BIOCHEMICAL ANALYSIS OF SPIRULINA BIOMASS THROUGH FTIR, TGA , CHN Abstract

Microalgae has become one of the potential feedstock for biomass production. Present
study investigates thecharacterisation done through thermogravimetric analysis,
elemental analysis, FTIR spectroscopy. These charaterisation techniques were
implemented to study the bio-chemical characteristics of spirulina biomass .

Identification and observation of the protein content, lipids and carbohydrates content
of microalgae were done. The various frequency ranges and related functional groups
were analyzed during the study period. FT-IR spectra of Spirulina have been recorded in
the region of 3870-3448 cm-1 to 695-545 cm-1 in the various frequency ranges.

FTIR spectroscopy examination of spirulina dry biomass revealed the presence of various
functional groups as -OH, -COOH, NH, C-H and C=O organic compound groups. The
investigated results clearly states that the dry biomass of Spirulina sp. is sustainable
green energy and the richest protein source. The main components, protein (3453 and
1645 cm(-1)) and carbohydrate (1032 and 1033 cm(-1)), had distinct fingerprint
characteristics of IR spectra.

Keywords: Microalgae, Spirulina, Protein, TGA, FTIR Contents Introduction Materials and
Methods Spirulina biomass and culture medium Thermo Gravimetric Analysis (TGA)
Infrared Analysis Spectroscopy (FTIR) Elemental Analysis Nutritional Composition:
Results Thermogravimetric analysis Spirulina FTIR absorption fingerprint CHN Elemental
Analysis Conclusions References Introduction Spirulina platensis is a filamentous, spiral-
shaped multicellular algae (1).

Microalgae are studied on the basis of high-protein and low-lipid content, which are
cultivated and harvested round the globe for foods and biofuels. The growth and
biomass productivity of Spirulina totally depend on nutrients availability, pH, light
intensity, and temperature of medium along with the surroundings. Economically , Cost
and media composition are major challenges for feasible and large scale production of
Spirulina.

Presently Spirulina has become a kind of universal health food product. Spirulina is
having fundamental and all the basic nutrients as Vitamin A (as ß -carotene), Vitamin
B12. Spirulina is also an extremely rare source of GLA (Gamma Linolenic Acid) which is
an essential fatty acid. Moreover it is also a mahjor source of phytochemicals.

Spirulina has potential advantages in the significant zones as biochemical, antioxidant

and anti-inflammatory protection, cardiovascular health, detoxification Spirulina cultures
are also an important source of aquaculture feeds, human food supplements, and
pharmaceuticals [2-4], and they have additionally been proposed as an exceptionally
decent choice for fuel production [5].

A few authors have demonstrated the potential of the use of Thermogravimetric

Analysis (TGA) for the investigation of microalgal species. TGA was applied in an air
atmosphere and studied the effects of temperature range and reported the different
phases of growth, related to the presence of various molecules which was produced
during the microalgal growth and to the differences in the thermal properties of these
intracellular molecules[6-7].

The weight loss TGA curves at different temperature ranges were differentiated in three
steps.[8-11]. FTIR is widely used fingerprint methods for the identification of the
chemical constituents and exemplify the compounds structures and this has been
implemented as a requisite method mostly to identify medicinal compounds in
Pharmacopoeia of different countries [12-13].

The utilization oFT IR spectroscopy for analyzing the biological samples was first
proposed during the 1940s the strategy was as a rule effectively explored for the
analysis of organic or biological materials. IR spectroscopy has turned into an
acknowledged tool for the characterization of biomolecules Characterization of
microalgae biomass is enforced for the identification of biochemical composition,
protein or lipid content.

The economics aspects of substantial scale advancement of microalgae unequivocally

relies upon the biochemical composition of the microalgal biomass. The FTIR
spectroscopy studies in microalgal biomass investigations has been utilized in
characterizing and monitoring the chemically complex substance compositions, mainly
as microalgae [14]. Thermogravimetric analysis (TGA) and FTIR has been studied to
characterize microalgal Nannochloropsis sp. [7].
In this work, Spirulina sp,. Blue-green algae, were cultivated in different media to
determine their potential as a source of protein as nutraceuticals as well as
pharmaceuticals The present examination is aimed to discover simple, quick, and
vigorous investigation strategies for assessing the thermal and biochemical properties
of microalgae Spirulina sp.

