ARTICLE High Cell Density Propionic Acid Fermentation with an Acid Tolerant Strain of Propionibacterium acidipropionici Zhongqiang Wang, Ying Jin, Shang-Tian Yang William G. Lowrie Department of Chemical & Biomolecular Engineering, The Ohio State University, 140W. 19th Ave, Columbus, OH 43210; telephone: þ1-614-292-6611; fax: þ1-614-292-3769; e-mail: [email protected]
ABSTRACT: Propionic acid is an important chemical with wide applications and its production via fermentation is of great interest. However, economic production of bio-based propionic acid requires high product titer, yield, and productivity in the fermentation. A highly efficient and stable high cell density (HCD) fermentation process with cell recycle by centrifugation was developed for propionic acid production from glucose using an acid-tolerant strain of Propionibacterium acidipropionici, which had a higher specific growth rate, productivity, and acid tolerance compared to the wild type ATCC 4875. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a high titer of 40 g/L and productivity of 2.98 g/L h, with a yield of 0.44 g/g. The product yield increased to 0.53–0.62 g/g at a lower pH of 5.0–5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/L h was obtained in fed-batch HCD fermentation at pH 6.5. A 3-stage simulated fed-batch process in serum bottles produced 49.2 g/L propionic acid with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free-cell propionic acid fermentation. Biotechnol. Bioeng. 2014;9999: 1–10. ß 2014 Wiley Periodicals, Inc. KEYWORDS: acid tolerant strain; high cell density fermentation; propionic acid; Propionibacterium acidipropionici; sequential batch fermentation
Introduction Propionic acid is a 3-carbon fatty acid with various industrial applications, including uses as preservatives in animal feed Zhongqiang Wang and Ying Jin contributed equally to this paper. Correspondence to: S.-T. Yang Contract grant sponsor: The Dow Chemical Company Received 16 May 2014; Revision received 28 July 2014; Accepted 16 September 2014 Accepted manuscript online xx Month 2014; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25466
ß 2014 Wiley Periodicals, Inc.
and dairy and bakery products, and in manufacturing herbicide intermediate and cellulose acetate propionate. Currently, propionic acid is mainly produced by petrochemical-based processes, such as the oxo process with ethylene and carbon monoxide as feedstocks. With the depletion of petroleum, rising oil price, environmental burden of fossil fuel production and public’s preference for biobased chemicals, the production of propionic acid from renewable biomass via fermentation has gained large attention in recent years. Many bacteria are capable of producing propionic acid. Particularly, the species in the genus of Propionibacterium, including P. acidipropionici, P. freudenreichii, and P. shermanii that have been widely used in industrial production of Swiss cheese and vitamin B12 (Gardner and Champagne, 2005), have been intensively studied for propionic acid fermentation (Barbirato et al., 1997; Dishisha et al., 2012; Huang et al., 2002; Liang et al., 2012; Liu et al., 2011; Paik and Glatz, 1994; Wang and Yang, 2013; Yang et al., 1994, 1995; Zhu et al., 2012). However, propionic acid fermentation usually suffers from low productivity, yield and titer due to the strong inhibition of propionic acid and the coproduction of acetate and succinate, making it difficult to compete with petrochemical processes. Numerous research efforts have thus focused on increasing the reactor productivity by elevating viable cell density (see Table I) or removing propionic acid in situ to alleviate product inhibition (Jin and Yang, 1998; Lewis and Yang, 1992; Yang et al., 2006). Cell recycle, retention and immobilization are three efficient ways to achieve high density of active cells. Continuous fermentation processes with cell retention using an in situ spin filter (Gupta and Srivastava, 2001) or cell recycle via an external ultrafiltration unit have been used to increase cell density and reactor productivity, achieving the highest volumetric productivity of 14.3 g/L h ever reported (Boyaval and Corre, 1987). However, these continuous fermentation processes were not stable, because of membrane fouling, and suffered from a low final product titer, usually less than 20 g/L, which would greatly increase the cost for downstream product recovery. The high equipment and operation costs with ultrafiltration and spin filter for continuous cell recycle or retention also limit their
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
1
Table I. Comparison of high cell density propionic acid fermentation processes with P. acidipropionici and various substrates.
Fermentation mode
Substrate
Cell recycle (ultrafiltration) Continuous Whey Continuous Whey Continuous Glucose/xylose Sequential-batch Whey Cell recycle (centrifugation) Sequential batch Glycerol SB (pH 6.5) Glucose SB (pH 5.5) Glucose Fed-batch (pH 6.5) Glucose Fed-batch (pH 5.5–6.5) Fed-batch (pH 5.5) Sim. fed-batch (pH 5.0) Glucose Cell retention (in situ spin filter) Continuous Lactose Continuous Whey permeate
Cell density (g/L) 80 100 95 50 23.7 36.9 32.5 29.6 35.2 35.6 20.9 21.2 11.5
Final titer (g/L) 7 25 18 35 27.3 39.7 34.5 55.7 58.5 42.6 49.2 18.5 14.5
Propionate yield (g/g)
Productivity (g/L h)
0.43 — 0.42 0.54
2.14 14.3 2.2 1.2
0.63 0.44–0.48 0.62 0.43 0.46 0.53 0.53
1.42 2.98 1.28 2.23 1.44 0.57 0.66
Dishisha et al. (2013) This study
0.40 0.54
0.9 0.72
Goswami and Srivastava (2001) Gupta and Srivastava (2001)
Refs Blanc and Goma (1987) Boyaval and Corre (1987) Carrondo et al. (1988) Colomban et al. (1993)
This study
This study
SB, sequential batch.
application. On the other hand, sequential or repeated batches with cell recycle for high cell density fermentation is relatively simple to operate, and can give a high final product concentration and productivity (Colomban et al., 1993). More recently, cell recycle by centrifugation was used in sequential batch (SB) fermentation that produced propionic acid at a titer of 25–50.8 g/L, yield of 0.6 g/g and productivity of 0.29–1.42 g/L h (Dishisha et al., 2013). The goal of this study was to develop high cell density (HCD) fermentation for propionic acid production from glucose with an acid-tolerant strain of P. acidipropionici isolated in our laboratory. Compared to its parental strain ATCC 4875, this acid-tolerant strain had a much higher tolerance to propionic acid inhibition and would form large cell aggregates that facilitated cell recycle by centrifugation. The effects of seeding density on propionic acid fermentation productivity and yield were first studied in serum bottles. Then, HCD sequential batch and fed-batch fermentations with cell recycle by centrifugation were studied at various pHs, between 5.0 and 6.5 in stirred-tank bioreactors. The results demonstrated that a stable, high-titer, high-rate and high-yield process can be developed for propionic acid production.
and concentrated glucose solution were autoclaved separately at 121 C for 30 min and then mixed aseptically before use. Batch Fermentation in Serum Bottles The seed culture was prepared in a stirred-tank bioreactor containing 1 L medium (50 g/L glucose) at 32 C and pH 6.5, which was controlled by adding ammonium hydroxide. When the cell density, which was periodically monitored by measuring the optical density (OD), had reached the highest value in the bioreactor in 48 h, cells were collected by centrifugation at 4,000g for 5 min. After removing the supernatant, cells were resuspended in 200 mL of fresh medium and used to seed serum-bottle fermentations. To study the effect of seeding cell density on the fermentation, various amounts of cells (2, 4, 8, 14, and 20 mL) were added to serum bottles containing sterile medium to a total volume of 20 mL, and 1.2 mL of sterile concentrated glucose solution (500 g/L) was then added to a final concentration of 30 g/L. Each serum bottle also contained 50 g/L CaCO3 to buffer the pH at 5.0. These bottles were incubated at 32 C and liquid samples were withdrawn regularly and stored at 20 C. Duplicated bottles were used for each condition studied.
Materials and Methods Sequential Batch Fermentation in Bioreactor Culture and Media An acid-tolerant strain of P. acidipropionici ACT-1 derived from ATCC 4875 after a serial adaptation in a fibrous-bed bioreactor (Zhang and Yang, 2009) followed with screening and isolation of single colonies on propionate-containing agar plates was used in this study. The fermentation was studied in a synthetic medium containing 10 g/L yeast extract (Difco, Detroit, MI), 5 g/L Trypticase (BBL), 0.25 g/ L K2HPO4, 0.05 g/L MnSO4, and 30–50 g/L glucose as carbon source, unless otherwise noted. The medium without glucose
2
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
Sequential batch (SB) fermentation was performed in a 1-L bioreactor containing 500 mL medium with agitation at 100 rpm. The bioreactor temperature was maintained at 32 C and the pH was controlled at 6.5, 5.5, or 5.0 by the addition of ammonia hydroxide. Anaerobic conditions were established by purging the medium with nitrogen for 20 min. The reactor was then inoculated with an overnight culture at 30% (v/v). The batch fermentation was continued until glucose in the medium was almost depleted. At the end of the batch fermentation, cells were collected from the
fermentation broth by centrifugation at 4,000g for 5 min and resuspended in fresh medium to start the next batch. The bioreactor was sparged with nitrogen between batches to maintain anaerobiosis. A total of 11 consecutive batches with cell recycle were carried out in medium containing 70–80 g/L glucose at pH 6.5 in 230 h. Thereafter, three sequential batches at pH 5.5, followed with one batch at 5.0 and a final batch with 50 g/L CaCO3 for buffering the pH were carried out to study the pH effect. Sequential Batch and Fed-Batch Fermentations With High Cell Density The SB fermentation at pH 6.5 was repeated with a higher initial seeding density of about OD 100. Cells in the late exponential phase cultured in 3 L medium with 50 g/L glucose at pH 6.5 were collected by centrifugation and used to seed the batch fermentation in a 1-L bioreactor containing 500 mL medium with agitation at 100 rpm. A total of four sequential batches were performed at pH 6.5 in 65 h. The fermentation was then studied in the fed-batch mode to reach a high final propionic acid titer of 50 g/L. In fed-batch fermentation, an initial glucose concentration of 50 g/L was used and a concentrated glucose solution was pulse fed into the reactor when glucose was lower than 15 g/L or about to deplete. Three sequential fed-batch fermentations were performed: the first at pH 6.5, the second at pH 5.5 initially and then shifted to pH 6.5, and the third at pH 5.5.
