최희정
(Hee-Jeong Choi)
*†
이정민
(Jung-Min Lee)
**
Copyright © 2016, Korean Society on Water Environment
Key words
Acorn-starch, Biomass, Glucose, Microalgae, Mixotrophic, Oil content
1. Introduction
Algae strains that are robust and highly productive are selected for the conversion
of biomass into energy (Spolaore et al., 2006), and strains with relatively high lipid contents are very attractive for biodiesel
fuel production (Choi, 2015b; Rudolfi et al., 2009). Microalgae have received considerable interest as a source of renewable energy;
however, further optimization of the mass culture conditions is necessary to make
microalgal biofuel production economically viable and sustainable (Choi, 2014; Pittman et al., 2011).
Many algal organisms can use either autotrophic, heterotrophic or mixotrophic metabolic
processes for growth. The growth rate and biomass production for some algae in mixo-or
heterotrophic conditions can be several times higher than in photoautotrophic-only
conditions (Qiao et al., 2009; Yang et al., 2000; Zhang et al., 2011). Moreover, the synthesis of metabolic products, such as lipids and pigments, is influenced
by the quality and quantity of organic carbon. The use of organic carbon in mixotrophic
cultures reduces the need for carbon dioxide in the culture and facilitates the growth
of algal species that are sensitive to agitation (Andrade and Costa, 2007; Chojnacka and Noworyta, 2004). Bouarab et al. (2004) reported that Micractinium pusillum grew in the presence of organic substrates, such as glucose and acetate, under both
mixotrophic and heterotrophic conditions. It can be concluded from the above that
mixotrophism is an ideal nutritional mode for high density cultivation of microalgae
for the production of biofuels and functional components. However, even though the
biomass and lipid productivities are significantly higher compared with those from
autotrophic growth, the cost of organic carbon sources (usually in the form of glucose
or acetate) is high when compared to all other added nutrients. To overcome this high
carbon cost, a cheap resource must be found.
Acorns are capable of providing such a supply. Acorns are much cheaper than other
organic carbon sources and are found everywhere in the world. The acorn is the nut
from oaks and their close relatives (genera Quercus and Lithocarpus, in the family Fagaceae). It usually contains a single seed (rarely two seeds), enclosed
in a tough, leathery shell and borne in a cup-shaped cupule. Acorns vary from 1-6
cm in length and 0.8-4 cm in width. Acorns are also rich in nutrients. Percentages
vary from species to species, but all acorns contain large amounts of water (10.6%),
protein (0.8%), carbohydrates (87.3%) and fats (1.2%), as well as various minerals
(e.g., calcium, phosphorus, magnesium, potassium and the vitamin niacin) and sugar
(e.g., rhamnose, galactose, arainose, mannose, fucose and xylose) (Choi, 2015a). Glucose is especially rich in the acorn, representing 97.9% of the sugar composition.
Glucose is an important carbohydrate in biology, indicated by the fact that cells
use it as a secondary source of energy and a metabolic intermediate. Glucose is one
of the main products of photosynthesis and fuels for cellular respiration (Pyle et al., 2008). In mixotrophic growth, there are two distinctive processes: photosynthesis and aerobic
respiration. The former is influenced by light intensity, and the latter is related
to the organic substrate (glucose) concentration (Andruleviciute et al., 2014; Heredia-Arroyo et al., 2011). The high cell density in mixotrophic cultures has demonstrated that the growth stimulating
effects of light and CO2 in mixotrophic cultures are as strong as the effects of glucose
(Kong et al., 2013).
The above findings suggest that acorn-glucose is a potential substrate for the mixotrophic
cultivation of microalgae that may reduce the production cost of microalgal biodiesel.
However, there are few reports on the use of acorn-glucose in biomass production and
algal cell components under mixotrophic conditions. Therefore, the effects of various
concentrations of saccharified acorn-starch (acorn-glucose) on the biomass growth
and triacylglycerols (TAGs) content of Chlorella vulgaris (C. vulgaris) were evaluated under mixotrophic conditions in this study.
2. Materials and Methods
2.1. Microalgae Cultures and Medium
The investigated microalgae were isolated from KMMCC (Korea Marine Microalgae Culture
Center). The C. vulgaris (FC-16, KMMCC-135) cells were round in shape and 3-8 μm in diameter. C. vulgaris cells were cultivated in Jaworski’s medium (JM), which was prepared using deionised
water under LED lamps at ambient temperature. Jaworski’s medium is comprised of 4.0
g Ca(NO3)2H2O, 2.48 g KH2PO4, 10.0 g MgSO4·7H2O, 3.18 g NaHCO3, 0.45 g EDTAFeNa, 0.45 g EDTANa2, 0.496 g H3BO3, 0.278 g MnCl2·4H2O, 0.20 g (NH4)6Mo7O24·4H2O, 0.008 g cyanocobalamin, 0.008 g thiamine HCl, 0.008 g biotin, 16.0 g NaNO3 and 7.2 g Na2HPO4·12H2O in 200 ml of deionised water. The initial concentration of C. vulgaris was 0.357 g/L, and the pH was adjusted to 7.3±0.3. Each culture was grown at room
temperature (23±2℃) under 200~250 μmol photons/m 2s illumination using white LEDs
(light emitting diodes) for dark and light cycles of 12 h each. Light intensity was
measured with a data logger (model LI-1400) using an LI-190SA quantum sensor. For
batch cultivation in laboratory conditions, Erlenmeyer glass flasks with a working
volume of 5 L were used, and a 15-day cultivation period was chosen. The cultivation
period of 15 days was not chosen randomly; the highest biomass concentration is reached
during the stationary growth phase, and the lipid content increases with increasing
cultivation time.
