Process Biochemistry 49 (2014) 14091414

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Short communication

Production of polyhydroxyalkanoates using hydrolysate of spent

coffee grounds
Stanislav Obruca a, , Pavla Benesova a , Sinisa Petrik a , Jana Oborna b ,
Radek Prikryl c , Ivana Marova a
a

Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic
Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno,
Czech Republic
c
Institute of Material Chemistry, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic
b

a r t i c l e

i n f o

Article history:
Received 24 February 2014
Received in revised form 20 May 2014
Accepted 21 May 2014
Available online 29 May 2014
Keywords:
Spent coffee grounds
Polyhydroxyalkanoates
PHA
Burkholderia cepacia
Detoxication

a b s t r a c t
Spent coffee grounds (SCG) are solid fraction wastes deriving from coffee industries, the disposal of which
represents a serious environmental issue. This work aims at the conversion of hydrolysate of SCG (SCGH)
into polyhydroxyalkanoates (PHA) by Burkholderia cepacia. The bacteria was capable of SCGH utilization
and production of copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(HB-co-HV]. Levulinic acid
present in SCGH probably served as the precursor of 3HV for the copolymer biosynthesis. To improve
the PHA yields, various detoxication methods were tested. The extraction of polyphenols from SCG by
ethanol prior to the hydrolysis seems to be the most promising, since, apart from the fact that it enhanced
the PHA yields by about 25%, polyphenols extracted from SCG may represent important side products,
because they might be used for the production of functional foods and other high value products.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Polyhydroxyalkanoates (PHA) are polyesters synthesized and
stored in bacterial cells in the form of intracellular granules. Bacteria use PHA as carbon, energy and reducing power storage
materials. PHA have attracted much attention as biodegradable
alternative to traditional petrochemical plastics, their properties
and potential applications were extensively reviewed by Philip
et al. [1].
Among PHA, the homopolymer of 3-hydroxybutyrate, poly(3hydroxybutyrate) (PHB), is the most abundant and the best
characterized polymer to date. However, PHB possesses several
properties, such as high crystallinity, melting point temperature
and low exibility, which complicate its processing and also limit
the range of its application. The mechanical properties of this
polymer can be signicantly improved by the incorporation of
3-hydroxyvalerate (3HV) units into the PHA structure resulting in
the formation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
[P(HB-co-HV)] with better mechanical properties and
processability [2].

Corresponding author. Tel.: +420 541 149 486; fax: +420 541 211 697.
E-mail address: Stana.O@seznam.cz (S. Obruca).
http://dx.doi.org/10.1016/j.procbio.2014.05.013
1359-5113/ 2014 Elsevier Ltd. All rights reserved.

Since about 45% of the total costs of PHA production are ascribed
to carbon sources, such as rened glucose or sucrose [3], cheap
wastes or side products of agriculture and food industry are used
as inexpensive carbon substrates improving thus the economic
feasibility of the PHA production [4]. Moreover, cellulosic and hemicellulosic biomass are considered being very promising renewable
sources for the biotechnological production of fuels and chemicals,
including PHA [5]. Therefore, the PHA production from Burkholderia
cepacia, Burkholderia sacchari [6] and Ralstonia eutropha [7] has been
reported by utilizing sugarcane bagase hydrolysate. More recently,
Pan and his colleagues studied the PHA production from detoxied
sugar maple hemicellulosic hydrolysate by B. cepacia [8,9], Zhang
et al. utilized oil palm empty fruit bunch for the PHB production
using Bacillus megatrerium [10] and B. sacchari was employed for
the PHA production from wheat straw hydrolysate [11].
Coffee is one of the world most popular beverages and has
grown steadily in commercial importance during the last 150 years.
Nowadays, coffee is, after petroleum, the second largest traded
commodity in the world. In 2010 the worldwide annual production
of coffee beans exceeded 8 million tons [12]. During the preparation
of coffee beverage or the manufacturing of instant coffee, raw coffee powder is mixed with hot water or steam under the conditions
of favoring the release of aroma compounds and other coffee-bean
constituents into the liquid, which generates solid residues known

