Biosynthesis of polyhydroxyalkanoate by Gamma proteobacterium WD-3 from volatile fatty acids

Chemosphere 82 (2011) 1209–1213

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Technical Note

Biosynthesis of polyhydroxyalkanoate by Gamma proteobacterium WD-3 from volatile fatty acids Zhiqiang Chen a,b, Yunbei Li a, Qinxue Wen a,⇑, Huichao Zhang a a b

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (SKLUWRE, HIT), Harbin 150090, PR China National Engineering Research Center of Urban Water Resources, Harbin Institute of Technology, Harbin 150090, PR China

a r t i c l e

i n f o

Article history: Received 8 September 2010 Received in revised form 9 November 2010 Accepted 9 November 2010 Available online 3 December 2010 Keywords: Poly-b-hydroxyalkanoate (PHA) Volatile fatty acids (VFAs) Fermentation Carbon source Substrate Bacteria

a b s t r a c t The production of copolymers of poly-b-hydroxyalkanoates (PHA) is generally a high cost process. To reduce the production costs, inexpensive carbon sources such as volatile fatty acids (VFAs) from acidified wastewater can be used. Therefore, isolation of bacterial strains that can produce PHA copolymers using VFAs as a sole carbon source would be a beneficial alternative. In this study, a strain of PHA accumulating bacterium was isolated from the wastewater treatment plant of a soybean processing facility in Harbin. The strain was identified as c-proteobacterium according to its 16S rDNA information and was originally named as strain WD-3. The strain accumulated a mass of PHA up to 45% of its dry cell weight when it was cultured under the optimum fermentation condition in this study when butyrate was used as the carbon source. In addition, WD-3 could synthesize PHA copolymers of poly-hydroxybutyrate and poly-hydroxyvalerate (PHV) either from C-even substrates or from C-odd substrates, and one-third of the copolymer was PHV. Results from this study demonstrated that small molecule organic acids can be used by the strain of WD-3 as the carbon source for growth and PHA production. The maximum PHA yield in the study was 0.45 g g 1 dry cell. Ó 2010 Published by Elsevier Ltd.

1. Introduction Poly-b-hydroxyalkanoates (PHAs) are polyesters accumulated by various bacteria under unbalanced growth conditions when the carbon substrate is in excess of other nutrients such as nitrogen, sulfur, phosphorus or growth factors (Chien et al., 2007). Under such conditions, PHA is accumulated as a storage material which provides an intracellular carbon and energy storage by many microorganisms including photosynthetic bacteria. Since the discovery of poly-hydroxybutyrate (PHB) as storage inclusions in Bacillus megaterium in 1926, bacteria belonging to more than 90 genera have been identified which accumulate these polyesters (Luengo et al., 2003). The most studied microorganisms for PHA production are Ralstonia eutropha (formerly Alcaligenes eutrophus) (Kim et al., 2005), Pseudomonas spp. (Daniel et al., 1992; Fuller et al., 1992; Kimura et al., 1992) and Azotobacter spp. (Page, 1992; Pozo and Martinez-Toledo, 2002). Each of these organisms can accumulate a considerable amount of PHA in the presence of a carbon source in excessive concentrations. For example, the accumulation of PHA by Alcaligenes eutropha can reach levels higher ⇑ Corresponding author. Address: School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China. Tel.: +86 451 8628 3008; fax: +86 451 8628 2103. E-mail address: [email protected] (Q. Wen). 0045-6535/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.chemosphere.2010.11.030

than 80% of the dry cell weight (DCW) using fructose or glucose as a carbon source (Jung et al., 2002; Reddy et al., 2003). Although there is considerable interest in PHA due to its potential as a biodegradable material, the wider use of PHA has been inhibited by high production costs (Choi and Lee, 1999; Nath et al., 2008). Previous authors have reported that the main reasons for high PHA production costs are the need for effective carbon substrates and extraction processes (Yu et al., 2008). Almost 30% of the total PHA production cost is attributed to the acquisition of carbon substrates (Salehizadeh and Van Loosdrecht, 2004). Research into different types of PHA is beginning to reveal materials with varying potential. For example, PHB and polyhydroxyvalerate (PHV), each has a wide range of physical properties (Reis et al., 2003). The homopolymer PHB is known to have high crystallinity, stiffness and brittleness, and is therefore of limited practical use. However, the inclusion of a fraction of hydroxyvalerate (HV) or other monomers in PHB significantly increases the softness and flexibility of this biopolymer (Rhu et al., 2003). The copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) is produced industrially using A. eutrophus from two carbon sources: glucose and propionic acid. The ratio of the carbon sources determines the ratio between the monomers incorporated. The production of this copolymer is generally a high cost process due to the requirement for pure culture cultivation and for a specific mixture of carbon sources (Choi and Lee, 2000).

