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Utilization of desugarized sugar beet molasses for the production of poly(3-hydroxybutyrate) by halophilic Bacillus megaterium uyuni S29
Process Biochemistry 86 (2019) 9–15
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Utilization of desugarized sugar beet molasses for the production of poly(3hydroxybutyrate) by halophilic Bacillus megaterium uyuni S29
T
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 AGRANA Research & Innovation Center GmbH, Tulln, Austria c University of Technology, Graz, Institute for Chemistry and Technology of Materials, Graz, Austria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus megaterium Desugarized sugar beet molasses Poly(3-hydroxybutyrate) Halophilic fermentation Sugar industry by-product
Desugarized sugar beet molasses is a high-salinity side stream after fractionation of beet molasses by chromatography. It could be demonstrated that this by-product of the sugar industry is a suitable substrate for the production of poly(3-hydroxybutyrate) with the halophilic bacterium Bacillus megaterium uyuni S29. 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 24 h of batch cultivation. Utilization of a desugarized sugar beet molasses medium resulted in a two- to threefold 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 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 production, while the remaining 80% are produced from sugar cane [1]. On the 30th of September 2017 the European quota system for sugar production 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 produced 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 pharmaceutical 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
⁎
molasses as it undergoes an additional chromatographic desugarization 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 economic value than regular sugar beet molasses and is currently used as fertilizer and as nutrient additive for animal feed [2]. AGRANA processes 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 content 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 (approximately 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: [email protected] (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.
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2.2. Medium for cultivation
Table 1 Composition of regular sugar beet molasses and batches of desugarized sugar beet molasses from 2016 (DSBM16) and 2017 (DSBM17). Unit
Sugar beet molasses 8.3
DSBM16 6.6
DSBM17 7.5
% % % % % % % g kg−1 g kg−1 mg kg−1 % %
82.0 50.5 10.9 1.8 0.07 11.0 0.02 13.4 32.8 111 2.7 0.5
70.7 17.6 20.4 2.1 0.05 13.0 < 0.02 25.2 50.3 235 6.2 1.3
71.6 13.2 25.5 1.8 0.05 11.4 < 0.02 26.2 74.2 255 4.0 0.9
pH Dry matter (Brix) Sucrose Ash Nitrogen Ammonium Protein Phosphorus Sodium Potassium Calcium Lactate Acetate
For comparison, mineral salt medium (MSM) as described by Kulpreecha et al. [19] was used, containing: CaCl2: 0.02 g L−1, MgSO4·7 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 solution composed of ZnSO4·7 H2O: 0.1 g L−1, H3BO3: 0.3 g L−1, CoCl2·6 H2O: 0.2 g L−1, CuSO4: 0.006 g L−1, NiCl2·6 H2O: 0.02 g L−1, Na2MoO4·2 H2O: 0.03 g L−1, MnCl2·2 H2O: 0.025 g L−1. In order to ensure phosphorous limitation the concentration of KH2PO4 was reduced from 2.0 to 0.3 g L−1. The pH was adjusted to 7.0 before inoculation. Desugarized sugar beet molasses was provided by AGRANA (Tulln, Austria). Batches from 2016 (DSBM16) and 2017 (DSBM17) were used. The compositions are presented in Table 1. Due to its low water activity the material is stable and can be stored at room temperature for a longer period of time. For the use as fermentation substrate desugarized sugar beet molasses was diluted with deionized water to a concentration of 15% (w/w) based on DSBM wet mass and the pH was set to 7.0 with 5 M H2SO4 and 5 M NaOH. No additional nutrients were added. The DSBM medium was sterilized at 121 °C for 20 min.
