Article Type: Original Article

Accepted Article
Production of succinic acid from sugarcane molasses supplemented with a mixture of corn steep liquor

powder and peanut meal as nitrogen sources by Actinobacillus succinogenes

Naikun Shen1,2, Yan Qin2, Qingyan Wang1,2, Siming Liao1,2, Jing Zhu2, Qixia Zhu2, Huizhi Mi2, Benu Adhikari3,

Yuto Wei1 and Ribo Huang1,2,*1

1 Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization, College of Life Science and Technology, Guangxi

University, Nanning, Guangxi 530005, China

2 State Key Laboratory of Non-Food Biomass and Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery,

Guangxi Academy of Sciences, Nanning, Guangxi 530007, China

3 School of Applied Sciences, RMIT University, City Campus, Melbourne, VIC 3001, Australia

A concise and informative title: Succinic acid from cane molasses

Significance and Impact of the Study: Succinic acid (SA) is commonly used as a platform chemical to

produce a number of high value derivatives. Yeast extract (YE) is used as a nitrogen source to produce SA. The

high cost of YE is currently the limiting factor for industrial production of SA. This study reports the use of a

mixture of corn steep liquor powder (CSLP) and peanut meal (PM) as an inexpensive nitrogen source to

1*
Corresponding author. Tel. : +86-771-2503902;
Fax: +86-771-2503908;
E-mail: rbhuang@gxas.cn (Ribo Huang)
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/lam.12399
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 substitute YE. The results showed that this CSLP-PM mixed formulation can be used as an effective and
Accepted Article
economic nitrogen source for the production of SA.

Abstract

The potential of using corn steep liquor powder (CSLP), peanut meal (PM), soybean meal (SM), cotton meal

(CM) and urea as the substitute of yeast extract (YE) as the nitrogen source was investigated for producing

succinic acid (SA). Actinobacillus succinogenes GXAS137 was used as the fermenting bacterium and sugarcane

molasses was used as the main substrate. None of these materials were able to produce SA as high as YE did.

The CSLP could still be considered as a feasible and inexpensive alternative of YE as the yield of SA produced

by using CSLP was second only to the yield of SA obtained by using YE. The use of CSLP-PM mixed

formulation (CSLP to PM ratio =2.6) as nitrogen source produced SA up to 59.2 g l-1 with a productivity of 1.2 g

l-1 h-1. A batch fermentation using a stirred bioreactor produced up to 60.7 g l-1 of SA at the same formulation.

Fed-batch fermentation that minimized the substrate inhibition produced 64.7 g l-1 SA. These results suggest that

sugarcane molasses supplemented with a mixture of CSLP and PM as the nitrogen source could be used to

produce SA more economically using A. succinogenes.

Key words: succinic acid, Actinobacillus succinogenes, sugarcane molasses, corn steep liquor powder, peanut

meal

Introduction

Succinic acid (SA) is a member of four-carbon dicarboxylic acid family in tricarboxylic acid cycle (TCA).

It has a broad application as a surfactant, a green solvent and a pharmaceutical intermediate (Song and Lee

2006). SA has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals

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 (Werpy and Petersen 2004). Currently, the SA is produced mainly by hydrogenation of petroleum-derived
Accepted Article
maleic anhydride. However, the increase in the price of oil and petroleum derivatives has made the production

of SA through microbial fermentation an economically attractive option (Chen et al. 2014). SA can be produced

through microbial fermentation by using a number of microorganisms such as Actinobacillus succinogenes

(McKinlay et al. 2007; Carvalho et al. 2013), Mannheimia succiniciproducens (Lee et al. 2008),

Anaerobiospirillum succiniciproducens (Lee et al. 2003) and recombinant Escherichia coli (Chan et al. 2012).

Among these microorganisms, A. succinogenes is one of the most promising SA producers due to its ability to

produce SA with high yield from a broad range of carbon sources.

