Nessa pesquisa foram testados separadamente dois inóculos solo/compostagem e lodo termofílico de reator anaeróbio de manta de lodo e fluxo ascendente (UASB) do tratamento de vinhaça em relação ao potencial de produção de compostos de valor agregado a partir do bagaço de cana-de-açúcar (BCA) em reatores em batelada em condição mesofílica e termofílica, respectivamente. Pré-tratamentos térmico, ácido e diluição seriada foram aplicados ao inóculo termofílico, como tentativa de inibir as arqueias metanogênicas. A diluição seriada foi o pré-tratamento aplicado em ambos os inóculos, mesofílico e termofílico, para obtenção de consórcio de bactérias fermentadoras. Cinco meios de culturas foram testados como fonte de nutrientes para o crescimento de bactérias celulolíticas e fermentadoras. Dentre esses foi selecionado para os ensaios mesofílicos e termofílicos, o meio de cultura mais complexo suplementado com extrato de levedura. O BCA foi submetido à pré-tratamento biológico, térmico, explosão a vapor, deslignificação alcalina e hidrotérmico sendo o último utilizado nos ensaios do planejamento fatorial. O efeito das variáveis independentes de concentração de extrato de levedura e temperatura foi avaliado na produção de hidrogênio, metano e ácidos orgânicos a partir do inóculo termofílico. O efeito da concentração de extrato de levedura e substrato (BCA) foi avaliado na produção de bioprodutos a partir do inóculo mesofílico (37ºC). A maior produção de hidrogênio foi de 17,30 mmol/L à 60ºC e 3,42 g/L de extrato de levedura para a condição termofílica. Em relação aos ensaios mesofílicos observou-se 1,53 mmol/L com 3,42 e 5,00 g/L de extrato de levedura e BCA, respectivamente. Caracterização taxonômica e funcional dos microrganismos dos reatores de melhor desempenho de produção de hidrogênio dos planejamentos fatoriais termofílico e mesofílico foi realizada por análise Metagenômica (Illumina HiSeq). Nestas condições foram identificados microrganismos dos domínios Archaea, Bacteria, Eukarya, além de vírus. Para o domínio Bacteria foram identificados microrganismos celulolíticos e fermentadores como Coprothermobacter e Clostridium, enquanto para o domínio Archaea, foram identificadas metanogênicas hidrogenotróficas e acetoclásticas, como Methanothermobacter e Methanosarcina. Foram ainda identificados genes codificantes de enzimas catalisadoras da degradação de celulose, hemicelulose e lignina, constituintes principais da biomassa lignocelulósica utilizada como substrato, tais como celulase, carboxylesterase e 2-ácido hidroxi oxidase, respectivamente. Microrganismos aderidos no BCA in natura foram observados por microscopia eletrônica de varredura (MEV) e identificados por sequenciamento do RNAr 16S, e semelhantes a Streptomyces, Paenibacillus, Stenotrophomonas, Sphingomonas. Os microrganismos do lodo termofílico, solo e compostagem foram caracterizados por sequenciamento do RNAr 16S, e foram semelhantes a Clostridium, Thermoanaerobacter, Caloramator, Anaerobaculum, Tatlockia, Coprothermobacter, Dysgonomonas, Coprococcus, Sporomusa, Methanobacterium, Methanothermobacter, Methanosaeta, dentre outros. / In the present study, two inoculum (soil/compost and thermophilic sludge from upflow anaerobic sludge blanket from vinasse treatment) were separately evaluated as potential to production of value-added products from sugarcane bagasse in mesophilic and thermophilic conditions, respectively. Thermal, acid and serial dilution pretreatments were performed in the thermophilic inoculum to inhibition of methanogenic archaea. Serial dilution was applied into the mesophilic inoculm. Five culture medium were evaluated as nutritional source to enrichment of cellulolytic and fermenters bacteria; between then, the most complex one, supplemented with yeast extract was selected for the mesophilic and thermophilic bioassays. The sugarcane bagasse (SCB) was submitted to biological, thermal, stem explosion alkaline delignification and hydrothermal pretreatments, and the last one was used as substrate for the factorial designs. The effect of independent variables, such as yeast extract and temperature were evaluated on the hydrogen, methane and organic acids production from the thermophilic inoculum. The effect of yeast extract and substrate (SCB) concentrations were evaluated on the bioproducts generation from the mesophilic inoculum. The highest hydrogen production of 17.3 mmol/L was obtained at 60ºC and 3.42 g/L of yeast extract, on the thermophilic factorial design. In relation to the mesophilic factorial design, obtained 1.53 mmol/L of hydrogen with 3.42 