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Keywords:

  • 16S rDNA;
  • glycerol;
  • identification;
  • PHB/PHBV accumulation;
  • poly(3-hydroxybutyrate);
  • Zobellella denitrificans

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol.

Methods and Results:  Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram-negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth-associated. Although it was not an absolute requirement, 20 g l−1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol.

Conclusions:  A new PHB-accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry.

Significance and Impact of the Study:  This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Polyhydroxyalkanoates (PHAs) are aliphatic polyesters accumulated intracellularly by numerous bacteria as carbon and energy reserves under conditions of nutrient limitation and carbon excess (Anderson and Dawes 1990). Approx. 150 different constituents of PHAs have been identified (Steinbüchel and Valentin 1995; Steinbüchel and Füchtenbusch 1998). All PHAs are completely degradable to carbon dioxide and water through natural microbiological mineralization (Jendrossek and Handrick 2002). Two different groups of PHAs are distinguished: short-chain length PHA (scl-PHA) such as poly(3-hydroxybutyrate) (PHB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], which are produced by several bacteria like Ralstonia eutropha, and medium-chain-length PHA (mcl-PHA) such as poly(3-hydroxyoctanoate), or poly(3-hydroxyundecanoate), which are composed either of saturated or unsaturated 3-hydroxyfatty acids ranging in length from C6 to C14, and which are synthesized in many Pseudomonas species. In general, scl-PHAs usually exhibit thermoplastic properties like conventional petroleum-based plastics, while mcl-PHAs behave as elastomers or adhesives (Van der Walle et al. 2001). Several PHAs were produced by microbial fermentation; however, the main obstacle that hinders the economic feasibility of PHAs production is the cost of the carbon substrate [28–50% of total production cost (Choi and Lee 1997, 1999)].

In the last decades, the sharp increase in oil prices made it necessary to identify alternatives to fossil fuels like ‘bioethanol’ and ‘biodiesel’. Many studies have been conducted to achieve decent substitutes not only for the crude oil but also for the petroleum-based materials such as plastics. Promising new materials for such purposes are PHAs, which in addition to their physical properties mentioned above are produced from renewable raw materials. Moreover, they are biocompatible and could therefore be used in biomedical and pharmaceutical applications (Holmes 1985).

Unfortunately, the prices of renewable carbon sources like glucose are rising rapidly as a result of the increased demand for bioethanol production. However, at the same time, significant quantities of glycerol are produced by the biodiesel industry as a coproduct (Pachauri and He 2006; Solaiman et al. 2006). This crude glycerol will provide a cheap alternative to the conventional fermentation substrates.

Recently, a new genus, Zobellella comprising the two species Zobellella denitrificans ZD1 and Zobellella taiwanensis ZT1, has been established (Lin and Shieh 2006); however, there have been no reports on the accumulation of PHAs by members of this genus. In this study, a new PHA-accumulating strain was isolated and affiliated to this new genus. This strain is able to utilize glycerol as sole carbon source for growth and PHB accumulation. Optimum conditions for growth and PHB accumulation were investigated.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Isolation of PHB-accumulating bacteria

Samples were collected from different German (DE) and Egyptian (EG) environments: agriculture draining water DE1, sewage DE2, sludge EG1, compost EG2, thermocopost EG3, wastewater sediments EG4, soil EG5 and Nile river water EG6. One gram or one millilitre of each sample was incubated for 3 days at 37°C and 200 rev min−1 in 100 ml mineral salt medium (MSM) (Schlegel et al. 1961) containing (g l−1) Na2HPO4·12H2O, 9·0; KH2PO4, 1·5; MgSO4·7H2O, 0·2; NH4Cl, 1·0; CaCl2·2H2O, 0·02; Fe(III)NH4-citrate, 0·0012; 1 ml of trace elements solution containing (g l−1): EDTA, 50·0; FeCl3, 8·3; ZnCl2, 0·84; CuCl2·2H2O, 0·13; CoCl2·6H2O, 0·1; MnCl2·6H2O, 0·016; H3BO3, 0·1. Glycerol (20 g l−1) was used as the sole carbon source. The pH of the medium was adjusted to 7·3 before sterilization.

Aliquots of 0·1 ml from each enrichment culture were plated onto MSM agar containing glycerol as sole carbon source. The hydrophobic dye, Nile red [0·25 mg dissolved in 1 ml dimethyl sulfoxide (DMSO)], was added to a final concentration of 0·5 μg ml−1 to apply the viable colony staining method (Spiekermann et al. 1999). After 72 h incubation at 37°C, the agar plates were exposed to UV light (312 nm). Fluorescent colonies were transferred again to the same medium to find single colonies until pure cultures were obtained.

Fluorescence microscopy

The presence of cytoplasmic PHA inclusions was evidenced by staining the biopolymer with Nile red and by observing the cells under the fluorescence microscope (López-Cortés et al. 2008).

Physiological and biochemical characterization

The biochemical properties were tested using the API 20 NE (BioMérieux, France) and BBL Oxi/Ferm Tube II (Becton Dickinson, USA) identification system according to the manufacturer’s instructions. Cytochrome oxidase and l-alanine aminopeptidase activities were determined by Bactident Oxidase and Bactident Amidopeptidase strips respectively (Merck KGaA, Darmstadt, Germany).

