Appl Microbiol Biotechnol (2008) 80:665–673
DOI 10.1007/s00253-008-1593-0
APPLIED MICROBIAL AND CELL PHYSIOLOGY
The conversion of BTEX compounds by single and defined
mixed cultures to medium-chain-length
polyhydroxyalkanoate
Jasmina Nikodinovic & Shane T. Kenny &
Ramesh P. Babu & Trevor Woods & Werner J. Blau &
Kevin E. O’Connor
Received: 27 March 2008 / Revised: 20 June 2008 / Accepted: 23 June 2008 / Published online: 16 July 2008
# Springer-Verlag 2008
Abstract Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological
conversion into valuable biodegradable plastic polymers—
polyhydroxyalkanoates (PHAs). We assessed the ability of
the known Pseudomonas putida species that are able to
utilize benzene, toluene, ethylbenzene, p-xylene (BTEX)
compounds as a sole carbon and energy source for their
ability to produce PHA from the single substrates. P. putida
F1 is able to accumulate medium-chain-length (mcl) PHA
when supplied with toluene, benzene, or ethylbenzene. P.
putida mt-2 accumulates mcl-PHA when supplied with
toluene or p-xylene. The highest level of PHA accumulated
by cultures in shake flask was 26% cell dry weight for P.
putida mt-2 supplied with p-xylene. A synthetic mixture of
benzene, toluene, ethylbenzene, p-xylene, and styrene
(BTEXS) which mimics the aromatic fraction of mixed
plastic pyrolysis oil was supplied to a defined mixed culture
of P. putida F1, mt-2, and CA-3 in the shake flasks and
fermentation experiments. PHA was accumulated to 24%
and to 36% of the cell dry weight of the shake flask and
fermentation grown cultures respectively. In addition a
three-fold higher cell density was achieved with the mixed
culture grown in the bioreactor compared to shake flask
experiments. A run in the 5-l fermentor resulted in the
J. Nikodinovic : S. T. Kenny : K. E. O’Connor (*)
School of Biomolecular and Biomedical Sciences,
Ardmore House, University College Dublin,
Belfield,
Dublin 4, Ireland
e-mail: [email protected]
R. P. Babu : T. Woods : W. J. Blau
Polymer Research Centre, School of Physics,
Trinity College Dublin,
Dublin 2, Ireland
utilization of 59.6 g (67.5 ml) of the BTEXS mixture and
the production of 6 g of mcl-PHA. The monomer
composition of PHA accumulated by the mixed culture
was the same as that accumulated by single strains supplied
with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer
was partially crystalline with an average molecular weight
of 86.9 kDa. It has a thermal degradation temperature of
350 °C and a glass transition temperature of −48.5 °C.
Introduction
Petrochemical non-oxygenated monoaromatic hydrocarbons, in particular, benzene, toluene, ethylbenzene, and
xylenes (BTEX compounds) are widely used in the industry
as solvents and as starting materials for the production of
pharmaceuticals, polymers, and paints (APA 2001). Indeed,
BTEX compounds are among the top 50 chemicals
produced and used worldwide (Energetics 2000). As a
consequence of their wide usage, they are common waste
materials from the industry (Caprino and Togna 1998;
ATSDR 2004). The metabolism of BTEX compounds by
microorganisms is well known with physiological, biochemical, and molecular investigations of their degradation
reported (Smith 1990; Pieper et al. 2004). However, the
focus over the last three to four decades has been towards
BTEX biodegradation, and not their conversion to valuable
end products. We view BTEX compounds as a potentially
valuable feedstock for the biosynthesis of biodegradable
plastic polyhydroxyalkanoate (PHA) by bacteria. Polyhydroxyalkanoates represent a class of natural polyesters that
are biodegradable thermoplastics with a broad range of uses
(Anderson 1990). While many bacteria (and in particular
666
Pseudomonas strains) can consume a large number of
aromatic compounds, they are not always able to accumulate PHA (Zylstra and Gibson 1989; Lee et al. 1995;
Hoffmann et al. 2000).
