Polyhydroxyalkanoate biosynthesis and ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Polyhydroxyalkanoate biosynthesis and simplified polymer recovery by a novel moderately halophilic bacterium isolated from hypersaline microbial mats D.-N. Rathi1, H.G. Amir2, R.M.M. Abed3, A. Kosugi4, T. Arai4, O. Sulaiman5, R. Hashim5 and K. Sudesh1 1 2 3 4 5

Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia Soil Science Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia Biology Department, College of Science, Sultan Qaboos University, Al-Khoud, Oman Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki, Japan School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia

Keywords Halomonas sp., halophilic micro-organisms, oil palm trunk sap, osmotic lysis, polyhydroxyalkanoate. Correspondence Kumar Sudesh, Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia. E-mail: [email protected] 2013/1564: received 3 September 2012, revised 4 November 2012 and accepted 6 November 2012 doi:10.1111/jam.12083

Abstract Aims: Halophilic micro-organisms have received much interest because of their potential biotechnological applications, among which is the capability of some strains to synthesize polyhydroxyalkanoates (PHA). Halomonas sp. SK5, which was isolated from hypersaline microbial mats, accumulated intracellular granules of poly(3-hydroxybutyrate) [P(3HB)] in modified accumulation medium supplemented with 10% (w/v) salinity and 3% (w/v) glucose. Methods and Results: A cell density of approximately 30 g l 1 was attained in this culture which yielded 48 wt% P(3HB). The bacterial strain was also capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HBco-3HV)] when cofed with relevant precursors. Feeding with sodium valerate (07 mol l 1 carbon) at various time intervals within 36 h resulted in 3HV molar fractions ranging from 6 up to 54 mol%. Oil palm trunk sap (OPTS) and seawater as the carbon source and culture medium respectively facilitated a significant accumulation of P(3HB). Simplified downstream processing based on osmotic lysis in the presence of alkali/detergent for both dry and wet biomass resulted in approximately 90–100% recovery of polymers with purity as high as 90%. Weight-average molecular weight (Mw) of the polymers recovered was in the range of 1–2 9 106. Conclusions: Halomonas sp. SK5 was able to synthesize P(3HB) homopolymer as well as P(3HB-co-3HV) copolymer from various carbon sources. Significance and Impact of the Study: This is the first time a comprehensive study of both production and downstream processing is reported for Halomonas spp.

Introduction Polyhydroxyalkanoate (PHA) is an intracellular storage compound accumulated as energy reserve by numerous micro-organisms under certain stressful environmental conditions (Doi 1990; Byrom 1994). Many bacteria accumulate PHA when carbon source supply is in excess but 384

other essential nutrients for cellular growth such as nitrogen, phosphorus, magnesium, sulfur and/or oxygen undergo depletion (Doi 1990; Byrom 1994). The vast attention towards PHAs is mainly attributed to its properties that resemble some petrochemical plastics, as well as its biocompatibility and complete biodegradability. Microbial activity in the natural environment breaks

Journal of Applied Microbiology 114, 384--395 © 2012 The Society for Applied Microbiology

D.-N. Rathi et al.

down such bioplastics to release carbon dioxide and water or methane under aerobic and anaerobic conditions, respectively (Naik et al. 2008). Apart from a few well-known PHA producers such as Cupriavidus necator, Alcaligenes latus, Pseudomonas and Escherichia coli transformants (Lee 1996), certain halophilic micro-organisms are also known to accumulate PHA granules (Quillanguaman et al. 2010). Halophiles are salt-loving micro-organisms, which require sodium chloride (NaCl) to grow. They are able to balance environmental osmotic pressure and resist the denaturing effects of salts. Halophiles are found in two of the three domains of life, Archaea and Bacteria, within the family Halobacteriaceae and Halomonadaceae, respectively. Archaea are generally extremophiles with requirement of NaCl as high as 15–30% (w/v), whereas moderate halophilic bacteria are less stringent and grow in the range of 3–15% (w/v) NaCl (Kushner 1978; Ventosa et al. 1998). In recent years, extensive research on halophilic microorganisms has identified Halomonas genera as prominent PHA producers among moderately halophilic bacteria of Halomonadaceae family (Mata et al. 2002). There were many previous studies on PHA production by halophilic micro-organisms that were fed on simple sugars, with several attempts having been made to culture PHA-producing micro-organisms on agricultural and glycerol residues (Quillanguaman et al. 2005, 2006, 2007, 2008; Van-Thuoc et al. 2008; Kawata and Aiba 2010). There is current interest to identify other cheap and renewable carbon resources for more sustainable PHA production. Recently, an interesting carbon source reportedly suitable for PHA production is oil palm trunk sap (OPTS) that is readily obtainable from felled oil palms (Lokesh et al. 2012). Being rich in sugars, such sap may be suitable as a feedstock in fermentation processes. The successful utilization of OPTS as fermentation feedstock was reported with the accumulation of about 30 wt % P(3HB) by Bacillus megaterium MC1 within 16 h of cultivation (Lokesh et al. 2012). Poly(3-hydroxybutyrate), P(3HB), the most commonly synthesized form of PHA, has certain unfavourable properties such as high crystallinity, brittleness, stiffness and low elongation to break. However, these P(3HB) polymers could be processed with favourable properties when it is of high molecular weight (Kusaka et al. 1999). In order to improve polymeric properties of P(3HB), co-monomers such as 3-hydroxyvalerate (3HV) (Steinb€ uchel et al. 1993), 3-hydroxyhexanoate (3HHx) (Doi et al. 1995) or 4-hydroxybutyrate (4HB) (Nakamura et al. 1992) are commonly incorporated to mitigate these problems. While the syntheses of these copolymers are usually initiated by the addition of structurally related precursor compounds (Sudesh and Doi 2005), the toxicity of these