1410

S. Obruca et al. / Process Biochemistry 49 (2014) 14091414

as spent coffee grounds (SCG). On an average, the manufacturing of

one ton of green coffee generates about 650 kg of SCG [12]. Despite
the fact that this waste stream of coffee industry has been considered as a raw substrate for the production of various compounds
such as polyphenols [13,14], bioethanol [15,16], biodiesel [16,17]
or mannooligosaccharides [18], the most of this residue remains
unutilized, being discharged to the environment or burned for
elimination, which can be considered neither as environmentally
friendly nor economically feasible techniques [12].
We have recently investigated the utilization of oil extracted
from SCG for the PHA production [19]. Hence, we aimed at the
valorization of residual SCG after the oil extraction. To our best
knowledge, there is no report on the utilization of cellulosic
biomass of SCG for the biosynthesis of polyhydroxyalkanoates.
Therefore, in the present work, the ability of B. cepacia to produce
PHA from SCG was tested. Moreover, various detoxication methods were used to improve the fermentability of SCG based media
and thus, the PHA yields obtained on this substrate.

Table 1
Composition of SCGH used in this study (150 g SCG per liter).
Parameter

Concentration/value

Dry solid matter (g l1 )

Ash (g l1 )
Conductivity (mS cm1 )
PO4 3 (g l1 )
Total nitrogen (wt.%)
Proteins (N 6.25) (g l1 )
Polyphenols (g l1 )
Levulinic acid (g l1 )
5-Hydroxymethyl furfural (g l1 )
Furfural (g l1 )
Total sugars (g l1 )
Cellobiose (g l1 )
Galactose (g l1 )
Mannose (g l1 )
Arabinose (g l1 )
Glucose (g l1 )

98.4 0.1
27.6 0.8
23.2
0.32 0.03
0.162 0.1
10.2 0.6
3.6 0.1
1.72 0.19
0.15 0.03
n.d.
50.1 2.2
2.7 0.3
17.3 0.3
23.6 0.6
2.8 0.2
3.9 0.2

Results are in form average standard deviation.

n.d. not detected.

2. Materials and methods

2.4. PHA analysis

2.1. Raw material preparation

The biomass concentration was analyzed gravimetrically, the samples were centrifuged and the cells were washed with 5% (vol/vol) Triton X (10 ml) and distilled
water, respectively, and afterwards they were dried (105 C) to the constant weight.
The PHB content of dried cells was analyzed by gas chromatography (Trace GC Ultra,
Thermo Scientic, USA) as reported by Brandl et al. [23]. Commercially available
P(HB-co-HV) (SigmaAldrich, Germany, HV content 12 mol.%) was used as a standard; benzoic acid was used as an internal standard. The molecular weight of PHB
was determined by gel permeation chromatography (Agilent 1100 Series; column
PLgel Mixed B (300 9 7.5 mm; 10 lm)).
The Differential Scanning Calorimetry (DSC) (204 F1, Netzsch) was used for
determination of thermal properties of produced material as described elsewhere
[2]. Thermogravimetric apparatus (TGA) (Q500; TA Instruments) was used for evaluation of thermal stability of the PHB.

Spent coffee grounds (SCG) were obtained from a coffee automat machine at
the Faculty of Chemistry, Brno University of Technology, Czech Republic. The waste
material was rstly dried to the constant weight (80 C for 24 h). The coffee oil
extraction was performed with n-hexane as described by Al-Hamamre et al. [17]. To
hydrolyze the hemicelluloses of raw material, 15% (w/v) SCG were rstly treated by
1 vol.% H2 SO4 for 90 min at 121 C [16]. In the next step, the enzymatic digestion was
used to break up cellulose releasing fermentable saccharides. Therefore, pH of the
suspension after the acidic hydrolysis was set to 4.5 (10 M NaOH) and cellulose was
treated by 0.5% (vol/vol) of Celluclast 1.5 L (Novozymes A/S, Bagsvrd, Denmark) at
50 C under permanent shaking (150 rpm) for 24 h [16]. At the end of the process
the solids obtained after enzyme treatments (representing approx. 30% of SCG after
oil extraction) were removed by ltration and the permeate, called spent coffee
grounds hydrolyzate (SCGH), was used for the cultivation media preparation and
the PHA production.

