Natural Resources, 2011, 2, 173-179
doi:10.4236/nr.2011.23023 Published Online September 2011 (http://www.SciRP.org/journal/nr)
173
Trehalose and Sucrose Osmolytes Accumulated by
Algae as Potential Raw Material for Bioethanol
Ma. del Pilar Bremauntz1, Luis G. Torres-Bustillos1, Rosa-Olivia Cañizares-Villanueva2, Enrique
Duran-Paramo1, Luis Fernández-Linares1
1Laboratory of Bioprocesses, Department of Bioprocesses, Unidad Profesional Interdisciplinaria de Biotecnología UPIBI-Instituto
Politécnico Nacional, Mexico City, Mexico; 2Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios
Avanzados- IPN, Mexico City, Mexico.
Received April 29th, 2011; revised June 30th, 2011; accepted July 10th, 2011.
ABSTRACT
Currently, obtaining sustainable fuels, such as biodiesel and bioethanol, from cheap and renewable materials is a
challenge. In recent years, a new approach being developed consists of producing, sugars from algae by pho tosyn the sis.
Sugar accumulation can be increased under osmotic stress (osmoregulation). The aim of this study is to show the pro-
duction of sugars from algae, isolated from natural sources, and the effect of osmotic stress on fermentable sugars ac-
cumulation. S train isolation, produ ction of sugars from each alga and th e effect of osmotic stress on gro wth and sugar
production are described. Twelve algal strains were isolated, showing growths between 0.6 and 1.8 g of biomass dry
weight /L, all with production of intra cellu lar and extra cellular su ga rs. The strain iden tified a s Ch lorella sp. showed an
increase in sugar production from 23.64 to 421 mg of sugars/g of biomass dry weight after 24 h of osmotic stress with
0.4 M NaCl. Sucrose and trehalose, both fermentable sugars, were the compatible osmolytes accumulated in response
to the osmotic stress. The isolated strains are potential producers of fermentable sugars, using the photosynthetic
pathway and osmotic stress.
Keywords: Algae, Biofuels, Osmoregulators, Carbohydrates, Bioethanol
1. Introduction
Carbon neutral renewable fuels, as bioethanol, are deci-
sive in contributing to the replacement of petroleum-
derived fuels that contribute to global warming. Produc-
tion of biodiesel from oil crops and bioethanol from sug-
arcane and crops in large amounts are not sustainable. An
alternative offered by microalgae results from photosyn-
thesis-produced sugars. This production can be increased
via osmotic stress as osmolytes accumulation in algae.
One mechanism developed by microorganisms as a re-
sponse to osmotic stress, is the ability to accumulate com-
patible low-molecular-weight organic solutes such as car-
bohydrates [1-5].
Three categories of microalgae have been proposed
according to their response to salt, a) low stress tolerance
to salinity (0.7 M NaCl), with production of sucrose and
trehalose, b) medium tolerance to salinity (0.7 - 1.8 M
NaCl) with production of glucosylglycerol, and c) high
tolerance to salinity (more than 2.7 M NaCl) with produc-
tion of amino compounds such as glycine, betaine, or
glutamate [6-9]. The mechanism by which microalgae
produce sucrose is through the production of a complex
enzyme sucrose-phosphate synthetase/phosphatase (SPS/
SPP) [10].
The aim of this study is to show the effect of osmotic
stress in algae fermentable sugars accumulation, as a
potential supply source of raw material for alcoholic
fermentation.
2. Material and Methods
2.1. Algae Isolation and Culture
Samples were taken from four different places: the Lake
of Texcoco, located in the Metropolitan area of Mexico
City (salinity 2000 ppm, pH 8 - 10) [11]; the Lake of
Guadalupe, in the State of Mexico (fresh water, pH 7.2,
salinity 0.3 ppm); a lake in Tabasco, southeastern Mex-
ico (pH 7.2 - 7.8, salinity 2000 ppm); and the river
Thames, in Ontario, Canada (pH 6.7, salinity 0.5 ppm).

