>Magister Scientiae - MSc / Lignocellulose has a complex structure composed mainly of lignin, hemicellulose and cellulose. Several enzymes are needed for the degradation of lignocellulose into simple sugars. Actinomycetes are known to produce laecases which are able to degrade lignin. Laccase activities were detected in actinomycete strains MS26 isolated from soil collected from the Zambian Copperbelt and DFNR17 isolated from soil collected from a New Zealand farm. Morphological .studies showed that the strains produced extensively branched substrate mycelia and aerial hyphae. Micromorphological characteristics were consistent with the assignment of these strains to the genus Streptomyces. Isolates were found to be mesophiles, with growth occurring in a temperature range of 16 and 45°C. Optimal growth occurred at temperatures between 30 and 37°C. Analysis of the 16S rRNA gene sequences of the strains showed that strain MS26 had the highest sequence similarity (99%) to Streptomyces atrovirens strain NRRLB-16357 and Streptomyces viridodiastaticus strain IFO 13106. Strain DFNR17 had the highest 16S rRNA gene sequence similarity (99%) to Streptomyces althioticus strain KCTC9752. The strains shared several physiological and biochemical characteristics with their closest neighbours which, along with 16S rRNA gene sequences analysis, confirmed that the strains were members of the genus Streptomyces. Attempts to identify the laecase genes from these isolates by screening a fosmid library failed. Subsequently isolates were screened by PCR using laccase-like cooper oxidase degenerate primers designed from several Streptomyces strains. A 300 bp amplicon was obtained from both isolates. Phylogenetic analysis was performed and both amplicons from strains MS26 and DFNR17 had the highest similarities with the copper oxidase gene from Streptomyces griseoflavus strain Tu4000. Therefore it is probable that the laecase activity observed for these strains is due to the activity of copper oxidase gene products.

The research focuses on the synthesis and application of multifunctional lignocellulosic biomass bioadsorbent and nanobiocomposites for water purification. A bioadsorbent was prepared from kenaf fiber by self-activation without the use of any toxic chemicals in an innovative method. Silver nanoparticles were synthesized by the green route and then impregnated on the surface of kenaf-based activated carbon (KAC), and hemp fibers by heating and photoirradiation. The formation of hemp-based and kenaf-based silver nanocomposites was confirmed using an environmental scanning electron microscope and energy-dispersive x-ray spectroscopy. Low-cost benign nanoadsorbents demonstrated excellent capabilities for the anionic dye Congo red (CR) and cationic dye brilliant green (BG) degradation, inorganic heavy metals [Cu (II), Pb (II), and Cd (II)] adsorption and antibacterial activities. Antibacterial test via a modified disc diffusion method and minimum inhibitory concentrations was assessed towards the pathogenic strains of bacteria, E. coli and S. aureus. A working portable point-of-use filter was designed and developed, with the filter column encapsulated with nanobiocomposites for the removal of multi-metals and dye. Water samples collected from a wastewater treatment plant in Texas and a mining site in Mexico were used to determine the efficacy of the nanobiocomposites columned in the filter. A comparative analysis was also conducted with the popular commercial water filter material. This research contributes to improved water treatment by the use of benign engineered nanomaterials for pollutants removal that are cost-effective. The technological innovation will also alleviate the financial burden of maintaining centralized wastewater treatment plants by the employment of sustainable and point-of-use methods.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24906号 / 農博第2569号 / 新制||農||1102(附属図書館) / 学位論文||R5||N5469 / DGAM / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 大村 和香子, 教授 髙野 俊幸, 教授 飛松 裕基 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

