Isolation and Purification of Lyngbya majuscula on Nutrient-enriched Agar Plates

A Special Problem

ABSTRACT

Lyngbya majuscula samples were obtained from the Phycology Laboratory stock culture of the UPV Institute of Aquaculture. Five (5) mm fragments of these were inoculated into agar plates that used 1% agar concentration enriched with varying concentrations (1.0%, 1.5%, and 2.0%) of Hughes, et. al. (1958) Mineral Medium No. II.

After ten (10) days of culture, the Trichome Length (TL), Trichome Width (TW), Sheath Width (SW) and Total Length of the Lyngbya filaments were measured from photomicrographs of the samples using Image Tool (Version 3.00) developed by the University of Texas Health Science in San Antonio (UTHSCSA).

Lyngbya filaments in all cultures enriched with the Hughes medium obtained significantly higher TL and TW over those of the control treatment. But enrichment of the agar medium did not result in an increase of sheath widths.

Filaments cultured in 1.5% and 2% enriched agar showed contamination. Those in 1% nutrient concentration had comparable growth, but no contamination. Furthermore, Lyngbya cultured in enriched agar did not grow beyond the area of inoculation. Isolation was also more difficult in agar than in the liquid control medium. INTRODUCTION

As early as the 1900s, Lyngbya sp. blooms were documented in the areas of Eastern Moreton Bay and Bribie Island in Australia (Abal & Lawn, 2004). Lyngbya is a genus which is composed of species which are usually inhabitants of the sea. Numerous species reside in estuarine places and in brackish channels. Others are found in freshwater areas and thermal springs (Harvey, 1857). Lyngbya has been found to be a nuisance in the areas of South Eastern United States. It is a cyanobacteria which lives naturally in the benthic zone (Gross & Martin, 1996). In recent studies, it has been found out that cyanobacteria have reacted to human modification of aquatic environments, especially in the most notably nutrient enhanced primary production which is euthrophication. Lyngbya, specifically, is able to develop into thick mats which are responsible for euthrophication in freshwater and these causes the lakes to be over fertilized (Perovich, 2008; Gross & Martin, 1996). L. majuscula is not desirable to consume, and many pay no attention to it, allowing it to grow rapidly and have advantage over other benthic species found on coastlines. This permits them to grow uncontrollably in favorable conditions. Regulating its population is necessary to manipulate the chemicals or substances it generates, which are usually harmful and may give negative effects to humans, and to aquatic animals at the same time (Paul, et. al., 2007). To be able to manage its growth, further studies regarding its characteristics, in terms of its physiology, structure, and biology, must be conducted. These studies can be done efficiently through pure cultures in the laboratory. Pure cultures do not occur naturally since the process involves a completely sterile environment in which there are no contaminants. In microbiology, isolation is performed to be able to produce a population that consists of a single clone, one which is derived from a single cell and one which is free of other microorganisms. This can be done by streaking on solid media such as on agar plates. The strains acquired through this manner are important in studies of the physiology, molecular biology, and genetics of microorganisms (Overmann, 2006). There has been no recorded research regarding the culture of L. majuscula in nutrient-enriched agar. Its survival in gel is still questionable. However, it may be possibly grown in agar being a cyanobacterium, which characteristically is a microbe (Abal & Lawn, 2004; Perovich, 2008). This study explores the possibility of culturing L. majuscula in agar.

OBJECTIVES OF THE STUDY

The general objectives of this study are to isolate strands of L. majuscula, grow them in agar plates enriched with Mineral Medium No. II, a nutrient source developed by Hughes, et. al (1958), and purify them for culture in the laboratory. It also aims to measure the survival and growth of L. majuscula at three concentrations of the nutrient medium with 12 replicates each.

REVIEW OF RELATED LITERATURE

Lyngbya majuscula: Physiology and Morphology

Lyngbya majuscula is a filamentous, nonheterocystrous cyanobacteria belonging to the Family Oscillatoriaceae (Garibay, et. al., 2007). Cyanobacteria are known to be one of the most varied prokaryotes. They range from unicellular to complex filamentous forms (Waterbury, 2006). They evolved approximately 3.5 billion years ago and added oxygen to the anaerobic atmosphere which existed at that time. They are capable of surviving in extreme conditions, including dessication, hypersalinity, high UV radiation, and hyperthermal circumstances (Perovich, 2008). Cyanobacteria comprise the major source of biotoxins found in supplies of water (Carmichael, 1997). Colloquially known as “Mermaid’s hair” (Neilan, et. al., 2000), L. majuscula develops in clumps and looks like dark masses of hair. It is usually blackish green or olive green, but also grows in shades of yellow, red, or gray. Its filaments can grow up to four inches long (Scott & Thomas, 1997). It lives in rocks covered with mud or sand at or below half-tide level in the sea. It can reach the coastline after storms, when it is thrown up from deep waters up to 100 feet (Harvey, 1851; Scott &Thomas, 1997). L. majuscula filaments are thick, very long and firm, and occur in long crisped bundles, with green or purple tubes which are continuous (Harvey, 1857, 1851). The endochromes of L. majuscula are of the color dull green and its tube has a broad and colorless border. Sometimes the endochromes look as if it is broken and sometimes it separates into two distinct portions and it is highly possible that at a longer period in time, the uppermost portion separates and forms a new filament (Harvey, 1851). It has been observed to be the major species in algal blooms that occurred in Harvey and Moreton Bay, also in cases in Guam, Hawaii, and Florida. Upon the inspection of the affected areas, presence of abnormally high amounts of iron has been acknowledged (Neilan, et. al., 2000). Research has shown that L. majuscula has high requirement for biologically available iron (Neilan, et. al., 2000). Nitrogen, phosphorus, iron and molybdenum can be potential limiting factors for its growth (Watkinson, 2000). Lyngbya spp. has also become a major problem in coastal areas sited in Florida. It has been said that this happens because of the nutrient enhancement in the highly industrialized shoreline (Arthur, et.al, 2009).

