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.