ORIGINAL RESEARCH
Metabolomics and Nutritional Profiling
of Azolla microphylla: Insights from Amino
Acid Quantification and GC-MS Analysis
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1
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan
2
Institute of Pharmacology and Toxicology, University of Veterinary and Animal Sciences, Lahore, Pakistan
3
Dr. Punjwani Centre for Molecular Medicine and Drug Research (PCMD), International Center for Chemical
and Biological Sciences, University of Karachi, Pakistan
Submission date: 2025-12-05
Final revision date: 2026-05-21
Acceptance date: 2026-08-25
Online publication date: 2026-10-08
Corresponding author
Rahat Naseer
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Outfall Road, 54000, Lahore, Pakistan
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ABSTRACT
Azolla microphylla, a free-floating nitrogen-fixing aquatic fern, has emerged as a promising
sustainable bioresource owing to its rapid biomass accumulation, high protein content, and rich
phytochemical composition. The present study investigated the seasonal growth dynamics, biomass
productivity, nutritional composition, amino acid profile, mineral content, and phytochemical
constituents of A. microphylla cultivated under controlled pond conditions across three climatic
quarters over one annual cycle. Species authentication through molecular barcoding confirmed 99%
homology with Azolla microphylla. Biomass productivity exhibited significant seasonal variation
(p<0.05), with maximum wet biomass yield (40,584.25±2,093.37 kg ha⁻¹) and dry matter yield
(8,116.85±418.60 kg ha⁻¹) recorded during Q1 (May-August), characterized by elevated temperature,
humidity, and rainfall, whereas reduced yields were observed during Q2 (September-December) under
comparatively cooler and drier environmental conditions. Despite fluctuations in biomass production,
proximate analysis demonstrated substantial nutritional stability throughout the year, with crude protein
content consistently maintained at approximately 30%, indicating resilience of nitrogen assimilation
and protein biosynthesis under varying climatic conditions. Amino acid profiling revealed significant
seasonal modulation (p<0.05), with elevated concentrations of leucine, lysine, glutamic acid, glycine,
threonine, and isoleucine during Q2, while arginine and methionine reached maximum levels during
Q1. Mineral analysis confirmed the presence of appreciable concentrations of calcium, phosphorus,
potassium, magnesium, sodium, and zinc, supporting the nutritional suitability of the biomass for
feed applications. GC–MS analysis of the n-hexane extract identified twelve major phytochemical
constituents, predominantly fatty acids, phytosterols, and long-chain hydrocarbons, including
stigmasterol (42.30%), phytol (23.79%), n-hexadecanoic acid (12.35%), oleic acid, campesterol, and gamma-sitosterol, compounds previously associated with antimicrobial, antioxidant, antiinflammatory,
and pharmacological activities. Collectively, the findings indicate that optimum
cultivation of A. microphylla is achieved at temperatures ranging from 29 to 33ºC and pH 6.3-6.8,
while its stable nutritional quality and diverse bioactive profile establish this species as a sustainable
alternative feed resource and a potential reservoir of industrially and pharmacologically valuable
metabolites.
CONFLICT OF 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.
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