Mass Production of Manure Fertilizer

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ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTENEW DELHI-110012. . . प. - प110012Title of the study: Mass Production of Manure / Fertilizer from Agricultural Biomass
Final Draft ReportPeriod of Study: May 2021 to March 2022Submitted toNational Institution for Transforming India (NITI), Aayog,Governance and Research Vertical,Sansad Marg New Delhi-110 001 44
Project Research TeamProject Investigator-1. Dr. K Annapurna, Head, Division of Microbiology, ICAR-IARI, New DelhiCo-Investigator-1.Dr. Livleen Shukla, Principal Scientist, Division of Microbiology, ICAR-IARI, New Delhi2.Dr. Satish Lande, Senior Scientist, Division of Agricultural engineering, ICAR-IARI, New Delhi3.Dr. Lata, Principal Scientist, Division of Microbiology, ICAR-IARI, New Delhi4.Ms. Anju Arora, Scientist (SG), Division of Microbiology, ICAR-IARI, New DelhiObjectives / TORs•To develop a technology to convert crop bio waste (particularly paddy) into farm compost in less than six months period with economically efficient methods•To convert bio waste into wealth and offer economically viable alternative to prevent burning of crop residues, stubble etc.•Create possibility of giving an added value to the agricultural activity through the availability of an additional source of income for managing the treatment and selling resultant compost.•Availability of a new material to improve the soil fertility with the application of compost (in substitution of chemical fertilizers). 44
CONTENTSl. No.ParticularsPage No.Project Title and objectives1-2Research team2Content3List of Table4List of figures5Chapter 1Introduction6-8Chapter 2Review of literature9-29Chapter 3Data and Methodology 30-34Chapter 4Results and discussion 35-56Chapter 5Conclusions and Limitations 57-58Bibliography59-78 44
LIST OF TABLESTABLE NO.TITLE OF TABLESPAGE NO.1.Various enzymes involved in lignin degradation152.Standards of compost as described in Fertilizer Control Order (1985)333.Comparative cost of different paddy straw management systems374.Changes in various enzyme activities during composting of paddy straw415.Evaluation of Mesophilic and thermophillic bacterial and fungalPopulation416.List of Punjab and Haryana Farmers for ex-situ paddy straw decomposition in year 2021-2022397List of Punjab Farmers for in situ paddy straw decomposition508.Range of percent increase in Soil OC, available N, Soil dehydrogenase activity and Microbial Biomass C in field samples where PD was applied after 25 DAS.559.Changes in NPK content during in-situ decomposition at farmer’s field49 44
LIST OF FIGURESFIGURE NO.TILTLE OF FIGUREPageNo.1.Disposal of paddy straw by burning in field62.Lignocellulolytic material (Plant derived)103.Chemical structure of lignin174.Village Kattiyanwali, Shri Muktsar sahib345.Location map of Punjab districts where Pusa Decomposer wasapplied by the research team in farmers’ fields516.Application of Pusa Decomposer in farmers field527.Field demonstration of PUSA Decomposer and farmerworkshops in Punjab State538.Soil biological parameters over a period of time in PD treated and untreated fields549.CO2 emissions in PD treated and untreated plots55 44
1.1Introduction:Chapter 1 44
India generates 686.0 MT dry biomass annually from various crops of which, 234.5 MT is considered as surplus crop residue (Cardoen et al., 2015). Rice residues alone contribute 34% of the total crop residues in India. A major share of farmers from various parts of India, utilize rice residue for livestock feed, soil mulching and composting. They also use rice residue as a substrate for mushroom cultivation and energy production. However, disposal of crop residues is still a challenging mission since a large quantity of these are left unutilized in the fields. In India, paddy stubble burning is predominant in northern states like Punjab, Haryana and Uttar Pradesh and other states like West Bengal (Prasad et al., 2012; DAC, 2014; Kumar et al., 2016). Where the subsequent crop is grown in a short period. Straw burning (Fig. 1) contributes to the release of trace gases such as CO2, CH4, CO, N2O, SO2 and vast amounts of particulates, causing harmful effects on human health. India is expected to emit 144719 mg of total particulate matter yearly from the open field burning of rice straw (Kumar et. al., 2015). When it comes to soil-related loss, crop residue burning leads to the loss of beneficial microbes as well as nutrients in the soil like C, N, P, K (DAC, 2014; Prasad et al., 2020).
Fig. 1. Disposal of paddy straw by burning in fieldRice straw contains approximately 40% cellulose, 20% hemicellulose and 12% lignin (Juliano, 1985). Microorganisms quickly degrade cellulose and hemicellulose. However, lignin, a chemically complex aromatic biopolymer, makes a covalent bond with cellulose and makes itself resistant to degradation. Lignin is a complex polymer of three different phenylpropanoid alcohols, viz., coniferyl, coumaryl, and sinapyl alcohols. Other than these three components, feed stocks are also made up of small quantity of pectin, mineral residues and some nitrogenous compounds (Pollegioni, et.al. 2015). Degradation of lignin is slower and complicated due to its structural complexity and macromolecular features. Since a large part of the organic carbon in lignin and in other compounds is shielded by lignin, research on lignin biodegradation is important and essential. Over the past few decades, research on microbial degradation of lignin has gained momentum to 44
break the accessibility barrier and use the carbohydrates released. Fungal species capable of decomposing lignin can be divided into three major groups based on their morphology viz; brown rot fungi, white rot fungi and soft rot fungi. Further, there are some groups of fungi that are dung-dwelling (coprophilic) fungi, and litter- decomposing fungi also can degrade lignin effectively (Blanchette 1995; Liers et al. 2011). 44
South-east Asia is the principle niche of rice crop, and in this subcontinent rice and wheat occupy nearly 59.16 and 42.55 million ha, respectively, and annual grain output is around 181.35 and109.07 million tonnes, respectively (RWC-CIMMYT,2003). The common farming system in the Indo-Gangetic Plain (IGP) is the rice-wheat rotation system. With the introduction of combine harvesters, more than 75% of the rice area is harvested mechanically in north-western parts of the Indo-Gangetic Plains. Most of the farmers remove wheat straw for feeding the animals. However, management of the rice straw is a major challenge as it is considered to be a poor feed for the animals owing to high silica content. Although direct transformation of rice straw as mulch in fields is an alternative for its cost-effective utilization, soil application of large doses of undecomposed rice straw can lead to unfavourable effects on successive plant growth and crop yields due to production of certain phytotoxic allelochemicals (Chung et al., 2001; Inderjit et al., 2004; Lee et al., 1999).Under these circumstances it is imperative to develop technologies which are eco-friendly, economically viable and easy to use. The technologies must be demonstrated under farmer’s field conditions for them to adopt. A fundamental shift in farmer’s behaviour is possible at large scale, if actionable and affordable solutions are made available to them on time. Other than machinery, biological interventions are feasible.Development and application of effective microbial products for accelerated decomposition of paddy residue is one such solution. In the three Indo-Gangetic States, viz: Punjab, Haryana and UP, this practice of burning has become more prevalent in recent years, as for the next crop to be sown in time, the window for field preparation is very small and the farmer does not have any other option than burning. This leads to increase in air pollution due to GHG emissions, loss in soil nutrients leading to reduced fertility, deterioration of soil health and loss of microbial diversity. It has become a major concern and efforts by the Agriculture Ministry, GOI, ICAR and IARI are being made to come up with a multiple solutions/strategies to curtail burning and make the farmers aware of the hazards of the same. 44
Chapter 22. Review of literatureRice (Oryza sativa) occupies a pivotal place in Indian agriculture and is the staple food for more than 70% of population. Rice is grown in almost all the states covering an area of 44.6 m ha with annual production of 87 million tonnes. Straw is a major by-product of rice cultivation with an annual production of 120 million tonnes. With the emergence of intensive dairying, the focus shifted from rice straw as fodder, as it possesses lesser nutritive value in comparison to feed concentrates, contains excessive amounts of silica (11-15%) and has very less digestibility (Juliano, 1985). In the changing scenario rice straw started being considered as a waste product to be disposed by burning (Plate 1). The presence of lignin-cellulose complex in straw makes degradation process by microorganisms arduous. Hence, pre-treatment of lignocellulosic substrates is a prerequisite for the enhancement of their susceptibility to hydrolytic agents. Pretreatment involves partial delignification by protein enrichment of crop residue and requires alkali and urea treatment, but still exhibits only 55-60 per cent digestibility (Singh and Shiere, 1993). In contrast, composting of crop residue through the action of lignocellulolytic microorganisms is easier to manage.2.1Lignocellulose degrading microorganismsThe organic substrate, bulking agents and the amendments used in composting are mostly derived from plant material. Lignocellulose, the composite of the predominant polymers of vascular plant biomass, is composed of polysaccharides like cellulose and hemicellulose and the phenolic polymer lignin (Fig.2). Hence, the capacity of microorganisms to assimilate organic matter depends on their ability to produce the enzymes needed for degradation of the substrate components i.e., cellulose, hemicellulose and lignin. The more complex the substrate, the more extensive and comprehensive is the enzyme system required. Through the synergistic action of microorganisms, complex organic compounds are degraded to smaller molecules, which can then be utilized by microbial cells (Golueke, 1991, Shukla et al., 2014, 2016). 44
