Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity
© Manso Cobos et al. 2015
Received: 9 February 2015
Accepted: 25 May 2015
Published: 10 June 2015
Cyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN−) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required.
In this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha−, deleted in putative mcl-PHA synthases; Spha−, deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC–MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha− and Spha−, respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers.
These results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain benefits of this type of approaches.
Cyanide is produced at high concentrations by anthropogenic sources like electroplating, jewelry and mining activities. Residues that contain high concentrations of cyanide must be remediated in order to remove cyanide, and biological approaches may display advantages over physicochemical treatments. The alkaliphilic bacterium P. pseudoalcaligenes CECT5344 is able to utilize cyanide and cyano-derivatives as the sole nitrogen source . In the CECT5344 strain, cyanide induces various mechanisms for cyanide resistance and assimilation, such as cyanide-insensitive respiration , mechanisms for iron homeostasis  and synthesis of the specific nitrilase involved in the cyanide assimilation pathway . Recently, the complete genome sequence of this bacterium has been reported [5, 6]. In addition to genes involved in cyanide assimilation and resistance, such as the nit genes encoding nitrilases and the cio genes coding for alternative cyanide-insensitive oxidases, this strain harbors some genes potentially involved in other processes with a great biotechnological potential, such as the production of polyhydroxyalkanoates (PHAs). The cost-efficient production of PHAs is averted partly due to the high costs of the carbon sources supplied to the production medium. Valorization and utilization of wastes via their bioconversion into bioplastics is one of the most distinctive strategies to unlock the PHAs production at industrial scale. In this sense, side production of PHAs as an extra-income (by-product) of non-strictly cost-dependent processes such as those driven to strategies for bioremediation of toxic compounds has not been extensively considered.
Many bacteria accumulate PHAs in the cytoplasm as carbon and energy storage material when growing under nutrient imbalanced conditions, but with an excess of a carbon source [7–10]. PHAs are biopolyesters that consist of 3-hydroxycarboxylic acids that are classified into two major groups displaying different material properties: the short chain length (scl-) with 3–5 carbon atoms or the medium chain length (mcl-) with 6–14 carbons [9–11]. Different proteins associated to the PHAs granules have been identified [11–15], such as PHA synthases for polymerization, PHA depolymerases involved in bioplastic degradation and monomer mobilization, and phasins with structural and regulatory functions [13, 16, 17]. Essential steps for PHAs biosynthesis are generation of the hydroxyacyl-CoA (HA-CoA) and the PHA synthase-catalyzed HA-CoAs polymerization into PHAs [18–20]. PHA synthases are classified into four types, of which class I and class II PHA synthases are composed of one subunit (PhaC). However, type I enzymes accept scl-HA-CoA for polymerization [21–28] whereas type II synthases, mainly found in pseudomonads, display substrate specificity towards mcl-HA-CoA . Most pseudomonads produce polymers containing mcl-PHAs. However, a few strains like Pseudomonas sp. 61-3 , Pseudomonas oleovorans strain B-778 , Pseudomonas pseudoalcaligenes YS1  and Pseudomonas stutzeri 1317  synthesize a mixture of scl- and mcl-PHAs.
Three gene clusters putatively involved in the metabolism of scl- and mcl-PHAs have been identified in the genome of the cyanide-degrading bacterium P. pseudoalcaligenes CECT5344 . The phbRphaBAC gene cluster includes the phaB gene that codes for an NADPH-dependent acetoacetyl coenzyme A reductase, the phaA gene encoding a β-ketothiolase, and the phaC gene coding for a class I scl-PHA synthase. The phbR gene shows similarity to members of the AraC/XylS transcriptional activators family. In the same locus, an additional cluster comprises the phaP and phaR genes that code for a phasin and a regulatory protein, respectively. In a different locus, a third gene cluster is similar to those found in other mcl-PHAs producers [revised in 10]. It comprises the phaC1 and phaC2 genes that code for class II PHA synthases, the regulatory phaD gene, the phaF and phaI genes encoding a phasin and a regulatory protein, respectively, and the phaZ gene, which is located between the phaC1 and phaC2 genes and encodes a putative depolymerase responsible for PHAs mobilization .
