Presented by:Shraddha Bhatt

PhD (Microbiology)AAU,Anand

1. Habitat2. Morphology3. Cultivation4. Reproduction5. Evolution6. Genetics7. Photosynthesis

Cyanobacteria terminology

Division CyanophytaCyanobacteria ‘formerly known as’

BlueGreen Algae

- Cyano = blue- Bacteria – acknowledges that they are

more closely related to prokaryotic bacteria than eukaryotic algae

• cyanobacteria can be found in almost every conceivable environment, from oceans to fresh water to bare rock to soil.

• They can occur as planktonic cells or form phototrophic biofilms in fresh water and marine environments,

• they occur in damp soil, or even temporarily moistened rocks indeserts.

• A few are endosymbionts in lichens, plants, various protists, or sponges and provide energy for the host.

• Some live in the fur of sloths, providing a form of camouflage.• Aquatic cyanobacteria are probably best known for the extensive

and highly visible blooms that can form in both freshwater and the marine environment and can have the appearance of blue-green paint or scum.

Habitat

Basic Morphology of Bacteria

Growth Forms

Synechococcus

Crococcus Cells of Anacystis

Oscillatoria

Spirlina spp.Anabaena

Fossil Cyanobacteria

"Ooid" sand grain containing Endolithic Cyanobacterium

(CB). Erosion caused by Endolithic Cyanobacteria

Hot Spring at Yellowstone Park: The dark color is due to the presence of

Cyanobacteria.

Limestone deposit at Yellowstone Park: The localized areas of green are due to

the presence of Cyanobacteria

A schematic outline of the acquisition, reduction, and loss of genomes and compartments during evolution. Black arrows indicate evolutionarypathways; white arrows indicate endosymbiotic events in the host cell. Endosymbiotic event 1 occurred at the origin of eukaryotes. The proteobacterial endosymbiont gave rise to mitochondria (the smaller organelles in the bottom part of the diagram). Endosymbiotic event 2 occurred at the origin of plastid-containing cells. Endosymbiotic event 3 represents the secondary and higher-order endosymbioses giving rise to numerous algal phyla, as well as apicomplexans (such as Plasmodium), which have residual plastids, and to trypanosomes, which have no plastid at all. Black, filled circles indicate nuclei or nucleomorphs; ellipses within organelles indicate bacterially derived genomes, which may be reduced or lost completely.More than one kind of host cell and of endosymbiont is involved in the secondary, and in the higher-order, symbioses. The genome of theArchaebacterium is not represented in the diagram.

Structural drawing of the fine structural features of

a cyanobacterial cell. (D) DNA fibrils;

(G) gas vesicles; (Gl) glycogen granules;

(P) plasmalemma; (PB) polyphosphate

body; (Ph) polyhedral body; (Py) phycobilisomes;

(R) ribosomes; (S) sheat;

(SG) structured granules (cyanophycin granules);

(W) wall.

Evolution• Old 3.5 billion years• Dominated as biogenic reefs• During Proterozoic – Age of Bacteria (2.5 bya – 750 mya) they were wide spread• Then multicellularity took over• Cyanobacteria were first algae!

Cyanobacteria Production

• For BGA production dig a small pit of 6x3x9 feet size in the soil and lay down a polythene sheet in the pit to check to percolation of water. Large galvanized steel tray containing soil can also be used for this purpose. Now 10 Kg soil, 200 gm of super phosphate, 6 litre water and 100 gm BGA dry flakes containing wooden dust or mother culture of BGA are added into the prepared pit. If found any pest in the pit, spray melathion solution ( 1 ml melathion in one litre water) to destroy pests.

• If green algae and diatoms are found in pit, use 0.05% CuSO4 solution which will kill the green algae. After 12-15 days, a thick layer of BGA will be found floating on the water surface of the pit. You can easily collect/harvest the BGA directly from the pit or let the pit dry after water evaporation and take out dry flakes of BGA and fill it in small polypack (100-200 gm) for sale.

• By this simple method BGA culture/incoulum can be prepared for use in the paddy fields. The same methods may be used in the paddy fields for the large scale productions of BGA culture.

CULTIVATION STOCK CULTURE •The stock culture for maintenance of laboratory culture, 2- 3 mL of a 3 weeks old cyanobacterial stock culture was used as inoculum in 50 mL of autoclaved BG 11 medium in 150 mL Erlenmeyer flasks. •The cultivation was carried out at 20 ±2°C, under continuous illumination of 8gmol/m2 by cool fluorescence lamps. •The stock cultures were maintained for 20-30 days.

