Research Article | Open Access
Hema N. and K.P. Sreenat
Department of Botany, Bangalore University, Bangalore – 560 056, Karnataka, India.
J. Pure Appl. Microbiol., 2018, 12 (4): 2313-2319 | Article Number: 5356
https://doi.org/10.22207/JPAM.12.4.73 | © The Author(s). 2018
Received: 23/09/2018| Accepted: 07/11/2018 |Published: 31/12/2018
Abstract

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.

Keywords

Succinic acid production, 16S rDNA analysis, Phylogenetic tree, Enterobacter cloacae, Bacillus sp.

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 – butyrolactone1. 1, 4, BDO is the largest segment of the succinic acid market. High demand from 1,4, BDO is driving the industrial material segment. The succinic acid application market is  segmented in seven major categories which are 1,4, BDO, Plasticizer, PBS, Solvent & lubricant, Polyols, Pharmaceutical application and food & beverages applications (Fig.1)2. Due to this wide applicability, Bio-succinic Acid Wins “Biofuels Digest, Chemical Of The Year Award” in 2017.

Fig. 1. Applications  of  Succinic acid (Source: Laufuhr-test.info, Images: Succinic acid)

Increasing interest, demand and environmental benefits associated with fermentative succinic acid production called up for the challenge of producing succinic acid economically. Several organisms such as Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, E.coli, Clostridium thermosuccinogenes, Corynebacterium glutamicum, Enterococcus faecalis, Bracteroids fragilis, Enterococcus flavescens, Klebsiella pneumoniae, Saccharomyces cerevisiae, Yarawo lipolitica, Aspergillus niger, Penicillium simplicissium etc were reported for the production of succinic acid3. Among them Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens,  Escherichia coli and Corynebacterium glutamicum  are some of the widely studied strains4.

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 succinogenes5,6, Manheimia succiniproducens7, Anaerobiospirillum succiniproducens8, recombinant Escherichia coli9,10, Corynebacterium glutamicum11,12,13, Saccharomyces cerevisiae14,15,16  were carried out leading to commercialization of the fermentation processes using the mutant strains17,18. Thus all the succinate producers used in industries are isolated and genetically modified for improved production19. 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.

Materials and Methods

Source of the strains
The strains used for the study were obtained by screening more than 90 isolates from various sources for succinic acid production20. Fourteen strains producing succinic acid were obtained. Six high yielding bacterial strains RFC(P3), BS(D), BS(MC), CRF(S), RFC(W), and RFC(C) of the fourteen have been used as isolates from Indian sources for identification studies.

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 Neighbour-joining 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 :

Fig. 2. gDNA and 16S Amplicon QC data

Fig. 3. Phylogenetic tree of RFC(P3)

Fig. 4. Phylogenetic tree of BS(D)

Fig. 5. Phylogenetic tree of BS(MC)

Fig. 6. Phylogenetic tree of CRF(S)

Fig. 7. Phylogenetic tree of RFC(C)

Fig. 8. Phylogenetic tree of RFC(W)

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.

Table (1):
Accession numbers of the newly isolated six strains for succinic acid production.

Isolates
and Source
Identification of isolates through 16S rDNA sequencing and phylogenetic analysis
Code given to Isolates for Submission
GenBank Accession Number
Date of publish in NCBI database
RFC(P3)
Rumen fluid of cow
Enterobacter cloacae
Enterobacter cloacae HMI57
MH570202
11-JUL-2018
BS(D)
Biogas slurry
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens HMI57
MH819555
07-SEP-2018
BS(MC)
Rumen fluid of cow
Enterobacter cloacae
Enterobacter cloacae HEMI057
MK209628
28-NOV-2018
CRF(S)
Rumen fluid of cow
Enterobacter cloacae
Enterobacter cloacae  HIND7557
MK212385
30-NOV-2018
RFC(C)
Rumen fluid of cow
Bacillus subtilis
Bacillus subtilis HMRB715
MK123381
10-NOV-2018
RFC(W)
Rumen fluid of cow
Bacillus subtilis
Bacillus subtilis MHIRFC75
MK156714
18-NOV-2018
DISCUSSION

Of the six succinic acid producing strains, three isolates RFC(P3), BS(MC) & CRF(S) are three different strains belonging to Enterobacter cloacae; RFC(C), RFC(W) & BS(D) are three different strains of  Bacillus species. Among six strains, five are isolated from rumen fluid of cow wherein Enterobacter cloacae being dominant species. Enterococcus flavescens (21) and  Klebsiella pneumoniae (22) are the only strains till date, reported for succinic acid production from Indian sources. None of the strains 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, RFC(C)  – Bacillus subtilis MHIRFC75 identified have not been reported prior for succinic acid production from Indian sources.

Declarations

Acknowledgements
The authors gratefully acknowledge Principal, St. Joseph’s College (Autonomous), Bangalore,  for providing laboratory facilities for the above work. Our heartfelt  thanks  to Dr. Purushothama, Professor, Department of Botany, St Joseph’s College for his guidance and support. We are also thankful to Dr. Thamizhseran,  Professor, Department of Botany, for his help during the research investigation.

Conflict Of Interest
The authors declares that there is no conflict of interest.

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