Characterization and in vitro antibacterial activity of sulfated polysaccharides from freshwater alga Cladophora crispata

Graphical Abstract Sulfated polysaccharides (SPs) from Cladophora crispata were extracted by ultrasonic bath: purify the crude SPs. Compounds of SPs were identified, and we investigated the effect of SPs as antibacterial agents.


INTRODUCTION
Green algae (Chlorophyta) are the primary producers in aquatic ecosystems, with an estimated count of 6000 to 8000 species. Most of these are macroalgae, while others are microalgae such as Cladophora (Ulvophyceae, Cladophorales) [1]. These are multinucleate filamentous algae, and their cells contain a parietal perforate or reticulated chloroplast. The presence of pigments such as chlorophyll (a and b), xanthophylls and β-carotene give it its bright green colour. Cladophora is predominantly benthic [2], and it is usually found in the region of unidirectional flow or periodic wave action in fresh water and marine habitats. Cladophora is a mid to late successional species [1,2].
Algae can be cultivated in open and closed ponds, and photobioreactors for biomass production [3]. Microbial polysaccharides are of great biotechnological and commercial interest, and have a wide application in the food, cosmetics and medical industries due to their emulsifying, thickening, flocculating, stabilizing, anti-oxidizing and antimicrobial properties. It is much easier and advantageous to use them because of the short life cycle of the microbes that allows quick production under controlled environmental conditions. Polysaccharides have been extracted from fungi, bacteria and yeasts [4][5][6], and they are the most important products of algae, representing 38 to 54 % of algal dry weight [7,8]. They show important biochemical properties, which require further investigation to understand their structure and biochemical functions. Several studies have reported the antibacterial, antifungal and antiviral properties of sulfated polysaccharides (SPs) [9][10][11], as well as their scavenging activity against superoxide, hydroxyl, DPPH (1,2-diphenylpicrylhydrazyl) and ABTS radicals [2,2′-azino-bis (3ethylbenzothiazoline-6-sulfonicacid) diammonium salt] [12, 13], and they have commercial applications in food industries [14]. Moreover, the SPs from Cladophora oligoclada have been reported to have anticoagulant properties [15]. Polysaccharides from green algae are heteropolysaccharides that are composed of different monosaccharides [10,16].
Algae polysaccharides can be extracted by several green techniques such as microwave, enzyme and ultrasonic-assisted extraction (UAE). UAE reduces energy consumption and solvent use. In addition, the low temperatures and short times used in UAE processes can maintain the functionalities of the bioactive compounds [12,17,18]. The objectives for this study were (a) to isolate and cultivate Cladophora crispata to understand its growth dynamics, and (b) to study the chemical and functional properties of the crude and purified SPs produced by this species.

Isolation and biomass production of algae
Algae samples were collected from the Barada River, Rabweh, North-West of Damascus city, Syria ( Fig. 1), on 20 April 2021. Samples were kept in 5 l polyethylene bottles at 4 °C until they were shipped to the laboratory (Plant Biology Department, Faculty of Science, Damascus University, Syria), where they were washed with distilled water several times before identification; Cladophora cells were collected by grabber. Morphological diagnosis was performed as described by Prescott [19] by examination under a light microscope (Olympus CX41).
The identified isolates were purified by repeated cultivation in algae culture broth (Fluka medium; macronutrients NaNO 3 , KH 2 PO 4 , alkaline EDTA, acidified iron solution, boron, trace metals solution) as indicated by West [20]. A single-batch system was used for this purpose, where 10 g algae was inoculated into 100 ml medium in a 250 ml conical flask, and incubated for 14 days at 23 °C and pH 8. Cultures were aerated by aerating pumps, and illuminated by fluorescent tubes with an intensity of 1800 lux, and a light/dark cycle of 16/8 h. Algae were transferred for outdoor cultivation for biomass production during the summer using open ponds with control of the nitrogen-to-phosphorus ratio [21], as shown in Table 1; a DR 2800 Lange spectrophotometer was used to estimate these elements in the water.
Algal growth rate was estimated by the method of Stein [22] based on measuring the optical density by spectrophotometer (UV/ vis spectrophotometer model Optizen 2120 UV plus) at a wavelength of 650 nm. The algae biomass was isolated from the culture medium by centrifugation method at a speed of 3000 r.p.m. for 30 min, for the purpose of disassembling the strands into cells, sediment was discarded and the supernatant liquid was taken for measurement. Culture medium was used as the control solution, and the growth rate was calculated and expressed as the specific growth rate. Generation time was calculated according to the two equations developed by Huang and Wang [23]: K = log ODT − log OD0 T X3.322 G = 0.301 K K is the specific growth rate (cells h −1 ); ODT, optical density at the end of the experiment; OD0, optical density at the start of the experiment; T, time of the experiment; G, generation time (h).
Biomass was harvested at the stationary phase (where optical density was constant for a period of time then declined).

