Muitos organismos vivos, tal como seus constituintes, são formados por sistemas químicos complexos que envolvem a interação entre compostos orgânicos e inorgânicos ligados química e/ou fisicamente. Nestes sistemas as matrizes orgânicas são geralmente compostas por macromoléculas como polissacarídeos e proteínas. Essas moléculas têm o papel de direcionar a nucleação e o crescimento da porção inorgânica. O uso de superfícies metálicas adequadas recobertas por este tipo de filme híbrido tem potencial aplicação em implantes de substituição óssea, no qual são requeridas superfícies quimicamente inertes, mas que ao mesmo tempo estimulem processos de calcificação. Nesta tese estudou-se o crescimento de CaCO3 sobre superfícies metálicas de alumínio e aço inox recobertas por matrizes orgânicas compostas por diferentes poliânions e pelo policátion quitosana, na forma de filmes montados camada-a-camada (do inglês LbL), na presença ou não de fosfolipídeos (filmes Langmuir-Blodgett), formando um meio confinado para o crescimento do mineral. Diferentes técnicas foram utilizadas: microscopia eletrônica, microscopia de força atômica, espectroscopias de reflexão nas regiões do Uv-vis, e do infravermelho, Raman, espalhamento e difração de raios-X. Estudou-se a influência de diferentes grupos carregados dos fosfolipídeos e dos poliânions, tal como sua conformação, no crescimento de CaCO3. O tipo de interação entre o poliânion e a quitosana leva ao crescimento de matrizes poliméricas com diferenças em suas espessuras e capacidade de retenção de líquido, modificando as condições de supersaturação local e influenciando no tipo de estrutura de CaCO3. Puderam ser identificados dois polimorfos formados sobre os filmes orgânicos de poli(ácido acrílico) e quitosana, sugerindo que existem dois diferentes sítios onde a nucleação pode ser iniciada: a partir da solução de CaCl2 aprisionada na matriz polimérica e o outro a partir dos íons cálcio ligados como contra-íons aos grupos negativamente carregados do poliânion. Na presença do pré-recobrimento LB, a natureza da cabeça polar do fosfolipídeo direciona o tipo de ligação e crescimento da matriz polimérica, que levam ao crescimento de partículas de CaCO3 com morfologia e tamanho variados, explicados em termos da presença de ambientes com diferenças de concentrações locais de Ca2+. Além disso, verificou-se que a rugosidade superficial dos suportes metálicos pode favorecer a formação do polimorfo de CaCO3 cineticamente mais estável, mostrando que o processo de cristalização sobre estes suportes é um processo governado por difusão. A hidrofilicidade dos suportes é aumentada pela presença da matriz orgânica e pela presença de CaCO3 sobre as matrizes. O crescimento de CaCO3 em meios confinados tridimensionais, formados por membranas de policarbonato modificadas com filme finos de polieletrólitos, também foi estudado. Este tipo de molde leva à formação de estruturas cilíndricas que seguem a morfologia dos poros da membrana. A presença de poli(ácido acrílico) leva a formação de estruturas cilíndricas ocas, enquanto que cilindros completamente preenchidos foram formados nos poros contendo quitosana na última camada. Estes resultados foram explicados com base em diferenças na etapa de nucleação: na presença de PAA a nucleação de CaCO3 deve iniciar-se a partir dos íons Ca2+ ligados ao poliânion que, por sua vez, está ligado diretamente às paredes do molde; já na presença que quitosana, com maior capacidade de retenção de liquido e sem interação específica com Ca2+ a nucleação e seqüente cristalização devem ocorrer por todo o poro da membrana. As estruturas formadas são em sua maioria monocristais de calcita hexagonal orientadas na direção cristalográfica <2 -2 1>. / Some living organisms as well as their constituents are formed by complex chemical systems which involves the interaction among organic and inorganic compounds bounded physically or chemically. In these systems the organic matrices are usually composed by macromolecules like polysaccharides and proteins. These molecules have an important hole in tailoring the nucleation and the sequent growth of the inorganic portion. Metallic surfaces coated with