Bioresource Technology 114 (2012) 711–714

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Bioresource Technology
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Short Communication

Efficient utilization of hemicellulose hydrolysate for propionic acid production using

Propionibacterium acidipropionici
Zhen Liu a, Cuiqing Ma a,⇑, Chao Gao a, Ping Xu b,a
a
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China
b
State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Hemicellulose, which contains glucose, xylose, and arabinose as the 3 main sugars, is an important
Received 21 October 2011 renewable source for biorefinery. In this study, propionic acid production from glucose, xylose, or arab-
Received in revised form 23 February 2012 inose using Propionibacterium acidipropionici ATCC 4875 was investigated. Using xylose, the predominant
Accepted 24 February 2012
sugar in hemicellulose, a final propionic acid concentration of 53.2 g l–1 was obtained via fed-batch fer-
Available online 4 March 2012
mentation. Using corncob molasses, a waste by-product from xylitol production as a representative of
hemicellulose hydrolysate, the final concentration of propionic acid was 71.8 g l–1, with a corresponding
Keywords:
productivity of 0.28 g l–1 h–1. The present study suggests that hemicellulose hydrolysate is an excellent
Hemicellulose
Propionic acid
carbon source for efficient propionic acid production by this strain.
Propionibacterium acidipropionici Ó 2012 Elsevier Ltd. All rights reserved.
Xylose

1. Introduction 1995), Propionibacterium freudenreichii (Feng et al., 2011), and Pro-

With the exhaustion of petroleum and the serious environmen-
tal pollution caused by the utilization of fossil resources, more and
more attention has been focused on biorefinery for obtaining bio-
fuels and other chemicals from renewable resources (Chew and
Bhatia, 2008). Lignocellulose is the most abundant renewable re-
source (Aristidou and Penttilä, 2000), and a major raw material
in the biorefinery. As an important part of lignocellulose, hemicel-
lulose could be used as a substrate for the production of many
valuable chemicals such as ethanol (Asghari et al., 1996), lactate
(Gao et al., 2011) and butanol (Qureshi et al., 2006).
Propionic acid and its associated calcium, sodium, and potas-
sium salts are ‘‘generally recognized as safe’’ by the Food and Drug
Administration (Colomban et al., 1993), and are chiefly used as
antifungal agents in human food and grain preservatives, as well
as additives in animal food (Boyaval and Corre, 1995; Suwannak-
ham, 2005). In other fields, propionic acid can be used for animal
therapy, and for the manufacture of perfume, herbicides, cellulose
plastics, and flavoring agents (Boyaval and Corre, 1995).
Currently, propionic acid is mainly produced via petrochemical
routes using propane, propionaldehyde, or propanol as raw mate-
rial (Boyaval and Corre, 1995; Suwannakham, 2005). There is
increasing demand for natural propionic acid for use as an impor-
tant preservative, which can be produced through fermentation by
bacteria such as Selenomonas ruminantium (Eaton and Gabelman,
⇑ Corresponding author. Tel: +86 531 88364003; fax: +86 531 88369463.
E-mail address: macq@sdu.edu.cn (C. Ma).
pionibacterium acidipropionici (Suwannakham, 2005). P. acidipropi-
onici has been long used in the cheese industry and has been
‘‘generally recognized as safe’’ (Brede et al., 2005). The purpose of
this study is to determine whether hemicellulose can be used as
a potential carbon source for propionic acid production using P.
acidipropionici in biorefinery processes.
2. Methods
2.1. Chemicals
Corncob molasses containing glucose (90 g l–1), xylose
(450 g l–1), and arabinose (146 g l–1) was supplied by Longlive
Bio-technology Co., Ltd. (Shandong, China). Glucose, xylose, arabi-
nose, propionic acid, acetic acid, and succinic acid were all of ana-
lytical grade, and were purchased from Guoyao Reagent Co., Ltd.
(Shanghai, China). Tryptone and yeast extracts were purchased
from Oxoid Ltd. (Basingstoke, Hampshire, England).
2.2. Culture and medium
Stock culture of P. acidipropionici ATCC 4875 was stored at
80 °C. The culture grown at 30 °C for 48 h (exponential phase)
was used as inoculum.
The medium used in this study contained (per liter): 5.0 g tryp-
tone, 10.0 g yeast extract, 0.25 g K2HPO4, 0.05 g MnSO4, and 50 ml
of corncob molasses or sugars with desired concentrations.