and 5.00 g/L of yeast extract and SCB, respectively. Taxonomical and functional characterizations from the microorganisms were performed in the reactors with highest hydrogen production on the factorial designs using Metagenomics analysis (Illumina HiSeq). In both condition, mesophilic and thermophilic were found microorganisms from Archaea, Bacteria, Eukarya domin, besides Viruses. Concerning the Bacteria domain were found cellulolytic and fermenters microorganisms similar to Coprothermobacter and Clostridium, whiles for Archaea domain were identified hydrogenotrophic and acetoclastic methanogenic similar to Methanothermobacter and Methanosarcina. There were obtained genes coding to enzymes related to the cellulose, hemicellulose and lignin degradation such as carboxylesterase e 2-acid-hydroxioxidase, respectively. Microorganisms adhered into the in natura SCB fiber were observed by scanning electronic microscopy (SEM) and identified by 16 S rRNA sequencing, mainly as similar to Streptomyces, Paenibacillus, Stenotrophomonas, Sphingomonas. The microorganisms from the thermophilic sludge, soil and composting were also characterized by 16S RNAr sequencing and were similar to Clostridium, Thermoanaerobacter, Caloramator, Anaerobaculum, Tatlockia, Coprothermobacter, Dysgonomonas, Coprococcus, Sporomusa, Methanobacterium, Methanothermobacter, Methanosaeta, among others.

A exploração de fontes alternativas para a produção de energia e produtos químicos ganha cada vez mais relevância devido à crescente demanda mundial por energia, combustíveis e produtos sintéticos. Nesse contexto, a biomassa lignocelulósica passa a ser importante matéria prima e o uso de catalisadores heterogêneos uma via atrativa para a transformação química da biomassa. A associação do Ni com Nb2O5 é promissora para obtenção de um sistema catalítico multifuncional com propriedades ácidas e de hidrogenação. O Brasil é o maior produtor mundial de nióbio e possui enormes quantidades de biomassa lignocelulósica. Assim, o uso de catalisadores à base de nióbio para valorização da biomassa é uma maneira de se agregar ciência e tecnologia a estas matérias primas abundantes em nosso país. Dessa forma, o objetivo deste trabalho foi o desenvolvimento de catalisadores heterogêneos de Ni/Nb2O5 para a exploração da biomassa lignocelulósica como matéria prima para produção de combustíveis e produtos químicos. Nb2O5 foi preparado por duas rotas de síntese, hidrólise básica (HB) que produziu um material amorfo com partículas sem morfologia definida e a síntese hidrotérmica (HT), que produziu um material cristalino com morfologia de nano-bastões. O método HT gerou uma nióbia estável em condições hidrotérmicas e com propriedades texturais e ácidas bastantes superiores do que HB. Foi depositado Ni (5, 10, 15 e 25% m/m) em Nb2O5 e através de experimentos de redução monitorados in situ por técnicas de luz síncrotron ficaram estabelecidas as condições de ativação como sendo temperatura de 320oC e tempo de isoterma de no mínimo 1 h sob fluxo de H2. Isso para obtenção de cristalitos pequenos de Ni0 (2 a 25 nm) e para preservar as propriedades estruturais do suporte. Os catalisadores foram avaliados em reações de hidrodesoxigenação de éter difenílico e o sistema Ni/Nb2O5(HT) apresentou atividade para hidrogenólise da ligações éter, hidrogenação do anel aromático e hidrodesoxigenação, apresentando conversão completa do substrato e seletividade maior que 99% para cicloexano, além de poder ser reciclado por cinco ciclos de reação. 15%Ni/Nb2O5(HT) foi ativo e apresentou boa estabilidade na hidrodesoxigenação de um substrato real de lignina e produziu uma mistura de cicloalcanos e álcoois cíclicos em fase líquida com potencial para ser utilizada como biocombustíveis devido a sua baixa razão O/C. Experimentos exploratórios de conversão de celulose indicaram que o sistema catalítico Ni/Nb2O5(HT) também apresenta potencial para obtenção de polióis e glicóis a partir da fração de carboidratos da biomassa. / The exploitation of alternative resources for the production of energy and chemical products is gaining more and more relevance due to the growing world demand for energy, fuels and synthetic products. In this context, lignocellulosic biomass become an important raw material and the use of heterogeneous catalysts a very attractive way for biomass chemical transformation. The association of Ni with Nb2O5 is promising for obtaining a multifunctional catalytic system with acidic and hydrogenation properties. Brazil is the world\'s largest producer of niobium and has enormous amounts of