Bioresource Technology 174 (2014) 190–197

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using Corynebacterium glutamicum immobilized in porous polyurethane filler Xinchi Shi 1, Yong Chen 1, Hengfei Ren, Dong Liu, Ting Zhao, Nan Zhao, Hanjie Ying ⇑ State Key Laboratory of Materials-Oriented Chemical Engineering, College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, PR China

h i g h l i g h t s  CBH was used as carbon source for fermentation of succinic acid.  Mixed alkalis (NaOH and Mg(OH)2) were used as a novel method for regulating pH.  The C. glutamicum strains were first immobilized in porous polyurethane filler.  Using CBH and mixed alkali in the immobilized system for succinic acid production.

a r t i c l e

i n f o

Article history: Received 4 August 2014 Received in revised form 25 September 2014 Accepted 26 September 2014 Available online 5 October 2014 Keywords: Cassava bagasse hydrolysate Corynebacterium glutamicum Mixed alkaline neutralizers Porous polyurethane filler (PPF) Succinic acid

a b s t r a c t An immobilized fermentation system, using cassava bagasse hydrolysate (CBH) and mixed alkalis, was developed to achieve economical succinic acid production by Corynebacterium glutamicum. The C. glutamicum strains were immobilized in porous polyurethane filler (PPF). CBH was used efficiently as a carbon source instead of more expensive glucose. Moreover, as a novel method for regulating pH, the easily decomposing NaHCO3 was replaced by mixed alkalis (NaOH and Mg(OH)2) for succinic acid production by C. glutamicum. Using CBH and mixed alkalis in the immobilized batch fermentation system, succinic acid productivity of 0.42 g L 1 h 1 was obtained from 35 g L 1 glucose of CBH, which is similar to that obtained with conventional free-cell fermentation with glucose and NaHCO3. In repeated batch fermentation, an average of 22.5 g L 1 succinic acid could be obtained from each batch, which demonstrated the enhanced stability of the immobilized C. glutamicum cells. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Recently, succinic acid has gained attention as an important chemical because it can be used for the synthesis of many basic general chemicals. At present, the primary method for succinic acid production is chemical synthesis, using fossil fuels, which is associated with a high environmental cost, particularly higher CO2 emissions. As the price of fossil fuel has skyrocketed and the consciousness of environmental awareness has increased, biological processes for succinic acid production will become more economical and acceptable (Shohei et al., 2008).

Abbreviations: CBH, cassava bagasse hydrolysate; PPF, porous polyurethane filler; PPFS, porous polyurethane fillers; DCW, dry cell weight. ⇑ Corresponding author. Tel./fax: +86 25 86990001. E-mail address: [email protected] (H. Ying). 1 These authors are equally contributed to this work. http://dx.doi.org/10.1016/j.biortech.2014.09.137 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Succinic acid is an end product during anaerobic fermentation by some anaerobic and facultative anaerobic microorganisms. Strain is one of the key points of succinic acid biosynthesis; most bacteria and fungi can produce succinic acid (Song and Lee, 2006). At present, research on industrial succinic acid fermentation strains are mainly focused on anaerobic bacteria, such as Anaerobiospirillum succiniciproducens (Lee et al., 2003), gram-negative bacteria, such as Actinobacillus succinogenes (Guettler et al., 1999), and Escherichia coli (Lin et al., 2005; Skorokhodova et al., 2013). Moreover, Corynebacterium glutamicum has received much attention in this field in recent decades. Under anaerobic conditions, cell growth of C. glutamicum was inhibited, but the cells remained capable of metabolizing carbohydrate to produce organic acid, such as L-lactic acid, succinic acid, and acetic acid. The metabolic pathway used by C. glutamicum to produce lactic acid and succinic acid under anaerobic condition is shown in Fig. 1 (Chen et al., 2012). Large amounts of alkaline neutralizer are required

X. Shi et al. / Bioresource Technology 174 (2014) 190–197

to maintain the pH during succinic acid fermentation. The majority of studies on succinic acid production by C. glutamicum have used NaHCO3 as alkaline neutralizer to achieve high product concentrations (Inui et al., 2004; Li et al., 2010). Nevertheless, NaHCO3 is not stable when temperature is over 50 °C, so use of NaHCO3 as a neutralizer is limited in industrial succinic acid fermentation. There have been no reports regarding the use of mixed alkalis (e.g., NaOH and Mg(OH)2) as alkaline neutralizers for regulating pH for succinic acid production by C. glutamicum. Carbon sources often constitute a large portion of the raw material costs in biological fermentation, and much effort has gone into identifying cheap and renewable sources that can replace such expensive sources. Cassava bagasse, the fibrous residue from the industrial processing of cassava for starch extraction, is generated in large quantities in many countries and is treated as solid waste, because bagasse can be used only as low-value animal feed or must be disposed into landfills. Biological conversion of cassava bagasse has been previously studied for production of organic acids, butanol, and aromatic compound production (Bramorski et al., 1998; Carta et al., 1999; Lu et al., 2012; Thongchul et al., 2009), but has never been reported for succinic acid production by C. glutamicum. Utilizing cassava bagasse for succinic acid production could lower the substrate costs and could add value to the cassava processing industry while reducing environmental pollution caused by bagasse disposal. Immobilization of cells onto inert materials has been an alternative means of high biomass retention. When provided with nutrients, the cells divide within and on the core of the matrix, also releasing some of the progeny in the medium. To date, studies of succinic acid production by C. glutamicum relied on centrifugation of the aerobic medium to collect the cell mass (Shohei et al., 2005,