We report on the accumulation of PHA from single
aromatic substrates by single cultures (P. putida F1:
benzene, toluene, ethylbenzene, and P. putida mt-2:
toluene, p-xylene) as well as mixed aromatic substrates by
defined mixed cultures. While BTEX compounds frequently
co-occur as wastes, they are also major components of oils
(55.6%, v/v) generated from the heat treatment (pyrolysis)
of mixed plastic waste (Kaminsky and Kim 1999; Angyal
et al. 2007). This oil also contains styrene in high amounts
(18.7%, v/v). The mixture of aromatic hydrocarbons (BTEX
and styrene (BTEXS)) can be easily separated from the
other components of the pyrolysis oil by the existing
distillation system in petrochemical plants (Kaminsky and
Kim 1999). We propose that this aromatic fraction could
be used as a feedstock for bacterial synthesis of PHA. We
have previously demonstrated the conversion of polystyrene to PHA through pyrolysis of polystyrene and feeding
of the pyrolysis oil to P. putida CA-3 (Ward et al. 2006). A
synthetic mixture, mimicking the ratio of aromatic compounds found in the product of mixed plastic pyrolysis,
was supplied to mixed cultures of Pseudomonas putida
F1, mt-2, and CA-3 to determine if mixed aromatic
substrates could be used by mixed cultures for PHA
biosynthesis.
Materials and methods
Chemicals and materials
Benzene, toluene, ethylbenzene, p-xylene, styrene, benzoic
acid and (R)-3-hydroxyalkanoic acids were analytical grade
and supplied from Sigma-Aldrich (Steinheim, Germany).
Chloroform, methanol, and other solvents were of GC
grade and supplied from Fluka (Buchs, Switzerland). A
synthetic mixture composed of benzene, toluene, ethylbenzene, p-xylene, and styrene in a ratio of 16:11:1.5:1:15 was
used to mimic the aromatic components of a mixed plastic
pyrolysis sample.
Microorganisms
Reference Pseudomonas strains were obtained from the
Deutsche Sammlung von Mikroorganismen und Zellculturen
GmbH or NCIMB culture collection (Aberdeen, Scotland,
UK) for their ability to degrade aromatic hydrocarbons. P.
putida F1 (DSM 6899) is a benzene, toluene, and ethylbenzene degrader (Zylstra and Gibson 1989); P. putida mt-2
(NCIMB10432) is a toluene and m- and p-xylene degrader
Appl Microbiol Biotechnol (2008) 80:665–673
(Worsey and Williams 1975); P. putida CA-3 (NCIMB41162)
is a styrene degrader (O’Connor et al. 1995).
Culture conditions for growth and PHA accumulation
by bacterial cultures in shake flasks experiments
Strains were grown in mineral MSM broth (Schlegel et al.
1961), and were stored frozen at −20 °C in 15% glycerol/
MSM medium stocks. The utilization by the isolates of the
single BTEX compounds as a sole carbon an energy source
was determined by growth on MSM mineral medium plates
solidified with 1% (w/v) agarose (Sigma) and supplemented
via the vapor phase by placing 40 μl of benzene, toluene,
ethylbenzene, styrene, or p-xylene into Eppendorf pipette
tips inside the Petri dishes. After 2–4 days of incubation at
30 °C, plates were screened for presence of colonies.
Growth was confirmed by comparison with control plates
without substrate and 20 mM glucose as carbon source,
respectively.
Single colonies of P. putida strains grown on solid media
were transferred to 3 ml of MSM media (pH 7.0) containing
20 mM benzoic acid as a carbon source and grown
overnight at 30 °C. Conical flasks (250 ml) containing
50 ml of MSM liquid media containing 0.25 g/l NH4Cl
(nitrogen-limited conditions) were then inoculated with
bacterial cells (1% inoculum). A corresponding BTEX
compound (350 μl) or a mixture (350 μl) was placed into a
central column and flasks were tightly closed using sterile
cotton plugs and a triple layer of aluminum foil as
previously described (Ward et al. 2005). Cultures were
grown shaking at 200 rpm in an incubator at 30 °C for
48 h during which time, 350 μl partitioned from the central
column into the air and subsequently into the liquid
medium where it is utilized by the bacteria (Ward et al.
2005).
Fermentation conditions
One hundred milliliters of a precultured mixed cell culture
(as described in the shake flasks experiments section) was
used as the inoculum for the 7.5-l (5 l working volume)
stirred tank reactor (fermentor; Electrolab, Tewkesbury,
UK) containing 5 l of MSM medium. Fermentations were
performed for 50 h at 30 °C with air supplied at 5 l/min,
agitation rate of 500 rpm and the pH controlled at 6.8. For
mcl-PHA accumulation, nitrogen was limited with a
starting concentration of 65 mg/l, and a feeding rate of
1.5 mg/l/h as previously described (Goff et al. 2007).
