Assessment of the Pb 2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions

doi:10.21203/rs.3.rs-4836282/v1

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Assessment of the Pb 2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions

https://doi.org/10.21203/rs.3.rs-4836282/v1

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One solution for reducing the scaling risk of lead (Pb)-containing phases is to remove the aqueous Pb²⁺ ions from the brine by a sorption process before oversaturation of Pb²⁺ phases at unwanted locations within the geothermal fluid loop. This study investigated the known capacity of fungal biomass to bind Pb²⁺ ions (biosorb) and thus remove Pb²⁺ 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 Pb²⁺ 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 a geothermal power plant in which Pb²⁺ scaling occurs. The 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 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. Moreover, the potential for biosorption of P. citrinum HEK1 biomass was not affected by changes in temperature, time of exposure or by the presence of organic acids within the fluids. However, the biosorption potential was overall low (1.4% of total Pb²⁺). It nevertheless increased to about 5 to 10% of Pb²⁺ removal in experiments with 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

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 Pb²⁺, scalings such as PbSO₄, PbS, PbCO₃, Pb(OH)Cl, PbOI or native Pb can be considered (Regenspurg et al., 2016). These different scales form depending on the Pb²⁺ concentration present in the targeted 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 ²¹⁰Pb, 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, for 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 biosorbent used and the experimental conditions.

More precisely, Pb²⁺ biosorption has been broadly studied in fungi, especially in mold 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). 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. 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 (Pb²⁺) and (Cd²⁺) by living cells was equal at 25°C and 35°C, and even slightly better at 45°C for Pb²⁺ (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 of the fungus Rhizopus arrizhus dropped by 28% when the salinity was increased from 0 to 50 g/L (Aksu and Balibek, 2010) and the Cr²⁺ biosorption capacity of the same fungus dropped by 17% with the same salinity change (Aksu and Balibek, 2007). Biosorption of Pb²⁺ and Cu²⁺ also decreased with higher concentrations of NaCl and CaCl₂ for brewer yeasts (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 the 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 Cd²⁺ 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. Adapting such 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, recovering various metal ions through biosorption or bioaccumulation (biorecovery) is promising for fully exploiting geothermal fluids (Lo et al., 2014). For instance, the co-production of lithium from geothermal fluids is one of the processes used 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 aims of the present study were to assess the stability of dead biomass of the fungus Penicillium citrinum HEK1 strain as well as its Pb²⁺ biosorption capacity under conditions simulating the physico-chemical parameters of the geothermal power plant from which this fungus was isolated.

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 Fischer Scientific) on several points of the surface of the dried biomass to obtain relative element concentrations.

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. Growth was done on MA plates at 30°C with an agar plug of 0.5 cm of diameter as a starting inoculum. To produce biomass, the fungus was grown on MA at 30°C. Once the fungus had colonized the entire Petri dish, the mycelium was cut with the agar and put in a Falcon tube with 25 ml of malt-broth (MB). 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 biomass was rinsed with deionized water to remove the remaining growing medium. The biomass was then autoclaved once more, frozen at -80°C for 20 minutes and then dried for 32 h in a desiccator at room temperature (LyoQuest, Telstar®, condenser at -50°C). The dry biomass was powdered with a sterile pestle and mortar and kept at room temperature until use.

2.3 Assessment of the biomass stability at extreme conditions and the biosorption capacity of the biomass

2.3.1 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 Pb²⁺, in form of lead nitrate (Pb(NO₃)₂, Merck, DE) were added to ultrapure water. To assess the impact of acetic acid on Pb²⁺ biosorption, acetic acid was added to half of the solutions in concentrations of 60 mg/L. The pH value was stabilized to pH 5 in all solutions by adding NaOH. Finally, biomass was added at a concentration of 4 g/L (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.

2.3.2 Evaluation of the biomass effect on the fluid

To assess the stability of the biomass, 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 KH₂PO₄, 1.6 g/L Na₂HPO₄) 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 CO₂ 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.

2.3.2 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 (Bregnard and Leins et al., 2024) was digested with 10 mL of nitric acid (HNO₃) (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 HNO₃-digested solutions were further diluted at a 1:40 ratio. The amount of Pb²⁺ was measured byatomic absorption spectrometry (contra 800G High-End AAS). In addition, the presence of Pb²⁺ in the original biomass prepared for biosorption experiment was assessed. This biomass, not exposed to Pb²⁺, 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.

