Cellulose, the most abundant biopolymer on earth, is composed of β – 1,4 – linked glucose units, which in turn form a highly ordered crystalline structure that is insoluble and recalcitrant to degradation. It is the world’s most attractive, abundant and renewable energy resource, representing the bioconversion of carbon dioxide into green plants. Cellulosic biomass, such as agricultural and forestry residues, waste paper and industrial waste can therefore be used as an inexpensive and abundantly available source of sugar for fermentation into fuel ethanol. The combustion of biofuels releases carbon dioxide which is thus recycled and hence the use of these fuels in transportation provides an alternative to fossil fuels, solving many environmental problems. The ability to degrade crystalline cellulose seems to be restricted to a specialized group of microorganisms which includes for example Clostridium, Cellulomonas, Cytophaga, Trichoderma etc. Hence the aim of this project was to create BioBricks using different cellulases from cellulose degraders and express them in different expression hosts like Escherichia coli, Bacillus subtilis, Citrobacter freundii etc., using two different promoters, spac and lac. It was observed that the expression of Cytophaga hutchinsonii cellulases (CHU_2103 and CHU_2802) and dehydrogenases (CHU_1944 and CHU_2315) was toxic to the E. coli host for some unknown reason. Therefore it was decided to use cellulases from Cellulomonas fimi, which are well characterized. BioBricks of cellulases (cenA and cex) from C. fimi were introduced into different expression hosts. It was observed that under our experimental conditions Citrobacter freundii SBS197 gave the best results. Both Pspac and Plac were functional in this organism with expression being higher when Pspac was used. When E. coli JM109 was used as an expression host, activity was only detected when the lac promoter was used to control the expression. Although the expression was higher when E. coli JM109 (containing Plac) was used as an expression host, almost all of this activity was residing within the cells, whereas when C. freundii SBS197 was used as an expression host, considerable activity was detected in the surrounding medium, which is essential for cellulose degradation. Growth curve studies were done to see if heterologous cellulases enable the host to use cellulosic substrates as a source of carbon. It was observed that C. freundii SBS197 expressing cenA and cex was able to use filter paper and Avicel as a source of carbon with maximum growth of up to 8.8×108 cfu/ml and 1.2×109 cfu/ml respectively. This was about 2 – 5 fold higher when compared to the control (vector and/or negative) strains. Filter paper completely disappeared within 3 – 4 days when C. freundii SBS197 was used. Slight degradation was observed when E. coli JM109 was used but there was no physical degradation seen when B. subtilis 168 was used as an expression host. Hence it was concluded that heterologous cellulases impart to C. freundii SBS197 with the ability to use cellulosic substrates as a source of carbon. The maximum growth obtained using these cultures is to our knowledge higher than what has been reported so far for recombinant organisms expressing heterologous cellulases using cellulosic substrates as a source of carbon.

Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Biochar (charcoal) is lauded by many scientists as an effective way to remove carbon dioxide from the atmosphere and storing it in a very stable form in the soil for hundreds to thousands of years, whilst promoting soil fertility and productivity. Considering that no significant amounts of charcoal are presently accumulating in the environment, despite considerable amounts produced globally in natural and man-made fires, this study focuses on understanding the degradation of biochar and its interactions with plants and soil organisms. The following experiments were conducted to achieve this goal. Controlled chemical oxidation of biochar, using different concentrations of hydrogen peroxide, was conducted in an attempt to mimic the enzymatic degradation of biochar by basidiomycetes. The changes occurring in biochars structure and chemistry were assessed afterwards. Furthermore, aerobic and anaerobic digestion of biochar was conducted in vitro, and in vivo to investigate the changes occurring in biochar‘s elemental composition and chemistry during oxidation and factors that play a determining role in the rate of biochar degradation. The influence of biochar in soil on free-living and symbiotic microbial communities as