Accepted Manuscript Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis Dong-Hyun Kim, Sang-Bum Lee, Gwi-Taek Jeong PII: DOI: Reference:
S0960-8524(14)00383-6 http://dx.doi.org/10.1016/j.biortech.2014.03.078 BITE 13209
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
16 January 2014 11 March 2014 16 March 2014
Please cite this article as: Kim, D-H., Lee, S-B., Jeong, G-T., Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis, Bioresource Technology (2014), doi: http://dx.doi.org/ 10.1016/j.biortech.2014.03.078
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1 2
Production of reducing sugar from Enteromorpha intestinalis
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by hydrothermal and enzymatic hydrolysis
4 5
Dong-Hyun Kim, Sang-Bum Lee, Gwi-Taek Jeong*
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Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea
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The two authors equally contributed to this work.
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*Corresponding author: Gwi-Taek Jeong
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Department of Biotechnology,
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Pukyong National University, Busan 608-737, South Korea
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Tel: +82-51-629-5869
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Fax: +82-51-629-5863
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E-mail: [email protected]
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1
Abstract
2
In this work, to evaluate the efficacy of marine macro-algae Enteromorpha intestinalis as a
3
potential bioenergy resource, the effects of reaction conditions (solid-to-liquid ratio, reaction
4
temperature, and reaction time) on sugars produced by a combined process of hydrothermal
5
and enzymatic hydrolysis were investigated. As a result of the hydrothermal hydrolysis, a 7.3
6
g/L (8% yield) total reducing sugar was obtained under conditions including solid-to-liquid
7
ratio of 1:10, reaction temperature of 170°C, and reaction time of 60 min. By subsequent
8
(post-hydrothermal) enzymatic hydrolysis of samples treated at 170°C for 30 min, a 20.1 g/L
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(22% yield) was achieved.
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Keywords : Enteromorpha intestinalis, hydrothermal treatment, enzymatic hydrolysis, total
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reducing sugar, severity factor, bioenergy
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2
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1. Introduction
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The requirement for fossil fuel alternatives has received significant attention. Indeed, the
3
discovery and utilization of new resources that can replace fossil resources is emerging as a
4
major global sustainability issue (Adams et al., 2009; Lee et al., 2011). Biomass use in
5
industrial applications is spreading around the world. As utilized, biomass typically is
6
categorized into three generations: starch-based (first generation), wood-based (second
7
generation), and marine seaweed (third generation) (Jeong and Park, 2010; Meinita et al.,
8
2012, 2013). Marine biomass is attracting increasing worldwide interest for its potential as an
9
environmental-friendly and economically sustainable resource. Marine algae have a number
10
of advantages over other resources. They do not compete with food resources; also, given
11
their limited lignin and cellulose contents compared with starch-based or lignocellulosic
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materials, their pretreatment and saccharification is simple and easy (Goh and Lee, 2010; Lee
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et al., 2011). And this is not to mention the fact that most of algae grow faster than land plants
14
and offer higher productivity per cultivation area. These important advantages
15
notwithstanding, algae has yet to be applied as a raw material in chemical industry
16
applications (Adams et al., 2009; Lee et al., 2011). Micro-algae cultivation and its application
17
are occupied majority of algae field. On the other hand, a few studies has been done or
18
reported on biofuel- or chemical production from macro-algae (Adams et al., 2009; Jeong and
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Park, 2010; Meinita et al., 2012, 2013).
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Macro-algae are classified as green-algae, red-algae or brown-algae. These algae live in
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shallow seas and on coastal rock (Blomster et al., 2002; Kim, 2010). Green-algae are
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composed of cellulose, mannose and xylan as the cell-wall carbohydrates, and starch as the
23
storage carbohydrate. Unlike the two other macro-algae species, it has a high cellulose
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content in the cell wall. Moreover, it compares favorably with land plants in its similar
25
amounts of photosynthetic-pigment carotene and xanthophylls (Feng et al., 2011; Kim, 2010; 3
1
Suganya et al., 2013). The Enteromorpha genus, a green macro-algae belonging to the
2
phylum chlorophyta and the family ulvaceae, comprises E. intestinalis, E. prolifera, E. linza,
3
and E. compressa (Blomster et al., 2002; Suganya et al., 2013). It is distributed widely in
4
inter-tidal zones and on coastal rocks worldwide (Feng et al., 2011; Suganya et al., 2013).
5
Enteromorpha have many polysaccharides containing large amounts of glucose, xylose, D-
6
glucuronic acid, and L-rhamnose (Feng et al., 2011). These polysaccharides are a great
7
potential resource for biofuel and chemical production.
8
For conversion of biomass into biofuels or chemicals, its hydrolysis is essential except for
9
combustion, incineration, pyrolysis, gasification, solubilization and so on (Feng et al., 2011).
10
Pretreatment, thus, is one of the core processes in the bioconversion of biomass (Jeong et al.,
11
2013; Kang et al., 2013). Pretreatment reduces the structural and compositional obstacles of
12
biomass in order to maximize enzymatic conversion to mono-sugars. Many pretreatment
13
processes for enzymatic hydrolysis (degradation) of biomass are applied; they include
14
hydrothermal hydrolysis, steam explosion, acid, alkaline, and ammonia fiber expansion, as
15
well as soaking in aqueous ammonia and inorganic salts (Kang et al., 2013; Nitsos et al.,
16
2013). Actually, some work on the pretreatment of macro-algae already has been conducted
17
(Feng et al., 2011; Jeong and Park, 2011; Lee et al., 2011; Meinita et al., 2012, 2013; Park
18
and Jeong, 2013; Yoon et al., 2011). The proper pretreatment process is a function of the
19
biomass characteristics, catalysts, operation factors, and strength (Kang et al., 2013; Jeong et
20
al., 2013; Meinita et al., 2012). During pretreatment, numerous degradation products such as
21
5-HMF, levulinic acid, formic acid, furfural, and phenolic compounds, which can inhibit cell
22
growth, are formed under severe operation conditions (Kang et al., 2013; Jeong et al., 2013;
23
Meinita et al., 2012). Because hydrothermal pretreatment does not involve expensive or
24
hazardous chemicals, it is simple, safe, economic, and environmentally friendly (Nitsos et al.,
25
2013). 4
1
In the present study, for the purposes of minimizing inhibitor formation and enhancing
2
mono-sugar recovery, a combined process of hydrothermal and enzymatic hydrolysis was
3
evaluated. Biomass pretreatment efficiencies in terms of reaction conditions were evaluated
4
and compared on the basis of the severity factor (log (R0)) as a function of pretreatment
5
temperature and time (Brosse et al., 2010), which approach is widely applied to the
6
evaluation and comparison of lignocellulosic pretreatments (Brosse et al., 2010; Pedersen et
7
al., 2010). The overall objective was to determine the potential of marine macro-algae
8
Enteromorpha intestinalis as a bioenergy resource.
