Environmental Technology & Innovation 35 (2024) 103667

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Influence of different pyrolysis temperatures on chemical

composition and graphite-like structure of biochar produced from
biomass of green microalgae Chlorella sp
Monika Makowska *, Karolina Dziosa
Bioeconomy and Ecoinnovation Centre, Łukasiewicz Research Network – Institute for Sustainable Technologies, K. Pułaskiego 6/10, Radom, Poland

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

Keywords: There is a great demand for environmentally friendly carbon materials as an alternative to fossil
Microalgae biomass raw materials in industrial production. Microalgae biomass can be a valuable renewable source
Chlorella sp for the production of biochar. The study examined the effect of the pyrolysis temperature on the
Pyrolysis process
chemical composition, morphological features and the degree of order of the carbon structure of
Biochar
Graphite-like structure
biochar based on green microalgae – Chlorella sp. Thermochemical conversion was carried out at
400÷900 ◦ C in a CO2 atmosphere. The obtained biochar material had a porous structure. It was
found that as the temperature increased, the biochar yield decreased from 38.4 to 21.9 wt%,
while its aromaticity and stability, determined by the H/C molar ratio, increased. Based on the
results of FTIR and Raman spectroscopy, it was evidenced that the biochar produced at
400÷500 ◦ C was distinguished by the presence of functional groups and double bonds (i.e. –NH2,
C–O, C––C, C––N, N– –O) as well as a high degree of order in the carbon structure (ID/IG=0.2). As
the temperature increases, the amorphous nature of biochar began to develop. This study could
provide data for the proper selection of temperature for pyrolysis of green algae to obtain biochar
with the desired graphite-like structure and properties.

1. Introduction

Biomass, as a very promising renewable raw material, intended mainly for the production of energy and useful bioproducts (e.g.
biodiesel, bioethanol and biogas), is becoming increasingly popular (Devi et al., 2021; Qian et al., 2015; Sathasivam et al., 2019). It can
be produced from living and dead organisms, as well as from non-living materials, most of which are non-fossil. It is also neutral in
terms of carbon dioxide emissions (Abioye and Ani, 2015; Zhang et al., 2023; Zhou et al., 2021). Generally, biomass consist mainly of
carbon, hydrogen, oxygen, nitrogen, sulphur and chlorine (Lee et al., 2020). Depending on the chemical composition, biomass is
divided into: (i) non-lignocellulosic and waste biomass (mainly: proteins, lipids, polysaccharides/carbohydrates, inorganic substances,
minerals and photosynthetic pigments, and small lignocellulosic fraction) and (ii) lignocellulosic biomass (mainly: lignin, hemicel­
lulose, cellulose, extracts and ash) (Hokkanen et al., 2016). The most abundant renewable carbon source on Earth is biomass based on
cellulose and lignin (González-García, 2018).
Depending on its origin, biomass can be divided into woody, herbaceous and aquatic biomass (Zhang et al., 2020). Its main sources
are agricultural residues and waste, industrial residues and municipal waste (Sankaranarayanan et al., 2021). Growing economic

* Corresponding author.
E-mail address: monika.makowska@itee.lukasiewicz.gov.pl (M. Makowska).

https://doi.org/10.1016/j.eti.2024.103667
Received 5 March 2024; Received in revised form 6 May 2024; Accepted 7 May 2024
Available online 11 May 2024
2352-1864/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
M. Makowska and K. Dziosa Environmental Technology & Innovation 35 (2024) 103667

