Efficient Lignin Decomposing Microbial Consortium to Hasten Rice-Straw Composting with Moderate GHGs Fluxes

We hypothesized that lignin decomposition microbial consortium would make rice-straw decomposition faster as straw contain around 15–24% lignin. In this study, we isolated aerobic lignin degrading microbes from four natural sources and based on their ability towards lignin degradation four microbial strains and their combination (2 bacteria (LB 8, LB 18) and 2 fungi (LF 3, LF 9) were selected for rice straw decomposition. The greenhouse gases emission, enzymatic activities (β-glucosidase, cellulase, laccase), reduction in lignin content, weight loss and carbon nitrogen ratio (C:N) were quantified during the process of decomposition. The β-glucosidase, cellulase and laccase activities were higher in compost where LB 18 + LF 3 consortium was applied as compared to others. The lignin content was also decreased (8.9–9.5 to 6.6–7.9%) gradually from initial to 28th days of composting under LB 18 + LF 3. We found the microbial consortium LB 18 + LF 3 decomposed the rice straw relatively faster than other strains/consortium tested as indicated by lowering of C:N ratio and reduction of lignin, hemicellulose, and cellulose contents of 60, 19.2, 41.5 and 10.3%, respectively at 28th day of composting compared to initial values. However, higher, methane and carbon dioxide fluxes were also observed at 28th days of composting (1.36 and 200.7 mg m−2 h−1) with no significant trend in nitrous oxide fluxes. Further, the consortium identified could be tested for in-situ straw decomposition with proper moisture management to evaluate its potential in field condition. Therefore, we conclude that use of lignin decomposing microbial consortium has the potential to hasten the composting of rice straw in large scale.


Introduction
India is the prime producer of rice (Oryza sativa) globally, accounting for about 20% of world rice production. In India 43.2 m ha of area is under rice cultivation. The main byproducts of rice are straw, rice-husk and rice-bran. Approximately, 760 million tons of rice straw produced per year globally which is the 1.5 times greater than per ton of rice-grain production [23 ,55]. The disposal of this surplus straw creates a major concern now a day in all the rice growing regions [10]. Moreover, since last two decade farmers prefer to burn this straw to clear the field for the timely sowing of wheat in northern and north-western parts of India. Open field-burning of straw release a large quantity of pollutants including methane (CH 4 ) and fine/ inhalable particles, toxic gases such as carbon monoxide (CO), carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds (VOCs) [60] which are responsible for environmental pollution and human health hazards. Burning of rice straw emits 0.7-4.1 g of CH 4 and 0.019-0.057 g of nitrous oxide (N 2 O) per kg of dry rice straw and other gaseous pollutants such as Sulphur dioxide (SO 2 ), nitrogen oxides (NOx ) , hydrochloric acid (HCl), and to some extent, dioxins and furans [48].
Rice straw is primarily a carbohydrate-polymers with heterogeneous compounds. It is a lignocellulosic material consists of three major biochemical compounds namely, cellulose, hemicellulose and lignin. Cellulose is composed of glucose units, which are linked together by β-1, 4-glycosidic bonds in linear and crystalline forms. Hemicellulose is a hetero-polymer consisting of xylose, mannose, arabinose, galactose and glucose in short and highly branched chain. Lignin is made of the structurally complex-carbohydrate, consisting of various bio-chemically stable linkages having high molecular weight [29]. We know that after cellulose, lignin is the second most abundant renewable biopolymer in nature and also the most abundant aromatic polymer in the biosphere [50].
Ex-situ rice straw composting is a good option to tackle the issues like straw-burning and soil health management simultaneously. The main bottle-neck of rice straw decomposition both in in-situ and ex-situ condition is higher lignin content (5-24%) of straw that takes more time for its composting [26]. In this context, microbes originally from natural sources and with higher lignin decomposing potential could be an environment friendly and economic option for large-scale straw decomposition. Further, it is known that microbes that produce higher ligninolytic enzymes hasten the rice-straw composting. However, enzymes which degrading lignin are extra-cellular in nature and lignin having structurally heterogeneous and chemically more complex that cannot enter the cell for intracellular action [50]. Apart from that, ligno-cellulolytic enzymes have substantial other applications in various industries including chemicals, brewery and wine, food, fuel, textile and laundry, animal feed, paper-pulp and also in agriculture [31].
Incorporation of rice straw directly to the soil is associated with immobilization of plant nutrients. High C:N ratio of straw and having considerable amount of silica, lignin makes it difficult to decompose. Therefore, farmers favour in-situ burning of rice straw in the field causes great nutrient and economic losses. Rice straw burning also causes serious environmental problems which emits aerosols and greenhouse gases [13]. However, composting of rice straw could bring much needed organic manure to the soils that improves soil fertility as well as productivity.
Therefore, in our study we focused on the isolation of lignin degrading microbial from natural lignin rich sources, such as elephant dung, dead plant-bark, vermicomposting pits and compost pits. After that most efficient bacterial and fungal strains as well as their consortium were evaluated for straw decomposition and composting. So, the objective of our study was to use the efficient lignin degrading microbial strains for rice straw decomposition in shorter time in ecofriendly manner.

