Optimizing Xylindein from Chlorociboria Spp. for (Opto)Electronic Applications

processes

Article Optimizing Xylindein from Chlorociboria spp. for (Opto)electronic Applications

R.C. Van Court 1 , Gregory Giesbers 2, Oksana Ostroverkhova 2 and Seri C. Robinson 1,*

1 Department of Wood Science and Engineering, Oregon State University, Corvallis, OR 97331, USA; [email protected] 2 Department of Physics, Oregon State University, Corvallis, OR 97331, USA; [email protected] (G.G.); [email protected] (O.O.) * Correspondence: [email protected]

Received: 26 October 2020; Accepted: 12 November 2020; Published: 17 November 2020

Abstract: Xylindein, a stable quinonic blue-green fungal pigment, has shown potential for use not only as a colorant but also as an (opto)electronic material. As no method presently exists to synthesize the pigment, organic production by slow-growing fungi from the genus Chlorociboria is the only method to obtain it. This has resulted in limited quantities of impure xylindein, hampering research. In order to improve quantity and quality of pigment for optoelectronic applications, speed of xylindein production by Chlorociboria aeruginosa and its relative purity were compared across liquid and solid-state fermentation conditions on selected nutrient sources. Liquid 2% malt shaking cultures produced the same amount of pigment in 5 weeks that previous testing produced in 2 months. Xylindein generation speed, purity, and conductive properties of produced pigment for (opto)electronics was then compared between two Chlorociboria species native to North America, Chlorociboria aeruginosa and Chlorociboria aeruginascens. Differences were seen in the conductivity of extracted pigment between species and strains, with xylindein from C. aeruginascens strain UAMH 7614 producing films with the highest effective electron mobility. The identification of the most effective growth conditions and the strain with highest purity xylindein production should support further development of sustainable organic (opto)electronics. Future work identifying new strains with reduced production of interfering metabolites and new extraction methodologies will help to produce very low cost xylindein, supporting sustainable technologies based on the pigment.

Keywords: spalting; Chlorociboria; organic semiconductor; (opto)electronics; fungal pigment; secondary metabolites; green energy

1. Introduction The pigment xylindein is a blue-green quinonic secondary metabolite produced by soft rot decay fungi within the genus Chlorociboria. Wood colored by Chlorociboria has a long history of use in art, with pieces dating back to the 15th century retaining their blue-green coloration today [1,2]. In modern times, pigment extracted from Chlorociboria spp. has been investigated for use as a coloration agent for wood [3], for paints [4], and for dying textiles [5–9]. In addition, xylindein has unique physical properties that are of interest to the ﬁeld of optoelectronics. Xylindein possesses a π-conjugated core structure and exhibits aggregate formation likely due to hydrogen bonding and molecular π–π stacking [10]. This enables electron transport with mobility of up to 0.4 cm2/(Vs) [11] in amorphous − xylindein ﬁlms and potentially higher in high-purity [12] and/or crystalline xylindein. The observed semiconducting properties, in addition to excellent photostability of xylindein, make it a candidate for green technologies such as sustainable organic electronics [10–13].

Processes 2020, 8, 1477; doi:10.3390/pr8111477 www.mdpi.com/journal/processes Processes 2020, 8, 1477 2 of 23

Despite multiple investigations into laboratory synthesis of xylindein, none have thus far been successful [14–16]. This makes synthesis by Chlorociboria spp. The only way to obtain the pigment. Unfortunately, the fungi grow slowly under most laboratory conditions [17] and can lose their pigmentation in culture [18,19]. Research into the relationship between culture conditions and pigment production of Chlorociboria cultures has been the subject of multiple investigations (Table S1). The first was in 1928, where growth substrates such as beech wood and organic medias such as malt extract and plum decoction were found to result in blue-green pigmentation [20]. Later work identified sugar-based media as showing the best pigmentation, with organic nitrogen sources inhibiting pigmentation [19]. Recent work has suggested that this effect may be due to nitrogen limitation leading to stimulation of pigment production, with high available nitrogen growth conditions leading to high biomass production but no pigmentation [21]. The standard methodology of acquiring xylindein for present day (opto)electronic research has relied upon use of 2% malt agar plates amended with Acer saccharum (Marshall) woodchips. This media has been found to increase colony size and improve pigmentation across Chlorociboria aeruginascens isolates [17,22]. It also allows for straightforward extraction of the pigment into a solvent [23]. However, recent work has also shown that liquid media growth conditions also produce strong pigmentation results [24–26] and may be easier to scale for industrial production. While multiple publications have studied pigmentation in Chlorociboria cultures, methodologies have been highly varied and results have been difficult if not impossible to directly compare. They have ranged from morphological descriptions to extracted pigment color (Table S1). No studies have compared colony diameter to extractable pigment amounts, or directly compared xylindein production between liquid and solid medias. This makes it difficult to identify which tested growth conditions actually result in the best pigment production. In addition, it is unknown which growth conditions result in the purest extracted xylindein solution. Current extraction methodology from xylindein does not separate it from a variety of other metabolites that have been reported from Chlorociboria cultures extracted into dichloromethane [24], or components of media. These contaminants have been found to interfere with the electronic potential of xylindein, reducing conduction [12]. No work has been performed to investigate which growth conditions are associated with more impurities. Previous work on purification of the pigment used centrifugal partition chromatography [27]; however, specialized equipment was required and only 70% extraction efficiency was achieved. In addition to variation in pigment production in response to environmental conditions, production differences between fungal species and strains may also be significant. In North America, two species in the Chlorociboria genus appear sympatrically, Chlorociboria aeruginascens and the slightly less common Chlorociboria aeruginosa [18]. The two species are morphologically similar although genetically quite distinct, both showing more genetic similarities to southern hemisphere Chlorociboria species than to each other [22,28]. The primary physical differences between the two species are that C. aeruginascens has smaller spores and smooth instead of rough tomentum hyphae [18,29]. Different species of pigmenting fungi within the same genus are known to show variation in pigment production. Notably, two pigmenting spalting fungi, Scytalidium cuboideum and Scytalidium ganodermophthorum, show very different pigmentation patterns, with S. cuboideum producing a naphthoquinonic red pigment [30] and S. ganodermophthorum producing an unknown yellow pigment [8,31]. However, production of blue-green pigment is conserved within the Chlorociboria genus [32], although variation in pigment production between species and strain has been noted [22,24]. In addition to growth speed differences between species, there is also a high likelihood of secondary metabolite production variation between species and strains. Variation in pigmentation between fungal strains is well known. For example, strains of Monascus purpureus have shown variation in production of pigment and toxic secondary metabolite citrinin [33], and multiple mutants produce variations in pigmentation [34,35]. Chlorociboria species produce other secondary metabolites including a yellow compound [24], which, in addition to other metabolites from the fungi, may be responsible for reducing the conductivity of films made from extracted xylindein [12]. Pigment extracted from Processes 2020, 8, 1477 3 of 23 fungi shows a great deal of variability, as they are biological organisms that respond differently to their environments. The inhomogeneity associated with biological production is an obstacle to research into (opto)electronics, where purity and consistency are desired. Xylindein extract from one fungus or growth condition may have a different profile of secondary metabolites that interfere with conductivity, complicating interpretations of results into (opto)electronic applications. However, no previous research has been conducted comparing xylindein quality from different sources. Identification of the pigment source with best (opto)electronic capabilities is therefore important to support further research into technologies based on the pigment. Minimization of these interfering metabolites should also simplify purification methodologies, allowing for cheaper mass production of purified pigment for optoelectronic technologies. This research sought to identify Chlorociboria spp. growth conditions for the highest xylindein production with the fewest contaminants. This will support further research into xylindein’s use in (opto)electronics. Sustainable, natural product-derived materials such as xylindein have been a focus of research for organic electronics [36,37]. Organic semiconductors have economic and material advantages for photovoltaics in addition to their sustainability [38,39]. Pigments in particular have been shown to be effective organic semiconductors [40–44]. Identification of growth conditions that result in pigment extracts with purity high enough to support direct use in (opto)electronics could allow for low-cost production of technologies built on xylindein. As an organic optoelectronic material with good light stability [13] it could be used in devices such as organic solar cells, which have the potential to replace the environmental issues associated with traditional solar cell technology [45]. In order to obtain quality material to support this research, the systemic comparison of existing production methods to new processes is critical to establish the best growth conditions. Tailoring production of secondary metabolites from fungi for (opto)electronics is a novel approach in the field of organic semiconductors. Pigment production was compared in multiple media across liquid shaking, liquid static, and solid woodchip-amended plate cultures, and parallels were drawn with all previous stimulatory Chlorociboria growth/pigment generation research (the two are often interchangeable in publications despite not meaning the same thing). Next, to compare speed, quantity, and quality of xylindein production, this study compared growth of two C. aeruginascens strains (UAMH 11655 and UAMH 7614) and one C. aeruginosa strain (UAMH 11657)—all commonly used in U.S. spalting research. Identification of the species and strain of Chlorociboria that produces the highest extractable quantities of the pigment xylindein, with best purity, in the quickest time, will allow for the most efficient production of the pigment. This budding technology could allow for development of truly sustainable energy production, and identification of “best” species and strain (in terms of xylindein synthesis) is critical for expansions of research, purification studies, and technological adoption of xylindein into commercial green energy fields.

2. Materials and Methods

2.1. Preliminary Media Screen Chlorociboria aeruginosa (UAMH 11657, isolated from a rotting hardwood log in Halliburton, ON, Canada) was inoculated into 50 mL of sterilized media in 250 mL borosilicate Mason jars with plastic screw top lids. All media were inoculated using single point inoculation (6 mm diameter round) from cultures grown on 2% malt agar, sealed with Paraﬁlm “M” and allowed to grow at ambient room conditions (21 ◦C and 35% relative humidity (RH)) for 2, 3, 5, or 23 weeks with a total of 5 repetitions of each tested condition. Mediums tested included Czapek Dox Liquid Medium (M1170A, HIMEDIA, Mumbai, India), low pH fungal broth (M265, HIMEDIA, Mumbai, India), potato dextrose broth (254920, Difco, Franklin Lakes, NJ, USA), 2% brewer’s malt extract (Briess CBW Sparkling Amber Dry Malt Extract, Chilton, WI, USA), 2% bacteriological malt extract (VWRVJ873, VWR Life Science, Radnor, PA, USA), 2% honey (Glory Bee Raw Paciﬁc Northwest Blackberry Blossom Honey, Eugene, OR, Processes 2020, 8, 1477 4 of 23

USA), 2% of 10.4% cellulose nanocrystal suspension (University of Maine Process Development Center, ME, USA), peptone yeast glucose (PYG) media (30 g/L dextrose (BDH9230, VWR Analytical, Radnor, PA, USA), 1.25 g/L ultrapure bacteriological peptone (J20048-P2, Thermo Scientiﬁc, Waltham, MA, USA), 1.25 g/L yeast extract(103303, Mp Biomedicals, China)), yeast peptone soluble starch (YPSS) media (4 g/L yeast extract(103303, Mp Biomedicals, China), 15 g/L solubilized starch (470302-754, Ward’s Science, Henrietta, NY, USA), 1 g/LK2HPO4(0781, VWR Life Science, Radnor, PA, USA), 0.5 g/L MgSO4(BDH9246, VWR Analytical, Radnor, PA, USA)). Media for further experimentation were selected at week 5 on the basis of growth morphology, although the experiment was continued until week 23 to see if extracellular secretion of pigment would occur. Bacteriological malt, brewer’s malt, potato dextrose broth (PDB), and honey were selected for further testing, as these media showed pigment production in hyphae by week 5 of growth.

