Process Biochemistry 86 (2019) 9–15

Contents lists available at ScienceDirect

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

Utilization of desugarized sugar beet molasses for the production of poly(3- T

hydroxybutyrate) by halophilic Bacillus megaterium uyuni S29
Maximilian T. Schmida, Hyunjeong Songa, Michaela Raschbauera, Florian Emerstorferb,
⁎
Markus Omannb, Franz Stelzerc, Markus Neureitera,
a
University of Natural Resources and Life Sciences, Vienna, Institute of Environmental Biotechnology, Tulln, Austria
b
AGRANA Research & Innovation Center GmbH, Tulln, Austria
c
University of Technology, Graz, Institute for Chemistry and Technology of Materials, Graz, Austria

A R T I C LE I N FO A B S T R A C T

Keywords: Desugarized sugar beet molasses is a high-salinity side stream after fractionation of beet molasses by chroma-
Bacillus megaterium tography. It could be demonstrated that this by-product of the sugar industry is a suitable substrate for the
Desugarized sugar beet molasses production of poly(3-hydroxybutyrate) with the halophilic bacterium Bacillus megaterium uyuni S29.
Poly(3-hydroxybutyrate) Cell dry mass concentrations of up to 16.7 g L−1 with a P(3HB) content of 0.6 g g−1 were achieved after only
Halophilic fermentation
24 h of batch cultivation. Utilization of a desugarized sugar beet molasses medium resulted in a two- to threefold
Sugar industry by-product
increase in biomass production compared to a conventional mineral based medium. Depending on the extraction
method, the molecular mass (Mn) of the P(3HB) varied between 119 kDa and 722 kDa.
The high P(3HB) productivity of 0.42 g L−1 h−1 during batch experiments combined with desugarized sugar
beet molasses as inexpensive substrate suggests a simple and robust process for cost effective P(3HB) production.

1. Introduction molasses as it undergoes an additional chromatographic desugarization

The European Union is the world’s largest producer of beet sugar
resulting in an annual production of approximately 17 million t.
However, beet sugar represents only 20% of the global sugar produc-
tion, while the remaining 80% are produced from sugar cane [1]. On
the 30th of September 2017 the European quota system for sugar pro-
duction was terminated. As a consequence the price of sugar dropped
due to overproduction. The development of high value products derived
from by-products of the sugar industry is therefore receiving increasing
attention. Sugar beets typically contain between 15–20% sucrose per kg
of beet. About 85 to 90% of the extracted sugar can be recovered by
crystallization, while the rest remains in a non-crystallized syrup called
molasses. Based on literature it can be calculated that 7 t of sugar beets
are processed to obtain 1 t of sugar and between 0.25 and 0.35 t of
molasses [2]. Based on approximately 17 × 106 t of beet sugar pro-
duced an estimated amount of 6 × 106 t of molasses is produced per
year. Beet molasses is currently mainly used as a fermentation feedstock
for the production of baker’s yeast [3], in the chemical or pharma-
ceutical industry for the production of e.g. amino acids, antibiotics or
citric acid and as animal feed in agriculture [4].
Desugarized sugar beet molasses differs from regular sugar beet
process, resulting in the separation of three fractions: the sugar fraction,
the betaine fraction and the raffinate fraction. This process increases
the yield of sugar per ton of beet. In addition, betaine is obtained as an
added-value product. Raffinate is concentrated by evaporation of water,
resulting in desugarized sugar beet molasses, which has a lower eco-
nomic value than regular sugar beet molasses and is currently used as
fertilizer and as nutrient additive for animal feed [2]. AGRANA pro-
cesses around 85,000 t of molasses annually in Austria, leading to the
production of around 35,000 t of desugarized sugar beet molasses at
AGRANA’s sugar factory in Tulln.
In comparison to molasses, desugarized sugar beet molasses is
characterized by a lower sucrose concentration, an increased salt con-
tent and much darker color. The composition can vary depending on
the origin of the sugar beets, the quality and consistence of the molasses
and the performance of the desugarization process. A comparison of
beet molasses and two different batches of desugarized sugar beet
molasses (DSBM) from 2016 and 2017 is shown in Table 1. Desugarized
sugar beet molasses still has a relatively high sugar content (approxi-
mately 15% w/w) and it also contains nutrients that can act as growth
factors for microorganisms. This makes it interesting as a substrate for
biotechnological processes; however, due to its salinity it is unsuitable

⁎
Corresponding author.
E-mail address: markus.neureiter@boku.ac.at (M. Neureiter).

https://doi.org/10.1016/j.procbio.2019.08.001
Received 9 April 2019; Received in revised form 31 July 2019; Accepted 1 August 2019
Available online 07 August 2019
1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
M.T. Schmid, et al. Process Biochemistry 86 (2019) 9–15