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2. Materials and methods
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2.1. Materials
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Dried Enteromorpha intestinalis was harvested in Jindo (Jeonnam, Korea) in October 2010.
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The dried biomass was pulverized in disintegrator until it passed through a 140 mesh sieve
14
and kept in a sealed bag at room temperature. E. intestinalis is composed of 42.8% total
15
carbohydrate, 31.6% crude protein, 1.3% crude lipid and 24.3% crude ash. Viscozyme L and
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Cellic® CTec2 were obtained from Novozymes A/S (Denmark). All of the other chemicals
17
were of analytical grade.
18 19
2.2. Batch hydrothermal experiment procedure
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Preparatory to a batch hydrothermal reaction, a certain quantity of biomass and 40 mL of
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distilled water were filled into a stainless steel reactor of 50 mL size. The reaction
22
temperature (130-210°C) was controlled by means of an oil bath equipped with a PID
23
temperature controller and magnetic stirring of approximately 200 rpm. After completion of
24
the reaction, the reactor was quickly cooled to room temperature using tap water (Jeong and
25
Park, 2011). The pretreated biomass solution was used in subsequent analyses of total 5
1
reducing sugar (TRS) and enzymatic hydrolysis.
2 3
2.2.1. Effect of solid-to-liquid ratio
4
In order to investigate the effect of the solid-to-liquid ratio (S/L ratio) on the production of
5
TRS from E. intestinalis, an S/L ratio experiment (range: 1:5 ~ 1:20; mass per volume) was
6
conducted. The liquid amount was fixed at 40 mL, and designed amounts of solid (biomass)
7
were added at different S/L ratios. The hydrothermal hydrolysis was conducted to obtain TRS
8
under 170°C reaction temperature and 30 or 60 min reaction time conditions.
9 10
2.2.2. Effects of reaction temperature
11
In order to investigate the effect of reaction temperature on TRS production under the
12
condition of an S/L ratio of 1:10 (Jang et al., 2012; Kang et al., 2013; Meinita et al., 2012),
13
hydrothermal hydrolysis was conducted to obtain TRS at reaction temperatures ranging from
14
130 to 210°C and a 30 or 60 min reaction time.
15 16
2.3. Effect of enzymatic hydrolysis
17
In order to investigate the effect of enzymatic hydrolysis on TRS production, the biomass
18
solution pretreated by hydrothermal hydrolysis between 130 and 210°C was mixed with 10%
19
enzyme mixture (Viscozyme L and Cellic CTec2 = 1:1, v/v) based on biomass weight in a
20
flask, and then reacted in a 170 rpm shaking incubator at 45°C for 48 hr (Song et al., 2011).
21
Upon completion of the enzymatic hydrolysis, supernatant samples were obtained for TRS
22
analysis by 4,000 rpm centrifugation for 15 min.
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2.4. Definition of severity factor The hydrothermal pretreatment was evaluated according to the severity factor, which 6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
represents the pretreatment severity as a function of pretreatment temperature and time
2
(Brosse et al., 2010; Pedersen et al., 2010). Severity factor is log (R0).
3
,
4
where t is the pretreatment time (min), T(t) is the pretreatment temperature (°C), 100 is the
5
reference temperature, and 14.75 is the fitted value of the arbitrary constant.
6 7
2.5. Analysis method
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TRS yields were determined by the modified 3,5-dinitrosalicylic acid (DNSA) method
9
(Miller, 1959). Typically, 200 μL of properly diluted sample and 3 mL of DNS reagent were
10
mixed and heated in a boiling water bath for 5 min, then cooled immediately in a cold-water
11
bath. Upon completion of the reaction, the absorbance was measured by spectrophotometry
12
(Spekol 1300, Analytik Jena, Germany) at 540 nm, using glucose as the standard. The TRS
13
yield was calculated as the produced TRS concentration per initial biomass concentration.
14 15
3. Results and discussion
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3.1. Production of total reducing sugar by hydrothermal reaction
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Hydrothermal pretreatment/hydrolysis utilizes water with or without acid/alkali as the
18
solvent at between 120 and 230°C. This range is beneficial to hydrolytic reactions of
19
biomass; at the same time, it promotes the formation of sugar degradation products. In such
20
reactions, the operating factors and their values are carefully selected to maximize sugar
21
contents and enzymatic digestibility (Nitsos et al., 2013). In this work, the production of TRS
22
from E. intestinalis by hydrothermal hydrolysis was evaluated. The experimental reaction
23
factors included the S/L ratio, reaction temperature, and reaction time.
24 25
7
1 2
3.1.2. Effect of solid-to-liquid ratio
3
In order to investigate the effect of the S/L ratio on the production of TRS from E.
4
intestinalis, ratios within the 1:5 - 1:20 range were tested. Fig. 1A shows the TRS produced
5
under the 170°C, 60 min condition. With increasing S/L ratio, the TRS decreased. The 30
6
min reaction showed a decreasing TRS pattern similar to that for 60 min. With an S/L ratio of
7
1:5, an approximately 11.8 g/L TRS yield was obtained to 60 min. For comparison, Fig. 1B
8
plots the TRS yields for different S/L ratios. After 30 min reaction time, the highest yield was
9
obtained at S/L ratios between 1:7.5 and 1:10. However, increasing the S/L ratio beyond 1.10
10
caused a slight decrease in yield. For the 60 min reaction time, the yield increased with
11
increasing S/L ratio until 1:10, whereas at ratios over 1:10, the yields were relatively constant.
12
At the 1:10 S/L ratio, the TRS yield was 8.26%. At lower S/L ratios, despite the high TRS
13
concentrations, the yields were relatively low. As a similar results, Jang et al. (2012) reported
14
that in a conventional simultaneous saccharification and fermentation processes of
15
Saccharina japonica, solid content (S/L ratio) was usually limited to 10% (w/v) due to the
16
high viscosity, which caused the difficulty to handle the slurry. Also, the optimum
17
Kappaphycus alvarezii content for both H2SO4 and HCl hydrolysis were 10% (Meinita et al.,
18
2012). In light of all of these results, the 1:10 S/L ratio was applied in further experiments.
