1 Fungal peptidomelanin: a novel metabolite for the

2 amelioration of soil heavy metal toxicity

3 Rakshita Sukruth Kolipakala1 , Suranjana Basu1,+ , Senjuti Sarkar1,+ , Beneta Merin Biju1,+ ,
4 Daniela Salazar2 , Likhit Reddy1 , Harshitha Balaji1 , Shrijita Nath1 , Anish Hemanth
5 Samprathi1,3 , Aparna Shetye4 , and Deepesh Nagarajan1,4,*

6
1 Department of Biotechnology, M.S. Ramaiah University of Applied Sciences, Bangalore - 560054, India
7
2 Ecology and Genetics Research Unit, University of Oulu, Oulu - 90014, Finland.
8
3 Department of Biotechnology, Fergusson College (Autonomous), Pune - 411004, India

9
4 Department of Microbiology, St. Xavier’s College, Mumbai - 400001, India

10
* [email protected], [email protected], [email protected]

11
+ these authors contributed equally to this work

12 ABSTRACT

Heavy metal contamination of agricultural soils reduces crop yields, contaminates groundwater and disrupts local ecosystems.
Here, we describe a novel, water-soluble form of melanin (peptidomelanin) capable of chelating heavy metals in large quantities.
Peptidomelanin is composed of an L-DOPA core polymer that is solubilized via short, heterogeneous peptide chains with a
13 mean amino acid length of ∼2.6. It is secreted by the spores of Aspergillus niger melanoliber during germination. It was found
to chelate large quantities of lead, mercury, and uranyl. It increased the germination rate, seed mass, and shoot length of wheat
planted in substrate contaminated with 100 ppm mercury. Therefore, peptidomelanin may increase crop yields in contaminated
agricultural soils treated in situ with the substance.

14 Introduction
15 The contamination of agricultural soils with heavy metals represents a major impediment to food production worldwide1 .
16 The presence of heavy metals within the soil, beyond acceptable limits, can detrimentally affect germination, photosynthetic
17 efficiency and crop yields, while also contaminating groundwater and disrupting local ecosystems2 . Additionally, heavy metals
18 can contaminate groundwater and disrupt local ecosystems3 . Globally, 5 million sites comprising 20 million hectares of land
19 are contaminated with heavy metals and metalloids4 . The rates of heavy metal contamination vary between countries and
20 regions, ranging from 3% in Poland5, 6 to 19% in China7 . Accelerated mining, urbanization, power generation, industrialization
21 and vehicular activity are the predominant anthropogenic causes of heavy metal contamination. Heavy metals enter agricultural
22 soils from these sources through leaching from mine tailings8 , atmospheric deposition9 , application of phosphate fertilizers10 ,
23 untreated industrial effluents11 , and untreated sewage sludge12 . The United States Agency for Toxic Substances and Disease
24 Registry (ATSDR) lists upwards of 20 toxic heavy metals1 , of which four are listed as being particularly detrimental to human
25 health: arsenic, lead, cadmium and mercury.
26

27 Unlike other chemical contaminants, heavy metals cannot be degraded and will remain in agricultural soils indefinitely
28 unless removed. Conventional soil remediation strategies greatly differ and are typically tailored to suit specific local con-
29 ditions4 . Such strategies vary in cost with an upper bound of USD 100/m24 . Of particualr interest to this study are in situ
30 immobilization techniques via chemical fixation. Immobilization of heavy metals involves their in situ conversion into insoluble
31 or poorly soluble forms, thereby decreasing their mobility in water and their uptake by roots. EDTA13 , NTA14 , CaCO3 , iron
32 grit, conocarpus biochar, and phosphate rock15 have all been characterized as heavy metal immobilizers.
33

34 Interestingly, melanin purified from A. resinae was capable of immobilizing heavy metals such as copper, zinc, lead,
35 cadmium in metal-contaminated effluents16 . Melanin is an ancient pigment present in all kingdoms of life. It is a large,
36 heterogenous, amorphous polymer of phenolic and indole components17 . Its extreme heterogeneity and variability has made the
37 structural and chemical characterization of the substance difficult. Fungi are a desirable source of melanin due to their greater
38 availability, higher melanin yields, and easier purification protocols18 .
39

40 The vast majority of reports pertaining to melanin describe an insoluble polymer present as intracellular granules19 . Only a

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 41 handful of reports describing soluble forms of melanin can be found in the literature. A. fumigatus, Madurella mycetomatis,
42 Yarrowia lipolytica20 , and Bacillus thuringiensis21 , for example, secrete soluble melanin. Aspergillus nidulans22 , Streptomyces
43 cavourensis SV 2123 and Streptomyces lusitanus DMZ-324 produce both insoluble and soluble of melanin. Soluble melanin
44 extracted from Inonotus hispidus25 , squid ink26 , and vinary waste27 possess excellent antioxidant activity while extracts from
45 Inonotus obliquus28 possess in vitro immunomodulatory activity. The production of synthetic soluble melanin possessing a
46 larger number of carbonyl groups has also been reported29 .
47

48 In this study we have discovered and biochemically characterized a novel form of soluble melanin that we term as peptidome-
49 lanin. Peptidomelanin was liberated into the surrounding medium during the germination of Aspergillus niger melanoliber
50 spores. It was found be an amorphous, heterogenous polymer containing an insoluble L-DOPA core. It was solubilized via
51 short heterogenous peptides with a mean residue length of 2.63 amino acids anchored to the core polymer via peptide bonds.
52 It was found to be a potent heavy metal chelating agent, possessing the ability to chelate large quantities lead, mercury, and
53 uranyl. It was found to ameliorate the toxic effects of 100 ppm mercury on the germination and growth of wheat. The ease of
54 preparation of peptidomelanin and the potential to produce it in situ lead us to believe that it may improve crop yields in soils
55 contaminated with heavy metals.
56

57 Results
58 Here we describe the discovery and extraction of peptidomelanin, its biochemical composition, and its ability to chelate metals
59 thereby ameliorating heavy metal toxicity for germinating seeds.
60

Figure 1. Morphology of Aspergillus niger strains used in this study. (A-C) The A. niger type strain (MTCC 281) (A) dense hyphal mass
possessing abundant conidiophores. (B) Conodiophore possessing relatively fewer conidiospores (C) Large conidiospores with echinulations
evenly distributed across their surface. Polar regions are not discernible. (D-F) The A. niger melanoliber (MTCC 13366). (D) Sparse hyphal
mass possessing fewer large conidiophores. (E) Conodiophore possessing a large number of small conodospores. (F) Small conidospores
possessing deep, longitudinal striations originating from discernible polar regions.

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 61 We discovered secreted peptidomelanin in a strain of Aspergillus niger isolated from a soil sample in Mumbai, India. We
62 named the strain Aspergillus niger melanoliber and deposited it in the Microbial Type Culture Collection (MTCC), Chandigarh
63 (strain ID: MTCC 13366). Species identification was performed through ITS sequencing. The ITS sequence was deposited into
64 GenBank (accession ID: PP077302). A. niger melanoliber differs in morphology from the type strain of A. niger (MTCC 281 /
65 ATCC 9029). The type strain possesses a dense hyphal, abundant conidiophores, and large conidiospores (or simply, spores)
66 with non-discernible polar regions and evenly distributed echinulations (Figure 1A-C). A. niger melanoliber possesses a sparse
67 hyphal mass, fewer large conidiophores, and small conodiospores with deep longitudinal striations originating from the poles
68 (Figure 1D-F).