Fourier transform infrared spectroscopy (FT-IR)
FT-IR was used to recognize the functional groups in purified SPs. Each fraction (2 mg) from polysaccharide was dried in an oven at 40 °C for 12 h, and then mixed with potassium bromide (KBr) powder. FT-IR spectra were measured in the frequency range of 400 to 4000 cm −1 using a FT-IR-106 4200 type A-C077661018 instrument [12].

Determination of SP components
The phenol/sulfuric acid method was used to determine the carbohydrate content of each fraction of SPs The monosaccharides were selected by high performance liquid chromatography (HPLC) according to the assay of Xu et al.
[30], with some modifications; the temperatures of the column and refractive index detector (RID) were set at 30 °C, the mobile phase was composed of acetonitrile and deionized water (85:15, v/v), flow rate was 1.0 ml min −1 and the injection volume was 20 µl. A standard solution (Sigma-Aldrich) was injected into the chromatography equipment at the concentration range of 0.5-50 mg in 1 ml deionized water. All samples were diluted with deionized water and filtered through 0.45 µm nylon filters prior to HPLC analysis. The experiments were repeated three times. The column was washed with the mobile phase at the end of each experiment period for more than 20 min.

Antibacterial activity of SPs
Analysis of the antimicrobial activity of crude SP and its fractions was carried out against both Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative bacteria (Enterobacter aerogenes and Pseudomonas aeruginosa), which were obtained from the Microbiology and Algae Laboratory, Damascus University, where they were identified according to Bergey's manual [31]. The strains were grown on nutrient agar for 24 h at 37 °C, a part of each bacterial inoculum was then taken into a sterile tube containing 5 ml nutrient broth and incubated for 4 h, with shaking at 100 r.p.m. at 37 °C [32].

Preparation of inoculum
The turbidity of bacterial suspension was adjusted to 0.5 McFarland scale (at OD 600 ) using sterile physiological saline solution, equal to 1×10 8 c.f.u. ml −1 using the turbidity method by McFarland assay; followed by dilution to a concentration of 10 5 c.f.u. ml −1 after that. The bacterial suspension was then inoculated onto Mueller-Hinton agar (MHA) plates [33].

Antibacterial activity assay
Antibacterial activity was determined for each fraction. Firstly, 50 µl standardized inoculum of the bacterial suspension (10 5 c.f.u. ml −1 ) was inoculated onto the MHA surface. Then, wells were made in the MHA medium using a drill bit no. 3, where the agar plugs were removed with a sterile needle [34]. A known concentration of each fraction from SPs (20, 30, 40, 50 mg ml −1 ) was inoculated into the wells. The positive control was ceftriaxone (at a concentration of 0.45%), while distilled water was the negative control. Then, the agar plates were incubated at 37 °C for 24 h, and the bacterial sensitivity was recorded.
Inhibition measurement was carried out using a ruler, as the diameter of the zone sizes to the nearest millimetre [33].

In vitro minimum inhibitory concentration (MIC) determination
The MIC of SPs on the bacterial growth was determined by the agar dilution method. The SPs were dissolved in MHA medium, and serial dilution of SPs were prepared (concentration of SP solutions ranged from 0.5 to 50 mg ml −1 ). SP solutions were placed in Petri dishes and exposed to drying; there were also Petri dishes without SP solutions. Fresh bacterial suspension (10 5 c.f.u. ml −1 ) was used (1 µl each microbial strain was added to each Petri dish), and placed in an incubator at 37 °C for 24 h. The experiments were performed in triplicate [35].

Statistical analysis
All experiments were performed in triplicate, and experimental data are represented by the mean value ±sd of each sample. Statistical analyses were done by IBM spss version 20 using one-or three-way ANOVA at a level of significance of 0.01.