lignocellulosic biomass. So, the use of niobium-based catalysts for biomass valorisation is a way for adding science and technology to these abundant raw materials in our country. Thus, the aim of this work is the development of heterogeneous Ni/Nb2O5 catalysts for the exploitation of lignocellulosic biomass as raw material for the production of fuels and chemicals. Nb2O5 was prepared by two routes of synthesis, basic hydrolysis (HB) that produced an amorphous material with particles with non-defined morphology and hydrothermal (HT) synthesis that produced a crystalline material with morphology of nano-rods. The HT method produced a stable niobia in hydrothermal conditions and with textures and acidic properties quite higher than HB. Ni (5, 10, 15 and 25 wt.%) was deposited on Nb2O5 and by in situ experiments of reduction monitored by synchrotron light techniques the activation conditions were established as being temperature of 320oC for at least 1 h under H2 flow. This condition enables the production of small crystallites of Ni0 (2 at 25 nm) and for preserving the structural properties of the support. The catalysts were evaluated in hydrodeoxygenation reactions of diphenyl ether and the Ni/Nb2O5(HT) system showed activity for hydrogenolysis of the ether linkages, hydrogenation of aromatic rings and hydrodeoxygenation, converting the substrate completely with selectivity higher than 99% for cyclohexane and being recyclable for five reaction cycles. 15% Ni/Nb2O5(HT) was active for hydrodeoxygenation of a real lignin substrate and exhibited good stability, producing a mixture of cyclic cycloalkanes and alcohols in a liquid phase with potential to be used as biofuels due to their low O/C ratio. Exploratory experiments of cellulose conversion indicated that the Ni/Nb2O5(HT) catalyst also has the potential for obtaining polyols and glycols from the carbohydrate fraction of the biomass.

A biomassa lignocelulósica é uma alternativa atrativa para o aumento na oferta de biocombustíveis, uma vez que é constituída de celulose e hemicelulose. Esses polímeros são constituídos principalmente de unidades menores de glicose e xilose, os quais por meio de bactérias anaeróbias termófilas, podem ser metabolizados em etanol. Portanto, estabeleceu-se o objetivo desse trabalho, em utilizar as principais fontes de carbono da biomassa lignocelulósica (celulose, glicose e xilose), e produzir etanol por meio da ação de consórcio microbiano selecionado a partir de inóculo termófilo e anaeróbio. O inóculo foi submetido a condição de crescimento com variação de pH (2,3,4,5,6,e 7) e variação de dois meios de cultivo em reatores em batelada, visando favorecer bactérias celulolíticas e fermentativas produtoras de etanol. Para a produção de etanol, o pH e meio de cultivo mais adequados foram 7,0 e Meio Thermoanaerobacter ethanolicus, respectivamente. A partir do inóculo enriquecido nas condições nutricionais de pH e meio de cultivo, prosseguiu-se a realização dos ensaios de produção de etanol a partir de celulose, glicose e xilose (1g/L de cada substrato), em pH 7 e meio T. ethanolicus. Os ensaios foram realizados em reator em batelada, em triplicata, a 55 ºC, ambos seguidos de um reator controle, sem adição desses substratos orgânicos. Os rendimentos de etanol foram de 1,73 mol etanol/mol glicose e 1,33 mol de etanol /mol de xilose. Para o substrato celulose obteve-se 1,88 mmol de etanol/g de celulose. Para os reatores controle de glicose, celulose e xilose, no qual o extrato de levedura foi a única fonte orgânica adicionada, a produção de etanol foi 1,27 mmol/L, 0,39 mmol/L e 1,65 mmol/L, respectivamente. Em todos os reatores foi detectado produção de ácido acético, ácido butírico e ácido propiônico. A produção de ácido acético foi de 5,73 mmol/L, 9,73 mmol/L e 14,45 mmol/L, para os reatores de glicose, celulose e xilose, respectivamente. No reator com glicose, observou-se baixo rendimento de hidrogênio (0,31 mol hidrogênio/mol glicose), e nos demais reatores não foi constatado produção desse gás. Em contrapartida, observou-se rendimentos de 6,6 mmol de metano/g de celulose e 0,68 mol de metano/mol de xilose para os respectivos reatores. Dessa forma, pode-se mencionar que em função das características do consórcio microbiano foi possível obter a degradação da celulose e metabolização da glicose e xilose em etanol. / Lignocellulosic biomass is an attractive alternative to increase biofuels proposal, as its composed of cellulose and hemicellulose. These polymers are consisted in individual molecules of glucose and xylose, through some thermophilic bacteria, can