Harmful Algal Blooms and Its Impacts on Humans Algal blooms have catastrophic short-term and long-term consequences to their affected areas. These blooms also have adverse effects not only to animals but also to the health of human beings (Molley & Rose, 2007). Impacts of Harmful Algal Blooms (HABs) range from human diseases to death from direct or indirect exposure to the toxins produced by harmful algae. Ecosystems are also equally affected by these impacts. Environmental damage reduces the sustainability of life in ecosystems increases the susceptibility of animals to disease and alters community structures. HABs may also lead to economic hardships to coastal communities and aquaculture businesses (Ramsdell, et.al., 2005). Recently, HABs have increased in number of occurrence and severity of the cases. Among these blooms, cyanobacteria blooms, specifically that of Lyngbya spp., are increasing in number and are continuing to persist in tropical and subtropical marine and estuarine settings (Arthur, et.al, 2009). L. majuscula is the only algae known to cause rash in humans. It contains potent toxins that cause skin damage upon contact (Scott & Thomas, 1997). In lieu with this, experiments and skin testings that were conducted showed that isolated compounds, Lyngbyatoxin A (LA), phenolic bis-lactones aplysiatoxin (AT), and debromoaplysiatoxin (DAT), from L. majuscula extracts were found to be the major agents capable of instigating a severe contact dermatitis on animals and humans (Falconer & Stewart, 2008). L. majuscula contains an irritant that generates erythema, blisters, necrosis, and a burning sensation (Iversen & Skinner, 2006). Its filaments may get caught inside swimming suits, rubbing against the skin (Scott & Thomas, 1997). A swimmer can have a contact with the floating algae that discharges the irritants and can suffer from irritation for a number of days (Iversen & Skinner, 2006). In the late 1950s, an encounter with Lyngbya majuscula came about at a Hawaiian beach. At least 100 swimmers made a complaint of dermatitis that was thought to be caused by exposure to L. majuscula (Falconer & Stewart, 2008). On March 2005, an algal bloom occurred at Miag-ao, Iloilo, Philippines, which stretched up to a kilometer. It was discovered that L. majuscula was the dominant species. Local residents experienced direct and indirect effects of the bloom, such as eye, skin, and nasal irritations and breathing difficulty (Garibay, et. al., 2007). Inhalation of the fragments of L. majuscula can cause severe inflammation of the lungs. Chewing of these blue green algae can cause mouth or lip burns. According to some experiments on laboratory mice, swallowing these filaments can cause animal deaths and may also instigate fatality to humans (Scott & Thomas, 1997).

Agar as a Solidifying Media Culture on agar plates is a means of isolation and separation of bacteria and prokaryotic cells (Irving, 2005; Overmann, 2006). Its use can be dated back to 1911 when growth of different cells in agar was observed. However, agar was not widely used until recently (Kuroki, 1975). Agar is used in the culture of filamentous blue green algae at conecnetrations of 1-2% to obtain optimum growth. In the medical field, agar is used as a laxative, as a coating for some types of capsules, and as a vessel for suppositories (Anonymous, 1951). In microbiology, agar is used as a gelling agent to which nutrients such as blood, peptone and sugars, and other factors such as buffers, salts, and indicators, is added (Stein, 1973; Irving, 2005). Agar has a strong gel structure and has a stable gelling temperature making it suitable for solidifying growth media. It is also used for techniques such as immunodiffusion and electrophoresis (Stein, 1973). Hughes, et. al. (1958) Mineral Medium No. II as Nutrient Enrichment Mineral Medium No. II has been extensively used for the isolation and sustenance of cyanobacteria and it supports growth of a wide variety of blue-green algae as well as other algae. It was formulated by Hughes, Gorham, and Zender in 1958 and was modified by Allen in 1968. It has also been suggested for culture of freshwater algae (Stein, 1973; Waterbury, 2006; Sharma, 1986). METHODOLOGY

Experimental Site This study was conducted at the laboratory and hatchery of the Institute of Aquaculture (IA) at the College of Fisheries and Ocean Sciences (CFOS), University of the Philippines Visayas (UPV) in Miagao, Iloilo.

