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Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis
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JTICE-843; No. of Pages 6 Journal of the Taiwan Institute of Chemical Engineers xxx (2014) xxx–xxx
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Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis Chih-Ching Chien *, Li-Jung Wang, Wei-Ran Lin Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Chung-Li 32003, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 September 2013 Received in revised form 28 January 2014 Accepted 5 February 2014 Available online xxx
Polyhydroxybutyrate (PHB) is usually produced in various microorganisms in response to the environmental conditions of physiological stress. Cupriavidus taiwanensis is known to accumulate significant amounts of PHB and is phylogenetically related to Ralstonia eutropha (Cupriavidus necator), which is also well known for producing PHB. In this study, an environmental isolate of C. taiwanensis resistant to heavy metals (strain EJ02) was evaluated for its ability to accumulate PHB when grown in the presence of cadmium ions. C. taiwanensis EJ02 tolerated much higher concentrations of cadmium (up to 5 mM) when grown in complex (Luria-Bertani) media compared to C. taiwanensis strains BCRC 17206T and BCRC 17208 (1 mM). The growth of the strain EJ02 showed more resistance to cadmium (up to 7 mM) when grown in LB medium containing sodium gluconate than that in LB only. There was no significant effect on cadmium resistance for C. taiwanensis BCRC 17206T and BCRC 17208 when sodium gluconate was supplemented in LB medium. Although the production of PHB by C. taiwanensis EJ02 is enhanced by the presence of cadmium, the mRNA expression of the key genes for PHB biosynthesis (i.e., phaCAB) is lower than in the EJ02 cells not exposed to cadmium determined by real-time PCR analysis. The tolerance of C. taiwanensis EJ02 to the presence of heavy metals might be associated with its ability to accumulate PHB and to adapt to the stress of an environment with heavy metals. ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Polyhydroxybutyrate Stress resistance Heavy metal Cupriavidus
1. Introduction Polyhydroxyalkanoates (PHAs) are polymers synthesized by a variety of environmental microorganisms under unbalanced growth conditions or physiological stress [1,2]. PHAs are classified according to the length of their carbon chains. Short chain-length PHA (scl-PHA) polymers contain three to five C-atoms, and medium chain-length PHA (mcl-PHA) polymers contain six or more C-atoms [3,4]. The most commonly found monomer in an sclPHA is 3-hydroxy fatty acid, which is a short chain-length PHA. Microorganisms belonging to the genus Cupriavidus including Cupriavidus necator and C. taiwanensis are well known for being able to synthesize polyhydroxybutyrate (PHB), and the amount of PHB accumulation can be as high as 80–90% of the cell’s dry weight [5–8]. Because the properties of PHAs are similar to those of polyethylene (PE) and polypropylene (PP), PHAs have been
* Corresponding author at: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 32003, Taiwan. Tel.: +886 3 4638800x2184; fax: +886 3 4334667. E-mail address: [email protected] (C.-C. Chien).
explored as alternatives to petrochemical plastics [9,10]. In addition to their broad biotechnological applications, PHAs may play an important ecologic role by regulating microbial survival and tolerance in stressed environments [11]. For example, PHAs improved starvation tolerance [12], and increased resistance to various stress agents such as ethanol, heat shock and cold [13– 16]. Although the isolation of PHA-producing bacteria from environments contaminated with different pollutants has been studied, only a few reports describe the microbial production of PHA in response to environmental heavy metal stress [17]. In nature, strains of C. taiwanensis and C. necator are able to accumulate a significant amount of PHB. Unlike the related species C. metallidurans, which possesses the ability to tolerate the stress induced by high concentrations of several forms of heavy metals [18], laboratory strains of C. taiwanensis generally cannot survive in high concentrations of heavy metal. We have isolated a strain of C. taiwanensis (known as strain EJ02) from sediments contaminated with various pollutants including heavy metals [19]. The bacteria were able to tolerate a high concentration of cadmium. Because C. taiwanensis EJ02 accumulates PHB, it is an exceptional candidate for studying the biosynthesis of PHB under the conditions of stress induced by high concentrations of the heavy metal cadmium.
http://dx.doi.org/10.1016/j.jtice.2014.02.004 1876-1070/ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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2. Materials and methods 2.1. Strain isolation and identification C. taiwanensis BCRC 17206T and BCRC 17208 were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). Strain EJ02 was isolated from sediments taken from the Er-Jen River in southern Taiwan using an enrichment culture technique of inoculating sediment (ca. 0.5 g) into Luria Bertani (LB) medium (trypton 10 g/L; yeast extract 5 g/L and NaCl 5 g/L) supplemented with 1 mM cadmium. The Er-Jen River is well known to be heavily contaminated by runoff from local industrial activities including electroplating factories and smelters [20]. Strain EJ02 was selected from the isolates because of its ability to grow well in the presence of cadmium ions in the culture medium. The initial characterization of strain EJ02 included analyzing the 16S-rRNA gene sequence as well as determining the bacterial staining characteristics and morphology. The bacterial DNA was extracted using the tissue and cell genomic DNA Purification Kit (Gene-Mark, Hopegen Biotechnology Development Enterprise, Taiwan). The 16S rRNA gene was amplified with the universal bacterial primers F24 and F25, as described in a previous study [21]. The purified polymerase chain reaction (PCR) products (1.5 kb) were then sequenced and compared with the 16S rRNA gene sequences available in the database at the National Center for Biotechnology Information (NCBI). The phylogenetic analyses of strain EJ02 and related taxa were performed by the ClustalV method included in the Lasergene software (DNASTAR) based on the 16S-rRNA gene sequences [22]. 2.2. Bacterial cultivation and cadmium tolerance C. taiwanensis BCRC 17206T, C. taiwanensis BCRC 17208 and strain EJ02 were routinely grown in 250-ml Erlenmeyer flasks containing 50 ml LB medium with or without sodium gluconate as an additional carbon source. To determine the extent of cadmium tolerance of the bacterial strains, the cells were grown in 250-ml Erlenmeyer flasks containing 50 ml LB medium with or without sodium gluconate as an additional carbon source and supplemented with different concentrations (500–7 mM) of cadmium [CdCl2]. The cultures were incubated at 32 8C while being shaken to maintain aerobic conditions. The cell growth was determined by measuring optical density at 600 nm (OD600).