To date research efforts are largely devoted in producing SA from inexpensive and renewable carbon

sources such as starch (Dorado et al. 2009), whey (Wang et al. 2008), sucrose (Jiang et al. 2014), bakery waste

(Zhang et al. 2013), hemicellulose (Borges et al. 2011), and cane molasses (Agarwal et al. 2006; Liu et al. 2008;

Barrosa et al. 2013) through batch or continuous fermentation. Cane molasses is a byproduct of the sugar

industry, which contains approximately 50% sugars and the remaining being water, suspended colloids, heavy

metals, vitamins, and amino acids, etc. (Kotzamanidis et al. 2002). Sugarcane molasses contains relatively high

concentration of metal ions and suspended colloids, which could be toxic or produce inhibitory effect on the

microbial cells. Hence, sugarcane molasses requires pretreatment to remove these hazardous substances. In this

regard, the sulfuric acid treatment was found to be effective and inexpensive compared to other pretreatment

methods such as potassium ferrocyanide, cation exchange resin and activated carbon (Liu et al. 2008).

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 In addition, A. succinogenes is a fastidious organism (McKinlay et al. 2005) with high demand for
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nitrogen for which higher input of nitrogen source is required. Yeast extract (YE) is the best choice among the

various nitrogen sources for both microbial growth and SA production (Chen et al. 2012). Currently, the high

cost of YE is hindering its greater application in in large-scale industrial production of SA. To reduce the

production costs, the use of various low-cost nitrogen sources such as corn steep liquor powder (CSLP) (Xi et al.

2013) and spent brewer's yeast hydrolysate (Jiang et al. 2010) has been extensively investigated. The use of the

above mentioned ingredients resulted into lower yield of SA compared to when YE was used, except when

growth factors, such as biotin, heme or vitamins were added. The by-products of oil industry such as soybean

meal (SM), cottonseed meal (CM), and peanut meal (PM) are relatively inexpensive and are readily available in

the market (Sharma et al. 2002). These by-products are rich in amino acids and polypeptides and are excellent

source of organic nitrogen. However, to the best of our knowledge, there are no studies on the use of these

substrates as the nitrogen source for SA production.

In the above context, we studied the potential of using sulfuric acid pretreated sugarcane molasses as a

feedstock for production of SA. As finding of the alternative of YE as a nitrogen source is important for

producing SA more economically, we assessed the potential of a number of materials as potential alternatives

including CSLP, PM, SM, CM and urea. We confirm that if a mixed formulation of CSLP and PM is used as

nitrogen source, comparable productivity of SA can be achieved.

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 Results and discussion
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Effect of YE concentration on the production of SA

In order to determine the effect of concentration of YE on SA production, different concentrations of YE (0, 8.0,

10.0, 12.0, 14.0 g l-1) were tested at an initial total sugar concentration of 70.0 g l-1 using an anaerobic

fermentation. As shown in Fig. 1, both the dry cell weight and SA yield increased with the increase of YE up to

12.0 g l-1. When the YE was above 12.0 g l-1, the dry cell weight and acetic acid contents increased with the

increase of YE, while there was no significant (p>0.05) increase in the SA yield. A supplementation of 12.0 g l-1

of YE was required to completely utilize the 70.0 g l-1 of total sugar which yielded 54.6 g l-1 of SA. A decent

amount of SA (22.0 g l-1) and dry cell weight (0.5 g l-1) were produced even when the YE was not added. The

above observation suggests that the sugarcane molasses contains appreciable amount of nitrogenous substances

required for the growth of A. succinogenes. The addition of 12.0 g l-1 YE produced 3.2 times higher SA

compared to when it was not added. However, the high cost of YE means that industrial production of SA using

YE as the sole nitrogen source becomes quite expensive. It is, therefore, desirable to find other more

cost-effective and functionally comparable nitrogen sources.

