Medium chain length polyhydroxyalkanoates consisting primarily of unsaturated 3-hydroxy-5-cis-dodecanoate synthesized by newly isolated bacteria using crude glycerol

Abstract

Background: Our study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates.

Results: Newly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. (1)H NMR, two dimensional-(1)H-correlation spectroscopy (2D-(1)H-COSY) and (13)C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9% similarity); ASC2, Pseudomonas genus (99.2% similarity); ASC3, Enterobacter genus (99.2% similarity); ASC4, Bacillus genus (98.4% similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3% wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol%), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (M N) of 3.6 × 10(4) Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h.

Conclusions: Novel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.

Keywords: 3-Hydroxy-5-cis-dodecanoate (3H5DD); 3-Hydroxyoctanoate; Acinetobacter sp.; Bacillus sp.; Crude glycerol; Enterobacter sp.; Medium chain length PHAs; Pseudomonas sp.; Used cooking oil.

Fig. 1

Phylogenetic tree constructed based on 16S rDNA sequences. a Acinetobacter sp. ASC1. b Pseudomonas sp. ASC2. c Enterobacter sp. ASC3. d Bacillus sp. ASC4. The trees were constructed by the neighbor-joining method and rooted by referring to Pseudomonas oleovorans or Cupriavidus necator A-04. The numbers at branches refer to the percentage confidence estimated by a bootstrap analysis with 100 replications. The analysis was performed by including 16S rDNA gene sequences from GenBank (accession numbers indicated in parentheses). Bar = 0.1 estimated substitutions per sequence position

Fig. 2

Schematic of the biodiesel production process in which used cooking oils are the substrate

Fig. 3

Time courses of cell dry mass (g/L), mcl-PHA levels (g/L) and total organic carbon (TOC) concentration (g/L) in cultures. a Acinetobacter sp. ASC1. b Pseudomonas sp. ASC2. c Enterobacter sp. ASC3. d Bacillus sp. ASC4. The cells were grown on crude glycerol with a TOC concentration of 10 g/L and a C/N ratio of 200 in a bioreactor in batch mode. Cultures were performed in triplicate. The error bars represent standard deviations

Fig. 4

Transmission electron micrographs of an ultrathin section. a Acinetobacter sp. ASC1. b Pseudomonas sp. ASC2. c Enterobacter sp. ASC3. d Bacillus sp. ASC4. Bars 200 nm

Fig. 5

Effect of the C/N ratio on the specific growth rate of bacteria and specific production rate of PHA. a Acinetobacter sp. ASC1. b Pseudomonas sp. ASC2. c Enterobacter sp. ASC3. d Bacillus sp. ASC4

Fig. 6

500 MHz ¹H-NMR spectrum of P(5 % 3HO-co-95 % 3H5DD) synthesized by Pseudomonas sp. ASC2. Crude glycerol with a TOC concentration of 10 g/L and a C/N molar ratio of 200 were used in the culture. Letters on the spectrum indicate peaks arising from the protons marked in the corresponding structure. 3HO, 3-hydroxyoctanoate; 3H5DD, 3-hydroxy-5-cis-dodecanoate

Fig. 7

2D-COSY-¹H NMR spectrum of P(5 % 3HO-co-95 % 3H5DD) synthesized by Pseudomonas sp. ASC2. Crude glycerol with a TOC concentration of 10 g/L and a C/N molar ratio of 200 were used in the culture. Letters on the spectrum indicate peaks arising from the protons marked in the corresponding structure. 3HO, 3-hydroxyoctanoate; 3H5DD, 3-hydroxy-5-cis-dodecanoate

Fig. 8

500 MHz ¹³C-NMR spectrum of P(5 % 3HO-co-95 % 3H5DD) synthesized by Pseudomonas sp. ASC2. Crude glycerol with a TOC concentration of 10 g/L and a C/N molar ratio of 200 were used in the culture. 3HO, 3-hydroxyoctanoate; 3H5DD, 3-hydroxy-5-cis-dodecanoate

Fig. 9

Time course profiles of mcl-PHA content (wt %) and monomer composition (3HO, 3-hydroxyoctanoate; 3H5DD, 3-hydroxy-5-cis-dodecanoate) (mol %). a mcl-PHA produced by Acinetobacter sp. ASC1. b Pseudomonas sp. ASC2. c Enterobacter sp. ASC3. d Bacillus sp. ASC4. Cells were grown on crude glycerol with a TOC concentration of 10 g/L and a C/N molar ratio of 200. Cultures were performed in triplicate. The error bars represent standard deviations