60 minutes contact time, 4 g/L of biomass, and a biomass aged of 5 days. However, in this study, the amount of Pb\u003csup\u003e2+\u003c/sup\u003e in the biomass was calculated using the biosorption capacity of the biomass (q) that considers the initial and final concentration of metal ions in the liquid cultures, alongside the initial dry weight of biomass used. Here the amount of Pb\u003csup\u003e2+\u003c/sup\u003e in the biomass was measured directly by considering the dry weight of the biomass after Pb\u003csup\u003e2+\u003c/sup\u003e exposure and the consecutive Pb\u003csup\u003e2+\u003c/sup\u003e measure. The values of both studies should thus be compared with caution. Moreover, the biosorption experiment conducted by (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) did not conduct experiments under saline conditions. If considering the biosorption performed without salt, the maximal biosorption values from (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) were roughly 6.85 times higher than in the present study (329 mg Pb\u003csup\u003e2+\u003c/sup\u003e per g of biomass for (Wahab et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) compared to a maximal mean of 48.05 ± 28.96 mg Pb\u003csup\u003e2+\u003c/sup\u003e/g at 25°C, 2 h contact time, no acetic acid, and no salt in the present study). This suggests that the conditions used here are not optimal for Pb\u003csup\u003e2+\u003c/sup\u003e biosorption and should be adapted to increase the biosorption yield. Nevertheless, a striking difference in the Pb\u003csup\u003e2+\u003c/sup\u003e content of the biomass was noticed between the biomass treated with the salt-containing fluid compared to the one treated without salt. Furthermore, considering geothermal fluids in general, the composition of these fluids will add another layer of complexity as many other ions may be present within the fluids (Kovács et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These ions may compete with the Pb\u003csup\u003e2+\u003c/sup\u003e ions, for instance by competing for binding sites of the dead biomass and thus interfering with the Pb\u003csup\u003e2+\u003c/sup\u003e biosorption capacity of the biosorbent (Michalak et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sağ, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and this may explain why salinity is an issue for biosorption processes. It was indeed assessed that when biosorption is done with different heavy metals, including Pb\u003csup\u003e2+\u003c/sup\u003e, and different types of biosorbents, the adsorption capacity of the biosorbent was reduced compared to systems where only one metal was present (Mahamadi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the latter case, lead ions tended to outcompete the other metals assessed and lead was the only metal being successfully sorbed during most of the tested multi-metals biosorption experiments (Mahamadi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It however highlights the fact that multi-metals interactions must be considered (Mahamadi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sağ, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) before upscaling any biosorption method to an industrial scale, in our case, the geothermal power plant scale. Indeed, the amount of Pb\u003csup\u003e2+\u003c/sup\u003e sorbed by the biomass without salt was drastically higher as compared to the biomass with a high concentration of salt. This highlights the fact that working with highly saline fluids may be challenging for biosorption, decreasing the absolute quantity of metals that can be biosorbed by the dead biomass. This is consistent with a chromium biosorption study using the fungus \u003cem\u003eRhizopus arrhizus\u003c/em\u003e, where increasing salt (NaCl) concentrations gradually reduced the biosorption capacity, leading to a maximum of 28% biosorption decrease (Aksu and Balibek, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In our case, the addition of 260 g/L of NaCl induced a 96.25% decrease of biosorption capacity compared to the no salt condition. Moreover, SEM microscopy images coupled to EDX analysis of the biomass showed the presence of NaCl crystals associated with fungal biomass. This can easily explain why no Pb\u003csup\u003e2+\u003c/sup\u003e was detected with EDX analysis, as Pb was probably not sorbed due to competition with sodium ions and NaCl oversaturation. Nonetheless, SEM analysis showed that the biomass structure was not destroyed by heat and pressure, as hyphal structures were still observable after exposure, which confirms its stability at high temperature and pressure conditions.