Structural characterization of cellulosic materials using x-ray and neutron scattering

Cellulosic biomass can be used as a feedstock for sustainable production of biofuels and various other products. A complete utilization of the raw material requires understanding on its structural aspects and their role in the various processes. In this thesis, x-ray and neutron scattering methods were applied to study the structure of various cellulosic materials and how they are affected in different processes. The obtained results were reviewed in the context of a model for the cellulose nanostructure. The dimensions of cellulose crystallites and the crystallinity were determined with wide-angle x-ray scattering (WAXS), whereas the nanoscale fibrillar structure of cellulose was characterized with small-angle x-ray and neutron scattering (SAXS and SANS). The properties determined with the small-angle scattering methods included specific surface areas and distances characteristic of the packing of cellulose microfibrils. Also other physical characterization methods, such as x-ray microtomography, infrared spectroscopy, and solid-state NMR were utilized in this work. In the analysis of the results, a comprehensive understanding of the structural changes throughout a range of length scales was aimed at. Pretreatment of birch sawdust by pressurized hot water extraction was observed to increase the crystal width of cellulose, as determined with WAXS, even though the cellulose crystallinity was slightly decreased. A denser packing of microfibrils caused by the removal of hemicelluloses and lignin in the extraction was evidenced by SAXS. This resulted in the opening of new pores between the microfibril bundles and an increase of the specific surface area. Enzymatic hydrolysis of microcrystalline cellulose (MCC) did not lead to differences in the average crystallinity or crystal size of the hydrolysis residues, which was explained to be caused by limitations due to the large size of the enzymes as compared to the pores inside the fibril aggregates. The SAXS intensities of rewetted samples suggested a modest opening of the fibrillar structure during hydrolysis, but no changes could be observed in the dry state. Nanofibrillated cellulose (NFC) made of birch pulp with original and reduced xylan content was used as the substrate for enzymatic hydrolysis to reduce the effects of fibrillar aggregation. The results showed that the xylan present in the NFC with original xylan content limited the hydrolysis and caused an increase in cellulose crystallinity and crystal width, whereas the hydrolysis of NFC with reduced xylan content was more efficient and no increase in crystallinity or crystal size was detected. According to the SANS and SAXS results, the fibril network retained its shape in the NFC with original xylan content, whereas it gradually broke down during the hydrolysis in the NFC with reduced xylan content. Cellulose whiskers were prepared from MCC without any major change in the cellulose crystallinity, as determined with WAXS and solid-state NMR. The drying behaviour of the whiskers was studied with SAXS by characterizing the structures formed under