S. SHIVAKUMAR

Turk J Biol 36 (2012) 225-232 © TÜBİTAK doi:10.3906/biy-1103-2

Accumulation of poly(3-hydroxybutyrate) by DSM 20145

Srividya SHIVAKUMAR Department of Microbiology, Centre for PG Studies, Jain University, Bangalore 560011 - INDIA

Received: 02.03.2011

Abstract: A gram-positive bacterium that accumulated poly(3-hydroxybutyric) acid (PHB) was isolated from petrol bunk soil. From its morphological and physiological properties and the nucleotide sequence (about 1.5 kb) of its 16S rDNA, it was identifi ed as Microbacterium barkeri DSM 20145. Based on the available literature, this is the fi rst report of PHB accumulation by Microbacterium sp. Th e isolate accumulated 0.6 g/L, or 29.7%, of PHB under growth conditions in a nitrogen-defi cient medium containing glucose and yeast extract (4:1 ratio), with maximum production by 48 h. Th e capacity of the isolate to produce PHB under diff erent carbon and nitrogen sources was also investigated. Th e strain produced appreciable levels of PHB by utilizing hemicellulosic sugars such as glucose, xylose, and arabinose, as well as disaccharides such as lactose and sucrose, alternative carbon sources like wheat bran and carboxymethyl cellulose, and organic acids such as propionic acid, with maximum production achieved using xylose (1.7 g/L, 68%). Among the nitrogen sources tested, peptone supported maximum production (1.4 g/L, 16%), followed by casein (0.9 g/L, 22.6%) and sodium nitrate (0.4 g/L, 26.4%), although the PHB yield was not appreciably high. Th e addition of xylose to the nitrogen-defi cient medium increased the values of PHB yield (g/L) by 2.5 and the PHB accumulation (% dry cell weight) by 2.3; the replacement of yeast extract in the medium with peptone increased the PHB yield (g/L) by 2.05. Th e isolated polymer was highly soluble in chloroform and peaked sharply at 235 nm upon digestion with concentrated H2SO4. Th is study adds a new species to the family of PHB producers.

Key words: Microbacterium barkeri DSM 20145, poly(3-hydroxybutyrate) formation, carbon and nitrogen sources

Introduction genera of have been known to accumulate Poly(3-hydroxybutyric acid) (PHB), the best- PHB as intracellular granules (1). PHB production has known member of the polyhydroxyalkanoate most commonly been studied in microorganisms (PHA) group of polymers, is produced as an belonging to the genera Alcaligenes, Azotobacter, intracellular carbon and energy reserve by a wide Bacillus, and Pseudomonas (1,3-9). Th is polymer is variety of bacteria when grown under conditions synthesized under limited culture conditions and is that limit nutrients other than carbon (1,2). Due to used as a carbon and energy reserve. Microbacterium their biodegradability and biocompatibility, poly(3- species are widely distributed in various environments hydroxybutyrate) and copolymers of 3-hydroxybutyrate (10-23). (3HB) and 3-hydroxyvalerate (3HV), P(3HB-co-3HV), Th e accumulation of PHB by members of have attracted attention as possible alternatives to the genus Microbacterium has thus far not been nonbiodegradable thermoplastics. At least 75 diff erent reported in the literature. In the present study, a

225 Accumulation of poly(3-hydroxybutyrate) by Microbacterium barkeri DSM 20145

Microbacterium species capable of producing PHB Inoculum preparation was identifi ed during screening for PHB-producing For all of the experiments, the inoculum was prepared bacteria. Recent research has focused on the use of in 250-mL Erlenmeyer fl asks containing 50 mL of alternative substrates, novel extraction methods, sterile nutrient broth. Th e fl asks were incubated at 37 genetically enhanced species, and mixed cultures in °C for 24 h on a rotary shaker at 120 rpm. Th e culture order to make PHB more commercially attractive. broth was centrifuged at 8000 × g for 15 min, washed Th e aim of the present research was to determine twice, and suspended in sterile distilled water. Th e PHB production by Microbacterium sp. and to test bacterial cell suspensions were inoculated into the for further PHB production in diff erent nitrogen and nitrogen-defi cient media for PHB production studies. carbon sources. PHB production studies in nitrogen-defi cient media Materials and methods Unless otherwise stated, the nitrogen-defi cient Isolation of PHB-producing microorganisms medium used for cell growth and PHB accumulation

