Molecular identification of cyanobacteria was based on PCR amplification and 16S rRNA gene sequencing. The waters of the Limpopo River Basin also provide drinking water for wildlife and a habitat for aquatic organisms/animals.
BACKGROUND
Recently published methods and improved DNA reference libraries have shown great promise for algal identification (Kermarrec et al., 2013). Different forms of each gene sequence called alleles are used in analysis based on their difference during DNA fingerprinting (Jeffreys et al., 1985).
STATEMENT OF THE PROBLEM
Currently, two methods of algal species identification are used: morphological; which involves the observation of various features by microscope and molecular;.
MOTIVATION
RESEARCH QUESTIONS
RESEARCH OBJECTIVES
Main objective
Specific objectives
THE STUDY AREA
Nearly 14 million people live in the Limpopo River Basin in four riparian states (DWA, 2011/12). The Mzingwane catchment generates almost a quarter of the discharge in the Limpopo basin (Görgens and Boroto, 1997).
RESEARCH DESIGN
One of the major rivers adjacent to the capital of Botswana (Gaborone) with the largest areas of urban growth is in South Africa's southeastern corridor, the Notwane River (Mladenov et al., 2005). To harness the water from the river and its tributaries for use as a water supply for Gaborone, the Gaborone Reservoir was built in the 1970s; and this changed the Notwane River (Senai, 1999).
Thesis outline
A review of the Mzingwane watershed in Zimbabwe, contribution to WaterNet Challenge Program 17 "Integrated water resources management for improved rural livelihoods: managing risk, mitigating drought and improving water productivity in the water-scarce Limpopo Basin". A review of the current status of water quality and eutrophication in South African rivers and reservoirs.
Algae in fresh waters
Multiplication of algae and cyanobacteria in freshwater has been added to by human activities (agricultural runoff, road runoff, and inadequate sewage treatment), which also results in eutrophication of many water bodies and significantly affects recreational water quality (Vollenweider, 1992). Human activities that disrupt the balance of an ecosystem have been linked to the increase in some freshwater HAB outbreaks (Pearl, 2008).
The Occurrence of cyanobacteria blooms in the tributaries of the Limpopo River
Hartbeespoort Dam and the Crocodile River
Occurrence of some freshwater HABs has been reported as a result of human activities and these include increased nutrient loading and pollution, modified hydrology and introduced species (Lopez et al., 2008). Many species of freshwater algae proliferate well in eutrophic waters, but they do not form blooms, so the toxins produced do not accumulate to concentrations harmful to humans and animals (Chorus & Bartram, 1999).
Notwane River
Mzingwane River
Cyanobacteria and its toxins
Floating mechanism
HABs are considered one of the most visible indicators of nutrient over-enrichment (Pearl and Fulton, 2006). On sunny days they photosynthesize and these can result in high rates of oxygen production, forming bubbles that break part of the mat and release them to the surface (WHO, 2003).
Production of cyanotoxins
Gaborone 'Water and waste economics in three African capitals, Ash gate, Aldershot, England. Cyanotoxins have been implicated in negative plant growth (stunting), which can have serious consequences for irrigated farmers (McCollough, 2016).
Dermatoxins
Effects of cyanotoxins on human and the environment
Impact on humans
The main pathway for microcystins to enter cells is through the bile acid carrier, which is present in liver cells and to a lesser extent in the intestinal epithelium (Falconer, 1993). In vertebrates, a lethal dose causes death by hepatic necrosis within hours to days.
Impact on aquatic ecosystem
1994) illustrate the disruption of nasal tissue by the common hydrophilic analogue microcystin-LR, whereas oral toxicity is generally at least less than toxicity by intraperitoneal (i.p.) injection. 1994) also demonstrated that the toxicity of microcystin is cumulative, meaning that a single oral dose cannot increase liver weight (which is a measure of liver damage), whereas if the same dose is used daily for seven days, it can cause liver enlargement. 84% and thus had the same effect as a single oral dose, 16 times greater. This can be explained by the irreversible covalent bond between microcystin and protein phosphatases and the subsequent significant damage to the cell structure (Falconer, 1993).
