Abstract
Traces of heavy metals found in water resources, due to mining activities and e-waste discharge, pose a global threat. Conventional treatment processes fail to remove toxic heavy metals, such as lead, from drinking water in a resource-efficient manner when their initial concentrations are low. Here, we show that by using the yeast Saccharomyces cerevisiae we can effectively remove trace lead from water via a rapid mass transfer process, achieving an uptake of up to 12 mg lead per gram of biomass in solutions with initial lead concentrations below 1 part per million. We found that the yeast cell wall plays a crucial role in this process, with its mannoproteins and β-glucans being the key potential lead adsorbents. Furthermore, we discovered that biosorption is linked to a significant increase in cell wall stiffness. These findings open new opportunities for using environmentally friendly and abundant biomaterials for advanced water treatment targeting emerging contaminants.
One-Sentence Summary Removing toxic heavy metals from water at challenging trace levels in an environmentally friendly, resource-efficient manner.
Heavy metals are highly water-soluble and non-biodegradable, tending to persist indefinitely when released into water bodies. Electronic waste (e-waste) discharge and mining are the most dominant anthropogenic activities responsible for heavy metal contamination of water resources(1). Acid mine drainage (AMD), i.e., leakage of highly acidic water rich in metals, is a global environmental threat(2). In the United States (US) alone, AMD is the main source of water pollution, impacting currently over 20,000 km of streams(3), deriving from the 13,000 active and the 500,000 abandoned mines(4), which continue generating AMD for centuries after their closure(5). In addition, around 50 million tons of e-waste was globally produced in 2018, expected to reach 120 million tons per year by 2050. Over 80% of this e-waste either ends up in landfills or is being recycled under poorly regulated conditions, using primitive and pollutive methods, hence, seriously contaminating water resources(6).
Lead (Pb) is one of the most widely used heavy metals; its production increased by about 20% during the last decade, reaching around 11.7 million tons globally in 2020(7). Pb is highly toxic, even at trace concentrations, with deleterious effects on organs and tissues of the human body(8). It can enter drinking water either due to inadequate water treatment, or due to chemical reactions with Pb-containing components of water distribution systems(9, 10). After numerous incidents of Pb contamination(9, 11), with most recent the water crisis in the city of Flint, Michigan, USA in 2014, limits of Pb in drinking water are becoming more stringent: in 2018, the European Commission proposed reducing Pb limits to 5 parts per billion (ppb)(12), while in 2020, the US Environmental Protection Agency determined that no level of Pb in drinking water is safe(13).
Conventional water treatment methods either fail to completely remove trace Pb amounts or result in significant financial and environmental costs to do so(14–16). Biosorption, a mass transfer process by which heavy metal ions bind onto inactive biological materials by physicochemical interactions, is a competitive alternative to conventional processes, as abundant biomass sources can be effective, practical, and sustainable adsorbents(17). Although biosorption of heavy metals has been studied at the parts per million (ppm) contaminants scale, there is a significant research gap at the challenging trace initial concentrations of ppb and below.
In this study, the unexplored Pb biosorption mechanisms at the ppb scale are investigated using inactive yeast biomass as the biosorbent. A strain of the common yeast, Saccharomyces cerevisiae (S. cerevisiae), was selected, as it is a biodegradable adsorbent, widely used in various industrial settings(15, 18), that effectively remove Pb at ppm initial concentrations(19). The yeast cells were harvested at the peak of their exponential growth phase (Fig. 1A), for optimal biosorptive capacity(19). The harvested cells were washed to remove culture medium residues and metabolites (Fig. 1B), before being lyophilized (freeze-dried) and converted to powder (Fig. 1C). Kinetic and equilibrium experiments were conducted by adding this yeast powder biomaterial to ultrapure water spiked with lead(II) nitrate (Pb(NO3)2). After the required contact time, liquid and solid phases were separated and analyzed to measure residual Pb concentrations and identify potential biomass sites responsible for Pb uptake (Fig. 1D). Overall, this work showcases the use of an effective trace heavy-metal removal biomaterial, made from an environmentally friendly, inexpensive, benign to human health, and easy-to-mass-produce microorganism.
