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Bioconversion and biodynamics of Eisenia foetida in different organic wastes through microbially enriched vermiconversion technologies
Ecological Engineering 86 (2016) 154–161
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Bioconversion and biodynamics of Eisenia foetida in different organic wastes through microbially enriched vermiconversion technologies D. Das a , P. Bhattacharyya b , B.C. Ghosh c , P. Banik a,∗ a b c
Agricultural and Ecological Research Unit, Indian Statistical Institute, 203 Barrackpore Trunk Road, Kolkata 700108, India Agricultural and Ecological Research Unit, Indian Statistical Institute, Giridih, 815301, Jharkhand, India Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, India
a r t i c l e
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Article history: Received 14 January 2015 Received in revised form 14 October 2015 Accepted 10 November 2015 Keywords: Vermicomposting Eisenia foetida Microorganisms Organic wastes Nutrients
a b s t r a c t The present investigation is an attempt to convert organic wastes like water hyacinth, paddy straw and sawdust to organic manure through vermicomposting by Eisenia foetida together with the help of some beneficial microbes such as Trichoderma viride (cellulolytic), Azotobacter chroococcum (nitrogen fixing), Bacillus polymixa (phosphate solubilizing) and Bacillus firmus (potassium solubilizing). Chemical and biochemical properties of vermicompost prepared from the aforementioned organic wastes were studied. Earthworm biomass, growth performance, and cocoon production were higher in vermicompost prepared from water hyacinth and paddy straw as compared to vermicompost prepared from sawdust. All vermibeds showed significant reduction in pH, organic carbon, cellulose content and C/N ratio and substantial increase in humic acid, total N, available P and exchangeable K. Vermistabilization also increased the microbial population and enzyme activities of all organic wastes. Inoculation of A. chroococcum, B. polymixa and B. firmus alone or in combination with T. viride significantly increased nitrogen, available P and K content. Significant increases in microbial population and enzyme activities in vermicomposts were recorded with inoculation of T. viride either alone or in consortia. The best quality compost was prepared when the substrate was treated with all the four microorganisms together followed by vermicomposting. Results indicated that the combination of earthworms and microorganisms reduced the overall time required for composting besides producing a nutrient-enriched compost product. © 2015 Published by Elsevier B.V.
1. Introduction About 3000 million tons of organic wastes are produced every year in India (Bharadwaj, 2010). Disposal of increasing quantities of organic wastes is becoming a serious problem. Aquatic waste like water hyacinth is a free-floating aquatic weed which creates major problems in the aquatic system due to its rapid growth and the robustness of its seeds. Paddy straw is a post-harvest residue of the agricultural field whereas saw dust, produced by the sawmill, is found in large quantities in the environment. In many countries traditional management practice of wastes is open air burning and this leads to release of greenhouse gases, and production of particulate matter. As an alternative to burning of wastes vermiculture technology for recycling these organic residues yields valuable manure within a short period. Vermicomposting is an ecobiotechnological process that transforms energy rich and complex
∗ Corresponding author. E-mail address: [email protected] (P. Banik). http://dx.doi.org/10.1016/j.ecoleng.2015.11.012 0925-8574/© 2015 Published by Elsevier B.V.
organic substances into stabilized humus like product with the help of microorganisms. Vermicomposting is accelerated by the combined activity of a diverse group of microorganisms. One of the possible ways of increasing the nutrient content of vermicompost is a microbial enrichment technique with nitrogen fixers, and phosphorous solubilisers (Kumar et al., 2010; Bustao et al., 2012). The incorporation of vermicompost along with microbial fertilizers had a beneficial effect on the crop yield. Microorganisms produce enzymes that cause biochemical decomposition of organic matter, but earthworms are the crucial driver of the process as they are involved in the indirect stimulation of microbial population through fragmentation and ingestion of fresh organic matter. This, in turn, results in a greater surface area available for microbial colonization, thus dramatically increasing microbiological activity (Aira et al., 2002). Inoculation of suitable cellulolytic and lignolytic strains like Trichoderma viridae (TV) is able to accelerate the composting rate of organic substrates because the lignin-cellulose complex is transformed with their cellulolytic enzymes, thus enriching the final product with nutrients (Tiwari et al., 1989). Inoculation of N2 fixing
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Table 1 Physico-chemical properties of organic wastes. Organic wastes
pH
EC (ds/m)
Organic C (%)
Total N (%)
C/N ratio
Total P (%)
Total K (%)
Cow dung Water hyacinth Paddy straw Saw dust
6.2 7.3 7.8 7.5
1.3 0.623 0.742 0.82
20.3 29.6 52.6 68
0.68 0.62 0.78 0.18
29.9 47.8 67.4 377.8
0.4 0.35 0.27 0.009
0.65 0.82 0.62 0.23
bacteria and phosphorus solubilizing bacteria (PSB) during vermicomposting increased nitrogen content and phosphorus content and N2 fixing ability of bacteria depended upon the use of organic wastes (Kumar et al., 2010; Bustao et al., 2012; Olivares et al., 2012). The effect of body growth of earthworm by the inoculation of phosphate solubilizing and potassium releasing bacteria during vermicomposting process has been scantly studied. Sheng and He (2006) reported that solubilization of illite and feldspar by potassium releasing microorganisms is due to the production of organic acids like oxalic acid and tartaric acid and also due to production of capsular polysaccharides which helps in dissolution of minerals to release potassium. Therefore, potassium releasing bacteria, Bacillus firmus might be added during vermicomposting to solubilize potassium and increasing potassium content in the final product of vermicompost. It is well known that the food source influences not only the size of earthworm population, but also their growth and reproduction rates. There is abundant literature on the response of earthworms to different type of vegetable substrate in the field (Bansal and Kapoor, 2000; Bharadwaj, 2010), but there is a lack of information on the effect of additional microbial strains in the feed of earthworms during vermicomposting. Keeping this in mind, the present study was conducted to assess the potential of Eisenia foetida in vermicomposting of different types of organic materials and quality of vermicompost thus produced. An attempt was also made to bring changes in nitrogen, phosphorus and potassium components, humic acids and some enzyme activities of resulting vermicompost which is produced from three different C/N ratio based organic wastes by inoculating of N2 fixing, Phosphate solubilizing, potassium releasing bacteria and cellulose decomposing fungi viz. T. viridae.
bacteria) and Bacillus polymixa (phosphate solubilising bacteria) were used. Bacillus firmus (potassium releasing bacteria) were procured from Indian Agricultural Research Institute, New Delhi. The fungal cultures were maintained by sub culturing on potato dextrose agar, while bacteria A. chroococcum, B. Polymixa, B. firmus were sub culturing on Jensen’s Agar Media, Pikovaskaya medium and Aleksandrov medium, respectively.
