Bagasse hydrolyzates from Agave tequilana as substrates for succinic acid production by Actinobacillus succinogenes in batch and repeated batch reactor

Bioresource Technology 205 (2016) 15–23

Contents lists available at ScienceDirect

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

Bagasse hydrolyzates from Agave tequilana as substrates for succinic acid production by Actinobacillus succinogenes in batch and repeated batch reactor Rosa Isela Corona-González a,⇑, Karla María Varela-Almanza a, Enrique Arriola-Guevara a, Álvaro de Jesús Martínez-Gómez a, Carlos Pelayo-Ortiz a, Guillermo Toriz b a b

Departamento de Ingeniería Química, CUCEI-Universidad de Guadalajara, Blvd. M. García Barragán 1421, C.P. 44430 Guadalajara, Jalisco, Mexico Departamento de Madera Celulosa y Papel, Universidad de Guadalajara, km. 15.5 carretera Guadalajara-Nogales, C.P. 45020, Las Agujas, Zapopan, Jalisco, Mexico

h i g h l i g h t s
Lignocellulosic residues can be used as a source of hydrolyzates for fermentation.
Enzymatic hydrolyzates were free of furfural and hydroxymethylfurfural.
Production of succinic acid was faster by using enzymatic hydrolyzates.
33.6 g/L succinic acid was obtained from 87.2 g/L fermentable sugars by repeated batch.
Immobilization of cells reduced significantly the time of fermentation.

a r t i c l e

i n f o

Article history: Received 4 November 2015 Received in revised form 18 December 2015 Accepted 28 December 2015 Available online 11 January 2016 Keywords: Actinobacillus succinogenes Acid hydrolyzates Enzymatic hydrolyzates Succinic acid Repeated batch fermentations

a b s t r a c t The aim of this work was to obtain fermentable sugars by enzymatic or acid hydrolyses of Agave tequilana Weber bagasse in order to produce succinic acid with Actinobacillus succinogenes. Hydrolyses were carried out with mineral acids (sulfuric and hydrochloric acids) or a commercial cellulolytic enzyme, and were optimized statistically by a response surface methodology, having as factors the concentration of acid/ enzyme and time of hydrolysis. The concentration of sugars obtained at optimal conditions for each hydrolysis were 21.7, 22.4 y 19.8 g/L for H2SO4, HCl and the enzymatic preparation respectively. Concerning succinic acid production, the enzymatic hydrolyzates resulted in the highest yield (0.446 g/ g) and productivity (0.57 g/L h) using A. succinogenes in a batch reactor system. Repeated batch fermentation with immobilized A. succinogenes in agar and with the enzymatic hydrolyzates resulted in a maximum concentration of succinic acid of 33.6 g/L from 87.2 g/L monosaccharides after 5 cycles in 40 h, obtaining a productivity of 1.32 g/Lh. Ó 2016 Published by Elsevier Ltd.

1. Introduction The high demand for biodegradable polymers has stimulated the production of succinic acid (Song and Lee, 2006; Cheng et al., 2012). Succinic acid is commercially produced from butane through maleic anhydride, and is an important intermediate in the preparation of antiseptics, cosmetics, drugs, sutures, solvents, synthetic resins, herbicides, fungicides, inks, detergents and as a growth stimulant for plants and animals (Cheng et al., 2012; Zeikus et al., 1999). However, the high cost of converting maleic anhydride to succinic acid limits the use of this process in ⇑ Corresponding author. Tel./fax: +52 33 13785900x27536. E-mail address: [email protected] (R.I. Corona-González). http://dx.doi.org/10.1016/j.biortech.2015.12.081 0960-8524/Ó 2016 Published by Elsevier Ltd.

numerous applications (Song and Lee, 2006; Zeikus et al., 1999). Furthermore, environmental and economic concerns have spurred producing succinic acid from renewable resources, especially by bacterial fermentation (Urbance et al., 2004). Microorganisms with high potential for production of succinic acid include Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens and A. succinogenes (Li et al., 2010). A. succinogenes has interesting features that make it well suited to produce relatively large amounts of succinic acid (Song and Lee, 2006). It could use a wide range of carbohydrates as carbon sources, including lactose, xylose, arabinose, cellobiose, and other reduced sugars, having as the major end product succinic acid (Guettler et al., 1999; Van der Werf et al., 1997). Lignocellulosic materials hydrolyzed into mixed sugars are an interesting option for production of

