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The ultimate objective of this research is the elucidation of the mechanism of the resorcinol-formaldehyde condensation. Whilst a vast literature has been built up about the phenol- formaldehyde condensation, that of resorcinol has received but scant attention, probably because the high reactivity of resorcinol rendered the following of the condensation extremely difficult. The earliest observation concerning the resorcinol-formaldehyde condensation was due to Baeyer who, in 1872, condensed resorcinol with various aldehydes. When the aldehyde was relatively inactive crystalline compounds were obtalned but with acetaldehyde or formaldehyde a resinous product resulted. In 1892 Caro condensed an excess ot resorcinol with formaldehyde in the presence of dilute hydrochloric acid; the product obtained recrystallised from alcohol as white microsoopic crystals, decomposed without melting at 250° and was stated to be bis (2- 4 dihydroxyphenyl) methane ... In decidlng upon the most suitable lines of attack it would seem that a survey of the literature relating to the phenol-formaldehyde concentration which might be expected to be closely related to that of resorcinol, would be of value. Summary of p. 1-5

by Hon-chiu Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Bibliography: leaves 122-126. / Abstract --- p.i / Acknowledgement --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / Introduction --- p.1 / Literature Review --- p.4 / Chapter 1) --- Ethanol production in bacteria / Chapter 1.1) --- Zymomonas mobilis --- p.4 / Chapter 1.2) --- Clostridium species --- p.7 / Chapter 1.3) --- "Enterobacter, Klebsiella, Serritia and Erwinia sp" --- p.9 / Chapter 1.4) --- Escherichia coli and Salmonella typhimurium --- p.10 / Chapter 2) --- Pyruvate decarboxylase of Z. mobilis / Chapter 2.1) --- Enzyme properties --- p.13 / Chapter 2.2) --- Cloning and expression of pdc gene --- p.15 / Chapter 3) --- Alcohol dehydrogenase (adh) gene / Chapter 3.1) --- "Cloning, chararterization and expression of adh genes" --- p.17 / Chapter 4) --- Gene transfer systems in Vibrio species --- p.21 / Chapter 5) --- Rationale and objectives of this study --- p.22 / Chapter Part I) --- Ethanol Production in terrestrial enteric bacteria / Chapter A) --- Introduction --- p.24 / Chapter B) --- Materials and Methods / Chapter 1) --- Bacterial strains and plasmids --- p.25 / Chapter 2) --- Media --- p.26 / Chapter 3) --- Solutions --- p.27 / Chapter 4) --- Isolation of plasmids / Chapter 4.1) --- Small Scale Isolation of plasmids --- p.30 / Chapter 4.2) --- Large Scale Isolation of plasmids --- p.32 / Chapter 5) --- Construction of a broad-host-range plasmid harbouring Zymomonas mobilis genes --- p.35 / Chapter 6) --- Transformation --- p.35 / Chapter 7) --- High Performance Liquid Chromatography of Organic Acids --- p.36 / Chapter 8) --- Maintenace of plasmids harbouring the genes of Zymomonas mobilis genes --- p.38 / Chapter 9) --- Ethanol tolerance of S. typhimurium strains --- p.38 / Chapter C) --- Results / Chapter 1) --- Construction of Salmonella typhimurium strains harbouring Z. mobilis genes --- p.39 / Chapter 2) --- Fermentative end products in culture medium --- p.48 / Chapter 3) --- Growth of hosts and transformants --- p.61 / Chapter 4) --- Ethanol tolerance of S. typhimurium strains --- p.65 / Chapter 5) --- Maintenance of plasmids --- p.67 / Chapter 6) --- Construction of broad-host-range plasmid harbouring Z. mobilis genes --- p.69 / Chapter D) --- Discussions / Chapter 1) --- Comparison of ethanol production in Escherichia coli and Salmonella typhimurium --- p.72 / Chapter 2) --- Ethanol tolerance of S. typhimurium strains --- p.74 / Chapter 3) --- Maintenance of plasmids --- p.76 / Chapter 4) --- Construction of broad-host-range plasmids harbouring Z. mobilis genes --- p.78 / Chapter