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An alternative production method which may reduce PHA production costs is the use of inexpensive carbon sources as feed stocks for the bacteria, such as volatile fatty acids (VFAs) from acidified wastewater from anaerobic systems (Choi and Lee, 1999; Tobella et al., 2005). High strength organic wastewater contains large amounts of carbonaceous organic material. This organic material can be converted to VFAs under anaerobic conditions, which can then be used as the carbon source for biosynthesis of PHA by special microorganisms. Despite this, fructose and glucose are reported to be the carbon source by a great number of researchers investigating the production of PHA (Suchada and Songsri, 2006). Few researchers have investigated the use of VFAs, such as acetic, propionic and butyric acids from post-fermentation wastewater effluent as a carbon source to produce PHA other than A. eutrophus (Ruan et al., 2003). Isolation of bacterial strains that can produce PHA copolymers using VFAs as a sole carbon source would be a beneficial alternative for the economical production of PHA copolymers. In this study, a strain of c-proteobacterium that can produce PHA from VFAs was isolated. The strain was able to synthesize PHA containing both 3-hydroxybutyrate (3HB) and 3HV monomer through a single carbon source. The primary properties of the fermentation process for PHA production were tested within shaker flasks containing the liquor medium. 2. Materials and methods 2.1. Microorganisms The bacteria used in all experiments was c-proteobacterium WD-3 isolated from the activated sludge of a soybean wastewater treatment plant in Harbin. 2.2. Medium The seed culture medium was 5 g L 1 yeast extract, 10 g L 1 peptone, 5 g L 1 NaCl. The pH of the medium was neutralized with 3 M NaOH and 3 M HCl and the medium was sterilized before use. Pure cultures were isolated on agar with the same medium containing Nile blue (50 lg mL 1, Sigma) (Spiekermann et al., 1999). The medium used for fermentation was composed of a carbon source, a nitrogen source and a trace metal solution. The compositions of the mineral solution were: Na2HPO4
7H2O 8.9 g L 1, KH2PO41.5 g L 1, MgSO4
7H2O 0.2 g L 1, ferric ammonium citrate 60 mg L 1, CaCl2
2H2O 10 mg L 1, and 10 mL L 1 trace element solution. Trace element solution contains: H3BO3 0.3 g L 1, CoCl2
6H2O 0.2 g L 1, ZnSO4
7H2O 0.1 g L 1, MnCl2
4H2O 30 mg L 1, NaMoO4
2H2O 30 mg L 1, NiCl2
6H2O 20 mg L 1, CuSO4
5H2O 10 mg L 1. Distilled water was used in all media. Three different VFAs, acetate, propionate and butyrate, were added as carbon sources, and ammonia sulfate was added as a nitrogen source. 2.3. Isolation and identification Pure cultures were isolated on agar with the same seed medium containing Nile blue (50 lg mL 1). Colonies were directly examined through fluorescence by exposure to ultraviolet light to examine the accumulation of lipid storage compounds including PHA. The sequences of partial 16S rDNA (about 1500 bp) of the strain were analyzed. The sequencing work was completed by Shanghai Sangon Biological Engineering Technology and Service Co. The sequences were compared against those available in the public databases of the National Center for Biotechnology Information using their internet database (http://www.ncbi.nlm.nih.gov), and the Basic Local Alignment Search Tool algorithm.

2.4. Electron microscopy Morphology was examined by transmission electron microscopy. For transmission microscopy, centrifuged cell pellets were fixed with 4% glutaraldehyde and 1% osmium tetroxide. Ultra-thin sections of the sample embedded in epoxy resin were prepared with an ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a JEM-1200EXII transmission electron microscope (JEOL, Tokyo, Japan).

2.5. Flask culture The effects of alterations to the C/N ratio and variations in the carbon source on PHA production were investigated. For the C/N ratio experiment, butyrate acid was used as the sole carbon source. The C/N ratios investigated included 35, 40 and 45. The carbon source experiment was undertaken to compare the use of butyrate, acetate and propionate acid as a carbon source for PHA production. Ammonia sulfate was used as the nitrogen source in all cases. To begin each experiment, the well growth slant culture was inoculated in the seed medium. The seed culture was then cultivated in 250 mL flasks containing 60 mL medium each on a rotary shaker at 180 rpm for 24 h. The temperature was kept at 30 °C. The cells were harvested after centrifugation at 8000g for 2 min and washed with 0.9% NaCl solution, then inoculated to the fermentation medium with 5% (v/v) inoculums and fermented for another 72 h (for the C/N ratio experiment) and 48 h (for the carbon source experiment).

