Fuel 305 (2021) 121592

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Full Length Article

Nitrogen content reduction on scenedesmus obliquus biomass used to

produce biocrude by hydrothermal liquefaction
Alejandra M. Miranda a, David Ocampo b, Gabriel J. Vargas c, Luis A. Ríos b, Alex A. Sáez a, *
a
Biological Sciences and Bioprocesses Group, School of Biological Sciences, Universidad de EAFIT Carrera 49, # 7 sur 50, Medellín, Colombia
b
Industrial and Chemical Processes Group, School of Engineering, Universidad de Antioquia, Calle 70 # 52 - 21, Medellin, Colombia
c
I&D Cementos Argos S.A, Centro de Argos para la Innovación, Carrera 49, número 7 sur 50, Medellín, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: The use of Microalgae biomass for biofuel production by hydrothermal liquefaction (HTL) has been proposed as
Microalgae an alternative to traditional fossil fuels. When comparing fossil fuels, Microalgae biocrude has a high nitrogen
Bio-oil content, which is one of the challenges that affect this technology. This factor causes operational issues because it
Hydrothermal liquefaction
lowers the carbon fraction in the chemical composition, which reduces the calorific value and, as a result, raises
Biocrude
NOx emissions during combustion.
Scenedesmus obliquus
In this work, conditions were established for obtaining Microalgae biomass to allow the production of bio­
crude with low nitrogen content by HTL. The effect of the variation in nitrogen levels on the culture medium, on
cell growth and nitrogen content, was evaluated in the final biomass for the Scenedesmus obliquus strain; at a
laboratory scale, the best results (around 0.25 g/L NaNO3) were used at a bench scale with 20 L tubular pho­
tobioreactors (PBRs). The remaining biomass was processed through HTL then the resulting biocrude was
characterized. The results show that as the nitrogen source in the culture medium increases, the percentage of
nitrogen in the remaining biomass and the biocrude produced also increases. It was possible to establish a
concentration of nitrogen source in the culture medium that promotes the accumulation of lipids, decreases the
protein content in the final biomass and consequently, in the biocrude, without affecting production and yield, a
critical factor to take into consideration when assessing the viability of the proposed process.

1. Introduction
The current global energy crisis is driving the search for alternative
energy sources, with biomass showing promise as a substitute for fossil
fuels [1]. Microalgae biomass has generated significant interest among
various types of raw materials due to its multiple advantages (fast
growth rate, high productivity levels, does not compete for land, among
others) [2], in addition to its ability to capture greenhouse gases (GHGs),
mainly carbon dioxide (CO2), which accounts for 68% of these emissions
and is considered one of the main causes of global climate change [3,4].
To produce biofuels from Microalgae biomass, various conversion
techniques have been developed (fermentation, pyrolysis, gasification
and HTL) [5]. HTL is considered a promising technology for biocrude
production because it converts wet biomass (80%–95% by weight) into
energy-dense biocrude, meaning that the significant amounts of energy
required by other processes to dry the biomass are no longer required
[6–8]. It has been reported that LHT can convert both carbohydrates and
proteins into biocrude, with a higher energy density compared to the
first- or second-generation materials [9–11].
When comparing technologies such as lipid extraction, using sol­
vents and subsequent transesterification to produce fatty acid methyl
esters (FAMEs), against LHT it has been shown that the latter is much
more efficient in the use of reagents, energy requirements can be
reduced by 50%, freshwater consumption by 33% and nitrogen and
phosphorus demand were reduced by 44% [12].
LHT compared to fast pyrolysis can achieve higher biocrude yields,
lower coke production and energy consumption. Furthermore, LHT
biocrude has a higher energy density and superior quality in terms of its
thermal and storage stability [13]. In addition to this, the biocrude from
pyrolysis have a higher oxygen content, which reduces the quality of the
biocrude and results in inconveniences for the improvement process
[13].
One of the main technical issues affecting the biocrude produced by
HTL from Microalgae biomass is the presence of heteroatoms (nitrogen,

