Thesis (doctoral)--Helsinki University of Technology, 2004. / Includes bibliographical references. Also available on the World Wide Web.

Th. doct.-ing.--Nancy, I.N.P.L., 1982.

There is increasing interest in producing biofuels; biofuels are preferable to fossil fuels as the biomass from which they are derived is seen as a renewable source, as opposed to fossil fuels which are a finite resource. “First Generation” biofuels are derived from food crops such as grains and sugar cane. The use of food crops is not sustainable in this age of increasing food insecurity. A promising alternative appears to be what is termed “Second Generation” feedstocks, such as energy crops like Miscanthus spp., and agricultural by-products. The problem with the use of second generation feedstocks is firstly that the sugars are locked up in the cell wall polymers (CWP), which need to be released by physio-chemical pre-treatments, that are costly and time consuming. The second problem is that not all the sugars that are released from CWP are able to be utilised by wild type product-forming organisms. However, model chassis organisms can be genetically modified to utilise these sugars and /or produce enzymes to degrade biomass which reduces the time and costs involved in the process. While engineering these organisms to utilise a range of monosaccharides has already been successful, engineering them to produce degradation enzymes is proving to be problematic. A potentially more effective system is to use co-cultures of both cellulose-degrading and product-forming organisms. Since this is a novel approach it is not known whether the two organisms are able to live together without any adverse effects. The aims of this study were firstly to determine whether mixed cultures of both cellulose-degrading and potential product-forming organisms could survive in the presence of one another, secondly whether the cellulose-degrading organisms could degrade potential feedstock down into their monosaccharide building blocks and thirdly whether the potential product-forming organisms could survive and utilise these monosaccharides for growth and potential fermentation. It was discovered that C. hutchinsonii can degrade both paper and Triticum aestivum straw polymers into their monosaccharide components and that B. subtilis can survive on the sugars released by C. hutchinsonii. It was also discovered that C. hutchinsonii and B. subtilis 168 can only tolerate an ethanol concentration of up to 2% (v/v) and that this is below the baseline for a biofuel system to be economically viable. Likewise, C. hutchinsonii and B. subtilis 168 have an even poorer tolerance for butanol; growth is inhibited by < 1% butanol in its growth media. A series of physio-chemical pre-treatments were developed in order to make the monosaccharides present in the cell wall polymers more accessible to microbial saccharification. Sequential pre-treatments, both physical milling and chemical hydrolysis in tandem, had the greatest effect on the bio chemistry of the biomass, but that these physio-chemical pre-treatments produced inhibitory compounds in the medium that retarded microbial growth. Attempts were made to genetically modified Bacillus subtilis 168 to produce lactic acid and ethanol by over expressing the native ldh gene under the highly-expressed promoter of the cspD gene and by integrating the fused pdc:adh gene from Z. mobilis under the same promoter. Transformation of B. subtilis to over express LDH was successful, with PCR confirmation of the correct insertion and enzyme activity for the ldh both in vitro and in vivo, with the latter producing more lactic acid aerobically than the wild type. Transformation of B. subtilis to express pdc:adh and subsequent production of ethanol was not successful.