L'amélioration des processus de dégradation de la biomasse lignocellulosique est un enjeu capital du développement des biotechnologies vertes. Au cours de ces travaux de thèse, nous nous sommes intéressés à deux sujets centrés autour de l'exploitation des souches fongiques pour la dégradation de la lignocellulose. Tout d'abord, nous avons étudié la diversité taxonomique et enzymatique d'un environnement original : la mangrove. Une campagne de prélèvement en Nouvelle-Calédonie à été organisée, afin d'étudier la diversité taxonomique des communautés fongiques établies sur les palétuviers. L'utilisation d'une approche « Tag-Pyrosequencing » a permis de mettre en lumière l'existence d'une taxonomiques extrêmement importante puisque plusieurs milliers d'espèces ont été détectées dans les différentes niches écologiques de cet écosystème. Ces travaux ont également permis d'établir le rôle primordial de la spécificité d'hôte dans l'établissement des communautés fongiques. Nous avons par la suite isolé différentes souches de champignons à partir d'échantillons de palétuvier et avons procédé au criblage de leurs activités lignocellulolytiques (oxidase, cellulase, mannanase, xyalanase). A l'issue de ce crible, une souche, identifiée comme un Pestalotiopsis sp, présentant la plus grande polyvalence en termes d'activités enzymatiques a été sélectionnée. / The improvement of lignocellulosic biomass degradation processes is a key aspect of the development of “green” biotechnologies. During this thesis, we focused on two subjects centred on the usage of fungal strains for the degradation of lignocellulose. First, we studied the taxonomic and enzymatic diversity in an original ecosystem: mangroves. A sampling was performed in New-Caledonia in order to study the fungal communities colonizing mangrove trees. By using Tag-Pyrosequencing, we showed the existence of very large communities harbouring several thousand species in the different microhabitats of the ecosystem. This work also revealed the key role of host specificity as a factor driving the fungal colonisation of mangrove trees. We then isolated several fungal strains from various mangrove tree samples, and performed a screening of their lignocellulolytic activities (oxidase, cellulase, mannanase, xylanase). A single strain was selected fromthis screening, identified as Pestalotiopsis sp., which showed the most complete diverse enzymatic activities. A de novo transcriptome was assembled from mRNA sequences, which allowed highlighting a wide array of transcripts encoding biomass degradation enzymes, as well as the existence of a mechanism of adaptation to salt based on the secretion of salt-tolerant lignocellulolytic enzymes. Secondly, we studied the limitations of fungal co-culture linked to competitive interactions mechanisms. The RNA-Seq analysis of genetic expression during the interaction between Pycnoporus coccineus and Coniophora puteana or Botrytis cinerea indicates that different mechanisms are used depending on the opponent.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR

Im Rahmen dieser Arbeit erfolgte am Beispiel von Weizenstroh die Entwicklung und Optimierung eines Prozesses zur Produktion von Bioethanol. Die Synergie aus effizienter Vorbehandlung mit NaOH, Reaktortyp (Freifallmischer) und einem effizienten Enzymkomplex aus Penicillium verruculosum ermöglichte die Herstellung von bis zu 13 Vol.-% Ethanol. Die Hydrolyse und Fermentation wurden simultan durchgeführt (SSF-Prozess), woraus eine Vermeidung der Produktinhibierung sowie eine Erhöhung der Ethanolproduktivität um 94 % resultierten. Eine in situ-Ethanolentfernung mittels Stripping gewährleistete einen kontinuierlichen SSF-Prozess und ermöglichte die Folgekonversion des Bioethanols mittels katalytischer Dehydratisierung zu Bioethylen. Dadurch wurde eine Wertsteigerung des Ethanols erzielt und eine energie- und kostenintensive Abtrennung, Reinigung und Konzentrierung des Ethanols umgangen. Außerdem gelang eine Übertragung des Prozesses zur Ethanolproduktion vom Labormaßstab (2 L) in den Technikumsmaßstab (200 L). Auch die Anwendung auf andere Biomassen (Miscanthus, Luzerne) sowie Rohstoffe (Weizenkleie) wurde erfolgreich gezeigt.