16

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

succinic acid, due to their enormous abundance and the fact that they do not compete with agricultural land (Li et al., 2010; Zheng et al., 2009; Jiang et al., 2013; Ribeiro-Borges and Pereira, 2011). Biomass feedstock that grow on semiarid lands, such as the Agave species, could be a sustainable answer to increasing demands for renewable fuels that do not conflict with food and feed production, require minimal inputs of water and nutrients and can be grown on lands that are marginal for food crops (Davis et al., 2011). Agave tequilana bagasse is one of the main residues generated by the tequila industry, and represents 40 wt.% from the agave plant with a high content of polysaccharides (specifically cellulose and hemicellulose), thus resulting in an inexpensive feedstock. The consumption of tequila, the national Mexican liquor, has increased worldwide, rising significantly the generation of residues. It is estimated that nearly one million tons of agave plants will be used yearly, generating about 400,000 tons of agave bagasse (CRT, 2012). This fact poses an environmental problem because composting agave bagasse takes long times and other traditional uses such as the manufacture of bricks and mattresses do not have a significant impact on its utilization. Therefore, hydrolysis of this lignocellulosic residue for production of succinic acid is desirable. Several studies have reported hydrolysis of different types of lignocellulosic biomass (Kumar et al., 2009), and dilute acid catalyzed hydrolysis could be performed easily at low cost. However, generation of toxic byproducts, such as furfural and hydroxymethyl furfural, is often found. A. tequilana bagasse has been used as a feedstock for production of ethanol (Saucedo-Luna et al., 2011; Caspeta et al., 2014) and methane (Arreola-Vargas et al., 2015). Pretreatments include the use of dilute sulfuric and hydrochloric acid at high temperatures (Saucedo-Luna et al., 2011; Arreola-Vargas et al., 2015), and use of ionic liquids (Pérez-Pimienta et al., 2013). These studies have focused on the production of fuel ethanol and methane and the microorganisms used can tolerate the presence of furfural and hydroxymehthylfurfural, which in the case of A. succinogenes inhibit growth by lowering cell’s membrane permeability (Chen et al., 2010). Therefore, it is necessary to optimize hydrolysis conditions to increase the concentration of monosaccharides and decrease undesirable byproducts for the fermentative production of succinic acid. The aim of this contribution was to optimize the hydrolysis of agave bagasse, both acid or enzymatic, to evaluate the production of succinic acid in batch cultivation with free cell and immobilized A. succinogenes. Immobilized cells were also tested, because these can be easily separated from products in solution without losing activity, have more physical stability and can be re-utilized (Kikani et al., 2013). In the best of our knowledge this is the first report for production of succinic acid with agave bagasse hydrolyzates as substrate by immobilized A. succinogenes.

2. Methods 2.1. Materials 2.1.1. Culture strain A. succinogenes ZT-130 (ATCC 55617) was used in this study. This microorganism was cultivated every other week in vials using trypticasein soy broth (TSB) (Difco).

2.1.2. Agave bagasse Agave bagasse (A. tequilana Weber var. azul) was collected from a tequila distillery in the village Arandas Jalisco, Mexico. The bagasse was profusely washed, then dried at 50 °C for 24 h and

thereafter ground and meshed (#40 mesh) to obtain homogeneous particle size (0.5 mm). 2.2. Optimization of hydrolyses of agave bagasse 2.2.1. Exploratory trials on hydrolysis of agave bagasse In order to find appropriate hydrolysis conditions both types of hydrolyses (acid or enzymatic) were carried out at the following ratios of bagasse weight to volume of hydrolysis solution: 1:2, 1:5, 1:10 and 1:15. The enzymatic hydrolyses were carried out with a mixture of cellulolytic enzymes from Aspergillus niger and Trichoderma reesei (Macerex PM, activity 17.4 U/mL cellulase and 39.8 U/mL xilanase), by varying the temperature at 40, 50 and 60 °C and the ratio bagasse weight to 1% enzymatic solution. The acid hydrolyses were carried out at 1.5 wt.% H2SO4 or HCl for 30 min at 121 °C. The release of fermentable sugars (glucose, xylose and arabinose) from bagasse was quantified as described in Section 2.6.1. 2.2.2. Statistical optimization of hydrolyses Optimal hydrolysis conditions were sought by applying a response surface methodology. Two factors were evaluated for each type of hydrolysis, i.e. time and concentration of hydrolyzing agent, having as the response the concentration of fermentable sugars (g/L). A central composite experimental design with 4 factorial, 4 axial, and five central points at solids to liquid ratio 1:10 was carried out using coded values (the relationship of the coded values with actual values is depicted in Table 1). The temperature for the enzymatic hydrolysis was selected at 50 °C, whereas 121 °C was chosen for acid hydrolyses. The time range evaluated for acid hydrolysis was 30.2–151.8 min, whereas for the enzymatic hydrolysis was 14.3–25.7 h. The concentration of acid (sulfuric/ hydrochloric) was in the range 0.79–2.21 wt.%, while the concentration of enzyme varied from 3.17 to 8.83 wt.% as seen in Table 1. The central composite experimental designs can be described by the following second degree polynomial quadratic equations:

C k ¼ b0k þ b1k xi þ b2k xj þ b3k xi xj þ b4k x2i þ b5k x2j þ ek where k represents sulfuric acid (SA), hydrochloric acid (HA) or enzymatic (E); the constant terms are represented with bs, e represents the experimental error, xi and xj are the coded independent variables. Data analysis, response surface plots and ANOVA were realized using Design-Expert v. 9.0.6.2 (trial version, Stat-Ease Inc., Minneapolis, MN, USA) software. The effects were considered to be of statistical significance at pvalue < 0.05. 2.3. Neutralization of mineral acids after acid hydrolyses In order to prevent inhibition of bacterial growth during fermentations, the neutralization of hydrolyzates was pursued by using an anion exchange column (Amberlite 96). The amount of anion exchange resin needed to increase the pH was 350 g per liter of hydrolyzates. This treatment allowed the removal of 99% SO=4 and 80% Cl having a total carbohydrate loss of only 2.56% and 14.56% respectively. 2.4. Succinic acid production Succinic acid was produced with free cells of A. succinogenes by fermentation of agave hydrolyzates by applying the optimal values (time and concentration of hydrolyzing agents) found through the response surface methodologies. The hydrolyzates were sterilized at 110 °C for 15 min in an autoclave and then the media was complemented by adding the following components, to reach a final concentration in the medium (in g/L): yeast extract (10), NaHCO3

17

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23 Table 1 Central composite design for acid or enzymatic hydrolyses with coded and actual values along with the responses. Run number

xj

tA (min)

wA (wt.%)

tE (h)

wE (wt.%)