DNA extraction and 16S rRNA gene analysis

Strain MW1 was cultivated on MSM plate with glycerol, and its genomic DNA was extracted from a single colony. The 16S rRNA gene was amplified by PCR from total DNA using standard oligonucleotide primers (MWG-BIOTECH AG, Ebersberg, Germany) (Rainey et al. 1996). PCR products were purified using the Easy Nucleic Acid Gel Extraction (EZNA) kit (OMEGA Bio-Tek, Doraville, USA) and were then directly sequenced. rRNA gene sequencing was performed in custom at the Institut für Klinische Chemie und Laboratoriumsmedizin (WWU Münster, Münster, Germany) on a capillary sequencer (ABI Prism 3730 DNA analyser); sequences were analysed by data collection software ver. 3.0 (both from Applied Biosystems, Darmstadt, Germany). Sequence reactions were prepared using the BigDye® terminator ver. 3.1 cycle sequencing kit (Applied Biosystems) according to procedures provided by the manufacturer and by using the following oligonucleotides: 27f (5′-GAGTTTGATCCTGGCTCAG-3′), 343r (5′-CTGCTGCCTCCCGTA-3′), 357f (5′-TACGGGAGGCAGCAG-3′), 519r [5′-G(T/A)-ATTACCGCGGC(T/G)GCTG-3′], 536f [5′-CAGC(C/A)GCCGCGGTAAT(T/A)C-3′], 803f (5′-ATTAGATACCCTGGTAG-3′), 907r (5′-CCGTCAATTCATTTGAGTTT-3′), 1114f (5′-GCAACGAGCGCAACCC-3′), 1385r [5′-CGGTGTGT(A/G)CAAGGCCC-3′] and 1525r (5′-AGAAAGGAGGTGATCCAGCC-3′) (MWG-BIOTECH AG).

Sequence analysis and alignment, as well as the construction of the phylogenetic tree, were carried out as described previously (Sallam and Steinbüchel 2008): nucleic acid sequence data were analysed with the Contig Assembly Program online software (Huang 1992). Sequences were aligned with previously published sequences of representative strains and other bacteria using the blast function available on the National Center for Biotechnology Information (NCBI) database. Reference sequences were aligned using the ClustalX 1.8 software (Thompson et al. 1997). Positions of sequence and alignment uncertainty were omitted from the analysis using the programme BioEdit ver. 7.0.5 (T. Hall, North Carolina State University [http://www.mbio.ncsu.edu/BioEdit/bioedit.html]). Phylogenetic trees were constructed using the programs TreeView 1.6.5 (Page 1996) and NJplot (Perrière and Gouy 1996). Bootstraping was applied to evaluate the tree topology by performing 100 resemblings.

Production of PHB

Erlenmeyer flasks (250 ml) containing 50 ml MSM with the carbon source (20 g l−1) indicated in the text were inoculated with overnight preculture (4%, v/v). Unless stated otherwise, flasks were incubated at 41°C and 200 rev min−1 for 96 h. Cells were harvested by 20 min centrifugation at 1200 g and 4°C, washed with distilled water, frozen and lyophilized. Cell concentration, defined as dry cell weight per litre of culture broth, was determined by weighing lyophilized cells.

Production of other PHAs

To investigate the ability of strain MW1 to accumulate PHAs others than PHB, the isolate was cultivated in MSM containing sodium octanoate, ammonium octanoate, triolein, octanoic acid or hexanoic acid as the sole carbon source or with glycerol as a cosubstrate. Sodium propionate and levulinic acid were used as cosubstrates to investigate whether 3HV and 4HB were incorporated into the accumulated PHAs respectively.

PHB extraction

Lyophilized cells were treated with acetone, dried and then stirred in 50 volumes of chloroform for 48 h at 30°C. After filtration, the extracted PHA was concentrated by partially evaporating the solvent, and five volumes of cold methanol was used for precipitation. The precipitated PHA was filtered and dried at 37°C for 24 h (Hahn et al. 1995). PHB was repurified, and it was like purified P(3HV) homopolymer, which was available from our laboratory (Steinbüchel and Schmack 1995), used as a standard for calibration.

GC and GC/MS analyses

To determine the PHA content of the cells and the composition, samples were subjected to methanolysis in the presence of 15% (v/v) sulfuric acid. The resulting 3-hydroxybutyric acid methyl esters were analysed by gas chromatography (Huijberts et al. 1994).

PHB content (%, w/w) was defined as a percentage of cell dry weight (CDW). 3-HV contents in copolyesters are given as molar percentage (mol%) of the polyester constituents. All presented results are from duplicate or triplicate measurements.

Coupled GC/MS analysis was carried out in a Series 6890 GC system equipped with a Series 5973 EI MSD mass-selective detector (Hewlett Packard). A 3-μl portion of the organic phase was analysed after splitless injection on a BPX 35 capillary column (60 m × 250 μm, film thickness 250 nm; SGE, Griesheim, Germany). Helium (constant flow 0·6 ml min−1) was used as carrier gas. The temperatures of the injector and detector were 250 and 240°C respectively. The following temperature programme was applied: 120°C for 5 min, increasing by 3°C min−1 to 180°C and by 10°C min−1 to 220°C, then the temperature was kept for 31 min at 220°C. Data were evaluated by using the NIST-Mass Spectral Search Program (Stein et al. 1998).

