Diversity of Marine 1,3-Xylan-Utilizing Bacteria and Characters of Their Extracellular 1,3-Xylanases

Abstract

1,3-xylan is present in the cell walls of some red and green algae and is an important organic carbon in the ocean. However, information on its bacterial degradation is quite limited. Here, after enrichment with 1,3-xylan, the diversity of bacteria recovered from marine algae collected in Hainan, China, was analyzed with both the 16S rRNA gene amplicon sequencing and the culture-dependent method. Bacteria recovered were affiliated with more than 19 families mainly in phyla Proteobacteria and Bacteroidetes, suggesting a high bacterial diversity. Moreover, 12 strains with high 1,3-xylanase-secreting ability from genera Vibrio, Neiella, Alteromonas, and Gilvimarinus were isolated from the enrichment culture. The extracellular 1,3-xylanases secreted by Vibrio sp. EA2, Neiella sp. GA3, Alteromonas sp. CA13-2, and Gilvimarinus sp. HA3-2, which were taken as representatives due to their efficient utilization of 1,3-xylan for growth, were further characterized. The extracellular 1,3-xylanases secreted by these strains showed the highest activity at pH 6.0-7.0 and 30-40°C in 0-0.5M NaCl, exhibiting thermo-unstable and alkali-resistant characters. Their degradation products on 1,3-xylan were mainly 1,3-xylobiose and 1,3-xylotriose. This study reveals the diversity of marine bacteria involved in the degradation and utilization of 1,3-xylan, helpful in our understanding of the recycling of 1,3-xylan driven by bacteria in the ocean and the discovery of novel 1,3-xylanases.

Keywords: 1,3-xylan; diversity; extracellular 1,3-xylanases; marine algae; marine bacteria.

Figure 1

Geographic location of the sampling station (A) and sample images (B). At the sampling station, fresh and rotten seaweeds were collected from a Caulerpa lentillifera aquaculture base (samples C, E, and H) and the nearby beach (sample G) in Wenchang, Hainan, China. Samples C, E, G, and H are Caulerpa sertularioides, rotten Caulerpa lentillifera, unknown rotten algae and Chaetomorpha sp., respectively.

Figure 2

The bacterial communities recovered from algal samples with 1,3-xylan based on 16S rRNA gene amplicon sequencing. (A) Relative abundance of recovered bacteria at the family level. (B) A venn diagram analysis of recovered families from algal samples. (C) A venn diagram analysis of recovered families from media A and B. (D) Principal component analysis (PCA) of the bacterial communities.

Figure 3

Hydrolytic zones of the isolated 1,3-xylanase-secreting bacteria cultured on plates containing 0.2% 1,3-xylan (A) and the neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences of these strains and their closest neighbors (B). These strains were cultured at 30°C for 5days for the observation of their hydrolytic zones. The neighbor-joining tree was built using the Kimura 2-parameter model (Kimura, 1980). A bootstrap test of 1,000 replicates was conducted, and values above 50% are shown. Representative strains for detailed study are marked with solid circles.

Figure 4

Growth and the extracellular 1,3-xylanase activities of Vibrio sp. EA2 (A), Neiella sp. GA3 (B), Alteromonas sp. CA13-2 (C), and Gilvimarinus sp. HA3-2 (D) cultured with 0.2% 1,3-xylan as the carbon source. The 1,3-xylanase activities were determined at 30°C in PBS (pH 7.0), and the highest activity was taken as 100%. The highest activities were 0.25±0.01U/mL (for Vibrio sp. EA2), 0.06±0.001U/mL (for Neiella sp. GA3), 0.21±0.01U/mL (for Alteromonas sp. CA13-2), and 0.10±0.01U/mL (for Gilvimarinus sp. HA3-2). Cultures without 1,3-xylan were treated as controls. The data shown in the graph are from triplicate experiments (mean±S.D.).

Figure 5

The products released from 1,3-xylan by the hydrolysis of the extracellular 1,3-xylanases secreted by Vibrio sp. EA2 (A), Neiella sp. GA3 (B), Alteromonas sp. CA13-2 (C), and Gilvimarinus sp. HA3-2 (D). Extracellular 1,3-xylanases from each strain were incubated with 1,3-xylan at 30°C for 24h. Then, the products were analyzed by HPLC-ELSD. The intensity of the 1,3-xylotriose in each chromatogram was normalized as 100%. The reaction system without 1,3-xylanases was used as the control. The data are representative of the results of triplicate experiments.