Molecular Identification of Bacterial Strains Producing Succinic Acid from Indian Sources

The present study includes molecular characterization of six bacterial strains isolated for succinic acid production from Indian region. RFC(P3), BS(D), BS(MC), CRF(S), RFC(W) and RFC(C) are the strains which were identified by 16S rDNA sequencing and phylogenetic analysis. All the strains have been proved to be newly isolated strains of the species through genbank accession number from NCBI. RFC(P3) Enterobacter cloacae HMI57, BS(D) Bacillus amyloliquefaciens HMI57, BS(MC) Enterobacter cloacae HEMI057, CRF(S) Enterobacter cloacae HIND7557, RFC(W) Bacillus subtilis HMRB715 and RFC(C) Bacillus subtilis MHIRFC75 are the identified six organisms to be first reported for succinic acid production from Indian sources.


INTRODUCTION
Bio-based products are gaining wide acceptability and demand due to its sustainable benefits. Succinic acid has been proved to be one such bio-based chemical that can be produced through fermentation using renewable feedstock. Due to increased prices of limited petroleum feedstock for chemical production and its harmful effects, biological production of succinic acid wherein green house gas carbon dioxide is utilized, has attracted researches worldwide from past two decades.
According to report on "2018-2023 Bio-Succinic Acid Market Global Key Player, Demand, Growth, Opportunities and Analysis Forecast" by Wise Guy Reports.com the Bio Succinic Acid Market is expected to grow at CAGR of 23.2% from 2018 to 2023.
Succinic acid, one of the most important platform chemicals is used as a precursor for many industrial important chemicals such as adipic acid, 1,4-butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, succinate salts and gamma -butyrolactone 1  Strain capability, feedstock cost and fermentation technology are the key aspects for biological succinic acid production. This led to massive developments in the last few decades with respect to strain improvement, advancements in fermentative technologies, efforts to utilize various waste substrates efficiently to meet the needs for commercial scale production in an economically feasible way. Among which, strain development plays a prominent role in enhancing the efficiency and reducing the costs of the overall costs of the bioprocess. This has been accomplished by metabolic engineering of the organisms for maximizing succinic acid yield and minimizing the by-products.
Metabolic engineering of succinate producing strains such as Actinobacillus succinogenes 5,6 , Manheimia succiniproducens 7 , A n a e r o b i o s p i r i l l u m s u cc i n i p r o d u c e n s 8 , recombinant Escherichia coli 9,10 , Corynebacterium glutamicum 11,12,13 , Saccharomyces cerevisiae 14, 15,16 were carried out leading to commercialization of the fermentation processes using the mutant strains 17,18 . Thus all the succinate producers used in industries are isolated and genetically modified for improved production 19 . Isolation of new strains opens up more opportunities for technology development for succinic acid production. Owing to this fact as objective, a study focusing on screening for isolation of new strains for succinic acid production was carried out. The present work reports the results of characterization of the isolated six strains which has wide opportunities to be explored for its industrial applications.

Source of the strains
The strains used for the study were obtained by screening more than 90 isolates from various sources for succinic acid production 20 .

Molecular characterization and phylogenetic analysis
The DNA was isolated from the culture isolates. Its quality was evaluated on 1.0% Agarose Gel, a single band of high-molecular weight DNA has been observed. Fragment of 16S rDNA gene was amplified by 27F and 1492R primers. A single discrete PCR amplicon band of 1500 bp was observed when resolved on Agarose gel. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forward primer and reverse primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rDNA gene was generated from forward and reverse sequence data using aligner software.
The 16S rDNA gene sequences were compared with existing sequences in the NCBI database using the BlastN program.. Based on maximum identity score sequences were selected and aligned using multiple alignment software program Clustal W. Phylogenetic tree for strain RFC(P3) with different species of Enterobacteriaceae was constructed by Neighbourjoining method from 100 bootstrapping replicates using MEGA 5 software. Distance matrix of the other strains were generated and the phylogenetic tree was constructed by Neighbor-joining method from 1000 bootstrapping replicates using MEGA 7 software.

RESULTS
The molecular characterization of all the six strains carried out through 16S rDNA sequencing and phylogenetic analysis lead to the nucleotide sequences and construction of the phylogenetic tree of strains as given below : gDNA 16S PCR amplicon Ladder specification  Distance matrix for sequences producing significance alignment were obtained for RFC(P3), BS(D), BS(MC), CRF(S), RFC(C) and RFC(W). Based on the greatest homology which is indicated by high score and low expect value the best match for the strains were found. The DNA sequence of RFC(P3) matches best with that of Enterobacter cloacae ATCC 13047. Sequence of BS(D) showed best with Bacillus amyloliquefaciens strain NBRC 15535 with 98% similarity. Enterobacter cloacae strain TLS20 was the best match when compared with DNA sequence of BS(MC). Similarly CRF (S) showed best match with Enterobacter cloacae strain SBP-8 showing 99% similarity. The DNA sequences of the strains RFC(C) and RFC(W) matches best with Bacillus subtilis strain NBRC 13719 and with Bacillus subtilis strain DSM 10 respectively showing 99% similarity.
None of them showed 100% similarity to any of the strains in distance matrix generated. Thus the nucleotide sequences of the newly isolated strains were submitted to NCBI Genbank submission portal and accession numbers were obtained for all the six strains i.e RFC(P3), BS(D), BS(MC), CRF(S), RFC(C) and RFC(W). The details of the six strains isolated, source of isolation, identification, accession number obtained from NCBI Genbank and date of publish of the same in NCBI database has been summarized in Table 1.