Appl Biochem Biotechnol DOI 10.1007/s12010-015-1479-4
Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Production by a Moderate Halophile Yangia sp. ND199 Using Glycerol as a Carbon Source Doan Van-Thuoc & Tran Huu-Phong & Dang Minh-Khuong & Rajni Hatti-Kaul
Received: 26 October 2014 / Accepted: 1 January 2015 # Springer Science+Business Media New York 2015
Abstract Yangia sp. ND199, a moderate halophile isolated from mangrove soil sample in Vietnam, was found to accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources in a medium with 4.5 % (w/v) NaCl. Cultivation with glycerol as carbon source and yeast extract as nitrogen source resulted in maximum cell dry weight of 5.7 g/l and PHBV content of 52.8 wt% (containing 2.9 mol% of 3HV) after 40 h. The 3HV content of the PHBV was the highest during initial stages of copolymer production and decreased with increase in the copolymer amount with time, but was not affected by changing the pH of the culture medium. Only homopolymer poly(3-hydroxybutyrate) was synthesized when monosodium glutamate was used as the nitrogen source. Fed-batch cultivation of Yangia sp. ND199 with glycerol and yeast extract gave PHBV content and productivity of 53.2 wt% and 0.44 g/l/h, respectively, which were reduced to 40.6 wt% and 0.25 g/l/h, respectively, with crude glycerol as carbon source. Both the copolymer content and productivity were improved to 56 wt% and 0.61 g/l/h, respectively, by using 1:1 mixture of crude glycerol and high fructose corn syrup. This is the first report of PHBV production by a wild-type halophilic bacterium using glycerol as carbon source. Keywords Yangia sp. ND199 . Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) . Glycerol . Carbon source . 3HV content
Introduction Polyhydroxyalkanoates (PHAs) are a group of biodegradable polyesters of biological origin, which have attracted industrial interest as potential alternatives for fossil-based plastics [1]. PHAs D. Van-Thuoc : T. Huu-Phong : D. Minh-Khuong Department of Microbiology and Biotechnology, Faculty of Biology, Hanoi National University of Education, 136 XuanThuy, CauGiay, Hanoi, Vietnam R. Hatti-Kaul (*) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden e-mail: [email protected]
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are accumulated intracellularly as carbon and energy storage materials in several microorganisms, usually when grown under the limitation of an essential nutrient such as oxygen, nitrogen, phosphorous, sulphur or magnesium and in the presence of excess and suitable carbon source [2]. There have so far been relatively limited reports available on PHA accumulation by halophilic microorganisms that have nevertheless shown these organisms to hold promise as potential industrial PHA producers. Several genera of halophilic archaea including Halobacterium, Haloferax, Haloarcula and Haloquadratum have been found to accumulate PHA [3–6]. Haloferax mediterranei is the best studied and was shown to accumulate up to 65 wt% PHA with respect to cell dry weight from starch or glucose in batch cultures under phosphorus limited conditions, while about 45 wt% was accumulated in continuous cultivations [7, 8]. The PHA formed by H. mediterranei from the carbohydrates was the copolymer poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) [9]. Moderate halophiles, e.g. Halomonas boliviensis [10], Halomonas hydrothermalis [11], Halomonas TD01 [12] and Halomonas campaniensis [13], isolated from saline habitats in Bolivia, India and China, respectively, have also demonstrated high polymer accumulation. H. boliviensis could produce PHB from different sugars, sodium acetate and butyric acid and agricultural residues. It could accumulate over 80 wt% PHB in fed-batch cultivations [10, 14]. The major advantages of halophilic organisms are the possibility of growing them under unsterile conditions [12], using seawater and cheap carbon sources to constitute the cultivation medium [13], and also the relative ease of extracting the biopolymer from the cells by hypoosmotic shock of the cells [15]. Mangroves are recognized to be highly productive ecosystems covering approximately 60– 75 % of the world’s tropical and subtropical coastlines [16]. Although rich in organic matter, mangrove ecosystems are generally nutrient deficient, especially in nitrogen and phosphorus. The productivity is ascribed to the microbial activity that is responsible for major nutrient transformations. Bacteria and fungi constitute 91 % of the total microbial biomass; the bacteria being responsible for most of the carbon- and energy flux. In an earlier study, several halophilic and halotolerant bacterial strains isolated from mangrove soil samples in Nam Dinh province, Northern Vietnam, were shown to have the ability to accumulate PHAs [17]. Two of the moderately halophilic Gram-negative strains that were identified as Yangia sp. ND199 and ND218 were found to synthesize the copolymer PHBV when grown on glucose as carbon source. Yangia sp. ND199 is able to grow at NaCl concentration up to 15 % (w/v), with an optimum of about 4–5 % (w/v), but exhibits no growth in the absence of salt. In the present work, we have evaluated the cell growth and PHA production by Yangia sp. ND199 on different carbon sources in the saline medium and subsequently continued the studies on PHA accumulation using glycerol as carbon source. Biodiesel production during the past decade has resulted in ready availability of the co-product glycerol (about 1224 million tonnes was produced in 2008). This has led to growing interest in its use as raw material for conversion to different value-added products including PHA [18]. As the crude glycerol obtained has a higher salt concentration, ranging from 3 to 7 %w/v, the possibility of using halophilic organisms could potentially provide an attractive production system for PHA.
Materials and Methods Bacterial Strain and Maintenance Yangia sp. ND199 was maintained on moderate halophiles (HM) agar plates at 4 °C [19], containing per litre: 45 g NaCl, 0.25 g MgSO4·7H2O, 0.09 g CaCl2·2H2O, 0.5 g KCl, 0.06 g NaBr, 5 g peptone, 10 g yeast extract, 1 g glucose and 20 g granulated agar. The pH of the medium was adjusted to 7.0.