development generates more bio-waste of this type; it is commonly composted or open burned, which produces greenhouse gases and
polluting gases. It is estimated that the amount of crops wasted by Europeans reaches 700 million tons per year (Zhou et al., 2021).
Unfortunately, this happens despite the fact that biomass has an enormous application potential in the production of functional
materials (Abioye and Ani, 2015; Barizão et al., 2023; González-García, 2018).
Due to the necessary protection of farmland and woodland, biomass from non-agricultural sources is increasingly used for utility
purposes, based on raw materials such as energy crops grown on low-quality land, lignocellulosic materials or raw materials of aquatic
origin, i.e. chitinous materials and algae (macro- and microalgae) (Zhang et al., 2023; Zhu et al., 2018). Globally, approximately 9200
tons of dry microalgal biomass are harvested annually from wild habitats and aquaculture farms (Pathy et al., 2020; Vassilev and
Vassileva, 2016). Compared to lignocellulosic biomass, it is characterised by higher photosynthetic efficiency, high survivability, short
reproductive cycle, and high growth dynamics; additionally, it plays a significant role in the process of sequestering atmospheric
carbon dioxide and minimising the greenhouse effect (Krishnamoorthy et al., 2023; Yu et al., 2017a; Yu et al., 2018; Zhang et al.,
2020). For example, production of 1 kg of microalgae binds ~1.83 kg of CO2 (Cheah et al., 2015). In addition, it is a raw material for
the production of many biomaterials, the advantages and disadvantages of which are widely discussed in numerous publications,
including: pigments and fatty acids for the pharmaceutical and food industries (Sathasivam et al., 2019; Ummalyma et al., 2017;
Widyaningrum and Prianto, 2021), sugars and enzymes (Brasil et al., 2017), animal feed (Gondi et al., 2022), biofertilizers and
biopolymers (Costa et al., 2023), third generation biofuels (Bach et al., 2017; Grace et al., 2020), bio-oil (Costa et al., 2023), and biogas
(Gutiérrez et al., 2015).
Microalgae effectively use micro- and macroelements from wastewater (mainly nitrogen and phosphorus) for the development of
their cells (Barizão et al., 2023). Thanks to such a non-synthetic environment, microalgae convert pollutants into biomass in an
environmentally friendly way (up to 1 kg of dry biomass from 1 m3 of wastewater) (Costa et al., 2023; Fernandez et al., 2018), from
which value-added bioproducts can be produced (Yu et al., 2017a). Valuable secondary metabolites can be extracted from biomass,
among others: lipids, proteins, and carbohydrates (Grima et al., 2003; Lieutaud et al., 2019; Na et al., 2015; Sathasivam et al., 2019; Yu
et al., 2017b). However, large amounts of extraction residues often become waste that is problematic for the environment and requires
management (Bryant et al., 2012; Kim et al., 2021). The solution to this problem may be pyrolysis (slow or fast (Lee et al., 2020; Oochit
et al., 2017; Sekar et al., 2021; Yang et al., 2019)), as one of the thermochemical conversion methods, next to torrefaction (Bach et al.,
2017; Viegas et al., 2021) and hydrothermal carbonisation (Alazaiza et al., 2023; Ho et al., 2020; Leong and Chang, 2023; Marcilla
et al., 2013). In the case of algae, it is an extremely complex process of thermal degradation of biomass in an anaerobic environment,
resulting in the production of liquid fuels (bio-oil) and gaseous fuels (syngas), as well as solid carbon-rich materials (i.e. carbon
aerogels, activated carbon or biochar) (Aravind et al., 2020; Bird et al., 2012; Lee et al., 2020; Sankaranarayanan et al., 2021). The
latter are by-products with a graphite-like structure (Yahya et al., 2015).
A lot of research on the characterisation and application of algae-based biochar has been conducted around the world. The main
areas of its potential application are:

− sorbents for wastewater treatment, among others from organic pollutants, microplastics, pesticides, dyes, antibiotics, HMs, PAHs,
and PCBs (Antunes et al., 2022; Jagadeesh and Sundaram, 2023; Law et al., 2022; Singh et al., 2021; Wu et al., 2021; Yu et al.,
2022);
− soil improvers (fertility, reclamation, bioremediation, melioration, structure, pH, stability, organic matter content) (Binda et al.,
2020; Kapoor et al., 2023; Leong and Chang, 2023);
− supercapacitors (Sankaranarayanan et al., 2021; Tsarpali et al., 2021);
− fertilizers (Gao et al., 2022; Karthik et al., 2021; Rombel et al., 2022; Wang et al., 2022);
− CO2 adsorbents (Alazaiza et al., 2023; Krishnamoorthy et al., 2023);
− odour absorbers (Conte et al., 2021);
− biopolymers and biocomposites (Tsarpali et al., 2021);
− activated carbon (Tsarpali et al., 2021);
− activation of persulfates (Alazaiza et al., 2023);
− biochar aerogels (Sankaranarayanan et al., 2021; Schuepfer et al., 2020);
− catalysis (Conte et al., 2021; Ibrahim et al., 2020);
− animal feeding [(Conte et al., 2021)];
− biofuel production (Vassilev and Vassileva, 2016; (Zhu et al., 2018);
− pigments and fatty acids (Yu et al., 2017);
− hydrogen production (Pathy et al., 2020);
− carbon quantum dots (Leong and Chang, 2023).

Mathematical modelling can provide very valuable information for predicting biochar yield and quality, but most models devel­
oped so far concern the lignocellulosic biomass. Therefore, various attempts are made to properly manage algae pyrolysis, whose
mechanisms are extremely complex (Kapoor et al., 2023; Marcilla et al., 2013): (i) chemometric analyses, (ii) multivariate statistical
analyses and (iii) modelling that employs machine learning methods (e.g. using XGB algorithms). The latest studies have shown that
the carbon content in the biomass and the pyrolysis temperature have the major impact on the algae-based biochar yield (Pathy et al.,
2020; Zhou et al., 2021). However, the results of the research on the impact of process temperature on carbon content are ambiguous
and contradictory. There are no research results in the literature regarding biochar derived from a specific species of popular
microalgae obtained under the same experimental conditions. Therefore, there is a great need to expand knowledge in this area.