freeze-drying and under ambient conditions. The resulting structures were observed to be influenced by both the drying method and by surface charge neutralization.Selluloosaisten materiaalien nanometritason rakennetta ja sen muuttumista erilaisissa prosesseissa tutkittiin röntgen- ja neutronisironnalla. Saadut tulokset lisäävät ymmärrystä selluloosapohjaisen biomassan rakenteesta ja edistävät ympäristöystävällisten polttoaineiden, kemikaalien ja materiaalien valmistamista eloperäisistä, uusiutuvista raaka-aineista. Työssä selvitettiin muun muassa, kuinka koivupurun rakenne muuttaa muotoaan esikäsittelyssä ja millaiset tekijät vaikeuttavat selluloosan hajottamista entsyymeillä. Pitkät, ketjumaiset selluloosamolekyylit muodostavat luonnossa kuitumaisia mikrofibrillejä, joissa ketjut ovat osittain järjestyneet kiteiksi. Selluloosamikrofibrillit ovat puolestaan liittyneet yhteen suuremmiksi kimpuiksi, jotka lopulta muodostavat kasvisoluseinän jäykän tukirangan yhdessä hemiselluloosien ja ligniinin kanssa. Bioetanolin valmistaminen selluloosasta edellyttää tämän monimutkaisen rakenteen rikkomista ja selluloosamolekyylien pilkkomista glukoosiksi. Jalostusprosessin alussa biomassa täytyy saattaa sellaiseen muotoon, että selluloosaa pilkkovat entsyymit pääsevät siihen helposti käsiksi. Tässä työssä havaittiin, kuinka paineistetussa kuumassa vedessä tehty esikäsittely suurensi mikrofibrillikimppujen välisiä huokosia koivusahanpurussa, mikä todennäköisesti helpottaa entsyymien toimintaa. Samalla selluloosakiteiden koko kasvoi hieman ja mikrofibrillikimput tiivistyivät hemiselluloosien poistuessa mikrofibrillien välistä. Työssä saadut tulokset auttavat myös ymmärtämään entsyymien toimintaa ja tehostamaan selluloosan pilkkomisprosessia. Tiiviisti yhteen liittyneiden mikrofibrillien hajottaminen mikrokiteisessä selluloosassa oli hidasta, koska entsyymit eivät päässeet tunkeutumaan mikrofibrillien väleihin. Tämän vuoksi monet keskiarvoiset ominaisuudet, kuten selluloosan kiteisyys ja kidekoko, säilyivät. Kun yksittäiset mikrofibrillit oli erotettu toisistaan mekaanisesti, voitiin havaita myös hemiselluloosien, tarkemmin sanottuna ksylaanin, vaikeuttavan entsyymien toimintaa. Runsaasti ksylaania sisältäneessä nanoselluloosassa selluloosakiteiden koko kasvoi ja fibrillien muodostama verkkomainen rakenne säilytti muotonsa, kun taas vähemmän ksylaania sisältänyt nanoselluloosa hajosi helpommin, niin kidetasolla kuin laajemmassakin mittakaavassa. Myös selluloosasta valmistettujen muutamien nanometrien paksuisten jäykkien tikkujen, whiskereiden, ominaisuuksia ja kuivumista tutkittiin. Eroja havaittiin eri kuivausmenetelmien välillä ja rakenteelliset erot heijastuivat muun muassa whiskereiden lämmönsietokykyyn. Whiskereitä voidaan käyttää esimerkiksi lujittamaan komposiittimateriaaleja, jolloin kyseisillä ominaisuuksilla on suuri merkitys. Röntgensirontamenetelmistä laajakulmasironnalla saadaan tietoa selluloosakiteiden koosta ja niiden suhteellisesta osuudesta näytteessä, kun taas pienkulmasironta kuvaa mikrofibrillien keskinäistä järjestystä ja esimerkiksi huokosrakenteen ominaisuuksia. Röntgenmenetelmien lisäksi työssä sovellettiin pienkulmaneutronisirontaa

