Bioresource Technology 135 (2013) 191–198

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

Bioethanol production using carbohydrate-rich microalgae biomass as feedstock

Shih-Hsin Ho a, Shu-Wen Huang a, Chun-Yen Chen b, Tomohisa Hasunuma c, Akihiko Kondo c,
Jo-Shu Chang a,b,d,⇑
a
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan, ROC
b
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, ROC
c
Department of Chemical Science and Engineering, Kobe University, Kobe, Japan
d
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan, ROC

h i g h l i g h t s

" A sugar-rich Chlorella vulgaris FSP-E strain was used as feedstock for ethanol production.
" Enzymatic and acidic hydrolyses can efficiently saccharify the microalgae biomass.
" SHF & SSF processes produced ethanol from the microalgae biomass with high yield.
" SSF process gave better ethanol production performance with a 92% theoretical yield.

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to evaluate the potential of using a carbohydrate-rich microalga Chlorella vulgaris FSP-E
Available online 16 October 2012 as feedstock for bioethanol production via various hydrolysis strategies and fermentation processes.
Enzymatic hydrolysis of C. vulgaris FSP-E biomass (containing 51% carbohydrate per dry weight) gave a
Keywords: glucose yield of 90.4% (or 0.461 g (g biomass) 1). The SHF and SSF processes converted the enzymatic
Microalgae microalgae hydrolysate into ethanol with a 79.9% and 92.3% theoretical yield, respectively. Dilute acidic
Chlorella vulgaris hydrolysis with 1% sulfuric acid was also very effective in saccharifying C. vulgaris FSP-E biomass, achiev-
Carbohydrate
ing a glucose yield of nearly 93.6% from the microalgal carbohydrates at a starting biomass concentration
Enzymatic hydrolysis
Acid hydrolysis
of 50 g L 1. Using the acidic hydrolysate of C. vulgaris FSP-E biomass as feedstock, the SHF process pro-
duced ethanol at a concentration of 11.7 g L 1 and an 87.6% theoretical yield. These findings indicate
the feasibility of using carbohydrate-producing microalgae as feedstock for fermentative bioethanol
production.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2011). Currently, bioethanol is mainly derived from sucrose

and starch crops (e.g., sugarcane and corn) as well as lignocellu-
The fast growth of the world population and rapid development losic materials (e.g., rice straw and switchgrass) (Nigam and Singh,
of a number of emerging economies have both led to sharp in- 2011). However, using agricultural crops or agricultural waste as
crease in global energy consumption (Harun et al., 2010). However, feedstock for bioethanol production still presents a number of
the use of fossil fuels is associated with environmental pollution, problems, due to the increasing demands this makes on the limited
the greenhouse effect, and climate change (Ho et al., 2011; arable lands and water supply, as well the high costs involved in
Sivakumar et al., 2010), and thus many countries are now increas- converting lignocellulosic materials into ethanol. The major cause
ing their efforts with regard to developing renewable energy of the latter is the high lignin content in the lignocellulosic bio-
sources, which are both more economic and environmentally mass, making the saccharification process very difficult (Sun and
friendly (Mussatto et al., 2010). Biomass is one of the most Cheng, 2002).
promising renewable resources used to generate different types Microalgae have recently been considered as a third generation
of biofuels, such as biodiesel (Ho et al., 2010) and bioethanol (John feedstock for biofuel production (Nigam and Singh, 2011), with the
focal fuel in such processes being biodiesel (Chen et al., 2011;
⇑ Corresponding author at: Department of Chemical Engineering, National Cheng
Chisti, 2007; Ho et al., 2010). However, since some microalgae
Kung University, No. 1 University Road, Tainan, Taiwan, ROC. Tel.: +886 6 species have high carbohydrate content, in terms of starch and cel-
2757575x62651; fax: +886 6 2357146. lulose, they are also excellent substrates for bioethanol production.
E-mail address: [email protected] (J.-S. Chang). Using carbohydrate-rich microalgal biomass for bioethanol pro-

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.015
192 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198

