Energies 10 00224
Energies 10 00224
Energies 10 00224
4 Laboratorio de Biotecnología Acuática, Instituto del Mar del Perú (IMARPE), Esquina Gamarra y General
Valle S/N Chucuito, Callao 07021, Peru; [email protected] (L.F.); [email protected] (C.P.A.)
5 Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía
(CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Psje. Los Paujiles S/N, Iquitos 16024, Peru;
[email protected]
* Correspondence: [email protected] (M.C.); [email protected] (J.C.C.);
Tel.: +51-065-261074 (M.C.); +51-065-263569 (J.C.C.)
Abstract: Biodiesel production from microalgae triacylglycerols is growing, because this feedstock
is a more sustainable and advantageous alternative. In this study, we isolated and identified
fourteen strains of native microalgae from the Peruvian Amazon. These strains showed great
heterogeneity in biomass productivity, lipid productivity and lipid content, and thus, three of them
(Acutodesmus obliquus, Ankistrodesmus sp. and Chlorella lewinii) were selected for further evaluation
under culture of nitrogen-sufficient (+N) and nitrogen-deficient (−N) Chu medium No. 10. These
microalgae species showed modifications in biomolecule content (protein, lipid and carbohydrate)
with a pronounced increase of lipids and carbohydrate and a decrease of protein content under
stress culture. Furthermore, the fatty acid profile was peculiar for each species, and these patterns
showed evident changes, particularly in the proportion of saturated and monounsaturated fatty
acids. The results of this research suggest that the isolated native microalgae, from the Peruvian
Amazon, could be suitable candidates for biodiesel production.
Keywords: biodiesel; fatty acid profiling; lipid content; oleaginous microalgae; Peruvian Amazon
1. Introduction
The global increase of energy demand, the depletion and increasing of the costs of fossil fuels
[1], and climate change [2] are problems that require urgent solutions. In order to mitigate these
problems, Peru has legal dispositions (Law 28054 of the promotion of the biofuel market, and their
regulation with Supreme Decree No. 013-2005-EM), so that the energy matrix of the country depends
on renewable resources [3,4]. These legal dispositions have increased the importation of biodiesel,
due to insufficient national production, which is currently obtained from plant oils, such as Jatropha
curcas “white pinion” and Elaeis guineensis “palm”, both cultured in large areas of the Peruvian
Amazon [5].
Biodiesel obtained from crop-based plant oils, however, has major drawbacks. These include
low yields, high land and water requirements, detrimental effects on food supplies and associated
Energies 2017, 10, 224; doi:10.3390/en10020224 www.mdpi.com/journal/energies
Energies 2017, 10, 224 2 of 16
extensive deforestation of the rainforest, which pose threats to native biodiversity and ecosystem
functions, goods and services [6–8]. These limitations can be overcome by the next generation of
microalgae-based biofuels. Compared to terrestrial crops, the main advantages of microalgal systems
are that they have a higher photon conversion efficiency, can be harvested batch-wise nearly
all-year-round, can utilize salt and waste water streams, can couple CO2-neutral fuel production with
CO2 sequestration and produce non-toxic and highly biodegradable biofuels [7].
The isolation, selection and culturing of robust oleaginous microalgae with desirable attributes
(e.g., high triacylglycerol content, appropriate fatty acid profiles, high growth rate and resistance to
invasion of local microorganism), however, are the principal challenges associated with microalgae
biofuel production [9,10]. Although such microalgae strains are currently unavailable, optimism
persists due to the fact that there is an abundance of species in the wild yet to be isolated [11]. Indeed,
only a few thousand (~50,000) are conserved worldwide in microalgal culture collections [9,12], and
of those, only a few hundred (<10%) have been investigated for their biochemical composition [13].
Consequently, the isolation, selection, biochemical and molecular characterization of microalgae
from aquatic environments must be a continuous effort of screening for strains with the potential for
biodiesel production [14]. Therefore, a sustainable development of microalgal-based biodiesel and
bioactive chemicals production in Peru requires the bioprospecting and exploitation of native
microalgae bioresources as the most viable option. The northeastern region of Peru, particularly the
Loreto region, contains a great variety of freshwater aquatic environments (i.e., rivers, lagoons, etc.)
that harbor a rich biodiversity of microalgae. In this study, 14 strains of native microalgae from the
Peruvian Amazon were isolated, identified and tested for their biomass productivity, lipid
productivity and total lipid content. In addition, the growth profile, total carbohydrate, total protein,
total lipid content and fatty acids profiles of three selected strains were also investigated and
compared under nitrogen-sufficient and -deficient culture conditions.
2. Experimental Section
15 min at 37 °C. After washing once, stained microalgal cells were observed by Carl Zeiss fluorescent
microscopy and photographed with a digital camera AxioLab.A1 AxioCamERc real time 5 s. Images
were obtained at a magnification of 1000× with visible light and epifluorescence (excitation 510–560,
emission 590).
within the MEGA 6.06 software [28]. The bootstrap analysis [29] was used with 1000 replications to
test the relative support for the branches produced by NJ analysis.