colonies on propionate-containing agar plates. Compared to the wild type P. acidipropionici ATCC 4875, ACT-1 showed a much higher tolerance to propionic acid inhibition in a growth test with various initial concentrations of propionic acid in the medium (Fig. 1A). For the wild type, the specific growth rate decreased 80% from 0.19 to 0.04 h1 in the presence of 20 g/L propionic acid. In contrast, ACT-1 maintained 60% of its growth rate (0.15 vs. 0.24 h1) at 20 g/L propionic acid. In addition, ACT-1 also grew much faster than the wild type and had a high volumetric productivity of 0.62 g/L h (data not shown), which was more than twice of the highest productivity (0.25 g/L h) reported so far (Woskow and Glatz, 1991; Zhu et al., 2012), in conventional free-cell batch fermentation. The ACT-1 also formed large cell aggregates in fermentation (see Fig. 1B), which could facilitate its recycle by centrifugation (or microfiltration) for use in HCD SB fermentation.
Analytical Methods Cell growth was monitored by measuring the optical density (OD) at 600 nm in a 1.5-mL cuvette (light path length: 1 cm) using a spectrophotometer (UV-16-1, Shimazu, Columbia, MD). Samples with suspended cells were diluted with distilled water to give an OD reading of less than 0.8. To determine the cell dry weight (CDW), cells in fermentation broth with known OD and volume were collected by centrifugation and washed with distilled water. Then, the collected cells were dried at 110 C overnight or until the cell dry weight (CDW) was constant. Five samples were analyzed to establish the relationship between OD and CDW by plotting OD against CDW. One OD unit at 600 nm was found to be equivalent to 0.20 g/L CDW. The concentrations of glucose and acid products (acetic, succinic, and propionic acids) in fermentation broth samples were analyzed by high performance liquid chromatography (HPX-87H, Bio-Rad, Hercules, CA) at 45 C and 0.005 M H2SO4 as the mobile phase at 0.6 mL/min (Suwannakham and Yang, 2005).
Results and Discussion Acid Tolerant Strain of P. acidipropionici The acid-tolerant P. acidipropionici ACT-1 was obtained after a serial adaptation in a fibrous-bed bioreactor (Zhang and Yang, 2009) followed with screening and isolation of single
Figure 1.
Comparison of P. acidipropionici ACT-1 and the wild type ATCC 4875 in their acid tolerance and cell morphology. A: Non-competitive inhibition of propionic acid on the specific growth rate. ACT-1 had higher specific growth rates and propionic acid tolerance than the wild type (WT) as indicated by the higher values of mm and Kp in the non-competitive product inhibition model. A higher Kp value means less sensitive to propionic acid inhibition. mm and Kp were determined from the intercept and slope in the linear plot of 1/m versus propionic acid concentration (P). Symbols show the data and curves show the model predictions. B: Microscopic images showing large cell aggregates of ACT-1 and well dispersed individual cells and small cell clusters of wild type found in fermentation. ACT-1 cells were smaller (shorter) but tended to form large aggregates (40–160 mm in diameter), especially toward the end of each batch fermentation, whereas the wild type cells were larger (longer) and often with several cells connected together but did not form large aggregates.
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
3
High Cell Density Fermentation in Serum Bottles HCD fermentation was first studied in serum bottles with P. acidipropionici ATCC 4875 wild type and the acid tolerant ACT-1 with 30 g/L glucose as carbon source and 50 g/L CaCO3 to buffer the pH. Five different initial cell densities were tested for each strain and the fermentation results are summarized and compared in Figure 2. As expected, propionic acid productivity increased with increasing the initial cell optical density (OD) for both strains and there was a clear linear correlation between productivity and OD except for the last one or two points (Fig. 2A). The increase in productivity with OD leveled off at the higher cell density can
Productivity (g/L/h)
A
be attributed to stronger product inhibition at a higher propionic acid concentration reached in the HCD fermentation, especially at a low pH of 4.9 in serum bottles. Compared to the wild type, the acid tolerant ACT-1 had significantly higher productivity at the same cell density, confirming that ACT-1 is a better strain for propionic acid production. A high productivity of 1.46 g/L h was obtained with ACT-1 at an initial cell density of 24 g/L (OD 120). Interestingly, propionic acid yield also increased with increasing the initial cell density for both strains (Fig. 2B). When the initial cell density increased ten-fold, propionic acid yield increased from 0.51 g/g to 0.64 g/g for the wild type and from 0.525 g/g to 0.634 g/g for ACT-1. It is noted
1.8
B 0.80
1.6
0.75
1.4
0.70
mutant
Yield (g/g)
1.2 1.0 0.8 0.6
0.65 0.60 0.55 0.50
0.4
0.45
0.2 0.0
WT
0.40 0
50
100
150
200
0
50
C
D
18 16
12
200
30
20
P/S (g/g)
P/A (g/g)
150
25
14
10 8 6
15 10
4
5
2 0
100
OD
OD
WT 0
50
100
mutant 150
OD
WT 200
0
0
50
100
mutant 150
200
OD
Figure 2. Effects of cell density on productivity, yield, P/A ratio and P/S ratio in propionic acid fermentation of glucose by P. acidipropionici ATCC 4875 and ACT-1 in serum bottles. Each serum bottle contained 30 g/L glucose as carbon source and 50 g/L CaCO3 for buffering pH at 5.0.
4
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
þ Acetic acid þ CO2 þ H2 O HMP : 3 Glucose þ NADþ ! 5 Propionic acid
Sequential Batch Fermentation in Bioreactor at pH 6.5 Figure 3 shows the kinetics of SB fermentation in bioreactor at pH 6.5. A total of 11 consecutive batches with cell recycle were carried out in medium containing 70–80 g/L glucose in 230 h (Fig. 3A). The first batch was inoculated at 30% (v/v), which gave an initial OD of 0.564. After a 7-h lag phase, rapid
succinic acid OD
acec acid 200
70 60
150
50 40
100
30 20
50
10 0
þ 3 CO2 þ NADH
0
25
50
75
100
125
150
175
200
225
0
Time (h)
B 90
glucose propionic acid
80
Concentration (g/L)
The theoretical maximum propionic acid yield from glucose is thus between 0.55 and 0.69 g/g depending on if glucose is oxidized via EMP or HMP pathway. More propionic acid and less acetic acid are produced from glucose via the HMP pathway because it can generate more reducing power or NADH needed for propionic acid biosynthesis. The high propionic acid yield of >0.55 g/g obtained in the HCD fermentation clearly indicated that a significant amount of glucose was oxidized via the HMP pathway. The fact that increasing the initial cell density also increased propionic acid yield also suggested that more glucose was metabolized through the HMP pathway at the higher cell density. Also, at a higher cell density, cell growth would be slower and thus less glucose would be used for cell biomass, which also contributed to the increased propionic acid yield. In addition, less ATP would be required for cell growth at a higher cell density, which not only would allow more glucose to be oxidized through the HMP pathway but also would reduce the carbon flux through the ATPgenerating acetate biosynthesis pathway, resulting in a higher propionic acid/acetic acid ratio (P/A). For the wild type, P/A increased from 4.3 at OD 16.7 to 6.3 at OD 33.4, whereas for ACT-1 P/A increased from 10.2 to 14.1 when OD increased from 12 to 120 (Fig. 2C). Similar findings have also been reported in a recent paper (Stowers et al., 2014). Succinate was also produced as a by-product in the fermentation. Succinic acid is an intermediate metabolite in the dicarboxylic acid pathway, but its accumulation can lead to its secretion to the fermentation broth. However, only a small amount of succinate was produced in the HCD fermentations as indicated by the high propionic acid/succinic acid (P/S) ratio of 9–17 for ACT-1 and >20 for the wild type (Fig. 2D). It is thus clear that HCD fermentation can provide a highly efficient propionic acid production process with high productivity, yield and product purity.
250
glucose propionic acid
80
OD
1:5 Glucose ! 2 Propionic acid
90
succinic acid OD
300
acec acid
250
70
200
60 50
150
40 30
100
20
50
10 0
OD
EMP :
A Concentration (g/L)
that these two strains had almost the same propionic acid yields at the same cell density. Theoretically, propionibacteria will convert each mol of glucose to 4/3 mol propionic acid and 2/3 mol acetic acid via the Embden-Meyerhof-Parnas (EMP) pathway, and 5/3 mol propionic acid but no acetic acid via the Hexose Monophosphate (HMP) pathway, as shown in the following equations (Playne, 1985).