2.2. Growth Conditions Testing Chlorociboria aeruginosa growth was compared across solid-state fermentation, static liquid state fermentation, and shaking liquid state fermentation states for 4 media selected on the basis of preliminary results. Bacteriological malt, brewer’s malt, and honey were all analyzed for main sugars by the Linus Pauling Core Laboratories at Oregon State University, with the following results: bacteriological malt showed 16.1% glucose, 3.22% fructose, 76.93% maltose, and 3.65% sucrose; brewer’s malt showed 18.12% glucose, 2.19% fructose, 73.35% maltose, and 6.33% sucrose; and honey contained 53.75% glucose, 41.10% fructose, 4.57% maltose, and 0.59% sucrose. Liquid media cultures were grown and extracted following the general method laid out in Weber et al. (2016), and solid cultures followed the general method laid out in Robinson et al. (2012). Single point inoculation of Chlorociboria aeruginosa (strain UAMH 11657, isolated from a rotting hardwood log in Halliburton, ON, Canada) was used for all samples. All media was inoculated from cultures grown on 2% malt agar for 6 weeks at ambient room conditions (21◦ C and 35% RH). Cultures were then sealed with Paraﬁlm “M” and allowed to grow at ambient room conditions for 3, 5, 7, or 9 weeks with a total of 5 repetitions of each tested condition. Cultures were then extracted into 30 mL of HPLC grade dichloromethane (DCM) (BDH23373, VWR, Radnor, PA) and color read, calculating color diﬀerence between all samples and dichloromethane alone. Due to a lack of any visible pigmentation, static brewer’s malt and potato dextrose broth (PDB) in week 3 and static PDB in week 5 were ascribed a ∆E value of zero. Before inoculation and at week 9, liquid cultures were tested with pH strip (VWR pH-Test 0–14, BDH35309, VWR, Radnor, PA, USA) to identify pH. In addition to comparison of extracted pigment production, C. aeruginosa growth was also compared between plates amended with woodchips and those with only tested media. Pigment cannot be extracted from non-amended plates following the standard procedure laid out by [23], but the growth radius was measured from point of inoculation to colony edge, and growth morphologies were compared.

2.2.1. Liquid Shaking Inoculated 250 mL borosilicate Mason jars containing 50 mL of sterile media were placed on a shake table (Orbital Gene Scientific Industries, Bohemia, NY, USA) and shaken at 99 rpm for the incubation period. This speed was selected as the fastest speed that would securely support the moving weight of shaking cultures. Contents of the jars were individually blended on the ice crush setting for 2 min with an Oster classic series blender (Oster, Atlanta, GA, USA) to break up hyphae and improve pigment yield, as described in Weber et al. (2016). Blended liquid and 30 mL of DCM was added per 50 mL jar of media in a 250 mL Erlenmeyer flask. Flasks were corked and manually swirled for 3 min to fully mix water and DCM layers, then allowed to settle for 27 min. Contents of flasks were then placed in a 250 mL separatory funnel and DCM layer was collected. DCM layer was then color read as described below. Processes 2020, 8, 1477 5 of 23

2.2.2. Liquid Static Inoculated 250 mL borosilicate Mason jars containing 50 mL of sterile media were grown statically for set incubation period. Cultures were then blended and extracted, and pigment production was measured via color reading as described for liquid shaking cultures.

2.2.3. Solid State Woodchip Plates Chlorociboria aeruginosa was cultured on sterile disposable Petri plates for each tested media using methodology laid out in Robinson et al. [17]. Each tested liquid media was made up with the addition of sterilized 15 g bacteriological agar (J637, VWR Life Science, Radnor, PA, USA) per 1 L of deionized water, and amended with sterilized white-rotted sugar maple (Acer saccharum Marsh.) shavings processed through #20 mesh in a Wiley mill. Cultures were allowed to grow at ambient room conditions (21 ◦C and 35% RH) for incubation period, then were opened, lids were removed, and the cultures were allowed to dry under ambient conditions in a fume hood for 48 h prior to extraction. Once dry, cultures were broken up into small ( 1 mm) pieces using a blender on the ice ≈ crush setting for 2 min in order to break down hyphal walls and release intra-cellular pigment in addition to extra-cellular pigment already present in media. Ground culture material was placed in a 250 mL Erlenmeyer flask with 30 mL of dichloromethane (DCM), and a 7.0 mm 24.5 mm magnetic stir × bar was added. A rubber stopper was placed on top of the flask to prevent evaporation, and the flask was stirred at 230 rpm for 30 min. After stirring, the solution was filtered through laboratory grade Whatman no. 1002150 (Whatman, Madistone, UK) filter paper and color read as described below.

2.3. Analysis of Pigment Concentration for Growth Conditions Testing A glimmix model followed by Tukey’s Honestly Significant Difference (HSD) test was performed on SAS version 9.4, using the independent variables media, week, and growth condition (shaking, static, or woodchip plates) and the dependent variable color difference (∆E). Color readings of controls extracted into DCM were subtracted from ∆E values to account for variation in pigmentation resulting from the different media. To account for heteroscedasticity of data, we relaxed the assumption of constant variance for both media and week variables using the random option.

2.4. Comparisons of Species and Strain Pigmentation Fungal cultures were grown in liquid shaking media and on woodchip-amended plates (specific protocols below). Single point inoculation from cultures grown on 2% malt agar was used, including Chlorociboria aeruginosa (UAMH 11657, isolated from a rotting hardwood log in Halliburton, ON, Canada) and two strains of Chlorociboria aeruginascens, UAMH 11655 (derived from rotting hardwood in Halliburton, ON, Canada) and UAMH7614 (isolated from single ascospore in Lake District, UK). After inoculation, plates and jars were sealed with Parafilm “M” and allowed to grow at ambient room conditions (21 ◦C and 35% RH, grown in the dark) for 1 to 9 weeks, with 3 cultures from each test condition harvested and extracted each week. Cultures were then extracted into 30 mL of HPLC-grade dichloromethane (DCM) (VWR BDH23373) and color read as described below, calculating color difference between all samples and dichloromethane alone. In addition, colony radius was measured for each species in woodchip plates via the colony edge, identifying largest diameter going through colony center and averaging this value with smallest diameter for total colony diameter.

2.4.1. Liquid Shaking Cultures for Strain Comparison Liquid shaking cultures were prepared for strain comparison as described above. However, after blending culture liquid and 30 mL of dichloromethane (DCM) (BDH23373) was stirred at 100 rpm on ≈ a 15 point multiposition stir plate (Variomag Telesystem, Thermo Scientiﬁc) for 30 min using a 2 cm × 7 mm stir bar. Contents of ﬂasks were then placed in a 250 mL separatory funnel and the DCM layer was collected. This layer was then color read as described below. Processes 2020, 8, 1477 6 of 23

2.4.2. Solid State Woodchip Plates for Strain Comparison Woodchip plates with 2% bacteriological malt extract (VWR Life Science VJ873) amended with white-rotted sugar maple (Acer saccharum Marsh.) were made up, inoculated, and extracted using methods described above for media comparisons. Extracted pigment was color read as described below.

2.5. Data Analysis for Comparisons of Species and Strain A glimmix model followed by Tukey’s HSD test was performed on SAS version 9.4, using the independent variables species, growth condition, and week, and dependent variable color difference (∆E). To account for heteroscedasticity of data, the assumption of constant variance was relaxed for species and week variables using the random option. The tested hypothesis was that there would be a significant difference between color differences (∆E) as a result of the effects of species and week. An additional glimmix model with Tukey’s HSD was run, comparing radius of colony growth, with independent variables week and species. Assumption of constant variance was relaxed using the random option for variable week. The tested hypothesis was that there would be a significant difference between colony radii as a result of the effects of species and week.

2.6. Color Reading and Difference Calculation Color reading was carried out using a Konica Minolta CR-5 Chroma meter, following standard methodology using the CIE L*a*b color space, which measures color in three-dimensional space with an axis running from green ( a) to red (a), another running from blue ( b) to yellow (+b), and a − − vertical axis (L*) running from black (0) to white (100) [42]. Color difference (∆E) was calculated with the CIE76 formula (Equation (1), below), which is more suitable for liquids than CIE00 and has been used in previous studies as an indirect measure of pigment concentration for spalting fungal pigments [10,38,40,43]. The validity of this method has also been confirmed in comparison to HPLC data [22]. Extracted pigment samples were individually color read in 3 mL VWR Spectrophotometer Cell (414004-067). r 2 2 2 ∆E = L L + a a + b b (1) 2∗ − 1∗ 2∗ − 1∗ 2∗ − 1∗

2.7. Optical Absorption of Extracted Pigment Solutions After color reading data was taken, optical absorption spectra were measured using a Xe lamp (96000 Oriel, Stratford, CT, USA) and a ﬁber coupled Ocean Optics USB2000 spectrometer (Ocean Optics, Largo, FL, USA) to determine relative purity. Absorbance (A) was calculated from the incident (I ) and transmitted (I) beam intensities as A = log(I/I0), then normalized at the peak absorbance 0 − around 670 nm. Reﬂection losses were taken into account by referencing with respect to a cuvette with pure DCM. For comparisons of growth conditions, we dropped PDA samples from comparison due to lack of pigmentation, and a control of ethanol-washed xylindein was included, prepared following methods used in a previous study investigating xylindein purity [12].

2.8. Measurement of Electronic Properties For measurements of current-voltage (I-V) characteristics, voltage was applied to the samples as described in our previous publications [14,15] using a Keithley 237 source-measure unit. Current was measured as a function of applied voltage in the 0–300 V range, under ambient conditions in the dark. Samples of each tested strain grown to 12 weeks in liquid cultures and extracted as described above were tested before and after an ethanol wash, which has been shown to improve conductivity in previous work [14]. This wash consisted of rinsing vials of solid xylindein adhered to the vial with multiple passes of clean ethanol (Sigma-Aldrich) until the ethanol wash [14] no longer showed measurable fluorescence under UV light. Xylindein powder was then dissolved in DCM to form 10 mg/mL solutions, which were drop-cast onto glass substrates with interdigitated Au electrodes with Processes 2020, 8, 1477 7 of 23 a 25 µm gap. Substrates were patterned using conventional lift-off photolithography, and Au with a 5 nm Cr adhesion layer was deposited using a Veeco 7700 thermal evaporator. The effective mobilities were calculated from the space-charge limited current (SCLC) regime of the I-V characteristics (when I is proportional to the voltage squared V2), in the thin-film approximation [14,15] (Equation (2)). 2µe f f εε0 V2 j = (2) π L2 where j is the linear current density, j = I/d; I is the measured current; and d is the length of the electrode. V is the applied voltage, L is the gap between the electrodes, ε0 is the vacuum permittivity, ε is the dielectric constant (assumed to be equal to 3), and µeff is the effective mobility.