Results
Effect of solution pH on Pb speciation and uptake
Solution pH is a key parameter for biosorption, as it affects the chemistry and speciation of both the metal-uptaking functional groups in the biomass, and of the hydrolyzed Pb ionic forms. Pb speciation in the solution is also affected by the Pb concentration at any given pH and oxidation state(17, 20). To quantify the resulting Pb speciation after the hydrolysis of Pb(NO3)2 in a wide pH range (3-13), at 25 °C, and at a given initial concentration (C0) of 1 μM Pb(NO3)2, the Eawag ChemEQL v3.2 software(21) was used. Pb2+ is the dominant species until pH reaches 5.8, where lead hydroxides (e.g., PbOH+) are beginning to form (Fig. 2A).
Solution pH increases with the addition of biomass and plateaus for biomass values greater than 100 mg (Fig. 2B). Therefore, to assess the effect of solution pH on the biomass Pb2+ uptake capacity (q, in μg of Pb2+ by g of biomass), the initial pH of the aqueous solution (before biomass addition) was adjusted to pH values within the range of 3-7, and then 5 mg of yeast biomass were added to moderate the anticipated pH increase due to biomass addition. Pb2+ concentrations and pH values were measured both before biomass addition and after Pb2+ biosorption (contact time of 24 h). The biomass Pb2+ uptake capacity increased significantly as the initial solution pH was increased from 3 to 5 (Fig. 2C). For pH ≥ 6.0 the measured initial Pb2+ concentration was significantly lower than the known amount added to the solutions, indicating loss of soluble Pb analytes due to precipitation. This is validated by the formation of lead hydroxides after pH 5.8 (Fig. 2A). The increase of solution pH by biomass addition, as well as the increase of q with increasing pH values could be attributed to a potential protonation of the functional groups of yeast biomass at pH values below the pH point zero, i.e., the pH at which the overall biomass surface charge is zero. At pH values below the pH point zero, the biomaterial may exhibit an overall positive charge, thus attracting negatively charged species and not adsorbing Pb2+cations resulting in lower q values, while at higher pH values the biomass surface could acquire negative charges leading to increased Pb2+ uptake. However, such an approach assumes a rather simplistic electrostatic attraction driving mechanism, which has been shown not to be the only case in biosorption(17).
Based on these results, pH 5 was proven to be the most suitable for Pb biosorption, where soluble Pb2+ is the most dominant species in the solution and q is maximized. All experiments described in the following sections were performed by adding 5 mg of yeast biomass (unless otherwise specified) in aqueous solutions, after adjusting their initial pH to 5 at 25 °C, agitated at 200 rpm.
Adsorption kinetics & growth analysis of lyophilized yeast
Kinetic experiments were conducted to determine the change in Pb2+ concentration in the liquid phase as a function of contact time and identify the contact time required to attain equilibrium. Lyophilized yeast cells were added in aqueous solutions with C0 of 100 ppb Pb2+ and were incubated for 24 hours. Samples were taken at specific time intervals (i.e., 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h) and analyzed using inductively coupled plasma mass spectrometry (ICP-MS). As observed, biosorption is a rapid process, with equilibrium being reached within the first five minutes of contact (Fig. 2D). Kinetics data fit accurately with the pseudo-first-order model(22), (R2: 0.99), with the pseudo-first-order rate constant (k1) equal to 111.98 h-1.
In parallel the freeze-dried S. cerevisiae cells were incubated for 24 h under conditions identical to the kinetic experiments and observed by phase-contrast microscopy, while acquiring optical density (OD) measurements, to validate that the cells remain inactive during biosorption. Indeed, after 24 h there was neither cell growth nor cell division observed (Fig. 2D); time-lapse video and further information confirming these results are included in the Supplementary Materials of this work.
