Bioresource Technology 196 (2015) 2227

Contents lists available at ScienceDirect

Bioresource Technology

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

Bioethanol production from carbohydrate-enriched residual biomass

obtained after lipid extraction of Chlorella sp. KR-1

Ok Kyung Lee , You-Kwan Oh , Eun Yeol Lee

a,

Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Republic of Korea
Research, Daejeon 305-343, Republic of Korea

Clean Fuel Department, Korea Institute of Energy

highlights

Chlorella sp. KR-1 was accumulated 36% (w/w) of carbohydrates and 38% (w/w) of lipids.

The lipid was converted to biodiesel while lipid-extracted residual biomass was used for bioethanol fermentation. A saccharification yield of
almost 100% was obtained using dilute acid method.

A 0.4 g ethanol/g fermentable sugar and 0.16 g ethanol/g residual biomass were produced.
Received in revised form 8 July 2015 Accepted 10 July 2015

article info
Available online 20 July 2015
Article history:

Keywords:
Received 11 June 2015

Chlorella sp. KR-1

Residual biomass

Saccharification

Bioethanol fermentation

abstract

The residual biomass of Chlorella sp. KR-1 obtained after lipid extraction was used for
saccharification and bioethanol production. The carbohydrate was saccharified using simple
enzymatic and chemical methods using Pectinex at pH 5.5 and 45 LC and 0.3 N HCl at 121
LC for 15 min with 76.9% and 98.2% yield, respec-tively, without any pretreatment. The
residual biomass contained 49.7% carbohydrate consisting of 82.4% fermentable sugar and
17.6% non-fermentable sugar, which is valuable for bioethanol fermentation. Approximately
98.2% of the total carbohydrate was converted into monosaccharide (fermentable + nonfermentable sugar) using dilute acid saccharification. The fermentable sugar was
subsequently fermented to bioethanol through separate hydrolysis and fermentation with a
fermentation yield of 79.3%. Overall, 0.4 g ethanol/g fermentable sugar and 0.16 g ethanol/g
residual biomass were produced.

2015 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.biortech.2015.07.040 0960-8524/ 2015

Elsevier Ltd. All rights reserved.

1. Introduction

Concerns over fossil fuel shortages and increasing CO2

emis-sions have increased interest in the production of
biofuels derived from biomass (Harun et al., 2011; Lin and
Tanaka, 2006). Biodiesel and bioethanol are considered
promising alternatives to petroleum-based diesel and
gasoline, respectively (Chisti, 2007; Ho et al., 2013).
Biofuel production from first-generation biomass, such as
sugarcane and corn, has evoked concerns over using
food for energy production. The use of second-generation,
lignocellu-losic materials, such as rice straw and switch
grass, is still problem-atic owing to the pretreatment
needed to process lignin (Naik et al., 2010; Nigam and
Singh, 2011).

Microalgae have recently been considered as a promising

third-generation feedstock for biofuel production (Lam and
Lee, 2012; Norsker et al., 2011). Generally, microalgae
are used for

Corresponding author. Tel.: +82 31 201 3839; fax: +82 31 204 8114. Email address: eunylee@khu.ac.kr (E.Y. Lee).

biodiesel production because of their high lipid content, up

to 80% of the dry mass (Hu et al., 2008; Libessart et al.,
1995). However, mainly due its high production cost,
production of bio-diesel from microalgae has not been
implemented on an industrial scale (Singh and Gu, 2010;
Lardon et al., 2009). One solution for reducing the cost of
biofuel production is the biorefinery that can convert all
cellular components of the microalgae into biofuels and
various valuable chemicals (Wijffels et al., 2010; Wijffels
and Barbosa, 2010).

Some microalgae, like Chlorella, Dunaliella, and

Tetraselmis, have been shown to accumulate a large
amount of carbohydrates (>40% of the dry weight), thus
they are considered as good candidates for bioethanol
feedstock (Ho et al., 2012, 2013; John et al., 2011). Most
of the carbohydrates contained in microalgae are in the
form of starch and cellulose. The absence of lignin
indicates this material can be easily converted into usable
monosaccharides (Harun and Danquaha, 2011; Harun et
al., 2011). Residual microalgae biomass after lipid
extraction for biodiesel production can also be used for
bioethanol fermentation due to the significant amount of

O.K. Lee et al. / Bioresource Technology 196 (2015) 2227

23

remaining carbohydrates. In the case of

Dunaliella, the carbohy-drate is mostly
present as starch (Lee et al., 2013a). These
carbohy-drates have been successfully
saccharified into glucose, and the resulting
saccharification broth was used for bioethanol
fermenta-tion without any pretreatment (Lee
et al., 2013a). This kind of sequential
production of biodiesel and bioethanol can
improve the overall cost-effectiveness of
microalgae-based production of biofuels.

