Efficient production of 1,3-propanediol from ... - Springer Link

Bioprocess Biosyst Eng (2014) 37:225–233 DOI 10.1007/s00449-013-0989-0

ORIGINAL PAPER

Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium Donna Dietz • An-Ping Zeng

Received: 4 April 2013 / Accepted: 27 May 2013 / Published online: 9 June 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract The objective of this study was to examine the applicability of mixed cultures for 1,3-propanediol (1,3PDO) production from crude glycerol. Three different sources of mixed cultures were tested, where the mixed culture from a municipal wastewater treatment plant showed the best results. 1,3-PDO can be produced as the main product in this mixed culture with typical organic acids like acetic and butyric acids as by-products. The yield was in the range of 0.56–0.76 mol 1,3-PDO per mol glycerol consumed depending on the glycerol concentration. A final product concentration as high as 70 g/L was obtained in fed-batch cultivation with a productivity of 2.6 g/L h. 1,3-PDO can be kept in the culture several days after termination of the fermentation without being degraded. Degradation tests showed that 1,3-PDO is degraded much slower than other compounds in the fermentation broth. In comparison to 1,3-PDO production in typical pure cultures, the process developed in this work with a mixed culture achieved the same levels of product titer, yield and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used and nitrogen gassing can be omitted during cultivation, leading to a strong reduction of investment and production costs.

D. Dietz (&)  A.-P. Zeng Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestr.15, 21071 Hamburg, Germany e-mail: [email protected] Present Address: D. Dietz Leibniz-Institut fu¨r Agrartechnik (ATB), Max-Eyth-Allee 100, 14469 Potsdam, Germany

Keywords Mixed cultures  1,3-propanediol  Crude glycerol  Non-sterile  Anaerobic degradation  Biogas

Introduction Polymers are part of our daily life: from plastic bottles to airplanes are made of composite polymers. Different properties are needed for different applications [27]. Polytrimethyleneterephthalate (PTT), a new group of polyester, is particularly appealing for textile and fiber industries. 1,3-Propanediol (1,3-PDO) serves as a monomer for the production of this polymer, which can be produced either via chemical or biotechnological routes. The oldest known 1,3-PDO fermentation pathway was mentioned by August Freund [7] with glycerol and a mixed culture containing probably Clostridium pasteurianum. Later, in 1990s, research in 1,3-PDO started again after Shell announced the commercialization of a new polymer based on polycondensation of terephthalic acid and 1,3PDO [4]. Several bacteria genera are known for their ability to produce this chemical: Klebsiella, Enterobacter, Citrobacter, Clostridium and Lactobacillus [2, 3, 8, 11, 16, 18, 22, 27]. Up to date, most researches have focused on fermentation optimization of 1,3-PDO using pure culture or genetically modified microorganisms. Among them, K. pneumoniae and C. butyricum are mostly studied [30, 31, 32, 33]. Because the production cost strongly depends on the availability and price of the substrate, a lot of efforts have been undertaken to develop a system suitable for crude glycerol [6, 13, 14, 16, 17]. Glycerol is a by-product of biodiesel production; 1 tons of raw glycerol accumulates for every 10 tons biodiesel produced. Since European Union set up a mandatory