Figure 2. Peptidomelanin release assay for Aspergillus spps. spores. (A) A. niger melanoliber (MTCC 13366) and A. niger (MTCC 281)
spores were inoculated into Sabouraud broth. A. niger melanoliber rapidly released soluble peptidomelanin into the medium. In contrast, A.
niger released no soluble peptidomelanin into the medium. (B) Peptidomelanin retention assay for A. niger melanoliber spores in Sabouraud
broth. At predetermined timepoints, samples were isolated and fixed using 8% glutaraldehyde to halt peptidomelanin release. Samples were
treated with 10% NaOH to release retained peptidomelanin. Retained peptidomelanin decreased with time, indicating that new
peptidomelanin is not synthesized during germination, and only existing peptidomelanin is released. (C) Melanin release assay for
UV-irradiated A. niger melanoliber spores. Irradiated spores did not release peptidomelanin into the Sabouraud’s broth, demonstrating that
only live spores release melanin. (Inset) Petri plate confirming UV irradiated spores do not germinate. All experiments were performed in
quadruplicate. Lines represent means. The shaded area represents standard deviation.

Figure 3. Spectral analysis of peptidomelanin. (A) Peptidomelanin appears dark brown in color. Peptidomelanin can be concentrated
using a 30 kD centrifugal concentrator. Absorbance at 420 nm revealed that ≥99% of peptidomelanin was retained within the centrifugal
concentrator, indicating that the substance is a macromolecule. (B) UV-visible spectra of melanin. Peptidomelanin displays an absorbance
spectrum characteristic of melanin. It displays the greatest absorbance in the UV region but absorbs light across the entire spectrum tested.
Synthetic L-DOPA melanin displays a similar absorbance spectrum. Acid-hydrolyzed melanin (lacking a peptide component) also displays a
similar curve. (C) FTIR spectra of peptidomelanin and synthetic L-DOPA melanin. Both spectra have been vertically offset to ease
comparison. Both spectra appear similar. Both display a broad dip between 3500 and 3100 cm−1 assignable to the O–H or N–H stretch
vibration, and a sharp dip between 1800 to 1600 cm−1 assignable to C-C or C=O stretch vibration.

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 69 While germinating, A. niger melanoliber spores release peptidomelanin rapidly into Sabouraud broth, resulting in a ∼5-fold
70 increase in absorbance (at 420 nm) in 1 hour (Figure 2A). In contrast, spores of the type culture of A. niger (MTCC 281 / ATCC
71 9029) do not release any form of melanin into Sabouraud broth while germinating (Figure 2A). The melanin content within A.
72 niger melanoliber spores decreases during germination (Figure 2B), indicating that peptidomelanin is not synthesized during
73 germination; only existing peptidomelanin is released. Spores killed via UV-irradiation do not secrete peptidomelanin (Figure
74 2C). Therefore, peptidomelanin release is a biochemical process that requires a live organism to perform. Morphological
75 changes associated with germination such as the disruption of the spore pellicle and hyphal growth occur at timepoints long
76 after peptidomelanin release (Figure S1).
77

Figure 4. PAGE gels indicate the biochemical composition of peptidomelanin. (A) Native PAGE of peptidomelanin. A brown smear from
∼20 to ≥250 kD is observed. Coomassie blue staining darkens the smear, indicating the presence of an amino acid component. (B) SDS
PAGE and (C) 8M Urea PAGE of peptidomelanin. Coomassie blue staining reveals that the amino acid component does not separate from the
melanin component, indicating a covalent linkage between the two components (ladder not resolved). The observation of smears rather than
discrete bands on gels A-C confirms that peptidomelanin is an amorphous polymer. (D) Native PAGE of resolubilized acid-hydrolyzed
melanin displays a smear that runs slower on a gel. The smear was only marginally stained by Coomassie blue. Both these observations
indicate the loss of the amino acid component upon acid-hydrolysis. (E) Native PAGE of synthetic L-DOPA melanin. The smear does not
uptake Coomassie blue as it lacks an amino acid component.

78 Peptidomelanin released into Sabouraud broth was precipitated at pH 2 (HCl-KCl buffer) and resuspended at pH 8.8 (Tris
79 buffer) for further experiments. The buffer system can be exchanged via dialysis through a 3.5 kD membrane. Peptidomelanin
80 concentrated using a 30 kD centrifugal concentrator displayed a dark brown color, characteristic of melanin polymers (Figure

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 81 3A). Peptidomelanin displayed a UV-vis spectrum characteristic of L-DOPA melanin, absorbing across the entire UV-visible
82 spectrum (200-1000 nm) but displaying a maxima in the UV region at ≤ 200 nm (Figure 3B). Peptidomelanin also possesses
83 a broad local spectral maxima at 420-460 nm which was used for the colorimetric estimation of melanin content (Figure 2).
84 Acid-hydrolyzed melanin (lacking an amino acid component) displays a similar spectrum, indicating that the melanin core
85 polymer is responsible for the absorbance spectrum. Synthetic L-DOPA melanin also displays a similar spectrum, indicating
86 that the melanin core polymer is composed of L-DOPA. An FTIR spectral comparison of peptidomelanin with synthetic
87 L-DOPA melanin revealed that both substances display similar spectra (Figure 3C). Both spectra contain a broad dip between
88 3500 and 3100 cm−1 assignable to the O–H or N–H stretch vibration, and a sharp dip between 1800 to 1600 cm−1 assignable
89 to C-C or C=O stretch vibration. Such spectra are characteristic of polymers belonging to the L-DOPA melanin family.
90 Allomelanin extracted from the black knot fungus30 , melanin extracted from Pseudomonas sp. WH0015531 , and melanin
91 extracted from Bacillus cereus (Bc58)32 display similar spectra. Melanin synthesis inhibition experiments using tropolone lead
92 to the formation of albino spores lacking any pigmentation. Tropolone inhibits melanin synthesis via the DOPA pathway33
93 (Figure S2), confirming the L-DOPA composition of the peptidomelanin core polymer.
94

95 Preliminary experiments using PAGE and agarose gels provided insights into the biochemical composition of peptidome-
96 lanin. Peptidomelanin runs as a smear, rather than a discrete band, on a native PAGE, SDS PAGE, and urea PAGE gels (Figure
97 4A-C). This indicates that peptidomelanin is an amorphous polymer with molecular weights ranging from ∼20 to ≥250 kD.
98 Synthetic L-DOPA melanin, a known amorphous polymer, also runs as a smear on a native PAGE gel (Figure 4E). The native
99 PAGE peptidomelanin smear is stained by Coomassie blue 4A), indicating that it possesses an amino acid component that
100 we later determined to be composed of short peptides rather than long proteins. Peptidomelanin smears on SDS and Urea
101 PAGE gels 4B,C) are also stained by Coomassie blue, indicating that they retain their amino acid component even under strong
102 denaturing conditions. This indicates that the amino acid component is covalently bound to peptidomelanin. Peptidomelanin
103 was acid-hydrolyzed using 6N HCl under a N2 atmosphere. The melanin hydrolysate was insoluble and had to be resolubilized
104 in water by dissolving in DMSO and dialysing against deionized water. The resulting substance runs slower on a native PAGE
105 gel but still forms a smear. The smear does not readily uptake Coomassie blue, indicating that acid-hydrolysis removed the
106 amino acid component (Figure 4D). This substance is therefore referred to simply as acid-hydrolyzed melanin. The synthetic
107 L-DOPA melanin smear run on a native PAGE, known to lack a peptide component, also does not readily uptake Coomassie
108 blue (Figure 4E).
109

