Microbial Enrichment of Vermicompost

The present study has been conducted to explore the possibility of enrichment of vermicompost with microbial inoculants (i.e., biofertilizer organisms), Azospirillum brasilense and Rhizobium leguminosarum, optimization of inoculum level, and time of inoculation during vermicomposting. The survival rate of each microbial inoculant, total microbial population in vermicompost, and their correlation with the microbial inoculants during the storage period (180 days) were assessed. The change in population of A. brasilense and R. leguminosarum in vermicompost (at 30, 35, and 40 mL/175 g substrates) with reference to storage period showed highly significant negative correlation (P < 0.001). The total microbial population in A. brasilense and R. leguminosarum inoculated vermicompost was high during initial phases of storage and then total microbial population declined towards the end. The inoculum level of A. brasilense and R. leguminosarum at 35 mL per 175 g vermibed substrate is sufficient to maintain 1× 10 viable cells up to 160 days after ther harvesting of vermicompost. The inoculum of these two biofertilizer organisms into vermibed on the 30th day showed increased survival rate and, hence, the optimized inoculation of 35 mL of inoculum per 175 g substrate on the 30th day of vermicomposting is helpful for the maintenance of sufficient viable population for more than five months in the enriched vermicompost.


Introduction
The compost prepared from organic materials using earthworms is a low cost and ecofriendly technology called vermicomposting.The fine granular peat-like end product, vermicompost that is produced is reported to contain elevated levels of nitrogen, phosphorus, and potassium (NPK) in available form, micronutrients, microflora, enzymes, and growth regulators [1,2].Because of this, the vermicompost when applied or supplemented in soil improves crop growth and yield [3,4].The earthworms, the drivers of many processes in soil, apart from the known vermicomposting, are also found to enhance phytoextraction of metals from contaminated soils [5].In addition, vermicompost, produced by the joint action of earthworms and microbes, contains nutrients in available form with increased microbial activity [6].
The use of biofertilizers is nowadays known to bring out several benefits to soil: solubilization of essential minerals, get hold of nutrients, offering micronutrients in more utilizable form for plants, and taking part in biological nitrogen fixation.Microorganisms of this group are generally known as plant growth promoting microorganisms (PGPMs) which include Azospirillum, Azotobacter, phosphobacteria, Rhizobia, and cyanobacteria.The PGPMs are capable of putting forth advantageous properties on growth and yield characteristics of several cultivable crops in different parts of the world [7][8][9].Rhizosphere bacteria promote plant growth by improving the availability of nutrients suppressing the growth of plant pathogens or by production of hormones such as auxins [10].In laboratory, the biofertilizers are mass multiplied in large scale using traditional culture medium and/or chief source supplemented medium for agricultural purposes [11][12][13].Recently, Raja Sekar and Karmegam [14] reported that the vermicasts are able to increase the survival rate of biofertilizer organisms for more than a year when used as carrier material.The process of vermicomposting results in the increase of microbial diversity and activity dramatically and the vermicompost produced could be a source of plant growth regulators produced by interactions between microorganisms and earthworms, which could contribute significantly to increased plant growth, flowering, and yields [15].So, the addition or enrichment of microbial inoculants such as biofertilizers would provide an increased plant growth and yield.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 optimize the inoculum level and time of inoculation of biofertilizers in enrichment process of vermicompost and assessing the survival rate of A. brasilense, and R. leguminosarum in enriched vermicompost in relation to total microbial population.

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.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 and E. eugeniae was introduced in all the vermibeds.
The vermicompost was collected from all the vermibeds after 40 days.The mass multiplied biofertilizer organisms at the rate of 30, 35, and 40 mL per 175 g of vermibed substrates were added to each of the experiments, T1, T2, T3, and T4 on the 0th, 10th, 20th and 30th day, respectively, to find out the optimum inoculum level and time of inoculation that results in the maintenance of 1 × 10 7 viable cells per gram of vermicompost during storage.

