No el mycelium-based biocomposi es om nu shell pa icles p oduced h ough a biop ocess
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enginee ing app oach combining liquid- and solid-s a e e men a ion
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Š ěpán Hýsek1,2,*, Pa aske i Cha alambous3, Ch is oph P eimesbe ge 2,4, Luboš P okůpek5,
3
No bu ga Gie linge 3,*, Rupe Wimme 2,6
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1 - Facul y o Fo es y and Wood Sciences, Czech Uni e si y o Li e Science P ague, Kamýcká
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129, P ague, 165 00, Czech Republic
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2 - BOKU Uni e si y, Ins i u e o Wood Technology and Renewable Ma e ials, Depa men o
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Na u al Sciences and Sus ainable Resou ces, Kon ad-Lo enz-S aße 24, 3430 Tulln, Aus ia
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3 - BOKU Uni e si y, Ins i u e o Biophysics, Depa men o Na u al Sciences and Sus ainable
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Resou ces, Mu hgasse 11, 1190 Vienna, Aus ia
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4 - Wood K plus - Compe ence Cen e o Wood Composi es & Wood Chemis y,
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Kompe enzzen um Holz GmbH, Al enbe ge S aße 69, A-4040 Linz, Aus ia
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5 - Ins i u e o Chemis y and Technology o Mac omolecula Ma e ials, Facul y o Chemical
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Technology, Uni e si y o Pa dubice, S uden ská 573, 532 10 Pa dubice, Czech Republic
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6 - Depa men o Wood Science and Technology, Facul y o Fo es y and Wood Technology,
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Mendel Uni e si y in B no, Zemědělská 1, 613 00 B no, Czech Republic
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*Co esponding au ho : bu gi.gie li[email p o ec ed].a ; hyse[email p o ec ed].cz
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Abs ac
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This s udy p esen s he de elopmen and cha ac e isa ion o mycelium-based biocomposi es
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(MBBs) ein o ced wi h g inded walnu and pis achio nu shells. A combined liquid- and solid-
20
s a e e men a ion p ocess employing Ganode ma sessile was used o bind he lignocellulosic
21
pa icles wi hou syn he ic adhesi es. The in luence o nu species and glyce ol ea men on
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composi e p ope ies was s udied h ough dynamic mechanical analysis (DMA), Sho e A
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ha dness, dynamic apou so p ion (DVS), Fou ie - ans o m in a ed spec oscopy (FTIR),
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simul aneous he mal analysis (STA), and scanning elec on mic oscopy (SEM). Resul s
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showed ha walnu -based MBBs exhibi ed highe s i ness and Sho e A ha dness compa ed
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o pis achio-based a ian s, wi h he glyce ol- ea ed walnu MBB eaching he highes
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mechanical pe o mance. DVS analysis con i med educed mois u e up ake and hys e esis in
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glyce ol- ea ed samples. SEM e ealed a dense hyphal ne wo k in e connec ing nu shell
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pa icles and a su ace ungal skin. FTIR spec a indica ed composi ional di e ences ela ed
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o lignin and chi in con en , while STA e ealed ypical he mal decomposi ion p o iles o
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lignocellulosic composi es wi h addi ional deg ada ion s ages a ibu ed o glyce ol. Resul s
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showed ha walnu -based MBBs exhibi ed highe s i ness and Sho e A ha dness compa ed
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o pis achio-based a ian s, wi h he glyce ol- ea ed walnu MBB eaching he highes
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mechanical pe o mance. I s Sho e A ha dness was 71.4, compa ed o 63.8 in he un ea ed
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pis achio MBB. DVS analysis con i med educed equilib ium mois u e con en in glyce ol-
36
ea ed samples and a dec ease in hys e esis. STA showed a esidual mass o 25.6% and
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main DTG peaks a 300.6 °C and 354.6 °C o he glyce ol- ea ed walnu MBB, compa ed o
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29.9% esidual mass and main DTG peaks a 281.5 °C and 335.1 °C o he un ea ed pis achio
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a ian . FTIR spec a indica ed highe lignin-associa ed abso bance in walnu composi es,
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consis en wi h hei mechanical pe o mance. SEM e ealed a dense hyphal ne wo k
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in e connec ing he nu shell pa icles and a su ace ungal skin, wi h no s uc u al di e ences
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obse ed due o glyce ol ea men .