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biofuel target of 10 % for the European transport sector in 2020 [12], the amount of crude glycerol increases and its price decreases. After several subsequent treatment processes, the purity of the raw glycerol ranges between 60 and 88 % (w/w) [12]. The rest consists of water, free-fatty acids, salts and methanol, which may cause growth inhibition for microorganisms. So far, the application of mixed cultures is still mostly limited to the fields of traditional foods, beverages/alcohols, waste water treatment and biogas production [21]. But mixed cultures have large potentials for the production of bulk chemicals. Some research groups already showed the possibility of converting glycerol to 1,3-PDO by mixed cultures [5, 19, 23, 25, 26]. Temudo et al. [26] performed glycerol fermentation by mixed cultures originated from distillery wastewater and a sludge solution from a potato starch processing acidification tank. Selembo et al. [23] used mixed cultures from two different soils, compost and sludge, to enhance hydrogen and 1,3-PDO production. However, in the previous studies, either the glycerol concentration utilized (e.g., 3 g/L by [23]) or the 1,3-PDO titer and yield achieved (e.g., 0.14 mol/mol in [26]) are relatively low. Many 1,3-PDO producers have been isolated from different environmental sources: sediments and sewage sludge [11], compost, decaying straw, and mud [3], anaerobic distillery wastewater [1], and soil samples [20]. 1,3-PDO producers generally exist in environment with facultative and obligate anaerobic conditions. Therefore, mesophilicdigested sludge from biogas degradation process would also be an interesting source for obtaining 1,3-PDO producers. On the other hand, as with other organic compounds, there exists a very real possibility that 1,3-PDO would not only be produced but also anaerobically degraded during fermentation with mixed cultures. The mechanism and kinetics of 1,3-PDO degradation are still not fully understood. Qatibi et al. [19] suggested that 1,3-PDO degradation requires either a terminal electron acceptor such as sulfate or syntrophic cooperation with methanogenic hydrogenotrophic bacteria. A possible group of bacteria that can degrade 1,3-PDO is sulfate-reducing bacteria. In this work, we also investigated the possibility of 1,3-PDO degradation and ways to avoid the degradation. Glycerol fermentation under unsterile conditions has been recently successfully demonstrated with pure cultures of C. butyricum [6, 28], K. oxytoca [13], and Citrobacter freundii [14]. However, the fermentations were performed with complex media consisting of meat extract and yeast extract. The aim of this work was to develop a 1,3-PDO production process with mixed culture(s) which should result in comparable product concentration and yield as in pure culture processes, but significantly reduced production costs by working with a simplified medium and under unsterile conditions.

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Materials and methods Mixed culture inoculum The mixed cultures were obtained initially as sludge from different full-scale biogas plants in Germany: Hamburg (HH), Fu¨rstenwalde (FW) and Hagen (HA). All biogas plants were operated at a mesophilic temperature (35–37 °C) and a pH between 7.5 and 7.6. The mixed cultures were stored for a week at 37 °C to reduce their biogas production. The biogas plant in Hamburg was fed with municipal wastewater. In Fu¨rstenwalde, the biogas plant was fed with a mixture of household bio-waste, commercial waste and agricultural waste. The Hagen biogas plant was fed only with agricultural waste. Mixed cultures from the HH biogas plant were also used for the degradation experiments. Pure culture inoculum Clostridium butyricum VPI 1718 (now: ATCC 860) was provided by the former Gesellschaft fu¨r Biotechnologische Forschung, Braunschweig, Germany. The strain was maintained in reinforced Clostridial Medium (RCM) in serum bottles at 4 °C. Pre-cultures of pure culture inoculum were incubated at 37 °C for 24 h. Culture media and substrate The composition of the pre-culture and fermentation medium (per liter of water) was: 5 g NH4Cl; 0.75 g KCl; 0.6 g NaH2PO4H2O; 1.54 g Na2HPO47H2O; 0.28 g Na2SO4; 0.42 g citric acid; 0.2 g L-cysteine; 0.024 mg biotin; 0.015 mg pantothenate; 1 mg FeCl3; 0.26 g MgCl26H2O; 2.9 mg CaCl22H2O; 2 mL trace element solution (70 mg/L ZnCl2; 100 mg/L MnCl24H2O; 200 mg/L CoCl26H2O; 20 mg/L CuCl22H2O; 35 mg/L Na2MoO42H2O; 25 mg/L NiCl26H2O; 60 mg/L H3BO3; 0.9 mL/L HCl). In addition, 2.45 g/L NaH2PO4H2O and 4.58 g/L Na2HPO4 were added to the pre-culture medium. No yeast extract was added. Sterilization of the medium was not necessary. Crude glycerol was obtained from ADM Industries, Hamburg (Germany) with following composition (in w/w): 81 % glycerol, 10–12 % water, 5–6 % potassium salts, 1 % freefatty acids, and\0.1 % methanol. Analytical grade glycerol (99.5 %) was obtained from Carl Roth, Germany. Pre-culture and fermentation Serum bottles were used for preliminary experiments and all pre-cultivations. Each bottle had a volume of 100 mL and working volume of 50 mL. After the medium was filled, each bottle was sparged with pure nitrogen for 5 min