Part II) --- Ethanol Production in marine enteric bacteria / Chapter A) --- Introduction --- p.79 / Chapter B) --- Materials and Methods / Chapter 1) --- Bacterial strains and plasmids --- p.80 / Chapter 2) --- Media --- p.80 / Chapter 3) --- Solutions --- p.80 / Chapter 4) --- Routine Identification Processes --- p.81 / Chapter 5) --- Systematic studies by Arbitrarily- Primed Polymerase Chain Reaction --- p.86 / Chapter 6) --- Optimal growth conditions --- p.88 / Chapter 7) --- Isolation of broad-host-range plasmid pIOl ( 64kb) --- p.89 / Chapter 8) --- Transformation of Vibrio sp. strain 60 --- p.90 / Chapter 9) --- Production of ethanol using different carbon sources in fermentation --- p.91 / Chapter C) --- Results / Chapter 1) --- Identification of Vibrio sp. strain 60 --- p.92 / Chapter 2) --- Optimal growth conditions --- p.101 / Chapter 3) --- Isolation of high molecular weight plasmid --- p.105 / Chapter 4) --- Ethanol production from different carbon sources --- p.107 / Chapter 5) --- Ethanol tolerance of Vibrio sp. strain 60 --- p.109 / Chapter 6) --- Salt tolerance of Vibrio sp. strain 60 --- p.111 / Chapter 7) --- Transformation of Vibrio sp. strain 60 --- p.113 / Chapter D) --- Discussions / Chapter 1) --- Strain identification by arbitrarily-primed PCR --- p.116 / Chapter 2) --- Isolation of high molecular weight plasmid --- p.118 / Chapter 3) --- Ethanol production of Vibrio sp. strain60 --- p.120 / References --- p.122

This thesis focuses on the effects of lactobacilli and their end-products, lactic acid and acetic acid, on 'Saccharomyces cerevisiae' growth and fermentation, and on antimicrobials used to manage such contaminants. To assess the effects of the bacteria, normal gravity (22-24 g/100 ml dissolved solids) wheat mashes inoculated with yeast at ~106 colony forming units (CFU)/ml were deliberately infected (coinoculated) with each of five industrially important strains of lactobacilli at ~10 5, ~106, ~107, ~10 8, and ~109 CFU/ml. Controls with yeast alone or with bacteria alone (~107 CFU/ml) were included. End-products, yeast growth and fermentation rates were monitored. Results indicated that production of lactic acid by lactobacilli and suspected competition of the bacteria with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and decreases in final ethanol yield. A chemically defined minimal medium was used to determine the effects of added acetic and lactic acid, and their mode of action on two strains of ' S. cerevisiae'. The effects of these two acids on yeast intracellular pH (pHi), plasma membrane H+-ATPase activity and on the plasma membrane lipid composition were studied. It was found that the specific growth rates ([mu]) of the two yeast strains decreased exponentially (R2 > 0.9) as the concentrations of acetic or lactic acid were increased. Acetic and lactic acids synergistically reduced the specific growth rate of yeast. Acetic acid caused the yeast cell to expend ATP to pump out excess protons that result from the passive diffusion of the acid into the cell at medium pH (pHe) followed by its dissociation within the cell as a result of higher pHi. Lactic acid (0.5 % w/v) caused intracellular acidification (which could lead to arrest in glycolytic flux) as a result of a significant decrease (P = 0.05) in the plasma membrane H +-ATPase activity. Moreover, the plasma membrane fluidity was reduced due to decrease in unsaturated fatty acyl residues. Among the antimicrobials studied, urea hydrogen peroxide (UHP) was superior compared to stabilized chlorine dioxide and nisin, but its bactericidal activity was greatly affected by the presence of particulate matter. When used near 30 mmoles/L (in unclarified mash), in addition to its bactericidal effect, UHP provided near optimum levels of assimilable nitrogen and oxygen that aided in vigorous yeast fermentation. This process was patented.

