Modified cellulase enzyme assay methods were developed to determine the potential of using enzyme activities to evaluate the decomposition of organic matter in carbon amended mine tailings systems. Cellulase assays are commonly performed in soil science applications, industrial production and bio-energy research to determine organic matter response to physical, chemical or temporal variation but, they have not been applied in a mine waste environment. Heavy metal content is considered to be a potential inhibitor to cellulase enzyme activity. Using samples collected from Greens Creek Mine in Juneau, Alaska, USA, the modified assay was evaluated to develop a method that provided reproducible results. It was determined that a sample mass of three grams is sufficient to provide consistent enzyme measurements. Each sample location was characterized by four replicates to ensure statistically representative data. Matrix interferences were determined to be inconsequential in this system. Despite the low organic content amended to the tailings, heavy metal content and potentially low enzyme activity, the modified enzyme assay method provided reproducible enzyme measurements. Following the development of the cellulase assays, spatial and temporal variations in cellulase enzymes were investigated from carbon amended tailings samples collected at the Greens Creek Mine. Six test cells, containing a mixture of tailings and a combination of peat, spent brewery grain and/or municipal biosolids were sampled in the fall of 2005, 2007 and 2009. Exo-(1,4)-β-D-glucanase (EC 3.2.1.91), Endo-(1,4)-β-D-glucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) enzymes were assayed from core sections at five different depths. Enzyme activities were compared to sulfate reducing and acid producing bacterial enumerations, sulfide trends and carbon content. General trends were consistent between enzyme activity and SRB enumerations. The range of total carbon values fell between 3 and 5 wt % in each test cell while the average inorganic carbon content was 3.5 wt %. The range of organic carbon content was between 0.2 and 1.2 wt %. Total, inorganic and organic carbon values were more characteristic of test cell carbon distribution. Cellulase enzyme assays provide valuable information regarding the degradation of cellulose and hemi-cellulose. This study demonstrates that enzymes can be monitored in a tailings environment and that enzyme assays conducted for monitoring purposes may be a useful practice to indicate the sustained or declining performance of organic matter in a carbon amended remedial system.

Modified cellulase enzyme assay methods were developed to determine the potential of using enzyme activities to evaluate the decomposition of organic matter in carbon amended mine tailings systems. Cellulase assays are commonly performed in soil science applications, industrial production and bio-energy research to determine organic matter response to physical, chemical or temporal variation but, they have not been applied in a mine waste environment. Heavy metal content is considered to be a potential inhibitor to cellulase enzyme activity. Using samples collected from Greens Creek Mine in Juneau, Alaska, USA, the modified assay was evaluated to develop a method that provided reproducible results. It was determined that a sample mass of three grams is sufficient to provide consistent enzyme measurements. Each sample location was characterized by four replicates to ensure statistically representative data. Matrix interferences were determined to be inconsequential in this system. Despite the low organic content amended to the tailings, heavy metal content and potentially low enzyme activity, the modified enzyme assay method provided reproducible enzyme measurements. Following the development of the cellulase assays, spatial and temporal variations in cellulase enzymes were investigated from carbon amended tailings samples collected at the Greens Creek Mine. Six test cells, containing a mixture of tailings and a combination of peat, spent brewery grain and/or municipal biosolids were sampled in the fall of 2005, 2007 and 2009. Exo-(1,4)-β-D-glucanase (EC 3.2.1.91), Endo-(1,4)-β-D-glucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) enzymes were assayed from core sections at five different depths. Enzyme activities were compared to sulfate reducing and acid producing bacterial enumerations, sulfide trends and carbon content. General trends were consistent between enzyme activity and SRB enumerations. The range of total carbon values fell between 3 and 5 wt % in each test cell while the average inorganic carbon content was 3.5 wt %. The range of organic carbon content was between 0.2 and 1.2 wt %. Total, inorganic and organic carbon values were more characteristic of test cell carbon distribution. Cellulase enzyme assays provide valuable information regarding the degradation of cellulose and hemi-cellulose. This study demonstrates that enzymes can be monitored in a tailings environment and that enzyme assays conducted for monitoring purposes may be a useful practice to indicate the sustained or declining performance of organic matter in a carbon amended remedial system.

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.