duction is advantageous, since microalgae grow faster and fix CO2 of approximately 60 lmol m 2 s 1. The light intensity was mea-
at a higher rate than terrestrial plants. In addition, microalgae- sured using an Li-250 light meter with a Li-190SA pyranometer
based carbohydrates are mainly in the form of starch and cellulose sensor (Li-COR, Inc., Lincoln, Nebraska, USA).
(with the absence of lignin), are thus much easier to convert to
monosaccharides when compared with lignocellulosic materials 2.2. Operation of photobioreactor
(Harun et al., 2010; Ho et al., 2012; John et al., 2011). Microalgae
like Chlorella, Chlamydomonas, Dunaliella, Scenedesmus, and Tetra- The photobioreactor (PBR) used to grow the microalga was a 1-L
selmis have been shown to accumulate a large amount of carbohy- glass vessel illuminated with an external light source (14 watt TL5
drates (>40% of the dry weight) (John et al., 2011). For example, tungsten filament lamps; Philips Co., Taipei, Taiwan) mounted on
many researchers have reported that the genus of Chlorella possess both sides of the PBR. The microalgal strains were pre-cultured
has a high carbohydrate content, especially the species of C. vulga- and inoculated into the photobioreactor with an inoculum size of
ris, with carbohydrates being 37–55% of its dry weight (Brennan 0.14 g L 1. The PBR was operated at 28 °C, pH 6.2, and an agitation
and Owende, 2010; Dragone et al., 2011; Illman et al., 2000). rate of 300 rpm. Serving as the sole carbon source, 2.0% CO2 was
The carbohydrates in green algae mainly come from starch in fed into the microalgae culture continuously during cultivation
chloroplasts and cellulose/polysaccharides on cell walls with an aeration rate of 0.2 vvm. The liquid sample was collected
(Domozych et al., 2012; Richmond, 2004), which are not readily from the sealed glass vessel with respect to time to determine mic-
fermentable for ethanol production by microorganisms. Therefore, roalgae cell concentration, pH, lipid/carbohydrate content, and
prior to ethanol fermentation, the polysaccharides of microalgae residual concentration of the nitrogen source.
should be hydrolyzed to fermentable sugars (Hahn-Hagerdal
et al., 2007). In general, chemical (acid and alkaline) or enzymatic 2.3. Determination of microalgae cell concentration
hydrolysis are common methods used for this purpose. While acid
hydrolysis is faster, easier and cheaper than other types of hydro- The cell concentration of the microalgae culture was deter-
lysis, the acidic conditions may lead to decomposition of the sugars mined regularly by measuring optical density at wavelength
into unwanted compounds that inhibit the fermentation process 688 nm (denoted as OD688) using a UV/VIS spectrophotometer
(Girio et al., 2010; Harun et al., 2010; Moxley and Zhang, 2007). (model U-2001, Hitachi, Tokyo, Japan) after proper dilution with
In contrast, enzymatic hydrolysis is slower and much more expen- deionized water to give an absorbance range of 0.05–0.90. The
sive than acidic hydrolysis (Lynd et al., 2002), but it is an environ- dry cell weight (DCW) of the microalgae biomass was estimated
mentally benign process and can obtain higher glucose yields by filtering 50 ml aliquots of culture through a cellulose acetate
without producing inhibitory products. In addition to the cost is- membrane filter (0.45 lm pore size, 47 mm in diameter). Each
sue, enzymatic hydrolysis has another drawback of requiring costly loaded filter was dried at 105 °C until the weight was invariant.
or energy-consuming physical or chemical pretreatments of the The dry weight of the blank filter was subtracted from that of
biomass to enhance the hydrolysis efficiency. To date, few studies the loaded filter to obtain the microalgae dry cell weight. The
have reported using microalgae for ethanol production (Harun and OD688 values were converted to biomass concentration via appro-
Danquah, 2011; Harun et al., 2010), while there are some examples priate calibration between OD688 and dry cell weight. The conver-
describing the use of macroalgae (e.g., seaweed) for ethanol fer- sion factor was determined as follows: 1.0 OD688 equals
mentation (John et al., 2011; Wargacki et al., 2012). approximately 0.20 g DCW L 1.
In this study, an indigenous microalgal isolate (identified as
C. vulgaris FSP-E), with carbohydrates accounting for over 50% of 2.4. Determination of residual concentration of nitrogen source
its dry weight, was used as feedstock for producing ethanol via
fermentation with Zymomonas mobilis. The effects of various The urea concentration of the culture medium was analyzed
hydrolysis methods and conditions on saccharification of the mic- with manual urease assay procedures proposed by Butler
roalgal biomass were investigated. Several factors influencing the et al.(1996). Samples (1.0 mL each) were taken from the microal-
hydrolysis efficiency were examined, including hydrolysis strate- gae culture, and were then centrifuged at 10,000 rpm for 5 min.
gies (enzymatic or acidic), hydrolytic enzyme composition, sulfuric The supernatant was taken (100 lL) and added to 0.5 mL urease
acid concentration, hydrolysis time, and microalgae loading. The solution. The mixture was placed in a water bath set at 37 °C for
efficiency of converting the hydrolyzed microalgal biomass to bio- 10 min. After that, the reaction mixture was removed from the
ethanol was also assessed by separate hydrolysis and fermentation water bath, and the reaction was stopped with 1.0 mL phenol
(SHF) and simultaneous saccharification and fermentation (SSF) nitroprusside (enzyme terminator).1.0 mL alkaline hypochlorite
processes. The aim of this work was to evaluate the feasibility of (chromogenic agent) was then added with mixing. After setting
using high-sugar-content microalgal biomass as feedstock for at room temperature for 30 min, the optical density of the sample
bioethanol production. was measured at a wavelength of 570 nm (i.e., OD570) using a spec-
trophotometer (model U-2001, Hitachi, Tokyo, Japan). The urea
concentration was estimated by the OD570 value based on the fol-
2. Methods lowing correlation: 1.0 OD570 = 68.5 mg L 1 urea.