2.2. Analysis of SCGH composition

The concentration of dry matter was estimated by drying 10 ml of SCGH to constant weight at 105 C. Ash content was determined as the weight of solids obtained
after the incubation of 2 ml of SCG-media at 800 C for 2 h. Subsequently, the phosphorus content of ash was measured by Ion Chromatography (850 Professional IC,
Metrohm, Switzerland) using Metrosep A Supp 7-250/4.0 column. Particular sugars
contents (cellobiose, glucose, mannose, galactose and arabinose) were estimated by
liquid chromatography (pump LCP 4020, thermostat LCO 101, degasser DG-1210,
refractometric detector RIDK 102; Ecom, Czech Republic) with REZEX-ROA column
(150 mm 4.6 mm, 5 m; Phenomex, USA). The determination of total sugars by
Somogyi-Nelson method was employed for routine analysis of sugars. The concentrations of levulinic acid, 5-hydroxymethyfurfural and furfural were determined
by gas chromatography equipped with ame ionization detector (GC-FID, Trace GC
Ultra, Thermo Scientic) according to Omari et al. [20]. The concentration of nitrogen compounds was determined by Kjehdal method and the overall protein content
was calculated using the conversion factor of 6.25. The total phenolics content was
estimated using the FolinCiocalteu reagent method [21]. All analyses were performed at least in triplicate and the results were evaluated as mean values with SD
values.

2.3. Microorganisms and cultivations

B. cepacia CCM 2656 (ATCC 17759) was purchased from the Czech Collection
of Microorganisms. The bacterial culture was cultivated in mineral salt medium
described by Bertrand et al. [22]. SCGH, salt solutions and microelement solutions
were autoclaved separately (115 C, 20 min) and then aseptically reconstituted at
room temperature prior to the inoculation to reach desired portion of SCGH in
the medium and nal concentration of salts. The cultivations were performed in
Erlenmeyer asks (volume 250 ml) containing 50 ml of the cultivation media. The
temperature was set to 30 C and the agitation to 180 rpm. After 72 h of cultivation, the cells were harvested (centrifugation, 8000 g, 5 min) and the biomass as
well as the PHB yields were determined as described below. All cultivations were
performed in triplicate.

2.5. Detoxication of SCGH

Several methods which should reduce the amount of toxic substances in SCGH
were tested in this study. Polyphenols were extracted from SCG prior to its hydrolysis using various mixtures of distilled water and ethanol (0, 30, 45 and 60 vol.% of
ethanol). The polyphenol extraction from 20 g of SCG was performed using 40 ml
of particular solvent for 2 h at 50 C under constant shaking (150 rpm). After the
extraction, SCG was ltered, dried and hydrolyzed as described above. The control
was prepared identically; however, SCG was not subjected to the extraction.
Further, the overliming was performed with SCGH (after hydrolysis) as
described by [8], also the activated charcoal (AC) detoxication was employed; AC
was mixed with SCGH in the ratio of 1:20 (w/v) and stirred for 1 h at 60 C [8].

3. Results and discussion

3.1. Composition of SCGH
To hydrolyze SCG, the combination of diluted acid hydrolysis
(1% H2 SO4 ) of hemicelluloses and enzymatic execution of cellulose was applied. This approach yielded liquid hydrolysate of SCG
(SCGH) the composition of which is shown in Table 1. The total
concentration of sugars was determined to be 50.1 g l1 (by the
hydrolysis of 150 g l1 of SCG), the major sugars identied in SCGH
are mannose (23.6 g l1 ) and galactose (17.3 g l1 ) followed by glucose (3.9 g l1 ), arabinose (2.8 g l1 ) and cellobiose (2.7 g l1 ). Unlike
in other hemicellulose hydrolysates such as wheat straw [13] or
maple hemicellulosic hydrolysate [10] containing signicant portion of pentoses, SCGH contains only low portion of arabinose. The
fact that hexoses are substantially dominating sugars of SCGH may
be an important factor positively inuencing the PHA production.
The metabolic pathways of hexoses utilization generates larger
amount of energy, which might, in consequence, enhance the PHB
yields. Lopes et al. reported the theoretical ATP/3HB monomer ratio
being 3 mol/mol in case of xylose compared to the value of 7 mol
ATP/mol 3HB in glucose [24].