Synthesis and recovery of PHA using Halomonas sp.

precursors limits the cell growth (Steinb€ uchel and L€ utkeEversloh 2003). Toxicity effects exerted by these substances could be minimized via manipulated feeding strategies, with the addition of precursors at lower concentration or at specific time intervals during suitable growth phases (Du et al. 2001; Zhao and Chen 2007; Bhubalan et al. 2008). Certain micro-organisms such as Rhodobacter ruber, Nocardia corallina, C. necator-SH-69, Agrobacterium sp. strain (SH1, GW-014) and Bacillus cereus SPV are capable of synthesizing 3HB and 3HV copolymers from simple carbon sources, without the addition of precursor substances (Madison and Huisman 1999; Valappil et al. 2008). Similarly, a halophilic archaea (Haloferax mediterranei) can synthesize P(3HB-co-3HV) copolymer from glucose, extruded starch, extruded rice bran and corn starch (Lillo and Rodriguez-Valera 1990). Several Halomonas species were also reported to incorporate 3HV monomers. For example, Halomonas profundus synthesized P(3HB-co-3HV) from various carbon sources such as glucose, glycerol, propionic or valeric acid (SimonColin et al. 2008), whereas Halomonas campisalis synthesizes P(3HB-co-3HV) copolymer with about 4 mol% 3HV from maltose (Kulkarni et al. 2010). PHA production by halophilic micro-organisms is presumed to be advantageous for industrial applications because the saline environment only favours the growth of halophiles, but not other contaminating organisms (Don et al. 2006; Quillanguaman et al. 2010). A recent study reported a possible approach of non-sterile continuous production of P(3HB) by Halomonas TD01 (Tan et al. 2011). The extraction and purification processes of PHA can account for up to 50% of the production cost (Choi and Lee 1999). Currently, solvent extraction is one of the most commonly used recovery method for large-scale applications. Although polymer degradation is insignificant, this method is costly and hazardous (Byrom 1994; Jacquel et al. 2008). Studies have identified various other recovery methods such as chemical/enzymatic digestion, thermolysis, osmotic shock, high-pressure homogenization, glass bead milling or even ultrasonication (Middelberg 1995; Jacquel et al. 2008; Kunasundari and Sudesh 2011). Irrespective of the recovery method used, purity as well as molecular weights of recovered polymers should not be compromised. We report here the biosynthesis of P(3HB) and P (3HB-co-3HV) from various carbon sources, including OPTS, by a moderately halophilic bacterium (Halomonas sp. SK5). In order to develop a sustainable process, natural seawater was also used as an alternative to artificial salt water. The PHA synthesized was recovered by osmotic lysis, which is a simple, environment-friendly procedure.


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metrically using a 6505 UV-Vis spectrophotometer at 600 nm.

Materials and methods Media formulation A total of four media formulations were used, namely modified growth medium (MGM), nutrient-rich (NR) medium, lysogenic broth (LB) medium and modified accumulation medium (MAM). MGM was comprised of 5 g l 1 peptone and 1 g l 1 of yeast extract, whereas NR medium was made up of 10 g l 1 peptone, 10 g l 1 meat extract and 2 g l 1 yeast extract. LB medium was prepared according to manufacturer’s instructions (Sigma, India) and was comprised of 10 g l 1 casein enzyme hydrolysate type Ι, 5 g l 1 yeast extract and 10 g l 1 NaCl. MAM was comprised of 05 g l 1 yeast extract and 2% (w/v) glucose. The composition of all the media was as mentioned above in all the experiments, unless otherwise stated. For all media formulations, salinity (0–25% w/v) was introduced into each medium based on a 30% (w/v) stock self-mixed salt water made up of 240 g l 1 NaCl, 30 g l 1 MgCl26H2O, 35 g l 1 MgSO47H2O and 7 g l 1 KCl. Natural seawater source was collected from Shamrock Beach, Batu Ferringi, Malaysia, in August 2011. The collected seawater was used as an alternative to self-mixed salt water for a more sustainable approach. Solid medium was prepared by the addition of 15 g l 1 bacteriological agar powder (Himedia Laboratories, Bombay, India). Preliminary assessment of PHA accumulation The strain used in this study was isolated from hypersaline microbial mats in Oman (Abed et al. 2011) and was identified as Halomonas sp. SK5 based on its 16S rRNA sequence. The strain was initially grown on 15% (w/v) solid MGM as well as MAM (Abed et al. 2011) at 30°C for approximately 1 week and routinely streaked to acclimatize the cells. The isolate grown on MAM was then used in Nile Red staining assay (Spiekermann et al. 1999), to screen for its potential to produce PHA. Stained and non-stained cells were observed under phase-contrast and fluorescence microscopy, respectively, to determine the presence of PHA granules.