Fig 2. Lignocellulolytic material (Plant derived)Fungi: Hundreds of species of fungi are able to degrade lignocellulose. There are mainly three types of fungi living on dead wood that preferentially degrade one or more wood components viz. soft rot fungi, brown rot fungi and white rot fungi (Kirk, 1983). Soft rot fungi (Ascomycetes and fungi imperfecti) can efficiently decompose cellulose but are reported to degrade lignin slowly and incompletely. The brown rot fungi (Basidiomycetes) generally exhibit preference for the carbohydrate components of wood (Janshekar and Friecher, 1983; Kirk, 1983) with activity towards lignin largely confined to demethylation (Kirk, 1983). White rot fungi are capable of degrading both lignin and cellulose. The most extensively studied lignocellulolytic fungi are Trichoderma and Phanerochaete. Lignolytic and cellulolytic Lynch et al. (1981) observed that Cladosporium sp., Alternaria sp. and Fusarium sp. were more active decomposers than Phoma sp. Nigam and Parvu (1985) reported the cellulolytic activity of Basidiomycetes sp., Pleurotus ostreatus and Polyporus versicolor. 44
BacteriaCellulolytic bacteria are ubiquitous in nature. Under appropriate conditions bacteria produce cellulase and hence many bacterial strains are known to solubilize and modify the lignocellulosic structures extensively. But their ability to mineralize lignin is limited (Ball et al., 1989; Eriksson et al., 1990; Godden et al., 1992). Cellulomonas and Cytophaga are the aerobic mesophilic bacteria able to produce cellulose degrading enzymes (Thayer et al., 1984; Rajoka and Malik, 1986). More than one-half of the Bacillus sp. examined to date produces extracellular cellulases. Mesophilic, aerobic and anaerobic forms of Bacillus, B. subtilis B. polymyxa, B. licheniformis, B. pumilus, B. brevis, B. firmus, B. circulans, B. megaterium and B. cereus are known to be cellulose and hemicellulose degraders. Thermophilic cellulolytic B. stearothermophilus, B. brevis, B. sphaericus, B. subtilis and two other species of Bacillus were isolated by Strom (1985) from soil waste composter. Ray et al. (2007) isolated two bacteria viz. Bacillus subtilis CY5 and Bacillus circulans TP3 from fish gastrointestinal tracts which were able to produce cellulase optimally at 40 °C. Walker et al. (2006) isolated an alkaliphilic cellulase producing bacteria Nocardiopsis sp. from an indoor contaminated agar plate during a screening program which showed prominent clear hydrolysis of the CM cellulose even at pH 10.ActinomycetesActinomycetes isolated from soil and related substances show primary biodegradative activity, secreting a range of extracellular enzymes and exhibiting the capacity to metabolize recalcitrant molecules. In neutral and alkaline environment, Streptomyces viridosporus is likely to be dominant over fungi as a decomposer of lignin and cellulose (Pometto and Crawford, 1986). Thermophilic cellulase producing Thermoactinomyces, Streptomyces and Thermomonospora were found to be present in dry, warm land and also where salt concentrations are too high and soil pH is 44
Alkaline (Stutzenberger, 1972). From Indian desert soil of Jodhpur, Rao and Venkateswaralu (1983) isolated Streptomyces, Micromonospora and Thermoactinomyces. These organisms were found to depolymerize crystalline celluloses by two cellulase enzyme systems and glucosidase. Strom (1985) isolated thermophillic and highly cellulolytic Streptomyces, Thermoactinomyces sp. from solid waste composter. Jang and Chen (2003) isolated eighteen strains of actinomycetes from the compost of agricultural wastes (vegetable residues supplemented with corncob, straw and rice hull) and cultivated them at 50 oC for the thermostable cellulase production. Chellapandi and Himanshu (2008) isolated two cellulolytic Streptomyces sp. from garden soil and they were found to be good producer of endoglucanase under SmF.2.2Degradative enzymesCellulose degrading enzymes: In nature, cellulose components of biomass are hydrolysed to sugars by a complex enzyme system called 'Cellulase’ Cellulase is a hydrolytic enzyme complex containing chiefly endo- and exo-β- glucanases and cellobiase. Reese (1956) concluded that a true cellulolytic microbes possess two enzymes termed C1 and Cx. C1 was postulated to attack crystalline cellulose, either producing short chains or decrystallising glucan chains so that the cellulose was then susceptible to attack by hydrolytic Cx enzymes.A name of ‘Cellulase complex’ has been proposed and it refers to a system of three different enzymes whose combined action leads to the efficient degradation of cellulose, endo-β-1, 4 glucanase, exo-β-1, 4 glucanase and β-glucosidase (Ladisch et al., 1983). β-Glucosidases, appear to be exclusively cell-bound (Choi et al., 1978; Stoppok et al., 1982). The presence of cellulose in growth media usually gives the greatest yield of enzyme, however, in some species the enzymes are always present regardless of the carbon source (Prasertsan and Doelle (1987). All the three distinct enzymatic activities are required for the hydrolysis of cellulose to glucose. 44
Hemicellulose degrading enzymes: Hemicellulose component of a biomass can be degraded by different kinds of enzymes viz. (i) endoenzymes - which randomly cleave the bonds between the building blocks of a polymer (ii) exoenzymes - which cleave either a single dimer or monomer from the end of the polysaccharide chain and (iii) glycosidases- hydrolyse the oligomers or disaccharides of hemicellulose polymers. According to Dekker and Richards (1976) hemicellulase can be classified into L-arabinase, D-galactanase, D-mannanase and D- xylanase. Of the four types of hemicellulases, D-xylanase is seen in many types of microorganisms. These bring about the hydrolysis at β-D-(1-3) linkages of xylan. Xylanolytic enzymes include endo- 1-4-β-xylanases (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) which are produced by many microorganisms including fungi A. niger (Conrad, 1981; Gokhale et al., 1986), Aspergillus sp. and T. reesei (Dekker, 1983; Chahal, 1985). Of the thermophiles, Thermomyces lanuginosus RM-B performed best producing 154 U xylanase. The enzyme was reported to have optimum temperature 60-700C at neutral pH (Bakalova et al., 2002).Lignin degrading enzyme system: Enzymes involved in lignin degradation can be grouped into two categories. First group of enzyme called phenol oxidases. Mutants of Sporotrichum pulverulentum lacking phenol oxidase could not degrade lignin while its revertant degraded all wood components. Another enzyme ‘cellobiase oxidoreductase’ from Sporotrichum pulverulentum and Polyporus versicolor was involved in lignin degradation. The investigation on Pleurotus ostreatus wild type with a cellulase less mutant of S. pulverulentum suggested that the phenol oxidase activity was not necessary for lignin degradation (Liwicki et al., 1985). Laccase is another kind of phenol oxidase that has been widely associated with lignin degradation. Laccase causes free radical formation of cinnamyl alcohol and this non- enzymatic polymerization may induce cleavage of bonds between aromatic rings and propane side chain as well as the formation of the carboxyl group in the side chain, which enhance the rate of lignin degradation. Green (1977) observed associated enzyme laccase glucose:quinone oxidoreductase in lignin degradation. Ishihara (1980) observed demethylation activity of laccase in both lignin and lignin model compounds and concluded that depolymerization activity of laccase could be the first step in lignin degradation. 44
Peroxidase may contribute its hydroxyl ion (- OH) formation, which has been suspected in lignin biodegradation (Dordick et al., 1986). However, the exact role of peroxidase in lignin degradation is yet to be established. Meanwhile, hydrogen peroxide dependent oxygenase enzyme has been identified and proved to be involved in the initial depolymerisation of lignin degradation. Hydrogen peroxide dependent oxygenase enzyme from lignolytic cultures of wood degrading Phanerochaete chrysosporium formed radical oxygen by the generation of ethylene from 2-keto-3- thiomethyl butyric acid (KTBA). The lignin degradation is not catalyzed by any particular enzyme but concerted action of oxidative coupling of phenol oxidase, peroxidase, glucose oxidase and ligninase. Ligninase catalyze breakdown of the ether linkages with glucose oxidase providing necessary co-substrate peroxide, then peroxidase is involved in radical formation, phenoloxidases polymerize and depolymerize phenols often initial degradation depends on the level of glucose oxidases. Table 1 gives the lignin degrading enzyme system details. 44
Table 1: Various enzymes involved in lignin degradation (Janusz et al., 2017)EnzymeEC NumberCompoundsoxidizedCommonlyproducedOrganismsLignin peroxidaseEC 1.11.1.14Phenolic aromatic compounds and non- phenolic lignin model compoundsPhanerochaete, Chrysosporium, Trametes versicolor and Phlebia tremellosaManganese peroxidaseEC 1.11.1.13Monomeric phenols and lignin modelcompoundsP. chrysosporium, Panus tigrinus andAgaricus bisporusVersatile peroxidaseEC 1.11.1.16Methoxybenzenes andnon-phenolic modellignincompounds
Pleurotus eryngii, Pleurotus ostreatusand Bjerkandera adusta
Dye-decolorizing peroxidaseEC 1.11.1.19Lignin and dyesGeotrichumcandidum, Termitomyces albuminosus and Rhodococcus jostiLaccaseEC 1.10.3.2Phenolicmoieties in lignin, aromatic amine benzenothiolsandhydroxyindols
All white rot fungi, Streptomyces griseus and some bacteria
Glyoxal oxidaseEC 1.2.3.5Glyoxaland methylglyoxalcompoundsPhanerochaete chrysosporium
Aryl alcohol oxidaseEC 1.1.3.7Phenolic andnon-phenolicaryl- alcoholsAgaricalesspecies,Aspergillus, and FusariumHeme-thiolate haloperoxidasesEC 1.11.1.10Organic sulfides, olefins and aromaticringsCaldariomyces fumago 44 44
Lignin, a chemically complex aromatic biopolymer (Fig. 3), makes a covalent bond with cellulose and makes it resistant to degradation. Cellulose, hemicellulose and lignin are the major constituents of any lignocellulosic raw materials in which, strands of cellulose and hemicellulose are bound together by lignin.