Effect of different materials as nitrogen source on SA production

The efficacy of different nitrogen sources (CSLP, PM, CM, SM, and urea) on the yield of SA was compared

with the yield of SA obtained from using 12.0 g l-1 of YE. This is because, it has shown in preceding section that

12.0 g l-1 of YE produces 54.6 g l-1 of SA by consuming 70.0 g l-1 of total sugar. The concentrations of CSLP,

PM, CM, SM, and urea used in these tests were 20.6, 18.8, 19.1, 20.1, and 3.3 g l-1, respectively which

corresponded to the nitrogen concentration of 1.5 g l-1. To improve the utilization of PM, CM, and SM, the

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 materials were simultaneous enzymatic hydrolysis and fermentation, with the neutrase (0.5 g l-1) added to the
Accepted Article
medium after filtration through a 0.22 μm filter (Wang et al. 2011). As shown in Fig. 2, the production of SA

was markedly affected by the type of the nitrogen source used. YE was found to be the best nitrogen source for

the production of SA which led to the best cell growth and the highest production of SA (54.6 g l-1). The

application of urea as the sole nitrogen source resulted into the lowest production of SA (20.7 g l-1). Although

the nitrogen content of CSLP, PM, CM, and SM in these tests was equal to that of YE, they produced SA values

of 47.9, 43.3, 38.7, and 37.5 g l-1, respectively, which were significantly (p<0.05) lower than the SA content

delivered by YE. At the same nitrogen content, the SA content delivered by the CSLP was second only (12%

less) to the SA content delivered by YE indicating that it could be considered as an inexpensive alternative

organic nitrogen source. This may be due to the fact that CSLP typically contains approximately 1 mg kg-1

biotin and either provides sufficient biotin or makes a significant contribution to the nutritional requirement of A.

succinogenes (Nghiem et al. 1996). An appreciable amount of residual total sugar (about 6.5 g l-1) was observed

at the end of fermentation when CSLP was used as the nitrogen source. Furthermore, the increase of CSLP

concentration in the fermentation medium to obtain the nitrogen concentration of 2.5 g l-1 (equivalent to 20.0 g

l-1 of YE) did not produce significantly (p>0.05) higher SA (data not shown). This might be due to the fact that

the biotin or other trace substances required for the growth of A. succinogenes was not adequately provided by

CSLP alone. Biotin is a cofactor of carboxylases, which is vitally important for metabolism of protein, lipid,

carbohydrate and electron transfer in the anaerobic respiratory chain of A. succinogenes. However, A.

succinogenes lacks several genes such as bioA, bioF, and bioW involved in the synthesis of biotin (Nghiem et al.

1996). Therefore, the supplementation of biotin is essential for meeting the essential physiological demands of A.

succinogenes. In this regard, Xi et al. (2012) found that the lowest concentration of biotin in a chemically

defined medium which was able to provide good growth of A. succinogenes was 100 μg l-1. PM is rich in biotin

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 and contains biotin up to 1.79 mg kg-1 and its polypeptides are also excellent source of organic nitrogen (Jacob
Accepted Article
and Elemer 1975). Although the CSLP alone could perform as YE does in terms of production and yield of SA;

however, when other ingredients such as biotin or PM are added, it may be able to meet the physiological

demands of A. succinogenes required to obtain high yield of SA.

Effect of combining CSLP with biotin or PM on SA production

The effect of concentration of biotin or PM when used together with CSLP on the production of SA was

investigated using anaerobic batch fermentation. In these tests, the concentrations of CSLP and total sugar were

fixed at 20.6 g l-1 and 70.0 g l-1, respectively. As shown in Table 1, the production of SA and the dry cell weight

increased with the increase of biotin content up to 0.2 mg l-1. There was no significant (p>0.05) increase in these

two parameters when the biotin was > 0.2 mg l-1. The acetic acid concentration increased with the increase of

biotin concentration. Interestingly, the biotin content had little effect on the succinic acid to acetic acid (SA/AC)

ratio which ranged from 7.1 to 7.7. This phenomenon was also reported earlier when YE (Nghiem et al. 1996)

and yeast cell hydrolysate (Chen et al. 2013) supplemented by biotin were used as nitrogen source. The highest

SA production (52.6 g l-1) was obtained after 48 h of fermentation at biotin concentration of 0.2 mg l-1 which

was 12.3% increase compared to the batch in which biotin was not added. At this formulation, the dry cell

weight and utilization of sugar were increased by 16.7% and 8.2% compared to the corresponding values when

biotin was not added. SA concentration and dry cell weight were also increased when the concentration of PM

was increased up to 8.0 g l-1 above which no significant (p>0.05) increase in the production of SA was observed.