3. Results and Discussion

3.1. Pigment Variation across Growth Conditions Pigment production varied across growth conditions and between species. In the first phase of the experiment, C. aeruginosa UAMH 11657 showed different pigment production in response to growth conditions. This was apparent in culture morphology (Figure1) and significant di fferences in colony radii (Figure S1). Chlorociboria aeruginosa grown in lab-grade malt extract under shaking conditions resulted in the highest mean extracted pigment production, as measured by color difference (∆E), in the shortest amount of time (Figure2). The interaction between media, week, and growth conditions was highly significant (df = (18,150), F-value = 13.47, p < 0.0001). However, while shaking lab grade malt colonies grown for 9 weeks yielded the largest ∆E, it was not significantly larger than all other growth conditions. It was significantly different compared to all PDB (potato dextrose broth) conditions (p < 0.0001) and brewer’s malt conditions (p < 0.0001), other than woodchip amended plates grown for 7 and 9 weeks. Shaking lab grade malt cultures at week 9 were also significantly different in comparison to all honey growth conditions, apart from week 9 woodchip-amended plates (p = 1) and static honey cultures (p = 1). Lab-grade malt shaking cultures showed the highest ∆E value in week 3 and were significantly different from all other conditions in that week (p > 0.07). While overall xylindein production in liquid cultures was comparable to production in woodchip plates, higher levels of pigment were produced in liquid cultures in earlier growth stages. The growth rate in shaking lab grade malt cultures was sufficient to meet the concentration of standard xylindein solution (∆E = L* = 82.28 ( 2.0), a* = 11.06 ( 2.0), and b* = 5.40 ( 2.0)) used in previous ± − ± − ± publications [7,46] after only 5 weeks of incubation. In week 5, color values were L* = 83.92, a* = 9.83, b* = 6.20 for shaking cultures, equal to the standard, and by week 9 these values had − − exceeded the standard, reaching the considerably darker L* = 77.64, a* = 7.61, b* = 5.178. As the − − standard was based upon mean values of cultures grown for 2–3 months and then triple-extracted [7], this growth method showing higher concentrations in a fraction of the time is considerably faster. In addition, extracted pigment from the same strain in malt shaking cultures at week 5 (∆E = 21.1 10.8) ± was roughly equivalent to previous work where ∆E of 17 [24]. ≈ Chlorociboria colony sizes seen were also comparable to those reported in previous literature. Robinson et al. [17] achieved full plate colonization of Chlorociboria aeruginascens in 26 days, with colony diameters limited by plate size of 17.6 7.7 mm. This was equivalent to the maximum colony ± diameter seen in Robinson and Laks, which was obtained in 4 weeks but persisted over a period of 4 months [47]. Tudor et al. [22], using the same methodology and strain, had cultures over twice that size, which may be ascribed to differences in species or strain tested. This decrease in speed of growth may be due to the effects of laboratory storage, which has been seen to affect growth rate [17]. The increase in colony diameter shown in these results upon addition of woodchips also mirrors previous results [17,22], although it was only significant for three varieties of media after 9 weeks of growth. Finally, Stange et al. [25] reported larger colonies after a period of 10 weeks of growth; Processes 2020, 8, 1477 8 of 23

Processes 2020, 8, x FOR PEER REVIEW 8 of 23 however, they did not begin to pigment until late in their growth, and a later paper focusing on pigmentationdid not begin reported to pigment a colony until radius late inof their22 mm growth, after and 8 weeks a later [ 48 paper]. focusing on pigmentation ≈ Whilereported comparisons a colony radius of colony of ≈22 mm diameter after 8 haveweeks been [48]. used in most historical work and are therefore used as aWhile point comparisons of comparison, of col pigmentony diameter production have been was used found in most to historical be only work inconsistently and are therefore related to used as a point of comparison, pigment production was found to be only inconsistently related to colony size. Week 9 woodchip lab-grade malt cultures were not significantly different in terms of colony size. Week 9 woodchip lab-grade malt cultures were not significantly different in terms of colonycolony radius radius in comparison in comparison with with brewer’s brewer’s malt at at week weekss 7 and 7 and 9 (p 9 = ( p0.99,= 0.99, 1), or 1), honey or honey at week ats weeks5, 7, 5, 7, andand 9 (9p (=p =0.54, 0.54, 0.48,0.48, 1.0). Extracted Extracted pigment pigment from from the lab the-grade lab-grade malt plates malt platesat week at 9 weekwas also 9 was not also not significantlysignificantly didifferentfferent ( (pp == 1)1) from from brewer brewer’s’s malt malt at week at week 7 and 7 and9 (p = 9 1), (p =but1), was but only was equivalent only equivalent to to honeyhoney at at week week 99 ((pp = 1),1), and and also also lab lab-grade-grade malt malt at week at week 7 (p = 71). (p In= addition,1). In addition, despite a despitehigh mean a high meancolony colony size size in in honey honey plates plates at week at week 5, no 5, similar no similar increase increase in pigment in pigment production production was seen. was This seen. This suggestssuggests surface surface colonization colonization of ofmedia media only. only. Hence Hence,, while while colony colony diameter diameter generally generally corresponds corresponds to to extractableextractable pigment pigment inin cultures,cultures, this this relationship relationship is not is not a reliable a reliable measure. measure.

FigureFigure 1. Cultures 1. Cultures of Chlorociboria of Chlorociboria aeruginosa aeruginosain in allall testedtested media media at at 9 9wee weeks.ks. All All liquid liquid cultures cultures were were shaken at 99 rpm. (a) Static honey liquid culture; (b) static potato dextrose broth (PDB) liquid culture; shaken at 99 rpm. (a) Static honey liquid culture; (b) static potato dextrose broth (PDB) liquid culture; (c) static brewer’s malt liquid culture; (d) static lab-grade malt liquid culture; (e) shaking honey liquid (c) static brewer’s malt liquid culture; (d) static lab-grade malt liquid culture; (e) shaking honey culture; (f) shaking PDB liquid culture; (g) shaking brewer’s malt liquid culture; (h) shaking lab-grade liquidmalt culture; liquid (culture;f) shaking (i) honey PDB agar liquid culture; culture; (j) PDB (g) agar shaking culture; brewer’s (k) brewer’s malt malt liquid agar culture; culture; ( h(l) lab shaking- lab-grade malt liquid culture; (i) honey agar culture; (j) PDB agar culture; (k) brewer’s malt agar culture; ( l) lab-grade malt agar culture; (m) honey woodchip agar culture; (n) PDB woodchip agar culture; (o) brewer’s malt woodchip agar culture; (p) lab-grade malt agar culture. Processes 2020, 8, x FOR PEER REVIEW 9 of 23

Processesgrade2020, 8 malt, 1477 agar culture; (m) honey woodchip agar culture; (n) PDB woodchip agar culture; (o9) of 23 brewer’s malt woodchip agar culture; (p) lab-grade malt agar culture.

FigureFigure 2. 2.Mean Mean color color di ﬀdifferenceerence (∆E) (ΔE) resulting resulting from from variation variation of media of media and cultivation and cultivation technique. technique Error . barsError represent bars represent standard standard error. Interaction error. Interaction between between media, media, week, andweek, growth and growth conditions conditions tested withtested awith Proc a Glimmix Proc Glimmix model model was found was found to be to highly be highly signiﬁcant significant (df = (df18,150, = 18,150,F-value F-value= 13.47, = 13.47,p

In addition, previous research has also shown pH to be a significant factor in pigmentation In addition, previous research has also shown pH to be a significant factor in pigmentation of of Chlorociboria aeruginascens, with the highest pigmentation reported at pH 2.5 and 5 in malt agar Chlorociboria aeruginascens, with the highest pigmentation reported at pH 2.5 and 5 in malt agar plates plates [49] and at pH of 4 in orange juice medium [48]. Here, the pH of liquid cultures was tested [49] and at pH of 4 in orange juice medium [48]. Here, the pH of liquid cultures was tested before before inoculation and at the end of nine weeks of growth. Overall, media became less acidic after inoculation and at the end of nine weeks of growth. Overall, media became less acidic after fungal fungal growth (Table1). Pigment formation in malt extract agar (MEA) at di fferent pH values in this growth (Table 1). Pigment formation in malt extract agar (MEA) at different pH values in this range range (as tested by Tudor, Robinson and Cooper [49]) showed brownish pigmentation at a pH of 7, and (as tested by Tudor, Robinson and Cooper [49]) showed brownish pigmentation at a pH of 7, and dark green pigmentation at pH values of 5 and 6. Overall, this aligns well with morphology seen in dark green pigmentation at pH values of 5 and 6. Overall, this aligns well with morphology seen in malt media. However, the lack of pigmentation seen in pH 5 PDB, and the dark pigmentation in honey, malt media. However, the lack of pigmentation seen in pH 5 PDB, and the dark pigmentation in show that the differences were not due to pH changes but down to the differences in growth conditions. honey, show that the differences were not due to pH changes but down to the differences in growth conditions. Table 1. pH of liquid media before and after Chlorociboria growth.

Growth ConditionTable 1. pH of Honey liquid media before Brewer’s and Malt after Chlorociboria Lab-Grade growth Malt. PDB Pre-growthGrowth Condition 6Honey Brewer’s 6 Malt Lab-Grade 6 Malt PDB 5 Static post-growth 7 6 5 5 Pre-growth 6 6 6 5 Shaking post-growth 7 6 5 6 Static post-growth 7 6 5 5 Shaking post-growth 7 6 5 6 The superior performance of shaking liquid cultures may have been due to multiple factors. As an aerobic organism, increased oxygen availability in liquid culture may have facilitated higher levels of growth. While the volumetric oxygen transfer coefficient (kLa) was not determined, agitation Processes 2020, 8, 1477 10 of 23 of cultures is standard in bioreactors to facilitate increased gas exchange, and both agitation and oxygenation have been found to influence secondary metabolite production in filamentous fungi [50,51]. However this explanation may not fully explain the results, as previous work has found that increased level of oxygen was not associated with increases in pigment production [48]. In addition to the possible influence of increased oxygen availability, the shear stresses experienced by the fungi in shaking liquid cultivation may have led to breakdown of fungal hyphae. This could have led to increased pigmentation as a stress response. The superior pigmentation of Chlorociboria aeruginosa grown on malt extract confirmed results from other researchers [19,20]. It was hoped that there would not be a significant difference between the bacteriological malt extract and the more economical brewer’s malt extract, allowing for cheaper pigment production. However, while brewer’s malt performed well in woodchip plates, it did not compare to the pigment production from shaking lab-grade malt cultures. Overall, ratios of four tested sugars were similar between malt varieties, with bacteriological-grade malt containing a slightly higher percent maltose (76.93% vs. 73.35%). As maltose has been identified as a preferred carbon source, this may help account for color discrepancies, although this may also be down to nitrogen availability, which has been suggested to have more of an effect on pigmentation of Chlorociboria species [21]. The minimal pigmentation seen in the PDB cultures’ solid culture was also reflective of previous research, with potato starch described as producing brown and light green coloration of colonies in solid growth substrate [20,22]. Honey resulted in good growth and pigmentation, equivalent to or better than all other growth medias and conditions. The honey mostly contained glucose and fructose, with smaller amounts of maltose and sucrose. The pigmentation produced by this combination is consistent with studies showing pigmented Chlorociboria growth in fruit juices [20,21,25,26,48], which normally contain glucose, fructose, and sucrose. Glucose media in particular have been described leading to pigmented growth [19–21]. Variations of growth morphology are more likely indicative of varying levels of nitrogen present in media, and fungal stress responses. While nitrogen content was not analyzed in these tested media, other work on media where nitrogen is not a limiting factor has shown fungal metabolism, focusing on biomass production with pale growth until late in colony life when available nitrogen has likely been depleted [21]. Determination of sugar concentration and biomass were also not assessed due to the blending and extraction methodology interfering with measurements. Biomass in particular was not feasible due to the very fine texture of fungal particles and their tendency to stick to separatory funnel walls. Other work [21] has shown that pigment production in Chlorociboria does not directly relate to biomass, and the pattern of high biomass not being associated with high pigmentation was seen in the preliminary media screen, where multiple media only started to show pigmentation after months of colony growth, and in pale growth in early weeks for all media apart from honey. Honey showed high levels of pigmentation and visibly low levels of biomass growth, supporting a metabolic switch from growth to pigmentation. However, this switch was also seen to be associated with other growth conditions, as shown in PDA plates, which began to pigment only on addition of woodchips. As xylindein production has been seen to occur in response to environmental stressors [48], it is likely that these woodchips provided the stress that induced pigmentation, perhaps through containing secondary metabolites of other decay fungi once present in the wood.

