The electron optical limits of performance of single-polepiece magnetic electron lenses

The thesis is concerned with the electron properties of single-polepiece magnetic electron lenses especially under conditions of extreme polepiece saturation. The electron optical properties are first analysed under conditions of high polepiece permeability. From this analysis, a general idea can be obtained of the important parameters that affect ultimate lens performance. In addition, useful information is obtained concerning the design of improved lenses operating under conditions of extreme polepiece saturation, for example at flux densities of the order of 10 Tesla. It is shown that in a single-polepiece lens , the position and shape of the lens exciting coil plays an important role. In particular, the maximum permissible current density in the windings,rather than the properties of the iron, can set a limit to lens performance. This factor was therefore investigated in some detail. The axial field distribution of a single-polepiece lens, unlike that of a conventional lens, is highly asymmetrical. There are therefore two possible physical arrangements of the lens with respect to the incoming electron beam. In general these two orientations will result in different aberration coefficients. This feature has also been investigated in some detail. Single-pole piece lenses are thus considerably more complicated electron- optically than conventional double polepiece lenses. In particular, the absence of the usual second polepiece causes most of the axial magnetic flux density distribution to lie outside the body of the lens. This can have many advantages in electron microscopy but it creates problems in calculating the magnetic field distribution. In particular, presently available computer programs are liable to be considerably in error when applied to such structures. It was therefore necessary to find independent ways of checking the field calculations. Furthermore, if the polepiece is allowed to saturate, much more calculation is involved since the field distribution becomes a non-linear function of the lens excitation. In searching for optimum lens designs, care was therefore taken to ensure that the coil was placed in the optimum position. If this condition is satisfied there seems to be no theoretical limit to the maximum flux density that can be attained at the polepiece tip. However , under iron saturation condition, some broadening of the axial field distribution will take place, thereby changing the lens aberrations . Extensive calculations were therefore made to find the minimum spherical and chromatic aberration coefficients . The focal properties of such lens designs are presented and compared with the best conventional double-polepiece lenses presently available.

The design of gas chromatography ionisation detectors:a study of the electron capture detector and the home ionisation detector

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The influence of defects on the electron-transfer and magnetic properties of RbxMn[Fe(CN)6]y·zH2O

The synthesis and detailed characterization of a few samples of the compound RbMn[Fe(CN)]·zHO are described. The composition of the materials significantly depends on the applied preparative conditions. Analysis of spectroscopic results (FTIR, Raman, Fe Mössbauer, XPS) and X-ray powder-diffraction data yielded a further assessment of the difference in structural features in terms of the amount of Fe(CN)6 vacancies and the associated number of water molecules. The characteristic individual magnetic behavior, as well as the metal-to-metal charge-transfer capabilities of the various samples, could be related to significant changes within the structures that appear to be associated with the synthetic method used.

On chlorosulphonation and the electron microscopy of polyethylene

It is shown that chlorosulphonation is a major aid to the electron microscopy of polyethylene for various samples which had mostly been crystallized at high pressures and included at least a proportion of the so-called chain-extended form. It is confirmed that sheets of excess electron density are produced at lamellar surfaces, but also including lateral surfaces. This is due primarily to the incorporation of chlorine and sulphur rather than to added uranium. The time to achieve an overall reaction varies sensitively with morphology, decreasing as the number of diffusion channels increases. Crystallinity is gradually lost, but sufficient crystals remain when a sample has become uniform, and in their initial orientations, for diffraction studies to be possible. The technique has been used to demonstrate that, during melt crystallization, the thickness of one lamella changes in response to altered growth conditions. This is direct confirmation that lamellar thickness is determined by secondary nucleation at the growth front. The tapered profile of a growing lamella previously observed in thick crystals of various polymers has been observed for chain-folded polyethylene lamellae, providing further evidence that this is a general feature of melt growth. © 1977 Chapman and Hall Ltd.