110 Peptidomelanin does not possesses chitin, as evidenced the inability of the native PAGE peptidomelanin smear to uptake
111 Calcofluor White (Figure S3A). Peptidomelanin does not possesses nucleic acids, as evidences by the inability of an agarose gel
112 peptidomelanin smear to uptake ethidium bromide (Figure S3B). The elemental composition of peptidomelanin was obtained
113 using SEM-EDS (Table S1, Dataset S1). The absence of phosphorous confirms the absence of nucleic acids. Peptidomelanin
114 was found to contain K, Na, and Ca, indicating it possesses some ability to chelate these metals.
115

116 The amino acid component of peptidomelanin was confirmed via LC-MS of the acid-hydrolysate. Peptidomelanin was
117 acid-hydrolyzed using 6N HCl under a N2 atmosphere. The resulting insoluble acid-hydrolyzed melanin was centrifuged
118 to obtain an insoluble pellet. The solvent was evaporated to remove HCl and the resulting residue was resuspended in
119 deionized water. LC-MS was performed on this solution to obtain its amino acid composition (Table 1). It is worth noting
120 that the amino acid component is predominantly composed of glycine (∼33%) and negatively charged residues (aspartate
121 and glutamate, ∼21%). The percentage amino acid composition of peptidomelanin was calculated by simply dividing this
122 mass by the total mass of amino acids with the total mass of peptidomelanin prior to acid-hydrolysis, and was found to be
123 22.98 ±1.84 %. LC-MS reports and analyses are provided in the supplementary information (Dataset S2, Text S1, and Table S2).
124

125 Despite possessing amino acids, papain-hydrolyzed peptidomelanin did not display peptide fragments in the molecular
126 weight range of 400 to 2000 Dalton (Figure S4). This indicates that the amino acids in peptidomelanin are polymerized as
127 short peptides too small to be cleaved by papain, rather than as long proteins. We used dansyl chloride in order to determine
128 the mean amino acid length of the peptides comprising peptidomelanin. Dansyl chloride is a sulfur-containing fluorophore
129 that can be covalently linked to the N-terminal of amino acids and peptides. We successfully dansylated peptidomelanin and
130 acid-hydrolyzed melanin (Figure 5A,B). Dansylated peptidomelanin remained soluble whereas acid-hydrolyzed melanin did
131 not. We subjected four substances: peptidomelanin, dansylated peptidomelanin, acid-hydrolyzed melanin, and dansylated
132 acid-hydrolyzed melanin, to SEM-EDS in order to quantify their sulfur content (% weight). The chemical nature of these
133 substances is depicted in Figure 6A. The sulfur originating from the dansyl group alone (∆S1 ) can be quantified using SEM-EDS
134 by normalizing and subtracting the sulfur content (% weight) of dansylated peptidomelanin from the sulfur content (% weight)
135 of peptidomelanin (Figure 6B). We found ∆S1 to be 2.12% (% weight). The dansyl content (% molar) could then be calculated

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 136 from ∆S1 (where % molar = % weight of substance / MW). As dansyl groups covalently link to N-terminals in a 1:1 molar
137 stoichiometric ratio, the dansyl content (% molar) corresponds to the N-terminal content (% molar). The amino acid content (%
138 molar) could be calculated from LC-MS data (Table 1). The mean number of amino acids per peptide chain was then simply the
139 amino acid content (% molar) divided by the N-terminal content (% molar), or the number of amino acids for every N-terminal.
140 This value was found to be 2.63 amino acids. Figure 6C illustrates the experiments and calculations performed. All calculations
141 are provided in Text S2. All raw SEM-EDS data is provided in Dataset S3.
142

Amino acid Absolute mass % Relative mass %

(mean ±SD) (mean ±SD)
Glycine 7.73 ±0.82 33.62 ±0.92
Glutamate 2.49 ±0.05 10.85 ±0.61
Proline 2.45 ±0.09 10.69 ±0.46
Alanine 2.39 ±0.12 10.42 ±0.27
Aspartate 2.26 ±0.19 9.83 ±0.06
Arginine 1.42 ±0.10 6.19 ±0.05
Leucine 0.90 ±0.02 3.93 ±0.21
Lysine 0.78 ±0.07 3.39 ±0.03
Serine 0.61 ±0.01 2.67 ±0.16
Phenylalanine 0.51 ±0.03 2.23 ±0.04
Threonine 0.47 ±0.18 2.03 ±0.62
Histidine 0.25 ±0.11 1.08 ±0.40
Tyrosine 0.21 ±0.01 0.93 ±0.03
Valine 0.18 ±0.01 0.80 ±0.01
Methionine 0.16 ±0.01 0.69 ±0.03
Isoleucine 0.13 ±0.02 0.55 ±0.15
Cysteine 0.01 ±0.00 0.06 ±0.01
Tryptophan 0.01 ±0.00 0.04 ±0.00
Asparagine 0.00 ±0.00 0.01 ±0.01
Glutamine 0.00 ±0.00 0.00 ±0.00
Total 22.98 ±1.84 100

Table 1. Amino acid composition of peptidomelanin acquired via

LC-MS (DNA Labs India, Hyderabad). 19 of 20 canonical amino
acids were detected in the peptidomelanin acid-hydrolysate.
Absolute mass % refers to the mass of each amino acid as a
percentage of the total mass of peptidomelanin. The sum total of the
absolute mass % of all amino acids is 22.98 ±1.84 %, which is the
total % amino acid composition of peptidomelanin. Relative mass %
refers to the mass of each amino acid as a percentage of the total
mass of all amino acids present.

Figure 5. Dansylation of peptidolelanin and its derivatives.

Dansyl chloride is a sulfur-containing fluorophore that can be
covalently linked to the N-terminal of amino acids and peptides. (A)
SDS PAGE of peptidomelanin and dansylated peptidomelanin. Both
substances are stained by Coomassie blue. However, only
dansylated peptidomelanin displays fluorescence under UV
transillumination. (B) SDS PAGE of resolubilized acid-hydrolyzed
melanin and dansylated acid-hydrolyzed melanin. Both substances
are poorly stained by Coomassie blue as they lack their amino acid
component. Only dansylated acid-hydrolyzed melanin displays
fluorescence under UV transillumination. Dansylated
acid-hydrolyzed peptidomelanin could not be resolubilized and
therefore does not migrate on an SDS PAGE gel.

