Waste management is a problem to municipal authorities in many developing
countries where the facilities available to municipal authorities for
collection of waste are usually not enough as well as inadequate. As a
result, wastes are seen choking storm drainages, on the streets, scattered
around collection points or are dumped at unauthorized places which eventually
pose a threat to the environment and human health. There is therefore
the need to develop alternative approaches or methods of managing waste
in developing countries. One of such approaches of waste management is
to convert waste into compost/co-compost which is reported to support
plant growth (Nardi et al., 1994). Compost prepared from municipal
solid waste and co-compost, municipal solid waste blended with human excreta
are made up of humic substances that contribute to plant growth and development
(Piccolo et al., 1992; Chen and Aviad, 1990). Generally, the application
of organic waste to the soil causes an increase in soil chemical,
physical and biological properties (Tejada and Gonzalez, 2004).
The significant growth exhibited by plants grown in compost/co-compost
suggests that compost/co-compost provide more than just plant nutrients
(nitrogen, phosphorus and potassium) for growth (Atiyeh et al.,
It is envisaged that compost/co-compost probably contains plant growth
hormones, although this has not been firmly established. Whereas other
authors believe that the plant tissues cannot store these plant growth
hormones due to their unstable nature, another school of thought also
proposes that plant growth hormones are also present in compost, but are
synthesized by microorganism in the compost (Pizzeghello et al.,
2001; Glick, 2003). Another group of workers also proposed that the humic
acid and fulvic acid present in the compost/co-compost rather contain
or behave like plant growth hormones (Nardi et al., 1994). However,
it appears that there has not been much attention given to the extraction
and identification of the hormones from compost/co-compost.
Consequently, this study was carried out to isolate and identify plant
growth hormones present in compost and co-compost.
MATERIALS AND METHODS
Extraction of Plant Growth Hormones from Recycled Waste Materials
The study was carried out at Ecological Laboratory of the University
of Ghana, Legon between August, 2006-May, 2007 to isolate and identify
plant growth hormones in co-compost and compost. Plant growth hormones
were extracted from samples of municipal solid waste materials which have
been recycled into compost and dewatered human excreta mixed with the
recycled municipal solid waste to form co-compost. Samples of the compost
and co-compost were obtained from a composting site at Buobai near Kumasi.
Distilled water was first used to extract the plant growth hormones from
the compost and the co-compost, followed by the use of chilled 80% methanol.
The procedure described by Witham et al. (1971) was used in
the extraction. Ten grams of the material (either compost or co-compost
alone) was put into extraction bottle and 150 mL of distilled water was
added. The slurry was placed on a mechanical shaker for approximately
3 h. The water extract was allowed to stand and partitioned into solid
and liquid phases, after which the supernatant was decanted and centrifuged
for 6 min at 4000 rpm. The same volume of distilled water was added to
the solid residue in the extraction bottle shaken for 3 h on the mechanical
shaker before decanting and centrifuged at the same time and speed. This
process/procedure was repeated a third time and the total volume of the
extracts was bulked. In all 60 g of the ground materials was extracted
with 900 mL of distilled water.
Eighty Percent ( 80%) Methanol Extraction
In order to obtain a high concentration of any potential plant growth
hormones present in compost or co-compost, the procedure described by
Badr et al. (1971) and modified by Taylor et al. (2004)
was adopted. Fifteen grams of either compost or co-compost alone was weighed
into an extraction bottle and 150 mL of chilled 80% methanol was added.
This process was repeated six times to obtain a final/ total volume of
900 mL of 80% methanol. One hundred and fifty mililitres of 80% methanolic
slurry of either compost or co-compost alone was placed on a mechanical
shaker for approximately 24 h. The slurry was allowed to stand and partitioned/separated
out into the liquid and solid phases prior to decanting the supernatant.
The filtrate was then centrifuged for 6 min at 4000 rpm, filtered and
stored at -5°C in a freezer. The same volume of 80% methanol was added
to the residue in the extraction bottle and shaken for another 24 h after
which the solution was again decanted, centrifuged for 6 min at 4000 rpm
and filtered. The extraction process was repeated a third time following
which all the extracts were bulked and reduced to aqueous phase (48 mL)
using a rotary evaporator prior to storage in a refrigerator.
The pH of the aqueous extract from either the compost or co-compost
alone was adjusted to 2.5 by adding few drops of 1N H2SO4.