191

2008). This procedure was time consuming and liable to be contaminated by strains suspended in the air. To solve this problem, some researchers have attempted the use of immobilization techniques with C. glutamicum (Amin and Al-Talhi, 2007; Chu et al., 1996; Jun et al., 2007; Vijayaraghavan et al., 2008). Most studies employed entrapment methods using a polyelectrolyte complex gel and polysulfone matrix (Chu et al., 1996; Vijayaraghavan et al., 2008). In gel-entrapping methods, however, the limitation of carbon dioxide supply by diffusional resistance of the polyelectrolyte complex gel and polysulfone matrix may decrease the fermentation rate and/or succinic acid transformation efficiency (Corona-González et al., 2014). Hence, in this work, we proposed a natural attachment method by using porous polyurethane filler (PPF) as a carrier for C. glutamicum immobilization. PPF has macropores larger than hundreds of microns and a pore volume fraction greater than 0.9. To our knowledge, succinic acid production has not been achieved using C. glutamicum with PPF (John et al., 2007; Zhu et al., 1996) to date. Moreover, in this study, mixed alkalis (NaOH and Mg(OH)2) were used as a novel method for the first time for regulating pH for succinic acid production by C. glutamicum. A system using cassava bagasse hydrolysate (CBH) as the carbon source and mixed alkalis as the alkaline neutralizer was developed here to achieve economical succinic acid production by C. glutamicum immobilized in PPF. 2. Methods 2.1. Preparation of CBH Cassava bagasse, obtained from a cassava-processing factory in Guangdong, China, was dried and mechanically milled to a fine powder (about 50–100 lm in diameter). Before saccharification, 200 g of dried cassava bagasse powder was mixed with 1800 mL water (corresponding to a 10% (w/w) solid loading) and liquefied (using a-amylase 20,000 U mL 1 and 15 U g 1 dry cassava bagasse) at 86 °C for 2 h in a 5-L conical flask. Then, the liquefied cassava was cooled to 55 °C, after which commercial glucoamylase (100,000 U mL 1, from Zhiyi, Shanghai, China) was aseptically added at a 200 U g 1 dry cassava bagasse loading, to hydrolyze the cooked starch content into glucose. This enzymatic hydrolysis process was operated at 55 °C, pH 4.5, and 200 rpm for 2 h. Then, cellulase (10,000 U g 1, from Zhiyi, Shanghai, China) was added into the mixture at a loading rate of 0.1 mL g 1 dry cassava bagasse to hydrolyze the remaining cellulose into glucose. This process was operated at 50 °C, pH 5.0, and 200 rpm for 24 h. 2 M HCl and 4 M NaOH solutions were used for pH adjustments only prior to each enzymatic hydrolysis. No pH adjustment or buffer solution was used during the enzymatic hydrolysis process, as no significant pH change was observed. After hydrolysis, the mixture was centrifuged at 7000g for 15 min to remove the insoluble substances. The clear liquid, CBH, contained 43.5 g L 1 glucose, 1.4 g L 1 xylose, and trace amounts of arabinose, acetic acid, and lactic acid. 2.2. Microorganism and culture medium

Fig. 1. Metabolic pathways of C. glutamicum under oxygen-deprivation conditions. L-Lactic acid, succinic acid, and acetic acid are the main products during the anaerobic fermentation.

A hyper-succinic acid-producing C. glutamicum strain, strain 534, derived from ATCC 13032 through mutagenesis and adaptation in PPF, was used in this study. The stock culture of this mutant strain was stored in a 15% glycerol stock solution in a 80 °C freezer. To prepare the seed culture for fermentation studies, the strain was transferred from a Luria broth (LB) culture plate into an Erlenmeyer flask (500 mL) containing 30 mL seed medium, and incubated aerobically at 30 °C for 12–15 h, until the cells were highly active. The seed culture medium, containing a carbon source (20 g L 1 glucose),

192

X. Shi et al. / Bioresource Technology 174 (2014) 190–197

urea (25 g L 1), phosphate buffer (2 g L 1 KH2PO4 and 6 g L 1 K2HPO4
3H2O), vitamins (2 mg L 1 thiamin and 1 mg L 1 biotin), and mineral salts (2 g L 1 MgSO4
7H2O, 0.2 g L 1 MnSO4
H2O, 0.2 g L 1 FeSO4
7H2O). Succinic acid fermentation using CBH (or glucose) as the carbon source requires minerals (1 g L 1 NaCl) and neutralizer for the fermentation medium. CBH (or glucose) and the mineral solution were sterilized by autoclaving at 115 °C for 20 min. To ensure anaerobiosis, all solutions were purged with nitrogen for 1 h through a sterile 0.2-lm filter.

periodically taken from the bioreactor until the end of the batch fermentation process (namely, a glucose concentration below 1 g L 1 was determined). Repeated batch fermentations were performed as follows: a first fermentation was carried out under the conditions described above. Once the fermentation cycle was completed, the bioreactor was drained (using a peristaltic pump under sterile conditions) and then replenished with fresh medium. The repeated batch fermentations were maintained for 272 h to evaluate long-term performance and stability.

