Journal Review

Journal Review of Cyanobacteria

Alexander Richard Congreng (1400610001), Michael Pangestu (1400610010),

Stephanie Christy (1400610006), Vania Eveline (1400610005), and Yoana
Aprillia (1400610021)
Departement of Chemical Engineering, Faculty of Clean and Climate Change,
Surya University
Abstract: Cyanobacteria, also known as blue-green algae, are the oldest
photosynthetic organisms. It has become the subject of substantial amount
of research due to its multi usages and advantages which orginate from its
unique anatomy and properties. It also eliminate extensive carbon sources
into reduced form. Thus, Cyanobacteria are ideal biosynthetic instruments
for sustainable chemical synthesis. Cyanobacteria contain high contents of
nutrition such as proteins, pigments, lipids, carbohydrates, and biomass
resulting a mass production and research development on extraction.
Cyanobacteria has been used as food additives, food supplements, paints
and dyes due to its high-value of proteins. It also contain lipid and
carbohydrates which can be reduced into biofuels such as biodiesel and
ethanol. Several studies also suggest antimicrobial and anticancer
activities. Thus, Cyanobacteria has a promising future in pharmaceutical
and energy industries.
Keywords: Cyanobacteria, nutrition, industry, bioproduct

INTRODUCTION
Cyanobacteria, also known as the blue-green algae, are the oldest
photosynthetic organisms on earth that originated approximately 2.63.5 billion
years ago (Hedger, et al. 2001). These names are used because of the presence of
a bluegreen colored pigment, c-phycocyanin (C-PC), which is a pigment used for
photosynthesis. Cyanobacteria have multipurposes and advantages due to their
unique anatomy and properties. They are prokaryotic morphologically exist and
diverse in forms including unicellular, filamentous, planktonic or benthic, and
colonial (Lau, Matsui and Abdullah 2015).
Cyanobacteria, gifted with photosynthesis system to fix carbon dioxide into
reduced form, are ideal biosynthetic instruments for sustainable synthesis of
chemicals and bioproducts. To enact such processes, Cyanobacteria require only
sunlight, carbon dioxide, water, and low concentration of nutrients for growth,
eliminating the cost of extensive carbon sources and complex growth media. It
can also convert solar energies into biomass at 3-9% efficiency (compared with
terrestrial plants at 0.25-3%) (Dismukes, et al. 2008). Not only that, they are
carbon neutrals organism which fixate the same amount of CO2 that it released

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(Ducat, Way and Silver 2011).

Figure 1. Biofuel Production by photosynthesis (Ducat, Way and Silver 2011)

As prokaryotes, they have a simple genetic markers that eases

manipulation. In addition, even after the extraction process, Cyanobacteria still
contain high-value nutritions which can be converted into organic fertilizer or
animal feed. During the past years, researches focused on high yields methods of
extraction and production (Garcia-Mier, et al. 2014). Cyanobacteria contains high-
value nutritions, such as proteins, fatty acids, carbohydrates, vitamins, minerals,
etc. For example, Spirulina, a well-known cyanobacteria, contain 65% protein
compared to soybean flour with 37% of proteins (Ali and Saleh 2012). Either than
higher content of protein (6070 %), Spirulina is also a rich source of vitamins,
especially vitamin B12 and pro-vitamin A (- carotene) as well as minerals. Thus,
Spirulina are also used as food supplements due to its nutritions, compounds, and
its digestibility (El-Sayed, et al. 2010). Other cyanobacteria may contain materials
for biofuels, such as bioethanols, alkanes and biodiesel. Experiments shows that
Synechocystis sp. PCC 6803, a cyanobacteria, produce bioethanol with 5500
mg/mL yield (Lau, Matsui and Abdullah 2015).
Cyanobacteria also require less area of land for cultivation (Lau, Matsui and
Abdullah 2015). Their natural diversity make them capable to live in an area that
is uninhabitable for terrestrial plants. For example, the cyanobacteria Spirulina
maxima can develop well under alkaline conditions (pH 11) and high salinity
(1.2M Sodium carbonate) (Ducat, Way and Silver 2011). Hence, cyanobacteria
does not have to grow with other terrestrial plants and can live at non-productive
lands. Therefore, Cyanobacteria will not compete with daily crops.
A major concern of photosynthesis and green-based process is the
requirement of fresh water supplies. The application of cyanobacteria in green-
based process support water conservation. As a matter of fact, Arthrospira