2.4 Statistical analyses

A two-way Anova followed by a post-hoc Tukey test was performed on the Pb²⁺ 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. 5 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.

3.1 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. 1A). The EDX analysis revealed that these crystals correspond to the elements Pb and S, suggesting that these precipitates correspond to galena (PbS) crystals (Fig. 1B). 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. 2A). The chromatogram of the filtered exudates 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. 2B).

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. Independently 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.

3.2.2 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 Pb²⁺ 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

The average amount of Pb²⁺ present in the biomass after the experiment with the presence of 260 g/L NaCl was 1.80 mg of Pb²⁺ per g of biomass. A blank biomass showed a concentration of 0.01 mg of Pb²⁺ per g of biomass. Therefore, the Pb²⁺ natively present in the biomass was considered as negligible compared to the values measured in the biomass after biosorption. The Pb²⁺ content in the biomass ranged from 1.43 mg Pb²⁺ per g of biomass to 2.72 mg Pb²⁺ per g of biomass. The lowest mean amount of Pb²⁺ in the biomass was detected in the experiments at 25°C, for 1 h and without acid acetic (mean 1.43 mg Pb²⁺ per g of biomass), closely followed by the biomass exposed to 98°C, for 3 h and without acetic acid (mean 1.43 mg Pb²⁺ per g of biomass) (Fig. 6). The highest mean amount of Pb²⁺ in the biomass was found in the experiments at 25°C, for 2 h and without acetic acid (mean 2.72 mg Pb²⁺ per g of biomass), followed by the same conditions but with 3 h contact (mean 2.26 mg Pb²⁺ 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. 6A). 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 Pb²⁺ 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 Pb²⁺ present in the biomass. Overall, the biosorption capacity was low, with only 1.4% of the total Pb²⁺ in solution sorbed in the biomass.

3.2.2 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 Pb²⁺ per g of biomass to 79.76 mg Pb²⁺ per g of biomass while the amount of lead detected in the biomass in the presence of salt ranged from 1.43 mg Pb²⁺ 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 uptake 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

Many geothermal sites such as Heemskerk in the Netherlands have to deal with Pb²⁺ scales. The presence of Pb²⁺ within the scaling precipitates was confirmed in this study by SEM microscopy. These analyses confirmed galena (PbS) as a major constituent of the scales. The main aim of this study was to test the feasibility of Pb²⁺ removal from the fluids by biosorption. The biosorption potential for Pb²⁺ in dead biomass by the fungus P. citrinum HEK1, isolated from the Heemskerk geothermal power plant, was investigated under 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. Afterwards, 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 and 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 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. 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 22°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 deriving from the biomass are more easily dissolved into the fluid at elevated temperatures. However, at 60°C and 98°C, respectively, the DOC appeared to decrease after a run-time of 3 h. Thermal degradation of DOC is likely responsible. 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). 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 (Pb²⁺) and salt (NaCl), the amount of Pb²⁺ present varied greatly (from 0.85 mg to 3.35 mg of Pb²⁺ 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 (2.724 mg Pb²⁺ 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 had a significant impact on the biosorption capacity of the biomass, however, only a significant difference between the biomass at 25°C without acetic acid and the biomass exposed at 98°C with acetic acid was noticed. Indeed, these two combinations of variables are those that resulted in the highest and lowest mean amount of lead present within the biomass.

In another study, the optimization of biosorption processes using P. citrinum (Wahab et al., 2017) resulted in an average of o 329 ± 33.4 mg of Pb²⁺ per mg of biomass at 30°C, 150 rpm, 60 minutes, 4 g/L of biomass and a biomass aged of 5 days. However, in this study, the amount of Pb²⁺ 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 Pb²⁺ in the biomass was measured directly by considering the dry weight of the biomass after Pb²⁺ exposure and the consecutive Pb²⁺ 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 Pb²⁺ per g of biomass for (Wahab et al., 2017) compared to a maximal mean of 47.39 mg Pb²⁺/g at 25°C, 2 h contact time and no acetic acid, in the present study). This suggests that the conditions used here are not optimal for Pb²⁺ biosorption. Nevertheless, a striking difference in the Pb²⁺ content of the biomass was noticed between the biomass containing salt and the biomass without salt. Furthermore, considering geothermal fluids in general, the composition of these fluids will add another layer of complexity as many other ions are present within the fluids (Kovács et al., 2023). These ions may compete with the Pb²⁺ ions, for instance by competing for binding sites on the dead biomass and thus interfering with the Pb²⁺ 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 Pb²⁺, 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 a geothermal power plant scale. Indeed, the amount of Pb²⁺ 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 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 (Aksu and Balibek, 2007). Moreover, SEM microscopy images coupled to EDX analysis of the biomass showed the presence of NaCl crystals, which easily prevented the detection of Pb²⁺ using EDX analysis. Nonetheless, the same 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 already higher than temperatures usually tested in biosorption studies. It is consistent with the temperature of fluids used for heat production, 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. However, a biosorption process at lower temperature could be applicable after the heat exchange for instance. 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.

