10

CHAPTER 2

BIODEGRADATION OF COIR WASTE BY

MARINE CYANOBACTERIA

Cyanobacteria are a phylum of aquatic bacteria that obtain their

energy through photosynthesis. They are often referred to as blue green algae

or blue bacteria, which are common in fresh water, brackish water and marine

environment as well as in different soils and ecosystems. They are

specialized for nitrogen fixation, and are gram-negative. Photosynthesis in

cyanobacteria generally uses water as an electron donor and oxygen as a by

product and some may also use hydrogen sulfide as found among other

photosynthetic bacteria.

Most cyanobacteria contain chlorophyll, which give the cells a

typical blue green to grayish brown colour. Cyanobacteria are utilized in the

form of biofertilizers because of their unique capacity to fix both carbon and

nitrogen from the atmosphere. These biofertilizers are also effective in

reducing the pollution.

11

2.1 Morphology of Phormidium sp.

Figure 2.1 Electron Microgram of Phormidium Sp.

Phormidium usually forms flat, slimy mats of tangled filaments, and

is similar in morphology to Lyngbya and Oscillatoria. However, Phormidium

(Fig. 2.1) mats do not dissociate as easily as those of Oscillatoria, and the

sheaths on Phormidium filaments are looser than the rigid sheaths of Lyngbya.

The mats are usually attached to benthic substrates, and can detach and float

to the surface. Occasionally, the filaments may be solitary or arranged in tufts

(Failk, 2004).

12

2.2 Morphology of Oscillatoria sp.

Figure 2.2 Electron Microgram of Oscillatoria Sp.

Oscillatoria is blue green algae common in freshwater

environments. The Fig.2.2 shows unbranched filamentous alga, occurring

singly or in tangled mats, derives its name from its slow, rhythmic oscillating

motion, which is thought to result from a secretion of mucilage that pushes

the filament away from the direction of excretion. Reproduction is by

fragmentation in which dead concave cells (separation discs) separate sections

of the filament (hormogonia).

13

2.3 Morphology of Anabaena sp.

Figure 2.3 Electron Microgram of Anabaena Sp.

Anabaena (Fig. 2.3) is a genus of filamentous cyanobacteria, or

bluegreen algae, found as plankton. It is known for its nitrogen fixing

abilities, and they form symbiotic relationships with certain plants. They are

one of four genera of cyanobacteria that produce neurotoxins, which are

harmful to local wildlife, as well as farm animals and pets. Production of

these neurotoxins is assumed to be an input into its symbiotic relationships,

protecting the plant from grazing pressure.

A DNA sequencing project was done by Herrero and Flores

(2008) which mapped the complete genome of Anabaena, which is 7.2

million base pairs long. The study focused on heterocysts, which convert

14

nitrogen into ammonia. Certain species of Anabaena have been used on rice

paddy fields, proving to be an effective natural fertilizer.

2.4 Biodegradation of lignin

Studies on plant evolution suggested that land plants originated from

simpler aquatic plants, which were exposed to a uniform hydrostatic pressure

from all sides. Land plants are always subjected to strong mechanical stresses

of gravity, wind and rain and thus they have acquired strong supporting

organs, such xylem cells reinforced with lignin against these stresses. The

stems of land vascular plants are composed of phloem and xylem tissues, and

the latter is comprised of lignified supporting conducting organs such as wood

fibers, tracheids and vessels.

Recent studies on the distribution of lignin in the respective layers

of the completely lignified cell walls of tracheids of black spruce have shown

that the average lignin concentration in the compound middle lamella is about

twice that in the secondary wall, but the volume of the secondary wall is

much greater than the volume of the middle lamella, and thus, 70-80% of the

total lignin is in the secondary wall, leaving only 30-20% in the compound

middle lamella and cell corner middle lamella regions. The syringyl lignin is

concentrated in fiber secondary walls, whereas the guaiacyl lignin is

concentrated in vessel walls (Angadi and Eshwarlal, 2004).

The lignin always occurs in intimate associations with the cell wall

polysaccharide and it is difficult to isolate chemically unchanged lignin

materials from plant materials. The lignocellulose degradation has focused on

15

the mechanisms of the process rather than the eco-physiology of the organism

involved. Lignin resists attack by most microorganisms and anaerobic

catabolism tend not to attack the aromatic rings. Aerobic breakdown of lignin

is slow and may take many days. Lignin is nature’s cement along with

hemicelluloses to exploit the strength of cellulose while conferring flexibility

(Ardelean and Zamea, 1996).

2.5 Biofertilizer

Biofertilizers are eco-friendly that supplies all the nutrient input of

biological origin for plant growth. Normally plant needs nitrogen and other

nutrients for its growth. As bacteria or cyanobacteria which fix atmospheric

nitrogen are widely used as biofertilizer they are called microbial

biofertilizers.Nowadays cyanobacterium is used in paddy fields as

biofertilizer in water logged condition. The cyanobacteria multiply, fix

atmospheric nitrogen and release it into its surroundings in the form of amino

acids, proteins and other growth promoting substance.

The bioorganic fertilizer can increase the quality and improve the

output to develop a green and sustainable agriculture in all kinds of plants.

Finally the production of biofertilizer is done by very easy and simple

techniques. Biofertilizer will help in saving our environment and conserving

the biodiversity.

The salient features of biofertilzers are

Low cost

Improving crop production

16

Highly biodegradable nature

Non pollutant to both aquatic and terrestrial ecosystem

Eco-friendly and helps small farmers

Biofertilizers from coir waste

The coconut palm is a monocot belonging to the genus Cocos. It is

a one monotypic genus having only one species ‘nucifera’. The coconut palm

is referred to as Kalpathara. The coconut fruit is encased within a hard shell

and outside the shell there is a thick covering of husk that consists of a

smooth water proof skin called the epicarp and a mesocarp which consist of

fibro-vascular bundles of coir embedded in a non-fibrous parenchymatous

corky connective tissue called as pith.