PHA biosynthesis Preliminary analysis was carried out in MAM, and suitable carbon sources (fructose, glucose, sucrose, sodium acetate) were used for PHA biosynthesis. OPTS and seawater were prefiltered using Whatman no. 3 filter paper, followed by filter sterilization using a 020-lm Minisart® cellulose acetate membrane (Sartorius, Göttingen, Germany), except for unsterile cultivation studies. OPTS was used as carbon source as well as growth and production medium (Lokesh et al. 2012). For medium formulation studies, filter-sterilized OPTS was directly used or diluted accordingly with sterile distilled water or artificial salt water. OPTS–seawater mixture studies were carried out similarly, under both sterile and unsterile conditions. Cultures were incubated aerobically at 30°C for 48 h on an orbital shaker and shaken at 200 rev min 1. Cultures were harvested by centrifugation at 9370 g, 4°C for 10 min. The cell pellets were then washed once with approximately 50 ml phosphate-buffered saline (pH 74) consisting of 80 g l 1 NaCl, 2 g l 1 KCl, 144 g l 1 Na2HPO4 and 24 g l 1 KH2PO4. The cell pellets were then lyophilized and used for subsequent analysis. Polymer recovery studies from wet and dry biomass Recovery of PHA granules from the strain was carried out using both lyophilized cells and freshly harvested culture. PHA recovery was initiated by adding approximately 05 g of freeze-dried cells directly into 50 ml distilled water (dH2O). As for freshly harvested cultures, approximately 250 ml of culture was centrifuged and cell pellets were resuspended in dH2O (1 : 1). Stirring time, heat and additional effect of mild detergent/alkali were also studied in order to enhance the purity and yield of PHA. The treated mixtures were then centrifuged at 6000 g and 10°C for 20 min followed by washing with dH2O. The final product was then oven-dried at 60°C. The recovery yield was determined gravimetrically, whereas purity was determined by gas chromatography (GC) analysis.

Growth profile analysis Preliminary evaluation of Halomonas sp. SK5 growth in MGM was not satisfactory; therefore, another two commonly used growth media (NR and LB) were used. The media were adjusted to a suitable salinity prior to autoclaving at 121°C for 15 min. Cultures were grown in 50 ml culture medium in 250 ml flask at 30°C and 200 rev min 1. Cell density was measured spectrophoto386

Analytical procedures Gas chromatography Polyhydroxyalkanoate quantification was carried out via GC analysis using caprylic methyl ester (CME) as the internal standard for the determination of PHA content, monomer composition and purity determination of the extracted polymers. Approximately 20 mg of lyophilized


D.-N. Rathi et al.

cells was weighed into screw-cap test tubes and subjected for methanolysis (140 min, 100°C) in the presence of 85% (v/v) methanol and 15% (v/v) sulphuric acid (Braunegg et al. 1978). The resulting methyl esters were then analysed by GC (Shimadzu GC-2010 AF 230LV) equipped with a capillary column SPB-1 (30 m length, 025 mm internal diameter and 025 mm film thickness; Supelco, Bellefonte, PA, USA) connected to a flame ionization detector. Nitrogen gas was used as the carrier gas (1 ml min 1), and the chloroform-dissolved sample (2 ml) was injected using an autoinjector (Shimadzu AOC-20i). The injector and detector temperatures were set at 270 and 280°C, respectively. The column temperature was increased from 70 to 280°C at 10°C min 1. Gel permeation chromatography Molecular weight (Mw) data were obtained by gel permeation chromatography (GPC) analysis at 40°C, using Shimadzu 10A GPC system (Shimadzu, Tokyo, Japan) equipped with a 10A refractive index detector and Shodex K-806M and K-802 columns. Approximately 10 mg of polymer sample was dissolved in 1 ml of HPLC-grade chloroform in order to obtain a sample concentration of 10 mg ml 1. The polymer solution was then filtered using Minisart® SRP15 020-lm PTFE filter (Sartorius). HPLC-grade chloroform was used as the eluent at a flow rate of 08 ml min 1. Polystyrene standards (Polymer Laboratories, Amherst, MA, USA) with a low polydispersity were used to draw a calibration curve. Results Preliminary assessment of PHA accumulation Phase-contrast microscopy observation showed the presence of PHA-like granules within the cells of Halomonas sp. SK5 (Fig. 1a). Further confirmation was carried out by Nile Red staining under UV light microscopy (Fig. 1b). The strain was then used for growth profile and PHA biosynthesis experiments.


