Conversion of lignocellulosic biomass via hydrolysis of cellulose to simple sugars has failed to achieve economic competitiveness to produce renewable fuels and chemicals partly due to the inherent recalcitrance of the substrate and partly due to the use of non-recyclable catalysts. Solid acids have been proposed for cellulose hydrolysis as a recyclable alternative to enzymes and homogeneous acids. However, their catalytic mechanism has not been elucidated partly due to incomplete structural characterization. We focused on elucidating the structure of chloromethyl polystyrene based catalysts which exhibit remarkable activity towards hydrolyzing cellulose. By carrying out spatially resolved analysis of CMP-SO3H-0.3, a catalyst decorated with benzyl chloride and benzyl sulfonic acid groups, we discovered that the external surface of the catalyst is devoid of any chloride groups, which were hypothesized to interact with cellulose. Despite apparent greater reactivity than sulfonated-only catalysts, we found the CMP-SO3H-0.3 reacts with water at the reaction conditions used for cellulose hydrolysis, resulting in leaching of homogeneous hydrochloric acid, which in turn is responsible for the observed cellulose hydrolysis. Building on these results we investigated whether catalysts from various structural classes are stable in the hydrothermal environment or leach homogeneous acid. Surprisingly, we discovered that materials commonly used for cellulose hydrolysis are hydrothermally unstable and the leached homogeneous acid they produced was responsible for their apparent catalytic activity. On the other hand, hydrothermally stable materials did not exhibit greater hydrolysis activity than water. Cellulose crystallinity has been theorized for decades as a structural parameter determining the reactivity of cellulose, which motivated decrystallization pretreatment processes. However, water-induced recrystallization had not been accounted for in hydrolysis models, albeit being a well-documented phenomenon, and all hydrolysis processes use water as a reaction medium. By carrying out detailed structure-reactivity analysis we concluded that decrystallized cellulose undergoes a rapid transformation to an active crystalline cellulose, characterized by allomorphs I and II and greater content of surface polymer chains. Water-induced recrystallization reduced the reactivity of cellulose and prevented conversion of highly reactive amorphous regions. To circumvent the recrystallization pathway, we used ethanolysis as a means for rapid and selective depolymerization of amorphous cellulose. Ethanolysis of ball-milled cellulose for 30 minutes at 410 K resulted in 38% conversion, while hydrolysis at the same conditions in only 15%. Scission-relaxation caused recrystallization and limited conversion via ethanolysis. By using co-solvents capable of swelling cellulose, we were able to increase cellulose conversion to 48%. The results presented in those studies can guide future development of catalysts and depolymerization processes that circumvent the inhibiting effects caused by the use of water.

In order to produce ethanol derived from lignocellulosic feeds at a cost that is competitive with current gasoline prices, the fermentation process, converting sugars to produce ethanol and the subsequent purification steps, must be enhanced. Due to their comparatively lower costs, the widespread availability across a range of climates, and their status as a dedicated energy crop, lignocellulosic biomass feeds are ideal raw materials that can be used to produce domestic fuels to partly displace our dependence on non-renewable sources. Currently, a major drawback of the technology is the relatively low ethanol tolerance of the micro-organisms used to ferment xylose and glucose. To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124) during fermentation, online ethanol sequestration was achieved through the implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal (using F-600 activated carbon). By removing ethanol from the broth during the fermentation, inhibition due to the presence of ethanol could be alleviated, enhancing the substrate utilization and fermentation rate and the ethanol production of the fermentation. This study details a comprehensive adsorbent screening to identify ethanol selective materials, modelling of multi-component adsorption systems, and the design, implementation and modelling of a fermentation unit coupled with an externally located packed bed adsorber.

In order to produce ethanol derived from lignocellulosic feeds at a cost that is competitive with current gasoline prices, the fermentation process, converting sugars to produce ethanol and the subsequent purification steps, must be enhanced. Due to their comparatively lower costs, the widespread availability across a range of climates, and their status as a dedicated energy crop, lignocellulosic biomass feeds are ideal raw materials that can be used to produce domestic fuels to partly displace our dependence on non-renewable sources. Currently, a major drawback of the technology is the relatively low ethanol tolerance of the micro-organisms used to ferment xylose and glucose. To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124) during fermentation, online ethanol sequestration was achieved through the implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal (using F-600 activated carbon). By removing ethanol from the broth during the fermentation, inhibition due to the presence of ethanol could be alleviated, enhancing the substrate utilization and fermentation rate and the ethanol production of the fermentation. This study details a comprehensive adsorbent screening to identify ethanol selective materials, modelling of multi-component adsorption systems, and the design, implementation and modelling of a fermentation unit coupled with an externally located packed bed adsorber.