The coirwaste contains high lignin content, organic content and

water holding capacity. It can absorb water eight times its own weight. It

does not burn in its natural state and has low bulk capacity.

2.6 Review of Literature

Coir waste decomposition was enhanced by amending with urea

(Nagaraj et al., 1990). Uses of microbes such as mushroom fungi

(Ragunathan et al., 1996) and mushroom fungi in combination with

chemical amendments, (Ejini et al., 1995) have given promising results for

recycling of coir wastes. Pleurotus sp was tested for their ability to reduce

organic carbon and to increase the nitrogen content of plant residues like

paddy straw, sorghum stalks, cotton stalks, waste cotton, parthenium, bagasse,

17

coir pith, sawdust and paper waste (Nallathambi and Marimuthu, 1993).

Coir waste compost using ligno-cellulolytic fungi such as Pleurotus sps and

Calocybe indica was used as bio control agents (Ramamoorthy et al., 1999).

Udahyakumar (1984) had found the degradation of coir waste by

Streptomyces to be unsuccessful. However, Sivaprakasam (1984) suggested

that white rot Basidomycetes were known to grow on coir waste.

Biodegradation of coir waste by Pleurotus sajorcaju shows a drastic

reduction in lignin, cellulose and hemicellulose degradation of coir waste and

its conversion to organic manure was tested on the yield of bhendi (Suharban

et al., 1997).

Organic additives were used for composting coir waste such as fresh

cowdung, garden weeds, and sunhemp and this was enriched by using

inorganic additives, phosphate, micro-nutrients and also lignin degrading

inoculum of Pleurotus sajorcaju (Kadalli and Sussela Nair, 2000).

Application of composted coir waste with amendments had enhanced the

performances of bhendi mainly due to increased water holding capacity and

nutrient supplying ability of coir waste (Ramasamy and Kothandaraman,

1991).

Coir waste was used as a bedding material in homestead poultry

farm and analysis was made for coir waste enriched with poultry droppings

for its composition with respect to manurial value (Maheswarappa et al.,

2008).

18

Ramesh and Gunathilagaraj (1996) studied the degradation of

coir waste using earth worm along with chemical and microbial amendments.

Coir waste was decomposed using various starter materials such as Biogas

slurry (BGS), Cowdung slurry (CDS), Farm Yard Manure (FYM),

Mechanical Compost (MC) and soil (Ravichandra et al., 1996).

Experiments were conducted on the growth of tomato fruit by

applying different fertilizers in which coir waste as organic manure showed

highest ascorbic acid content. (Selvi and Ram Perumal, 1997).

Mathew et al. (2006) reported the technology to produce biogas

from coir waste. The highest quantity of gas was produced from the mixture

containing 80% cow dung and 20% coir waste.

A lignin degrading bacterium Pseudomonas KV03 was isolated

through enrichment technique with lignin/tannic acid as a sole carbon source,

for decaying coir wastes (Uma et al., 2007). Muthukaruppan et al. (1997)

studied the effect of aerobic and anaerobic treatment of coir pith and

groundnut shell produced 45% biogas production. Residual effect of fertilizer

nitrogen, coir waste and biofertlizers on availability of nutrients in soybean in

a mixed black soil was studied by Duraisamy and Mari (2001).

Saxena and Rai (2002) studied the effect of nitrogen on the

production of extra cellular degradative enzymes by Pleurotus sajorcaju (Fr)

on wheat straw and coir waste. Dakshinamoorthy (2001), carried out the

phosphorus use efficiency in finger millet with different sources of manure

along with the composted coir waste on calcareous and Non-calcareous soils.

19

Removal of fluoride by coconut coir waste carbon was studies by Dahiya and

Amarjeet Kaur (1999). Anita Das Ravindranath and Sarma (1998) studies

the application of microorganisms to enhance biodegradation of phenolic

compounds and to improve retting coir.

Ansu Joseph et al. (2001) studied the lignocellulose degradation by

oyster mushroom using different substrates like paddy straw, non retted coir

pith and retted coir.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and

Cyathus stercoreus ATCC36910 were evaluated for their ability to delignify

Bermuda grass (Cyanodon dactylon) stems and improve biodegradability.

Compositional and structural alterations in plant cell walls affected by the

fungi were determined by nuclear magnetic resonance spectroscopy, gas

chromatography of alkali-treated residues, microspectrophotometry and

electron microscopy (Akin et al., 1995). Potent cellulolytic fungal strains

Phanerochaete chrysosporium and Cladosporium sp. BK-II were isolated

from soil by carboxy methy1 cellulose enrichment. The strains were capable

of utilizing lignocellulosic wastes ie, rice husk, wheat bran and baggase as

substrate; untreated, steam treated and alkali treated at room temperature and

100°C (Koijam et al., 2000).

Lignin degradation by Paecilomyces inflatus, isolated form compost

samples consisting of municipal wastes, paper and wood chips was studied

following the mineralization of a synthetic C -labelled lignin (side-chain

labelled dehydrognenation polymer, DHP). Approximately 6.5% of the

synthetic lignin was mineralized during solid-state cultivation of the fungus in

20

autoclaved compost and 15.5% was converted into water soluble fragments.

Two strains of the Deuteromycete Paecilomyces inflatus were isolated from

compost samples consisting of municipal wastes, paper and wood chips.

Lignin degradation studies following the mineralization of a synthetic C

labelled lignin (side chain labelled dehydrogenation polymer, DHP)

(Turpienen et al., 2003).

