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Objective
The aim of our study was to expose the lignolytic activity of Phormidium sp. BDU5 on coir pith. In addition the growth promoting ability of degraded coir pith and cyanobacteria as basal fertilizer and the supernatant as foliar spray was investigated.
Summary
Lignin is the most abundant aromatic material on earth. Lignin is degraded by a narrower array of microbes than the other major biopolymers. Lignin biodegradation is central to the earth's carbon cycle because iignin is second only to cellulose in abundance and, perhaps more significantly, because lignin physically protects most of the world's cellulose and hemicellulose from enzymatic hydrolysis.
Research on lignin biodegradation has accelerated greatly, mainly because of the substantial potential applications of biolignolytic systems in pulping, bleaching, converting lignins to useful products etc. But to the best of our knowledge there is no attempt to use it in biofertiler application.
Despite numerous studies it is not entirely clear which microbes, other than certain fungi degrade the lignin polymer. The apparent inability of microorganisms to use lignin as sole carbon/energy source for growth precludes the isolation of lignin degraders by standard enrichment procedures and the use of growth on lignin a criterion for degradative ability.
Cyanobacteria are one of the oldest groups of organisms on earth. They are free living, photoautotrophic Gram negative prokaryotes well known for their adaptability to adverse environments. The viability and metabolic activity of these cyanobacteria, unlike those of heterotrophic organisms, are not subjected to reduction by the increase in the concentration of the pollutants that they may break down. Cyanobacteria have been shown to degrade both naturally occurring aromatic hydrocarbons like naphthalene and xenobiotics. They have been shown to degrade phenol also. So far there has been only

one report on the lignin degrading ability of cyanobacteria. That was done using the N2 fixing Anabaena azollae Ml 2 in coir pith. However, in this study the detection of lignolytic enzymes only was performed. The level of degradation of lignin and its further application was not performed.
Coconut (Cocus nucifera Linn.) is one of the most useful and extensively cultivated palm in tropical countries. Coir, the fibrous mesocarp of its fruit is a useful byproduct from this palm. The waste products of coir yarn industry are coir dust and coir pith which constitute about 70% of the husk. In spite of their limited use as soil conditioners, the quantity of coir dust produced is so enormous making its disposal difficult because of its lignocellulosic nature and slow degradation in natural environment. Because of the high lignin content and slow decomposition, coir pith has limited use in agriculture. It is estimated that the total quantity of coir dust produced in India is around 5 lakh tones annually.
Biofertilizers are eco-iriendly and are also called as microbial inoculants. Biofertilizers, as the name implies is of biological origin and serves the function of supplying the nutrients for effective plant growth. They are the most advanced biotechnology, necessary to support developing organic agriculture, sustainable agriculture and non-pollution agriculture. It is well known that the continued use and overuse of petrochemical based fertilizers and toxic pesticides have caused a detrimental effect to our soils, water supplies, food, animals and even human. The advantages of biofertilizers over chemical fertilizers are
a) low cost
b) enhancement of soil fertility
c) no change in the soil texture for longer period
d) no health hazard
Detailed description
Synopsis Process 1 - Study on the lignolytic activity of Phormidium sp. BDU5 on coir pith
a) Procurement of coir pith
b) Inoculation of cyanobacteria with coir pith
c) Chlorophyll estimation

d) Reducing sugar estimation
e) Spectral analysis
f) Lignin estimation
g) Holocellulosc estimation
Process 2 - Growth promoting effect of basal application and foliar application of coir pith based cyanobacterial biofertilizer on Coriandum sativum Linn. (Coriander)
a) Seed germination study
b) Field study

1. basal application
2. basal + foliar application
3. foliar application
4. control

c) Effect of biofertilizer on morphological parameters
d) Effect of biofertilizer on yield
Process 3 - Growth promoting effect of basal application and foliar application of coir pith based cyanobacterial biofertilizer on Vigna ungiculata Linn. (Cow pea)
a) basal application
b) basal ■+• foliar application
c) foliar application in different concentrations
d) control
e) Morphological parameters analysis
f) Yield comparative analysis
Process 1 - Study on the lignolytic activity of Phormidium sp. BDU5 on coir pith a) Procurement of coir pith
Coir pith was collected from a coir factory in Tiruchirapalli, Tamilnadu, India. After thorough washing under running tap water, detergent and double distilled water it was oven dried at 60 ° C overnight. Dried coir pith was autoclaved at 121 ° C 15psi for 20 min. in SANYO MLS-3788 autoclave. The purpose of autoclaving the coir pith was not