Statistical Analyses.
Data were subjected for analysis of variance (ANOVA) followed by Duncan's 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.between earthworm and microorganisms in deriving nutrients from harder materials like cellulose and hemicellulose [16].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" [17][18][19][20].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. brasilense and R. leguminosarum, at the rate of 30, 35, and 40 mL/175 g of substrate.This was done to find out the optimum level of inoculum required for the maintenance of 1 × 10 7 viable cells in the vermicompost.The inoculation was done on the 0th, 10th, 20th and 30th days 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.Microbial inoculation on 0th, 10th, 20th, and 30th days showed decrease in the viable population of A. brasilense and R. leguminosarum inoculated at the rate of 30, 35, and 40 mL/175 g of substrate, towards the progression of storage period of vermicompost, uniformly.The change of A. brasilense and R. leguminosarum in the vermicompost (at 30, 35, and 40 mL/175 g substrate) with reference to the storage period (180 days) showed a highly significant negative correlation (P < 0.001).In all the treatments, the viable population of A. brasilense and R. leguminosarum at the rates of 30, 35, and 40 mL/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 all the four microbial inoculants, ionoculated on the 0th, 10th, 20th, and 30th days of vermicomposting.

Results and Discussion
The viable population of A. brasilense inoculated at the rate of 30 mL/175 g of substrate on the 30th day of vermicomposting showed 11, 9, 7, 5, and 1 × 10 7 g −1 CFU during the 30th, 60th, 90th, 120th, and 150th days, respectively, which was negatively significant at P < 0.001 (y = −0.0696x+ 12.71, R 2 = 0.9067) (Table 1; Figure 1).Similar results were obtained for 35 and 40 mL/175 g of substrate inoculation (Tables 2 and 3; Figures 2 and 3).The viable population of R. leguminosarum inoculated at the rates of 30, 35, and 40 mL/175 g substrate in different intervals showed decrease in viable population during the storage of vermicompost (Tables 4, 5, and 6).The decrease of viable population upon storage of vermicompost which received R. leguminosarum inoculants at different intervals was negatively correlated (Figures 4, 5, and 6).The population of R. leguminosarum at the rate of 40 mL/175 g substrate inoculated on the 0th day and 10th day of vermicomposting showed 1 and 3 ×10 7 g −1 CFU on the 120th day of storage after harvest.There after no viable cells were observed up to 180 days.On the 20th and 30th days of inoculation, the survival of R. leguminosarum was observed up to the 135th and 150th day, and the subsequent observations showed no viable population up to 180 days.The enrichment of vermicompost with the addition of nutrient rich substrates was demonstrated by Daniel et al. [21], 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 [19] studied the effect of ironenriched vermicompost on growth and nutrition of tomato and reported that total and available forms of iron in ironenriched vermicomposts as well as in tomato tissues increased by an increase in the proportion of iron refuse in vermicompost.Some 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 [22].The inoculation of consortium of microorganisms Aspergillus niger, P. sajor-caju, Azotobacter chroococcum, Trichoderma harzianum not only accelerated vermicomposting of crop residues and farm yard manure but also enriched the quality of product [17].During the incubation period the inoculated bacterial strains proliferated rapidly, fixed nitrogen, solubilised, and added native phosphate [23].Pressmud alone and in combination with other byproducts 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 combination of both treatments reduced the overall time required for composting to 20 days and accelerated the degradation process, thereby producing a nutrient-enriched compost product [20].The study conducted by Padmavathiamma et al. [24] reported that the enrichment generally had a significant effect on the nutrient contents, especially for N, P, K, Mg, and Mn.Eudrilus compost, when treated with Azospirillum and P-solubilising organisms, gave an 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 [25].An increase in N-content due to microbial inoculation was reported by Rasal et al. [26].The P-contents were significantly higher when inoculated with Azospirillum and P-solubilising organisms (1.76%) than in uninoculated compost (0.72%).The mechanisms 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 H 2 S under aerobic and anaerobic conditions and by mineralizing organic compounds, with the release of inorganic phosphate [26].
Kumar and Shweta [25] studied the enhancement of wood waste decomposition by microbial inoculation prior to vermicomposting.The timber wastes which were inoculated 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 1 × 10 7 g −1 viable population of A. brasilense and R. leguminosarum 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. brasilense and R. leguminosarum, at the rate 35 mL/175 g substrate on the 30th day of vermicomposting are the optimum inoculation level and time for the maintenance of 1 × 10 7 viable cells in the vermicompost is the maximum number of days during storage (Figure 7).The study also reveals that the microbial inoculants inoculated at the later stage of vermicomposting survive for long period.

