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Scientific
Publications - Work Done by Microbiology Reader
ABSTRACT The sensitivity of industrial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici to osmotic stress was studied. Growth of A. aceti and G. frateurii was totally inhibited at 0.4 M NaCl concentration, but P. acidipropionici was able to grow on a medium containing 1.2 M NaCl. Addition of glycine betaine to the medium had no detectable osmoprotective effect on A. aceti and G. frateurii cultivations in elevated NaCl concentrations, but it enabled cells of P. acidipropionici to achieve faster the maximum specific growth rate after the prolonged lag phase and therefore to gain faster the final biomass and product concentrations. The final concentrations of biomass and product of P. acidipropionici were the same as for the cultivations of the bacterium without NaCl and glycine betaine present in the medium. Intracellular accumulation of glycine betaine was detected in P. acidipropionici cells cultivated in the medium containing glycine betaine. The amount accumulated increased with NaCl concentration, suggesting that glycine betaine plays an important role in the osmoadaptation.
Introduction The economic feasibility of fermentation processes is often limited by low productivity due to low cell densities. In fermentation processes, cells are exposed to osmotic stress caused by medium components and metabolic products. Cells respond to increased osmolarity by spontaneous water loss, which may inactivate the enzymatic reactions inside the cells and thus be a limiting factor for both growth and production. Several process alternatives have been proposed to avoid substrate and product inhibition, including stepwise substrate addition by a fed batch process and continuous withdrawal of the product by a cell recycle process. Although improvements in productivity have been achieved, the cell loss due to osmotic stress has not been avoided. Bacteria minimize water loss by adjusting their turgor pressure to the external osmotic pressure through the accumulation of inorganic ions and compatible organic solutes. Compatible organic solutes, called osmolytes, when accumulated at high intracellular concentrations are not deleterious to essential biochemical and metabolic functions of the cells. Osmolytes are either synthesized de novo (endogenous osmolytes) or taken up from the environment (exogenous osmolytes). Glycine betaine, choline, proline, ectoine, trehalose, and 3-dimethylsulfoniopropionate (DMSP) are the most studied among the known exogenous osmolytes (Beumer et al. 1994; Cosquer et al. 1999; Jebbar et al. 1995; Skjerdahl et al. 1996). Endogenous osmolytes synthesized by bacteria include a few amino and imino acids such as glycine betaine, glutamate, proline, and ectoine; polyol glucosylglycerol; and the disaccharides trehalose and sucrose (Gouffi et al. 1999). Glycine betaine is a compatible solute found in large amounts in some bacterial cells living in environments with high osmolarity. A transport system specific for glycine betaine has been found in both halophilic (Canovas et al. 1996) and nonhalophilic bacteria (Bae et al. 1993; Park et al. 1995; Peter et al. 1998; Pichereau et al. 1999; Skjerdal et al. 1995) as well as in methanogenic archae (Proctor et al. 1997). Accumulation of glycine betaine increases cell tolerance to high salinity and drought (Beumer et al. 1994; Jebbar et al. 1995; Kets et al. 1996; Peter et al. 1998; Skjerdal et al. 1996). Besides acting as a compatible solute, glycine betaine has an osmoprotective function in some bacteria, stimulating their growth in hyperosmotic environments (Csonka 1989). In addition to glycine betaine transport, Bacillus subtilis (Boch et al. 1994) and Halomonas elongata (Canovas et al. 1996, 1998) reportedly are able to syn‑ thesize glycine betaine from exogenously added precursor choline, and they possess an uptake system specific for choline. Improvement of the productivity of fermentation processes requires an enhancement of the osmotolerance of the production strains. It is of utmost importance therefore to understand the mechanism of osmoadaptation of the bacteria and the osmoprotective effect that the osmolytes have on these bacteria. The objective of the present study was to establish the effects of glycine betaine addition on bacterial cell cultivations in elevated osmolarities created through the addition of NaCl. We examined the osmosensitivity of commercially promising bacterial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici and the effect of glycine betaine addition on their osmotolerance. In addition, we studied the uptake of glycine betaine by resting cells of P. acidipropionici with 14C-labeled glycine betaine.
