Bioresource Technology 233 (2017) 4450

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

Comparison of red microalgae (Porphyridium cruentum) culture

conditions for bioethanol production
Ho Myeong Kim a, Chi Hoon Oh b, Hyeun-Jong Bae a,b,
a
Bio-Energy Research Center, Chonnam National University, Gwangju 61186, Republic of Korea
b
Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 61186, Republic of Korea

h i g h l i g h t s

 Porphyridium cruemtum (PC) can grow in seawater and freshwater conditions.

 Freshwater P. cruemtum (FPC) biomass can efficiently produce the bioethanol.
 SSF process was superior to SHF process for bioethanol production from PC.

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

Article history: Microalgae biomass are useful resources in biofuel production. The objective of this study was to evaluate
Received 23 January 2017 bioethanol production in response to Porphyridium cruemtum culture conditions. Enzymatic hydrolysis of
Received in revised form 9 February 2017 seawater P. cruemtum (SPC) and freshwater P. cruemtum (FPC, 1% substrate loading, w/v) resulted in
Accepted 10 February 2017
glucose conversion yields of 89.8 and 85.3%, respectively, without any pretreatment. However, FPC
Available online 12 February 2017
hydrolysate was more efficiently converted to ethanol about 7.1% than SPC hydrolysate. The comparison
of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF)
Keywords:
showed that SSF processing is a superior method for bioethanol production from both SPC and FPC.
Porphyridium cruemtum
Microalgae
Though SSF processing (5% substrate loading, w/v) in a 500-mL twin-neck round bottom flask, we
Bioethanol achieved ethanol conversion yields of 65.4 and 70.3% from SPC and FPC, respectively, after 9 h. These find-
Simultaneous saccharification and ings indicate that P. cruemtum can grow in freshwater conditions and is an efficient candidate for bioetha-
fermentation (SSF) nol production.
2017 Elsevier Ltd. All rights reserved.

1. Introduction
According to the US Department of Energy (DOE, Independent
Statistics & Analysis), world biofuel production will increase from
approximately 1.3 million barrels per day in 2010 to approxi-
mately 3.0 million barrels per day in 2040 (International Energy
Outlook, 2014). New, renewable energy sources, such as microal-
gae and lignocellulosic biomass, have attracted increasing atten-
tion and become an important issue related to the generation of
alternative energy (Ho et al., 2013; Kim et al., 2012; Mussatto
et al., 2010). Specifically, microalgae may become useful resources
in biofuel and bio-industrial applications due to their many advan-
tages, including rapid and sustainable growth. Some Chlamydo-
manos species have been reported to double their mass within
6 h, with carbon saving effects, and they have a high CO2 fixation
Corresponding author at: Department of Bioenergy Science and Technology,
Chonnam National University, Gwangju 500-757, Republic of Korea.
E-mail address: baehj@chonnam.ac.kr (H.-J. Bae).
ability, i.e., more than 10-fold that terrestrial plants (Chen et al.,
2009; Mostafa, 2012). Some microalgae have high a carbohydrate
content that exceeds 50% of their dry weight (John et al., 2011).
For these reasons, many studies have investigated the application
of microalgal resources to bioethanol production (Ho et al., 2013;
Markou et al., 2013). Many studies have been conducted on the
optimal culture conditions of microalgae, investigating the influ-
ence of nutrients such as nitrogen, sulfur, and phosphate on
microalgal chemical composition (Kim et al., 2014; Razaghi et al.,
2014; Wang et al., 2015). For example, in Chlorella vulgaris and Por-
phyridium cruentum, nitrogen deficiency in the growth medium can
increase carbohydrate content (Kim et al., 2014; Razaghi et al.,
2014).
Many studies involving microalgae have focused on their utility
in biodiesel production, due to their high lipid content, environ-
mental advantages, and economical production (Hill et al., 2006;
Hannon et al., 2010). Recent studies have focused on cost-
effective bioethanol production methods, particularly efficient bio-
mass harvesting methods, and low enzyme loading, and rapid