these hybrid films have potential application as implants for bone substitution for which the surfaces must be chemically inert but at the same time they should stimulate calcification processes. In this present thesis we studied the growth of CaCO3 over aluminium and stainless steal surfaces coated with layer-by-layer films composed by different polyanions and chitosan as polycation, in the presence or not of phospholipids (Langmuir-Blodgett films). These organic matrices formed a confined medium within which CaCO3 particles were growth. Different techniques were applied in order to understand these systems: electronic microscopy, atomic force microscopy, UV-Vis and infrared reflection spectroscopy, Raman, and X-ray scattering and diffraction. We studied the influence of the different charged groups of the phospholipids and the polyanion as well as their conformation on CaCO3 growth. The type of interaction between the polycation and the polyanions tailors the growth of the organic matrices, forming films with different thickness and different water retention abilities which change the local supersaturation conditions changing the structure of the CaCO3 formed. Two types of CaCO3 polymorphs were growth over poly(acrylic acid) (PAA) and chitosan films suggesting that there are two sites where the nucleation can be started: the CaCl2 solution retained in the gel-like organic films and the Ca2+ ions bounded to the negative groups of the polyanion. In the presence of the LB pre-coating, the nature of the phospholipid polar head tailors the binding and the growth of the polymeric matrices leading to the formation of CaCO3 particles with difference in their sizes and morphologies. This result was explained in basis of the differences in the Ca2+ local concentrations in each situation. Moreover, it was observed that the surface roughness of the supports can favour the formation of vaterite, the kinetically most stable CaCO3 polymorph, showing that the crystallization may be guided by diffusion processes. The hidrophilicity of the supports was improved by the presence of both organic and hybrid films. The growth of CaCO3 in tridimentional confined mediums was done using LbL modified polycarbonate membranes as template. This template leads to the formation of cylindrical CaCO3 particles following the morphology of the membrane pores. CaCO3 tube-like structures were formed in presence of PAA, while rod-like structures were formed in presence of chitosan in the top LbL layer. These results were explained on basis of the difference in the nucleation stages: in the presence of PAA the nucleation starts on the Ca2+ ions bounded to the polyanion that is linked to the walls of the template; in the presence of chitosan that presents higher water retention ability and has no specific interaction with Ca2+ ions, the nucleation and sequent crystallization should occur through the entire pore of the membrane. The electron diffraction patterns showed that the CaCO3 structures are single crystals of the calcite polymorph oriented in < 2 -2 1> crystallographic direction.

O processo de biomineralização óssea é a deposição de cristais de fosfato de cálcio, na forma de hidroxiapatita formando o tecido ósseo. São mediados pelas vesículas da matriz (MVs) que são liberadas no local específico do início da biomineralização. As MVs contêm altas concentrações de íons Ca2+ e fosfato inorgânico (Pi), proporcionando um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita. Para que isso ocorra corretamente são necessárias diversas proteínas/enzimas, bem como microambientes com condições específicas. Neste trabalho uma atenção especial foi dada a duas proteínas presentes nas MVs: Anexina V (AnxA5) e a nucleotídeo pirofosfatase/fosfodiesterase-1 (NPP1). A Anexina V é responsável pela formação de um canal de cálcio por meio da associação desta proteína tanto com a face externa quanto interna da membrana das MVs. A NPP1 possui a função principal de hidrolisar adenosina trifosfato (ATP), formando adenosina monofosfato (AMP) e pirofosfato (PPi). Assim, experimentos de expressão de Anexina V e NPP1 foram realizados com o intuito de iniciar os estudos de interação