The chemistry of acid catalyzed delignification of sugarcane bagasse in the ionic liquid trihexyl tetradecyl phosphonium chloride

This article reports on the cleavage of lignin ß-aryl ether bonds in sugarcane bagasse by the ionic liquid (IL) trihexyl tetradecyl phosphonium chloride [P66614] Cl, in the presence of catalytic amounts of mineral acid fca. 0.4%). The deligniflcation process of bagasse was studied over a range of temperatures (120°C to 150°C) by monitoring the production of ß-ketones (indicative of cleavage of ß-aryl ethers) using FTIR spectroscopy and by compositional analysis of the undissolved fractions. Maximum deligniflcation was obtained at 150°C, with 52% of lignin removed from the original lignin content of bagasse. No deligniflcation was observed in the absence of acid, which suggests that the reaction is acid catalyzed with the IL solubilizing the lignin fragments. The rate of deligniflcation was significantly higher at 150°C, suggesting that crossing the glass transition temperature of lignin effects greater freedom of rotation about the propanoid carbon-carbon bonds and leads to increased cleavage of ß-aryl ethers. An attempt has been made to propose a probable mechanism of deligniflcation of bagasse with the phosphonuim IL. © Taylor & Francis Group, LLC.

Ionic liquid pre-treatment and fractionation of sugarcane bagasse for the production of bioethanol

Pretretament is an essential and expensive processing step for the manufacturing of ethanol from lignocellulosic raw materials. Ionic liquids are a new class of solvents that have the potential to be used as pretreatment agents. The attractive characteristics of ionic liquid pretreatment of lignocellulosics such as thermal stability, dissolution properties, fractionation potential, cellulose decrystallisation capacity and saccharification impact are investigated in this thesis. Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) at high temperatures (110 �‹C to 160 �‹C) is investigated as a pretreatment process. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 �‹C for 90 min). At these conditions, the dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is efficiently hydrolysed (93 %, 3 h, 15 FPU). At pretreatment temperatures < 150 �‹C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures . 150 �‹C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material (100 %, 3h, 15 FPU). Complete dissolution is not necessary to maximize saccharification efficiency at temperatures . 150 �‹C. Fermentation of [C4mim]Cl-pretreated, enzyme-saccharified bagasse to ethanol is successfully conducted (85 % molar glucose-to-ethanol conversion efficiency). As compared to standard dilute acid pretreatment, the optimised [C4mim]Cl pretreatment achieves substantially higher ethanol yields (79 % cf. 52 %) in less than half the processing time (pretreatment, saccharification, fermentation). Fractionation of bagasse partially dissolved in [C4mim]Cl to a polysaccharide rich and a lignin rich fraction is attempted using aqueous biphasic systems (ABSs) and single phase systems with preferential precipitation. ABSs of ILs and concentrated aqueous inorganic salt solutions are achievable (e.g. [C4mim]Cl with 200 g L-1 NaOH), albeit they exhibit a number of technical problems including phase convergence (which increases with increasing biomass loading) and deprotonation of imidazolium ILs (5 % - 8 % mol). Single phase fractionation systems comprising lignin solvents / cellulose antisolvents, viz. NaOH (2M) and acetone in water (1:1, volume basis), afford solids with, respectively, 40 % mass and 29 % mass less lignin than water precipitated solids. However, this delignification imparts little increase in saccharification rates and extents of these solids. An alternative single phase fractionation system is achieved simply by using water as an antisolvent. Regulating the water : IL ratio results in a solution that precipitates cellulose and maintains lignin in solution (0.5 water : IL mass ratio) in both [C4mim]Cl and 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc)). This water based fractionation is applied in three IL pretreatments on bagasse ([C4mim]Cl, 1-ethyl-3-methyl imidazolium chloride ([C2mim]Cl) and [C2mim]OAc). Lignin removal of 10 %, 50 % and 60 % mass respectively is achieved although only 0.3 %, 1.5 % and 11.7 % is recoverable even after ample water addition (3.5 water : IL mass ratio) and acidification (pH . 1). In addition the recovered lignin fraction contains 70 % mass hemicelluloses. The delignified, cellulose-rich bagasse recovered from these three ILs is exposed to enzyme saccharification. The saccharification (24 h, 15 FPU) of the cellulose mass in starting bagasse, achieved by these pretreatments rank as: [C2mim]OAc (83 %)>>[C2mim]Cl (53 %)=[C4mim]Cl(53%). Mass balance determinations accounted for 97 % of starting bagasse mass for the [C4mim]Cl pretreatment , 81 % for [C2mim]Cl and 79 %for [C2mim]OAc. For all three IL treatments, the remaining bagasse mass (not accounted for by mass balance determinations) is mainly (more than half) lignin that is not recoverable from the liquid fraction. After pretreatment, 100 % mass of both ions of all three ILs were recovered in the liquid fraction. Compositional characteristics of [C2mim]OAc treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are opposite to those of chloride IL treated solids. The former biomass characteristics resemble those imparted by aqueous alkali pretreatment while the latter resemble those of aqueous acid pretreatments. The 100 % mass recovery of cellulose in [C2mim]OAc as opposed to 53 % mass recovery in [C2mim]Cl further demonstrates this since the cellulose glycosidic bonds are protected under alkali conditions. The alkyl chain length decrease in the imidazolium cation of these ILs imparts higher rates of dissolution and losses, and increases the severity of the treatment without changing the chemistry involved.