RT Premix kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For real-time PCR, the primers phaA, phaB, phaC and rrs (ribosomal RNA 16S, housekeeping gene for normalization) were selected and designed by the software Primer3 (http://primer3.ut.ee/) according to the whole genome of C. taiwanensis [8,24]. The primer sequence Primer sequences were as follows: phaA, 50 -CCATGACCATCAACAAGGTG-30 (forward) and 5 0 -ATGCCCATGTGGTACTGGTT-3 0 (reverse); phaB, 5 0 AAAAGTGGCTGGAACAGCAG-3 0 (forward) and 5 0 -GAGGTCAGGTTGGTGTCGAT-30 (reverse); phaC, 50 -GAACGACCTGGTGTGGAACT-3 0 (forward) and 5 0 -TCGTTCTGCAGGTAGGTGTG-3 0 (reverse); and rrs, 50 -AGGCCTTCGGGTTGTAAAGT-30 (forward) and 50 -CGGGGATTTCACATCTGACT-30 (reverse). A real-time PCR reaction with the SYBR green mix (Thermo Fisher Scientific, San Diego, CA, USA) was carried out on an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA, USA). The specificity of the primers was confirmed by the presence of a single peak of the melting curve. Each target mRNA level was evaluated from the real-time threshold cycle and compared with the amount of rrs as an internal control. 3. Results and discussion 3.1. Strains isolation and identification Strain EJ02 was isolated from the sediment of the Er-Jen River in Tainan County, Taiwan [20]. The genus and species of the bacteria were identified as Cupriavidus taiwanensis according to the morphology, the staining characteristics, and the sequence of 16S rRNA gene. The strain used in the present study was designated EJ02. The phylogeny of C. taiwanensis EJ02 and some known related bacteria based on the alignment of their 16S rRNA gene sequences are shown in Fig. 1. The nucleotide sequence accession number of 16S rDNA of strain EJ02 determined in the present study has been deposited in the GenBank database (www.ncbi.nlm.nih.gov/Genbank; National Institutes of Health, Rockville, MD, USA) under accession number KF646764. 3.2. Growth and cadmium tolerance of C. taiwanensis EJ02, BCRC 17206T and BCRC 17208 The results of the growth of C. taiwanensis EJ02, C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 in LB medium
2.3. Evaluation of PHB accumulation by C. taiwanensis in the presence of cadmium Cells of C. taiwanensis BCRC 17206T, BCRC 17208 and EJ02 were cultivated in 500 ml Erlenmeyer flasks containing 100 ml LB medium supplemented with different concentrations of cadmium and incubated in a rotary shaker at 200 rev/min at 32 8C. The cells were harvested after 24 h, 48 h, 72 h or 96 h incubation, and washed with phosphate-buffered saline (pH 7.4). The collected cell pellets were then dried at 105 8C in an oven and weighed to obtain the dry cell mass. The amount of PHB (wt%) in the cells was determined by gas chromatography (GC) [23] and expressed as a percentage of the mass of PHB in dry cell weight. 2.4. Expression of phaA, phaB and phaC by real-time PCR analysis The total RNA was isolated from the cells of C. taiwanensis EJ02 using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cells were grown in LB medium supplemented with 1.5% sodium gluconate either with or without cadmium (1.0 mM or 3 mM). The cells were collected in the exponential phase and early stationary phase, respectively. First-strand cDNA synthesis was carried out using an
Fig. 1. The phylogenetic analysis based on the near-complete 16S rDNA sequences of Cupriavidus taiwanensis EJ02 and related taxa. The alignment was performed by the ClustalV method included in Lasergene software (DNASTAR). The scale beneath the tree indicates the number of nucleotide substitutions. GenBank accession numbers are listed in parentheses.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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Fig. 2. Growth curves of Cupriavidus taiwanensis EJ02 in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 1 mM (-*-), 2 mM (-!-), 3 mM (-~-), 4 mM (-&-), 5 mM (-&-), 6 mM (-^-), 7 mM (-^-) and 8 mM (-~-), respectively.
containing different concentrations of cadmium are shown in Fig. 2A (C. taiwanensis EJ02), Fig. 3A (C. taiwanensis BCRC 17206T) and Fig. 4A (C. taiwanensis BCRC 17208). The growth curves of C. taiwanensis EJ02, C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 in LB medium with 1.5% sodium gluconate as an additional carbon source containing different concentrations of cadmium are shown in Fig. 2B (C. taiwanensis EJ02), Fig. 3B (C.
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Fig. 3. Growth curves of Cupriavidus taiwanensis BCRC 17206T in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 0.5 mM (-*-), 1 mM (-!-), 1.5 mM (-~-).
taiwanensis BCRC 17206T) and Fig. 4B (C. taiwanensis BCRC 17208), respectively. Because PHB accumulates in bacteria under unbalanced growth conditions such as when the carbon substrate is in excess of the other nutrients (e.g., nitrogen, sulfur, phosphorus or oxygen), the addition of an extra carbon source could improve the accumulation of PHB in the cells. Sodium gluconate was chosen as an extra
Table 1 Accumulation of PHB by C. taiwanensis BCRB 17206T in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48 72
1.01 0.35 1.32 0.06 1.25 0.02
19.89 0.02 2.44 1.37 2.14 1.55
4.24 0.21 2.99 0.71 2.49 0.22
54.50 1.00 53.50 1.65 43.00 1.00
0.5
24 48 72
1.20 0.08 1.17 0.34 0.93 0.26
33.98 3.48 2.51 0.26 2.92 0.74
2.52 0.22 1.40 0.10 1.28 0.11
45.80 0.10 41.80 0.10 32.35 2.00
1.0
24 48 72
0.49 0.07 0.52 0.21 0.32 0.16
32.79 2.05 16.96 3.46 15.03 1.62
1.32 0.32 0.78 0.02 0.76 0.04
50.65 0.20 49.55 1.45 40.30 1.30
1.5
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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cells and the content of PHB synthesized by these strains when grown in LB medium (maximum content of PHB ranging between 15% and 20%) were much lower compared to cells grown in LB supplemented with sodium gluconate (maximum content of PHB ranging between 50% and 65%). When cadmium was added to the LB medium, cell growth (measured by cell dry weight) was reduced up to 60% in C. taiwanensis BCRC 17206T and BCRC 17208
Fig. 4. Growth curves of Cupriavidus taiwanensis BCRC 17208 in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 0.5 mM (-*-), 1 mM (-!-), 1.5 mM (-~-).