2.3. Immobilization of C. glutamicum 534

2.5. Analytical methods

The 7.5-L bioreactor (BioFlo 115, New Brunswick Scientific, Edison, NJ, USA) (Fig. 2) containing 3.5 L of seed culture medium and 30 PPFS was flushed with air for 30 min, until the bioreactor was filled with air. Each PPF consisted of a porous hollow ball (1.6 cm i.d.  2 cm o.d.) and a spherical polyurethane filler (1.4-cm diameter). The bioreactor was then inoculated with 350 mL of actively growing cells (12 h) and then maintained at 30 °C and pH 7.5 (with the addition of 4 M NaOH), and agitated at 300 rpm for 18–24 h until the cell density in the broth ceased to decrease. The seed solution was replaced with fresh seed culture medium to allow growth of the cells in these PPFS. The medium was changed again and the process repeated several times until a stable and high cell density was reached in these PPFS. Throughout the process, the temperature was maintained at 30 °C and the pH was controlled automatically at 7.5.

The cell density was measured using a BioMate™ 3 spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 600 nm. A small volume (5 mL) of culture was centrifuged at 4000g for 10 min; the precipitate was then washed with deionized water, and dried at 80 °C until the weight remained constant. Cell mass was calculated from a calibration curve that related the culture OD to cell concentration (1 OD600 = 0.35 g dry cell weight L 1). Moreover, the supernatants were used to determine the concentration of glucose, succinic acid, lactic acid, and acetic acid. The sugar, succinic acid, lactic acid, and acetic acid concentrations were measured by high performance liquid chromatography (Agilent 1100 series; Hewlett–Packard, Palo Alto, CA, USA) with a refractive index detector, using an Aminex HPX-87H ion exclusion column (300  7.8 mm; Bio-Rad Laboratories, Hercules, CA, USA), with 5.0 mM H2SO4 used as the mobile phase (0.4 mL min 1) at 80 °C.

2.4. Batch fermentation and repeated batch fermentation 3. Results and discussion After the cell-immobilization procedure, seed cultures were drained (with a peristaltic pump, under sterile conditions), while these PPFS were retained and fresh fermentation medium was then pumped into the bioreactor. Batch fermentation kinetics was then studied in 7.5-L bioreactors with an initial working volume 3.5 L (Fig. 2). The fermentation medium was used for succinic acid fermentation and was purged with nitrogen gas for 30 min to remove oxygen. The pH of the medium was maintained with the addition of various alkaline neutralizers. When CaCO3 or MgCO3 was used as pH regulators, they were added initially to the medium. When Na2CO3, NaOH, NaHCO3, NH3
H2O, Mg(OH)2, or Ca(OH)2 was used as pH regulators, they were first prepared as solutions at the appropriate concentrations by means of exogenous feeding during the course of fermentation. All fermentations were performed with agitation speed of 200 rpm and a CO2 flow rate of 0.5 vvm. All experiments were repeated three times. Broth samples were

3.1. Hydrolysis of cassava bagasse The dried cassava bagasse used in this study contained (w/w): starch, 45% ± 3%; cellulose, 23% ± 4%; hemicellulose, 9% ± 5%; acetyl bromide-soluble lignin, 9% ± 2%; and water, 14 ± 3%. The hemicellulose was mainly made up of glucose (26.1%), xylose (66.4%), and arabinose (7.5%). The starch and cellulose components of the bagasse were converted into fermentable sugars after the two-step enzymatic hydrolysis, with a total sugar yield of 0.457 g g 1 cassava bagasse. The resulting CBH contained 44.3 g L 1 glucose, 1.64 g L 1 xylose, 0.056 g L 1 arabinose, 0.33 g L 1 lactic acid, and 0.44 g L 1 acetic acid. Thus, less than 68.3% of the polysaccharides present in the cassava bagasse were hydrolyzed to fermentable sugars by the enzymatic hydrolysis process without requiring acid pretreatment. However, as no acid pretreatment was used in

Temperature pH controller controller peristaltic pump

Air/CO2 tank

Metal film air distributor Neutralizer bottle

Spinner flask porous polyurethane filler Fig. 2. Immobilized fermentation system.

