1. Professor, Northern Christian College, Inc. Laoag City, Philippines.

*             Correspondence: admirantejosue@gmail.com

Keywords: bioremediation, decomposed, mangrove leaves

1. INTRODUCTION

The unique physio-chemical properties of the marine environment confer marine organisms with physiological adaptations which could be exploited for biotechnological applications. In the mangrove environment, fallen leaves are colonized and decomposed by microorganisms [1,2]. Research shows that the undecomposed leaves are poor in nutrients, and they become nutritious due to the microbial enrichment process during decomposition process. Microorganisms play an important ecological role in decomposing organic matter and producing protein-rich detritus that serves as food to fishes especially in detritus-based marine ecosystems like mangroves [3,4]. Bacteria are particularly important in the marine environment as decomposers of dead organic substrates. The dead organic matter and the associated microorganisms form the base of the food webs of commercially important fishes and crustaceans [5,6]. Bacteria occurring in decomposing plant organic material or detritus like that of decomposing mangrove leaves have been shown to be source of enzymes that are of importance in industry and bioremediation [7,8]. An author was able to isolate two genera of total heterotrophic bacteria (THB), four species of Lactobacillus, two species of Azotobacter, two species of Actinobacteria, three isolates of fungus, four species of yeasts, two species of Thraustochytrids and four species of Trichoderma from decomposing mangrove leaves and these microorganisms’ exhibited capability of producing extracellular enzymes like proteases, chitinases, lipases, amylases and cellulases [8]. The microbial decomposition of mangrove litter has been extensively studied. However, very little information is available about the industrial and bioremediation potential of microbial enzymes produced during the mangrove litter decomposition [9,10]. Bacteria are minute organisms but are of great importance if they are properly utilized. They are not just agents in causing disease but are widely used in industries as well [11]. Results of the study, therefore, could serve as a stepping stone in the discovery of halophilic bacteria, a microorganism that could produce enzymes for industrial and biotechnological applications. The results could also provide a vital reference resource for microbial researchers and other specialists on the importance of biological control strategic value of microbial isolates in bioremediation and improvement, thereby contributing to social and environmental benefits especially in regions with contaminated lands and waters [12,13]. The study was conducted at the Molecular Microbiology and Biotechnology Laboratory, College of Arts and Sciences, Mariano Marcos State University, City of Batac. In general, the study aimed to isolate, characterize and determine the industrial and bioremediation potential of bacterial isolates from decomposing mangrove leaves from Naga Bungan Bay, Pasuquin, Ilocos Norte [14]. Specifically, it aimed to: describe the physio-chemical characteristics of the collection site in terms of: temperature, pH, and salinity; isolate marine bacteria from decomposing mangrove leaves in the area; characterize the bacterial isolates in terms of their: morphological characteristics: shape (coccus, rod, spiral),colony characteristics (form, elevation, margin, color), and slant growth type; biochemical characteristics: Gram staining affinity, casein/protease hydrolysis test, starch hydrolysis test, lipid hydrolysis test, gelatin hydrolysis test and hemolysin test determine bioremediation potential of the bacterial isolates by determining their tolerance when grown in media inoculated with different concentrations of lead[15].     

2. MATERIALS AND METHODS

Samples were collected from the Mangrove Natural Stand in Naga Bungan Bay, Barangay Davila, Pasquini, I locos Norte. Naga Bungan Bay is located at the northernmost tip of the municipality of Pasquini. Figure 01 shows the locale of the study.