2. Materials and methods
2.4. Treatment details
2.1. Organic wastes and earthworm species used for vermiconversion The organic wastes, like water hyacinth (WH), paddy straw (PS) and sawdust (SD) were collected from a local farm of Kharagpur, India. Before carrying out the experiment some common physicochemical characteristics of organic wastes were analyzed (Table 1). Urine free cow dung, collected from a nearby dairy farm was used as the organic source to initiate as well as expedite the biological conversion process. Juvenile, non-clitellated specimens of epigeic earthworm E. foetida, weighing about 200–250 mg, were obtained from our experimental vermiculture unit and used for composting on organic mixed wastes. The experiment on biodegradable organic wastes was conducted in 2.0 l earthen pots with a small hole at the bottom. Three different biodegradable organic wastes, viz. Water hyacinth, paddy straw and sawdust with variable C/N ratios were used as food for E. foetida. Water hyacinth and paddy straw were chopped to reduce their size and volume by about 50–70%. Finely chopped substrates were dipped overnight in 0.1% formalin. 2.2. Microbial source Pure cultures of microbial inoculants, viz. T. Viridae (lignolytic and cellulolytic fungi), A. chrococcum (free living nitrogen fixing
2.3. Experimental design 50 adult earthworms of E. foetida were placed in each container and supplied with 2 kg of organic wastes (WH, PS or SD), 600 g of Cow Dung (30% w/w) was mixed with each organic waste to provide an initial favorable environment for the earthworm. Trichoderma viride (cellulolytic, M2 treatment), Azotobacter chroococcum (nitrogen fixing, M3 treatment), Bacillus polymixa (phosphate solubilizing, M4 treatment) and Bacillus firmus (potassium solubilizing, M5 treatment) and mixed all microbes (M6) were inoculated as 50 mL of 7 days old broth culture per kg (106 cells per ml) of these organic substrates M1 is control, i.e., no microbial inoculate in this treatment. Moisture content was maintained at about 60% (dry weight basis) during the vermicomposting process of periodic sprinkling water. The pots were kept in dark at maximum laboratory temperatures ranging between 27 and 30 ◦ C during the whole experimental period. The worms were not supplied with additional food for the duration of the experiment that went on for 16 weeks. The experiment was conducted following complete randomized block design with three replications. The sampling for analysis was made after stabilization of vermicomposting (84–112 days), which varied depending upon the nature of the feed.
T1—Organic wastes + cowdung T2—Organic wastes + cowdung + Trichoderma viridae T3—Organic wastes + cowdung + Azatobactor chroococcum T4—Organic wastes + cowdung + Bacillus polymixa T5—Organic wastes + cowdung + Bacillus firmus T6—Organic wastes + cowdung + Trichoderma viridae + Azatobactor chroococcum + Bacillus polymixa + Bacillus firmus 2.5. Physico-chemical and biochemical analysis Physico-chemical analysis of wastes, vermicompost was done according to the methods proposed by Page et al. (1982). Enzymes such as urease, phosphatase, and amylase activities of soils were estimated following the methods suggested by Tabatabai (1994). These measurements were carried out using the field moist condition. However, the measured activities were expressed on moisture-free basis. 2.6. Microbial assays Microbial plate counts were made following the method of Travors and Cook (1992). Vermicompost (1 g) from the same sample employed for microbial respiration was taken and stirred with 100 ml sterile distilled water in a conical flask and the supernatant
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Fig. 1. Earthworm growth rate, reproduction rate and cocoon production in different organic wastes during vermicomposting by using different microbes. Error bars represent standard deviation. Arithmetic means with the same letter are not significantly different from each other at 5% probability level.
was serial dilution method to estimate the population of total bacteria, total fungus, cellulolytic fungi, phosphorus solubilising bacteria (PSB), azatobactor (AZC) and potassium solubilising bacteria (KSB) in nutrient agar medium, potato dextrose agar medium, asparagines medium, azatobactor medium, pikovskaya agar medium and aleksandrov medium, respectively. Soil respiration was determined following the method of Alef (1995). 2.7. Statistical analysis Statistical analyses such as the least significant difference (LSD) and Duncan multiple range test (DMRT) were carried out using the SPSS statistical package. 3. Results and discussions Table 1 represents the physico-chemical properties of the organic wastes used in this study. pH of all the wastes is alkaline in nature. Organic C, and total N content were higher in saw dust and paddy straw, respectively. The C/N ratio of the sawdust was substantially higher compared to other organic wastes. Water hyacinth contains highest total P and K content. The results of earthworm growth rate and cocoon production are presented in Fig. 1. Many current workers followed a long duration composting process having time span not less than 90 days (Suthar, 2010; Deka et al., 2011; Fornes et al., 2012). However, we have adopted a simple and rapid process of vermitechnology under natural ambient environment as described in the earlier section.
The earthworm growth rate (mg/worm/day) has been considered a useful comparative index to compare the growth of earthworm in different feed wastes (Edwards et al., 1998). Biomass of earthworm in all the experimented wastes increased significantly. The overall growth rate was maximum in paddy straw (19.977 mg/worm/day) followed by water hyacinth (17.937 mg/worm/day) and sawdust (7.233 mg/ worm/day). Among the microbes inoculated in the organic substrates, M6 treatment showed the most effective growth rate. The growth rate in M6 and M2 treatments was significantly higher over M1, M3, M4 and M5 treatments. Earthworms utilize microorganisms in their substrates as a food source and can digest them selectively (Edwards et al., 1998). Cocoon production was significantly higher in paddy straw than water hyacinth and saw dust wastes. Treatment M6 was significantly higher in cocoon production over other respective treatments. The increase in earthworm’s growth may also be attributed to a low C/N ratio (Ndegwa and Thompson, 2000) of the pre-decomposed substrate. The use of bulk agents sawdust waste does not adequately improve cocoon production due to higher C/N ratio. The results indicated that the quality and palatability of food directly affect the survival, growth rate and reproduction potential of earthworms (Suthar, 2009, 2010). The difference in growth rate and cocoon production in different wastes for vermicomposting could be related to biochemical quality of the feed, which is one of the important factors in determining the time taken to reach sexual maturity onset cocoon production (Flack and Hartenstein, 1984). The changes in individual biomass during the vermicomposting process in different wastes with the addition of microbes are
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Fig. 2. Earthworm growth patterns in different organic wastes during vermicomposting by using different microbes. Error bars represent standard deviation. LSD = least significant deviation.
described in Fig. 2. Among different wastes, the highest biomass achieved was 829.9 mg adult−1 in paddy straw treatment and the lowest in saw dust. There was a rapid increase in individual biomass of inoculated earthworms in all vermibeds during initial 21 to 25 days, 25 to 30 days and 33 to 40 days of vermicomposting in case of water hyacinth, paddy straw and sawdust wastes, respectively. The maximum weight gain was followed by weight loss by the time of termination of the experiment. The loss in earthworm weight was also reported by a few other scientists (Suthar, 2007; Singh et al., 2010). They have attributed the weight loss to the food shortage in vermicompost beds. It may be due to the conversion of most of the used substrate material into vermicompost, which cannot further support the growth in earthworms. When E. foetida received food below the maintenance level, it lost weight at a rate which depended upon the quantity and nature of its ingestible substrates (Neuhauser et al., 1988). The feeds that provide earthworms with a sufficient amount of easily metabolize organic matter and non-assimilated carbohydrates favor the growth and reproduction of the earthworms (Edwards et al., 1998). Among the microbial inoculation, TV (M2) and mix (M6) treated wastes were noted higher individual biomass than other microbial inoculants. Trichoderma viridiae is a cellulolytic decomposing fungus. It decomposes the cellulolytic material more rapidly than other microbial treatments. With the progress of vermicomposting, palatability and release of locked up nutrients from the wastes increased, thereby showing maximum biomass, as well as, survival
of earthworms at a later period compared to other different waste treatment. Substrates such as water hyacinth, paddy straw and sawdust had a slightly alkaline pH value at the initial stage. After vermicomposting, pH shifted towards neutrality, irrespective of the initial pH of the organic substrates (Fig. 3). Vermicompost obtained from saw dust recorded lowest pH (7.11), followed by that from paddy straw (7.31) and water hyacinth (7.36). Microbial inoculants showed the variable influence of pH in the resulting vermicompost depending on the nature of microbes and the biodegradable organic wastes used for vermicomposting. In T. viridae (M2) and mix (M6) treatments the lowest pH value (towards the neutral range) were recorded. The neutral pH recorded in the final products might have been due to production of CO2 and organic acids by microbial metabolism during decomposition of different organic substrates (Garg et al., 2006). The highest organic carbon value was recorded in saw dust wastes (526.63 mg/g) followed by paddy straw (339.86 mg/g) and water hyacinth (243.43 mg/g) (Fig. 3). After vermicomposting, all the treatments showed significantly lower organic carbon. The treatment M2 and M6 were recorded lowest organic carbon content compared to other microbial inoculants treatments. The lower organic carbon percentage in vermicomposts obtained from organic sources inoculated with microorganisms might be attributed to a higher rate of decomposition facilitated by the microbes (Bhardwaj, 1999). Moreover, losses of carbon in the form
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Fig. 3. Physico-chemical properties of vermicomposts as affected by different treatments. Error bars represent standard deviation. LSD = least significant deviation.
