EXTRACELLULAR PIGMENT PRODUCTION FROM WOOD-INHABITING FUNGI: DEVELOPING A COLOR PALETTE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1009811
• Project Start Date: Jul 1, 2016
• Project End Date: Jun 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
Wood Science and Engineering

Non Technical Summary
There is increasing interest from the biobased materials industry for environmentally derived and environmentally conscious dyes. Current dye usage in the textile industry alone contributes to substantial environmental impact, including the use of peripheral chemicals, water, and synthetic dyestuffs, many of which are petroleum based.While fungal pigments have been experimented with as dyes, historically these dyes have not been seen as viable commercial options due to the low volume of dye produced or the poor durability of the dye. Recently, a new class of dyes has been developed from a specific group of fungi known as spalting fungi. Spalting is defined as any penetrating color produced inside wood from a fungus, and has been used historically in woodcraft since at least the 1400s. The dyes derived from spalting fungi have proven to be easy to grow in large batch cultures and durable across a large number of tests, including UV, washing, and crocking. As an added bonus, the fungi responsible for spalting are native to the Pacific Northwest (PNW) and grow in abundance in the local forests. Their use has the potential to bring added value to PNW forests and landowners looking to add another 'crop' to their forests for harvest.While spalting fungi show strong potential to replace synthetic dyes across numerous industries, there are currently only three colors available - red, blue-green, and yellow. These pigments have unique chemical properties and are not additive - one cannot simply make a new color by adding one to the other. In order for the dyes to be marketable, a full color palette is needed. Research has shown that the fungi will alter their pigment color in response to pH changes in the media, however those colors produced from this effect are simply shades of the base color. Preliminary research in the Robinson lab has shown that growing the fungi that produce these pigments together in the same batch reactor may, under certain conditions, result in blended color. If the current colors (red, blue-green, yellow) could be blended, a full color palette could be available and the commercialization of these dyes into the textile and printing markets could be realized.Currently the dyes are being tested in large scale textile dye applications through use of inkjet printers. Knowledge of what color combinations are possible, as additive colors cannot be created through dye blends in the print head, and what conditions are required to acquire said colors, is needed. This will involve growing the fungi together in batch culture and assessing their color change over time, across different environmental effects.

Animal Health Component

80%

Research Effort Categories

Basic

10%

Applied

80%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	4020	1102	100%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1102 - Mycology;

Keywords

fungi

chlorociboria species

scytalidium species

spalting

dyes

pigments

Goals / Objectives
The overarching goal of this project is to develop a full range color palette available from the fungiChlorociboria aeruginascens, Chlorociboria aeruginosa, Scytalidium cuboideumandScytalidium ganodermophthorum,?and blends of each.

Project Methods
Methods and ProcedureThe fungi will be grown in a batch culture reactor as defined in Weber et al. 2017 (in press). This reactor involves a clear glass tank and small propeller to keep the 2% malt solution in perpetual agitation. Under normal circumstances, a monoculture plug of one fungus is added to the sterile solution and the fungus grows and produces pigment. The pigment is then harvested through extraction with dichloromethane (DCM) and used as a dye.The proposed method will grow two species together in an attempt to create a uniform dye blend. This will be done through several approaches:inoculating the solution at the same time with two fungi and allowing them to growinoculating the solution first with one fungus, waiting a set period of time (testing intervals of two days, one week, two weeks) and then inoculating with the second fungusTemperature and humidity will be altered to determine effect on pigment colormedia (malt, potato dextrose, wood chip enhanced) will be altered to determine effect on pigment colorColor change will be measured through a small amount of solution being removed from the reactor and color read in a colorimeter. The batch cultures will be run until color change no longer occurs. Color change will be calculated using the delta E calculation, which is the standard for the CIE Lab colorspace. We anticipate a minimum of five replicates per growth blend, and will be using ANOVA followed by Tukey HSD for analysis.The efforts used to cause a change in knowledge will be scientific publication of findings, and use of the new dyes immediately in the ongoing inkjet printing test at OSU. Outputs will be evaluated based upon the performance of the dyes in the printing, as well as the successful publication of the methodology in peer-reviewed literature. Key milestones will be in two stages. Stage I will be initiation of the batch reactors and stage II will be the conclusion of data collection. Indicators of success will be CIE Lab values from the colorimeter that deviate plus or minus two from the standard color values of the unblended colorants.

