Bioresource Technology 197 (2015) 1–6
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Continuous propionic acid production with Propionibacterium acidipropionici immobilized in a novel xylan hydrogel matrix Janne Wallenius a,⇑, Nikolaos Pahimanolis a, Justin Zoppe a, Petri Kilpeläinen b, Emma Master c, Hannu Ilvesniemi b, Jukka Seppälä a, Tero Eerikäinen a, Heikki Ojamo a a b c
Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P.O. Box 6100, FIN-02015, Finland Finnish Natural Resources Institute (Luke), Jokiniemenkuja 1, P.O. Box 18, 01301 Vantaa, Finland Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada
h i g h l i g h t s Cell immobilization possibilities of novel xylan-based hydrogel are introduced. Continuous fermentation of P. acidipropionici with packed immobilization is studied. Very high cell density within the immobilization material is achieved.
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Article history: Received 18 June 2015 Received in revised form 31 July 2015 Accepted 1 August 2015 Available online 21 August 2015 Keywords: Cell immobilization Propionic acid Xylan Hydrogel P. acidipropionici
a b s t r a c t The cell immobilization potential of a novel xylan based disulfide-crosslinked hydrogel matrix reinforced with cellulose nanocrystals was studied with continuous cultivation of Propionibacterium acidipropionici using various dilution rates. The cells were immobilized to hydrogel beads suspended freely in the fermentation broth or else packed into a column connected to a stirred tank reactor. The maximum propionic acid productivity for the combined stirred tank and column was 0.88 g L1 h1 and the maximum productivity for the column was determined to be 1.39 g L1 h1. The maximum propionic acid titer for the combined system was 13.9 g L1 with a dilution rate of 0.06 h1. Dry cell density of 99.7 g L1 was obtained within the column packed with hydrogel beads and productivity of 1.02 g L1 h1 was maintained in the column even with the high circulation rate of 3.37 h1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Propionic acid is an important C-3 platform chemical (Werpy et al., 2004). Roughly half of the produced propionic acid is used in salt form as preservatives by food industries and in animal feed. It is also used in baking and dairy products, herbicides, and in the synthesis of cellulose acetate propionate used in printing inks. Propionic acid is mainly produced via petrochemical synthesis from ethylene, CO and steam (Reppe process) or from ethanol and CO (Larson process) (Wang et al., 2013). The possibility to produce propionic acid via fermentation from renewable biomass provides an alternative route to the refining of diminishing petrochemical resources. Many bacteria are capable of producing propionic acid, including the Gram positive Propionibacteria. Generally Recognized as ⇑ Corresponding author. Tel.: +358 504160139. E-mail address: [email protected] (J. Wallenius). http://dx.doi.org/10.1016/j.biortech.2015.08.037 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Safe (GRAS) organisms, Propionibacterium acidipropionici and Propionibacterium freudenreichii, are the two most industrially important facultative anaerobes from this genus (Poonam et al., 2012). P. acidipropionici and P. freudenreichii as well as other species within this genus produce propionic acid via the dicarboxylic acid pathway from a variety of carbon sources (Wang et al., 2013). For example, crude glycerol that forms as a side stream during biodiesel production is a potential feedstock for propionic acid fermentation (Zhang and Yang, 2009; Zhuge et al., 2014). Glycerol induces nearly homopropionic acid fermentation in contrast to other possible substrates such as lactose, sucrose, glucose, or xylose (Dishisha et al., 2012). Notably however, glycerol suffers from comparatively low volumetric productivities in suspended cell fermentations, although this can be overcome through cell immobilization (Zhang and Yang, 2009; Dishisha et al., 2012; Wang et al., 2013). Cell immobilization has been used for biotransformations, production of antibiotics, detoxification of biochemical compounds, biosensors, water and air purification (Mishra et al., 2012). Immo-
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bilization techniques can provide higher cell density, longer usability, recoverability and higher productivities compared to applications that employ suspended cells. The immobilization may be carried out as adsorption directly to a solid carrier surface, entrapment, encapsulation or cell flocculation (Manojlovic et al., 2013). P. acidipropionici cells have been immobilized e.g. on fibrous bed bioreactors and calcium alginate beads (Wang et al., 2013). Recently, also polyethylenimine (PEI) – treated Poraver glass beads and Luffa has been studied as immobilization materials for P. acidipropionici (Dishisha et al., 2012). Different approaches to propionic acid production via immobilized fermentations are further reviewed by Wang et al. (2013). Hydrogels are three-dimensional networks of hydrophilic polymers capable of retaining large amount of water, and have been investigated for use in numerous biomedical applications from drug delivery to tissue engineering, as well as cell immobilization matrices (Coviello et al., 2007; Moreno-Garrido, 2008; Yu and Ding, 2008). They offer diffusion of nutrients and other small molecules to and from the cells and give a structural matrix for cell attachment. Recently, cell immobilization to hydrogels have been studied e. g. by Mishra et al. (2012) for use in biosensor applications, and xylan-based hydrogels for mesenchymal stem cell immobilization by Kuzmenko et al. (2014). Polysaccharide based hydrogels have some advantages over synthetic polymers, since in addition to their abundant availability, they are non-toxic and biologically compatible (Coviello et al., 2007). Hydrogel formation can be achieved by various chemical or physical methods (Van Tomme et al., 2008), among which the oxidative thiol-thiol coupling is particularly attractive given its biologically relevant chemistry for covalent crosslinking under benign conditions (Shu et al., 2002). Köhnke et al. (2014) produced cross-linked xylan hydrogels reinforced with cellulose nanocrystals via ice templating. Despite this, the use of disulfide coupling for developing hydrogel materials for immobilization has not been reported so far. In this study propionic acid production and cell adsorption in a xylan-based hydrogel matrix with P. acidipropionici was investigated. Fermentations were carried out as a continuous process with glycerol as the feedstock. P. acidipropionici cells were immobilized to hydrogel beads freely in the cultivation broth or else packed in a column construct. 2. Methods 2.1. Organism and media composition P. acidipropionici NRRL B-3569 was maintained as frozen stock cultures containing 15% (w/v) glycerol and stored in 1 mL ampoules at 80 °C. The pre-cultivation medium contained 50 g L1 sucrose, 12 g L1 yeast extract, 1 g L1 KH2PO4, 1 g L1 K2HPO4, 0.2 g L1 MgSO47H2O, 0.02 g L1 CoCl7H2O, 0.0025 g L1 FeSO47H2O, and 0.002 g L1 5,6-dimethylbenzimidazole. For batch-phase cultivations the same medium was used and for the continuous cultivation, 50 g L1 sucrose was replaced by 50 g L1 glycerol. All media were sterilized by autoclaving at 105 Pa (121 °C) for 20 min and cooled prior use. 2.2. Precultivations Pre-cultivation medium of 100 mL was inoculated with 1 mL of P. acidipropionici NRRL B3569 glycerol stock solution. The preculture was incubated at 32 °C on a rotary shaker for 48 h. For the suspended cell and hydrogel beads in a stirred tank reactor (STR) cultivations, 20 mL of pre-culture was used to inoculate 350 mL of the medium in a STR and for the packed bed experiment
