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Article

Yellow Pigment Isolation During Optimization of Extracted Xylindein from Chlorociboria aeruginascens

by
Padraic Duggan
,
Bo MacGill
,
Olivia Queisser
,
Cole Cerrato
,
Hayden Houck
and
Seri C. Robinson
*
Department of Wood Science & Engineering, Oregon State University, Corvallis, OR 97331, USA
*
Author to whom correspondence should be addressed.
Colorants 2026, 5(3), 31; https://doi.org/10.3390/colorants5030031
Submission received: 30 July 2026 / Revised: 8 September 2026 / Accepted: 10 September 2026 / Published: 11 September 2026
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Abstract

The blue-green fungal pigment xylindein, extracted from species of the Chlorociboria genus, has a long history of use in the arts and is of growing interest to material scientists as a component in photovoltaic cells, textile dyes, and semiconductors. Although there is a plethora of fundamental research on xylindein, commercial scale-up of pigment production has not yet occurred, and the methodology for reliable batch culture growth is still evolving. To help aid in eventual commercial batch culturing and processing of xylindein, this research explored additional mechanical processing methods and solvent combinations. None of the physical processing steps (ultrasonication, centrifugation, and drying) produced significantly more xylindein than any other. However, all solvent combinations that contained benzyl alcohol extracted a visually significant amount of yellow color—a compound that was determined to be xylindein as well. Specifically, solvent combinations of dicholormethane (DCM) and benzyl alcohol (BA), methylethylketone (MEK) and BA, and tetrahydrofuran (THF) and MEK showed a significantly greater color shift toward the yellow spectrum. The results of this research, while unexpected, allow for control over the relative blue–yellow balance in xylindein pigment and for a reliable yellow pigment production from xylindein.

1. Introduction

Synthetic color development is similar to chemical agriculture and fossil-fuel industrialization; whenever humans have found a cheaper way to produce colorants in greater abundance, they have generally done so swiftly. Similar to chemical agriculture and fossil fuels, this has had some significant drawbacks, and just as with chemical agriculture and fossil fuels, modern science is now capable of improving the efficiency of forgotten or overlooked natural solutions. One such potential alternative is the application of modern cellular cultivation and processing techniques to the blue-green pigmenting soft-rot fungi in the genus Chlorociboria, which produce the pigment xylindein.
Xylindein was a popular colorant in marquetry and intarsia art of the 1500s–1900s—notable for its longevity in a time when no colorant, natural or synthetic, could reliably hold a blue color [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. The greatest examples of intarsia art were distinguished by their use of striking blue-green segments, offsetting the brown and gold tones of the more common colorants. Over time, the colors used in these panels faded; however, the blue-green remained vibrant and unfaded. By 1979, multiple research groups had attempted to synthesize the fungal pigment, and one group, led by Giles, even succeeded in producing a precursor that showed promise [16]. Unfortunately, the system used was determined to be incapable of complete synthesis, as the high proportion of free oxygen required to create the precursor inhibited further necessary reactions [17].
Following this near-success, increasing natural biosynthesis was investigated as a potential means of xylindein production. Testing showed that intensive culturing increased growth only slightly and often resulted in low pigmentation or even complete lack of color. The reason for the greatly reduced pigmentation of Chlorociboria spp. in a growth-friendly environment, its two-stage pigmenting metabolism, would not be discovered for almost thirty years [18].
By the turn of the millennium, more advanced methods became available. In 2000, a Japanese group around Saikawa utilized heavy-atom analysis X-ray crystallography to definitively illustrate the structure of pure xylindein [19]. It was around this time that another Japanese group led by Shibata began testing it as an alternative plant-growth inhibitor, hoping for lower mammalian toxicity than traditional pesticides [20]. Research continued on applications for xylindein, ranging from textile [21] and wood dyes [22], to thin films for photovoltaic cells [23].
A consistent issue with xylindein has been the inability to synthesize the molecule. Chlorociboria species are slow growing and slow to pigment, even in optimized systems. This has made scale-up industrial processes impossible. The majority of xylindein research currently focuses in this area in the hopes that xylindein can be produced in sufficient quantities to compete with the synthetic dye market and/or produce enough to be viable in the optoelectronic market. The largest success in this area has been by Stange et al. [24,25], who optimized its growth and metabolic parameters via current industry-standard biotechnology practices. Stange et al. characterized an ideal set of growth conditions in media composition, along with preference for a complex yeast-extract nitrogen source and 0.01 M citrate pH buffer. They also detailed for the first time the dual metabolism at work in Chlorociboria species’ xylindein production. These results outlined an ideal 2-stage xylindein production pipeline wherein exponential growth is reached as soon as possible, before pigmentation is induced by nitrogen limitation and light exposure, followed by harvesting and return to a growth stage.
In 2021, Zschatzsch [26] released a case study detailing the construction of a liquid fermentation bioreactor using their previously discovered optimized growth and pigmentation parameters for Chlorociboria aeruginascens. Along with scaling up their production from a table-top 3 L reactor to a 70 L design more comparable to the industrial scale, they also tested new parameters for optimal pigment production. The tested parameters were shear stress/tip speed, as well as a set of extraction solvents that had yet to be investigated in relation to xylindein specifically. Along with determining several potential improvements to DCM in the form of acetylacetone and MEK, the study also found that a very low but constant impeller velocity of <0.5 m/s, along with their previous nutrient and environmental parameters, allowed them to reduce cultivation time down to 2 weeks per batch. This was also accompanied by a significant increase in pigment production, allowing a product yield of 5%, which is more than an order of magnitude higher than comparable natural dye producers such as woad, which have yields around 0.3% with current techniques.
These gains were accompanied by significantly improved processing methodology, more in step with current biotechnology standards. The submerged culture was first separated into wet biomass and supernatant; the first was dried and ground, while the second was subjected to ultrafiltration. Both were subsequently dissolved in MEK and processed by rotary evaporation which, after washing and drying, produced theoretically pure xylindein. While seemingly innocuous, this utilization of more advanced processing equipment is significantly advanced from what has been the standard for the last 10–20 years of xylindein production, which has been the simple grinding of cultured biomass and filter extraction and subsequent storage by DCM.
While this is a foundational work in the study and production of fungal pigments, establishing approximations of ideal growth parameters is only the first step in applying modern biocultivation and processing methods to this space. Stange et al. have created a bioreactor larger than any to date and produced a correspondingly significant amount of xylindein for further testing, but their processing methods, despite utilizing modern techniques and equipment, were not one of the variables tested in that study. They also explored a range of solvents as potential alternatives to DCM, but many complex molecules, such as xylindein, require combined or multi-stage solvent stages to achieve full extraction. There is potential in the exploration of these more complex processing methodologies to increase the production of xylindein to commercially viable levels, but the behaviors of the pigment, in particular solvents, as they relate to different physical processing methods, must be investigated in detail. This study sought to understand if lysing of cellular structure before or after drying influences the availability to bind to the solvent, or whether centrifugation is sufficient to separate fungal proteins and other metabolites from the pigment of interest.
Given the potential shown in previous studies for xylindein to function as a replacement for any number of synthetic colorants, preservative treatments, and even fully organic electronic or photovoltaic components, any improvement in the efficiency of production should be pursued. While the methods outlined here are a proposed significant advancement over the previous standard, there are many more combinations of solvents and physical processing methods that might yield even more effective production systems waiting to be tested and implemented.