Made available in DSpace on 2017-07-10T18:08:12Z (GMT). No. of bitstreams: 1 Caroline Mariana de Aguiar.pdf: 5014872 bytes, checksum: 64beb98ea03bbb601831a3ce234b8c31 (MD5) Previous issue date: 2010-02-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lignocellulosic materials are the most abundant residues in the world and there is a worldwide concern to use them as raw material for bioethanol production. This is possible because these materials are rich in cellulose. Cellulose is a biopolymer composed of glucose molecules linked by ß-1-4 glycosidic bonds. Glucose can be converted into ethanol by fermentation and can be obtained from cellulose by enzymatic hydrolysis using cellulases. The cellulases can be produced by several microorganisms under appropriate environmental conditions. Amongst these microorganisms is the fungus Aspergillus niger. In this work, cellulases were obtained by fermentation cultivating A. niger in broth containing pretreated lignocellulosic materials such as sugarcane bagasse, corn straw or wheat straw as the only carbon source. The fermentation kinetic was observed when the pretreated sugarcane bagasse was used as the carbon source. Several variables that affect the enzymatic hydrolysis were analyzed using the three pretreated lignocellulosic materials as hydrolysis substrate. The variables analyzed were: pH, temperature, time of the hydrolysis, mass fraction of the substrate and dilution of the enzymatic broth. The pretreatment of the lignocellulosic materials is paramount for exposing the cellulose chain. Pretreatment consisted of using 4%w/w NaOH solution or 1%w/w H2O2 and their efficiency for removing the lignin from the residues were evaluated. The enzymatic activity also was evaluated by submeting the lignocellulosic materials to successive enzymatic hydrolysis. The enzyme deactivation was evaluated by cooling or freezing the enzymatic broth. It was concluded that Aspergillus niger produces cellulases when grown on medium with pretreated lignocellulosic materials as carbon source. Considering the fermentation kinetic, the ideal time to collect the enzymatic broth with maximum productivity was about 7 days. The cellulase complex does not suffer considerable deactivation when stored at -18°C (freezer) for 43 days, however, the broth activity drops by 43% after 48 hours when stored at 4°C (fridge). The corn straw showed better results as carbon source in fermentation and as substrate hydrolysis, compared with the other materials, with enzymatic activity of 0.895 U/ml. The ideal pH to conduct the enzymatic hydrolysis was 4.8 at 50°C for 50 minutes. The mass fraction of the substrate and enzyme concentration affects the enzymatic activity by a linear dependence. The pretreated materials provided higher enzymatic activity results than the untreated materials. The highest activity enzymatic results were obtained with H2O2 treated substrates, with enzymatic activity of 0.655 U/ml for the sugarcane bagasse, 0.892 U/ml for the corn straw and 0.801 U/ml for the wheat straw. Also, the results show that the H2O2 pretreated materials can be submitted up to, at least, four successive hydrolysis with the second one yielding the highest enzymatic activity for all pretreated residues. / Os resíduos lignocelulósicos são os mais abundantes no mundo e atualmente há uma preocupação mundial em aproveitá-los como matéria-prima na produção de bioetanol. Isto é possível visto que tais resíduos são ricos em celulose. A celulose é um biopolímero composto por moléculas de glicose unidas por ligações glicosídicas ß-1-4. A glicose pode ser transformada em etanol por via fermentativa e pode ser obtida da celulose via hidrólise enzimática utilizando as enzimas celulases. As celulases podem ser produzidas por diversos micro-organismos sob condições adequadas. Dentre esses micro-organismos, destaca-se o fungo Aspergillus niger. Neste trabalho, celulases foram obtidas cultivando-se A. niger em meio de cultura com os resíduos lignocelulósicos bagaço de cana-de-açúcar, palha de milho e palha de trigo pré-tratados com NaOH 4% como única fonte de carbono. Observou-se a cinética da fermentação com bagaço de cana-de-açúcar pré-tratado com NaOH 4% como fonte de carbono. Foram analisadas diversas variáveis que afetam a hidrólise enzimática utilizando os três resíduos lignocelulósicos pré-tratados com NaOH 4% como substrato. As variáveis analisadas foram: pH, temperatura, tempo de hidrólise enzimática, fração mássica de substrato e diluição do caldo enzimático. Avaliou-se a eficiência dos pré-tratamentos dos resíduos com NaOH 4% e com H2O2 1%. Avaliou-se o comportamento da atividade enzimática submetendo os resíduos lignocelulósicos a hidrólises enzimáticas sucessivas. A desativação enzimática foi avaliada nas condições de resfriamento e congelamento do caldo enzimático. Nas condições estudadas, foi concluído que o Aspergillus niger produz celulases quando cultivado em meio com resíduos lignocelulósicos pré-tratados como fonte de carbono. O tempo ideal para coleta do caldo enzimático, com produtividade máxima, foi de aproximadamente 7 dias. O complexo celulásico não sofre desativação se armazenado a temperatura de -18°C (freezer) por 43 dias, mas perde sua atividade em 43% após 48 h se armazenado a 4°C (geladeira). A palha de milho apresentou melhores resultados como fonte de carbono na fermentação e como substrato na hidrólise, comparada com os outros resíduos, com atividade enzimática de 0,895 U/mL. O pH ideal para se conduzir a hidrólise foi 4,8 na temperatura de 50ºC por 50 minutos. A fração de substrato e a concentração das enzimas afetam linearmente a atividade enzimática. Os resíduos pré-tratados proporcionaram melhores resultados de atividade enzimática do que os resíduos não tratados. Os melhores resultados de atividade foram obtidos com os resíduos tratados com solução de H2O2 1%, com atividade de 0,655 U/mL para o bagaço de cana, 0,892 U/mL para a palha de milho e 0,801 U/mL para a palha de trigo. Além disso, os resíduos tratados com H2O2 podem sofrer quatro processos de hidrólise sucessivos, com o segundo processo rendendo a maior atividade enzimática para todos os resíduos pré-tratados.