INTRODUCTION
Freshwater macroalgae and microalgae are among the most essential primary producers in the biosphere. They are recognized as commercially important, living, and renewable resources. These algae contain over sixty trace elements, including vital minerals, proteins, iodine, bromine, and a wide array of bioactive compounds¹. As a highly diverse group of organisms, freshwater algae have the capacity to produce an extensive variety of chemical substances. The declining effectiveness of antibiotics—regardless of their mechanism of action—highlights the urgent need for effective alternative treatments. The widespread and increasing use of antibiotics and chemotherapeutic agents in disease control has contributed to the rise of drug-resistant strains and has had negative impacts on ecosystems².
Numerous chemically distinct compounds with notable biological activities have been extracted from both macroalgae and microalgae³. Some of these compounds are currently under research, while others are being developed into new pharmaceutical agents.
The rising scientific and commercial interest in the exploitation of genetic resources has become a significant issue for international policy discussions. Freshwater algae are notable for their unique and intriguing biochemical properties, which are fundamental to their antibacterial and antifungal efficacy⁴. In addition, many studies have reported the in vitro antibacterial and antifungal activities of freshwater algal extracts⁵. These extracts, including their active constituents, have shown activity against both Gram-positive and Gram-negative bacteria. As such, algal extracts present immense therapeutic potential as antibacterial agents, offering effective treatment options with fewer side effects compared to conventional synthetic drugs.
MATERIALS AND METHODS
Preparation of Algal Extracts
The algal biomass that had been shade-dried was weighed and then ground into a fine powder using a mortar and pestle. Then, ten grams of the powdered algae were extracted using a variety of solvents, including acetone, methanol, ethanol and distilled water. Each solvent extraction materials were soaked in 100 milliliters of the respective solvent for 48 hours at room temperature, with intermittent shaking to ensure maximum extraction of bioactive compounds. The mixtures were filtered through Whatman filter paper after extraction to provide clean filtrates. Through the use of a rotating evaporator, these filters were concentrated at lower pressure and stowed at low Temperature in airtight container for study of antibacterial assays.
ANTIBACTERIAL ASSAY
Test of Micro Organisms
Three bacterial strains were used to assess the algal extracts’ antibacterial efficacy and they are Serratia marcescens (ATCC 13880), Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). These strains were obtained from the MTCC, ensuring the use of standardized reference strains.
Preparation of Inoculum
To prepare the bacterial inoculum, a loopful of each microorganism from a Nutrient broth (pH 7.4) was used to inoculate a 24-hour-old nutrient agar slant, which was then incubated for 24 hours at 37°C. This development period allowed the bacteria to reach the exponential (log) phase of growth, ensuring a high density of viable cells6. The cultures’ optical density (OD) was calibrated to the 0.5 McFarland standard in order to standardize the inoculum density for the antibacterial laboratory experiment. Uninoculated nutrient broth served as a negative control throughout the experiments.
Antimicrobial Activity Assessment Using the Disc Diffusion Method
Preparation of Agar Plates
The agar well diffusion method was utilized to assess the antibacterial activity of the fresh water algal extracts. The pH of the produced MHA medium was adjusted to 7.4. After that, medium was sterilized by autoclaved for 15 minutes at 121°C and 15 pressure. After autoclaving, Twenty milliliters of the sterilized MHA were transferred into aseptic Petri dishes and left to harden at room temperature7.
Inoculation of Test Microorganisms
The test microorganisms, which included S. aureus, S. marcescens, and E. coli, were cultivated in nutritional broth and incubated for a whole day at 37°C.Once incubated, sterile cotton swabs were used to evenly spread the bacterial suspensions onto the surface of the solidified MHA plates. Each bacterial strain was inoculated on separate MHA dish. To make sure the bacterial inoculum was absorbed into the agar, the infected dish was let to stand for a short while8.
Well Preparation and Application of Algal extracts
Sterile cork borers were used to punch wells of 5 mm diameter into the centre of the inoculated MHA plates. Each well was carefully loaded with fifteen microliter of the respective fresh water algal biomass extract. Than extracts were allowed to diffuse into the agar at room temperature for 30 minutes. Control plates, with wells containing no extracts, were also prepared to compare and ensure the validity of the results by excluding any potential contamination.
