We initially studied the treatment of acid rock drainage using a sulfate-reducing bioreactor with zero-valent iron as the electron donor. The results demonstrate that this electron donor can serve as the sole exogenous slow-release electron donor to drive sulfate reduction over 400 operational days at short HRTs (1-3 days). The synthetic acid rock drainage contained high heavy metal concentrations (up to 50 mg/L of copper) and pH values ranging from 3.0 to 7.0. Treatment of this acid rock drainage efficiently removed Cu, Cd and Pb (>99.7%) and increased pH to circumneutral values (7.3-7.7). Elemental analysis indicated that formation of insoluble metal sulfides was responsible for the effective metal removal in the zero valent iron columns. In the second study, three inoculated columns containing anaerobic granular sludge were fed a synthetic medium containing H₂SO₄ and Cu²⁺ during the experimental period of 4 months. Algae biomass promoted 80% of sulfate removal (12.7 mg SO₄²⁻ d-1), enabling near complete Cu removal (>99.5 %), and alkalinity generation, raising the effluent pH to 6.5. In the algae amended columns Cu²⁺ was precipitated with biogenic H2S produced by sulfate reduction. Whole cell algae and lipid extracted algae biomasses were both shown to be effective e-donors in driving sulfate reduction of ARD, thus enabling the precipitation and removal of Cu²⁺. The precipitate retained in the columns was composed mostly of insoluble copper sulfide formed from the biogenic sulfide, as shown by sequential extraction and X-ray diffraction. In the third study, several pretreatments, i.e., thermal, chemical, sonication and combinations thereof, that enhance anaerobic biodegradability of Chlorella protothecoides biomass were evaluated. The results demonstrate that anaerobic digestion of pretreated Chlorella protothecoides biomass generates energy-rich methane and recovers nitrogen nutrients. Sonication of algal biomass under optimized conditions provided a significant increase in the methane yield (327 mL STP CH₄ g⁻¹ VS) compared to untreated algae (146 mL STP CH₄ g⁻¹ VS), as demonstrated in anaerobic digestion experiments incubated for 41 days. In contrast, thermal pretreatment provided only a moderate increase of the methane yield and alkaline treatment led to a decrease of the methane yield compared to the untreated algal biomass. Additionally, sonication treatment provided a 4.1-fold increase in the release of ammonia nitrogen during anaerobic digestion of the algal biomass. In the fourth study, the nutrient recovery and biogas generation from the anaerobic digestion of waste biomass from algal biofuel production was investigated. Anaerobic digestion of whole cell and lipid extracted Chlorella sorokiniana-1412 released 48.1 and 61.5% of the total algal nitrogen as NH₄⁺-N, and 87.7 and 93.6% of the total algal P as soluble P, respectively. The biochemical methane potential, quantified through the methane yield of whole cell algae and lipid extracted algae, was 0.298 and 0.253 L methane/g algal volatile solids, respectively. The conversion of lipid extracted algae and whole cell algae biomasses to methane was very similar (38 and 41% on a COD basis, respectively), indicating that the energy yield was not significantly lowered by extraction of the lipid fraction (which accounted for 9% of algal dry weight). Sonication improved the access of hydrolytic enzymes to algal biopolymers, compensating in part for the energy lost due to lipid extraction. The above results demonstrate that algal waste from the biodiesel industry has the potential to be recycled through anaerobic digestion into valuable nutrients and energy. These studies indicate that zero valent iron and algae biomass are promising reactive materials for the treatment of acid rock drainage in sulfate-reducing permeable reactive barrier systems. Additionally, to promote algae cultivation for the biodiesel industry, the anaerobic digestion of algae residues can generate nutrients and energy, making algae cultivation more fiscally attractive.