Sample Collection
The samples were collected from four natural lignocellulosic sources like, elephant dung, dead plant-bark, vermicomposting pits and compost pits. The samples were collected in sterilized polythene bags and transported to the laboratory for immediate storing in refrigerator at 4 °C.

Alkali-Lignin Extraction
The lignin was extracted from the bark of the woody plant as described by Howard et al. [31]. The bark used for extraction 1 3 of lignin was dried and grinded to powder. Ten grams of powdered-bark was taken in a beaker and 5 mL of 1% sulfuric acid was added to it. Then, it was heated in hot air oven at 80 °C for 20 min. After heating, 100 mL of 4% sodium hydroxide was added and the suspension was boiled for 30 min. The dark brown colored alkali-lignin was thus obtained, then it was filtered and autoclaved at 15 lbs for 10 min [54]. After autoclave, the soluble lignin was used as the selective media for isolation of lignin degrading bacteria.

Isolation of Lignin Degrading Bacteria and Fungus
The lignin degrading bacteria and fungus were isolated by using nutrient agar medium (NA) and potato dextrose agar (PDA), respectively in which lignin provided the sole carbon and energy source. In the medium, 1% alkaline lignin was added before autoclaving. Serial diluted collected samples were spreaded in the respective media for bacteria and fungus isolated from different sources. The bacterial plates were incubated at 37 °C for 24 h, whereas the fungal plates at 30ºC for 48 h until colonies were developed. After incubation, the colony forming units (CFU) were counted. These isolated bacteria and fungus of different morphology and characters were plated onto fresh nutrient agar (NA) and potato dextrose agar (PDA), respectively to obtain pure cultures [50].

Lignolytic Activity of Isolated Microbes
The microbial isolates were transferred to plats having methylene blue dye as an indicator for further screening. The microbes possess lignolytic enzymes undergoes oxidation of indicator dye. The isolated bacteria and fungus were streaked on methylene blue indicator dye (0.25 g L −1 ) containing NA and PDA plates. The plates were incubated and placed under observation daily for growth and decolorization zone Bandounas et al. [5]. The decolorized microbial colonies were processed for identification.

Efficiency Test of the Isolates
The efficiency test of the bacterial and fungal isolates to utilize lignin-substrate was performed in minimal salt media containing lignin (MSM-L). The MSM-L consisted of 1% alkaline-lignin minimum salt medium solution (K 2 HPO 4 -4.55 g; KH 2 PO 4 -0.53 g; MgSO 4 -0.5 g; NH 4 NO 3 -5 mL in 1 L deionized water) [12]. The isolates were inoculated in 50 mL broth and placed in shaker. Dinitrosalicylic acid solution (Ingredients, g L −1 ; Dinitrosalicylic Acid-10 mL; Phenol-2.0 mL; Sodium Sulphite-0.5 g; Rochelle salt (Potassium sodium tartrate)-200 g) was used for estimating reducing sugar. The glucose standard solution was prepared for this. The 2 mL of culture solution was taken from each conical flask and was transferred to test tubes and then 3 mL of DNS solution and distilled water was added to each test tube and made up the volume to 10 mL. After that test tubes were heated 5 min in water bath and cooled down then the absorbance was taken at 540 nm.