set 30 mL of pre-culture was used to inoculate 500 mL of the medium in STR. Batch fermentation was carried out for all of the cases for 24 h with 100 rpm stirring with a magnetic stirrer (ø 45 mm) in a 1 L jacketed glass bioreactor. The fermentation pH was set to 6.6 and it was kept constant with 5 M NaOH. The temperature was maintained at 32 °C during the batch phase. 2.3. Bead material Hydrogel beads having a diameter of approximately 3 mm were used as the immobilization matrix for P. acidipropionici cells. Beads consisted of xylan cross-linked with disulfide bonds (Pahimanolis et al., 2015) having a water content of 94 wt.% and reinforced with cellulose nanocrystals 11 wt.% of solids content see Supplementary material (Appendix A) for detailed synthesis and analysis. 2.4. Continuous process setup and cell immobilization 2.4.1. The suspended cell and hydrogel beads in STR cultivations In STR cultivations comprising suspended cells and hydrogel beads, the 24 h batch phase was followed by the continuous process illustrated in Fig. 1A. Briefly, fresh feed medium was pumped into the head-space of fermentation vessel with a peristaltic pump through a glass pipet device to prevent growth inside the feed line. The dilution rate was varied using different feed rates. The working volume of the process was maintained constant with an overflow mechanism by pumping fermentation broth out from the reactor with a slightly higher flow rate than that used to introduce fresh medium. During the continuous process pH and temperature were controlled similar to the batch phase. For STR cultivations comprising hydrogel beads, the approximate ratio beads to medium was 1:4, where 73.5 g beads were used. The continuous fermentation with suspended cells was carried out with dilution rates 0.048, 0.084, and 0.116 h1. Slightly higher dilution rates were used for the experiment with hydrogel beads freely in the STR medium to study the stability of productivity at higher dilution rates. The dilution rates were 0.49, 0.106, and 0.160 h1. Between the sampling of the different dilution rates the total flow through was at least three times the working volume to ensure the steady state. 2.4.2. Packed bed experiments For the packed bed experiments the hydrogel beads were packed in a Pharmacia XK 26 purification column (Pharmacia, Sweden) with a plastic jacket. The inner diameter of the column was 25 mm and 73.5 g of beads were used for the packing. The whole immobilization matrix was disinfected by circulating 70% ethanol in Milli-Q water through the column overnight after which the column was flushed with sterilized water. The free liquid space was determined to be 33 mL. The column was pre-heated to 32 °C using a water bath connected to the column jacket. After 24 h of batch the cultivation phase in the STR, the packed bed column was connected to the STR through the bottom end of the column and the medium was introduced to the column with a flow rate of 115.2 mL h1. The excess water from flushing was first removed to avoid dilution of the medium before circulating the medium back to the STR. The column was loaded for 24 h before the continuous process began. The continuous process setup with packed bed column is illustrated in Fig. 1B. A similar setup has been applied earlier by (Dishisha et al., 2012). The same control scheme and STR volume was used as for the continuous STR cultivations using suspended cell and hydrogel beads. The medium from the STR was circulated through the packed bed column from bottom to top. The sampling was carried out from the return flow of the column to the STR and from the effluent flow of the STR. Five different dilution rates for the whole packed bed and STR system was studied. The dilution
J. Wallenius et al. / Bioresource Technology 197 (2015) 1–6
(A)
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(B)
Fig. 1. (A) Schematic illustration of continuous process setup used in suspended cell and hydrogel beads in a STR cultivations. (B) Schematic illustration of the continuous process setup used in packed bed experiments.
rates for the STR were varied between 0.06 and 0.17 h1. Also the efficiency of the packed bed column part was further studied varying the column circulation rates. Determination of process parameters for the packed bed column is explained in Section 2.6. Four different circulation rates were used and at the end of the experiments the feed medium was directly connected to the column inlet instead of the STR to run one more experiment. 2.5. Analytical procedures The concentrations of propionic acid, acetic acid, succinic acid, butyric acid, and glycerol were determined by HPLC (Waters Alliance 2690, USA) equipped with an RI detector (Waters 2414, USA) and Hi-Plex H column (300 7.7 mm, Agilent Technologies, USA). The column was heated at 65 °C. The fermentation samples were diluted with Milli-Q water and filtered through 0.2 lm filters prior the injection into 5 mM H2SO4 mobile phase flowing at a rate of 0.6 mL min1. The dry weight of immobilized cells was determined by sampling the content of the packed bed column at the end of the set of experiments. A representative sample was washed with saline water. The wet weight of the washed sample was determined on a pre-dried glass plate. The sample was dried at 110 °C overnight and prior to dry weight determination the sample was cooled down in a desiccator. To determine the actual cell dry weight (CDW) the dried sample was resuspended and washed with 0.5 M NaOH. The sample was sonicated for 5 min and washed again with 0.5 M NaOH and finally with Milli-Q water. The sample was dried again on a pre-dried glass plate at 110 °C overnight and the weight of the washed hydrogel beads was determined after cooling in a desiccator. The cell attachment to the hydrogel beads was confirmed with scanning electron microscopy (SEM). The hydrogel samples were instantly frozen with liquid nitrogen and freeze-dried. The freeze-dried beads were cut in halves and made conductive by Pt sputtering to study the inner side of the beads. The SEM images were obtained using a Zeiss Sigma VP instrument operating at 3 kV. 2.6. Calculation of process parameters The dilution rate (D, h1) was calculated dividing the feed rate with the reactor volume and in the case of packed bed experiments for the whole system the volume of the STR and free liquid volume in the column together were used as divisor. The overall productivity (Q, g L1 h1) was calculated by multiplying the effluent product concentration by the overall dilution rate. The substrate
utilization was determined from the initial glycerol concentration of the feed and the fermentation sample glycerol concentrations. The product yields (Y, w-%) were calculated dividing the product concentration (g L1) by utilized glycerol (g L1). The productivity and yield for the column part in packed bed experiments were determined according to (Villadsen et al., 2011):
qs V þ v ðsi;feed si Þ ¼ 0
ð1Þ
qp V þ v ðpi;feed pi Þ ¼ 0
ð2Þ
q Y ji ¼ i qj
ð3Þ
where qs is the volumetric production rate (g L1 h1) of substrate s and qp is the volumetric production rate (g L1 h1) of product p in the column, V is the free liquid volume of the column, v is the flow rate through the column, si is the effluent concentrations of the substrate and pi the product from the column, and si,feed and pi,feed are the inlet concentrations, respectively. The inlet concentrations are considered to be equal to those in the STR while the STR volume is remarkably higher than the column free liquid volume. Yji in Eq. (3) is the product yield. 3. Results and discussion 3.1. Continuous fermentation with suspended cells and hydrogel beads in a STR The study of a continuous process with beads in a STR provided a method to investigate if the novel hydrogel material had any cell immobilization potential. Although the relative immobilization material volume remained low (1:4) the process efficiency could be compared to a regular continuous fermentation with suspended cells in terms of propionic acid productivity. Increasing the dilution rate led to increased propionic acid yield for STR comprising suspended cells and cells immobilized to hydrogel beads (Fig. 2). Also for comparison propionic acid productivities of suspended cells, from earlier study by Dishisha et al. (2012), decreased rapidly as the dilution rate was increased (Fig. 2). Moreover, increasing the dilution rate increased propionic acid productivity in the STR holding the suspended hydrogel beads. The productivity remained at 0.82 g L1 h1 when the dilution rate was increased from 0.106 to 0.160. It can be concluded that the productivity cannot be increased more only by increasing dilution rate for this kind of setup. In contrast to previous studies where the productivity of suspended cells dropped quickly with increased dilution rate (Dishisha et al., 2012), reference experiments per-
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Fig. 2. Productivities and concentrations of propionic acid in continuous process experiments with suspended cells and cells immobilized to hydrogel beads in a STR. Also reference literature (Dishisha et al., 2012) values for propionic acid productivity (blue ‘r’). Green ‘4’ refers to propionic acid concentrations in non-packed hydrogel bead experiment, black ‘+’ to propionic acid concentration with suspended cells, red ‘h’ to propionic acid productivity in non-packed hydrogel bead experiment, and purple ‘x’ to propionic acid productivity with suspended cells.
