The chewed waste of RPW (Figure 1) was used in all the experiments those were collected (28th December 2017) from trunk of infested date palm plantation located in Hoorat A’ali, Northern Governorate, Kingdom of Bahrain (Latitude: 26.167612 Longitude: 50.527532). Collected chewed materials stored at 4˚C for analysis.

One gram of chewed waste was homogenized in 10 ml sterile distilled water following three different serial dilutions for plating in two different culture media; potato dextrose agar, Sigma Aldrich (PDA) and Luria-Bertani Agar, Difco^TM Miller (LBA).

Inoculum from three different dilutions was plated on PDA and LBA media. All plates were incubated at 37˚C for 72 hrs. Different bacterial colonies were clearly observed on all the plates (Figure 2(a)). Bacteria from the individual colony were isolated and sub-cultured separately in corresponding PDA and LBA media overnight (Figure 2(b)).

Bacteria from each individual colony were further allowed to grow in LB broth and incubated on shaker at 37˚C at a speed of 150 rpm for overnight growth. Culture from each isolate was stored in 50% glycerol and stored in −80˚C for further use.

At the first step of identification, all of the bacterial isolates were examined morphologically using standard Gram staining technique. Stained slides of bacteria were differentiated as Gram positive/negative class of bacteria using microscopic technique under (100×) with oil immersion.

DNA was isolated from freshly grown overnight culture. The genomic DNA was isolated using kit (XG-2411-00), following the manufacturer’s instructions. The purity of the extracted DNA was confirmed by 1% agarose gel by visualization of DNA bands.

Isolated DNAs of all bacteria were amplified with 16S rRNA specific universal primers 8F and 1492R (Table 1). The amplification was carried out in ABI Veriti

Thermal Cycler at initial denaturation 94˚C for 3 min, 35 cycles of 94˚C 30 sec, 52˚C 30 sec, and 72˚C 1 min, and a final extension at 72˚C for 7 min (Table 2). 1.2% Agarose gel was used to check the PCR amplified products. After visualization of amplified products under UV light using the gel documentation system (Zenith Biosciences, Figure 3(a), Figure 3(b)) were processed further for sequencing.

*Bi-directional sequencing.

PCR amplicons were purified and sequenced using BDT v3.1, cycle sequencing kit on ABI 3730xl Genetic Analyzer (Applied Biosystems, USA), bi-directionally using 8F and 1492R primers. The sequencing used was set as 95˚C for 3 min and 25 cycles of (95˚C for 5 sec, 55˚C for 5 sec, and 60˚C for 4 min). Sequences were aligned using Codoncode aligner.

The obtained 16S rRNA gene sequences of each isolate were used to carry out the BLAST analysis in NCBI Genbank database. Based on the maximum identity score, using multiple alignment software program ClustalW, sequences were compared. The 16S rRNA gene sequences of successful isolates were deposited in GenBank (Table 4).

RPW feeding waste contained an average of 1.2 - 1.9 × 10⁹ CFU/ml of bacteria in the serially diluted culture used for study. Twenty four culturable bacterial isolates were studied by Gram staining, among which 16 were Gram-positive (Figure 4(a)) and 8 were Gram-negative (Figure 4(b)). Gram-positive bacteria appeared as a purple-colored stain (Figure 4(a)) by taking crystal violet stain after washing, due to thick peptidoglycan layer of their cell wall. On the other hand, Gram-negative bacteria cannot retain the crystal violet stain after washing, due to their much thinner and sandwiched nature of peptidoglycan layer of their cell wall (Figure 4(b)).

DNA were isolated and PCR amplified from all the culturable bacteria using 16S rRNA specific universal primers 8F and 1492R for identification as mentioned in

Table 1. The PCR amplification was carried out following the conditions as mentioned in Table 2. 1.2% Agarose gel showed a single amplified DNA products of 1500 bp of all different bacterial isolatesas shown in Figure 3(a) and Figure 3(b) (only two different bacteria has presented). But, the bidirectional DNA sequencing products of PCR amplicons (1500 bp) of different bacterial isolates varied in sequencing results from 1316 bp to 1461 bp and showed in Table 3. Bacterial DNA isolated from chewed waste of RPW was identified using 16S rRNA gene sequence by BLAST alignment in NCBI site. The success of obtaining of sequence was 91.66% in this study. Bacteria were identified based on maximum sequence identity level in GenBank. Finally, 79.16% bacteria were identified using the BLAST program in reference to GenBank accession numbers (NCBI) which have been presented. The level of sequence identity percent varied from 94 to 100.