The acidified aqueous extract (48 mL) was transferred into a separatory
funnel and 380 mL of ethyl acetate was added, shaken and allowed to partition
into the organic and aqueous phases. The organic phase (ethyl acetate)
was separated from the aqueous phase into a clean conical flask before
the same volume of ethyl acetate (45 mL) was again added to the aqueous
phase, shaken, allowed to stand and the organic phase separated from the
aqueous phase. This process was repeated a third time, before the three
(3) aliquot of the organic phases were pooled together. The pH of the
remaining aqueous phase was adjusted to 7 by adding a few drops of 1 N
NaOH. This was then transferred into separatory funnel and the same volume
of water saturated n-butanol (40 mL) added. The funnel was shaken gently
and the solution allowed to separate into less dense water saturated n-butanol
phase (organic) and denser aqueous phases, before the two phases were
separated. This process was repeated three times using the aqueous phase.
All the water saturated n-butanol organic phases (which were presumed
to contain the cytokinins) were bulked, stored at -5°C for further
analysis and the aqueous phase was discarded. The ethyl acetate phase
(125 mL) was also transferred into a separatory funnel and an equal volume
of 5% NaHCO3 was added, shaken and allowed to partition into
organic (ethyl acetate) and aqueous (NaHCO3) phases. The two
phases were separated from each other and the process was repeated four
times. All the aqueous phases were bulked, stored at -5°C and the
organic phases (ethyl acetate) were discarded. The aqueous phase was adjusted
to pH 2.5 with drops of 1 N H2SO4. It was transferred
into a separatory funnel and an equal volume (45 mL) of dry diethyl ether
was added. The separatory funnel was shaken and the solution separated
into organic (diethyl ether) and aqueous (NaHCO3) phases on
standing before the aqueous phase was separated from the organic phase.
This step was repeated four times with the aqueous phase and each phase
bulked separately and stored at -5°C. The ether phase was presumed
to contain auxins. The stored acidic aqueous phase was poured into a separatory
funnel and the same volume (45 mL) of water saturated n-butanol was added.
The content was shaken and allowed to partition into organic (n-butanol
phase) and aqueous phases before the two phases were separated. The process
was repeated four times using the aqueous phase. All the organic phase
or the water saturated n-butanol phase were combined and stored at -5°C
in a refrigerator (this was presumed to contain gibberellins). The aqueous
phase obtained was discarded. All the partitioned stored extracts were
dried at 40°C using a rotary evaporator. These samples were then subjected
to thin layer chromatography for further purification and identification,
bioassay to determine the concentration of each extracted plant growth
hormones and spectra analysis.
Identification of the Plant Growth Hormones
Identification of plant growth hormones was done using bioassay, co-chromatography
(Co-TLC), colour of sprayed spots with reagents and spectrophotometer
Bioassay Using Water Extract
Serial dilutions of the water extract (100%) were prepared to obtain
C75 (75 mL extract +25 mL distilled water), C50 (50 mL extract +50 mL
distilled water), C25 (25 mL extract +75 mL distilled water) solutions.
Distilled water was used as control. Three petri dishes were prepared
for each concentration and the control. Each petri dish was lined with
filter paper and 10 grains of maize variety (Obaatanpa) were placed in
the dish and replicated three times. Five millilitres of the diluted extract
and the control (distilled water) was added to the corresponding labelled
petri dish. Elongation of the radicle and coleoptiles was measured at
24 h intervals.
Co-Chromatography of Extracts and their Standards
Dried extracts from the solvent partitioning were dissolved in 1 mL
of methanol, except the cytokinins fraction which was dissolved in 80%
methanol Standards for plant growth hormones: Benzyl Amino Purine (BAP),
Indole-3-Acetic Acid (IAA) and Gibberelleric Acid (GA3) for
each extract were also prepared. Each extract and its prepared standard
were spot loaded onto thin layer chromatographic plates of dimension 20x20
cm and silica gel (60254) of thickness 0.25 mm and developed
in isopropanol: ammonium hydroxide: water (84:4:4 v/v/v) to about eighteen
centimeters (18 cm) in a vertical direction, except for the cytokinins
which were run in butanol:ethyl acetate: water (90:10:10 v/v/v) solvent
system. The distance moved by the solvent system (solvent front) and the
spots were measured, after which the relative fluidity, Rf
values were calculated by dividing the distance moved by the solvent system
by the distance moved by the spots. The Rf values of the extracted
plant growth hormones were compared to the Rf values of the
Colour Reactions of the Separated Plant Growth Hormones from the Co-Chromatography
Following the procedure described by Herborne (1998) for identification
of cytokinins, each developed TLC plate containing the separated spots
of the standard and extracted hormones was sprayed with bromophenol blue,
observed when dried and again observed under the UV light or wavelength
254 nm. Furthermore, another developed TLC plates containing the separated
spots from both the standard and extracted hormones were each sprayed
with H2SO4: water (7:3) and heated at 120°C.
The plates were then observed under the UV light at wavelength 254 nm
(Unyayar et al., 2002). The other spot separated spots from the
TLC were exposed to ammonia vapour to form ammonia complex. The plates
were then observed under the UV light at a wavelength of 254 nm.