for conventional applications like yeast fermentation. It has also been suggested as a substrate for biohydrogen production [5]. Polyhydroxyalkanoates (PHA) are microbial polyesters synthesized by Archaea and various Gram-positive and Gram-negative bacteria under unbalanced nutrient conditions [6]. PHA can be used for the production of biobased and biodegradable materials. Due to its biocompatibility, applications range from agriculture, packaging material to medical applications [7–9]; however, high production costs currently reduce the commercial competitiveness of the polymer. Around 40–48% of the costs arise from the raw material providing the carbon source [10]. The usage of conventional starch and/or sugar containing raw materials is increasingly criticized as they compete with food production. As a replacement, low-cost by-products from biomass processing, such as crude glycerol [11], chicory roots [12], grape pomace [13], fruit processing water [14] or by-products of the cider industry [15] have been suggested to reduce production costs. Many alternative carbon sources, however, are often not available in sufficient quantities and may also require intensive pre-treatment. This does not apply for desugarized sugar beet molasses, since it can be provided in large quantities and is not used for nutritional purposes. Bacillus megaterium uyuni S29 CECT 7922 was selected for this study because the high salinity requires the usage of halotolerant bacteria. This strain was isolated from the Bolivian salt lake Uyuni and is reported to accumulate solely poly(3-hydroxybutyrate) at concentrations up to 60 g per 100 g cell dry mass (CDM) [16]. It has been described to grow at NaCl concentrations varying from 0.5 to 15% [17]. In addition to these unique abilities, the lack of endotoxins in the Gram-positive cell wall may be an advantage for biomedical applications of the polymer [18,9]. In this study we evaluated, whether desugarized sugar beet molasses is a suitable substrate for the production of P(3HB) with B. megaterium uyuni S29. In addition, we proposed a simple and reproducible fermentation process and characterized the produced polymer.
2.3. Cultivation Shake flask experiments were carried out in 300 mL shake flasks with 100 mL fermentation medium. The inoculation was conducted by transferring 1 mL of pre-culture to each flask. The cultures were incubated at 35 °C and 130 rpm. All experiments were performed as triplicates. Fermentations in bioreactors were conducted in a parallel benchtop system (DASGIP, Eppendorf) with a total reactor volume of 1.4 L and a working volume of 800 mL. For inoculum preparation, 300 mL shake flasks with 100 mL fermentation medium were inoculated from a liquid pre-culture and incubated over night at 35 °C in a rotary incubator at 130 rpm (Infors Multitron Incubator shaker). For the inoculation of 750 mL of fermentation medium (MSM or DSBM medium), 50 mL of inoculum were transferred into the bioreactor. Relevant process parameters like pH-value, dissolved oxygen level (DO), consumption of acid or base were continuously monitored. For pH-control, 5 M NaOH and 5 M H2SO4 were used. The DO (percentage of maximum oxygen saturation) was set at 20% and controlled by varying the stirrer speed while the airflow (compressed air) was fixed at 30.5 L h−1. The experiments were conducted in duplicates. 2.4. Extraction of P(3HB) For extraction the biomass was harvested by centrifuging for 20 min at 10,000 g (Sorvall Lynx4000 centrifuge). Untreated biomass and cells pretreated by ultrasonication were compared regarding the extraction 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 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) 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 performed overnight with 90 mL of chloroform (VWR; ≥ 99.8%) in an automatic extraction unit (Soxtherm 2000). The extraction was conducted at a temperature of 240 °C and was divided in a cooking phase (1 h) and an extraction phase (1.5 h). After extraction P(3HB) was precipitated by adding 96% ethanol (10 times the amount of chloroform) and harvested by filtration.
2. Materials and methods 2.1. Microorganism Bacillus megaterium uyuni CECT 7922 was cultivated at 35 °C on a modified beef extract medium (meat extract: 10 g L−1, peptone: 10 g L−1; NaCl: 5 g L−1) with 1% agar, and transferred to a new plate once a week. Cultures can be stored at 4 °C for at least one month. Pre-cultures were prepared in 300 mL baffled shake flasks by inoculation with single colonies from a solid plate to 100 mL modified beef extract medium. 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
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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 phosphorous 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 refractive 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, respectively. 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 (SPD20A) 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.
