Shewanella algae and Microbulbifer elongatus from marine macro-algae – isolation and characterization of agar-hydrolysing bacteria

Macro-algae are a good source of agar oligosaccharides, which can be obtained through bacterial enzymatic hydrolysis. The agarase enzyme secreted by the micro-organisms cleaves the cell wall of the algae and releases agar oligosaccharides as degradation products with various applications. Agarolytic bacteria were isolated from the marine algae Kappaphycus sp., and Sargassum sp., and studied for their agar-degrading properties. Among the 70 isolates, 2 isolates (A13 and Sg8) showed agarase activity in in vitro assays. The maximum agarolytic index was recorded in the isolate Sg8 (3.75 mm and 4.29 µg ml−1 agarase activity), followed by the isolate A13 (2.53 mm and 2.6 µg ml−1 agarase activity). Optimum agarase production of isolate Sg8 was observed at pH7 and at a temperature of 25 °C in 24–48 h, whereas for isolate A13 the optimum production was at pH7 and at a temperature of 37 °C in 48 h. The identities of the agarolytic isolates (Sg8 and A13) were confirmed based on microscopy, morphological, biochemical and molecular analysis as Shewanella algae [National Center for Biotechnology Information (NCBI) GenBank accession number MK121204.1] and Microbulbifer elongatus [NCBI GenBank accession number MK825484.1], respectively.

Isolation of agarase-producing bacteria from seawater and examination of the enzyme activity

This study aimed to isolate agarase-producing bacteria from seawater, and then determine activity of the agarase. Eight coastal surface seawater samples were collected from Ba Ria - Vung Tau province. Twenty-one bacterial strains that are capable of liquefying agar were isolated. These isolates produced disintegration zones around their colonies on agar plates with diameters ranging from 4.0 to 7.0 cm after an incubation period of 2 days at room temperature. Five bacterial strains (M1, M5, M7, M62B, and M71) that produced large halos on plates were identified belonging to Vibrio genus with identity > 96%. The crude enzyme activities of these strains ranged from 0.15 to 0.22 U/mL in reaction with agarose as substrate. Among isolated strains, the strain M71 showed the highest agarase activity, and was used to examine the degradation of seaweed. The hydrolysis of dried Gracilaria seaweed by the crude enzyme of M71 at concentration of 5% (v/v) released 915 μM/mL reducing sugar after a 24-h incubation period at 40oC.

Flavobacterium faecale sp. nov., an agarase-producing species isolated from stools of Antarctic penguins

Taxonomic studies were performed on an agarase-producing strain, designated WV33T, isolated from faeces of Antarctic penguins. Cells of strain WV33T were Gram-staining-negative, strictly aerobic, orange and rod-shaped. Strain WV33T displayed agarase activity and was able to utilize galactose as a sole carbon source. 16S rRNA gene sequence analysis revealed that strain WV33T was closely related to Flavobacterium algicola TC2T (98.0 % similarity), F. frigidarium ATCC 700810T (96.9 %) and F. frigoris LMG 21922T (96.1 %). The predominant cellular fatty acids were iso-C15 : 1 G, iso-C15 : 0, C15 : 0, C16 : 0 and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c). Menaquinone 6 (MK-6) was the sole quinone identified, and the major pigment was zeaxanthin. The major polar lipid was phosphatidylethanolamine. DNA–DNA relatedness of strain WV33T with respect to its closest phylogenetic neighbours was 25 % for F. algicola NBRC 102673T, 23 % for F. frigidarium DSM 17623T and 21 % for F. frigoris DSM 15719T. The DNA G+C content of strain WV33T was 37±0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain WV33T is concluded to represent a novel species of the genus Flavobacterium , for which the name Flavobacterium faecale sp. nov. is proposed. The type strain is WV33T ( = KCTC 32457T = CECT 8384T).

Enhanced Activity of Intracellular Agarase from a Novel Marine Strain Agarivorans gilvus WH0801

A marine bacterium strain Agarivorans gilvus WH0801 with the efficient agar degradation ability isolated from fresh seaweed samples of Weihai coast was found to be potential in producing agarase. We studied on the optimal medium composition and culture conditions of Agarivorans gilvus WH0801 by statistical methods in shake flasks. First, several more important factors influencing agarase activity were selected by Plackett-Burman design. They are agar concentration, yeast extract concentration and seed age. Then the optimum levels of these three variables were further determined using Box-Behnken design. The highest agarase activity is obtained in the medium consisting of 2.49 g L-1 agar and 0.88 g L-1 yeast extract when the seed age is 25.64 h. The levels of other factors are 1 g L-1 peptone, 0.01 g L-1 ironic citrate at initial pH 7.0 and 28 °C. The whole optimization strategy results in the activity of agarase reaches 1.158 U mL-1, which is about 6.2-fold increase compares with the control.

Simiduia areninigrae sp. nov., an agarolytic bacterium isolated from sea sand

During a study intended to screen for agar-degrading bacteria, strain M2-5T was isolated from black sand off the shore of Jeju Island, Republic of Korea. Strain M2-5T exhibited agarase activity; the β-agarase gene of the isolate had 62 % amino acid sequence identity to the β-agarase gene of Microbulbifer thermotolerans JAMB A94T. The isolate was closely related to members of the genus Simiduia but was clearly discernible from reported Simiduia species, based on a polyphasic analysis. Cells of strain M2-5T were Gram-negative, catalase- and oxidase-positive, motile rods. The DNA G+C content was 53.3 mol%. The predominant isoprenoid quinone was Q-8. The major cellular fatty acids were C17 : 1ω8c (25.9 %), summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c; 17.2 %) and C17 : 0 (15.0 %). Phylogenetic analysis using 16S rRNA gene sequences showed that strain M2-5T had 96.6 % gene sequence similarity to Simiduia agarivorans SA1T, the most closely related type strain of the genus Simiduia . These results suggest that strain M2-5T represents a novel species in the genus Simiduia , for which the name Simiduia areninigrae sp. nov. is proposed; the type strain is M2-5T ( = KCTC 23293T = NCAIM B 02424T).

Sequence Analysis of a 101-Kilobase Plasmid Required for Agar Degradation by a MicroscillaIsolate

ABSTRACT An agar-degrading marine bacterium identified as aMicroscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.