DNA finger printing
Cyanotoxins can come into contact with humans, animals, and the environment through drinking contaminated water from a lake or reservoir with HABs, drinking untreated water, engaging in recreational activities in HAB-contaminated waters, inhaling aerosols from activities related to water, such as such as jet skiing or boating, inhaling aerosols while watering lawns, watering golf courses, etc.
Toxic genes identification
It was discovered that the detection unit of this method was 8.8 cells per reaction and that cell concentration determined by real-time PCR correlated positively with the cell concentration determined from direct microscopic counting. It was discovered to be a powerful tool to detect and quantify potentially toxic cyanobacteria in the laboratory and field samples (Al-Tebrineh et al., 2011).
Conclusion
A quantification study of microcystin-producing cyanobacteria was carried out by Furukawa et al. The study revealed the presence of the mcyE gene, which was found in one strain of Leptolyngbya and one strain of Oscillatoria and is responsible for the production of cyanotoxins.
Harmful cyanobacterial algal blooms – State of the science and research needs: Advances in Experimental Medicine and Biology, v. Interagency Working Group on Harmful Algal Blooms, Hypoxia, and Human Health of the Joint Subcommittee on Ocean Science and Technology. Quantitative assessment of daily phytoplankton ingestion by Tilapia nilotica and Haplochromis nigripinnis in Lake George, Uganda.
INTRODUCTION
Therefore, the main objective of this chapter is to evaluate the physicochemical characteristics of river sediments and how these contribute to the recovery and growth of cyanobacteria if ideal conditions, especially river flow, return. The ability of cyanobacteria to survive periods of drought allows them to live in habitats with fluctuating conditions (Perez et al., 2016). Thus, the main objective of the study is to assess the physicochemical characteristics of river sediments and how these contribute to the revival and growth of cyanobacteria if ideal conditions, especially river flow, return (Figure 3.1).
MATERIALS AND METHODS
SAMPLING
Sand River downstream (at bridge on road N1 towards Musina) S10 Crocodile River downstream (at bridge on road D1235) near Thabazimbi S11 Nzhelele River upstream near Mphephu resort (downstream from Siloam. oxidation ponds).
PHYSICAL MEASUREMENTS
NUTRIENTS ANALYSES
Total phosphorus was determined using the perchloric acid digestion method as described by APHA (1998): 2 g of air-dried sediment was acidified to methyl orange with concentrated HNO3, another 5 ml of concentrated HNO3 was added and evaporated on a hot plate until dense smoke to appear. Total nitrogen was determined according to APHA (1998) as ammonia: 1 g of each air-dried sediment sample was treated with 2 ml of sulfuric acid. The filtrate of each sample was made up to 250 ml with deionized water and 55 ml of 1 M sodium hydroxide solution.
DATA ANALYSIS
10 mL each of concentrated HNO3 and HClO4 were added and gently evaporated until dense white vapors of HClO4 appeared. TOC extraction was performed by adding 200 ml of deionized water to 6 g of sediment in a plastic bottle and shaken for 24 h on an orbital shaker at 24 0C after shaking, the suspension was centrifuged at 900 xg for 30 min and filtered. through the 0.45 µm pore size filter. TOC was determined using TOC cell boxes following the manufacturer's instructions and analyzed by Merck Spectroquant® Pharo 100 spectrophotometer Merck (Darmstadt, Germany) with a wavelength of 320–1100 nm.
RESULTS AND DISCUSSION
The physical characteristics of the river sediments
The abundance of nutrients in the river sediments
The nitrate concentration values in the river sediments varied from 1.5 mg/l to 6.5 mg/l (Figure 3.10). The local municipality of Musina has drilled 8 boreholes in the Limpopo riverbed and most of these boreholes are located near S16 (Figure 3.11). Presence of TOC influences the growth of cyanobacteria by proving food when the rivers start to flow or when the production of cyanobacteria increases (Figure 3.12).