2.1. Microalga strain and growth medium 2.5. Determination of biochemical composition of C. vulgaris FSP-E

Microalgal C. vulgaris FSP-E was isolated from a freshwater area The carbohydrate content and composition in microalgae were
located in southern Taiwan. Modified Basal medium was used for determined using the modified quantitative saccharification (QS)
both pre-culture and culture of this strain, consisting of (g/L): urea, method reported by the National Renewable Energy Laboratory
0.56; KH2PO4, 1.25; MgSO4
7H2O, 1.00; CaCl2, 0.0835; H3BO3, (NREL), USA (Moxley and Zhang, 2007). A small amount of dry
0.1142; FeSO4
7H2O, 0.0498; ZnSO4
7H2O, 0.0882; MnCl2
4H2O, algae powder was added to 3 ml 72% (w/w) sulfuric acid and incu-
0.0144; MoO3, 0.0071; CuSO4
5H2O, 0.0157; Co(NO3)2
6H2O, bated for 20 min at 30 °C for the primary hydrolysis. The hydroly-
0.0049; EDTA
2Na, 0.50. Under the pre-culture condition, the mic- sate was then diluted to 4% (w/w) sulfuric acid and incubated for
roalgal strain was grown with continuous CO2 (2%) feeding and 20 min at 121 °C (sterilization) as the secondary hydrolysis. The
under illumination with a TL5 tungsten lamp at a light intensity supernatant was neutralized and analyzed by high performance
 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198 193