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platensis uses 2000L of water to produce 1 kg of dry proteins. This is only 25%,
17%, and 2% of the amount that needed to produce 1 kg of dry proteins in
soybean, corn and beef respectively (Ducat, Way and Silver 2011). Latest
research also found a possibility of anti-cancer substances produced by
cyanobacteria. A number of studies suggests that cryptophycins, an anti-cancer
agent is produced by cyanobacteria Nostoc Sp.
There are also other substances that were found by Singh et al (Singh, et
al. 2011). Therefore, Cyanobacteria have a possibility of being used as precursor
to many bioproducts and chemicals. This journal will focus on reviewing
cyanobacteria and its bioproducts. As stated before, the bioproducts are proteins
and pigments, fatty acids and lipids, biomasses, and carbohydrates. This review
will also discuss the products derived from the bioproducts and the methods of
extracting and purifying.

THE STRUCTURAL AND FUNCTIONS OF CYANOBACTERIA

The constituent cell structure of Cyanobacteria is similar to Gram-negative
bacterial cell, with the main characteristics of Cyanobacteria is to have cell walls
that contains peptidoglycan thin layer (Baulina 2012). The cell wall is the one that
is responsible for giving the permanent shape of algae and also protecting all
contents of the cell. On the surface, the spines, sheath, pili, and glycoprotein S-
layer can be found (see Figure 2).

Figure 2. General scheme of a cyanobacteria vegetative cell in section,

glycogen -granules, high electron density lipid -granules, CG
cyanophycin granule, CM cytoplasmic membrane, Cs carboxysome, CQ

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sell wall, GV gas vesicles, N nucleoid, OM outer membrane, Pg

peptidoglycan, PBS phycobilisome, Phb poly--hydroxybutyrate granules,
Pi pili, PP polyphosphate granules, PS periplasmic space, R ribosomes, S
S-layer, Sh sheath, Sp spines, T thylakoid(s), TM thylakoid membrane
(Baulina 2012)
The intracytoplasmic membrane structures responsible for energy supply
(thylakoids) ll most of the cytoplasm. These membrane structures may form a
single system, called as thylakoid networks (Nevo et al. 2012). The thylakoids are
formed by paired membranes containing chlorophyll a as a component of the
photosystems. Chlorophyll a is the major photosynthetic pigment in cyanobacteria
(Miyashita, et al. 1997).
On the outer side of the membrane elements of the photosynthetic
apparatus, phycobilisomes are located. The nucleoid with (poly) ribosome are
located between the thylakoids, almost in the central part of the cell. Other various
structures and inclusions are present in the cells, their set depending on the species
and growth conditions of cyanobacteria. The main components include gas
vesicles, which promote oatation of the cell within the water column in order to
get sunlight to pursue photosynthesis; carboxysomes (polyhedral bodies), deposits
of the key enzyme catalyzing CO2 xation, ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO); cyanophycin granules, consisting of a unique
polypeptide built of the L-arginine and L-aspartate residues and acting as an
alternative nitrogen source; granules of glycogen (-granules), lipid granules (-
granules), and granules of poly--hydroxybutyrate acting as sources of carbon and
energy; and polyphosphate granules as the sources of phosphorus. There are also
other inclusions, as well as various microtubules and microlaments (Berner
1993).

TAXONOMY AND CLASSIFICATION

Several systems of higher level classification of Cyanobacteria have been
published (Komarek, et al. 2014). It took several years from 1925-1979 for the
best classification to occur. Rippka et al. (1979) recommended five sections,
which became the primary basis for the non-nomenclatural classification in
Bergeys Manual of Systematic Bacteriology, which recognized five subsections
instead of orders, I (=Chroococcales), II(=Pleurocapsales), III (= Oscillatoriales),
IV (= Nostocales) and V (=Stigonematales) (Komarek et al. 2014).
Rippka divides the cyanobacteria into five sections. She describes her first
two sections, I and II, as Unicellular; cells single or forming colonial aggregates
held together by additional outer cell wall layers. In the other three sections, III to
V, she describes as Filamentous; a trichome (chain of cells) which grows by
intercalary cell division (Rippka et al. 1979).