Elevated uric acid correlates with wound severity

Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research. Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise through better understanding the processes involved in the formation of chronic wounds. We have for the first time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may play a critical role in the formation of chronic wounds by prolonging the inflammatory process.

ESEM study of authigenic chlorite acid sensitivity in sandstone reservoirs

The effect of HCl on authigenic chlorite in three different sandstones has been examined uisng an Environmental Scanning Electron Microscope (ESEM), together with conventional analytical techniques. The ESEM enabled chlorites to be directly observed in situ at high magnifications during HCl treatment, and was particularly effective in allowing the same chlorite areas to be closely compared before and after acid treatment. Chlorites were reacted with 1M to 10M HCl at temperatures up to 80°C and for periods up to five months. After all treatments, chlorites show extensive leaching of iron, magnesium and aluminum, and their crystalline structure is destroyed. However, despite these major compositional and structural changes, chlorites show little or no visible evidence of acid attack, with precise morphological detail of individual plates preserved in all samples following acid treatments. Chlorite dissolution, sensu stricto, did not occur as a result of acidization of the host sandstones. Acid-treated chlorides are likely to exits in a structurally weakened state that may make them susceptible to physical disintegration during fluid flow. Accordingly, fines migration may be a significant engineering problem associated with the acidization of chlorite-bearing sandstones. © 1993.

Influence of fatty acid structure on fuel properties of algae derived biodiesel

Physical and chemical properties of biofuel are influenced by structural features of fatty acid such as chain length, degree of unsaturation and branching of the chain. A simple and reliable calculation method to estimate fuel property is therefore needed to avoid experimental testing which is difficult, costly and time consuming. Typically in commercial biodiesel production such testing is done for every batch of fuel produced. In this study 9 different algae species were selected that were likely to be suitable for subtropical climates. The fatty acid methyl esters (FAMEs) of all algae species were analysed and the fuel properties like cetane number (CN), cold filter plugging point (CFPP), kinematic viscosity (KV), density and higher heating value (HHV) were determined. The relation of each fatty acid with particular fuel property is analysed using multivariate and multi-criteria decision method (MCDM) software. They showed that some fatty acids have major influences on the fuel properties whereas others have minimal influence. Based on the fuel properties and amounts of lipid content rank order is drawn by PROMETHEE-GAIA which helped to select the best algae species for biodiesel production in subtropical climates. Three species had fatty acid profiles that gave the best fuel properties although only one of these (Nannochloropsis oculata) is considered the best choice because of its higher lipid content.

