Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis

Ulla Moilanen

Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis

Julkaisun otsikon käännös: Ligniiniä muokkaavat entsyymit – tuotanto kiinteällä alustalla, käyttö väriaineiden poistossa ja lignoselluloosan entsymaattisessa hydrolyysissä

Ulla Moilanen

Tutkimustuotos: Doctoral Thesis › Collection of Articles

Abstrakti

Lignoselluloosaa sisältävä kasvibiomassa koostuu pääasiassa kolmesta polymeeristä: selluloosasta, hemiselluloosasta ja ligniinistä. Toisiinsa tiukasti sitoutuneina nämä kompo-nentit muodostavat mikrobien hajotukselle vastustuskykyisen rakenteen. Lahottajasienet hajottavat kasvibiomassaa erittämällä solunulkoisia entsyymejä, jotka joko hajottavat tai muokkaavat hiilihydraatteja ja ligniiniä. Tässä väitöskirjassa keskityttiin ligniiniä muokkaavien entsyymien tuotantoon ja sovelluksiin. Entsyymejä tuotettiin kiinteillä lignoselluloosaa sisältävillä kasvatusalustoilla Kun Cerrena unicolor -valkolahosientä kasvatettiin laboratoriomittakaavan (20 l) bio-reaktorissa, saavutettiin korkeita entsyymiaktiivisuuksia. Mangaaniperoksidaasiaktiivisuus (340 nkat g-1 ka) oli korkeampi kuin aiemmin raportoiduissa kiinteäalusta- tai neste-kasvatuksissa. Lakkaasiaktiivisuus (470 nkat g-1 ka) oli samaa tasoa kuin aiemmin saavutetut. Näiden entsyymien tuotto oli ainakin osittain riippuvaista kasvusta ja entsyyminmuodostus viittasi useiden isoentsyymien erittymiseen. Lakkaasit ovat hapettavia entsyymejä, joita voidaan laajan substraattivalikoimansa vuoksi hyödyntää useissa sovelluksissa. Tässä työssä käytettiin kahden valkolahosienen (C. unicolor ja Trametes hirsuta) lakkaaseja tekstiiliteollisuudessa syntyvien jätevesien värinpoistossa ja lignoselluloosaa sisältävien raaka-aineiden entsymaattisen hydrolyysin tehostamisessa. Tekstiilivärien liuoksia käsiteltiin lakkaaseilla, mikä johti jopa 75 %:n värinpoistoon jo 7,5 tunnissa. Tekstiiliteollisuuden jätevesiä simuloitaessa väriliuoksiin lisättiin useita lakkaaseille mahdollisesti haitallisia aineita, mikä hidasti värinhajotusta. Siitä huolimatta 50-90 %:n värin-poisto saavutettiin 19,5 tunnissa. T. hirsuta -lakkaasin suorituskyky parani huomattavasti mediaattoreita lisäämällä, kun taas C. unicolor -lakkaasi ei tarvinnut mediaattoreita riittävän värinpoiston saavuttamiseen. Lakkaasien ja mediaattorien käyttöä tutkittiin termisesti esikäsiteltyjen lignoselluloosa-raaka-aineiden entsymaattisen hydrolyysin tehostamisessa. Pelkkä lakkaasikäsittely paransi kuusen, mutta huononsi jättiruo'on hydrolyysiä. Mediaattorien lisäys kasvatti kuusen sokeri-saantoa entisestään yli 50 %:lla. Lisäksi havaittiin, että TEMPO, joka on yksi käytetyimmistä lakkaasin mediaattoreista, hapetti lakkaasin avulla selluloosaa. Hapetus kuitenkin heikensi selluloosan entsymaattista hydrolyysiä. Lisäksi osoitettiin ensimmäistä kertaa, että lakkaasi-mediaattorikäsittelyjen aiheuttama kuusen hydrolyysisaannon kasvu johtui selluloosaa hajottavien entsyymien vähäisemmästä sitoutumisesta ligniiniin, mikä mahdollisti selluloosan tehokkaamman hydrolyysin.

