Mold Fungi Syncephalastrum contaminatum STG-160 and Cladosporium sphaerospermum STG-161 from 16 th century icon—Risks of Tempera Pigments Degradation

Technical Summary: Risks of Tempera Pigment Degradation by Syncephalastrum contaminatum and Cladosporium sphaerospermum

Problem Statement
Tempera paintings on wood, particularly 16th-century Russian icons, possess a complex stratigraphy comprising organic binders (animal glues, egg yolk, resins) and inorganic mineral pigments. While restoration workshops frequently encounter biodeterioration, the susceptibility of specific pigment areas to fungal degradation varies significantly. The study addresses the lack of understanding regarding why certain pigments remain intact while others degrade, specifically investigating the interaction between mold fungi and heavy metal ions inherent in traditional pigments (Fe, Zn, Co, Cd). The goal is to evaluate biodegradation risks to inform preventive conservation strategies for tempera paintings.

Methodology
The research focused on two fungal strains isolated from the 16th-century icon "Descent into Hell" at the State Tretyakov Gallery: Syncephalastrum contaminatum STG-160 and Cladosporium sphaerospermum STG-161. The investigation employed a multi-faceted approach:

1. Genomic Analysis: Whole-genome sequencing (Illumina technology) was performed to identify secondary metabolite gene clusters, specifically focusing on siderophore synthesis pathways.
2. Mock Layer Experiments: Prototype miniature icons were constructed using traditional materials (linen, gesso, sturgeon glue, egg yolk emulsion) and specific pigments (ochres, siennas, zinc white, cadmium yellow, cobalt green, etc.). These were inoculated with the fungal strains, and biodeterioration rates were monitored over 30 days via colony diameter measurement and microscopic observation (SEM/EDX).
3. In Vitro Metal Tolerance Testing: The fungi were cultured on Chapek-Dox agar (CDA) supplemented with varying concentrations of Fe³⁺, Zn²⁺, Co²⁺, and Cd²⁺. Growth inhibition was measured to determine the 50% growth inhibitory concentration (IC₅₀).
4. Elemental Analysis: Energy-dispersive X-ray spectroscopy (EDX) was used to characterize the elemental composition of the pigments used in the mock layers.

Key Results

• Genomic Findings:

  • S. contaminatum STG-160 possesses two gene clusters (regions 30.1 and 67.1) responsible for the synthesis of NRPS-independent (NI) siderophores. These molecules facilitate the chelation and uptake of Fe³⁺ from inert sources like ochres and earth pigments.
  • C. sphaerospermum STG-161 lacks these specific siderophore gene clusters.
  • S. contaminatum showed a total of 31 predicted secondary metabolite regions, while C. sphaerospermum showed 53, including various NRPS and PKS clusters, but no siderophore regions.

• Biodegradation on Mock Layers:

  • S. contaminatum STG-160 exhibited rapid growth on organic binders (egg yolk, sturgeon glue) and iron-rich pigments (English red, grey-green, ochres). It caused significant surface damage within 10 days.
  • C. sphaerospermum STG-161 degraded organic binders first but showed minimal impact on earth pigments and zinc white. It is characterized by melanotic (black) pigmentation.
  • Zinc White Resistance: Both fungi demonstrated negligible growth on zinc white and pigments mixed with zinc white, indicating high resistance to zinc toxicity in a solid matrix.
  • Cadmium Yellow: Growth occurred on cadmium yellow mock layers, likely due to the rich organic medium beneath the pigment facilitating mycelial penetration, despite the presence of cadmium.

• Metal Tolerance (IC₅₀) and Toxicity:

  • Iron (Fe³⁺) and Zinc (Zn²⁺): S. contaminatum STG-160 demonstrated significantly higher tolerance (IC₅₀: 4.26 mmol/L for Fe³⁺; 10.8 mmol/L for Zn²⁺) compared to C. sphaerospermum STG-161 (IC₅₀: 0.81 mmol/L for Fe³⁺; 2.16 mmol/L for Zn²⁺). S. contaminatum showed a parabolic growth response to Fe³⁺ and Zn²⁺, with a burst of growth between 0.5 and 3 mmol/L, suggesting an adaptive utilization of these metals.
  • Cobalt (Co²⁺): Both fungi showed sensitivity, with S. contaminatum having an IC₅₀ of 0.22 mmol/L and C. sphaerospermum 0.11 mmol/L.
  • Cadmium (Cd²⁺): S. contaminatum was highly sensitive, showing no growth even at the lowest tested concentrations (0.01–0.1 mmol/L). Conversely, C. sphaerospermum exhibited a degree of tolerance (IC₅₀: 0.017 mmol/L), likely due to the formation of metallothionein complexes that sequester toxic metals.

Significance and Claims
The paper claims that the differential degradation of tempera pigments is directly linked to the specific metabolic capabilities of the contaminating fungi and the chemical composition of the pigments.

1. Mechanism of Iron Utilization: The high affinity of S. contaminatum for iron-rich pigments (ochres, siennas) is attributed to its genomic capacity to produce siderophores, allowing it to solubilize and transport iron from mineral pigments.
2. Predictive Conservation: The study establishes that areas containing zinc white and cobalt green pigments are naturally resistant to these specific fungal strains due to heavy metal toxicity, whereas iron-rich earth pigments and organic binders are high-risk zones.
3. Conservation Strategy: The authors propose that restoration strategies should be tailored based on pigment composition. Areas with low metal toxicity (iron-rich zones) require more thorough treatment with biocides to prevent aesthetic damage and structural loss. Conversely, zinc-rich areas may require less aggressive intervention regarding fungal growth.
4. Environmental Context: The paper notes that while current museum conditions (State Tretyakov Gallery) are excellent, fungal spores remain dormant. Changes in temperature, humidity, or transportation could reactivate these strains, making the identification of vulnerable pigment zones critical for long-term preservation.

The authors conclude that understanding the specific IC₅₀ values and genomic traits of local fungal isolates allows for the prediction of biodegradation risks, thereby enabling more effective preventive and interventive conservation measures for tempera paintings.