Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions
© Poblete-Castro et al.; licensee BioMed Central. 2014
Received: 8 October 2014
Accepted: 11 December 2014
Published: 23 December 2014
Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source.
Among all tested strains, P. putida KT2440 most efficiently synthesized mcl-PHA under nitrogen-limiting conditions, amassing more than 34% of its cell dry weight as PHA. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% PHA (%wt/wt). The low biomass and PHA titer found in the mutant strain and the wild-type strain KT2440 seems to be triggered by the high production of the side-product citrate during the fermentation process which shows a high yield of 0.6 g/g.
Overall, this work demonstrates the importance of choosing an appropriate microbe for the synthesis of mcl-PHA from waste materials, and a close inspection of the cell metabolism in order to identify undesired compounds that diminish the availability of precursors in the synthesis of biopolymers such as polyhydroxyalkanoates. Future metabolic engineering works should focus on reducing the production of citrate in order to modulate resource allocation in the cell’s metabolism of P. putida, and finally increase the biopolymer production.
The consumption of non-renewable materials for industrial production of chemicals has given rise to environmental and energy concerns within society. Plastic products are considered essential materials to meet the needs of our current lifestyle and modern manufacturing. As an attempt to move towards a sustainable society, research has been undertaken to develop the microbial fermentation of renewable resources for the synthesis of biopolymers ,. An example of the sustainable production of biopolymers is the large-scale production of polyhydroxyalkanoates (PHAs), which exhibit similar physical and mechanical properties to oil-based thermoplastics . Bacteria have the capability to naturally accumulate PHAs as carbon/energy storage materials in their cytoplasmic space, usually when there is a nutrient imbalance in the environment: a high carbon concentration accompanied by the limitation of an inorganic nutrient . Taking advantage of this evolutionary bacterial trait, commercialized PHAs are currently being produced by Metabolix (USA), Meridian Inc. (USA), Biocycle (Brazil), and Biomer (Germany), to name a few. They use sugars (cane or beet) and plant-based fatty acids as carbon feedstocks for the fermentation process in stirred-tank bioreactors, and obtain a diverse assortment of biopolymers. Despite the advances, one of the main drawbacks of PHAs for better market positioning is the high costs related to the production process ,. To truly develop the synthesis of these polyesters and make them more cost-competitive against petroleum-based thermoplastics, it is crucial to find novel sustainable alternatives for large-scale production. Recently, production of PHAs using waste materials from agriculture residues and waste water treatment streams as a carbon substrate has shown to be a very promising alternative -, since raw materials account for the majority of production costs in the bacterial synthesis of PHAs ,. These processes have mainly focused on the synthesis of Poly(3-hydroxybutyrate) (PHB), the first and best-characterized PHA . Despite the large range of applications possible with PHB and its co-polymer Poly(3-hydroxybutyrate-valerate) (PHBV), there is still an urgent demand for other types of biopolymers with unique physical and mechanical properties, with a particular emphasis on medical applications . Medium chain length polyhydroxyalkanoates (mcl-PHAs) are polyesters synthesized mainly by gram-negative bacteria; most studies on their synthesis have focused on the use of Pseudomonas species as microbial cell factories -. By applying different metabolic engineering strategies - and fermentation conditions , the titer of this biopolymer was successfully improved, and it was demonstrated also that the monomer composition of the produced mcl-PHAs can be tuned -. In addition, given the high metabolic versatility shown by some Pseudomonas putida strains , waste materials from different streams have been used as carbon substrate to produce mcl-PHAs. Efficient synthesis of mcl-PHAs was obtained from animal wastes , PET  and raw glycerol . Also, the conversion of biomass-derived compounds into mcl-PHA has been recently achieved ,. One of the most promising raw materials for the synthesis of biobased chemicals is glycerol. The commercial price of this polyol has drastically decreased in the last decade due to the large industrial production of biodiesel from fatty acids, where glycerol is produced as a byproduct of the esterification process. For every 10 tons of biodiesel, 1 ton of glycerol is formed. Because of this, there is currently an oversupply of this commodity worldwide which has resulted in its classification as a waste product instead of a valuable good . P. putida strains have been recently described to be capable of synthesizing mcl-PHAs from raw glycerol ,. In this work, several P. putida strains were challenged to growth in raw glycerol as carbon and energy source and compared for their capability to produce mcl-PHA. The best PHA-producing P. putida strain was further engineered to enhance the synthesis of mcl-PHA in the cell. An exo-metabolome analysis was performed to identify and quantify byproduct formation during the fermentation process in bioreactors, and finally the causes for the low yield of biomass and biopolymers in Pseudomonas putida fed with glycerol were elucidated.
