Beruflich Dokumente
Kultur Dokumente
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand
c
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b
a r t i c l e
i n f o
Article history:
Received 15 May 2009
Received in revised form 21 July 2009
Accepted 24 August 2009
Available online xxxx
Keywords:
Efficiency of acid hydrolysis
Sugarcane bagasse
Lactic acid
Xylose
a b s t r a c t
In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid
hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of
hydrochloric (HCl) or sulfuric (H2SO4) concentration (0.55%, v/v), reaction time (15 h) and incubation
temperature (90120 !C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of
HCl at 100 !C for 5 h, which the main components (in g l!1) in the hydrolysate were glucose, 1.50; xylose,
22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from
the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l!1 of xylose and 7 g l!1 of yeast extract. The main products (in g l!1) of the fermentation
were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24.
" 2009 Elsevier Ltd. All rights reserved.
1. Introduction
In 2007, Thailand produced approximately 70 million tons of
sugarcane and became the world third sugar producer following
Brazil and Australia, respectively. Sugarcane bagasse, a byproduct
of the sugar production industry, consists of cellulose 43.6%, hemicellulose 33.8%, lignin 18.1%, ash 2.3% and wax 0.8% on a dry
weight basis (Sun et al., 2004). It is an abundant source of lignocellulose that can be hydrolysed to yield fermentable sugars for the
production of value added bio-products such as lactic acid, thus
increasing the economy of the process. Other applications of sugarcane bagasse are as sources of animal feed, energy, pulp, paper and
boards (Banerjee and Pandey, 2002).
Acids can be used as catalysts for sugarcane bagasse hydrolysis
because they can break down heterocyclic ether bonds between
sugar monomers in the polymeric chains, which are formed by
hemicellulose and cellulose (Aguilar et al., 2002). H2SO4 (Nguyen
et al., 1999, 2000; Sun and Cheng, 2002; Kumar et al., 2009) and
HCl (Sun and Cheng, 2002; Herrera et al., 2004; Hernndez-Salas
et al., 2009) are potential acids which can be used to hydrolyse
sugarcane bagasse. The hydrolysate of lignocellulosic materials
mainly consists of fermentable sugars such as xylose, glucose and
arabinose (Rodrguez-Chong et al., 2004). Accompanying these
0960-8524/$ - see front matter " 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.08.091
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doi:10.1016/j.biortech.2009.08.091
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P
S
P
E
1 I
P
where
S is the sum of sugar concentrations in the hydrolysates
P
(glucose, xylose and arabinose) and
I is the sum of inhibitor concentrations in the hydrolysates (acetic acid and furfural).
2.2. Lactic acid fermentation
2.2.1. Basal medium and inoculum preparation
The basal medium was composed of (in g l!1) yeast extract (Oxoid, England), 5; peptone (Oxoid, England), 5 and NaCl (BDH, UK),
5. The medium was supplemented with 22 or 30 g l!1 of xylose
(Fluka, Switzerland) and adjusted to pH 6.0 with 2 M HCl.
The stock culture of L. lactis IO-1 (JCM 7638) was rejuvenated by
incubation in 5 ml of thioglycolate medium (Difco, USA) for 16 h at
37 !C in a static incubator. One ml of the culture was transferred
into 150 ml of the basal medium and incubated at 37 !C, 150 rpm
for 3 h. An inoculum (5%, v/v) was used to initiate the batch
cultures.
16
(a)
glucose
arabinose
xylose
acetic acid
furfural
efficiency
14
12
10
8
16
14
12
10
8
0
16
(b)
14
0
16
14
12
12
10
10
0
16
(c)
14
0
16
14
12
12
10
10
0
16
0
16
(d)
14
14
12
12
10
10
Time (h)
Efficiency
-1
2. Methods
Fig. 1. The main components of sugarcane bagasse hydrolysate and acid hydrolysis
efficiency (E) under 0.5% of HCl and different temperatures: (a) 90, (b) 100, (c) 110
and (d) 120 !C.
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Table 1
Soluble fraction of sugarcane bagasse hydrolysed at different temperatures and HCl concentrations giving the maximum xylose concentration.
Temperature (!C)
Xylose (g l!1)
Glucose (g l!1)
Arabinose (g l!1)
Furfural (g l!1)
90
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
5
5
5
3
5
4
4
2
4
3
2
1
4
2
1
2
7.55 0.38
7.33 0.37
9.46 0.47
8.56 0.43
13.09 0.65
7.56 0.38
8.97 0.45
8.94 0.45
13.33 0.67
9.40 0.47
8.93 0.45
11.18 0.56
15.16 0.76
7.69 0.38
10.50 0.53
11.89 0.59
0.21 0.01
1.21 0.06
1.64 0.08
1.18 0.06
2.66 0.13
1.67 0.08
2.23 0.11
2.00 0.10
3.28 0.16
1.87 0.09
1.79 0.09
1.20 0.06
2.85 0.14
1.41 0.07
1.69 0.08
5.47 0.27
0.93 0.05
1.04 0.05
1.16 0.06
1.13 0.06
1.22 0.06
1.23 0.06
1.43 0.07
1.11 0.06
1.12 0.06
1.24 0.06
1.31 0.07
1.30 0.07
1.35 0.07
1.22 0.06
1.22 0.06
1.11 0.06
0
0
0
0
0.06 0.00
0
0
0
0
0
0
0
0.04 0.00
0
0
0
0
0
0
0
0
0
0.20 0.01
0
0.28 0.01
0
0
0
0.66 0.03
0
0
0.75 0.04
100
110
120
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Table 2
Soluble fraction of sugarcane bagasse hydrolysed at different temperatures and H2SO4 concentrations giving the maximum xylose concentration.
