Molecular Identification of the Iraqi Aquatic Fern Azolla pinnata and Evaluation of its Efficiency in Reducing CO₂ under Various Environmental Conditions

Azolla is an aquatic fern under the family Azollaceae which can easily be grown in a variety of water types including fresh water of lakes, ponds, swampy lands, rice paddies and slow moving waterways. It is described by the fact that it develops on water with the formation of small flat structure akin to leaves floating on the water surface. Azolla is a heterosporous fern and is mutualistic with bacteria related to cyanobacteria, nitrogen-fixing bacteria that dwell within the dorsal cavities of Azolla’s fronds (Rames, 2019). It is characterized by its ability to float on the water’s surface, forming small, flat, leaf-like structures that rest atop the water. Azolla is classified as a heterosporous fern and establishes a symbiotic relationship with nitrogen-fixing bacteria (cyanobacteria), which reside within the dorsal cavities of its fronds (Rames, 2019). Tropical and subtropical environments are suitable for the growth and reproduction of Azolla, however, it is distributed almost all over the region of North and South, America, Indian continent, African countries and Australia as mentioned by Korsa et al., (2024). From among such varieties of Azolla, the most common are Azolla pinnata, Azolla nilotica and Azolla mexicana. Among them, Azolla pinnata has been considered the most important biofertilizer due to its varieties, short Generation time and requirement of nutrients (Nasir et al., 2022).
       
Azolla is considered a promising application of biotechnology that can be scientifically utilized to mitigate environmental pollution. Its rapid growth rate and low production cost make it highly efficient, with the ability to double its biomass within a period of 3 to 6 days (Rehman et al., 2022). Due to its symbiotic relationship with the nitrogen-fixing cyanobacterium Anabaena azollae, Azolla efficiently captures CO₂ and converts it into organic matter through rapid growth. Studies indicate that Azolla can sequester up to 1.1 tons of CO₂ per hectare annually, making it a promising tool for climate change mitigation. Additionally, its high photosynthetic rate and ability to thrive in aquatic environments enhance its potential for bioremediation and sustainable agriculture (Wagner, 1997; Brouwer et al., 2018).
       
The photosynthetic rates obtained in different experiments vary widely, probably due to differences in species or strain, biomass per unit area and other conditions of experimentation. Reported photosynthetic rates for A. caroliniana ranged from 40 to 123, µmoles CO₂ mg^-1 chlorophyll  hr^-1. Net photosynthesis of A. pinnata varied from 2.0 to 12 ,µmol CO₂ m^-2 S^-l (Daniel and Bartholomew, 1993). By reducing carbon dioxide emissions, Azolla plays a crucial role in mitigating air pollution and enhancing overall environmental quality (Hamdan and Hour, 2022). Additionally, Azolla serves as a valuable dietary supplement for various livestock species due to its rich content of essential vitamins and minerals, providing vital nutrition for animals (Ahmed et al., 2016; Bhatt et al., 2020).
       
In many developed and developing countries, Azolla is used as a substitute for chemical and nitrogen-based fertilizers, owing to its symbiotic relationship with nitrogen-fixing bacteria. This unique trait has positioned Azolla as a crucial component in agricultural practices (Chandrababu and Parvathy, 2025). Moreover, Azolla biomass can be used as a biofertilizer to increase the organic content of certain soil types, contributing to improved soil quality and enhanced agricultural productivity. This application also reduces agricultural costs and alleviates the need for the production and use of organic fertilizers, which may have negative environmental impacts due to their high nitrogen content. Additionally, Azolla’s ability to absorb nitrogen from the atmosphere further strengthens its role in promoting environmental sustainability and minimizing the negative impacts of agriculture (Baviskar et al., 2023). Carbon dioxide levels in the atmosphere can be reduced by absorbing large amounts through major ecosystems such as forests, which play a crucial role in lowering emissions. Additionally, several strategies can be employed to mitigate elevated carbon dioxide levels, including controlling and reducing emissions from fossil fuels, or removing carbon dioxide from the atmosphere through various methods known as carbon sequestration. This process encompasses both biological and non-biological techniques (Ussiri and Lair, 2017). Among the biological methods, Azolla, in its various species, plays a significant role in regulating the Earth’s climate by absorbing large amounts of atmospheric carbon. Azolla is characterized by its rapid growth and ability to double its biomass within a short period, ranging from five to six days, offering a substantial opportunity to sequester some of the carbon dioxide emitted into the atmosphere. It is noted that the flourishing of Azolla in ancient times significantly contributed to the reduction of carbon dioxide levels, making it an effective tool for capturing large quantities of carbon and lowering its concentration in the environment (Kosesakal and Yildiz, 2019). The biomass produced by Azolla can subsequently be stored to sequester carbon and remove it from the active carbon cycle. Studies have indicated that 1 hectare of Azolla can capture approximately 21,266 kg of carbon dioxide. Research also shows that carbon dioxide emissions have slowed in their growth since the early 21^st century (Hamdan and Hour, 2022).
               