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. Between 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 molassesmedium 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 maximum 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 (consumed) 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 phosphorus concentration available in DSBM of 0.02% (Table 1) allows employing a phosphorus limitation strategy. The phosphate
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 fermentation substrate. In order to determine the optimum level of dilution, 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 concentrations 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
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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.
3.3. Process stability aspects
Table 2 Influence of pH on the formation of biomass (CDM) and poly(3-hydroxybutyrate) P(3HB) of B. megaterium cultivated on DSBM (15%) in bioreactors. Optimal growth conditions lie in the range between pH 7 and 8. No growth was observed at pH 6 and 8.5.
Ensuring consistent biomass and P(3HB) formation is crucial in order to establish a process with consistent yields and product properties. Endospore formation and variation of substrate composition were identified as major potential factors affecting process stability. Bacteria of the genus Bacillus are defined by their ability to form endospores [23]. Spore formation during cultivation implies serious issues regarding the stability and reproducibility of the production process. During the experiments, no endospore formation of B. megaterium uyuni S29 CECT 7922 was observed. This is in accordance with the observations of Rodríguez-Contreras et al. [21]. It can therefore be assumed that no unwanted sporulation will occur during large scale production under these conditions. The composition of desugarized sugar beet molasses can vary between different batches (Table 1). Therefore, additional experiments were conducted to compare batches from 2016 and 2017. No significant difference in biomass and P(3HB) formation could be observed (Table 3). The maximum biomass and P(3HB) content was reached after 24 h, confirming the preceding results. This indicates that variations in quality of desugarized sugar beet molasses, as they can be expected during normal processing, may only have a minor impact on growth and P(3HB) production in the bioprocess. However, the concentration of carbon sources can vary depending on the performance of the molasses desugarization process and the amount of organic acids that are formed during the sugar production, which end up in molasses. It is therefore recommended to adjust the sucrose concentration to a value between 26 g L−1 and 30 g L−1 to ensure similar conditions for each fermentation. Based on these findings desugarized sugar beet molasses appears to be a promising substrate for industrial scale production of P(3HB) with B. megaterium, because it is available in large amounts and variations in composition can be easily handled. Furthermore, compared to other alternative substrates, it can be used directly without pre-treatment or additional nutrients, further simplifying the process and improving the economical viability.
Cultivation time pH
6.0 7.0 8.0 8.5
12 h
24 h
45 h
CDM [g L−1]
P(3HB) [g L−1]
CDM [g L−1]
P(3HB) [g L−1]
CDM [g L−1]
P(3HB) [g L−1]
0.4 17.5 15.4 0.2
0 6.6 4.5 0
0.4 27.0 26.7 0.4
0 14.0 14.1 0
0.4 22.9 21.8 0.4
0 8.7 9.2 0
concentration was below the detection limit of the applied method after 1 h of cultivation. When cultivated on desugarized sugar beet molasses medium, B. megaterium accumulated P(3HB) also during exponential growth, maintaining a P(3HB) content of approximately 50 g per 100 g CDM (see Fig. 2, B). In contrast, on mineral salt medium a distinct growth phase could be observed, where only low amounts of P(3HB) (0.5 g L−1) were formed. The actual phase of polymer formation only began after the phosphate had been depleted. The ability of B. megaterium to produce P(3HB) during growth on mineral salt medium with glucose as carbon source was also observed by Rodríguez-Contreras et al. [21]. The observations above suggest that low phosphate concentrations in desugarized sugar beet molasses medium a priori elicit limiting conditions for B. megaterium. In an additional bioreactor cultivation the phosphate concentration in desugarized sugar beet molasses medium was adjusted to that of the mineral salt medium by adding 0.18 g L−1 KH2PO4. This resulted in an increased CDM of 19.46 g L−1 with a reduced P(3HB) content of 8.19 g L−1 (Table 2). Based on these results, we suggest focusing further research toward fed-batch cultivation, which has been demonstrated to improve biomass concentration and overall PHB productivity in similar processes [19]. However, there may be limitations due to the high salt content and viscosity of DSBM.
