10/30/23, 12:41 PM Study of Sulfite Pretreatment to Prepare Bamboo for Enzymatic Hydrolysis and Ethanol Fermentation | SpringerLink

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CURRENT PROBLEMS. Alternative Feedstock Published: 31 July 2014

Study of Sulfite Pretreatment to Prepare Bamboo for

Enzymatic Hydrolysis and Ethanol Fermentation
Zhiqiang Li , Benhua Fei & Zehui Jiang

Chemistry and Technology of Fuels and Oils 50, 189–196 (2014)

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We have studied the change in composition for three bamboo species of

different ages with sulfite pretreatment and treatment with dilute sulfuric acid.
Enzymatic hydrolysis and ethanol fermentation of the hydrolyzate were carried
out (sequentially and simultaneously). We show that sulfite pretreatment
significantly increases the conversion of cellulose to glucose during enzymatic
hydrolysis, and the conversion depends on the type and age of the bamboo.
When 2-year moso bamboo is treated at a temperature of 180 °C for 30
minutes with a solution containing 5 % sulfuric acid and 9 % sodium sulfite,
conversion of cellulose to glucose during hydrolysis is as high as 89.3 %. A
higher ethanol yield results from sequential separate hydrolysis and
fermentation than for simultaneous saccharification (hydrolysis) of cellulose
and fermentation.

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Bamboo is the second most important forest resource and source of cellulose,
ranking just behind wood. About 1200 species of bamboo grow in the tropics
and subtropics [1]. In China, the annual production of bamboo is more than
1.539 billion stems. Bamboo is an ideal renewable resource because of its rapid
growth and easy propagation. Furthermore, bamboo has high cellulose and
hemicellulose content, which are sources of sugars for production of ethanol
and other chemical products.

Bioethanol production from bamboo, as from other types of biomass, includes

three steps: pretreatment of the biomass, hydrolysis, and fermentation. The

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first step is needed to ensure that the cellulose is accessible to reaction with
cellulase, a saccharifying enzyme. Pretreatment is particularly important for
recalcitrant (degradation-resistant) bamboo [2]. Sulfite pretreatment is an
effective method to prepare softwoods for enzymatic hydrolysis. The cellulose-
to-glucose conversion is greater than 90 % or as high as 100 % [3–6]. However,
when 4-year moso bamboo is treated by this method, the conversion is no
greater than 25 % [7].

Bamboo, in contrast to wood trees, reaches its mature height and stem
thickness within a few days, and then growth involves only increase in density
and development of the internal structure of the stem; the plant matures in 3 to
5 years. Therefore the effectiveness of the pretreatment for hydrolysis stage to a
significant extent depends on the age of the plant. In this paper, we have
obtained the dependence of the cellulose-to-glucose conversion and ethanol
yield for processing three species of bamboo of different ages.

We used samples of 2-year moso bamboo (Phyllostachys heterocycla), 2-year

and 5-year Bambusa blumeana bamboo, and 2-year and 5-year Bambusa
pervariabilis McClure bamboo, all obtained from Guangxi, China. The air-
dried bamboo was milled and the <1 mm fraction was screened. The milled
bamboo powder was stored in leaktight plastic bags.

Analytical grade reagents were used: sulfuric acid, sodium sulfite, sodium
acetate, peptone, potassium dihydrogen phosphate, magnesium sulfate,
anhydrous calcium chloride, microcrystalline cellulose, glucose. The cellulose
was purchased from Sunsonenzymes (China); Saccharomyces cerevisiae yeast
was purchased from Angel Yeast Co., Ltd.