S. Obruca et al. / Process Biochemistry 49 (2014) 14091414

1411

The analysis also focused on chemical compounds which might

complicate the fermentability of SCGH. SCGH contains signicant
portion of ash (27.6 g l1 ) which may be attributed to the application of H2 SO4 during acidic hydrolysis and NaOH for subsequent
neutralization of SCGH. High concentration of salts may cause the
inhibition of bacterial growth due to the induction of osmotic stress.
On the contrary, it was observed that mild osmotic stress may support PHA biosynthesis [25]. Moreover, SCGH contains also high
amounts of potentially toxic substances such as polyphenols, 5hydromethylfurfural (5HMF) and levulinic acid (LA). 5HMF and LA
are the products of hexoses degradation under high temperature
and acidic conditions [20].
3.2. PHB production from SCGH employing B. cepacia
B. cepacia ATCC 17759 is a gram-negative bacterium which is
generally considered as one of the most promising candidates for
the PHA production from hemicellose and cellulose hydrolysates
because, apart from the fact that it is able to utilize various hexoses and pentoses, it also tolerates antimicrobial substance [6,8,9].
Therefore, B. cepacia was employed for the PHA production from
SCGH.
At rst, B. cepacia was cultivated in media with various portions
of SCGH for 72 h. The results are provided in Table 2. The highest
biomass and also PHA yields (4.91 and 2.69 g l1 ) were achieved
when the medium contained 30 vol.% of SCGH. Lower portion of
SCGH probably did not provide sufcient amount of the substrates,
on the contrary, higher amount of SCGH in the cultivation media
revealed rather inhibition effect on the biomass as well as PHB
yields. LA and 5HMF are not present in SCGH in the amounts which
could result in strong inhibition of the cell growth because it was
reported that the growth of B. cepacia was not affected by LA up
to 5 g l1 and by 5HMF up to 1.0 g l1 [8], which is far above their
concentration in SCGH. Oppositely, polyphenolics are in SCGH in
the concentration (3.6 g l1 ) that can cause toxic effect, because
Pan et al. [8] reported than the growth of B. cepacia was strongly
inhibited by the presence of 2.0 g l1 of polyphenol vanillin. Moreover, in their further publication Pan et al. observed that synergistic
inhibitory effects occurred in the presence of multiple inhibitors
when B. cepacia was grown on wood hydrolysate [9]. This may be
an explanation for the B. cepacia growth inhibition in the presence
of higher amount of SCGH and why simple dilution may solve the
problems accompanying the presence of inhibitors in SCGH. Since
all the production parameters including yield coefcient YP/S were
the highest when the SCGH portion in media was set at 30 vol.%,
this dilution of SCGH was used as optimal in further experiments.
Furthermore, B. cepacia was capable of accumulation of copolymer P(HB-co-HV). It is likely that LA present in SCGH, which was
already reported to be transformed into 3HV by B. cepacia [26],
served as the primary precursor of 3HV. The portion of 3HV in
copolymer increased with the amount of SCGH in the cultivation
media, which may be attributed to rising concentration of the 3HV
precursors.

Fig. 1. Time course of biomass, PHA and particular sugars concentrations during
cultivation of B. cepacia on SCGH.

3.3. Time course of PHA production by B. cepacia on SCGH

In order to get deeper insight into the process of PHA production from SCGH by B. cepacia, the biomass, PHB yields, individual
sugar concentrations and other parameters were recorded during
the cultivation, the results of the experiments are shown in Fig. 1.
During the time course of cultivation, the biomass concentration
was steadily growing accompanied by the increase of PHA content in the biomass. The cultivation was nished after 77 h, at this
cultivation time the biomass concentration, the PHA content in
the biomass and the PHA yields reached 4.90 g l1 , 57.26 wt.% and
2.81 g l1 , respectively.

LA was completely exhausted during initial 48 h of cultivation,

which corresponded with the development of ratio of 3HB to 3HV
in copolymer, because the portion of 3HV in copolymer decreased
during the course of cultivation. Therefore, it is likely that at the
beginning of the cultivation the copolymer with high portion of 3HV
was synthetized, the amount of 3HV decreased with the reduction
of levulinic acid level and, once the LA was completely utilized, the
bacterial culture synthetized PHB homopolymer. This explanation
is in accordance with the results of DSC study, which suggested,
that at the 77th h of cultivation the polymer was a blend of PHB
homopolymer and P(HB-co-HV) copolymer (see below).