0
10
20
30
40
50
60
70
0
Time (h)
Figure 3. Sequential batch fermentations of glucose by P. acidipropionici ACT-1 in bioreactor at pH 6.5. A: Fermentation with a low initial seeding cell density; (B) Fermentation with a high initial seeding cell density.
cell growth and propionic acid production were observed. Cells entered the stationary phase after 32.5 h but propionic acid production continued and reached 30 g/L by 52 h when the first batch was stopped. In the following 10 sequential batches, no lag phase was observed and cell density continued to increase and reached a high OD value of 210 (42 g/L CDW) at the end of the 11th batch. Clearly, cells recycled by centrifugation remained active. As the cell density increased, the glucose consumption rate also increased in each subsequent batch. Consequently, the time for each batch fermentation also shortened incrementally to 1.8 g/L h but lower average yield of 0.44 g/g. The P/A ratio obtained in the SB fermentation
At the end of the 11th batch of the SB fermentation at pH 6.5, cells were recycled by centrifugation to start sequential batch HCD fermentations at lower pHs. Three sequential batches at pH 5.5, one batch at pH 5.0 and one batch with 50 g/L CaCO3 for buffering the reactor pH at 5.0 were performed. The time-course data are shown in Figure 5A and the kinetic parameters are listed in Table II. For the three batches at pH 5.5, both propionic acid productivity and yield increased progressively from 1.07 g/L h and 0.528 g/g to 1.28 g/L h and 0.616 g/g, respectively, while the cell density decreased from OD 190 at the beginning to OD 170 at the end of the 3rd batch. The P/A ratio increased slightly from 5.7 to 6.6, while the P/S ratio decreased from 9.4 to 7.9. The increased propionic acid yield thus can be attributed to the gradually reduced cell growth, as indicated by the lowered OD and increased P/A, whereas the increased productivity might be
Table II. Kinetics of high cell density sequential batch and fed-batch fermentations at lower pHs. Mode
Batch
pH
OD
Titer (g/L)
Yield (g/g)
Productivity (g/L h)
P/A (g/g)
P/S (g/g)
PA/TA (%)
Batch
1 2 3 4 5 1 2 3 Stage 1 Stage 2 Stage 3 Overall
5.5
194–167 150–178 162–165 116–147
33.1 35.6 34.5 23.8 33.5 55.7 58.5 42.6 21.2 38.2 49.2 49.2
0.53 0.57 0.62 0.55 0.54 0.43 0.46 0.53 0.66 0.50 0.44 0.53
1.07 1.23 1.28 0.49 0.71 2.23 1.44 0.57 0.75 0.70 0.46 0.66
5.71 6.21 6.59 6.69 7.29 12.24 8.44 5.38 16.0 9.2 7.8 11.0
9.41 8.52 7.89 27.24 10.06 6.66 5.81 10.85 97.4 7.1 2.8 7.0
78.0 78.2 78.2 84.3 80.9 75.0 78.0 78.2 93.1 80.9 67.2 81.0
Fed-batch
“Fed-batch”
5.0 CaCO3 (5.0) 6.5 5.5–6.5 5.5 5.4–4.8 5.9–5.4 6.1–5.6
148–147 177–176 213–143 120 120 120
The initial glucose concentration was 70 g/L in the batch fermentation, 50 g/L for each batch in the fed-batch fermentation, and 30 g/L in each stage of the simulated “fed-batch” fermentation.
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
7
attributed to cell adaptation to the lower pH environment. The propionic acid yield of 0.53–0.62 g/g and productivity of 1.28 g/L h obtained at pH 5.5 were comparable to those obtained at pH 5.0 in serum bottles at a similar cell density. The results are consistent with the observation that higher propionic acid yield and P/A ratio are favored at lower pH of 5.5 (Hsu and Yang, 1991). For the subsequent batch operated at pH 5.0, the productivity decreased drastically to 0.5 g/L h and yield also decreased to 0.55 g/g. The decreased productivity was caused by the stronger propionic acid inhibition at pH 5.0 and lower initial cell density (OD 115), whereas the propionic acid yield was lowered because there was significant cell growth as evidenced by the OD increase to 150. To improve propionic acid yield and productivity, the fermentation was then conducted with 50 g/L CaCO3 in the bioreactor to mimic the conditions of HCD fermentation in serum bottles. With CaCO3, not only the pH would be maintained at 5.0, propionic acid yield could also be increased for more CO2, released from the acid neutralization reaction, would be available for the CO2 fixation reaction catalyzed by pyruvate carboxylase. Indeed, the productivity increased 43% to 0.71 g/L h; however, the propionic acid yield remained unchanged at 0.54 g/g. Both the productivity and yield from this batch fermentation were significantly lower than those in serum bottles with a comparable cell density, suggesting that not all recycled cells were viable or active due to prolonged exposure to the acidic pH and possible cell damage in repeated treatments with centrifugation. It should be noted, however, that the static cultivation mode and the accumulated high CO2 pressure within the sealed bottles, as well as the lower glucose concentration (30 vs. 70–80 g/L) used in serum bottle fermentation might have also contributed to the higher propionic acid production. Among all different pH conditions studied in the bioreactor, pH 5.5 was the best because it can give both a high yield (>0.6 g/g) and a relatively high productivity (1.28 g/L h).
inhibition. Compared to the fed-batch at constant pH 6.5, the overall propionic acid yield improved 7.3% to 0.46 g/g while the overall productivity decreased to 1.44 g/L h. For the fed-batch at constant pH 5.5, fermentation almost ceased at 40 g/L propionic acid, and the prolonged fermentation time resulted in a significantly lower productivity of 0.57 g/L h although the overall yield was higher at 0.53 g/g (see Table II). Since higher productivity and yield were obtained in HCD fermentation carried out in serum bottles with CaCO3 for pH buffering, simulated “fed-batch” fermentation was carried out in serum bottles in three stages to progressively increase propionic acid titer to 20 g/L, then to 40 g/L, and finally to 50 g/L. Each stage with 30 g/L of glucose was seeded with a high cell density of OD 104.4 and run for 23 h. The fermentation kinetics is shown in Figure 6, with key performance parameters listed in Table II. The first stage reached 21 g/L of propionic acid, with a high yield of 0.66 g/g and productivity of 0.75 g/L h; the second stage reached 39 g/L with a yield of 0.50 g/g and productivity of 0.70 g/L h; the final stage reached 49 g/L with a yield of 0.44 g/g and productivity of 0.46 g/L h. Both the productivity and yield decreased as the propionic acid concentration increased because of the stronger propionic acid inhibition at higher concentration. Overall, the simulated “fed-batch” fermentation produced 49 g/L propionic acid from 90 g/L glucose in 69 h, with a propionic acid yield of 0.53 g/g and productivity of 0.66 g/L h, which was significantly higher than that in the bioreactor with pH controlled at 5.0. Effect of Glucose Concentration on Propionic Acid Yield Higher propionic acid yields were obtained in HCD fermentation in serum bottles than in bioreactor. The surprisingly high yields of more than 0.6 g/g were achieved when the initial OD was higher than 50 in serum bottles. However, the average yield of HCD fermentation in
High Cell Density Fed-Batch Fermentation
8
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
60
7 Stage I
Stage III
Stage II
50
6
40
glucose succinic acid lacc acid acec acid propionic acid pH
30 20 10 0
5 4
pH
Concentration (g/L)
Fed-batch fermentation has been widely used to produce propionic acid at a high final titer, which is inversely proportional to the cost of downstream product recovery and purification. HCD fermentation at the fed-batch mode was studied at three pH conditions: pH 6.5, pH shift from 5.5 to 6.5, and pH 5.5. The results are shown in Figure 5B and Table II. The final propionic acid titer at pH 6.5 reached 55.7 g/L with a high productivity of 2.23 g/L h and a propionic acid yield of 0.43 g/g. Both the productivity and yield were comparable to those of HCD sequential batch fermentation at pH 6.5, suggesting that ACT-1 can tolerate more than 50 g/L propionic acid at pH 6.5. Propionic acid fermentation with pH shift could give both a high final titer and a high yield (Feng et al., 2010; Zhuge et al., 2014). The pH was set at 5.5 at first for higher yield and then shifted to 6.5 when propionic acid had reached 33 g/L to alleviate propionic acid
3 2
0
15
30
45
60
75
1
Time (h)
Figure 6.
Kinetics of three-stage simulated fed-batch fermentation in serum bottles with CaCO3 for pH buffering.
bioreactor at pH 6.5 was only 0.44 g/g. The yield was still lower than 0.55 g/g even when pH dropped to 5.0. In order to increase the yield in bioreactor, it is of great interest to find the reasons for this discrepancy. Since the initial glucose concentration was different, its impact on propionic acid yield was studied. Serum bottles containing 30, 50, and 70 g/L glucose were inoculated at two different initial cell densities (OD 68.5 and 137) and the fermentation lasted 30 h. Glucose at 30 g/L was depleted in 24 h while a significant amount of glucose was left at the end point for 50 and 70 g/L bottles. As shown in Figure 7, generally the same results were obtained for both cell density conditions: the yields for 30 g/L glucose were similar to those from the previous serum bottle test (>0.6 g/g) but decreased with an increase in the initial glucose concentration. The results clearly showed that a higher glucose concentration (70 g/L) would give a lower propionic acid yield (0.48–0.50 g/g), which could account for the lower yield in bioreactor which also contained 70–80 g/L glucose. Detailed analysis revealed that propionic acid titer increased between 24 and 30 h, even though glucose was depleted in 24 h, which greatly contributed to the higher yield. Pyruvate is a central metabolite in the metabolic pathway of propionibacteria and its secretion and reassimilation in propionic acid fermentation have been reported (Goswami and Srivastava, 2001; Hsu and Yang, 1991). Therefore, when glucose was completely used, probably pyruvate accumulated within propionibacteria cells would be converted to propionic acid via either transcarboxylation with methylmalonyl-CoA or carboxylation with CO2 fixation (Parizzi et al., 2012). It is also likely that the trehalose accumulated during cell growth (Ruhal and Choudhury, 2012) could be reassimilated and used when glucose was depleted. It should be noted that some propionic acid might also be produced from amino acids present in yeast extract and trypticase, which would significantly increase the
0.7 OD 68.5 0.6
OD 137
Yield (g/g)
0.5 0.4 0.3 0.2 0.1 0 30
Figure 7.