Temperature (!C)
Xylose (g l!1)
Glucose (g l!1)
Arabinose (g l!1)
Furfural (g l!1)
90
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
4
5
5
5
5
5
4
3
4
2
3
2
4
2
2
1
7.62 0.38
6.02 0.30
5.84 0.29
11.63 0.58
10.96 0.55
6.25 0.31
6.12 0.31
7.59 0.38
12.64 0.63
6.86 0.34
7.91 0.40
11.48 0.57
10.12 0.51
6.48 0.32
8.87 0.44
8.44 0.42
0.17 0.00
0.66 0.03
0.71 0.04
1.43 0.07
2.01 0.10
1.08 0.05
0.71 0.04
0.86 0.04
2.28 0.11
1.54 0.08
1.59 0.08
1.34 0.07
1.76 0.09
0.88 0.04
1.22 0.06
1.23 0.06
1.03 0.05
0.70 0.04
0.65 0.03
1.34 0.07
1.18 0.06
1.06 0.05
1.20 0.06
0.51 0.03
1.33 0.07
0.98 0.05
1.13 0.06
1.17 0.06
0.86 0.04
1.06 0.05
0.93 0.05
1.08 0.05
0
0
0.04 0.00
0
0
0
0
0
0.06 0.00
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.18 0.00
0
0
0
0
0
0
0
0
100
110
120
Table 3
Catalytic efficiencies of acid hydrolysis (E) under various conditions.
Temperature (!C)
90
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
0.5
2
3
5
100
110
120
HCl hydrolysis
H2SO4 hydrolysis
5
5
5
3
5
4
4
2
4
3
2
1
4
2
1
2
8.69 0.43
9.58 0.48
12.26 0.61
10.87 0.54
16.01 0.40
10.46 0.52
10.53 0.53
12.05 0.60
13.85 0.69
12.51 0.63
12.03 0.60
13.68 0.68
11.39 0.57
10.32 0.52
13.41 0.67
10.55 0.53
4
5
5
5
5
5
4
3
4
2
3
2
4
2
2
1
8.82 0.44
7.38 0.37
6.92 0.35
14.40 0.72
14.15 0.71
8.39 0.42
8.03 0.40
7.59 0.38
15.33 0.20
9.38 0.47
10.63 0.53
13.99 0.70
12.74 0.64
8.42 0.42
11.02 0.55
10.75 0.54
Table 4
Comparison of the main components of the hydrolysates of sugarcane bagasse using
0.5% of HCl at 100 !C for 5 h in a 30 ml test tube and a 50 l reactor.
Components (g l!1)
30 ml Test tube
50 l Reactor
Xylose
Glucose
Arabinose
Acetic acid
Furfural
E
13.09 0.65
2.66 0.13
1.22 0.06
0.06 0.00
0
16.01 0.40
22.59 1.13
1.50 0.08
1.29 0.06
0.15 0.06
1.19 0.02
10.85 0.50
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Table 5
Comparison of the main components of the acid-treated hydrolysates of sugarcane bagasse and the E values under various conditions from several studies.
Xylose
(g l!1)
Glucose
(g l!1)
Arabinose
(g l!1)
Total sugar
(g l!1)
Acetic acid
(g l!1)
Furfural
(g l!1)
22.59
21.6
18.60
1.50
3.00
2.87
1.29
ND*
2.04
25.38
24.60
23.51
0.15
3.65
0.90
1.19
0.52
1.32
10.85
4.76
7.30
17.60
21.50
17.10
3.00
5.84
7.20
2.60
2.95
2.00
23.20
30.29
26.30
4.00
5.45
4.00
1.20
ND
1.40
3.74
4.70
4.11
Not determined.
2.0
1.8
1.6
1.0
2.0
1.8
1.6
1.4
1.2
1.0
.8
.6
.4
.2
0.0
1
0
0
0
10
20
30
40
50
60
70
80
25
8
7
(b)
20
6
5
-1
)
10
-1
Product concentrations (g l )
-1
)
Dry cell weight (g l
0.0
15
.8
.2
20
1.2
.4
(a)
1.4
.6
25
Sugar concentrations (g l
15
4
10
3
2
1
0
0
0
10
20
30
40
50
60
70
80
Time (h)
Fig. 2. Batch culture profiles of L. lactis IO-1 grown on the non-detoxified hydrolysate (a) and the detoxified hydrolysate (b) of sugarcane bagasse supplemented with 7 g l!1 of
yeast extract: lactic acid (d), formic acid (s), acetic acid (.), ethanol (4), dry cell weight (j), glucose (h), xylose (}), arabinose (q).