Investigation of the effects of low energy high dose ion bombardment in metals and compound semiconductors

The effect of low energy nitrogen molecular ion beam bombardment on metals and compound semiconductors has been studied, with the aim to investigate at the effects of ion and target properties. For this purpose, nitrogen ion implantation in aluminium, iron, copper, gold, GaAs and AIGaAs is studied using XPS and Angle Resolve XPS. A series of experimental studies on N+2 bombardment induced compositional changes, especially the amount of nitrogen retained in the target, were accomplished. Both monoenergetic implantation and non-monoenergetic ion implantation were investigated, using the VG Scientific ESCALAB 200D system and a d. c. plasma cell, respectively. When the samples, with the exception of gold, are exposed to air, native oxide layers are formed on the surfaces. In the case of monoenergetic implantation, the surfaces were cleaned using Ar+ beam bombardment prior to implantation. The materials were then bombarded with N2+ beam and eight sets of successful experiments were performed on each sample, using a rastered N2+ ion beam of energy of 2, 3, 4 and 5 keV with current densities of 1 μA/cm2 and 5 μA/cm22 for each energy. The bombarded samples were examined by ARXPS. After each complete implantation, XPS depth profiles were created using Ar+ beam at energy 2 ke V and current density 2 μA/cm2 . As the current density was chosen as one of the parameters, accurate determination of current density was very important. In the case of glow discharge, two sets of successful experiments were performed in each case, by exposing the samples to nitrogen plasma for the two conditions: at low pressure and high voltage and high pressure and low voltage. These samples were then examined by ARXPS. On the theoretical side, the major problem was prediction of the number of ions of an element that can be implanted in a given matrix. Although the programme is essentially on experimental study, but an attempt is being made to understand the current theoretical models, such as SATVAL, SUSPRE and TRIM. The experimental results were compared with theoretical predictions, in order to gain a better understanding of the mechanisms responsible. From the experimental results, considering possible experimental uncertainties, there is no evidence of significant variation in nitrogen saturation concentration with ion energy or ion current density in the range of 2-5 ke V, however, the retention characteristics of implantant seem to strongly depend on the chemical reactivity between ion species and target material. The experimental data suggests the presence of at least one thermal process. The discrepancy between the theoretical and experimental results could be the inability of the codes to account for molecular ion impact and thermal processes.

Seeing and believing: three episodes in the history of neurophysiology

In this contribution I look at three episodes in the history of neurophysiology that bring out the complex relationship between seeing and believing. I start with Vesalius in the mid-sixteenth century who writes that he can in no way see any cavity in nerves, even in the optic nerves. He thus questions the age-old theory (dating back to the Alexandrians in the third century BC) but, because of the overarching psychophysiology of his time, does not press his case. This conflict between observation and theory persisted for a quarter of a millennium until finally resolved at the beginning of the nineteenth century by the discoveries of Galvani and Volta. The second case is provided by the early history of retinal synaptology. Schultze in 1866 had represented rod spherules and bipolar dendrites in the outer plexiform layer as being separated by a (synaptic) gap, yet in his written account, because of his theoretical commitments, held them to be continuous. Cajal later, 1892, criticized Schultze for this pusillanimity, but his own figure in La Cellule is by no means clear. It was only with the advent of the electron microscopy in the mid-twentieth century that the true complexity of the junction was revealed and it was shown that both investigators were partially right. My final example comes from the Hodgkin-Huxley biophysics of the 1950s. Their theory of the action potential depended on the existence of unseen ion pores with quite complex biophysical characteristics. These were not seen until the Nobel-Prize-winning X-ray diffraction analyses of the early twenty-first century. Seeing, even at several removes, then confirmed Hodgkin and Huxley’s belief. The relation between seeing and believing is by no means straightforward.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Development of a sediment yield model for South East Ghana in the regions around Lake Volta

Soil erosion is one of the most pressing issues facing developing countries. The need for soil erosion assessment is paramount as a successful and productive agricultural base is necessary for economic growth and stability. In Ghana, a country with an expanding population and high potential for economic growth, agriculture is an important resource; however, most of the crop production is restricted to low technology shifting cultivation agriculture. The high intensity seasonal rainfall coincides with the early growing period of many of the crops meaning that plots are very susceptible to erosion, especially on steep sided valleys in the region south of Lake Volta. This research investigated the processes of soil erosion by rainfall with the aim of producing a sediment yield model for a small semi-agricultural catchment in rural Ghana. Various types of modelling techniques were considered to discover those most applicable to the sub-tropical environment of Southern Ghana. Once an appropriate model had been developed and calibrated, the aim was to look at how to enable the scaling up of the model using sub-catchments to calculate sedimentation rates of Lake Volta. An experimental catchment was located in Ghana, south west of Lake Volta, where data on rainstorms and the associated streamflow, sediment loads and soil data (moisture content, classification and particle size distribution) was collected to calibrate the model. Additional data was obtained from the Soil Research Institute in Ghana to explore calibration of the Universal Soil Loss Equation (USLE, Wischmeier and Smith, 1978) for Ghanaian soils and environment. It was shown that the USLE could be successfully converted to provide meaningful soil loss estimates in the Ghanaian environment. However, due to experimental difficulties, the proposed theory and methodology of the sediment yield model could only be tested in principle. Future work may include validation of the model and subsequent scaling up to estimate sedimentation rates in Lake Volta.