Thesis (MScEng (Process Engineering))--Stellenbosch University, 2008. / A substance called CRUD (Chalk River Unidentified Deposit) was deposited together with gold tailings to the East Paydam tailings dam. Previous research conducted on the material has shown that the crud leaches Mn and Ni at concentrations that are above their acceptable risks limits as well as Zn which leaches at concentration slightly below its acceptable limits thereby posing an environmental risk. The main objective of the research was to test the hypothesis stating that soil washing in series with bioremediation can be used to remove the heavy metals from the material from the East Paydam tailings dam. Various laboratory and pilot scale tests were conducted to investigate critical soil washing and bioremediation parameters and their respective influence on the treatment process. Laboratory work involved column tests and batch tests. These tests were crucial in determining the critical parameters for the pilot scale tests such as the selection of the suitable lixiviant from the four that were investigated. The optimal concentration of the lixiviant was also determined together with the optimum soil: liquid ratio. These parameters were employed in the pilot scale tests. Pilot scale tests involved soil washing in series with bioremediation. The bacterial growth over the bioremediation period was also determined. Precipitation of the heavy metals from leachate was investigated by varying the pH and temperature. Results showed that the soil from the East Paydam can effectively be treated by soil washing in series with bioremediation. Oxalic acid was selected for soil washing of the payable slimes at a concentration of 0.001M. However, material that contains high amount of CRUD (deeper down the slime dam) required the relatively concentrated 0.1M oxalic acid and mechanical agitation. Bioremediation was determined to increase the amount of heavy metals that was leached from the material from the East Paydam slimes dam. Precipitation of the heavy metals at a pH of 12 achieved up to 98% removal of heavy metals from leachate.

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

<font face="Times New Roman"><font face="Times New Roman"> <p align="left">The popularization and health benefits associated with the &ldquo / Mediterranean diet&rdquo / saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds. <font face="Times New Roman">In order to identify organisms which could potentially be used in the bioremediation of olive wastewater, 36 microbial strains were isolated from evaporation ponds in the Boland region of South Africa. Twenty five isolates were capable of growth on 50% olive wastewater and their bioremediation potential as well as their ability to produce valuable intermediate compounds were subsequently characterized. Based on the RPHPLC results, which showed that a number of chemical intermediates were produced in fermentation of olive wastewater, isolate AS-35 was selected for further analysis. Strain AS-35, identified as a </font><font face="Times New Roman"><em>Bacillus megaterium,</em> </font><font face="Times New Roman">was significantly influenced by the exposure to olive waste. The total cellular protein profile, generation time and cellular morphology of this isolate were dramatically affected by the introduction of olive waste. <font face="Times New Roman">This study investigated the differential gene display of </font><font face="Times New Roman"><font face="Times New Roman"><em>Bacillus megaterium</em></font> </font><font face="Times New Roman">following exposure to olive wastewater. Proteomic and transcriptomic differences of the organism cultured in nutrient rich LB and olive wastewater were compared. These results indicated that AS-35 expressed genes involved in glycolysis, tryptophan and nucleotide synthesis as well as the chaperones GroEL and DnaK during its growth in LB. In contrast, genes induced following the abolishment of glucose dependent catabolite repression, genes involved in biotin synthesis and &szlig / -oxidation of fatty or organic acids as well as a gene whose expression is regulated by stress induced s</font><font face="Times New Roman" size="1">B</font><font face="Times New Roman">-dependent regulon were expressed during olive waste growth.</font></font></p> </font></font>