Mixotrophic conditions for microalgae cultivation were achieved with acorn-glucose.
This acorn contained considerable amounts of starch and glucose. The corresponding
amount of acorn-glucose was added to the JM growth medium to achieve the desired mixotrophic
medium. The cultures were shaken by hand several times a day to avoid sticking.
2.2. Pretreatment of Acorn
Acorns were collected in Ganuneung City in Korea. To remove adherent and interference
materials, such as organics and salts, the acorns were rinsed several times with deionised
water. After cleaning, the hard shell of the acorn was removed, and the nuts were
dried in an oven at 105℃ for 24 h then crushed into a fine powder using a mortar and
pestle. Tannins were removed by soaking chopped acorns in several changes of water
until the water no longer turned brown. The acorn powder was extracted using ultrasonic
cycle extraction equipment (JYD-US01, Shenzhen Jiayuanda Technol. Co., Ltd, Guangdong,
China) with 20-kHz ultrasonic frequency in an 80% ethanol solution for 120 min with
a liquid-to-solid ratio of 15:1 (Pan et al., 2014).
2.3. Saccharification of Acorn-starch
Acorn powder was added to deionized water at a ratio of 1:5 and mixed with calcium
chloride and α-amylase (30 U/g dry acorn) for 2h at 90℃. The liquefied mixture was
saccharified with glucoamylase (150 U/g dry acorn) for 4h at pH 4.0 and 60℃ (Tang et al., 2011). Acorn powder was treated as above for the liquefaction step; however, the saccharification
step was allowed to continue for 12 h to completely convert all the starch to glucose.
The glucose was measured to determine the total starch in the powder.
2.4. Analytical Methods
The experiment was carried out five times, and the mean values and standard deviations
were calculated. Saccharification of starch represents a normal glucose equivalent
(Dextrose Equivalent, DE).
DE = (Glucose form saccharified starch/ total solid) × 100
2.4.1. Proximate Compositions
The moisture content of the acorns was determined by drying the various parts in an
oven at 105℃ until a constant weight was obtained. The crude protein content was calculated
by converting the nitrogen content, which was determined using the method proposed
by Kjeldahl (6.25 × N). The fat content was determined with the acid saccharification
Soxhlet system using the method described by the AOAC (2005). The ash content was
determined by dry ashing in a furnace oven at 600℃ for 10 h. The carbohydrate content
was estimated by subtracting the sum of the weights of the protein, fibre and ash
from the total dry matter. All determinations were performed in triplicate.
2.4.2. Measurement of Minerals
The samples used for mineral determination were first digested in HNO3/HCl. The elements K, Ca, Fe, Na and Mg were measured with atomic absorption spectrophotometry
(AAS) using a Varian Spectra atomic absorption spectrophotometer, Buck Scientific
210 GVP model. All determinations were performed in triplicate, and we also added
spike samples to verify the accuracy of the procedure. P was analysed according to
the Mo-blue method using a UV/Vis Spectrophotometer DU800 (Beckman Coulter, USA).
2.4.3. Determination of Monosaccharides for Acorn
The monosaccharide content was determined by a method of separation described by Blakeney et al. (1983). The 10-mg sample was placed into a Teflon-lined screw cap tube and mixed with 125
μL of 73% (w/w) H2SO4. After 45 minutes, the solution was saccharified with 1.35 mL of distilled water
at 100℃ for 3 hour, and then it was neutralised by adding 320 μL of 15M NH4OH. After neutralization, 1 mL of 2% NaBH4 in DMSO was added to the mixture to react for 90 minutes at 40℃. Next, 100 μL of
18 M glacial acetic acid, 200 μL of 1-methylimidazole and 2.0 mL of acetic anhydride
were added to the reaction mixture and allowed to stand at room temperature for 10
minutes. After decomposing, the excess acetic anhydride was separated into a microcentrifuge
tube for analysis by GLC. The GLC analysis conditions are shown in Table 1.
Table 1.
Instrument and operation conditions for monosaccharide analysis by GLC
2.4.4. Measurement of Cell Weight and Specific Growth Rate
Acorn-glucose at various concentrations (0, 1, 2, 3 and 5 g/L) was added during the
initial growth phase and the growth of the algae biomass as well as the lipid accumulation
was evaluated. To determine the biomass concentration, a sample of microalgae in growth
medium was centrifuged for 10 min at 628 g, washed with distilled water and dried
in an oven at 105ºC for 24 h to constant weight. The biomass productivity P (g/(L·day))
was calculated from the variation in biomass concentration X (g/L) within a cultivation time t (in days), according to the following equation:
The specific growth rate μ (in days) was calculated using equation (2):
where X1 and X0 are the biomass concentration (g/L) on days t1 and t0, respectively.
