J. L. Vinke

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G.

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.

Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.

Abrasion of suspended biofilm pellets in airlift reactors: importance of shape, structure, and particle concentrations.

The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. (c) 1997 John Wiley & Sons, Inc.

Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor

The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.

Influence of temperature on growth and ajmalicine production by Catharantus roseus suspension cultures

Industrial production of valuable secondary metabolites by plant cell cultures is generally hampered by low productivity. This productivity is controlled by several factors, one of these is temperature. Secondary metabolites are in most cases produced in a two-stage process: biomass growth, followed by secondary metabolite production. In part I of this study the optimal temperature for biomass growth was investigated aiming at: maximal formation of biosynthetic active biomass, minimal formation of useless by-products. These processes each had their characteristic temperature dependence. The growth of Catharanthus roseus biomass for the production of ajmalicine was found to be optimal at 27.5°C. The effects of oxygen limitation are discussed. In part II the temperature effect on ajmalicine production was investigated. The productivity was governed by two processes: induction and production. Induction and production were both optimal at 27.5°C. The length of the induction period was easily estimated from a rapid concentration decrease of the precursor tryptamine.

Gaseous metabolites and the ajmalicine production rate in high density cell cultures of Catharanthus roseus

An important factor in the scale-up of plant cell processes for the production of secondary metabolites is the gas exchange between the gas and liquid phases. In aerobic fermentations, the gas exchange involves mainly two processes: the supply of oxygen and the removal of gaseous metabolites (CO,, ethylene, etc.). The concentrations of dissolved oxygen (DO) and dissolved gaseous metabolites (DGM) which are critical parameters in large-scale fermentations are dependent on mass transfer and mixing in the bioreactor as well as the biomass concentration. Since mass transfer and mixing are dependent on the size of the bioreactor, DO and DGM change during scale-up of the process. Consequently, it is essential for process design, optimization, operation, and scale-up to establish separate relationships between DO and DGM on the one hand and cell growth and secondary metabolite production on the other hand. In the present study, the relation between the DGM concentration and the specific ajmalicine production rate by Catharanthus roseus was investigated. DGM is a collective noun for the various gaseous compounds produced by plant cells. Carbon dioxide and ethylene are important representatives of this group. The effect of the individual compounds

Biofilm abrasion by particle collisions in airlift reactors

Biofilm development is determined by the balance between growth and detachment. The detachment of biomass from suspended biofilm pellets was investigated in three-phase internal loop airlift reactors under non-growth conditions, and in the presence of bare carrier particles. Particle collisions were found to dominate the detachment. These collisions caused an on-going abrasion of the biofilm pellets. The abrasion rate was linear with both the concentration of biofilm pellets and the bare carrier particles up to a solids hold-up of 30%. An increase in particle size drastically increased the abrasion rate. Carrier roughness also strongly influenced the detachment rate. Experimental results were interpreted in terms of collision frequency and collision impact, and could partly be described by conventional collision theory. Particle responses to flow fluctuations, the effect of carrier roughness and the effect of growth rate on biofilm strength and elasticity need to be included. Some consequences for reactor operation and start-up are discussed.