193

X. Shi et al. / Bioresource Technology 174 (2014) 190–197

hydrolyzing the cassava bagasse, a little furfural, hydroxymethylfurfural, and lignin degradation products, such as hydroquinone and ferulic acid, would be present in the CBH. Cassava bagasse from different regions varied markedly in its composition, with starch ranging from 40% to 60% and fibers from 14% to 50% (Carta et al., 1999; Pandey et al., 2000). After removing ca. 92% of its water by evaporation under vacuum, the CBH was concentrated to a thick syrup containing approximately 567 g L 1 glucose, 12.7 g L 1 xylose, 0.4 g L 1 arabinose, 1.7 g L 1 acetic acid, and 1.3 g L 1 lactic acid. It is noted that evaporation also removed significant amounts of volatile compounds such as acetic acid and furfural generated from the dehydration of xylose (Larsson et al., 1999; Mussatto and Roberto, 2004; Parawira and Tekere, 2011). The high glucose content with little acids makes CBH a suitable carbon source for succinic acid fermentation. 3.2. Utilization of CBH as the carbon source The cassava bagasse represents a large reservoir of potentially fermentable carbohydrates. These carbohydrates, obtained via either acid-promoted hydrolysis or by enzymatic hydrolysis, are intrinsically a mixture of pentoses such as xylose and arabinose and hexoses such as glucose, among others (Lu et al., 2012; Stephanopoulos, 2007). Generally, complete hydrolysis of cassava bagasse ideally generates a solution primarily containing glucose (97.8% w/w), xylose (2% w/w), and arabinose (0.2% w/w) (Carta et al., 1999). Our research showed that C. glutamicum 534 could only take up glucose from CBH to produce succinic acid. Nonetheless, glucose accounted for a large proportion (97.8%) of the total sugar, effective fermentation of glucose from CBH may be achieved with C. glutamicum 534. As shown in Fig. 3, succinic acid production from CBH, with glucose concentrations from 20 to 50 g L 1, was investigated by anaerobic bottle cultivation. A total glucose utilization of approximately 99% was achieved at glucose concentrations ranging from 20 to 40 g L 1. However, glucose utilization decreased when its concentration exceeded 40 g L 1. Succinic acid concentration increased with increasing glucose concentrations ranging from 20 to 35 g L 1. Further increases in glucose concentrations caused no marked change in cell growth or succinic acid concentration, and

even led to a decrease in these parameters at a glucose concentration of 50 g L 1. When the glucose concentration was 35 g L 1, succinic acid production peaked at 20.6 g L 1. The results of free-cell fermentation using glucose and CBH as carbon sources were compared (Fig. 4). CBH was diluted to a concentration of 35 g L 1 of the initial glucose concentration. As shown in Fig. 4, the lag phase of CBH fermentation was longer than that of glucose fermentation. The residual glucose was only 0.5 g L 1 after 46 h, whereas the residual sugar of CBH fermentation was 10.3 g L 1 after this time period. This phenomenon may be caused by the inhibitors present in the enzymatic hydrolysates, including hydrocyanic acid, acetic acid, lactic acid, furfural, hydroxymethylfurfural, and lignin degradation products (Abdel-Rahman et al., 2011; Behera et al., 2014). However, 17.4 g L 1 of succinic acid and 15.6 g L 1 of DCW attained from fermentation of CBH was similar to 19.6 g L 1 of succinic acid and 16.6 g L 1 of DCW obtained from fermentation of a 35 g L 1 glucose solution at the end of batch fermentation (Fig. 4). These results indicated that this sugar mixture could be efficiently fermented by C. glutamicum 534 to produce succinic acid and that CBH could be economically used as a carbon source for succinic acid production. 3.3. Comparisons of free and immobilized cells for succinic acid batch fermentation The fermentation was conducted using a two-step process, in which an aerobic growth phase was followed by an anaerobic production phase (Cavinato et al., 2011; Litsanov et al., 2012; Ma et al., 2010). To study the feasibility of immobilizing C. glutamicum 534 to be used as succinic acid-producing strain, we assessed three models in the bioreactor (Fig. 2): model 1 (free cells), model 2 (porous polyurethane added in the beginning), and model 3 (free-cell incubation in the early stage, and then porous polyurethane was added into the bioreactor when the cell growth reach the middle of the exponential phase). After incubation for 12 h, the medium of model 1 was centrifuged (5000g, 4 °C, 10 min) to harvest the cell mass. Fresh sterile fermentation broth was used to wash and suspend the cell mass (twice) in order to eliminate the seed medium before adding the cell mass into the fermentation broth for succinic acid production. For the two immobilized models, the seed

Glucose utilization

Succinic acid

DCW

30 100

80 20 60 15 40

10

20

5

0

Glucose utilization (%)

DCW (g L-1) Succinic acid (g L-1)

25

0 20

25

30

35

40

45

50

Glucose concentration from CBH (g L-1) Fig. 3. Succinic acid production at varying total sugar concentrations of CBH (20–50 g L

1

). CBH: cassava bagasse hydrolysate; DCW: dry cell weight.

194

X. Shi et al. / Bioresource Technology 174 (2014) 190–197

Glucose

40

DCW

Succinic acid

B

20

16

12

20 8

15 10

4

Glucose (g L -1 )

25

16

30

Succinic acid (g L -1 ) DCW (g L -1 )

-1

)

30

Glucose (g L

20

35

35

25

12

20 8

15 10

5 0

Glucose concentration of CBH DCW Succinic acid

40

4

Succinic acid (g L -1 ) DCW (g L -1 )

A

5

0

10

20

30

0 50

40

0

0

10

20

Fermentation time (h)

30

40

50

60

0

Fermentation time (h)

Fig. 4. Results of fermentation with glucose and cassava bagasse hydrolysate (CBH) as carbon sources. (A) Succinic acid production with glucose, (B) succinic acid production with glucose derived from CBH. CBH: cassava bagasse hydrolysate; DCW: dry cell weight.