formed herein showed that the productivity remained high at the studied dilution rate range but was nevertheless lower than that observed for cultivations using hydrogel beads (below 0.6 g L1 h1). The differences between our reference experiments and the earlier study by Dishisha et al. (2012) are likely to result from differing mixing conditions or medium composition. The glycerol utilization remained low for suspended cell and immobilized STR cultivations (between 9% and 36%), and decreased in both cases as the dilution rate increased (Appendix B). Acetic acid was produced in the cultivations in small concentrations, 1.2–1.3 g L1 in reference experiments and 0.4–0.5 g L1 in hydrogel bead experiments, regardless of the dilution rates. 3.2. Continuous fermentation with packed hydrogel beads The impact of cell immobilization to the hydrogel beads was further studied with combined system of STR and column comprising packed hydrogel beads. The approach permitted to control pH properly and to study the performance of the both parts separately and as a whole, i.e. the flow-through rates (dilution rates) were varied for the column part and for the whole system (Table 1). The adsorption of the cells within the beads was further studied with SEM (Appendix C). The cells completely covered the bead surface and can be observed inside the gel structure, indicating some migration also into the hydrogel structure during cultivation. The
cell concentration was determined to be 99.7 g L1 within the column material. Compared to earlier studies, similar magnitude of propionibacteria cell densities have only been obtained with cell recycling combined with ultrafiltration (Blanc and Goma 1987a, 1987). While the column circulation rate was kept constant at 3.37 h1, the propionic acid yield was relatively constant, between 0.56 and 0.58 g g1. The maximum propionic acid productivity of 0.88 g L1 h1 for the STR and column system was reached with the dilution rate of 0.11 h1. Considering the low column volume per STR volume ratio the achieved productivity is relatively high. For example, STR experiments using similar dilution rates but with suspended cells, only reached a productivity of 0.54 g L1 h1. The highest propionic acid titer of 13.9 g L1 was with the lowest dilution rate, 0.06 h1. The glycerol utilization for the whole system varied between 6.7 and 24.2 g L1 within the experiments. In general the produced titers were decreased when the dilution rate was increased. Acetic acid concentrations remained approximately at the same level, 0.6–0.7 g L1 through the experiments. Succinic acid concentrations varied between 0.3 and 1.4 g L1, respectively. Also low titers of butyric acid and traces of propanol were detected in the experiments. Considering the propionic acid production industrially homofermentation is preferred. The ratio between propionic acid yield and by product acid yields reached the value of 7.7 mol mol1
Table 1 Process parameters and measured concentrations from continuous fermentation experiments with packed hydrogel beads. The given yields and productivities are in respect of propionic acid. System
STR + packed bed column part
D, column part (h1) D, STR (h1) Acetic acid (g L1) Butyric acid (g L1) Final glycerol (g L1) Propionic acid (g L1) Succinic acid (g L1) Y (g g1) Q (g L1 h1) Utilized glycerol (g L1)
3.37 0.06 0.7 0.1 27.3 13.9 1.4 0.57 0.77 24.2
3.37 0.11 0.6 0.1 39.8 7.7 0.6 0.58 0.88 13.4
3.37 0.17 0.7 0.1 46.6 3.8 0.3 0.56 0.66 6.7
3.37 0.08 0.6 0.2 32.0 11.1 1.0 0.57 0.85 19.6
Packed bed column part 2.24 0.07 0.6 0.4 30.9 10.5 1.1 0.51 0.75 20.7
1.09 0.07 0.6 0.5 32.0 9.6 1.1 0.46 0.69 20.7
D: Dilution rate; Y: Yield; Q: Productivity. * In this experiment the feed medium was directly connected to the column inlet.
0.36 0.07 0.6 0.5 34.1 8.7 0.9 0.47 0.62 18.4
3.37 0.06 0.7 0.1 26.8 14.0 1.5 0.29 0.49 0.5
3.37 0.11 0.6 0.1 39.3 7.9 0.6 0.35 0.59 0.5
3.37 0.17 0.7 0.1 45.9 4.1 0.3 0.44 1.02 0.7
3.37 0.08 0.6 0.2 31.1 11.2 1.1 0.11 0.33 0.9
2.24 0.07 0.6 0.5 30.0 10.8 1.2 0.40 0.73 0.8
1.09 0.07 0.6 0.7 28.1 10.8 1.4 0.11 1.27 4.0
0.36 0.07 0.7 0.8 26.0 12.1 1.3 0.41 1.23 8.1
0.38* 0.5 0.2 45.8 3.7 0.3 0.49 1.39 7.4
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for the whole system when STR dilution rate was 0.06 h1 and column part dilution rate was 3.37 h1 and remained below that in the rest of the experiments. Higher ratios have been achieved by Liu et al. (2011), magnitude of 12 mol mol1, with glycerol/glucose co-fermentation, and with ack (acetakinase) knockout mutant by Zhang and Yang (2009), magnitude of 31 mol mol1. The yield of acetic acid from the whole system increased as the STR dilution rate was increased. Though biomass yields were not determined in this study, yet in general the biomass yield decreases when the dilution rate is increased which can also be observed from the results by Dishisha et al. (2012). Apparently, acetic acid is being produced more to maintain the NADH/NAD+ ratio when less biomass is formed from glycerol. Propionic acid production from glycerol does not change the NADH/NAD+ ratio while metabolic pathways toward biomass or acetic acid formation accumulate NADH (Liu et al., 2011; Zhang and Yang, 2009). Succinic acid per propionic acid ratio varied between 0.5 and 0.7 mol mol1 throughout the experiments for the whole system without apparent coupling with the process parameters. The circulation rate through the hydrogel packed column, i.e. the column dilution rate was kept high (between 0.36 and 3.37 h1) to prevent high pH or substrate gradients occurring in the column. Also due to the small working volume of the column it was challenging to reliably reach lower dilution rates. However, even though the dilution rates for the column were high, and thus the titers in the effluent from the column were close to titers in the STR, the productivity remained above 0.33 g L1 h1 even in at the highest flow rates attempted. Considering the column separately, the peak value of 1.39 g L1 h1 for productivity was reached with yield of 0.49 g g1 with respect to propionic acid and 14.0% glycerol utilization when the feed medium was connected directly to the column. In general, high column productivities and yields were reached at higher dilution rates of the STR. Thus, the results indicate that the higher the inlet concentration of propionic acid to the column, the more inhibited the propionic acid production is within the column. Reductions in propionic acid production rate at different propionic acid concentrations was earlier observed by Blanc and Goma (1987b). In general, propionic acid yield in the column was lower than in the STR. Similar propionic acid productivities to earlier study by Dishisha et al. (2012) with PEIPoraver immobilized cells (0.83–1.44 g L1 h1) were reached, although the yields remained lower. As observed in the earlier study by Dishisha et al. (2012) with PEI-Poraver matrix, considering the high cell density, the productivity remained relatively low. The hydrogel provides high potential as a cell immobilization matrix material when considering the possibilities to engineer the properties of the material. Hydrogel property engineering potential is further covered by Peak et al. (2013) in their review. At the end of continuous packed bed experiments some deterioration, i.e. shape distortion and cleavage to smaller particles, of hydrogel beads was observed. However, the process was run over a month with a considerably high dilution rate. The beads withstand at least mild mixing conditions considering that no visible deterioration was observed after the hydrogel beads in STR cultivation. The material could also be further enhanced to tolerate mechanical stress better, e.g. by double network formation (Peak et al., 2013).