Fig: 3 Chemical structure of lignin 44
2.3Lignin degrading microorganismsMicrobial delignification is one of the most efficient mechanisms for natural degradation of lignin. Complete degradation of lignin is a result of cooperative action of a wide variety of microorganisms such as bacteria and fungi (Janshekar and Fiechter, 1983). Degradation of lignin mediated by bacteria is slow and limited, while fungi are more efficient (Sigoillot et al., 2012).2.3.1BacteriaBacteria that are reported to degrade lignin mainly belong to three classes, α-Proteobacteria, γ – Proteobacteria and actinobacteria (Bugg et al., 2011). Many strains of bacteria belonging to the genera Ochrobactrum, Brucella, Sphingobium and Sphingomonas are reported to degrade lignin. Pseudomonas fluorescens is known to be most efficient bacterium for lignin peroxidase production (Tian et al., 2014).So far, numerous filamentous bacterial species capable of lignin degradation have been isolated and identified from Streptomyces group, and about ten enzymes that participate in lignin degradation have been characterized till now (Fernandes et al., 2014). Jing and Wang (2012) observed that the actinobacterial species, Streptomyces cinnamomeus produces both laccase and lignin peroxidase enzymes. Laccase activity and lignin peroxidase activity were also observed in another strain of Streptomyces viridosporus (Bugg et al. 2011; Tian et al. 2014). In another study, Větrovský et al. (2014) isolated Streptomyces spp. from forest soil and meadow. These strains were able to solubilize up to 4% of poplar lignin and 64% of catechol. Majumdar et al. (2014) studied the lignin degradation activity of laccases from Streptomyces lividans TK24, Streptomyces coelicolor A3 (2), Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using ethanosolv lignin and some lignin model compounds. All four laccases were capable of effectively degrading various lignin model compounds. 44
2.3.2FungiThere are a large number of fungal species capable of decomposing lignocellulotic substrates. In natural and human- affected forest environments, wood-degrading fungi live mainly as saprotrophs or weak parasites (Couturier et al. 2012). They can be divided into three major groups based on morphology viz; brown rot fungi, white rot fungi and soft rot fungi. Further, there are some groups of fungi that are dung-dwelling (coprophilic) fungi, and litter-decomposing fungi also can degrade lignin effectively (Blanchette 1995; Liers et al. 2011). Many fungi belonging to orders Agaricales and Polyporales viz: Ganoderma spp., Lentinula edodes, Phlebia radiata or Pleurotus spp. are known as white rot fungi. However, fungal species such as Gloeophyllum trabeuma and Coniophora puteana are categorized as brown rot fungi (Blanchette 1995). Suhara et al. (2012) screened 51 fungal strains isolated from bamboo culms for lignin degradation. After 12 weeks, Punctularia sp. TUFC20056, a white rot fungus and TUFC20057 (unidentified fungi) exhibited more than 50% lignin degradation and high lignin/holocellulose loss ratios (>6). Chang et al. (2012) collected fungal samples from woody surfaces and screened for lignolytic property on rice straw. One of the isolates (No 812 identified as Fusarium moniliforme exhibited maximum lignin degradation (34.7%). This was much more than the traditional wood degrading fungi Phanerochaete chrysosporium (28.3% lignin degradation). Zhang et al. (2012) investigated the lignin removal efficiency of Phanerochaete chrysosporium from rice straw. After ten days of solid state fermentation, the lignin removal efficiency was observed to increase sharply to 50.13%. In another study, Liang et al. (2010) observed that lignin degradation by Phanerochaete chrysosporium improved by 54% in the presence of 0.007% dirhamnolipid. At the same concentration, dirhamnolipid also increased lignin peroxidase activity of Phanerochaete chrysosporium by 86%. 44
Pildain et al. (2005) reported that several Eutypella species produce white rot decay in the late stages of wood decay and soft rot in the early stages. Mustafa et al. (2016) used Pleurotus ostreatus and Trichoderma reesei for pretreatment of rice straw to enhance methane production. Pretreatment with P. ostreatus at 75% moisture content removed 33.4% lignin after 20 days, and Trichoderma reesei removed 23.6% lignin at same conditions.2.5Enzymes involved in lignin degradationLignin degrading enzymes are broadly classified into two classes, lignin-oxidizing enzymes and lignin-degrading auxiliary enzymes. Commonly known enzymes such as laccases, lignin peroxidase, versatile peroxidases, manganese peroxidases and chloroperoxidases belong to lignin- oxidizing enzymes (Levasseur et al. 2008).2.5.1Lignin peroxidase (LiP; EC 1.11.1.14)Lignin peroxidase (LiP) oxidizes phenolic aromatic substrates and several non-phenolic lignin model compounds non- specifically in the presence of H2O2 and is secreted by microorganisms as isozymes, and its relative composition as well as isoelectric points are decided by the nutrient conditions (Santhanam et al. 2012). Sigoillot et al. (2012) stated that the three-dimensional structure of LiP is constituted with two Ca2+ binding sites, two glycosylation sites and four disulfide bridges. Isoelectric point of this enzyme varies from 3.1 and 4.7, makes lignin peroxidase capable of oxidizing a wide variety of substrates that are not oxidized by other classes of peroxidases. Kumari et al. (2002) demonstrated secretion of LiP from various fungi such as Aspergillus terreus, Penicillium citrinum and Fusarium oxysporum. They observed that induction of these enzymes was highest in liquid broth in which coir dust was added.In a recent study, Fan et al. (2019) used lignin peroxidase produced from Aspergillus oryzae for degradation of corn stover lignin. The apparent maximum rate of degradation reaction of lignin at an enzyme concentration of 3.75 U/100 mL was found to be 1.68 (mg/mL)/min. 44
2.5.2 Laccase (EC 1.10.3.2)Laccase, one of the oldest enzymes, is a multi-copper enzyme that is ubiquitous in white-rot fungi. Laccase, being an essential part of lignolytic system, can have extracellular, intracellular or periplasmic location, depending upon its physiological functions. However, in contrast to the other ligninolytic enzymes, it is mostly reported as extracellular proteins. Kumar et al. (2016) isolated laccase producing strain of Aspergillus flavus. They observed that the yield of laccase is highest with cellulose (8%), peptone (2%) and incubation at 35 °C. Ghosh and Ghosh (2017) optimized laccase production from another strain of Aspergillus flavus PUF5. They used agro-waste including ribbed gourd peel as a substrate and the fermentation was performed under submerged conditions. At yeast extract concentration of 0.3% and pH 4, laccase production was improved by 4.6-fold (15.96 U/ml). Aspergillus fumigatus strain VkJ2.4.5 was a potential source of laccase, when banana peel was used as substrate in solid state fermentation. While growing on banana peel, the fungal strain produced significant amount of laccase (6281.4 ±63.60 U l−1) and notable levels of manganese peroxidase (1339.0 ± 131.23 U l−1) (Vivekanand et al., 2011). Jin and Ning (2013) studied laccase production from a different strain of Aspergillus fumigatus AF1. The research revealed that optimized conditions for the highest laccase (142,198 ± 3586 U L−1) are NaOH at 0.39 mol L−1, pH 3.12 and temperature 25.43 °C. 44
2.5.3 Manganese peroxidase (EC 1.11.1.13)Manganese peroxidase is another vital lignin modifying enzyme secreted in multiple isoforms and detected in species such as Panus tigrinus, Lenzites betulinus, Nematoloma frowardii, Bacillus pumilus and Azospirillum brasilense (Janusz et al., 2017). The catalytic mechanisms of manganese peroxidase are similar to LiP, but utilize Mn (II) as their reducing substrate and generate Mn (III) (Martinez et al., 2005).2.6Degradation of rice residueRice straw decomposition depends on certain microbes that can attack and degrade its different components, including cellulose, holocellulose and lignin (Coronel et al., 1991). Kumar et al. (2008) applied a consortium of three fungi, namely, Cytalidium thermophilum (Th5), Aspergillus nidulans (Th4) and Humicola sp. (Th10) to degrade a mixture of paddy straw and soybean trash. The C: N ratio of the material was reduced to 9.5:1 after composting for three months. Pandey et al., (2009) studied the effect of a hyperlignolytic fungal consortium containing Aspergillus awamori F-18, Aspergillus nidulans ITCC 2011, Trichoderma viride ITCC 2211 and Phanerochaete chrysosporium NCIM 1073 on rice straw degradation. Native microbial community with externally applied consortium accelerated the degradation of paddy straw and reduced its C: N ratio to an acceptable level within one month. Moreover, supplementation of poultry manure improved the degradation of paddy straw.Kausar et al. (2010) also developed a lignocellulolytic fungal consortium targeting rapid composting of paddy straw. Two promising cultures identified as, Aspergillus niger (F44) and Trichoderma viride (F26) were tested for rice straw degradation in- vitro. The results revealed that cellulose, hemicellulose, lignin, and total carbon were significantly decomposed by the fungal consortium over control. In the three weeks of the decomposition processes, the C/N ratio was decreased to 19.5 from an initial value of 29.3, thus demonstrating the potential of this method for use in large-scale rice straw composting.Wei et al. (2019) augmented various lignocellulosic materials, including rice straw and wheat straw with thermophilic actinomycetes. The results showed that inoculation of actinomycetes not only altered the composition of the actinomycetes and bacterial community but also enhanced the 44