Table 1 2.2. Medium for cultivation

Composition of regular sugar beet molasses and batches of desugarized sugar
beet molasses from 2016 (DSBM16) and 2017 (DSBM17). For comparison, mineral salt medium (MSM) as described by
Unit Sugar beet molasses DSBM16 DSBM17 Kulpreecha et al. [19] was used, containing: CaCl2: 0.02 g L−1, MgSO4·7
pH 8.3 6.6 7.5 H2O: 1 g L−1, citric acid: 0.75 g L−1, (NH4)2SO4: 5 g L−1, sucrose: 30 g
L−1, FeSO4: 0.025 g L−1, NaCl: 10 g L−1 and 1 mL of trace element
Dry matter (Brix) % 82.0 70.7 71.6
solution composed of ZnSO4·7 H2O: 0.1 g L−1, H3BO3: 0.3 g L−1,
Sucrose % 50.5 17.6 13.2
Ash % 10.9 20.4 25.5 CoCl2·6 H2O: 0.2 g L−1, CuSO4: 0.006 g L−1, NiCl2·6 H2O: 0.02 g L−1,
Nitrogen % 1.8 2.1 1.8 Na2MoO4·2 H2O: 0.03 g L−1, MnCl2·2 H2O: 0.025 g L−1. In order to
Ammonium % 0.07 0.05 0.05 ensure phosphorous limitation the concentration of KH2PO4 was re-
Protein % 11.0 13.0 11.4
duced from 2.0 to 0.3 g L−1. The pH was adjusted to 7.0 before in-
Phosphorus % 0.02 < 0.02 < 0.02
Sodium g kg−1 13.4 25.2 26.2
oculation.
Potassium g kg−1 32.8 50.3 74.2 Desugarized sugar beet molasses was provided by AGRANA (Tulln,
Calcium mg kg−1 111 235 255 Austria). Batches from 2016 (DSBM16) and 2017 (DSBM17) were used.
Lactate % 2.7 6.2 4.0 The compositions are presented in Table 1. Due to its low water activity
Acetate % 0.5 1.3 0.9
the material is stable and can be stored at room temperature for a
longer period of time. For the use as fermentation substrate desugarized
for conventional applications like yeast fermentation. It has also been sugar beet molasses was diluted with deionized water to a concentra-
suggested as a substrate for biohydrogen production [5]. tion of 15% (w/w) based on DSBM wet mass and the pH was set to 7.0
Polyhydroxyalkanoates (PHA) are microbial polyesters synthesized with 5 M H2SO4 and 5 M NaOH. No additional nutrients were added.
by Archaea and various Gram-positive and Gram-negative bacteria The DSBM medium was sterilized at 121 °C for 20 min.
under unbalanced nutrient conditions [6]. PHA can be used for the
production of biobased and biodegradable materials. Due to its bio- 2.3. Cultivation
compatibility, applications range from agriculture, packaging material
to medical applications [7–9]; however, high production costs currently Shake flask experiments were carried out in 300 mL shake flasks
reduce the commercial competitiveness of the polymer. Around with 100 mL fermentation medium. The inoculation was conducted by
40–48% of the costs arise from the raw material providing the carbon transferring 1 mL of pre-culture to each flask. The cultures were in-
source [10]. The usage of conventional starch and/or sugar containing cubated at 35 °C and 130 rpm. All experiments were performed as tri-
raw materials is increasingly criticized as they compete with food plicates.
production. As a replacement, low-cost by-products from biomass Fermentations in bioreactors were conducted in a parallel benchtop
processing, such as crude glycerol [11], chicory roots [12], grape po- system (DASGIP, Eppendorf) with a total reactor volume of 1.4 L and a
mace [13], fruit processing water [14] or by-products of the cider in- working volume of 800 mL. For inoculum preparation, 300 mL shake
dustry [15] have been suggested to reduce production costs. Many al- flasks with 100 mL fermentation medium were inoculated from a liquid
ternative carbon sources, however, are often not available in sufficient pre-culture and incubated over night at 35 °C in a rotary incubator at
quantities and may also require intensive pre-treatment. This does not 130 rpm (Infors Multitron Incubator shaker). For the inoculation of
apply for desugarized sugar beet molasses, since it can be provided in 750 mL of fermentation medium (MSM or DSBM medium), 50 mL of
large quantities and is not used for nutritional purposes. inoculum were transferred into the bioreactor. Relevant process para-
Bacillus megaterium uyuni S29 CECT 7922 was selected for this study meters like pH-value, dissolved oxygen level (DO), consumption of acid
because the high salinity requires the usage of halotolerant bacteria. or base were continuously monitored. For pH-control, 5 M NaOH and
This strain was isolated from the Bolivian salt lake Uyuni and is re- 5 M H2SO4 were used. The DO (percentage of maximum oxygen sa-
ported to accumulate solely poly(3-hydroxybutyrate) at concentrations turation) was set at 20% and controlled by varying the stirrer speed
up to 60 g per 100 g cell dry mass (CDM) [16]. It has been described to while the airflow (compressed air) was fixed at 30.5 L h−1. The ex-
grow at NaCl concentrations varying from 0.5 to 15% [17]. In addition periments were conducted in duplicates.
to these unique abilities, the lack of endotoxins in the Gram-positive
cell wall may be an advantage for biomedical applications of the 2.4. Extraction of P(3HB)
polymer [18,9].
In this study we evaluated, whether desugarized sugar beet molasses For extraction the biomass was harvested by centrifuging for 20 min
is a suitable substrate for the production of P(3HB) with B. megaterium at 10,000 g (Sorvall Lynx4000 centrifuge). Untreated biomass and cells
uyuni S29. In addition, we proposed a simple and reproducible fer- pretreated by ultrasonication were compared regarding the extraction
mentation process and characterized the produced polymer. efficiency. For ultrasonication (Branson Digital Sonifier 250) 3 g of
dried biomass were dissolved in 30 mL of deionized water and treated 5
times with 60% of the maximum amplitude (120 W) for 60 s with 2 min
2. Materials and methods breaks in-between. For extraction 2–4 g of dehydrated biomass were
weighed in a beaker and 96% Ethanol (10 times the amount of biomass)
2.1. Microorganism was added. The biomass extracted with alcohol was filtered through a
0.2 μm filter and the filter cake was dried at 80 °C. The extraction was
Bacillus megaterium uyuni CECT 7922 was cultivated at 35 °C on a performed overnight with 90 mL of chloroform (VWR; ≥ 99.8%) in an
modified beef extract medium (meat extract: 10 g L−1, peptone: 10 g automatic extraction unit (Soxtherm 2000). The extraction was con-
L−1; NaCl: 5 g L−1) with 1% agar, and transferred to a new plate once a ducted at a temperature of 240 °C and was divided in a cooking phase
week. Cultures can be stored at 4 °C for at least one month. Pre-cultures (1 h) and an extraction phase (1.5 h). After extraction P(3HB) was
were prepared in 300 mL baffled shake flasks by inoculation with single precipitated by adding 96% ethanol (10 times the amount of chloro-
colonies from a solid plate to 100 mL modified beef extract medium. form) and harvested by filtration.
The culture was incubated in an Infors Multitron Incubator shaker for
24 h at 35 °C and 130 rpm. 2.5. Analytical methods