entre as duas proteínas, por meio de reconstituição em lipossomos. A expressão de Anexina V foi realizada em células E. coli BL21(DE3) e induzida por isopropil -D-1-tiogalactopiranosídeo (IPTG); para purificação, três procedimentos foram necessários, utilizando coluna de níquel, coluna Desalting e coluna de troca iônica (Mono-Q). Experimentos de dicroísmo circular foram realizados com amostras de Anexina V após purificação e mostraram que todas as amostras apresentavam estruturas em -hélice. Pelo método da gota pendente foi estudada a interação de Anexina V com íons Ca2+ (10 mM) em monocamadas constituídas por dipalmitoil fosfatidilcolina (DPPC) e de uma mistura de composição lipídica 9:1 de DPPC e dipalmitoil fosfatidilserina (DPPS). Os dados obtidos mostraram alta afinidade de Anexina V por monocamadas constituídas de (9:1) DPPC:DPPS na presença de íons Ca2+. A expressão de NPP1 foi realizada com transfecção por meio de eletroporação do DNA recombinante em células COS-1, e seleção com antibiótico G418 após 24 horas de cultivo. Amostras de fração de membrana controle e NPP1 recombinante foram preparadas após 60 horas de cultivo celular e foi observada atividade catalítica na amostra de fração de membrana da NPP1. Todas as amostras de expressão, tanto de Anexina V e NPP1, foram analisadas por eletroforese em gel de poliacrilamida. A padronização da Anexina V foi obtida com sucesso, porém com relação à NPP1, experimentos ainda devem ser realizados a fim de padronizar a obtenção desta proteína recombinante em quantidade suficiente para continuar os estudos. / The bone biomineralization process is the deposition of calcium phosphate crystals in the form of hydroxyapatite forming the bone tissue. They are mediated by matrix vesicles (MVs) that are released at the specific site of the onset of biomineralization. MVs contain high concentrations of Ca2+ ions and inorganic phosphate (Pi), providing a suitable microenvironment for the initial formation and propagation of hydroxyapatite crystals. To occur properly, several proteins/enzymes are needed, as well as microenvironments with very particular conditions. In this work, special attention should be given to two proteins present in the MVs: Annexin V (AnxA5) and nucleotide pyrophosphatase/ phosphodiesterase (NPP1). Annexin V is responsible for the formation of a calcium channel through the association of this protein with both the outer and the inner face of the MVs membrane. NPP1 has the main function of hydrolyzing adenosine triphosphate (ATP), adenosine monophosphate (AMP) and pyrophosphate (PPi).Thus, experiments of expression of Annexin V and NPP1 were performed with the aim of initiating the interaction studies between the two proteins, through reconstitution in liposomes. Annexin V expression was performed in E.coli BL21 (DE3) and the cells were induced by isopropyl -D-1-thiogalactopyranoside (IPTG); for purification, three procedures were required using a nickel column, a Desalting column and an ion exchange column (Mono-Q). Circular dichroism experiments were performed with Annexin V samples after purification and showed that all samples contain -helix structures. Using the pendant drop method, the interaction of Annexin V with (10 mM) Ca2+ ions was studied in monolayers composed of dipalmitoyl-phosphatidyl-choline (DPPC) and a mixture of lipid composition 9:1 DPPC and dipalmitoyl-phosphatidyl-serine (DPPS). Data showed high affinity of Annexin V by monolayers constituted of (9: 1) DPPC:DPPS in the presence of Ca2+ ions. NPP1 expression was performed with transfection by electroporation of the recombinant DNA into COS-1 cells and selection with G418 antibiotic after 24 hours of culture. Samples of the control membrane fraction and recombinant NPP1 were prepared and the activity of the membrane fraction of NPP1 was observed in the samples. All expression samples, both AnxA5 and NPP1, were analyzed by polyacrylamide gel electrophoresis. The standardization of Annexin V has been obtained with success, but regarding NPP1, experiments have yet to be performed to standardize the production of this recombinant protein and to obtain enough quantity to continue the study.