carbon source for these experiments because C. taiwanensis EJ02, similar to C. necator (a known PHB accumulator), is not able to metabolize glucose but utilizes various organic carbon and energy sources for heterotrophic growth including gluconic acid [25]. The amount of growth of C. taiwanensis EJ02 in LB medium (with or without 1.5% (w/v) gluconate) was comparable to that of C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208. However, when grown in LB medium containing cadmium, C. taiwanensis EJ02 was able to tolerate much higher concentrations of the heavy metal (up to 5 mM) compared to C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 (1 mM). C. taiwanensis EJ02 was able to tolerate 7 mM cadmium when sodium gluconate (1.5%, w/v) was added to the medium as an additional carbon source, but no significant increase in resistance to cadmium by C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 was observed when grown in the same conditions. In addition, the growth of C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 measured by OD600 was reduced in the presence of low concentrations of cadmium (i.e., 0.5 mM or 1 mM). 3.3. PHB production in the presence of cadmium by C. taiwanensis The intracellular accumulation of PHB was observed in the three strains of C. taiwanensis we examined. The dry weight of the
Fig. 5. Analysis of the expression of phaA, phaB and phaC of C. taiwanensis EJ02 grown in the medium with and without cadmium by real-time PCR. The expression fold change were relative to cells exponentially growing in the medium without cadmium: (A) mRNA expression level of cells grown in the medium without cadmium at early-stationary phase; (B) with cadmium (1 mM) at exponential phase (black bars) and early stationary phase (gray bars) and (C) with cadmium (3 mM) at exponential phase (black bars) and early stationary phase (gray bars).
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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(in the presence of 1 mM Cd) (Tables 1 and 2). However, in the presence of cadmium (0.5 mM and 1 mM), the amount of PHB that accumulated in C. taiwanensis BCRC 17206T and BCRC 17208 appeared to be much higher than in cells grown in LB medium without cadmium (Tables 1 and 2). An increase in PHB content was not observed in the cells grown in the LB medium containing sodium gluconate even in the presence of cadmium. Unlike C. taiwanensis BCRC 17206T and BCRC 17208, growing C. taiwanensis EJ02 in the LB medium in the presence of cadmium (0– 2 mM) or in LB medium with sodium gluconate in the presence of cadmium (0–4 mM) did not result in an appreciable difference in the cell dry weight and PHB content (Table 3). However, a decrease in the resultant cell dry weight and PHB content became apparent when the concentrations in the media of cadmium increased (3 mM and above in LB medium and 5 mM and above in LB medium with sodium gluconate) (Table 3). The accumulation of PHA might play a protective role for cellular survival under stress conditions [26]. An increase in the microbial accumulation of PHA was also demonstrated when bacterial cells were subjected to metal stress [17]. Application of the use of mild stress for improvement of PHB production has also been described [27]. In addition, pollutant-degrading bacteria such
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as dye-decolorizing bacteria were also shown to be able to produce PHB wastewater decolorization [28]. Although PHB accumulation was poor in LB medium and there was no significant increase in PHB synthesis by C. taiwanensis BCRC 17206T and BCRC 17208 under mild cadmium stress, the content of PHB accumulation was higher when the cells were grown in LB medium with cadmium. In addition, C. taiwanensis EJ02 showed more tolerance to cadmium when grown in LB medium supplemented with sodium gluconate. The addition of sodium gluconate as an extra carbon source could improve PHB accumulation in cells. The results in the present study indicate that PHB synthesis in C. taiwanensis EJ02 might enhance cellular resistance to heavy metal stress including cadmium stress. 3.4. Expression of phaA, phaB and phaC by real-time PCR analysis The expression of phaA, phaB and phaC was evaluated by detecting the mRNA transcripts of these genes using real-time PCR. The mRNA was harvested from cells in the exponential and earlystationary phases after growth in LB medium supplemented with 1.5% sodium gluconate with (1 mM or 3 mM) and without cadmium. The results showed that the expression of phaC, phaA and phaB
Table 2 Accumulation of PHB by C. taiwanensis BCRC 17208 in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48 72
1.45 0.12 1.40 0.12 0.41 0.07
18.44 1.64 2.19 0.77 1.85 0.41
4.76 0.16 3.54 0.97 2.85 0.28
60.29 4.70 64.00 2.00 51.79 2.79
0.5
24 48 72
1.40 0.22 0.80 0.16 0.53 0.11
31.96 1.28 5.51 2.75 7.15 2.87
1.32 0.32 1.45 0.25 1.49 0.21
43.19 3.81 36.28 4.27 26.40 4.27
1.0
24 48 72
0.56 0.24 0.41 0.07 0.37 0.10
33.95 2.44 26.30 4.45 26.20 4.31
1.05 0.30 0.70 0.30 0.72 0.28
40.85 3.34 41.00 0.15 35.80 2.77
1.5
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable. Table 3 Accumulation of PHB by C. taiwanensis EJ02 in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48
1.14 0.13 1.18 0.07
13.86 2.75 13.29 0.29
2.05 0.29 2.52 0.32
53.97 2.61 66.00 2.50
1
24 48
1.16 0.13 1.25 0.11
19.46 2.42 11.34 3.54
1.75 0.40 1.90 0.18
50.27 3.41 55.64 3.48
2
24 48
0.98 0.18 1.17 0.21
14.13 3.68 13.60 3.50
1.75 0.33 1.41 0.34
64.79 1.87 56.07 3.48
3
24 48
NG 0.69 0.13
ND 8.29 3.27
1.95 0.21 1.59 0.39
59.06 1.67 51.45 0.10
4
24 48
NG 0.42 0.12
ND 8.66 3.60
1.63 0.08 1.86 0.15
53.54 3.07 47.87 3.50
5
48 72
NG 0.60 0.05
ND 11.50 2.13
1.87 0.30 1.67 0.21
42.42 4.05 43.31 3.20
6
72 96
NG NG
ND ND
1.38 0.32 1.61 0.36
39.82 2.82 27.79 1.94
7
72 96
NG NG
ND ND
1.21 0.37 1.57 0.32
31.47 0.03 19.27 3.22
8
