Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water–gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B₁₂ (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B₁₂ was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.     

1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026

We report a Klebsiella pneumoniae DSM2026 fermentation procedure for the efficient production of a key enzyme of 1,3-propanediol formation: 1,3-propanediol oxidoreductase (E.C. 1.1.1.202). The fermentation process is composed of an aerobic batch phase on glucose and glycerol and an anaerobic phase on glycerol. The role of the aerobic phase is to produce sufficiently high cell mass (12.9–14.6 g/l dry weight) and to activate the aerobic branch of the Klebsiella glycerol pathway, whereas in the anaerobic phase there is a rapid initiation of 1,3-propanediol oxidoreductase formation. A fast change from an aerobic to an anaerobic environment led to a redox imbalance, which resulted in the abrupt activation of the anaerobic branch of glycerol utilization, with the occurrence of a high 1,3-propanediol-oxidoreductase activity. A mathematical model with substrate inhibition showed that the adequate glycerol concentration for enzyme production was 14–16 g/l. The combination of the optimal substrate concentration together with the subsequent use of glucose and glycerol resulted in 90.6 ± 11.6 U enzyme activity referred to 1 l of fermentation broth and 10.3 ± 0.9 U/(1 h) productivity.     

Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source

Klebsiella pneumoniae is a Gram-negative facultative anaerobe that metabolizes glycerol efficiently under both aerobic and anaerobic conditions. This microbe is considered an outstanding biocatalyst for transforming glycerol into a variety of value-added products. Crude glycerol is a cheap carbon source and can be converted by K. pneumoniae into useful compounds such as lactic acid, 3-hydroxypropionic acid, ethanol, 1,3-propanediol, 2,3-butanediol, and succinic acid. This review summarizes glycerol metabolism in K. pneumoniae and its potential as a microbial cell factory for the production of commercially important acids and alcohols. Although many challenges remain, K. pneumoniae is a promising workhorse when glycerol is used as the carbon source.     

Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> under micro-aerobic conditions

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.     

1,3-Propanediol production by Klebsiella pneumoniae under different aeration strategies

1,3-Propanediol production by Klebsiella pneumoniae was studied in batch cultures under N2 flow and four airflow systems. Different byproducts were formed under different aeration conditions. An anaerobic/aerobic combined fed-batch culture was developed giving 70 g 1,3-propanediol l(-1) and 16 g 2,3-butanediol l(-1) with total diol yield of 0.6 mol(-1) glycerol.     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Dielectric barrier discharge plasma as a novel approach for improving 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Dielectric barrier discharge plasma was used to generate a stable strain of Klebsiella pneumoniae (designated to as Kp-M2) with improved 1,3-propanediol production. The specific activities of glycerol dehydrogenase, glycerol dehydatase and 1,3-propanediol oxidoreductase in the crude cell extract increased from 0.11, 9.2 and 0.15 U mg⁻¹, respectively, for wild type to 0.67, 14.4 and 1.6 U mg⁻¹ for Kp-M2. The glycerol flux of Kp-M2 was redistributed with the flux to the reductive pathway being increased by 20% in batch fermentation. The final 1,3-propanediol concentrations achieved by Kp-M2 in batch and fed-batch fermentations were 19.9 and 76.7 g l⁻¹, respectively, which were higher than those of wild type (16.2 and 49.2 g l⁻¹). The results suggested that dielectric barrier discharge plasma could be used as an effective approach to improve 1,3-propanediol production in K. pneumoniae.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD⁺-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B₁₂, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD⁺ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD⁺ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD⁺ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.