Algae species description and biomass production
Freshwater green macroalga C. crispata is multicellular, filamentous, truly branched, with cell patterns that are cylindrical, and the diameter of the head branches is about 50-70 μm, while ranging between 20 and 35 μm in the lateral branches, as seen in Fig. 2. The same microscopy observations have been reported by others [36]. Algae structure and a large amount of biomass production are related to habitat conditions. Depth of water, total dissolved salts, orthophosphate, nitrate chloride and Chlorophyll-a pigment content in water are key parameters to ubiquitous algae colonies [37].
In conditions such as a water temperature at 26 °C, weather temperature at 35 °C, an illumination duration of 14.30 h, the intensity of illumination 790 lux and N:P ratio 3:1, the specific growth rate was 0.3 cells h −1 and the generation time 0.97 h. So, our results indicate that the algae biomass was exposed to a higher temperature causing stress to the algae, this led to an increase in the carbohydrate content within their biomass; as carbohydrate synthesis requires less energy, carbohydrates were synthesized before lipids in a rapid response to environmental stress [38]. The synthesis of carbohydrates was affected by nitrogen concentration. The highest carbohydrate content (75.23%) was obtained in the culture supplemented with a higher nitrogen concentration at high temperatures, and these results were consistent with similar research [39]. A culture that was P limited (low P) led to the accumulation of carbohydrate content, this is in agreement with other work [40].

Purification of crude SP from C. crispata
A sequential extraction method was used after sonication to extract a maximum amount of SPs. The SPs yield was 7.14 %, which consisted of 74.12 % carbohydrate and 9.08 % sulfate group. Purification of SPs from C. crispata gave purified SPs that were well characterized (high carbohydrate and low protein contents), crude SPs were purified by gel chromatography, and fractions of SPs were selected based on the total carbohydrate elution profile (Fig. 3). The crude SP extract gave two fractions, this result is in agreement with other studies [12, 25]: F 1 (fraction numbers 10-17) and F 2 (fraction numbers 22-27).

FT-IR spectra
The infrared spectra of each of the two fractions obtained from the purification of crude polysaccharides are shown in Fig. 4

Chemical contents of purified SPs
The chemistry of the contents of crude extract and fractions F 1 and F 2 is shown in Table 2; we noted the carbohydrate content was associated with protein content (r=0.73; R 2 =0.54) but not for the sulfate (r=−0.39; R 2 =0.15) and uronic acids (r=−0.99; R 2 =0.99). Carbohydrate, protein, sulfate groups and uronic acids contents for crude extract and fractions are not the same. A protein is contamination of cell wall SPs, because protein is a part of the structure of cell walls and was closely associated with polysaccharides. All SP extracts were contaminated with protein because of the ester sulfate moieties, which can form strong anions and attract positively charged proteins [43], after purification, the high sulfate groups are tightly linked to the SP chains [44].

Monosaccharide composition
The monosaccharides in the crude extract and fractions of SPs extracted from C. crispata were: rhamnose, galactose, xylose and ribose, based on the HPLC analysis (Fig. 5); this result is in agreement with other research [36]. The polysaccharide from green algae is ulvan. Ulvan is a heteropolysaccharide; and the composition is rhamnose, xylose, sulfate groups and uronic acids, such as iduronic acid or glucuronic acid. The composition of ulvan depends on the processing procedures of the biomass, algae species and eco-physiology. Ulvan has three types based on linked rhamnose to uronic acids; nuclear magnetic resonance (NMR) spectroscopy has been used for analysis, and other monosaccharides were reported (e.g. galactose, glucose, arabinose and mannose). This structure is common in marine and freshwater algae [9, 43,45].