Quantitative analyses of ammonium and carbohydrates

The concentrations of ammonium in cell-free supernatants were determined by employing a gas-sensitive type 152303000 ammonium electrode (Mettler Toledo GmbH, Greifensee, Switzerland). Analysis of residual carbon sources was carried out with a LaChrom Elite HPLC apparatus (VWR-Hitachi International GmbH, Darmstadt, Germany) consisting of a Metacarb 67H advanced C column (Varian, Palo Alto, CA, USA; Bio-Rad Aminex equivalent) and a 22350 VWR-Hitachi column oven. The column (300 mm by 6·5 mm) consisted of a sulfonated polystyrene resin in the protonated form. The primary separation mechanism included ligand exchange, ion exclusion and adsorption. A VWR-Hitachi refractive index detector (type 2490) with an active flow cell temperature control and automated reference flushing eliminating temperature effects on the refractive index baseline was used for detection. Aliquots of 20 μl were injected and eluted with 0·005 N sulfuric acid in double-distilled water at a flow rate of 0·8 ml min−1. Online integration and analysis were carried out with EZ Chrome Elite software (VWR International GmbH).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Screening for bacteria accumulating PHA from glycerol

Eight axenic cultures were obtained from various environmental samples, which were able to grow in MSM with glycerol as the sole carbon source. When applying the viable colony staining method (Spiekermann et al. 1999), three of them exhibited strong fluorescence under UV light, (MW1, MW2 and MW7). TLC analysis showed that MW2 and MW7 accumulated lipids, however, without displaying much cytoplasmic inclusions (data not shown). GC and GC/MS analyses revealed that only isolate MW1 (from Egyptian waste water sediments sample, EG4) accumulated PHB. According to TLC analysis, this strain did not accumulate lipids.

Physiological and biochemical characterization of isolate MW1

Strain MW1 is a Gram-negative, rod-shaped, nonspore-forming bacterium (Fig. 1). Both cytochrome oxidase and l-alanine aminopeptidase tests were positive. Data from the API 20NE and BBL Oxi/Ferm Tube II tests (Table 1) showed that strain MW1 utilizes d-mannitol, N-acetylglucosamine, d-maltose, potassium gluconate, malate, citrate, xylose, lactose and sucrose as carbon sources. Glucose was utilized aerobically and anaerobically. Strain MW1 reduced nitrate to the gaseous nitrogen. The strain did not hydrolyse gelatin, arginine or urea.

image

Figure 1.  Micrographs of cells of strain MW1 cultivated in mineral salts medium supplemented with 20 g l−1 glycerol, incubated at 41°C, pH 7·3 and 200 rev min−1 for 96 h. (a) Phase contrast light microscopy showing cells containing bright cytoplasmic inclusions. (b) Differential interference contrast micrograph mimicking cells stereo vision. (c) Inclusions stained with Nile red.

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Table 1.   Biochemical characterization of isolate MW1
TestResult
  1. Incubations were carried out at 37°C.

  2. (+), Weak result.

  3. Results were recorded after 24 and 48 h of incubation.

  4. *Result from both API 20 NE and Oxi/Ferm tube identification systems.

  5. PNPG: 4-nitrophenyl-βD-galactopyranoside.

Nitrate reduction
 NO3 to NO2
 NO3 to N2+*
l-tryptophan−*
Glucose fermentation+*
Arginine dihydrolase−*
Urease−*
Esculin (β-glucosidase)(+)
Gelatinase (protease)
β-galactosidase (PNPG)
Glucose assimilation+*
l-arabinose assimilation
d-mannose assimilation
d-manitol assimilation+*
N-acetyl-glucosamine assimilation+
d-maltose assimilation+
Potassium gluconate assimilation+
Capric acid assimilation
Adipic acid assimilation
Malate assimilation+*
Citrate assimilation+*
Phenylacetic acid assimilation
Phenylalanine
Xylose oxidation+
Sucrose oxidation+
Lactose oxidation+
Lysine decarboxylation

16S rRNA gene sequence and phylogenetic analysis

The almost complete 16S rRNA gene sequence of strain MW1 (1541 bp) was determined. The sequence was aligned and compared with the bacterial sequences available in the GenBank database. The highest similarity level detected was for the 16S rDNA sequence of Z. denitrificans strain ZD1 (98·5%). Lower sequence similarities occurred with 16S rRNA gene sequences of Z. taiwanensis ZT1 (96·2%), Oceanimonas doudoroffii (95·3%) and Oceanimonas smirnovii (94·7%).

The consensus sequence of strain MW1 and strains belonging to species of Oceanimonas and Oceanisphaera and other related taxa belonging to the Alteromonas-like Gammaproteobacteria were aligned, and the phylogenetic tree shown in Fig. 2 was constructed. Our isolate will therefore be refered as Z. denitrificans strain MW1. The 16S rRNA gene sequence for strain MW1 was deposited in the NCBI database under the accession number: EU569287.

image

Figure 2.  Unrooted phylogenetic tree derived from neighbour-joining analysis of the 16S rRNA gene sequences of strain MW1 and of species from related genera of the Alteromonas-like Gammaproteobacteria. GenBank accession numbers are given in parentheses. Numbers above nodes represent bootstrap confidence values obtained with 100 resamplings; values below 70 are not shown. Bar, 1% estimated sequence divergence.