http://dx.doi.org/10.1016/j.biortech.2017.02.040
0960-8524/ 2017 Elsevier Ltd. All rights reserved.
 H.M. Kim et al. / Bioresource Technology 233 (2017) 4450
growth processes (Sathe and Durand, 2015; Kim et al., 2015). Addi-
tionally, environmentally sound practices are required in biofuel
production. The use of certain chemicals may lead to fermentation
inhibition and the production of environmental pollutants (Bensah
and Mensah, 2013). Thus, eco-friendly processes may increase fer-
mentation yield and reduce external environmental costs.
The red algae P. cruentum (PC) is one of the most promising can-
didate organisms for producing fatty acids, lipids, carbohydrates,
and pigments (Plaza et al., 2009). In a previous study, PC biomass
was found to contain up to 57% carbohydrate (Becker, 1994). In this
study, we investigated bioethanol production from PC by compar-
ing culture conditions and morphological changes according to the
nitrogen concentration. We also evaluated ethanol production of
FPC and SPC by compared to the separate hydrolysis and fermenta-
tion (SHF) and simultaneous saccharification and fermentation
(SSF) methods.
2. Material and methods
2.1. Microalgae cultivation and growth conditions
P. cruentum (KMMCC-1061) was purchased from the Korea Mar-
ine Microalgae Culture Center (Pusan, Korea). The microalgae were
pre-cultured in a 100 mL flask at 20 C with a filtered air pump for
aeration (168 h, lightdark cycle), and then inoculated into 1 L of
Jaworskis medium (JM) consisting of (mg/L) Ca(NO3)2
4H2O (20),
KH2PO4 (12.4), MgSO4
7H2O (50), NaHCO3 (15.9), FeNa-EDTA
(2.25), EDTA-Na2 (2.25), H3BO3 (2.48), MnCl2
4H2O (1.36), (NH4)6-
Mo7O24
4H2O (1), cyanocobalamin (0.04), thiamine HCI (0.04), bio-
tin (0.04), NaNO3 (80), and Na2HPO4
12H2O (36) with deionized
water and f/2 medium consisting of (mg/L) NaNO3 (75), NaH2PO4-