19 20
3.1.2. Effect of reaction temperature
21
In order to investigate the effect of reaction temperature on TRS production under the
22
condition of a 1:10 S/L ratio, the TRS produced, the pH change and the solid residue were
23
analyzed after hydrothermal hydrolysis under reaction temperatures ranging from 130 to
24
210°C. Fig. 2A shows the TRS concentrations produced at different reaction temperatures. At
25
the lowest reaction temperature, 130°C, the TRS concentration fell within the 1.57-1.72 g/L 8
1
range. With increasing reaction temperature, the TRS also was increased. With a reaction
2
time of 30 min, the highest TRS, 7.16 g/L, was produced at 190°C. For the 60 min reaction
3
time, the highest TRS, 7.3 g/L, was obtained at 170°C. Under the 190°C temperature, the
4
TRS concentration was decreased from 7.16 g/L (at 30 min) to 4.37 g/L (at 60 min). It is
5
estimated that decomposition (or dehydration) of sugars, which, again, results in low TRS
6
concentrations, occurs at 210°C. This indicated that over-decomposition of TRS was incurred
7
by its long-duration exposure to high temperature. In fact, it has been reported that in the
8
acidic hydrolysis of lignocellulosics or marine algae, such exposure results in over-
9
dehydration (i.e. over-decomposition) of sugars which is formed by-products such as 5-HMF,
10
levulinic acid, formic acid and char (Jeong and Park, 2010; Meinita et al., 2012, 2013). Kim
11
(2010) reported the effect of hydrothermal and microwave pretreatment on the production of
12
reducing sugar from Ulva pertusa. In hydrothermal pretreatment at 100-150°C, the highest
13
value was obtained at 150°C. In the case of the microwave pretreatment, the effect was more
14
highly influenced by the pretreatment temperature than by the microwave power (50W,
15
100W). However, Yoon et al. (2011) reported that their highest glucose yield was obtained by
16
pretreatment of U. pertusa with 5% hydrogen peroxide at 60°C for 3 h. Interestingly, Lee et
17
al. (2011) reported bioethanol production from Pichia stipitis using 30 g/L hydrolysate
18
obtained by condensation after 190°C, 15 min hydrolysis of 100 g/L of U. pertusa. Its main
19
sugar is glucose and xylose.
20
Fig. 2B plots the TRS yields for different reaction temperatures. The yield was
21
proportional to the TRS concentration produced. Under the 170°C, 60 min condition, an 8.0%
22
yield obtained; however, the yield was only 7.87% under the 190°C, 30 min condition. Fig.
23
2C indicates the pH change at different reaction temperatures after hydrothermal hydrolysis.
24
The pH of the reactant before the hydrothermal hydrolysis was 5.3; after the reaction, the pH
25
was decreased under all of the tested conditions. The lowest pH was 4.05, at 190°C. This low 9
1
pH promoted the hydrolysis of E. intestinalis without the addition of a catalyst. Also, it is
2
estimated that dehydrated products such as levulinic acid, formic acid and others, formed by
3
high reaction temperature, might decrease the pH of a product. On the other hand, the pH
4
increase at 210°C is estimated that the condensation products (humins and other byproducts)
5
formed from dehydrated products (5-HMF, levulinic acid and formic acid) by high
6
temperature, might increase the pH of solution (Deng et al., 2014; Shi et al., 2013; Wang et
7
al., 2013). Fig. 2D shows the amounts of solid residue after hydrothermal hydrolysis at
8
different reaction temperatures. As can be seen, the solid residue was linearly decreased by
9
reaction temperature increases. Over the course of the 30 and 60 min reaction durations, the
10
remaining solid residue decreased with increasing reaction time.
11 12
3.2. Production of total reducing sugar by post-hydrothermal enzymatic hydrolysis
13
In order to investigate the effect of enzymatic treatment, the biomass solution treated by
14
hydrothermal hydrolysis at 130-210°C for 30 min (see Fig. 2) was again hydrolyzed with the
15
enzyme mixture (Viscozyme L and Cellic® CTec2 = 1:1, v/v) in a 170 rpm shaking incubator
16
at 45°C for 48 hr (Fig. 3). Fig. 3A plots the results. It can be seen that initially, the TRS
17
concentration increased with increasing reaction time. The highest TRS was obtained at
18
170°C, and then 20.1 g/L TRS was produced by 48 hr enzymatic hydrolysis. For high
19
reaction temperatures in the post-hydrothermal enzymatic hydrolysis, overall low TRS
20
concentrations were obtained. Comparing enzymatic hydrolysis with hydrothermal hydrolysis
21
(see Fig. 2A) for low reaction temperatures, it is apparent that more TRS was obtained by
22
enzymatic hydrolysis. As shown in Fig. 2D, it was estimated that due to the large amount of
23
solid residue remaining at low reaction temperatures, the TRS concentration was increased by
24
the enzymatic hydrolysis. In other words, the amount of TRS produced by enzymatic
25
hydrolysis was proportional to the amount of solid residue remaining after the initial 10
1
hydrothermal hydrolysis. Fig. 3B plots the results of 48 hr enzymatic hydrolysis using
2
biomass solution obtained from the hydrothermal hydrolysis at 130-210°C for 60 min. By the
3
24 and 48 hr enzymatic hydrolysis at low reaction temperatures, the TRS yield increased with
4
increasing reaction time. The highest TRS yield, 20.1 g/L, was obtained at 130°C. This is a
5
higher value than the enzymatic hydrolysis result after hydrothermal hydrolysis at 130°C for
6
30 min (Fig. 3A). It was demonstrated that the solid residue remaining after low-temperature
7
hydrothermal hydrolysis can easily be hydrolyzed in subsequent enzymatic hydrolysis. As
8
shown in Fig. 3A, with increasing reaction temperature, a low TRS yield was obtained.