Results and discussion
Features of Pseudomonas putida strains used in this study
Genome size (bp)
GC content (%)
P. putida KT2440
Bioremediation Synthesis of biomaterials and chemicals
P. putida KT2442
Spontaneous rifampicin resistant
Bioremediation Synthesis of biomaterials and chemicals
P. putida F1
Degradation of chloroaromatic compounds
P. putida S12
Synthesis of added-value chemicals
Evaluation of PHA synthesis on different Pseudomonas putidastrains in shake flask experiments
Monomer composition of medium chain length PHA produced by different P. putida strains
Monomer composition (%)
P. putida KT2440
4.23 ± 0.11
1.46 ± 0.21
P. putida KT2442
3.45 ± 0.07
0.91 ± 0.13
P. putida F1
3.50 ± 0.14
0.36 ± 0.01
P. putida S12
3.20 ± 0.07
0.40 ± 0.05
4.20 ± 0.21
1.94 ± 0.17
Deletion of the phaZ gene in P. putidaKT2440 enhances mcl-PHA synthesis using raw glycerol as a carbon and energy source
One of the first strategies for maintaining the titer of PHA inside of cells during the fermentation process is to remove the gene responsible for the depolymerization process of PHA (PHA depolymerase). However, the role of PhaZ proteins in the synthesis of mcl-PHA in P. putida strains is not well understood. In a previous work, disruption of the PHA depolymerase gene (PP_5004, phaZ) in P. putida KT2442 led to an increase in PHA synthesis using fatty acids as carbon source, but not when growing on glucose or gluconate , where the mutant strain produced less PHA in comparison to the wild-type strain. One could argue then, since glycerol is also a non-related PHA carbon source, it should show the same PHA-accumulation pattern as glucose or gluconate, resulting ultimately in no increase of the biopolymer. Nevertheless, in the study done by , they generated a phaZ mutant strain in KT2442, and as demonstrated in the research presented in this paper, this strain produces less mcl-PHA from no-alkanoate substrates. Therefore we generated a knockout mutant strain in the best PHA-producing strain, P. putida KT2440. The chromosomal deletion of phaZ (PP_5004) in P. putida KT2440 was performed as described in Methods. The ΔphaZ mutant strain was then subjected to PHA-production conditions in flask cultures in the same manner as described above with the wild type strain KT2440. There was no difference in total biomass yield (Table 2) between ΔphaZ and P. putida KT2440 wild type, however and most importantly, the first improved the PHA titer by 34% (Table 2). PHA depolymerase is believed to be required for the efficient production of the polyester —under PHA-accumulating conditions in batch culture— in P. putida KT2442 and P. putida U using fatty acids as the carbon substrate -. However, the opposite result has been reported for the same mutant strain (ΔphaZ) in P. putida KT2442 , and P. putida U . Because we measured more mcl-PHA in ΔphaZ than its parent strain KT2440, we conclude that PHA depolymerase does not impose a negative effect on the PHA synthesis machinery. The PHA cluster genes orchestrates a simultaneous process of PHA synthesis and hydrolysis , making it impossible for the cell to degrade the PHA granule and further mobilize 3-hydroxyalkanoic acids for energy generation; this may be the primary reason for the greater accumulation of mcl-PHA in the ΔphaZ mutant strain when grown on glycerol.
PHA morphology and monomer composition of PHA in P. putidastrains grown on pure and raw glycerol
Influence of aeration on the production of mcl-polyhydroxyalkanoates in P. putida ΔphaZmutant strain
Comparison of mcl-PHA synthesis by P. putida KT2440 and ΔphaZmutant strain on mcl-PHA synthesis in well-controlled batch bioreactor
Citric acid-producing microorganisms using different carbon sources
Y. lipolytica mutant
Y. lipolytica A-101
A. niger mutant
Beet, Cane Molasse
P. putida KT2440
P. putida ΔphaZ
This study shows that bacteria from the genus Pseudomonas can cope with the adverse conditions imposed by the chemical mixture of which raw glycerol from biodiesel production is composed. It also demonstrates the use of this cheap material in the efficient synthesis of mcl-PHA. Among all tested strains, P. putida KT2440 was the most efficient at mcl-PHA synthesis, amassing more than 34% of its CDW as PHA using raw glycerol as its only energy/carbon source. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% (%PHA/wt). The low biomass and PHA titer found in P. putida KT2440 and the ΔphaZ strain on glycerol seems to be caused by the production of high levels of citrate during the fermentation process. Future metabolic engineering works should focus on eliminating or reducing the production of this organic acid in order to modulate resource allocation in the cell’s metabolism, and finally increase the specific volumetric productivity of PHA that is crucial for a cost-effective biopolymer production.