Adsorption isotherm
Aqueous solutions with different initial Pb2+ concentrations (C0: 20, 40, 100, 200, 300, 500, 700, and 1,000 ppb) were incubated for 1 h. The equilibrium Pb2+ concentrations (Ceq) were measured after biosorption, and the Pb2+ uptake capacity of yeast biomass at equilibrium (qe) was calculated (using Equitation 1 of Supplementary Materials) to develop the adsorption isotherm (Fig. 2E). The maximum qe measured is equal to 12 mg/g, for aqueous solutions with C0of 1,000 ppb Pb2+. The experimental data are accurately fitting with the Langmuir adsorption isotherm model(23) (R2: 0.98), with the ratio of the adsorption and desorption rates (KL) equal to 1.5 L/mg, and the maximum estimated adsorption capacity (qm) equal to 21 mg/g. However, this fit cannot provide any meaningful insight into the biosorption mechanism(17, 24).
Pb2+ percentage removal versus Pb2+ C0 was also measured (Fig. 2F). Pb2+ removal for C0 20 ppb Pb2+ is approximately 25% and increases with the increase of C0, reaching a maximum of 43% at C0 300 ppb Pb2+. After this point, Pb2+ removal decreases gradually with the decrease of C0, indicating that the optimum uptake capacity of the yeast biomass quantity (5 mg) is reached around C0 300 ppb Pb2+.
Yeast biomass imaging
Extracellular and intracellular imaging of yeast cells was performed to observe potential changes in their structure after biosorption, using SEM and transmission electron microscope (TEM) imaging, respectively. Yeast cells harvested from ultrapure water (C0: 0 ppb Pb2+), served as control cells and were compared with yeast cells harvested from aqueous solutions of C0 100 ppb Pb2+. No morphological change was observed in the yeast cells after Pb2+ biosorption, with the structure and dimensions of the cell wall and cytoplasm remaining the same (Fig. 3A, B). Yeast cell walls were ∼180 nm thick, which is the typical cell-wall thickness of the yeast strain used(25). However, yeast cell walls became more electron-dense after Pb2+ biosorption, indicating the binding of Pb2+ ions on them, and in particular on the outer part of the cell wall (Fig. 3C, D).
Yeast biomass spectroscopy
Metal biosorption is thought to occur through interactions with functional groups native to the biomass cell wall(26). Attenuated-total-reflectance enhanced Fourier transformed infrared spectroscopy (ATR-FTIR) was performed to identify functional groups present in the yeast cell wall and detect changes in them after biosorption, indicating their involvement in Pb2+ adsorption. Freeze-dried control (C0: 0 ppb Pb2+) and Pb2+-exposed yeast cells (C0: 100 and 1,000 ppb Pb2+) were analyzed using ATR-FTIR (Fig. 4A). Changes were observed after biosorption in peaks representing C≡N and C≡C stretches, while peak shifts were detected corresponding to N-H in-plane bending from secondary protein amides, which overlaps with the C–N and NO2 asymmetric stretching, to vibrational changes of the C-N amide group, and to C–O stretching in the esters and carboxylic acid groups. These changes indicate the contribution of amide and carboxylic acid groups to Pb2+ biosorption and the potential role of N in the yeast cell wall on Pb2+ binding.
In addition, the chemical composition of the yeast surface before (C0: 0 ppb Pb2+) and after biosorption (C0: 100 and 1,000 ppb Pb2+) was analyzed by X-ray photoelectron spectroscopy (XPS) to further explore potential changes in the functional groups of the yeast cell walls. The yeast surface is mainly composed of carbon (C), oxygen (O), and nitrogen (N)(27, 28).
