Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
Influence of Molasses on Growth, Biochemical
Composition and Ethanol Production of the Green
Algae Chlorella vulgaris and Scenedesmus obliquus
Mostafa M. El-Sheekh*, Mohamed Y. Bedaiwy, Mohamed E. Osman, Mona M. Ismail
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt
*
[email protected]
Abstract-Two green algae (Chlorella vulgaris and Scenedesmus obliquus) were grown under mixotrophic and heterotrophic
conditions using molasses as carbon source. Under both growth conditions, the growth rate, carbohydrate and protein contents
increased by the increased concentrations of molasses. The pigment fractions and lipid contents increased in response to increasing
molasses concentrations under mixotrophic conditions for both algae, whereas they decreased under heterotrophic conditions till the
algal cells become bright yellow. The biomass of C. vulgaris and S. obliquus grown on molasses under heterotrophic conditions were
used to protein fractionation and production of ethanol.
Keywords- Microalgae; Molasses; Mixotrophic Growth; Heterotrophic; Ethanol Fermentation
I
INTRODUCTION
Algae can be cultivated all year round under autotrophic, mixotrophic or heterotrophic conditions. Mixotrophic and
heterotrophic cultures have a place as alternative modes of producing algae biomass. These methods can yield an extremely
high final biomass, but it is not appropriate for all algae or their products. Some algae cannot utilize organic substrates [1],
either because they lack appropriate uptake mechanisms [2], or alternatively, because they lack fully functional metabolic
pathways required for effective dissimilation of the substrate [3]. Another constraint is the inability of algae to produce some
metabolites in the dark [4]. The ability to utilize organic substrate appears subject to wide variation between species and strains
[5].
For an economical biomass cultivation of algae it is necessary to explore the possibilities of utilizing low cost carbon
sources e.g. molasses as industrial by products [6]. Molasses can be used to grow the algae either heterotrophically or
mixotrophically [7]. Molasses contain 29.64 % sucrose, 24.18 % glucose, and 24.18 % fructose, raffinose is varying amounts.
Total nitrogen contained in molasses ranges from 0.82 % to 2.2 % [8]. Nitrogen is a component of various substances such as
protein, amino acids, amides, ammonium salts, nitrates and nitrites [9].
Chlorella vulgaris and Scenedesmus obliquus have the ability to utilize organic substrates under both light and dark
conditions [10]. Bai [11] reported that a higher growth rate of S. acutus can be obtained under mixotrophic conditions with
beet sugar molasses as substrates. Becker [12] stated that optimal growth rates were obtained from S. obliquus supplied with
CO2 and molasses as carbon sources; hence combined supplementation (CO2 and molasses) to cultures appears advisable and
increases the algal biomass.
The highest cost of microalgal culture systems relates to the need of light and the relatively slow growth rate of the algae.
Although this problem has been avoided in some instances by growing the algae heterotrophically [13], while, heterotrophic
growth in the dark supported by a carbon source replacing the traditional support of light energy and in most cases,
heterotrophic cultivations is far cheaper, simpler to construct facilities, and easier than autotrophic cultivation to maintain on a
large scale [14].
Heterotrophic culture has several advantages: (I) fermentation systems are well understood and there is wide experience in
their design and operation; (II) high cell densities between 20 and 100 g L-1 can be achieved reducing harvesting costs of the
cultivation vessels [15]; and (III) elimination of the requirement for light [16]. However, there are disadvantages as well: (i)
increased potential of contamination by bacteria; (ii) inhibition of growth by organic substrates at low concentrations [17]; (iii)
the inability to produce some light induced products, such as pigments; and (iv) a limited number of available heterotrophic
algal species [18].
Algal strains able to a heterotrophic growth have been listed by [1]. The first commercial venture using heterotrophic
cultures to be reported was by the Grain Processing Corporation [19]; the green alga Spongiococcum was used as poultry feed.
Heterotrophic cultivation on glucose or acetate as carbon sources has been used for some time for Chlorella [20] and [21] with
approximately 550 tonnes produced in Japan in 1996 [22]. Acetone, butanol, and ethanol (ABE) fermentation by Clostridium
saccharoperbutylacetonicum N1–4 using wastewater algae biomass as a carbon source was demonstrated [23]. This work
aims at studying the effect of molasses which is the waste of the sugar cane factories on cultivation of microalgae for its use as
- 20 DOI: 10.18005/JAEB0202002
Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
protein source and for production of ethanol.
II
MATERIALS AND METHODS
A Waste
Molasses, obtained from El-Hawamdia factory of sugar at Upper Egypt.
