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Research Article
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Enrichment of Biogas Slurry Vermicompost with Azotobacter chroococcum and Bacillus megaterium |
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Natchimuthu Karmegam
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Kuppuraj Rajasekar
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ABSTRACT
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The present study has been conducted on the enrichment of biogas slurry vermicompost with microbial inoculants Azotobacter chroococcum and Bacillus megaterium, optimization of inoculum level and time inoculation during vermicomposting along with the survival rate of each microbial inoculant, the total microbial population and their correlation during the storage. On 0, 10, 20 and 30th day of inoculation showed decrease in the viable population of A. chroococcum and B. megaterium inoculated at the rate of 30, 35 and 40 mL-1 175 g of substrate, towards the progression of storage period of vermicompost, uniformly. The change of A. chroococcum and B. megaterium in the vermicompost (at 30, 35 and 40 mL-1 175 g substrate) with reference to the storage period (180 days) showed highly significant negative correlation (p<0.001). In all the four treatments, the viable population of A. chroococcum and B. megaterium at the rate of 30, 35 and 40 mL-1 175 g substrate from 0th day (after harvest) onwards showed statistically significant decline with that of storage period of vermicompost. This trend was observed uniformly for both the microbial inoculants, inoculated on 0, 10, 20 and 30th day of vermicomposting. In the present study, total microbial population in A. chroococcum and B. megaterium inoculated vermicompost was high during the initial phases of storage and then total microbial population declined towards the end. The vermicompost inoculated on 20th day of vermicomposting with A. chroococcum at the rate of 35 mL-1 175 g of substrate showed 11, 9, 8, 6, 3 and 0x107 cfu g-1 population of total microorganisms, respectively during 0, 15, 45, 75, 105 and 135th day of storage. Similar trend of results were obtained for B. megaterium inoculated vermicompost suggesting that the overall maintenance of total microbial population in vermicompost is similar in the vermicomposts with any microbial inoculants.
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Received: August 20, 2011;
Accepted: December 06, 2011;
Published: January 23, 2012
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INTRODUCTION
The global scientific community today is searching for a technology which should
be economically viable, environmentally sustainable and socially acceptable.
Vermiculture technology combines all these virtues and qualities together (Sinha
et al., 2010; Ansari, 2011). Vermicomposting
is a controlled biological process that involves the joint action of earthworms
and microorganisms to biotransform organic wastes. Vermicomposting is generally
more efficient than composting because earthworms feed on organic matter and
create conditions that favor the colonization and degradation of partially-decomposed
and fragmented organic material by aerobic microorganisms (Edwards,
2004). Worms have been found to proliferate Actinomycetes, Azotobacter,
Rhizobium, Nitrobacter and phosphate solubilizing bacteria significantly
in their end products. The study conducted by Singh (2009)
indicated higher values of Azotobacter (the nitrogen fixing bacteria)
and the Actinomycetes (the bacteria that increase biological resistance in plants
against pests and diseases) in vermicompost as compared to the conventional
aerobic and anaerobic composts (Singh, 2009). Combining
composting and vermicomposting also yields good results (Alidadi
et al., 2007). Vermistabilization is known to bring down the level
of pathogens in the raw material (Kumar and Shweta, 2011a).
It is known fact that from municipal solid waste including municipal sewage
sludge can be vermicomposted with earthworms (Zularisam
et al., 2010; Paul et al., 2011).
Number of researchers has observed increased proliferation of a variety of
microorganisms in the gut of earthworms, viz., bacteria, fungi and actinomycetes.
The organic matter that passes through earthworm gut results in increased levels
of microbial populations, activity and respiration and of enzymatic activity
and micro and macro nutrients (Kalam et al., 2004;
Prakash and Karmegam, 2010). Biofertilizers are the
Plant Growth Promoting Microorganisms (PGPMs) which by several mechanisms augment
plant growth (Mostafa and Abo-Baker, 2010; Abo-Baker
and Mostafa, 2011; Yadav et al., 2011). On
the soil, plant growth promoting microorganisms like Azospirillum, Azotobacter,
Phosphobacteria and Rhizobia could contribute to solubilize and/or to acquire
essential minerals, making scarce nutrients more available to the plant. Bacteria
of the genus Azospirillum are associative nitrogen-fixing rhizobacteria
that are found in close association with plant roots. They are able to exert
beneficial effects on plant growth and yield of many agronomic crops under a
variety of environmental and soil conditions. Soil bacteria of the genera Rhizobium,
Azorhizobium and Bradyrhizobium (collectively referred to as rhizobia)
interact with leguminous plants to form nitrogen-fixing nodules (Barassi
et al., 2007; Saikia and Jain, 2007). Recent
evidences on the use of biofertilizers for enriching the vermicompost showed
promising results on the enhanced quality of vermicompost (Kumar
and Singh, 2001; Kaushik et al., 2008).