H2O (5), Na2SiO3
9H2O (30), FeCl3
6H2O (3.15), Na2EDTA
2H2O
(4.36), CuSO4
5H2O (0.0098), Na2MoO4
2H2O (0.0063), ZnSO4
7H2-
O (0.022), CoCl2
6H2O (0.01), MnCl2
4H2O (0.18), cyanocobalamin
(0.0005), biotin (0.0005), and thiamine HCl (0.0001) with filtered
seawater. After 12 days cultivation, the microalgae were harvested
by centrifugation for 10 min at 26,000g (Avanti J-E, Beckman,
Fullerton, CA, USA).
2.2. Comparison of mass productivity, cell number, and growth
according to the cultivation period
After 5, 10, and 15 days cultivation, the FPC and SPC were har-
vested by centrifugation for 10 min at 26,000g (Avanti J-E, Beck-
man, Fullerton, CA, USA). The harvested FPC and SPC were
washed in water several times, and then dried at 105 C. The cell
number of FPC and SPC were counted using a hemocytometer.
The change in growth of FPC and SPC were observed by light
microcopy after staining with 0.1% Toluidine blue.
2.3. Chemical composition analysis
After organic solvent extraction of SPC and FPC using ethanol-
benzene, the neutral sugar contents (glucose, xylose, arabinose,
mannose, galactose, and rhamnose) were analyzed using gas chro-
matography (GC; Choi et al., 2013).
2.4. Enzyme activity and optimization of enzyme
Cellulase (Celluclast 1.5 L) and pectinase (Pectinex SP-L) were
purchased from Novozyme A/S (Bagsvaerd, Denmark). The cellu-
lase activity was determined by the National Renewable Energy
Laboratory (NREL) method (Adney and Baker, 2008) and the pecti-
nase activity was measured with method described by Kittur et al.
(2003). The cellulase and pectinase activities were 0.122 filter
45
paper unit (FPU)/mg protein and 240 international unit (IU)/mg
protein, respectively. We carried out the enzymatic hydrolysis of
1% (w/v) dry matter SPC and FPC with varying loadings (2.4, 4.8,
9.6, 14.4, 19.2, and 28.8 mg/g SPC or FPC) of pectinase at 37 C
for confirmation of optimal enzyme loadings. After 24 h, the reduc-
ing sugars were measured using a 3,5-dinitrosalicylic acid (DNS)
reagent and a standard glucose curve (Miller, 1959).
2.5. Scanning electron microscopy (SEM)
In preparation for SEM analysis, the raw SPC and enzyme-
treated SPC samples were fixed with 2% (v/v) paraformaldehyde
and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2).
The washed samples were dehydrated in a graded ethanol series
(50, 70, 90, 95, and 100%), and then lyophilized. Samples were
coated with a thin layer of gold (20 nm) and observed via SEM
(S2400; Hitachi, Tokyo, Japan).
2.6. Separate hydrolysis and fermentation (SHF) and simultaneous
saccharification and fermentation (SSF)
Enzymatic hydrolysis was conducted with SPC and FPC samples
in a 5 mL total volume containing 1% (w/v) dry matter, pectinase
(4.8 mg/g PC), cellulase (7.2 mg/g PC), 0.1% (w/v) yeast extract,
0.2% (w/v) peptone, and 50 mM citrate buffer (pH 4.8) at 37 C
for 7 h in a 15-mL conical tube. After enzymatic hydrolysis, fer-
mentation was performed using 3 mL hydrolysate with 3 mg dry
yeast (Saccharomyces cerevisiae, KCTC 7906) at 30 C for 7 h.
SSF was conducted for SPC and FPC samples in a 5 mL total vol-
ume containing 1% (w/v) dry matter, pectinase (4.8 mg/g PC), cel-
lulase (7.2 mg/g PC), 5 mg dry yeast (S. cerevisiae KCTC 7906),
0.1% (w/v) yeast extract, 0.2% (w/v) peptone, and 0.05 M citrate
buffer (pH 4.8) at 37 C for 10 h in a 15-mL conical tube. To
increase ethanol concentration, SSF was performed in a 100 mL
total volume with 5% substrate of SPC and FPC in a 500-mL twin-
neck round bottom flask at 37 C for 12 h. All SHF and SSF pro-
cesses were conducted with three replicates.
2.7. Sugar and ethanol analysis by high-performance liquid
chromatography (HPLC)
After SHF and SSF reactions, the contents of sugars and ethanol
were analyzed by HPLC using a refractive index detector (2414;
Waters, Milford, MA, USA). The REZEX RPM (Phenomenex, Tor-
rance, CA, USA) column (300 mm  7.8 mm) was used at 85 C,
and samples were eluted with deionized water at a flow rate of
0.6 mL min1.
3. Results and discussion
3.1. Culture condition and chemical composition
Sufficient carbohydrate content and efficient biomass harvest
are required for economical bioethanol production from microal-
gae. Therefore, many studies have reported increase of carbohy-
drate content through the control of nutrient stress, as well as
harvesting methods to reduce cost and energy consumption
(Morales-Snchez et al., 2014; Milledge and Heaven, 2013). In one
such study, bicarbonate supplementation for microalgae increased
biomass and biochemical content (Pancha et al., 2015). In general,
PC is known to grow in seawater (Golueke and Oswald, 1962).
The neutral sugar content of seawater P. cruentum (SPC) and fresh-
water P. cruentum (FPC) was determined by gas chromatography
(Table 1). SPC and FPC had total sugar compositions of 27.0 and
28.8%, respectively. Notably, PC showed the low carbohydrate
46 H.M. Kim et al. / Bioresource Technology 233 (2017) 4450

Table 1
Chemical composition of SPC and FPC.

(% Dry matter) Rhamnose Arabinose Xylose Mannose Galactose Glucose Total

SPC 0.0 0.0 0.0 0.0 4.7 0.0 0.1 0.0 5.3 0.0 16.9 0.2 27.0 0.3
FPC 0.0 0.0 0.0 0.0 6.4 0.2 0.2 0.0 5.5 0.2 16.6 0.7 28.8 1.1

Values represent the average over three replicates.