2. Materials and Methods

2.1. Overview

Three physical processes and 15 chemical processes were tested for a total of 45 process cases at 21 °C. In order to moderate the variance natural to biological growth, the cultures were grown in four batches, each sufficient for a single test of all 45 factor combinations, giving four replicates per test case. The batches were cultivated from strain UAMH 11657 (originally isolated on a hardwood log in Haliburton, ON, Canada) of C. aeruginascens in growth conditions approximating those used by Stange et al. [24,25]. This strain of Chlorociboria was chosen due to its history of strong pigmentation in media [27]. The major difference was in using a standard 2% honey medium (Glory Bee Raw Honey Blackberry Blossom, Junction City, OR, USA) rather than the orange juice-based medium used in the paper, as the specific juice brand used—which the authors of the Stange study specifically noted as being different from others—is unavailable in the United States. Honey medium was used as a replacement, as studies have shown 2% honey medium to generate both significantly more mycelium and more pigment generation in Chlorociboria strains than in malt or other complex sugars [17]. The ‘benchmark’ xylindein sample in this experiment came from 1 mL of xylindein in dicholormethane (DCM) with L*a*b* values (L = brightness, a = red/green, b = yellow/blue) of L* = 82.28, a* = −11.06, and b* = −5.40 and has been used in previous publications as a benchmark ‘standard’ xylindein concentration. This standard was originally based on the maximum xylindein extraction from one 2% malt agar plate of Chlorociboria aeruginascens at six months’ growth and has been used as a baseline comparative number for xylindein color testing since 2014 [22].