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 Figure 6. Dansylation experiments using peptidomelanin and its derivatives. (A) Schematic showing all the substances under study.
Dansyl chloride is a sulfur-containing fluorophore that can be covalently linked to the N-terminal of amino acids and peptides.
Peptidomelanin will possess an amino acid component, whereas acid-hydrolyzed melanin will not. (B) SEM-EDS was used to estimate the
sulfur (weight %) content of all four substances depicted in panel A. Sulfur acts as a proxy for the dansyl group. The increase in sulfur (∆S1 =
2.12%) between peptidomelanin and dansylated peptidomelanin represents sulfur originating only from dansyl linked to N-terminals.
Likewise, the increase in sulfur (∆S2 = 2.75%) between acid-hydrolyzed melanin and dansylated acid-hydrolyzed melanin represents sulfur
originating only from dansyl linked to N-terminals. ∆S1 is not significantly different from ∆S2 (p = 0.98), indicating that peptides are linked
to the melanin core polymer via a peptide bond (see text). Statistical significance testing was performed using the Welch 2-sample t-test.
Error bars represent the standard deviation from the mean. (C) The mean peptide chain length can be calculated using data from dansylation
experiments (panel B) as well as LC-MS amino acid quantification experiments (Table 1). Placeholder data is used here to explain the
rationale. The actual mean peptide chain length was calculated to be 2.63 amino acids. All calculations are provided in Text S2.

143 Each short peptide chain possesses an individual sequence. This can be inferred simply by observing that the number
144 of canonical amino acids detected in peptidomelanin (19 of 20, Table 1) is larger than the mean number of amino acids per
145 peptide chain (2.63). Therefore all the amino acids observed cannot possibly be contained within a single chain. The dansylated
146 amino acid mixture from acid-hydrolyzed dansylated peptidomelanin was subjected to electrophoresis in order to resolve the
147 dansylated N-terminal amino acid (Figure S5). We observed a broad smear, indicating the presence of multiple N-terminal
148 amino acids which further confirms that every peptide chain possesses an individual sequence.
149

150 The difference in sulfur content originating only from the dansyl group for dansylated peptidomelanin (∆S1 = 2.12%) and
151 dansylated acid-hydrolyzed melanin (∆S2 = 2.75%) is not statistically significant (p = 0.98, Figure 6B, Text S2). Therefore,
152 the total number of dansylatable N-terminals (% molar) remains very similar in both peptidomelanin and acid-hydrolyzed
153 melanin despite the latter lacking a peptide component. From this we infer that the the loss of the peptide N-terminus during
154 acid-hydrolysis is compensated for by the appearance of an N-terminus on the surface of acid-hydrolyzed melanin in a ∼1:1
155 ratio. This indicates that peptides are linked to the melanin core polymer via a peptide bond, which will appear as an N-terminus
156 after hydrolysis.
157

158 We assayed the ability of peptidomelanin to chelate metals. The ability of other forms of melanin to chelate heavy
159 metals16, 34, 35 and the presence of amino acids on peptidomelanin capable of forming complexes with metals lead us to believe
160 peptidomelanin would possesses similar abilities. We performed experiments to determine the metal:substance stoichiometric
161 ratios (w%:100%) for 4 substances against 9 metals. The substances chosen were (1) peptidomelanin; (2) synthetic L-DOPA
162 melanin, serving as a positive control; (3) activated charcoal, serving as standard metal chelating agent36–39 to benchmark
163 peptidomelanin against and (4) silica, expected to act as an inert substrate. The metals chosen ranged from light to heavy, and
164 were: sodium, chromium, nickel, copper, zinc, cadmium, mercury, lead, and uranyl (UO2 ) (Figure 7). A solution containing
165 excess metal salt was introduced to a solution containing any one of these four substances and allowed to react. Excess metal
166 salt was then removed via dialysis or pelleting, depending on the solubility of the metal complex (Figure 7A). The elemental
167 compositions of all metal complexes were assayed using SEM-EDS.
168

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 Figure 7. Metal-binding stoichiometric assays used to determine which substance tested has the greatest capacity to chelate metal ions.
Four substances were tested: silica, activated charcoal, synthetic L-DOPA melanin, and peptidomelanin. (A) A visual depiction of our
protocol. An excess of metal salt is added to all substances tested. The unbound metal salt was then removed via dialysis or centrifuging and
washing, depending on the solubility of the metal complex. SEM-EDS was used to estimate the percentage metal in all complexes.
Placeholder data is used to describe how the stoichiometric ratio (wt%:100%) and stoichiometric equivalent ratio are calculated. (B) The
mean metal-binding stoichiometric ratio of all substances tested across 9 metals, ranging from sodium to uranyl. Peptidomelanin can chelate
significantly larger ratios of metal compared to silica and activated charcoal. Synthetic melanin does not display a significant difference in
metal chelation compared to peptidomelanin. Here, a molar stoichiometric ratio is used to normalize our data across metals with different
molecular weights. (C) The ratios of metal chelated by peptidomelanin, synthetic melanin, and activated charcoal is positively correlated with
the molecular weight of the metal. Silica displays no strong correlation. Regression lines for all substances tested are shown. (D-L)
Metal-binding stoichiometric assays used to determine which substance tested has the greatest capacity to chelate a given metal ion. The
means obtained from these experiments were used as data for the summary statistics in panels B and C. The individual metal ions tested were:
(D) sodium, from NaCl, (E) chromium, from CrCl3 .6H2 O, (F) nickel, from Ni(NO3 )2 , (G) copper, from CuSO4 .5H2 O, (H) zinc, from
ZnSO4 , (I) cadmium, from Cd(NO3 )2 , (J) mercury, from HgCl2 , (K) lead, from Pb(NO3 )2 , and (L) uranyl, from UO2 (CH3 COO− ).2H2 O.
For panels B to L, statistical significance testing was performed using the Welch 2-sample t-test for all panels. All substances were compared
to peptidomelanin. P-values ≤0.05 are shown. Error bars represent the standard deviation from the mean. Raw data is provided in Dataset S4.

169 Peptidomelanin was observed to chelate significantly larger ratios of metals compared to silica and activated charcoal
170 (Figure 7B). Synthetic melanin did not display a significant difference in metal chelation ratios compared to peptidomelanin.
171 Silica did not chelate heavy metals. However, it is interesting to note that both peptidomelanin and synthetic melanin were
172 able to chelate larger ratios of metals compared to activated charcoal, which is extensively used in agricultural settings and
173 was expected to chelate heavy metals36–39 . The ratios of metal chelated by peptidomelanin, synthetic melanin, and activated
174 charcoal is positively correlated with the molecular weight of the metal (Figure 7C). For example, peptidomelanin chelates a
175 smaller ratio of MW-adjusted sodium (0.05 wt%/MW:100%, MW = 23 Da) compared to uranyl (0.18 wt%/MW:100%, MW
176 = 270 Da). Silica displays no strong correlation. This phenomenon may simply be explained by the fact that heavier metals
177 possesses higher coordination numbers40 and would therefore be more easily chelated than lighter metals.
178