1412

S. Obruca et al. / Process Biochemistry 49 (2014) 14091414

Table 2
Production of PHB from SCGH employing B. cepacia.
SCGH portion in media (vol.%)

CDW (g l1 )

15
30
50
65

3.24
4.91
2.78
2.71

0.34
0.10
0.27
0.11

PHA (wt.%)
25.36
54.79
34.04
29.74

PHA (g l1 )

1.61
1.23
0.10
3.21

0.82
2.69
0.94
0.81

3HV (mol.%)

0.10
0.07
0.08
0.09

4.5
5.0
8.7
9.4

0.1
0.2
0.1
0.4

Sugars consumed (g l1 )

YP/S

7.52
11.93
16.85
18.37

0.11
0.23
0.06
0.04

Results are in form average standard deviation.

CDW stands for cell dry weight.
3HV represents content of 3-hydroxyvalerate in P(HB-co-HV) copolymer.

The carbon catabolite repression (CCR) represents a serious

problem when dealing with lignocellulosic hydrolysates as fermentation substrates. CCR is caused by preferential consumption of one
of the sugars, which may in turn result in the accumulation of other
sugars (usually pentoses) to potentially inhibiting concentrations
when the fed-batch cultivation strategy is applied [11]. The growth
of B. cepacia on SCGH was accompanied by the utilization of sugars
present in SCGH. All the sugar of SCGH were consumed gradually
during the whole course of cultivation, however, glucose and cellobiose were exhausted as the rst (Fig. 1C). Since SCGH contains
only low portion of pentose, in particular arabinose, and; moreover,
this sugar is consumed along with mannose and galactose, CCR
should not occur during the PHA production from SCGH employing
B. cepacia.
When the cultivation was nished, polymer was extracted from
the cells to study its properties thoroughly. The molecular weight
(Mw ) and the polydispersity index of the polymer were determined
to be 2.62 105 and 3.33, respectively. The DSC study revealed
two peaks corresponding to melting temperatures of the polymer:
170.2 and 162.3 C (see Supplementary materials). This indicates
that the polymer might be a blend of P(HB-co-HV) (lower melting
temperature) and homopolymer PHB (higher melting temperature)
[2]. The TGA analysis shown that polymer started to degrade and
lose weight at a temperature of about 230 C, the maximum weightloss rate was determined as 285.3 C.

methods on total sugars and inhibitors concentrations in SCGH is

shown in Table 3.
The application of post-hydrolysis detoxication methods
resulted in signicant removal of polyphenols where the introduction of activated charcoal seems to be more efcient than the
overliming. However, both approaches also caused signicant loss
of fermentable sugars (from 19.3% to 34.6%), which is undesirable
side effect of the detoxication procedure. On the other hand, when
polyphenols were extracted from SCG prior to the hydrolysis, no
signicant elimination of sugars was observed. The efciency of
removal of phenolic compounds increased with rising concentration of ethanol in the extraction mixture from 18.3% to 36.9%. All the
detoxication methods tested in this work reduced also the amount
of LA (from 5.2% to 64.8%) and 5HMF (from 10.2% to 84.3%).
In order to further investigate the effect of detoxication methods on the PHA production from SCGH, the set of shake-ask
experiments was performed using non-detoxied SCGH as a control, the results are provided in Table 4. Among the detoxication
methods tested, the extraction of polyphenols from SCG by 30%
ethanol prior to their hydrolysis seems to be the most efcient,
because this approach improved the PHA yields more than about
25% in comparison with the control culture. Aside from the positive
effect of phenolic compounds extraction on the PHA production
from SCG prior its hydrolysis, this detoxication strategy offers
also additional advantage, because extracted phenolic compounds
can represent important side-product of the intended technology.
SCG contain large amounts of chlorogenic acid and its derivatives collectively referred to as chlorogenic acids which provide
many health benets to humans [27]. For instance the pharmacokinetic studies demonstrated that about one-third of ingested
chlorogenic acids are absorbed in the stomach and proximal
duodenum, while the remaining are cleaved by gut microora
into caffeic and quinic acids [28]. Interestingly, the latter compounds have recently been shown to possess the neurotrophic
and neuroprotective properties [29,30]. Hence, phenolics extracted
from SCG prior to its hydrolysis can be used for the production of high value products such as functional foods or dietary
supplements.
The product yield coefcient YP/S obtained in this study for PHA
production on SCGH employing B. cepacia reached 0.27, which is