2.8. Determination of Total Protein, Total Carbohydrate, Ash Content, Esterification and Fatty
Acids Profiling
The microalgae cultures maintained for seven days under +N and −N Chu medium No. 10 were
harvested by centrifugation, rinsed and dried as previously described (Section 2.5). From the
Energies 2017, 10, 224 5 of 16
microalgae powders, total protein content was determined according to the Hartree–Lowry
method [32]. To 5 mg of dry microalgal biomass was added 5 mL of 0.5 M NaOH and incubated for
10 min at 80 °C, then centrifuged for 5 min at 14,000× g. Protein content in 1 mL of the supernatant
was determined by adding 0.5 mL of 0.5 M sodium carbonate, 0.5 mL of 0.5 M sodium potassium
tartrate, 0.5 M of copper sulfate and 2 mL of Folin-Ciocalteu reagent. The blue complex was analyzed
in a Varian Cary 50 Bio UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA)
set at 650 nm against a calibration curve based on a known concentration of BSA as the standard.
Total carbohydrate content of the microalgal biomass was determined with the phenol-sulfuric
acid method [32,33]. For this purpose, 5 mg of dry microalgae biomass were added to 5 mL of water.
After this, a 1-mL aliquot of the sample was added to 3 mL of sulfuric acid and 1 mL of 5% aqueous
solution of phenol, and the mixture was stirred and incubated for 5 min at 90 °C. The yellow-brown
complex was analyzed by spectrophotometric analysis at 488 nm against a calibration curve based
on a known concentration of glucose.
Ash content was done according to the AOAC procedure [34]. One hundred milligrams of dry
material were ashed in a muffle furnace Thermolyne™ F6010 (Thermo Fisher Scientific, Walthman,
MA, USA) at 550 °C for 16 h, and the content was determined gravimetrically. All analyses were
carried out in triplicate, and the data are expressed as the mean ± SD.
Fatty acids methyl esters (FAME) were prepared by acid transesterification according to Ichihara
and Fukubayashi [35]. The crude lipid extract (1 mg) was dissolved in 0.2 mL of toluene, 1.5 mL of
methanol and 0.3 mL of 0.8% HCl (prepared in methanol:water 85:15 v/v), transferred into a capped
test tube, mixed for 5 min and then incubated at 45 °C for 12 h for derivatization reaction. Then, 2 mL
of n-hexane and 2 mL of distilled water were added to the tube. After vortexing, the hexane phases
containing FAMEs were transferred into a 1.5-mL tube and dried under a stream of nitrogen. Finally,
the FAMEs were redissolved in 10 µL acetonitrile and analyzed by using a gas chromatograph Varian
CP-3800 GC (Agilent Technologies, Santa Clara, CA, USA) equipped with an automated sampler and
injector, a flame ionization detector and using a 30 m × 0.32 mm × 0.25 µm Stabilwax® capillary
column (Restek, Bellefonte, PA, USA). The GC conditions were as follows: injector temperature: 250
°C; the column temperature gradient was: 120 °C for 1 min, followed by an increase to 160 °C at the
rate of 30 °C/min, 160 °C for 1 min, followed by an increase to 240 °C at the rate of 4 °C/min and 240
°C for 7 min; detector temperature: 260 °C. Gas pressures for He, H2 and synthetic air were
maintained at 40, 80 and 60 psi, respectively. The carrier gas (He) flow was maintained at 1 mL/min.
The fatty acids were analyzed by comparing their retention time of the corresponding peaks with a
known standard mixture of FAMEs (Nu-Check Prep, Elysian, MN, USA), and methyl tricosanoate
(Sigma-Aldrich, Saint Louis, MO, USA) was added to each sample as the internal standard. All
chromatograms of the microalgal samples were analyzed by using the Galaxie™ Chromatography
Data System Version 1.9.3.2 (Agilent Technologies, Santa Clara, CA, USA). All peaks spanning a peak
area of more than 50 units were integrated. All analyses were carried out in triplicate, and the data
are expressed as the mean ± SD.
products [36–38]. Furthermore, native microalgae strains can have potential applications for efficient
biological sequestration of CO2, wastewater treatment and other environmentally-friendly
applications [38–40]. Consequently, isolation is a fundamental process to obtain pure cultures and is
the first phase towards the screening and selection of microalgae strains with the potential for
biodiesel production and the aforementioned applications.
According to these demands, in this research, samples were collected from three different types
of water bodies, the Amazon River (white-water type), the Itaya and Nanay Rivers, both of the
black-water type. The white-water rivers originate from the Andean catchments bringing high
amounts of nutrient-rich sediments. In contrast, the black-water rivers drain geochemically-poor
watersheds and are characterized by their high content in humic acids, low pH and poor nutrient
status [41]. A total of 14 microalgal strains were isolated using the standard cell washing technique,
and based on distinguishable morphological characters under light microscopic examination, these
strains were preliminary ascribed to the genera: Acutodesmus (Hegewald) Tsarenko, 2001;
Ankistrodesmus Corda, 1838; Chlorella Beyerinck Beijerinck, 1890; Desmodesmus (Chodat) S.S. An, T.
Friedl, and E. Hegewald, 1999; and Tetradesmus G.M. Smith, 1913 (Table 1). These genera are green
algae that belong to division Chlorophyta, class Chlorophyceae and orders Chlorellales and
Sphaeropleales. Although morphological analysis is frequently used to identify microalgae, it is
inaccurate and very difficult for the identification at the species level, because the relationship
between diagnostic morphology and biological species boundaries are largely unknown in many
micro-eukaryotes [42]. Furthermore, according to Yu et al. [31], the microalgae morphology for the
same strain varies in relation to age and culture conditions. Consequently, in microalgae, the classic
taxonomy is supported by molecular tools [42]. For example, the ITS2 rDNA region is thought to be
an excellent marker for molecular phylogenetics studies in lower taxonomic levels, due to being
highly divergent and fast-evolving, which can discriminate among closely-related organisms, which
otherwise display almost identical sequences [43,44]. For this reason, the molecular identification
based in phylogenetic analysis of ITS2 rDNA gene sequences is commonly used to identify and/or
delineate species [20,45,46].
