Bioresource Technology 133 (2013) 3844

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Valorization of glycerol through the production of biopolymers: The PHB

case using Bacillus megaterium
Javier M. Naranjo, John A. Posada, Juan C. Higuita, Carlos A. Cardona
Instituto de Biotecnologa y Agroindustria, Departamento de Ingeniera Qumica, Universidad Nacional de Colombia sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia

h i g h l i g h t s

" Polyhydroxybutyrate production from glycerol with Bacillus megaterium was evaluated.
" Polyhydroxybutyrate production at industrial conditions using glycerol was simulated.
" Polyhydroxybutyrate production costs and glycerol valorization were assessed.

a r t i c l e i n f o a b s t r a c t

Article history: In this work technical and economic analyses were performed to evaluate the glycerol transformation
Received 12 December 2012 into Polyhydroxybutyrate using Bacillus megaterium. The production of PHB was compared using glycerol
Received in revised form 22 January 2013 or glucose as substrates and similar yields were obtained. The total production costs for PHB generation
Accepted 23 January 2013
with both substrates were estimated at an industrial scale. Compared to glucose, glycerol showed a 10%
Available online 30 January 2013
and 20% decrease in the PHB production costs using two different separation schemes respectively. More-
over, a 20% prot margin in the PHB sales price using glycerol as substrate resulted in a 166% valorization
Keywords:
of crude glycerol. In this work, the feasibility of glycerol as feedstock for the production of PHB at labo-
Bacillus megaterium
Glycerol
ratory (up to 60% PHB accumulation) and industrial (2.6US$/kgPHB) scales is demonstrated.
Adaptation 2013 Elsevier Ltd. All rights reserved.
PHB bioproduction
Simulation process

1. Introduction glycerol. PHAs are homo or heteropolyesters and can be synthe-

Crude glycerol is generated as a co-product in the production of
biodiesel. For every 100 lbs of biodiesel produced, 10 lbs of crude
glycerol are generated (10% weight) (Yazdani and Gonzalez,
2007). Although pure glycerol is an important industrial feedstock
used in food, drugs, cosmetics, pharmaceuticals, pulp and paper,
leather, textile and tobacco industries, the growth of the biodiesel
industry has carried out a glycerol surplus causing a major de-
crease in the market price of glycerol (Posada et al., 2011). Thus,
the economy of the biodiesel industry has been directly affected.
In order to convert glycerol to added-value products, chemical
and biological transformations have been analyzed (Wu et al.,
2011). Biological conversions offer the opportunity to synthesize
a large array of products with diverse functionalities. Glycerol
can be used as a carbon source substituting sugars in several
microbiological processes. Polyhydroxyakanoate (PHA) production
is an interesting alternative for the biological transformation of
Corresponding author. Tel.: +57 6 8879300x50417; fax: +57 6 8859300x50199.
E-mail address: ccardonaal@unal.edu.co (C.A. Cardona).
sized and intracellularly stored by many prokaryotes in the form
of granules and can account for up to 80% of the total bacterial
dry weight (Khanna and Srivastava, 2005; Mahishi et al., 2003).
PHAs can be produced from renewable sources through a fermen-
tation process under restricted growth conditions for nitrogen,
phosphorus, sulfur and/or oxygen in the presence of an excess car-
bon source and can be biocompatible and completely biodegraded
(Steinbchel, 2001; Lee, 1996). Polyhydroxybutyrate (PHB) was the
rst type of PHAs discovered and the most widely studied. PHB has
similar mechanical properties to conventional plastics like poly-
propylene or polyethylene, but its production costs are higher than
the petrochemical plastics. The total PHB production cost depends
on the microorganism (yield and productivity), the carbon and
nitrogen sources (substrates), the fermentation conditions (tem-
perature, aeration, pH-value) and the recovery and purication
processes. The carbon source could account for 2545% of the total
production costs (Nath et al., 2008) hence the necessity to nd
cheaper carbon sources. Agroindustrial wastes are attractive candi-
dates since they have some of the desired characteristics namely
low prices and high availability. Moreover, when agroindustrial