Identification of Lignin Degrading Bacteria
Molecular identification of the efficient lignin degrading bacterial and fungal isolates were done by polymerase chain reaction (PCR) amplification of 16S rDNA and 18S rDNA, respectively. Amplified DNA were purified using a DNA purification kit (Promega, Madison, WI), and di-deoxy chain termination method was used for individual nucleotide sequences. The forward and reverse DNA sequences of each were confirmed and compared by using database Basic Local Alignment Search Tool (BLAST) (http:// www. ncbi. nlm. nih. gov/ BLAST/).

Preparation of Consortium Followed by Compatibility Test
The most efficient two bacterial (LB 8, LB 18 (MN784664 & MN784667)) and two fungal (LF 3, LF 9 (MK855473 & MK855476)) strains were selected for rice straw decomposition. For preparing the consortium, at first compatibility test of selected microorganisms were done, because incompatibility of the co-isolates may also inhibit the growth of each other. After compatibility test, the bacterial and fungal isolates were inoculated into autoclaved nutrient broth (NB) and potato dextrose broth (PDB) separately. Then bacterial culture was incubated at 37 °C for 24 h whereas, fungal culture at 30 °C for 48 h. After incubation, consortiums were prepared by addition of both bacterial and fungal broth. The bacteria and fungus strains alone and their respective consortium were taken for composting of rice straw.

Evaluation of Straw Decomposition Capabilities of the Microbial Consortium
An experiment was conducted to study the efficacy of the isolated strains of bacteria, fungus and their consortium for decomposition of rice straw. In this experiment, 27 numbers of 10 kg capacity pots (9 treatments × 3 replications) were taken. Nine treatments included 2 bacteria; 2 fungi; four bacteria-fungi consortium and one control (no microbial strain) were imposed. In each pot 600 g of oven dried rice straw (2-3 cm size) were added. One litre of liquid fungal broth was comprised of freshly prepared spore solution (10 6 -10 7 spores mL −1 ). Similarly, 1 L of liquid bacterial broth contains (10 6 -10 8 viable cells mL −1 ). Finally, these liquid broths used single inoculum and also prepared the consortia by mixing thoroughly. Then the 1 3 broths were transferred to jiggery solution for rapid multiplication of microbes for 5-7 days and 200 mL of jiggery based microbial inoculum applied to the rice straw. Then all the microbial broth having bacteria, fungus and their consortium were inoculated to different pots having straw. Optimum moisture content was maintained during the decomposition process (15-20%). The samples were collected during straw decomposition from different treatments at seven day interval for estimating the enzymatic activities (β-glucosidase, cellulase, laccase), lignin contents, C:N ratio and weight loss.

β-Glucosidase Activities
The β-glucosidase activities were estimated based on the detection of p-nitrophenol (PNP) from p-nitro phenyl-β-Dglucopyranoside (PNG, 0.05 M) as substrate [22]. Briefly, 1 g of sample from different treatments were added with 0.25 mL of toluene, 4 mL of MUB buffer (pH-6.5) and 1 mL of PNG solution and mixed with in a 50 mL Erlenmeyer flask. Then flasks were mixed properly, stoppered and incubated for 1 h at 37 °C. After that, 1 mL of 0.5 M CaCl 2 was added and tris-hydroxymethyl amino methane (THAM) was added stop the reaction. Flasks were swirled for few second and the suspension was filtered through Whatman no. 41. The concentrations of the filtrates were measured at 420 nm on a spectrophotometer.

Cellulase Activities
In order to estimate the cellulase enzymatic activity, first 1 gm sample was taken. After that 10 mL of 1% carboxy methyl cellulose (CMC) was added as substrate followed by 10 mL of acetate buffer (0.2 M solution of acetic acid + 0.2 M solution of sodium acetate, pH 5.9). After that, the solution was incubated for 24 h and the released sugar was estimated by di-nitrosalicylic (DNS) acid method [46].

Laccase Activities
Laccase activity of the samples were done by preparing the crude enzyme extract from moist samples of rice straw (during composting) [58,68]. First, 10 mL of distilled water was added to 1 g of sample and shaken for 30 min. After shaking, the samples were filtered and centrifuged at 10,000 rpm for 15 min. Then, 25 µl of the supernatant was taken and 1.5 mL of 0.2% ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) in sodium acetate buffer was added. It was incubated at 30 °C for 30 min. After incubation, absorbance was taken at 436 nm by using spectrophotometer.