Ion plating

Under ideal conditions ion plating produces finely grained dense coatings with excellent adhesion. The ion bombardment induced damage initiates a large number of small nuclei. Simultaneous coating and sputtering stimulates high rates of diffusion and forms an interfacial region of graded composition responsible for good adhesion. To obtain such coatings on components far industrial applications, the design and construction Of an ion plater with a 24" (O.6rn) diameter chamber were investigated and modifications of the electron beam gun were proposed. A 12" (O.3m) diameter ion plater was designed and constructed. The equipment was used to develop surfaces for solar energy applications. The conditions to give extended surfaces by sputter etching were studied. Austenitic stainless steel was sputter etched at 20 and 30 mTorr working pressure and at 3, 4 and 5 kV. Uniform etching was achieved by redesigning the specimen holder to give a uniform electrostatic field over the surfaces of the specimens. Surface protrusions were observed after sputter etching. They were caused by the sputter process and were independent of grain boundaries, surface contaminants and inclusions. The sputtering rate of stainless steel was highly dependent on the background pressure which should be kept below 10-5 Torr. Sputter etching improved the performance of stainless steel used as a solar selective surface. A twofold improvement was achieved on sputter etching bright annealed stainless steel. However, there was only slight improvement after sputter etching stainless steel which had been mechanically polished to a mirror finish. Cooling curves Were used to measure the thermal emittance of specimens.The deposition rate of copper was measured at different levels of power input and was found to be a maximum at 9.5 kW. The diameter of the copper feed rod was found to be critical for the maintenance of a uniform evaporation rate.

Spray dried combinations of lactoferrin with antibiotics appear superior to monotherapy for reducing biofilm formation by pseudomonas aeruginosa