0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.01.129
 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844
wastes are used as substrates for the production of PHB, their di-
rect nal disposal is avoided thus solving an environmental prob-
lem. A wide variety of substrates such as whey, lignocellulosic
materials and glycerol (obtained from the biodiesel production)
have been used with different microorganisms to improve the
yields and production of PHBs (Nath et al., 2008; Koller et al.,
2008, 2005; Pantazaki et al., 2009; Kim, 2000; Silva et al., 2004;
Yu and Stahl, 2008; Cavalheiro et al., 2009; Bormann and Roth,
1999; Nikel et al., 2008).
A variety of microorganisms are able to produce PHB from di-
verse agroindustrial wastes. These microorganisms can be wild
strains such as Cupriavidus necator (Cavalheiro et al., 2009), Meth-
ylobacterium rhodesianum (Borman et al., 1999) or recombinant
microorganisms such as Escherichia coli CT1061 (Nikel et al.,
2008). Both Gram positive and Gram negative bacterial strains
are in the group of PHB producing bacteria. Currently, PHB is pro-
duced at an industrial scale using Gram negative bacteria. Never-
theless, Gram negative organisms contain lipopolysaccharides
(LPS) which co-purify with the PHAs and induce a strong immuno-
genic reaction (Valappil et al., 2007) which is therefore undesirable
for biomedical applications. Gram positive bacteria lack LPS and
are potentially better sources of PHAs when used for biomedical
purposes (Chen and Wu, 2005).
Bacillus megaterium is a Gram positive, strict aerobic, non mo-
tile, rod shaped, spore forming bacterium that has the ability to
produce PHB using glucose or glycerol as carbon sources (Lpez
et al., 2012). In this paper, the inuence of different fermenting
conditions for the production of PHB using B. megaterium were
studied. Moreover, a techno-economic analysis of the production
of PHB at an industrial scale was performed and a valorization of
crude glycerol is demonstrated.
2. Methods
2.1. Bacterial strains
The Bacillus megaterium strain used in this work is a wild strain
isolated from supercial sediments of the Bahia Blanca Estuary (Bue-
nos Aires, Argentina) and characterized as a PHB producer in the
presence of an excess carbon source and nitrogen restriction (Lpez
et al., 2012). Stock cultures were grown at 33 C in nutrient broth
and maintained at 4 C after growth on nutrient agar. The stock cul-
ture adapted to glycerol as the sole carbon source was maintained at
4 C after growth on a formulated agar medium. The adapted B.
megaterium cells were stored at 80 C in 2 ml cryo-vials containing
30% glycerol and 70% of the grown liquid culture.
2.2. Culture media
The seeding medium was prepared with the following concen-
trations: (NH4)2SO4, 1 g/l; KH2PO4, 1.5 g/l; Na2HPO4, 9 g/l; MgSO4-