4. Conclusions The novel xylan-based hydrogel matrix reinforced with cellulose nanocrystals shows potential as a biological immobilization matrix. In the continuous fermentation with packed hydrogel beads very high cell density was achieved. Low productivity per cell mass was observed. Also with our experimental setup it could
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be observed that the immobilized cells were inhibited by the product even though the titers were low. Process-wise the material would benefit from further development enhancing tolerance towards mechanical stress. The achieved productivities were comparable to those reported earlier. However, the relatively high productivities were maintained even with the extreme dilution rates indicating good cell adhesion. Acknowledgements This work has been partly funded by a FiDiPro Fellowship from the Finnish Funding Agency for Technology and Innovation (Tekes) project ‘‘Chemo-Enzymatic Modification of Underutilized Plant Biopolymers.” We wish to thank Ms. Tiia Juhala for elemental analyses and Dr. Nguyen Dang Luong for SEM imaging. The authors acknowledge the Laboratory of Inorganic Chemistry of AaltoUniversity for access to X-ray diffraction equipment and Dr. Markus Valkeapää and Dr. Matti Lehtimäki for their assistance with the measurements. This work made use of Aalto University Bioeconomy and Aalto University Nanomicroscopy Center (AaltoNMC) facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08. 037. References Blanc, P., Goma, G., 1987a. Propionic acid fermentation: improvement of performances by coupling continuous fermentation and ultrafiltration. Bioprocess Eng. 2, 137–139. Blanc, P., Goma, G., 1987b. Kinetics of inhibition in propionic acid fermentation. Bioprocess Eng. 2, 175–179. Boyaval, P., Corre, C., 1987. Continuous fermentation of sweet whey permeate for propionic acid production in a CSTR with UF recycle. Biotechnol. Lett. 9, 801– 806. Coviello, T., Matricardi, P., Marianecci, C., Alhaique, F., 2007. Polysaccharide hydrogels for modified release formulations. J. Control. Release 119, 5–24. Dishisha, T., Alvarez, M.T., Hatti-Kaul, R., 2012. Batch- and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici. Bioresour. Technol. 118, 553–562. Köhnke, T., Elder, T., Theliander, H., Ragauskas, A.J., 2014. Ice templated and crosslinked xylan/nanocrystalline cellulose hydrogels. Carbohydr. Polym. 100, 24– 30. Kuzmenko, V., Hägg, D., Toriz, G., Gatenholm, P., 2014. In situ forming spruce xylanbased hydrogel for cell immobilization. Carbohydr. Polym. 102, 862–868. Liu, Y., Zhang, Y.-G., Zhang, R.-B., Zhang, F., Zhu, J., 2011. Glycerol/glucose cofermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr. Microbiol. 62, 152–158. Manojlovic, V., Bugarski, B., Nedovic, V., 2013. Immobilized cells. In: Flickinger, M.C. (Ed.), Upstream Industrial Biotechnology. John Wiley & Sons Inc, Somerset, New Jersey, pp. 1179–1199. Mishra, S., Scarano, F.J., Calvert, P., 2012. Entrapment of Saccharomyces cerevisiae and 3T3 fibroblast cells into blue light cured hydrogels. J. Biomed. Mater. Res. A 100, 2829–2838. Moreno-Garrido, I., 2008. Microalgae immobilization: current techniques and uses. Bioresour. Technol. 99, 3949–3964. Pahimanolis, N., Kilpeläinen, P., Master, E., Ilvesniemi, H., Seppälä, J., 2015. Novel thiol- amine- and amino acid functional xylan derivatives synthesized by thiolene reaction. Carbohydr. Polym. 131, 392–398. Peak, C.W., Wilker, J.J., Schmidt, G., 2013. A review on tough and sticky hydrogels. Colloid Polym. Sci. 291, 2031–2047. Poonam, Pophaly, S.D., Tomar, S.K., De, S., Singh, R., 2012. Multifaceted attributes of dairy propionibacteria: a review. World J. Microbiol. Biotechnol. 28, 3081–3095. Shu, X.Z., Liu, Y., Luo, Y., Roberts, M.C., Prestwich, G.D., 2002. Disulfide cross-linked hyaluronan hydrogels. Biomacromolecules 3, 1304–1311. Van Tomme, S.R., Storm, G., Hennink, W.E., 2008. In situ gelling hydrogels for pharmaceutical and biomedical applications. Int. J. Pharm. 355, 1–18. Wang, Z., Sun, J., Zhang, A., Yang, S., 2013. Propionic acid fermentation. In: Yang, S.T., El-Ensashy, H., Thongchul, N. (Eds.), Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers. American Institute of Chemical Engineers, Somerset, NJ, USA, pp. 331–349. Werpy, T., Petersen, G., Aden, A., Bozell, J., Holladay, J., White, J., Manheim, A., Elliot, D., Lasure, L., Jones, S., Gerber, M., Ibsen, K., Lumberg, L., Kelley, S., 2004. Top
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Value Added Chemicals from Biomass Volume I – Results of Screening for Potential Candidates from Sugars and Synthesis Gas. doi: 10.2172/15008859. Villadsen, J., Nielsen, J., Lidén, G., 2011. Bioreaction Engineering Principles. Springer Science & Business Media. Yu, L., Ding, J., 2008. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 37, 1473–1481.