Chlorella sp. KR-1 accumulates total lipids at

up to 40% of its dry mass (Lee et al., 2013c;
Na et al., 2011). Lee et al. were able to completely extract the lipids from Chlorella sp.
KR-1 biomass using a (7:3, v/v) mixture of
dimethyl carbonate (DMC) and methanol, followed by transesterification into fatty acid
methyl esters (FAMEs) with a productivity of
293.8 mg/g biomass (Lee et al., 2013b). In
the present study, the total lipid-extracted
residual biomass of Chlorella sp. KR-1, in
which carbohydrates represent greater than
49.7% of its dry mass, was used as feedstock
for bioethanol produc-tion. Various enzymatic
and dilute acid saccharifications of the
residual biomass were investigated without
any pretreatment. Saccharomyces cerevisiae
was used for bioethanol fermentation using
the saccharification broth made from the
residual biomass of Chlorella sp. KR-1
directly without any pretreatment.

rate of 0.75 L/min (Lee et al., 2013c). Cells

were harvested by centrifugation (4000 rpm,
10 min), washed with DI water, and then
freeze-dried (FD5512, Il Shin BioBase Co.,
Korea) for four days or longer. The harvested
cells were stored at 20 LC until use.

2.2. Lipid extraction and lipase-catalyzed

transesterification of triglycerides from
Chlorella sp. KR-1 for biodiesel production

The total lipid extraction from the freeze-dried

Chlorella sp. KR-1 was carried out using
dimethyl carbonate and methanol (7:3, v/v)
under magnetic stirring at 60 LC for 12 h (Lee
et al., 2013b). After lipid extraction, the
extraction mixture was filtered to separate the
lipid solution and lipid-extracted residual
biomass. The residual biomass was dried at
room temperature for three days.

For the synthesis of FAMEs, a 20% (w/w)

Novozyme 435 was used based on the
amount of extracted lipid in 10 mL of reaction
mixture for 6 h at 60 LC. Samples were
periodically taken and fil-tered using syringe
filters (porosity 0.20 lm, hydrophobic) in order
to remove reaction residues and lipase.
These samples were then analyzed using gas
chromatography (GC) to determine the
degree of FAME conversion.

2. Methods

2.1. Cultivation and growth conditions of

Chlorella sp. KR-1

The culture medium compositions for

Chlorella sp. KR-1 were as follows (Na et al.,
2011): KNO3, 3 mM; KH2PO4, 5.44 mM;

2.3. Determination of biochemical

composition of whole Chlorella sp. KR-1 cells
and lipid-extracted residual biomass

The lipid content of the dried Chlorella sp.

KR-1 was determined using the Soxhlet
method (AOAC, 2000; method 920.39). The

Na2HPO4, 1.83 mM; MgSO4 7H2O, 0.20 mM;

CaCl2, 0.12 mM; FeNaEDTA, 0.03 mM;
ZnSO4 7H2O, 0.01 mM; MnCl2 4H2O, 0.07
mM; CuSO4, 0.07 mM; Al2(SO4)3 18H2O, 0.01
mM. Chlorella sp. KR-1 was culti-vated using
a Pyrex bubble-column reactor (working
volume: 6 L) equipped with 12 fluorescent
lamps on the front, right, and left sides (light
2

intensity: 80 lmol/m s) at 30 LC. The reactor

was sup-plied with 10% (v/v) CO2 in air at a

carbohydrate content was determined using

analytical methods of the National Renewable
Energy Laboratory (NREL) (Sluiter, 2011).

The carbohydrate content and composition

from the microalgae were also determined
using methods established by NREL (Moxley

and Zhang, 2007). A small amount of freezedried microalgae bio-mass was added to 0.5
mL of 72% (w/w) sulfuric acid and then
incubated for 30 min at 30 LC. The
hydrolysate was diluted to 4% (w/w) sulfuric
acid and incubated for 20 min at 121 LC
(steriliza-tion). The supernatant was
neutralized and analyzed using high-pressure
liquid chromatography (HPLC). Using the
direct transesterification method proposed by
Jo et al. (2014), the lipids were determined to
be composed of FAMEs. The protein content
was analyzed using a micro-Kjedahl method
(AOAC, 2000; method 976.05). The ash
content was determined by comparing the
sample weight before and after heating in a
furnace at 600 LC for 12 h.