CSA (g/L)

CHA (g/L)

CE (g/L)

1 1 1 1 0

48 134 48 134 91

1.00 1.00 2.00 2.00 1.50

16.0 24.0 16.0 24.0 14.3

4.00 4.00 8.00 8.00 6.00

12.8 20.3 18.6 21.4 12.9

14.0 21.5 21.0 21.6 16.6

13.6 16.0 17.2 16.5 14.6

0

91

1.50

25.7

6.00

21.9

20.2

17.0

7

1 1 1 1 pffiffiffi  2 pffiffiffi 2 0

30.2

0.79

20.0

3.17

17.7

16.9

13.9

8

0

151.8

2.21

20.0

10.83

19.9

22.0

17.1

9 10 11 12 13 Subscript codes

0 0 0 0 0

91 91 91 91 91

1.50 1.50 1.50 1.50 1.50

20.0 20.0 20.0 20.0 20.0 i:{1, 3} j = i + 1:{2, 4}

6.00 6.00 6.00 6.00 6.00

20.8 20.0 19.7 20.1 19.6

20.4 21.0 20.3 20.9 20.5

20.3 19.9 19.3 19.6 19.2

Codes

x1 ¼ ðtA 19ÞðminÞ 43 min

6

x3 ¼

2

Responses2

xi 1 2 3 4 5

1

Actual values1

Coded value

pffiffiffi  2 pffiffiffi 2 0 0 0 0 0

ðt E 20ÞðhÞ 4ðhÞ

x2 ¼ ðwA 1:50Þðwt:%Þ 0:5ðwt:%Þ x4 ¼ ðwE 6:00Þðwt:%Þ 2ðwt:%Þ

tA: acid hydrolysis time; wA: acid concentration; tE: enzymatic hydrolysis time; wE: enzyme concentration. Concentration of monosaccharides (g/L) obtained with: sulfuric acid CSA; hydrochloric acid CHA; or enzyme preparation CE.

(10), NaCl (1), MgSO4 (0.05), K2HPO4 (6.4), and NaH2PO4 (15.4). The added components were previously sterilized at the same conditions as the hydrolyzates. Comparative fermentations were also carried out with a synthetic medium having as carbon source 15 g/L glucose. All experiments were performed under the same conditions: pH (6.7 ± 0.1), temperature (37 °C), stirring (150 rpm), and CO2 flow (0.3 vvm) in duplicate. Sugars and generated acids were quantified according to Section 2.6.

2.6.2. Determination of substrate and products of fermentations Glucose, xylose, arabinose, succinic, formic, and acetic acids were measured by high-performance liquid chromatography with a fermentation monitoring column Aminex HPX-87H (300  7.8 mm) at 50 °C. The eluent was 5 mM H2SO4 at a flow rate of 0.6 mL/min. The apparatus was integrated with a Waters 600 controller, Waters 717 plus autoinjector, and RI detector (Waters 2410).

2.5. Repeated batch fermentations

2.7. Calculation of kinetic parameters

Immobilization of A. succinogenes was carried out as described elsewhere (Corona-González et al., 2014). The fermentations in repeated batch with immobilized cells in agar were performed with the hydrolyzates that showed higher production in batch as follows: a first fermentation was carried out under the conditions described above (succinic acid production). Once the fermentation cycle was complete, the bioreactor was placed in a laminar flow cabinet (to avoid contamination), the medium was drained (with a peristaltic pump under sterile conditions) and refilled three times with a sterile isotonic solution (NaCl 0.85 wt.%) to rinse free cells from the support, and the reactor was then replenished with fresh medium. The fermentation cycle was completed when the amount of sugars was constant. Carbohydrates and produced acids were quantified according to Section 2.6.

The rates of production for succinic acid (rS), formic acid (rF), and acetic acid (rA) are expressed in g/L h and were obtained by plotting the respective variable (glucose, succinate, formate, etc.) against time and determining the slope of the resulting curve. The yields of fermentation products (succinic, formic and acetic acids) were calculated by plotting the relative differences in the products for each time period as a function of the relative difference of monosaccharides for the same time period and determining the slope of the resulting curve.

2.6. Analytical methods For sugar or acid quantification the samples were centrifugated at 4000 rpm for 15 min, the supernatant was collected and filtered in a membrane (0.45 lm) and thereafter analyzed by HPLC as described below. 2.6.1. Determination of hydrolyzate sugar composition Glucose, xylose and arabinose, were measured by highperformance liquid chromatography with an Aminex HPX-87C (300  7.8 mm) column; a RI detector (Waters 2410), Waters 600 controller, Waters 717 plus autoinjector, at 85 °C using water as the mobile phase at a flow rate of 0.6 ml/min.

3. Results and discussion In the production of tequila, agave plants are generally steamed at 120 °C during 24 h to hydrolyze the carbohydrates, then the juices from the plants are extracted and the solid residue, agave bagasse, is obtained. The chemical composition of agave bagasse consists of 42 wt.% cellulose, 22 wt.% hemicellulose, 18 wt.% lignin, and 18 wt.% extractives. Steam processing affects the lignocellulosic structure and thus can be considered as a pretreatment. In order to obtain fermentable sugars, the agave bagasse was subjected to hydrolysis by two different means, i.e. mineral acids (sulfuric and hydrochloric acids) or a commercial cellulolytic enzyme preparation. Optimization of the production of fermentable sugars was achieved by applying a response surface methodology. Then, the hydrolyzates were used to evaluate the production of succinic acid with free cells, and thereafter the best conditions were selected to carry out repeated batch fermentations. In the following sections the discussion is structured in the order described.