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Keywo ds: composi e ma e ial, mycelium, walnu , pis achio, e men a ion
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1. In oduc ion
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Mycelium-based biocomposi es (MBB) a e composi e ma e ials om lignocellulosic
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(LC) aw ma e ials bonded wi h ungal ib es. As mycelium is used in o de o in e connec he
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LC pa icles, no adhesi es a e used o bonding he composi e ma e ial (Mui u i e al. 2023).
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Mycelium, he li ing o ganism, can be unde s ood as a big ad an age in he p ocess o ma e ial
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p oduc ion. The li ing mycelium in ce ain s ages o ma e ial p oduc ion o ma e ial use
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p o ides a possibili y o ma e ial pa ial egene a ion du ing MBB p oduc ion and e en po en ial
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egene a ion o damaged mycelium du ing p oduc use. These abili ies can inally p olong
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ma e ials' li e cycle and signi ican ly in luence i s unc ional p ope ies (Elsacke e al. 2021;
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Sandak 2023).
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LC aw ma e ial used o MBB p oduc ion is mos ly wood o s aw and ep esen s he
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main subs a e componen o MBB (Alaneme e al. 2023). Addi ionally, ag icul u al esidues
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om co on p oduc ion, hemp s aw and seeds, lax shi es, kena ib es, suga cane bagasse
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o ice husks we e success ully used in subs a es o MBB p oduc ion since hey a e ich in
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cellulose, lignin o lowe molecula -weigh suga s (Sydo e al. 2021). These ag icul u al
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esidues a e also commonly u ilised as ein o cing ille s o ma ix componen s in o he ypes
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o biocomposi es (Hýsek e al. 2016; Gajdačo á e al. 2018; Němec e al. 2025). O he aluable
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sou ces o LC biomass a e nu shell pa icles, which ha e been p e iously used o he
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p oduc ion o a ious ypes o composi es (Su i isedsak e al. 2012; Dong e al. 2017; Zhang
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and Yu 2022; Sahin e al. 2024), bu no ye o he p oduc ion o MBB. Nu shells a e no only
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composed o ib es, bu also hick-walled ligni ied scle enchyma cells o di e en shape (Huss
65
e al. 2020; Huss and Gie linge 2021). Among he di e en nu shells, walnu and pis achio
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shells s and ou by being solely composed o polyloba e puzzle cells (An eich e al. 2019, Xiao
67
e al. 2021).
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Solid-s a e e men a ion is widely used o manu ac u e MBB. This me hod is based on
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LC subs a e colonisa ion wi h ungal mycelium. To p oduce pa icles o ib es o subs a es,
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LC aw ma e ials ha e o be disin eg a ed, and he subs a e is u he hyd a ed wi h wa e and
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s e ilised o pas eu ised, usually wi h hea . A e his hea ea men , he subs a e is eady o
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be inocula ed by he selec ed ungus. Fungi used o MBB p oduc ion a e wood-decaying ungi
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ha a e able o decompose lignin, cellulose, and hemicelluloses. The e o e, LC subs a e
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se es as a sou ce o nu i ion o ungus and p o ides i wi h enough nu ien s o build a
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ne wo k o ungal ib es ha in e connec he pa icles. When he mycelium colonizes he
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subs a e, he LC subs a e is, howe e , no ully deg aded and, he e o e, p o ides s uc u al
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s abili y o he MBB (A ias e al. 2020; Elsacke e al. 2020; Sydo e al. 2021). The p ope ies
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o MBB a e subs an ially a ec ed by he ungus used (Appels e al. 2019). The esul ing MBB
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p ope ies a e, howe e , no he only c i e ia used o he selec ion o ungus. G ow h a e,
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esis ance o con amina ion and undemanding g ow h condi ions ha e also o be conside ed.
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The mos sui able ungal species o he p oduc ion o MBB, he e o e, belong o he Pleu o us
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genus, ollowed by Ganode ma and T ame es (Aiduang e al. 2022; Ce imi e al. 2019).