Lignin is phenyl-propanoid monomer. The process of lignification is

initiated when a phenolic hydrogen atom is removed by peroxidase to form

phenoxy free radical. The monomers such as p-coumary1 alcohol, sinapy1

alcohol are converted to lignin. The radical centre can be decolorized to

aromatic and side chain carbons. Such radicals when coupled together

leading to polymerization, a beta 1- 4 bond is the most common inter unit

linkage in lignin (Rohella et al., 1997). Lignin degrading enzymes from fungi

can also used to degrade pollutants from industries such as chlorophenols,

nitrophenols and poly aromatic hydrocarbons. (Bogan and Lamar, 1995)

Gutierrez et al. (1996) analyzed lignin-polysaccharide complexes.

It was found that the aromatic fractions of lignin polysaccharide complexes

were derived from lignin and the break down of H (p-hyrdroxypheny1

propane), G (guaiacy1propane) and S (Syringy1 propane) lignin amounts

were identified.

A marine cyanobacterial strain with reasonable high growth rate,

nutritive value and acceptability by fishes and prawns had been identified and

formulated as an aquaculture feed that had been successfully tested in the lab

and being tested in the field (Subramanian, 1996). Cyanobacteria are of

21

particular interest in view of their extensive occurrence in paddy fields as

natural biofertilizers, helping to maintain higher nitrogen fertility (Singh

et al., 1996).

Cyanobacteria play a spectrum of remarkable roles in agriculture

especially in sustainable integrated agro ecosystems. As biofertilizer they can

contribute around 30kg of fixed nitrogen per hectare in flooded paddy fields

each season, the value could be increased dramatically with up-to-date

biotechniques. (Venkataraman and Shanmugasundaram, 1992).

The increase in yield and parameters of rice was observed with

consecutive application of cyanobacterial biofertilizers at reduced levels of

commercial nitrogen (Kannaiyan, 1981). Soil aggregation was reported to be

improved due to algal biomass (Kannaiyan, 1989).

Water soluble products isolated from Calothrix Sp. and Anabaena

Sp. had a rhizogenous effect and stimulated plant organs. The probable

nature of these substances has been likely that of gibberellin (Gupta and

Shukla, 1972). Cyanobacteria can be used for the reclamation of saline and

alkaline soils. (Kannaiyan, 1989).

Plot experiments conducted by Lavanya Priya (1997) and

Krishnaveni (1999) showed a considerable increase in the growth of rice

plants with coir waste based cyanobacterial biofertilizers. Srivastava et al.

(1996) reported that algae had a bigger history as food supplement, since the

nutritional properties have proved to be equal to conventional plant proteins.

Leena Tanja and Tasneem Fatma (1996) reported that cyanobacteria are

22

unique photosynthetic prokaryotes known to accumulate metal from the

surrounding media and may serve as models for treatment of water bodies.

Prunus and tomato plants pretreated in nutrient solutions showed a significant

increase of potassium in leaves and roots (Rosen and Carlson, 1984).

Hamed et al. (1988) reported that salinity effect on tomato resulted in the

decreased productivity.

Ardelean and Zamea (1996) reported that cyanobacteria have a

very versatile metabolism (photosynthesis either oxygenic or anoxygenic,

respiration, fermentation, nitrogen fixation etc.) thereby generating a response

against many environmental factors.

Suseela Bai et al. (1996) observed that the cyanobacteria had an

integral part in aquaculture, since these organisms are the biofertilizing agent.

They are potentially useful as food for human consumption in the

bioconversion of energy. Kulasooriya (1996) reported that Azolla had a

significant potential as a biofertilizer for rice, particularly in low-country wet

zone of Srilanka.

Ayman et al. (1988) used clay, sand, peat moss and saw dust in

various proportions as media for seed germination of tomato and observed

that saw dust decreased the time for germination.

The blue green algae fix N2 and also secrete Vit B12 , auxins and

ascorbic acid which may also contribute to the growth of rice plants (Singh,

1998). Zenat and Sharma (1990) observed the effect of application of

23

cyanobacteria in combination with the chemical fertilizer, diammonium

phosphate on the growth and yield of tomatoes.

Satapathy (1999) had reported that blue green algae contains

organic carbon and N2, that are considered to be efficient biofertilizers in

increasing soil fertility as well as productivity of rice. Blue green algal

extract are known to stimulate the growth of plants. This may be due to the

presence of growth hormone (Borowiztka et al. 1988)

Dutta et al. (1999) observed that recycling and reuse of coir pith to

promote plant growth in crops such as cowpea, green gram, cluster beans,

soyabean and mulberry was found to be useful to enhance crop and soil

productivity. An appropriate method was employed to utilize the coir waste

in a profitable manner to reduce the environmental hazards.

The combination of nitrogen fixing and non-nitrogen fixing algae

give more effect in terms of growth and yield of paddy. The non-nitrogen

fixing algae have also independently played an important role in the growth

and yield of paddy. The combined effect of fixing and non-nitrogen fixing

cyanobacteria have over all beneficial effects on soil enrichment, growth and

yield of paddy. The non-nitrogen fixing cyanabacteria, which enriched the

phosphorus and potassium content in the soil also, played a major role. This

may be due to release of growth promoting substance from cyanobacteria

(Selvarani, 1983).

There is a report on increase in germination percentage, shoot and

root length and biochemical contents like protein, carbohydrates and amino

24

acids in the seedlings Helianthus annus L. grown in effluent amended with

cyanobacteria than in the effluent without cyanobacteria. Hence, the above

investigation concludes that the cyanobacteria can be used to promote growth

of the plants (Rajula and Padmavathi, 2008).

Satapathy(2006) observed that the organic carbon, total nitrogen

and available phosphate of soil were increased due to the application of azolla

and bluegreen algae.

Prasad (1996) observed that the grain yield of rice was increased by

the use of Azolla in Nepal. At present nitrogen fixing cyanobacteria are used

as biofertilizer for rice cultivation in the state of Orissa (Nayak et al., 1996).

Angadi and Eshwarlal (2004) observed that the inoculation of cultured

cyanobacterial and mycorrhizal inoculum recorded highest shoot length, dry

weight, nitrogen and phosphate of Cajanus cajan.