2.4.5. Extraction of Lipids
The algal biomass for lipid extraction was prepared by centrifugation and drying.
After oven drying, the algae were pulverised and subjected to Soxhlet extraction.
All Soxhlet extractions were performed for 72 h using 500 mL of solvent for 1 g of
pulverised dry algae with a cycle time of 10-15 min. The Soxhlet extraction with hexane
was selected because the Bligh and Dyer (1959) extraction method is suitable for the extraction of all lipids, including triglycerides,
phospholipids and other pigments (Sobczuk and Chisti 2010). The lipid content does not reflect the exact amount of triacylglycerols (TAGs) because
only triglycerides are used in the synthesis of biodiesel, and other components are
undesirable. The excess hexane was evaporated by rotary evaporation until the total
volume reached 30-40 mL. The solutions were diluted to 50 mL and used to determine
the TAG content. The amount of TAGs was determined using a Fourier transform infrared
(FTIR) spectrometer Spectrum RX 1 (Perkin Elmer) according to the carbonyl stretching
absorption at 1740/cm (Stehfest et al., 2005). The amount of TAGs in the extract solutions was determined using a standard graph,
and the amount of TAGs was calculated in the dry algae (%, w/w). The experiments were
performed five times, and the mean values and standard deviations were calculated.
3. Results and Discussion
3.1. Saccharification of Acorn Powder
The composition of raw acorn and saccharified acorn is represented in Table 2. The proximate compositions such as moisture, crude ash, crude protein, crude fat
and carbohydrate decreased from 70.2-78.8% to 14.7-20.5%. In contrast, sugar composition
(Glucose, Rhamnose, Galactose, Arabinose, Mannose, Fructose and Xylose) increased
after sacchrification of the acorns, from 23.2-28.6% to 75.3-91.4%. These results
indicated that most of the starch in the acorns was converted to glucose through saccahrification.
Mineral contents (K, P, Ca, Na, Mg and Fe etc.) and fatty acid composition decreased
slightly from 4.3-5.2% to 3.8-4.9% and 5.6-6.2% to 5.1-5.4%, respectively, with saccharification.
Table 2.
The acorn composition
Fig. 1 illustrates glucose release during the saccharification of acorn starch from the
pretreated materials. Starch concentration decreased to 14.50 g/L from 123.50 g/L.
While starch concentration decreased by 88.3%, glucose increased 9.8 fold in 5 hours
after saccharification. The saccharification rate reached 81.3% within 5 hours but
the starch could not be converted completely. After 5 hours of saccharification, the
glucose and starch concentrations did not change further. α-amylase and glucoamylase,
saccharified enzymes, convert the α-1,4- bond polymer in starch to form low molecular
weight dextrins such as glucose, maltose, oligosaccharides. The activities of α-amylase
and glucoamylase are inhibited by tannins (Pan et al., 2014), which comprise about 6.2% of acorns. After the saccharification enzymes saccharified
amylose to dextrins (6 to 8 form glucose), the dextrin is decomposed into oligosaccharides
such as maltoteratose, maltopentaose and maltotriose by the enzyme. Thereafter, oligosaccharides
are slowly broken down to glucose and maltose by the enzyme and the concentration
becomes diluted (Charef et al., 2008). However, if the concentration of oligosaccharides is high, isomaltose and panose
can be regenerated using saccharified energy from maltose and glucose. In particular,
since the concentration of glucose and maltose increase rapidly in accordance with
the progress of sacchrification, the regeneration of isomaltose and panose also occurs
rapidly (Tang et al., 2011). Thus, obtaining a large amount of glucose from starch saccharification is difficult.
Glucose is a very important carbon source for the growth of microalgae under mixotrophic
conditions and was the main sugar composition in the acorns evaluated. Chaudhary et al. (2012) reported that glucose is a very important factor for microalgae growth and that E.
coli growth with glucose is 3 times faster than with glycerol. The results of the
current study indicated that the acorns contained a considerable amount of carbonate
and glucose, which positively affected the growth of microalgae.
Fig. 1.
Process of glucose and starch concentration during saccharification of acorn starch.
3.2. Effect of Acorn-glucose Dosage on the Growth of Algal Species
Figure 2 shows the effect of different acorn-glucose concentrations on the growth of C. vulgaris compared to growth in the autotrophic condition. On the first day, the microalgae
grew at the same rate in all concentrations of acorn-glucose. After 2 days, microalgae
growth in 5 g/L acorn-glucose was significantly higher than the other samples. The
biomass increased for the first 6 days in the sample containing 5 g/L acorn-glucose.
Thereafter, the rate of biomass production was similar to the 3 g/L acorn-glucose
sample, indicating that high glucose concentrations inhibit the growth of microalgae.
Maximum biomass productions were 0.76±0.03, 3.49±0.05, 8.06±0.35, 12.44±1.34 and 10.62±0.75
g/L in the media containing 0, 1, 2, 3 and 5 g/L acorn-glucose, respectively. This
was 4.6, 10.6, 16.4 and 14.0 fold higher than the concentration achieved with C. vulgaris in autotrophic medium, respectively. The highest biomass production in the mixotrophic
condition was obtained with 3 g/L acorn-glucose. All media with acorn-glucose yielded
a higher biomass than the autotrophic condition.