The process of vermicomposting results in the increase of microbial diversity
and activity dramatically and the vermicompost produced could be a definitive
source of plant growth regulators produced by interactions between microorganisms
and earthworms which could contribute significantly to increased plant growth,
flowering and yields (Arancon and Edwards, 2009; Jayashree
et al., 2011). So, the addition or enrichment of microbial inoculants
such as biofertilizers definitely would provide an increased plant growth and
yield.
The studies on the survivability of the biofertilizers, Azotobacter chroococcum
and Bacillus megaterium are available (Sekar
and Karmegam, 2010). However, The studies on the microbial enrichment of
vermicompost with reference to the amount of inoculum required, time of inoculation,
survival rate of inoculated microorganisms in vermicompost during storage and
the relation of total microbial population with that of inoculated microorganisms
are not documented. Hence, the present study has been undertaken to enrich biogas
slurry vermicompost with the biofertilizers, A. chroococcum and B.
megaterium and to optimize the biofertilizer inoculum level and time of
inoculation during enrichment process of vermicompost and to assess the survival
rate of A. chroococcum and B. megaterium in relation to total
microbial population in the vermicompost.
MATERIALS AND METHODS
Collection of biogas slurry: Biogas slurry for the study was collected
from the biogas plant situated at Kottagoundanpatty, Salem (Dt.), Tamil Nadu
and used for the preparation of vermibed. One week aged biogas slurry was collected,
air dried and stored in polyethylene bags until use.
Collection of earthworms: The earthworm, Eudrilus eugeniae Kinberg for the study, originally collected from culture bank of the Department of Biology, Gandhigram Rural University, Tamil Nadu, India was mass multiplied in cowdung and used for the study. Mass multiplication of biofertilizers: The cultures of Azotobacter chroococcum (MTCC 446) and Bacillus megaterium (MTCC 453) were procured from Microbial Type Culture Collection (MTCC), Chandigarh and used for the present study. The organisms were revealed in the suggested broth medium and sub-cultured in Jensons and Nutrient Agar media, respectively. A loop full of A. chroococcum and B. megaterium was transferred, respectively to 100 mL of respective selective medium and incubated. After incubation, 10 mL of the inoculum was transferred to 1000 mL of respective broth and kept in shaking incubator for mass multiplication. Enrichment of vermicompost with biofertilizers: For enrichment studies, four different vermicomposting trials, each with six replicates were carried out by preparing the vermibeds. In this, the vermicompost was collected from all the vermibeds after 40 days and subjected to physico-chemical and microbiological analysis as per the standard procedures. E. eugeniae was introduced in all the vermibeds. The mass multiplied biofertilizer organisms at the rate of 30, 35 and 40 mL-1 175 g of vermibed substrates were added to each of the experiments, T1, T2, T3 and T4 on 0, 10, 20 and 30th day, respectively to find out the optimum inoculum level and time of inoculation that results in the maintenance of 1x107 viable cells per gram of vermicompost during storage. Statistical analyses: Data were subjected for Analysis of Variance (ANOVA) followed by Duncans multiple-range test to differentiate the significant difference between different treatments at the probability level of p<0.05 using SPSS® computer software for Windows (version 9.05). The relation between viable cell counts in different carrier materials and incubation days were carried out using Microcal Origin Computer Software (Version 6.1) and correlation coefficient (r) was calculated to know the level of significance of correlation.