well as its impact on total plant biomass production and root development was assessed in three greenhouse pot trials using wheat and green beans as test plants It was proven that biochar is almost fully H2O2-degradable, mostly through hydroxylation and carboxylation reactions which led to the formation of various short chained carboxylic acids, surface saturation with acidic functional groups as determined by the surface acidity measurements and proven by the increase in the intensity of FT-IR peaks associated with carboxyl and phenolic C-O groups. Furthermore, hydrogen peroxide treatment resulted in preferential removal of volatile organic carbons and led to the purification of biochar as evident by the new, more intense and sharper peaks in the region of 1600-1000 cm-1. These FT-IR peaks are considered as the more recalcitrant fraction of biochar and were shown to be mostly associated with transformation products of lignin and cellulose formed during pyrolysis. The incubation trial confirmed that biochar cannot be utilized as a sole carbon source without the addition of nutrients or glucose, to activate microbial activity within the columns. Furthermore, abiotic oxidation can be facilitated by oxidative soil minerals such as birnessite, but oxidation with atmospheric oxygen did not result in the evolution of CO2 from biochar. The average CO2 production in pot trials without plants in both the fertilized and unfertilized treatments increased linearly (R2= 0.80; 0.79 respectively) with increasing biochar application rates when biochar was the main carbon sources. Anaerobic degradation of biochar by a methanogenic consortium was much more efficient in utilizing biochar as a carbon source, compared to aerobic digestion. The anaerobic digesters maintained a chemical oxygen demand (COD) removal efficiency of 30% per week with continuous production of CO2, whilst methane production was very erratic. We proposed that better control over pH and alkalinity as well as an increase in hydraulic retention time would improve both the COD removal efficiency and methane production. Field incubations resulted in various degrees of oxidation at different incubation sites. An increase in the oxygen content and a decreased in the carbon content of biochar‘s elemental composition and also an increase in the surface acidity due to a larger amount of carboxyl acid groups on the surface as seen in the increase in the FT-IR peak at 1700 cm-1 confirmed that biochar are susceptible to oxidation under field conditions. We came to the conclusion that oxidation and mineralization of biochar in this trial occurred at a faster rate in soils with a higher microbial activity. The pot trials, confirmed that biochar does not serve as a fertilizer even though it did increase total biomass production between biochar application rates of 0.05-2.5 % (w/w). For agricultural purposes the addition of biochar should always be applied together with NPK fertilizer. In both the wheat and green bean trials it was confirmed that biochar application rates of 0.05-0.5% (w/w) on the sandy, slightly acidic soil used in this trial resulted in the greatest biomass production and fertilizer use efficiency. Biochar additions resulted in considerable increases in soil pH and C/N ratios which were considered as the main reasons for the decrease in microbial biomass in the unfertilized green bean treatments as it made the uptake of N more limited. The addition of fertilizer however, alleviated N-supply constraints and as a result promoted microbial growth at all biochar application rates of pot trial 1. However, biochar did not promote mycorrhyzal colonization and caused a decrease in the mycorrhizal colonization of roots with increasing biochar application rates and within biochar layers. Biological nitrogen fixation, however, reacted positively to the addition of biochar. High biochar application rates significantly enhanced the plants reliance on these symbiotic relationships. We hypothesized that biochar physically immobilized N into its microvoids through capillary suction and then served as a physical barrier between plant roots and absorbed N. However, immobilzation of N by microbes could also have contributed to the decrease in N uptake if one takes into account that microbial activity was higher (respiration data) at the higher biochar application rates. Further investigations are needed to warrant this hypothesizes. / AFRIKAANSE OPSOMMING: Biochar (houtskool) is deur talle wetenskaplikes die lof toegeswaai as ‘n doeltreffende manier om koolstofdioksied uit die atmosfeer te verwyder en in ‘n baie