giving the physical pretreatmcnt but to ensure that the degradative action oa the coir pith
was exclusively by cyanobacteria.
Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stainer, R.Y.: Genetic
assignments, strain histories and properties of pure cultures of cyanobacteria. - J.
Gen. Microbiol. Ill: 1-61. 1979.
b) Cyanobacterial culture source and maintenance
Phormidium sp. BDU-5 was obtained from the germplasm collection of National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirapalli, Tamilnadu, India. This strain was selected since it was fast growing and they were able to grow luxuriantly in presence of lignoccllulosic material. For maintenance of the culture and the experiment BG-11 medium (Rippka et al. 1979) was used.
c) Inoculation of cyanobacteria with coir pith
The degradation experiments were carried out in 250 ml sterile Erlenmeyer flasks. Each flask received 70 ml of autoclaved BG-11 medium, 10 ml of culture suspension (of approximately 50 mg of dry weight) and 500 mg of coir pith (dry weight). Control cyanobacteria and coir pith alone were also maintained separately. All flasks were maintained at 25 ± 2 ° C and a light regime of 14 hr light and 10 hr dark. The light intensity was 28.5 (_) mol (photons) m"2 s"1 set using a Kyoritzu, illuminator, Japan. The experiment was carried out for 30 days and analytical experiments were performed periodically in triplicates.
d) Chlorophyll estimation (Mackinney 1941)
To washed cyanobacterial pellets 5 ml methanol was added and then incubated for 1 hr at 60 ° C. after incubation the samples were centrifuged and the supernatant was collected and absorbance was read at 663nm in Jasco V- 550 spectrophotometer, Japan.
Mackinney, G.: Absorption of light by chlorophyll sol. - J. Biol. Chem. 140: 314 -322, 1941.

e) Reducing sugar estimation (Miller 1959)
lml of supernatant was added to 1ml of dinitrosalicyclic acid reagent and boiled for 10 min. then it was allowed to cool and then the absorbance was taken at 540nm in Jasco V- 550 spectrophotometer, Japan.
Miller.: Dinitrosalicylic acid reagent for determination of reducing sugar. - Anal. Chem. 31:426-428, 1959.
f) Lignin estimation
Klason lignin estimation was performed to determine the extent of lignin degradation. 1 g sample was taken and lml of 72 % H2SO4 was added and incubated for lhr at room temperature with intermittent stirring. Then 560 ml of distilled water was added and then the sample was autoclaved. The samples were allowed to cool down and then they were filtered using Whattman No. 1 filterpaper. The samples were washed to remove the acid content and kept for drying. The weight of the samples was noted as the acid insoluble lignin content. Lignin content in both control coir pith and coir pith with cyanobacteria were performed and the reduction in lignin content was calculated comparing the initial lignin content.
Crawford, R.L., Crawford. D.L.: HC Lignin- Labelled lignocelluloses and 14C labelled Milled wood lignins: preparation, characterization and uses In: Wood, W.A. and Kellog. S.T. (ed.): Methods in Enzymology. Biomass Part B lignin, pectin and chitin. Pp.23-24. Acedemic press. Inc. 1988.
g) Holocelluose estimation
5g coir pith was taken in 100ml of distilled water to which lg sodium chlorite and lml acetic acid was added. It was subjected to rcllux lor lhr and again the same amount of sodium chlorite and acetic acid was added. This process was repeated five times. Then the sample was filtered and dried in hot air oven overnight and the weight of the holocellulose was noted.
TAPPI method, T 203 cm 99. Alpha-, beta- and gamma cellulose in pulp.