Materials and methods Organisms, maintenance, and inocula preparation Propionibacterium acidipropionici ATCC 4875, Acetobacter aceti IFO 3281, and Gluconobacter frateurii IFO 3254 were stored as frozen stock cultures containing 10% (v/v) glycerol in 2-ml ampoules at -80 °C. Inocula for osmosensitivity studies was prepared by cultivating P. acidipropionici, A. aceti, and G. frateurii in 250-m1 shake flasks containing 50 ml complex medium at 30 °C for 20-24 h. A. aceti and G. frateurii were shaken at 200 rpm during cultivation in a rotary shaker (Certomat R, B. Braun Biotech International, Germany). Inocula for bioreactor cultivation were prepared by cultivating P. acidipropionici first in 10-m1 test tubes on complex medium for 24 h at 30 °C and then in 250-m1 shake flasks containing 50 ml complex medium for 24 h without shaking. Cells were separated by centrifugation (8,000 rpm, 10 min, Sorval RC 5 C Plus, Du Pont, USA), washed and suspended in 30 ml deionized water to give an initial biomass concentration of 0.14-0.34 g cell dry weight (CDW) per liter in a bioreactor. Inocula for glycine betaine uptake studies with resting cells were prepared by cultivating P. acidipropionici on 50 ml complex medium for 48 h at 30 °C. Cells were separated by centrifugation (8,000 rpm, 10 min, Sorval RC 5 C Plus, Du Pont, USA). Glycine betaine taken up by the cells from the complex medium was washed off with Sorensen buffer solution and washed cells were suspended in deionized water.
Media Complex medium contained yeast extract (Difco, USA) 10 g/1, trypticase peptone (BBL = Becton Dickinson and Company, USA) 10 g/1, KH2PO4 0.125 9/1, MgSO4.7H2O 0.25 g/1 and glucose (Fluka, Switzerland) or glycerol (BBL = Becton Dickinson and Company, USA) 10 g/1. The pH of the medium was adjusted to 5.8 for A. aceti and G. frateurii and to 6.8 for P. acidipropionici cultivations. Mineral medium was prepared according to Verduyn et al. (1992) and contained NH4(SO4)2 5.0 g/1, KH2P04 3.0 g/1, MgSO4 7H2O 0.5 g/1, EDTA 15.0 mg/1, ZnSO4 7H2O 4.5 mg/l, COC12 6H2O 0.3 g/1, MnC12.4H2O 1.03 g/1, CUSO4 5H2O 0.3 g/1, CaC122142O 4.5 g/1, FeSO4.7H2O 3.0 g/1, NaMo042H2O 0.4 g/1, H3BO4 1.0 mg/1, KI 0.1 mg/1, and silicone antifoaming agent 331512 K 0.05 mg/l (BDH = BDH Laboratory Supplies, England). Glycerol (10 g/1) was added as a carbon source for A. aceti and G. frateurii cultivations, while glucose (10 g/1) was used in P. acidipropionici cultivations. After sterilization (20 min at 120 °C), a filter-sterilized vitamin solution was added, giving a final concentration of biotin 0.05 mg/1, calcium pantothenate 1.0 mg/1, nicotinic acid 1.0 mg/1, myoinositol 25 mg/1, pyridoxal hydrochloride 1.0 mg/1, and para-aminobenzoic acid 0.2 mg/1. An additional vitamin supplement was used for acetic acid bacteria cultivation to give final concentrations of biotin 1.05 mg/1, nicotinic acid 1.5 mg/l, pyridoxal hydrochloride 1.5 mg/1, para-aminobenzoic acid 1.0 mg/ 1, riboflavin 1.5 mg/l, folic acid 1.0 mg/1, and vitamin B12 1.0 mg/1. The pH of the medium was adjusted to 5.8 for A. aceti and G. frateurii and to 6.5 for P. acidipropionici cultivations.