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Shake Flask Cultivation of Yangia sp. ND199 with Different Carbon Sources Yangia sp. ND199 was grown in 20 ml HM medium in 100-ml shake flasks at 32 °C with rotary shaking at 180 rpm for 13 h (optical density (OD)600 =5.5). Subsequently, 2.5 ml aliquots of the culture broth were inoculated in 250-ml Erlenmeyer flasks with 50 ml HM-1 medium containing per litre: 45 g NaCl, 0.25 g MgSO4·7H2O, 0.09 g CaCl2·2H2O, 0.5 g KCl, 0.06 g NaBr, 0.25 g KH2PO4, 2 g yeast extract or monosodium glutamate and 20 g of a carbon source. The pH of the medium was initially adjusted to 7.2 using 1 M NaOH. The cultures were incubated at 32 °C with rotary shaking at 180 rpm, and samples were withdrawn at different time intervals for monitoring cell dry weight (CDW) and PHA content. Batch Cultivation of Yangia sp. with Glycerol as Carbon Source at Controlled pH Yangia sp. ND199 was initially grown in 150 ml HM medium in 1-l flask at 32 °C with rotary shaking at 180 rpm for 13 h (OD600 =4.5). The medium was then used to inoculate 1.35 l of HM-1 medium with 20 g/l glycerol as carbon source in a 3 l bioreactor. The cultivations were performed in batch mode during which temperature was kept constant at 32 °C and pH was maintained at 6.7, 7.2 or 7.7 by adding 5 M HCl/NaOH. Stirring velocity and aeration, initially set at 300 rpm and 0.5 l/min, were increased during the fermentation to maintain the dissolved oxygen concentration above 40 %. The highest speed for agitation and air inflow attained were 700 rpm and 2.5 l/min, respectively. After initial 9 h of cultivation, samples were taken every 3 h for CDW and PHA analysis. PHA Production by Fed-Batch Cultivation of Yangia sp. Fed-batch fermentation was performed at 30 °C, pH 7.0 using glycerol, crude glycerol (containing 44.89 % glycerol, 24.5 % biodiesel, 10.2 % methanol, 15.4 % fatty acid) or a mixture of crude glycerol and high fructose corn syrup (HFCS) (Daesang, South Korea) as carbon sources. The feed solution containing 10x concentrated HM-1 medium with 20 % (w/v) of the carbon sources was pumped into the bioreactor during first 20 h of cultivation to maintain the concentration of carbon source above 10 g/l with the help of offline analysis. After that, a feed solution containing only 20 % (w/v) carbon source was added to the bioreactor. Antifoam was added to the bioreactor when needed. After initial 6 h of cultivation, samples were taken every 3 h for CDW and PHA analysis. Quantitative Analyses CDW was determined by centrifuging 3 ml of the culture samples at 2000×g for 15 min in preweighed centrifuge tubes; the pellet was washed once with 3 ml distilled water, centrifuged and dried at 105 °C until constant weight was obtained. CDW was obtained by the difference in the final and initial weight of the centrifuge tube. The concentrations of glycerol and sugars (glucose and fructose) were determined by HPLC using a chromatography system (Jasco, Tokyo, Japan) equipped with RI-detector (ERC, Taguchi, Japan). Glycerol was separated on an Aminex HPX-87H column at 55 °C using a mobile phase of 5 mM H2SO4 at flow rate of 0.6 ml/min. The sugars were separated on an Aminex HPX-87P column, using MiliQ water as mobile phase at a rate of 0.6 ml/min and column temperature of 65 °C. PHA concentration was determined using a gas chromatographic method [20]. For this, about 10 mg of freeze-dried cells were mixed with 1 ml chloroform and 1 ml methanol solution containing 15 % (v/v) sulphuric acid and 0.4 % (w/v) benzoic acid. The mixture was
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incubated at 100 °C for 3 h to convert the constituents to their methyl esters. After cooling to room temperature, 0.5 ml of distilled water was added, and the mixture was shaken for 30 s. The lower chloroform layer was transferred to a fresh tube and used for gas chromatography analysis. Sample volume of 2 μl was injected into the gas chromatography column (VARIAN, Factor Four Capillary Column, CP8907). The injection temperature was 250 °C, the detector temperature was 240 °C and the column temperature was 60 °C for the initial 5 min and then increased at 3 °C/min to 120 °C. PHB and PHBV containing 12 % valerate (Sigma) were used as standards for calibration. All analyses were performed in triplicates. Residual cell mass (RCM) was defined as CDW minus PHA concentration, while PHA content was obtained as the percentage of the ratio of PHA concentration to CDW. The polymer yield was calculated as gram PHA produced per gram of carbon source utilized. NMR Analysis of PHAs NMR analysis of the PHAs produced by Yangia sp. was performed as described previously [10]. The polymer containing bacterial cells were harvested from 300 ml of culture broth by centrifugation at 6000g for 10 min, washed twice with distilled water and lyophilized. PHA was recovered from the lyophilized cells by extraction for 30 h with chloroform in a Soxhlet apparatus (Duran, Germany) and concentrated by evaporating the solvent under vacuum. The polymer was precipitated from the concentrated solution with 10 volumes of ethanol, and the resulting PHA granules were filtered twice. The 1H nuclear magnetic resonance (NMR) spectrum was recorded at 500 MHz with a Bruker ARX500 Spectrometer (Bruker, Sikerstrifen, Germany) at room temperature using deuterated chloroform as internal reference solvent. The spectrum was evaluated using standard BrukerUXNMR software.
Results and Discussion PHA Production by Yangia sp. ND199 from Different Carbon Sources Yangia sp. ND199 was grown in HM-1 medium with yeast extract as nitrogen source and with different carbon sources in order to investigate the effect on cell growth and PHA production. The results of CDW, PHA content and concentration, RCM and monomer Table 1 Effect of different carbon sources on cell growth and PHA accumulation by Yangia sp. ND199 Carbon sourcea
CDW (g/l)
PHA content (wt%)
PHA conc. (g/l)
RCM (g/l)
Monomer composition (mol%) 3HB
3HV
Glucose
5.0±0.1
48.9±1.4
2.5±0.2
2.5±0.2
98.7
1.3
Maltose Xylose
3.9±0.1 1.9±0.2
22.3±0.7 0
0.9±0.1 0
3.0±0.1 1.9±0.2
92.6 0
7.4 0
Sucrose
4.4±0.1
26.7±0.7
1.2±0.1
3.2±0.1
94.7
5.3
Fructose
5.6±0.2
53.8±1.3
3.0±0.2
2.6±0.2
100
Trace
Dextrin
2.0±0.1
0
0
2.0±0.1
0
0
Glycerol
4.5±0.1
44.8±1.2
2.0±0.2
2.5±0.1
94.6
5.4
a
All the carbon sources were of analytical grade and were obtained from standard sources
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composition of the PHAs in the samples obtained after 30 h of cultivation are summarized in Table 1. Among the seven carbon sources tested, fructose, glucose and glycerol were found to be the most suitable substrates for both cell growth and PHA accumulation. Maximum CDW of 5.6 g/l and PHA content of 53.8 wt% were obtained with fructose, followed by glucose (CDW of 5.0 g/l and PHA content of 48.9 wt%) and glycerol (CDW of 4.5 g/l and PHA content of 44.8 wt%). Sucrose and maltose led to lower accumulation of PHA, while xylose and dextrin served as poor substrates for cell growth and also did not result in PHA synthesis.