2.4.2. Dilute acid saccharification

For dilute acid saccharification, 5% (w/v) of

the residual biomass or of whole cells were
autoclaved at 121 LC for 15 min in the presence of HCl or H2SO4 (0.1, 0.3, 0.5, 0.7, 1 N).
After dilute acid hydrolysis, the samples were
cooled to room temperature and cen-trifuged
at 7000g for 20 min. The supernatant
containing the released reducing sugars was
collected as dilute acid hydrolysate, the sugar
content and composition of which were
determined using the DNS method.

2.4. Saccharification of lipid-extracted

residual biomass and whole Chlorella sp. KR1 cells

2.4.1. Enzymatic saccharification

The enzymatic saccharification of Chlorella

sp. KR-1 was con-ducted using various
commercial enzymes. Polygalacturonase
(Pectinex Ultra SP-L with 9500
polygalacturonase units (PGU)/mL),
amyloglucosidase (AMG 300L with 300
amyloglucosi-dase units (AGU)/mL), cellulase
(Celluclast 1.5L with 700 endoglu-canase
units (EGU)/mL) and Viscozyme L (with 100
fungal b-glucanase units (FBG)/g) were
purchased from Novozymes (Denmark). For
enzymatic saccharification, 5% (w/v) of the
residual biomass or whole cells were
incubated at various temperatures (3555
LC) and pH (3.56.5) in the presence of
Celluclast 1.5L, AMG 300L, Viscozyme L or
Pectinex Ultra SP-L. Enzymes were used in
the range of 0.082.4 mL/g based on the dry
mass of the residual biomass. The batch
saccharification reaction was performed in a 1
L Erlenmeyer flask (at 45 LC, pH 5.5, 0.8 mL
enzyme/g residual biomass) while shaking at
230 rpm. Next, 1 mL of the reaction mix-ture
was periodically sampled, and the amount of
reducing sugars was determined using the
DNS method.

2.5. Bioethanol fermentation

2.5.1. Separate hydrolysis and fermentation

(SHF) processes

S. cerevisiae KCTC 7931 (ATCC 20626) was

used for bioethanol fermentation. To produce
a seed culture, S. cerevisiae was cultured in a
50 mL falcon tube with 5 mL culture medium
at 30 LC and 180 rpm for 16 h. The
composition of the culture medium was as
follows: yeast extract, 3 g/L; malt extract, 3
g/L; peptone, 5 g/L; dextrose, 10 g/L (Lee et
al., 2011). For SHF processing of enzymatic
hydrolysate, 3 mL of the pre-cultured S.
cerevisiae was inoculated with 200 mL of the
enzymatic hydrolysate containing reducing
sugars in a 500 mL culture flask at 30 LC and
180 rpm for 20 h. SHF processing was also
conducted using dilute acid hydrolysis. The
acid hydrolysate was collected via
centrifugation (7000 rpm for 20 min) and the
pH of the hydrolysate was then adjusted to
6.0 with a 4 N NaOH solution to reach a
suitable pH range for etha-nol fermentation
with S. cerevisiae. The fermentable sugar

24

O.K. Lee et al. / Bioresource Technology 196 (2015) 2227

3.6

Table 1

5.8
Total
100

Compositions of whole Chlorella sp. KR-1 cells and the

lipid-extracted residual biomass.

100

Components
Whole cell
Lipid-extracted residual biomass

consumption and amount of produced

ethanol were determined periodically.

Carbohydrates

2.5.2. Simultaneous saccharification and

fermentation (SSF)

36.1
49.7
Glucose
(29.9)
(40.9)
Galactose/xylose
(2.3)

As the SSF medium, 5% of the residual

biomass was mixed with a 50 mM sodium
acetate buffer solution (pH 6.0). S. cerevisiae
was inoculated with 1.5% (v/v) of the SSF
medium and the filter-sterilized Pectinex
solution (0.8 mL/g biomass). The sodium
acetate buffer medium containing the residual
biomass was then added to this mixture.

(3.1)
Rhamnose

2.6. Analysis

(1.9)
(2.8)
Arabinose
(2.0)
(2.8)
Lipid
38.0
6.5
Protein
16.6
28.5
Ash
5.9
9.5
Moisture