Climate change is an environmental global event that has a great impact on the ecosystem and human health. Arguably one of the main drivers of climate change through carbon dioxide (CO₂) emissions with the removal or reduction in these CO₂ emissions using absorption of excess CO₂ being a vital solution to climate change. So it is highly required, you find other alternatives to remove CO₂ and Azolla offers one, various environmental conditions are key factors influencing Azolla’s growth. Typically, rapid growth is associated with Azolla’s production of carbon dioxide. Therefore, the current study aims to assess the impact of different light intensities, temperature levels and exposure durations on the effectiveness of Azolla in reducing CO₂ emissions.

This study was conducted at the College of Agriculture - Tikrit University - Iraq, during 2022-2024.
 
Isolation  of Azolla pinnata
 
A. pinnata was collected from the Tigris River-IRAQ , It was diagnosed phenotypically to the genus level by the herbarium of the College of Agriculture, Tikrit University
 
Molecular identification of   A. pinnata
 
For DNA extraction 0.5 g of  fresh, young leaves of for A. pinnata were extracted following the protocol of the Genomic DNA mini kit (plant) extraction kit provided  from Geneaid. The entire ITS region (ITS1, 5.8S gene and ITS2) was amplified via polymerase chain reaction (PCR) using primers ITS1 and ITS4 (White et al., 1990). The mixture of reaction (25 μL) consisted of the following components: 1.5 µl DNA, 5 µl Taq PCR PreMix, forward and reverse primers each at a concentration of 10 pc/ml (1 µl) then make up to 25 µl with distilled water. PCR system consisted of a total of 37 cycles, with each round including the following:

1. Initial denaturation at 95^oC for 5 minutes-one cycle.
2. Denaturing at 95^oC for 45 seconds + annealing at 58^oC for 45 seconds + first extension at 72^oC for 45 seconds-35  cycles.
3. Final extension at 72^oC for 7 minutes- one cycle (Hassan and Al-Qiassi, 2022).
       
Gene amplification was performed using a thermocycler (Applied Biosystem Gene-amp PCR System 9700). After amplification, PCR products were analyzed by electrophoresis using a 1.5% agarose gel, then the bands were visualized under ultraviolet (UV) light at a wavelength of 302 nm after treating with dye (Intron Korea red stain). Nucleotide sequences of genes were determined in the Korean company Biotechnology Lab using (Applied Biosystem 3730XL, DNA Sequencer). The results were compared to the database at the National Center for Biotechnology Information (NCBI) using a specialized local alignment search tool (BLAST) to align with nucleotide sequences of the targeted gene from plant isolates in this study and identify their species based on matches in the aforementioned database. After completing the diagnosis of Azolla spp., the identified species was recorded in NCBI and a phylogenetic tree was constructed using the Maximum Likelihood method via MEGA 11 software (Kumar et al., 2018).
 
Nutrient culture medium and growth conditions
 
60 ×  40 × 40 cm ) length × width × depth (of plastic container was used for growth of A. pinnata,  the container was supplied by  Espinas and Watanabe’s medium (EWM), consisted of macronutrients  included; CaCl₂ (40 ppm), KH₂PO₄ (20 ppm), MgSO₄ (40 ppm) and KCl (40 ppm) and micronutrients MnSO₄ (0.5 ppm), Na₂MoO₄ (0.15 ppm), H₃BO₃ (0.2 ppm), ZnSO₄ (0.01 ppm), CuSO₄ (0.01 ppm) and Ferric citrate (2 ppm) the pH was adjusted at 6.5 (Dawar and Singh, 2002). 10 g fresh wt. of washed A. pinnata plants were taken as initial inoculum.
       
