Hanno Biebl

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.

Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends.

The need for a sustainable resource supply, the rapid advances in plant biotechnology and microbial genetics and the strategic shift of major chemical companies into the area of life sciences are some of the driving forces for renewed interest in producing bulk chemicals from renewable resources by biological processes. The microbial production of 1,3-propanediol as briefly reviewed in this article and compared with the competing chemical processes demonstrates the promise and constraints of bioprocesses for bulk chemicals. The new concept of biorefinery and biocommodity engineering and future research needs in this area are also outlined.

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

SummaryGlycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %.

Glycerol conversion to 1,3-propanediol by newly isolated clostridia

SummaryFrom pasteurized mud and soil samples glycerol-fermenting clostridia that produced 1,3-propanediol, butyrate and acetate were obtained. The isolates were taxonomically characterized and identified as Clostridium butyricum. The most active strain, SH1 = DSM 5431, was able to convert up to 110 g/l of glycerol to 56 g/l of 1,3-propanediol in 29 h. A few Clostridium strains from culture-collections (3 out of 16 of the C. butyricum group) and some isolates of Kutzner from cheese samples were also able to ferment glycerol, but the final concentration and the productivity of 1,3-propanediol was lower than in strain SH1. Strain SH1 grew well in a pH range between 6.0 and 7.5, with a weak optimum at 6.5, and was stimulated by sparging with N2. Best overall productivity was obtained in fed-batch culture with a starting concentration of 5% glycerol. In all fermentations the yield of 1,3-propanediol in relation to glycerol was higher than expected from NADH production by acid formation. On the other hand the H2 production was lower than expected, if per mole of acetyl coenzyme A one mole of H2 is released. The observations point to a substantial transfer of reducing potential from ferredoxin to NAD, which finally results in increased 1,3-propanediol production.

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.

Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat

SummaryThe fermentation of glycerol to 1,3-propanediol, acetate, and butyrate by Clostridium butyricum was studied with respect to growth inhibition by the accumulating products. The clostridia were grown in a pH-auxostat culture at low cell density and product concentration and near maximum growth rate. The products were then added individually to the medium in increasing concentrations and the resulting depression of growth rate was used as a quantitative estimate of product inhibition. Under these conditions growth was totally inhibited at concentrations of 60 g/l for 1,3-propanediol, 27 g/l for acetic acid and 19 g/l for butyric acid at pH 6.5. Appreciable inhibition by glycerol was found only above a concentration of 80 g/l. In a pH-auxostat without added products but with high cell density as well as in batch cultures the product proportions were different. The 1,3-propanediol concentration may approach the value of complete inhibition while the concentrations of acetic and butyric acids remained below these values by at least one order of magnitude. It was therefore concluded that 1,3-propanediol is the first range inhibitor in this fermentation.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: I. The phenomena and characterization of oscillation and hysteresis.

Oscillation and hysteresis phenomena are observed in the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae in long‐term cultivations under a variety of conditions. In this work, the conditions for the occurrence of these phenomena are reported and the patterns of cell growth and metabolism under oscillation are characterized. During an oscillation period, the formation rates of CO2, H2, and formate and the consumption rate of alkali periodically pass values of maxima and minima, the latter being close to zero. The formation of biomass and fermentation products such as 1,3‐propanediol, acetate, and ethanol also undergo periodic changes which shift maxima and minima. Sustained oscillation occurs only under conditions of substrate excess within a distinct regime. At pH 7.0, it is only found at dilution rates above 0.15 h−1 under the experimental conditions. At lower pH values, oscillations are more likely to happen, even at a relatively low dilution rate and low substrate excess. Whereas the amplitude of oscillations at pH 7.0 depends on both the dilution rate and the residual glycerol concentration (CGlyc) the interval of oscillations appears to be only a function of CGlyc. An increase of CGlyc in culture damps the oscillation and leads to its disappearance at CGlyc = 1100 to 1200 mmol/L (pH 7.0). The operation mode was also found to be an important parameter in determining the stability and actual state of the culture, resulting in hysteresis under certain conditions, particularly at low pH values. Generally, a large perturbation of cultivation conditions tends to cause oscillation and hysteresis. The results unambiguously demonstrate that the oscillation and hysteresis phenomena shown in this work are bound to genuine metabolic fluctuations of the microorganism. They reveal several differences and new features compared with those reported in the literature and cannot be readily explained by the mechanisms known so far.

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture

SummaryThe effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h−1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l−1 h−1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of the cells decreased. In the pH range 5.5–6.5 the product concentration (butanediol + acetoin) was maximal and nearly constant. However, the specific production continuously declined with increasing pH. Experiments with addition of acetic acid showed that the various effects of pH are due to inhibition of the by-product acetic acid on cell growth. The strength of the acetic and inhibition depended only on the concentration of its undissociated form [HAc]. The biomass concentration and the specific OUR were also only functions of [HAc], irrespective of the pH. Although the specific ATP requirement (qATP) strongly depended on the pH, [HAc] at constant pH.

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%.