P. putida KT2440 (DSM 6125) and F1 (DSM 6899) were obtained from the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, Germany). P. putida KT2442  and P. putida S12  were obtained from CSIC Madrid (Spain) and RWTH Aachen (Germany), respectively.
Construction of the phaZ knock-out mutant strain in P. putidaKT2440
Primers used in this study
To generate a single mutant of gene PP5004, genome editing was applied . Therefore, vector pEMG_ΔPP5004 was co-integrated by a single crossover into the chromosome of P. putida KT2440 using tri-parental mating with E. coli HB101 as helper strain, as well as the donor strain E. coli CC118λpir pEMG_ΔPP5004. Successful homologous integration of the vector DNA and the successful genomic deletions was confirmed by PCR (data not shown).
P. putida strains, kept as frozen stock in 25% glycerol at −80°C, were streaked on Luria Bertani agar plates and incubated for one day at 30°C. Single colonies were then picked from the plate and inoculated it into a 50 mL shake flask containing 10 ml of the above described medium (M9) and incubated overnight under aerobic conditions at 30°C and 180 rpm (Innova, Enfield, USA) set. By taking a calculated volume of the obtained cell suspension from the pre-culture (to begin the PHA-accumulating process with an initial OD of 0.05), 500 mL baffled shake flasks containing 100 mL of culture medium were inoculated and placed in a rotary shaker under aerobic conditions at 30°C. Each culture was carried out by triplicate. P. putida strains were grown in a defined M9 mineral medium consisting of (per liter) 12.8 g Na2HPO4.7H2O, 3 g KH2O4, 1 g NH4Cl, and 0.5 g NaCl. This basic solution was autoclaved and subsequently supplemented with 0.12 g of MgSO4.H2O, trace elements (mg/L): 6.0 FeSO4.7H2O, 2.7 CaCO3, 2.0 ZnSO4.H2O, 1.16 MnSO4.H2O, 0.37 CoSO4.7H2O, 0.33 CuSO4.5H2O, 0.08 H3BO3 (all filter-sterilized). 30 g/L of raw glycerol was then added to the medium which was previously autoclaved. The raw glycerol used in all experiments was obtained from the company Cremer Oleo (Hamburg, Germany). It contained 80% glycerol, 0.5% methanol, 10% ash, 3% organic matter, and 6.5% water.
P. putida strains were grown in M9 medium supplemented with 30 g/L raw glycerol. Bioreactor batch fermentations were carried out in a 2 L top-bench BIOSTAT B1 bioreactor (Sartorius B Systems GmbH, Melsungen, Germany) with a working volume of 1.5 L, at 30°C. The aeration rate was set to 500 mL/Lmin using a mass flow controller (PR4000, MKS Instruments, Wilmington, MA, USA). The dissolved oxygen level was kept above 20% air saturation by control of the agitation speed up to a maximum of 700 rpm. The pH was maintained at 7.0 by automatic pH controlled addition of 0.5 M H2SO4 or 1 M of KOH.
Cell growth was recorded as optical density (OD) at 600nm (Ultraspec 2000, Hitachi, Japan). The cell dry weight was determined gravimetrically after collection of 10 mL culture broth for 10 min at 4°C and 9,000 × g (Eppendorf 5810 R, Hamburg, Germany) in pre-weighed tubes, including a washing step with distilled water, and drying of the obtained pellet at 100°C until constant weight. The ammonium concentration in cell-free supernatant was measured by a photometric test (LCK 303 kit, Hach Lange, Danaher, USA).
The glycerol and organic acids concentration (citrate, isocitrate, succinate, fumarate, malate, pyruvate, and citrate) in cultivation supernatant was analyzed by HPLC Agilent 1260 (Agilent, Krefeld, Germany) equipped with an 8 mm Rezex ROA-organic acid H column (Phenomenex, USA) at 65°C with 0.013 N H2SO4 as the mobile phase (0.5 mL·min−1) followed by detection using a RID detector (Agilent serie1260).