For analysis 5 mL sample of fermentation broth were taken in

10
M.T. Schmid, et al.
duplicate. An aliquot of 20 μL was diluted 1:50 and used to determine
the optical density at a wavelength of 600 nm (DR 3900 Hach-Lange
photometer). The remaining sample was centrifuged at 2500 g for
20 min (Eppendorf, Centrifuge 5810). The pellet was washed two times
and dried at 105 °C for 48 h to determine the cell dry mass (CDM). The
supernatant was stored at −20 °C for subsequent analysis of nitrogen
and phosphorus concentrations as well as the determination of sugar
and organic acids content.
Nitrogen was determined as total Kjeldahl nitrogen with a
AutoKjeldahl Unit K-370 (Buechi, Flawil, Switzerland). Total phos-
phorous concentration was analyzed with a colorimetric method based
on the formation of phospho-molybdenum blue using a LCK 350
Phosphate kit.
The carbon source was determined by HPLC (1100 series with re-
fractive index detector, Agilent; ION 300 column, Transgenomic;
column temperature: 45 °C; 0.005 M H2SO4 as eluent, flow rate
0.325 mL min−1; 46 bar). Since the system is not suitable to determine
sucrose directly, it was converted to fructose and glucose by invertase
(SIGMA, I9253) before analysis. 33 μL sample were mixed with 67 μL
invertase solution (10 mg mL−1) and incubated for 30 min at 50 °C.
Remaining proteins were removed by Carrez precipitation.
PHA was quantified according to a method by Furrer et al., which
was modified by doubling the amount of methylene chloride solution
and the iso-propanol/HCl solution [20]. The samples were analyzed by
gas chromatography (Agilent Technologies 7890B, Column: Agilent
19091G-133: HP-35 column (30 m ×0.25 mm) with a thickness of
0.25 μm, flame ionization detector). Helium with a flow rate of 1.75 mL
min−1 was used as mobile phase and nitrogen as makeup gas, respec-
tively. The measurement started at a temperature of 50 °C, which was
raised with a rate of 15 °C min−1 to 150 °C, 10 °C min−1 to 200 °C and
25 °C min−1 to 280 °C.
GPC-HPLC measurements in chloroform were performed on a LC-20
AD system from Shimadzu equipped with separation columns (MZ-Gel
Sdplus Linear 5 μm separation columns from MZ Analysentechnik) in
line and a refractive index (RD-20A) as well as a UV/Vis detector (SPD-
20A) at a wave length of 254 nm. Polystyrene standards purchased from
Polymer Standard Service were used for calibration.
All NMR spectra were recorded on a Bruker Avance III 300 MHz
spectrometer. For 1H spectra, 10 mg of polymer were dissolved in
850 μL of CDCl3 and transferred to an NMR-tube. 1H spectra of polymer
samples were recorded with a delay of 10 s and 32 scans, APT spectra
with a delay of 2 s and 256 scans, 1H, 1H-COSY spectra with a delay of
1.371 s and 8 scans, HSQC spectra with a delay of 1.442 s and 2 scans
and HMBC spectra with a delay of 1.2698s and 8 scans. As solvents,
CDCl3 with 0.03% TMS as internal standard was used. All spectra were
referenced to the residual solvent peaks.
3. Results and discussion
3.1. Evaluating growth on desugarized sugar beet molasses medium
Desugarized sugar beet molasses shows a very high viscosity and
salt content. Hence, dilution is necessary to make it suitable as a fer-
mentation substrate. In order to determine the optimum level of dilu-
tion, concentrations ranging from 15% (w/w) to 40% (w/w) were
tested in shake flasks. Fig. 1 shows that there was no significant growth
within a time frame of 72 h at desugarized sugar beet molasses con-
centrations above 35% (w/w). Generally, it can be said that higher
desugarized sugar beet molasses concentrations significantly increased
the lag phase. If growth was observed, the pH decreased in the first
8–10 h and increased afterwards (data not shown). The shortest lag
phase and consistent growth were observed at concentrations of 15%
(w/w) and 17% (w/w) desugarized sugar beet molasses. Therefore, it
was decided to use a concentration of 15% (w/w) for the subsequent
experiments.
Experiments at various pH-conditions ranging from 6.0 to 8.5 were
11
Process Biochemistry 86 (2019) 9–15
conducted in bioreactors in order to determine the optimum pH for