* Corresponding author.
E-mail address: asaez@eafit.edu.co (A.A. Sáez).

https://doi.org/10.1016/j.fuel.2021.121592
Received 26 January 2021; Received in revised form 21 July 2021; Accepted 28 July 2021
Available online 6 August 2021
0016-2361/© 2021 Elsevier Ltd. All rights reserved.
A.M. Miranda et al.
oxygen, sulphur up to 7%, 9% and 1.5%, respectively) originating from
proteins and nucleotides [9–11]. These compounds generate sediments
and rubbers, reduce fuel quality and have low thermal stability [6,9,10].
Additionally, the high nitrogen content hinders the refining of biocrude
for biofuel production, deactivating the catalysts used and causing the
formation of ammonia, which is difficult to manage at the plant.
Different strategies have been studied to solve the problem of nitrogen
content in biocrude, such as the two-step approach to HTL and the
evaluation of different catalysts, among others [6,10]. However, the
results have not been satisfactory, due to more research must conduct on
processes that make it possible to produce biocrude with a low nitrogen
content. One possible alternative to this problem is to reduce the ni­
trogen content in the Microalgae biomass used in HTL. Chew et al. [12]
report that limiting the supply of nitrogen during cultivation increases
up to 50% in lipid productivity and reduces the synthesis of proteins in
the microalgae. Yeesang and Cheirsilp [13] claim that microalgae
respond to nitrogen limitation in the culture medium, degrading nitro­
gen macromolecules and accumulating carbon reserve compounds, such
as polysaccharides and lipids, which decrease the percentage of nitrogen
present in the final biomass. In several species of microalgae, the vari­
ation of the nitrogen content in the culture medium is an important
factor in productivity levels and in the production of metabolites such as
proteins, lipids and carbohydrates, among others [1,14]. This work es­
tablishes the effect of the variation of nitrogen levels in the culture
medium on cell growth; on the nitrogen content of the final biomass; and
on the biocrude produced by HTL for the Scenedesmus obliquus
(S. obliquus) strain at a laboratory scale and at a bench scale with 20 L
tubular photobioreactors (PBRs).
2. Materials and methods
2.1. Materials
The S. obliquus strain (ATCC® 11457) was provided by the American
Type Culture Collection (ATCC) and preserved at the biotechnology
laboratory of the Centro Argos para la Innovación (CApI) located at the
Medellín campus of Universidad EAFIT. Bench scale tests were per­
formed using 20 L cylindrical PBRs made of transparent acrylic. Gases
dispersed using a membrane diffuser located at the bottom of the PBR.
The culture medium used at the laboratory and bench scale tests was the
Bristol’s Modified Medium (BMM) in which the nitrogen concentration
(NaNO3) was modified, and the other components remained constant
[20].
2.2. Methods
2.2.1. Cell propagation
The inoculum used in the laboratory scale tests was grown in 5 L of
sterile BBM - liquid, no modifications, at room temperature, with a lu­
minous intensity of 52 μmol/m2s, a 12:12 photoperiod and CO2-
enriched air bubbling. The inoculum used in the bench scale test was
developed in 20 L cylindrical PBRs, with an airflow of 2.5 Lpm, 3% CO2-
enriched air bubbling, natural photoperiods, an initial cell concentration
of 0.2 g/L and unsterilized liquid BBM.
The nitrogen concentrations evaluated at a laboratory scale were
0.80–0.65–0.40–0.25–0.10 g/L of NaNO3. Based on these tests, the
concentrations to be evaluated at the bench scale were established at
0.25 and 1.0 g/L NaNO3. Additionally, both tests evaluated the effect of
initial cell concentration (Xo) in 0.2–0.4 g/L values (laboratory scale)
and 0.2–1.0 g/L values (bench scale)
2.2.2. Cell growth kinetics
A 2-mL sample was taken every 48 h to establish cell growth kinetics
for each treatment at the laboratory scale. The cell concentration was
quantified through the optical density method, using a 680-nm wave­
length. The spectrophotometer used was a HALO RB-10 UV–VIS Ratio
2
Fuel 305 (2021) 121592
Beam Spectrophotometer. The final cell concentration (in g/L) was
determined by dry weight correlation using cellulose ester filters