broth was poured out and then the fermentation broth was added for the succinic acid production step (Chen et al., 2012, 2013). The sugar consumption rate was the fastest and the concentration of succinic acid was the highest in model 2 (Table 1 and Table 2). The OD600 of the seed broth in model 3 was higher than that in model 2, which indicated that the immobilization methods used in model 2 were more optimal than those used in model 3. Moreover, the productivity of model 2 was 1.1 times higher than that of model 3 and 1.2 times higher than that of model 1.

was lowest with NaOH as the regulator, at 82.4%. In cell metabolism, Na+ plays a very important role in maintaining the transmembrane pH gradient, cell osmotic pressure, and regulation of intracellular pH (Liu et al., 2008). With Na+ alkaline neutralizers as regulators, the high Na+ level resulted in a hypertonic environment during the latter part of the fermentation, which had a negative effect on cell growth and metabolism. When Mg(OH)2 was fed into the solution, a succinic acid concentration of 21.4 g L 1 was achieved, which was more than that attained when MgCO3 (20.9 g L 1) was added initially to the medium as the regulator. Mg2+ is an activator of many enzymes; a key enzyme in the pathway of succinic acid, phosphoenolpyruvate carboxykinase, also requires Mg2+ as a cofactor (Bazaes et al., 2007; Podkovyrov and Zeikus, 1993). Using NH3
H2O as regulator, cell growth was also significantly suppressed and only a few organic acids were produced. Thus, to complement the strong alkalinity and high solubility of NaOH with the indispensability of Mg2+, NaOH and Mg(OH)2 were chosen as a mixed alkaline neutralizer to regulate the fermentation pH.

3.4. Effects of different alkaline neutralizers on cell growth and succinic acid production During immobilized fermentation, organic acids gradually accumulate and the pH decreases correspondingly. To control the pH, large amounts of alkaline neutralizers are required; thus, the cost of the alkaline neutralizer accounts for a significant portion of the raw material costs. To identify the most efficient and economical neutralizer, the effects of different alkaline neutralizers on cell growth and succinic acid production were examined. The glucose consumption for fermentation using Na2CO3, NaHCO3, Mg(OH)2, or MgCO3 as alkaline neutralizers was much higher than that using CaCO3, NaOH, and NH3
H2O (Table 1). When Ca(OH)2 and CaCO3 were used as alkaline neutralizers, cell growth was severely suppressed, and little succinic acid was produced. Some reports have shown that Ca2+ is toxic to C. glutamicum during succinic acid production (Song et al., 2007). Ca2+ can disturb the normal fluidity and permeability of cell membranes, which in turn can affect cell growth and metabolism. Among the three Na+ alkaline neutralizers, a succinic acid concentration of 21.2 g L 1 with NaOH as the regulator was higher than that achieved using Na2CO3 (18.4 g L 1) and NaHCO3 (21.1 g L 1), whereas glucose utilization

3.5. Utilization of NaOH and Mg(OH)2 mixture as alkaline neutralizers In a 3.5-L batch immobilized fermentation process, a mixed NaOH and Mg(OH)2 alkaline neutralizer was used to regulate pH at an initial glucose concentration of 35 g L 1 obtained from CBH. Different mass ratios (3:1, 2:1, 1:1, 1:2, 1:3) of NaOH and Mg(OH)2 were investigated. As shown in Fig. 5, with a decreasing proportion of NaOH in the mixture, the residual glucose level gradually decreased. When the mass ratio of NaOH and Mg(OH)2 was 2:1, the glucose was completely consumed and the succinic acid concentration increased up to 22.3 g L 1. Although the glucose level was also completely consumed at NaOH and Mg(OH)2 mass ratios

Table 1 Residual sugars and yield of succinic acid of the three models. Model

Seed culture Initial sugar (g L

Model 1 Model 2 Model 3

20 20 20

Fermentation 1

)

Residual sugar (g L 4.5 2 4

1

)

DCW (g L 3.432 0.113 0.584

1

)

Initial sugar (g L 35 35 35

The strain was incubated under aerobic conditions at 30 °C for 12 h. Fermentation was conducted for 60 h.

1

)

Residual sugar (g L 1.7 0.2 1.1

1

)

Succinic acid (g L 18.2 21.12 19.37

1

)

195

X. Shi et al. / Bioresource Technology 174 (2014) 190–197 Table 2 Effect of different alkaline neutralizers on succinic acid production. Alkaline neutralizer

Initial total sugar (g L 1)

Residual total sugar (g L 1)

Succinic acid (g L 1)

Lactic acid (g L 1)

Acetic acid (g L 1)

Total sugar utilizationa (%)

Succinic acid yieldb (%)

Ca(OH)2 CaCO3 NaOH NaHCO3 Na2CO3 Mg(OH)2 MgCO3 NH3
H2O

34.7 ± 1.1 34.8 ± 1.2 35.2 ± 1.2 34.9 ± 1.4 34.9 ± 1.3 35.1 ± 1.5 35.3 ± 1.5 34.8 ± 1.2