As an important chemical raw material, alcohols can be used as fuels, solvents and chemical feedstocks to produce a variety of downstream products. With limited fossil fuel resources, alcohol synthesis from syngas reactions can be a potential alternative to the traditional petroleum based alcohol synthesis. Among many catalysts active for syngas to alcohol processes, alkali promoted Mo2C has shown promising performance. More interestingly, the alkali promoter was found to play an important role in shifting the reaction selectivity from hydrocarbons to alcohols. However, limited understanding of the mechanism of this alkali promoter effect is available due to the complexity of syngas reaction mechanism and low content of alkali added to the catalysts. In this thesis, we performed a comprehensive investigation of the alkali promoter effect with density functional theory (DFT) calculations as our primary tool. We first examine various Mo2C surfaces to determine a representative surface structure active to alkali adsorption. On this particular surface, we develop a syngas reaction network including relevant reaction mechanisms proposed in previous literature. With energetics derived from DFT calculations and a BEP relation, we predict the syngas reaction selectivity and find it to be in excellent agreement with experimental results. The dominant reaction mechanism and selectivity determining steps are determined from sensitivity analysis. We also propose a formation mechanism of alkali promoters on Mo2C catalysts that shows consistency between experimental IR and DFT computed vibrational frequencies. Finally, the effect of alkali promoters on the selectivity determining steps for syngas reactions are investigated from DFT calculations and charge analysis. We are able to rationalize the role of alkali promoters in shifting the reaction selectivity from hydrocarbons to alcohols on Mo2C catalysts.

The use of biofuels as an alternative to gasoline in the transportation sector is seen by policy makers as an important strategy to reduce global greenhouse gas emissions. Biobutanol is one such biofuel that is gathering increasing attention in the biofuel community, because of its preferable fuel qualities over bioethanol. However, despite increasing research into biobutanol production, the thermochemical route for biobutanol production has not been adequately studied in the peer-reviewed literature. In light of this motivation, this thesis considers the design, and economic and environmental assessment of thermochemical plants for producing second and third generation biobutanol. In addition, the potential for using process intensification technology such as dividing wall columns (DWC) in place of conventional distillation columns is also investigated as a way to improve thermochemical biobutanol plants. As a first step, a novel thermochemical plant for producing second generation biobutanol is developed. Detailed economic analysis of this plant show that it is competitive with gasoline under certain process, and market conditions. The designed plant is then extended, with some modifications, to evaluate the economic and environmental potential of a thermochemical plant for producing third generation biobutanol from macroalgae. It was concluded from the results that the thermochemical route is preferable for producing second generation biobutanol over third generation biobutanol. The novel thermochemical plant design is then updated by using a kinetic model of a pilot-scale demonstrated catalyst to represent the critical mixed alcohol synthesis reaction step. This change allows optimal unreacted syngas recycle configurations for maximizing butanol yield to be established. Furthermore, integrating a DWC, designed using a methodology developed in the thesis, into the updated thermochemical plant leads to additional plant improvements. Overall, the work carried out in this thesis demonstrates that the thermochemical route is a viable option for producing second generation biobutanol. / Thesis / Doctor of Philosophy (PhD)

The nucleophilic addition of organomagnesium/lithium reagents to aldehydes and ketones has long enabled the synthesis of valuable alcohol derivatives; however, these types of transformations are often plagued by poor functional group tolerance and require harsh reaction conditions. The direct coupling of carbonyls and alcohols with aryl halides is an appealing alternative to access secondary alcohol products. However, this necessitates a formal C-H bond activation which is not well established in the literature. Chapter 1 provides a detailed literature background of the transition metal-catalyzed functionalization of carbonyls and alcohols. The work discussed in Chapter 2 of this thesis demonstrates the addition of aryl halides to aryl and aliphatic aldehydes and alcohols providing secondary alcohol products in moderate to high yields. Key to the success of this transformation was the implementation of underexplored and readily synthesized 1,5-diaza-3,7-diphosphacyclooctane (P2N2) ligands. Chapter 3 extends the methodology established in chapter 2 and aims to get a preliminary understanding of the application and mechanism of the reaction described above. For this purpose, pharmaceutically relevant isatin substrates are derivatized, providing access to substitution at the 3-position. Coupling isatins with aryl halides yields 3-aryl-3-hydroxy-2-oxindole products which are scaffolds for many natural product derivatives. Through high-throughput experimentation (HTE), we were able to discover that 1,2-addition at the carbonyl position of isatins is highly compatible with our established system and led us to develop a modest scope as well as gain useful mechanistic insights for this coupling.