Sulfite pretreatment of bamboo. An oven-dried sample of bamboo

weighing 8 g was placed in a 100 mL vessel, and then 50 mL of a sulfuric acid
solution (2 % or 5 % w/w anhydrous acid/bamboo) was poured into the vessel
and sodium sulfite (5 % or 9 % w/w bamboo) was added. The weight ratio of
liquor to bamboo was 6:1. The vessel was placed in a microwave oven (MARS
model, CEM Corporation, USA) at the center of a rotating circular ceramic
plate. The temperature was raised to 180 °C for about 10 minutes and held
constant for 30 minutes. When the pretreatment was complete, we waited a
few minutes until the temperature dropped down below 80 °C and then the
substrate and liquor were separated by filtration. The liquor was stored at a
temperature of 4 °C for later analysis for sugars and fermentation inhibitors by
high performance liquid chromatography (HPLC). The substrate was washed
with water until the pH of the wash water was neutral, and then also stored at a
temperature of 4 °C for enzymatic hydrolysis and analysis of its composition.
Each bamboo pretreatment experiment was conducted twice; in this paper, we
give the average values.

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Enzymatic hydrolysis was carried out in 100 mL plastic flasks at 50 °C on

an orbital shaking incubator (KYC-100C, Shanghai Fuma Laboratory
Instrument Co., Ltd., China) at 200 rpm. The substrate, in an amount
equivalent to 0.8 g glucan, was placed in 40 mL of buffer solution (0.05 M
sodium a cetate, pH = 4.8). ~1.5 mg tetracycline chloride was added to the
mixture to control the growth of microorganisms and to prevent consumption
of sugars liberated during hydrolysis. The enzymes cellulase (15 FPU/g glucan,
where FPU = filter paper units) and β-glucosidase (30 IU/g glucan) were added
to the contents of the flask. The hydrolyzate was sampled after 1, 3, 6, 12, 24,
and 48 hours for analysis of the glucose content. Each hydrolysis experiment
was conducted twice; in this paper, we give the average values.

Ethanol fermentation. Saccharomyces cerevisiae yeast were prepared as

follows. The yeast powder was placed in a 100 mL glass flask containing 40 mL
of a 2 % glucose solution. The flask was held in a water bath at 38 °C for 15-20
minutes, and then was transferred to another water bath at 33 °C and held for
90 minutes.

For sequential separate hydrolysis and fermentation, peptone (5 g/L), KH2PO4

(2 g/L), MgSO4 (1 g/L), and CaCl2 (0.25 g/L) were added to the hydrolyzate
and glucose (the reference). The solutions were autoclaved at 121 °C for 20
minutes for sterilization. We used a 0.6 M solution of sodium hydroxide or 6 %
sulfuric acid to maintain the pH of the hydrolyzate at 5.5 ± 0.1. The hydrolyzate
was inoculated with the precultured yeast solution. The flasks with the samples
were placed in the incubator, and the contents were shaken at 200 rpm, 37 °C
for 24 hours. In order to ensure anaerobic fermentation conditions, the flasks
were sealed with plastic food wrap [8]. If possible, immediately after
completion of fermentation, samples were withdrawn to determine the ethanol
content [9].

For simultaneous hydrolysis (saccharification) and fermentation, the

substrates and microcrystalline cellulose (the reference) were placed in 40 mL
of buffer solution (pH = 5.25) and the following were added: peptone (5 g/L),
KH2PO4 (2 g/L), MgSO4 (1 g/L), and CaCl2 (0.25 g/L). The solutions were
autoclaved. The enzymes cellulase (15 FPU/g glucan) and β-glucosidase (30
IU/g glucan) were added to the mixture, and it was inoculated with the
precultured yeast solution. The flasks with the samples were placed in the
incubator, and the contents were shaken at 150 rpm, 37 °C for 48 hours. As
indicated above, the flasks were also sealed with plastic food wrap. If possible,
immediately after completion of fermentation, samples were withdrawn to
determine the ethanol content.

