Composting of Rice Straw Using Different Inocula and Analysis of Compost Quality

MATERIALS AND METHODS

This study was carried out in the Department of Microbiology, CCS Haryana Agricultural University, Hisar on the straw obtained from rice crop during the months of October 2008 to December 2010.

Inoculum used for composting: Paddy straw was collected from the fields of Regional Rice Research Station, Kaul of CCS Haryana Agricultural University, Hisar, India. Cattle dung was collected from Animal Science Department and biogas slurry was collected from biogas plant of Department of Microbiology of the University. Organic carbon and total nitrogen contents of the three inocula were determined. The inoculum containing a fungal consortium of three fungi Aspergillus awamorii, Paecilomyces fusisporous and Trichoderma viride, isolated from our previous experiment was used as inoculum (Goyal et al., 2005). Composting of unchopped paddy straw was carried out in windrows with following treatments:

Preparation of compost: The unchopped paddy straw was soaked in 0.1% urea solution for 3 min in a drum of 200 Litres capacity and then stacked in windrows (1 m heightx1 m widex2 m long). It was covered with thick polythene sheets to conserve moisture. The different inocula containing biogas slurry and cattle dung were added at 1.0% on dry weight basis to the urea solution. Charcoal based consortium of three fungi (100 g) containing 10⁹ spores mL^-1 was suspended in 1000 Litre of urea solution. The level of moisture was maintained to 60% WHC by adding water at different intervals of composting. The material was allowed to decompose for three months and two turnings were given at 15 and 30 days of composting. Sampling was done after 0, 15, 30, 60 and 90 days.

Chemical analysis: The compost samples were dried and grounded to pass through 2 mm sieve for chemical analysis of carbon, nitrogen, phosphorus and potassium. Total organic C in the compost was measured by the method of Nelson and Sommers (1982) and total N was measured by Kjeldahl’s digestion method (Bremner and Mulvaney, 1982). Total phosphorus content was determined by the method of John (1970). The potassium content in the digest was estimated on flame photometer by direct feeding. Total C in compost water extract was measured by the titration method (Kalembasa and Jenkinson, 1973). The cellulase and xylanase activities involved in the degradation of organic materials (Tiquia, 2002) were estimated by the method of Schinner and Von Mersi (1990) using carboxymethyl cellulose and birch wood xylan, respectively as substrates. Carbon dioxide evolution in finished product was determined by the method of Pramer and Schmidt (1964). Humic substances in compost were determined according to the method of Kononova (1961).

Seed germination: The germination index of wheat and mustard seeds was determined by taking 30 seeds of wheat and mustard on a sterile Petri plate containing a sterilized ordinary filter paper disc (El Hammadi et al., 2007). Compost water extract was prepared by adding 10 g of finished compost in 90 mL of distilled water, shaken for 30 min and then filtered through Whatman no 1 filter paper. Eight mL of compost water extract from each treatment was added to the above plates and incubated in a BOD incubator at 30°C. The germination of seeds in each treatment was counted. The numbers of seeds germinated in sterilized distilled water were taken as control for calculation of germination percentage.

Statistical analysis: All chemical measurements were carried out in triplicate and LSD values at p = 0.05 were used to determine the significant differences between treatment means. Statistical analysis for determination of CD was done by using SPSS software programme (OPSTAT). Linear correlation coefficient between different compost maturity parameters was also determined.

RESULTS

Determination of organic C, total N and C:N ratio in paddy straw compost: Paddy straw contained 51.76% organic C, 0.65% nitrogen, 0.20% phosphorus, 0.30% potassium where as biogas slurry showed 44.0% organic C, 1.60% total N, 0.6 total P and 0.93 K. Cattle dung contained 48.14% organic C, 1.42% total N, 0.4% total P and 0.90% K. The initial C:N ratio of paddy straw, biogas slurry and cattle dung was found 80:1, 27:5 and 33:9, respectively.