The amount of reducing sugar was

determined using the DNS (3,5-dinitrosalicylic
acid) method with glucose (from 0.1 to 2.0
mg/mL) used as the standard. After mixing a
0.5 mL sample with 1.5 mL of the DNS
reagent, the mixture was heated at 100 LC
for 10 min, and 8 mL of water was added.
The absorbance of the mixture was analyzed
using a spectrophotometer at a wave-length
of 550 nm. The compositions of sugars and
ethanol were identified and quantified using
HPLC (Jasco, Japan). The chro-matography
was equipped with an Aminex HPX-87H
column (Bio-rad) and a refraction index
detector. A 5 mM H2SO4 solution at a flow
rate of 0.6 mL/min was used as the eluent. A
40 lL sample was injected at 60 LC. The
FAMEs within the sample were analyzed
using a gas chromatograph (M600D, Young
Lin Instrument, Korea) equipped with an
InterCap WAX column (GL Sciences, 30 m

length 0.53 mm diameter) and a flame

ionization detector (FID).

while protein constituted 28.5% of the lipidextracted residual biomass, followed by ash
at 9.5%. Both the whole cell and residual
biomass contained a significant amount of
carbohydrates and thus can be used for
bioethanol fer-mentation after
saccharification.

3. Results and discussion

3.1. Two-step method for biodiesel production

from Chlorella sp. KR-1

FAMEs were synthesized using a two-step

method consisting of DMC-mediated lipid
extraction, followed by Novozyme 435catalyzed transesterification of the extracted
lipids into FAMEs. A mixture of DMC and
methanol was used as the extraction solvent
for the microalgae lipids; DMC was then
employed as the

substrate for the synthesis of FAMEs from

triglycerides. The FAMEs were synthesized in
a yield of 250298 mg FAMEs per gram of
microalgae biomass.

3.2. Composition analysis of whole Chlorella

sp. KR-1 cells and residual biomass after lipid
extraction

The compositions of whole Chlorella sp. KR-1

cell and the lipid-extracted residual biomass
are shown in Table 1. Using only the DMC
and methanol mixture (7:3, v/v), the total
lipids extracted was approximately 38.0%
(w/w). The carbohydrates account for 36.1%
of the dry weight in whole cells of Chlorella
sp. KR-1. Glucose, galactose, xylose,
arabinose, and rhamnose represented
82.8%, 5.5%, 0.9%, 5.3% and 5.4% of the
weight, respectively. Thus, the amount of
fermentable sugars was 82.8% of the total
monosac-charides, suggesting that Chlorella
sp. KR-1 is suitable for bioetha-nol
fermentation. The carbohydrate content in the
lipid-extracted residual biomass was 49.7%,

3.3. Saccharification of the lipid-extracted

residual biomass

Enzymatic saccharification and dilute acid

saccharification were used to convert the
carbohydrates from the lipid-extracted residual biomass into sugars for bioethanol
fermentation. Several enzymes were
evaluated for saccharification capability while
vary-ing pH (3.56.5) and temperature. As
shown in Table 2, when the AMG 300L was
used to hydrolyze the residual biomass of
Chlorella sp. KR-1, 13.7 g/L of reducing
sugars were produced after 1 h of reaction
time. Meanwhile, the saccharification yield
was only 55.1% (based on the total
carbohydrate of the residual bio-mass),
indicating that the efficiency of AMG 300L for
saccharifica-tion is not satisfactory. It is likely
that the activity of amyloglucosidase was not
sufficient since the major carbohydrate
contained in Chlorella sp. is starch, followed
by galactan, xylan, ara-binan and rhamnose
(Gerken et al., 2013; Kim et al., 2014).
Pectinex exhibited the best saccharification
efficiency under the given conditions. For 1 h
of reaction time, a sugar yield of 70.4%
(based on the total carbohydrate of the
residual biomass) was obtained at pH 5.5 and
45 LC. Pectinex has the ability to break down
undisrupted cell wall carbohydrates of
residual biomass. Also, to hydrolyze the
major carbohydrate (starch), no additional
enzymes were needed since Pectinex from
Aspergillus contains a-amylases, cellobiases,
and xylanases (Botella et al., 2007; Mamma et
al., 2008; Tengerdy and Szakacs, 2003).
Because the bio-mass to enzyme ratio is an
important factor, the Pectinex concen-tration for
enzymatic saccharification was optimized for the
lipid-extracted residual biomass of Chlorella sp.
KR-1. The concen-tration of Pectinex was varied
in the range of 0.082.4 mL/g resid-ual biomass.
The maximum yield of saccharification was
approximately 75% in the presence of 0.8 mL
Pectinex/g residual

Table 2

Effects of enzyme, reaction temperature and pH on the enzymatic saccharification of the lipid-extracted residual
biomass from Chlorella sp. KR-1. Enzymatic saccharification condition: 0.8 mL enzyme/g residual biomass and 1 h.