Bone is the most commonly replaced organ, with nearly 1 million grafting procedures performed annually in the United States. Inherent limitations associated with bone grafts, such as graft availability and donor site morbidity, leave room for alternative grafting solutions. Current mineralized tissue engineering approaches include the use of synthetic hydroxyapatite as cement or as nano- or micro-particles pre-incorporated into a tissue engineering scaffold prior to cell seeding or implantation. While promising results have been reported with such methods, these constructs are not biomimetic as they fail to replicate neither the size, distribution, nor density of mineral inherent in the native bone, leading to inferior mechanical properties and supra-physiologic levels of calcium phosphate that can disrupt healing, alter cell response and inhibit normal tissue homeostasis. To address these issues, inspiration is taken from the native biomineralization process which is often facilitated by matrix vesicles, a lipid-based nanocarrier within which calcium and phosphate ions are combined to form calcium phosphate mineral in hard tissues such as bone. Synthetic matrix vesicles (SMV) formulated from self-assembling liposomes have emerged as a promising model both for studying the biomineralization process as it relates to matrix vesicles and for use in regenerative medicine. The ideal SMV system is defined as follows: the mineral formed should match the native calcium phosphate in both structure and chemistry, the mineral must be stable in the physiological environment and can continue to grow in size when necessary and the matrix vesicles should also be able to work in conjunction with a scaffold tailored for bone tissue engineering. It is hypothesized that the formation of native bone-like calcium phosphate can be achieved with the controlled optimization of matrix vesicles in terms of fabrication parameters, ion transport, cell response and interactions with a gelatinous matrix. To this end, a liposome-based, biomimetic matrix vesicle system was designed to facilitate vesicle-mediated biomineralization for regeneration of calcified tissues. Synthetic matrix vesicles were fabricated from two different phospholipids, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DMPC (1,2-bis(myristoyl)-sn-glycero-3-phosphocholine) and optimized in terms of membrane composition, alkaline phosphatase bioconjugation, and ion encapsulation. Calcium (Ca2+) and phosphate (Pi) ions were successfully encapsulated within the liposomes. Ion permeability across the bi-layer membrane, which is necessary for Ca2+ and Pi to combine within the SMV for mineralization, was found to increase with increasing DMPC composition, validated through ion release studies and diffusion modeling through Fick's 2nd Law. In addition, alkaline phosphatase (ALP), an enzyme which cleaves Pi from organic phosphate molecules for mineral formation with Ca2+, was successfully conjugated to the SMV membrane through the use of biotin-functionalized phospholipids and streptavidin-ALP. Human osteoblast-like cells were dosed with the optimized SMV and the effects of SMV type and dosage on mineralization response was evaluated. Mineralization potential of human osteoblast-like cells was found to decrease through exposure to Pi-encapsulated SMV similar to the response found for human osteoblast-like cells supplemented with beta-glycerophosphate (beta-GP), an organic phosphate source typically used in mineralization in vitro studies. Human osteoblast-like cells were also dosed with two different configurations of ALP SMV liposomes with ALP bound within (ALP-inside SMV) and liposomes with ALP bound to the membrane on the outside (ALP-outside SMV). ALP-outside SMV were ultimately selected for further study since the location of the ALP in the outside configuration more closely mimics the structure of native matrix vesicles. While mineral-like structures were observed in several types of SMV under cryo-electron microscopy, no bulk mineralization was observed by human osteoblast-like cells from SMV supplementation alone. This motivated a dosage study conducted with the Pi SMV which optimized the cell-to-liposome ratio and the concentration of Pi encapsulated. The optimized ALP-outside SMV and Pi SMV were individually combined with an electrospun gelatin nanofiber scaffold to further promote cell mineral deposition by acting as a biomimetic substrate for calcium phosphate nucleation. It was demonstrated that in the absence of growth factor stiumulation, culture of human osteoblast-like cells with SMV+beta-GP and Pi SMV resulted in mineral deposition on the gelatin nanofiber scaffold. Human mesenchymal stem cells (hMSC), a more clinically relevant cell type, were also cultured on the SMV-gelatin scaffold system. Mineralization potential was found to increase for hMSC cultured with ALP SMV, and the osteogenic marker osteocalcin was upregulated for cultures with Pi SMV. Dosage of hMSC with SMV+beta-GP and Pi SMV alone resulted in the formation of a mineralized matrix. In summary, this thesis focuses on the design of a biomimetic, liposome-based synthetic matrix vesicle system and elucidates the compositional and dosage parameters for the formation of calcified tissue by human osteoblast-like cells and MSCs. The synthetic matrix vesicle system developed in this thesis can be utilized for further investigation into the mechanisms of biomineralization, in addition to its potential for use in promoting cell-mediated regeneration of a variety of calcified tissues, including bone, teeth and mineralized soft-to-hard tissue interfaces.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 119-136).