Therefore, C 1s, O 1s, N 1s core levels spectra were recorded together with the Pb 4f. Significant changes among the control and the Pb2+-exposed yeast were only observed for the C 1s spectrum, while changes in the Pb spectra could not be detected as the analyzed trace Pb2+ concentrations were below the detection limit of the instrument. Deconvolution of the C 1s spectrum into Gaussian-shaped lines was performed to identify possible chemical bonds between C, O, and N (Fig. 4B). In all three samples, the C 1s peaks are decomposed to peaks at 284.3 eV, 285.9 eV, 287.5 eV, and 288.6 eV representing C-C (sp3 C), C-N, O-C=O, and C=O respectively. The magnitude and shape of all observed bonds have radically changed after Pb2+ biosorption, particularly for C-C, C-N, and O-C=O bonds. These changes indicate the contribution of carboxylic acid and amide groups on Pb2+ adsorption, which is consistent with the ATR-FTIR results.
Chitin’s contribution to Pb2+ adsorption
The above analyses indicate that the cell wall of S. cerevisiae plays a vital role in Pb2+ biosorption. The yeast cell wall has a complex macromolecular structure with a layered organization, including an amorphous inner and a fibrillar outer layer(29). The inner layer mainly consists of β-glucans and chitin. The outer layer consists predominantly of mannan polymers, highly glycosylated and linked to proteins (mannoproteins)(30).
Several sources suggest that the chitin amine nitrogen is responsible for heavy metals sequestering at the ppm scale(17, 31, 32). To further investigate this, we assessed the Pb2+ uptake capacity of chitin from shrimp shells, which is similar to that found in the yeast cell wall. We used 20 times more chitin, i.e., 1.8 mg, than the maximum equivalent amount present in the 5 mg of yeast, considering a 30% dry weight of yeast cell wall and a 6% contribution of chitin per mass to it(30). We added this amount to an aqueous solution of 0.2 L with C0 1,000 ppb Pb2+ for 24 h at 200 rpm and 25°C. We ran the same experiments with 5 mg of yeast biomass and with 5 mg of chitin. It was shown that chitin’s Pb2+ uptake is negligible, as C0 was reduced by less than 0.3% when we added the 1.8 mg of chitin, which is within the measurement error. Even when the chitin amount added was equal to the total yeast mass (5 mg), C0 was only reduced by 3%, compared to the ∼30% reduction achieved by the yeast biomass (Fig. 5A). Hence, it can be concluded that chitin alone is not contributing to the Pb2+ biosorption process.
Yeast biomass nanomechanical characterization
We employed nanomechanical characterization to investigate biosorption. We assessed the stiffness of the yeast cells before and after Pb2+ exposure. Single-cell mechanical testing by atomic force microscopy (AFM) showed a significant increase in the stiffness of samples following Pb2+ uptake (Fig. 5B). However, when yeast biomass is treated with solutions containing higher Pb2+ levels, mechanical stiffness is not noticeably increased (C0 500 vs 1,000 ppb Pb2+). This cannot be attributed to a potential saturation of the yeast cell wall binding sites, as Pb2+uptake increases significantly with the increase of C0 from 500 to 1,000 ppb Pb2+ (Fig. 2E). While the exact mechanism for this stiffness change is yet to be determined, it is possible that adsorption of even a thin layer of Pb2+, as evidenced by the TEM data (Fig. 3D), can act as a film on the cell surface that fuses the fibrillar structures together, which then effectively resists deformation more than the untreated cell wall.
Discussion
This work reports the biosorption isotherm and kinetics of initial Pb2+ concentration at the ppb scale, using lyophilized S. cerevisiae yeast cells as biosorbents. Comparing our results with prior studies of similar systems at the ppm scale it can be concluded that the biosorption processes at the ppb scale happen faster; the fastest equilibrium attainment reported at the ppm scale is 10 min(18), while our results show that equilibrium is achieved in the first 5 minutes of contact. The adsorption isotherm reported in our study (Fig. 2E) follows the same pattern as the adsorption isotherms reported in the ppm literature (e.g., fig. S1). Interestingly, the maximum Pb2+ uptake capacity of 12 mg/g reported in this study is in the same range as the uptake capacities reported in the ppm literature for untreated inactive S. cerevisiae yeast, i.e., 2-30 mg/g (e.g., table S3), proving the suitability of this biomaterial as a biosorbent at the ppb scale. pH is a significant factor in the biosorption process in both scales. The rapid biosorption and high Pb2+ uptake are advantageous for the large-scale application of this inexpensive and abundant biomaterial suitable for the removal of trace heavy metals from water.