27.6 ± 1.4 24.3 ± 1.2 6.2 ± 0.7 2.4 ± 0.6 1.8 ± 0.3 0.7 ± 0.2 2.2 ± 0.3 22.8 ± 1.2

2.1 ± 0.3 6.2 ± 0.6 21.2 ± 1.5 21.0 ± 1.3 18.4 ± 1.3 21.4 ± 0.9 20.9 ± 1.1 7.1 ± 0.7

0.4 ± 0.1 1.3 ± 0.2 1.7 ± 0.3 3.4 ± 0.5 4.3 ± 0.4 2.5 ± 0.3 1.9 ± 0.1 1.1 ± 0.1

1.3 ± 0.2 4.4 ± 0.3 4.7 ± 0.4 9.3 ± 0.5 10.8 ± 0.6 9.5 ± 0.5 8.3 ± 0.4 2.6 ± 0.2

20.5 ± 0.6 30.2 ± 0.8 82.4 ± 1.3 88.0 ± 1.4 94.8 ± 1.5 98.0 ± 1.1 93.8 ± 0.9 34.5 ± 0.8

6.05 ± 0.3 17.8 ± 0.5 60.2 ± 1.1 60.5 ± 0.7 52.7 ± 0.7 60.9 ± 1.2 59.2 ± 0.8 20.4 ± 0.5

Each value is an average of three parallel replicates. a Glucose utilization (%) is defined as the percentage of the concentration of total sugar utilized by the bacteria in the initial total sugar. b Succinic acid yield (%) is defined as the percentage of the concentration of succinic acid in the initial total sugar.

30

Residual glucose succinic acid Other organic acids

Concentration (g/L)

25

20

15

10

5

0 3:1

2:1

1:1

1:2

1:3

Ratio of NaOH and Mg(OH)2 (g/g) Fig. 5. Comparison of the effect of using different mass ratios of NaOH and Mg(OH)2 in the production of succinic acid during batch fermentation.

of 1:2 and 1:3, the concentration of succinic acid was lower than that attained at a 2:1 ratio, because of the higher Mg(OH)2 dilution rate. When the mass ratio of NaOH and Mg(OH)2 was 2:1, the excessive accumulation of Na+ was eased, enhancing glucose utilization, and the high dilution of Mg(OH)2 was also decreased. Therefore, 2:1 is the optimal mass ratio of NaOH and Mg(OH)2 for succinic acid production. The results obtained using a 2:1 NaOH and Mg(OH)2 mass ratio were compared with those obtained when using NaHCO3 (Table 3). Glucose utilization and succinic acid production using the mixed neutralizer were higher than those achieved with NaHCO3. Thus, NaHCO3 as alkaline neutralizer could be completely replaced with a NaOH and Mg(OH)2 mixture. As shown in Table 3, the immobilized fermentation of succinic acid from CBH using a mixture of NaOH and Mg(OH)2, at a mass ratio of 2:1, as the alkaline neutralizer was compared with free-cell fermentation using glucose and NaHCO3. Immobilized fermentation of succinic acid from CBH using a mixture of NaOH and Mg(OH)2 as alkaline neutralizer resulted in a succinic acid productivity of 0.42 g L 1 h 1, which was similar to that obtained with free-cell fermentation utilizing glucose and NaHCO3. These results demonstrated that immobilized fermentation from CBH using a mixture of NaOH and Mg(OH)2 could replace free cell fermentation using glucose and NaHCO3, resulting in more economical production of succinic acid.

3.6. Repeated immobilized batch fermentation of succinic acid from CBH using mixed alkalis (NaOH and Mg(OH)2) Repeated batch fermentations were carried out using PPFimmobilized cells to assess the mechanical stability of the support for reutilization and to evaluate the time required for cell adaptation in each repetition. The initial total sugar concentration was approximately 35 g L 1. All fermentations were performed using immobilized cells from the previous fermentation. Fig. 6 depicted the fermentation profile for five repeated batches. In the first batch, the fermentation time was longer than that of the other batches. The time to reach the stationary phase was 60 h for the first batch, whereas for batches 2–5, the time to reach the stationary phase was only 48–54 h. The final concentration of succinic acid was similar in all five batches (22.4 g L 1), and glucose in the CBH was depleted (below 1 g L 1 glucose) in all batches. Lactic acid and acetic acid were produced at 3.7 and 9.2 g L 1, respectively. It was observed that the concentrations of these acids were slightly increased in repeated batches. The production of lactic acid indicated that the capability of C. glutamicum 534 to product succinic acid was not strong enough to use all the NADH generated in acetate formation. The yields of succinic acid were approximately 64.8% (w/w) for each batch, as seen in Fig. 6. The productivity of succinic acid was 0.41 g L 1 h 1 in the first batch, whereas it was approximately

196

X. Shi et al. / Bioresource Technology 174 (2014) 190–197

Table 3 Results of fermentation using different carbon sources, alkaline neutralizers, and fermentative modes.