Fig. 1 1H NMR spectra of PHBV isolated from Yangia sp. ND199 grown on a glucose and b maltose, respectively, as carbon source
Appl Biochem Biotechnol
W CDW
PHA content
HV fraction
7
60
6
50
CDW (g/l)
5
40
4
30
3
20
2
PHA content (% CDW) HV fraction (mol%)
(A)
10
1
0
0
0
0 10
30
20
40
50
Time (h)
(B) 3.5
25
CDW (g/l)
3
20
2.5
15
2 1.5
10
1
PHA content (% CDW) HV fraction (mol%)
30
4
5
0.5
0
0
0
10
20
30
40
50
Time (h) Fig. 2 Profiles of CDW, PHA content and PHA composition during cultivation of Yangia sp. ND199 in shake flasks in the medium with glycerol as carbon source and a yeast extract and b monosodium glutamate, respectively, as nitrogen source
The composition of PHA is known to depend on the microorganism and the substrate used [2]. The 1H NMR analysis of the PHA isolated from the Yangia sp. ND199 cells grown on glucose and maltose, respectively, showed major peaks corresponding to PHBV (Fig. 1a, b). The 3HV content in the copolymer synthesized varied with the carbon source used; the highest 3HV content of 7.4 mol% was obtained when the organism was grown on maltose followed by glycerol and sucrose giving 5.4 and 5.3 mol%, respectively (Table 1). PHBV is less crystalline, more elastic
Fig. 3 Effect of varying pH on a CDW, b PHBV content and c 3HV mol% of PHBV accumulated by Yangia sp. ND199 during cultivation in the medium with glycerol and yeast extract as carbon and nitrogen sources, respectively
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(A)
pH 6.7
8
pH 7.2
pH 7.7
7
CDW (g/l)
6 5 4 3 2 1 0 0
5
10
15 20 Time (h)
25
30
(B) PHA content (% CDW)
50 40 30 20 10 0 9
12
15
18
21
24
27
30
Time (h)
(C) HV fraction (mol%)
100 80 60 40 20 0 9
12
15
18
21
Time (h)
24
27
30
Appl Biochem Biotechnol Fig. 4 Profiles of cell dry weight and PHBV concentration and content in a fed-batch fermentation mode using a glycerol, b crude glycerol and c 1:1 mixture of crude glycerol and high fructose corn syrup, as carbon sources. The concentrations of the carbon sources are also shown
and tougher than PHB and has broader range of applications. The increase in the 3HV content is correlated with decrease in crystallinity and increased elasticity of the copolymer [2]. As glycerol was found to be a suitable substrate for both cell growth and PHBV synthesis with relatively high 3HV content by Yangia sp. ND199, it was used for further studies. Glycerol in crude as well as refined form is considered as relatively cheap and abundant substrate for industrial microbiology, especially since its increased availability as a co-product of biodiesel production [21]. Integration of glycerol conversion to PHA will not only provide added value to biodiesel production but should also reduce the polymer production cost. Glycerol has earlier been used as carbon source for PHB production by Ralstonia eutropha [22], Zobellella denitrificans [23] and Pseudomonas oleovorans and for medium chain length PHA by Pseudomonas corrugata [24]. Salmonella enterica and Escherichia coli have been metabolically engineered for the production of PHBV and poly(3-hydroxypropionate) (PHP), respectively, from glycerol [25, 26]. Effect of Nitrogen Source on PHA Production Preliminary studies in the lab on cultivation of Yangia sp. ND199 showed yeast extract and monosodium glutamate (MSG), respectively, to be the most suitable nitrogen sources for cell growth and that polymer synthesis was facilitated by nitrogen limitation. As seen in Fig. 2, PHA synthesis followed the same trend as the cell growth. When grown on glycerol and yeast extract, the cells grew faster and accumulated higher amount of PHA giving maximum CDW of 5.7 g/l and PHA content of 52.8 wt% after 40 h of cultivation (Fig. 2a), while only 3.3 g/l CDW and 24 wt% PHA were obtained with MSG as nitrogen source (Fig. 2b). Furthermore, while the copolymer PHBV was synthesized in the medium containing yeast extract, only homopolymer PHB was synthesized with MSG (Fig. 2a, b). This would suggest that Yangia sp. ND199 is able to metabolize amino acids present in the yeast extract like aspartate, threonine and methionine through pathways generating propionyl-coenzyme A, an important precursor for 3HV monomer. H. mediterranei has been shown to possess multiple propionyl-CoA supplying pathways for the production of PHBV [27]. One such metabolic pathway via threonine biosynthesis has been engineered in E. coli for the production of PHBV from an unrelated carbon source like xylose [28]. As seen in Fig. 2a, the highest 3HV content of 9 mol% was obtained in the PHBVafter 24 h of cultivation in the yeast extract medium and was then decreased to 2.8 mol% after 48 h, with concomitant increase in 3HB content of the copolymer that uses acetyl-CoA as precursor. PHBV Production at Different pH Batch cultivation of Yangia sp. ND199 with glycerol and yeast extract in a bioreactor at controlled pH (6.7, 7.2 or 7.7) showed highest rate of cell growth and PHA accumulation at pH 6.7 reaching a maximum CDW of 7.1 g/l and PHBV content of 45 wt% after 24 h of cultivation (Fig. 3a, b). With increase in pH to 7.2, maximum CDW obtained was the same but was achieved at a slightly reduced growth rate and the cells accumulated lower amount of PHA (Fig. 3b). Further increase in pH to 7.7 resulted in a significant decrease in both CDW and PHBV content (Fig. 3a, b). The polymer synthesis thus appears to follow the same trend as the cell growth. These results are in accordance with earlier reports showing that changing the
Appl Biochem Biotechnol CDW
PHBV conc.
Glycerol
PHBV content
50 30 40 20
30 20
10 10 0
0 0
(B)
5
CDW
10
15 20 Time (h)
PHBV conc.