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Time course of growth and PHB accumulation by Zobellella denitrificans MW1

The time course of growth and PHB accumulation by Z. denitrificans MW1 was studied in 1-l flasks containing 300 ml MSM, supplemented with 20 g l−1 glycerol. Cultures were incubated at 37°C and 200 rev min−1, and samples were taken at different intervals under sterile conditions for analysis. Figure 3 shows that growth-associated PHB accumulation occurred during exponential phase of growth, and the maximum cell density (3·7 g CDW per litre) and a PHB content of 73·5%, (w/w) were reached after 100 h of incubation. At this time, ammonium was consumed, and no further decrease in glycerol concentration was detected. Prolonged further incubation of up to 336 h was not accompanied by any increase in PHB content despite the availability of glycerol (about 10 g l−1). Also, no considerable decreases in CDW were detected. The specific growth rate (μ) and doubling time (td) were about 0·053 h−1 and 13 h respectively.

image

Figure 3.  Time course of growth and polyhydroxybutyrate (PHB) accumulation: cells of Zobellella denitrificans strain MW1 were cultivated in 1-l flask containing 300 ml mineral salts medium with 20 g l−1 glycerol. Cultures were incubated at 37°C and 200 rev min−1.

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Production of PHB at various temperatures

The effect of temperature on growth and PHB contents was investigated. As shown in Fig. 4, the cell density (3·6 g CDW per litre) and PHB content (81·2% of CDW, w/w) became maximal at 41°C. Both decreased slightly at 37°C, and much lower growth and polymer contents were obtained at 30°C. Although incubation at a temperature of 45°C affected the growth of Z. denitrificans MW1 negatively, the PHB biosynthesis enzymes of this strain may exhibit moderate thermostability because at this temperature the cells accumulated PHB up to 38·5% (w/w) of the CDW, which is 47·4% of the maximum PHB content.

image

Figure 4.  Effect of incubation temperature on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1. Cultures were inoculated in 250-ml flasks containing 50 ml mineral salts medium with 20 g l−1 glycerol at pH 7·3. Cultures were incubated for 96 h at 200 rev min−1 at different temperatures.

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Biosynthesis of other PHAs

The ability of strain MW1 to accumulate PHAs other than PHB was investigated at a temperature of 41°C. As shown in Fig. 5, octanoate or triolein alone (3·0 g l−1) did not support growth of Z. denitrificans MW1. Substantial inhibition of growth was observed when sodium propionate, levulinic acid, sodium octanoate, ammonium octanoate, octanoic acid or hexanoic acid were used as cosubstrates at concentrations of 1·0 g l−1 together with glycerol (20 g l−1). No considerable inhibition was observed when triolein (1·0 g l−1) was fed together with glycerol (20 g l−1). Sodium oleate (6·0 g l−1) was utilized by the strain, and a PHB content of 47·0% (w/w) was obtained. From all the substrates used, Z. denitrificans MW1 did not produce PHAs others than PHB except P(3HB-co-3HV) with a 3HV content of 22·7 mol% when sodium propionate (1·0 g l−1) was co-fed with glycerol (20 g l−1).

image

Figure 5.  Growth and PHA accumulation by Zobellella denitrificans MW1 utilizing different substrates. Cultivations were carried out in 250-ml flasks containing 50 ml mineral salts medium with the indicated carbon source (see below) at pH 7·3. Cultures were incubated for 96 h at 200 rev min−1 at 41°C. The following carbon sources (concentration) were used and are indicated on the X-axis: Gly, glycerol (20 g l−1); Oct, octanoate (20 mmol l−1 as sole carbon and 3 mmol l−1 when co-fed); Ole, oleate (6·0 g l−1); Pro, propionate (1·0 g l−1); Lev a, levulinic acid (1·0 g l−1); Oct a, octanoic acid (1·0 g l−1); Hex a, hexanoic acid (1·0 g l−1); Tri, triolein (10 g l−1 as sole carbon and 1·0 g l−1 when co-fed). CDW, cell dry weight; PHA, polyhydroxyalkanoate contents; and 3HV (mol%), 3-hydroxyvalerate mol fraction, were determined as described in Materials and methods.

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Effect of sodium chloride concentration

To estimate the effect of sodium chloride on growth and PHB accumulation by Z. denitrificans MW1, different concentrations of NaCl were added to MSM containing 20 g l−1 glycerol. Analyses were carried out after 48- and 96-h incubation. Sodium chloride at a concentration of 20 g l−1 enhanced both growth and polymer contents of the cells. In comparison to a control culture without additionally added NaCl, cell density and PHB productivity increased from 3·52 to 4·78 g l−1 and from 2·82 to 4·16 g l−1, respectively, after 96 h of incubation. Negative effects were observed at higher concentrations of sodium chloride. For example, in the presence of 50 g l−1 NaCl, only 1·77 g l−1 PHB was obtained after 96 h (Table 2).

Table 2.   Effect of sodium chloride concentration on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1
NaCl concentration (g l−1)Incubation time (h)Final pH OD600 nm CDW (g l−1) PHB% (w/w) PHB (g l−1)Residual glycerol (g l−1)
  1. Cells of Z. denitrificans MW1 were cultivated in mineral salts medium containing 20-g l−1 glycerol plus the indicated concentration of NaCl at 41°C and 200 rev min−1. The initial pH was 7·3. Samples were analysed after 48- and 96-h incubation. Analyses were carried out in duplicate; average and standard deviations are presented.

