C-lignin (caffeyl-lignin) is a novel linear lignin polymer found in the seed coats of several non-crop plants, notably Vanilla planifolia (Vanilla), Jatropha Curcas (Jatropha), and Cleome hassleriana (Cleome). C-lignin has several advantages over normal G/S-lignin, found in the majority of lignocellulosic biomass, for valorization in the context of bioprocessing: less cross-linking to cell wall polysaccharides (less recalcitrant biomass), ordered linkages between monomers (homogeneous polymer), and no branching points (linear polymer). These properties make C-lignin an attractive replacement for native lignin in lignocellulosic biomass crops. The seed coats of Cleome hassleriana (Cleome) synthesize G-lignin during early seed maturation, then switch to synthesis of C-lignin during late maturation. This switch to C-lignin in Cleome seed coats is accompanied by loss of caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) and caffeic acid 3-O-methyltransferase (COMT) activities, along with changes in transcript abundance of several lignin related genes. The focus of this research thesis is to understand the biochemical changes leading to C-lignin deposition in Cleome hassleriana seed coats, and to explore the ability of Arabidopsis thaliana seedlings to polymerize caffeyl alcohol to C-lignin. In this thesis, candidate transcripts were implicated in C-lignin biosynthesis by differential gene expression analysis of transcripts in seed coat tissues at 8-18 days after pollination (DAP) and in non-seed coat tissues. Three candidate genes were selected for recombinant expression and their in vitro kinetic properties were measured with potential substrates. Of the three candidates, a cinnamyl alcohol dehydrogenase (ChCAD5) was found to have high transcript levels during C-lignin formation and have a novel preference for converting caffealdehyde to caffeyl alcohol, the precursor of C-lignin. To determine if accumulation of caffeyl alcohol is sufficient for polymerization of C-lignin, Arabidopsis seedlings grown in a xylem induction system were supplied caffeyl alcohol. Polymerization of caffeyl alcohol was not found to occur in this Arabidopsis system, suggesting the need for a C-lignin specific polymerization mechanism.

Soil is one of the natural habitats of microorganisms that are significantly involved in the continuous transformation of organic and inorganic substances. The issues currently faced by this area of research are based on the occurrence of extraneous synthetic substances (xenobiotics) in the environment. These xenobiotics do not have a natural origin but have been artificially synthesized by humans. The contamination of the xenobiotic environment occurs with the development of human economic activity (industry and agriculture); xenobiotics are resistant to degradation processes, accumulate in soil and act on microorganisms. Biogenic substances are, by way of exception, easily decomposable. Few studies have been conducted to analyze the relationship between the occurrence of biogenic and xenobiotic substances in soil and their effects on microorganisms. The aim of this dissertation was to gain knowledge about the influence of biogenic and xenobiotic substances on the metabolism of soil microorganisms and to improve the procedures used to evaluate the influence of chemicals applied to the soil. This dissertation is also devoted to the study of soil enzymes. The dissertation is divided into two parts. The first theoretical part is further divided into: a) a review article on adsorption of enzymes to the solid soil component and factors limiting their activity. This review article summarizes some of the important findings published in the scientific literature and provides information on adsorption of enzymes to the solid soil component and factors limiting their activity. The first part of this review article discusses in particular the problems of enzyme interactions with the solid soil component and enzyme adsorption kinetics. In the second part of this paper I deal with the issue of factors that can affect the activity of adsorbed enzymes (b) An overview on lignin. This review article summarizes the knowledge about the chemical composition of lignin and its content in forest soils, lignin biodegradation and microorganisms producing ligninolytic enzymes; lignin peroxidase, manganese-dependent peroxidase and other versatile peroxidases are amongst the most conspicuous ligninolytic enzymes. (c) An article on the influence of agrochemicals on soil and soil micro-organisms. The aim of this review article was to summarize the knowledge of the influence of agrochemicals on sustainability and soil health. The second experimental part contains knowledge about the effects of xenobiotic (chlortetracycline and sulfapyridine) and biogenic substances (ascorbic acid and amino acids) on soil microorganisms. The results obtained showed the effect of chlortetracycline and sulfapyridine on the activity of soil enzymes depending on the enzyme studied and the antibiotic used. Within this experimental part, the influence of oxytetracycline and sulfamethoxazole on the structure and function of the microbial community in the soil was also studied. The results obtained showed the different effects of both tested antibiotics on cumulative respiration and carbon microbial biomass in soil. In the framework of this dissertation, a study of respiration of forest soils after application of ascorbic acid and amino acids was also carried out. The results obtained from these experiments showed an increase in respiratory activity after administration of ascorbic acid or ascorbic acid in combination with amino acids, depending on the properties of the soil samples tested; maximum respiratory activity in acidic soils was induced after the addition of L-glutamic acid and alkaline soils after the addition of beta-alanine. The size of the mesh holes has a significant effect on soil disturbance that affects pore structure, fungal hyphae, fungal fraction and bacterial fraction. The effects are dependent on the soil type and the vegetation cover. Sifting through 2 mm sieve increases the mineralization of exogenously supplied carbohydrates and phenolic substances compared to 5 mm and the sieve of significant importance (p <0.05), especially in organic horizons, due to increased microbial metabolism and changes in other soil properties.