50 70 Inial glucose concentraon (g/L)
Effect of glucose concentration on propionic acid yield in high cell density fermentations in serum bottles with two different initial cell densities.
product yield based on glucose consumption, especially when less glucose was present in the medium. Comparison to Other Studies Table I summarizes and compares propionic acid production from various substrates with high-density P. acidipropionici cells attained by cell recycle or retention. Boyaval and Corre (1987) used ultrafiltration to reach a high cell density of 100 g/L in a continuous fermentation process and reported a high volumetric productivity of 14.3 g/L h at the end of the study, which was stopped due to severe clogging of the ultrafiltration membrane. The maximum productivity of 2.1–2.2 g/L h was attained in another two continuous processes with ultrafiltration for cell recycle (Blanc and Goma, 1987; Carrondo et al., 1988). However, the final propionic acid titer was low, less than 20 g/L. A continuous fermentation process with cell retention facilitated by an in situ spin filter gave a moderate productivity of 0.40–0.54 g/L h at the final titer of less than 20 g/L (Goswami and Srivastava, 2001; Gupta and Srivastava, 2001). Recently, sequential batch high cell density fermentation was investigated with cells recycled by centrifugation and glycerol as carbon source (Dishisha et al., 2013). Productivity increased as the cell density continued to increase in sequential batches and reached the maximum cell density of 23.7 g/L, which gave the maximum productivity of 1.42 g/L h, by the seventh batch. However, both cell density and productivity ceased to increase after the 7th batch, perhaps due to the redox imbalance caused by the lower reducing state of glycerol (Chang et al., 1999; Himmi et al., 2000; Wang and Yang, 2013). The average propionic acid titer reached in their sequential batch fermentations was 27 g/L, although a higher propionic acid titer of 50 g/L was obtained from 120 g/L glycerol at a lower productivity of 0.29 g/L h. In general, the maximum cell density and volumetric productivity obtained with glycerol were much lower than those attained with glucose in our study. We achieved a high cell density of 40 g/L, productivity of 2.98 g/L h, and yield of 0.44–0.48 g/g in SB fermentation at pH 6.5, with a final propionic acid titer of 40 g/L. The yield increased to 0.62 g/g when the reactor pH was controlled at 5.5, while the productivity was 1.28 g/L h. In addition, a high final propionic acid titer of >55 g/L and a high productivity of 2.23 g/L h were obtained in fed-batch HCD fermentation at pH 6.5. Finally, 49.2 g/L propionic acid was produced in a 3-stage simulated fed-batch process in serum bottles with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free-cell propionic acid fermentation.
Conclusions A highly efficient and stable HCD fermentation process was developed for propionic acid production from glucose using an acid tolerant P. acidipropionici strain. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
9
high titer of 40 g/L and productivity of 2.98 g/L h. The propionic acid yield was 0.44 g/g at pH 6.5 and increased to 0.53–0.62 g/g at a lower pH of 5.0–5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/ L h was obtained in fed-batch HCD fermentation at pH 6.5. Since the productivity increases with cell density and yield would be higher at lower cell growth, it is desirable to operate the fed-batch fermentation at the highest possible cell density and low glucose concentration and pH. Also, operating the fermentation under a high CO2 partial pressure may increase CO2 fixation and propionic acid production, which should be studied in a pressurized bioreactor. Finally, co-fermentation with a more reduced substrate such as glycerol can increase product yield (Wang and Yang, 2013), and thus should also be considered in future study. This study was supported in part by The Dow Chemical Company.
References Barbirato F, Chedaille D, Bories A. 1997. Propionic acid fermentation from glycerol: Comparison with conventional substrates. Appl Microbiol Biotechnol 47:441–446. Blanc P, Goma G. 1987. Propionic acid fermentation: Improvement of performances by coupling continuous fermentation and ultrafiltration. Bioprocess Eng 2:137–139. 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. Carrondo MJT, Crespo JPSG, Moura MJ. 1988. Production of propionic acid using a xylose utilizing Propionibacterium. Appl Biochem Biotechnol 17:295–312. Chang D-E, Shin S, Rhee JS, Pan JG. 1999. Acetate metabolism in a pta mutant of Escherichia coli W3110: Importance of maintaining acetyl coenzyme a flux for growth and survival. J Bacteriol 181:6656–6663. Colomban A, Roger L, Boyaval P. 1993. Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling. Biotechnol Bioeng 42:1091–1098. Dishisha T, Alvarez MT, 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. Dishisha T, Ståhl A, Lundmark S, Hatti-Kaul R. 2013. An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation. Bioresour Technol 135:504–512. Feng X, Xu H, Yao J, Li S, Zhu H, Ouyang P. 2010. Kinetic analysis and pHshift control strategy for propionic acid production with Propionibacterium freudenreichii CCTCC M207015. Appl Biochem Biotechnol 160:343– 349. Gardner N, Champagne CP. 2005. Production of Propionibacterium shermanii biomass and vitamin B12 on spent media. J Appl Microbiol 99:1236– 1245. Goswami V, Srivastava AK. 2001. Propionic acid production in an in situ cell retention bioreactor. Appl Microbiol Biotechnol 56:676–680. Gupta A, Srivastava AK. 2001. Continuous propionic acid production from cheese whey using in situ spin filter. Biotechnol Bioprocess Eng 6:1–5. Himmi EH, Bories A, Boussaid A, Hassani L. 2000. Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. Appl Microbiol Biotechnol 53:435–440.
10
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
Hsu S-T, Yang S-T. 1991. Propionic acid fermentation of lactose by Propionibacterium acidipropionici: Effects of pH. Biotechnol Bioeng 38:571–578. Huang YL, Wu Z, Zhang L, Cheung CM, Yang S-T. 2002. Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresour Technol 82:51–59. Jin Z, Yang S-T. 1998. Extractive fermentation for enhanced propionic acid production from lactose by Propionibacterium acidipropionici. Biotechnol Prog 14:457–465. Lewis VP, Yang S-T. 1992. A novel extractive fermentation process for propionic acid production from whey lactose. Biotechnol Prog 8:104–110. Liang Z, Li L, Li S, Cai Y, Yang ST, Wang J. 2012. Enhanced propionic acid production from Jerusalem artichoke hydrolysate by immobilized Propionibacterium acidipropionici in a fibrous-bed bioreactor. Bioprocess Biosyst Eng 35:915–921. Liu Y, Zhang Y, Zhang R, Zhang F, Zhu J. 2011. Glycerol/glucose cofermentation: One more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr Microbiol 62:152–158. Paik H-D, Glatz BA. 1994. Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici. Appl Microbiol Biotechnol 42:22–27. Parizzi LP, Grassi MC, Llerena LA, Carazzolle MF, Queiroz VL, Lunardi I, Zeidler AF, Teixeira PJ, Mieczkowski P, Rincones J, Pereira GA. 2012. The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential. BMC Genomics 13:562. Playne MJ. 1985. Propionic and butyric acids. In: Moo-Young M, editor. Comprehensive biotechnology. New York: Pergamon Press. p 731–759. Ruhal R, Choudhury B. 2012. Use of an osmotically sensitive mutant of Propionibacterium freudenreichii subspp. shermanii for the simultaneous productions of organic acids and trehalose from biodiesel waste based crude glycerol. Bioresour Technol 109:131–139. Stowers CC, Cox BM, Rodriguez BA. 2014. Development of an industrializable fermentation process for propionic acid production. J Ind Microbiol Biotechnol 41:837–852. Suwannakham S, Yang ST. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng 91:325–337. Wang Z, Yang S-T. 2013. Propionic acid production in glycerol/glucose cofermentation by Propionibacterium freudenreichii subsp. shermanii. Bioresour Technol 137:116–123. Woskow SA, Glatz BA. 1991. Propionic acid production by a propionic acidtolerant strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation. Appl Environ Microbiol 57:2821–2828. Yang S-T, Huang Y, Hong G. 1995. A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose. Biotechnol Bioeng 45:379–386. Yang S-T, Zhu H, Li Y, Hong G. 1994. Continuous propionate production from whey permeate using a novel fibrous bed bioreactor. Biotechnol Bioeng 43:1124–1130. Yang ST, Huang H, Tay A, Qin W, De Guzman L, San Nicolas EC. 2006. Extractive fermentation for the production of carboxylic acids. In: Yang ST, editor. Bioprocessing for value-added products from renewable resources. Amsterdam, The Netherlands: Elsevier. p 421–446. Zhang A, Yang S-T. 2009. Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentation. Biotechnol Bioeng 45:79–386. Zhu L, Wei P, Cai J, Zhu X, Wang Z, Huang L, Xu Z. 2012. Improving the productivity of propionic acid with FBB-immobilized cells of an adapted acid-tolerant Propionibacterium acidipropionici. Bioresour Technol 112:248–253. Zhuge X, Liu L, Shin H-D, 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.