Fig. 2.
Growth of C. vulgaris in various acorn-glucose concentrations.
The oxidation of glucose in microalgae contributes to a series of complex biochemical
reactions that provide the energy needed by cells (Choi and Yu, 2015; Liang et al., 2009). The first step in the breakdown of glucose in all cells is glycolysis to produce
pyruvate, which is the starting point for all other processes in cellular respiration.
In cells where oxygen is present (aerobic respiration), these processes are modelled
in the tricarboxylic acid cycle (TCA) or the Krebs cycle. The majority of the energy
generated from glucose oxidation is used in the conversion of adenosine diphosphate
(ADP) to adenosine triphosphate (ATP), with the energy-rich molecule ATP used subsequently
as the energy currency of the cell (Mitra et al., 2012; Perez-Garcia et al., 2010). Bouarab et al. (2004) reported that Micractinium pusillum grew in the presence of organic substrates, i.e., glucose and acetate, under mixotrophic
conditions, as well as they did under heterotrophic conditions. The growth of M. pusillum was much more eugenic in the light than in the dark and was higher in the presence
of glucose than acetate. It can be concluded from the above that mixotrophism is an
ideal nutritional model for the production of biofuels and functional components.
In the present study, the acorn-glucose concentration influenced the biomass production
of C. vulgaris. The results of this study suggest that the investigated algae species may be excellent
biofuel producers because organic materials stimulate the growth rate of these strains.
Growth of C. vulgaris in various acorn-glucose concentrations depicted in Figure 3. C. vulgaris achieved a maximum biomass productivity of 0.342±0.015 g/L·day and a maximum specific
grow rate of 0.367±0.021 g/L·day with 3 g/L acorn-glucose. The maximum biomass productivity
and maximum specific growth rate in samples with acorn-glucose were higher than the
authotrophic condition.
Fig. 3.
Pmax and μmax with various acorn-glucose concentrations.
An early report indicated that mixotrophic growth had the potential to greatly increase
the microalgal cell concentration and volumetric productivity in a batch system (Yamane et al., 2001). The report established that the adenosine triphosphate formed in photochemical reactions
accelerated glucose anabolism in the mixotrophic culture of Euglena gracilis and was the reason why growth in the mixotrophic culture increased. The results of
our study suggest that C. vulgaris has the potential to be an excellent biofuel producer because the growth rate of
the strains can be stimulated by organic materials.
3.3. Total TAGs (triacylglycerols) Content in Algae Species
The total TAGs content with different acorn-glucose concentrations are represented
in Table 3. The highest TAGs content was 32.9% for C. vulgaris with 3 g/L acorn-glucose. Furthermore, 3 g/L acorn-glucose resulted in 29.2% more
TAGs content in C. vulgaris compared with the autotrophic condition. The TAGs content increased approximately
2 fold with each 1 g/L acorn-glycerol increment, up to 3 g/L acorn-glucose. However,
slight inhibition was observed during the initial cultivation when the acorn-glucose
concentration reached 5 g/L. Therefore, to obtain high TAGs content, the recommended
mixotrophic condition for the microalgae species is 3 g/L acorn-glucose.
Table 3.
Total TAGs content in dry biomass for different acorn-glucose concentrations
Liang et al. (2010) observed an increase in lipid content with increasing concentrations of glucose.
The lipid content increased from 22% with 1 g/L glucose to 32% with 2 g/L glucose;
however, the highest amount (10 g/L) of glucose had an inhibitory effect on the growth
of algae and on TAGs content. Another study demonstrated that Chlorella protothecoides was slightly inhibited by glucose and could still grow even when the salinity of
the culture medium reached 35 g/L (Chaudhary et al., 2012). Chlorella vulgaris, however, had a much lower tolerance of glucose and its growth was inhibited when
the glucose concentration reached 15 g/L. In this study, slight inhibition was observed
during the initial cultivation when the acorn-glucose concentration reached 5 g/L.
The TAGs content and biomass production with acorn-glucose increased with increasing
acorn-glucose concentration. The results showed that using acorn-glucose as a carbon
source for the mixotrophic cultivation of C. vulgaris is a feasible way to solve the problem of low algal cell density when the acorn-glucose
is the sole carbon source, which stimulates additional utilization of the acorn-glucose.
Furthermore, this study demonstrated the feasibility of acorn-glucose as an alternative
carbon substrate to glucose for microalgae cultivation, and cost reduction of the
carbon substrate feed for microalgal lipid production is expected. The TAGs content
and efficiency of microalgae growth are important for biodiesel production (Ramos et al., 2009). Improved lipid accumulation with slower microalgae growth may result in lower oil
yields compared to faster growing microalgae with less lipid accumulation.