Zhang, A., Yang, S.-T., 2009. Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici. Process Biochem. 44, 1346–1351. Zhuge, X., Liu, L., Shin, H., Li, J., Du, G., Chen, J., 2014. Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy. Bioresour. Technol. 152, 519–525.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Continuous propionic acid production with Propionibacterium acidipropionici immobilized in a novel xylan hydrogel matrix Janne Wallenius a,⇑, Nikolaos Pahimanolis a, Justin Zoppe a, Petri Kilpeläinen b, Emma Master c, Hannu Ilvesniemi b, Jukka Seppälä a, Tero Eerikäinen a, Heikki Ojamo a a b c
Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P.O. Box 6100, FIN-02015, Finland Finnish Natural Resources Institute (Luke), Jokiniemenkuja 1, P.O. Box 18, 01301 Vantaa, Finland Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada
h i g h l i g h t s Cell immobilization possibilities of novel xylan-based hydrogel are introduced. Continuous fermentation of P. acidipropionici with packed immobilization is studied. Very high cell density within the immobilization material is achieved.
a r t i c l e
i n f o
Article history: Received 18 June 2015 Received in revised form 31 July 2015 Accepted 1 August 2015 Available online 21 August 2015 Keywords: Cell immobilization Propionic acid Xylan Hydrogel P. acidipropionici
a b s t r a c t The cell immobilization potential of a novel xylan based disulfide-crosslinked hydrogel matrix reinforced with cellulose nanocrystals was studied with continuous cultivation of Propionibacterium acidipropionici using various dilution rates. The cells were immobilized to hydrogel beads suspended freely in the fermentation broth or else packed into a column connected to a stirred tank reactor. The maximum propionic acid productivity for the combined stirred tank and column was 0.88 g L1 h1 and the maximum productivity for the column was determined to be 1.39 g L1 h1. The maximum propionic acid titer for the combined system was 13.9 g L1 with a dilution rate of 0.06 h1. Dry cell density of 99.7 g L1 was obtained within the column packed with hydrogel beads and productivity of 1.02 g L1 h1 was maintained in the column even with the high circulation rate of 3.37 h1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Propionic acid is an important C-3 platform chemical (Werpy et al., 2004). Roughly half of the produced propionic acid is used in salt form as preservatives by food industries and in animal feed. It is also used in baking and dairy products, herbicides, and in the synthesis of cellulose acetate propionate used in printing inks. Propionic acid is mainly produced via petrochemical synthesis from ethylene, CO and steam (Reppe process) or from ethanol and CO (Larson process) (Wang et al., 2013). The possibility to produce propionic acid via fermentation from renewable biomass provides an alternative route to the refining of diminishing petrochemical resources. Many bacteria are capable of producing propionic acid, including the Gram positive Propionibacteria. Generally Recognized as ⇑ Corresponding author. Tel.: +358 504160139. E-mail address: [email protected] (J. Wallenius). http://dx.doi.org/10.1016/j.biortech.2015.08.037 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Safe (GRAS) organisms, Propionibacterium acidipropionici and Propionibacterium freudenreichii, are the two most industrially important facultative anaerobes from this genus (Poonam et al., 2012). P. acidipropionici and P. freudenreichii as well as other species within this genus produce propionic acid via the dicarboxylic acid pathway from a variety of carbon sources (Wang et al., 2013). For example, crude glycerol that forms as a side stream during biodiesel production is a potential feedstock for propionic acid fermentation (Zhang and Yang, 2009; Zhuge et al., 2014). Glycerol induces nearly homopropionic acid fermentation in contrast to other possible substrates such as lactose, sucrose, glucose, or xylose (Dishisha et al., 2012). Notably however, glycerol suffers from comparatively low volumetric productivities in suspended cell fermentations, although this can be overcome through cell immobilization (Zhang and Yang, 2009; Dishisha et al., 2012; Wang et al., 2013). Cell immobilization has been used for biotransformations, production of antibiotics, detoxification of biochemical compounds, biosensors, water and air purification (Mishra et al., 2012). Immo-
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bilization techniques can provide higher cell density, longer usability, recoverability and higher productivities compared to applications that employ suspended cells. The immobilization may be carried out as adsorption directly to a solid carrier surface, entrapment, encapsulation or cell flocculation (Manojlovic et al., 2013). P. acidipropionici cells have been immobilized e.g. on fibrous bed bioreactors and calcium alginate beads (Wang et al., 2013). Recently, also polyethylenimine (PEI) – treated Poraver glass beads and Luffa has been studied as immobilization materials for P. acidipropionici (Dishisha et al., 2012). Different approaches to propionic acid production via immobilized fermentations are further reviewed by Wang et al. (2013). Hydrogels are three-dimensional networks of hydrophilic polymers capable of retaining large amount of water, and have been investigated for use in numerous biomedical applications from drug delivery to tissue engineering, as well as cell immobilization matrices (Coviello et al., 2007; Moreno-Garrido, 2008; Yu and Ding, 2008). They offer diffusion of nutrients and other small molecules to and from the cells and give a structural matrix for cell attachment. Recently, cell immobilization to hydrogels have been studied e. g. by Mishra et al. (2012) for use in biosensor applications, and xylan-based hydrogels for mesenchymal stem cell immobilization by Kuzmenko et al. (2014). Polysaccharide based hydrogels have some advantages over synthetic polymers, since in addition to their abundant availability, they are non-toxic and biologically compatible (Coviello et al., 2007). Hydrogel formation can be achieved by various chemical or physical methods (Van Tomme et al., 2008), among which the oxidative thiol-thiol coupling is particularly attractive given its biologically relevant chemistry for covalent crosslinking under benign conditions (Shu et al., 2002). Köhnke et al. (2014) produced cross-linked xylan hydrogels reinforced with cellulose nanocrystals via ice templating. Despite this, the use of disulfide coupling for developing hydrogel materials for immobilization has not been reported so far. In this study propionic acid production and cell adsorption in a xylan-based hydrogel matrix with P. acidipropionici was investigated. Fermentations were carried out as a continuous process with glycerol as the feedstock. P. acidipropionici cells were immobilized to hydrogel beads freely in the cultivation broth or else packed in a column construct. 2. Methods 2.1. Organism and media composition P. acidipropionici NRRL B-3569 was maintained as frozen stock cultures containing 15% (w/v) glycerol and stored in 1 mL ampoules at 80 °C. The pre-cultivation medium contained 50 g L1 sucrose, 12 g L1 yeast extract, 1 g L1 KH2PO4, 1 g L1 K2HPO4, 0.2 g L1 MgSO47H2O, 0.02 g L1 CoCl7H2O, 0.0025 g L1 FeSO47H2O, and 0.002 g L1 5,6-dimethylbenzimidazole. For batch-phase cultivations the same medium was used and for the continuous cultivation, 50 g L1 sucrose was replaced by 50 g L1 glycerol. All media were sterilized by autoclaving at 105 Pa (121 °C) for 20 min and cooled prior use. 2.2. Precultivations Pre-cultivation medium of 100 mL was inoculated with 1 mL of P. acidipropionici NRRL B3569 glycerol stock solution. The preculture was incubated at 32 °C on a rotary shaker for 48 h. For the suspended cell and hydrogel beads in a stirred tank reactor (STR) cultivations, 20 mL of pre-culture was used to inoculate 350 mL of the medium in a STR and for the packed bed experiment