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This p oduc ion me hod based on solid-s a e e men a ion p o ides MBB wi h po ous
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s uc u e cha ac e ised by densi y anging mos ly om 110 kg/m3 o 330 kg/m3, he mal
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conduc i i y compa able o he commonly used he mal insula ion ma e ials (0.05 - 0.07
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W/m.K) and g ea acous ic abso p ion p ope ies. Howe e , he composi es a e so ,
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cha ac e ised by low mechanical p ope ies and high wa e abso p ion (Alaneme e al. 2023;
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Madusanka e al. 2024). Fo ins ance, MBB p oduced by he mycelium o P. os ea us g own
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on s aw subs a e eached a comp essi e s eng h o 0.02 MPa; when g own on he subs a e
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om oak sawdus , he comp essi e s eng h was 0.15 MPa (Ghaz inian e al. 2019). The
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bending s eng h o MBB p oduced om wood pa icles en iched wi h cellulose nano ibe s
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(CNF) was 3.5 MPa (Sun e al. 2019) and he ensile s eng h o MBB om wood sawdus was
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epo ed o be om 0.05 MPa o 0.18 MPa, whe eas he composi es g own on s aw eached
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subs an ially lowe ensile s eng h (Jones e al. 2020). All s udies clea ly con i med ha he
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mechanical p ope ies a e in luenced mainly by MBB densi y, size o pa icles used and ungal
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s ain (Sydo e al. 2021; Alaneme e al. 2023).
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The wa e abso p ion a e o MBB anges om 40% o 580% and is a ec ed by
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subs a e composi ion, manu ac u ing pa ame e s, and me hod o measu emen (A ias e al.
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2020; Madusanka e al. 2024). Besides his, he su ace o MBB s ongly a ec s he wa e
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abso p ion a e. In he case ha s ong mycelium, called ungal skin, is o med on he su ace
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o he sample, he wa e abso p ion d ama ically dec eases. Howe e , gi en he hyd ophilic
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p ope ies o LC subs a es used o MBB p oduc ion, he bulk o no modi ied MBB is always
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epo ed o be hyd ophilic, which also de e mines MBB du abili y and biodeg adabili y (Hýsek
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e al. 2023; Madusanka e al. 2024).
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Liquid-s a e e men a ion has al eady been employed in o de o p oduce pu e
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mycelium ( ungal skin). As he ungal skin is g own on he su ace o he liquid cul u e, he
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ad an age o his me hod is ha i enables he p oduc ion o pu e mycelium wi hou mixing he
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mycelium wi h LC subs a e (Appels e al. 2018; Appels e al. 2020; Say u dino a e al. 2023).
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Howe e , no app oach has been epo ed in scien i ic pape s so a whe e liquid-s a e
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e men a ion is used o bond LC pa icles wi h mycelium and subsequen ly bond a composi e.
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The objec i e o his s udy was o de elop MBB om pis achio and walnu nu shell
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pa icles using an inno a i e me hod composed o a combina ion o solid- and liquid-s a e
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e men a ion. We hypo hesise ha he used p oduc ion me hod p o ides he no el MBB wi h
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a na u e-inspi ed s uc u e, esul ing in MBB wi h unique p ope ies compa ed o he cu en
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s a e o he a .
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2. Ma e ials and Me hods
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2.1 Ma e ials
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Pis achio (Pis acia e a) and walnu (Juglans egia) nu shell pa icles wi h a pa icle
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size lowe han 100 mic ome es we e used in o de o p oduce mycelium-based
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biocomposi es and he selec ed s ain was Ganode ma sessile (“No h Blai ”, Te es ial Fungi,
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USA). Mal ex ac (ME) was pu chased om Ca l Ro h GmbH + Co. KG, Ge many, and a 2%
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wa e solu ion o ME (2%ME) was used in o de o p oduce liquid cul u e as well as liquid
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inoculum. Glyce ol (≥99 %) was pu chased om Ca l Ro h GmbH + Co. KG, Ge many. Mal
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ex ac aga (MAE) was pu chased om Ca l Ro h GmbH + Co. KG, Ge many and was used
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in o de o cul i a e he cul u es on aga placed in Pe i dishes.
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2.2 Me hods
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MBB p oduc ion
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Fi e squa es (5 mm x 5 mm in dimensions) o mycelium g own on MEA we e shaken
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(150 pm) wi h 200 ml 2%ME a 22°C o 13 days in o de o p epa e he liquid inoculum o G.