18

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

increasing the reaction time and the acid concentration, resulted in a higher amount of sugars. Furthermore, Anova analysis indicated that both factors and their interactions were significant at a pvalue < 0.05 (see Supplementary material Table S1). The response surface of sulfuric acid hydrolysis shows a maximum at medium acid concentration and high reaction time. On the other hand, the response surface for hydrochloric acid shows a saddle, which means that the response can be maximized either at high acid concentration and low reaction times or low acid concentration and high reaction times. Fig. 1B also suggests that a higher concentration of acid is required for obtaining a higher concentration of fermentable sugars as reported by Kumar et al. (2009). However, high acid concentration results in the generation of toxic byproducts, i.e. hydroxymethyl furfural (HMF) and furfural, as already shown in the hydrolysis of sugar cane bagasse for production of succinic acid (Xi et al., 2013). According to the mathematical models obtained with the response surface methodology, optimal conditions for sulfuric acid hydrolysis were 135–145 min at 1.5–2.2 wt.% acid to obtain

3.1. Acid hydrolysis of agave bagasse

Concentration of fermentable sugar ( )(g/L)

Exploratory trials on agave bagasse hydrolysis (at 1.5 wt.% H2SO4 or HCl and 30 min reaction time at 121 °C) were evaluated regarding the concentration of released monosaccharides. Several bagasse weight: acid volume ratios (1:2, 1:5, 1:10 and 1:15, g: mL) were tested. It was found that the bagasse weight:acid ratios 1:2 and 1:5 resulted in a paste that was impractical to use for fermentation. The 1:10 ratio was selected because yielded 17.8 g/L fermentable sugars versus 14.2 g/L obtained with a ratio 1:15 with sulfuric acid. Similar results were obtained with hydrochloric acid. The response surface methodology was applied at a solid: liquid ratio 1:10 and the time of acid hydrolysis was varied between 30 and 152 min because time can significantly affect the final concentration of monosaccharides. The acid concentration (0.79–2.21 wt. %) was kept low to avoid undesirable products such as furfural and hydroxymethyl furfural. Fig. 1 shows the response surfaces for hydrolyses with sulfuric acid (1-A) and with hydrochloric acid (1-B). It can be seen that

A 25 20 15 10 5 0

14 15

10 11 12 13

-1.4

16

18

17

19

1.4

20

1

-1

0.6

21.65

-0.6

0.2

-0.2

21

0.2

21.5

-0.2 -0.6

0.6 -1

1 1.4

-1.4

Maximun concentration 21.7 g/L

Concentration of fermentable )(g/L) sugar (

B 25 20 15 10 5 0 22

-1.4

11 12 13

14

15

16

17

18

19

20

21.5

20

21 21

-1

1.4

1 -0.6

0.6 -0.2

0.2 0.2

-0.2 0.6

-0.6 1

-1 1.4

-1.4

Maximized concentration 22.4 g/L Fig. 1. Response surface for acid hydrolyses: effects of reaction time and acid concentration on the release of fermentable sugars of agave bagasse with H2SO4 (A) and HCl (B).

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

21.7 g/L fermentable sugars, which is similar to optimal results reported elsewhere (Xi et al., 2013). Mathematical optimal conditions for fermentable sugar release with hydrochloric acid were calculated at 73 min and 2.2 wt.% acid for 22.4 g/L total sugar (Table 2). These results reflect the selectivity of the different acids towards the hydrolysis, since with sulfuric acid a longer reaction time at moderate acid concentration is required to obtain a similar concentration of fermentable sugars as compared to hydrochloric acid hydrolysis. On the other hand, HCl hydrolysis requires a relatively higher concentration of acid but a significantly lower reaction time, probably due to the fact that chlorine is more electronegative and undergoes irreversible dissociation (ClavijoDíaz, 2002). Conversely sulfuric acid requires two steps for complete dissociation, which might require longer hydrolysis time. Also the surface responses show that for sulfuric acid the selection of hydrolysis conditions near to the mathematical optimum is required, whereas the saddle obtained with hydrochloric acid hydrolysis allows for an ampler variety of conditions, such as high acid concentration with lower reaction time. Therefore, the condition for hydrolysis of bagasse with sulfuric acid were selected near the mathematical optimum (137 min and 2.2 wt.% acid), whereas for hydrochloric acid were chosen at 40 min and 2.2 wt.% acid since these conditions fall at the contour that allows high concentration of sugars. Under these conditions for agave bagasse hydrolyses the sugar concentrations obtained were 20.6 and 22.6 g/L for sulfuric and hydrochloric acid hydrolyses respectively. Chen et al. (2010) applied an experimental orthogonal design and reported the optimum conditions for production of corn fiber acid hydrolyzates as 1 wt.% sulfuric acid, 120 min and 121 °C and Xi et al. (2013) reported optimal conditions for sugar cane bagasse at 2 wt.% H2SO4 for 150 min. In this contribution it was found that optimal conditions for releasing of fermentable sugars were similar to previous studies with sulfuric acid, but that hydrolysis with hydrochloric acid can reduce the time of hydrolysis to one third. It is worth noticing that neither furfural nor hydroxymethylfurfural were detected in this work.