Cyanide degradation by the strain CECT5344 under alkaline conditions was previously optimized in a batch reactor loaded with a minimal medium containing acetate as carbon source and 2 mM NaCN as nitrogen source . In this work we analyse the ability of this strain to bioremediate an industrial cyano-waste through a biological treatment that concomitantly generates PHAs as by-product.
Results and discussion
Cyanide assimilation and simultaneous synthesis of PHAs by P. pseudoalcaligenes CECT5344
PHAs accumulation in the wild-type and PHA synthase defective mutants of P. pseudoalcaligenes CECT5344
CDW (g L−1)
scl-PHAs (% CDW)
mcl-PHAs (% CDW)
mcl-PHA monomer composition (%)
1.18 ± 0.1
64.95 ± 3.60
20.12 ± 0.08
5.75 ± 0.49
94.25 ± 0.35
0.76 ± 0.05
66.14 ± 1.95
0.48 ± 0.03
36.81 ± 1.90
9.16 ± 0.08
90.84 ± 0.10
0.34 ± 0.03
0.22 ± 0.04
31.02 ± 3.25
16.37 ± 2.81
5.97 ± 0.11
94.03 ± 0.14
0.28 ± 0.02
55.47 ± 4.29
0.18 ± 0.02
15.44 ± 1.05
6.08 ± 0.08
93.92 ± 0.06
0.22 ± 0.03
0.34 ± 0.03
29.29 ± 0.74
25.63 ± 1.27
5.58 ± 0.07
94.42 ± 0.05
0.36 ± 0.04
39.92 ± 2.12
0.28 ± 0.04
19.94 ± 0.02
5.43 ± 0.06
94.57 ± 0.13
0.28 ± 0.05
By-product accumulation by P. pseudoalcaligenes CECT5344 under cyanide detoxification culture conditions
Broadening the portfolio of PHA by-products from cyano-wastes
The ability of these mutant strains to accumulate PHAs was also tested in batch cultures on shake flasks with 12.5 mM octanoate as carbon source and either ammonium chloride, sodium cyanide or cyanide from the jewelry residue, each at 2 mM initial concentration, as the sole nitrogen source (Table 1). The PHA type accumulated by P. pseudoalcaligenes CECT5344 during degradation of the cyanide-containing industrial residue has been found to be different depending on the bacterial strain used; scl-PHAs (Mpha− mutant), mcl-PHAs (Spha− mutant) or a mixture of both scl- and mcl-PHAs (wild-type). By contrast to wild-type, the Mpha− strain defective in the phaC1ZC2 genes only accumulated scl-PHAs, whereas the Spha− strain deficient in the phaC gene only synthesized mcl-PHAs. The Mpha−/Spha− double mutant was unable to accumulate PHAs. The scl-PHAs content in Mpha− cells cultured with sodium cyanide was higher than in cells grown with jewelry residue, but the mcl-PHAs content in Spha− cells was lower with NaCN than in the presence of the cyanide-containing residue (Table 1). The scl-PHAs obtained in the Mpha− mutant were homopolymers of 3-hydroxybutyrate whereas the mcl-PHA monomer composition accumulated by Spha− mutant was similar to those observed in wild type cells, with about 95% 3-hydroxyoctanoate and 5% 3-hydroxyhexanoate monomers (Table 1). Although PHAs content of cyanide-grown cells was not very high for an industrial purpose by itself, it may be attractive as a value-added of the cyanide detoxification process. It is worth to mention that the jewelry industry located in the city of Córdoba, Spain, produces 4–5 tons per year of an alkaline residue containing up to 26 g L−1 of free cyanide (around 1 M) together with high amounts of heavy metals , thus making this effluent highly toxic and environmentally hazardous.