Antibacterial activities
The block randomization method was used in this research, not only Gram-positive but also Gram-negative bacteria strains were inhibited by purified SPs, and the antibacterial activity (inhibition zone) of the purified fractions of SPs increased with their increasing concentration, as shown in Table 3. S. aureus was the most sensitive to this SP, with an inhibition zone ranging from 7 to 27 mm. These results were similar to those in other reports [33]. The positive control had an effect in all bacterial strains, but not the negative control, as shown in Fig. 6.  The antibacterial activity of algae extracts against Enterobacter sp. was classified as resistant or active. The purified ulvan from the green alga Ulva reticulata also had an antibacterial activity with an inhibition zone diameter of about 20 mm [45]. S. aureus and B. anthracis bacteria are more sensitive to antibacterial agents than E. aerogenes and P. aeruginosa, as a result of having the additional protection afforded by an outer membrane, such as lipopolysaccharide and phospholipids [46]. According to other studies, the ester sulfate of polysaccharides is related to their biological activity, such as antibacterial activities and preventing preformed biofilms [47,48]. S. aureus had the lowest MIC (6 mg l −1 ) when we used F 2 ; while P. aeruginosa had the highest MIC (32 mg ml −1 ) when we used F 1 (Table 4). Our results suggested that the SPs might be able to activate intestinal epithelial cells to produce cell-mediated immune response cytokines that initiate and amplify protective immune responses of the host [33].  Table 3. Antibacterial activity of the purified fractions of SPs from C. crispata A, B, C, D, E, F, G, H, the same alphabetical capital letters in the same column indicate no significant differences at 1%.

Conclusions
C. crispata is simple to classify because it has regular branches, and its growth in the outdoor ponds systems is an important feature for this species, because of the cheap equipment required for this purpose. Cladophora biomass is an unlimited, easily cultivated low-cost and valuable resource for different applications. Ultrasonic waves were considered a useful method for extracting SPs from C. crispata, with the following conditions: ratio of 30:1 ml g −1 , 60 °C, 120 min.
SPs from C. crispata are heteropolysaccharides, both F 1 and F 2 from purified SPs have the same monosaccharides, consisting of carbohydrate, protein, sulfate groups and uronic acids based on FT-IR analysis. It could be interesting to better purify the SP fractions by using ion-exchange resins to eliminate the presence of proteins. Purified SPs exhibit antibacterial activity because of their viscosity, water-solubility, hydroxyl and sulfate group. Mechanisms of antibacterial action for SPs are due to glycoprotein receptors present on the cell surface of polysaccharides that bind with compounds in the bacterial cell wall, cytoplasmic membrane and DNA, and cause increased permeability of the cytoplasmic membrane, protein leakage and binding of bacterial DNA. The antibacterial activity of SPs suggested possible use within the food industry, animal diets and pharmaceutical industries. We advise investigation of the antioxidant properties for SPs as further research, because the ester sulfate in SPs can donate an electron, which can give it antioxidant properties.

Funding information
This research is part of a Ph.D. thesis. This work was supported by Damascus University, Syria, which supplied the raw materials and laboratory equipment. The authors received no specific grant from any funding agency.

Letter for reviewers
Dear Professor I did all the required modifications from reviewers,Editor and Editorial Office requirements briefly: 1. I have corrected the grammatical , typographical, and formatting errors were corrected.
2. Abbreviations were defined when they are first appearance.
3. I added graphical abstract with legend needed in my article.
4. I provide more detail in the Introduction and Methods Section.

1.In the Abstract Section:
I have corrected the grammatical , typographical, and formatting errors were corrected had been emerged(line 37 )=>have been emerged

In the Introduction Section:
I have corrected the grammatical errors: İt is multinucleate filamentous algae. its cells contain a parietal perforate or reticulate chloroplast. The presence of pigments such as chlorophyll (a and b), xanthophylls, and β-carotene give it its bright green color(line 67-70)=>İt is multinucleate filamentous algae, its cells contain a parietal perforate or reticulated chloroplast. The presence of pigments such as chlorophyll (a and b), xanthophylls and β-carotene, give it its bright green color quick production under controlled environmental conditions.polysaccharides were extracted from fungi, bacterial and yeasts [4][5][6], and from algae; polysaccharides are the most important products of algae(line78-79 )=>quick production under controlled environmental conditions. Polysaccharides were extracted from fungi, bacteria and yeasts [4][5][6], and they are the most important products of algae, which represent 38 % to 54 % of algal dry weight [7-8],

in the Results andDiscussionSection:
I have corrected the grammatical , typographical, and formatting errors

In section Algae species Description and biomass production:
the diameter about 50 -70 micrometers, while the lateral branches are small diameter ranged(line229-231 )=>the diameter of the head branches is about 50 -70micrometers, while ranged between 20 -35 micrometers in the lateral branches, as seen in Figure  2, same microscopic observation were found with [36].

