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MATERIALS AND METHODS

Selection of Marigold wastes


The methods of waste disposal are inefficient and yet
remain a major problem in many of the developing countries.
Utilization of these wastes profitably for the production of
value added products would make us realize their full
potential and avoid pollution too.
The materials selected for the study includes
a) Cow dung:
b) Cow dung was collected in and around Tennur,
Trichy.
c) Solid waste namely the Ash waste was collected from
Neyveli Lignite corporation, Cuddalore district near
neyveli.
d) Collection of earthworm
The epigeic (or) surface dwelling earthworms
Eudrilluseugineae were obtained from Periyar
Maniammai University, Thanjavur.
Preparation of experimental media:
The cow dung, Ash waste and two different earthworm
species were used for the waste degradation process. The
process was carried out for a period of 20, 40, 60, days. The
different kinds of waste preparation are as follows
Trough:- Ash waste + Soil + Flower waste + Earthworm
Fig.4: Ash Wastes subjected for vermicomposting
Vermicomposting of the ash waste:
The mixture was t ea over manually for 15 days in order
to eliminate volatile substances toxic to earthworms. After 15
days, 50 non-ciliated earthworms were introduced into
vermicompost plastic containers. The moisture content weas
maintained at 60-80%throughout the study period by periodic
sprinkling of adequate quantities of water. The containers
were kept in the dark under identical ambient conditions
(room temperature;25+30 C). Leaf additional waste was added
at starting stage during the study period.
Characterization of Vermicompost:
The entire process of vermicomposting of Ash waste was
carried out with the addition of cow dung and earthworm. The
process was carried out for a period of 60 days. During the
process, the variations in the physicochemical parameters
were observed.
The physic chemical parameters suchas pH, EC,
Alkalinity, TOC, TOM, TKN and C: N were analysed.
pH and electrical conductivity:
pH is a term used universally to express the intensity of
the acid or alkaline condition of the solution. It is a measure
of hydrogen ion concentration or precisely the hydrogen ion
activity. For determination of pH, 1:10 suspension of
Vermicompost and Bio composts were prepared by adding
100 ml distilled water to 10 gram of biocompost in a beaker,
the contents were stirred for half an hour at regular intervals.
The pH and EC were estimated using the pH meter (model:
Elico) and Electrical conductivity meter (Model: L180 Elico)
respectively.
Estimation of available nutrients
Estimation of organic carbon:
Walky and Black (1934) method: (Muthuvel &
Udayasoorian, 1998). The organic matter of the compost is
oxidized by potassium dichromate utilizing the heat of
dilution of sulphuric acid (130+500 C) . The unreacted
(excess) dichromate is determined by back titration with
standard ferrous ammonium sulphate solution using
diphenylamine as an internal indicator and total organic
matter calculated method described below:
Percentage of organic matter=% of carbon * 1.274
Estimation of Alkalinity:
1gm of sample with 50ml distilled water (1:5). These
50ml soil solution was taken in a conical flask and 2-3 drop of
phenolphthalein indicator was added. If the solution remains
colour less so alkalinity is 0. In case the colour changed to
pink titrated with 0.1NHCL in the burette untilled the colour
disappeared. Then further 2-3 drops methyl orange was added
to the same sample and titration was continued until the
yellow colour changed to pink colour at end point. The total
alkalinity of the soil was estimated by the direct titration of
the soil solution (1:10 ratio), with a strong acid using
phenolphthalein and methyl orange as indicators.
Estimation of Phosphorous:
1gm of sample with 100ml distilled water. This solution
taken in a conical flask and then 10ml of acid mixture (9:4
mixture of HNO3:HCLO4 ). The content transferred into swirl
it red NO2 fumes comes. The contents are further evaporate
until the volume is reduced to about 3-5ml but not to After
cooling add 20ml of distilled water. The solution is filtered
through whatman no.1 filter paper. Then add 10ml of
venadonmarlbdate reagent. Then make up to 50ml by using
the distilled water by using the distilled water. Read the
transmittance (or) obserbance of solution after 30 minutes at
420nm in spectrophotometer.

