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College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, PR China
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA
a r t i c l e
i n f o
Article history:
Received 12 May 2014
Received in revised form
21 November 2014
Accepted 26 November 2014
Available online 27 November 2014
Keywords:
Cellulose
Fermentation
Filamentous fungi
Lactic acid
Rhizopus oryzae
Xylo-oligosaccharides
a b s t r a c t
High substrate cost and low lactic acid yield are the most pressing concerns in fermentative production
of l-lactic acid by Rhizopus oryzae. In this study, waste residue from corncob after xylo-oligosaccharides
(XOS) manufacturing was used as an alternative abundant, renewable, and inexpensive substrate for
l-lactic acid production. After enzymatic hydrolysis, both glucose and xylose in the hydrolysate were
converted to 34.0 g L1 of l-lactic acid, equivalent to a yield of 0.34 g g1 dry waste residue, by R. oryzae
in separate hydrolysis and fermentation. In contrast, a higher l-lactic acid titer (60.3 g L1 ) and yield
(0.60 g g1 dry waste residue) were achieved in simultaneous saccharication and fermentation (SSF)
with 10% (w/v) substrate loading at 40 C, demonstrating, for the rst time, the feasibility of l-lactic acid
production from XOS manufacturing waste residues. The SSF process for l-lactic acid production from
XOS waste residues was also demonstrated in a 5-L stirred-tank bioreactor, although further optimization
would be necessary.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Lactic acid is a commonly occurring organic acid that can be
produced biologically from renewable carbohydrates, and is valuable due to its wide use in food and related industries [1]. In
addition, a variety of useful chemicals, including plastics, bers,
solvents, and oxygenated chemicals, can be produced from lactic
acid derived from renewable feedstocks by sustainable biotechnological routes [2]. More recently, bio-based l-lactic acid has
attracted increasing attention for its use as a starting material in the
synthesis of poly-lactic acid (PLA) polymers, which are biodegradable and biocompatible with wide applications that conventional
petroleum-based plastics such as polyesters are not suitable or
unfavorable due to environmental concerns [3]. Today, lactic acid
produced in fermentation has become one of the most promising
feedstock monomers in the chemical industry.
Abbreviations: R. oryzae, Rhizopus oryzae; XOS, xylo-oligosaccharides; SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharication
and fermentation.
Corresponding author. Tel.:+86 25 85427587; fax: +86 25 85427587.
Corresponding author. Tel.: +1 614 2926611; fax: +1 614 2923769.
E-mail addresses: swhx@njfu.com.cn (Q. Yong), yang.15@osu.edu (S.-T. Yang).
http://dx.doi.org/10.1016/j.bej.2014.11.020
1369-703X/ 2014 Elsevier B.V. All rights reserved.
93
94
3. Results
Fig. 1. Time course proles of sugars released in enzymatic hydrolysis of xylooligosaccharides waste residues at 5%, 10%, 15%, and 20% (w/v) substrate loading.
A: glucose; B: xylose. Data shown are the average with error bar representing the
standard deviation from duplicate runs. Error bar is not visible for some data points
because the standard deviation is smaller than the size of the symbol.
95
Table 1
Effects of substrate loading on enzymatic hydrolysis of XOS waste residue.
Substrate loading (%)
Glucose
Xylose
1
Titer (g L
5
10
15
20
31.68
61.32
88.89
104.97
0.00
0.35
0.07
1.12
Yield (%)
87.13
84.32
81.49
72.17
Productivity (g L
0.01
0.49
0.06
0.77
0.66
1.28
1.48
1.75
Titer (g L1 )
0.00
0.01
0.00
0.02
8.10
15.90
23.46
29.38
Productivity (g L1 h1 )
Yield (%)
0.02
0.05
0.39
0.12
64.24
63.06
62.02
58.26
0.16
0.18
1.04
0.23
0.17
0.33
0.39
0.49
0.00
0.00
0.01
0.00
Data shown are the average standard deviation from duplicated runs.