in section Purification of Crude Sulfated polysaccharide from Cladophoracrispata:
Purification of SPs from Cladophoracrispata gave purified SPs with well-characterized (high carbohydrate and low protein contents), we purified the crude SPs by gel chromatography, fractions of SPs were selected based on the total carbohydrate elution profile  It was very difficult to follow as no line numbering was used and the language made it hard and this affected the reviewing process as elaborated by Reviewer 1. Although the author made the required revision requested by the 2 reviewers, and to help guide that revision, I've added a few notes on the revised manuscript and highlighted points that seem to warrant special attention. I still think there is a need to improve the language for effective communication of your science (e.g. better figures) and throughout the manuscript, it is difficult to follow where a sentence starts and stops and where the next one starts. Throughout the manuscript, there is a need for better sentence construction such as proper tense namely at: Line 3: "had been emerged" Line 68:"or reticulatechloroplast. " Line 69:"carotene give it its " Line 78-79: It appears something is missing from this sentence., so please check "quick production under controlled environmental conditions polysaccharides were extracted from fungi, bacterial and yeasts [4][5][6], and from algae; polysaccharides are the most important Line 142:"modifications, Briefly; Dry powder " Line 158 (disolved -> dissolved). This also contained a hyperlink as it this was copy-pasted from a website. Line 194-105:"they were identified it's according to Bergey's manual" Line 205: "Antimicrobial activity determined for each fraction" Line 227: Spss Line 233: "branches are small the diameter ranged". Something is missing here. Line 238: "illumination duration of 14.30 hours." Is this correct? Line 256-258: Please rewrite this. Line 272: "it indicates to present uronic acids. " Please rewrite. Line 289: Please rewrite "proteins [41], after purification, the high sulfate groups are tightly linked to the SPs chains [43]." Line 321" Please write proper tense "spectroscopy had used for this " Line 324: Rewrite "polysaccharide; inhibit " Line 364: Rewrite "The ultrasonic waves consider a useful " Line 374:"which caused increased " Section 3.2 is irrelevant and can be merged with another section. The language is also confusing. Please clarify. Figure 1 needs to be of better quality with a proper legend. Table 1 needs to be designed better so as to make it easy understand what the control and distil water are for. Table 2 top legend: "Uroinc acids -> uronic"

Anonymous.
Date report received: 18 April 2023 Recommendation: Minor Amendment

Comments:
The corresponding author responded to reviewer comments well. But I think that the resubmitted manuscript is not revised.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data? Partially support

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
Author response to reviewers to Version 2

Letter for reviewers
Dear Professor a.maybe it is the most ubiquitous freshwater macroalgae worldwide . Barada River is one of the most important aqueous habitat for algae in Syria.
b.In this study, UAE was used to extract SPs from Cladophora crispata that had been isolated from the Barada River and grown outdoors. We have further investigated the chemical composition, and in vitro antibacterial activity of crude and purified SPs.
I added and show important biochemical properties which require thorough investigations to understand their structure and biochemical functions I replace there is increasing concern in studying the structure and biochemical functions of polysaccharides' -by several studies have reported antimicrobial properties of sulfated polysaccharides I added The objectives for this study were a) to isolate and cultivate Cladophora crispata to understand its growth dynamics b)to study the chemical and functional properties of the crude and purified SPs produced by this species GPS of sampling site was provided , the distance between river and lab is 10 km, so condition during storageisn't needed.
Cladophoracells was Collected by grabber I added table 1.treatments of outdoors cultivation for biomass production, and we used we used DR 2800-lange to estimate elements (N,P)in the water The pure ethanol (99.9%).
Sephadex gel G-100 gel(Sigma, Japan); the column distance 2.6 × 20 cm I added more details of Xu's assay I did not study the effect sulfated polysaccharides on Ecoli and Bacillus subtilis because it was studied previously McFarland work at OD= 600 nm I made a photopanel.

Reviewer 1
I correct the typographical, and formatting errorsas: Carbohydrates => Carbohydrate in Table 1 "Inhabit zone" => "Inhibition zone in Table 2 I correct the formatting errors of the reference as: The I added an explanation for peak 1 (2.800) and peak 2 (3.517); the peak 1 (2.800) and peak 2 (3.517) are mobile phase.