(Advantec MFS, Inc) with a pore size of 0.45 μm, a diameter of 47 mm
and Sartorius Mark 3 moisture analyzers [20].
2.2.3. Biomass separation, drying and bromatological analysis
The final biomass produced from S. obliquus was separated in all tests
using an Eppendorf centrifuge at 4200 rpm for five minutes. The pre­
cipitate obtained was washed three times with type I distilled water and
dried in a forced convection furnace for 48 h at 40 ◦ C. Additionally, the
biomass produced at a bench scale was analysed for moisture, ash, total
fat, raw fibre and percentage of nitrogen using AOAC 945.15, 923.03,
920.39, 962.09 and 990.03, respectively.
2.3. Biocrude production
The biomass liquefaction was conducted in a 250 mL PARR4576 B
reactor, which was loaded with 25 g of biomass. Deionized water was
used as the solvent and the catalyst was K2CO3 in a proportion of about
50% (w / dw biomass). The atmosphere was pressurized with 10 bars of
argon and brought up to approximately 300◦ C. The reaction time was
60 min with constant stirring at 360 rpm. The reaction products were
separated at room temperature.
For the recovery of the biocrude in the solid and aqueous products,
acetone and dichloromethane are used, respectively, at a ratio of 18.2%
wt, In Fig. 1 is shown the flow process of this biocrude production and
the separation of the different products [15 –18]. Likewise, the biocrude
yield was quantified in the different fractions using the following
equations:
final dry weight biocrude
Biocrude yield(%) = *100 (1)
loaded dry weight biomass
solid fraction dry weight
Solid yield(%) = *100 (2)
loaded dry weight biomass
Furthermore, the biocrude obtained after the reaction was charac­
terized by thermogravimetric analysis (TGA). The calorific value was
determined using a calorimeter (Automatic Isoperibol Calorimeter Parr
6400) under ASTM D240 (Heat of Combustion of Liquid Hydrocarbon
Fuels by Bomb Calorimeter) and API gravity was determined by corre­
lation based on density (15 ◦ C) (g/mL) ASTM D 4052. The percentage of
nitrogen in the biocrude was quantified using the Kjeldahl method [19].
In Fig. 1 the flow process of the biocrude production in this study is
shown.
2.4. Product characterization
The elemental compositions of the biocrude were determined using a
Leco brand TruSepc CHNS-O micro-elemental analyzer. The higher
calorific value of the biocrude was determined using the Dulong for­
mula, as it is shown in equation (3) [15,20,21,22,23,24].
( )
MJ O
HHV = 0.338*C + 1.428((H − ) (3)
kg 8
where C, H, O, correspond to the composition (%) of each element in the
final biocrude obtained for the different treatments.
The chromatographic results of the biocrude were determined using
Agilent Technology 7890A Chromatograph with a mass detector|5975C.
a column no polar DB-5HT (30 m, 0.25 mm y 0.10 mm) was used to
separate the components. Tetradecane was used as an internal standard
to quantify the main compounds of the biocrude. According for database
(NIST 2005), for coincidences of signals >95%.
A.M. Miranda et al.
Fig. 1. Process flow for obtaining the different stages and products resulting from the HTL method using Microalgae biomass.
2.5. Experiment design and statistical analysis
To establish the effect of the variations in the nitrogen concentration
in the culture medium on cell growth, nitrogen content in the final
biomass and in the biocrude for the S. obliquus strain cultivated at lab­
oratory scale, a 5 × 2 factorial design was used with five levels of factor
I, NaNO3 nitrogen source (0.10–0.25–0.40–0.65–0.80 g/L), and two
levels of factor II, initial cell concentration (0.20–0.40 g/L), performing
three replicates for each treatment for 30 experimental units. At a bench
scale a 2 × 2 factorial design was developed, comprised of two factors:
factor I, a nitrogen source of 0.25–1.00 g/L NaNO3; and factor II, an
initial cell concentration of 0.20–1.00 g/L. Treatments were performed
in triplicate with 12 experimental units. Statistical analysis of the results
was conducted using analysis of variance (ANOVA) with a reliability of
95%. The ANOVA assumptions were verified through Shapiro - Wilk,
Kolmogorv, Levene and Durbin–Watson tests. The LSD (Least Square