Cellulose, Hemicellulose and Lignin Content
Ultrasound assisted alkaline extraction method (gravimetric) was used for the estimation of cellulose, hemicellulose lignin content of samples collected during straw decomposition [65]. The samples were collected at regular intervals during rice-straw decomposition were dried and grinded into powder. One gram of sample was taken in centrifuge tube. Then samples were treated with 2 M NaOH solution, and there after it were placed in water bath at 90 °C for 1.30 h. Then each tube was placed in the ultra sonicator for 30 min. After sonication, those samples were centrifuged and the residue and filtrate were separated out. Then residue was washed with water for repeated times until clear or transparent filtrate was reached. After washing the tubes were placed in inverted position for 24 h and the residues were transferred to petriplates and placed in hot air oven at 50-60 °C for estimation of cellulose. After the cellulose was collected, the filtrate was acidified with HCl to pH 5.5. Then it was precipitated with 90% ethanol solution (1:3: sample: ethanol). It was again centrifuged and residue and filtrate were separated out. Residues were washed with 70% ethanol solution and after washing the tubes were placed inverted position for 24 h and then similar to cellular estimation the residues were transferred to petriplates and placed in hot air oven at 50-60 °C and hemicellulose content were obtained. Then filtrates were acidified with HCl to pH 1.5 and placed for evaporation of ethanol. Samples were centrifuged after evaporation of ethanol. The residues were washed with HCl (pH 2.0) and lyophilized. After lyopholization, lignin contents were obtained after weighing of tubes taking empty tube as control.

Estimation of Carbon: Nitrogen (C:N) Ratio
Total carbon and nitrogen contents were estimated by using the CN Analyzer (Model No. SNC100-IC, Skalar Primacs). The samples were air dried and grinded by using 2 mm sieve. After that, 0.5 g of samples were taken in the auto sampler.

Weight Loss
At the beginning of the composting 600 g of oven dried rice straw were taken in each pot. After adding constant amount of water in each treatment for moisture maintenance, the weight was measured. At an interval of seven days during composting, the weight loss of the compost was measured.

Greenhouse Gas (GHGs) Fluxes Measurement
The gas samples were collected from different treatments at seven days interval by using 'plastic funnel chamber' 1 3 (specifically designed; with volume 5306.6 cm 3 and area of 0.053 m 2 ) fitted with rubber cork at the top. The funnel is air tight with the upper part of the pot (Fig. 1). The funnel chamber was put in the respective pots during the gas-sampling time only. The gas was collected by using 50 mL syringe and measured the GHGs i.e. methane (CH 4 ), carbon dioxide (CO 2 ) and nitrous oxide (N 2 O) in gas chromatograph (GC) (Model no. Trace 1110, Thermo Scientific). This GC was fitted with Porapak Q column and having flame ionization detector (FID) and electron capture detector (ECD) for measuring the GHGs concentrations. The GHGs fluxes were calculated by using this formula (Eqs. 1, 2, 3).

Statistical analysis
Analysis of variance (ANOVA) and critical difference (CD) at a p ≤ 0.05 level using two factorial completely random design (CRD). The treatments and days after composting were considered as two factors for different variables and all the analyses were done by using SAS software (Ver. 9.2, SAS Institute, Cary, NC.

Isolation of Microbes from Potential Sources
In order to identify lignin decomposing microbial population, culturable bacterial and fungal population was isolated from potential natural sources like, elephant dung, vermicompost, compost, and dead plant bark by using nutrient agar (for bacteria) and potato dextrose agar (for fungi). Bacterial population (10 -3 and 10 -4 dilution) was highest in   (Table 1). The fungal population (10 -2 and 10 -3 dilution) was also highest under elephant dung (5.4 and 5.2 Log CFU g −1 ) and lowest in dead plant bark (5.1 and 4.9 Log CFU g −1 ) ( Table 1).