Determination of Concentrations of Plant Growth Hormones in Co-Compost/Compost
The methods used by Hedden (1993) and Witham et al. (1971)
were employed to determine the concentration of plant growth hormones
in the compost and co-compost.
The dried extracts were each dissolved in 2 mL of methanol and diluted
to different solutions of unknown concentrations with deionized water
(test solution). Standard GA3, BAP and IAA of different concentrations;
100 ppm (10-4 M), 10 ppm (10-5 M), 1 ppm (10-6
M), 0.1 ppm (10-7 M), 0.01 ppm (10-8M) were prepared.
Methanol and deionzed water were used as controls. Petri dishes were lined
with filter paper and arranged three each for a treatment in three replicates.
There were a total of 72 petri dishes for the test solution, 45 dishes
for the standard hormones and 18 for the controls.
Maize grains were soaked in tap water for about five hours to remove
any dirt or greasy substances. Fifteen grains of maize were placed in
each petri dish and placed in the dark at 25°C and 85-95% humidity
to germinate. Two days after germination, 10 uniform seedlings were selected
from each petri dish and 3 mL of each treatment solution was added to
each petri dish. The length of coleoptile, radicle and weight of cotyledon
(cytokinin) were measured after 5 days of growth in continuous florescence
light. The concentrations of the standard IAA and GA3 were
plotted against coleoptile length, while the concentrations of the BAP
were plotted against the weight of cotyledon to obtain standard response
curves. The concentration of each test solution was determined from the
Bioassay for Identification of Plant Growth Hormones
Figure 1-4 show the effect of water extract of compost
and co-compost diluted to different concentrations, on the elongation
of radicle and coleoptile of maize. There were significant differences
among treatments in the length of coleoptile and radicle of the seedlings
treated with C100, C75, C50, C25 and the control (p<0.05). It was observed
that C25 the most diluted solution of the extracts stimulated coleoptile
and radicle elongation more than the other solutions (Fig.
2-4) except in Fig. 1 where C50 performed better
than C25. The highly concentrated solutions, C75 and C100, inhibited coleoptile
and radicle elongation.
||Radicle elongation of maize treated with water extract
||Radicle elongation of maize treated with water extract
||Coleoptile elongation of maize treated with water extracts
||Coleoptile elongation of maize treated with water extracts
Confirmatory Test for the Separated Plant Growth Hormones
Table 1 shows the calculated Rf values, colour reactions
of isolated spots treated with the vapour of ammonia, bromophenol blue
and H2SO4: water (7:3 v/v).
The co-compost extract of auxin gave two Rf zones on TLC whiles
the compost extract gave only one. One of the Rf zones from
the co-compost extract (spot 2) had Rf value, 0.8475 which
was close to the Rf value of the standard IAA (0.8203). This
spot also produced the same colours as the standard when treated with
Bromophenol blue and ammonia. The other Rf zones also produced
colours similar to that of the standard. The co-compost and compost extracts
of cytokinins produced three and two spots, respectively. One spot each
from both the co-compost (spot 3) and the compost (spot 2) produced Rf
values of 0.7235 and 0.7156, respectively, as compared to the Rf
value of 0.7765 from the standard, BAP. All these spots produced blue
colouration with bromophenol blue. In another development, the spots from
the gibberellins gave Rf values that were different from the
standard, GA3. However spot 1 from co-compost (Rf
value 0.4133) and spot 2 from compost (Rf value 0.4334) were
almost the same and produced a yellow colour as compared to the yellow
to green colour of the standard.
||Co-chromatographic and colour formation from reagents
result (Confirmatory test)
||Standard curve showing the effect of IAA on elongation
of coleoptile of maize
Determination of Concentrations of Plant Growth Hormones in Co-Compost
and Compost Using Bioassay
Figure 5 shows the responses of coleoptile elongation
to the various concentrations of auxins (standard). The solution with
concentration of 5.71x10-5 M stimulated coleoptile elongation
more than the other solutions. The standard curve was used to calculate
the auxin concentration in the extracts of the compost and co-compost.
||Standard curve showing the effect of BAP on weight of
|| Standard curve showing the effect of GA3 on elongation
of coleoptile of maize
The auxins concentration was estimated from the curve to be 68.3 to 345.1
mg kg-1 in the co-compost and 42.0 to 248.8 mg kg-1
in the compost.
Figure 6 also shows the response of weight of cotyledon
to various concentrations of BAP (standard). The 1.47x10-6
M solution responded to highest production of weight of cotyledon than
the remaining solutions. Again the standard curve was used to calculate
the cytokinin concentration in the extracts of the compost and co-compost.
The concentration of cytokinins in co-compost was found to be 61.9 to
185.8 mg kg-1 and 33.1 to 198.3 mg kg-1 in the compost.