The changes in C, N and C/N ratio were determined at different intervals of paddy straw composting (Table 1). Carbon content of the compostable material decreased from 52.3 (at 0 day) to 34.9% (at 90 days) in the treatment with inoculation of fungal cultures in the paddy straw. After 90 days of decomposition, lowest amount of organic C was found in paddy straw inoculated with consortium of fungi and cattle dung in comparison to uninoculated straw. Nitrogen content per unit of material increased from 0.70 to 2.17% that resulted in the decrease of C:N ratio of the compostable material. Inoculation of paddy straw with biogas slurry, consortium of fungi and cattle dung showed 66.6, 194.3 and 77.9% more N in comparison to uninoculated paddy straw after 90 days of composting. Thus, maximum increase in N content was observed by inoculation of fungal consortium followed by cattle dung application. However, in the beginning of the composting process, biogas slurry and cattle dung treatments showed more total N content than the consortium of three fungi which was due to presence of 1.6 and 1.4% N in biogas slurry and cattle dung, respectively.

The low C:N ratio is considered as one of the parameter for estimating the compost maturity and stability. In the treatment having fungal inoculum, the C:N ratio of compostable material decreased from 73.7 to 36.1 after 15 days of composting showing that microorganisms developed during the composting process decomposed the material efficiently. The microorganisms present in biogas slurry treatment lowered down C:N ratio from 50.9 to 34.0 and from 41.7 to 26.3 in cattle dung treatment after 15 days of composting. A non significant difference in C:N ratio was observed with cattle dung and consortium of three fungi than using biogas slurry as inoculum. After 90 days of composting, C:N ratio varied from 34.5 to 16.6 in different treatments. Thus, inoculation with consortium of fungi resulted in compostable material with lowest C:N ratio of 16.6 followed by cattle dung having C:N ratio of 17.0.

Table 1:	Changes in organic carbon, total nitrogen and C:N ratio at different intervals of paddy straw composting

Total nitrogen, phosphorus and potassium content of the compost: In the prepared compost, the total Nitrogen (N) content varied from 1.15 in paddy straw alone to 2.17% in paddy straw amended with cattle dung after 90 days of decomposition (Table 2). Maximum increase in total Phosphorus (P) content was found with fungal culture treatment followed by inoculation of cattle dung and it varied from 0.062 in paddy straw alone to 0.164 by inoculation of fungal consortium. The total potassium (K) content also varied from 0.134 to 0.169% and maximum increase in total K was observed by inoculation of the fungal consortium. Thus, amount of N, P and K contents were found more in compost prepared with consortium of fungi and cattle dung than the compost prepared from biogas slurry.

Cellulase and xylanase activities: Cellulases and xylanases are among the various hydrolytic enzymes which are involved in degradation of organic materials Cellulase activity increased from 6 mg (in uninoculated paddy straw at 0 day) to 252 mg reducing sugar kg^-1 dry matter h^-1 in treatment having fungal inoculum at 30 days of composting (Table 3). Highest amount of cellulase activity was observed at 30 days of composting in all the treatments (expect the treatment with fungal culture inoculum) and then declined at later stages of composting. Paddy straw inoculated with consortium of fungal cultures showed 65% higher cellulase activity in comparison to uninoculated paddy straw. On the other hand, addition of biogas slurry resulted in 48% increase whereas, only 13% increase in cellulase activity was observed with amendment of cattle dung at 30 days of composting. The results showed that these fungi used for inoculation were efficient in decomposing the lignocelluloses present in the paddy straw. After 90 days of decomposition, no significant difference in cellulase activity was observed in the treatments in which paddy straw was inoculated with consortium of fungi or amended with cattle dung. Xylanase activity also varied from 9 to 111 mg reducing sugar kg^-1 dry matter h^-1 (Table 3). The activity increased to maximum at 60 days and thereafter, it declined. Highest amount of xylanase activity was observed by using consortium of fungi and cattle dung as inoculum than the treatment amended with biogas slurry.

Table 2:	Plant nutrient contents (N, P, K) of paddy straw compost after 90 days of paddy straw composting

Table 3:	Changes in cellulases and xylanases activity during paddy straw composting at different intervals

Humic acid and fulvic acid C content in the compost: During composting, the complex organic material lignin is polymerized into fulvic acid and humic acids due to the humification process. These humic substances become stable fraction of soil organic matter, provide energy for the growth of microorganisms and regulate the carbon cycle.

The amount of humic acid in the finished compost varied from 91 to 110 mg g^-1 of compost and fulvic acid from 3 to 23 mg g^-1 of compost (Table 4). Highest amount of humic substances were present in the prepared compost using cattle dung and consortium of fungi as inoculum. There was no significant difference in the amount of humic substances in these two kinds of composts. However, the compost prepared from paddy straw using biogas slurry as inoculum or paddy straw alone showed lower amount of humic substances. Thus, inoculation of paddy straw with fungal consortium and cattle dung showed 21.4 and 25.1% more total humic substances carbon in finished product in comparison to untreated control. The present studies showed that the total amount of humic substances generated by lignocellulolytic fungi and cattle dung were more than using biogas slurry as inoculum or to the uninoculated control.