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Coating urea to prepare controlled release N-fertilizer has been considered as an effective way to increase its nutrient use efficiency, thus reduce its waste and the consequent harmful environmental impacts. Inorganic sulphur and synthetic polymers have been used in the industry as coating materials together with utilization of various types of expensive coating equipment which commonly requires also complicated technical setup and controls. As development trends, biopolymers are attention-grabbing to replace the synthetic polymers. Alternative simple coating technique is also desired. So far, polylactic acid (PLA) has been reported as a more promising biopolymer than several synthetic polymers for coating. On the other hand, highly purified industrial softwood kraft lignin (SKL) produced after LignoBoost process is now available in a large quantity, which should also be a promising biopolymer for the coating application. Aiming at increase of the efficiency of PLA-coated urea and development of alternative coating technique to generally make the preparation of control-released fertilizer more effective, economic and environmentally sustainable, in this study, SKL has been used in a PLA-SKL blending form as complex coating material and simple dip-coating technique has been investigated and applied. In order to lower the wettability of PLA-SKL coat layer, four different anhydrides, namely acetic anhydride, palmitic anhydride, lauric anhydride and trifluoroacetic anhydride, were used to esterify SKL to form AcSKL, PaSKL, LauSKL and TFASKL respectively before its utilization. NMR and FTIR analyses showed that the esterification reactions have been completed for AcSKL and PaSKL. LauSKL was partly esterified due to the low charge of lauric anhydride regent, while TFASKL was not esterified expectantly due to the steric hindrance between the three F atoms and polymeric SKL. In order to obtain organically bound nitrogen structure to also create slow-release type of N-fertilizer, Mannich reaction on SKL using diethylamine was also conducted to prepare ManSKL. The reaction was completed as shown by NMR and FTIR spectroscopy. To bring further functionality of metal chelation to open the possibility to also bring essential trace element into the final fertilizer, ethylenediaminetriacetic acid (ED3A) was synthesized and further used via Mannich reaction to modify SKL to form ED3ASKL. ED3A is not commercially available and it was synthesized successfully with an environmentally friendly method from commercial EDTA and the structure was verified by NMR spectroscopy. However, the Mannich reaction using ED3A was not very successful as shown by product’s NMR and FTIR spectra. In a comparison experiment using vanillyl alcohol as a lignin model structure, ED3A was successfully coupled onto the vanillyl alcohol structure as shown by NMR and FTIR spectra. Apparently there was a severe steric hindrance from SKL for the Mannich reaction using the larger molecule of ED3A than diethylamine for Mannich reaction. For utilization of dip-coating technique, dichloromethane(DCM) and tetrahydrofuran (THF) were chosen to dissolve PLA and SKL or the modified SKL respectively. Cast films of PLA/modified lignin complex were prepared using Teflon Petri-dishes. The optimal concentration of PLA in DCM and the effect of DCM/THF ratios on the prepared cast film which expectantly represents the quality of the complex coat in the coated urea were compared with SEM images and contact angle determination. It has been found that a 30 wt% of PLA in DCM was the best and this solution mixed with modified lignin solution (6 % in THF) in a ratio of DCM/THF =3/2 (v/v) had the best film performances and water barrier properties. Generally, the cast films from PLA/modified lignin complexes showed better properties compared with the neat PLA cast film. No pores and cracks were found on the surface. Comparatively, the LauSKL film showed the most homogeneous surface. But the AcSKL film had the best water barrier properties. The PLA/modified lignin complex coated urea was then prepared by dip-coating process. The coat thickness and weight increase showed statistically positive correlations against the repeating times of the dip-coating process. The coating layer also showed one single layer structure. The speed of urea releasing for coated urea was tested and the results showed that it was much slower