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Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins

Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins,10.1128/AEM.69.1.252-257.2003,Applied and Environmental Mic

Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins   (Citations: 6)
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An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium compo- nents. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins. Geothermal environments are often highly acidic. Such ex- treme environments harbor a wide range of acidophilic hyper- thermophilic organisms including members of both the bacte- rial and archaeal prokaryotic subdivisions. Among the archaeal representatives, members of the Sulfolobus genus have been most intensively characterized. These organisms can grow che- moheterotrophically on reduced carbon compounds (6, 9, 17) and lithoautotrophically on reduced sulfur and carbon dioxide (3, 23). This metabolic versatility is thought to contribute at least in part to the low pH of their growth environment (2), and this together with the high temperature of cultivation creates unique challenges for their physiological manipulation. Studies of the biomolecules produced by acidophilic hyper- thermophiles require sufficient biomass to enable biochemical studies (7, 13, 16, 20, 21). However, large-scale systems suitable for the cultivation of these organisms are not generally avail- able. Conventional microbial fermentors or bioreactors are fabricated out of stainless steel. Stainless steel is not appropri- ate for cultivation of thermoacidophiles, however, because it rusts. To avoid this problem, it is necessary to minimize steel or stainless steel surface area, particularly for reactor components that cannot be replaced, such as the reactor itself. Fermentors must instead be lined with glass or ceramic materials to cir- cumvent metal oxidation, resulting in significant added reactor cost. One solution to this problem has been the use of plastic or rubber containers placed within secondary containment ves- sels to allow for reactor insulation. A fabricated lid provides reactor access, and temperature is controlled externally with a hot plate or heating jacket or internally with an immersible heater and thermostat (8, 14, 19). Both Sulfolobus shibatae and Sulfolobus acidocaldarius were cultivated in this manner with complex media and prolonged incubation periods. The result- ing biomass was used for the isolation and analysis of intracel- lular proteins. However, as yet unexamined in such alternative cultivation systems are process control parameters like pH and temperature variation, physiological indicators including growth rate and specific yield, and cultivation criteria such as medium composition. Perhaps because of these issues, the application of these alternative cultivation methods to studies of physiologically regulated processes has remained untested. Enzymes secreted by hyperthermophiles seldom accumulate in culture supernatants to significant levels (5, 18). These se- creted enzymes include various hydrolytic activities including glycosyl hydrolases like the secreted alpha-amylases from Sul- folobus solfataricus (10-12). Synthesis of this enzyme is highly regulated and is dependent upon the maintenance of specific growth temperature and pH values as well as medium compo- sition. The low abundance of such proteins, however, presents a significant technical barrier complicating detailed analysis of their structure, function, and regulation. To overcome this constraint, simplified methods for the preparation and process- ing of acidophilic hyperthermophilic cultures were developed. These included the development of a low-cost bioreactor, a modified culture medium enabling the use of concentrated stocks, and a high-throughput purification step based on the use of a strong cation-exchange resin.
Journal: Applied and Environmental Microbiology - AEM , vol. 69, no. 1, pp. 252-257, 2003
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    • ...Some have cited difficulties with continuous measurements of pH and redox potential (Nemati et al. 2000) or with pH and oxygen supply regulations (Nicolaus et al. 1991) due to the harshness of the environment for probes, while others have cited problems of evaporation of liquid medium (Park and Lee 1997; Worthington et al. 2003) and a fortiori relatively volatile...
    • ...bioreactors (Nicolaus et al. 1991; Park and Lee 1997; Nemati et al. 2000; Worthington et al. 2003) were successfully overcome...

    Pierre Christenet al. Phenol biodegradation by the thermoacidophilic archaeon Sulfolobus sol...

    • ...Sulfolobus solfataricus strains were grown with aeration at 80°C in the medium of Allen (1) as modified by Brock et al. (8) at a pH of 3.0 in 250-ml screw-cap flasks as described previously (22, 30)...

    James Schelertet al. Regulation of Mercury Resistance in the Crenarchaeote Sulfolobus solfa...

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