7H2O, 0.2 g/l; and 1 ml of a trace element solution composed
by: FeSO4


7H2O, 10 g/l; ZnSO4


7H2O, 2.25 g/l; CuSO4


5 H2O,
1 g/l; MnSO4


4H2O, 0.5 g/l; CaCl2


2H2O, 2 g/l; H3BO4, 0.23 g/l;
(NH4)2Mo7O24, 0.2 g/l; and HCl, 10 ml. The analyzed carbon sources
were glycerol and glucose. The carbon source and the MgSO4
7H2O
were autoclaved separately and added aseptically to the medium
after cooling.
2.3. Adaptation of microorganisms and cultivation conditions
B. megaterium cells grown in nutrient broth at 30 C were used
to inoculate nutrient agar plates supplemented with 20 g/l of glyc-
erol. Single colonies from the grown nutrient agar plates supple-
mented with glycerol were inoculated in 100 ml erlenmeyer
39
asks containing 10 ml of sterile nutrient broth medium supple-
mented with 20 g/l of glycerol and incubated at 30 C in an orbital
shaker at 200 rpm for 23 days. One milliliter of these cultures was
again transferred to 10 ml of the above described seeding medium
with glycerol. 23 successive subcultures were performed to as-
sure a good adaptation of cells to the seeding medium with glyc-
erol as the sole carbon source.
2.4. Batch cultivations
The fermentations to produce PHB were carried out for 42 h in a
3.7 l Lab Fermenter (Bioengineering, Switzerland) with a working
volume of 1.5 l, 8% v/v of a grown formulated medium was used
as preinoculum. Several variables were analyzed such as: initial
concentration of substrate, temperature of the fermentation, pH
and the dissolved oxygen concentration. Moreover, two different
carbon sources were studied: glucose and glycerol. The initial con-
centrations of glycerol were: 10, 20 and 50 g/l. The initial glucose
concentration was 20 g/l. The analyzed temperatures were: 30 C
and 33 C. The dissolved oxygen concentration was controlled at
three levels of saturation: 30%, 60% and 80%. The oxygen saturation
in the medium was controlled by agitation (between 200 and
300 rpm) and with a constant air ow of 12 l/min. The inuence
of pH was analyzed by fermentation without pH control, and at
three different controlled pH values 5.0, 7.0 and 9.0 pH was con-
trolled with NaOH 1 N and H2SO4 1 M solutions.
2.5. Analytical methods
2.5.1. Biomass
Biomass was measured using the dry mass technique. Briey,
1 ml samples were collected in previously dried and weighed mi-
cro-centrifuge tubes and centrifuged at 12,000 rpm for 10 min.
Then, the resulting supernatant was discarded and the pellet was
washed with distilled water, centrifuged again and the excess of
water discarded. The nal biomass was weighed after drying for
48 h at 60 C.
2.5.2. PHB extraction
After fermentation, cells were harvested by centrifugation at
18 C and 6.000 rpm for 20 min and then the intracellular PHB
was extracted using the Chloroformhypochlorite dispersion
extraction method. Briey, the dispersion media containing 50 ml
of chloroform and 50 ml of a diluted (30% weight) sodium hypo-
chlorite solution in water was treated in an orbital shaker at
100 rpm and 30 C for 1 h. The mixture obtained was then centri-
fuged at 4000 rpm for 10 min, which resulted in three separate
phases. PHB was recovered from the bottom phase that contains
PHB dissolved in chloroform. PHB is then precipitated using 10 vol-
umes of ice-cold methanol (Hahn et al., 1995).
2.5.3. PHB quantication
Dried biomass is used for methanolysis of monomers according
to the method described by Braunegg et al. (1978) and modied by
Lageveen et al. (1998). Approximately 10 mg of cell mass was re-
acted in a small screw-cap test tube with a solution containing
1 ml of chloroform, 0.85 ml of methanol, 0.15 ml of sulfuric acid
and 0.2 ml of internal standard (benzoic acid in methanol) for
140 min at 100 C. After reaction, 0.5 ml of distilled water was
added and the test tube was shaken vigorously for 1 min. After
phase separation, the organic phase (bottom layer) was removed
and transferred to a small screw-cap glass vial. 500 ll from this
organic phase were taken and added to a test tube and injected
in the Gas ChromatographMass Spectrometer. An Agilent Tech-
nologies 6850 series II gas chromatographer was used. The gas
chromatographer was equipped with a HP-5MS capillary column
40 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844

(25 m length, 0.32 mm internal diameter). Helium (velocity at

5 cm/min) was used as the carrier gas. The injector and detector
were operated at 230 and 275 C, respectively. A temperature pro-
gram was used for efcient separation of the esters (120 C for
5 min, temperature ramp of 8 C per min, 180 C during 12 min).
An Agilent Technologies 5975B mass spectrometer was used for
identication and quantication of the derivatized PHB.

2.5.4. Substrate quantication

Glycerol and glucose concentration were determined off-line by
HPLC (Hitachi LaChrom Elite) equipped with an auto sampler (Hit-
achi LaChrom Elite L-2200), a Bio-Rad Aminex Fermentation Mon-
itoring Column (150 mm  7.8 mm), a column oven (Hitachi
LaChrom Elite L-2300), a HPLC pump (Hitachi LaChrom Elite L-
2130) and a Hitachi LaChrom Elite L-2490 refraction index detec-
tor. Injection volume was 20 ll and elution was achieved using a
50 mM solution of H2SO4. The column was kept at 65 C and the
pump was operated at a ow rate of 0.3 ml/ min.