2.2. Growth Stage

Batches were grown in four, 1 L two-armed borosilicate glass spinner flasks with magnetic paddle-type impellers (Corning brand, Glendale, AZ, USA). Each was autoclaved with 1 L of 2% honey liquid medium following the methodology in [18] and inoculated with a 5 mm piece of biomass from an actively growing culture of Chlorociboria aeruginascens strain UAMH 11657 (first isolated from Haliburton, ON, Canada, on a decaying log). After 16 days of growth in culturing chambers at ambient indoor temperature (21 °C), oxygenated via a stirrer at 150 rpm under low-light conditions, in a 2% honey liquid nutrition medium at a test pH of 4.1 (1 L distilled water, 20 mL honey), the samples were then physically processed. Sixteen days was chosen, as it is noted by Stange [24,25] as the earliest day of verifiable xylindein in liquid growth media [24,25].

2.3. Physical Processing

Three methods of physical processing were used (Figure 1):
  • The media and biomass were centrifuged at 5000 rpm for 60 min in a Beckman Coulter Allegra 64 R Centrifuge (Indianapolis, IN, USA), followed by the pigmented layers being extracted by Fisher Scientific 13-678-5 B 1.5 mL glass pipettes (Greenville, NC, USA) and deposited in the solvents discussed below.
  • The growth media and biomass were ultrasonicated for 60 min in a Hielscher UIP1000HdT (Tetlow, Germany) at 1000 watts and 100% amplitude, and the cell contents then extracted by glass pipettes and deposited in the solvents discussed below.
  • Growth media and biomass were dried at room temperature (21 °C) for 24 h, then deposited using glass pipettes in a solvent (solvents discussed below).
Colorants 05 00031 g001
Figure 1. Outline of methodology flow.
Figure 1. Outline of methodology flow.
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Each batch was then tested using either one (10 mL) or a combination (5 mL each) of the following five solvents: DCM, MEK (methyl ethyl ketone), ACN (acetonitrile), THF (tetrahydrofuran), and benzyl alcohol (BA). These were tested both alone, and in a 1:1 ratio mixture with each other solvent being tested, creating a total of 15 solvent-combination test cases. With three physical processing methodologies, this resulted in 45 distinct test conditions to compare the efficacy of xylindein extraction. Four replicates were repeated per test condition, for a final number of 180 samples tested. Blanks of each solvent combination in matching vials, along with a purified sample of xylindein in DCM, were also measured by reflectance colorimetry, as outlined in 3.4 below for comparison. Each replicate batch was grown in its own bioreactor to limit interference from growth differences influencing between-replicate analysis. The solvent combinations will be referred to by number, as displayed in Table 1 below.

2.4. Xylindein Analysis (Chemical Processing)

The color analysis was done by depositing 5 mL of each solvent via glass pipette into matched test vials, for a total of 10 mL per vial of either 1:1 combinations of each solvent, or a larger portion of a single solvent (always using the same total mL of solvent). Portions of the biomass resulting from each physical processing methodology were then randomly assigned to vials. After 24 h of extraction time, 1 mL of xylindein-bearing solvent was extracted using a pipette from the vials of combined mechanically processed biomass and solvent and placed into 3 mL opticlear Fisher-Scientific glass cuvettes for colorimetric reading using a Konika Minolta CR-5 colorimeter (Tokyo, Japan). Opticlear glass cuvettes with a 3 mL capacity were filled with each xylindein–solvent combination and tested via reflectance on a liquid sample. This gave a color-space reflectance value for each sample, which was then run through the ΔE76 formula shown in Equation (1) Figure 2 for color-space determination. ΔE76 was chosen over more recent color-change ΔE formulas for ease of comparison with previous studies, which have predominantly used that method. ΔE values require a ‘starting’ color to use to determine color change. Two different ΔEs were calculated: The first, hereafter ‘Test A,’ calculated the color change of each sample-extracted solvent from the base color of each solvent alone. The second, hereafter ‘Test B’, calculated the color change from a benchmark of concentrated xylindein in DCM used in previous studies [9]. The benchmark offers a way to compare the results from this work with previously published solvent extraction work on xylindein.
∆ E a b * = ( L 2 * − L 1 * ) 2 + ( a 2 * − a 1 * ) 2 + ( b 2 * − b 1 * ) 2
A second method to determine color change was the JND, the ‘just noticeable difference.’ This is a measurement standard defined by the CIE lab, the producers of the ΔE formula, and represents the minimum amount of difference in color detectable by color measurement and the human eye. This was included in addition to statistical analysis for two reasons. First, the highly dilute solutions tested were unlikely to produce such high variation as to be easily measured as statistically significant, regardless of the actual difference in color distance. Second, when measuring relative change in pigmentation, any physically detectable difference in color is potentially relevant to discussion and further experimentation, particularly for artistic applications.
It is important to note here that volumetric and weight comparisons are not generally used for spalting pigment generation due to the “sticky” nature of the pigments. Concentration in early growth stages in particular is best measured via color change spectrometry rather than by weight and volume, as discussed in [28].