Growth profile analysis Cell growth showed insignificant difference when grown in NR or commercial LB medium (Fig. 2a,b). The strain generally tolerated various levels of salinity, although salinity above 20% (w/v) slightly impaired its growth, with the effect being more obvious in LB medium. A stringent requirement of salinity for the growth of this strain was proven as there was no growth observed over the 36 h duration in control medium in the absence of added salt. Although the growth pattern was almost similar at 5 and 10% (w/v) salinity, further experiments to identify suitable culture parameters for PHA biosynthesis were carried out with minimal salinity (5% w/v NR medium). The culture media used in previous studies generally contained yeast extract and peptone combined with mineral salts. However, SK5 grew better with the addition of meat extract that resulted in a medium resembling NR medium. PHA biosynthesis Studies on the bacterial strain’s ability to synthesize PHA were initiated with the screening for the most suitable carbon source. Four carbon sources were evaluated, namely fructose, glucose, sucrose and sodium acetate. Yeast extract, initially fixed at 05 g l 1, provided the nitrogen source. The results showed better growth of the bacterial strain in a medium supplemented with glucose and sucrose (Fig. 3). However, the different concentrations of the supplemented carbon source did not produce significant changes in cell growth. As shown in Fig. 3, 3% (w/v) of glucose was chosen as the main carbon source. At this concentration, approximately 23 07 g l 1 CDW was synthesized of which 12 03 g l 1 comprised P(3HB) granules. Initial studies on the optimal nitrogen source concentration and primary inoculum load showed cell growth and P(3HB) accumulation to be best at 07 g l 1 yeast extract and 9% (v/v) inoculum density (results not shown). The effects of different

(a)

Figure 1 Micrograph of Halomonas sp. SK5 cells grown on 15% (w/v) modified accumulation medium with 2% (w/v) glucose upon 48 h of cultivation (a) Phase-contrast microscopy and (b) fluorescence microscopy.


10 µm

(b)

10 µm

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salinity on cell growth and P(3HB) synthesis were also tested. The best cell growth and P(3HB) production were obtained at 10% (w/v) salinity. The bacterium could grow at higher salinities, but CDW was observed to be slightly reduced at the extremes of the salinity range tested (Table 1). P(3HB) content, however, gradually decreased with an increase in salinity. The results indicated that 10% (w/v) salinity was the optimal saline level that produced relatively high amounts of CDW and P (3HB) content (Table 1).

(a)

(b)

Evaluation of the potentials of OPTS in mixtures of artificial salt water and seawater as growth and production medium

Figure 2 Growth profile analysis (OD600 nm) of Halomonas sp. SK5 grown at 30°C in two types of media with varied salinity (a) lysogenic broth and (b) nutrient-rich medium (control: media without salinity). ( ) 5%; ( ) 10%; ( ) 15%; ( ) 20%; ( ) 25% and ( ) control.

(a) CDW (g l–1)

4

Effect of different carbon sources and concentrations on CDW (g l–1)

3

1

P(3HB) content (wt%)

80

a

a

0

(b)

b

b

2

1

a

a

b

b a

a

b

b

b a

a

a

2 3 4 Carbon source concentration (% w/v)

r

Table 1 Effects of varying salinity on the cell growth and P(3HB) biosynthesis in Halomonas sp. SK5

5

r

r p

40

0

a

b

Effect of different carbon sources and concentrations on P(3HB) content (wt%)

60

20

b

b

q p 1

p q p 2

p

q

r p

p 3

p

r

q

4

p

q

p

5

Carbon source concentration (% w/v) Figure 3 (a) Cell growth and (b) P(3HB) accumulation in Halomonas sp. SK5 using various types and concentration of carbon sources (glucose, fructose, sucrose, sodium acetate) in 5% (w/v) modified accumulation medium supplemented with 05 g l 1 yeast extract upon 48 h of cultivation at 30°C and 200 rev min 1. All carbon sources were added during inoculation (0 h). Yeast extract was added into the medium prior to autoclaving. Data shown are means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD, P < 005). ( ) Fructose; ( ) Glucose; (&) Sodium acetate and ( ) Sucrose.

388

The suitability of OPTS for cell growth and PHA production was evaluated via supplementation of OPTS as carbon source. CDW was observed to be in the range of 37 –51 g l 1 (Table 2), although not much variation was observed with the P(3HB) content. This positive outcome led us to further experiment on utilizing OPTS as PHA accumulation medium under saline and non-saline conditions. The initial sugar content in OPTS was 64% (w/v) with glucose and sucrose as its primary sugars. Besides glucose and sucrose, OPTS also contains trace amounts of other sugars such as fructose, xylose, arabinose, galactose, inositol and maltose (Lokesh et al. 2012). The OPTS was diluted (1 : 1 and 1 : 3) by using sterile distilled water or artificial salt water. Under non-saline

Salinity (w/v%)*

CDW (g l 1)

5 10 15 20 25

24 35 36 34 26

01p 02q 02q 03q 01p

PHA content (wt%)† 40 41 30 26 15

4 4 5 4 4

PHA concentration (g l 1)‡ 09 14 11 09 04

01q 02r 02qr 01q 01p

Residual biomass (g l 1)‡ 14 02p 21q 25qr 25 03r 22 01qr

PHA, polyhydroxyalkanoates Data shown are means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD, P < 005). *Incubated for 48 h at 30°C and 200 rev min 1, in modified accumulation medium (initial pH 75, 9% (v/v) preculture) supplemented with 3% (w/v) glucose and 07 g l 1 yeast extract as the carbon and nitrogen source, respectively. Glucose was added during inoculation (0 h). Yeast extract was added into the medium prior to autoclaving. †PHA content in freeze-dried cells was determined via gas chromatography (GC). ‡PHA concentration = PHA content 9 cell concentration; residual biomass = cell dry weight PHA concentration.