than the un-coated or PLA-coated urea. The single-layered PLA/AcSKL and PLA/ManSKL were both observed with sound properties in delaying the release of urea cores in water. Conclusively, the PLA/modified SKL coated urea fertilizers prepared by dip-coating technique demonstrated in this study have highly efficiency with better effects of water barrier, organically N slow release, and nitrification inhibiting (due to free phenolic functional groups) properties. Both SKL and the dip-coating technique are promising in the fertilizer applications. / Att ytbelägga urea för att skapa kontrollerad frisläppning av kväverika gödningsmedel har ansetts vara en effektiv metod för att öka användandet av näringsämnena från urea och dessutom minska den möjliga miljöpåverkan urea har. Kommersiellt har oorganiskt svavel och syntetiska polymerer använts för att ytbelägga olika material och detta är kopplat till olika typer av dyra ytbeläggningsutrustningar som ofta kräver komplicerade tekniska lösningar och kontroller. För att förbättra dagens lösningar är en intressant trend användandet av biopolymerer och en annan viktig aspekt är att utveckla nya enklare ytbeläggningstekniker än vad som finns på marknaden idag. Polylaktid (PLA) har till exempel rapporterats som en mer lovande förnybar polymer än flera av de syntetiska polymererna för ytbeläggnig. En annan intressant förnybar polymer är lignin, som idag tillverkas med hög renhet industriellt som barrveds kraft lignin (SKL) ur LignoBoost processen. Med målet att öka effektiviteten hos urea belagd med PLA och att utveckla en alternativ ytbeläggningsmetod för att göra den generellt mer effektiv, ekonomisk och miljövänlig har SKL använts i en PLA-SKL blandning för att ytbelägga med urea och en enkel doppbeläggningsmetod utvecklats och applicerats. För att minska vätbarheten av PLA-SKL ytbeläggningen har fyra anhydrider, ättiksyraanhydrid, palmitisk anhydrid, lauric anhydrid, och trifluoroacetisk anydrid, använts för att esterifiera SKL och bilda AcSKL, PaSKL, LauSKL och TFASKL. NMR och FTIR användes för att verifiera esterifieringsreaktionerna. Fullständig reaktion kunde konstateras för AcSKL och PaSKL, LauSKL hade bara delvis esterifierat pga den låga mängd lauric anydrid som användes medan TFASKL inte ledde till den tilltänkta esterifieringen pga steriska hinder mellan de tre flor-atomerna och SKL polymeren. För att tillverka en organiskt bunden kvävestruktur, som dessutom har en långsam frisättning av N-rika gödningsmedel genomfördes en Mannich-reaktion på SKL med dietylamin som katalysator för att framställa ManSKL. Reaktion gick till full omsättning, enligt NMR- och FTIR-spektroskopi. För att få ytterligare funktionalitet såsom metallkelation, vilken öppnar möjligheten att tillföra väsentliga spårämnen till gödningsmedlet, syntetiserades och användes etylendiamintriättiksyra (ED3A) för att modifiera SKL för att bilda ED3ASKL. ED3A finns inte kommersiellt tillgängligt utan syntetiserades framgångsrikt med en miljövänlig metod från kommersiell EDTA, varefter strukturen verifierades genom NMR-spektroskopi. Mannich-reaktionen på SKL med ED3A var emellertid inte särskilt framgångsrik, vilket NMR- och FTIR-spektra av produkten visade, Som modellexperiment användes vaniljalkohol som en ligninmodellstruktur till vilken ED3A framgångsrikt kopplades. Orsaken till denna stora skillnad i reaktivitet tros vara steriska hinder från SKL. För att utveckla en doppbeläggningsteknik valdes diklormetan (DCM) och tetrahydrofuran (THF) som lösningsmedel för att lösa upp PLA, SKL och den modifierade SKL. Gjutna filmer av PLA/modifierat lignin tillverkades i Teflon Petri-skålar. Den optimala koncentrationen av PLA i DCM och effekten av DCM/THF-förhållandet på filmens morfologi förväntas representerar kvaliteten för den framtida ytbeläggningen på urea, därför jämfördes filmernas SEM-bilder och kontaktvinkel. Det kunde konstateras att en 30 vikt% PLA i DCM var optimal och att denna lösning blandad med modifierad ligninlösning (6% i THF) i ett förhållande av DCM/THF = 3/2 (v/v) hade den bästa filmprestanda och vattenbarriäregenskaper. Generellt visade filmer av PLA/modifierade lignin bättre egenskaper jämfört med den rena PLA-filmen då inga porer och sprickor hittades på ytan. LauSKL-filmen uppvisade den mest homogena ytan medan AcSKL-filmen hade de bästa vattenbarriäregenskaperna. Urea ytbelagdes med PLA och PLA/modifierade lignin genom en doppbeläggningsprocessen. Beläggningstjockleken