growth and P(3HB) formation. CDM formation is presented in Table 2.
No growth was observed at pH-values below 6.5 and above 8.5. Be-
tween pH 7.0 and 8.2 there is no significant difference in biomass and P
(3HB) formation. As the pH-value of desugarized sugar beet molasses-
medium is already 7.0 after preparation it was decided to set the pH at
7.0 for all experiments.
3.2. Biomass and P(3HB) formation on desugarized sugar beet molasses
medium and mineral salt medium
B. megaterium was cultivated on desugarized sugar beet molasses
and mineral salt medium at controlled conditions in bioreactors and
formation of CDM and P(3HB) was compared to evaluate the potential
of desugarized sugar beet molasses as a fermentation substrate.
Cultivating B. megaterium on mineral salt medium yielded a max-
imum biomass concentration of 11.3 g L−1 CDM and a P(3HB) content
of 2.8 g L−1 after 13 h (Fig. 2, A). P(3HB) formation was induced after
9 h due to phosphorous limitation, as the phosphate concentration had
dropped below 1 mg L−1 at this point. The peak concentration of P
(3HB) was reached after 13 h and remained constant during further
40 h of cultivation. In this case a biomass yield of 0.35 g CDM g−1
sucrose (consumed) and a P(3HB) yield of 0.09 g g−1 sucrose (con-
sumed) was achieved at an average productivity of 0.09 g L−1 h−1 over
a period of 24 h. Only addition of NaOH was required to maintain the
pH at 7.0. Rodríguez-Contreras et al. reported a higher P(3HB)/CDM
ratio of 0.41 with the same strain compared to a value of 0.27 in our
experiments [17]; however, they performed a batch cultivation under
nitrogen limiting conditions with excess glucose as sole carbon source
resulting in a maximum CDM of 5.42 g L−1 with 2.22 g L−1 P(3HB).
Fig. 2 (B) presents the results of the cultivations on desugarized
sugar beet molasses. Compared to the experiments on mineral salt
medium a higher CDM and P(3HB) formation was observed (Fig. 2). A
maximum CDM of 18.7 g L−1 with a P(3HB) content of 10.2 g L−1 was
obtained after 30 h. It was observed that B. megaterium inverted sucrose
from the start and that the glucose uptake is more rapid compared to
fructose. In addition, these experiments illustrated that lactate that is
already present in DSBM is metabolized as well. Remaining fructose
and lactate present in desugarized sugar beet molasses are consumed
simultaneously in a later stage of the process. These results demonstrate
that it is possible to increase the concentration of P(3HB) by a factor of
approximately 3.5 when using desugarized beet molasses instead of
mineral salt medium.
Notably, volumetric productivity of P(3HB) on desugarized sugar
beet molasses is approximately 0.42 g L−1 h−1. This is significantly
higher compared to batch experiments conducted on mineral salt
medium (0.09 g L−1 h−1).
A maximum theoretical yield of 0.5 g g−1 has been calculated for P
(3HB) on sucrose [22]. Kulpreecha et al. [19] reported a yield of 0.37 g
P(3HB) per g sucrose with B. megaterium on cane molasses. Based on the
sugar content, a yield of 0.41 g P(3HB) per g sucrose can be calculated
for our experiments on DSBM; however, this is rather an estimation,
since there are – to a minor extent – additional carbon sources available
in DSBM, such as lactic acid, but probably also other, yet unidentified,
organic compounds.
As expected, addition of NaOH was required for pH control during
the first 8–10 h due to metabolic activity of B. megaterium. It can be
assumed that the decline in pH was due to the consumption of free
ammonium present in DSBM (Table 1). During the later stage of the
process, the increased alkalinity due to lactate consumption had to be
compensated with H2SO4.
The nitrogen content of desugarized sugar beet molasses is usually
between 18 and 20 g L−1 (Table 1). Therefore, it is impracticable to
induce PHA synthesis by nitrogen limitation; however, the low phos-
phorus concentration available in DSBM of 0.02% (Table 1) allows
employing a phosphorus limitation strategy. The phosphate
M.T. Schmid, et al. Process Biochemistry 86 (2019) 9–15