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol 174
Samples were taken in bottles and, upon arriving to the
laboratory, pH and salinity were determined through
conductivity.
Samples were enriched through cultivation in different
media according to their salinity, either BG11, BG11
with 25% NaCl, or Guillard [12]. The culture conditions
were, in all experiments: fluorescent light in periods of
12:12 h (100 mol/m2 s); 25˚C and aeration 4 vvm (in
liquid medium). After two replanting procedures, they
were plated onto solid medium (the same culture medium
with 10% agar-agar). After 15 days of incubation, sam-
ples of each separate colony were taken from the plate
and seeded again in a liquid medium culture at the same
above mentioned conditions. Isolated strains were con-
served in liquid medium with natural light at room tem-
perature [12]. The strains were identified by optical mi-
croscopy using a LEICA-DMLB microscope. Each strain
was observed and compared with a taxonomy library [13].
2.2. Growth Kinetics
A 250-mL flask with 150 mL of selected medium was
inoculated (10% inoculum) and cultivated under the pre-
viously fixed conditions. Growth was monitored by ab-
sorbance (O.D. 600 nm) every 24 h in a colorimeter
Genesis 10 UV and by dry weight (dw): culture was fil-
tered through a 0.42 m pre-weighted filter, dried at 80˚C
for 24 h, and weighted. Culture purity was monitored by
optical microscopy.
2.3. Intra- and Extra-Cellular Sugars Analysis
The 15-day cell culture was centrifuged at 5000 rpm, for
20 min at 4˚C, in a Beckman J2-MC centrifuge. Liquid
phase was separated for total carbohydrate determination.
For intracellular sugars, biomass was lyophilized, the dry
cells (0.01 - 0.05 g) were extracted with 10 mL of 70%
ethanol and incubated at 65˚C during 4 h, then they were
centrifuged at 10,000 rpm for 15 min in a Sorvall Bio-
fugeprimo Centrifuge. The supernatants were collected
and dried at 40˚C in an oven. The obtained material was
redissolved in 10 mL of distilled water [8,14]. The total
carbohydrate content, in both intra- and extra-cellular
fractions, was established by the phenol-sulphuric acid
method (Dubois Method); and identified by HPLC as
established by Müller [8,14], The supernatants were col-
lected and deionised by shaking with mixed-bed resin
(Bio-Rad 501 X8), the solute was filtered through a mi-
crofilter (22 μm) prior to injection. A Varian 9002 HPLC
with refractive index detector, using a column Phenome-
nex REYEX organic acid 300X7.8 75985 00H-0138-KO,
mobile phase H2O/H2SO4 at a flow rate 0.3 mL/min, was
employed.
2.4. Effect of Salt Stress on Growth and Osmotic
Shock on Sugar Production
To assess the NaCl concentration effect on growth, iso-
lated strains were grown in BG11 medium with 0, 0.2,
0.4, and 0.6 M NaCl under the same conditions men-
tioned above. Growth, intra- and extra-celullular, and
total carbohydrates were determined during 15 days as
indicated before.
The effect of osmotic shock on sugar production was
completed by stressing the growth cultures (biomass at
1.5 OD600nm) with the addition of NaCl, as concentrated
solution, to achieve 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 M;
subsequently shocked cultures were incubated during
three additional days, growth and intra- and extra-cellular
sugars were determined every 24 h, as indicated before.
3. Results and Discussion
3.1. Photosynthetic Microorganisms Isolation
and Its Growth
In the initial stage of this work, twelve different types of
photosynthetic microorganisms were isolated and identi-
fied, they showed different growths and biomass produc-
tions (Table 1).
Table 1. Identification of isolated algae, place of origin, and biomass produced (g dw/L).