Azolla can grow on a medium containing ammonium or nitrate as

well as on a N2 free medium. Nitrate seems to be a milder inhibitor of

nitrogen fixation and on the fourth day of incubation nitrogen fixing activity

was at nearly the same level as that without combined nitrogen (Ohmori and

Hattori, 1972).

The relative growth rate of the nitrogen fixing water fern is high and

generally it doubles its weight in 2-4 days. Because of its rapid growth, it

produces very high biomass in a short period (Kannaiyan, 1992).

Plant growth regulators isolated from blue green algae

are named as Algorum and they promote seed germination

25

(Fatima and Venkataraman, 1999). Twenty five strains of marine

cyanobacteria were subjected to screening for their ability to promote carrot

somatic embryogenesis (Takeyama and Matsunaga, 1996). Effect of extract

on the growth of shoot and roots has also been shown by Gupta and Shukla

(1969).

Immobilized as well as free living cyanobacterial application was

found to be distinctly advantageous over control, as it enhanced significantly

the various parameters of growth of rice plants such as shoot length and root

length, fresh and dry weight of the plants chlorophyll and protein content over

a period of 30 days (Sophia Rajini, 1955).

Primary leaves of bean plants showed an increase in protein content,

when treated with 2, 4-D (Shell et al., 1949) where as in normal leaves and

roots, the protein and free amino acid contents were reduced (Weller et al.,

1950).

Malliga et al. (2007) reported that Anabaena azollae while being

used as a biofertilizer exhibited lignolysis and released phenolic compounds

which induced profuse sporulation of the organism. This report gives the

usefulness of coir waste as carrier for cyanobacterial biofertilizer with

supporting enzyme studies on lignin degrading ability of cyanobacteria and

use of lignocellulosic coir waste as an excellent and inexpensive carrier for

cyanobacterial biofertilizer.

26

2.7 Materials and Methods

Organism used

Cyanobacterial strains of Phormidium sp. BDU-2, Oscillatoria sp.

BDU-5, and Anabaena azollae sp. ML-2, were obtained from the germplasm

collection of National Facility for Marine Cyanobacteria and cultured in

haffkins flask as stock (Fig 2.4) and coir waste was collected from coir

industry near Nagercoil (Fig 2.5).

Log phase culture of cyanobacteria was taken and mixed with coir

waste (1:5) and incubated for degradation process (Fig 2.6 – 2.11). During

this period the mixture was observed under microscope. The cyanobacterial

filaments were entrapped and immobilized in the coir particles as the

degradation process started (Fig 2.6).

27

DEGRADATION OF COIR WASTE BY USING CYANOBACTERIA

Figure 2.4 Cyanobacteria

Figure 2.5 Coir Waste

28

Figure 2.6 Immobilized Cyanobacteria with Coir Waste

Figure 2.7 Phormidium Sp. (BDU-2)

29

Figure 2.8 Oscillatoria Sp.( BDU – 5 )

Figure 2.9 Anabaena Sp. (ML- 2)

30

Figure 2.10 Mass Cultivation in Tray

Figure 2.11 Mass Cultivation in Fiber Tanks

31

Medium and growth condition

Cyanobacteria were grown in BG-11 medium under white

fluorescent light of 1500 lux at 25 ± 2°C. Table 2.1 shows the Composition of

BG-11 Medium and Trace Metal mix.

Table 2.1 Composition of BG-11Medium (g/l) (Bothe et al., 1968)

Name of Chemicals Concentration g/l

NaNO3 1.5

K2HPO4.3H2O 0.040

MgSO4.7H2O 0.075

CaCl2.2H2O 0.036

Citric acid 0.006

Ferric ammonium citrate 0.006

EDTA (Disodium salt) 0.001

Trace metal mix 1ml

Distilled water 1000ml

pH 7.2

Trace metal mix (g/l)

H3BO3 2.86

MnCl2.4H2O 1.81

ZnSO4.7H20 0.222

Na2MoO4.2H2O 0.390

CuSO4.5H2O 0.079

Co(NO3)2.6H2O 0.0494

Distilled water 1000ml

32

2.7.1 Estimation of organic carbon (Mc Cready, 1950)

Reagents

a. Potassium Dichromate (0.1 N)

b. Diphenylamine indicator

c. Ferrous ammonium sulphate (0.5 N)

d. Concentrated sulphuric acid

e. Orthophosphoric acid (85%)

Methodology

0.5g of dried homogenized soil sample was taken in a conical flask

and 10 ml of 1 N potassium dichromate was poured into the flask. It was

shaken well and then 20 ml of concentrated sulphuric acid was added. The

contents were mixed well by swirling the flask gently for 3 min. After

30 min, it was diluted with 200 ml of distilled water and 10 ml of phosphoric

acid and 1ml of diphenylamine indicator were added. The solution was

titrated with 0.5 N ferrous ammonium sulphate. The green color was changed

into blue in the middle and bright green at the end. The volume of ferrous

ammonium sulphate consumed was used to determine the organic carbon

content of the sample.

33

2.7.2 Estimation of nitrogen by Microkjeldal Method

Reagents

a. Sodium hydroxide (2N)

b. Potassium iodide (1%)

c. Mercuric iodide (0.2%)

d. Ammonium sulphate (3%)

Methodology

1g of Coir waste sample was dissolved in 0.5 ml of concentrated

sulphuric acid. 50 mg of catalyst was added along with the sample in

digestion flask and heated at 160°C – 180°C until apple green color was

developed. 2 ml of color reagent and 3 ml of 2 N sodium hydroxide were

added to the sample and incubated for 15 min at room temperature. The

yellow color developed was read at 490 nm using spectrophotometer.