25
30
Glycerol
35
PHBV content
60 50
30 40 30
20
20 10 10
PHA content (% CDW)
CDW, PHA conc., CG (g/l)
40
0
0 0
(C) CDW, PHA conc., Sugars. Glycerol (g/l)
PHA content (% CDW)
60
40
5
10
CDW Glycerol
15 20 Time (h)
25
PHBV conc. PHBV content
30
35
Sugars
60
40
50 30 40 30
20
20 10 10 0
0 0
5
10
15 20 Time (h)
25
30
35
PHA content (% CDW)
CDW, PHA conc., Glycerol (g/l)
(A)
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pH of the culture medium could be used to control not only the cell growth rate but also the polymer synthesis, regardless of the medium composition [29]. Monitoring the 3HV content of PHBV formed with time during cultivation of Yangia sp. ND199 showed that the organism uses only 3HV monomers at the start to form the polymer (e.g. 100 % 3HV content recorded at 0.5 wt% PHA at pH 7.7), but the 3HV content decreases sharply as increasing number of 3HB units are incorporated into the polymer (Fig. 3c). The 3HV content of the copolymer followed a similar trend irrespective of the pH during cultivation. For example, at PHBV content of 1.19 wt%, 3HV content was found to be 59.27 mol% after 9 h of cultivation at pH 7.2, while at pH 7.7, nearly similar values for PHBV content of 1.63 wt% and 3HV content of 63.1 mol% were obtained but after 21 h of cultivation. Similarly, after 12-h cultivation at pH 7.2 and 27-h cultivation at pH 7.7, PHBV contents of about 7.78 and 7.11 wt%, respectively, with 3HV contents of about 21.3 and 21.5 mol%, respectively, were achieved at the two pH values (Fig. 3b, c). In contrast, the composition of PHA synthesized in a mixed culture was strongly affected by pH, as observed by the increase in 3HV content of PHBV from about 13 mol% up to about 48 mol% with increase in pH from 8.5 to 9.5 [30]. PHBV Production by Fed-Batch Cultivation of Yangia sp. ND199 Yangia sp. ND199 was then cultivated in a fed-batch fermentation mode using glycerol and crude glycerol, respectively, as carbon source. Figure 4a shows that CDW reached a maximum value of 27.6 g/l with PHA content of 53.2 wt% at cultivation time of 33 h, resulting in PHA concentration of 14.68 g/l and productivity of 0.44 g/l/h. PHBVyield with respect to glycerol was 0.29 g per gram glycerol, which is comparable to the PHB productivity obtained (0.25 g/g) for Z. denitrificans [31]. When crude glycerol was used as the carbon source, the growth and PHBV accumulation in Yangia sp. ND199 were reduced due to the inhibitory effect of salt and other components. CDW, PHBV content and PHBV productivity and yield of 20.5 g/l, 40.6 %, 0.25 g/l/h and 0.2 g/g, respectively, were obtained after 33 h of cultivation (Fig. 4b). The inhibition does not seem to be as severe as reported earlier for cell growth and PHB production in Paracoccus denitrificans and Cupriavidus necator JMP134 grown on crude glycerol; the PHB yield coefficient for the latter organism was reduced from 0.37 to 0.14 g/g on replacing pure glycerol by salt-contaminated crude glycerol [32]. More recently, a halophilic bacterium H. hydrothermalis, isolated from a marine environment, using Jatropha biodiesel by-product as carbon source gave a PHB content of 75.8 wt% but with cell dry weight of 0.4 g/l after 72 h of cultivation [11]. Table 2 provides a comparison of PHA production from glycerol by different organisms and shows that production by Yangia sp. ND199 in this study is in the same range as that by other wild-type PHA producers, C. necator JMP134 and Zobellella denitrificans MW1. However, the cell density and PHA concentration in Yangia sp. are relatively lower, suggesting that improvement in cell growth should lead to increased polymer yield and productivity. The inhibitory effect of crude glycerol can be reduced by combining it with other cheap carbon substrate(s), especially the ones that can contribute to increased intracellular NAD(P)H concentration and/or high NAD(P)H/NAD(P) ratio [33]. Synthesis of PHB was significantly influenced by using mixtures of substrates such as fructose and methanol in case of Methylobacterium rhodesianum [34] and glucose and xylose for H. boliviensis [35]. Using crude glycerol and high fructose corn syrup (HFCS; containing approximately 55 % fructose and 42 % glucose) at a ratio of 1:1 as carbon source for Yangia sp. ND199 led to significant increase in the CDW, PHBV content and PHBV concentration of Yangia sp. ND199 reaching maximum values of 36.2 g/l, 56 wt% and 20.3 g/l, respectively after 33 h cultivation (Fig. 4c). As also seen in Fig. 4c, the bacteria utilized the sugars more efficiently than glycerol. PHBV productivity obtained in this fermentation was 0.61 g/l/h, which is 1.4
Glycerol
Crude glycerol
Glycerol
Crude glycerol
Glycerol
Crude glycerol
Yangia sp. ND199
Yangia sp. ND199
Recombinant E. coli
Recombinant E. coli
Z. denitrificans MW1
C. necator JMP 134
Carbon source
Organism
PHB
PHB
PHP
PHP
PHBV
PHBV
PHA type
50
81.2
5.15
11.98
20.5
27.6
CDW (g/l)
48
66.9
5.2
11.98
40.6
53.2
PHA content (wt%)
24
54.3
0.27
1.42
8.3
14.7
PHA conc. (g/l)
Not reported
1.09
0.003
0.015
0.25
0.44
PHA productivity (g/l/h)
Table 2 Comparison of PHA production from glycerol or crude glycerol in fed-batch cultivation by Yangia sp. ND199 with other bacterial species
0.14
0.25
0.004
0.017
0.2
0.29
PHA yield (g/g C source)
[32]
[31]
[26]
[26]
This work
This work
Reference
Appl Biochem Biotechnol
Appl Biochem Biotechnol
and 2.4 times higher than fermentations with glycerol and crude glycerol, respectively, as carbon source.
Conclusions The study shows Yangia sp. ND199 to be a novel microorganism with significant potential for production of the copolymer PHBV from unrelated carbon sources including glycerol. Accumulation of PHA by Yangia sp. ND199 is an adaptation of the organism to the nutrient-poor environment of the mangroves. This is the first report of a wild-type halophilic bacterium using glycerol as carbon source for PHBV production. The organism was able to use crude glycerol as carbon source without significant inhibition on cell growth and polymer production. Mixing another cheap carbon source providing higher reducing equivalents could be used to advantage for achieving improved cell growth and polymer accumulation. Yangia sp. ND199 can potentially grow under unsterile conditions and may be used directly with the by-product streams from biodiesel and other processes, thus reducing the process costs. For improving cell growth as well as PHBV production, studies are currently being pursued on metabolic flux analysis in Yangia sp. ND199 with selected carbon sources and on optimizing cultivation conditions. Acknowledgments The authors are grateful to the National Foundation for Science and Technology Development (Grant no. 106.03-2010.64) and the Swedish Research Council through the Swedish Research Links programme for supporting this work.