Analysis methods. The content of water solubles or ethanol solubles was

determined by exhaustive Soxhlet extraction. The carbohydrate and lignin
contents (acid-soluble and acid-insoluble) in the original bamboo and in the
bamboo after sulfite pretreatment were analyzed by the National Renewable

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Energy Laboratory (NREL) method, “Determination of structural

hydrocarbons and lignin in biomass.” The method is based on degradation of
carbohydrates down to monosaccharides during two-step sulfuric acid
hydrolysis and then determination of the sugar content in the hydrolyzate. The
acid-soluble lignin was determined pectrophotometrically at the wavelength
205 nm. The acid-insoluble lignin was isolated by drying the residue of acid
treatment under vacuum at a temperature of 60 °C for 10 hours.

Liquid samples were analyzed by high performance liquid chromatography

with a refractometric detector (Waters Corporation, USA). The sugar content
in the hydrolyzate, including glucose, xylose, mannose, galactose, and
arabinose, was determined using an anion-exchange column (Aminex HPX-
87P, Bio-Rad, USA) at 85 °C with 0.6 mL/min water flow rate.

The course of the hydrolysis and fermentation reactions were monitored from
the change over time in the glucose and ethanol concentrations in the reaction
mixture, using a commercial biosensor analyzer (SBA-40E, Shandong
Academy of Science, China) for fast analysis. According to the manufacturer’s
specifications, the instrumental uncertainty is ~2 %. The data presented in this
paper represent the arithmetic mean of two measurements.

Composition of the original and pretreated bamboo. Table 1 presents

the composition of the analyte bamboo samples. As we see, the cellulose and
lignin content is higher in 4-year and 5-year bamboo than in 2-year bamboo.
The content of extractables depends on the species of bamboo. Phyllostachys
heterocycla contains more extractables than the other two species. In [10], a
cellulose content of 42.06 %-47.32 % is reported in the bamboo Phyllostachys
heterocycla. The discrepancy between the results and our data may be due to
the different analysis methods.

Table 1

Table 2 gives the composition of the substrates obtained by pretreatment of the

analyte bamboo samples with dilute acid and (or) sodium sulfite. No
delignification occurs for pretreatment with dilute acid. Substrates obtained by
sulfite pretreatment, on the other hand, contain more cellulose and less lignin
than the original bamboo samples. The solid recovery for pretreatment with
more concentrated solutions (5 % sulfuric acid and 9 % sodium sulfite) is less
than for pretreatment with less concentrated solutions.

Table 2

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Sequential hydrolysis and fermentation calls for enzymatic hydrolysis of

the cellulose in the substrate to glucose, followed by its fermentation to
ethanol. Both steps were carried out under conditions (temperature, pH) that
were optimal for each. Figure 1 shows the change in glucose and ethanol
content in the fermented solution vs. fermentation time for the hydrolyzate of
the 2-year Phyllostachys heterocycla bamboo. We see that at the beginning of
fermentation, the glucose content sharply declines, with a simultaneous rapid
rise in ethanol content. 12 hours after the beginning of fermentation, the curves
flatten to a plateau, and the ethanol concentration reaches a maximum. When
the fermentation time approaches 48 hours, the ethanol content decreases a
little; the optimal fermentation time is 24 hours.

Fig. 1

Glucose (1) and ethanol (2) content vs. fermentation time.

Table 3 gives the cellulose-to-glucose conversion values for 48 h hydrolysis and

the ethanol yield for 24 h fermentation for the different bamboo samples.
Sulfite pretreatment (2 % sulfuric acid + 4 % sodium sulfite) of the bamboo did
not result in as effective hydrolysis as pretreatment with just 2 % acid, which is
consistent with the results of our previous studies. At the same time, sulfite
pretreatment with more concentrated solutions (5 % sulfuric acid and 9 %
sodium sulfite) results in significantly higher cellulose-to-glucose conversion
than the above-indicated pretreatment variants. Conversion of the cellulose in
2-year Phyllostachys heterocycla bamboo is as high as 89.3 %; this value is
consistent with the results of sulfite pretreatment of biomass for other species
(spruce, switchgrass and poplar).

