Evaluation of Microalgae Diversity From Fresh Water Within Centenary City Abakaliki, Ebonyi State

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Evaluation of Microalgae Diversity From Fresh Water Within Centenary City Abakaliki, Ebonyi State

Microalgae generally referred to as microscopic plants due to the inability to see them with the unaided eyes grow suspended in water and are capable of the same photosynthetic process as that of higher plants (Haneltet al., 2007). Microalgae typically comprises of bacteria, diatoms, other protists and higher plants.

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However, in comparison with higher plants, microalgae have no need for a vascular system for its nutrient transport as each cell is photoautotrophic directly absorbing nutrients (Bahadar and Khan, 2013). The microalgal cells are sunlight stimulating cells with the capacity of converting carbon dioxide (CO2) into raw materials for the production of biofuels (e.g biodiesel and bioethanol), animal feed-stocks and high valuably bioactive compounds (Razzaket al., 2013).
Microalgae either eukaryotes or prokaryotes are oxygenic autotrophs (that is they evolve oxygen during photosynthesis) that lives in aquatic ecosystems ranging from fresh water and brackish water to oligotrophic water. Different biological, ecological and functional traits characterise the microalgal world which represent rich biodiversity. The number of species ranges from 30,000 species to one million, and some studies report more than 200,000 species only for the Bacillariophyceae (Guiry, 2012).Based on recent studies, this group is the most recent and variegated group, and up until now, more than 8000-10000 diatoms species have been discovered, while just a few species have been used for biotechnological applications (Lebeauet al., 2003).
The uses of microalgal by native populations have been carried out for centuries.

Edible blue green algae which include Nostoc, Arthrospira (Spirulina) and Aphanizomenon species have been used as a source of food for thousands of years (Jensen et al., 2001). However, the cultivation of microalgae is only but a few decades old (Borowitzka, 1999). In the early 1950s as the world population increases and predictions of an inadequate supply of proteins led to a search for new alternative and different protein sources. Algal biomass as at that time appeared as a good experiment for this purpose (Borowitzka, 1995).
There exists an extensive scientific literature for microalgae which is known as phycology, due to their vital roles in the natural and human ecosystems. Half of the global primary production is being accounted for by microalgae and they are the basic source of most aquatic food chains. However, their excess quantities is often a symptom of in land or near shore eutrophication, which promotes fish kills, dead zones, red tides etc. The ecological roles of phytoplankton, including the effects of pollution have been reviewed by most research studies of microalgae, and much has been of a basic nature (physiology, metabolism, photosynthesis, genetics, etc.) (Falkowski,2007). In the study of microalgae, advanced genetics and other recently developed molecular tools in the fields of biology have been applied, from genomics to metabolomics (Gouveia and Oliveira 2009).

AIM
This study aims to evaluate the diversity of microalgae in fresh water from a building site at Centenary City Abakaliki, Ebonyi State.
1.2: OBJECTIVES
1. To isolate pure microalgae samples.
2. To identify isolates through microscopic identification.
3. To identify isolates by molecular analysis.
4. To compare the growth of the microalgae cells with different media.

CHAPTER TWO
LITERATURE REVIEW

ALGAE
Algae which comprises of diverse group of organisms make up the lower phylogenetic echelon (ranks) of the plant kingdom. An exact definition of this group is difficulty: in comparison with higher plants, they share manky characteristics, whereas their distinguishing features from other plant groups are different and more subtle. Most of the algae are either photosynthetic or they are related to organisms that are. 50% of the photosynthetic processes on this planet are performed by the algae and thus are vital in supporting the biosphere (John 2004). Algae include the macroalgae, or seaweeds and microalgae.

DIVERSITY OF ALGAE
The algae which have been long recognized as a group of heterogeneous organisms, ranging from the blue-green microscopic algae, closely related to the gram-negative bacteria to the large, complex kelps, which goes beyond 10m in length.Molecular biology data in the recent studies supports the idea that algae are a group of organisms that have independently acquired chloroplasts (intra- cellular bodies that contain the photosynthetic machinery; Gibbs 1992).