Orden y Género Lugar de origen Máxima biomasa (g/L)Factor de eficiencia
Chlorococcales Chlorella Lago de Guadalupe 1.36 29.11
Chysospheaceae Chrysosphaera Lago en Tabasco 0.32 5.44
Chlorococcales Trebouxia Lago de Guadalupe 0.6 6.22
Chlorococcales Chlorella (T) Lago en Tabasco 0.64 2.70
Chlorococcales Scenedesmus Río Tamesi, Canadá 1.58 5.37
Chroococcales Synechocystis Lago de Texcoco 1.74 1.81
Chroococcales Synechocystis (T) Lago en Tabasco 1.18 0.83
Nostocales Nodularia Lago de Texcoco 2.16 1.30
Oscillattoriales Lyngbya Lago de Texcoco 2.56 1.02
Chroococcales Gloecapsa Lago en Tabasco 0.9 0.27
Chroococcales Cistococcus Lago de Texcoco 1.76 0.70
Oscillattoriales Oscillatoria Río Tamesi Canadá 1.24 2.61
*mg of total sugars/L culture medium

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol 175
Among the isolated strains, maximum growth (bio-
mass) occurred in increasing order: Lyngbya, Synecho-
cistis, Cystecocus, Scenedesmus, Chlorella, and Oscilla-
toria; they showed a final biomass (dw) higher than 1
g/L (Figures 1(a) and 1(b)). A second group of strains:
Gloecapsa, Chlorella (T), Trebouxia, Nodularia, and
Chrysosphaera showed the lowest biomass dw (<1), i.e.,
50% less than the Lyngbya strain (Figure 1(c)).
In order to establish the sugars-production efficiency
for each strain, the yield of total sugars production per
gram of dried biomass per liter of algae culture was cal-
culated for each strain. Chlorella sp., Chysospheaceae,
Chrysosphaera, Trebouxia, and Scenedesmus were the
most efficient producer strains (Table1).
No relationship was found between salinity of the
strains place of origin and the natural production (with-
out osmotic stress) of total sugars; for example, strains
Chlorella sp. and Trebouxia, which were isolated from
the Guadalupe Lake (fresh water), showed different
sugar production rates; from these algae, Chlorella pro-
duced 4-fold more sugars than Trebouxia (Figure 2).
Given its high efficiency in sugar production, Chlorella
sp. was selected for the latter osmotic stress assays.
3.2. Effect of NaCl Concentration on the Growth
and Sugar Production of Chlorella
Biomass growth was inhibited at 0.4 and 0.6 M of NaCl,
and decreased 28% at 0.2 M (Figure 3). Brown [15] re-
ported a similar phenomenon with Nannochloris bacil-
laris (Chlorophyceae), whose growth was inhibited
300% in seawater (0.8 M). Other studies in cyanobateria
mention that this type of photosynthetic microorganism
presents growth inhibition at 0.4 M NaCl [16]. Chlorella
vulgaris decreases growth 50% at 0.6 M, 30% at 0.4, and
10% at 0.2 M [17]. All these species, including Chlorella
isolated in this study, undergo a strong growth inhibition
even under moderate osmotic stress, which means that
they are stenohaline.
3.3. Osmotic Shock Effect on Sugar Production
When Chl orella was treated under osmotic shock, in-
crements in the production of sugars were observed
(Figure 4). Total sugar production was 18-fold higher
after 24 h of osmotic shock using 0.4 M NaCl (421 mg/g
of biomass dw).
Page et al. [8] reported a 15-fold increment of total
sugar production under osmotic stress for a strain of Sy-
tonema, after 24 h in 0.15 M NaCl. When Synechocyst i s
PCC 6714 was grown in BG11, total carbohydrate pro-
duction increased above 13-fold when the medium was
changed to a BG11/100% seawater [18].