D.-N. Rathi et al.


Table 2 Evaluation on the potential of oil palm trunk sap (OPTS) as carbon source as well as sole growth medium in Halomonas sp. SK5 OPTS concentration*

CDW (g l 1)

PHA content (wt%)†

PHA concentration (g l 1)‡

Supplementation of sap (%) as carbon source 1 38 01p 34 2 15 48 03q 33 3 2 42 04pq 36 8 Sap (64 w/v%) as growth and production medium (without dilution) Sap 03p 1 Sap and salt water 18 01r 14 2 Sap (64 w/v%) as growth and production medium (1 : 1 dilution) 24 4 Sap 17 02r Sap and salt water 28 02t 44 6 Sap (64 w/v%) as growth and production medium (1 : 3 dilution) Sap 09 01q 1 Sap and salt water 23 01s 42 1

Residual biomass (g l 1)‡

13 01p 16 02p 15 04p

25 01p 32 02q 27 03p

0003p 025 003q

03p 16 01r

04r 12 01t

13 02qr 16 03r

001p 10s

08 01q 13 01r

PHA, polyhydroxyalkanoates Data shown are means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD, P < 005). *Incubated for 48 h at 30°C and 200 rev min 1, in modified accumulation medium [initial pH 75, 9% (v/v) preculture] supplemented with 07 g l 1 yeast extract as the nitrogen source. Filter-sterilized OPTS was added during inoculation for carbon source experiments. For media experiments, filter-sterilized OPTS was directly used without the addition of external nitrogen source. Dilutions were performed by the addition of sterile distilled water or artificial salt water. Yeast extract was added into the medium prior to autoclaving. †PHA content in freeze-dried cells was determined via gas chromatography (GC). ‡PHA concentration = PHA content 9 cell concentration; residual biomass = cell concentration PHA concentration.

conditions, it was observed that cell growth was slightly impaired under both undiluted (64% w/v sugar content) and 1 : 3 dilution (16% w/v sugar content). The highest cell biomass was attained with 32% (w/v) sugars, although cell growth at the other two sugar contents (16 and 64% w/v) showed improvements with introduction of saline conditions. It was observed that bacterial growth and PHA biosynthetic ability were significantly better in a mixture of OPTS and salt water (Table 2). An almost similar yield of cell biomass and P(3HB) content was observed under saline conditions at 16% (1 : 3 dilution) and 32% (w/v) sugar content (1 : 1 dilution), in comparison with PHA accumulation medium with glucose as the carbon source. The highest cell biomass (28 02 g l 1) was attained at 32% (w/v) sugar content, while 23 01 g l 1 was achieved at 16% (w/v) sugar content. P(3HB) contents were 44 6 and 42 1 wt%, respectively (Table 2). In addition to the utilization of OPTS, the potential of natural seawater as growth medium was also evaluated. Figure 4 shows that the mixtures of seawater and OPTS produced better cell growth in comparison with seawater medium alone and in mixtures with glucose. The utilization of various OPTS and seawater mixture did not show much difference. Cell growth was similar under sterile and unsterile conditions, with no signs of contamination as observed under microscope (results not shown). Time profile studies of cell growth and P(3HB) accumulation

utilizing mixtures of OPTS and seawater showed a steady increase, yielding approximately 24 02 g l 1 cell biomass and P(3HB) content (24 8 wt%) at the end of 48 h of cultivation (Fig. 5). Similar cultivation in artificial salt water gave significantly higher P(3HB) content (44 6 wt%), with an almost comparable cell biomass (28 02 g l 1) (Table 2). PHA recovery by simplified extraction process and molecular weight (Mw) analysis of the recovered polymers Besides identifying cheap, renewable and easily accessible carbon substrates for PHA production, downstream processing for PHA purification also requires serious attention to control the total production cost. In this study, recovery of PHA granules was attempted by osmotic lysis of the dry and wet cell biomass (Table 3). As shown in Table 3, variation in stirring time gave rise to polymer purity as high as 90% upon 6 h of stirring in distilled water from dry cell biomass. The highest possible purity was observed after stirring for 18 h, with 94 1% purity and 98% recovery yield. The addition of detergent was tested to obtain higher purity of the PHA granules. Stirring the dry cell biomass in the presence of 01% (w/v) sodium dodecyl sulphate (SDS) for 18 h resulted in almost similar purity (94 1%) and approximately 82% recovery yield, with successful removal of impurities.


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Log OD600

(a) 10·00

1·00

0·10

0·01

0

4

8

12

16

20

24

12 16 Time (h)

20

24

(b)

10

Log OD600

Time (h)

1

0·1

0

4

8

CDW (g l–1)

3

2 a

a

a

1 b

a

a

a

a

a

b

b

b

b

b

b

18

24

30

36

42

48

b 0

6

12

40 35 30 25 20 15 10 5 0

P(3HB) content (wt%)

Figure 4 Growth profile analysis (OD600 nm) of Halomonas sp. SK5 grown in (a) seawater and in mixtures of glucose; (b) in different media combination with natural seawater source at 30°C and 200 rev min 1. ( ) Seawater; ( ) seawater and glucose; ( ) sterile oil palm trunk sap (OPTS) (peristaltic followed by syringe filtration) and seawater; ( ) sterile OPTS (peristaltic filtration) and seawater; ( ) unsterile OPTS and seawater; ( ) sterile OPTS and ( ) nutrient rich and seawater.