Several nanomaterials systems, both inorganic and organic in nature, have been extensively investigated by a number of characterization techniques including atomic force microscopy (AFM), electron microscopy, Fourier transform infrared spectroscopy (FTIR), and energy dispersive x-ray spectroscopy (EDX). The first system consists of boron nitride nanotubes (BNNTs) synthesized via two different methods. The first method, silica-assisted catalytic chemical vapour deposition (SA-CVD), produced boron nitride nanotubes with different morphologies depending on the synthesis temperature. The second method, growth vapour trapping chemical vapour deposition (GVT-CVD), produced multiwall boron nitride nanotubes (MWBNNTs). The bending modulus of individual MWBNNTs was determined using an AFM three-point bending technique, and was found to be diameter-dependent due to the presence of shear effects. The second type of nanomaterial investigated is the biomineralized calcareous shell of the serpulid Hydroides dianthus. This material was found to be an inorganic-organic composite material composed of two different morphologies of CaCO3, collagen, and carboxylated and sulphated polysaccharides. The organic components were demonstrated to mediate the mineralization of CaCO3 in vitro. The final system studied is the proteinaceous cement of the barnacle Amphibalanus amphitrite. The secondary structure of the protein components was investigated via FTIR, revealing the presence of β-sheet conformation, and nanoscale rod-shaped structures within the cement were identified as β-sheet containing amyloid fibrils via chemical staining. These rod-shaped structures exhibited a stiffer nature compared with other structures in the adhesive, as measured by AFM nanoindentation.

Diatoms are unicellular photosynthetic algae that display intricately patterned cell walls made of amorphous silicon dioxide (silica). Long-chain polyamines and highly phosphorylated proteins, silaffins and silacidins, are believed to play an important role in biosilica formation. The phosphate moieties on silaffins and silacidins play a significant role in biomineral formation, yet no kinase has been identified that phosphorylates these biomineral forming proteins. This dissertation describes the characterization of a novel kinase from the diatom Thalassiosira pseudonana, tpSTK1, which is upregulated during silica formation. A recombinantly expressed histidine-tagged version of tpSTK1 was capable of phosphorylating recombinant silaffins but not recombinant silacidin in vitro. Through establishing methods for subcellular fraction of T. pseudonana membranes in combination with antibody inhibition assay, it was discovered that native tpSTK1 phosphorylates silaffins but not silacidins in vitro (i.e. it exhibits the same substrate specificity as recombinant tpSTK1). As tpSTK1 is an abundant protein in the ER lumen (~ 0.5 % of total ER protein) it seems highly likely to function as a silaffin kinase in vivo. TpSTK1 lacks clear sequence homologs in non-diatom organisms and is the first molecularly characterized kinase that appears to be involved in biomineralization. The predicted kinase domain (KD) of tpSTK2, the only T. pseudonana homolog of tpSTK1, was recombinantly expressed and tested for phosphorylation activity. Recombinant tpSTK2-KD and native tpSTK2 exhibited detectable activity with myelin basic protein, but did not phosphorylate silaffins or silacidins in vitro. Western blot analysis demonstrated that native tpSTK2 was not present in the ER, but associated with the cytosol and Golgi membrane containing subcellular fractions.

Thesis (Ph.D)--Earth and Atmospheric Sciences, Georgia Institute of Technology, 2010. / Committee Chair: Taillefert, Martial; Committee Member: DiChristina, Thomas; Committee Member: Sobecky, Patricia; Committee Member: Van Cappellen, Philippe; Committee Member: Webb, Samuel. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Seven novel, amphiphilic AApeptides were prepared. Two cationic, lipo-α-AApeptides, NA-75 and NA-77 were found to possess potent antimicrobial activity against Gram-positive bacteria, with almost no hemolytic activity. In addition to NA-75 and 77, four amphiphilic, γ-AApeptides, NA-133, 135, 137, and 139, and one anionic lipo-α-AApeptide, NA-81, were prepared for molecular self-assembly studies, with several interesting nanostructures observed by TEM. Mineralization of calcium carbonate from gaseous CO2 and Ca2+ in the presence of the 7 AApeptide amphiphiles was also observed by optical microscopy. Several AApeptides were found to be able to influence CaCO3 crystal morphology. Another α-AApeptide, NA-63, was synthesized by a novel, alternative method, which has several potential advantages over the previous synthesis methods.