Total Microbial Population and the Microbial Inoculants.
In the present study, total microbial population in A. brasilense and R. leguminosarum inoculated vermicompost was high during the initial phases of storage and then total microbial population declined towards the end (Tables 7 and 8).
No viable population of total microorganisms in 10 7 dilution on the135th, 150th, 265th and 165th days of storage was observed, respectively, in the 0th, 10th, 20th, and 30th days inoculation of A. brasilense at the rate of 30 mL/175 g substrate.The vermicompost inoculated on the 20th day of  The correlation of total microbial population with the individual microbial inoculants, A. brasilense and R. leguminosarum in respective vermicompost during storage period, showed significant positive correlation, that is, the increase/ decrease of individual inoculants and total microbial population were parallel (Table 9).The viable population of R. leguminosarum inoculated (40 mL/175 g) on the 0th day of vermicomposting was significantly correlated with total microbial population recorded during storage (r = 0.960573; y = 0.5456x+1.3712;P < 0.001).Similar positive correlation was recorded for different time of inoculation and with different inoculum level (data not shown).These results clearly show that the population of microbial inoculants and   surface area for microbial activity, and significantly altering biological activity of the process [30].The vermicasts when used as carrier material for biofertilizers supported the survival rate for more than one year [14].The survival and increase of microbial population in vermicasts and wormworked compost (vermicompost) falls in line with present study results.The findings of the present study also showed similar results where the vermicompost served as a substrate for the survival and viability of the biofertilizer inoculants, A. brasilense and R. leguminosarum, for long period during storage.

Conclusion
The enhancement of nutrients and beneficial microbial population in the vermicompost is yet another important evolving trend where the vermicompost is value added with nutrients and or microorganisms resulting in improved growth and yield of crop plants.The inoculum level of A brasilense. and R. leguminosarum at the rate of 35 mL per 175 g of vermibed substrate is sufficient to maintain 1 × 10 7 viable cells up to 160 days after harvesting of vermicompost.The inoculum of biofertilizer organisms into vermibed on the 30th day showed increased survival rate and hence, the optimized inoculation of 35 mL of inoculum per 175 g of substrate on the 30th of vermicomposting is helpful for the maintenance of sufficient viable population for more than five months in the enriched vermicompost.

Figure 3 :
Figure 3: The change of A. brasilense population in the vermicompost (at 40 mL per 175 g of substrate) during storage period (180 days).Error bars indicate ± SD; time of inoculation of A. brasilense during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day and (d) 30th day.

Figure 4 :
Figure 4: The change of R. leguminosarum population in the vermicompost (at 30 mL per 175 g of substrate) during storage period (180 days).Error bars indicate ± SD; time of inoculation of R. leguminosarum during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day, and (d) 30th day.

Figure 5 :
Figure 5: The change of R. leguminosarum population in the vermicompost (at 35 mL per 175 g of substrate) during storage period (180 days).Error bars indicate ± SD; time of inoculation of R. leguminosarum during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day, and (d) 30th day.

Figure 6 :
Figure 6: The change of R. leguminosarum population in the vermicompost (at 40 mL per 175 g of substrate) during storage period (180 days).Error bars indicate ± SD; time of inoculation of R. leguminosarum during vermicomposting: (a) 0th day, (b) 10th day, (c) 20th day, and (d) 30th day.

Table 1 :
The viable population of A. brasilense 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 Duncan's multiple-range test.

Table 2 :
The viable population of A. brasilense 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 Duncan's multiple-range test.Storage period of vermicompost (after harvest, in days)Viable population of A. brasilense (CFU × 10 7 g −1 )

Table 3 :
The viable population of A. brasilense 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 Duncan's multiple-range test.

Table 4 :
The viable population of R. leguminosarum 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 Duncan's multiple-range test.

Table 5 :
The viable population of R. leguminosarum 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 Duncan's multiple-range test.

Table 6 :
The viable population of R. leguminosarum 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 Duncan's multiple-range test.Storage period of vermicompost (after harvest, in days)Viable population of R. leguminosarum (CFU × g −1 )Time of inoculation (vermicomposting days

Table 7 :
The total microbial population change in A. brasilense inoculated vermicompost during storage period.Values are rounded of mean values of three replicates.

Table 8 :
The total microbial population change in R. leguminosarum inoculated vermicompost during storage period.Values are rounded of mean values of three replicates.

Table 9 :
Correlation of population dynamics between total microflora and A. brasilense and R. leguminosarum during storage of enriched vermicompost (180 days).