2.5. Statistical Analysis

A single-factor ANOVA was performed on the four batches to determine if a significant difference existed between them. There was found to be no statistically significant difference; as such, the factor was discarded and treated as a single batch in the remainder of the statistical analysis.
The raw data met neither the normal distribution nor homogeneity of variance assumptions required for significance testing with an ANOVA. As such, a transformation was performed using cubed root on the final ΔE values prior to further analysis. After transformation, a single-factor ANOVA was performed on the ΔE values from Test A (where the final xylindein color was compared to the original color of each solvent) to determine if any solvents were unable to carry significant xylindein.
Finally, a two-factor ANOVA was performed on the transformed ΔEs from Test B (color distance from xylindein benchmark), followed by a Tukey HSD. When the 2-way ANOVA showed no significant interaction between the two terms, a single-factor ANOVA and accompanying Tukey HSD were run on each independent variable in isolation.

2.6. Follow Up on Yellow Pigment

A chromatogram analysis was run on the extracts containing benzyl alcohol to determine if xylindein was indeed present in the yellow extract. To do this, the extracts were run through a 1290 Infinity II LC (Agilent Technology, Inc., Santa Clara, CA, USA) by injecting 5 uL of sample onto a Poroshell 120 EC-C18 column (1.9 um, 2.1 × 50 mm, Agilent). The compound was separated with water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) using a binary pump program of 5% solvent B, increased linearly to 100% at 30 min at a flow rate of 0.4 mL/min. The compound was detected using an Agilent 6546 QToF (quadrupolar time-of-flight) mass spectrometer in positive mode, with parameters for drying gas (N2) temp, drying gas flow, nebulizer pressure, sheath gas temp, and sheath gas flow set to 320 C, 8 L/min, 35 psi, 350 C, and 11 L/min, respectively. Source and collision cell parameters included capillary, nozzle, fragmentor, skimmer, and collision voltages at 3500 V, 1000 V, 175 V, 65 V, 750 V, and 0 V, respectively. Data was acquired for 40–700 m/z at a rate of 1 spectrum/s and 9295 transients/spectrum.
As spalting pigments are primarily used for art coloration, especially of wood, the purity of the xylindein extract was not important at this stage, as much as simply confirming its presence in the yellow extract. Purification of xylindein extracts has been done for toxicity studies [29] but has not yet been done for artistic uses.

3. Results

3.1. Summary

Test A, which used the ΔE value that measures the ability of the solvents to extract Chlorociboria metabolites in general, produced meaningful results in that all the solvents were able to extract a significant amount of color. However, this measured the color distance entirely from the empty solvents, and not every test case changed towards the blue-green that is characteristic of xylindein.
In Test B, based on ΔE values representing the color distance between the tested samples and the benchmark value for xylindein, the two-factor ANOVA did not show a statistically significant interaction between the physical processing method and solvent combination, at p = 0.47. Physical processing alone had a p value of 0.07, while solvent combinations alone had a p value of <0.0001. The R2 value for this test was low however, at 0.42, and a three-factor analysis was untenable due to insufficient degrees of freedom. The follow-up single-factor analysis of solvent combinations had an even lower R2 value of 0.28. Tukey’s HSD showed that Solvent 15 had a significantly higher change in color; however, it was not significantly different from solvents 15, 5, 12, 9, and 1. The follow-up single-factor analysis of physical processing gave a p value of only 0.13 and as such, no further analysis of the physical processing factor was performed. A general summary of the untransformed data from Test B is shown below in Figure 2, illustrating the visual differences represented by color-read data instead of statistical differences.