Fractured rock formations represent a valuable source of groundwater and can be highly susceptible to contamination by dense, non-aqueous phase liquids (DNAPLs). The goal of this research is to evaluate the effectiveness of three accepted remediation technologies for addressing DNAPL contamination in fractured rock environments. The technologies under investigation in this study are chemical oxidation, bioremediation, and surfactant flushing. Numerical simulations were employed to examine the performance of each of these technologies at the field scale. The numerical model DNAPL3D-RX, a finite difference multiphase flow-dissolution-aqueous transport code that incorporates RT3D for multiple species reactions, was modified to simulate fractured rock environments. A gridding routine was developed to allow the model to accurately capture DNAPL migration in fractures and aqueous phase diffusion gradients in the matrix while retaining overall model efficiency. Reaction kinetics code subroutines were developed for each technology so as to ensure the key processes were accounted for in the simulations. The three remedial approaches were systematically evaluated via simulations in two-dimensional domains characterized by heterogeneous orthogonal fracture networks parameterized to be representative of sandstone, granite, and shale. Each simulation included a DNAPL release at the water table, redistribution to pools and residual, followed by 20 years of ‘ageing’ under ambient gradient conditions. Suites of simulations for each technology examined a variety of operational issues including the influence of DNAPL type and remedial fluid injection protocol. Performance metrics included changes in mass flux exiting, mass destruction in the matrix versus the fractures, and percentage of injected remedial fluid interacting with the target contaminant. The effectiveness of the three remediation technologies covered a wide range; the mass of contaminants destroyed were found to range from 15% to 99.5% of the initial mass present. Effectiveness of each technology was found to depend on a variety of critical factors particular to each approach. For example, in-situ chemical oxidation was found to be limited by the organic material present in the matrix of the rocks, while the efficiency of enhanced bioremediation was found to be related to factors such as the location of indigenous bacteria present in the domain and rate of bioremediation. In the chemical oxidation study, the efficiency of oxidant consumption was observed to be poor across the suite of scenarios, with greater than 90% of the injected permanganate consumed by natural oxidant demand. This study further revealed that the same factors that contributed to forward diffusion of contaminants prior to treatment are critical to this remediation method as they can determine the extent of contaminant destruction during the injection period. Bioremediation in fractured rock was demonstrated to produce relatively good results under robust first-order decay rates and active microorganisms throughout the fractures and matrix. It was demonstrated that under ideal conditions, of the total initial mass present, up to 3/4 could be reduced to ethene, indicating bioremediation may be a promising treatment approach due to the effective penetration of electron donor into the matrix during the treatment period and the ongoing treatment that occurs after injection ceases. However, when indigenous bacteria was assumed to exist only within the fractured walls of sandstone, it was found that under the same conditions, the rate of dechlorination was 200 times less than the Base Case. Since the majority of the mass resided in the matrix, lack of bioremediation in the matrix significantly reduced the effectiveness of treatment. Surfactant treatment with Tween-80 was proven to be a relatively effective technique in enhanced solubilisation of DNAPL from the fractures within the domain. However, by comparing the aqueous and sorbed mass at the start and end of the Treatment stage, it is revealed that surfactant treatment is not efficient in removing these masses that reside within the matrix. Furthermore, DNAPLs identified in dead end vertical fractures were found to remain in the domain by the end of the simulations across all scenarios studied; indicating that the injected surfactant experiences difficulty in accessing DNAPLs entrapped in dead end fractures. Altogether, the results underscore the challenge of restoring fractured rock aquifers due to the field scale limitations on sufficient contact between remedial fluids and in situ contaminants in all but the most ideal circumstances.

Chlorinated solvents such as trichloroethene (TCE) and perchloroethene (PCE) are commonly found in industrialized areas and can have major impact on human health and groundwater quality. The techniques for removing these substances from the subsurface environment is constantly being tuned and revised, and as such, the need for monitoring at such remediation sites is crucial. To find important correlations and hidden patterns between variables principle component analyses (PCA) and correlations matrixes were used on sets of field data from an existing remediation site in southern Sweden. Four important components were extracted in the following order; End products of dechlorination (EPD), second wave of dechlorination (SWD), first wave of dechlorination (FWD) and indicators of dechlorination (ID). The underlying pattern found in the data set was most likely derived from thermodynamic preference, explaining important correlations such as the correlation between iron and sulfate, the correlation between redox and degree of dechlorination. The law of thermodynamic preference means that we can (roughly) estimate the level of difficulty and/or the time it will take to remediate a polluted site.  These findings show that similar results shown in theory and laboratory environments also applies in the field and also that PCA is a potent tool for evaluating large data sets in this field of science. However, it is of great importance that the correlations are examined thoroughly, as correlation it not equal to causation.

Biostimulation is the use of in-situ microorganisms and added reagents in order to biosequester, precipitate, or absorb contaminants from contaminated groundwater and sediment. To test the effectiveness of this remediation approach at a particular site, small scale experiments, such as miscible-displacement, batch, or microcosm experiments, should be performed before a large-scale in-situ biosequestration electron donor injection. In this study, electron donor solutions containing contaminated groundwater and ethanol, acetate, benzoate, or glucose were injected into aquifer sediments collected from a UMTRCA Title 1 Site in Monument Valley, AZ. These experiments showed that ethanol, acetate, and glucose were effective electron donors for the stimulation of microbial activity in order to sequester uranium and reduce nitrate and sulfate concentrations. Conversely, benzoate was not effective at sequestering or reducing the contaminants. After electron-donor deficient groundwater was injected into the columns, a rebound of nitrate, sulfate, and uranium concentrations was observed. Due to this rebound, it was inferred that the mechanism of sequestration of uranium and hence reduction of nitrate and sulfate was due to the creation of reducing conditions via microbial activity. The insoluble reduced uranium was hypothesized to have precipitated or adsorbed to surrounding sediments. Incoming groundwater contained dissolved oxygen and therefore oxidized the reduced contaminants, consequently returning them into solution. It was hypothesized that a similar rebound would occur if ethanol, acetate, or glucose were to be injected in-situ due to sustained groundwater flow through the aquifer sediments on site.