Soil samples from diff erent places in and around studies contained (in g/L): glucose, 10; MgSO4, 0.2; Bangalore were screened for PHB-positive colonies NaCl, 0.1; KH2PO4, 0.5; and yeast extract, 2.5. PHB by plating them on nutrient agar and then fl ooding production studies were carried out in 250-mL the colonies with Sudan Black (SB). Briefl y, a 0.02% fl asks containing 50 mL of the culture medium and SB solution in 95% ethanol was spread over the incubated at 37 °C on a rotary shaker at 120 rpm for colonies for 30-60 min, discarded, and washed with 48 h (with the exception of the time-course study). 95% ethanol (24). Bluish-black colonies were selected Time-course study of PHB formation for PHB production studies. Th e isolated colonies Th e inoculum was prepared as described above and were streaked on nutritive agar until pure cultures PHB production as a function of time (0, 24, 48, could be obtained for use in the identifi cation of 72, and 96 h) was determined during growth in the each separate morphotype from the colonies. Gram nitrogen-defi cient media. staining was used to determine the form, size, and type of aggregation as well as the presence of Eff ect of diff erent carbon and nitrogen sources on characteristic structures such as reserve granules, PHB production endospores, and cysts. Glucose in the nitrogen-defi cient medium was M. barkeri DSM 20145, isolated from a petrol replaced by 10 g/L (w/v) of hemicellulosic sugars bunk soil sample and identifi ed by the established such as glucose, arabinose, and xylose in pure method, was used in this study. Stock cultures were forms, as well as disaccharides such as sucrose and grown and maintained at 4 °C on nutrient agar slants. lactose, organic acids like propionic acid, and the Characterization of the isolated bacteria sugar alcohol mannitol. Additionally, wheat bran and carboxymethyl cellulose (CMC) were used as Th e morphological and physiological properties of carbon sources, while yeast extract was variously the isolate were investigated according to Bergey’s replaced by 0.25% (w/v) peptone, tryptone, casein, Manual of Determinative Bacteriology (25). Th e NH4(SO4)2, NH4Cl, and NaNO3 as nitrogen sources. sequencing of 16S rDNA and the taxonomic studies Th e experiments were conducted in duplicates and of strain DSM 20145 were performed at Chromous the averages of the readings were calculated for dry Biotech Pvt. Ltd. in Bangalore, India. A partial cell weight (DCW) and PHB content measurements. 16S rDNA fragment of approximately 1.5 kb was amplifi ed using high-fi delity polymerase chain Dry cell weight reaction (PCR) polymerase. Th e PCR product was Growth of the organisms was determined by sequenced bidirectionally using the forward, reverse, measuring the dry weight of the bacterial pellet. Th e and internal primers. Th e sequence data were then bacterial pellet was separated by centrifugation at aligned and analyzed to identify the bacterium and 10,000 × g for 10 min, washed twice with distilled fi nd the most closely related strains. water, and dried to a constant weight at 80 °C.

226 S. SHIVAKUMAR

PHB extraction and estimation least 2 independent experiments run in duplicates. For the extraction of poly(3-hydroxybutyrate), the Th e correlation between bacterial DCW (g/L) and the bacterial pellet was lysed in sodium hypochlorite at PHB production (g/L) of the bacteria was determined 37 °C for 2 h and centrifuged at 10,000 × g, and the according to Spearman’s correlation. residue was washed twice with each of the following: water, acetone, ethanol, and diethyl ether. Finally, the Results and discussion residue was extracted with boiling chloroform and fi ltered through Whatman No. 1 fi lter paper. Th e Of the 30 total bacteria screened from diff erent soil chloroform extract was evaporated to dryness, and the samples, the isolate M. barkeri DSM 20145, isolated amount of PHB was quantifi ed following the method from petrol bunk soil, was identifi ed as a PHB- of Law and Slepecky (26) with an ELICO SL150 UV- producing bacterium on the basis of the bluish-black VIS spectrophotometer. For dry weight estimation, colored colonies present upon fl ooding with Sudan the pellet aft er extraction was dried to constant weight. Black B. Th e strain accumulated 0.25 g/L PHB with Th e production of PHB was expressed as PHB yield an 18.3% yield in nutrient broth by 48 h and was (g/L) and PHB accumulation (% DCW). selected for further studies. Th e morphological and taxonomic features of the isolate were examined Statistical analysis (Table). Based on the biochemical characteristics Th e mean and standard deviation values of the DCW, and an analysis of the partial nucleotide sequence PHB yield, and PHB content were calculated from at of the 16S rDNA (approximately 1.5 kb), the isolate

Table. Physiobiochemical characteristics of isolate M. barkeri DSM 20145.