3.4. Detoxication of SCGH

Several detoxication methods were applied to improve the fermentability of SCGH. At rst, phenolic compounds can be extracted
from SCG prior to their hydrolysis which should reduce their
amount in SCGH. Zuoro and Lavecchia reported that coffee phenolics could be extracted by the mixture of water and ethanol [13];
therefore several concentrations of ethanol were tested. Furthermore, apart from the extraction of polyphenols prior to hydrolysis,
also the methods of SCGH detoxication after the hydrolysis were
tested. The overliming (OL) can be effective due to the precipitation or chemical destabilization of inhibitors and activated charcoal
(AC) can improve the fermentability of SCGH by the absorbtion of
phenolic compounds [8]. The inuence of tested detoxication

Table 3
Effect of different SCGH detoxication methods on concentration of total sugars and microbial inhibitors.
Total sugars
1

Control
EtOH 0%
EtOH 30%
EtOH 45%
EtOH 60%
AC
OL

c (g l

49.4
53.0
52.6
47.3
46.0
39.9
34.6

4.9
1.3
0.6
8.7
0.2
2.1
1.1

Polyphenols
1

E* (%)

c (g l

7.4
6.6
4.4
6.9
19.3
30.0

3.68
3.01
2.84
2.41
2.32
1.66
2.96

0.03
0.04
0.11
0.01
0.06
0.02
0.06

Results are in form average standard deviation.

E* stands for elimination of particular substance in comparison with control in %.

Levulinic acid
1

E* (%)

c (g l

18.3
22.7
34.5
36.9
55.0
19.7

1.58
0.56
0.79
0.57
0.59
0.63
1.49

0.13
0.03
0.05
0.02
0.07
0.20
0.36

5HMF
E* (%)

c (g l1 )

64.8
49.9
64.0
62.8
60.3
5.2

0.147
0.109
0.114
0.090
0.132
0.070
0.023

E* (%)
0.060
0.002
0.019
0.066
0.001
0.004
0.000

25.9
22.5
38.9
10.2
52.6
84.3

S. Obruca et al. / Process Biochemistry 49 (2014) 14091414

1413

Table 4
PHA production by B. cepacia on detoxied SCGH.
CDW (g l1 )
Control
EtOH 0%
EtOH 30%
EtOH 45%
EtOH 60%
AC
OL

5.42
4.86
5.49
5.20
5.02
4.78
7.40

0.48
0.23
0.40
0.32
0.32
0.34
0.47

PHA (wt.%)
49.08
52.37
56.01
49.99
51.33
56.79
23.47

0.70
2.47
1.79
3.20
4.80
0.00
6.56

PHA (g l1 )
2.66
2.54
3.08
2.60
2.58
2.72
1.74

3HV (mol.%)

0.24
0.17
0.24
0.23
0.29
0.19
0.50

8.1
5.6
4.2
6.0
5.1
6.1
11.6

0.8
0.4
0.2
0.7
0.6
0.5
0.5

Sugars consumed (g l1 )

YP/S

9.77
12.83
12.97
10.83
10.45
10.45
6.77

0.27
0.20
0.24
0.24
0.25
0.26
0.26

Results are in form average standard deviation.