liquid chromatography for sugar assays. Moreover, the starch con- the pre-cultured Z. mobilis cells were centrifuged at 10,000 rpm
tent in the microalgal biomass was determined by the hydrolysis of for 10 min and added to the solution containing the hydrolyzed
starch to glucose with amylolytic enzymes (a-amylase and amylo- microalgal biomass at an inoculum size of 10% (optical density
glucosidase) according to the Megazyme total starch analysis pro- OD600 was about 2.0) to carry out ethanol fermentation at 30 °C
cedures (AA/AMG) (McCleary et al., 1997). in a desktop fermentor.
The total nitrogen content of the microalga was first detected by The SHF process was also conducted by using dilute acid for
an elemental analyzer (Elementar vario EL III), and then the crude hydrolysis. The pretreated microalgal biomass (50 g L 1) was
protein concentration was obtained according to the correlation mixed with 1.0% sulfuric acid to carry out acid hydrolysis based
reported in the literature (i.e., protein concentration = nitrogen on the procedures mentioned earlier. After that, the acidic hydroly-
content  6.25) (Becker, 1994). sate was collected via centrifugation (10,000 rpm for 10 min), and
The lipid composition was determined as fatty acid methyl es- the pH of the hydrolysate was then adjusted from 5.0 to 6.0 with
ters (FAMEs) through the direct transesterification method pro- CaCO3 to reach a suitable pH range for ethanol fermentation with
posed by Lepage and Roy (Yeh and Chang, 2011). The FAMEs in Z. mobilis. The fermentation was performed at 30 °C in a desktop
the sample were analyzed using a gas chromatograph (GC-2014, fermentor.
Shimadzu, Kyoto, Japan) equipped with a flame ionization detector
(FID).
2.8.2. Procedures of simultaneous saccharification and fermentation
(SSF)
2.6. Enzymatic hydrolysis
For the SSF operation, the sonication-pretreated microalgal bio-
mass in an acetate buffer solution (pH 6.0) with a biomass concen-
The enzymes used to hydrolyze carbohydrate components
tration of 20 g L 1 was autoclaved and the solution was used as SSF
(mainly, starch and cellulose) in the microalgae feedstock were ob-
medium. Z. mobilis was inoculated at an inoculum size of 10% (or
tained from an isolated cellulose-hydrolyzing bacterium – Pseudo-
optical density, OD600 = 2.0) and the filter-sterilized enzyme solu-
monas sp. CL3 (Cheng and Chang, 2011). BHM medium was used
tion (consisting of: endoglucanase, 0.61 U mL 1; b-glucosidase,
for the cultivation of Pseudomonas sp. CL3. The BHM medium con-
0.30 U mL 1; amylase, 0.75 U mL 1) were simultaneously added
sisted of (g/L): 0.2; MgSO4
7H2O, 1.0; K2HPO4, 1.0; KH2PO4, 0.05;
into the acetate buffer medium containing pretreated microalgal
FeCl3
6H2O, 0.02; CaCl2, 2.0; Starch (with or without for producing
biomass. SSF was then operated at a temperature 30 °C in a desk-
enzyme mixture 2 and enzyme mixture 1, respectively), 10.0; CMC,
top fermentor.
13.2; Rice Straw, 3.17; Yeast Extract. To prepare the hydrolytic en-
zymes, Pseudomonas sp. CL3 was grown on BHM medium at 37 °C
and 200 rpm for 4 days. A designated amount of the culture broth 3. Results and discussion
was taken and centrifuged (4 °C, 9000 g) for 20 min. The superna-
tant containing a mixture of cellulases and amylase enzymes were 3.1. Effect of nitrogen starvation on carbohydrate accumulation in
concentrated prior to being used for hydrolysis of microalgae bio- C. vulgaris FSP-E
mass. For the enzymatic hydrolysis experiments, dry microalgae
powder was first loaded into an acetate buffer solution (pH 6) with To obtain a large amount of sugar-rich microalgal biomass as
a concentration of 10, 20, 30, and 40 g/L, and slurry was pre-treated feedstock for bioethanol production at a low cost, a carbohydrate
by sonication (Homogenizatory Fsat Prep-24, MP Biomedicals, productivity of C. vulgaris FSP-E (i.e., high biomass productivity
USA) for 10 min. After that, the sample was autoclaved at 121 °C with sufficient carbohydrate content) much be obtained. How to
for 20 min, and then a fixed amount of enzyme mixture was added trigger the synthesis of carbohydrates in microalgae has attracted
into the pretreated microalgae biomass to conduct enzymatic considerable research interest worldwide. Several recent reports
hydrolysis at an agitation rate of 200 rpm and constant tempera- (Dragone et al., 2011; Ho et al., 2012; Yeh and Chang, 2011) have
ture of 45 °C. The enzyme mixture consisted of: endoglucanase, demonstrated that cultivation under nitrogen deficient conditions
0.65 U mL 1; b-glucosidase, 1.50 U mL 1; amylases, 0.09 U mL 1. leads to a sharp increase in the lipid or carbohydrate content, be-
cause under the nitrogen-depletion condition, the proteins or pep-
2.7. Acid hydrolysis tides accumulated inside the microalgae cells may be transformed
to lipids or carbohydrates (Ho et al., 2012; Rismani-Yazdi et al.,
The different loadings (10–80 g/L) of lyophilized microalgae 2011). However, from the engineering aspect, it is necessary to ex-
powders were mixed with sulfuric acid to reach at a final acid con- plore the optimal time length of cultivating the microalgae cells
centration of 0.1–5.0% (v/v). The resulting slurries were then auto- under nitrogen-starvation conditions to achieve the highest carbo-
claved at 121 °C for 20 min. After hydrolysis, the samples were hydrate production efficiency. In this work, the C. vulgaris FSP-E
cooled to room temperature, centrifuged at 4 °C and 9000g for cells were cultivated under a light intensity of 450 lmol m 2 s 1
20 min, and the supernatant containing the released sugars was for around 5.3 days with nitrogen deficient conditions, and the pro-
collected as acidic hydrolysate, the sugar content and composition tein, lipid, and carbohydrate contents were monitored with respect
of which was measured. to cultivation time. As shown in Fig. 1, during the nitrogen starva-
tion period, the protein content decreased from 58.8% to 20.1%,
2.8. Bioethanol production processes whereas the carbohydrate content increased from 13.3% to 54.4%.
The carbohydrate content increased as the starvation time in-
2.8.1. Procedures of separate hydrolysis and fermentation (SHF) creased until reaching a maximum value of 54.4% after 3 days.
The efficiency of the fermentative conversion of hydrolyzed On the other hand, the lipid content did not vary significantly dur-
microalgae biomass to ethanol was examined with an ethanol-pro- ing the nitrogen starvation period. Some studies have shown that
ducing bacterium Z. mobilis ATCC 29191. The pretreated microalgal the carbon flow in microalgae is allocated to energy-rich com-
biomass (at a concentration of 20 g L 1) was first hydrolyzed by en- pounds, such as lipids and carbohydrates, when they are under
zyme 2 (endoglucanase: 0.61 U mL 1, b-glucosidase: 0.30 U mL 1, stress, such as nitrogen deficient conditions, and there is a compe-
Amylase: 0.75 U mL 1) at 45 °C with an agitation rate of 200 rpm tition between synthesis of lipid and starch (Rismani-Yazdi et al.,
until a constant monosaccharide concentration was achieved, as 2011; Siaut et al., 2011). However, the increase in lipid or carbohy-
process that typically needed 2 days. After enzymatic hydrolysis, drate synthesis under stress conditions seems to differ from strain
194 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198