RESULTS AND DISCUSSION
Microbial enrichment of vermicompost: Enrichment studies with A.
chroococcum: A. chroococcum inoculated at the rate of 30 mL-1
175 g of substrate showed the viable cells in 10-7 dilution upto
75, 90 and 105th day of storage in 10, 20 and 30th day of inoculation, respectively
(Table 1). A. chroococcum inoculated on 0, 10 and 20th
day showed decline in viable population during storage period. In 30th day of
inoculation, the viable population initially increased, then decreased. The
viable population of 5, 9 and 7 cfu x107 g-1 were observed
on 0, 15 and 30th day of storage of vermicompost which received A. chroococcum
inoculum on 30th day of vermicomposting. Then, colonies in the same vermicompost
showed decline upto 105 days of storage. The change of A. chroococcum
population in vermicompost which received the inoculum level of 30 mL-1
175 g substrate during the storage period showed negative correlation. The 30th
day of inoculation of A. chroococcum during vermicomposting showed viable
population in 107 dilution for a maximum storage period i.e., 105
days. The decrease of A. chroococcum population showed significant negative
correlation with storage period (p<0.001) (Fig. 1). The
viable population of A. chroococcum inoculated at the rate of 35 mL-1
175 g of substrate in different intervals showed decrease in viable population
during the storage of vermicompost. The 30th day inoculation of A. chroococcum
showed survival upto 105 days whereas, the A. chroococcum inoculated
on 10 and 20th day showed the viable population upto 90 and 105th day, respectively.
Table 1: |
The viable population of A. chroococcum inoculated
at the rate of 30 mL per 175 g of substrate in different intervals along
the storage period of vermicompost. Values are rounded of mean values of
three replicates |
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The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
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Fig. 1(a-d): |
The change of A. chroococcum population in the vermicompost
(at 30 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of A. chroococcum
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
Table 2: |
The viable population of A. chroococcum inoculated
at the rate of 35 mL per 175 g of substrate in different intervals along
the storage period of vermicompost. Values are rounded of mean values of
three replicates |
 |
The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
The 0th day inoculation showed viable population upto 90 days of storage period
(Table 2). The decrease of viable population upon storage
of vermicompost which received A. chroococcum inoculum at different
intervals were negatively correlated (Fig. 2) A. chroococcum
population at 35 mL-1 175 g substrate, inoculated on 30th day showed
negative correlation with a R2 value of 0.682 (y = -0.067+11.17)
which was highly significant at 0.1% level. The viable population of A. chroococcum
inoculated at the rate of 40 mL-1 175 g of substrate on the 0th day
of storage was 10, 10, 11 and 8x107 g-1 cfu in the vermicompost,
inoculated on 0, 10, 20 and 30th day of vermicomposting, respectively (Table
3). The population of A. chroococcum inoculated on 0 and 10th day
of vermicomposting showed 2 and 1 x107 g-1 cfu, on 105
and 120th day of storage, respectively. There after no viable cells were observed
upto 180 days. On 20 and 30th day of inoculation the survival of A. chroococcum
was observed upto 135th day and the subsequent observations showed no viable
population upto 180 days. The change of A. chroococcum population in
vermicompost (at 40 mL-1 175 g of substrate) with reference to the
storage period (180 days) showed negative correlation with the storage period
(p<0.001, Fig. 3).
Enrichment studies with B. megaterium: The viable population of B. megaterium inoculated at the rate of 30 mL-1 175 g of substrate in different intervals showed decrease in viable population during the storage of vermicompost. The 30th day inoculation of B. megaterium showed survival upto 150 days whereas, the B. megaterium inoculated on 10 and 20th day showed the viable population upto 120th and 135th day, respectively; 0th day inoculation showed viable population upto 105 days of storage period (Table 4).
The decrease of viable population upon storage of vermicompost which received
B. megaterium inoculum at different intervals was negatively correlated
(Fig. 4). B. megaterium population at 30 mL-1
175 g substrate, inoculated on 30th day showed negative correlation with a R2
value of 0.850 (y = -0.072+14) which was highly significant at 0.1% level.
The B. megaterium inoculated at the rate of 35 mL-1 175 g
substrate showed the viable cells in 10-7 dilution upto 135, 150
and 165th day of storage in 10, 20 and 30th day of inoculation, respectively
(Table 5).