B Microorganisms
The tested microalgae C. vulgaris and S. obliquus were isolated from different channels and sewage in Gharbia
Governorate, Egypt. The isolated algae were identified according to Watanabe and Niiyama [24]. Purification of the organisms
was done by subculturing and antibiotic treatment according to Venkataraman [25]. The algae inoculated on Kühl medium
[26] agar slants and left in diffused light at room temperature to grow for several days, thereafter; they were kept in a
refrigerator at 4 ºC. Subcultures were made regularly, nearly, every month.
Saccharomyces cerevisiae was kindly taken from laboratory of Mycology, Botany Department, Faculty of Science, Tanta
University and maintained on Czapex's medium [27] agar slants and plates, respectively and incubated at 25 ºC to establish
growth, and stored at 5 ºC in refrigerator.
C Cultivation Condition of Green Algae
Pre cultivation of microalgae was carried out in Kühl medium. The medium was autoclaved at 121 ºC and 1.5 atm. for 20
min. After cooling the Erlenmeyer flasks were inoculated by one loop of 7 day old cultures. The culture flasks were aerated by
air pumps and incubated at 25 ± 1 ºC under continuous illumination provided from day light fluorescent lamps (80 µmol m-2 s1
) [28] for seven days.
The main cultivation was performed using molasses as carbon source which were added in different concentrations (0.05,
0.15, 0.25, 0.35 and 0.45% (v/v)) then the pH of the medium was adjusted at 6.8. The complete culture medium was sterilized,
and later inoculated with 20% (v/v) of exponentially growing inoculum (O.D at 560 nm =0.28). Growth vessels were bubbled
with air pumps and incubated at 25 ± 1ºC for 10 days under continuous illumination for mixotrophic conditions, and another
test flasks were wrapped with aluminum foil to shade of the light for heterotrophic conditions. Experiments were carried out
triplicate.
D Growth Measurements
Growth of the algae was measured spectrophotometrically as optical density at 560nm Wetherell [29].
E Metabolic Activities
The cells were collected by centrifugation (3000 rpm for 10 min) every 2 days and washed with distilled water. The
washed cells were used to estimate total carbohydrate content as described by [30], total protein content [31], chlorophyll a and
b according to Jeffrey and Humphrey [32], carotenoids according to Jensen and Liaan [33] and lipid [34].
Under the optimum conditions, algal cells were subjected to protein fractionation [35] and ethanol fermentation [36].
F Statistical Analysis
All the results reported are the means of three replicates. One way analysis of variance (ANOVA) and Pearson correlation
coefficient were done using (SPSS, 1999) computer program of biostatistics [37].
III
RESULTS
A. Mixotrophic and Heterotrophic Growth of Some Microalgae on Molasses
Results compiled in Figs. 1. A, B, C and D show that the growth of C. vulgaris and S. obliquus increased appreciably under
both mixotrophic and heterotrophic conditions, on supplementing sugarcane molasses as carbon source. The increase in growth
depended on the concentration of molasses and culture condition. The growth of C. vulgaris after 6 days and S. obliquus after
8 days increased by about 94.4 and 70.02 % corresponding to 0.45% (v/v) molasses under mixotrophic conditions and 119 and
107% under heterotrophic conditions.
- 21 DOI: 10.18005/JAEB0202002
Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
Fig. 1 Growth of C. vulgaris (A, B) and S. obliquus (C, D) on molasses under mixotrophic and heterotrophic conditions, respectively. Measured as optical
density at 560 nm
B. Carbohydrate Contents
The effect of different concentrations 0.05, 0.15, 0.25, 0.35 and 0.45% (v/v) of molasses caused highly significant increase
in the carbohydrate contents of C. vulgaris at P < 0.001 under mixotrophic conditions by about 64.08, 78.87, 81.69, 87.32 and
94.37 %, respectively (Fig. 2), and they caused also highly significant increase at P < 0.001 in carbohydrate contents of C.
vulgaris under heterotrophic conditions by about 57.78, 72.22, 76.30, 80 and 92.22 %, above the control value after 6 days of
incubation (Fig. 2).
It is clear that cultures of S. obliquus treated with 0.05, 0.15, 0.25, 0.35 and 0.45 % (v/v) molasses caused highly significant
increase in the amount of carbohydrate contents at P< 0.001 by about 64.96, 76.07, 82.05, 90.60 and 96.30 %, under
mixotrophic conditions (Fig. 2), and also they increased highly significantly by 42.37, 60.17, 78.81, 80.51 and 85.60 % at P <
0.001, under heterotrophic conditions after 8 days of incubation (Fig. 2).
