Background

Polyhydroxyalkanoates (PHA) are macromolecules produced from microbiota by consuming different carbon sources. These are ecofriendly and can be used to make biodegradable plastics. Degradable polymeric biomaterials are preferred because of their biological, biomechanical, and physiochemical properties. For a few decades, petroleum-based plastics have been produced and its resistance to biodegradation has created a severe environmental challenge for solid waste management [1]. Biotechnological research is increasingly focusing on the creation of biodegradable polymers as an alternative to petrochemical plastics which are still in wide usage [2].

Over the past few decades, researchers have been working on developing biodegradable polymers using various marine microbial species because of their potential to reduce fermentation and recovery costs of biopolymer. Microbial polymers have the potential to mold into an application, there are already many intriguing applications in the medical and other domains [3, 4]. Microbial bioplastics made up of polylactic acid (PLA) and polyhydroxyalkanoates (PHA) are completely degradable and provide a variety of unique material properties and commercial opportunities [5].

PHAs are the most promising biopolymers because they are nontoxic, biodegradable, and biocompatible and have qualities similar to traditional plastics [6]. PHAs are the polyesters of various hydroxyalkanoates synthesized by microorganisms such as marine actinomycetes, algae, and halophile bacteria. They are strong candidates as biodegradable polymers because they are completely degraded into water and carbon dioxide by microorganisms when they are discarded into nature [7]. Polymer ranges in number of carbons (4 to 14 atoms) and the sort of monomeric units determines structurally whether they are homopolymers or heteropolymers [8]. In circumstances like nutritional imbalance and in stress, the bacterial cells synthesize PHA as stored energy granules in its cytoplasm as a part of defence mechanism [9]. PHAs are implicated in medical, pharmacy, agriculture, food processing, and paint industries [10]. Poly-3-hydroxybutyrate (P3HB or PHB), the most well-known type of PHA, accumulates in many microbes by binding hydroxybutyrate monomers with ester bonds. They are completely linear isotactic structure with 60–70% crystalline content [11]. The analytical studies on physiochemical properties reveal the nature and structural composition of the PHA derivatives.

Marine bacterial species such as Vibrio harveyi [12], Micrococcus species [13], Halomonas elongate [14], and Bacillus megaterium [15] have been found to be potent producers of PHB. Brachybacterium paraconglomeratum is a species of Gram positive, non-motile, facultative anaerobe, yellowish brown pigmented bacterium which belongs to the phylum Actinobacteria. The cells appear coccoid during the stationary phase and irregular rods during the exponential phase. Although B. conglomeratum and B. paraconglomeratum are similar in their physiological characteristics, they can be differentiated on the basis of their cellular fatty acid patterns and low levels of DNA-DNA hybridization. To date, only a few reports on the commercialization of PHAs produced by marine microbes and halophiles are available. As a result, the current study was undertaken to determine the strain’s novelty in producing biopolymer.

Methods

Isolation and screening of PHA-producing bacteria

Bacterial strains are isolated from the mangrove region of Bay of Bengal, Suryalanka, India. The collected soil samples were transferred to laboratory and stored at −20°C until further use. The soil samples were subjected for serial dilution; initial isolation was done on nutrient agar medium with dextrose. PHA producers were further screened on minimal media, the composition includes dextrose 10.0g/L, ammonium sulfate 1.0g/L, dipotassium phosphate 7.0g/L, monopotassium phosphate 2.0g/L, sodium citrate 0.5g/L, magnesium sulfate 0.1g/L, and final pH (at 25°C) 7.0±0.2 and incubated at 37°C.

Presumptive test

Isolation and detection for PHA producers using lipophilic stain Sudan black B is a presumptive test [16]. Stain was prepared by dissolution of 0.3 g of dye in 100 ml of 70% ethanol. Stain was flooded on the colonies in selective medium; after 30min, the excess dye was decanted and washed with 100% ethanol, and colonies appear bluish black and accredited as PHA-positive strains.

Confirmative test

A selective stain, Nile blue A, fluorescent dye is used for the detection of PHB granules [17]. Dye at 0.5μg/ml of selective media was composed and sterilized. The initially screened isolate M4 was streaked on the plates and incubated at 35°C for about 96h. As a positive mark, the colonies emit orange yellow fluorescence under a UV transilluminator.

Biochemical characterization

The series of biochemical tests were done for all the isolates according to Bergy’s manual, and the activities of M4 were tabulated.