Antifoam (polypropylene glycol, P2000) was supplied at the
start of fermentation (1 ml/l). BTEXS mix was supplied using
the external single channel peristaltic pump (Electrolab,
Tewkesbury, UK) at a substrate flow rate of 12.5 μl/min for
the first 6 h of the growth. This was increased to 25 μl/min for
Appl Microbiol Biotechnol (2008) 80:665–673
the remainder of the fermentation. Forty milliliter samples
were taken at 6, 24, 30, and 48 h to determine PHA, biomass,
and nitrogen concentration.
Nitrogen assay
The nitrogen concentration in the fermentation medium
was determined by phenol–hypochlorite method
as previously described (Scheiner 1976).
Analysis of mixed culture growth (colony forming units/ml)
when supplied with mixed substrates
The colony forming units of various Pseudomonas strains
when supplied with the synthetic mixed substrate (BTEXS)
was determined over 48 h by periodically sampling from
the liquid culture in shake flasks and the fermenter. The
total cell count was determined by plating culture dilutions
onto LB solid medium (Sambrook et al. 1989). The specific
cell count was determined by plating culture dilutions onto
MSM plates supplemented with ethylbenzene for P. putida
F1, p-xylene for P. putida mt-2, and styrene for P. putida
CA-3. Plates were incubated for 4 days at 30 °C before the
enumeration of the colonies.
PHA content and monomer determination from bacterial
cultures
After 48 h incubation at 30 °C, cells were then harvested by
centrifugation at 4,000 rpm for 20 min in benchtop 5810R
centrifuge (Eppendorf). The pellet was washed twice with 1
ml of 50 mM phosphate buffer (pH 7.4) and freeze dried.
To determine the polymer content of lyophilized whole
cells, approximately 5 mg of the cells was subjected to
acidic methanolysis according to previously described
protocols (Brandl et al. 1988; Lageveen et al. 1988). This
method degrades the intracellular PHA to its constituent
3-hydroxyalkanoic acid methyl esters. Cell material (5–10
mg) or PHA standard was resuspended in 2 ml acidified
methanol (15% H2SO4, v/v) and 2 ml of chloroform
containing 6 mg/l benzoate methyl ester as an internal
standard. The mixture was placed in 15 ml Pyrex test tubes
and incubated at 100 °C for 3 h (with frequent inversions).
The solution was extracted with 1 ml of water (vigorous
vortex 2 min). The phases were allowed to separate before
removing the top layer (water). The organic phase (bottom
layer) was dried with Na2SO4 before further analysis.
The 3-hydroxyalkanoic acid methyl esters were assayed
by gas chromatography (GC) using Hewlett-Packard
HP6890 chromatograph equipped with a BP21 capillary
column (25 m by 0.25 mm, 0.32-μm film thickness; SGE
Analytical Sciences) and a flame ionization detector (FID).
A temperature program was used to separate the different 3-
667
hydroxyalkanoic acid methyl esters (120 °C for 5 min;
temperature ramp of 3 °C per min; 180 °C for 10 min). For
the peak identification, commercially available 3-hydroxyalkanoic acid was methylated as described above for PHA
samples.
PHA polymer isolation and analysis
PHA polymer was isolated from the lyophilized cells
containing about 37% (w/w) of polymer by solvent
extraction using a slightly modified method from that
previously reported (Jiang et al. 2006). Cells (15 g, dry
mass) were resuspended in 300 ml of acetone. The
suspension was vigorously stirred at room temperature for
12 h, after which suspension was filtered twice using firstly
Whatman filter paper No.1 (Whatman Ltd., Maidstone,
UK) followed by filtration through 0.45 μm PTFE filter
(Pall Corporation, New York, USA). After evaporation to
30 ml, the filtrate was slowly added to 200 ml of ice-cold
methanol. The white PHA precipitate was collected by
centrifugation at 5,000 rpm for 30 min at 4 °C in Sorvall
RC5CPlus centrifuge (Kendro Lab Products, Newtown, CT,
USA). Methanol was decanted and PHA was dissolved in
20 ml of acetone. The washing procedure in acetone and
precipitation in methanol was repeated three times. The
polymer was finally resuspended in 15 ml of acetone and
cast on a glass plate where it was allowed to dry for 48 h at
room temperature prior to analysis.
Molecular weight distribution of the PHA sample was
obtained by gel permeation chromatography (GPC) using
PL gel 5 mm mixed-C+PL gel column (Perkin Elmer) with
PELV 290 UV–Vis detector set at 254 nm. Sample
concentration of 1% (w/v), injection volumes of 500 μl
and spectroscopic grade choloroform as the eluent at flow
rate of 1 ml/min were used. A molecular weight calibration
curve was generated with polystyrene standards with low
polydispersity using the Turbochrom 4.0 software.