Materials deposited in low-temperature environments are characterized by their small particle size. The activity of microorganisms in aqueous sedimentary environments may have effective impacts on the geochemical parameters, which consequently change the original mineralogical records. The characterization of the fine environmental materials, especially those related to the microbial mediation is usually overlooked, but is essential for studies in the field of environmental microbiology or records of environmental change. The Kamchatka volcanic hot springs have been extensively studied for their microbiology because of their unique geological setting and applications to our understanding of life in the extreme environment. In those hot springs, minerals commonly appear with various morphologies. The hot springs have been existed for 40,000 years. Authigenic minerals, such as clay minerals, silica, sulfur, sulfide, and sulfate were characterized. Two types of silica and silicified biota were observed. Elemental sulfur crystals were observed although they are thermodynamically unstable. Pyrite and gypsum showed high diversities in morphologies and crystal sizes. Single forms of pyrite crystals included: cube, pyritohedron, octahedron, and sphericities. Prismatic, prismatic pseudo-hexagonal, fibrous, tubular, lenticular and twinned gypsum crystals were observed. The co-existence of diverse crystal habits of gypsum implies a long-term interaction between hot spring geochemistry and the metabolisms of the microbial community. The morphology of gypsum in hot springs was compared with that of gypsum with hydrothermal genesis in Lower Cambrian black shale. The crystallization of gypsum in the black shale of the Lower Cambrian, which shows similar but less varied morphology, was influenced by post-depositional hydrothermal fluids. I suggest that the high diversities of the morphology and crystal size of gypsum in those hot springs represent the continuous mediation of microorganism, which could be used as mineral ecophysiological records of life not only on Earth, but on Mars. Comparatively, the Pearl River Delta sedimentary environments are characterized by low-temperature and different mineralogical assemblages. Based on lithological records and dating data, C/N and organic carbon isotope are used to identify alterations between terrestrial and marine depositional environments in borehole transect that build up seven stages of sedimentary records. Secondly, element intensities taken by X-ray fluorescence core scanner and %Fe(III) variation implied different sedimentary environment records (terrestrial and marine) in HKUV15. Detrital minerals and authigenic minerals (gypsum, opal, and pyrite) were observed and characterized by scanning electron microscope. Diatom and coccolithophorid genera were identified. Most of mineralogical and biological records showed depositional environments consistent with geochemical records. In the Pearl River Delta sedimentary samples, the microbial mediated mineralization only contributed a small fraction to the mineral assemblages, while in the hot springs, the microbial mediation had more influences on the nucleation of authigenic minerals, which was reflected by the high diversity of crystal size and morphology of sulfates, sulfides, and siliceous minerals. / published_or_final_version / Earth Sciences / Doctoral / Doctor of Philosophy

Arsenic (As) is a highly toxic chemical that is widely distributed in groundwater around the world. As-bearing sulfide minerals (ASM) are known to contribute to high background concentrations of As in groundwater in regions where the geochemistry of the parent material is dominated by sulfide minerals. The fate of As in groundwater depends on the activity of microorganisms which can oxidize arsenite (Asᴵᴵᴵ), or reduce arsenate (Asᵛ). In oxidizing environments, Asᵛ is the predominant species, and the accumulation of As is limited by the sorption of As onto iron (Fe) oxides and hydroxides. Under reducing environments, Asᴵᴵᴵ is the predominant specie, and while the sorption strength of Asᴵᴵᴵ on the Fe-surface of Fe (oxy)hydroxides is weaker, the accumulation of As in water can be limited by the precipitation of As as part of an ASM. The main aim of this research is to study the impact of microbial activity on the mobilization and immobilization of As in the environment. The first objective of this research was to characterize the metabolic activity of three Asᴵᴵᴵ-oxidizing bacteria, Azoarcus sp. pb-1 strain EC1, Azoarcus sp. pb-1 strain EC3 and Diaphorobacter sp. pb-1 strain MC, isolated from a non-contaminated, pristine environment. These Asᴵᴵᴵ-oxidizing bacteria demonstrated a great metabolic flexibility to use oxygen and nitrate to oxidize Asᴵᴵᴵ as well as organic and inorganic substrates as alternative electron donors (e-donors) explains their presence in non-As-contaminated environments. The