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Recently, the authors of the paper (Leong and Chang, 2023) conducted an in-depth literature review and found that different studies
showed different trends: a decrease or an increase in the C content with increasing process temperature, but also no effect. The pa­
rameters of the pyrolysis process determine the structure (surface architecture), physicochemical properties, and efficiency of the
produced biochar from algae (Kapoor et al., 2023). And this, in turn, plays a huge role when looking for directions of application
(Oliveira et al., 2017).
The main aim of this research was to compare the chemical composition of microalgae-based biochar produced under different
thermal conditions and to investigate the impact of the pyrolysis temperature on the degree of order/disorder of its graphite-like
structure. The novelty of this work is a comprehensive study of the chemistry and structure of biochar obtained in a wide range of
pyrolysis temperatures (from 400 to 900 ◦ C). The results obtained in this study, due to the lack of analogous data for microalgae, were
referred to biomass and biochar from lignocellulose.

2. Materials and methods

2.1. Materials

The object of the research was a dried biomass of green microalgae Chlorella sp. (humidity 5.89 wt%; alkaline pH<8). The chemical
composition of the microalgae biomass is: proteins (amino acids), lipids, and carbohydrates containing functional groups, i.e. C–
–C,
C–
–O, C–O, C–N, N–H, –OH (Chen et al., 2017). Additionally, in smaller amounts: pigments (chlorophyll), moisture, ash, and vitamins.

2.2. Pyrolysis process

Biochar was produced from the biomass (sample weight 100±1 g) during slow pyrolysis process in a Czylok FCF-V12RM retort
furnace equipped with a PID MRT-4 controller. The process was carried out in the absence of oxygen, using CO2 (5 dm3/min) for
105 min. To ensure greater efficiency of the process, the biomass was subjected to preliminary thermal treatment, the so-called tor­
refaction, at 200÷250◦ C (removal of volatile substances), and then the refined biomass was pyrolyzed (Aravind et al., 2020).
Therefore, during the first 30 min of the process, the temperature increase was identical for all pyrolysis variants, i.e. heating from
20◦ C to 200◦ C (12 ◦ C/min) and stabilisation at this temperature for 15 min. Further heating took place under six thermally different
conditions: to a temperature lower than the set temperature by 50 ◦ C for 30 min and stabilisation for 15 min, and then heating to final
temperature (i.e. 400; 500; 600; 700; 800 or 900◦ C) for 15 min and the isothermal state was maintained for 15 min. After this time, the
furnace was turned off and the sample was left in the chamber under CO2 atmosphere to cool down to ambient temperature. The
obtained sample was then weighed. In this work, only the solid pyrolysis product (biochar) was investigated.
The biochar yield was determined from the formula:

biochar yield = biochar weight [g] /initial biomass weight [g] x 100 [%]

A Testchem LMWs vibratory grinder with a tungsten carbide grinding vessel was used to grind and simultaneously blend the
biochar samples. The obtained powders were then sieved on a steel sieve (mesh size of 40 microns) and stored in a tightly closed
container.

2.3. Scaning electron microscope

The morphology of biomass/biochar microstructure was also analysed using a Hitachi SU-70 Schottky field-emission Scanning
Electron Microscope (SEM) under the following conditions: magnification 500x and 2,000x, vacuum 10− 8 Pa, accelerating voltage
15 kV, take-off angle 30◦ . To perform the analysis, the powdered sample was attached to a piece of an adhesive tape.

2.4. Organic elemental analysis

A Thermo Scientific Flash 2000 CHNS/O analyzer with an oxidation/reduction reactor (quartz crucible; ϕ18 mm) was used for the
purpose of an organic elemental analysis of the microalgae biomass and biochar. The content of carbon (C), hydrogen (H), nitrogen
(N), and sulphur (S) was determined simultaneously (in a single analysis run) by dynamic combustion of biomass/biochar at 960 ◦ C.
The samples were weighed (~3 mg; accuracy ±0.01 mg) in tin capsules and introduced into the reactor via a Thermo Scientific
MAS 200 R autosampler at oxygen flow 250 cm3/min by 5 s. The option of delaying sampling by 12 s was used. After combustion, the
resulted gases were carried by a helium flow (140 cm3/min) to a layer filled with copper, then swept through a GC column that enables
the separation of the combustion gases, and finally, they were detected by a TCD. Data analysis was performed using Thermo Scientific
Eager Xperience software. The cycle time was 520 s. Three measurements were performed for each sample. The obtained results were
averaged and the standard deviations were calculated.

2.5. Fourier-transform Infrared spectroscopy

Fourier-transform infrared (FTIR) spectroscopy was used to investigate surface chemistry by presence of functional groups in the
structure of the biochar. Reflection spectra were recorded using a Jasco FT/IR 6200 spectrometer fitted with a TGS detector, in the

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4000÷650 cm− 1 wavenumber range (spectral resolution of 4 cm− 1, 30 scans at point). This system was equipped with PIKE Tech­
nology MIRacle single reflection ATR accessory containing a plate for powdered samples (contact area: ϕ 1.8 mm) and a high pressure
clamp (10,141 psi upon a sample). The biochar sample was evenly distributed on the diamond crystal in the form of a compact layer. At
least three spectra were recorded for each sample. The spectra were subjected to minor corrections using Spectra Manager software: a
noise elimination/smoothing function (Means-Movement method; width 15). Selected overlapping bands were processed (deconvo­
lution; FWHM 120) to separate individual signals.