Various carbon sources, such as sodium acetate (Qiao et al., 2009), fructose (Gao et al., 2009), glucose (Yeh and Chang, 2012), glycerol (Heredia-Arroyo et al., 2010), sucrose (Gao et al., 2009), and acetate (Heredia-Arroyo et al., 2010), have been successfully applied to increase
the rate of growth and lipid content of microalgae. However, these methods are cost-intensive
(Borowitzka and Moheimani, 2013; Lin and Wu, 2015; Vidotti et al., 2014). The carbon source used in this study is simple and cost effective. The prices of
glucose (obtained from starch produced from plants that are cultivated under phototrophic
conditions, e.g. corn), glycerol and acetate are in the range of 0.5-0.8, 0.6-0.7
and 0.9-0.94 US dollars per kg, respectively. While the use of carbon dioxide from
flue gases has an additional bonus due to the reduction of emissions to the atmosphere
(Gouveia and Oliveira, 2009), additional cleanup steps are likely to be required for
the flue gas. In contrast, acorns are inexpensive, costing approximately $0.15-0.3
USD per kilogram (Leon-Camacho et al., 2004; Shim et al., 2005). Acorn-glucose does not contaminate the growth medium, which can be recycled to reduce
not only the cost and the demand for water but also the extra operational costs for
reusing growth medium. This cost effective carbon source will help reduce the production
cost of using algae for biodiesel.
4. Conclusions
The growth of the algae strain C. vulgaris under mixotrophic conditions in the presence of saccharified acorn-starch (acorn-glucose)
was investigated with the objective of increasing the biomass growth and TAGs content.
Acorn-starch concentration decreased by 88.26% and glucose increased 9.8 fold within
5 hours after saccharification. The saccharification reached 81.3% in 5 hours. Biomass
production from 0.357 g/L of C. vulgaris was determined to be 0.76, 3.49, 8.06, 12.44 and 10.62 g/L with 0, 1, 2, 3 and 5
g/L acorn-glucose, respectively. Biomass production with 3 g/L acorn-glucose was 16.4
fold higher than that of the autotrophic growth condition. In addition, the amount
of TAGs in the algal strains was 3.7, 8.7, 18.2, 32.9 and 18.4% for 0, 1, 2, 3 and
5 g/L acorn-glucose, respectively. The 3 g/L acorn-glucose concentration under the
mixotrophic conditions was most effective for maximum increases in biomass production/productivity
and TAGs content. The acorn-glucose enhanced the investigated microalgae growth, biomass
productivity and TAGs content.
Acknowledgements
This study was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (2013006899).
References
(2007), Mixotrophic Cultivation of Microalga Spirulina platensis using Molasses as
Organic Substrate, Aquaculture, Andrade, M. R. and Costa, J. A. V. (2007). Mixotrophic
Cultivation of Microalga Spirulina platensis using Molasses as Organic Substrate,
Aquaculture, 264, pp. 130-134., Vol. 264, pp. 130-134
(2014), Biomass and Oil Content of Chlorella sp., Haematococcus sp., Nannochloris
sp. and Scenedesmus sp. under Mixotrophic Growth Conditions in the Presence of Technical
Glycerol, Journal of Applied Phycology, Andruleviciute, V., Makareviciene, V., Skorupskaite,
V., and Gumbyte, M. (2014). Biomass and Oil Content of Chlorella sp., Haematococcus
sp., Nannochloris sp. and Scenedesmus sp. under Mixotrophic Growth Conditions in the
Presence of Technical Glycerol, Journal of Applied Phycology, 26, pp. 83-90., Vol.
26, pp. 83-90
(1980), Official Methods of Analysis, Association of Official Analytical Chemist (AOAC).
(1980). Official Methods of Analysis, 14th ed, Association of official analytical
chemist, Washington DC, pp. 129-133., pp. 129-133
(1983), A Simple and Rapid Preparation of Alditol Acetate for Monosaccharide Analysis,
Carbohydrate Research, Blakeney, A. B., Harris, P. J., Henry, R. J., and Stone, B.
A. (1983). A Simple and Rapid Preparation of Alditol Acetate for Monosaccharide Analysis,
Carbohydrate Research, 113(2), pp. 291-299., Vol. 113, No. 2, pp. 291-299
(1959), A Rapid Method of Total Lipid Extraction and Purification, Canadian Journal
of Biochemistry and Physiology, Bligh, E. G. and Dyer, W. J. (1959). A Rapid Method
of Total Lipid Extraction and Purification, Canadian Journal of Biochemistry and Physiology,
37, pp. 911-917., Vol. 37, pp. 911-917
(2004), Heterotrophic and Mixotrophic Growth of Micractinium pusillum fresenius in
the Presence of Acetate and Glucose: Effect of Light and Acetate Gradient Concentration,
Water Research, Bouarab, L., Dauta, A., and Loudiki, M. (2004). Heterotrophic and
Mixotrophic Growth of Micractinium pusillum fresenius in the Presence of Acetate and
Glucose: Effect of Light and Acetate Gradient Concentration, Water Research, 38, pp.