The highest SA concentration was 55.7 g l-1 with the yield of 83.4%, which were 5.9% higher than biotin (0.2

mg l-1) supplemented CSLP and 16.5% higher compared to when CSLP alone was used as the nitrogen source.

The increased yield of SA when PM was used together with CSLP is not solely due to the presence of biotin in
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 PM. It is expected that there are other unknown or uncharacterized nutrients in PM which increase the
Accepted Article
fermentation activity of A. succinogenes. A detailed study to identify these nutrients and their mechanism of

action has not been undertaken so far. A number of low-cost industrial byproducts such as CSLP (Xi et al. 2013),

dry yeast cells (Liu et al. 2008) and spent yeast cell hydrolysate (Jiang et al. 2010; Chen et al. 2011) have been

used to improve the production of SA at the same time substituting the commercial YE as the nitrogen source.

For example, Xi et al. (2013) reported that the use of heme supplemented CSLP as a nitrogen source for A.

succinogenes NJ113 produced the SA content and yield of 37.9 g l-1 and 81.5%, respectively. Jiang et al. (2010)

reported that the use of enzymatically produced hydrolysate of spent brewer's yeast supplemented by a mixture

of vitamins resulted into SA production and yield of 46.8 g l-1 and 68.82%, respectively using 68 g l-1 glucose.

Chen et al. (2011) reported that when spent yeast cell hydrolysate was used as a nitrogen source, SA

concentration of up to 35.5 g l-1 and the corresponding yield of 71% were obtained at glucose concentration of

50 g l-1. When biotin (0.15 mg l-1) was added in this formulation, the glucose utilization and SA concentration

were increased by 37.6% and 49.0%, respectively. Our results (Table 1) show that a formulation involving 70.0

g l-1 sugarcane molasses together with the mixture of CSLP (20.6 g l-1) and PM (8.0 g l-1) as nitrogen source

produced 55.7 g l-1 of SA and achieved an yield of 79.6%, both of which are much higher than currently

reported in the literature .

Effect of initial total sugar concentration on SA production

To assess the substrate tolerance of the strain, different initial total sugar concentrations (60.0, 65.0, 70.0,

75.0, and 80.0 g l-1) were used to produce SA. As shown in Fig. 3, when the A. succinogenes GXAS137 was

grown in a medium containing 80.0 g l-1 of total sugar the SA still remained as the main product of fermentation.

When the total sugar concentration was below 75.0 g l-1, the production of SA increased significantly (p<0.05)
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 with the increase in total sugar content. The highest values of SA content, yield and dry cell weight of 59.2 g l-1,
Accepted Article
78.9 % and 4.0 g l-1, respectively were obtained at the initial total sugar content of 75.0 g l-1 and when the

fermentation was carried out for 48 h. On the contrary, SA content and yield decreased when the initial total

sugar was higher than 75.0 g l-1. This can be attributed to the increased substrate concentration, which has been

shown to extend the lag time prior to the exponential growth of A. succinogenes (Lin et al. 2008; Gunnarsson et

al. 2014). Many reports have shown that the concentration of sugar in the medium affects the growth and

metabolite production of A. succinogenes (Lin et al. 2008; Jiang et al. 2014). Urbance et al. (2004) reported that

A. succinogenes could tolerate up to 160 g l-1 of initial glucose in batch fermentation. In this study, significant

decrease in both the SA production and utilization of sugar was observed at initial sugar concentrations above

75.0 g l-1. Our results showed that the highest amount of SA was produced when the initial total sugar content

was 75.0 g l-1, which indicated that this particular formulation required higher initial sugar content compared to

that reported in the literatures (65 g l-1) (Lin et al. 2008; Liu et al. 2008). This might be due to difference in

strain and prevailing experimental conditions.