Table 2 Polynomial equations for the quadratic models, regression coefficients and optimal conditions for hydrolyses of agave bagasse1. H2SO4

HCl

Enzymatic

Quadratic model equation ^ SA ¼ 20:04 þ 2:88x1 þ 1:25x2  1:18x1 x2  1:28x2  0:58x2 C 1 2 R2 Adjusted Predicted Standard Adequate 2 2 R R deviation precision 0.9653 0.9405 0.8024 0.71 18.100 Optimal values of the test variables in coded and actual units ^ SA x1 x2 tA wA C (min) (mass) (g/L) 1.2142 0.1571 143.2 1.42 21.7 Quadratic model equation ^ HA ¼ 20:62 þ 1:65x1 þ 1:79x2  1:73x1 x2  0:96x2  0:43x2 C 1 2 R2 Adjusted Predicted Standard Adequate 2 2 R R deviation precision 0.9967 0.9440 0.7990 0.5700 21.378 Optimal values of the test variables in coded and actual units ^ HA x1 x2 tA wA C (min) (mass) (g/L) 0.4143 1.4142 73.20 2.21 22.40 Quadratic model equation ^ E ¼ 19:66 þ 0:64x3 þ 1:08x4  0:78x3 x4  1:89x2  2:04x2 C 3 4 R2 Adjusted Predicted Standard Adequate 2 2 R R deviation precision 0.9803 0.9662 0.9306 0.42 22.29 Optimal values of the test variables in coded and actual units ^E x3 x4 tE wE C (h) (mass %) (g/L) 0.1286

1

0.2428

20.50

6.50

19.80

For description of the symbols in this table please refer to Table 1.

19

The conditions described above were used to obtain hydrolyzates for fermentation with A. succinogenes for production of succinic acid. 3.2. Enzymatic hydrolysis of agave bagasse Enzymatic hydrolysis of agave bagasse was carried out with a mixture of commercial cellulolytic enzymes from A. niger and T. reesei by considering their low cost and stability. From the preliminary trials, the higher release of monosaccharides (g/L) was found at 50 °C at a bagasse weight: enzyme solution volume 1:10 (g:mL). Under these conditions optimization of enzymatic hydrolysis was realized by varying the reaction time (14.3–25 h) and enzyme concentration (3.17–10.83 wt./v%). Fig. 2 shows the response surface for both factors, which were significant at a pvalue < 0.05 (see Supplementary material Table S1). Fig. 2 shows a maximum at the center of the response surface, meaning that the most favorable conditions of hydrolysis were reached at the medium values of the factors tested. According to the model obtained with the response surface it was calculated that a region for optimal conditions in the enzymatic hydrolysis were 5.6–7.6 wt.% enzyme concentration and 18–22 h. The conditions used for generation of hydrolyzates for production of succinic acid with A. succinogenes were 6.8 wt.% lignocellulolytic enzyme concentration and 19.7 h to obtain 19.0 g/L fermentable sugars. The release of fermentable sugars obtained in this study (19.0 g sugars/100 g bagasse), using the enzymatic cocktail of industrial grade can be compared to results (12.2 g sugar/50 g bagasse) obtained with agave bagasse using Celluclast 1.5 L from T. ressei (Sigma–Aldrich) and a bglucosidase 188 preparation from A. niger (Novozymes), both highly purified enzymes (Saucedo-Luna et al., 2011) and quite expensive. Furthermore, in this study we achieved these results in a significantly shorter time (19.7 h) as compared to the cited work (72 h). 3.3. Comparison between enzymatic and acid hydrolyses The concentration of sugars obtained directly with the optimal conditions for hydrolyses with the acids or the enzymatic preparation can be seen in Fig. 3A. It can be seen that the hydrolysis with both acids resulted mainly in xylose and in a minor arabinose quantity (17.1 and 2.1 g/L respectively) because hemicelluloses were the main source of fermentable sugars. The amount of pentoses obtained corresponds to 87% of the total hemicellulose in agave bagasse. Glucose was obtained at 3.4 g/L, which corresponds to 8% of the cellulose originally present in the agave bagasse. Hydrolysis of cellulose most likely occurred at the amorphous regions, which is in agreement with the findings of Lu et al. (2007) using corn stover. In contrast, the enzymatic hydrolysis resulted in a preferential release of glucose (12.2 g/L, which corresponds to 29 wt.% recovery of glucose from cellulose) because the enzymatic cocktail contains mainly cellulase. Also, due to the presence of a minor amount of hemicellulase, xylose and arabinose were obtained as well (5 and 1.85 g/L respectively, 31 wt.% pentose recovery from hemicellulose). Saccharification yields of 39.63%, 36.35% and 32.15% for H2SO4, HCl and MacerexÒ, respectively, are comparable to results reported elsewhere with agave bagasse (Saucedo-Luna et al., 2011; Arreola-Vargas et al., 2015). However, in order to carry out fermentations to produce succinic acid, sulfates and chlorides were exchanged thereby neutralizing the acid hydrolyzates. Also, both acid and enzymatic hydrolyzates need to be sterilized and then supplemented with nutrients. This handling of the samples resulted in a loss of carbohydrates for fermentation as seen in Fig. 3B, likely due to precipitation and caramelization of sugars during both anion exchange and

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

Concentration of fermentable sugar ( )(g/L)

20

20 15 10 5 15

0

19.7

-1.4

10 11 12 13 14 15 16

17

18

19.5

19

16

15 14

1.4

1

-1 0.6

-0.6 0.2

-0.2 -0.2

0.2 0.6

-0.6

14

1 1.4

-1 -1.4

Maximun concentration 19.8 g/L

Fig. 2. Response surface for enzymatic hydrolysis: effects of hydrolysis time and enzyme concentration on release of fermentable sugar from agave bagasse.