Table 1. Genera, GenBank accession number of ITS2 sequences, biomass productivity, lipid
productivity and lipid content of isolated microalgae strains.
AMA001 Ankistrodesmus sp. KP878496.1 12.2 ± 0.3 3.7 ± 0.1 30.7 ± 1.5
Amazon River
AMA002 Ankistrodesmus sp. KP878497.1 3.6 ± 0.4 1.0 ± 0.2 27.7 ± 2.1
(03°41′0.6′′ S,
AMA003 Chlorella lewinii KP878501.1 22.3 ± 0.7 5.1 ± 0.5 22.7 ± 1.5
73°14′8.9′′ W)
AMA004 Acutodesmus abliquus KP878505.1 31.6 ± 0.4 6.4 ± 0.7 20.3 ± 2.1
ITA001 Ankistrodesmus sp. KP878499.1 5.9 ± 0.2 4.0 ± 0.3 43.7 ± 1.7
ITA002 Ankistrodesmus sp. KP878500.1 13.6 ± 0.4 5.7 ± 0.5 30.6 ± 2.0
Itaya River ITA003 Chlorella lewinii KP878503.1 12.9 ± 0.3 4.0 ± 0.5 22.4 ± 2.7
(03°43′1.4′′ S, ITA004 Chlorella lewinii KP878504.1 22.1 ± 0.7 3.1 ± 0.4 21.5 ± 2.3
73°14′17.8′′ W) ITA005 Scenedesmus regularis KP878508.1 9.3 ± 0.4 5.6 ± 0.4 24.2 ± 1.4
ITA006 Scenedesmus dimorphus KP878509.1 18.6 ± 0.6 3.2 ± 0.3 26.0 ± 1.7
ITA007 Acutodesmus abliquus KP878510.1 17.9 ± 0.4 2.2 ± 0.3 15.6 ± 1.9
Nanay River NAN001 Ankistrodesmus sp. KP878498.1 14.3 ± 0.6 2.8 ± 0.2 46.7 ± 1.5
(03°42′0.2′′ S, NAN002 Desmodesmus tropicus KP878506.1 12.2 ± 0.4 2.3 ± 0.2 17.5 ± 1.1
73°15′32′′ W) NAN003 Tetradesmus sp. KP878507.1 13.9 ± 0.4 3.0 ± 0.3 13.5 ± 1.2
To realize phylogenetic analysis, genomic DNA of each isolate strain was purified and used for
PCR amplification of ITS2 rDNA. The universal primers used to amplify the ITS2 rDNA successfully
amplified the desired DNA fragments (~300 bp) from all microalgal strains (Figure S2). Further, these
amplicons were sequenced, published in the NCBI databases, and the corresponding GenBank
accession numbers are presented in Table 1. Figure 1 shows a neighbor-joining phylogenetic tree
depicting the relatedness of the native microalgae strain isolates with selected sequences from the
Energies 2017, 10, 224 7 of 16
public GenBank database. This phylogram contained three clusters, each of which included different
microalgae genera. For example, the first cluster consisted of microalgae of the genera Acutodesmus,
Scenedesmus, Tetradesmus and Desmodesmus and six isolated strains (ITA007, AMA004, ITA006,
NAN003, ITA005 and NAN002). The second cluster consisted of a microalgae of the genus
Ankistrodesmus and the isolated strains AMA001, ITA001, ITA002, NAN001 and AMA002. The third
one consisted of a microalgae of the genus Chlorella and three isolated strains (ITA004, AMA003 and
ITA003). Overall, the identification of native microalgae strains presented a good correspondence
between morphological characters and sequence-based phylogenetic analysis. Microalgae species of
these genera were also isolated from different countries of the world, such as Brazil [47],
Argentina [46], United States [48], India [20], Egypt [49], Taiwan [50], South Korea [45] and
China [51]. Some of these microalgae genera, such as Ankistrodesmus, Chlorella, Desmodesmus and
Scenedesmus, were used in large-scale biodiesel production using photobioreactors and raceway
pond systems [52,53].
Figure 1. Unrooted phylogenetic tree of isolated microalgae strains using the ITS2 sequences. The tree
was constructed by the neighbor-joining method and Kimura’s two-parameter algorithm in
MEGA 6.0 software based on the multiple sequence alignment by Clustal Omega. Bootstrap values of
1000 replicates (%) are shown at the branches.
from 30 to 120 mg·L−1·d−1) in the same and other microalgae genera [45,55,56]. These discrepancies
may be attributed to differences in the culture conditions, in the culture media composition and in
the microalgae genotypes. Lipid productivity also exhibited statistically-significant differences (F =
47.74, df = 13, p < 0.001) and ranged from 1.0 ± 0.4 mg·L−1·d−1 to 6.4 ± 0.7 mg·L−1·d−1 with strains AMA002
and AMA004, respectively. These lipid productivities were low in comparison to other reports, which
reported values from 19.0 to 43.3 mg·L−1·d−1 [45,55]. Furthermore, lipid content as a percentage of the
microalgal biomass dry weight showed statistically-significant differences (F = 85.09, df = 13, p < 0.001)
and ranged from 13.5 ± 1.2 (Tetradesmus sp. strain NAN003) to 46.7 ± 1.5 (Ankistrodesmus sp. strain
NAN001). These percentage values in total lipid content are similar to several microalgae species of
these genera isolated and characterized by other researchers worldwide [20,45,46,48–50,57].
