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The first part of the dissertation presented a potentially novel aerobic, thermophilic, and cellulolytic bacterium identified as Brevibacillus sp. Strain JXL which was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between the effects caused by these two simple sugars. After the end of the 24 hour period, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also observed when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and optimal pH range of 6-8. These cellulases also had high thermotolerance as demonstrated by retaining more than 50% activity after 1 h at 100°C. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose. In the second part of the dissertation, the effect of Cellulose- and Organic-Solvent based Lignocellulose Fractionation (COSLIF) (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng.2007, 97 (2), 214−223) pretreatment conditions on sweet sorghum bagasse (SSB) feedstock was studied using Response Surface Methodology (RSM). Batch experimental matrix was set up based on response surface method's central composite design in two factors to determine the effects of reaction time and temperature on the yield of simple sugars after a sequential pretreatment-enzyme hydrolysis process. Accordingly, changes in delignification, total reducing sugar (TRS) yield, glucan retention, digestibility and overall sugar yields resulting from various combinations of reaction times and temperatures were determined. The results suggested that both pretreatment temperature and reaction time were significant factors, although temperature was more so than reaction time. COSLIF pretreatment conditions of 50°C and 40 min were found to be the optimum pretreatment conditions for the saccharification of SSB. At the end of pretreatment and enzymatic hydrolysis, maximum values of 51.4% delignification, 85% overall glucose yield, and 44% overall xylose yield at an ACCELERASE®1500 loading of 0.25 mL/g sweet sorghum bagasse were achieved. Optimum ACCELERASE®1500 dosage of 0.1 mL/g of sweet sorghum bagasse was identified which resulted in an overall glucose yield of 82.2%±1.05. An effort has also been made to prescribe predictive models which represented the correlation between independent variables (reaction time and temperature), and dependent variables (delignification, and overall glucose yield) using RSM. The significance of the correlations and adequacy of these models were statistically tested for the selected objective functions. The outcomes suggested very competent and statistically adequate regression models which provided quantitative information both for delignification and overall glucose yield for the batch experiments studied.

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Manuscripts have an essential importance as being irreplaceable parts of our cultural heritage. It has long been known that these works have been suffered from serious damages due to the corrosive effects of the inks. In this study, the influence of inks prepared according to historical recipes on the degradation of model paper has been investigated. Totally, sixteen ink solutions were prepared and applied on Whatman No. 41 filter papers. The mostly examined ink is the iron-gall-ink which is known as very corrosive. Changing the ratios of the ingredients in the ink, it is aimed to understand the degradation mechanism of the cellulose caused by the iron-gall-ink. Influence of iron to gallic acid ratio, copper to iron ratio and the effect of saffron on ink corrosion were examined. Also the influence of colored inks (red and green) / prepared according to the historical recipes on cellulose degradation was studied. In order to determine the changes in the cellulose in time, dry heat accelerated ageing was performed on the paper samples. UV-Vis spectroscopy, FTIR-ATR and Color spectrophotometry were used to investigate the changes in the chemical composition of the cellulose after accelerated ageing.

The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps in the conversion of lignocellulosic biomass to biofuel. This hydrolysis by cellulases, a heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion in the case of crystalline cellulose. Various rate-limiting factors were investigated employing experimental as well as computational studies. Cellulose accessibility and the hydrolysable fraction of accessible substrate (a previously undefined and unreported quantity) were shown to decrease steadily with conversion, while cellulose reactivity, defined in terms of hydrolytic activity per amount of actively adsorbed cellulase, remained constant. Faster restart rates were observed on partially converted cellulose as compared to uninterrupted hydrolysis rates, supporting the presence of an enzyme clogging phenomenon. Cellulose crystallinity is a major substrate property affecting the rates, but its quantification has suffered from lack of consistency and accuracy. Using multivariate statistical analysis of X-ray data from cellulose, a new method to determine the degree of crystallinity was developed. Cel7A CBD is a promising target for protein engineering as cellulose pretreated with Cel7A CBDs exhibits enhanced hydrolysis rates resulting from a reduction in crystallinity. However, for Cel7A CBD, a high throughput assay is unlikely to be developed. In the absence of a high throughput assay (required for directed evolution) and extensive knowledge of the role of specific protein residues (required for rational protein design), the mutations need to be picked wisely, to avoid the generation of inactive variants. To tackle this issue, a method utilizing the underlying patterns in the sequences of a protein family has been developed.