Reviewer 2
well-characterized(high carbohydrate and low protein contents), ' and add -and show important biochemical properties which require thorough investigations to understand their structure and biochemical functions 74 -'there is increasing concern in studying the structure and biochemical functions of polysaccharides' -several studies have reported antimicrobial properties of sulfated polysaccharides 79 -for e.g. polysaccharides from green algae.... 85 -'In this study' …. The objectives for this study were a) to isolate and cultivate Cladophora crispata to understand its growth dynamics b)to study the chemical and functional properties of the crude and purified SPs produced by this species 91 -which month? Saying 2021 spring is not enough 92 -GPS coordinates of sampling sites are needed. Please provide study site map as Fig. 1  Comments: The manuscript has been revised well, including the corrections of grammatical errors. However, additional errors were found in revised sentences. My concerns are shown below. Specific comments: There are a lot of typos. Examples are below. Line 45: "crud" => "crude" Line 64: "amultinucleate" => "multinucleate" Line 97: "Kawach" => "Kawachi" Line 106: "Song" => "Song et al." Lines 123-125: "PBS saline" => "PBS" Line 173: "Cefteriaxons" => "Ceftriaxons" Line 186: "2.9." => "2.8." Table 2: "Inhabit zone" => "Inhibition zone". "samalphabetical" is a typo? Regarding format errors, incorrect use of capitalization is found in many places. Some examples are shown below. Line 38: "sulfated Polysaccharides" Line 45: "infrared Spectroscopy" Line 47: "High performance" Additionally, it would be nice to define what authors are omitting. Line 76: It would be better to spell out "DPPH" and "ABTS". Lines 132-133: KBr powder => Potassium bromide (KBr) powder Line 148: "RID" might be spelled out. Lines 48-51: In the abstract, MIC values do not correspond to results shown in Fig. 5. The data will need to be checked again. It would be good to indicate whether each bacterium is gram-positive or gram-negative. It makes the third sentence in the highlights section easier to understand. Lines 72-74: Yeasts also belong to fungi. It would be better to explain why polysaccharides are the most important products. "There are increasing interests" might be better than "there is increasing concern". Lines 97-99: It would be good to explain the composition of fermentation medium.  [43] does not include the description of the ulvan. Should be confirmed references. Figure 4: It would be nice to add an explanation for peak 1 (2.800) and peak 2 (3.517). Table 1: "Total" for "Carbohydrates" is needed? Total Carbohydrates => Carbohydrate I do not understand the meaning of the same alphabetical letters. It would be good to discuss the results in the text.

Please rate the manuscript for methodological rigour Satisfactory
Please rate the quality of the presentation and structure of the manuscript Satisfactory To what extent are the conclusions supported by the data? Partially support filamentous algae. It's cells contain a parietal perforate or reticulatechloroplast. The presence of pigments such as chlorophyll (a and b), xanthophylls,and β-carotene give it its bright green color.Cladophorais predominantly benthic, and is usually found in the region of unidirectional flow or periodic wave action in freshwater and marine habitats.Cladophorais a mid to late successional species, maybe it is the most ubiquitous freshwater macroalgae worldwide [1,2] We add new sentences in line numbers no 70.
Barada River is one of the most important aqueous habitat for algae in Syria.
I have corrected the grammatical errors in line numbers no 71.
Algae can be cultivatedin open and closed ponds.
I have corrected the grammatical, typographical, and formatting errors there is increasing concern in line numbers no73.
as well as their scavenging activity against in line numbers no76.
However, polysaccharides characteristics in line numbers no 79.
Polysaccharides from green algae are heteropolysaccharides which are composed of different monosaccharides in line numbers from 80-81.
can be extracted by several green in line numbers no 82.
UAE reduce energy consumption and solvent use. In addition, the low temperatures and short times used in UAE processes can keep the functionalities of the bioactive compounds in line numbers from 83-85.
We add new sentences in line numbers from 85-88.
In this study, UAE was used to extract SPs from Cladophoracrispatathat had been isolated from the Barada River and grown outdoors. We have further investigated the chemical composition, and in vitro antibacterial activity of crude and purified SPs.