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Fig. 2(a-d): |
The change of A. chroococcum population in the vermicompost
(at 35 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of A. chroococcum
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
Table 3: |
The viable population of A. chroococcum inoculated
at the rate of 40 mL per 175 g of substrate in different intervals along
the storage period of vermicompost. Values are rounded of mean values of
three replicates |
 |
The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
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Fig. 3(a-d): |
The change of A. chroococcum population in the vermicompost
(at 40 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of A. chroococcum
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
Table 4: |
The viable population of B. megaterium inoculated at
the rate of 30 mL per 175 g of substrate in different intervals along the
storage period of vermicompost. Values are rounded of mean values of three
replicates |
 |
The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
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Fig. 4(a-d): |
The change of B. megaterium population in the vermicompost
(at 30 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of B. megaterium
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
Table 5: |
The viable population of B. megaterium inoculated at
the rate of 35 mL per 175 g of substrate in different intervals along the
storage period of vermicompost. Values are rounded of mean values of three
replicates |
 |
The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
B. megaterium inoculated on 0, 10 and 20th day showed a decline in viable
population during storage period. In 30th day of inoculation, the viable population
initially increased, then decreased.
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Fig. 5(a-d): |
The change of B. megaterium population in the vermicompost
(at 35 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of B. megaterium
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
The viable population of 10, 15 and 13 cfux107 g-1 were observed on 0, 15 and 30th day of storage of vermicompost which received B. megaterium inoculum on 30th day of vermicomposting. Then, colonies in the same vermicompost showed decline upto 165 days of storage. The change of B. megaterium population in vermicompost which received the inoculum level of 35 mL-1 175 g substrate during the storage period showed negative correlation. The 30th day of inoculation of B. megaterium during vermicomposting showed viable population in 10-7 dilution for a maximum storage period i.e., 165 days. The decrease of B. megaterium population showed significant negative correlation with storage period (p<0.001) (Fig. 5).
The viable population of B. megaterium inoculated at the rate of 40
mL-1 175 g of substrate on the 0th day of storage was 12,13, 14 and
11x107 g-1 cfu in the vermicompost, inoculated on 0, 10,
20 and 30th day of vermicomposting, respectively (Table 6).
The population of B. megaterium inoculated on 0th day and 10th day of
vermicomposting showed 1 and 2x107 g-1 cfu on 120 and
135th day, respectively there after no viable cells were observed upto 180 days.
On 20 and 30th day of inoculation the survival of B. megaterium was observed
upto 150 and 165th day, respectively and the subsequent observations showed
no viable population upto 180 days. The change of B. megaterium population
in vermicompost (at 40 mL-1 175 g of substrate) with reference to
the storage period (180 days) showed that negative correlation with storage
period (p<0.001, Fig. 6). The viable population of B.
megaterium inoculated at the rate of 40 mL-1 175 g of substrate
on 30th day of vermicomposting showed 14, 11, 9, 7, 4 and 2x10-7
g-1 cfu during 30, 60, 90, 120, 150 and 165th day, respectively which
was negatively significant at p<0.001 (y = -0.081x +14.29, R2 =
0.876) (Fig. 6). On 0th, 10th and 20th day of inoculation,
decrease in the viable population of B. megaterium towards the progression
of storage period of vermicompost was observed.
Table 6: |
The viable population of B. megaterium inoculated at
the rate of 40 mL per 175 g of substrate in different intervals along the
storage period of vermicompost. Values are rounded of mean values of three
replicates |
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The values with same superscript letters between columns are
not significantly different at 5% level (p<0.05) by Duncans Multiple
range test |
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Fig. 6(a-d): |
The change of B. megaterium population in the vermicompost
(at 40 mL-1 175 g of substrate) during storage period (180 days).
Error bars Indicate±SD; Time of inoculation of B. megaterium
during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d)
30th day |
Whereas, the 30th day inoculation of B. megaterium showed increase in
viable population within 15 days and declined gradually towards the termination
of the study. Among the different time of inoculation of B. megaterium,
the 30th day showed viable population of 2x107 g-1 cfu
upto 165 days. In all the four treatments, the viable population of B. megaterium
from 0th day onwards showed statistically significant decline with that of storage
period of vermicompost (Table 6, Fig. 6).
Earthworm activity is closely associated with microbial activity. Lavelle
(1983), is of the opinion that there may exist competition between microorganisms
and earthworms for easily digestible and energy rich substrates. Such competition
may depend on availability of nutrients in the medium. Contrary to this, earthworms
may derive benefit from microorganisms when they have to survive on materials
rich in cellulose or hemicellulose. So, there exists mutualistic relation between
earthworms and microorganisms. The enrichment of vermicompost with nutrients
and microorganisms using different organic and inorganic materials and microbial
inoculants is now popularizing, due to the advantage of using the enriched
vermicompost (Singh and Sharma, 2002; Anilkumar
et al., 2007; Hashemimajd and Golchin, 2009).