stabiele vorm in die grond vir honderde tot duisende jare te stoor, terwyl dit die grondvrugbaarheid en produktiwiteit bevorder. As daar in ag geneem word dat geen beduidende hoeveelheid houtskool in die omgewing opgaar nie ondanks groot hoeveelhede wat wêreldwyd deur natuurlike en mensgemaakte brande gevorm word, is die doel van hierdie studie om die afbraak en die interaksie van biochar met plante en grondmikrobes beter te verstaan. Om hierdie doel te bereik is die volgende eksperimente uitgevoer: Beheerde chemiese oksidasie is op die biochar toegepas deur gebruik te maak van verskillende konsentrasies waterstofperoksied in 'n poging om die ensiematiese afbraak van biochar deur basidiomysete na te maak. Die veranderinge wat plaasvind in die struktuur en chemie van biochar is daarna bestudeer. Daarbenewens is die aerobiese and anearobiese afbraak van biochar toegepas beide in vitro- en in vivo-, om die veranderinge wat in biochar se elementele samestelling en chemie plaasvind gedurende oksidasie en ook die faktore wat 'n bepalende rol in die tempo waarteen biochar afbreek, te ondersoek. Die invloed van biochar in die grond op vrylewende en simbiotiese mikrobiese populasies, sowel as die impak daarvan op die totale plant biomassa produksie en ontwikkeling van plantwortels, is vasgestel tydens drie groeitonnel potproewe waarby koring en boontjies as planttoetsspesies gebruik is Dit is bewys dat biochar byna volledig deur H2O2 afgebreek kan word, meestal deur hidroksilasie en karboksilasie reaksies wat gelei het tot die vorming van 'n verskeidenheid kort ketting karboksielsure, 'n biochar oppervlak versadig met suurvormende funksionele groepe soos bepaal en bewys deur die toename in intensiteit van die FT-IR (Fourier Transvorm Infrarooi Spektroskopie) pieke geassosieer met karboksiel en fenoliese C-O groepe. Die behandeling van biochar met H2O2 het by voorkeur die vlugtige organise koolstof verwyder wat gelei het tot suiwering van die biochar, wat bevestig is deur die nuwe, meer intens en skerper FT-IR pieke in die area tussen 1600-1000 cm-1. Die FT-IR pieke word beskou as die meer weerstandbiedende fraksie van biochar en daar is bewys dat die pieke meestal met getransformeerde produkte van lignien en sellulose wat tydens pirolise gevorm is, geassosieer word. Die inkubasie proef het bevestig dat biochar nie deur mikrobes benut kan word as enigste bron van koolstof sonder die byvoeging van nutriente of glukose nie, om die mikrobes binne die inkubasie kolom te aktiveer. Daarbenewens kan abiotiese oksidasie van biochar deur oksidatiewe grondminerale soos birnessite (δ-MnO2) gefasiliteer word, terwyl oksidasie van biochar deur atmosferiese suurstof nie tot enige CO2 produksie gelei het nie. Nogtans het die gemiddelde CO2 produksie in die boontjie potproef, sonder die plante, in beide die onbemeste en bemeste behandelings linieer toegeneem (R2= 0.80; 0.79 onderskeidelik) met toenemende aanwendingskoers van biochar, toe biochar die dominante bron van koolstof was. Anaerobiese afbraak van biochar deur 'n metanogeniese konsortium was heelwat meer effektief in die benutting van biochar as enigste koolstofbron in vergelyking met aerobiese afbraak. Die anaerobiese verteertoestel het konstant 30% van die chemiese suurstof behoefte (CSB) weekliks verwyder, gepaardegaande met die voortdurende produksie van CO2, terwyl metaangasproduksie baie onegalig was. Dit word voorgestel dat met beter beheer oor pH en alkaliniteit en ook 'n langer hidrouliese retensie tyd, kan beide die CSB verwyderingseffektiwiteit en metaangasproduksie verbeter kan word. Veld inkubasies het verskeie mates van oksidasie meegebring tussen die verskillende inkubasie liggings. 'n Toename in die suurstofinhoud en 'n afname in die koolstof inhoud van biochar se elementele samestelling sowel as 'n toename in die oppervlak suurheid weens die groter hoeveelheid karboksielsure aan die oppervlak soos blyk uit die FT-IR piek by 1700 cm-1, het bevestig dat biochar wel vatbaar is vir oksidasie onder veld kondisies. Die gevolgtrekking was dat biochar oksidasie en mineralisasie in hierdie proef teen 'n vinniger tempo plaasgevind het in die gronde met hoer mikrobiese aktiwiteit. Die potproewe het bevestig dat biochar nie as bemestingsstof sal dien nie, alhoewel dit tot 'n toename in die biomassa produksie gelei het tussen die biochar aanwendingskoerse van 0.05-2.5% (w/w). Vir landbou doeleindes moet die aanwending van biochar altyd gepaardgaan met NPK bemesting. Beide die koring- en boontjie proewe het bevestig dat die biochar aanwendingskoerse tussen 0.05-0.5% (w/w) op die sanderig, effens suur grond wat