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Methods

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

Penny Worthington, Paul Blum, Francisco Perez-Pomares, Tom Elthon
Penny Worthington
School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, Nebraska
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Paul Blum
School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, Nebraska
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  • For correspondence: pblum@biocomp.unl.edu
Francisco Perez-Pomares
School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, Nebraska
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Tom Elthon
School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, Nebraska
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DOI: 10.1128/AEM.69.1.252-257.2003
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  • FIG. 1.
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    FIG. 1.

    Bioreactor schematic.

  • FIG. 2.
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    FIG. 2.

    Bioreactor temperature regulation. Shown are results with temperature control with prefabricated surface-mounted thermostat and circuit (open circles) and temperature control with immersion thermostat and modified heating control circuit (solid circles).

  • FIG. 3.
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    FIG. 3.

    Bioreactor heating control circuit. Symbols used represent standard electrical devices.

  • FIG. 4.
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    FIG. 4.

    Dependence of S. solfataricus growth on medium pH. (A) Growth curve of S. solfataricus with anode rod in place. (B) Growth curve of S. solfataricus without anode rod. (C) Growth curve of S. solfataricus without anode rod and with acid addition. Symbols: open circles, pH; solid circles, culture absorbance.

  • FIG. 5.
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    FIG. 5.

    Induction of alpha-amylase secretion. The arrow indicates the time of addition of starch to the culture medium. Symbols: solid circles, culture absorbance; open circles, alpha-amylase activity.

  • FIG. 6.
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    FIG. 6.

    SDS-PAGE of fractions eluted from High-S. (A) SDS-PAGE of fractions containing alpha-amylase activity eluted from High-S with an increasing linear sodium chloride gradient. Proteins were visualized by Coomassie blue staining. The arrow indicates the position of the alpha-amylase. (B) Alpha-amylase activity in active fractions. Symbols: open circles, densitometric band intensity of the alpha-amylase; solid circles, alpha-amylase activity.

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  • TABLE 1.

    Yields of hyperthermophilesa

    OrganismDirect cell countOD540Protein (g)
    S. solfataricus 7.0 × 10112,2602.23
    S. shibatae 6.6 × 10111,3201.60
    S. acidocaldarius 2.4 × 10101,1101.28
    • ↵ a Values indicated are normalized to liter culture volumes.

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Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins
Penny Worthington, Paul Blum, Francisco Perez-Pomares, Tom Elthon
Applied and Environmental Microbiology Jan 2003, 69 (1) 252-257; DOI: 10.1128/AEM.69.1.252-257.2003

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Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins
Penny Worthington, Paul Blum, Francisco Perez-Pomares, Tom Elthon
Applied and Environmental Microbiology Jan 2003, 69 (1) 252-257; DOI: 10.1128/AEM.69.1.252-257.2003
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KEYWORDS

Archaeal Proteins
bioreactors
Sulfolobus
alpha-Amylases

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