Time (h) Figure 5 Time profile analysis of P(3HB) biosynthesis by Halomonas sp. SK5 grown in oil palm trunk sap seawater medium at 30°C and 200 rev min 1. Data shown are means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD, P < 005). ( ) P(3HB) content (wt%) and ( ) CDW g l 1.

In the case of wet cell biomass, stirring in distilled water alone was not sufficient to achieve good polymer purity. As shown in Table 3, stirring of the wet cells at 30°C and 60°C in distilled water produced PHA granules with approximately 50–70% purity. A similar approach at both temperatures stirred in a mixture of distilled water and mild detergent (01% w/v SDS) resulted in an 390

improved purity (70–90%), with the best results achieved at higher temperatures. The highest purity of 90 5% was attained with 120 min of stirring. Further prolonged stirring for 240 min at 60°C decreased the purity (76 1%). A comparison of the effects of alkali and detergent showed the superiority of SDS (detergent) in recovering highly pure polymers even with the lowest concentration (005% w/v), whereas relatively higher concentrations of NaOH (alkali) was necessary to achieve comparable recovery yield and purity. The best recovery efficiency was attained at 01% (w/v) SDS (98 1% yield, 94 7% purity), while 02% (w/v) of NaOH was necessary to achieve 86 2% yield and 84 6% purity. The various treatments used for PHA granules’ recovery affected their molecular weight (Table 3). SDS treatment resulted in polymers with Mn and Mw in the range of 7–9 9 105 and 14–19 9 106 Da, respectively. On the other hand, PHA granules recovered via NaOH treatment of wet cell biomass were of lower Mn and Mw, with 4–7 9 105 and 10–14 9 106 Da, respectively. PHA granules recovered from lyophilized cells using distilled water and with the addition of SDS resulted in insignificant changes in both Mn and Mw. The polydispersity index (D) was approximately 2 with various treatments. Discussion Moderate halophilic bacteria are preferred for various biotechnological applications because of their flexibility to use wide range of substrates and also because they are amenable to culture in bioreactors (Ventosa et al. 1998). Various modified growth media have been studied and reported for their culture. Among the essential components required for cell growth are sodium chloride, magnesium salts, potassium and calcium chlorides, as well as peptone and yeast extract. These are culture requirements reported in most studies, although some experiments have included sodium bromide or sodium hydrogen carbonate in small amounts (Quillanguaman et al. 2005, 2006, 2007, 2008; Van-Thuoc et al. 2008; Kulkarni et al. 2010). In the present study, we employed the combination of NaCl, MgSO47H2O, MgCl26H2O, KCl, CaCl2H2O, along with peptone, yeast extract and meat extract in the culture media. The growth parameters used in this study (30°C, pH 75, 200 rev min 1) were comparable with most other studies of Halomonas spp. The utilization of a higher temperature and pH, as well as lower agitation, are variations that have also been reported (Quillanguaman et al. 2005, 2006, 2007, 2008; Van-Thuoc et al. 2008; Kulkarni et al. 2010). However, the main difference between the reports on halophiles has been in relation to the NaCl concentrations used. Halomonas boliviensis


D.-N. Rathi et al.


Table 3 Effects of various factors (stirring time, stirring temperature, solvent/detergent type and concentration) on the yield (%), purity (%) and molecular weight (Mw) of the polymers recovered from both lyophilized cells and fresh cultures

Sample Dry

Stirring temperature (°C)

Solvent

Stirring time (h)

Room Chloroform 3 days temperature Dry§ 30 dH2O 1 3 Dry§ 30 dH2O Dry§ 30 dH2O 6 12 Dry§ 30 dH2O Dry§ 30 dH2O 18 Dry§ 30 dH2O 24 Dry§ 30 01% (w/v) SDS 18 Effect of stirring temperature and solvent/detergent addition Wet¶ 30 dH2O 1 Wet¶ 30 01% (w/v) SDS 1 Wet¶ 60 dH2O 1 Wet¶ 60 01% (w/v) SDS 1 Effect of stirring time Wet¶ 60 01% (w/v) SDS 1 Wet¶ 60 01% (w/v) SDS 2 Wet¶ 60 01% (w/v) SDS 3 Wet¶ 60 01% (w/v) SDS 4 Effect of alkali/detergent type and concentration Wet¶ 60 005% (w/v) NaOH 2 Wet¶ 60 01% (w/v) NaOH 2 Wet¶ 60 02% (w/v) NaOH 2 Wet¶ 60 005% (w/v) SDS 2 Wet¶ 60 01% (w/v) SDS 2 Wet¶ 60 02% (w/v) SDS 2

Yield (%)*

Purity (%)†

Number average molecular weight (Mn)