Phylogenetic analysis of the P. pseudoalcaligenes CECT5344 PHA synthases
The phbRphaBAC gene cluster of P. pseudoalcaligenes CECT5344 shows similar gene arrangement to those found in other bacteria, like Pseudomonas sp. 61-3 , Pseudomonas sp. USM 4-55 , Azotobacter sp. FA8  and Azotobacter vinelandii . In P. extremaustralis 14-3 the phbRphaBAC gene cluster is located within the phaGI genomic island, which is flanked by an 8 bp direct repeat sequence, 5′-TTTTTTGA-3′, and shares strong similarity with the genomic islands found in diverse Proteobacteria, including Azotobacter vinelandii and Burkholderiales species . The G + C content, phylogeny inference and codon usage analysis show that the phaBAC genes have a complex mosaic structure and suggest that phaB and phaC genes could be acquired by horizontal gene transfer, probably from Burkholderiales . The P. pseudoalcaligenes CECT5344 phbRphaBAC gene cluster might be also acquired by horizontal gene transfer, although it lacks the direct repeat sequence and the composition of the neighbor genes is also different.
Our results demonstrate that the cyanotrophic bacterium P. pseudoalcaligenes CECT5344 is able to carry out the biodegradation of toxic cyanide-rich jewelry wastewater associated with production of PHAs as by-product. Experiments performed in flasks or in bioreactor cultures support that the type of PHA accumulated can be tailored. Both Mpha− (mcl-PHAs minus) and Spha− (scl-PHAs minus) mutants are able to assimilate cyanide, as well as wild-type, but Mpha− strain produces exclusively scl-PHAs and Spha− only produces mcl-PHAs, whereas the wild-type strain accumulates both types of PHAs. This constitutes a proof of concept to design more profitable processes to biodegrade cyanide-containing wastes.
Bacterial strains, growth conditions and plamids
P. pseudoalcaligenes CECT5344 (CECT: Spanish Type Culture Collection) was isolated by cyanide-enrichment cultivation from samples of the Guadalquivir River (Córdoba, Spain). This strain was able to assimilate cyanide as sole nitrogen source under alkaline conditions and it was classified as Pseudomonas pseudoalcaligenes by its 16S RNA sequence analysis . The bacterial strains of Escherichia coli and P. pseudoalcaligenes were grown in LB rich medium  at 37 and 30°C, respectively. The appropriate antibiotics, nalidixic acid (10 μg mL−1), ampicillin (150 μg mL−1), gentamicin (20 μg mL−1) and kanamycin (25 μg mL−1), were added when required.
For the analysis of PHAs production in flask cultures, P. pseudoalcaligenes CECT5344 was first grown in LB media in order to obtain a large biomass. These cultures were centrifuged and the cells were suspended in M9 minimal medium  as a source of inoculums. The media were adjusted to pH 9.5 and inoculated with the appropriate cell amount to reach an initial absorbance at 600 nm (A600) of about 0.1. Cells were grown in 500-mL Erlenmeyer flasks containing 200 mL of M9 minimal media with 12.5 mM octanoate, but when indicated, sodium acetate (50 mM) was also used as the sole carbon source. Ammonium chloride, sodium cyanide or cyanide-containing jewelry residue (2 mM initial concentration) was added as the sole nitrogen source. Therefore, in all media, a C/N ratio of 50/1 was used. The jewelry industry residue, which contains cyanide in addition to metals like iron, copper and zinc, was supplied by the companies GEMASUR S.L. and SAVECO S.L., which handle the industrial residues from Córdoba.
Batch reactor culture conditions
Experiments were carried out in a Biostat® B plus (Sartorius BBI Systems) 5 L bioreactor, using the following operational procedure based on . The reactor was loaded with M9 minimal medium and further autoclaved. The working volume was 5 L. Sodium octanoate (12.5 mM) and jewelry residue (2 mM cyanide) were used as carbon and nitrogen sources, respectively. MgSO4 and FeSO4 solutions were sterilized by filtration and added to the M9 trace solution after autoclaving. Antifoam and the appropriate antibiotics were also added to the media. The reactor was then inoculated with the appropriate bacterial strain to reach an initial A600 of about 0.2 (25 ml of a bacterial suspension obtained from an overnight 500 ml LB culture). Temperature was maintained at 30°C and pH was initially adjusted to 9.5 and kept constant by automatic addition of 1 M NaOH. Continuous agitation at 450 rpm and dissolved oxygen saturation at 10% were controlled automatically. To prevent cyanhydric acid (HCN) losses, a bioreactor exhaust cooler was connected to a washing flask containing a concentrated NaOH solution. The absence of cyanide in samples from this flask was confirmed during the process.