och viktökningen visade statistiskt positiva korrelationer gentemot antalet upprepningar av doppbeläggningsprocessen. Beläggningsskiktet visade också en enda skiktstruktur. Hastigheten det tog för urea att frigöra sig från den ytbelagda urean undersöktes och resultaten visade att den var mycket långsammare än den obehandlade urean eller den PLA-belagda urean. Enkelskiktad PLA/AcSKL och PLA/ManSKL uppvisade båda goda fördröjningsegenskaper för frisättningen av urea i vatten. Sammanfattningsvis kan man säga att PLA/modifierade SKL-belagda ureagödningsmedel framställda genom en utvecklad doppbeläggningsteknik, uppvisar hög effektivitet med bättre egenskap som vattenbarriär, långsam frisättning av organiskt kväve och nitrifikationshämmande egenskaper (beroende på fria fenoliska funktionella grupper). Både SKL och doppbeläggningstekniken är lovande i för gödningsmedelstillämpningarna.

Copolymerization reaction of propylene oxide and Kraft lignin with various catalysts, catalyst concentrations, at several temperature levels were studied. The reaction rate of propylene oxide was estimated from the pressure vs time diagram of the reaction and the copolymerization extent was evaluated on the basis of H-NMR spectroscopy in terms of molar substitution of purified lignin/propylene oxide copolymers. The investigation included studies with lignin-like model compounds under conditions of varying KOR concentrations at 190°C. Reaction rate and molar substitution were determined. Homogeneous ( completely liquefied ) Kraft lignin products were . 0 only obtained at reaction temperatures above 190 C or temperatures above the glass transition temperature of the particular lignin preparation. Various lignins including Kraft lignin, methylated Kraft lignin, and demethylated Kraft lignin were employed to reacted with propylene oxide at 190°c. The effect of lignin structure variations on both reaction rate and molar substitution were determined. Results revealed that homopolymerization was the major reaction although copolymerization also took place during the hydroxypropylation. Reactions with model compounds indicated that the reaction rate depended on the ratio of total alkalinity to total acidity in the system which was found to be related with KOH concentration, lignin content, and lignin structure. Particularly, carboxyl groups of lignin resulted in high acidity and low homopolymerization rate. It was also found that molar substitution varied between zero and three and results from model compound study indicated that conjugated aliphatic hydroxyl and certain phenolic hydroxyl groups reacted with particular ease with propylene oxide. Therefore, reaction products from lignins rich in these types of functional groups can be expected to produce derivatives with low glass transition temperature and improved solubility. / Master of Science

Spruce wood meal (Picea pungens), sequentially solvent pre-extracted with water, ethyl ether, and ethanol were subjected to acidolytic delignification treatments of increasing severity, and the lignin sol fractions were analyzed with regard to chemical structure and molecular architecture. Treatments with 0.2 N HCL in 90% aqueous dioxane ranged between 12 hr. at room temperature and 8 hrs. at reflux. The analysis of the dissolved lignin (sol) fractions indicate significant changes in the chemical structures only with regard to aryl-alkyl ether and phenolic hydroxyl content, and with regard to molecular weights and molecular weight distributions. While alkyl-aryl ether content decreases rapidly with acidolysis treatment under reflex conditions, phenolic hydroxyl content increases conversely. Molecular weight increases as delignification procedes. As crosslink density is a function of alkyl-aryl ether content, these results suggest that the 6 7 crosslink density of the sol fraction decreases while molecular weight increases. This contradicts to the theoretical behavior for the degradation of an infinite network and suggests that lignin in wood exists as a gel (network polymer) of extremely low crosslink density or as an ungelled, branched polymer only. Delignification is therefore not controlled by degelation but rather by interaction with carbohydrates via covalent or hydrogen bonds and/or by physical entrapment in the cell wall matrix. This is in accord with earlier hypothesis on the controlling effects of pore structure by Goring et. al. and transient ether linkages by Glasser. / M.S.