3.In the Materials and Methods Section:
I have corrected the grammatical(in red color), typographical, and formatting errors(sentences highlighted in yellow), and both errors (red color andsentences highlighted in yellow).
Isolation and biomass production of algae in Line numbers no 90.
Algae samples were in line numbers no 91.
Samples were kept in 5-L polyethylene bottles at 4 °C until they were shipped to the in Line numbers no 92.
where they were in line numbers no 94.
was performed in line numbers from 95.
The identified isolates were purified by repeated cultivation on algae culture broth (Fluka medium, India) as indicated by Andersen and Kawach [20]. The single-batch system was used for this purpose, where 10 g of the algae were inoculated into 100 ml of the fermentation medium in 250-mL conical flask, and incubated for 14 days at 23 °C and pH 8. Cultures were aerated by aerating pumps, and illuminated by fluorescent tubes with a light intensity of 1800 Lux, and a light/dark cycle of 16/8 h. Algae were transferred for outdoors cultivation for biomass production during summer using open ponds with controlling the nitrogen-tophosphorus ratio [21]. The biomass was harvested at the stationary phase in line numbers from 95-103.
Polysaccharides were extracted using UAE technique as described by Esmaeili et al [22], followed by the sequential extraction according to the method of Song [11] with some modifications. Dry powder of the algae was mixed with distilled water with the ratio of30:1)ml: g), and the mixture was put in an ultrasonic bath (Ultrasonic cleaner model:ps-60ar, 360 W,40 kHz frequencies, jeken, China) for 120 min at 60 °C. Algae biomass was separated from the extract by centrifugation at 5000 rpm for 10 min. Polysaccharides in the supernatants was precipitated by the addition of pure ethanol which volume is three times that of extract in line numbers from 105-111.
Lipids were removed by the addition of three volumes of absolute chloroform and absolute acetone mixture (3:1). The residual proteins were removed by the addition of Sevage reagent (1:4 v/v mixtures of n-butanol and chloroform) and centrifugation at 4000 rpm for 15 min, and the precipitate was discarded in line numbers from 113-116.
where 0.5 g of the crude SPs was dissolved in line numbers no 121-122.
The eluted fractions were collected with the volume of 700 μL in line numbers from 125-126.
The SPs were then crystallized by incubation in line numbers no 128.
FT-IR was used to recognize the functional groups in purified SPs. Each fraction (2 mg) from polysaccharide was dried in an oven at 40 °C for 12 h, and then mixed with KBr powder in line numbers from 132-133.
Protein content was determined according to the method of Lowry in line numbers no 139.
30 mg from each sample (crude SPs, F1, and F2) was in line numbers no 142. and the mixture then was diluted by the addition of 8.4 mL of distilled water and incubated at 121 °C for 1 h in line numbers from 143-144.
The monosaccharides were in line numbers no 146.
with some modifications; the temperatures of the column and RID were set at 30°C, The mobile phase was composed of acetonitrile and deionized water (85:15, v / v), and the flow rate was 1.0 mL/min, and the injection volume was 20 μL. A standard solution (Sigma-Aldrich) was injected into the chromatographic equipment at the concentration range of 0.5-50 mg in 1 mL of deionized water. All samples were diluted with deionized water and filtered through 0.45μm Nylon filters prior to HPLC-analysis. The experiments were repeated three times. Column was washed with the mobile phase at the end of each experiment period for more than 20 min in line numbers from 147-154.
against both gram-positive in line numbers no 157.
where they were identified it's in line numbers no 159.
The strains were grown in line numbers no 160.
with shaking in line numbers no 162.
The turbidity of bacterial suspension in line numbers no 164.
suspension was then inoculated in line numbers no 167.
50 μl of the standardized inoculum of the bacterial suspension (10 5 cfu/ml) in line numbers from 169-170.
agar plugs were removed in line numbers no 171.
A known concentration of each fraction from SPs (20-50 mg/ mL) was inoculated into the wells in line numbers from 172-173.
bacterial growth was in Line numbers no 179.
concentration of SPs solutions range from 0.5 to 50 mg/mL (1 μl of each microbial strain was added to each wells in Petri dish) in line numbers from 181-182.

in the Results andDiscussionSection:
I have corrected the grammatical(in red color), typographical, and formatting errors(sentences highlighted in yellow), and both errors (red color andsentences highlighted in yellow).
Abbreviations were defined when they are first appearance( in blood).
Algae structure in line numbers no 196.   A protein is contamination in line numbers no 246.

Morphology of
was closely associated in line numbers no 247.