Le tétrachloroéthène (PCE) et les éthènes chlorés qui lui sont apparentés ont été abondamment utilisés pour plusieurs applications en industrie dès le début du 20e siècle. Ils sont cependant comptés parmi les polluants les plus communs des sols et de l’eau et beaucoup d’efforts sont déployés afin de les éliminer. Nous croyons que la conversion des éthènes chlorés en éthènes par des microorganismes est une solution prometteuse. Le premier aspect du projet visait donc à établir les conditions pour lesquelles un consortium enrichi en Dehalococcoides ethenogenes permettrait la conversion complète de PCE en éthène. Les expériences réalisées nous ont permis de souligner le rôle de l’acide lactique ajouté aux cultures comme source de carbone et source indirecte d’électrons pour la déhalorespiration. Nous avons également pu établir l’effet de la concentration initiale de biomasse dans les cultures sur le profil de déhalogénation du PCE. Le deuxième aspect du projet visait à développer un protocole d’encapsulation du consortium dans une matrice polymérique afin de profiter des nombreux avantages potentiels de l’encapsulation. Nous avons testé trois montages d’encapsulation différents : atomisation avec jet d’air, atomisation avec vibrations ultrasoniques et « drop-wise ». Le dernier montage prévoyait l’encapsulation des cultures dans des billes d’alginate enrobées de chitosane gélifié par du lignosulfonate. C’est le seul montage qui nous a permis d’encapsuler le consortium de façon efficace sans effet significatifs négatifs sur son activité de déchlorination. Aussi, la comparaison des profils de déhalogénation du PCE de cellules encapsulées et cellules libres a montré une plus faible accumulation de TCE, 1,2-DCE et VC dans les échantillons de cellules encapsulée et, par conséquent, une conversion plus rapide et plus complète du PCE en éthène. Finalement, nous avons observé une tendance favorable à l’idée que les microorganismes encapsulés bénéficient d’un effet de protection contre de faibles concentrations d’oxygène. / Tetrachloroethylene (PCE) and other chlorinated ethenes have been used for industrial purposes since the beginnning of 20th century. However, they are now considered common pollutants of soil and water. A lot of efforts are directed toward elimination of these compounds and we believe degradation of these chlorinated ethenes by microorganisms is the best solution. The first step of this project was to establish a complete conversion of PCE to its non-toxic product ethylene using an enriched consortium of Dehalococcoides ethenogenes. Our results show the importance of lactic acid as a carbon source and indirect source of electrons in a reaction known as dehalorespiration. We have been able to establish the effect of initial biomass on the biodegradation profile of PCE. The second step of the project was to obtain a working protocol for encapsulation of the consortium in a polymeric matrix. Such immobilization procedure would then allows numerous possible advantages as shown in the literature. We tested three encapsulation setups: air atomization, ultrasonic atomization and drop-wise technique. In the last setup, we successfully encapsulated the bacterial consortium into particles made of an alginate core surrounded by a chitosan layer. Thus the drop-wise technique allowed encapsulation of the consortium without negative effects on its dechlorination activity. In addition, the dechlorination profiles of encapsulated cells showed a lower accumulation of chlorinated intermediates TCE, 1,2-DCE and VC which yield a more rapid and complete conversion of PCE to ethylene. Finally, our results support the idea that encapsulated microorganisms may benefit from a protective effect when oxygen is present in the medium.

Factory legacy pollutants are an increasing concern for waterways as old infrastructure deteriorates and contaminates nearby environments. The Fisherville Mill in Grafton, Massachusetts, USA exemplifies this problem since it has now fallen into disrepair and is leaking Bunker C crude oil into the adjoining Blackstone River, a third order stream. Our research examines how effectively an ecologically engineered system (EES), consisting of anaerobic bacteria environments, fungal microcosms, and aquatic plant environments, can break down petroleum hydrocarbons, specifically aliphatic and polycyclic aromatic hydrocarbons (PAH), in this river environment. Our testing protocol involved taking water samples before and after each filtration stage monthly from June through October 2012. Water samples were analyzed at the Brown University Superfund Research Lab using mass spectrometry to determine aliphatic and PAH concentrations. Post-treatment aliphatic oil concentrations were significantly different from baseline concentrations (p=0.005), with an average reduction of 95.2%. Post-treatment PAH concentrations were also significantly different from baseline concentrations (p=0.001), with an average reduction of 91%. We conclude that this EES provided effective treatment for Bunker C crude oil, even though some filtration stages did not achieve their intended objectives. This type of filtration arrangement might be scaled up for use in larger remediation efforts regarding Bunker C crude oil.