3.2. Pigment Variation across Chlorociboria Species and Strains Xylindein production was seen to vary over time, between strains, with a significant difference seen for the interaction between growth condition (liquid media vs. woodchip), week, and fungal strain ((df = 27, 144), F = 47.93, p < 0.0001). The highest mean color difference (∆E) of 26.45 ( 0.44) was ± seen in extracted pigment from C. aeruginascens UAMH 11655 grown on woodchip-amended plates for 9 weeks (Figure1). However, while significantly di fferent from all values in weeks 1 through 4, and Processes 2020, 8, 1477 11 of 23

Processes 2020, 8, x FOR PEER REVIEW 11 of 23 all control values (p < 0.0001), it did not significantly differ after week 5 (aside from C. aeruginascens UAMHand all 7614 control woodchip values plates (p < at0.0001), week 5 it (p did< 0.0001)). not significantly differ after week 5 (aside from C. aeruginascensWhile cultures UAMH resulted 7614 woodchip in similar plates levels at of week extracted 5 (p < pigment 0.0001)). in later weeks, early weeks showed differentWhile pigment cultures production resulted in patterns. similar levels By week of extracted 2, ∆E of C.pigment aeruginosa in laterUAMH weeks, 11657 early was weeks significantly showed didifferentfferent than pigment the liquid production media controlpatterns. (p By< 0.0001). week 2,C. ∆E aeruginosa of C. aeruginosaUAMH UAMH 11657 also11657 showed was significantly a significant didifferentfference from than C. the aeruginascens liquid mediaUAMH control 7614 (p < (p 0<.0001).0.0001). C. Inaeruginosa weeks 3 andUAMH 4, liquid 11657 cultures also showed generally a performedsignificant better difference than woodchipfrom C. aeruginascens plate cultures, UAMH with 7614C. aeruginascens (p < 0.0001). InUAMH weeks 7614 3 and yielding 4, liquid the cultures highest meangenerally color performed changes (Figure better tha3).n In woodchip week 3, plateC. aeruginascens cultures, withUAMH C. aeruginascens 7614 in liquid UAMH media 7614 showedyielding a significantlythe highest highermean color value changes than in its(Figure pigment 3). In production week 3, C. in aeruginascens wood (p < 0.0001) UAMH or compared7614 in liquid to woodchip media culturesshowed of a significantlyC. aeruginosa higherUAMH value 11657 than and in its control pigment (p =production0.001). In in week wood 4, (pC. < aeruginascens0.0001) or comparedUAMH 7614to woodchip still had thecultures highest of C mean. aeruginosa of 20.76 UAMH ( 1.00), 11657 and and showed control a ( significantp = 0.001). In di weekfference 4, C. compared aeruginascens to all ± otherUAMH tested 7614 conditions still had the apart highest from meanC. aeruginosa of 20.76 UAMH(±1.00), and 11657 showed culture a significant (p = 0.3764 difference liquid media, comparedp = 0.51 solidto all media). other tested conditions apart from C. aeruginosa UAMH 11657 culture (p = 0.3764 liquid media, p = 0.51 solid media).

Figure 3. Differences in color (∆E) produced by Chlorociboria sp. over 9 weeks growth. Strain UAMH Figure 3. Differences in color (∆E) produced by Chlorociboria sp. over 9 weeks growth. Strain UAMH Chlorociboria aeruginosa Chlorociboria 1165711657 was was Chlorociboria aeruginosa,, and and strains strains UAMH 11655 11655 and and UAMH UAMH 7614 7614 were were Chlorociboria aeruginascens.aeruginascens. Error bars represent one standard error. error. A A significant significant difference difference was was seen seen for for the the interactioninteraction between between growth growth substrate substrate (liquid(liquid mediamedia vs. woodchip), woodchip), week, week, and and fungal fungal strain strain ((df ((df = =27,27 , 144),144),F F == 447.93,7.93, pp << 0.00010.0001).). No Nosignificant significant differences differences were wereseen after seen week after 5 week in ∆E5 between in ∆E betweenfungal fungalcultures. cultures.

CulturesCultures showed showed consistent consistent morphology morphology when grown grown in in liquid liquid culture, culture, with with color color variation variation (Figure(Figure4). 4). Most Most notably notablyC. C. aeruginascensaeruginascens UAMH 11655 by week week 9 9 had had media media that that appeared appeared somewhat somewhat yellow,yellow, whereas whereas the the other other cultures cultures were morewere consistently more consistently blue/green. blue/green. Upon blending, Upon blending, these diﬀ erences these weredifferences no longer were as no apparent. longer as These apparent. visual These diﬀerences visual differences indicate variability indicate variability in secondary in secondary metabolite productionmetabolite betweenproduction cultures, between and cultures, inhomogeneity and inhomogeneity of extractions. of extractions.

Processes 2020, 8, 1477 12 of 23 Processes 2020, 8, x FOR PEER REVIEW 12 of 23

Figure 4. Liquid cultures of tested Chlorociboria species and strains in lab-gradelab-grade malt extract liquid media at at 99 weeks weeks of of growth. growth. (a ) (a Shaking) Shaking cultures; cultures; (b) the (b) samethe same shaking shaking cultures cultures after blending after blending 3 min to3 minutbreakes up to hyphae. break up hyphae.

Extraction results mirrored colony radius on amended woodchip plates (Figure 55)),, asas waswas seenseen in comparisons of growth conditions. A A significant significant difference difference was seen between species radii over time (df(df (16, 54), F == 4.34, p < 0.001)0.001)).). However However,, on on the the basis basis of of Tukey comparisons,comparisons, we found no significancesignificance within within each each week week between between strains strains until until week week 8; there 8; was there a significant was a significant difference difference between C.between aeruginascens C. aeruginascensUAMH 7614UAMH and 7614C. aeruginascens and C. aeruginascensUAMH 11655UAMH (p 11655= 0.0037), (p = where0.0037), strain where UAMH strain 11655UAMH had 11655 the had highest the meanhighest radius mean overallradius withoverall a valuewith a of value 19.5 (of 19.54.3). (±C. 4.3). aeruginosa C. aeruginosaUAMH UAMH 11657 ± was11657 found was found to have to thehave highest the highest mean mean size in size week in week 9, although 9, although this was this not was significantly not significantly different different from weekfrom week 5 (p = 50.25 (p =), 0.25), or from or fromC. aeruginascens C. aeruginascensUAMH UAMH 11655 11655 and andC. aeruginosa C. aeruginosaUAMH UAMH 11657 11657 in anyin any of ofthe the following following weeks weeks (p > (p0.1). > 0.1). Week Week 9 also 9 also showed showed variations variations in colony in colony morphology morphology (Figure (Figure6), with 6), C.with aeruginascens C. aeruginascensUAMH UAMH 7614 7614 lacking lacking a dark a dark border border around around the colonythe colony edge, edge which, which was was seen seen in the in theother other two two strains. strains. These results confirm previous research showing variation in colony size between pigmenting species and strains of Chlorociboria [17,22]. In addition, it lends support to the assumption that larger colony sizes between different species and strains are associated with increases in actual extractible pigment. This was demonstrated by C. aeruginascens UAMH 7614, which showed a consistently smaller colony diameter in comparison with the other species/strains tested, and showed lower mean ∆E values in woodchip plates also, although this difference was not significant. As it has been hypothesized that pigment production redirects metabolic energy away from biomass production [21], this result was important to confirm.

Processes 2020, 8, x FOR PEER REVIEW 13 of 23 Processes 2020, 8, 1477 13 of 23

Processes 2020, 8, x FOR PEER REVIEW 13 of 23

Figure 5. Comparison of colony radius and Chlorociboria variety in lab-grade malt agar media. UAMH Figure7614Figure and 5. 5.Comparison UAMH Comparison 11655 of of colony werecolony both radius radius C. and and aeruginascens, Chlorociboria with varietyvariety UAMH in in lab lab-grade - 11657grade maltC. malt aeruginosa. agar agar media. media. Errors UAMH UAMH bars 7614 and UAMH 11655 were both C. aeruginascens, with UAMH 11657 C. aeruginosa. Errors bars 7614represent and UAMHone standard 11655 error. were bothA significantC. aeruginascens, differencewith was UAMH seen over 11657 timeC. ( aeruginosa.df (16, 54), FErrors = 4.34, bars p < represent one standard error. A significant difference was seen over time (df (16, 54), F = 4.34, p < represent0.001). one standard error. A signiﬁcant diﬀerence was seen over time (df (16, 54), F = 4.34, p < 0.001). 0.001).