TYPES OF MICROALGAE
Typically, microalgae cannot be seen by the unaided eye, but if the water is eutrophic, huge algae blooms takes place, turning the water in a brown, blue, orange or green liquid mass. Just a few tens of thousands, out of a number of 200,000 to 800,000 diverse species have been described in literature. The ranking and genetic analysis of species of microalgae is still under study and the classification still remains incomplete and inconsistent (Tredici, 2010). Based on recent studies, taxonomists have differentiated the following main groups:

Bacillariophyta(Diatoms)
Having close to 100,000 described species, diatoms are the most common group of algae in existence and tend to overthrow the phytoplankton in the oceans, but are usually seen in fresh and salty water habitats. The golden brown colour is as a result of the presence of fucoxanthin and -carotene which covers the green colour chlorophyll a and chlorophyll cSheehan et al., 2008). There are two major distinguished groups of diatoms which are the Penates having bilateral symmetry and the centrals with radical symmetry.

Chlorophyta(Green Algae)
This group is also among the common microalgae found in freshwater. Approximately 8,000 species are thought to be in existence (Sheehan et al., 2008). Chlorophyll a and chlorophyll b and other carotenoids can be found to be contained in this group and they can be produced under stress conditions and can be responsible for the alga change in colour. Their basis storage componecolour. Their basis storage component is starch. Higher plants are considered lower families of the green algae and because of that reason; they have been given more attention than the other algal groups with Chlorella and Chlamydomonas the most studied genera (Sheehan et al., 2008).

Chrysophyta(Golden-Brown Algae)
This is also referred to as chrysophytes and shares some similarities with diatoms in comparison with the pigment and biochemical composition. About 1,000 species have been identified, and are basically present in freshwater habitats (Sheehan et al., 2008), mostly in oligotrophic waters derived of calcium.

Cyanobacteria(Blue-green)
These are prokaryotic organisms having chlorophyll a. Due to the presence of phycocyanin and phycoerythrin, it was given the name of blue-green and it covers the chlorophyll pigmentation. They are identical to bacteria in that they lack a nucleus, chloroplasts and also have a separate gene structure in their cells. A random figure of 2,000 species is in this group. Some cyanobacteria are known to take in atmospheric nitrogen (N) thus removing the need to produce fixed nitrogen (N) to the cells. None of them produces a good amount of lipid as storage (Sheehan et al., 2008).

Rhodophyta (Red algae)
These are a group of 5000 mostly multicellular marine species, existing in the tidal zone of the sea.

Yellow green algae
They are similar to the brown algae being close relatives, but most of the 600 species exists in fresh water and are unicellular. Nannochloropsisis a good example being a fast growing species found in the sea. Large amount of oils are being produced by this algae as a food reserve and are quite good for the production of raw material for biodiesel (Sheehan et al., 2008).

MORPHOLOGY
The microalgae as a group is made up of single cell forms, but some are multicellular in the fact that the cells may be somewhat connected. Those with chains or filaments are usually referred to as multicellular microalgae. The length of the individual cells ranges from less than one micron to above 2000 microns (Palmer, 2005). The common shapes are round, oval and cylindrical, though the shapes can be quite complex. The external cell coverings are made up of common polysaccharides to silica and calcium carbonate. The silica coverings referred to as frustules form complex and beautiful structures on most members of the Bacillariophyta. The calcium carbonate external coverings (coccolithophorids) formed by those species transform the calcium carbonate into ornate scales with many types of shapes and sizes (Weyeret al., 2010). These scales have the possibility of falling off the cells and because of their special reflective qualities; blooms of the species can be captured by satellite in space. Microalgae species have flagella while others have flagella-like structures (e.g. haptonema). Naturalist have been overwhelm for hundreds of years by the diversity of the morphology (since the first microscopes) and needs to be seen to be appreciated (Wang et al., 2008).