2706-2712., Vol. 38, pp. 2706-2712
(2013), Sustainable Biofuels from Algae, Mitigation and Adaptation Strategies for
Global Change, Borowitzka, M. A. and Moheimani, N. R. (2013). Sustainable Biofuels
from Algae, Mitigation and Adaptation Strategies for Global Change, 18, pp. 13-25.,
Vol. 18, pp. 13-25
(2008), Determineation of the Fatty Acid Composition of Acorn (Quercus), Pistacia
Lentiscus Seeds Growing in Algeria, Journal of the American Oil Chemists' Society,
Charef, M., Yousfi, M., Saidi, M., and Stocker, P. (2008). Determineation of the Fatty
Acid Composition of Acorn (Quercus), Pistacia Lentiscus Seeds Growing in Algeria,
Journal of the American Oil Chemists' Society, 85, pp. 921-924., Vol. 85, pp. 921-924
(2012), Comparison of Glucose, Glycerol and Crude Glycerol Fermentation by Echerichia
Coli K12, Journal of Bioprocessing and Biotechniques, Chaudhary, N., Ngadi, M. O.,
and Simpson, B. (2012). Comparison of Glucose, Glycerol and Crude Glycerol Fermentation
by Echerichia Coli K12, Journal of Bioprocessing and Biotechniques, S1:001 doi:10.4172/2155-9821
(2014), Efficiency of Nutrient Removal and Biomass Productivity in the Wastewater
by Microalgae Membrane Bioreactor Process, Journal of Korean Society on Water Environment,
Choi, H. J. (2014). Efficiency of Nutrient Removal and Biomass Productivity in the
Wastewater by Microalgae Membrane Bioreactor Process, Journal of Korean Society on
Water Environment, 30(4), pp. 386-393. [Korean Literature], Vol. 30, No. 4, pp. 386-393
(2015a), Effect of Acorn Powder on the Biomass Productivity of Microalgae, Journal
of Korean Society on Water Environment, Choi, H. J. (2015a). Effect of Acorn Powder
on the Biomass Productivity of Microalgae, Journal of Korean Society on Water Environment,
31(2), pp. 134-141. [Korean Literature], Vol. 31, No. 2, pp. 134-141
(2015b), A Comparative Study on Microalgae Recovery Rates in Response to Different
Low Cost Bio-flocculant Applications, Journal of Korean Society on Water Environment,
Choi, H. J. (2015b). A Comparative Study on Microalgae Recovery Rates in Response
to Different Low Cost Bio-flocculant Applications, Journal of Korean Society on Water
Environment, 31(6), pp. 625-631. [Korean Literature], Vol. 31, No. 6, pp. 625-631
(2015), Influence of Crude Glycerol on the Biomass and Lipid Content of Microalgae,
Biotechnology and Biotechnological Equipment, Choi, H. J. and Yu, S. W. (2015). Influence
of Crude Glycerol on the Biomass and Lipid Content of Microalgae, Biotechnology and
Biotechnological Equipment, 29(3), pp. 506-513., Vol. 29, No. 3, pp. 506-513
(2004), Evaluation of Spirulina sp. Growth in Photoautotrophic, Heterotrophic and
Mixotrophic Cultures, Enzyme and Microbial Technology, Chojnacka, K. and Noworyta,
A. (2004). Evaluation of Spirulina sp. Growth in Photoautotrophic, Heterotrophic and
Mixotrophic Cultures, Enzyme and Microbial Technology, 34, pp. 461-465., Vol. 34,
pp. 461-465
(2009), Application of Sweet Sorghum for Biodiesel Production by Heterotrophic Microalga
Chlorella protothecoides, Applied Energy, Gao, C., Zhai, Y., Ding, Y., and Wu, Q.
(2009). Application of Sweet Sorghum for Biodiesel Production by Heterotrophic Microalga
Chlorella protothecoides, Applied Energy, 87, pp. 756-761., Vol. 87, pp. 756-761
(2009), Microalgae as a Raw Material for Biofuels Production, Journal of Industrial
Microbiology and Biotechnology, Gouvela, L. and Oliveira, C. (2009). Microalgae as
a Raw Material for Biofuels Production, Journal of Industrial Microbiology and Biotechnology,
36, pp. 269-274., Vol. 36, pp. 269-274
(2011), Mixotrophic Cultivation of Chlorella vulgaris and its Potential Application
for the Oil Accumulation from non-sugar Materials, Biomass Bioenergy, Heredia-Arroyo,
T., Wei, W., Ruan, R., and Hu, B. (2011). Mixotrophic Cultivation of Chlorella vulgaris
and its Potential Application for the Oil Accumulation from non-sugar Materials, Biomass
Bioenergy, 5, pp. 2-10., Vol. 5, pp. 2-10
(2013), Effects of Glycerol and Glucose on the Enhancement of Biomass, Lipid and Soluble
Carbohydrate Production by Chlorella vulgaris in Mixotrophic Cultures, Food Technology
and Biotechnology, Kong, W. B., Yang, H., Cao, Y. T., Song, H., Hua, S. F., and Xia,
C. G. (2013). Effects of Glycerol and Glucose on the Enhancement of Biomass, Lipid
and Soluble Carbohydrate Production by Chlorella vulgaris in Mixotrophic Cultures,
Food Technology and Biotechnology, 51, pp. 62-69., Vol. 51, pp. 62-69
(2004), Acorn (Quercus Spp) Fruit Lipids: Saponifiable and Unsaponifiable Fractions:
A detailed Study, Journal of the American Oil Chemists' Society, Leon-Camacho, M.,
Viera-Alcaide, I., and Vicario, I. M. (2004). Acorn (Quercus Spp) Fruit Lipids: Saponifiable
and Unsaponifiable Fractions: A detailed Study, Journal of the American Oil Chemists'
Society, 81, pp. 447-453., Vol. 81, pp. 447-453
(2009), Biomass and Lipid Productivities of Chlorella vulgaris under Autotrophic,
Heterotrophic and Mixotrophic Growth Conditions, Biotechnology Letters, Liang, Y.,
Sarkany, N., and Cui, Y. (2009). Biomass and Lipid Productivities of Chlorella vulgaris
under Autotrophic, Heterotrophic and Mixotrophic Growth Conditions, Biotechnology
Letters, 3, pp. 1043-1049., Vol. 3, pp. 1043-1049
(2010), Batch Stage Study of Lipid Production from Crude Glycerol derived from Yellow
Grease or Animal Fats through Microalgal Fermentation, Bioresource Technology, Liang,
Y., Sarkany, N., Cui, Y., and Blackburn, J. M. (2010). Batch Stage Study of Lipid
Production from Crude Glycerol derived from Yellow Grease or Animal Fats through Microalgal
Fermentation, Bioresource Technology, 101, pp. 6745-6750., Vol. 101, pp. 6745-6750
(2015), Effect of Carbon Source on Growth and Lipid Accumulation of newly isolated
Microalga cultured under Mixtrophic Condition, Bioresource Technology, Lin, T. S.