Difference) multi-range test was used to determine whether there are
significant differences among treatments. The error bars in the graphs
correspond to the statistical error in the replicate of each treatment [25].
STATGRAPHICS Centurion XVI was used for the statistical analysis.
Fig. 2. S. obliquus growth curves at different concentrations (NaNO3) with an initial cell concentration of (a) 0.2 g/L and (b) 0.4 g/L.
3
Fuel 305 (2021) 121592
3. Results
3.1. Laboratory scale
Fig. 2 shows the cell growth kinetics of the S. obliquus strain
measured at laboratory scale by the spectrophotometric method at
different treatments of the experimental design. The growth pattern of
the different treatments exhibits an adaptation phase in the first six days,
followed by an exponential phase which ranged between seven and 28
days of cultivation, ending with a stationary phase. The treatments that
yielded the highest cell concentration of 1.29 and 1.27 g/L were 0.25 g/
L NaNO3–0.2 g/L X0 and 0.4 g/L NaNO3–0.2 g/L Xo, respectively.
Based on the analysis of variance, it was found that there is a sta­
tistically significant difference with a p < 0.05 between the mean cell
growth for the treatments using 0.10, 0.65 and 0.80 g/L NaNO3
compared to 0.25 and 0.40 g/L NaNO3 (Fig. 3), where the initial cell
concentration was 0.2 g/L with a confidence level of 95%. Likewise, for
the main effect analysis no interaction between factors was found.
This result indicates that the different concentrations of the nitrogen
source influence the final cell concentration. The results obtained with
an initial biomass concentration of 0.4 g/L under different nitrogen
concentrations exhibited significant differences in the final cell con­
centration (X) dependent variable (Fig. 3) (matching letters in Fig. 3
correspond to statistically homogeneous groups according to the LSD
A.M. Miranda et al.
Fig. 3. Final cell concentration of S. obliquus for treatments with an initial cell
concentration of 0.2 and 0.4 g/L at different NaNO3 concentrations.
multi-range test, with a confidence level of 95%).
Based on the above, it is inferred that the variation in the concen­
tration of nitrogen source affects the final cell concentration, also
justified in the fact that nitrogen is considered an essential factor in
microalgae growth and that its variation impacts the biosynthesis of
proteins, nucleic acids and pigments, such as chlorophylls, among others
[26].
The treatments with 0.25 and 0.40 g/L NaNO3 exhibit the greatest
cell growth, compared to 0.10 g/L NaNO3. These results are likely due to
the limitation of the nitrogen source in the culture medium, decreasing
biomass productivity levels to the extent that the depletion of nitrogen
levels prevents cell division and maintenance in the microalgae [26].
Jiang et al. [27] report that cell growth could be sensitive to high
deficiency levels in the nitrogen source of the culture medium. Instead,
when nitrogen deficiency is less severe, the photosynthesis mechanism
could be functioning, resulting in high cell growth and lipid production
rates [28].
The treatments with 0.65–0.80 g/L NaNO3 had higher cell growth
levels compared to the treatment with 0.10 g/L NaNO3, but less than
treatments with 0.25–0.40 g/L NaNO3. It is possible because the growth
rate tends to increase only to a certain extent with the increase in ni­
trogen concentration, which is probably an indication of cell growth
inhibition [28–30].
As for the variation in initial cell concentration, no statistically sig­
nificant differences were found (p > 0.05), which means that the vari­
ation in the initial concentration of the range under evaluation had no
impact on the final cell concentration of the microalgae with a confi­
dence level of 95 %.
Nitrogen amounts observed in the final biomass in Fig. 4 (matching
letters correspond to statistically homogeneous groups according to the
LSD multi-range test, with a confidence level of 95 %) showed a
Fig. 4. Nitrogen content in the final biomass of S. obliquus in treatments with
an initial biomass concentration of 0.2 g/L and 0.4 g/L at different NaNO3
concentrations.
4
Fuel 305 (2021) 121592