 0486·09 ± 0·0112·8 ± 0·63·10 ± 0·653·6 ± 4·81·68 ± 0·515·9 ± 1·4
966·02 ± 0·0014·9 ± 0·83·52 ± 0·180·1 ± 3·02·82 ± 0·212·9 ± 1·0
10485·61 ± 0·0313·7 ± 0·13·16 ± 0·776·1 ± 5·92·40 ± 0·715·3 ± 1·3
964·80 ± 0·0418·0 ± 0·44·08 ± 0·383·3 ± 2·73·40 ± 0·312·2 ± 3·0
20485·51 ± 0·0615·2 ± 0·83·62 ± 1·079·7 ± 4·52·89 ± 1·016·2 ± 2·7
964·80 ± 0·0322·8 ± 0·64·78 ± 0·687·0 ± 3·74·16 ± 0·711·1 ± 0·7
30485·19 ± 0·0014·7 ± 0·13·50 ± 0·371·0 ± 5·82·49 ± 0·415·1 ± 0·4
964·52 ± 0·0719·9 ± 1·04·30 ± 0·884·8 ± 2·13·65 ± 0·812·2 ± 1·4
50484·77 ± 0·0410·4 ± 0·12·60 ± 0·369·1 ± 3·41·80 ± 0·315·9 ± 0·8
964·40 ± 0·0113·2 ± 0·72·58 ± 0·868·5 ± 2·51·77 ± 0·615·6 ± 1·7

Production of PHB from various carbon sources

The ability of strain MW1 to utilize different cheap carbon sources for PHB accumulation was investigated in MSM in the presence of 20 g l−1 NaCl. Table 3 shows that the strain can utilize glycerol, sodium gluconate, glucose, sucrose and sodium acetate as carbon sources for growth.

Table 3.   Effect of carbon sources and their concentrations on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1
Carbon sourceCarbon source concentration (g l−1)Final pH OD600 nm CDW (g l−1) PHB% (w/w) PHB (g l−1)Residual carbon source (g l−1) Product yield (YP/S)*
  1. Zobellella denitrificans MW1 was cultivated in mineral salts medium containing 20 g l−1 NaCl plus the indicated carbon source at 41°C, pH 7·3 and 200 rev min−1 for 96 h. Analyses were carried out in duplicate; average and standard deviations are presented.

  2. ND, not determined.

  3. *Product yield (YP/S), gram PHB per gram substrate used.

Glycerol56·27 ± 0·067·4 ± 1·32·28 ± 0·261·3 ± 2·71·40 ± 0·2 0·00·28 ± 0·03
106·15 ± 0·0716·8 ± 0·33·94 ± 0·179·0 ± 1·33·11 ± 0·2 0·00·31 ± 0·02
155·20 ± 0·0821·8 ± 0·65·04 ± 0·184·8 ± 0·64·27 ± 0·12·8 ± 0·30·29 ± 0·01
204·94 ± 0·0625·2 ± 1·34·89 ± 0·185·6 ± 2·84·19 ± 0·29·5 ± 0·80·21 ± 0·01
304·85 ± 0·0120·7 ± 1·04·21 ± 0·383·6 ± 3·53·52 ± 0·421·1 ± 1·10·12 ± 0·01
504·86 ± 0·0812·5 ± 0·12·72 ± 0·155·8 ± 1·41·52 ± 0·145·1 ± 2·80·03 ± 0·00
Gluconate57·56 ± 0·144·3 ± 0·11·38 ± 0·417·2 ± 2·10·24 ± 0·1 0·00·05 ± 0·02
108·75 ± 0·139·0 ± 0·42·52 ± 0·153·3 ± 0·71·34 ± 0·1 0·00·13 ± 0·00
158·87 ± 0·0612·6 ± 0·33·24 ± 0·372·0 ± 1·32·33 ± 0·2 0·00·16 ± 0·02
208·80 ± 0·0317·2 ± 0·13·91 ± 0·176·4 ± 3·32·99 ± 0·2 0·00·15 ± 0·01
308·47 ± 0·0421·1 ± 0·64·75 ± 0·184·5 ± 1·04·01 ± 0·20·3 ± 0·30·13 ± 0·01
507·82 ± 0·0123·1 ± 0·35·15 ± 0·183·4 ± 1·74·30 ± 0·27·7 ± 0·80·09 ± 0·00
Glucose205·16 ± 0·014·7 ± 0·11·24 ± 0·450·6 ± 4·40·63 ± 0·312·1 ± 0·60·03 ± 0·01
Sucrose205·23 ± 0·044·8 ± 0·81·25 ± 0·138·1 ± 2·30·48 ± 0·1 0·00·02 ± 0·00
Starch206·70 ± 0·061·3 ± 0·30·41 ± 0·11·6 ± 0·7<0·01NDND
Acetate109·14 ± 0·005·4 ± 0·11·59 ± 0·138·8 ± 2·80·62 ± 0·1 2·0 ± 0·80·06 ± 0·01
209·11 ± 0·088·2 ± 0·32·07 ± 0·151·9 ± 1·31·07 ± 0·1 3·6 ± 1·00·05 ± 0·00
Methanol106·77 ± 0·010·8 ± 0·10·36 ± 0·1<1<0·01NDND
206·76 ± 0·030·7 ± 0·40·26 ± 0·1NDNDNDND

Glycerol and gluconate were the most suitable substrates to obtain high cell density as well as high PHB contents of the cells. PHB yield could be enhanced by the increase in gluconate concentration (4·3 g l−1 PHB at 50 g l−1 gluconate), but in case of glycerol, cell growth and PHB accumulation were inhibited at high concentrations (30–50 g l−1) of this substrate (Table 3). The substrate conversion factor values (product yield YP/S) for gluconate were significantly lower than that for glycerol at the same substrate concentrations (i.e. 30 and 50 g l−1). The highest product yield (0·31 g g−1) was obtained at a concentration of 10 g l−1 glycerol. Figure 6 shows that the optimum concentration of glycerol is 15 g l−1 at which the highest cell density (5·04 g l−1) and volumetric PHB productivity (4·27 g l−1) were obtained, together with an appropriately high substrate yield coefficient (0·29 g PHB per gram glycerol). Glucose, sucrose and acetate were also utilized; however, cell densities and PHB yields were lower than those with gluconate or glycerol. Starch and methanol did not support cell growth or polymer accumulation by Z. denitrificans MW1 (Table 3).

image

Figure 6.  Effect of glycerol concentration on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1. Cultivations were studied in 250-ml flasks containing 50 ml mineral salts medium with 20 g l−1 NaCl and with different concentrations of glycerol at 41°C and pH 7·3. Cultures were incubated for 96 h at 200 rev min−1. Cell dry weight (CDW), PHB, contents of cells and product yield were determined as described in materials and methods.