Figure 01: Map of the Collection Site, Naga Bungan Bay, Brgy. Davila, Pasquini, I locos Norte

Senescent (yellow to brown) fallen leaves of mangrove trees were collected at the Mangrove Natural Stand in Naga Bungan Bay, Davila, Pasquini, I locos Norte, 30-40 leaves were collected using sterile forceps. These leaf samples were put into sterile Ziplock plastic bags and taken back to the Molecular and Biotechnology Laboratory in ice chest for isolation of bacteria. Sample collections were done at 6 in the morning. The temperature, salinity, and percentage of Hydrogen or pH of the mangrove area, specifically the seawater, were determined. Thermometer was used in getting the temperature that is measured in degrees Celsius (⁰C), as well as salinometer measured in parts per thousand (ppt) and pH meter was used in the determination of salinity and pH, respectively. Glassware’s and apparatus used in the experiment were washed with antibacterial dishwashing soap and water, and were air dried. After air drying the glassware’s were directed into the oven at 100⁰C for one hour. Marine Agar is prepared by 55 grams of marine agar was weighed and dissolved in one liter of distilled water. The mixture then was heated by constant stirring until solutions became clear. The agar was transferred into sterilized Erlenmeyer flasks, covered with cotton plug and sterilized in an autoclave/sterilizer at 15 psi for 1 hour. Finally, the sterilized MA was allowed to cool and then poured into sterilized petri dishes.  Marine Broth (MB) is prepared by forty grams of marine broth was dissolved in one liter of distilled water, placed in a sterilized Erlenmeyer flasks covered with cotton plug. The medium was heated to facilitate the dissolution of the MB until the solution became clear yellow. MB was sterilized in an autoclave at 121⁰C (15 lbs. pressure) for 15 minutes. The sterilized MB was used in the inoculation of samples. Casein Agar is prepared by forty grams of Casein Agar was dissolved in one liter of distilled water. The solution was heated in moderate flame temperature to allow complete dissolution. The casein agar was sterilized using pressure cooker at 15 psi for 30 minutes and later was poured on petri dishes. Decomposing mangrove leaves were washed with sterile seawater three times with an interval of one minute. The leaves were cut in one centimeter length and were soaked in the freshly prepared test tubes with marine broth. To thoroughly separate the bacteria from the leaves, these were shaken in Vortex for 15 minutes and incubated for 1 ½ hours. These were immediately spread onto sterile marine agar plates using pour plate method and then incubated at 28 to 30⁰C inverted, corresponding to the ambient seawater temperature for 24 to 48 hours. After the period of incubation, the plates were observed for the presence of bacterial colonies. Isolation of the bacterial colonies was done and pure culture was used in the characterization. Pure cultures were obtained by streaking a colony on marine agar plates and subsequently stored as marine agar slant culture. The isolated bacteria were characterized in terms of their morphology and biochemical properties. Shape. Samples from the pure culture were smeared into a clean glass slide. The smears were passed on a flame. A drop of crystal violet was used in staining the sample for one-minute, excess stain was removed with a tap. The smear was attained again with iodine for another minute then was washed with tap water, a drop of 95 % ethyl alcohol was added for 10-20 seconds, rinsed again with water. Drops of safranin were put into the slide for 20 seconds and were washed gently for few seconds and dried. The slides were examined to determine the shapes of the bacteria whether they are coccus, bacillus or spirillum. Characterization of colony. Bacterial colonies in plated sample were characterized according to form, elevation, margin and color of the bacteria. Slant Growth. Colonies were touched by sterile wire loop and streaked to the solidified slant agar aseptically. Colonies were taken from the previous culture. The slants were incubated for 24-48 hours. Slant growth types were observed. Gram staining affinity. A loopful of bacterial sample from the pure culture was used and obtained using a sterilized wire loop and was spread evenly on a clean glass slide. The smear was passed over a flame three to five times. Drops of crystal violet were added over the smear and after one minute, washed again with running water. The smear was stained with iodine and after one minute washed again, then a drop of 95 % ethyl alcohol was added for 10-20 seconds, and then rinsed again with water. The smear was stained again and was washed again with running water after one minute. Microscopic examination was done to determine whether the bacteria were gram positive or gram negative. Casein/Protease hydrolysis test. Forty grams of casein agar were weighed and dissolved for one liter of distilled water. The solution was heated in microwave to allow dissolution and the solution was sterilized in an autoclave for 15 psi for 1 hour. Then ten ml of the sterile solution were poured in each petri dish. Plates were made and inoculated with the isolates. These were incubated overnight and observed for clearing zone. The presence of clearing zone indicated a positive result. Starch hydrolysis test. A total of 0.14 grams of starch agar was weighed and added to 14 grams of agar powder, and this was dissolved with one liter of distilled water. The solution was heated in a microwave to allow dissolution; then this was sterilized in an autoclave at 15 psi for one hour. A total volume of 10 ml of the solution were poured to each petri dish. This was cooled until the prepared media solidifies. The starch agar plates were inoculated with the isolates and incubated at 37⁰C for 24 hours. After incubation, the plates were flooded with iodine. Formations of clearing zone along the growth of the bacteria will be the indication of starch hydrolysis. Lipid Hydrolysis Test. Fourteen grams of agar powder was weighed and dissolved in one liter of distilled water. To allow complete dissolution, the solution was heated in a microwave. The solution was sterilized in an autoclave at 15 psi at one hour. After sterilization, five percent of tween 80 of the total volume of the solution was added, and were mixed. Then ten ml of the solution was poured to each petri dish. After the plates have solidified, these were inoculated with the isolates and incubated for 37⁰C for 24 hours. The production of opalescence precipitate was observed and this was indicative of positive result. Gelatin Hydrolysis Test. A half gram of gelatin powder was weighed and added to 14 grams of agar powder and were dissolved in one liter of distilled water. The solution was heated in microwave to allow dissolution, this was then sterilized in an autoclave, and afterwards, ten ml of the solution was poured in each petri dish. Gelatin plates were made and were inoculated with the isolates. These were incubated for 24 hours and were observed for the presence of clearing zones. Hemolysin Test. One hundred ml of 0.9 % NaCl solution was added to 1.8 g of agar powder and the solution was stocked for 30 minutes and heated at 80⁰C on a water bath with stirring. After which, the solution was cooled at 40⁰C and one ml of blood was added and stirred then ten ml of the solution was poured in each petri dish. Pure culture was streaked over the plates to observe RBC lyses. The plates were incubated for 24-48 hours at 30⁰C. The presence of clearing zone indicated that the organisms were of hemolysis. Range Finder Test for Treatment Formulation. Five (5) treatments in three replicates were used as initial treatments to evaluate bioremediation potential of the bacterial isolates. From these concentrations, the amount was increased until there was no more growth of bacteria observed.