Furthermore, it is important to emphasize that lipid content is dependent on the different growth
phases. Commonly, in several microalgae taxa, the lipid content increases gradually from logarithmic
to stationary growth phases [58–60]. For example, Kaur et al. [20] reported that the total lipid content
of Scenedesmus sp. DRLMA9 demonstrated values of 20.7% in the late exponential phase and 39.7%
in the stationary phase; and Desmodesmus elegans DRLMA13 displayed values of 10.3% in the late
exponential phase and 16.9% in the stationary phase. Likewise, Chiu et al. [61] found that the total
lipid content of Nannochloropsis oculata was 30.8% in the logarithmic phase, 39.7% in the early
stationary phase and 50.4% in the stationary phase. These changes in lipid content with culture age
have been associated with nitrate depletion [61], which causes a channeling of photosynthetic ATP
to carbohydrate and lipid biosynthesis [60]. Finally, Pearson correlation analysis of biomass
productivity and lipid content was inversely related (r = −0.37, p < 0.01), which is similar to the results
obtained by Liang et al. [62] and Rodolfi et al. [52]. In contrast, Li et al. [51] and Hempel et al. [63]
found no relationship between these parameters. The negative correlation obtained is likely due to
the high metabolic cost of lipid biosynthesis [62].
Figure 2. Growth profiles of three microalgal strains cultivated under +N (filled boxes) and −N (empty
boxes) Chu medium No. 10. The culture was incubated in a controlled culture room at 25 ± 1 °C with
12:12-h light-dark cycles using 80 µE·m−2·s−1 intensity of cool-white fluorescent light and continuous
agitation at 150 rpm. Experiments were carried out in triplicate.
Figure 3. Biomolecule content (ash, carbohydrate, total lipid and protein) of three microalgal strains
cultivated under +N and −N Chu medium No. 10. The culture was incubated in a controlled culture
room at 25 ± 1 °C with 12:12-h light-dark cycles using 80 µE·m−2·s−1 intensity of cool-white fluorescent
light and continuous agitation at 150 rpm. Experiments were carried out in triplicate.
in cetane number [84–86]. Considering the SFA and unsaturated fatty acids (UFA) components,
approximately 20%–31% are SFA and 69%–80% are UFA. Hence, the SFA/UFA ratio in the microalgae
strains evaluated ranges between 0.25 and 0.45, which is similar to the reports of several microalgae
species isolated in different latitudes worldwide [20,31,48,49,70,72].
It is important to highlight that the fatty acid profile determination is an essential step in the
process of the characterization of microalgae strains, because it ultimately affects the quality of the
biodiesel product [84,85]. The quality properties of biodiesel, such as high cetane number, oxidative
stability and cold-flow, are dependent on the hydrocarbonated chain length of saturated and
unsaturated fatty acids [85,87]. For example, biodiesel composed of a mixture of a high percentage of
SFA and MUFA is preferred for increased energy yield, superior oxidative stability and higher cetane
numbers, but is prone to solidify at low temperature. In contrast, biodiesel with high levels of PUFA
has decreased energy yield, low cetane numbers and excellent cold-flow properties, but insufficient
oxidative stability [54,85,88]. Therefore, to solve these performance problems, five approaches have
been developed, such as changing the fatty acids profile by physical means, genetic modification of
the feedstock or the use of alternative feedstocks with different fatty acids profiles [89].
Table 2. Effect of nitrogen deficiency on fatty acid profiling in three native microalgal species from
the Peruvian Amazon.
4. Conclusions
Fourteen microalgae were isolated from three river basins from the Peruvian Amazon and were
identified by morphological features and molecular phylogeny approaches. These strains were
initially characterized in the stationary growth phase and exhibited significant heterogeneity in key
parameters, such as biomass productivity (3.6–31.6 mg·L−1·d−1), lipid productivity (1.0–6.4 mg·L−1·d−1)
and total lipid content (13.5%–46.7%). Furthermore, three selected strains, based on elevated biomass
and lipid productivities, were cultured under nitrogen-sufficient and nitrogen-deficient medium to
boost lipid accumulation. Under these culture conditions, all strains showed noticeable decreases in
total protein content and increases in total lipid and total carbohydrate contents. In addition, these
strains presented modifications in the fatty acids profiles with improvement in biodiesel properties,
due to a predominance of SFA and MUFA, which corresponds to an increase in cetane number. The
results of this research suggest that the isolated native microalgae, from the Peruvian Amazon, could
be suitable renewable feedstocks for the production of both biodiesel and bioethanol, which could
contribute to sustainable development at the local, regional and national levels as a viable alternative
to petroleum exploitation. However, it will be necessary to develop open, closed or hybrid systems
Energies 2017, 10, 224 12 of 16
for large-scale biomass production of selected native microalgae strains and/or de novo microalgae
strains designed to produce biofuels and with the abilities to synthesize useful bioactive compounds
to be efficiently exploited using biorefinery approaches.