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Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
Fig. 2 Effect of molasses concentrations % (v/v) on the carbohydrate contents of C. vulgaris and S. obliquus (mg/g dry weight) after 6 and 8 days of
application, respectively
C. Protein Contents
The results show that under mixotrophic and heterotrophic conditions, the total soluble proteins of C. vulgaris were
increased as the concentration of molasses increased. Thus, concentrations 0.05, 0.15, 0.25, 0.35 and 0.45% (v/v) of molasses
showed very highly significant increase in the total soluble proteins under mixotrophic conditions by about 7.80, 11.46, 25.61,
38.68 and 47.80 %, respectively after 6 days of incubation (Fig. 3), and also showed highly significant increase by 37.20,
55.56, 61.83, 75.20 and 94.20% at P< 0.001, above the control value under heterotrophic conditions (Fig. 3).
Fig. 3 Effect of molasses concentrations % (v/v) on the total soluble protein of C. vulgaris and S. obliquus (mg/g dry weight) after 6 and 8 days of application,
respectively
The total soluble protein of S. obliquus was significantly increased according to the concentration of molasses and culture
conditions (light or dark).
Thus, concentrations 0.05, 0.15, 0.25, 0.35 and 0.45 % (v/v) of molasses induced very highly significant increase in the
protein content at P < 0.001 by about 8.3, 14.94, 26.01, 35.79 and 79.50 %, under mixotrophic conditions respectively (Fig. 3)
and also 29.71, 41.68, 64.30, 79.94 and 98.23 % under heterotrophic conditions after 8 days of incubation (Fig. 3).
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May 2014, Vol. 2 Iss. 2, PP. 20-28
D. Pigment Contents
Tables 1 and 2 indicate that chlorophyll (a+b) and carotenoids contents of C. vulgaris and S. obliquus varied according to
the concentrations of molasses in medium. Under mixotrophic condition, chlorophylls (a+b) and carotenoids content of C.
vulgaris increased by about (83.41 and 71.12%) after 6 days of incubation, in contrast to the pigment contents of S. obliquus
which decreased by about (71.12 and 25.66%) after 8 days of 0.45 (v/v) % incubation.
Under heterotrophic conditions, pigments content of cultures decreased with increase time of incubation, whereas that
heterotrophically cultured cells were bright yellow in color with increasing the time of incubation. Addition of molasses led to
significant increase of the chlorophyll (a+b) and Carotenoids content above the control level at p < 0.05 by about (74.93 and
36.67%) after 6 days of incubation for C. vulgaris and by (57.95 and 61.11%) for S. obliquus after 8 days of 0.45 (v/v)%
incubation.
E. Estimation of Lipid Contents
Table 1 and 2 shows that lipid contents of molasses treated C. vulgaris and S. obliquus were increased with increasing
molasses concentrations after 6 and 8 days of incubation under mixotrophic conditions, thus 0.05, 0.15, 0.25, 0.35 and 0.45%
(v/v) of molasses induced highly significant increase in the lipid contents by about 12, 24, 30, 48.8 and 52% for C. vulgaris
(F=419.54, P < 0.001) and 12.30, 14.37, 25.93, 33.33 and 57.04% for S. obliquus (F=619.39, P < 0.001), respectively above
the control culture. In contrast to the heterotrophic conditions, the lipid contents of treated C. vulgaris cultures showed highly
significant decrease by about 11.16, 17.40, 34.35, 54.59 and 62.22% corresponding to the above mentioned concentrations of
molasses (F=737.69, P < 0.001) and also lipid contents of S. obliquus reduced by about 24.70, 28.16, 33.88, 49.70 and 34.34%
below the control level at (F=194.93, p 0.001).
From the previous results, it is evident that the optimum conditions for growth and productivity of C. vulgaris and S.
obliquus was medium contain 0.45% (v/v) molasses under heterotrophic conditions. This amount of algal cells was subjected
to protein fractionation and ethanol fermentation.
TABLE 1 EFFECT OF DIFFERENT CONCENTRATIONS OF MOLASSES % (V/V) ON THE PIGMENTS AND LIPID CONTENTS OF C. VULGARIS AFTER 6 DAYS UNDER
MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Conc. (v/v)%
Cont.
0.05
Mixotrophic
condition
0.15
Heterotrophic
0.35
0.45
F-value
Cont.
0.05
0.25
condition
0.15
0.35
0.45
F-value
0.25
Chl (a+b) µg/ml 32.85±1 37.6±0. 45.83±0
60.25±0
3.64±0. 3.97±0. 3.90±0. 4.21±0. 5.76±0. 6.35±0.
52.88±0.40 59.24±1.20
20.67**
.60
40
.00
.90
11
45
60
60
32
10
algal suspension
Chl (a/b) µg/ml
0.98±0. 0.93±0. 0.95±0.
0.90±0.
0.90±0. 0.90±0. 0.93±0. 0.92±0. 0.93±0. 0.93±0.
