DEVELOPMENT OF A MECHANISTIC MODEL OF BACTERIAL GROWTH ON METHANOL IN STEADY STATE, CONTINUOUS CULTURE

ELIZABETH LOUISE RYKER, Purdue University

Abstract

A mechanistic model was formulated to describe bacterial growth on methanol. The methylotrophic bacteria of interest were the subgroup which followed the ribulose monophosphate cycle for carbon assimilation. The specific bacterium investigated was an obligate methylotroph (L3) isolated in this laboratory. The growth situation to be described by the model was restricted to steady state growth in continuous cultures with methanol as the sole feed substrate and sole limiting nutrient. In final form, the steady state model contained (1) five mass balance equations for the metabolic species methanol dehydrogenase, hexulose phosphate synthase, cell mass, methanol, and formaldehyde; (2) six reaction rates for the metabolic reactions represented in the model; and (3) sixteen kinetic parameters. The model included cellular regulation in the form of induction and repression of enzyme synthesis, inhibition of enzyme activity, and enzyme degradation. Based on previous observations, formaldehyde was given an important role in the regulatory and metabolic processes. The parameters were estimated by nonlinear regression analysis, effecting a fit of the mass balance equations to data taken from both stable and unstable steady state cultures. Experimental studies were made of a continuous-flow bioreactor with methanol as the feed substrate. The bioreactor was implemented with capabilities of feedback control on cell density by manipulation of feed flow rate. Measurements were made of residual methanol, residual formaldehyde, cell mass concentration, specific methanol dehydrogenase activity, and specific hexulose phosphate synthase activity. After the parameter values were estimated, simulations of steady state behavior were examined. Good qualitative agreement and adequate quantitative agreement were observed between model simulations and the experimental data used in the parameter estimation procedure. As a result, it was concluded that the mechanistic model successfully represented steady state, continuous culture behavior where methanol was the sole feed substrate. The model predicted multiple steady states for a given dilution rate, a growth phenomenon characteristic of methanol-utilizing organisms. In addition, measurements of key enzyme activities in the steady state cultures supported the basic premise in the model that a repression or inhibition of the metabolic machinery in the cell accompanied the occurrence of the unstable steady state.

Degree

Ph.D.

Subject Area

Chemical engineering

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