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and sealed with butyl rubber septa and aluminum caps. Since no yeast extract and carbon source were added, the pre-cultures can be stored for several months at room temperature without autoclaving beforehand. The pre-cultures were inoculated with 10–20 % (v/v) of inoculum. After inoculation, the serum bottles were incubated at 37 °C for 24 h. Fermentations were conducted in 2-L stirred tank foil bioreactors (Bioengineering AG, Switzerland). One liter of medium was prepared for each batch culture and 10–20 % (v/v) of pre-culture was added. Crude glycerol was used as the sole carbon source at an initial concentration of 25 g/L. The temperature was controlled at 37 °C and the pH was regulated at 7.0 by adding 5 M NaOH. The base consumption was followed using a balance (Sartorius AG, Go¨ttingen, Germany). To ensure anaerobic conditions, the fermentation medium was sparged with O2-free nitrogen for 10 min before and after the inoculation. The stirring rate was kept constant at 500 rpm. Fermentations were also carried out in fed-batch mode. In fed-batch fermentations, after the substrate was consumed to a very low but not limiting level, the feeding of raw glycerol solution without any supplement was initiated. The feeding rate was adjusted to the substrate consumption rate. The feeding rate was regulated and controlled by a peristaltic pump (Watson Marlow GmbH, Germany) and a balance (Sartorius AG, Go¨ttingen, Germany). Three different feeding strategies were tested: pulse feeding, feeding coupled with base consumption, and continuous feeding. By the pulse feeding method, the pH value was used as reference. The pH increased slightly after the substrate was completely consumed and was thereby an indicator to start feeding.

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Glycerol, 1,3-PDO and fermentation broth were used as substrate for the degradation tests. Each set of the experiments was investigated in triplicate. Cellulose was used as reference to ensure the biological activity of mixed culture. One set of triplicate tests was also prepared only with inoculum without any substrates. Hence, the net gas volume reported for each degradation test is the result of the difference between the gas volume of the degradation test with substrates (or products) and the gas volume of inoculum without the substrate. Analytics Samples were withdrawn from the reactors and centrifuged at 13,000 rpm for 10 min. The supernatant was then filtered with 0.2-lm filter and diluted before chromatography measurement. The concentrations of substrate, product and by-products were measured with HPLC (Kontron Instruments, Switzerland) with a flow rate of 0.6 mL/min and a 5-mM sulfuric acid as eluent. An Aminex 300 mm 9 7.8 mm HPX-87H column (Bio-Rad Laboratories, USA) was used for the separation with a column temperature of 60 °C. The column was coupled to a differential refractometer RI-detector (Kontron Instruments, Switzerland) and a UV-detector (Shimadzu, Japan) at 210 nm. In the degradation experiments, the biogas was removed via a Hamilton syringe once a day and directly injected in a gas chromatograph (HP 6890, Agilent, USA). The composition of CH4 and CO2 was determined with a thermal conductivity detector equipped with a Porapack Q column (2 m, 1/800 , 80/100 mesh) at 210 °C. Helium functioned as carrier gas at constant flow of 45 mL/min.

Degradation experiments Results and discussion To determine the degradability of substrate and products of glycerol conversion by mixed cultures, two types of degradation experiments were carried out. The first one was carried out based on the Guideline VDI 4630 ‘‘Fermentation of organic materials: characterization of the substrate, sampling collection of material data, fermentation tests’’. The experimental set up was configured according to DIN 31414-S8 of the German Institute for Standardization, where the volume of the gas produced was equivalent to the displaced barrier solution. As test equipments, 500-mL flasks with 450 mL working volume were placed on a stirrer. 400 mL of inoculum and 50 mL of substrate were used in each set of experiments. The flasks were sealed with butyl rubber septa and placed in a 37 °C incubation room. Ambient temperature and pressure were measured daily. The pH was determined at the beginning and the end of each experiment.

