isolation of micro algae from fresh water
- 1. Isolation of microalgae from fresh water (summer internship programme) IIT (ISM) Dhanbad PRESENTED BY:- Govind Kumar Gulashan Biotechnology 3rd Year NIT Arunachal Pradesh
- 2. Algae • A group of photosynthetic organisms that in general possess pigments such as chlorophyll but lack true roots, stems and leaves characteristic of terrestrial plants. • Algae are typically aquatic and members of the traditionally recognized eukaryotic Kingdom Protista(All single celled organisms are placed under the Kingdom Protista ).
- 3. Classification of algae The 7 groups numbered below are the 7 major lineages of photosynthetic organisms. 1. Cyanobacteria (blue‐green algae) 2. Dinoflagellates 3. Euglenoids 4. Brown, Golden‐brown, and Yellow‐brown Algae 5. Glaucophytes 6. Red Algae 7. Green Algae
- 4. Cyanobacteria (blue‐green algae) • Cyanobacteria are photosynthetic bacteria and were sometimes called “blue‐green algae” by earlier botanists. • cyanobacteria are prokaryotes
- 5. Dinoflagellates • Dinoflagellates are not bacteria but unicellular protists. • It have olive‐brown color.
- 6. Euglenoids • Euglena are interesting because they combine superficial characteristics of both plants and animals. • They can make their own food like a plant, through photosynthesis, but they can also eat other things “like” an animal. • They can also swim and move by their flagellum.
- 7. Brown, Golden‐brown, and Yellow‐brown Algae A. Diatoms :- • Diatoms are unicellular or colonial organisms In addition to their chlorophyll, diatom chloroplasts contain the accessory pigment fucoxanthin . • Diatoms lack flagella and are non‐motile. • chlorophyll plus fucoxanthin makes them appear golden‐brown or brown‐yellow in color. B. Brown Algae:- • Most browns are relatively big, and all are multicellular. • they have chlorophyll, their special carotenoid fucoxanthin impart a brown color to them.
- 8. Glaucophytes • Extremely small group of algae closely related to red and green algae.
- 9. Red Algae • The red algae are largely marine and multicellular “seaweeds”. • A class of accessory pigments (phycobilins) makes them reddish. • Red algae have cellulosic cell walls.
- 10. Green Algae • The green algae are a very large and diverse group. They range from unicellular organisms such as Chlamydomonas or Chlorella to colonial organisms. i. Unicellular forms ii. Colonial forms iii. Filamentous forms i ii iii
- 11. A phylogenetic tree based on DNA sequences, showing 7 major groups of photosynthetic organisms.
- 12. Use of algae 1. Fuel source • Algae can be used to make Biodiesel , Bioethanol and biobutanol and by some estimates can produce vastly superior amounts of vegetable oil. • Algae can be grown to produce hydrogen. In 1939 a German researcher named Hans Gaffron, while working at the University of Chicago, observed that the algae Chlamydomonas reinhardtii (a green-algae), would sometimes switch from the production of oxygen to the production of hydrogen. • Algae can be grown to produce biomass, which can be burned to produce heat and electricity.
- 13. Use of algae 2. Food supplement • Algae have been in use as human food for centuries in various parts of the world. • It is a complete protein with essential amino acids (unlike most plant foods) that are involved in major metabolic processes such as energy and enzyme production. • It contains high amounts of simple and complex carbohydrates which provide the body with a source of additional fuel. • It contains an extensive fatty acid profile, These essential fatty acids also play a key role in the production of energy. • It has an abundance of vitamins, minerals, and trace elements in naturally- occurring synergistic design.
- 14. Use of algae 3. Fertilizers • The large Brown and Red algae are used as organic fertilizers, especially on land close to the sea. • A concentrated extract of seaweed is also sold as a liquid fertilizer. • Coralline algae Lithothamnion calcareum and Lithophyllum sp. are used profusely for liming the soil. • They are cultured in clear tanks or ponds and either harvested or used to treat effluents pumped through the ponds.
