Luonnosta peräisin olevien fenolisten välittäjäaineiden toiminta hapettavien entsyymien katalysoimassa ligniinin malliyhdisteiden ja synteettisen ligniinin kemiallisessa muokkaamisessa

Eco-efficient biotechnological applications are important innovations for modern industrial developments, such as production of chemicals and fuels from renewable biomass. Lignocellulosic biomass is the only sustainable organic resource that can be converted to both energy and various value-added products. The effective use of wood and plants for feedstock in biorefineries is in most cases dependent on the plant cell wall recalcitrance and especially the extent of lignification of the cell wall. Oxidative enzymes such as laccases and manganese peroxidases (MnPs), provide interesting possibilities for plant biomass modification as they are the key catalysts in modification and degradation of the most recalcitrant component of the lignocellulose biopolymers, lignin, in nature. Both peroxidases and laccases exhibit low substrate specificity and produce organic radicals through one-electron abstraction reactions. In many cases these enzymes require low molecular weight co-substrates to mediate oxidation to the more recalcitrant parts of lignin polymer. In the first study of this thesis, various types of lignin model compounds were investigated both as mediators and as final targets to screen their effect and potential in laccase-catalyzed oxidation reactions. Especially acetosyringone and methyl syringate were found to mediate the laccase-catalyzed oxidation even with low-redox potential laccase that generally shows only fair oxidation activity on lignocellulosic oxidation. Secondly, in two of the articles, the mediating rate and effectivity were enhanced by using high redox potential laccases and suitable reaction conditions to retain the stability of the mediator. Oxidation potentials, pH and chemical stability of the mediator were found to affect the oxidation efficiency. These simple phenolics were, for the first time, also proved to mediate the MnP-catalyzed reactions of non-phenolic compounds. The problematic stability of peroxidases was circumvented by utilizing reaction system with no external addition of hydrogen peroxide. Finally, to prove the effect on polymer material a new reaction set-up was developed for oxidation of synthetic lignin (DHP, dehydrogenation polymer) and the structural analysis of the oxidized polymers showed clear modifications in the polymer outcome. In all cases, mediated reactions gave selective alpha-oxidation products of benzylic alcohol moieties, the prevailing structural moiety in lignin. These oxidations provide valuable intermediates for further treatments in various lignin valorization processes. In the near future, this oxidative laccase-mediator oxidative system will be explored in an EU Horizon 2020 project for valorization of biorefinery lignin by combination of biotechnical and chemical means to added-value products such as marine biofuel, engine octane boosters and valuable low-molecular weight chemicals.Fenyylipropaaniyksiköistä koostuva ligniini on selluloosan jälkeen toiseksi yleisin biopolymeeri maapallolla. Metsäpuiden ja kasvien solunseinämissä ligniinillä on useita merkittäviä tehtäviä. Ligniini sitoo kasvien kuituja toisiinsa lisäten rungon mekaanista lujuutta. Hydrofobisena ja antibakteerisena aineena ligniinillä on myös keskeinen merkitys kasvien vedenkuljetuksessa sekä kasvien suojautumisessa mikrobeja ja taudinaiheuttajia vastaan. Ligniinin osuus metsäpuissa ja ruohoissa on 15-35 % kuivapainosta, joten suuren hiilipitoisuutensa vuoksi sen taloudellinen merkitys on huomattava etsittäessä korvaavia raaka-ainelähteitä fossiilisille raaka-aineille. Valkolahosienet tuottavat ligniiniä hajottavia ja muokkaavia entsyymejä, oksidoreduktaaseja, jotka käsittävät erilaisia peroksidaaseja ja lakkaaseja. Synteettisten tai luonnosta peräisin olevien yhdisteiden, ns. mediaattoreiden, avulla lakkaasit hapettavat myös yhdisteitä, joita ne eivät tavallisesti ole sen luonnollisia substraatteja. Sienten lakkaasi-mediaattoripari muodostaa siten hyvin tehokkaan luonnollisen hapetus-systeemin, jolla voi olla monenlaisia bioteknisiä sovelluksia. Näitä sovelluksia on jo tutkittu mm. paperimassan valkaisussa, biomassan esikäsittelyssä biopolttoaineiden tuotantoon, jätevesien ja vaikeasti hajoavien ympäristömyrkkyjen käsittelyssä, tekstiilikuitujen muokkauksessa ja sekä muissa kohteissa kuten erilaisten materiaalien bioteknisessä muokkauksessa. Jotta näitä lupaavia sovelluksia voitaisiin optimoida, erilaisten entsyymien sekä entsyymi-mediaattorisysteemien katalyyttiset mekanismit tulisi tuntea, varsinkin niiden hapetuspotentiaali sekä substraattispesifisyys. Välittäjäaineiden kohdalla puolestaan on substraattiominaisuuksien ohella tunnettava myös niiden stabiilisuus sekä katalyyttinen tehokkuus. Työssä tutkittiin erilaisten oksidoreduktaasi-mediaattori -parien katalyyttisen systeemin tehokkuutta lignoselluloosan muokkauksessa käyttäen erilaisia ligniinin malliyhdisteitä sekä synteettistä ligniiniä substraatteina. Työn tarkoituksena on ollut osoittaa, että erilaiset luonnosta eristetyt fenoliset yhdisteet tai niiden johdannaiset, luonnolliset mediaattorit , pystyvät välittämään entsymaattisen hapetuksen myös molekyyleille, joiden hapetuspotentiaali on liian korkea itse entsyymin hapetettavaksi tai joiden koko on liian suuri jotta ne voisivat toimia entsyymin substraattina. Vertailuna käytettiin kaupallisia synteettisiä mediaattoreita, joiden hapetusteho on ennestään tunnettu, mutta jotka haitallisuutensa ja kalleutensa vuoksi eivät sovellu teollisen mittakaavan prosesseihin. Kun katalyyttina käytettiin matalan hapetuspotentiaalin omaavaa lakkaasia, havaittiin, että fenoliset mediaattorit toimivat joko yhtä hyvin tai jopa paremmin kuin kaupalliset mediaattorit etenkin pienemmässä mediaattoripitoisuudessa. Toisaalta käytettäessä katalyyttinä korkean hapetuspotentiaalin lakkaaseja, sekä kaupallisten että fenolisten mediaattoreiden tehokkuus lisääntyi. Reaktio-olosuhteilla havaittiin olevan myös merkittävä vaikutus käytettäessä fenolisia mediaattoreita, ja tässä yhteydessä todettiin että pH-olosuhteet tulisi optimoida pikemminkin käytetyn mediaattorin mukaan kuin katalyytin toiminnallisen optimin mukaan. Työssä osoitettiin lopuksi, että fenoliset mediaattorit soveltuvat lakkaasihapetusten lisäksi tehostamaan erilaisten peroksidaasien radikaalihapetusreaktioita. Viimeksi mainituissa tutkimuksissa peroksidaasi-mediaattorisysteemin teho ligniinin muokkauksessa osoitettiin kehittämällä yksinkertainen reaktiosysteemi synteettisen ligniinin hapettamiseksi