2.6. Raman spectroscopy

Raman spectroscopy was used to determine the order degree of the graphite-like structure of the biochar and the presence of defects
in this structure. The spectra were recorded with a Jasco NRS-5100 laser Raman spectrometer with a Micro Spectra Measurement
software package, in the 100÷3700 cm− 1 wavenumber range (spectral resolution of 1.4 cm− 1), using a 532 nm laser excitation source.
The spectra are presented in the range of 300÷2500 cm− 1, because only in this range significant peaks appeared.
The system was equipped with a 4-stage Peltier cooled Charge-Couple Device (CCD), Czerny-Turner monochromator with a focal
length of 300 mm and an LWD 50x microscope objective. All measurements were performed under the following conditions: exposure
time 60 s x 20 accumulation, slit 10 ×1000 µm, aperture 4000 µm, grating L600, and attenuator OD4. The sample of the powdered
biochar was evenly distributed on a microscope slide in the form of a compact layer, then placed on an automatic table and the
measurement point was determined using a built-in high-resolution CMOS camera. To ensure the reliability of the results, spectra for
each sample were recorded – five times from different locations.
All Raman spectra were subjected to mathematical processing using Spectra Manager software. To remove noise and smooth the
curve, the following function was applied: noise elimination/smoothing (Means-Movement method; width 15). Additionally, Spectra
Manager software calculated the areas and heights of the D and G peaks, as well as their full width at half maximum (FWHMD and
FWHMG, respectively) in the declared spectral range. The intensity of the D and G bands was then determined on the basis of the peak
area or peak height in spectral region of interest between about 1270 and 1440 cm− 1 as well as about 1480 and 1640 cm− 1. The ratio of
D-band intensity to G-band intensity (ID/IG) was calculated as ratio of their area (AD/AG) or height (hD/hG). Extreme results were
eliminated and the arithmetic mean of the three remaining results and standard deviation were calculated.

3. Results and discussion

3.1. Effect of pyrolysis temperature on biochar yield

The conventional pyrolysis process of Chlorella sp. microalgae biomass, resulting in the production of biochar, was conducted each
time at a different temperature, ranging from 400 ◦ C to 900 ◦ C. The determination of the pyrolysis conditions was based on the authors
own experience and on the test results published in the literature (Costa et al., 2023; Kapoor et al., 2023; Krishnamoorthy et al., 2023;
Leong and Chang, 2023; Sekar et al., 2021; Yahya et al., 2015), but none of the publications covers such a range as in this work.
It was found that the yield of the biochar production decreased as the process temperature increased. At of 400 ◦ C, the highest result
of 38.4 wt% was recorded, and the lowest at 900 ◦ C, i.e. 21.9 wt% (Table 1). Similar observations concerned the pyrolysis of the
lignocellulose-based biochar: high temperatures (>600 ◦ C) resulted in decreased yield (González-García, 2018). Other studies showed
that microalgae-based biochar yields ranged from 28% to 31% for slow pyrolysis (Jagadeesh and Sundaram, 2023) or within a wider
range (44÷66%), depending on the input parameters (Pathy et al., 2020).
The tendency for microalgal biochar yield to decrease with increasing temperature can be explained by the rapid depolymerization
and thermal decomposition of unstable organics (i.e. proteins, carbohydrates), as well as the loss of non-condensable gases and volatile
substances resulting from dehydration. For comparison, in the study (Leong and Chang, 2023) Chlorella sp.-based biochar yield
decreased from 57.5% (at 200◦ C) to 30.5% (at 600◦ C). Finally, six solid samples (showed an alkaline pH≤9) were prepared in the form
of homogeneous powdered biochar and subjected to microscopic and spectral analysis.

3.2. Characterisation of biochar morphology

The porous structure of the dry Chlorella sp. biomass is evidenced by SEM images for magnification 500x (Fig. 1a) and 2,000x
(Fig. 1b). It was found that the cells of microalgae were spherical, had a slightly rough surface (as effect of drying (Binda et al., 2020)),

Table 1
Effect of pyrolysis temperature on biochar yield.
Pyrolysis temperature [◦ C] Biochar yield [wt%]