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Table 1. Major sub-groups of cyanobacteria (Rippka et al. 1979)

Unicellular; cells Reproduction by binary fission or Section I
single of forming by budding
colonial aggregates Reproduction by multiple fission
held together by giving rise to small daughter cells Section II
additional outer cell (baecytes), or by both multiple
wall layers fission and binary fission
Trichome
always Division is only one
Reproduction by composed only plane
random trichome of vegetative Section III
breakage, by cells
Filamentous; a
formation of In the absence Division in only
trichome (chain of
hormogonia of combined one plane
cells) which grows
(Section IV and nitrogen, Section IV
by intercalary cell
V only) and trichome
division
sometimes by contains
Division in more
germination of heterocysts;
than one plane
akinetes some also
Section V
produce
akinetes

Figure 3. Ultrastructure of Anabaena variabilis, Chlorogloeopsis fritschii, and

Synechococcus sp (Baulina 2012)

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Table 2. Identifications Groups of Cyanobacteria (Rippka et al. 1979)

Group Identifications

Trichome helical, cells composing trichome are

Spirulina isodiaetric, cylindrical or disc-shaped, reproduction by
transcellular trichome breakage

Trichome straight, cells composing trichome are disc

Oscillatoria shaped, reproduction by transcellular trichome breakage

Trichome straight, cells composing trichome are

isodiametric or cylindrical, reproduction by transcellular
Pseudanabaena or intercellular trichome breakage, cells contain polar
gas vacuoles

Reproduction by random trichome breakage and by

germination of akinetes, heterocyst are intercalary or
Anabaena terminal, position of akinetes is variable, vegetative
cells are spherical, ovoid, or cylindrical

Reproduction by random trichome breakage and by

germination of akinetes, heterocyst are intercalary or
Nodularia terminal, position of akinetes is variable, vegetative
cells are disc-shaped

Reproduction by random trichome breakage and by

germination of akinetes, heterocyst are exclusively
Cylindrosperum terminal, akinetes are always adjacent to heterocysts,
vegetative cells are isodiametric or cylindrical

Reproduction by random trichome breakage, by

germination of akinetes, and by formation of
hormogonia, hormogonia give rise to young filaments
Nostoc that bear a terminal heterocyst at both ends of the
cellular chain, vegetative cells are spherical, ovoid or
cylindrical, akinetes are not initiated adjacent to
heterocysts

Reproduction by random trichome breakage, by

Scytonema germination of akinetes, and by formation of
hormogonia, hormogonia give rise to young filaments
that bear a terminal heterocyst at only one end of the
cellular chain, mature trichome is composed of cells of
even width, heterocysts are predominantly intercalary,
vegetative cells are disc-shaped, ispdiametric or
cylindrical

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Calothrix Reproduction by random trichome breakage, by

germination of akinetes, and by formation of
hormogonia, hormogonia give rise to young filaments
that bear a terminal heterocyst at only one end of the
cellular chain, mature trichome tapers grom base (which
bears a terminal heterocyst), vegetative cells are disc
shaped, ispdiametric or cylindrical

Chlorogloeopsis Reproduction by random trichome breakage and by

formation of hormogonia, hormogonia composed of
small cylindrical cells which enlarge and become
spherical heterocysts develop in terinal and intercalary
positions, hormogonia are produced within terminal
heterocysts, cells in the mature trichome divide in more
than one plane

Fischerella Reproduction by random trichome breakage and by

formation of hormogonia, hormogonia composed of
small cylindrical cells which enlarge and become
rounded, hormogonia are produced from the ends of
trichomes, cells in th mature trichome divide more than
one plane