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2012.02.118
712 Z. Liu et al. / Bioresource Technology 114 (2012) 711–714

Fig. 1. Time course of batch fermentation with glucose (a), xylose (b) or arabinose (c) as the substrate by P. acidipropionici ATCC 4875 at 30 °C in 100-ml bottles. DCW (j),
glucose (s), xylose (d), arabinose (h), propionic acid (w), acetic acid (), succinic acid (N). Data are presented as the average ± standard deviation values of 3 parallel
replicates.

For fermentation in 100-ml bottles, the culture was incubated

without agitation, and 1 g of CaCO3 was added. pH was determined
at each sampling, and if lower than 6.0, it would be adjusted to
approximately 6.0 by injecting 10 M NaOH into the medium.
For fermentation in the bioreactor, the culture was incubated
with agitation at 100 rpm and controlled pH at 6.0 by automati-
cally adding 10 M NaOH, and slow nitrogen flux was used to main-
tain anaerobic conditions. During fed-batch fermentation in the
bioreactor, sugar powder was directly fed at desired time.
With a single sugar as the substrate, the initial concentration of
glucose, xylose, or arabinose in the medium was 30 g l–1. With
corncob molasses as the substrate, the initial concentrations of glu-
cose, xylose, and arabinose in the medium were 4.5, 22.5, and
7.3 g l–1, respectively.

Fig. 2. Time course of fed-batch fermentation with xylose as the substrate by P.

acidipropionici ATCC 4875 at pH 6.0 and 30 °C in a 5-l bioreactor. DCW (j), xylose
(d), propionic acid (w), acetic acid (), succinic acid (N).

Corncob molasses was autoclaved alone at 121 °C for 30 min, and

added to the medium on inoculation.
Air in the 100-ml screw-capped bottle containing 50 ml of the
medium was removed by creation of vacuum, followed by purging
3 times with high purity nitrogen. Then, the bottles were sealed
with butyl rubber caps in an anaerobic chamber (Model 1029, For-
ma Scientific, Inc., USA), and then autoclaved at 121 °C for 30 min.

2.3. Batch and fed-batch fermentations

Fermentation by P. acidipropionici ATCC 4875 was carried out in

100-ml bottles containing 50 ml of the medium or a 5-l stirred bio-
reactor with 3 l of the medium. After inoculation with the seed cul-
ture (5%, v/v), fermentation was carried out at 30 °C at an initial pH
of 6.0.
Fig. 3. Time course of fed-batch production of propionic acid using corncob
molasses as the substrate by P. acidipropionici ATCC 4875 with an initial cells
density of 14.4 g DCW l–1 at 30 °C in 100-ml bottles. DCW (j), glucose (s), xylose
(d), arabinose (h), propionic acid (w), acetic acid (), succinic acid (N). Data are
presented as the average ± standard deviation values of 3 parallel replicates.
 Z. Liu et al. / Bioresource Technology 114 (2012) 711–714 713

Table 1
Comparison of propionic acid production with xylose or hemicellulose hydrolysate as substrate.

Strain System Carbon source Propionic acid Productivity Reference

(g l–1) (g l–1 h–1)
P. acidipropionici ATCC 25562 Batch Glucose + xylose (3:1) 22.9 0.30 Carrondo et al. (1988)
P. acidipropionici ATCC 25562 CSTRa+UFb+cell recycle Glucose + xylose (3:1) 18 2.7 Carrondo et al. (1988)
P. acidipropionici ATCC 4875 Batch Hemicellulose hydrolysate 18.3 0.23 Ramsay et al. (1998)
P. acidipropionici ATCC 4875 Batch Xylose 18.5 0.24 Suwannakham (2005)
P. acidipropionici ATCC 4875 adapted mutant Batch Xylose 19.5 0.33 Suwannakham (2005)
P. acidipropionici ATCC 4875 Fed-batch Xylose 53.2 0.23 This work
P. acidipropionici ATCC 4875 Fed-batch Corncob molasses 71.8 0.28 This work
a
CSTR: Continuous stirred tank reactor.
b
UF: Ultrafiltration.