Osmosensitivity studies Growth of P. acidipropionici, A. aced, and G. frateurii was studied in Bioscreen C (Bioscreen C, Labsystems, Finland) microcultivation equipment. In Bioscreen C, the changes in optical density in the culture medium due to growth of micro-organism are measured kinetically with a vertical photometer in which the light beam passes up through the bottom of the cuvette and through the sample suspension to a detector. The incubator of the Bioscreen C consists of an incubator tray, a cover for the incubator tray, a temperature control system, and a shaker. Bioscreen C permits 200 samples to be run at a time, with samples placed in the wells of two honeycomb plates, each with 100 wells of 500 0 volume. In our studies 50 0 inoculum was added to 350 0 mineral medium and incubated at 30 °C for 4 days. The optical density of the cultivation medium was measured automatically at 600 nm once an hour. P. acidipropionici cultivation plates were shaken 20 s before each optical density measurement and A. aced and G. frateurii cultivations were continuously shaken. The NaCI concentration of the medium was adjusted to 0, 0.29 0.49 0.8, or 1.2 M and glycine betaine concentration to 0 or 20 mM. Glycine betaine (Betafin, anhydrous, pharmaceutical grade) was a kind gift from Finnfeeds, Finland. Results of these experiments are averages of measurements in five to ten different wells. The standard deviation of the optical density measurements of the samples was less than 10%.
Bioreactor cultivations of P. acidipropionici Bioreactor cultivations were carried out in a 1-dm3 bioreactor (Biostat Q. Braun Biotech International, Germany) with a working volume of 500 ml and automatic pH and temperature control. The temperature of the cultivation was 30 °C and stirrer speed 400 rpm. The culture pH was set to 6.5. The NaCI concentration of the medium was adjusted to 0, 0.4 or 0.8 M. The growth was measured as cell dry weight from the culture medium samples. All experiments were done in duplicate. The deviation between the dry weight samples of the parallel cultivation was less than 10%.
Dry weight measurement Culture samples (10 ml) were vacuum-filtered through a predried and weighed nitrocellulose filter (0.45 µm, Schleicher & Schuell, Germany), washed with Milli-p water, and dried in a microwave oven for 20 min (Ignis, Japan). Two parallel measurements were done from one sample and the standard deviation of the technique was determined to be less than 3%.
Substrate and metabolite analysis Samples of 1 ml from the bioreactor cultivations were centrifuged at 10,000 rpm for 10 min (Heraeus Sepatech, Biofuge A. Germany) and the supernatant was stored at -20 °C for further analysis. Glucose, acetate, and propionate concentrations were determined by high-performance liquid chromatography (HPLC). Sample components were separated on an HPX-87H Aminex ion-exclusion column (300 x 7.8 mm, Bio-Rad, USA) and detected with a Waters 410 refractive index detector and a Waters 486 UV detector. The column was eluted at 65 °C with 5 mM H2SO4 at a flow rate of 0.6 ml/min. The standard error of the analytical method was less than 5%.
Intracellular glycine betaine analysis Culture samples (20 ml) from bioreactor cultivations were centrifuged (5500 rpm, 15 min, Heraeus Sepatech, Megafuge 1.0, Germany) and the biomass was washed once with a fresh mineral medium without carbon source. Washing of the cells was done immediately with a mineral medium of the same osmotic strength as the cultivation medium to avoid the loss of intracellular glycine betaine by osmotic downshock. Washed cells were suspended in 5 ml of Milli-p water and stored at -20 °C for further analysis. Washed cell suspension (2 ml) was incubated at 100 °C for 10 min and then mixed vigorously with glass beads for 2-10 s in a test tube shaker (Vortex-genie 2, USA). Supernatant was separated by centrifugation (10 min, 10,000 rpm, Heraeus Sepatech, Biofuge A, Germany) and glycine betaine concentration was determined by HPLC. The sample components were separated on an HPX-87 C Aminex ion-exclusion column (300 x 7.8 mm, Bio-Rad, USA) and detected with a Shimadzu refractive index detector. The column was eluted at 85 °C with H2O at a flow rate of 0.6 ml/min. The standard error of the analytical method was less than 5%.