Test Observation Genus Microbacterium Gram staining Gram-positive + Endospores _ _ Cell shape Rods + Rod-coccus style _ _ Arranged at angle to give V-formation ++ Colony color Yellow + Motility Nonmotile -/+ Catalase + + Oxidase _ _ Methyl red + + Voges-Proskauer _ + Casein hydrolysis _ _ Esculin hydrolysis + + Starch hydrolysis + + Pectin hydrolysis + + Tween 20 & 80 hydrolysis + + Acid-fast _ _ Growth in 2% NaCl + + Oxygen requirement + +

H2S _ +/- Acid from glucose + +

227 Accumulation of poly(3-hydroxybutyrate) by Microbacterium barkeri DSM 20145

was determined to be Microbacterium barkeri DSM When tested on the various carbon and nitrogen 20145, with a close homology to M. arborescens DSM sources, the strain exhibited nutritional versatility 20754 (Figure 1). in terms of varied growth and PHB formation. Th e Time-course PHB formation by M. barkeri DSM highest level of PHB accumulation was observed

20145 was investigated in production media. Th ere in the N2-defi cient medium with xylose (DCW 1.7 was a gradual increase in cell biomass until 24 h, aft er g/L, PHB accumulation 68%), followed by the media which it remained constant until 72 h; meanwhile, featuring sucrose (0.8 g/L, 57%), propionic acid PHB formation and accumulation increased (0.8 g/L, 57%), and arabinose (1.0 g/L, 40%). Wheat signifi cantly and showed a maximal value by 48 h bran, CMC, mannitol, and lactose were also found (from 0.18 g/L and 11.96% to 0.683 g/L and 29.69%), to support PHB formation by this strain (Figure followed by a gradual decrease by 72 h (Figure 2). 3). Of the various nitrogen sources tested, peptone Th e decrease in PHB accumulation observed aft er supported maximum PHB formation (1.4 g/L, 16%), the cessation of cell growth could be attributed to followed by casein (0.95 g/L, 22.61%) and NaNO3 the intracellular consumption of PHB as a source of (0.45 g/L, 26.4%), although the PHB yield was not energy and carbon (27). Th e time profi le of growth appreciably high (Figure 4). and PHB accumulation by this strain indicated that maximum PHB formation occurred during the Th e purifi ed polymer was highly soluble in stationary phase, which was followed by a decline in chloroform and 1 N NaOH and moderately soluble in the polymer content. Th e existence of a relationship dioxane, pyridine, and toluene, but insoluble in water, between the DCW and PHB production was also sodium hypochlorite, acetone, ethanol, methanol, investigated, but statistical analysis showed that there and diethyl ether. Digestion of the polymer with was no correlation between DCW (g/L) and the PHB concentrated H2SO4 gave a sharp peak at 235 nm, (g/L) content of the cultures (q = 0.099). characteristic of crotonic acid (Figure 5).

Aub.resist Mbm.thlass Mbm.testac Y17231 AF154098 Mbm.ketred PJ.txt Mbm.barker Mbm.imper2 Mbm.arbor2 AJ249780 Scale: 0.01 Figure 1. Phylogenetic tree depicting close homologs of M. barkeri DSM 20145. Th e tree was constructed by neighbor-joining on a p distance matrix. PJ.txt - strain investigated. Reference strains: Aub.resist - Aureobacterium resistens DMMZ 1710 (T) Mbm.thlass - Microbacterium thalassium IFO 16060 (T) Mbm.testac - Microbacterium testaceum DSM 20166 (T) Y17231 - Microbacterium esteraromaticum DSM 8609 AF154098 - clone BPC090 Mbm.ketred - Microbacterium ketosireducens IFO 14548 (T) Mbm.barker - Microbacterium barkeri DSM 20145 (T) Mbm.imper2 - Microbacterium imperiale DSM 20530 (T) Mbm.arbor2 - Microbacterium arborescens DSM 20754 (T) AJ249780 - str. P 333/02 DSM 12966