PHYSIOLOGY
There are many exceptions in regards to the photosynthetic nature of microalgae. For example, about 50% of the Dinophytaare non-photosynthetic. Oxygenis formed as a primary waste product by most microalgae while H2S is formed by some. Microalgae are a much better source of protein than most land crops, even after they are processed. Few species of microalgae are been grown for the human health food market as a source of micronutrient dietary supplements (Paashe, 2000).
Lipids and pigments are the major components that are currently been extracted on a large scale. To be precise, the long chains of polyunsaturated omega three fatty acids that are quite beneficial to the human health were gotten from microalgae. Other pigments such as beta-carotene along with fatty acids can be produced from algae grown in large scale culture (Rutledge et al., 2002). The potential value and range of products is very exciting. One fascinating new development is the adaptation of toxins for medicinal use. The toxins found in microalgae can currently be a cause of environmental problems.

REPRODUCTION
Reproduction in microalgae is via binary fission which yields two daughter cells. A few species can form four or eight cells when dividing. Normally a species will produce many cells via binary fission (vegetative growth). Harvestable biomass can be produced by most commercial operations based on this fact. Environmental conditions become less suitable for growth when the physiological trigger into another life history phase for most species, though this is not well understood. The next stage in the normal life history often involves sexual recombination of DNA sometimes before and after the formation of a cyst (Tran et al., 2010). The physiology of cysts are different from that of the vegetative cell, but it is yet unknown if their biochemistry is different fundamentally. The investigation of cysts for special biomolecules is yet to begin, but it seems likely that the cell types will yield potent anti-bacterial compounds. Cysts can survive for many years and they are often found in sediments prior to germination carrying out a similar ecological function as bacterial cysts or seeds from land plants (Demirbas, 2010). Sexual reproduction in some microalgal species can be relatively simple having only two mating types, but populations can be made up of eight or more mating types that do not all interbreed. Sexual reproduction stage occurs in most species in their life cycle, but life cycles and their stages are explained for only a few species. It can be true that many small flagellates that come out occasionally in natural communities are lying history stages of some other form of microalgae (Ugwuet al., 2008).

MICROALGAE CULTIVATION METHODS
Many methods on how to grow microalgae exits due to the difference in environments from place to place. The methods includes closed pond system, open ponds system, wastewater, marine environment and desert environment and under all the conditions (Ginzburg, 2007).

OPEN PONDS SYSTEM
Lakes, lagoons, ponds and artificial ponds or containers are examples of open ponds system that can be used in the proportion of microalgae. The growth of microalgae cannot be under control in open ponds system. One of the advantages of open ponds system to that of closed pond system is that they are easier to construct and operate (Borowitzka, 2007). However, major disadvantages in open ponds system include diffusion of CO2 to the atmosphere, poor light utilization by the cells, requirement of large areas of land and evaporative losses. The growth of microalgae is changed by all pollutants in open ponds system. However, the possibility of controlling the productivity and the quantity of the microalgae is not certain. Furthermore, microalgae can only be grown when all the conditions required for their growth are available during certain period of the year. In addition to the major disadvantages of microalgae cultivation in open ponds system is the difficulty in harvesting the microalgae (Gonzales et al., 2012).

CLOSED POND SYSTEM
Closed ponds system such as photo-bioreactor and other types can be used to grown microalgae. One of the major characteristics of closed pond is that all conditions can be monitored like percentage of light utilization, carbon dioxide and not required for large areas of land. However, one of the major shortcomings in closed ponds is the difficulty in constructing, operating and very costly (Vasudevanet al., 2008).

PHOTOBIOREACTORS
Photo bioreactor which is an example of a closed ponds system can be used to grow microalgae. There are several reasons for the growth in photo bioreactors such as, its growth can be controlled and it produces high productivity, for example: carbon dioxide supply, gas supply, standard temperature and pressure, water supply, suitable lights, pH levels, mixing regime and culture density (Stephenson et al., 2010).
The harvesting of microalgae cultivation can be easier than open ponds system. However, there is availability of all the requirements to microalgae growth. Thus, it can be grown anywhere and anytime unlike open ponds system. There are many shortcomings in photo bioreactors such as, the capital cost is much (industrial production), a lot of hinders in sterilizing these photo bioreactor and other defects (Richmond, 2004). Many types of photo bioreactors exist in the industry operation: Flat-plate photo bioreactor and tubular bioreactor.