CULTIVATION SHAKE CULTURE

Aliquots of 50 mL from the stationary phase stock cultures were used to inoculate 500 mL of autoclaved BG11 medium in 1.5 liter Fehrnbach flasks. These samples were cultivated at 20 ±20°C, under continuous illumination of 8gmol/m2 bycool fluorescence lamps. The cyanobacterial cultures were harvested after 4-6 weeks.The cells were separated from the medium by centrifugation (4000 rpm/ 10 min/ 100C) followed by filtration with filter paper. The biomasses were lyophilized and stored at -20°C until use while cultivation media were concentrated to 1/10 (v/v) by rotary evaporation in vacuum at 400°C and extracted immediately with EtOAc solvent.

The large scale cultivation was carried out in a 45 liter-glass fermentor. The fermentor was cleaned by distilled water and 70% isopropanol before use. At the beginning, the fermentor was filled with 15 L of medium and after 1- 2 hours 1.5 L of growing culture (after 20 days of cultivation in three Fehrnbach flasks) was added. Afterwards, every day 5 L of medium were added into the fermentor until 35 L of medium were reached. The cultures were illuminated continuously with banks of cool white fluorescent tubes of 8gmol/m2 and incubated at temperature of 26°C to 28°C adjusted using a heater. The pH-value of the large scale culture was adjusted to 7.4-8.5 using CO2

supplementation.

The biomass was collected by centrifugation at 6500 rpm in a refrigerated continuous-flow centrifuge and

lyophilized, then stored at -20°C.

Composition :Magnesium Sulphate

(MgSO4 .7H2O)0.025 g

Calcium chloride 0.05 gSodium chloride 0.20 g

Dipotassium hydrogen phosphate

0.35 g

A5 trace elements stock solution

1.0 ml

Glass-distilled water 1,000 ml

General purpose media for cyanobacteria (blue green algae) : Allen and Arnon's Medium (modified):

This medium is generally used for nitrogen-fixing cyanobacteria. If 0.20 g of potassium nitrate is added, the medium supports the growth of many non-nitrogen-fixing cyanobacteria.

Life cycle of Cyanobacteria

Heterocysts are the sites

for Nitrogen fixation.

Akinetes are asexual propagules which derived from the vegetative cells. It

protects in unfavourable condition this aids in asexual reproduction

Provides a buffering microenvironment.

Pilli : secretion

Storage products1. Phosphate Storage:• Polyphosphate bodies contain metals, mostly potassium,

calcium, and magnesium. Polyphosphate bodies in heterocysts of Anabaena showed an increased content of S and Mo and a reduction in the Ca content. The fact that polyphosphate bodies can take up heavy metals led to the hypothesis that polyphosphate bodies may be important in heavy-metal accumulation.

• The enzyme involved in the synthesis of polyphosphates in cyanobacteria is thought to be polyphosphate synthetase (polyphosphate kinase), which catalyses the formation of polyphosphate from ATP. No polyphosphate is required as a primer but the enzyme requires magnesium.Polyphosphates are broken down by alkaline polyphosphatase.

Storage products2. Cyanophycin• Nitrogen can be stored in cyanobacteria in the

form of electron-dense granules, called structured granules, containing cyanophycin granule polypeptide (CGP) .

• CGP consists of a simple polypeptide, composed of arginine and aspartic acid in a 1 : 1 molar ratio and is called multi-L-arginil-poly(L-aspartic acid), or cyanophycin.

• Cyanophycin is unique to cyanobacteria ,but is not found in all species.

3. Phycobilin pigments:• Phycobilisomes are composed of light-harvesting

phycobilin pigments that transfer absorbed light to photosystem II reaction centres.

• Phycocyanin is sometimes regarded as a nitrogen storage compound.

• It is rapidly degraded during N starvation and the apoprotein synthesis is specifically repressed.

• However, kinetic analyses have shown that when N is available, phycocyanin is always synthesised after the formation of cyanophycin and when N is limiting, phycocyanin is degraded after CGP.

Storage products

Photosynthesis

The compartmentalization of the cyanobacterial cell:The thylakoid membrane,the internal membrane system that separates the cytoplasm from the lumen and that is present invirtually all cyanobacteria,contains both photosynthetic and respiratory electron transport chains. These electron transport chains intersect,and in part utilize the same components in the membrane. Note that oxygenic photosynthesis (conversion of CO2 and water to sugars using the energy from light) essentially is the reverse of respiration (conversion of sugars to CO2 and water releasing energy). The cytoplasmic membrane,separating the cytoplasmfrom the periplasm,contains a respiratory electron transport chain but not photosynthetic complexes in most cyanobacteria. Therefore,in most cyanobacteria,photosyntheticelectron transport occurs solely in thylakoids, whereas respiratory electron flow takes place in both the thylakoid and cytoplasmic membrane systems.