 T1: nutrient agar (10 ml) + 40 ppm PbNO₃ (10 ml)

T2: nutrient agar (10 ml) + 80 ppm PbNO₃ (10 ml)

T3: nutrient agar (10 ml) + 120 ppm PbNO₃ (10 ml)

T4: nutrient agar (10 ml) + 160 ppm PbNO₃ (10 ml)

T5: nutrient agar (10 ml) + 200 ppm PbNO₃ (10 ml)

A loopful of the isolate from the pure cultures was inoculated into each treatment. After one day of incubation, microbial growths were monitored. Tolerance Test. From the initial treatments of the Range Finder Test, the isolates were allowed to grow in nutrient agar with increasing concentration of Lead Nitrate until there was no more growth of bacteria observed. The treatments that were used ranged from 40ppm to 1,500 ppm Lead Nitrate.

3. RESULT & DISCUSSION

To fully understand the physio-chemical of the seawater sample used in the study, its physio-chemical characteristics, specifically its temperature, pH and salinity were measured.

Table 01: Physio-chemical Characteristics of the Seawater of Naga Bungan Bay

Sample	Temperature (0C)	pH	Salinity (ppt)
Seawater from Naga Bungan Bay	30	8.3	34

Table 01 shows the physio-chemical characteristics of the seawater from Naga Bungan Bay, Pasquini, I locos Norte. The temperature of the seawater is 30⁰C; its pH is 8.3, and its salinity measures 34 pp. Temperature affects directly every function of bacteria by controlling the ratio of metabolism. Most organisms function within a temperature range between 0⁰C and 50⁰C. Within this temperature range, organisms have minimum, maximum and optimum requirements for them to perform their functions [16]. Another factor that may affect the bacteria is ph. pH affects the function of the protein and enzyme which are important in the metabolism, growth and development of the bacteria. The pH of the seawater was found to be 8.3, which indicates that it is basic. According to a study moderately halophilic bacteria could also be enriched from seawater by its salinity, therefore the salinity of seawater is determined to know its level for bacterial enrichment [17]. The decomposing mangrove leaves from Naga Bungan Bay was the source of bacterial isolates in this study. Table 2 shows the bacteria that were isolated from decomposing mangrove leaves. The isolates were designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