Figure 6. Woodchip-amended lab-grade malt media plate cultures of species and strains at 9 weeks FigureFigure 6.6. Woodchip-amendedWoodchip-amended lab-gradelab-grade maltmalt mediamedia plateplate culturescultures ofof speciesspecies andand strainsstrains atat 99 weeksweeks of growth. ofof growth.growth. Comparisons of extracted pigment (color only) were similar to what was seen by Weber et al. TheseComparisons results conﬁrmof extracted previous pigment research (color showing only) were variation similar in to colony what sizewas betweenseen by pigmentingWeber et al. [22], who compared only one strain of C. aeruginascens and C. aeruginosa in liquid cultures alone. In species[22], who and compared strains of onlyChlorociboria one strain[17 of,22 C.]. aeruginascens In addition, it and lends C. supportaeruginosa to thein liquid assumption cultures that alone. larger In

Processes 2020, 8, 1477 14 of 23 colony sizes between different species and strains are associated with increases in actual extractible pigment. This was demonstrated by C. aeruginascens UAMH 7614, which showed a consistently smaller colony diameter in comparison with the other species/strains tested, and showed lower mean ∆E values in woodchip plates also, although this difference was not significant. As it has been hypothesized that pigment production redirects metabolic energy away from biomass production [21], this result was important to confirm. Comparisons of extracted pigment (color only) were similar to what was seen by Weber et al. [22], who compared only one strain of C. aeruginascens and C. aeruginosa in liquid cultures alone. In that study, C. aeruginosa UAMH 11657 produced a ∆E of 17 after 6 weeks and 6 days (48 total days), ≈ compared to C. aeruginascens UAMH 7615, which showed little increase in color change after 4 weeks (final ∆E of 12) [24]. In comparison, in this study liquid cultures at 7 weeks (C. aeruginosa strain ≈ UAMH 11657) had a comparable ∆E value of 17.36 7.3. However, C. aeruginascens cultures performed ± equally well, instead of exhibiting plateauing pigment production (UAMH 7614 with mean of 17.24 ± 8.13 and UAMH 11655 of 23.42 5.04). While no significant differences were seen between species or ± strains, 5 weeks was the cut off above which no significant differences were seen between radius size and final colony radius for C. aeruginosa UAMH 11657 and C. aeruginascens UAMH 11655. It is notable that Weber et al. [24] described pigmentation for C. aeruginascens strain 7615 tapering off after 4 weeks, as this also correlates fairly well to weeks 4 and 5, marking a point at which differences between strains are no longer significant. However, this may also be due to limitations in the extraction method used, with pigment not able to transfer from media to DCM after a certain saturation point (lower than xylindein total saturation point). Despite no statistically significant difference between samples after week 6, we found that L*a*b* color values showed strain-based variation in comparison to the standard color values used for standardizing xylindein extraction. The standards were originally developed as an indirect method of quantifying pigment concentration in extract solutions on the basis of a triple extraction of a single plate of C. aeruginosa UAMH 11657 grown in woodchip plates for 2–3 months, in 50 mL of DCM [6], with solutions considered standardized if color values fell within 2.0 of the values L* = 82.28, ± a = 11.06, and b = 5.40. It should be noted that at present there is no method of converting color − − value to concentration. Mathematical relationships have been established for the pigment produced by Scytalidium cuboideum, anther spalting fungi [52]. Use of color values as a measure of concentration has been validated [24]; however, limited amounts of xylindein and a lack of purification techniques have prevented modeling this relationship thus far. Using the above as a measurement bar, average values of C. aeruginosa UAMH 11657 woodchip plate cultures at week 7 were the only test condition that “met” this standard (Table2). C. aeruginosa UAMH 11657 woodchip cultures (weeks 8 and 9) showed lower L* values (the extract was darker, which indicates a higher concentration of xylindein) but otherwise fell within the standard range. However, on the basis of average values alone, several other cultures showed variation in the a* value, while L* and b* values were within range, resulting in “greener” colors (Table2). These included C. aeruginosa UAMH 11657 in liquid media by week 9 and C. aeruginascens UAMH 11655 in all conditions. Woodchip C. aeruginascens UAMH 11655 were also slightly out of range in terms of b* values. However, range of samples shown in standard deviation values showed that even though average values did not fall within the range of the sample, this difference may not be supported statistically. As fungi are biological organisms, variation and inhomogeneity in extracts from them is expected. However, the fact that so many values appear “greener” suggests that there is variation in pigment production between samples. Using the lab standard color chart and previous literature on Chlorociboria color variation, it is possible to hypothesize about color variance in xylindein and other secondary metabolites. The “greener” color likely indicates production of additional pigmented secondary metabolites occurring at different rates, resulting in differences in overall culture coloration. L* values, representing the darkening of solution from increased pigmentation, have been noted to be the most associated with fungal pigment Processes 2020, 8, 1477 15 of 23 concentration in previous work [52]. However, variation of culture color is well known in Chlorociboria sp. Culture colors ranging from blue-green to lime were reported by Frenzel [20], implying the presence of a yellowProcesses compound, 2020, 8, x and FOR PEER Edwards REVIEW and Kale [53] reported a yellow compound they identified15 of 23 as a Processes 2020, 8, x FOR PEER REVIEW 15 of 23 xylindein quinol.Processes A2020 yellow,, 8,, xx FORFOR pigment PEERPEER REVIEWREVIEW was also noted by Weber et al. [24], the amount of which decreased15 of 23 Processes 2020,, 8,, xx FORFOR PEERPEER REVIEWREVIEW 15 of 23 ProcessesThe 20 20variation, 8, x FOR inPEER colors REVIEW seen between strains suggests that using standard color values, as15 ofhas 23 with colony age,The and variation it was suggestedin colors seen to bebetween a potential strains breakdown suggests that or using precursor standard of xylindein.color values, Variation as has been Thedone variation in multiple in colors publications seen between [3,7,9,54] strains, may suggests not be appropriate that using standardfor use on color pigment values, extracted as has in productionbeen of Thedone this variation yellowin multiple in compound colors publications seen between between [3,7,9,54] speciesstrains, may suggests not may be beappropriate that responsible using standardfor use for on the color pigment color values, values extracted as has with beenfrom donecultures in multiplethat are notpublications C. aeruginosa [3,7,9,54] UAMH,, maymay 11657 notnot grown bebe appropriateappropriate on woodchips, forfor useuse or onon at pigmentpigmentthe very extractedextractedleast, the more negativefrombeen a* culturesdone values. in multiplethat The are di notpuff blicationserenceC. aeruginosa is [3,7,9,54] not UAMH seen, may in11657the not grown b*be values,appropriate on woodchips, likely for use because or on at pigmentthe b* very values extractedleast, rangethe froma* values cultures should that have are anot wider C. aeruginosa range of acceptability UAMH 11657 for grown research on or woodchips, applications or wathere the anyvery coloration least, the a*from values cultures should that have are anot wider C. aeruginosa range of acceptability UAMH 11657 for grown research on or woodchips, applications or w athere the anyvery coloration least, the from yellowa*produced to values blue, should andby Chlorociboria presence have a wider ofsp. arange isyellow acceptable. of acceptability compound Indirect for may measuresresearch be outweighed or of applications concentration by w thehere should bluer any coloration be xylindein, based produceda* values should by Chlorociboria have a wider sp. range is acceptable. of acceptability Indirect for measuresresearch or of applications concentration where should any coloration be based resulting inproducedprimarily no overall on by changesL* Chlorociboria values onsimilar this sp. to scale.is S. acceptable. cuboideum [52] Indirect, and specific measures strains of concentration and growth conditions should be should based primarilyproduced on by L* Chlorociboria values similar sp. to is S. acceptable. cuboideum [52] Indirect, and specific measures strains of concentration and growth conditions should be should based primarilybe used if onachieving L* values a specific similar colorto S. cuboideum is desired. [52] ,, andand specificspecific strainsstrains andand growthgrowth conditionsconditions shouldshould beprimarily used if onachieving L* values a specific similar colorto S. cuboideum is desired. [52] , and specific strains and growth conditions should Table 2.beChlorociboria used if achievingextraction a specific colors color with is L*desired. values matching or below standard. Extractions within be used if achieving a specific color is desired. the lab standardTable are2. Chlorociboria denoted withextraction an asterisk,colors with and L* values values matching out of or range below are standard. in bold Extractions. Average within color Table 2. Chlorociboria extraction colors with L* values matching or below standard. Extractions within Tablethe lab 2. standard Chlorociboria are extractiondenoted with colors an withasterisk, L* values and valuesmatching out or of below range standard. are in bold Extractions. Average within color values of a*theTable were lab 2. seen standard Chlorociboria to go are out denotedextraction of range with colors of an standard, withasterisk, L* values and while valuesmatching L* andout or of b* below range values standard. are conformed, in bold Extractions. Average apart within color from theTable lab 2. standard Chlorociboria are denotedextraction with colors an withasterisk, L* values and valuesmatching out or of below range standard. are inin bold Extractions.. AverageAverage within colorcolor valuesthe lab ofstandard a* were areseen denoted to go out with of rangean asterisk, of standard and values, while outL* andof range b* values are ininconformed, bold.. AverageAverage apart colorfromcolor C. aeruginascensvaluesthe labUAMH of standard a* were 11655 areseen woodchipdenoted to go out with of plate rangean asterisk, samples. of standard and values, while outL* and of range b* values are inconformed, bold. Average apart fromcolor valuesC. aeruginascens of a* were UAMH seen to 11655 go out woodchip of range plateof standard samples.,, while L* and b* values conformed, apart from C.values aeruginascens of a* were UAMH seen to 11655 go out woodchip of range plateof standard samples., while L* and b* values conformed, apart from C.values aeruginascens of a* were UAMH seen to 11655 go out woodchip of range plateof standard samples., while L* and b* values conformed, apart from ChlorociboriaC. aeruginascensSource UAMH Week 11655 woodchip L* plate samples. a* b* Color C. aeruginascensChlorociboria UAMH Source 11655 woodchipWeek plate samples.L* a* b* Color Chlorociboria Source Week L* a* b* Color Chlorociboria Source Week L* a* b* Color Chlorociboria Source Week 82.28L* −11.06a* −5.40b* Color Standard (HinschStandard 2015) * (Hinsch 2015) 8–12 * 82.288– (122.00) 82.2811.06 ( 2.00)−11.06 5.40 ( −5.402.00) Standard (Hinsch 2015) * 8–12± (±2.00)82.28− ± (±2.00)−11.06 − (±2.00)±−5.40 Standard (Hinsch 2015) * 8–12 (±2.00)82.28 (±2.00)−11.06 (±2.00)−5.40 Standard (Hinsch 2015) * 8–12 (±2.00)82.28 (±2.00)−11.06 (±2.00)−5.40 Standard (Hinsch 2015) * 8–12 (±2.00)82.28 (±2.00)−11.06 (±2.00)−5.40 Standard (Hinsch 2015) * 8–12 (±2.00) (±2.00) (±2.00) (±2.00) (±2.00) (±2.00) Standard (Hinsch 2015) * 8–12 (±2.00) (±2.00) (±2.00) (±2.00) (±2.00) (±2.00) C. aeruginosa UAMH 11657 woodchip 82.83 −10.43 −6.68 C. aeruginosaC. aeruginosaUAMH 11657 UAMH 11657 woodchip 7 82.83 −10.43 −6.68 C. aeruginosa UAMHplate *11657 woodchip7 82.837 ( 7.18) (±7.18)82.8310.43 ( 3.08)(±3.08)−10.43 6.68 ((±1.40)−6.681.40) woodchipC. aeruginosa plate * UAMHplate *11657 woodchip 7± (±7.18)82.83− ± (±3.08)−10.43 − (±1.40)±−6.68 C. aeruginosa UAMHplate *11657 woodchip 7 (±7.18)82.83 (±3.08)−10.43 (±1.40)−6.68 C. aeruginosa UAMHplate *11657 woodchip 7 (±7.18)82.83 (±3.08)−10.43 (±1.40)−6.68 plate * 7 (±7.18) (±3.08) (±1.40) plate * (±7.18) (±3.08) (±1.40) plate * 7 (±7.18) (±3.08) (±1.40) plate * (±7.18) (±3.08) (±1.40) C. aeruginosa UAMH 11657 woodchip 77.36 −6.47 C. aeruginosaC. aeruginosaUAMH 11657 UAMH 11657 woodchip 8 77.36 −7.68 (±2.62) −6.47 C. aeruginosa UAMHplate 11657 woodchip8 77.36 (8 1.37) (±1.37)77.367.68 ( −7.682.62) (±2.62) 6.47 ((±1.04)−6.471.04) C. aeruginosa UAMHplate 11657 woodchip 8 (±1.37)77.36 −7.68 (±2.62) (±1.04)−6.47 woodchipC. aeruginosa plate UAMHplate 11657 woodchip 8± (±1.37)77.36− ±−7.68 (±2.62)− (±1.04)±−6.47 C. aeruginosa UAMHplate 11657 woodchip 8 (±1.37)77.36 −7.68 (±2.62) (±1.04)−6.47 plate 8 (±1.37) −7.68 (±2.62) (±1.04) plate (±1.37) (±1.04) plate 8 (±1.37) −7.68 (±2.62) (±1.04) plate (±1.37) (±1.04) C. aeruginosa UAMH 11657 woodchip 75.96 −6.17 C. aeruginosa UAMH 11657 woodchip 9 75.96 −6.96 (±2.04) −6.17 C. aeruginosaC. aeruginosaUAMH 11657 UAMHplate 11657 woodchip 9 (±1.61)75.96 −6.96 (±2.04) (±0.75)−6.17 C. aeruginosa UAMHplate 11657 woodchip9 75.96 (9 1.61) (±1.61)75.966.96 ( −6.962.04) (±2.04) 6.17 ((±0.75)−6.170.75) C. aeruginosa UAMHplate 11657 woodchip 9 (±1.61)75.96 −6.96 (±2.04) (±0.75)−6.17 woodchipC. aeruginosa plate UAMHplate 11657 woodchip 9± (±1.61)75.96− ±−6.96 (±2.04)− (±0.75)±−6.17 plate 9 (±1.61) −6.96 (±2.04) (±0.75) plate (±1.61) (±0.75) plate 9 (±1.61) −6.96 (±2.04) (±0.75) plate (±1.61) (±0.75) C. aeruginosa UAMH 11657 liquid 83.05 −13.32 −6.62 C. aeruginosa UAMH 11657 liquid 9 83.05 −13.32 −6.62 C. aeruginosa UAMHC. aeruginosa 11657liquid UAMHmedia 11657 liquid 9 (±4.45)83.05 (±3.20)−13.32 (±2.09)−6.62 C. aeruginosa UAMHmedia 11657 liquid 9 (±4.45)83.05 (±3.20)−13.32 (±2.09)−6.62 C. aeruginosa UAMHmedia 116579 liquid 83.05 (9 4.45) (±4.45)83.0513.32 ( 3.20)(±3.20)−13.32 6.62 ((±2.09)−6.622.09) C. aeruginosa UAMHmedia 11657 liquid 9 (±4.45)83.05 (±3.20)−13.32 (±2.09)−6.62 media media 9± (±4.45)− ± (±3.20) − (±2.09)± media (±4.45) (±3.20) (±2.09) media 9 (±4.45) (±3.20) (±2.09) C. aeruginascensmedia UAMH 11655 (±4.45) (±3.20) (±2.09) C. aeruginascens UAMH 11655 C. aeruginascens UAMH 11655 83.97 −13.47 −7.65 C. aeruginascens(chenc hUAMHen) 11655 7 83.97 −13.47 −7.65 C. aeruginascens(chenc hUAMHen) 11655 7 (±6.40)83.97 (±4.70)−13.47 (±0.76)−7.65 C. aeruginascensC.UAMH aeruginascens 11655(chenc hUAMHen) 11655 7 (±6.40)83.97 (±4.70)−13.47 (±0.76)−7.65 woodchip(chench eplaten) 7 (±6.40)83.97 (±4.70)−13.47 (±0.76)−7.65 woodchip(chench eplaten) 7 83.977 ( 6.40) (±6.40)83.9713.47 ( 4.70)(±4.70)−13.47 7.65 ((±0.76)−7.650.76) woodchip(chench eplaten) 7 (±6.40) (±4.70) (±0.76) woodchip plate ± (±6.40)− ± (±4.70) − (±0.76)± woodchip(chench eplaten) 7 (±6.40) (±4.70) (±0.76) C. aeruginascenswoodchip UAMH plate 11655 (±6.40) (±4.70) (±0.76) C. aeruginascenswoodchip UAMH plate 11655 C. aeruginascenswoodchip UAMH plate 11655 83.62 −13.74 −7.44 C. aeruginascens(chenc hUAMHen) 11655 8 83.62 −13.74 −7.44 C. aeruginascens(chenc hUAMHen) 11655 8 (±5.22)83.62 (±3.87)−13.74 (±0.04)−7.44 C. aeruginascens(chenc hUAMHen) 11655 8 (±5.22)83.62 (±3.87)−13.74 (±0.04)−7.44 C. aeruginascens UAMHwoodchip 11655(chench eplaten) 8 (±5.22)83.62 (±3.87)−13.74 (±0.04)−7.44 woodchip(chench eplaten) 8 (±5.22)83.62 (±3.87)−13.74 (±0.04)−7.44 woodchip(chench eplaten) 8 83.62 (8 5.22) (±5.22)13.74 ( 3.87)(±3.87) 7.44 ((±0.04)0.04) (±5.22) (±3.87) (±0.04) woodchip platewoodchip(chench eplaten) 8± (±5.22)− ± (±3.87) − (±0.04)± C. aeruginascenswoodchip UAMH plate 11655 (±5.22) (±3.87) (±0.04) C. aeruginascenswoodchip UAMH plate 11655 C. aeruginascenswoodchip UAMH plate 11655 83.06 −13.28 C. aeruginascens(chenc hUAMHen) 11655 7 83.06 −13.28 6.67 (±0.89) C. aeruginascens(chenc hUAMHen) 11655 7 (±6.74)83.06 (±0.05)−13.28 6.67 (±0.89) C. aeruginascens(chenc hUAMHen) 11655 7 (±6.74)83.06 (±0.05)−13.28 6.67 (±0.89) liquid(chen cmediahen) 7 (±6.74)83.06 (±0.05)−13.28 6.67 (±0.89) C. aeruginascens UAMHliquid 11655(chen cmediahen) 7 (±6.74)83.06 (±0.05)−13.28 6.67 (±0.89) liquid(chen cmediahen) 7 (±6.74) (±0.05) 6.67 (±0.89) (±6.74) (±0.05) liquidliquid(chen cmediamediahen) 7 83.06 (7 6.74) (±6.74)13.28 ( 0.05)(±0.05) 6.676.67 ( 0.89) (±0.89) liquid media liquid media ± (±6.74)− ± (±0.05) ± liquid media 3.3. Comparison of Extracted Xylindein Purity and Electronic Activity 3.3. Comparison of Extracted Xylindein Purity and Electronic Activity 3.3. Comparison of Extracted Xylindein Purity and Electronic Activity 3.3. ComparisonIn order to of assess Extracted purity Xylindein of xylindein Purity producedand Electronic by different Activity strains/species and across growth 3.3. ComparisonIn order to of assess Extracted purity Xylindein of xylindein Purity producedand Electronic by different Activity strains/species and across growth The variationconditionsIn order in, colors we to assess compared seen purity between absorption of xylindein strains spectra.suggests produced For that by comparisons different using standard strains/species of C. aeruginosa color and values, acrossacross as growth has been conditionsIn order, we to comparedassess purity absorption of xylindein spectra. produced For comparisonsby different strains/species of C. aeruginosa and acrossacross growth done in multipleconditionsconditions, publications,, we we we took compared compared spectra [3,7, 9absorption ,on54 ],week may 9 notspectra.xylindein be appropriate For extractions comparisons (Figure for use of 7)C. on, with aeruginosa pigment the exception across extracted growthof PDB from conditions,conditions, we we took compared spectra absorptionon week 9 xylindein spectra. For extractions comparisons (Figure of 7)C., with aeruginosa the exception across of growth PDB conditions,conditions, we we took compared spectra absorptionon week 9 xylindein spectra. For extractions comparisons (Figure of 7)C.,, with aeruginosa the exception across of growth PDB cultures thatculturesconditions, are not thatC. we aeruginosaproduced took spectra soUAMH little on week xylindein 11657 9 xylindein grown they could on extractions woodchips, not be (Figure tested or effectively.at7),, the with very the Xylindein least,exception the shows a*of PDB values a culturesconditions, that we produced took spectra so little on week xylindein 9 xylindein they could extractions not be (Figure tested effectively.7), with the Xylindein exception shows of PDB a culturesdominant that peak produced at the ≈660 so nm little wavelength xylindein theyregion could in DCM, not bewith tested S0–S effectively.1 absorption Xylindein in the 550 – shows700 nm a should havedominantcultures a wider that peak range produced at ofthe acceptability ≈660 so nm little wavelength xylindein for research theyregion could or in applicationsDCM, not bewith tested S0– whereS 1 effectively. absorption any coloration Xylindeinin the 550 – showsproduced700 nm a dominantcultures that peak produced at the ≈660 so nm little wavelength xylindein theyregion could in DCM, not bewith tested S0–S 1 effectively. absorption Xylindeinin the 550 – shows700 nm a rangedominant [10,12] peak, which at the could ≈660 benm seen wavelength in all samples. region Purityin DCM, of sampleswith S0– Swas1 absorption determined in the on 55the0– basis700 nm of rangedominant [10,12] peak, which at the could ≈660 nmbe seen wavelength in all samples. region Purityin DCM, of withsamples S0– Swas1 absorption determined in the on 55the0– basis700 nm of by Chlorociboriarangedominantsp. [10,12] ispeak acceptable., which at the could ≈660 Indirect benm seen wavelength in measures all samples. region of Purityin concentration DCM, of sampleswith S0– shouldSwas1 absorption determined be based in the on primarily55the0– basis700 nm ofon rangeabsorption [10,12] in,, whichthe 400 could nm region, be seen where in all samples.contaminants Purity in of xylindein samples solutionswas determined have been on thepreviously basis of absorptionrange [10,12] in, whichthe 400 could nm region, be seen where in all samples.contaminants Purity in of xylindein samples solutionswas determined have been on thepreviously basis of L* values similarabsorptiondescribed to asS. in causing cuboideum the 400 increased nm [region,52], absorption and where specific contaminants [12]. strains Comparisons andin xylindein growth showed solutions conditionsthat lab- gradehave should been(bacteriological) previously be used if describedabsorption as in causing the 400 increased nm region, absorption where contaminants [12]. Comparisons in xylindein showed solutions that lab- gradehave been(bacteriological) previously achieving adescribedmalt specific cultures coloras causing grown is desired. inincreased shaking absorption conditions [12]. showed Comparisons the lowest showed absorption that aroulab-gradend 400 (bacteriological) nm, appearing maltdescribed cultures as causing grown inincreased shaking absorption conditions [12].showed Comparisons the lowest showed absorption that aroulab-gradend 400 (bacteriological) nm, appearing maltquite cultures close to grown control in ethanolshaking- washedconditions xylindein showed samplesthe lowest [26]. absorption Brewer’s arou maltnd cultures 400 nm, inappearing general quitemalt cultures close to grown control in ethanolshaking-washed conditions xylindein showed samplesthe lowest [26]. absorption Brewer’s arou maltnd cultures 400 nm, inappearing general 3.3. Comparisonquiteshowed of close Extracted high to contaminant control Xylindein ethanol levels Purity-washed as reflected and xylindein Electronic in high samples absorption Activity [26]. around Brewer’s this area, malt with cultures honey in cultures general showedquite close high to contaminant control ethanol levels-washed as reflected xylindein in high samples absorption [26]. around Brewer’s this area, malt with cultures honey in cultures general showedshowing high reduced contaminant contaminants levels in as compar reflectedison. in high It is absorptionnotable that around cultures this associated area, with with honey the cultures highest In ordershowingshowed to assess high reduced contaminant purity contaminants of xylindein levels in as compar reflected producedison. in high It by is notableabsorption different that around strains cultures this/species associated area, with and with honey across the cultures highest growth showingpigmentation reduced levels contaminants (lab-grade maltin compar and honeyison. Itcultures) is notable showed that cultures lower overallassociated levels with of theabsorption highest pigmentationshowing reduced levels contaminants (lab-grade malt in compar and honeyison. Itcultures) is notable showed that cultures lower overallassociated levels with of theabsorption highest conditions, wepigmentationshowing compared reduced levels absorption contaminants (lab-grade spectra. malt in compar and For honey comparisonsison. Itcultures) is notable ofshowed C.that aeruginosa cultures lower overallassociatedacross levels growth with of theabsorption conditions, highest pigmentation levels (lab-grade malt and honey cultures) showed lower overall levels of absorption we took spectrapigmentation on week levels 9 xylindein (lab-grade extractions malt and honey (Figure cultures)7), with showed the lower exception overall of levels PDB of cultures absorption that produced so little xylindein they could not be tested effectively. Xylindein shows a dominant peak