PROTEIN, GLUCOSE, AND LIPID CONTENT

Cyanobacteria is a photosynthetic organisms that use the suns energy, H2O
and CO2 to synthesize their energy storage components, i.e. carbohydrates, lipids
and proteins. Carboxysomes, one of cyanobacteria organelles that consist of
polyhedral protein shells filled with the enzyme Ribulose-1,5-biphosphate
carboxylase/oxygenase (RuBisCO) is the living proof that protein is one of
biological molecules that exist in cyanobacteria. Cyanobacteria also have granules
of glycogen (-granules) which is the source of glucose and lipid granules (-
granules) that are responsible for producing lipid (Berner 1993). These are the
energy storage components that form a potential feedstock which can be
converted into bioenergy (see Table 3). Of these three biochemical fractions,
lipids have the highest energy content (Quintana et al. 2011).

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PROTEIN AND PIGMENTS

Proteins, nitrogen containing substances, are one of the most essential
nutrition a human body needs. Proteins are precursor to many vital biological
substances, such as antibodies, pigments, enzymes, blood plasma, hemoglobin,
muscles, etc (Nelson and Cox 2004). They are also crucial chemicals that form
DNA. Cyanobacteria mostly compose of proteins and pigments. For instance,
Spirulina, a well-known cyanobacteria, contain 65% protein compared to soybean
flour with only 37% of proteins (Ali and Saleh 2012). During photosynthesis,
sunlight is captured by light-harvesting pigments, such as phycocyanin and
chlorophyll (Satoh, et al. 2000). While chlorophyll exist in many other plants,
phycocyanin only exist in cyanobacteria.
Cyanobacteria stored proteins in inner cells, particularly in carboxysome.
Cell lysis is a principal method of proteins extraction from cyanobacteria. Cells
need to be broken down to extract proteins. As stated before, Cyanobacteria are
known as blue-green algae which contain C-phycocyanin (blue-green pigments)
and a small amount of Allophycocyanin (purple pigments). In general,
phycocyanins and phycoerythrins are part of phycobiliproteins which have certain
pharmacological properties. Romay, et al found that phycocyanin has an
antioxidant activity. It was shown that C-phycocyanin could reduce peroxyl
radicals by one half (Romay, et al. 2003). C-phycocyanin are discovered to be a
good inhibitor for free radicals such as cyclooxygenase (Hosseini, et al. 2013).
Due to this discovery, there has been an urgency to find best methods to
extract C-phycocyanin. Studies have discovered various ways to extract
phycocyanin, particularly C-phycocyanin, mainly from Spirulina Platensis.
Silveira et al who studied the C-phycocyanin concentration in Spirulina Platensis
through cell lysis. Sample was homogenized, frozen, and stirred for 30 hour.
Experiments were varied over biomass concentration and stirring duration. C-
phycocyanin concentration (C-PC) was investigated through sampling after some
hours. The C-phycocyanin concentration (C-PC) was evaluated through optical
density. The results indicated that the best CPC was achieved at 4 hrs and 0.08 g
mL-1 (Silveira, et al. 2007).
Moraes, et al also investigated the best conditions that would result in the
best yields. They conducted 4 different types of extraction. The samples were
treated with inorganic acid, organic acid, pH 7 buffer, and ultrasound. The result
indicated that the highest C-PC was obtained using with ultrasound treatment
while the lowest C-PC was acquired by inorganic acid extraction (Moraes, et al.
2011). Ultrasound emits a high frequency waves which causes cells to break
resulting a very high yield PC. Inorganic acid causes proteins to denature resulting
in low PC.
After extraction, the samples must be purified. As stated before,
phycocyanin is a phycobilinproteins which can be purified with proteins
precipitation technique. Silva, et al investigate the C-PC and EP (Extract Purity)