Table 2
Comparison of cell number, cell size, and mass productivity according to the culture condition.

(Day) Fresh water Sea water

Cell number (mL 1
) Cell size (lm) Mass productivity (mg L 1
) Cell number (m L 1
) Cell size (lm) Mass productivity (mg L 1
)
5 1.62( 0.62)  105 8.53 1.28 36.0 3.5 3.05( 0.33)  106 6.27 0.58 168.3 14.0
10 3.97( 0.65)  106 6.83 1.02 315.7 24.7 5.82( 0.38)  106 7.44 0.66 600.3 59.5
15 4.74( 0.17)  106 8.12 0.98 635.0 51.7 7.60( 0.40)  106 8.02 0.88 978.7 81.6

contents compared to the previous study, which is influenced by 3.4. Optimization of pectinase loading
the culture conditions and environment (Becker, 1994). Glucose
was the major component of SPC and FPC, comprising approxi- Eco-friendly processes are important for biofuels production for
mately 16.9 and 16.6% of the total dry mass, respectively; xylose environmental protection and the removal of inhibitors (Naik et al.,
and galactose were minor components. These results suggest that 2010). Therefore, enzymatic hydrolysis to produce fermentable
culture condition (fresh water and sea water) did not significantly
affect the chemical composition of PC; additionally, PC can grow
in freshwater conditions.

3.2. Comparison of cell number, cell size, mass productivity and

growth condition in SPC and FPC

Chemical stress (due to varying level of nitrogen, phosphate,

and salinity) and physical stress (due to varying levels of pH, tem-
perature, and light intensity) were effected to induce an increase in
carbohydrate content (Pancha et al., 2014). We cultivated SPC and
FPC to compare cell number, cell size, mass productivity, and
growth. SPC grows rapidly compared to FPC in initial cultivation
conditions (5 days). However, many cells died after 10 days
because SPC cells begin the process of aging after approximately
7 days. Very few cells survived beyond 15 days. Conversely, FPC
begins to grow later, which may receive the stress according to
the different culture conditions. FPC cell size increased by approx-
imately 36% due to carbohydrate storage for the cell survival
(Table 2). Additionally, FPC grew rapidly after 8 days of cultivation.
Also, many FPC cells were survived beyond 15 days (Fig. S1).
Cell number and mass productivity of FPC and SPC increased as
the cultivation period increased (Table 2). SPC mass productivity
was more efficient than that of FPC until 15 days of cultivation.
However, after 20 days, FPC had increased mass productivity by
approximately 19% compared to 15 days cultivated SPC (data not
shown). These results suggest that the mass productivity should
depend on harvest time of FPC and SPC.