9
Whereas, the TRS yield (IA after EA) firstly increased and then decreased again when
10
comparing 24 and 48 hr enzymatic hydrolysis of substrate pretreated at 170 to 210 °C for 60
11
min (Fig. 3B). This phenomenon is estimated that the enzymatic hydrolysis
12
inhibited by dehydration byproducts (5-HMF, levulinic acid, formic acid) and
13
condensation byproduct (humans, and other products) (Feng et al., 2012). Kim
14
(2010), after a comparable study, reported the production of reducing sugar from U. pertusa
15
by microwave pretreatment and enzymatic hydrolysis. In their enzymatic hydrolysis, 10%
16
higher reducing sugar yield was produced in the case of an enzyme mixture of α-amylase,
17
cellulose, and β-glucosidase than in the case of only α-amylase. Also, Jung et al. (2012)
18
reported on acid-pretreatment conditions for enzymatic hydrolysis of U. pertusa. Their
19
hydrolysis rate, 58.8%, was achieved with 0.1M HCl at a pretreatment temperature of 121°C
20
and a 15 min reaction time, as followed by enzymatic hydrolysis using a 2:1 mixture of
21
Viscozyme and xylanase.
22 23 24
3.3. Effect of severity factor The effect of the severity factor on TRS production in hydrothermal and enzymatic
11
1
hydrolysis of E. intestinalis was investigated (Fig. 4). The severity factor is the combined
2
reaction temperature and reaction time in hydrothermal hydrolysis (pretreatment). In the
3
hydrothermal hydrolysis (pretreatment), the TRS concentration linearly increased to the
4
severity factor of 3.84. Over 4.1, the TRS concentration decreased. The hydrothermal
5
reaction conditions at severity factors between 3.6 and 4.1 were 190-210°C and 30-60 min.
6
This result indicated that the higher severity factor can cause the over-degradation of
7
carbohydrates, as shown in Fig. 3A. The severity factors of post-hydrothermal enzymatic
8
hydrolysis are calculated based on the conditions of hydrothermal hydrolysis (pretreatment).
9
In the post-hydrothermal enzymatic hydrolysis, the TRS concentration was maintained at
10
nearly 20 g/L to the severity factor of 3.84. Over 3.84, it sharply decreased. As shown in Fig.
11
3A, with increasing pretreatment temperature and time, the severity factor increased, until the
12
severe conditions incurred a decrease in the TRS concentration. Moreover, the solid residue
13
remaining at the low severity factors prevailing during the hydrothermal reaction can easily
14
be hydrolyzed in enzymatic hydrolysis.
15 16
4. Conclusions
17
In this work, the potential of E. intestinalis as a bioenergy resource was investigated. The
18
TRS was produced by combined process of hydrothermal and enzymatic hydrolysis. By
19
hydrothermal hydrolysis, 7.3 g/L of TRS was produced under 1:10 S/L ratio, 170°C and 60
20
min. By post-hydrothermal enzymatic hydrolysis, 20.1 g/L of TRS was produced at samples
21
of 170°C and 30 min. Also, higher severity factor can cause the over-degradation of
22
carbohydrates in hydrothermal hydrolysis. These results will provide basic information
23
applicable to further studies on the use of E. intestinalis for bioenergy production.
24 25
Acknowledgements 12
1
This research was supported by Basic Science Research Program through the National
2
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
3
Technology (2012R1A1A2006718).
4
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marine macroalgae Enteromorpha compressa by two step process: Optimization and
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kinetic study. Bioresource Technology 128, 392-400.
20
26. Wang, H., Deng, T., Wang, Y., Qi, Y., Hou, X., Zhu, Y., 2013. Efficient catalytic system
21
for the conversion of fructose into 5-ethoxymethylfurfural. Bioresource Technology 136,
22
394-400.
23 24
27. Yoon, B.T., Kim, Y.W., Chung, K.W., Kim, J.S., 2011. Enzymatic hydrolysis of pretreated Ulva pertusa with alkaline peroxide. Appl. Chem. Eng. 22(3), 336-339.
15
1
Figure Legends
2 3 4
Fig. 1. Effect of solid-to-liquid (S/L) ratio on hydrothermal hydrolysis of E. intestinalis. (A) Total reducing sugar (TRS), (B) Yield
5 6 7
Fig. 2. Effects of reaction temperature and time on hydrothermal hydrolysis of E. intestinalis. (A) TRS, (B) Yield, (C) pH, (D) Solid residue
8 9
Fig. 3. Post-hydrothermal enzymatic hydrolysis of E. intestinalis hydrolysate. (A) After 30
10
min hydrothermal pretreatment; (B) After 60 min hydrothermal pretreatment; EH
11
(Enzymatic Hydrolysis); IA after EH (Increasing Amount after Enzymatic
12
Hydrolysis)
13 14 15
Fig. 4. Effect of severity factor on TRS production by hydrothermal hydrolysis and posthydrothermal enzymatic hydrolysis of E. intestinalis.
16 17 18
16
14 30 min 60 min
Total reducing sugar (g/L)
12
10
8
6
4
2
0 5
10
15
20
Solid-to-liquid ratio (-)
(A)
10 30 min 60 min
Yield (%)
8
6
4
2
0 5
10
15
Solid-to-liquid ratio (-)
(B)
Fig. 1.
17
20
10
8
30 min 60 min
30 min 60 min
Yield (%)
Total reducing sugar (g/L)
8 6
4
6
4
2 2
0
0 130
150
170
190
130
210
150
170
190
210
Reaction temperature (oC)
Reaction temperature (oC)
(A)
(B)
100 30 min 60 min
30 min 60 min
6
Solid residue (%)
80
pH
4
60
40
2 20
0
0 130
150
170
190
130
210
150
170
190
Reaction temperature (oC)
o
Reaction temperature ( C)
(C)
(D)
Fig. 2.
18
210
25 EH for 24 hr EH for 48 hr IA after EH for 24 hr IA after EH for 48 hr
Total reducing sugar (g/L)
20
15
10
5
0 130
150
170
190
210
o
Temperature ( C)
(A)
25 EH for 24 hr EH for 48 hr IA after EH 24 hr IA after EH 48 hr
Total reducing sugar (g/L)
20
15
10
5
0 130
150
170
190 o
Temperature ( C)
(B)
Fig. 3.
19
210
30 Hydrothermal pretreatment (HP) Post-hydrothermal enzymatic hydrolysis
Total reducing sugar (g/L)
25
20
15
10
5
0 2.0
2.5
3.0
3.5
4.0
Severity factor
Fig. 4.