Screening and Efficiency Test of Lignin Degrading Microbes
Screening was performed by using methylene blue; after screening 21 bacterial and 9 numbers of fungal isolates were selected. The efficacy of lignin decomposing bacterial isolates were judged by measuring the quantity of glucose produced during decomposition process through DNS method. The sugar production was increased from 1st days of incubation to 7 days and then it was declined. Compared to all the bacterial isolates, LB 18 and LB 8 degraded the lignin more during incubation. The highest sugar was produced by LB 18 (154.8 μg mL −1 ) and LB 8 (149.9 μg mL −1 ) at the 7th days of incubation (Fig. 2a). However, the glucose production of the fungal isolates was increased from initial days and was observed maximum at 9th days of incubation. Therefore, the two efficient fungi were selected for further study i.e. LF 3 and LF 9, which produced 171.3 and 147.0 μg mL −1 respectively (Fig. 2b).

Morphological Characterization of the Isolates
The bacterial and fungal isolates were morphologically evaluated ( Table 2). The sequences of selected efficient microbial strains were registered to NCBI. The NCBI accession numbers of LB 8, LB 18 and LF 3, LF 9 were MN784664, MN784667 and MK855473, MK855476, respectively.

Evaluation of Efficient Microbes/ Microbial Consortium for Rice-Straw Decomposition
The efficient bacterial (LB 8, LB 18), fungal (LF 3, LF 9) strains and their consortium were tested in net house Glucose (μg ml -1 )  Enzymatic Activities β-Glucosidase Activities The β-glucosidase activities were increased in the treatments where microbial consortium were used up to 28th ' days of composting' (DOC) and then steadily decreased up to 42th DOC in all the treatments (Fig. 3a). In 28th DOC, the β-glucosidase activities were ranged from 423.3 to 630.1 µg PNP g −1 h −1 in the treatments. The higher β-glucosidase activities were observed under the microbial consortium, LB 18 + LF 3 (290.4 µg PNP g −1 h −1 ) followed by LB 8 + LF 3 (280.3 µg PNP g −1 h −1 ) than the other treatments.

Days of incubation
Cellulase Activities Similar to β-glucosidase activities, the cellulase activities were higher at 28th DOC and then decreased up to 42th DOC in all the treatments (Fig. 3b). The average cellulase activity throughout the decomposition process, was also higher in LB 18 + LF 3 (64.7 μg glucose g −1 h −1 ) followed by LB 8 + LF 3 (62.8 μg glucose g −1 h −1 ) than the other treatments and least in control (without microbial inoculation) (45.4 μg glucose g −1 h −1 ).

Laccase Activities
The laccase activities were ranged from 4.9 to 73.8 µg g −1 h −1 in all the treatments (Fig. 3c). Higher laccase activities were observed at 28th DOC followed by 21 st DOC. At 28th DOC, higher laccase activities were observed in LF 18 + LF 3 (73.8 µg g −1 h −1 ) as compared to other treatments and least in control (14.04 µg g −1 h −1 ).

Cellulose, Hemicellulose and Lignin Content
The initial content of cellulose, hemicellulose and lignin of the rice straw were 39.4, 20.4 and 9.3%, respectively. After inocu-lation of microbes and their consortium to the pots having straw, the cellulose, hemicellulose and lignin contents were decreased significantly by 18.4, 17.8 and 7.2%, respectively at 28th DOC ( Fig. 4a-c).

C:N Ratio
The C and N contents were ranged from 45.6 to 52.6% and 0.71 to 0.82%, respectively at initial date of composting and 31.8 to 36.1% and 1.27 to 1.42%, respectively at 28th DOC (Table 3). Similarly, the C:N ratio were reduced from initial day of composting (60.0 to 67.3%) to 28th DOC composting (24.8 to 27.7%) in all the treatments. Moreover, highest degree of reduction of C:N ratio was found at 28th DOC in the consortium LB 18 + LF 3 (60%) as compared to control treatment.

The GHGs Fluxes During Composting
The GHGs fluxes were measured during rice straw composting in all the treatments. The CH 4 fluxes increased from 7th DOC to 28th DOC and then decreased (Fig. 6a). Maximum CH 4 flux was observed at 28th DOC in all the treatments. At 28th DOC, the CH 4 flux was ranged from 0.11 to 1.36 mg m −2 h −1 . Higher CH 4 flux was observed in LB 18 + LF 3 treatment (1.36 mg m −2 h −1 ) followed by LB 8 + LF 3 (1.13 mg m −2 h −1 ). Higher CO 2 flux was also observed at 28th DOC followed by 14th DOC (Fig. 6b). At 14th and 28th DOC the CO 2 fluxes were in the range of 37.9 to 191.9 mg m −2 h −1 and 84.5 to 201.4 mg m −2 h −1 , respectively. However, there was no significant differences were observed in N 2 O fluxes (Fig. 6c).