Progress 07/01/16 to 06/30/21

Outputs
Target Audience:The target audience for the duration is academic journals, where we hope to publish the remainder of the findings. From there we hope to also put out a technical bulletin for the members of the Spalting Cooperative, who are primarily small business owners and high-end crafters. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the years a number of graduate and undergraduate students worked on the project. This gave them all a chance to learn about culture maintainence, color extraction, and applications. These are critical, basic lab skills they can transfer to other lab jobs. How have the results been disseminated to communities of interest?Peer-reviewed publications, conference proceedings, book chapters, and presentations have all included aspects of the research. A photo of the full color range is included in almost all public presentations to woodworkers and woodturners to showcase the diversity of pigmenting fungi. They love this photo and it is a great graphical representation of the work. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Yes, this was accomplished and can be seen in the publication record. From there, we had some extra time and so expanded into really investigating the yellow pigment and it properties, and also looked a bit into toxicity of the pigments.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2021 Citation: Suthar, M., Lagshetti, A., Rais�nen, R., Singh, P.N., Dufoss�, L., Robinson, S.C., Singh, S.K. 2021. Advances in Macrofungi: Industrial Avenues and Prospects. Chapter contributed: Industrial applications of pigments from macrofungi. CRC Press
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Hassan Almurshidi, B., Van Court, R.C., Vega Gutierrez, S.M., Harper, B., Harper, S., Robinson, S.C. Toxicity of spalting fungal pigments: a comparison between extraction methods. Journal of Fungi in review

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience for the duration is academic journals, where we hope to publish the remainder of the findings. From there we hope to also put out a technical bulletin for the members of the Spalting Cooperative, who are primarily small business owners and high-end crafters. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the years a number of graduate and undergraduate students worked on the project. This gave them all a chance to learn about culture maintainence, color extraction, and applications. These are critical, basic lab skills they can transfer to other lab jobs. How have the results been disseminated to communities of interest?Peer-reviewed publications, conference proceedings, book chapters, and presentations have all included aspects of the research. A photo of the full color range is included in almost all public presentations to woodworkers and woodturners to showcase the diversity of pigmenting fungi. They love this photo and it is a great graphical representation of the work. What do you plan to do during the next reporting period to accomplish the goals?We're hoping to gain a more fundamental understanding of the yellow and red pigments. We are currently looking at what causes a crystal formation from S. cuboideum to be red or orange, and how to control this behavior. We hope to publish on that by the end of the year. We also hope to begin elucidation of the yellow compound, however this may take several years to full understand the structure.

Impacts
What was accomplished under these goals? Yes, this was accomplished and can be seen in the publication record. From there, we had some extra time and so expanded into really investigating the yellow pigment and it properties, and also looked a bit into toxicity of the pigments.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2021 Citation: Suthar, M., Lagshetti, A., Rais�nen, R., Singh, P.N., Dufoss�, L., Robinson, S.C., Singh, S.K. 2021. Advances in Macrofungi: Industrial Avenues and Prospects. Chapter contributed: Industrial applications of pigments from macrofungi. CRC Press
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Hassan Almurshidi, B., Van Court, R.C., Vega Gutierrez, S.M., Harper, B., Harper, S., Robinson, S.C. Toxicity of spalting fungal pigments: a comparison between extraction methods. Journal of Fungi in review

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audience for this reporting period was mostly those who read peer-reviewed journal articles (scientists), as well as wood artists who utilize these fungal pigments. We expanded a bit to textile manufacturers as well with our newest paper. Changes/Problems:The pigments cannot readily be mixed. This was a goal of the research and as it has not worked, we have had to backstep to more fundamental research. What opportunities for training and professional development has the project provided?Two graduate students got an opportunity to look at data analysis and how to format a paper for publication, which they had not done before. How have the results been disseminated to communities of interest?Once published, the color results will be shared via spalting facebook groups and the spalting co-op What do you plan to do during the next reporting period to accomplish the goals?We're hoping to gain a more fundamental understanding of the yellow and red pigments. We are currently looking at what causes a crystal formation fromS. cuboideumto be red or orange, and how to control this behavior. We hope to publish on that by the end of the year. We also hope to begin elucidation of the yellow compound, however this may take several years to full understand the structure.