Development of the ammonia emission inventory in Finland. Revised model for agriculture

Agriculture is the main source of ammonia (NH3) emissions in Finland comprising ca. 90% of the total emissions annually. Agriculture is also an important source of nitrous oxide (N2O), a greenhouse gas for which agriculture is responsible for ca. 50% of emissions. The main source for ammonia is livestock manure whereas for N2O its importance is much smaller. However, the same activity data are needed to assess both NH3 and direct N2O emissions from animal husbandry. In addition to this, indirect emissions of N2O are calculated based on NH3 and NO emissions. NH3 and N2O emissions are annually reported according to international reporting classifications. The aims of the study were 1) to construct a calculation model for gaseous agricultural nitrogen emissions thereby developing and updating the emission calculation procedure to better reflect the development of these emissions in Finland, and 2) to improve correspondence of the emission inventory reporting with the reporting classifications. In 2007, the Finnish emissions of ammonia from agricultural sources totalled 30,686 tonnes, of which more than 60% originated from cattle manure. Time series for ammonia emissions from agriculture show that there have been no large changes in the total emissions during the last two decades. Despite the decreased number of cattle during that period the emissions have remained near the present level, mainly because of the increased nitrogen excretion of cattle. Emission projections for the years 2008–2050 show no significant changes in emissions in the future. As for ammonia, no significant changes for nitrous oxide emissions from animal husbandry have taken place, and no big changes can be expected in the future as long as there are no drastic alterations in animal production. Despite the development of emission modelling, the emission estimates still include significant sources of uncertainty, which is mainly related to information on the distribution of manure management systems and the use of different manure application methods as well as to information on ammonia evaporation in different manure management phases in Finland

Infrared and Raman spectra of lignin substructures : Dibenzodioxocin

Vibrational spectroscopy is a very suitable tool for investigating the plant cell wall in situ with almost no sample preparation. The structural information of all different constituents is contained in a single spectrum. Interpretation therefore heavily relies on reference spectra and understanding of the vibrational behavior of the components under study. For the first time, we show infrared (IR) and Raman spectra of dibenzodioxocin (DBDO), an important lignin substructure. A detailed vibrational assignment of the molecule, based on quantum chemical computations, is given in the Supporting Information; the main results are found in the paper. Furthermore, we show IR and Raman spectra of synthetic guaiacyl lignin (dehydrogenation polymer-G-DHP). Raman spectra of DBDO and G-DHP both differ with respect to the excitation wavelength and therefore reveal different features of the substructure/polymer. This study confirms the idea previously put forward that Raman at 532 nm selectively probes end groups of lignin, whereas Raman at 785 nm and IR seem to represent the majority of lignin substructures.Peer reviewe

Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N–OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)–(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.Peer reviewe

Fractionation of Technical Lignin from Enzymatically Treated Steam-Exploded Poplar Using Ethanol and Formic Acid