Table 02: Isolated Bacteria from Decomposing Mangrove Leaves from Naga Bungan Bay

Sampling Area	Isolated Bacteria
Mangrove Area – Naga Bungan Bay	1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

Bacterial isolates were characterized according to their form, elevation, margin, color, and slant growth type. Table 03 shows the morphological characteristics of the bacteria isolated from decomposing mangrove leaves at the mangrove area of Naga Bungan Bay.

Table 03: Morphological Characteristics of the Bacterial Isolates

Isolates	Colony Characteristics:					Slant
	Shape	Form	Elevation	Margin	Color	Growth Type
1	Coccus	Irregular	Raised	Undulate	Red	Filiform
2	Coccus	Circular	Raised	Entire	White	Filiform
3	Coccus	Irregular	Flat	Undulate	Red	Filiform
4	Coccus	Circular	Raised	Entire	Red	Echinulate
5	Coccus	Circular	Flat	Entire	Yellow	Filiform
6	Coccus	Irregular	Flat	Undulate	Violet	Diffuse
7	Coccus	Circular	Raised	Entire	White	Echinulate
8	Coccus	Circular	Flat	Entire	White	Echinulate
9	Coccus	Irregular	Flat	Undulate	Yellow	Beaded
10	Coccus	Circular	Flat	Entire	Yellow	Filiform
11	Coccus	Punctiform	Convex	Entire	White	Echinulate
12	Coccus	Punctiform	Raised	Entire	White	Filiform
13 14	Coccus Rod	Punctiform Rhizoid	Flat Flat	Entire Filamentous	White White	Filiform Filiform

The shape of bacteria determines its ability to get food and attachment. Most of the isolates are cocci except isolate 14 which was observed to be rod in shape. The elevations of the bacterial isolates are mostly flat or raised with only isolate 11 having a convex elevation. Their growth in plate is circular, irregular, or punctiform with isolate 14 being the only rhizoid among all the isolates. The margins of the isolates are mostly undulate and entire with isolate 14 being the only filamentous in margin. In terms of color, isolates 2, 7, 8, 11, 12, 13 and 14 are white color while isolates 5, 9 and 10 are yellow, isolates 1, 3 and 4 are red, and only isolate 6 is violet. In terms of slant growth type, most are filiform (isolates 1, 2, 3, 5, 10, 12, 13 and 14), four are echinulate (isolates 4, 7, 8 and 11), isolate 6 diffused and isolate 9 with beaded slant growth type. Six tests were conducted to characterize the isolates biochemically. Table 04 presents the biochemical characteristics of the bacterial isolates taken from decomposing mangrove leaves at the mangrove area of Naga Bungan Bay.

Table 04: Biochemical Characteristics of the Bacterial Isolates

Isolates	Gram Staining Affinity	Protease Test	Starch Test	Lipid Test	Gelatin Test	Hemolysin Test
1	+	–	–	–	–	–
2	+	–	–	–	–	–
3	+	–	–	–	–	–
4	+	–	+	–	–	–
5	+	–	–	–	–	–
6	+	+	–	–	–	–
7	+	+	+	–	–	+
8	+	–	–	–	–	+
9	+	–	–	–	–	–
10	+	–	–	–	–	–
11	+	–	+	–	–	–
12	+	+	+	–	–	–
13	+	–	+	–	–	–
14	+	+	+	–	–	–

Gram staining test is used to identify two major groups of bacteria based on their different cell wall constituents, the bacteria is either thick layer of peptidoglycan that retain the crystal violet after staining (gram positive, violet/blue) or thinner peptidoglycan which do not retain the crystal violet during decolorizing process (gram negative, pink/red) [18]. The results show that all the bacterial isolates are Gram positive. Figure 02 shows the Gram positive and Gram-negative results.