2.4. Analytical methods
The optical density (OD) was measured at 620 nm using a 2100
spectrophotometer (Unico Instruments Co., Ltd., Shanghai, China),
and the dry cell weight (DCW) was calculated from the OD value
by using a calibration equation (DCW = 0.2893  OD620 nm g l–1).
The concentrations of glucose, xylose, arabinose, propionic acid,
acetic acid, and succinic acid were determined by high perfor-
mance liquid chromatography (HPLC) in an Agilent 1100 series,
equipped with a Bio-Rad Aminex HPX-87H column (300 
7.8 mm) and a refractive index detector. Operating conditions were
as follows: mobile phase, 10 mM H2SO4; flow rate, 0.4 ml min–1;
and column temperature, 55 °C.
The productivity of propionic acid fermentation was calculated
as the ratio of the amount of propionic acid produced to the total
time required for fermentation.
3. Results and discussion
3.1. Batch fermentation with glucose, xylose or arabinose
Utilization of glucose, xylose or arabinose by P. acidipropionici
ATCC 4875 was investigated, as these are the 3 main sugars in
hemicellulose (Jones et al., 1979). As shown in Fig. 1, the highest
cell density attained was 7.6, 6.3 and 5.0 g DCW l–1 with glucose,
xylose, and arabinose as substrate, respectively. Propionic acid
with the final concentrations of 13.4, 13.3 and 13.8 g l–1 could be
produced from glucose, xylose, and arabinose, respectively. The re-
sults indicate that glucose, xylose, and arabinose are all good sub-
strates for production of propionic acid.
3.2. Fed-batch fermentation with xylose
Xylose, the predominant sugar in hemicellulose, should be
effectively utilized (Jones et al., 1979). To study whether xylose
could be effectively utilized by P. acidipropionici ATCC 4875, fed-
batch fermentation using xylose as the sole carbon source was
investigated. In the process of fed-batch fermentation, proper
amounts of xylose powder were added as supplements to the bio-
reactor when the xylose concentration was lower than 15 g l–1. As
shown in Fig. 2, cell growth entered stationary phase at approxi-
mately 4 d of fermentation, with the cell density fluctuating be-
tween 9 and 11 g DCW l–1 after that. The maximum propionic
acid concentration was 53.2 g l–1 at 9.5 d, with a corresponding
productivity of 0.23 g l–1 h–1, and the maximum concentrations of
acetic acid and succinic acid were 10.3 and 14.4 g l–1, respectively.
3.3. Fed-batch production of propionic acid with corncob molasses
Xylose accounts for about 78% of the total reducing sugars in
corncob hydrolysate (Cheng et al., 2010). In xylitol production, a
part of xylose is extracted from corncob hydrolysate, and the resid-
ual part is termed ‘‘corncob molasses’’. Although corncob molasses
is a waste by-product, it still contains mixed sugars with high su-
gar concentrations (total sugar is about 60 70% of the total bio-
mass). Furthermore, the ratio of xylose to the total reducing
sugars is about 66%, which is comparable to those in many hemi-
celluloses such as corn stover (61%) (Asghari et al., 1996). Corncob
molasses could be regarded as a representative of hemicellulose
hydrolysate. Therefore, propionic acid production using corncob
molasses as the sole carbon source was investigated in this study.
Propionic acid production was conducted using corncob molas-
ses as the substrate by P. acidipropionici ATCC 4875 with an initial
cell density of 14.4 g DCW l–1 in the medium at 30 °C, pH 6.0. As
shown in Fig. 3, the final propionic acid concentration was
71.8 g l–1 with a corresponding productivity of 0.28 g l–1 h–1.
As shown in Table 1, the 53.2 g l–1 of propionic acid obtained in
this study is the highest propionic acid concentration obtained
with xylose as the substrate so far (Carrondo et al., 1988; Suwan-
nakham, 2005). On the other hand, the propionic acid concentra-
tion (71.8 g l–1) with corncob molasses as the substrate is higher
than other reports (Carrondo et al., 1988; Ramsay et al., 1998).
These results suggest that corncob molasses is a good carbon
source for the propionic acid production. Therefore, hemicellulose
hydrolyaste is quite promising as a potential carbon source for the
production of propionic acid.
4. Conclusion
Utilization of glucose, xylose, and arabinose for propionic acid
production was investigated, and a high concentration (71.8 g l–1)
of propionic acid was produced from a hemicellulose hydrolysate,
corncob molasses. Thus, the results of this study suggest that hemi-
cellulose hydrolysate can serve as a good carbon source for effi-
cient propionic acid production by the P. acidipropionici strain.
Acknowledgements
The work was partially supported by the China Postdoctoral Sci-
ence Foundation (20100480600), and the Research Fund for the
Doctoral Program of Higher Education of China (20090131110036).
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