2.6. Simulation methodology

The simulation process was based on a previously described

methodology (Posada and Cardona, 2010; Cardona and Snchez,
2006; Quintero et al., 2008; Gutirrez et al., 2009). The approach
used for the process design was a knowledge-based strategy that
considers both heuristic rules and researchers experience. The
PHB production processes were simulated using Aspen Plus (Aspen
Technologies Inc., USA). The economic analysis was performed
using the Aspen Economic Analyzer (Aspen Technology, Inc.,
USA) package. This software estimates the capital costs of process
units as well as the operating costs, among other valuable data, uti-
lizing the design information provided by Aspen Plus and the data
introduced by the user for specic conditions such as project loca-
tion among others. This analysis was estimated in US dollars for a
10-year period at an annual interest rate of 16%, considering the
straight-line depreciation method and a 33% income tax.
3. Results and discussion
Although pure glycerol has been used as a sole carbon source
for the production of PHB, there is evidence that some Bacillus spe-
cies have the ability to produce PHB from crude glycerol. Sindhu
et al. (2011) worked with Bacillus sphaericus NII 0838 and the
PHB accumulation was 31% of cell dry weight. Deepthi et al.
(2011) worked with Bacillus rmus NII 0830 and the PHB accumu-
lation was 53% of cell dry weight. Nevertheless, Cavalheiro et al.
(2009) demonstrated that the PHB yields and productivities using
Cupriavidus necator can be improved using pure glycerol (a 78%
improvement). Posada et al. (2011) veried that the prot margin
obtained when glycerol was puried before the fermentation step
is higher compared to crude glycerol and supplied the costs gener-
ated by the purication steps.
3.1. Experimental
The productivity and yield of PHB production by B. megaterium
from pure glycerol (98% weight) was studied at different levels
such as: initial concentration of substrate, fermentation tempera-
ture, pH and the dissolved oxygen concentration.
After the adaptation of B. megaterium to glycerol as substrate,
the inuence of the initial substrate concentration was analyzed.
Three different initial concentrations were used: 10, 20 and
50 g/l. The fermentation conditions were: temperature 30 C, air
ow 12 l/min, 200 rpm, and uncontrolled pH. Fig. 1 shows the
Fig. 1. Comparison of biomass (a) and PHB (b) proles from B. megaterium with
different initial concentrations of glycerol, T = 30 C, air ow = 12 l/min, 200 rpm.
comparison of biomass (a) and PHB production (b) using glycerol
as the sole carbon source.
The inuence of temperature in the PHB production was stud-
ied. Fermentations with an initial glycerol concentration of 20 g/l,
air ow 12 l/min, 200 rpm, and uncontrolled pH, were carried out
at three different temperatures: 30, 33 and 35 C. The proles of
biomass and the PHB obtained are shown in Fig. 2.
The pH of the medium varies during growth of the B. megateri-
um strain, and it affects the PHB production as discussed by Mona
et al. (2001). The inuence of pH in this strain of B. megaterium was
analyzed at three different pH values (i.e., 5.0, 7.0 and 9.0) and
were compared with the fermentation performed with no pH con-
trol. The results obtained show that the PHB produced is higher at
pH 7.0 followed by the uncontrolled conditions > pH 5.0 > pH 9.0.
On the other hand, the production of PHB was also analyzed using
different levels of oxygen saturation in the cultures (i.e., 30%, 60%
and 80%). The saturation was controlled by agitation, with a con-
stant air ow of 12 l/min. The results showed that the major pro-
duction of PHB was seen at 80% of oxygen saturation.
Finally, a fermentation using glucose as substrate was per-
formed and compared with the fermentations using glycerol as
carbon source. When glucose was used as carbon source, the work-
ing conditions where the best fermentation conditions found when
glycerol was used as substrate. Table 1 shows a comparison of the
experimental results obtained for PHB production using either glu-
cose or glycerol as substrates. As shown in Table 1, the best culture
conditions in terms of PHB yield and productivity were: tempera-
ture 33 C, initial glycerol concentration 20 g/l, controlled pH 7.0
and 80% of oxygen saturation. The production of PHB is improved
by approximately 10% (compared with the uncontrolled approach)
 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844 41

Thus, the purication step used for LPS depletion in the PHB pro-
duced by Gram-negative bacteria is not necessary.

3.2. Simulation

3.2.1. Process description

The process simulation is a useful tool to get an idea of the total
production costs in a process including raw materials, energy con-
sumption and the installation costs to start the production of a de-
sired product. The blocks process analysis was chosen as the best
technological scheme for the production of PHB (glycerol purica-
tion, fermentation, separation and purication).