20
4.5
5.0
5.5
Highlights
z Marine green-algae Enteromorpha intestinalis converted to reducing sugar. z 7.3 g/L TRS obtained under hydrothermal condition (1:10 S/L ratio, 170°C, 60 min). z By post-hydrothermal enzymatic hydrolysis, 20.1 g/L yield was achieved. z The higher severity factor can cause the over-degradation of carbohydrates.
21
S0960-8524(14)00383-6 http://dx.doi.org/10.1016/j.biortech.2014.03.078 BITE 13209
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
16 January 2014 11 March 2014 16 March 2014
Please cite this article as: Kim, D-H., Lee, S-B., Jeong, G-T., Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis, Bioresource Technology (2014), doi: http://dx.doi.org/ 10.1016/j.biortech.2014.03.078
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1 2
Production of reducing sugar from Enteromorpha intestinalis
3
by hydrothermal and enzymatic hydrolysis
4 5
Dong-Hyun Kim, Sang-Bum Lee, Gwi-Taek Jeong*
6
Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea
7 8 9 10 11 12
The two authors equally contributed to this work.
13 14 15
*Corresponding author: Gwi-Taek Jeong
16
Department of Biotechnology,
17
Pukyong National University, Busan 608-737, South Korea
18
Tel: +82-51-629-5869
19
Fax: +82-51-629-5863
20
E-mail: [email protected]
1
1
Abstract
2
In this work, to evaluate the efficacy of marine macro-algae Enteromorpha intestinalis as a
3
potential bioenergy resource, the effects of reaction conditions (solid-to-liquid ratio, reaction
4
temperature, and reaction time) on sugars produced by a combined process of hydrothermal
5
and enzymatic hydrolysis were investigated. As a result of the hydrothermal hydrolysis, a 7.3
6
g/L (8% yield) total reducing sugar was obtained under conditions including solid-to-liquid
7
ratio of 1:10, reaction temperature of 170°C, and reaction time of 60 min. By subsequent
8
(post-hydrothermal) enzymatic hydrolysis of samples treated at 170°C for 30 min, a 20.1 g/L
9
(22% yield) was achieved.
10 11
Keywords : Enteromorpha intestinalis, hydrothermal treatment, enzymatic hydrolysis, total
12
reducing sugar, severity factor, bioenergy
13
2
1
1. Introduction
2
The requirement for fossil fuel alternatives has received significant attention. Indeed, the
3
discovery and utilization of new resources that can replace fossil resources is emerging as a
4
major global sustainability issue (Adams et al., 2009; Lee et al., 2011). Biomass use in
5
industrial applications is spreading around the world. As utilized, biomass typically is
6
categorized into three generations: starch-based (first generation), wood-based (second
7
generation), and marine seaweed (third generation) (Jeong and Park, 2010; Meinita et al.,
8
2012, 2013). Marine biomass is attracting increasing worldwide interest for its potential as an
9
environmental-friendly and economically sustainable resource. Marine algae have a number
10
of advantages over other resources. They do not compete with food resources; also, given
11
their limited lignin and cellulose contents compared with starch-based or lignocellulosic
12
materials, their pretreatment and saccharification is simple and easy (Goh and Lee, 2010; Lee
13
et al., 2011). And this is not to mention the fact that most of algae grow faster than land plants
14
and offer higher productivity per cultivation area. These important advantages
15
notwithstanding, algae has yet to be applied as a raw material in chemical industry
16
applications (Adams et al., 2009; Lee et al., 2011). Micro-algae cultivation and its application
17
are occupied majority of algae field. On the other hand, a few studies has been done or
18
reported on biofuel- or chemical production from macro-algae (Adams et al., 2009; Jeong and
19
Park, 2010; Meinita et al., 2012, 2013).
20
Macro-algae are classified as green-algae, red-algae or brown-algae. These algae live in
21
shallow seas and on coastal rock (Blomster et al., 2002; Kim, 2010). Green-algae are
22
composed of cellulose, mannose and xylan as the cell-wall carbohydrates, and starch as the
23
storage carbohydrate. Unlike the two other macro-algae species, it has a high cellulose
24
content in the cell wall. Moreover, it compares favorably with land plants in its similar
25
amounts of photosynthetic-pigment carotene and xanthophylls (Feng et al., 2011; Kim, 2010; 3
1
Suganya et al., 2013). The Enteromorpha genus, a green macro-algae belonging to the
2
phylum chlorophyta and the family ulvaceae, comprises E. intestinalis, E. prolifera, E. linza,
3
and E. compressa (Blomster et al., 2002; Suganya et al., 2013). It is distributed widely in
4
inter-tidal zones and on coastal rocks worldwide (Feng et al., 2011; Suganya et al., 2013).
5
Enteromorpha have many polysaccharides containing large amounts of glucose, xylose, D-
6
glucuronic acid, and L-rhamnose (Feng et al., 2011). These polysaccharides are a great
7
potential resource for biofuel and chemical production.
8
For conversion of biomass into biofuels or chemicals, its hydrolysis is essential except for
9
combustion, incineration, pyrolysis, gasification, solubilization and so on (Feng et al., 2011).
10
Pretreatment, thus, is one of the core processes in the bioconversion of biomass (Jeong et al.,
11
2013; Kang et al., 2013). Pretreatment reduces the structural and compositional obstacles of
12
biomass in order to maximize enzymatic conversion to mono-sugars. Many pretreatment
13
processes for enzymatic hydrolysis (degradation) of biomass are applied; they include
14
hydrothermal hydrolysis, steam explosion, acid, alkaline, and ammonia fiber expansion, as
15
well as soaking in aqueous ammonia and inorganic salts (Kang et al., 2013; Nitsos et al.,
16
2013). Actually, some work on the pretreatment of macro-algae already has been conducted
17
(Feng et al., 2011; Jeong and Park, 2011; Lee et al., 2011; Meinita et al., 2012, 2013; Park
18
and Jeong, 2013; Yoon et al., 2011). The proper pretreatment process is a function of the
19
biomass characteristics, catalysts, operation factors, and strength (Kang et al., 2013; Jeong et
20
al., 2013; Meinita et al., 2012). During pretreatment, numerous degradation products such as
21
5-HMF, levulinic acid, formic acid, furfural, and phenolic compounds, which can inhibit cell
22
growth, are formed under severe operation conditions (Kang et al., 2013; Jeong et al., 2013;
23
Meinita et al., 2012). Because hydrothermal pretreatment does not involve expensive or
24
hazardous chemicals, it is simple, safe, economic, and environmentally friendly (Nitsos et al.,
25
2013). 4
1
In the present study, for the purposes of minimizing inhibitor formation and enhancing
2
mono-sugar recovery, a combined process of hydrothermal and enzymatic hydrolysis was
3
evaluated. Biomass pretreatment efficiencies in terms of reaction conditions were evaluated
4
and compared on the basis of the severity factor (log (R0)) as a function of pretreatment
5
temperature and time (Brosse et al., 2010), which approach is widely applied to the
6
evaluation and comparison of lignocellulosic pretreatments (Brosse et al., 2010; Pedersen et
7
al., 2010). The overall objective was to determine the potential of marine macro-algae
8
Enteromorpha intestinalis as a bioenergy resource.