The current study showed the potential of dead biomass of the fungus P. citrinum to be used as a lead biosorbent at conditions simulating geothermal fluids. Maximum Pb²⁺ biosorption by P. citrinum in highly saline fluids was 3.35 mg/g and the average across all treatments was 1.80 mg Pb²⁺/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 without salt had a mean biosorption of 47.39 mg Pb²⁺/g biomass thus indicating that the most 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 Pb²⁺ with other ions in geothermal brines. 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 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.

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

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Not applicable

Funding

Supported within the funding programme "Open Access Publikationskosten" Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project Number 491075472

Author Contribution

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.

Acknowledgement

We wish to thank Florian Lüdicke (GFZ Potsdam) for the technical assistance and Kristin Günther (GFZ Potsdam) for the laboratory analyses of the samples. This work has been funded by the European Union’s Horizon 2020 research and innovation program within the framework of the REFLECT project under the grant agreement No 850626.

Data Availability

All results of the batch experiments with synthetic brines and the dead biomass are available as a data publication using the following link: https://gfzpublic.gfz-potsdam.de/pubman/item/item_5025807.

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. Effect of salinity on metal-complex dye biosorption by Rhizopus arrhizus. J. Environ. Manage. 91, 1546–1555. https://doi.org/10.1016/j.jenvman.2010.02.026
Aksu, Z., Balibek, E., 2007. Chromium(VI) biosorption by dried Rhizopus arrhizus: Effect of salt (NaCl) concentration on equilibrium and kinetic parameters. J. Hazard. Mater. 145, 210–220. https://doi.org/10.1016/j.jhazmat.2006.11.011
Amini, M., Younesi, H., Bahramifar, N., Lorestani, A.A.Z., Ghorbani, F., Daneshi, A., Sharifzadeh, M., 2008. Application of response surface methodology for optimization of lead biosorption in an aqueous solution by Aspergillus niger. J. Hazard. Mater. 154, 694–702. https://doi.org/10.1016/j.jhazmat.2007.10.114
Barbosa, J.R., Carvalho Junior, R.N. de, 2020. Occurrence and possible roles of polysaccharides in fungi and their influence on the development of new technologies. Carbohydr. Polym. 246, 116613. https://doi.org/10.1016/j.carbpol.2020.116613
Bregnard, D., Leins, A., Vieth-Hillebrand, A., Junier, P., Regenspurg, S., van Zonnefeld, W., Stammeier, J., Iannotta, J., Günther, K., 2024. Lead(II) biosorption experiments of the fungus Penicillium citrinum under geothermal conditions. https://doi.org/10.5880/FIDGEO.2024.006
Congeevaram, S., Dhanarani, S., Park, J., Dexilin, M., Thamaraiselvi, K., 2007. Biosorption of chromium and nickel by heavy metal resistant fungal and bacterial isolates. J. Hazard. Mater. 146, 270–277. https://doi.org/10.1016/j.jhazmat.2006.12.017
Demir, M.M., Baba, A., Atilla, V., Inanlı, M., 2014. Types of the scaling in hyper saline geothermal system in northwest Turkey. Geothermics 50, 1–9.
Filella, M., Buffle, J., Parthasarathy, N., 2005. HUMIC AND FULVIC COMPOUNDS, in: Worsfold, P., Townshend, A., Poole, C. (Eds.), Encyclopedia of Analytical Science (Second Edition). Elsevier, Oxford, pp. 288–298. https://doi.org/10.1016/B0-12-369397-7/00260-0
Han, R., Li, H., Li, Y., Zhang, J., Xiao, H., Shi, J., 2006. Biosorption of copper and lead ions by waste beer yeast. J. Hazard. Mater. 137, 1569–1576. https://doi.org/10.1016/j.jhazmat.2006.04.045