Figure 02: Grams Staining Affinity of the Different Isolates

 Casein/Protease hydrolysis test is used to determine the proteolytic activity of the isolates. Isolates 6, 7, 12 and 14, were observed to have clearing zones around the growth of the bacteria [19]. This indicates that the four isolates can produce protease and thus are capable of hydrolyzing casein, an important consideration for bacteria that have industrial potential. Figure 03 shows the positive and negative response of isolates to Casein hydrolysis test.

Figure 03: Casein/Protease Hydrolysis Test of the Different Isolates

Figure 04: Starch Hydrolysis Test of the Different Isolates

Table 04 shows that isolates 4, 7, 11, 12, 13 and 14 are the only bacteria that are able to hydrolyze starch as revealed by their positive response to the test. Clearing zone around the bacterial growth in the starch agar plates indicates that the organism can produce amylase and maltase. It could be inferred that these bacteria contain the industrially important enzymes amylase and maltase. Figure 4 shows the positive and negative response of isolates to Starch hydrolysis test. Lipid hydrolysis test is performed to know the ability of the isolates to hydrolyze lipid by the production of an enzyme called lipase [20]. It is shown in Table 04 that all the isolates are negative in the lipid hydrolysis test. This means that no one from among the isolates produces lipase. Figure 05 shows the negative result of isolates to Lipid hydrolysis test.

Figure 05: Negative Result in Lipid Hydrolysis Test of the Different Isolates

Gelatin test was also conducted to know if the isolates produce gelatinase and are able to hydrolyze gelatin. Table 04 also shows that all the isolates showed negative response in the gelatin test which indicates that hydrolysis did not take place and that all the isolates do not produce gelatinase, a very important enzyme necessary for the breakdown of proteins into amino acids. Figure 06 shows the negative result of isolates to Gelatin hydrolysis test.

Figure 06: Negative Result in Gelatin Hydrolysis Test of the Different Isolates

Hemolysin test is a test to know if an organism is able to lyse red blood cells. Isolates 7 and 8 were found to be positive and all the rest are negative. This means that isolates 7 and 8 are putative pathogens and could not be tapped for industrial purposes. All the other isolates, on the other hand, have the potential utilization in the industry.

Figure 07: Hemolysin Test of the Different Isolates

Table 05 reveals the summary of the morphological and biochemical characteristics of the isolated bacteria from decomposing mangrove leaves. As shown in the table, bacterial isolate shapes are mostly cocci, only one (isolate 14) is rod. In gram staining affinity, all isolates are Gram positive. In the Protease test isolates 6, 7, 12 and 14 have a positive result. This indicates that the four isolates are capable of producing proteases and degrade casein into peptones. In starch test, isolates 4, 7, 11, 12, 13 and 14 are positive which means that they produce the enzyme amylase and are capable of hydrolyzing starch into its simple glucose units. They are, therefore, important bacteria with industrial application. In hemolysin test, only isolates 7 and 8 are positive, meaning they are putative pathogens and could not be tapped for industrial purposes. All the other isolates have the potential utilization in the industry as shown by their positive response to the other tests and negative response to the hemolysin test.

Table 05: Summary of the Morphological and Biochemical Characterization of the Bacterial Isolates

Isolates	Shape	Gram Staining Affinity	Positive results in the different tests
1	Coccus	Gram +	–
2	Coccus	Gram +	–
3	Coccus	Gram +	–
4	Coccus	Gram +	S
5	Coccus	Gram +	–
6	Coccus	Gram +	P
7	Coccus	Gram +	P, S, H
8	Coccus	Gram +	H
9	Coccus	Gram +	–
10	Coccus	Gram +	–
11	Coccus	Gram +	S
12	Coccus	Gram +	P, S
13	Coccus	Gram +	S
14	Rod	Gram +	P, S

Legend, Protease test: P                                Starch test: S                      Hemolysin test: H

Bacterial isolates from decomposing mangrove leaves at the mangrove area of Naga Bungan Bay were allowed to grow in growth media inoculated with different Lead (Pb) concentration to determine the point where they can no longer tolerate a certain lead concentration. Table 06 shows the different bacterial isolates with the point where they can no longer tolerate Lead (Pb) in ppm.