set 30 mL of pre-culture was used to inoculate 500 mL of the medium in STR. Batch fermentation was carried out for all of the cases for 24 h with 100 rpm stirring with a magnetic stirrer (ø 45 mm) in a 1 L jacketed glass bioreactor. The fermentation pH was set to 6.6 and it was kept constant with 5 M NaOH. The temperature was maintained at 32 °C during the batch phase. 2.3. Bead material Hydrogel beads having a diameter of approximately 3 mm were used as the immobilization matrix for P. acidipropionici cells. Beads consisted of xylan cross-linked with disulfide bonds (Pahimanolis et al., 2015) having a water content of 94 wt.% and reinforced with cellulose nanocrystals 11 wt.% of solids content see Supplementary material (Appendix A) for detailed synthesis and analysis. 2.4. Continuous process setup and cell immobilization 2.4.1. The suspended cell and hydrogel beads in STR cultivations In STR cultivations comprising suspended cells and hydrogel beads, the 24 h batch phase was followed by the continuous process illustrated in Fig. 1A. Briefly, fresh feed medium was pumped into the head-space of fermentation vessel with a peristaltic pump through a glass pipet device to prevent growth inside the feed line. The dilution rate was varied using different feed rates. The working volume of the process was maintained constant with an overflow mechanism by pumping fermentation broth out from the reactor with a slightly higher flow rate than that used to introduce fresh medium. During the continuous process pH and temperature were controlled similar to the batch phase. For STR cultivations comprising hydrogel beads, the approximate ratio beads to medium was 1:4, where 73.5 g beads were used. The continuous fermentation with suspended cells was carried out with dilution rates 0.048, 0.084, and 0.116 h1. Slightly higher dilution rates were used for the experiment with hydrogel beads freely in the STR medium to study the stability of productivity at higher dilution rates. The dilution rates were 0.49, 0.106, and 0.160 h1. Between the sampling of the different dilution rates the total flow through was at least three times the working volume to ensure the steady state. 2.4.2. Packed bed experiments For the packed bed experiments the hydrogel beads were packed in a Pharmacia XK 26 purification column (Pharmacia, Sweden) with a plastic jacket. The inner diameter of the column was 25 mm and 73.5 g of beads were used for the packing. The whole immobilization matrix was disinfected by circulating 70% ethanol in Milli-Q water through the column overnight after which the column was flushed with sterilized water. The free liquid space was determined to be 33 mL. The column was pre-heated to 32 °C using a water bath connected to the column jacket. After 24 h of batch the cultivation phase in the STR, the packed bed column was connected to the STR through the bottom end of the column and the medium was introduced to the column with a flow rate of 115.2 mL h1. The excess water from flushing was first removed to avoid dilution of the medium before circulating the medium back to the STR. The column was loaded for 24 h before the continuous process began. The continuous process setup with packed bed column is illustrated in Fig. 1B. A similar setup has been applied earlier by (Dishisha et al., 2012). The same control scheme and STR volume was used as for the continuous STR cultivations using suspended cell and hydrogel beads. The medium from the STR was circulated through the packed bed column from bottom to top. The sampling was carried out from the return flow of the column to the STR and from the effluent flow of the STR. Five different dilution rates for the whole packed bed and STR system was studied. The dilution
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(A)
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(B)
Fig. 1. (A) Schematic illustration of continuous process setup used in suspended cell and hydrogel beads in a STR cultivations. (B) Schematic illustration of the continuous process setup used in packed bed experiments.
rates for the STR were varied between 0.06 and 0.17 h1. Also the efficiency of the packed bed column part was further studied varying the column circulation rates. Determination of process parameters for the packed bed column is explained in Section 2.6. Four different circulation rates were used and at the end of the experiments the feed medium was directly connected to the column inlet instead of the STR to run one more experiment. 2.5. Analytical procedures The concentrations of propionic acid, acetic acid, succinic acid, butyric acid, and glycerol were determined by HPLC (Waters Alliance 2690, USA) equipped with an RI detector (Waters 2414, USA) and Hi-Plex H column (300 7.7 mm, Agilent Technologies, USA). The column was heated at 65 °C. The fermentation samples were diluted with Milli-Q water and filtered through 0.2 lm filters prior the injection into 5 mM H2SO4 mobile phase flowing at a rate of 0.6 mL min1. The dry weight of immobilized cells was determined by sampling the content of the packed bed column at the end of the set of experiments. A representative sample was washed with saline water. The wet weight of the washed sample was determined on a pre-dried glass plate. The sample was dried at 110 °C overnight and prior to dry weight determination the sample was cooled down in a desiccator. To determine the actual cell dry weight (CDW) the dried sample was resuspended and washed with 0.5 M NaOH. The sample was sonicated for 5 min and washed again with 0.5 M NaOH and finally with Milli-Q water. The sample was dried again on a pre-dried glass plate at 110 °C overnight and the weight of the washed hydrogel beads was determined after cooling in a desiccator. The cell attachment to the hydrogel beads was confirmed with scanning electron microscopy (SEM). The hydrogel samples were instantly frozen with liquid nitrogen and freeze-dried. The freeze-dried beads were cut in halves and made conductive by Pt sputtering to study the inner side of the beads. The SEM images were obtained using a Zeiss Sigma VP instrument operating at 3 kV. 2.6. Calculation of process parameters The dilution rate (D, h1) was calculated dividing the feed rate with the reactor volume and in the case of packed bed experiments for the whole system the volume of the STR and free liquid volume in the column together were used as divisor. The overall productivity (Q, g L1 h1) was calculated by multiplying the effluent product concentration by the overall dilution rate. The substrate
utilization was determined from the initial glycerol concentration of the feed and the fermentation sample glycerol concentrations. The product yields (Y, w-%) were calculated dividing the product concentration (g L1) by utilized glycerol (g L1). The productivity and yield for the column part in packed bed experiments were determined according to (Villadsen et al., 2011):
qs V þ v ðsi;feed si Þ ¼ 0
ð1Þ
qp V þ v ðpi;feed pi Þ ¼ 0
ð2Þ
q Y ji ¼ i qj
ð3Þ
where qs is the volumetric production rate (g L1 h1) of substrate s and qp is the volumetric production rate (g L1 h1) of product p in the column, V is the free liquid volume of the column, v is the flow rate through the column, si is the effluent concentrations of the substrate and pi the product from the column, and si,feed and pi,feed are the inlet concentrations, respectively. The inlet concentrations are considered to be equal to those in the STR while the STR volume is remarkably higher than the column free liquid volume. Yji in Eq. (3) is the product yield. 3. Results and discussion 3.1. Continuous fermentation with suspended cells and hydrogel beads in a STR The study of a continuous process with beads in a STR provided a method to investigate if the novel hydrogel material had any cell immobilization potential. Although the relative immobilization material volume remained low (1:4) the process efficiency could be compared to a regular continuous fermentation with suspended cells in terms of propionic acid productivity. Increasing the dilution rate led to increased propionic acid yield for STR comprising suspended cells and cells immobilized to hydrogel beads (Fig. 2). Also for comparison propionic acid productivities of suspended cells, from earlier study by Dishisha et al. (2012), decreased rapidly as the dilution rate was increased (Fig. 2). Moreover, increasing the dilution rate increased propionic acid productivity in the STR holding the suspended hydrogel beads. The productivity remained at 0.82 g L1 h1 when the dilution rate was increased from 0.106 to 0.160. It can be concluded that the productivity cannot be increased more only by increasing dilution rate for this kind of setup. In contrast to previous studies where the productivity of suspended cells dropped quickly with increased dilution rate (Dishisha et al., 2012), reference experiments per-
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Fig. 2. Productivities and concentrations of propionic acid in continuous process experiments with suspended cells and cells immobilized to hydrogel beads in a STR. Also reference literature (Dishisha et al., 2012) values for propionic acid productivity (blue ‘r’). Green ‘4’ refers to propionic acid concentrations in non-packed hydrogel bead experiment, black ‘+’ to propionic acid concentration with suspended cells, red ‘h’ to propionic acid productivity in non-packed hydrogel bead experiment, and purple ‘x’ to propionic acid productivity with suspended cells.