228 S. SHIVAKUMAR

2.5 30 1.6 PHB (g/L) 30 1.4 PHB yield (%) 25 25 2 1.2 20 1 20 1.5 0.8 15 PHB (%)

DCW (g) 15 PHB (g/L) 0.6 10 PHB (g/L) 1 0.4 PHB content (%)

10 PHB conent (%) 5 PHB (g/L)/DCW (g) 0.2 0.5 0 0 5 te Casein Peptone Tryptone 0 0 Yeast extract 0 h 24 h 48 h 72 h Sodium nitra Time (h) Ammonium sulfate Ammonium chloride Figure 2. A time-course growth study of M. barkeri DSM 20145 Nitrogen sources showing cell biomass in terms of dry cell weight (DCW), PHB content (%), and PHB accumulation Figure 4. Eff ect of various nitrogen sources on PHB formation (g/L) in nitrogen-defi cient medium. in M. barkeri DSM 20145.

0.5 1.8 PHB (g/L) 80 0.45 1.6 PHB Yield (%) 70 0.4 1.4 60 0.35 1.2 50 1 0.3 40 0.8 0.25 PHB (%)

PHB (g/L) 30 0.6 0.2

0.4 20 Optical density 0.15 0.2 10 0 0 0.1 0.05 Xylose CMC Glucose Sucrose Lactose 0 Arabinose Mannitol Wheat bran 200 220 240 260 280 300 Propionic acid Absorbance (nm) Carbon sources Figure 3. Eff ect of various carbon sources on PHB formation in Figure 5. Ultraviolet absorption spectra [ ] of isolated M. barkeri DSM 20145. PHB following depolymerization and dehydration in concentrated sulfuric acid.

Species from more than 75 genera are known to be genus is also gram-positive and catalase-positive, not capable of synthesizing PHB (28). Th e thermoplastic acid-fast, and not spore-forming. As shown in the properties of the polymer and its biodegradability Table, the taxonomic properties of the strain were determine its importance as a substitute for consistent with those distinguishing characteristics petrochemical plastics (11). of Microbacterium (18,19) and the strain should According to the description of the genus therefore be assigned to that genus. Th e sequence Microbacterium (13,18,19), it has the following comparison results of the 16S rRNA gene of the determinative phenotypic characteristics: irregular strain with that of the related genera also supported rods, but no marked rod-coccus cycle as exhibited the view that the strain may be a member of the genus by some genera of coryneform bacteria (14); the Microbacterium. Comparisons of the 16S rRNA gene

229 Accumulation of poly(3-hydroxybutyrate) by Microbacterium barkeri DSM 20145

sequences between the strain and the close relatives used has a huge infl uence on PHB productivity. retrieved from the databases showed a clear affi liation Th e carbon source must always be provided in with the genus Microbacterium. A phylogenetic tree excess to allow for maximum PHB accumulation of the strain, based on the 16S rRNA gene sequences, in the biomass. M. barkeri DSM 20145 exhibited was constructed using the neighbor-joining method. high nutritional versatility by showing a capacity Th ese data, presented in Figure 1, strongly suggest to utilize hemicellulose sugars, pentoses, hexoses, that strain DSM 20145 should belong to the genus disaccharides, sugar alcohol, wheat bran, CMC, and Microbacterium. organic acid to form PHB. Th e addition of xylose to