Fig. 1. Time-course profiles of nitrate concentration, protein content, carbohydrate content, and lipid content during the growth of C. vulgaris FSP-E. (light source: TL5; total
light intensity = 450 lmol m 2 s 1; CO2 aeration = 2.0%; CO2 flow rate = 0.2 vvm).

to strain (Ho et al., 2012), and the key factors determining which 100 100
metabolic pathway will be induced under stress would require fur-
ther investigations. In the case of C. vulgaris FSP-E, carbohydrate

Reducing sugars yield (%)

accumulation is predominant under nitrogen starvation. Based on 80 80
Glucose yield (%)

the data shown in Fig. 1, the maximum biomass productivity

(1.363 g L 1d 1), carbohydrate productivity (0.687 g L 1d 1), and 60 60
carbohydrate content (50.39 ± 0.44%) were obtained after a 2-day
nitrogen-starvation period with a total cultivation time of
4.25 days. The chemical composition of the resulting carbohy- 40 Control (glucose) 40
Enzyme 1 (glucose)
drate-rich C. vulgaris FSP-E biomass was characterized. It shows Enzyme 2 (glucose)
that the carbohydrates contributed to 50.39 ± 0.44% (w/w) of dry Control (reducing sugars)
biomass weight, with a starch content of 31.25 ± 1.28% per dry 20 Enzyme 1 (reducing sugars) 20
Enzyme 2 (reducing sugars)
weight. The carbohydrates contain mostly glucose, as the glucose
content was 46.92 ± 0.44% per dry weight (or 93.1% of total carbo- 0 0
hydrates). On the other hand, the protein and lipid content was 0.0 0.5 1.0 1.5 2.0 2.5 3.0
much less than that of the carbohydrates, accounting for Hydrolysis time (days)
23.28 ± 0.03% and 11.59 ± 1.55%, respectively.
Fig. 2. Effect of enzymatic composition on glucose and total sugars yield (Enzyme 1
contains endoglucanase: 0.65 U mL 1, b-glucosidase: 1.50 U mL 1, amylases:
3.2. Effect of enzyme composition on microalgae hydrolysis efficiency 0.09 U mL 1; Enzyme 2 contains endoglucanase: 0.65 U mL 1, b-glucosidase:
0.30 U mL 1, amylases: 0.75 U mL 1; control means that non-added enzyme and
without pretreatment).
The composition of hydrolytic enzymes may significantly affect
the efficiency of enzymatic hydrolysis of microalgae biomass for
obtaining the fermentable sugars, due to the specific structures enzymatic hydrolysis. Enzyme 2 was obtained from the superna-
and proportions of cellulose and starch in C. vulgaris FSP-E. In this tant of the Pseudomonas sp. CL3 strain grown on starch-enriched
study, the enzyme mixtures derived from an isolated Pseudomonas medium, and thus had a higher amylases activity. The composition
sp. CL3 strain were used for enzymatic hydrolysis, which was of enzyme 2 was endoglucanase (0.65 U mL 1), b-glucosidase
conducted at 45 °C, pH 6, 200 rpm agitation rate with sonication- (0.30 U mL 1), and amylases (0.75 U mL 1), with a ratio of
pretreated C. vulgaris FSP-E biomass (at a concentration of 1.00:0.46:1.15 for the three enzymes, respectively. Fig. 2 shows
10 g L 1). The first enzyme mixture used (denoted as enzyme 1) that the hydrolysis efficiency with enzyme 2 was much higher than
consisted of endoglucanase (0.65 U mL 1), b-glucosidase that of enzyme 1. After 1 day of enzymatic hydrolysis, the glucose
(1.50 U mL 1), and amylases (0.09 U mL 1) (the ratio of the three yield reached 79.1%, which was nearly twice that of enzyme 1.
enzymes was 1.00:2.31:0.14). As depicted in Fig. 2, when enzyme Therefore, a higher glucose production rate was obtained when
1 was used to hydrolyze the biomass of C. vulgaris FSP-E, after reac- enzyme 2 was used, and the maximum glucose yield of 90% was
tion for 1 day, the reducing sugar yield reached about 69.8%, while obtained after hydrolysis for 3 days.
the glucose yield was only 30.5%, indicating that the efficiency of
enzyme 1 for obtaining glucose from the microalgae feedstock is
not satisfactory. It is likely that the activity of amylases was not 3.3. Effect of microalgae biomass loading on enzymatic hydrolysis
sufficient, since the majority of carbohydrates in the microalgae efficiency
were starch (accounting for nearly 62% of total carbohydrates).
Therefore, to enhance glucose yield from C. vulgaris FSP-E, a From the prospective of enzyme kinetics, both the substrate
modified enzyme mixture (denoted as enzyme 2) was used for concentration (microalgal cell loading) and enzyme loading are
 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198 195

0.5%, 1.0%, 2.0%, 3.0% and 5.0% (v/v). Hydrolysis of the samples
Maximum glucose concentration (g L )