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sessile. Liquid cul u es we e p epa ed by inocula ion o 200 ml 2%ME by 40 ml o inoculum,
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and he cul u es we e cul i a ed a 20°C o 7 days by cons an shaking a 150 pm. A e 7
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days, 5% (d y weigh / olume) o g inded nu shells we e added o he liquid cul u e and shaken
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a he same condi ion o ano he 4 days. A e his pe iod, 40 g o he cul u e was il e ed using
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a polye hylene (PE) il e wi h po es 29 μm in diame e , he p essu e du ing il a ion was 20
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mba . The il e cake was placed on a pe i dish and ligh ly co e ed in o de o allow ai o en e
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he pe i dish and enable u he me abolic ac i i y o he mycelium. This expe imen design
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enabled an addi ional 7-day-long solid-s a e e men a ion, a e which he g owing composi e
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was u ned o e in o de o allow he mycelium o g ow p ope ly on bo h sides. A e ano he 7
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days, he composi es we e ully g own, and he g ow h was e mina ed by acuum d ying a
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70°C and 700 mba o 2 days. In o de o de e mine he d y mass o he p oduced composi es,
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all samples we e o en-d ied a 103°C. This p ocedu e esul ed in he p oduc ion o pa icle-
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based composi es bonded wi h mycelium non-wo en s uc u e; he weigh pe uni a ea o he
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esul ing composi es om walnu shell pa icles was 1035 g/m2, composi es om pis achio
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nu shells eached 935 g/m2 and e e ence wi hou nu shell pa icles 230 g/m2. The mycelium-
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based biocomposi es we e ea ed wi h glyce ol. Glyce ol ea men was conduc ed as
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imme sion o he samples in 20% wa e solu ion o glyce ol o 96 hou s a 20°C. The lis o
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p oduced a ian s is p o ided in Table 1.
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Table 1 Va ian s o mycelium-based biocomposi es
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Va ian
Nu shell
T ea men
W0
Walnu
-
WG
Walnu
Glyce ol
P0
Pis achio
-
PG
Pis achio
Glyce ol
R0
-
-
RG
-
Glyce ol
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Dynamic Vapou So p ion (DVS)
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So p ion/deso p ion iso he ms we e de e mined using a DVS Ad an age appa a us (Su ace
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Measu emen Sys ems L d., London, Uni ed Kingdom). All measu emen s we e conduc ed a
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a cons an empe a u e o 25 °C. The measu emen p og am is illus a ed in Figu e 1A. B ie ly,
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he samples we e ini ially p e-d ied a 0% ela i e humidi y (RH) o 9 hou s. Following his, he
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RH was inc eased in 20% inc emen s, each main ained o 3 hou s, un il eaching 100% RH.
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The deso p ion cycle ollowed he same s eps in e e se, dec easing he RH in 20%
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inc emen s om 100% back o 0%.
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Figu e 1 The measu emen p og am o so p ion/deso p ion iso he ms de e mina ion (A), he
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measu emen p og am used in STA expe imen s (B)
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Dynamic Mechanical Analysis
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Dynamic mechanical analysis (DMA) was conduc ed using DMA 303 Explexo (Ne zsch, Selb,
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Ge many). The samples we e es ed using a p essu e mode, he equency was 1 Hz, he
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dynamic de o ma ion was 0.03 mm, he dynamic o ce was 10 N, and he s a ic o ce was 13
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N. The es ed empe a u e in e al was om 20°C o 230°C wi h an inc emen o 3°C/min. The
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es ed MBB samples had dimensions o 5 mm × 5 mm × 1 mm (leng h × wid h × hickness). In
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con as , he e e ence samples R0 and RG we e signi ican ly hinne , and i was no easible
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o es a single laye o hese ma e ials. The e o e, hese samples o DMA we e p epa ed by
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s acking i e laye s o R0 and RG o achie e compa able hickness. I should be no ed ha
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his laye ed s uc u e di e s om he s uc u e o he MBB samples con aining nu shell
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pa icles. This s uc u al di e ence mus be aken in o accoun when in e p e ing he DMA
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esul s.
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Sho e Ha dness
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Sho e ha dness was measu ed using a Sho e du ome e (PTC Ins umen s, USA), in
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acco dance wi h he EN ISO 868. Sho e A me hod wi h a la ened ip o he inden e was
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employed. Al hough he Sho e A me hod is p ima ily in ended o es ing lexible ma e ials, i
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was employed in his s udy because he p oduced composi es we e b i le, and al e na i e
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ha dness es ing me hods led o ma e ial chipping. Each a ian was measu ed 10 imes.