3.3. Growth Comparison of P. cruentum at different nitrogen

concentrations

Nitrogen limitation in microalgae growth media has been previ-

ously reported to cause high accumulation of carbohydrates or
lipids in the cell wall (Kim et al., 2014; Sharma et al., 2012). In this
study, we observed PC growth in f/2 medium for 12 days at varying
nitrogen concentration (NaNO3, 16144 mg/L). Nitrogen concen-
tration in the media caused morphological changes in PC
(Fig. S2). PC experienced rapid growth with an increase in nitrogen Fig. 1. Pectinase optimization for SPC and FPC degradation. (A) SPC. (B) FPC. The
concentration, whereas a low concentration of nitrogen reduced enzymatic hydrolysis was conducted at 37 C for 24 h. The enzymatic hydrolysis
the PC growth or led to aging. was conducted with three replicates.
 H.M. Kim et al. / Bioresource Technology 233 (2017) 4450
sugars is a preferable method compared to the addition of chemi-
cals. Pectinase is a major enzyme for the degradation of algal bio-
mass to produce monosaccharides, and has been shown to be
required for the production of glucose from Chlorella vulgaris
(Kim et al., 2014). Therefore, we optimized the pectinase loading
for the saccharification of SPC and FPC, which is necessary for eco-
nomical bioethanol production due to high enzyme cost. After 12 h
of saccharification of SPC and FPC, we analyzed the reducing sugar
content using the DNS method, and then established 4.8 mg pecti-
nase/g SPC or FPC as the optimal pectinase loading volume for sac-
charification (Fig. 1).
3.5. Light microscopy and scanning electron microscopy (SEM)
analysis
Light microscopy and SEM analysis were conducted to deter-
mine the morphological changes in enzyme-treated SPC (Fig. S3).
The surfaces of enzyme-treated SPC were more heavily degraded
compared to those of raw SPC due to the removal of cellulose
and pectic polysaccharides. This result indicates that an enzyme
mixture was required to produce monosaccharides from SPC cell
wall.
3.6. Ethanol production in SPC and FPC
SHF and SSF processes are both commonly used in bioethanol
production (Daroch et al., 2013). Many studies have reported that
the SSF process is superior to SHF for bioethanol production
because it can increase the ethanol conversion yield (Kim et al.,
2015; Sarkar et al., 2012). In this study, we conducted both SHF
Fig. 2. Separate hydrolysis and fermentation processes with 1% (dry matter, w/v) SPC and FPC biomass. Change over time in (A) glucose concentration, (B) glucose conversion
yield, (C) glucose utilization and ethanol production by Saccharomyces cerevisiae, and (D) ethanol conversion yields. All SHF processes were conducted with three replicates.
47
and SSF to determine which is the more efficient method for the
production of bioethanol.
Enzymatic hydrolysis was conducted with SPC and FPC. After
saccharification for 7 h, the glucose concentration and glucose con-
version yield of the SPC and FPC based on the initial glucose con-
tent were 1.52 and 1.42 mg/mL, and 89.8 and 85.3%, respectively
(Fig. 2A and B). We conducted fermentation at 30 C for 7 h. All
hydrolysate fermentation processes were completed within 4 h.
The ethanol conversion yields were calculated as a percentage of
the maximal theoretical yield for initial glucose content (0.51 g/
g). The maximum ethanol concentration and ethanol conversion
yield for the SPC and FPC from the initial glucose content were
0.61 and 0.66 mg/mL, and 70.4 and 77.5%, respectively
(Fig. 2C and D). Cost-effective ethanol production requires efficient
pretreatment, low enzyme loading and rapid processes of sacchar-
ification and fermentation (Ding et al., 2012; Kim et al., 2015). In
this study, we demonstrated efficient bioethanol production with-
out pretreatment, using rapid processing compared to the previous
studies (Table 3). FPC biomass appears to use resources more effi-
ciently than SPC in bioethanol production.
We also conducted the SSF process, which took 4 h to complete
for both SPC and FPC. The ethanol concentration and ethanol con-
version yield of the SPC and FPC from the initial glucose content
were 0.64 and 0.68 mg/mL, and 74.2 and 80.3%, respectively
(Fig. 3A and B). In our previous study, we reported that bioethanol
production using SSF processing from autoclave-treated Gelidium
amansii samples occurred rapidly (Kim et al., 2015). Similarly, the
current study shows that the SSF process was more efficient
method to produce bioethanol from SPC and FPC. To obtain a high
ethanol concentration, we also conducted SSF for SPC and FPC at a
5% concentration completing the SSF processes for SPC and FPC
48 H.M. Kim et al. / Bioresource Technology 233 (2017) 4450

Table 3
Comparison of the pretreatment method, SHF process, and SSF process with previous studies.