3.4. Polymer characteristics 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
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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.
When using untreated biomass, only 30% of the P(3HB) could be extracted. GPC analysis found that two fractions of P(3HB) were present (Mn): one at a low concentration with a very high molecular mass of 722 kDa and a second one with 189 kDa as main fraction. RodríguezContreras et al. also describe the presence of two P(3HB) fractions, with molecular masses of 600 kDa and 125 kDa, with the same strain [21]. Biomass pre-treatment by ultrasonication resulted in the extraction of 90% of the P(3HB). In this case, only one fraction with a molecular mass of 119 kDa was obtained. It is assumed that ultrasonication breaks down the two fractions resulting in a more homogenous molecular mass distribution and increasing the effectiveness of the chloroform extraction. A summary and comparison of these results and polydispersity is shown in Table 4. Analysis revealed that the extracted polymer from
Table 3 Cultivation of B. megaterium on two batches of desugarized sugar beet molasses (DSBM16 and DSBM17) in parallel bioreactors. Values after 24 h are similar, indicating a negligible effect of different batches on accumulation of biomass (CDM) and poly(3-hydroxybutyrate) (P(3HB)). Duration
Unit [h]
DSBM16 24
DSBM17 24
CDM P(3HB) P(3HB) per CDM
[g L−1] [g L−1] [g g−1]
16.5 ± 0.32 9.2 ± 0.05 0.55 ± 0.61
17.2 ± 0.33 10.2 ± 0.04 0.60 ± 0.08
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 554 339 600–125 1000
1.4 2.9 2.8 1.2–1.5 3.2
Sucrose + lactate
Present work
Sucrose + lactate Glucose Glucose + fructose
Present work [16] [30]
B. megaterium uyuni S29 with pretreatment B. megaterium uyuni S29 Cupriavidus necator DSM 545
13
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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.
(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).
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 between 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 processing 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 material is therefore interesting for medical applications [8]. Other important 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.
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 financial 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.
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
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Utilization of desugarized sugar beet molasses for the production of poly(3hydroxybutyrate) by halophilic Bacillus megaterium uyuni S29
T
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 AGRANA Research & Innovation Center GmbH, Tulln, Austria c University of Technology, Graz, Institute for Chemistry and Technology of Materials, Graz, Austria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus megaterium Desugarized sugar beet molasses Poly(3-hydroxybutyrate) Halophilic fermentation Sugar industry by-product
Desugarized sugar beet molasses is a high-salinity side stream after fractionation of beet molasses by chromatography. It could be demonstrated that this by-product of the sugar industry is a suitable substrate for the production of poly(3-hydroxybutyrate) with the halophilic bacterium Bacillus megaterium uyuni S29. 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 24 h of batch cultivation. Utilization of a desugarized sugar beet molasses medium resulted in a two- to threefold 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 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 production, while the remaining 80% are produced from sugar cane [1]. On the 30th of September 2017 the European quota system for sugar production 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 produced 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 pharmaceutical 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
⁎
molasses as it undergoes an additional chromatographic desugarization 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 economic value than regular sugar beet molasses and is currently used as fertilizer and as nutrient additive for animal feed [2]. AGRANA processes 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 content 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 (approximately 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: [email protected] (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.