La lignocellulose est le composant principal des parois végétales et donc le biopolymère végétal le plus abondant sur Terre. Sa transformation en molécules d’intérêt industriel est donc une voie prometteuse pour diminuer la consommation de ressources fossiles. Au sein de la plateforme des carboxylates, la transformation de la lignocellulose repose sur l’utilisation de communautés bactériennes. Mais s’ils sont augmentés par des approches de prétraitement du substrat, les rendements sont encore faibles. Afin de les améliorer, nous avons ici testé les capacités de dégradations de communautés microbiennes issues de l’enrichissement de rumen bovin et d’intestin de termites. Afin de caractériser l’effet de la source d’inoculum et du prétraitement du substrat sur le fonctionnement des communautés sélectionnées, une approche de séquençage 16S a été utilisée. Celle-ci a permis la comparaison des compositions de communautés obtenues, mais également de leurs dynamiques au cours de la transformation du substrat lignocellulosique. Les conditions de culture imposées semblent avoir un effet très fort sur la composition des communautés sélectionnées puisque malgré leurs différences, celles-ci présentent d’importantes similitudes et sont bien plus proches que ne l’étaient les inocula initiaux. Enfin, les communautés associées à la dégradation du substrat lignocellulosique montrent des dynamiques très marquées, caractérisées par une importante baisse de diversité et la dominance de quelques populations bactériennes seulement lors du maximum de dégradation. / Lignocellulose is the main component of vegetal cell wall and is thus the most abundant biopolymer on Earth. Its conversion into industrially relevant molecules is of concern to reduce fossil resources consumption. In the dedicated carboxylates platform, lignocellulose conversion relies on the metabolic potential of microbial consortia, but lignocellulose transformation rates can still be improved, despite substrate pretreatment approaches. In order to improve these rates, we here tested the transformation capacities of microbial communities originated from cow rumen and termite guts. 16S sequencing was used to characterize the effects of inoculum source and substrate pretreatment on the selected communities’ functioning. It allowed the comparison between obtained communities, but also between their dynamics during lignocellulose transformation. Culture conditions appeared to have a strong effect on the selected communities, which presented high similarities despite differences between initial inocula. Finally, communities associated to lignocellulose degradation showed marked dynamics, with a strong decrease in diversity indexes and the dominance of a few bacterial populations during the degradation maximum.

La fermentation en milieu solide est un bioprocédé pouvant éventuellement être utilisé comme technologie de rupture pour diminuer le coût des biocatalyseurs utilisés dans la conversion de biomasse lignocellulosique comme le son de blé. La première partie de ces travaux de recherche a donc étudié le potentiel de cette technologie par rapport à celle de fermentation en milieu submergé lors d'une comparaison en application. Plusieurs tests de saccharification ont ainsi été réalisés sur différentes matières premières (cellulose, son de blé) et ont permis de montrer l'avantage différentiateur des biocatalyseurs produits par fermentation en milieu solide. Ensuite, la deuxième partie de ces travaux de recherche a porté sur l'étude des facteurs de la récalcitrance du son de blé à l'hydrolyse enzymatique. Deux principaux facteurs ont ainsi pu être démontrés : un facteur physique, lié à l'accessibilité des biocatalyseurs aux polysaccharides, et un facteur biochimique, lié à l'absence ou à la faible présence de certaines activités enzymatiques (féruloyl estérase,…) dans le complexe enzymatique de Trichoderma reesei Rut-C30. Cette étude a également permis d'identifier l'origine des différentes fractions glucidiques hydrolysées et de déterminer le potentiel glucidique actuellement hydrolysable à partir de cette biomasse. Enfin, la dernière partie de ces travaux de recherche a été consacrée à l'étude pratique d'un concept innovant de procédé permettant de favoriser la conversion des polysaccharides contenus dans le son de blé. Une levée de la barrière physique au transfert de masse et par conséquent une validation de ce concept a finalement pu être réalisée. / Solid-state fermentation is a bioprocess that can optionally be used as disruptive technology to reduce the cost of biocatalysts used in the lignocellulosic biomass conversion like wheat bran. The first part of this research has explored the potential of this technology compared to submerged fermentation in an application comparison. Several saccharification tests have thus been carried on different feedstocks (cellulose, wheat bran) and have shown the differentiator advantage of biocatalysts produced by solid state fermentation. Then, the second part of this research has investigated the recalcitrance factors of wheat bran to enzymatic hydrolysis. Two main factors have thus been demonstrated: a physical factor, related to the accessibility of biocatalysts to the polysaccharides, and a biochemical factor, related to the absence or the low presence of some enzymatic activities (feruloyl esterase, ...) in the enzymatic complex of Trichoderma reesei Rut-C30. This study has also identified the origin of the various carbohydrate moieties hydrolyzed and has determined the carbohydrate potential currently releasable from this biomass. Finally, the last part of this research has been devoted to the practical study of an innovative concept of process to promote the conversion of polysaccharides in wheat bran. A removal of the physical barrier to mass transfer and therefore a validation of this concept has finally been achieved.

