Isolation and enumeration of microorganisms
Gram staining of the predigested slurry showed presence of variety of gram-positive rods in singles, pairs and chains. All the cells were in their vegetative form and no spores were observed. This was reflective of fast growing cells in nutritionally rich environment. The total bacterial count of slurry samples collected from predigester 1 and 2 was found to be 2.5x107 and 1.96 x107 respectively, suggesting extensive growth of bacteria. Few colonies were developed on Kenknight Munaier’s and Wickerham’s agar. However, they showed characteristics of Bacillus sp. and not of actinomycetes. No growth was observed on Rose Bengal potato dextrose agar plates even after 7 days of incubation. The MacConkey’s and cetrimide agar plates also showed absence of colonies. These observations indicated that fungi, coliforms and Pseudomonas sp. were absent in the predigested slurry.
Cultural and morphological characteristics of organisms isolated from the predigester
Twenty colonies from predigester 1 (Supplementary data, Table 1) sample and twenty four colonies from predigester 2 (Supplementary data, Table 2) sample showing different morphological and cultural characteristics were selected for identification in our study. Gram staining and spore staining were also performed for each isolate and all of them occurred as Gram-positive and spore forming rods of variable sizes (Supplementary data Fig. 2). Thus, based on morphological and cultural characteristics, it was concluded that all isolates belonged to the genus Bacillus.
In Nisargruna biogas plant, the biodegradable wastes mainly consist of kitchen wastes from houses, restaurants and hotels, and other green wastes like foliage, papers etc. are treated. These materials are mainly composed of carbohydrates, proteins and lipids. Thus, the heterogeneous nature of the waste allows the introduction of diverse microorganisms in the predigester of Nisargruna biogas plant. However, the direct microscopic examination of the predigested slurry showed only one type of organism i.e. gram-positive rods of various dimensions. All the isolates were identified as Bacillus species in our study. Similar to our study, Ghosh et al.  also indicated the predominance of Bacillus sp. in the pre-digester of Nisargruna biogas plant in a previous study. The selective enrichment of Bacillus sp. was presumed to be the result of moderately high temperature of the predigester (between 45°C to 50°C) that killed pathogenic bacteria but supported the growth of spore-bearing Bacillus sps.
Biochemical characteristics of organisms isolated from the predigester
Surprisingly, microbiological analysis of the predigested slurry revealed that the entire aerobic degradation process was carried out exclusively by ten different species of genus Bacillus and their innumerable variants isolated from predigester 1 (Supplementary data, Tables 3) and predigester 2 (Supplementary data, Tables 4). For identification of Bacillus, the morphology of the rods, spore shape, structure and its location have considerable significance. The preliminary identification was done based on their gram nature and morphology (Supplementary data, Fig. 2). The phase contrast microscopy was particularly helpful in the study of spores. A simple identification key consisting of commonly used biochemical tests was helpful in easy identification of Bacillus sp. with precision . Specially prepared and stained sections of different species of Bacillus were viewed under transmission electron microscope (TEM) to illustrate their ultrastructure and thus reveal structural differences within different species. Fig. 1 demonstrates the TEM micrographs observed for different Bacillus sp.
Studies on the microbiological analysis of anaerobic digesters have been reported as early as 1967. In a study undertaken by Toerien , specific emphasis was given to determine the aerobic and facultative anaerobic participants of early anaerobic digestion process, in the degradation of cellulose, starch, casein, peptone and sunflower oil. In their study, the facultative anaerobic bacteria such as Bacillus sp. were suggested to play a major role in the primary liquefaction of macromolecules. Later in the same year, Toerien et al.  extended the scope of their study towards several laboratory-scale digesters and concluded that aerobic and facultative anaerobic bacteria occur in all digesters at all stages. However, they are negligible in number in the further stages of digestion. These observations thus indicated the importance of obligate anaerobic bacteria in acid and gas production. It also indicated the importance of aerobic bacteria in initial decomposition of waste material.
Molecular identification of Bacillus sp. by 16S rDNA sequencing technique
Twenty three cultures that were identified by morphological, cultural and biochemical characterization were selected for 16S rDNA sequencing and the sequences of these organisms were submitted with the GenBank. The PCR amplification of 16S rDNA gene of 23 Bacillus sp. are represented in Fig. 2. The accession numbers of these identified organisms are given in Table 3. The different species of Bacillus identified were B. subtilis, B. pumilus, B. megaterium, B. thuringenesis, B. cereus, B. licheniformis, B. velezensis, B. amyloliquifaciens, B. silvestris and B. firmus. All these species belong to the B. subtilis cluster group I .