Supplementary Materials: The following are available online at www.mdpi.com/1996-1073/10/2/224/s1, Figure
S1: Results of electrophoretic and spectrophotometric analysis of the genomic DNA isolated from the fourteen
microalgae, Figure S2: ITS2-rDNA structure (A) and amplicons obtained (B) from the microalgae isolated, Figure
S3: Microphotographic images of the three microalgae strains analyzed under light and fluorescence stained
with Nile Red, Figure S4: Spectrophotometric scanning for the determination of the maximum absorption peak
for each microalgae strain.
Acknowledgments: This research was supported by the Peruvian funding agencies Consejo Nacional de
Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC) with Grant Contract No. 364-CONCYTEC-OAJ and
Fondos para la Innovación, Ciencia y Tecnología (FINCyT) with Grant Contract No. 383-PNICP-PIBA-2014.
Author Contributions: Marianela Cobos and Juan C. Castro obtained funds for the research, participated in the
study design, coordinated activities, and participated in the preparation of the manuscript. Jae D. Paredes
participated in sample collection, microalgae isolation, and helped to draft the manuscript. J. Dylan Maddox
performed the bioinformatics analysis and participated in the preparation of the manuscript. Gabriel Vargas-Arana
and Leenin Flores participated in biochemical analysis and helped to draft the manuscript. Carla P. Aguilar
participated in the study design, supervised biochemical analysis, and helped to draft the manuscript.
Jorge L. Marapara participated in molecular analysis and helped to draft the manuscript.
References
1. BP Global BP Statisical Review of World Energy 2014. Available online: http://www.bp.com/content/
dam/bp/pdf/Energy-economics/statistical-review-2014/BP-statistical-review-of-world-energy-2014-full-
report.pdf (accessed on 20 June 2014).
2. Pachauri, R.K.; Reisinger, A. Cambio Climático 2007: Informe de Síntesis. Contribución de los Grupos de Trabajo
I, II y III al Cuarto Informe de Evaluación del Grupo Intergubernamental de Expertos Sobre el Cambio Climático;
IPCC: Geneva, Switzerland, 2007; p. 104.
3. Perú. Comisión Permanente del Congreso de la República. Ley de Promoción del Mercado de
Biocombustibles. Available online: http://www2.congreso.gob.pe/sicr/cendocbib/con4_uibd.nsf/
9E95620CC059138105257C9E005AB2F9/$FILE/28054.pdf (accessed on 12 February 2017).
4. Perú. Comisión Permanente del Congreso de la República. Reglamento de la Ley de Promoción del
Mercado de Biocombustibles. Available online: http://www2.osinerg.gob.pe/MarcoLegal/docrev/DS-013-
2005-EM-CONCORDADO.pdf (accessed on 12 February 2017).
5. Arévalo, L.F.; Nalvarte, J.; Torres, J.; Ramírez, Y. Impactos Socio-Económicos de la Producción de Biocombustibles
en la Amazonía Peruana; Primera; SNV-WWF: Lima, Perú, 2009.
6. Sharma, K.K.; Schuhmann, H.; Schenk, P.M. High Lipid Induction in Microalgae for Biodiesel Production.
Energies 2012, 5, 1532–1553.
7. Schenk, P.M.; Thomas-Hall, S.R.; Stephens, E.; Marx, U.C.; Mussgnug, J.H.; Posten, C.; Kruse, O.;
Hankamer, B. Second Generation Biofuels: High-Efficiency Microalgae for Biodiesel Production. BioEnergy
Res. 2008, 1, 20–43.
8. Foley, J.A.; Asner, G.P.; Costa, M.H.; Coe, M.T.; de Fries, R.; Gibbs, H.K.; Howard, E.A.; Olson, S.; Patz, J.;
Ramankutty, N.; et al. Amazonia revealed: Forest degradation and loss of ecosystem goods and services in
the Amazon Basin. Front. Ecol. Environ. 2007, 5, 25–32.
9. Duong, V.T.; Li, Y.; Nowak, E.; Schenk, P.M. Microalgae Isolation and Selection for Prospective Biodiesel
Production. Energies 2012, 5, 1835–1849.
10. Georgianna, D.R.; Mayfield, S.P. Exploiting diversity and synthetic biology for the production of algal
biofuels. Nature 2012, 488, 329–335.
11. Wijffels, R.H.; Barbosa, M.J. An Outlook on Microalgal Biofuels. Science 2010, 329, 796–799.
12. Richmond, A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology; Blackwell Sci. Ltd.:
Hudson, NJ, USA, 2004.
13. Menetrez, M.Y. An overview of algae biofuel production and potential environmental impact. Environ. Sci.
Technol. 2012, 46, 7073–7085.
Energies 2017, 10, 224 13 of 16
14. Khoo, H.H.; Sharratt, P.N.; Das, P.; Balasubramanian, R.K.; Naraharisetti, P.K.; Shaik, S. Life cycle energy
and CO2 analysis of microalgae-to-biodiesel: Preliminary results and comparisons. Bioresour. Technol. 2011,
102, 5800–5807.
15. Chu, S.P. The Influence of the Mineral Composition of the Medium on the Growth of Planktonic Algae:
Part I. Methods and Culture Media. J. Ecol. 1942, 30, 284–325.
16. Carlos, E.M.B.; Mariãngela, M. Gêneros de Algas de Águas Continentais do Brasil—Chave Para Identificação e
Descrições, 2nd ed.; Rima: Sao Carlos, Brasil, 2006.
17. Greenspan, P.; Mayer, E.P.; Fowler, S.D. Nile red: A selective fluorescent stain for intracellular lipid
droplets. J. Cell Biol. 1985, 100, 965–973.
18. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem.
Bull. 1987, 19, 11–15.
19. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor
Laboratory Press: New York, NY, USA, 1989.
20. Kaur, S.; Sarkar, M.; Srivastava, R.B.; Gogoi, H.K.; Kalita, M.C. Fatty acid profiling and molecular
characterization of some freshwater microalgae from India with potential for biodiesel production. New
Biotechnol. 2012, 29, 332–344.
21. Benson, D.A.; Cavanaugh, M.; Clark, K.; Karsch-Mizrachi, I.; Lipman, D.J.; Ostell, J.; Sayers, E.W. GenBank.
Nucleic Acids Res. 2013, 41, D36–D42.
22. NCBI Resource Coordinators. Database Resources of the National Center for Biotechnology Information.
Nucleic Acids Res. 2015, 43, D6–D17.
23. Ye, J.; McGinnis, S.; Madden, T.L. BLAST: Improvements for better sequence analysis. Nucleic Acids Res.
2006, 34, W6–W9.
24. BioEdit Sequence Alignment Editor for Windows 95/98/NT/XP/Vista/7. Available online:
http://www.mbio.ncsu.edu/bioedit/bioedit.html (accessed on 2 December 2015).
25. Sievers, F.; Higgins, D.G. Clustal omega, accurate alignment of very large numbers of sequences. Methods
Mol. Biol. (Clifton NJ) 2014, 1079, 105–116.
26. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 1987, 4, 406–425.
27. Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative
studies of nucleotide sequences. J. Mol. Evol. 1980, 16, 111–120.
28. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics
Analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729.
29. Felsenstein, J. Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution 1985, 39,
783–791.
30. Bligh, E.G.; Dyer, W.J. A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Physiol.
1959, 37, 911–917.
31. Yu, X.; Zhao, P.; He, C.; Li, J.; Tang, X.; Zhou, J.; Huang, Z. Isolation of a novel strain of Monoraphidium sp. and
characterization of its potential application as biodiesel feedstock. Bioresour. Technol. 2012, 121, 256–262.
32. Hartree, E.F. Determination of protein: A modification of the lowry method that gives a linear photometric
response. Anal. Biochem. 1972, 48, 422–427.
33. DuBois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric Method for Determination of
Sugars and Related Substances. Anal. Chem. 1956, 28, 350–356.
34. AOAC International. AOAC Official Methods of Analysis of the Association of Official Analytical Chemists
(AOAC), 15th ed.; AOAC International: Washington, DC, USA, 1990; Volume 1.
35. Ichihara, K.; Fukubayashi, Y. Preparation of fatty acid methyl esters for gas-liquid chromatography. J. Lipid
Res. 2010, 51, 635–640.
36. Brennan, L.; Owende, P. Biofuels from microalgae—A review of technologies for production, processing,
and extractions of biofuels and co-products. Renew. Sustain. Energ Rev. 2010, 14, 557–577.
37. Raja, R.; Hemaiswarya, S.; Kumar, N.A.; Sridhar, S.; Rengasamy, R. A perspective on the biotechnological
potential of microalgae. Crit. Rev. Microbiol. 2008, 34, 77–88.
38. Mata, T.M.; Martins, A.A.; Caetano, N.S. Microalgae for biodiesel production and other applications: A
review. Renew. Sustain. Energy Rev. 2010, 14, 217–232.
Energies 2017, 10, 224 14 of 16
39. De Wilt, A.; Butkovskyi, A.; Tuantet, K.; Leal, L.H.; Fernandes, T.V.; Langenhoff, A.; Zeeman, G.
Micropollutant removal in an algal treatment system fed with source separated wastewater streams. J.
Hazard. Mater. 2015, 304, 84–92.
40. Li, T.; Lin, G.; Podola, B.; Melkonian, M. Continuous removal of zinc from wastewater and mine dump
leachate by a microalgal biofilm PSBR. J. Hazard. Mater. 2015, 297, 112–118.
41. Sioli, H. The Amazon and its main affluents: Hydrography, morphology of the river courses, and river
types. In Monographiae Biologicae; Sioli, H., Ed.; Springer: Dordrecht, The Netherlands, 1984; pp. 127–165.
42. Moniz, M.B.J.; Kaczmarska, I. Barcoding of Diatoms: Nuclear Encoded ITS Revisited. Protist 2010, 161, 7–34.
43. Coleman, A.W. Is there a molecular key to the level of “biological species” in eukaryotes? A DNA guide.
Mol. Phylogenet. Evol. 2009, 50, 197–203.
44. Mai, J.C.; Coleman, A.W. The Internal Transcribed Spacer 2 Exhibits a Common Secondary Structure in
Green Algae and Flowering Plants. J. Mol. Evol. 1997, 44, 258–271.
45. Abou-Shanab, R.A.I.; Matter, I.A.; Kim, S.-N.; Oh, Y.-K.; Choi, J.; Jeon, B.-H. Characterization and
identification of lipid-producing microalgae species isolated from a freshwater lake. Biomass Bioenergy 2011,
35, 3079–3085.
46. Do Nascimento, M.; Ortiz-Marquez, J.C.F.; Sanchez-Rizza, L.; Echarte, M.M.; Curatti, L. Bioprospecting for
fast growing and biomass characterization of oleaginous microalgae from South-Eastern Buenos Aires,
Argentina. Bioresour. Technol. 2012, 125, 283–290.