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with ammonium sulfate precipitation. Spirulina biomass were stirred with 50%
and 70% saturation of ammonium sulfate at low temperature. The results showed
EP at 0.8 (Silva, et al. 2009).
All of the extraction and purification techniques have the same purposes.
Cyanobacteria have plenty proteins that can be commercialized and used for
health benefits. Proteins can be converted into food supplements, food additives
and precursor for chemicals. As food supplements, spirulina can be packed as
tablets or capsules that are made for human consumption. As food additives,
microalgae like Cynaobacteria were added to improve the nutritional value of
foods such as bread and cookies (Hosseini, et al. 2013). The phycocyanin and
other phycobilinproteins can also provide color and health benefits, such as paints
and dyes. It can be used as food coloring agents such as candies and bakery
products (Hosseini, et al. 2013). Romay et al also found an anti-cancer activity
which is a promising future for pharmaceutical industries (Romay, et al. 2003).

LIPID
Cyanobacteria contains lipids (fats and oil) with compositions similar to
vegetable oils. The lipids of some cyanobacteria species are rich of fatty acids
such as linoleic and gamma linoleic acids (Mongra and Agrawal 2014). The fatty
acids are essential components of the diet of humans and animals and it is
important as feed additives in aquaculture (Borowitzka 1988). The lipids of
Cyanobacteria are esters of glycerol and fatty acids (saturated or unsaturated) that
comprise of organic compounds including fats, waxes, phospholipids, glycolipids,
etc. Some of cyanobacteria have large quantities of unsaturated fatty acids (25 to
60 % of the total) (Parker, Van Baalen and Maurer 1967).
Cyanobacteria are characterized by rapid photoautotrophic growth and high
speed of biomass accumulation that are important as renewable energy
alternatives for petroleum-based fuels, such as biofuels-biogas, bioethanol,
butanol, or biodiesel (Nicole, et al. 2013). Biodiesel is a biofuel that comprises of
mono alkyl esters that are derived from organic oils, plants, or animals (Sakthivel,
Elumalai and Mohommad Arif 2011) and can be produced from biological lipids
through transesterification (Williams and Laurens 2010). Lipid content in
cyanoacteria forms 20 to 50 % of the weight of dry biomass. Sometimes this can
be higher than 80 %. Thus it is showed the high lipid productivity that are the
most desirable for producing biodiesel (Spolaore, et al. 2006).

LIPID CONTENT
About 80 % of the total lipid fraction in cyanobacteria are triglycerides and
fatty acid as shown in Table 4 (Klyachko-Gurvich 1974). Other than these, the
other major lipids of cyanobacteria are sulphoquinovosyl diglycerides (SQDG),

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 Table 4. Composition of Fatty Acid in Cyanobacteria

(C. Singh, P. Sinha and P. Hader 2002)

14 : 0 16 : 0 16 : 1 16 : 2 16 : 2 17 : 1 18 : 0 18 : 1 18 : 2 18 : 3 18 : 3 18 : 4 20 : 1

6, 9, 9, 12, 6, 9,
- - 9 6, 9 9, 12 - - 9 9, 12 11
12 15 12, 15

40 60 50 20 50 10 30 40 40 30 40 30 10
content, and DGD, SQDG, and PG each represent only 10-20 % (Murata 1989).
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phosphatidyl glycerol (PG). MGDG represents 50-60 % of the total glycerolipid

monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG) and

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 Fatty Acids

Position of
Double
Bond
Percentage
of Total
Fatty Acids
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The quantification of the total lipid content of some species of cyanobacteria

has been illustrated in Table 5. The percentage of lipids is different in each
species of cynobacteria, depends on the storage structure of the organism
(Rittmann 2011), the enzymes (Wada and Sato 2010), and the environmental
conditions (Karatay and Donmez 2011). Spirulina platensis as an example,
contains lipids and fatty acids, that particularly contain large amount of gamma
linolenic acid (Mahajan and Kamat 1995).
Table 5. Percentage of Lipid (Beetul, et al. 2014)
Microalgae Percentage of Lipids

Leptolyngbya sp. 2.75 0.21

(Albion)

Leptolyngbya sp. 19.09 0.26

(Belle Mare)

Nodularia harveyana 10.12 0.19

(Albion)

Micro-phytoplankton ND

(Flic en Flac)

Symbiodinium clade C 38.39 6.58

(Flic en Flac)