and Wu, J. Y. (2015). Effect of Carbon Source on Growth and Lipid Accumulation of
newly isolated Microalga cultured under Mixtrophic Condition, Bioresource Technology,
184, pp. 100-107., Vol. 184, pp. 100-107
(2012), Heterotrophic/Mixotrophic Cultivation of Oleaginous Chlorella vulgaris on
Industrial Co-products, Algal Research, Mitra, D., van Leenwen, J., and Lamsal, B.
(2012). Heterotrophic/Mixotrophic Cultivation of Oleaginous Chlorella vulgaris on
Industrial Co-products, Algal Research, 1, pp. 40-48., Vol. 1, pp. 40-48
(2014), Effect of Ultrasonic-assisted Pretreatment on Hydrolysis and Fermentation
of Acorn Starch, Bioresources, Pan, P., Tang, Y., Sun, D., Jiang, J., and Song, X.
(2014). Effect of Ultrasonic-assisted Pretreatment on Hydrolysis and Fermentation
of Acorn Starch, Bioresources, 9(2), pp. 2705-2716., Vol. 9, No. 2, pp. 2705-2716
(2010), Efficiency of Growth and Nutrient Uptake from Wastewater by Heterotrophic,
Autotrophic, and Mixotrophic Cultivation of Chlorella vulgaris immobilized with Azospirillum
brasilense, Journal of Phycology, Perez-Garcia, O., de-Bashan, L. E., Hernandez, J.
P., and Bashan, Y. (2010). Efficiency of Growth and Nutrient Uptake from Wastewater
by Heterotrophic, Autotrophic, and Mixotrophic Cultivation of Chlorella vulgaris immobilized
with Azospirillum brasilense, Journal of Phycology, 46, pp. 800-812., Vol. 46, pp.
800-812
(2011), The Potential of Sustainable Algal Biofuel Production using Wastewater Resources,
Bioresource Technology, Pittman, J. K., Dean, A. P., and Osundeko, O. (2011). The
Potential of Sustainable Algal Biofuel Production using Wastewater Resources, Bioresource
Technology, 102(1), pp. 17-25., Vol. 102, No. 1, pp. 17-25
(2008), Docosahexaenoic Acid (DHA)-rich Algae from Biodiesel-derived Crude Glycerol:
Effects of Impurities on DHA Production and Algal Biomass Composition, Journal of
Agricultural and Food Chemistry, Pyle, D. J., Garcia, R. A., and Wen, Z. (2008). Docosahexaenoic
Acid (DHA)-rich Algae from Biodiesel-derived Crude Glycerol: Effects of Impurities
on DHA Production and Algal Biomass Composition, Journal of Agricultural and Food
Chemistry, 56, pp. 3933-3939., Vol. 56, pp. 3933-3939
(2009), Isolation and Characterization of Chlorella sorokiniana GXNN01(Chlorophyta)
with the Properties of Heterotrophic and Microaerobic Growth, Journal of Phycology,
Qiao, H., Wang, G., and Zhang, X. (2009). Isolation and Characterization of Chlorella
sorokiniana GXNN01(Chlorophyta) with the Properties of Heterotrophic and Microaerobic
Growth, Journal of Phycology, 45, pp. 1153-1162., Vol. 45, pp. 1153-1162
(2009), Influence of Fatty Acid Composition of Raw Materials on Biodiesel Properties,
Bioresource Technology, Ramos, M. J., Fernández, C. M., Casas, A., Rodríguez, L.,
and Pérez, A. (2009). Influence of Fatty Acid Composition of Raw Materials on Biodiesel
Properties, Bioresource Technology, 100, pp. 261-268., Vol. 100, pp. 261-268
(2009), Microalgae for Oil: Strain Selection, Induction of Lipid Synthesis and Outdoor
Mass Cultivation in a Low-cost Photobioreactor, Biotechnology and Bioengineering,
Rudolfi, L., Zittelli, G. C., Bassin, N., Padovani, G., Biondi, N., Bonini, G., and
Tredicil, M. R. (2009). Microalgae for Oil: Strain Selection, Induction of Lipid Synthesis
and Outdoor Mass Cultivation in a Low-cost Photobioreactor, Biotechnology and Bioengineering,
102, pp. 100-112., Vol. 102, pp. 100-112
(2005), Studies on the Components and Evaluation of Antioxidation Activity in Acorn
Powder, Report, Institute of Health and Environment, Shim, T. H., Jin, Y. S., Sa,
J. H., Shin, I. C., Heo, S. I., Lee, T. W., Kim, T. W., Park, K. Y., Jeong, K. J.,