Fermentation in stirred bioreactors

To confirm the performance of nitrogen source, batch and fed-batch fermentation experiments were

performed in 1.3 l bioreactors at 37ºC. In these tests, the initial total sugar content of 75.0 g l-1 was used and the

fermentation was carried out for 48 h. The concentrations of CSLP and PM in these tests were 20.6 g l-1 and 8.0

g l-1, respectively. Samples were taken from these reactors every 4 h during the entire 48-h long fermentation

cycle.

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 The batch fermentation (Fig. 4a) results showed that the fermentation process was completed within 48 h as
Accepted Article
evidenced by the complete depletion of the glucose content. At the end of the 48-hour fermentation period the

final SA concentration reached 60.7 g l-1 and the sugar supply was completely exhausted. At this point, the

productivity and yield of SA were 1.3 g l-1 h-1 and 80.9%, respectively. These results clearly demonstrate that A.

succinogenes GXAS137 can efficiently utilize CSLP and PM as nitrogen source it can produce SA from the

sugarcane molasses. In fed-batch fermentation (Fig. 4b), the production of SA, its yield and productivity reached

64.7 g l-1, 86.2%, 1.4 g l-1 h-1, respectively all of which were higher than their corresponding values in the batch

fermentation. The above observation suggests that the substrate inhibition clearly limits the production of SA by

A. succinogenes in batch fermentation. When the sugar concentration was maintained at a lower level during the

fed-batch fermentation process, cells grew faster and the steady phase became longer due to the elimination of

the substrate inhibition (Liu et al. 2008). Thus, the fed-batch process was proven to be more efficient

fermentation system compared to the batch fermentation for the production of succinic acid using A.

succinogenes.

Conclusions

A detailed study was carried out aiming to replace the commercial YE by a mixture of CSLP and PM as a

nitrogen source in a process that produced SA by using A. succinogenes GXAS 137. When the CSLP was used

alone as the supplement, it produced smaller amount of SA and it was less effective compared to the YE.

However, when CSLP and PM were combined as mixed supplements, this formulation was found to meet the

physiological demands of A. succinogenes and produced up to 55.7 g l-1 SA in batch process which was

comparable to the amount of SA produced by YE supplemented sugarcane molasses formulation. When batch

fermentation was carried out, the same CSLP+PM (CSLP to PM ratio=2.6) formulation produced 60.7 g l-1 of

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 SA. Fed batch fermentation was able to minimize the substrate inhibition and produced 64.7 g l-1 of SA with a
Accepted Article
yield of 86.2%. These results suggest that sugarcane molasses supplemented with a mixture of CSLP and PM as

nitrogen sources could be for the production of SA more economically.

Materials and methods

Materials and chemicals

Sugarcane molasses was obtained from Fenghao sugar-refinery (Guangxi, China). Yeast extract (YE) was

purchased from OXOID (Hampshire, England). Corn steep liquor powder (CSLP), soybean meal (SM),

cottonseed meal (CM) and peanut meal (PM) were purchased from Comwin Pharm-culture Co., Ltd. (Beijing,

China). According to the product information data sheet, the total nitrogen content (w w-1) in YE, CSLP, SM,

CM, PM, urea was 12.7%, 7.4%, 8.1%, 8.0%, 7.6%, and 46.0%, respectively. Commercially available neutral

protease (Neutrase EC 3.4.24.28) was purchased from Novozymes (Tianjin, China). All other chemicals used

throughout this study were obtained from Sinochem (Shanghai, PR China), except otherwise specified.

Microorganism and growth conditions

A. succinogenes GXAS137 was isolated in our laboratory and stored at China Center for Type Culture

Collection (No. CCTCC M 2011399). This strain was shown to produce high amount of SA (Shen et al. 2014).

Cells were grown in 100 mL sealed anaerobic bottles containing 50 mL of medium. The medium for inoculum

preparation contained the following (in g l-1): 20.0 glucose, 5.0 YE, 2.5 CSLP, 8.5 NaH2PO4·H2O, 15.5 K2HPO4,

and 5 NaHCO3. All liquid cultures were incubated in rotary shaker (100 rpm) at 37˚C.