C-Phycocyanins Absorb Green-Yellow

Light (615-620A).

Allophycocyanins Absorb

Orange-Red (650-670A)

C- Phycoerythrin Absorbs Green Light (495-570)

(PS II) uses light energy to split water and to reduce the PQ pool. Electrons are transported from the PQ pool to the cytochrome b6f complex and from there to a soluble electron carrier on the luminal side of the thylakoid membrane. In cyanobacteria this soluble carrier may be plastocyanin or cytochrome c553,depending on the species and on the availability of copper (plastocyanin is a copper containing enzyme). Either of these soluble one-electron carriers can reduce the oxidized PS I reaction centre chlorophyll,P700 . This oxidized form of the reaction centre chlorophyll is formed by a light-induced transfer of an electron from PS I to ferredoxin (Fd) and eventually to NADP. Reduced NADP can be used for CO2 fixation.Photosynthetic electron transfer leads to a proton gradient across the thylakoid membrane. In PS II,protons are released into the lumen upon water splitting,and protons formed upon plastoquinol oxidation by the cytochrome b6f complex are released into the lumen as well. The proton gradient across the thylakoid membrane is used for ATP synthesis by the ATP synthase in the thylakoid; this ATP may be applied for CO2 fixation and for other cell processes.

REGULATION OF PHOTOSYNTHESIS

• If light is abundant,the photosynthetic electron transport chain has a much higher capacity of electron flow than has the respiratory chain,

• but at very low light intensity or in darkness respiratory rates are higher than those of photosynthesis.

Phycobilisomes which are attached to the Surface of the Thylakoids . Phycobilisomes contain Accessory Pigments for Photosynthesis . These are Water Soluble and are stabilized by bonds to Proteins.

PS I activity is abundant relative to that of the cytochrome b6f complex

Reproduction

Conditions affect on blooms

A juicy, yummy microbial mat, full of cyanobacterial

goodness from Yellowstone Park.

Nutrient Availability: Nutrients are a limiting factor for cyanobacteria populations. As long as the correct nutrients are in excess, they can grow until some other factor, often light or temperature, becomes limiting.Competition: Ability to adapt to the environment is a big factors determining whether a bloom will form. Many blue-greens are less edible, have gas vacuoles that help them float, can sequester nutrients at the sediment water interface, or can fix dissolved nitrogen, any of which can give them a competitive advantage over other algae and lead to bloom formation.Light Intensity: Since cyanobacteria are phytoplankton, light is important and different species thrive under different light intensities. If light is not extinguished by particles or color in the water, a bloom is more likely. Many blue-greens thrive under low light, and so may be favored unless light is nearly absent (such as in some high particulate reservoir systems).Mixing: Mixing allows nutrients to be more evenly distributed and affects other aspects of water quality that in turn affect algal abundance and composition. Mixing can also move algae to depths with less light, limiting growth and survival. In general, blue-greens do better wtih less mixing (Cylindrospermopsis is one taxon that seems to do well in mixed systems, though). Temperature: Surface water temperatures consistently above 28 degrees Celsius (82 degrees Fahrenheit) encourage blue-green blooms, although blooms may still occur in late fall (October, November) in the Northern U.S.Species: The above factors influence different species very differently, because each species or taxon has a unique way of dealing with their environment. There are generalizations that apply to blooms and blue-green dominance, but ther are exceptions in most cases. Algal bloom formation is a complicated ecological process. Toxicity: Not all blue-greens are toxic, so while risk may be higher during a bloom, high biomass does not necessarily result in toxicity. Also, although many toxin producing algae produce taste and odor compounds, the presence or absence of geosmin or MIB is not a predictor of the presence of toxins.

The pH and moisture of soil and population of cyanobacteria in four

seasons of the year

Effects of cyanobacteria on plant and soil. Analysis was performed with independent

Samples t-test

Algalization in paddy field

(1) Increase in soil pores with having filamentous structure and production of adhesive substances. (2) Excretion of growth-promoting substances such as hormones (auxin, gibberellin), vitamins, amino acids (Roger and Reynaud 1982, Rodriguez et al. 2006). (3) Increase in water- holding capacity through their jelly structure (Roger and Reynaud 1982). (4) Increase in soil biomass after their death and decomposition. (5) Decrease in soil salinity. (6) Preventing weeds growth. (7) Increase in soil phosphate by excretion of organic acids (Wilson 2006)

Rice growth with Cyanobacteria

Mishra and Pabbi (2004)

Mishra and Pabbi (2004)

Effect of cyanobacterial biofertilizer inoculation on rice grain yield at a farmer’s field

the open-air algal biofertilizer production technologyfor production at farmers’ level is not popular among the

farming community.