Processes 2020, 8, 1477 16 of 23 at the 660 nm wavelength region in DCM, with S –S absorption in the 550–700 nm range [10,12], ≈ 0 1 which could be seen in all samples. Purity of samples was determined on the basis of absorption in the 400 nm region, where contaminants in xylindein solutions have been previously described as causing increased absorption [12]. Comparisons showed that lab-grade (bacteriological) malt cultures grown in shaking conditions showed the lowest absorption around 400 nm, appearing quite close to control ethanol-washed xylindein samples [26]. Brewer’s malt cultures in general showed high contaminant levels as reﬂected in high absorption around this area, with honey cultures showing reduced contaminants in comparison. It is notable that cultures associated with the highest pigmentationProcesses 2020, 8, x levels FOR PEER (lab-grade REVIEW malt and honey cultures) showed lower overall levels of absorption16 of at23 400 nm. Finally, woodchip cultures generally showed higher absorption around 400 nm, in comparison at 400 nm. Finally, woodchip cultures generally showed higher absorption around 400 nm, in to liquid cultures of the same media, likely due to extractives from the wood. comparison to liquid cultures of the same media, likely due to extractives from the wood.

Figure 7. Xylindein extract from C. aeruginosa (UAMH 11657) absorption across growth conditions conditions.. Control sample sample was was ethanol-washed ethanol-washed solidiﬁed solidified xylindein. xylindein. (a) Absorbance (a) Absorbance of extracted of extracted pigment solutions pigment showingsolutions characteristicshowing characteristic xylindein peakxylindein at 660 peak nm at and ≈660 variation nm and in variation absorbance in atabsorbance shorter wavelengths at shorter ≈ duewavelengths to varying due concentration to varying concentration of non-xylindein of non interferents-xylindein interferents known to reduce known electronic to reduce properties. electronic (propertiesb) Close up. (b absorbance) Close up absorbance around 400 arou nm,nd where 400 largestnm, where variation largest was variation seen in was terms seen of otherin terms interfering of other compoundsinterfering compounds in solution. in Higher solution. absorbance Higher isabsorbance associated is with associated higher concentrations with higher concentrations of contaminants, of withcontaminants, lab-grade with shaking lab- culturesgrade shaking showing cultures lowest showing contamination. lowest contamination.

Optical data provided limited information on variation in production of secondary metabolites for comparisons comparisons of of species species and and strain, strain, and therefore and therefore xylindein xylindein quality (Figurequality8 ). (Figure Xylindein’s 8). Xylindein’s dominant peak was seen in the 660 nm wavelength region in DCM, with S –S absorption in the 550–700 nm dominant peak was seen≈ in the ≈660 nm wavelength region in DCM,0 with1 S0–S1 absorption in the 550– range700 nm [13 range,14]; [13,14] however,; however the diff, erencesthe differences in the absorptionin the absorption at around at around 400 nm 400 was nm relatively was relatively small. Therefore,small. Therefore, no determinations no determinations of purity of di purityfferences differences between strainsbetween were strains made were on themade basis on of the these basis data. of theseConductivity data. of xylindein extract from tested species and strains were compared, and differences in conductivity were seen in samples both before and after an ethanol wash (Figure9), with the post-wash C. aeruginascens UAMH 7614 showing the highest electric currents in the current–voltage (I-V) characteristics in produced thin films. It is notable that these clear differences between strains were not seen in optical data, indicating other interfering compounds are likely present in samples that were not contributing to the enhanced absorption in the 370–450 nm range previously described. C. aeruginascens UAMH 11656 showed less improvement when subjected to an ethanol wash compared to C. aeruginosa UAMH 11657, which may indicate increased production of secondary metabolites not soluble in ethanol. Finally, effective electron mobilites were calculated using fits of the post-wash data (Figure 10) to Equation (2) (see Materials and Methods), with C. aeruginascens strain UAMH 7614 showing the best effective electron mobility at 0.012 cm2/(Vs), followed by C. aeruginosa strain UAMH 11657 at 0.0035 cm2/(Vs) and finally C. aeruginascens strain UAMH 11655 at 8.2 10 4 cm2/(Vs). × −

Figure 8. Xylindein absorption across growth strains and growth conditions for week 9 samples. Chlorociboria cultures (C. aeruginosa strain UAMH 11657 and C. aeruginascens strains UAMH 11655 and UAMH 7614) grown in lab-grade malt media, in either liquid culture or woodchip-amended agar plates (denoted “wood” in legend). Absorbance of extracted pigment solutions showed a

Processes 2020, 8, x FOR PEER REVIEW 16 of 23 at 400 nm. Finally, woodchip cultures generally showed higher absorption around 400 nm, in comparison to liquid cultures of the same media, likely due to extractives from the wood.

Processes 2020, 8, 1477 17 of 23

The highest effective mobility of 0.012 cm2/(Vs) is comparable to 0.01 cm2/(Vs) previously obtained in post-wash wild-type xylindein films in the same device geometry [15]. However, these values are considered to be a lower limit to the intrinsic mobility, as considerably higher values of up to 2 0.4 cmFigure/(Vs) 7. were Xylindein reported extract in similarfrom C. filmsaeruginosa in a (UAMH more optimized 11657) absorption device geometry across growth [15]. conditions. ControlWhile C. sample aeruginascens was ethanolUAMH-washed 7614 showed solidified the xylindein. slowest pigment(a) Absorbance production of extracted and smallest pigment colony sizes,solutions it was alsoshowing associated characteristic with highestxylindein quality peak at xylindein, ≈660 nm and as variation shown in in (I-V) absorbance characteristics at shorter from xylindeinwavelengths films. due This to may varying be dueconcentration to larger of allocation non-xylindein of resources interferents away known from to growthreduce electronic and towards productionproperties of. pigment,(b) Close up a patternabsorbance that arou hasnd been 400 nm, suggested where largest elsewhere variation [21 was]. It seen is notable in terms thatof other medias seeninterfering to produce compounds higher levels in solution. of pigmentation, Higher absorbance lab-grade is malt,associated and honeywith higher were concentrations also associated of with fewercontaminants, contaminants with absorbing lab-grade in shaking the 40m cultures range. showing This suggests lowest contamination. that these contaminants are primarily fungal secondary metabolites associated with growth or biomass production, production of which is likelyOptical reduced data with provided higher energylimited allocationinformation to pigmenton variation production. in production Decreased of secondary biomass metabolites production forby C. comparisons aeruginascens ofUAMH species 7614 and in strain, early weeks and thereforecould have xylindein resulted in quality reduced (Figure pigmentation 8). Xylindein’s overall; dominanthowever, the peak pigment was seen produced in the ≈ appears660 nm wavelength to have been region less contaminated in DCM, with with S0–S other1 absorption products in ofthe fungal 550– 700metabolism. nm range It [13,14] is interesting; however to,note the differences that this strain in the had absorption a different at morphology around 400 ofnm growth was relatively than the small.other two,Therefore, lacking no a deeplydeterminations pigmented of purity outer zone. differences between strains were made on the basis of these data.

Figure 8. XylindeinXylindein ab absorptionsorption across across growth growth strains strains and and growth growth conditions conditions for for week week 9 9 samples. samples. Chlorociboria cultures (C. aeruginosa strain UAMH 11657 and C. aeruginascens strains UAMH 11655 and UAMH 7614) grown in lab-gradelab-grade malt media, in either liquid culture or woodchip-amendedwoodchip-amended agar plates (denoted (denoted “wood” “wood” in legend). in legend). Absorbance Absorbance of extracted of extracted pigment solutions pigment showed solutions a characteristic showed a xylindein peak at 660 nm and variation in absorbance at shorter wavelengths. Averages are given for ≈ each strain; however, a large amount of variability was seen between samples of each strain around the 400 nm mark. Therefore, no strain could be clearly identiﬁed as producing highest quality pigment using optical data.