SD Apo Lactoferrin-Tobramycin/Gentamicin Combinations are superior to monotherapy in the eradication of Pseudomonas aeruginosa Biofilm in the lungs Wilson Oguejiofor1, Lindsay J. Marshall1, Andrew J. Ingham1, Robert Price2, Jag. Shur2 1School of Life and Health Sciences, Aston University, Birmingham, UK. 2School of Pharmacy and Pharmacology, University of Bath, Bath, UK. KEYWORDS: lactoferrin, apo lactoferrin, spray drying, biofilm, cystic fibrosis Introduction Chronic lung infections from the opportunistic pathogeen Pseudomonas aeruginosa has been recognised as a major contributor to the incidences of high morbidity and mortality amongst cystic fibrosis (CF) patients (1,2). Currently, strategies for managing lung infections in CF patients involves the aggressive use of aerosolised antibiotics (3), however, increasing evidence suggests that the biofilm component of P. aeruginosa in the lower airway remains unperturbed and is associated with the development of antibiotic resistance. If this is so then, there is an urgent need to suitably adjust the current treatment strategy so that it includes compounds that prevent biofilm formation or disrupt established biofilms. It is well understood that biofilm formation is strongly dependent on iron (Fe3+) availability (4), therefore aerosolised anti-infective formulations which has the ability to chelate iron may essentially be a well suited therapy for eliminating P. aeruginosa biofilms on CF airway epithelial cells (5). In this study, we report the use of combination therapy; an aminoglycosides (tobramycin and gentamicin) and an antimicrobial peptide (lactoferrin) to significantly deplete P. aeruginosa biofilms. We demonstrate that lactoferrin-tobramycin and lactoferrin-gentamicin combinations are superior to the single antibiotic regime currently being employed to combat P. aeruginosa biofilms. MATERIALS AND METHOD Antibiotics: The antibiotics used in this study included gentamicin and tobramycin supplied by Fagron, UK. Bacterial strain and growth conditions: Pseudomonas aeruginosa strain PAO1 was provided by Prof. Peter Lambert of Aston University, Birmingham UK. The Strains were routinely grown from storage in a medium supplemented with magnesium chloride, glucose and casamino acids. Dialysis of lactoferrin: Apo lactoferrin was prepared by dialyzing a suspension of lactoferrin for 24 hrs at 4 °C against 20 mmol/L sodium dihydrogen phosphate, 20 mmol/L sodium acetate and 40 mmol/L EDTA (pH 3.5). Ferric ion (Fe3+) removal was verified by atomic absorption spectroscopy measurements. Spray drying of combinations of lactoferrin and apo lactoferrin with the different aminoglycosides: Combinations of tobramycin and gentamicin with the different preparations of lactoferrin were spray dried (SD) as a 2% (w/v) aqueous suspension. The spray drying parameters utilized for the production of suitable micron-sized particles includes: Inlet temperature, 180°C, spray flow rate, 606 L/hr; pump setting, 10%; aspirator setting, 85% (34m3/hr) to produce various outlet temperatures ranging from 99 - 106°C. Viability assay: To test the bactericidal activity of the various combinations, a viability assay was performed as previously described by Xu, Xiong et al. (6) with some modifications. Briefly, 10µL of ~ c. 6.6 x 107 CFU mL-1 P. aeruginosa strain PAO1 suspension were incubated (37°C, 60 mins) with 90 µL of a 2 µg/mL concentration of the various combinations and sampled every 10 mins. After incubation, the cells were diluted in deionised water and plated in Mueller hinton agar plates. Following 24 h incubation of the plates at 37°C, the percentage of viable cells was determined relative to incubation without added antibiotics. Biofilm assay: To test the susceptibility of the P. aeruginosa strain to various antibiotics in the biofilms mode of growth, overnight cultures of P. aeruginosa were diluted 1:100 into fresh medium supplemented with magnesium chloride, glucose and casamino acids. Aliquots of the dilution were dispensed into a 96 well dish and incubated (37°C, 24 h). Excess broth was removed and the number of colony forming units per milliliter (CFU/mL) of the planktonic bacteria was quantified. The biofilms were then washed and stained with 0.1% (w/v) crystal violet for 15 mins at room temperature. Following vigorous washing with water, the stained biofilms were solubilized in 30% acetic acid and the absorbance at 550nm of a 125 µL aliquot was determined in a microplate reader (Multiskan spectrum, Thermo Scientific) using 30% acetic acid in water as the blank. Aliquots of the broth prior to staining were used as an indicator of the level of planktonic growth. RESULTS AND DISCUSSION Following spray drying, the mean yield, volume weighted mean diameter and moisture content of lactoferrin powder were measured and were as follows (Table 1 and table 2); Table 1: Spray drying parameters FormulationInlet temp (°C)Outlet temp (°C)Airflow rate (L/hr)Mean yield (%)Moisture content (%) SD Lactoferrin18099 - 10060645.2 ±2.75.9 ±0.4 SD Apo Lactoferrin180100 - 10260657.8 ±1.85.7 ±0.2 Tobramycin180102 - 10460682.1 ±2.23.2 ±0.4 Lactoferrin + Tobramycin180104 - 10660687.5 ±1.43.7 ±0.2 Apo Lactoferrin + Tobramycin180103 - 10460676.3 ±2.43.3 ±0.5 Gentamicin18099 - 10260685.4 ±1.34.0 ±0.2 Lactoferrin + Gentamicin180102 - 10460687.3 ±2.13.9 ±0.3 Apo Lactoferrin + Gentamicin18099 -10360680.1±1.93.4 ±0.4 Table 2: Particle size distribution d10 d50d90 SD Lactoferrin1.384.9111.08 SD Apo Lactoferrin1.284.7911.04 SD Tobramycin1.254.9011.29 SD Lactoferrin + Tobramycin1.175.2715.23 SD Apo Lactoferrin + Tobramycin1.115.0614.31 SD Gentamicin1.406.0614.38 SD Lactoferrin + Gentamicin1.476.2314.41 SD Apo Lactoferrin + Gentamicin1.465.1511.53 The bactericidal activity of the various combinations were tested against P. aeruginosa PAO1 following a 60 minute incubation period (Figure 1 and Figure 2). While 2 µg/mL of a 1:1 combination of spray dried apo lactoferrin and Gentamicin was able to completely kill all bacterial cells within 40 mins, the same concentration was not as effective for the other antibiotic combinations. However, there was an overall reduction of bacterial cells by over 3 log units by the other combinations within 60 mins. Figure 1: Logarithmic plot of bacterial cell viability of various combinations of tobramycin and lactoferrin preparations at 2µg/mL (n = 3). Figure 2: Logarithmic plot of bacterial cell viability of various combinations of gentamicin and lactoferrin preparations at 2µg/mL (n = 3). Crystal violet staining showed that biofilm formation by P. aeruginosa PAO1 was significantly (ANOVA, p < 0.05) inhibited in the presence of the different lactoferrin preparations. Interestingly, apo lactoferrin and spray dried lactoferrin exhibited greater inhibition of both biofilm formation and biofilm persistence (Figure 2). Figure 2: Crystal violet staining of residual biofilms of P. aeruginosa following a 24hr incubation with the various combinations of antibiotics and an exposure to 48 hr formed biofilms. CONCLUSION In conclusion, combination therapy comprising of an antimicrobial peptide (lactoferrin) and an aminoglycosides (tobramycin or gentamicin) provides a feasible and alternative approach to monotherapy since the various combinations are more efficient than the respective monotherapy in the eradication of both planktonic and biofilms of P. aeruginosa. ACKNOWLEDGEMENT The authors would like to thank Mr. John Swarbrick and Friesland Campina for their generous donation of the Lactoferrin. REFERENCES 1.Hassett, D.J., Sutton, M.D., Schurr, M.J., Herr, A.B., Caldwell, C.C. and Matu, J.O. (2009), "Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways". Trends in Microbiology, 17, 130-138. 2.Trust, C.F. (2009), "Antibiotic treatment for cystic fibrosis". Report of the UK Cystic Fibrosis Trust Antibiotic Working Group. Consensus document. London: Cystic Fibrosis Trust. 3.Garcia-Contreras, L. and Hickey, A.J. (2002), "Pharmaceutical and biotechnological aerosols for cystic fibrosis therapy". Advanced Drug Delivery Reviews, 54, 1491-1504. 4.O'May, C.Y., Sanderson, K., Roddam, L.F., Kirov, S.M. and Reid, D.W. (2009), "Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions". J Med Microbiol, 58, 765-773. 5.Reid, D.W., Carroll, V., O'May, C., Champion, A. and Kirov, S.M. (2007), "Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis". European Respiratory Journal, 30, 286-292. 6.Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y. and Xiong, S. (2010), "Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa". J Appl Microbiol, 109, 1311-1318.