The 16S rRNA gene sequence of identified bacteria has been submitted to NCBI GenBank and their sequence size, name and accession numbers have been presented in Table 4. The comparison of sequences with the global multiple sequence alignment (MSA), and the identification and submission of sequences in NCBI GenBank were adopted in this study. Sixty six percent of the identified bacteria were Gram-positive while the rest of them were Gram-negative. In the study, 86% of the bacteria were of different Bacillus species of different strains (Phylum: Firmicutes) and 14% were other bacteria (Achromobacter xylosoxidans strain MPF-B6, Acetobacter ghanensis strain MPF-C5, Vibrio alginolyticus strain MPF-D3). The Gram-positive Bacillus spp. (Family, Bacillaceae) belongs to Phylum Firmicutes and Gram-negative bacteria were of Proteobactera from three different families. The molecular phylogenic affinity was analyzed using sequences of 16S rRNA and a phylogenetic tree was constructed in Figure 5. The

phylogenic relationship and evolutionary history was drawn using neighbor-joining method [27] in MEGA7 software [28]. According to the 16S rRNA gene sequences, B. amyloliquefaciens strain MPF-C2, B. siamensis strain MPF-C3, B. methylotrophicus strain MPF-C1, B. tequilensis strain MPF-A2, B. wiedmannii strain MPF-B1a, all showed closeness and came in one clade in the phylogenic tree. The analysis of pairwise distance data and evolutionary divergence between the sequences uses the Kimura-2-parameter following transitions and transversions of the codons as mentioned in program. It showed 67%, 70% and 70% distance between B. amyloliquefaciens and the sequences of V. alginolyticus strain MPF-D3, Acetobacter ghanensis strain MPF-C5, Achromobacter xylosoxidans strain MPF-B6 respectively within their evolutionary history.

The phylogenetic relationship was studied using 16S rRNA gene sequences, the Gram-negative bacteria came in one clade while Gram-positive different Bacillus sp. of different strains showed evolutionary closeness to each other and accordingly, they came in one major clade under three different sub-clades in the phylogenetic tree (Figure 5).

The identification of bacteria was done by using 16S rRNA gene sequence analysis using NCBI site with comparison (Table 3 and Table 4). Comparison of the bacterial 16S rRNA gene sequence has characterized as a preferred genetic technique to identify poorly described, rarely isolated, or phenotypically aberrant strains of bacteria from any source [29] [30]. Sequence identity searching, specifically using the BLAST program in NCBI, is the most widely used and most reliable, for identifying and characterizing newly determined sequences with great reliability [31]. Among the culture-dependent isolated bacteria, 80% were identified by comparing 16S rRNA gene sequence in NCBI database, using BLAST. Among the identified bacteria, 85% were Gram-positive while the rest of them were Gram-negative. A high abundance of bacteria from the Bacillaceae family of Firmicutes phylum and sixteen different species of Bacillus were identified in comparison with NCBI GenBank. Other Gram-negative bacteria belong to Phylum Proteobacteria of three different families.

The highest percentage of identified bacteria was of different Bacillus sp. of different strains that were present in feeding waste of RPW. There are several reports on RPW gut microbiota where mostly Bacillus sp. together with other bacteria observed. Those bacteria play an important role in RPW’s digestive system, nutrient absorption, degradation of lignocellulose, and their survival [13] [23] [32]. Several Bacillus sp has great medical, biotechnological and economic importance [33]. They are resistant to heat, radiation, chemicals, oxidizing agents and desiccation, due to spore production, which is an important condition for their adaptation to different ecological niches [34]. The identified organism B. tequilensis strain MPF-A2 showed a 99% sequence identity with B. tequilensis strain HBUM07078 and Bacillus subtilis strain 5723. Similarly, B. tequilensis sp. nov., showed close realation with Bacillus subtilis [35]. In another report, B. tequilensis GYLH001 regarded as endophytic bacteria and was used as biocontrol agent of fungus Magnaporthe oryzae causing rice blast [36]. Moreover, Bacillus altitudinis P-10 worked as potential bioprotectant against Xanthomonas oryzae pv. oryzae, isolated from rice rhizosphere in Java, Indonesia [37]. B. altitudinis, B. xiamenensis and B. pumilus are the phenotypically and genotipically very closely related species and they produce acid from cellobiose, glucose and mannose [38]. B. aerius and B. stratosphericus are recognized as enzyme producers with probiotic activity inindustrial uses [37]. Several Bacillus sp. viz. B. megaterium, B. laterosporus and B. sphaericus were isolated from natural habitats associated with RPW insect-damaged date palms in Egypt and their bioassay showed 40% - 60% killing of RPW larvae in feeding study [23]. Considering the huge economic and environmental damage caused by RPW, some attention has been paid to the efficacy of different chemical and biocontrol strategies [39] in regard to several bacterial species to use as biological control [10] [20].

In the phylogenetic study, the Gram-negative bacteria came in one clade while Gram-positive Bacillus sp. showed evolutionary closeness to each other and accordingly they came under one main clade with three different sub-clades in the phylogenetic tree.