Experimental Treatments Three nutrient-enriched agar culture media using Mineral Medium No. II developed by Hughes, et. al. (1958) as nutrient source, and a control were tested in this study. The treatment formulations are shown in Table 1:
Table 1: Formulations of Agar and Hughes Per Treatment
Treatment Agar Concentration Mineral Medium No. II (Hughes, et. al. (1958))
A 1% 1.0%
B 1% 1.5%
C 1% 2.0%
Control 0 % 0 % (grown in seawater)

Lyngbya majuscula filaments in the control treatment were grown and cultured in seawater so as to provide the conditions in the natural environment of the plant. Disinfection of Glassware All necessary glassware was prepared by washing with soap and rinsing with tap water. An acidified sodium dichromate cleansing solution was applied after which and the glassware was rinsed with distilled water. These were then sterilized at 121°C for 20 – 35 minutes in an autoclave and set aside while the media was prepared.
Media Preparation One hundred milliliters of the culture medium was prepared for each treatment. This was composed of 1% plain agar enriched with varying quantities of Mineral Medium No. II of Hughes, et. al., (1958). The mineral medium was prepared by combining, dissolving, and diluting to one liter the following reagents (Table 2).
Table 2: Mineral Medium No. II (Hughes, Gorham, and Zehnder 1958)
NaNO3 1.5 g
K2HPO4 0.369 g
MgSO4 • 7H2O 0.075 g
CaCl2 • 2H2O 0.036 g
Na2CO3 0.020 g
Na2SiO3 • 9H2O 0.058 g
Ferric Citrate* 0.006 g
Citric Acid 0.006 g
EDTA 0.001 g
Minor Element Solution** 0.08ml
*Autoclave iron separately
**Minor Element Solution in g/L: H3BO3 - 3.1 g; ZnSO4 - 0.287 g; (NH4)6Mo7O24 • 4H20 - 0.088 g; KI - 0.083 g

For all agar treatments, separate preparations of the agar solutions were made by weighing and dissolving 1 g of plain agar in 75 ml of distilled water. These were pre-cooked while continuously stirring for five minutes until most of the plain agar was dissolved (Figure 1).

Figure 1: Flasks Containing Mixture of 1% Agar and 75 ml Distilled Water The agar solutions were each subsequently combined with 25 ml of water containing 1 ml, 1.5 ml, and 2.0 ml of the nutrient medium for treatments A, B, and C, respectively (Figure 2).

Figure 2: Flasks Containing Mixture of Varying Amounts of Hughes, et al. (1958) Mineral Medium No. II and 25 ml Distilled Water

The solutions in the two flasks labeled A were mixed together. The same was followed for flasks labeled B and C, respectively (Figure 3). The three mixtures were put in the autoclave for 30 minutes at 121°C. These were then cooled down to 50°C, poured into three Petri dishes per treatment, and stored at room temperature for 24 hours before the inoculation.

Figure 3: Flasks Containing Mixture of Varying Amounts of Hughes, et al. (1958) Mineral Medium No. II and 1% Agar

Algal Sample Preparation and Inoculation The Lyngbya majuscula filaments used in this study were taken from the stock culture of the Phycology Laboratory of the IA Hatchery. The filaments were separated one by one using a microscope and a pair of forceps. After separation, the strands were cut into the approximately 5 mm fragments using a surgical blade under a microscope. Each agar plate was divided into four quadrants and each quadrant inoculated with a single 5 mm fragment. The plates were stored at room temperature with one 220V fluorescent bulb that served as bright lighting throughout the whole experiment. Observations were done after 24 hours and every day thereafter for the duration of the culture period which lasted for 10 days.

Growth Measurement After ten days of culture, the survival, length, and appearance of the filaments were observed from photographs (10 x magnification) taken using a photomicroscope. The trichome length (TL), trichome width (TW), upper sheath width (SW1) and lower sheath width (SW2) of each filament were measured using Image Tool (Version 3.00) developed by the University of Texas Health Science in San Antonio (UTHSCSA), a free image processing and analysis program (Figure 4). Total Length was also computed by adding the two Sheath Widths (SW 1 and 2) the Trichome Length (TL).

Figure 4: Parameters Measured to Determine Growth of L. majuscula

Statistical Analysis Means of the TL, TW, SW1, SW2, and Total Length were calculated for all treatments. The data were analyzed using the model Analysis of Variance (ANOVA) with the aid of the software Statistical Package for the Social Sciences (SPSS). Post Hoc analysis was also used to determine the significant differences between means. Differences were considered significant at P0.05) (Tables 7 & 8).

Figure 13: Mean Upper Sheath Width of L. majuscula at the End of the Culture Period

Table 7: Results of One-Way ANOVA on the Upper Sheath Width (SW1) of L. majuscula Sum of Squares df Mean Square F Sig.
Between Groups 1.133 3 .378 2.392 .086
Within Groups 5.209 33 .158
Total 6.342 36

Table 8: Post Hoc Analysis for Upper Sheath Width of L. majuscula
Treatment N Subset for alpha = 0.05 1
1.5% 11 1.5026
2.0% 10 1.8330
1.0% 11 1.8753
Control 5 1.9626
Sig. .105

Similarly, there were also no significant differences (P