In almost all cases the molecular identification matched the conventional one which used morphological, cultural and biochemical characterization. However, there were some exceptions and the identification by the two methods did not match. A culture identified as B. laterosporus (PI15) by conventional methods was recognized as B. pumilus by molecular method. Similarly, B. subtilis (PI11) isolate was identified as B. velezensis and B. sphaericus (PII13) as B. silvertis. Among these, B. velezensis is a recently known species of Bacillus, closely related to B. subtilis . In addition, the molecular identification by 16S rDNA gene could not distinguish between the closely related species like B. thuringenesis and B. cereus. The only differentiating character between these two species was the presence of parasporal bodies observed with the help of Ziehl Neelsen staining method (Supplementary data Fig. 3). B. thuringenesis produced the crystalline parasporal bodies which were absent in B. cereus. A special staining procedure thus proved valuable in identification of Bacillus sp. microscopically. These observations promptly suggests that identification of an organism should be considered authentic only when it is done by cultural as well as molecular methods, and not by either of the two alone.
Molecular identification of unculturable bacteria
The 16S rDNA gene was successfully amplified from DNA isolated from predigested slurry, ligated and cloned into T/A vector. Over 500 colonies were transformed and screened for clones. Around 100 clones confirmed the presence of desired inserts, when 16S rDNA fragments were amplified using insert specific primers. These clones were grouped into different categories based on their RFLP profile. Thirty nine sequence of clones obtained in this culture independent molecular identification study of predigester were deposited with the Genebank (Table 4). Figures 3 and 4 indicate representative RFLP profiles of PCR amplified fragments digested with TaqI and Sau3A respectively.
The PCR and RFLP analysis of mixed populations of predigested slurry showed presence of many species of Lactobacillus and Leuconostoc. Three species of the genus Enterobacter and two species of Pseudomonas were also detected. In addition, single species of genera viz., Citrobacter, Klebsiella, Cytophaga, Erwinia, Pediococcus, Geobacter, Brucella and Vibrio were identified. Although isolation of these bacteria was attempted on selective media in the present study, none of them showed characteristic growth. Hence, it could be suggested that these organisms were present in the raw waste. However, the addition of hot water and maintenance of predigester at moderately high temperature (45°C) eliminated these bacteria during processing of wastes. Development of acidic conditions in 96 h and the predominance of spore bearing Bacillus sp. may also be responsible for these observations. During the culture independent approach, clones of anaerobic bacteria were also identified. They included Pantoea, Eubacterium, Methanosarcina, Caloramator, Asteroplasma, Exiguobacterium, Clostridium, Ochrobacter, Butyrivibrio, Acinetobacter, Sphingomonas, Syntrophomonas, Megasphaera and Olsenella. These bacteria may be present as non-viable forms in the predigester slurry due to its strong aerobic character. Presence of these anaerobic clones in the aerobic predigester slurry was presumed to be as a result of recirculation of water separated from manure pit into predigester tank. Our previous study suggested low BOD (100ppm) and absence of coliforms, fungi and other microorganisms in the liquid obtained on dewatering the manure collected after complete digestion of wastes in the Nisargruna biogas plant. Hence this liquid was recycled back into the predigester to maintain the consistency of slurry and, at the same time, save water .
The predominance of different types of bacterial profiles at different stages of microbiological process has also been observed by other researchers. For this purpose, the PCR-RFLP analysis of 16S rDNA was first attempted by Hiraishi et al.  to identify methanogenic bacteria from mixed populations of anaerobic sludge. They amplified the 16S rDNA fragments of 0.4 kb size from the bulk DNA extracted from anaerobic digester using methanogen-specific primers and cloned it directly using the T/A cloning vector. It proved to be a rapid culture independent approach and has since become a popular method for microbiological analysis of anaerobic digesters. Using similar technique, a study identified over 1129 bacterial operational taxonomic units from anaerobic digester fed with dairy manure and wheat distillery is reported. They reported dominance of different genus during different phases of their analysis regardless of the raw material used, and indicated the prevalence of Clostridium sp. by day 7 and Acetivibrio related sp. by day 35 . These observations were reported to be true not only the anaerobic digesters but also for wastewater treatment systems, where significant difference was observed in enriched cultures (depending on the phase of wastewater treatment), irrespective of the microbial profiles of inoculums .