Higher alcohol synthesis (HAS) from syngas or biomass-derived syngas is an important process for the production of oxygenate fuels, fuel additives and other intermediates for valueded chemical feedstock to produce medicine, cosmetics, lubricants, detergents, and polyesters. Chapter I reviews biomass to liquid fuels technology, higher alcohols being used as alternative fuels and fuel additives, the historical perspective and commercial status of higher alcohols, the catalyst system and the reaction mechanism for HAS from syngas. Chapter II discusses the Zn-Mn promoted Cu-Fe based catalyst that was synthesized by the co-precipitation method. The reaction temperature has been tested to study the influence on the catalytic performance. The maximal CO conversion rate was 72%, and the yield of alcohol and hydrocarbon was also very high. Cu was the active site for alcohol synthesis, iron carbide was the active site for olefin and paraffin synthesis. The reaction mechanism of HAS from syngas over Zn-Mn promoted Cu-Fe based catalyst was proposed. Chapter III documents the three-dimensionally ordered macroporous (3DOM) Cu-Fe catalyst developed using a glyoxylate route colloidal crystal template method. The high intrinsic activity was ascribed to three factors. First, the unique ordered structure has a large pore size and interconnected macroporous tunnels of the catalyst with a large accessible surface area to improve the catalytic activity. Second, a high density of uniformly distributed defective Cu0 and Fe5C2 nanoparticles derived from the glyoxylate route helps to provide abundant, active, and stable dual sites. Third, atomic steps on the Cu surface, induced by planar defects and lattice strain, serve as high-activity oxygenation sites. Active Fe5C2 chain-growth sites intimately surround the defective and strained form of the Cu surface, which results in a synergetic effect between the active and stable Cu–FexCy dual site for HAS. In Chapter IV, in situ ambient pressure x-ray photoelectron spectroscopy and in situ synchrotron powder diffraction were applied to identify the active site of 3DOM Cu-Fe catalyst for HAS. The results show that after syngas pretreatment of the 3DOM Cu-Fe catalyst, Cu0 is the active oxygenation site for alcohol synthesis, and Fe5C2 is the active site for carbon chain growth.

The production of higher alcohols from synthesis gas over Cu-Co-Cr-K catalysts has been studied. The production rate of alcohol was measured in the flow reactor, operating at 250 to 350°C, 3500 to 8000 gas hourly space velocity, and 900 to 1800 psig. The productivity as a function of temperature, pressure, gas hourly space velocity, carbon dioxide content of the feed, and reaction time was also examined. Physisorption data have been analyzed using the Langmuir model, the Brunauer-Emmett-Teller (BET) method, the Barret-Joyner-Halenda (BJH) method, and the de Boer and Halsey t-method. The surface areas of catalysts CB1(1), CB1(3), and CB1(1) after reaction were 39.9 ± .9 m2/g, 28.9 ± 1.7 m2/g, and 26.5 ± 0.3 m2/g, respectively. Moreover, information such as pore size distribution, pore shape, monolayer volume, micropore volume and thickness of adsorption layer were also obtained. The atomic concentration and oxidation states of near surface species were established by X-ray Photoelectron Spectroscopy.