formed herein showed that the productivity remained high at the studied dilution rate range but was nevertheless lower than that observed for cultivations using hydrogel beads (below 0.6 g L1 h1). The differences between our reference experiments and the earlier study by Dishisha et al. (2012) are likely to result from differing mixing conditions or medium composition. The glycerol utilization remained low for suspended cell and immobilized STR cultivations (between 9% and 36%), and decreased in both cases as the dilution rate increased (Appendix B). Acetic acid was produced in the cultivations in small concentrations, 1.2–1.3 g L1 in reference experiments and 0.4–0.5 g L1 in hydrogel bead experiments, regardless of the dilution rates. 3.2. Continuous fermentation with packed hydrogel beads The impact of cell immobilization to the hydrogel beads was further studied with combined system of STR and column comprising packed hydrogel beads. The approach permitted to control pH properly and to study the performance of the both parts separately and as a whole, i.e. the flow-through rates (dilution rates) were varied for the column part and for the whole system (Table 1). The adsorption of the cells within the beads was further studied with SEM (Appendix C). The cells completely covered the bead surface and can be observed inside the gel structure, indicating some migration also into the hydrogel structure during cultivation. The
cell concentration was determined to be 99.7 g L1 within the column material. Compared to earlier studies, similar magnitude of propionibacteria cell densities have only been obtained with cell recycling combined with ultrafiltration (Blanc and Goma 1987a, 1987). While the column circulation rate was kept constant at 3.37 h1, the propionic acid yield was relatively constant, between 0.56 and 0.58 g g1. The maximum propionic acid productivity of 0.88 g L1 h1 for the STR and column system was reached with the dilution rate of 0.11 h1. Considering the low column volume per STR volume ratio the achieved productivity is relatively high. For example, STR experiments using similar dilution rates but with suspended cells, only reached a productivity of 0.54 g L1 h1. The highest propionic acid titer of 13.9 g L1 was with the lowest dilution rate, 0.06 h1. The glycerol utilization for the whole system varied between 6.7 and 24.2 g L1 within the experiments. In general the produced titers were decreased when the dilution rate was increased. Acetic acid concentrations remained approximately at the same level, 0.6–0.7 g L1 through the experiments. Succinic acid concentrations varied between 0.3 and 1.4 g L1, respectively. Also low titers of butyric acid and traces of propanol were detected in the experiments. Considering the propionic acid production industrially homofermentation is preferred. The ratio between propionic acid yield and by product acid yields reached the value of 7.7 mol mol1
Table 1 Process parameters and measured concentrations from continuous fermentation experiments with packed hydrogel beads. The given yields and productivities are in respect of propionic acid. System
STR + packed bed column part
D, column part (h1) D, STR (h1) Acetic acid (g L1) Butyric acid (g L1) Final glycerol (g L1) Propionic acid (g L1) Succinic acid (g L1) Y (g g1) Q (g L1 h1) Utilized glycerol (g L1)
3.37 0.06 0.7 0.1 27.3 13.9 1.4 0.57 0.77 24.2
3.37 0.11 0.6 0.1 39.8 7.7 0.6 0.58 0.88 13.4
3.37 0.17 0.7 0.1 46.6 3.8 0.3 0.56 0.66 6.7
3.37 0.08 0.6 0.2 32.0 11.1 1.0 0.57 0.85 19.6
Packed bed column part 2.24 0.07 0.6 0.4 30.9 10.5 1.1 0.51 0.75 20.7
1.09 0.07 0.6 0.5 32.0 9.6 1.1 0.46 0.69 20.7
D: Dilution rate; Y: Yield; Q: Productivity. * In this experiment the feed medium was directly connected to the column inlet.
0.36 0.07 0.6 0.5 34.1 8.7 0.9 0.47 0.62 18.4
3.37 0.06 0.7 0.1 26.8 14.0 1.5 0.29 0.49 0.5
3.37 0.11 0.6 0.1 39.3 7.9 0.6 0.35 0.59 0.5
3.37 0.17 0.7 0.1 45.9 4.1 0.3 0.44 1.02 0.7
3.37 0.08 0.6 0.2 31.1 11.2 1.1 0.11 0.33 0.9
2.24 0.07 0.6 0.5 30.0 10.8 1.2 0.40 0.73 0.8
1.09 0.07 0.6 0.7 28.1 10.8 1.4 0.11 1.27 4.0
0.36 0.07 0.7 0.8 26.0 12.1 1.3 0.41 1.23 8.1
0.38* 0.5 0.2 45.8 3.7 0.3 0.49 1.39 7.4
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for the whole system when STR dilution rate was 0.06 h1 and column part dilution rate was 3.37 h1 and remained below that in the rest of the experiments. Higher ratios have been achieved by Liu et al. (2011), magnitude of 12 mol mol1, with glycerol/glucose co-fermentation, and with ack (acetakinase) knockout mutant by Zhang and Yang (2009), magnitude of 31 mol mol1. The yield of acetic acid from the whole system increased as the STR dilution rate was increased. Though biomass yields were not determined in this study, yet in general the biomass yield decreases when the dilution rate is increased which can also be observed from the results by Dishisha et al. (2012). Apparently, acetic acid is being produced more to maintain the NADH/NAD+ ratio when less biomass is formed from glycerol. Propionic acid production from glycerol does not change the NADH/NAD+ ratio while metabolic pathways toward biomass or acetic acid formation accumulate NADH (Liu et al., 2011; Zhang and Yang, 2009). Succinic acid per propionic acid ratio varied between 0.5 and 0.7 mol mol1 throughout the experiments for the whole system without apparent coupling with the process parameters. The circulation rate through the hydrogel packed column, i.e. the column dilution rate was kept high (between 0.36 and 3.37 h1) to prevent high pH or substrate gradients occurring in the column. Also due to the small working volume of the column it was challenging to reliably reach lower dilution rates. However, even though the dilution rates for the column were high, and thus the titers in the effluent from the column were close to titers in the STR, the productivity remained above 0.33 g L1 h1 even in at the highest flow rates attempted. Considering the column separately, the peak value of 1.39 g L1 h1 for productivity was reached with yield of 0.49 g g1 with respect to propionic acid and 14.0% glycerol utilization when the feed medium was connected directly to the column. In general, high column productivities and yields were reached at higher dilution rates of the STR. Thus, the results indicate that the higher the inlet concentration of propionic acid to the column, the more inhibited the propionic acid production is within the column. Reductions in propionic acid production rate at different propionic acid concentrations was earlier observed by Blanc and Goma (1987b). In general, propionic acid yield in the column was lower than in the STR. Similar propionic acid productivities to earlier study by Dishisha et al. (2012) with PEIPoraver immobilized cells (0.83–1.44 g L1 h1) were reached, although the yields remained lower. As observed in the earlier study by Dishisha et al. (2012) with PEI-Poraver matrix, considering the high cell density, the productivity remained relatively low. The hydrogel provides high potential as a cell immobilization matrix material when considering the possibilities to engineer the properties of the material. Hydrogel property engineering potential is further covered by Peak et al. (2013) in their review. At the end of continuous packed bed experiments some deterioration, i.e. shape distortion and cleavage to smaller particles, of hydrogel beads was observed. However, the process was run over a month with a considerably high dilution rate. The beads withstand at least mild mixing conditions considering that no visible deterioration was observed after the hydrogel beads in STR cultivation. The material could also be further enhanced to tolerate mechanical stress better, e.g. by double network formation (Peak et al., 2013).
