Metabolic responses ofPseudomonas stutzeriandEscherichia colisubjected to starvation and surplus of carbon/energy substrates in bioreactors

Sammanfattning: Transient nitrite accumulation during anaerobicdenitrification is a widespread phenomenon for manydenitrifying bacteria includingPseudomonasstutzeri. Nitrate starvation caused the degradation ofthe second denitrifying enzyme nitrite reductase (NiR) inP.stutzeri. A relationship exists between the nitratestarvation time (0-24 hr) and the lost NiR activity, whichaccounts for the varying degrees of nitrite accumulation(0-100%). When nitrate was re-supplied, the cell synthesizednew NiR and thus its activity was restored. A possible releaseof gaseous intermediates (nitric oxide and nitrous oxide) underthese conditions was examined by detecting the NOaqusing an ISO-NO sensor. No significant amount ofNOaqwas found. An integrated nitrate over-flow model,which included both conventional enzymatic kinetics and dynamicenzyme degradation/re-synthesis, was developed and used tosimulate the variable degrees of transient nitrite accumulationbyP.stutzeri.Another metabolic over-flow phenomenon, the aerobic acetateformation inEscherichiacolibatch and fed-batch culture, was also investigatedand modeled. Acetate inhibited the specific growth rate in anon-competitive manner. In the presence of growth-limitingglucose, the cell co-utilized previously formed acetate. Adynamic glucose over-flow model, which described the differentintracellular carbon flows and included the inhibitory effectsof acetate on substrate and oxygen uptake, is presented. Thismodel accurately describes cell growth, respiration, acetateformation and re-consumption during batch cultures, thetransitions of batch to fed-batch and the fed-batchcultures.Metabolic and stress response to hypothetical oxygendeficient zones in a large-scale fed-batch cultivation wasinvestigated. By using a scale-down two-compartment reactor,typical mixed-acid products such as D-lactate, formate andacetate were produced at high rates in the high glucoseconcentration zone (PFR reactor). All D-lactate, almost allacetate and 90% of formate produced in the PFR werere-assimilated by the glucose-limited and oxygen-sufficientcells in the STR. The steadily accumulating formate wassuggested to be an indicator of local oxygen depletion in anaerobic fed-batch process. The commonly observed loweredbiomass yield in large scale reactors, as compared to that of alab scale reactor, is suggested to be at least partly due tothe repeated production and re-assimilation of mixed-acidproducts. Under these heterogeneous conditions, also mRNAlevels of four stress genes (clpB,dnaK,uspA andproU) and three genes responding to oxygen limitationand/or glucose excess (pfl,frd, andackA) were investigated. In the STR reactor, theproU mRNA level was high when glucose uptake rate washigh; theackA mRNA level correlated with the production andconsumption phases of acetate. When batch glucose and acetatewere completely consumed,proU andackA mRNA levels dropped to zero. In the PFR reactor,the mRNA levels of genes that respond to glucose excess (ackA), to oxygen limitation (ackA,pfl) and to heat shock (dnaK andclpB) were significantly elevated within seconds. In thelarge-scale reactor, the level of stress mRNAs was, in mostcases, highest in the middle and lowest at the bottom of thefermentor. It is suggested that also these stress responsescontribute to a reduced biomass yield in large scaleprocesses.Key words:Pseudomonasstutzeri,Escherichiacoli, denitrification, over-flow metabolism, batch,fed-batch, aerobic, anaerobic, glucose, acetate, mathematicalmodel, simulation, scale-down, concentration gradients,mixed-acid fermentation, mRNA, stress genes.