Glycine betaine uptake experiments with resting cells The experiment was initiated by adding 3 ml cell suspension to test tubes containing 10 ml mineral medium. To ensure nongrowth conditions the medium contained no carbon source. NaCI concentration in the medium was adjusted to 0.0 M, 0.2 M, 0.4 M, or 0.8 M and glycine betaine concentration to 0 mM, 1 mM, 5 mM, or 10 mM. Fifty to one hundred microliters of 14C-labeled glycine betaine, prepared by oxidation reaction from 14C-labeled choline, was added to the test tubes to adjust the activity in the medium to 24.3-31.5 nCi/ml (54,000-70,000 cpm/ml). Labeled glycine betaine was kindly provided by Danisco-Cultor Kantvik Research Center, Finland. Medium samples of 1 ml were taken immediately after the addition of cell suspension and after 45 and 90 min of incubation at 30 °C without shaking. Samples were filtered through a cellulose nitrate membrane (0.45 µm, Sartorius, Germany) and washed with a mineral medium in which the glycine betaine was replaced with 0.5 M sucrose. Sucrose was added to the washing medium to prevent the loss of intracellular glycine betaine during washing by osmotic downshock. Membrane filters were analyzed for /i-radiation by liquid scintillation counting (LS 6000 IC, Beckman, USA). The number of counts per minute in the sample was proportional to the amount of glycine betaine taken in by the cells in the sample. All uptake experiments were done in duplicate. The deviation between the liquid scintillation counts of parallel samples was less than 10%.
Results Osmosensitivity studies A. aced (IFO 3281) and G. frateurii (IFO 3254) were very sensitive to elevated osmolarities. A. aced did not grow when the NaCI concentration of the medium was 0.2 M or higher. G. frateurii could grow on medium containing 0.2 M NaCl, but not when the NaCI concentration was 0.4 M or higher. Glycine betaine addition had no effect on the growth of these bacteria in elevated osmolarities.
Fig. 1 Effect of increasing osmolarity and glycine betaine on the growth of P. acidipropionici (ATCC 4875). Growth was measured as change in optical density of the cultivation medium at 600 nm. The NaCI concentration in the medium was adjusted to 0.0 M NaCI (n), 0.8 M NaCI (¨), or 1.2 M NaCI (n). Closed symbols are for cultivations without glycine betaine and open symbols for cultivations with 20 mM glycine betaine
As can be seen in Fig. 1, the lag phase of the growth of P. acidipropionici increased and the maximum growth rate decreased with increasing NaCI concentration. P. acidipropionici (ATCC 4875) was able to grow on a medium containing 1.2 M NaCl, although the maximum growth rate was significantly lower (0.009 absorbance units (AU h-1)) than that in a medium without NaCI (0.040 AU h-'). Achievement of the maximum growth rate after the lag phase was faster in cultivations where glycine betaine was present in the medium. No growth was detected for any of the three bacteria on mineral medium containing glycine betaine as the only carbon source, verifying that glycine betaine could not be used as a sole carbon source for growth by these strains.
Bioreactor cultivations of P. acidipropionici P. acidipropionici (ATCC 4875) adapted faster to elevated NaCI concentration when glycine betaine was present in the medium. This result, obtained in the small-scale cultivations, was confirmed in bioreactor cultivations with automatic pH control. The lag phase of the P. acidipropionici cultivation increased to 36 h in a medium containing 0.8 M NaCI compared to the 12 h in medium without NaCI (Fig. 2). Although the presence of glycine betaine in the medium had no effect on the lag phase of the cultivation, it did inhibit the effect of elevated NaCI concentration on the specific growth rate. The effect of glycine betaine was seen as faster achievement of the maximum specific growth rate after the lag phase relative to cells cultivated without glycine betaine. Specific biomass yield (YSx) and propionic acid yield (YXp) (Table 1) were not affected by either NaCI or glycine betaine concentration in the medium.