of CO2 as well as the conversion of any part of the organic fraction into worm biomass are the cause of carbon loss from the substrate (Suthar, 2009). Among the three organic wastes, paddy straw recorded the maximum increase in nitrogen content in the vermicompost over its initial raw material (Fig. 3). Inoculation of microorganism in the biodegradable organic wastes increased Total N (TKN) content of vermicompost, but its effect varied widely (M6: 52–72%, M5: 3–25%, M4:8–37%, M3: 43–64%, M2: 22–32% over control treatment). Among different microbial inoculation treatments Azotobactor (M3) and mix microbial treatments (M6) contain the highest value of TKN content. TKN content in the vermicompost was primarily governed by the initial nitrogen content of the organic wastes used as feed to the earthworm and also by the extent of decomposition (Suthar, 2009). The rate of increase of TKN in the vermicompost from its corresponding organic wastes was well correlated to the C/N ratio of organic substrates. Narrower C/N ratio, greater was the decomposition rate of organic wastes and increases in the TKN content in vermicompost. Earthworms prefer organic wastes with a narrower C/N ratio as they feed (Moody et al., 1995). This explained the greater increase in TKN in vermicompost prepared from water hyacinth as compared to vermicompost prepare from sawdust. TV (M2) and mix (M6) treatments had a low C/N ratio and a higher nitrogen content. Increase in TKN content in the vermicompost obtained on inoculation of cellulolytic cum lignolytic fungi (T. viridae) might be due to enhanced decomposition of the organic matter (Viel et al., 1987), leading to decrease in the organic carbon
content. Thus a decrease in the C/N ratio in the vermicompost as compared to the initial organic substrate, which might be due to a relative increase in the TKN on loss of organic carbon as CO2 as well as water loss by evaporation during mineralization (Viel et al., 1987). The correlation between TKN of vermicompost and a number of free living nitrogen fixing bacteria (Azatobactor chroococcum) [r = 0. 69] suggested that TKN content of vermicompost depended not only on the quality and quantity of initial organic substrate, but also to the extent N2 fixed by free living N2 -fixing bacteria (Kale et al., 1982). Data reveals that total phosphorus (TP) and total potassium (TK) content increased in the resulting vermicompost as compared to the initial organic substrates (Fig. 3). Vermicompost prepared from paddy straw had the highest TP content (8.58 mg/g) followed by water hyacinth (8.0 mg/g) and sawdust (1.46 mg/g) wastes. Among the microbes inoculated in the organic substrates, M6 and M4 treatments were most effective in increasing TP (34–80% and 23–31%, respectively) content. Vermicompost prepared from water hyacinth substrate increased total potassium content as compared to other organic wastes paddy straw and sawdust wastes. Among the microbes inoculated in the organic substrates, M6 and M5 treatments are most effective over control treatments (45.38–80.72% and 33.62–56.66%) in the vermicompost obtained from the different substrates. Phosphorus and potassium mineralization depends on the quality of bulking materials. Acid production during organic matter decomposition of the microorganism is the major mechanism of solubilisation of insoluble phosphorus and potassium.
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Fig. 4. Population of different microorganisms, enzyme activity and microbial respiration of vermicomposts as affected by different treatments. Error bars represent standard deviation. LSD = least significant deviation.
The enhanced number of the microflora present in the gut of earthworms in the case of vermicomposting might have played an important role in this process and increased K2 O over the raw material (Kaviraj and Sharma, 2003). Earthworm primes its symbiotic gut microflora with secreted mucus, water and gut enzymes, i.e. phosphatase enzymes enhance their degradation of ingested organic matter and release of available metabolites. Therefore, directly or indirectly earthworm enriched the substrate material with exchangeable potassium and phosphorus contents. Phosphorus solubilising bacteria increased the phosphorus concentration in vermicompost due to the presence of phosphatase enzyme which enhances the solubility of phosphorus. The increase of phosphorus content could be attributed to the phosphorus solubilising microorganisms present in the worm cast (Suthar, 2009). Potassium solubilising bacteria, increase potassium content in vermicompost could be attributed by the secretion of acid from bacteria and which was solubilised potassium. Vermicompost prepared from paddy straw waste showed the highest humic acid content (0.810 mg/g) followed by water hyacinth (0.533 mg/g) and sawdust (0.343 mg/g) (Fig. 3). Microbial inoculation increased the humic acid content (18.14 to 64.19%)
of vermicompost depending on the organic substrates used. Vermicompost obtained from organic substrates inoculated with T. viridae (M2) and mix (M6) microbes recorded highest humic acid content. T. viridae related treatment was the most effective. Earthworm fragments the organic substrates, thus largely enhancing microbial activities and increasing rates of mineralization, and rapidly converting the wastes into humus like substances (Atiyeh et al., 2001). Hence the narrower C/N ratio of organic matter facilitates higher rate of decomposition (Brady, 1985), organic wastes with a lesser C/N ratio showed higher humic acid content. Preparation of vermicompost by inoculation of microbes in organic wastes hastened the decomposition of organic wastes. The high humic acid content in vermicompost from organic waste inoculated with T. viridae might be due to the fact that the ability of these cellulolytic and lignolytic fungi to decompose organic matter was maximum, but it is statistically at par with the other microbial inoculants. On the other hand, the simultaneous activity of microflora present in the gut of earthworms and in waste might have intensified cellulolysis and lignolysis (Edwards et al., 1998). Earthworms digest long chains of polysaccharides, enhancing microbial colonization. Simultaneously the structure of lignin changes, probably due to