3.2. Processing in Detail

A more granular representation of that visual color difference is shown in a series of figures. Figure S1 illustrates the lack of a statistically significant difference in xylindein extraction, as measured by color change across the three types of physical processing methodologies. However, there were meaningful differences between solvent combinations as shown in Supplemental Figures S2–S4. An image of the L*a*b* color space is provided below to aid in reader comprehension of the data (Figure 3).
ΔE measures the overall distance between two points in this color space, but equally relevant is which colors are changing, to what degree, and in which direction. Along the red-green (*a) color space plane on Test A (the comparison between test sample and blank solvent), Figure 4 shows green being extracted by the solvent. Although green in the L*A*B* color space is represented by ‘−a’, the ΔA calculation wherein the ‘target’ is the base color of the pure solvent and the ‘sample’ is the final color after extraction (atarget-asample) turns negative numbers positive. Given this, Figure 4 should be read as positive numbers equating to more green color, or an ‘increase of −a’. In the same vein, Figure 5 shows positive numbers equating to more blue color removed by the solvent (ΔB). These graphs illustrate that the solvent extract was blue and green, characteristic of xylindein.
Test B, ΔA (red/green) overall can be seen in Figure 6. The negative numbers here represent increased red color, indicating the extraction was less like the benchmark in terms of red/green content. A direct comparison between a concentrated benchmark and diluted solvent combinations is not a complete summary. The benchmark was based on a highly concentrated DCM extraction. Within this test, Solvent 1 is the DCM-only extraction that received the same concentration of initial xylindein-bearing biomass as the other solvent cases. Interpreted with this relationship between Solvent 1 and the benchmark in mind, the data from Figure 6 and Figure 7 showing less red and yellow suggest greater extraction of xylindein as compared to DCM alone (as the negative numbers get closer to 0, they indicates less red and less yellow in the sample). This greater extraction is simply not enough to overcome the difference in concentration.
The only statistically significant independent variable was solvent. Broken down by these alone, ΔA (red/green) (Figure 8) shows additional red color being pulled from the solutions. More simply, the more negative the number shown in Figure 8, the farther away from the benchmark color the extract was, and the more red it possessed (as opposed to the target green). The same applies to Figure 9, with more negative numbers equating to increased yellow content. Once again however, the benchmark is concentrated xylindein in DCM. When compared to the DCM extraction within this test, all solvents had less red. Figure 9 indicates notably more yellow being extracted by solvents 5, 9, 12, and 15 than by Solvent 1, the same solvents that exhibited statistically significant differences.

3.3. Analysis of Yellow Solution

The chromatograph (Figure 10) and mass spectrum (Figure 11) confirmed the presence of xylindein in the benzyl extracts, indicating that a yellow xylindein was indeed being extracted, and the color did not come from oxidated benzyl alcohol.

4. Discussion

4.1. Statistical Discussion

The statistically significant differences in this study were limited, with the R values for all the tests indicating that factors outside of the tested independent variables were likely at play. Additionally, the low number of replicates along with the high number of tested conditions likely had a reducing effect on the degrees of freedom. Given that, solvent type did have a significant effect on color change when compared both to the bare solvent alone and to a xylindein benchmark. Solvents that can carry xylindein in visually discernible amounts have been known to be few and far between, with xylindein best carried in DCM, chloroform, and pyridine [30], and finding an optimal compromise between solvent efficacy and chemical danger from toxicity or other factors has been an ongoing struggle in xylindein extraction since the first attempt at extraction. In this study, benzyl alcohol (BA) (Solvent 15) had the greatest color change by far, but interestingly did not show the greatest blue-green increase. While BA is hardly benign with regard to human health, it is far less toxic, volatile, and generally dangerous than the current standards for extracting xylindein, ether MEK, or DCM. DCM is also a suspected human carcinogen, while BA presently has no known carcinogenicity concerns. Lastly, BA generally costs less than DCM.
Also of note is that MEK (Methyl-Ethyl-Ketone, aka Butanone), the solvent declared preferable for extraction of xylindein by the Stange research group in two different papers [9,10], only performed well in this study when used in combination with BA, statistically speaking. As the performance of MEK in this study was similar to DCM and other solvents, it is possible that BA is simply so much more effective than MEK that they were only comparable when combined. However, MEK is also only miscible in ~27.5% water; this study used a higher proportion of wet biomass taken directly from liquid culture, while the 2021 study that arrived at MEK as the superior solvent used biomass that had undergone centrifugal grinding and was very dry. This may also contribute to the difference in observed behavior.