Zur stereoselektiven Herstellung von α-Hydroxyketonen aus prochiralen Ketonen stellen die Alkoholdehydrogenasen eine ökologische als auch ökonomische Alternative zu den verfügbaren industriellen Syntheserouten dar. Zurzeit stoßen sowohl die Biokatalyse als auch die organische Katalyse bei der Herstellung von sterisch anspruchsvollen α-Hydroxyketonen an ihre Grenzen. Die Synthese von enantiomerenreinem (R)-Phenylacetylcarbinol [(R)-PAC] und S- Phenylacetylcarbinol [(S)-PAC] aus dem prochiralen α-Diketon Phenylpropan-1,2-dion (PPD) stellt eine anspruchsvolle Synthese sowohl für akademische als auch für industrielle Zwecke dar. Diese chiralen Bausteine dienen als Vorgänger bei der Synthese von (‒)-Ephedrin und (+)-Pseudoephedrin. (‒)-Ephedrin und (+)-Pseudoephedrin werden jährlich in großen Mengen hergestellt, was zunehmend ein ernsthaftes ökologisches Problem darstellt. Aufgrund ihrer Toxizität als auch ihre Persistenz in der Umwelt, beispielsweise in Abwasserkläranlagen, wurden sie kürzlich als neu auftauchende Kontaminanten eingestuft. In dieser Arbeit wurde die Biodegradierung der Isomere von Ephedrin untersucht. Dabei wurde der neue Stamm Arthrobacter sp. TS-15 isoliert, welcher mit Ephedrin als einzige Kohlenstoffquelle wachsen kann. Dieser Stamm wurde bei der DSMZ unter der Nummer (DSM 32400) hinterlegt. Das Genom dieses Stammes wurde sequenziert und unter der Zugangsnummer (SDXQ00000000) in der Genbank verwahrt. Anhand verschiedener phylogenetischer Untersuchungen wurde TS-15 als eine Subspezies von Arthrobacter aurescens eingeordnet. Des Weiteren wurde der Einfluss der Isomerie von Ephedrin auf dessen Biodegradierung sowie auf die Wachstumsrate von TS-15 untersucht. Es wurde festgestellt, dass das Isomer (‒)-Pseudoephedrin am langsamsten abgebaut wird und dementsprechend einen negativen Einfluss auf das Kulturwachstum hat. Hingegen zeigte sein Enantiomer (+)-Pseudoephedrin die schnellste Biodegradierung mit einem positiven Effekt auf das Wachstum von TS-15. Anhand der Analyse der Metabolite im Kultivierungsmedium als auch aus den Zellextrakten von TS-15 wurde ein neuer katabolischer einleitender Schritt detektiert, in dem das Ephedrin zu Methcathinon VII oxidiert wird. Zur Bestimmung der oxidierenden Enzyme wurden Proteinanreicherungsverfahren eingesetzt. Mittels Peptidmassenfingerprints wurden 51 Proteinhits ermittelt. Nach einer kombinierten Analyse mittels der Proteinhits und des rationalen Genomminings wurde ein neues Gencluster zum Abbau von Ephedrin identifiziert. Zwei postulierte Dehydrogenasen wurden aus dem Genom isoliert, kloniert und in dem E. coli T7 SHuffle Stamm heterolog exprimiert. Dadurch wurden neue enantiokomplementäre Enzyme entdeckt. Die Pseudoephedrin Dehydrogenase (PseDH) ist enantiospezifisch für (+)-S,(N)-(Pseudo-)-Ephedrin, während die Ephedrin Dehydrogenase (EDH) nur die enantiospezifische Oxidation von (‒)-R,(N)-(Pseudo-)-Ephedrin katalysieren kann. Beide Dehydrogenasen sind NADH-abhängig und der Superfamilie der kurzkettigen Dehydrogenasen untergeordnet. Bei der Charakterisierung dieser Dehydrogenasen konnte gezeigt werden, dass das Substratspektrum wertvolle chirale Produkte umfasst. Beide Dehydrogenasen zeigen strikte Regio- und Enantioselektivität gegenüber dem α-Diketon Phenylpropan-1,2-Dion (PPD). Somit wurde PPD zu (S)-PAC (ee >99%) und (R)-PAC (ee >99%) mittels PseDH bzw. EDH mit vollem Umsatz reduziert. Darüber hinaus wurde die Kristallstruktur der PseDH im Rahmen einer Zusammenarbeit mit der Universität von York mit einer Auflösung von 1,8 Å aufgeklärt. Die Kristallstruktur wurde in PDB unter der Zugangsnummer (6QHE) hinterlegt. Mittels der Kristallstruktur der PseDH und des Homologiemodells der EDH wurden Strukturanalysen durchgeführt und die ersten Hypothesen zur Funktionsweise dieser Enzyme aufgestellt. Des Weiteren wurden über Peptidsequenzanalysen zu diesen Enzymen Rückschlüsse auf ihren evolutionären Ursprung gezogen. Die Stabilität der Dehydrogenasen wurde mit unterschiedlichen Lösungsmitteln bestimmt. CPME wurde als geeignetstes organisches Lösungsmittel für die Biokatalyse mit diesen Enzymen ermittelt. Beide Enzyme wurden mittels eines organisch-wässrigen Zweiphasensystems unter enzymgekoppelter Cofaktorregenerierung getestet. Dadurch wurde der Zugang zur Produktion von (S)-PAC und (R)-PAC aus PPD mittels PseDH bzw. EDH geschaffen.