Han, K. S., Oh, H. S., and Wang, M. H. (2005). Studies on the Components and Evaluation
of Antioxidation Activity in Acorn Powder, Report, Institute of Health and Environment,
16, pp. 46-52., Vol. 16, pp. 46-52
(2010), Potential Fuel Oils from the Microalga Choricystis minor, Journal of Chemical
Technology and Biotechnology, Sobczuk, T. M., and Chisti, Y. (2010). Potential Fuel
Oils from the Microalga Choricystis minor, Journal of Chemical Technology and Biotechnology,
85, pp. 100-108., Vol. 85, pp. 100-108
(2006), Commercial Applications of Microalgae, Journal of Bioscience and Bioengineering,
Spolaore, P., Joannis-Cassan, C., Duran, E., and Isambert, A. (2006). Commercial Applications
of Microalgae, Journal of Bioscience and Bioengineering, 10, pp. 87-96., Vol. 10,
pp. 87-96
(2005), The Application of Micro-FTIR Spectroscopy to analyze Nutrient Stress-related
Changes in Biomass Composition of Phytoplankton algae, Plant Physiology and Biochemistry,
Stehfest, K., Toepel, J., and Wilhelm, C. (2005). The Application of Micro-FTIR Spectroscopy
to analyze Nutrient Stress-related Changes in Biomass Composition of Phytoplankton
algae, Plant Physiology and Biochemistry, 43, pp. 717-726., Vol. 43, pp. 717-726
(2011), Simultaneous Saccharification and Cofermentation of Lignocellulosic Residues
from Commercial Furfural Production and Corn Kernels using Different Nutrient Media,
Biotechnology for Biofuels, Tang, Y., Zhao, D. Q., Cristhian, C., and Jiang, J. X.
(2011). Simultaneous Saccharification and Cofermentation of Lignocellulosic Residues
from Commercial Furfural Production and Corn Kernels using Different Nutrient Media,
Biotechnology for Biofuels, 4(22), doi: 10.1186/1754-6834-4-22., Vol. 4, No. 22
(2014), Miniaturized Culture for Hetrotrophic Microalgae using Low Cost Carbon Sources
as a Tool to isolate fast and economical Strains, Chemical engineering transactions,
Vidotti, A. D. S., Coelho, R. S., Franco, L. M., and Franco, T. T. (2014). Miniaturized
Culture for Hetrotrophic Microalgae using Low Cost Carbon Sources as a Tool to isolate
fast and economical Strains, Chemical engineering transactions, 38, pp. 325-330.,
Vol. 38, pp. 325-330
(2001), Biomass Production in Mixotrophic Culture of Euglena gracilis under Acidic
Conditions and its Growth Energetic, Biotechnology Letters, Yamane, Y., Utsunomiya,
T., Watanabe, M., and Sasaki, K. (2001). Biomass Production in Mixotrophic Culture
of Euglena gracilis under Acidic Conditions and its Growth Energetic, Biotechnology
Letters, 23, pp. 1223-1228., Vol. 23, pp. 1223-1228
(2000), Energetics and Carbon Metabolism during Growth of Microalgal Cells under Photoautotrophic,
Mixotrophic and Cyclic Light-autotrophic/Dark-heterotrophic Conditions, Biochemical
Engineering Journal, Yang, C., Hua, Q., and Shimizu, K. (2000). Energetics and Carbon
Metabolism during Growth of Microalgal Cells under Photoautotrophic, Mixotrophic and
Cyclic Light-autotrophic/Dark-heterotrophic Conditions, Biochemical Engineering Journal,
6, pp. 87-102., Vol. 6, pp. 87-102
(2012), Effects of Cultivation Conditions and Media Composition on Cell Growth and
Lipid Productivity of Indigenous Microalga Chlorella vulgaris ESP-31, Bioresource
Technology, Yeh, K. L. and Chang, J. S. (2012). Effects of Cultivation Conditions
and Media Composition on Cell Growth and Lipid Productivity of Indigenous Microalga
Chlorella vulgaris ESP-31, Bioresource Technology, 105, pp. 120-127., Vol. 105, pp.
120-127
(2011), Mixotrophic Cultivation of Botryococcus braunii, Biomass Bioenergy, Zhang,
H., Weiliang, L. Y., Yang, W., and Shen, G. (2011). Mixotrophic Cultivation of Botryococcus
braunii, Biomass Bioenergy, 35, pp. 1710-1715., Vol. 35, pp. 1710-1715