Fig. 1. Growth curves (OD600) of B. megaterium on desugarized sugar beet molasses (DSBM) in different concentrations cultivated in shaking flasks. Concentrations in
the range of 15–17% (w/w) appear to be optimal. Above 30% DSBM (w/w) growth is inhibited.

Table 2 3.3. Process stability aspects

Influence of pH on the formation of biomass (CDM) and poly(3-hydro-
xybutyrate) P(3HB) of B. megaterium cultivated on DSBM (15%) in bioreactors. Ensuring consistent biomass and P(3HB) formation is crucial in
Optimal growth conditions lie in the range between pH 7 and 8. No growth was order to establish a process with consistent yields and product prop-
observed at pH 6 and 8.5. erties. Endospore formation and variation of substrate composition
Cultivation time were identified as major potential factors affecting process stability.
Bacteria of the genus Bacillus are defined by their ability to form
pH 12 h 24 h 45 h endospores [23]. Spore formation during cultivation implies serious
CDM P(3HB) CDM P(3HB) CDM P(3HB)
issues regarding the stability and reproducibility of the production
[g L−1] [g L−1] [g L−1] [g L−1] [g L−1] [g L−1] process. During the experiments, no endospore formation of B. mega-
terium uyuni S29 CECT 7922 was observed. This is in accordance with
6.0 0.4 0 0.4 0 0.4 0 the observations of Rodríguez-Contreras et al. [21]. It can therefore be
7.0 17.5 6.6 27.0 14.0 22.9 8.7
8.0 15.4 4.5 26.7 14.1 21.8 9.2
assumed that no unwanted sporulation will occur during large scale
8.5 0.2 0 0.4 0 0.4 0 production under these conditions.
The composition of desugarized sugar beet molasses can vary be-
tween different batches (Table 1). Therefore, additional experiments
concentration was below the detection limit of the applied method after were conducted to compare batches from 2016 and 2017. No significant
1 h of cultivation. difference in biomass and P(3HB) formation could be observed
When cultivated on desugarized sugar beet molasses medium, B. (Table 3). The maximum biomass and P(3HB) content was reached after
megaterium accumulated P(3HB) also during exponential growth, 24 h, confirming the preceding results. This indicates that variations in
maintaining a P(3HB) content of approximately 50 g per 100 g CDM quality of desugarized sugar beet molasses, as they can be expected
(see Fig. 2, B). In contrast, on mineral salt medium a distinct growth during normal processing, may only have a minor impact on growth
phase could be observed, where only low amounts of P(3HB) (0.5 g and P(3HB) production in the bioprocess. However, the concentration
L−1) were formed. The actual phase of polymer formation only began of carbon sources can vary depending on the performance of the mo-
after the phosphate had been depleted. The ability of B. megaterium to lasses desugarization process and the amount of organic acids that are
produce P(3HB) during growth on mineral salt medium with glucose as formed during the sugar production, which end up in molasses. It is
carbon source was also observed by Rodríguez-Contreras et al. [21]. therefore recommended to adjust the sucrose concentration to a value
The observations above suggest that low phosphate concentrations between 26 g L−1 and 30 g L−1 to ensure similar conditions for each
in desugarized sugar beet molasses medium a priori elicit limiting fermentation.
conditions for B. megaterium. In an additional bioreactor cultivation the Based on these findings desugarized sugar beet molasses appears to
phosphate concentration in desugarized sugar beet molasses medium be a promising substrate for industrial scale production of P(3HB) with
was adjusted to that of the mineral salt medium by adding 0.18 g L−1 B. megaterium, because it is available in large amounts and variations in
KH2PO4. This resulted in an increased CDM of 19.46 g L−1 with a re- composition can be easily handled. Furthermore, compared to other
duced P(3HB) content of 8.19 g L−1 (Table 2). alternative substrates, it can be used directly without pre-treatment or
Based on these results, we suggest focusing further research toward additional nutrients, further simplifying the process and improving the
fed-batch cultivation, which has been demonstrated to improve biomass economical viability.
concentration and overall PHB productivity in similar processes [19].
However, there may be limitations due to the high salt content and
3.4. Polymer characteristics
viscosity of DSBM.