Water soluble carbon and carbon dioxide evolution in finished compost: Changes in water soluble carbon in relation to total carbon content in the prepared compost varied from 2.69 to 5.92% by using different inocula (Table 4). The compost prepared from paddy straw alone and biogas slurry showed more amount of dissolved organic carbon than compost prepared from cattle dung and using consortium of fungi. Lesser amount of water soluble C (2.69%) was observed with the consortium of fungi inoculated paddy straw. However, biogas slurry and cattle dung inoculated paddy straw showed 4.01 and 2.74% water soluble C, respectively in comparison to uninoculated paddy straw (5.92%). The presence of more amount of water soluble carbon in compost prepared from paddy straw alone or by amendment of biogas slurry showed that these two composts were not stable.

Measurement of CO₂-C evolved from 90 days old compost obtained after inoculation of cattle dung and consortium of three fungi showed less amount of CO₂-C evolution than compost prepared with biogas slurry or without any inoculum (Table 4). The evolution of CO₂-C in four weeks varied from 174 to 333 mg 100 g^-1 compost. The three different inocula helped in the breakdown of complex organic matter present in paddy straw. In untreated paddy straw, CO₂-C evolution was 333 mg 100 g^-1 compost. The higher amount of CO₂ evolution suggested that material is not stabilized enough and needs further decomposition. During the present studies, the evolution of CO₂-C in 90 days old compost was less than above limit indicating that these composts were stabilized after the 90 days of decomposition.

Germination index of wheat and mustard: The application of unstable and immature compost in soil can lead to reduction of oxygen concentration, immobilization of important plant nutrients and may result in inhibition of seed germination due to its phytotoxic effect.

Table 4:	Humic and fulvic acid C in paddy straw compost after 90 days of composting

Table 5:	Germination index of wheat and mustard seeds in compost water extract

Table 6:	Correlation coefficient between compost maturity parameters
Correlation coefficient at 0.05% level, * and ** : Level of correlation with each other, NS : Non significant correlation

To find out the phytotoxic effect of the compost prepared by using different inocula, per cent germination of wheat and mustard seeds were tested under laboratory conditions. Germination of seeds in sterilized water was used as control. Compost prepared without any inoculum showed only 69 to 68% seed germination of wheat and mustard, respectively (Table 5). Whereas, the paddy straw compost prepared by inoculation of biogas slurry, cattle dung and consortium of three fungi showed higher percent germination of seeds which varied from 78 to 87% in wheat and 79 to 86% in mustard. Thus, compost prepared without any inoculum was not fully mature and therefore, it inhibited germination of seeds. The poor quality of compost resulting in the inhibition of seeds is also supported by the amount of CO₂-C evolved, water soluble C and humic substances present in the paddy straw compost prepared without any inoculum.

Correlation coefficient: Correlation coefficient among various parameters was determined which could be used as indicator of compost maturity and stability (Table 6). Some parameters were positively correlated with each other in this study. For example, there was statistically significant positive correlation between C:N ratio and humic substances, water soluble C and CO₂ evolution. Whereas, no significant correlation was observed among water soluble C, humic substances, CO₂ evolution and germination index of wheat and mustard. However, there was no single parameter that can be used to determine compost quality and maturity.

DISCUSSION

Rice is a major crop grown worldwide and generates large production of straw annually. Paddy straw contains 51.76% organic C, 0.65% nitrogen, 0.20% phosphorus, 0.30% potassium and has high C:N ratio. It is also rich in silica and lignin which makes it difficult to be degraded (Kumar et al., 2006). The composting of rice straw results in reusability of the nutrients contained in the residue and also avoids air pollution caused by residue burning (Dileep and Dixit, 2005; Sidhu and Beri, 2008).

During decomposition of paddy straw, carbon content of the compostable material decreased from 52.3 to 34.9% in the different treatments (Table 1). Maximum 17.4% decrease in organic C was observed in the treatment containing consortium of three fungi, whereas only 14.0% decrease in organic C was observed with cattle dung at 90 days of composting. The lowering of carbon contents during the process of composting is due to the intense microbial activity of the microbiota present in the compostable material or that has been inoculated as inoculum. During composting, there is a degradation of complex organic matter into simple ones with the evolution of CO₂ gas and energy (Adani et al., 1997). Vuorinen and Saharinen (1997) have also reported that approximately 11-27% of the total C is lost during initial stage of active composting and about 62-66% during the whole composting time.