47. Araujo, G.S.; Matos, L.J.B.L.; Gonçalves, L.R.B.; Fernandes, F.A.N.; Farias, W.R.L. Bioprospecting for oil
producing microalgal strains: Evaluation of oil and biomass production for ten microalgal strains. Bioresour.
Technol. 2011, 102, 5248–5250.
48. Zhou, W.; Li, Y.; Min, M.; Hu, B.; Chen, P.; Ruan, R. Local bioprospecting for high-lipid producing
microalgal strains to be grown on concentrated municipal wastewater for biofuel production. Bioresour.
Technol. 2011, 102, 6909–6919.
49. Mahmoud, E.A.; Farahat, L.A.; Abdel Aziz, Z.K.; Fatthallah, N.A.; Salah El Din, R.A. Evaluation of the
potential for some isolated microalgae to produce biodiesel. Egypt. J. Pet. 2015, 24, 97–101.
50. Ho, S.; Lai, Y.-Y.; Chiang, C.-Y.; Chen, C.-N.N.; Chang, J.-S. Selection of elite microalgae for biodiesel
production in tropical conditions using a standardized platform. Bioresour. Technol. 2013, 147, 135–142.
51. Li, L.; Cui, J.; Liu, Q.; Ding, Y.; Liu, J. Screening and phylogenetic analysis of lipid-rich microalgae. Algal
Res. 2015, 11, 381–386.
52. Rodolfi, L.; Chini Zittelli, G.; Bassi, N.; Padovani, G.; Biondi, N.; Bonini, G.; Tredici, M.R. Microalgae for
oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.
Biotechnol. Bioeng. 2009, 102, 100–112.
53. Li, P.; Miao, X.; Li, R.; Zhong, J. In situ biodiesel production from fast-growing and high oil content
Chlorella pyrenoidosa in rice straw hydrolysate. J. Biomed. Biotechnol. 2011, 2011, 141207.
54. Doan, T.T.Y.; Sivaloganathan, B.; Obbard, J.P. Screening of marine microalgae for biodiesel feedstock.
Biomass Bioenergy 2011, 35, 2534–2544.
55. Abou-Shanab, R.A.I.; Hwang, J.-H.; Cho, Y.; Min, B.; Jeon, B.-H. Characterization of microalgal species isolated
from fresh water bodies as a potential source for biodiesel production. Appl. Energy 2011, 88, 3300–3306.
56. Chaichalerm, S.; Pokethitiyook, P.; Yuan, W.; Meetam, M.; Sritong, K.; Pugkaew, W.; Kungvansaichol, K.;
Kruatrachue, M.; Damrongphol, P. Culture of microalgal strains isolated from natural habitats in Thailand
in various enriched media. Appl. Energy 2012, 89, 296–302.
57. Zhang, S.; Liu, P.; Yang, X.; Hao, Z.; Zhang, L.; Luo, N.; Shi, J. Isolation and identification by 18S rDNA
sequence of high lipid potential microalgal species for fuel production in Hainan Dao. Biomass Bioenergy
2014, 66, 197–203.
58. Chiu, S.-Y.; Kao, C.-Y.; Tsai, M.-T.; Ong, S.-C.; Chen, C.-H.; Lin, C.-S. Lipid accumulation and CO2
utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 2009, 100, 833–838.
59. Huerlimann, R.; de Nys, R.; Heimann, K. Growth, lipid content, productivity, and fatty acid composition
of tropical microalgae for scale-up production. Biotechnol. Bioeng. 2010, 107, 245–257.
60. Liang, Y.; Sarkany, N.; Cui, Y. Biomass and lipid productivities of Chlorella vulgaris under autotrophic,
heterotrophic and mixotrophic growth conditions. Biotechnol. Lett. 2009, 31, 1043–1049.
61. Hempel, N.; Petrick, I.; Behrendt, F. Biomass productivity and productivity of fatty acids and amino acids
of microalgae strains as key characteristics of suitability for biodiesel production. J. Appl. Phycol. 2012, 24,
1407–1418.
Energies 2017, 10, 224 15 of 16
62. Yeesang, C.; Cheirsilp, B. Effect of nitrogen, salt, and iron content in the growth medium and light intensity
on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 2011,
102, 3034–3040.
63. Msanne, J.; Xu, D.; Konda, A.R.; Casas-Mollano, J.A.; Awada, T.; Cahoon, E.B.; Cerutti, H. Metabolic and
gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae
Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 2012, 75, 50–59.
64. Lin, Q.; Lin, J. Effects of nitrogen source and concentration on biomass and oil production of a Scenedesmus
rubescens like microalga. Bioresour. Technol. 2011, 102, 1615–1621.
65. Sheehan, J.; Dunahay, T.; Benemann, J.; Roessler, P. A Look Back at the U.S. Department of Energy’s Aquatic
Species Program—Biodiesel from Algae; U.S. Department of Energy’s Office of Fuels Development: Golden,
CO, USA, 1998; p. 328.
66. Vaulot, D.; Olson, R.J.; Merkel, S.; Chisholm, S.W. Cell-cycle response to nutrient starvation in two
phytoplankton species, Thalassiosira weissflogii and Hymenomonas carterae. Mar. Biol. 1987, 95, 625–630.
67. Olson, R.J.; Chisholm, S.W. Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate
Amphidinium carteri. J. Plankton Res. 1986, 8, 785–793.