FLAT-PLATE PHOTO BIOREACTOR (FPPB)
One of the most important photo bioreactors is the flat-plate bioreactor. The major features of flat-plate photo bioreactor is that it is suitable for outdoor cultivation, good light path, good biomass productivities, immobilization of algae, very cheap, low oxygen build and can be read, easy to clean (Miao and Wu 2004).

TUBULAR PHOTO BIOREACTOR(TPB)
Tubular bioreactor is quite necessary in the industry for many reasons such as: very large illumination surface area, suitable for outdoor cultures, very cheap, good biomass productivities and other properties in tubular photo bioreactor (Cronarand Fallow-field 2007).

MICROALGAE IDENTIFICATION USING MOLECULAR TECHNIQUES
POLYMERASE CHAIN REACTION (PCR)
The polymerase chain reaction (PCR) is a technique quite strong and sensitive which amplifies exact DNA sequences exponentially done in multiple cycles through a three-step process (Saiki et al., 2005). Firstly, high temperature can bring about the denaturing of the double-stranded DNA. Later there is addition of thermostable DNA polymerase, such as Taq DNA polymerase right after the annealing of the sequence-specific primers to the right sequence (Chienet al., 2006). The extension of the primers and making twice the amount of the original DNA sequence is carried out by the action of the enzyme. The product produced then becomes an extra template for continuous cycles of amplification. The three steps are normally carried out in cycles for 20 to 30 times, leading to an increase of target DNA concentration of 105 – 109 times the previous amount. There are both positive and negative sides in using PCR to get huge amounts of a desired product. The production of unwanted products resulting even to the exclusion of the target product can be possible if the application is unsuccessful. The opposite would be that no product may be amplified. Several variables have been identified to contribute to this effect as regards to optimization (Saiki et al., 2005). Magnesium (Mg2+) concentrations, cycling conditions, buffer pH are the main variables. The annealing condition is of utmost importance inside the cycling conditions. The interdependence between the variables adds difficulty to the situation. For instance, adding more amounts of deoxynucleotide triphosphates (dNTPS) reduces the concentration of the free Mg2+ available to put an effect on polymerase function because dNTPS directly chelate a proportional number of Mg2+ ions (Lyamichevet al., 2003).

ENHANCING AGENTS
With the aim of adding more yield, specificity and consistency, many additives and enhancing agents which includes dimethyl sulfoxide (DMSO), N, N, N,-trimethylglycine (betaine), glycerol, formamide, bovine serum albumin, non-ionic detergents,tetramethyl ammonium chloride and polyethylene glycol can be included in PCR reactions (Ono andCuello, 2007). The elimination of the need of a full multivariate matrix analysis for each of the variables tested can become a burdensome and costly task through the application of the Taguchi method (Tang et al., 2011), which is based only on the main effects and two factor interactions. Using this method, the size of the matrix can be reduced significantly and there would be simultaneously alterations of the several key variables (Cobb and Clarkson, 2004).

MAGNESIUM CONCENTRATION
The optimization for every primer/template should always be attained and magnesium chloride acts as an essential co-factor for the DNA polymerase. Magnesium ion is tightly bound to the phosphate-sugar backbone in nucleotides and nucleic acids; therefore many quantities can have strong and complex effects on experiments on which the nucleic acids partake in (Borowitzka, 2000). Because of the vital role of free magnesium as enzyme co-factor in PCR, its total ion concentration has to be above the total dNTP concentration. Generally, magnesium ions concentration differs in series of 1.5 – 4 mM with 0.5mM step increments (Komarek, 2006).