Huber, S.A., Balz, A., Abert, M., Pronk, W., 2011. Characterisation of aquatic humic and non-humic matter with size-exclusion chromatography–organic carbon detection–organic nitrogen detection (LC-OCD-OND). Water Res. 45, 879–885.
Iram, S., Shabbir, R., Zafar, H., Javaid, M., 2015. Biosorption and Bioaccumulation of Copper and Lead by Heavy Metal-Resistant Fungal Isolates. Arab. J. Sci. Eng. 40, 1867–1873. https://doi.org/10.1007/s13369-015-1702-1
Kharaka, Y.K., Hanor, J.S., 2003. Deep Fluids in the Continents: I. Sedimentary Basins. Treatise Geochem. 5, 605. https://doi.org/10.1016/B0-08-043751-6/05085-4
Kindler, R., Siemens, J., Kaiser, K., Walmsley, D.C., Bernhofer, C., Buchmann, N., Cellier, P., Eugster, W., Gleixner, G., Grũnwald, T., Heim, A., Ibrom, A., Jones, S.K., Jones, M., Klumpp, K., Kutsch, W., Larsen, K.S., Lehuger, S., Loubet, B., Mckenzie, R., Moors, E., Osborne, B., Pilegaard, K., Rebmann, C., Saunders, M., Schmidt, M.W.I., Schrumpf, M., Seyfferth, J., Skiba, U., Soussana, J.-F., Sutton, M.A., Tefs, C., Vowinckel, B., Zeeman, M.J., Kaupenjohann, M., 2011. Dissolved carbon leaching from soil is a crucial component of the net ecosystem carbon balance. Glob. Change Biol. 17, 1167–1185. https://doi.org/10.1111/j.1365-2486.2010.02282.x
Kovács, K., Seres, A., Hartai, É., 2023. The H2020 REFLECT project: Deliverable 3.2–The REFLECT European Fluid Atlas.
Laurent, J., Casellas, M., Dagot, C., 2010. Heavy metals biosorption on disintegrated activated sludge: Description of a new equilibrium model. Chem. Eng. J. 164, 63–69. https://doi.org/10.1016/j.cej.2010.08.023
Lebedev, L., 1972. Minerals of contemporary hydrotherms of Cheleken. Geochem. Int. 9, 485–504.
Leins, A., Bregnard, D., Vieth-Hillebrand, A., Junier, P., Regenspurg, S., 2022. Dissolved organic compounds in geothermal fluids used for energy production: a review. Geotherm. Energy 10, 9. https://doi.org/10.1186/s40517-022-00220-8
Leins, A., Vieth-Hillebrand, A., Günther, K., Regenspurg, S., 2023. Dissolved organic compounds in geothermal fluids used for energy production – part II. https://doi.org/10.5880/GFZ.4.8.2023.005
Lo, Y.-C., Cheng, C.-L., Han, Y.-L., Chen, B.-Y., Chang, J.-S., 2014. Recovery of high-value metals from geothermal sites by biosorption and bioaccumulation. Bioresour. Technol., Special Issue on Biosorption 160, 182–190. https://doi.org/10.1016/j.biortech.2014.02.008
Lu, N., Hu, T., Zhai, Y., Qin, H., Aliyeva, J., Zhang, H., 2020. 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
Mahamadi, 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
Martins, L.R., Lyra, F.H., Rugani, M.M.H., Takahashi, J.A., 2016. Bioremediation of Metallic Ions by Eight Penicillium Species. J. Environ. Eng. 142, C4015007. https://doi.org/10.1061/(ASCE)EE.1943-7870.0000998
Michalak, I., Chojnacka, K., Witek-Krowiak, A., 2013. State of the Art for the Biosorption Process—a Review. Appl. Biochem. Biotechnol. 170, 1389. https://doi.org/10.1007/s12010-013-0269-0
Mouchot, 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. 12–14.
Nakhavali, 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, 1083–1096.
Nitschke, 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-Forêts. Neues Jahrb. Für Mineral. - Abh. 145–156. https://doi.org/10.1127/0077-7757/2014/0253
Noormohamadi, H.R., Fat’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, 124–130. https://doi.org/10.1016/j.chemosphere.2018.10.113
Pang, 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 Penicillium citrinum. Chem. Eng. J. 170, 1–6. https://doi.org/10.1016/j.cej.2010.10.068
Regenspurg, 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. Earth Sci. 72, 4131–4140. https://doi.org/10.1007/s12665-014-3306-6
Regenspurg, S., Feldbusch, E., Byrne, J., Deon, F., Driba, D.L., Henninges, J., Kappler, A., Naumann, R., Reinsch, T., Schubert, C., 2015. Mineral precipitation during production of geothermal fluid from a Permian Rotliegend reservoir. Geothermics 54, 122–135.