85

93 1

543

108

199

80 89 96 82 98 75 82

65 80 84 92 94 94 94

4 4 4 5 1 6 1

ND* ND ND ND 549 ND 522

ND ND ND ND 132 ND 120

ND ND ND ND 215 ND 228

3 2 1 1

58 74 68 86

3 6 3 5

ND ND ND 906

ND ND ND 183

ND ND ND 202

98 1 91 1 91 1 88

84 90 74 76

8 5 4 1

ND ND ND ND

ND ND ND ND

ND ND ND ND

58 68 84 72 94 96

9w 9w 6wx 1wx 7x 4x

745 564 464 744 755 827

139 110 104 144 157 165

187 195 225 193 193 199

80 98 92 98

92 76 86 97 98 80

1y 2wx 2w 1y 1y 1x

l

Weight-average molecular weight (Mw)

Polydispersity index (D)‡

ND, not determined; PHA, polyhydroxyalkanoates. Data shown are means of triplicate. *Yield (%) was determined gravimetrically [weight of air-dried/wet polymer samples/weight of lyophilized/wet cells 9 PHA content (wt%)]. †Purity (%) was determined via gas chromatography (GC). ‡Polydispersity index (D) = Mw/Mn. §05 g freeze-dried cells were resuspended in 50 ml distilled water and with the addition of 01% (w/v) SDS where necessary. Mixtures were stirred for varied time at 30°C. ¶Wet cells from 250-ml culture were immersed (approximately 2 g wet cells) into 250 ml distilled water and with the addition of detergents, where necessary. Mixtures were stirred at varied temperature, time and alkali/detergent concentrations.

requires approximately 45% (w/v) NaCl to produce high cell densities and P(3HB) content (Quillanguaman et al. 2006). In the case of Halomonas sp. SK5, the most appropriate NaCl concentration was in the range of 4–8% (w/v), which facilitated the production of a reasonably high cell biomass and P(3HB) content. This NaCl concentration is consistent with the common encountered range of 3–15% (w/v) reported to sustain the growth of moderately halophilic bacteria (Kushner 1978; Ventosa et al. 1998). Halophilic bacteria of Halomonas sp. have demonstrated their ability to utilize diverse carbon sources. This ranges from simple sugars, agricultural residues, starch hydrolysates, sodium acetate and even waste glycerol

(Quillanguaman et al. 2005, 2006, 2007, 2008; Van-Thuoc et al. 2008; Kawata and Aiba 2010; Kulkarni et al. 2010). With regard to nitrogen sources, yeast extract is the most commonly utilized, although some researchers prefer ammonium sulfate or ammonium chloride. Yeast extract contains amino acids and their derivatives (ectoine, glycine-betaine, glutamate) that are essential for maintaining cell osmolarity under changing salinity pressure. The availability of these osmoprotectants within the culture medium eliminates the necessity for high-energy production of such solutes, thus enhancing cell survivability under harsh environmental surroundings (daCosta et al. 1998; Oren 1999; Kunte 2005). In their presence, bacterial cells were able to grow at higher salinity to attain reason-


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ably high cell biomass (Table 1). A previous study also reported that ectoine might serve as an intracellular source of nitrogen; hence, its presence might affect PHA biosynthesis (Mothes et al. 2008; Strazzullo et al. 2008). Shake flask studies revealed the ability of several Halomonas spp. to accumulate PHA. Among these, H. boliviensis LC1 was reported to synthesize P(3HB) from starch hydrolysates and maltose, with a maximal yield of 56 wt % and 588 wt%, respectively (Quillanguaman et al. 2005). In addition, the same strain was also reported to utilize a mixture of butyric acid and sodium acetate, as well as sucrose solely, yielding approximately 54 wt% P (3HB) under both conditions (Quillanguaman et al. 2006, 2007). Similarly, Halomonas marina accumulated about 59 wt% P(3HB) from glucose upon 50 h of cultivation. In contrast, a longer cultivation period (120 h) was required by Halomonas elongata to synthesize approximately similar PHA content (55 wt%) as that of H. marina (Mothes et al. 2008; Biswas et al. 2009). In the present study, an almost comparable P(3HB) synthesis (50 wt%) was observed within 48 h of cultivation using glucose as the carbon source. In addition to P(3HB) synthesis, the ability of halophilic micro-organisms to synthesize copolymers of P(3HB-co-3HV) has attracted much interest due to the desirable properties of the copolymers. Sodium propionate and sodium valerate are the two commonly used 3HV-precursors for this purpose (Bhubalan et al. 2008; Lee et al. 2008). Apart from these, sodium heptanoate was also reported to generate 3HV as well as 3-hydroxyheptanoate (3HHp) monomers for PHA synthesis by C. necator harbouring the PHA biosynthetic genes of Aeromonas caviae (Fukui et al. 1997). In this study, Halomonas sp. SK5 showed the ability to biosynthesize P(3HB-co-3HV) copolymer from all three types of 3HVprecursors. Sodium valerate resulted in better cell growth, PHA synthesis and 3HV monomer production in comparison with sodium propionate and heptanoate (Tables S1 and S2). The findings were comparable with those of a previous report that showed a higher concentration of sodium propionate was required to generate a comparable amount of 3HV monomers that could be achieved with lower concentrations of sodium valerate (Bhubalan et al. 2008). This study demonstrated the ability of Halomonas sp. SK5 to use the combination of OPTS and seawater for cell growth and PHA accumulation. As the oil palm has an economic life span of 20–25 years, it is replanted in cycles to maintain crop productivity in the plantation (Basiron and Chan 2006). It is estimated that roughly 64 –80 million palms are felled every year in Malaysia and Indonesia, the two largest producers of palm oil in the world (Husin 2000). The weak structure of the trunks of 392

