Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

Sustainable Energy Production in the United States: Life Cycle Assessment of Biofuels and Bioenergy

The United States of America is making great efforts to transform the renewable and abundant biomass resources into cost-competitive, high-performance biofuels, bioproducts, and biopower. This is the key to increase domestic production of transportation fuels and renewable energy, and reduce greenhouse gas and other pollutant emissions. This dissertation focuses specifically on assessing the life cycle environmental impacts of biofuels and bioenergy produced from renewable feedstocks, such as lignocellulosic biomass, renewable oils and fats. The first part of the dissertation presents the life cycle greenhouse gas (GHG) emissions and energy demands of renewable diesel (RD) and hydroprocessed jet fuels (HRJ). The feedstocks include soybean, camelina, field pennycress, jatropha, algae, tallow and etc. Results show that RD and HRJ produced from these feedstocks reduce GHG emissions by over 50% compared to comparably performing petroleum fuels. Fossil energy requirements are also significantly reduced. The second part of this dissertation discusses the life cycle GHG emissions, energy demands and other environmental aspects of pyrolysis oil as well as pyrolysis oil derived biofuels and bioenergy. The feedstocks include waste materials such as sawmill residues, logging residues, sugarcane bagasse and corn stover, and short rotation forestry feedstocks such as hybrid poplar and willow. These LCA results show that as much as 98% GHG emission savings is possible relative to a petroleum heavy fuel oil. Life cycle GHG savings of 77 to 99% were estimated for power generation from pyrolysis oil combustion relative to fossil fuels combustion for electricity, depending on the biomass feedstock and combustion technologies used. Transportation fuels hydroprocessed from pyrolysis oil show over 60% of GHG reductions compared to petroleum gasoline and diesel. The energy required to produce pyrolysis oil and pyrolysis oil derived biofuels and bioelectricity are mainly from renewable biomass, as opposed to fossil energy. Other environmental benefits include human health, ecosystem quality and fossil resources. The third part of the dissertation addresses the direct land use change (dLUC) impact of forest based biofuels and bioenergy. An intensive harvest of aspen in Michigan is investigated to understand the GHG mitigation with biofuels and bioenergy production. The study shows that the intensive harvest of aspen in MI compared to business as usual (BAU) harvesting can produce 18.5 billion gallons of ethanol to blend with gasoline for the transport sector over the next 250 years, or 32.2 billion gallons of bio-oil by the fast pyrolysis process, which can be combusted to generate electricity or upgraded to gasoline and diesel. Intensive harvesting of these forests can result in carbon loss initially in the aspen forest, but eventually accumulates more carbon in the ecosystem, which translates to a CO2 credit from the dLUC impact. Time required for the forest-based biofuels to reach carbon neutrality is approximately 60 years. The last part of the dissertation describes the use of depolymerization model as a tool to understand the kinetic behavior of hemicellulose hydrolysis under dilute acid conditions. Experiments are carried out to measure the concentrations of xylose and xylooligomers during dilute acid hydrolysis of aspen. The experiment data are used to fine tune the parameters of the depolymerization model. The results show that the depolymerization model successfully predicts the xylose monomer profile in the reaction, however, it overestimates the concentrations of xylooligomers.

Examining the use of Simarouba glauca Seed Oil as a Feedstock for the Production of Biodiesel using a Small Scale Model Developed in India

Simarouba glauca, a non-edible oilseed crop native to South Florida, is gaining popularity as a feedstock for the production of biodiesel. The University of Agriculture Sciences in Bangalore, India has developed a biodiesel production model based on the principles of decentralization, small scales, and multiple fuel sources. Success of such a program depends on conversion efficiencies at multiple stages. The conversion efficiency of the field-level, decentralized production model was compared with the in-laboratory conversion efficiency benchmark. The study indicated that the field-level model conversion efficiency was less than that of the lab-scale set up. The fuel qualities and characteristics of the Simarouba glauca biodiesel were tested and found to be the standards required for fuel designation. However, this research suggests that for Simarouba glauca to be widely accepted as a biodiesel feedstock further investigation is still required.

Patterns in the space use of the Bearded Reedling, Panurus biarmicus, on the Tay Reedbeds, Scotland