0.94±0.10 0.90±0.06
24.31*
01
05
04
01
01
01
03
02
03
00
algal suspension
6.94*
6.09*
Carotenoid µg/ml
algal suspension
Lipid content
(mg/g DW)
4.12±0. 6.28±0. 6.44±0.
7.05±0.
6.64±0.10 6.77±0.06
20
05
04
12
29.29*
0.30±0. 0.32±0. 0.34±0. 0.35±0. 0.40±0. 0.41±0.
01
02
03
02
05
01
7.31*
12.5±0.
15.5±0.
14±0.11
16.25±0.05 18.6±0.00 19±0.04
05
15
124**
9.14±0. 8.12±0. 7.55±0.
4.15±0. 3.45±0.
6±0.06
04
02
00
05
00
158***
Values represent mean values ±standard deviation (n = 3). * Significant P < 0.05, **highly significant P <0.01, highly significant P < 0.001
TABLE 2 EFFECT OF DIFFERENT CONCENTRATIONS OF MOLASSES % (V/V) ON THE PIGMENTS AND LIPID CONTENTS OF S. OBLIQUUS AFTER 8 DAYS UNDER
MIXOTROPHIC AND HETEROTROPHIC CONDITIONS
Heterotrophic
condition
Mixotrophic
condition
F-value
Conc. (v/v)%
Chl (a+b) µg/ml
algal suspension
Chl (a/b) µg/ml
Cont.
0.05
0.15
0.25
0.35
0.45
Cont. 0.05
0.15
0.25
0.35
0.45
9.28*
26.87±19.71±18.31±17.44± 15.22 18.99
0.10 0.90 0.10 0.20 ±0.30 ±1.00
3.52±0 4.21±0 5.00±0.2 5.25±0.5 5.37±0.1
.54
.07
4
0
2
5.56±0.14
F- value
11.5**
0.93±0 0.88±0 0.89± 0.82±0 0.82±0 0.75±0 5.27* 082±0 0.94±0 0.89±0.0 0.86±0.0 0.84±0.0 0.98±0.01
.02
.02 0.01 .01
.03
.05
.18
.02
4
4
0
4.12*
algal suspension
3.78±0 3.38±0 2.80± 2.72±0 2.01±0 2.81±0 7.12* 0.27±0 0.34±0 0.42±0.0 0.42±0.0 0.42±0.0 0.43±0.06
.77
.14 0.04 .06
.00
.34
.02
.02
6
6
5
9.15**
Lipid content
(mg/g DW)
6.75±0 7.58±0 7.72± 8.05±0
10.6±0 84.1*** 6.64±0 5±0.0 4.77±0.0 4.39±0.0 3.34±0.0 4.36±0.04
.05
.07 0.03 .00 9±0.00 .01
.01
5
8
1
1
211***
algal suspension
Carotenoid µg/ml
- 24 DOI: 10.18005/JAEB0202002
Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
Values represent mean values ±standard deviation (n = 3).
Values represent mean ±standard deviation (n = 3). *Significant P < 0.05, **highly significant P < 0.01, ***very highly significant P < 0.001.
TABLE 3 PROTEIN FRACTIONATION OF C. VULGARIS AND S. OBLIQUUS (MG/G DRY WEIGHT)
Fraction %
0.45% (v/v)
Molasses
Control
Control
0.45% (v/v)
molasses
Water-soluble
"albumin"
28.70 ± 0.30
46.50 ± 0.80
25.76 ±0.46
45.44 ± 0.70
Salt -soluble
"globulin"
10.30 ± 1.40
12.01 ± 0.56
14.65 ±0.36
14.30 ± 0.70
18.20 ± 0.45
18.66 ± 0.20
17.72 ±0.10
18.04 ± 0.70
Alkali- soluble
"glutilin"
38.05 ± 0.85
21.50 ±1.0
37.43 ±1.60
20.78 ± 0.60
Insoluble protein
4.75
1.33
4.34
1.44
Total
100.00
100.00
100.00
100.00
Alcohol- soluble
"protamine"
Values represent mean values ±standard deviation (n = 3).
TABLE 4 ABILITY OF C. VULGARIS AND S. OBLIQUUS FOR PRODUCTION OF ETHANOL UNDER HETEROTROPHIC CONDITIONS.MEANS ±SD (N=3)
Untreated
Chlorella
vulgaris
Molasses
treated C. vulgaris
(0.45 %(v/v))
Untreated
Scenedesmus
obliquus
Molasses
treated Sc.
obliquus (0.45 %
(v/v))
Concentrations of glucose before
fermentation (mg/g dry cell
weight)
62.98 ± 2.40
26.84 ± 3.3
38.75 ± 0.90
22.57 ± 0.35
Concentration of glucose after
fermentation (mg/g dry cell
weight)
30.28 ± 1.21
13.50 ± 3.95
14.40 ± 1.21
8.69 ± 0.69
Concentration of ethanol (mg/g dry cell
weight)
38.23 ± 3.50
15.02 ± 1.80
17.22 ± 0.60
10.69 ± 1.20
Percentage of glucose consuming during
fermentation (%)
51.90
49.70
62.8
61.5
Values represent mean values ±standard deviation (n = 3).