Differential Scanning Calorimetry (DSC) was performed with Perkin Elmer Pyris-Diamond Calorimeter
calibrated to indium standards. The samples weighing 7–
8 mg were encapsulated in hermetically sealed aluminum
pans and heated from −70 to 100 °C at a rate of 10 °C/min.
To determine the glass transition temperature (Tg) the
samples were held at 100 °C for 1 min and rapidly quenched
to −70 °C. The samples were then re-heated from −70 to
100 °C at 10 °C/min to determine the melting temperature
(Tm) and Tg. The melting temperature (Tm) was taken at
the peak of the melting endotherm, while the Tg was taken
as the mid point of heat capacity change, respectively.
To determine the thermal stability and decomposition
temperature (DT) as well as decomposition profile of the
PHA sample, thermogravimetric analysis (TGA) was
carried out on a Perkin Elmer Pyris 1 Thermogravimetric
668
Appl Microbiol Biotechnol (2008) 80:665–673
Analyzer calibrated using nickel and iron standards. Each
sample was weighed to 7 mg and placed in a platinum pan
and heated from 30 to 700 °C at the heating rate of 10 °C/min
under an air atmosphere.
Dynamic Mechanical Analysis (DMA) was carried out on
a Perkin Elmer Mechanical Analyzer. Dynamic measurements
were made in extension mode on clamped film samples with
dimensions of 5×2.8×0.5 mm. The experiments were
performed under nitrogen atmosphere at a temperature range
of −100 to 50 °C at a heating rate of 2 °C/min and frequency
of 0.1, 1, and 10 Hz.
Solution nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DPX400 with 1H at 400.13 MHz and
13
C at 100.62 MHz. The solvent chloroform-d and
tetramethylsilane (TMS) were used as internal references
for chemical shifts in 13C and 1H NMR, respectively.
Fourier transform infrared spectroscopy (FTIR) was
carried out on a Nicolet Nexus Spectrometer in transmission mode over a range of 4,000 to 400 cm−1 and with a
resolution of 4 cm−1. One hundred scans were recorded for
the sample and machine calibration was checked to
polystyrene standards.
Results
PHA accumulation from single BTEX compounds
by single cultures
P. putida strains F1 and mt-2 were examined for their
ability to grow and accumulate PHA from a variety of
aromatic hydrocarbons (Table 1).
P. putida F1 when supplied with 350 μl of toluene,
benzene, or ethylbenzene accumulates PHA to 22%, 15%, and
14% of cell dry weight (CDW), respectively (Fig. 1), under
nitrogen limitation (65 mg N/l). However, the strains failed
to grow with p-xylene or styrene as a sole source of carbon
and energy. P. putida F1 achieved a similar cell dry weight
when supplied with either toluene or ethylbenzene as the sole
source of carbon and energy (Table 1). A two-fold lower cell
dry weight was achieved with benzene as the carbon source.
Thus, the PHA productivity (g/l) was four-fold and 2.1-fold
higher for toluene- and ethylbenzene-grown cells, respectively, compared to benzene-grown cells (Table 1). The PHA
accumulated by strain F1 is almost identical from all three
aromatic carbon sources supplied. The PHA is composed
predominantly of 3-hydroxydecanoic acid with 3-hydroxyoctanoic acid and 3-hydroxydodecanoic acid occurring at
similar levels, while 3-hydroxydodecenoc acid appears in
substantially lower amounts (Table 1).
P. putida mt-2 when supplied with 350 μl toluene or
p-xylene accumulates PHA up to 22% and 26% of CDW
respectively. Strain mt-2 failed to grow with either benzene,
ethylbenzene, or styrene as the sole carbon and energy
source. The cell dry weight achieved by P. putida mt-2
when supplied with toluene was almost half that achieved
by P. putida F1 and, thus, despite a similar PHA content per
cell, the PHA productivity was 2.2-fold lower for P. putida
mt-2. P. putida mt-2 achieves a higher cell dry weight and
PHA content per cell dry weight with p-xylene as the
substrate compared to toluene (Table 1, Fig. 1). Thus, a 1.9fold higher PHA productivity is achieved with this substrate
compared to toluene.