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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was higher in cells grown in the medium without cadmium and harvested at stationary phase (1.7 for phaA, 1.1 for phaB and 2.3 for phaC, respectively) compared to the cells harvested while exponentially growing in the same medium (Fig. 5A). The expression of these genes in the cells grown in the medium with low concentration of cadmium (1 mM) had only approximately 34% and 50% (exponential grown cells and stationary grown cells; phaA), 23 and 28% (phaB) and 38 and 92% (phaC) of the activity of those cells growing exponentially in the medium without cadmium (Fig. 5B). However, the expression of these genes in the cells grown in the medium with higher concentration of cadmium (3 mM) decreased to only approximately 22 and 9% (phaA), 10 and 5% (phaB) and 40 and 21% (phaC) of the activity of those cells growing exponentially in the medium without cadmium (Fig. 5C). Although the expression of the genes required for PHB biosynthesis did not increase in the cells of the strain EJ02 when exposed to cadmium, the biomass and PHB accumulation were comparable to those cells that were not exposed to the metal ion. The production of PHB by C. taiwanensis EJ02 is enhanced by the presence of cadmium, even though the mRNA expression of the necessary key players is lower than in the EJ02 cells not exposed to cadmium. Besides, many other components of PHA metabolism have been identified in both C. necator and C. taiwanensis and the analysis of their genomes extended a comprehensive view of PHB metabolism in these microorganisms [24,29]. Some examples include the existence of a second gene for a PHB synthase, phaC2 and isologs of phaA and phaB [29]. The activities of these additional genes might also affect biosynthesis of PHB by C. taiwanensis in the presence of heavy metal stress. In addition, genes encode PHB depolymerase such as phaZ also involved in mobilization of PHB during carbon starvation [29]. The effects of these gene activities and the impact on the bacterial biosynthesis under cadmium stress remains to be investigated. 4. Conclusion We have isolated a strain of bacteria resistant to the presence of a heavy metal from the sediments of a river heavily contaminated by the runoff of the local industrial activities. The bacteria, designated C. taiwanensis EJ02, tolerated the presence of cadmium up to a concentration of 7 mM. These bacteria, which accumulate PHB, provided us with an exceptional candidate for studying the biosynthesis of PHB under the stress of high concentrations of a heavy metal (cadmium). The results in the present study suggest that the resistance of C. taiwanensis EJ02 to cadmium stress might stem from its ability to synthesize PHB. Acknowledgements We thank Jian-Haw Chen, Ya-Ting Lin and professor Chao-Ling Yao, Department of Chemical Engineering and Materials Science, Yuan Ze University for help with real-time PCR analysis. This work was partially supported by grants (contract numbers NSC 1012632-E-155-001-MY3 and N101-2221-E-155-031-MY3) from the National Science Council, Taiwan. References [1] Luengo JM, Garcı´a B, Sandoval A, Naharro G, Olivera ER. Bioplastics from microorganisms. Curr Opin Microbiol 2003;6:251–60.
[2] Lu J, Tappel RC, Nomura CT. Mini-review. Biosynthesis of poly(hydroxyalkanoates). Polym Rev 2009;49:226–48. [3] Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates. Process Biochem 2005;40:607–19. [4] Steinbu¨chel A, Valentin HE. Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 1995;128:219–28. [5] Schlegel HG, Gottschalk G, Von Bartha R. Formation and utilization of poly-bhydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature 1961;191:463–5. [6] Kim YB, Lenz RW. Polyesters from microorganisms. Adv Biochem Eng Biotechnol 2001;71:51–79. [7] Vandamme P, Coenye T. Taxonomy of the genus Cupriavidus: a tale of lost and found. Int J Syst Evol Microbiol 2004;54:2285–9. [8] Chien C-C, Hong C-C, Soo P-C, Wei Y-H, Chen S-Y, Cheng M-L, et al. Functional expression of phaCAB genes from Cupriavidus taiwanensis strain 184 in Escherichia coli for polyhydroxybutyrate production. Appl Biochem Biotechnol 2010;162:2355–64. [9] Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 2000;25:1503–55. [10] Reddy CSK, Ghai R, Rashmi Kalia VC. Polyhydroxyalkanoates: an overview. Bioresour Technol 2003;87:137–46. [11] Kadouri D, Jurkevitch E, Okon Y, Castro-Sowinski S. Ecological and agricultural significance of bacterial polyhydroxyalkanoates. Crit Rev Microbiol 2005;31:55–67. [12] Ruiz JA, Lo´pez NI, Me´ndez BS. rpoS gene expression in carbon-starved cultures of the polyhydroxyalkanoate-accumulating species Pseudomonas oleovorans. Curr Microbiol 2004;48:396–400. [13] Ayub ND, Tribelli PM, Lo´pez NI. Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 143 during low temperature adaptation. Extremophiles 2009;13:59–66. [14] Ayub ND, Pettinari MJ, Me´ndez BS, Lo´pez NI. The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island. Plasmid 2007;58:240–8. [15] Zhao YH, Li HM, Qin LF, Wang HH, Chen G-Q. Disruption of the polyhydroxyalkanoate synthase gene in Aeromonas hydrophila reduces its survival ability under stress conditions. FEMS Microbiol Lett 2007;276:34–41. [16] Soto G, Setten L, Lisi C, Maurelis C, Mozzicafreddo M, Cuccioloni M, et al. Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium Pseudomonas sp. CT13 under abiotic stress. Extremophiles 2012;16:455–62. [17] Pal A, Paul AK. Accumulation of polyhydroxyalkanoates by Rhizobacteria underneath nickel-hyperaccumulators from serpentine ecosystem. J Polym Environ 2012;20:10–6. [18] Nies DH. Heavy metal resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia spec. CH34. Extremophiles 2000;4:77–82. [19] Chien C-C, Jiang M-H, Tsai M-R, Chien C-C. Isolation and characterization of an environmental cadmium- and tellurite-resistant Pseudomonas strain. Environ Toxicol Chem 2011;30:2202–7. [20] Chien C-C, Han C-T. Tellurite resistance and reduction by a Paenibacillus sp. isolated from heavy metal-contaminated sediment. Environ Toxicol Chem 2009;28:1627–32. [21] Dewhirst FE, Chien C-C, Paster BJ, Ericson RL, Orcutt RP, Schauer DB, et al. Phylogeny of the defined murine microbiota: altered Schaedler flora. Appl Environ Microbiol 1997;65:3287–92. [22] Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS 1989;5:151–3. [23] Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas chromatographic method for determination of poly-b-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol 1978;6:29–37. [24] Amadou C, Pascal G, Mangenot S, Glew M, Bontemps C, Capela D, et al. Genome sequence of the b-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Res 2008;18:1472–83. [25] Raberg M, Peplinski K, Heiss S, Ehrenreich A, Voigt B, Do¨ring C, et al. Proteomic and transcriptomic elucidation of the mutant Ralstonia eutropha G+1 with regard to glucose utilization. Appl Environ Microbiol 2011;77:2058–70. [26] Pavez P, Castillo JL, Gonza´lez C, Martı´nez M. Poly-b-hydroxyalkanoate exert a protective effect against carbon starvation and frozen conditions in Sphingopyxis chilensis. Curr Microbiol 2009;59:636–40. [27] Obruca S, Marova I, Svoboda Z, Mikulikova R. Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator. Folia Microbiol 2010;55:17–22. [28] Chen B-Y, Shiau T-J, Wei Y-H, Chen W-M. Feasibility study on polyhydroxybutyrate production of dye-decolorizing bacteria using dye and aminebearing cultures. J Taiwan Inst Chem Eng 2012;43:241–5. [29] Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, et al. Genome sequence of the bioplastic-producing Knallgas bacterium Ralstonia eutropha H16. Nat Biotechnol 2006;24:1257–62.
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Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis Chih-Ching Chien *, Li-Jung Wang, Wei-Ran Lin Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Chung-Li 32003, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 September 2013 Received in revised form 28 January 2014 Accepted 5 February 2014 Available online xxx
Polyhydroxybutyrate (PHB) is usually produced in various microorganisms in response to the environmental conditions of physiological stress. Cupriavidus taiwanensis is known to accumulate significant amounts of PHB and is phylogenetically related to Ralstonia eutropha (Cupriavidus necator), which is also well known for producing PHB. In this study, an environmental isolate of C. taiwanensis resistant to heavy metals (strain EJ02) was evaluated for its ability to accumulate PHB when grown in the presence of cadmium ions. C. taiwanensis EJ02 tolerated much higher concentrations of cadmium (up to 5 mM) when grown in complex (Luria-Bertani) media compared to C. taiwanensis strains BCRC 17206T and BCRC 17208 (1 mM). The growth of the strain EJ02 showed more resistance to cadmium (up to 7 mM) when grown in LB medium containing sodium gluconate than that in LB only. There was no significant effect on cadmium resistance for C. taiwanensis BCRC 17206T and BCRC 17208 when sodium gluconate was supplemented in LB medium. Although the production of PHB by C. taiwanensis EJ02 is enhanced by the presence of cadmium, the mRNA expression of the key genes for PHB biosynthesis (i.e., phaCAB) is lower than in the EJ02 cells not exposed to cadmium determined by real-time PCR analysis. The tolerance of C. taiwanensis EJ02 to the presence of heavy metals might be associated with its ability to accumulate PHB and to adapt to the stress of an environment with heavy metals. ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Polyhydroxybutyrate Stress resistance Heavy metal Cupriavidus
1. Introduction Polyhydroxyalkanoates (PHAs) are polymers synthesized by a variety of environmental microorganisms under unbalanced growth conditions or physiological stress [1,2]. PHAs are classified according to the length of their carbon chains. Short chain-length PHA (scl-PHA) polymers contain three to five C-atoms, and medium chain-length PHA (mcl-PHA) polymers contain six or more C-atoms [3,4]. The most commonly found monomer in an sclPHA is 3-hydroxy fatty acid, which is a short chain-length PHA. Microorganisms belonging to the genus Cupriavidus including Cupriavidus necator and C. taiwanensis are well known for being able to synthesize polyhydroxybutyrate (PHB), and the amount of PHB accumulation can be as high as 80–90% of the cell’s dry weight [5–8]. Because the properties of PHAs are similar to those of polyethylene (PE) and polypropylene (PP), PHAs have been
* Corresponding author at: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 32003, Taiwan. Tel.: +886 3 4638800x2184; fax: +886 3 4334667. E-mail address: [email protected] (C.-C. Chien).
explored as alternatives to petrochemical plastics [9,10]. In addition to their broad biotechnological applications, PHAs may play an important ecologic role by regulating microbial survival and tolerance in stressed environments [11]. For example, PHAs improved starvation tolerance [12], and increased resistance to various stress agents such as ethanol, heat shock and cold [13– 16]. Although the isolation of PHA-producing bacteria from environments contaminated with different pollutants has been studied, only a few reports describe the microbial production of PHA in response to environmental heavy metal stress [17]. In nature, strains of C. taiwanensis and C. necator are able to accumulate a significant amount of PHB. Unlike the related species C. metallidurans, which possesses the ability to tolerate the stress induced by high concentrations of several forms of heavy metals [18], laboratory strains of C. taiwanensis generally cannot survive in high concentrations of heavy metal. We have isolated a strain of C. taiwanensis (known as strain EJ02) from sediments contaminated with various pollutants including heavy metals [19]. The bacteria were able to tolerate a high concentration of cadmium. Because C. taiwanensis EJ02 accumulates PHB, it is an exceptional candidate for studying the biosynthesis of PHB under the conditions of stress induced by high concentrations of the heavy metal cadmium.