4.2. Color Discussion

The use of statistical significance in measuring visual color and types of color is an increasingly complex discussion in color theory communities [31]. Test B, the comparison of sample extracts to a benchmark of concentrated xylindein extracted with DCM, was certainly informative, but it is not a 1:1 comparison. The b value in particular showed an unexpected rise in yellow color, which could be for several reasons. The first may be related to the ‘yellow oil’ found by the Donner group when attempting to synthesize xylindein [32]. Another option might be a redox optical modulation, which has previously been observed when xylindein was subjected to bias under moderate voltage [33]. However, in the voltage case, xylindein did return to its original blue-green after the external influence ended, and the yellow pigments extracted herein remained stable.
The initial hope of this research was to find a solvent combination that produced an increase in blue in samples, as prior research has suggested that isolated xylindein is closer to a pure blue color and only appears more green due to being on wood (adding yellow color) or due to another molecule: either a xylindein precursor with yellow coloration, or a different metabolite altogether with a yellow color profile, being extracted along with the xylindein. The chromatogram results in this study indicate that the yellow is indeed xylindein, confirming that xylindein does have a yellow phase. It is also known from previous research that xylindein can “turn” yellow under certain external electrical conditions, although this color state is not permanent and is unlikely to be the cause in this current study. The difference in color performance between solvents appears to revolve at least partially around what ratio of blue xylindein to yellow xylindein they extract. Solvents 1 (DCM), 5 (DCM/BA), 9 (MEK/BA), 12 (THF/BA), and 15 (BA/BA) had significantly greater color change, but they also appeared to extract more yellow. These solvents therefore may not be better at extracting the more coveted blue xylindein, but rather better at extracting the yellow precursor.
This unexpected result could have beneficial effects. Within the spalting palette, yellow is a notoriously difficult color to reliably generate. While xylindein has been found to have utility in materials as diverse as solar cells, fabric dyes, etc., the origin and main bulk of its use has remained as a blue-green colorant. Outside of applications that necessitate total purity like photovoltaic cells and semiconductors, the addition of yellows to the blue of xylindein, and the variety of beautiful greens and teals the combination produces, have long been seen as a benefit rather than a detriment. The potential capacity to control the amount of yellow in a given xylindein solution, and thus the proportional amount of green to blue within a given extract, is a useful tool for those seeking more reliable colors or even particular shades in that range. There is a growing body of research on the reasons and methods by which so many spalting fungi make such a wide variety of colors, particularly around competition and environmental protection [34,35,36]. The importance of these colors in art and other dye work is evident, as xylindein has been used in wood art since at least the 1400s [37,38], and a not-insignificant portion of that work has simply been attempting to limit or eliminate color variation across any number of factors from growth time and environmental conditions to subspecies, strain, and even batch. The ability to influence color change or separate the blue and yellow color components is significant.
Additionally, while the majority of the solvent combinations tested did not have a statistically significant effect on color change, that is not the only metric of relevance. The CIE L*A*B* color measurement formula uses a “Just Noticeable Difference” standard for the minimum color space distance detectable between two points, the minimum difference between two samples to physically measure a difference in color (ΔB of 2.3 being the threshold). Several of the solvents that did not show a statistically significant change did pass the JND threshold, namely Solvents 6 (MEK/MEK), 8 (ACN/MEK), 11 (THF/ACN), and 14 (BA/ACN). While statistical significance is an important qualifier, a physically detectable difference in color when investigating pigmenting substances is still useful information, even if it does not reach statistical significance. These solvent combinations showed a detectable color change and may be worthy of further investigation. The book Colorimetry: Understand the CIE System [31] contains robust discussions on human perception of color versus machine perception of color. It is worth noting that while a color change may not have been statistically significant, for an artist, a visual difference may be enough to choose one pigment, brand, or strain over another. As a final note, xylindein has been extracted by many different solvents and at many different concentrations and is remarkably stable in its blue-green coloration, although previous research [39] has noted a shift toward green in solvents like tetrahydrofuran when stored in solution. The reasons behind this shift are currently unknown.

4.3. Physical Processing Significance Discussion

Of particular surprise in this study, no statistically significant relationship was shown between xylindein extraction and any physical processing method. While the methodology used in the Stange et al. studies [24,25] did both isolate significantly greater quantities of xylindein than previous studies and utilize physical processing techniques, namely centrifugal grinding, that does not necessitate a causal relationship between physical processing and greater xylindein yields. Their process also utilized an advanced cellular bioreactor of a size unprecedented in xylindein production, as well as solvents used for extraction that had not been studied in depth before. As such, it is possible that the increased production demonstrated was unrelated to physical processing as seen here.
Spalting fungi often secrete these compounds to outcompete neighbors and protect resources [34,35,40,41]. As such, it may not be an evolutionarily effective tactic to produce such pigments if they are likely to remain bound in the cell; such large, energetically expensive, complex molecules would be more useful if utilized soon after creation, or only created when immediately needed. Similarly, one of the traits of xylindein that has made it such an attractive focus of research is the incredible degree to which it binds to its target, with xylindein-stained wood remaining vibrant and colorful for centuries [42]. With such a strong pigmenting ability from a soft-rotting wood decay fungus [43], it would follow that Chlorociboria species likely produce other metabolites related to digestion and resource capture. Given this, the lack of a significant effect of physical processing may simply be due to the long arms of evolution having ensured that a self-defense molecule intended to spread out from the cells producing it would not remain bound within them, and, as such, rupturing them would not release a meaningfully greater amount of pigment than simply collecting that which was secreted.