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2.2. Medium for cultivation
Table 1 Composition of regular sugar beet molasses and batches of desugarized sugar beet molasses from 2016 (DSBM16) and 2017 (DSBM17). Unit
Sugar beet molasses 8.3
DSBM16 6.6
DSBM17 7.5
% % % % % % % g kg−1 g kg−1 mg kg−1 % %
82.0 50.5 10.9 1.8 0.07 11.0 0.02 13.4 32.8 111 2.7 0.5
70.7 17.6 20.4 2.1 0.05 13.0 < 0.02 25.2 50.3 235 6.2 1.3
71.6 13.2 25.5 1.8 0.05 11.4 < 0.02 26.2 74.2 255 4.0 0.9
pH Dry matter (Brix) Sucrose Ash Nitrogen Ammonium Protein Phosphorus Sodium Potassium Calcium Lactate Acetate
For comparison, mineral salt medium (MSM) as described by Kulpreecha et al. [19] was used, containing: CaCl2: 0.02 g L−1, MgSO4·7 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 solution composed of ZnSO4·7 H2O: 0.1 g L−1, H3BO3: 0.3 g L−1, CoCl2·6 H2O: 0.2 g L−1, CuSO4: 0.006 g L−1, NiCl2·6 H2O: 0.02 g L−1, Na2MoO4·2 H2O: 0.03 g L−1, MnCl2·2 H2O: 0.025 g L−1. In order to ensure phosphorous limitation the concentration of KH2PO4 was reduced from 2.0 to 0.3 g L−1. The pH was adjusted to 7.0 before inoculation. Desugarized sugar beet molasses was provided by AGRANA (Tulln, Austria). Batches from 2016 (DSBM16) and 2017 (DSBM17) were used. The compositions are presented in Table 1. Due to its low water activity the material is stable and can be stored at room temperature for a longer period of time. For the use as fermentation substrate desugarized sugar beet molasses was diluted with deionized water to a concentration of 15% (w/w) based on DSBM wet mass and the pH was set to 7.0 with 5 M H2SO4 and 5 M NaOH. No additional nutrients were added. The DSBM medium was sterilized at 121 °C for 20 min.
for conventional applications like yeast fermentation. It has also been suggested as a substrate for biohydrogen production [5]. Polyhydroxyalkanoates (PHA) are microbial polyesters synthesized by Archaea and various Gram-positive and Gram-negative bacteria under unbalanced nutrient conditions [6]. PHA can be used for the production of biobased and biodegradable materials. Due to its biocompatibility, applications range from agriculture, packaging material to medical applications [7–9]; however, high production costs currently reduce the commercial competitiveness of the polymer. Around 40–48% of the costs arise from the raw material providing the carbon source [10]. The usage of conventional starch and/or sugar containing raw materials is increasingly criticized as they compete with food production. As a replacement, low-cost by-products from biomass processing, such as crude glycerol [11], chicory roots [12], grape pomace [13], fruit processing water [14] or by-products of the cider industry [15] have been suggested to reduce production costs. Many alternative carbon sources, however, are often not available in sufficient quantities and may also require intensive pre-treatment. This does not apply for desugarized sugar beet molasses, since it can be provided in large quantities and is not used for nutritional purposes. Bacillus megaterium uyuni S29 CECT 7922 was selected for this study because the high salinity requires the usage of halotolerant bacteria. This strain was isolated from the Bolivian salt lake Uyuni and is reported to accumulate solely poly(3-hydroxybutyrate) at concentrations up to 60 g per 100 g cell dry mass (CDM) [16]. It has been described to grow at NaCl concentrations varying from 0.5 to 15% [17]. In addition to these unique abilities, the lack of endotoxins in the Gram-positive cell wall may be an advantage for biomedical applications of the polymer [18,9]. In this study we evaluated, whether desugarized sugar beet molasses is a suitable substrate for the production of P(3HB) with B. megaterium uyuni S29. In addition, we proposed a simple and reproducible fermentation process and characterized the produced polymer.
