Experimental And Theoretical Study On Multiplicity And Oscillation In Continuous Microbial Cultures

Multiplicity and oscillation in continuous microbial cultures were studied in this work. The work was divided into two parts: experimental results and theoretical analysis. In the first part, the microbial conversion of glycerol to 1,3- propanediol by Klebsie//a pneumoniae was used to investigate the effects of three monovalent cations in media and bases being used to control pH on metabolic flow, to verify multiple steady states and transient response from one steady state to another. In addition, the excessive kinetics was used to simulate the experimental results. The influences of three monovalent cations (Nat, K, and NH4) on bioconversion of glycerol to 1 ,3-propanediol by K/ebsiella pneumoniae were investigated by designing three different media and pH control strategies and discussed in terms of metabolic control. Experimental results showed that glycerol dehydrogenase, one of the key enzymes of glycerol conversion, was inhibited by high concentration of Nat, leading to a decline of yields of biomass and l,3-propanediol on glycerol. It is feasible to adjust pH by using KOH in a medium consisting of enough NH4 or using ammonia in a medium without NH4~ and Na~ in continuous culture. Under substrate sufficient conditions, the generation of bioenergy ATP, cell growth and the production of I ,3-propanediol were increased by adding dihydroxyacetone into glycerol fermentation. This showed that the activity of glycerol dehydrogenase was strongly inhibited under substrate excess. At a dilution rate of 0.35 h1, two steady states occurred under the same conditions when the initial glycerol concentration changed from low to high or from high to low in the range of 400 to 1400mM. If the dilution rate shifted from 0.1 hup to 0.22 fr1 at an initial glycerol concentration of 40 gIL, the residual glycerol concentration in reactor initially increased and thendecreased, and the concentrations of l,3-propanediol and acotate alwaysincreased, but the ethanol concentfation declined.The excess kinetic models fOr substrate consumPtion and product formationwere reexamined for the bioconversion of glycerol to 1,3-propanediol byKlebsiella pneumoniae. Whereas these kinetic models described the substrateconsumption and the formaton of l,3Tropanediol and acetic acid reasonablywell, relative large deviations were observed for the formation of ethanol,especially at low substrate concentrations. A new term, i.e. the substrateconcentration change ratio (Cs. D) was found in this work to be useful fOrdescribing ethanol fOrmation in continuous culture. The resulting kinetic modelsand parameters satisfactorily described both continuous and batch cultures ofglycerol fermentation by K pneumoniae under various conditions. The modelalso predicted the occurence of multiplicity in the bioprocess. Alteration ofmetabolic pathways associated with ethanol fOrmation aPpeared to be themain reason causing multiplicity in a fermentation When transferring fromsubstrate-limiting to substrate-sufficient conditions.The transient responses of Klebsiella pneumoniae to dilUtion rate and glycerolconcentration step changes in continuous bioconversion of glycerol to l,3-propanediol showed that distinguished differences lay in cell growth andintracellular metabolism under differeni conditions. The tfansient behaviordepended on the both states before and after a step change of operationconditions. When substrate concentration transferred from a limiting conditionto a excessive condition, the residual concentration of glycerol in reactorconstamly increased, bat ethanol conceotration decreased gradually asfermelltation time. Simultaneously biomass, l,3-propanediol and acetateconcentration increased firstIy and then decreased.