2.6. Fed-batch culture The relationship between cell growth, PHA accumulation and depletion of substrates was investigated through fed-batch culture in the culture flasks. The seed culture was prepared in a 250 mL flask at 30 °C for 24 h. The washed cells were then transferred into a nitrogen-limited medium containing 17.01 g L 1 of sodium butyrate as the carbon source and 1 g L 1 ammonia sulfate as the nitrogen source, respectively, with an inoculation volume 0.5% (v/v). The C/N ratio was 35 and the temperature was controlled at 30 °C. Samples were withdrawn at regular intervals of 10–12 h for analysis.

2.7. Analysis Microbial growth was monitored by measuring the cell density of the culture at 600 nm after suitable dilution with distilled water. The ammonium sulfate concentration was measured using the Kemper method (Kemper, 1974). DCW was measured after vacuum drying at 50 °C for 24 h. The amount of PHA was measured using a gas chromatograph (GC) according to the following process: 2 mL of chloroform and 2 mL of methanol solution were added into the incubator at 105 °C. The methanol solution was composed of 3% (v/v) of concentrated sulfuric acid and 1 g L 1 of benzoic acid. The mixture was then shaken periodically during the incubation. After cooling to room temperature, 1 mL of distilled water was added, and the mixture was shaken for 20–30 s. The heavier under layer was injected directly into the GC (GC6890 N/FID, Agilent, United States) with an HP-5 column (30 m length, 320 lm internal diameter, 0.25 lm film thickness) to determine the PHA content. VFAs were also measured using the GC. All experiments were performed in triplicate. The significance of results was determined using standard deviation.

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(b)

(a)

3. Results and discussion 3.1. Strain isolation and identification Forty two different colonies were selected from Nile blue agar plates, and named as ST1–ST11 (where ST means from soil), WD1–WD10 (where WD means from soybean wastewater treatment plant), WZ1–WZ17 (where WZ means from brewery wastewater treatment plant) and WS1–WS4 (where WS means from the sequencing batch reactor of a municipal wastewater treatment plant). Six colonies were picked out for further fermentation study according to the results of the GC analysis. Cells of the six selected colonies were grown in the fermentation medium and were collected after 48 h culture. PHA contents and DCWs of the 6 strains are shown in Table 1. Table 1 shows that strain WD-3 grew well and, of the 6 strains tested, accumulated the most PHA (up to 21% of its DCW) under these experimental conditions. Therefore, the WD-3 strain was used in further experiments. The sequences of partial 16S rDNA (about 1500 bp) from strain WD-3 were compared against those available in the public databases. It was found that WD-3 was closely related to c-proteobacterium spp. (98% identical to 16S rDNA sequences). WD-3 was thus identified as c-proteobacterium spp. The phylogeny based on these partial 16S rDNA sequences and related c-proteobacterium spp. is shown in Fig. 1. The nucleotide sequences of 16S rDNA from the WD-3 strain determined in this study have been deposited in the GenBank database under the accession number FJ440556. 3.2. Microbiological characteristics by transmission electron microscopy PHA exists as discrete inclusions that are typically 0.2–0.5 lm in diameter localized in the cell cytoplasm and may be visualized

Table 1 PHA content and the DCW of the 6 strains. 1

Bacterial strain

DCW (g L

WD-1 WD-3 ST-4 ST-8 WS-10 WS-11

2.5 ± 0.17 6.25 ± 0.21 3.57 ± 0.13 1.8 ± 0.18 1.67 ± 0.16 2.87 ± 0.12

)

PHA content (%) 18 ± 1 21 ± 1 11 ± 1 11 ± 1 6±1 9±1

PHA content: PHA/CDW (w/w).

Uncultured Spirochaeta sp. (DQ218325) Borrelia anserina (U42284) S.africana (X93928) gamma proteobacterium SSCT59(AB210976) gamma proteobacterium WD 3 (FJ440555) Planctomyces maris (AJ231184) P.marina (X62912) Thermodesulfobacterium (AF418169) Geothermobacterium ferrireducens(AF411013) Thermotoga maritima (AJ401021) Fervidobacterium islandicum (AF434670) Methanothermococcus okinawensis (AB057722) Desulfurococcus fermentans (AY264344) 0.05

Fig. 1. 16S rDNA phylogenetic tree of the strain WD-3.