Degradation of cellulose, hemicellulose and lignin and intensified the activities of key enzymes, including xylanase, CMCase, lignin peroxidase, manganese peroxidase and laccase, primarily from rice straw and wheat straw throughout composting process. Finally, the study concluded that inoculation of actinomycetes enhanced lignocellulose degradation by 34.3 per cent and enzyme activity by 8.3 per cent.2.7Factors affecting degradation of lignocelluloseDegradation of lignocellulosic biomass is associated with several factors which influence the process. In order to incorporate into the soil or boost the decomposition rate, straw chopping is required as a pretreatment. It decreases the straw length, preventing long pieces of material from fouling on cultivation. It also simplifies the process of soil mixing and increases the successful biological breakdown of the straw (Muzamil et al., 2015). Silva et al. (2012) studied the effect of grinding on enzymatic degradation of wheat straw. Various grinding size (ranges between 50 μm to 800 μm) of wheat straw was investigated using Trichoderma reesei enzymatic cocktail. The results revealed that the degradability of wheat straw was increased up to 100 μm size reduction. Dai et al. (2019) also studied the effect of rice straw particle size on its degradation and methane production. Different particle sizes (20, 1, 0.15, and 0.075 mm) were studied, and methane yield was observed to increase as the size reduced (from 107 mL g−1 VS to 197 mL g−1 VS). The rate of degradation of cellulose was also increased from 27% to 93%. These results indicated that particle size reduction in rice straw could boost the methane yield in anaerobic digestion processes in conjunction with optimized microbial growth. The large particle size restricts the entry of fungi into the biomass and inhibits water, air and intermediate metabolites from spreading into the particles. Whereas, tiny particles reduce the size of the inter-specific channel, which would adversely affect the circulation of the inter-particle gas. Therefore, for successful biological pretreatment, ideal particle size has to be used (Sindhu et al., 2016).The composition of microbial communities and enzymatic activities vary with nutrient availability and environmental factors like temperature and moisture. It is known that, nitrogen influence rate of decomposition. Recently, a detailed litter bag study was conducted to estimate rice straw decomposition and microbial community dynamics under different levels of nitrogen. L- leucine aminopeptidase and N- acetyl-glucosamidase activities associated with rice straw were maximum 44
at 180 kg N ha-1 and 270 kg N ha-1 with higher activities at early stages of decomposition. The straw associated actinobacterial gene (GH48) and cellulolytic fungi gene (cbh1) abundance also varied with the stage of decomposition in such a manner that, during the middle stage of decomposition, higher abundance of these genes was recorded. Fungi and actinobacteria played a key role in the degradation of recalcitrant compounds at the later stage of decomposition (Guo et al., 2018).C/N ratio is another important factor for lignocellulose degradation. Several studies had been conducted to know the extent to which C/N ratio influence the microbial decomposition of rice stubble. Asgher et al. (2016) reported that production of various lignolytic enzymes such as manganese peroxidase, laccase and lignin peroxidase from a white-rot fungus Schizophyllum commune IBL-06 produced maximally, when the C:N ratio of rice straw was adjusted to 20:1. The resulting crude ligninolytic extract was used to delignify various agro-industrial residues. The enzyme extract induced lignin removal from the banana stalk, sugarcane bagasse, corn cobs and wheat straw by 61.7%, 72.3 %, 47.5 % and 67.2 %, respectively. Yan et al. (2015) studied the effect of C: N ratio on anaerobic digestion of rice straw. The study revealed that maximum biogas production happens when the C:N ratio was 29.6:1. Moreover, significant interactive effect of temperature, initial substrate concentration and C/N ratio was found on the biogas production from rice straw. Khudzari et al. (2016) checked the effect of different C/N ratios on power generation in compost microbial fuel cells. They used vegetable fruit mix and soil with different C/N ratios. The results indicated that lower C/N ratio (C/N ratio 24) had a higher power output with a maximum power density, signifying a more favourable microbial growth condition.For successful establishment of microbial growth in the biomass, the initial moisture content is important. The initial moisture content profoundly influences the development of fungal growth as well as enzyme production and directly improves the degradation of lignin (Sindhu et al., 2016). The initial moisture content of lignocellulosic biomass has an important impact on the rate of microbial degradation. Lignocellulosic substrate at moisture level 12% and 30% were degraded under solid-state fermentation conditions after steam explosion. The results indicated that higher moisture content enhanced lignin removal and sugar recovery from biomass (Cullis et al., 2004). Reid (1989) reported that several white-rot fungi degrade lignin optimally at moisture content ranges from 70% to 80%. Later, Fujian et al. (2001) stated that Manganese peroxidase and lignin peroxidase enzymes production is favored by lower solid liquid ratio. Shi et al. (2008) pretreated cotton stalks using Penicillium chrysogenum at different moisture levels. The results indicated that 44
Moisture content between 75-80% resulted in more lignin degradation than moisture at a low level (65%). However, optimum moisture content for degradation depends on the species of microorganism and type of biomass.Concentration and the type of inoculum influence rate of decomposition considerably. This is mainly because inoculum size can influence the time required by the organism to colonize the substrate. Generally, spores are used as inoculum and large size of inoculum will shorten the time required for colonization on substrates (Sindhu et al., 2016). Recently, Li et al. (2018a) carried out solid-state anaerobic digestion of tomato residues along with corn stover and dairy manure. They reported that anaerobic digestion was quickly initiated at a substrate inoculum ratio of 6. Maximum production of methane was observed when a substrate inoculum ratio of 2 was applied. Li et al. (2018b) studied the effect of moisture and inoculum size on delignification and subsequent saccharification of switch grass after solid state fermentation with Pleurotus ostreatus. After 80 days, highest degradation of lignin (52%) and maximum ethanol yield (31%) were recorded in treatment containing 75% moisture content and 5 mL inoculum.Optimum incubation temperature is important during biological pretreatment of lignocellulosic residues. Though the optimum temperature is different for each fungus, majority of ascomycetes white-rot fungi grow optimally around 390C, and white-rot fungi belonging to basidiomycetes grow optimally between 25 and 300C. Fungi generate a considerable amount of heat during metabolism and develop temperature gradients in solid-state media. Fungal physiology, fungal strain, and type of substrate are responsible for the differences in optimal temperature for biological degradation of biomass. 2.8In situ degradation of rice stubbleThere are scanty publications related to in situ degradation of rice stubble using bio-inoculants. Earlier, Darmwal and Gaur (1988) studied the effect of cellulolytic fungi Aspergillus awamori andA. niger along with nitrogen fixer Azospirillum lipoferum in wheat soil supplemented with rice straw. The soil inoculated with microorganism’s recorded highest yield and fixed nitrogen. Among the inoculants, Aspergillus awamori recorded promising results followed by A. niger and. lipoferum. Gaind and Nain (2007), incorporated paddy straw in field and applied Aspergillus awamori F18 (phosphate dissolving and cellulolytic) and Trichoderma reesei MTCC164. The study finalized that, in situ incorporation of paddy straw combined with T. reesei and N60P60 was effective for proper disposal of paddy straw as well as to improve soil health. 44
Recently, Borah et al. (2018), compared degradation in two different varieties of rice namely, Mahsuri and Ranjit. The stubbles of the two varieties after harvest were sprayed with laboratory culture of cellulose degrading microorganism (CDM) or commercial yoghurt or mixture of CDM and yoghurt with glyphosate. The variety Mahsuri showed significant decreases in dry biomass (61.1%) and per cent organic carbon (45.3%) than Ranjit (47.1% and 46.4%) with stubbles, following treatment with CDM culture or yoghurt with glyphosate solution.Patel et al. (2016) conducted a field experiment to study effects of in situ decompositions of paddy straw inoculated with composting culture in an onion field. They observed an overall significant change in properties such as pH and EC as well as mineral composition of soil after harvest on onion crop. In another study, Bhattacharjee et al. (2013) evaluated the changes in the nitrogen profile during in situ management of rice stubble. Various treatments such as lignocellulolytic fungal inoculated stubble, phosphocompost blended uninoculated stubble and uninoculated stubble were evaluated. They observed that stubble blended with phosphocompost along with nitrogen fertilizer recorded highest total, nitrate and ammonical nitrogen as well as biomass carbon (MBC). The urease activity in wheat rhizosphere soil was also recorded the most elevated in phosphocompost blended rice stubble. Meena et al. (2016) conducted an open pit field 44