Fig. 2 Effect of increasing osmolarity and glycine betaine on the growth and glycine betaine uptake of P. acidipropionici (ATCC 4875) in bioreactor cultivations. The growth was measured as cell dry weight (CDW) on a mineral medium containing 0.0 M NaCI (n), 0.4 M NaCI (A), or 0.8 M NaCI (0). Closed symbols are for cultivations without glycine betaine and open symbols for cultivations with 20 mM glycine betaine. Intracellular glycine betaine concentration was measured as the difference between the original glycine betaine concentration in the medium and the concentration at the time of measurement in a mineral medium containing 0.0 M (+) or 0.8 M NaCI (x) and 20 mM glycine betaine
Fig.3 The uptake of glycine betaine by P. acidipropionici cells cultivated in a bioreactor. The uptake of glycine betaine is calculated per gram CDW on a mineral medium containing 0.0 M NaCI (q) or 0.8 M NaCI (O). The growth measured as CDW in the same media is represented by closed symbols
The uptake of glycine betaine was determined directly by measuring the intracellular glycine betaine concentration of the cells and indirectly as the difference between the initial concentration of glycine betaine in the medium and the glycine betaine concentration at the time of measurement (Fig. 2). During the cultivation, glycine betaine was taken up by P. acidipropionici until the stationary phase was reached. Glycine betaine was present in excess in the medium (_13 mM) and was at no time exhausted, suggesting that the uptake was not limited by glycine betaine concentration. As can be seen in Fig. 3, the cells cultivated in a medium without NaCI achieved the maximum level of intracellular glycine betaine concentration, 0.50 mmol/g CDW, at the end of the lag phase. During the growth phase the intracellular glycine betaine concentration decreased to 0.37 mmol/g CDW, but increased again to near maximum level, 0.43 mmol/g CDW, in the stationary phase. The maximum specific uptake rate of glycine betaine achieved during the lag phase was 0.84 x 10-6 mol/g per minute. A similar trend in the intracellular glycine betaine concentration was observed with cells culti- vated in a medium containing 0.8 M NaCl: the max- imum intracellular glycine betaine concentration, 4.08 mmol/g CDW, was achieved at the end of the lag phase. During the growth phase the intracellular gly- cine betaine concentration decreased linearly to 1.46 mmol/g CDW, which also was the concentration in the stationary phase. The initial specific uptake rate of glycine betaine achieved during the lag phase was 1.00 x 10-6 mol/g per minute and comparable to the uptake rate in the medium without NaCl. The maxi- mum specific uptake rate of glycine betaine, 3.74 x 10-6 mol/g per minute, was achieved at the end of the lag phase.
Table 1 Maximum specific growth rates, biomass yield on substrate, and propionic acid yield on biomass on mineral medium containing 0, 0.4, and 0.8 M NaCI with and without 20 mM glycine betaine addition. The yields are calculated at the point of cultivation where the concentration of P. acidipropionici (ATCC 4875) biomass was 2 g/1
Glycine betaine uptake by resting cells Resting cells of P. acidipropionici (ATCC 4875) were able to take in glycine betaine from the incubation medium. As can be seen in Fig. 4, the amount of glycine betaine taken up by the resting cells increased with the amount of NaCI in the medium. Even in the medium containing no NaCI some glycine betaine was taken up by the resting cells when these were added to the incubation medium after washing of the cells with buffer solution and suspension in deionized water. During 90 min incubation the uptake rate of glycine betaine per cell dry weight was constant and higher in the cells incubated under osmotic stress than in the cells incubated in a medium containing no NaCl. The uptake rate of glycine betaine was not directly proportional to the NaCI concentration in the medium since a plateau in the uptake rate (values shown in Fig. 4) was achieved in the medium containing 0.4 M NaCl. Uptake of glycine betaine by the resting cells was independent of the glycine betaine concentration in the range of 1-10 mM in the medium, indicating that the uptake of glycine betaine was dependent only on the osmolarity created through the addition of NaCl.
