The Predominance of Bacillus Species and non- Culturable Microaerophilic/Anaerobic Micro ora in the Aerobically Operated Predigester of Nisargruna Biogas Plant


 In an attempt to convert biodegradable solid wastes into methane gas, the Nisargruna biogas technology is developed by the Bhabha Atomic Research Center, Mumbai and is functioning at more than 300 places in India. This plant is uniquely designed to allow microbiological degradation in a two-step process, where breakdown of waste material is facilitated under aerobic conditions prior to anaerobic digestion. Introduction of aerobic predigestion helps to overcome the drawbacks of solely anaerobic degradation like scum formation and high retention time of waste material. The aim of the present study was to identify the culturable as well as non-culturable microorganisms from the predigester of two phase Nisargruna biogas plant. Surprisingly, only Bacillus sp. were prevalent in the predigester and, among them, B. subtilis and B. pumilus were predominated. The spores, parasporal bodies and TEM micrographs of Bacillus sp. were also studied. Molecular techniques like PCR and RFLP analysis identified non culturable bacteria like Citrobacter, Klebsiella, Cytophaga, Erwinia, Pediococcus, Geobacter, Brucella and Vibrio sp. and clones of 14 different genera of anaerobic bacteria in the predigester slurry. The current study introduces the relatively unique microflora of two-phase Nisargruna biogas plant as compared to other anaerobic digesters, and relates its efficiency to diverse metabolic activity of Bacillus sp.


Introduction
The increasing population continuously challenges the waste management systems especially in countries with faster urban development. In spite of the radical measures undertaken, the millions of tonnes of waste collection and disposal annually is an arduous task. Moreover, the approaches of waste reduction, recycling and other socio-economic aspects of solid waste management become impractical in this scenario [1,2]. Hence, effective solutions and improvement in existing technologies are continuously in demand. In addition, proper solid waste management practices can be economically favourable since wastes are nothing but vital reserve of precious biochemical elements [3]. If handled intelligently, it can lead to production of valuable bioenergy like methane, and manure that can be applied as a fertilizer and soil conditioner. The biodegradable waste generally consists of carbohydrates, proteins and lipids in variable proportions. These compounds contribute to more than half of the solid wastes generated and can be composted under appropriate conditions for its management. The kitchen wastes generated from household, restaurants and food industries can be repurposed for the above processes [4]. In this regard, the use Nisargruna biogas plant, introduced by Bhabha Atomic Research Centre, Mumbai, has proven to be bene cial. It combines the advancement in methods of biological treatment of wastes with biogas production technology to provide a productive and environment friendly approach for management of biodegradable solid waste.
The most remarkable feature of Nisargruna system is the aerobic decomposition of wastes in the predigester prior to anaerobic degradation in the main digester of the plant. The raw slurry is rst collected in the predigester 1 and after initial decomposition and acquiring a owing consistency, it moves to predigester 2. During the processing in this stage, the temperature is maintained between 40°C to 45°C to promote the degradation of waste material into their soluble monomeric components by extracellular enzymes produced by bacteria naturally introduced in the predigester. The moderately high temperature further facilitates the conversion of degraded compounds into short-chain fatty acids, alcohols, CO 2 and H 2 by fermentative bacteria. Our previous study indicated that the raw waste containing proteins, carbohydrates and lipids are broken down to their respective monomers that are fermented to produce volatile fatty acids like formic acid, acetic acid, propionic acid and butyric acid along with liberation of CO 2 . The volatile acids produced as a result of fermentation in the predigester remain in the aqueous phase and enter the main digester (anaerobic) to promote acetogenesis and methanogenesis [5]. The anaerobic digestion is a slow process and hence the hydrolysis and fermentation becomes a rate limiting step in methane production. The Nisargruna plant is designed to overcome this shortcoming by partitioning the reactor into predigester and main digester that operates under aerobic and anaerobic conditions respectively. This allows the initial hydrolysis and fermentation, to occur under aerobic conditions in the predigester. The above step not only reduces the retention time of wastes but also provides ready nutrients as raw materials for methanogenesis that occurs in the main The biodegradation of organic matter is a complex phenomenon involving diverse microorganisms exhibiting various enzyme activities. Thus, elaborate characterization of microbial ora in any microbiological process is essential for improvising the technology for better and wider applications.
Hence the current study was carried out with an objective to analyze the metabolically dynamic system of the predigester by isolation and enumeration of its culturable and non-culturable microbial ora.