The containers were provide with white UV-Free Sun Lamp ( verilux light bulbs) United States Digital lux metrer and Air quality detector (RoSH model JSM-131-China) for measurement of CO₂ (Fig 1).


 
Effect of temperature on the CO₂ level
 
The containers contained nutrient liquid medium separately incubated at 10, 20,30 and 40C for 10 days with 0 (dark) -10000 lux. CO₂ was measured at interval 2 days for 10 days.
 
Determination of growth parameters
 
According to (Brouwer et al., 2018), the absolute growth rate (AGR) was determined as (dry matter weight (g)/m²/day).
 

y_t =  Standing A. pinnata crop = AGR.t+b

 
b = Standing crop at the start of the linear growth (g/m²). 
t = Time (days).
 
Statistical analysis
 
Factorial experiments were carried out in this study and analysis of variance was conducted using the program (SPSS). The means were compared according to Duncan’s multiple range test at a level of significance of 0.05 (Al-Rawi and Khalaf Allah, 1980).

Molecular identification of Azolla pinnata
 
Azolla pinnata was molecularly identified using ITS within 5.8S rRNA gene, Azolla pinnata coded Azolla pinnata strain IRAQ-Tikrit.Has,A.A. registered in NCBI under the accession number PQ836460.1, the phylogentic tree on Fig 2 showed the Azolla pinnata strain IRAQ-Tikrit.Has,A.A. was the nucleotide sequence of the ITS region of Azolla was most identical to the American Azolla isolate (Azolla pinnata subsp. pinnata voucher Ap7, accession number  JN604551.1) with a match percentage of 99.27. E-value and per cent identity were zero and more than 90% with all Azolla pinnata  registered in NCBI, while they were more than zero and less than 80% with other Azolla spp. like Azolla nilotica, Azolla mexicana, Azolla caroliniana, Azolla microphylla, Azolla cristata and  Azolla rubra. These results confirm that the precise classification of Azolla isolated for the first time from the Iraqi environment belongs to the Azolla pinnata.


 
Effect of temperature and Light Intensity on CO₂ Concentration for 10 days growth of A. pinnata
 
Fig 3 shows the effect of temperature and light intensity on CO₂ concentration  produced from A. pinnata in the culture medium at a depth of 20 cm for 10 days. The results showed no significant differences in CO₂ concentrations during incubation in darkness for 10 days, with the highest concentrations recorded ranging from 827.43 to 894.74 ppm across temperatures from 10^oC to 40^oC. In contrast, incubation under light conditions revealed a consistent decrease in CO₂ concentrations with increasing light intensity, reaching the lowest concentration at 10,000 lux., at this intensity, temperatures of 20^oC and 30^oC had a significant effect in reducing CO₂ levels, with the lowest values recorded at 375.14 and 362.38 ppm, respectively, compared to 416.81 and 410.25 ppm at 10^oC and 40^oC.


       
When comparing the percentages of CO₂ reduction, it is noted from Fig 4 that the highest percentage of CO₂ reduction was when A. pinnata grew at temperatures of 20 and 30^oC, while the lowest percentage of CO₂ reduction was when growing at temperatures of 10 and 40^oC. On the other hand, the same figure shows that the highest percentage of reduction was when A. pinnata grew at a light intensity of 10,000 lux compared to other light intensities.


 
Effect of nutrient medium depth on CO₂ concentration and growth parameters of A. pinnata
 
The effect of the depth of the nutrient medium on the concentration of CO₂ gas and growth indicators of A. pinnata. Table 1 shows the effect of the depth of the liquid nutrient medium on the growth of A. pinnata and its effect on the concentrations of CO₂ gas. The results showed that there was an increase in the growth of A. pinnata with an increase in the depth of the liquid nutrient medium up to 50 cm, as the value of the growth indicators AGR and yt reached 10.53 g/m²/day and 106.42 g/m² with a decrease in CO₂ gas to 325.64 ppm, with no significant effects on the growth indicators and gas concentrations when growing at depths of 60-80 cm.