ABSTRACT: Propionic acid is an important chemical with wide applications and its production via fermentation is of great interest. However, economic production of bio-based propionic acid requires high product titer, yield, and productivity in the fermentation. A highly efficient and stable high cell density (HCD) fermentation process with cell recycle by centrifugation was developed for propionic acid production from glucose using an acid-tolerant strain of Propionibacterium acidipropionici, which had a higher specific growth rate, productivity, and acid tolerance compared to the wild type ATCC 4875. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a high titer of 40 g/L and productivity of 2.98 g/L h, with a yield of 0.44 g/g. The product yield increased to 0.53–0.62 g/g at a lower pH of 5.0–5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/L h was obtained in fed-batch HCD fermentation at pH 6.5. A 3-stage simulated fed-batch process in serum bottles produced 49.2 g/L propionic acid with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free-cell propionic acid fermentation. Biotechnol. Bioeng. 2014;9999: 1–10. ß 2014 Wiley Periodicals, Inc. KEYWORDS: acid tolerant strain; high cell density fermentation; propionic acid; Propionibacterium acidipropionici; sequential batch fermentation
Introduction Propionic acid is a 3-carbon fatty acid with various industrial applications, including uses as preservatives in animal feed Zhongqiang Wang and Ying Jin contributed equally to this paper. Correspondence to: S.-T. Yang Contract grant sponsor: The Dow Chemical Company Received 16 May 2014; Revision received 28 July 2014; Accepted 16 September 2014 Accepted manuscript online xx Month 2014; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25466
ß 2014 Wiley Periodicals, Inc.
and dairy and bakery products, and in manufacturing herbicide intermediate and cellulose acetate propionate. Currently, propionic acid is mainly produced by petrochemical-based processes, such as the oxo process with ethylene and carbon monoxide as feedstocks. With the depletion of petroleum, rising oil price, environmental burden of fossil fuel production and public’s preference for biobased chemicals, the production of propionic acid from renewable biomass via fermentation has gained large attention in recent years. Many bacteria are capable of producing propionic acid. Particularly, the species in the genus of Propionibacterium, including P. acidipropionici, P. freudenreichii, and P. shermanii that have been widely used in industrial production of Swiss cheese and vitamin B12 (Gardner and Champagne, 2005), have been intensively studied for propionic acid fermentation (Barbirato et al., 1997; Dishisha et al., 2012; Huang et al., 2002; Liang et al., 2012; Liu et al., 2011; Paik and Glatz, 1994; Wang and Yang, 2013; Yang et al., 1994, 1995; Zhu et al., 2012). However, propionic acid fermentation usually suffers from low productivity, yield and titer due to the strong inhibition of propionic acid and the coproduction of acetate and succinate, making it difficult to compete with petrochemical processes. Numerous research efforts have thus focused on increasing the reactor productivity by elevating viable cell density (see Table I) or removing propionic acid in situ to alleviate product inhibition (Jin and Yang, 1998; Lewis and Yang, 1992; Yang et al., 2006). Cell recycle, retention and immobilization are three efficient ways to achieve high density of active cells. Continuous fermentation processes with cell retention using an in situ spin filter (Gupta and Srivastava, 2001) or cell recycle via an external ultrafiltration unit have been used to increase cell density and reactor productivity, achieving the highest volumetric productivity of 14.3 g/L h ever reported (Boyaval and Corre, 1987). However, these continuous fermentation processes were not stable, because of membrane fouling, and suffered from a low final product titer, usually less than 20 g/L, which would greatly increase the cost for downstream product recovery. The high equipment and operation costs with ultrafiltration and spin filter for continuous cell recycle or retention also limit their
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
1
Table I. Comparison of high cell density propionic acid fermentation processes with P. acidipropionici and various substrates.
Fermentation mode
Substrate
Cell recycle (ultrafiltration) Continuous Whey Continuous Whey Continuous Glucose/xylose Sequential-batch Whey Cell recycle (centrifugation) Sequential batch Glycerol SB (pH 6.5) Glucose SB (pH 5.5) Glucose Fed-batch (pH 6.5) Glucose Fed-batch (pH 5.5–6.5) Fed-batch (pH 5.5) Sim. fed-batch (pH 5.0) Glucose Cell retention (in situ spin filter) Continuous Lactose Continuous Whey permeate
Cell density (g/L) 80 100 95 50 23.7 36.9 32.5 29.6 35.2 35.6 20.9 21.2 11.5
Final titer (g/L) 7 25 18 35 27.3 39.7 34.5 55.7 58.5 42.6 49.2 18.5 14.5
Propionate yield (g/g)
Productivity (g/L h)
0.43 — 0.42 0.54
2.14 14.3 2.2 1.2
0.63 0.44–0.48 0.62 0.43 0.46 0.53 0.53
1.42 2.98 1.28 2.23 1.44 0.57 0.66
Dishisha et al. (2013) This study
0.40 0.54
0.9 0.72
Goswami and Srivastava (2001) Gupta and Srivastava (2001)
Refs Blanc and Goma (1987) Boyaval and Corre (1987) Carrondo et al. (1988) Colomban et al. (1993)
This study
This study
SB, sequential batch.
application. On the other hand, sequential or repeated batches with cell recycle for high cell density fermentation is relatively simple to operate, and can give a high final product concentration and productivity (Colomban et al., 1993). More recently, cell recycle by centrifugation was used in sequential batch (SB) fermentation that produced propionic acid at a titer of 25–50.8 g/L, yield of 0.6 g/g and productivity of 0.29–1.42 g/L h (Dishisha et al., 2013). The goal of this study was to develop high cell density (HCD) fermentation for propionic acid production from glucose with an acid-tolerant strain of P. acidipropionici isolated in our laboratory. Compared to its parental strain ATCC 4875, this acid-tolerant strain had a much higher tolerance to propionic acid inhibition and would form large cell aggregates that facilitated cell recycle by centrifugation. The effects of seeding density on propionic acid fermentation productivity and yield were first studied in serum bottles. Then, HCD sequential batch and fed-batch fermentations with cell recycle by centrifugation were studied at various pHs, between 5.0 and 6.5 in stirred-tank bioreactors. The results demonstrated that a stable, high-titer, high-rate and high-yield process can be developed for propionic acid production.
and concentrated glucose solution were autoclaved separately at 121 C for 30 min and then mixed aseptically before use. Batch Fermentation in Serum Bottles The seed culture was prepared in a stirred-tank bioreactor containing 1 L medium (50 g/L glucose) at 32 C and pH 6.5, which was controlled by adding ammonium hydroxide. When the cell density, which was periodically monitored by measuring the optical density (OD), had reached the highest value in the bioreactor in 48 h, cells were collected by centrifugation at 4,000g for 5 min. After removing the supernatant, cells were resuspended in 200 mL of fresh medium and used to seed serum-bottle fermentations. To study the effect of seeding cell density on the fermentation, various amounts of cells (2, 4, 8, 14, and 20 mL) were added to serum bottles containing sterile medium to a total volume of 20 mL, and 1.2 mL of sterile concentrated glucose solution (500 g/L) was then added to a final concentration of 30 g/L. Each serum bottle also contained 50 g/L CaCO3 to buffer the pH at 5.0. These bottles were incubated at 32 C and liquid samples were withdrawn regularly and stored at 20 C. Duplicated bottles were used for each condition studied.
Materials and Methods Sequential Batch Fermentation in Bioreactor Culture and Media An acid-tolerant strain of P. acidipropionici ACT-1 derived from ATCC 4875 after a serial adaptation in a fibrous-bed bioreactor (Zhang and Yang, 2009) followed with screening and isolation of single colonies on propionate-containing agar plates was used in this study. The fermentation was studied in a synthetic medium containing 10 g/L yeast extract (Difco, Detroit, MI), 5 g/L Trypticase (BBL), 0.25 g/ L K2HPO4, 0.05 g/L MnSO4, and 30–50 g/L glucose as carbon source, unless otherwise noted. The medium without glucose
2
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
Sequential batch (SB) fermentation was performed in a 1-L bioreactor containing 500 mL medium with agitation at 100 rpm. The bioreactor temperature was maintained at 32 C and the pH was controlled at 6.5, 5.5, or 5.0 by the addition of ammonia hydroxide. Anaerobic conditions were established by purging the medium with nitrogen for 20 min. The reactor was then inoculated with an overnight culture at 30% (v/v). The batch fermentation was continued until glucose in the medium was almost depleted. At the end of the batch fermentation, cells were collected from the
fermentation broth by centrifugation at 4,000g for 5 min and resuspended in fresh medium to start the next batch. The bioreactor was sparged with nitrogen between batches to maintain anaerobiosis. A total of 11 consecutive batches with cell recycle were carried out in medium containing 70–80 g/L glucose at pH 6.5 in 230 h. Thereafter, three sequential batches at pH 5.5, followed with one batch at 5.0 and a final batch with 50 g/L CaCO3 for buffering the pH were carried out to study the pH effect. Sequential Batch and Fed-Batch Fermentations With High Cell Density The SB fermentation at pH 6.5 was repeated with a higher initial seeding density of about OD 100. Cells in the late exponential phase cultured in 3 L medium with 50 g/L glucose at pH 6.5 were collected by centrifugation and used to seed the batch fermentation in a 1-L bioreactor containing 500 mL medium with agitation at 100 rpm. A total of four sequential batches were performed at pH 6.5 in 65 h. The fermentation was then studied in the fed-batch mode to reach a high final propionic acid titer of 50 g/L. In fed-batch fermentation, an initial glucose concentration of 50 g/L was used and a concentrated glucose solution was pulse fed into the reactor when glucose was lower than 15 g/L or about to deplete. Three sequential fed-batch fermentations were performed: the first at pH 6.5, the second at pH 5.5 initially and then shifted to pH 6.5, and the third at pH 5.5.