Thesis (PhD)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Lignocellulose is the principal form of biomass in the biosphere and therefore the predominant renewable source in the environment. However, owing to the chemical and structural complexity of lignocellulose substrates, the effective and sustainable utilization of lignocellulose wastes is limited. Many environments where lignocellulose residues are ordinarily stored can be highly acidic (e.g. landfills), and under these circumstances biodegradation of the lignocellulose is slow and unhygienic. Owing to the metabolic activities of the micro-organisms, the initially acidified habitats rapidly undergoes self-neutralization. A number of pathogenic bacteria (coliforms and Salmonella sp.) are present during this slow degradation process and it is therefore imperative to improve the efficiency and hygienic effects of the biodegradation of the lignocellulose. Although the fundamentals of biodegradation of lignocellulose have been widely investigated, many issues still need to be resolved in order to develop commercially viable technology for the exploitation of these waste products. For example, owing to the complex, heterogeneous structure of lignocellulose, the degree of solubilization, modification and conversion of the different components are not clear. Likewise, the overall anaerobic degradation of lignocellulose is not understood well as yet. In this study, the emphasis was on the promotion of solid anaerobic digestion of lignocellulose wastes for environmental beneficiation and waste reutilization. The degradation of lignocellulose in landfill environments was first simulated experimentally. Once the microbial populations and the degradation products of the system were characterized, the promotion of anaerobic digestion by use of activated sludge was studied. This included acidogenic fermentation, as well as recovery of the methanogenic phase. Moreover, special attention was given to the further disposal of humic acids or humic acid bearing leachates formed in the digestive system, since these acids pose a major problem in the digestion of the lingocellulose. With ultrasonication, approximately 50% of the lower molecular weight fraction of humic acids could be decomposed into volatile forms, but the higher molecular weight fraction tended to aggregate into a colloidal form, which could only be removed from the system by making use of ultrasonically assisted adsorption on preformed aluminium hydroxide floes. This was followed by an investigation of the microbial degradation of humic acids and the toxicity of these acids to anaerobic consortia. Further experimental work was conducted to optimize the biological and abiological treatment of lignocellulose in an upflow anaerobic sludge blanket (DASB) reactor fed with glucose substrate. The humic acids could be partially hydrolysed and decomposed by the acid fermentative consortia of the granules in the DASB reactor. Finally, solid mesothermophilic lignocellulose anaerobic digestive sludge can be viewed as a humus-rich hygienic product that can improve the fertility and water-holding capacity of agricultural soil, nourish plants and immobilize heavy metals in the environment as a bioabsorbent. / AFRIKAANSE OPSOMMING: Lignosellulose is die hoofbron van biomassa in die biosfeer en is daarom ook die belangrikste hernubare bron in die omgewing. As gevolg van die chemiese en strukturele kompleksiteit van lignosellulose substrate, is die doeltreffende en volhoubare benutting van lignosellulose afval egter beperk. Die suurgehalte van die omgewings waar lignosellulose reste gewoonlik gestoor word, soos opvullingsterreine, kan hoog wees en onder hierdie omstandighede is die biodegradasie van die lignosellulose stadig en onhigiënies. As gevolg van die metaboliese aktiwiteite van die mikro-organismes ondergaan die aanvanklik aangesuurde habitatte vinnig self-neutralisasie. 'n Aantal patogeniese bakterieë (koliforme en Salmonella sp.) is deurgaans gedurende dié stadige natuurlike proses teenwoordig en dit is dus van die grootste belang om die effektiwiteit en die higiëne van die bioafbreking van die lignosellulose-substraat te verhoog. Alhoewel die grondbeginsels van die bioafbreking van lignosellulose reeds wyd ondersoek is, moet verskeie probleme nog opgelos word ten einde kommersieel haalbare tegnologie te ontwikkel vir die ontginning van afvalprodukte. Byvoorbeeld, as gevolg van die komplekse, heterogene struktuur van lignosellulose, is die graad van solubilisering en die modifikasie en omskakeling van verskillende komponente nog onduidelik. Net so word die algehele anaerobiese afbreking van lignosellulose ook nog nie ten volle verstaan nie. In hierdie ondersoek het die klem geval op die bevordering