Impacts
What was accomplished under these goals? We finally have the full color range for all three fungi listed, the blue-green Chlorociboria aeruginascens/aeruginosa (shades of blue to green), the 'yellow' Scytalidium ganodermophthorum (shades of yellow, green, purple, and red), and the 'red' Scytalidium cuboideum (shades of orange, red, blue, and purple). Blends have thus far not proven effective, so we are going back to basic understanding of the compounds to see why that is.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Vega Gutierrez, P., Almurshidi, B., Huber, M., Andersen, C., Van Court, R.C., Robinson, S.C. Understanding color variation in the pigment produced by Scytalidium ganodermophthorum for artistic applications. International Wood Products Journal in review

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:We continue to share our research with woodworkers--our target demographic. This is mostly done through Facebook group dissemination and teaching at a woodworking school, and including our findings in with the course material. Changes/Problems:We had a major mite infestation that destroyed all our cultures last year. The cultures had to be repurchased from culture banks and we had to start a lot of things from scratch. We now have the mite issue under control. Publication is proceeding at an expected rate, and we hope to have a number of additional papers out this year now that most of the experimental data have been gathered. What opportunities for training and professional development has the project provided?We continue to host a menegerie of undergraduate volunteer students who learn basic mycological skills while working with our fungi. The yellow project will likely turn into a PhD thesis after this preliminary test is complete, as the chromophore is unknown and needs to be elucidated. How have the results been disseminated to communities of interest?We continue to dissseminate regularly through woodworking channels, such as Marc Adams School of Woodworking. We also publish in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?It is our hope to get the yellow paper submitted and published during this next reporting period, and to finally start work on an overarching color blend paper.

Impacts
What was accomplished under these goals? We have completed the color palettes for the red fungus and the blue-green fungus. The yellow fungus has had all its data collected and we are in the process of writing the paper. We hope to have it submitted to a journal next week. The blends have proven troublesome due to carrier differences. We have published a paper on the color palette of the pigments in oils, and a color palette of the pigments in paint, including blends. We are still working on color blends in just raw DCM, but hope to have this completed soon.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:During lab tours we have started to discuss the cultures and their importance. These tours have been for small woodland owners, woodturning professionals, and university/high school students. Changes/Problems:No changes have occured. Major problems continue to be the equipment. Right after our color reader came back, our shaker table broke and we had to wait two months to get it repaired. We now have two, but with them running constantly it is very hard on the bearings. We have learned that all the instruments used in this test must be callibrated yearly, so we will have at least one month of down time every year, during which we will have to send in all the machines for maintenence. What opportunities for training and professional development has the project provided?The undergraduate and graduate student working on the project continue to have a very in-depth look at study design and pure culture work. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are now working on the color blends, especially within the orange range. We hope to have the range of at least one blend figured out before the next reporting period.

Impacts
What was accomplished under these goals? Our intial round of testing yielded an orange color from the combination of S. cuboideum and S. ganodermophtorum. While we are still awaiting results on the other combinations, we are now expanding the testing for the above pairing and color reading week by week, so we can get the range of orange hues available. We have finished testing the base range of color for each pigment and have the full range ofunblendedcolors now. A total of eight colors can be achieved without blends: red, orange, yellow, three shades of green, blue, and purple.

Publications

Progress 07/01/16 to 09/30/16

Outputs
Target Audience:We're still in experimental design phase, so there has been no outreach yet. Changes/Problems:As noted early, we had contamination issues with our base culture for our yellow fungus, and our color reader broke and had to be sent out for repairs. These have not changed the approach, but have significantly delayed the timeline. All is on track now, and we should be able to start growing the cultures once the colorimeter is returned. What opportunities for training and professional development has the project provided?The undergraduate and graduate student working on the project have had a very in-depth look at study design and pure culture work, and the inherent frustrations and repetition that come with science. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During this next period we hope to inoculate the media and actually begin the testing cycle.

Impacts
What was accomplished under these goals? We have explored replication issues and time issues related to growing all the cutlures at the same time, and have finally acquired the necessary glassware and shake table to do so. Our S. ganodermophthorum isolate became contaminated in the base culture, and we finally have it mite and contaiminant free. Our color reader also broke this cycle, and we've only just been scheduled to get it back. Hopefully once it returns we can actually initiate the project.

Publications