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Appl Biochem Biotechnol 16. Holguin, G., Vazquez, P., & Bashan, Y. (2001). Biology and Fertility of Soils, 33, 265–278. 17. Van-Thuoc, D., Huu-Phong, T., Thi-Binh, N., Thi-Tho, N., Minh-Lam, D., & Quillaguamán, J. (2012). Microbiology Open, 1, 395–406. 18. Yang, F., Hanna, M. A., & Sun, R. (2012). Biotechnology for Biofuels, 5, 13. 19. Quillaguamán, J., Hatti-Kaul, R., Mattiasson, B., Alvarez, M. T., & Delgado, O. (2004). International Journal of Systematic and Evolutionary Microbiology, 54, 721–725. 20. Huijberts, G. N. M., van der Wal, H., Wilkinson, C., & Eggink, G. (1994). Biotechnology Techniques, 8, 187–192. 21. da Silva, G. P., Mack, M., & Contiero, J. (2009). Biotechnology Advances, 27, 30–39. 22. Taidi, B., Anderson, A. J., Dawes, E. A., & Byrom, D. (1994). Applied Microbiology and Biotechnology, 40, 786–790. 23. Ibrahim, M. H. A., & Steinbüchel, A. (2010). Journal of Applied Microbiology, 108, 214–225. 24. Ashby, R. D., Solaima, D. K. Y., & Foglia, T. A. (2005). Biomacromolecules, 6, 2106–2112. 25. Aldor, I. S., Kim, S. W., Prather, K. L., & Keasling, J. D. (2002). Applied and Environmental Microbiology, 68, 3848–3854. 26. Andreeβen, B., Kange, A. B., Robenek, H., & Steinbuchel, A. (2010). Applied and Environmental Microbiology, 76, 622–626. 27. Han, J., Hou, J., Zhang, F., Ai, G., Li, M., Cai, S., Liu, H., Wang, Z., Zhang, S., Cai, L., Zhao, D., Zhou, J., & Xiang, H. (2013). Applied and Environmental Microbiology, 79, 2922–2931. 28. Chen, Q., Wang, Q., Wei, G., Liang, Q., & Qi, Q. (2011). Applied and Environmental Microbiology, 77, 4886–4893. 29. Suzuki, T., Tsygankov, A. A., Miyake, J., Tokiwa, Y., & Asada, Y. (1995). Biotechnology Letters, 17, 395– 400. 30. Villano, M., Beccari, M., Dionisi, D., Lampis, S., Miccheli, A., Vallini, G., & Majone, M. (2010). Process Biochemistry, 2010, 714–723. 31. Ibrahim, M. H. A., & Steinbüchel, A. (2009). Applied and Environmental Microbiology, 75, 6222–6231. 32. Mothes, G., Schnorpfeil, C., & Ackermann, J. U. (2007). Engineering in Life Sciences, 7, 475–479. 33. Babel, W., Ackermann, J. U., & Breuer, U. (2001). Advances in biochemical engineering biotechnology, vol. 71. In T. Scheper, W. Babel, & A. Steinbüchel (Eds.), Biopolyesters (pp. 125–157). Berlin: Springer Press. 34. Ackermann, J. U., & Babel, W. (1997). Applied Microbiology and Biotechnology, 47, 144–149. 35. Van-Thuoc, D., Quillaguamán, J., Mamo, G., & Mattiasson, B. (2008). Journal of Applied Microbiology, 104, 420–428.
Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Production by a Moderate Halophile Yangia sp. ND199 Using Glycerol as a Carbon Source Doan Van-Thuoc & Tran Huu-Phong & Dang Minh-Khuong & Rajni Hatti-Kaul
Received: 26 October 2014 / Accepted: 1 January 2015 # Springer Science+Business Media New York 2015
Abstract Yangia sp. ND199, a moderate halophile isolated from mangrove soil sample in Vietnam, was found to accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources in a medium with 4.5 % (w/v) NaCl. Cultivation with glycerol as carbon source and yeast extract as nitrogen source resulted in maximum cell dry weight of 5.7 g/l and PHBV content of 52.8 wt% (containing 2.9 mol% of 3HV) after 40 h. The 3HV content of the PHBV was the highest during initial stages of copolymer production and decreased with increase in the copolymer amount with time, but was not affected by changing the pH of the culture medium. Only homopolymer poly(3-hydroxybutyrate) was synthesized when monosodium glutamate was used as the nitrogen source. Fed-batch cultivation of Yangia sp. ND199 with glycerol and yeast extract gave PHBV content and productivity of 53.2 wt% and 0.44 g/l/h, respectively, which were reduced to 40.6 wt% and 0.25 g/l/h, respectively, with crude glycerol as carbon source. Both the copolymer content and productivity were improved to 56 wt% and 0.61 g/l/h, respectively, by using 1:1 mixture of crude glycerol and high fructose corn syrup. This is the first report of PHBV production by a wild-type halophilic bacterium using glycerol as carbon source. Keywords Yangia sp. ND199 . Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) . Glycerol . Carbon source . 3HV content
Introduction Polyhydroxyalkanoates (PHAs) are a group of biodegradable polyesters of biological origin, which have attracted industrial interest as potential alternatives for fossil-based plastics [1]. PHAs D. Van-Thuoc : T. Huu-Phong : D. Minh-Khuong Department of Microbiology and Biotechnology, Faculty of Biology, Hanoi National University of Education, 136 XuanThuy, CauGiay, Hanoi, Vietnam R. Hatti-Kaul (*) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden e-mail: [email protected]
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are accumulated intracellularly as carbon and energy storage materials in several microorganisms, usually when grown under the limitation of an essential nutrient such as oxygen, nitrogen, phosphorous, sulphur or magnesium and in the presence of excess and suitable carbon source [2]. There have so far been relatively limited reports available on PHA accumulation by halophilic microorganisms that have nevertheless shown these organisms to hold promise as potential industrial PHA producers. Several genera of halophilic archaea including Halobacterium, Haloferax, Haloarcula and Haloquadratum have been found to accumulate PHA [3–6]. Haloferax mediterranei is the best studied and was shown to accumulate up to 65 wt% PHA with respect to cell dry weight from starch or glucose in batch cultures under phosphorus limited conditions, while about 45 wt% was accumulated in continuous cultivations [7, 8]. The PHA formed by H. mediterranei from the carbohydrates was the copolymer poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) [9]. Moderate halophiles, e.g. Halomonas boliviensis [10], Halomonas hydrothermalis [11], Halomonas TD01 [12] and Halomonas campaniensis [13], isolated from saline habitats in Bolivia, India and China, respectively, have also demonstrated high polymer accumulation. H. boliviensis could produce PHB from different sugars, sodium acetate and butyric acid and agricultural residues. It could accumulate over 80 wt% PHB in fed-batch cultivations [10, 14]. The major advantages of halophilic organisms are the possibility of growing them under unsterile conditions [12], using seawater and cheap carbon sources to constitute the cultivation medium [13], and also the relative ease of extracting the biopolymer from the cells by hypoosmotic shock of the cells [15]. Mangroves are recognized to be highly productive ecosystems covering approximately 60– 75 % of the world’s tropical and subtropical coastlines [16]. Although rich in organic matter, mangrove ecosystems are generally nutrient deficient, especially in nitrogen and phosphorus. The productivity is ascribed to the microbial activity that is responsible for major nutrient transformations. Bacteria and fungi constitute 91 % of the total microbial biomass; the bacteria being responsible for most of the carbon- and energy flux. In an earlier study, several halophilic and halotolerant bacterial strains isolated from mangrove soil samples in Nam Dinh province, Northern Vietnam, were shown to have the ability to accumulate PHAs [17]. Two of the moderately halophilic Gram-negative strains that were identified as Yangia sp. ND199 and ND218 were found to synthesize the copolymer PHBV when grown on glucose as carbon source. Yangia sp. ND199 is able to grow at NaCl concentration up to 15 % (w/v), with an optimum of about 4–5 % (w/v), but exhibits no growth in the absence of salt. In the present work, we have evaluated the cell growth and PHA production by Yangia sp. ND199 on different carbon sources in the saline medium and subsequently continued the studies on PHA accumulation using glycerol as carbon source. Biodiesel production during the past decade has resulted in ready availability of the co-product glycerol (about 1224 million tonnes was produced in 2008). This has led to growing interest in its use as raw material for conversion to different value-added products including PHA [18]. As the crude glycerol obtained has a higher salt concentration, ranging from 3 to 7 %w/v, the possibility of using halophilic organisms could potentially provide an attractive production system for PHA.