F. Protein Fractionation
It is obvious that glutilin is the highest protein fraction in untreated cultures, but in treated albumin is the highest one
followed by glutilin, protamine, globulin and smallest amount of insoluble fraction.
G. Ethanol Fermentation
The saccharified solution which was consumed during ethanol fermentation of treated cultures were below the level of
untreated cultures by about 57.38 % for C. vulgaris and 41.75% for S. obliquus before fermentation process, so the ethanol
concentration produced from treated cultures was lower than untreated cultures by about 61.71 and 37.92% for C. vulgaris and
- 25 DOI: 10.18005/JAEB0202002
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May 2014, Vol. 2 Iss. 2, PP. 20-28
S. obliquus, respectively.
IV
DISCUSSION
The tested alga C. vulgaris and S. obliquus were suitable for mixotrophic and heterotrophic mass cultivation by different
rate depending on dose of molasses, algal species and illumination of culture. This observation has been emphasized by [38]
who recorded that assimilation of organic carbon sources depend on the cell strain and culture conditions. Shamala et al., [6]
demonstrated that the increase in algal growth depended on the inoculum level of the algae as well as the amount of molasses
added to the cultures and stated that in mixotrophic cultures with carbon dioxide and molasses, the biomass production of S.
acutus increased from 46 g/m2/3days to 98 g/m2. Xua et al., [39] showed that cell density of C. protothecoides significantly
increased under the heterotrophic condition. Bhatnagar et al., [40] detected that the mixotrophic growth of Chlamydomonas
globosa, C. minutissima and S. bijuga resulted in 3–10 times more biomass production relative to phototrophy. Liu et al. [41]
stated that biomass production of C. zofingiensis mutant on molasses increased 2 fold under heterotrophic condition more than
phototrophy conditions. On the other hand, Leesing and Kookkhunthod [42] stated that no significant difference in Chlorella
sp. KKU-S2 growth biomass using different molasses concentration.
It can be concluded from our results that the increase in carbohydrate contents as result of treatment molasses may be
correlated with increase in algal dry weight. This observation is similar to that observed by [7] who stated that the increase in
carbohydrate content of S. acutus in cultured supplied with molasses and CO2 may be attributed to higher cell division rate,
Chu et al., [43] concluded that inclusion of glucose in culture of Ankistrodesmus convolutus induced increase in carbohydrate
contents. On other hand, the carbohydrate contents of both tested microalgae also increased under heterotrophic conditions,
this phenomenon is in agreement with the results of Griffiths [44] who stated that heterotrophically grown cells was diverted
toward carbohydrate synthesis rather than toward the synthesis of other cellular constituents. Shamala et al., [45] showed that
heterotrophic growth lead to accumulation of carbohydrate.
Our results indicated that the quantity of protein accumulated by both organisms depended on the applied concentrations of
molasses and culture conditions and these results are in agreement with [44] who stated that cultivation of Scenedesmus on
molasses increased the protein production. Becker and Venkataraman [7] recorded that the addition of molasses increases
carbohydrate and protein contents of Scenedesmus grown under different culture conditions.
It could be deduced from this result that molasses act as stimulator for pigment biosynthesis in C. vulgaris. This may be
due to the effect of nitrogenous content of molasses. Similar observation were obtained by Piorreck et al., [46] who found that
increasing N concentrations in the nutrient medium led to a big increase in chlorophyll content in the green algae. The
pigments content of treated S. obliquus decreased with increase the concentrations of molasses. Under heterotrophic
conditions, the effect of molasses on the pigments content of both tested algae showed a similar pattern of changes in response
to the increase in molasses concentrations and cultured cells were bright yellow in color at the end of incubation period. This
observation coincides with Chen’s [18] who stated that under heterotrophic conditions, the algal cells characterized by inability
to produce some light induced products such as pigments.
With regard to the effect of molasses on lipid content of tested algae, the results showed that application of molasses
increased the lipid contents of both microalgae under mixotrophic conditions. Xu et al., [47] stated that the yield of lipid
content (Eicosapentaenoic acid) of Microcystis sp. was 22 mg L-1 in the mixotrophic cultivation and 20 mg L-1 under
photoautotrophic conditions. Liu et al., [48] demonstrated that cane molasses provided better productivities of lipid, and
astaxanthin of Chlorella zofingiensis 0.71 g L-1 day-1 than glucose.