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Effect of initial pH

The influence of the initial pH on growth and PHB accumulation by Z. denitrificans MW1 was further investigated. For this, the pH values of the culture medium were adjusted to 6·0, 6·7, 7·3, 7·7, 8·3 or 8·7 with concentrated HCl or NaOH solutions prior to sterilization. As shown in Fig. 7, an initial pH of 7·3 was optimal for PHB accumulation (4·16 g l−1 PHB). Higher pH values slightly decreased the PHB accumulation activity. The most negative effect on growth and PHB accumulation occurred at pH 6·0. At this pH, the cell density and volumetric PHB production decreased by about 82% and 77% respectively; this may be attributed to the low bioavailability of some trace elements. Generally, the final pH was lower than the initial pH. It was also noted during the time course of growth (Fig. 3) that the decrease in the final pH is correlated to cell growth.

image

Figure 7.  Effect of initial incubation pH on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1. Cultivations were carried out in 250-ml flask containing 50 ml mineral salts medium with 15 g l−1 glycerol and 20-g l−1 NaCl at 41°C at pH values between 6·0 and 9·0. Cultivations were carried out for 96 h at 200 rev min−1. Cell dry weight (CDW) and PHB contents were determined as described in materials and methods.

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Effect of nitrogen sources

Ammonium chloride, ammonium sulfate and yeast extract were used at different concentrations in MSM containing glycerol to identify a nitrogen source optimum for both, growth and PHB accumulation activity (Fig. 8). The highest PHB yield (4·3 g l−1) was obtained when 20 mmol l−1 NH4Cl was used, which is almost identical with the basic medium (approx. 1 g l−1) that was used as control. However, with 10 mmol l−1 NH4Cl, slightly higher PHB contents of the cells were obtained. Lower cell densities and lower PHB yields were obtained when yeast extract was used as sole nitrogen source.

image

Figure 8.  Effect of nitrogen source and its concentration on growth and polyhydroxybutyrate (PHB) accumulation by Zobellella denitrificans MW1. Cultivations were carried out in 250-ml flask containing 50 ml mineral salts medium with 15-g l−1 glycerol, 20-g l−1 NaCl and supplemented with different nitrogen sources at the indicated concentrations at 41°C, pH 7·3. Cultivations were carried out for 96 h at 200 rev min−1.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

In this study, we succeeded in isolating a new bacterium that grows well in a chemically defined mineral salts medium containing glycerol as sole carbon source for growth. The new isolate MW1 was affiliated to the new genus Zobellella, and according to the similarity of its 16S rRNA gene sequence to that of Zobellella denitrificans ZD1 (98·5%), it was classified as a strain of the species Z. denitrificans (Fig. 2). Although there have been no previous reports on the accumulation of PHA in this new genus, Z. denitrificans MW1 is capable of accumulating PHB amounting to 80·4% (w/w) of the cellular dry weight. This makes this bacterium to be one of the best PHB producers reported so far (Braunegg et al. 1998; Reddy et al. 2003; Khanna and Srivastava 2005; Suriyamongkol et al. 2007).

PHB accumulation by Z. denitrificans MW1 is associated with cell growth (Fig. 3) and occurs in parallel with the consumption of glycerol and ammonia, i.e. without obvious nutrient limitation. So far, Alcaligenes latus served as a model organism for growth-associated PHB accumulation, and the behaviour of A. latus in fed-batch culture was therefore studied in detail (Yamane et al. 1996; Grothe and Chisti 2000). Growth-associated PHB and poly(3HB-co-3HV) accumulations were also reported for the new methylotrophic bacterium Methylobacterium sp. GW2 (Yezza et al. 2006). With Z. denitrificans MW1, it will be possible to obtain high cell densities accompanied by high polymer contents in a simple one-step fermentation process without the need to apply a nutrient limitation.

Cultivation of Z. denitrificans MW1 on a variety of short and long carbon-chain length fatty acids like octanoic acid salts or triolein demonstrated that the strain can obviously not utilize them for growth or for synthesizing mcl-PHA (Fig. 5). Beside PHB, this bacterium is only able to synthesize the copolymer P(3HB-co-3HV) during co-feeding of sodium propionate together with glycerol, supposing that the strain possesses the scl-PHA biosynthesis pathway (Suriyamongkol et al. 2007). Although decent 3HV yield was reached by Z. denitrificans MW1 (0·279 g 3HV per gram propionate), a low 3HB yield of 0·060 g 3HB per gram glycerol was attained. However, the molar 3HV content of 22·7% confers to the polymer appropriate physical properties distinguishing it from the brittle PHB homopolymer. P(3HB-co-3HV) is less fragile and has a lower melting point than PHB, thereby offering improved thermal processing and enhanced mechanical properties (Loo and Sudesh 2007). Cell densities and total PHA contents obtained under conditions yielding P(3HB-co-3HV) were lower than those for PHB. This is probably because of the toxic effect of propionate on growth, which needs very tight control to reach appropriate molar ratio of HV while also keeping decent polymer productivity (Ramsay et al. 1990; Son and Lee 1996; Kim and Lenz 2001; Yu et al. 2005). Usually, the costs of cosubstrate increase the production costs of P(3HB-co-3HV) especially if a high 3HV content is intended (Choi and Lee 2000).