But these studies have not described the standardized protocol for enriching
vermicompost with microbial inoculants such as biofertilizers.
The enrichment of vermicompost was done in the present study by inoculating the biofertilizer inoculants, A. chroococcum and B. megaterium at the rate of 30, 35 and 40 mL-1 175 g of substrate. This was done to find out the optimum level of inoculum required for the maintenance of 1x107 viable cells in the vermicompost. The inoculation was done on 0, 10, 20 and 30th day of vermicomposting to assess whether the time of inoculation had any effect on survival rate of biofertilizer inoculants during storage. At the same time, the survival rates of each inoculant were correlated with total microbial population in the vermicompost to study the influence of other microbial groups on biofertilizer inoculants. The change of A. chroococcum and B. megaterium in the vermicompost (at 30, 35 and 40 mL-1 175 g substrate) with reference to the storage period (180 days) showed highly significant negative correlation (p<0.001). In all the four treatments, the viable population of A. chroococcum and B. megaterium at the rate of 30, 35 and 40 mL-1 175 g substrate from 0th day (after harvest) onwards showed statistically significant decline with that of storage period of vermicompost.
The enrichment of vermicompost with the addition of nutrient rich substrates
was demonstrated by Daniel et al. (2010) where
their results reveal that the leaves of Gliricidia sepium and Leucaena
leucocephala can be converted into microbial-and nutrient-rich vermicompost
using E. fetida. Hashemimajd and Golchin (2009)
studied the effect of iron-enriched vermicompost on growth and nutrition of
tomato and reported that total and available forms of iron in iron-enriched
vermicomposts as well as in tomato tissues increased by an increase in the proportion
of iron refuse in vermicompost. Some of the authors tried microbial inoculants
for hastening the process of vermicomposting or for enriching the vermicompost.
The inoculation of microbial consortia like jeevamrutha and
cow dung together with organic substrates significantly enhances the microbial
density throughout the process of decomposition during vermicomposting (Veeresh
et al., 2010). The inoculation of consortium of microorganisms Aspergillus
niger, P. sajor-caju, Azotobacter chroococum, Trichoderma
harzianum not only accelerated vermicomposting of crop residues and farm
yard manure but also enriched the quality of product (Singh
and Sharma, 2002). During the incubation period the inoculated bacterial
strains proliferated rapidly, fixed nitrogen and solublised added and native
phosphate (Kumar and Singh, 2001).
Press-mud alone and in combination with other by-products of sugar processing
industries was pre-decomposed for 30 days by inoculation with combination of
Pleurotus sajorcaju, Trichoderma viridae, Aspergillus niger
and Pseudomonas striatum, followed by vermicomposting for 40 days with
the native earthworm, Drawida willsi. The study conducted by Prabha
et al. (2007) reported that the enrichment generally had a significant
effect on the nutrient content, especially for N, P, K, Mg and Mn. Eudrilus
compost, when treated with Azospirillum and P-solubilising organisms,
gave a N-content of 2.08% which was significantly higher than the N-content
of uninoculated Eudrilus compost (1.8%). The nitrogen was enriched appreciably
by Azospirillum. The enrichment increased progressively when Azospirillum
inoculation was supplemented with phosphate solubilising culture, a beneficial
additive to obtain good quality compost, rich in N (Tiwari
et al., 1989). An increase in N-content due to microbial inoculation
was reported by Rasal et al. (1988). The P-contents
were significantly higher when inoculated with Azospirillum and P-solubilising
organisms (1.76%) than in uninoculated compost (0.72%). The mechanism of conversion
of insoluble P by P-solubilising organisms to available forms include altering
the solubility of inorganic compounds to the ultimate soluble form by production
of acids and H2S under aerobic and anaerobic conditions and by mineralizing
organic compounds, with the release of inorganic phosphate (Rasal
et al., 1988).
Kumar and Shweta (2011b) studied the enhancement of
wood waste decomposition by microbial inoculation prior to vermicomposting.