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Simul aneous The mal Analysis (STA)
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The he mal decomposi ion beha iou o he ma e ial was analysed by simul aneous he mal
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analysis (STA) using a Ne zsch STA 409 PG unde py oly ic condi ions wi h a ni ogen low o
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60 mL/min. P io o measu emen , he sample was g ound o a pa icle size below 0.5 µm using
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a Re sch Ul a Cen i ugal Mill ZM 200. The analysis was pe o med in wo s eps. In he i s
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s ep, he sample was hea ed o 105 °C, ollowed by a 10-minu e iso he mal d ying phase.
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A e wa d, he sample was cooled o 20 °C using liquid ni ogen. In he second s ep, he
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ma e ial was hea ed o 600 °C a a hea ing a e o 10 K/min. An illus a ion o he measu emen
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p og am can be ound in Figu e 1B.
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Fou ie -T ans o m In a ed Spec oscopy (FT-IR)
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To conclude on di e ences in he chemical composi ion o he ilms, FT- IR spec a we e
191
acqui ed wi h 32 scans using an ATR-uni a ached o an FT-IR spec ome e (Ve ex 70,
192
B uke , Bille ica, USA). The d y samples we e milled o ha e a homogeneous mix u e and ou
193
spec a measu ed, a e aged and cu o he mos ele an wa enumbe ange om 1800 cm-1
194
o 850 cm-1 using OPUS 7.5 so wa e (B uke , USA).
195
196
197
Scanning Elec on Mic oscopy (SEM)
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SEM was pe o med o examine he a chi ec u e o he composi es. The samples we e gold
199
spu e ed (4nm) wi h a spu e coa e (LEICA EM SCD005). High- acuum seconda y elec on
200
imaging was pe o med using an Ap eo VS SEM (The mo Scien i ic, The Ne he lands) a 5 and
201
10 kV and 0.10 nA cu en , using he Op iPlan use case, de ec o T1, and mode A+B.
202
3. Resul s and discussion
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Dynamic Vapou So p ion
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The un ea ed MBB om walnu and pis achio nu shells exhibi ed so p ion and deso p ion
205
beha iou compa able o ha o con en ional wood-based composi es (Wu 1999). The highes
206
so p ion hys e esis was obse ed in he un ea ed e e ence samples, while he lowes
207
hys e esis was ound in glyce ol- ea ed MBB made om pis achio nu shells (Figu e 2A-B).
208
Glyce ol- ea ed samples gene ally eached a lowe equilib ium mois u e con en han hei
209
un ea ed coun e pa s. Al hough his obse a ion does no ully co espond wi h ou STA
210
esul s (p esen ed la e in his manusc ip ), i should be emphasised ha he DVS me hod
211
o e s highe p ecision and is speci ically designed o he de e mina ion o his cha ac e is ic.
212
Fu he mo e, he glyce ol ea men led o a la ening o he so p ion/deso p ion iso he ms,
213
sugges ing a modi ied in e ac ion be ween he ma e ial and ambien humidi y caused by he
214
blocking o ee hyd oxyl g oups, which co esponds o al eady published s udies (Essoua e
215
al. 2016; Appels e al. 2020). A nega i e change in mass du ing deso p ion was obse ed in
216
he glyce ol- ea ed samples. This beha iou is caused by esidual ME in he MBB samples
217
and can be explained by hyd a e o ma ion (Scholl and Schmid 2014).
218
219
220
304
Figu e 4 TG, DTG and DSC cu es o he mycelium-based composi es
305
Fou ie -T ans o m In a ed Spec oscopy
306
The FT-IR spec a o he e e ence pu e ungi ma e ials (RO, RG) showed bands a ibu ed o
307
ß-chi in: he s ong amide I band a 1645 cm-1 and high in ensi y skele al C-O-C s e ching
308
bands a ound 1032 cm-1 (Fig. 5, black spec a) (Kumi ska e al. 2010). Typical glyce ol bands
309
a e ound in none o he a ian s (RG, PG, WG), explained by he ac ha i was jus a e y
310
hin ou e laye . In all walnu and pis achio composi es he bands ela ed o chi in we e mixed
311
wi h ypical bands o ligno-cellulosic ma e ials. An ace yl g oup (C=O ib a ion) a ibu ed o
312
hemicelluloses was ound a 1733 cm-1 and an a oma ic s e ching ib a ion ypical o lignin
313
a ound 1505 cm-1 (Faix 1991). Based on he band in ensi y o he la e one we can conclude
314
on lowe lignin con en in pis achio (P0, PG, blue spec a) han in walnu shell composi es
315
(WO, WG). This is in ag eemen wi h o me spec oscopic esul s (Xiao e al. 2021) and
316
chemical analysis poin ing up o 50-% lignin o he o al weigh o he ui (Zhao e al. 2019,
317
Mo ales e al. 2022). The highe lignin con en in walnu shell composi es could explain he
318
highe sho e ha dness.