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 Treatment of molasses
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The crude molasses was diluted with distilled water to obtain 250 g l-1 total sugar concentration. For

sulfuric acid treatment, the above molasses solution was adjusted to pH 3.5 with 5 mol l-1 H2SO4, and heated at

60˚C for 2 h. After centrifuging at 8000× g for 15 min, the supernatant was adjusted to pH 7.0 with 10 mol l-1

NaOH (Liu et al. 2008).

Anaerobic batch fermentation

In this fermentation, the cells in exponential growth phase were inoculated in 150 ml sealed anaerobic

bottles containing 50 ml of the medium. The basic fermentation medium contained the following (in g l-1) was

composed of 10.0 YE, 3.0 K2HPO4, 2.0 NaH2PO4·H2O, 1.0 NaCl, 0.21 MgCl2·6H2O, and CaCl2·2H2O. The pH

of the medium was maintained by adding 70 g l-1 of MgCO3. Separately autoclaved sugarcane molasses was

added aseptically to the medium to maintain the desired sugar concentration. The materials used as nitrogen

source were also separately autoclaved. The sterile medium was sparged with CO2, and 0.2 g l-1 Na2S·9H2O was

added before inoculation to maintain strictly anaerobic condition. For the fermentation experiments, the above

mentioned medium was inoculated with 5% of seed culture. This fermentation was carried out in a rotary shaker

(100 rpm) at 37˚C for 48 h. The variation of pH during the fermentation was maintained at 6.5-7.0 by adding

MgCO3.

Fermentation in a stirred bioreactor

To further assess the effect of materials used as nitrogen source on the production of SA and in an attempt

to achieve higher production of SA than in anaerobic bottles, fermentation was carried out using a 1.3 l

fermenter (Eppendorf BioFlo/CelliGen 115) with a broth volume of 0.8 l. Triplicate runs were performed at

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 37˚C at an agitation speed of 100 rpm. The flow rate of CO2 was maintained at 0.3 l min-1. The fermentation
Accepted Article
medium was the same as in the anaerobic bottles and the pH during the fermentation was controlled using

MgCO3. Biomass, residual sugar and organic acid contents of the broth were determined at 4 h interval.

Fermentation was also carried out in fed-batch mode. The medium and the condition in the fed-batch

experiments were the same as that in batch experiment, except for the fact that the initial total sugar content was

30.0 g l-1. When the concentration of total sugar was lower than 20.0 g l-1, sugarcane molasses containing 300 g

l-1 total sugar was fed into the stirred bioreactor using a peristaltic pump to maintain the concentration of sugar

within 20.0-30.0 g l-1 during the course of fermentation.

Analytical methods

The dry cell weight (DCW) was determined using the curves which plotted optical density at 660 nm

against biomass dry weight. An OD660 of 1.0 represented 520 mg of dry cell weight per liter. The culture

biomass was diluted with 0.2 mol l-1 HCl, and centrifuged at 10, 000× g for 10 min, in order to ensure that no

residual MgCO3 remained with the cell mass. The cell pellets were washed for three times with distilled water to

remove all the residues coming from the fermentation medium.

The organic acids and sugar contents were determined by using high-performance liquid chromatography

(HPLC) equipped with a tunable UV detector set at 210 nm. A Rezex ROA-Organic Acid H+ (Phenomenex) was

eluted with 0.005 mol l-1 of H2SO4 as a mobile phase at a flow rate of 0.6 ml min-1. The column temperature was

maintained at 45ºC and a refractive index (RI) detector was used. The temperature of RI detector was

maintained at 55ºC. The residual total sugar content was determined by the DNS method (Dubois et al. 1956)

after acid hydrolysis. For the hydrolysis the pH was adjusted to 1.0 with HCl and heated to 100ºC for 30 min.
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 Statistical analysis
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Experiments were performed in triplicate except indicated otherwise. The results are represented as

mean ± SD. The yield of SA was defined as the amount of SA produced per gram of sugar consumed, and

expressed as a percentage. The productivity of SA was calculated as the SA content of broth divided by the

fermentation time.

One-way analysis of variance (ANOVA) was performed among the mean values to determine whether or

not significant difference existed among them. The difference was considered significant at 95% confidence

level (p<0.05).