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microbial oxidation and demethylation. Microbial cleavage of the aromatic rings of lignin leads to new polysaccharide and humins in the organic matter (Edwards et al., 1998). Vermicompost prepared from water hyacinth waste showed significantly lowest cellulose content compared to other organic substrates (Fig. 3). Microbial inoculation increases the cellulose degradation. Treatment of M2 and M6 showed the lowest value of cellulose content. The production of cellulose degrading enzymes by the inoculated microbes during vermicomposting might have accelerated the degradation process (Singh and Sharma, 2002). Similar results were also reported by Rasal et al. (1988) who reported rapid decomposition of sugar cane trash with a mixture of cellulolytic fungi; T. viride, Trichurus spiralis, Paecelomyces fusisporus and Aspergillus sp. along with nitrogen fixing bacteria Azotobacter sp. Microbial colony estimation revealed that the total bacteria (26 × 108 CFU) as well as nitrogen fixing bacteria (AZC count) (6 × 103 CFU), PSB (9 × 106 ) and KSB (7 × 104 CFU) populations were lost in vermicompost obtained from saw dust wastes and it differed significantly (P < 0.05) from vermicomposts prepared from other organic wastes (Fig. 4). Among the microbial inoculation treatments, M6 treatments showed a higher population for total bacterial counts, AZC, PSB and KSB count. Total fungal count (8.17 × 106 CFU) as well as cellulolytic fungal (10 × 105 CFU) population were least in vermicompost obtained from saw dust followed by water hyacinth (38 × 106 and 42 × 105 CFU) and paddy straw (39 × 106 and 45 × 105 CFU). Though vermicompost obtained from paddy straw was showing a higher abundance, population, but it was statistically significant (P < 0.05) from other vermicompost which is prepared by other organic wastes. Urease activity in vermicompost varied depending on the organic wastes used and showed the highest activity in vermicompost obtained from water hyacinth wastes (128.70 g NH4 /g/h) followed by paddy straw (121.33 g NH4 /g/h) and saw dust wastes (33.81 g NH4 /g/h) (Fig. 4). Among the microbial treatments, Azotobacter inoculation in organic wastes significantly increased urease activity. The urease activity was well correlated to the nitrogen content (r = 0.691), Azotobactor chroococum (N2 fixing bacteria) (r = 0.743) of the vermicompost. The positive correlation between urease activities of the vermicompost from different organic wastes and the humic acid content (r = 0.826) suggested that humic acids might be responsible for preserving the urease enzyme as humicenzyme complex in vermicompost. The variation in urease activity among the vermicompost obtained from different organic sources might be due to variation in the organic matter content of the initial substrates (Syers et al., 1979). High correlation value (r = 0.83) between the urease activity and total microbial respiration in the vermicompost suggested that microorganisms, activity of which were enhanced by the earthworm, while passing the substrates through the worm guts (Fischer et al., 1997) might be solely responsible for urease activities in the vermicompost. Fig. 4 showed that higher phosphatase activity in vermicompost prepared from paddy straw wastes followed by water hyacinth (238.33 g p-nitro phenol/g/h) and sawdust (87.23 g p-nitro phenol/g/h) wastes. Among the microbial inoculation treatments M2 and M6 showed higher phosphatase activity. Phosphatase activity was significantly and positively correlated with humic acid extracted from vermicompost (r = 0.87) and phosphorus solubilising bacterial count (r = 0.737). Earthworms are used to feed fungi, for their reproduction and growth (Parthasarathi and Ranganathan, 1999). Microbes entering the worm guts, consume nitrogenous compounds of the mucus (Zhang et al., 2000), which largely increase their activity, which in turn enables them to contribute enzymes in the digestive process of the earthworm. Microorganisms not only mineralize complex substance into plant available form but also can synthesize biologically active substances. Hence
phosphatase activity in vermicompost was increased as compared to their raw materials. The positive correlation of acid phosphatase enzyme activities with microbial respiration in vermicompost (r = 0.836) confirmed the fact that the enhanced enzyme activity in the cast was due to microbes, rather than the epithelium of the gut (Vinotha et al., 2000). Amylase activity of vermicompost varied depends upon the organic wastes used and showed the highest activity in vermicompost obtained from water hyacinth wastes followed by paddy straw and sawdust wastes (Fig. 4). Treatment M2 and MS showed the higher amylase activity compared to other treatments. Inoculation of microorganisms significantly (P < 0.05) increased respiration of both the organic substrates and their effect varied being dependent on the nature of the organic wastes and microorganisms (Fig. 4). The data suggested that vermicompost prepared from paddy straw wastes was recorded significantly higher respiration as compared to other vermicomposts. In general, fungi decompose organic matter faster than bacteria. Among these microbial inoculants treatments, M2 and M6 treatments were recorded a higher microbial respiration than other inoculants treatments. Vermicompost prepared from water hyacinth waste was recorded higher respiration rate than other vermicompost that might be due to higher mineralization rate and lowest C/N ratio than other vermicompost. 4. Conclusions The results demonstrated that the organic wastes can be converted into an environmentally-friendly nutrient source by vermicomposting, using different microorganisms. Growth and population of E. foetida were influenced by different waste materials. Among waste materials, paddy straw was preferred by E. foetida, followed by water hyacinth, and saw dust. Inoculation of microorganisms with earthworms accelerated the decomposition process. Vermicompost using T. viride showed the highest nutrient content as well as enzymatic and microbial status, but the combined effect of all inoculated microorganisms proved to have a significant effect on the quality of vermicompost. Acknowledgements Authors thankfully acknowledge Prof. R. L. Brahmachary for his help to improve the writing style of the article. References Aira, M., Moroy, F., Dominguez, J., Mato, S., 2002. How earthworm density affects microbial biomass and activity in pig manure. Eur. J. Soil Biol. 38, 7–10. Alef, K., 1995. Estimation of soil respiration. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London. Atiyeh, R.M., Edwards, C.A., Suber, S., Metzger, J.D., 2001. Pig manure vermicompost as a component of horticultural bedding plant medium: effects on physicochemical properties and plant growth. Bioresour. Technol. 78, 11–20. Bansal, S., Kapoor, K.K., 2000. Vermicomposting of crop residues and cattle dung with Eisenia foetida. Bioresour. Technol. 73, 95–98. Bhardwaj, K.K.R., 1999. Significance of microbial inoculants in organic matter decomposition. In: Jha, M.N. (Ed.), Agromicrobes, Current Trends in Life Sciences. Today and Tomorrow’s Printers and Publisher, New Delhi, pp. 235–247. Bharadwaj, A., 2010. Management of kitchen waste material through vermicomposting. Asian J. Exp. Biol. Sci. 1, 175–177. Brady, N.C., 1985. The Nature and Properties of soils, eighth ed. Colier Mcmillan, London. Bustao, J.G., Lima, L.S., Aguiar, N.O., Canellas, L.P., Olivares, F.B., 2012. Changes in labile phosphorous forms during maturation of vermicompost enriched with phosphorus solubilizing and diazotrophic bacteria. Bioresour. Technol. 110, 390–395. Deka, H., Deka, S., Baruah, C.K., Das, J., Hoque, S., Sarma, N.S., 2011. Vermicomposting of distillation waste of citronella plant employing Eudrilus eugeniae. Bioresour. Technol. 102, 6944–6950. Edwards, C.A., Dominguez, J., Neuhauser, E.F., 1998. The potential use of Perionyx excavates (Perr.) (Megascolecidae) in organic waste management. Biol. Fertil. Soils 27, 155–161.