Th e occurrence of PHB has been reported from the N2-defi cient medium increased the values of PHB a wide variety of bacteria (1,2,29), but strains of yield (g/L) 2.5-fold and PHB accumulation (% DCW) Microbacterium spp. have not been investigated 2.3-fold. Replacing the yeast in the medium with for this ability until now. A review of the available peptone increased the PHB yield (g/L) 2.05-fold. Th is literature indicates that this is the fi rst report on PHB observation indicates the strain’s potential for use production by Microbacterium sp. in the industrial production of PHB using cheaper alternative carbon sources such as hemicellulosic Some of the A. eutrophus strains used for waste. M. barkeri DSM 12045 showed varied levels commercial PHB production have a PHB of PHB production under diff erent nitrogen sources concentration that is approximately 80% (w/w) of with maximum values obtained in the presence of the DCW (29). Chen et al. (30) studied PHA in 11 peptone. Th e enhancement of PHB accumulation may diff erent Bacillus spp. and found PHB at up to 50% result from the presence of amino acids and peptides (w/v) of the DCW of the bacteria. Microbacterium in peptone, tryptone, and casein. Such stimulation barkeri DSM 20145 accumulated 29.69% PHB by 40 of biosynthesis might be due to the channeling of h. When compared to related studies in the literature, the carbon source into PHB accumulation. Similar these results show a lower PHB production. Th e enhancement of PHB accumulation was reported in A. disparity may result from diff erences in the strains, vinelandii, A. chroococcum, A. beijerinckii, and E. coli types of media, or the cultivation methods used in when the organisms were grown in media containing the individual studies. organic nitrogen sources (31-36). Several renewable Bacteria able to synthesize PHA can be divided materials, including casein hydrolysates and gluten, into 2 groups (29). Th e fi rst group, accumulating are known to be available from agriculture as PHA during the stationary phase, requires the nitrogen sources that can be used as cheap substrates limitation of N, P, Mg, and oxygen, for example, and (37). Th e addition of inorganic nitrogen sources an excess of the carbon sources. Th e most important also supported PHB production. In this study, the microorganism for industrial PHA production, ratio of carbon and nitrogen was always maintained Ralstonia eutropha, belongs to this group. Th e second at 4:1 to allow for maximum PHB accumulation in group, which accumulates PHA during the growth the biomass. Further optimization of the C:N ratio phase, includes Alcaligenes latus, a mutant strain may encourage increased PHB formation by this of Azotobacter vinelandii, A. beijerinckii (27), and strain. Xi et al. (20) determined that PHB synthesis recombinant strains of Escherichia coli bearing the was highly dependent on the nitrogen source. While PHA operon of R. eutropha. Our strain, M. barkeri PHB synthesis by Pseudomonas oleovorans was not DSM 20145, belongs to the fi rst group, because signifi cantly stimulated by nitrogen limitation, P. PHB accumulates during the stationary phase. Th e resinovorans responded to nitrogen limitation by physiological characteristics of the organism are greatly increasing the PHB production rate (16). important for the bioprocess to produce polymer. Th ese observations corroborate our results. Th e Several factors infl uence the economics of identity and purity of the PHB obtained from M. biodegradable polymer production. Th ese factors barkeri DSM 20145 were confi rmed by solubility include substrate cost and the ability to produce properties and UV absorption spectra. Th e Law and biodegradable polymers from inexpensive or Slepecky method detects only PHB and no other renewable substrates (14). Th e type of carbon source polyhydroxyalkanoates. Future studies will focus on

230 S. SHIVAKUMAR

investigating the ability of this strain to accumulate provide new opportunities for the production of various other polyhydroxyalkanoates. cost-eff ective biodegradable plastics. Th e present study highlights the accumulation and partial characterization of PHB by a new strain, M. Acknowledgements barkeri DSM 20145, and examines the infl uence of a variety of carbon and nitrogen sources on the growth Th e fi nancial support of this project, provided by the and PHB production of this strain. PHB accumulation management of Jain University, Bangalore, is highly by this strain is comparable to most other PHB- appreciated. producing gram-positive Bacillus spp. and much higher than the results of Streptomyces, Clostridium, and Nocardia sp. previously reported (38). M. barkeri Corresponding author: DSM 12045 may be an attractive candidate for the Srividya SHIVAKUMAR production of biodegradable plastics, but further Department of Microbiology, research is needed to characterize the type of PHA produced by this strain on glucose and diff erent Centre for PG Studies, Jain University, carbon feeds in order to verify its potential use in the 18/3, 9th Main Jayanagar 3rd block, commercial production of biodegradable polymers. Th e addition of a new species to the existing list Bangalore 560011 - INDIA of PHB-producing microorganisms will certainly E-mail: [email protected]

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