Molecular Identification of Isolated Bacteria from Soils in Likouala Peat Bog Area, Republic of Congo ()
1. Introduction
The research of microorganisms in peatlands has been of increasing interest in the last five years, with the aim of understanding their influence in the functioning and stability of peatlands and the interaction between microbial activities and the effects of climate change. Several studies carried out on the role of peatlands in the global carbon cycle in the face of global warming show a rise in temperatures leading to a strong degradation of organic matter following an increase in microbial activity, resulting in a significant release of CO2 and carbon fixation by photosynthesis [1] . Microflora studies have revealed the presence of high amounts of molds in the peat with concentrations of molds close to 107 per gram of dry peat; up to 23 species of yeasts belonging to the genera Trichosporon, Candida, Rhodotorula and others [2] , and other species of pathogenic mycobacteria such as Mycobacterium fortuitum [3] . Although the study of the diversity of microorganisms in peatlands is the subject of growing interest, the processes maintaining this diversity and its role in the functioning and stability of peatlands remain to date very little explored. This lack of knowledge is even greater if we consider the interaction between the effect of diversity and the effects of climate change [4] . Indeed, despite advances in knowledge of current carbon storage, there are still considerable knowledge gaps on the microbiology of these ecosystems [5] . The best understanding lies mainly in the ability to better characterize communities of microorganisms both at the taxonomic and functional level. To date, in the Republic of Congo, no identification of microbial strains has been carried out on the microorganisms of the peat soils of Likouala. In order to assess the evolution of its quality and its impact on the environment, we proposed to identify after having characterized the bacteria contained in the soils of the Likouala peat bog area.
2. Materials and Methods
2.1. Materials
The biological materials used in this study were three (3) composite soil samples collected in the Likouala peat bog area. A total of 9 soil samples collected. We used the samples collected according to [6] . After collection of soils samples, all the samples were transferred to a molecular microbiology Laboratory for analyses.
2.2. Methods
2.2.1. Enumeration and Isolation
The samples were cultured on Mossel media and TSB enriched with petroleum and vegetable oil [7] [8] . After culture, the colonies were purified on Mossel and TSB enriched. A colony is removed using a loop or Pasteur pipette using the striation technique to obtain isolated colonies. The dishes were incubated in an oven at 37˚C, and then observed after 24 h. The isolates were considered pure and then stored in sterile Eppendorf tubes containing 900 µl of liquid TBS and 100 µl of glycerol, then kept cool at −20˚C [9] .
2.2.2. Phenotypic Characterization of Isolates
The phenotypic characterization of the strains was carried out by applying classic microbiology techniques based on the search for phenotypic characters: cultural characters, morphological characters (colony and cell morphology), Gram type and catalase production [10] [11] .
2.2.3. Molecular Identification
1) DNA extraction
The extraction was done using the Qiagen kit whose execution was carried out as indicated by the manufacturer.
2) Evaluation of DNA concentration
DNA concentration and purity were measured using a Biorad spectrophotometer, using the A 260/A280 ratio to assess protein contamination in the DNA solution DNA electrophoresis was performed on 1% agarose gel [12] .
3) PCR amplification of the 16S rRNA gene
a) Primers design
Universal primers were used to amplify genes encoding 1500 bp ribosomal RNA [12] . See in Table 1.
b) Mix and PCR conditions
The PCR reaction was carried out in a final volume of 50 μL according to the conditions in Table 2. 1 μL for each primer and 5 μLfor the buffer, all concentrations of all PCR mix are given in Table 2.
The PCR amplification was carried out in a Thermocycler as follows: first
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Table 1. Universal primers for amplification of 16S RNA.
initial denaturation at 95˚C for 5 min; 30 cycles for each of the following steps; denaturation at 95˚C for 30 seconds; Hybridization was carried out at 55˚C for 30 seconds; elongation at 72˚C for 1 min 30 s, and a final elongation at 72˚C for 5 min, followed by storage of the PCR fragments (amplicons) at 4˚C.
c) DNA Electrophoresis and Sequencing
DNA electrophoresis was performed on 1% agarose gel using TBE buffer DNA was revealed with Syber green then visualized using a computer combined with a band visualization device of the Gel Doc EZ imager type (UV device). The size of the DNA amplicon is estimated using a molecular weight marker. For sequencing, the sequence library was prepared according to the Oxford Nanopore Technology protocol. Sequences were purified with the AMpurex kit, then quantified using the spectrophotometer, normalized, coded, and sequenced using the GRiDION Oxford Nanopore tool.