Process 2 - Growth promoting effect of basal application and foliar application of coir pith based cyanobacterial biofertilizer on Coriandum sativum Linn. (Coriander)
a. Field study
Cyanobacteria grown with coir pith were subjected to filtration to separate supernatant and pellet and were allowed to air dry. Basal application of cyanobacteria with coir pith was performed at a ratio 1 g/pot. Foliar spray was prepared by diluting the dried supernatant in water. The experimental set up was split into groups as given below
1) basal application
2) basal ; foliar application
3) foliar application
4) control
b. Effect of biofertilizer on morphological parameters of Coriandum sativum
Linn. (Coriander)
Once fully grown the effect of the coir pith based cyanobacterial biofertilizer was verified by analyzing the following physical parameters of the plant.
1) length of shoot
2) length of roots
3) length of leaves
4) breadth of leaves
5) number of leaves
6) number oi" branches
7) number of root hairs
c. Effect of biofertili/er on yield of Coriandttm sativum Linn. (Coriander)
The effect of the biofertilizer on yield was also studied by comparing the following parameters of control and test samples.
1) number of flowers
2) number of pods /plant
3) number of seeds / pod
4) weight of each pod
5) weight of seed / pod

Process 3 - Growth promoting effect of basal application and foliar application of coir pith based cyanobactcrial biofertilizer on Vigna ungiculata Linn. (Cow pea)
a) Field application
1) basal application
2) basal * foliar application
3) foliar application in different concentrations
4) control

b) Morphological parameters analysis
c) Yield comparative analysis
RESULTS AND DISCUSSION
Phormidium sp. showed good growth in the presence of the wood material and also degraded it substantially. The filamentous cyanobacterium showed higher chlorophyll content (Fig.l) in presence of lignoccllulosic waste when compared to control. This indicated mainly two facts. Firstly, the presence of coir pith was not inhibiting the growth of cyanobacteria. Secondly, the better growth than control shows that cyanobacteria is receiving some sort of nutrient from the coir pith which is yet to be studied.
The introduction of cyanobacteria with coir pith resulted in greater extent of reducing sugar release into the medium (Fig.2) when compared to the control samples (cyanobacteria and coir pith separately in media). Verification of composition of control and test sample revealed 22% lignin degradation whereas in control sample the degradation was negligible. However, the holoullulose estimation revealed that only 6.2% was degraded (Fig.3). These three results are interlinked. Lignocellulosic material is composed of lignin and holocellulose, which inclunes cellulose and hemicellulose. If the interlinking bonds are broken then the basic units of the holocellulosic part will be released as reducing sugars. Thus Fig 3 acts as a supp'.vting data to prove the increase in reducing sugar in test sample supernatant (Fig 2) and viceversa. Similarly El-Gammel et al. (1998) has reported variation in percentage of sugar released by the lignolytic activity of a Streptomyces sp. and three white rot fungi on various lignocellulosic substances.

Lignolytic activity of our study was well supported by the report of Malliga et al who revealed the presence of lignolytic enzymes in Anahaena azollae ML2 which were
induced on introduction to coir pith. These analysis performed on
cyanobacteria and coir pith proved the lignolytic'hormidium sp. BDU5.
In field study the morphological pararcompared as mentioned in
Table-1. Among all the parameters comparedh of leaves combination of
basal coir pith based biofertilizer and foliar application showed significant increase to
control samples. This proved that growth promowas present both in the basal
and foliar halves. Comparison of the yield (table-2)also revealed the same result. Thus
we were able to conclude that the combinan of basal and foliar foliar spray was best for
biofertilizer application. The interesting study was that the dried pellet (lg/pot) containing cyanobacteria and coir pithcame in touch with water
shoed growth of cyanobacteria. This is a characteris feature of cyanobacteria to escape
from the drought condition, which came handy in the field application. Once reactivated
daily moistening was sufficient to maintain the growth of cyanobacteria.
To confirm the growth promoting ability in the field study was further carried out in
Vigna ungiculata L. Further to study the effectay varying concentration of
foliar spray was applied along with basal application the results indicated that 1% dilution of foliar spray with lg/pot of basal application were the best for Vigna
ungiculata L as it showed significant increase ihological characters as well as yield.
From these results we conclude that BDU5 with coir pith is
suitable to be applied as both basal and foliar will help in reducing the
application of chemical fertilizers. It can be stored n the form of dried pellet (cyanobacteria with coir pith).
El-Gammel, A.A., Kamcl, Z., Adeep, Z., Helinygnocellulosicsubstances and production of sugars andiation intermediates by four selected strains. - Polymer degradation ar.v. 61: 535-542, 1998.

Fig.—3 Degradation of lignin and holoceilulose in the presence and absence of cyanobacteria. % of degradation calculated as the difference in lignin and holocellulose content between initial and the final. Results are average of three replicates.