Fatty acid Analysis and Poly-β-hydroxybutyrate (PHB) optimization in

Limnospira/Arthrospira Species

Microalgae, ranging from 200,000 to 800,000 species, are known for their economic importance
and are often called "green gold" due to their bioactive compounds used in various industries.
They are rich in proteins, carbohydrates, lipids, vitamins, and minerals, making them valuable
resources. In regions like Chad and Burkina Faso, microalgae have improved nutrition for
vulnerable populations. However, in Eastern Africa, the nutritional benefits of Arthrospira are
not fully utilized, highlighting the need to unlock microalgae's potential to combat malnutrition
in the region. Studying the fatty acid profiles of these cyanobacterial species in lakes can offer
insights into their nutritional value, ecological roles, and potential applications (Damessa, 2021;
Hegazi et al., 2024; Piccolo, 2012; Simpore et al., 2005). Poly-β-hydroxybutyrate (PHB) can be
used as an effective thermoplastic and has many characteristics similar to those of standard
commercial plastics like polypropylene. Among cyanobacterial strains known to produce PHB,
A. platensis is currently under large scale production.

Methodology

Sampling and mesurments

Water samples was collected from Chitu shores in January and July, with measurements taken
for conductivity, pH, temperature, salinity, and water transparency.

Isolation, identification and harvesting of Arthrospira

The water sample was placed in Zarrouk’s liquid medium and cultured for two weeks before
centrifugation at 2000 rpm to remove impurities. Following serial dilution, Arthrospira samples
were spread on Zarrouk’s solid plates and incubated at 22 ± 2°C for three weeks. Isolated
colonies were then purified for purity. Identification of the species was done using taxonomic
methods with an inverted microscope. A single filament was isolated from the agar plate and
cultured in fresh liquid medium to obtain a pure isolate after five weeks. This isolate was used
for further testing and identification (Nowicka-Krawczyk et al., 2019). The pure culture was
allowed to grow in liquid media for two weeks before being centrifuged. Once Arthrospira
floated to the surface, it was harvested and dried for fatty acid analysis.

Optimization and PHB determination

The effect of different pH and nutrient conditions, pH (8, 9, 10), NaCl addition (0.5, 1.0, 1.5, g/l)
and Na acetate addition (0.5, 1.0, 1.5, g/l) on PHB yield by Arthrospira species was determined.

10 ml of Arthrospira was centrifuged at 6000 rpm, dried at 100ºC, and its dry weight determined.
The pellet was then suspended in sterile water, homogenized, and mixed by vortexing. After acid
treatment and chloroform extraction, PHB content was measured by UV spectrophotometry for
calculating the percentage produced by the organisms. Arthrospira, acting as a biopolymer, was
harvested from the surface after drying (Ansari and Fatma, 2016)..
Determination of fatty acid composition

To characterize the overall lipid composition, dried biomass of Limnospira extract was subjected
to saponification followed by esterification to convert free fatty acids into their corresponding
methyl esters, hereafter referred to as fatty acid methyl esters (FAMES). All derivatization
experiments were repeated at least twice, and each sample was injected twice into the GC–MS,
resulting in at least quadruplicate results for every sample.

Result and discussion

Identification and harvesting of Arthrospira
Arthrospira appears as a coiled thread of cells (trichomes) and are unicellular under the
microscope. Individual trichomes are basically made up of single cells that coil up the entire
length. The species differ partly on the basis of the tightness of the spiral. Arthrospira species
forms mucilage which flows along the length of the thread. This mucilage creates movement
which can be viewed microscopically (Serediak and Huynh, 2011).
Fig 1. Harvesting process Fig 2. The Arthrospira on a) agar plate b) Microscopic appearance

PH and nutrient optimization for PHB accumulation

PH is an important parameter in the synthesis of metabolites and the growth of cyanobacteria

because it influences metabolic processes. In present study the highest accumulation of PHB
(0.09 g/ml) observed at pH 9.0. Previous studies have demonstrated Spirulina platensis exhibited
significant PHB accumulation at pH 9, while Synechocystis sp. showed high PHB accumulation
at pH 8.5 (Ansari and Fatma, 2016).