9 10
2. Materials and methods
11
2.1. Materials
12
Dried Enteromorpha intestinalis was harvested in Jindo (Jeonnam, Korea) in October 2010.
13
The dried biomass was pulverized in disintegrator until it passed through a 140 mesh sieve
14
and kept in a sealed bag at room temperature. E. intestinalis is composed of 42.8% total
15
carbohydrate, 31.6% crude protein, 1.3% crude lipid and 24.3% crude ash. Viscozyme L and
16
Cellic® CTec2 were obtained from Novozymes A/S (Denmark). All of the other chemicals
17
were of analytical grade.
18 19
2.2. Batch hydrothermal experiment procedure
20
Preparatory to a batch hydrothermal reaction, a certain quantity of biomass and 40 mL of
21
distilled water were filled into a stainless steel reactor of 50 mL size. The reaction
22
temperature (130-210°C) was controlled by means of an oil bath equipped with a PID
23
temperature controller and magnetic stirring of approximately 200 rpm. After completion of
24
the reaction, the reactor was quickly cooled to room temperature using tap water (Jeong and
25
Park, 2011). The pretreated biomass solution was used in subsequent analyses of total 5
1
reducing sugar (TRS) and enzymatic hydrolysis.
2 3
2.2.1. Effect of solid-to-liquid ratio
4
In order to investigate the effect of the solid-to-liquid ratio (S/L ratio) on the production of
5
TRS from E. intestinalis, an S/L ratio experiment (range: 1:5 ~ 1:20; mass per volume) was
6
conducted. The liquid amount was fixed at 40 mL, and designed amounts of solid (biomass)
7
were added at different S/L ratios. The hydrothermal hydrolysis was conducted to obtain TRS
8
under 170°C reaction temperature and 30 or 60 min reaction time conditions.
9 10
2.2.2. Effects of reaction temperature
11
In order to investigate the effect of reaction temperature on TRS production under the
12
condition of an S/L ratio of 1:10 (Jang et al., 2012; Kang et al., 2013; Meinita et al., 2012),
13
hydrothermal hydrolysis was conducted to obtain TRS at reaction temperatures ranging from
14
130 to 210°C and a 30 or 60 min reaction time.
15 16
2.3. Effect of enzymatic hydrolysis
17
In order to investigate the effect of enzymatic hydrolysis on TRS production, the biomass
18
solution pretreated by hydrothermal hydrolysis between 130 and 210°C was mixed with 10%
19
enzyme mixture (Viscozyme L and Cellic CTec2 = 1:1, v/v) based on biomass weight in a
20
flask, and then reacted in a 170 rpm shaking incubator at 45°C for 48 hr (Song et al., 2011).
21
Upon completion of the enzymatic hydrolysis, supernatant samples were obtained for TRS
22
analysis by 4,000 rpm centrifugation for 15 min.
23 24 25
2.4. Definition of severity factor The hydrothermal pretreatment was evaluated according to the severity factor, which 6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
represents the pretreatment severity as a function of pretreatment temperature and time
2
(Brosse et al., 2010; Pedersen et al., 2010). Severity factor is log (R0).
3
,
4
where t is the pretreatment time (min), T(t) is the pretreatment temperature (°C), 100 is the
5
reference temperature, and 14.75 is the fitted value of the arbitrary constant.
6 7
2.5. Analysis method
8
TRS yields were determined by the modified 3,5-dinitrosalicylic acid (DNSA) method
9
(Miller, 1959). Typically, 200 μL of properly diluted sample and 3 mL of DNS reagent were
10
mixed and heated in a boiling water bath for 5 min, then cooled immediately in a cold-water
11
bath. Upon completion of the reaction, the absorbance was measured by spectrophotometry
12
(Spekol 1300, Analytik Jena, Germany) at 540 nm, using glucose as the standard. The TRS
13
yield was calculated as the produced TRS concentration per initial biomass concentration.
14 15
3. Results and discussion
16
3.1. Production of total reducing sugar by hydrothermal reaction
17
Hydrothermal pretreatment/hydrolysis utilizes water with or without acid/alkali as the
18
solvent at between 120 and 230°C. This range is beneficial to hydrolytic reactions of
19
biomass; at the same time, it promotes the formation of sugar degradation products. In such
20
reactions, the operating factors and their values are carefully selected to maximize sugar
21
contents and enzymatic digestibility (Nitsos et al., 2013). In this work, the production of TRS
22
from E. intestinalis by hydrothermal hydrolysis was evaluated. The experimental reaction
23
factors included the S/L ratio, reaction temperature, and reaction time.
24 25
7
1 2
3.1.2. Effect of solid-to-liquid ratio
3
In order to investigate the effect of the S/L ratio on the production of TRS from E.
4
intestinalis, ratios within the 1:5 - 1:20 range were tested. Fig. 1A shows the TRS produced
5
under the 170°C, 60 min condition. With increasing S/L ratio, the TRS decreased. The 30
6
min reaction showed a decreasing TRS pattern similar to that for 60 min. With an S/L ratio of
7
1:5, an approximately 11.8 g/L TRS yield was obtained to 60 min. For comparison, Fig. 1B
8
plots the TRS yields for different S/L ratios. After 30 min reaction time, the highest yield was
9
obtained at S/L ratios between 1:7.5 and 1:10. However, increasing the S/L ratio beyond 1.10
10
caused a slight decrease in yield. For the 60 min reaction time, the yield increased with
11
increasing S/L ratio until 1:10, whereas at ratios over 1:10, the yields were relatively constant.