metabolize these carbohydrates in ethanol. Therefore, this study reports on using the principals carbon sources of lignocellulosic biomass (cellulose, glucose, and xylose), and producing ethanol through microbial consortium from anaerobic and thermophilic inoculum. The biomass was submitted to variation of pH (2,3,4,5,6, and 7) and two kinds of medium, due to ethanol production in batch reactors. For ethanol production, the optimized pH and medium were 7,0 and Thermoanaerobacter ethanolicus medium, respectively. The enriched culture was being cultivated in pH and medium experiments were used to ethanol production experiments that carried out in batch reactors, from cellulose, glucose and xylose were realized in triplicate and were maintained at 55 °C, in both batches had a control reactor (without these organics substrates). Positive results in ethanol yields were 1,73 mol ethanol/ mol glucose and 1,33 mol ethanol/ mol xylose. In celluloses reactors the microbial consortium was efficient in substrate degradation, however, was obtained lower ethanol yields (1,88 mol ethanol/ g cellulose). In control reactors from glucose, cellulose and xylose, that yeast extract was the unique organic source, ethanol production was 1,27 mmol/L, 0,39 mmol/L e 1,65 mmol/L, respectively. In all reactors were detected acetic, butyric and propionic acids. The acetic acid production was 5,73 mmol/L, 9,73 mmol/L e 14,45 mmol/L in glucose, cellulose and xylose reactors, respectively. For glucoses reactors were observed lower hydrogen production (0,31 mol hydrogen/ mol glucose), in the other reactors did not observed gases production. Instead of the following yields were obtained: 6,6 mmol methane/ g cellulose and 0,68 mol methane/ mol xylose. Taking this into account, microbial consortium enriched had characteristics to degrade cellulose and metabolize glucose and xylose to ethanol.

A geração de energia a partir de biomassa lignocelulósica pode contribuir de maneira significativa com a ampliação do escopo energético, no que se refere ao uso de fontes alternativas voltadas à geração de combustíveis renováveis. Porém, a utilização destas fontes enfrenta ainda desafios relacionados à exploração de diferentes matérias-primas com potencial de utilização e o desenvolvimento de tecnologias de processos que garantam a viabilidade técnica e econômica da geração do etanol 2G. Neste estudo foi estimada a produção de etanol da biomassa aérea da batata-doce (Ipomoea batatas (L.) Lam.) e da biomassa do capim-elefante (Pennisetum purpureum Schumach), cv. Roxo e cv. Napier após pré-tratamento ácido e básico, sob hidrólise de dois complexos enzimáticos comerciais (CTec2 e HTec2) e o extrato enzimático obtido a partir de fungo isolado de ninhos de Acromyrmex balzani. O delineamento experimental utilizado foi o de blocos inteiramente casualizados e o arranjo dos tratamentos o Esquema Fatorial 3x3, em três repetições. Foi verificado, para a batata-doce, o rendimento estimado médio de 37,42 mL/Kg de MS hidrolisada com os complexos enzimáticos comerciais e que o capim-elefante, nesse experimento, confirmou seu potencial para ser utilizado na produção de energia renovável, pois apresentou o rendimento médio de 123,5 mL/Kg de MS hidrolisada nas mesmas condições da hidrólise da biomassa aérea da batata-doce. / Power generation from lignocellulosic biomass can contribute significantly to the energy scope expansion, with regard to the alternative sources use focused on the renewable fuels generation. However, the use of these sources still faces challenges related to different raw materials exploration with potential use and technologies processes development that ensure the technical and economic viability of generating 2G ethanol. The aim of this study was to evaluate the ethanol production potential from aerial sweet potato (Ipomoea batatas (L.) Lam.) (B) crop residues compared to elephantgrass materials (Pennisetum purpureum Schum), cultivar Roxo and cultivar Napier by the hydrolytic action of three enzyme solutions: two commercial enzymatic complexes (CTec2 e HTec2) and an enzyme extract obtained from filamentous fungi isolated from Acromyrmex balzani. The experimental design was the Factorial Scheme 3x3, in triplicate. It was checked on the sweet potato, the average yield of 37.42 mL/kg MS hydrolyzed with commercial enzymatic complexes and the elephantgrass, this experiment confirmed its potential to be used in the renewable energy production, as it presented average yield of 123.5 mL/kg MS hydrolyzed under the same conditions of biomass sweet potato hydrolysis.