Cell growth was determined by following absorbance at 600 nm. To estimate biomass calculation, cell densities (expressed in grams of CDW per liter) were determined gravimetrically by using 50 mL Falcon tubes. Ammonium concentration was determined by the Nessler reagent as previously described . Cyanide concentration was determined colorimetrically .
DNA manipulations and generation of mutant strains
DNA manipulations and other molecular biology techniques were essentially performed as described previously . The mutant strain Mpha− of P. pseudoalcaligenes was constructed by deletion of the phaC1ZC2 genes and insertion of a kanamycin cassette resistance gene. In this mutant, deletion of both phaC1 and phaC2 genes was partial, whereas phaZ gene was completely deleted. A central region of 619 bp in the phaC1 gene was cloned into pBluescript II KS (±) after PCR amplification with primers phaC1E (5′-GAAGCCTTCGAATTCGGCAAGAAC-3′; EcoRI restriction site is underlined) and phaC1S (5′-GCAGGTAGTTGTCGACCCAGTAGTTC-3′; SalI restriction site is underlined) to yield the pBKS-A construct. An internal fragment of 506 bp of the phaC2 gene was amplified by PCR using genomic DNA from P. pseudoalcaligenes CECT5344 as template and the oligonucleotides phaC2S (5′-TCGAAGTCGACCGCAATCTGG-3′; SalI restriction site is underlined) and phaC2X (5′-CTTGGCTGACTCGAGGGTTTCC-3′; XhoI restriction site is underlined). This fragment was digested with the appropriate restriction enzymes and cloned into the unique SalI and XhoI sites of the pBKS-A plasmid to yield pBKS-AB. The 2.2 kb XhoI/SalI fragment that contains the kanamycin cassette resistance gene from pSUP2025  was ligated into the pBKS-AB vector previously digested with SalI to generate pBKS-AKB. This construct was digested with EcoRI and KpnI, yielding a 3.3 kb fragment that contains the kanamycin cassette resistance gene flanked by the internal region of the phaC1 and phaC2 genes. The resulting fragment was cloned into pK18mob, a suicide plasmid for Pseudomonas that confers kanamycin resistance but lacks of a functional replication origin for this bacterium . The final construct pK18mob-AKB was used to deliver the ΔphaC1ZC2 mutation to the host chromosome via homologous recombination. Biparental mating was performed using E. coli S17-1 (pK18mob-AKB) as the donor strain and a spontaneous nalidixic acid resistant mutant of P. pseudoalcaligenes CECT5344 as the recipient strain. Transconjugants were selected in M9 media with nalidixic acid and kanamycin. Disruption of phaC1ZC2 genes was confirmed by PCR sequencing analysis.
To generate the Spha− mutant of P. pseudoalcaligenes, the phaC gene was amplified by PCR using the oligonucleotides phbB (5′-GCCTGCTGCACGAGATCTTCCGCC-3′; BglII restriction site is underlined) and phbH (5′-ATTGGCGCTGGCGAAGCTTGAACCC-3′; HindIII restriction site is underlined) and P. pseudoalcaligenes CECT5344 genomic DNA as template. The amplified DNA fragment was then inserted into pBluescript II KS (±) previously digested with EcoRV and SmaI to generate the pBKS-phaC construct. The 1 kb EcoRI/PstI fragment that contains the gentamicin resistance cassette from pMS255  was ligated into the pBKS-phaC vector to generate the pBKS-ΔphaC construct. The restriction enzymes SpeI and HindII were used to digest pBKS-ΔphaC, generating a 2.1 kb fragment containing the phaC gene disrupted by the gentamicin resistance cassette. This fragment was then ligated into the pK18mob vector previously digested with HindIII and XbaI to generate pK18mob-ΔphaC. Biparental mating was performed using E. coli S17-1 (pK18mob-ΔphaC) as donor strain and the nalidixic acid resistant mutant of P. pseudoalcaligenes CECT5344 as recipient strain. Transconjugants (nalidixic acid and gentamicin resistant, and kanamycin sensitive) were isolated. Disruption of phaC gene was confirmed by PCR sequencing.