Selection of mixed cultures Three different mixed cultures were chosen to determine their suitability and ability to convert crude glycerol to 1,3PDO. The experiments with mixed cultures were performed in batch fermentations in 2-L bioreactors with crude glycerol. To compare the performance, fermentations were also carried out with a pure culture of C. butyricum and analytical grade glycerol under sterile conditions. The initial substrate concentration was 30 g/L. Figure 1 shows the glycerol consumption by different mixed cultures. The mixed culture HA did not show any degradation in the first 8 h; and after more than 24 h fermentation, glycerol concentration decreased only marginally and was not consumed completely. The second mixed culture HH showed a complete substrate consumption after 12 h, almost at the

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provided additional reduction equivalents for 1,3-PDO production from glycerol. Effect of initial substrate concentration in batch culture

Fig. 1 Glycerol consumption in fermentations with three different mixed cultures (HA biogas plant Hagen, HH biogas plant Hamburg, FW biogas plant Fu¨rstenwalde) in comparison with that of a pure culture of C.butyricum VPI 1718

same rate as the pure culture. The inoculum from biogas plant FA took 4.5 h longer than the inoculum from HH to consume the substrate completely. Table 1 summarizes the product concentrations of the fermentations by the different mixed cultures and pure culture of C. butyricum. Except for the mixed culture HA, all the other cultures formed 1,3PDO as main product of the glycerol consumption. The major by-product was acetate. While butyrate was formed by the mixed cultures from biogas plant HH and C. butyricum, the mixed culture of biogas plant FW produced lactate, ethanol and formate as by-products. The 1,3-PDO yields from the mixed cultures of HH and FW and from the pure culture were 0.76, 0.51, and 0.73 mol 1,3-PDO per mol glycerol consumed, respectively. Since the mixed culture HH showed the best yield, a high glycerol consumption rate and a similar by-product composition as the pure culture, it was thus chosen as the mixed culture for subsequent investigations. The mixed culture HH achieved a 1,3-PDO yield slightly higher than the theoretical yield of 0.72 mol/mol in pure culture [30, 31]. It could be assumed that other unknown carbon source existed in the sludge, which Table 1 Product and byproducts of fermentation with three different mixed cultures and pure culture of C.butyricum

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The effects of initial crude glycerol concentrations in the range of 5–95 g/L were studied in batch fermentation. 10 % (v/v) of the mixed culture HH served as inoculum (Fig. 2). It was observed that up to an initial concentration of 50 g/L of glycerol, the substrate can be consumed relatively quickly and completely. Above this concentration, the glycerol consumption seemed to be significantly inhibited. At an initial glycerol concentration of 65 g/L, the substrate can still be completely consumed, but with a much longer fermentation time. At higher initial substrate concentrations (75, 85 and 95 g/L), glycerol was consumed only to a certain extent, either due to substrate inhibition [33] or depletion of nutrients because of the use of a simple medium without yeast extract. Fermentations with newly isolated strains of K. oxytoca [13] and C. freundii [14] showed that substrate inhibition in batch fermentation processes started at a much higher glycerol initial concentration of over 100 g/L. This could be attributed to the complex nutrients in these fermentations. To achieve high end product concentrations, fed-batch fermentations are preferred to avoid substrate inhibition. However, a simplified medium was also used for fed-batch fermentations in this work. The main product in all the fermentations was 1,3-PDO, with acetate and butyrate as major by-products, as exemplified in Fig. 3 for a fermentation with 25 g glycerol/L as initial substrate concentration. The product spectrum is similar to that of conventional fermentations with a pure culture of C. butyricum under sterile conditions [4, 9]. Preliminary characterization of the culture showed that the dominating 1,3-producer in our selected mixed cultures is probably a Clostridium species like C. pasteurianum (data not shown). Fed-batch fermentation To avoid substrate inhibition and to achieve a higher final concentration of 1,3-PDO, fed-batch fermentations were Concentration [g/L] 1,3-PDO

Acetate

Butyrate

Lactate

Ethanol

Formate –

Mixed culture of biogas plant HA

–

–

–

–

–

Mixed culture of biogas plant HH

15.21

3.10

1.56

–

–

–

Mixed culture of biogas plant FW

12.54

5.84

–

1.79

3.13

2.77

Pure culture C.butyricum

19.50

1.60

3.43

0.43

–

–

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Fig. 2 Glycerol consumption at different initial substrate concentrations in fermentations with a selected mixed culture

