Kinetic and Stoichiometric Analysis of Hairy Roots in a Segmented Bubble Column Reactor

Abstract

Hairy roots of Atropa belladonna were cultured in a modified 2.5‐L multicompartment bubble column for analysis of growth kinetics, stoichiometry, and atropine production. Average biomass density reached 9.9 g L−1 dry weight after 43 days of batch culture; local root densities in some parts of the vessel were considerably higher, up to 17 g L−1. Bulk mixing in the reactor was very poor: after 14 days of culture, the time taken to reach 95% of the equilibrium value after a concentration pulse in the vessel was 12 min. Growth and specific sugar uptake rates declined continuously throughout the culture even though adequate sugar and nitrogen remained in the medium. The observed biomass yield from sugar was approximately constant at 0.35 g g−1; biomass yields from ammonia and nitrate were 0.44 and 0.35 g mmol−1, respectively. Specific atropine content in the roots varied from 4.1 mg g−1 dry weight at the beginning of the culture to 1.4 mg g−1 after 28 days; 35 mg or 14 mg L−1 atropine was produced over the 43‐day culture period. Biomass composition was represented by the elemental formula CH1.63O0.80N0.13, plus 9.8% (w/w) ash. A balanced stoichiometric equation was developed for hairy root growth; this indicated that 8.3% of carbon supplied to the culture was excreted into the medium as byproducts.

Using an aerosol deposition model to increase hairy root growth in a mist reactor

“…The reasons why the growth rate slows at this early stage (4-6 days) in the small mist reactor are not known; however, it is not completely unexpected. Hairy roots of a range of plant species have been reported to show exponential growth only during the first few days of culture (Bhadra et al, 1993;Doran, 1994;Hilton and Rhodes, 1993;Kanokwaree and Doran, 1997;Kwok and Doran, 1995).…”

Growth dynamics of Artemisia annua hairy roots in three culture systems

“…Although a similar approach can be used in reactors (Kwok and Doran, 1995), it is generally too expensive in terms of time, labor, equipment, and space. For hairy root cultures, where it is not possible to directly sample a representative population, developing reliable aseptic methods to estimate biomass in a growing culture has been a challenge (Ramakrishnan et al, 1999).…”

“…Nutrient utilization rates for hairy root cultures are usually reported as the apparent biomass yield-the ratio of the amount of nutrient consumed to the increase in biomass (Bhadra and Shanks, 1997;Kwok and Doran, 1995). The apparent yield can be further subdivided into the actual yield, a conversion efficiency of the nutrient consumed for biomass gain, and the maintenance coefficient, the nutrient required to maintain viability of existing biomass (Schnapp et al, 1991).…”

Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations

“…Because dissolved oxygen tensions may be signi®cantly lower inside hairy root clumps than in the bulk medium (Yu et al, 1997), and as hairy roots have relatively high external critical oxygen tensions for growth and respiration (Shiao and Doran, 2000;Williams and Doran, 1999), delivery of oxygen is likely to become inadequate in submerged root systems. The consequences of oxygen limitation in hairy root cultures include reduced nutrient uptake and lower rates of growth and product synthesis (Kanokwaree and Doran, 1998;Kwok and Doran, 1995;McKelvey et al, 1993).…”

“…Accordingly, a range of prototype reactor con®gurations has been described and tested for hairy roots. These include liquid-dispersed or nutrient mist reactors (Buer et al, 1996;Liu et al, 1999;Williams and Doran, 2000;Wilson, 1997), reactors with multiple aeration outlets or distributed aeration throughout the vessel volume (Kanokwaree and Doran, 1998;Kwok and Doran, 1995), rotating drum and radial-¯ow (Kino-oka et al, 1999) reactors, and reactors with internal supports for immobilizing hairy roots (Holmes et al, 1997;Kwok and Doran, 1995;Mukundan et al, 1998;Taya et al, 1989;Wilson, 1997).…”