Materials and Methods Bacterial Strain and Maintenance Yangia sp. ND199 was maintained on moderate halophiles (HM) agar plates at 4 °C [19], containing per litre: 45 g NaCl, 0.25 g MgSO4·7H2O, 0.09 g CaCl2·2H2O, 0.5 g KCl, 0.06 g NaBr, 5 g peptone, 10 g yeast extract, 1 g glucose and 20 g granulated agar. The pH of the medium was adjusted to 7.0.
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Shake Flask Cultivation of Yangia sp. ND199 with Different Carbon Sources Yangia sp. ND199 was grown in 20 ml HM medium in 100-ml shake flasks at 32 °C with rotary shaking at 180 rpm for 13 h (optical density (OD)600 =5.5). Subsequently, 2.5 ml aliquots of the culture broth were inoculated in 250-ml Erlenmeyer flasks with 50 ml HM-1 medium containing per litre: 45 g NaCl, 0.25 g MgSO4·7H2O, 0.09 g CaCl2·2H2O, 0.5 g KCl, 0.06 g NaBr, 0.25 g KH2PO4, 2 g yeast extract or monosodium glutamate and 20 g of a carbon source. The pH of the medium was initially adjusted to 7.2 using 1 M NaOH. The cultures were incubated at 32 °C with rotary shaking at 180 rpm, and samples were withdrawn at different time intervals for monitoring cell dry weight (CDW) and PHA content. Batch Cultivation of Yangia sp. with Glycerol as Carbon Source at Controlled pH Yangia sp. ND199 was initially grown in 150 ml HM medium in 1-l flask at 32 °C with rotary shaking at 180 rpm for 13 h (OD600 =4.5). The medium was then used to inoculate 1.35 l of HM-1 medium with 20 g/l glycerol as carbon source in a 3 l bioreactor. The cultivations were performed in batch mode during which temperature was kept constant at 32 °C and pH was maintained at 6.7, 7.2 or 7.7 by adding 5 M HCl/NaOH. Stirring velocity and aeration, initially set at 300 rpm and 0.5 l/min, were increased during the fermentation to maintain the dissolved oxygen concentration above 40 %. The highest speed for agitation and air inflow attained were 700 rpm and 2.5 l/min, respectively. After initial 9 h of cultivation, samples were taken every 3 h for CDW and PHA analysis. PHA Production by Fed-Batch Cultivation of Yangia sp. Fed-batch fermentation was performed at 30 °C, pH 7.0 using glycerol, crude glycerol (containing 44.89 % glycerol, 24.5 % biodiesel, 10.2 % methanol, 15.4 % fatty acid) or a mixture of crude glycerol and high fructose corn syrup (HFCS) (Daesang, South Korea) as carbon sources. The feed solution containing 10x concentrated HM-1 medium with 20 % (w/v) of the carbon sources was pumped into the bioreactor during first 20 h of cultivation to maintain the concentration of carbon source above 10 g/l with the help of offline analysis. After that, a feed solution containing only 20 % (w/v) carbon source was added to the bioreactor. Antifoam was added to the bioreactor when needed. After initial 6 h of cultivation, samples were taken every 3 h for CDW and PHA analysis. Quantitative Analyses CDW was determined by centrifuging 3 ml of the culture samples at 2000×g for 15 min in preweighed centrifuge tubes; the pellet was washed once with 3 ml distilled water, centrifuged and dried at 105 °C until constant weight was obtained. CDW was obtained by the difference in the final and initial weight of the centrifuge tube. The concentrations of glycerol and sugars (glucose and fructose) were determined by HPLC using a chromatography system (Jasco, Tokyo, Japan) equipped with RI-detector (ERC, Taguchi, Japan). Glycerol was separated on an Aminex HPX-87H column at 55 °C using a mobile phase of 5 mM H2SO4 at flow rate of 0.6 ml/min. The sugars were separated on an Aminex HPX-87P column, using MiliQ water as mobile phase at a rate of 0.6 ml/min and column temperature of 65 °C. PHA concentration was determined using a gas chromatographic method [20]. For this, about 10 mg of freeze-dried cells were mixed with 1 ml chloroform and 1 ml methanol solution containing 15 % (v/v) sulphuric acid and 0.4 % (w/v) benzoic acid. The mixture was
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incubated at 100 °C for 3 h to convert the constituents to their methyl esters. After cooling to room temperature, 0.5 ml of distilled water was added, and the mixture was shaken for 30 s. The lower chloroform layer was transferred to a fresh tube and used for gas chromatography analysis. Sample volume of 2 μl was injected into the gas chromatography column (VARIAN, Factor Four Capillary Column, CP8907). The injection temperature was 250 °C, the detector temperature was 240 °C and the column temperature was 60 °C for the initial 5 min and then increased at 3 °C/min to 120 °C. PHB and PHBV containing 12 % valerate (Sigma) were used as standards for calibration. All analyses were performed in triplicates. Residual cell mass (RCM) was defined as CDW minus PHA concentration, while PHA content was obtained as the percentage of the ratio of PHA concentration to CDW. The polymer yield was calculated as gram PHA produced per gram of carbon source utilized. NMR Analysis of PHAs NMR analysis of the PHAs produced by Yangia sp. was performed as described previously [10]. The polymer containing bacterial cells were harvested from 300 ml of culture broth by centrifugation at 6000g for 10 min, washed twice with distilled water and lyophilized. PHA was recovered from the lyophilized cells by extraction for 30 h with chloroform in a Soxhlet apparatus (Duran, Germany) and concentrated by evaporating the solvent under vacuum. The polymer was precipitated from the concentrated solution with 10 volumes of ethanol, and the resulting PHA granules were filtered twice. The 1H nuclear magnetic resonance (NMR) spectrum was recorded at 500 MHz with a Bruker ARX500 Spectrometer (Bruker, Sikerstrifen, Germany) at room temperature using deuterated chloroform as internal reference solvent. The spectrum was evaluated using standard BrukerUXNMR software.