\u003c/p\u003e \u003cp\u003eStill, several limitations of the current study should be indicated. First of all, for biosorption experiments at 60°C and 98°C, it needs to be taken into consideration that the fluids and biomass were not directly at the right temperature, as the preparation took place at room temperature (25°C) and as the autoclaves took around 30 minutes to reach the desired temperature. It can therefore not be excluded that the biosorption observed had happened during that time. However, even if biosorption occurred during that time frame, our results showed that it did not seem to be reversed by exposure to high temperature or high pressure as the results at higher temperatures were not significantly different from those at room temperature in most cases. Desorption processes, used to recover the metal ions sorbed by the biomass, usually involve the use of solvents (Sen and Dastidar, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Şenol et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) rather than temperature treatment of the biomass. This suggests that biosorption processes may not be reversed by higher temperature treatments. Moreover, it is also important to consider that the maximum temperature tested in this study was 98°C, which is higher than temperatures usually tested in biosorption studies. However, it is consistent with the temperature of fluids used for heat production in the geothermal context, but it is still low when considering geothermal fluids used for electricity production. In fact, geothermal fluids used for electricity production are usually fluids above 150°C, with some exceptions. Nevertheless, a biosorption process at lower temperature could be applicable after the heat exchange for instance. Secondly, the manipulation steps of the biomass after exposure, such as the separation of the liquid phase and the biomass by filtration and the subsequent biomass removal from the filters, can induce experimental biases as some variable portions of lead could desorb and sorb again on the filter material, therefore induce losses during the process. This is especially true while working with highly saline fluids, which could lead to salt precipitation on filters. These biases were attenuated by using triplicates. However, in future the experimental design should be improved and better adapted to highly saline fluids such as by using decantation processes to separate the biomass from the liquid phase or by keeping the biomass on the filters for lead measurements. Moreover, the filtration processes, here done on 20 mL samples, proved to be impractical and should be adapted to scale experiments up to industrial processes for biosorption to be used in geothermal plants settings. This participates in the challenge to adapt biosorption processes to industrial scale processes in general (de Freitas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Finally, in this study, only dead biomass of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecitrinum\u003c/em\u003e was used as a biosorbent. Nevertheless, testing dead biomass from other fungi in the context of geothermal fluids could allow to find optimal organisms for different fluid conditions. Testing other types of biomass use, such as immobilized biomass, for instance in calcium alginate beads, could also help to improve the uptake capacity of the biomass (Verma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which would be a necessary step to advance toward an application of such processes at a larger scale.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study investigated the potential of dead biomass \u003cem\u003eof the fungus P. citrinum\u003c/em\u003e to be used as a lead biosorbent at conditions simulating geothermal fluids. To the best of our knowledge, this is the first study assessing the biosorption potential of a fungus under geothermal conditions. Such preliminary study is a necessary step to assess the feasibility of using biosorption to reduce scaling in geothermal plants. Maximum Pb\u003csup\u003e2+\u003c/sup\u003e biosorption by \u003cem\u003eP. citrinum\u003c/em\u003e in highly saline fluids was 3.35 mg/g and the average across all treatments was 1.84 ± 0.51 mg Pb\u003csup\u003e2+\u003c/sup\u003e/g. The process was slightly impacted by temperature but was not influenced by changes in contact time and presence or absence of acetic acid. However, at 25°C and 2 h of contact time, biomass treated with the fluid without salt had a mean biosorption of 48.05 ± 28.96 mg Pb\u003csup\u003e2+\u003c/sup\u003e/g