LIPID EXTRACTION METHOD

Lipid Extraction from Biomass
Lipid can be extracted from biomass of cyanobacteria strains (Lyngbya sp.
and Synechococcus sp.,) culture in different media. Cyanobacteria cell pellet is
ground using a mortar and pestle by adding pulverized glass powder and
extraction solvent (chloroform : methanol, 2:1). The extract filtered with filter
paper and add distilled water to remove water-soluble impurities. It is then
allowed to stand for the separation of two layers. The lower lipid layer is
separated by sodium sulfate crystals and then dried. The lipid content and weight
is calculate by the following equations :
Weight of lipid = (weight of container + extracted lipid) weight of container (1)
!"#$%& !" !"#"$ !"#$%&#!' !
Lipid content (%) = !"#$%& !" !"#$#%&' !"#$%& !
x 100 % (Selvan, et al. 2013) (2)

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Transesterification
Transesterification of the dried crude lipid can be carried out using a method
from Sato and Murata (1988) and Liu et al. (2005). Transesterification could be
processed without prior extraction (Sheng, Vannela and E. Rittmann 2011). The
diagram process of transesterification is illustrated in Figure 4.This method is an
important step in the overall process of lipid extraction and biodiesel production.
Transesterification is catalyzed by alkali and acid catalysts. Alkali catalysts which
are commonly used are NaOH, CH3ONa, CH3OK, and KOH (Kasendo, K. T. and
Bhatia 2009). For acid catalyzed systemscan be used sulfuric acid, HCl, BF3,
H3PO4, and organic sulfonic acid (E., et al. 2005).

Acid Transesterification
(MeOH : HCl : CHCl3 ; 10 : 1 : 1 v/v/v) Analysis of Fatty Acid
Treatments : Methyl Esters
a. Sample Size : 1, 3, 5, 10, 20 mg (GC/CG-MS)
b. Reaction Duration : 15, 30, 60, 120
minutes, at 90oC

Acid Transesterification
(MeOH : HCl : CHCl3 ; 10 : 1 : 1 v/v/v)
BIOMASS 60 minutes, at 90oC

Lipid Extraction
(Dryer)
Treatments :
a.Solvent addition sequence :
- CHCl3 then MeOH then
H2O (1:2:0.8 v/v/v) or ;
- H2O then MeOH then CHCl3.
b. MeOH concentration : CHCl2-2(MeOH)-Water:2
* standard concentration.
c. Sonication : CHCl2-MeOH-W-S, Water-MeOH-
CHCl2-S, CHCl2-2MeOH-W-S:2*1 minute

Figure 4. Flow Diagram of Transesterification

(Lewis, Nichols and A. Mcmeekin 2000)
Alcohol quantity, reaction temperature, reaction time, and catalyst
concentration are some factors that influence the transesterification reaction.
Theoretically, a transesterification reaction requires 3 mol of alcohol for 1 mol of
triglyceride to produce 3 mol of fatty acid ester and 1 mol og glycerol. Typically,
biodiesel production used an excess of alcohol to ensure the oils or lipids are
completely converted to esters (D'Oca, et al. 2011).

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GLUCOSE AND CELLULOSE

Cellulose is a polysaccharide which is made of sugar molecular that are
linked and then form a long chain. It is usually found in plants and become a
major food source. (Senese 2015). Besides plants, cellulose is also found in
prokaryotes and eukaryotes including cyanobacteria.

Figure 5. The structure of cellulose (Gibson 2012)

Cyanobacteria produce extracellular polysaccharide (EPS) which can be
used to protect them from dehydration and to maintain their cells (Jia, et al. 2007).
By using cellobiohydrolase I (CBHI)-gold labeling and x-ray diffraction, the
presence of cellulose in cyanobacteria can be detected (Nobles, Romanovicz and
R. Malcolm Brown 2001). The method can only detect the presence of cellulose,
but the amount of cellulose is unknown, probably because of lack of abundance
(Kawano, et al. 2011).
Cyanobacteria has been known to store the energy at night but needs the
sun for energy source. Because of this, the green-blue algae store the energy as the
glycogen. It means that all glucose which has been produced during the day will
be changed into glycogen at night, and then, it needs Glycogenin enzyme. When
glycogen turns back into glucose, it needs enzyme called glycogen phosphorylase
(Schutten n.d.). According to Quintana, et al. (2011), Glycogen is the major
carbon in cyanobacteria. It functions as the energy storage polysaccharide and will
be synthesized during light period in the cell.