http://dx.doi.org/10.1016/j.jtice.2014.02.004 1876-1070/ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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2. Materials and methods 2.1. Strain isolation and identification C. taiwanensis BCRC 17206T and BCRC 17208 were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). Strain EJ02 was isolated from sediments taken from the Er-Jen River in southern Taiwan using an enrichment culture technique of inoculating sediment (ca. 0.5 g) into Luria Bertani (LB) medium (trypton 10 g/L; yeast extract 5 g/L and NaCl 5 g/L) supplemented with 1 mM cadmium. The Er-Jen River is well known to be heavily contaminated by runoff from local industrial activities including electroplating factories and smelters [20]. Strain EJ02 was selected from the isolates because of its ability to grow well in the presence of cadmium ions in the culture medium. The initial characterization of strain EJ02 included analyzing the 16S-rRNA gene sequence as well as determining the bacterial staining characteristics and morphology. The bacterial DNA was extracted using the tissue and cell genomic DNA Purification Kit (Gene-Mark, Hopegen Biotechnology Development Enterprise, Taiwan). The 16S rRNA gene was amplified with the universal bacterial primers F24 and F25, as described in a previous study [21]. The purified polymerase chain reaction (PCR) products (1.5 kb) were then sequenced and compared with the 16S rRNA gene sequences available in the database at the National Center for Biotechnology Information (NCBI). The phylogenetic analyses of strain EJ02 and related taxa were performed by the ClustalV method included in the Lasergene software (DNASTAR) based on the 16S-rRNA gene sequences [22]. 2.2. Bacterial cultivation and cadmium tolerance C. taiwanensis BCRC 17206T, C. taiwanensis BCRC 17208 and strain EJ02 were routinely grown in 250-ml Erlenmeyer flasks containing 50 ml LB medium with or without sodium gluconate as an additional carbon source. To determine the extent of cadmium tolerance of the bacterial strains, the cells were grown in 250-ml Erlenmeyer flasks containing 50 ml LB medium with or without sodium gluconate as an additional carbon source and supplemented with different concentrations (500–7 mM) of cadmium [CdCl2]. The cultures were incubated at 32 8C while being shaken to maintain aerobic conditions. The cell growth was determined by measuring optical density at 600 nm (OD600).
RT Premix kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For real-time PCR, the primers phaA, phaB, phaC and rrs (ribosomal RNA 16S, housekeeping gene for normalization) were selected and designed by the software Primer3 (http://primer3.ut.ee/) according to the whole genome of C. taiwanensis [8,24]. The primer sequence Primer sequences were as follows: phaA, 50 -CCATGACCATCAACAAGGTG-30 (forward) and 5 0 -ATGCCCATGTGGTACTGGTT-3 0 (reverse); phaB, 5 0 AAAAGTGGCTGGAACAGCAG-3 0 (forward) and 5 0 -GAGGTCAGGTTGGTGTCGAT-30 (reverse); phaC, 50 -GAACGACCTGGTGTGGAACT-3 0 (forward) and 5 0 -TCGTTCTGCAGGTAGGTGTG-3 0 (reverse); and rrs, 50 -AGGCCTTCGGGTTGTAAAGT-30 (forward) and 50 -CGGGGATTTCACATCTGACT-30 (reverse). A real-time PCR reaction with the SYBR green mix (Thermo Fisher Scientific, San Diego, CA, USA) was carried out on an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA, USA). The specificity of the primers was confirmed by the presence of a single peak of the melting curve. Each target mRNA level was evaluated from the real-time threshold cycle and compared with the amount of rrs as an internal control. 3. Results and discussion 3.1. Strains isolation and identification Strain EJ02 was isolated from the sediment of the Er-Jen River in Tainan County, Taiwan [20]. The genus and species of the bacteria were identified as Cupriavidus taiwanensis according to the morphology, the staining characteristics, and the sequence of 16S rRNA gene. The strain used in the present study was designated EJ02. The phylogeny of C. taiwanensis EJ02 and some known related bacteria based on the alignment of their 16S rRNA gene sequences are shown in Fig. 1. The nucleotide sequence accession number of 16S rDNA of strain EJ02 determined in the present study has been deposited in the GenBank database (www.ncbi.nlm.nih.gov/Genbank; National Institutes of Health, Rockville, MD, USA) under accession number KF646764. 3.2. Growth and cadmium tolerance of C. taiwanensis EJ02, BCRC 17206T and BCRC 17208 The results of the growth of C. taiwanensis EJ02, C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 in LB medium
2.3. Evaluation of PHB accumulation by C. taiwanensis in the presence of cadmium Cells of C. taiwanensis BCRC 17206T, BCRC 17208 and EJ02 were cultivated in 500 ml Erlenmeyer flasks containing 100 ml LB medium supplemented with different concentrations of cadmium and incubated in a rotary shaker at 200 rev/min at 32 8C. The cells were harvested after 24 h, 48 h, 72 h or 96 h incubation, and washed with phosphate-buffered saline (pH 7.4). The collected cell pellets were then dried at 105 8C in an oven and weighed to obtain the dry cell mass. The amount of PHB (wt%) in the cells was determined by gas chromatography (GC) [23] and expressed as a percentage of the mass of PHB in dry cell weight. 2.4. Expression of phaA, phaB and phaC by real-time PCR analysis The total RNA was isolated from the cells of C. taiwanensis EJ02 using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cells were grown in LB medium supplemented with 1.5% sodium gluconate either with or without cadmium (1.0 mM or 3 mM). The cells were collected in the exponential phase and early stationary phase, respectively. First-strand cDNA synthesis was carried out using an
Fig. 1. The phylogenetic analysis based on the near-complete 16S rDNA sequences of Cupriavidus taiwanensis EJ02 and related taxa. The alignment was performed by the ClustalV method included in Lasergene software (DNASTAR). The scale beneath the tree indicates the number of nucleotide substitutions. GenBank accession numbers are listed in parentheses.
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Fig. 2. Growth curves of Cupriavidus taiwanensis EJ02 in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 1 mM (-*-), 2 mM (-!-), 3 mM (-~-), 4 mM (-&-), 5 mM (-&-), 6 mM (-^-), 7 mM (-^-) and 8 mM (-~-), respectively.
containing different concentrations of cadmium are shown in Fig. 2A (C. taiwanensis EJ02), Fig. 3A (C. taiwanensis BCRC 17206T) and Fig. 4A (C. taiwanensis BCRC 17208). The growth curves of C. taiwanensis EJ02, C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 in LB medium with 1.5% sodium gluconate as an additional carbon source containing different concentrations of cadmium are shown in Fig. 2B (C. taiwanensis EJ02), Fig. 3B (C.