Biomass Substrate & Pretreatment SHF SSF References

concentration (%)
Saccharification time Fermentation time Reaction time (h) &
(h) & Sugar (h) & EtOH EtOH conversion
conversion yield (%) conversion yield (%) yield (%)
Lignocellulose Jerusalem artichoke & 10 Hydrogen peroxide- 48 & 86.0 48 & 84.0 Non Song et al. (2016)
acetic acid
Wheat straw & 10 Acid 48 & 48.3 48 & 30.2 48 & 54.4 Singhania et al.
(2014)
Rice straw & 15 Popping 48 & 87.2a 24 & 80.9c Non Wi et al. (2013)
Corn stover & 3 Magnesium bisulfite 48 & 93.8 12 & 93.5 Non Yu et al. (2016)
Seaweeds Sargassum spp. & 10 Dilute acid 96 & Non 72 & 89.1 Non Borines et al.
(2013)
Chlorella vulgaris & 10 Bead beating (Milling) 72 & 79.0 24 & 89.0 Non Kim et al. (2014)
Gelidium amansii & 2 Autoclaving (60 min) 24 & 90.7 6 & 74.7c 12 & 84.9c Kim et al. (2015)
Porphyridium cruentum Non 7 & 85.3b 4 & 77.5c 4 & 80.3c This study
(FPC) & 1
Porphyridium cruentum Non 7 & 89.8b 4 & 70.4c 4 & 74.2c This study
(SPC) & 1
c
Porphyridium cruentum Non Non Non 9 & 70.3 This study
(FPC) & 5
c
Porphyridium cruentum Non Non Non 9 & 65.4 This study
(SPC) & 5
a
Glucose conversion yield based on the glucose content in raw material.
b
Glucose conversion yield.
c
From initial glucose content.

Fig. 3. Time courses of simultaneous saccharification and fermentation process. (A) Ethanol concentration and (B) ethanol conversion yields for 1% (dry matter, w/v) FPC and
SPC biomass. (C) Ethanol concentration and (D) ethanol conversion yields for 5% (dry matter, w/v) FPC and SPC biomass. All SSF processes were conducted with three
replicates.

after 9 h. The ethanol concentration and ethanol conversion yield The ethanol conversion yield of the SPC and FPC (5% substrate,
of the SPC and FPC from the initial glucose content were 2.77 w/v) by SSF are significantly different at the 5% significance level.
and 2.98 mg/mL, and 65.4 and 70.3%, respectively (Fig. 3C and D). The SSF process resulted in a high ethanol conversion yield and
 H.M. Kim et al. / Bioresource Technology 233 (2017) 4450
Fig. 4. Overall mass balance for bioethanol production from SPC and FPC.
rapid processing compared to the SHF process. Therefore, this pro-
cess is an ideal method for bioethanol production from PC.
3.7. Overall mass balance
After optimizing each process, we designed an overall mass bal-
ance for bioethanol production based on SSF process of SPC and
FPC (5% substrate, w/v, Fig. 4). The 100 g of SPC consists of 16.9 g
glucose, 5.3 g galactose, and 4.7 g xylose, whereas 100 g of FPC
consists of 16.6 g glucose, 5.5 g galactose, and 6.4 g xylose. The
SSF processing (5% substrate loading, w/v) of SPC and FPC was con-
ducted with pectinase (4.8 mg/g PC), cellulase (7.2 mg/g PC), and
Saccharomyces cerevisiae at 37 C for 12 h, resulting in ethanol pro-
duction of 5.58 g and 5.90 g, respectively. These results suggest
that FPC biomass is a more efficient candidate for bioethanol pro-
duction than SPC biomass.
4. Conclusion
Culture condition are very important factors in microalgae cul-
tivation. PC experienced rapid growth in high nitrogen concentra-
tion, whereas a low concentration of nitrogen reduced the PC
growth or led to aging. In this study, PC was grown in freshwater
and seawater conditions, and FPC biomass produced ethanol more
efficiently than SPC biomass. SSF processing was superior to SHF
processing for bioethanol production from PC. The ethanol produc-
49
tion for SPC and FPC (5% substrate, w/v) by SSF process were 2.77
and 2.98 mg/mL, respectively. This study reports rapid processing
for ethanol production and demonstrates a variety of culture con-
ditions for PC.
Acknowledgements
This work was supported by Priority Research Centers Program
(2010-0020141) through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology, Republic of Korea. Also, this work was supported by
the National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIP) (No. 2015R1A2A2A01004594).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.02.
040.
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