0.5um

0.2um

Fig. 2. Electron microscopy of the thin section of strain WD-3: (a) magnified by 125,000 and (b) magnified by 50,000.

Table 2 PHA yield and cell growth of WD-3 in the substrate with different C/N ratios. C/N ratio (mol mol 35 40 45

1

)

Sodium butyrate (g L 17.01 17.84 18.74

1

)

Ammonia sulfate (g L

1

1 1 1

)

DCW (g L 1)

PHA content (%)

6.68 ± 0.18 4.00 ± 0.17 9.00 ± 0.19

22 ± 1 14 ± 1 17 ± 1

quite clearly with a phase contrast light microscope due to their high refractivity (Kourmentza et al., 2009). In this study, cells were grown in fermentation medium with butyrate acid as the carbon source and were collected after 48 h cultivation. When thin sections of WD-3 were observed by transmission electron microscopy, gray granules of accumulated PHA polymers were observed, as shown in Fig. 2. 3.3. Effect of C/N ratio on PHA production The effect of nitrogen limitation on PHA synthesis was investigated in this study. The experiment conditions were described in 2.5. The PHA content of the bacteria was analyzed after 72 h cultivation. The results are shown in Table 2. The results focus on two concentrations, those of the DCW and PHA, which are of primary interest in production scale fermentation. The maximum PHA accumulation, which was 22% of DCW, was obtained when the C/N ratio was 35. However, the maximum dry cell weight of up to 9 g L 1 was obtained with a C/N ratio of 45. This phenomenon was inconsistent with previous studies which indicated that higher C/N ratios can increase the accumulation of PHA (Sharma and Mallick, 2005). When the C/N ratio increased to 45, the cell grew well, however, it was considered that the microorganism may need more time to accumulate PHA. It therefore may be that if the culture process was prolonged, the content of PHA may increase further. Based on this, a subsequent experiment was undertaken using a 150 h culture period. The WD-3 strain accumulated PHA as high as 41% of its DCW under a C/N ratio of 45. 3.4. Effect of carbon source on PHA production Since the identification of other components besides 3HB in PHA more than two decades ago, it is known that the enzyme responsible for PHA synthesis within a bacteria shows a broad substrate specificity and therefore a wide variety of monomers can be polymerized (Steinbuèchel et al., 1992). One of the factors that determines the nature of PHA constituents produced by a bacteria is the carbon source (Sudesh et al., 2000). Depending on PHA accumulation kinetics, bacteria can be divided in two groups. The first group is formed by bacteria that require the limitation of some nutrients. R. eutropha and Pseudomonas oleovorans belong to this group. Bacteria of the second group do not depend on nutritional

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Table 3 Effect of carbon source on PHA accumulation in WD-3 after 48 h of incubation.

pH

12.2 ± 0.79 8.76 ± 0.21 9.66 ± 0.12

Dry cell weight

PHA

1

)

PHB:PHV

PHA content (%)

53:47 35:65 77:23

25 ± 1 21 ± 1 34 ± 1

Ammonium Sulfate

VFA

180 160

-1

pH & Dry cell weight (g L )

8

140 6

120 100

4

80 60

2

40 20

0

0

20

40

60

80

100 120 140 160

incubation time (h)

0

12000 10000 8000 -1

Acetate Propionate Butyrate

Dry cell weight (g L

VFA (g L )

)

-1

1

ammonia sulfate (mg L ) & PHA content (%)

Carbon source (g L

6000 4000 2000 0

Fig. 3. Accumulation of PHA in the fed-batch fermentation experiment of WD-3 with reference to pH, VFA, ammonia sulfate and the cell growth.