van soliede anaerobiese digestie van lignosellulose afval vir omgewingsverbetering en die benutting van die afval. Die afbreking van lignosellulose in opvullingsterreine is eers eksperimenteel gesimuleer. Nadat die mikrobiese populasies en die afbrekingsprodukte gekarakteriseer is, is die bevordering van anaerobiese digestie deur die gebruik van geaktiveerde slyk bestudeer. Dit het asidogeniese fermentasie ingesluit, sowel as herwinning van die metanogeniese fase. Spesiale aandag is gegee aan die verdere verwerking van humus sure en humussuurbevattende legate wat in die digestiewe stelsel gegenereer is, aangesien die sure probleme veroorsaak het met die vertering van die lignosellulose. Met ultrasoniese straling is nagenoeg 50% van die lae-molekulêre massafraksie van die humussure ontbind in vlugtige vorm, maar die hoë-molekulêre massafraksie het geneig om in 'n kolloïdale vorm te aggregeer, wat slegs uit die stelsel verwyder kon word deur middel van ultrasonies ondersteunde adsorpsie op voorafgevormde aluminiumhidroksiedvlokkies. Dit is gevolg deur 'n ondersoek na die mikrobiese afbreking van humus sure en die toksisiteit van die sure ten opsigte van anaerobiese konsortia. Verdere eksperimentele werk is gedoen ten opsigte van die biologiese en abiologiese behandeling van lignosellulose in 'n opwaartsvloeiende anaerobiese slikkombersreaktor (OASK) gevoer met glukosesubstrate. Die humus sure kon gedeeltelik gehidroliseer en ontbind word deur die suurgistende konsortia van die granules in die OASK reactor. Ten slotte kan die vaste termofiliese-mesofiliese anaerobiese lignosellulose verteringslik ook gesien word as 'n humusryke higiëniese produk wat die vrugbaarheid en die waterhoudende vermoë van landbougrond kan verhoog, plante kan voed en kan funksioneer as bioabsorbeerder van swaarmetale in die omgewing.

Biogas production from organic materials can be used as a renewable vehicle fuel, provide heat and generate electricity and can thereby reduce the greenhouse gas emissions. This thesis focuses on the biogas production based on lignocelluloses. There is an abundant availability of lignocelluloses, constituting 50% of the total biomass worldwide. However, the biomass recalcitrance limits the microbial degradation as well as the biogas production from these types of materials. In the present work different pretreatment methods have been performed in order to decrease the biomass recalcitrance and improve the biogas production. Steam explosion pretreatment, together with the addition of sodium hydroxide and hydrogen peroxide, has been performed on lignocellulosic-rich paper tube residuals. The pretreatment has resulted in methane yields of up to 493 NmL/gVS, which is an increase by 107% compared with untreated material. Furthermore, the use of an organic solvent, N-methylmorpholine-N-oxide (NMMO), was evaluated as a pretreatment method for spruce (both chips and milled), rice straw, and triticale straw. The NMMO pretreatment resulted in 202, 395, 328, and 362 NmL CH4/g carbohydrates produced of these substrates, respectively, corresponding to an increase of between 400-1,200% compared with the untreated version of the same material. Moreover, the paper tube residuals have been co-digested with an unstable nitrogen-rich substrate mixture, mainly based on municipal solid waste. The addition of the lignocellulosic-rich paper tubes in a co-digestion process showed stabilizing effects and prevented the accumulation of volatile fatty acids with a subsequent reactor failure. Additionally, synergistic effects have been found leading to between 15-33% higher methane yields when paper tubes were added to the co-digestion process compared with the yields calculated from the methane potentials of the two substrates. Substrate characterization analysis can be used to study the changes on the lignocellulosic components after the pretreatment, relating the changes to the performance in the anaerobic digestion. Increased accessible surface area, measured by the Simons’ stain and the enzymatic adsorption methods, as well as decreased crystallinity, determined by using the Fourier Transform Infrared Spectroscopy, can all be linked to improved biogas production after pretreatment. Finally, the NMMO pretreatment on forest residues has been financially evaluated for an industrial scale process design. The base case that was evaluated simulated a case where pretreated forest residues were co-digested with the organic fraction of municipal solid waste to obtain optimal nutritional balance for the anaerobic digestion. This process has been found to be economically feasible with an internal rate of return of 20.7%. / <p>Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 24 maj 2013, klockan 10.00 i KA,Kemigården 4, Göteborg</p>