D. Das et al. / Ecological Engineering 86 (2016) 154–161 Fischer, K., Hahn, D., Honerlage, W., Zeyer, J., 1997. Effect of passage through the gut of earthworm Lumbricus terrestris L. on Bacillus megaterium studied by whole cell hybridization. Soil Biol. Biochem. 29, 1149–1152. Flack, F.M., Hartenstein, R., 1984. Growth of the earthworm Eisenia foetida on microorganisms and cellulose. Soil Biol. Biochem. 16, 491–495. Fornes, F., Mendoza-Hernández, García-de-la-Fuente, R., Abad, M., Belda, R.M., 2012. Composting versus vermicomposting: a comparative study of organic matter evolution through straight and combined processes. Bioresour. Technol. 118, 296–305. Garg, P., Gupta, A., Satya, S., 2006. Vermicomposting of different types of waste using Eisenia foetida: a comparative study. Bioresour. Technol. 97, 391–395. Kale, R.D., Bano, K., Krishnamoorthy, R.V., 1982. Potential of Perionyx Excavates for utilization of organic wastes. Pedobiology 23, 419–425. Kaviraj, Sharma, S., 2003. Municipal Solid waste management through vermicomposting employing exotic and local species of earthworms. Bioresour. Technol. 90, 169–173. Kumar, R., Verma, D., Singh, B.L., Kumar, U., Shweta, 2010. Composting of sugar cane waste by products through treatment with microrganisms and subsequent vermicomposting. Bioresour. Technol. 101, 6707–6711. Moody, S.A., Briones, M.J.I., Piearce, T.G., Dighton, J., 1995. Selective consumption of decomposing wheat straw by earthwoms. Soil Biol. Biochem. 27, 1209–1213. Neuhauser, E.F., Loehr, R.C., Malecki, M.R., 1988. The potential of earthworms for managing sewage sludge. In: Edwards, C.A., Neuhauser, E.F. (Eds.), Earthworms and Waste Management. SPB Acad. Publ., The Netherlands. Ndegwa, P.M., Thompson, S.A., 2000. Effect of C and N Ratio on Vermicomposting in the Treatment and Bioconversion of Biosolids. Bioresour. Technol. 76, 7–12. Olivares, F.L., Canellas, L.P., Aguiar, N.O., Lima, L.S., Busato, J.G., 2012. Changes in labile phosphorus forms during maturation of Vermicompost enriched with phosphorus-solubilizing and diazotrophic bacteria. Bioresour. Technol. 110, 390–395. Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis. Part 2, SSSA, Madison, WI. Parthasarathi, K., Ranganathan, L.S., 1999. Longevity of microbial and enzyme activity and their influence on NPK content in pressmud vermicasts. Eur. J. Soil Biol. 35, 107–113. Rasal, P.H., Kalbhar, H.B., Shingte, V.V., Patil, P.L., 1988. Development of technology for rapid composting and enrichment. In: Sen, S.P., Palit, P. (Eds.), Biofertilizers:
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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Bioconversion and biodynamics of Eisenia foetida in different organic wastes through microbially enriched vermiconversion technologies D. Das a , P. Bhattacharyya b , B.C. Ghosh c , P. Banik a,∗ a b c
Agricultural and Ecological Research Unit, Indian Statistical Institute, 203 Barrackpore Trunk Road, Kolkata 700108, India Agricultural and Ecological Research Unit, Indian Statistical Institute, Giridih, 815301, Jharkhand, India Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, India
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 14 October 2015 Accepted 10 November 2015 Keywords: Vermicomposting Eisenia foetida Microorganisms Organic wastes Nutrients
a b s t r a c t The present investigation is an attempt to convert organic wastes like water hyacinth, paddy straw and sawdust to organic manure through vermicomposting by Eisenia foetida together with the help of some beneficial microbes such as Trichoderma viride (cellulolytic), Azotobacter chroococcum (nitrogen fixing), Bacillus polymixa (phosphate solubilizing) and Bacillus firmus (potassium solubilizing). Chemical and biochemical properties of vermicompost prepared from the aforementioned organic wastes were studied. Earthworm biomass, growth performance, and cocoon production were higher in vermicompost prepared from water hyacinth and paddy straw as compared to vermicompost prepared from sawdust. All vermibeds showed significant reduction in pH, organic carbon, cellulose content and C/N ratio and substantial increase in humic acid, total N, available P and exchangeable K. Vermistabilization also increased the microbial population and enzyme activities of all organic wastes. Inoculation of A. chroococcum, B. polymixa and B. firmus alone or in combination with T. viride significantly increased nitrogen, available P and K content. Significant increases in microbial population and enzyme activities in vermicomposts were recorded with inoculation of T. viride either alone or in consortia. The best quality compost was prepared when the substrate was treated with all the four microorganisms together followed by vermicomposting. Results indicated that the combination of earthworms and microorganisms reduced the overall time required for composting besides producing a nutrient-enriched compost product. © 2015 Published by Elsevier B.V.
1. Introduction About 3000 million tons of organic wastes are produced every year in India (Bharadwaj, 2010). Disposal of increasing quantities of organic wastes is becoming a serious problem. Aquatic waste like water hyacinth is a free-floating aquatic weed which creates major problems in the aquatic system due to its rapid growth and the robustness of its seeds. Paddy straw is a post-harvest residue of the agricultural field whereas saw dust, produced by the sawmill, is found in large quantities in the environment. In many countries traditional management practice of wastes is open air burning and this leads to release of greenhouse gases, and production of particulate matter. As an alternative to burning of wastes vermiculture technology for recycling these organic residues yields valuable manure within a short period. Vermicomposting is an ecobiotechnological process that transforms energy rich and complex
∗ Corresponding author. E-mail address: [email protected] (P. Banik). http://dx.doi.org/10.1016/j.ecoleng.2015.11.012 0925-8574/© 2015 Published by Elsevier B.V.
organic substances into stabilized humus like product with the help of microorganisms. Vermicomposting is accelerated by the combined activity of a diverse group of microorganisms. One of the possible ways of increasing the nutrient content of vermicompost is a microbial enrichment technique with nitrogen fixers, and phosphorous solubilisers (Kumar et al., 2010; Bustao et al., 2012). The incorporation of vermicompost along with microbial fertilizers had a beneficial effect on the crop yield. Microorganisms produce enzymes that cause biochemical decomposition of organic matter, but earthworms are the crucial driver of the process as they are involved in the indirect stimulation of microbial population through fragmentation and ingestion of fresh organic matter. This, in turn, results in a greater surface area available for microbial colonization, thus dramatically increasing microbiological activity (Aira et al., 2002). Inoculation of suitable cellulolytic and lignolytic strains like Trichoderma viridae (TV) is able to accelerate the composting rate of organic substrates because the lignin-cellulose complex is transformed with their cellulolytic enzymes, thus enriching the final product with nutrients (Tiwari et al., 1989). Inoculation of N2 fixing
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Table 1 Physico-chemical properties of organic wastes. Organic wastes
pH
EC (ds/m)
Organic C (%)
Total N (%)
C/N ratio
Total P (%)
Total K (%)
Cow dung Water hyacinth Paddy straw Saw dust
6.2 7.3 7.8 7.5
1.3 0.623 0.742 0.82
20.3 29.6 52.6 68
0.68 0.62 0.78 0.18
29.9 47.8 67.4 377.8
0.4 0.35 0.27 0.009
0.65 0.82 0.62 0.23
bacteria and phosphorus solubilizing bacteria (PSB) during vermicomposting increased nitrogen content and phosphorus content and N2 fixing ability of bacteria depended upon the use of organic wastes (Kumar et al., 2010; Bustao et al., 2012; Olivares et al., 2012). The effect of body growth of earthworm by the inoculation of phosphate solubilizing and potassium releasing bacteria during vermicomposting process has been scantly studied. Sheng and He (2006) reported that solubilization of illite and feldspar by potassium releasing microorganisms is due to the production of organic acids like oxalic acid and tartaric acid and also due to production of capsular polysaccharides which helps in dissolution of minerals to release potassium. Therefore, potassium releasing bacteria, Bacillus firmus might be added during vermicomposting to solubilize potassium and increasing potassium content in the final product of vermicompost. It is well known that the food source influences not only the size of earthworm population, but also their growth and reproduction rates. There is abundant literature on the response of earthworms to different type of vegetable substrate in the field (Bansal and Kapoor, 2000; Bharadwaj, 2010), but there is a lack of information on the effect of additional microbial strains in the feed of earthworms during vermicomposting. Keeping this in mind, the present study was conducted to assess the potential of Eisenia foetida in vermicomposting of different types of organic materials and quality of vermicompost thus produced. An attempt was also made to bring changes in nitrogen, phosphorus and potassium components, humic acids and some enzyme activities of resulting vermicompost which is produced from three different C/N ratio based organic wastes by inoculating of N2 fixing, Phosphate solubilizing, potassium releasing bacteria and cellulose decomposing fungi viz. T. viridae.
bacteria) and Bacillus polymixa (phosphate solubilising bacteria) were used. Bacillus firmus (potassium releasing bacteria) were procured from Indian Agricultural Research Institute, New Delhi. The fungal cultures were maintained by sub culturing on potato dextrose agar, while bacteria A. chroococcum, B. Polymixa, B. firmus were sub culturing on Jensen’s Agar Media, Pikovaskaya medium and Aleksandrov medium, respectively.