experiment using ICAR-IARI compost inoculants contain four hyper lignocellulolytic fungi namely, Trichoderma viride, Aspergillus nidulans, Phanerochaete chrysosporium and Aspergillus awamori for degradation of rice straw. As compared to control, a treatment containing compost inoculants along with molasses 5% spray recorded the lowest value of C/N ratio. This may be due to rapid multiplication of applied compost inoculants in the presence of 5% molasses. 3.0 Alternate approaches of managing agri-residueThe Govt. of India has attempted to manage this problem, through numerous measures and campaigns designed to promote sustainable management methods such as converting crop residue into energy. Government has already taken steps to encourage production of cellulosic ethanol from agricultural wastes and residues that would otherwise be burnt. The mandate for National Biofuel Policy (NBP) is to produce 10 million litres of E10 biofuel which would further save Rs 28 crore in forex and around 20,000 tonnes of carbon dioxide emissions. However, biomass removal put additional burden to replenish soil with nutrients. Conversion of agro-waste to organic fertilizer is extremely important into the framework of circular economy compared to other approaches as it addresses ground level issues on agriculture, environment and socio economy of Indian farmers.Composting, biochar production, conservation agriculture, etc. are a few effective sustainable techniques that can help to curtail the issue while recycle and retain the nutrients present in the crop residue and improving soil health.Composting technologies: Ex-situ managementPhospho-Sulpho-Nitro Compost TechnologyIndian soils are widely deficient in phosphorus a major soil nutrient responsible for crop productivity. On the other hand, rock phosphate (containing 11-32 % P2O5) deposits are present in different parts of Indiaviz., Udaipur (Rajasthan), Jhabua (Madhya Pradesh), Visakhapattanam (Andhra Pradesh), Purulia (West Bengal), Mussori (Uttaranchal) etc. Low-grade rock phosphate can be used for preparation of nutrient enriched “Phospho-Sulpho-Nitro Compost” and applied as a source of organic matter and phosphorus for crop production. This technology includes the use of rock phosphate, pyrites, and P solubilizing organisms including P solubilizing fungi Aspergillus awamori) and P- solubilizing bacteria (Bacillus polymyxa, Pseudomonas striata) as bioinoculum for solubilizing P from rock phosphate. P-S-N compost can be prepared by pit and heap methods. To avoid the leaching of nutrients, the floor should be cemented for heap method. In pit (10 Ft length x 5 ft width x 3 ft deep) and in heap (7.5 Ft length x 6 ft width x 3 ft height) methods about 500 kg of wastes can be used for decomposition. 44
VermicompostingVermicomposting is a mesophilic process of composting using epigeic earthworms and differs from ordinary or conventional composting in several ways. It contains cocoons, excreta and undigested feed of earthworms which is highly rich in antibiotics, vitamins and enzymes like cellulase, protease, amylase, chitinase and lipase. These enzymes continue disintegration of organic matter after excretion from the worms as casts. Though nutrient value of vermicompost and vermicast is always lower than any standard chemical fertilizer but the nutrient value in this compost is better than conventional compost. Advances in vermiculture technology have recently led to novel products like vermiwash. This product has now not only caught the attention of commercial vermiculturists but also the farmers. Farmers in their own way have started collecting vermiwash for foliar application.Vermicompost Preparation by Pit and Heap MethodsOpen permanent pits of 10 feet length 3 feet width 2 feet deep are constructed under the tree shade, which is about 2 feet above ground to avoid entry of rainwater into the pits.Brick walls are constructed above the pit floor and perforated into 10 cm diameter 5-6 holes in the pit wall for aeration. The holes in the wall are blocked with nylon screen (100 mesh) so that earthworms may not escape from the pits.Partially decomposed dung (dung about 2 months old) is spread on the bottom of the pits to a thickness of about 3-4 cm. This was followed by addition of layer of litter/residue and dung in the ratio of 1:1 (w/w).A second layer of dung is then applied followed by another layer of litter/crop residue in the same ratio up to a height of 2 feet.Three species of epigeic earthworm’s viz., Eisenia foetida, Eudrillus eugineae and Perionyx excavatus are inoculated in the pit.Moisture content is maintained at 60-70% throughout the decomposition period.Jute bag (gunny bags) are spread uniformly on the surface of the materials to facilitate maintenance of suitable moisture regime and temperature conditions.Watering by sprinkler is often done.The materials are allowed to decompose for 15-20 days to stabilize the temperature because to reach the mesophilic stage, the process has to pass the thermophilic stage, which comes in about 3 weeks. Earthworms are inoculated in the pit or heap with 10 adult earthworms per kg of waste material and a total of 500 worms are added to each pit or heap. The materials are allowed to decompose for 110 44
days. The forest litter was decomposed little earlier (75 to 85 days) than farm residue (105 days). About 450-500 kg of vermicast can be harvested from one ton of residue mixture within 3 months.Table 2. Standards of compost as described in Fertilizer Control Order (1985)ParameterCompostMoisture percent by weight15.0-25.0ColourDark brown to blackOdourAbsence of foul odourParticle sizeMinimum 90% material should pass through 4.0mm IS sieveBulk density (g/cm3)<1.0Total organic carbon, percent by weight, minimum16.0Total nitrogen (as N), percent by weight, minimum0.5Total phosphates (as P2O5), percent by weight, minimum0.5Total potash (as K2O), percent by weight, minimum1.0C:N ratio20:1 or lesspH6.5-7.5Conductivity (as dsm-1), not more than4.0PathogensNilArsenic (as As2O3)10.0Cadmium (as Cd)5.0Chromium (as Cr)50.0Copper (as Cu)300.0Mercury (as Hg)0.15Nickel (as Ni)50.0Lead (as Pb)100.0Zinc (as Zn)1000.0 44
Chapter 3Methodology3.1Development of microbial consortiaPusa Decomposer: A consortium of seven hypercellulolytic fungal cultures, namely A. awamori ITCC 8945; A. clavatus ITCC 8306; T. harzanium ITCC 8946; A. niger ITCC 7790; T. asperellum ITCC 7793; P. oxalicum ITCC 6587 and Ch. globossum ITCC 3680 was optimized for compost production on the basis of their lignocellulolytic enzyme production potential. The consortium has been effectively used for composting of diverse agricultural wastes such as paddy straw, fruit waste, vegetable waste and garden waste.3.2Qualitative screening of cellulolytic isolates:The cellulolytic ability of the fungal isolates was assessed by Congo Red test on the basis of zone of clearing on Carboxy methyl cellulose (CMC) agar plates.Congo Red Test (Teather and Wood, 1982): The CMC agar plates were prepared by adding 1%carboxymethyl cellulose (low viscosity) and 2% agar to the following basal medium (NH4)2 SO4:0.5 g; KH2 PO4 : 1.5 g 24 ; K2 HPO4 : 5.0 g ; MgSO4 .7H2 O : 0.1 g; Yeast extract : 0.1 g; NaCl : 0.2g ). The final volume of medium was made to 1liter with distilled water. Medium was autoclaved at 15 psi for 15 minutes. The organisms were point inoculated on the CMC agar plates andincubated at 300C. After five days, the plates were flooded with Congo Red solution (1mg/ml indistilled water). The dye was decanted after 20 minutes and the plates were flooded with 5M NaCl. After 20-30 minutes, the NaCl was decanted and CMCase producing colonies were seen to be surrounded by a pale orange to clear zone against an otherwise red background.3.2 Quantitative screening of cultures : For this purpose, the cultures were grown in Reese’s mineral medium with chopped paddy straw as sole carbon source.Preparation of fungal inoculum: A bit of fungal mycelium was aseptically transferred to a petri dish having 30 ml of potato dextrose agar medium. After 48 hours of incubation at 30°C, one agar 44
plug (6 mm) from the growing edge of the colony was scooped out using a sterilized cork borer and used as an inoculum.Production of crude enzyme: Reese's (1956) mineral medium (100 ml) supplemented with rice straw (1%) as carbon source was sterilized in 250 ml Erlenmeyer flask at 15 psi for 30 minutes and used for submerged fermentation (SmF). For fungi, each flask was inoculated with one agar plug of diameter 6.0 mm taken from the edge of 48 hours old fungal colonies and incubated at 30°C on a rotary shaker. Two sets of flasks were used for each fungus and one set was withdrawn after 7 days and other set after 15 days and filtered through Whatman filter paper No. 1 to collect the filtrate for estimating enzymatic activity. The filtrate was stored at 4°C until use for estimating the activity of extracellular lignocellulolytic enzymes.Determination of Carboxymethyl Cellulase (CMCase) activity: The carboxymethyl cellulase (Endo- β -1-4 glucanase) activity of cell free culture filtrates was estimated by the method described by Ghose et al. (1983). An aliquot of 0.5 ml of enzyme filtrate was incubated with 2% caboxymethylcellulose (0.5 ml) at 50°C for 30 minutes. The reducing sugar was estimated as described for the FPase activity. An enzyme blank was prepared similarly without the substrate. The enzymatic activity of filtrate was expressed as unit per ml (U/ml) which is defined as the amount of enzyme, which liberates one μg of reducing sugars per minute under assay conditions.Determination of cellobiase activity: The Cellobiase (β-glucosidase) activity of cell free culture filtrates was estimated by the method described by Wood and Bhat (1988). To 0.5 ml of culturefiltrate in test tubes, 0.5 ml of substrate solution was added. Enzyme blanks were also preparedosimilarly without the substrate. All the tubes were incubated at 50 C for 30 minutes. Afterincubation, 1.5 ml of Glycine buffer was added to each tube and the absorbance was taken at 430 nm. The amount of p-nitrophenol produced was determined spectrophotometrically at 430 nm from a standard curve of p-nitrophenol (20-200 μg). One unit of cellobiase enzyme is defined as the amount of enzyme required to release one μg of p-nitrophenol per ml per minute.Determination of xylanase activity: The xylanase activity of cell free culture filtrates was estimated by the method described by Ghose and Bisaria (1987).3.5Estimation of total Nitrogen (AOAC) in soil:Nitrogen content in the sample was determined by micro-kjeldahl method. Sample (100mg) was weighed and taken in a digestion flask. Concentrated sulfuric acid (5ml) and catalyst mixture 44