significant difference between treatments (p < 0.05) based on the
analysis of variance. The highest percentage of nitrogen in the final
biomass found in treatments with 0.65, in the culture medium 0.80 g/L
NaNO3 while the lowest final percentage of nitrogen in the biomass was
achieved by treatments from 0.10 to 0.25 g/L NaNO3. The former sug­
gests that higher levels of NaNO3 in the culture medium favors the
assimilation of nitrogen by the microalgae for use in the synthesis of
proteins and other biomolecules [26].
In addition, as the different concentration of fed NaNO3 showed a
significant effect on microalgae growth, meanwhile for the initial cell
concentrations did not show differences between treatments. Then, the
second phase of experiments was conducted in FBRs using the concen­
trations that produced the highest cell growth.
3.2. Bench scale
Fig. 5 shows the S. obliquus cell growth curves of the different
treatments through day 23 of cultivation in PBRs.
The results obtained from the initial cell concentrations of 0.2 and
1.0 g/L are shown in Fig. 6, where the treatments with 0.20 g/L NaNO3
yielded higher final cell concentration values. The analysis of the dif­
ference in the final cell concentration, setting the initial concentration at
0.2 g/L, shows that the nitrogen concentration has a positive effect. This
is confirmed through statistically significant differences (p < 0.05),
which appear by applying the analysis of variance to final cell concen­
trations. The results show that an increase in the concentration of ni­
trogen source in the culture medium in turn increases cell growth, which
indicates that nutrient availability is a crucial factor in regulating the
growth and development of microalgae as reported by other authors
[31–32].
From the results obtained, it is clear that the treatments carried out in
BRF presented a higher final cell concentration (>50%) compared to the
procedure at a laboratory scale. It is possibly due to conditions such as
light and natural light intensity, which favor cell growth [33].
The results of the quantification of the percentage of nitrogen in the
final biomass (Fig. 7- Matching letters correspond to statistically ho­
mogeneous groups according to the LSD multi-range test, with a 95%
confidence level) show that a greater amounts of nitrogen source in the
culture medium results in a higher percentage of nitrogen in the final
biomass due to an increase in protein formation and assimilation by the
metabolism of microalgae, in contrast to what happens with nitrogen
starvation in most species of microalgae, which promoted the accumu­
lation of lipids or carbohydrates (Table 1), and decreases protein con­
tent, as indicated in other publications [26,9,31]. In terms of the
variation in initial cell concentration, no statistically significant differ­
ences were found (p > 0.05) during the procedure, which means that the
variation in the initial concentration had not impacted the nitrogen
content in the biomass upon completion of the tests.
The total fat content of the different treatments (Table 1) includes
triglycerides and terpenes, which are compounds essential to the HTL
process to the extent they lead to the hydrogenation of triglycerides to
generate fatty acids, as well as hydrocracking and hydrogenation of
terpenes when subjected to high temperature and pressure conditions in
a reducing atmosphere (H2). These products are the precursors of
typical diesel alkanes. Therefore, Table 1 shows that the lowest con­
centration of NaNO3 is favorable from this standpoint.
The percentage of fiber obtained from the different treatments
amounted to 15.4–16.4 %, which is similar to the percentages reported
by Andrade et al. [34] for the Scenedesmus sp. strain.
The ash content obtained in each test are very similar to one another,
due to which organic matter that can be similarly transformed is ex­
pected. Proteins in microalgae are transformed into pyridine compounds
and quinolines.
Table 2 shows a general composition of the main functional groups
for the biocrudes obtained through HTL by GC–MS analyses, in which
the different compounds obtained by families were grouped. These
A.M. Miranda et al. Fuel 305 (2021) 121592