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Fig. 3. Growth curves of Cupriavidus taiwanensis BCRC 17206T in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 0.5 mM (-*-), 1 mM (-!-), 1.5 mM (-~-).
taiwanensis BCRC 17206T) and Fig. 4B (C. taiwanensis BCRC 17208), respectively. Because PHB accumulates in bacteria under unbalanced growth conditions such as when the carbon substrate is in excess of the other nutrients (e.g., nitrogen, sulfur, phosphorus or oxygen), the addition of an extra carbon source could improve the accumulation of PHB in the cells. Sodium gluconate was chosen as an extra
Table 1 Accumulation of PHB by C. taiwanensis BCRB 17206T in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48 72
1.01 0.35 1.32 0.06 1.25 0.02
19.89 0.02 2.44 1.37 2.14 1.55
4.24 0.21 2.99 0.71 2.49 0.22
54.50 1.00 53.50 1.65 43.00 1.00
0.5
24 48 72
1.20 0.08 1.17 0.34 0.93 0.26
33.98 3.48 2.51 0.26 2.92 0.74
2.52 0.22 1.40 0.10 1.28 0.11
45.80 0.10 41.80 0.10 32.35 2.00
1.0
24 48 72
0.49 0.07 0.52 0.21 0.32 0.16
32.79 2.05 16.96 3.46 15.03 1.62
1.32 0.32 0.78 0.02 0.76 0.04
50.65 0.20 49.55 1.45 40.30 1.30
1.5
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable.
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cells and the content of PHB synthesized by these strains when grown in LB medium (maximum content of PHB ranging between 15% and 20%) were much lower compared to cells grown in LB supplemented with sodium gluconate (maximum content of PHB ranging between 50% and 65%). When cadmium was added to the LB medium, cell growth (measured by cell dry weight) was reduced up to 60% in C. taiwanensis BCRC 17206T and BCRC 17208
Fig. 4. Growth curves of Cupriavidus taiwanensis BCRC 17208 in LB medium containing different concentrations of CdCl2 without (A) and with (B) sodium gluconate (1.5%, w/v) as a supplemental carbon source. The concentrations of CdCl2 in the media were 0 mM (-*-), 0.5 mM (-*-), 1 mM (-!-), 1.5 mM (-~-).
carbon source for these experiments because C. taiwanensis EJ02, similar to C. necator (a known PHB accumulator), is not able to metabolize glucose but utilizes various organic carbon and energy sources for heterotrophic growth including gluconic acid [25]. The amount of growth of C. taiwanensis EJ02 in LB medium (with or without 1.5% (w/v) gluconate) was comparable to that of C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208. However, when grown in LB medium containing cadmium, C. taiwanensis EJ02 was able to tolerate much higher concentrations of the heavy metal (up to 5 mM) compared to C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 (1 mM). C. taiwanensis EJ02 was able to tolerate 7 mM cadmium when sodium gluconate (1.5%, w/v) was added to the medium as an additional carbon source, but no significant increase in resistance to cadmium by C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 was observed when grown in the same conditions. In addition, the growth of C. taiwanensis BCRC 17206T and C. taiwanensis BCRC 17208 measured by OD600 was reduced in the presence of low concentrations of cadmium (i.e., 0.5 mM or 1 mM). 3.3. PHB production in the presence of cadmium by C. taiwanensis The intracellular accumulation of PHB was observed in the three strains of C. taiwanensis we examined. The dry weight of the
Fig. 5. Analysis of the expression of phaA, phaB and phaC of C. taiwanensis EJ02 grown in the medium with and without cadmium by real-time PCR. The expression fold change were relative to cells exponentially growing in the medium without cadmium: (A) mRNA expression level of cells grown in the medium without cadmium at early-stationary phase; (B) with cadmium (1 mM) at exponential phase (black bars) and early stationary phase (gray bars) and (C) with cadmium (3 mM) at exponential phase (black bars) and early stationary phase (gray bars).
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(in the presence of 1 mM Cd) (Tables 1 and 2). However, in the presence of cadmium (0.5 mM and 1 mM), the amount of PHB that accumulated in C. taiwanensis BCRC 17206T and BCRC 17208 appeared to be much higher than in cells grown in LB medium without cadmium (Tables 1 and 2). An increase in PHB content was not observed in the cells grown in the LB medium containing sodium gluconate even in the presence of cadmium. Unlike C. taiwanensis BCRC 17206T and BCRC 17208, growing C. taiwanensis EJ02 in the LB medium in the presence of cadmium (0– 2 mM) or in LB medium with sodium gluconate in the presence of cadmium (0–4 mM) did not result in an appreciable difference in the cell dry weight and PHB content (Table 3). However, a decrease in the resultant cell dry weight and PHB content became apparent when the concentrations in the media of cadmium increased (3 mM and above in LB medium and 5 mM and above in LB medium with sodium gluconate) (Table 3). The accumulation of PHA might play a protective role for cellular survival under stress conditions [26]. An increase in the microbial accumulation of PHA was also demonstrated when bacterial cells were subjected to metal stress [17]. Application of the use of mild stress for improvement of PHB production has also been described [27]. In addition, pollutant-degrading bacteria such
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as dye-decolorizing bacteria were also shown to be able to produce PHB wastewater decolorization [28]. Although PHB accumulation was poor in LB medium and there was no significant increase in PHB synthesis by C. taiwanensis BCRC 17206T and BCRC 17208 under mild cadmium stress, the content of PHB accumulation was higher when the cells were grown in LB medium with cadmium. In addition, C. taiwanensis EJ02 showed more tolerance to cadmium when grown in LB medium supplemented with sodium gluconate. The addition of sodium gluconate as an extra carbon source could improve PHB accumulation in cells. The results in the present study indicate that PHB synthesis in C. taiwanensis EJ02 might enhance cellular resistance to heavy metal stress including cadmium stress. 3.4. Expression of phaA, phaB and phaC by real-time PCR analysis The expression of phaA, phaB and phaC was evaluated by detecting the mRNA transcripts of these genes using real-time PCR. The mRNA was harvested from cells in the exponential and earlystationary phases after growth in LB medium supplemented with 1.5% sodium gluconate with (1 mM or 3 mM) and without cadmium. The results showed that the expression of phaC, phaA and phaB