Fig. 3 Typical results of glycerol fermentation by a selected mixed culture

carried out with the mixed culture HH. An initial glycerol concentration of 25 g/L was applied. In a fed-batch process with pulse feeding, the substrate consumption rate and the 1,3-PDO production rate decreased after two feed additions. The end product concentration was 40 g/L with a yield of 0.55 mol/mol. An even lower end concentration was obtained with feeding coupled to base consumption. Only 20 g/L of 1,3-PDO was produced with a yield of 0.52 mol/mol. A high 1,3-PDO final concentration was achieved with continuous substrate feeding. Crude glycerol solely was fed continuously with a feeding rate set at 20 g/L h. The results are shown in Fig. 4. After several hours, glycerol accumulated in the culture. Hereupon, the feeding rate was reduced to 10 g/L h. 1,3-PDO increased steadily until it reached a high end concentration of 70 g/L, with the yield

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Fig. 4 Fed-batch fermentation of raw glycerol in the selected mixed culture with continuous substrate feeding

and productivity being 0.56 mol/mol and 2.60 g/L h, respectively. Acetate and butyrate were the major by-products of the fermentation. As 1,3-PDO concentration increased sharply from 35 to 60 g/L, the production of butyrate was preferred over acetate formation. The end product titer of the fed-batch fermentation in this work is among the highest 1,3-PDO concentrations achieved so far, especially when considering the fact that no complex nutrient was added in the feeding in our work. Obviously, in the case of fed-batch fermentations with mixed cultures, the absence of complex nutrients in the fermentations medium and feeding did not affect the production of 1,3-PDO. Hirschmann et al. [10] reached 100 g/ L of 1,3-PDO final concentration with Clostridium species, but the feeding contained 80 % (w/v) glycerol and 40 g/L yeast extract. Wilkens et al. [28] reached 76.2 g/L of 1,3PDO with C. butyricum and crude glycerol, however, with a sterile feeding with 40 g/L of yeast extract as well. A relatively high final product concentration was also obtained with pure cultures of K. oxytoca (50.1 g/L) and C. freundii (68.1 g/L) [13, 14] under non-sterile conditions with crude glycerol. Degradation of 1,3-PDO The high yield and concentration of 1,3-PDO production in the selected mixed culture cannot be taken as granted. Since most organic compounds can be degraded anaerobically, theoretically 1,3-PDO would also be degraded in the mixed cultures eventually. Degradation experiments did confirm this. But the degradation rate of different compounds in the culture varied significantly, because some organic compounds are much easier to be degraded and, thus have a higher degradation rate than other compounds. First, the

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degradability of glycerol and 1,3-PDO was tested separately. After that, a mixture of glycerol and 1,3-PDO and a fermentation broth from glycerol fermentation were investigated. Figure 5 shows the cumulative biogas production in the different degradation tests. CH4 and CO2 were the end products of anaerobic degradation. The gas composition is summarized in Table 2. The pH was stable during the whole degradation process. The degradation profiles for the first 9 days showed significant differences in the degradation rate of each compound (Fig. 5, right). Glycerol was degraded quickly. No biogas was produced anymore after 4 days. 1,3-PDO, on the other hand, started to be degraded after 5 days of incubation. It was further degraded until day 12. These results agree well with those of Oppenberg and Schink [15], where the gas production started after 3–10 days. The mixture of glycerol and 1,3-PDO was also degraded, but the biogas production increased again on the sixth day. This was obviously due to subsequent degradation of 1,3-PDO. Clearly, glycerol was first degraded and 1,3-PDO was degraded later. The fermentation broth showed a similar degradation pattern as those of glycerol and glycerol plus 1,3-PDO. 1,3-PDO is a reduced product and not easy to be degraded. Its degradation requires either an electron acceptor such as sulfate or a syntrophic co-culture, to favor the degradation thermodynamically by direct hydrogen consumption [19]. Molybdate is a structural analog to sulfate and, therefore, a competitive inhibitor for the sulfate uptake system. At an elevated amount, molybdate inhibited sulfate respiration of sulfate-reducing bacteria [24]. In this work, 4 mM of sodium molybdate (Na2MoO42H2O, Sigma-Aldrich, Munich, Germany) was added to fermentation medium to study its effect on 1,3-PDO degradation. As a control, fermentations without addition of molybdate were also performed. The results are shown in Figs. 6 and 7.