2.2.4. Results Analysis
The results were represented, illustrated and statistically analyzed by Microsoft Excel 2016 software. For the Bioinformatics analysis, all the sequences were the subject of an in-silico study in BLASTn which revealed allowed us to identify the eight sequences and two homologues from the bank. The comparison of the partial nucleotide sequences of the gene encoding 16S rRNA using the GenBank portal by alignment allowed us to obtain identification rates that vary from 96.75 to 100%. Using percentage of similarity, E value, ten sequences were aligned with ClustalW and we the inference phylogenetic tree was performed with PhyML [8] . The eight new sequences were definitely submitted to GenBank.
3. Results
3.1. Enumeration
Table 3 shows the bacterial loads of samples 1, 2 and 3 on Mossel medium. The total load of bacteria of the Bacillus genus depends on the sample. Samples 1 and 2 present loads of (5.81 ± 1.08) × 104 and (6.64 ± 1.94) × 104 CFU/g respectively against the load of sample 3 where it is (8.56 ± 1.19) × 103 CFU/g.
Table 4 presents the bacterial loads on TSB medium enriched with petroleum
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Table 3. Loads (CFU/g) of bacteria of the Bacillus genus in the samples.
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Table 4. Loads (CFU/g) of hydrocarbonoclasts bacteria of sample 2.
and vegetable oil mainly with the sample 2 because of its load on Mossel medium. The bacterial load is higher with the TSB medium enriched with petroleum with (2.12 ± 4.1) × 108 CFU/g against (8.15 ± 10.1) × 107 CFU/g with the TSB medium enriched with vegetable oil.
3.2. Isolation and Characterization
Table 5 shows the phenotypic characteristics of the sequenced strains. Microscopic examination reveals the rod-shaped bacteria, the majority of which were Gram positive (Gram+) and catalase positive. These 10 isolates were selected and identified by molecular biology.
3.3. Molecular Identification by 16S rDNA Analysis
3.3.1. Extracted DNA Concentrations
After extraction of the genomic DNA, the concentration and purity of the extracts were evaluated using the spectrophotometer. DNA purity is acceptable when the A260nm/A280nm ratio is between 1.8 and 2.0 [13] . If R is less than 1.8, contaminating proteins are probably available in the solution. A value greater than 2 indicates probable RNA contamination. The A 260nm/280nm ratio varies from 1.86 to 2.09 and varies from one DNA extract to another, because it depends on the sequence composition. The resulting results are in Table 6.
3.3.2. Extracted DNA Electrophoresis
After extraction and quantification of DNA extracts, electrophoresis on 1% agarose gel. was made. Of the 10 isolates, 9 bands were observed and one band was not observed. These results are shown in Figure 1.
3.3.3. Electrophoresis of PCR Fragments
The identification of the isolates, by amplification of the purified 16S gene revealed by DNA bands of approximately 1500 bp as indicated in Figure 2.
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Table 5. Phenotypic characteristics of the isolates.
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Table 6. DNA concentration and purity after extraction.
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Figure 1. 1% agarose gel electrophoresis of genomic DNA extracts from isolates.
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Figure 2. Electrophoresis on a 1% agarose gel of the PCR fragments.
3.3.4. Bioinformatics Analysis of 16S rRNA Sequences
The identification of the different isolates by 16S rDNA sequencing made it possible to differentiate the genus and the species of the isolates. A total of 10 sequences corresponding to the ten isolates were identified, eight of which could be submitted to GenBank. Table 7 indicates the various sequences and corresponding isolates.
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Table 7. Correspondence of isolate codes and strains identified by 16S rRNA.
3.3.5. Multiple Sequence Alignment of Identified Strains with Some GenBank Homologs
The sequences of the identified strains were aligned in order to analyze the distance and the rate of similarity between strains and to understand the events that occurred during evolution. Similarly, these analyzes make it possible to test the efficiency of the 16S rRNA gene to discriminate between strains sharing a similar polymorphism. Figure 3 illustrates the multiple alignment of the studied sequences and their homologs. A strong sequence similarity was observed between the three strains of B. thuringiensis (GEB22, GE3B22 and GE4B22) and the homologous strain B. thuringiensis (B.t10). This observation is the same between the two strains of P. megaterium (GE7B22 and zs-3); the two strains of B. subtilis (GE9B22 and MK736123); the two strains of Entobacter and between the strains of B. cereus; B. anthracis.
3.3.6. Phylogenetic Inference
Figure 4 shows the dendrogram of the sequences of the identified strains and their homologs obtained from GenBank. The resulting phylogenetic tree is divided into two clusters; Enterobacteriaceae species on the one hand and Bacillus species on the other. The species composing each cluster have a strong similarity between them.