Data from GC analysis and NMR reveal that a P(3HB) homopolymer

was produced in all experiments. The amount of extractable P(3HB)
and the molecular mass varied dependent on the extraction method.

12
M.T. Schmid, et al. Process Biochemistry 86 (2019) 9–15

Fig. 2. Cultivation of B. megaterium on MSM (A) and DSBM-Medium (B) cultivated in parallel bioreactors. The comparison of biomass (CDM), substrate and P(3HB)
concentrations shows that DSBM-medium results in higher biomass and P(3HB) formation.

Table 3 When using untreated biomass, only 30% of the P(3HB) could be ex-
Cultivation of B. megaterium on two batches of desugarized sugar beet molasses tracted. GPC analysis found that two fractions of P(3HB) were present
(DSBM16 and DSBM17) in parallel bioreactors. Values after 24 h are similar, (Mn): one at a low concentration with a very high molecular mass of
indicating a negligible effect of different batches on accumulation of biomass 722 kDa and a second one with 189 kDa as main fraction. Rodríguez-
(CDM) and poly(3-hydroxybutyrate) (P(3HB)). Contreras et al. also describe the presence of two P(3HB) fractions, with
Unit DSBM16 DSBM17 molecular masses of 600 kDa and 125 kDa, with the same strain [21].
Duration [h] 24 24 Biomass pre-treatment by ultrasonication resulted in the extraction
of 90% of the P(3HB). In this case, only one fraction with a molecular
CDM [g L−1] 16.5 ± 0.32 17.2 ± 0.33
P(3HB) [g L−1] 9.2 ± 0.05 10.2 ± 0.04 mass of 119 kDa was obtained. It is assumed that ultrasonication breaks
P(3HB) per CDM [g g−1] 0.55 ± 0.61 0.60 ± 0.08 down the two fractions resulting in a more homogenous molecular mass
distribution and increasing the effectiveness of the chloroform extrac-
tion. A summary and comparison of these results and polydispersity is
shown in Table 4. Analysis revealed that the extracted polymer from

Table 4
Polymer characteristics with reference values for poly(3-hydroxybutyrate) (P(3HB)) producing strains. The properties of the produced polymer (Mw: average mass
distribution of molecular weight; Mn: average number of the molecular mass; PDI: polydispersity index) are comparable to standard literature values.
Organism Mw [kDa] PDI (Mw/Mn) Carbon source Reference

B. megaterium uyuni S29 1001 1.4 Sucrose + lactate Present work

554 2.9
B. megaterium uyuni S29 with pretreatment 339 2.8 Sucrose + lactate Present work
B. megaterium uyuni S29 600–125 1.2–1.5 Glucose [16]
Cupriavidus necator DSM 545 1000 3.2 Glucose + fructose [30]