35 LC

45 LC

55 LC

pH 3.5
pH 4.5
pH 5.5
pH 6.5
pH 3.5
pH 4.5
pH 5.5
pH 6.5

pH 3.5
pH 4.5
pH 5.5
pH 6.5

Pectinex Ultra SP-L

13.3
12.9
12.5
12.2

15.0
16.4
17.5
14.9

14.3
14.9
15.3
14.8

Viscozyme L
10.6
10.9
9.4
8.4

7.6
11.5
12.5
12.4

13.5
11.0
11.6
8.6

AMG 300L
9.4

8.2
2.6
1.1

10.5
11.1
6.0
4.9

13.5
13.7
7.58
5.68

Celluclast 1.5L
0.2
0.3
0.2
0.2

0
0.6
0.7
0.7

0
2.06
2.49
0.73

The numbers in the table represent reducing sugar concentration (g/L).

O.K. Lee et al. / Bioresource Technology 196 (2015) 2227

25

yields
w/w)

60

100

80

Saccharification
(%,

40

20

Whole
Lipid-

cell
extracted

residual

0.1

biomass

0.3
0.5
0.7
1

Total carbohydrate

Acid concentration (N)

36.1
49.7
Fig. 1. Effect of dilute acid concentrations on chemical
saccharification of the lipid-extracted residual biomass of
Chlorella sp. KR-1. Dilute acid hydrolysis saccharification condition: 121 LC and 15 min.

Enzymatic
Sugar concentration (g/L)
4.09

Table 3

19.1
saccharification
Saccharification yield

Saccharification of whole cells and the lipid-extracted

residual biomass of Chlorella sp. KR-1 under optimal
conditions. Enzymatic saccharification condition: 0.8 mL
enzyme/g biomass, pH 5.5, 45 LC, 3 h. Dilute acid
saccharification condition: 0.3 N HCl, 121 LC, 15 min.

22.7
76.9

Saccharification yield

27.4
93.4
Dilute acid
Sugar concentration (g/L)
16.8
24.4
saccharification
Saccharification yield

biomass. Further increase in Pectinex

concentration did not enhance the
saccharification yield. Thus, the most suitable
enzyme concentration was found to be 0.8
mL/g residual biomass. The Pectinexcatalyzed saccharification of the lipidextracted residual biomass took place over 3
h, and the final saccharification yield was
approximately 76.8% based on total
carbohydrates of the residual biomass. The
reducing sugar concentration was 19.1 g/L.

93.3
98.2

Saccharification
yield = (sugar
concentration/total
carbohydrate
of
biomass) * 100.

Saccharification

For dilute acid saccharification, several

concentrations of HCl and H2SO4 ranging
from 0.1 to 1 N were evaluated in an
autoclave at 121 LC for 15 min. Fig. 1 shows
the acid concentration effect on
saccharification efficiency. For
saccharification of 5% (w/v) residual biomass,
0.3 N of HCl produced a saturated
saccharification effi-ciency of 98.2% based on
the amount of total carbohydrate. The most
favorable solution is that with the lowest
possible acid con-centration producing the
highest possible saccharification yield. A
saccharification yield of nearly 100% (based
on fermentable sugar) was obtained using 0.3
N HCl, while the saccharification yield (based
on total carbohydrate) was 98.2%. With
regard to the environmental impact and
operating costs, a hydrochloric acid
concentration of 0.3 N (approximately 2.5%
(v/v)) is cost-effective for dilute acid
saccharification.

The Pectinex-catalyzed saccharification broth

was analyzed using HPLC in order to
determine the monosaccharide composi-tion.
Glucose was the main fermentable sugar,
constituting

yield = (sugar
concentration/fermentable
sugar
of
biomass) * 100.

(A)

20

100

60

10

80

(%)

Concentration (g/L)
15

Ethanol conversion yield

40

20

20

Time (h)

(B)

20

100
0

80
0

(%)

5
10
15

Concentration (g/L)

15

Ethanol conversion yield

40

20

60

10

100

80
0

(%)

5
10

(g/L)
15
20

15

Time (h)

yield

(C)

20

60

20

Concentration

Ethanol conversion

10

40

Time (h)

0
5

Fig. 2. Bioethanol fermentation of the enzymatic

hydrolysates of the lipid-extracted residual biomass of
Chlorella sp. KR-1 using S. cerevisiae and different
processes. (A) SHF using enzymatic hydrolysate, (B)
SSF using enzymatic hydro-lysate, and (C) SHF using
dilute acid hydrolysate (symbol; d: fermentable sugar, s:
bioethanol, j: bioethanol conversion yield).