Understanding mild acid pretreatment of sugarcane bagasse through particle scale modeling

Sugarcane bagasse is an abundant and sustainable resource, generated as a by-product of sugarcane milling. The cellulosic material within bagasse can be broken down into glucose molecules and fermented to produce ethanol, making it a promising feedstock for biofuel production. Mild acid pretreatment hydrolyses the hemicellulosic component of biomass, thus allowing enzymes greater access to the cellulosic substrate during saccharification. A particle-scale mathematical model describing the mild acid pretreatment of sugarcane bagasse has been developed, using a volume averaged framework. Discrete population-balance equations are used to characterise the polymer degradation kinetics, and diffusive effects account for mass transport within the cell wall of the bagasse. As the fibrous material hydrolyses over time, variations in the porosity of the cell wall and the downstream effects on the reaction kinetics are accounted for using conservation of volume arguments. Non-dimensionalization of the model equations reduces the number of parameters in the system to a set of four dimensionless ratios that compare the timescales of different reaction and diffusion events. Theoretical yield curves are compared to macroscopic experimental observations from the literature and inferences are made as to constraints on these “unknown” parameters. These results enable connections to be made between experimental data and the underlying thermodynamics of acid pretreatment. Consequently, the results suggest that data-fitting techniques used to obtain kinetic parameters should be carefully applied, with prudent consideration given to the chemical and physiological processes being modeled.

Uric acid and xanthine oxidoreductase in wound healing

Chronic wounds are an important health problem because they are difficult to heal and treatment is often complicated, lengthy and expensive. For a majority of sufferers the most common outcomes are long-term immobility, infection and prolonged hospitalisation. There is therefore an urgent need for effective therapeutics that will enhance ulcer healing and patient quality of life, and will reduce healthcare costs. Studies in our laboratory have revealed elevated levels of purine catabolites in wound fluid from patients with venous leg ulcers. In particular, we have discovered that uric acid is elevated in wound fluid, with higher concentrations correlating with increased wound severity. We have also revealed a corresponding depletion in uric acid precursors, including adenosine. Further, we have revealed that xanthine oxidoreductase, the enzyme that catalyses the production of uric acid, is present at elevated levels in wound fluid. Taken together, these findings provide evidence that xanthine oxidoreductase may have a function in the formation or persistence of chronic wounds. Here we describe the potential function of xanthine oxidoreductase and uric acid accumulation in the wound site, and the effect of xanthine oxidoreductase in potentiating the inflammatory response.

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

An investigation into the production of chloromethly furfual from bagasse and bagasse pulp

There are many attractive alternatives to produce chemicals similar to those currently produced from fossil fuel resources. The most viable renewable resource of fixed carbon is biomass. This paper examines processing conditions for the production and recovery of furanics from bagasse as well as bagasse pulp. It is shown that bio-oil consisting mainly of furanics (~84% chloromethly furfural) may be obtained in yields of ~78% and ~87% by weight from bagasse and bagasse pulp respectively using a biphasic acid hydrolysis system. The biphasic system consists of an organic layer of dichloroethane and an aqueous phase of concentrated hydrochloric acid. Generally the lower the impurity content and the higher the cellulose content, the higher the furanics yield.

Evaluation of a pilot-scale oil extraction from microalgae for biodiesel production

Biodiesel derived from microalgae is one of a suite of potential solutions to meet the increasing demand for a renewable, carbon-neutral energy source. However, there are numerous challenges that must be addressed before algae biodiesel can become commercially viable. These challenges include the economic feasibility of harvesting and dewatering the biomass and the extraction of lipids and their conversion into biodiesel. Therefore, it is essential to find a suitable extraction process given these processes presently contribute significantly to the total production costs which, at this stage, inhibit the ability of biodiesel to compete financially with petroleum diesel. This study focuses on pilot-scale (100 kg dried microalgae) solvent extraction of lipids from microalgae and subsequent transesterification to biodiesel. Three different solvents (hexane, isopropanol (IPA) and hexane + IPA (1:1)) were used with two different extraction methods (static and Soxhlet) at bench-scale to find the most suitable solvent extraction process for the pilot-scale. The Soxhlet method extracted only 4.2% more lipid compared to the static method. However, the fatty acid profiles of different extraction methods with different solvents are similar, suggesting that none of the solvents or extraction processes were biased for extraction of particular fatty acids. Considering the cost and availability of the solvents, hexane was chosen for pilot-scale extraction using static extraction. At pilot-scale the lipid yield was found to be 20.3% of total biomass which is 2.5% less than from bench scale. Extracted fatty acids were dominated by polyunsaturated fatty acids (PUFAs) (68.94±0.17%) including 47.7±0.43 and 17.86±0.42% being docosahexaenoic acid (DHA) (C22:6) and docosapentaenoic acid (DPA) (C22:5, ω-3), respectively. These high amounts of long chain poly unsaturated fatty acids are unique to some marine microalgae and protists and vary with environmental conditions, culture age and nutrient status, as well as with cultivation process. Calculated physical and chemical properties of density, viscosity of transesterified fatty acid methyl esters (FAMEs) were within the limits of the biodiesel standard specifications as per ASTM D6751-2012 and EN 14214. The calculated cetane number was, however, significantly lower (17.8~18.6) compared to ASTM D6751-2012 or EN 14214-specified minimal requirements. We conclude that the obtained microalgal biodiesel would likely only be suitable for blending with petroleum diesel to a maximum of 5 to 20%.