P. putida CA-3 is a well-known styrene degrader capable
of accumulating PHA from styrene (Ward et al. 2005; Ward
et al. 2006). This strain, however, can not metabolize any
other aromatic hydrocarbon investigated in this study
(Table 1).
Table 1 PHA accumulation from benzene, toluene, ethylbenzene, xylene (BTEX) and styrene by Pseudomonas putida strains
Substrate
P. putida strain
F1
mt-2
CA-3
F1+mt-2+CA-3
CDW
PHA
C8:C10:
CDW
PHA
C8:C10:
CDW
PHA
C8:C10:
CDW
PHA
C8:C10:
(g/l)
(g/l)
C12:C12:1a
(g/l)
(g/l)
C12:C12:1
(g/l)
(g/l)
C12:C12:1
(g/l)
(g/l)
C12:C12:1
Benzene
0.340.03
0.048±0.002 17:65:14:4 NG
Toluene
0.72±0.11
0.16± 0.02
NG
18:62:16:4 0.37±0.03 0.081±0.003 17:63:15:5 NG
Ethylbenzene 0.67±0.04 0.098±0.001 17:64:15:4 NG
p-Xylene
NG
0.53±0.01
Styrene
NG
NG
BTEXS mix
NG
0.14±0.02
16:66:14:4 NG
0.79±0.02 0.26±0.03 18:65:11:6
1.03±0.04 0.25±0.04 16:64:15:4
a
Traces of C6 monomer were also detected, but these were not accounted for in the calculation of the total yield of the polymer.
Cell yield is given as cell dry weight (CDW); the PHA yield is calculated by multiplying the cell yield (g/l) by the PHA content (% cell dry
weight) of the cells. Monomeric unit composition of PHA is given as percentage ratio of C8:C10:C12:C12:1. C8, 3-hydroxyoctanoate; C10, 3hydroxydecanoate; C12, 3-hydroxydodecanoate; C12:1, 3-hydroxydodecenoate. All data is an average of three independent measurements.
NG no growth.
Appl Microbiol Biotechnol (2008) 80:665–673
Fig. 1 PHA accumulation by P. putida strains from petrochemical
monoaromatic hydrocarbons (BTEX and styrene). Pure cultures were
grown in MSM mineral medium under nitrogen-limiting conditions
(67 mg N/l) in the presence of a single carbon source for 48 h. All data
are the average of at least three independent experiments
PHA accumulation from BTEXS mixture by mixed culture
Given the individual abilities of these strains to accumulate
PHA from the various aromatic substrates tested, it is
possible that a mixed culture will grow and accumulate
PHA when supplied with a mixture of the aromatic
compounds. However, the presence of compounds that are
not metabolized by a bacterium may cause toxic effects and
hinder growth of that bacterium (Reardon et al. 2000). The
ability of a defined mixed culture of P. putida F1, mt-2, and
CA-3 (log10 5 to log10 6 CFU/ml of each and a total 1%
inoculum) to metabolize a mixture (350 μl) of aromatic
hydrocarbons (benzene, toluene, ethylbenzene, p-xylene,
and styrene in a ratio of 16:11:1.5:1:15) was examined with
a view to assessing PHA accumulation. The ratio of each
substrate to the other was based on the composition of the
mixed plastic pyrolysis oil reported by Kaminsky and Kim
(1999). The final cell dry weight achieved after 48 h of
growth was 1.03 g/l. Twenty-four percent of the cell dry
weight was composed of PHA representing a PHA
productivity of 0.25 g/l (Table 1). The ratio of PHA
monomers was almost identical to that achieved by
individual strains with individual substrates (Table 1). An
analysis of PHA accumulation over the 48-h growth period
showed PHA was detected at low levels after 6 h of growth
and increased 13-fold by T24. A further two- and 2.5-fold
increase in PHA production was observed over the next 6
and 24 h, respectively (Fig. 2a).
To determine the fate of each of the individual strains in
this mixed culture, samples were taken periodically over the
48-h growth period and analyzed for total colony forming
units (TCFU) on LB as well as CFU for each individual
strain on selected aromatic carbon substrates (see “Materials
and methods”; Fig. 2a). The TCFU/ml increased from 9×
106 to 9×1010 over the first 24 h of growth but did not
increase after this time point. Indeed, TCFU/ml dropped
669
between T30 and T48 despite an increase in PHA
accumulation over this period (Fig. 2a). P. putida F1 is
the predominant strain in this mixed culture for the first
24 h of growth. However, the CFU/ml for this organism
decreased approximately 1,000-fold over the next 24 h. P.
putida mt-2 and CA-3 had identical CFU/ml values
6 h after inoculation. However, P. putida CA-3 outgrew
strain mt-2 and was the predominant organism in the mixed
culture after 30 h of growth. P. putida F1 and mt-2 had
similar CFU/ml at T48 (Fig. 2a).