Cellulosic thermoplastics are anticipated as promising replacements to petroleum-based thermoplastics, but their high manufacturing costs have limited wide-spread application. The primary objectives of this thesis were to use low-cost lignocellulose, practically forestry waste, as the raw material rather than more expensive purified cellulose in the preparation of new plastics and, consequently, to develop an economical reactive process focused on diminishing the use of expensive solvents in the thermoplasticization of lignocellulose. The thermoplasticization of lignocellulosic fibers started by developing a high solids content (60 wt%) twin-screw extrusion technique to defibrillate the raw material for the subsequent chemical modification. By this approach, the received lignocellulosic fibers showed improving handling as a feedstock for extrusion as well as chemical accessibility. To effectively wet the lignocellulosic fibers for chemical modification and avoid using expensive and largely ineffective solvents, a low-cost additive was derived by mimicking aspects of an ionic liquid using benzethonium chloride (hyamine) and sulfuric acid. The effectiveness of the hyamine/sulfuric acid wetting agent was demonstrated initially in a bench-top method where the additive also became chemically bonded to the lignocellulose and strongly contributed to its thermoplasticity. During acetylation, this new and low-cost wetting/functionalizing agent converted the lignocellulosic fibers into a compression-moldable thermoplastic. The molar ratio of benzethonium chloride to sulfuric acid was found to be the most significant variable to determine grafting behaviour as well as degradation of the polymer chains. Subsequently, this new modification chemistry was translated over to the environment of a twin-screw extruder to devise a continuous, greener method of thermoplasticization for lignocellulose. The new reactive extrusion process had a short reaction time of 45-90 s and yet showed a good tendency for producing a flowable thermoplastic suitable for melt molding without plasticizers. A notable benefit to the method was the moldable lignocellulosic bioplastic maintained the excellent stiffness inherent to cellulose. Moreover, by the reactive extrusion method, the properties of the lignocellulosic thermoplastics were found to be tunable with the selected esterifying agents (butyric anhydride versus acetyl anhydride) and the molar ratio of benzethonium chloride to sulfuric acid. A statistical analysis based on a Design of Experiment method revealed details on desirable extrusion conditions. The project concluded with improvements to the high solids-content process was exploring the novel concept of a recycle stream for reactive extrusion. The excessive esterifying agent content used in the initial studies was necessary to lubricate the fibrous mass inside the extruder else it would jam the process. This meant that the extrudate left the extruder with an unnecessary amount of reactant and required costly cleaning. The idea of recycling a portion of the newly made cellulosic thermoplastic was to add a natural lubricant and thereby lower the content of the esterifying agent in the extruder. Under optimal recycling conditions, a significant 50% decline in reactant was possible without decreasing the degree of modification or harming the thermoplasticity of the modified lignocellulose. / Thesis / Doctor of Philosophy (PhD) / Wood biomass is the most abundant renewable material on the planet and comprises polymer chains like plastics. These polymer chains in wood biomass are locked by strong bonds, which limits their mobility. It is for this reason that wood biomass can not “melt” like commercial plastics such as polyethylene, and thus limits its application in the manufacture of objects with complex shapes. Using chemical modifiers to react with the wood biomass can unlock those bonds among the chains and convert it into a “meltable” thermoplastic. The current preparation of thermoplastics from wood biomass is very costly because of using expensive purified cellulose and solvents to assist the reaction. This thesis describes the development of an economical reactive process for converting less purified wood biomass into thermoplastics. It used low-cost lignocellulose, practically forestry waste, and discovered a low-cost but effective reaction method for using less expensive reactants. Finally, a rapid mechanically assisted reaction process (called reactive extrusion) was adopted based on the new chemistry to convert the lignocellulose biomass with significantly fewer reactants than needed in a batch system.

Thesis (Ph.D.)--Baylor University, 2007. / Includes bibliographical references (p. 127-135).

Thesis (M.S.)--Baylor University, 2009. / Includes bibliographical references (p. 54-61).

A significant trend in the modern day industrial biotechnology is the utilization and application of renewable resources, and ecofriendly approach to industrial processes and waste management. As a consequence thereof, the biotechnology of holocellulases: cellulase and xylanase and, enzymatic hydrolysis of renewable and abundant lignocellulosic biomass to energy and value added products are rapidly increasing; hence, cost effective enzyme system is imperative. In that context, exploration of microbiota for strains and enzymes with novel industrial properties is vital for efficient and commercially viable enzyme biotechnology. Consequent on the complex characteristics of high salinity, variable pressure, temperature and nutritional conditions, bacterial strains from the marine environment are equipped with enzyme machinery of industrial importance for adaptation and survival. In this study, bacterial strains were isolated form Nahoon beach and optimized for holocellulase production. Three isolates selected for lignocellulolytic potential were identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequence analysis. Isolate FS1k had 98 percent similarity with Streptomyces albidoflavus strain AIH12, was designated as Streptomyces albidoflavus strain SAMRC-UFH5 and deposited in the GenBank with accession number KU171373. Similarly, isolates CS14b and CS22d with respective percentage similarity of 98 and 99 (percent) with Bacillus cereus strains and Streptomyces sp. strain WMMB251 were named Bacillus cereus strain SAMRC-UFH9 and Streptomyces sp. strain SAMRC-UFH6; and were deposited in the GenBank with accession number KX524510 and KU171374 respectively. Optimal pH, temperature and agitation speed for cellulase production by S. albidoflavus strain SAMRC-UFH5, and B. cereus strain SAMRC-UFH9 were 6 and 7; 40 and 30 (°C); and 100 and 150 (rpm) respectively; while xylanase production was optimal at pH, temperature and agitation speed of 8 and 7; 40 and 30 (°C); and 150 and 50 (rpm) respectively. Maximum cellulase activity of 0.26 and 0.061(U/mL) by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 were attained at 60 h respectively, while maximal xylanase activity of 18.54 and 16.6 (U/mL) was produced by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 at 48 h and 60 h respectively. Furthermore, xylanase production by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 was maximally induced by wheat straw and xylan respectively, while cellulase production was best induced by mannose and carboxymethyl cellulose respectively. On the other hand, cellulase and xylanase production by Streptomyces sp. strain SAMRC-UFH6 was optimal at pH, temperature and agitation speed of 7 and 8, 40 °C and 100 rpm, respectively. Highest production of cellulase and xylanase was attained at 84 and 60 h with respective activity of 0.065 and 6.34 (U/mL). In addition, cellulase and xylanase production by the strain was best induced by beechwood xylan. Moreover, xylanase produced by Streptomyces sp. strain SAMRC-UFH6 at optimal conditions was characterized by optimal pH and temperature of 8 and 80-90 °C respectively; retaining over 70 percent activity at pH 5-10 after 1 h and 60 percent of initial activity at 90 °C after 90 min of incubation. In all, optimization improved cellulase and xylanase production yields, being 40 and 95.5, 10.89 and 72.17, and 10 and 115- fold increase by S. albidoflavus strain SAMRC-UFH5, B. cereus strain SAMRC-UFH9 and Streptomyces sp. SAMRC-UFH6 respectively. The results of this study suggest that the marine bacterial strains are resource for holocellulase with industrial applications.