Fig. 5. These graphs show the growth curves of S. obliquus at different concentrations (NaNO3) with an initial cell concentration of (a) 0.25 g/L and (b) 1.0 g/L.

Fig. 6. This graph shows the final cell concentration of S. obliquus with an
initial cell concentration of 0.2 and 1.0 g/L at different NaNO3 concentrations.
Matching letters correspond to statistically homogeneous groups according to
the LSD multi-range test, with a 95% confidence level.
Fig. 7. This graph shows the percentage of nitrogen in the final biomass of
S. obliquus with an initial cell concentration of 0.2 and 1.0 g/L at different
NaNO3 concentrations. Matching letters correspond to statistically homoge­
neous groups according to the LSD multi-range test, with a 95% confi­
dence level.
compositions could vary, in terms of the protein content in the micro­
algae. Higher contents of quinolines and pyridine compounds are ex­
pected with higher protein contents. These results according to the
database installed in the chromatograph (NIST 2005), for coincidences
of signals>95%.
Authors like H. Durak et al. [35–36] obtained the highest HHV value
30.66 MJ/kg, at the highest carbon ratio H/C 0,11. In our case, part of
the oxygen is replaced by nitrogen (Table 3). Biocrudes obtained from
the biomass produced in the bench-scale treatments exhibited similar
calorific values around 30 MJ/kg.
However, there is a marked difference in biocrude yields (Table 4).
The biomass produced in treatment with 0.9 g/L NaNO3 − 1.0 g/L Xo
generates the highest biocrude yield, with a value of 41.1% and a solid
phase yield of 21%. The above means that about 79% was transformed
into liquefaction products such as biocrude and gas.
The biomass obtained in the treatment with 0.9 g/L NaNO3 − 1.0 g/L
Xo has one of the highest concentrations of protein and exhibited the
highest biocrude yield. However, it also had a higher content of quin­
olines and pyridine compounds. The protein content of the biomass
obtained in the treatment with 0.9 g/L NaNO3 and 0.2 g/L Xo is similar
to the protein content of the treatment with 0.9 g/L NaNO3 1.0 g/L Xo,
with a value close to 42%; however, the biocrude yield is much lower,
with a value of 18%, showing that fewer compounds of interest (bio­
crude in this case) are produced, and that much of the Microalgae
biomass did not react or was coked. The treatments with 0.2, 0.9 g/L
NaNO3 and 1.0 g/L Xo behave in a similar way. It should be noted that a
timely evaluation of the HTL process was performed and the conditions
in the HTL process, such as temperatures, S/L ratios and reaction times,
were not modified for every test.
The transformation of carbohydrates and lipids into biocrude re­
quires less drastic conditions than proteins, which may explain the low
biocrude yields in some tests, with an increase in solid-phase yields due
to the carbonization of the biomass.
The higher the protein content, the greater the amount of low-quality
biocrude produced from a chemical viewpoint, as indicated by Haverly
et al. [37]. A higher nitrogen content is obtained despite having similar
physicochemical characteristics, such as API density and calorific value,
which means that in subsequent refinement processes, more drastic
conditions will be required to remove it. Authors like Dabros et al. [38],
Sun, Yang and Shi [39] indicate that the use of fuels with high nitrogen
content would produce higher NOx emissions.
Meanwhile, authors like H. Durak [35,36,40] group GC–MS analyses
by different families, which allows better identification of the charac­
teristics of the biocrude obtained. A similar GC–MS analysis in Table 2,
confirms the nitrogen content in the biocrude showing a higher number