10
15
20

approximately 93.8% of the total

monosaccharides. Xylose was pre-sent at
about 5.9%, followed by arabinose. The
dilute-acid saccha-rification broth contained
glucose as the main fermentable sugar

26

O.K. Lee et al. / Bioresource Technology 196 (2015) 2227

(82% of the total monosaccharides). Xylose

and galactose were pre-sent at 0.9% and
5.9% weight percent, respectively, followed
by ara-binose (5.7%) and rhamnose (5.9%).

3.4. Saccharification of whole Chlorella sp.

KR-1 cells

For comparison with the saccharification yield

from lipid-extracted residual biomass, whole
cells of Chlorella sp. KR-1 were subjected to
saccharification using enzymatic and dilute
acid methods (Table 3). A yield of 22.7% was
obtained through enzy-matic saccharification,
while a yield of 93.3% was obtained using the
dilute acid saccharification with an acid
concentration of 0.3 N HCl. With regard to
enzymatic saccharification, a high saccharification yield was obtained using residual
biomass rather than the whole cells since the
residual biomass had already been dis-rupted
by DMC and methanol mixture during lipid
extraction. The cell disruption after lipid
extraction was confirmed by compar-ing the
cell morphology of intact cell and solventtreated cell based on scanning electron
microscopic analysis (data not shown).

3.5. Bioethanol fermentation using

saccharification broth of lipid-extracted
residual biomass

Bioethanol fermentation of fermentable

sugars from the resid-ual biomass of
Chlorella sp. KR-1 was investigated using S.
cere-visiae with separate hydrolysis and
fermentation (SHF) process (Fig. 2). The
residual biomass of Chlorella sp. KR-1 had a
glucose content of 82.4%, based on total
carbohydrate of residual biomass. After 3 h of
enzyme saccharification at 45 LC and pH 5.5,
the glu-cose concentration of 17.8 g/L. S.
cerevisiae was then inoculated at 30 LC for
ethanol fermentation. The glucose was
consumed rapidly (initial glucose
concentration: 14.75 g/L), resulting in an
ethanol concentration and yield of 5.9 g/L and
79.3%, respectively.

Bioethanol production from the residual

biomass of Chlorella sp. KR-1 was also
performed via simultaneous saccharification

and fermentation (SSF) process. At a final

glucose concentration of nearly zero, the
ethanol yield was 82.3%. The SSF process
seems more suitable for the lipid-extracted
residual biomass of Chlorella

sp. KR-1 for bioethanol fermentation since it

is a simpler reaction and has a lower reaction
time and a higher ethanol yield, compared to
the SHF process.

The dilute acid hydrolysate from the residual

biomass of Chlorella sp. KR-1 was also used
for bioethanol fermentation via SHF process.
Dilute acid saccharification, however, may
lead to the formation of fermentationinhibitory byproducts due to the high acid
concentration to biomass ratio (Girio et al.,
2010; Moxley and Zhang, 2007). Dilute acid
saccharification products from lignocellulosic
biomass contain several fermentation inhibitors like formic acid or hydroxymethylfurfural,
and fermentative yeast did not completely
consume sugars from the saccharification
broth (Wikandari et al., 2010; Biswas et al.,
2013). In contrast to lignocellulosic biomass,
simple SHF process achieved an ethanol
yield of 79.3% from the diluted acid
saccharification broth of the lipid-extracted
residual biomass of Chlorella sp. KR-1,
indicating that dilute acid saccharification
does not form fermentation inhibi-tors. This is
an advantage of using residual microalgaebased car-bohydrates for ethanol
fermentation compared with lignocellulosic
biomass.

3.6. Mass balance of biodiesel and bioethanol

production from Chlorella sp. KR-1
microalgae

A mass balance of biodiesel and bioethanol

production from 1 kg of Chlorella sp. KR-1
biomass was analyzed (Fig. 3). Whole cells
contain total lipids and carbohydrates of
38.0% and 36.1% (w/w), respectively. From
this sample, 0.3 kg of biodiesel and 0.62 kg of
the lipid-extracted residual biomass were
produced. The residual biomass contained
49.7% carbohydrates (82.4% fermentable
sugar and 17.6% non-fermentable sugar).
Approximately 98.2% of the total
carbohydrates were converted into
monosaccharides (fer-mentable + nonfermentable sugar) using dilute acid

saccharifica-tion. The fermentable sugars

were then subsequently fermented into
bioethanol with a fermentation yield of 79.3%
using the SHF process.

In this study, bioethanol was sequentially

produced using the lipid-extracted residual
biomass of Chlorella sp. KR-1 as

Fig. 3. Mass balance of the production of biodiesel and bioethanol from Chlorella sp. KR-1 microalgae.