Fig. 3. l-lactic acid fermentation proles of R. oryzae using XOS waste residue hydrolysates as substrate in shake-asks at 40 C and various substrate loadings. A: 5%; B: 10%;
C: 15%; and D: 20% (w/v) loading. Data shown are the average with error bar representing the standard deviation from duplicated runs. Error bar is not visible for some data
points because the standard deviation is smaller than the size of the symbol.
fermentation, xylose was also consumed simultaneously with glucose, but at a much slower rate. As can be seen in Fig. 3A, xylose
consumption rate increased signicantly 12 h after glucose depletion, suggesting that xylose uptake by the cells was inhibited or
repressed by glucose [9]. The slower xylose uptake by cells might
also be because xylose transport was much slower than glucose or
was strongly inhibited by glucose. Increasing the substrate loading
from 5% to 15% also increased lactic acid production titer (from
16.5 g L1 to 36.5 g L1 ) and productivity (from 0.34 g L1 h1 to
0.76 g L1 h1 ) (see Table 2). Further increasing the substrate loading to 20% signicantly decreased lactic acid production, which
might be attributed to the limitation in oxygen transfer at the
higher solid loading. The lactic acid yield was 0.330.34 g g1
dry waste residue when the substrate loading was 510%, but
Table 2
Effects of substrate loading on l-lactic acid fermentation by R. oryzae with the enzymatic hydrolysates of XOS waste residues.
Substrate loading (%)
Lactic acid (g L1 )
5
10
15
20
16.53
34.00
36.50
24.44
0.38
0.04
0.15
0.01
Ethanol (g L1 )
5.04
9.50
12.00
15.85
0.45
0.25
0.19
0.71
Data shown are the average standard deviation from duplicated runs.
3.28
3.58
3.04
1.54
0.33
0.34
0.24
0.12
0.01
0.00
0.00
0.00
0.01
0.00
0.00
0.00
96
Fig. 4. Effect of temperature on l-lactic acid production from XOS waste residues
at 5% (w/v) substrate loading in simultaneous saccharication and fermentation in
shake-asks. Data shown are the average with error bar representing the standard
deviation from duplicated runs.
Fig. 5. Proles of simultaneous saccharication and fermentation of XOS waste residues by R. oryzae in shake-asks at 40 C and various substrate loadings. A: 5%; B: 10%; C:
15%; and D: 20% (w/v) loading. CaCO3 was added at 12 h, which caused an increase in the pH. Data shown are the average with error bar representing the standard deviation
from duplicated runs. Error bar is not visible for some data points because the standard deviation is smaller than the size of the symbol.
97
Table 3
Effects of substrate loading on simultaneous saccharication and fermentation for l-lactic acid production from XOS waste residue by R. oryzae.
Substrate loading (%)
Lactic acid (g L1 )
5
10
15
20
24.87
60.29
47.72
33.75
0.10
0.26
0.19
1.01
Ethanol (g L1 )
4.64
5.71
7.28
8.13
0.17
0.32
0.21
1.03
5.36
10.56
6.55
4.15
0.50
0.60
0.32
0.17
Data shown are the average standard deviation from duplicated runs.
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.01
98
Table 4
Comparison of l-lactic acid production from lignocellulosic biomass by R. oryzae.
Strain
Substrate
Titer (g L1 )
Yield (g g1 )
Productivity (g L1 h1 )
References
SHF
R. oryzae HZS6
R. oryzae UMIP 4.77
R. oryzae NRRL 395
R. oryzae NBRC 5378
R. oryzae CBS 112.07
R. oryzae NRRL 395
R. sp. MK-96-1196
R. oryzae NRRL 395
R. oryzae GY 18
R. oryzae NLX-M-1
Corncob
Wheat straw
Waste ofce paper
Wheat straw
Wheat straw
Cassava pulp
Corncob
Wheat bran
Corncob
XOS waste residue
77.2
10
49.1
2
6.8
16.7
26
6
34.0
0.26
0.23
0.24
0.26
0.36
0.34
0.99
0.27
0.51
0.29
0.71
[12]
[22]
[23]
[24]
[25]
[27]
[28]
[29]
[30]
This study
SSF
R. oryzae UMIP 4.77
R. oryzae NBRC 5378
R. sp. MK-96-1196
R. oryzae NRRL 395
R. oryzae NLX-M-1
Paper pulp
Wheat straw
Corncob
Corncob
XOS waste residue
24.1
6
24
60.3
0.23
0.24
0.30
0.60
0.5
1.0
[22]
[24]
[28]
[35]
This study
SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharication and fermentation.