PHA characterization and quantification
PHA compositions of the polymer produced, as well as the cellular PHA content concentration were determined by gas chromatography (GC) and mass spectrometry (MS) of the methanolyzed polyester. First, 10 mL of the culture broth was placed in a falcon tube and centrifuged for 10 min at 4°C and 9,000 × g (Eppendorf 5810 R, Hamburg, Germany), following by a washing step with distilled water. The supernatant was poured away by pipetting and the cell pellet kept at −20°C for further process. Methanolysis was carried out by suspending 5–10 mg of lyophilized aliquots in 2 mL of chloroform and 2 mL of methanol containing 15% sulfuric acid and 0.5 mg/mL 3-methylbenzoic acid as internal standard, respectively, followed by incubation at 100°C for 4 h. After cooling, 1 mL of demineralized water was added and the organic phase containing the resulting methyl esters of monomers was analyzed by GC-MS. Analysis was performed in Varian GC-MS system 450GC/240MS ion trap mass spectrometer (Varian Inc., Agilent Technologies) and operated by the software MS Workstation 6.9.3 (Varian Inc., Agilent Technologies). An aliquot (1 mL) of the organic phase was injected into the gas chromatograph at a split ratio of 1:10. Separation of compounds of interest (i.e. the methyl esters of 3-hydroxyexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecanoate, 3-hydroxy-5-cis-dodecanoate, 3-hydroxytetradecanoate) was achieved by a FactorFour VF-5 ms capillary column (30 m × 0.25 mm i.d. × 0.25 mm film thickness). Helium was used as carrier gas at a flow rate of 0.9 mL/min. The injector and transfer line temperature were 275°C and 300°C respectively. The oven temperature program was: initial temperature 40°C for 2 min, then from 40°C up 150°C at a rate of 5°C min−1 and finally up to 280°C at a rate of 10°C min−1. Positive ions were obtained using electron ionization at 70 eV and mass spectra were generated by scanning ions of m/z 50 to m/z 650. The PHA content (wt%) was defined as the percentage of the cell dry weight (CDW) represented by the polyhydroxyalkanoate.
Transmission electron microscopy
Bacteria were fixed by chilling the cultures to 4°C and addition of glutaraldehyde (2%) and formaldehyde (5%). They were then washed with cacodylate buffer (0.01 mol l)1 cacodylate, 0.01 mol l)1 CaCl2, 0.01 mol l)1 MgCl2 6H2O, 0.09 mol l)1 sucrose, pH 6/9) and stained with 1% aqueous osmium for 1 h at room temperature. Samples were then dehydrated with a graded series of acetone (10, 30, 50, 70, 90 and 100%) with incubation for 30 min at each concen- tration, except for the 70% acetone, which contained 2% uranyl acetate and was performed overnight. Samples were infiltrated with an epoxy resin, according to the Spurr for- mula for hard resin, for several days with pure resin. Ultrathin sections were cut with a diamond knife, counterstained with uranyl acetate and lead citrate and examined in a TEM910 transmission electron micro- scope (Carl Zeiss, Oberkochen, Germany) at an accelera- tion voltage of 80 kV. Images were taken at calibrated magnifications using a line replica. Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Munster, Germany).
Field emission scanning electron microscopy
Samples were fixed as above, washed with cacodylate buf- fer and then washed with TE-buffer (20 mmol l)1 TRIS, 1 mmol l)1 EDTA, pH 6.9). 50 ll of washed bacteria were applied to poly-l-lysine precoated cover slips (12 mm in diameter), which were left for 5 min, washed in TE-buffer, incubated with 2% glutaraldehyde in TE- buffer for 15 min and washed again with TE-buffer. Dehydration was carried out with a graded series of ace- tone (10, 30, 50, 70, 90, 100%) on ice for 15 min for each step, followed by 100% acetone at room temperature and critical-point drying with liquid CO2 (CPD 30; Bal-Tec, Balzers, Liechtenstein). Samples were then gold shadowed by sputter coating (SCD 500; Bal-Tec) and examined with a field emission scanning electron microscope Zeiss DSM 982 Gemini (Carl Zeiss, Oberkochen, Germany), using the Everhart Thornley SE detector and the inlens detector in a 50: 50 ratio at an acceleration voltage of 5 kV. Images were recorded onto a MO-disc. Contrast and brightness were adjusted with Adobe Photoshop CS3.
We would like to thank Dr. Nick Wierckx for providing the P. putida S12 strain used in this study. The authors thank Carie M. Frantz for carefully reading this manuscript. Ignacio Poblete-Castro acknowledges the financial support by the program “Convenio de Desempeño Apoyo a la Innovación en Educación Superior” (PMIUAB 1301).
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