Julkaisun otsikon käännös	Ligniiniä muokkaavat entsyymit – tuotanto kiinteällä alustalla, käyttö väriaineiden poistossa ja lignoselluloosan entsymaattisessa hydrolyysissä
Alkuperäiskieli	Englanti
Pätevyys	Tohtorintutkinto
Myöntävä instituutio	Aalto-yliopisto
Valvoja/neuvonantaja	Ojamo, Heikki, Vastuuprofessori Viikari, Liisa, Ohjaaja, Ulkoinen henkilö
Kustantaja	Aalto University
Painoksen ISBN	978-952-60-6195-5
Sähköinen ISBN	978-952-60-6196-2
Tila	Julkaistu - 2015
OKM-julkaisutyyppi	G5 Artikkeliväitöskirja

Tutkimusalat

kiinteäalusta
värinpoisto
entsymaattinen hydrolyysi
lakkaasi
mediaattori

Pääsy asiakirjaan

http://urn.fi/URN:ISBN:978-952-60-6196-2Lisenssi: Määrittelemätön

Biotalousinfrastruktuuri
Seppälä, J. (Manager)
Kemian tekniikan korkeakoulu
Laitteistot/tilat: Facility

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@phdthesis{caa8f6888c3f41b2b2b488621e3eb640,

title = "Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis",

abstract = "Lignocellulosic plant biomass is composed of three main polymers: cellulose, hemicelluloses, and lignin. These components are bound together tightly forming a resistant structure. In nature, wood-rotting fungi efficiently decompose plant biomass by secreting extracellular enzymes that degrade or modify carbohydrates and lignin. This thesis focuses on the production and application of lignin-modifying enzymes. The enzymes were produced in solid-state cultivations on a lignocellulosic substrate. High enzyme activities were observed when the white-rot fungus Cerrena unicolor was cultivated in a larger laboratory-scale bioreactor (20 L), with which manganese peroxidase activity (340 nkat g-1 DM) was higher than what was obtained in submerged or solid-state cultures of the same scale and laccase activity (470 nkat g-1 DM) was comparable to activities reported previously. Production of the two enzymes appeared to be at least partially associated with growth and the enzyme production pattern indicated the formation of several isoforms of the enzymes. Laccases are oxidative enzymes that have a broad substrate range and thus can be utilized in several applications. In this study, two fungal laccases, from C. unicolor and Trametes hirsuta, were used for decolorizing textile dye baths and improving enzymatic hydrolysis of lignocellulosic feedstocks. Textile dye solutions were treated with laccases, leading to up to 75% color removal already in 7.5 h. When industrial textile dye effluents were simulated, the added, potentially inhibitory components slowed down the decolorization. Nevertheless, a 50 to 90% color removal was reached with C. unicolor laccase in 19.5 h. The addition of mediators enhanced the action of T. hirsuta laccase to a remarkable degree. Notably, C. unicolor laccase did not require mediators to degrade all the tested dyes satisfactorily. Laccases and mediators were studied for the modification of lignin and cellulose to enhance the saccharification yields of lignocellulosic substrates. It was demonstrated that laccase treatment alone increased the hydrolysis of steam pre-treated spruce (SPS) but decreased that of steam pre-treated giant reed. With added mediators, the hydrolysis of SPS was increased even further and saccharification yields improved by more than 50%. In addition, it was shown that one of the most common laccase mediators, TEMPO, oxidized cellulose when applied together with laccase, but that the oxidation impaired the enzymatic hydrolysis of cellulose. Finally, it was demonstrated for the first time that the increased hydrolysis of SPS, treated with laccases and mediators, was caused by reduced non-productive binding of cellulose-degrading enzymes on lignin, which enabled more efficient hydrolysis of cellulose.",

keywords = "solid-state, dye decolorization, enzymatic hydrolysis, laccase, mediator, lignin, kiinte{\"a}alusta, v{\"a}rinpoisto, entsymaattinen hydrolyysi, lakkaasi, mediaattori, solid-state, dye decolorization, enzymatic hydrolysis, laccase, mediator, lignin",