colonies on propionate-containing agar plates. Compared to the wild type P. acidipropionici ATCC 4875, ACT-1 showed a much higher tolerance to propionic acid inhibition in a growth test with various initial concentrations of propionic acid in the medium (Fig. 1A). For the wild type, the specific growth rate decreased 80% from 0.19 to 0.04 h1 in the presence of 20 g/L propionic acid. In contrast, ACT-1 maintained 60% of its growth rate (0.15 vs. 0.24 h1) at 20 g/L propionic acid. In addition, ACT-1 also grew much faster than the wild type and had a high volumetric productivity of 0.62 g/L h (data not shown), which was more than twice of the highest productivity (0.25 g/L h) reported so far (Woskow and Glatz, 1991; Zhu et al., 2012), in conventional free-cell batch fermentation. The ACT-1 also formed large cell aggregates in fermentation (see Fig. 1B), which could facilitate its recycle by centrifugation (or microfiltration) for use in HCD SB fermentation.
Analytical Methods Cell growth was monitored by measuring the optical density (OD) at 600 nm in a 1.5-mL cuvette (light path length: 1 cm) using a spectrophotometer (UV-16-1, Shimazu, Columbia, MD). Samples with suspended cells were diluted with distilled water to give an OD reading of less than 0.8. To determine the cell dry weight (CDW), cells in fermentation broth with known OD and volume were collected by centrifugation and washed with distilled water. Then, the collected cells were dried at 110 C overnight or until the cell dry weight (CDW) was constant. Five samples were analyzed to establish the relationship between OD and CDW by plotting OD against CDW. One OD unit at 600 nm was found to be equivalent to 0.20 g/L CDW. The concentrations of glucose and acid products (acetic, succinic, and propionic acids) in fermentation broth samples were analyzed by high performance liquid chromatography (HPX-87H, Bio-Rad, Hercules, CA) at 45 C and 0.005 M H2SO4 as the mobile phase at 0.6 mL/min (Suwannakham and Yang, 2005).
Results and Discussion Acid Tolerant Strain of P. acidipropionici The acid-tolerant P. acidipropionici ACT-1 was obtained after a serial adaptation in a fibrous-bed bioreactor (Zhang and Yang, 2009) followed with screening and isolation of single
Figure 1.
Comparison of P. acidipropionici ACT-1 and the wild type ATCC 4875 in their acid tolerance and cell morphology. A: Non-competitive inhibition of propionic acid on the specific growth rate. ACT-1 had higher specific growth rates and propionic acid tolerance than the wild type (WT) as indicated by the higher values of mm and Kp in the non-competitive product inhibition model. A higher Kp value means less sensitive to propionic acid inhibition. mm and Kp were determined from the intercept and slope in the linear plot of 1/m versus propionic acid concentration (P). Symbols show the data and curves show the model predictions. B: Microscopic images showing large cell aggregates of ACT-1 and well dispersed individual cells and small cell clusters of wild type found in fermentation. ACT-1 cells were smaller (shorter) but tended to form large aggregates (40–160 mm in diameter), especially toward the end of each batch fermentation, whereas the wild type cells were larger (longer) and often with several cells connected together but did not form large aggregates.
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
3
High Cell Density Fermentation in Serum Bottles HCD fermentation was first studied in serum bottles with P. acidipropionici ATCC 4875 wild type and the acid tolerant ACT-1 with 30 g/L glucose as carbon source and 50 g/L CaCO3 to buffer the pH. Five different initial cell densities were tested for each strain and the fermentation results are summarized and compared in Figure 2. As expected, propionic acid productivity increased with increasing the initial cell optical density (OD) for both strains and there was a clear linear correlation between productivity and OD except for the last one or two points (Fig. 2A). The increase in productivity with OD leveled off at the higher cell density can
Productivity (g/L/h)
A
be attributed to stronger product inhibition at a higher propionic acid concentration reached in the HCD fermentation, especially at a low pH of 4.9 in serum bottles. Compared to the wild type, the acid tolerant ACT-1 had significantly higher productivity at the same cell density, confirming that ACT-1 is a better strain for propionic acid production. A high productivity of 1.46 g/L h was obtained with ACT-1 at an initial cell density of 24 g/L (OD 120). Interestingly, propionic acid yield also increased with increasing the initial cell density for both strains (Fig. 2B). When the initial cell density increased ten-fold, propionic acid yield increased from 0.51 g/g to 0.64 g/g for the wild type and from 0.525 g/g to 0.634 g/g for ACT-1. It is noted
1.8
B 0.80
1.6
0.75
1.4
0.70
mutant
Yield (g/g)
1.2 1.0 0.8 0.6
0.65 0.60 0.55 0.50
0.4
0.45
0.2 0.0
WT
0.40 0
50
100
150
200
0
50
C
D
18 16
12
200
30
20
P/S (g/g)
P/A (g/g)
150
25
14
10 8 6
15 10
4
5
2 0
100
OD
OD
WT 0
50
100
mutant 150
OD
WT 200
0
0
50
100
mutant 150
200
OD
Figure 2. Effects of cell density on productivity, yield, P/A ratio and P/S ratio in propionic acid fermentation of glucose by P. acidipropionici ATCC 4875 and ACT-1 in serum bottles. Each serum bottle contained 30 g/L glucose as carbon source and 50 g/L CaCO3 for buffering pH at 5.0.
4
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
þ Acetic acid þ CO2 þ H2 O HMP : 3 Glucose þ NADþ ! 5 Propionic acid
Sequential Batch Fermentation in Bioreactor at pH 6.5 Figure 3 shows the kinetics of SB fermentation in bioreactor at pH 6.5. A total of 11 consecutive batches with cell recycle were carried out in medium containing 70–80 g/L glucose in 230 h (Fig. 3A). The first batch was inoculated at 30% (v/v), which gave an initial OD of 0.564. After a 7-h lag phase, rapid
succinic acid OD
acec acid 200
70 60
150
50 40
100
30 20
50
10 0
þ 3 CO2 þ NADH
0
25
50
75
100
125
150
175
200
225
0
Time (h)
B 90
glucose propionic acid
80
Concentration (g/L)
The theoretical maximum propionic acid yield from glucose is thus between 0.55 and 0.69 g/g depending on if glucose is oxidized via EMP or HMP pathway. More propionic acid and less acetic acid are produced from glucose via the HMP pathway because it can generate more reducing power or NADH needed for propionic acid biosynthesis. The high propionic acid yield of >0.55 g/g obtained in the HCD fermentation clearly indicated that a significant amount of glucose was oxidized via the HMP pathway. The fact that increasing the initial cell density also increased propionic acid yield also suggested that more glucose was metabolized through the HMP pathway at the higher cell density. Also, at a higher cell density, cell growth would be slower and thus less glucose would be used for cell biomass, which also contributed to the increased propionic acid yield. In addition, less ATP would be required for cell growth at a higher cell density, which not only would allow more glucose to be oxidized through the HMP pathway but also would reduce the carbon flux through the ATPgenerating acetate biosynthesis pathway, resulting in a higher propionic acid/acetic acid ratio (P/A). For the wild type, P/A increased from 4.3 at OD 16.7 to 6.3 at OD 33.4, whereas for ACT-1 P/A increased from 10.2 to 14.1 when OD increased from 12 to 120 (Fig. 2C). Similar findings have also been reported in a recent paper (Stowers et al., 2014). Succinate was also produced as a by-product in the fermentation. Succinic acid is an intermediate metabolite in the dicarboxylic acid pathway, but its accumulation can lead to its secretion to the fermentation broth. However, only a small amount of succinate was produced in the HCD fermentations as indicated by the high propionic acid/succinic acid (P/S) ratio of 9–17 for ACT-1 and >20 for the wild type (Fig. 2D). It is thus clear that HCD fermentation can provide a highly efficient propionic acid production process with high productivity, yield and product purity.
250
glucose propionic acid
80
OD
1:5 Glucose ! 2 Propionic acid
90
succinic acid OD
300
acec acid
250
70
200
60 50
150
40 30
100
20
50
10 0
OD
EMP :
A Concentration (g/L)
that these two strains had almost the same propionic acid yields at the same cell density. Theoretically, propionibacteria will convert each mol of glucose to 4/3 mol propionic acid and 2/3 mol acetic acid via the Embden-Meyerhof-Parnas (EMP) pathway, and 5/3 mol propionic acid but no acetic acid via the Hexose Monophosphate (HMP) pathway, as shown in the following equations (Playne, 1985).