The enhanced growth and polymer accumulation by Z. denitrificans MW1 in the presence of 20 g l−1 NaCl is in accordance with the findings of Lin and Shieh (2006), who found enhanced growth of Z. denitrificans ZD1 at NaCl concentrations of up to 30 g l−1; however, NaCl was not indispensable for growth. That may explain the existence of this genus in different environments with or without salinity like in the case of Z. denitrificans MW1 that was isolated from a wastewater sediment sample.

Sodium chloride is the main impurity in crude glycerol derived from biodiesel production during alkaline-catalysed transesterification of animal fats or vegetable oils. It occurs at a concentration that – if accumulated during feeding of glycerol – may inhibit microbial growth and may thus prevent its usage as substrate for large-scale microbial fermentation. Crude glycerol from different biodiesel sources was investigated by Mothes et al. (2007), and it was shown that crude glycerol contaminated with NaCl had a significant negative effect on PHB productivity and on product yield.

Further experiments with Z. denitrificans MW1 were carried out in the presence of NaCl (20 g l−1) to show whether this isolate is a potent strain for PHB production from low-value glycerol. Obviously, glycerol appeared to be the best substrate for growth and PHB accumulation by Z denitrificans MW1, and the highest conversion rate was 0·31 g PHB per gram glycerol. The maximal PHB productivity (4·27 g l−1) was reached with glycerol at a concentration of 15 g l−1. Similar PHB productivity (4·30 g l−1) was obtained with 50 g l−1 gluconate (Table 3). The conversion rate of glycerol to PHB, which was obtained at the optimum glycerol concentration of 15 g l−1 (0·29 g PHB per gram glycerol), was significantly higher than those reported for glycerol by de Almeida et al. (2007) (0·08 g PHB per gram glycerol), Bormann and Roth (1999) (0·17 PHB per gram glycerol) and Koller et al. (2005) (0·23 g PHB per gram glycerol). This conversion rate is also comparable to that obtained by Mothes et al. (2007) (0·37 g PHB per gram glycerol), in particular when the significantly decreased conversion rate obtained in the presence of NaCl-contaminated crude glycerol is considered (0·14 g PHB per gram glycerol).

The pH of the medium and the concentration of the nitrogen source and of other macroelements have to be controlled to reach higher cell densities while retaining the very high PHB contents of the cells in the future. Although growth-associated PHB accumulation in cells of Zdenitrificans MW1 did not require nitrogen-limited conditions, the PHB content increased slightly by 5·8% reaching 89·4% (w/w) at the lower concentration of NH4Cl in comparison to the optimum concentration (20 mmol l−1 NH4Cl) for growth (Fig. 8). However, because a 34% decrease in cell density occurred, the volumetric productivity decreased to 69% of the maximum. Enhanced productivity of growth-associated PHB accumulation was also reported for A. latus at low nitrogen concentration (Wang and Lee 1997).