There are about 100,000 described fungal species, however, the diversity could be higher because conventional techniques do not allow identification of all groups of fungi and there are still unexplored geographical areas. High-throughput DNA sequencing methods provide the opportunity to resolve the diversity and distribution of mycelia in soil. Soils are the largest pool of terrestrial carbon and macromolecular materials, such as lignin and cellulose, form an important part of this soil carbon. Saprotrophs (decomposers) fungi degrade lignin and cellulose that is important to the global carbon cycle, although lignin is highly resistant to degradation if compared with cellulose. In this work, we investigated the diversity of fungi in sand-dune soils and their involvement in the decomposition of lignin and cellulose. The key findings of this work were:•A comparison of sand-dune ecosystems from two reserves in the UK showed differences in the ion concentrations, pH and total organic carbon in soils, suggesting that there were different environmental conditions that could potentially affect the distribution/presence of microbial communities in soils, e.g. fungal communities.•Fungi from field samples were identified using 454 pyrosequencing. The identified fungal species belong to groups with different ecologies, among which are wood-rotting fungi that are the main agents responsible for the lignin breakdown. The fungal communities were distributed differently across the different sand-dune ecosystems, sampling times and type of bait materials.•Lignin and cellulose can be degraded in field samples over time. Lignin degradation was shown by the shifts in the [Ac/Al]S, [Ac/Al]G and [S/G] relative lignin decomposition state proxies, and cellulose degradation by the shifts in the [cellulose:cellulose+lignin] ratio. Cellulose degradation was faster than lignin, thus confirming previous studies.•The degradation of both lignin and cellulose was different depending on the type of plant material, ecosystem/soil characteristics where the material was buried and fungal communities present on the bait materials.•Lignin breakdown was most likely to be by white-rot fungi that were identified colonising the bait materials.

History teaches us that novel materials, such as chlorofluorocarbon and asbestos, can have dire unintended consequences to human and environmental health. The exponential growth of the field of nanotechnology and the products developed along the way provide the opportunity for a new paradigm of design thinking, in which human and environmental impacts are considered early on in product development. In particular, nanocellulose is touted as a promising green nanomaterial, as it is sourced from an effectively inexhaustible feedstock of wood-based cellulose and is assumed to be harmless to the environment since it is derived from a natural material and assumed to be biodegradable. The various forms of nanocellulose possess an impressive diversity of properties, making it suitable for a wide variety of applications such as drug delivery, reinforcement, food additives, and iridescent make-up. However, as nanomaterials can have different properties relative to their bulk form, it is questionable whether they are truly environmentally friendly, particularly in terms of their biodegradability and potential impacts to receiving environments. Given the projected mass-scale application of nanocellulose and the inevitability of its subsequent release into environment, the purpose of this study was to determine the biodegradability of nanocellulose and the response of environmentally-relevant microbial communities. Specifically, it was hypothesized that cellulose in the nano size range would display distinct biodegradation patterns and rates, relative to larger forms of cellulose. Further, it was hypothesized that modification of nanocellulose, in terms of morphology and surface properties (e.g., charge), would further influence its biodegradability. Wetlands and anaerobic digesters were selected as two environmentally-relevant receiving environments that also play critical roles in global carbon turnover. To examine the biodegradability of nanocellulose, two distinct microbial consortia were enriched from wetland (W) and anaerobic digester (AD) inocula and applied in parallel experiments. The consortia were grown under anaerobic conditions with microcrystalline cellulose as the sole carbon substrate over a period of 246 days before being aliquoted to microcosms for subsequent biodegradation assays. Various forms of nanocellulose were spiked into the microcosms and compared with microcrystalline cellulose as a non nano reference. Microcosms were sacrificed in triplicate with time to monitor cellulose degradation as well as various measures of microbial community response. Microbial communities were characterized in terms of gene markers for total bacteria (16S rRNA genes) and anaerobic cellulose degraders (glycoside hydrolase family 48 genes, i.e., cel48) as well as high throughput amplicon sequencing of 16S rRNA genes (V4 region). A series of three studies examined: 1) the effect of nanocrystalline versus microcrystalline cellulose; 2) the effects of nanocellulose morphology (crystalline rod versus filament) and surface functionalization (cationic and anionic); and 3) metagenomic characterization of cellulose degrading communities using next-generation DNA sequencing. It was found that the nano- size range did not hinder cellulose degradation, in fact, nanocrystalline cellulose degraded slightly faster than microcrystalline cellulose according to 1st order kinetics (1st order decay constants: 0.62±0.08 wk-1 for anionic nanocrystalline cellulose versus 0.39±0.05 wk-1 for microcrystalline cellulose exposed to AD culture; 0.69±0.04 wk-1 for anionic nanocrystalline cellulose versus 0.58±0.05 wk-1 for microcrystalline cellulose exposed to W). Experiments comparing the effects of surface functionalization indicated that anionic nanocellulose degraded faster than cationic cellulose (1st order decay constants for cationic nanocrystalline cellulose: 0.48±0.06 wk-1 and 0.58±0.07 wk-1 on exposure to AD and W cultures respectively). Measurements of 16S rRNA and cel48 genes were consistent with this trend of greater biological growth and cellulose-degrading potential in the anionic nanocellulose condition, suggesting that surface properties can influence biodegradation patterns. Taxonomic characterization of 16S rRNA gene amplicons suggested that taxa known to contain anaerobic cellulose degraders were enriched in both W and AD consortia, which shifted in a distinct manner in response to exposure to the different cellulosic materials. This suggests that distinct groups of microbes may drive the biodegradation of different forms of cellulose. Further, metagenomic investigation provided new insight into taxonomic and functional aspects of anaerobic cellulose degradation, including identification of enzymatic families associated with degradation of the various forms of cellulose. Overall, the findings of this study advance understanding of anaerobic cellulose degradation and indicate that nanocellulose is likely to readily degrade in receiving environments and not pose an environmental concern. / Ph. D.