       
When Azolla was incubated under dark conditions, an increase in carbon dioxide (CO₂) levels was observed throughout the experiment period (ten days) across all studied temperatures. This can be explained firstly by the non-photosynthetic character of A. pinnata in conditions of the darkness and secondly, by other physiological processes in Azolla’s metabolism, predominantly respiratory, involved in the increases of CO₂ amount. Conversely, under light conditions, the results showed a significant and continuous decrease in CO₂ levels with increasing temperature and light intensity up to 10,000 lux throughout the incubation period (ten days).
               
This continuous decrease in carbon dioxide may be attributed to the photosynthesis process, as previous studies have proven an increase in carbon dioxide retention from the atmosphere with the increase in the biomass of Azolla, which results in an increase in the photosynthesis process, which works to convert carbon dioxide into glucose and water with the release of oxygen in the presence of light. Therefore, there is a direct relationship between the rates of photosynthesis of Azolla and the increase in CO₂ retention in the atmosphere, which made Azolla An important vital factor in combating global warming and controlling climate change, in addition to its effective role in improving the quality of the water in it and the sustainability of biodiversity (Hamdan and Hour, 2022). The results showed that the best vegetative growth of Azolla was recorded at temperatures of 20-30^oC, as slow growth of Azolla was observed at temperatures below 20^oC, while the effect of temperatures above 30°C is negative on the vegetative growth of Azolla in the presence of light. The results of our study agreed with Cheng et al., (2010), De et al., (2015) and Hossain et al., (2021), who stated that the optimum temperatures for Azolla growth are in the range of 20-30^oC and that temperatures above 30^oC negatively affect the growth of Azolla, as previous studies recorded the death of Azolla at a temperature of 45^oC (Pereira and Carrapico, 2009). Photosynthesis occurring in the optimal conditions (20-30^oC) for the growth of Azolla is the main activity that works to sequester carbon dioxide by increasing the biomass of the plant (Pereira and Carrapico, 2009; Hamdan and Hour, 2022; Sarkar et al., 2023). A significant increase in carbon dioxide consumption was recorded with increasing light intensity during the incubation period (ten days). A significant decrease in CO₂ levels was observed with increasing light intensity up to 10,000 lux. This is in agreement with Hossain et al., (2021) and Sara et al., (2023), who recorded a decrease in CO₂ levels with increasing light intensity levels between 8,000-10,000 lux. The importance of light intensity lies in its direct effect on the photosynthesis process of Azolla and the process of building carbohydrates that are important in plant metabolism, in addition to Its direct effect on the symbiotic relationship between Azolla and nitrogen-fixing bacteria, which leads to an increase in the vegetative growth of Azolla plants, which is noticeable at radiation intensity ranging between 5000-10000 lux. This is what our study proved, which recorded the highest reduction in CO₂ levels at light intensity of 8000-10000 lux, while recording the best vegetative growth of Azolla plants at these ranges. The depth of the medium represents the nutritional content available for the growth of the Azolla plant. The higher the nutritional content, the more we expect the growth to increase and thus the biomass of the Azolla plant to increase, which is reflected in an increase in the photosynthesis process and an increase in the consumption of carbon dioxide. The results showed that the best depth of the medium was 50 cm, which recorded the best vegetative growth of Azolla, in addition to recording the highest percentage of decrease in CO₂ levels, while depths greater than 50 cm did not affect the vegetative growth of Azolla negatively or positively or on the consumption of carbon dioxide, which is consistent with the results of Sadeghi et al., (2012).

The results  from this study refer to  the promising potential of the fern plant Azolla pinnata in reducing CO₂. In the case of indoor cultivation of Azolla, the growth of A. pinnata can increase, producing high biomass while consuming high levels of CO₂ if it is incubated at a light intensity of 8000-10000 lux, at temperatures of 20-30^oC and at a depth of 50 cm. In the future studies, it is planned to implement this study in large basins on a large scale in geographical areas suffering from high levels of carbon dioxide, especially in industrial areas and we will monitor the levels of this gas throughout the year.

The present study was supported by College of Agriculture, Tikrit University, Iraq.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The manuscript does not contain animal experiments.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.