2. Materials and methods
2.4. Treatment details
2.1. Organic wastes and earthworm species used for vermiconversion The organic wastes, like water hyacinth (WH), paddy straw (PS) and sawdust (SD) were collected from a local farm of Kharagpur, India. Before carrying out the experiment some common physicochemical characteristics of organic wastes were analyzed (Table 1). Urine free cow dung, collected from a nearby dairy farm was used as the organic source to initiate as well as expedite the biological conversion process. Juvenile, non-clitellated specimens of epigeic earthworm E. foetida, weighing about 200–250 mg, were obtained from our experimental vermiculture unit and used for composting on organic mixed wastes. The experiment on biodegradable organic wastes was conducted in 2.0 l earthen pots with a small hole at the bottom. Three different biodegradable organic wastes, viz. Water hyacinth, paddy straw and sawdust with variable C/N ratios were used as food for E. foetida. Water hyacinth and paddy straw were chopped to reduce their size and volume by about 50–70%. Finely chopped substrates were dipped overnight in 0.1% formalin. 2.2. Microbial source Pure cultures of microbial inoculants, viz. T. Viridae (lignolytic and cellulolytic fungi), A. chrococcum (free living nitrogen fixing
2.3. Experimental design 50 adult earthworms of E. foetida were placed in each container and supplied with 2 kg of organic wastes (WH, PS or SD), 600 g of Cow Dung (30% w/w) was mixed with each organic waste to provide an initial favorable environment for the earthworm. Trichoderma viride (cellulolytic, M2 treatment), Azotobacter chroococcum (nitrogen fixing, M3 treatment), Bacillus polymixa (phosphate solubilizing, M4 treatment) and Bacillus firmus (potassium solubilizing, M5 treatment) and mixed all microbes (M6) were inoculated as 50 mL of 7 days old broth culture per kg (106 cells per ml) of these organic substrates M1 is control, i.e., no microbial inoculate in this treatment. Moisture content was maintained at about 60% (dry weight basis) during the vermicomposting process of periodic sprinkling water. The pots were kept in dark at maximum laboratory temperatures ranging between 27 and 30 ◦ C during the whole experimental period. The worms were not supplied with additional food for the duration of the experiment that went on for 16 weeks. The experiment was conducted following complete randomized block design with three replications. The sampling for analysis was made after stabilization of vermicomposting (84–112 days), which varied depending upon the nature of the feed.
T1—Organic wastes + cowdung T2—Organic wastes + cowdung + Trichoderma viridae T3—Organic wastes + cowdung + Azatobactor chroococcum T4—Organic wastes + cowdung + Bacillus polymixa T5—Organic wastes + cowdung + Bacillus firmus T6—Organic wastes + cowdung + Trichoderma viridae + Azatobactor chroococcum + Bacillus polymixa + Bacillus firmus 2.5. Physico-chemical and biochemical analysis Physico-chemical analysis of wastes, vermicompost was done according to the methods proposed by Page et al. (1982). Enzymes such as urease, phosphatase, and amylase activities of soils were estimated following the methods suggested by Tabatabai (1994). These measurements were carried out using the field moist condition. However, the measured activities were expressed on moisture-free basis. 2.6. Microbial assays Microbial plate counts were made following the method of Travors and Cook (1992). Vermicompost (1 g) from the same sample employed for microbial respiration was taken and stirred with 100 ml sterile distilled water in a conical flask and the supernatant
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Fig. 1. Earthworm growth rate, reproduction rate and cocoon production in different organic wastes during vermicomposting by using different microbes. Error bars represent standard deviation. Arithmetic means with the same letter are not significantly different from each other at 5% probability level.
was serial dilution method to estimate the population of total bacteria, total fungus, cellulolytic fungi, phosphorus solubilising bacteria (PSB), azatobactor (AZC) and potassium solubilising bacteria (KSB) in nutrient agar medium, potato dextrose agar medium, asparagines medium, azatobactor medium, pikovskaya agar medium and aleksandrov medium, respectively. Soil respiration was determined following the method of Alef (1995). 2.7. Statistical analysis Statistical analyses such as the least significant difference (LSD) and Duncan multiple range test (DMRT) were carried out using the SPSS statistical package. 3. Results and discussions Table 1 represents the physico-chemical properties of the organic wastes used in this study. pH of all the wastes is alkaline in nature. Organic C, and total N content were higher in saw dust and paddy straw, respectively. The C/N ratio of the sawdust was substantially higher compared to other organic wastes. Water hyacinth contains highest total P and K content. The results of earthworm growth rate and cocoon production are presented in Fig. 1. Many current workers followed a long duration composting process having time span not less than 90 days (Suthar, 2010; Deka et al., 2011; Fornes et al., 2012). However, we have adopted a simple and rapid process of vermitechnology under natural ambient environment as described in the earlier section.
The earthworm growth rate (mg/worm/day) has been considered a useful comparative index to compare the growth of earthworm in different feed wastes (Edwards et al., 1998). Biomass of earthworm in all the experimented wastes increased significantly. The overall growth rate was maximum in paddy straw (19.977 mg/worm/day) followed by water hyacinth (17.937 mg/worm/day) and sawdust (7.233 mg/ worm/day). Among the microbes inoculated in the organic substrates, M6 treatment showed the most effective growth rate. The growth rate in M6 and M2 treatments was significantly higher over M1, M3, M4 and M5 treatments. Earthworms utilize microorganisms in their substrates as a food source and can digest them selectively (Edwards et al., 1998). Cocoon production was significantly higher in paddy straw than water hyacinth and saw dust wastes. Treatment M6 was significantly higher in cocoon production over other respective treatments. The increase in earthworm’s growth may also be attributed to a low C/N ratio (Ndegwa and Thompson, 2000) of the pre-decomposed substrate. The use of bulk agents sawdust waste does not adequately improve cocoon production due to higher C/N ratio. The results indicated that the quality and palatability of food directly affect the survival, growth rate and reproduction potential of earthworms (Suthar, 2009, 2010). The difference in growth rate and cocoon production in different wastes for vermicomposting could be related to biochemical quality of the feed, which is one of the important factors in determining the time taken to reach sexual maturity onset cocoon production (Flack and Hartenstein, 1984). The changes in individual biomass during the vermicomposting process in different wastes with the addition of microbes are
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Fig. 2. Earthworm growth patterns in different organic wastes during vermicomposting by using different microbes. Error bars represent standard deviation. LSD = least significant deviation.
