Recent interest in Thiobacillus thiooxidans has arisen from its central role in rapid, costly corrosion of concrete sewers. This study focuses on biochemical and ultrastructural responses of the intact cells and isolated carboxysomes, the polyhedral inclusions and CO₂ fixation sites, of the chemolithoautotroph to chemical inhibitors. Inhibition experiments were conducted in pure, batch cultures, grown in a basal salts medium using elemental sulfur as the energy source. D-ribulose 1,5-bisphosphate carboxylase (RuBPCase), the key enzyme of CO2 assimilation in T. thiooxidans, was chosen as the target for chemical inhibition. Observations were based on multiple metabolic measurements of cell growth, acid production, O2 respiration, CO₂ assimilation, intracellular ATP, and subcellular ultrastructure. Weak organic acids proved capable of inhibiting thiobacillus metabolism. Bacterial sensitivity was strongly dependent upon culture pH relative to the respective pKₐ values. Pyruvate and oxaloacetate were strong growth inhibitors. 2-c-carboxy-Darabinitol 1,5-bisphosphate (CABP) and hydroxylamine blocked in vivo CO2 assimilation and growth of T. thiooxidans without affecting on bacterial respiration. Evidence that the primary site of the selective inhibition lies on the biosynthetic side was supported by measurements of intracellular ATP and transmission electron microscopy (TEM). Dimethyl sulfoxide (DMSO) substantially promoted CABP inhibition of CO2 fixation by increasing cell membrane permeability. Carboxysomes were observed in intact cells of T. thiooxidans and characterized in the isolated form. Cell partitioning experiments showed that RuBPCase is sequestered and concentrated in these polyhedral inclusions. TEM observations were performed in conjunction with inhibitor studies. Treatment with the specific inhibitors, such as CABP and hydroxylamine, more than doubled the numbers of carboxysomes per cell without altering the shape and structure of the inclusion bodies, while effectively blocking both in vivo and in vitro CO₂ fixation. In contrast, non-specific inhibitors (cyanide, etc.) caused general intracellular disorder in thiobacilli and structural damage among isolated carboxy somes at concentrations that inhibited metabolic activities. Results suggest that by targeting critical, unique biochemical features of the acidophilic thiobacilli, it is possible to selectively inhibit these organisms, thereby mitigating the severity of sewer corrosion, without affecting general sewer biota or endangering down-stream biological wastewater treatment operations.
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