biomass, thus indicating that the main limiting factor under these tested conditions is the high salinity of the fluids. DOC ranging from LMWN’s (\u0026lt; 350 Da) to Makro 1 fraction compounds (10,000 Da) were released from the biomass into the fluids. This increases with temperature but does not appear to affect the stability of the biomass with regard to its biosorption potential. It can be assumed that lead extraction using \u003cem\u003eP. citrinum\u003c/em\u003e biomass on a larger scale (e.g., in geothermal facilities) would be more effective with low salinity fluids. Enhancement of biosorption processes is needed even for low salinity waters in order to test competition of Pb\u003csup\u003e2+\u003c/sup\u003e with other ions in geothermal brines and to improve the overall biosorption capacity. Moreover, large amounts of biomass would be needed for an effective solution for geothermal operators, thus requiring an optimization of the fungal culture conditions to ensure a larger biomass production. Further research in conditions simulating different geothermal fluids, combined with other types of biomass, other organisms, and other metals is required to enhance our knowledge on biosorption processes under extreme environmental conditions. In addition, to upscale our finding, on-site test pipes at selected geothermal facilities would be crucial to assess the feasibility to integrate biosorption systems into geothermal facilities. Despite the limited potential use of \u003cem\u003eP. citrinum\u003c/em\u003e biomass, this study highlights that microbial strains coming directly from the targeted geothermal fluids might be part of the solution to deal with scaling issues within geothermal power plants. The isolation of more strains from these extreme environments could then not only be beneficial for the biosorption in various contexts, but also within the geothermal industry itself.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDOC:\u0026nbsp;\u003c/strong\u003eDissolved organic carbon\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDX:\u003c/strong\u003e energy dispersive X-ray\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFE-SEM:\u003c/strong\u003e Field emission scanning electron microscope\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHOC:\u003c/strong\u003e Hydrophobic organic carbon\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHSS:\u003c/strong\u003e International Humic Substances Society\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-OCD:\u0026nbsp;\u003c/strong\u003eLiquid chromatography organic carbon detection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMWA:\u003c/strong\u003e Low molecular weight acids\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLMWN:\u003c/strong\u003e Low molecular weight neutrals\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMA:\u003c/strong\u003e Malt-agar\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMB:\u003c/strong\u003e Malt-broth\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEC:\u003c/strong\u003e Size-exclusion-chromatography\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM:\u0026nbsp;\u003c/strong\u003escanning electron microscopy\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe wish to thank Florian L\u0026uuml;dicke (GFZ Potsdam) for the technical assistance and Kristin G\u0026uuml;nther (GFZ Potsdam) for the laboratory analyses of the samples.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll results of the batch experiments with synthetic brines and the dead biomass are available as a data publication\u0026nbsp;(Bregnard and Leins et al., 2024)\u0026nbsp;using the following link: https://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eSupported within the funding programme \u0026quot;Open Access Publikationskosten\u0026quot; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project Number 491075472 and the project CRM geothermal EU HORIZON program under grant number 101058163.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eContributions\u003c/h2\u003e\n\u003cp\u003eAVH, PJ and SR designed the project. AL and DB designed and conducted the experiment. IS performed the SEM microscopy and EDX analysis. AL and DB analyzed the data. AL and DB wrote the manuscript (equal contribution). AVH, PJ, SB, WZ and SR reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkar, T., Tunali, S., \u0026Ccedil;abuk, A., 2007. Study on the characterization of lead (II) biosorption by fungus Aspergillus parasiticus. Appl. Biochem. Biotechnol. 