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 Figure 8. Wheat germination dose-response experiments show that peptidomelanin can ameliorate soil mercury toxicity in agricultural
contexts. (A) Wheat seed mass shows a strong inverse correlation (log-linear) with mercury concentration (r = -0.85) across a range of 0 ppm
to 10,000 ppm mercury. (B) When germinated in substrate containing 100 ppm mercury, wheat seed mass shows a strong positive correlation
(log-linear) with increasing concentrations of peptidomelanin (r = 0.80). Wheat seeds germinated in substrate containing 100 ppm mercury
completely neutralized by peptidomelanin (at 1:1 and 1:10 stoichiometric equivalent ratios) display significantly larger seed masses compared
to wheat seeds germinated in 100 ppm mercury alone (1:0). (C) When germinated in substrate containing 100 ppm mercury, wheat shoot
length shows a strong positive correlation (log-log) with increasing concentrations of peptidomelanin (r = 0.63). (D) Wheat seeds grown in
substrate containing 100 ppm mercury completely neutralized by peptidomelanin (at 1:1 and 1:10 stoichiometric equivalent ratios) display
significantly longer shoot lengths compared to wheat seeds germinated in 100 ppm mercury alone (1:0). (E) ICP-OES quantification of the
mercury concentration of wheat seed homogenates. When germinated in substrate containing 100 ppm mercury, plant tissue mercury
concentrations show a strong inverse correlation (log-log) with increasing concentrations of peptidomelanin (r = 0.98). Wheat seeds
germinated in substrate containing 100 ppm mercury either partially or completely neutralized by peptidomelanin (at 1:0.1, 1:1, and 1:10
stoichiometric equivalent ratios) display significantly lower tissue mercury concentrations compared to wheat seeds germinated in 100 ppm
mercury alone (1:0). Statistical significance testing was performed using the Welch 2-sample t-test for all panels. For panels A,B, and E, error
bars represent the standard deviation from the mean. For panels C and D, error bars represent the interquartile range. Here, the median and
quartiles were considered as shoot lengths do not follow a gaussian distribution. Raw data (images) are provided in Figure S6.

179 Stoichiometric data for individual metals ranging from sodium to uranyl is provided in (Figure 7D-L). Both peptidomelanin
180 and synthetic melanin were able to chelate all metals tested, including agriculturally-relevant heavy metals (chromium to uranyl),
181 in significantly larger stoichiometric ratios compared to activated charcoal. For example, peptidomelanin chelates mercury,
182 lead and uranyl at stoichiometric ratios (wt%:100%) of greater than 10% (Figure 7J,K,L). Due to the ease of peptidomelanin
183 preparation and solvation, we tested its ability to remediate substrates contaminated with mercury expecting to observe an
184 improvement in seed germination and growth. All raw SEM-EDS data for metal binding experiments is provided in Dataset S4.
185

186 We performed germination experiments on wheat seeds planted in silica substrate intentionally contaminated with mercury.
187 Mercury from HgCl2 was prepared at concentrations ranging from 0.01 ppm to 10,000 ppm, at 10-fold concentration intervals.
188 An uncontaminated control was also prepared. 10 wheat seeds per concentration were introduced into this substrate and
189 incubated at ambient temperature and lighting conditions for 7 days. At the end of this time-interval, we observed a significant
190 decrease in both seed mass and shoot length with increasing concentrations of mercury (Figure 8A,C). Wheat seed mass was
191 found to be inversely correlated with increasing mercury concentration (r = -0.85, log-linear correlation, Figure 8A). Wheat
192 shoot length was found to be inversely correlated with increasing mercury concentration (r = -0.94, log-log correlation, Figure
193 8C). These observations were expected as mercury is known to be toxic to plants41, 42 .

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 194

195 We then attempted to remediate the effects of mercury toxicity on germinating wheat seeds planted in silica substrate
196 intentionally contaminated with 100 ppm mercury using different stoichiometric equivalent ratios of peptidomelanin. Here, a
197 stoichiometric equivalent is defined as the mass of peptidomelanin needed to completely chelate and neutralize one unit mass of
198 mercury (data obtained from Figure 7J). Silica substrate containing peptidomelanin:mercury stoichiometric equivalent ratios of
199 0:0 (growth control), 0:1 (toxicity control), 0.1:1, 1:1, and 10:1 were prepared and planted with wheat seeds. Incubation was
200 performed at ambient temperature and lighting conditions for 7 days. At the end of this time-interval, we observed a statistically
201 significant increase in seed mass of seeds planted in 1:1 and 10:1 peptidomelanin:mercury stoichiometric equivalent ratios
202 (Figure 8B), compared to the seeds planted in only 100 ppm mercury (0:1). The increase in seed mass was strongly correlated
203 with the increasing stoichiometric equivalent ratio of melanin (r = 0.80, log-linear correlation, Figure 8B).
204

205 Likewise, we observed a statistically significant increase the shoot lengths of seeds planted in 1:1 and 10:1 peptidome-
206 lanin:mercury stoichiometric equivalent ratios (Figure 8D), compared to the seeds planted in only 100 ppm mercury (0:1). The
207 increase in shoot length was strongly correlated with the increasing stoichiometric equivalent ratio of melanin (r = 0.63, log-log
208 correlation, Figure 8D).
209

210 ICP-OES was used to quantify the concentration of mercury in germinating wheat seeds planted in all peptidome-
211 lanin:mercury stoichiometric equivalent ratios tested. We observed a statistically significant decrease in mercucy concentrations
212 of seeds across 0.1:1, 1:1 and 10:1 peptidomelanin:mercury stoichiometric equivalent ratio (Figure 8E), compared to seeds
213 planted in only 100 ppm mercury (0:1). The internal mercury concentration was inversely correlated with the increasing
214 stoichiometric equivalent ratio of melanin (r = -0.98, log-log correlation, Figure 8E). This indicates that peptidomelanin present
215 in the silica substrate is able to chelate a sufficient quantity of mercury, preventing it from being absorbed by germinating wheat
216 seeds.
217

218 Overall, wheat germination experiments indicate that peptidomelanin may have applications in the remediation of agricul-
219 tural soils contaminated with heavy metals. Pictures of all wheat seeds and seedlings experimented upon can be found in Figure
220 S6. The ICP-OES report for seedling mercury concentration can be found in Dataset S5.
221

222 Discussion

Figure 9. Our model of the biochemical composition of peptidomelanin. Claims 1-7 are supported by experimental data as described in
the text. *: We also hypothesize that tyrosine residues may serve as ’bridges’: linking to the peptide component via the backbone and linking
to the L-DOPA core polymer via the hydroxylated sidechain.

