Isolation of Toxin Producing Cyanobacteria from Aquatic Samples with Lyngbya

Abstract

Cyanobacteria are represented by morphologically diverse, ecologically important, phylogenetic Gram-negative prokaryotes. They constitute a phylogenetically coherent group of evolutionarily ancient phototrophic bacteria. The toxins produced by cyanobacteria are classified into endotoxins, hepatotoxins, lipopolysaccharides, and neurotoxins. Some species of Lyngbya can have Lyngbyatoxin-a and Aplysiatoxins, causing dermatitis with itching, burning, pain, rash, and blisters when in contact with human skin and could lead to loss of superficial layers of the skin by cell death. The effect of cyanobacterial intoxication includes epithelial cell damage in the digestive and respiratory tracts promoting tumour development. This chapter aims to elucidate the methodology for isolating the cyanotoxins from Lyngbya.

1 Introduction

Lyngyba is filamentous blue-green algae that flourish in freshwater. The unbranched filaments are covered by firm polysaccharide sheaths and are seen covering both water surfaces and benthic sediments (Speziale et al. 1988). The consumption of water contaminated with cyanobacteria will call gastroenteritis. Cyanobacteria can produce diverse toxins that may affect the central nervous system like anatoxin-a, anatoxin-a(s), and saxitoxins. How cyanotoxins have their toxic effects can vary among different groups. The principal toxins primarily affect liver, such microcystins, nodularin, and cylindrospermopsin (Otero and Silva 2022). The marine Lyngbya majuscula produces a several bioactive compounds including Dermatotoxins. Lyngbyatoxin-a a potent irritant and vesicant produce rashes and other skin reactions. Aplysiatoxins considered promoting tumour development. Similarly, Lyngbya wollei can produce a potent neurotoxin like saxitoxins. The toxic effects include damage to the digestive and respiratory tract’s epithelial cells. The toxins produced by Lyngbya promote the development of a tumour and the development of fibropapillomatosis (Beasley 2020). Cylindrospermopsin is another important alkaloid cyanotoxin produced by Lyngbya consisting of tricyclic guanidine (Ohtani et al. 1992; Guzmán-Guillén et al. 2013). The structural variant 7- epicylindrospermopsin is highly toxic (Banker et al. 2001). Toxins released from the algal cells to the surroundings are accumulated in the organisms in the lower level of the food chain (Rücker et al. 2007; Kinnear 2010). Cyanobacterial intoxication requires accurately identifying the algal species in the contaminated water (Van der Merwe 2015). Toxicological analyses of biological specimens contracted with algal toxins are recommended. Since the toxicity of these cyanobacteria is strain specific, more than mere identification is needed to predict their hazardous level. Liquid chromatography and tandem mass spectrometry are the best methodologies adapted for the analysis (Puschner and Moore 2013).

2 Materials

• Z-8 medium

• Modified seawater BG-11 medium

• ASN-III medium

• Sterile agar

• Cycloheximide

• Germanium dioxide

• Cycloserine

• N-formimidoyl thienamycin monohydrate

• Distilled water

• Sterile containers

• Petri dishes

• Erlenmeyer flasks (125 mL, 500 mL, and 1 L)

• Culture vessels

• Dive knife

• Autoclave

• Incubator

• Cool white fluorescence tube

3 Methods

3.1 Sample Collection (Burja et al. 2002)

• 1. Collect the samples with a clean dive knife.

• 2. Place them in sterile sample containers and seal them underwater.

• 3. Ship to the laboratory within a day and process them within 1 week of collection.

3.2 Z-8 Medium

• 1. Wash the filaments with sterile Z-8 medium (Kotai 1972; Carmichael 1986).

• 2. Autoclave the sterile agar and wash it thrice over a period of 72 h with distilled water. Then, mix with the filter-sterilized nutrient solution.

• 3. Add 50 mg L⁻¹ and 100 μg mL⁻¹ of cycloheximide and germanium dioxide, respectively, to eliminate the growth of eukaryotes. Add cycloserine and N-formimidoyl thienamycin monohydrate (Imipenem) to prevent bacterial growth (Vaara et al. 1979).

• 4. Store the agar plates at 4 °C before use.

• 5. Cut 0.5 cm long segments and plate at the centre of Petri dishes containing 1% w/v agar and Z-8 medium. Prepare in triplicate.

• 6. Maintain the cultures in an incubator under 26 °C under 16/8 light and dark photoperiod.

• 7. Use cool white fluorescence tube with an irradiance of 22 μmol m⁻² s⁻¹ photon flux as a light source (Speziale and Dyck 1992).

• 8. Cut new trichomes that grow out of old ones away from the region of contamination. Transfer the trichome to another Petri dish containing the same medium. Repeat until unialgal culture is obtained.

• 9. Then, transfer the trichomes to 125 mL Erlenmeyer flasks containing 100 mL of Z-8 medium. Allow the filaments to adapt to the Z-8 medium for 2 weeks.

• 10. After 2 weeks, transfer to the 1-L Erlenmeyer flasks containing 800 mL of Z-8 medium.

• 11. Aerate the cultures at the rate of 43–71 Lh⁻¹.

3.3 Modified Seawater BG-11 Medium (Burja et al. 2002)

• 1. Subculture the samples in 500 mL Erlenmeyer flasks containing 200 mL of modified seawater BG-11 medium (Rossi et al. 1997).

• 2. Let the cultures to achieve equilibrium with their new environment at 20 °C and then inoculate in large-scale culture vessels.

• 3. Maintain the cultures under a 12-h light/dark regime (Lux 250) and at a temperature of 20 °C.

• 4. Aerate with 0.2 mm filtered air and subculture every 2 months.

3.4 ASN-III Medium (Rippka et al. 1979)

• 1. Culture the filaments in ASN-III medium and subculture repeatedly to obtain axenic culture.

• 2. Maintain the cultures under 25 ± 1 °C and 7.5 W/m² of temperature and light intensity, respectively.

• 3. Use cool-white, fluorescent tubes as the light source with a photoperiod of 14:10 h.

4 Observation (Burja et al. 2002)

• 1. Use light microscopy to study the external morphology of Lyngbya. Autofluorescence images of filamentous Lyngbya visualized at the emission range of 400–500 nm under a fluorescence microscopy.