Lignocellulosic biorefineries produce lignin-rich side streams with high valorization potential concealed behind their recalcitrant structure. Valorization of these residues to chemicals, materials, and fuels increases the profitability of biorefineries. Fractionation is required to reduce the lignins’ structural heterogeneity for further processing. We fractionated the technical biorefinery lignin received after steam explosion and saccharification processes. More homogeneous lignin fractions were produced with high β-O-4′ and aromatic content without residual carbohydrates. Non-toxic biodegradable organic solvents like ethanol and formic acid were used for fractionation and can be adapted to the existing biorefinery processes. Macromolecular properties of the isolated fractions were carefully characterized by structural, chemical, and thermal methods. The ethanol organosolv treatment produced highly soluble lignin with a reasonable yield, providing a uniform material for lignin applications. The organosolv fractionation with formic acid and combined ethanol-formic acid produced modified lignins that, based on thermal analysis, are promising as thermoresponsive materials.Lignocellulosic biorefineries produce lignin-rich side streams with high valorization potential concealed behind their recalcitrant structure. Valorization of these residues to chemicals, materials, and fuels increases the profitability of biorefineries. Fractionation is required to reduce the lignins' structural heterogeneity for further processing. We fractionated the technical biorefinery lignin received after steam explosion and saccharification processes. More homogeneous lignin fractions were produced with high beta-O-4 ' and aromatic content without residual carbohydrates. Non-toxic biodegradable organic solvents like ethanol and formic acid were used for fractionation and can be adapted to the existing biorefinery processes. Macromolecular properties of the isolated fractions were carefully characterized by structural, chemical, and thermal methods. The ethanol organosolv treatment produced highly soluble lignin with a reasonable yield, providing a uniform material for lignin applications. The organosolv fractionation with formic acid and combined ethanol-formic acid produced modified lignins that, based on thermal analysis, are promising as thermoresponsive materials.Peer reviewe

Exploring laccase mediator systems with acidic recombinant enzymes from Obba rivulosa

Peer reviewe

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories

Background The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. Results Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. Conclusions This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.Peer reviewe

On the Effect of Hot-Water Pretreatment in Sulfur-Free Pulping of Aspen and Wheat Straw

Published under an ACS AuthorChoice LicenseIn modern biorefineries, low value lignin and hemicellulose fractions are produced as side streams. New extraction methods for their purification are needed in order to utilize the whole biomass more efficiently and to produce special target products. In several new applications using plant based biomaterials, the native-type chemical and polymeric properties are desired. Especially, production of high-quality native-type lignin enables valorization of biomass entirely, thus making novel processes sustainable and economically viable. To investigate sulfur-free possibilities for so-called "lignin first" technologies, we compared alkaline organosolv, formic acid organosolv, and ionic liquid processes to simple soda "cooking" using wheat straw and aspen as raw materials. All experiments were carried out using microwave-assisted pulping approach toy enable rapid heat transfer and convenient control of temperature and pressure. The main target was to evaluate the advantage of a brief hot water extraction as a pretreatment for the pulping process. Most of these novel pulping methods resulted in high-quality lignin, which may be valorized more diversely than kraft lignin. Lignin fractions were thoroughly analyzed with NMR (C-13 and HSQC) and gel permeation chromatography to study the quality of the collected lignin. The cellulose fractions were analyzed by determining their lignin contents and carbohydrate profiles for further utilization in cellulose-based products or biofuels.Peer reviewe

Samapalkkaisuuden perusteet ja edistäminen

Naisten ja miesten välinen palkkaero kaventuu Suomessa hitaasti. Palkkatasa-arvon saavuttaminen edellyttää eriarvoistavien palkkarakenteiden korjaamista, palkkasyrjintään puuttumista sekä tasa-arvon aktiivista edistämistä. Samapalkkaisuus ja syrjimättömyys ovat ihmis- ja perusoikeuksia, jotka perustuvat kansainvälisiin sopimuksiin, EUoikeuteen sekä Suomen kansalliseen lainsäädäntöön. Julkaisu kokoaa viiden asiantuntijan ajankohtaiset puheenvuorot työelämän keskeisestä tasa-arvo-ongelmasta. Kirjoittajat lähestyvät kysymystä ensisijaisesti oikeudellisesta, mutta myös rakenteellisesta näkökulmasta sekä ottavat kantaa palkkaeron kaventamiskeinoihin. Outi Viitamaa-Tervonen luo katsauksen sukupuolten työmarkkina-asemaan ja rakenteisiin, jotka ylläpitävät palkkaeroja sekä pohtii sitä, millaisin keinoin samapalkkaisuuteen tulisi pyrkiä. Niklas Bruun käsittelee Suomen kansainvälisiä palkkatasa-arvovelvoitteita erityisesti CEDAW- ja ILO-sopimusten valossa. Anja Nummijärvi arvioi kansallista palkkakartoitussääntelyä ja sen toimivuutta. Kevät Nousiainen puolestaan käsittelee palkka-avoimuuden oikeudellisia edellytyksiä ja merkitystä samapalkkaisuusperiaatteen toteuttamisessa. Lopuksi Paula Koskinen Sandberg avaa suomalaisen samapalkkaisuuspolitiikan ja korporatistisen järjestelmän yhteyksiä