2.3. Cultivation Shake flask experiments were carried out in 300 mL shake flasks with 100 mL fermentation medium. The inoculation was conducted by transferring 1 mL of pre-culture to each flask. The cultures were incubated at 35 °C and 130 rpm. All experiments were performed as triplicates. Fermentations in bioreactors were conducted in a parallel benchtop system (DASGIP, Eppendorf) with a total reactor volume of 1.4 L and a working volume of 800 mL. For inoculum preparation, 300 mL shake flasks with 100 mL fermentation medium were inoculated from a liquid pre-culture and incubated over night at 35 °C in a rotary incubator at 130 rpm (Infors Multitron Incubator shaker). For the inoculation of 750 mL of fermentation medium (MSM or DSBM medium), 50 mL of inoculum were transferred into the bioreactor. Relevant process parameters like pH-value, dissolved oxygen level (DO), consumption of acid or base were continuously monitored. For pH-control, 5 M NaOH and 5 M H2SO4 were used. The DO (percentage of maximum oxygen saturation) was set at 20% and controlled by varying the stirrer speed while the airflow (compressed air) was fixed at 30.5 L h−1. The experiments were conducted in duplicates. 2.4. Extraction of P(3HB) For extraction the biomass was harvested by centrifuging for 20 min at 10,000 g (Sorvall Lynx4000 centrifuge). Untreated biomass and cells pretreated by ultrasonication were compared regarding the extraction 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 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) 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 performed overnight with 90 mL of chloroform (VWR; ≥ 99.8%) in an automatic extraction unit (Soxtherm 2000). The extraction was conducted at a temperature of 240 °C and was divided in a cooking phase (1 h) and an extraction phase (1.5 h). After extraction P(3HB) was precipitated by adding 96% ethanol (10 times the amount of chloroform) and harvested by filtration.
2. Materials and methods 2.1. Microorganism Bacillus megaterium uyuni CECT 7922 was cultivated at 35 °C on a modified beef extract medium (meat extract: 10 g L−1, peptone: 10 g L−1; NaCl: 5 g L−1) with 1% agar, and transferred to a new plate once a week. Cultures can be stored at 4 °C for at least one month. Pre-cultures were prepared in 300 mL baffled shake flasks by inoculation with single colonies from a solid plate to 100 mL modified beef extract medium. 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
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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 phosphorous 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 refractive 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, respectively. 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 (SPD20A) 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.
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. Between 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 molassesmedium 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 maximum 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 (consumed) 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 phosphorus concentration available in DSBM of 0.02% (Table 1) allows employing a phosphorus limitation strategy. The phosphate
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 fermentation substrate. In order to determine the optimum level of dilution, 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 concentrations 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
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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.
3.3. Process stability aspects
Table 2 Influence of pH on the formation of biomass (CDM) and poly(3-hydroxybutyrate) P(3HB) of B. megaterium cultivated on DSBM (15%) in bioreactors. Optimal growth conditions lie in the range between pH 7 and 8. No growth was observed at pH 6 and 8.5.
Ensuring consistent biomass and P(3HB) formation is crucial in order to establish a process with consistent yields and product properties. Endospore formation and variation of substrate composition were identified as major potential factors affecting process stability. Bacteria of the genus Bacillus are defined by their ability to form endospores [23]. Spore formation during cultivation implies serious issues regarding the stability and reproducibility of the production process. During the experiments, no endospore formation of B. megaterium uyuni S29 CECT 7922 was observed. This is in accordance with the observations of Rodríguez-Contreras et al. [21]. It can therefore be assumed that no unwanted sporulation will occur during large scale production under these conditions. The composition of desugarized sugar beet molasses can vary between different batches (Table 1). Therefore, additional experiments were conducted to compare batches from 2016 and 2017. No significant difference in biomass and P(3HB) formation could be observed (Table 3). The maximum biomass and P(3HB) content was reached after 24 h, confirming the preceding results. This indicates that variations in quality of desugarized sugar beet molasses, as they can be expected during normal processing, may only have a minor impact on growth and P(3HB) production in the bioprocess. However, the concentration of carbon sources can vary depending on the performance of the molasses desugarization process and the amount of organic acids that are formed during the sugar production, which end up in molasses. It is therefore recommended to adjust the sucrose concentration to a value between 26 g L−1 and 30 g L−1 to ensure similar conditions for each fermentation. Based on these findings desugarized sugar beet molasses appears to be a promising substrate for industrial scale production of P(3HB) with B. megaterium, because it is available in large amounts and variations in composition can be easily handled. Furthermore, compared to other alternative substrates, it can be used directly without pre-treatment or additional nutrients, further simplifying the process and improving the economical viability.