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14
30
(a)
12
25
10
20
8
15
6
2
0
10
14
20
30
40
50
60
70
30
(b)
12
25
10
20
-1
10
Sugar concentrations (g l )
Our preliminary study revealed that L. lactis IO-1 could not grow
and produce in the sugarcane bagasse hydrolysate without nutrient supplementation (data not shown). Similar results were
observed by Nancib et al. (2001) who found that nitrogen supplementation in dates hydrolysate had a significant effect on growth
and lactic acid production of L. casei subsp. rhamnosus. Olsson
and Hahn-Hgerdal (1996), Garde et al. (2002) and Martin et al.
(2002) also found that lignocellulosic hydrolysates contained some
inhibitors of bacterial growth e.g. furfural, 5-hydroxymethylfurfural (HMF), levulunic acid and phenol compounds. In addition, Mussatto and Roberto (2004) reported that anion-exchange resins
removed high percentages of the toxic compounds (96% of acetic
acid, 91% of phenolic compounds, 73% of furfural and 70% of
HMF) from lignocellulosic hydrolysates. Therefore, in this study
the sugarcane bagasse hydrolysate was detoxified by passing the
broth through the amberlite (modified from Domnquez et al.
(1997)) before use. It was found that the concentrations of xylose
glucose, arabinose and acetic acid before and after detoxification
were equal, whereas that of furfural was decreased from 1.19 to
0.09 g l!1.
The effects of nitrogen supplementation were investigated by
the addition of 0, 3, 5 and 7 g l!1 of yeast extract, peptone or
ammonium sulphate or both 5 g l!1 of yeast extract and 5 g l!1 of
peptone (as used in the basal medium) into the detoxified hydrolysate. The results showed that 7 g l!1 of yeast extract gave the
highest yield of lactic acid (0.26 g lactic per g sugar utilised), while
microbial growth and lactic acid production were not detected in
the broth containing ammonium sulphate at all concentrations
(data not shown). The absence of ammonium assimilation by L. lactis IO-1 suggests that small peptides may be required by this
microorganism.
Fig. 2 shows the batch culture profiles of L. lactis IO-1 grown on
the non-detoxified and the detoxified hydrolysate containing
7 g l!1 of yeast extract. The results clearly showed that L. lactis
could slightly grow in the non-detoxified broth but lactic acid
was not produced (Fig. 2a). This might be due to the effects of some
inhibitors in the medium as previously described. When the hydrolysate was detoxified, the results clearly showed that the detoxification improved the fermentability of the hydrolysate by L. lactis
IO-1 compared to the non-detoxified hydrolysate, since without
detoxification there was no lactic acid production (Fig. 2a). Xylose
utilisation was started after glucose was completely consumed
(Fig. 2b). Xylose was almost completely utilised, while L. lactis
was not able to utilise arabinose. Products from the fermentation
were increased against time and relatively constant at 40 h. The
highest main product concentrations detected were acetic acid,
formic acid, lactic acid and ethanol, respectively.
In Fig. 2a, a weak growth is observed, however without any substrate consumption. This phenomena was explained by Benthin and
Villadsen (1996), Loubire et al. (1997), Novk and Loubire (2000),
who reported that for lactic acid bacteria, sugar is only a catabolic
substrate leading to catabolic end-products whereas the biomass
is built from anabolic precursors, e.g., amino acids, nucleotides,
etc., present in yeast extract and peptone in the medium. Similar results were observed by Plihon et al. (1995) who performed batch
experiments with Leuconostoc mesenteroids in de Man, Rogosa, and
Sharpe (MRS) medium and concluded that from the carbon balance,
biomass was not created from sugar metabolism.
Thani et al. (2006) revealed that in xylose batch cultures, the
molar product (lactic acid, acetic acid, formic acid and ethanol)
yields were dependent on the initial xylose concentration. At high
initial xylose concentrations of 30 and 50 g l!1, the molar product
yield of lactic acid was the highest with the values of 0.71 and
0.87 mol product mol!1 xylose utilised, respectively while at low
-1
8
15
6
10
2
0
10
20
30
40
Time (h)
50
60
70
Fig. 3. Batch culture profiles of L. lactis IO-1 grown on the detoxified hydrolysate of
sugarcane bagasse containing 30 g l!1 of xylose and 7 g l!1 of yeast extract (a) and
the basal medium containing 30 g l!1 of xylose (b): lactic acid (d), formic acid (s),
acetic acid (.), ethanol (4), dry cell weight (j), glucose (h), xylose (}), arabinose
(q).
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Lactic acid
Formic acid
Acetic acid
Ethanol
1.34 0.20
0.74 0.13
10.85 0.51
12.67 0.62
6.04 0.30
4.72 0.23
7.87 0.44
7.66 0.38
5.24 0.61
3.36 0.33
64
37
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