Made available in DSpace on 2016-08-17T18:39:30Z (GMT). No. of bitstreams: 1 2540.pdf: 2404853 bytes, checksum: 5e1d90d2a3d16f30a431e16e644da26d (MD5) Previous issue date: 2009-04-28 / Financiadora de Estudos e Projetos / The sugarcane weevil, Sphenophorus levis, is an important pest in sugarcane culture in São Paulo state, Brazil. To complete its life cycle, S. levis may depends on microorganisms that inhabit its intestinal tract and play an important key in the insect physiology and nutrition. In this study we report the characterization of the intestinal microbiota from population of insect larvae from field and laboratory. Analysis of 16S rDNA sequences revealed a total of fourteen genera, one group from Candidatus category and two uncultivable groups represented by Alfa-Proteobacteria, Beta-Proteobacteria, Gamma-Proteobacteria, Firmicutes and Bacteroidetes phylum. Microorganisms isolated through culture-dependent methods were classified according morphological parameters and using 16S rDNA molecular marker. In addition to bacteria, four filamentous fungi were isolated. It was observed a slightly higher bacterial diversity in field than in laboratory according to Shannon index (Field H'= 3,36; Laboratory H'= 3,26). It is also our objective in this work to search for microorganisms capable to degrade cellulose, an important event in the insect attack. From the cultivable microorganisms, five genera of bacteria and two filamentous fungi presented cellulolytic activity. This is the first study about S. levis microbiota which may contribute to understand the interaction plant-pathogen and also be useful for future development of new strategies for control of S. levis in sugarcane cultivation. / A cana-de-açúcar é uma das mais importantes culturas no Brasil. No entanto, muitas pragas atacam esta cultura causando prejuízos econômicos. O gorgulho da cana-deaçúcar, Sphenophorus levis, é uma importante praga na cultura canavieira no Estado de São Paulo. Para completar o seu ciclo de vida, S. levis parece depender de microorganismos que habitam o seu trato intestinal e desempenham um importante função na fisiologia e nutrição do inseto. Neste estudo, nós realizamos a caracterização da microbiota intestinal de população de larvas de inseto campo e de laboratório. As análises das seqüências de 16S rDNA revelaram um total de catorze gêneros, um grupo da categoria Candidatus e dois grupos nãocultiváveis representados pelos filos Alfa-Proteobacteria, Beta-Proteobacteria, Gamma- Proteobacteria, Firmicutes e Bacteroidetes tanto em larvas do campo quanto nas do laboratório. Os microrganismos isolados através dos métodos dependentes de cultura foram agrupados de acordo com parâmetros morfológicos e através do marcador moléculas 16S rDNA. Além das bactérias também foram isolados quatro fungos filamentosos. Observou-se uma diversidade bacteriana levemente superior no campo do que no laboratório de acordo com o índice de Shannon (Campo H '= 3,36; Laboratório H' = 3,26). É também nosso objetivo neste trabalho encontrar microorganismos capazes de degradar celulose, um importante evento no ataque do inseto. Dos microorganismos cultiváveis, cinco gêneros de bactérias e dois fungos filamentosos apresentaram atividade celulolítica. Este é o primeiro estudo sobre a microbiota do S. levis que pode contribuir para a compreensão da interação planta-patógeno e também ser útil para o futuro desenvolvimento de novas estratégias de controle de S. levis no cultivo da cana-de-açúcar.

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