Figure 7 shows the response of coleoptile elongation
to the various concentrations of gibberellins (standard). The solution
with concentration of 5.71x10-5 M stimulated coleoptile elongation
more than the other solutions and a standard curve was used to calculate
the gibberellins concentration in the extracts of the compost as well
as the co-compost. The extracted gibberellins from co-compost and compost
were estimated as, 250.4 to 312.7 and 10.1 to 200.2 mg kg-1,
Methanol was used in the extraction of the plant hormones since alcohol
is a good all purpose solvent for preliminary extraction because it can
extract both polar and non-polar constituents (Harborne, 1998). Piccolo
et al. (1992) reported that the constituents of compost or co-compost
which are humins, fulvic acid and humic acid, each dissolves in different
solvents at different pH. Humins are not soluble in alkali (high pH),
acid (low pH) and in water (at any pH) since they are considered macro
organic substances due to their higher molecular weight. Humic acids are
organic acids which are soluble in water under alkaline condition only
and acid is precipitated in aqueous solution under acidic condition. Fulvic
acid on the other hand, is soluble in water under all pHs since it has
more hydrophilic (water-loving) end than lypophilic. Thus, 80% methanol
stands to be the best solvent since it is a polar organic solvent which
can dissolve both organic and inorganic substances. Partitioning the extracts
into acidic, neutral and basic pHs enabled the dissolution of the basic,
neutral and acidic components in the compost or co-compost such as humins,
humic acid, fulvic acid and their hormonal content (Harborne, 1998).
Of the several solvent systems used for the thin layer chromatography,
Isopropanol: Ammonium hydroxide: Water (90/10/10, v/v/v) gave the best
separation for auxins and gibberellins (Taylor et al., 2004 ).
Butanol: acetic acid: water (80/10/10, v/v/v) was selected for cytokinins
since it proved to be the best solvent system. Thin layer chromatography
apart from being used for purification and identification of compounds
also gives an idea of the number of components present in a sample (Sherma,
For auxins, two spots and one spot were identified from the co-compost
and compost, respectively. Gibberellins and cytokinins both gave three
well separated spots each for co-compost as compared to two each in the
The Rf (relative fluidity or Retardation factor) can be used
to identify the types of component or plant hormones present in an extract
(Sherma, 2002). Identification of the plant hormones was based on co-chromatography
with the authentic plant hormones. IAA standard produced Rf
value of 0.8203 as compared to 0.8475 of the extracted auxin spot from
Rf zone 2 of the co-compost. This indicates that the co-compost
contains a hormone which is similar to the IAA standard. Similarly, the
Rf values of the separated components from co-compost (0.7235)
and compost (0.7156) of the extract of cytokinins, compared favourably
to the standard BAP (0.7765). The Rf values for co-compost
and compost separated spots were not similar to the Rf value
(0.7376) of the standard gibberllins (GA3) and implies that
the isolated hormones may be other forms of gibberellins other than GA3.
The types of hormones extracted were confirmed by the colour of the spot
of hormones sprayed with bromophenol blue, sulphuric acid: water (7:3,
v/v) and exposure to ammonia and observed under UV light. The auxins spots
from co-compost and compost gave different shades of brown colour with
ammonia complex which closely matched the deep brown colour from the standard
(IAA). The standard, GA3 and BAP produced colours which matched
TLC spots from compost and co-compost. According to Harborne (1998), cytokinins
can be detected as blue spots after spraying with bromophenol blue or
silver nitrate reagent. Similarly, the standard method of detecting gibberellins
is by spraying plates of the separated spots or chromatogram with sulphuric
acid-water (7:3, v/v) and heating at 120°C; gibberellins appear as
yellow-green spots (Harborne, 1998).
The most diluted water extracts from the compost and co-compost were
more effective at stimulating growth than the other diluted extracts.
The 25% concentration of the water extract was the most effective in stimulating
the elongation of coleoptiles and radicles in both maize and cowpea. Plant
growth hormones are required in minute quantities to elicit particular
response (Moore et al., 1995). The extracts from both compost and
co-compost diluted to 25% concentration might have contained the optimal
amount of plant growth hormones needed to stimulate the elongation of
coleoptile and radicle in both maize and cowpea.
The study confirmed the presence of auxins, gibberellins and cytokinins
in both co-compost and compost. The effect of compost and co-compost on
crop growth and development may be the result of the interaction between
the nutrients present and plant growth hormones.
We wish to thank the Swiss National Centre for Competence in Research
North South ( NCCR NS) and International Water Research Management Institute
(IWMI) for providing funds for the research. We are also grateful to Mr.
Noah Adamtey for supplying the compost and co-compost used in the study.
The contribution by workers of the University of Ghana farm, Legon to
the successful completion of the study is also very much appreciated.