3.2.1.1. Glycerol purication. A typical composition for a crude glyc-

erol stream obtained from the biodiesel production process is as
follows (% w/w): 32.59% methanol, 60.05% glycerol, 2.62% NaOCH3,
1.94% fats, and 2.8% ash. Although there are studies that use con-
sortia of microorganisms for the production of PHB from crude
glycerol (Dobroth et al., 2012), in this work glycerol is puried be-
fore the fermentation step by physical methods up to 98% weight
according to the methodology described by Posada et al. (2009)
(Fig. 3). Briey, crude glycerol is initially evaporated and 90% of
the methanol is recovered at 99% weight. This resulting methanol
stream meets the necessary conditions to be reused in the transe-
sterication process for the production of biodiesel. The resulting
bottom stream from the evaporation stage is neutralized using
an acid solution where both the salts produced during the neutral-
ization and the remaining ashes are removed by centrifugation.
The uid stream is washed with water using a weight ratio of 2.4
(water/glycerol stream), and thus an aqueous glycerol free of salts
Fig. 2. Comparison of biomass (a) and PHB (b) proles from B. megaterium with and solids, with a low concentration of methanol and triglycerides
different temperatures of fermentation, glycerol initial concentration 20 g/l, air is obtained. More than 90% of water and the remaining methanol
ow = 5 l/min, 200 rpm.
are then removed by evaporation. At this point the purity of the ob-
tained glycerol is 80% weight, and the glycerol stream is nally
when pH is controlled at 7.0 but the dissolved oxygen concentra- puried through a distillation column up to 98% weight.
tion is not constant. Nevertheless, an increase in about 25% of
the PHB production is reached when both pH and oxygen satura- 3.2.1.2. Fermentation process. Table 3 shows the PHB production,
tion are kept constant (7.0 and 80%, respectively) compared with substrate consumption and biomass concentration from the fer-
the uncontrolled scenario. Hence, these results show the capability mentation process using either glucose or glycerol as substrates.
of B. megaterium to produce PHB at similar yields and productivi- The fermentation step is carried out in a single stage fermenter.
ties using both a common substrate as glucose and an important In this level, cell growth and PHB accumulation occurs. The glyc-
industrial waste like glycerol. erol stream is diluted at 20 g/l in the input fermenter and the out-
Table 2 shows the percentage of PHB accumulation by some put glycerol concentration from the fermenter is 8 g/l and the
microorganisms with different agroindustrial wastes used as sub- residence time is 42 h. The results conrm the ability of B. megate-
strates. The % of PHB accumulation showed by B. megaterium can rium to use glycerol as the only carbon source. The yields obtained
be compared with other microorganisms that use glycerol as sub- are very similar compared to the PHB production from glucose.
strate. One of the main features of this bacterial strain is that it pro-
duces PHB free of LPS and can be used for medical applications. 3.2.1.3. Separation and purication. The PHB separation and puri-
cation processes from a fermentation broth can be divided in three
steps: pretreatment, extraction, and purication. Economically
wise, Posada et al. (2011) demonstrated that the most appropriate
downstream process is based on the BIOPOL owsheet. This pro-
Table 1
cess starts with a heat pretreatment stage at 85 C, followed by a
Comparison of the PHB production using glucose or glycerol as carbon sources.
simultaneous chemical-enzymatic digestion with both NaOCl
GLYCEROL 20 g/l GLUCOSE 20 g/l (30% weight) and the pancreatin enzyme Burkholderia sp. PTU9
Uncontrolled Controlled Controlled (2% weight), at 50 C and pH 9.0. Then the disrupted cell mass is
Conditionsa Conditionsb Conditions discarded by a centrifugation process. The suspended PHB stream
CDWc(g/l) 5.7 7.7 7.1 is then washed with a H2O2 diluted stream (1.2 v/v%). Finally, the
PHB production (g/l) 3.4 4.8 4.2 PHB is puried up to 99.9% weight by evaporation and spray
PHB accumulation (%) 60 62.4 59.1
drying.
Total substrate 59 71.5 57.5
consumption (%)
For the separation of the PHB produced, two different down-
Yield P/Sd (g/g) 0.3 0.3 0.4 stream processes were considered. For the two different Down
a
Stream Processes (DSP), called DSP A (Fig. 4) and DSP B (Fig. 5),
Uncontrolled conditions: without control of both pH and oxygen saturation.
b
Controlled conditions: pH = 7, oxygen saturation = 80%, temperature = 33 C.
both of the used operating conditions and solvent ratio to aqueous
c
CDW = cell dry weight. phase were the same as described by Posada et al. (2011). Never-
d
P/S = product/substrate consumed. theless, there are some differences in the separation congurations
42 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844

Table 2
Some PHB producing microorganisms from different agroindustrial wastes.