Incubation and Measurement of Inhibition Zones
The plates loaded with algal extracts were incubated at 37°C for whole day. Post incubation, the appearance of antibacterial activity was showed by clear zones of inhibition surrounding the wells. These zones were calculated in millimetres, together of diameter of the well, using a calibrated ruler. The diameters of the inhibition zones were recorded to evaluate the antibacterial efficacy of the algal extracts against the test microorganisms.
FTIR Spectroscopy Methodology
The functional groups found in the algal extracts of Cladophora crispata and Chara fibrosa were described using FTIR spectroscopy. The algal biomass was initially shade-dried and Using an agate mortar and pestle, grind into a fine powder to avoid contamination. To make a homogenous mixture, 100 milligrams of dry KBr was mixed with around 10 milligrams of the powdered sample. This mixture was then compressed into a thin, transparent pellet using a hydraulic press under a pressure of 10 tons for about 5 minutes. The prepared KBr pellets were immediately analyzed using an FTIR spectrometer, scanning in the spectral range of 4000-400 cm⁻¹ with a resolution of 4 cm⁻¹. The prepared KBr pellets were immediately analyzed using an FTIR spectrometer, scanning in 4 cm⁻¹ resolution in the 4000-400 cm⁻¹ spectral range. The resulting spectra point recorded and analyzed to identify the specifical absorption bands corresponding to various element found in the algal extracts, providing insight into their chemical composition and potential bioactive constituents.
Qualitative Phytochemical Analysis (FTIR)
The qualitative phytochemical analysis of Chara fibrosa and Cladophora crispata extracts was conducted using FTIR spectroscopy to detect the element present in the extracts. FTIR is a powerful analytical technique used to determine the types of chemical bonds (functional groups) within a compound based on the specific frequencies of light absorbed by these bonds.
The FTIR spectra of the algal extracts revealed distinct absorption bands, each corresponding to different functional groups. Characteristic absorption peaks were identified and annotated on the spectrum, allowing for the determination of specific chemical bonds present in the algal extracts9. The presence of broad absorption bands around 3400 cm⁻¹ indicated O-H stretching vibrations, suggestive of hydroxyl groups, commonly found in phenolic compounds. Peaks observed near 2920 cm⁻¹ and 2850 cm⁻¹ were ascribe to C-H stretching vibrations, indicative of aliphatic chains. Some absorption bands around 1650 cm⁻¹ was found C=C stretching vibrations, typical of alkenes or aromatic rings. Additionally, the presence of peaks near 1100 cm⁻¹ and 1050 cm⁻¹ suggested C-N and C-O stretching vibrations, consequently, indicating the existence of ether, or amine groups. Peaks in the region of 650-500 cm⁻¹ were attributed to C-Br stretching vibrations, suggesting halogenated compounds.
The detailed results of the functional group analysis for Chara fibrosa and Cladophora crispata are presented in Table 1, highlighting the diverse range of bioactive compounds present in these algal species. This comprehensive identification of functional groups underscores the potential of these algae as sources of novel bioactive compounds for pharmaceutical applications.
FTIR spectroscopy analysis of Chara fibrosa extract revealed a variety of functional groups and compound classes, each contributing to the chemical profile of the extract. The spectrum exhibited a C=C bending vibration at 713.66 cm⁻¹, indicating the presence of alkene groups. Fluoro compounds were identified through a C-F stretching peak at 1008.77 cm⁻¹, confirming their presence in the extract. Additionally, amines were detected with C-N
stretching vibrations at 1024.2 cm⁻¹ and 1244.09 cm⁻¹, suggesting that multiple amine groups are present.
Figure 1: FTIR Spectra of Algal Extracts from Chara fibrosa.
The extract also contained sulfoxides, as evidenced by an S=O stretching peak at 1037.7 cm⁻¹. Aliphatic ethers were identified through a C-O stretching peak at 1147.65 cm⁻¹. Carboxylic acids were confirmed by O-H bowing down to 1433.11 cm⁻¹ and O-H stretching at 3273.2 cm⁻¹. Alkane groups were detected with C-H bending vibrations at 1444.68 cm⁻¹ and 1454.33 cm⁻¹, and a C-H stretching peak at 2848.86 cm⁻¹.