Cultivation time pH
6.0 7.0 8.0 8.5
12 h
24 h
45 h
CDM [g L−1]
P(3HB) [g L−1]
CDM [g L−1]
P(3HB) [g L−1]
CDM [g L−1]
P(3HB) [g L−1]
0.4 17.5 15.4 0.2
0 6.6 4.5 0
0.4 27.0 26.7 0.4
0 14.0 14.1 0
0.4 22.9 21.8 0.4
0 8.7 9.2 0
concentration was below the detection limit of the applied method after 1 h of cultivation. When cultivated on desugarized sugar beet molasses medium, B. megaterium accumulated P(3HB) also during exponential growth, maintaining a P(3HB) content of approximately 50 g per 100 g CDM (see Fig. 2, B). In contrast, on mineral salt medium a distinct growth phase could be observed, where only low amounts of P(3HB) (0.5 g L−1) were formed. The actual phase of polymer formation only began after the phosphate had been depleted. The ability of B. megaterium to produce P(3HB) during growth on mineral salt medium with glucose as carbon source was also observed by Rodríguez-Contreras et al. [21]. The observations above suggest that low phosphate concentrations in desugarized sugar beet molasses medium a priori elicit limiting conditions for B. megaterium. In an additional bioreactor cultivation the phosphate concentration in desugarized sugar beet molasses medium was adjusted to that of the mineral salt medium by adding 0.18 g L−1 KH2PO4. This resulted in an increased CDM of 19.46 g L−1 with a reduced P(3HB) content of 8.19 g L−1 (Table 2). Based on these results, we suggest focusing further research toward fed-batch cultivation, which has been demonstrated to improve biomass concentration and overall PHB productivity in similar processes [19]. However, there may be limitations due to the high salt content and viscosity of DSBM.
3.4. Polymer characteristics 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
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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.
When using untreated biomass, only 30% of the P(3HB) could be extracted. GPC analysis found that two fractions of P(3HB) were present (Mn): one at a low concentration with a very high molecular mass of 722 kDa and a second one with 189 kDa as main fraction. RodríguezContreras et al. also describe the presence of two P(3HB) fractions, with molecular masses of 600 kDa and 125 kDa, with the same strain [21]. Biomass pre-treatment by ultrasonication resulted in the extraction of 90% of the P(3HB). In this case, only one fraction with a molecular mass of 119 kDa was obtained. It is assumed that ultrasonication breaks down the two fractions resulting in a more homogenous molecular mass distribution and increasing the effectiveness of the chloroform extraction. A summary and comparison of these results and polydispersity is shown in Table 4. Analysis revealed that the extracted polymer from
Table 3 Cultivation of B. megaterium on two batches of desugarized sugar beet molasses (DSBM16 and DSBM17) in parallel bioreactors. Values after 24 h are similar, indicating a negligible effect of different batches on accumulation of biomass (CDM) and poly(3-hydroxybutyrate) (P(3HB)). Duration
Unit [h]
DSBM16 24
DSBM17 24
CDM P(3HB) P(3HB) per CDM
[g L−1] [g L−1] [g g−1]
16.5 ± 0.32 9.2 ± 0.05 0.55 ± 0.61
17.2 ± 0.33 10.2 ± 0.04 0.60 ± 0.08
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 554 339 600–125 1000
1.4 2.9 2.8 1.2–1.5 3.2
Sucrose + lactate
Present work
Sucrose + lactate Glucose Glucose + fructose
Present work [16] [30]
B. megaterium uyuni S29 with pretreatment B. megaterium uyuni S29 Cupriavidus necator DSM 545
13
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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.
(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).
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 between 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 processing 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 material is therefore interesting for medical applications [8]. Other important 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.
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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
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