This was evaluated by: Studying the thermal and biochemical composition of algal
biomass; Studying the protein yield of algae; The following five methods were therefore
included in the study for evaluation: FTIR analysis; Thermogravimetric analysis; Elemental
analysis. Proximate analysis Nutritional composition 2. Materials and Methods (i)
Spirulina biomass and culture medium The Spirulina mother culture was collected from
Gerophyta Nutraceuticals, illupur, Tamil Nadu and developed at Bioenergy lab at Energy
Centre, MANIT, Bhopal.

The mother culture was maintained in Zarrouk's media at ambient temperature, with 12
hours both of light and dark photoperiod with normal white light and the flask were
given manual aeration hourly. All the reagents used for preparing media were of food
grade. For cultivation of Spirulina two different growth media were used` as Zarrouk
media and modified media.

(ii) Thermo Gravimetric Analysis (TGA) TGA measures the difference in weight during
heating constant temperature , under oxidative (air) or inert (nitrogen or helium)
atmosphere. This method is viable for quantitative analysis of the thermal behavior of
various samples that are accompanied by weight. (iii) Infrared Analysis Spectroscopy
(FTIR) A Nicolet FT-IR spectrometer (Madison, USA) was utilized to acquire spectrum
from microalgae sample.

FTIR spectroscopy can be used as physiological to contemplate the structure and

bonding of the algal biomass, particularly to recognize the functional groups. This
investigation was done on a MAGNA 550 Nicolet Instruments spectrometer (Madison,
USA). The instrument was equipped with mercury cadmium telluride (MCT) detector and
the spectra were recorded in the frequency range of 400 to 4000 cm-1 .

For FTIR analysis, the microalgae samples of approximately 1.5 mg were ground with
100 mg of potassium bromide (KBr) powder which was spectroscopic grade in an agate
mortar. In order to obtain 1 mm of transparent pellets, 10-12 tonnes of pressure was
applied to this mixture for 5 min.

(iv) Elemental Analysis Qualitative elemental analysis of spirulina biomass was

determined and analyzed by CHNO elemental analyzer using Thermo Finnigan, FLASH
EA 1112 series, Italy. The dried solid content was calculated as the dry residue at 105 0C
for 24 h. The ash content was determined as the solid residue at 550 0C for 3 hours
after the combustion of feedstock .

The contents of crude protein was studied by kjeldahls method, crude fat by soxhlet
apparatus extraction method, and carbohydrate by phenol-sulfuric acid methodwere
measured. Table 1 presents the composition of both the samples.[15] (v) Nutritional
Composition: Spirulina have been attracting the most consideration as potential, which
interms is indeed actual, sources of proteins and other natural nutrients as fatty acids,
macro- and trace elements).

Among various microalgae, the filamentous cyanobacterium Spirulina gives off an

impression of being ubiquitous component of the phytoplankton can be easily grown in
ocean and seawaters, springs or saline lakes. This microalgae is having highest protein
content (65- 70% protein by dry weight), also contains essential amino acids, which is
easily digestible and is bulier than unicellular algae which simplifies harvesting.

This is useful in human nutrient as it has a high quality as well as quantity of its protein
content. The nutritional content of protein is analogous to the quality of amino acid
digestibility coefficient as well as by its biological value considered it constant and used
it as an internal standard for calculating band intensities. [16-17]. Spirulina consist of
essential amino acids the highest values of leucine as 10.9% of total amino acids,
Valaine being 7.5% and isoleucine as of about 6.8%, [18]. Denaturation of Spirulina
protein occurs when its heated above 680C[19-20].

Results (i) Thermogravimetric analysis The initial application of Fourier transforms

infrared spectroscopy Rapid, non-destructive evaluation of the quality of spirulina
powder. In this process, the cell structure is accelaretory destroyed, and phenomena
such as alteration of lipid structures and thermal unfolding of protein takes place.
The second step occurs at 180-400 0C range, and involves the deterioration of proteins
and carbohydrates. This step involves the major weight loss. Finally, the third step occurs
in the 400-760 0C range and corresponds to the complete oxidation of the organic
matter.[7]. three stages were of · decomposition, i.e., dehydration, · devolatilization and ·
solid decomposition.