APPLICATIONS OF MICROALGAE
BIOFIXATION OF CARBON DIOXIDE BY MICROALGAE
Microalgae are microorganisms that are used in the elimination of carbon dioxide emissions into the atmosphere, where this compound is bio-fixed in the presence of light energy. Findings of a highly developed photosynthetic system has led to the contribution of using microalgae in the treatment of gaseous wastes with elevated CO2 concentrations, generated through industrial discharges (Hsuehet al., 2007).There was the development of research, especially in Japan, based on the processes for the bio-fixation of carbon dioxide using microalgae. There are many increases in the studies, aiming at projecting systems that operate efficiently and economically with the objective of bringing more technologies for the decrease of gaseous pollutants (Cheng et al., 2006). The use of microalgae in carbon dioxide fixation processes is considered a better alternative, since the element carbon can be transformed by several mechanisms. Firstly, the carbon dioxide dissolved in the aqueous phase of the system can be sequestered by chemical precipitation as a result of the reaction of the ions bicarbonate and carbonate with elements present in the culture medium, such as calcium and magnesium. These reactions are catalysed by the growth and physiology of the microalgae (Jaiswal and Kashyap2002).
Calvin-Benson cycle is another carbon-fixing pathway, where specialised enzymes available in these organisms catalyse reactions that incorporate carbon atoms coming from the CO2 involved in photosynthesis (Franciscoet al., 2010). Products of the photosynthetic metabolism as results of the biological conversion of carbon dioxide are cells, oxygen, biopolymers soluble in the culture medium and volatile organic compounds (VOC’s) (Jacob-Lopes et al., 2010). The CO2 transformation into biomass is high only under conditions where the CO2 mass loading rate is reduced. The formation of volatile organic compounds at a high CO2 mass loading rate, is the main CO2 biotransformation route.

MICROALGAE AS A SOURCE OF FOOD AND DIETARY SUPPLEMENTS
Due to their high nutritional value, some of the microalgae have been eaten as food or health food. For example, Spirulinahave been harvested for food by the people in Mexico and Chad (Africa) since old times. In Mexico, Spirulina is gotten from Lake Texcoco and used for preparing dry cake called tecuitlatl. In Chad, Spirulina is gotten from the alkaline lake Kossorom for the making of dry cake referred to as dihe. In the recent times, Spirulinastill contributes greatly to the economy of Chad as the local trading value of dihe is worth much more than US$ 100,000 (Abdulqaderet al., 2000). Right now, Spirulinais being cultured on a large scale using open ponds for commercial manufacture of the biomass as dietary supplements in countries such as Thailand, China, United States and India. The estimated annual worldwide production of Spirulinaranges from 3000 to 4000 metric tons. Due to the high content of proteins, g-linolenic acid, vitamins and minerals, Spirulinais regarded as a nutritious food. Furthermore to being eaten as a food product, Spirulinais seen to have therapeutic implications, including for health problems such as diabetes, arthritis, anaemia, cardiovascular diseases and cancer. Spirulinais also vital as a useful ingredient as it is infused into different food products to improve their nutritional qualities and for therapeutic management of chronic disorders such as diabetes, hypertension and heart disease. Spirulina is also popular for its antioxidant compounds such as phycocyaninand vitamin E (Chu et al., 2010).
Another microalga that has been cultured for commercial preparation of health food in the forms of pills and powder is Chlorella. Its nutritional value is due to their large contents of proteins (51-58% dry weight) and carotenoids, with a range of vitamins (Becker, 2004). Also, the alga contains b-glucan, which is an active immune-stimulator, and has several beneficial effects in searching for unused radicals and decreasing blood lipids.

MICROALGAE AS ANIMAL FEEDS
Many toxicological and nutritional research have shown the possibility of algal biomass as feed supplement (Pulz and Gross 2004).Arthrospira is widely used in this domain and is about many types of animals: cats, dogs, aquarium fish, ornamental birds, horses, cows and breeding bulls. Algae affect the physiology in a good way (by creating a large profile of natural vitamins, essential fatty acids and minerals; better immune response and fertility; and better weight control) and their outward look (resulting in healthy skin and a soft and shinning coat) of animals (Certik and Shimizu 2000). Partial replacement for conventional proteins can be achieved by safely using algae up to 5-10% in poultry rations. Continuous feeding of algae at large concentrations produces side effects. The yellow colour of broiler skin and the shanks as well as that of the egg yolk is the major feature that can be influenced by feeding algae (Pulz and Gross 2004).