O.K. Lee et al. / Bioresource Technology 196 (2015) 2227

27

fermentation feedstock. The lipid-extracted

residual biomass was directly saccharified
and fermented with high efficiency since the
cell wall was disrupted during lipid extraction
step in biodiesel production process. In
another manner, the saccharification broth
generated by enzymatic method was directly
used for bioethanol fermentation without any
pretreatment. The waste residual biomass
generated from biodiesel production process
could be used as feedstock for bioethanol
fermentation, which contributes to economic
feasibility of biofuel production from
microalgae.

4. Conclusion

AOAC (Association of Analytical Chemists),

2000. In: Horwitz, W. (Ed.), Official Methods of
Analysis of Association of Analytical Chemist.
Gaithersburg, Maryland, USA.

Biswas, R., Uellendahl, H., Ahring, B.K., 2013.

Conversion of C6 and C5 sugars in undetoxified
wet exploded bagasse hydrolysates using
Scheffersomyces (Pichia) stipitis CBS6054. AMB
Express 3, 42.

Botella, C., Diaz, A., de Ory Webb, C., Blandino,

A., 2007. Xylanase and pectinase production
by Aspergillus awamori on grape pomace in solid
state fermentation. Process Biochem. 42, 98
101.
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol.
Adv. 25, 294306.

Pectinex-catalyzed and dilute acid

saccharification of lipid-extracted residual
biomass from Chlorella sp. KR-1 generated
19.1 and 24.4 g/L of reducing sugars,
respectively. The dilute acid hydrolysate from
the lipid-extracted residual biomass was
converted into bioethanol by SHF with a yield
of 0.4 g ethanol/g fermentable sugar. The
bioethanol yield based on the lipid-extracted
residual biomass was 0.16 g ethanol/g
biomass. In this study, we demonstrated the
use of lipid-extracted residual bio-mass for
improved economic feasibility of microalgae
biomass for the production of biodiesel and
bioethanol.

Gerken, H.G., Donohoe, B., Knoshaug, E.P.,

2013. Enzymatic cell wall degradation of
Chlorella vulgaris and other microalgae for
biofuels production. Planta 237, 239 253.

Girio, F.M., Fonseca, C., Carvalheiro, F., Duarte,

L.C., Marques, S., Bogel-ukasik, R., 2010.
Hemicellulose for fuel ethanol: a review.
Bioresour. Technol. 101, 4775 4800.

Harun, R., Danquaha, M.K., 2011. Enzymatic

hydrolysis of microalgal biomass for bioethanol
production. Chem. Eng. J. 168, 10791084.

Acknowledgements

This research was supported by the Basic

Science Research Program at the National
Research Foundation of Korea (NRF),
Ministry of Science, ICT and Future Planning
(NRF-2013R1A2A2A01068863). This work
was also supported by a grant from the
Marine Biotechnology Program funded by the
Ministry of Oceans and Fisheries of the
Korean government.

References

Harun, R., Jason, W.S.Y., Cherrington, T.,

Danquah, M.K., 2011. Exploring alkaline pretreatment of microalgal biomass for bioethanol
production. Appl. Energy 88, 34643467.

Ho, S.H., Chen, C.Y., Chang, J.S., 2012. Effect of

light intensity and nitrogen starvation on CO2
fixation and lipid/carbohydrate production of
an indigenous microalga Scenedesmus obliquus
CNW-N. Bioresour. Technol. 113, 244252.

Ho, S.H., Huang, S.W., Chen, C.Y., Hasunuma, T.,

Kondo, A., Chang, J.S., 2013. Bioethanol
production using carbohydrate-rich microalgae
biomass as feedstock. Bioresour. Technol. 135,
191198.

Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M.,

Posewitz, M., Seibert, M., Darzins, A., 2008.
Microalgal triacylglycerols as feedstocks for
biofuel production: perspectives and advances.
Plant J. 54, 621639.

Jo, Y.J., Lee, O.K., Lee, E.Y., 2014. Dimethyl

carbonate-mediated lipid extraction and lipasecatalyzed in situ transesterification for
simultaneous preparation of fatty acid methyl
esters and glycerol carbonate from Chlorella sp.
KR-1 biomass. Bioresour. Technol. 158, 105110.

John, R.P., Anisha, G.S., Nampoothiri, K.M., Pandey, A.,

2011. Micro and macroalgal biomass: a renewable source
for bioethanol. Bioresour. Technol. 102, 186193.

Kim, K.H., Choi, I.S., Kim, H.M., Wi, S.G., Bae, H.J.,
2014. Bioethanol production from the nutrient
stress-induced microalga Chlorella vulgaris by
enzymatic hydrolysis and immobilized yeast
fermentation. Bioresour. Technol. 153, 4754.

Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: a

critical review of issues, problems and the way
forward. Biotechnol. Adv. 30, 673690.

Lardon, L., Hlias, A., Sialve, B., Steyer, J.P., Bernard, O.,
2009. Life-cycle assessment of biodiesel production from
microalgae. Environ. Sci. Technol. 43, 64756481.

Lee, S.J., Oh, Y.H., Kim, D.H., Kwon, D.Y., Lee,

C.G., Lee, J.W., 2011. Converting carbohydrates
extracted from marine algae into ethanol using
various ethanolic Escherichia coli strains. Appl.
Biochem. Biotechnol. 164, 878888.

Lee, O.K., Kim, A.L., Seong, D.H., Lee, C.G., Jung,

Y.T., Lee, J.W., Lee, E.Y., 2013a. Chemo-enzymatic
saccharification and bioethanol fermentation of
lipid-extracted residual biomass of the
microalgae, Dunaliella tertiolecta. Bioresour.
Technol. 132, 197201.

Lee, O.K., Kim, Y.H., Na, J.-G., Oh, Y.-K., Lee, E.Y.,
2013b. Highly efficient extraction and lipase-

catalyzed transesterification of triglycerides

from Chlorella sp. KR-1 for production of
biodiesel. Bioresour. Technol. 147, 240245.

Lee, Y.C., Huh, Y.S., Farooq, W., Chung, J., Han,

J.I., Shin, H.J., Jeong, S.H., Lee, J.S., Oh, Y.K.,
Park, J.Y., 2013c. Lipid extractions from
docosahexaenoic acid (DHA)-rich and
oleaginous Chlorella sp. biomasses by organicnanoclays. Bioresour. Technol. 137, 7481.

Libessart, N., Maddelein, M.L., Koornhuyse, N.,

Decq, A., Delrue, B., Mouille, G., DHulst, C.,
Ball, S., 1995. Storage, photosynthesis and
growth: the conditional nature of mutations
affecting starch synthesis and structure in
Chlamydomonas. Plant Cell 7, 11171127.

Lin, Y., Tanaka, S., 2006. Ethanol fermentation from

biomass resources: current state and prospects. Appl.
Microbiol. Biotechnol. 69, 627642.

Mamma, D., Kourtoglou, E., Christakopoulos,

P., 2008. Fungal multienzyme production on
industrial by-products of the citrus-processing
industry. Bioresour. Technol. 99, 23732383.

Moxley, G., Zhang, Y.H.P., 2007. More accurate

determination of acid-labile carbohydrates in
lignocellulose by modified quantitative
saccharification. Energy Fuels 21, 36843688.

Na, J.G., Lee, H.S., Oh, Y.K., Park, J.Y., Ko, C.H.,
Lee, S.H., Yi, K.B., Chung, S.H., Jeon, S.G.,
2011. Rapid estimation of triacylglycerol
content of Chlorella sp. by thermogravimetric
analysis. Biotechnol. Lett. 33, 957960.

Naik, S.N., Goud, V., Rout, P.K., Dalai, A.K.,

2010. Production of first and second
generation biofuels: a comprehensive review.
Renewable Sustainable Energy Rev. 14, 578
597.

Nigam, P.S., Singh, A., 2011. Production of

liquid biofuels from renewable resources. Prog.
Energy Combust. Sci. 37, 5268.

Norsker, N.H., Barbosa, M.J., Vermu, M.H., Wijffels,

R.H., 2011. Microalgal production a close look at the
economics. Biotechnol. Adv. 29, 2427.

Wijffels, R.H., Barbosa, M.J., 2010. An outlook

on microalgal biofuels. Science 329, 796799.
Singh, J., Gu, S., 2010. Commercialization potential of
microalgae for biofuels production. Renewable
Sustainable Energy Rev. 14, 25962610.
Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2010.
Microalgae for production of bulk chemicals and
biofuels. Biofuels, Bioprod. Biorefin. 4, 287295.

Sluiter, A., 2011. Determination of structural

carbohydrates and lignin in biomass. National
Renewable Energy Laboratory, USA.
<http://www.nrel.gov/biomass/ pdfs/42618.pdf>.

Tengerdy, R.P., Szakacs, G., 2003. Bioconversion

of lignocellulose in solid substrate fermentation.
Biochem. Eng. J. 13, 169179.

Wikandari, R., Millati, R., Syamsiyah, S.,

Muriana, R., Ayuningsih, Y., 2010. Effect of
furfural, hydroxymethylfurfural and acetic acid
on indigenous microbial isolate for bioethanol
production. Agric. J. 5, 105109.