<p>Lignocellulosic biomass represents an important renewable resource for biofuels production. Lignocellulosic biomass is comprised of cellulose, hemicellulose and lignin. Lignocellulosics are highly recalcitrant to enzymatic degradation and due to its complex nature a range of enzymes are required to synergistically hydrolyse biomass. Many microorganisms are capable of producing these enzymes as part of their hemicellulolytic hydrolysis system(s). The aim of this study was the characterisation of a thermophilic actinobacterial isolate (ORS10), capable of producing hemicellulosic enzymes, and the cloning and characterization of a hemicellulosic enzyme produced by the isolate. Phylogenetic analyses clustered ORS10 with species of the genus Streptomyces. BLAST analysis revealed that ORS10 was most closely related to Streptomyces achromogenes (99% identity). A small-insert genomic library was constructed and a putative acetylxylan esterase (AXEase) gene, axe10, was identified. The enzyme, Axe10, has moderate similarity to &alpha / /&beta / hydrolase proteins, and contains an esterase/lipase superfamily conserved domain and a typical AXEase catalytic triad. The axe10 gene was sub-cloned into an expression vector [pET21a(+)] and a 28.7 kDa protein with demonstrated AXE activity was purified from E. coli Rosetta (DE3) pLysS. Axe10 displayed optimum activity at 37oC and pH 7.0. Despite being derived from a thermophilic Streptomyces species Axe10 was not thermostable. However, given the relative novelty of Axe10, further characterisation and assessment of this enzyme is warranted.</p>

Lignocellulosic biomass is under extensive investigation as a bioethanol and bio-based materials feedstock. However, the complex structural and chemical mechanisms of lignocellulosic plant, which cause resistance to deconstruction during saccharification, require a pretreatment process. In this study, raw materials (corn bran, wheat bran and wheat straw) were selected because of their production and consumption in Turkey and also their accessibilities to be used as bioethanol source. Microfluidization pretreatment (high-pressure fluidization), which stands as a new approach for nano-cellulosic fibers production, was studied at 500 bar and 2000 bar to observe the qualitative and quantitative modifications in enzymatic hydrolysis depending on its effects on lignocellulosic structure. Optimum cellulase concentrations were determined for microfluidized samples as 4.5 U/g dry biomass for wheat bran, corn bran and 6.0 U/g dry biomass for wheat straw samples for the first 150 min interval. Effective usage of solid loads were found as 5.0 %, 2.5 %, and 7.5 % (dw/v) for wheat bran, wheat straw and corn bran, respectively. X-ray diffraction and SEM results of the microfluidized samples have indicated that the pretreatment has increased crystallinity index of all the samples and resulted in a scattered structure. Comparisons with other methods (softening, dilute-acid and lime pretreatments) have shown that microfluidization is advantageous over others by reducing the time required for enzymatic hydrolysis and thus can be a promising alternative pretreatment.

Microbial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individ- ual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.