Avec l'augmentation de niveau de vie des populations, de plus en plus déchets urbains sont produits chaque jour en grande quantité. Ces déchets non seulement occupent une place importante mais dégradent l'environnement en particulier parce qu'ils contiennent divers polluants, qui peuvent porter atteinte à la santé humaine. Le compostage permet aux déchets urbains d'être recyclés en transformant la matière organique en engrais pour les sols agricoles. Par ailleurs, ce procédé peut aussi être utilisé comme méthode de biorestauration pour les sols pollués. Les modèles mathématiques sont des outils qui permettent de comprendre et éventuellement de modifier le processus de compostage. Ils pourraient également prédire la stabilité des produits issus du compostage et quantifier la nocivité des polluants qu'ils contiennent. L'objectif dans cette étude était de construire un modèle décrivant la dynamique des micropolluants organiques au cours du compostage et paramétrer le modèle sous l'interface de MATLAB. Ce modèle peut être utilisé selon les besoins des usagers avec 3 modules: le module de carbone organique, le module de polluants organiques et le module de couplage. Afin de calibrer et valider notre modèle, un ensemble de données de 12 différents mélanges de déchets et 4 différents types de polluants ont été utilisés. Pour l'application de la technique de compostage dans le domaine de la bioremédiation des sols contaminés, une expérience de compostage en laboratoire a été conçue dans lequel les déchets urbains organiques ont été mélangés avec un sol contaminé par des HAP. Nous avons étudié la dynamique des matières organiques, des micropolluants organiques et des populations microbiennes. Les résultats ont montré que la quantité des HAP dégradée a été de 50-60% lorsque le sol est composté avec des déchets organiques. En outre, des populations microbiennes successives ont été observées pendant le processus de compostage. Ces résultats expérimentaux ont été simulés par notre modèle afin de tester sa capacité dans un système sol-compostage. Cependant, quelques questions de recherches se posent encore: en ce qui concerne la modélisation, la simulation des substrats organiques solubles n'est pas assez bonne, ce qui est probablement dû à des limites sur l'étude des caractéristiques biochimiques des fractions organiques soluble; par ailleurs, seule la fonction température a été considérée comme facteur de l'environnement dans notre modèle, des limitations dues à l'l'humidité ou à l'oxygène, par exemple, doivent être étudiées dans des études ultérieures. L'activité microbienne pendant chaque phase de dégradation des HAPs n'a pas encore été assez clairement établie pour être incorporée dans notre modèle. / With the increasing of people's living standards, more and more urban wastes are produced everyday with a huge quantity. The big amount of wastes have not only occupied precious land resource, influenced the industry seriously and agricultural production, but also introduced various pollutants, which will harm human's life and health. Compost processing can help urban wastes to be recycled by transforming the organic matter into fertilizer and be returned to nature. Also it can be used as a bioremediation method for the polluted soil. The mathematical model is an efficient tool for understanding and modifying the composting process, predicting the stability of compost products, and then assuring the harmfulness of pollutants for agriculture. Our objective in this study was to construct a model describing the dynamics of organic micro-pollutants during the composting and parameterize the model under the interface of MATLAB. This model should be used according to users' needs with 3 modules: organic carbon module, organic pollutants module and coupling module. In order to calibrate and validate our model, the datasets from 12 different waste mixtures and 2 different types of pollutants with different initial conditions were applied. On the other hand, for applying the composting technique in the field of bioremediation of contaminated soil, an in-vessel composting experiment was designed in which the organic urban wastes mixed with soil contaminated by PAHs were used as materials. We studied the dynamics of organic matter, organic micro-pollutants and the microbial populations. The conclusion showed that the concentration of PAHs could be decrease by 50-60% with the effects of organic matters and the accelerated microbial activities compared to bare soil. Furthermore, the successive microbial populations have been observed during the composting process. These experimental results could be well simulated by our model which indicates its capacity in the soil-composting system. However, we still came across some questions during my research. The simulations for soluble organic substrates were not accurate enough, due to the limits of the biochemical characterization of organic fractions. In our model, only the function of temperature has been considered as the environmental factor, more limiting factors such as moisture, oxygen, … need to be investigated in the further studies. The microbial diversity of each decomposition phase has been investigated in the experiment but was not added in the model because its impact on PAHs degradation is not clear enough. In order to answer all these questions, further researches needs to be done, although the study in the field of microbial flora has begun to be carried out.