In order to improve the PHA yield from the BTEXS
substrate, the mixed culture was grown in the fermentor
(5-l working volume). The BTEXS mixture (substrate) was
supplied as a liquid and a nitrogen-feeding strategy that was
previously developed by Goff et al. (2007), was used. A
1.5-fold increase in the level of PHA accumulation and a
three-fold increase in cell dry weight (biomass) were
observed relative to that obtained in the shake flask
experiments (Fig. 2b). During the 48-h fermentation, a
total of 67.5 ml (59.6 g) of BTEXS mixture was utilized,
resulting in the formation of 16.5 g of biomass (cell dry
Fig. 2 PHA accumulation (bars) and mixed culture population
analysis when supplied with a mixture of aromatic hydrocarbons in
a shake flasks and b stirred tank bioreactor (fermentor). Total cfu/ml
(filled diamond), cfu/ml for P. putida CA-3 (filled upright triangle), P.
putida F1 (filled square) and P. putida mt-2 (filled circle). Mixed
cultures were grown for 48 h in MSM mineral medium under
nitrogen-limiting conditions (65 mg N/l). Nitrogen levels (empty
upright triangle) and optical density (OD540, empty diamond) were
monitored during fermentation. All shake flasks experiments are an
average of at least three independent determinations while the values
in bioreactor study are an average of two independent determinations
670
Appl Microbiol Biotechnol (2008) 80:665–673
weight) and 6.02 g of PHA. A similar pattern of strain
distribution (CFU/ml) was observed in the fermentor as for
shake flask cultures (data not shown). PHA accumulation
started when the nitrogen levels reached a concentration of
28 mg/l (T6). PHA accumulation continued throughout the
fermentation during which time the nitrogen concentration
was kept at 2–3 mg/l. PHA levels increased 8.5-fold from 6
to 24 h, with a further 1.8- and 2.1-fold increase over the
subsequent 6 and 18 h of incubation, respectively (Fig. 2b).
Properties of the mcl-PHA accumulated from BTEXS
Using the acetone extraction, about 5 g of mcl-PHA was
obtained from the 15 g of lyophilized cells grown in the
fermentor. The average molecular weight (Mw) and the
polydispersity (Q, degree of distribution) of the polymer
extracted were 86.9 kDa and 3.76, respectively (Table 2).
Polymer is thermo-stable bioplastic with the decomposition
temperature (DT) of 350 °C. The polymer is also partially
crystalline with a melting temperature of 43.95 °C (Table 2).
The rapidly cooled PHA polymer is totally amorphous,
with a glass transition temperature of −48.46 °C.
The 1H NMR spectrum of PHA sample was determined
to establish the structure of the monomers unambiguously
(Fig. 3a). Peak assignments are typical of medium-chainlength PHA derivates (Huijberts et al. 1992). Terminal CH3
protons are detected by the strong resonance at 0.88 ppm.
Another strong signal from the methylene hydrogens
associated with saturated side chains occurs at 1.28 ppm,
whereas those methylenes associated with double bonds are
detected at 2.0 ppm. The resonance at 1.59 ppm is
associated with the methylene protons on C4. Methine
protons are assigned to the quadruplet resonance located at
5.2 ppm. Finally, the weak signal at 5.5 ppm is assigned to
side chains with –CH=CH– moieties (Huijberts et al. 1992).
Chemical shift assignments for 13C spectrum of PHA are
prominently associated with medium-chain-length monomer
structural units of 3-hydroxydodecanoate (C12), 3-hydroxydecanoate (C10) and 3-hydroxyoctanoate (C8; Fig. 3b). The
percentage of unsaturated side chains is estimated to be in
the order of approximately 5% by comparison to the methine
Table 2 Polymer properties of the mcl-PHA accumulated from
benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) by
Pseudomonas putida strains
Mw (Da)
Mn
Q (Mw/Mn)
Tg (°C)
Tm (°C)
DT (°C)
86,990
23,110
3.76
−48.46
43.95
350
All values are the means of two independent determinations.