Fig. 4 Glycine betaine uptake (mmol/g CDW) by resting cells of P. acidipropionici (ATCC 4875) in a medium containing 10 mM glycine betaine. NaCI concentration of the medium was adjusted to 0.0 M (black bars), 0.2 M (white bars), 0.4 M (light gray bars), or 0.8 M (dark gray bars). The glycine betaine uptake rate was linear in the studied range, regression coefficient 0.980 < R2 > 0.999
Discussion Bacteria are capable of living in a broad spectrum of environmental conditions, and there is wide variety even among nonhalophilic bacteria in the osmolarities they tolerate. The lower sensitivity of P. acidipropionici than of A. aced and G. frateurii to NaCI suggests that P. acidipropionici has a mechanism for osmoadaptation that enables its growth in elevated osmolarities. P. acidipropionici was able to grow on a medium containing 1.2 M NaCl, which is relatively low osmolarity compared to that reported for the nonhalophilic bacterium Staphylococcus aureus, which was found to grow on a defined medium containing 2.5 M NaCI (Vijaranakul et al. 1997). On the other hand, growth of the nonhalophilic soil bacterium Bacillus subtilis was inhibited in a medium containing more than 0.6 M NaCI (Boch et al. 1994). The effect of glycine betaine on growth characteristics of P. acidipropionici cultivations in elevated osmolarities was less dramatic than expected. According to Vijaranakul et al. (1997), osmoprotectants glycine betaine and choline significantly shorten the lag phase of the NaCl-sensitive S. aureus mutants. This was also expected in P. acidipropionici cultivations, but no such effect was seen. The results we have presented show that P. acidipropionici cells in elevated osmolarities achieved maximum specific growth rate after the prolonged lag phase faster when glycine betaine was present in the medium. Other growth characteristics were not affected by either NaCI or glycine betaine in the medium. The maximum levels of biomass and product concentrations obtained in the stationary phase were ultimately the same in media with and without glycine betaine addition, although the stationary level was achieved faster when glycine betaine was present. Beumer et al. (1994) found, on the other hand, that the final amount of colony-forming units (CFU) of Listeria monocytogenes per milliliter cultivated in a medium containing 3 % (0.52 M) NaCI was ten times as great when glycine betaine (1 mM) was present as when it was not. The CFU of the cultivation were not measured in our experiments. Interestingly, two different rates of uptake of glycine betaine were detected during the lag phase of P. acidipropionici cultivation in the medium containing 0.8 M NaCl, suggesting that some cellular reactions are needed for full function of an active transport of glycine betaine. As the prolonged lag phase could not be overcome by exogenously added glycine betaine, it can be presumed that other cellular reactions besides glycine betaine uptake are needed for total adaptation of the cells. The time needed to achieve the maximum growth rate in the medium may be essential for the synthesis of compatible solutes not present in the medium. Experiments with 14C-labeled glycine betaine and resting cells of P. acidipropionici confirmed that the uptake of exogenously added glycine betaine was not a growth-dependent phenomenon. Glycine betaine was not metabolized and the amount of glycine betaine taken in by the cells, as also the uptake rate, increased with the osmolarity in the incubation medium. We therefore assume that the transport was osmotically induced and glycine betaine acted as a nonmetabolizable compatible solute in adjusting the osmolarity inside the cell to the osmolarity in the environment, in order to maintain the turgor pressure. Glycine betaine has been found to have similar osmotically induced transport in a number of organisms, including Enterococcus faecalis, Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium linens, and Yersinia enterocolitica (Jebbar et al. 1995; Kappes et al. 1996; Park et al. 1995; Peter et al. 1998; Pichereau et al. 1999). The maximum specific uptake rate of glycine betaine was much lower in the resting than the growing cells of P. acidipropionici. Possibly the transport of glycine betaine requires some energy-dependent cellular reactions. Osmotically induced glycine betaine transport mechanism in P. acidipropionici was again suggested by the immediate ejection of intracellular glycine betaine from the cells when these were exposed to osmotic downshock by washing with water (results not shown here). A small amount of glycine betaine was also found in cells incubated without osmotic stress by NaCl, perhaps in response to the osmotic stress on the cells by other components of the medium. This is consistent with the results obtained for the cultivations of P. acidipropionici in elevated osmolarities due to glucose, where no NaCI was added. The lag phase of the growth was extended with increased glucose concentration of the medium (results not shown here). On the other hand, it may well be that betaine has other functions in the cell besides acting as an osmolyte. Boch et al. (1994) assumed that the choline uptake and conversion to betaine found in B. subtilis on a low-osmolarity medium was due to the maintenance of the very high turgor in B. subtilis. In conclusion, exogenously added glycine betaine enabled P. acidipropionici to achieve faster its maximum specific growth rate after the prolonged lag phase in elevated osmolarities and so to achieve faster its final biomass and product concentrations. The final concentrations of biomass and product were the same as when NaCI and glycine betaine were not present in the medium. A major improvement in fermentation processes might thus be achieved through the elevated substrate and product concentrations allowed by bacteria when their osmotolerance is enhanced by the addition of glycine betaine to the medium. Acknowledgements We gratefully thank Katja Ranta for assistance with the bioreactor cultivations. We would also like to thank Finnfeeds, Finland for providing the glycine betaine and Danisco-Cultor Kantvik Research Center, Finland for providing the 14C-labelled glycine betaine used in our experiments. The project was carried out with the financial support from the Graduate School of Chemical Engineering, Finland.
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