- 15. Use of algae 4. Pollution control • Algae are used in Wastewater Treatment facilities, reducing the need for greater amounts of toxic chemicals than are already used. • Algae can be used to capture fertilizers in runoff from farms. When subsequently harvested, the enriched algae itself can be used as fertilizer. • Algae Bioreactors are used by some power plants to reduce CO2 emissions. The CO2 can be pumped into a pond, or some kind of tank, on which the algae feed. Alternatively, the Bioreactor can be installed directly on top of a smokestack
- 16. Use of algae 5. Stabilizing agent • Chondrus crispus is also used as "carrageen". It is an excellent stabiliser in milk products it reacts with the milk protein . • It has several important industrial applications for e.g., in textile industry, in paper making to give body to the paper, in the manufacture of straw and felt hats as a stiffening agent. • it used as an emulsifying and suspending agent, in the baking, dairy industries and in clarifying liquors.
- 17. Use of algae 6. Sewage Disposal • The most common algal species present in the sewage oxidation ponds are Chlamydomonas, Scenedesmus, Chlorella etc. • Aerobic bacteria breakdown products of sewage, from complex organic substrates into simple inorganic products, fulfill the primary-requirements of these photosynthetic algae and in return the bacteria receive the necessary oxygen for their activity. • The relationship existing between algae and bacteria in a stabilization oxidation pond.
- 18. Use of algae 7. Commercial Products • Many forms of marine algae, Phaeophyceae and Rhodophyceae, are highly valuable for certain commercial products, chiefly agar-agar, algin or alginic acid and carrageenin. • Agar-agar is obtained from various species of red algae for e.g., Gelidium corneum, G. cartilageneum. • It is a non-nitrogenous extract obtained almost in a pure mucilaginous form. The chief constituent of agar is a carbohydrate galactan. • Algin and Alginates-Algin is a calcium magnesium salt of alginic acid Because of its special colloidal properties alginic acid and its derivatives find considerable use in industry. • Its salts are used in the manufacture of variety of goods ranging from ice- cream, salad cream, custard and jams to cosmetics, films, fabrics, ceramics and textiles.
- 19. Use of algae 8. Medicinal use: • Alaria was once used for strengthening the stomach and restoring the appetite after sickness. • Alginates are used for their haemostatic nature. • Antibiotics:-The antibacterial product chlorellin, obtained from Chlorella. • Extracts from Rhodomela larix and Ascophyllum nodosum are effective against both gram positive and gram negative bacteria.
- 20. Use of algae 9.Binding of Soil Particles • Algae act as an important binding agent on the surface of the soil. • Disturbed or burnt soils are soon covered with a growth of green and blue- green algae. • The role of Cyanophycean members as a pioneer in colony formation and thus in soil formation is well known.
- 21. Harmful Effects of algae 1.Blooms • When blue-green algae spread or "bloom," thanks to optimal weather conditions, the algae can turn the water a greenish, bluish, reddish or brownish color, and create a scummy film on the surface area. 2. Toxicity • Cyanobacteria blooms are made up of tiny cells. These cells can house various "cyanotoxins" or "cyanobacterial toxins." When cells break, the toxins are released into the water, and can be harmful to humans and animals. • blue-green algae include endotoxins and cytotoxins, which along with the other types of toxins, can irritate the skin. 3. Symptoms • Drinking cyanobacteria-tainted water or eating cyanobacteria-infested fish could result in headaches, stomach aches, fever, diarrhea, nausea or vomiting.
- 22. Algal Culturing Introduction:- • Photosynthetic microorganisms play an important role in the conversion of solar energy into chemical energy. • Algal biomass has historically served as fertilizer and a food source for both humans and animals for secondary waste water treatment and bioremediation. • Algae grow in almost every habitat in every part of the world. • The ubiquity of these organisms together with the plasticity of their metabolic requirements make many algal species easily available for investigation, collection, or simple observation.
- 23. Steps 1. Collection:- • Floating microalgae can be collected with a mesh net or, if in sufficient quantity (i.e., coloring the water), by simply scooping a jar through the water. • Some algae live attached to other types of substrate, such as dead leaves, twigs, and any underwater plants, which may be growing in the water. • Macroalgae and the attached microalgae can be collected by hand or with a knife, including part or all of the substrate (rock, plant, wood, etc.) if possible. fig:- algal sample
- 24. Steps 2.Labeling • Any sample should be labeled with standard information such as the locality, date of collection, and as many of the following features as possible. 3. Storage • Algae can be stored initially in a glass jar, plastic bottle or bag, or in a vial with some water from the collecting site. • The container should be left open or only half filled with liquid and wide shallow containers are better than narrow deep jar.