(100mg) were added to the flask and digestion process were performed until the solution became colourless. The content of flask was transferred from digestion flask and made up the volume to 25ml. Distillation was performed in distillation unit with 5 ml digested sample and 10 ml of 40% NaOH solution. Further, ammonia released were trapped in 25 ml of 1% boric acid solution containing mixed indicator. Once the ammonia was trapped, this was back titrated with 0.01N hydrochloric acid solution. A blank was maintained only with distilled waterN% = ml of HCl in sample solution- ml of HCl in blank sample /weight of sample (100mg) X Normality of HCl X 143.6Estimation of soil dehydrogenase activity (Casida et al. 1964)Air dried soil (20g) was mixed with 0.2g CaCO3. Distributed 6g of this mixed soil in screw cap tube, added 1 ml of 3% TTC aqueous solution and 2.5ml distilled water. The contents were mixed thoroughly with glass rods. Tubes were covered properly and incubated at 370C for 24 hours. The cover was removed after 24 hours and added 10 ml methanol. The mixture was filtered after vortexing the mixture for 1 minute. Again added 10ml methanol and repeated the same step. Absorbance was recorded at 485nm. Concentration of TPF released3.7Determination of soil alkaline phosphatase activity was done following the method of Tabatabai and Bremner (1969). The concentration of PNP released was calculated by referring standard curve prepared with different concentrations of p-nitrophenol. Alkaline phosphatase activity was expressed as µg PNP released g-1 hr-1.3.8Enumeration of total fungi and Bacteria in the fieldThe soil samples were serially diluted and plated on Nutrient agar and Potato dextrose agar for enumeration of total bacterial and total fungal count respectively.Compatibility test: Compatibility test was performed by the modified method of Tehrani et al. (2001). Selected fungi showing the maximum enzymatic activity were tested for their compatibilit to grow together, by point inoculation on single PDA plate. The plates were incubated at 30oC for 4 days in a BOD incubator.Pusa Decomposer (PD); A microbial consortium has been developed for rapid decomposition of paddy straw, both for in-situ and ex-situ decomposition. All the selected fungi were produced in mass using modified jaggery medium and packed in the form of capsules. Liquid consortium was provided for demonstrations at farmer’s field. 44
Four capsules of this product can be scaled up to 25L liquid formulation which can be applied in- situ to 1.0 ha of combine fitted with SMS harvested rice field having 5-6tonnes of paddy straw. It accelerates process of paddy straw decomposition and field ready for potato, peas and wheat sowing in 20-25 days following conventional tilling (CT) practices. CT is one of the major activities in the Govt. funded CRM scheme. Its use enriches the soil with organic carbon (OC) and nutrients while improving the soil biological properties. Pusa Decomposer is a long term sustainable solution for management of paddy straw in conjunction with CT, Happy Seeder and Super Seeder options. We worked out a tentative comparative cost of PDSM systems as below.Table 3. Comparative cost of different paddy straw management systemsS.No.OperationsCost (Rs./acre) of different optionsHappy SeederSuper SeederConventional Tillage (CT)CT with PD1.Happy Seeding1300---2.Super seeding-2000--3.Chopper--120012004.Rotavator (mixing residue)--100010005.Irrigation4004004004006.Rotavator (before sowing)--100010007.Seed cum Ferti-drill--6006008.Cost of PD and application---300Total17002400420045003.9Selection of study area:In situ locations: Five districts of Punjab viz: Gurdaspur, Mukerian, Amritsar, Srimuktsar sahib and Fazalika and one of Haryana which was Village Anwal, Rohtak.Ex –situ locations:Twelve districts viz: VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab; VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab; VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab; Village-Machhiwala, Near Ramdas, Teh. Ajnala; Village-Machhiwala, Near Ramdas, Teh. Ajnala; Vill. Tuto Mazara, Mahilpur, Distt. Hoshiarpur; Vill. Hayatpur Teh. Mukerian Distt. Hoshiarpur; Vill. Kingra, Distt. Malout Balim, Gurdaspur; Vill. Anwal, Distt. Rohtak, Haryana; Bhavdin Kheda Sadh. Trial was also carried out at Sonipat, village Badwasini and Sirsa, Sardulgarh village. 44Fig 4. Village Kattiyanwali, ShriMuktsar
Soil samples were collected from each study field from 5 points 100g each, packed in polythene bags and brought to the laboratory where composite was made. The soil was homogenized, sieved and then analyzed for different parameters.Field application methodology: A standard protocol was followed for the in situ straw degradation using Pusa Decomposer. Pusa Decomposer in 10 L was mixed in 200L of water and was applied/sprayed on one acre field having approximately 2-2.5 ton straw. The SOPs followed was to first spray by tractor driven, followed by incorporation using Rotavator and the irrigating the filed to ensure moisture. This procedure allowed the degradation to take place in the shortest possible time and enabled the farmer to go for his wheat sowing. Below are the farmers field photos (Fig 4).

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Chapter 4Result and DiscussionOur major focus was on agri-residue management using Pusa Decomposer under in situ farmer field conditions. However, as an in-house research program studies on ex-situ management using compost technologies using pit and windrow method and developing machinery for agri-residue management were carried out.All the staff was recruited after advertisement in newspaper and IARI web site on time, most of the chemicals, glassware were procured on time and for travel to villages the vehicle was hired from IARI rate contract travel agency. 4.1Laboratory studiesPaddy straw collection was done from the IARI experimental fields. Paddy straw of eight different varieties namely Pusa Basmati 1, Pusa Basmati 1121, Pusa Basmati 1509, Pusa Basmati 1637, Basmati 1407 and Tarawari Basmati 1 was collected after harvesting. Unchopped paddy straw of each variety was inoculated with liquid inoculum of Pusa decomposer @ 5 litres per tonne. The material was mixed properly along with cowdung and then kept in polythene bags and kept for decomposition. After 15 days the material in the bags was mixed properly and kept further for decomposition. The straw of all the varieties degraded within 25-30 days of inoculation and the decomposed material was collected in trays. This was a preliminary laboratory study to ascertain the degradation potential of Pusa Decomposer, a microbial consortium developed at Division of Microbiology, IARI, on different varieties of paddy. Phytotoxicity tests have shown the compost to be of good quality.4.2Rapid decomposition of paddy straw using pit methodThe crop residues have been traditionally used for preparing compost. For this, crop residues are used as animal bedding and are then heaped in dung pits. In the animal shed each kilogram of straw absorbs about 2-3 kg of urine, which enriches it with N. The residues of rice crop from one hectare land, on composting, give about 3 tons of manure as rich in nutrients as farmyard manure (FYM). Indian Agricultural Research Institute (IARI), New Delhi, has successfully 44
developed a biomass-compost unit for making of good quality compost. This mechanized unit efficiently uses waste biomass and crop residues generated in the IARI farm. The decomposition process, which is hastened by a consortium of microorganisms, takes 75-90 days. During the year 2019-20, the unit prepared about 5000 tons of compost.For pit method: 100 kilograms of compostable material for each treatment T1 and T2 was thoroughly mixed and moisture was adjusted to 80% water holding capacity (WHC). The material was put in cemented pits at Division of Microbiology, IARI and allowed to decompose. The contents were turned after 15,30,60 and 75 days and samples were drawn at each interval and analysed for mesophilic and thermophillic bacterial and fungal population, multifunctional bacterial population mainly P solubilisers, N fixers, lipolytic, amylolytic, Gram positive, and Gram negative bacterial population. Different enzyme activities cellulases, xylanases and proteases were also estimated. The cellulases activity increased during decomposition and was found maximum at 30 days and declined at 60 days (Table 4) for uninoculated treatment whereas a decline in cellulose activity was observed after 60 days in inoculated treatment and it was found higher than control due to the inoculation of fungal consortium. Similarly, the initial xylanase and protease activity increased in inoculated treatment as compared to uninoculated treatment.Population of mesophilic bacteria and fungi was found highest at 30 days in inoculated treatment (Table 5) while bacterial population declined after 30 days but fungal population showed an increase till 60 days and declined after 60 days whereas thermophillic bacteria and fungi were found maximum at 15 days and bacterial population declined at 30 days but fungal population increased at 60 days and declined at 75 days. Among bacterial population, gram positive bacteria predominated the population as compared to Gram negative population. 44
Table 4: Changes in various enzyme activities during composting of paddy strawTreatmentCellulase activity(mg reducing sugar kg -1 dry matter h-1)15306075Xylanase activity (mg reducing sugar kg -1 dry matter h-1)15306075Protease activity (mg tyrosine kg -1 dry matter h-1)15306075T1 Paddy straw + Cow dung2681513658655330309530954797T2 Paddy straw + Cow dung +Pusa decomposer4119727763711081278355212401520907Initial cellulase activity : 8 mg reducing sugar kg -1 dry matter h-1 ; Initial xyalanase activity: 22 mg reducing sugar kg -1 dry matter h-1 ; Initial Protease activity: 159 mg tyrosine kg -1 dry matter h-1Table 5: Evaluation of Mesophilic and thermophillic bacterial and fungal populationTreatmentMesophilic bacteriaX 109 g-103060Thermophilic bacteriaX 105 g-103060Mesophilic fungi X 108g-103060Thermophilic fungiX 105 g-103060Paddy straw + Cow dung3016475010521417046Paddy straw + Cow dung + Pusa decomposer30207114024205253202138
The range of initial pH in pits was from 4.5 to 5.5 and it started dropping till 7 days and reached less than4.0 within 7 days and after 7 days again it started increasing and a maximum of 7.9 was observed after 30 days. Electric conductivity: The EC range was 3 - 6 U siemens / cm was observed in finished product while it fluctuated when thermophilic phase was observed. The initial moisture content was recorded 60.8% and after 15 days it increased to 68.9 % and later in final product it decreased to 45 .2% in all the treatments. The result of germination test with Lepidum sativum seeds showed that all the samples taken at the maturation period had GI values of greater than 50%, which indicates a phytotoxin-free compost product. All the germination rates treated with 1% extraction of composting products were above 95%, indicating the compounds present in the raw wastes or produced during the first days of composting as intermediate products of microbial metabolism, were degraded within 30 days of composting. 44
Development of Compost Turner cum Mixer: A powerful technology to convert the biomass into nutrient rich compost by windrow / pit method using microbial culture and mechanical intervention. Suitable for thorough turning and mixing of cow dung, farm residues and biomass for compost preparation.