CONCLUSION
Since food will be easily obtained, cyanobacteria return to high-density proliferation in rivers leading to a decrease in water quality. Ozonation has been shown to be a more effective method for removing cyanotoxins, and if the TOC in the raw water is high, the efficiency of ozone in killing cyanobacteria decreases dramatically (Hoeger et al., 2002). When TOC levels are higher than 3.0 mg/l, ozone concentrations of <1.0 mg/l cannot completely remove or destroy cyanotoxins (Hoeger et al., 2002).
Detection of microcystin-producing cyanobacteria in Finnish lakes by sex-specific microcystin synthetase Gene E (mcyE) PCR and associations with environmental factors. Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413. Growth and vertical movement of the cyanobacterium Microcystis in stable and artificially mixed water columns.
INTRODUCTION
MATERIAL AND METHODS
THE CULTURE OF CYANOBACTERIA SPECIES IN RIVER SEDIMENTS
THE IDENTIFICATION OF CYANOBACTERIAL SPECIES USING THE FLOW-
RESULTS AND DISCUSSION
THE PRESENCE OF CYANOBACTERIA IN THE RIVER SEDIMENTS
The FlowCam showed the following cyanobacteria species: Microcystis aeruginosa, Lyngbya, Calothrix, Anabeana, Oscillatoria, Phormidium and Chroococcus. Some cyanobacteria can produce neurotoxin, dermatotoxins, hepatotoxin or other bioactive compounds; and toxic cyanobacterial blooms pose a threat if present in drinking water sources (Steidinger et al., 1997). A guideline limit of 1 μg L-1 for microcystin-LR (most common cyanobacterial toxins) has been issued by the World Health Organization for drinking water (Lopez et al., 2008).
CONCLUSION
INTRODUCTION
TOXIC GENES IDENTIFICATION
The main objective of the study is to use molecular techniques to identify toxic and non-toxic cyanobacterial genes in the river sediments and to use 16S rRNA to identify the cyanobacterial species and explore the relationships between the cyanobacterial species in the river sediments.
MATERIALS AND METHODS
- MOLECULAR CHARACTERIZATION
- DETECTION AND AMPLIFICATION OF 16S rRNA BY PCR
- TOXIN GENES DETECTION
- PCR PURIFICATION AND SEQUENCING
- PHYLOGENETIC RELATIONSHIP
- DIVERGENCE MATRIX
The conditions of the PCR reactions are similar to those described for the amplification of the 16S rRNA gene. The gel slice was placed in a pre-weighed 1.5 ml tube and the weight of the gel was weighed and recorded. Then, 700 µl of wash buffer was added to the purification column and centrifuged at 12 000 x g for one minute and the flow-through was discarded.
RESULTS AND DISCUSSION
- PCR ANALYSIS OF 16S rRNA GENE
- DETECTION OF GENES INVOLVED IN TOXIN PRODUCTION
- PHYLOGENETIC RELATIONSHIP
- DIVERGENCE MATRIX
Evolutionary distances were calculated using the two-parameter method of Kimura (Kimura, 1980) and are in units of the number of base substitutions per site. Gene mutation can result in reduced toxicity of strains when maintained in culture. Microcystins have already been implicated in wildlife mortality in the Kruger National Park (Oberholster et al., 2009).
CONCLUSION AND RECOMMENDATION
CONCLUSION
RECOMMENDATION
Molecular identification and evolution of cyclic peptide hepatotoxins, microcystin and nodularin synthetase genes in three orders of cyanobacteria. Characterization of the nodularin synthetase gene cluster and proposed theory of cyanobacterial hepatotoxin evolution. The polyketide synthase gene associated with the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin.