Leesing and Kookkhunthod [42] detected that the increase in molasses concentration beyond 30g/L resulted in a slight drop
in lipid content of Chlorella sp. KKU-S2.
On the other hand, the lipid contents were reduced with increase concentrations of molasses under heterotrophic
conditions. Our results are in conformity with Becker [49] who demonstrated that the heterotrophic culture of Tetraselmis had
low lipid levels (especially fatty acids) compared to phototrophically cultured cells.
Relatively to the effect of molasses as carbon source of protein fractions, molasses elevated the value of albumin as
compared to the control value in both tested algae. Its amounted ranged from 45.70 to 47.30% in C. vulgaris and 44.74 to
45.14% in S. obliquus. In human nutrition, the most important protein is the water- soluble fraction due to its high digestibility;
therefore the biological value of protein depends on the amount of albumin fraction.
Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal
carbohydrates can be derived from atmospheric CO2 and their harvesting methods are simple [50]. The present results show
that the both tested algae have high content of carbohydrate under heterotrophic conditions which consumed during ethanol
fermentation. Algae capable of accumulating high starch/cellulose can serve as an excellent alternative to food crops for
bioethanol production, a green fuel for sustainable future in addition, certain species of algae can produce ethanol during darkanaerobic fermentation and thus serve as a direct source for ethanol production [51]. Some microalgae can accumulate high
starch content (about 44% of dry base) via photosynthesis [52]. In general, our results showed that the concentrations of
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May 2014, Vol. 2 Iss. 2, PP. 20-28
ethanol increased by raising the molasses concentration in saccharified solution of algae. Miyamoto and Hirata [53] reported
that Chlamydomonas reinhardtii was fermented using Zymomonas mobilis, ethanol conversion of glucose was 97%. Choi et
al., [52] detected approximately 235 mg of ethanol was produced from 1.0 g of Chlamydomonas reinhardtii UTEX 90 biomass
by two commercial hydrolytic enzymes.
REFERENCES
[1] M. R. Droop, “Heterotrophy of carbon,” in: W.D.P. Stewart, (Ed), Algal Physiology and Biochemistry, Blackwell, Oxford, pp. 530-559,
1974.
[2] A. H. Nelison and R. A. Lewin, “The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry”,
Phycologia, vol. 13, pp. 227-264, 1974.
[3] A. J. Smith, J. London, and R.Y. Stanier, “Biochemical basis of obligate autotrophy in blue-green algae and thiobacilli,”. J. Bacteriol,
vol 94, pp. 972-983, 1967.
[4] N. D. E. Pauw and G. Persoone, “Micro-algae for aquaculture”, in: M. A. Borowitizka, L. J. Borowitzka, (Eds.), Micro-algal
Biotechnology, Cambridge University Press, Cambridge, pp. 197-221, 1988.
[5] A. Killam and J. A. Myers, “Special effect of light on the growth of Chlorella vulgaris,” Amer. J. Bot, vol. 43, pp. 569-572, 1956.
[6] T. R. Shamala, F. Drawert, and G. Leupold,“Studies on Scenedesmus acutus growth, I. Effect of autotrophic and mixotrophic conditions
on the growth of Scenedesmus acutus”, Biotechnol., Bioengin., vol. 24, pp. 1287-1299, 1982a.
[7] E. W. Becker and L. V. Venkataraman, “A manual on the cultivation and processing of algae as a source of single cell protein”, Algae
Feed Food, pp. 1-36, 1979.
[8] J. Malanowska and S. Labendzinski, “Changes in the chemical composition of molasses depending on period of year, in which the sugar
beet was processed” Prace inst. Lab. Bad. Przem. Spoz, vol. 19, pp. 27-42, 1969.
[9] A. Ginterova, “Dedikaryotization of higher fungi in submerged culture”, Folia Microbiol., (Praha) vol. 18, no.4, pp. 277-280, 1973.
[10] C. Combres, G. Laliberte, J. S. Reyssac and J. Lanoue, “Effect of acetate on growth and ammonium uptake in the microalga
Scenedesmus obliquus”, Plant Physiol., vol. 91, pp. 729-734, 1994.
[11] J. Bai, “Tätigkeitsbericht, Kohlenstoffbiologische Forschungsstation” E. V. Dortmund, In: T. R Shamala, F. Drawert, and G. Leopold,
1982a. “Studies on Scenedesmus acutus growth I. Effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus
acutus”, Biotechnol. Bioengin., vol. 24, pp. 1287-1299, 1973.