Genomic profiling of bacteria

The Zymo research (ZR) fungal/bacterial DNA Mini Prep kit was used to isolate genomic DNA from the M4 pure culture. The 16S rRNA gene was amplified by PCR using universal primers 27F AGAGTTTGATCCTGGCTCAG, 357F CTCCTACGGGAGGCAGCA and 786R GATTAGATACCCTGGTAG, 1492R TACGGYTACCTTGTTACGACTT respectively. The PCR conditions for amplification of the 16S rRNA gene were as follows: 5 min of initial denaturation at 94°C, followed by 35 cycles of 1min denaturation at 94°C, 1 min annealing at 56°C, 2min extension at 72°C, and 10 min final extension at 72 C. On a 1% agarose gel, the PCR product was visualized. QIA quick Gel Extraction Kit was used to gel elute and purify the PCR amplicon. The Sanger DNA sequencing method was used to sequence the purified PCR product [14]. Finch TV software version 1.4 was used to visualize and analyze the obtained sequences. The nucleotide sequences of the 16S rRNA gene were compared using the BLAST tool in NCBI and EzBiocloud (http://www.ezbiocloud.net).

Fatty acid methyl ester analysis (FAME) of M4

The extraction and analysis of the fatty acid methyl ester profile of M4 was done using GCMSQP2010, SHIMADZU. First, the isolate was grown on Nutrient Agar for 24 h at 35 °C. Forty milligrams of culture was transferred to a Teflon-lined, screw-caped tube and mixed with 1 ml of saponification solution containing 3.75 M NaOH in MeOH:H20 to extract whole lipids. The test tube was vortexed for 5 min and placed in a water bath at 100°C for 25min. Following the incubation period, the test tubes were cooled immediately. Then, 2 ml of methylation solution containing 6.00 M HCl: MeOH (1: 0.85) was added, vortexed, and incubated for 10 min at 80 °C in a water bath before being immediately rinsed with cool tap water. Following that, 1.25 ml of the extraction solution (1:1) hexane: methyl-tert butyl ether was added to the extract. Three milliliters of the washing solution containing 0.3 M NaOH was added and gently mixed for 5 min to remove acidic residual reagents. Finally, the organic phase was transferred from the glass tube to a vial for automated sample injection with the carrier gas alone. Internal standard was prepared to a concentration of 1mg/ml from 10mg of heneicosanoic acid methyl ester (C21:0) [18]. For about 3 h, the column runs at high temperature. The obtained data was searched in the NIST107.LIB Library.

Extraction of PHA

The isolate M4 was cultured in dextrose-rich and nitrogen-deficit minimal media for about 96h at 35°C at constant 150rpm. Now 5ml culture was taken and centrifuged at 10,000rpm for 10min. The supernatant was discarded the pellet was treated with 2.5ml of 4% sodium hypochlorite for digestion and 2.5ml of hot chloroform, incubated at 37°C/H. Three different phases were observed the upper phase contains sodium hypochlorite was discarded, middle phase contain chloroform with cell debris, and the bottom phase PHA with chloroform was collected, further followed by extraction with hot chloroform and precipitated with 1:1 ethanol and acetone. The precipitate was evaporated to attain PHA crystals or powder [16].

Crotonic acid assay

This was done according to the procedure described by Law and Slepecky. The extracts of PHA were treated with con.H2SO4 and heated at 100°C for 10min; as a result crotonic acid was formed. The concentration of crotonic acid was observed at 200 to 600 nm in a UV spectrophotometer [16].

GC-MS confirmation of PHA

The final extract is prepared for GC-MS. Methanolysis method described by Juengert et al. [11] was chosen for the GC–MS analysis. In a screw-capped glass bottle (20 mL capacity) with a polytetrafluoroethylene cap, 10 mg of extracted PHB, 1 mL chloroform, and 1 mL acidified methanol (15% methanol in H2SO4) are mixed, heated in a water bath at 100 °C for 2 h. After incubation, the bottle was filled with 1 mL chloroform containing an internal standard 0.2% methyl benzoate v/v and 1 mL deionized water for phase separation. The bottom organic phase was collected and dehydrated with anhydrous Na2SO4, and an aliquot of 1μl was injected into the Shimadzu GC–MS QP2010S gas chromatograph, which was equipped with a Rxi-5Sil MS (30 m × 0.25 mm × 0.25 μm).