At present, the world's transportation sector is being principally supplied by fossil fuels. However, energy consumption in this sector is drastically increasing and there are concerns with supply, cost, and environmental issues with the continuing use of fossil fuels. Utilizing non-petroleum ethanol in the transportation sector reduces the dependence on oil, and allows for cleaner burning of gasoline. Lignocellulose materials are structurally composed of five types of polymeric sugars, glucan, galactan, mannan, arabinan, and xylan. NREL has developed a quantitative saccharification (QS) method for determining carbohydrate composition. We proposed a new protocol based on the NREL 2006 Laboratory Analytical Procedure "Determination of Structural Carbohydrates and Lignin in Biomass" (Sluiter et al. 2006a) with a slight modification, in which xylose concentration was determined after the secondary hydrolysis by using 1% sulfuric acid rather than 4% sulfuric acid. We found that the current NREL protocol led to a statistically significant overestimation of acid-labile xylan content ranging from 4 to 8 percent. Lignocellulosic biomass is naturally recalcitrant to enzymatic hydrolysis, and must be pretreated before it can be effectively used for bioethanol production. One such pretreatment is a fractionation process that separates lignin and hemicellulose from the cellulose and converts crystalline cellulose microfibrils to amorphous cellulose. Here we evaluated the feasibility of lignocellulose fractionation applicable to the hurds of industrial hemp. Hurds are the remaining material of the stalk after all leaves, seeds, and fiber have been stripped from the plant. After optimizing acid concentration, reaction time and temperature, the pretreated cellulosic samples were hydrolyzed to more than 96% after 24 hours of hydrolysis (enzyme loading conditions of 15 FPU/g glucan Spezyme CP and 60 IU/g glucan Novozyme 188) at the optimal pretreatment condition (> 84% H₃PO₄, > 50 °C and > 1 hour). The overall glucose and xylose yields were 89% (94% pretreatment; 96% digestibility) and 61%, respectively. All data suggest the technical feasibility of building a biorefinery based on the hurds of industrial hemp as a feedstock and a new lignocellulose fractionation technology for producing cellulosic ethanol. The choice of feedstock and processing technology gives high sugar yields, low processing costs, low cost feedstock, and low capital investment. / Master of Science