felled oil palms restricts their use in structural applications. For example, only the outer part of the trunk is useful for plywood manufacture, whereas the brittle inner part of the trunk is normally discarded (Dalibard 1999). Oil palm sap obtained from felled trunk was evaluated for its sugar content following extraction procedures that included the pressing, collection and storage stages. The sugar content was highest in the sap obtained from the innermost part of the felled trunk where moisture content was higher (Yamada et al. 2010; Kosugi et al. 2010). Hence, the oil palm sap collected from the inner depth of the felled trunk has the potential to become a source material for ethanol and lactic acid production. Although for the time-being, sugarcane is an established industrial source material for bioethanol production, studies conducted using oil palm sap have shown it to be a viable source material for the production of bioethanol (Kosugi et al. 2010; Yamada et al. 2010). The present study showed that the use of OPTS could be extended to the development of culture media for micro-organisms. Oil palm trunk sap is a rich source of amino acids, organic acids, minerals, vitamins and sugars (Kosugi et al. 2010). The presence of these additional nutrients in combination with high sugar content explains its suitability for use as a bacterial cell growth and PHA biosynthesis medium. It is of interest to also note that there are earlier reports of better growth of halophilic microorganisms using a combination of sugars, instead of a sole carbon source (Quillanguaman et al. 2005; VanThuoc et al. 2008). Interestingly, the OPTS medium has also supported bacterial cell growth despite the absence of salts from the formulation (Table S1). On the other hand, a similar attempt to grow the strains in NR medium under non-saline conditions has attained insignificant growth (Fig. 2). The high sugar content in OPTS along with other nutrients could have substituted the role of salts, thereby supporting the growth of Halomonas sp. SK5. Although the strain was capable of growing in the absence of salt, better growth and P(3HB) accumulation were attained with the addition of salt water. This reflects the necessity of salinity to maximize its growth potential. Halomonas sp. SK5 was unable to grow in seawater alone because of the limitations of carbon and nitrogen sources. However, when seawater was mixed with NR or OPTS, cell growth was observed. In addition, Halomonas sp. SK5 could be cultivated under non-sterile conditions with no signs of contamination throughout the cultivation period. Essentially, there was no significant difference in the growth of the cells in sterile and non-sterile conditions. The possibility of non-sterile cultivation was proven in a recent study whereby approximately 40 g l 1 CDW with 60 wt % P(3HB) was produced in 14 days. Analysis by the


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polymerase chain reaction (PCR) confirmed that there were no contaminating organisms at 60 g l 1 NaCl concentration (Tan et al. 2011). Thus, the absence of contamination allows for less-stringent sterile conditions. Accordingly, large-scale cultivation of Halomonas sp. SK5 in the mixture of OPTS and seawater without stringent sterilization may be an attractive option for low-cost PHA production. Various approaches are also necessary to reduce the costs involved with downstream processing. This study demonstrated a simplified recovery process based on osmotic lysis. Small amounts of SDS (005–02%) in distilled water were sufficient to recover PHA granules in high purity. We believe this is the first time a comprehensive study of both production and downstream processing is reported for Halomonas spp. This study demonstrated the ability of a moderately halophilic bacterium strain Halomonas sp. SK5 to synthesize P(3HB) from various sugars and OPTS. The bacterium has the ability to synthesize copolymers in the presence of appropriate precursor substrates. Higher and controlled molar fractions of 3HV were produced using sodium valerate as the precursor. The adaptation of a simplified recovery technique based on osmotic lysis resulted in effective recovery of polymer with high Mw. This study showed the potential of OPTS and seawater for use in low-cost culture medium to support bacterial cell growth and PHA production. In conclusion, halophilic micro-organisms portray great potential as industrial-scale PHA producers. As shown in this study, Halomonas sp. SK5 opens up new possibilities to evaluate larger-scale unsterile PHA production in combination of OPTS and seawater collected under various environmental conditions. Acknowledgements This study was supported in part by FRGS (Fundamental Research Grant Scheme) provided by Ministry of Higher Education (MOHE) of Malaysia and by New Energy and Industrial Technology Development Organization (NEDO) of Japan. R.D-N. acknowledges University Postgraduate Research Scholarship Scheme (PGD) awarded by Ministry of Science, Technology and Innovation (MOSTI) of Malaysia for financial support. We are grateful to Dr Kesaven Bhubalan and Mr Bhadravathi Eswara Lokesh for their valuable suggestions and ideas. Conflict of interest All authors have agreed with this submission, and there is no conflict of interest.


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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Biosynthesis of P(3HB-co-3HV) copolymer with cofeeding of relevant precursors by the Halomonas sp. SK5 Table S2 Effects of varying sodium valerate feeding time on the P(3HB-co-3HV) copolymer synthesis in Halomonas sp. SK5


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