described in Fig. 2. Among different wastes, the highest biomass achieved was 829.9 mg adult−1 in paddy straw treatment and the lowest in saw dust. There was a rapid increase in individual biomass of inoculated earthworms in all vermibeds during initial 21 to 25 days, 25 to 30 days and 33 to 40 days of vermicomposting in case of water hyacinth, paddy straw and sawdust wastes, respectively. The maximum weight gain was followed by weight loss by the time of termination of the experiment. The loss in earthworm weight was also reported by a few other scientists (Suthar, 2007; Singh et al., 2010). They have attributed the weight loss to the food shortage in vermicompost beds. It may be due to the conversion of most of the used substrate material into vermicompost, which cannot further support the growth in earthworms. When E. foetida received food below the maintenance level, it lost weight at a rate which depended upon the quantity and nature of its ingestible substrates (Neuhauser et al., 1988). The feeds that provide earthworms with a sufficient amount of easily metabolize organic matter and non-assimilated carbohydrates favor the growth and reproduction of the earthworms (Edwards et al., 1998). Among the microbial inoculation, TV (M2) and mix (M6) treated wastes were noted higher individual biomass than other microbial inoculants. Trichoderma viridiae is a cellulolytic decomposing fungus. It decomposes the cellulolytic material more rapidly than other microbial treatments. With the progress of vermicomposting, palatability and release of locked up nutrients from the wastes increased, thereby showing maximum biomass, as well as, survival
of earthworms at a later period compared to other different waste treatment. Substrates such as water hyacinth, paddy straw and sawdust had a slightly alkaline pH value at the initial stage. After vermicomposting, pH shifted towards neutrality, irrespective of the initial pH of the organic substrates (Fig. 3). Vermicompost obtained from saw dust recorded lowest pH (7.11), followed by that from paddy straw (7.31) and water hyacinth (7.36). Microbial inoculants showed the variable influence of pH in the resulting vermicompost depending on the nature of microbes and the biodegradable organic wastes used for vermicomposting. In T. viridae (M2) and mix (M6) treatments the lowest pH value (towards the neutral range) were recorded. The neutral pH recorded in the final products might have been due to production of CO2 and organic acids by microbial metabolism during decomposition of different organic substrates (Garg et al., 2006). The highest organic carbon value was recorded in saw dust wastes (526.63 mg/g) followed by paddy straw (339.86 mg/g) and water hyacinth (243.43 mg/g) (Fig. 3). After vermicomposting, all the treatments showed significantly lower organic carbon. The treatment M2 and M6 were recorded lowest organic carbon content compared to other microbial inoculants treatments. The lower organic carbon percentage in vermicomposts obtained from organic sources inoculated with microorganisms might be attributed to a higher rate of decomposition facilitated by the microbes (Bhardwaj, 1999). Moreover, losses of carbon in the form
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Fig. 3. Physico-chemical properties of vermicomposts as affected by different treatments. Error bars represent standard deviation. LSD = least significant deviation.
of CO2 as well as the conversion of any part of the organic fraction into worm biomass are the cause of carbon loss from the substrate (Suthar, 2009). Among the three organic wastes, paddy straw recorded the maximum increase in nitrogen content in the vermicompost over its initial raw material (Fig. 3). Inoculation of microorganism in the biodegradable organic wastes increased Total N (TKN) content of vermicompost, but its effect varied widely (M6: 52–72%, M5: 3–25%, M4:8–37%, M3: 43–64%, M2: 22–32% over control treatment). Among different microbial inoculation treatments Azotobactor (M3) and mix microbial treatments (M6) contain the highest value of TKN content. TKN content in the vermicompost was primarily governed by the initial nitrogen content of the organic wastes used as feed to the earthworm and also by the extent of decomposition (Suthar, 2009). The rate of increase of TKN in the vermicompost from its corresponding organic wastes was well correlated to the C/N ratio of organic substrates. Narrower C/N ratio, greater was the decomposition rate of organic wastes and increases in the TKN content in vermicompost. Earthworms prefer organic wastes with a narrower C/N ratio as they feed (Moody et al., 1995). This explained the greater increase in TKN in vermicompost prepared from water hyacinth as compared to vermicompost prepare from sawdust. TV (M2) and mix (M6) treatments had a low C/N ratio and a higher nitrogen content. Increase in TKN content in the vermicompost obtained on inoculation of cellulolytic cum lignolytic fungi (T. viridae) might be due to enhanced decomposition of the organic matter (Viel et al., 1987), leading to decrease in the organic carbon
content. Thus a decrease in the C/N ratio in the vermicompost as compared to the initial organic substrate, which might be due to a relative increase in the TKN on loss of organic carbon as CO2 as well as water loss by evaporation during mineralization (Viel et al., 1987). The correlation between TKN of vermicompost and a number of free living nitrogen fixing bacteria (Azatobactor chroococcum) [r = 0. 69] suggested that TKN content of vermicompost depended not only on the quality and quantity of initial organic substrate, but also to the extent N2 fixed by free living N2 -fixing bacteria (Kale et al., 1982). Data reveals that total phosphorus (TP) and total potassium (TK) content increased in the resulting vermicompost as compared to the initial organic substrates (Fig. 3). Vermicompost prepared from paddy straw had the highest TP content (8.58 mg/g) followed by water hyacinth (8.0 mg/g) and sawdust (1.46 mg/g) wastes. Among the microbes inoculated in the organic substrates, M6 and M4 treatments were most effective in increasing TP (34–80% and 23–31%, respectively) content. Vermicompost prepared from water hyacinth substrate increased total potassium content as compared to other organic wastes paddy straw and sawdust wastes. Among the microbes inoculated in the organic substrates, M6 and M5 treatments are most effective over control treatments (45.38–80.72% and 33.62–56.66%) in the vermicompost obtained from the different substrates. Phosphorus and potassium mineralization depends on the quality of bulking materials. Acid production during organic matter decomposition of the microorganism is the major mechanism of solubilisation of insoluble phosphorus and potassium.
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Fig. 4. Population of different microorganisms, enzyme activity and microbial respiration of vermicomposts as affected by different treatments. Error bars represent standard deviation. LSD = least significant deviation.