The depletion of fossil fuel reserves and global warming are the two main factors contributing to the current demand in clean and renewable energy resources. Biofuels are renewable energy resources and have an advantage over other renewable resources due to biofuels having a zero carbon footprint and most feedstock is abundant. The use of biofuels brought about major concerns and these include food, water and land security. The use of lignocellulose as bioethanol feedstock can provide a solution to the food, water and security concerns. Biofuels such as bioethanol can be produced from lignocellulose by breaking down the structure of lignocellulose liberating fermentable sugars. Amaranth lignocellulose has a potential to be used as a feedstock for bioethanol production because amaranth plants has a high yield of biomass per hectare, require very little to no irrigation and have the ability to withstand harsh environmental conditions. The aim of this study was to investigate the viability of amaranth as a feedstock for bioethanol production by using alkaline assisted microwave pretreatment. Alkaline pretreatment of amaranth using Ca(OH)2, NaOH and KOH at various concentrations (10-50 g kg-1 of alkaline solution in water) was carried out at different energy input (6-54 kJ/g). The pretreated broth was enzymatically hydrolysed using Celluclast 1.5L, Novozyme 188 and Tween 80 at pH 4.8 and 50oC for 48 hours. The hydrolysate was further fermented to ethanol using Saccharomyces cerevisiae at a pH of 4.8 and 30oC for 48 hours. The effect of microwave pretreatment on amaranth lignocellulose was evaluated using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The monomeric sugars and ethanol were quantified using high performance liquid chromatography (HPLC). A maximum sugar yield of 0.36 g/g of biomass was obtained for pretreatment with 30 g kg-1 Ca(OH)2 solution in water, 0.24 g/g of biomass was obtained for pretreatment with 50 g kg-1 NaOH solution in water and 0.21g/g of biomass was obtained for pretreatment with 50 g kg-1 KOH solution in water at 32 kJ/g of energy input. After enzymatic hydrolysis the yields increased to 0.43 g/g, 0.63 g/g and 0.52 g g-1 of biomass for Ca(OH)2 , KOH and NaOH pretreated biomass respectively. The highest ethanol yield obtained was found to be 0.18 g/g of biomass from fermentation of KOH pretreated broth. The ethanol yield obtained from fermentation of Ca(OH)2 and NaOH pretreated broth was 0.13 g/g of biomass and 0.15 g/g of biomass respectively. The results showed that an increase in concentration of alkaline solution and an increase in energy input liberate more sugars. A decrease in biomass loading was found to increase the total sugar yield. Pretreatment with KOH was found to liberate more pentose sugars than the other alkaline solutions. The morphological changes shown by the SEM images showed that microwave irradiation is effective in breaking the structure of amaranth lignocellulose. The structural changes shown by the FTIR also validated that alkaline bases were effective in breaking the lignin, cellulose and hemicellulose linkages and liberating more sugars in the process. This work has demonstrated the enormous potential that amaranth lignocellulose has on being a feedstock for bioethanol production. / MSc (Engineering Sciences in Chemical Engineering), North-West University, Potchefstroom Campus, 2014

The depletion of fossil fuel reserves and global warming are the two main factors contributing to the current demand in clean and renewable energy resources. Biofuels are renewable energy resources and have an advantage over other renewable resources due to biofuels having a zero carbon footprint and most feedstock is abundant. The use of biofuels brought about major concerns and these include food, water and land security. The use of lignocellulose as bioethanol feedstock can provide a solution to the food, water and security concerns. Biofuels such as bioethanol can be produced from lignocellulose by breaking down the structure of lignocellulose liberating fermentable sugars. Amaranth lignocellulose has a potential to be used as a feedstock for bioethanol production because amaranth plants has a high yield of biomass per hectare, require very little to no irrigation and have the ability to withstand harsh environmental conditions. The aim of this study was to investigate the viability of amaranth as a feedstock for bioethanol production by using alkaline assisted microwave pretreatment. Alkaline pretreatment of amaranth using Ca(OH)2, NaOH and KOH at various concentrations (10-50 g kg-1 of alkaline solution in water) was carried out at different energy input (6-54 kJ/g). The pretreated broth was enzymatically hydrolysed using Celluclast 1.5L, Novozyme 188 and Tween 80 at pH 4.8 and 50oC for 48 hours. The hydrolysate was further fermented to ethanol using Saccharomyces cerevisiae at a pH of 4.8 and 30oC for 48 hours. The effect of microwave pretreatment on amaranth lignocellulose was evaluated using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The monomeric sugars and ethanol were quantified using high performance liquid chromatography (HPLC). A maximum sugar yield of 0.36 g/g of biomass was obtained for pretreatment with 30 g kg-1 Ca(OH)2 solution in water, 0.24 g/g of biomass was obtained for pretreatment with 50 g kg-1 NaOH solution in water and 0.21g/g of biomass was obtained for pretreatment with 50 g kg-1 KOH solution in water at 32 kJ/g of energy input. After enzymatic hydrolysis the yields increased to 0.43 g/g, 0.63 g/g and 0.52 g g-1 of biomass for Ca(OH)2 , KOH and NaOH pretreated biomass respectively. The highest ethanol yield obtained was found to be 0.18 g/g of biomass from fermentation of KOH pretreated broth. The ethanol yield obtained from fermentation of Ca(OH)2 and NaOH pretreated broth was 0.13 g/g of biomass and 0.15 g/g of biomass respectively. The results showed that an increase in concentration of alkaline solution and an increase in energy input liberate more sugars. A decrease in biomass loading was found to increase the total sugar yield. Pretreatment with KOH was found to liberate more pentose sugars than the other alkaline solutions. The morphological changes shown by the SEM images showed that microwave irradiation is effective in breaking the structure of amaranth lignocellulose. The structural changes shown by the FTIR also validated that alkaline bases were effective in breaking the lignin, cellulose and hemicellulose linkages and liberating more sugars in the process. This work has demonstrated the enormous potential that amaranth lignocellulose has on being a feedstock for bioethanol production. / MSc (Engineering Sciences in Chemical Engineering), North-West University, Potchefstroom Campus, 2014