The effect of antifoam addition on protein production yields

Pichia pastoris is a widely used host for recombinant protein production. The foaming associated with culturing it on a large scale is commonly prevented by the addition of chemical antifoaming agents or "antifoams." Unexpectedly, the addition of a range of antifoams to both shake flask and bioreactor cultures of P. pastoris has been shown to alter the total yield of the recombinant protein being produced. Possible explanations for this are that the presence of the antifoam increases the total amount of protein being produced and secreted per cell or that it increases the density of the culture. Antifoaming agents may therefore have specific effects on the growth and yield characteristics of recombinant cultures, in addition to their primary action as de-foamers.

A comparative study on the pyrolysis of metal- and ash-enriched wood and the combustion properties of the gained char

This study aims to investigate the pyrolysis behaviour of metal-contaminated wood and the combustion properties of char derived from wood pyrolysis. Seven metals (Na, Mg, Ca, Zn, Cd, Pb and Fe(III)) were introduced to willow in cation form by ion-exchange and the thermal behaviour of demineralised samples and samples with additional ash were also investigated. The results show that the char yield increased from 21% to 24-28% and levoglucosan yield in vapour phase decreased from 88% to 62-29% after the addition of inorganic compounds, even though the metal binding capacity of wood varied from one metal ion to another. While char yield seems to be effected mainly by the concentration of the metal ions, levoglucosan yield was more dependent on the ionic species especially when sodium ions were present. When combustion experiments were carried out with char made of the metal enriched wood, two consecutive steps were observed, both effected by the presence of inorganic compounds. The first step was identified as the release and combustion of volatiles, while the second peak of the burning profile is the actual combustion of the fixed carbon. The burnout temperatures, estimated ignition indices and the conversion indicate that the type and not the amount of metal ions were the determining factors during the second step of combustion. © 2012 Published by Elsevier B.V.