Initial glucose (g L 1) Residual glucose (g L 1) Succinic acid (g L 1) Other organic acid (g L 1) Fermentation time Succinic acid productivity (g L 1 h 1) Succinic acid yield (%)

Immobilized fermentation from CBH using a mixture of NaOH and Mg(OH)2 (mass ratio of 2:1)

Immobilized fermentation from CBH using NaHCO3

Free-cell fermentation using glucose and NaHCO3

35 ± 0.3 0.6 ± 0.1 22.3 ± 0.3 11.6 ± 0.2

35 ± 0.4 1.8 ± 0.2 20.8 ± 0.2 10.7 ± 0.1

35 ± 0.5 0.2 ± 0.2 19.6 ± 0.2 12 ± 0.1

54 0.42 ± 0.13

54 0.35 ± 0.12

46 0.43 ± 0.12

63.7 ± 0.3

59.4 ± 0.2

56.0 ± 0.2

Each value is an average of three parallel replicates.

Glucose conentration of CBH Succinic acid

40

Batch 1

Batch 2

Batch 3

Acetic acid Batch 4

Batch 5

26 24 22 20

30

18 25

16 14

20 12 10

15

8 10

6

Organic acid (g/L)

Glucose conentration of CBH (g/L)

35

Lactic acid

4

5

2 0 0

50

100

150

200

250

0 300

Fermentation time (h) Fig. 6. Production of succinic acid with C. glutamicum 534-immobilized cells for repeated batch fermentation, using mixed alkalis (NaOH and Mg(OH)2) and cassava bagasse hydrolysate (CBH).

0.42 g L 1 h 1 in batches 2–5. Repeated batches were carried out in a total time of 267 h, and the total sugar consumed was 173 g L 1, while the amount of succinic acid produced was 112.1 g L 1. 4. Conclusions In this study, we developed an approach for economical succinic acid production through the fermentation of CBH, by C. glutamicum 534 immobilized in PPF, using a mixed neutralizer consisting of NaOH and Mg(OH)2 (2:1, w/w). Using a cheap medium and optimally mixed alkaline neutralizers in the immobilized fermentation system enhanced yield, productivity, and the final concentration of succinic acid. From an industrial standpoint, use of a cheap carbon source with mixed alkaline neutralizers in the immobilized fermentation system reduced the raw material cost. The process is useful for efficient, economically stable, large-scale production of succinic acid. Acknowledgements This work was supported by the National Outstanding Youth Foundation of China (21025625), the National High-Tech Research and Development Program of China (863) (2012AA021203), the

National Basic Research Program of China (973) (2013CB733602), the Major Research Plan of the National Natural Science Foundation of China (21390204), the National Technology Support Program (2012BAI44G01), the National Natural Science Foundation of China, General Program (21376118), the National Natural Science Foundation of China, Youth Program (21106070), the Jiangsu Provincial Natural Science Foundation of China (SBK 201150207), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Abdel-Rahman, M.A., Tashiro, Y., Sonomoto, K., 2011. Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: overview and limits. J. Biotechnol. 156, 286–301. Amin, G.A., Al-Talhi, A., 2007. Production of L-glutamic acid by immobilized cell reactor of the bacterium Corynebacterium glutamicum entrapped into carrageenan gel beads. World Appl. Sci. J. 2, 62–67. Bazaes, S., Toncio, M., Laivenieks, M., Zeikus, J.G., Cardemil, E., 2007. Comparative kinetic effects of Mn(II), Mg(II) and the ATP/ADP ratio on phosphoenolpyruvate carboxykinases from Anaerobiospirillum succiniciproducens and Saccharomyces cerevisiae. Protein J. 26, 265–269. Behera, S., Arora, R., Nandhagopal, N., Kumar, S., 2014. Importance of chemical pretreatment for bioconversion of lignocellulosic biomass. Renew. Sust. Energ. Rev. 36, 91–106.