68. Li, Y.; Fei, X.; Deng, X. Novel molecular insights into nitrogen starvation-induced triacylglycerols
accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum.
Biomass Bioenergy 2012, 42, 199–211.
69. Abdelaziz, A.E.M.; Leite, G.B.; Belhaj, M.A.; Hallenbeck, P.C. Screening microalgae native to Quebec for
wastewater treatment and biodiesel production. Bioresour. Technol. 2014, 157, 140–148.
70. Valdez-Ojeda, R.; González-Muñoz, M.; Us-Vázquez, R.; Narváez-Zapata, J.; Chavarria-Hernandez, J.C.;
López-Adrián, S.; Barahona-Pérez, F.; Toledano-Thompson, T.; Garduño-Solórzano, G.; Escobedo-Gracia
Medrano, R.M. Characterization of five fresh water microalgae with potential for biodiesel production.
Algal Res. 2015, 7, 33–44.
71. Shrivastav, A.; Mishra, S.K.; Suh, W.I.; Farooq, W.; Moon, M.; Kim, T.-H.; Kumar, K.; Choi, G.-G.; Park,
M.S.; Yang, J.-W. Characterization of newly isolated oleaginous microalga Monoraphidium sp. for lipid
production under different conditions. Algal Res. 2015, 12, 289–294.
72. Yen, H.-W.; Hu, I.-C.; Chen, C.-Y.; Ho, S.-H.; Lee, D.-J.; Chang, J.-S. Microalgae-based biorefinery—From
biofuels to natural products. Bioresour. Technol. 2013, 135, 166–174.
73. Li, J.; Liu, Y.; Cheng, J.J.; Mos, M.; Daroch, M. Biological potential of microalgae in China for biorefinery-
based production of biofuels and high value compounds. New Biotechnol. 2015, 32, 588–596.
74. El-Sheekh, M.; Abomohra, A.E.-F.; Hanelt, D. Optimization of biomass and fatty acid productivity of
Scenedesmus obliquus as a promising microalga for biodiesel production. World J. Microbiol. Biotechnol. 2013,
29, 915–922.
75. Singh, P.; Guldhe, A.; Kumari, S.; Rawat, I.; Bux, F. Investigation of combined effect of nitrogen,
phosphorus and iron on lipid productivity of microalgae Ankistrodesmus falcatus KJ671624 using response
surface methodology. Biochem. Eng. J. 2015, 94, 22–29.
76. Kilham, S.; Kreeger, D.; Goulden, C.; Lynn, S. Effects of nutrient limitation on biochemical constituents of
Ankistrodesmus falcatus. Freshw. Biol. 1997, 38, 591–596.
77. Breuer, G.; Lamers, P.P.; Martens, D.E.; Draaisma, R.B.; Wijffels, R.H. The impact of nitrogen starvation on the
dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 2012, 124, 217–226.
78. Breuer, G.; de Jaeger, L.; Artus, V.G.; Martens, D.E.; Springer, J.; Draaisma, R.B.; Eggink, G.; Wijffels, R.H.;
Lamers, P.P. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II)
evaluation of TAG yield and productivity in controlled photobioreactors. Biotechnol. Biofuels 2014, 7, 70.
79. Nigam, S.; Prakash-Rai, M.; Sharma, R. Effect of Nitrogen on Growth and Lipid Content of Chlorella
pyrenoidosa. Am. J. Biochem. Biotechnol. 2011, 7, 126–131.
80. Piligaev, A.V.; Sorokina, K.N.; Bryanskaya, A.V.; Peltek, S.E.; Kolchanov, N.A.; Parmon, V.N. Isolation of
prospective microalgal strains with high saturated fatty acid content for biofuel production. Algal Res. 2015,
12, 368–376.
81. Weiss, S.B.; Kennedy, E.P. The enzymatic synthesis of triglycerides. J. Am. Chem. Soc. 1956, 78, 3550–3550.
82. Miller, R.; Wu, G.; Deshpande, R.R.; Vieler, A.; Gärtner, K.; Li, X.; Moellering, E.R.; Zäuner, S.; Cornish,
A.J.; Liu, B.; et al. Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen
Deprivation Predict Diversion of Metabolism. Plant Physiol. 2010, 154, 1737–1752.
Energies 2017, 10, 224 16 of 16
83. Rismani-Yazdi, H.; Haznedaroglu, B.Z.; Bibby, K.; Peccia, J. Transcriptome sequencing and annotation of
the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of next-
generation biofuels. BMC Genom. 2011, 12, 148.
84. Ramos, M.J.; Fernández, C.M.; Casas, A.; Rodríguez, L.; Pérez, A. Influence of fatty acid composition of raw
materials on biodiesel properties. Bioresour. Technol. 2009, 100, 261–268.
85. Knothe, G. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process.
Technol. 2005, 86, 1059–1070.
86. Knothe, G. “Designer” Biodiesel: Optimizing Fatty Ester Composition to Improve Fuel Properties. Energy
Fuels 2008, 22, 1358–1364.
87. Smith, P.C.; Ngothai, Y.; Dzuy Nguyen, Q.; O’Neill, B.K. Improving the low-temperature properties of
biodiesel: Methods and consequences. Renew. Energy 2010, 35, 1145–1151.
88. Dunn, R.O. Effect of antioxidants on the oxidative stability of methyl soyate (biodiesel). Fuel Process.
Technol. 2005, 86, 1071–1085.
89. Knothe, G. Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ. Sci.
2009, 2, 759–766.
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