13
M.T. Schmid, et al.
Fig. 3. NMR spectra of P(3HB) extracted form untreated biomass (A) and pretreated biomass (B). The data show that both samples contained P(3HB) with a 99%
purity.
pre- and untreated biomass consisted of nearly pure P(3HB) as it can be
seen in NMR of Fig. 3.
Molecular mass is an important factor to determine potential
polymer applications. In general, molecular masses in the range be-
tween 50–1000 kDa for P(3HB) have been reported for non-GMO
strains, depending on the organism [24]. Cultivation stage, cultivation
conditions and the type of substrate have been identified as parameters
influencing the molecular mass [25,26]. High molecular weight P(3HB)
has a high industrial potential as it can be melt-processed into various
final forms [27]. Blending PHB with beech wood floor by melt pro-
cessing resulted in the production of biodegradable films and improved
material performance [28]. PHB polymers with a molecular mass of
260 kDa have been demonstrated to work for in-vivo controlled release
applications [29]. Generally, PHB is compatible with blood and tissues
as hydroxybutyrate is a metabolite also found human blood. The ma-
terial is therefore interesting for medical applications [8]. Other im-
portant parameters that determine the polymer properties are glass
transition temperature, melting temperature, degree of crystallinity,
tensile strength and elongation at break [7]. A final general assessment
for the potential applications of P(3HB) produced by B. megaterium from
desugarized sugar beet molasses will only be possible after a complete
characterization of the polymer properties.
4. Conclusion
A novel process for the production of P(3HB) with B. megaterium
uyuni S29 based on desugarized sugar beet molasses was developed.
Desugarized sugar beet molasses proved to be superior to mineral-based
cultivation media. It was demonstrated that consistent CDM and P
14
Process Biochemistry 86 (2019) 9–15
(3HB) production is possible without additional nutrient supply. The
polymer produced consisted of two fractions with a Mn of 722 kDa and
189 kDa, respectively. Differences in composition between batches of
desugarized sugar beet molasses have a negligible effect on growth and
polymer formation. This study shows that DSBM is a potential substrate
for the development of a simple and robust process for the production
of P(3HB).
Acknowledgements
The authors gratefully acknowledge the Austrian Ministry for
Transport, Innovation and Technology (FFG project No. 853424) and
AGRANA Research & Innovation Center GmbH, Tulln, Austria for fi-
nancial support.
References
[1] M.A. Rajaeifar, S. Sadeghzadeh Hemayati, M. Tabatabaei, M. Aghbashlo,
S.B. Mahmoudi, A review on beet sugar industry with a focus on implementation of
waste-to-energy strategy for power supply, Renew. Sustain. Energy Rev. 103 (2019)
423–442, https://doi.org/10.1016/j.rser.2018.12.056.
[2] M. Asadi, Beet-Sugar Handbook, John Wiley & Sons, Hoboken, New Jersey, 2007, p.
480, https://doi.org/10.1002/9780471790990.ch4.
[3] G.M. Walker, Yeast Physiology and Biotechnology, John Wiley & Sons, Chichester,
1998, pp. 265–301.
[4] V. Bollert, R. Csuk, F. Hirsinger, F. Schierbaum, B. Zoebelein, H. Zoebelein,
Dictionary of Renewable Resources, 2nd edition, Wiley-VCH Verlag GmbH, 2001, p.
287.
[5] P. Kongjan, S. O-Thong, I. Angelidaki, Hydrogen and methane production from
desugared molasses using a two-stage thermophilic anaerobic process, Eng. Life Sci.
13 (2013) 118–125, https://doi.org/10.1002/elsc.201100191.
[6] M. Koller, L. Maršálek, M.M. de Sousa Dias, G. Braunegg, Producing microbial
polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner, Nat.
M.T. Schmid, et al.
Biotechnol. 37 (2017) 24–38, https://doi.org/10.1016/j.nbt.2016.05.001.
[7] E. Bugnicourt, P. Cinelli, A. Lazzeri, V. Alvarez, Polyhydroxyalkanoate (PHA): re-
view of synthesis, characteristics, processing and potential applications in packa-
ging, Express Polym. Lett. 8 (2014) 791–808, https://doi.org/10.3144/
expresspolymlett.2014.82.
[8] K.P. Luef, F. Stelzer, F. Wiesbrock, Poly(hydroxy alkanoate)s in medical applica-
tions, Chem. Biochem. Eng. Q. 29 (2015) 287–297, https://doi.org/10.15255/
CABEQ.2014.2261.
[9] S.P. Valappil, A.R. Boccaccini, C. Bucke, I. Roy, Polyhydroxyalkanoates in Gram-
positive bacteria: Insights from the genera Bacillus and Streptomyces, Antonie van
Leeuwenhoek, Int. J. Gen. Mol. Microbiol. 91 (2007) 1–17, https://doi.org/10.
1007/s10482-006-9095-5.
[10] S. Rodriguez-Perez, A. Serrano, A.A. Pantión, B. Alonso-Fariñas, Challenges of
scaling-up PHA production from waste streams. A review, J. Environ. Manage. 205
(2018) 215–230, https://doi.org/10.1016/j.jenvman.2017.09.083.
