Cobetia sp. Bacteria, Which Are Capable of Utilizing Alginate or Waste Laminaria sp. for Poly(3-Hydroxybutyrate) Synthesis, Isolated From a Marine Environment

Abstract

We isolated the Cobetia sp. strains IU 180733JP01 (5-11-6-3) and 190790JP01 (5-25-4-2) from seaweeds and showed that both strains accumulate poly(3-hydroxybutyrate) [P(3HB)] homopolymer in a nitrogen-limiting mineral salt medium containing alginate as a sole carbon source. Genome sequence analysis of the isolated strains showed that they have putative genes which encode enzymes relevant to alginate assimilation and P(3HB) synthesis, and the putative alginate-assimilating genes formed a cluster. Investigation of the optimum culture conditions for high accumulation of P(3HB) showed that when the 5-11-6-3 strain was cultured in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and 3% (w/v) alginate as a sole carbon source for 2 days, the P(3HB) content and P(3HB) production reached 62.1 ± 3.4 wt% and 3.11 ± 0.16 g/L, respectively. When the 5-25-4-2 strain was cultured in a nitrogen-limiting mineral salt medium (pH 4.0) containing 5% NaCl and 3% (w/v) alginate for 2 days, the P(3HB) content and P(3HB) production reached 56.9 ± 2.1 wt% and 2.67 ± 0.11 g/L, respectively. Moreover, the 5-11-6-3 strain also produced P(3HB) in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and freeze-dried and crushed waste Laminaria sp., which is classified into brown algae and contains alginate abundantly. The resulting P(3HB) content and P(3HB) productivity were 13.5 ± 0.13 wt% and 3.99 ± 0.15 mg/L/h, respectively. Thus, we demonstrated the potential application of the isolated strains to a simple P(3HB) production process from seaweeds without chemical hydrolysis and enzymatic saccharification.

Keywords: alginic acid brown algae; biopolymer; marine biomass resources; polyhydroxyalkanoate; seaweeds.

FIGURE 1

¹H-NMR spectra of a commercial P(3HB) and P(3HB) produced from the isolated strains grown in the MM medium containing 3% (w/v) alginate at pH 5.0 and 30°C for 2 days. (A) P(3HB) produced from Cobetia sp. IU180733JP01 (5-11-6-3); (B) P(3HB) produced from Cobetia sp. IU190790JP01 (5-25-4-2); (C) commercial P(3HB).

FIGURE 2

Phylogenetic tree of isolated strains with alginate-degrading bacteria (alginate lyase-producing bacteria). The trees were constructed by the neighbor-joining method with MEGA-X software. The GenBank accession no. was described after each microorganism name. The numbers at the branch nodes represent the levels of bootstrap support based on the analyses of 500 replicates. Bar 0.05 substitutions per site.

FIGURE 3

Schematic representation of the putative gene cluster encoding enzymes relevant to alginate assimilation in Cobetia sp. IU180733JP01 (5-11-6-3) and Cobetia sp. IU190790JP01 (5-25-4-2). The gene cluster was 13.5 kbp. Oligoalginate transporter and outer membrane porins genes: gray; alginate lyase genes, black; DEH reductase gene, vertical stripe; Kdg F, horizontal stripe; transcription regulator, white.

FIGURE 4

Predicted biosynthetic pathway of P(3HB) from alginate in Cobetia sp. IU180733JP01 (5-11-6-3) and Cobetia sp. IU190790JP01 (5-25-4-2). PhaA, β-ketothiolase; PhaB, acetoacetyl-coenzyme A reductase; PhaC, PHA synthase.

FIGURE 5

Multiple alignments of putative Class I PHA synthase (PhaC) from Cobetia sp. IU180733JP01 (5-11-6-3) and Cobetia sp. IU190790JP01 (5-25-4-2) with PhaC from Ralstonia eutropha (accession no. WP_078196023) and Chromobacterium sp. USM2 (accession no. ADL70203). Asterisks, identical residues; white box, α/β hydrolase domain of PhaC from R. eutropha and Chromobacterium sp. USM2; black box, catalytic triad residues; gray box, lipase-like box residues. The multiple alignments were performed with Constraint-based Multiple Alignment Tool (COBALT) (Papadopoulos and Agarwala, 2007) at NCBI.

FIGURE 6

Effects of alginate concentration, NaCl concentration, and pH on growth and P(3HB) production in Cobetia sp. IU180733JP01 (5-11-6-3) and Cobetia sp. IU190790JP01 (5-25-4-2). (A) Effect of alginate concentration on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU180733JP01 (5-11-6-3) in the MM medium containing 2% NaCl at 30°C, pH 5.0 for 3 days. (B) Effect of alginate concentration on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU190790JP01 (5-25-4-2) in the MM medium containing 2% NaCl at 30°C, pH 5.0, for 2 days. (C) Effects of NaCl concentration on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU180733JP01 (5-11-6-3) in the MM medium containing 3% (w/v) alginate at 30°C, pH 5.0 for 3 days. (D) Effect of NaCl concentration on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU190790JP01 (5-25-4-2) in the MM medium containing 3% (w/v) alginate at 30°C, pH 5.0, for 2 days. (E) Effects of pH on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU180733JP01 (5-11-6-3) in the MM medium containing 3% (w/v) alginate and 6% NaCl at 30°C for 3 days. (F) Effect of pH on growth, P(3HB) production, and P(3HB) content of Cobetia sp. IU190790JP01 (5-25-4-2) in the MM medium containing 3% (w/v) alginate and 5% NaCl at 30°C for 2 days. Closed bars, dry cell weight; gray bars, P(3HB) production; open bars, P(3HB) content. Dry cell weight was measured after freeze-drying. The P(3HB) content of freeze-dried cells was determined by the weight of the produced P(3HB). The data represent means ± S.D (n = 3).

FIGURE 7

Time profiles of growth and P(3HB) synthesis by Cobetia sp. IU180733JP01 (5-11-6-3) and Cobetia sp. IU190790JP01 (5-25-4-2). (A) Cobetia sp. IU180733JP01 (5-11-6-3) was cultured in the MM medium containing 3% (w/v) alginate and 6% NaCl at pH 5.0 and 30°C. (B) Cobetia sp. IU190790JP01 (5-25-4-2) was cultured in the MM medium containing 3% (w/v) alginate and 5% NaCl at pH 4.0 and 30°C. Filled circles, dried cell weight; opened triangles, P(3HB) content; closed squares, P(3HB) production. The data represent means ± S.D (n = 3). Dry cell weight was measured after freeze-drying. The P(3HB) content of freeze-dried cells was determined by the weight of the produced P(3HB).

FIGURE 8

Time profiles of growth and P(3HB) synthesis by Cobetia sp. IU180733JP01 (5-11-6-3) from waste Laminaria species. Cobetia sp. IU180733JP01 (5-11-6-3) was cultured in the MM medium containing 6% NaCl and 5% (w/v) Laminaria sp. at pH 5.0 and 30°C. After Laminaria sp. was lyophilized and crushed into small chips, the small chips Laminaria sp. were added to the MM medium. Filled circles, dried cell weight; opened triangles, P(3HB) content; closed squares, P(3HB) productivity. The data represent means ± S.D (n = 3). Dry cell weight was measured after freeze-drying. The P(3HB) content of freeze-dried cells was determined by the weight of the produced P(3HB).