From the performed analyses, it can be concluded that the cell wall of S. cerevisiae contributes significantly to Pb2+ biosorption, and in particular its carboxylic acid and amide groups. By excluding chitin as a biosorbent, mannoproteins and β-glucans are the potential key S. cerevisiae cell wall components, which should be further analyzed to elucidate the biosorption mechanisms involved. The combined outcomes of the TEM, the spectroscopic analyses, and the cellular nanomechanical characterization, validate the likelihood of N-linked σ-hole attraction to Pb2+ species as a possible mechanism of biosorptive Pb retention by the mannoprotein/β-glucan cell wall fraction(33, 34), leading to supramolecular assemblies that make yeast cells stiffer after biosorption. These findings open new experimental pathways for approaching the challenging task of biosorption investigation at the ppb scale.
The results showed herein, together with the fact that 3 million tons of yeast are used annually by the global fermentation industry(35) and that the yeast market is expected to grow by 35% in the next 5 years(36), indicate that exploiting this biosorbent is practically feasible and economically attractive. In addition, due to its simplicity, this approach can be easily reproduced, locally sourced, and applied at scale. The approach described here compares favorably to many of the highly sophisticated synthetic biology and advanced nanomaterials approaches that have also been examined as candidates for heavy metal removal from water(37, 38). Applying such a low-value resource to remove trace contaminants from water could lead to significant environmental benefits for the water and wastewater treatment utilities, including their decarbonization due to limited energy use, and waste reduction as yeast cells are biodegradable. Moreover, potential desorption processes would allow for heavy metals reclamation, enhancing the application of circular economic models.
Funding
Bodossaki Foundation, Stamatis G. Mantzavinos’s Memorial Postdoctoral Scholarship (P.M.S.). Standard Banking Group sponsorship to the MIT CBA (A.M.). This work was also supported by MIT CBA Consortia funding.
Author contributions
Conceptualization: P.M.S., C.E.A., M.T., H.G., N.G.
Methodology: P.M.S., C.E.A., M.T., J.G., E.M.D.
Investigation: P.M.S., C.E.A. J.G., C.B.
Visualization: P.M.S., C.E.A.
Funding acquisition: P.M.S., C.E.A., F.T., A.M., N.P.P., B.W.S., N.G.
Project administration: P.M.S., C.E.A.
Supervision: C.E.A., M.T., H.G., N.G.
Writing – original draft: P.M.S., C.E.A.
Writing – review & editing: All authors
Competing interests
Authors declare that they have no competing interests.
Data and materials availability
All data are available in the main text or the supplementary materials. Additional data supporting the results presented herein can be provided upon request.
Acknowledgments
We thank the MIT Koch Institute’s Robert A. Swanson (1969) Biotechnology Center for technical support, and specifically the Nanotechnology Materials Lab for assisting in TEM sample preparation and imaging. We acknowledge the MIT Materials Research Science and Engineering Center (MRSEC) for assisting with TEM imaging and ATR-FTIR analyses. We also thank the MIT Center for Environmental Health Sciences (CEHS) and in particular Dr. Bogdan Fedeles for the helpful discussions and troubleshooting concerning the ICP-MS analyses of our samples. We are grateful to Dr. Constantinos Katsimpouras from the MIT Metabolic Engineering Laboratory for assisting with the HPLC analyses. We also thank Lorena Altamirano for her assistance with biological methods and protocols.