After salt stress, sucrose and trehalose concentrations
increased from 1.13 to 1.95 and from 0.012 to 0.019
0
0.5
1
1.5
2
2.5
3
3.5
0123456789101112131415
Grow th OD 600 n m
t (d ays)
Cistococcus Scenedesmus
Synechocystis Chlorella
(a)
0
0.5
1
1.5
2
2.5
3
3.5
0 1 2 3 4 5 6 7 8 9101112131415
Grow th OD 600nm
t (d ays )
S
ynechocystis Oscillatoria NodulariaLyngbya
(b)
0
0.5
1
1.5
2
2.5
3
3.5
0 1 2 3 4 5 6 7 8 9101112131415
Grow th OD 600 n m
t (d ays)
Trebouxia Chlorella
Gloecapsa Chrysosphaera
(c)
Figure 1. Kinetic growth of isolated strain cultures in
BG11medium.
mg/g biomasse dw, respectively, in Chlorella. Under salt
stress, Scytonema produced sucrose and trehalose, and
trehalose concentration was higher than sucrose [8]; per-
haps because trehalose has shown superior protein stabi-
lisation capacity [21]. Chlorella, under osmotic stress,
produced proline and sucrose as osmolytes [19-21],
aeroterrestrial “Chlorella” trebouxioides SAG2142 and
“Chlorella” luteoviridis SAG2196 accumulate the poly-
ols, ribitol and sorbitol [20]. The present study is the first
report of trehalose synthesis by Chlorella as osmotic
response; nevertheless, in this study sucrose concentra-
tion was higher than that of trehalose, being the main
osmolyte in Chlorella.
Algae have been used to obtain different products of
interest for humans. Microalgae can provide several diff-
erent types of renewable biofuels. These include meth-

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol 176
0
5
10
15
20
25
T otal su gar (m g/ g b i o m as s d w )
Strain
Extracellular sugarsIntracellular sugars
Figure 2. Intra- and extra-cellular sugar production for each isolated strain, grown in BG11 medium without NaCl.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0123456789101112
Growth OD (60 0nm )
T i m e (d ays )
0 M N aC l0.2 M N aCl
0.4 M N aCl0.6 M N aCl
Figure 3. Effect of NaCl concentration on growth of Chlor-
ella sp.
ane produced by anaerobic digestion of the algal biomass
and biohydrogen produced photobiologically, as well as
biodiesel derived from microalgal oil [22], bioethanol
from sugars and cellulose [23,24], biomass for combus-
tion or hydrogen and methane production.
Aside from using the sugars produced by algae to ob-
tain bioethanol, other celular components as lipids and
protein can be used for the synthesis of biodiesel and
fodder, respectively; making it more feasible economi-
cally to obtain biofuels from algae. When Botryococcus
braunii is grown at different NaCl concentrations, in-
creasing salinity induces a decrease in protein content, but
the carbohydrate and lipid contents remain unchanged [25].
Considering the results obtained in the present work re-
24 h48 h72 h
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
375
400
425
450
475
0.1 0.2 0.3 0.4 0.5 0.6 0.1 0.2 0.3 0.4 0.5 0.6 0.1 0.2 0.3 0.4 0.5 0.6
0
NaClconcentration( M)
Total sugars (mg/g biomass dw)
Intracellular sugars Extracellular sugars
Figure 4. Osmotic stress effect in total sugar production in
Chlorella sp grown in BG me dium .
garding sugars production by Chlorella, we estimated the
annual production of fermentable sugars, considering a
hectare of raceways surface of 0.3 m depth (6000 m3),
and a harvested biomass of 20% of the volume per day,
under four different scenarios: 50% of the maximum
biomass production achieved (equivalent to 0.7 g/L) and
the maximum biomass (equivalent to 1.36 g/L ), both
with a sugars yield without osmotic shock (equivalent to

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol177
0.02 kg sugar/kg biomass dw), corresponding to scenar-
ios 1 y 2, respectively. A 50% and 100% of the maxi-
mum biomass production achieved with a sugars yield
after osmotic shock (equivalent to 0.25 kg sugar/kg dw),
corresponds to scenarios 3 and 4, respectively (Table 2).