In the beginning of the composting process, biogas slurry and cattle dung treatments showed more total N content than the consortium of three fungi which was due to presence of 1.6 and 1.4% N in the inocula consisting of biogas slurry and cattle dung, respectively. At 90 days of composting, increase in N content was found more by inoculation of fungal consortium (194.3%) followed by cattle dung application (77.9%) whereas, inoculation of paddy straw with biogas slurry showed only 66.6% more N in comparison to uninoculated paddy straw. Thus, maximum decrease in C:N ratio of compostable material from 73.7 to 16.6 was observed with consortium of fungi. In different treatments, C:N ratio varied from 34.5 to 16.6 after 90 days of composting. The C:N ratio of compostable material inoculated with consortium of fungi (16.6) and cattle dung (17.0) was lowest in comparison to uninoculated paddy straw or paddy straw inoculated with biogas slurry. Gaind et al. (2005) also reported that composting of wheat straw by inoculation of fungi along with application of 1% urea resulted into an end product with C:N ratio of 10:7 within three months in pits. Similarly, Brito et al. (2008) also observed a decline in the C:N ratio at the end of composting from 36 to a value of 14, by the addition of solid fraction of cattle slurry. Compost production from sugar cane organic waste was performed using Trichoderma fungi with different levels of urea and pH (Torkashvand et al., 2008) and it was observed that C:N ratio reduced below 30 in 5 weeks and below 20 in 10 weeks after composting.

Maximum increase in total N content in the prepared compost was observed by amendment with cattle dung after 90 days of decomposition whereas maximum increase in total P content was found with fungal culture treatment followed by inoculation of cattle dung (Table 2). Similarly, maximum increase in total K was observed by inoculation of the fungal consortium and it varied from 0.134 to 0.169%. A non significant difference in the N, P and K content was observed in paddy straw compost prepared by using consortium of fungi and cattle dung. Similarly, Saludes et al. (2008) also reported that the total N, P and K concentrations of the dairy cattle manure/wallboard compost mixture did not change significantly after 28 days of composting. Application of 250 t ha^-1 of fresh organic amendments in nutrient depleted soil of South-Western Nigeria was found to increase the soil N, P, K, Ca, Mg, Cu, Zn, Mn and Fe (Chukwuka and Omotayo, 2008). Moreover, the compost prepared from the uprooted Parthenium (before flowering) was found to contribute 1.58, 0.33 and 1.64% of total N, P and K, respectively and also provided Fe, Mn, Zn and Cu micronutrients (Kishor et al., 2010).

The compost prepared by using fungal consortium as inoculum showed maximum cellulase activity at 30 days of composting and it increased from 88 mg (control untreated paddy straw) to 252 mg reducing sugar kg^-1 dry matter h^-1 in fungal inoculated compostable material (Table 3). Xylanase activity varied from 9 to 111 mg reducing sugar kg^-1 dry matter h^-1 and highest amount of xylanase activity was observed by using consortium of fungi and cattle dung as inoculum. Addition of consortium of fungal cultures, biogas slurry and cattle dung resulted in 65, 48 and 13% higher cellulase activity at 30 days of composting. Similarly, maximum cellulase activity was observed at 30 days during composting of organic waste (Goyal et al., 2005) and using bark of pine and eucalyptus (Cunha-Queda et al., 2002).

Paddy straw is recalcitrant and contains high amount of silica. Due to the humification process carried out by lignocellulolytic fungi, lignin present in the paddy straw is decomposed into fulvic acid and humic acids which become stable fraction of soil organic matter (Crawford and Crawford, 1980; Huang et al., 2008). These humic substances act as permanent source of energy for the growth of microorganisms and regulate the carbon cycle (Veeken et al., 2000). In the finished compost, amount of humic acid varied from 91 to 110 mg g^-1 of compost and fulvic acid from 3 to 23 mg g^-1 of compost (Table 4). The inoculation of paddy straw with fungal consortium and cattle dung showed 21.4 and 25.1% more total humic substances carbon in finished product in comparison to untreated control. Different composting strategies have been reported to have impact on the degree of humification during composting process (Alburquerque et al., 2009). Lopez et al. (2006) evaluated the degradation of lignin in the mixtures of horticultural plant residues having different C:N ratios by the inoculation of different lignocellulolytic fungi namely Coriolus versicolor, Phanerochaete flavido-alba and Trichoderma koningii. Inoculation with these three fungi enhanced the formation of humic substances by degradation of lignin.