Discussion
Huge amount of lignocellulosic rice straw is generated worldwide have the potential to reuse for bioethanol, biochar and pulp production [7,32]. Lignin is a polymer with high molecular weight that gave it rigidity and resistant against decomposition by different kinds of microorganisms [63]. So, it causes hindrance for making compost or the value-added product due to their slower decomposition rate. However, addition of rice-straw compost in field and insitu decomposition of straw could result soil carbon enrichment and improvement in soil health and productivity [27,52,71]). Therefore, microbial decomposition is a viable option to make use of this resource (rice straw) for better alternatives in eco-friendly manner. White-rot and brownrot fungi are the well-known for lignin-degradation by generating extracellular oxidative enzymes. White-rot fungi like Ceriporia lacerata, Phanerochaete chrysosporium, Fig. 3 a β-glucosidase and b cellulase (C) laccase activity of the rice straw at different 'days of composting' (DOC) by using bacterial and fungal strains alone and also their respective consortium  [57]. It was reported that, the fungi have higher potential but sometimes natural or extreme environmental conditions and substrate restriction is not suitable for lignin degradation [56]. Another school of thought that the bacteria, in particular are better microbes having immense environmental adaptability and biochemical versatility for lignin degradation [20,42]. A variety of bacterial species have been reported to be able to degrade lignin and cellulosic materials such as Bacillus, Pseudomonas and Acintobacteria [43], Bacillus, Clostridium [63], and Acintobacteria [64]. So there is a potential to use microbial consortium using both fungi and bacteria for eco-friendly decomposition of rice straw for cleaning up environments and to use the value added end products like, bio-compost, biofuel, lignin for producing dyes, paper pulp and generating silica. Lignin protects cellulose, hemicelluloses and other cell wall constituents in rice straw for easy decomposition but a few microorganisms can act on complex structure of lignin by secreting extra cellular enzymes. Ligno-cellulolytic fungi  have benefits in bio-conversion of solid waste as they are filamentous and produce high-volume of spores [18]. Microbial consortium (bacteria and fungi) have greater effect on substrate accumulation through resistance to environmental contamination and increased enzyme production [19]. Compatibility of microbial consortium is another important aspect which influences the distribution, density, association and ecological steadiness of communities [44,47,61]. Thus, a better compatible microbial consortium might have plays an important part for hastening rice straw bio-conversion. In our study, the lignin degrading bacterial and fungal strains were isolated from the natural sources like, elephant dung, dead plant-bark, vermicomposting pits and compost pits. We got two efficient bacterial (LB 8, Bacillus cereus; LB 18, Enterobacteriaceae bacterium) and two fungal (LF 3, Penicillium sp.; LF 9, Alternaria alternata) isolates, which were used for rice straw composting. These isolates alone and their consortium were used for composting. Hence, isolation of the lignin degrading microbes from natural lignin rich sources is effective due to higher lignin content. The elephants are herbivorous and their dung is reach in lignin. Similarly, the dead plant bark is slowly degraded by the lignin decomposing microbes. Hence, there is a possibility to get efficient isolates from this lignin rich sources. Composting of agricultural wastes through bio-augmentation of biodegradable microbes is an efficient process [41,53,56,62,66]. In recent years, social demand for economic and eco-friendly decomposing technology of rice straw have drawn attention to researcher. Bhattacharjya et al. [6] revealed that there was significant reduction in cellulose and lignin content of rice and wheat residues, as compared to the initial content, after 30 days of in situ decomposition by using microbial consortium (bacteria, fungus and actinomycetes). The sustainable resources to produce biofuel have also increased interest because of cost effectiveness and readiness in the recent past [15]. Among them, agricultural byproducts are the renewable and eco-friendly raw materials which could provide substitute biomass for high-demanded woody materials 15,40]. As rice straw is a renewable agricultural residue and rich in nutrients and lignocellulosic materials, hence, could be used efficiently for the future energy requirements [73]. Recycling of organic matter through agriculture residues (rice straw) also highly adopted methods [4].