Regenspurg, S., Feldbusch, E., Norden, B., Tichomirowa, M., 2016. Fluid-rock interactions in a geothermal Rotliegend/Permo-Carboniferous reservoir (North German Basin). Appl. Geochem. 69, 12–27. https://doi.org/10.1016/j.apgeochem.2016.03.010
Sağ, Y., 2001. Biosorption of Heavy Metals by Fungal Biomass and Modeling of Fungal Biosorption: A Review. Sep. Purif. Methods 30, 1–48. https://doi.org/10.1081/SPM-100102984
Scheiber, J., Seibt, A., Birner, J., Genter, A., Moeckes, W., 2013. Application of a scaling inhibitor system at the geothermal power plant in Soultz-sous-Forêts: laboratory and on-site studies. Presented at the Proceedings of the European Geothermal Congress, pp. 3–7.
Sen, M., Dastidar, M.G., 2010. Adsorption-desorption studies on Cr (VI) using non-living fungal biomass. Asian J. Chem. 22, 2331.
Şenol, Z.M., Gül, Ü.D., Gurbanov, R., Şimşek, S., 2021. Optimization the removal of lead ions by fungi: Explanation of the mycosorption mechanism. J. Environ. Chem. Eng. 9, 104760. https://doi.org/10.1016/j.jece.2020.104760
TNO-GDN, 2023. Slochteren Formation | DINOloket [WWW Document]. URL https://www.dinoloket.nl/en/stratigraphic-nomenclature/slochteren-formation (accessed 7.23.24).
Tranvik, L.J., 2009. Dystrophy, in: Likens, G.E. (Ed.), Encyclopedia of Inland Waters. Academic Press, Oxford, pp. 405–410. https://doi.org/10.1016/B978-012370626-3.00202-7
Verma, A., Shalu, Singh, A., Bishnoi, N.R., Gupta, A., 2013. Biosorption of Cu (II) using free and immobilized biomass of Penicillium citrinum. Ecol. Eng. 61, 486–490. https://doi.org/10.1016/j.ecoleng.2013.10.008
Wahab, A.A., Awang, A.S. a. H., Azham, Z., Tay, M.G., Adeyemi, F.M., 2017. Biosorption of lead (II) ion using Penicillium citrinum KR706304 isolated from the mangrove soil environment of southeast Borneo. Ife J. Sci. 19, 341–351. https://doi.org/10.4314/ijs.v19i2.14
Weinand, J.M., Vandenberg, G., Risch, S., Behrens, J., Pflugradt, N., Linßen, J., Stolten, D., 2023. Low-carbon lithium extraction makes deep geothermal plants cost-competitive in future energy systems. Adv. Appl. Energy 11, 100148. https://doi.org/10.1016/j.adapen.2023.100148
Wolfgramm, M., Rauppach, K., Seibt, A., 2009. Langfristige Betriebsführung und monitoring geothermischer Anlagen in Deutschland, in: Proceedings of the 2009 Geothermal Congress, Bochum, Germany. p. 12.
Zotzmann, 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

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Assessment of the Pb 2+ biosorption potential of the fungus Penicillium citrinum under geothermal conditions

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Abstract

Figures

1. Introduction

2. Methods

2.1 Site description

2.2 Scaling analysis

2.3 Fungal strain and biomass production

2.3 Assessment of the biomass stability at extreme conditions and the biosorption capacity of the biomass

2.3.1 General preparation

2.3.2 Evaluation of the biomass effect on the fluid

2.3.2 Assessment of the biosorption capacity of the biomass

2.4 Statistical analyses

2. 5 Data availability

3 Results

3.1 Characterization of the scales recovered from the geothermal power plant

3.2 Effect of the biomass on the fluid: DOC and bulk fractions

3.2.2 Impact of temperature and acetic acid on the structure of the biomass itself

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

4 Discussion

4.1 DOC composition and impact of the biomass at elevated temperatures

4.2 Effect of temperature, contact time and the presence or absence of NaCl and acetic acid on biosorption

Conclusion

Abbreviations

Declarations

Competing interests

Ethics approval and consent to participate

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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