400 38.4
500 29.3
600 27.4
700 26.2
800 24.2
900 21.9

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and were attached to each other, forming agglomerates structures (Fig. 1). The images also show salt crystals from the culture medium.
The main structure of microalgae underwent significant changes as a result of thermal decomposition. After pyrolysis of the
biomass, an irregular, porous structure with numerous cavities on the biochar surface was discovered, as presented in Fig. 1c-e. These
images clearly show that significant changes occur in the biochar morphology as a result of this process. Moreover, it was observed that
the structure of biochar changed depending on the pyrolysis temperature. This is visible in the SEM images, corresponding to bio­
products obtained at 400 ◦ C, 700 ◦ C and 900 ◦ C (Fig. 1c-e).
At 400 and 500 ◦ C, a product with irregular porosity and the largest pore sizes was obtained (exemplary image in Fig. 1c). The pore
shapes were formed as irregular geometries in a stochastic arrangement. At higher temperatures, the pore sizes were significantly
smaller (Fig. 1d). Moreover, the structure of the biochar produced at 900 ◦ C (Fig. 1e) differed from the others in that it had a relatively
poorly developed surface, containing mainly cavities and heterogeneous cracks.
An important factor determining the porosity of biochar was the pyrolysis temperature. First, moisture and low molecular weight
volatile substances were released, then light aromatic compounds, and finally hydrogen gas (Ali et al., 2012). The pore structure
development at lower temperatures can be explained by the opening of the pores as a result of the burn-up of the trapped reactive
products and the release of gases. At higher temperatures, when the emission of gases was less intense, new pores of slightly smaller
sizes developed and the internal pores of the carbon skeleton expanded (Devi et al., 2021). At a temperature of 900 ◦ C, deformations of
some pores were observed, which could be filled with tarry pyrolysis residues (González-García, 2018).

3.3. Organic elemental composition of microalgae-based biochar

The organic elemental composition of the produced biochar differed from the composition of the initial biomass (Table 2). The
content of C, H, and N in the biomass is slightly higher than the values typical for algae reported in the literature (Yang et al., 2019).
The source of nitrogen are proteins, i.e. the basic components of the biomass of microalgae Chlorella sp., therefore, its content is several
times higher than in lignocellulosic biomass (1÷2 wt%) (Pathy et al., 2020). Released gaseous products (burn-off gases) such as CO,
CO2, H2 or CH4 are an important factor influencing the development of initial porosity, which is well illustrated in Fig. 1c. This resulted
in an increase in the content of carbon bonded in the biochar (aromatic carbon) compared to the biomass before pyrolysis: from initial
value ~49% to ~60% at 400÷500 ◦ C, which is up to 25% of the initial value. The next results are: ~56% C at 600÷800 ◦ C and ~53% C
at 900 ◦ C. For comparison, Chlorella vulgaris-based biochar contains 61.3 wt% organic carbon, which is an increase in relation to
biomass (49.6 wt%) (Yu et al., 2018). On the other hand, after pyrolysis, the content of hydrogen, nitrogen and sulphur decreased due

Fig. 1. SEM images of: the Chlorella sp. biomass for magnification 500x (a) and 2,000x (b) and biochar produced during pyrolysis at 400 ◦ C (c),
700 ◦ C (d) and 900 ◦ C (e) for magnification 500x.

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Fig. 2. FTIR spectrum of biochar produced from Chlorella sp. microalgae biomass by pyrolysis: at 400 ◦ C (a) with deconvoluted bands at
~1596 cm− 1 (b), ~1118 cm− 1 (c), and at 600 ◦ C (d).

Table 2
Elemental composition of microalgae-based biochar produced at different pyrolysis temperature.
Pyrolysis temperature [◦ C] Elemental composition [wt%]

C H N S H/C (molar)

before pyrolysis 49.10 ± 0.14 8.60 ± 0.24 7.23 ± 0.01 1.09 ± 0.13 2.10
400 58.77 ± 0.37 4.19 ± 0.07 9.72 ± 0.37 <0.01 0.86
500 61.63 ± 0.26 3.76 ± 0.24 8.68 ± 0.68 <0.01 0.73
600 56.85 ± 1.80 1.53 ± 0.37 6.73 ± 0.44 <0.01 0.32
700 55.67 ± 2.54 1.32 ± 0.05 7.72 ± 0.46 <0.01 0.28
800 56.68 ± 0.68 0.88 ± 0.12 6.72 ± 0.92 <0.01 0.19
900 53.18 ± 1.21 0.85 ± 0.07 4.72 ± 0.46 <0.01 0.19

to the reduction in the number of functional groups containing these elements. These results are within the guidelines for a sustainable
production of biochar included in the European Biochar Certificate, i.e. in the range from 35% to 95%, depending on the raw material
(EBC, 2012, 2022).
Simultaneously, there was a systematic decrease in the hydrogen content, i.e. from ~7% to ~4% at 400◦ C and even to <1% at
800÷900◦ C. In the case of nitrogen, an increase in its content was observed in the biochar produced at 400 ◦ C (9.72%) compared to the
initial biomass (8.60%). This was probably due to the incorporation of this element into the structure of biochar. However, at tem­
peratures in the range of 600÷900 ◦ C, a decreasing trend in nitrogen content was visible. During the carbonisation process, pyrolytic
decomposition of biochar precursor (e.g. protein depolymerisation) occurs together with the parallel release of most non-carbon
species (S, H, N, O) (Acién et al., 2012; Ali et al., 2012; Leong and Chang, 2023). This process creates a carbon skeleton that forms
aromatic sheets (Yahya et al., 2015). On the other hand, no sulphur was detected in any of the biochar samples.
Based on the data presented in Table 2, the H/C molar ratios in the biomass and produced biochar samples were determined. They
were much lower than in the biomass (H/C=2.10) and ranged from 0.19 (for 900 ◦ C) to 0.86 (for 400 ◦ C). It is generally recognised
that the H/C ratio in biochar should not exceed 0.7 according to the recommendations of the European Biochar Certificate (EBC, 2012,
2022) and according to some researchers even 0.6 (Mierzwa-Hersztek et al., 2019). Only the biochar produced at a temperature of
400÷500 ◦ C (H/C=0.73 at 500 ◦ C) exceeded these indicators. At temperatures of 600÷800 ◦ C, increasingly lower values were ob­
tained, respectively: 0.32, 0.28, and 0.19, due to the removal of hydrogen atoms during pyrolysis. This indicates a gradual increase in
hydrophobicity (decrease in polarity) and aromaticity of biochar with increasing pyrolysis temperature. This also means an increase in
the potential stability of biochar (Oliveira et al., 2017). These features determine the adsorption mechanisms involving biochar.