[11] A. Burniol-Figols, C. Varrone, A.E. Daugaard, S.B. Le, I.V. Skiadas, H.N. Gavala,
Polyhydroxyalkanoates (PHA) production from fermented crude glycerol: study on
the conversion of 1,3-propanediol to PHA in mixed microbial consortia, Water Res.
128 (2018) 255–266, https://doi.org/10.1016/j.watres.2017.10.046.
[12] C. Haas, V. Steinwandter, E. Diaz De Apodaca, B. Maestro Madurga, M. Smerilli,
T. Dietrich, M. Neureiter, Production of PHB from chicory roots – comparison of
three Cupriavidus necator strains, Chem. Biochem. Eng. Q. 29 (2015) 99–112,
https://doi.org/10.15255/CABEQ.2014.2250.
[13] S. Follonier, R. Riesen, M. Zinn, Pilot-scale production of functionalized mcl-PHA
from grape pomace supplemented with fatty acids, Chem. Biochem. Eng. Q. 29
(2015) 113–121, https://doi.org/10.15255/CABEQ.2014.2251.
[14] A. Elain, A. Le Grand, Y.-M. Corre, M. Le Fellic, N. Hachet, V. Le Tilly, P. Loulergue,
J.-L. Audic, S. Bruzaud, Valorisation of local agro-industrial processing waters as
growth media for polyhydroxyalkanoates (PHA) production, Ind. Crops Prod. 80
(2016) 1–5, https://doi.org/10.1016/j.indcrop.2015.10.052.
[15] L. Urbina, P. Wongsirichot, M.Á. Corcuera, N. Gabilondo, A. Eceiza, J. Winterburn,
A. Retegi, Application of cider by-products for medium chain length poly-
hydroxyalkanoate production by Pseudomonas putida KT2440, Eur. Polym. J. 108
(2018) 1–9, https://doi.org/10.1016/j.eurpolymj.2018.08.020.
[16] A. Rodríguez-Contreras, M. Koller, M. Miranda-de Sousa Dias, M. Calafell-Monfort,
G. Braunegg, M.S.S. Marqués-Calvo, High production of poly(3-hydroxybutyrate)
from a wild Bacillus megaterium Bolivian strain, J. Appl. Microbiol. 114 (2013)
1378–1387, https://doi.org/10.1111/jam.12151.
[17] A. Rodríguez-Contreras, M. Koller, G. Braunegg, M.S. Marqués-Calvo, Poly[(R)-3-
hydroxybutyrate] production under different salinity conditions by a novel Bacillus
megaterium strain, Nat. Biotechnol. 33 (2016) 73–77, https://doi.org/10.1016/j.
15
Process Biochemistry 86 (2019) 9–15
nbt.2015.08.006.
[18] S. Taguchi, T. Ooi, K. Mizuno, H. Matsusaki, Advances and needs for endotoxin-free
production strains, Appl. Microbiol. Biotechnol. 99 (2015) 9349–9360, https://doi.
org/10.1007/s00253-015-6947-9.
[19] S. Kulpreecha, A. Boonruangthavorn, B. Meksiriporn, N. Thongchul, Inexpensive
fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate
of Bacillus megaterium, J. Biosci. Bioeng. 107 (2009) 240–245, https://doi.org/10.
1016/j.jbiosc.2008.10.006.
[20] P. Furrer, R. Hany, D. Rentsch, A. Grubelnik, K. Ruth, S. Panke, M. Zinn,
Quantitative analysis of bacterial medium-chain-length poly([R]-3-hydroxyalk-
anoates) by gas chromatography, J. Chromatogr. A 1143 (2007) 199–206, https://
doi.org/10.1016/j.chroma.2007.01.002.
[21] A. Rodríguez-Contreras, M. Koller, M. Miranda, D.S. Dias, Novel poly[(R)-3-hy-
droxybutyrate]-producing bacterium isolated from a Bolivian hypersaline lake,
Food Technol. Biotechnol. 51 (2013) 123–130.
[22] T. Yamane, Yield of poly-D-(-)-3-hydroxybutuyrate from various carbon sources: a
theroratical study, Biotechnol. Bioeng. (1993) 165–170.
[23] P. Setlow, Germination of spores of Bacillus species: what we know and do not
know, J. Bacteriol. 196 (2014) 1297–1305, https://doi.org/10.1128/JB.01455-13.
[24] L.L. Madison, G.W. Huisman, Metabolic engineering of poly (3-hydroalkanoates):
from DNA to plastic, Microbiol. Mol. Biol. (1999) 21–53.
[25] G. Chen, W.J. Page, The effect of substrate on the molecular weight of poly-β-hy-
droxybutyrate produced by Azotobacter vinelandii UWD, Biotechnol. Lett. 16 (1994)
155–160, https://doi.org/10.1007/BF01021663.
[26] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalk-
anoates: biological polyesters, Prog. Polym. Sci. 25 (2000) 1503–1555, https://doi.
org/10.1016/S0079-6700(00)00035-6.
[27] K.D. Snell, O.P. Peoples, PHA bioplastic: a value-added coproduct for biomass
biorefineries, Biofuels, Bioprod. Biorefining. (2009) 456–467, https://doi.org/10.
1002/bbb.161.
[28] A. Gregorova, R. Wimmer, M. Hrabalova, M. Koller, T. Ters, N. Mundigler, Effect of
surface modification of beech wood flour on mechanical and thermal properties of
poly (3-hydroxybutyrate)/wood flour composites, Holzforschung 63 (2009)
565–570, https://doi.org/10.1515/HF.2009.098.
[29] C.W. Pouton, S. Akhtarb, Biosynthetic polyhydroxyalkanoates and their potential in
drug delivery, Adv. Drug Deliver. Rev. 18 (1996) 133–162, https://doi.org/10.
1016/0169-409X(95)00092-L.
[30] M.L. Fiorese, F. Freitas, J. Pais, A.M. Ramos, G.M.F. De Aragão, M.A.M. Reis,
Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by sol-
vent extraction with 1,2-propylene carbonate, Eng. Life Sci. 9 (2009) 454–461,
https://doi.org/10.1002/elsc.200900034.