100
-1

12 Glucose concentration was conducted in an autoclave vessel at 121 °C for 20 min. A glu-

Glucose production yield (%)

Yield cose yield of nearly 100% was obtained at 2.0% or higher acid con-
90 centrations, while a yield of approximately 96.0% was obtained
10
when the acid concentration was 1.0%. This means that the glucose
8 80
yield was only improved by 3.5–4.0% when the acid concentration
was doubled from 1.0% to 2.0%. With regard to the environmental
6 impact and operating cost, a sulfuric acid concentration of 1.0%
70 seems to be preferable for a cost-effective acid hydrolysis process.
4 Moreover, the effect of microalgae biomass loading on the
hydrolysis efficiency is also vital for the commercial process.
60
2 Fig. 4 shows that when using a 1.0% sulfuric acid concentration
as the hydrolysis reagent (i.e., the preferable acid concentration
0 50 based on the result of the acid concentration effect on hydrolysis
10 20 30 40
-1 yield shown above), the glucose yield slightly decreased from
Microalgal biomass concentration (g L ) 98.0% to 95.9% when the microalgae biomass concentration was in-
creased from 10 to 50 g L 1 (or a biomass-to-acid ratio of 1–5)
0 5 10 15 20 25
(Fig. 4). However, the glucose yield decreased dramatically from
Biomass-to-enzyme ratio (g mL-1) 93.6% to 75.8% when the biomass loading was further increased
Fig. 3. Effect of microalgal biomass concentration (or biomass-to-enzyme ratio) on
from 60 to 80 g L 1. Therefore, the most suitable microalgae bio-
glucose concentration and glucose production yield via enzymatic hydrolysis using mass concentration was around 50 g L 1 (or a biomass-to-acid ra-
enzyme 2. (Enzyme 2 consists of endoglucanase: 0.65 U mL 1, b-glucosidase: tio of 50) (glucose yield is over 95%). The reason for this poor
0.30 U mL 1, amylase: 0.75 U mL 1). hydrolysis performance could be due to the high biomass-to-acid
ratio, which may lead to lower exposure (or insufficient contact)
of biomass to the hydrolysis reagents.
critical factors determining the performance of enzymatic hydroly-
The surprisingly high sugar (glucose) yield obtained via dilute
sis. Therefore, the biomass-to-enzyme ratio is an important operat-
acid hydrolysis could be due to the chemical form of the microal-
ing factor, which can be optimized to obtain the highest glucose
gae-based carbohydrates. It is known that starch can be easily
production rate and yield, thus markedly reducing the sugar pro-
hydrolyzed with dilute acids (John et al., 2011), but for lignocellu-
duction cost. The enzymatic hydrolysis efficiency was thus investi-
losic materials derived from terrestrial plants, dilute acid usually
gated using a microalgae biomass concentration of 10–40 g L 1 (or
acts as a form of pretreatment rather than hydrolysis, due to their
a biomass-to-enzyme ratio of 5–20 g biomass/ml enzyme). The
complex structures arising from the presence of lignin (John et al.,
enzymatic hydrolysis conditions were controlled at 45 °C, pH 6,
2011). However, since the cellulose primarily located in the inner
and 200 rpm agitation, with the addition of a fixed amount
membrane of C. vulgaris species is free of lignin, the microalgal cel-
(2 ml) of enzyme 2 consisting of endoglucanase (0.65 U mL 1),
lulose seems to be readily hydrolysable with the dilute acid. This
b-glucosidase (0.30 U mL 1), and amylase (0.75 U mL 1). The re-
makes the saccharification of microalgae biomass much easier than
sults are shown in Fig. 3. When the microalgae biomass concentra-
that of lignocellulosic materials, and this is a clear advantage of
tion was increased from 10 to 40 g L 1 (or a biomass-to-enzyme
using microalgae as the sugar substrate for fermentation compared
ratio of 5–20 g biomass/ml enzyme), the maximum glucose con-
to using lignocellulosic feedstock. There are few studies that focus
centration increased from 3.94 to 10.9 g L 1. When the microalgal
on using microalgae as sugar feedstock for fermentation process
biomass concentration was increased from 10 to 20 g L 1, the glu-
(Harun et al., 2010; John et al., 2011). The advantages of using
cose yield remained constant at approximately 90%. However,
when the microalgae biomass concentration was increased from
20 to 40 g L 1, the glucose yield decreased sharply, from 90% to
about 60%. When considering both the glucose production yield
and operating cost, the most suitable microalgae biomass loading
for the enzyme loading used (i.e., enzyme 2 with a composition
of endoglucanases, 0.65 U mL 1; b-glucosidase, 0.30 U mL 1; amy-
lase, 0.75 U mL 1) is around 20 g L 1. Under this optimal biomass-
to-enzyme ratio, the glucose production titer and glucose yield
were 7.86 g L 1 and 90.4%, respectively (Fig. 3).