3.4. FTIR identification of surface functional groups in the biochar structure

FTIR spectroscopy is one of the most effective methods for measuring functional groups in biochar (Cole et al., 2019). It was found

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that the pyrolysis temperature has a significant impact on the structure and probably also on the properties of biochar. As the pyrolysis
temperature increased, the intensity of individual absorption peaks in the spectrum gradually decreased until they completely dis­
appeared. Only in the case of the biochar sample obtained during pyrolysis at 400 ◦ C, relatively intense absorption bands of surface
functional groups were visible in the FTIR spectrum (Fig. 2a). In some spectra of the biochar produced at 500 ◦ C, these bands appeared,
but they were very weak. However, for the remaining samples (produced at 600÷900 ◦ C), gradually disappeared infrared signals were
recorded (Fig. 2d).
The wide band ~1596 cm− 1 (Fig. 2a) corresponds mainly to C– –C stretching vibrations in the aromatic ring, but also C––N, –NH2
and N–O vibrations. The occurrence of aliphatic and aromatic hydrocarbons was supported by the presence of weak peaks at 2920 and
–
2851 cm− 1. The spectral range of 1500÷650 cm− 1 contained stretching vibration bands of single bonds, i.e. C–C, C–O, C–N, and bands
corresponding to C–H bending vibrations. Almost all the bands that appear are the evidence of the existence of aromatic carbon:
~2920 cm− 1, ~1596 cm− 1, ~993 cm− 1, ~897 cm− 1, ~741 cm− 1. Moreover, quite high intensity at 897÷741 cm− 1 compared to
1596 cm− 1 indicates some degree of condensation (smaller, substituted aromatic units condense into larger sheets (Keiluweit et al.,
2010)).
The overlapping bands centred at ~1596 cm− 1 and at ~1118 cm− 1 were deconvoluted. After separating these bands, new signals
appeared: 1698, 1639, 1586, and 1511 cm− 1 (Fig. 2b) as well as 1163 and 1110 cm− 1 (Fig. 2c). This facilitated the interpretation of the
spectrum in these ranges, presented in Table 3.
Nitrogen compounds were formed as a result of breaking the protein chains and its dehydration (Muzyka et al., 2018). The presence
of characteristic absorption peaks is consistent with the results of the elemental analysis. This corresponds to an increase in nitrogen
content in the biochar produced at 400 ◦ C (Table 2), compared to the initial microalgae biomass.
In summary, the FTIR spectral analysis showed that with the increase in pyrolysis temperature, the components of microalgae
biomass were gradually decomposed and dehydrated, and the easily decomposed surface functional groups were removed by pyrolysis.
In addition, the presence of aromatic carbon-rich structures and transformation products of microalgal biomass, i.e. mainly proteins,
fats, and carbohydrates, as well as a relatively high degree of condensation, were detected.

3.5. Assessment of the structural properties of microalgae-based biochar using Raman spectroscopy

Raman spectroscopy has now been recognised as one of the most powerful techniques for examining the structure of carbon
materials, including graphite. Based on the combination of the position of individual Raman bands, their intensity (e.g. peak area, peak
height) and FWHM, we can obtain important qualitative and quantitative information, in particular regarding the number of layers,
chirality, arrangement order, degree of functionalisation and the presence of defects (Li et al., 2023; Tai et al., 2006). Such knowledge
of the structure enables the prediction of some properties and determination of the potential areas of application of such materials.
The D band, known as the disorder band (or the defect band), represents a ring breathing mode with A1g symmetry from sp2 carbon
rings, but to be active the ring must be adjacent to the defect (Thapliyal et al., 2022; Umada et al., 2023). The band is typically weak in
graphite and graphene and significant in the material containing numerous defects, e.g. holes, edges, grain boundaries, substitutional
atoms, stacking faults and cracks where the lattice symmetry breaks down, which introduce some disorder and enhance
double-resonant Raman scattering (electron-phonon-defect) (Li et al., 2023; Skrzypek, 2021).
The G band, the so-called order band (or the graphitic band), represents the planar configuration of the sp2 bonded carbon found in
graphitic materials and double bonds (Chia et al., 2012). It is the primary mode in graphene and graphite with E2g stretching vi­
brations, both in rings and chains (Ferrari and Robertson, 2000; Moseenkov et al., 2023; Wu et al., 2021).
During carbonisation, carbon atoms are rearranged into a graphite-like structure (Yahya et al., 2015). The intensity ratio between
the bands (ID/IG) is related to the in-plane length (La) of the graphene-like layers (González-García, 2018). This relationship was used
in this study to investigate the impact of the pyrolysis temperature of microalgae biomass on the degree of order/disorder of the
biochar structure. The D peak means the disorder carbon bonding and the G peak means the graphite carbon bonding.