Effects of enzymatic hydrolysis on the structure of microcrystalline cellulose

Cellulose can be used as a renewable raw material for energy production. The utilization requires degradation of cellulose into glucose, which can be done with the aid of enzymatic hydrolysis. In this thesis, various x-ray methods were used to characterize sub-micrometer changes in microcrystalline cellulose during enzymatic hydrolysis to clarify the process and factors slowering it. The methods included wide-angle x-ray scattering (WAXS), small-angle x-ray scattering (SAXS) and x-ray microtomography. In addition, the samples were studied with transmission electron microscopy (TEM). The studied samples were hydrolyzed by enzymes of the Trichoderma reesei species for 6, 24, and 75 hours, which corresponded to 31 %, 58 %, and 68 % degrees of hydrolysis, respectively. Freeze-dried hydrolysis residues were measured with WAXS, SAXS and microtomography, whereas some of them were re-wetted for the wet SAXS and TEM measurements. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures similar cylindrical and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results were ambiguous and partly imprecise, but showed a change in the structure of wet samples in scale of 10-30 nm. According to the WAXS results, the degrees of crystallinity and the crystal sizes remained the same. The gained results support the assumption, that the cellulosic particles are hydrolyzed mostly on their surface, since the enzymes are unable to penetrate into the nanopores of wet cellulose. The hydrolysis therefore proceeds quickly in easily accessible particles and leaves the unaccesible particles almost untouched. The structural changes observed in the SAXS measurements might correspond to slight loosening of the microfibril aggregates, which was seen only in the wet samples because of their different pore structure.Selluloosaa voidaan käyttää energiatuotannon uusiutuvana raaka-aineena. Hyötykäyttö vaatii selluloosan hajottamista glukoosiksi, mikä voidaan tehdä entsyymien katalysoimien hydrolyysireaktioiden avulla. Tässä työssä tutkittiin entsyymien vaikutusta mikrokiteisen selluloosan mikrometritasoa pienempiin rakenteisiin. Samalla yritettiin selvittää hydrolyysin kulkua ja syitä sen vähittäiseen hidastumiseen. Tutkimuksessa käytettiin ensisijaisesti röntgenfysiikan menetelmiä, laajakulmasirontaa (WAXS) ja pienkulmasirontaa (SAXS) sekä röntgenmikrotomografiaa. Näiden lisäksi näytteitä tutkittiin läpäisyelektronimikroskoopilla (TEM). Työssä tutkittuja näytteitä oli hydrolysoitu Trichoderma reesei -lajin entsyymeillä 6, 24 ja 75 tuntia, jolloin ne vastasivat hydrolyysiasteita 31 %, 58 % ja 68 %. Pakastekuivatut hydrolyysin jäännökset mitattiin WAXS:lla, SAXS:lla ja mikrotomografialla, minkä lisäksi osa näytteistä kasteltiin uudelleen SAXS- ja TEM-mittauksia varten. Tuloksissa korostuivat tutkimusmenetelmien erot. Mikrotomografiakuvissa näkyi selvä partikkelikoon pienentyminen kymmenien mikrometrien kokoluokassa. Kaikkien näytteiden TEM-kuvissa havaittiin hydrolyysiajasta riippumatta samantapaisia sylinterimäisiä ja osittain haaroittuneita rakenteita. SAXS-tuloksia oli vaikea tulkita ja ne olivat osittain epätarkkoja, mutta märkien näytteiden rakenteessa nähtiin kuitenkin muutos 10-30 nm:n kokoluokassa. WAXS-tulosten perusteella kiteisyysasteet ja kidekoot eivät muuttuneet. Työssä saadut tulokset tukevat käsitystä, jonka mukaan selluloosapartikkelit hydrolysoituvat lähinnä pinnaltaan, koska entsyymit eivät pääse tunkeutumaan märän selluloosan nanohuokosiin. Hydrolyysi etenee nopeasti helppopääsyisissä partikkeleissa ja jättää vaikeapääsyisemmät lähes koskemattomiksi. SAXS-mittauksissa havaitut rakenteelliset muutokset voisivat viitata mikrofibrilliaggregaattien lievään löyhentymiseen. Tämä havaittiin ainoastaan märissä näytteissä, koska niiden huokosrakenne poikkesi kuivista näytteistä

Greenhouse gas balance of forestry-drained boreal peatlands: Sinks or sources?

Non peer reviewe

Do logging residue piles trigger extra decomposition of soil organic matter?

Logging residue piles have been suggested to markedly increase the decomposition of the underlying peat soil leading to large carbon dioxide emissions. We aimed at scrutinizing this postulate with straightforward decomposition (mass loss) measurements. For the purpose, authentic soil organic matter (humus and peat) was incubated in mesh bags under piles and at control plots. The effect of piles was assumed to result from physical (shading and insulation on soil surface) and chemical-biological (leaching of nutrients and fresh organic matter) sources. To distinguish between the two, artificial piles of inorganic matter were established to mimic the bare physical effects. Enhancement of decomposition in the soil under the real and artificial piles was assessed by measuring the mass loss of cellulose strips. Logging residue piles had clear physical effects on soil: temperatures were lowered and their diurnal variation subdued, and relative humidity at the soil surface was higher. The effect on soil moisture was also evident, but more variable, including both decreases and increases. These effects, mimicked by the artificial piles, decreased rather than increased cellulose mass loss. As the real piles, on the other hand, increased mass loss, we conclude that logging residue piles may enhance decomposition in soil due to chemical-biological mechanisms. Also the results on humus and peat mass loss indicate that piles can both increase and decrease decomposition. Consistent, remarkable increase in mass loss was not observed. Thus, our results do not support the postulate of logging residue piles dramatically increasing decomposition of soil organic matter. Rather, they hint that the effect of logging residue piles on soil is an interplay of physical and chemical-biological effects and carbon transport via roots and fungi. To fully understand and quantify these effects, vertical C fluxes between piles and soil and horizontal C fluxes within soil need to be assessed in addition to decomposition in soil and piles.Peer reviewe