Increasing substrate concentration, especially with Na acetate, significantly boosted PHB yield.
PHB production increased with different concentrations of Na acetate and NaCl at pH levels 8
and 9. PHB accumulation is influenced by the species' capacity to thrive in the presence of
acetate. Typically, the organism's PHB production is stimulated by the α-ketothiolase enzyme,
with the addition of Na acetate in the medium monitoring acetyl CoA activity. Elevated acetate
levels lead to the production of PHB with a lower molecular weight (Myshkina et al., 2008).
When Nacl concentration increases, the osmotic pressure also increases, this leads to the
dehydration of cells. Though, dehydration provides more space for the accumulation of PHB
(Wang et al., 2021).

Na acetate (PH 8) Na acetate (PH 9) Na acetate (PH 10)

0.02
0.15 0.12 dry
d 0.015 ma
0.09 r
0.1 ss
0.06 y 0.01
0.05 PH 0.03 .
B 0.005
.
0 (g) 0 .
0.5g/l 1g/l 1.5g/l 0.5g/l 1g/l 1.5g/l 0
0.5g/l 1g/l 1.5g/l
Concentration concentration Concentration
A B C
 Nacl (PH 8) Nacl (PH 9)
0.12 0.1
Nacl (PH 10)
0.05
0.1 dr
0.08 y. 0.04
0.08 dry 0.06 .. 0.03
0.06 ma dry mass
ss 0.04 0.02
0.04 PHB (g)
0.02 0.01
0.02
0 0
0 0.5g/ 1g/l 1.5g/ 0.5g/l 1g/l 1.5g/l
0.5g/l 1g/l 1.5g/l l l
Concentration
Concentration Concentration
D E F

Fig. 3. Production of PHB by Arthrospira species using two nutrient sources (Na acetate and
Nacl) and concentrations, A, B, C. PHB content (g) and dry cell weight (g/l) at PH 8,9,10 and
using Na acetate as nutrient sources, D, E, F. PHB content (g) and dry cell weight (g/l) at PH
8,9,10 and using Na acetate as nutrient sources

PHB spectrophotometric assay

The spectrophotometric assay of the polymer extracted from Arthrospira species and PHB were
analyzed after sulphuric acid digestion. In the present study, pH 9.0 Arthrospira species showed
maximum intensity measured in terms of absorbance (Table 1, 2). The zarrouk’s media having
Nacl displays high absorbance measurment at 235nm using spectrophotometer when compared
with media having Na acetate at different PH and concentration. Absorbance decreased with
increase in Nacl concentrations 1.5g/l at PH 9 and highest measurement observed at media
having Na acetate when the concentration was 1g/l at PH 10 zarrouk’s media. Addition of low
concentrations of external organic carbon enhances PHA production by cyanobacteria. This
result was similar with the works with freshwater strains that showed enhanced PHA
accumulation under salinity stress (Mendhulkar and Shetye, 2017; Shrivastav et al., 2010).

Table 1. % PHB using Na acetate nutrient

No nutrient concentration PH dry weight PHB (g) %PHB Absorbance

1 Na acetate 0.5g/l 8 0.002 0.005 250 0.823
2 Na acetate 0.5g/l 9 0.02 0.01 50 0.946
3 Na acetate 0.5g/l 10 0.002 0.004 200 0.906
4 Na acetate 1g/l 8 0.005 0.012 240 0.640
5 Na acetate 1g/l 9 0.006 0.024 400 0.735
6 Na acetate 1g/l 10 0.014 0.017 121.4 0.414
 7 Na acetate 1.5g/l 8 0.012 0.106 883.3 0.864
8 Na acetate 1.5g/l 9 0.012 0.092 766.6 1.000
9 Na acetate 1.5g/l 10 0.001 0.014 1400 0.960