Table 3
Assignment of characteristic vibrations to individual peaks in FTIR spectrum of biochar produced at 400 ◦ C (references adopted from
(Aboulkas et al., 2017; Arnold et al., 2018; Bach et al., 2017; Binda et al., 2020; Chen et al., 2008; Chia et al., 2012; Cole et al., 2019;
Keiluweit et al., 2010; Muzyka et al., 2018; Nasir et al., 2023; Zhu and Zou, 2021)).
Wavenumber [cm− 1] Characteristic vibrations Band assignment

2920 –CH3 asymmetric stretching –CH3 attached to benzene ring, lipid

2851 –CH2 symmetric stretching aliphatic, lipid
1596 C–
–C stretching aromatic, benzene ring
C–
–N stretching in benzene ring, protein amide
–NH2 scissoring protein amine
N–
–O stretching aromatic, nitro compounds
1118 C–O–C symmetric stretching aromatic, aliphatic, carbohydrate
C–N stretching (polysaccharides)
protein amide
993 C–H out of plane bending –C––C–H, substituted aromatic,
C–O stretching
897 C–H out of plane bending –C––C–H, substituted aromatic
741 C–H out of plane bending substituted aromatics

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M. Makowska and K. Dziosa Environmental Technology & Innovation 35 (2024) 103667

In the Raman spectra of the biochar produced during pyrolysis at 400÷900 ◦ C, the D band (centred at ~1355 cm− 1) and the G band
(centred at ~1588 cm− 1) were clearly visible, as shown in Fig. 3. These were the strongest and most widely studied Raman bands for
sp2 carbon materials (Li et al., 2023). The D and G single peaks were sharp, relatively symmetrical, repeatable in shape, clearly
separated from each other in all spectra, i.e. they did not overlap. The G peak dominated, which means that the share of ordered
structures in the microalgae-based biochar in each thermal variant of the pyrolysis process was greater than that of disordered
structures. The differences between the positions of individual bands were almost independent of the pyrolysis temperature used.
Based on the recorded Raman spectra of the biochar produced in the temperature range from 400 ◦ C to 900 ◦ C, the heights (h) and
areas (A) of the D and G peaks, FWHM and the peak intensity ratio ID/IG (calculated from peak areas and peak heights: AD/AG and hD/
hG, respectively) were determined. The obtained results are summarized in Table 4. For all biochar samples analysed, the intensity of
the G peak was higher than the intensity of the D peak, which means that their structures were quite well ordered. This difference was
more visible at lower temperatures.
The intensities of individual peaks are not reliable (height, area, and FWHM); therefore, it is not possible to directly conclude about
the structure of these materials based on them. It was necessary to precisely determine the relationship between the intensities of both
D and G bands, i.e. AD/AG (Fig. 4a) or hD/hG (Fig. 4b). The values of these parameters did not exceed 1 (Table 4), which was also
confirmed by the results presented in (Schuepfer et al., 2020). It was found that the lower pyrolysis temperature, the higher the ID/IG
value. For a pyrolysis temperature of 400 ◦ C, the G peak area was 5 times larger than the D peak area. For comparison, in the case of
graphite, the G peak area is approximately 7–8 times larger than the D peak area (Muzyka et al., 2018). As the pyrolysis temperature
increases, this difference gradually decreased to ~1.4 times (at 900 ◦ C).
Fig. 4 shows the mean values and standard deviations of the ID/IG ratio. Regardless of whether the ratio was determined on the basis
of the peak areas (Fig. 4a) or peak heights (Fig. 4b), a significant impact of the pyrolysis temperature on the changes in the content of
the ordered/disordered structures in the tested materials is visible. As the temperature increased, the content of graphite-like struc­
tures in biochar gradually decreased. The ID/IG ratio for biochar produced at 900 ◦ C increased approximately 3 times compared to
400 ◦ C. For temperatures of 400 and 500 ◦ C, it was practically at the same level (ranged from 0.2 to 0.3). A similar situation occurred in
the case of pyrolysis at 800 and 900 ◦ C (in the range of 0.6÷0.7 for peak areas and 0.5÷0.6 for peak heights; e.g. for commercial
activated carbon: 0.9÷1,0 and 1.5÷1.7, respectively). At a temperature of 600 ◦ C, the value differed significantly from the values given
above and it reached a level of approximately 0.4. However, taking into account the non-ideal symmetry of the peaks, it was found that
the ID/IG ratio calculated on the basis of the peak areas (AD/AG) was a more reliable parameter.