The timber wastes which were inoculated with different combinations of the fungi
Phanerochete chrysosporium, Trichoderma reesei, Aspergillus
niger and the bacteria Azotobacter chroococcum (MTCC 3853) and Bacillus
cereus (MTCC 4079) and incubated at 28-30°C in a mechanical composter.
The inoculation enhanced the degradation of timber wastes, increased total nitrogen
and improved the quality and enhanced production of vermicompost generated with
the native earthworm Drawida willsi Michelsen. Their study showed that
microbial predecomposition of timber wastes to produce quality vermicompost
is a feasible technology.
However, the above studies were focused on either the enhancement of vermicomposting process or the nutrients. These studies have not described the standardization of amount of inoculum and time of inoculation for the maintenance of 1x107 g-1 viable population of A. chroococcum and B. megaterium and their survival during storage in comparison with the total microbial population. From the results of this study, it is concluded that the biofertilizer inoculants, A. chroococcum and B. megaterium at the rate 35 mL-1 175 g substrate on 30th day of vermicomposting is the optimum inoculation level and time for the maintenance of 1x107 viable cells in the vermicompost is maximum number of days during storage. The study also reveals that the microbial inoculants inoculated at the later stage of vermicomposting survive for long period.
TOTAL MICROBIAL POPULATION IN THE VERMICOMPOST
Total microbial population in the vermicompost inoculated with A. chroococcum
at 30, 35 and 40 mL-1 175 g substrate: The total microbial population
in A. chroococcum inoculated vermicompost was high during the initial
phases of storage and then total microbial population (1x107 cfu
g-1) declined towards the end (Table 7). The bioinoculation
on 0th day of vermicomposting with A. chroococcum at the rate of 30 mL-1
175 g substrate showed 14, 11, 6, 1 and 0x107 g-1 cfu
population of total microorganisms, respectively during 0, 30, 60, 90 and 120th
day of storage. No viable population of total microorganisms in 10-7
dilution on 105, 120, 135 and 150th day of storage was observed, respectively
in 0, 10, 20 and 30th day inoculation of A. chroococcum at the rate of
30 mL-1 175 g substrate. On 20th day of vermicomposting with A.
chroococcum at the rate of 35 mL-1 175 g of substrate showed
15, 11, 8, 4, 1 and 0x107 g-1 cfu population of total
microorganisms, respectively during 15, 45 75, 105, 135 and 165th day of storage
(Table 7). Similar observations were made for the inoculum
level of 40 mL-1 175 g substrate.
Table 7: |
The total microbial population change in A. chroococcum
and B. megaterium inoculated vermicompost during storage period.
Values are rounded of mean values of three replicates |
 |
Total microbial population in the vermicompost inoculated with B. megaterium at 30, 35 and 40 mL-1 175 g substrate: B. megaterium inoculated at the rate of 30 mL-1 175 g of substrate on 0th day of vermicomposting showed 16, 13, 9, 5, 2 and 0x107 g-1 cfu population of total microorganisms, respectively during 0, 30, 60, 90, 120 and 150th day of storage. The total microbial population in B. megaterium inoculated vermicompost was high during the initial phases of storage and then total microbial population (1x107 g-1 cfu) declined towards the end (Table 7). The microbial inoculants inoculated on 20th day of vermicomposting with B. megaterium at the rate of 35 mL-1 175 g of substrate showed 17, 15, 11, 7, 3 and 0x10-7 g-1 cfu population of total microorganisms, respectively during 15, 45 75, 105, 135 and 165th day of storage. B. megaterium at the rate of 35 mL-1 175 g of substrate inoculated on 20th day of vermicomposting showed 16, 18, 15, 12, 8, 3 and 0x10-7 g-1 cfu population of total microorganisms, respectively during 0, 30, 60, 90, 120, 150 and 165th day of storage. CORRELATION OF TOTAL MICROBIAL POPULATION WITH STORAGE PERIOD The change of total microbial population in A. chroococcum inoculated vermicompost (at 30 mL-1 175 g substrate) as a function of storage period (180 days) showed negative correlation in all the four treatments