- 25. Steps • Fig:-algal sample stored in a deep jar
- 26. 4. Enrichment of a culture • Enrichment is the process of providing a suitable environment for the growth and reproduction of a special group of microalgae while being inhibitory or lethal for nontarget organisms. 5. Establishing Unialgal Cultures Streaking and successive plating on agar media • Streaking is useful for single-celled, colonial, or filamentous algae that will grow on an agar surface. • Algal cells can be concentrated and then plated on agar medium.
- 27. Fig:-Algae plated on agar medium.
- 28. Steps 6. Isolation • Direct isolation:-Filaments can be grabbed with a slightly curved pipette tip and dragged through soft agar (less than 1%) to remove contaminants. It is best to begin with young branches or filament tips. • Single-cell isolations using capillary pipettes:-Single cells or filaments can be picked up under a dissecting microscope, using micropipettes. The individual cells are transferred to agar medium or fresh sterile medium for isolation. 7. Serial dilution techniques • A simple technique to purify a contaminated strain is to proceed with repeated subcultures obtained by progressive dilutions of the original sample.
- 30. 8. PRODUCING AXENIC CULTURES • Axenic cultures should contain only one alga without bacteria,fungi, or protozoa. • Biological contamination of algal cultures by other eukaryotes and prokaryotic organisms in most cases invalidates experimental work, and may lead to the extinction of the desired algal species in culture through outcompetition or grazing. i.Extensive cell washing:-Under a dissecting microscope, an individual algal cell is picked up using a micropipette and placed in a sterile liquid medium in a spot plate. The organism is then transferred through a series of sterile media. ii. Density gradient centrifugation:-Microalgae can be separated from bacteria using density gradient centrifugation. The algae at a particular position within the gradient are collected by fractionation of the gradient.
- 31. iii. UV irradiation:-Most algae are slightly more resistant to ultraviolet light than bacterial cells. iV. Filtration:-Filamentous algae can be separated from bacteria using membrane filters. Sonication is often employed to break up the algae into small length filaments. The diluted sample is then vacuum filtered. V. Antibiotics:-Axenic cultures can be obtained by treating isolated algae with one or more antibiotics to discourage growth of contaminating cyanobacteria and other bacteria. • Various antibiotics have been effectively used in removing bacteria from algae. • Eg:-antibiotics nystatin and cycloheximide were used to eliminate fungal contaminants from cyanobacteria
- 32. Steps 9. Checking the Sterility of cultures • Sterility of cultures should be checked by microscopic examination or by adding a small amount of sterile bacterial culture medium to a microalgal culture, incubated in dark and observing regularly for two to three days for bacterial growth.
- 33. CULTURE PARAMETERS • A culture has three distinct components: (1) a culture medium contained in a suitable vessel (2) the algal cells growing in the medium (3) air, to allow exchange of carbon dioxide between medium and atmosphere. • Parameters regulating algal growth a.Temperature:-The temperature at which cultures are maintained should ideally be as close as possible to the temperature at which the organisms were collected. • Most commonly cultured species of microalgae tolerate temperatures between 290K and 300K. b. Light:-requirements greatly vary with the culture depth and the density of the algal culture • At higher depths and cell concentrations the light intensity must be increased to penetrate through the culture.
- 34. c. PH:-The pH range for most cultured algal species is between 7 and 9, with the optimum range being 8.2–8.7.Complete culture collapse due to the disruption of many cellular processes can result from a failure to maintain an acceptable pH. d. Mixing:-it is necessary • to prevent sedimentation of the algae. • to ensure that all cells of the population are equally exposed to the light and nutrients. • to improve gas exchange between the culture medium and the air.
- 35. Tap media prepration Reagents used for 1 Lt. tap media. I. 2.42gm Tris Base. II. 25 ml Tap salt. III. Phosphate buffer 0.375 ml (colour of solution changed in turbid colour). IV. Tris salt (red) 1ml. V. Glacial acetic acid 1 ml.