Results and Discussion PHA Production by Yangia sp. ND199 from Different Carbon Sources Yangia sp. ND199 was grown in HM-1 medium with yeast extract as nitrogen source and with different carbon sources in order to investigate the effect on cell growth and PHA production. The results of CDW, PHA content and concentration, RCM and monomer Table 1 Effect of different carbon sources on cell growth and PHA accumulation by Yangia sp. ND199 Carbon sourcea
CDW (g/l)
PHA content (wt%)
PHA conc. (g/l)
RCM (g/l)
Monomer composition (mol%) 3HB
3HV
Glucose
5.0±0.1
48.9±1.4
2.5±0.2
2.5±0.2
98.7
1.3
Maltose Xylose
3.9±0.1 1.9±0.2
22.3±0.7 0
0.9±0.1 0
3.0±0.1 1.9±0.2
92.6 0
7.4 0
Sucrose
4.4±0.1
26.7±0.7
1.2±0.1
3.2±0.1
94.7
5.3
Fructose
5.6±0.2
53.8±1.3
3.0±0.2
2.6±0.2
100
Trace
Dextrin
2.0±0.1
0
0
2.0±0.1
0
0
Glycerol
4.5±0.1
44.8±1.2
2.0±0.2
2.5±0.1
94.6
5.4
a
All the carbon sources were of analytical grade and were obtained from standard sources
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composition of the PHAs in the samples obtained after 30 h of cultivation are summarized in Table 1. Among the seven carbon sources tested, fructose, glucose and glycerol were found to be the most suitable substrates for both cell growth and PHA accumulation. Maximum CDW of 5.6 g/l and PHA content of 53.8 wt% were obtained with fructose, followed by glucose (CDW of 5.0 g/l and PHA content of 48.9 wt%) and glycerol (CDW of 4.5 g/l and PHA content of 44.8 wt%). Sucrose and maltose led to lower accumulation of PHA, while xylose and dextrin served as poor substrates for cell growth and also did not result in PHA synthesis.
Fig. 1 1H NMR spectra of PHBV isolated from Yangia sp. ND199 grown on a glucose and b maltose, respectively, as carbon source
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7
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Time (h) Fig. 2 Profiles of CDW, PHA content and PHA composition during cultivation of Yangia sp. ND199 in shake flasks in the medium with glycerol as carbon source and a yeast extract and b monosodium glutamate, respectively, as nitrogen source
The composition of PHA is known to depend on the microorganism and the substrate used [2]. The 1H NMR analysis of the PHA isolated from the Yangia sp. ND199 cells grown on glucose and maltose, respectively, showed major peaks corresponding to PHBV (Fig. 1a, b). The 3HV content in the copolymer synthesized varied with the carbon source used; the highest 3HV content of 7.4 mol% was obtained when the organism was grown on maltose followed by glycerol and sucrose giving 5.4 and 5.3 mol%, respectively (Table 1). PHBV is less crystalline, more elastic
Fig. 3 Effect of varying pH on a CDW, b PHBV content and c 3HV mol% of PHBV accumulated by Yangia sp. ND199 during cultivation in the medium with glycerol and yeast extract as carbon and nitrogen sources, respectively
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(A)
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100 80 60 40 20 0 9
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Appl Biochem Biotechnol Fig. 4 Profiles of cell dry weight and PHBV concentration and content in a fed-batch fermentation mode using a glycerol, b crude glycerol and c 1:1 mixture of crude glycerol and high fructose corn syrup, as carbon sources. The concentrations of the carbon sources are also shown
and tougher than PHB and has broader range of applications. The increase in the 3HV content is correlated with decrease in crystallinity and increased elasticity of the copolymer [2]. As glycerol was found to be a suitable substrate for both cell growth and PHBV synthesis with relatively high 3HV content by Yangia sp. ND199, it was used for further studies. Glycerol in crude as well as refined form is considered as relatively cheap and abundant substrate for industrial microbiology, especially since its increased availability as a co-product of biodiesel production [21]. Integration of glycerol conversion to PHA will not only provide added value to biodiesel production but should also reduce the polymer production cost. Glycerol has earlier been used as carbon source for PHB production by Ralstonia eutropha [22], Zobellella denitrificans [23] and Pseudomonas oleovorans and for medium chain length PHA by Pseudomonas corrugata [24]. Salmonella enterica and Escherichia coli have been metabolically engineered for the production of PHBV and poly(3-hydroxypropionate) (PHP), respectively, from glycerol [25, 26]. Effect of Nitrogen Source on PHA Production Preliminary studies in the lab on cultivation of Yangia sp. ND199 showed yeast extract and monosodium glutamate (MSG), respectively, to be the most suitable nitrogen sources for cell growth and that polymer synthesis was facilitated by nitrogen limitation. As seen in Fig. 2, PHA synthesis followed the same trend as the cell growth. When grown on glycerol and yeast extract, the cells grew faster and accumulated higher amount of PHA giving maximum CDW of 5.7 g/l and PHA content of 52.8 wt% after 40 h of cultivation (Fig. 2a), while only 3.3 g/l CDW and 24 wt% PHA were obtained with MSG as nitrogen source (Fig. 2b). Furthermore, while the copolymer PHBV was synthesized in the medium containing yeast extract, only homopolymer PHB was synthesized with MSG (Fig. 2a, b). This would suggest that Yangia sp. ND199 is able to metabolize amino acids present in the yeast extract like aspartate, threonine and methionine through pathways generating propionyl-coenzyme A, an important precursor for 3HV monomer. H. mediterranei has been shown to possess multiple propionyl-CoA supplying pathways for the production of PHBV [27]. One such metabolic pathway via threonine biosynthesis has been engineered in E. coli for the production of PHBV from an unrelated carbon source like xylose [28]. As seen in Fig. 2a, the highest 3HV content of 9 mol% was obtained in the PHBVafter 24 h of cultivation in the yeast extract medium and was then decreased to 2.8 mol% after 48 h, with concomitant increase in 3HB content of the copolymer that uses acetyl-CoA as precursor. PHBV Production at Different pH Batch cultivation of Yangia sp. ND199 with glycerol and yeast extract in a bioreactor at controlled pH (6.7, 7.2 or 7.7) showed highest rate of cell growth and PHA accumulation at pH 6.7 reaching a maximum CDW of 7.1 g/l and PHBV content of 45 wt% after 24 h of cultivation (Fig. 3a, b). With increase in pH to 7.2, maximum CDW obtained was the same but was achieved at a slightly reduced growth rate and the cells accumulated lower amount of PHA (Fig. 3b). Further increase in pH to 7.7 resulted in a significant decrease in both CDW and PHBV content (Fig. 3a, b). The polymer synthesis thus appears to follow the same trend as the cell growth. These results are in accordance with earlier reports showing that changing the