The enhanced number of the microflora present in the gut of earthworms in the case of vermicomposting might have played an important role in this process and increased K2 O over the raw material (Kaviraj and Sharma, 2003). Earthworm primes its symbiotic gut microflora with secreted mucus, water and gut enzymes, i.e. phosphatase enzymes enhance their degradation of ingested organic matter and release of available metabolites. Therefore, directly or indirectly earthworm enriched the substrate material with exchangeable potassium and phosphorus contents. Phosphorus solubilising bacteria increased the phosphorus concentration in vermicompost due to the presence of phosphatase enzyme which enhances the solubility of phosphorus. The increase of phosphorus content could be attributed to the phosphorus solubilising microorganisms present in the worm cast (Suthar, 2009). Potassium solubilising bacteria, increase potassium content in vermicompost could be attributed by the secretion of acid from bacteria and which was solubilised potassium. Vermicompost prepared from paddy straw waste showed the highest humic acid content (0.810 mg/g) followed by water hyacinth (0.533 mg/g) and sawdust (0.343 mg/g) (Fig. 3). Microbial inoculation increased the humic acid content (18.14 to 64.19%)
of vermicompost depending on the organic substrates used. Vermicompost obtained from organic substrates inoculated with T. viridae (M2) and mix (M6) microbes recorded highest humic acid content. T. viridae related treatment was the most effective. Earthworm fragments the organic substrates, thus largely enhancing microbial activities and increasing rates of mineralization, and rapidly converting the wastes into humus like substances (Atiyeh et al., 2001). Hence the narrower C/N ratio of organic matter facilitates higher rate of decomposition (Brady, 1985), organic wastes with a lesser C/N ratio showed higher humic acid content. Preparation of vermicompost by inoculation of microbes in organic wastes hastened the decomposition of organic wastes. The high humic acid content in vermicompost from organic waste inoculated with T. viridae might be due to the fact that the ability of these cellulolytic and lignolytic fungi to decompose organic matter was maximum, but it is statistically at par with the other microbial inoculants. On the other hand, the simultaneous activity of microflora present in the gut of earthworms and in waste might have intensified cellulolysis and lignolysis (Edwards et al., 1998). Earthworms digest long chains of polysaccharides, enhancing microbial colonization. Simultaneously the structure of lignin changes, probably due to
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microbial oxidation and demethylation. Microbial cleavage of the aromatic rings of lignin leads to new polysaccharide and humins in the organic matter (Edwards et al., 1998). Vermicompost prepared from water hyacinth waste showed significantly lowest cellulose content compared to other organic substrates (Fig. 3). Microbial inoculation increases the cellulose degradation. Treatment of M2 and M6 showed the lowest value of cellulose content. The production of cellulose degrading enzymes by the inoculated microbes during vermicomposting might have accelerated the degradation process (Singh and Sharma, 2002). Similar results were also reported by Rasal et al. (1988) who reported rapid decomposition of sugar cane trash with a mixture of cellulolytic fungi; T. viride, Trichurus spiralis, Paecelomyces fusisporus and Aspergillus sp. along with nitrogen fixing bacteria Azotobacter sp. Microbial colony estimation revealed that the total bacteria (26 × 108 CFU) as well as nitrogen fixing bacteria (AZC count) (6 × 103 CFU), PSB (9 × 106 ) and KSB (7 × 104 CFU) populations were lost in vermicompost obtained from saw dust wastes and it differed significantly (P < 0.05) from vermicomposts prepared from other organic wastes (Fig. 4). Among the microbial inoculation treatments, M6 treatments showed a higher population for total bacterial counts, AZC, PSB and KSB count. Total fungal count (8.17 × 106 CFU) as well as cellulolytic fungal (10 × 105 CFU) population were least in vermicompost obtained from saw dust followed by water hyacinth (38 × 106 and 42 × 105 CFU) and paddy straw (39 × 106 and 45 × 105 CFU). Though vermicompost obtained from paddy straw was showing a higher abundance, population, but it was statistically significant (P < 0.05) from other vermicompost which is prepared by other organic wastes. Urease activity in vermicompost varied depending on the organic wastes used and showed the highest activity in vermicompost obtained from water hyacinth wastes (128.70 g NH4 /g/h) followed by paddy straw (121.33 g NH4 /g/h) and saw dust wastes (33.81 g NH4 /g/h) (Fig. 4). Among the microbial treatments, Azotobacter inoculation in organic wastes significantly increased urease activity. The urease activity was well correlated to the nitrogen content (r = 0.691), Azotobactor chroococum (N2 fixing bacteria) (r = 0.743) of the vermicompost. The positive correlation between urease activities of the vermicompost from different organic wastes and the humic acid content (r = 0.826) suggested that humic acids might be responsible for preserving the urease enzyme as humicenzyme complex in vermicompost. The variation in urease activity among the vermicompost obtained from different organic sources might be due to variation in the organic matter content of the initial substrates (Syers et al., 1979). High correlation value (r = 0.83) between the urease activity and total microbial respiration in the vermicompost suggested that microorganisms, activity of which were enhanced by the earthworm, while passing the substrates through the worm guts (Fischer et al., 1997) might be solely responsible for urease activities in the vermicompost. Fig. 4 showed that higher phosphatase activity in vermicompost prepared from paddy straw wastes followed by water hyacinth (238.33 g p-nitro phenol/g/h) and sawdust (87.23 g p-nitro phenol/g/h) wastes. Among the microbial inoculation treatments M2 and M6 showed higher phosphatase activity. Phosphatase activity was significantly and positively correlated with humic acid extracted from vermicompost (r = 0.87) and phosphorus solubilising bacterial count (r = 0.737). Earthworms are used to feed fungi, for their reproduction and growth (Parthasarathi and Ranganathan, 1999). Microbes entering the worm guts, consume nitrogenous compounds of the mucus (Zhang et al., 2000), which largely increase their activity, which in turn enables them to contribute enzymes in the digestive process of the earthworm. Microorganisms not only mineralize complex substance into plant available form but also can synthesize biologically active substances. Hence
phosphatase activity in vermicompost was increased as compared to their raw materials. The positive correlation of acid phosphatase enzyme activities with microbial respiration in vermicompost (r = 0.836) confirmed the fact that the enhanced enzyme activity in the cast was due to microbes, rather than the epithelium of the gut (Vinotha et al., 2000). Amylase activity of vermicompost varied depends upon the organic wastes used and showed the highest activity in vermicompost obtained from water hyacinth wastes followed by paddy straw and sawdust wastes (Fig. 4). Treatment M2 and MS showed the higher amylase activity compared to other treatments. Inoculation of microorganisms significantly (P < 0.05) increased respiration of both the organic substrates and their effect varied being dependent on the nature of the organic wastes and microorganisms (Fig. 4). The data suggested that vermicompost prepared from paddy straw wastes was recorded significantly higher respiration as compared to other vermicomposts. In general, fungi decompose organic matter faster than bacteria. Among these microbial inoculants treatments, M2 and M6 treatments were recorded a higher microbial respiration than other inoculants treatments. Vermicompost prepared from water hyacinth waste was recorded higher respiration rate than other vermicompost that might be due to higher mineralization rate and lowest C/N ratio than other vermicompost. 4. Conclusions The results demonstrated that the organic wastes can be converted into an environmentally-friendly nutrient source by vermicomposting, using different microorganisms. Growth and population of E. foetida were influenced by different waste materials. Among waste materials, paddy straw was preferred by E. foetida, followed by water hyacinth, and saw dust. Inoculation of microorganisms with earthworms accelerated the decomposition process. Vermicompost using T. viride showed the highest nutrient content as well as enzymatic and microbial status, but the combined effect of all inoculated microorganisms proved to have a significant effect on the quality of vermicompost. Acknowledgements Authors thankfully acknowledge Prof. R. L. Brahmachary for his help to improve the writing style of the article. References Aira, M., Moroy, F., Dominguez, J., Mato, S., 2002. How earthworm density affects microbial biomass and activity in pig manure. Eur. J. Soil Biol. 38, 7–10. Alef, K., 1995. Estimation of soil respiration. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London. Atiyeh, R.M., Edwards, C.A., Suber, S., Metzger, J.D., 2001. Pig manure vermicompost as a component of horticultural bedding plant medium: effects on physicochemical properties and plant growth. Bioresour. Technol. 78, 11–20. Bansal, S., Kapoor, K.K., 2000. Vermicomposting of crop residues and cattle dung with Eisenia foetida. Bioresour. Technol. 73, 95–98. Bhardwaj, K.K.R., 1999. Significance of microbial inoculants in organic matter decomposition. In: Jha, M.N. (Ed.), Agromicrobes, Current Trends in Life Sciences. Today and Tomorrow’s Printers and Publisher, New Delhi, pp. 235–247. Bharadwaj, A., 2010. Management of kitchen waste material through vermicomposting. Asian J. Exp. Biol. Sci. 1, 175–177. Brady, N.C., 1985. The Nature and Properties of soils, eighth ed. Colier Mcmillan, London. Bustao, J.G., Lima, L.S., Aguiar, N.O., Canellas, L.P., Olivares, F.B., 2012. Changes in labile phosphorous forms during maturation of vermicompost enriched with phosphorus solubilizing and diazotrophic bacteria. Bioresour. Technol. 110, 390–395. Deka, H., Deka, S., Baruah, C.K., Das, J., Hoque, S., Sarma, N.S., 2011. Vermicomposting of distillation waste of citronella plant employing Eudrilus eugeniae. Bioresour. Technol. 102, 6944–6950. Edwards, C.A., Dominguez, J., Neuhauser, E.F., 1998. The potential use of Perionyx excavates (Perr.) (Megascolecidae) in organic waste management. Biol. Fertil. Soils 27, 155–161.
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