CDW stands for cell dry weight.
3HV represents content of 3-hydroxyvalerate in P(HB-co-HV) copolymer.
SCGH portion in cultivation media was set to be 30 vol.%.

more than comparable to data reported for PHA production on

other lignocellulose hydrolysates such as bagasse hydrolysate using
B. cepacia (0.23) [6] or R. eutropha (0.08) [7]. Similar results were
also obtained on oil palm empty fruit bunch by Bacillus megaterium
(0.27) [10] and on wood hydrolysate by B. cepacia (0.19) [8].
Thus, this study suggested a novel promising waste substrate
for the PHA production spent coffee grounds. World-wide coffee
production has been steadily growing and it is accompanied by the
production of waste-products, especially SCG. Therefore SCG are
available in millions of tons, because, on an average, the processing of one ton of green coffee generates about 650 kg of SCG [13].
Hence, there is high amount of potential substrate for the PHA production from SCG, especially in coffee producing countries. On the
other hand, also Europe belongs among regions with strong coffee industry and high per capita consumption [31]. Furthermore,
SCG are highly pollutant due to the presence of organic material
that requires a great quantity of oxygen in order to degrade. Being
discharged to the environment, they can cause contamination and
environmental pollution problems and, furthermore, simply piled
up, they can ferment and produce spontaneous combustion [15].
Therefore, the conversion of SCG into PHA by B. cepacia can represent theoretically economically feasible process (due to low cost
of entering substrate); and, moreover, also ecologically friendly
approach to the elimination of potentially problematic wastes.
Acknowledgement
This work was supported by project Centre for Materials
Research at FCH BUT No. CZ.1.05/2.1.00/01.0012 from European
Regional Development Fund (ERFD) and by the project Excellent young researcher at BUT No. CZ.1.07./2.3.00/30.0039. Authors
kindly thank to Dr. Tomas Opravil (Brno University of Technology,
Czech Republic) for collection of spent coffee grounds.

[6]

[7]
[8]

[9]

[10]

[11]

[12]
[13]
[14]

[15]

[16]
[17]

[18]

[19]

[20]

Appendix A. Supplementary data

[21]

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.procbio.2014.05.013.

[22]

References

[23]

[1] Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers

with a range of applications. J Chem Technol Biotechnol 2007;82:23347.
[2] Lee WH, Loo CY, Nomura CT, Sudesh K. Biosynthesis of polyhydroxyalkanoate
copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors.
Bioresour Technol 2008;99:684451.
[3] Choi J, Lee SY. Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl Microbiol Biotechnol 1999;51:1221.
[4] Koller M, Bona R, Braunegg G, Hermann C, Horvat P, Kroutil M, et al. Production of polyhydroxyalkanoates from agricultural waste and surplus materials.
Biomacromolecules 2006;6:5615.
[5] Wyman CE, Decker SR, Himmel ME, Brady JW, Skopec CE, Viikari L. Hydrolysis of
cellulose and hemicellulose. In: Dumitriu S, editor. Polysaccharides: structural

[24]

[25]

[26]

[27]