Greenhouse gases emissions could also increase with low C:N ratio of substrate-biomass during biological composting processes [35]. Biomass burning is a global phenomenon which responsible towards air pollution in worldwide [67]. Globally, forest burning is the major biological source of carbon loss, GHGs emissions and air pollution followed agricultural biomass burning (approximately 2020 Tg) [ [3], 17], 34]. Generally, frequency of bio-conversion of rice straw is slow and it takes more time to composting in natural field condition [11]. Increase use combine-harvester further aggravated the problem of in-situ straw decomposition by spreading the straw all over the field. Many a times these cause field burning of straw on large scale. In those context, ex-situ decomposition of rice straw through lignin degrading microbial inoculation is an effective way to hasten the composting process with ecofriendly manner. Straw-compost is also a valued source of nutrients to agricultural soil due to its local availability [36]. The inoculation of lingo-cellulolytic microorganisms to straw-compost has been widely used as an worthy approach that could potentially hasten up the decomposition [49,69] and ultimately improve the quality of compost [28,45,63,70].
Carbon provides the source of energy to microbes during composting, and CO 2 emission is the byproduct of the processes [38]. In our study, initially the CO 2 emission was more (highest at 28th DOC) and then reduced. The practice of fresh rice straw incorporation to the soil significantly increases methane emissions, whereas decomposed straw emits relatively less emissions [8,9,16,25].
Data on GHGs emissions related to in-situ straw decomposition is limited. Similarly, emissions of GHGs during straw composting processes is also sparse. We found, similar to CO 2 , the CH 4 fluxes also increased from 7th DOC to 28th DOC and then decreased gradually. One peak of CO 2 at 14 days of composting may be due to higher respiration because of higher availability of labile carbon and then decreased at 21 days and again increased at 28 days when complex carbon sources break into simpler form and make available to the microorganisms. Applying of straw-compost in the field reduced GHGs emission as compared to fresh straw incorporation and open-field-straw burning ( Table 4). The reason behind this as an aerobic process, composting converts organic substrates into a humus-like material which impedes methanogenesis and also increases CH 4 oxidation [14]. At the same time, as the compost application is gradually decreasing in agriculture, there is a need to recycling the straw to restore soil-carbon and nutrients and also to get rid of straw burning menace. Although burning of straw in the field condition ensures the quick land preparation to subsequent crops and also restricts the nitrogen immobilization during straw decomposition with wider C/N ratio, yet incomplete carbon-combustion generates large amounts of GHGs and adversely affects the air quality [1,33]. Zschornack et al. [72] indicated that CH 4 and N 2 O emission decreased by 69 and 81%, respectively in surface retention when compared to straw incorporation in rice fields [33]. Apart from this, open straw burning is an unrestrained combustion practice, in which CO 2 , N 2 O, CH 4 , CO, other hydrocarbons (NMHC), NOx, SO 2 , particulate matter (PM) were also emitted to the atmosphere. It is estimated that quantity of annual rice straw open burnt (13.92 Tg) in India would represent about 15% of the total amount of crop residues (84 Tg) [59]. Study revealed application of microbial consortia along with crop residues had significantly reduce the cumulative CO 2 -C loss than residue burning [6]. Further, it was found that application of rice-straw-compost could reduce the CH 4 emissions from irrigated paddy fields compared with other fertilizers application and also sustain the yield. Therefore, composting of rice straw by use of efficient lignin degrading microbial consortium is an intelligent step towards mitigating GHGs emission and combat with the menace of straw burning.

Conclusion
Isolation of lignin decomposing microbial strain from natural sources namely, elephant dung, dead plant-bark, vermicomposting pits and compost pits found effective for rice straw decomposition in shorter time i.e. 28 days. Combination of fungus (LF 3, Penicillium sp.; LF 9, Alternaria alternata) and bacteria (LB 8, Bacillus cereus; LB 18, Enterobacteriaceae bacterium) for hastening ex-situ rice straw decomposition is more effective than single microbial inoculation. Production of β-glucosidase, cellulase and laccase during decomposition were positively corelated with better quality compost (suitable C:N ratio, lignin and cellulose content). However, quicker decomposition relatively triggers higher CO 2 and CH 4 fluxes, but if we take the process as a whole, it could be balanced. Future endeavor should be in the direction of in-situ straw decomposition in shorter time with this valuable microbial consortium in order to curb the menace of on-field straw burning.