Physical parameters

Thermogravimetric analysis (TGA)

The analysis was carried out with an indium-calibrated TGA apparatus (Mettler-Toledo, TGA/SDTA 851, Columbus, OH, USA). About 5 mg of dried extract was used in the study. The analysis was conducted by increasing temperature from 30 to 400°C at 10°C/min under nitrogen flow [19].

Viscosity

The viscosity was measured for culture broth as well as for the extracts of M4 according to the Lovis method using a rolling ball viscometer (Antan paar, Germany). The capillary tube was filled with the sample and allowed to measure the viscosity at 37°C.

Results

Isolation and screening of PHA producers

The mud samples were collected from the estuary (15.858952 N, 80.511521 E) at coastal region of Bay of Bengal, Suryalanka, Bapatla, South India. Ten bacterial isolates with varied features in size shape color were identified and cultured on minimal media, only six isolates were found to be active on minimal media. On this selective media, M4 shows peculiar features like color (Fig. 1) and irregular cell shapes during differential staining. The M4 isolate was tested for PHA granules using Sudan black B stain in presumptive test. The colonies remain bluish black color (Fig. 2) after washing with 99% alcohol. The isolate M4 was cultured on Nile blue A agar plates, and after 3days of incubation at 37°C, the colonies emit orange yellowish (Fig. 3) fluorescence under UV transilluminator. The colonial features of isolate M4 are described in Table 1.

Fig. 1
figure 1

Slimy yellowish colonies of Brachybacterium paraconglomeratum MTCC13074 (M4) on minimal media (before Sudan staining)

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Fig. 2
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Selective staining with Sudan black B which showed positive for PHA formation with bluish black color colonies

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Fig. 3
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Selective screening of bacteria with PHA granules showed positive results by emitting yellow orange fluorescence with Nile blue A stain on minimal agar

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Table 1 Phenotypical features of M4 strain
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Biochemical activities of the isolate M4

The biochemical activities of the isolate M4 are shown in Table 2. The oxidase, urease, vogues proskauer, nitrate reduction, and motility tests all came back negative, but acid production was observed with 6 sugars during fermentation. At 37°C, maximum growth was observed. M4 has a high ability to hydrolyze substrates (starch, gelatin, casein). M4 is slightly halophilic that allows it to grow up to 3.5% of NaCl concentration. The same strain exhibits γ-hemolysis. The results were compared to the data in Bergey’s Manual of Determinative Bacteriology and the isolate was identified as a facultative anaerobe, non-motile, and coccoid in shape. The isolate M4 was then subjected to 16S rRNA molecular sequencing for further characterization.

Table 2 Biochemical activities of M4
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Fatty acid methyl ester analysis

FAME analysis distinguishes bacterial species more quickly and easily than biochemical characterization. The analysis was performed using GC; FAMEs (fatty acid methyl esters) were formed by methanolysis in acidic conditions. The principal cellular fatty acids were C14:0, C15:0, C16:0, C18:1cis-9, C18:0, iso-C15: 0, iso-C14: 0, anteiso-C17: 0, and C18:1ω-7, and 2 unidentified phospholipids are detected and listed in Table 3. In this study, basing on area %, octane 9 enoic acid was identified as major lipid and (11E)-11- octadecanoic acid was detected as less % of lipid in the M4 strain

Table 3 Fatty acid methyl ester profile of B. paraconglomeratum MTCC13074 (M4)
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16SrRNA sequence of M4 strain

DNA was extracted from M4, and 1% agarose gels (Fig. 4) was used to check the homogeneity. 16S rRNA analysis is an unambiguous identification to classify bacteria at the species level. Using universal primers, the 16S rRNA sequence was amplified. On agarose gels, a single separated PCR product of 1500 bp was detected and the PCR product was refined to remove impurities. The sequence obtained was BLAST searched to retrieve the first ten hits and tabulated (Table 4). The % identity among the top ten closely related species after the BLAST ranged from 99.72 to 97.67%. Using aligner software, the generated consensus sequence was created using forward and reverse sequence data. A phylogenetic tree (Fig. 5) was designed for bacterial culture M4 (Brachybacterium paraconglomeratum) based on 16S rRNA gene sequences using neighbor joining method in MEGA7 software. Isolate M4 was deposited in CSIR-IMTech and accessed as Brachybacterium paraconglomeratum MTCC 13074 (M4). The 16S ribosomal sequence was deposited in GenBank and NCBI, and the accession number assigned is MW899045.