Bioprocess Biosyst Eng (2014) 37:225–233 Table 2 Degradability of different compounds: biogas composition Substrate

CH4 [vol %]

CO2 [vol %]

Glycerol

65.5

25.0

1,3-PDO

75.9

18.4

Glycerol/1,3-PDO

75.3

20.0

Fermentation broth

67.5

25.0

Glycerol assimilation to 1,3-PDO was not affected by the addition of molybdate. However, molybdate delayed 1,3PDO degradation. At a lower 1,3-PDO concentration of 15 g/L (Fig. 6), 1,3-PDO (without molybdate addition) was degraded after 50 h. When sodium molybdate was added, 1,3-PDO was stable for 150 h. As 1,3-PDO was degraded, propionate concentration increased concomitantly: after the degradation of 14 g/L of 1,3-PDO, 14 g/L of propionate was measured at the end of the fermentation. A high propionate concentration, in turn, inhibited further degradation steps: other by-products (acetate, butyrate, lactate) could not be further degraded and thus the fermentation process ceased. At a high 1,3-PDO concentration (Fig. 7), its degradation still occurred even with the addition of molybdate. However, the degradation did not occur for the first 10 days. In comparison, 1,3-PDO in fermentation medium without molybdate was already degraded after 5 days. As before, the acetate and butyrate concentrations were not affected. But eventually, the same amount of 1,3-PDO was degraded, with or without molybdate. It can be assumed that the concentration of molybdate was in this case too low to prevent a complete inhibition on 1,3-PDO degradation. These results show that even though 1,3-PDO was degraded, its degradation started only after the degradation of glycerol and other typical organic compounds in the fermentation broth. It should be mentioned that an active

Fig. 5 Degradability of different compounds (glycerol, 1,3-PDO and fermentation broth) in standardized tests according to the Guideline VDI 4630. Left complete degradation profiles. Right a segment of degradation tests of the first 7 days

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Fig. 6 1,3-PDO production and the effect of molybdate on 1,3-PDO degradation at lower 1,3-PDO concentration (15 g/L). Left 1,3-PDO degradation without molybdate. Right Effect of molybdate addition

Fig. 7 1,3-PDO production and the effect of molybdate on 1,3-PDO degradation at high 1,3-PDO concentration (60 g/L). Left 1,3-PDO degradation without molybdate. Right Effect of molybdate addition

biogas-producing mixed culture with a very high ratio of inoculum (400 ml inoculum for 50 ml test substrate) was applied in the degradation tests. In our fermentation process with the selected mixed culture the conversion of glycerol and 1,3-PDO into biogas must be very low or negligible due to the short fermentation time (Fig. 5) and the low amount of inoculums. Even if methanogenic microorganisms may exist in our mixed culture, they may first convert the by-products, organic acids like acetate and butyrate, into biogas. Because the organic acids are inhibitive for cell growth and 1,3-PDO production, the simultaneous conversion of organic acids into biogas can be considered as an advantage of our fermentation process with mixed culture [5]. This effect could be even used to remove the organic acids in the fermentation broth to simplify the downstream processing, since the organic acids and their salts represent a major burden for the recovery of 1,3-PDO [29].

Conclusion In this study, we successfully achieved 1,3-PDO production at high concentration and yield with a selected mixed culture grown on raw glycerol. The process can be carried out under unsterile working conditions and with a simple medium without the use of yeast extract. The mixed culture is robust and easier to handle. Raw glycerol was efficiently converted to 1,3-PDO with a comparable yield (0.56–0.76 mol/mol) and productivity to those reported for pure cultures. In a fed-batch fermentation, a high 1,3-PDO concentration of 70 g/L was obtained with a productivity of 2.60 g/L h. Even though 1,3-PDO could be degraded in mixed cultures with an active population of biogas-producing microorganisms, its degradation process is considerably slower than that of glycerol and other compounds in culture. In our fermentation process with a selected mixed

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culture 1,3-PDO can be produced in a time window which prevents 1,3-PDO degradation. The process developed in this work can significantly reduce the costs of investment and operation for microbial production of 1,3-PDO from raw glycerol. Acknowledgments This work was financially supported by 7th Framework research program of the European Union through the project Propanergy (grant agreement no. 212671). We thank the project partners, especially Mr. Klaus Rohbrecht-Buck and Mr. Ralf Gabler from the company BKW Fu¨rstenwalde for their help and support in the pilot plant fermentation.

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