diversity and functional versatility. second ed. New York: CRC Press; 2004. p.
9951034.
Silva LF, Taciro MK, Ramos MEM, Carter JM, Pradella JCG, Gomez JGC. Poly3-hyroxybutyrate (P3HB) production by bacteria from xylose, glucose and
sugarcane bagasse hydrolysate. J Ind Microbiol Biotechnol 2004;31:24554.
Yu J, Stahl H. Microbial utilization and biopolyester synthesis of bagasse
hydrolysates. Bioresour Technol 2008;99:80428.
Pan W, Perrotta JA, Stipanovic AJ, Nomura CT, Nakas JP. Production of
polyhydroxyalkanoates by Burholderia cepacia ATCC 17759 using detoxied
sugar maple hemicellulosic hydrolysate. J Ind Microbiol Biotechnol 2012;39:
45969.
Pan W, Nomura CT, Nakas JP. Estimation of inhibitory effects of hemicellulosic wood hydrolysate inhibitors on PHA production by Burkholderia
cepacia ATCC 17759 using response surface methodology. Bioresour Technol
2012;125:27582.
Zhang Y, Sun W, Wang H, Geng A. Polyhydroxybutyrate production from oil
palm empty fruit bunch using Bacillus megaterium R11. Bioresour Technol
2013;147:30714.
Cesario MT, Raposo RS, de Almeida MCMD, van Keulen F, Ferreira BS, de Fonseca
MMR. Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw
lignocellulosic hydrolysates. New Biotechnol 2014;31:10413.
Murthy PS, Naidu MM. Sustainable management of coffee industry by-products
and value addition a review. Resour Conserv Recycl 2012;66:4558.
Zuorro A, Lavecchia R. Spent coffee grounds as a valuable source of phenolic
compounds and bioenergy. J Clean Prod 2012;34:4956.
Machado EMS, Rodriguez-Jasso RM, Teixeira JA, Mussatto SI. Growth of fungal
strains on coffee industry residues with removal of polyphenolic compounds.
Biochem Eng J 2012;60:8790.
Mussatto SI, Machado EMS, Carneiro LM, Teixeira JA. Sugars metabolism and
ethanol production by different yeast strains from coffee industry wastes
hydrolysates. Appl Energy 2012;92:7638.
Kwon EE, Yi H, Jeon YJ. Sequential co-production of biodiesel and bioethanol
with spent coffee grounds. Bioresour Technol 2013;136:47580.
Al-Hamamre Z, Foerster S, Hartmann F, Kroger M, Kaltschmitt M. Oil extracted
from spent coffee ground as a renewable source for fatty acid methyl ester
manufacturing. Fuel 2012;96:706.
Asano I, Umemura M, Fujii S, Hoshino H, Iino H. Effects of mannooligosaccharides from coffee mannan on fecal microora and defecation in healthy
volunteers. Food Sci Technol Res 2004;10:937.
Obruca S, Petrik S, Benesova P, Svoboda Z, Eremka L, Marova I. Utilization of oil extracted from spent coffee grounds for sustainable
production of polyhydroxyalkanoates. Appl Microbiol Biotechnol 2014,
http://dx.doi.org/10.1007/s00253-014-5653-3 (in press).
Omari KW, Besaw JE, Kerton FM. Hydrolysis of chitosan to yield levulinic acid
and 5-hydroxymethylfurfural in water under microwave irradiation. Green
Chem 2012;14:14807.
Marova I, Parilova K, Friedl Z, Obruca S, Duronova K. Analysis of phenolic
compounds in lager beers of different origin: a contribution to potential determination of the authenticity of Czech beer. Chromatographia 2011;73:S8395.
Bertrand JL, Ramsay BA, Ramsay JA, Chavarie C. Biosynthesis of poly-phydroxyalkanoates from pentoses by Pseudomonas pseudoava. Appl Environ
Microb 1990;56:31338.
Brandl H, Gross RA, Lenz RW, Fuller RC. Pseudomonas oleovorans as a source
of poly(beta-hydroxyalkanoates) for potential application as a biodegradable
polyester. Appl Environ Microb 1988;54:197782.
Lopes M, Rocha R, Zanotto S, Gomez J, Silva L. Screening of bacteria to
produce polyhydroxyalkanoates from xylose. World J Microbiol Biotechnol
2009;25:17516.
Obruca S, Marova I, Svoboda Z, Mikulikova R. Use of controlled exogenous
stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus
necator. Folia Microbiol 2010;55:1722.
Keenan TM, Tanenbaum SW, Stipanovic AJ, Nakas JP. Production and characterization of poly-beta-hydroxyalkanoate copolymers from Burkholderia cepacia
utilizing xylose and levulinic acid. Biotechnol Prog 2004;20:1697704.
Brezova V, Slebodova A, Stasko A. Coffee as a source of antioxidants: an EPR
study. Food Chem 2009;114:85968.

1414

S. Obruca et al. / Process Biochemistry 49 (2014) 14091414

[28] Stalmach A, Steiling H, Williamson G, Crozier A. Bioavailability of chlorogenic

acids following acute ingestion of coffee by humans with an ileostomy. Arch
Biochem Biophys 2010;501:98105.
[29] Sul DG, Kim HS, Lee DH, Joo SS, Hwang KW, Park SY. Protective effect of caffeic
acid against beta-amyloid-induced neurotoxicity by the inhibition of calcium
inux and tau phosphorylation. Life Sci 2009;84:25762.

[30] Jeong CH, Jeong HR, Choi GN, Kim DO, Lee U, Heo HJ. Neuroprotective and
anti-oxidant effects of caffeic acid isolated from Erigeron annuus leaf. Chin Med
2011;24:625.
[31] European Coffee Report. European Coffee Federation; 2012/2013. Available online at http://www.ecf-coffee.org/publications