5. Conclusions

One of the primary focuses of this study was whether the effect of physical processing and cell disruption on cultured Chlorociboria species had an influence on the extraction of pigment from those cultures. Unexpectedly, not only was there no difference between different processing methods, but no meaningful difference from processing was found whatsoever. This suggests that, whatever other difficulties there are in extracting xylindein, being too tightly bound up in the cell is not a meaningful part of them. Hence, physical processing had no significant effect on xylindein extraction.
What did affect xylindein extraction was the solvent utilized, with combinations containing benzyl alcohol pulling significantly more yellow xylindein from solution. Previous research suggested that a yellow metabolite in Chlorociboria aeruginascens was likely present, and this study confirms the presence of the yellow xylindein although follow-up testing will be required in order to determine the specific nature of the pigment. These solvents suggest a method to reliably produce yellow pigment from Chlorociboria species. As yellow is one of the least reliable spalting pigments to generate, a stable method of producing yellow pigment will further spalting as an art form and may open new doors for chemical testing for optoelectronics as well.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/colorants5030031/s1. Figure S1: The full set of ΔE values in Test B across physical processing methods performed. The black central bars represent the mean of each processing case, with the grey regions to either side representing 50% of the scores. The error bars beyond show the more distant 25 of scores, with the remaining dots representing outliers in each case. Higher ΔE values here depict color-distance from the benchmark, suggesting dissimilarity from concentrated xylindein. This uses untransformed data in the interest of readability, as no significance was found in the transformed data; Figure S2: Transformed ΔE (via cube-root transformation) values from the single-factor ANOVA. The X axis shows solvent-combination (as enumerated in Table 1) and the Y axis shows ΔE values. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, Solvent 15 = BA/BA; Figure S3: Raw ΔE values from the single-factor ANOVA of solvent-combinations, independent of batch or physical processing method. The X axis shows solvent-combination (as enumerated in Table 1) and the Y axis shows ΔE values. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, Solvent 15 = BA/BA; Figure S4: Untransformed ΔE Test B values (comparing tested samples to the xylindein benchmark) from the single-factor ANOVA. The X axis shows solvent-combination (as enumerated in Table 1) and the Y axis shows ΔE values. Error bars represent one standard deviation for each solvent-combination case. The A, B, and AB letters signify statistically significant differences at alpha = 0.05. Visual differences occur at ΔE 2.3 or higher, with those below the JND bar representing no visible difference between that case and the xylindein benchmark. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, Solvent 15 = BA/BA.

Author Contributions

Conceptualization, P.D. and S.C.R.; methodology, P.D. and H.H.; software, P.D. and C.C.; validation, S.C.R.; formal analysis, P.D. and S.C.R.; investigation, P.D., B.M., O.Q. and H.H.; resources, S.C.R.; data curation, P.D. and C.C.; writing—original draft preparation: P.D.; writing—review and editing, S.C.R.; visualization, P.D. and S.C.R.; supervision, S.C.R.; project administration, S.C.R.; funding acquisition, S.C.R. All authors have read and agreed to the published version of the manuscript.