Algal sugar production was compared to the sugar cane
productivity in Colombia, where the best yield is 80 and
9.6 ton/ha of sugar cane and sugar, respectively [26-29].
As for production of sugars, in the case of Chlorella in
the most conservative condition (scenario 1), the produc-
tion of sugars is lower (31%) than sugar cane. Neverthe-
less, for scenario 2, it is 13% higher than that of sugar
cane. The two scenarios with osmotic shock, scenarios 3
and 4 are 7 and 14-fold higher than sugar cane, respec-
tively. However, the dilution of sugars in the culture
broth is a limiting factor that needs to be considered con-
trary to sugar cane juice.
As for Chlorella’s by-products, lipids, proteins and re-
sidual biomass after extraction (cake) were estimated in a
very conservative way (based on our results), the per-
centage of yields were 10%, 20%, and 50%, respectively.
The values of the by- products estimated with Chlorella
are more than one fold higher than those obtained with
African oil palm (Elaeis guineensis) (Table 2). Never-
theless, many technical challenges remain in regarding to
scaling up and adequate process to make the use of algae
feasible for the production of biofuels.
4. Conclusions
Twelve strains were isolated from different ecosystems,
which produced intra- and extra-cellular carbohydrates.
Chlorella sp. showed the highest total sugars production,
29.1 mg of sugars/g dw biomass. When salinity level in
culture media was increased, culture growth decreased
Table 2. Comparative scenarios production of sugars, lipids and protein, considering a hectare of raceways surface and 0.3 m
depth, and a harvested biomass of 20% of the volume per day. Sce nario (1) 50% of the maximal biomass production achieved
(0.7 g/L); (2) the maximal biomass (1.36 g/L ) both with sugars yield without osmotic shock (0.02 kg sugar/kg dw), (3) and (4)
50% and 100% of the maximum biomass production with sugars yield after osmotic shock (0.25 kg sugar/kg dw), scenarios 3
and 4, respectively. Sugar cane from Colombia and Oil Palm form Africa.
Biomass Sugars Lipids Protein Biomass after extraction
Feedstock(scenario) (Ton/Ha/year)
Chlorella (1) 294 5.9 29.4 59 147
Chlorella (2) 571 11 57.1 114 286
Chlorella (3) 294 73.5* 29.4 59 147
Chlorella (4) 571 142.8* 57.1 114 286
Sugar cane 80 9.6 - 5.2(32) 26
Oil palm 22(29) 2.6(29) 5.7(29) 3.5(30) 5
(31)
Figure 5. HPLC chromatograms from sacarose standard, A; trehalose standard, B; Chlorella’s extract without osmotic stress,
C; and Chlorella’s extract after 24 h of osmotic stress with 0.4 M NaCl, D.

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol 178
with increasing NaCl concentration, and was inhibited at
concentrations higher than 0.4 M for all strains.
After the osmotic stress of Chlorella culture, sugar
production increased 14-fold in 0.4 M NaCl after 24 h.
The main osmolyte identified was sucrose; however, tre-
halose was also accumulated (1.93 and 0.019 mg/ bioma-
sse dw, respectively). These results show the potential use
of algae as sugar resource of fermetable sugars and the
use of osmotic stress to increase the total sugar produc-
tion. This work shows also trehalose accumulation as
osmolyte by Chlorella.
5. Acknowledgements
Funding by Instituto Politecnico Nacional PIFI-20100242.
The microscopy studies and strain identificactions
were led by Dr. Ma. Esther Meave from Univesidad Au-
tónoma Metropolitana. Iztapalapa, Mexico.
HPLC analyses were made at the Central Analítica,
Depto. de Biotecnología. CINVESTAV-IPN and directed
by Elvira Rios, Cirino Rojas, and Gustavo Medina.
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