Appl Biochem Biotechnol CDW
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PHBV content
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(A)
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pH of the culture medium could be used to control not only the cell growth rate but also the polymer synthesis, regardless of the medium composition [29]. Monitoring the 3HV content of PHBV formed with time during cultivation of Yangia sp. ND199 showed that the organism uses only 3HV monomers at the start to form the polymer (e.g. 100 % 3HV content recorded at 0.5 wt% PHA at pH 7.7), but the 3HV content decreases sharply as increasing number of 3HB units are incorporated into the polymer (Fig. 3c). The 3HV content of the copolymer followed a similar trend irrespective of the pH during cultivation. For example, at PHBV content of 1.19 wt%, 3HV content was found to be 59.27 mol% after 9 h of cultivation at pH 7.2, while at pH 7.7, nearly similar values for PHBV content of 1.63 wt% and 3HV content of 63.1 mol% were obtained but after 21 h of cultivation. Similarly, after 12-h cultivation at pH 7.2 and 27-h cultivation at pH 7.7, PHBV contents of about 7.78 and 7.11 wt%, respectively, with 3HV contents of about 21.3 and 21.5 mol%, respectively, were achieved at the two pH values (Fig. 3b, c). In contrast, the composition of PHA synthesized in a mixed culture was strongly affected by pH, as observed by the increase in 3HV content of PHBV from about 13 mol% up to about 48 mol% with increase in pH from 8.5 to 9.5 [30]. PHBV Production by Fed-Batch Cultivation of Yangia sp. ND199 Yangia sp. ND199 was then cultivated in a fed-batch fermentation mode using glycerol and crude glycerol, respectively, as carbon source. Figure 4a shows that CDW reached a maximum value of 27.6 g/l with PHA content of 53.2 wt% at cultivation time of 33 h, resulting in PHA concentration of 14.68 g/l and productivity of 0.44 g/l/h. PHBVyield with respect to glycerol was 0.29 g per gram glycerol, which is comparable to the PHB productivity obtained (0.25 g/g) for Z. denitrificans [31]. When crude glycerol was used as the carbon source, the growth and PHBV accumulation in Yangia sp. ND199 were reduced due to the inhibitory effect of salt and other components. CDW, PHBV content and PHBV productivity and yield of 20.5 g/l, 40.6 %, 0.25 g/l/h and 0.2 g/g, respectively, were obtained after 33 h of cultivation (Fig. 4b). The inhibition does not seem to be as severe as reported earlier for cell growth and PHB production in Paracoccus denitrificans and Cupriavidus necator JMP134 grown on crude glycerol; the PHB yield coefficient for the latter organism was reduced from 0.37 to 0.14 g/g on replacing pure glycerol by salt-contaminated crude glycerol [32]. More recently, a halophilic bacterium H. hydrothermalis, isolated from a marine environment, using Jatropha biodiesel by-product as carbon source gave a PHB content of 75.8 wt% but with cell dry weight of 0.4 g/l after 72 h of cultivation [11]. Table 2 provides a comparison of PHA production from glycerol by different organisms and shows that production by Yangia sp. ND199 in this study is in the same range as that by other wild-type PHA producers, C. necator JMP134 and Zobellella denitrificans MW1. However, the cell density and PHA concentration in Yangia sp. are relatively lower, suggesting that improvement in cell growth should lead to increased polymer yield and productivity. The inhibitory effect of crude glycerol can be reduced by combining it with other cheap carbon substrate(s), especially the ones that can contribute to increased intracellular NAD(P)H concentration and/or high NAD(P)H/NAD(P) ratio [33]. Synthesis of PHB was significantly influenced by using mixtures of substrates such as fructose and methanol in case of Methylobacterium rhodesianum [34] and glucose and xylose for H. boliviensis [35]. Using crude glycerol and high fructose corn syrup (HFCS; containing approximately 55 % fructose and 42 % glucose) at a ratio of 1:1 as carbon source for Yangia sp. ND199 led to significant increase in the CDW, PHBV content and PHBV concentration of Yangia sp. ND199 reaching maximum values of 36.2 g/l, 56 wt% and 20.3 g/l, respectively after 33 h cultivation (Fig. 4c). As also seen in Fig. 4c, the bacteria utilized the sugars more efficiently than glycerol. PHBV productivity obtained in this fermentation was 0.61 g/l/h, which is 1.4
Glycerol
Crude glycerol
Glycerol
Crude glycerol
Glycerol
Crude glycerol
Yangia sp. ND199
Yangia sp. ND199
Recombinant E. coli
Recombinant E. coli
Z. denitrificans MW1
C. necator JMP 134
Carbon source
Organism
PHB
PHB
PHP
PHP
PHBV
PHBV
PHA type
50
81.2
5.15
11.98
20.5
27.6
CDW (g/l)
48
66.9
5.2
11.98
40.6
53.2
PHA content (wt%)
24
54.3
0.27
1.42
8.3
14.7
PHA conc. (g/l)
Not reported
1.09
0.003
0.015
0.25
0.44
PHA productivity (g/l/h)
Table 2 Comparison of PHA production from glycerol or crude glycerol in fed-batch cultivation by Yangia sp. ND199 with other bacterial species
0.14
0.25
0.004
0.017
0.2
0.29
PHA yield (g/g C source)
[32]
[31]
[26]
[26]
This work
This work
Reference
Appl Biochem Biotechnol
Appl Biochem Biotechnol
and 2.4 times higher than fermentations with glycerol and crude glycerol, respectively, as carbon source.
Conclusions The study shows Yangia sp. ND199 to be a novel microorganism with significant potential for production of the copolymer PHBV from unrelated carbon sources including glycerol. Accumulation of PHA by Yangia sp. ND199 is an adaptation of the organism to the nutrient-poor environment of the mangroves. This is the first report of a wild-type halophilic bacterium using glycerol as carbon source for PHBV production. The organism was able to use crude glycerol as carbon source without significant inhibition on cell growth and polymer production. Mixing another cheap carbon source providing higher reducing equivalents could be used to advantage for achieving improved cell growth and polymer accumulation. Yangia sp. ND199 can potentially grow under unsterile conditions and may be used directly with the by-product streams from biodiesel and other processes, thus reducing the process costs. For improving cell growth as well as PHBV production, studies are currently being pursued on metabolic flux analysis in Yangia sp. ND199 with selected carbon sources and on optimizing cultivation conditions. Acknowledgments The authors are grateful to the National Foundation for Science and Technology Development (Grant no. 106.03-2010.64) and the Swedish Research Council through the Swedish Research Links programme for supporting this work.
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