from XOS waste residues in our study. For SSF, most of the previous studies with R. oryzae produced only up to 24 g L1 of lactic acid
with a moderate yield of 0.230.30 g g1 substrate and a productivity of 0.5 g L1 h1 . On the other hand, our SSF process produced
a high lactic acid titer of 60.3 g L1 with a yield of 0.6 g g1 and
productivity of 1.0 g L1 h1 from XOS waste residues, which were
among the highest ever reported with R. oryzae using lignocellulosic biomass as raw material. It is noted that the lactic acid yield
from XOS waste residues by R. oryzae was comparable to those
obtained from similar lignocellulosic biomass with lactic acid bacteria. Sreenath et al. [15] used Lactobacillus plantarum to produce
lactic acid from alfalfa ber in a SSF process, achieving a yield of
0.61 g g1 dry matter of ber. A coculture of Lactobacillus rhamnosus
and Lactobacillus brevis was used in a SSF process, which produced
lactic acid from corn stover with a yield of 0.70 g g1 cellulose and
hemicellulose [16].
For the same lignocellulosic materials, R. oryzae could produce
more l-lactic acid at a higher nal titer in SSF than in SHF. For example, only 2 g L1 of lactic acid was produced from wheat straw in
SHF, while lactic acid production reached 6 g L1 in SSF [24]. This is
because the inhibition of cellulasecatalyzed reaction by glucose
can be eliminated or greatly alleviated by simultaneously using
glucose in the fermentation, which prevents the accumulation of
glucose generated from the hydrolysis of cellulosic materials in the
SSF process [38]. However, Miura et al. [28] reported that 26 g L1
l-lactic acid was produced from the enzymatic hydrolysate of
corncob, while in the SSF process using a mixed culture of cellulaseproducing Acremonium thermophilus and Rhizopus sp. MK-96-1196
at 35 C, 24 g L1 of l-lactic acid was produced from 100 g L1 of
untreated raw corncob. It is thus, clear that the performance of SSF
will also be dependent on the raw materials used, and each SSF
process will have to be optimized for the conditions used in the
process, including solid loading and agitation and aeration rates.
Further studies including process optimization, lactic acid conversion from xylose and economic analysis will have to be performed
before the process can be commercialized. Better understanding of
the metabolic pathways involved in xylose utilization by R. oryzae is
also needed in order to maximize l-lactic acid production in terms
of nal product titer, yield, and productivity [9].
5. Conclusion
The waste residues from corncobs used in XOS manufacturing
can be used as a low-cost substrate for l-lactic acid production
by R. oryzae. The solid waste residues can be efciently treated
and hydrolyzed with commercial cellulase enzymes to release fermentable sugars, glucose, and xylose, for lactic acid production. A
high cellulose-to-lactic acid conversion and yield can be obtained
in a SSF process with R. oryzae at a relatively high temperature of
40 C. Although lactic acid production in an SSF process has been
demonstrated with starchy materials and lactic acid bacteria [39],
this study is the rst demonstration of SSF for high-titer and highyield l-lactic acid production from lignocellulosic materials by R.
oryzae. The utilization of cheap XOS waste residues for l-lactic acid
production can provide a cost-effective approach for organic acid
production as well as in waste management.
Acknowledgements
This work was supported by the International Advanced
Forestry Technology Introduction Project Funding (Grant No. 20124-18), Natural Science Foundation of Jiangsu Province (Grant No.
BK20131426), Excellent Youth Foundation of Jiangsu Province of
China (BK2012038), the Doctorate Fellowship Foundation of Nanjing Forestry University, Graduate Research Innovation Projects of
Jiangsu Province Ordinary University (CXZZ13 0544), and the Priority Academic Program Development of Jiangsu Higher Education
Institutions.
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