The new strain MW1 of Z. denitrificans can be regarded as a strain of choice for conversion of glycerol into PHB. This strain utilized glycerol as sole carbon source for growth and PHB biosynthesis exhibiting relatively good growth on glycerol (td = 13 h) and accumulating PHB up to 87% of the cell dry mass. The strain grows and accumulates PHB even in the presence of considerable concentrations of NaCl. As a conclusion, Z. denitrificans MW1 is recommended as a good candidate for industrial production of PHB from technical-grade glycerol as it occurs as a residue during industrial biodiesel production. This strain is currently investigated to strongly increase the volumetric productivity of PHB by fed-batch fermentation.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Financial support of this study by BASF AG (Ludwigshafen, Germany) is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
  • De Almeida, A., Nikel, P.I., Giordano, A.M. and Pettinari, M.J. (2007) Effects of granule-associated protein PhaP on glycerol-dependent growth and polymer production in poly(3-hydroxybutyrate)-producing Escherichia coli. Appl Environ Microbiol 73, 79127916.
  • Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54, 450472.
  • Bormann, E.J. and Roth, M. (1999) The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates. Biotechnol Lett 21, 10591063.
  • Braunegg, G., Lefebvre, G. and Genser, K.F. (1998) Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects. J Biotechnol 65, 127161.
  • Choi, J.I. and Lee, S.Y. (1997) Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Biosyst Eng 17, 335342.
  • Choi, J. and Lee, S.Y. (1999) Factors affecting the economics of polyhydroxyalkanoates production by bacterial fermentation. Appl Microbiol Biotechnol 51, 1321.
  • Choi, J. and Lee, S.Y. (2000) Economic considerations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterial fermentation. Appl Microbiol Biotechnol 53, 646649.
  • Grothe, E. and Chisti, Y. (2000) Poly(beta-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: behavior of fed-batch cultures. Bioprocess Biosyst Eng 22, 441449.
  • Hahn, S.K., Chang, Y.K. and Lee, S.Y. (1995) Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli. Appl Environ Microbiol 61, 3439.
  • Holmes, P.A. (1985) Applications of PHB – a microbially produced biodegradable thermoplastic. Phys Technol 16, 3236.
  • Huang, X. (1992) A contig assembly program based on sensitive detection of fragment overlaps. Genomics 14, 1825.
  • Huijberts, G.N.M., Van Der Wal, H., Wilkinson, C. and Eggink, G. (1994) Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria. Biotechnol Tech 8, 187192.
  • Jendrossek, D. and Handrick, R. (2002) Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 56, 403432.
  • Khanna, S. and Srivastava, A.K. (2005) Recent advances in microbial polyhydroxyalkanoates. Process Biochem 40, 607619.
  • Kim, Y.B. and Lenz, R.W. (2001) Polyesters from microorganisms. In Advances in Biochemical Engineering/Biotechnology ed. Steinbüchel, A. and Babel, W. Vol. 71, pp. 5179. Berlin: Springer.
  • Koller, M., Bona, R., Braunegg, G., Hermann, C., Horvat, P., Kroutil, M., Martinz, J., Neto, J. et al. (2005) Production of polyhydroxyalkanoates from agricultural waste and surplus materials. Biomacromolecules 6, 561565.
  • Lin, Y.T. and Shieh, W.Y. (2006) Zobellella denitrificans gen. nov., sp. nov. and Zobellella taiwanensis sp. nov., denitrifying bacteria capable of fermentative metabolism. Int J Syst Evol Microbiol 56, 12091215.
  • Loo, C.Y. and Sudesh, K. (2007) Polyhydroxyalkanoates: bio-based microbial plastics and their properties. Malaysian Polym J 2, 3157.
  • López-Cortés, A., Lanz-Landázuri, A. and Garacía-Maldonado, J.Q. (2008) Screening and isolation of PHB-producing bacteria in a polluted marine microbial mat. Microb Ecol 56, 112120.
  • Mothes, G., Schnorpfeil, C. and Ackermann, J.U. (2007) Production of PHB from crude glycerol. Eng Life Sci 7, 475479.
  • Pachauri, N. and He, B. (2006) Value-added utilization of crude glycerol from biodiesel production: a survey of current research activities. ASABE Paper no. 066223. St Joseph, MI: ASABE.
  • Page, R.D.M. (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.
  • Perrière, G. and Gouy, M. (1996) WWW-query: an on-line retrieval system for biological sequence banks. Biochimie 78, 364369.
  • Rainey, F.A., Ward-Rainey, N., Kroppenstedt, R.M. and Stackebrandt, E. (1996) The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 10881092.
  • Ramsay, B.A., Lomaliza, K., Chavarie, C., Dube, B., Bataille, P. and Ramsay, J.A. (1990) Production of poly-(β-hydroxybutyric-co-3-hydroxyvaleric) acids. Appl Environ Microbiol 56, 20932098.
  • Reddy, C.S.K., Ghai, R., Rashmi and Kalia, V.C. (2003) Polyhydroxyalkanoates: an overview. Bioresour Technol 87, 137146.
  • Sallam, A. and Steinbüchel, A. (2008) Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1 and role of three other coisolates in a mixed bacterial consortium. Appl Environ Microbiol 74, 34343443.
  • Schlegel, H.G., Kaltwasser, H. and Gottschalk, G. (1961) Ein Submersverfahren zur Kultur Wasserstoff oxydierender Bakterien: wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38, 209222.
  • Solaiman, D.K.Y., Ashby, R.D., Foglia, T.A. and Marmer, W.N. (2006) Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Appl Microbiol Biotechnol 71, 783789.
  • Son, H. and Lee, S. (1996) Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from structurally unrelated single carbon sources by newly isolated pseudomonas sp. El-2. Biotechnol Lett 18, 12171222.
  • Spiekermann, P., Rehm, B.H.A., Kalscheuer, R., Baumeister, D. and Steinbüchel, A. (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroyalkanoic acids and other lipid storage compounds. Arch Microbiol 171, 7380.
  • Stein, S., Levitsky, A., Fateev, O. and Mallard, G. (1998) The NIST Mass Spectral Search Program, ver. 1.6d. Gaithersburg, MD: National Institute of Standards and Technology.
  • Steinbüchel, A. and Füchtenbusch, B. (1998) Bacterial and other biological systems for polyester production. Trends Biotechnol 16, 419427.
  • Steinbüchel, A. and Schmack, G. (1995) Large scale production of poly(3-hydroxyvaleric acid) by fermentation of chromobacterium violaceum, processing, and characterization of the homopolyester. J Environ Polym Degrad 3, 243258.
  • Steinbüchel, A. and Valentin, H.E. (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128, 219228.
  • Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M. and Shah, S. (2007) Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants – a review. Biotechnol Adv 25, 148175.
  • Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 48764882.
  • Van der Walle, G.A.M., De Koning, G.J.M., Weusthuis, R.A. and Eggink, G. (2001) Properties, modifications and applications of biopolyesters. In Advances in Biochemical Engineering/Biotechnology ed. Steinbüchel, A. and Babel, W. Vol. 71, pp. 263291. Berlin: Springer.
  • Wang, F. and Lee, S.Y. (1997) Poly(3-hydroxybutyrate) production with high productivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation. Appl Environ Microbiol 63, 37033706.
  • Yamane, T., Fukunaga, M. and Lee, Y.W. (1996) Increased PHB productivity by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer. Biotechnol Bioeng 50, 197202.
  • Yezza, A., Fournier, D., Halasz, A. and Hawari, J. (2006) Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2. Appl Microbiol Biotechnol 73, 211218.
  • Yu, S.T., Lin, C.C. and Too, J.R. (2005) PHBV production by Ralstonia eutropha in continuous stirred tank reactor. Process Biochem 40, 27292734.