A double mutant Mpha−/Spha− was obtained by biparental mating using E. coli S17-1 (pK18mob-ΔphaC) as donor strain and the P. pseudoalcaligenes Mpha− mutant as recipient strain. Tranconjugants were selected in M9 with nalidixic acid, kanamycin and gentamicin. Disruptions of phaC1ZC2 and phaC genes were confirmed by PCR sequencing.
Transmission electron microscopy
Cells were harvested, washed twice in M9 minimal medium and fixed in 2% (w/v) glutaraldehyde in the same solution. Then, cells were suspended in 1% (w/v) OsO4 for 1 h, gradually dehydrated in acetone 30, 50, 70, 90 and 100% (v/v), 30 min each, and finally treated with propylene oxide (two changes, 10 min each). Afterwards, cells were embedded sequentially into 2:1, 1:1, 1:2 propylene oxide-resin. Ultrathin sections (thickness 50 nm) were cut with a Leica Ultracut R ultramicrotome (Leica Inc, Buffalo, USA) using a diatome diamond knife. The sections were picked up with 200 mesh cupper grids coated with a layer of carbon and subsequently observed in a Jeol JEM-1400 (Tokyo, Japan) electron microscope. These analyses were carried out by using the microscopy facilities at the central services for research support (SCAI) of the University of Córdoba (Spain).
Gas chromatography-mass spectrometry (GC–MS) analysis for PHAs and octanoate determinations
Polyhydroxyalkanoate monomer composition and cellular PHAs content were determined by GC–MS of the methanolysed polyester. Samples of 50–150 mL culture medium were centrifuged for 20 min at 12,000×g and 4°C. Cell pellets were freeze-dried for 24 h in a lyophilizer and weighed. Methanolysis procedure was carried out by suspending 5–10 mg of lyophilized aliquots in 0.5 mL chloroform and 2 mL methanol containing 15% sulfuric acid and 0.5 mg mL−1 3-methylbenzoic acid (internal standard) and then incubated at 80°C for 7 h. After cooling, 1 mL demineralized water and 1 mL chloroform were added, and the organic phase containing the resulting methyl esters of monomers was analyzed by GC–MS . An Agilent series 7890A coupled with 5975C MS detector (EI, 70 eV) and a split-splitless injector were used for analysis. An aliquot (1 µL) of organic phase was injected into the gas chromatograph at a split ratio 1:20. Separation of compounds was achieved using an HP-5 MS capillary column (5% phenyl-95% methyl siloxane, 30 m × 0.25 mm film thickness). Helium was used as carrier gas at a flow rate of 1 mL min−1. The injector and transfer line temperature were set at 275 and 300°C, respectively. Oven temperature was initially 80°C for 2 min, then rose from 80°C up to 150°C at 5°C min−1, and kept at 150°C for 1 min. The mass spectra were recorded in full scan mode (m/z 40–550). 3-hydroxybutyric acid methyl ester was resolved using selected ion monitoring mode (SIM).
Octanoate concentration in the medium was analyzed by GC–MS following the procedure described by Escapa et al. .
IM performed the experiments and wrote the manuscript. II and LPS carried out reactor experiments. MAP and FP carried out the GC-MS analysis. VMLA participated in the phylogenetic analysis. CMV, MDR, FC and MAP participated in designing the experiments and in revising the manuscript. All authors read and approved the final manuscript.
This work was funded by Ministerio de Economia y Competitividad (Grants PET2008_0048, BIO2011-30026-C02-02 and BIO2013-44878-R) and by Junta de Andalucía (Grant CVI-7560). We also thank the companies GEMASUR, SAVECO and AVENIR for their fruitful collaborations.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interest.
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