that received A. chroococcum during 0, 10, 20 and 30th day of vermicomposting. The vermicompost inoculated with A. chroococcum on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient (r): -0.9840 (y = -0.1344x+14.4). Similar results were recorded for 10th day inoculation: r = -0.9794, y = -0.1119x+15.231; for 20th day inoculation: r = -0.9794, y = -0.1224x+16.909; for 30th day inoculation: r = -0.9541, y = -0.1152x+17.833. The total microbial population change in A. chroococcum inoculated vermicompost (at 35 mL-1 175 g substrate) as a function of storage period (180 days) showed negative correlation in all the four treatments that received A. chroococcum during 0, 10, 20 and 30th day of vermicomposting. The vermicompost inoculated with A. chroococcum on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient of r = -0.9913, y = -0.1089x+13.422. Similar results were recorded for 10th day inoculation: r = -0.9757, y = -0.1079x+14.874; for 20th day inoculation: r = -0.9948, y = -0.1188x+16.818; for 30th day inoculation: r = -0.9542, y = -0.127x+20.731. The vermicompost inoculated with A. chroococcum at the rate of 40 mL-1 175 g substrate on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient of r = -0.991, y = -0.1089x+13.422. Similar results were recorded for 10th day inoculation: r = -0.9710, y = -0.1008x+14.421; for 20th day inoculation: r = -0.994, y = -0.1255x+18.136; for 30th day inoculation: r = -0.9558, y = -0.1193x+18.679. Similar trend of results were observed for B. megaterium during vermicomposting. The vermicompost inoculated with B. megaterium (at 30 mL-1 175 g substrate) on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient of r = -0.9142, y = -0.0861x+12.132. Similar results were recorded for 10th day inoculation: r = -0.942, y = -0.1011x+14.637; for 20th day inoculation: r = -0.972, y = -0.1051x+16; for 30th day inoculation: r = -0.9538, y = -0.1088x+17.484. B. megaterium inoculated at 35 mL-1 175 g substrate on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient of r = -0.9913, y = -0.1089x+13.422. Similar results were recorded for 10th day inoculation: r = -0.9757, y = -0.1079x+14.874; for 20th day inoculation: r = -0.9948, y = -0.1188x+16.818; for 30th day inoculation: r = -0.9543, y = -0.127x+20.731. The vermicompost inoculated with B. megaterium at 40 mL-1 175 g substrate on 0th day of vermicomposting showed significant (p<0.001) negative correlation with a correlation coefficient of r = -0.9913, y = -0.1089x+13.422. Similar results were recorded for 10th day inoculation: r = -0.9790, y = -0.1008x+14.421; for 20th day inoculation: r = -0.9941, y = -0.1255x+18.136; for 30th day inoculation: r = -0.9558, y = -0.1193x+18.679.
CORRELATION OF POPULATION DYNAMICS BETWEEN TOTAL MICROFLORA AND INDIVIDUAL
MICROBIAL INOCULANTS
Total microbial population vs A. chroococcum: Total microbial
population in the vermicompost inoculated with A. chroococcum on 0, 10,
20 and 30th day of vermicomposting at the rate 30, 35 and 40 mL-1
175 g each showed a significant positive correlation (Table 8).
Similar positive correlation was recorded for different time of inoculation
and with different inoculum levels as indicated in the table. The viable population
of A. chroococcum inoculated (35 and 40 mL-1 175 g substrate)
on 0th day of vermicomposting was significantly correlated with total microbial
population recorded during storage (Table 8).
Total microbial population vs B. megaterium: The viable population of B. megaterium inoculated (30 mL-1 175 g) on 0th day of vermicomposting was significantly correlated with total microbial population recorded during storage (r = 0.9534; y = 0.6328x+0.648; p<0.001). Similar positive correlation was recorded for different time of inoculation and with different inoculum levels (Table 8). In the present study, total microbial population in A. chroococcum and B. megaterium inoculated vermicompost was high during the initial phases of storage and then total microbial population declined towards the end suggesting that the overall maintenance of total microbial population in vermicompost is similar in the vermicomposts with any microbial inoculant.