Table 06: Bacterial Isolates and their Maximum Range of Lead Tolerance in ppm

Bacterial Isolates	Maximum Range of Lead Tolerance in ppm
1 2 3 4 5 6 7 8 9 10 11 12 13 14	<991 <1,121 <1,121 <1,041 <981 <1,065 <1,081 <501 <1,121 <898 <1,123 <1,111 <1,122 <1,041

The table shows that the tolerance values for lead nitrate in the different bacterial isolates. The bacterial colonies that grew on the agar plates with Lead Nitrate indicate the tolerance of bacteria to the Lead [21]. Almost all of the isolates grew and tolerated high Lead Nitrate (PbNO₃) concentrations of up to 1,000 to 1,123ppm. Their ability to tolerate and survive when grown in as high as 500 ppm Lead Nitrate concentration indicates that they have potential to bioremediate [22].According to DENR Administrative Order no. 34, series of 1990, Class SB Water Bodies (Recreational Water Class I) such as those used for primary contact recreation such as bathing, swimming skin diving and Class SC Water which are intended for Fishery purposes have maximum limits of 0.05 mg/L or 0.05 ppm of Lead [23,24]. Considering the tolerance values obtained for the bacterial isolates which ranged from 40 to 1,123 ppm of lead, the values are far higher than the allowable limit. It is seldom in nature that led contamination reaches that high [25]. The possible mechanisms presented that explains the ability of the bacterial isolates to bioremediate lead contamination is their ability to extract heavy metals and sequester them inside their cell membranes [26]. Their mechanism of metal resistance includes formation of precipitates of the metal in the form of phosphates, carbonates, sulphates and nitrates and become efficient in Lead Bio adsorption.

4. CONCLUSION

Based from the results, it can be concluded that the bacteria isolated in decomposing mangrove leaves in the mangrove natural stand at the Naga Bungan Bay, Pasquini, I locos Norte are distinct. Some of the bacterial isolates are protease and amylase producer. Out of the 14 isolates, 8 were enzyme producers, however 2 of them are putative pathogens, isolate 7 and isolate 8, because they are able to lyse red blood cells and therefore not recommended for industrial purposes. In the determination of bioremediation potential of bacterial isolates, it is concluded that there are 14 bacteria that can be isolated from decomposing mangrove leaves in the mangrove natural stand at the Naga Bungan Bay, Pasquini, I locos Norte which can be tapped as possible phytoremediators to accumulate heavy metals like Lead (Pb) in contaminated lands and waters.            Based from the findings and conclusions, it is recommended that further identification and search for applications of the bacteria be made. Moreover, expression genes involved in the enzyme production be studied for further biotechnological and industrial exploration except for isolate 7 and isolate 8 which are putative pathogens. It is recommended also that further studies should be done to prove the bioremediation potential of the bacterial isolates to other type of heavy metals like mercury (Hg).