gebruik is in die proef, gelei het tot die hoogste biomassa produksie en bemestingseffektiwiteit. Die toediening van biochar het gelei tot merkbare toenames in grond pH en C/N verhoudings en hierdie toestande was beskou as die hoof redes vir die afname in mikrobiese biomassa in die onbemeste boontjie behandelings omdat dit die opname van N meer beperk. Die toediening van bemesting het egter die beperkings op N voorsiening opgehef en as gevolg hiervan die mikrobiese biomassa bevorder by alle biochar aanwendingskoerse. Biochar het egter nie mikorrisa kolonisasie bevorder nie en het gelei tot =n afname in die mikorrisa kolonisasie van die wortels met toenemende biochar aanwendingskoerse en binne in die biochar lae van potproef 1. Biologiese stikstof vaslegging het egter positief reageer op die toediening van biochar. Hoë biochar aanwendingskoerse het beduidend die plant se afhanklikheid op hierdie simbiotiese verhouding verhoog. Ons hipotese is dat die biochar fisies N immobiliseer binne in die mikro-ruimtes deur kapillêre suigaksie en dan as 'n fisiese versperring dien tussen die plantwortels en die geabsorbeerde N. Die immobilisasie van minerale N deur mikrobes kon egter ook grootliks bygedra het tot die afname in N opname as daar in ag geneem word dat mikrobiese aktiwiteit (respirasie data) hoër was by die hoër biochar aanwendingskoerse. Verdere ondersoeke moet daarom uitgevoer word om hierdie hipotese te bevestig.

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway.

Paclitaxel is a very important anticancer drug commonly called Taxol®. We know from previous work (Hoffman, et al., 1998) that Taxol exists in filbert, Corylus avellana L. material. Quantification of Taxol in filbert plant material is painstaking and hitherto was accomplished by rapidly processing single batches in a complicated procedure (Hoffman, et al., 1998), which seemingly unavoidably, was accompanied by some degradation. However for extraction, testing, plant physiological and horticultural purposes a simplified method of determining Taxol yield is required. All simplified methods tested were found limited by rapid degradation of Taxol. Under these conditions not only is the sought product broken down, but we were unable to distinguish Taxol precursors from degradation products. Thus it was decided to go back to first principles and study the degradation of Taxol in vitro. Degradation kinetics of Taxol was studied using electrospray ionization mass spectrometry (ESIMS) to identify possible Taxol adducts and degradation products. Our preparation for ESIMS analysis by experimental necessity involved various other components. Since some of these components interacted with the degradation products, we developed a program to distinguish these putative adducts from spurious components found in the system and we were able to plot the pH dependence of Taxol degradation in this system at room temperature (approximately 25oC). The results of the mass spectrometric analysis of these degradants were found dependent on pH and time. Our results show major degradation at pH 9 and beyond, plus minor degradation at pH 5. Two optimum pHs for stability were found at pH 4 and pH 7. This data varies slightly from the published results Dordunoo and Burt (1996). Our smoother curves define two pHs events in this pH range which to our knowledge, have not been reported and our temperature was lower. We hope that this information will help us extract Taxol more efficiently with greater yield from novel plant sources, e.g. hazel (filbert) tree, Corylus avellana L. The possibility of Taxol dimer formation in solution and perhaps in vivo can be inferred, but not proven, in this work. This dimer, may exist, in dynamic equilibrium with parent compound, Taxol. / Graduation date: 2006

Glyphosate (N-(phosphonomethyl)glycine) is the phytotoxic reagent in the widely used Roundup® herbicide. Its mode of action in plants is the disruption of the Shikimate pathway, part of an important route to the biosynthesis of essential aromatic amino acids. It is well documented that glyphosate can be degraded by soil microorganisms after contact of the herbicide solution with the soil. It is also accepted that glyphosate, an excellent ligand, is readily absorbed to metal ions, such as Fe(III), that can be abundant in soils. There have been many accounts on the microbial degradation of glyphosate, and several metal-glyphosate complexes have been synthesised and characterised. Surprisingly, given the degree of adsorption to metal ions in the soil, there have not, to date, been any reports in the literature on the reactivity