Biodegradation of phenol and metabolic intermediates by the haloalkaliphile Halomonas campisalis in saline and alkaline solutions

The first part of the present dissertation is devoted to the description of halophilic and alkaliphilic bacteria and the impact and potential use of these unique microorganisms in environmental biotechnology.; The second part of this dissertation is the result of the investigation performed with Halomonas campisalis to transform a model waste compound, phenol, in alkaline and saline conditions. Phenol was degraded as the sole source of carbon and energy at pH 8 through 11 and 0 through 150 g/L NaCl. Metabolic intermediates catechol, cis, cis-muconic acid, and (+) muconolactone were identified, thus indicating that phenol was degraded via the β-ketoadipate metabolic pathway. Although phenol and catechol were completely degraded in all cases, small amounts of the valuable chemical cis, cis-muconic acid accumulated proportionally to increases in pH. These results indicate that it may be feasible to use haloalkaliphiles for the treatment of saline and/or alkaline phenolic wastewater.; The third part of this dissertation presents the result of research done with the muconate lactonizing enzyme (MLE) from H. campisalis. This enzyme plays an important role in the biodegradation of aromatic compounds, by transforming cis, cis-muconic acid into (+) muconolactone. Cis, cis-muconic acid was completely transformed into (+) muconolactone at pH 7 through 9 and 0 through 0.6 M NaCl. It appears that both NaCl and pH exert an inhibitory effect on MLE activity since V_maxapp, obtained from the Michaelis-Menten equation, decreased and K_mapp values increased with increasing pH and salinity. When the ionic strength was increased (i.e. NaCl), enzyme activity decreased following a mixed inhibitory model where chloride ions seem to be the cause of inhibition, perhaps by competing with the negatively charged cis, cis-muconic acid for the positively charged active site. These results indicate that the H. campisalis MLE is neither alkaliphilic nor halophilic. This is not surprising; since Halomonas use osmoregulatory solutes (e.g. ectoine) to balance osmotic pressure and internal cell pH is typically 2 units below external for haloalkaliphiles.