MICROALGAE AND BIOFUEL
Microalgae have since been used as potentially good sources for biofuel production due to their high oil content and fast biomass production. Recently, the use of microalgae as an alternative biodiesel feedstock has gotten renewed interest from researchers, entrepreneurs and the general public at large (Katirciogluet al., 2006). Potential advantages of algae include:
1. Not been traditional foods and feeds, eliminates their need to compete with traditional agriculture and they can be cultivated in large open ponds or in closed photo-bioreactors.

2. Their growth is not affected by wide variety of climate and water conditions; they can sequester and utilise CO2 from different sources.

3. Algae can strongly manufacture 1000-4000 gallon which is significantly higher than soybeans and other oil crops

4. Lastly, they can be processed into a wide spectrum of products including biodiesel via trans-esterification, green diesel and gasoline replacements through direct catalytic hydrothermal conversion, and catalytic upgrading, and bioethanol through fermentation, methane through anaerobic digestion, heat through combustion, bio-oil and bio-char through thermochemical conversion, and high protein animal feed.

There are several ways to transform microalgal biomass to energy sources, which can be classified into biochemical conversion,direct combustion, thermochemical conversion and chemical reaction. Therefore, microalgae can provide feedstock for renewable liquid fuels such as biodiesel and bioethanol (Chisti, 2007).

MICROALGAE IN WASTEWATER TREATMENT
Due to their ability to accumulate plant nutrients, heavy metals, pesticides, organic and inorganic toxic substances and radioactive matters in their cells/bodies, microalgae have become significant organism for biological purification of wastewater (Kalesh and Nair, 2005). The biological waste water treatment systems using microalgae have received importance in the last 50 years and it is now largely accepted that algal wastewater treatment systems are as good as conventional treatment systems. These specific characteristics have made algal wastewaters treatment systems and important low-cost alternatives to complex expensive treatment systems just for purification of municipal wastewaters. Furthermore, algae gotten from treatment ponds are widely used as nitrogen and phosphorus supplement for agriculture use and can be carried to fermentation in order to get energy from methane.
Algae can also accumulate highly toxic substances such as selenium, zinc and arsenic in their cells and bodies therefore reducing such substances from aquatic environments. Some water contains naturally radioactive materials, and other become radioactive through contamination resulting in the radiation as an important type of pollution. Many algae can collect and store many radioactive materials in their cells even from larger concentrations in water.MacKenthun emphasized that spirogyra can store radio-phosphorus by a factor 850,000 times that of water (MacKenthun, 2000).
Taking note of the abilities of algae o purify the polluted water of many types, it is wise to say that algal technology in wastewater treatment systems are expected to get more popular in future years. Wastewater treatment which is applied to upgrade the quality of a wastewater involves chemical, biological and physical processes in primary, secondary or tertiary stages. The materials that will either float or readily settle out by gravity are removed by primary treatment. It includes the physical processes of screening, contamination, grit removal, and sedimentation. While the secondary treatment is usually done by biological processes and the soluble organic matter and suspended solids left from primary treatment is removed. Tertiary or advanced treatment is process for the removal of nitrates and phosphates as well as fine particles through purification (Droste, 2007).
However, the basic cost as well as operating cost of wastewater treatment plant including primary, secondary or advanced stages is highly expensive. It is well observed that algae have a vital role in self-purification of organic pollution in natural waters. Moreover, different research showed that algae remove nutrients especially nitrogen and phosphorus, heavy metals, pesticides, organic and inorganic toxins, pathogens from surrounding water by storing and using them in their cells(Guhaet al., 2001). Also, research shows that as a result oftheir bioaccumulation, algae may be used successfully for wastewater treatment (Oswald, 2008).

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