Table 1: Range of major FTIR band assignments reported for Chara fibrosa analysis
| S. No. | Wavenumber range | Assignments (stretches/bends) | Functional Group | Comments |
| 1 | 713.66 | C = C | Alkene | Alkene |
| 2 | 1008.77 | C – F | Fluoro compound | Fluoro compound |
| 3 | 1024.2 | C – N | Amine | Amine |
| 4 | 1037.7 | S = O | Sulfoxide | Sulfoxide |
| 5 | 1147.65 | C – O | Aliphatic ether | Aliphatic ether |
| 6 | 1244.09 | C – N | Amine | Amine |
| 7 | 1433.11 | O – H | Carboxylic acid | Carboxylic acid |
| 8 | 1444.68 | C – H | Alkane | Alkane |
| 9 | 1454.33 | C – H | Alkane | Alkane |
| 10 | 1795.73 | C = O | Conjugated acid halide | Conjugated acid halide |
| 11 | 2848.86 | C – H | Alkane | Alkane |
| 12 | 3273.2 | O – H | Carboxylic acid | Carboxylic acid |
Furthermore, a C=O stretching peak at 1795.73 cm⁻¹ indicated the presence of conjugated acid halides. These results highlight the diverse chemical composition of the Cladophora crispata extract, reflecting a range of functional groups that may contribute to its biological activity and potential applications in various fields.
The FTIR analysis of Cladophora crispata extract, as detailed in Table 2, identified a range of functional groups and compound classes with distinct absorption peaks. Notably, the extract displayed multiple peaks corresponding to halo compounds, with strong C-Br stretching vibrations observed at 526.57, 555.5, 615.29, and 667.37 cm⁻¹. These peaks suggest the presence of brominated halo compounds in the extract. Additionally, the spectrum revealed an alkene group, characterized by a C=C bending vibration at 713.66 cm⁻¹. Fluoro compounds were identified with a C-F stretching peak at 1008.77 cm⁻¹, although no antibacterial activity was associated with this group. The analysis also identified several amine groups, with C-N stretching vibrations observed at 1022.27, 1035.77, 1076.28, and 1242.16 cm⁻¹. This indicates the presence of various amine derivatives, including aromatic amines, which were identified with a C-N stretching vibration at 1336.67 cm⁻¹.
Figure 2: FTIR Spectrum of Cladophora crispata Extract
Functional groups associated with alcohols and esters were also present. Secondary alcohols were indicated by a C-O stretching peak at 1112.93 cm⁻¹, while tertiary alcohols were detected at 1161.15 cm⁻¹. An ester group was identified by a C-O stretching peak at 1207.44 cm⁻¹. An O-H bending peak at 1382.86 cm⁻¹ indicated the presence of phenol and carboxylic acids were detected with O-H stretching at 3269.34 cm⁻¹ and O-H bending at 1433.11 cm⁻¹
Table 2: Range of major FTIR band assignments reported for Cladophora crispata analysis
| S. No. | Wavenumber range | Assignments (stretches/bends) | Functional Group | Comments |
| 1 | 526.57 | C – Br | Halo compound | Halo compound |
| 2 | 713.66 | C = C | Alkene | Alkene |
| 3 | 1008.77 | C – F | Fluoro compound | Fluoro compound |
| 4 | 1022.27 | C – N | Amine | Amine |
| 5 | 1112.93 | C – O | Secondary alcohol | Secondary alcohol |
| 6 | 1161.15 | C – O | Tertiary alcohol | Tertiary alcohol |
| 7 | 1207.44 | C – O | Ester | Ester |
| 8 | 1242.16 | C – N | Amine | Amine |
| 9 | 1336.67 | C – N | Aromatic amine | Aromatic amine |
| 10 | 1382.86 | O – H | Phenol | Phenol |
| 11 | 1433.11 | O – H | Carboxylic acid | Carboxylic acid |
| 12 | 2852.72 | C – H | Aldehyde | Aldehyde |
| 13 | 3269.34 | O – H | Carboxylic acid | Carboxylic acid |
Furthermore, the extract contained aldehyde groups, indicated by C-H stretching at 2852.72 cm⁻¹. Overall, the FTIR results demonstrate a diverse range of functional groups in Cladophora crispata extract, which may contribute to its bioactivity and potential applications.