2.7.3 Estimation of moisture content of coir waste

An empty porcelain crucible with lid was cleaned well and heated to

red hot over a Bunsen burner. The crucible was cooled in a dessicator and

weighed. 1g of coir waste sample was taken and heated in an oven at 100°C

for 6 hrs. The crucible was cooled in a dessicator and weighed. The

difference in weight showed the moisture content of a sample.

34

2.7.4 Estimation of ash content

An empty porcelain crucible was cleaned and heated over a Bunsen

burner to red hot. The crucible was cooled in dessicator and weighed. 1g of

sample was heated over a Bunsen burner till it was turned to ash. The

difference in weight of crucible with ash and empty crucible was calculated.

2.7.5 Estimation of cellulose (Updegroff, 1969)

a. Acetic/nitric Reagent (10:2)

b. Anthrone Reagent:200 mg in 600 ml of sulphuric acid

c. Sulphuric acid (67%)

Methodology

3 ml of acetic/nitric reagent was added to a known amount

(0.5g or 1g) of the sample in a test tube and mixed in a vortex mixer and

placed in a water bath at 100°C for 30 min, cooled and centrifuged for 15-20

min and the supernatant was discarded. Then the pellet was washed with

distilled water. 10 ml of 67% sulphuric acid was added and was allowed it to

stand for 1hr. 1 ml of the above solution was diluted to 100 ml .To 1 ml of

this diluted solution; 10mL of anthrone reagent was added and mixed well.

The tubes were kept in a boiling water bath for 10 min, cooled and the color

was measured at 630 nm using spectrophotometer.

35

2.7.6 Estimation of hemicellulose (Chang and Hudson, 1967)

Reagents

Potassium hydroxide (24%)

Methodology

Known weight of the coir waste sample was taken and this was

extracted with 24% potassium hydroxide for 4 hr at room temperature. This

was washed with water and allowed for drying. Weight of the dried material

was taken and the hemicellulose content was calculated.

2.7.7 Estimation of lignin (Bhat and Narayan, 2003)

Reagents

a. Sulphuric acid (72%)

b. Acid: water (5:1)

Methodology

200 mg of sample was weighed and 2ml of 72% sulphuric acid was

added. The mixture was kept in a water bath at 30-35°C and stirred

frequently. After one hour, the sample was diluted with 28 ml of acid -water

and was transferred into a 125 ml flask. This was hydrolyzed again by

autoclaving at 120°C for 1 hr. The hot solution was filtered through an

alundum or fritted glass crucible. The lignin residue was washed with water to

remove acid. The crucible containing the sample was dried to measure the

36

constant weight at 105°C and lignin was expressed as percent of the original

sample.

Dry ashing

5g of plant sample was accurately weighed in a crucible sample. It

was heated in an incinerator to completely volatilize much of the organic

matter (until no more of smoke is given out by the material as possible). The

crucible was transferred to a temperature control muffle furnace and the

temperature was adjusted to 550-600°C for 4-hrs. The crucible was removed

from the muffle furnace, allowed to cool and then the contents in crucible was

washed with 40-50 ml of dilute hydrochloric acid with the help of a pipette

and made up to 100 ml with dilute hydrochloric acid. Then the solution was

used for the estimation of minerals.

2.7.8 Estimation of potassium

Reagents

a. Ash solution

b. Potassium chloride solution: 74.6 mg of potassium chloride was

dissolved in 100 ml of distilled water.

Methodology

First the blank solution (double distilled water) was fed into the

flame photometer and the optical density was set to zero. Then the maximum

concentrated solution was fed and the optical density was set to 100. Then the

37

intermediate concentrated solution and the unknown solution was fed in to the

flame photometer and the amount of potassium present in the sample was

calculated and expressed in mg/g of dried plant sample.

2.7.9 Estimation of iron

Reagents

a. 3N Potassium thiocyanate

b. Saturated potassium persulphate

c. Concentrated Sulphuric Acid

d. Ferrous ammonium sulphate (5%)

Methodology

0.5 ml of ash solution was taken and made up to 7.7 ml using

distilled water. Then 0.4 ml of saturated potassium persulphate, 0.3 ml of

concentrated sulphuric acid and 1.6 ml of 3N potassium thiocyanate was

added and incubated for 10 min at room temperature. 7.7 ml of water, 0.4 ml

of saturated potassium persulphate, 0.3 ml of concentrated sulphuric acid and

1.6 ml of potassium thiocyanate was used as the blank. The red color

developed was read in a colorimeter against a reagent blank at 540 nm within

10 minutes.

38

2.7.10 Estimation of phosphorus

Reagents

a. Ammonium molybdate solution (1%)

b. Aminonaphthol sulphonic acid Solution: 0.5 g of-1-amino-2-

naphthol-4-Sulphuric acid, 30g sodium bisulphite and 6 g

sodium sulphite were dissolved in 100 ml of water.

c. Potassium dihydrogen phosphate (0.4g) was dissolved in 10 ml

of 10 N sulphuric acid and 1ml of chloroform was added as

preservative

Methodology

10 ml of standard potassium phosphate solution was diluted to 50 ml

with water. The aliquotes of standard solution (5-40 ml) was pipetted out into

50 ml volumetric flasks. 5 ml of molybdate reagent was added and mixed.

Then 2ml of aminonaphthol sulphonic acid reagent was added and the volume

was made up to 50 ml and the color was measured at 650 nm. 5 ml of ash

solution obtained by dry ashing, 5 ml of molybdate reagent was added and

mixed. 2ml of aminonaphthol sulphonic acid solution was added, mixed and

made up the volume to 50 ml. Similarly a reagent blank was prepared using

water in the place of the sample. It was allowed to stand for 10 min and the

colour developed was measured at 650 nm. From the standard curve the

phosphorus content was calculated and expressed in milligram per 100 gram

fresh tissue.