In-situ demonstrationsUnder the Niti Aayog banner a slogan was prepared “Jalao Nahi Galao” and as the harvesting season of paddy started many trials at IARI and farmers field were organized in various villages of Punjab. The trial was initiated at IARI field where after harvesting of paddy, Pusa Decomposer was sprayed @ 10litre/acre and whole straw was turned in the soil using Rotavator and the field was irrigated. Soil samples were collected periodically from initial till 25 days to estimate the various soil health parameters and study the degradation of straw. Simultaneously tours were also conducted to various villages of Punjab to demonstrate the application of Pusa Decomposer at farmer’s field with the aim to make them aware about ill- effects of burning and how they can benefit by decomposing the straw into nutrient rich manure by use of Pusa Decomposer. The villages covered were from district Gurdaspur, Mukerian, Amritsar, Moga, Srimuktsar sahib and Fazalika (Fig. 5). All progressive farmers (Table 4) were provided with Pusa Decomposer capsules as well as liquid solution for spraying in field (Fig 6). Soil samples were collected. The role of Niti Aayog was also highlighted in all the farmers meet. We organized three workshops with Punjab farmers in the fields by close interactions (Fig.7).Ex-situ DemonstrationsWe conducted Ex-situ management of paddy straw in different villages of Haryana and Punjab at twelve sites at farmer’s field. At each site a good number of farmers were present having interest in composting and they were explained the benefits of Pusa Decomposer for ex-situ management of paddy straw along with cow dung. All the three methods of composting namely pit, heap, and windrow were explained in detail and farmers were 44
demonstrated pit as well as heap method. The farmers were highly satisfied with the technology as a low cost economically viable as the only output needed was Pusa Decomposer and rest of the things like Paddy straw and cow dung were already available with them.Windrow methodIn village Anwal, District Rohtak, Haryana, windrow method of composting was demonstrated by the team on Shri. R. N. Arichwal farm where he used machines for making windrows and sprayed Pusa Decomposer and applied the ready compost for vegetables. In the village Kingra a good gathering of farmers was observed and both methods: heap and pit were demonstrated. Farmers themselves suggested to use tractor operated machines to mix the straw and cow dung along with decomposer and prepared a small pile and used another small machine to prepare the heap to convert the rice straw into compost. Therefore, all methods of composting was demonstrated and farmers were advised to give turning after every fifteen days. The evaluation of complete decomposition was also provided to farmers in detail. The farmers reciprocated the team efforts and wanted continuous monitoring by the team. Table 6: List of Punjab and Haryana Farmers for ex-situ paddy straw decomposition in year 2021-2022S.NoNameMobile numberAddress1.Gurmeet Singh9872397000VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab2.Ranjeet Singh8847454682VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab3.Manpreet Singh7307200077VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab4.Jasmittarjit Singh9855461914Village-Machhiwala, Near Ramdas, Teh. Ajnala5.Sukhwant SinghVillage-Machhiwala, Near Ramdas, Teh. Ajnala6.Sukhwinder Singh S/o Balwant Singh9888972473Vill. Tuto Mazara, Mahilpur, Distt. Hoshiarpur7Onkar Singh S/O Harbinder Singh9914503010Vill. Hayatpur Teh. Mukerian Distt. Hoshiarpur8Lakhwinder Singh6284531232Vill. Kingra, Distt. Malout9Cap. Kashmir Singh 8054013648Balim, Gurdaspur10 R. N. Arichwal9711190973Vill. Anwal, Distt. Rohtak, Haryana 44
11Baljeet9813080997Bhavdin12Rajesh9991648719Kheda Sadh

Village: Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab
Village: Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab 44
VPO- Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab
Village: Hayatpur Teh. Mukerian Distt. Hoshiarpur
Village-Machhiwala, Near Ramdas, Teh. Ajnala 44
Village: Tuto Mazara, Mahilpur, Distt. Hoshiarpur
Village- Anwal, Distt. Rohtak, Haryana
Field DayA field day was organized on 12th March, 2022 at village Anwal, Distt. Rohtak, Haryana. A progressive farmer Sh. R. N. Arichwal is using Pusa Decomposers technology for both in-situ and ex-situ management of paddy straw. A few farmers from Haryana, Punjab also accompanied IARI team consisting of Nodal Officer Dr. I M Mishra, Head, Microbiology Division Dr. Sunil Pabbi, Principal Scientist Dr. Livleen Shukla and Senior Scientist Dr. Satish Lande and Supporting staff Sh. Goverdhan Thakur and SRF & YP II. During the interactive discussions farmers were apprised about the advantage of applying Pusa 44
Decomposer for faster degradation of agri residue, explained the in situ and ex-situ methods as well as SOPs. All were aware of ill-effects of paddy straw burning and appreciated the work done by Sh. Ram Niwas. Compost Sh. Ram Nivas ji prepared compost from paddy straw using heap and windrow method applying Pusa Decomposer and suggested other farmers to do the composting using Pusa Decomposer. Quality compost shall be a revenue generating means to the farmer.

Village- Anwal, Distt. Rohtak, Haryana Success Story of Farmers:1.Gurmeet Singh: Sh. Gurmeet Singh is the resident of village Kattianwali, Teh. Malout, Distt-Sri Muktsar Sahib Punjab. He has total 16 acre land out of which 4 acre land was totally barren, only wild sarkanda was growing, then after interaction with pusa scientists, he start using bio fertilizers, decomposers such as Azotobacter, VAM and phosphate solubilizing bacteria and Pusa Decomposers since 2016. He used in-situ method of decomposing of paddy straw and produce 2.5 quintal per acre which was not possible before. He also suggested other farmers of village to follow this practice and helped them getting benefit of this technology. More than 200 farmers adopted in-situ Pusa Decomposer technology and incorporated residue in more than 1000acres in village kattianwali out of total 2285 44
acres (43.8%).
44 44

44


2.R. N. Arichwal: Sh. Arichwal is the resident of village Anwal, Distt-Rohtak, Haryana. He told to IARI team that he was not getting proper crop even after giving proper manure, IARI team suggest him to use Pusa Decomposer and told him every method of making compost like, pit, heap and windrow, since then Sh. R. N. Arichwal is using Pusa Decomposers technology for both in-situ and ex-situ management of paddy straw. He used windrow and heap method for producing compost using Pusa Decomposer. His land had high pH, high salinity and low N, P, K content but after using Pusa Decomposer and other product like VAM, phosphate solubilizing bacteria, potash solubilizing bacteria, Azotobacter his field quality was improved. In the end he told that, I am grateful to using Pusa Decomposers and other products, which decrease the pH, salinity and increase the porosity of soil, however 44
improved the overall yield of field. He also produced 10 acre seedling of sugarcane using compost prepared from Pusa Decomposer.


3.Rajesh Saini: Sh. Rajesh Saini is the resident of Village: Meerpur, Block-Mukerian, Distt. Hoshiarpur, Punjab. He said management of paddy residue is of utmost important and also discussed other problems related to low yield in his 6 acres of land. He was advised to use of Pusa Decomposer, explained and demonstrated the protocols of composting through pit, heap and windrow methods. Understanding the benefits he started spraying the Pusa Decomposer on his paddy residue. He observed fast degradation in the field with in 25 to 30 days. He is now regularly using the decomposer since 2019. He reports an increase in crop yields, better tillering and reduced chemical inputs. For his low cost and innovative 44
cultivation practices he has been awarded by Krishi Vigyan Kendra, Hoshiarpur.