Figure 6. Glycogen phosphorylase breaks glycogen down to Glucose 1-phosphate

(Schutten n.d.)
Having use the glucose as a substrate for fermentation, the organism can
extend dark anaerobic survival. The survivals time is prolonged when sodium
thioglycolate or titanium (III) citrate is increasing and then lowering the
environmental redox potential (Richardson and Castenholz 1987). Ethanol and

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lactate are the fermentation product formed from the degradation of glycogen.
Besides ethanol and lactate, carbon dioxide, hydrogen, formate and acetate are
also the fermentation product of cyanobacteria. (Stal and Moezelaar 1997). A
sucroseD-fructoseD-glucose assay kit can be used for determining the sugar
production by cyanobacteria. Sugar production is optimally going on at an optical
density at 750nm (Niederholtmeyer, et al. 2010). As stated before, cyanobacteria
store the energy at night. The dark phase of the light-dark cycle is the state when
sugar catabolic pathways are active. (Quintana, et al. 2011).

Figure 7. Pathway of heterolactic in fermentation in Oscillatoria limosa

(Stal and Moezelaar 1997)
Nowadays, the world has paid many attention to biological ethanol
production. Cyanobacteria produce ethanol in a small amount because the
efficiency of photosynthesis is quite low. The efficiency is about 10% of the total
energy which will be stored as chemical energy (Brenner, et al. 2006). Pyruvate
decarboxylase (PDC) and alcohol dehydrogenase (ADH) are two enzymes that
catalyzed the process of ethanol synthesis. Acetaldehyde and carbon dioxide will
be produced by PDC by catalyzing the non-oxidative decarboxylation of pyruvate.
Moreover, ADH will turn acetaldehyde into ethanol (Deng and Coleman 1999).

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Table 6. Ethanol yields production by cyanobacteria

No Species Times Production References

1 Synechococcus sp. strain PCC Day 3-6 54 nmol unit-1 liter-1 day-1 (Deng and Coleman
7942 at OD730 1999)

Synechocystis sp. PCC6803 212 mg liter-1 day-1 (Gao, et al. 2012)

2
Synechocystis sp. PCC6803 5.2 mmol unit-1 liter-1 day-1 (Dexter and Fu
3 at OD730 2009)

4700 mg liter-1 (Dienst, et al. 2014)

Synechocystis sp. PCC6803
4 Day 18

According to the table of ethanol yields production by cyanobacteria, it

can be concluded that Synechocystis sp. PCC6803 is the best for ethanol
production because it can produce about 200 milligram per liter per day which is
economically good for future development.
Furthermore, hydrogenase activity is reversible which means that many
strains of cyanobacteria can produce hydrogen. (Quintana, et al. 2011). However,
according to Ducat, Way and Silver (2011), oxygen sensitivity of hydrogenases
make the production of hydrogen from cyanobacteria to become low. Other than
ethanol and hydrogen, biofuel that are can also be produced by cyanobacteria.
Besides, biochemical natural product has been produced by marine cyanobacteria
(Burja, et al. 2001). Furthermore, this green-blue algae also produced
mycosporines (Ducat, Way and Silver 2011), a tool against UV radiation.
Cyanobacteria can produce some product that can be useful in industrial
area. It can produce isoprene which is a volatile precursor that can be polymerized
into synthetic rubber (Ducat, Way and Silver 2011). Moreover, cyanobacteria can
produce biopolymers which can replace petroleum-based plastics that has a
promising application in biomedical and pharmaceutical industry (Lau, Matsui
and Abdullah 2015). Finally, this green-blue algae also can synthesized alkanes or
alkenes which can be used for combustion.
Currently, according to Food and Agriculture Organization (FAO) of the
United Nations, Spirulina is an ideal food which is very popular as a dietary
supplement (Ali and Saleh 2012) . Spirulina is used in beverages and bakery
products. It can be added to bread, healthy drink such as sour milk and green tea
to increase their nutritional value. (Hosseini, et al. 2013). In pharmaceutical
industries, spirulina functions as an anti-cancer effects. An evidence shows that
the polysaccharides of Spirulina can repair the damaged DNA which prevent
tumor growth in hamster cheek pouch mucosa (Hosseini, et al. 2013).