39

2.7.11 Estimation of total carbohydrate by Anthrone method (Hedge

Hofreiter, 1962)

Reagents

a. 2.5 N HCl

b. Anthrone Reagent: Dissolve 200 mg anthrone in 100 ml water.

c. Glucose (0.1%)

Methodology

100 mg of the sample was weighed in a boiling tube and was

hydrolyzed by keeping it in a boiling water bath for three hours with 5 ml of

2.5 N HCl and cooled to room temperature. Then it was neutralized with solid

sodium carbonate until the effervescence ceases and the volume was made up

to 10 ml and centrifuged. The supernatant was collected and 0.5 ml aliquots

were taken for analysis. 4 ml of anthrone reagent was added and kept for

eight min in a boiling water bath. The green color was read at 630 nm.

2.7.12 Estimation of sugar (Miller, 1952)

Reagents

a. Dinitro salicylic acid (0.5%)

b. Sodium hydroxide (2N)

c. Sodium potassium tartarate (1%).

40

Methodology

1 ml of reagent solution was added to 1 ml of cell free culture

filtrate and mixed well. The reaction mixture was kept in boiling water bath

for 10 min. The volume was made up to 10 ml using distilled water. The

optical density was read at 540 nm after cooling and the concentration of

sugar was determined by computing optical density against the standard curve

which was prepared using sugar concentration from 0.1 mg/ml to 10 mg/ml.

2.7.13 Estimation of ethanol (Caputi et al., 1968)

Reagents

a. Potassium dichromate (3.4 g)

b. Sulphuric acid (6.6N).

Methodology

5ml of potassium dichromate in sulphuric acid solution was added

to 1 ml of cell free culture filtrate and mixed well. The reaction mixture was

kept at 60°C for 20 min in water bath. The optical density was read at 600 nm

after cooling and concentration of ethanol was determined by computing

optical density against the standard curve, which was prepared using ethanol

concentration from 0.2 to 2.0 mg/ml.

2.7.14 Thin layer chromatography (TLC) (Wood and Kellogg, 1988)

To separate the compound, thin layer chromatography technique

was adopted.

41

Preparation of TLC Plate

Silica gel was mixed with distilled water (1:2) in order to make the

slurry. The slurry was poured into the hopper which was present on the

movable spreader. About 10 cleaned glass plates were placed on a plastic

template. The applicator was placed on the first plate and when the hopper

was rotated, the slurry was released. The thickness was adjusted to 0.5 mm

and the applicator was pushed uniformly over the plates. The plates were air

dried for 15-30 minutes and then was activated overnight in an oven at

110° C. 25 ml of culture filtrates of cyanobacteria alone, coir waste without

cyanobacteria and coir waste with cyanobacteria were dried and scraped and

phenolic compounds were extracted before loading on to TLC plates.

Phenol extraction

Reagent

80% ethanol.

Methodology

The dried powder was ground with 10 times the volume of 80%

ethanol. It was centrifuged at 10,000 rpm for 10 min and the supernatant was

collected and the pellet was re-extracted with 5 volumes of 80% ethanol. The

supernatants were pooled and dried in a speed vac concentrator.

Then the dried powder was reconstituted in hexane and was loaded

onto TLC plates. The mobile phase in TLC was ethy1 acetate: n-hexane

42

(1:10) and the stationary phase was silica gel. The TLC plates were dried and

then exposed to iodine vapor to confirm the presence of organic compounds.

2.8 Results and Discussion

The biodegradation of coir waste by three different species of

marine cyanobacteria such as Phormidium sp (BDU-2) Oscillatoria sp

(BDU-5) Anabaena azollae sp (ML-2) were monitored at regular intervals.

The changes in pH and temperature of coirwaste treated with cyanobacteria

were indicated the maturity of compost. The biochemical constituents of coir

waste such as lignin, cellulose and hemicellulose were analysed.

Table 2.2 Organic carbon content (%) of coir waste treated with three

different types of marine cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 41.25 ---- --- ---Coir waste + Phormidium (BDU-2) 41.25 40.4 38.7 30.2Coir waste + Oscillatoria (BDU-5) 41.25 39 35.8 25.7Coir Waste + Anabaena azollae (ML 2) 41.25 38.2 29.4 20.5

Table 2.2 shows that organic manure carbon content of coir waste

gradually reduced on 60 days of incubation with three different species of

Marine cyanobacteria. Maximum reduction was found out in Anabaena

azollae sp. followed by Oscillatoria sp. Initially fast reduction was also

observed in Anabaena azollae treated coir waste, compared to other

43

cyanobacterial species. Only limited reduction was noted in Phormidium sp

between 20 to 40 days of incubation.

This result was been supported by the view given by Nallathambi

and Marimuthu (1993), that Pleurotus platypus caused a great reduction in

organic carbon of cotton stalk and coir waste which has higher initial organic

carbon. The organic carbon varied depending upon the place and source of

coir waste. Pleurotus sajor caju also showed marked reduction of organic

carbon when compared with Trichoderma viridae and Bacillus sp

(Lakshmishree, 2001).

Table 2.3 Nitrogen content (%) of coir waste treated with three

different types of marine cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 0.45 ---- --- ---Coir waste + Phormidium (BDU-2) 0.45 0.76 1.92 5.86Coir waste + Oscillatoria (BDU-5) 0.45 0.86 2.43 7.52Coir Waste + Anabaena azollae (ML 2) 0.45 0.92 3.86 9.52

Table 2.3 describes the increase in the total nitrogen content in

treated coir waste in the order of Anabaena azollae sp > Oscillatoria sp >

Phormidium sp. This result shares the evidence given by Solon (2004) who

observed increase in nitrogen content when coir waste treated with Pleurotus

platypus and Polyporus species. It was also supported with Lakshmi Shree

(2001) who observed high nitrogen content in Pleurotus sajor caju.