Funding

Funding was supplied by The Spalting Cooperative.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. Data are not publicly available, as further research is still being conducted.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 2. Untransformed ΔE values across physical processing and solvent/solvent combinations (represented by numbers from Table 1). The red line indicates the CIELAB recognized JND value—the just-noticeable difference to the human eye.
Figure 2. Untransformed ΔE values across physical processing and solvent/solvent combinations (represented by numbers from Table 1). The red line indicates the CIELAB recognized JND value—the just-noticeable difference to the human eye.
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Figure 3. The CIE L*A*B color space. Negative ‘a’ numbers indicate a greater presence of green, and correspondingly positive ‘a’ numbers indicate a greater presence of red. The same relationship is seen with positive and negative ‘B’ numbers, with negatives indicating blue presence and positive indicating yellow presence. ‘L’ numbers indicate lightness, with zero representing black (Konica Minolta Sensing Americas).
Figure 3. The CIE L*A*B color space. Negative ‘a’ numbers indicate a greater presence of green, and correspondingly positive ‘a’ numbers indicate a greater presence of red. The same relationship is seen with positive and negative ‘B’ numbers, with negatives indicating blue presence and positive indicating yellow presence. ‘L’ numbers indicate lightness, with zero representing black (Konica Minolta Sensing Americas).
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Figure 4. ΔA (green/red) color change from Test A (sample compared to the base solvent), by solvent and physical processing. Larger ΔA numbers indicate greater green color being extracted by the solvent. Means are calculated from the replicates within each processing group. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 4. ΔA (green/red) color change from Test A (sample compared to the base solvent), by solvent and physical processing. Larger ΔA numbers indicate greater green color being extracted by the solvent. Means are calculated from the replicates within each processing group. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 5. ΔB (yellow/blue) color change from Test A (sample compared to the base solvent), by solvent and physical processing. Larger ΔB numbers indicate greater blue color being extracted by the solvent. Means are calculated from the replicates within each processing group. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 5. ΔB (yellow/blue) color change from Test A (sample compared to the base solvent), by solvent and physical processing. Larger ΔB numbers indicate greater blue color being extracted by the solvent. Means are calculated from the replicates within each processing group. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 6. ΔA values for Test B (sample comparison to benchmark xylindein) across physical processes. Negative values indicate greater red. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 6. ΔA values for Test B (sample comparison to benchmark xylindein) across physical processes. Negative values indicate greater red. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 7. ΔB values for Test B (sample comparison to benchmark xylindein) across physical processes. Negative numbers indicate greater yellow. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 7. ΔB values for Test B (sample comparison to benchmark xylindein) across physical processes. Negative numbers indicate greater yellow. Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 8. Test B, ΔA (red/green) by solvent. Negative numbers indicate red color (higher negatives indicate more red). Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 8. Test B, ΔA (red/green) by solvent. Negative numbers indicate red color (higher negatives indicate more red). Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 9. Test B, ΔB (yellow/blue) by solvent. Negative numbers indicate yellow color (higher negative numbers indicate more yellow). Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
Figure 9. Test B, ΔB (yellow/blue) by solvent. Negative numbers indicate yellow color (higher negative numbers indicate more yellow). Solvent 1 = DCM/DCM, Solvent 2 = DCM/MEK, Solvent 3 = DCM/THF, Solvent 4 = DCM/ACN, Solvent 5 = DCM/BA, Solvent 6 = MEK/MEK, Solvent 7 = MEK/THF, Solvent 8 = MEK/ACN, Solvent 9 = MEK/BA, Solvent 10 = THF/THF, Solvent 11 = THF/ACN, Solvent 12 = THF/BA, Solvent 13 = ACN/ACN, Solvent 14 = ACN/BA, and Solvent 15 = BA/BA.
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Figure 10. Extracted chromatogram at 569.14 m/z showing a signal at 15.014 min (xylindein).
Figure 10. Extracted chromatogram at 569.14 m/z showing a signal at 15.014 min (xylindein).
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Figure 11. Mass spectrum for the extracted compound from 14.93 to 15.198 min, revealing a parent ion of at 569.1448 m/z (xylindein).
Figure 11. Mass spectrum for the extracted compound from 14.93 to 15.198 min, revealing a parent ion of at 569.1448 m/z (xylindein).
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Table 1. Solvent combination chart. Numbers are used for ease of future readings so that solvent combinations are not continually repeated. DCM = dichloromethane, MEK = methyl-ethyl-ketone, THF = tetrahydrofuran, ACN = acetonitrile, BA = benzyl alcohol.
Table 1. Solvent combination chart. Numbers are used for ease of future readings so that solvent combinations are not continually repeated. DCM = dichloromethane, MEK = methyl-ethyl-ketone, THF = tetrahydrofuran, ACN = acetonitrile, BA = benzyl alcohol.
SolventDCMMEKTHFACNBA
DCM12345
MEK26789
THF37101112
ACN48111314
BA59121415
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MDPI and ACS Style

Duggan, P.; MacGill, B.; Queisser, O.; Cerrato, C.; Houck, H.; Robinson, S.C. Yellow Pigment Isolation During Optimization of Extracted Xylindein from Chlorociboria aeruginascens. Colorants 2026, 5, 31. https://doi.org/10.3390/colorants5030031

AMA Style

Duggan P, MacGill B, Queisser O, Cerrato C, Houck H, Robinson SC. Yellow Pigment Isolation During Optimization of Extracted Xylindein from Chlorociboria aeruginascens. Colorants. 2026; 5(3):31. https://doi.org/10.3390/colorants5030031

Chicago/Turabian Style

Duggan, Padraic, Bo MacGill, Olivia Queisser, Cole Cerrato, Hayden Houck, and Seri C. Robinson. 2026. "Yellow Pigment Isolation During Optimization of Extracted Xylindein from Chlorociboria aeruginascens" Colorants 5, no. 3: 31. https://doi.org/10.3390/colorants5030031

APA Style

Duggan, P., MacGill, B., Queisser, O., Cerrato, C., Houck, H., & Robinson, S. C. (2026). Yellow Pigment Isolation During Optimization of Extracted Xylindein from Chlorociboria aeruginascens. Colorants, 5(3), 31. https://doi.org/10.3390/colorants5030031

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