of metal-glyphosate complexes. The behaviour of these types of complexes under various reaction conditions may give us an insight into the mechanisms present when glyphosate degrades. In order to explore the behaviour of metal-glyphosate adducts, we have prepared several new cobalt-PMG complexes in the lab. These complexes have been characterised by NMR, mass spectrometry, elemental analysis, and in some cases X-ray crystal structure determination. We chose to synthesise complexes where the PMG ligand is bidentate or tridentate, filling the remaining four or three (respectively) coordination sites with an ancillary, nitrogen-containing ligand. We have subjected the complexes to photolytic and basic conditions, as we are interested in ascertaining how coordinated PMG might behave when irradiated with UV light, and when it is C-deprotonated. Metal-glyphosate complexes in nature may be exposed to UV light, so we are concerned with how the coordinated ligand might react under these circumstances. We have found that the prepared cobalt-PMG complexes are reactive when exposed to UV light, and that this appears to result in the degradation of the complex, and in some cases, the PMG ligand itself. The reactivity of C-deprotonated PMG is also an area of interest to us. It is possible that elevations in soil pH can lead to C-deprotonation of glyphosate, then further reactivity that may contribute to the degradation of the compound. Furthermore, when the herbicide is held in the active site of an enzyme within a microbe, it may become deprotonated, and this may aid in its microbial degradation. We have found that, under basic conditions, the reactivity of the prepared PMG complexes depends on the ancillary ligand attached - π-acidic, pyridine-containing ancillary ligands appear to increase the reactivity of coordinated PMG. It seems that amine-containing ligands hinder the reactivity of the coordinated PMG such that the complex remains intact. It is hoped that the results of the research described in this thesis will assist in the future investigations into the reactivity of the herbicide glyphosate.

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No description available.

Fenton-dependent recovery of carbon initially saturated with one of several chlorinated aliphatic contaminants was studied in batch and continuous-flow reactors. A specialty carbon, URV-MOD 1 (Calgon) was employed to minimize non-productive H2O2 demand - that which does not yield hydroxyl or superoxide radicals.Enhancement of PCE degradation kinetics by ferric iron addition is limited by iron solubility, even at relatively low pH. Quinone addition increased the pseudo-first-order rate constant for PCE loss temporarily. Only copper addition sustainably enhanced the specific rate of PCE loss. For copper-to-iron molar ratios of 0.25 to 5, the pseudo-first-order rate constant for PCE transformation was increased by a factor of 3.5. It is apparent that the effect of copper addition on Fenton-dependent reaction rates is complex, and involves a shift in chemical mechanism, as indicated by the differing slopes in the Arrhenius plot (with and without copper).A mathematical model was developed to evaluate the effect of operational parameters ([Fe(III)]T:[H2O2]o ratio and pH) on degradation kinetics and optimize the PCE degradation process in homogeneous reaction mixtures. The model simulated experimental degradation of the organic target in a homogeneous Fenton-reaction system. The model requires further refinement to simulate Fenton's systems in which ions in solution (such as sulfate and chloride) play significant roles.In continuous-flow reactors, Fenton's reagents were cycled through spent GAC in columns to degrade one of seven chlorinated compounds tested. The contaminant with the weakest adsorption characteristics, methylene chloride, was 99% lost from the carbon surface during a 14-hour regeneration period. At the field site, the GAC was saturated with gases containing TCE and PCE from a soil vapor extraction (SVE) system. In the field, up to 95% of the sorbed TCE was removed from GAC during regeneration periods of 50-60 hours. Recovery of PCE-loaded GAC was significantly slower. Column experiments show that there is minimal loss of carbon adsorption capacity during Fenton treatment and that the rate of GAC regeneration is compound specific. Scoping-level cost estimates indicated that field use of Fenton regeneration is not cost effective without optimization and/or iron surface amendments, except in the case of the most soluble VOCs.

No description available.

No description available.