findings suggest that at least some Asᴵᴵᴵ-oxidizing bacteria are flexible with respect to electron-acceptors and e-donors and that they are potentially widespread in low As concentration environments. The second objective of this research was to investigate the stability of orpiment (As₂S₃) and arsenopyrite (FeAsS), at circumneutral pH and 30°C, under aerobic- and or anoxic conditions (nitrate amended as electron acceptor (e-acceptor)), in order to assess the feasibility of immobilizing As by formation of ASM as a long-term option for the bioremediation of As contamination. The percentage of As released from the minerals ranged from zero when FeAsS was biologically incubated to 87% for As₂S₃(s) under anoxic abiotic conditions. While the dissolution of ASM was greater in biological conditions, the presence of inoculum provided as sludge served as a sink for As, limiting the mobilization of As into aqueous phase. Thus, the mobilization of As from ASM can be controlled by altering the environmental conditions such as the redox conditions or by stimulating microbial activity. Further research investigated the formation of ASM catalyzed by biological reduction of Asᵛ and sulfate (SO₄²⁻). In particular, the third objective of this research was to study the effect of the pH on the removal of As due to the biological-mediated formation of ASM in an iron-poor system. A series of batch experiments were performed to study the reduction of SO₄²⁻ and Asᵛ by an anaerobic mixed culture in a range of pH conditions (6.1-7.2), using ethanol as the e-donor. A marked decrease of the total aqueous concentrations of As and S and the formation of a yellow precipitate was observed in the inoculated treatments amended with ethanol, but not in the non-inoculated controls, indicating that the As-removal was biologically mediated. The pH dramatically affected the extent and rate of As removal, as well as the stoichiometric composition of the precipitate. The precipitate was composed of a mixture of orpiment and realgar, and the proportion of orpiment in the sample increased with increasing pH. The results suggest that ASM formation is greatly enhanced at mildly acidic pH conditions. The fourth objective was to investigate the biomineralization of As through simultaneous Asᵛ and SO₄²⁻ reduction in a minimal iron environment for the As-contaminated groundwater bioremediation. A continuous bioreactor, inoculated with an anaerobic sludge was maintained at circumneutral pH (6.25-6.50) and fed with Asᵛ and SO₄²⁻, utilizing ethanol as an e-donor for over 250 d. A second bioreactor running under the same conditions but lacking SO₄²⁻ was operated as a control to study the fate of As removal. The reactor fed with both Asᵛ and SO₄²⁻ removed on the average 91.2% of the total soluble As, while less than 5% removal was observed in the control bioreactor without S. The biomineralization of As in the bioreactor was also evident from the formation of a yellow precipitate made of a mixture of As₂S₃ and AsS minerals. These results taken as a whole indicate that a bioremediation process relying on the addition of a simple, low-cost e-donor offers potential to promote the removal of As from groundwater by precipitation of ASM. The fifth objective was to evaluate the toxic impact that the exposure to soluble As or the formation of ASM could have on the anaerobic mixed culture used as inocula. The methanogenic community on the reactors was impacted by addition of As. The biogenic ASM inhibited the acetoclastic methanogens causing an accumulation of acetate. In the SO₄²⁻-free bioreactor, the methanogens were initially highly sensitive to Asᴵᴵᴵ (formed from Asᵛ reduction) but quickly adapted to its toxicity. Consequently, the formation of ASM would impact the methanogenic activity of an anaerobic biofilm, while the exposure to Asᴵᴵᴵ would not have a negative impact if the biofilm undergoes adaptation. The sixth and final objective was to study the stability of a biogenic ASM at two different pH values (6.5 and 7.5) and under different redox conditions. The long-term stability was evaluated in three different bioreactors that operated for 145 d: aerobic (R1), anoxic (nitrate as alternative e-acceptor (R2) and anaerobic (R3). The dissolution of ASM was greatly affected by the pH, and slightly by the presence and nature of the e-acceptor. The ASM was very stable at pH 6.5, however, the As mobilization rate was up to 7-fold higher at pH 7.5, likely due to the formation of thioarsenic species. The stability of ASM was also impacted by the e-acceptor present. The As mobilization rate was 77% higher under anaerobic conditions than under aerobic conditions, most likely due to the formation of secondary As-bearing minerals. Therefore, the stability of ASM depends on the conditions of the operation, and it can be controlled by altering the environmental conditions, such as the pH or the presence of the e-acceptor.