It can be observed that the TGA curves for the Spirulina biomass are similar, and
demonstrated three stages during the thermal process. Specifically, the first stage (stage
I) was from 10 to 2500C. This stage of decrease in mass presumably results from the
evaporation/dehydration as the elimination of water in the microalgal cells and external
water bounded by surface tension [18]. This is a stage of little decrease in mass due to
the lower moisture content of the two samples of Spirulina.

It was observed that there was major decrease in mass which was due to the
decomposition volatile matters or devolatisation present in both the samples.Second
stage started at about 2500C and upto 6900C. It exhibited a major weight loss for the
two samples of Spirulina, which corresponds to the decomposition/depolymerization of
Spirulina organic substances including proteins, carbohydrates and lipids. The third
stage was ranged from 7000C to the final temperature 8900C. A very slight mass loss
could be observed during this stage.

This is basically due to the decomposition of carbonaceous materials retained in the

solid residues . There were some differences observed between both the samples of
spirulina. The thermal degradation of spirulina biomass can best be understood from
the weight loss at different temperatures.

The weight loss curves obtained for the spirulina sp. algal biomass are shown in Figure 1
and 2. / Figure 1: TGA results of Spirulina Plantesis in modified media with urea Heat
from 30.00°C to 900.00°C at 10.00°C/min / Figure 2: TGA results of Spirulina Plantesis in
modified media with KNO3 Heat from 30.00°C to 900.00°C at 10.00°C/min The three
main decomposition steps observed for the species are: ? The first step (dehydration);
the weight loss was due to moisture removal from the sample: For the first sample:
1100C For the second sample.
1000C ? The second step (devolatilization); vaporization of organic matter and initiation
of thermal Degradation of the sample: three overlapped peaks at For first sample:
2600C, 3500C, 6800C For the second sample: 2700C, 3650C, 6800C ? The third step
(solid residue decomposition); slow decomposition of the solid residue resulting from
the previous step: For the first sample: 7400C For the second sample: 7400C (ii) Spirulina
FTIR absorption fingerprint Infrared spectroscopy is the absorption spectrum of
molecular vibrational levels, and any functional group has Infrared absorption.
Spectrums of Spirulina sp. present in the Fig.

3 and 4 revealed the presence of various functional groups as hydroxyl, amino,

carboxylic and carbonyl groups. The presence of OH group along with carbonyl group
confirmed the presence of carboxylic acid groups in the biosorbent. The presence of NH
group and OH group along with carbonyl group might be attributed to the presence of
amino acid groups in the biosorbent.

.Strectching vibrations of OH, NH and CO groups were shifted and this was indicative of
the presence and involvement of these three groups in biosorption. Assignment of
these bands to a functional group on the surface of Spirulina sp. as observed from FTIR
spectroscopy is summarized. Table 1. Assignment of bands to the functional group on
the surface of Spirulina sp. as observed from FTIR spectroscopy.[12] S.No.

_Frequency Range (cm-1) _Functional groups _ _ _3870-3500 _O-H stretching vibration

presence of carbohydrate amino acid _ _ _3500-3300 _N-H stretching vibration presence
of secondary amines (protein, lipid) _ _ _3300-2925 _O-H bending vibration _ _ _1650-
1580 _N-H bending vibration carbonyl ß- Unsaturated Ketone amide _ _ _1435-1400
_CH2 bending vibration CH2-CO- the presence of carbonyl compounds _ _ _1300-1240
_C=O asymmetric stretching, the presence of esters _ _ _1120-1030 _Symmetric O-H
stretching vibration _ _ _700-500 _Symmetric C-H stretching vibration, the presence of
the antioxidant enzyme _ _ The FT-IR analyzes of Spirulina represent the following
functional groups.

The infrared spectrum reveals frequency ranges from 3870-3500, 3300-2925 cm-1
representing the O-H stretching and bending vibration, the presence of carbohydrate
and amino acid. The frequency ranges from, 3500-3300 cm-1 peaks are representing in
the N-H stretching vibration presence of secondary amines (protein, lipid) The following
peaks 1650-1580 cm-1 are present in the N-H bending vibration present in the carbonyl
ß unsaturated ketone amide.

The frequency ranges from 1435-1405 cm-1 peak are present in the CH2 bending
vibration. The particular frequency ranges from 1350-1260 cm-1 C-O stretching, O-H
bending vibration presence of alcohol. The following frequency ranges from 1300-1250
cm-1, the presence of C-O asymmetric C-O-C stretching presence of esters, the peak
range 1120-1030 cm-1 present in symmetric C-H stretching, the presence of antioxidant
enzymes, [7].