Mw Molecular weight, Mn molecular number, Q (Mw/Mn) polydispersity, Tg glass transition temperature, Tm melting temperature, DT
decomposition temperature
Fig. 3 Spectra of PHA polymer isolated from mixed P. putida culture
grown on BTEXS in the bioreactor, a 400-MHz 1H NMR spectrum
NMRs; b 13C NMR; c FTIR spectrum
signal intensities (134 to 123 ppm) to those of the methylene
groups (10–40 ppm). GC analysis of the monomer composition also confirmed the presence of the unsaturated
3-hydroxydodecenoate (C12:1; Table 1).
FTIR spectrum of the PHA sample is indicative of the
typical structure of a highly saturated polyester (Fig. 3c).
The spectrum is mainly characterized by the sharp high
Appl Microbiol Biotechnol (2008) 80:665–673
intensity band due to the amorphous stretching vibration of
carbonyl groups (C=O) at about 1739–1743 cm−1 and two
bands at about 1,164 and 1,100 cm−1, due to –C–C–O–
stretching vibrations. A band at 970 cm−1 is attributed to
the bending vibrations of aliphatic C–H. The strong
methylene C–H stretching vibrations were observed at
2,955, 2,928 and 2,856 cm−1. The pronounced vibration
band at about 722 cm−1 is typically associated with –CH2
side chains (Randriamahefa et al. 2003).
Discussion
The objective of this study was to convert petrochemical
aromatic hydrocarbons such as BTEX compounds into
biodegradable PHA by bacterial fermentation. BTEX
compounds are very valuable and relatively inexpensive
petrochemicals with broad uses in synthetic chemistry
(APA 2001). Due to their high usage, they are also major
waste compounds that can give rise to pollution, but are
predominantly burnt for energy (EPA 2001). Petrochemical
aromatic hydrocarbons such as BTEX compounds are also
the major constituents of product of mixed plastic pyrolysis
(Kaminsky and Kim 1999). We are proposing that aromatic
hydrocarbons coming from either of these two waste
streams could also be converted into the valuable polymer
PHA. PHAs are well known to be biodegradable, nontoxic,
biocompatible, elastomeric, and suitable for biomedical
applications (Valappil et al. 2006). Thus, the possibility
exists to convert a mixed petrochemical waste into an
environmentally friendly product as well as add value to the
recycling (pyrolysis) of petrochemical plastics.
From the numerous studies that were carried out on the use
of intrinsic bioremediation processes to manage BTEXcontaminated environments, many organisms (predominantly
Pseudomonas) have been isolated and described (Zylstra
and Gibson 1989; Reardon et al. 2000; Popp et al. 2006).
However, no microbial strain, to date, has been shown to
accumulate PHA from these substrates. Indeed, a limited
number of microorganisms have been reported to accumulate aliphatic mcl-PHA from other aromatic hydrocarbons
(Tobin and O’Connor 2005; Ward et al. 2005). The best
studied and most successful PHA-accumulating bacterium
from an aromatic hydrocarbon (styrene) is P. putida CA-3
which can accumulate mcl-PHA at up to 27% CDW in
shake flasks and 43% of CDW in a bioreactor with a
specific nitrogen-feeding strategy (O’Connor et al. 1995;
Goff et al. 2007). Thus, the level of PHA accumulated by P.
putida F1 (toluene 22%) and mt-2 (p-xylene 26%) is very
promising as higher PHA and biomass can be achieved in a
bioreactor. However, low levels of the PHA accumulation
from benzene and ethylbenzene by P. putida F1 and from
toluene by mt-2 will require further improvement.