Table 1
This table shows the results of the bromatological analysis conducted on the final biomass of S. obliquus. before its conversion to biocrude.
Treatment NaNO3 g/L Initial cell concentration (g/L) % Fiber % Protein – (% Nitrogen) % Total Fat % Ash % Carbohydrates

A 0.25 0.2 16.4 16.8 (2.7) 10.4 12.3 44.1

B 0.90 0.2 15.4 40.5 (6.5) 3.2 12.1 28.8
C 0.25 1.0 15.8 30.0 (4.8) 4.2 12.4 37.6
D 0.90 1.0 15.8 38.5 (6.2) 2.5 13.0 30.2

5
A.M. Miranda et al. Fuel 305 (2021) 121592

Table 2 Table 2 (continued )

General composition of functional groups of the biocrudes produced. Compound % Area
Compound % Area
A B C D
A B C D
Tridecane, 5-propyl 1.21 1.13 2.28 –
Ketones 2-Acetonylcyclopentanone 3.47 – 1.68 – Tridecane, 7-methyl 1.68 1.05 – –
2-Cyclopenten-1-one, 2,3- – – 1.15 –
dimethyl
2-Cyclopenten-1-one, 3-methyl – – 1.09 –
Butyrolactone 1.25 – – – Table 3
Ethanone, 1-(3-hydroxyphenyl) 2.87 – – – Comparison of the elemental composition and the higher heating value (HHV)
Aromatics 1,2-Benzenediol – – 2.61 – for biocrude obtained at each experiment.
1-Bromobenzene, 4-(4- – – – –
Biocrude %C %H %O %N H/C O/C HHV (MJ/
bromobenzyl)
from kg)
Benzene, ethoxy 5.8 – – –
Chondrillasterol – – – 2.81 A 80.2 8.86 8.18 2.76 0.11 0.10 30.12
Cyclo-(l-leucyl-l-phenylalanyl) 1.08 1.1 2.12 – B 64.02 8.73 20.83 6.42 0.14 0.33 30.48
Morphinan-6-ol, 4,5-epoxy-N- – 1 – 1.52 C 67.43 8.92 19.04 4.61 0.13 0.28 31.02
methyl D 67.08 9.02 17.21 6.69 0.13 0.26 29.97
Oxirane, [4-(1,1-dimethylethyl)] – 2.01 – –
Oxirane, 2-decyl-3-(5-methy) – 2.43 – –
Phenol, 3,5-dimethoxy – – – 2.21
p-Hydroxybiphenyl – – – – Table 4
Stigmasta-7,16-dien-3-ol – 4.11 – – Biocrude production by HTL with different treatments.
Stigmasterol – – – 1.2
Pyridines 1,2,5-Oxadiazole – – – 2.08 Biomass Biocrude Solid HHV API Nitrogen in
2-Ethyl-1,3,4-trimethyl-3-pyrazo – 2.83 – – yield (% wt.) yield (% (MJ/ density Biocrude (%
2-Piperidinone – 1.66 – 1.26 wt.) Kg) wt.)
3,6-Diisopropylpiperazin-2,5- – – 1.41 – A 18.9 28.1 30.12 3.90 2.8
dione B 18.0 32.9 30.48 4.82 6.4
Azacyclohexane, 3-[1-pyrrolidyl] 2.38 – – – C 29.3 19.5 31.02 4.55 4.6
N,N’-Dibutyl-N,N’-dimethyl 1.88 – – – D 41.1 21.0 29.97 4.95 6.7
Pyridine, 2,4,6-trimethyl – 5.01 – 1.44
Pyrido[2,3-d]pyrimidine, 4- – 1.72 – 1.6
methyl
of compounds such as quinoline and pyridines when the biocrude has a
Pyrimidine, 4,6-dimethyl – – 1.45 –
Pyrrolo[1,2-a]pyrazine] 2.99 4.99 2.93 2.85 higher nitrogen content.
Tetradecanamide – 2.31 – 1.06 Therefore, it is necessary to establish appropriate operating condi­
Furans 1,3-Dioxolane, 2-(3,4-dihydroxy) 1.97 – – 1.26 tions for the HTL process (temperature, pressure, microalgae/solvent
Furan, 2-methyl-5-(methylthio) 9.73 – 5.6 – ratios and residence time) after modifying the culture medium of the
Quinolines 1H-Indole, 1-methyl-2-phenyl 1.36
microalgae to produce the highest amount of biocrude with the lowest
– – –