Table 3

Based on the results obtained, we can assume that sulfite pretreatment is

effective for relatively high concentrations of acid and sodium sulfite.
Delignification is higher for higher concentration of acid and sodium sulfite. In
addition, sulfonation makes lignin less hydrophobic, which then helps reduce
adsorption of hydrophobic enzymes on the lignin and enzyme losses. However,

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sulfite pretreatment was not as effective for the bamboo species Bambusa
blumeana and Bambusa pervariabilis, where the cellulose-to-glucose
conversion was no greater than 60 %. This means that the residual lignin
content in the substrate is only one of the factors determining the effectiveness
of the hydrolysis.

The ethanol yield from the hydrolyzate is 74.1 %-96.5 %; from glucose, 85.4 %.
After 24 h fermentation, no glucose is detected in solution, i.e., all the glucose
was consumed in the fermentation process. Most of the glucose is converted to
ethanol, and a small portion of it is used as nutrient by the yeast.

Simultaneous hydrolysis and fermentation. Figure 2 shows the change

in ethanol and glucose content for simultaneous hydrolysis (saccharification)
and fermentation of the substrate after sulfite pretreatment (5 % sulfuric acid
and 9 % sodium sulfite) of the 2-year Phyllostachys heterocycla bamboo. The
glucose content in solution was low during the entire process, while the ethanol
content increased over time and a significant increase in ethanol content
occurred in the first few hours from the beginning of the experiment.
Consequently, the rate of ethanol formation is determined by the rate of
glucose formation from the substrate, i.e., hydrolysis of the substrate is the
rate-determining step.

Fig. 2

Glucose (1) and ethanol (2) content vs. process time for simultaneous hydrolysis
(saccharification) and fermentation.

Table 4 shows the data on glucose yield and cellulose-to-ethanol conversion.

The conversion of microcrystalline cellulose to ethanol is only 46.8 %. The
cellulose-to-ethanol conversion for all the bamboo samples with simultaneous
hydrolysis and fermentation was lower than for sequential separate hydrolysis
and fermentation. The maximum ethanol yield is provided by the
Phyllostachys heterocycla bamboo after sulfite pretreatment (5 % sulfuric acid
and 9 % sodium sulfite).

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Table 4

We note again that the temperature and pH of the solution were not optimal
for conversion of cellulose to glucose, and so this step determined the rate of
ethanol formation. It will be helpful to further investigate the possibility of
improving the ethanol yield by means of preliminary hydrolysis of cellulose
under optimal conditions for several hours, followed by addition of yeast to the
solution.

Therefore, the species and age of the bamboo are the main factors affecting the
hydrolyzability of the substrate. The maximum hydrolyzability was seen for the
substrate obtained by pretreatment of 2-year Phyllostachys heterocycla
bamboo with a solution containing 5 % sulfuric acid and 9 % sodium sulfite.
Generally ethanol can be successfully produced from bamboo hydrolyzate by
fermentation using Saccharomyces cerevisiae yeast.
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This work was supported by the National Project for Scientific and Technical
Programs (2012BAD54G01) and the Basic Research Fund of the International
Centre for Bamboo and Rattan (Grant No. 1632012001.

Author information

Authors and Affiliations

International Center for Bamboo and Rattan, Key Lab of Bamboo
and Rattan Science and Technology, Beijing, 100102, China
Zhiqiang Li, Benhua Fei & Zehui Jiang

Corresponding author
Correspondence to Zhiqiang Li.

Additional information

Translated from Khimiya i Tekhnologiya Topliv i Masel, No. 3, pp. 9 – 13, May
– June, 2014.

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Li, Z., Fei, B. & Jiang, Z. Study of Sulfite Pretreatment to Prepare Bamboo for Enzymatic
Hydrolysis and Ethanol Fermentation. Chem Technol Fuels Oils 50, 189–196 (2014).
https://doi.org/10.1007/s10553-014-0507-3

Published Issue Date

31 July 2014 July 2014

DOI
https://doi.org/10.1007/s10553-014-0507-3

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Keywords
bamboo sulfite pretreatment of cellulose enzymatic hydrolysis

fermentation bioethanol

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