>MTCC13074 Brachybacterium paraconglomeratum strain M4 16S ribosomal RNA sequenceCACATGCAAGTCGAACGATGACGGTGGTGCTTGCACCGCCTGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCCCACTTCGGGATAACCTCGGGAAATCGTGGCTAATACCGGATATGAGCACTCATCGCATGGTGAGTGCTGGAAAGATTTATCGGTGGGGGATGGGCTCGCGGCCTATCAGTTTGTTGGTGAGGTGATGGCTCACCAAGACGATGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCCCTTTCAGTAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTTGTAGGTGGCTTGTCGCGTCTGCCGTGAAAACCCGAGGCTCAACCTCGGGCGTGCGGTGGGTACGGGCAGGCTAGAGTGTGGTAGGGGAGACTGGAACTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCATTACTGACACTGAGAAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCTGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGCACTGGACGGCTGCAGAGATGTGGCTTTCTTTGGACTGGTGCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTGATGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCAAGCATGCTACAATGGTCGGTACAATGGGTTGCGAAACTGTGAGGTGGAGCGAATCCCAAAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCAGTGGCCCATCCTCGTGAGGGAGCTGTCGAAGGTGGGATCGGTGATTGGGACTAAGTCGTAACAGGGTTAACCCGTAA

Fig. 4
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Agarose gel electrophoresis of PCR amplified product (standard DNA ladder of 10kb)

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Table 4 BLAST results of M4 16SrRNA sequence
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Fig. 5
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Phylogenetic tree design for bacterial culture M4 (Brachybacterium paraconglomeratum) using the neighbor joining method in MEGA7 software using 16S rRNA gene sequence.

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Estimation of crotonic acid and PHB

The PHA extracts treated with conc.H2SO4 were measured at the range of 200–600nm according to Slepecky R A method. The concentration was estimated as 2.1 μg/ml in the extracts of B. paraconglomeratum at 237nm (Fig. 6). The total estimated biomass in 1L of culture media was 11.31g/L. Finally, the yield of PHB was estimated to be 129 mg/gdcw (w/w) represented in Table 5. The yield of PHB would be increased by optimizing the parameters of the culture media.

Fig. 6.
figure 6

UV spectra confirmation for crotonic acid at 237nm

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Table 5 PHB production from B. paraconglomeratum
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GC-MS detection of biopolymers

GC–MS analysis was used to confirm the derivatives of PHA/PHB. The outcomes were compared to those of standards. The chromatogram (Fig. 7) of the tested PHA extract revealed four major peaks with retention times of 21.140, 22.635, 30.096, and 35.441min, respectively. The derivatives like Tetradecanoic acid, 13-Docosenoic acid methyl ester, (Z)- Hexadecanoic acid methyl ester, and 9-Octadecenoic acid (Z)- 2-hydroxy-3-[(1-oxohexadecyl) oxy] propyl ester are identified by comparing with GC–MS library, and the same is depicted in the Table 6. This confirms that B. paraconglomeratum is one of the producers of polyhydroxybutyric acid (PHB).

Fig. 7
figure 7

Chromatogram representing the GC confirmation for the derivatives of PHA in the extracts of B. paraconglomeratum

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Table 6 Derivatives of PHA confirmed in the GC-MS analysis
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Physical properties of biopolymer

Thermogravimetric analysis

The thermogram (Fig. 8) showed the melting temperature of extracted PHA at 175.7°C, the PHA in the extract began to lose the mass significantly from 240.2°C and decomposed completely (thermolysis) around 283.4 °C.

Fig. 8
figure 8

Thermogram representing the thermal nature of PHA produced by B. paraconglomeratum

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Viscosity

The viscosity was measured in 1ml of extract of different incubation hours to check the optimum time for PHA production. Data was collected and described in Table 7. Viscosity is the best physical method for detecting the presence of biopolymers in the extract. This could be considered as screening method for PHB.