Hydroxypropyl lignin derivative block (HPL) was reacted with low DP cellulose triacetate block (CTA). The synthesis of this block copolymer involves the following steps. • Synthesis of an hydroxyl-terminated CTA oligomer through the depolymerization of high molecular weight cellulose triacetate under conditions that deacetylation is prevented. • Reaction of this block with 2,4 toluene diisocyanate (TDI) to form an isocyanate-capped CTA prepolymer under conditions that chain extension is limited and a sufficient NCO equivalent/mole is achieved (ie: 1 or 2). • Reaction in solution of this low DP CTA prepolymer with the HPL block. Films were directly obtained by solution casting of the mixture and cured. The parameters of investigation were HPL, or CTA block content and size of CTA block. Morphological characteristics and thermal analysis were investigated as a function of these parameters. Analysis of the results showed that all the films were brittle. They exhibited single T_g values and were crystalline even for short size cellulose blocks. Network structure was not the predominant factor influencing the properties as was expected from the multi-hydroxyl functionality of the HPL component but rather the crystalline character observed in the copolymers at all compositions. / M.S.

Thesis (doctoral)--University of Helsinki, 1999. / Includes bibliographical references. Also available on the World Wide Web.

RISE Bioeconomy is working with the biorefinery concept. That is, for the company, to utilize all wood derivatives in effective processes, resulting in unique products. RISE Bioeconomy is investigating the possibilities of producing holocellulose through an oxidative delignification process of wood, using peracetic acid (PAA). The product, high-purity holocellulose, can be used for the production of for example cellulose nanofibers, a product that in recent years have increased in interest. The PAA-delignification process relies on oxidative treatment with peracetic acid, where the lignin is solubilized in water through the formation of carboxylic groups in the lignin, yielding cellulose of very low lignin content and minimal degradation of the cellulose. In addition to the holocellulose, a solution containing water, acetic acid, salts and highly oxidized lignin is obtained. The PAA-delignification has yet only been performed in lab-scale, however, the aim is to scale up the process. There has not yet been any further utilization of the oxidized lignin and it has been proposed that esterification of the carboxylic groups with an alcohol is a possible valorization route. Due to the up-scaling of the PAA-delignification, it is of large interest to find a valorization route for the lignin by-product to fulfill the biorefinery concept. This master thesis focuses on evaluating esterification of an oxidized lignin, with the aim to propose a process suitable for up-scaling. The lab trials for the PAA-delignification of wood yielded limited amounts of lignin and due to difficulties while isolating this lignin, it was not partially characterized until months into the project. Therefore, model compounds were initially used to evaluate the process setup. Compounds with muconic acid type structures are some of the expected products from PAA-delignification of wood and muconic acid was therefore used as a model compound. In addition to this, vanillic acid was used to represent the phenolic lignin compounds. Gas Chromatography/Mass Spectrometry (GC/MS) analysis of the vanillic acid esterification was shown to be a suitable method for analyzing the conversion of this reaction. Due to the difficulties with the analysis of the model compounds, no results regarding the reaction procedures were obtained. As a “proof-of-concept”, a kraft lignin was oxidized, isolated and then esterified. Two different methods of esterification were performed, with and without a molecular sieve. It was assumed that the amount of carboxylic groups would increase after oxidation and decrease after a successful esterification. The amount of carboxylic groups after each process was analyzed with Phosphorus Nuclear Magnetic Resonance (31P-NMR). For the process without molecular sieve, the 31P-NMR analysis indicated that esterification of the lignin had occurred, to a limited extent. This was confirmed by Fourier Transform Infrared Spectrometry (FTIR). The process with molecular sieve showed no indication of esterification of the lignin. The project did prove the concept of esterifying an oxidized lignin and led to several different proposals for further work in the area. Due to the very wide scope of this project and several unexpected obstacles, this project did not fully answer the research questions given.