671
The investigation of aromatic hydrocarbon biodegradation has often focused on single substrates alone, neglecting
the fact that these compounds rarely occur alone as either a
waste or as a pollutant. Having successfully established that
P. putida F1 and mt-2 were capable of accumulating PHA
when supplied with single aromatic hydrocarbon substrates,
we assessed the ability of these strains to grow and
accumulate PHA when supplied with a mixture of aromatic
hydrocarbons. We used a synthetic mixture of aromatic
hydrocarbons composed of benzene, toluene, ethylbenzene,
xylene, and styrene in the proportions found in the
pyrolysis oil of the mixed plastic waste. Recently, thermal
decomposition of municipal plastic waste (pyrolysis) has
received much attention as part of the efforts for environmentally sound sustainable plastic recycling (Hall and
Williams 2007; Lee 2007). The obtained oil can be burnt
for energy but could also be used as a feedstock for bacteria
to make PHA. We have demonstrated this principle with
polystyrene where pyrolysis combined with bacterial
fermentation has resulted in the production of PHA from
polystyrene (Ward et al. 2006). We included P. putida CA-3
in the mixed culture in the present study as the other two
cultures (F1 and mt-2) are not capable of growth with
styrene. A BTEXS mixture (350 μl; ratio 16:11:1.5:1:15)
was placed in the central glass column in the shake flask
containing 50 ml of liquid medium. The volatile liquid
partitions into the air and subsequently into the liquid
where the bacteria utilize the mixture of compounds as
carbon an energy sources. A mass of 309.5 mg of the
substrate is present in the 350 μl BTEXS mixture. A total
of 51.5 mg of cell dry weight (1.03 mg/ml) was achieved
by the mixed culture supplied with BTEXS. This equates to
a growth yield of approximately 0.17 (grams CDW/grams
substrate supplied). This low figure is most likely explained
by the fact that the volatile substrates absorb onto the cotton
wool seal in the flask and, thus, not all the substrate is
available to the bacteria. This was also observed in previous
studies with P. putida CA-3 supplied with styrene in the
same way (Ward et al. 2005). The supply of volatile
substrates in a bioreactor generates much high cell growth
yields due to much more efficient feeding of the substrate to
the liquid medium (Ward et al. 2005; Goff et al. 2007) and,
thus, we predicted higher cell growth yields when these
strains are grown in a bioreactor.
In the bioreactor study, when the mixed P. putida-mixed
culture was supplied with the BTEXS mixture as a liquid
(fed-batch), we achieved 1.5-fold improvement in the PHA
yield (percent cell dry weight) which was comparable to
our previous study of PHA production from the polystyrene
pyrolysis oil (Ward et al. 2006). Furthermore, we also
achieved a three-fold increase in the biomass yield, which
resulted in 4.5-fold overall PHA productivity increase (from
0.25 to 1.16 g/l). The growth yield of 0.27 (grams CDW/
672
grams substrate supplied), represents a 1.6-fold improvement in comparison to shake flask experiments. Further
improvements will be achieved when we optimize the feed
of BTEXS as a liquid to the culture medium in the
fermentor.
The monomer composition of PHA accumulated by
all strains was very similar (Table 1). The monomer
composition of the PHA did not change when the strains
were switched from growth on a single substrate to the
aromatic mixture. This is advantageous for the further
process development and for the future application of this
technology to different substrate mixtures. PHA accumulation from aromatic hydrocarbons proceeds through the
fatty acid biosynthetic pathway where 3-hydroxyacylCoA intermediates serve as the precursors for the PHA
synthesis (Rehm et al. 1998). It has been shown by Rehm
et al. that the enzyme responsible for transferring (R)-3hydroxyacyl-ACP’s to their corresponding CoA equivalents has a preference for 3-hydroxydecanoyl units
(Rehm et al. 1998). It appears that from the present study
and other studies that this preference for ten carbon units
predominates in Pseudomonas species (Tobin et al.
2007).
The mcl-PHA polymer obtained from the cells grown in
the fermentor is a partially crystalline bioplastic, as
evidenced by the presence of a melting peak (Table 2). It
is interesting to note that for PHA samples quenched from
the melt with rapid cooling, a residual crystalline endothermic
peak is detected during the subsequent heating cycle. This was
not present in PHA accumulated by P. putida CA-3 grown on
styrene alone (Ward et al. 2005; Ward and O’Connor 2005).
The low Tg of the polymer may be attributed to internal
plasticizer effect of randomly placed side chains. However,
the molecular weight of the PHA accumulated from BTEXS,
although comparable to that obtained from styrene, is
relatively low and the polydispersity is relatively high
(Table 2), when compared to other mcl-PHAs or synthetic
polyesters (Preusting et al. 1990; van der Walle et al. 2001).
We shall attempt, in the future, to improve the polydispersity
value through optimization of the PHA extraction method as
well as examining fermentation parameters.
In conclusion, we have demonstrated the conversion of
petrochemical aromatic hydrocarbons (BTEXS) to mclPHA by Pseudomonas species either as single strains or
defined mixed cultures. Furthermore, we have demonstrated
that supply of the BTEXS through liquid feeding in a
fermentor improved growth yield and PHA accumulation
rates compared to shake flask experiments. As BTEXS are
the major components of the oil generated from pyrolysis of
mixed plastic, the conversion of mixed plastic waste to value
added mcl-PHA is now possible.
Appl Microbiol Biotechnol (2008) 80:665–673
Acknowledgments This research was funded by the Environmental
Protection Agency (EPA) of Ireland (Project Code 2005-ET-LS-9-M3).
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