This thesis presents research into the space use of a specialist reedbed Passerine, the Bearded Reedling, or Bearded Tit, Panurus biarmicus, with a view to inform the conservation of this species and reedbeds as a whole. How a species uses space, and how space use changes between individuals or over time, can influence: the ability to forage and hunt effectively, breeding success, susceptibility to predation, genetic health, disease spread, robustness against environmental change and ultimately, colonisation or extinction. Thus, understanding the space use of animals can provide critical insight into ecological systems. Birds offer interesting models when studying animal space use, as, by being intrinsically mobile, many bird species can occupy multiple spatial scales. As a consequence of being completely dependent on patchy and ephemeral reedbed habitats, the Bearded Reedling, has a clustered, inhomogeneous distribution throughout its range. This drives the existence of distinct spatial scales upon which space use studies should be characterised. Distribution and movement within a single reedbed can be considered local-scale, while spatial processes between reedbeds can be considered wide-scale. Temporal processes may act upon both of these scales. For example, changing interactions with predators may influence nest positioning at a local-scale, while seasonal changes in resource requirements might drive processes such as migration at a wide-scale. The Bearded Reedling has a wide temperate breeding range, extending over much of Eurasia. On the IUCN’s red list, it is listed as ‘of least concern’, with an estimated European population between 240,000-480,000 breeding pairs. Despite its relatively favourable conservation status, its dependence on reedbed habitats drives a fragmented distribution, with populations being concentrated in small, isolated, stands. Over the last century reedbed wetlands have suffered rapid declines caused by drainage schemes undertaken to improve land for development or agriculture. Additionally, many remaining reed stands are subject to extensive commercial management to produce thatch or biofuel. Conversely, in other areas, management is driven by conservation motives which recognise the present threats to reedbeds, and aim to encourage the diversity of species associated with these habitats. As the Bearded Reedling is fundamentally linked to the quality and structure of a reed stand, understanding the space use of this species will offer information for the direct conservation of this specialist species, and for the effects of reedbed management as a whole. This thesis first presents studies of space use at a local-scale. All local-scale research is conducted at the Tay Reedbeds in eastern Scotland. Mist netting and bird ringing data are used within capture recapture models, which include an explicit spatial component, to gain insight into the abundance of the Bearded Reedling on the Tay. This abundance estimation approach suggests the Tay reedbeds are a stronghold for this species on the British Isles, and that, as a high latitude site, the Tay may have importance for range expansion. A combination of transect surveys and radio-tracking data are then used to establish the local-scale space use of this species during the breeding and autumnal seasons. These data are related to changes in the structure of reed caused by local management in the form of mosaic winter reed cutting. Results suggest that birds exploit young and cut patches of reed as foraging resources when they are available, and that old, unmanaged reed is critical for nesting and winter foraging. Further local-scale studies concern the spatial patterns in the nesting habits of this species. Mosaic reed cutting creates clear edges in a reedbed. Artificial nests placed in the Tay Reedbeds demonstrate increased nest predation rates closer to the edges of cut patches. Additionally, high predation rates become reduced as the cut reed re-grows, suggesting that reed cutting may increase accessibility of the stand to predators. As Bearded Reedling nests are uncommon and difficult to locate, the timing, site selection and structure of a sample of real nests from the Tay is then detailed. These demonstrate an early, and relatively rigid breeding onset in this species, the importance of dense, compacted reeds as nesting sites and a degree of flexibility in nest structure. Conservation efforts will also benefit from studies into wide-scale spatial processes. These may be important when establishing how colonisation events occur and when predicting the effects of climatic change. The Bearded Reedling has been traditionally considered a resident species which only occasionally undertakes wide-scale, between-reedbed, movements. Indeed, the ecology of this species suggests strict year round local residency to reedbeds, with distinct seasonal changes in diet allowing occupation of these habitats year round. The European ringing recoveries of this species, since the 1970s are investigated to better characterise the wider movements of specialist resident. These suggest residency in southern populations, but higher instances of movement than expected in more northerly regions. In these regions wide-scale movement patterns resemble those of partial regular migratory species. An understanding of local and wide-scale spatial processes can offer a strong foundation on which to build conservation strategies. This thesis aims to use studies of space use to provide this foundation for the Bearded Reedling and offer further insight into the ecology of reedbed habitats as a whole. The thesis concludes by proposing an effective strategy for the conservation management of reedbeds that will especially benefit the Bearded Reedling.

Expansion of sunflower crop production in Brazil: a survey of future trends.

Sunflower cropped area in Brazil has been showing potential possibilities to be increased in a short period of time for biofuel production. Planning the activities is one of the requirements for the success of future cropped area expansion. This requires a previous survey that identifies future trends in the transformation and rearrangement of the sunflower agro-industry sector and also identifies technological needs that may affect this process. With the objectives of identify future trends and technological needs, a value production chain was built and a questionary was applied to agents of all the sectors participating at the V National Brazilian Symposium of Sunflower and at the XVII Sunflower National Research Meeting Network. The results pointed out a strong tendency for area expansion in the next two to five years (75%); being as a secondary follow-up crop (83%) specially after soybean and top be used for biofuel (77%). The main research needs were linked with disease control, crop zoning and varietal improvement for disease resistance and high oleic oil content. Also considering the vision of and concerns regarding the future expansion and transformation of the sunflower productive complex, it is believed that the expansion is a consolidated trend, requiring a strategic sector planning associated with an economic and technological police for its success within the Brazilian agribusiness.

Agronomic performance of selected sweet potato cultivars under greenhouse and field infested with meloigogyne incognita and soilborne insect pests.

The root knot nematode (RKN), Meloidogyne incognita, is widespread worldwide and a major pathogen of several cultivated crops. The use of resistant genotypes is the most effective and environmentally sound way to manage RKN. In this study, we screened 16 selected sweet potato cultivars including Amanda, Bárbara, Beatriz, Beauregard, Brazlândia Branca, Brazlândia Rosada, Brazlândia Roxa, BRS Amélia, BRS Cuia, BRS Rubissol, Carolina Vitória, Duda, Júlia, Marcela, PA-26/2009, and Princesa obtained from Embrapa and Universidade Federal do Tocantins? germplasm bank. Studies were conducted under greenhouse and field conditions and the agronomic performance of the cultivars was evaluated in a nematode and soilborne insect-infested field. All 16 sweet potato cultivars tested were rated as resistant to this nematode both under greenhouse and field conditions with reproduction factors < 1. In the field infested with M. incognita, sweet potato cultivars Duda, BRS Amélia, Beauregard, Brazlândia Rosada, and Brazlândia Roxa stood out as superior cultivars, with average yield ranging from 26 to 47 tons per ha. Overall, most cultivars exhibited a fusiform to near fusiform root shape, a good characteristic for the market, and were moderately affected by insects (attack incidence 1 to 30%). As global demand for energy continues to rise, selecting new cultivars of sweet potatoes with increased resistance to nematode diseases and with high yield will be important for food security and biofuel production.