25

A Concentration (g/L)

20

15

10

5

0 Glucose

Xilose

Arabinose

TM

25

B

3.4. Succinic acid production under batch fermentation with A. succinogenes free cells on agave bagasse hydrolyzates

Concentration (g/L)

20

15

10

5

0 Glucose

pretreatment for fermentation the concentration of sugars in the hydrolyzates from sulfuric acid decreased in about 43% to 11.8 g/L. Similarly, the concentration of sugars from the enzymatic hydrolyzates also decreased about 25% after sterilization. In the case of acid hydrolyzates, part of the sugar loss might be attributed to some retention of sugars in the ion exchange columns. Sterilization of both acid and enzymatic hydrolyzates might have promoted formation of new chemical compounds at high temperature/ moderate pressure, which precipitated and colored the fermentation media (so called Klason lignin). In spite of sugar losses, pretreatment of acid hydrolyzates by anion exchange cannot be avoided due to the low pH conditions that make impossible the fermentation, as well as the need to sterilize the media, which is essential for fermentation. However, the enzymatic hydrolysis is more adequate for this type of fermentation because it does not require exchanging ions and resulted in a higher concentration of glucose and other fermentable sugars.

Xilose

Arabinose

TM

Fig. 3. (A) Concentration of carbohydrates obtained at optimal conditions for hydrolysis of agave bagasse; (B) Concentration of carbohydrates after ion-exchange for fermentation to produce succinic acid. Symbols represent hydrolysis with H2SO4 (h), HCl ( ) and enzymatic (j), TM: total monosaccharides.

sterilization. Optimal conditions showed that hydrochloric acid produced the higher concentration of fermentable sugars (22.6 g/L from 100 g of agave bagasse), however, when the hydrolyzates were ion exchanged and sterilized, the concentration of fermentable sugars decreased about 32% to 15.5 g/L. After

Fermentations with A. succinogenes were carried out both with the enzymatic and the acid hydrolyzates and also with a synthetic medium. The initial concentrations of monosaccharides can be seen in Fig. 3B. Consumption of monosaccharides was similar, regardless the type of hydrolysis (see Supplementary material Fig. S1). Glucose from all hydrolyzates was consumed faster followed by xylose and then arabinose. Glucose from the enzymatic extract showed a degradation profile similar to the degradation profile obtained from the synthetic medium and its consumption was complete (see Supplementary material Fig. S1). Arabinose consumption was slightly faster in the enzymatic hydrolyzates, because the acid hydrolyzates might have inhibitory compounds. The total consumption of sugars was completed in 25 h (Fig. S1) although the stationary phase for production of acids was reached at up to 18 h (Fig. 4). The production of succinic, acetic and formic acid can be seen in Fig. 4. The productivity (rS = 0.573 g/Lh Table 3) and production of succinic acid (Fig. 4A,) were higher and faster with the enzymatic hydrolyzates, although the final concentration of succinic acid was higher in the synthetic medium. This can be due to the fact

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

8

A

Succinic acid (g/L)

7 6 5 4 3 2 1 0 0

5

10 15 Time (h)

20

25

8

B

Formic acid (g/L)

7 6 5 4

they are composed mainly of xylose and arabinose (pentoses) that need to enter to the pentose-phosphate pathway. The production and productivity of succnic acid were higher with hydrolyzates from the hydrochloric acid as compared to the sulfuric acid hydrolyzates due to a higher initial concentration of monosaccharides (36% higher) and probably an inhibitory effect of residual compounds as seen before with corn stover hydrolyzates (Chen et al., 2010). Fig. 4B and C show that the production of formic and acetic acids followed the same trend as the production of succinic acid, i.e. the enzymatic hydrolyzates promoted a faster production of these acids and their final concentration was similar to the synthetic medium but higher as compared to the acid hydrolyzates. The yields for the produced acids (succinic, formic and acetic) were similar for the enzymatic hydrolyzates and the synthetic medium and slightly lower for both acid hydrolyzates (Table 3). This study shows that A. succinogenes is able to utilize the sugars from the hydrolysis of agave bagasse and that to obtain hydrolyzates by enzymatic means is a great alternative, because a higher concentration of substrate can be obtained, there is no need to pretreat such hydrolyzates before fermentation, and by applying this methodology the production and productivity of succinic acid was higher. 3.5. Succinic acid production with immobilized A. succinogenes by fermentation in repeated batches

3 2 1 0 0

8

5

10

15 Time (h)

20

25

5

10 15 Time (h)

20

25

C

7 6 Acetic acid (g/L)

21

5 4 3 2 1 0 0

Fig. 4. Production of (A) succinic acid, (B) formic acid, and (C) acetic acid in batch fermentations with A. succinogenes from hydrolyzates of: H2SO4 (N), HCl (d), enzymatic (j) and a synthetic medium (r).

that even if the initial concentration of monosaccharides is similar in all fermentations, the synthetic medium is composed only of glucose, the enzymatic hydrolyzates contain mainly glucose and the acid hydrolyzates contain xylose as the main component. It has been found that a higher initial concentration of glucose (in the range 5–30 g/L) increased the concentration and productivity of succinic acid (Corona-González et al., 2008). Furthermore, the natural ability of this microorganism to convert glucose to succinic acid by glycolysis explains the higher production with enzymatic and synthetic medium hydrolyzates, which contain glucose in higher amounts. On the other hand, productivity of succinic acid with acid hydrolyzates was lower, probably due to the fact that

