W.-D. Deckwer

Microbial production of 1,3-propanediol.

Abstract 1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae

In a continuous fermentation of glycerol by Klebsiella pneumoniae, a final propanediol concentration of 35.2–48.5 g l−1 and a volumetric productivity of 4.9–8.8 g l−1 h−1 can be obtained at dilution rates between 0.1 and 0.25 h−1. These results correspond to about 80–96% of the theoretical maxima under ideal conditions (no ethanol and hydrogen formation). The highest propanediol concentration achieved in this study is close to teh maximum propanediol concentration (50–60 g l−1) found in batch and fed-batch cultures. The productivity of the continuous culture is, however, about 2–3.5-fold higher. Further improvement of the glycerol fermentation by K. pneumoniae should be focused on increasing the product tolerance of the strain and reducing the ethanol formation.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation

The initial steps of glycerol dissimilation and 1,3-propanediol (1, 3-PD) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enzymes in continuous culture under conditions of steady state and oscillation and during transient phases. The enzymes studied included glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3-propanediol oxidoreductase (PDOR). Three conclusions can be drawn from the steady-state results. First, glycerol concentration in the culture is a key parameter that inversely affects the in vitro activities (concentrations) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro and in vivo enzyme activities under low glycerol concentrations, but not under glycerol excess. Second, whereas the flux through the oxidative pathway of glycerol dissimilation is governed mainly by the regulation of in vivo enzyme activity on a metabolic level, the flux through the reductive pathway is largely controlled by the synthesis of enzymes. Third, GDHt is a major rate-liming enzyme for the consumption of glycerol and the formation of 1,3-PD in K. pneumoniae at high glycerol concentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions. Experiments with step changes in the feed concentration of glycerol demonstrated that growth and product formation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the genetic and metabolic networks. They should be considered when modeling the dynamics of the culture and attempting to improve the formation of 1,3-PD.

Pathway analysis of glycerol fermentation by Klebsiella pneumoniae : regulation of reducing equivalent balance and product formation

The method of metabolic pathway stoichiometry was used to analyze the fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae DSM 2026, with particular emphasis on the regulation of hydrogen formation and balance of reducing equivalents (NADH2). Under conditions of glycerol limitation, H2 formation was found to be higher than the maximum amount that could be generated from the splitting of pyruvate to acetyl-CoA. Simultaneously, a lower recovery of reducing equivalents was found in the reduced product 1,3-propanediol. Under conditions of glycerol excess, formation of H2 was drastically reduced and a surplus of NADH2 was generated for the formation of 1,3-propanediol. These findings indicate the existence of enzymes in K. pneumoniae that transfer reducing equivalents from NADH2 to H2 and 1,3-propanediol flexibility. The physiological flexibly. The physiological meanings of these regulatory mechanisms are discussed from the viewpoint of bioenergetics and product inhibition. Taking these regulation mechanisms into account, the maximum energy and product yields of this fermentation were calculated, which agreed well with experimental results. A corrected NADH2 balance equation was derived which can be used to calculate the formation of hydrogen under different conditions.

Bacterial alginate: physiology, product quality and process aspects.

Abstract. Alginate, a copolymer of β-D-mannuronic acid and α-L-guluronic acid and currently commercially produced from the marine brown algae, can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas. The ever-increasing applications of this polymer in the food and pharmaceutical sectors have led to continuing research interest aimed at better understanding the metabolic pathways, the physiological or biological function of this polymer, the regulation of its formation and composition, and optimising the microbial production process. These aspects are reviewed here, with particular attention to alginate formation in the soil bacterium A. vinelandii. In addition, the biotechnological and industrial applications of alginate are summarised.

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase

ABSTRACT The activity of nitrogenase in the nitrogen-fixing bacteriumAzotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O2 by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O2 tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O2 tension led to the formation of alginate with a higher molecular weight and a greater l-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O2 transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism

The activities of pyruvate kinase (PK), pyruvate: formate‐lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in the anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, and PDH are strongly affected by the substrate concentration and its uptake rate, as is the in vitro activity of CS. The flux from phosphoenolpyruvate to pyruvate is found to be mainly regulated on a genetic level by the synthesis rate of PK, particularly at low substrate concentration and low growth rate. In contrast, the conversion of pyruvate to acetyl‐CoA is mainly regulated on a metabolic level by the in vivo activities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH under the experimental conditions. The regulation of in vivo activity and synthesis of these enzymes is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo activities. In particular, PFL is strongly affected during oscillations; its average in vitro activity is only about half of its corresponding steady‐state value under similar environmental conditions. The average in vitro activities of PDH and PK under oscillations are close to their corresponding steady‐state values. In contrast to all other enzymes measured for the glycerol metabolism by K. pneumoniae PFL and PDH are more effectively in vivo utilized under oscillations than under steady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture.

Growth inhibition by ammonia and use of a pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum.

The inhibitory effect of ammonia on the growth of the polychlorinated xenobiotic-degrading bacterium Mycobacterium chlorophenolicum was examined. The strain is inhibited by both the ionized and nonionized forms of ammonia. At pH 6.9 50% reduction of the growth rate was observed at 6.8 g l−1 total ammonium. For 23 experiments performed in shake-flask culture at different pH values and ammonium concentrations a growth model based on the extended Monod kinetic fits the data with a deviation of 5.3%. To overcome growth inhibition in bioreactors a pH-controlled feeding strategy was developed for effective cultivation of M. chlorophenolicum at an ammonium level below 0.3 g l−1. The ammonium addition was controlled on-line by the stoichiometric interdependence of ammonium consumption and pH decline. With this on-line control strategy a biomass concentration as high as 26.2 g l−1 can be achieved within less than 1 week of cultivation, compared to a biomass concentration of 15.5 g l−1 in normal batch culture after 2 weeks of cultivation. The yield is also increased from 0.32 g to 0.43 g biomass (g glucose)−1. The strategy developed provides an effective method for the production of biomass of M. chlorophenolicum serving as the inoculum in remediation technologies.

Comparison of the energetic efficiencies of hydrogen and oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during anaerobic growth on glycerol.

Data for the anaerobic growth of Klebsiella pneumoniae DSM 2026 and Clostridium butyricum DSM 5431 on glycerol have been analyzed using the concept of material and available electron balances with consideration for hydrogen production. Models for the kinetics of energetic efficiencies of product formation under low residual glycerol are presented. For Klebsiella pneumoniae, the specific rates of electron transfer to the products were mainly significantly dependent on specific growth rate with the exception of ethanol and hydrogen which were also significantly non-growth associated. In the case of Clostridium butyricum, the rates were only growth rate dependent, except for hydrogen formation. The analysis also indicated that the production of 1,3-propanediol by Klebsiella pneumoniae was favoured by limitations other than glycerol limitation, while hydrogen generation was best under low residual glycerol and particularly in the presence of external 1,3-propanediol. Klebsiella pneumoniae appeared to be able to incorporate more of the available electrons of glycerol into hydrogen as compared with the Clostridium butyricum. The study demonstrates the need for properly considering H2 in models describing anaerobic processes.

PRODUCTION OF 2,3-BUTANEDIOL IN A MEMBRANE BIOREACTOR WITH CELL RECYCLE

SummaryThe production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.