In order to produce ethanol derived from lignocellulosic feeds at a cost that is competitive with current gasoline prices, the fermentation process, converting sugars to produce ethanol and the subsequent purification steps, must be enhanced. Due to their comparatively lower costs, the widespread availability across a range of climates, and their status as a dedicated energy crop, lignocellulosic biomass feeds are ideal raw materials that can be used to produce domestic fuels to partly displace our dependence on non-renewable sources. Currently, a major drawback of the technology is the relatively low ethanol tolerance of the micro-organisms used to ferment xylose and glucose. To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124) during fermentation, online ethanol sequestration was achieved through the implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal (using F-600 activated carbon). By removing ethanol from the broth during the fermentation, inhibition due to the presence of ethanol could be alleviated, enhancing the substrate utilization and fermentation rate and the ethanol production of the fermentation. This study details a comprehensive adsorbent screening to identify ethanol selective materials, modelling of multi-component adsorption systems, and the design, implementation and modelling of a fermentation unit coupled with an externally located packed bed adsorber.

In order to produce ethanol derived from lignocellulosic feeds at a cost that is competitive with current gasoline prices, the fermentation process, converting sugars to produce ethanol and the subsequent purification steps, must be enhanced. Due to their comparatively lower costs, the widespread availability across a range of climates, and their status as a dedicated energy crop, lignocellulosic biomass feeds are ideal raw materials that can be used to produce domestic fuels to partly displace our dependence on non-renewable sources. Currently, a major drawback of the technology is the relatively low ethanol tolerance of the micro-organisms used to ferment xylose and glucose. To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124) during fermentation, online ethanol sequestration was achieved through the implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal (using F-600 activated carbon). By removing ethanol from the broth during the fermentation, inhibition due to the presence of ethanol could be alleviated, enhancing the substrate utilization and fermentation rate and the ethanol production of the fermentation. This study details a comprehensive adsorbent screening to identify ethanol selective materials, modelling of multi-component adsorption systems, and the design, implementation and modelling of a fermentation unit coupled with an externally located packed bed adsorber.

>Magister Scientiae - MSc / Lignocellulosic biomass, a waste component of the agricultural industry, is a promising source for use in bioethanol production. Due to a complex structure, the synergistic action of lignocellulosic enzymes is required to achieve complete digestion to fermentable sugars. This study aimed to isolate, identify and characterise novel lignocellulase producing bacteria from thermophilic straw-based compost (71°C). Colonies with different morphological characteristics were isolated and screened for lignocellulosic activity. A facultative aerobic isolate RZ1 showed xylanase, cellulase and lipase/esterase activity. In addition to these activities, it was also able to produce proteases, catalases, amylases and gelatinases. RZ1 cells were motile, rod-shaped, Gram positive and endospore forming. The growth temperature of isolate RZ1 ranged from 25-55°C with optimal growth at 37°C. The 16S rRNA gene sequence was 99% identical to that of Bacillus subtilis strain MSB10. Based on the biochemical and physiological characteristics and 16S rRNA gene sequence, isolate RZ1 is considered a member of the species B. subtilis. A small insert genomic library with an average insert size of 5 kb was constructed and screened for lignocellulosic activity. An E.coli plasmid clone harbouring a 4.9 kb gDNA fragment tested positive for xylanase activity. The xyl R gene was identified with the aid of transposon mutagenesis and the deduced amino acid sequence showed 99% similarity to an endo-1-4-β-xylanase from B. pumilus. High levels of xylanases were produced when isolate RZ1 was cultured (37°C) with beechwood xylan as a carbon source. On the other hand, the production of xylanases was inhibited in the presence of xylose. Marked xylanase activity was measured in the presence of sugarcane bagasse, a natural lignocellulosic substrate. While active at 50°C, higher xylanase activity was detected at 37°C. Isolate RZ1 also produced accessory enzymes such as β-xylosidases and α-L-arabinofuranosidases, able to hydrolyse hemicellulose.

The rising price of oil is hurting consumers all over the world. There is growing interest in producing biofuels from non-food crops, such as sugarcane trash. Lignocellulosic biomass (e.g., sugarcane trash) is an abundant, inexpensive, and renewable resource. The patented MixAlco process is a cost-effective solution, which does not require sterility or the addition of expensive enzymes to convert lignocellulosic biomass to transportation fuels and valuable chemicals. In this study, the MixAlco process was used to convert sugarcane trash to carboxylic acids under thermophilic conditions. Lime-treated sugarcane trash (80%) and chicken manure (20%) was used as the feedstock in rotary 1-L fermentors. Ammonium bicarbonate buffer was used to mitigate the effects of product (carboxylic acid) inhibition. Marine inoculum was used because of the high adaptability of the mixed culture of microorganisms present. Iodoform solution was added to inhibit methanogenesis. Preliminary batch studies over a 20-day period produced 19.7 g/L of carboxylic acids. Sugarcane trash had the highest average yield (0.31 g total acid/g VS fed) and highest average conversion (0.70 g VS digested/g VS fed) among the three substrates compared. Countercurrent fermentations were performed at various volatile solid loading rates (VSLR) and liquid residence times (LRT). The highest acid productivity of 1.40 g/(L�d) was at a total acid concentration of 29.9 g/L. The highest conversion and yield were 0.64 g VS digested/g VS fed and 0.36 g total acid/g VS fed, respectively. The continuum particle distribution model (CPDM) was used to predict acid concentration at various VSLR and LRT. The average error in between the predicted and experimental acid concentration and conversion were 4.62% and 1.42%, respectively. The effectiveness of several pretreatment methods was evaluated using the CPDM method. The best-performing method was short-term, no-wash, oxidative lime pretreatment with ball milling. At an industrial-scale solids loading of 300 g VS/L liquid, the CPDM ?map? predicts a total acid concentration of 64.0 g/L at LRT of 30 days, VSLR of 7 g/(L�d), and conversion of 57%. Also high conversion of 76% and high acid concentration of 52 g/L are achieved at a VSLR of 4 g/(L�d) and LRT of 30 days.