Sample collection
Representative samples were collected from two different compartments of the predigester of Nisargruna biogas plant located at Govandi, Mumbai, Maharashtra and labeled as predigester 1 and predigester 2. The homogenized samples were collected in sterile plastic bottles of 1 L capacity and processed immediately.
Enumeration and identi cation of isolates from predigester Selective media were used for isolation and enumeration of bacteria (Nutrient agar), fungi (Rose Bengal potato dextrose agar) and actinomycetes (Kenknight Munaier's and Wickerham's agar). In addition, the presence of coliforms and pseudomonads were also screened on Mac Conkey's and cetrimide agar plates respectively. All the isolation techniques were performed in triplicates and the plates were incubated for 24-48h at 45°C. This is because the temperature in predigester is maintained between 45°C to 50°C. After incubation, colonies were counted and colony-forming units per mL (cfu/mL) were estimated. All the isolates were maintained on nutrient agar slants.
A stepwise biochemical tests were performed for identi cation of Bacillus sp. as indicated in Table 1 since most of the isolates showed cultural and morphological characteristic of the genus Bacillus [7].
Molecular identi cation of culturable and non-culturable bacteria The con rmation of species was done by 16S rDNA sequencing technique. The primers used for identi cation of culturable and non-culturable bacteria are indicated in Table 2. PCR ampli ed products were puri ed using Genei quick PCR puri cation kit (Bangalore Genei, India) and sequenced bidirectionally speci c primers. For this purpose, ABI PRISM 3100 (Applied Biosystems/Hitachi) was used as a sequencer. The sequence data thus obtained was aligned to obtain the complete fragment sequence and analyzed for homology in GenBank by BLASTn search to identify the bacteria. The phylogenetic trees were also constructed for the identi ed bacteria [12]. The PCR product was ligated into T/A vector and E. coli DH5α cells were transformed with the recombinant DNA. The T/A vector is a high copy number vector with pMB1 origin of replication and carries ampicillin resistance marker. The genetic map of T/A vector is represented in Supplementary data, Fig. 1. Plasmids were then isolated from the transformed cells and cloning was con rmed by checking the release of insert by treatment with NcoI. The clones that showed release of insert and hence con rmed cloning were selected for re-ampli cation and puri ed using plasmid puri cation kit (KT 61, Bangalore, Genei). The re-ampli ed inserts were subjected to RFLP using two restriction endonucleases-Sau3AI and Taq I. Clones that showed different RFLP patterns were selected for sequencing and identi ed using the ABI PRISM 3100 sequencer (Applied Biosystems/Hitachi). The data obtained after sequencing was aligned to get the complete sequence of the 16S rDNA gene. This was done by using Bioedit software. The sequences were then analyzed for homology in GenBank by using BLASTn search. Phylogenetic trees of all the isolates were constructed for their identi cation.

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 re ective 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 2.5x10 7 and 1.96 x10 7 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 identi cation 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. grampositive rods of various dimensions. All the isolates were identi ed as Bacillus species in our study. Similar to our study, Ghosh et al. [13] 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 identi cation of Bacillus, the morphology of the rods, spore shape, structure and its location have considerable signi cance. The preliminary identi cation 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 identi cation key consisting of commonly used biochemical tests was helpful in easy identi cation of Bacillus sp. with precision [7]. 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 [14], speci c 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 sun ower 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. [15] 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 identi cation of Bacillus sp. by 16S rDNA sequencing technique
Twenty three cultures that were identi ed 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 ampli cation of 16S rDNA gene of 23 Bacillus sp. are represented in Fig. 2. The accession numbers of these identi ed organisms are given in Table 3. The different species of Bacillus identi ed were B. subtilis, B. pumilus, B. megaterium, B. thuringenesis, B. cereus, B. licheniformis, B. velezensis, B. amyloliquifaciens, B. silvestris and B. rmus. All these species belong to the B. subtilis cluster group I [7].
In almost all cases the molecular identi cation matched the conventional one which used morphological, cultural and biochemical characterization. However, there were some exceptions and the identi cation by the two methods did not match. A culture identi ed as B. laterosporus (PI15) by conventional methods was recognized as B. pumilus by molecular method. Similarly, B. subtilis (PI11) isolate was identi ed 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 [7]. In addition, the molecular identi cation 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 identi cation of Bacillus sp. microscopically. These observations promptly suggests that identi cation 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 identi cation of unculturable bacteria
The 16S rDNA gene was successfully ampli ed 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 con rmed the presence of desired inserts, when 16S rDNA fragments were ampli ed using insert speci c primers. These clones were grouped into different categories based on their RFLP pro le. Thirty nine sequence of clones obtained in this culture independent molecular identi cation study of predigester were deposited with the Genebank (Table 4). Figures 3 and 4 indicate representative RFLP pro les of PCR ampli ed 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 identi ed. 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 identi ed. 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 [5].
The predominance of different types of bacterial pro les at different stages of microbiological process has also been observed by other researchers. For this purpose, the PCR-RFLP analysis of 16S rDNA was rst attempted by Hiraishi et al.
[16] to identify methanogenic bacteria from mixed populations of anaerobic sludge. They ampli ed the 16S rDNA fragments of 0.4 kb size from the bulk DNA extracted from anaerobic digester using methanogen-speci c 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 identi ed 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 [17]. These observations were reported to be true not only the anaerobic digesters but also for wastewater treatment systems, where signi cant difference was observed in enriched cultures (depending on the phase of wastewater treatment), irrespective of the microbial pro les of inoculums [18].

Conclusion
The current study indicated that the complicated degradation process that takes place in the predigester of Nisargruna biogas plant is essentially carried out by ten species of the genus Bacillus. The cultures obtained from predigester 1, where the waste is rst collected showed less variation in the Bacillus sp. Here, only four different sp. were found and amongst them B. subtilis and B. pumilus were predominant. In the predigester 2, where partially hydrolyzed waste from predigester 1 is concentrated, showed much more variation in identi ed Bacillus sp., as compared to that in predigester 1. This observation proposes that the rst attack on the complex biomolecules is done primarily by B. subtilis and B. pumilus. Subsequently, the breakdown steps are then taken over by other Bacillus sp., leading to generation of mixtures of acids.   RFLP pro le of non culturable bacteria in predigester slurry using Sau3A digestion (1L-100 bp ladder; 2L -500 bp ladder)