4. Discussion
This work aimed to the molecular identification of bacteria isolated from samples soil of the Likouala peat bog area (Congo-Brazzaville). Counting the bacteria on Mossel medium reveals bacterial loads of (5.81 ± 1.08) × 104 and (6.64 ± 1.94) × 104 CFU/g for samples 1 and 2 respectively. Whereas, sample 3 presented a load of (8.56 ± 1.19) × 103 CFU/g (Table 3). The count of sample 2 on the TSB medium, enriched with petroleum and vegetable oil, shows higher bacterial loads than on the Mossel medium. Indeed, on the TSB medium enriched with
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Figure 3. Part of Multiple sequence alignment of identified isolates with their homologs.
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Figure 4. Phylogenetic tree of sequences in this study and their homologs.
petroleum, the bacterial load is (2.12 ± 4.1) × 108 CFU/g and (8.15 ± 10.1) × 107 CFU/g on the same medium enriched with vegetable oil. These results suggest that the total load of bacteria of the genus Bacillus and hydrocarbonoclasts is a function of the sample, the medium and in varying concentrations of the used source of carbon. These results are close to those obtained by [14] with samples soil of Brazzaville where the bacterial load after counting was around 9.3 × 104 CFU/g. The microscopic and macroscopic examinations made it possible to distinguish variable characteristics. Two forms of bacteria were characterized: cocci and rods which were more abundant, mostly gram positive (Gram+) and catalase positive thus suggesting bacteria of the genus Bacillus. These results corroborate those of several studies which have already shown that bacteria of the Bacillus genus can be isolated from food and soil [15] [16] . In order to carry out the molecular identification of bacteria isolated from peat bog soils; we ensured the efficiency of DNA amplification by different PCR reactions allowing to amplify different fragments up to 1500 bp. The 260/280 ratio was determined, which made it possible to detect contamination of the nucleic acids by proteins. Its value varied between 1.8 and 2.0 for DNA and between 2.0 and 2.2 for RNA. The A260nm/A280nm ratio varied from one fragment to another, respectively from 1.74 to 1.88 because it depends on the sequence composition, these results are in accordance with the statement of Jay A. [13] . Most sequences showed 99% similarity to the closest sequences already presented in NCBI databases. The more significant the alignment score, the results are expressed as a percentage of similarity of the strains identified for our study with the closest species with regard to the intervals. In Figure 3 the bloc from 841 to 854 is showing the conserved region of ADNr16S, all identified sequences and homologs present a highly this conserved region. This evidence corroborates the postulate in which all procaryotes specially bacteria can be identified by the sequencing of the conserved region of ADNr16S. Authors [8] [17] have already found the same results. When watching the bloc [855 - 861] of Figure 3 the two sequences of Enterobacter sp are different from those of all Bacillus strains. Phylogenetic Inference has confirmed the phylogeny of reference which is well established between the Bacillus strains and the Enterobacter sp strains. The phylogenetic tree dispays two monophyletic groups, these results have been already found by others authors [17] [18] [19] .
PCR amplification and then sequencing of the gene encoding rRNA 16S identified eight (08) species of bacteria including three (3) species of Bacillus thuringiensis, Bacillus cereus, Priestia megaterium, Bacillus antracis, and Bacillus subtilis and one species Enterobacter sp., isolate whose species could not be determined because of the low rate of similarity, this observation has already been made by other auhors [8] . The association of other more discriminating molecular markers in addition to the gene encoding 16S rRNA is possible in the future in order to assess the microbial biodiversity colonizing peatland ecosystems.
5. Conclusion
This study contributes to the molecular identification of bacteria isolated in soil samples constituting the surface part of the peat bog in the Likouala area in the Republic of Congo. The results of the microbiological analyses of this present study showed that the soils of the peat bogs of the Likouala zone are rich in bacteria of the Bacillus genus. PCR amplification, followed by sequencing of the 16S rRNA gene allowed the identification of eight (8) species of bacteria including six (6) of the Bacillus genus: Bacillus thuringiensis, Bacillus cereus Bacillus thuringiensis; Bacillus thuringiensis; Bacillus anthracis; Bacillus subtilis; a strain of Enterobacter sp and a strain of Priestia megaterium. This present work constitutes a scientific support in the study, the understanding and the interaction of the bacterial diversity colonizing the soils of the peat bogs in the Republic of Congo.
Acknowledgements
We were grateful to the members of the molecular and cellular biology Laboratory of the Faculty of Sciences and Techniques for providing used laboratory materials.