One-Step Process for Environment-Friendly Preparation of Agar Oligosaccharides From Gracilaria lemaneiformis by the Action of Flammeovirga sp. OC4

Abstract

Oligosaccharides extracted from agar Gracilaria lemaneiformis (G. lemaneiformis) have stronger physiological activities and a higher value than agar itself, but the pollution caused by the extraction process greatly restricts the sustainable use of agar. In this study, four bacterial strains with a high ability to degrade G. lemaneiformis were isolated from seawater by in situ enrichment in the deep sea. Among them, Flammeovirga sp. OC4, identified by morphological observation and its 16S rRNA sequencing (98.07% similarity to type strain JL-4 of Flammeovirga aprica), was selected. The optimum temperature and pH of crude enzyme produced by Flammeovirga sp. OC4 were 50°C and 8, respectively. More than 60% of the maximum enzyme activity remained after storage at pH 5.0-10.0 for 60 min. Both Mn²⁺ and Ba²⁺ could enhance the enzyme activity. A "one-step process" for preparation of oligosaccharides from G. lemaneiformis was established using Flammeovirga sp. OC4. After optimization of the Plackett-Burman (PB) design and response surface methodology (RSM), the yield of oligosaccharides was increased by 36.1% from 2.71 to 3.09 g L^-1 in a 250-mL fermenter with optimized parameters: 30 g L^-1 G. lemaneiformis powder, 4.84 g L^-1 (NH4)₂SO₄, 44.8-mL working medium volume at 36.7°C, and a shaking speed of 200 × g for 42 h. The extracted oligosaccharides were identified by thin layer chromatography (TLC) and ion chromatography, which consisted of neoagarobiose, agarotriose, neoagarotetraose, agaropentaose, and neoagarohexaose. These results provided an alternative approach for environment-friendly and sustainable utilization of algae.

Keywords: Flammeovirga sp. OC4; Gracilaria lemaneiformis; oligosaccharides; one-step process; optimization; response surface methodology.

FIGURE 1

Temperature effect on the activity of the crude enzyme solution.

FIGURE 2

Colony morphology and cell morphology identification of strain OC4. (A) Colony morphology. (B) Cell morphology.

FIGURE 3

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Flammeovirga sp. OC4 and representatives of related taxa. Bootstrap values based on 100 replications are shown at branch nodes. The scale bar indicates the average number of substitutions per site.

FIGURE 4

Effect of temperature and pH on enzyme activity of Flammeovirga sp. OC4. (A) The effect of temperature on enzyme activity. The activity was detected at the temperature ranges from 30 to 80°C at pH 7.4. (B) The effect of temperature on the stability of the enzyme. The remaining activity was measured after incubating crude enzyme in the absence of substrate at 30°C, 40°C, and 50°C for various durations. (C) pH effects on the activity of crude enzyme. pH profiles were determined by incubating crude enzyme at 50°C in the following buffers: Na2HPO4/citric acid solution (pH 3.0–8.0), Tris-HCl buffer (pH 7.0–9.0), and Gly/NaOH buffer (pH 9.0–11.0). (D) pH effects on the stability of crude enzyme. Prior to determination of residual activity, the crude enzyme was first incubated in buffers of desired pH (pH 3.0–11.0) at 50°C for 60 min. For all of the above plots, values are presented as percentages of the maximum activity of crude enzyme (taken as 100%) and are expressed as mean of three parallel trials with standard deviation.

FIGURE 5

Effects of each single variable on the production of oligosaccharides. (A) Effect of G. lemaneiformis. The concentration was from 10 to 40 g L⁻¹. (B) Effect of inorganic nitrogen sources. 5 g L⁻¹ of KNO₃, NaNO₃, (NH₄)₂SO₄, NH4NO₃ were tested. (C) Effect of (NH₃)₂SO₄. The concentration of (NH₄)₂SO₄ was from 2 to 10 g L⁻¹. (D) Effect of organic nitrogen sources. 4 g L⁻¹ of yeast extract, peptone, beef extract, bean pulp were tested. (E) Effect of peptone. The concentration of peptone was from 2 to 11 g L⁻¹. (F) Effect of inorganic salts. 10 g L⁻¹ of NaCl, KCl, MgCl₂, MgSO₄ and sea water were tested. (G) Effect of KCl. The concentration of KCl was from 5 to 30 g L⁻¹. (H) Effect of temperature. Different temperatures (25, 28, 30, 33, 37, and 40°C) were tested. (I) Effect of initial pH. Different initial pH (5, 6, 7, 8, 9) were tested. (J) Effect of inocula. Different inocula (1%, 3%, 5%, 8%, 10%) were tested. (K) Effect of medium volume. Different medium volume (30, 40, 50, 60, and 70 mL) were tested.

FIGURE 6

T-value of the medium constituents for oligosaccharide production by Flammeovirga sp. OC4 based on the Plackett–Burman (PB) experimental results. (A) Peptone; (B) KCl; (D) (NH₄)₂SO₄; (E) initial pH; (G) inocula; (H) working medium volume; and (K) temperature.

FIGURE 7

The three-dimensional (3D) response surface curves of the combined effects of medium volume, temperature, and (NH₄)₂SO₄ on oligosaccharide production. (A) Medium volume and temperature at the fixed level of (NH₄)₂SO₄. (B) Medium volume and (NH₄)₂SO₄ at the fixed level of temperature. (C) (NH₄)₂SO₄ and temperature at the fixed level of medium volume.

FIGURE 8

Thin layer chromatography (TLC) analysis (A) and high-performance liquid chromatography (HPLC) chromatograms (B,C) of purified fermentation products of Flammeovirga sp. OC4. (A) Lane 1, purified fermentation products; lane 2, neoagarobiose; lane 3, agarotriose; lane 4, neoagarotetraose; lane 5, agaropentaose; lane 6, neoagarohexaose; lane 7, agaroheptaose; lane 8, neoagarooctaose; lane 9, agarononaose; lane 10, neoagarodecaose; lane 11, agaroundecaose; and lane 12, neoagarododecaose.