3.4. Effect of acid concentration and microalgae biomass loading on

acidic hydrolysis efficiency

In addition to enzymatic hydrolysis, acid hydrolysis was also

used to convert the carbohydrates (mainly starch and cellulose)
in microalgae biomass to simple sugars for fermentative ethanol
production using fermenting microorganisms (e.g., yeast) (John
et al., 2011). The effect of acid concentration on the efficiency of
microalgae biomass hydrolysis was first investigated. Theoretically
and practically, the lowest acid concentration that can reach the
highest hydrolysis performance would be the most favorable. The
lyophilized C. vulgaris FSP-E powders were slurried into water at Fig. 4. Effect of microalgal biomass concentration (or biomass-to-acid ratio) on the
a 1% solid to liquid ratio (w/v), and different amounts of sulfuric acid hydrolysis efficiency of C. vulgaris FSP-E. The acid concentration used for
acid were added to obtain final acid concentrations of 0.1%, 0.2%, hydrolysis was 1%.
196 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198

microalgae, instead of macroalgae, as sugar feedstock are as fol- ethanol concentration of 3.55 g L 1 and ethanol yield of 89.3% (ver-
lows. First, microalgae have much higher sugar productivity than sus the theoretical ethanol yield based on the amount of glucose
macroalgae, due mainly to their faster cell growth rate. Second, released from microalgae after enzymatic hydrolysis). In summary,
the major monosaccharide derived from the carbohydrates pro- the enzymatic hydrolysis of C. vulgaris FSP-E for ethanol production
duced from some microalgae species (such as C. vulgaris) is glucose. via the SHF process achieved an overall yield of 79.9%, which is a
For example, it was found in this study that glucose accounts for percentage of the maximal theoretical ethanol yield based on the
over 90% of total carbohydrates in C. vulgaris FSP-E. In contrast, available glucose in the microalgal biomass.
the major building blocks of polysaccharides found in seaweed Bioethanol production from C. vulgaris FSP-E was also con-
are alginate and mannitol, which are much more difficult to fer- ducted via the SSF process. The biomass of C. vulgaris FSP-E used
ment with yeasts, making it more challenging in converting sea- for the SSF operation contained carbohydrate and glucose contents
weed to bioethanol (Wargacki et al., 2012). of 50.9% and 48.0%, respectively. Fig. 5(b) shows the results of this.
The residual glucose concentration remained at about 0.5 g L 1 for
the first 12 h, and then gradually decreased to nearly zero after SSF
3.5. Bioethanol production using biomass of C. vulgaris FSP-E with operation for 36 h. Meanwhile, the ethanol concentration in-
acidic or enzymatic hydrolysis via SHF and SSF processes creased sharply to 4.17 g L 1 within 36 h, with nearly complete
consumption of glucose, giving an ethanol yield of 0.209 g
Fermentative conversion of hydrolyzed microalgae biomass to ethanol/g biomass (or 87.1% versus theoretical yield). The ethanol
ethanol was investigated by using the ethanol-producing strain Z. production in the SSF process did not vary significantly from 36
mobilis ATCC 29191 with Separate Hydrolysis and Fermentation to 60 h (Fig. 5(b)). The maximum ethanol concentration and etha-
(SHF) and Simultaneous Saccharification and Fermentation (SSF) nol yield were 4.27 g L 1 and 92.3%, respectively. These results
processes. The C. vulgaris FSP-E biomass used in this work con- show that the SSF process seems to be a better one for enzymatic
tained carbohydrate and glucose contents of 46.86 ± 0.45% and hydrolysis-based bioethanol production, since it not only simpli-
43.5 ± 0.34%, respectively. Fig. 5(a) shows the time course profile fies the reaction procedures and shortens the reaction time re-
of residual sugar and ethanol concentrations during the SHF pro- quired to reach maximum ethanol production, but also achieves
cess. After 48 h of enzymatic hydrolysis at 45 °C, pH 6, and a higher ethanol yield (92.3% of theoretical yield), in contrast to
200 rpm agitation, the glucose concentration and glucose yield an 79.9% yield obtained from using the SHF process.
were 7.78 g L 1 and 87.17%, respectively. Z. mobilis was then inoc- The acid-hydrolyzed microalgae biomass was also used for
ulated at 48 h to perform ethanol fermentation at a constant tem- ethanol production via the SHF process using the optimal acidic
perature of 30 °C. Shortly after Z. mobilis inoculation, the glucose hydrolysis conditions (1% sulfuric acid, 121 °C, 20 min) (Fig. 6).
concentration was consumed significantly, which was accompa- As the control experiment, pure glucose with a concentration
nied by a sharp increase in ethanol concentration, achieving an equivalent to that in the acidic hydrolysate (i.e., 22–24 g L 1). This
glucose concentration was based on complete hydrolysis of the
microalgal biomass (50 g/L; 0.47–0.48% glucose content), which
10 5
a. SHF gives the theoretically highest glucose concentration of around
22–24 g L 1. The initial pH levels during hydrolysis were controlled
Glucose concentration (g L )