<p>Lignocellulosic biomass represents an important renewable resource for biofuels production. Lignocellulosic biomass is comprised of cellulose, hemicellulose and lignin. Lignocellulosics are highly recalcitrant to enzymatic degradation and due to its complex nature a range of enzymes are required to synergistically hydrolyse biomass. Many microorganisms are capable of producing these enzymes as part of their hemicellulolytic hydrolysis system(s). The aim of this study was the characterisation of a thermophilic actinobacterial isolate (ORS10), capable of producing hemicellulosic enzymes, and the cloning and characterization of a hemicellulosic enzyme produced by the isolate. Phylogenetic analyses clustered ORS10 with species of the genus Streptomyces. BLAST analysis revealed that ORS10 was most closely related to Streptomyces achromogenes (99% identity). A small-insert genomic library was constructed and a putative acetylxylan esterase (AXEase) gene, axe10, was identified. The enzyme, Axe10, has moderate similarity to &alpha / /&beta / hydrolase proteins, and contains an esterase/lipase superfamily conserved domain and a typical AXEase catalytic triad. The axe10 gene was sub-cloned into an expression vector [pET21a(+)] and a 28.7 kDa protein with demonstrated AXE activity was purified from E. coli Rosetta (DE3) pLysS. Axe10 displayed optimum activity at 37oC and pH 7.0. Despite being derived from a thermophilic Streptomyces species Axe10 was not thermostable. However, given the relative novelty of Axe10, further characterisation and assessment of this enzyme is warranted.</p>

The focus of this dissertation centers on the development and applications of gas chromatography/mass spectrometry and capillary electrophoresis methodologies to quantify monomeric compositions of the β-O-4 linkages in lignin. Pretreatment is a required step in the utilization of lignocellulosic biomass for biofuels. Lignin is the target of pretreatment because it hinders the accessibility of enzymes and chemicals to cellulose. The effects of pretreatment are commonly assessed utilizing enzymatic saccharification and lignin assays. However, these techniques do not elucidate the effects of pretreatment on the monomeric make up of lignin. The overarching hypothesis of this dissertation is that changes in individual monolignol content upon pretreatment can be observed from quantification. To test the hypothesis, a pretreatment, solution phase Fenton chemistry, was conducted on various lignocellulosic biomass feedstocks. Enzymatic saccharification studies showed a significant increase in glucose production upon Fenton pretreatment, however, lignin assays did not show a significant decrease in lignin content. Project two of this dissertation aimed to synthesize analytical standards in order to develop a quantitative thioacidolysis technique. The successful synthesis of the three arylglycerols were conducted utilizing and epoxidation reaction scheme which was hypothesized to produce a single diastereomer, as supported by GC/MS and chiral CE analysis. Upon method development, a quantitative thioacidolysis GC/MS method was applied to untreated and Fenton treated biomass. Results from this project revealed there was no significant change in the three lignin monomers. To verify the method, quantitative thioacidolysis GC/MS method was applied to a pretreatment method known to degrade lignin, alkaline peroxide pretreatment. The results of this project showed a significant change in monolignol concentrations upon alkaline peroxide pretreatment. Analytical degradative techniques, such as thioacidolysis, has traditionally assessed lignin as monomeric ratios. However, as this dissertation showed, upon alkaline peroxide pretreatment, no significant change was seen in the monomeric ratios, but there was a significant difference in all three monolignol concentrations. These results support the overall hypothesis that changes in individual monolignol content upon pretreatment can be observed from quantification. The works of this dissertation provides an analytical method which contributes to the elucidation of lignin.

This study investigated the fate of lignocellulosic biomass (wheat-feed and perennial rye grass) in different anaerobic digestion systems, evaluating the role of substrate specificity on the pattern of degradation. The two-stage (biohydrogen-biomethane) anaerobic system was found to be more effective in the degradation of lignocellulose, when compared to the conventional single-stage system. The perennial rye grass substrate possessed about 21% higher holocellulose concentration when compared to the wheat-feed; its exploitation in the acidogenic digestion was however poor, resulting in a 2.9% lower biogas yield in a equivalent two-stage system. The study therefore developed a treatment technique involving the use of cellulase and ferulic acid esterase enzyme combinations for the treatment of perennial rye grass. The enzyme cocktail at 0.202 ml enzyme/g VS added resulted in efficient bioconversion of the complex polymers to soluble carbohydrates, evident in the yield increase of soluble COD, to 321.0±10.9 mg/gVS, a 393.2% yield increase, when compared to the no enzyme added control. The yield of bio-hydrogen after enzymatic addition was 48ml/gVS, 335% higher when compared to the alkaline treatment; and more than seven fold higher than the yield obtained from the fermentation with no pre-treatment. The acetate to butyrate ratio varied from 4:1, when no pre-treatment was used, to 2:1when alkaline pre-treatment was used, then to 1:1 after the enzymatic treatment. The downstream effect of the prior hydrolysis on the subsequent processes to acidogenic fermentation like biomethane and PHA production and lignin recovery were also investigated. The hydrogenic/acidogenic fermentation resulted in methane yield improvement of 45.7%. The study shows that the more effective a hydrolysis procedure is in the depolymerisation of complex polymers, the greater the accumulation of PHA in the PHA biosynthesis operations. The enhanced hydrogenic /acidogenic fermentation having effectively degraded the holocellulose component of the perennial rye grass substrate ensured that relatively high quality lignin was obtained in an Organosolv lignin-extraction procedure. FT-IR profile show less contamination of polysaccharides and proteins in the lignin extracted from the enzymatically enhanced acidogenic fermentation. An evaluation of the economic viability of the investigated secondary processes showed that direct integrations of those processes to the biohydrogen process may not be as economically advantageous, when compared to a 2nd -stage biomethanation system.