limitation as they accumulate PHA during cell growth. Some examples are Alcaligenes latus, Azotobacter vinelandii, Pseudomonas putida, Pseudomonas aeruginosa 47T2 and r- Escherichia coli (Fernández et al., 2005). In this study, PHB and PHV are the most common constituents in the PHA product, as shown in Table 3. The results indicate that the biosynthetic pathway of the WD-3 strain belongs to the first group of bacteria described above. However, there is some difference between the biosynthetic pathway of WD-3 and R. eutropha. Akiyama et al. (1992) reported that R. eutropha was capable of producing the PHB homopolymer from even-carbon numbered (C-even) n-alkanoates, while using odd-carbon numbered (C-odd) n-alkanoates (such as propionic acid or valeric acid) as the carbon source resulted in the accumulation of copolymers of 3HV. Table 3 demonstrates that in contrast to R. eutropha, WD-3 can synthesize PHA copolymers of 3HB and 3HV from either C-even substrates or C-odd substrates. This implies that the WD-3 strain can accumulate PHA containing both the 3HB and 3HV monomers in the presence of a single carbon source and it is the only reported bacteria ever shown to be able to produce both monomers in the presence of a single carbon source. When propionate was employed as the sole carbon source, the 3HB:3HV monomer composition of the polymer was 35:65, however, when acetate and butyrate were used as carbon sources, the 3HB:3HV monomer compositions of the polymer were 53:47 and 77:23, respectively. 3.5. Fed-batch culture The relationship of cell growth, PHA production and the depletion of substrates was studied through fed-batch culture. Butyrate acid was used as a carbon source in this experiment. Fig. 3 shows the profiles of pH, DCW, ammonia and VFA in the medium and PHA production during the fed-batch fermentation process of the WD-3 strain. It was observed that the PHA production, DCW and pH increased with time during the fermentation process, while VFA and ammonia sulfate concentration declined, especially during the cell growth phase. During the fermentation, the DCW increased significantly

from 0.1 to 6.45 g L 1 between 6 and 100 h, and after 100 h, the cells grew slowly. In contrast to cell growth, the PHA content was observed to increase slowly in the first 90 h of the fermentation while increasing significantly from 19% to 45% after 100 h. The result shows that PHA accumulation occurred mainly during the stationary phase of growth rather than during the logarithmic growth phase of the cells. This finding is in agreement with the results reported by Campbell et al. (1982) and Stal (1992), who observed that the maximum accumulation of PHB occurred during the stationary phase of Spirulina platensis and Oscillatoria limosa. The most probable reason for this finding is that, during fermentation, normal microbial growth and reproduction are limited when the concentrations of the carbon source and the nitrogen source decrease while the C/N ratio is still relatively high at later periods of the fermentation. Bacteria produce and store PHA when they lack the complete range of nutrients required for cell division but have generous supplies of carbon. The pH of the system, which was not controlled, increased from 7.0 to 8.6 during the fermentation process. The maximum PHA accumulation was measured at the end of the fermentation when pH increased to approximately 8.5. The maximum PHA content accumulated by WD-3 in this study was 45% of its DCW, equivalent to a yield of 0.45 g g 1 dry cell. The proportion of PHV occupied one-third of the PHA end product. This significant observation provided a wide spectrum of polymers with varying physical properties, because PHA copolymers composed of primarily 3HB with a fraction of longer chain monomers, such as 3HV, are more flexible and tougher plastics. Such plastics typically have a wider range of applications. 4. Conclusions A bacterium, isolated from the wastewater treatment plant of a soybean processing facility in Harbin was identified as c-proteobacterium spp., strain WD-3. It was able to produce PHAs using small molecule organic acids as a carbon source, including butyrate, acetate and propionate. The C/N ratio had mixed results on the fermentation process. When sodium butyrate was used as the carbon source, a best PHA accumulation of 22% of DCW was obtained when the C/N ratio was 35 after 72 h cultivation, but higher PHA production of 41% of DCW under a C/N ratio of 45 was achieved after a 150 h cultivation. Use of different carbon sources produced a different combination of monomers in the PHA. When propionate was employed as the sole carbon source, the 3HB:3HV monomer composition of the polymer was 35:65, however, when acetate and butyrate were used as carbon sources, the 3HB:3HV monomer compositions of the polymer were 53:47 and 77:23, respectively. WD-3 strain can accumulate PHA containing both the 3HB and 3HV monomers in the presence of a single carbon source Results from the fed-batch culture experiment showed that the maximum PHA accumulation occurred at the stationary phase of cell growth. The maximum PHA accumulated by the WD-3 strain in this study was 45% of its DCW, equivalent to a yield of 0.45 g g 1 dry cell. This work shows that residues generated from the acid hydrolysis of high strength organic wastewater or waste sludge, such as small molecule organic acids, can be used for the bacterial biosynthesis of PHA by the isolated strain of c-proteobacterium WD-3. This process has the potential to reduce the cost of PHA production while also offering environmental benefits through the reuse of sludge from wastewater treatment processes. Acknowledgements The authors acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 50807026), China

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