Two webinars were conducted on line medium during the lock down linking 100 farmers from Punjab in collaboration with Khaiti Virasat Mission. Under the Niti Aayog banner a slogan was prepared “Jalao Nahi Galao”. 44
Table 7: List of Punjab Farmers for in situ paddy straw decompositionS.NoNameMobile numberAddress1.Tarsem Singh9914609544Village, Bhagwa, Kalanaur Amritsar2.Gurmeet Singh Sandhu9872397000Sri Mukstar Saheb, Punjab3.Sukhdeep Hayer9404600003Sri Mukstar Saheb, Punjab4.Rajesh Saini9815169598Village:kotli khas, hoshiarpur5.Capt. Kashmir Singh8054013648Gurdaspur, Punjab6.Balwinder Singh9569236000Fazilka, Punjab7Tarsem Singh Saran9463073787Moga, Punjab 44
Fig 5. Location map of Punjab districts where Pusa Decomposer was applied by the research team in farmers fields (in- situ) 44
Spraying: 17/10/2020
After 25 days
Germination- 23/11/2020 (Seed was broadcasted on 11/11/2020)Fig 6. Application of Pusa Decomposer in farmers field 44
Village: Balim (Gurdaspur)Village: Salina, Moga
Fig.7. Field demonstration of PUSA Decomposer and farmer workshops in Punjab StateVillage : Kattianwali, Shri Muktsar SahibVillage : Kohali, Amritsar 44
Soil health parameters like microbial population, soil enzymes and soil OC and available N were estimated (Fig 8 and Table 7, 8). The soil dehydrogenase activity improved from 13µg TPF g- 1/24 hr at 0 days to 22 µg TPF g-1/24 hrs after 25 days of application of Pusa Decomposer. Similarly the bacterial and fungal count improved from 25 x 108 to 200 x 108 cfu/g soil and 10 x 104 cfu/g soil to 30 x 104 cfu/g soil respectively from 0 days to 25 days of application of Pusa Decomposer. (Fig 8)
Fig. 8: Soil biological parameters over a period of time in PD treated and untreated fields.Soil samples were analysed for organic C (%) and available N (kg/ha) at zero day and after 25 days of Pusa Decomposer application. Increase in the above parameters was found in samples collected after 25 days of Pusa Decomposer application. Soils collected from Punjab and IARI fields, showed an increase in microbial biomass carbon (MBC). Wheat was the rabi season crop in all Punjab farmers’ fields where Pusa Decomposer was applied. The farmers reported wheat yield 3226 variety 26q /acre on an average where Pusa Decomposer was sprayed in Kattianwali, Shri Muktsar Sahib Kohali and Amristar . 44
Table 8: Range of percent increase in Soil OC, available N, Soil dehydrogenase activity and Microbial Biomass C in field samples where PD was applied after 25 DAS.Village sSamplesOrgan ic C (%)incr
Available N (kg/ha)IncrDHA µg TPFg-1 soil 24hrs-1incr
MBC µ gm of biomass/ gm of soil0D - 25DPunjab106 - 15%05 - 20%2 - 5 µg49 - 149IARI103 - 5%8.7 - 21.8%1 - 3 µg35 - 192
At IARI fields, effect of Pusa Decomposer on CH4, CO2 and N2O emissions for the first 12 days was measured. It was found that CO2 emissions increased in Pusa Decomposer treated plots compared to non-treated plots (Fig. 9) indicating the microbial activity. This is an indication that degradation is being carried out by the microbial consortium N2O emissions were found negligible.
Fig 9: CO2 emissions in PD treated and untreated plots
CO2 emission 44
Table 9: Changes in NPK content during in-situ decomposition at farmer’s fieldField NameN (kg/ha)P (kg/ha)K (kg/ha)0 days25 days0 days25 days0 days25 daysBALIM 1Control209.663229.92710.32512.043116.683149.840BALIM 2209.067250.87010.97112.450116.550151.280BALIM 3214.677271.68010.38312.540116.700151.290BALIM 4198.160213.24310.37012.610116.800151.227BALIM 5183.600209.06710.37012.627116.697151.257KOHALI 1Control206.607223.60020.15021.560268.867324.700KOHALI 2229.957271.00020.23321.787268.800330.267KOHALI 3250.067334.00020.20020.820268.800331.400KOHALI 4271.000292.00020.18721.820268.710331.893KOHALI 5239.000251.00020.24321.600268.800330.267Katianwali157.333157.00060.00090.900642.000825.000Mukerian209.000209.00024.45022.700105.323127.000CD @ 5%4.0444.8332.972.5094.3215.372S.Em5.7598.2263.1052.2176.57610.160 44
Chapter 5Executive SummaryTOR I:To develop a technology to convert crop bio waste (particularly paddy) into farm compost in less than six months period with economically efficient methodA microbial solution known as Pusa Decomposer has been developed by the scientists of Division of Microbiology, Indian Agricultural Research Institute, New Delhi, to convert paddy residue into manure in 20-25 days.It is an Economically Efficient Technology. Using cheap source of C like jaggery, 25L of the microbial solution can be developed. 50g of besan (Rs 6.0) and 750g of jaggery is needed for 25L preparation @40/- per kg. Total cost for preparation of one ha solution of Pusa Decomposer is approx. Rs. 86.0 (Rs. 50 for 4 capsules, Rs.30 jaggery, Rs. 6.0 besan).Along with Pusa Decomposer a standard operating protocol (SOPs) has also been developed for In-situ management of agri-residue using Pusa Decomposer. The protocol involves application of the Pusa Decomposer spray, followed by rotavator for proper mixing of the spray with the straw and light irrigation to ensure moisture in the field.Pusa Decomposer is a promising low cost technology for accelerated degradation of paddy straw in the field. The study proved the effectiveness of Pusa Decomposer for accelerated degradation of the paddy residue in Punjab. However, for successful and wide scale benefit of the impact of this technology, Punjab State Govt. must adopt it in a bigger scale. It should be made an integral part of the agriculture programs in the State.TOR II: To convert bio waste into wealth and offer economically viable alternative to prevent burning of crop residues, stubble etcOne ton of paddy residue burning releases 1391.9 Kg of carbon dioxide (CO2), over 82.62 Kg of carbon monoxide (CO), 0.36 Kg oxides of sulphur (SOX), 0.06 Kg of N2O, and 4.21 Kg of particulate matter. Our preliminary studies have shown that application of Pusa Decomposer had shown only 0.222 g/t of biomass of CO2 and 4.6 mg/t of biomass of N2O evolution.The farmers who had used Pusa Decomposer observed the benefits in terms of higher yields and better tillering in wheat which was the next crop. This is an economically viable alternative to prevent burning. Farmers could save one bag of urea. This approach for at least three years should help the farmers in the ease of the burden, discourage them from burning and convince them of the benefits of the application in terms of saving inputs as well as soil health and fertility. 44
We have a 300 odd farmers in our group who are in constant touch with us for updates on PD and other aspects. Regular interactions with farmers telling them about the pollution hazards due to burning and saving on the chemicals for crop productivity for sustainable cultivation has made them understand the problem and acceptance of the technology.TOR III:Create possibility of giving an added value to the agricultural activity through the availability of an additional source of income for managing the treatment and selling resultant compost.Under ex-situ conditions, the farm residue can be decomposed using Pusa Decomposer and nutrient enriched compost which is generated can be used by the farmer for his own requirements. However, if this activity is carried out as a community venture, it has the potential to generate self-sustainability to village youth and generate income, besides managing the agri residue effectively.The ex-situ demonstrations were carried out at 12 different sites, viz: Kattianwali (3), Machiwala, TutoMazara (2), Hayatpur, Kingra, Balim, Anwal, Bhavdin, and Kheda Sahib. Pit method was common to all sites, at some locations windrow and heap methods were also demonstrated. Ex-situ is an alternative method of residue management by which farmers can prepare quality compost which can become as a source of income by the sale of the same. At each demonstration, farmers from the village and nearby villages gathered.The selling rate of compost by farmers is @Rs 13-15 per kg of the compost. The direct sale would be an additional source of income to the farmer.TOR IV:Availability of a new material to improve the soil fertility with the application of compost (in substitution of chemical fertilizers).Pusa Decomposer mediated compost/manure from agri-residue can be applied for improving the soil fertility & health. Many farmers have reported to have cut down on their use of urea by one bag.The application of Pusa decomposer for in-situ decomposition of paddy straw enhanced the fertility of soil. Organic C increased in the range of 6-15% and available N in the range of 5-20%.•Soil health parameters like microbial population, soil enzymes and soil OC and available N were estimated from the soils in which Pusa Decomposer was sprayed, results indicate improvement of all the parameters.•The soil dehydrogenase activity improved from 13µg TPF g-1 /24 hr at 0 days to 22 µg TPF g-1 /24 hrs after 25 days of application of Pusa Decomposer approx. 69% increase.•Similarly, the bacterial and fungal count improved from 25 x 108 to 200 x 108 cfu/g soil and 10 x 104 cfu/g soil to 30 x 104 cfu/g soil respectively from 0 days to 25 days of application of Pusa Decomposer. 44
•Soil samples were analysed for organic C (%) and available N (kg/ha) at zero day and after 25 days of Pusa Decomposer application. Increase in the above parameters was found in samples collected after 25 days of Pusa Decomposer application. 44
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