Table 2. % PHB using Nacl nutrient

No nutrient concentration Ph dry mass PHB (g) %PHB Absorbance

1 Nacl 0.5g/l 8 0.011 0.104 945.4 1.722
2 Nacl 0.5g/l 9 0.014 0.084 600 1.881
3 Nacl 0.5g/l 10 0.011 0.022 200 1.699
4 Nacl 1g/l 8 0.043 0.051 118.6 1.667
5 Nacl 1g/l 9 0.014 0.031 221.4 1.643
6 Nacl 1g/l 10 0.025 0.035 140 1.606
7 Nacl 1.5g/l 8 0.022 0.017 77.2 1.520
8 Nacl 1.5g/l 9 0.005 0.031 620 1.469
9 Nacl 1.5g/l 10 0.027 0.043 159.2 1.503
Fatty acid composition analysis
Fifteen fatty acids were separated and identified from Spirulina samples using GC-MS/ FID
methods. FA serve as storage materials in cells; and FA derivatives are involved in cell signaling.
In the present study essential fatty acid that the human body cannot synthesize were detected
(palmic acid, gama linolenate, oleic acid, steric acid, eicostatrienoic acid). GLA (omega-6 fatty
acids) play essential roles in the function of all cells in the body. When the body converts, it
produces substances with anti-inflammatory and anticancer effects. Most of the fatty acids
detected from the study have important roles in maintaining cellular function, supporting heart
health, reducing inflammation, and promoting overall well-being.

Table 4. GC-MS qualitative compound report

Fig 3. Base Peak Chromatogram

Conclusion
The species showed potential as PHB-producing cyanobacteria, with positive UV-
spectrophotometer results at 235nm using concentrated sulfuric acid as a blank. Arthrospira
demonstrated PHB accumulation at alkaline pH levels (8-10) with increased Na acetate
concentration (1.5g/l) and reduced NaCl concentrations (0.5g/l). Essential fatty acids were
detected, offering various health benefits.

Refernces

Ansari, S. and Fatma, T. (2016). Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and
characterization. PLoS One, 11(6), e0158168.
Damessa, F. (2021). Nutritional and functional values of microalgae (spirulina) naturally found in East Africa. NM-
AIST,
Hegazi, N., Khattab, A.R., Saad, H.H., Abib, B. and Farag, M.A. (2024). A multiplex metabolomic approach for
quality control of Spirulina supplement and its allied microalgae (Amphora & Chlorella) assisted by
chemometrics and molecular networking. Sci Rep, 14(1), 2809. doi:10.1038/s41598-024-53219-5
Lloyd, C., Tan, K.H., Lim, K.L., Valu, V.G., Fun, S.M.Y., Chye, T.R., Mak, H.M., Sim, W.X., Musa, S.L., Ng,
J.J.Q., Bte Nordin, N.S., Bte Md Aidzil, N., Eng, Z.Y.W., Manickavasagam, P. and New, J.Y. (2021).
Identification of microalgae cultured in Bold’s Basal medium from freshwater samples, from a high-rise
city. Scientific Reports, 11(1), 4474. doi:10.1038/s41598-021-84112-0
Nowicka-Krawczyk, P., Muhlsteinova, R. and Hauer, T. (2019). Detailed characterization of the Arthrospira type
species separating commercially grown taxa into the new genus Limnospira (Cyanobacteria). Scientific
Reports, 9, 694. doi:10.1038/s41598-018-36831-0
Piccolo, A. (2012). Spirulina—A Livelihood and a Business Venture. REPORT/RAPPORT: SF//16.
Rajasekaran, C., Ajeesh, C.M., Balaji, S., Shalini, M., Ramamoorthy, S., Ranjan, D., Fulzele, D.P., Kalaivani,
T.J.W.J.o.S. and Technology. (2016). Effect of modified Zarrouk’s medium on growth of different
Spirulina strains. 13(1), 67-75.
Serediak, N. and Huynh, M.-L. (2011). Algae identification lab guide: accompanying manual to the algae
identification field guide: Agriculture and Agri-Food Canada.
Simpore, J., Zongo, F., Kabore, F., Dansou, D., Bere, A., Nikiema, J.-B., Pignatelli, S., Biondi, D.M., Ruberto, G.,
Musumeci, S. and metabolism. (2005). Nutrition rehabilitation of HIV-infected and HIV-negative
undernourished children utilizing spirulina. Annals of nutrition, 49(6), 373-380.