12
At the 1:10 S/L ratio, the TRS yield was 8.26%. At lower S/L ratios, despite the high TRS
13
concentrations, the yields were relatively low. As a similar results, Jang et al. (2012) reported
14
that in a conventional simultaneous saccharification and fermentation processes of
15
Saccharina japonica, solid content (S/L ratio) was usually limited to 10% (w/v) due to the
16
high viscosity, which caused the difficulty to handle the slurry. Also, the optimum
17
Kappaphycus alvarezii content for both H2SO4 and HCl hydrolysis were 10% (Meinita et al.,
18
2012). In light of all of these results, the 1:10 S/L ratio was applied in further experiments.
19 20
3.1.2. Effect of reaction temperature
21
In order to investigate the effect of reaction temperature on TRS production under the
22
condition of a 1:10 S/L ratio, the TRS produced, the pH change and the solid residue were
23
analyzed after hydrothermal hydrolysis under reaction temperatures ranging from 130 to
24
210°C. Fig. 2A shows the TRS concentrations produced at different reaction temperatures. At
25
the lowest reaction temperature, 130°C, the TRS concentration fell within the 1.57-1.72 g/L 8
1
range. With increasing reaction temperature, the TRS also was increased. With a reaction
2
time of 30 min, the highest TRS, 7.16 g/L, was produced at 190°C. For the 60 min reaction
3
time, the highest TRS, 7.3 g/L, was obtained at 170°C. Under the 190°C temperature, the
4
TRS concentration was decreased from 7.16 g/L (at 30 min) to 4.37 g/L (at 60 min). It is
5
estimated that decomposition (or dehydration) of sugars, which, again, results in low TRS
6
concentrations, occurs at 210°C. This indicated that over-decomposition of TRS was incurred
7
by its long-duration exposure to high temperature. In fact, it has been reported that in the
8
acidic hydrolysis of lignocellulosics or marine algae, such exposure results in over-
9
dehydration (i.e. over-decomposition) of sugars which is formed by-products such as 5-HMF,
10
levulinic acid, formic acid and char (Jeong and Park, 2010; Meinita et al., 2012, 2013). Kim
11
(2010) reported the effect of hydrothermal and microwave pretreatment on the production of
12
reducing sugar from Ulva pertusa. In hydrothermal pretreatment at 100-150°C, the highest
13
value was obtained at 150°C. In the case of the microwave pretreatment, the effect was more
14
highly influenced by the pretreatment temperature than by the microwave power (50W,
15
100W). However, Yoon et al. (2011) reported that their highest glucose yield was obtained by
16
pretreatment of U. pertusa with 5% hydrogen peroxide at 60°C for 3 h. Interestingly, Lee et
17
al. (2011) reported bioethanol production from Pichia stipitis using 30 g/L hydrolysate
18
obtained by condensation after 190°C, 15 min hydrolysis of 100 g/L of U. pertusa. Its main
19
sugar is glucose and xylose.
20
Fig. 2B plots the TRS yields for different reaction temperatures. The yield was
21
proportional to the TRS concentration produced. Under the 170°C, 60 min condition, an 8.0%
22
yield obtained; however, the yield was only 7.87% under the 190°C, 30 min condition. Fig.
23
2C indicates the pH change at different reaction temperatures after hydrothermal hydrolysis.
24
The pH of the reactant before the hydrothermal hydrolysis was 5.3; after the reaction, the pH
25
was decreased under all of the tested conditions. The lowest pH was 4.05, at 190°C. This low 9
1
pH promoted the hydrolysis of E. intestinalis without the addition of a catalyst. Also, it is
2
estimated that dehydrated products such as levulinic acid, formic acid and others, formed by
3
high reaction temperature, might decrease the pH of a product. On the other hand, the pH
4
increase at 210°C is estimated that the condensation products (humins and other byproducts)
5
formed from dehydrated products (5-HMF, levulinic acid and formic acid) by high
6
temperature, might increase the pH of solution (Deng et al., 2014; Shi et al., 2013; Wang et
7
al., 2013). Fig. 2D shows the amounts of solid residue after hydrothermal hydrolysis at
8
different reaction temperatures. As can be seen, the solid residue was linearly decreased by
9
reaction temperature increases. Over the course of the 30 and 60 min reaction durations, the
10
remaining solid residue decreased with increasing reaction time.
11 12
3.2. Production of total reducing sugar by post-hydrothermal enzymatic hydrolysis
13
In order to investigate the effect of enzymatic treatment, the biomass solution treated by
14
hydrothermal hydrolysis at 130-210°C for 30 min (see Fig. 2) was again hydrolyzed with the
15
enzyme mixture (Viscozyme L and Cellic® CTec2 = 1:1, v/v) in a 170 rpm shaking incubator
16
at 45°C for 48 hr (Fig. 3). Fig. 3A plots the results. It can be seen that initially, the TRS
17
concentration increased with increasing reaction time. The highest TRS was obtained at
18
170°C, and then 20.1 g/L TRS was produced by 48 hr enzymatic hydrolysis. For high
19
reaction temperatures in the post-hydrothermal enzymatic hydrolysis, overall low TRS
20
concentrations were obtained. Comparing enzymatic hydrolysis with hydrothermal hydrolysis
21
(see Fig. 2A) for low reaction temperatures, it is apparent that more TRS was obtained by
22
enzymatic hydrolysis. As shown in Fig. 2D, it was estimated that due to the large amount of
23
solid residue remaining at low reaction temperatures, the TRS concentration was increased by
24
the enzymatic hydrolysis. In other words, the amount of TRS produced by enzymatic
25
hydrolysis was proportional to the amount of solid residue remaining after the initial 10
1
hydrothermal hydrolysis. Fig. 3B plots the results of 48 hr enzymatic hydrolysis using
2
biomass solution obtained from the hydrothermal hydrolysis at 130-210°C for 60 min. By the
3
24 and 48 hr enzymatic hydrolysis at low reaction temperatures, the TRS yield increased with
4
increasing reaction time. The highest TRS yield, 20.1 g/L, was obtained at 130°C. This is a
5
higher value than the enzymatic hydrolysis result after hydrothermal hydrolysis at 130°C for
6
30 min (Fig. 3A). It was demonstrated that the solid residue remaining after low-temperature
7
hydrothermal hydrolysis can easily be hydrolyzed in subsequent enzymatic hydrolysis. As
8
shown in Fig. 3A, with increasing reaction temperature, a low TRS yield was obtained.