author = "Ulla Moilanen",

year = "2015",

language = "English",

isbn = "978-952-60-6195-5",

series = "Aalto University publication series DOCTORAL DISSERTATIONS",

publisher = "Aalto University",

number = "64",

address = "Finland",

school = "Aalto University",

}

TY - BOOK

T1 - Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis

AU - Moilanen, Ulla

PY - 2015

Y1 - 2015

N2 - Lignocellulosic plant biomass is composed of three main polymers: cellulose, hemicelluloses, and lignin. These components are bound together tightly forming a resistant structure. In nature, wood-rotting fungi efficiently decompose plant biomass by secreting extracellular enzymes that degrade or modify carbohydrates and lignin. This thesis focuses on the production and application of lignin-modifying enzymes. The enzymes were produced in solid-state cultivations on a lignocellulosic substrate. High enzyme activities were observed when the white-rot fungus Cerrena unicolor was cultivated in a larger laboratory-scale bioreactor (20 L), with which manganese peroxidase activity (340 nkat g-1 DM) was higher than what was obtained in submerged or solid-state cultures of the same scale and laccase activity (470 nkat g-1 DM) was comparable to activities reported previously. Production of the two enzymes appeared to be at least partially associated with growth and the enzyme production pattern indicated the formation of several isoforms of the enzymes. Laccases are oxidative enzymes that have a broad substrate range and thus can be utilized in several applications. In this study, two fungal laccases, from C. unicolor and Trametes hirsuta, were used for decolorizing textile dye baths and improving enzymatic hydrolysis of lignocellulosic feedstocks. Textile dye solutions were treated with laccases, leading to up to 75% color removal already in 7.5 h. When industrial textile dye effluents were simulated, the added, potentially inhibitory components slowed down the decolorization. Nevertheless, a 50 to 90% color removal was reached with C. unicolor laccase in 19.5 h. The addition of mediators enhanced the action of T. hirsuta laccase to a remarkable degree. Notably, C. unicolor laccase did not require mediators to degrade all the tested dyes satisfactorily. Laccases and mediators were studied for the modification of lignin and cellulose to enhance the saccharification yields of lignocellulosic substrates. It was demonstrated that laccase treatment alone increased the hydrolysis of steam pre-treated spruce (SPS) but decreased that of steam pre-treated giant reed. With added mediators, the hydrolysis of SPS was increased even further and saccharification yields improved by more than 50%. In addition, it was shown that one of the most common laccase mediators, TEMPO, oxidized cellulose when applied together with laccase, but that the oxidation impaired the enzymatic hydrolysis of cellulose. Finally, it was demonstrated for the first time that the increased hydrolysis of SPS, treated with laccases and mediators, was caused by reduced non-productive binding of cellulose-degrading enzymes on lignin, which enabled more efficient hydrolysis of cellulose.

AB - Lignocellulosic plant biomass is composed of three main polymers: cellulose, hemicelluloses, and lignin. These components are bound together tightly forming a resistant structure. In nature, wood-rotting fungi efficiently decompose plant biomass by secreting extracellular enzymes that degrade or modify carbohydrates and lignin. This thesis focuses on the production and application of lignin-modifying enzymes. The enzymes were produced in solid-state cultivations on a lignocellulosic substrate. High enzyme activities were observed when the white-rot fungus Cerrena unicolor was cultivated in a larger laboratory-scale bioreactor (20 L), with which manganese peroxidase activity (340 nkat g-1 DM) was higher than what was obtained in submerged or solid-state cultures of the same scale and laccase activity (470 nkat g-1 DM) was comparable to activities reported previously. Production of the two enzymes appeared to be at least partially associated with growth and the enzyme production pattern indicated the formation of several isoforms of the enzymes. Laccases are oxidative enzymes that have a broad substrate range and thus can be utilized in several applications. In this study, two fungal laccases, from C. unicolor and Trametes hirsuta, were used for decolorizing textile dye baths and improving enzymatic hydrolysis of lignocellulosic feedstocks. Textile dye solutions were treated with laccases, leading to up to 75% color removal already in 7.5 h. When industrial textile dye effluents were simulated, the added, potentially inhibitory components slowed down the decolorization. Nevertheless, a 50 to 90% color removal was reached with C. unicolor laccase in 19.5 h. The addition of mediators enhanced the action of T. hirsuta laccase to a remarkable degree. Notably, C. unicolor laccase did not require mediators to degrade all the tested dyes satisfactorily. Laccases and mediators were studied for the modification of lignin and cellulose to enhance the saccharification yields of lignocellulosic substrates. It was demonstrated that laccase treatment alone increased the hydrolysis of steam pre-treated spruce (SPS) but decreased that of steam pre-treated giant reed. With added mediators, the hydrolysis of SPS was increased even further and saccharification yields improved by more than 50%. In addition, it was shown that one of the most common laccase mediators, TEMPO, oxidized cellulose when applied together with laccase, but that the oxidation impaired the enzymatic hydrolysis of cellulose. Finally, it was demonstrated for the first time that the increased hydrolysis of SPS, treated with laccases and mediators, was caused by reduced non-productive binding of cellulose-degrading enzymes on lignin, which enabled more efficient hydrolysis of cellulose.

KW - solid-state

KW - dye decolorization

KW - enzymatic hydrolysis

KW - laccase

KW - mediator

KW - lignin

KW - kiinteäalusta

KW - värinpoisto

KW - entsymaattinen hydrolyysi

KW - lakkaasi

KW - mediaattori

KW - solid-state

KW - dye decolorization

KW - enzymatic hydrolysis

KW - laccase

KW - mediator

KW - lignin

M3 - Doctoral Thesis

SN - 978-952-60-6195-5

T3 - Aalto University publication series DOCTORAL DISSERTATIONS

PB - Aalto University

ER -

Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis

Abstrakti

Tutkimusalat

Pääsy asiakirjaan

Sormenjälki

Laitteet

Biotalousinfrastruktuuri

Siteeraa tätä