Acknowledgements

This work was supported by the Natural Science Foundation of Guangxi Province (No. 2013GXNSFBA019102,

2014GXNSFAA118102), Science foundation of Guangxi Academy of Science (No.13YJ22SW) and BaGui

Scholars Program Foundation of Guangxi Province, China.

Conflict of interest

There is no conflict of interest to declare.

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Figure Captions

Fig. 1. Effect of supplementing sugarcane molasses with different concentration of yeast extract on succinic acid

content, biomass, and main by-products produced by A. succinogenes GXAS 137. Cells were grown with initial

total glucose concentration of 70.0 g l-1 for 48 h. Each value is an average of three parallel replicates and is

represented as mean ± SD. ( ) SA, succinic acid; ( ) AC, acetic acid; ( ) FA, formic acid; ( ) DCW, dry

cell weight; YE, Yeast extract.

Fig. 2. Effect of supplementing sugarcane molasses with steep liquor powder (CSLP) and other nitrogen

sources on the succinic acid content, biomass, and main by-products produced by A. succinogenes GXAS 137.

Cells were grown at a fixed intial total glucose concentration of 70.0 g l-1 for 48 h. Each value is an average of

three parallel replicates and is represented as mean ± SD. ( ) SA, succinic acid; ( ) AC, acetic acid; ( ) FA,

formic acid; ( ) DCW, dry cell weight; YE, Yeast extract; PM, peanut meal; CM, cottonseed meal; SM,

soybean meal.

Fig. 3. Effect of supplementing sugarcan molasses CSLP and different initial total sugar concentration on the

succinic acid conent, biomass, and main by-products produced by A. succinogenes GXAS 137. Each value is an

average of three parallel replicates and is represented as mean ± SD. ( ) SA, succinic acid; ( ) AC, acetic

acid; ( ) FA, formic acid; ( ) DCW, dry cell weight; YE, Yeast extract.

Fig. 4 Time course of cell growth of A. succinogenes GXAS 137 and production of succinic acid in a batch (a)

and a fed-batch (b) fermationan processes where sugarcane molasses was supplemented by 20.6 g l-1 corn steep

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 liquor powder and 8.0 g l-1 peanut meal as mixed nitrogen source. Fermentation was carried out using a 1.3 l
Accepted Article stirred bioreactor. Cells were grown using initial total sugar concentration of 75.0 g l-1 for 48 h. The plotted data

points were an average of three parallel replicates and are represented as mean ± SD. ( ) TS, total sugar; ( )

SA, succinic acid; ( ) AC, acetic acid; FA, ( ) DCW, dry cell weight.

Table 1 Effect of combining corn steep liquor powder (CSLP) with different amount of biotin or peanut meal

(PM) as nitrogen source on SA production*

Nitrogen Biotin (mg l-1) PM (g l-1)

source 0 0.1 0.2 0.3 0.4 4.0 6.0 8.0 10.0

SA (g l-1)** 47.8±0.5 49.3±0.4 53.6±0.7 53.9±0.6 53.5±0.3 50.9±0.5 53.4±0.4 55.7±0.5 55.6±0.6

AC (g l-1) 6.30±0.6 6.5±0.8 7.0±1.0 7.2±0.7 7.3±0.5 6.8±0.9 7.3±0.6 7.5±0.5 7.8±0.8

SA/AC 7.6 7.6 7.7 7.5 7.3 7.5 7.3 7.4 7.1

FA (g l-1) 1.1±0.4 1.2±0.5 1.3±0.3 1.4±0.7 1.4±0.9 1.2±0.5 1.3±0.7 1.5±0.6 1.5±0.3

DCW (g l-1) 3.0±0.5 3.4±0.7 3.5±1.1 3.7±0.9 3.6±0.8 3.5±1.3 3.6±1.0 3.8±0.9 3.6±0.9

RTS (g l-1) 12.6±0.4 8.3±0.6 4.5±0.4 4.5±0.9 4.6±0.5 7.2±1.1 4.8±0.5 3.2±1.0 3.3±0.4

SA yield (%) 68.4±0.4 70.4±0.6 76.6±0.7 77.0±0.5 76.4±0.6 72.7±1.1 76.3±0.7 79.6±0.5 79.4±1.2

*
The concentrations of initial total sugar and CSLP were fixed at 70.0 g l-1 and 20.6 g l-1, respectively. The

concentration of biotin and PM are allowed to vary. Cells were grown in anaerobic bottles for 48 h. Each value

is an average of three parallel replicates and is represented as mean ± SD.