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 223 Heavy metal contamination in agricultural soils reduces crop yields, contaminates groundwater, and disrupts local ecosys-
224 tems1–3 . In this study we report the discovery of peptidomelanin, a novel macromolecule secreted during the germination of
225 Aspergillus niger melanoliber spores. Although previous reports describing soluble melanins isolated from varying sources
226 exist in the literature20–29 , none describe the substances’ biochemical composition in sufficient detail.
227

228 Based on our experimental data, we can draw the following inferences about the biochemical composition of peptidomelanin:
229 1: Peptidomelanin is an amorphous polymer with molecular weights ranging from ≤10 kDa to ≥ 250 kDa, as determined
230 by native PAGE (Figure 4A). 2: Peptidomelanin is composed of an L-DOPA core polymer as it possesses a similar FTIR
231 spectrum to synthetic L-DOPA melanin (Figure 3C), as well as natural samples of L-DOPA melanin30–32 . 3: The peptide
232 component constitutes ∼23% of peptidomelanin, as determined by LC-MS (Table 1). 4: The mean length of these peptide
233 chains is 2.63 amino acids. This was determined by a combination of the aforementioned LC-MS data and the elemental
234 composition (SEM-EDS) of dansylated peptidomelanin (Figure 6C). 5: Peptide chains do not possess a single sequence. This
235 was determined by cellulose thread electrophoresis of acid-hydrolyzed dansylated peptidomelanin (Figure S5), the absence of
236 peptide hydrolysate fragments upon papain proteolysis of peptidomelanin (Figure S4), as well as simply observing the number
237 of amino acids present in peptidomelanin (19 of 20, Table 1 > 2.63, Figure 6C). 6: Amino acid chains are covalently linked to
238 peptidomelanin via a peptide bond, as inferred from the elemental composition (SEM-EDS) of dansylated peptidomelanin
239 (Figure 6B). 7: Metal ions and oxides can be chelated to peptidomelanin via functional groups on both the melanin core
240 L-DOPA polymer and peptide chains. This was determined via the metal-chelating ability of both peptidomelanin and synthetic
241 L-DOPA melanin (Figure 7). The increased uranyl-chelating ability of peptidomelanin compared to synthetic L-DOPA melanin
242 (Figure 7L) may be a consequence of the disproportionate presence of uranyl-chelating Asp and Glu residues43–45 (Table 1,
243 20.68% relative mass of all amino acids). Our model of the biochemical composition of peptidomelanin is illustrated in Figure 9.
244

245 Based on the L-DOPA composition of the peptidomelanin core polymer, and based on the linkage of peptide chains to
246 peptidomelanin via a peptide bond, we hypothesize that tyrosine residues (the precursor of L-DOPA) may serve as ’bridges’:
247 linking to the peptide component via the backbone and linking to the L-DOPA core polymer via the hydroxylated sidechain.
248 Further experiments involving the incorporation of 13 C- and 15 N-labeled tyrosine into peptidomelanin, detectable via isotope
249 ratio mass spectrometry (IRMS), can help validate this hypothesis. During sporogenesis, the simplest biosynthetic approach to
250 produce peptidomelanin would be to proteolytically cleave excess cytoplasmic proteins and incorporate the resulting peptides
251 into the growing peptidomelanin polymer via tyrosyl side chains. Such a biosynthetic approach would also help explain why
252 each peptide chain covalently linked to the melanin L-DOPA core polymer possesses a different amino acid sequence. However,
253 further experiments are required to validate this hypothesis.
254

255 Peptidomelanin was observed to ameliorate the toxicity of 100 ppm mercury during the germination of wheat, improving the
256 crop’s germination rate, seed mass, and shoot length (Figure 8), indicating potential agricultural applications for the substance.
257 Since peptidomelanin is secreted by germinating A. niger melanoliber spores, the substance can potentially be produced in situ,
258 by providing the conditions necessary for spores to germinate and propagate on agricultural land. Peptidomelanin may serve as
259 a cost-effective alternative to increase crop yields on soils contaminated with heavy metals. Future in situ experiments will be
260 performed to assay the efficacy of peptidomelanin in this role.
261

262 Methods
263 Scanning electron microscopy - energy-dispersive X-ray spectroscopy (SEM-EDS)
264 SEM / SEM-EDS experiments were performed using a FEI Quanta 200 Scanning electron microscope at Icon Labs Pvt. Ltd.,
265 Mumbai.
266

267 Peptidomelanin exudation assay

268 Fungal spores (A. niger strains) were added to 5 mL of Sabouraud broth till the absorbance at 420 nm reached 0.6. A further 10
269 mL of Sabouraud’s broth was added to make a total of 15 ml. Absorbance readings at 420 nm were recorded at t = 0, 10 min,
270 20 min, 30 min, 40 min, 50 min, 60 min, 1.5 hr, 2 hr, 3 hr, and 4 hr intervals. Absorbance was recorded using a Systronics
271 Digital colorimeter type 112 (Sr. No. 12275).
272

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 273 Peptidomelanin retention assay
274 Fungal spores (A. niger melanoliber) were added to 5 mL of Sabouraud broth till the absorbance at 420 nm reached 0.6. A
275 further 10 mL of Sabouraud’s broth was added to make a total of 15 ml. Absorbance readings at 420 nm were recorded at t = 0,
276 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 1.5 hr, 2 hr, 3 hr, and 4 hr intervals. At every time interval 1 mL of mixture
277 was pipetted out and 0.2 mL of 8% glutaraldehyde was immediately added to fix the spores and stop melanin release. Each
278 sample was then centrifuged at 10,000 rpm for 10 min. The pellet was then resuspended in 100ul of 0.1M NaOH to extract
279 melanin from the spores. The sample was then incubated for 1hr. Absorbance at 420 nm for each sample was measured in 96
280 well plates.
281

282 UV-vis spectroscopy

283 UV-vis spectroscopy was performed using a Shimadzu UV-Vis Spectrophotometer UV - 1900i spectrophtometer. The spectrum
284 of peptidomelanin was recorded in deionized water (Milli-Q). The spectra of synthetic L-DOPA melanin (Merck: M8631-
285 100MG, CAS: 8049-97-6) and acid-hydrolyzed melanin were recorded in DMSO.
286

287 FTIR spectroscopy

288 FTIR spectra for peptidomelanin and synthetic L-DOPA melanin were recorded on a Bruker Alpha II Compact FT-IR Spec-
289 trometer. Peptidomelanin was dialyzed against deionized water (Milli-Q) and dried overnight at 80◦ C to convert into a dry
290 powder prior to FTIR spectra collection. Synthetic L-DOPA melanin was also analyzed as a dry powder.
291

292 Peptidomelanin extraction

293 A. niger melanoliber was cultured on Sabouraud dextrose agar. Spores were harvested and transferred to Sabouraud dextrose
294 broth to induce peptidomelanin release. The broth with spores was centrifuged and the supernatant containing peptidomelanin
295 was subjected to syringe filtration (0.2 µm pore size). Peptidomelanin in the filtrate was acid precipitated using 100mM
296 HCl-KCl (pH 2) buffer. The peptidomelanin filtrate was washed thrice in excess HCl-KCl buffer, added at a volume 5× that of
297 the peptidomelanin filtrate’s volume. The melanin concentrate was dissolved in 100 mM Tris buffer (pH 8.8) and stored at 4◦ C.
298 Before use, the melanin concentrate was dialyzed against deionized water (Milli-Q) through SnakeSkin™.3.5 kDa molecular
299 weight cutoff dialysis tubing.
300

301 Peptidomelanin could be concentrated using a 30 kDa centrifugal concentrator. Centrifugation at 10,000 rpm / 30 minutes
302 concentrated peptidomelanin. >99% of the melanin was retained behind the filter membrane, with <1% present in the
303 flowthrough, suggesting a molecular weight of ≥30 kDa for the secreted melanin.
304