REFERENCES

1. Alcamo, P. (1997). Fundamentals of Microbiology, Third edition.
2. (2010). Morphology of Bacteria. Retrieved from http://ygoy.com/2010/10/15/bacteria/
3. Md Arafat Al Ajmir Sarker, Ayon Sen & Amit Barai (2025). Fields of Future: Utilizing Supervised Ml for Barley Yield Prediction. Dinkum Journal of Natural & Scientific Innovations, 4(01):01-10.
4. Blood Agar. (2003). Hemolysis. Retrieved from http://www.enotes.com/blood-agarhemolysis-hemolytic-reactions-reference/blood-agar-hemolysis-hemolytic-reactions­
5. (2012). Bioremediation of Lead by Lead-resistant Microorganisms Isolated from Industrial Sample. Retrieved from http://www.SciRP.org/journal/abb/
6. DENR Administrative Order No. 34, Series of 1990
7. (1998). The Physical Characteristics of the Environment. Retrieved from http://dx.doi.org/10.4236/abb.2012.33041
8. Jasmin Adhikari, Pravin Mann Shakya, Prativa Shrestha, Rameswor Aryal & Damodar Sedai (2024). The Isolation & Identification of Escherichia Coli in the Chicken Sausages Samples with its Microbial Quality Analysis Study Collected From Bhaktapur District, Nepal. Dinkum Journal of Natural & Scientific Innovations, 3(03):385-393.
9. Gram Staining. (2013). Gram Staining. Retrieved from http://serc.carleton.edu/microbelife/researchmethods/microscopy/gramstain.html
10. (2009). Hemolysin. Retrieved from http://www.merriamwebster.com/medical/hemolysin
11. (2011).Isolation of Different Marine Organisms. Retrieved from http://bipublication.com/files/IJABRv2i32011011.pdf
12. Prativa Shrestha, Damodar Sedai, Pragya koirala, Tulsi ram Gompo & Diker Dev Bhatt (2024). Study on Enrofloxacillin Antibiotic Residue in Chicken Meat Marketed Of Kathmandu Valley. Dinkum Journal of Natural & Scientific Innovations, 3(02):240-250.
13. Nester, Eugene W. (2001). Microbiology: A Human Perspective. 3rd ed. New York: McGraw-Hill.
14. Durga Karki & Arun Kumar Shrestha (2024). An Analysis of the Ground water for Irrigation Purpose of Morang, Nepal. Dinkum Journal of Natural & Scientific Innovations, 3(03):316-333.
15. Perfumo, Amedea. (2007). Thermally Enhanced Approaches for Bioremediation of Hydrocarbon-Contaminated Soils. Chemosphere 66: 179-184.
16. Pettersson, Marie. (2004). Factors affecting rates of change in soil bacterial communities. Doctoral thesis. Lund University, Sweden. Retrived from http://www.lub.lu.se/luft/diss/sci_649/sci_649.pdf
17. (2012). Protease. Retrieved from http://en.wikipedia.org/wiki/Protease
18. Rajendran, N. and Kathiresan, K. (2007). Microbial flora associated with submerged mangrove leaf litter in India. Int. J. Trop. Biol 55(2): 393-400.
19. Prativa Shrestha, Damodar Sedhai, Jasmine Adhikari & Tulsi Ram Gompo (2023). Study on Antibiotic Resistance in Escherichia Coli Isolates in Chicken Meat Marketed in Kathmandu Valley. Dinkum Journal of Natural & Scientific Innovations, 2(12):870-881.
20. (2012). Starch. Retrieved from http://www.eng.umd.edu/̴ nsw/ench485/lab5.html
21. State of Mississippi. Department of Environmental Quality. (1998). Fundamental Principles of Bioremediation. Retrieved from http://www.deq.state.ms.us/MDEQ.nsf/pdf/GARD_Bioremediation/$File/Bioremediation.pdf
22. Walsh, G. E. (1974). Mangroves: A Review. In: “Ecology of Halophytes”(R J Remold and W H Queen, eds). pp 51-174. Academic Press, NewYork.
23. Walworth, James, Andrew Pond, Ian Snape, John Rayner, Susan Ferguson, and Paul Harvey. (2005). Fine Tuning Soil Nitrogen to Maximize Petroleum Bioremediation. ARCSACC (2005): 251-257.
24. Williams, Jera, (2006). Bioremediation of contaminated soils: A comparison of in situ and ex situ techniques. Retrieved from http://home.eng.iastate.edu/~tge/ce421-521/jera.pdf
25. (n.d). The Salinity of Seawater. Retrieved from http://water.epa.gov/drink/contaminants/basicinformation/lead.cfm
26. Vidali, M. (2001). Bioremediation. an overview. Pure appl. chem.IUPAC. 73(7),1163–1172.