>Magister Scientiae - MSc / Biofuels are currently recognised as the most viable source of energy to replace depleting fossil fuel reserves, with bioethanol the most popular alternative alcohol fuel. Producing bioethanol from agricultural waste residues is a feasible socio-economic industrial process. Lignocellulose, from which plant material is composed, is highly recalcitrant to enzymatic degradation and therefore requires a suite of enzymes for complete hydrolysis of the biomass. Metagenomes, particularly from extreme environments, represent an unlimited resource for the discovery of novel biocatalysts for inclusion in industrial processes. Here we report on the cloning and functional characterisation of a novel thermophilic cellulase identified by the functional screening of a Malawian, hotspring sediment metagenomic library. The gene encoding the cellulase, celMHS, composed of 2,705 nucleotides and encoded a polypeptide of 905 amino acids with a predicted molecular mass of about 98 kDa. The in silico translated protein, CelMHS, contained a putative transmembrane domain, a family 4 carbohydrate binding motive (CBM 4), a truncated glycoside hydrolase family 42 (GH42) domain and a N-terminal region that does not have sequence similarity to any previously described domains. Functional characterisation of the recombinant CelMHS demonstrated that the protein displayed an optimal pH of 6.0 and temperature of 100°C. CelMHS had high specific activity toward substrates comprising of β-1,4 linked glucose subunits such as carboxymethyl cellulose, β-D-glucan from barley and lichenan, however, some activity was also observed against avicel, a crystalline cellulose substrate. HPLC analysis of the hydrolysis products produced by CelMHS indicates that this particular enzyme prefers longer chain oligosaccharides. This is, to the best of our knowledge, the first investigation describing the cloning and characterization of a carbohydrate hydrolysing enzyme comprised of the unique sequence architecture: a partial GH42 catalytic domain, a CBM 4 and a unique N-domain sequence. Key words: cellulose, cellulases, lignocellulosic biomass, bioethanol, saccharification, hydrolysis, metagenomic library, thermophilic

Oleaginous microbes can accumulate over 20% of their cell dry weight as lipids that are stored as intracellular energy reserves. The characterization of other oleaginous bacteria creates opportunities for the development of alternative feedstocks and technologies. Rhodococcus rhodochrous is a gram-positive bacterium known for its biodegradation capabilities, but little is known about its ability to accumulate lipids. As R. rhodochrous is capable of degrading hydrocarbon gasses and other aromatics, this study aims to investigate any associated lipid production during the conversion of waste and nontraditional carbon sources, such as model lignocellulosic inhibitors. Lignocellulosic biomass is the most abundant and renewable organic material in the world and is composed of cellulose, hemicellulose, and lignin, which can be pretreated to release sugars from the complex, and often recalcitrant, lignin polymer for microbial fermentation. R. rhodochrous was cultivated with various carbon sources, including glucose, xylose, acetic acid, furfural, phenol, vanillic acid, hydroxybenzoic acid, and propane. The results suggest that R. rhodochrous can survive in the presence of these compounds, achieving almost 7g/L cell dry weight after 168 hours and still accumulate up to 40-50% of cell dry weight as lipid in glucose supplemented media. Furthermore, the aromatic compounds are undetected after 48 hours indicating that R. rhodochrous was able to tolerate these compounds and accumulate lipids. Fatty acid methyl ester profiles show a prevalence of palmitic and oleic methyl esters. Overall, these studies are contributing to a better understanding and characterization of another oleaginous Rhodococcus species, Rhodococcus rhodochrous.

Production of liquid fuels is a high national priority to provide transporation fuels. Production of liquid biouels from biomass has been idenfied as a viable goal over the next decades. Fast pyrolysis is the rapid thermal degradation of lignocellulosic biomass in the absence of oxygen. Levoglucosan, which can be hydrolyzed and fermented into bio-ethanol, is produced during the pyrolysis process of the cellulose contained in biomass. Pure cellulose results in the production of levoglucosan of more than 50% by feedstock weight while woody biomass typically produces about 3% during pyrolysis. Researchers have performed significant research into methods to increase yields of levoglucosan and other associated anhydrosugars during pyrolysis. Most research has focused on mild acid pretreatment of biomass feedstocks prior to pyrolysis. Such treatment demineralizes and removes hemicellulose that appears to hinder the production of levoglucosan during pyrolysis. The objective of this study is to move beyond simple acid pretreatment to increase pyrolytic anhydrosugars yields during fast pyrolysis.