136, 389\u0026ndash;405. https://doi.org/10.1007/s12010-007-9032-8\u003c/li\u003e\n\u003cli\u003eAksu, Z., Balibek, E., 2010. Effect of salinity on metal-complex dye biosorption by \u003cem\u003eRhizopus arrhizus\u003c/em\u003e. J. Environ. Manage. 91, 1546\u0026ndash;1555. https://doi.org/10.1016/j.jenvman.2010.02.026\u003c/li\u003e\n\u003cli\u003eAksu, Z., Balibek, E., 2007. 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Fungal cell with artificial metal container for heavy metals biosorption: Equilibrium, kinetics study and mechanisms analysis. Environ. Res. 182, 109061. https://doi.org/10.1016/j.envres.2019.109061\u003c/li\u003e\n\u003cli\u003eMahamadi, C., 2019. On the dominance of Pb during competitive biosorption from multi-metal systems: A review. Cogent Environ. Sci. 5, 1635335. https://doi.org/10.1080/23311843.2019.1635335\u003c/li\u003e\n\u003cli\u003eMartins, L.R., Lyra, F.H., Rugani, M.M.H., Takahashi, J.A., 2016. Bioremediation of Metallic Ions by Eight \u003cem\u003ePenicillium\u003c/em\u003e Species. J. Environ. Eng. 142, C4015007. https://doi.org/10.1061/(ASCE)EE.1943-7870.0000998\u003c/li\u003e\n\u003cli\u003eMichalak, I., Chojnacka, K., Witek-Krowiak, A., 2013. State of the Art for the Biosorption Process\u0026mdash;a Review. Appl. Biochem. Biotechnol. 170, 1389. https://doi.org/10.1007/s12010-013-0269-0\u003c/li\u003e\n\u003cli\u003eMouchot, Justine, Genter, Albert, Cuenot, Nicolas, Scheiber, J., Seibel, O., Bosia, C., Ravier, G., Mouchot, J, Genter, A, Cuenot, N, 2018. First year of operation from EGS geothermal plants in Alsace, France: Scaling issues. Presented at the Proceedings of the 43rd Workshop on Geothermal Reservoir Engineering, Stanford, CA, USA, pp. 12\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eNakhavali, M., Lauerwald, R., Regnier, P., Guenet, B., Chadburn, S., Friedlingstein, P., 2021. Leaching of dissolved organic carbon from mineral soils plays a significant role in the terrestrial carbon balance. Glob. Change Biol. 27, 1083\u0026ndash;1096.\u003c/li\u003e\n\u003cli\u003eNitschke, F., Scheiber, J., Kramar, U., Neumann, T., 2014. Formation of alternating layered Ba-Sr-sulfate and Pb-sulfide scaling in the geothermal plant of Soultz-sous-For\u0026ecirc;ts. Neues Jahrb. F\u0026uuml;r Mineral. - Abh. 145\u0026ndash;156. https://doi.org/10.1127/0077-7757/2014/0253\u003c/li\u003e\n\u003cli\u003eNoormohamadi, H.R., Fat\u0026rsquo;hi, M.R., Ghaedi, M., Ghezelbash, G.R., 2019. Potentiality of white-rot fungi in biosorption of nickel and cadmium: Modeling optimization and kinetics study. Chemosphere 216, 124\u0026ndash;130. https://doi.org/10.1016/j.chemosphere.2018.10.113\u003c/li\u003e\n\u003cli\u003ePang, C., Liu, Y.-H., Cao, X.-H., Li, M., Huang, G.-L., Hua, R., Wang, C.-X., Liu, Y.-T., An, X.-F., 2011. Biosorption of uranium(VI) from aqueous solution by dead fungal biomass of \u003cem\u003ePenicillium citrinum\u003c/em\u003e. Chem. Eng. J. 170, 1\u0026ndash;6. https://doi.org/10.1016/j.cej.2010.10.068\u003c/li\u003e\n\u003cli\u003eRegenspurg, S., Dilling, J., Mielcarek, J., Korte, F., Schkade, U.-K., 2014. Naturally occurring radionuclides and their geochemical interactions at a geothermal site in the North German Basin. Environ. 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Energy 11, 100148. https://doi.org/10.1016/j.adapen.2023.100148\u003c/li\u003e\n\u003cli\u003eWolfgramm, M., Rauppach, K., Seibt, A., 2009. Langfristige Betriebsf\u0026uuml;hrung und monitoring geothermischer Anlagen in Deutschland, in: Proceedings of the 2009 Geothermal Congress, Bochum, Germany. p. 12.\u003c/li\u003e\n\u003cli\u003eZotzmann, J., Feldbusch, E., Kruppke, I., Aibibu, D., Regenspurg, S., 2023. Removal of lead and copper ions from geothermal brine at various temperatures and salinities using chemically crosslinked chitosan. Appl. Geochem. 155, 105733. https://doi.org/10.1016/j.apgeochem.2023.105733\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"geothermal-energy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"geen","sideBox":"Learn more about [Geothermal Energy](https://geothermal-energy-journal.springeropen.com/about)","snPcode":"40517","submissionUrl":"https://submission.springernature.com/new-submission/40517/3","title":"Geothermal Energy","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Geothermal energy, Biosorption, Fungal Biomass, Pb2+ scaling, Penicillium citrinum","lastPublishedDoi":"10.21203/rs.3.rs-4836282/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4836282/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOne solution for reducing the scaling risk of lead (Pb)-containing phases consists of removing the aqueous Pb\u003csup\u003e2+\u003c/sup\u003e ions