[12] E. W. Becker, “Biotechnology and exploitation of the green alga Scenedesmus obliquus in India”, Biomass, vol 4, no. 1, pp. 1-19, 1984.
[13] M. A. Borowitzka, “Commercial production of microalgae: ponds, tanks, tubes and fermenters”, J. Biotechnol., vol. 70, pp. 313-321,
1999.
[14] O. Perez-Garciaa, F. M. E. Escalantea, L. E. De-Bashana, and Y. Bashana “Heterotrophic cultures of microalgae: Metabolism and
potential products”, Water Res., vol. 45, no. 1, pp. 11-36, 2011.
[15] J. A. Running, R. J. Huss, and P. T. Olson, “Heterotrophic production of ascorbic acid by microalgae”, J. Appl. Phycol., vol. 6, pp. 99104, 1994.
[16] M. R Johns, “Heterotrophic culture of microalgae”, in: S.M. Phang, M. A. Borowitzka, B. A. Whitton, (Eds.), Algal Biotechnology in
Asia-Pacific Region, University Malaya, Kuala Lumpur, pp. 150-154, 1994.
[17] F. Chen and M. R. Johns, “A strategy for high cell density culture of microalgae with inhibitory substances”, J. App. Phycol., vol. 7, pp.
43- 46, 1995.
[18] F. Chen, “High cell density culture of microalgae in heterotrophic growth”, Trends Biotechnol., vol. 14, pp. 421- 426, 1996.
[19] A. M. Hanson, “Microbial production of pigments and vitamins”, in: H. J. Peppler, (Ed.), Microbial Technol., pp. 222-250, 1967.
[20] K. Kawaguchi, “Microalgae production systems in Asia”, in: G. Shelef, C. J. Soeder, (Eds.), Algae Biomass Production and Use,
Elsevier/North Holland Biomedical Press, Amsterdam, pp. 25-33, 1980.
[21] P. Soong “Production and development of Chlorella and Spirulina in Taiwan”, in: G. Shelef, C. J. Soeder, (Eds.) Algae Biomass,
Elsevier/North Holland Biomedical Press, Amsterdam, pp. 97-113, 1980.
[22] Y. K. Lee “Commercial production of microalgae in Asia Pacific rim”, J. Appl. Phycol., vol. 9, pp. 403-411, 1997.
[23] J. T. Ellis, N. N. Hengge, R. C. Sims, C. D. Miller, “Acetone, butanol, and ethanol production from wastewater algae,” Biores.
Technol., vol. 111, pp. 491-495, 2012.
[24] M. Watanabe and Y. Niiyama, “Fresh water algae from Lake Akan (5)”, Bulletin of National Science, Nus. Tokyo, ser., vol. 16, no. 1,
pp. 29-39, 1990.
[25] G. S. Venkataraman, “The cultivation of algae. Published by Indian Council of Agricultural Research”, New Delhi, 1969.
[26] A. Kühl, “Zur physiologie der speicherung kondersierter an organischer phosphate in Chlorella”, Vortr. Botan. Hrsg. Deut. Botan. Ges.
(N.F.), vol. 1, pp. 157-166, 1962.
[27] P. Starr, H. G. Stolp, A. Balows, and H. G. Schelegel, “The prokaryoter springer”, Verlag Berlin, Heidelberg, U.S.A, 1981.
[28] H. Lorenzen, “Synchronization of Chlorella with light-dark changes and periodical dilution to a standard cell number”, in: E. ZEITEN,
(Ed.), Inersci, Synchrony of cell division and growth, Publ., NewYork, p. 571, 1964.
[29] D. F. Wetherell, “Culture of fresh water algae in enriched natural seawater”, Plant Physiol. (Copenh), vol. 14, pp. 1-6, 1961.
[30] M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, “Colorimetric method for determination of sugars and related
- 27 DOI: 10.18005/JAEB0202002
Journal of Agricultural Engineering and Biotechnology
May 2014, Vol. 2 Iss. 2, PP. 20-28
substances”. Anal.Chem., vol. 28, pp. 350-356, 1956.
[31] O. M. Lowry, N. J. Roserbrough, L. A. Farr, and R. J. Randall, “Protein measurements with the folin phenol reagent”, J. Biol. Chem.,
vol. 193, pp. 265-275, 1951.
[32] S. W. Jeffrey and G. F. Humphrey, “New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants,
algae and natural phytoplankton”, Biochemie Physiolgie der Pflanzen, vol. 167, pp. 191-194, 1975.
[33] A. Jensen and J. S. Liaan, “Qualitative paper chromatography of carotenoids”, Acta Chemica Scandinavica, vol. 13, pp. 1863-1868,
1959.