9
Whereas, the TRS yield (IA after EA) firstly increased and then decreased again when
10
comparing 24 and 48 hr enzymatic hydrolysis of substrate pretreated at 170 to 210 °C for 60
11
min (Fig. 3B). This phenomenon is estimated that the enzymatic hydrolysis
12
inhibited by dehydration byproducts (5-HMF, levulinic acid, formic acid) and
13
condensation byproduct (humans, and other products) (Feng et al., 2012). Kim
14
(2010), after a comparable study, reported the production of reducing sugar from U. pertusa
15
by microwave pretreatment and enzymatic hydrolysis. In their enzymatic hydrolysis, 10%
16
higher reducing sugar yield was produced in the case of an enzyme mixture of α-amylase,
17
cellulose, and β-glucosidase than in the case of only α-amylase. Also, Jung et al. (2012)
18
reported on acid-pretreatment conditions for enzymatic hydrolysis of U. pertusa. Their
19
hydrolysis rate, 58.8%, was achieved with 0.1M HCl at a pretreatment temperature of 121°C
20
and a 15 min reaction time, as followed by enzymatic hydrolysis using a 2:1 mixture of
21
Viscozyme and xylanase.
22 23 24
3.3. Effect of severity factor The effect of the severity factor on TRS production in hydrothermal and enzymatic
11
1
hydrolysis of E. intestinalis was investigated (Fig. 4). The severity factor is the combined
2
reaction temperature and reaction time in hydrothermal hydrolysis (pretreatment). In the
3
hydrothermal hydrolysis (pretreatment), the TRS concentration linearly increased to the
4
severity factor of 3.84. Over 4.1, the TRS concentration decreased. The hydrothermal
5
reaction conditions at severity factors between 3.6 and 4.1 were 190-210°C and 30-60 min.
6
This result indicated that the higher severity factor can cause the over-degradation of
7
carbohydrates, as shown in Fig. 3A. The severity factors of post-hydrothermal enzymatic
8
hydrolysis are calculated based on the conditions of hydrothermal hydrolysis (pretreatment).
9
In the post-hydrothermal enzymatic hydrolysis, the TRS concentration was maintained at
10
nearly 20 g/L to the severity factor of 3.84. Over 3.84, it sharply decreased. As shown in Fig.
11
3A, with increasing pretreatment temperature and time, the severity factor increased, until the
12
severe conditions incurred a decrease in the TRS concentration. Moreover, the solid residue
13
remaining at the low severity factors prevailing during the hydrothermal reaction can easily
14
be hydrolyzed in enzymatic hydrolysis.
15 16
4. Conclusions
17
In this work, the potential of E. intestinalis as a bioenergy resource was investigated. The
18
TRS was produced by combined process of hydrothermal and enzymatic hydrolysis. By
19
hydrothermal hydrolysis, 7.3 g/L of TRS was produced under 1:10 S/L ratio, 170°C and 60
20
min. By post-hydrothermal enzymatic hydrolysis, 20.1 g/L of TRS was produced at samples
21
of 170°C and 30 min. Also, higher severity factor can cause the over-degradation of
22
carbohydrates in hydrothermal hydrolysis. These results will provide basic information
23
applicable to further studies on the use of E. intestinalis for bioenergy production.
24 25
Acknowledgements 12
1
This research was supported by Basic Science Research Program through the National
2
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
3
Technology (2012R1A1A2006718).
4
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1
Figure Legends
2 3 4
Fig. 1. Effect of solid-to-liquid (S/L) ratio on hydrothermal hydrolysis of E. intestinalis. (A) Total reducing sugar (TRS), (B) Yield
5 6 7
Fig. 2. Effects of reaction temperature and time on hydrothermal hydrolysis of E. intestinalis. (A) TRS, (B) Yield, (C) pH, (D) Solid residue
8 9
Fig. 3. Post-hydrothermal enzymatic hydrolysis of E. intestinalis hydrolysate. (A) After 30
10
min hydrothermal pretreatment; (B) After 60 min hydrothermal pretreatment; EH
11
(Enzymatic Hydrolysis); IA after EH (Increasing Amount after Enzymatic
12
Hydrolysis)
13 14 15
Fig. 4. Effect of severity factor on TRS production by hydrothermal hydrolysis and posthydrothermal enzymatic hydrolysis of E. intestinalis.
16 17 18
16
14 30 min 60 min
Total reducing sugar (g/L)
12
10
8
6
4
2
0 5
10
15
20
Solid-to-liquid ratio (-)
(A)
10 30 min 60 min
Yield (%)
8
6
4
2
0 5
10
15
Solid-to-liquid ratio (-)
(B)
Fig. 1.
17
20
10
8
30 min 60 min
30 min 60 min
Yield (%)
Total reducing sugar (g/L)
8 6
4
6
4
2 2
0
0 130
150
170
190
130
210
150
170
190
210
Reaction temperature (oC)
Reaction temperature (oC)
(A)
(B)
100 30 min 60 min
30 min 60 min
6
Solid residue (%)
80
pH
4
60
40
2 20
0
0 130
150
170
190
130
210
150
170
190
Reaction temperature (oC)
o
Reaction temperature ( C)
(C)
(D)
Fig. 2.
18
210
25 EH for 24 hr EH for 48 hr IA after EH for 24 hr IA after EH for 48 hr
Total reducing sugar (g/L)
20
15
10
5
0 130
150
170
190
210
o
Temperature ( C)
(A)
25 EH for 24 hr EH for 48 hr IA after EH 24 hr IA after EH 48 hr
Total reducing sugar (g/L)
20
15
10
5
0 130
150
170
190 o
Temperature ( C)
(B)
Fig. 3.
19
210
30 Hydrothermal pretreatment (HP) Post-hydrothermal enzymatic hydrolysis
Total reducing sugar (g/L)
25
20
15
10
5
0 2.0
2.5
3.0
3.5
4.0
Severity factor
Fig. 4.
20
4.5
5.0
5.5
Highlights
z Marine green-algae Enteromorpha intestinalis converted to reducing sugar. z 7.3 g/L TRS obtained under hydrothermal condition (1:10 S/L ratio, 170°C, 60 min). z By post-hydrothermal enzymatic hydrolysis, 20.1 g/L yield was achieved. z The higher severity factor can cause the over-degradation of carbohydrates.
21