0
10
20
30
40
50
60
70
0
Time (h)
Figure 3. Sequential batch fermentations of glucose by P. acidipropionici ACT-1 in bioreactor at pH 6.5. A: Fermentation with a low initial seeding cell density; (B) Fermentation with a high initial seeding cell density.
cell growth and propionic acid production were observed. Cells entered the stationary phase after 32.5 h but propionic acid production continued and reached 30 g/L by 52 h when the first batch was stopped. In the following 10 sequential batches, no lag phase was observed and cell density continued to increase and reached a high OD value of 210 (42 g/L CDW) at the end of the 11th batch. Clearly, cells recycled by centrifugation remained active. As the cell density increased, the glucose consumption rate also increased in each subsequent batch. Consequently, the time for each batch fermentation also shortened incrementally to 1.8 g/L h but lower average yield of 0.44 g/g. The P/A ratio obtained in the SB fermentation
At the end of the 11th batch of the SB fermentation at pH 6.5, cells were recycled by centrifugation to start sequential batch HCD fermentations at lower pHs. Three sequential batches at pH 5.5, one batch at pH 5.0 and one batch with 50 g/L CaCO3 for buffering the reactor pH at 5.0 were performed. The time-course data are shown in Figure 5A and the kinetic parameters are listed in Table II. For the three batches at pH 5.5, both propionic acid productivity and yield increased progressively from 1.07 g/L h and 0.528 g/g to 1.28 g/L h and 0.616 g/g, respectively, while the cell density decreased from OD 190 at the beginning to OD 170 at the end of the 3rd batch. The P/A ratio increased slightly from 5.7 to 6.6, while the P/S ratio decreased from 9.4 to 7.9. The increased propionic acid yield thus can be attributed to the gradually reduced cell growth, as indicated by the lowered OD and increased P/A, whereas the increased productivity might be
Table II. Kinetics of high cell density sequential batch and fed-batch fermentations at lower pHs. Mode
Batch
pH
OD
Titer (g/L)
Yield (g/g)
Productivity (g/L h)
P/A (g/g)
P/S (g/g)
PA/TA (%)
Batch
1 2 3 4 5 1 2 3 Stage 1 Stage 2 Stage 3 Overall
5.5
194–167 150–178 162–165 116–147
33.1 35.6 34.5 23.8 33.5 55.7 58.5 42.6 21.2 38.2 49.2 49.2
0.53 0.57 0.62 0.55 0.54 0.43 0.46 0.53 0.66 0.50 0.44 0.53
1.07 1.23 1.28 0.49 0.71 2.23 1.44 0.57 0.75 0.70 0.46 0.66
5.71 6.21 6.59 6.69 7.29 12.24 8.44 5.38 16.0 9.2 7.8 11.0
9.41 8.52 7.89 27.24 10.06 6.66 5.81 10.85 97.4 7.1 2.8 7.0
78.0 78.2 78.2 84.3 80.9 75.0 78.0 78.2 93.1 80.9 67.2 81.0
Fed-batch
“Fed-batch”
5.0 CaCO3 (5.0) 6.5 5.5–6.5 5.5 5.4–4.8 5.9–5.4 6.1–5.6
148–147 177–176 213–143 120 120 120
The initial glucose concentration was 70 g/L in the batch fermentation, 50 g/L for each batch in the fed-batch fermentation, and 30 g/L in each stage of the simulated “fed-batch” fermentation.
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
7
attributed to cell adaptation to the lower pH environment. The propionic acid yield of 0.53–0.62 g/g and productivity of 1.28 g/L h obtained at pH 5.5 were comparable to those obtained at pH 5.0 in serum bottles at a similar cell density. The results are consistent with the observation that higher propionic acid yield and P/A ratio are favored at lower pH of 5.5 (Hsu and Yang, 1991). For the subsequent batch operated at pH 5.0, the productivity decreased drastically to 0.5 g/L h and yield also decreased to 0.55 g/g. The decreased productivity was caused by the stronger propionic acid inhibition at pH 5.0 and lower initial cell density (OD 115), whereas the propionic acid yield was lowered because there was significant cell growth as evidenced by the OD increase to 150. To improve propionic acid yield and productivity, the fermentation was then conducted with 50 g/L CaCO3 in the bioreactor to mimic the conditions of HCD fermentation in serum bottles. With CaCO3, not only the pH would be maintained at 5.0, propionic acid yield could also be increased for more CO2, released from the acid neutralization reaction, would be available for the CO2 fixation reaction catalyzed by pyruvate carboxylase. Indeed, the productivity increased 43% to 0.71 g/L h; however, the propionic acid yield remained unchanged at 0.54 g/g. Both the productivity and yield from this batch fermentation were significantly lower than those in serum bottles with a comparable cell density, suggesting that not all recycled cells were viable or active due to prolonged exposure to the acidic pH and possible cell damage in repeated treatments with centrifugation. It should be noted, however, that the static cultivation mode and the accumulated high CO2 pressure within the sealed bottles, as well as the lower glucose concentration (30 vs. 70–80 g/L) used in serum bottle fermentation might have also contributed to the higher propionic acid production. Among all different pH conditions studied in the bioreactor, pH 5.5 was the best because it can give both a high yield (>0.6 g/g) and a relatively high productivity (1.28 g/L h).
inhibition. Compared to the fed-batch at constant pH 6.5, the overall propionic acid yield improved 7.3% to 0.46 g/g while the overall productivity decreased to 1.44 g/L h. For the fed-batch at constant pH 5.5, fermentation almost ceased at 40 g/L propionic acid, and the prolonged fermentation time resulted in a significantly lower productivity of 0.57 g/L h although the overall yield was higher at 0.53 g/g (see Table II). Since higher productivity and yield were obtained in HCD fermentation carried out in serum bottles with CaCO3 for pH buffering, simulated “fed-batch” fermentation was carried out in serum bottles in three stages to progressively increase propionic acid titer to 20 g/L, then to 40 g/L, and finally to 50 g/L. Each stage with 30 g/L of glucose was seeded with a high cell density of OD 104.4 and run for 23 h. The fermentation kinetics is shown in Figure 6, with key performance parameters listed in Table II. The first stage reached 21 g/L of propionic acid, with a high yield of 0.66 g/g and productivity of 0.75 g/L h; the second stage reached 39 g/L with a yield of 0.50 g/g and productivity of 0.70 g/L h; the final stage reached 49 g/L with a yield of 0.44 g/g and productivity of 0.46 g/L h. Both the productivity and yield decreased as the propionic acid concentration increased because of the stronger propionic acid inhibition at higher concentration. Overall, the simulated “fed-batch” fermentation produced 49 g/L propionic acid from 90 g/L glucose in 69 h, with a propionic acid yield of 0.53 g/g and productivity of 0.66 g/L h, which was significantly higher than that in the bioreactor with pH controlled at 5.0. Effect of Glucose Concentration on Propionic Acid Yield Higher propionic acid yields were obtained in HCD fermentation in serum bottles than in bioreactor. The surprisingly high yields of more than 0.6 g/g were achieved when the initial OD was higher than 50 in serum bottles. However, the average yield of HCD fermentation in
High Cell Density Fed-Batch Fermentation
8
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
60
7 Stage I
Stage III
Stage II
50
6
40
glucose succinic acid lacc acid acec acid propionic acid pH
30 20 10 0
5 4
pH
Concentration (g/L)
Fed-batch fermentation has been widely used to produce propionic acid at a high final titer, which is inversely proportional to the cost of downstream product recovery and purification. HCD fermentation at the fed-batch mode was studied at three pH conditions: pH 6.5, pH shift from 5.5 to 6.5, and pH 5.5. The results are shown in Figure 5B and Table II. The final propionic acid titer at pH 6.5 reached 55.7 g/L with a high productivity of 2.23 g/L h and a propionic acid yield of 0.43 g/g. Both the productivity and yield were comparable to those of HCD sequential batch fermentation at pH 6.5, suggesting that ACT-1 can tolerate more than 50 g/L propionic acid at pH 6.5. Propionic acid fermentation with pH shift could give both a high final titer and a high yield (Feng et al., 2010; Zhuge et al., 2014). The pH was set at 5.5 at first for higher yield and then shifted to 6.5 when propionic acid had reached 33 g/L to alleviate propionic acid
3 2
0
15
30
45
60
75
1
Time (h)
Figure 6.
Kinetics of three-stage simulated fed-batch fermentation in serum bottles with CaCO3 for pH buffering.
bioreactor at pH 6.5 was only 0.44 g/g. The yield was still lower than 0.55 g/g even when pH dropped to 5.0. In order to increase the yield in bioreactor, it is of great interest to find the reasons for this discrepancy. Since the initial glucose concentration was different, its impact on propionic acid yield was studied. Serum bottles containing 30, 50, and 70 g/L glucose were inoculated at two different initial cell densities (OD 68.5 and 137) and the fermentation lasted 30 h. Glucose at 30 g/L was depleted in 24 h while a significant amount of glucose was left at the end point for 50 and 70 g/L bottles. As shown in Figure 7, generally the same results were obtained for both cell density conditions: the yields for 30 g/L glucose were similar to those from the previous serum bottle test (>0.6 g/g) but decreased with an increase in the initial glucose concentration. The results clearly showed that a higher glucose concentration (70 g/L) would give a lower propionic acid yield (0.48–0.50 g/g), which could account for the lower yield in bioreactor which also contained 70–80 g/L glucose. Detailed analysis revealed that propionic acid titer increased between 24 and 30 h, even though glucose was depleted in 24 h, which greatly contributed to the higher yield. Pyruvate is a central metabolite in the metabolic pathway of propionibacteria and its secretion and reassimilation in propionic acid fermentation have been reported (Goswami and Srivastava, 2001; Hsu and Yang, 1991). Therefore, when glucose was completely used, probably pyruvate accumulated within propionibacteria cells would be converted to propionic acid via either transcarboxylation with methylmalonyl-CoA or carboxylation with CO2 fixation (Parizzi et al., 2012). It is also likely that the trehalose accumulated during cell growth (Ruhal and Choudhury, 2012) could be reassimilated and used when glucose was depleted. It should be noted that some propionic acid might also be produced from amino acids present in yeast extract and trypticase, which would significantly increase the
0.7 OD 68.5 0.6
OD 137
Yield (g/g)
0.5 0.4 0.3 0.2 0.1 0 30
Figure 7.