It has been shown that a high initial concentration of substrate (30 g/L) favors the production of succinic acid with A. succinogenes (Corona-González et al., 2008), which can be achieved by evaporation of water from the hydrolyzates. However, water evaporation also increases the concentration of undesirable inhibitory compounds (Xi et al., 2013). A better alternative is to implement repeated batch fermentations with immobilized bacteria, which can increase succinic acid production and productivity. In this case the enzymatic hydrolyzates were utilized in a 5 consecutive batches with immobilized A. succinogenes in agar (CoronaGonzález et al., 2014). Fig. 5 shows monosaccharide (glucose, xylose and arabinose) consumption, production of acids, yield and also the productivity of succinic acid in each batch. During the first batch, the consumption of monosaccharides was similar to fermentations with free cells, i.e. a 5 h lag phase and the time to halt consumption at 11 h. In the following batches the fermentation time was reduced to half because the adaptation time of the cells was suppressed. However, it is worth to point out that the consumption of xylose and arabinose was not complete (residual sugar concentration 3–4 g/L). The production of acids is also depicted in Fig. 5. It can be observed that the production of acids and the consumption of sugars were not coupled because the production of acids reached the stationary phase before the consumption of sugars stopped, because this microorganism prefers to feed on glucose. The yield of succinic acid in each batch was around 0.44 g/g even if in each consecutive batch the production was slightly higher, which was owed to a higher initial sugar concentration. Productivity, in the other hand, was increased 2.5-fold, reducing the time of acid production. The yield for production of formic acid and acetic acid in this regime was in average 0.14 g/g and 0.32 g/g respectively. It was observed that the production of acetic acid increased with each fermentation batch, whereas formic acid production was kept constant as observed in synthetic medium (Corona-González et al., 2014). This fact could be owed to alterations in the intracellular redox potential for ATP synthesis (Barbirato et al., 1997), because formation of one mole of succinate requires 2 mol of NADH (Cheng et al., 2012). It was also observed that after 5 repeated batches the cells continued viable and had the capacity to produce

22

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23

Table 3 Yields, productivities, acids production and initial substrate concentration from fermentations with A. succinogenes using acid or enzymatic hydrolyzates of agave bagasse. Substrate

SM

EH

SH

HH

YS/M (g/g) YA/M (g/g) YF/M (g/g) rS (g/L-h) rA (g/L-h) rF (g/L-h) Succinic acid (g/L) Formic acid (g/L) Acetic acid (g/L) Monosaccharides (g/L)1

0.451 ± 0.012 0.210 ± 0.006 0.207 ± 0.009 0.524 ± 0.018 0.159 ± 0.04 0.241 ± 0.07 6.7 ± 0.15 3.3 ± 0.10 3.3 ± 0.12 14.6 ± 0.16

0.446 ± 0.015 0.262 ± 0.021 0.344 ± 0.012 0.573 ± 0.022 0.379 ± 0.044 0.467 ± 0.038 5.2 ± 0.18 3.8 ± 0.23 3.2 ± 0.37 14.5 ± 0.42

0.398 ± 0.042 0.188 ± 0.005 0.176 ± 0.012 0.316 ± 0.022 0.141 ± 0.024 0.138 ± 0.018 4.6 ± 0.19 1.9 ± 0.12 2.4 ± 0.42 11.4 ± 0.28

0.341 ± 0.052 0.196 ± 0.036 0.175 ± 0.019 0.248 ± 0.034 0.150 ± 0.045 0.196 ± 0.032 5.2 ± 0.35 3.3 ± 0.017 3.1 ± 0.22 15.5 ± 0.19

SM: synthetic medium, EH: enzymatic hydrolyzates, SH: sulfuric acid hydrolyzates, HH: hydrochloric acid hydrolyzates 1 Monosaccharides concentration represents the total amount of initial glucose, xylose and arabinose.

r S (g/L-h) Y X/S (g/g)

0.49 0.43

20

1

18

1.13 0.45

1.33 0.44

1.26 0.45

1.28 0.46

2

3

4

5

Concentration (g/L)

16 14 12 10 8 6 4 2 0 0

10

20

30

40

Formic acid

Acetic acid

Time (h) Monosaccharides

Succinic acid

Fig. 5. Succinic acid production in repeated batch fermentations by immobilized A. succinogenes from enzymatic hydrolyzates of agave bagasse.

acids. Under repeated batch conditions we obtained 33.6 g/L succinic acid in 40 h from 87.2 g/L total monosaccharides and 18.7 g/L residual sugars, which is an attractive methodology as compared to previous works where lignocellulosic waste was used (Li et al., 2010, 2011; Zheng et al., 2009; Jiang et al., 2013; Ribeiro-Borges and Pereira, 2011; Chen et al., 2010).

Acknowledgements This work was supported by Fondo Mixto CONACYT-Gobierno del Estado de Jalisco, México (Project No. 2012-07/190100).

Appendix A. Supplementary data 4. Conclusions It was found that by applying mild acid hydrolyses or an enzymatic preparation on agave bagasse, furfural and/or HMF were not found in the obtained hydrolyzates. This fact is advantageous to avoid inhibitory effect on the microorganism used to produce succinic acid. This contribution demonstrates that it is feasible to produce succinic acid with the enzymatic hydrolyzates from agave bagasse by using immobilized cells under repeated batch fermentation. Simultaneous saccharification and fermentation studies are on progress to assess the simultaneous enzymatic saccharification and fermentation of agave bagasse for production of succinic acid.