Table 8: |
Correlation of population dynamics between total microflora
and A. chroococcum and B. megaterium during storage of enriched
vermicompost (180 days) |
 |
Eq: Regression equation; r: Correlation co-efficient |
The current study results on the change of total microbial population in A. chroococcum and B. megaterium inoculated vermicompost (at 30, 35 and 40 mL-1 175 g substrate) as a function of storage period (180 days) showed negative correlation in all the four treatments received the microbial inoculants during 0, 10, 20 and 30th day of vermicomposting. The same trend of results was obtained in the total microbial population change in B. megaterium inoculated vermicompost which also showed negative correlation with storage period in all treatments. The results on the correlation of total microbial population with the individual microbial inoculants, A. chroococcum and B. megaterium in respective vermicompost during storage period showed significant positive correlation, i.e., the increase/decrease of individual inoculants and total microbial population were parallel. These results clearly show that the population of microbial inoculants and the total microbial population in the vermicompost are dependents of each other. However, the competition between these two groups for nutrients requires further insight. There are many studies focusing the increase of microbial population in earthworm excreted or processed material than the parent material. Recent developments in the country as well as at the global level is the application of detritivorous epigeic earthworms for organic manure/vermicompost production from biodegradable organic materials recovered from agricultural lands, agro-based industries and municipal solid waste. This field of study is closely associated with earthworm microbe interaction. The quality of the manure or vermicompost depends on microorganisms associated with the process of decomposition. Earthworm activity is closely associated with microbial activity.
Lavelle (1983), is of the opinion that there may exist
competition between microorganisms and earthworms for easily digestible and
energy rich substrates. Such competition may depend on availability of nutrients
in the medium. Contrary to this, earthworms may derive benefit from microorganisms
when they have to survive on materials rich in cellulose or hemicellulose. So,
there exists mutualistic relation between earthworms and microorganisms. Tiunov
and Scheu (2004) have shown that earthworms deprive easily available carbon
to microorganisms and availability of carbon increases effective mobilization
of N and P by earthworms. Earthworms are mainly responsible for fragmentation
and conditioning of the substrate, increasing surface area for microbial activity
and significantly altering biological activity of the process (Dominguez
et al., 2003). The survival and increase of microbial population
in vermicasts and worm-worked compost (vermicompost) falls in line with regard
to the present study results.
The enhanced survival rate of A. brasilense, A. chroococcum, B. megaterium
and R. leguminosarum in the present study might be due to the presence
of nutrients and other factors reported in the following studies. There are
reports that earthworms influence the abundance and activity of soil microorganisms
(Brown et al., 2000; Scheu
et al., 2002), either by changes in the physical structure of the
soil, e.g., soil aggregation (Winding et al., 1997)
or by direct interactions when microorganisms are ingested together with mineral
soil and organic material. During gut passage, some microbes are digested while
others survive and may be stimulated by the rich nutrient supply (Winding
et al., 1997; Brown et al., 2000).
The correlation between the physico-chemical parameters and microbial populations
of the casts of P. corethrurus showed that the establishment of microbial
population requires optimum moisture, organic carbon and nitrogen content (Karmegam
and Daniel, 2000). The incubation of vermicasts (45 days) showed significant
correlation with that of the increase in fungal population (r = 0.720; p<0.05),
decrease in moisture content (r = -0.984; p<0.001) and the decrease in moisture
content statistically had no effect on the total fungal population in the vermicasts
of P. ceylanensis (Prakash et al., 2008).
Further, their study showed that the total microbial population, viz., bacteria,
fungi and actinomycetes were found to be many-fold higher than in the initial
vermibed substrate and in substrate without earthworms (control). Similar studies
by Sekar and Karmegam (2009) and Prakash
and Karmegam (2010) with different vermibed substrates revealed that the
microbial population increased in vermicompost than in compost. These studies
well support well the findings of the present study which showed the similar
results where the vermicompost served as a substrate for the survival and viability
of the biofertilizer inoculants, A. chroococcum and B. megaterium
for long period.
CONCLUSION In the present study, the inoculum level of A. chroococcum and B. megaterium at the rate of 35 mL-1 175 g of vermibed substrate is sufficient to maintain 1x107 viable cells up to 160 days after harvesting of vermicompost. The inoculum of biofertilizer organisms into vermibed on 30th day showed increased survival rate and hence, the optimized inoculation of 35 mL of inoculum per 175 g of substrate on 30th of vermicomposting is helpful for the maintenance of sufficient viable population for more than five months in the enriched vermicompost. ACKNOWLEDGMENT The authors sincerely thank Prof. Thilagavathy Daniel, Department of Biology, Gandhigram Rural Institute, Gandhigram, India for her suggestions and constant encouragements.
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