Lesser amount of water soluble C (2.69%) was observed in the compost prepared with inoculation of consortium of fungi (Table 4). However, biogas slurry and cattle dung inoculated paddy straw showed 4.01 and 2.74% water soluble C, respectively in comparison to uninoculated paddy straw (5.92%). Said-Pullicino et al. (2007) reported that increase of value greater than unity in the ratio of hydrophobic to hydrophilic carbon, is an indicator of stabilized compost. Castaldi et al. (2005) also reported that water-solube organic C concentration rapidly increased to a maximum at day 18 and declined thereafter during 122 days of composting. Garcia et al. (1992) found that water soluble carbon content prepared from municipal waste reached from 0.41 to 1.19 of total organic C in 90 days. On the other hand, these values ranged from 2.69 to 5.92% in the present studies. Moreover, the evolution of CO₂-C in four weeks incubation varied from 174 to 333 mg 100 g^-1 compost (Table 4). Inoculation of cattle dung and consortium of three fungi showed less amount of CO₂-C evolution than compost prepared with biogas slurry or without any inoculum. It has been suggested that good quality compost should have carbon dioxide evolution less than 500 mg 100 g^-1 of total organic C (Garcia et al., 1992). The evolution of CO₂-C in 90 days old compost was less than above limit during the present studies, indicating that these composts were stabilized after the 90 days of decomposition (Bernal et al., 1998).

Stability of compost is very important before its application into field and it should not have phytotoxic effect (Hue and Liu, 1995; El Hammadi et al., 2007). The paddy straw compost prepared by use of different inocula showed 78 to 87% germination of seeds in wheat and 79 to 86% germination in mustard, as compared to 69 to 68% seed germination of wheat and mustard with the compost prepared without any inoculum (Table 5). Cunha-Queda et al. (2002) also tested the stability and maturity of compost by measuring the phytotoxicity towards germination of Lipidum sativum seeds. Evaluation of compost maturity prepared from poultry droppings, neem cake, castor cake, jatropha cake and grass clippings by inoculation of different fungal consortium showed that mixture of wheat straw, poultry dropping and jatropha cake had the lowest C:N ratio of 10:1 and showed germination index exceeding 80% in 60 days of decomposition (Gaind et al., 2009). Similarly, wheat straw-poultry dropping mixture inoculated with fungal consortium and grass clippings resulted in compost with C:N ratio of 13.5 and showed germination index of 59.66%. Al-Turki (2010) analyzed 14 commercially produced composts in Saudi Arabia and their germination index indicated that 64% of examined samples were below 80%, indicating that these composts could exhibit phytotoxicity.

In this study, a statistically significant positive correlation was observed between C:N ratio and humic substances, water soluble C and CO₂ evolution (Table 6). Similarly, Zmora-Nahum et al. (2005) reported a highly positive correlation between C:N ratio and dissolve organic carbon during estimation of compost maturity. Goyal et al. (2005) also observed that there was statistically significant correlation between C:N ratio and CO₂ evolution, water soluble C and humic substances. On the other hand, no significant correlation was observed among water soluble C, humic substances, CO₂ evolution and germination index of wheat and mustard.

CONCLUSION

The present study showed that good quality compost could be prepared from paddy straw within three months by using consortium of three fungi as inoculum. Maximum 17.4% decrease in organic C and in C:N ratio of compostable material from 73.7 to 16.6 was observed with consortium of fungi at 90 days of composting. Cellulase activity increased in fungal inoculated compostable material at 30 days of composting whereas highest amount of xylanase activity was observed by using consortium of fungi and cattle dung as inoculum. Total humic substances in finished product were 121 and 127 mg g^-1 compost with consortium of three fungi and with cattle dung, respectively. The resulting compost product is stable and mature and does not show any phytotoxic effect on seed germination of wheat and mustard. Thus, composting of rice straw resulted in reusability of the nutrients contained in the residue and this process will avoid air pollution caused by residue burning as well as loss of plant nutrients.