1H-Indole, 3-methyl – – – 1.17
1-Pentanamine, N,N-dipentyl – – 2.71 3.72 nitrogen content as indicated by Thilakaratne, Brown and Wright [41],
2,5-Piperazinedione, 3- – – 2.49 – stressing that it is necessary to combine the microalgae cultivation
(phenylme) processes and HTL to ensure industrial viability.
2,5-Piperazinedione, 3,6-bis(2- 2.53 1.81 1.83 –
methyl)
2,5-Piperazinedione, 3-benzyl 2.86 2.59 5.19 6.21 4. Conclusions
Indole – 1.63 – 3.04
Alkanes 17-Pentatriacontene – – – 1.32 The stress generated by limiting the nitrogen source and an excessive
1-Heptadecanol – 2.83 – – increase in its availability harms cell growth. When seeking a balance
1-Hexadecanol 1.49 – 1.43 –
1-Hexadecyne – 1.52 – –
between higher cell growth and low nitrogen content in the biocrude
2-Decene, 3-methyl-, (Z) – – 7.26 1.74 produced, desirable for subsequent refining processes that lead to the
2-Hexadecene, 3,7,11,15- 2.58 1.15 1.38 1.72 production of biofuels, the NaNO3 concentration, which yielded the best
tetramethyl results was 0.25 g/L. At this nitrogen level, the final concentration of
2-Pentadecanone, 6,10,14- 4.62 4.41
– –
biomass during cultivation was not influenced by the initial cell con­
trimethyl
3,7,11,15-Tetramethyl-2- – – – 1.74 centration in the range under evaluation.
hexadecane As the nitrogen concentration in the culture medium decreases, the
4-Decene, 8-methyl-, (E)- 5.27 – – – percentage of nitrogen in the final biomass and the biocrude produced
5-Fluoro-m-xylene 3.29 – – – from this biomass decreases, decreasing the costs of refining processes to
5-Octadecene, (E)- 1.16
produce Diesel-type fuel, requiring lower catalyst ratios and less severe
– – –
9,12-Octadecadienoic acid (Z,Z) 5.64 – 8.26 –
9-Hexadecenoic acid 1.81 – 3.73 – conditions. The importance of establishing joint cultivation and HTL
Cyclohexene, 3-(2-methylpropyl) – – – 1.11 conditions was demonstrated to balance the lower nitrogen content in
Dodecane 3.12 – 1.64 – microalgae and the higher biocrude production yields.
Heptadecane 3.18 1.86 2.21 1.65
Hexadecanamide 1.44 – 2.9 1.36
Hexadecane 3.11 2.08 2.79 – Declaration of Competing Interest
Isophytol – 5.94 – 5.35
Nonadecanoic acid, methyl ester 1.66 – 3.59 – The authors declare that they have no known competing financial
Octadecane, 1-(ethenyloxy) 1.62
interests or personal relationships that could have appeared to influence
– – –
Octadecane, 2,6-dimethyl 3.12 – –
Pentadecane 1.29 10.02 4.81 12.51 the work reported in this paper.
Phytol – 12.77 4.21 12.23
Tetratetracontane 1.1 – – – Acknowledgments
Trichloroacetic acid, pentadecyl – – – 2.76

We would like to thank the Ministry of Science and the Mining and

6
A.M. Miranda et al.
Energy Planning Unit – UPME as co-financers of the project “Effect of the
culture medium on nitrogen content in microalgae used in the bio­
fixation of CO2.”
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