from the brine by sorption before oversaturation of Pb\u003csup\u003e2+\u003c/sup\u003e phases at unwanted locations within the geothermal fluid loop. Hence, this study investigated the known capacity of fungal biomass to biosorb Pb\u003csup\u003e2+\u003c/sup\u003e ions to remove Pb\u003csup\u003e2+\u003c/sup\u003e from the brine. So far, biosorption studies have neither been done at high temperatures or salinity, nor under high pressure, three conditions that have to be considered within geothermal power plants. Thus, the overall goal of this study was to assess the Pb\u003csup\u003e2+\u003c/sup\u003e biosorption potential of dead biomass of the fungus \u003cem\u003ePenicillium citrinum\u003c/em\u003e strain HEK1 under conditions mimicking those of natural highly saline geothermal fluids. This specific strain was isolated from geothermal brine circulating in a plant in which Pb\u003csup\u003e2+\u003c/sup\u003e scaling occurs. To assess biosorption, dead biomass of \u003cem\u003eP. citrinum\u003c/em\u003e was added to synthetic solutions containing 260 g/L NaCl, 1g/L Pb, and (in half of the treatments) 60 mg/L acetic acid. These synthetic solutions, including the dead biomass, were then incubated at high pressure (8 bar), at different temperatures (25\u0026deg;C, 60\u0026deg;C, 98\u0026deg;C), and for different time intervals (1 h, 2 h, 3 h). Results showed that the structure of the biomass was stable in such conditions, at all temperatures tested, but small amounts of organic compounds, with a wide variety of low molecular weight (\u0026lt;\u0026thinsp;350 Da to 10,000 Da) have been released into the fluids from the biomass. In general, increased temperature resulted in an increase of dissolved organic carbon (DOC) concentration. The biosorption potential of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecitrinum\u003c/em\u003e HEK1 biomass was overall low (0.72% of total Pb\u003csup\u003e2+\u003c/sup\u003e). While it was not affected by changes in temperature, time of exposure or by the presence of organic acids within the fluids, salinity showed to be influential as biosorption increased up to 19.22% of Pb\u003csup\u003e2+\u003c/sup\u003e removal in non-saline conditions. Therefore, the high salinity of the fluids was the factor limiting the biosorption to the highest extent, highlighting that working with highly saline geothermal fluids might be limiting for biosorption processes to happen efficiently.\u003c/p\u003e","manuscriptTitle":"Assessment of the Pb2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-04-29 07:52:22","doi":"10.21203/rs.3.rs-4836282/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T00:07:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T14:41:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T17:20:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T02:43:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296086958869692781065260883796044614283","date":"2025-08-27T08:14:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275855022103143193335853323554379688282","date":"2025-08-26T23:46:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130967420459571486912877812704257995312","date":"2025-08-24T16:36:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-01T21:30:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-12T12:49:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-12T12:46:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Geothermal Energy","date":"2025-04-02T10:21:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"geothermal-energy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"geen","sideBox":"Learn more about [Geothermal Energy](https://geothermal-energy-journal.springeropen.com/about)","snPcode":"40517","submissionUrl":"https://submission.springernature.com/new-submission/40517/3","title":"Geothermal Energy","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da4f95e4-f2a1-4f37-b9b3-1e7d62532eb9","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:12:11+00:00","versionOfRecord":{"articleIdentity":"rs-4836282","link":"https://doi.org/10.1186/s40517-025-00368-z","journal":{"identity":"geothermal-energy","isVorOnly":false,"title":"Geothermal Energy"},"publishedOn":"2025-12-02 15:58:25","publishedOnDateReadable":"December 2nd, 2025"},"versionCreatedAt":"2025-04-29 07:52:22","video":"","vorDoi":"10.1186/s40517-025-00368-z","vorDoiUrl":"https://doi.org/10.1186/s40517-025-00368-z","workflowStages":[]},"version":"v2","identity":"rs-4836282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4836282","identity":"rs-4836282","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}