[34] A. K. Varma and P. N. Tiwari, “Rhizobium inoculation and oil content of soy bean seeds (Glycine max)”, Current Science, vol. 20, pp.
275, 1967.
[35] L. V Shoch, C. W. Deyoe, F. K.Shoup, J. Bathurst, and D. Liang, “Protein fractionation of Sorghum grains”, Cereal Chem., vol. 47, pp.
472, 1970.
[36] A. Polkis and M. A. Mackell,“Quantitative estimation of ethyl alcohol”, Clinical Chem., vol. 28, pp. 2125-2127, 1982.
[37] A. A. Ghonam and N. M. Sabre, “Statistical analysis of data by using SPSS”, Dar Kappa Publishing, Cairo, 2000, pp. 300, 1999.
[38] F. Martinez and M. I. Orus, “Interactions between glucose and inorganic carbon metabolism in Chlorella vulgaris strain UAM 101”,
Plant Physiol., vol. 95, pp. 1150-1155, 1991.
[39] H. Xua, X. Miao, and Q. Wu, “High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in
fermenters”, J. Biotechnol., vol. 126, vol. 4, pp. 499-507, 2006.
[40] A. S. Bhatnagar, M. Chinnasamy, Singh and K. C. Das, “Renewable biomass production by mixotrophic algae in the presence of
various carbon sources and wastewaters”, Appl. Energy, vol. 88, no.10, pp. 3425-3431, 2011.
[41] J. Liu, Z. Sun, Y. Zhong, H. Gerken, J. Huang, and F. Chen, “Utilization of cane molasses towards cost-saving astaxanthin production
by a Chlorella zofingiensis mutant,” J Appl Phycol, DOI 10.1007/s10811-013-9974-x, 2013.
[42] R. Leesing and S. Kookkhunthod, “Heterotrophic Growth of Chlorella sp. KKU-S2 for Lipid Production using Molasses as a Carbon
Substrate,” International Conf. Food Eng. Biotechnol, 2011.
[43] W. L. Chu, S. M. Phang, and S. I. Hock, “influence of carbon source on growth, biochemical composition and pigmentation of
Ankistrodesmus convolutes”, J. Appl. Phycol., vol. 7, pp. 59-64, 1995.
[44] D. J. Griffiths, “The accumulation of carbohydrate in Chlorella vulgaris under heterotrophic conditions”, Annals Botany, vol. 115, pp.
347-357, 1965.
[45] T. R. Shamala, F. Drawert, and G. Leupold, “Studies on Scenedesmus acutus growth. II. Effect of autotrophic and mixotrophic growth
on the amino acid and carbohydrate composition of Scenedesmus acutus”, Biotechnology and bioengineering, vol. 24, pp. 1301-1317,
1982b.
[46] M. Piorreck, K. H. Baasch, and P. Pohl, “Biomass production, total protein, chlorophylls, lipids and fatty acids of freshwater green and
blue-green algae under different nitrogen regimes,” Photochem, vol. 23, vol.2, pp. 207-216, 1984.
[47] F. Xu, H. H. Hu, W. Cong, Z. L. Cai, and F. Ouyang, “Growth characteristics and eicosapentaenoic acid production by Nannochloropsis
sp. in mixotrophic conditions”, Biotechnol. Letter, vol. 26, no. 1, pp. 51-53, 2004.
[48] J. Liu, J. Huang, Y. Jiang, and F. Chen, “Molasses-based growth and production of oil and astaxanthin by Chlorella zofingiensis,”
Bioresour Technol., vol.17, no.107, pp. 393-398, 2012.
[49] E. W. Becker, “Microalgae, Biotechnology and microbiology”, Cambridge University Press, Cambridge, pp. 293, 1994.
[50] T. D. Nguyen, K-P. Kim, M-T. Nguyen, M. S. Kim, D. Kim, and S. J. Sim, “Enhancement of fermentative hydrogen production from
green algal biomass of Thermotoga neapolitana by various pretreatment methods”, Int. J. Hydrogen, vol. 35, no. 23, pp. 13035-13040,
2010.
[51] R. P. John, G. S. Anisha, K. M. Nampoothiri, and A. Pandey, “Micro and macroalgal biomass: A renewable source for bioethanol”,
Biores. Technol., vol. 102, no.1, pp. 186-193, 2011.
[52] S. P. Choi, M. T. Nguyen, and S. J. Sim, “Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production”,
Biores. Technol., vol. 101, no. 14, pp. 5330-5336, 2010.
[53] K. Miyamoto and K. Hirata, “Microalgal CO2 fixation and biological recycling of resources. Seisan to Gijutsu (in Japanese)”, vol. 48,
pp. 59- 63, 1996.
- 28 DOI: 10.18005/JAEB0202002