Biodiesel production from non-edible beauty leaf (Calophyllum Inophyllum) oil : process optimization using response surface methodology (RSM)

In recent years, the beauty leaf plant (Calophyllum Inophyllum) is being considered as a potential 2nd generation biodiesel source due to high seed oil content, high fruit production rate, simple cultivation and ability to grow in a wide range of climate conditions. However, however, due to the high free fatty acid (FFA) content in this oil, the potential of this biodiesel feedstock is still unrealized, and little research has been undertaken on it. In this study, transesterification of beauty leaf oil to produce biodiesel has been investigated. A two-step biodiesel conversion method consisting of acid catalysed pre-esterification and alkali catalysed transesterification has been utilized. The three main factors that drive the biodiesel (fatty acid methyl ester (FAME)) conversion from vegetable oil (triglycerides) were studied using response surface methodology (RSM) based on a Box-Behnken experimental design. The factors considered in this study were catalyst concentration, methanol to oil molar ratio and reaction temperature. Linear and full quadratic regression models were developed to predict FFA and FAME concentration and to optimize the reaction conditions. The significance of these factors and their interaction in both stages was determined using analysis of variance (ANOVA). The reaction conditions for the largest reduction in FFA concentration for acid catalysed pre-esterification was 30:1 methanol to oil molar ratio, 10% (w/w) sulfuric acid catalyst loading and 75 °C reaction temperature. In the alkali catalysed transesterification process 7.5:1 methanol to oil molar ratio, 1% (w/w) sodium methoxide catalyst loading and 55 °C reaction temperature were found to result in the highest FAME conversion. The good agreement between model outputs and experimental results demonstrated that this methodology may be useful for industrial process optimization for biodiesel production from beauty leaf oil and possibly other industrial processes as well.

Development and characterization of a novel, antimicrobial, sterile hydrogel dressing for burn wounds : single-step production with gamma irradiation creates silver nanoparticles and radical polymerization

Patients with burn wounds are susceptible to wound infection and sepsis. This research introduces a novel burn wound dressing that contains silver nanoparticles (SNPs) to treat infection in a 2-acrylamido-2-methylpropane sulfonic acid sodium salt (AMPS-Na(+) ) hydrogel. Silver nitrate was dissolved in AMPS-Na(+) solution and then exposed to gamma irradiation to form SNP-infused hydrogels. The gamma irradiation results in a cross-linked polymeric network of sterile hydrogel dressing and a reduction of silver ions to form SNPs infused in the hydrogel in a one-step process. About 80% of the total silver was released from the hydrogels after 72 h immersion in simulated body fluid solution; therefore, they could be used on wounds for up to 3 days. All the hydrogels were found to be nontoxic to normal human dermal fibroblast cells. The silver-loaded hydrogels had good inhibitory action against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Results from a pilot study on a porcine burn model showed that the 5-mM silver hydrogel was efficient at preventing bacterial colonization of wounds, and the results were comparable to the commercially available silver dressings (Acticoat(TM) , PolyMem Silver(®) ). These results support its use as a potential burn wound dressing.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.