Table 7 Viscosity of M4 extracts at different incubation times
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Discussion

Ever since petroleum-based plastics have become a major source of pollution, biodegradable plastics have gotten a lot of interest for their unique thermoplastic qualities all over the world. However, due to their greater manufacturing costs, the manufacture of these bioplastics is limited. There are already over 300 distinct bacterial species identified from various environmental conditions that produce PHA. In the current research, partial halophile B. Paraconglomeratum was isolated, sequenced, and screened for PHB production. As reported so far, Sudan black B and Nile blue A staining are viable colony methods that can be employed for the fast detection and isolation of PHA-producing bacteria. When stained with Sudan black B, a preliminary screening agent for lipid bodies in the bacteria, the isolates showed a black-blue coloration [19, 20] and when stained with Nile blue A, a particular dye for PHA granules, the isolates showed positive orange yellowish color [21]. Scanning electron microscopic analysis was done before and reported that Brachybacterium sp. are oval-shaped cells [22, 23]. Similar, biochemical tests were done and reported for Brachybacterium sp. [24]. The fame analysis on different Brachybacterium sp. was done and reported by Takeuchi et al. [22]. In this study, 21 to 22% of peak area for octane 9 enoic acid was identified as major lipid and with less peak area for (11E)-11-octadecanoic acid was detected and shown in the Table 3. The similar pattern of fatty acid methyl ester analysis has been specified in levan-producing Brachybacterium sp. [25]. Brachybacterium saurashtrense sp. nov., a halotolerant root-associated bacterium with plant growth-promoting potential, was also subjected to methyl ester analysis [26]. In the study, neighbor joining (NJ) phylogenetic analysis method was done. NJ is algorithmic and statistically consistent under many models of evolution, also it will reconstruct the true tree with high probability [27]. The phylogenetic relationship between strain KWS-1T and other related members of the genus Brachbacterium is shown by a NJ tree based on 16S rRNA gene (1423 base) sequences and the outgroup was Dermabacter hominis ATCC 49369T (=DSM 7083T) (X91034) [28]. Likewise, in Bacillus and Pseudomonas spp. 16S rRNA sequencing was done and accession numbers are assigned from NCBI [29]. At 235nm, the alpha, beta-unsaturated acids, and beta-hydroxy acids converted into crotonic acid on treatment with conc.H2SO4; this is a rapid UV spectrophotometric assay reported so far [30]. Likewise, crotonic acid is estimated in the production of short side chain-polyhydroxyalkanoates from the Ralstonia pickettii [31]. Cell culture was cultivated as reported previously, and the cell pellet was dried to measure the dry weight of the cell (DCW) in g/L. The difference between dry weight of the cell and dry weight of extracted PHA was used to calculate the residual biomass and % of PHA produced [32]. Similar reports, such as 3-hydroxybutyric acid methyl ester and pentadecanoic acid methyl ester, were found in the chromatogram of the PHBV standard [33]. Likewise, the 3-hydroxybutyric acid methyl ester is a monomer methyl ester of 3-HB, whereas the trimer and tetramer methyl esters of 3-HV and 3-HB are pentadecanoic acid methyl ester and hexadecanoic acid methyl ester, respectively [34]. It was reported that GC detected [butanoic acid, 2-amino-4-(methylseleno); hexanoic acid, 4-methyl-, methyl ester and hexanedioic acid, monomethyl ester] as three distinct peaks of various butenoic acid derivatives from the PHB of P. xiamenensis [35]. In the present study, the presence of PHB in M4 extract was confirmed by GC-MS analysis, by detecting four monomer derivatives of PHB. In TGA, the thermal stability was similar to that of PHB reported by Pillai et al. [19]. The results were consistent with those obtained from the regular PHB (Tm-176.29°C) as well as other investigations. The Tm of Bacillus sp. NA10 is 182.34°C and the enthalpy of PHA fusion is 83.62 J/g, according to TGA findings. The observed thermal stability of the copolymer can be attributed to the sample’s higher hydroxyvalerate content, which may improve its ductility and flexibility [34]. An increase in viscosity indicates the increased production of biopolymers. However, better viscosity values appear at optimized conditions due to high yields of PHA [36].

Conclusion

In the present study, Brachybacterium paraconglomeratum a novel strain was identified from the estuary near Suryalanka Bapatla, India. 16srRNA sequencing and fatty acid methyl ester analysis was used to characterize the strain. B. paraconglomeratum, an actinobacter, has the unprecedented feature of producing biopolymer (PHB). It is reported for the first time that a few derivatives of polyhydroxybutyric acid (PHB) were produced by this bacterium. The strain has promising features of producing bioactive compounds which were revealed by biochemical activities. This research also helps to understand the diversity of PHA-producing bacteria isolated from marine source. Future reporting would focus on the optimization, physiochemical characteristics, and biotechnological applications of PHB produced by the B. paraconglomeratum.