Biological molecules such as microalgae represents complex vibrational spectrum that

include overtones and combination of several bands. [21-24] The FT-IR analysis of
Spirulina shows the presence of high quantity of proteins, vitamins, phycocyanin and
antioxidants substances.
/ Figure 3: FTIR of open pond system with urea / Figure 4: FTIR of open pond system of
with KNO3 (iii) CHN Elemental Analysis The proximate and ultimate analysis of
microalgae biomass is given in Table 2.

Organic matter lost below 110 0 C is the moisture content and from 110 0C – 7000C is
the volatile matterThe organic matter lost between 110 and 700° C is the volatile matter
and the weight left after 900°C is the ash content[25-27]. The fixed carbon content (FC)
is determined by the difference of total sample and the moisture content, volatile matter
(VM), and ash contents. The major elements and their approximate composition (wt%)
were carbon (21.45%), oxygen (22.45%), hydrogen (6.57%), with high nitrogen contents
(15.42%) due to protein.

The high nitrogenous content highly distinguishes algal biomass from lignocellulosic
feedstocks, which typically contain less than 1% N. It is seen from the results that the
carbon contents (in %) of microalgae are much lower due to the high ash content. Table
2: Elemental, Proximate and Nutritional composition of spirulina species Elemental
Analysis _ _C% _H% _N% _O% _H/C% _ _21.45 _6.57 _15.42 _22.45 _1.43 _ _Proximate
Analysis _ _Moisture content _Volatile matter _Ash content _Fixed carbon _ _6.30%
_59.32% _17.67% _16.71% _ _Nutritional composition _ _ _Proteins _Carbohydrates
_Lipids _Fats _ _ _60-70% _23-28% _6-7% _7-8.5% _ _Sample 1 _43.17% _26% _1.7% _4%
_ _Sample 2 _47.3% _21.7% _1.98% _4.35% _ _ The combination of CHN/TGA/FTIR is a
viable option for determining thermal and biochemical composition in dried biomass
from microalgae.

The long term benefit of both these methods is that it provides a wide scope of
qualitative and quantitative information. Microalgae species spirulina used in this
experiment had 59.32% of the sample composed of volatile components that included
mostly oxygenated and nitrogenated products. The FTIR analysis of dried spirulina
samples clearly marked the presence of surface-bound amide, alcoholic groups,
carboxyl groups, and lipid functional groups.

FTIR spectroscopy offers a practical and real alternative for routine identification proof
of microalgae, as it is less time to devour and more affordable contrasted with molecular
methodologies. Substantial scale microalgae development requires an immense
speculation because of the costly start-up and operation costs[28]. Commercialization of
microalgae for protein-rich source production will rely upon pilot-scale preliminaries
and field demonstrations to understand its actual potential [15].

Conclusions From the experimental study and results,the following canclusions are
drawn: Analysis of standards for spirulina, Infrared spectral fingerprints of the product
determine the major component of the Spirulina powder as protein Quality Thermal
decomposition behavior of two samples of spirulina was analyzed using TGA at different
heating rates.

The results revealed that the reaction rate of thermal degradation of the prior sample
tends to be greater than that of the second sample of spirulina. The decomposition of
the microalgae that occurs in three important degradation steps can also be seen in the
IR spectra. FTIR spectral analysis has confirmed the functional groups present in algae
biomass.

The FTIR analysis of dry algae clearly indicated the presence of amide groups, alcoholic
groups, and carboxyl groups. FTIR/TGA studies could be used to estimate the
approximate protein proportion of a given microalga; therefore, a combination of
FTIR/TGA offers an excellent tool for the characterization of microalgae. Microalga
spirulina sp. is a beneficial raw material for sustainable protein-rich source and biofuel
production.
However, to improve the desired protein yield and quality, more research work is
needed. Based on the systematic analysis, Spirulina contains protein, lipid, carbohydrate,
aliphatic (C-H), Carbonyl (esters and acid), Carbonyl Beta Unsaturated Ketone amide
(C=N), ester, symmetric C-H stretching vibration,.

The distinctive biochemical components of the microalgae appear to be deteriorated in

the accompanying order: starting with the polysaccharides and proteins and lastly lipids.
These contemplations could be utilized to evaluate the estimated lipid content of a
given microalga. Further investigations and researches must be carried for its
clarification.