44

Table 2.4 C: N ratio of coir waste treated with three marine

cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 75:1 ---- --- ---Coir waste + Phormidium (BDU-2) 75:1 70:1 53:1 48:1Coir waste + Oscillatoria (BDU-5) 75:1 67:1 48:1 32:1Coir Waste + Anabaena azollae (ML 2) 75:1 62:1 44:1 30:1

C: N ratio of the compost was considered as an index for assessing

the maturity of compost. Table 2.4 shows the reduction of C: N ratio

compared to that of control. In the present study C: N ratio was decreased

from 2nd week onwards. A better final reduction was noted on 60th day.

Higher amount of reduction was noted with Anabaena azollae coir waste and

slow reduction was observed in Phormidium sp.

This report was falling in line with the report given by Theradimani

and Marimuthu (2000) that C:N ration was 18:1 in coir pith decomposed by

Pleurotus platypus and Ramamoorthy et al. (2001) who recorded C:N ratio

of 25:1 with Trichoderma harzierum. Nagarajan et al (1998) and Jothi

Mari (2000) reported that C: N ratio of 24:1 would be used a good source of

organic manure for field crops.

45

Table 2.5 Moisture content (%) of coir waste in control and

experimental group

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 47.6 ---- --- ---Coir waste + Phormidium (BDU-2) 47.6 70.1 73.9 84.1Coir waste + Oscillatoria (BDU-5) 47.6 69.3 71.5 83.1Coir Waste + Anabaena azollae (ML 2) 47.6 67.9 78.1 80.1

Table 2.5 depicts that increase in moisture content of coir waste was

observed from 20 days of incubation. The high moisture content was observed

in Phormidium treated coir waste. The level of 1-3% of moisture content was

increased in coir waste treated Oscillartoria and Anabaena sp between 20

days to 40 days of incubation. The initial moisture content was uniformly

maintained about 45-50% during the inoculation of sample and it was

observed without sprinkling the water during 8th week. Thus the

cyanobacteria itself maintained the moisture content.

Table 2.6 Ash content of coir waste treated with three different species

of cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 99.6 99.4 --- ---Coir waste + Phormidium (BDU-2) 99.6 98.3 97.5 88.4Coir waste + Oscillatoria (BDU-5) 99.6 99.2 98.4 90.6Coir Waste + Anabaena azollae (ML-2) 99.6 98.7 97.5 94.8

46

Table 2.6 shows that ash content decreased in respective treatment

days. Ash content decreased in the preposition of coir waste + Phormidium

< coir waste+ Oscillatoria < Coir waste + Anabaena azollae. Coir waste was

rich in ash content having increased micronutrients such as potassium.

Table 2.7 Cellulose content (%) of coir waste in control and

experimental group

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 42 ---- --- ---Coir waste + Phormidium (BDU-2) 42 40 38 36Coir waste + Oscillatoria (BDU-5) 42 40 37 30Coir Waste + Anabaena azollae (ML-2) 42 38 35 32

Table 2.7 clearly indicates that the rate of degradation of coir waste

was faster in all treatments comparing to that of control. The degradation of

cellulose was in the order of 12% reduction for Oscillatoria treated coir

waste, followed by 6-10% reduction from the initial value of cellulose by

other treatments. This result was supported by Ansu Joseph (2001) that

P. sajorcaju was inferred to be the most efficient lignocellulose degrader.

Ramamoorthy (2003) found that Trichoderma haiziarum was capable of

producing maximum cellulose activity which was comparable with the results

of Trichoderma viridae.

47

Table 2.8 Hemicellulose content (%) of coir waste inoculated with

marine cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 47 ---- --- ---Coir waste + Phormidium (BDU-2) 47 26 12 1.28Coir waste + Oscillatoria (BDU-5) 47 30 18 1.3Coir Waste + Anabaena azollae (ML-2) 47 29 14 1.12

The Hemicellulose degradation of coir waste by marine

cyanobacteria was found to be faster when compared with other biochemical

constituents of coir waste. Anabaena azollae sp showed 92% degradation of

hemicellulose followed by Phormidium sp degraded 87% of hemicellulose.

This result was supported by Viswajith Malliga (2008) that the hemicellulose

content was drastically reduced at the end of 60 days. 48% degradation in

hemicellulose content of coir waste was reported in Fungi (Bhat and

Narayan, 2003).

Table 2.9 Lignin content (%) coir waste treated with cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 32 ---- --- ---

Coir waste + Phormidium (BDU-2) 32 31 29 14

Coir waste + Oscillatoria (BDU-5) 32 30 18 15

Coir waste + Anabaena azollae (ML-2) 32 30 26 21

48

Table 2.9 shows that lignin content decreased in all treated coir

waste compared to that of control. The lignin content was also reduced to

around 50-70% after 60 days period of degradation process by cyanobacteria.

This result was supported by Bhat and Narayan (2003) that lignin content of

coir waste was reduced from 48% to 32% by the action of Pleurotus

sajorcaju.

Coir waste is the product of coir industry which posses the high

amount of lignin (Rohella et al., 1977). Some actinomycetes degrade lignin

(Coling and Kirk, 1976). Pleurotus can degrade lignin and form a brown

precipitate (Gutierraz et al., 1996). Pseudomonas sp was able to degrade

acid, dioxin and fibre lignin which are the representatives of native lignin

(Uma et al., 2007). The hemicellulose and lignin content of coir waste were

degraded only by other microorganisms such as bacteria (Perestelo et al.,

1994) actinomycetes (Perestelo et al. 1994) fungi (Bhat and Narayan 2003)

and cyanobacteria (Malliga, 2008).

Table 2.10 Potassium content (ppm) of coir waste subjected to

cyanobacterial degradation

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 0.01 ---- --- ---Coir waste + Phormidium (BDU-2) 0.01 0.03 0.03 0.04Coir waste + Oscillatoria (BDU-5) 0.01 0.13 0.05 0.06Coir Waste + Anabaena azollae (ML-2) 0.01 0.02 0.035 0.05

Table 2.10 shows that potassium content of coir waste gradually

increased while treated with marine cyanobacteria. Potassium content was

49

slightly increased in the proportion of (coir waste + Oscillatoria ) > (Coir

waste + Anabaena azollae) > (Coir waste + Phormidium). Coir waste was rich

in ash content having increased micro nutrients such as potassium.