Substrate Microorganism PHB accumulation (%) References

Whey Methylobacterium sp. ZP24 64 Khanna and Srivastava (2005)
Pseudomonas hydrogenovora 28 Koller et al. (2008)
Glycerol Bacillus megaterium 62 This work
Cupriavidus necator DMS 545 62 Cavalheiro et al. (2009)
Ralstonia eutropha 55 Gzde et al. (2012)
Methylobacterium rhodesienum MB 126 50 Cavalheiro et al. (2009)
Escherichia coli CT1061 51 Nikel et al. (2008)
Sugarcane bagasse Burkholderia sacchari IPT 101 62 Silva et al. (2004)
Burkholderia cepacia IPT 048 41 Silva et al. (2004)

Fig. 3. Simplied owsheet of the glycerol purication process up to 98% weight. E-1: evaporator I, R-1, neutralizing reactor I, cen-1: centrifuge I, D-1: decanter I, E-2:
evaporator II, DC-1: Distillation Column I.

compared with the Posada et al. (2011) work. When Gram negative
bacteria like C. necator is used, the washing step with a H2O2 di-
Table 3
luted stream is necessary since the PHB produced contains lipo-
PHB concentration, substrate consumption and biomass production in the fermen-
tation step.
polysaccharides (LPS) and this H2O2 treatment eliminates these
toxic compounds. When Gram positive bacteria are used, the puri-
Feature Glycerol Glucose
cation with H2O2 is omitted.
Initial substrate concentration (g/l) 20 20 Fig. 4 shows the DSP A conguration. This separation scheme
Final substrate concentration (g/l) 8.2 10.7 has a solvent recuperation in the 13 and 14 units. Fig. 5 shows
Conversion 0.6 0.5
Concentration of PHB output (g/l) 4.8 4.8
the DSP B where there is no solvent recuperation. Table 4 shows
Concentration of biomass output (g/l) 7.7 7.1 some yields in the separation and purication stages using glycerol
Fermentation time (h) 42 42 or glucose as substrates with the DSP A or DSP B congurations.
Although the process scheme used was the same for both sub-

Fig. 4. Flowsheet for PHB production from glycerol using the Down Stream Process A (DSP A). (1) Pump; (2) Sterilizer; (3) heat exchanger I; (4) fermenter; (5) heater; (6)
digestor; (7) centrifuge; (8) heat exchanger II; (9) heat exchanger III; (10) decanter I; (11) distillation column I; (12) spray drier; (13) distillation column II; (14) decanter II;
(15) mixer I; (16) mixer II.
 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844 43

Fig. 5. Flowsheet for PHB production from glycerol using the Down Stream Process B (DSP B) (1) pump; (2) sterilizer; (3) heat exchanger I; (4) fermenter; (5) homogenizer;
(6) centrifuge I; (7) heat exchanger II; (8) heat exchanger III; (9) extractor; (10). centrifuge II; (11) heat exchanger IV; (12) decanter; (13) distillation column; (14) spray drier;
(15) mixer.

Table 4 Table 5
Yields in the separation and purication stages using glycerol or glucose as substrates Comparison of the total PHB production costs from both glycerol and glucose with
with the DSP A or DSP B congurations. two different downstream processes.