4. Conclusions

In this work, biochar was produced from the biomass of green microalgae Chlorella sp. through a slow pyrolysis process at different
temperatures (in the range of 400÷900 ◦ C). Microalgae biomass is an appropriate precursor for the production of a material with a
relatively high carbon content of 50÷60 wt%. It was found that the morphology, structure and chemical composition of the produced
biochar depended largely on the thermal conditions of the process. In the next stages of work, it is planned to analyze the crystallinity

Fig. 3. Series of typical Raman spectra recorded for biochar produced during microalgae pyrolysis at 400÷900 ◦ C.

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M. Makowska and K. Dziosa Environmental Technology & Innovation 35 (2024) 103667

Table 4
Results of Raman spectral analysis of biochar produced during pyrolysis at 400÷900 ◦ C.
Temperature [◦ C] G band intensity D band intensity ID/IG intensity ratio

hG FWHMG AG hD FWHMD AD AD/AG hD/hG

400 60.20 74.73 4527.77 16.32 68.35 1137.17 0.25 0.27

57.29 71.52 4155.31 10.61 69.31 656.12 0.16 0.19
81.05 72.00 6010.92 15.84 63.54 1058.88 0.18 0.20
500 78.66 69.11 5646.99 20.42 69.15 1420.61 0.25 0.26
71.20 69.70 4992.93 17.11 63.52 1120.51 0.22 0.24
72.61 70.25 4879.20 19.73 62.82 1248.46 0.26 0.27
600 76.67 67.82 5423.27 33.85 73.61 2516.03 0.46 0.44
84.01 68.37 5876.38 32.65 74.96 2468.26 0.42 0.39
46.73 72.58 3426.00 18.72 74.99 1437.58 0.42 0.40
700 41.79 68.76 2881.76 21.41 77.75 1629.80 0.57 0.51
39.59 66.20 2694.50 20.25 79.10 1559.26 0.58 0.51
49.48 69.36 3455.88 25.48 72.36 1853.66 0.54 0.51
800 51.61 66.53 3533.16 24.91 75.73 1906.30 0.54 0.48
45.73 67.01 3090.06 26.25 71.98 1958.72 0.63 0.57
52.35 66.13 3508.79 30.57 84.24 2605.32 0.74 0.58
900 43.85 65.03 2905.51 25.50 81.52 2055.83 0.71 0.58
43.41 61.16 2730.76 21.94 79.50 1759.56 0.64 0.51
27.39 63.86 1735.57 14.99 89.35 1243.51 0.72 0.55

Fig. 4. Impact of microalgae pyrolysis temperature on the ratio of the D band to G band intensity (ID/IG) measured by: a) peak area (AD/AG), b) peak
height (hD/hG).

of inorganic compounds as well as specific surface area and pore volume distribution. The increase in the pyrolysis temperature
resulted in: a decrease in biochar efficiency and deterioration of its porosity, a higher degree of decomposition of the basic biomass
components, an increased content of aromatic carbon, and the development of disorders in the turbostratic structure of sp2-hybridized
carbon material. Out of all the materials examined, the biochar produced at 400÷500 ◦ C had high-carbon content and was charac­
terised by the presence of functional groups, i.e. –CH3, –CH2, –CH, –NH2 and double bonds C– –C, C– –N, N––O, as well as the most
ordered graphite-like structure (ID/IG=0.2, calculated from peak areas). The H/C molar ratio in the biochar produced at temperatures
600÷900 ◦ C did not exceed 0.32, which proves it high aromaticity. Knowledge of the composition and structure of the microalgae-

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M. Makowska and K. Dziosa Environmental Technology & Innovation 35 (2024) 103667

based biochar is very important when searching for areas of its future application. These include: a potential sustainable precursor for
the production of activated carbon, as non-graphitic carbon (NGC), or a substitute for the critical graphitic carbon (CRM, 2023;
Schuepfer et al., 2020). It can be a cheap, green alternative to high-emission fossil materials, which emphasises its potential in a
circular economy.
In summary, the future prospects for microalgae-based biochar are promising. Focusing on directions regarding research gaps and
emerging new trends can ensure its wide-scale commercialisation in various sectors. However, we should not forget about the need to
conduct in-depth analyses of the risks related to the impact of biochar on the natural environment. The worst-case scenario may be one
in which biochar turns out to be toxic to the ecosystem, causing a change in the native population of microorganisms, or harmful to
human health due to insufficient testing of, for example, cosmetic products containing biochar. Many publications and patents should
also be approached with caution, especially those that do not describe in detail the type of biocomponents and the mechanisms
responsible for their action.

CRediT authorship contribution statement

Karolina Dziosa: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Monika Makowska:
Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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