Table 2.11 Phosphorus content (%) of coir waste treated with three

different species of marine cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 0.78 ---- --- ---Coir waste + Phormidium (BDU-2) 0.78 0.79 0.81 0.84Coir waste + Oscillatoria (BDU-5) 0.78 0.82 1.00 1.48Coir Waste + Anabaena azollae (ML-2) 0.78 0.81 1.26 1.92

Table 2.11 showed that among all treatments, Anabaena azollae

treated coir waste increased phosphorus content. This result was supported by

Kadalli and Suseela Nair (2001) that Pleurotus sajorcaju showed higher rate

of phosphorus on 8th week of incubation.

Table 2.12 The iron content (ppm) of coir waste treated with three

species of marine cyanobacteria

Days of incubationTreatments0th

Day20th

Day40th

Day60th

DayCoir waste 0.07 ---- --- ---Coir waste + Phormidium (BDU-2) 0.07 0.07 0.08 0.08Coir waste + Oscillatoria (BDU-5) 0.07 0.072 0.08 0.09Coir Waste + Anabaena azollae (ML-2) 0.07 0.071 0.07 0.08

50

Table 2.12 shows the iron content (ppm) of coir waste treated with

three different species of marine cyanobacteria. The iron content of coir waste

slowly increased on 60 days of incubation. High iron content was observed in

Oscillatoria treated with coir waste. This result was supported by Lakshimi

shree (2001) that Pleurotus sajorcaju showed high rate of iron content of cow

dung.

Table 2.13 Carbohydrate content (%) in control and treated groups

of coir waste

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste 35.7 ---- --- ---Coir waste + Phormidium (BDU-2) 35.7 32.9 31.5 29.6Coir waste + Oscillatoria (BDU-5) 35.7 34.1 32.1 31.3Coir Waste + Anabaena azollae (ML-2) 35.7 33.9 30.8 27.6

Table 2.13 shows the reduction of total carbohydrate slowly. The

maximum reduction was observed in Anabaena azollae treated variable.

Pleurotus sajorcaju and P.amsidine degrading bacteria had high level of total

carbohydrate reduction (Malliga et al., 2007).

51

Table 2.14 Sugar content (%) of coir waste treated with three marine

cyanobacteria

Days of incubationTreatments

0th Day 20th Day 40th Day 60th DayCoir waste ---- --- ---Coir waste + Phormidium (BDU-2) 4 6.5 8.3 10.1Coir waste + Oscillatoria (BDU-5) 4 5 8 10Coir Waste + Anabaena azollae (ML-2) 4 6.9 8.5 10.5

Table 2.14 shows the production of reducing sugar in to the coir

waste treated sample since the microorganisms reduce the cellulose and total

carbohydrates into reducing sugar for their growth, the reducing sugar is

utilized which in turn gets oxidized. The formation of reducing sugar was

highly observed in Anabaena azollae. The rapid degradation of coir waste

indicates the presence of reduction in sugar and alcohols.

52

Table 2.15 Separation of components in coir waste using Thin Layer

Chromatography

S.No Sample Distance traveledby solvent (cm)

Distance traveledby solute (cm) Rf Value

1 Coir Waste 16

(1)(2)(3)(4)(5)

2.33.68.6

14.615.7

0.1440.2250.5370.9120.98

2 BDU-2 16.1

(1)(2)(3)(4)(5)(6)(7)(8)(9)

1.32.12.63.95.18.6

14.615.516

0.080.13

0.1610.2420.3170.5340.9070.9630.994

3 BDU-2+CW 15.4

(1)(2)(3)(4)(5)(6)(7)(8)

1.52.53.97.98.8

13.514.214.8

0.0970.160.25

0.5130.57

0.8770.9220.961

4 BDU-5 15.3

(1)(2)(3)(4)

1.92.88.8

14.8

0.1240.1830.5750.967

5 BDU-5 + CW 14.4

(1)(2)(3)(4)(5)(6)(7)(8)

1.82.43.44.47.59.8

12.314.4

0.1250.1660.2360.3050.520.680.851.00

6 ML 215.0

(1)(2)(3)(4)(5)

1.82.87.4

13.815.0

0.120.180.490.921.00

7 ML 2 + CW 14.7

(1)(2)(3)(4)

1.82.87.5

13.5

0.1220.190.51

0.918

53

The Table 2.15 highlights the TLC pattern of coir waste with

different cyanobacterial species confirming the degradation and release of

intermediate products. The poor degradation was observed in Phormidium

species. This was due to the presence of large number of intermediate

products.

5

5.5

6

6.5

Control 20 Days 40 Days 60 Days

Days of Incubation

pH

PhormidiumOscillatoriaAnabaena Azollae

Figure 2.12 The effect of biodegradation of coir waste on pH by three

different cyanobacteria

Figure 2.12 highlights the effect of biodegradation of coir waste on

pH when inoculated with different cyanobacterial species. It has been found

that the pH of the treated waste gradually increased on 40 days of incubation

but rapidly declined on 60 days of incubation. It indicates the maturity of the

compost.

54

05

101520253035404550

Control 20 Days 40 Days 60 Days

Days of Incubation

Tem

pera

ture

Phormidium Oscillatoria Anabaena Azollae

Figure 2.13 The effect of biodegradation of coir waste on temperature

by three different cyanobacteria species

The above figure highlights the effect of biodegradation of coir

waste on temperature when inoculated with different cyanobacteria species.

It has been found that the temperature of the treated waste gradually increased

up to 42°C on 40 days of incubation but rapidly declined on 60 days of

incubation. It indicates the maturity of the compost.