X. Shi et al. / Bioresource Technology 174 (2014) 190–197 Bramorski, A., Soccol, C.R., Christen, P., Revah, S., 1998. Fruity aroma production by Ceratocystis fimbriata in solid cultures from agro-industrial wastes. Rev. Microbiol. 29, 208–212. Carta, F.S., Soccol, C.R., Ramos, L.P., Fontana, J.D., 1999. Production of fumaric acid by fermentation of enzymatic hydrolysates derived from cassava bagasse. Bioresour. Technol. 68, 23–28. Cavinato, C., Bolzonella, D., Fatone, F., Cecchi, F., Pavan, P., 2011. Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation. Bioresour. Technol. 102, 8605–8611. Chen, X., Jiang, S., Zheng, Z., Pan, L., Luo, S., 2012. Effects of culture redox potential on succinic acid production by Corynebacterium crenatum under anaerobic conditions. Process Biochem. 47, 1250–1255. Chen, Y., Liu, Q.G., Zhou, T., Li, B.B., Yao, S.W., Li, A., Wu, J.L., Ying, H.J., 2013. Ethanol production by repeated batch and continuous fermentations by Saccharomyces cerevisiae Immobilized in a Fibrous Bed Bioreactor. J. Microbiol. Biotechnol. 23, 511–517. Chu, C.H., Kumagai, H., Nakamura, K., 1996. Application of polyelectrolyte complex gel composed of xanthan and chitosan to the immobilization of Corynebacterium glutamicum. J. Appl. Polym. Sci. 60, 1041–1047. Corona-González, R.I., Miramontes-Murillo, R., Arriola-Guevara, E., GuatemalaMorales, G., Toriz, G., Pelayo-Ortiz, C., 2014. Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid. Bioresour. Technol. 164, 113–118. Guettler, M.V., Rumler, D., Jain, M.K., 1999. Actinobacillus succinogenes sp. Nov., a novel succinic-acid-producing strain from the bovine rumen. Int. J. Syst. Bacteriol. 49, 207–216. Inui, M., Murakami, S., Okino, S., Kawaguchi, H., Vertès, A.A., Yukawa, H., 2004. Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microb. Biotech. 7, 182–196. John, R.P., Nampoothiri, K.M., Pandey, A., 2007. Polyurethane foam as an inert carrier for the production of L (+)-lactic acid by Lactobacillus casei under solidstate fermentation. Lett. Appl. Microbiol. 44, 582–587. Jun, Y.S., Huh, Y.S., Park, H.S., Thomas, A., Jeon, S.J., Lee, E.Z., Hong, Y.K., 2007. Adsorption of pyruvic and succinic acid by amine-functionalized SBA-15 for the purification of succinic acid from fermentation broth. J. Phys. Chem. C 111, 13076–13086. }sson, J.J., 1999. Comparison of different Larsson, S., Reimann, A., Nilvebrant, N., Jo methods for the detoxification of lignocellulose hydrolysates of spruce. Appl. Biochem. Biotechnol. 77–79, 91–103. Lee, P.C., Lee, S.Y., Hong, S.H., Chang, H.N., Park, S.C., 2003. Biological conversion of wood hydrolysate to succinic acid by Anaerobiospirillum succiniciproducens. Biotechnol. Lett. 25, 111–114. Li, Q., Wang, D., Wu, Y., Yang, M., Li, W., Xing, J., Su, Z., 2010. Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130ZT. J. Microbiol. 48, 290–296. Lin, H., Bennett, G.N., San, K.Y., 2005. Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab. Eng. 7, 116–127.

197

Litsanov, B., Brocker, M., Bott, M., 2012. Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate. Appl. Environ. Microb. 78, 3325–3337. Liu, Y.P., Zheng, P., Sun, Z.H., Ni, Y., Dong, J.J., Wei, P., 2008. Strategies of pH control and glucose-fed batch fermentation for production of succinic acid by Actinobacillus succinogenes CGMCC1593.J. Chem. Technol. Biotechnol. 83, 722– 729. Lu, C.C., Zhao, J.B., Yang, S.T., Wei, D., 2012. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387. Ma, J.F., Jiang, M., Chen, K.Q., Xu, B., Liu, S.W., Wei, P., Ying, H.J., 2010. Succinic acid production with metabolically engineered E. coli recovered from two-stage fermentation. Biotechnol. Lett. 32, 1413–1418. Mussatto, S.I., Roberto, I.C., 2004. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresour. Technol. 93, 1–10. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., Vandenberghe, L.P., Mohan, R., 2000. Biotechnological potential of agro-industrial residues. II: cassava bagasse. Bioresour. Technol. 74, 81–87. Parawira, W., Tekere, M., 2011. Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review. Crit. Rev. Biotechnol. 31, 20–31. Podkovyrov, S.M., Zeikus, J.G., 1993. Purification and characterization of phosphoenolpyruvate carboxykinase, a catabolic CO2-fixing enzyme, from Anaerobiospirillum succiniciproducens. J. Gen. Microbiol. 139, 223–228. Shohei, O., Masayuki, I., Hideaki, Y., 2005. Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl. Microbiol. Biotechnol. 68, 475–480. Shohei, O., Ryoji, N., Masako, S., Toru, J., Masayuki, I., Hideaki, Y., 2008. An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl. Microbiol. Biotechnol. 81, 459–464. Skorokhodova, A.Y., Gulevich, A.Y., Morzhakova, A.A., Shakulov, R.S., Debabov, V.G., 2013. Metabolic engineering of Escherichia coli for the production of succinic acid from glucose. Appl. Biochem. Microbiol. 49, 629–637. Song, H., Lee, S.Y., 2006. Production of succinic acid by bacterial fermentation. Enzyme Microb. Tech. 39, 353–361. Song, H., Lee, J.W., Choi, S., You, J.K., Hong, W.H., Lee, S.Y., 2007. Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production. Biotechnol. Bioeng. 98, 1296–1304. Stephanopoulos, G., 2007. Challenges in engineering microbes for biofuels production. Science 315, 801–804. Thongchul, N., Navankasattusas, S., Yang, S.T., 2009. Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis. Bioprocess Biosyst. Eng. 33, 407–416. Vijayaraghavan, K., Mao, J., Yun, Y.S., 2008. Biosorption of methylene blue from aqueous solution using free and polysulfone-immobilized Corynebacterium glutamicum: Batch and column studies. Bioresour. Technol. 99, 2864–2871. Zhu, Y., Knol, W., Smits, J.P., Bol, J., 1996. Medium optimization for nuclease P1 production by Penicillium citrinum in solid-state fermentation using polyurethane foam as inert carrier. Enzyme Microb. Technol. 18, 108–112.