Feature Glycerol Glucose GLYCEROL GLUCOSE

Yield DSP A (%) 98.0 97.99 DSP A DSP B DSPA DSP B
DES losses in DSP A (%) 0.3 0.2
Raw materials (US$/kg PHB) 0.2 0.2 0.7 0.7
PHB purity (%) 99.9 99.9
Utilities (US$/kg PHB) 1.3 0.8 1.2 0.9
Yield DSP B (%) 98.9 98.9
Operating labor (US$/kg PHB) 0.1 0.1 0.1 0.1
DES* losses in DSP B (%) 0.3 0.3
Maintenance (US$/kg PHB) 0.4 0.3 0.4 0.4
Purity PHB (%) 99.9 99.9
Operating charges (US$/kg PHB) 0.2 0.2 0.2 0.2
*
DES: diethylsuccinate. Plant overhead (US$/kg PHB) 0.06 0.04 0.07 0.05
G and A cost (US$/kg PHB) 0.3 0.2 0.3 0.3
Depreciation of capital (US$/kg PHB) 1.0 0.7 1.0 0.7
Total (US$/kg PHB) 3.5 2.6 3.9 3.3
strates, DSP A showed more diethylsuccinate (DES) losses when
glycerol was the carbon source compared to glucose. However,
the yield of the separation process was the same in both cases
(98% approximately). In DSP B, the yields and DES losses were visors was USD$ 2.1/h and USD$ 4.3/h, respectively. Also, the prices
the same with both substrates. used for electricity, water and low pressure vapor were USD$ 0.03/
kWh, USD$ 1.252/m3 and USD$ 8.2/ton, respectively. All of these
3.2.2. Simulation procedure values are based on Colombian conditions.
The PHB production processes were simulated using Aspen Plus
(Aspen Technologies Inc., USA). Thus, the design of the distillation 3.2.3. Economic analysis
columns in all cases required the denition of the preliminary The total PHB production costs from two different substrates
specications using the DSTWU shortcut method included in As- such as glycerol and glucose are shown in Table 5. The two down-
pen Plus. This method uses the WinnUnderwoodGilliland proce- stream processes were compared (DSP A and DSP B). With DSP A,
dure providing an initial estimate of the minimum number of the PHB total production costs were 3.9 US$/kg and 3.5 US$/kg
theoretical stages, the minimum reux ratio, the localization of for glucose and glycerol respectively. In turn, with DSP B, the
the feed stage, and the products split. To perform the rigorous cal- PHB total production costs were 3.3 US$/kg and 2.6 US$/kg for glu-
culation of the distillation columns, the Rad-Frac module (based on cose and glycerol respectively. In general terms the lower PHB pro-
the MESH equations) was used. duction costs were obtained when there is no solvent recuperation
B. megaterium was simulated as a solid compound while the en- in the separation scheme and glycerol is used as substrate.
zymes were simulated as non-conventional compounds. For the The current sale prices of PHB are between 3.1 and 4.4 USD/kg,
thermodynamic analysis the UNIFAC model was used. The enzy- using glucose and/or sucrose as carbon sources. The total produc-
matic digestion was simulated based on a stoichiometric approach. tion cost of the PHB calculated in this work using glycerol as car-
Calculation of energy consumption was based on the thermal en- bon source is 2.6 USD/kg having approximately a 20% of prot
ergy required by heat exchangers, reboilers, ash drier units and margin (0.5 USD/kg). Hence, the impact of glycerol in the biodiesel
the power supply required by the pumps. supply chain is analyzed as follows: the sale price of the crude
The economic analysis was performed using the Aspen Eco- glycerol obtained in the biodiesel production is 0,1 USD/kg. The
nomic Analyzer (Aspen Technology, Inc., USA) package. This soft- conversion of glycerol into PHB using the conditions established
ware estimates the capital costs of process units as well as the in this work is 0,6 kg PHB/kg glycerol. Taking into consideration
operating costs, among other valuable data, utilizing the design the prot margin demonstrated in the production of PHB from
information provided by Aspen Plus and the data introduced by crude glycerol, a valorization of approximately 166.2% of this com-
the user for specic conditions such as project location among oth- pound is then obtained. Even though B. megaterium demonstrated
ers. This analysis was estimated in US dollars for a 10-year period to produce PHB using glycerol as carbon source, other producing
at an annual interest rate of 16%, considering the straight-line microorganisms (such as C. necator) should be studied and their
depreciation method and a 33% income tax. The cost of crude glyc- PHB production analyzed using the process schemes proposed in
erol was USD$ 0.1/l. The labor cost used for operatives and super- this study.
44 J.M. Naranjo et al. / Bioresource Technology 133 (2013) 3844
4. Conclusions
The microbiological conversion of glycerol into PHB is an inter-
esting alternative to obtain added value products using this by
product of the biodiesel industry. The PHB production using B.
megaterium could be improved when pH and oxygen saturation
are controlled in the fermentation conditions. Using simulation
programs, the PHB total production costs were estimated at an
industrial scale with both substrates (glycerol and glucose). PHB
production using glycerol showed a 1020% lower production cost
compared with glucose. The crude glycerol generated in the biodie-
sel production can be valorized in 166.2% through the production
of PHB.
Acknowledgements
To the National University of Colombia at Manizales for funding
this research and to Dr. M.A. Villar and his group at PLAPIQUI
(UNS-CONICET) in Argentina for the B. megaterium strain.
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