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Furthermore, spirulina have a role in prevention of cellular ageing and infectious

diseases (Ali and Saleh 2012).

RESIDUAL BIOMASS
Residual biomass is a term that is used to describe waste products from
biomass resources (Mona 2013). There are many way to valorize the residual
biomass of algae. We can use the residual biomass to produce fertilizer (Spalaore
et al. 2006), feed or food, chemical feedstocks, fuel (Hirano et al. 1997), and
many more valuable product depending on how the residual biomass condition.
From the lipid extracted biomass we could convert the residuals to fertilizer by
anaerobic digestion. From the de-fatted biomass, we can convert to food to
recover the remaining protein, and many more. In this case, we only focus on the
lipid-extracted residual biomass. To convert the residual biomass into a fertilizer,
it must be treated by anaerobic digestion i.e. a series of biological processes in
which microorganisms break down the biodegradable materials in the absence of
oxygen. In anaerobic digestion there are 4 main stages, i.e. hydrolysis,
acidogenesis, acetogenesis, and methanogenesis. The most simple way to convert
the remaining biomass slurry is do an ferment. Fermentation is also an anaeorbic
disgestion process which the process is kept at pH between 6-9 and it will produce
methane that can be converted into electricity (Ueda et al. 1996). Other than this,
the residual slurry of microalgae biomass which contains high nutritional value
can also become food for animals.
Furthermore, residual biomass also still contains vitamins and minerals. Spirulina,
for example, contain 2.33 gr provitamin per 100 gram biomass. For comparison,
Spirulina is ten time higher on vitamin A than carrots. Another concern is the
antioxidant such as, tocopherol (Ali and Saleh 2012). These high value
antioxidants is a promosing future in pharmaceutical industries. This will make
Cyanobacteria a valuable commodity even after the extraction process. Thus
biomass can still provide the necessary nutritions to used as food additives at the
end of processing stage. The biomass, for example, can add nutritional values,
such as Vitamin C and A in beverages like tea (Hosseini et al. 2013).

FUTURE DIRECTION
Cyanobacteria is an autotrophs organisms which needs photosynthesis
process which really affect the production of ethanol. Ethanol production by
metabolites in cyanobacteria has been explored since 1999. Even though there has
been an elaboration of target metabolites, the product from metabolites in
cyanobacteria is not as economical as fossil fuel which come from bio-industrial
processes (Dexter, et al. 2015). Furthermore, the photosynthesis process is not
optimal so that the yields of products produced by cyanobacteria are relatively

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low in amount compared to the ones which produced by the industries or by

heterotrophic processes. Hence, a development of synergic design principle which
integrate more heterologous pathways in cyanobacteria metabolism will be
require (Dexter, et al. 2015). Heterologous pathways means that cyanobacteria
needs another systematic procedure or process that is more effective in
metabolism to produce more ethanol than its own photosynthetic process. In
industrial scale, this needs a good comprehension, not only from cyanobacteria
intracellular, but also from the ecological point of view.
In conclusion, cyanobacteria contains cellulose and glucose which can be
detected using a certain method. Glucose in cyanobacteria can be used as a
substrate for fermentation. The fermentation process can produce some products
such as ethanol, lactate, sugar and hydrogen. Ethanol produced by cyanobacteria
is not as optimal and economical as the one produced by the industry or
heterotrophic process. It is because the process also depends on metabolism of
this green-blue algae. Therefore, it needs a metabolic engineering for
cyanobacteria to produce ethanol optimally and economically good.

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