4. Conclusions The novel xylan-based hydrogel matrix reinforced with cellulose nanocrystals shows potential as a biological immobilization matrix. In the continuous fermentation with packed hydrogel beads very high cell density was achieved. Low productivity per cell mass was observed. Also with our experimental setup it could
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be observed that the immobilized cells were inhibited by the product even though the titers were low. Process-wise the material would benefit from further development enhancing tolerance towards mechanical stress. The achieved productivities were comparable to those reported earlier. However, the relatively high productivities were maintained even with the extreme dilution rates indicating good cell adhesion. Acknowledgements This work has been partly funded by a FiDiPro Fellowship from the Finnish Funding Agency for Technology and Innovation (Tekes) project ‘‘Chemo-Enzymatic Modification of Underutilized Plant Biopolymers.” We wish to thank Ms. Tiia Juhala for elemental analyses and Dr. Nguyen Dang Luong for SEM imaging. The authors acknowledge the Laboratory of Inorganic Chemistry of AaltoUniversity for access to X-ray diffraction equipment and Dr. Markus Valkeapää and Dr. Matti Lehtimäki for their assistance with the measurements. This work made use of Aalto University Bioeconomy and Aalto University Nanomicroscopy Center (AaltoNMC) facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08. 037. References Blanc, P., Goma, G., 1987a. Propionic acid fermentation: improvement of performances by coupling continuous fermentation and ultrafiltration. Bioprocess Eng. 2, 137–139. Blanc, P., Goma, G., 1987b. Kinetics of inhibition in propionic acid fermentation. Bioprocess Eng. 2, 175–179. Boyaval, P., Corre, C., 1987. Continuous fermentation of sweet whey permeate for propionic acid production in a CSTR with UF recycle. Biotechnol. Lett. 9, 801– 806. Coviello, T., Matricardi, P., Marianecci, C., Alhaique, F., 2007. Polysaccharide hydrogels for modified release formulations. J. Control. Release 119, 5–24. Dishisha, T., Alvarez, M.T., Hatti-Kaul, R., 2012. Batch- and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici. Bioresour. Technol. 118, 553–562. Köhnke, T., Elder, T., Theliander, H., Ragauskas, A.J., 2014. Ice templated and crosslinked xylan/nanocrystalline cellulose hydrogels. Carbohydr. Polym. 100, 24– 30. Kuzmenko, V., Hägg, D., Toriz, G., Gatenholm, P., 2014. In situ forming spruce xylanbased hydrogel for cell immobilization. Carbohydr. Polym. 102, 862–868. Liu, Y., Zhang, Y.-G., Zhang, R.-B., Zhang, F., Zhu, J., 2011. Glycerol/glucose cofermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr. Microbiol. 62, 152–158. Manojlovic, V., Bugarski, B., Nedovic, V., 2013. Immobilized cells. In: Flickinger, M.C. (Ed.), Upstream Industrial Biotechnology. John Wiley & Sons Inc, Somerset, New Jersey, pp. 1179–1199. Mishra, S., Scarano, F.J., Calvert, P., 2012. Entrapment of Saccharomyces cerevisiae and 3T3 fibroblast cells into blue light cured hydrogels. J. Biomed. Mater. Res. A 100, 2829–2838. Moreno-Garrido, I., 2008. Microalgae immobilization: current techniques and uses. Bioresour. Technol. 99, 3949–3964. Pahimanolis, N., Kilpeläinen, P., Master, E., Ilvesniemi, H., Seppälä, J., 2015. Novel thiol- amine- and amino acid functional xylan derivatives synthesized by thiolene reaction. Carbohydr. Polym. 131, 392–398. Peak, C.W., Wilker, J.J., Schmidt, G., 2013. A review on tough and sticky hydrogels. Colloid Polym. Sci. 291, 2031–2047. Poonam, Pophaly, S.D., Tomar, S.K., De, S., Singh, R., 2012. Multifaceted attributes of dairy propionibacteria: a review. World J. Microbiol. Biotechnol. 28, 3081–3095. Shu, X.Z., Liu, Y., Luo, Y., Roberts, M.C., Prestwich, G.D., 2002. Disulfide cross-linked hyaluronan hydrogels. Biomacromolecules 3, 1304–1311. Van Tomme, S.R., Storm, G., Hennink, W.E., 2008. In situ gelling hydrogels for pharmaceutical and biomedical applications. Int. J. Pharm. 355, 1–18. Wang, Z., Sun, J., Zhang, A., Yang, S., 2013. Propionic acid fermentation. In: Yang, S.T., El-Ensashy, H., Thongchul, N. (Eds.), Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers. American Institute of Chemical Engineers, Somerset, NJ, USA, pp. 331–349. Werpy, T., Petersen, G., Aden, A., Bozell, J., Holladay, J., White, J., Manheim, A., Elliot, D., Lasure, L., Jones, S., Gerber, M., Ibsen, K., Lumberg, L., Kelley, S., 2004. Top
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Value Added Chemicals from Biomass Volume I – Results of Screening for Potential Candidates from Sugars and Synthesis Gas. doi: 10.2172/15008859. Villadsen, J., Nielsen, J., Lidén, G., 2011. Bioreaction Engineering Principles. Springer Science & Business Media. Yu, L., Ding, J., 2008. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 37, 1473–1481.
Zhang, A., Yang, S.-T., 2009. Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici. Process Biochem. 44, 1346–1351. Zhuge, X., Liu, L., Shin, H., Li, J., Du, G., Chen, J., 2014. Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy. Bioresour. Technol. 152, 519–525.