Competing interests The authors declare that they have no competing interests.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.12. 081.

References Arreola-Vargas, J., Ojeda-Castillo, V., Snell-Castro, R., Corona-González, R.I., Alatriste-Mondragón, F., Méndez-Acosta, H.O., 2015. Methane production from acid hydrolysates of Agave tequilana bagasse: evaluation of hydrolysis conditions and methane yield. Bioresour. Technol. 181, 191–199. Barbirato, F., Chedaille, D., Bories, A., 1997. Propionic acid fermentation from glycerol: comparison with conventional substrates. Appl. Microbiol. Biotechnol. 47, 441–446. Caspeta, L., Caro Bermudez, M.A., Ponce-Noyola, T., Martínez, A., 2014. Enzymatic hydrolysis at high-solids loadings for the conversión of agave to fuel etanol. Appl. Energy 113, 277–286. Chen, K., Zhang, H., Miao, Y., Jiang, M., Chen, J., 2010a. Succinic acid production from enzymatic hydrolysate of sake lees using Actinobacillus succinogenes 130Z. Enzyme Microb. Technol. 47, 236–240.

R.I. Corona-González et al. / Bioresource Technology 205 (2016) 15–23 Chen, K., Jiang, M., Wei, P., Yao, J., Wu, H., 2010b. Succinic acid production from acid hydrolysate of corn fiber by Actinobacillus succinogenes. Appl. Biochem. Biotechnol. 160, 477-48. Cheng, K.K., Zhao, X.B., Zeng, J., Zhang, J.A., 2012. Biotechnological production of succinic acid: current state and perspectives. Biofuels Bioprod. Biorefin. 6, 302–318. Clavijo-Díaz, A., 2002. Fundamentos de química analítica: equilibrio iónico y análisis químico. Universidad Nacional de Colombia, Bogotá, Primera Edición. Corona-González, R.I., Bories, A., Gonzalez-Alvarez, V., Pelayo-Ortiz, C., 2008. Kinetic study of succinic acid production by Actinobacillus succinogenes ZT-130. Process Biochem. 43, 1047–1053. 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. CRT (Consejo Regulador del Tequila). Estadísticas. Consejo Regulador del tequila. 17 jul 2012http://www.crt.org.mx/EstadisticasCRTweb/, 2012. Davis, S.C., Dohleman, F.G., Long, S.P., 2011. The global potential for Agave as a biofuel feedstock. GCB Bioenergy 3, 68–78. 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. Jiang, M., Xu, R., Xi, Y.L., Zhang, J.H., Dai, W.Y., Wan, Y.J., Chen, K.Q., Wei, P., 2013. Succinic acid production from cellobiose by Actinobacillus succinogenes. Bioresour. Technol. 135, 469–474. Kikani, B.A., Pandey, S., Singh, S.P., 2013. Immobilization of the a-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance. Bioprocess. Biosyst. Eng. 36, 567–577. Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P., 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48, 3713–3729. Li, Q., Yang, M.H., Wang, D., Li, W.L., Wu, Y., Zhang, Y.J., Xing, J.M., Su, Z.G., 2010. Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes. Bioresour. Technol. 101, 3292–3294.

23

Li, J., Zheng, X.Y., Fang, X.J., Liu, S.W., Chen, K., Jiang, M., Wei, P., Ouyang, P.K., 2011. A complete industrial system for economical succinic acid production by Actinobacillus succinogenes. Bioresour. Technol. 102, 6147–6152. Lu, X.B., Zhang, Y.M., Yang, J., Liang, Y., 2007. Enzymatic hydrolysis of corn stover after pretreatment with dilute sulfuric acid. Chem. Eng. Technol. 30 (7), 938– 944. Pérez-Pimienta, J.A., Lopez-Ortega, M.G., Varanasi, P., Stavila, V., Cheng, G., Singh, S., Simmons, B.A., 2013. Comparison of the impact of ionic liquid pretreatment on recalcitrance of agave bagasse and switchgrass. Bioresour. Technol. 127, 18–24. Ribeiro-Borges, E., Pereira, N., 2011. Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by Actinobacillus succinogenes. J. Ind. Microbiol. Biotechnol. 38, 1001–1011. Saucedo-Luna, J., Castro-Montoya, A., Martínez-Pacheco, M., Sosa-Aguirre, C., Campos-García, J., 2011. Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave tequilana bagasse to produce ethanol by Pichia caribbica. J. Ind. Microbiol. Biotechnol. 38, 725–732. Song, H., Lee, S.Y., 2006. Production of succinic acid by bacterial fermentation. Enzyme Microb. Technol. 39, 352–361. Urbance, S.E., Pometto, A.L., DiSpirito, A.A., Denli, Y., 2004. Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors. Appl. Microbiol. Biotechnol. 65, 664–670. Van der Werf, M.J., Guettler, M.V., Jain, M.K., Zeikus, J.G., 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167, 332–334. Xi, Y.I., Chen, K., Dai, W., Ma, J., Zhang, M., Jiang, M., Wei, P., Ouyang, P.K., 2013. Succinic acid production by Actinobacillus succinogenes NJ113 using corn steep liquor powder as nitrogen source. Bioresour. Technol. 136, 775–779. Zeikus, J.G., Jain, M.K., Elankovan, P., 1999. Biotechnology of succinic acid production and markets for derived industrial products. Appl. Microbiol. Biotechnol. 51, 545–552. Zheng, P., Dong, J., Sun, Z.H., Ni, Y., Fang, L., 2009. Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes. Bioresour. Technol. 100, 2425–2429.