The main limitations of this technology are:•due to open air nature of production it can be produced for only a limited period in a year (3-4 months in summer; Production has to be stopped during rainy and winter season),•high level of contamination due to open type of production,•slow production rate,• low population density and hence need for heavy inoculum per hectare.

1. The individual unit in the polyhouses can be of either RCC, brick and mortar, or even polythene lined pits in the ground. The algae are grown individually as species, by inoculating separate tanks with laboratory grown pure cultures, so as to ensure the presence of each required strain in the final product.

2. Once fully grown, the culture is harvested, mixed with the carrier material, presoaked overnight in water and multani mitti (in 1:1 ratio) and sun dried. The dried material is ground and packed in suitable size polythene bags, sealed and stored for future use.

3. The final product contains 10,000 to 1,00,000 units or propagules per gm of carrier material and, therefore,

500 g material is sufficient to inoculate one acre of rice growing area.

Mishra and Pabbi (2004)

Luxuriant growth of Cyanobacteria in a paddy field

Types of Photobioreactor• Tubular Reactors

–Horizontal–Vertical

• Flat panel reactors• Vertical column reactors• Bubble column reactors• Air lift reactors• Stirred tank photobioreactors• Immobilized bioreactors

Among the proposed photobioreactors, tubular photobioreactor is one of the most suitable types for outdoor mass cultures. Most outdoor tubular photobioreactors are usually constructed with either glass or plastic tube and their cultures are re-circulated either with pump or preferably with airlift system. They can be in form of horizontal / serpentine, vertical near horizontal, conical, inclined photobioreactor. Aeration and mixing of the cultures in tubular photobioreactors are usually done by air-pump or airlift systems.

Tubular photobioreactor are very suitable for outdoor mass cultures of algae since they have large illumination surface area. Tubular photobioreactors consist of straight, coiled or looped transparent tubing arranged in various ways for maximizing sunlight capture. Properly designed tubular photobioreactors completely isolate the culture from potentially contaminating external environments, hence, allowing extended duration monoalgal culture.

photoinhibition is very common in outdoor tubular photobioreactors .When a tubular photobioreactor is scaled up by increasing the diameter of tubes, the illumination surface to volume ratio would decrease. On the other hand, the length of the tube can be kept as short as possible while a tubular photobioreactor is scaled up by increasing the diameter of the tubes. In this case, the cells at the lower part of the tube will not receive enough light for cell growth (due to light shading effect) unless there is a good mixing system.

• Prospects Large illumination surface area, suitable for outdoor cultures, fairly good biomass productivities, relatively cheap.

• LimitationsGradients of pH, dissolved oxygen and CO2 along the tubes, fouling, some degree of wall growth, requires large land space.

Vertical Bioreactor Flate plate Bioreactor

Industrial size Photobioreactor

Seaweed cultivation

Seaweeds are macrophytic algae, a primitive type of plants lacking true roots, stems and leaves.

Most seaweeds belong to one of three divisions - the Chlorophyta (green algae), the Phaeophyta (brown algae) and the Rhodophyta (red algae).

There are about 900 species of green seaweed, 4000 red species and 1500 brown species found in nature.

The green seaweeds Enteromorpha, Ulva, Caulerpa and Codium are utilized exclusively as source of food.

Seaweed

Khan and Satam (2003)

1) the Single Rope Floating Raft (SRFR) technique developed by CSMCRI is suitable for culturing seaweeds in wide area and greater depth.

2) A long polypropylene rope of 10 mm diameter is attached to 2 wooden stakes with 2 synthetic fiber anchor cables and kept afloat with synthetic floats.

3) The length of the cable is twice the depth of the sea (3 to 4 m). Each raft is kept afloat by means of 25-30 floats. The cultivation rope (1 m long x 6 m diameter polypropylene) is hung with the floating rope.

4) A stone is attached to the lower end of the cultivation rope to keep it in a vertical position.

5) Generally 10 fragments of Gracilaria edulis are inserted on each rope. The distance between two rafts is kept at 2 m.

Chinese people provide fertilizer to seaweeds

Hungry fish and animals cause damage to seaweeds