305 Peptidomelanin acid-hydrolysis

306 An aqueous solution of peptidomelanin (500 µL) was mixed with 500 µL of 6N HCl containing 0.5% phenol. This reaction
307 mixture was subjected to acid-hydrolysis at 80◦ C for 24 hours under an inert N2 atmosphere. After the incubation period,
308 the reaction mixture was centrifuged at 10,000 rpm for 15 muinutes to separate the components. The pellet and supernatant
309 (containing hydrolyzed peptides) were separated and stored. Excess HCl was removed from the supernatant via heating at 80◦ C
310 for 24 hours or until complete removal of the HCl. The solid residue was resuspended in deionized water (Milli-Q) and used for
311 amino acid estimation via LC-MS (refer Text S1, Table S2, and Dataset S2 for all data).
312

313 Papain hydrolysis

314 Papain (Merck, P4762-25mg) at 0.1 mg/mL was prepared in 100 mM Tris (pH 7.5) (stock-P). BSA at 10 mg/mL was prepared
315 in deionized water (Milli-Q) (stock-B). Peptidomelanin at 4.5 mg/mL was prepared in 100 mM tris (pH 7.5) (stock-M). Three
316 reaction mixtures were also prepared: (1) Stock-P (450 µL) + stock-B (5 µL) + deionized water (45 µL); (2) stock-M (100
317 µL) + deionized water; (3) stock-P (900 µL) + stock-M (100 µL). Reaction mixture 1 served as a positive control. Reaction
318 mixture 2 served as a negative control. Reaction mixtures were incubated at 55◦ C in a water bath for 4 hours. Papain was
319 deactivated by heating all mixtures at 95◦ C for 15 minutes. All reaction mixtures were centrifuged at 10,000 rpm/10 minutes
320 and the supernatant was analyzed using MALDI.
321

322 Matrix-assisted laser desorption/ionization (MALDI) was performed in the Liquid Chromatography Mass Spectrometry
323 (LCMS) Facility (IISc, Bangalore) using a Bruker - rapifleX MALDI Tissuetyper instrument configured to operate in reflector

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 324 mode with positive polarity, acquiring 34,000 shots for data collection. The calibration was performed using the PeptideCalib-
325 Standard mono spectrum and reference list for accurate mass calibration and analysis of peptide signals in the sample.
326

327 Dansylation
328 Dansyl chloride was procured from Merck (D2625 - 1G). A dansyl chloride stock solution containing 2.5 mg/mL dansyl
329 chloride in acetone was prepared. The reaction mixture for dansylating peptidomelanin and acid-hydrolyzed melanin was
330 prepared as follows: 10 µL of a peptidomelanin stock (4.5 mg/mL) + 90 µL sodium bicarbonate (0.2M) + 100 µL Dansyl
331 chloride stock. This reaction mixture was incubate at 37◦ C for 3 hours in the absence of light. The dansylated reaction mix
332 was then dialyzed against deionized water (Milli-Q) through SnakeSkin™.3.5 kDa molecular weight cutoff dialysis tubing to
333 remove small molecules.
334

335 Metal binding stoichiometric assays

336 100 mM stock solutions of the following metal salts were prepared: NaCl, CrCl3 .6H2 O, Ni(NO3 )2 , CuSO4 .5H2 O, ZnSO4 ,
337 Cd(NO3 )2 , HgCl2 , Pb(NO3 )2 , and UO2 (CH3 COO− ).2H2 O. For all metals: a reaction mixture of 200 µL of fungal peptidome-
338 lanin (4.5 mg/mL) + 800 µL of metal salt stock was prepared. Incubation was performed at room temperature for 10 minutes
339 before centrifuging at 10,000 rpm for 15 minutes. Metal complexes that were soluble (only sodium) were dialyzed against
340 deionized water (Milli-Q) through SnakeSkin™.3.5 kDa molecular weight cutoff dialysis tubing to remove excess salt. Metal
341 complexes that were insoluble (all other metals) were centrufiged at 10,000 ppm for 15 minutes and washed with 10 mL
342 deionized water (Milli-Q) twice. All complexes were then heated at 80◦ C to evaporate remaining water. SEM-EDS was
343 performed on all residues to estimate metal content.
344

345 Mercury dose-response titration experiments on germinating wheat

346 Silica (SiO2 , extra pure, Loba Chemie Pvt. Ltd. Product code: 0571901000) was used as an inert substrate for mercury
347 dose-response titration experiments on germinating wheat. 1.25 g SiO2 and 5 g H2 O were added together to form 6.25 g of
348 substrate per condition. Tricyclazole (0.5 µg/ml) was added to all conditions to prevent fungal growth. Mercury (from HgCl2 )
349 concentrations of 0, 0.01, 0.1, 1, 10, 100, 1000, 10,000 ppm were prepared in this substrate as described in Table S3. 10
350 wheat seeds were planted into every condition and incubated for 7 days under ambient conditions in ambient sunlight and were
351 watered daily. Seed mass and shoot lengths were recorded at the end of this time interval.
352

353 Peptidomelanin:mercury dose-response titration experiments on germinating wheat

354 Silica substrate contaminated with 100 ppm mercury was prepared as described in the previous subsection. Peptidome-
355 lanin:mercury stoichiometric equivalent ratios of 0:0, 0:1, 0.1:1, 1:1, and 10:1 were prepared as described in Table S4. 10 wheat
356 seeds were planted into every condition and incubated for 7 days under ambient conditions in ambient sunlight and watered
357 daily. Seed mass and shoot lengths were recorded at the end of this time interval. Samples were prepared for ICP-OES at
358 Ramaiah Advanced Testing Lab (Bangalore) as described in Table S5.
359

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449 Acknowledgements
450 The authors thank the Research and Development Cell (R&D cell), St. Xavier’s College (Mumbai) for providing a seed grant to
451 the Department of Microbiology , St. Xavier’s College (Mumbai) to fund this project. The authors thank Mr. Buddy White for
452 his invaluable support. This project would not have been possible without his help. We would like to thank Kiran Rambhau
453 Bhotkar (Assistant Manager - Application Support) and Sunita Samgir (Senior Executive - Application support) from Icon
454 Labs Pvt. Ltd., Mumbai, for their excellent work as our SEM technicians. We thank Adidev Ajithkumar, Dorothy Martin, Gargi
455 Pandey, Srinidhi G. Santhanakrishnan, Divyani Singh, Neha Debbarma, Namrata Pandey, Nyaipriya Devi, and Rishika Pandey
456 for their technical assistance.

457 Author contributions statement

458 Must include all authors, identified by initials, for example: R.K. A.S, and D.N. conceived the experiment(s), R.K, S.S., B.B.,
459 D.S., L.R., H.B., S.N., and A.H.S. conducted the experiment(s), R.K and D.N. analysed the results. All authors reviewed the
460 manuscript.