Ethanol concentration (g L )
-1

-1

Enzymatic hydrolysis Fermentation at 5–6. After acidic hydrolysis of C. vulgaris FSP-E, the hydrolysate
8 4
with a glucose concentration of 23.6 g L 1 was obtained. Z. mobilis
cells were inoculated into the microalgae hydrolysate to carry out
6 3 ethanol fermentation at 30 °C. Shortly after inoculation, the glu-
cose concentration dropped rapidly along, with a sharp increase
in the ethanol concentration for both control and microalgae
4 2
group. For the microalgae group, although the ethanol production
rate is slightly lower than control in this first 4 h, a maximum eth-
2 1 anol concentration of 11.66 g L 1 and an ethanol yield of 1.93 mol
Glucose ethanol/mol glucose (or 96.95% out of the theoretical ethanol yield)
Ethanol
0 0
Glucose b. SSF
Glucose concentration (g L )

25 14
Ethanol concentration (g L )
-1

Ethanol
-1

4
3
Ethanol concentration (g L )
Glucose concentration (g L )

-1

12
-1

20
3 10
2
15
8
2
6
1 10
1
4
Control (ethanol conc.)
5 Microalgae (ethanol conc.)
0 0 Control (glucose conc.) 2
0 10 20 30 40 50 60 Microalgae (glucose conc.)
Reaction time (hour) 0 0
5 10 15 20 25
Fig. 5. Time courses of ethanol and glucose production by Z. mobilis cells via (a) the Reaction time (hour)
SHF and (b) SSF processes (substrate: 20 g L 1 of pretreated microalgal biomass;
enzyme dosage: endoglucanase: 0.65 U mL 1, b-glucosidase: 0.30 U mL 1, amylase: Fig. 6. Effect of acid hydrolysis on microalgal biomass for ethanol production by the
0.75 U mL 1). SHF process (control means that fermentation with original incubation medium).
 S.-H. Ho et al. / Bioresource Technology 135 (2013) 191–198 197

Table 1
Comparison of the ethanol concentration and yield of indigenous C. vulgaris FSP-E via different hydrolysis methods with the performance given in other studies.

Hydrolysis Hydrolysis sources Hydrolysis Initial algal biomass Glucose Ethanol Ethanol yield (g ethanol References
method type concentration (g L 1) (g L 1) (g L 1) (g algae)-1)
Enzymatic Cellulases + Amylases SHF 50 N.D. N.D. 0.080 Eshaq et al.
(2010)
a
Two-stage Sulfuric acid + Cellulases SHF N.D. 5.4 2.02 0.079 Wang et al.
(2011)
Physicalb Supercritical CO2 SHF 10 N.D. 3.83 0.383 Harun et al.
(2010)
Enzymaticc Amylases + Glucanase + Xylanase SHF 22 8.6 N.D. N.D. Marsalkova et al.
(2010)
Thermal - Sulfuric acid SHF 50 28.5 14.6 0.292 (Nguyen et al.,
chemical 2009)
Enzymatic Amylases SHF 50 N.D. 11.73 0.235 Sim et al. (2010)
Enzymatic Cellulases + Xylanases + Amylases SHF 100 23.3 N.D. N.D. Rodrigues and
Bon (2011)
Enzymatic Cellulases + Amylases SHF 20 7.78 3.55 0.178 (This study)
Enzymatic Cellulases + Amylases SSF 20 N.D. 4.27 0.214 (This study)
Chemical Sulfuric acid SHF 50 23.6 11.66 0.233 (This study)

N.D.: not determined.

a
Algal biomass was homogenized in a homogenizer.
b
The extracted microalgal biomass was obtained after supercritical oil extraction process.
c
Algal biomass was pretreated by mechanical disintegration.

were achieved within 12 h of ethanol fermentation. This SHF pro- Programs of the Japan Science and Technology Agency (JST).
cess achieved an overall yield of 87.59%, which is a percentage of Support from the Top University Project of NCKU and by Taiwan’s
the maximal theoretical ethanol yield based on the available glu- National Science Council under grant numbers NSC 101-3113-
cose in the microalgal biomass. Acidic hydrolysis usually generates P-110-002, NSC 101-3113-E-006-015, and NSC 101-3113-E- 006-
some unwanted compounds that may inhibit ethanol fermentation 016 is also acknowledged.
(Moxley and Zhang, 2007). Fortunately, the proposed dilute acidic
hydrolysis conditions did not lead to a significant inhibition of eth-
anol fermentation with the Z. mobilis strain, Therefore, dilute acid References
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