Rapid and Direct Preparation of Lignin Nanoparticles from Alkaline Pulping Liquor by Mild Ultrasonication

The production of lignin nanoparticles (LNPs) has opened new routes to the utilization of lignin in advanced applications. The existing challenge, however, is to develop a production method that can easily be adapted on an industrial scale. In this study, we demonstrated a green and rapid method of preparing LNPs directly from a sulfur-free alkaline pulping liquor by combining acid precipitation and ultrasonication. The combined method produced spherical LNPs, with a hierarchical nanostructure and a highly negative surface charge, within only 5 min of sonication. The mild, rapid sonication was achieved by sonicating directly without prior drying of the acid-precipitated and dialyzed lignin. Optimization of the method revealed the potential for minimizing acid consumption, shortening the dialysis time, and processing directly the alkaline liquor with as much as 20 wt % lignin. The isolated LNPs were stable during storage for 180 days, at a pH range of 4–7, and in a dispersing medium below 0.1 M NaCl. The LNPs also displayed excellent emulsifying properties, stabilizing oil-in-water emulsions. Thus, this simple and energy-efficient method opens a sustainable, straightforward, and scalable route to the production of organic solvent-free LNPs, with high potential as interface stabilizers of multiphase systems in the food and medical industries.Peer reviewe

Ultrastructural X-ray scattering studies of tropical and temperate hardwoods used as tonewoods

The structure of hardwoods representing eight tropical and five temperate species was characterized from the atomistic level up to the cellular level using X-ray scattering, X-ray microtomography and light microscopy. The species were chosen for this study based on their popularity as tonewoods. The ultrastructure of wood cell walls, including crystallite size, orientation and close-range order of cellulose microfibrils were determined by small- and wide-angle X-ray scattering (SAXS, WAXS). The SAXS patterns were interpreted by using an analytical model of cylinden packed in a hexagonal close-range order with paracrystalline distortion. The values for the cylinder diameters given by this model were compared to the average crystallite widths obtained by WAXS using the Scherrer equation. In six out of z6 samples, all of these representing tropical species used especially in fretboard parts of electric guitars, large differences between these two sizes were obtained. The WAXS and microscopy results of these samples corresponded to tension wood structures. These comparisons and interpretations of SAXS results have not been previously presented for any tropical hardwoods, especially related to those containing tension wood tissue. The importance of the ultrastructural characterization was highlighted in this study in the case of tropical hardwood samples.Peer reviewe

Lannoituksen pitkäaikaisvaikutukset vähäravinteisten ja ravinne-epätasapainoisten metsäojitusalueiden kasvihuonekaasupäästöihin

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The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS

AbstractThe reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50nm and 100nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a=9.29nm, b=8.03nm, and γ=84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a=9.41nm, b=6.48nm, and γ=77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92nm, corresponding to approximately ten aligned lattice planes

Carbon dioxide fluxes and vegetation structure in rewetted and pristine peatlands in Finland and Estonia

Vast areas of peatlands have been drained for forestry endangering their carbon sink function. Peatland rewetting aims at mitigating the situation through restoring the hydrology and vegetation of these areas. We compared the carbon dioxide (CO2) fluxes and phy-tomass on four pairs of rewetted and pristine peatland sites in Finland and Estonia, and described correlations between phytomass and CO2 fluxes. We measured the net ecosystem exchange of CO2 (NEE), respiration and photosynthesis over one growing season using manual chambers, and biomass of plant functional types (PFT) on rewetted sites and their pristine counterparts. Although pair-wise differences in the vegetation were small, pristine sites were on average stronger CO2 sinks than rewetted sites. Respiration was higher in hummocks while no differences were found in photosynthesis between hummocks and hollows. No clear relationship between the biomasses of PFTs and NEE was found. Generally, however, CO2 uptake decreased with increase in Sphagnum biomass. © 2019, Finnish Environment Institute. All rights reserved.Peer reviewe