RESULTS AND DISCUSSION
The agar well diffusion method was used to assess the antibacterial activity of the extracts of Chara fibrosa and Cladophora crispata. Table 3 provides an overview of the outcomes, which presents the zone of inhibition measured against three bacterial strains: Escherichia coli, Serratia marcescens, and Staphylococcus aureus.
As demonstrated in Table 3, both Chara fibrosa and Cladophora crispata extracts exhibited Serratia marcescens, Staphylococcus aureus and E. coli of the antibacterial action. They aqueous extracts showed varying degrees of inhibition. Specifically, Chara fibrosa yielded a zone of inhibition of 12 millimeter against Escherichia coli, while Cladophora crispata produced a slightly larger zone of 15 mm. For Serratia marcescens, Chara fibrosa exhibited a 12 mm inhibition zone, whereas Cladophora crispata demonstrated a more substantial inhibition with a zone measuring 17 mm.
Table 3: Data of antibacterial activity of algal extracts
| S.No. | Name of Sample | Concentration | Test Microorganism | Zone of Inhibition |
| 1 | Chara fibrosa | 15 µl | E. coli | 12 mm |
| 2 | Cladophora crispata | 15 µl | E. coli | 15 mm |
| 3 | Chara fibrosa | 15 µl | Serratia marcescens | 12 mm |
| 4 | Cladophora crispata | 15 µl | Serratia marcescens | 17 mm |
| 5 | Chara fibrosa | 15 µl | Staphylococcus aureus | No zone observed |
| 6 | Cladophora crispata | 15 µl | Staphylococcus aureus | No zone observed |
Neither Chara fibrosa nor Cladophora crispata showed any antibacterial activity against Staphylococcus aureus, as no inhibition zones were observed for this bacterial strain.
In conclusion, the extracts from both algal species effectively inhibited the growth of E. coli and Serratia marcescens, with Cladophora crispata showing superior activity compared to Chara fibrosa. The results suggest a higher antibacterial efficacy of both algal extracts against Serratia marcescens compared to E. coli, indicating that Serratia marcescens is more susceptible to the antibacterial effects of these algae.
Figure 3: Comparative antibacterial activity of Chara fibrosa and Cladophora crispata extracts against Escherichia coli, Serratia marcescens and Staphylococcus aureus
CONCLUSION
This research provides a preliminary investigation into the antibacterial potential of the freshwater microalgae Chara fibrosa and Cladophora crispata, assessing their efficacy against prominent bacterial pathogens including Escherichia coli, Serratia marcescens, and Staphylococcus aureus. The antibacterial activity of the algal extracts was assessed using the agar well diffusion method in this investigation.
which demonstrated notable effectiveness against E. coli and S. marcescens, but no activity against S. aureus.The results highlight the significant antibacterial properties of these microalgae, attributable to the presence of bioactive compounds such as phenolics, amines, aromatic cyclic compounds, and halo-compounds. These compounds were identified through FTIR analysis, revealing diverse functional groups that potentially contribute to the observed antimicrobial activity. Specifically, Cladophora crispata exhibited superior antibacterial activity compared to Chara fibrosa, particularly against S. marcescens, indicating a higher efficacy of the former in inhibiting bacterial growth. This study underscores the potential of utilizing natural phyco compounds from these algal species in the drug discovery process. The promising antibacterial activity observed suggests that Chara fibrosa and Cladophora crispata could serve as valuable sources of novel antimicrobial agents. Future studies will concentrate on doing additional pharmacological assessments, such as in silico analyses to evaluate the active compounds’ features related to absorption, metabolism, excretion, and ADMET, as well as molecular docking studies to explore interactions between these non-toxic compounds and target proteins. Comprehensive assessments like this will open the door for the creation of new, effective antimicrobial drugs derived from these algal resources, potentially contributing to more sustainable and eco-friendly alternatives in drug development.
ACKNOWLEDGEMENT
I am grateful to my Department of Chemistry and Cosmetics, Jeju National University, Jeju 63243, Republic of Korea, for providing me platform to carry out this work.
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