**
SA, succinic acid; AC, acetic acid; FA, formic acid; DCW, dry cell weight; RTS, residual total sugar.

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Accepted Article
Fig. 1.

60 10

50
8

40
SA, AC, FA ( g l )
-1

DCW ( g l )
-1
30

4
20

2
10

0 0
0 8 10 12 14
-1
YE ( g l )

Fig. 1. Effect of supplementing sugarcane molasses with different concentration of yeast extract on succinic

acid content, biomass, and main by-products produced by A. succinogenes GXAS 137. Cells were grown with

initial total sugar concentration of 70.0 g l-1 for 48 h. Each value is an average of three parallel replicates and is

represented as mean ± SD. ( ) SA, succinic acid; ( ) AC, acetic acid; ( ) FA, formic acid; ( ) DCW, dry

cell weight; YE, Yeast extract.

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 Fig. 2
Accepted Article
60 10

50
8

40
SA, AC, FA ( g l )
-1

DCW ( g l )
-1
6

30

4
20

2
10

0 0
YE CSLP PM CM SM Urea
Nitrogen source

Fig. 2. Effect of supplementing sugarcane molasses with corn steep liquor powder (CSLP) and other nitrogen

sources on the succinic acid content, biomass, and main by-products produced by A. succinogenes GXAS 137.

Cells were grown at a fixed intial total sugar concentration of 70 g l-1 for 48 h. Each value is an average of three

parallel replicates and is represented as mean ± SD. ( ) SA, succinic acid; ( ) AC, acetic acid; ( ) FA,

formic acid; ( ) DCW, dry cell weight; YE, Yeast extract; PM, peanut meal; CM, cottonseed meal; SM,

soybean meal.

This article is protected by copyright. All rights reserved.

 Fig. 3
Accepted Article
60 10

50
8

40
SA, AC, FA ( g l )
-1

DCW ( g l )
-1
6

30

4
20

2
10

0 0
60 65 70 75 80
-1
The initial total sugar ( g l )

Fig. 3. Effect of supplementing sugarcane molasses corn steep liquor powder and different initial total sugar

concentration on the succinic acid conent, biomass, and main by-products produced by A. succinogenes GXAS

137. Each value is an average of three parallel replicates and is represented as mean ± SD. ( ) SA, succinic

acid; ( ) AC, acetic acid; ( ) FA, formic acid; ( ) DCW, dry cell weight; YE, Yeast extract.

This article is protected by copyright. All rights reserved.

 Fig. 4
Accepted Article
a

80 6

70
5
Total sugar, origanic acid ( g l-1)

60

DCW ( g l-1)
50

40 3

30
2

20

1
10

0 0
0 4 8 12 16 20 24 28 32 36 40 44 48

Fermentation time (h)

b
80 6

70
5
Total sugar, origanic acid ( g l-1)

60

4
50
DCW ( g l-1)

40 3

30
2

20

1
10

0 0
0 4 8 12 16 20 24 28 32 36 40 44 48

Fermentation time (h)

Fig. 4 Time course of cell growth of A. succinogenes GXAS 137 and production of succinic acid in a batch (a)

and a fed-batch (b) fermationan processes where sugarcane molasses was supplemented by 20.6 g l-1 corn steep

liquor powder and 8.0 g l-1 peanut meal as mixed nitrogen source. Fermentation was carried out using a 1.3 l

stirred bioreactor. Cells were grown using initial total sugar concentration of 75.0 g l-1 for 48 h. The plotted data

points were an average of three parallel replicates and are represented as mean ± SD. ( ) TS, total sugar; ( )

SA, succinic acid; ( ) AC, acetic acid; FA, ( ) DCW, dry cell weight.

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