319
320
Figu e 5 FT-IR spec a o MBB a ian s
321
Scanning Elec on Mic oscopy
322
SEM con i med he hypo hesis ha il a ion o he liquid cul u e, consis ing o mycelium and
323
g inded nu shells in mal ex ac , esul ed in a composi e s uc u e o med by lignocellulosic
324
pa icles in e connec ed wi h ungal hyphae (Figu e 6A–D). On he su aces o he composi es
325
ha we e exposed o ai , a con inuous ungal skin was de eloped (Figu e 6B). As p e iously
326
epo ed, he ungal skin can p o ide addi ional unc ions, as egula coa ing o ma e ials
327
(Appels e al. 2020; Hýsek e al. 2023). G inded walnu and pis achio shells we e isibly bound
328
oge he by hyphae, o ming a po ous and in e connec ed ne wo k (Figu e 6A, C, D). The
329
dense p esence o ungal ma e ial is consis en wi h FTIR esul s indica ing a high chi in and
330
polysaccha ide con en . No signi ican s uc u al di e ences we e obse ed using SEM
331
be ween un ea ed and glyce ol- ea ed samples, sugges ing ha he glyce ol ea men did
332
no a ec he mo phology o he composi e a he mic oscale.
333
334
Figu e 6 S uc u e o c ea ed composi es; c oss sec ion o PG sample (A), c oss sec ion o
335
he su ace o PG sample (B), s uc u e o b eached W0 sample (C), c oss sec ion o WG
336
sample (D).
337
4. Conclusion
338
This s udy demons a ed he ab ica ion o MBBs ein o ced wi h g inded walnu and pis achio
339
nu shells using a combina ion o liquid- and solid-s a e e men a ion. The applied p ocess
340
enabled e ec i e binding o lignocellulosic pa icles by Ganode ma sessile wi hou syn he ic
341
adhesi es, esul ing in sel -binding composi e s uc u es. The ype o nu shell ille and pos -
342
ea men wi h glyce ol signi ican ly in luenced he p ope ies o he ma e ials. Walnu -based
343
MBBs ou pe o med pis achio-based a ian s in mechanical pe o mance, wi h he glyce ol-
344
ea ed walnu composi e achie ing he highes Sho e A ha dness and exhibi ing he bes
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s i ness and he mal s abili y cha ac e is ics. Mois u e- ela ed p ope ies we e also imp o ed.
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DVS measu emen s showed ha glyce ol- ea ed MBBs had a lowe equilib ium mois u e
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con en and a educ ion in so p ion hys e esis. FTIR spec a con i med composi ional
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di e ences, pa icula ly highe lignin signals in walnu -based samples and s ong chi in- ela ed
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bands ac oss all a ian s. SEM imaging e ealed a uni o m composi e mo phology, wi h no
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s uc u al deg ada ion due o glyce ol ea men , and he p esence o a ungal skin laye on
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su aces exposed o ai . These esul s demons a e ha nu shells a e iable ille s o MBBs
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and ha bio echnological app oaches, such as combined-s a e e men a ion, can be applied
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o enginee mechanically ein o ced bio-composi es o sus ainable applica ions.
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Acknowledgemen : This esea ch was suppo ed by HORIZON-MSCA-2022-PF-01-01, g an
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ag eemen ID: 101105443, p ojec ac onym: LignoMBB and ERC-POC g an ag eemen ID:
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101113395, p ojec ac onym PUZZLE MATERIALS.
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Da a A ailabili y S a emen : Da a can be downloaded a he ollowing link:
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h ps://doi.o g/10.5281/zenodo.17539612
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Decla a ion o gene a i e AI and AI-assis ed echnologies in he manusc ip p epa a ion
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