Table 2 Accumulation of PHB by C. taiwanensis BCRC 17208 in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48 72
1.45 0.12 1.40 0.12 0.41 0.07
18.44 1.64 2.19 0.77 1.85 0.41
4.76 0.16 3.54 0.97 2.85 0.28
60.29 4.70 64.00 2.00 51.79 2.79
0.5
24 48 72
1.40 0.22 0.80 0.16 0.53 0.11
31.96 1.28 5.51 2.75 7.15 2.87
1.32 0.32 1.45 0.25 1.49 0.21
43.19 3.81 36.28 4.27 26.40 4.27
1.0
24 48 72
0.56 0.24 0.41 0.07 0.37 0.10
33.95 2.44 26.30 4.45 26.20 4.31
1.05 0.30 0.70 0.30 0.72 0.28
40.85 3.34 41.00 0.15 35.80 2.77
1.5
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable. Table 3 Accumulation of PHB by C. taiwanensis EJ02 in LB medium with/without sodium gluconate in the presence of different conc. of cadmium. Conc. of cadmium (mM)
Culture time (h)
Without sodium gluconate
With 1.5% (w/v) sodium gluconate
CDW (g/L)
PHB content (%)
CDW (g/L)
PHB content (%)
0
24 48
1.14 0.13 1.18 0.07
13.86 2.75 13.29 0.29
2.05 0.29 2.52 0.32
53.97 2.61 66.00 2.50
1
24 48
1.16 0.13 1.25 0.11
19.46 2.42 11.34 3.54
1.75 0.40 1.90 0.18
50.27 3.41 55.64 3.48
2
24 48
0.98 0.18 1.17 0.21
14.13 3.68 13.60 3.50
1.75 0.33 1.41 0.34
64.79 1.87 56.07 3.48
3
24 48
NG 0.69 0.13
ND 8.29 3.27
1.95 0.21 1.59 0.39
59.06 1.67 51.45 0.10
4
24 48
NG 0.42 0.12
ND 8.66 3.60
1.63 0.08 1.86 0.15
53.54 3.07 47.87 3.50
5
48 72
NG 0.60 0.05
ND 11.50 2.13
1.87 0.30 1.67 0.21
42.42 4.05 43.31 3.20
6
72 96
NG NG
ND ND
1.38 0.32 1.61 0.36
39.82 2.82 27.79 1.94
7
72 96
NG NG
ND ND
1.21 0.37 1.57 0.32
31.47 0.03 19.27 3.22
8
NA
NG
ND
NG
ND
CDW, cell dry weight; PHB content, PHB/CDW (w/w). Cells were grown on LB medium with or without sodium gluconate (1.5%, w/v) as an extra carbon source and in the presence of different concentrations of cadmium. NA, not applicable; NG, no growth; ND, not detectable.
Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004
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JTICE-843; No. of Pages 6 C.-C. Chien et al. / Journal of the Taiwan Institute of Chemical Engineers xxx (2014) xxx–xxx
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was higher in cells grown in the medium without cadmium and harvested at stationary phase (1.7 for phaA, 1.1 for phaB and 2.3 for phaC, respectively) compared to the cells harvested while exponentially growing in the same medium (Fig. 5A). The expression of these genes in the cells grown in the medium with low concentration of cadmium (1 mM) had only approximately 34% and 50% (exponential grown cells and stationary grown cells; phaA), 23 and 28% (phaB) and 38 and 92% (phaC) of the activity of those cells growing exponentially in the medium without cadmium (Fig. 5B). However, the expression of these genes in the cells grown in the medium with higher concentration of cadmium (3 mM) decreased to only approximately 22 and 9% (phaA), 10 and 5% (phaB) and 40 and 21% (phaC) of the activity of those cells growing exponentially in the medium without cadmium (Fig. 5C). Although the expression of the genes required for PHB biosynthesis did not increase in the cells of the strain EJ02 when exposed to cadmium, the biomass and PHB accumulation were comparable to those cells that were not exposed to the metal ion. The production of PHB by C. taiwanensis EJ02 is enhanced by the presence of cadmium, even though the mRNA expression of the necessary key players is lower than in the EJ02 cells not exposed to cadmium. Besides, many other components of PHA metabolism have been identified in both C. necator and C. taiwanensis and the analysis of their genomes extended a comprehensive view of PHB metabolism in these microorganisms [24,29]. Some examples include the existence of a second gene for a PHB synthase, phaC2 and isologs of phaA and phaB [29]. The activities of these additional genes might also affect biosynthesis of PHB by C. taiwanensis in the presence of heavy metal stress. In addition, genes encode PHB depolymerase such as phaZ also involved in mobilization of PHB during carbon starvation [29]. The effects of these gene activities and the impact on the bacterial biosynthesis under cadmium stress remains to be investigated. 4. Conclusion We have isolated a strain of bacteria resistant to the presence of a heavy metal from the sediments of a river heavily contaminated by the runoff of the local industrial activities. The bacteria, designated C. taiwanensis EJ02, tolerated the presence of cadmium up to a concentration of 7 mM. These bacteria, which accumulate PHB, provided us with an exceptional candidate for studying the biosynthesis of PHB under the stress of high concentrations of a heavy metal (cadmium). The results in the present study suggest that the resistance of C. taiwanensis EJ02 to cadmium stress might stem from its ability to synthesize PHB. Acknowledgements We thank Jian-Haw Chen, Ya-Ting Lin and professor Chao-Ling Yao, Department of Chemical Engineering and Materials Science, Yuan Ze University for help with real-time PCR analysis. This work was partially supported by grants (contract numbers NSC 1012632-E-155-001-MY3 and N101-2221-E-155-031-MY3) from the National Science Council, Taiwan. References [1] Luengo JM, Garcı´a B, Sandoval A, Naharro G, Olivera ER. Bioplastics from microorganisms. Curr Opin Microbiol 2003;6:251–60.
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Please cite this article in press as: Chien C-C, et al. Polyhydroxybutyrate accumulation by a cadmium-resistant strain of Cupriavidus taiwanensis. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.02.004