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 461 Supplementary information
462 GenBank accession ID PP077302: ITS sequence for A. niger melanoliber (MTCC 13366).
463

464 Figure S1: Melanin biosynthesis inhibition experiments using tropolone. A. niger melanoliber spores sporulate normally in
465 the control (untreated) condition. A. niger melanoliber spores produce albino colonies in plates containing 0.25 mM to 1 mM
466 tropolone. Spores are completely inhibited in plates containing ≥ 2 mM tropolone.
467

468 Figure S2: Peptidomelanin does not contain chitin or nucleic acids. (A) Native PAGE gel containing a peptidomelanin
469 smear. This gel was soaked in Calcofluor White, washed with deionized water, and visualized under UV transillumination.
470 Calcofluor White selectively stains chitin, as evidenced by the chitin flake stained under the same conditions as the gel
471 displaying bright fluorescence. Calcofluor White did not stain the peptidomelanin smear. Therefore peptidomelanin does not
472 contain chitin. (B) Agarose gel (with 0.5 µg/ml ethidium bromide) containing a peptidomelanin smear. Peptidomelanin did not
473 display fluorescence. Therefore peptidomelanin does not contain nucleic acids (DNA/RNA).
474

475 Figure S3: Papain hydrolysis of peptidomelanin reveals the absence of proteins, despite the presence of amino acids in
476 peptidomelanin acid-hydrolysate (main article Table 1). (A) Bovine serum albumin (BSA) hydolyzed using papain. MALDI
477 displays numerous peaks in the 400-2000 Dalton molecular weight region (m/z) confirming BSA hydrolysis into peptides. (B)
478 Untreated peptidomelanin displays few peaks in the 400-2000 Dalton region. (C) Peptidomelanin treated with papain. The
479 MALDI spectrum does not show a substantially greater number of peaks compared to the spectrum of untreated peptidomelanin.
480 This indicates that peptidomelanin is not covalently linked to long, hydrolysable proteins.
481

482 Figure S4: Cellulose thread electrophoresis. (A) Raw data acquired from a UV-transilluminator (geldoc). Canonical
483 amino acids (G-H), labelled in 1-letter codes) were electrophorized to serve as controls. Blank- represents threads with no
484 sample loaded. ’*’ represents the acid-hydrolysate of dansylated peptidomelanin containing dansylated N-terminal amino
485 acids. The G-H and ’*’ were run simultaneously but visualized separately due to large differences in fluorescence intensity.
486 Horizontal thread marks the initial loading position. (B) The images depicted in panel A were contract-adjusted to normalize
487 for differences in fluorescence intensity. Amino acid standards (G-H) migrate as well-resolved spots, whereas ’*’ migrates as a
488 broad smear. This indicates that multiple different dansylated amino acids are present in ’*’, and therefore multiple amino acids
489 are present on the N-terminals of the peptide component of peptidomelanin.
490

491 Figure S5: Raw data for wheat germination dose-response experiments. These experiments reveal that peptidomelanin
492 can ameliorate soil mercury toxicity in agricultural contexts. (A) Dose-response standardization experiments show that the
493 proportion of wheat seeds that germinate declines with increasing concentrations of mercury. The shoot lengths of germinating
494 seeds also decreased with increasing concentrations of mercury. HgCl2 was used as a source of mercury, with concentrations
495 ranging from 0 ppm to 10,000 ppm at 10× increments. (B) Dose-response experiments show that peptidomelanin can ameliorate
496 toxicity induced by 100 ppm mercury during wheat seed germination. From main article Figure 7J, the mercury:peptidomelanin
497 stoichiometric ratio (w/w) was calculated to be 1:6.25 (∼16%:100%). This corresponds to a 1:1 mercury:peptidomelanin
498 stoichiometric equivalent ratio, where 1 equivalent of mercury (1 unit of mass) binds with 1 equivalent of peptidomelanin (6.25
499 units of mass). Wheat seeds were germinated in 1:0 to 1:10 stoichiometric equivalent ratios of mercury:peptidomelanin. The
500 proportion of wheat seeds that germinate in 100 ppm mercury increases with increasing concentrations of peptidomelanin. The
501 shoot lengths of germinating seeds also increased with increasing concentrations of peptidomelanin. A 0:0 ratio indicates a
502 growth control with no mercury or peptidomelanin.
503

504 Text S1: Raw data pertaining to the amino acid composition of peptidomelanin.
505

506 Text S2: SEM-EDS data normalization and processing for peptidomelanin, dansylated peptidomelanin, acid-hydrolyzed
507 melanin, and dansylated acid-hdyrolyzed melanin.
508

509 Table S1: Elemental composition of peptidomelanin acquired via SEM-EDS (Icon Labs, Mumbai). The presence of C, N,
510 and O is expected for any biological macromolecule. The low relative prevalence of cysteine (0.06%) and methionine (0.69%)
511 in peptidomelanin acid-hydrolysate (main article Table 1) indicates that sulfur occurs predominantly as a component of melanin.
512 The absence of P indicates the absence of nucleic acids (DNA/RNA). Alkali metals K, Na, and alkaline earth metal Ca were
513 detected. These elements may be present as coordinately bound ions. Peptidomelanin was dialyzed against deionized water
514 prior to analysis, eliminating the possibility that these ions are merely unbound buffer components. Cl may be present as a
515 component of a salt. SEM-EDS raw data is provided in Dataset S1.

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 516

517 Table S2: LC-MS quantification of the amino acid composition of peptidomelanin.
518

519 Table S3: Mercury dose-response titration experiments on germinating wheat: experimental setup. Stock A: 8.5 mg HgCl2
520 in 1 mL deionized water (Milli-Q). Stock B 10 µL Stock A + 990 µL deionized water (Milli-Q). Tricyclazole stock: 250
521 µg/mL diluted 50× to 0.5 µg/mL.
522

523 Table S4: Mercury dose-response titration experiments on germinating wheat: experimental setup. Stock-A: 8.5 mg
524 HgCl2 in 1 mL deionized water (Milli-Q). Stock-M: 5.55 mg/mL peptidomelanin in deionized water (Milli-Q). S.E. ratio:
525 stoichiometric equivalent ratio.
526

527 Table S5: ICP-OES Hg quantification in germinating wheat: experimental setup. All 10 seeds from a given condition were
528 thoroughly homogenize using a mortar and pestle. This homogenate was mixed with deionized water (Milli-Q) ranging from
529 ∼8-9 mL and sonicated. 1 mL of this sonicate was then dissolved in 14 mL of 20% HNO3 (15 mL total) and incubated at 80◦ C
530 for 7 days to solubilize all Hg. After incubation, the mixture was centrifuged at 10,000 rpm for 15 minutes and the supernatant
531 was sent to Ramaiah Advanced Testing Lab (Bangalore) for Hg quantification via ICP-OES. The ICP-OES report can be found
532 in Dataset S5.
533

534 Dataset S1: SEM-EDS raw data for the elemental composition of peptidomelanin.
535

536 Dataset S2: LC-MS reports for the amino acid composition of peptidomelanin.
537

538 Dataset S3: SEM-EDS raw data for the sulfur estimation (% weight) of peptidomelanin, dansylated peptidomelanin,
539 acid-hydrolyzed melanin, and dansylated acid-hydrolyzed melanin. Note that the SEM-EDS raw data for peptidomelanin is
540 identical in Datasets S1 and S3.
541

542 Dataset S4: SEM-EDS raw data for metal-chelating stoichiometric assays. Assays were performed using 4 substances:
543 silica, activated charcoal, synthetic L-DOPA melanin, and peptidomelanin. Assays were performed using 9 metals: sodium,
544 chromium, nickel, copper, zinc, cadmium, mercury, lead, and uranyl.
545

546 Dataset S5: ICP-OES report for mercury concentration in wheat seeds and seedlings, provided by Ramaiah Advanced
547 Testing Lab (Bangalore).
548

549 All supplementary information can be found inside our Google Drive folder.
550

551 Competing interests

552 The authors declare no competing interests.

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