In addition, the variation in values of a* despite L* and b* values in relation to the standard suggests that other secondary metabolites, including the yellow secondary metabolite, are being produced differentially among strains, species, and in response to different growing conditions. This is further supported by conductivity data, both before ethanol wash and in the different levels of improvement seen in response to ethanol washing. While data suggest that there is no significant difference between species in terms of extracted pigment production, differences in strain may have an effect on speed of xylindein production, especially in earlier weeks of growth. This suggests Processes 2020, 8, 1477 18 of 23 Processes 2020, 8, x FOR PEER REVIEW 17 of 23 that stimulatorycharacteristic mechanisms xylindein for peak pigment at ≈660 production nm and variation pathways in absorbance are highly at conserved shorter wavelengths. in the genus, Averages are given for each strain; however, a large amount of variability was seen between samples in addition to pigment production itself. Although the use of only three strains presents a somewhat of each strain around the 400 nm mark. Therefore, no strain could be clearly identified as producing limited picture,highest with quality the pigment two species using optical represented data. not being closely related in terms of phylogeny, it may be the case that most species in the genus Chlorociboria produce pigment at similar rates and in response to similarConductivity stimuli. of xylindein extract from tested species and strains were compared, and Whiledifferences this research in conductivity identified were growth seen conditionsin samples both leading before to and amounts after an of ethanol xylindein wash produced (Figure 9) in, only 5 weeks,with the equivalent post-wash C. to aeruginascens 2–3 months UAMH using 7614 previous showing methods, the highest it also electric indicated currents thatin the extraction current– methodologyvoltage needs (I-V) to characteristics be improved in to produced allow for th effiin cientfilms. xylindein It is notable collection. that these The clear statistical differences equivalence between strains were not seen in optical data, indicating other interfering compounds are likely present in of multiple media and culture conditions by week 9 for comparisons of growth, and after week 5 for samples that were not contributing to the enhanced absorption in the 370–450 nm range previously comparisonsdescribed. of strain C. aeruginascens indicate that UAMH there is11656 a limit showed to pigment less improvement extractable when into DCMsubjected using to an the ethanol current methodology.wash By compared week 9, to liquid C. aeruginosa cultures appearedUAMH 11657, almost which black maydue to indicate pigment increased concentration, production with of no visible disecondaryfference in metabolites color after not extraction. soluble in Significant ethanol. Finally, amounts effective of pigment electron produced mobilites by were the fungicalculated were thereforeusing not captured fits of the using post- thiswash extraction data (Figure methodology. 10) to Equation While (2) other (see Materials publications and haveMethods), used multiplewith C. extractionsaeruginascens and combined strain UAMHthe results 7614 to showing attempt the to best extract effective more electron of the pigmentmobility at from 0.012 the cm growth2/(Vs), substratefollowed [7], this processby C. aeruginosa is laborious. strain DevelopmentUAMH 11657 at of 0.0035 new processes, cm2/(Vs) and including finally C. experimentation aeruginascens strain with −4 2 2 new solvents,UAMH is 11655 important at 8.2 × to 10 effi cmciently/(Vs). The collect highest produced effective pigment. mobility of DCM 0.012 has cm been/(Vs) is the comparable best solvent to 2 thus far [0.0123], cm while/(Vs) other previously solvents obtained have been in post identified-wash wild to test-type using xylindein Hansen films solubility in the same parameters, device geometry [15]. However, these values are considered to be a lower limit to the intrinsic mobility, as including naphthalene, butyl acetate, and trichloroethylene [23]. Investigation into use of these and considerably higher values of up to 0.4 cm2/(Vs) were reported in similar films in a more optimized other solventsdevice maygeometry improve [15]. efficiency of extraction, in addition to procedural changes.

Figure 9.FigureCurrent–voltage 9. Current–voltage (I-V) characteristics (I-V) characteristics for “pre-wash” for “pre-wash” and and “post-wash” “post-wash” xylindein xylindein ﬁlmsfilms made made from extractsfrom of extracts diﬀerent of species different and species strains, and including strains, includingC. aeruginascens C. aeruginascensstrains UAMHstrains UAMH 7614 and 7614 UAMH and 11655 andUAMHC. aeruginosa 11655 andstrain C. aeruginosa UAMH 11657.strain UAMH Testing 11657. was carriedTesting outwas oncarried interdigitated out on interdigitated Au electrodes Au with a 25 µm gap. C. aeruginascens yielded higher quality xylindein, with strain UAMH 7614 showing the best results. Processes 2020, 8, x FOR PEER REVIEW 18 of 23

Processes 2020electrodes, 8, 1477 with a 25 μm gap. C. aeruginascens yielded higher quality xylindein, with strain UAMH19 of 23 7614 showing the best results.

FigureFigure 10. 10.The The current–voltage current–voltage (I-V) (I-V) characteristics characteristics plotted plotted as currentas current (I) vs.(I) vs voltage. voltage squared squared (V 2(V) in2) in xylindeinxylindein films films depending depending on on species species and and strains, strains, including includingC. aeruginascensC. aeruginascensstrains strains UAMH UAMH 7614 7614 and and UAMHUAMH 11655 11655 and andC. aeruginosa C. aeruginosastrain strain UAMH UAMH 11657. 11657. Lines representLines represent linear fits linear to Equation fits to Equation (2) from which(2) from effectivewhich mobilitieseffective mobilities were calculated. were calculated. Calculations Calculations indicate thatindicate xylindein that xylinde from C.in aeruginascens from C. aeruginascensstrain UAMHstrain 7614 UAMH showed 7614 the showed best effective the best electron effective mobility electron at 0.012 mobility cm2/(Vs), at followed 0.012 cm by2/(Vs),C. aeruginosa followedstrain by C. UAMHaeruginosa 11657 strain at 0.0035 UAMH cm2/ (Vs)11657 and at finally0.0035 C.cm aeruginascens2/(Vs) and finallystrain C. UAMH aeruginascens 11655 at strain8.2 10UAMH4 cm2 11655/(Vs). at × − 8.2 × 10−4 cm2/(Vs). Despite current inefficiencies in extraction methodology for liquid state growth, identification of growthWhile conditions C. aeruginascens leading to the UAMH fastest 7614 production showed of the quantities slowest ofpigment xylindein production has significant and smallest implications colony forsizes, research it was on thisalso compound.associated with Thus highest far, a number quality of xylindein potential, as uses shown for the in extracted(I-V) characteristics pigment have from beenxylindein identified, films. ranging This may from be use due as to a larger wood allocation stain [3] toof textileresources dye away [5–9], from with growth the most and exciting towards opportunitiesproduction inof thepigment, field of a optoelectronics. pattern that has All been of these suggested new applications, elsewhere [21] however,. It is requirenotable significantthat medias quantitiesseen to produce of pure xylindein.higher levels All of strains pigmentation, and growth lab-grade conditions malt,produced and honey inhomogeneous were also associated extracts with containingfewer contaminants a mix of secondary absorbing metabolites, in the 40m and range. while This this suggests is expected that from these biological contaminants organisms are itprimarily is not idealfungal for (opto)electronic secondary metabolites research. associated While some with work growth has been or biomass conducted production, to begin to production identify interfering of which is compoundslikely reduced [12], andwith a higher method energy has been allocation established to pigment to use production. centrifugal partition Decreased chromatography biomass producti foron purificationby C. aeruginascens [27], more UAMH research 7614 is in required early weeks to produce could largehave resulted amounts in of reduced pure pigment pigmentation required overall for ; industrialhowever adoption, the pigment of any produced technologies appears built on to xylindein.have been Production less contaminated of larger with amounts other of products pigment of willfungal allow metabolism. for development It is interesting of other purification to note that techniques this strain had a different morphology of growth than theIn other this two, study, lacking liquid a culturesdeeply pigmented were shown outer to zone. be competitive with solid cultures in terms of pigmentIn production, addition, the producing variation more in values pigment of ina* earlier despite stages L* and of growth b* values and in yielding relation a purerto the product. standard Thissuggests suggests that liquid other culture secondary growth metabolites, may have the including most potential the yellow for industrial secondary scale-up metabolite, and should are being be a focusproduced of future differentially research, among especially strains, as these species, cultures and arein response easier to to scale different up for growing industrial conditions. production This comparedis further to supported solid state by growth. conductivity While no data, diff erencesboth before in pigmentation ethanol wash concentration and in the different as measured levels by of colorimprovement difference ( ∆seenE) were in resp seenonse after to week ethanol 5 of Chlorociboriawashing. Whilegrowth, data electronic suggest that properties there is indicated no significant that C.difference aeruginascens betweenstrain UAMHspecies 7614in terms was of capable extracted of producing pigment theproduction, highest quality differences pigment. in strain Future may work have withan thiseffect strain on speed should of allowxylindein for the production, most efficient especially production in earlier of xylindein weeks of for growth. further This optoelectronic suggests that research.stimulatory In addition, mechanisms the knowledge for pigment that production large differences pathways exist betweenare highly xylindein conserved quality in the of di genus,fferent in speciesaddition and to strains pigment will driveproduction future itself. research Alt intohough collection the use and of only identification three strains of new presents strains a thatsomewhat may performlimited even picture, better. with Production the two species of quality represen xylindeinted not that being is straightforward closely related toin purifyterms of will phylogeny, allow for it anmay expansion be the case of research that most and species potential in the technological genus Chlorociboria adoption produce of xylindein pigment into at commercial similar rates green and in electronicsresponsefields. to similar stimuli.

Processes 2020, 8, 1477 20 of 23

4. Conclusions Previous work on Chlorociboria growth has primarily focused on comparisons of culture size and descriptions of pigmentation with limited comparisons of quantity of extractable xylindein. This study compared previous xylindein production research, and identified Chlorociboria aeruginosa grown under shaking conditions in bacteriological-grade malt as producing the largest mean color difference (∆E) of 26.3 5.9 in comparison to extracted pigment from shaking liquid, static liquid, ± and woodchip-amended agar plates, a value comparable with pigment produced in woodchip plates. Higher levels of pigmentation were seen in shaking liquid cultures in early weeks of growth, producing color values matching the standard concentration used in previous publications after only 5 weeks, instead of the normal 2 to 3 months. This result has great implications for industrial pigment production, as liquid cultures have more straightforward scalability. In addition, pigment production in C. aeruginosa and C. aeruginascens cultures was seen to be equivalent in color change (∆E) of extracted pigment after 5 weeks of growth, with earlier weeks showing variation based more on strain than on species. This may represent significant conservation of pigment metabolic pathway stimulation. C. aeruginascens strain UAMH 7614 was shown to have improved electronic properties, suggesting fewer contaminating secondary metabolites, which makes it preferred for future work on xylindein-based (opto)electronic devices. Identification of other strains and species that may outperform strains tested within this paper may further improve pigment production. This identification of ideal xylindein production by Chlorociboria spp. in laboratory settings should allow for improved xylindein production and expansion of new research into its use. With larger quantities of xylindein available, methods of purification can be developed to replace fungal extracts containing mixes of compounds with pure pigment for study. This will support further development of this promising organic semiconductor. Technologies using the electronic capabilities of xylindein could support green energy production through organic photovoltaics based on the pigment, as well as other green technologies for a more sustainable world.

Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9717/8/11/1477/s1: Figure S1: Investigations into pigment production of Chlorociboria sp. showing a lack of comparable results due to testing condition differences. Table S1: Colony radius for Chlorociboria aeruginosa on different media, with and without woodchips. Author Contributions: Conceptualization, R.C.V.C. and S.C.R.; methodology, R.C.V.C.; formal analysis, R.C.V.C.; investigation, R.C.V.C. and G.G.; writing—original draft R.C.V.C., G.G., and S.C.R.; writing—review and editing, S.C.R. and O.O.; visualization, R.C.V.C. and G.G.; supervision, S.C.R.; project administration, S.C.R. and O.O.; funding acquisition, S.C.R. and O.O. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Foundation “Energy for Sustainability” program (CBET-1705099). Acknowledgments: Authors would like to thank Linus Pauling Core Laboratories at Oregon State University for their analysis of major sugars in honey and malt samples, and Ariel Muldoon for statistical support. Conflicts of Interest: The authors declare no conflict of interest.

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