50 70 Inial glucose concentraon (g/L)
Effect of glucose concentration on propionic acid yield in high cell density fermentations in serum bottles with two different initial cell densities.
product yield based on glucose consumption, especially when less glucose was present in the medium. Comparison to Other Studies Table I summarizes and compares propionic acid production from various substrates with high-density P. acidipropionici cells attained by cell recycle or retention. Boyaval and Corre (1987) used ultrafiltration to reach a high cell density of 100 g/L in a continuous fermentation process and reported a high volumetric productivity of 14.3 g/L h at the end of the study, which was stopped due to severe clogging of the ultrafiltration membrane. The maximum productivity of 2.1–2.2 g/L h was attained in another two continuous processes with ultrafiltration for cell recycle (Blanc and Goma, 1987; Carrondo et al., 1988). However, the final propionic acid titer was low, less than 20 g/L. A continuous fermentation process with cell retention facilitated by an in situ spin filter gave a moderate productivity of 0.40–0.54 g/L h at the final titer of less than 20 g/L (Goswami and Srivastava, 2001; Gupta and Srivastava, 2001). Recently, sequential batch high cell density fermentation was investigated with cells recycled by centrifugation and glycerol as carbon source (Dishisha et al., 2013). Productivity increased as the cell density continued to increase in sequential batches and reached the maximum cell density of 23.7 g/L, which gave the maximum productivity of 1.42 g/L h, by the seventh batch. However, both cell density and productivity ceased to increase after the 7th batch, perhaps due to the redox imbalance caused by the lower reducing state of glycerol (Chang et al., 1999; Himmi et al., 2000; Wang and Yang, 2013). The average propionic acid titer reached in their sequential batch fermentations was 27 g/L, although a higher propionic acid titer of 50 g/L was obtained from 120 g/L glycerol at a lower productivity of 0.29 g/L h. In general, the maximum cell density and volumetric productivity obtained with glycerol were much lower than those attained with glucose in our study. We achieved a high cell density of 40 g/L, productivity of 2.98 g/L h, and yield of 0.44–0.48 g/g in SB fermentation at pH 6.5, with a final propionic acid titer of 40 g/L. The yield increased to 0.62 g/g when the reactor pH was controlled at 5.5, while the productivity was 1.28 g/L h. In addition, a high final propionic acid titer of >55 g/L and a high productivity of 2.23 g/L h were obtained in fed-batch HCD fermentation at pH 6.5. Finally, 49.2 g/L propionic acid was produced in a 3-stage simulated fed-batch process in serum bottles with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free-cell propionic acid fermentation.
Conclusions A highly efficient and stable HCD fermentation process was developed for propionic acid production from glucose using an acid tolerant P. acidipropionici strain. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a
Wang et al.: High Cell Density Propionic Acid Fermentation Biotechnology and Bioengineering
9
high titer of 40 g/L and productivity of 2.98 g/L h. The propionic acid yield was 0.44 g/g at pH 6.5 and increased to 0.53–0.62 g/g at a lower pH of 5.0–5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/ L h was obtained in fed-batch HCD fermentation at pH 6.5. Since the productivity increases with cell density and yield would be higher at lower cell growth, it is desirable to operate the fed-batch fermentation at the highest possible cell density and low glucose concentration and pH. Also, operating the fermentation under a high CO2 partial pressure may increase CO2 fixation and propionic acid production, which should be studied in a pressurized bioreactor. Finally, co-fermentation with a more reduced substrate such as glycerol can increase product yield (Wang and Yang, 2013), and thus should also be considered in future study. This study was supported in part by The Dow Chemical Company.
References Barbirato F, Chedaille D, Bories A. 1997. Propionic acid fermentation from glycerol: Comparison with conventional substrates. Appl Microbiol Biotechnol 47:441–446. Blanc P, Goma G. 1987. Propionic acid fermentation: Improvement of performances by coupling continuous fermentation and ultrafiltration. Bioprocess Eng 2:137–139. 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. Carrondo MJT, Crespo JPSG, Moura MJ. 1988. Production of propionic acid using a xylose utilizing Propionibacterium. Appl Biochem Biotechnol 17:295–312. Chang D-E, Shin S, Rhee JS, Pan JG. 1999. Acetate metabolism in a pta mutant of Escherichia coli W3110: Importance of maintaining acetyl coenzyme a flux for growth and survival. J Bacteriol 181:6656–6663. Colomban A, Roger L, Boyaval P. 1993. Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling. Biotechnol Bioeng 42:1091–1098. Dishisha T, Alvarez MT, 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. Dishisha T, Ståhl A, Lundmark S, Hatti-Kaul R. 2013. An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation. Bioresour Technol 135:504–512. Feng X, Xu H, Yao J, Li S, Zhu H, Ouyang P. 2010. Kinetic analysis and pHshift control strategy for propionic acid production with Propionibacterium freudenreichii CCTCC M207015. Appl Biochem Biotechnol 160:343– 349. Gardner N, Champagne CP. 2005. Production of Propionibacterium shermanii biomass and vitamin B12 on spent media. J Appl Microbiol 99:1236– 1245. Goswami V, Srivastava AK. 2001. Propionic acid production in an in situ cell retention bioreactor. Appl Microbiol Biotechnol 56:676–680. Gupta A, Srivastava AK. 2001. Continuous propionic acid production from cheese whey using in situ spin filter. Biotechnol Bioprocess Eng 6:1–5. Himmi EH, Bories A, Boussaid A, Hassani L. 2000. Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. Appl Microbiol Biotechnol 53:435–440.
10
Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2014
Hsu S-T, Yang S-T. 1991. Propionic acid fermentation of lactose by Propionibacterium acidipropionici: Effects of pH. Biotechnol Bioeng 38:571–578. Huang YL, Wu Z, Zhang L, Cheung CM, Yang S-T. 2002. Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresour Technol 82:51–59. Jin Z, Yang S-T. 1998. Extractive fermentation for enhanced propionic acid production from lactose by Propionibacterium acidipropionici. Biotechnol Prog 14:457–465. Lewis VP, Yang S-T. 1992. A novel extractive fermentation process for propionic acid production from whey lactose. Biotechnol Prog 8:104–110. Liang Z, Li L, Li S, Cai Y, Yang ST, Wang J. 2012. Enhanced propionic acid production from Jerusalem artichoke hydrolysate by immobilized Propionibacterium acidipropionici in a fibrous-bed bioreactor. Bioprocess Biosyst Eng 35:915–921. Liu Y, Zhang Y, Zhang R, Zhang F, Zhu J. 2011. Glycerol/glucose cofermentation: One more proficient process to produce propionic acid by Propionibacterium acidipropionici. Curr Microbiol 62:152–158. Paik H-D, Glatz BA. 1994. Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici. Appl Microbiol Biotechnol 42:22–27. Parizzi LP, Grassi MC, Llerena LA, Carazzolle MF, Queiroz VL, Lunardi I, Zeidler AF, Teixeira PJ, Mieczkowski P, Rincones J, Pereira GA. 2012. The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential. BMC Genomics 13:562. Playne MJ. 1985. Propionic and butyric acids. In: Moo-Young M, editor. Comprehensive biotechnology. New York: Pergamon Press. p 731–759. Ruhal R, Choudhury B. 2012. Use of an osmotically sensitive mutant of Propionibacterium freudenreichii subspp. shermanii for the simultaneous productions of organic acids and trehalose from biodiesel waste based crude glycerol. Bioresour Technol 109:131–139. Stowers CC, Cox BM, Rodriguez BA. 2014. Development of an industrializable fermentation process for propionic acid production. J Ind Microbiol Biotechnol 41:837–852. Suwannakham S, Yang ST. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng 91:325–337. Wang Z, Yang S-T. 2013. Propionic acid production in glycerol/glucose cofermentation by Propionibacterium freudenreichii subsp. shermanii. Bioresour Technol 137:116–123. Woskow SA, Glatz BA. 1991. Propionic acid production by a propionic acidtolerant strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation. Appl Environ Microbiol 57:2821–2828. Yang S-T, Huang Y, Hong G. 1995. A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose. Biotechnol Bioeng 45:379–386. Yang S-T, Zhu H, Li Y, Hong G. 1994. Continuous propionate production from whey permeate using a novel fibrous bed bioreactor. Biotechnol Bioeng 43:1124–1130. Yang ST, Huang H, Tay A, Qin W, De Guzman L, San Nicolas EC. 2006. Extractive fermentation for the production of carboxylic acids. In: Yang ST, editor. Bioprocessing for value-added products from renewable resources. Amsterdam, The Netherlands: Elsevier. p 421–446. Zhang A, Yang S-T. 2009. Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentation. Biotechnol Bioeng 45:79–386. Zhu L, Wei P, Cai J, Zhu X, Wang Z, Huang L, Xu Z. 2012. Improving the productivity of propionic acid with FBB-immobilized cells of an adapted acid-tolerant Propionibacterium acidipropionici. Bioresour Technol 112:248–253. Zhuge X, Liu L, Shin H-D, 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.