Isolation of microorganisms from vermicompostig samples


The isolation and identification of microorganisms from
the manure sample was done using the spread plate technique.
To 99ml of the sterile distilled water, 5g of the manure
sample was added aseptically and mixed thoroughly. From
this, 1 ml was transferred aseptically to 9ml of sterile distilled
water to obtain 10-1 – 10-6 dilutions. Then,1 ml from each
dilution was transferred aseptically into sterile petriplates.
Nutrienter agar medium was used to estimate the total
heterotrophic bacteria. The plates were further subjected for
incubation at 300C +20C for 24 hours for the isolation of
bacterial colonies.
Isolation of enzyme activities
Isolation of amylase producing bacteria from ash waste
1gm of compost was taken and was mixed with 9ml
sterile water and the dilution was noted as 10-1. 1ml of
suspension from dilution 10-1 was taken and was further
diluted up to 10-8 .0.1 ml suspension from dilution 106,107 and
108 were spread on the respective plates containing starch agar
by spread plate method. The plates were incubated at 370C for
24-48 hours. After incubation, iodine solution was added to
plates for detecting amylase producing microorganism.
Amylase producing microbes from a clear zone of hydrolysis
and their colonies were taken and stored in the slant.
Isolation of cellulose producing bacteria from ash waste
1gm of compost was taken and was mixed with 9ml
sterile water and the dilution was noted as 10-1. 1ml of
suspension from dilution 10-1 was taken and was further
diluted up to 10-8 .0.1 ml suspension from dilution 106,107 and
108 were spread on the respective plates containing cyapek
agar by spread plate method. The plates were incubated at
370C for 24-48 hours. After incubation, 1% congo red was
added to plates and allowed for 15 minutes and it washed
with water for three times. Then it was flooded with 1M
sodium chloride and allowed for 10minutes. Cellulose
producing microbes for the clear zone of hydrolysis and these
colonies were taken and stored in a slant.
Isolation of lipase producing microbes form Ash
1gm of compost was taken and was mixed with 9ml
sterile water and the dilution was noted as 10-1. 1ml of
suspension from dilution 10-1 was taken and was further
diluted up to 10-8 .0.1 ml suspension from dilution 106,107 and
were spread on the respective plates containing LB
tribulyrene agar by spread plate method. The plates were
incubated at 370C for 24-48 hours. The plates were observed
for zone of clearness.
Isolation of Protease producing bacteria from Ash waste
1gm of compost was taken and was mixed with 9ml
sterile water and the dilution was noted as 10-1. 1ml of
suspension from dilution 10-1 was taken and was further
diluted up to 10-8. 0.1 ml of suspension from dilution 106,107
and 108 were spread on the respective plates containing skim
milk agar by spread plate method. The plates were incubated
at 370C for 24-48 hours and observed for results.
Fourier-transforms spectrometers
Sample preparation
Sample preparationvary depending on the type of
sample. Liquid samples make good contact with the
germanium crystals and do not require any special treatment.
Solid samples, on the other hand, do not make good contact.
A Pressure Tower on the ATR accessory is used to squeeze
solid samples against the crystal surface.
Liquid samples: Apply 1-2 drops of liquid to the centre of
the germanium crystals with a disposable pipette (avoid
contact between the pipette and crystals). Allow the liquid to
spread out to make a thin film. Leave the pressure Tower
tilted back.
Solid samples: Place solids on the Centre of the germanium
crystal with a micro spatula (avoid contact between the
spatula and crystal). Carefully bring the pressure Tower
upright by pulling out the silver release knob and tilting the
Tower forward (do not let the tip fall on the crystal). Use the
spatula to position the sample underneath the Tower’s
pressure tip. Rotate the knob on top of the Tower clockwise so
that the pressure tip presses the sample onto the germanium
crystal. Stop rotating the knob when you hear a “click”. These
“clicks” are created by a slipclutch safety mechanism that
prevents the tip from applying too much pressure to the
crystal.
Typical operation involves the following steps:

 Setup spectrophotometer configuration


 Collect background spectrum
 Apply sample to ATR accessory
 Collect sample spectrum
 Find major peaks
 Print spectrum
 Clear sample spectrum and report
 Clean ATR accessory
Any waveform can be shown in one of two ways; either
in frequency domain or time domain. Dispersive IR
instruments operate in the frequency domain/ There are,
however, advantages to be gained from measurement in the
time domain followed by computer transformation into the
frequency domain.
If we wished to record a trace in the time domain, it
could possible to do so by allowing radiation to fall on a
detector and recording its response to over time. In practice,
no detector can respond quickly enough (the radiation has a
frequency greater than 1014Hz). This problem can be solved
by using interference to modulate the IR signal at a detectable
frequency. The Michelson interferometer is used to produce a
new signal of a much lower frequency which contains the
same information as the original IR signal. The output from
the interferometer is an interferogram.
The Michelson interferometer
Radiation leaves the source and is split. Half is reflected
to a stationary mirror and then back to the splitter. This
radiation has travelled a fixed distance. The other half of the
radiation from the source passes through the splitter and is
reflected back by a movable mirror. Therefore, the path
length of this beam is variable. The two reflected beams
recombine at the splitter, and they interface (e.g. for any one
wavelength, interface will be constructive if the difference in
path length is an exact multiple of the wavelength. If the
difference in path length is half the wavelength then
destructive interference will result. If the difference in path
length is half the wavelength then destructive interference
with result). If the movable mirror moves away from the beam
splitter at a constant speed, radiation reaching the detector
goes through a steady sequence of maxima and minima as the
interference alternates between constructive and destructive
phases.
Because all wavelengths emitted by the source are
present, the interferogram is extremely complicated. The
moving mirror must travel smoothly; a frictionless bearing is
used with electromagnetic drive. The position of the mirror is
measured by a laser shining on a corner of the mirror. A
simple sine wave interference pattern I produced. Each peak
indicates mirror travel of one half the wavelength of the laser.
The accuracy of this measurement system means that the IR
frequency scale is accurate and precise.
In the FT- IR instrument, the sample is placed between
the output of the interferometer and the detector. The sample
absorbs radiation of particular wavelengths. Therefore, the
interferogramcontains the spectrum of the source minus the
spectrum of the sample. An interferogram of a reference
(sample cell and solvent) is needed to obtain the spectrum of
the sample.
After an interferogram has been collected, a computer
performs a Fast Fourier Transformation, which results in a
frequency domain trace (i.e. intensity v/s wave number).
Pyroelytic detectors or liquid nitrogen cooled photon detectors
must be used. Thermal detectors are too slow.
Scanning Electron Microscope:
The control and final dried vermicompost samples were
analyzed to study the texture using the scaning electron
microscope. About 4-5 mg sample of particle size 300mm was
spread uniformly over the stub with the help of a double sided
adhesive tape and subsequently coated with gold using the
sputter coater at 2000X, 1000X and 500X magnification and
photography was done (Eldhoet al., 2012).